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3.12 Immunology of human saphenous vein allograft bypass grafting
Research I ranging from lo-’ to 10e6M. The supernatants and lysates were collected and tested for VEGF, bFGF, TGFPl , TNFol, and PDGF-AB using immunoassays. The cytokine levels were normalized to lo6 cells, compared to the albumin control, and analyzed as percent change of the control. ANOVA was used and P c 0.05 was taken as significant. Results: Cytokine production in cultured cells exposed to nicotine and cotinine expressed as percent change of the control. TNE‘(u PDGF AB
“I’ < 0.O.i. n = 4, c): no change.
Conclusion: These data suggest that N and C may promote SMC proliferation and development of intimal hyperplasia via endothelial cell stimulation in addition to the direct SMC stimulation we have previously demonstrated. Furthermore, smoking metabolites inhibit production of angiogenic cytokines by MVEC. This could negatively affect the development of collateral circulation in patients with arterial insufficiency who continue to smoke.
3.12 Immunology of Human SaphenousVein Allograft Bypass Grafting 1.p. CARPENTER and J.E. TOMASZE Philadelphia, Pennsylvania, USA
WSKZ,
Although it has been claimed that allografts of blood vessels might be successful because of minimal immunogenicity, they are subject to frequent and early failure, the etiology of which has not been thoroughly investigated. We sought to define the immune response to allograft bypass. In a prospective trial, 40 patients underwent cryopreserved vein allograft bypass. Anti-HLA antibody screens were obtained pre- and postoperatively. Allograft biopsies were performed at implantation and all subsequent opportunities and were scored in blinded fashion by standard microscopy and paraffin immunohistochemistry employing monoclonal antibodies. Preoperatively, eight patients had positive anti-HLA antibody screens and 30 were negative. Postoperatively, seven patients with previously negative anti-HLA antibody screens converted to positive and six patients with positive preoperative screens became more strongly positive. During the 31 month follow-up interval, 22 allografts were removed. Of these, 13 (59%) had moderate or severe infiltrates which were evenly distributed throughout the intima, media and adventia. Immunohistochemistry of 17 explants to date has demonstrated all of these infiltrates to be leukocytes (+LCA) which were predominantly T-lymphocytes (+CD3, CD8) containing cytotoxic granules (+TIA-1). Macrophages were uncommon (+CD68), B-cells (+L26, CD79) and NK-cells (+CD56) were rare. Severe infiltrates were associated with intramural hemorrhage (five) and capillary ingrowth (five) but little intimal thickening (two) or mural necrosis (one). Grafts not showing infiltrates (no cellular rejection) demonstrated thrombosis
CARDlOVASClLAR SURGERY
SEFTEMBER 1997
with intimal thickening (seven) and mural necrosis (three) but less intramural hemorrhage (one) and capitlary ingrowth (one). The degree of intimal thickening increased progressively with duration of allograft patency (P -z 0.01). No relationship was found between the development of anti-HLA antibodies and the presence of a cellular infiltrate, intimal thickening, intramural hemorrhage, necrosis or capillary ingrowth. Human vein allografts are immunogenic and prompt both a humoral (anti-HLA antibody) and T-cell mediated response. Allografts also fail without evidence of rejection presumably due to local injury, hypercoagulability or stasis. The contribution of rejection to vein allograft failure may be modifiable by immunosuppression, and that of local hypercoagulability by anticoagulation.
3.13 Characterization of Cellular Density and Determination of Neointimal Extr~&~lar Matrix Constituents in Human Lower Extremity Vein Graft Stenoses A.T. GENTILE, WESTERBAND, G.C. HUNTER Arizona, USA
J.L. MILLS, M.A. GOODEN, A. S.S. BERMAN, CA. BOSWELL, and S.K. WILLIAMS, Tucson.
Purpose: Arterial restenosis has been attributed to a hyperproliferative smooth muscle cell response. However, therapeutic strategies targeted to inhibit smooth muscle cell hyperplasia have failed in human trials. Studies of human coronary atherectomy and vein graft stenotic lesions have demonstrated a relatively low nuclear proliferative index with the majority of the myointimal mass consisting of extracellular matrix. The purpose of the present study was to characterize the cellular density and determine the relative composition of the extracellular matrix protein constituents in human lower extremity bypass graft stenoses. Methods and results: Duplex surveillance of 148 consecutive infrainguinal bypass grafts identified 17 patients with 22 preocclusive saphenous vein graft (GSV) stenoses (mean graft age 7 months, range 6 weeks to 13 months). These stenotic lesions were compared to excised samples of 20 saphenous vein segments taken at the time of graft implantation from matched control patients. Paraffin embedded micrograph cross sections of stenotic and control GSVs were digitized using computerized image analysis. Intimal and medial areas were compared and cell density per unit volume determined with fluorescent nuclear (Bisbenzimide) staining. Differential light microscopy using haematoxylin and eusin, trichrome, and pentachrome staining was performed to determine the relative percentage composition of intimal matrix constituents by stereologic morphometric (point-count) techniques.
Vessellayer
measurements
fntimal area ( X 10’ k m ’) Medial area (X 1 O6 em’) Intima/media ratio Intimal nuclear density (X lo3 cells/pm2) Medial nuclear density (X lo3 cells/pm2)
Control GSV Stenotic C&V t-test 1.64 3.x.5 P < 0.0001 5.01 3.3 1 P = 0.08 0.33 1.2 1.42 1.70 1’T: 0.03 2.27
5.29
I’ = 0.001
is
“I’ < 0.O.i. n = 4, c): no change.
Conclusion: These data suggest that N and C may promote SMC proliferation and development of intimal hyperplasia via endothelial cell stimulation in addition to the direct SMC stimulation we have previously demonstrated. Furthermore, smoking metabolites inhibit production of angiogenic cytokines by MVEC. This could negatively affect the development of collateral circulation in patients with arterial insufficiency who continue to smoke.
3.12 Immunology of Human SaphenousVein Allograft Bypass Grafting 1.p. CARPENTER and J.E. TOMASZE Philadelphia, Pennsylvania, USA
WSKZ,
Although it has been claimed that allografts of blood vessels might be successful because of minimal immunogenicity, they are subject to frequent and early failure, the etiology of which has not been thoroughly investigated. We sought to define the immune response to allograft bypass. In a prospective trial, 40 patients underwent cryopreserved vein allograft bypass. Anti-HLA antibody screens were obtained pre- and postoperatively. Allograft biopsies were performed at implantation and all subsequent opportunities and were scored in blinded fashion by standard microscopy and paraffin immunohistochemistry employing monoclonal antibodies. Preoperatively, eight patients had positive anti-HLA antibody screens and 30 were negative. Postoperatively, seven patients with previously negative anti-HLA antibody screens converted to positive and six patients with positive preoperative screens became more strongly positive. During the 31 month follow-up interval, 22 allografts were removed. Of these, 13 (59%) had moderate or severe infiltrates which were evenly distributed throughout the intima, media and adventia. Immunohistochemistry of 17 explants to date has demonstrated all of these infiltrates to be leukocytes (+LCA) which were predominantly T-lymphocytes (+CD3, CD8) containing cytotoxic granules (+TIA-1). Macrophages were uncommon (+CD68), B-cells (+L26, CD79) and NK-cells (+CD56) were rare. Severe infiltrates were associated with intramural hemorrhage (five) and capillary ingrowth (five) but little intimal thickening (two) or mural necrosis (one). Grafts not showing infiltrates (no cellular rejection) demonstrated thrombosis
CARDlOVASClLAR SURGERY
SEFTEMBER 1997
with intimal thickening (seven) and mural necrosis (three) but less intramural hemorrhage (one) and capitlary ingrowth (one). The degree of intimal thickening increased progressively with duration of allograft patency (P -z 0.01). No relationship was found between the development of anti-HLA antibodies and the presence of a cellular infiltrate, intimal thickening, intramural hemorrhage, necrosis or capillary ingrowth. Human vein allografts are immunogenic and prompt both a humoral (anti-HLA antibody) and T-cell mediated response. Allografts also fail without evidence of rejection presumably due to local injury, hypercoagulability or stasis. The contribution of rejection to vein allograft failure may be modifiable by immunosuppression, and that of local hypercoagulability by anticoagulation.
3.13 Characterization of Cellular Density and Determination of Neointimal Extr~&~lar Matrix Constituents in Human Lower Extremity Vein Graft Stenoses A.T. GENTILE, WESTERBAND, G.C. HUNTER Arizona, USA
J.L. MILLS, M.A. GOODEN, A. S.S. BERMAN, CA. BOSWELL, and S.K. WILLIAMS, Tucson.
Purpose: Arterial restenosis has been attributed to a hyperproliferative smooth muscle cell response. However, therapeutic strategies targeted to inhibit smooth muscle cell hyperplasia have failed in human trials. Studies of human coronary atherectomy and vein graft stenotic lesions have demonstrated a relatively low nuclear proliferative index with the majority of the myointimal mass consisting of extracellular matrix. The purpose of the present study was to characterize the cellular density and determine the relative composition of the extracellular matrix protein constituents in human lower extremity bypass graft stenoses. Methods and results: Duplex surveillance of 148 consecutive infrainguinal bypass grafts identified 17 patients with 22 preocclusive saphenous vein graft (GSV) stenoses (mean graft age 7 months, range 6 weeks to 13 months). These stenotic lesions were compared to excised samples of 20 saphenous vein segments taken at the time of graft implantation from matched control patients. Paraffin embedded micrograph cross sections of stenotic and control GSVs were digitized using computerized image analysis. Intimal and medial areas were compared and cell density per unit volume determined with fluorescent nuclear (Bisbenzimide) staining. Differential light microscopy using haematoxylin and eusin, trichrome, and pentachrome staining was performed to determine the relative percentage composition of intimal matrix constituents by stereologic morphometric (point-count) techniques.
Vessellayer
measurements
fntimal area ( X 10’ k m ’) Medial area (X 1 O6 em’) Intima/media ratio Intimal nuclear density (X lo3 cells/pm2) Medial nuclear density (X lo3 cells/pm2)
Control GSV Stenotic C&V t-test 1.64 3.x.5 P < 0.0001 5.01 3.3 1 P = 0.08 0.33 1.2 1.42 1.70 1’T: 0.03 2.27
5.29
I’ = 0.001
is