Download The effects of biomechanical forces on vascular cells

 The effects of biomechanical forces on vascular cells LOW BLOOD FLOW’S EFFECTS ON ENDOTHELIAL CELLS Student: Katharina Steiner – Gymnasium Interlaken | SJF Biology and Medicine | 13. – 19. March 2016 | | Tutor: Mannekomba Roxane Diagbouga, University of Geneva | | Head of Lab: Brenda Kwak, University of Geneva | Abstract Intracranial aneurysm (ICA) is a disease of the vascular wall resulting in abnormal enlargement of the arterial lumen Despite this massive threat to human life, only little is known about their cause or the process by which they form, grow, and eventually rupture. Blood flow is the continuous circulation of blood in the cardiovascular system. Normal blood flow in straight arteries is applied a high drag force of about 30 dynes per square centimeter on the vessel wall and is laminar. This force is called shear stress. In some pathological conditions as in aneurysm disease the vessel sustains a very low blood flow that is thought to be responsible of damages in the arterial wall. We hypothesize that hemodynamic forces, which are induced by blood pressure and the flowing blood, are of vital importance to maintain homeostasis in the structural organization of the vascular wall. We cultured endothelial cells (EC) to test how they will behave under low shear stress of 5 dynes per square centimeter, a force that is 6 times less than the normal force applying to the vessels wall. PAGE 1 Introduction Blood flow is the continuous circulation of blood in the cardiovascular system. Normal blood flow in straight arteries is applied a high dragging force of some 30 dynes per square centimetre on the vessel wall and is laminar. This force is called shear stress. In some pathological conditions as in aneurysm disease the vessel sustains a very low blood flow that is thought to be responsible of damages in the arterial wall. We cultured endothelial cells (EC) to test how they will behave under low shear stress of 5 dynes per square centimetre, a force that is 6 times less than the normal force applying to the vessel wall. Endothelial cells form the linings of the blood vessels. In general blood vessels, such as arteries and veins are composed of three layers which are concentrically arranged (Fig.1): 1. Tunica intima (EC’s) 2. Tunica media (smooth muscle) 3. Tunica adventitia or externa (Connective tissue and nerves) The layer of biggest interest for this research is the intima. It is the thinnest layer and is composed of a single layer of endothelial cells. They represent a Fig. 1: The structure of an artery wall barrier of the blood to the tissue. In the sacs of aneurysms, the shear stress level is reduced to a vast extent as it is clearly discernible in figure 2. In straight arteries the cells sustain high shear stress (in red) and in aneurysm pouch the cells sustain very low shear stress (green and blue). Fig. 2: Blood flow in aneurysm Aim The Aim of our project is to examine the behaviour of the EC in response to a low flow in terms of shape and orientation. We will also look at trans-­‐membrane proteins that are connexins and pannexin to compare their expression between cells were we applied low flow and cell were we didn’t apply flow (static). Material and Methods The procedure of examining the endothelial cells can be structured into following main steps: Cell culture
Immunostaining
Fluorescent Microscopy
Flow experiment
ECs were seeded on Ibidi slides (Fig. 3A) coated with 0.1% gelatin. When the cells reached confluency they were put under flow of 5 dynes/cm2 using the Ibidi system (Fig. 3B) for 24h. Then the cells were fixed with methanol at -­‐20 degrees for 5 min and permeabilized with 0.2% Triton x 100/PBS. The reactive aldehydes were quenched by incubating cells in a PBS-­‐NH4Cl (ammonium chloride) solution for 15 minutes. Then, the cells were incubated in 2% of PBS/BSA (=Bovine Serum Albumin) for 30 minutes to block non-­‐specific binding sites. A first Antibody (AB) is added to the ECs which will then hopefully attach specifically to the desired protein. Here 1 AB per channel was used, so only one specific protein was stained per channel. The f1rst AB is incubated overnight at 4°c. The antibodies used were for Connexin 37 diluted at 1/50, Connexin 40 diluted at 1/100, Connexin 43 diluted at 1/100, and Pannexin 1 diluted at 1/500. The following day the rest of the experiment was performed in the dark. Anti-­‐rabbit and anti-­‐chicken secondary antibodies were added respectively to detect Connexin and Pannexin antibody staining (Fig. 3C). The secondary antibodies were linked to the fluorescent dye Alexa 488. Then the cytoplasm was stained using 0,003% of Evans-­‐blue for 1 minute and finally the nucleus was stained using 1/20000 DAPI/PBS during 10 minutes. We put a drop of vectashield to stabilize the fluorescent dye. Fig. 2: A. Ibidi slide for cell cultures B. in vitro blood flow device C. Immunostaining Results and discussion Blood vessels are exposed to multiple mechanical forces that are exerted on the vessel wall (radial, circumferential and longitudinal forces) or on the endothelial surface (shear stress). The stresses and strains experienced by arteries influence the initiation of diseases like aneurysm, which develop at regions of arteries that are exposed to complex blood flow. Understanding how these forces regulate the behavior of the vascular endothelial and smooth muscle cells will help to get insight into the disease etiology. Negative control
Pannexin 1
Static
Flow
5 dynes/cm
2
Negative control
Connexin 37
Connexin 40
Connexin 43
Static
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5 dynes/cm
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Fig. 4: Pannexin or Connexins are marked in green. C ells are counterstained with Evans Blue (red). N uclei are marked with DAPI (blue). In this project, we used a good model to mimic blood flow in vitro. Our results show that under low flow of 5dynes/cm2 ECs lose Panx1 expression compared to static condition. We noticed a slight increase in Cx37 and Cx43 expression in ECs that sustained low flow and we had no expression of Cx40 in neither conditions. Moreover, we noticed no change in cells shape and orientation after 24h under low flow of 5dynes/cm2 (fig. 4). Further analyzes will be required to understand how the change in protein expression can interfere in ECs response to low flow. References 1. Hahn C, Schwartz MA. Mechanotransduction in vascular physiology and atherogenesis. Nature reviews Molecular cell biology. 2009 2. Xiang J et al, J NeuroIntervent Surg 2014 3. Zp://www.manchestercfd.co.uk/#!projects/cee5 4. hZp://radiopaedia.org/arAcles/histology-­‐-­‐-­‐of-­‐-­‐-­‐blood-­‐-­‐-­‐vessels Acknowledgements First of all I would like to thank the organisation “Schweizer Jugend forscht” for creating and planning a great week full of unique and new experiences in the aspect of science, opening a whole new world to me, but also in course of all the joy and friendship I had the pleasure to experience during a wonderful week in Geneva and Lausanne. I would like to express my very great appreciation to Mannekomba Roxane Diagbouga for supporting and helping me throughout the whole week. I am very grateful for, her taking the time to enable me such a valuable insight into current research and explaining and teaching to me current experiments and moreover for helping me create my poster and least but not last for helping me with my report. I am particularly grateful for the fact that I could come and actually work in the laboratory of the University of Geneva during this study week, therefore I want to thank the head of Lab Brenda Kwak for making this all possible. Thank you.