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Am J Physiol Heart Circ Physiol 292: H684-H693, 2007. First published September 15, 2006; doi:10.1152/ajpheart.00538.2006
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Induction of matrix metalloproteinase-2 enhances systemic arterial contraction after hypoxia

Jeff Z. He,1,2 Adrian Quan,1 Yi Xu,3 Hwee Teoh,1 Guilin Wang,1 Jason E. Fish,4 Brent M. Steer,4 Shigeyoshi Itohara,5 Philip A. Marsden,4 Sandra T. Davidge,3 and Michael E. Ward1,2

1Terrence Donnelly Laboratories, Division of Respirology and Department of Critical Care, St. Michael's Hospital and University of Toronto, Toronto, Ontario, Canada; 2Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; 3Departments of Obstetrics/Gynaecology and Physiology, University of Alberta, Edmonton, Alberta, Canada; 4Department of Medicine, St. Michael's Hospital and University of Toronto, Toronto, Ontario, Canada; and 5Laboratory for Behavioural Genetics, Riken Brain Science Institute, Wako City, Japan

Submitted 25 May 2006 ; accepted in final form 11 September 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was carried out to determine the role of increased vascular matrix metalloproteinase-2 (MMP-2) expression in the changes in systemic arterial contraction after prolonged hypoxia. Rats and mice were exposed to hypoxia (10% and 8% O2, respectively) or normoxia (21% O2) for 16 h, 48 h, or 7 days. Aortae and mesenteric arteries were either mounted in organ bath myographs or frozen in liquid nitrogen. MMP-2 inhibition with cyclic CTTHWGFTLC (CTT) reduced contraction to phenylephrine (PE) in aortae and mesenteric arteries from rats exposed to hypoxia for 7 days but not in vessels from normoxic rats. Similarly, CTT reduced contraction to Big endothelin-1 (Big ET-1) in aortae from rats exposed to hypoxia for 7 days. Responses to PE were reduced in hypoxic MMP-2–/– mice compared with MMP-2+/+ mice. Increased contraction to Big ET-1 after hypoxia was observed in MMP-2+/+ mice but not in MMP-2–/– mice. Rat aortic MMP-2 and membrane type 1 (MT1)-MMP protein levels and MMP activity were increased after 7 days of hypoxia. Rat aortic MMP-2 and MT1-MMP mRNA levels were increased in the deep medial vascular smooth muscle. We conclude that hypoxic induction of MMP-2 expression potentiates contraction in systemic conduit and resistance arteries. This may preserve the capacity to regulate the systemic circulation in the transition between the alterations in vascular tone and structural remodeling that occurs during prolonged hypoxic epochs.

vascular smooth muscle; endothelium; endothelin; vascular remodeling

HYPOXIA IS FREQUENTLY OBSERVED in patients with cardiopulmonary diseases and in normal subjects at high altitude. Studies done to date have primarily focused on the short-term systemic circulatory responses that redistribute blood flow (26) and enhance the capacity for oxygen extraction (8) in these conditions. As the duration of hypoxia increases, however, systemic vascular smooth muscle and endothelial function are impaired, limiting the efficacy of the acute responses (1, 47–49), whereas concurrent structural remodeling plays an increasing role in maintaining the balance between oxygen delivery and metabolic demand (26). Although the clinical and physiological relevance of these longer-term effects on vascular function is being increasingly recognized (39), the mechanisms that mediate them remain unknown.

Matrix metalloproteinase-2 (MMP-2) is a zinc-dependent proteinase secreted by both endothelial and smooth muscle cells, and its expression is increased in regions of matrix turnover and remodeling (7). Recently, it was discovered that MMP-2 can mediate the posttranslational modification of several vasoactive peptides (15, 16, 29), suggesting that it has a vasoregulatory role as well. MMP-2 protein and mRNA levels are increased after hypoxic incubation in endothelial cells (4). Its activating protease, membrane type 1-matrix metalloproteinase (MT1-MMP), and its endogenous inhibitors, tissue inhibitors of matrix metalloproteinase (TIMPs), are also oxygen regulated in some cell types (33, 36, 38). If hypoxia induces a functionally significant increase in MMP-2 expression in systemic arteries, modulation of MMP-2 activity may, in addition to mediating structural adaptations, contribute to the changes in vascular tone that occur during prolonged reductions in oxygen delivery. This study was, therefore, carried out to determine the role that altered MMP-2 expression and activity play in the changes in contractile responses observed in systemic conduit and resistance arteries after prolonged hypoxia in vivo.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Exposure to Hypoxia

All protocols were in compliance with standards set by the Canadian Council on Animal Care and were approved by the Institutional Animal Care Committee. Male Sprague-Dawley rats (200–250 g) and C56/B16J mice (20–25 g) were placed in a Plexiglas chamber into which the flow of air and nitrogen was controlled independently. In preliminary experiments, the arterial PO2 averaged 38 Torr (range 35–42 Torr) in rats breathing a gas mixture containing 10% O2 (1) and 38.1 Torr (range 35–40 Torr) in mice breathing 8% O2.

Rats and mice exposed to hypoxia breathed gas mixtures containing 10% or 8% oxygen, respectively, for 16 h, 48 h, or 7 days. Normoxic control animals breathed room air under otherwise identical conditions. At the end of the exposure period, rats were decapitated and mice were euthanized by cervical dislocation. Thoracic aortae were excised, cut into 4-mm segments and mounted in tissue bath myographs (Radnoti Glass Technology), frozen in liquid nitrogen, or fixed in 10% paraformaldehyde. Rat mesenteric arteries (100–200 µm internal diameter) were either mounted in wire myographs (Living Systems) or frozen in liquid nitrogen for later protein extraction.

Chemicals and Antibodies

The cyclic peptide MMP-2/9 antagonist CTTHWGFTLC (CTT) was purchased from Calbiochem. Polyclonal MMP-2- and TIMPs-1–3-specific antibodies and monoclonal MT1-MMP-specific antibody were purchased from Chemicon. Polyclonal TIMP-4-specific antibody was obtained from Biomol Research. Mca-RPPGFSAFK(Dnp)-OH and Mca-PLGL-Dpa-AR-NH were from R&D Systems. Endothelin-converting enzyme 1 (ECE-1)-specific polyclonal antibody was from Zymed. Histostain and PicoPure RNA Isolation Kit were purchased from Arcturus Bioscience. SYBR green universal master mix was from Applied Biosystems. Primers and Superscript II were from Invitrogen. All other reagents were from Sigma.

Rat Studies

Aortic and mesenteric artery contractile responses. Rat aortic segments were mounted in tissue bath myographs, and mesenteric arterial segments were mounted in wire myographs containing Krebs-Henseleit solution (KHS) composed of (in mmol/l) 120 NaCl, 25 NaHCO3, 11.1 glucose, 4.76 KCl, 1.18 MgSO4, 1.18 KH2PO4, and 2.5 CaCl2, aerated with 95% O2-5% CO2 at 37°C. Thoracic aortae and mesenteric arteries were equilibrated in warmed aerated KHS for 1 h under a resting tension of 2 g or 500 mg, respectively, before drug-induced changes in tension were monitored. When necessary, the endothelium was removed by gentle abrasion of the luminal surface. The failure of acetylcholine (1 µmol/l) to elicit relaxation after contraction with phenylephrine (PE; 1 µmol/l) was taken as evidence of functional endothelial ablation (47).

Redistribution of blood flow during hypoxia is mediated by neurohumoral stimulation of {alpha}-adrenoceptors (9). Cumulative concentration-response curves (CRCs) for the {alpha}1-adrenoceptor agonist PE (1 nmol/l to 10 µmol/l) were, therefore, generated in endothelium-intact aortic rings. MMP-2 cleaves Big endothelin-1 (ET-1) to release the potent vasoconstrictor ET-1-[1–32] (15). Since Big-ET-1 itself has minimal biological activity before proteolytic activation and since ECE-1 protein levels and ECE activity are unchanged after hypoxia, the contractile response to Big ET-1 was used as a bioassay for changes in vascular MMP-2 activity. To eliminate the confounding influence of endogenous endothelium-derived ET-1 (15), CRCs for rat Big ET-1 (1 nmol/l to 300 nmol/l) were generated in endothelium-denuded aortic rings from normoxic rats and rats exposed to hypoxia for 7 days after 45-min incubation with and in the continuous presence of CTT (30 µmol/l) or vehicle.

To assess the effect of hypoxia on contractile response of resistance vessels, CRCs for PE (10 nmol/l to 100 µmol/l) were generated in mesenteric arteries from rats exposed to normoxia or hypoxia for 7 days after 30-min incubation with and in the continuous presence of CTT (10 µmol/l) or vehicle.

Immunohistochemistry. Paraffin-embedded sections (5 µm) of rat aortae from normoxic rats and rats exposed to hypoxia for 7 days were analyzed using MMP-2-specific polyclonal antibodies as previously described (47). Preabsorption of the MMP-2 antibody with MMP-2 positive control peptide markedly inhibited or eliminated staining (data not shown). Slides processed in the identical manner, except incubated with nonspecific rabbit IgG instead of primary antibody, served as negative controls.

Western blots. Thoracic aortic proteins from rats exposed to normoxia or to hypoxia for 16 h, 48 h, and 7 days and mesenteric artery proteins from rats exposed to normoxia or to hypoxia for 7 days were extracted in 1% SDS, 0.001 mol/l sodium orthovanadate, and 0.01 mol/l Tris (pH 7.4). After protein concentrations in aortic and mesenteric arterial extracts were determined by the Lowry method, total proteins (60 µg for MMP-2 and MT1-MMP, 40 µg for TIMPs-1–4, and 100 µg for ECE-1) were resolved by 4–12% SDS-PAGE (Helixx Technologies) and transferred to nitrocellulose. MT1-MMP membranes were blocked in 3% milk-0.1% Tween Tris-buffered saline (TTBS). All other membranes were blocked in 5% milk-TTBS. Membranes were then incubated for 3 h at room temperature with goat polyclonal anti-MMP-2 (1:100); 1 h at room temperature with rabbit polyclonal anti-TIMP-1 (1:2,500) or anti-TIMP-4 (1:10,000); or overnight at 4°C with rabbit polyclonal anti-ECE-1 (1:400), anti-TIMP-2 (1:2,500), anti-TIMP-3 (1:2,500), or mouse monoclonal anti-MT1-MMP (1:400). Immunoblots were probed with horseradish peroxidase (HRP)-donkey anti-goat IgG (1:4,000 for MMP-2) or HRP-anti-rabbit IgG (1:4,000 for ECE-1, TIMP-1, TIMP-2, TIMP-3, and TIMP-4) and visualized by enhanced chemiluminescence (Amersham Biosciences). HRP-goat anti-mouse IgG (1:1,000) was used to probe for MT1-MMP, and the resulting bands were visualized by chemiluminescence (Sigma). Bands were quantified by densitometry. Samples from normoxic and hypoxic groups were paired on each gel to control for interexperimental variation. Protein loading and transfer efficiency were corroborated following transfer by using full-lane densitometry of the Ponceau red-stained membranes.

Gelatin zymography. Aortae from normoxic rats and rats exposed to hypoxia for 16 h, 48 h, and 7 days were extracted with 10 mmol/l Tris·HCl (pH 7.5) extraction buffer. Zymography was performed using 7.5% SDS-PAGE with copolymerized gelatin (2 mg/ml) as substrate. At the end of each run, gels were washed with 2.5% Triton X-100 and incubated for 48 h in an enzyme assay buffer (50 mmol/l Tris, pH 7.0, 5 mmol/l CaCl2, 0.15 mol/l NaCl, and 0.05% Na3N) to allow for the development of enzyme activity bands. Gels were stained with 0.05% Coomassie brilliant blue G-250 in a mixture of methanol: acetic acid: water (2.5:1:6.5) and destained in 4% methanol with 8% acetic acid. The gelatinolytic activity was detected as transparent bands against the background of Coomassie brilliant blue-stained gelatin. Gels were scanned using Fluor-S Multi-Imager (Bio-Rad) and analyzed for pro-MMP-2 and activated MMP-2 (72- and 64-kDa bands, respectively).

MMP and ECE activity. To further ensure that the change in the response to Big ET-1 was not attributable to a change in ECE activity, total MMP and ECE activity was measured in aortae from normoxic rats and rats exposed to hypoxia for 7 days. Thoracic aortic proteins (50 µg) from normoxic rats and rats exposed to hypoxia for 7 days were incubated for 1 h at 37°C with either 20 µmol/l of fluorogenic MMP substrate Mca-PLGL-Dpa-AR-NH (24) in 100 µl of reaction mixture (pH 7.5) composed of (in mmol/l) 50 Tris·HCl, 150 NaCl, and 1 CaCl2 or with 20 µmol/l of fluorogenic ECE substrate Mca-RPPGFSAFK(Dnp)-OH (22) in 100 µl of reaction mixture (pH 6.0) composed of (in mmol/l) 100 MES and 200 NaCl. Blanks containing the substrate dissolved in assay buffer were analyzed in parallel. Increases in fluorescence as a result of substrate cleavage were continuously measured using a fluorescence plate reader (Thermo Labsystems). Samples were run in triplicate, and final values were derived by subtracting the blank reading from the raw data.

Aortic MMP-2, MT1-MMP, MMP-7, and TIMPs-1–4 mRNA levels. Total aortic RNA was isolated as previously described (47). In addition, pure populations of aortic endothelial cells were isolated from aortae of rats exposed to normoxia or hypoxia for 7 days using the Hautchen technique (37). Pure cell populations of vascular smooth muscle cells (VSMC) from immediately below the endothelial cell layer (subendothelial VSMC) or from deep within the media of the vessel (deep medial VSMC; GoGoGoGoGoGoFig. 7) were obtained using the PixCell II Laser Capture Microdissection System according to the manufacturer's instructions, and RNA was extracted using the PicoPure RNA Isolation Kit. Aortic levels of specific mRNAs were measured by quantitative real-time RT-PCR [model ABI Prism 7900 HT, Applied Biosystems and the SYBR Green detection system (47)] using the following primers: MMP-2 (sense 5'-ACA CTG GGA CCT GTC ACT CC-3'; antisense 5'-ACA CGG CAT CAA TCT TTT CC-3'); MT1-MMP (sense 5'-TCC TGC TCC CCC TGC TCA CG-3'; antisense 5'-GTG ACT GGG GTG AGC GTT GTG T-3'); TIMP-1 (sense 5'-GGA TAT GTC CAC AAG TCC CAG AAC C-3'; antisense 5'-TTA TGC CAG GGA ACC AGG AAG C-3'); TIMP-2 (sense 5'-GGC CAA AGC AGT GAG CGA GAA-3'; antisense 5'-GGA GGG GGC CGT GTA GAT AAA T-3'); TIMP-3 (sense 5'-CCC TTT GGC ACT CTG GTC TAC ACT A-3'; antisense 5'-AGG CCA CAG AGA CTT TCA GAG GCT-3'); TIMP-4 (sense 5'-TAC ACG CCA TTT GAC TCT TCT CTC TG-3'; antisense 5'-CCT CCC AGG GCT CAA TGT AGT TG-3'); and MMP-7 (sense 5'-TGG GTC TGG GTC ACT CTT CTG TTC-3'; antisense 5'-TCA GGA AGG GCG TTT GCT CAT T-3'). The 18S (sense 5'-GAC GAT CAG ATA CCG TCG TAG TTC-3', antisense 5'-GTT TCA GCT TTG CAA CCA TAC TCC-3') and TATA binding protein (sense 5'-CCC CTA TCA CTC CTG CCA CAC C-3', antisense 5'-CGC AGT TGT TCG TGG CTC TCT T-3') transcripts were used as control genes for normalization, and the average change in the target gene with respect to 18S and TATA binding protein was determined.


Figure 1
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  		Fig. 1. A: concentration-response relationships for phenylephrine (PE) in the presence and absence of the matrix metalloproteinase-2/9 (MMP-2/9) inhibitor CTTHWGFTLC (CTT, 30 µmol/l) in endothelium-intact aortic rings from normoxic rats and rats exposed to hypoxia for 7 days (n = 8 per group). B: concentration-response relationships for PE in the presence and absence of CTT (10 µmol/l) in endothelium-intact mesenteric arteries from normoxic rats and rats exposed to hypoxia for 7 days (n = 5–6 per group). C: concentration-response relationships for Big endothelin-1 (ET-1) in the presence and absence of CTT (30 µmol/l) in endothelium-denuded aortic rings from normoxic rats and rats exposed to hypoxia for 7 days (n = 10 per group). *P < 0.05 for differences between CTT-treated and -untreated group. {dagger}P < 0.05 for difference from corresponding normoxic control group.

 

Figure 2
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  		Fig. 2. Immunohistochemistry for MMP-2 on aortic sections from normoxic rats and rats exposed to hypoxia for 7 days. Immunoreactivity (brown diaminobenzidine staining) is apparent in the aortic endothelium and media layer of both the normoxic (A) and hypoxic (C) groups but not in the normoxic (B) or hypoxic (D) negative controls (x40 objective).

 

Figure 3
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  		Fig. 3. A: aortic MMP-2 protein levels in normoxic rats and rats exposed to hypoxia for 16 h, 48 h, and 7 days (n = 9 per group). B: gelatin zymography showing the presence of MMP-2 activity in aortae of normoxic rats and from rats exposed to hypoxia for 16 h, 48 h, and 7 days (n = 6 per group). C: MMP-2 protein levels in mesenteric arteries from normoxic rats and rats exposed to hypoxia for 7 days (n = 8 per group). *P < 0.05 for differences from corresponding normoxic group.

 

Figure 4
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  		Fig. 4. MMP (A; n = 10) and endothelin-converting enzyme (ECE, B; n = 9) activity in aortae from normoxic rats and rats exposed to hypoxia for 7 days. *P < 0.05 for differences from corresponding normoxic group.

 

Figure 5
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  		Fig. 5. Levels of aortic membrane type 1 (MT1)-MMP (A), and tissue inhibitors of matrix metalloproteinases 1–4 (TIMPs-1–4; B–E) proteins in aortae from normoxic rats and rats exposed to hypoxia for 16 h, 48 h, and 7 days (n = 6 per group). *P < 0.05 for differences from corresponding normoxic group.

 

Figure 6
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  		Fig. 6. Levels of MMP-2 (A), MT1-MMP (B), TIMP-1 (C), TIMP-2 (D), TIMP-3 (E), and TIMP-4 (F) mRNAs in aortae from normoxic rats and rats exposed to hypoxia for 16 h, 48 h, and 7 days (n = 7 per group). *P < 0.05 for differences from corresponding normoxic group.

 

Figure 7
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  		Fig. 7. A–C: illustration of aortic regions where cells were collected for mRNA extraction. MMP-2 (D) and MT1-MMP (E) mRNA levels in endothelial, subendothelial vascular smooth muscle cells (VSMC), and deep medial VSMC from aortae of normoxic rats and from rats exposed to hypoxia for 7 days (n = 6 per group). *P < 0.05 for differences from corresponding normoxic group.

 
Studies in MMP-2–/– and MMP-2+/+ Mice

Animals. To corroborate the results of the pharmacological studies described above, aortic contraction was assessed in mice deficient in MMP-2. The MMP-2+/– mice on the C57/Bl6J background previously described (20) were interbred to generate MMP-2 knockout (MMP-2–/–) and littermate control (MMP-2+/+) groups. Mouse genotypes were assessed by polymerase chain reaction of genomic DNA. Primers for wild-type alleles were located in exon 1 (5'-CAA CGA TGG AGG CAC GAG TG-3' and 5'-GCC GGG GAA CTT GAT CAT GG-3'), and primers for the mutant allele were located in the neocassette (5'-CTT GGG TGG AGA GGC TAT TC-3' and 5'-AGG TGA GAT GAC AGG AGA TC-3').

Aortic contractile responses. Mouse aortic segments were equilibrated in warmed KHS aerated with 95% O2-5% CO2 at 37°C for 1 h under a resting tension of 1 g before drug-induced changes in tension were monitored. As in rat aortic segments, the endothelium was removed by luminal abrasion, and the success of endothelial ablation was assessed by acetylcholine-induced relaxation of PE-induced contraction (47). CRCs were generated for PE (1 nmol/l to 10 µmol/l) in endothelium-intact thoracic aortic rings, and the contractile response to human Big ET-1 (100 nmol/l) was determined in endothelium-denuded aortic rings from MMP-2+/+ and MMP-2–/– mice exposed to normoxia or hypoxia for 7 days.

Data Analysis

Results are presented as means ± SE for n number of animals with P < 0.05 representing statistical significance. Paired means were compared by two-tailed Student's t-test. Differences among multiple means were evaluated by analysis of variance corrected for multiple measures where appropriate, and, when overall differences were detected, individual means were compared post hoc using Dunnet's test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Rat Studies

Aortic and mesenteric artery contractile responses. Figure 1A shows the concentration-response relationship for PE in endothelium-intact aortic rings from normoxic rats and rats exposed to hypoxia for 7 days in the presence or absence of CTT (30 µmol/l). Inhibition of MMP-2 decreased the maximum tension generated during PE-induced contraction in aortic rings from rats exposed to hypoxia (Table 1) but had no effect in rings from normoxic rats. Figure 1B illustrates the response of endothelium-intact rat mesenteric artery segments to PE in the presence or absence of CTT. As in aortic rings, CTT had no effect on the response of mesenteric arteries from normoxic rats but reduced contraction in those from hypoxia-exposed animals (Table 1).


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  		Table 1. Effect of MMP inhibition on maximum contraction and EC50 values during PE and Big ET-1-induced rat aortic contraction

 
The contractile responses to Big ET-1 in endothelium-denuded aortic rings from rats exposed to normoxia or hypoxia for 7 days in the presence and absence of CTT (30 µmol/l) are illustrated in Fig. 1C. Maximal contractions achieved in rings from hypoxic animals were higher than those in the normoxic controls (Table 1). This hypoxia-dependent augmentation of Big ET-1-mediated contraction was abolished by CTT (Table 1). At this concentration, CTT had no effect on ET-1-induced contraction in aortic rings from either normoxic or hypoxia-exposed rats (data not shown), indicating that its effect is mediated by inhibition of Big ET-1 conversion.

Immunohistochemistry. Figure 2 illustrates the effect of hypoxic exposure for 7 days on the distribution of MMP-2 protein across the aortic wall. MMP-2 protein was detected in both the intima and media of the thoracic aorta from normoxic and hypoxic animals. Staining was more intense in the hypoxic group, with no apparent inhomogeneity in its distribution.

MMP-2 and ECE protein and activity levels. Western analysis shown in Fig. 3A indicates that rat aortic pro-MMP-2 (72 kDa) protein levels increased after prolonged hypoxia. These differences reached statistical significance after 48 h and 7 days of hypoxia. Activated MMP-2 (64 kDa) protein levels were also found to be elevated with increasing duration of hypoxia, achieving statistical significance after 7 days. Aortic MMP-2 activity, as determined by gelatin zymography, was also significantly greater after 7 days of hypoxia when compared with the normoxic control group (Fig. 3B). No bands corresponding to the expected molecular weight of MMP-9 were detected in these samples, suggesting that MMP-2 is the predominant source of gelatinase activity. Figure 3C illustrates that pro-MMP-2 protein is also increased in mesenteric arteries from rats exposed to hypoxia for 7 days when compared with the normoxic animals. Protein concentrations obtained from these small vessels were insufficient to quantify levels of the cleaved (activated) form. Aortic ECE-1 protein levels did not differ between normoxic and hypoxia-exposed rats (data not shown).

The results of fluorometric assays of total MMP and ECE activity are presented in Fig. 4. MMP activity was higher in aortae from hypoxia-exposed rats when compared with the normoxic group (Fig. 4A), whereas ECE activity was unchanged (Fig. 4B).

MT1-MMP and TIMP protein levels. MMP-2 activity is regulated by its activator protease MT1-MMP and its tissue inhibitors (TIMPs). Western analysis demonstrated that rat aortic MT1-MMP protein levels increased progressively with increasing duration of hypoxic exposure, reaching statistical significance after 7 days (Fig. 5A). Although aortic levels of TIMPs-1–4 exhibited an upward trend, these changes did not reach statistical significance during the same period of hypoxic exposure (Fig. 5, B–E).

MMP-2, MT1-MMP, MMP-7, and TIMP mRNA levels. Figure 6 illustrates MMP-2, MT1-MMP, and TIMPs-1–4 mRNA levels in aortae from normoxic rats and rats exposed to hypoxia for 16 h, 48 h, or 7 days. After exposure to hypoxia for 7 days, MMP-2 and MT1-MMP mRNA levels are increased compared with those of the normoxic control group. An increase in levels of TIMPs-1–3 mRNA was observed after 7 days of hypoxia, whereas TIMP-4 mRNA expression was upregulated at the earlier time points (16 h and 48 h) as well. Levels of aortic MMP-7 mRNA, another MMP shown to contribute to adrenoceptor-mediated contraction in rat arteries, were unchanged after hypoxic exposure (data not shown). To identify the cell type responsible for the observed increase in MMP-2 and MT1-MMP expression, MMP-2 and MT1-MMP mRNAs were quantified using RNA extracted from pure populations of aortic endothelial cells, subendothelial VSMC, and deep medial VSMC (Fig. 7, A–C). MMP-2 (Fig. 7D) and MT1-MMP mRNA levels (Fig. 7E) are increased in deep medial aortic VSMC from rats exposed to hypoxia for 7 days, whereas no change was observed in endothelial or subintimal smooth muscle cells.

Studies in MMP-2–/– and MMP-2+/+ Mice

Aortic contractile responses. Figure 8A presents the concentration-response relationships for PE in endothelium-intact aortic rings from mice exposed to normoxia or hypoxia for 7 days. In contrast to rats, hypoxia did not enhance the response to PE in MMP-2+/+ mice, possibly reflecting differences in the adaptive response to hypoxia between the two species. Nevertheless, after hypoxic exposure, the effect of MMP-2 deletion in mice mimics the effect of MMP inhibition in rats in that, after hypoxia, the maximum tensions generated during PE-induced contraction are reduced in MMP-2–/– compared with their MMP-2+/+ littermate controls (Table 2). The responses of endothelium-denuded aortic rings from MMP-2–/– and MMP-2+/+ mice to Big ET-1 (100 nmol/l) are illustrated in Fig. 8B. Similar to the results obtained in rat aortae, contraction to Big ET-1 is greater in aortic segments from MMP-2+/+ mice exposed to hypoxia than in segments from the corresponding normoxic control group (Table 2). In contrast, the aortic responses to Big ET-1 in normoxic and hypoxia-exposed MMP-2–/– mice do not differ.


Figure 8
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  		Fig. 8. A: concentration-response relationship for PE in endothelium-intact aortic rings from MMP-2-deficient (MMP-2–/–) and littermate control (MMP-2+/+) mice exposed to normoxia or hypoxia for 7 days (n = 8–9 per group). *P < 0.05 for differences between MMP-2+/+ and MMP-2–/– groups. B: contractile response to Big ET-1 (100 nmol/l) in endothelium-denuded aortic rings from MMP-2+/+ and MMP-2–/– mice exposed to normoxia or hypoxia for 7 days (n = 9 for each group). *P < 0.05 for difference from corresponding normoxic group.

 

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  		Table 2. Maximum contraction and EC50 values during PE-induced contraction and response to Big ET-1 in MMP-2–/– and MMP-2+/+ mice

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results of this study show that, after prolonged hypoxia, 1) MMP-2 inhibition (rat) or deletion (mouse) reduces aortic and mesenteric arterial contraction to PE; 2) the aortic contractile response to Big ET-1 is enhanced in rats and mice through an MMP-2-dependent mechanism; 3) MMP-2 protein levels in rat aortae and mesenteric arteries and MMP activity in rat aortae are increased; 4) aortic MT1-MMP protein levels are increased; 5) aortic MMP-2, MT-1-MMP, and TIMPs-1–4 mRNA levels are increased; and 6) the increase in rat aortic MMP-2 and MT1-MMP mRNA expression is localized to the deep medial vascular smooth muscle.

The 23 MMPs identified to date are divided on the basis of substrate preference into collagenases, gelatinases, stromelysins, matrilysins, and membrane-type MMPs. MMP-2 and MMP-9 are the gelatinases that efficiently degrade collagen type IV (40) and hence are involved in the restructuring of vascular basement membranes. A broader biological role for MMP-2 has become apparent with the recognition that its substrates also include a number of vasoregulatory peptides (14, 15, 18, 21, 28, 29). Inactivation of vasodilators [calcitonin gene-related peptide (16) and adrenomedullin (29)] and activation or release of vasoconstrictors [Big ET-1 (15), heparin-binding epidermal growth factors (18, 28), and integrin-binding Arg-Gly-Asp peptides (30)] all contribute to its vasoactive effects. Although the relative importance of each of these pathways and the possible existence of others remain to be explored, our present results emphasize that the net effect of increased MMP-2 activity in the systemic circulation, as occurs after hypoxia, is to potentiate vascular smooth muscle contraction.

In regions affected by arterial insufficiency, MMP-2-mediated enhancement of vascular contraction may be maladaptive since it will exacerbate ischemic injury. During global hypoxia, however, the primary defensive vascular response depends on the capacity of the adrenergic nervous system to regulate the regional distribution of blood flow and oxygen extraction (9). In previous studies in rats, systemic arterial smooth muscle contraction to adrenoceptor stimulation is impaired after 48 h of hypoxia due to induction of heme oxygenase and nitric oxide synthase expression and inhibition of myosin phosphorylation (17, 41, 47–49). Hence the ability to target oxygen delivery to areas of greatest metabolic demand (9, 19, 26) is impaired. Our present results indicate that, after 7 days of hypoxia, rat aortic and mesenteric arterial contractions are increased and that this is concomitant with and dependent on enhanced MMP-2 activity. In this setting, therefore, upregulation of vascular MMP-2 provides a mechanism to reinforce adrenergic regulation in the period during which maintenance of oxygen delivery to vital organs is mediated by changes in vascular tone before the structural change on which the redistribution of blood flow will ultimately depend.

In the rat aorta, MMP-2 and ECE-1 are the major enzymes that convert Big ET-1, the inactive prohormone, into the active vasoconstrictor ET-1. Activation of Big ET-1 by ECE-1 releases ET-1[1–21], whereas cleavage at Gly32-Leu33 by MMP-2 generates an isopeptide ET-1[1–32] with enhanced potency at the smooth muscle endothelin type A receptor (15). In the current study, the maximum contraction that could be elicited by Big ET-1 was increased after hypoxia. Since this was reversed by MMP inhibition or MMP-2 deletion, MMP-2-mediated formation of the more potent isopeptide ET-1-[1–32] appears to be a prominent pathway for Big ET-1 conversion during prolonged hypoxia.

MMP-2 is secreted as a zymogen, the activity of which is regulated by its activating protease MT1-MMP (7) and the endogenous TIMPs (40). An increase in MT1-MMP relative to the TIMPs is, therefore, requisite to any significant enhancement of bioactivity. The possibility that MT1-MMP activity may be regulated by an oxygen-sensitive mechanism has been suggested previously. MT1-MMP protein is increased after hypoxic incubation in HepG2 cells and in the myocardium after ischemia-reperfusion injury (12, 31), and the intracellular proprotein convertase furin, responsible for its activation, is transcriptionally regulated by hypoxia-inducible factor-1 (31). Our current results confirm the functional relevance of these findings in the systemic circulation and demonstrate that upregulation of MT1-MMP in the aorta parallels the expression of its substrate, MMP-2. We also considered the possibility that TIMPs may be oxygen regulated to provide an additional level of control. In cultured cells, TIMP-1 and -2 have been observed to increase (27, 33, 34), decrease (4, 10, 11), or remain unchanged (32) after hypoxic incubation. Our results confirm that aortic levels of TIMP mRNAs are sensitive to oxygen tension in vivo. However, in contrast to MT1-MMP and MMP-2, changes in TIMP protein levels did not reach statistical significance. Since MMP-2 activity was increased, this suggests that elevated TIMP expression is insufficient to offset the increase in MT1-MMP and MMP-2. Nevertheless, the fact that the expression of these endogenous inhibitors is hypoxia inducible suggests that they may provide an important negative feedback mechanism in some conditions.

The genes encoding MMP-2 and MT1-MMP contain consensus binding elements for a number of hypoxia-inducible transcription factors (5, 36), and their transcriptional regulation by oxygen tension would be anticipated. Nevertheless, the results of previous studies in cultured cells are in conflict on this point (4, 13, 25, 34, 35). Our present results provide both pharmacological and biochemical evidence that, in vivo, the expression of MMP-2 and MT1-MMP is upregulated in the systemic circulation after hypoxia as a result of increases in their steady-state mRNA levels and that this change in systemic vascular cell phenotype is functionally relevant. Vascular cells experience a broad range of oxygen tensions under physiological conditions. In the aorta, oxygen concentrations from 11.2% (90 mmHg) at the luminal surface to 2.2% (20 mmHg) at a depth of 150 µm (6) are reported, and longitudinal gradients of similar magnitude occur in the microcirculation (43). Since the severity of the hypoxic stimulus varies significantly across the aortic wall, production of MMP-2 and MT1-MMP would also be expected to demonstrate regional heterogeneity. Immunohistochemical analysis may be confounded because MMP-2 is secreted and hence distributed in the intracellular space across the aortic wall (see Fig. 2). Accordingly, we evaluated the regional expression of MMP-2 and MT1-MMP mRNA and found that these transcripts are selectively enriched in VSMC located deep within the aortic media, the most hypoxic region of the tissue (42). This supports our hypothesis that the expression of MMP-2 correlates with the severity of the hypoxic stimulus and suggests that proteolytic activation of MMP-2 proenzyme occurs as it is produced in the deep medial layer.

It is well recognized that changes in vascular tone precede the structural alterations that occur when changes in blood flow persist chronically (2, 3, 30). Such remodeling of the circulation is important in adapting the mature circulation to chronic changes in tissue perfusion as well as arterial growth to meet the changing blood flow demands of developing peripheral tissues. A role for MMP-2 in this process is supported by observations that MMP-2–/– mice demonstrate impaired angiogenesis (23) and that inhibitors of MMP reduce the pathological structural remodeling that accompanies monocrotaline-induced pulmonary arterial hypertension (44). Hypoxia is a potent stimulus for both changes in vascular tone and structural remodeling in the systemic circulation (45, 46). The results of the current study indicate that vascular MMP-2 levels and activity are tightly regulated by oxygen tension and hence represent a pivotal regulatory pathway by which the acute vascular responses associated with hypoxia may be integrated with the longer-term structural changes in both conduit and resistance arteries. Further investigation to determine the specific roles of MMP-2 and each of its newly identified substrates will advance our understanding of the pathobiology of this process in cardiopulmonary diseases and offer new therapeutic targets in their management.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was funded by a grant from the Canadian Institutes of Health Research (to M. E. Ward) and Kidney Foundation of Canada (to P. A. Marsden). The laser capture microdissection core facility is supported by a Heart and Stroke Foundation of Ontario group grant.


   ACKNOWLEDGMENTS
 
We thank Yupu Deng for expert technical assistance. J. Z. He was the recipient of a studentship from the Heart and Stroke/Richard Lewar Centre of Excellence and the Keenan Foundation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. E. Ward, Dept. of Critical Care, Rm. 4-015, St. Michael's Hospital, 30 Bond St., Toronto, ON, Canada M5B1W8 (e-mail: wardm{at}smh.toronto.on.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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