Purpose: Apoptosis is a physiologic mechanism of cell death that regulates mass and architecture in many tissues. Apoptosis has been described as a feature in human vascular atherosclerosis and large vessel structural integrity. We examined the extent of vascular smooth muscle cell (VSMC) apoptosis in aneurysmal, occlusive, and normal human aortic tissue. Methods: Tissue samples of aneurysmal, occlusive, and normal human infrarenal aorta were evaluated. DNA fragmentation detection methods, immunohistochemistry, and DNA electrophoresis determined VSMC density, VSMC apoptosis, and apoptosis markers. Apoptotic cells and VSMC nuclei were counted with the use of computer-generated image analysis. Aortic subtypes were compared statistically by analysis of variance. Results: Seventeen aneurysmal, ten occlusive, and five normal human aortas were evaluated. By α1-actin immunostaining, VSMC density was least in aneurysmal aortas (271.8 ± 13.5 cells/high-power field [HPF]) compared with occlusive aorta (278.2 ± 39.4 cells/HPF) and normal aortas (291.0 ± 25.4 cells/HPF; P = not significant). Presence of apoptotic VSMCs was demonstrated by terminal deoxynucleotidyl transferase fragment end labeling and propidium iodide nuclear staining. VSMC apoptosis was greatest within aneurysmal aortas with 11.7 ± 1.5 cells/HPF compared with occlusive aortas with 3.3 ± 0.8 cells/HPF (P <.05) and normal aortas with 3.75 ± 4.6 cells/HPF (P <.05). Significant differences in apoptosis markers, p53 or bcl-2, could not be demonstrated by immunohistochemistry or DNA electrophoresis in aortic subtypes. Conclusion: Apoptosis of VSMCs is increased and VSMC density is decreased within the medial layer of aneurysmal aortic tissue. Structural degeneration of aortic tissue at the cellular level contributes to aneurysmal formation. (J Vasc Surg 2000;31:567-576.)

423 We investigated the immune response to lyophilized allograft vein transplants implanted in the canine venous system without immunosuppression. Lyophilized veins (n=20) were prepared by vacuum freeze drying canine jugular veins within 24 hours of harvest. The grafts were stored at room temperature for two weeks before they were transplanted as interposition grafts into the femoral veins of 10 mongrel dogs. Each animal received an end to end interposition autograft (n=10) vein in one limb, and a randomly assigned allograft (n=10) in the opposite limb. Typing for three blood group factors (DEA-1.1/1.2/7) was obtained. Pre and posttransplant lymphocyte crossmatches were performed on each animal. Transplants were performed independent of blood type or crossmatch result. Aspirin was given perioperatively. Vein grafts were harvested after six weeks, and histologic evaluation was performed. Both allografts and autografts had a patency rate of 70%. Positive crossmatch was not associated with graft occlusion. None of the six negative preoperative crossmatches became positive postransplant. Lymphocytic CD3 + T cell infiltrate as measured by cell count per high power field, was not significantly different between allografts and autografts (1.44±0.82 vs. 1.08±0.42, p=N.S.). Cellular infiltrate was similar in allografts regardless of positive (n=4) or negative (n=6) crossmatch results (1.46±0.95 vs. 1.43±0.821). There was a trend for more cellular infiltrate in allografts implanted in blood type mismatched (n=3) recipients (1.97±1.17 vs. 1.22±0.61 p=N.S.). Lyophilized vein allografts implanted in the venous system without immunosuppression have the same patency rate as autografts. CD3 + T lymphocytic infiltrate and patency rates are similar regardless of positive pretransplant crossmatch or blood type mismatch. Lyophilized vein allografts represent an alternative for venous reconstruction. Supported in part by a grant from the Physicians Medical Education and Research Fund, Knoxville, TN.

Deregulated apoptosis among vascular smooth muscle cells (VSMC) can undermine the integrity of an atherosclerotic plaque and contribute to the rupture of its fibrous cap. Vascular endothelial growth factor (VEGF) is produced by VSMC and is present in atherosclerotic lesions. Although VEGF has well characterized interactions with endothelial cells, its effect on VSMC apoptosis has not been examined. We demonstrate that VEGF attenuates apoptosis in VSMC that have been exposed to UVB irradiation.

Adipose tissue offers an abundant source for isolation of microvascular endothelial cells (MVECs). Several cell types result from the enzymatic digestion of adipose tissue, including MVECs, mesothelial cells and fibroblasts. Pure populations of MVECs must be isolated from the mixed cultures or fibroblasts overgrow the population. Canine subcutaneous or mesenteric fat is obtained during elective surgical procedures and digested with collagenase. Microvascular endothelial cells are separated from capillary fragments by Percoll gradient centrifugation and plated in culture. At confluence, microscopic identification of most cultured cells indicate the typical cobblestone morphology of ECs, while other spindle shaped cells resemble fibroblasts. Micro- vascular endothelial cells are identified by their uptake of acetylated-low density lipoprotein (Ac-LDL). Mesothelial cells, which closely resemble MVECs in morphology, and fibroblasts are removed from cultures of MVECs using a fluorescent activated cell sorter (FACS). Acetylated-low density lipoprotein tagged with a fluorescent probe, 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl- indocarbocyanine perchlorate (DiI), is incubated with the mixed cultures and the cells are sorted using a FACS. The pure populations of MVECs that result are tested immunocytochemically and are identified by their positive staining with antibodies against factor VIII related antigen. Mesothelial cells are identified by positive staining for cytokeratin 18 antibody. Contaminating fibroblasts show negative staining for smooth muscle alpha actin, cytokeratin 18 and factor VIII related antigen antibodies. This study examines the efficiency of the fluorescent activated cell sorter to obtain pure populations of MVECs harvested from adipose tissue.

Genetically recombinant endothelial cells (rEC) may improve the patency of small diameter vascular grafts by preventing thrombosis or limiting neointimal hyperplasia. Previous work has shown that rEC have reduced adhesion to vascular bypass graftsin vivo.Poor adhesion may be due to altered adhesion (integrin) receptors. This study evaluated the expression of the α5β1(fibronectin), α2β1(collagen IV), and αvβ3(vitronectin) integrin subunits on rEC. Human umbilical vein EC or canine jugular vein EC were transduced with neoR, neoR and human tPA or hygromycin resistance genes using retroviral vectors. Naive EC and EC exposed to empty viral particles (mEC) were controls. Naive EC, mEC, and all rEC's were evaluated for α and β subunits for each integrin receptor studied using immunoblotting. Blotting for α2, α5, and αvexhibited expression of the α integrin subunits in all cells. The β1and β3subunits were present in mEC and nEC but were absent or truncated in all rEC. The decreased adhesion of rEC's to synthetic vascular grafts may be accounted for by their altered β1and β3integrin subunit expression. The β subunit is critical for organization of the cytoskeleton and cellular signal transduction. Diminished β subunit expression in rEC is neither vector specific nor related to retroviral exposure alone. Alteration of β integrin expression may be to associated with the over-expression of phosphotransferase genes such as neoR or hygromycin B used as selectable markers in gene transfer protocols.

Background. Incomplete luminal endothelialization may contribute to small diameter vascular graft failure. Vascular endothelial growth factor (VEGF) can be used to stimulate endothelialization without provoking smooth muscle cell (SMC) proliferation. Heparin and VEGF in a fibrin glue (FG) were investigated for their ability to promote selective human aortic endothelial cell (HAEC) proliferation and human aortic smooth muscle cell (HASMC) inhibition.Methods. HAECs and HASMCs were seeded on FG containing VEGF (2.5, 10, 30, 100 ng/ml) or VEGF and heparin (5, 50, 500 units/ml). Proliferation assays were performed with tritiated thymidine on days 1 and 3. Results were analysed by ANOVA, with p less than or equal to 0.05 significant.Results. HAEC proliferation on FG with 10, 30, and 100 ng/ml VEGF significantly increased HAEC proliferation to greater than FG with VEGF alone at day 1. Human aortic SMC proliferation was not stimulated by the addition of VEGF. The addition of 5, 50, and 500 units/ml heparin significantly inhibited HASMC proliferation regardless of VEGF concentration.Discussion. VEGF at 10 ng/ml combined with heparin at 50 units/ml exhibited maximal stimulation of HAECs with inhibition of HASMCs. VEGF and heparin in a biologic glue may improve patency by selectively promoting HAEC proliferation without HASMC growth on synthetic vascular bypass grafts.