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The inhibition of neointimal hyperplasia by gamma interferon
JOURNALOF VASCULARSURGERY Volume
18, Number
Meeting
2
abstracts
323
Results (Gertler et al.) PA*
HSV First 24 hr Second24 hr HUV First 24 hr Second24 hr
155%” J 52%* J 11% J 34%,
+l%/lW
MI*1
< 5% f 15%
+- 1 ratio
i 59%* J.5096*
J 15% 4 57%”
<5%
< 5%
*p < 0.01, Student’spaired t test.
Our findings in this kindred reconfirm an autosomal dominant inheritance pattern. Although the frequency of thromboembolic events was less than expected, event occurrence proved significantly related to levels of protein S functional activity.
The inhibition of neointimal hyperplasia by gamma interferon John Castronuovo, Jr., MD, Stephen B. Guss,MD, Anrita Sawhney, BA, Dmitry Mysh, MD, Marianne Wolff, MD, and Allen M. Gown, MD, Murristown Memorial Hospital, Mcvrimwn,
Ambient 0, tension modulates endothelial fibrinolysis Jonathan P. Gertler, MD, Leland K. Perry, BS, Nancy Chung-Welch, PhD, Gilbert L’Italien, BS, Richard I?. Cambria, MD, Roslyn W. Orkin, PhD, and Wiiam M. Abbott, MD, Massadwetts General Hospital, Boston, Mass.
Vascular procedures reoxygenate ischemic endothelial cells (EC) and arterialize saphenous vein (HSV) EC. The balance between the EC-derived fibrinolytic components, plasminogen activator (@A) and its inhibitor (PAI-l), contributes to maintaining thromboresistance. This balance also focuses proteolysis through plasmin generation, mediating matrix metabolism and thus affecting intimal hyperplasia, endothelial migration, and angiogenesis. To explore the impact of varying oxygen tensions on EC fibrinolysis, HSV and human umbilical vein (HUV) were subjected to PO, of 40 mm Hg for 24 hours with restoration of PO, to 150 mm Hg for 24 hours. Controls were maintained at PO, of 150 mm Hg for 48 hours. The concentrations of tPA and PAT-1 antigen (Ag) and tPA/PAI-1 Ag ratio in conditioned media, expressedas t or i % change, normalized for cell count, versus controls, ( were analyzed by enzyme-linked immunosorbent assay. Cellular tPA and PAI- mRNA were assessedby Northern analysis (see table). Messenger RNA for tPA was unchanged, but PAImRNA increased significantly for HSV and HUV after 24 hours of PO, at 40 mm Hg, returning to baselinewithin 24 hours of restoration of PO, to 150 mm Hg. These data support the hypothesis of a fibrinolytic component to postischemic hypercoaguability, regulated at both gene and protein level, and show that HSVEC are more sensitive to altered O, tensions than HWEC. Altered oxygen tensions depress EC fibrinolysis in this model. This alteration theoretically is a contributing , mechanism to late structural defects in arterialized autogenous vein grams.
NJ.
Gamma interferon has been shown to reduce neointimal hyperplasia (NH) by decreasing the proliferative response of medial smooth muscle cells to intirnal injury. We evaluated the inhibition of neointimal hyperplasia by gamma interferon-y (IFN-?I) treatment. Three groups of Sprague-Dawley rats (n = 24) weighing 500 grams underwent endothelial injury of the carotid artery with the Fogarty catheter (Baxter Healthcare Corp., Edwards Div., Irvine, Calif.) technique described by Clowes. The control group (group I rr = 8) received daily intraperitoneal (IP) injection of phosphate-buffered saline solution for 7 days after surgery. Group II (n = 8) received IFN-y 200,000 units IP daily for sevendays; group III (n = 8) was treated the same but, in addition, received pretreatment with 200,000 units IP IFN-7 the day before surgery. Animals were sacrificed at postoperative day 14, at which time the arteries were fixed with methyl Carnoy solution. Histologic sections were analyzed by computerized morphometric analysis for differences between the area of NH, and for differences between the ratio of the intimal area to the area bounded by the internal elastic lamina (NH/IEL). Mean neointimal hyperplasia was 0.16 f 0.04 mm’ in group I, 0.07 it 0.03 in group II, and 0.07 it 0.04 in group III. The NH/IEL was 0.49 t 0.11 in group I, 0.25 + 0.06 in group II, and 0.22 +- 0.12 in group III. Both IFN--r groups had a statistically significant reduction of the NH and NH/IEL compared to control (independent t test p < 0.001). Pretreatment with 1FN-y did not further enhance its effect on NH. Immunohistochemistry with proliferating cell nuclear antigen determined the degree of cellular proliferation in the media and neointima at day 14. At sacrifice the mean rate of medial smooth muscle cell proliferation was 6.3% in treated and 3.8% in the control group; the mean rate of cellular proliferation of the neointima was 38.5% in treated and 41.3% in control groups. Our results confirm that IFN-7 inhibits the development of neointima hyperplasia after arterial injury.
JOURNAL
324
Meeting
abstracts
The high rate of proliferation seen in the neointimal cells at day 14 compared with the rate seen for medial smooth muscle cells indicates that continuing IFN-y treatment for at least 14 days may improve its effect in reducing NH by decreasing the proliferative response that occurs in the neointimal at day 14 subsequent to the medial smooth muscle cell proliferative response.
Insulin-like growth factor-I receptors in human saphenous vein Anton N. Sidawy, MD, Fares Hakim, MD, Richard F. Neville, MD, and Louis Y. Korman, MD, VeteransA@irs Medical Center, Washington, D. C. The proliferation of medial smooth muscle cells is a very important step in the process of intimal hyperplasia. Veins exposed to arterial pressure develop intimal hyperplastic lesions that lead to failure of vein bypasses. Insulin-like growth factor-I (IGF-I) is a polypeptide hormone structurally related to insulin with insulin-like metabolic effects. In addition IGF-I has been found to work in concert with other growth factors, such as platelet derived growth factor in promoting the growth of vascular smooth muscle cells in culture. IGF-I exerts its effects via specific receptors located in the cell wall. We studied the in situ distribution of IGF-I receptors using receptor autoradiography and examined their binding characteristics in normal human greater saphenous vein (GSV). Twocentimeter segments of GSV were harvested from five patients at the time of bypass. Frozen sections, 20 pm thick, were prepared from the GSV specimens. The sections were incubated in a buffer containing iodine 125-labeled IGF-I in the presence of increasing concentrations of the unlabeled peptide. Autoradiographs were obtained by apposing the treated sections to LKB Ultrofilm. Analysis of the autoradiographs showed that IGF-I receptors were present predominantly in the muscular layer of human GSV. To characterize these binding sites, binding inhibition studies were performed and the dissociation constant (Kd) and maximum binding capacity (B,,) were obtained from Scatchard analysis (x 2 SEM, n = 5): K,(nmol) = 1.0 2 0.32; B,,(pmol/mg protein) = 0.46 ? 0.23. These values are consistent with a physiologic role for IGF-I in the tissue examined. The presence of high-affinity (Kd = 1.0 -t- 0.32) IGF-I receptors in the media of saphenous vein suggests that IGF-I plays an important role in regulating the multiplication of venous smooth muscle cells; an essential step in the process of venous intimal hyperplasia. Hemodynamics and aneurysm development in vascular alIografts Michael J. Petersen, MD, Peter B. H’Doubler, MD, Gilbert J. L’Italien, BS, Bernice E. Hoppel, MS, Bruce R. Rosen, MD, PhD, Roslyn W. Orkin, PhD, and William M. Abbott, MD, Massachusetts General Hospital, Boston, Mass. Mechanical and immunologic factors are believed to play a role in the development and enlargement rates of
OF VASCULAR
SURGERY August 1993
arterial and biologic graft aneurysms. We developed a rat aortic allografi aneurysm model in which segments of infrarenal aorta were transplanted between spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats and between SHR/SHR and WKY/WKY autografts at age 8 weeks when both are normotensive. Over the next 18 weeks systolic pressure (I’,,,) in the SHR host rose linearly to Psys > 200 mm Hg and an aneurysmal dilation of the transplanted segment occurred (n = 18). In contrast, the normotensive WKY host allograft (SHR segment) maintained normal diameter (n = 9, p < O.OOl), as did the SHR autograft (n= 6,p < 0.001) and WKY autograft (n = 2,p < 0.001). Histologic examination of allograft specimens noted a rejection reaction characterized by inflammatory cell infiltration and media smooth muscle cell loss. Autografts lacked evidence of immunologic rejection. Pretransplant sensitization with full thickness skin allografts accelerated aneurysm development in the SHR host (n = 6) by 6 weeks but had no effect on the normotensive WKY host (n = 6,p < 0.001). Seven SHR hosts with WKY aortic grafts (aneurysm formers) were treated with antihypertensive agents (chlorothiazide, hydralazine, and reserpine) beginning 1 day after allografting. Control aneurysm formers received none (n = 9). psys and heart rate were monitored with tail cuff plethysmography. Significant P,, differences occurred by 6 weeks after operation (Psys = 174 2 25 mm Hg [untreated] vs 113 2 7 mm Hg [treated], p < 0.001) and persisted. Allograft dilation was serially monitored with magnetic resonance imaging. At 18 weeks after transplantation, direct arterial pressure measurements were taken with a carotid artery catheter, and P/dT,,, was calculated in awake SHR hosts and in age-matched WKY rats (n = 9). Rates of allograft dilation as determined by magnetic resonance imaging and final allograft diameter were similar in treated and untreated groups (p > 0.2). The change in pressure per unit time (dP/dT,,,) in the untreated SHR host was 2166 2 950 mm/set, which differed significantly from the WKY (826 ? 362 mm/set, p = 0.002) but only marginally from the treated SHR host (1288 + 574 mm/sec,p = 0.056) (see table). In conclusion, we report a model in which both immunologic injury and abnormal hemodynamics are necessary for aneurysm development. DP/dT seems to be a greater contributor to aneurysm formation than the mere presence of hypertension, Cryopreserved saphenous vein allografks for infrainguinal bypass Rasesh M. Shah, MD, Gian Luca Faggioli, MD, Linda M. Harris, MD, Syde Taheri, MD, and John J. Ricotta, MD, State University of New York at Bu..lo, Buffalo, N.Y. Cryopreserved saphenous vein allografts (CSVA) are alternatives in arterial reconstructions when autogenous vein is unavailable. We reviewed our experience with 38 patients who underwent 43 infrainguinal bypass procedures with CSVA as the conduit when no acceptable autogenous greater saphenous vein was available. Mean follow-up is 7.8 t 5.2 months. The group includes 21
18, Number
Meeting
2
abstracts
323
Results (Gertler et al.) PA*
HSV First 24 hr Second24 hr HUV First 24 hr Second24 hr
155%” J 52%* J 11% J 34%,
+l%/lW
MI*1
< 5% f 15%
+- 1 ratio
i 59%* J.5096*
J 15% 4 57%”
<5%
< 5%
*p < 0.01, Student’spaired t test.
Our findings in this kindred reconfirm an autosomal dominant inheritance pattern. Although the frequency of thromboembolic events was less than expected, event occurrence proved significantly related to levels of protein S functional activity.
The inhibition of neointimal hyperplasia by gamma interferon John Castronuovo, Jr., MD, Stephen B. Guss,MD, Anrita Sawhney, BA, Dmitry Mysh, MD, Marianne Wolff, MD, and Allen M. Gown, MD, Murristown Memorial Hospital, Mcvrimwn,
Ambient 0, tension modulates endothelial fibrinolysis Jonathan P. Gertler, MD, Leland K. Perry, BS, Nancy Chung-Welch, PhD, Gilbert L’Italien, BS, Richard I?. Cambria, MD, Roslyn W. Orkin, PhD, and Wiiam M. Abbott, MD, Massadwetts General Hospital, Boston, Mass.
Vascular procedures reoxygenate ischemic endothelial cells (EC) and arterialize saphenous vein (HSV) EC. The balance between the EC-derived fibrinolytic components, plasminogen activator (@A) and its inhibitor (PAI-l), contributes to maintaining thromboresistance. This balance also focuses proteolysis through plasmin generation, mediating matrix metabolism and thus affecting intimal hyperplasia, endothelial migration, and angiogenesis. To explore the impact of varying oxygen tensions on EC fibrinolysis, HSV and human umbilical vein (HUV) were subjected to PO, of 40 mm Hg for 24 hours with restoration of PO, to 150 mm Hg for 24 hours. Controls were maintained at PO, of 150 mm Hg for 48 hours. The concentrations of tPA and PAT-1 antigen (Ag) and tPA/PAI-1 Ag ratio in conditioned media, expressedas t or i % change, normalized for cell count, versus controls, ( were analyzed by enzyme-linked immunosorbent assay. Cellular tPA and PAI- mRNA were assessedby Northern analysis (see table). Messenger RNA for tPA was unchanged, but PAImRNA increased significantly for HSV and HUV after 24 hours of PO, at 40 mm Hg, returning to baselinewithin 24 hours of restoration of PO, to 150 mm Hg. These data support the hypothesis of a fibrinolytic component to postischemic hypercoaguability, regulated at both gene and protein level, and show that HSVEC are more sensitive to altered O, tensions than HWEC. Altered oxygen tensions depress EC fibrinolysis in this model. This alteration theoretically is a contributing , mechanism to late structural defects in arterialized autogenous vein grams.
NJ.
Gamma interferon has been shown to reduce neointimal hyperplasia (NH) by decreasing the proliferative response of medial smooth muscle cells to intirnal injury. We evaluated the inhibition of neointimal hyperplasia by gamma interferon-y (IFN-?I) treatment. Three groups of Sprague-Dawley rats (n = 24) weighing 500 grams underwent endothelial injury of the carotid artery with the Fogarty catheter (Baxter Healthcare Corp., Edwards Div., Irvine, Calif.) technique described by Clowes. The control group (group I rr = 8) received daily intraperitoneal (IP) injection of phosphate-buffered saline solution for 7 days after surgery. Group II (n = 8) received IFN-y 200,000 units IP daily for sevendays; group III (n = 8) was treated the same but, in addition, received pretreatment with 200,000 units IP IFN-7 the day before surgery. Animals were sacrificed at postoperative day 14, at which time the arteries were fixed with methyl Carnoy solution. Histologic sections were analyzed by computerized morphometric analysis for differences between the area of NH, and for differences between the ratio of the intimal area to the area bounded by the internal elastic lamina (NH/IEL). Mean neointimal hyperplasia was 0.16 f 0.04 mm’ in group I, 0.07 it 0.03 in group II, and 0.07 it 0.04 in group III. The NH/IEL was 0.49 t 0.11 in group I, 0.25 + 0.06 in group II, and 0.22 +- 0.12 in group III. Both IFN--r groups had a statistically significant reduction of the NH and NH/IEL compared to control (independent t test p < 0.001). Pretreatment with 1FN-y did not further enhance its effect on NH. Immunohistochemistry with proliferating cell nuclear antigen determined the degree of cellular proliferation in the media and neointima at day 14. At sacrifice the mean rate of medial smooth muscle cell proliferation was 6.3% in treated and 3.8% in the control group; the mean rate of cellular proliferation of the neointima was 38.5% in treated and 41.3% in control groups. Our results confirm that IFN-7 inhibits the development of neointima hyperplasia after arterial injury.
JOURNAL
324
Meeting
abstracts
The high rate of proliferation seen in the neointimal cells at day 14 compared with the rate seen for medial smooth muscle cells indicates that continuing IFN-y treatment for at least 14 days may improve its effect in reducing NH by decreasing the proliferative response that occurs in the neointimal at day 14 subsequent to the medial smooth muscle cell proliferative response.
Insulin-like growth factor-I receptors in human saphenous vein Anton N. Sidawy, MD, Fares Hakim, MD, Richard F. Neville, MD, and Louis Y. Korman, MD, VeteransA@irs Medical Center, Washington, D. C. The proliferation of medial smooth muscle cells is a very important step in the process of intimal hyperplasia. Veins exposed to arterial pressure develop intimal hyperplastic lesions that lead to failure of vein bypasses. Insulin-like growth factor-I (IGF-I) is a polypeptide hormone structurally related to insulin with insulin-like metabolic effects. In addition IGF-I has been found to work in concert with other growth factors, such as platelet derived growth factor in promoting the growth of vascular smooth muscle cells in culture. IGF-I exerts its effects via specific receptors located in the cell wall. We studied the in situ distribution of IGF-I receptors using receptor autoradiography and examined their binding characteristics in normal human greater saphenous vein (GSV). Twocentimeter segments of GSV were harvested from five patients at the time of bypass. Frozen sections, 20 pm thick, were prepared from the GSV specimens. The sections were incubated in a buffer containing iodine 125-labeled IGF-I in the presence of increasing concentrations of the unlabeled peptide. Autoradiographs were obtained by apposing the treated sections to LKB Ultrofilm. Analysis of the autoradiographs showed that IGF-I receptors were present predominantly in the muscular layer of human GSV. To characterize these binding sites, binding inhibition studies were performed and the dissociation constant (Kd) and maximum binding capacity (B,,) were obtained from Scatchard analysis (x 2 SEM, n = 5): K,(nmol) = 1.0 2 0.32; B,,(pmol/mg protein) = 0.46 ? 0.23. These values are consistent with a physiologic role for IGF-I in the tissue examined. The presence of high-affinity (Kd = 1.0 -t- 0.32) IGF-I receptors in the media of saphenous vein suggests that IGF-I plays an important role in regulating the multiplication of venous smooth muscle cells; an essential step in the process of venous intimal hyperplasia. Hemodynamics and aneurysm development in vascular alIografts Michael J. Petersen, MD, Peter B. H’Doubler, MD, Gilbert J. L’Italien, BS, Bernice E. Hoppel, MS, Bruce R. Rosen, MD, PhD, Roslyn W. Orkin, PhD, and William M. Abbott, MD, Massachusetts General Hospital, Boston, Mass. Mechanical and immunologic factors are believed to play a role in the development and enlargement rates of
OF VASCULAR
SURGERY August 1993
arterial and biologic graft aneurysms. We developed a rat aortic allografi aneurysm model in which segments of infrarenal aorta were transplanted between spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats and between SHR/SHR and WKY/WKY autografts at age 8 weeks when both are normotensive. Over the next 18 weeks systolic pressure (I’,,,) in the SHR host rose linearly to Psys > 200 mm Hg and an aneurysmal dilation of the transplanted segment occurred (n = 18). In contrast, the normotensive WKY host allograft (SHR segment) maintained normal diameter (n = 9, p < O.OOl), as did the SHR autograft (n= 6,p < 0.001) and WKY autograft (n = 2,p < 0.001). Histologic examination of allograft specimens noted a rejection reaction characterized by inflammatory cell infiltration and media smooth muscle cell loss. Autografts lacked evidence of immunologic rejection. Pretransplant sensitization with full thickness skin allografts accelerated aneurysm development in the SHR host (n = 6) by 6 weeks but had no effect on the normotensive WKY host (n = 6,p < 0.001). Seven SHR hosts with WKY aortic grafts (aneurysm formers) were treated with antihypertensive agents (chlorothiazide, hydralazine, and reserpine) beginning 1 day after allografting. Control aneurysm formers received none (n = 9). psys and heart rate were monitored with tail cuff plethysmography. Significant P,, differences occurred by 6 weeks after operation (Psys = 174 2 25 mm Hg [untreated] vs 113 2 7 mm Hg [treated], p < 0.001) and persisted. Allograft dilation was serially monitored with magnetic resonance imaging. At 18 weeks after transplantation, direct arterial pressure measurements were taken with a carotid artery catheter, and P/dT,,, was calculated in awake SHR hosts and in age-matched WKY rats (n = 9). Rates of allograft dilation as determined by magnetic resonance imaging and final allograft diameter were similar in treated and untreated groups (p > 0.2). The change in pressure per unit time (dP/dT,,,) in the untreated SHR host was 2166 2 950 mm/set, which differed significantly from the WKY (826 ? 362 mm/set, p = 0.002) but only marginally from the treated SHR host (1288 + 574 mm/sec,p = 0.056) (see table). In conclusion, we report a model in which both immunologic injury and abnormal hemodynamics are necessary for aneurysm development. DP/dT seems to be a greater contributor to aneurysm formation than the mere presence of hypertension, Cryopreserved saphenous vein allografks for infrainguinal bypass Rasesh M. Shah, MD, Gian Luca Faggioli, MD, Linda M. Harris, MD, Syde Taheri, MD, and John J. Ricotta, MD, State University of New York at Bu..lo, Buffalo, N.Y. Cryopreserved saphenous vein allografts (CSVA) are alternatives in arterial reconstructions when autogenous vein is unavailable. We reviewed our experience with 38 patients who underwent 43 infrainguinal bypass procedures with CSVA as the conduit when no acceptable autogenous greater saphenous vein was available. Mean follow-up is 7.8 t 5.2 months. The group includes 21