MODULATION OF ENDOTHELIAL TIGHT JUNCTIONS AND VASCULAR PERMEABILITY BY VASCULAR ENDOTHELIAL GROWTH FACTOR

Poster Abstracts / Cardiovascular Pathology 13 (2004) S17–S79

P073 ROLE OF DIRECT INTERCELLULAR COMMUNICATION THROUGH GAP JUNCTIONS IN LYMPHOCYTE-MACROPHAGE INTERACTIONS IN VITRO. Ernesto Oviedo-Orta, Sharada Karanam, Ulrike Benbow, W Howard Evans, Andrew C. Newby. Bristol Heart Institute, Bristol, England, Dept. Medical Biochemistry & Immunology, University of Wales College of Medicine, Wales. Gap junctions are clusters of plasma membrane intercellular channels that permit direct cross talk between attached cells allowing the passage of small molecules and ions of less than 1kDa. Connexins (Cx), the protein building blocks of gap junction channels, are widely distributed, being found in all tissues and organs except striated muscle. Several studies have recently demonstrated that gap junction mediated communication operates between cells involved in atherosclerotic lesion development. Direct intercellular communication occurs between blood derived monocytes and endothelial and smooth muscle cells or between macrophage foam cells. These diverse observations strongly suggest that Cxs are important contributors to atherosclerosis. However, the mechanisms underlying their role remain to be elucidated. We have investigated the expression of Cx 43 by CD4 + T helper 1 (Th1) and 2 (Th2) lymphocytes, macrophages and co-cultures of both cell types. Cx 43 mRNA was studied by PCR. Our results show a significant upregulation of Cx 43 by pro-inflammatory Th1 cells (5.51 ± 0.4; p < .009) with respect to Th0 lymphocytes (3.08 ± 3.0) and also a complete abrogation of its expression by anti-inflammatory Th2 lymphocytes. Protein expression was measured by Western blot. While Cx 43 expression by Th2 lymphocytes remained unchanged Th1 lymphocytes significantly up-regulated it (47.74 ± 2.03; p < .006) with respect to naı¨ve Th0 cells (26.5 ± 1.3). The level of direct intercellular communication, measured by Calcein dye transfer, between macrophages and T helper cell subsets was assessed by FACS analysis. The results show an increased level of intercellular communication between macrophages and Th1 proinflammatory cells. Our studies suggest that Cx 43 is actively involved in inflammation in which it may play a crucial role in leukocyte activation and response. British Heart Foundation.

P075 ROLE OF CELL-CELL JUNCTIONS IN MEDIATING ENDOTHELIAL CELL RESPONSES TO FLUID SHEAR STRESS. Eleni Tzima, Mohamed Irani-Tehrani, Elizabetta Dejana, Martin Schwartz. The Scripps Research Institute, La Jolla, CA 92037, Istituto FIRC di Oncologia Molecolare, Milano, Italy, University of Virginia, Charlottesville, VA, USA. Fluid shear stress is a critical determinant of vascular remodeling, regulation of blood pressure and development of atherosclerosis. Endothelial cells (ECs) subjected to fluid flow show a rapid conversion of integrins to their high affinity state. These activated integrins then bind to available extracellular matrix proteins at the basal surface and initiate signals, including transient inactivation of the small GTPase Rho and transient activation of Rac and Cdc42. We now show that the pathway responsible for integrin activation in response to shear stress is initiated at intercellular junctions and that PI3K and Src are involved. PECAM-1 and VE-Cadherin null ECs failed to show shear stress-induced activation of Flk-1, PI3K and integrins or shear stress-dependent gene expression and were unable to align in the direction of flow. When magnetic beads coated with antibodies against the extracellular domain of PECAM-1 were attached to ECs and tugged by magnetic force, localized activation of integrins and PI3K was seen around the beads. Binding of the beads on VE-cadherin null cells or pulling on the cell surface using VE-cadherin-coated beads did not induce integrin or PI3K activation, suggesting that VE-cadherin is required but

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direct force application on it is insufficient to initiate shear stress signaling. In addition, VE-cadherin is required for the shear stress-induced association of PI3K with b-catenin and PECAM-1. Finally, transfection of Cos7 cells – which do not align in response to shear stress-with PECAM-1, VE-cadherin and Flk-1 facilitated alignment of their actin stress fibers in the direction of flow. These studies therefore define a novel mechanism by which shear stress initiates signaling through cell-cell junctions that leads to integrin activation and the modulation of downstream responses relevant to inflammatory pathology. American Heart Association Western States Affiliate.

P076 MODULATION OF ENDOTHELIAL TIGHT JUNCTIONS AND VASCULAR PERMEABILITY BY VASCULAR ENDOTHELIAL GROWTH FACTOR. Brian Wing Chi Wong, Maziar Rahmani, Zongshu Luo, Sylvia Loo, Donald Wong, Bobby Yanagawa, Decheng Yang, Bruce Maxwell McManus. The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, University of British Columbia, Vancouver, BC. Background: Vascular endothelial growth factor (VEGF) is specific mitogen for vascular endothelial cells and is a potent inducer of vascular permeability. Vascular permeability is a proposed mechanism for the rapid insudation of lipids and lipoproteins which occurs post-transplantation in the pathogenesis of transplant vascular disease. Our laboratory has previously demonstrated that VEGF can significantly reduce the integrity of tight junctions and increase the permeability of low density lipoproteins across the endothelial monolayer. The goal of our experiments was to determine the mechanism of VEGF-induced tight junctional disassembly and elucidate the route of induced vascular permeability. Hypothesis: Vascular endothelial growth factor induces phorsphorylationdependent disassociation of endothelial tight junctions, leading to increased permeability. Methods: Human coronary artery, cardiac microvessel, and umbilical vein endothelial cells were grown to 100% confluence on collagen-coated cultureware. Cells were treated with VEGF, then imaged using confocal microscopy in real time, using AlexaFluor 488-tagged phalloidin and Hoechst 33342 to visualize F-actin and nuclei. Cell lysates were obtained at 15, 30, 60, and 120 minutes and Western blot was used to assess the expression and phorsphorylation status of occludin, ZO-1, and claudin, complemented with alkaline phosphatase dephosphorylation analysis. As well, horseradish peroxidase (HRP) tracer was used to assess VEGFinduced permeability using transmission electron microscopy (TEM). Finally, we have optimized the transfection of a ZO1-GFP construct and experiments are currently underway to image the alterations in ZO1 localization in real time using confocal microscopy. Results: Upon stimulation with VEGF, cell retraction and intercellular gap formation was visualized in real time using differential interference contrast (DIC) microscopy and fluorescent visualization of F-actin and nuclei. Phosphorylation of occludin, ZO-1, and claudin was evident within 15 minutes as assessed by Western blot and alkaline phosphatase dephosphorylation analysis. As well, after VEGF-treatment, more HRP tracer was noticeable within intracellular vesicles upon TEM examination. Summary: We have demonstrated in human coronary artery, cardiac microvessel and umbilical vein endothelial cells that VEGF can induce cell retraction, actin disorganization, and phorsphorylation of tight junction proteins resulting in disruption of endothelial tight junctions. As well, TEM examination has demonstrated HRP tracer uptake in endothelial cells upon VEGF-stimulation. These data suggest two possible mechanisms of induced endothelial permeability upon VEGF stimulation. More experiments are currently underway to image low density lipoprotein transport across the endothelium after VEGF stimulation in real time using a ZO1GFP construct and confocal microscopy.

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Poster Abstracts / Cardiovascular Pathology 13 (2004) S17–S79

This work is supported by an operating grant from the Canadian Institutes of Health Research (BMM) and personal funding from the Heart and Stroke Foundation of Canada (BWCW, MR, BY) and the Canadian Institutes of Health Research (BY).

P077 THE DELETION OF CONNEXIN37 ENHANCES ATHEROSCLEROSIS IN APOLIPOPROTEIN E-DEFICIENT MICE. Cindy W Wong, Thomas Christen, Graziano Pelli, Francois Mach, Daniel A Goodenough, Brenda R Kwak. Division of Cardiology, University Hospital, Geneva, Switzerland, Dept. of Cell Biology, Harvard Medical School, Boston, MA 02115. Gap junctions, formed by the connexin (Cx) protein family, are intercellular channels that permit the direct cytoplasmic exchange of ions and small metabolites between cells, a process called gap junction intercellular communication (GJIC). There are presently over 20 connexins, each of which can create functional channels with unique properties including distinctive permeabilities for various signaling molecules. Growing evidence supports a role for GJIC in the development of atherosclerosis, a progressive immuno-inflammatory disease. For example, expression patterns of three vascular connexins (C  37, C  40 and C  43) are altered in mouse and human atherosclerotic lesions. Furthermore, reducing expression of C  43 in low-density lipoprotein receptor-deficient mice protects them against atherosclerosis. A genetic polymorphism in the human C  37 gene has been reported as a potential prognostic marker for atherosclerotic plaque development. Interestingly, we recently detected C  37 on macrophages of early atheromas and on macrophage foam cells of advanced plaques. Since C  37 is not present on circulating monocytes, its’ expression on monocytes/macrophages must be induced during atherogenesis. The purpose of this study was to examine the role of C  37 in the development of atherosclerosis in vivo. First, C  37-deficient (C  37 / ) and atherosclerosis-susceptible apolipoprotein E-deficient (ApoE / ) mice were interbred to generate double knock out mice (C  37 / ApoE / ). Male C  37 / ApoE / and C  37+/ + ApoE / mice (n = 10 per group) at 10 weeks of age were fed an atherogenic diet (1.25% cholesterol, no cholate) for 10 weeks. Atherosclerotic plaque development in these animals was then examined by quantifying the sudanophilic lesions found in their thoracicabdominal aortas and aortic roots. Both groups, before and after the atherogenic diet, had similar body weights as well as total plasma cholesterol and triglyceride levels. In contrast, progression of atherosclerosis was significantly faster in C  37 / ApoE / mice. Lipid depositions in the thoracic-abdominal aortas of C  37 / ApoE / and C  37+/ + ApoE / mice were 15.0 ± 1.4% and 10.6 ± 1.2% respectively (mean ± SEM, p < .03). Similar results were obtained from the aortic roots.\ In conclusion, these results demonstrate that eliminating C  37 expression in vivo accelerates the progression of atherosclerosis. Interestingly, this is the opposite effect observed in mice with reduced C  43 expression, confirming that gap junctions consisting of different connexins mediate unique intercellular communication. Again, our findings highlight the importance of direct cell-cell communication via gap junctions in atherogenesis. Perhaps, this will open up another therapeutic approach for atherosclerosis in which direct cell-cell rather than paracrine communication is targeted. This work was supported by the Swiss National Science Foundation (#PP00A-68883 and #3100-067777.02)

Vascular Cell Signal Transduction P078 ACTIVATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR-1 SUSTAINS ANGIOGENESIS AND BCL-2 EXPRESSION VIA THE PHOSPHOINOSITIDE 3-KINASE PATHWAY. Shakil Ahmad, Jun Cai, Mike Boulton, Asif Ahmed. Department of Reproductive and Vascular Biology, The Medical School, University of Birmingham, Birmingham, B15 2TT, UK, Department of Optometry & Vision Sciences, Cardiff University, Cardiff, CF10 3NB, UK. Vascular insufficiency and retinal ischemia precede many proliferative retinopathies and stimulate secretion of various vasoactive growth factors including vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF). It is unclear however how PlGF, which is elevated in proliferative diabetic retinopathy (PDR) and is a VEGF homolog that binds only to VEGFR-1, promotes pathological angiogenesis. When primary microvascular endothelial cells were grown on collagen gels, PlGF containing ligands up-regulated Bcl-2 expression and stimulated the formation of capillary like tube networks that were retained for up to 14 days in culture. Moreover, inhibition of VEGFR-1 resulted in a dramatic decrease in the number of capillary connections indicating that VEGFR-1 ligands promote branching angiogenesis. In contrast, VEGF-induced tube formations and Bcl-2 expression were significantly decreased at the end of this period. Flow cytometry analysis of annexin-V/propidium iodide stained cells revealed that PlGF and PlGF/VEGF heterodimer inhibited apoptosis in serum deprived endothelial cell. These two growth factors stimulated a survival signalling pathway phosphoinositide 3-kinase (PI3K) as identified by increased Akt phosphorylation and blocking PI3K with LY294002 abolished PlGF-induced survival. Moreover, inhibition of PI3K signalling by adenovirus-mediated overexpression of wild-type PTEN disrupted angiogenesis and decreased Bcl-2 expression by PlGF and PlGF/VEGF heterodimer, whereas a dominant negative PTEN mutant enhanced endothelial sprouts formation and Bcl-2 expression. Together these findings indicate that PlGF and PlGF/VEGF ability to enhance endothelial cell survival may come from their capacity to sustain angiogenesis and upregulate the antiapoptotic protein-Bcl-2 via the PI3K pathway thus contributing to the aberrant vascular growth observed during pathological angiogenesis. This study was supported by grants from the Wellcome Trust 053347/B/98/ Z (M.B. and A.A.) and British Heart Foundation Programme Grant RG8003 (A.A.).

P079 INHIBITION OF ANGIOTENSIN II-INDUCED VASCULAR SMOOTH MUSCLE CELL HYPERTROPHY BY DIFFERENT CATECHINS. Hee Y Ahn, Ying Zheng, Hye J Song, Jung C Park, Chan H Kim. Department of Pharmacology, College of Medicine, Chungbuk University. Growth (Hypertrophy and hyperplasia) of vascular smooth muscle cell (VSMC) is an important component in the initiation and progression of atherosclerosis and restenosis. In this study, our findings showed that treatment of rat VSMC with epicatechin (EC) and epigallocatechin (EGC) failed to inhibit 3H-leucine incorporation into VSMC by angiotensin II (Ang II). In contrast, epigallocatechin-3 gallate (EGCG), epicatechin-3 gallate (ECG) which all possess a galloyl group in the 3-position of the catechin structure effectively inhibited 3H-leucine incorporation into VSMC by Ang II. Ang II activated c-Jun N-terminal kinase (JNK), extracellular regulated kinase (Erk) 1/2 and p38 mitogen-activated protein kinase (MAPK) in VSMC. EGCG and ECG but not EGC and EC, only attenuated JNK activity by Ang II in VSMC. EGCG, ECG, EGC and EC did not affect Erk1/2 and p38 MAPK activity in VSMC. Ang II increased