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.
Received August 28, 2008; accepted January 9, 2009.
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.
Please see the online Data Supplement for the expanded Methods section.
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.
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.
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).
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.
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.22–24 Figure 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).
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).
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).
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).
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.
Biaggioni I. Should we target the sympathetic nervous system in the treatment of obesity-associated hypertension? Hypertension. 2008; 51: 168–171.
Fernandez-Patron C. Therapeutic potential of the epidermal growth factor receptor transactivation in hypertension: a convergent signaling pathway of vascular tone, oxidative stress, and hypertrophic growth downstream of vasoactive G-protein-coupled receptors? Can J Physiol Pharmacol. 2007; 85: 97–104.
Asakura M, Kitakaze M, Takashima S, Liao Y, Ishikura F, Yoshinaka T, Ohmoto H, Node K, Yoshino K, Ishiguro H, Asanuma H, Sanada S, Matsumura Y, Takeda H, Beppu S, Tada M, Hori M, Higashiyama S. Cardiac hypertrophy is inhibited by antagonism of ADAM12 processing of HB-EGF: metalloproteinase inhibitors as a new therapy. Nat Med. 2002; 8: 35–40.
Hao L, Du M, Lopez-Campistrous A, Fernandez-Patron C. Agonist-induced activation of matrix metalloproteinase-7 promotes vasoconstriction through the epidermal growth factor-receptor pathway. Circ Res. 2004; 94: 68–76.
Ohtsu H, Dempsey PJ, Frank GD, Brailoiu E, Higuchi S, Suzuki H, Nakashima H, Eguchi K, Eguchi S. ADAM17 mediates epidermal growth factor receptor transactivation and vascular smooth muscle cell hypertrophy induced by angiotensin II. Arterioscler Thromb Vasc Biol. 2006; 26: e133–e137.
Sunnarborg SW, Hinkle CL, Stevenson M, Russell WE, Raska CS, Peschon JJ, Castner BJ, Gerhart MJ, Paxton RJ, Black RA, Lee DC. Tumor necrosis factor-alpha converting enzyme (TACE) regulates epidermal growth factor receptor ligand availability. J Biol Chem. 2002; 277: 12838–12845.
Wilson CL, Heppner KJ, Labosky PA, Hogan BL, Matrisian LM. Intestinal tumorigenesis is suppressed in mice lacking the metalloproteinase matrilysin. Proc Natl Acad Sci U S A. 1997; 94: 1402–1407.
Wilson CL, Ouellette AJ, Satchell DP, Ayabe T, Lopez-Boado YS, Stratman JL, Hultgren SJ, Matrisian LM, Parks WC. Regulation of intestinal alpha-defensin activation by the metalloproteinase matrilysin in innate host defense. Science. 1999; 286: 113–117.
Zhang YC, Bui JD, Shen L, Phillips MI. Antisense inhibition of beta(1)-adrenergic receptor mRNA in a single dose produces a profound and prolonged reduction in high blood pressure in spontaneously hypertensive rats. Circulation. 2000; 101: 682–688.
Nava E, Noll G, Luscher TF. Increased activity of constitutive nitric oxide synthase in cardiac endothelium in spontaneous hypertension. Circulation. 1995; 91: 2310–2313.
Kawasaki H, Cline WH Jr, Su C. Involvement of the vascular renin-angiotensin system in beta adrenergic receptor-mediated facilitation of vascular neurotransmission in spontaneously hypertensive rats. J Pharmacol Exp Ther. 1984; 231: 23–32.
Lindsey ML, Escobar GP, Mukherjee R, Goshorn DK, Sheats NJ, Bruce JA, Mains IM, Hendrick JK, Hewett KW, Gourdie RG, Matrisian LM, Spinale FG. Matrix metalloproteinase-7 affects connexin-43 levels, electrical conduction, and survival after myocardial infarction. Circulation. 2006; 113: 2919–2928.
Fedak PW, Moravec CS, McCarthy PM, Altamentova SM, Wong AP, Skrtic M, Verma S, Weisel RD, Li RK. Altered expression of disintegrin metalloproteinases and their inhibitor in human dilated cardiomyopathy. Circulation. 2006; 113: 238–245.
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.