Roles of Flow Mechanics in Vascular Cell Biology in Health and Disease
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1 Roles of Flow Mechanics in Vascular Cell Biology in Health and Disease Shu Chien Dept. of Bioengineering & Medicine UC, San Diego Presented by Ming-Shaung Ju Dept. of Mech. Eng., NCKU, Tainan
2 Background Endothelial ( ) cell - inner lining blood vessel permeability barrier, regulate passage of molecules produce, secret, and metabolism of biochemical substances modulate contractility of smooth muscle cells
3 Atherosclerosis ( ) Accumulation of fatty materials as atheroma ( ) in artery wall Narrow of vessel lumen cause reduction of blood flow Problems: stroke, heart attack & walking difficulty
4 Role of LDL and monocyte in formation of foam cells Lipoprotein( ), monocyte( )
5 Whole artery tree has same LDL concentration. Atherosclerotic lesions show preferential distributions in branched points and curved regions. Local variations in hemodynamic forces may play a significant role in the focal nature of this lesion.
6 Components of hemodynamic forces
7 Aims Effects of shear stress on atherosclerosis. Fundamental mechanism of mechanochemical transduction. Biomechanical and molecular bases of preferential localization of atherosclerosis in arterial tree.
8 1. Role of Hemodynamic Factors in Transendothelial Permeability of Macromolecules At branch or curved, shear stress shows high spatial and temporal variations Complex flow patterns cause accelerated EC turnover (cell mitosis ) & death), increase permeability of large molecules (Weinbaum 85) EC mitosis & death associated w. albumin ( ) & low density lipoprotein (LDL)
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10 Mechanism of focal nature of atheroscrosis Local hemodynamic factors (complex flow pattern) EC turnover increase (mitosis & death) Local LDL permeability increase Focal lipid accumulation
11 2. Effects of Shear Stress on Monocyte Chemotatic Protein-1 (MCP-1) Gene Expression Process of gene expression proteins are encoded as genomic DNAs (2 strands, nucleic acids) promoter region cis-element, transcript factor, RNA (1 strand) RNA -> mrnas exit nucleus in ribosome( ) synthesize encoded proteins.
12
13 Gene Expression
14 Effects of shear stress on MCP-1 gene expression Monocyte/macrophages -> atherogenesis Macrophages found underneath EC at branch points -> local hemodynamic force can enhance monocyte entry. Chemotactic agent: monocyte chemotactic protein-1 (MCP-1) secreted by EC & other Phorbol ester TPA can stimulate EC
15 Effect of shear stress on MCP-1 secretion (human umbilical vein endothelial cells (HUVECs) Shear stress in arterial tree 12 dyn/cm^2,
16 Results Application of shear stress cause 2.5 fold increase in MCP-1 expression in 1.5 hours Sustained shear stress decrease gene expression even below pre-stressed level TPA responsive element (TRE) is a critical cis-element in shear stress activation. Transcription factor for TRE: dimer composed of cjun-cfos or cjun-cjun.
17 C-Jun and c-fos is mediated by mitogen activated protein kinase (MAPK) pathway MAPK pathway involves cell mitossis and programmed cell death (apoptossis) Pathways MAPK and attraction of monocytes by MCP-1 and modulation of LDL permeability by acceleration of cell turnover
18
19 3. Effects of Shear Stress on MARK Signaling Pathways MAPK signaling pathway protein kinases- enzyme that can do phosphorylation ( ) parallel pathways: JNK and ERK Ras common upstream molecular activated Ras bind GTP, phosphorylation of MAPK, JNK, ERK, activated c-jun & c- Fos & expression of MCP-1
20 1. Shear stress (12 dyn/cm^2) causes Ras bound to GTP 2. ERK and JNK are activated
21 Ras N17 - negative mutant molecule of Ras can suppress TRE adaptor molecules Shc, Grb2 and Sos upstream of Ras. (negative mutant suppress TRE activation)
22 What are the mechano-sensors responsible for initial conversion of mechanical signal to chemical changes?
23 4. Mechano-sensors for Shear Stress Two sets of membrane sensors: membrane receptors on luminal side of EC membrane integrin molecule on abluminal side facing the extracellular matrix ( ) (ECM)
24 Membrane receptor - receptor tyrosine kinase (RTK) Example: vascular endothelial growth factor receptor (VEGFR) Shear stress causes clustering and phosphorylation of VEGFR, binding of VEGF to Shc and other Activation of Ras and downstream molecules
25 Mechanical stimulation can activate VEGFR as chemical stimulation. VEGFR and G-protein coupled receptor can convert extracellular mechanical stimuli to intracellular chemical signals Shear stress cause membrane perturbation, changing conformation and/or interaction of membrane proteins such as VEGFR
26 Membrane integrins Integrins transmembrane receptors link intracellular cytoskeletal proteins w. proteins in ECM (2 way comm. Cell & ECM) > 20 types composed of two units α, β
27 α v β 3 integrin in endothelial cell bind to vitronetin and fibronectin in ECM shear stress causes association of α v β 3 w. Shc and subsequent activation of Ras pathway α v β 3 antibody attenuate shear-induced signaling.
28 Tension induced by shear stress on luminal side of EC membrane can be transmitted to integrins on abluminal side (mechanosensor) Signal initiated by membrane receptors transmitted by cytoskeleton to activate integrins (mechano-amplifier)
29 5. Effects of Complex Flow Patterns on Endothelial Cell Proliferation and Gene Expression Step flow to simulate flow condition at branch points eddy flow is induced and at reattachment region ( c ) shear stress is zero but gradient of stress is high. Proliferation of vascular ECs is assessed by using labeled nucleotide, BrdU.
30
31 After 24 h of laminar shear at 12 dyn/cm^2 BrdU is enhanced in re-attachment area and its vicinity Elsewhere are much less (down stream laminar flow) Same distribution for gene expression related to cell cycle control for proliferation and signaling molecule ERK Flow pattern in reattachment stimulate cell proliferation. Laminar area has low proliferation
32 Signal pathway & MCP-1 gene expression are down-regulated following sustained shearing. Sustained shearing in laminar flow region cause activation of growth-arrest genes (oppose proliferation) Complex flow pattern near branch points increases EC proliferation and does not down-regulate MCP-1 expression and placing the region at high risk of atherogenesis. Laminar flow region EC cell arrest.
33 Table 1 Comparison between straight part and branch points of arterial tree Flow Pattern Cell Turnover LDL Permeability Monocyte Infiltration Gene Expression Atherosclerosis Straight part Laminar Slow Low Few Growth Suppression Rare Branch Points Disturbed Rapid High Frequent Cell Proliferation Prevalent
34 6. Implications in Clinical Conditions The research has implications in prevention and treatment of cardiovascular diseases. Coronary artery disease: bypass surgery Balloon angioplasty Normal anatomy indication Procedure (1) Procedure (2) Procedure (3) Procedure (4)
35 Importance of mechanical matching between vascular bypass and native artery Vsacular bypass - segment of coronary artery w. atherosclerotic narrowing is replaced by artificial graft or vessel segment from patient. Saphenous vein( ) from patient s leg is often used. Veins normally exposed to low pressure (< 10 mmhg) artery (20 mmhg)
36 Importance of mechanical matching between vascular bypass and native artery (cnt d) Mechanical mismatching cause saphenous vein to bulge and sudden enlargement cause a geometric changes. Similar to step flow. Area of saphenous is subject to eddy flow and reattachment area is vulnerable to atherogenesis. Cell proliferation & MCP-1 expression. Need to reinforce saphenous vein prior to grafting. Important in tissue engineering.
37 Use of Ras negative mutant to prevent restenosis following balloon angioplasty Balloon angioplasty catheter w. a balloon at tip advanced to stenosis site under X-ray visualization. Inflation of balloon, press plaque, open vessel lumen. 1/3 re-stenosis within few months. Proliferation of smooth muscle cells, injury & chemical factors following endothelial denudation
38 Use of Ras negative mutant to prevent restenosis following balloon angioplasty Negative mutant RasN17 can be used to prevent vascular stenosis induced by balloon injury (animal studies) RasN17 was packaged to nonreplicating adenovirus to produce AdRasN17. LacZ was packaged to produce AdLaz. AdRasN17 treatment attenuate wall thickening due to balloon injury.
39 7. Summary and Conclusions Hemodynamic forces can modulate structure & function of endothelial cells and smooth muscle cells. Change in flow conditions can activate EC membrane receptors VEGFR & integrins to initiate transduction of Ras and JNK and ERK leading to increase in MCP1 expression.
40 7. Summary and Conclusions (cont d) Sustained laminar flow in artery tree down-regulate activation of MCP1 and minimize monocyte entry to vascular wall. Laminar flow associate w. low rate of cell turnover, growth-arrest genes are expressed. Atherosclerosis occurrs in arterial branch points and curved regions. Complex flow pattern, high turnover EC, permeability to LDL
41 7. Summary and Conclusions (cnt d) Widening of cell junction, monocyte accumulation due to high expression of MCP-1 Mechano-chemical transduction imply mechanical matching between grafting and vessel to prevent complex flow patterns Adenvirus-mediated transfer of RasN17 may become a prophylactic procedure against restenosis
42 By combining mechanics with biology (from molecules to tissues) bioengineering can contribute to better understanding of mechano-chemical transduction and improve methods of managing clinical conditions such as coronary artery disease.
43 Exercise due date 3/23/2006 What is the cell junction between endothelial cells? Find the ultra-structure of cell junction. What is gene expression? What is adenovirus-mediated transfer? How can this biotechnology be used to invent new drug?
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