Sandra T. Davidge

Vascular Matrix Metalloproteinase-2 Cleaves Big Endothelin-1 Yielding a Novel Vasoconstrictor

Matrix metalloproteinase-2 (MMP-2, gelatinase A) and its tissue inhibitor (TIMP-2) are mainly known for their roles in the (patho)physiological remodeling of the vasculature, angiogenesis, tissue repair, tumor invasion, inflammation, and atherosclerotic plaque rupture. A mechanism of action of MMP-2 is the proteolytic breakdown of specific extracellular matrix proteins. The amino acid sequences in interstitial collagen (Gly-Leu/Ile) and laminin-5 (Ala-Leu) that are cleaved by MMP-2 are homologous to a region (Gly(32)-Leu(33)) within human big endothelin-1[1 to 38] (big ET-1). Big ET-1 requires cleavage to an active form to produce vasoconstriction. We tested the hypothesis that vascular MMP-2 can cleave big ET-1, thus generating a vasoconstrictor peptide. In perfused rat mesenteric arteries with an intact endothelium, inhibition of vascular MMP-2 with TIMP-2 reduced (by 16.2+/-4.2%) the vasoconstrictor effects of big ET-1 (50 pmol). However, when the endothelium was mechanically removed, TIMP-2 abolished (>90%) the vasoconstriction of big ET-1, and this effect was mimicked by an anti-MMP-2 antibody. Incubation of big ET-1 with recombinant human MMP-2 resulted in the specific cleavage of the Gly(32)-Leu(33) bond of big ET-1. Moreover, the resultant peptide ET-1[1 to 32] exerted greater vasoconstrictor effects than big ET-1. We conclude that vascular MMP-2 contributes to the vasoconstrictor effects of big ET-1 by cleaving big ET-1 to yield a novel and potent vasoconstrictor, ET-1[1 to 32]. These data implicate, for the first time, the endogenous MMP-2/TIMP-2 system in the regulation of vascular reactivity.

Evidence for Peroxynitrite Formation in the Vasculature of Women With Preeclampsia

-Preeclampsia is a multisystemic disorder of pregnancy in which the normal vascular adaptations to pregnancy are compromised. Oxidative stress as well as endothelial cell dysfunction have been implicated as pathophysiological features of preeclampsia. Endothelial cells produce the vasorelaxant nitric oxide (NO). However, NO is also known to react with superoxide anions (produced under conditions of oxidative stress), yielding peroxynitrite that may impair vascular function. Our objective was to use immunohistochemical techniques to determine whether there is evidence of peroxynitrite formation in the maternal systemic vasculature of women with preeclampsia. Vessels were obtained from a biopsy of subcutaneous fat at the time of cesarean section from normal pregnant (n=7) and preeclamptic (n=7) women or at the time of hysterectomy from nonpregnant women (n=5). There were significantly more vessels staining with greater intensity for nitrotyrosine and endothelial NO synthase in the endothelium of vessels from women with preeclampsia compared with that of normal pregnant women or nonpregnant women. Both endothelial and smooth muscle cells from all vessels showed evidence for the presence of superoxide dismutase (SOD), an enzyme that scavenges superoxide anions. However, the intensity of staining for SOD in the endothelium was significantly lower in the preeclamptic and nonpregnant women than in normal pregnant women. These data of increased endothelial NO synthase, decreased SOD, and increased nitrotyrosine immunostaining in the maternal vasculature of women with preeclampsia suggest increased peroxynitrite formation. We speculate that peroxynitrite is involved in endothelial cell dysfunction in preeclamptic women and contributes to the pathophysiology of this pregnancy disorder.

Vascular matrix metalloproteinase-2-dependent cleavage of calcitonin gene-related peptide promotes vasoconstriction.

Matrix metalloproteinase (MMP)-2 has been historically associated with the process of vascular remodeling through the cleavage of extracellular matrix proteins. However, we recently found that MMP-2 also cleaves the endothelium-derived peptide big endothelin-1, ET-1[1–38] and yields the novel vasoconstrictor ET-1[1–32]. We therefore investigated the effects of MMP-2 inhibitors as potential vasodilators. MMP inhibition with ortho-phenanthroline (0.3 to 30 &mgr;mol/L) induced vasorelaxation of isolated rat mesenteric arteries (maximum of relaxation=74.5±27.6% at 30 &mgr;mol/L). However, phosphoramidon (0.3 to 30 &mgr;mol/L), which inhibits some metalloenzymes, but not MMP-2, did not dilate the arteries. Selective inhibition of endogenous MMP-2 with the novel tissue-permeable cyclic peptide CTTHWGFTLC (CTT, 10 &mgr;mol/L) also caused vasorelaxation (by 85±6%), whereas STTHWGFTLS (10 &mgr;mol/L), an inactive CTT analogue, did not dilate the arteries. Interestingly, the vasorelaxation that results from MMP-2 inhibition was endothelium-independent. Thus, we examined whether MMP-2 acted on peptides derived from the smooth muscle or the perivascular nerves. Recombinant human MMP-2 cleaved calcitonin gene-related peptide (CGRP) specifically at the Gly14-Leu15 peptide bond and reduced the vasodilatory potency of CGRP by 20-fold. Inhibition of MMP-2 increased the amount of intact CGRP in arteries and enhanced vasorelaxation induced by anandamide, which stimulates CGRP release. Vasorelaxation in response to MMP-2 inhibition was abolished by CGRP[8–37], a selective CGRP receptor antagonist, and by capsaicin, which depletes arterial perivascular nerves of CGRP. We conclude that vascular MMP-2 cleaves endogenous CGRP and promotes vasoconstriction. These data suggest a novel mechanism of regulating the vasoactive and, possibly, the neurohormonal actions of CGRP and establish MMP-2 as a modulator of vascular function.

Urine but not plasma nitric oxide metabolites are decreased in women with preeclampsia

OBJECTIVE Nitric oxide is a potent vasorelaxant produced by endothelial cells. We tested the hypothesis that urinary and perhaps plasma nitric oxide metabolites would be reduced in women with preeclampsia. STUDY DESIGN Plasma and urine from 14 women meeting strict clinical criteria for the diagnosis of preeclampsia and 20 normal nulliparous women were assayed for the stable metabolites of nitric oxide, nitrate and nitrite. RESULT There was no significant difference of plasma concentrations of nitrate and nitrite between women with preeclampsia and women with normal pregnancies (32.7 +/- 3.1 vs 25.8 +/- 2.4 micromol/L). Plasma creatinine levels were elevated in women with preeclampsia (0.85 +/- 0.09 vs 0.66 +/- 0.02 mg/dl, p<0.01), indicating a reduced glomerular filtration rate. Urine concentrations of nitrate and nitrite normalized by creatinine excretion were significantly lower in women with preeclampsia compared with normal pregnant women (0.37 +/- 0.06 vs 0.69 +/- 0.11 micromol of nitrite per milligram creatinine, p. <0.05). CONCLUSIONS Our study using concomitant measurement of plasma and urine nitrate and nitrite suggests a reduced production of nitric oxide in women with preeclampsia compared with normal pregnant women.

Impact of pregnancy-induced hypertension on fetal growth

OBJECTIVE The purpose of this study was to evaluate the effect of different types of pregnancy-induced hypertension on fetal growth. STUDY DESIGN A retrospective cohort study was conducted on the basis of 16,936 births from January 1, 1989, through December 31, 1990, by means of data from a population-based perinatal database in Suzhou, China. Pregnancy-induced hypertension was classified as gestational hypertension, preeclampsia, or severe preeclampsia-eclampsia. Univariate and multivariate regression analyses were performed to examine the effect of the various types of pregnancy-induced hypertension on gestational age, preterm birth, birth weight, low birth weight, and intrauterine growth restriction. RESULTS Gestation was 0.6 week shorter in women with severe preeclampsia than in normotensive women (P <.01). However, the risk of preterm birth was not increased with any classification of pregnancy-induced hypertension (for severe preeclampsia: adjusted odds ratio 1.75; 95% confidence interval, 0.88-3.47). After adjustment for duration of gestation and other confounders, preeclampsia and severe preeclampsia increased the risk of intrauterine growth restriction and low birth weight. The adjusted odds ratios of low birth weight were 2.65 (1.73-4.39) for preeclampsia and 2.53 (1.19-4.93) for severe preeclampsia. However, the risk of low birth weight was not increased significantly for gestational hypertension (adjusted odds ratio 1.56 [1.00-2.41]). CONCLUSION Preeclampsia increases the risk of intrauterine growth restriction and low birth weight.

Preeclampsia: current understanding of the molecular basis of vascular dysfunction.

Preeclampsia is a pregnancy-specific disorder characterised by hypertension and proteinuria occurring after the 20th week of gestation. Delivery of the placenta results in resolution of the condition, implicating the placenta as a central culprit in the pathogenesis of preeclampsia. In preeclampsia, an inadequate placental trophoblast invasion of the maternal uterine spiral arteries results in poor placental perfusion, leading to placental ischaemia. This could result in release of factors into the maternal circulation that cause widespread activation or dysfunction of the maternal endothelium. Factors in the maternal circulation might induce oxidative stress and/or elicit an inflammatory response in the maternal endothelium, resulting in the altered expression of several genes involved in the regulation of vascular tone. This review addresses the potential circulating factors and the molecular mechanisms involved in the alteration of vascular function that occurs in preeclampsia.

Matrix metalloproteinases regulate neutrophil-endothelial cell adhesion through generation of endothelin-1[1–32]

We recently reported that matrix metal‐loproteinase 2 (MMP‐2, gelatinase A) cleaves big endo‐thelin 1 (ET‐1), yielding the vasoactive peptide ET‐1[1–32]. We tested whether ET‐1[1–32] could affect the adhesion of human neutrophils to coronary artery endothelial cells (HCAEC). ET‐1[1–32] rapidly down‐regulated the expression of L‐selectin and up‐regulated expression of CD11b/CD18 on the neutrophil surface, with EC50 values of 1–3 nM. These actions of ET‐1[1–32] were mediated via ETA receptors and did not require conversion of ET‐1[1–32] into ET‐1 by neutrophil proteases, as revealed by liquid chromatography and mass spectroscopy. Moreover, ET‐1[1–32] evoked release of neutrophil gelatinase B, which cleaved big ET‐1 to yield ET‐1[1–32], thus revealing a positive feedback loop for ET‐1[1–32] generation. Up‐regula‐tion of CD11b/CD18 expression and gelatinase release was tightly associated with activation of extracellular signal‐regulated kinase (Erk). Stimulation of Erk activity was due to activation of Ras, Raf‐1, and MEK (MAPK kinase). ET‐1[1–32] also produced slight increases in the expression of ICAM‐1 and E‐selectin on HCAEC, and markedly enhanced ß2 integrin‐dependent adhesion of neutrophils to activated HCAEC. These results are the first indication that gelatinolytic MMPs via cleavage of big ET‐1 to yield ET‐1[1–32] activate neu‐trophils and promote leukocyte‐endothelial cell adhesion and, consequently, neutrophil trafficking into inflamed tissues.—Fernandez‐Patron, C., Zouki, C., Whittal, R., Chan, J. S. D., Davidge, S. T., Filep, J. G. Matrix metalloproteinases regulate neutrophil‐endothelial cell adhesion through generation of endothelin‐ 1[1–32]. FASEB J. 15, 2230–2240 (2001)

Estrogen modulation of left ventricular remodeling in the aged heart.

OBJECTIVE To investigate the effects of estrogen on left ventricle (LV) mass and collagen deposition, and on the expression of receptors for estrogen (ER alpha, ER beta) and Ang II (AT(2)R, AT(1)R) in the heart of aged female rats. METHODS Aged ( approximately 12 months old) intact (n=7), ovariectomized plus placebo (OVX, n=7), and estrogen-replaced (E2, n=6) as well as young ( approximately 3 months old, n=4) female Sprague-Dawley rats were used in this study. After 1 month of treatment, the left ventricular weight/body weight ratio (LVW/BW), changes in myosin heavy chain expression (MHC), matrix metalloproteinase (MMP)-2 activity, the collagen I/III ratio, and the expression of ERs and Ang II receptors in the LV were evaluated. RESULTS In aged rats, OVX increased LVW/BW associated with a higher expression of beta-MHC isoform, increased collagen I/III ratio, and decreased MMP-2 activity compared to intact rats. Furthermore, the OVX group had a decrease in ERs alpha and beta as well as AT(2)R but an increase in AT(1)R expression. Estrogen replacement prevented the effects of ovariectomy on heart remodeling as well as increased further expression of ER beta and decreased AT(1)R expression. CONCLUSION Removal of ovarian hormones increased LV remodeling in the aged rat, which could be attenuated by estrogen replacement. Moreover, regulation of Ang II receptor expression could be a mechanism by which estrogen may modulate heart remodeling.

Estrogen Reduces Angiotensin II-Induced Nitric Oxide Synthase and NAD(P)H Oxidase Expression in Endothelial Cells

Objective—Angiotensin II (AII) has been shown to increase endothelial NAD(P)H oxidase activity, which is a source of superoxide anion that in turn may induce the formation of peroxynitrite. Estrogen (E2) has been reported to have vascular protective effects. In this study, we hypothesized that E2 reduces the AII-induced expression of NAD(P)H oxidase and peroxynitrite in endothelial cells. Methods and Results—Endothelial cells were cultured and stimulated with AII in the absence or presence of E2. Western blots were used to assess nitric oxide synthase (NOS) and NAD(P)H oxidase expression. Immunofluorescence of nitrotyrosine provided evidence of peroxynitrite formation. Our data indicate that AII increased the expression of endothelial NOS, inducible NOS, and NAD(P)H oxidase in a dose-dependent manner, which was attenuated by incubation with either E2, superoxide dismutase, or the AII type 1 receptor (AT1R) inhibitor candesartan. Estrogen as well as superoxide dismutase also inhibited AII-induced AT1R expression and nitrotyrosine staining. The effects of E2 on the AII responses were not inhibited by the E2 receptor antagonist ICI-182,780. Conclusions—AII stimulation of endothelial cells increases expression of NAD(P)H oxidase and NOS, which may contribute to oxidative stress, as evidenced by peroxynitrite formation. E2 inhibits these AII effects, possibly through reduced AT1R expression.

Resveratrol prevents hypertension and cardiac hypertrophy in hypertensive rats and mice

Resveratrol (RESV) is a polyphenol with pleiotropic effects that include reduction of oxidative stress and increased vascular nitric oxide (NO) production. However, whether or not RESV can prevent rises in blood pressure (BP) is controversial and remains to be firmly established. The purpose of this study was to determine whether RESV attenuates elevated BP and subsequent adaptive cardiac hypertrophy and to better understand the mechanisms involved. The spontaneously hypertensive rat (SHR) and the angiotensin (Ang)-II infused mouse were used as hypertensive models. Compared to a standard control diet, consumption of diets containing RESV by SHRs and Ang-II hypertensive mice, markedly prevented rises in systolic BP. In addition, flow-mediated vasodilation was significantly improved by RESV in SHRs. RESV also reduced serum and cardiac levels of the lipid peroxidation by-product, 4-hydroxy-2-nonenal in the hypertensive rodents and inhibited the production of superoxide in human-derived endothelial cells. Analysis of mesenteric arteries from SHRs and Ang-II infused mice demonstrated that RESV increased endothelial NO synthase (eNOS) phosphorylation by enhancing the LKB1/adenosine monophosphate (AMP)-activated protein kinase (AMPK) signal transduction pathway. Moreover, RESV reduced hypertrophic growth of the myocardium through reduced hemodynamic load and inhibition of the p70 S6 kinase pro-hypertrophic signaling cascade. Overall, we show that high dose RESV reduces oxidative stress, improves vascular function, attenuates high BP and prevents cardiac hypertrophy through the preservation of the LKB1-AMPK-eNOS signaling axis.