The New Experimental Evidence To The Mechanism Of Collateral Vessel Growth

Objective The great progress has been made in human being in recongnizing the mechanism responsible for the collateral vessel growth after several decades'study. However lots of issues are still open. This project was designed to futher elluciate the mechanism for the collateral vessel growth by investigateing the remodeling of extemal iliac artery, the relation of MCP-1 expression to invasion of macrophages, invivo role of macrophage in collateral vessel growth and the effects of the degradation and components of extracellular matrix on proliferation and expression of focal adhesion kinase(FAK) in smooth muscle cells(SMC).Material and methods1. The preparation of animal modelEighteen adult New Zealand white rabbits weighing from 3-4kg were used in this study. The animals were anesthetized with an i. m. injection of midazolam (1 mg/kg)and xylazine (5mg/kg). Acute bilateral and unilateral femoral artery ligation was performed. Following immediately occlusion, an arteriovenous (AV) shunt was created unilaterally side-to-side, between the distal femoral artery stump and the accompanying femoral vein. Thereafter the animals were sacrified, and collateral arteries and skeleton muscles were dissected and snap-frozen in nitrigen at -196℃and stored until further processing at -80℃. In addition, femoral atrtery and its branches from normal rabbits without either ligation or shunt and normal skeleton muscles were also used as normal control.2. Cell cultureWe used primary cultures of rabbit aortic smooth muscle cells (SMCs), which were isolated and cultivated as described previously for porcine aortic SMCs [13]. Cells were cultivated routinely in medium 199 (PAA; Coelbe, Germany) with 20% FCS; when they reached confluence they were splitted 1:4. Cells between passage four and six were used for the described experiments.2.1 Preparation of SMC Extraceilular matrix (ECM) culturePlastic 12-well culture plates (Greiner; Frickendorf, Germany) containing 18-mm round glass coverslips were coated with either 5μg/cm² laminin (BD Biosciences) or 5μg/cm² fibronectin (BD Biosciences), for one hour at room temperature. Remaining material was aspirated and the plates were rinsed carefully. SMCs were seeded on top of the protein layer and incubated at 37℃, 5% CO₂ until they were 50-75% confluent. For cell culture in a three dimensional matrix, SMCs were added to 150μl/cm² Matrigel Basement Membrane Matrix (BD Biosciences) as instructed by the manufacturer. Plates were incubated for 30 minutes at 37℃and cell culture medium was added. Cell proliferation of smooth muscle cells cultured on different substrates, FN, LN and Matrigel Basement Membrane Matrix was detected with 5-Bromo-2'-deoxy-uridine Labeling and Detection Kit 1 (Roche Diagnostics GmbH, Germany) according to manufacturer protocol.3. ImmunofluorescenceImmunofluorescence for tissue and cultured cells was performed according to routine staining protocols. The proteins detected in this part included RAM11(a marker of macrophage, MCP-1, ICAM, VCAM, Ki67(an indicator of cell proliferation), FAK and FAK(pY397). In addition, the skeleton muscle was stained by FITC labeled actin-phalloidin. Capillaries were stained by Tritc labeled Lestin BS 1.4. Quantitative measurementsThe quantification of immunofluorescence intensity was performed with a Leica TCS SP confocal microscope,using the quantitation software of Leica. One channel with format 512 and appropriate filters was used. A full range of gray values from black to peak white (0-pixel to 255-pixel intensity level) was set during the whole process of measurements., The intensity of fluorescence was expressed as arbitrary units AU/μm². Quantitation of positive cells was performed with the confocal microscope. The counting was done at 40 X, and the ratio of positive nuclei to all nuclei of vascular wall cells or smooth muscle cells was considered as positive index. All data are presented as mean±SEM and analysized by SPSS software.5. Results5.1 The proliferation and expression of ICAM were rarely found in the ispolateral external iliac artery with simply femoral artery, but weak VCAM staining and a few of macrophages were detected in the adventitia. In contrast, A-V shunt led to a dramatical remodeling of external iliac artery morphologically and biochemically, including a 5% proliferation rate, induced expression of ICAM in endothelial cells, significant up-regulation of VCAM both in endothelial cell and adventitia; Accumulation of a number of macrophages in the advention was observed; In some parts of vascular wall, the presence of macrphages in the media was also noticed. There was no proliferation of smooth muscle cells in small arteries and arterioles distal to the ligated site in A-V shunt model, but degeneration of skeleton muscles and angiogenesis were observed.5.2 In normal vessels, expression of MCP-1 was very low. In growing collateral vessels, expression of MCP-1 was significantly up-regulated, 8 folds over that in normal vessels.This upregulation was demonstrated in all layers of the wall. In serial sections, the close relationship between MCP-1 expression and macrophage invasion, and betweem existence of macrophages and cell proliferation and elastic fiber degradation was observed. The invasion of macrophages was very active during early(2d) and active growth phases(7d) of arteriogenesis, and returned to similar state in maturation of collateral vessels.5.3 Smooth muscle cells cultured on fibronectin substrate highly expressed FAK and FAK (pY397), less amount on Laminin substrate, little inside Matrigel basememt membrane matrix. The index of cell proliferation in smooth muscle cells on fibronectin, laminin and insde matrigel basement membrane were 24%, 17% and 0%, respectively.Summary1. In this project, we found that elevated shear stress induced by A-V shunt led to rapid collateral vessel growth and external iliac artery remodeling; however severe ischemia didn't, but true for angiogenesis. This data provides powerful evidance for the notion that shear stress is the driving factor for collateral vessel growth, and makes it clear that ischemia is responsible for angiogenesis, but not for collateral vessel growth.2. The observation of upregulation of MCP-1 in collateral vessels and temporal pattern of macrophage invasion provides new information about inflammation formation during arteriogenesis; Moreover the data from serial sections about relationship between macrophages and cell proliferation and extracellular proteolysis add important invivo evidance to the knowledge that macrophage is a key player for collateral vessel growth.3. The findings that smooth muscle cells cultured on FN had a high proliferation rate and expressed high level of FAK, and those inside Matrigel gel had no proliferation and expressed low level of FAK make us better understanding the role of extracellular matrix played in smooth muscle cell proliferation and migration.