Download 1 ANATOMY RS#9 August 14, 2008 JR Churchill, Ph.D. 9:00

ANATOMY RS#9
J. R. Churchill, Ph.D.
August 14, 2008
9:00-10:00AM
CONNECTIVE TISSUE: TYPES OF ORDINARY CT & THE EXTRACELLULAR
MATRIX
Suggested Readings: Ross & Pawlina: Chapters 6 (Connective Tissue) & 9 (Adipose Tissue)
and Wheater: Chapter 4 (Supporting/Connective Tissues)
OBJECTIVES:
1.
2.
3.
4.
5.
6.
List the features that distinguish a connective tissue (CT) from the other three basic tissue
types (epithelium, muscle and nerve).
List the characteristics of embryonic CTs, loose CTs, dense irregular CT, dense regular
CT, adipose CTs & reticular CT.
List the body regions wherein one would find the various forms of ordinary CT, and
describe how these locations are related to the different functions of CTs.
Describe the three major classes of macromolecules that make up the extracellular
matrix: glycosaminoglycans and their derivatives (hyaluronic acid and proteoglycans),
fibers (collagen, reticular and elastic), and adhesive glycoproteins (fibronectin and
laminin).
Describe the structure of mature collagen & elastic fibers and recognize each in
appropriately stained light micrographs and in electron micrographs.
Describe how collagen types I-IV & VII differ from one another and where you would
expect to find them.
NOTES ON THE CONNECTIVE TISSUES:
I. General Information
Connective tissue is one of the four basic tissue types (epithelium, connective tissue, muscle and
nerve). Various combinations of these tissues form all the organs of the body, and an
understanding of the basic tissues is a necessary prerequisite for understanding the organs
formed from them.
A.
Distinguishing characteristics of a connective tissue (CT):
1.
an abundant extra-cellular matrix (ECM) formed of fluid, ground substance, and
fibers of various types
2.
relatively few cells which are widely scattered throughout the matrix
3.
most CTs contain quite a variety of different cell types
B.
Some Terminology
• The stroma is the supporting framework of an organ.
– Often formed by CT, but can also be formed by cells.
• The parenchyma of an organ is composed of the cells that perform the function of that
organ.
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C.
Functions of connective tissue:
1.
2.
3.
4.
5.
6.
7.
8.
D.
provides support for epithelia (the lamina propria for moist membranes and the
dermis for the skin) and an architectural framework (the stroma) for organs
protects and defines limits of organs (e.g., capsules, epimysium, epineurium)
serves as a medium of exchange through which metabolic wastes, nutrients, 02,
etc. diffuse (therefore CT is found around small blood vessels)
attaches elements of the musculoskeletal system to one another and resists
stretching forces (e.g., tendons & ligaments)
can store mechanical energy for future use (like a spring)
serves as a common site of inflammatory responses and immune reactions,
especially in the layers just beneath epithelia of mucosae [esp. the respiratory &
GI systems]
important in wound healing
storage of energy reserves in adipose cells & thermal regulation for the body via a
layer of subcutaneous fat
A classification of CTs:
1.
Embryonic CTs
a.
Mesenchyme [mesenchymal cells (stellate and spindle-shaped
pluripotential precursor cells for various adult tissues) in a gel-like ground
substance containing few fibers, mainly reticular]
1/
Mesenchyme is a precursor to many of the various adult tissues,
giving rise to some epithelia, to muscle, to some nervous tissue, to
connective tissue proper and specialized connective tissues.
2/
It is derived mainly from mesoderm, but some develops from
ectoderm via the neural crest.
b.
Mucous CT of the umbilical cord (Wharton's jelly; a jelly-like matrix
sparsely populated with large stellate fibroblasts, reticular fibers and
collagen fibers; helps to prevent knot formation in the human umbilical
cord)
2.
Connective tissue proper (“Ordinary” CT)
a.
Loose CT
1/
areolar or loose [irregular; superficial (papillary) dermis, around
ducts in mammary gland, lamina propria of some mucosae]
2/
cellular (lamina propria of some mucosae)
b.
Dense irregular CT [submucosae of tubular organs of the GI tract,
deep (reticular) dermis, capsules for many organs]
c.
Dense regular CT (tendons, aponeuroses, ligaments)
d.
Dense elastic CT [certain ligaments of vertebral column
(ligamentum flavum) and the nuchal ligament]
3.
Specialized CTs
a.
Cartilage (not this lecture)
b.
Bone (not this lecture)
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c.
d.
e.
E.
Blood (not this lecture)
Adipose tissue (white and brown fat)
1/
Distribution of brown fat: esp. around kidneys, adrenals, aorta &
suprascapular subcutaneous area.
Reticular tissue (in some lymphoid organs)
1/
has a stroma that is rich in reticular fibers. Reticular fibers are a
highly glycosylated form of collagen and thus can be stained by
the PAS method or via a silver stain. Thus reticular fibers are
argyrophilic.
Where in the body does one find connective tissue proper ("ordinary" CT)?
• Loose CT
• Dense Irregular CT
• Dense regular CT
See the CT Critter in Figure 1:
Where do you find ordinary CTs?
1.
2.
3.
4.
5.
CTs are found: just beneath epithelia and surrounding many organs (such as the
adventitia and surrounding CT)
forming the capsule of many organs, e.g., spleen, kidney, liver
forming the outer covering of muscles (epimysium) or nerves (epineurium)
within many organs where they form the stroma (i.e., the supporting framework of the
organ).
around small blood vessels such as capillaries & postcapillary venules
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6.
connecting muscles to bones (tendons are cord-like and aponeuroses are sheet-like) or
bones to other bones (ligaments)
II.
A.
A CT is composed of:
an EXTRACELLULAR MATRIX (ECM)
1.
formed of GROUND SUBSTANCE
2.
and insoluble FIBERS
with resident (fixed) and migratory cells (not this lecture).
B.
III.
GROUND SUBSTANCE - the stuff found between fibers and cells, is usually lost
during tissue preparation of ordinary CTs, thus is often under-appreciated in
routine tissue sections. It contains tissue fluid bound to polysaccharides.
A.
Glycosaminoglycans (GAGs) were previously called mucopolysaccharides. Most
GAGs are the polysaccharides of proteoglycans.
1.
GAGs are long, fairly inflexible, unbranched polysaccharide chains composed of
a repeating disaccharide unit. Most GAGs have fewer than 300 repeating
disaccharide units but hyaluronic acid can have as many as 25,000 repeating
disaccharide units.
2.
GAGs are highly hydrated due to an abundance of hydroxyl, carboxyl and sulfate
groups; they give the ECM a gel-like consistency and can prevent easy movement
of bacteria through the ECM.
3.
With one exception (hyaluronic acid or hyaluronan) GAGs are synthesized as
covalent, post-translational modifications of core proteins. Thus most GAGs are
components of proteoglycans. The core proteins are made in the RER, modified in
the Golgi by addition of the GAG which is then sulfated; the proteoglycans then
enter the secretory pathway of many cells (not just CT cells). Hyaluronic acid is
synthesized by cell-surface enzymes; it is not post-synthetically modified.
Hyaluronic acid is a GAG and not part of a proteoglycan.
4.
Table 6.3, p. 162 of Ross lists the major GAGs of connective tissues. Note that
hyaluronic acid is a very long GAG that is not sulfated, nor is it attached to a core
protein. Hyaluronic acid is found in most CTs & synovial fluid where a single
molecule can occupy a diameter of 200 nm. Heparin is a cleavage product from
heparan-sulfate. It also lacks a core protein. All of the other GAGs are present as
covalent attachments on the core protein of a proteoglycan. You should know the
names of the various GAGs, know which are sulfated, and which are found as
proteoglycans.
a.
b.
c.
heparan sulfate was first isolated from the liver
there are two forms of chondroitan sulfate; they were first isolated from
cartilage
keratan sulfate was first isolated from the cornea of the eye
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d.
B.
Proteoglycans consist of a core protein to which sulfated GAGs are covalently
bound. The GAG is the dominant component of the molecule in most descriptions of
function; only recently are the names of the specific proteoglycans used in descriptions of
GAGs.
1.
2.
3.
4.
5.
C.
2.
3.
A.
B.
The proteoglycan family of proteins is large and varied in structure and function.
Grouping them together is similar to grouping all phosphorylated proteins
together, however some general features make it convenient to consider them as a
group.
They are major constituents of cartilage, loose CT and basement membranes.
Mast cells package a proteoglycan to bind heparin in their secretory granules.
Most proteoglycans are secreted into the ECM but some remain bound to the
plasma membrane and are involved in cell adhesion to the ECM.
The number of GAGs attached to the core protein varies from one (decorin) to
more than 200 (aggrecan). Some proteoglycans have identical GAGs (perlecan)
while others are formed of a core protein linked to different GAGs (aggrecan,
syndecan).
Aggrecan has a large core protein (250 kD) with >100 keratan-sulfate and >150
chondroitin-sulfate GAGS. It is a proteoglycan found in cartilage. Decorin is
secreted by fibroblasts and it binds collagen fibrils and modifies their assembly.
Perlecan (the protein is like a string of pearls) is secreted from all cells that make
a basal lamina. It has 3 heparan-sulfate GAGs. It self-associates and also binds
laminin and basic fibroblast growth factor. Syndecan is an integral membrane
protein of embryonic and mesenchymal cells and is developmentally regulated in
B lymphocytes where it serves to anchor the cell to the ECM (only expressed in
developing cells bound to matrix fibers in the bone marrow and then again in
plasma cells in the matrix of lymph nodes).
Proteoglycan aggregates
1.
IV.
dermatan sulfate was first isolated from the dermis of the skin
consists of a hyaluronic acid molecule with which many proteoglycans are
noncovalently associated.
The enormous size of these aggregates present a physical barrier to the movement
of bacteria and metastatic cells though ground substance.
The virulence factors of some highly invasive bacteria include a secreted
hyaluronidase.
Glycoproteins have covalently attached carbohydrate with the protein component the
dominant portion of the molecule. Important glycoproteins of the ECM include:
Structural glycoproteins:
1.
Collagens
2.
Fibrillin component of elastic fibers
Adhesive glycoproteins
1.
Fibronectin
2.
Laminin
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V.
The Fibers of the ECM are made of glycoproteins.
A.
Collagen
1.
collagen is the major protein of the ECM (~30% of all the protein in the body)
where it provides great tensile strength when in high content
2.
collagen fibers stain pink (i.e., eosinophilic or acidophilic) with hematoxylin &
eosin (H&E), blue or green with most trichrome stains, and deep blue with Azan
stains
3.
types of collagen -- collagen is a family of structurally similar but not identical
proteins
a.
all collagen genes code for proteins with a repeating Gly-Pro-X
sequence; the Gly is needed to form the triple helix.
b.
all form a characteristic triple helical structure
c.
there are at least 25 types of triple helical collagen (30 different α chain
genes) but 90% is of type I.
d.
the various collagens differ in their ability to form fibrils or sheets or to
cross-link the fibril-forming collagens
e.
the major types of collagen and their characteristics are listed in Table 6.2,
p. 153-154 of Ross. You are responsible for collagen types I - IV & VII.
f.
in collagen nomenclature, the collagen type is in roman numerals and is
enclosed in parentheses. Although collagen chains are referred to as alpha
(α) chains, this does not denote the presence of an α -helical protein
secondary structure, indeed, collagen cannot assume an α-helix due to its
high content of proline residues.
1/
Type I collagen is composed of two α1(I) chains and one α2(I)
chain. It is written: [α1(I)]2α(2).
2/
Type II collagen: [α1(II)]3
3/
Type III collagen: [α1(III)]3
4/
Type IV collagen: [α1(IV)]2α2(IV)
5/
Type VII collagen: [α1(VII)]3
4.
structure of a collagen molecule
a.
has a unique composition with glycine in every third position and a high
content of proline, hydroxyproline and hydroxylysine. The hydroxylations
are done post-translationally. Assay for hydroxyproline content is often
used to quantitate collagen content in a tissue. Ascorbic acid (vitamin C) is
needed for the hydroxylating enzymes.
b.
composed of three polypeptide chains (α chains) that form a compact
triple helix
c.
a collagen molecule is also called tropocollagen
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5.
synthesis of tropocollagen - a triple-helical macromolecule formed by postsecretion cleavage
a.
in most ordinary CTs tropocollagen is produced by fibroblasts (but also
secreted by Schwann cells in nerves, by smooth muscle cells in the wall of
vessels)
b.
large precursors of the alpha chains (preproalpha chains) are synthesized
on the RER
c.
part of the amino acid chain (the signal sequence) is cleaved within the
lumen of the RER to produce a slightly smaller precursor (proalpha
chains)
d.
proalpha chains are modified (hydroxylated to form hyroxyproline and
hydroxylysine and glycosylated) within the RER
e.
the central parts of the three modified proalpha chains zipper together to
form a triple helix within the ER to produce a procollagen molecule;
sequences called propeptides at each end of the three proalpha chains aid
in alignment of the chains
f.
procollagen is transported to the Golgi where it is further modified and
packaged into secretory vesicles. The procollagen is constitutively
secreted (i.e., no storage of secretory vesicles prior to exocytosis of the
vesicle contents).
g.
outside the cell, the alignment sequences (called propeptides) are
enzymatically cleaved from the ends of the molecule by extracellular
enzymes to produce mature tropocollagen (collagen) macromolecules
h.
tropocollagen (collagen) molecules of types I, II, and III self-assemble
extracellularly within a cove (formed by the cell) to produce collagen
fibrils. An extracellular enzyme modifies the lysines and hydroxylysines
to promote the formation of covalent cross-links between tropocollagens
within the fibrils and thus stabilize the fibrils. This increases with time.
1/
fibrils are characterized by a distinctive banding pattern with a 6568 nm repeat
2/
fibrils are visible by electron microscopy, but NOT by light
microscopy
3/
fibrils become arranged in parallel to form fibers with types I and
III
4/
fibers are visible by light microscopy
6.
collagen type I is the most abundant (accounts for most collagen); forms fibrils
20-200 nm in diameter that combine into much larger fibers; the thick fiber
bundles in any CT seen via LM are usually composed mostly of type I fibrils
7.
collagen type II forms much thinner fibrils, and few if any fibers; found in the
matrix of hyaline and elastic cartilage, the nucleus pulposis of the intervertebral
disc and the vitreous body of the eye
8.
collagen type III makes up reticular fibers (see more below)
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B.
9.
collagen type IV is found in the basal laminae of epithelia and the external
laminae of certain cells (all muscle cells, adipocytes, Schwann cells); forms a
planar net of collagen molecules rather than fibers or fibrils
10.
collagen type VII forms anchoring fibrils that fasten the lamina densa of the basal
lamina of the epidermis to the underlying reticular lamina of the dermis; anchors
the basal lamina of stratified epithelia of skin, cornea, and esophagus to the
adjacent CT layer.
Reticular fibers (collagen type III)
1.
2.
3.
4.
C.
Reticular fibers cannot be distinguished from other collagen fibers using H&E.
Reticular fibers are very thin (0.5-2.0 µm diameter) formed from delicate 50 nm
diameter fibrils. They are highly glycosylated, giving them different staining
properties from other collagen types and they were originally thought to be
formed of a material different than collagen. Reticular fibers are PAS-positive
(colored magenta with PAS stain) and also stain black with certain silver stains
(they are argyrophilic).
reticular fibrils and fibers are very thin; they remain closely associated with the
fibroblast-like cells (reticular cells) that synthesize them in organs such as lymph
nodes
they form the stroma of organs such as liver, spleen, lymph nodes, some
endocrine organs and bone marrow, provide support for smooth muscle in the
wall of the gut and uterus, form the endoneurial layer outside of Schwann cells in
nerves and contribute to the ECM of embryonic CT.
in other locations they can be mixed with elastic fibers and collagen type I fibers
(e.g., in the wall of arteries, dermis of the skin, lungs)
Elastic fibers, sheets and laminae
1.
2.
3.
abundant in tissues which have to stretch often, e.g., dermis, elastic arteries,
lungs, vocal cords, urinary bladder, since elastic fibers can stretch to 50% longer
than their relaxed length
Elastic fibers are formed by fibrillin microfibrils and then filled in with the
protein elastin.
mature elastic fibers have 2 main components:
a.
an amorphous central region made up of the protein elastin. Elastin is a
randomly coiled protein, poor in hydroxyproline, lacking hydroxylysine,
but containing two unique amino acids, desmosine and isodesmosine.
Covalent cross-linking between the unique amino acids creates a network
capable of great elasticity.
b.
microfibrils composed of a glycoprotein called fibrillin; the microfibrils
are arranged around the periphery of the elastin core
c.
Elastic fibers stain poorly with eosin in H&E but have a different
refractive index than does collagen, thus can be distinguished from
collagen in LMs when present in thick layers or elastic lamella. Otherwise
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d.
require a special stain to see elastic fibers with light microscopy (Weigert's
resorcin-fuchsin, aldehyde-fuchsin, Verhoeff’s picro-ponceau).
EM appearance is unique since appear like a partially erased pencil
smudge surrounded by fibrillin microfibrils (similar to fat and glycogen in
LMs, they are sometimes noticed more by the void created in their place in
TEMs).
VI.
The Adhesive Glycoproteins of the ECM
A.
General
1.
2.
3.
4.
smaller molecules than the structural glycoprotein and include fibronectin,
laminin, and others
promote the attachment of ECM components to one another, and the attachment
of cells to the ECM
can perform these cross-linking functions because each molecule has several
different binding sites or domains; some domains bind to cells, others bind to
ECM components such as collagen or specific GAGs
the cell surface receptors for some of these adhesive molecules are members of
the integrin family ; integrins are transmembrane proteins with extracellular and
intracellular domains
a.
their extracellular domain binds some specific component of the ECM
(e.g., fibronectin or laminin); the extracellular domain of epithelial cell
integrins extends through the lamina lucida of basal laminae to bind to
laminin which is in the lamina densa portion (see Ross fig. 5.29a)
b.
their intracellular domain binds to some specific component of the
cytoskeleton (e.g., as part of focal adhesions or hemidesmosomes)
c.
their intracellular domain also interacts with internal signaling molecules
B.
fibronectin - a V-shaped molecule made up of two different polypeptide chains; has
different domains which bind to collagen, heparin, & CT cells - therefore facilitates the
binding of CT cells to the ECM. Found in CT and in blood (plasma fibronectin) and
marks paths for cell migration in embryos.
C.
laminin - a large complex made up of three different polypeptide chains arranged in the
shape of a cross; found in the basal lamina within the lamina lucida and densa; has
different domains which bind to collagen types IV and VII, the heparan sulfate of
perelecan and to epithelial cells; therefore facilitates the binding of epithelial cells to
basal laminae.
D.
entactin (nidogen) - cross-links laminin to the type IV collagen of the basal lamina
E.
tenascin - six polypeptide chains arranged to resemble a bug with six legs projecting
radially from a central body; binds to fibronectin and a cell-associated proteoglycan
(syndecan); usually limited to embryonic tissue, where it marks migratory pathways for
specific cells
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VII.
Basal Laminae and Basement Membranes
A.
Basal laminae are only visible via the electron microscope. A layer of ECM with a
similar appearance underlies epithelial sheets, surrounds myocytes, adipocytes, and
Schwann cells. The basal lamina is synthesized by the epithelial (muscle, fat, or
Schwann) cells. Basal laminae have two layers: adjacent to the epithelial sheet is an
electron lucent region termed the lamina lucida, adjacent to this is the electron-dense
region termed the lamina densa.
1.
The lamina lucida contains the extracellular domains of the integrins and an
anchoring collagen (type XVII).
2.
The lamina densa contains a meshwork of type IV collagen and the proteoglycan
perlecan. Perlecan has heparan sulfate GAGs. The CT-side of the lamina densa
contains fibronectin to facilitate adhesion to the next layer, the lamina reticularis
from the surrounding connective tissue.
A basement membrane is visible via light microscopy because the connective tissue
underlying an epithelial sheet contributes another layer termed the lamina reticularis
(reticular lamina).
1.
Reticular fibers (type III collagen), collagen fibers (type I collagen), and
anchoring fibrils (type VII collagen) aid in binding the basal lamina to the
adjacent connective tissue.
2.
The relative prominence of the basement membrane is dependent upon the
thickness of the reticular lamina. A prominent basement membrane is
characteristic of the epidermis and the trachea, two areas that are subjected to
frictional forces on the epithelial sheet.
B.
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