Anthony J. Arleth

Matrix Metalloproteinase Expression Increases After Cerebral Focal Ischemia in Rats: Inhibition of Matrix Metalloproteinase-9 Reduces Infarct Size

BACKGROUND AND PURPOSE Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade the extracellular matrix and are implicated in numerous pathological conditions including atherosclerosis, inflammation, and tumor growth and metastasis. In the brain, the endothelial cell wall, strengthened by tight junctions, defines the blood-brain barrier (BBB). The extracellular matrix molecules constitute the basement membrane underlying the vasculature and play a critical role in maintaining the integrity of the BBB. After focal stroke, there is a breakdown of the BBB with an associated increase in vascular permeability, inflammatory cell influx, and neuronal cell death. The present study was designed to investigate the effects of MMP expression after stroke. METHODS Focal stroke was produced by permanent middle cerebral artery occlusion (MCAO) in the rat, and MMP protein expression was measured by Western blot and zymogram analysis over a time course ranging from 6 hours to 30 days (n=32). Immunohistochemistry at 1 and 5 days (n=8 and 6, respectively) was also utilized to characterize the expression of several MMPs and related proteins after stroke, including their cellular source. To test the hypothesis that early increased MMP-9 expression is involved in ischemic brain injury, a neutralizing monoclonal antibody directed against MMP-9 was administered intravenously (n=7 per group) 1 hour before MCAO, and infarct size was measured 24 hours later. RESULTS MMP expression increased progressively over time after stroke. After 12 hours, significant (P<0.05) MMP-9 activity was observed that reached maximum levels by 24 hours (P<0.001), then persisted for 5 days at this level and returned to basal (zero) levels by 15 days. On the basis of morphological criteria, MMP-9 appeared to stain with endothelial cells and neutrophils identified both within and at the periphery of the infarct within 24 hours of focal ischemia. After 5 days, MMP-9 appeared to stain with macrophages present within the infarcted brain. MMP-2 activity was significantly (P<0.001) increased by 24 hours and was maximum after 5 days following MCAO. MMP-2 appeared to stain with macrophages present within the infarcted region. Unlike MMP-9 and MMP-2, tissue inhibitor of metalloproteinase-1 was identified at comparable levels in both control and ischemic tissue after MCAO. MMP-1 and MMP-3 could not be detected in the brain after focal stroke. When an MMP-9-neutralizing monoclonal antibody was administered systemically, animals exhibited significantly reduced infarct size (ie, a 30% reduction compared with non-immune antibody controls; P<0.05). CONCLUSIONS These results demonstrate that early increased MMP-9 expression in endothelial cells and infiltrating neutrophils is a significant response to cerebral focal ischemia and that selective inhibition of MMP-9 activity can significantly reduce brain injury after stroke.

The selective endothelin ETA receptor antagonist BQ123 antagonizes endothelin-1-mediated mitogenesis

The mitogenic effects of endothelin isopeptides and the selective ETA receptor antagonist BQ123 were evaluated in rat aortic vascular smooth muscle cells. Endothelin-1 (ET-1) and endothelin-3 (ET-3) produced concentration-dependent increases in [3H]thymidine incorporation (EC50 = 0.1 and 25 nM, respectively). The ETB-selective agonist sarafotoxin 6c did not produce significant effects on [3H]thymidine incorporation. BQ123 produced selective and concentration-dependent inhibition of ET-1-mediated [3H]thymidine incorporation. These data demonstrate that ET-1-mediated mitogenesis in vascular smooth muscle is mediated by ETA receptors.

Carvedilol inhibits vascular smooth muscle cell proliferation.

The antiproliferative properties of carvedilol, a newly developed multiple-action antihypertensive agent, were evaluated in early passage cultured rat aortic vascular smooth muscle cells. Carvedilol (10-7-10-5 M) produced concentration-dependent decreases in basal and endothelin-1-stimulated mitogenesis of rat aortic vascular smooth muscle cells. The IC50 for inhibition of [3H]thymidine incorporation by carvedilol in both basal and endothelin-1-stimulated rat aortic vascular smooth muscle cells was approximately 1 μM. Carvedilol (10 μM) inhibited basal mitogenesis by approximately 65%, and endothelin-1-stimulated mitogenesis by approximately 95%. Carvedilol (1–10 μM) also produced significant concentration-dependent inhibition of the mitogenic response mediated by thrombin (0.5 U/ml), epidermal growth factor (1 nM), platelet-derived growth factor (1 nM), and angiotensin II (5 nM). Endothelin-1− or PDGF A/B-induced increases in cell number were also significantly inhibited by carvedilol (10 μM). The antimitogenic effect of carvedilol on cell growth was reversible. The inhibitory effect of carvedilol was not shared by other β-adrenoceptor antagonists such as labetalol (10 μM), celiprolol (10 μM), or sotalol (10 μM), which did not significantly affect [3H]thymidine incorporation in rat vascular smooth muscle cells. Propranolol (10 μM) was the only β-adrenoceptor antagonist tested that inhibited [3H]thymidine incorporation, with effects of approximately 50 and 75% on basal and endothelin-1-mediated stimulation, respectively. In contrast, celiprolol (10 μM) produced significant stimulation of DNA synthesis (125% over basal). The calcium channel antagonist nifedipine (10 μM) inhibited basal and endothelin-1-mediated mitogenesis by 58 and 72%, respectively. The present studies demonstrate that the novel multiple-action antihypertensive agent carvedilol produced antimitogenic effects in rat vascular smooth muscle, a property that may be beneficial for suppressing the progression of vascular hypertrophy associated with hypertension.

CGRP stimulates the adhesion of leukocytes to vascular endothelial cells

Calcitonin gene-related peptide (CGRP) stimulates the adhesiveness of human umbilical vein endothelial cells for U937 cells and human neutrophils in a dose- and time-dependent manner. The onset of CGRP-induced adhesives of HUVEC was rapid (30 min), independent of protein synthesis, and lasted over 24 h in the continuous presence of the peptide. The stimulatory effect of CGRP was completely blocked by the CGRP antagonist, CGRP(8-37). The present study provides evidence in support of the potential role of sensory nerve-derived neuropeptides in the modulation of leukocyte adhesion to vascular endothelial cells.

Matrix Metalloproteinase Expression Increases After Cerebral Focal Ischemia in Rats

BACKGROUND AND PURPOSE Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade the extracellular matrix and are implicated in numerous pathological conditions including atherosclerosis, inflammation, and tumor growth and metastasis. In the brain, the endothelial cell wall, strengthened by tight junctions, defines the blood-brain barrier (BBB). The extracellular matrix molecules constitute the basement membrane underlying the vasculature and play a critical role in maintaining the integrity of the BBB. After focal stroke, there is a breakdown of the BBB with an associated increase in vascular permeability, inflammatory cell influx, and neuronal cell death. The present study was designed to investigate the effects of MMP expression after stroke. METHODS Focal stroke was produced by permanent middle cerebral artery occlusion (MCAO) in the rat, and MMP protein expression was measured by Western blot and zymogram analysis over a time course ranging from 6 hours to 30 days (n=32). Immunohistochemistry at 1 and 5 days (n=8 and 6, respectively) was also utilized to characterize the expression of several MMPs and related proteins after stroke, including their cellular source. To test the hypothesis that early increased MMP-9 expression is involved in ischemic brain injury, a neutralizing monoclonal antibody directed against MMP-9 was administered intravenously (n=7 per group) 1 hour before MCAO, and infarct size was measured 24 hours later. RESULTS MMP expression increased progressively over time after stroke. After 12 hours, significant (P<0.05) MMP-9 activity was observed that reached maximum levels by 24 hours (P<0.001), then persisted for 5 days at this level and returned to basal (zero) levels by 15 days. On the basis of morphological criteria, MMP-9 appeared to stain with endothelial cells and neutrophils identified both within and at the periphery of the infarct within 24 hours of focal ischemia. After 5 days, MMP-9 appeared to stain with macrophages present within the infarcted brain. MMP-2 activity was significantly (P<0.001) increased by 24 hours and was maximum after 5 days following MCAO. MMP-2 appeared to stain with macrophages present within the infarcted region. Unlike MMP-9 and MMP-2, tissue inhibitor of metalloproteinase-1 was identified at comparable levels in both control and ischemic tissue after MCAO. MMP-1 and MMP-3 could not be detected in the brain after focal stroke. When an MMP-9-neutralizing monoclonal antibody was administered systemically, animals exhibited significantly reduced infarct size (ie, a 30% reduction compared with non-immune antibody controls; P<0.05). CONCLUSIONS These results demonstrate that early increased MMP-9 expression in endothelial cells and infiltrating neutrophils is a significant response to cerebral focal ischemia and that selective inhibition of MMP-9 activity can significantly reduce brain injury after stroke.

Factor XIIIa Cross-links Lipoprotein(a) With Fibrinogen and Is Present in Human Atherosclerotic Lesions

During the development of atherosclerotic lesions, lipoprotein(a) [Lp(a)], a highly atherogenic lipoprotein, accumulates within fibrin clots attached to blood vessel walls. As Lp(a) accumulates within the fibrin clot with time, fatty streaks are formed that develop into occlusive atherosclerotic plaques. It is not understood, however, which mechanisms are involved in the binding of Lp(a) to fibrin and, hence, the stable incorporation of Lp(a) into the fibrin clot. The results of the present study demonstrate that factor XIIIa, a transglutaminase that catalyzes the formation of amide bonds between endo-gamma-glutaminyl and endo-epsilon-lysyl residues of proteins, is capable of cross-linking Lp(a) to fibrinogen, the soluble precursor of fibrin. Biochemical assays were conducted to demonstrate that factor XIIIa cross-links Lp(a) with fibrinogen in a time- and concentration-dependent manner. Additionally, immunohistochemical studies revealed that factor XIII protein expression colocalizes with Lp(a) expression in human atherosclerotic plaques. It is proposed that factor XIIIa-mediated cross-linking of Lp(a) to fibrin effectively increases the local concentration of Lp(a) within a fibrin clot. The accumulation of Lp(a) within the blood vessel promotes an antifibrinolytic environment, foam cell formation, the generation of a fatty streak, and an increase in smooth muscle cell content, all of which may contribute to the pathogenesis of atherosclerosis.

Matrix Metalloproteinase Expression Increases After Cerebral Focal Ischemia in Rats : Inhibition of Matrix Metalloproteinase-9 Reduces Infarct Size Editorial Comment: Inhibition of Matrix Metalloproteinase-9 Reduces Infarct Size

BACKGROUND AND PURPOSE Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade the extracellular matrix and are implicated in numerous pathological conditions including atherosclerosis, inflammation, and tumor growth and metastasis. In the brain, the endothelial cell wall, strengthened by tight junctions, defines the blood-brain barrier (BBB). The extracellular matrix molecules constitute the basement membrane underlying the vasculature and play a critical role in maintaining the integrity of the BBB. After focal stroke, there is a breakdown of the BBB with an associated increase in vascular permeability, inflammatory cell influx, and neuronal cell death. The present study was designed to investigate the effects of MMP expression after stroke. METHODS Focal stroke was produced by permanent middle cerebral artery occlusion (MCAO) in the rat, and MMP protein expression was measured by Western blot and zymogram analysis over a time course ranging from 6 hours to 30 days (n=32). Immunohistochemistry at 1 and 5 days (n=8 and 6, respectively) was also utilized to characterize the expression of several MMPs and related proteins after stroke, including their cellular source. To test the hypothesis that early increased MMP-9 expression is involved in ischemic brain injury, a neutralizing monoclonal antibody directed against MMP-9 was administered intravenously (n=7 per group) 1 hour before MCAO, and infarct size was measured 24 hours later. RESULTS MMP expression increased progressively over time after stroke. After 12 hours, significant (P<0.05) MMP-9 activity was observed that reached maximum levels by 24 hours (P<0.001), then persisted for 5 days at this level and returned to basal (zero) levels by 15 days. On the basis of morphological criteria, MMP-9 appeared to stain with endothelial cells and neutrophils identified both within and at the periphery of the infarct within 24 hours of focal ischemia. After 5 days, MMP-9 appeared to stain with macrophages present within the infarcted brain. MMP-2 activity was significantly (P<0.001) increased by 24 hours and was maximum after 5 days following MCAO. MMP-2 appeared to stain with macrophages present within the infarcted region. Unlike MMP-9 and MMP-2, tissue inhibitor of metalloproteinase-1 was identified at comparable levels in both control and ischemic tissue after MCAO. MMP-1 and MMP-3 could not be detected in the brain after focal stroke. When an MMP-9-neutralizing monoclonal antibody was administered systemically, animals exhibited significantly reduced infarct size (ie, a 30% reduction compared with non-immune antibody controls; P<0.05). CONCLUSIONS These results demonstrate that early increased MMP-9 expression in endothelial cells and infiltrating neutrophils is a significant response to cerebral focal ischemia and that selective inhibition of MMP-9 activity can significantly reduce brain injury after stroke.

Biosynthesis and modulation of endothelin from bovine pulmonary arterial endothelial cells

The biosynthesis and modulation of the vasoconstrictor peptide endothelin was studied in the conditioned medium from cultured bovine pulmonary artery endothelial (BPAE) cells. Conditioned medium from cultured BPAE cells produced contraction of isolated rabbit aortic rings. Incubation of BPAE cells with the protease inhibitors TPCK or isatoic anhydride attenuated the extent of conditioned medium-induced contractions. Incubation of BPAE cells with thrombin produced an enhancement of conditioned medium-induced contraction by approximately 25%. Endothelin levels in conditioned medium were measured by RIA and incubation of BPAE cells with TPCK or isatoic anhydride significantly reduced endothelin levels, whereas incubation with thrombin or transforming growth factor beta-1 stimulated the levels of endothelin in the conditioned medium. These data indicate that endothelin may be modulated by certain protease inhibitors and by platelet and immune cell mediators and suggest a potential new mode of vascular tone regulation.

Carvedilol, a novel cardiovascular agent, inhibits development of vascular and ventricular hypertrophy in spontaneously hypertensive rats

The effects of carvedilol, a novel cardiovascular agent, were evaluated in developing spontaneously hypertensive rats (SHR) for effects on hemodynamics, and the ability to effect the development of left ventricular, and vascular hypertrophy associated with chronic hypertension. Chronic oral administration of low dose carvedilol (20 mg/kg/day) was initiated when rats were 5 weeks of age, and experiments progressed until 14 weeks of age. Carvedilol-treated SHR had significantly reduced systolic blood pressures and heart rates throughout the duration of the experiment, and had significantly reduced ventricle/body weights by approximately 9.0%. Morphologic analysis of tertiary branches of the mesenteric artery revealed that carvedilol-treated SHR had significant reductions in medial cross-sectional area. Carvedilol produced concentration-dependent inhibition of basal [3H]thymidine incorporation in cultured SHR vascular smooth muscle cells, as well as by stimulation produced by PDGF (1 nM), EDGF (1 nM), thrombin (0.5 U/ml), or endothelin-1 (1 nM), indicating that carvedilol had direct anti-mitogenic activity. The present studies demonstrate that low dose carvedilol produced sustained reductions in blood pressure and heart rate in developing SHR that were accompanied by significant inhibition in the development of vascular and myocardial hypertrophy. The morphological changes induced by carvedilol may be mediated by a combination of hemodynamic effects, as well as by direct anti-mitogenic effects on vascular smooth muscle.

Involvement of Protein Kinase C in Cytokine-Induced Tissue Factor Production in Human Vascular Endothelial Cells

Cultured vascular endothelial cells demonstrate procoagulant activity following stimulation by cytokines and other inflammatory agents. Tissue factor is a cell surface membrane protein, which functions as a receptor and essential cofactor for Factor VII triggering the blood coagulation process. The mechanism(s) by which cytokine/inflammatory agents stimulate the production of tissue factor is still unclear. We hypothesized that protein kinase C (PKC) may be involved in cytokine/inflammatory agent-induced up-regulation of tissue factor in human vascular endothelial cells. The production of tissue factor by human umbilical endothelial cells (HUVEC) was enhanced markedly (5-10 fold) by TNFα (100 U/ml), IL-1 (10 U/ml) or LPS (100 ng/ml). Phorbol esters (PMA or PDBu) stimulated the production of tissue factor by HUVEC in a concentration-dependent manner, while the biologically inactive analog of PDBu, 4α-PDBu was inactive. Maximal stimulation by PMA (10-12 fold) was obtained at 200 nM and the ED50 was 50 nM. P...