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

Originally publishedhttps://doi.org/10.1161/CIRCULATIONAHA.108.835488Circulation. 2009;119:2480–2489

Abstract

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.

Hypertension, often termed the silent killer, is a systemic condition characterized by persistently elevated arterial blood pressure; it is typically associated with cardiovascular hypertrophy.1 More than 25% of the adult population in developed countries is hypertensive and therefore at risk of heart disease, peripheral vascular disease, end-stage renal disease, and cerebrovascular stroke. The pathogenesis of most hypertensive disorders is complex, because genetic, immune, and environmental factors all may predispose individuals to hypertension. A difficulty faced by physicians when deciding on a therapeutic strategy is that typically, the cause of the hypertension is unknown. Thus, treatment of hypertension remains rather empirical, with physicians choosing among many antihypertensive medications until a drug or drug combination is identified that effectively lowers the blood pressure in the patient. Of those individuals treated, 65% do not meet treatment goals.2 Therefore, treatment strategies are needed that (1) are preventative, (2) can stop pathological hypertrophy processes or induce the regression of preexisting cardiac hypertrophy, and (3) are efficacious in hypertensive disorders with multiple or unknown cause(s).

Clinical Perspective on p 2489

Recently, we proposed an approach to treat hypertension by blocking mediators that are commonly shared by many vasoconstrictors but significantly activated only in response to excessive agonist stimulation.3 The major vasoconstrictor systems discovered to date (catecholamines, endothelins, and angiotensin II) all use Gq protein–coupled receptors (GqPCRs) as their cognate receptors. GqPCRs act through the activation of the classic phospholipase C/protein kinase C pathway and downstream matrix metalloproteinases (MMPs, such as MMP-2, MMP-7, and MMP-9) and a disintegrin and metalloproteinases (such as [ADAM]-12 and ADAM-17/TACE [tumor necrosis factor-convertase]).4–7 Agonist-induced activation of these metalloproteinases is a rapid, posttranscriptional event mediated by protein kinase C, reactive oxygen species, and other metalloproteinases (such as membrane-type MMPs).4,8,9 Opening of a cysteine switch activates the prometalloproteinase, which sometimes results in autolysis.9 Once activated, metalloproteinases cleave a host of common substrates, including extracellular matrix proteins (eg, collagens), proinflammatory mediators (eg, tumor necrosis factor-α), and growth factors (eg, transforming growth factor-α and HB-EGF [heparin-binding epidermal growth factor–like growth factor]). Thus, an overabundance of vasoconstrictive agonists (as occurs in hypertensive disorders) results in the posttranscriptional activation of metalloproteinases, which next cleave and release (shed) substrates that signal through mitogen-activated protein kinases to transcriptionally activate immediate-early genes and reactivate fetal genes, including hypertrophy markers.10 This mechanism may signal multiple processes, including vascular smooth muscle and cardiomyocyte tone, cardiovascular hypertrophy, and tissue injury.5,6,11

The similar tissue localization, activation profile, substrates, and signaling pathways of many metalloproteinases, including MMP-7, MMP-2, ADAM-12, and ADAM-17/TACE,5–7,11–13 indicates a redundancy of their functions in vivo. However, the specific roles played by metalloproteinases, the hierarchical relationships that may coordinate their functions in vivo, and the therapeutic potential of these relationships remain poorly understood.

To start addressing these long-standing questions, we have focused on MMP-7. The present findings suggest the existence of hierarchical and agonist-dependent relationships between MMP-7 and ADAM-12, which suggests a novel central role of MMP-7 in agonist signaling of multiple cardiovascular processes, including hypertension and hypertrophy.

Methods

Please see the online Data Supplement for the expanded Methods section.

Animals

Animal protocols were conducted in accordance with institutional guidelines issued by the Canada Council on Animal Care. MMP-7−/− mice and age-matched C57BL/6 (wild-type) littermates (12 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, Me). The MMP-7−/− mice were generated by disrupting the MMP-7 gene through the insertion of a neomycin resistance cassette into the fragment spanning exon 3 and 4.14 Already-hypertensive (22-week-old) spontaneously hypertensive rats (SHRs) and age-matched Wistar-Kyoto (WKY) rats as well as Sprague Dawley rats (250–350 grams) were purchased from Charles River Laboratories Inc (Wilmington, Del).

Generation of MMP-7 Knockdown Models

The sequences of the MMP-7 antisense (active), MMP-7 scrambled (inactive) oligodeoxynucleotides, and MMP-7 small interfering RNA (siRNA) were derived from previous studies.15–17 The oligonucleotides were delivered with ALZET osmotic minipumps (DURECT Corp, Cupertino, Calif) implanted subcutaneously on the backs of the animals.

Data Analysis

Results are presented as mean±SEM and were analyzed with 1-way ANOVA or t test as appropriate with Jandel SigmaStat 3.5 statistical software. In the echocardiography studies, between-group comparisons of the means were performed by 1-way ANOVA followed by Scheffé’s F correction for multiple comparisons of the means. Statistical significance was considered when P≤0.05. Except where indicated otherwise, between 4 and 5 animals were used for each study.

The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

Results

MMP-7 as a Mediator in Pharmacologically Induced Acute Hypertension

To trigger an acute hypertensive response in otherwise normotensive Sprague Dawley rats and C57BL/6 mice, we injected intraperitoneally either PBS (vehicle) or (1) α-adrenergic agonists (phenylephrine and norepinephrine), (2) angiotensin II, or (3) NG-nitro-l-arginine methyl ester (L-NAME), which elevates blood pressure by blocking basal nitric oxide–dependent vasodilation, thus unmasking secondary vasoconstrictor mechanisms18,19 (Figure 1, top panels, and Figure 2).

Figure 1. MMP-7 as a mediator in rat models of acute hypertension. A through C, Top, Time course of systolic blood pressure of Sprague Dawley rats administered phenylephrine (PE; 3 mg/kg IP; A), angiotensin II (Ang II; 1.5 mg/kg IP; B) or NG-nitro-l-arginine methyl ester (L-NAME; 10 mg/kg IP; C) either alone or together with the broad-spectrum MMP blocker doxycycline (Dox). A through C, Bottom, Representative zymograms showing the effects of vasoconstrictors on vascular MMP-7 and MMP-2 activity in vivo (n=4 to 5 rats in each study group). MMP-7 zymography was conducted with casein used as substrate (this substrate was relatively more selective for the 25-kDa active form of MMP-7). MMP-2 zymography was conducted with gelatin as substrate. Sprague Dawley rats were administered PE (3 mg/kg IP), Ang II (1.5 mg/kg IP), or L-NAME (10 mg/kg IP) either alone or together with the broad-spectrum MMP blocker doxycycline (A, 120 mg/kg; B and C, 90 mg/kg). Vehicle was sterile PBS. *P<0.05 vs PBS. +P<0.05 vs agonist plus doxycycline (120 mg/kg; A). +P<0.05 vs agonist plus doxycycline (90 mg/kg; B, C). Results are mean±SEM of 4 to 5 rats in each study group.

Figure 2. Resistance to acute hypertension in mice lacking active MMP-7. A, Time course of systolic blood pressure in wild-type mice administered norepinephrine (NE; 1.5 mg/kg IP) alone or together with doxycycline (Dox; 90 mg/kg IP). *P<0.05 vs NE+Dox. Baseline refers to blood pressure before injection of norepinephrine (t=0). B, Protection from norepinephrine-induced acute hypertension in mice treated with MMP-7 antisense oligodeoxynucleotides (oligos; 0.6 mg · kg−1 · d−1 for 14 days) vs mice administered scrambled antisense oligodeoxynucleotides (0.6 mg · kg−1 · d−1 for 14 days). *P<0.05 vs scrambled oligodeoxynucleotides. C, Protection from norepinephrine-induced acute hypertension in MMP-7−/− mice. *P<0.05 vs wild-type. D, Effects of MMP-7 knockdown and MMP-7 gene knockout on resting blood pressure of mice. Left, Resting blood pressure of mice treated with MMP-7 antisense vs scrambled oligodeoxynucleotides for 14 days. Blood pressure at day 15. Right, Resting blood pressure of MMP-7−/− mice vs age-matched wild-type mice. E through G, Top, Time course of systolic blood pressure effects of norepinephrine (1.5 mg/kg IP), angiotensin II (Ang II; 1.5 mg/kg IP), or NG-nitro-l-arginine methyl ester (L-NAME; 15 mg/kg IP) in mice treated with MMP-7 antisense oligodeoxynucleotides (MMP-7 antisense; 0.6 mg · kg−1 · d−1 for 14 days) vs mice given PBS. E through G, Bottom, Quantitative analysis of the dose response for each vasoconstrictor. Baseline refers to blood pressure before injection of the vasoconstrictor (t=0). *P<0.05 vs PBS. Results are mean±SEM of 4 to 5 mice in each study group.

The involvement of MMPs in these experiments was suggested by effects of doxycycline, a broad-spectrum pharmacological inhibitor of MMP activity.20 Doxycycline (60 to 120 mg/kg IP) dose-dependently blocked the acute hypertensive responses to α-adrenergic agonists, angiotensin II, and L-NAME in rats (Figure 1, top panels) and mice (Figure 2A). High-performance liquid chromatography analyses indicated that doxycycline (90 mg/kg IP) resulted in plasma concentrations between 10−4 and 10−5 mol/L at 1 and 4 hours after IP injection, respectively (data not shown). These doxycycline concentrations are enough to relax small rat mesenteric arteries in isolation.6 When we examined arteries collected at a time point that coincided with the maximum elevation in systolic blood pressure induced by phenylephrine, angiotensin II, or L-NAME (ie, 4, 1, or 0.5 hours, respectively), the activity of vascular MMP-7 but not MMP-2 was elevated. The increase in MMP-7 activity was in all cases blocked by the coadministration of doxycycline (Figure 1, bottom panels).

We verified the link between MMP-7 expression and systemic blood pressure regulation in studies summarized in Figures 2A through 2C. In these studies, we examined both C57BL/6 mice in which the MMP-7 gene was disrupted by a neomycin resistance cassette to render them MMP-7−/− and wild-type C57BL/6 mice that were given MMP-7–specific antisense oligodeoxynucleotides (0.6 mg · kg−1 · d−1), scrambled (inactive) oligodeoxynucleotides (0.6 mg · kg−1 · d−1), or PBS for 14 days (through subcutaneous osmotic minipumps). The antisense sequence chosen for the present study was previously validated in vivo and has anticancer activity through the long-lasting knockdown of MMP-7.17 Antisense treatment resulted in a systemic downregulation of MMP-7 activity in various tissues, including aorta, heart, and small intestine (a tissue in which MMP-7 is normally expressed at very high levels21; supplemental Figure I).

Interestingly, resting systolic blood pressure was not significantly affected by doxycycline injections (data not shown), MMP-7 antisense oligodeoxynucleotides, or MMP-7 gene knockout (Figure 2D); however, mice that received MMP-7 antisense oligodeoxynucleotides displayed blunted acute hypertensive responses to norepinephrine, angiotensin II, and L-NAME (versus PBS and versus scrambled oligodeoxynucleotides; Figures 2B, 2E, 2F, and 2G). Similarly, MMP-7−/− mice showed attenuated acute responses to angiotensin II (data not shown) and norepinephrine (Figure 2C). Moreover, MMP-7−/− mice (but not wild-type mice) were resistant to chronic hypertension induced by repeated norepinephrine administration (Figure 3A). Isolated microperfused small mesenteric arteries from MMP-7−/− mice constricted less (versus wild-type mice) in response to luminally delivered boluses of the α-adrenergic agonist phenylephrine (0, 5, or 50 pmol per bolus; Figure 3B). Together, these in vivo and in vitro functional data strongly suggested that vasoconstrictors induce hypertension, at least in part, through the posttranscriptional activation of MMP-7.

Figure 3. Resistance to chronic hypertension in MMP-7−/− mice. A, MMP-7−/− and age-matched wild-type mice were injected with norepinephrine (NE; 1.5 mg/kg IP) once or twice daily for 9 days. Systolic blood pressure was measured on day 10 (ie, 24 hours after last injection of norepinephrine). *P<0.05 vs baseline. Results are mean±SEM of 4 to 5 mice in each study group. B, Attenuation of α1-adrenergic contractile responses in isolated small mesenteric arteries from MMP-7−/− vs wild-type mice. Representative traces of contractile response of small mesenteric arteries from wild-type mice and MMP-7−/− mice to increasing doses of the α1-adrenergic agonist phenylephrine. Small mesenteric arteries from mice were mounted on a microperfusion arteriograph (perfusion flow rate of 1 μL/min). To induce contraction of the arteries, the indicated amounts of phenylephrine (PE, in 5 μL) were injected in the line toward the artery. Unlike wild-type mice, MMP-7−/− mice had attenuated responses to PE (5 pmol). Traces are representative of 3 mice in each study group.

MMP-7 as a Mediator of Hypertension and Cardiac Hypertrophy in SHRs

We next examined whether blocking MMP-7 expression would decrease the systolic blood pressure of SHRs, a genetic model in which hypertension is caused by multiple mechanisms, including endothelial dysfunction and upregulated activities of catecholamines (ie, sympathetic system) and angiotensin II.22–24Figure 4 illustrates results obtained in already-hypertensive 22-week-old SHRs when we targeted the MMP-7 gene by RNA interference using an siRNA against the same mRNA sequence targeted by the MMP-7 antisense oligodeoxynucleotides (for an alignment of the sequences, please see supplemental Figure II). MMP-7 siRNA treatment significantly decreased the systolic blood pressure, producing an attenuation of the hypertension that lasted beyond the window of siRNA delivery (Figure 4A). The antihypertensive effects of MMP-7 siRNA treatment were associated with a significant decrease in MMP-7 activity in resistance arteries (Figure 4B). Interestingly, MMP-7 siRNA treatment stopped the progression of cardiac hypertrophy (Figure 5A and 5B; Table) in association with a downregulation of myocardial MMP-7 (Figure 5C). MMP-7 siRNA also decreased the number of nuclei/area unit in histological sections of hearts from treated SHRs, which further confirmed the prevention of cardiac hypertrophy (data not shown). Comparative gross pathology further revealed that treatment with MMP-7 siRNA resulted in an approximately 50% reduction in cardiac hypertrophy versus SHRs given PBS and versus untreated normotensive age- matched WKY rats: heart weight/body weight×1000 (WKY)=3.40±0.01, heart weight/body weight×1000 (SHR+MMP-7 siRNA)=3.99±0.05, heart weight/body weight(SHR+PBS)×1000=4.43±0.09; n=3 for WKY, n=4 for both SHR+PBS and SHR+MMP-7 siRNA. We excluded a major contribution of the inflammatory response in these antihypertensive and antihypertrophy effects of MMP-7 siRNA because we did not observe significantly elevated interferon-γ levels in plasma or in the left ventricle of the rats (supplemental Figure III, SHR).

Figure 4. MMP-7 as a mediator of hypertension in SHR. A, Time course of systolic blood pressure of already-hypertensive SHRs treated with PBS or siRNA to MMP-7 (n=4). Treatment 1: The rats were infused with either MMP-7 siRNA or PBS through minipumps for 14 days. Treatment 2: On day 23, treatment was restarted by implantation of new minipumps containing either MMP-7 siRNA or PBS. No treatment indicates period between treatments, ie, between day 15 and day 23, showing that protection lasted beyond the 14-day-window of siRNA delivery by the osmotic minipumps. B, Quantitative analysis indicates that in small (resistance) mesenteric arteries, MMP-7 activity was knocked down by siRNAs to MMP-7, as determined by substrate zymography. *P<0.05 vs PBS group. Results are mean±SEM of 3 to 4 rats in each group.

Figure 5. MMP-7 as a mediator of cardiac hypertrophy in SHR. A, Cardiac hypertrophy in SHRs treated with siRNA or PBS. Although the ratio of corrected left ventricle mass (corr. LV mass) to body weight (BW) increased over time in untreated rats, this ratio did not increase in rats that received siRNA. Time axis indicates when M-mode echocardiography analysis was conducted. B, Gross pathology analysis (conducted on day 41) indicated a significantly decreased heart weight (HW) to body weight (BW) ratio. C, Quantitative analysis and representative traces of zymography and Western immunoblotting indicating knockdown of MMP-7 expression. Zymography on casein gels was relatively selective for MMP-7 active form (25 kDa). Western blots show all immunoreactive bands detected with commercially available antibodies to MMP-7. *P<0.05 vs PBS group. Results are mean±SEM of 3 to 4 rats in each group.

Table. Involvement of MMP-7 in the Development of Cardiac Hypertrophy in the SHR Model: Morphometric, Hemodynamic, and Echocardiographic Results

Day 40PBSMMP-7 siRNA
IVS indicates interventricular septum; LVID, left ventricular inner diameter; and LVPW, left ventricular posterior wall dimension.
Results are mean±SEM. n=4 rats per group.
*P<0.05 vs PBS. Multiple ANOVA with Scheffé’s test.
IVS at diastole, mm1.90±0.031.57±0.05*
LVID at diastole, mm8.36±0.088.63±0.17
LVPW at diastole, mm1.74±0.041.60±0.03*
Ejection fraction, %65.73±1.1066.06±1.13
Heart rate, bpm353.7±14.8347.7±11.7
Body weight, g360.0±7.4363.7±9.9

MMP-7/ADAM-12 Signaling Axis

Administration of MMP-7 siRNA (0.4 mg · kg−1 · d−1) for 14 days resulted in a significant downregulation in myocardial MMP-7 mRNA levels in mice (Figure 6A). Interestingly, MMP-7 siRNA inhibited ADAM-12 transcription (Figure 6B) but had otherwise insignificant effects on other genes, including α-skeletal actin, TACE, TIMP-2 (tissue inhibitor of metalloproteinase-2), and MMP-9 (Figure 6C and 6D) and on interferon-γ levels (supplemental Figure III). Like the MMP-7 siRNA, MMP-7 gene knockout resulted in decreased levels of myocardial ADAM-12 mRNA (but normal levels of TACE; Figure 6E). Mice that received MMP-7 siRNA displayed no morphometric or echocardiographic abnormalities (supplemental Table).

Figure 6. Transcriptional relationships between MMP-7, ADAM-12, and hypertrophy marker genes define novel signaling pathways under basal conditions vs sustained agonist stimulation. A through D, Quantitative analysis of mRNA expression levels of indicated genes. Mice were administered either PBS or MMP-7 siRNA (0.4 mg · kg−1 · d−1 for 14 days) through a first osmotic minipump followed by the administration of either PBS (ie, basal conditions) or angiotensin II (1.4 mg · kg−1 · d−1, for 10 days, ie, from day 5 to day 15; sustained agonist stimulation) through a second osmotic minipump. The mice were euthanized on day 16, and mRNA levels were measured in myocardial tissue (left ventricle) by quantitative reverse-transcription polymerase chain reaction with TaqMan probes. A, B, Analysis of MMP-7 and ADAM-12 expression. C, Analysis of hypertrophy marker genes: β-myosin heavy chain (MHC), brain natriuretic peptide (BNP), and α skeletal actin (alpha-sk-actin). Mice were administered either PBS or MMP-7 siRNA (0.4 mg · kg−1 · d−1 for 14 days) through a first osmotic minipump followed by the administration of either PBS (ie, basal conditions) or angiotensin II (1.4 mg · kg−1 · d−1, for 10 days, ie, from day 5 to day 15; sustained agonist stimulation) through a second osmotic minipump. The mice were euthanized on day 16, and mRNA levels were measured in myocardial tissue (left ventricle) by quantitative reverse-transcription polymerase chain reaction with TaqMan probes. D, Examples of genes in which mRNA expression levels were unaltered by administration of siRNA to MMP-7. Mice were administered either PBS (ie, basal conditions) or MMP-7 siRNA (0.4 mg · kg−1 · d−1 for 14 days). The mice were euthanized on day 16, and mRNA levels were measured in myocardial tissue (left ventricle) by quantitative reverse-transcription polymerase chain reaction with TaqMan probes. E, Quantitative analysis of mRNA expression levels of TACE and ADAM-12 genes in MMP-7−/− mice vs age-matched wild-type mice. F, Pretreatment with MMP-7 siRNA before angiotensin II infusion significantly attenuated angiotensin II–induced hypertension in mice. The mice received a first minipump that delivered either vehicle (PBS) or siRNA (0.4 mg · kg−1 · d−1 for 14 days). On day 5, the mice were implanted with a second minipump loaded with either PBS or angiotensin II (1.4 mg · kg−1 · d−1). G, Pretreatment with MMP-7 siRNA prevented angiotensin II–induced left ventricular hypertrophy. Left, Ratio of corrected left ventricle mass (corr. LV mass; measured by M-mode echocardiography) to body weight (BW). Echocardiographic analysis was conducted 10 days after implantation of a second osmotic minipump (ie, on day 15). Right, Heart weight (HW) to body weight (BW) ratio. Mice were euthanized on day 16. *P<0.05 vs PBS. +P<0.05 vs (PBS+Ang II). Results are mean±SEM of 4 mice in each study group. Ang II indicates angiotensin II.

Mice given angiotensin II (1.4 mg · kg−1 · d−1 for 10 days) displayed hypertension and left ventricular hypertrophy (Figure 6F and 6G; supplemental Table). Interestingly, continuous angiotensin II infusion inhibited MMP-7 transcription (Figure 6A) but increased transcription of ADAM-12 and hypertrophy marker genes (β-myosin heavy chain, brain natriuretic peptide, and α-skeletal actin; Figure 6B and 6C). Pretreatment with MMP-7 siRNA attenuated angiotensin II–induced hypertension (as expected from studies in Figures 1 through 4), inhibited the angiotensin II–induced overexpression of both ADAM-12 and hypertrophy marker genes (Figure 6B and 6C), and prevented left ventricular hypertrophy (Figure 6F and 6G; supplemental Table). Supporting these observations, MMP-7−/− mice (but not age-matched wild-type mice) exhibited resistance to hypertension (Figure 3A), cardiac hypertrophy (supplemental Figure IVA), and the transactivation of cardiac growth factor receptors, which are purported mediators of agonist-activated ADAM-125 (supplemental Figure IVB).

Discussion

This investigation has resulted in 3 interrelated discoveries: (1) To the best of our knowledge, the present findings suggest for the first time that agonist signaling of both hypertension and cardiac hypertrophy depends on MMP-7 gene expression and activity. (2) We revealed a novel transcriptional link between MMP-7 and ADAM-12, the major disintegrin metalloproteinase implicated in the development of cardiac hypertrophy. (3) We have shown that disrupting the MMP-7/ADAM-12 axis at the level of MMP-7 protects against development of both cardiac hypertrophy and hypertension in simple models (such as mice infused with angiotensin II) and in a complex model (SHR). Thus, targeting the MMP-7/ADAM-12 axis (eg, at the level of MMP-7) could have general therapeutic potential in multiple hypertensive disorders caused by multiple or unknown agonists.

Prior to the present study, many characterizations of MMP-7 in vivo related to cancer25 or the innate immune response,21 with the exception of a few recent studies, including one that showed a novel interaction between MMP-7 and connexin-43 in cardiac failure.26 Our laboratory had proposed a role for MMP-7 in agonist-induced vasoconstriction of isolated arteries on the basis of broad-spectrum pharmacological inhibitor data6; however, none of our previous studies could establish its mediator role in agonist-induced hypertension nor its novel involvement in cardiac hypertrophy, a process that invariably develops subsequently to sustained vasoconstrictive agonist stimulation. Prior to this research, MMP-7 and ADAM-12 had been studied separately5–7,11–13; however, these separate studies suggested their involvement in cardiovascular hypertrophy processes through a common pathway. Accordingly, an overabundance of vasoconstrictor agonists (as occurs in hypertensive disorders) would enhance their activity through posttranscriptional pathways. Next, the activated MMP-7 and ADAM-12 would cleave and release substrates, including growth factors and inflammatory mediators (such as HB-EGF, transforming growth factor-α, and tumor necrosis factor-α). These mediators then trigger the mitogen-activated protein kinase cascade to promote cardiovascular hypertrophy through the transcriptional activation of immediate-early genes and fetal genes, often referred to as hypertrophy marker genes5–7,11–13 (Figure 7, module 1).

Figure 7. Proposed modules and network structure of the metalloproteinase signaling pathways. The diagram can explain the apparent functional redundancy of multiple metalloproteinases in the signaling of hypertension and hypertrophy processes. Module 1 derives from previous work by many groups.5–7,11–13 Activation of vasoconstrictive GqPCRs by cognate agonists posttranscriptionally induces the activity of metalloproteinases such as MMP-7 and ADAM-12. The activated metalloproteinases next transactivate growth factor receptor signaling to trigger the mitogen-activated protein kinase (MAPK) cascade. Module 2 illustrates transcriptional relationships between MMP-7 and ADAM-12 under basal conditions, which is fully supported by the quantitative reverse-transcription polymerase chain reaction data (Figure 6). Module 3 is an interpretation that can explain how and why sustained GqPCR agonist stimulation increases signaling through ADAM-12 while decreasing signaling through MMP-7. Accordingly, the high expression of ADAM-12 observed under sustained agonist stimulation acts in a negative feedback loop to inhibit MMP-7 transcription (Figure 6). The integration of the 3 modules can explain the complexity of metalloproteinase signaling during development of hypertension and hypertrophy processes. Sustained agonist stimulation regulates the development and progression of hypertension and hypertrophy processes through posttranscriptional (short-term) and transcriptional (long-term) mechanisms involving metalloproteinases such as MMP-7 and ADAM-12.5–7,11–13 The early signaling events by which vasoconstrictors trigger acute hypertension depend on MMP-7, but over time, signaling becomes increasingly dependent on expression of other metalloproteinases, including ADAM-12. Inhibition of MMP-7 transcription by ADAM-12 may be a compensatory mechanism to counter the development of hypertension and hypertrophy processes. Ang II indicates angiotensin II.

The data suggest that MMP-7 and ADAM-12 are connected in agonist-induced posttranscriptional and transcriptional events that may ultimately result in the development of hypertension and cardiovascular hypertrophy. We have further revealed novel hierarchical relationships between these metalloproteinases and observed that these relationships are dynamic, because they differ under basal conditions (Figure 7, module 2) versus agonist stimulation (Figure 7, module 3). Under basal conditions, MMP-7 transcriptionally controls the expression of ADAM-12 and downstream hypertrophy marker genes; however, under sustained agonist stimulation, MMP-7 mRNA levels and thereby the contribution of MMP-7 to signaling may decrease, whereas the expression and thereby the contribution of ADAM-12 to signaling may increase. We thus propose the following: (1) MMP-7 may mediate the early posttranscriptional events by which vasoconstrictor agonists trigger an acute elevation of blood pressure (in the short term) and the development of cardiovascular hypertrophy (which is a long-term process). (2) Under sustained agonist stimulation, the overexpression of ADAM-12 may act to inhibit MMP-7 transcription (in a negative feedback loop) while increasing transcription of hypertrophy marker genes. (3) The inhibition of MMP-7 transcription by sustained agonist stimulation may represent a novel physiological compensatory mechanism to counter hypertension and hypertrophy processes.

The therapeutic potential of disrupting the MMP-7/ADAM-12 axis at the level of MMP-7 was evidenced by studies in both mice with agonist-induced hypertension and SHR, a model in which hypertension has multiple or poorly understood causes.22–24 The present data clearly showed that blocking MMP-7 expression could be valuable for attenuating hypertension and preventing the development of cardiac hypertrophy.

Limitations and Future Studies

Although quantitative reverse-transcription polymerase chain reaction provided a reliable, highly sensitive, and quantitative tool, metalloproteinase quantitation by other complementary means remains challenging for various reasons. First, MMP-7 and ADAM-12 genes have very low tissue expression (particularly in the left ventricle). Second, commercial antibodies to these proteins have poor sensitivity or cross-react with many bands on Western immunoblotting, which hampers their unambiguous quantitation. Finally, activity-based determinations are potentially nonspecific and may favor the detection of the more active forms of these metalloproteinases, thus introducing a quantitation bias.

That vasoconstrictors signal through mutually regulated metalloproteinases (and not just through isolated metalloproteinases) is a novel observation that integrates and substantially expands previous research.5–7 This notion is in complete agreement with a previous investigation that detected a differential involvement of multiple metalloproteinases in various forms of cardiomyopathy, including hypertrophic obstructive cardiomyopathy and dilated cardiopathy in humans.27 Future studies should further dissect the dynamics of the metalloproteinase networks that may operate in various models of hypertension and cardiac hypertrophy and in different stages of the development of the disease. Such studies might enable the design of general treatments for hypertensive disorders with complex or unknown causes, such as preeclampsia, which complicates 5% of all pregnancies worldwide,28 and essential hypertension, which affects 25% of the adult population in developed countries.1

Sources of Funding

This work was supported by research grants of the Natural Sciences and Engineering Council (NSERC) and the Canadian Institutes of Health Research (CIHR) to Dr Fernandez-Patron, who is also a CIHR and Heart and Stroke Foundation of Canada New Investigator. This work was also supported by CIHR research grants to Drs Kassiri and Lopaschuk.

Disclosures

None.

Footnotes

Correspondence to Dr Carlos Fernandez-Patron, Department of Biochemistry, 3-19 Medical Sciences Bldg, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. E-mail

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circulationahaCirculationCirculationCirculation0009-73221524-4539Lippincott Williams & Wilkins
CLINICAL PERSPECTIVE12052009

Excessive stimulation of Gq protein–coupled receptors by cognate vasoconstrictor agonists induces a variety of cardiovascular processes, including hypertension and hypertrophy. Here, we observed that matrix metalloproteinase-7 and a disintegrin and metalloproteinase-12 (ADAM-12) may form a novel signaling axis in these processes. We suggest further that targeting the matrix metalloproteinase-7/ADAM-12 axis (eg, at the level of matrix metalloproteinase-7) with RNA interference–based approaches could have general therapeutic potential in multiple hypertensive disorders caused by multiple or unknown agonists.

The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.835488/DC1.

*The first 2 authors contributed equally to this work.

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