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Theses and Dissertations
Summer 2012
Cardiovascular end-organ damage in response to
increased blood pressure variability : impact of
oxidative stress
Kevin Richard Rarick
University of Iowa
Copyright 2012 Kevin Richard Rarick
This dissertation is available at Iowa Research Online: http://ir.uiowa.edu/etd/3370
Recommended Citation
Rarick, Kevin Richard. "Cardiovascular end-organ damage in response to increased blood pressure variability : impact of oxidative
stress." PhD (Doctor of Philosophy) thesis, University of Iowa, 2012.
http://ir.uiowa.edu/etd/3370.
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Part of the Systems and Integrative Physiology Commons
CARDIOVASCULAR END-ORGAN DAMAGE IN RESPONSE TO INCREASED
BLOOD PRESSURE VARIABILITY: IMPACT OF OXIDATIVE STRESS
by
Kevin Richard Rarick
An Abstract
Of a thesis submitted in partial fulfillment of the
requirements for the Doctor of Philosophy degree
in Integrative Physiology in
the Graduate College of
The University of Iowa
July 2012
Thesis Supervisor: Associate Professor Harald M. Stauss
1
ABSTRACT
Baroreflex sensitivity (BRS) is often reduced in elderly populations and patients
with chronic cardiovascular diseases leading to a concomitant rise in blood pressure
variability (BPV) that is associated with increased cardiovascular related morbidity and
mortality. Thus, there is a need to better understand the mechanisms by which BPV
causes cardiovascular end-organ damage. Animal studies using sinoaortic denervation
(SAD) to increase BPV have demonstrated pathologic changes in the structure of the
heart and blood vessels; however, there is a paucity of data investigating changes in
functional measures of the heart and smaller, resistance type arteries. Furthermore, the
pathogenic mechanisms involved in BPV-induced cardiovascular end-organ damage
remain unknown. Baroreceptor denervation results in multiple cardiac stressors, many of
which are associated with production of reactive oxygen species. Oxidative stress is
known to promote cardiovascular end-organ damage but it is unclear if it plays a role in
models of increased BPV. Thus, this study was designed to investigate the functional
responses of smaller resistance type arteries and the heart to chronic exposure to
enhanced BPV. In addition, the role of oxidative stress on these functional responses in a
normotensive rat model of increased BPV was also investigated. Rats were subjected to
either SAD surgery or a sham procedure and were observed for six weeks. To determine
the role of oxidative stress, SAD rats were either treated with the superoxide dismutase
mimetic tempol or left untreated. During the observation period, mean blood pressure
remained normotensive, whereas baroreflex sensitivity was reduced and BPV increased
two to three fold. Weekly in vivo assessment of vascular function of the long posterior
ciliary artery (LPCA) demonstrated a significant reduction in endothelial-dependent
dilation starting three weeks after SAD surgery compared to the sham group. Endothelial-
2
independent dilation was not affected by SAD. Structural changes were not evident in the
LPCA following SAD. However, structural (wall thickness, wall area, and wall
area/lumen area ratio) and functional (strain and distensibility) changes were observed in
the aorta. Cardiac structural (hypertrophy) and functional (diastolic dysfunction) effects
were also evident following six weeks of increased BPV. Antioxidant treatment with
tempol did not have any effect on the SAD-induced increase in BPV or decrease in BRS.
Nevertheless, chronic tempol treatment prevented or reduced the cardiovascular endorgan
damage
(endothelial-dependent
vascular
dysfunction,
decreased
aortic
distensibility, cardiac and vascular hypertrophy, and cardiac dysfunction) observed in the
untreated SAD group. These findings suggest that the pathology observed following SAD
is at least partly mediated by oxidative stress. Antioxidant treatment in patients with
increased BPV (e.g., hypertension, diabetes, heart failure) may prevent or ameliorate
cardiovascular end-organ damage and reduce the overall risk for cardiovascular disease
events.
Abstract Approved: ______________________________________________________
Thesis Supervisor
______________________________________________________
Title and Department
_______________________________________________________
Date
CARDIOVASCULAR END-ORGAN DAMAGE IN RESPONSE TO INCREASED
BLOOD PRESSURE VARIABILITY: IMPACT OF OXIDATIVE STRESS
by
Kevin Richard Rarick
A thesis submitted in partial fulfillment of the
requirements for the Doctor of
Philosophy degree in Integrative Physiology in
the Graduate College of
The University of Iowa
July 2012
Thesis Supervisor: Associate Professor Harald M. Stauss
Graduate College
The University of Iowa
Iowa City, Iowa
CERTIFICATE OF APPROVAL
_________________________
PH.D. THESIS
____________
This is to certify that the Ph. D. thesis of
Kevin Richard Rarick
has been approved by the Examining Committee
for the thesis requirement for the Doctor of
Philosophy degree in Integrative Physiology
at the July 2012 graduation.
Thesis Committee: _______________________________
Harald M. Stauss, Thesis Supervisor
_______________________________
Kevin C. Kregel
_______________________________
Warren G. Darling
_______________________________
Gary L. Pierce
_______________________________
Mark W. Chapleau
TABLE OF CONTENTS
LIST OF FIGURES .......................................................................................................... .iv
LIST OF ABBREVIATIONS ............................................................................................ vi
CHAPTER
I. INTRODUCTION ...............................................................................................1
Background ..................................................................................................1
Review of Literature ....................................................................................2
Clinical importance of BPV .............................................................2
Sinoaortic denervation as a normotensive model of BPV ...............3
SAD-induced cardiovascular end-organ damage.............................5
Cardiac changes following SAD ..........................................5
Vascular changes following SAD ........................................5
Mechanisms of SAD induced cardiovascular-end organ damage ...7
Renin angiotensin system ....................................................7
Mitogen activated protein kinases .......................................9
Oxidative stress ..................................................................10
Tempol as a potential treatment option ..........................................11
Effects of tempol on the heart ............................................11
Effects of tempol on the vessels.........................................13
Purpose of Study .......................................................................................14
II. METHODS.......................................................................................................16
Experimental Design ..................................................................................16
Animals ..........................................................................................16
Experimental protocol ...................................................................16
Surgical procedures .......................................................................17
Weekly Measures ......................................................................................18
Telemetric blood pressure recording ............................................18
Weekly in vivo vascular analysis of the LPCA ............................18
Determination of drug doses for LPCA functional analysis .........19
Endothelial dependent/independent dilation of the LPCA ...........20
LPCA image analysis ....................................................................20
Ultrasound of the ascending aorta .................................................21
Final End-Point Measures .........................................................................23
Cardiac function ............................................................................23
Final heart weight .........................................................................24
Left ventricle histology .................................................................24
Thoracic aorta histology ................................................................25
In vitro vascular function of the MCA ...........................................26
Lipid peroxidation assay ...............................................................27
ii
Statistical Analysis ....................................................................................28
III. RESULTS .......................................................................................................29
Weekly Measures ......................................................................................29
Water and tempol intake ................................................................29
Weekly body weight .....................................................................29
Weekly telemetric blood pressure recordings ...............................29
Determination of drug doses for LPCA functional analysis .........31
In vivo vascular analysis of the LPCA .........................................32
Ultrasound of ascending aorta ......................................................34
Final End-Point Measures .........................................................................36
Cardiac function ............................................................................36
Final heart weight .........................................................................37
Left ventricle histology .................................................................37
Thoracic aorta histology ...............................................................38
In vitro vascular function of the MCA ...........................................38
Lipid peroxidation assay ...............................................................39
IV. DISCUSSION .................................................................................................83
Major Findings ...........................................................................................83
Endothelial dysfunction in small resistance-type arteries ..............83
Role of oxidative stress ..................................................................84
Specific cardiac effects of enhanced BPV .....................................89
Specific vascular effects of enhanced BPV ...................................91
Importance of assessing the time course of BPV-induced effects .93
Study Considerations .................................................................................95
Summary and Conclusion .........................................................................96
Perspective and Future Research Directions..............................................97
REFERENCES ................................................................................................................100
iii
LIST OF FIGURES
Figure 1. Protocol timeline ................................................................................................41
Figure 2. Record of daily water intake...............................................................................42
Figure 3. Weekly changes in body weight .........................................................................43
Figure 4. Weekly telemteric blood pressure values ...........................................................44
Figure 5. Weekly BRS by the sequence technique ............................................................45
Figure 6. Weekly BPV calculated as variance in SBP.......................................................46
Figure 7. Weekly HR derived from the telemetric blood pressure ....................................47
Figure 8. Weekly HRV calculated as SDNN and RMSSD ...............................................48
Figure 9. Description of iris image analysis ......................................................................49
Figure 10. Time needed for maximal LPCA response to corneal application of SNP ......50
Figure 11. LPCA dose response curve to corneal application of SNP ..............................51
Figure 12. Weekly LPCA measures...................................................................................52
Figure 13. Image showing scale of dissected LPCA. ........................................................53
Figure 14. Images of the LPCA during the in vivo vascular function experiment ............54
Figure 15. Weekly LPCA response to pilocarpine ............................................................55
Figure 16. Weekly change in wall area/lumen area ratio in response to pilocarpine ........56
Figure 17. Weekly LPCA response to L-NAME ...............................................................57
Figure 18. Weekly LPCA response to SNP .......................................................................58
Figure 19. Weekly pilocarpine-induced dilation presented as % SNP response ...............59
Figure 20. Description of ultrasound imaging technique of the ascending aorta ..............60
Figure 21. Systolic and diastolic diameter of the ascending aorta .....................................61
Figure 22. Telemetric blood pressure values recorded during ultrasound .........................62
Figure 23. Weekly aortic strain calculated from ultrasound imaging ................................63
Figure 24. Weekly aortic distensibility calculated from ultrasound imaging. ...................64
iv
Figure 25. Aortic distensibility measured after nifedipine administration ........................65
Figure 26. Recording of LV and aortic pressure waveform ..............................................66
Figure 27. LV end diastolic pressure .................................................................................67
Figure 28. LV contractility ...............................................................................................68
Figure 29. LV contractility in response to increased afterload ..........................................69
Figure 30. BRS calculated in response to phenylephrine administration ..........................70
Figure 31. Heart weights normalized to body weight ........................................................71
Figure 32. Representative LV sections prepared with Van Gieson’s stain........................72
Figure 33. Representative histology images of the thoracic aorta .....................................73
Figure 34. Thoracic aorta wall thickness ...........................................................................74
Figure 35. Thoracic aorta wall area ...................................................................................75
Figure 36. Thoracic aorta lumen area ................................................................................76
Figure 37. Thoracic aorta wall area/lumen area ratio ........................................................77
Figure 38. Response of the MCA during the in vitro vascular function experiment .........78
Figure 39. Response of the MCA to ACh presented as % of SNP response. ....................79
Figure 40. Correlation of the endothelial-dependent responses of the MCA and LPCA ..80
Figure 41. Lipid peroxidation assay...................................................................................81
Figure 42. Potential pathways linking BPV, ROS, and cardiovascular damage ...............82
v
LIST OF ABBREVIATIONS
4-HAE: 4-hydroxyalkenals
ACE: angiotensin converting enzyme
ACh: acetylcholine
Ang II: angiotensin II
AT-1: angiotensin II receptor
BHT: butylated hydroxytoluene
BPV: blood pressure variability
BRS: baroreflex sensitivity
CaMKII: Ca2+/calmodulin-dependent protein kinase II
CRP: C-reactive protein
DBP: diastolic blood pressure
DDAH2: dimethylarginine dimethylaminohydrolase
eNOS: endothelial nitric oxide synthase
EPCs: endothelial progenitor cells
HR: heart rate
HRV: heart rate variability
i.p.: intraperitoneal
L-NAME: Nω-nitro-L-arginine methyl ester hydrochloride
LPCA: long posterior ciliary artery
LPO: lipid peroxidation
LV: left ventricle
LV dP/dtmax: left ventricular contractility
LV EDP: left ventricular end diastolic pressure
LVW: left ventricle weight
vi
MAP: mean arterial pressure
MAPKs: mitogen-activated protein kinases
MCP-1: monocyte chemoattractant protein-1
MDA: malondialdehyde
NADPH: nicotinamide adenine dinucleotide phosphate
NO: nitric oxide
Nox4: NADPH oxidase isoform 4
PP: pulse pressure
RAS: renin angiotensin system
RMSSD: square root of the mean squared difference of successive normal intervals
ROS: reactive oxygen species
RVW: right ventricle weight
SAD: sinoaortic denervation
SBP: systolic blood pressure
SBPvar: systolic blood pressure variance
SDNN: standard deviation of normal intervals
SNA: sympathetic neural activity
SNP: sodium nitroprusside
TAC: transverse aortic constriction
TGF-β: transforming growth factor-beta
THW: total heart weight
VSMC: vascular smooth muscle cell
vii
1
CHAPTER I: INTRODUCTION
Background
Blood pressure regulatory mechanisms maintain a relatively constant level of
mean arterial pressure from day to day; however, blood pressure does fluctuate
depending on the time of day, the phase of the respiratory cycle, or in response to
physical or psychological stressors. Having chronically elevated mean arterial pressure,
or hypertension, is positively associated with pathological changes to the structure and
function of the heart and blood vessels. Because of the physiological variation normally
seen in blood pressure, it has been suggested that cardiovascular end-organ damage may
be more closely associated with the average blood pressure obtained from a long-term
(i.e., 24-hour) or continuous mean blood pressure recording than with a single time point
measurement (Mancia 1983). In addition to the mean level of blood pressure, the amount
of spontaneous variability within the blood pressure is also positively correlated to the
severity of organ damage in humans (Parati, Pomidossi et al. 1987; Frattola, Parati et al.
1993). Having an abnormally elevated blood pressure variability (BPV) is considered a
cardiovascular risk factor independent of mean arterial pressure (Mancia, Frattola et al.
1994). In spontaneously hypertensive rats chronically treated to lower mean blood
pressure, cardiovascular damage was significantly decreased only when BPV was also
reduced (Liu, Xu et al. 2003). While it is understood that enhanced BPV is independently
associated with damage to multiple organ systems (Mancia, Frattola et al. 1994), the
physiological mechanisms involved (hemodynamic, neural, humoral, etc.) remain
unclear.
2
Review of Literature
Clinical importance of BPV
Enhanced BPV in hypertensive humans (Mancia, Ferrari et al. 1983) and animals
(Cheng, Kong et al. 1992) has been suggested to result from decreased baroreflex
sensitivity (BRS) (Piccirillo, Bucca et al. 1996). The arterial baroreflex represents an
important control system, buffering the magnitude of spontaneous blood pressure
fluctuations (Mancia, Parati et al. 1986). Reduced BRS can be the result of alterations in
the integrity of the autonomic nervous system (Robinson and Carr 2002) as well as
endothelial dysfunction (Chapleau, Cunningham et al. 1995). Attenuated baroreflex
function following endothelial cell damage is thought to result from a combination of the
reduced excitatory influence of prostacyclin as well as the increased formation of free
radicals and endothelin which contribute to decreased baroreceptor sensitivity (Chapleau
and Abboud 1994). Furthermore, endothelial damage could also reduce nitric oxide
release, which is thought to be an additional mechanism for short-term blood pressure
control independent of the baroreflex (Stauss and Persson 2000). Additional changes to
the mechanical properties of the arterial wall such as vascular calcification and arterial
stiffness may also contribute to reduced BRS (Chesterton, Sigrist et al. 2005).
The clinical importance of elevated BPV in normotensive populations is
highlighted by the many cardiovascular risk factors and diseases that are associated with
elevated BPV secondary to reduced BRS, such as aging (Monahan, Dinenno et al. 2001),
heart failure (Piccirillo, Nocco et al. 2003), post-myocardial infarction (La Rovere,
Bigger et al. 1998), chronic obstructive pulmonary disease (Patakas, Louridas et al.
1982), type II diabetes (Figueroa, Baynard et al. 2007), and chronic renal disease
3
(Johansson, Gao et al. 2007). Of course many additional mechanisms are involved in the
organ damage seen in these different clinical populations. It is unclear if enhanced BPV
primarily contributes to the cardiovascular end-organ damage in these conditions, or if
enhanced BPV occurs secondary as a result of these conditions. However, a negative
correlation has been found between baroreflex function and the New York Heart
Association heart failure (Rostagno, Felici et al. 1999) classification. This suggests that
enhanced BPV may very well contribute primarily to the disease progression or directly
contribute to additional organ system damage. The relevance of abnormal blood pressure
regulation is further indicated by an association between impaired BRS and cardiac
mortality and non-fatal cardiac arrests in patients following myocardial infarction (La
Rovere, Bigger et al. 1998). Furthermore, enhanced BPV has been identified as an
independent predictor of sudden death in chronic renal failure patients (Johansson, Gao et
al. 2007). Finally, in patients with uncomplicated type 2 diabetes, short-term nocturnal
BPV significantly predicts negative cardiovascular end-points such as stroke, myocardial
infarction, or sudden cardiac death even after adjusting for age, smoking, and the mean
level of blood pressure (Eguchi, Ishikawa et al. 2009).
Sinoaortic denervation as a
normotensive model of BPV
Sinoaortic denervation (SAD) is a surgical technique that causes a reduction in
BRS by interrupting the neural pathways from the baroreceptors in the carotid bifurcation
and the aortic arch to the cardiovascular centers in the brain (Krieger 1964). SAD results
in a transient (≤1 week) hypertension and increase in heart rate, but when observed
chronically (>1 week), baroreceptor-denervated animals have an average arterial pressure
4
and heart rate that is similar to baroreceptor intact animals (Sved, Schreihofer et al.
1997). The arterial baroreflex deficit associated with SAD is known to significantly
elevate BPV and plays an independent and important role in associated end-organ
damage observed in SAD animals (Shan, Dai et al. 2001).
Multiple mechanisms including hemodynamic, neural, and endocrine mechanisms
have been suggested to play a role in the increased variability of arterial pressure
observed following SAD. It has been suggested that vascular tone influences BPV in
SAD (Julien, Zhang et al. 1993). Increases in peripheral arterial resistance have been
demonstrated within the first seven to nine days following SAD but these changes in
vascular resistance did not correlate with the changes in BPV (Trapani, Barron et al.
1986). While sympathetic neural activity (SNA) has been shown to be increased in the
acute phase (<1 week) of SAD, chronically the average SNA discharge activity
normalizes. However, the pattern of discharge activity remains more labile than in sham
operated controls, possibly contributing to the increase in BPV (Irigoyen and Krieger
1998). It is likely that the variability seen in arterial pressure following SAD is produced
by the interaction of both neural and humoral factors that act on vascular smooth muscle
to alter vascular tone (Alper, Jacob et al. 1987). Such a mechanism has been suggested
based on experiments using ganglionic blockade which resulted in partial BPV reduction,
but only with the additional blockade of the endogenous humoral vasoconstrictors
vasopressin and angiotensin did BPV in sinoaortic denervated rats return to control levels
(Jacob, Barres et al. 1988). Interestingly, humoral blockade alone did not prevent the
SAD-induced increase in BPV, suggesting that neural activity may have a more
5
prominent or a permissive role in determining the severity of blood pressure lability
(Jacob, Barres et al. 1988).
SAD-induced cardiovascular end-organ damage
Cardiac changes following SAD
Enhanced BPV following SAD is associated with cardiac hypertrophy determined
as increased heart weight normalized to body weight (Miao and Su 2002; Xie, Miao et al.
2003; Zhang, Li et al. 2011). The increase in heart mass may be due to both increased
myocyte cross sectional area (Martinka, Fielitz et al. 2005; Takayama, Kai et al. 2011)
and increased collagen deposition (Martinka, Fielitz et al. 2005; Tao, Zhang et al. 2008;
Flues, Moraes-Silva et al. 2012). These changes have mostly been observed anywhere
from 10 to 16 weeks post SAD with the earliest significant increase in left ventricular
mass (+11%) demonstrated after only 6 weeks of SAD (Van Vliet, Hu et al. 1996). The
cardiac remodeling observed following SAD is associated with elevated left ventricular
end-diastolic pressure and decreased myocardial contractility, suggesting a loss of cardiac
function observed 10 weeks (Flues, Moraes-Silva et al. 2012) and 12 weeks (Martinka,
Fielitz et al. 2005) after SAD. These two studies are the only reports of changes in
cardiac function following SAD. It remains unclear if cardiac dysfunction is already
present after only 6 weeks of SAD, coinciding with the earliest structural cardiac
remodeling observed at this time point (Van Vliet, Hu et al. 1996).
Vascular changes following SAD
Vascular remodeling following SAD, mainly demonstrated in large conduit
arteries, is known to occur in as little as two (Miao and Su 2002) to four weeks (Shen,
Zhang et al. 2006). Similar to the heart, vascular changes have been determined as
6
increased vessel weight (Zhang, Li et al. 2011), increased wall thickness (Miao, Tao et al.
2001; Miao, Xie et al. 2002), and increased collagen deposition (Shen, Zhang et al. 2006;
Zhang, Li et al. 2011). While these changes have all been demonstrated in the aorta,
similar remodeling has also been shown in the pulmonary artery (Tao, Zhang et al. 2008).
The pattern of vascular remodeling is different based on the type of vessel. Large
conductance-type arteries show an increase in both internal and external vessel diameter
suggesting an increase in vascular smooth muscle cell (VSMC) mass. Small resistancetype arterioles respond to enhanced BPV with a different pattern of remodeling
characterized as inward remodeling (a decrease in internal diameter) and no change in
external diameter (Miao, Tao et al. 2001). This difference between large and small vessel
remodeling patterns has also been shown in hypertensive animals and is thought to be due
to the functional properties and blood flow patterns experienced in the different vessel
types (Mulvany 1999). The inward eutrophic remodeling in resistance-type arterioles
may result from a rearrangement of the existing VSMC mass with a proportionally
smaller contribution to VSMC growth (Mulvany 1996).
While the majority of studies using the SAD model have focused on structural
vascular remodeling, there is a paucity of data demonstrating functional vascular changes
following SAD, especially in vessels other than the aorta. Vascular relaxation in response
to acetylcholine (ACh) but not to sodium nitroprusside (SNP) is reduced after 4 to 16
weeks of SAD in isolated thoracic aorta preparations suggesting endothelial-dependent
dysfunction (Miao, Tao et al. 2001; Eto, Toba et al. 2003; Feng, Luo et al. 2011). The
reduced endothelial-mediated relaxation may be due to a decrease in the bioavailability of
NO as both dimethylarginine dimethylaminohydrolase (DDAH2) and endothelial nitric
7
oxide synthase (eNOS) expressions are decreased significantly in the aortae of SAD rats
(Feng, Luo et al. 2011). A decreased aortic distensibility for a given level of arterial
pressure occurs at least six weeks post SAD and may be due to an increased aortic wall
cross-sectional area, increased collagen content (Lacolley, Bezie et al. 1995), or a
decrease in elastin content (Miao, Xie et al. 2002). Interestingly, six weeks of enhanced
BPV following SAD surgery did not reduce endothelial-dependent relaxation observed in
isolated perfused mesenteric arteries (Van Vliet, Hu et al. 1996). The results of this study
would imply that in contrast to the aorta, smaller arteries are somehow protected against
the deleterious effects of SAD on endothelial-dependent dilation. Because the process of
vascular remodeling is different in vessels of varying sizes it may also be possible that
the functional consequences of enhanced BPV differ with respect to the magnitude and/or
time course of the response.
Mechanisms of SAD induced
cardiovascular-end organ damage
Renin angiotensin system
SAD is associated with increased expression of markers of inflammation, such as
cardiac monocyte chemoattractant protein-1 (MCP-1), transforming growth factor-beta
(TGF-β), and induction of macrophage infiltration (Kai, Kudo et al. 2009; Takayama, Kai
et al. 2011). Additionally, there is increased tissue expression of components of the renin
angiotensin system (RAS) while the circulating plasma concentrations remain unchanged
following SAD (Shan, Dai et al. 2003; Feng, Luo et al. 2011; Zhang, Li et al. 2011).
Treatment with an angiotensin receptor (AT-1) blocker prevents SAD-induced
inflammation and cardiovascular end-organ damage. Accordingly, it has been suggested
8
that activation of the cardiac RAS contributes to the pathogenesis of cardiovascular
remodeling and dysfunction following SAD (Kai, Kudo et al. 2009).
In SAD rats, there is increased endothelial cell apoptosis and degeneration (Shen,
Zhang et al. 2006; Feng, Luo et al. 2011). Endothelial progenitor cells (EPCs) preserve
the integrity of the endothelium by maintaining the balance between injury and the
capacity for repair. Reducing the functional capacity of EPCs through apoptosis can
result in a reduction in their ability to maintain the endothelium and ultimately lead to
endothelial-dependent dysfunction (Bao, Wu et al. 2010). It has been shown that eNOS
expression is significantly decreased in blood vessels of SAD rats (Shen, Zhang et al.
2006; Tao, Zhang et al. 2008; Feng, Luo et al. 2011). This reduction in eNOS has been
associated with an increase in apoptotic aortic endothelial cells. The increase in
endothelial cell degeneration and reduction in eNOS expression was prevented with the
coadministration of losartan (1 μM), an AT-1 receptor antagonist (Feng, Luo et al. 2011),
suggesting that activation of cardiac RAS leads to endothelial dysfunction possibly by
invoking an increase in endothelial cell apoptosis. In line with these findings, both AT-1
antagonists and angiotensin converting enzyme inhibitors have been used to successfully
prevent cardiovascular end-organ damage in SAD rats (Miao, Xie et al. 2002; Xie, Miao
et al. 2003; Tao, Zhang et al. 2008). However, RAS-based treatments also prevent any
increase in BPV following SAD (Miao, Xie et al. 2002; Xie, Miao et al. 2003; Tao,
Zhang et al. 2008). Because the stimulus of enhanced BPV is completely removed it is
unclear if there are additional important mechanisms involved in the beneficial effects of
RAS inhibition.
9
Mitogen activated protein kinases
Enhanced BPV following SAD is characterized by an increase in the number of
high pressure episodes and low pressure episodes without a change in the mean level of
blood pressure. It is thought that the increase in blood pressure fluctuations activate
mechanosensitive and autocrine pathways that result in the aforementioned
cardiovascular remodeling and dysfunction (Martinka, Fielitz et al. 2005). While there is
a large number of potential signal transduction pathways, the family of mitogen-activated
protein kinases (MAPKs), which include p38 MAPK, ERK1/2, and others, has been
implicated as critical mechanosensitive regulators involved in cardiovascular end-organ
damage (Molkentin 2004). Furthermore, MAPKs have been shown to be activated in the
SAD model of enhanced BPV (Martinka, Fielitz et al. 2005; Takayama, Kai et al. 2011).
The importance of the involvement of MAPKs has been recently shown by treating SAD
rats with simvastatin, a 3-hydroxy-3-methyl-glutaryl-CoA (HMGCoA) reductase
inhibitor. Simvastatin attenuated myocyte hypertrophy and this change was related to a
reduction in ERK1/2 expression (Takayama, Kai et al. 2011). The authors did not observe
any reduction in myocardial fibrosis, macrophage infiltration, or the expression of TGF-β
or MCP-1 suggesting only a partial involvement of ERK1/2. Nonetheless, this study is
important because it is the only one that demonstrates a reduction in cardiovascular endorgan damage in a treatment protocol that did not reduce the SAD-induced increase in
BPV. However, it also demonstrates the need to further investigate the mechanisms
involved in SAD-induced cardiovascular end-organ damage. In addition to its cholesterol
lowering effects, simvastatin is thought to possess antioxidant properties (Delbosc,
Cristol et al. 2002). Interestingly, nicotinamide adenine dinucleotide phosphate (NADPH)
10
oxidases have been shown to produce reactive oxygen species (ROS) that lead to the
phosphorylation and thereby activation of p38 MAPK (Griendling and Ushio-Fukai
2000). The results of these previous studies further implicate the family of MAPKs as
important intracellular messengers and imply that oxidative stress may act to mediate
MAPK involvement in SAD-induced cardiovascular end-organ damage.
Oxidative stress
AngII is known to play an important role in the development of cardiac
hypertrophy (Baker, Chernin et al. 1990), interstitial cardiac fibrosis (Tokuda, Kai et al.
2004), and left ventricular dysfunction (Schunkert, Dzau et al. 1990). Emerging evidence
indicates that ROS are important signaling molecules in cardiovascular cells and are
involved in transducing many of the effects of AngII such as mediating the activation of
important intracellular signals, such as p38 MAPK (Griendling and Ushio-Fukai 2000).
ROS have been linked to cardiac hypertrophy in response to both AngII (Nakamura,
Fushimi et al. 1998) and mechanical stretch (Pimentel, Amin et al. 2001). NADPH
oxidase has also been shown to contribute to the development of angiotensin II-induced
cardiac hypertrophy, independent of changes in blood pressure (Bendall, Cave et al.
2002).
ROS can also be generated in the blood vessels and are regulated by hormonesensitive enzymes such as the NADPH oxidases. Incubation of VSMC (Griendling,
Minieri et al. 1994) and aortic fibroblasts (Pagano, Chanock et al. 1998) with AngII has
been shown to induce production of ROS such as superoxide in these cells. As stated
above, a main function of ROS is to act as second messengers to activate multiple
intracellular proteins and enzymes which initiate signaling cascades leading to induction
11
of transcription factors involved in VSMC growth and migration, modulation of
endothelial-dependent relaxation, and modification of the extracellular matrix
(Griendling, Sorescu et al. 2000). For example, in endothelial cells it has been shown
that overexpression of NADPH oxidase isoform 4 (Nox4) enhances the phosphorylation
of p38 MAPK (Goettsch, Goettsch et al. 2009). Increased activation of p38 MAPK can
lead to enhanced EPC apoptosis (Bao, Wu et al. 2010) and has been implicated in the
pathogenesis of a variety of cardiovascular diseases (Wang 2007). As second messengers,
ROS can also modulate ion channels increasing intracellular free Ca2+ concentration, a
major determinant of vascular function (Touyz 2004). Conditions associated with
elevated circulating levels of AngII, such as enhanced BPV, may contribute to increased
superoxide production via activation by NADPH oxidases and thus may benefit by
antioxidant treatment (Rajagopalan, Kurz et al. 1996) with compounds such as tempol.
Tempol as a potential treatment option
Effects of tempol on the heart
Tempol (4-Hydroxy-2,2,6,6-tetramethylpiperidine) is a water soluble, membranepermeable superoxide dismutase mimetic that mainly acts as a free radical scavenger but
may also contribute to activating potassium channels or inhibiting the sympathetic
nervous system (Simonsen, Christensen et al. 2009). Two weeks of tempol administration
in the drinking water (1 mMol/L) has been shown to reduce oxidative stress in vivo and
significantly decrease mean arterial pressure (MAP) in spontaneously hypertensive rats
(SHR) (162 ± 8 to 134 ± 6 mmHg) without altering MAP in Wistar Kyoto normotensive
rats (WKY) (115 ± 5 vs. 118 ± 8 mmHg) (Schnackenberg, Welch et al. 1998). In rats with
chronic AngII infusion, Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME)
12
attenuated
tempol-mediated
anti-hypertensive
effects
and
improved
regional
hemodynamic control (Nishiyama, Fukui et al. 2001) suggesting that AngII-induced
oxidative stress inactivates NO (Yang, Li et al. 2011). Tempol has not previously been
used to treat sinoaortic denervated animals. It, therefore, remains unknown if tempol will
reduce enhanced BPV following SAD as is the case with other treatment options, such as
angiotensin converting enzyme (ACE) inhibitors (Tao, Zhang et al. 2008).
Several studies in other disease models of enhanced tissue RAS-induced oxidative
stress suggest a beneficial effect of tempol in preventing cardiac remodeling and
dysfunction (Hasegawa, Takano et al. 2006; Yagi, Akaike et al. 2010; Rizzi, Castro et al.
2011). Tempol also effectively reduced cardiac end-organ damage in mice on a highfructose diet subjected to transverse aortic constriction (TAC), also known as aortic
banding, for eight weeks (Chess, Xu et al. 2008) and in a diabetic murine model without
aortic banding (Ritchie, Quinn et al. 2007). In contrast, tempol did not reduce cardiac
hypertrophy despite reducing the cardiac tissue superoxide concentration in a rat model
of AngII-induced hypertension (Rugale, Delbosc et al. 2007) or in mice subjected to TAC
for four weeks prior to the start of tempol treatment (Moens, Takimoto et al. 2008). As
stated previously, ROS are known to activate cardiac MAPK signaling pathways leading
to cardiac hypertrophy and fibrosis. Tempol has been shown to effectively and
completely eliminate increased phosphorylation of MAPKs while preventing both cardiac
hypertrophy and fibrosis in the diabetic myocardium (Gurusamy, Watanabe et al. 2006).
In another study, tempol prevented cardiac fibrosis but did not reduce the enlarged
cardiac mass in chronic isoproterenol-infused rats (Zhang, Kimura et al. 2005). Because
of the discrepancies observed in previous studies the role of tempol in preventing cardiac
13
remodeling remains unclear. The results suggest that tempol treatment can prevent
cardiac fibrosis and may be able to prevent the onset of cardiac hypertrophy depending
on the study conditions but is less likely to successfully reverse a pre-existing pathologic
increase in cardiac mass.
Effects of tempol on the vessels
Similar to the heart, enhanced oxidative stress has been suggested to contribute to
vascular remodeling (Dikalova, Clempus et al. 2005). Accordingly, tempol treatment
decreases oxidative stress resulting in at least a partial reversal of vessel wall hypertrophy
while increasing luminal area (DeMarco, Habibi et al. 2008). Hypertension is associated
with increased amounts of ROS in the aortic wall which results in VSMC growth,
increased collagen deposition, and impairment of endothelial function thought to be due
to reduced NO bioavailability (Zicha, Dobesova et al. 2001; Castro, Rizzi et al. 2009;
Simonsen, Rodriguez-Rodriguez et al. 2009). Aging is also associated with marked
endothelial dysfunction that is mediated by ROS (Modrick, Didion et al. 2009; Trott,
Seawright et al. 2011; Fleenor, Seals et al. 2012). In both hypertensive and older animals,
eNOS activity was restored and the vascular response to ACh was improved with chronic
tempol treatment (Modrick, Didion et al. 2009; Durand, Moreno et al. 2010).
Additionally, tempol reduces large artery stiffening by normalizing arterial collagen
deposition (Fleenor, Seals et al. 2012).
As with the heart, there is evidence for a RAS/oxidative stress-induced MAPK
signaling cascade in blood vessels. A transgenic rat model (TG(mRen2)27), which
harbors the mouse renin transgene and has elevated tissue AngII levels exhibits increased
NADPH oxidase activity resulting in greater endothelial cell apoptosis and deactivation
14
of eNOS (Wei, Whaley-Connell et al. 2007). The importance of oxidative stress in this
model is exemplified by the normalization of apoptosis and eNOS activation following
tempol treatment. Furthermore, an association between ROS and p38 MAPK was
demonstrated by introducing elevated levels of C-reactive protein (CRP) into coronary
arteries (Blaschke, Bruemmer et al. 2004). CRP is known to increase as part of the
immune systems inflammatory response and is thought to be involved in cellular
apoptosis. Incubating arteries with CRP results in superoxide-mediated endotheliumdependent dysfunction (Blaschke, Bruemmer et al. 2004). This detrimental effect of CRP
on NO-mediated vasodilation can be prevented by treatment with tempol, the NADPH
oxidase inhibitor apocynin, or the p38 MAPK inhibitor SB203850 (Qamirani, Ren et al.
2005), suggesting p38 MAPK is intricately involved in oxidative stress signaling
pathways including those involved in NO bioavailability.
Purpose of Study
The hypothesis of this study is that SAD leads to increased production of ROS,
which mediate the subsequent cardiovascular remodeling and dysfunction. The specific
aims of this project will be to first determine the extent of cardiovascular remodeling and
dysfunction in response to enhanced BPV secondary to a partial loss of baroreflex
sensitivity. Unique to this project, we will be using an in vivo repeated measures
technique to determine the time course of changes in vascular function in small arterioles
and the ascending aorta. Secondly, we will investigate the role that oxidative stress may
play in SAD-induced cardiovascular end-organ damage by chronic administration of
tempol in the drinking water to prevent the accumulation of ROS.
15
Despite multiple published studies characterizing the cardiovascular end-organ
damage following SAD in normotensive rats, the pathogenesis remains unknown. While
the previous studies investigating SAD all tend to demonstrate similar findings, the time
course of SAD-induced cardiovascular end-organ damage is largely unknown. Previous
studies have used either a single end-point measurement (commonly at 2, 6, 10, 12, or 16
weeks) or were investigating changes at different weeks but still utilizing end-point
measurements obtained in different groups of animals for each time point. To my
knowledge, this project is the first to utilize repeated in vivo measures within the same
animal to determine the time course of cardiovascular end-organ damage following SAD.
Additionally, it remains to be seen if endothelial-dependent function is reduced in small
arteries or arterioles as BPV-induced endothelial-dysfunction has only previously been
observed in the thoracic aorta. Furthermore, antioxidant treatments have not been
investigated with regards to end-organ damage in response to enhanced BPV. The
evidence presented in the introduction to this thesis presents a strong case for an
important role of oxidative stress, possibly activated by the local tissue RAS, in the
cardiovascular end-organ damage observed following SAD.
16
CHAPTER II: METHODS
Experimental Design
Animals
All experiments were performed on 8-week-old male, normotensive Sprague
Dawley rats (Harlan Laboratories, Haslett, MI). Rats had free access to a standard rat
chow diet and drinking water. Rats were housed in a temperature (24±2°C), humidity
(60±10%), and 12-hour light cycle (lights on: 0600-1800) controlled environment. All
experiments have been approved by the Institutional Animal Care and Use Review
Committee at The University of Iowa.
Experimental protocol
The experimental protocol is illustrated in Figure 1. At an age of eight weeks, in
vivo imaging of the iris and ultrasound echocardiography were completed to determine
baseline conditions of vascular structure and function of the long posterior ciliary artery
(LPCA) and the ascending aorta, respectively. After baseline values were obtained,
normotensive Sprague Dawley rats had a telemetric blood pressure sensor implanted and
underwent either SAD surgery to induce an increase in BPV or a sham surgery to serve as
controls with normal BPV. The sinoaortic denervated rats were divided into untreated
(SAD) or tempol-treated groups (SAD+Tempol). The SAD+Tempol group received
chronic administration of tempol (1mMol/L, ~21mg·kg body weight-1·day-1) via the
drinking water throughout the entire time course of the protocol, starting immediately
after the SAD surgery was completed. The water bottles containing the tempol solution
were wrapped with aluminum foil because tempol is light sensitive. The tempol solution
was replaced three times per week. During the six-week observation period, blood
17
pressure (radio-telemetry) was recorded and in vivo imaging of the iris and ultrasound
imaging of the ascending aorta were performed once per week in all animals of all three
groups. After the six-week observation period, additional in vivo and in vitro end-point
parameters of cardiac and vascular structure and function were obtained.
Surgical procedures
Rats were anesthetized using isoflurane (~2% isoflurane in room air) throughout
all surgical procedures. A telemetric blood pressure sensor (TA11PA-C40; Data
Sciences, St. Paul, MN) was implanted with the tip of the catheter inserted into the
abdominal aorta and fixed into position at a level caudal to the renal arteries. SAD
surgery was performed bilaterally by exposing both common carotid arteries through a
midline incision in the neck region, removing the adventitia from each carotid bifurcation
and brushing its branches with a 10% phenol solution (phenol diluted with 70% ethanol).
Medial to the vagus nerve, the aortic depressor nerve was identified and sectioned
bilaterally. The laryngeal nerve that also contains aortic baroreceptor fibers was not cut.
This procedure is consistent with a complete carotid sinus denervation and a partial aortic
baroreceptor denervation. Thus, the BRS in the SAD rats is expected to only be partially
reduced thereby more closely mimicking the condition observed in elderly populations
and those with chronic diseases (Ketch, Biaggioni et al. 2002). For the control group, a
sham operation was performed by exposing each carotid artery but without applying
phenol and without cutting any nerves. Survival rate following SAD was ~69% while it
was 100% in the sham operated control group. This mortality rate is similar to what has
been previously reported for animals undergoing SAD surgery (Van Vliet, Chafe et al.
18
1999; Miao and Su 2002). The cause of mortality was related to respiratory
complications, with death occurring during surgery or within five days following surgery.
Weekly Measures
Telemetric blood pressure recording
Starting one week after SAD or sham surgery, weekly telemetric blood pressure
recordings were obtained throughout the six-week observation period. Blood pressure
was recorded for approximately one hour during the middle of the day (between the hours
of 1000-1400) while the rats were minimally active or resting. Average heart rate (HR),
systolic blood pressure (SBP), mean arterial pressure (MAP), and diastolic blood pressure
(DBP) were derived from the blood pressure waveform recordings. Systolic blood
pressure variance (SBPvar) and baroreflex sensitivity (sequence technique) were
calculated
using
the
freely
available
Hemolab
software
(http://www.haraldstauss.com/HemoLab/HemoLab.html) to confirm successful SAD
surgery. Additionally, time domain measures of heart rate variability (HRV) were also
calculated using the Hemolab software.
Weekly in vivo vascular analysis of the LPCA
The LPCA in the iris of the left eye of each rat was photographed through a slitlamp biomicroscope (model FS-2; Nikon Instruments, Melville, NY) using a 30x
objective lense and a Canon 50D (15 megapixels) digital camera (see Figure 9). To
increase optical resolution, a 3x teleconverter was inserted between the slit-lamp and the
digital camera, resulting in a final optical magnification of 90x and a final digital image
resolution of 0.53 µm/pixel. To obtain maximal image quality the camera was operated at
the highest resolution and highest quality setting, and the images were stored in lossless
Tagged Image File format. All of the imaging procedures were done in conscious rats
19
manually held in front of the slit-lamp biomicroscope by one investigator while a second
investigator operated the slit-lamp and the digital camera. The methods for this procedure
and some of the preliminary experiments were part of a separate study that has previously
been published (Stauss, Rarick et al. 2011).
Determination of drug doses for
LPCA functional analysis
In preliminary experiments, the time needed to elicit the maximal vasodilator
response following corneal drug application for pilocarpine (1% Pilocarpine
Hydrochloride Ophthalmic Solution, Falcon Pharmaceuticals, Fort Worth, TX) and SNP
(Sigma Aldrich) were determined. These experiments were performed on a separate
group of eight-week-old Sprague Dawley rats that were not used otherwise for the
chronic study. Iris imaging was carried out on different days to avoid drug interactions
and to allow time for the rats to recover. Thirty microliters of each drug solution was
applied on the cornea after a baseline image of the LPCA was taken. Subsequently,
images of the LPCA were taken every 2-3 min for approximately 30-45 minutes
depending on how long the dilator response was visible. After determining the time
required for each drug to elicit its maximal response, a dose response curve was
constructed for each drug. After a baseline image of the LPCA was taken, the lowest dose
of the drug was applied to the cornea, and after waiting the necessary amount of time to
obtain the maximal response a second set of images was taken. This procedure was then
repeated for each subsequent higher dose. This allowed for determination of the lowest
concentration of each drug that would still elicit a maximal vasodilation. In similar
experiments, the necessary dose of L-NAME needed to effectively inhibit pilocarpine-
20
induced vasodilation was determined. Briefly, a baseline image was taken and then the
LPCA was pretreated with one of several concentrations (0%, 1%, 2%, or 4%) of the NOsynthase inhibitor L-NAME (Sigma-Aldrich, St. Louis, MO). Five minutes after corneal
application of one of the L-NAME concentrations, pilocarpine was administered on the
cornea and after waiting 10 minutes (time point of maximum response) images of the
LPCA were taken to determine if pilocarpine administration was still able to elicit a
vasodilatory response if the LPCA was pre-treated with the respective dose of L-NAME.
Endothelial dependent/independent
dilation of the LPCA
Before application of any drugs, initial images of the LPCA were taken in order to
determine baseline vascular parameters that served as a reference for determining the
changes elicited by corneal application of the different drugs. First, to determine
endothelial-dependent dilation, a pilocarpine solution (30 µl, 1% solution in sterile saline)
was applied to the cornea and after waiting approximately 10 minutes for drug absorption
and action a second set of images was taken. Next, in order to block endogenous NO
production an L-NAME solution (30 µl, 4% solution in sterile saline) was instilled on the
cornea and a third set of images was taken after waiting approximately 10 minutes.
Finally, endothelium-independent maximal vasodilation of the LPCA was determined by
applying the exogenous NO donor SNP (30 µl, 0.1M diluted in 0.9% sterile saline) to the
cornea before a fourth set of images was taken.
LPCA image analysis
The photographs of the LPCA were analyzed using the image processing
software, Imager, which is included with the freely available HemoLab software package
21
(http://www.haraldstauss.com/HemoLab/HemoLab.html). This software visualizes the
images such that each pixel within the digital photograph is displayed at exactly one pixel
of the computer screen, thus avoiding scaling or interpolation and providing the highest
possible image resolution for data analysis. Using a crosshair cursor, the investigator
marks four points in perpendicular orientation to the longitudinal axis of the artery. Two
of the four points mark the outer edges of the vessel wall and two points mark the inner
edges of the vessel wall corresponding to the lumen of the artery. Based on these four
points the Imager software calculates the cross-sectional area of the artery (calculated as
an area of a circle with the diameter determined by the outer edges of the vessel wall) and
the lumen area (calculated as an area of a circle with the diameter determined by the inner
edges of the vessel wall). The Wall/Lumen ratio is then calculated as W/L ratio = [(area
of the artery – area of the lumen)/area of the lumen]. For each image, 10 measurements
are taken, and the average of the 10 measurements is used for statistical analysis (see
Figure 9).
Ultrasound of the ascending aorta
Weekly ultrasound imaging (7.5 MHz linear-array transducer; ultrasound model
CMS600B-3; Contec Medical Systems, Qinhuangdao China) was performed to assess the
time course of changes in vascular parameters of the ascending aorta following SADinduced increase in BPV. Imaging was performed with the rats under isoflurane
anesthesia (~2% in room air). The ascending aorta was imaged using a motion mode (Mmode) recording in the parasternal long-axis with the rat in the lateral decubitus position.
The M-mode image was analyzed in order to assess the systolic and diastolic diameters of
the
ascending
aorta
using
the
Imager
software
(HemoLab
software;
22
http://www.haraldstauss.com/HemoLab/HemoLab.html, see Figure 20). While the Mmode images were being taken, the telemetric blood pressure signal was simultaneously
recorded to obtain the aortic pulse pressure (Pressuresystole – Pressurediastole). The measured
diameters and pulse pressure were then used to calculate the aortic strain and aortic
distensibility as previously described by Stefanadis et al. (Stefanadis, Dernellis et al.
2000) using the following two equations:
1. Aortic Strain = [(systolic diameter – diastolic diameter)/diastolic diameter]
2. Aortic Distensibility = (2*Strain/pulse pressure)
Aortic strain and distensibility are commonly used as indices of aortic stiffness.
The aortic strain provides a measure of how much the aortic diameter increases in systole
compared to diastole. Aortic distensibility normalizes the aortic strain by the pulse
pressure to allow for comparison across groups that may have differences in their
distending pressures (such as SAD vs. Sham). A stiffer aorta, as experienced with age or
atherosclerosis, would be expected to have lower values for aortic strain and distensibility
when compared to a more compliant aorta. Additionally, in a small subset of animals
(SAD n=2; SAD+Tempol n=3) a second set of images were taken after intraperitoneal
(i.p.) administration of the calcium channel blocker nifedipine (5 mg/kg body weight) in
order to assess the aortic diameter under physiologic vascular tone (without nifedipine) as
well as in a relaxed vascular state (with nifedipine). The maximal response of the i.p.
nifedipine injection occurred within ~2 minutes at which point the M-mode image of the
ascending aorta was taken. The telemetric blood pressure signal was also recorded during
the imaging following nifedipine administration.
23
Final End-Point Measures
Cardiac function
Following completion of the six-week observation period, cardiac diastolic and
systolic function were determined by measuring left ventricular end diastolic pressure
(LV EDP) and left ventricular contractility (LV dP/dtmax), respectively. Experiments were
performed while the rats were anesthetized (~2% isoflurane in room air). A 23-gauge
needle-tipped, heparinized-saline filled catheter was inserted through the chest wall into
the left ventricle (LV). Placement of the catheter inside the LV was verified by visual
observation of the characteristic left ventricular pressure waveform. Once the catheter
was in place and flushed with heparinized-saline, a 10-minute baseline recording of the
LV pressure signal was obtained. After the baseline recording, intravenous bolus
injections of the selective α1-adrenergic receptor agonist phenylephrine (0.5ml of
10µg·ml-1) were given to determine changes in LV EDP and LV dP/dtmax following an
increase in cardiac afterload caused by elevated total peripheral resistance and mean
arterial pressure. Phenylephrine injections were repeated three times to verify the
response. LV pressure was allowed to return to baseline before each subsequent injection.
The
Analyzer
program
(HemoLab
software;
http://www.haraldstauss.com/HemoLab/HemoLab.html) was used to calculate LV EDP
and LV dP/dtmax on a beat-by-beat basis as described in the Hemolab manual. LV EDP
was determined using the zero-crossing of the second derivative of the LV pressure
waveform as a trigger. See Figure 26 for a representative pressure waveform and heart
rate recording during this experiment.
24
Final heart weight
Following completion of the left ventricular pressure experiment, rats were deeply
anesthetized using isoflurane (~5% isoflurane in room air) and euthanized by
exsanguination and subsequent removal of the heart. Blood was withdrawn by cardiac
puncture, collected into heparinized tubes, and immediately centrifuged (2000g for 20
minutes) at 4°C for separation of the plasma which was then flash frozen in liquid
nitrogen and stored at -80°C until analyzed. Upon removal, the hearts were placed into
cold (4°C) Krebs-Henseleit solution (D-Glucose 2.0g/L, Magnesium Sulfate 0.141g/L
Potassium Phosphate Monobasic 0.16g/L, Potassium Chloride 0.35g/L, Sodium Chloride
6.9g/L, Calcium Chloride 0.29g/L, and Sodium Bicarbonate g/L) and immediately
dissected by removal of any remaining vasculature or connective tissue, and any blood or
fluid from within the ventricles. The atria were removed before the total heart weight
(THW) which included only the left and right ventricles was measured and recorded. The
right and left ventricles were then separated; the left (LVW) and right (RVW) ventricles
were weighed, and both ventricles were stored for further analyses. The right ventricle
was kept whole and wrapped in aluminum foil and flash frozen in liquid nitrogen. The
left ventricle was further dissected into the base and apex. The base of the left ventricle
was freshly embedded in Tissue-Tek optimal cutting temperature compound (Sakura
Finetek, Torrance, CA) and slowly frozen in liquid nitrogen for histological analysis
while the apex was wrapped in aluminum foil and flash frozen in liquid nitrogen for
biochemical analysis.
Left ventricle histology
The base or superior half of the left ventricle, which was freshly embedded in
Tissue-Tek optimal cutting temperature compound (Sakura Finetek, Torrance, CA),
25
frozen in liquid nitrogen, and stored at -80°C was allowed to equilibrate to -20°C in a
freezer for 24 hours and then mounted in a microtome (model 855; American Optical
Scientific Instruments, Buffalo, NY) at -20°C. Three consecutive transverse sections (20
µm) starting from the middle of the left ventricle and working towards the base were
mounted on glass slides that had been coated with gelatin. The slides were then processed
with Van Gieson’s stain to differentiate the cardiac muscle tissue which stains yellow
from the connective tissue which stains pink (See Figure 32 for representative sections).
Three fields of vision (10x magnification) of each ventricular section were imaged using
a color camera (TCA-5.0) mounted on a microscope (Olympus CKX41). The images
were taken of both the free wall and the septal wall and were assessed via ImageJ
software (National Institutes of Health, Public Domain, Bethesda, MD). The percent area
of connective tissue per field was determined by using the ImageJ Threshold Color
plugin which allows for the selection and measurement of pixel area based on color
differences.
Thoracic aorta histology
After the rats were euthanized, the thoracic aorta was excised and placed in a
small dissecting dish filled with cold (4°C) Krebs-Henseleit solution (Sodium Chloride
6.9g/L, Magnesium Sulfate 0.141g/L Potassium Phosphate Monobasic 0.16g/L,
Potassium Chloride 0.35g/L, and Sodium Bicarbonate g/L without glucose and calcium
chloride). The thoracic aorta was dissected free of any remaining connective tissue and
gently flushed to remove any blood and then placed in a 10% neutral buffered formalin
fixative for histological analysis. Once fixed, the thoracic aortas were embedded in
Tissue-Tek optimal cutting temperature compound (Sakura Finetek, Torrance, CA) and
26
mounted in a microtome (model 855; American Optical Scientific Instruments, Buffalo,
NY) at -20°C. Approximately five consecutive cryosections (40 µm) were obtained for
analysis starting immediately at the point where the aortic arch was dissected from the
thoracic aorta. This ensured that the same section of each vessel was analyzed for each
rat. The sections were stained with Van Gieson’s stain (See Figure 33 for representative
sections) and imaged using a digital camera mounted on an inverted microscope. The
wall and lumen cross-sectional areas were determined using the ImageJ software
(National Institutes of Health, Public Domain, Bethesda, MD), and morphological
parameters were calculated.
In vitro vascular function of the MCA
After the rats were euthanized, the proximal section of the middle cerebral artery
(MCA) was removed from the brain in a small dissecting dish filled with cold (4°C)
Krebs-Henseleit solution (D-Glucose 2.0g/L, Magnesium Sulfate 0.141g/L Potassium
Phosphate Monobasic 0.16g/L, Potassium Chloride 0.35g/L, Sodium Chloride 6.9g/L,
Calcium Chloride 0.29g/L, and Sodium Bicarbonate g/L) bubbled with 95% O2/5% CO2
gas. The excised segment of the MCA was then moved to a vessel chamber containing
the same gassed Krebs-Henseleit solution that was warmed to 37°C. The MCA was
attached by silk suture material (7-0) to glass pipette cannulae and pressurized to 90
mmHg for all experiments. The viability of the vessel was determined by verifying that it
responded to changes in pressure and constricted in the presence of a high potassium
Krebs-Henseleit solution. The vessel was allowed to equilibrate for 30 minutes in the
regular potassium Krebs-Henseleit solution before any experiments were started. The
MCA was pre-constricted (~30% of the baseline diameter) by administration of
27
vasopressin (concentration = 1x10-10 to 1x10-11M) and allowed to re-equilibrate for
another 30 minutes. Under the continuous presence of vasopressin, an ACh dose response
curve (1x10-7 to 1x10-3 M) was performed to determine the endothelial-dependent
function. The endothelial-independent function was also determined by measuring the
response to a maximal dose of SNP (1x10-3M). In a small group of rats (SAD: N=3;
Sham: N=2), the above experiments were repeated in the presence of tempol (1x10-3M,
Sigma Aldrich) to determine if endothelial function would be restored following
pretreatment with the superoxide dismutase mimetic. In this experiment, the vessel was
again pre-constricted with vasopressin and allowed to equilibrate for 30 minutes with the
addition of tempol before the ACh dose response curve was repeated.
Lipid peroxidation assay
Frozen cardiac tissue samples from the ventricles were analyzed to determine the
tissue concentration of malondialdehyde (MDA) and 4-hydroxyalkenals (4-HAE) as an
indicator of lipid peroxidation and oxidative stress. Separate samples from the right and
left ventricle from each rat were analyzed to determine if there is a differential oxidative
stress-induced lipid peroxidation within the heart. A 100-mg tissue sample was weighed
and homogenized in 1 ml of ice-cold phosphate buffered saline solution (PBS, 20 mM,
pH 7.4) containing 10 µl 0.5 M butylated hydroxytoluene (BHT) in acetonitrile. The
homogenate was then centrifuged at 5000g for 10 minutes at 4°C. The supernatant was
removed and used for determination of MDA and 4-HAE as described in the
commercially available LPO-586 colorimetric assay kit (OXIS Health Products,
Burlingame, CA). Samples were read at 586 nm in a spectrophotometer (Biomate 3,
Thermo Electron Corporation, Madison, WI) and results are presented as lipid
28
peroxidation concentration per mg of protein. Protein concentration was determined from
a separate aliquot of each tissue homogenate sample using a colorimetric protein assay
(Bio-Rad Laboratories, Hercules, CA).
Statistical Analysis
A one-way ANOVA design of independent measures was used for single timepoint comparisons made between the three groups. A two-way ANOVA design of
independent/repeated measures was used to compare differences between the three
groups for any weekly repeated measures. Differences were considered significant at p <
0.05. When significant differences were observed in the ANOVA comparisons, post hoc
testing (Tukey HSD) was done to determine which groups or time-points contributed to
the significant differences. Pearson correlation coefficient was calculated to determine
linear dependence between sets of two variables. The calculated r value was considered
significant if equal to or greater than published critical values of r at α = 0.05. All data
are presented as Means ± SEM. Error bars used in the figures also represent the SEM.
29
CHAPTER III: RESULTS
Weekly Measures
Water and tempol intake
All rats had free access to a standard rat chow diet and drinking water. Food
intake was not monitored for any study group. Water intake was only monitored in the
SAD+Tempol group in order to determine the amount of tempol received during the
study (see Figure 2). Water intake remained relatively constant during the study with an
average value of 12.3 ± 0.7 ml/100 g body weight/day. With chronic administration of
tempol in the drinking water at a concentration of 1mMol/L, treated rats received ~6.4 ±
0.1 mg tempol/day or ~21 ± 1 mg/kg body weight/day.
Weekly body weight
Body weight was not significantly different between the three study groups at any
point during the study (see Figure 3). There was a time effect for all three groups to gain
weight each week during the study due to normal growth with the exception of week 1
(weight taken 1 week post-surgery) which was not different from week 0 (weight taken
the day of surgery). The lack of a weight gain in week 1 was most likely related to the
surgical procedures.
Weekly telemetric blood pressure recordings
A 30-minute section from each weekly telemetric BP recording was used to
derive SBP, MAP, DBP, and HR. Compared to sham surgery, SAD resulted in a mild and
non-significant elevation of arterial blood pressure at week one after surgery (SBP: Sham
= 121 ± 3 mmHg; SAD = 131 ± 2 mmHg; SAD+Tempol = 135 ± 15 mmHg) (MAP:
Sham = 102 ± 2 mmHg; SAD = 110 ± 2 mmHg; SAD+Tempol = 114 ± 15 mmHg) (DBP:
30
Sham = 84 ± 2 mmHg; SAD = 92 ± 2 mmHg; SAD+Tempol = 97 ± 15 mmHg).
Thereafter, no differences in BP were found between the three groups (see Figure 4).
The BRS calculated from SBP and pulse interval was significantly greater in the
Sham group compared to the SAD and SAD+Tempol groups during each week of the
study. This finding indicates successful baroreceptor denervation. BRS demonstrated a
time effect with a minimal sensitivity observed in the week immediately following
surgery, followed by a slight increase in weeks 2 - 3 and stable values from week 3 - 6
(see Figure 5). The greatest BRS gains were seen at week 6 in all groups and were still
significantly (p < 0.05) lower in the denervated groups (SAD = 1.1 ± 0.05 ms/mmHg;
SAD+Tempol = 1.1 ± 0.07 ms/mmHg) compared to the control group (Sham = 1.6 ± 0.1
ms/mmHg).
In line with the decreased BRS observed in the sinoaortic denervated groups,
BPV calculated as SBPvar was significantly increased in SAD and SAD+Tempol
compared to the Sham group during the six week observation period (see Figure 6).
Initially at week 1, SBPvar observed in the denervated groups was ~4 to 5 fold greater
than in the Sham group. BPV tended to decrease over the six week observation period in
the denervated groups but was still 2 to 3 fold greater at week 6 in the SAD and
SAD+Tempol groups compared to the Sham group. Notably, there was no significant
difference in either BRS or SBPvar between the tempol-treated and non-treated SAD
groups.
Similar to the BP values, HR tended to be slightly elevated at week 1 in SAD
(402 ± 13 bpm) and SAD+Tempol (380 ± 14 bpm) compared to Sham (358 ± 12 bpm) but
there were no significant differences between the three groups at any time point
31
throughout the study. However, there was a time effect with week 1 HR values being
greater than the values at weeks 2 – 6 (see Figure 7). Measures of HRV were also not
significantly different between the three groups (see Figure 8). However, a time effect
was observed for SDNN (standard deviation of normal intervals) with the first two weeks
being lower than weeks three to six (p < 0.05 vs. week one and two). RMSSD (square
root of the mean squared difference of successive normal intervals) also demonstrated a
time effect with weeks five and six being greater than week one (p < 0.05 vs. week one).
The pattern of lower overall HRV (SDNN) and lower vagally-mediated HRV (RMSSD)
immediately after surgery was most prevalent in the untreated SAD group.
Determination of drug doses
for LPCA functional analysis
The results of the pilocarpine and L-NAME experiments have been previously
published (Stauss, Rarick et al. 2011). All experiments were conducted in normotensive
Sprague Dawley rats. It was determined that a single corneal application of a 30-µl bolus
of a 1% pilocarpine solution resulted in a maximal vasodilator response within ~10
minutes. Pretreatment with corneal application of 30 µl of a 4% L-NAME solution was
able to prevent any significant increase in LPCA lumen diameter in response to the 1%
pilocarpine solution, suggesting that pilocarpine-mediated vasodilation of the LPCA is
mainly mediated by endothelial NO release. In the current study we wanted to include the
exogenous NO donor SNP to differentiate between endothelial-dependent and
independent vasodilator responses of the LPCA. The time to maximal vasodilation in
response to corneal application of a 30 µl bolus of a 0.01 M SNP solution was found after
~10 minutes (n = 2, see Figure 10). A dose response curve for SNP identified the
32
maximal vasodilator response of the LPCA at a dose of 30 µl of a 0.1 mM solution (n =
2, see Figure 11). At this dose the LPCA lumen diameter increased by 68 ± 7%.
Increasing the concentration to 1 M SNP did not result in any further dilation of the
LPCA (+58 ± 12%).
In vivo vascular analysis of the LPCA
The structural vascular parameters (wall thickness, lumen diameter, wall
area/lumen area ratio) of the LPCA were determined each week prior to the application of
any drugs. These values were then used as the baseline measures to determine the change
elicited by application of the experimental drugs (pilocarpine, L-NAME, and SNP) to
normalize for different starting lumen diameters. There were no significant changes in the
structural vascular parameters over the six week observation period for any of the three
groups compared to their respective pre-surgery values. Wall thickness, wall area, lumen
diameter, and lumen area were not significantly different between any of the three groups
pre-surgery or during the six week observation period. However, there was a group effect
for the wall area/lumen area ratio to be significantly increased in the SAD+Tempol (1.05
± 0.02) group compared to Sham (0.91 ± 0.02) and SAD (0.92 ± 0.01) due to the tendency
for SAD+Tempol to have a smaller lumen area throughout the study. For all three groups,
the lumen diameter and lumen area were significantly smaller when imaged pre-surgery
compared to weeks four, five, and six (see Figure 12). Figure 13 shows the wall thickness
and lumen diameter in an isolated, unpressurized LPCA. This LPCA was excised from
the iris muscle of a Sprague Dawley rat in order to compare the wall thickness and lumen
diameter of an isolated vessel with the values calculated from the slit-lamp images. The
isolated, unpressurized LPCA had a wall thickness of 8 to 10 µm and a lumen diameter
33
ranging from 35 to 40 µm, confirming the values obtained by in vivo imaging of the
LPCA utilizing the slit-lamp. This confirmation is important because it demonstrates that
the in vivo imaging is not affected by a potential magnifying effect of the cornea.
Functional vascular parameters were assessed as the change in LPCA lumen
diameter in response to corneal application of pilocarpine, L-NAME, and SNP (see
Figure 14). The dilatory response to pilocarpine was significantly different between the
three groups and demonstrated a time dependent decrease in the two denervated groups
compared to the sham group that was lessened with tempol treatment. Endothelialdependent dilation in response to pilocarpine was significantly reduced in the SAD group
(p < 0.05 vs. Sham) from week three to six. In tempol-treated SAD rats, the LPCA
response to pilocarpine was significantly less than in Sham rats at week one, three, and
six, but was still significantly greater than the SAD group at the end of the six week
observation period (see Figure 15). Similar to the change in lumen diameter, the
calculated wall area/lumen area ratio determined after corneal application of pilocarpine
significantly increased over time in the SAD group compared to the other two groups
starting at week three (see Figure 16). Notably, there was no difference between the
sham-operated control rats and tempol-treated SAD rats in this parameter. The wall
area/lumen area ratio was not different between the three groups following corneal
application of SNP (data not shown), indicating that the pilocarpine-induced response
was not due to a structural change in the vascular wall or to altered function of the
VSMCs.
Following the pilocarpine-induced vasodilation, corneal application of L-NAME
resulted in a similar lumen diameter in all three groups that was not significantly different
34
from the baseline lumen diameters measured prior to the application of any drugs (see
Figure 17). This finding indicates that there is very little (if any) endogenous basal NO
release in the LPCA of rats. There was no change in the vasoconstrictor response of the
LPCA to L-NAME during the six week observation period in any of the three groups.
The vasodilatory response to SNP was not significantly different between any of
the three groups during any week of the study (see Figure 18). However, the endothelialindependent dilation of the LPCA in response to SNP demonstrated a time dependent
effect and was lower at weeks 1 and 2 compared to the pre-surgery value (p < 0.05). This
initial reduction in the SNP response was only significant in the two denervated groups.
During the baseline measures taken pre-surgery, all three groups demonstrated a
similar dilatory response to pilocarpine and SNP. The pilocarpine-induced dilation
expressed as a percentage of the SNP-induced response was significantly reduced in the
untreated SAD group (p < 0.05 vs. Sham and SAD+Tempol) starting at week three but
not until week six in the tempol treated SAD group (p < 0.05 vs. Sham). At week six, the
pilocarpine-induced dilation in both denervated groups was ~20% lower than the dilation
seen after corneal application of SNP (Sham: 98.4 ± 2.8%; SAD: 81.1 ± 3.5 %;
SAD+Tempol: 84.9 ± 2.2) (see Figure 19).
Ultrasound of ascending aorta
Diameters of the ascending aorta during systole (Sham = 2.18 ± 0.03 mm; SAD =
2.07 ± 0.03 mm; SAD+Tempol = 1.99 ± 0.01 mm) and diastole (Sham = 1.68 ± 0.02 mm;
SAD = 1.83 ± 0.02 mm; SAD+Tempol = 1.63 ± 0.02 mm) did not differ among the three
groups (see Figure 21). The average telemetric blood pressure values, simultaneously
measured during the ultrasound imaging, were also not significantly different between the
35
three groups (SBP: Sham = 125 ± 1 mmHg; SAD = 127 ± 2 mmHg; SAD+Tempol = 120
± 3 mmHg) (DBP: Sham = 81 ± 1 mmHg; SAD = 84 ± 2 mmHg; SAD+Tempol = 78 ± 1
mmHg), see Figure 22. Pulse pressure, which was used to calculate aortic distensibility,
was similar between the three groups except during week 1 when it was significantly
lower in the SAD+Tempol group (p < 0.05 vs. Sham and SAD).
At the beginning of the protocol (pre-surgery) aortic strain was similar in the SAD
and Sham groups. Immediately starting one week after SAD surgery and persisting
throughout the observation period, the untreated SAD group demonstrated a significantly
reduced aortic strain compared to the Sham-operated animals (p < 0.05). The
SAD+Tempol group demonstrated a time-dependent change in aortic strain. One week
after denervation surgery, tempol-treated SAD rats had a significantly reduced aortic
strain (p < 0.05 vs. Sham) that temporarily improved during weeks two and three before
again significantly decreasing for the final three weeks of the study. Aortic strain in the
SAD+Tempol group never decreased to the same extent as the untreated SAD group as it
was still significantly greater even at week 6 (p = 0.03 vs. SAD) (see Figure 23).
Aortic distensibility was significantly decreased at all six weeks in the SAD group
when compared to the Sham group. In contrast, aortic distensibility in the SAD+Tempol
group was only significantly decreased at week five and six compared to the Sham group.
Tempol treatment not only delayed the onset of blunted distensibility but also reduced the
overall loss of aortic distensibility as compared to the untreated SAD group (see Figure
24). In a small subset of denervated rats (SAD: n = 2; SAD+Tempol: n =3) aortic
distensibility was also determined following the administration of the calcium channel
blocker nifedipine. In the absence of endogenous vascular tone due to calcium channel
36
blockade, the beneficial effect of tempol on aortic distensibility was no longer observed
(see Figure 25). This finding may indicate that the beneficial effect of tempol on aortic
distensibility is mediated through an effect on basal vascular tone. Statistics were not run
on the changes in aortic distensibility following nifedipine administration due to the small
sample size for each group.
Final End-Point Measures
Cardiac function
Six weeks after SAD surgery, LV EDP was significantly increased in both
untreated and tempol treated rats compared to the Sham group (p < 0.05) during resting
baseline conditions measured while the rats were under anesthesia. Following the
baseline recording, intravenous injections of phenylephrine were given to increase
cardiac afterload. All three groups had a similar increase in LV EDP in response to the
increased afterload. LV EDP measured during the peak increase in blood pressure was
significantly higher in the untreated SAD group, but not in the tempol-treated SAD group
when compared to the Sham group (see Figure 27).
LV contractility (LV dP/dtmax) was similar in all three groups during the baseline
recording as well as during the peak increase in blood pressure following phenylephrine
injection. In response to phenylephrine injection, the untreated SAD group only increased
LV dP/dtmax by 980.4 ± 181.7 mmHg/s compared to increases of 1437.9 ± 284.6 mmHg/s
for the Sham group and 1332.0 ± 166.1 mmHg/s for the SAD+Tempol group. However,
these delta values were not significantly different between the groups (see Figures 2829).
37
SBP and HR were derived from the aortic pressure waveform recorded during the
cardiac function analysis. BRS was calculated from the change in HR divided by the
change in SBP in response to phenylephrine injection. Both the untreated and tempoltreated SAD rats had a significantly decreased BRS compared to the Sham group (p <
0.05, see Figure 30). These observations of BRS taken at the end of the study further
confirm the BRS results calculated by the sequence technique from the weekly telemetric
blood pressure recordings in conscious rats.
Final heart weight
Six weeks of SAD resulted in significant cardiac hypertrophy as indicated by the
total weight of the heart normalized to body weight (Sham = 0.246 ± 0.008 g heart
tissue/g body weight*100; SAD = 0.281 ± 0.005 g heart tissue/g body weight*100;
SAD+Tempol = 0.265 ± 0.009 g heart tissue/g body weight*100). SAD also
demonstrated a significant increase in the weight of the LV while there was only a trend
(p = 0.09) for the weight of the RV to be increased compared to the sham group (see
Figure 31 for heart weights normalized to body weight). Total heart weight for the
SAD+Tempol group was not significantly different from either the Sham (p = 0.08) or
SAD group (p = 0.1). Similar tendencies were observed in the LV of the tempol-treated
group, whereas the weight of the RV was similar to the SAD group (p = 0.9) but tended
to be greater than the Sham group (p = 0.09).
Left ventricle histology
There was a trend for SAD to result in an increase in the percent area of
connective tissue in the wall of the left ventricle (see Figure 32). In seven of the ten SAD
rats there was a significant increase in connective tissue area that was 30 ± 2% greater
38
than the Sham group (n = 8), while three of the SAD rats had a percent area that was
lower than the Sham group. This variability caused the overall group averages to not be
significantly different. All six of the tempol-treated SAD rats had a similar percent area
of connective tissue in the LV wall compared to the Sham group (10 ± 1 % area for both
groups).
Thoracic aorta histology
Representative images of the thoracic aorta prepared with Van Gieson’s stain are
presented in Figure 33. Histology sections of the thoracic aorta revealed a significant
increase in the wall thickness and wall area following six weeks of SAD (p < 0.05 vs.
Sham). The increase in wall size, due to hypertrophy of the tunica media, was partially
prevented by chronic administration of tempol. In the SAD+Tempol group, the average
wall thickness was significantly less than in the untreated SAD group (88.9 ± 4.0 µm vs.
99.4 ± 2.5 µm); however, the wall area (Sham: 0.44 ± 0.03 mm2; SAD: 0.55 ± 0.02 mm2;
SAD+Tempol: 0.50 ± 0.03 mm2) tended to demonstrate some hypertrophy and was not
significantly different from either Sham (p = 0.17) or SAD (p = 0.15). Lumen areas of the
thoracic aortae did not differ between the three groups. Consequently, the wall
area/lumen area ratio followed the results observed for the wall area measures (see
Figures 34-37).
In vitro vascular function of the MCA
Figure 38 shows the absolute lumen diameters of the MCA in the isolated
microvessel experiments. The baseline lumen diameters after the vessels were
pressurized and allowed to equilibrate were similar in all three groups (~125 µm) as were
the vasopressin pre-constricted lumen diameters (~70% of baseline, vasopressin 10-10 to
39
10-11 M). In the untreated SAD group there was impaired endothelial-dependent dilation
in response to increasing doses of ACh (p < 0.05 vs. Sham). The loss of endothelialdependent function was almost fully prevented by chronic tempol treatment
(SAD+Tempol) with only the highest ACh dose eliciting a decreased response compared
to the Sham group. Endothelial-independent vascular function assessed by the response to
a maximal dose of SNP was not significantly different between any of the three groups.
The ACh-induced dilation of the MCA expressed as a percentage of the maximal
SNP response is presented in Figure 39. Only the Sham group had a maximal ACh–
induced dilation that was not significantly less than the SNP induced response (Sham:
92.3 ± 2.6 %; SAD: 27.7 ± 4.0 %; SAD+Tempol: 65.8 ± 4.5 %). Chronic tempol
treatment significantly improved endothelial function by significantly increasing the
dilatory response to all but the lowest dose of ACh administered compared to the
untreated SAD group.
There was a significant correlation observed between the maximal ACh-induced
dilation in the isolated MCA and the pilocarpine-induced dilation obtained in week 6
from the in vivo imaging of the LPCA in a pooled sample of individual rats from all three
groups (see Figure 40). The in vitro measures of vascular function in the MCA (lumen
diameter ~125 µm) confirm the results observed in the weekly in vivo imaging of the
LPCA (lumen diameter ~40 µm) and provide additional evidence of SAD-induced
endothelial-dependent dysfunction in small arteries and arterioles.
Lipid peroxidation assay
The tissue concentration of MDA and 4-HAE was determined in the left and right
ventricles as an indicator of oxidative stress. There was a trend for the combined tissue
40
concentration of MDA+4-HAE to be increased in the untreated SAD group in both
ventricles (LV: p = 0.09; RV: p = 0.068). Tempol treatment prevented this trend for an
increase in the measured indicators of oxidative stress. In rats from all three groups, lipid
peroxidation normalized for total protein content was significantly greater in the LV
compared to the RV (see Figure 41).
41
Figure 1. Protocol timeline and list of experiments performed at each time point. Baseline
measures were completed at an age of 8 weeks. Rats were randomized into three groups
and surgeries were performed the following week. The first of six weekly measurements
were taken approximately seven days following the completion of the surgical
procedures. The end-point measures were then taken in the seventh week post-surgery, at
an age of 16 weeks.
42
Water Intake (ml/100g/day)
30
24
18
12
6
0
Tempol (mg/kg/day)
40
30
20
10
0
0
1
2
3
4
Time (weeks)
5
6
7
Figure 2. Average daily water intake (top) and amount of tempol received (bottom) in the
SAD+Tempol group throughout the experimental protocol. Week 0 = week of surgery;
Week 1-6 = 6 week observation period; Week 7 = week of end-point measurements.
43
Sham
SAD
SAD+Tempol
500
Body Weight (g)
400
*
*
*
*
*
*
6
7
300
200
100
0
0
1
2
3
4
Time (weeks)
5
Figure 3. Change in body weight during the study starting the week of surgery (week 0).
There was no difference in body weight between the three groups. * p < 0.05 vs. week 0
for all three groups.
44
Sham
SAD
SAD+Tempol
SBP (mmHg)
150
120
90
60
30
0
MAP (mmHg)
150
120
90
60
30
DBP (mmHg)
0
150
120
90
60
30
0
1
2
3
4
Time (weeks)
5
6
Figure 4. Blood pressure values derived from the weekly telemetric aortic pressure
waveform recordings. Values were not significantly different between the three groups at
any time point.
45
Sham
SAD
SAD+Tempol
BRS gain (ms/mmHg)
2
1.6
1.2
*
*
0.8
*
*
*
*
#
#
#
#
3
4
Time (weeks)
5
6
0.4
0
1
2
Figure 5. Baroreflex sensitivity measured by the sequence technique using weekly one
hour telemetric blood pressure recordings. The Sham control group had a higher
baroreflex gain compared to both the untreated and tempol-treated SAD groups. * p <
0.05 SAD and SAD+Tempol vs. Sham. # p <0.05 vs. week one.
46
Sham
SBP variance (mmHg2)
160
SAD
SAD+Tempol
*
*
120
*
*
80
#
*
*
5
6
40
0
1
2
3
4
Time (weeks)
Figure 6. BPV calculated as the variance of SBP was greater in SAD and SAD+Tempol
compared to Sham (* p < 0.05). # p <0.05 week six vs. week one.
47
Sham
SAD
SAD+Tempol
500
#
HR (bpm)
400
#
#
#
3
4
Time (weeks)
5
#
300
200
100
0
1
2
6
Figure 7. HR was not significantly different between the three groups during the study
but tended to be elevated in the two denervated groups at week 1 compared to the Sham
group. A time effect was observed with HR being significantly greater in week one than
in weeks two through six (# p < 0.05).
48
Sham
15
SAD
#
SAD+Tempol
#
#
#
12
SDNN (ms)
9
6
3
0
8
#
RMSSD (ms)
6
#
4
2
0
1
2
3
4
Time (weeks)
5
6
Figure 8. Measures of HRV demonstrate a time effect for SDNN with the first two weeks
being lower than week three to six (# p < 0.05 vs. week one and two). RMSSD also
demonstrated a time effect with week one being less than week five and six (# p < 0.05
vs. week one).
49
Figure 9. Image displaying the four branches of the LPCA in the iris of a Sprague
Dawley rat (top panel). The lower right branch in the iris of the left eye was used for
imaging. The bottom panel shows a representative analysis using Imager (Hemolab
software) to place ten sets of four markers on the wall and lumen borders.
50
80
70
Lumen diameter (um)
60
50
40
30
20
10
0
BL
5
10
15
20
25
Time (minutes)
30
35
40
Figure 10. Preliminary experiment determining the time to maximum vasodilatory
response of the LPCA to SNP. Maximal vasodilation of the LPCA was seen around 10
minutes after corneal application of the SNP dose (30 µl of 0.01 M solution).
51
90
80
Lumen diameter (um)
70
60
50
40
30
20
10
0
00
-5
.00001
10
-4
1E-04
10
-3
1E-03
10
-2
1E-02
10
-1
1E-01
10
11
Dose (M)
Figure 11. SNP dose response curve to determine the minimal dose needed to elicit a
maximum vasodilatory response of the LPCA. Maximal vasodilation of the LPCA was
obtained with corneal application of the 30 µl 0.1M SNP dose.
Wall Thickness (µm)
52
Sham
10
SAD
SAD+Tempol
8
6
4
2
0
Wall Area/Lumen Area Ratio
Lumen Diameter (µm)
50
#
#
#
4
5
#
40
30
20
10
0
1.5
1.2
0.9
0.6
0.3
0
BL
1
2
3
Time (weeks)
6
Figure 12. Weekly imaging of the LPCA taken before the application of any drugs. Wall
thickness (top) and lumen diameter (middle) were not different between any of the
groups at any point during the study. There was a time effect for the lumen diameter
before surgery to be smaller than during week 1, 4, 5, and 6 (# p < 0.05 vs. BL). Wall
area/lumen area ratio (bottom) demonstrated a group effect with SAD+Tempol being
significantly greater than SAD and Sham (p <0.05). BL = week before surgery, 1-6 = the
six week observation period starting one week after surgery.
53
Figure 13. Isolated section of the LPCA from a Sprague Dawley rat. The LPCA was
removed from the iris and placed on a glass slide in saline solution and imaged at a
magnification of 40x to verify the wall and lumen values obtained with the slit-lamp
imaging technique. Wall thickness and lumen diameter values were comparable between
the images and the isolated vessel.
54
Baseline
Pilocarpine
L-NAME
SNP
Figure 14. Successive images taken of the same section of the LPCA in the iris of a
conscious rat using a slit-lamp biomicroscope before (Baseline, top left) and after corneal
application (30 µL) of the muscarinic agonist pilocarpine (top right), L-NAME (bottom
left), and SNP (bottom right).
55
Sham
SAD
SAD+Tempol
ΔLumen Diameter (um)
30
25
20
*
15
10
*
5
*
*
*†
0
BL
1
2
3
Time (weeks)
4
*†
5
*†
6
Figure 15. Change in lumen diameter from baseline (before any drug application) in
response to corneal application of pilocarpine. The LPCA response to pilocarpine was
significantly different between the three groups and demonstrated a time dependent
decrease in the two denervated groups that was lessened with tempol treatment. *p < 0.05
vs. Sham, †p < 0.05 SAD vs. SAD+Tempol. BL = week before surgery, 1-6 = the six
week observation period starting one week after surgery.
56
W/L ratio (% change from BL)
Sham
SAD
SAD+Tempol
40
*
30
*
*
*
20
10
0
-10
-20
BL
1
2
3
Time (weeks)
4
5
6
Figure 16. Data presented are for the wall area/lumen area ratio measured after corneal
application of pilocarpine over the course of the study and normalized to the pre-surgery
value (BL). The wall area/lumen area ratio significantly increased over time in the SAD
group compared to the other two groups (* p <0.05 vs. Sham and SAD+Tempol). BL =
week before surgery, 1-6 = the six week observation period starting one week after
surgery.
57
Sham
SAD
SAD+Tempol
10
ΔLumen Diameter (um)
8
6
4
2
0
-2
-4
-6
-8
-10
BL
1
2
3
Time (weeks)
4
5
6
Figure 17. Change in lumen diameter from baseline (before any drug application) in
response to corneal application of L-NAME. There were no significant differences
between the three groups. BL = week before surgery, 1-6 = the six week observation
period starting one week after surgery.
58
Sham
SAD
SAD+Tempol
ΔLumen Diameter (um)
30
25
20
15
10
*#
5
*
#
0
BL
1
2
3
Time (weeks)
4
5
6
Figure 18. Change in lumen diameter from baseline (before any drug application) in
response to corneal application of SNP. There were no significant group effects for the
LPCA response to SNP. There was a time effect for the average of all three groups to be
lower at weeks 1 and 2 compared to BL (p < 0.05). p < 0.05 *SAD weekly comparison
with BL value; #SAD+Tempol weekly comparison with BL value. BL = week before
surgery, 1-6 = the six week observation period starting one week after surgery.
59
Sham
SAD
SAD+Tempol
Pilocarpine Induced Dilation
(%of SNP response)
140
120
100
80
*†
60
*#
*†
40
20
0
BL
1
2
3
4
Time (weeks)
5
6
Figure 19. Pilocarpine-induced dilation of the LPCA presented as a percentage of the
vasodilatory response to exogenous NO. Endothelial-dependent dilation was impaired at
three weeks after SAD surgery. Tempol treatment resulted in improved endothelialdependent dilation and delayed the loss of endothelial-dependent function until week 6.
*p < 0.05 SAD vs. Sham, †p < 0.05 SAD vs. SAD+Tempol, #p < 0.05 SAD+Tempol vs.
Sham. BL = week before surgery, 1-6 = the six week observation period starting one
week after surgery.
60
Figure 20. Echocardiography image displaying the left ventricle in the parasternal long
axis (left) and the associated M-mode tracing (right). The trigger line (dotted white line)
marking the location of the ascending aorta used for the M-mode tracing is denoted by a
black arrow in the left panel. Determined from the M-mode tracing, the lumen diameter
of the ascending aorta was measured during systole (black marker) and during diastole
(white marker).
61
Sham
SAD
SAD+Tempol
Systolic Diameter (um)
0.3
0.25
0.2
0.15
0.1
0.05
0
Diastolic Diameter (um)
0.3
0.25
0.2
0.15
0.1
0.05
0
1
2
3
4
Time (weeks)
5
6
Figure 21. Weekly ultrasound measures of the lumen diameter of the ascending aorta
during systole (top panel) and diastole (bottom panel) were not different between the
three groups during the six week observation period that started one week post-surgery.
62
Sham
SAD
SAD+Tempol
3
4
Time (weeks)
5
SBP (mmHg)
150
120
90
60
30
0
DBP (mmHg)
100
80
60
40
20
Pulse Pressure (mmHg)
0
60
50
40
30
20
*
10
0
1
2
6
Figure 22. Telemetric blood pressure was simultaneously recorded during the weekly
ultrasound imaging of the ascending aorta. Blood pressure values for SBP (top) and DBP
(middle) were not different between the three groups during the six week observation
period that started one week post-surgery. Pulse pressure (bottom) was significantly
decreased in the SAD+Tempol group only at week 1 (* p < 0.05 vs. SAD and Sham).
63
Sham
SAD
SAD+Tempol
Strain [(sysø - diaø)/diaø]
0.4
0.32
#
0.24
#
#
#
0.16
*
0.08
*†
*†
*†
*†
*†
5
6
0
BL
1
2
3
4
Time (weeks)
Figure 23. Weekly ultrasound measures of aortic strain were similar pre-surgery (BL) but
then significantly decreased immediately one week following denervation surgery in both
the untreated and tempol-treated rats. Aortic strain stayed reduced in the untreated SAD
rats (* p < 0.05 SAD vs. Sham; † p < 0.05 SAD vs. Sad+Tempol), but was temporarily
restored by tempol treatment before partially decreasing in later weeks (# p <0.05
SAD+Tempol vs. Sham). sysø = lumen diameter during systole. diaø = lumen diameter
during diastole.
64
Sham
SAD
SAD+Tempol
Aortic Distensibility (mmHg-1)
0.02
0.016
0.012
#
#
*†
*†
5
6
0.008
0.004
*
*†
1
2
*†
*†
0
3
4
Time (weeks)
Figure 24. Weekly ultrasound measures of aortic distensibility were significantly
decreased throughout the six week observation period in the SAD group (* p < 0.05 SAD
vs. Sham; † p < 0.05 SAD vs. Sad+Tempol), but was temporarily prevented by tempol
treatment before partially decreasing in later weeks (# p <0.05 SAD+Tempol vs. Sham).
Aortic distensibility was not determined at baseline (pre-surgery) because the rats were
not yet instrumented with telemetric blood pressure sensors; therefore, pulse pressure
could not be measured.
65
SAD +Nif
Aortic Distensibility (mmHg-1)
0.020
SAD+Tempol +Nif
0.016
0.012
0.008
0.004
0.000
1
2
3
4
Time (weeks)
5
6
Figure 25. Administration of the calcium channel blocker nifedipine was able to restore
aortic distensibility in the SAD group (n = 2) to the level of the SAD+Tempol group
making it similar to the Sham group for the first four weeks of the six week observation
period. In the SAD+Tempol group (n = 3), nifedipine only improved the week one
response. Nifedipine restored aortic distensibility in the untreated SAD rats to the level of
distensibility in the tempol-treated SAD rats.
66
SBP
HR
Figure 26. Example of LV (red tracing) and aorta (blue tracing) pressure waveforms
recorded to determine the LV dP/dtmax and LV EDP after the six week protocol. Pressure
was recorded before and after intravenous administration of phenylephrine (0.5 ml, 10
µg/ml) to determine changes in cardiac function following an increase in afterload. SBP
and HR were derived from the aortic pressure waveform to determine BRS in response to
phenylephrine injection.
67
Baseline
Phenylephrine
40
#
*
LV EDP (mmHg)
32
24
*
*
16
8
0
Sham
SAD
SAD+Tempol
Figure 27. After six weeks of denervation, LV EDP was significantly greater in both
SAD groups compared to the Sham group during the baseline recording before
phenylephrine injection. Whereas during phenylephrine-induced increase in cardiac
afterload LV EDP was significantly higher in only the untreated SAD rats compared to
Sham-operated rats (*p < 0.05 vs. Sham). LV EDP was significantly increased by
phenylephrine injection in all rats (# p < 0.05 phenylephrine vs. baseline).
68
Baseline
Phenylephrine
LV dP/dtmax (mmHg/s)
9000
#
7500
6000
4500
3000
1500
0
Sham
SAD
SAD+Tempol
Figure 28. After six weeks of denervation, LV dP/dtmax was similar between all groups
during the baseline recording before as well as after phenylephrine injection. All groups
demonstrated a similar increase in LV EDP following the increase in afterload (# p < 0.05
phenylephrine vs. baseline).
69
1800
Δ LV dP/dtmax (mmHg/s)
1500
1200
900
600
300
0
Sham
SAD
SAD+Tempol
Figure 29. LV contractility tended to have a smaller increase in response to
phenylephrine injection in SAD compared to Sham (p = 0.14) following six weeks of
denervation. The tendency for this loss of contractility was not seen in tempol treated
rats.
70
ΔHR/ ΔSBP (bpm/mmHg)
1.2
0.9
*
*
SAD
SAD+Tempol
0.6
0.3
0.0
Sham
Figure 30. BRS as calculated from the ratio of change in HR to change in SBP following
phenylephrine injection was similarly decreased following six weeks of SAD in both
untreated and tempol-treated groups (* p < 0.05 vs. Sham).
71
(THW/BW)*100
0.30
*
0.27
0.24
0.21
0.18
0.15
(LVW/BW)*100
0.25
*
0.22
0.19
0.16
0.13
0.10
(RVW/BW)*100
0.07
0.06
0.05
0.04
0.03
0.02
Sham
SAD
SAD+Tempol
Figure 31. Total heart weight (top) and left ventricular weight (middle) normalized to
body weight were significantly increased in SAD vs. Sham (*p < 0.05) but not when
compared to SAD+Tempol. Tempol treatment tended to partially prevent cardiac
hypertrophy (THW: p = 0.08 vs. Sham; LV: p = 0.18 vs. Sham). Right ventricular weight
normalized to body weight for the SAD group tended to be increased vs. Sham (p =
0.09). SAD+Tempol was not significantly different from either group (p = 0.99 vs. SAD;
p = 0.2 vs. Sham).
72
Sham
SAD
SAD+Tempol
Figure 32. Representative histology sections of the left ventricle prepared with Van
Gieson’s stain to differentiate cardiac muscle (pale yellow color) from connective tissue
deposition (pink color). There was a trend for a greater percent area of connective tissue
in the untreated SAD group compared to Sham and the SAD+Tempol groups.
73
Sham
SAD
SAD+Tempol
Figure 33. Representative sections of the thoracic aorta prepared with Van Gieson’s stain.
The hypertrophic changes (increased wall thickness and wall area) seen with increased
BPV (SAD) were prevented by tempol treatment (SAD+Tempol).
74
120
*
Wall Thickness (µm)
100
80
60
40
20
0
Sham
SAD
SAD+Tempol
Figure 34. Wall thickness of the thoracic aorta was significantly increased in SAD (*p <
0.05 vs. Sham and SAD+Tempol). Tempol treatment limits the increase in wall thickness
in response to increased BPV (p = 0.13 vs. Sham).
75
0.6
*
Wall Area (mm2)
0.5
0.4
0.3
0.2
0.1
0
Sham
SAD
SAD+Tempol
Figure 35. Wall area of the thoracic aorta was significantly increased in SAD (*p < 0.05
vs. Sham). In the SAD+Tempol group, wall area tended to be greater than that of the
Sham group (p = 0.17) but was less than that of the SAD group (p = 0.15). Tempol
treatment limits the increase in wall area in response to increased BPV.
76
2.5
Lumen Area (mm2)
2
1.5
1
0.5
0
Sham
SAD
SAD+Tempol
Figure 36. Lumen area of the thoracic aorta was not different between the three
experimental groups.
77
0.30
Wall Area/Lumen Area ratio
*
0.24
0.18
0.12
0.06
0.00
Sham
SAD
SAD+Tempol
Figure 37. The wall area/lumen area ratio of the thoracic aorta was significantly increased
in SAD compared to the Sham group (*p < 0.05). In the SAD+Tempol group, wall
area/lumen area ratio tended to be greater than in the Sham group (p = 0.2) but also
tended to be less than the SAD group (p = 0.17). Tempol treatment limits the extent of
aortic hypertrophy in response to increased BPV.
78
Sham
SAD
SAD+Tempol
Lumen diameter (µm)
175
150
125
#
100
*
75
*
* *
*
50
25
0
-7
-7
-6
-6
-5
-5
-4
-4
-3
1x10 3x10 1x10 3x10 1x10 3x101x10 3x10 1x10
Drug Dose (M)
Figure 38. Response (absolute lumen diameter) of the middle cerebral artery to increasing
doses of ACh (10-7 to 10-3 M) and to a maximal dose of SNP (10-3 M) following
preconstriction with vasopressin (VP, 10-10 to 10-11 M). Increased BPV (SAD) resulted in
a blunted endothelial-dependent dilation (*p < 0.05 vs. Sham and SAD+Tempol) but in
no change in the endothelial-independent response. Tempol treatment almost completely
prevented the loss of endothelial-dependent function (# p < 0 .05: SAD+Tempol vs.
Sham).
79
SAD
SAD+Tempol
100
80
60
40
20
*
0
-7
1x10
-7
3x10
*
*
*
-6
1x10
-6
-5
*
*
-5
#
#
#
-4
3x10 1x10 3x10 1x10
*
Lumen diameter (% SNP response)
Sham
*
-4
3x10
-3
1x10
Drug Dose (M)
Figure 39. Response (expressed as a % of the maximal SNP-elicited dilation) of the
middle cerebral artery to increasing doses of ACh (10-7 to 10-3 M) following
preconstriction with vasopressin (VP, 10-10 to 10-11 M). Increased BPV (SAD) resulted in
a blunted endothelial-dependent dilation (*p < 0.05 vs. Sham and SAD+Tempol) that was
only 30% of the maximal response seen with the application of exogenous NO. Tempol
treatment was able to partially restore the endothelial-dependent dilation to 66 % of the
maximal SNP-elicited response (# p < 0 .05: SAD+Tempol vs. Sham).
80
90
y = 2.8833x + 15.116
R² = 0.6172
MCA Δ lumen diameter (µm)
80
70
60
50
40
30
20
10
0
0
5
10
15
LPCA Δ lumen diameter (µm)
20
25
Figure 40. There was a significant correlation (r = 0.8; p < 0.01 @ dfn-2 = 17, r =.575)
between the maximal endothelial-dependent response of the MCA to ACh (y-axis) and
the week six endothelial-dependent response of the LPCA to pilocarpine (x-axis). Each
data point represents an individual rat. Rats from all three groups are pooled for the
correlation analysis.
81
LV
RV
MDA + 4-HAE (nM/mg protein)
1.60
1.40
1.20
#
1.00
0.80
0.60
SAD+Tempol
SAD
Sham
0.40
0.20
0.00
Sham
SAD
SAD+Tempol
Figure 41. There was a trend for increased oxidative stress as measured by lipid
peroxidation in the left (p = 0.09) and right (p = 0.068) ventricles of SAD rats compared
to Sham rats. Tempol administration prevented any increase in oxidative stress
(SAD+Tempol vs. Sham: p = 0.99 for LV, p = 0.965 for RV). Indicators of lipid
peroxidation were significantly greater in the LV compared to the RV (p < 0.05).
82
Figure 42. Diagram demonstrating how cardiovascular end-organ damage resulting from
increased BPV can potentially be mediated through oxidative stress-dependent and
oxidative stress-independent mechanisms.
83
CHAPTER IV: DISCUSSION
Major Findings
There are two major novel findings of this study: First, we have demonstrated
that enhanced BPV causes a time-dependent reduction in endothelial-dependent dilation
in small resistance type arteries. Second, BPV-induced cardiovascular end-organ damage
can be ameliorated by chronic antioxidant treatment with the superoxide dismutase
mimetic tempol. These new findings are important because endothelial dysfunction of
small resistance-type arteries can have deleterious effects on regulation of
microcirculatory blood flow in vascular beds such as the cardiac and cerebral circulation
and contribute to development of secondary diseases, such as hypertension, coronary
heart disease, heart failure, or stroke. Finally, the beneficial effect of chronic antioxidant
treatment on BPV-induced cardiovascular end-organ damage provides important
mechanistic insights into the pathogenesis of BPV-associated cardiovascular morbidity
and mortality.
Endothelial dysfunction in small resistance-type arteries
While it has been demonstrated previously that enhanced BPV causes endothelial
dysfunction in large conduit arteries, such as the aorta (Miao, Tao et al. 2001; Eto, Toba
et al. 2003; Feng, Luo et al. 2011), the experiments described in this set of studies
unequivocally show that BPV-induced endothelial dysfunction also affects smaller
resistance-type arteries (LPCA = 40 µm; MCA = 125 µm). The small resistance vessels
of the microcirculation largely determine local tissue blood flow and total peripheral
resistance (DeLano, Schmid-Schonbein et al. 1991). Increased peripheral resistance
implies a narrowing of the resistance vessels which would occur with either structural
84
remodeling resulting in a reduction in lumen diameter (Mulvany 2012) or a functional
change, such as endothelial dysfunction (Deng, Li et al. 1995), resulting in greater
vascular tone. The ability of resistance vessels to appropriately alter vascular tone in
response to changes in blood flow/shear stress is essential to maintain adequate tissue
perfusion for nutrient and metabolic waste exchange as well as to protect smaller
downstream vessels from potentially damaging high blood flows. In the current study,
there was a progressive reduction in endothelial-dependent dilation without any evidence
for endothelial-independent dysfunction or structural remodeling of the LPCA. This
finding is in line with studies demonstrating that functional microvascular impairment
may act in a permissive manner to induce structural remodeling. A similar progression
from functional microvascular disturbances to structural vascular remodeling has been
observed in both diabetes and hypertension and may be associated with increasing
severity of the underlying disease (Levy, Schiffrin et al. 2008). For example, a reduction
in cerebral blood flow leads to energy depletion in neural cells and is thought to trigger a
cascade of events from inflammatory responses to ischemic stroke (Danton and Dietrich
2003). Similarly, impaired myocardial perfusion may lead to localized ischemia resulting
in impaired cardiac function and the development of heart failure or the potential to
provoke serious arrhythmias (Kaufmann and Camici 2005). Interestingly, reperfusion of
ischemic tissue can lead to accumulation of ROS which are known to further exacerbate
cardiovascular disease as will be described in the next section.
Role of oxidative stress
This study demonstrated for the first time that chronic antioxidant treatment using
the superoxide dismutase mimetic tempol prevents or ameliorates BPV-induced
85
cardiovascular end-organ damage. Specifically, tempol reduced the deleterious effects of
BPV on aortic stiffness (strain and distensibility) and aortic hypertrophy (wall to lumen
ratio) as well as cardiac function (LV EDP and LV dP/dtmax) and cardiac hypertrophy
(THW/BW and LVW/BW ratios and LV collagen deposition). Furthermore, tempol
treatment almost completely prevented endothelial dysfunction in small resistance
arteries (LPCA and MCA).
Importantly, the reduction in cardiovascular end-organ
damage observed with tempol treatment occurred without a reduction in the elevated
BPV or without improving the blunted baroreflex sensitivity following baroreceptor
denervation. Thus, the mechanisms of cardiovascular protection from BPV by tempol do
not include reduction of BPV or improvement of BRS. These findings provoke two
important questions: First, which mechanisms and/or pathways link elevated BPV with
increased oxidative stress? Second, which mechanisms and/or pathways link oxidative
stress with cardiovascular end-organ damage?
Regarding the first question, while a direct cause and effect relationship has yet to
be defined there are several potential mechanisms linking elevated BPV with increased
oxidative stress. First, activation of the RAS is considered a major pro-oxidant
neurohormonal system that is over activated in cardiovascular disease (Dhalla, Temsah et
al. 2000). Importantly, RAS activation has been demonstrated in models of chronic SAD
(Miao, Xie et al. 2002; Shan, Dai et al. 2003). Recently, SAD-induced elevated BPV has
been shown to increase the aortic tissue content of both AngII and the lipid peroxidation
by-product MDA, suggesting an association between activation of the RAS and increased
oxidative stress (Feng, Luo et al. 2011). A second potential mechanism would be related
to mechanosensitive or stress activated pathways. Oxidative stress can be a physiological
86
consequence of working cardiac muscle (Erickson, He et al. 2011). ROS are known to be
derived from the mitochondria at several points within the electron transport chain during
oxidative phosphorylation (Andreyev, Kushnareva et al. 2005). ROS production can also
be induced by mechanical stimuli such as shear stress, pulsatile strain, and stretch
(Lassegue and Clempus 2003). Once they are activated, ROS have been shown to induce
activation of multiple kinases and transcription factors through oxidation reactions
(Touyz, Tabet et al. 2003). Thus, increased BPV may create a greater mechanical
stimulus resulting in the accumulation of ROS. The increased oxidative stress would then
mediate the downstream activation of mechanosensitive second messengers such as
members of the MAPK family that have been demonstrated to be upregulated following
SAD (Martinka, Fielitz et al. 2005; Takayama, Kai et al. 2011). Finally, periods of
coronary ischemia/reperfusion may also result in accumulation of oxidative stress. While
SAD animals are considered to be normotensive (Norman, Coleman et al. 1981), the
higher BPV results in brief hypertensive as well as hypotensive episodes. Although it is
not clear if these brief hypotensive episodes are severe enough to result in myocardial
ischemia, the related changes in blood pressure and coronary flow could result in periods
of coronary hypo-perfusion. While ROS are normally formed during mitochondrial
respiration, it has been reported that ischemia and reperfusion of the ischemic
myocardium can result in an elevated production of ROS (Jennings, Schaper et al. 1985).
Thus, it is reasonable to speculate that brief hypertensive and hypotensive episodes,
associated with enhanced BPV, also result in the increased production of ROS.
Regarding the second question, there are several potential mechanisms and/or
pathways that link oxidative stress with cardiovascular end-organ damage. While there is
87
a lack of data investigating the impact of oxidative stress specifically in models of
increased BPV, it is generally thought that extraneous accumulation of ROS plays a
crucial role in the genesis of cardiovascular pathology or in mediating secondary effects
of established cardiovascular disease (Dhalla, Temsah et al. 2000). First, the results of
this study demonstrate increased lipid peroxidation in the left and right cardiac ventricles
after exposure to six weeks of enhanced BPV. Whereas the untreated SAD rats had signs
of cardiac remodeling (hypertrophy and fibrosis), cardiac dysfunction (increased LV
EDP), and increased cardiac tissue lipid peroxidation, all of these parameters were
reduced by chronic tempol treatment. Additionally, it has been shown that BPV-induced
lipid peroxidation in the aorta is associated with degeneration and apoptosis of the aortic
endothelial cells along with decreased eNOS expression and endothelial-dependent
dysfunction (Feng, Luo et al. 2011). Together, these findings would indicate that lipid
peroxidation contributes significantly to BPV-induced cardiovascular end-organ damage.
Lipid peroxidation is a hallmark of ROS-induced cellular injury (Dhalla, Temsah
et al. 2000). As a result, cellular membrane lipid bilayer arrangement becomes disrupted
which in turn can affect the functional properties of the cell (Grinna 1977). Furthermore,
lipid peroxidation produces unsaturated aldehydes, such as MDA and other metabolites
which are cytotoxic and mutagenic and capable of inactivating many cellular proteins by
forming protein cross-linkages (Siu and Draper 1982; Hagihara, Nishigaki et al. 1984). In
addition to the disruption of cell structure, ROS are known to regulate the activity of
several enzymes essential for cellular growth or apoptosis (Dhalla, Golfman et al. 1999;
Griendling,
Sorescu
et
al.
2000).
Several
neurohormonal
factors
including
catecholamines, AngII, and cytokines (TNF-α, TGF-β) are thought to convert cardiac
88
myocytes or fibroblasts into a pathologic phenotype by increasing intracellular oxidative
stress, either via increased release of ROS or by decreasing antioxidant enzyme levels
(Sorescu and Griendling 2002). For example, TGF-β is known to be released in response
to Ang II and is thought to be a potent stimulant for the conversion of fibroblasts into
active collagen producing myofibroblasts in a superoxide dependent pathway (Siwik,
Pagano et al. 2001). In that study, treatment with the superoxide dismutase inhibitor
diethyldithiocarbamic acid was able to decrease collagen synthesis. Upregulation of
TGF-β and an increase in LV collagen deposition has been shown previously in mice
following SAD (Martinka, Fielitz et al. 2005). Thus, tempol treatment may have
prevented cardiac fibrosis in the experiments described in this thesis by preventing ROSinduced activation of TGF-β.
Impaired endothelial-dependent dilation can be associated with decreased NO
bioavailability and an increase in vasomotor tone. Decreased NO bioavailability may
result from decreased NO synthesis or increased NO degradation through its interaction
with ROS (Landmesser, Dikalov et al. 2003). Vasomotor tone can also be increased by
the direct effects of ROS to increase intracellular calcium concentrations (Tabet, Savoia
et al. 2004).
The intracellular calcium-overload as a consequence of ROS may present
another potential link between oxidative stress and cardiovascular end-organ damage as it
can effect both pathologic cardiac remodeling (Dhalla, Temsah et al. 2000) and cardiac
dysfunction (Dhalla, Golfman et al. 1999). Specifically, RAS-induced oxidative stress is
thought to upregulate Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity,
which may be a downstream sensor of both oxidative stress and intracellular calcium
signals (Erickson, Joiner et al. 2008). CaMKII can activate the stress-activated and pro-
89
apoptotic p38MAPK (Takeda, Matsuzawa et al. 2004), which has been shown to be
upregulated in mice after 12 weeks of SAD (Martinka, Fielitz et al. 2005). Furthermore,
CaMKII is known to exert control over cardiac growth-related transcription factors in
models of heart failure (Passier, Zeng et al. 2000). Because of the complexity of potential
signal transduction pathways involved in cardiovascular remodeling it is likely that
treatments focused on one specific area would not be as effective as a combination of
therapies. This may explain why tempol treatment was only partially effective at
preventing the structural and functional cardiovascular end-organ damage observed in the
current study. See Figure 42 for a diagram summarizing the discussion of potential
pathways linking elevated BPV with oxidative stress/ROS and subsequent cardiovascular
damage. The specific cardiac and vascular changes observed in the experiments described
in this thesis will be further discussed in the next two sections.
Specific cardiac effects of enhanced BPV
The results of this study suggest that six weeks of increased BPV result in a
cardiac pathology that includes both structural remodeling and functional impairment.
These changes are at least partially associated with an increase in oxidative stress.
Cardiac weight was significantly increased by +14% compared to the Sham group
whereas the normalized heart weight in the tempol-treated rats was not significantly
greater (+7%) than the Sham group. Additionally, there was a trend (p= 0.2) for the
untreated SAD group to have a greater percent area of connective tissue in the wall of the
left ventricle compared to both the Sham and tempol-treated groups. This may suggest
that BPV-induced increases in cardiac fibrosis take longer to develop compared to
cardiac hypertrophy. The finding of an increase in cardiac weight is similar to previous
90
results that have observed an increase in the normalized weight of the LV of 11% after 6
weeks of SAD (Van Vliet, Hu et al. 1996). Similar increases in cardiac weight have also
been observed after a period of 10 to 16 weeks (Miao and Su 2002; Xie, Miao et al. 2003;
Zhang, Li et al. 2011) and have been associated with both increases in myocyte cross
sectional area (Martinka, Fielitz et al. 2005; Takayama, Kai et al. 2011) and increased
collagen deposition (Martinka, Fielitz et al. 2005; Tao, Zhang et al. 2008; Flues, MoraesSilva et al. 2012).
The structural modifications of the heart observed following six weeks of SAD
may be associated with a decrease in ventricular compliance. The loss of compliance or
increase in ventricular stiffness manifests as elevated LV EDP. In contrast to the
significant increase in LV EDP observed in this study, there was only a trend (p = 0.14)
for LV contractility to be reduced. This may suggest that six weeks of SAD is not long
enough to impair LV contractility. In studies that have investigated cardiac function after
10 (Flues, Moraes-Silva et al. 2012) or 12 weeks (Martinka, Fielitz et al. 2005) of SADinduced elevated BPV, evidence for both diastolic and systolic dysfunction were present.
Together, these findings suggest the loss of diastolic function occurs prior to any changes
in systolic function, which may only arise after seven to 10 weeks following SAD.
Tempol treatment of SAD rats limited many of the pathologic cardiac changes
observed in the untreated SAD group suggesting that oxidative stress may impact some
cardiac parameters more than others. Interestingly, tempol treatment was able to prevent
an increase in the amount of connective tissue observed in the wall of the left ventricle
but only partially limited the increase in cardiac hypertrophy. Conflicting results have
been described in the literature on the effects of tempol treatment on cardiac hypertrophy
91
and cardiac fibrosis (Zhang, Kimura et al. 2005; Moens, Takimoto et al. 2008; Rizzi,
Castro et al. 2011). This discrepancy in the literature may be related to the research
model used and other experimental conditions.
Specific vascular effects of enhanced BPV
Endothelial-dependent dilation of the LPCA in the denervated rats started to
deviate from the response of the control group at week one and got progressively worse
over the six week observation period. One of the major new findings in this study was the
ability of tempol treatment to limit the loss of endothelial-dependent dilation compared to
the untreated SAD group. During the initial week following the denervation surgery,
there was a similar reduction in the ability of the LPCA to respond to vasodilating agents
when comparing the untreated SAD rats and the tempol treated SAD rats. From week two
through six, tempol treated SAD rats had a greater LPCA dilatory response to pilocarpine
compared to untreated SAD rats. The endothelial-dependent dilation in the tempol treated
group was only slightly decreased from that observed in the LPCA of the control group.
Six weeks of SAD also resulted in endothelial-dependent vascular dysfunction in the
middle cerebral artery (MCA) without reduced endothelial-independent dilation. Again,
chronic tempol treatment was able to limit the endothelial-dependent vascular
dysfunction in the MCA compared to the untreated SAD group. Interestingly, incubating
the isolated MCA from chronically untreated rats (n = 2) in a tempol solution (10-3M) did
not improve the blunted ACh dose response curve (data not shown). This finding
suggests that chronically elevated BPV suppresses NO synthesis, which cannot be
restored by acute ROS scavenging. Furthermore, the lack of an effect of acute tempol
treatment, together with the beneficial effects of chronic tempol treatment to improve
92
endothelial-dependent dilation suggests that oxidative stress results in a more permanent,
presumably structural modification of the endothelial cells thereby reducing the synthesis
and release of NO. This may be related to ROS-induced endothelial progenitor cell
degeneration and apoptosis (Bao, Wu et al. 2010).
This is the first study showing a loss of endothelial-dependent function in small
resistance type arteries (LPCA = 40 µm; MCA = 125 µm) following SAD. Previous
studies have shown reduced endothelial-dependent dilation following SAD in large
conduit-type arteries (Miao, Tao et al. 2001; Eto, Toba et al. 2003; Feng, Luo et al.
2011). Eto et al. (Eto, Toba et al. 2003) have suggested that the blunted ACh-dependent
response they observed in the aorta following four weeks of SAD was due to a reduction
in NO release and remodeling of the endothelial cell layer to enhance neointimal
formation. Similarly, Feng et al. (Feng, Luo et al. 2011) have demonstrated reduced
endothelial-dependent dilation of the aorta that was associated with increased apoptosis
of endothelial cells and decreased eNOS expression in the aorta. Furthermore, their
laboratory has provided evidence for an increase in the aortic tissue concentrations of
both AngII and the lipid peroxidation byproduct MDA which would provide further
support for the findings in this thesis and suggest an important role for local tissue RASinduced oxidative stress in the SAD model.
While endothelial-dependent function was not investigated in the aorta in this
study, there was a decrease in aortic distensibility that was observed immediately one
week after SAD. Interestingly, inhibiting VSMC tone with an i.p. injection of the calcium
channel blocker nifedipine improved aortic distensibility back to the level of the Shamoperated group. The improvement of aortic distensibility was only observed during the
93
first two weeks at which point the distensibility progressively decreased but still
remained greater than when determined without inhibiting VSMC tone. This finding
would suggest that the initial decrease in distensibility was due entirely to an increase in
arterial tone. A combination of arterial tone and vascular remodeling may be responsible
for the long-term loss of aortic distensibility. Furthermore, in the presence of nifedipine
there was no difference in aortic distensibility between the untreated SAD rats and
tempol-treated SAD rats. This suggests that the beneficial effects of tempol to improve
aortic distensibility are related to reducing arterial tone. Thus, ROS may not be important
for the structural component but only for the functional component related to the BPVinduced reduction in aortic distensibility. A previous study has shown a similar loss of
aortic distensibility by investigating changes in the abdominal aorta after six weeks of
SAD and attributed the changes to a structural modification of the arterial wall that
included increases in collagen deposition and smooth muscle cell hypertrophy (Lacolley,
Bezie et al. 1995). In the current study there was a significant increase in the aortic wall
area suggesting the presence of structural vascular remodeling. There have been several
previous studies that have shown similar structural modifications of the aorta to include
medial smooth muscle cell growth and collagen accumulation in as little as two to four
weeks following SAD (Miao, Tao et al. 2001; Miao and Su 2002; Shen, Zhang et al.
2006; Zhang, Li et al. 2011).
Importance of assessing the time course
of BPV-induced effects
An important strength of this study is that the time course of hemodynamic
parameters and functional and structural cardiac and vascular indices have been obtained
94
in a repeated measures design. For example, this approach revealed that SAD-induced
BPV initially was 4-5 times higher than in the sham-operated group and gradually
declined towards the end of the six-week observation period where it was still 2-3 times
higher than in the control group. Interestingly, as BPV declined, BRS increased in a
closely matching time course. The magnitude of BPV observed at the end of the study as
well as the partial reduction of BPV over time is consistent with other reports (Miao and
Su 2002; Flues, Moraes-Silva et al. 2012).
In addition to the partial reduction in baroreflex buffering, the increase in BPV
following SAD is also thought to result from the interaction of both neural and humoral
factors acting on vascular smooth muscle to alter vascular tone (Alper, Jacob et al. 1987).
Thus, the larger increase in BPV as well as the lower BRS that was observed during week
one may reflect an initial over activation of the sympathetic nervous system. When
measured in chronic SAD studies, sympathetic activation is generally thought to return to
non-denervated levels after the first week (Irigoyen and Krieger 1998; Martinka, Fielitz
et al. 2005). In contrast, there is an increase in the cardiac tissue content of AngII (Miao,
Tao et al. 2001; Shan, Dai et al. 2003) and an increase in the circulating levels of
vasopressin (Callahan, Ludwig et al. 1997). This suggests an equal or more prominent
role for humoral factors such as the renin-angiotensin or vasopressin systems (Alper,
Jacob et al. 1987) in the maintenance of significantly elevated BPV in chronic (> 1 week)
SAD models. Alternatively, activation of humoral systems may be triggered as a
compensatory mechanism meant to buffer the SAD-induced BPV. However, activation of
these potential compensatory mechanisms – while initially improving blood pressure
regulation to some extent – may also elicit deleterious effects on the cardiovascular
95
system as indicated by the loss of endothelial-dependent dilation and impaired cardiac
function. Thus, loss of baroreceptor reflex function and increased BPV may trigger
compensatory mechanisms, such as the tissue RAS, which subsequently initiate the chain
of events leading to oxidative stress, lipid peroxidation, and ultimately cardiovascular
end-organ damage.
Study Considerations
BP was not significantly different between the three groups during the six week
observation period that started one week after SAD, confirming that the denervated rats
were indeed normotensive. However, during the first week of observation BP and HR
tended to be elevated in the two denervated groups compared to values in the Sham
group. BP is known to be elevated immediately following the SAD procedure; however
this acute hypertensive phase generally lasts less than seven days (Sved, Schreihofer et al.
1997). This initial hypertensive phase may have contributed to the observed changes in
cardiovascular remodeling and function as we did not control for this in the current study.
However, there has been some evidence to suggest the pathological changes observed
with increased BPV still occur even after preventing the initial hypertensive episode with
the ganglionic blocker hexamethonium bromide (Van Vliet, Hu et al. 1996; Van Vliet,
Chafe et al. 1999).
The significant reduction in the sensitivity of the baroreflex as well as the
significantly increased variability within the systolic blood pressure suggests successful
baroreceptor denervation in this study. It is important to note that the baroreceptor reflex
was not completely abolished in the denervated rats as there was still a partial reflex HR
response following changes in blood pressure. Alternatively, this could be viewed as a
96
strength of the study because a partial denervation would more closely mimic the
common human condition of subtle baroreflex impairment (Ketch, Biaggioni et al. 2002)
observed in elderly populations and those with chronic cardiovascular diseases such as
hypertension or heart failure.
The fact that aortic distensibility was the same in the tempol-treated and the
untreated SAD rats following administration of nifedipine suggests that chronic tempol
treatment can lower arterial tone. However, the precise mechanism relating tempol to the
lower arterial tone cannot be determined from this study. It is known that vasomotor tone
can be increased by the direct effects of ROS to increase intracellular calcium
concentrations (Tabet, Savoia et al. 2004). There is also evidence that tempol may
possess the ability to activate potassium channels or to act as a sympatholytic agent
(Wilcox and Pearlman 2008). Both of these actions, in addition to antioxidant scavenging
of ROS, may be involved in the reduction of arterial tone and other beneficial effects of
tempol observed in this study.
Summary and Conclusion
The results of this study provide greater insight into some of the potential
pathogenic mechanisms related to increased BPV-induced cardiovascular-end organ
damage. Through the use of a novel in vivo imaging technique this is the first study to
demonstrate a time course of endothelial-dependent dysfunction in resistance type
arteries within the same animals. The important role of smaller arteries (20 - 150 µm) to
regulate total peripheral resistance and the magnitude and distribution of tissue blood
flow explains the need to further understand how these resistance-type vessels are
impacted by pathologic stimuli such as increased BPV. The results of this study suggest
97
that chronically increased BPV results in a pro-oxidant environment in the heart and
blood vessels that leads to cardiovascular damage. With this regard, this study shows that
chronic treatment with the superoxide mimetic tempol is capable of limiting
cardiovascular damage in the normotensive SAD model of increased BPV presumably by
reducing oxidative stress. In conclusion, the cardiovascular pathology observed following
SAD appears to be at least partly mediated by oxidative stress. Specific treatments
(combination of scavenging and preventing ROS production) aimed at reducing the prooxidant environment in patients with increased BPV (e.g., hypertension, diabetes, heart
failure) may prevent or ameliorate cardiovascular end-organ damage and reduce the
overall risk of cardiovascular related morbidity and mortality.
Perspective and Future Research Directions
This study demonstrates the use of in vivo imaging of the LPCA to differentiate
between endothelial-dependent and endothelial-independent vascular function by using
corneal application of pilocarpine and SNP, respectively. It has been previously
suggested that the LPCA could be considered as a model for the resistance arteries of the
cerebral circulation because of its size and anatomical origin from the ophthalmic and
internal carotid arteries (Stauss, Rarick et al. 2011). In the current study there was a
significant correlation between the endothelial-dependent dilatory responses of the LPCA
and the MCA. Thus, assessing structure and function of the LPCA by in vivo imaging
may potentially be of considerable interest in research areas related to the cerebral
circulation or stroke.
Despite the fact that chronic tempol treatment has reduced or prevented
cardiovascular end-organ damage in the current study as well as in other animal models
98
of increased oxidative stress (Elmedal, de Dam et al. 2004; Ritchie, Quinn et al. 2007;
Rizzi, Castro et al. 2011; Fleenor, Seals et al. 2012), the use of chronic antioxidant
treatments in large clinical trials has mostly failed to demonstrate significant beneficial
effects on cardiovascular end points (Touyz 2004). This paradox may result from the
variety of ROS that can be generated in different cellular locations by a multitude of
enzymes (Dhalla, Temsah et al. 2000). Due to the different ROS characteristics it is
possible that certain antioxidants may not be able to access the source of ROS production
or may not have an equal scavenging effect on the different types of ROS produced
(Touyz 2004). It may be a more efficacious treatment option to prevent ROS generation
at the site of production as opposed to scavenging them after they have been produced.
Preventing ROS production is thought to be one of the additional mechanisms by which
some current cardiovascular drugs elicit their beneficial effects. For example, angiotensin
receptor blockers are thought to decrease oxidative stress by directly inhibiting the ROS
producing enzyme NADPH oxidase (Ghiadoni, Magagna et al. 2003).
Future studies investigating the effect of increased BPV-induced oxidative stress
on cardiovascular end-organ damage should determine both the source of oxidative stress
(cellular location/generating enzymes) and the main stimulus that initiates ROS
production (neural, humoral, stretch, intracellular calcium concentration). One important
pathway to consider would be the renin-angiotensin system and the subsequent activation
of CaMKII which is associated with increasing apoptosis and protein transcription.
Angiotensin II is likely an important contributor to ROS production and may also lead to
increased intracellular calcium concentration. It remains to be determined if the activity
of CaMKII, which is a downstream sensor of both ROS and calcium signals, remains
99
upregulated by calcium when the pro-oxidant environment created by increased BPV is
controlled by scavenging or preventing ROS production.
100
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