Shear force regulates matrix metalloproteinase activity in human saphenous vein organ culture.

BACKGROUND: Development of vein graft intimal hyperplasia has been related both to shear force and to the activity of matrix metalloproteinases (MMPs). Little data are available regarding the effects of shear on MMP expression and activity. The aim of this study was to examine the relationship among shear force, metalloproteinase activity, and intimal thickening in human saphenous vein segments maintained in organ culture. MATERIALS AND METHODS: Segments of human saphenous vein were cultured under static conditions, or perfused under low-flow and high-flow conditions in a perfusion apparatus for 7 days. Metalloproteinase levels and activities were measured using ELISA and substrate gel zymography, respectively. Intimal thickening was determined by morphometric analysis. Results were compared with control vein tissue, which was not subjected to organ culture, using a one-way ANOVA. RESULTS: A 13% increase in proteolytic activity was noted on substrate gel zymography at 68-72 kDa in high-flow vein tissue. The protein content of MMP-2, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2 was increased in high-flow vein tissue by 21%, 126%, more than 100-fold, and 86%, respectively. In culture media bathing the outside of the vein, TIMP-2 was increased in high-flow specimens, while TIMP-1 was inversely related to flow rate. Intimal thickening was directly related to flow rates, and was progressively increased in the low-flow and high-flow groups by 3-fold and 4-fold, respectively. CONCLUSIONS: Metalloproteinase levels in human saphenous vein cultures are related to shear force. MMP levels and activity correlate with the degree of intimal thickening. This model may provide a valuable tool for the analysis of physical forces and their influence on intimal thickening in human saphenous vein.

Metalloproteinase levels are decreased in symptomatic carotid plaques.

BACKGROUND: Matrix metalloproteinase enzymes (MMP) have been identified in carotid atherosclerotic plaques, but their role in the development of clinical symptoms remains ill defined. We correlated the activity and levels of metalloproteinase enzymes and their inhibitors in human carotid plaques to ischemic neurologic events. METHODS: Carotid plaques were collected at the time of endarterectomy from 23 patients with carotid stenosis. Sixteen patients were asymptomatic and 7 patients had symptoms of stroke or transient ischemic attack within 6 weeks of surgery. Protein was extracted from the plaques, proteolytic activity was determined by gelatin zymography, and pro-MMP and tissue inhibitor of metalloproteinase (TIMP) enzyme content were measured by ELISA assay. Macrophage accumulation in the plaque was determined using immunohistochemistry. RESULTS: Plaques from symptomatic patients had decreased proteolytic activity on substrate gel zymography at the 62- and 92-kDa regions (corresponding to active MMP-2 and pro-MMP-9). A decrease in pro-MMP-9 (8.21 +/- 2.35 vs 17.42 +/- 3.14 ng, P < 0. 05) and an increase in TIMP-2 protein (12.62 +/- 0.58 vs 10.56 +/- 0. 77 ng, P < 0.05) were noted on ELISA in plaques from symptomatic patients. No difference was noted in macrophage accumulation in the plaques between the two groups. CONCLUSIONS: Plaques from patients who present with ischemic neurologic symptoms have decreased proteolytic activity associated with decreased pro-MMP-9 and increased TIMP-2 protein levels. These data suggest that metalloproteinase enzymes are not responsible for plaque instability in the carotid circulation and may in fact promote plaque stability.

cGMP is decreased after acute ischemia in chronically ischemic canine limbs.

BACKGROUND: A chronic partially ischemic state may alter the skeletal muscle response to acute ischemia and free radical formation. METHODS: In order to investigate this hypothesis, a chronic ischemic state was established by ligating the right femoral artery of four mongrel dogs. ABIs were decreased from 1.05 +/- 0.25 preligation to 0.54 +/- 0.14 at 6 weeks (P = 0.04). At the end of 8 weeks, the hindlimb was subjected to 3 h of acute ischemia by clamping the iliac artery. The clamp was then released for 2 h of reperfusion. Plasma samples from the right iliac vein were taken during the ischemia-reperfusion period for analysis of cGMP. Tibialis anterior biopsies for Western analysis of eNOS and iNOS were taken upon completion of reperfusion. Comparisons to control dogs subjected to the acute ischemia and reperfusion without prior femoral artery ligation were made. RESULTS: cGMP levels were increased in the controls at 3 h of ischemia (3539 +/- 350) and 2 h of reperfusion (2880 +/- 269). The chronic ischemia group did not develop a corresponding increase in cGMP at 3 h of ischemia (2762 +/- 251) or after 2 h of reperfusion (2102 +/- 130). Western analysis of eNOS and iNOS revealed similar levels in both groups. Analysis of eNOS revealed 0.6429 +/- 0.086 and 0.5916 +/- 0.072 (densitometric units +/- SEM) for study and control dogs, respectively. Analysis of iNOS revealed 0.3401 +/- 0.067 and 0.2475 +/- 0.066 for study and control dogs, respectively. CONCLUSION: Previous ligation of the femoral artery resulting in chronic partial ischemia in this model demonstrated no increase in cGMP following acute ischemia that was not accompanied by a change in eNOS or iNOS levels. Nitric oxide activity is reflected by cGMP levels, which may increase in response to free radicals in the acute setting of complete ischemia.

Tissue inhibitor of metalloproteinase-1 is increased in the saphenofemoral junction of patients with varices in the leg.

PURPOSE: The goal of the present study was to examine the role of matrix metalloproteinase (MMP) activity in the development of varicose changes in the superficial veins of the lower extremity. METHODS: Normal-caliber vein segments from the saphenofemoral junction were harvested from patients undergoing saphenous vein ligation for varices and from patients undergoing infrainguinal bypass graft procedures. The activity and quantity of MMPs and their inhibitors (tissue inhibitors of metalloproteinases [TIMPs]) in the vein segments were compared. Vein segments were obtained from 13 patients. Seven patients had varicose disease in the leg, including 6 women and 1 man (average age, 48 years). Six patients had no evidence of varicose disease, including 2 women and 4 men (average age, 59 years). Proteolytic activity was determined with substrate gel zymography, and enzyme content was determined with Western immunoblotting using monoclonal antibodies directed against MMP-2, MMP-3, MMP-9, TIMP-1, TIMP-2, and alpha2-macroglobulin. Signals were quantified by scanning densitometry and normalized to a positive control (densitometric index [DI]). Immunohistochemistry was performed for enzyme localization. RESULTS: Zymography did not detect a difference between groups at loci consistent with the major MMPs; however, a small but significant decrease in proteolytic activity was noted in veins from patients with varices. TIMP-1 is increased in vein segments from patients with varices (DI 0.8 +/- 0.1 vs 0.2 +/- 0.05, P < .05) while MMP-2 levels were decreased (DI 1.5 +/- 0.3 vs 0.5 +/- 0.1, P < .05). Immunohistochemistry localized MMPs to the adventitia of the vein wall. CONCLUSION: A decrease in proteolytic activity may be responsible for the histological and structural alterations leading to varicose degeneration of superficial lower extremity veins.

Free intraperitoneal islet autografts in pancreatectomized dogs--impact of islet purity and posttransplantation exogenous insulin.

BACKGROUND: We compared the ability of impure, dispersed pancreatic islet tissue (DPIT) and purified islets to engraft in the peritoneal cavity of dogs. We also tested whether posttransplantation insulin therapy affects islet engraftment. METHODS: Thirty-two dogs underwent total pancreatectomy. DPIT was autotransplanted intraperitoneally in nine dogs. Purified islets were autotransplanted intraperitoneally in 13 dogs and intraportally in seven dogs. Dogs received comparable islet mass. One half of the recipients of intraperitoneal grafts received 12 days of posttransplantation exogenous insulin. Three dogs did not undergo transplantation. Intravenous glucose tolerance tests were done at 60 and 180 days. RESULTS: The long-term graft functional survival rate of intraperitoneal DPIT graft was significantly better than the rate of intraperitoneal purified islets (p < 0.01) and was as good as the rate of intraportal purified islets. Purified islets transplanted intraperitoneally failed early compared with other groups, with only 46% functioning at 3 weeks (p = 0.05); exogenous insulin reduced this early failure rate (p = 0.05). At 6 months 67% of intraperitoneal DPIT and 86% of intraportal purified grafts were functioning. Glucose disposal did not differ between groups. CONCLUSIONS: The peritoneal cavity is a safe, practical site for islet transplantation, particularly for high-volume DPIT grafts. DPIT may provide a larger, more viable beta-cell mass than purified islets, or the acinar-ductal tissue may have a positive effect on engraftment. Exogenous insulin may promote engraftment of transplanted islets with a marginal beta-cell mass.