Carlos Fernandez-Patron

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.

Agonist-Induced Activation of Matrix Metalloproteinase-7 Promotes Vasoconstriction Through the Epidermal Growth Factor–Receptor Pathway

Abstract— Matrix metalloproteinase (MMP)-dependent shedding of heparin-binding epidermal growth factor (HB-EGF) and subsequent activation of the EGF receptor (EGFR) in the cardiovasculature is emerging as a unique mechanism signaling growth effects of diverse G protein–coupled receptors (GPCRs). Among these GPCRs are adrenoceptors and angiotensin receptors that contribute to the pathogenesis of hypertension through their vasoconstrictive and growth effects. Focusing on &agr;1b-adrenoceptors, we suggest here that MMP-dependent activation of the EGFR promotes vasoconstriction as well as growth. We identified MMP-7 as a major HB-EGF sheddase in rat mesenteric arteries and &agr;1b-adrenoceptors, angiotensin receptors, and hypertension-stimulated MMP-7 activity. Adrenoceptors stimulated EGFR autophosphorylation in arteries, and this transactivation was opposed by the MMP-7 inhibitor GM6001 as well as MMP-7–specific antibodies. In isolated microperfused arteries, blockade of EGFR transactivation with inhibitors of the EGFR (AG1478 and PD153035), HB-EGF (CRM197 and neutralizing antibodies), or MMPs (doxycycline) inhibited adrenergic vasoconstriction. In spontaneously hypertensive rats but not in normotensive rats, the inhibition of MMPs with doxycycline (19.2 mg/d from week 7 until week 12) reduced systolic blood pressure and attenuated HB-EGF shedding in the mesenteric arteries. These findings suggest a previously unknown mechanism of vasoregulation whereby agonists of certain GPCRs (such as adrenoceptors and angiotensin receptors) activate MMPs (such as MMP-7) that shed EGFR ligands (such as HB-EGF), which then activate the EGFR, thereby promoting vasoconstriction as well as growth. Because this mechanism is triggered by agonists typically overexpressed in hypertension, its blockade may have therapeutic potential for simultaneously inhibiting pathological vasoconstriction and growth in hypertensive disorders.

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.

Tumor Necrosis Factor-α–Converting Enzyme Is a Key Regulator of Agonist-Induced Cardiac Hypertrophy and Fibrosis

Cardiac remodeling is associated with hypertrophy and fibrosis processes, which may depend on the activity of matrix metalloproteinases (MMPs) and “a disintegrin and metalloproteinases” (ADAMs). We investigated whether ADAM-17 (tumor necrosis factor-α–converting enzyme [TACE]) plays a role in agonist-induced cardiac remodeling and the relationships established among TACE, MMP-2, and ADAM-12. We targeted TACE in rodent models of spontaneous and agonist-induced hypertension using RNA interference combined with quantitative RT-PCR, activity determinations, and functional studies. Treatment of spontaneously hypertensive rats with previously validated TACE small-interfering RNA for 28 days resulted in systemic knockdown of TACE expression. TACE knockdown effectively stopped the development of cardiac hypertrophy. Mice receiving angiotensin II (1.4 mg/kg per day for 12 days) exhibited cardiac hypertrophy, as well as fibrosis, which was associated with elevated myocardial expression of molecular markers of hypertrophy (α-skeletal actin, β-myosin heavy chain, and brain natriuretic peptide) and fibrosis (collagen types I and III and fibronectin), as well as MMP-2 and ADAM-12. Treatment with TACE small-interfering RNA (but not with PBS or luciferase small-interfering RNA) inhibited TACE expression, thus preventing angiotensin II–induced cardiac hypertrophy and fibrosis. Moreover, knockdown of TACE inhibited angiotensin II–induced overexpression of markers of myocardial hypertrophy and fibrosis, as well as ADAM-12 and MMP-2. These findings provide the first in vivo evidence that agonist-induced cardiac hypertrophy and fibrosis processes are signaled through TACE, which acts through novel pathways involving transcriptional regulation of ADAM-12 and MMP-2. Targeting TACE has potential therapeutic importance for modulating agonist-induced cardiac remodeling.

Vascular Responses to α1-Adrenergic Receptors in Small Rat Mesenteric Arteries Depend on Mitochondrial Reactive Oxygen Species

Background—Agonists of G-protein–coupled receptors (eg, adrenoceptors and angiotensin receptors) signal, at least in part, through matrix metalloproteinases (such as matrix metalloproteinase [MMP]-7) that transactivate the epidermal growth factor receptor (EGFR). Focusing on adrenoceptors, we examined whether the MMP-dependent signaling pathway depends on reactive oxygen species (ROS). Methods and Results—In isolated rat mesenteric arteries, selective stimulation of &agr;1-adrenoceptors with phenylephrine induced MMP transactivation of the EGFR, mitochondrial ROS production (detected by MitoTrackerRed-CM-H2XRos-fluorescence and dihydroethidium-fluorescence and high-performance liquid chromatography [HPLC]/MS assay) and vasoconstriction. Inhibition of the synthesis of either MMP-7 or EGFR with anti-sense or siRNA oligonucleotides, respectively, decreased mitochondrial ROS production in response to phenylephrine. Targeted mitochondrial ROS scavenging with MitoTrackerRed-CM-H2XRos inhibited adrenergic vasoconstriction. Adrenoceptor-induced ROS increased mitochondrial membrane potential (&Dgr;&psgr;m), which was prevented by blockers of MMPs (GM6001, doxycycline), EGFR (AG1478), or complex I, all of which also prevented ROS production as well as vasoconstriction. Conclusions—Production of mitochondrial ROS is a new event in the pathway by which vasoactive agonists that induce MMP transactivation of the EGFR modulate vascular tone. Moreover, our findings suggest a connection between agonist-induced activity of MMPs, the promotion of oxidative stress, enhanced vascular tone, and hypertrophy, which are all implicated in the development and progression of vascular disease.

MATRIX METALLOPROTEINASE-2 IS ELEVATED IN THE PLASMA OF WOMEN WITH PREECLAMPSIA

Objective: We have recently demonstrated that matrix metalloproteinase-2 (MMP-2) alters vascular function through cleavage of vasoactive peptides, resulting in increased vasoconstriction and reduced vasodilation. We, therefore, hypothesized that MMP levels are increased in women with preeclampsia. In addition, because vascular endothelial growth factor (VEGF) has been implicated in the pathophysiology of preeclampsia and is involved in angiogenesis that requires the release of proteases to allow for migration of endothelial cells, we hypothesized that VEGF increases release of MMPs from endothelial cells. Methods: We used zymographic analysis to evaluate MMP-2/MMP-9 levels in plasma of women with preeclampsia (n=12) compared to women with uncomplicated pregnancies (n=12). In addition, we evaluated the changes in the levels of MMP-2 and MMP-9 as well as tissue inhibitors of MMPs (TIMP-1 and TIMP-2) released by cultured human umbilical vein endothelial cells in response to VEGF (0.1–10 ng/mL). Results: Our data showed that plasma MMP-2 levels were significantly higher in women with preeclampsia compared to women with uncomplicated pregnancies (arbitrary intensity units: 690 ±111 and 252 ±56, respectively, p<0.05). MMP-9 levels were below the level of detection. In addition, VEGF stimulated endothelial MMP-2 and MMP-9 release in a concentration- and time-dependent (6–24 h) manner. Moreover, VEGF stimulation of MMP release occurs without significantly affecting the release of TIMP-1 and TIMP-2. Conclusions: These data suggest that VEGF promotes secretion of MMPs from endothelial cells that, in turn, could alter vascular function in women with preeclampsia.

MMP-2 Mediates Angiotensin II–Induced Hypertension Under the Transcriptional Control of MMP-7 and TACE

Development of cardiovascular disease induced by excessive Gq protein–coupled receptor agonist stimulation depends on signaling networks involving multiple matrix metalloproteinases (MMPs) and metalloproteinase disintegrins (ADAMs). Here, we hypothesized that MMP-2, being a major gelatinase in cardiac and vascular tissue, was likely to play a key role in cardiovascular homeostasis. We targeted MMP-2 using complementary and overlapping approaches involving pharmacological inhibition and RNA interference in mice treated with angiotensin II (1.4 mg/kg per day) for 12 days. We studied the development of hypertension (by tail cuff plethysmography), cardiac hypertrophy (by M-mode echocardiography, cardiomyocyte cross-sectional area, and quantitative real-time polymerase chain reaction (qRT-PCR) analysis of hypertrophy marker genes), and fibrosis (by picrosirius red collagen staining and qRT-PCR analysis of fibrosis marker genes) in mice receiving angiotensin II. We found that angiotensin II infusion upregulated MMP-2 concurrent with the development of hypertension, hypertrophy, and fibrosis. This upregulation of MMP-2 depended on MMP-7 and TACE (tumor necrosis factor-&agr; convertase, ADAM-17). RNA interference targeting MMP-7 and TACE attenuated the angiotensin II–induced upregulation of MMP-2 and prevented the development of hypertension, as well as development of cardiac hypertrophy and fibrosis. In contrast, pharmacological inhibition and RNA interference of MMP-2 attenuated angiotensin II–induced hypertension, without influencing development of cardiac hypertrophy or fibrosis. Downstream of MMP-7 and TACE, MMP-2 mediated angiotensin II–induced hypertension, but did not mediate cardiac hypertrophy or fibrosis. This suggests a functional specialization of MMP-2 in agonist–induced cardiovascular disease development that has potential implications for the design of metalloproteinase-based therapeutic strategies.

Matrix Metalloproteinase-7 and ADAM-12 (a Disintegrin and Metalloproteinase-12) Define a Signaling Axis in Agonist-Induced Hypertension and Cardiac Hypertrophy

Background— Excessive stimulation of Gq protein–coupled receptors by cognate vasoconstrictor agonists induces a variety of cardiovascular processes, including hypertension and hypertrophy. Here, we report that matrix metalloproteinase-7 (MMP-7) and a disintegrin and metalloproteinase-12 (ADAM-12) form a novel signaling axis in these processes. Methods and Results— In functional studies, we targeted MMP-7 in rodent models of acute, long-term, and spontaneous hypertension by 3 complementary approaches: (1) Pharmacological inhibition of activity, (2) expression knockdown (by antisense oligodeoxynucleotides and RNA interference), and (3) gene knockout. We observed that induction of acute hypertension by vasoconstrictors (ie, catecholamines, angiotensin II, and the nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester) required the posttranscriptional activation of vascular MMP-7. In spontaneously hypertensive rats, knockdown of MMP-7 (by RNA interference) resulted in attenuation of hypertension and stopped development of cardiac hypertrophy. Quantitative reverse-transcription polymerase chain reaction studies in mouse models of MMP-7 knockdown (by RNA interference) and gene knockout revealed that MMP-7 controlled the transcription of ADAM-12, the major metalloproteinase implicated in cardiac hypertrophy. In mice with angiotensin II–induced hypertension and cardiac hypertrophy, myocardial ADAM-12 and downstream hypertrophy marker genes were overexpressed. Knockdown of MMP-7 attenuated hypertension, inhibited ADAM-12 overexpression, and prevented cardiac hypertrophy. Conclusions— Agonist signaling of both hypertension and hypertrophy depends on posttranscriptional and transcriptional mechanisms that involve MMP-7, which is transcriptionally connected with ADAM-12. Approaches targeting this novel MMP-7/ADAM-12 signaling axis could have generic therapeutic potential in hypertensive disorders caused by multiple or unknown agonists.

Mitochondrial dysfunction in the hypertensive rat brain: respiratory complexes exhibit assembly defects in hypertension.

The central nervous system plays a critical role in the normal control of arterial blood pressure and in its elevation in virtually all forms of hypertension. Mitochondrial dysfunction has been increasingly associated with the development of hypertension. Therefore, we examined whether mitochondrial dysfunction occurs in the brain in hypertension and characterized it at the molecular scale. Mitochondria from whole brain and brain stem from 12-week–old spontaneously hypertensive rats with elevated blood pressure (190±5 mm Hg) were compared against those from age-matched normotensive (134±7 mm Hg) Wistar Kyoto rats (n=4 in each group). Global differential analysis using 2D electrophoresis followed by tandem mass spectrometry–based protein identification suggested a downregulation of enzymes involved in cellular energetics in hypertension. Targeted differential analysis of mitochondrial respiratory complexes using the classical blue-native SDS-PAGE/Western method and a complementary combination of sucrose-gradient ultracentrifugation/tandem mass spectrometry revealed previously unknown assembly defects in complexes I, III, IV, and V in hypertension. Interestingly, targeted examination of the brain stem, a regulator of cardiovascular homeostasis and systemic blood pressure, further showed the occurrence of mitochondrial complex I dysfunction, elevated reactive oxygen species production, decreased ATP synthesis, and impaired respiration in hypertension. Our findings suggest that in already-hypertensive spontaneously hypertensive rats, the brain respiratory complexes exhibit previously unknown assembly defects. These defects impair the function of the mitochondrial respiratory chain. This mitochondrial dysfunction localizes to the brain stem and is, therefore, likely to contribute to the development, as well as to pathophysiological complications, of hypertension.

The tumor suppressor gene, RASSF1A, is essential for protection against inflammation -induced injury.

Ras association domain family protein 1A (RASSF1A) is a tumor suppressor gene silenced in cancer. Here we report that RASSF1A is a novel regulator of intestinal inflammation as Rassf1a+/−, Rassf1a−/− and an intestinal epithelial cell specific knockout mouse (Rassf1a IEC-KO) rapidly became sick following dextran sulphate sodium (DSS) administration, a chemical inducer of colitis. Rassf1a knockout mice displayed clinical symptoms of inflammatory bowel disease including: increased intestinal permeability, enhanced cytokine/chemokine production, elevated nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) activity, elevated colonic cell death and epithelial cell injury. Furthermore, epithelial restitution/repair was inhibited in DSS-treated Rassf1a−/− mice with reduction of several makers of proliferation including Yes associated protein (YAP)-driven proliferation. Surprisingly, tyrosine phosphorylation of YAP was detected which coincided with increased nuclear p73 association, Bax-driven epithelial cell death and p53 accumulation resulting in enhanced apoptosis and poor survival of DSS-treated Rassf1a knockout mice. We can inhibit these events and promote the survival of DSS-treated Rassf1a knockout mice with intraperitoneal injection of the c-Abl and c-Abl related protein tyrosine kinase inhibitor, imatinib/gleevec. However, p53 accumulation was not inhibited by imatinib/gleevec in the Rassf1a−/− background which revealed the importance of p53-dependent cell death during intestinal inflammation. These observations suggest that tyrosine phosphorylation of YAP (to drive p73 association and up-regulation of pro-apoptotic genes such as Bax) and accumulation of p53 are consequences of inflammation-induced injury in DSS-treated Rassf1a−/− mice. Mechanistically, we can detect robust associations of RASSF1A with membrane proximal Toll-like receptor (TLR) components to suggest that RASSF1A may function to interfere and restrict TLR-driven activation of NFκB. Failure to restrict NFκB resulted in the inflammation-induced DNA damage driven tyrosine phosphorylation of YAP, subsequent p53 accumulation and loss of intestinal epithelial homeostasis.