Background

Matrix metalloproteinases (MMPs) activity may modulate hypertension-related accumulation of extracellular matrix (ECM) in arteries. We tested whether estrogen deficiency induces alterations of vascular collagen, MMP-2, membrane-type 1-MMP (MT1-MMP), or tissue inhibitor of metalloproteinases-2 (TIMP-2) expression in ovariectomized rats, which may be associated with postmenopausal hypertension.

Methods

Estrogen deficiency was induced by ovariectomy (Ovx) in female rats. Time-course changes of aortic MMPs protein expression were evaluated. Treatment with tempol or aminoguanidine was used to examine the role of oxidative stress and nitric oxide (NO) on these changes.

Results

The level of the active-form MMP-2 was markedly reduced during 1–4 weeks after Ovx, with a significant increase in collagen accumulation and increased MT1-MMP expression. Although active-form MMP-2 and collagen progressively returned to normal levels, the markedly increased collagen deposition appeared again at 8 weeks and persisted until 12 weeks, followed by induction of MMP-2 and MT1-MMP at 12 weeks. The TIMP-2 level reduced for 2 weeks after Ovx, but soon returned to normal. Treatment with 17-estradiol (E₂), tempol, or aminoguanidine for 6 weeks prevented Ovx-induced blood pressure elevation and apparently reversed the MMPs changes.

Conclusions

In an initial period, E₂ deficiency induces a reduction of active-form MMP-2 leading to collagen accumulation, and induction of MT1-MMP, which may be a compensatory response to degrade collagen. At a latter stage, the concurrent elevation of active-form MMP-2 and MT1-MMP expression may be adaptive responses to regulate ECM composition in the vascular wall. Oxidative stress and NO contribute to activity modulation of vascular MMPs in Ovx rats.

Methods

Animal preparation. Female Sprague-Dawley rats were obtained from the National Laboratory Animal Breeding and Research Center of the National Science Council, Taiwan. Handling of the animals was in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). This study was approved by the National Defense Medical Center Institutional Animal Care and Use Committee, Taiwan. To produce the estrogen-deficient condition, young rats were anesthetized with sodium pentobarbital (50 mg/kg, IP) and underwent bilateral Ovx at 8 weeks of age. Small incisions were made bilaterally on the sides of their backs to expose the ovaries retroperitoneally. The ovaries were clamped and removed, and the uterine tubes were ligated. The muscle and skin were then sutured. The sham procedure consisted of anesthesia, visualization of the ovaries through incisions into the abdominal cavity, and closure of the wounds.

Experimental groups. One week after the operation, the rats were randomly divided into five groups: (i) sham group: rats had undergone sham operations (n = 21); (ii) Ovx group: rats were ovariectomized bilaterally (n = 18); (iii) Ovx+E₂ group: Ovx rats were injected with E₂ (50 g/kg per day, IM, once daily) for 1–6 weeks, beginning 1 week after Ovx (n = 18); (iv) Ovx+tempol group: Ovx rats were administered tempol (1 mmol/l in drinking water), a membrane-permeable superoxide dismutase mimetic,^15,16 for 1–6 weeks beginning 1 week after Ovx (n = 18); (v) Ovx+AG group: Ovx rats were administered aminoguanidine (AG, 1 mmol/l in drinking water), a selective iNOS inhibitor, for 1–6 weeks beginning 1 week after Ovx (n = 18).

Type I and III collagens are the major fibrillar collagens detectable in vessels, representing 60 and 30% of vascular collagens, respectively.¹⁷ They are the common substrates of MMP-2.¹⁸ Therefore, we chose collagen I as the representative to examine the possible role of vascular MMP-2 in the E₂-deficiency condition. To investigate the time courses of collagen type I, MMP-2, MT1-MMP, and TIMP-2 expression in the thoracic aortas, rats were reanesthetized with pentobarbital (60 mg/kg, IP) and killed each week after Ovx (n = 3 for each time point tested).

Plasma E₂ assay. Blood samples (1 ml) were withdrawn from the abdominal aorta in heparinized syringes under anesthesia at 7 weeks after Ovx and centrifuged for 10 min at 4 °C. Plasma concentration of E₂ was determined by ¹²⁵I radioimmunoassay using a commercially available kit (Diagnostic Products, Los Angeles, CA). The protocol provided by the manufacturer was strictly followed. All samples and standards were measured in duplicate and repeated twice.

Blood pressure measurements. The mean blood pressure was determined in conscious rats by a tail-cuff method using an automatic blood pressure monitoring system (MK-2000; Muromachi Kikai, Tokyo, Japan). In brief, conscious rats were placed in restraints and an occlusion cuff containing a piezoelectric pulse sensor was placed around the tail. The blood pressure was measured after 15 min of acclimatization. A minimum of 10 serial measurements was made and the average value was calculated.

Western blot analysis of collagen type I, MMP-2, MT1-MMP, and TIMP-2 protein expression in aortas. Aortas were ground in a mortar containing liquid nitrogen and pulverized using a pestle whereas the tissue was kept frozen by the addition of liquid nitrogen. The powdered tissue was suspended in 1 ml of lysis buffer (5 mmol/l EDTA, 50 mmol/l NaCl, and 50 mmol/l HEPES, pH 7.5) containing protease inhibitors (10 g/ml aprotinin, 1 mmol/l phenylmethanesulphonyl fluoride, and 10 g/ml leupeptin). Aliquots of aorta homogenates were subjected to electrophoresis in a 10% sodium dodecyl sulfate–polyacrylamide gel for 1 h at 100 V. The separated proteins were then transferred electrophoretically to a nitrocellulose membrane (Millipore, Billerica, MA). Immunodetection was as described previously.¹⁹ The blot was then incubated with mouse monoclonal antibodies directed against collagen type I, MT1-MMP, and TIMP-2, and rabbit polyclonal antibody against MMP-2, in Tris-buffered saline containing 0.1% Tween-20 overnight at 4 °C. The membrane was washed and incubated for 1 h at room temperature with horseradish-peroxidase-conjugated antimouse or antirabbit IgG antibody and enhanced chemiluminescent substrate (Pierce, Rockford, IL). The membrane was exposed to X-ray film for 5 min. The density of the individual bands was quantified by densitometric scanning of the blots using Image-Pro software, as described previously.

Statistical analysis. All measurements are expressed as group means s.e.m. Statistical evaluation was performed using a one-factor analysis of variance followed by the Newman–Keuls procedure. A P value of <0.05 was deemed statistically significant.

Results

Plasma levels of E₂

The plasma E₂ concentration averaged 120 4.7 pg/ml in the sham group. At 7 weeks after Ovx, the plasma E₂ concentration was 38 6.1 pg/ml in the Ovx group, 43.8 4.3 pg/ml in the Ovx+AG group, and 47.1 3.5 pg/ml in the Ovx+tempol group, and was significantly lower than that in the sham group (P < 0.05). After 6-weeks-replacement of E₂ (Ovx+E₂ group), the plasma level of E₂ was 124 5.2 pg/ml, which is significantly higher than that in the Ovx group (P < 0.05), but not significantly different from that in the sham group (P > 0.05).

Blood pressure measurements

The mean blood pressure in the sham group did not markedly change during the experimental period. In the Ovx group, mean blood pressure increased progressively and appeared dramatically elevated at 5 weeks after Ovx (P < 0.05), persisting at a high level until at least week 12 (Figure 1a). Treatment with E₂, tempol or AG for 4–5 weeks significantly prevented the elevation of mean blood pressure after Ovx, when compared with the Ovx group (Figure 1b).

Figure 1.

Time course of mean blood pressure measured in conscious rats by the tail-cuff method; (a) 1–12 weeks after ovariectomy (Ovx); (b) Ovx rats with treatment of E₂, tempol or aminoguanidine (AG) for 5 weeks beginning 1 week after Ovx. The initial number of animals is 18, and each week 3 rats were sacrificed for each time point. Data are given as means s.e.m., *P < 0.05 vs. sham. ^#P < 0.05 vs. Ovx.

Full figure and legend (16K)

Time-course changes in vascular collagen type I, MMP-2, MT1-MMP, and TIMP-2 protein expression after Ovx

Protein expression in the aortas of Ovx rats was studied by immunoblotting analysis. The levels of collagen type I, active form and latent form of MMP-2 (proMMP-2), MT1-MMP, and TIMP-2 proteins in sham-operated rats were not significantly different at the time points tested (1–7 weeks; data not shown). As shown in Figure 2a, collagen type I protein accumulated significantly at 1–4 weeks after Ovx when compared with the sham group (P < 0.05), returned to basal levels at 6 weeks, and was again elevated at 8 weeks. It persisted at high levels until at least 12 weeks post Ovx. Figure 2b demonstrates that protein expression of proMMP-2 was altered by Ovx. At 1 week after Ovx, the level of proMMP-2 protein reduced significantly when compared with the sham group (P < 0.05). It returned to basal levels at 2 weeks, and then rose again to a maximal level at 12 weeks. Interestingly, the levels of active-form MMP-2 decreased markedly during 1–4 weeks after Ovx, returned to basal levels during 6–8 weeks, and were significantly further elevated at 12 weeks. Figure 2c demonstrates that the levels of MT1-MMP increased markedly during 1–4 weeks after Ovx, returned to basal levels during 6–8 weeks, and were elevated again at 12 weeks. The protein expression of TIMP-2 was also immediately changed by Ovx, and was significantly attenuated at 1–2 weeks after Ovx. It returned to basal levels at 4 weeks, peaked at 6 weeks, and reverted during 8–12 weeks (Figure 2d).

Figure 2.

Time course of changes in (a) collagen type I, (b) pro- and active MMP-2, (c) MT1-MMP, and (d) TIMP-2 protein expression in the aorta 1–12 weeks after ovariectomy (Ovx) in rats. Data are given as means s.e.m. (n = 3 for each time point). *P < 0.05 vs. sham. MMP, matrix metalloproteinase; MT1-MMP, membrane type 1-MMP; TIMP-2, tissue inhibitor of metalloproteinases-2.

Full figure and legend (46K)

After E₂ replacement for 1–6 weeks, the accumulation of collagen type I was significantly reversed and was negligibly expressed during the experimental period (P < 0.05) (Figure 3a). E₂ prevented the reduction of TIMP-2 protein expression after Ovx and the loss of activity of MMP-2 (Figure 3b,d). Replacement of E₂ also reversed the increase of MT1-MMP levels after Ovx (Figure 3c). However, E₂ administered for >3 weeks caused an overt decrease of MT1-MMP, which was significantly less than that of the sham group.

Figure 3.

Effects of E₂ replacement on time course of changes in (a) collagen type I, (b) pro- and active MMP-2, (c) MT1-MMP, and (d) TIMP-2 protein expression in the aorta 2–7 weeks after ovariectomy (Ovx) in rats. Ovx: 2 weeks after Ovx. Ovx rats were injected with E₂ (50 g/kg per day, IM, once daily) for 1–6 weeks, beginning 1 week after Ovx. Data are given as means s.e.m. (n = 3 for each time point). *P < 0.05 vs. sham. ^#P < 0.05 vs. Ovx. MMP, matrix metalloproteinase; MT1-MMP, membrane type 1-MMP; TIMP-2, tissue inhibitor of metalloproteinases-2.

Full figure and legend (80K)

After administering the antioxidant tempol for 1–6 weeks, the accumulation of collagen type I after Ovx was significantly reduced (P < 0.05) (Figure 4a). The reduction of active-form MMP-2 after Ovx was also reversed (P < 0.05) (Figure 4b). The increase in MT1-MMP levels after Ovx was significantly prevented (P < 0.05) (Figure 4c). Treatment with tempol significantly prevented the decrease of TIMP-2 levels caused by Ovx, which showed statistically significant differences at 1 and 5 weeks when compared with the Ovx group (week 2) (P < 0.05) (Figure 4d).

Figure 4.

Effects of tempol treatment on time course of changes in (a) collagen type I, (b) pro- and active MMP-2, (c) MT1-MMP, and (d) TIMP-2 protein expression in the aorta 2–7 weeks after ovariectomy (Ovx) in rats. Ovx: 2 weeks after Ovx. Ovx rats were administered tempol (1 mmol/l in drinking water) for 1–6 weeks, beginning 1 week after Ovx. Data are given as means s.e.m. (n = 3 for each time point). *P < 0.05 vs. sham. ^#P < 0.05 vs. Ovx. MMP, matrix metalloproteinase; MT1-MMP, membrane type 1-MMP; TIMP-2, tissue inhibitor of metalloproteinases-2.

Full figure and legend (55K)

Treatment with the iNOS inhibitor AG also significantly inhibited the accumulation of collagen type I after Ovx (P < 0.05) (Figure 5a). The reduction of active-form MMP-2 after Ovx was also reversed (P < 0.05) (Figure 5b). The increase in MT1-MMP levels after Ovx was significantly attenuated after 1–6 weeks of AG treatment (P < 0.05) (Figure 5c). Treatment with AG for 1 week significantly prevented the decrease in TIMP-2 levels caused by Ovx (P < 0.05) (Figure 5d). Although longer treatment with AG elevated the levels of TIMP-2, it did not produce significant differences when compared with the Ovx group (week 2).

Figure 5.

Effects of aminoguanidine (AG) treatment on time course of changes in (a) collagen type I, (b) pro- and active MMP-2, (c) MT1-MMP, and (d) TIMP-2 protein expression in the aorta 2–7 weeks after ovariectomy (Ovx) in rats. Ovx: 2 weeks after Ovx. Ovx rats were administered AG (1 mmol/l in drinking water) for 1–6 weeks, beginning 1 week after Ovx. Data are given as means s.e.m. (n = 3 for each time point). *P < 0.05 vs. sham. ^#P < 0.05 vs. Ovx. MMP, matrix metalloproteinase; MT1-MMP, membrane type 1-MMP; TIMP-2, tissue inhibitor of metalloproteinases-2.

Full figure and legend (49K)

Discussion

In the initial period after Ovx, E₂ deficiency caused a marked reduction of vascular active-form MMP-2 and collagen accumulation, together with MT1-MMP induction. MT1-MMP induction may be a compensatory response to these changes, to convert more proMMP-2 to active MMP-2 or degrade collagen directly. Collagen accumulation occurred again beginning at 8 weeks, followed by increased active MMP-2 and MT1-MMP expression at 12 weeks, implying that collagen may induce synthesis and activation of proMMP-2 and MT1-MMP. E₂ replacement can reverse these alterations. Increased oxidative stress and NO is associated with suppression of MMP-2 activation and induction of MT1-MMP caused by E₂ deficiency. Impaired MMP-2 activation leading to vascular collagen accumulation may be involved in the elevation of blood pressure caused by Ovx.

There was a clear reduction in the activation of proMMP-2 1 week after Ovx that persisted for at least 4 weeks. Concurrently, collagen deposition increased during this period. Increased MMP-2 activity returned to basal levels at weeks 6–8, and collagen deposition was also reduced. This time course-matched evidence shows the relationship between the proteolytic activity of MMP-2 and ECM degradation in vivo. Interestingly, MT1-MMP, the activator of MMP-2, was also significantly elevated during the 1–4 weeks after Ovx. It has been shown that one of the important functions of MT1-MMP is its collagen degrading activity,²⁰ thus MT1-MMP induction may be an adaptive response for degradation of accumulated ECM. In addition, these changes of collagen and MMPs happened before blood pressure elevated (Ovx 1–4 weeks) indicating that the observations are not blood pressure-dependent. However, at week 8, collagen accumulation increased markedly again, followed by an increase in active-form MMP-2 and markedly increased expression of MT1-MMP at week 12. It is likely that collagen induces countervailing activation of MMP-2 and MT1-MMP expression to remove excess ECM deposition in the vascular wall. The compensatory capacity of MMP-2 activity became dampened when blood pressure is significantly elevated. The elevation of blood pressure caused by Ovx may participate in these latter changes (Ovx 8–12 weeks).

After 1–6 of weeks of estrogen replacement, the activity of MMP-2 was dramatically reversed, and deposition of collagen in aorta of Ovx rats was reduced. Similar evidence has also been reported. Estrogen replacement reduces age-associated remodeling in rat mesenteric arteries.¹¹ Estrogen exerts a beneficial effect on coronary vascular remodeling in the early stages of hypertension in spontaneously hypertensive rats.²¹ Estrogen can enhance the release of proMMP-2 from human vascular smooth muscle cells²² and increase the protein level of MMP-2 in mesangial cells via the transcription factor AP-2.²³ In this study, protein synthesis of proMMP-2 was significantly reduced 1 week after Ovx, and gradually increased and then returned to almost normal levels at 2 weeks (Figure 2b). E₂ replacement did not significantly affect the protein synthesis of proMMP-2 in Ovx rats, but dramatically elevated the content of active-form MMP-2, indicating that E₂ modulates the process of activation to maintain the normal function of MMP-2. Moreover, the levels of MMP-2 activator proteins MT1-MMP and TIMP-2, which are necessary for MT1-MMP to activate proMMP-2, were reversed by E₂ treatment (Figure 3c,d). Thus this effect of E₂ on MT1-MMP and TIMP-2 may be responsible for the maintenance of MMP-2 activity after Ovx.

Estrogen exerts genomic and nongenomic effects via estrogen receptor-dependent and -independent mechanisms, to confer protective effects on the cardiovascular system.²⁴ Of these biological effects, the antioxidant effects of E₂ may play a critical role in eliciting vasoprotective effects. Endogenous and exogenous estrogens have antioxidant potential in vitro^25,26 and in vivo.^27,28 Steroidal estrogens function as free radical scavengers under a variety of experimental conditions.^29,30 Oxidative stress has been shown to be increased in postmenopausal women³¹ and rats.¹⁹ Therefore, we chronically treated Ovx rats with the antioxidant tempol to observe the role of endogenous reactive oxygen species in the regulation of MMPs and to compare this with E₂ treatment. Tempol apparently reduced the accumulation of collagen, elevated the activity of MMP-2, and caused MT1-MMP to return to basal levels after Ovx, with significant increases in the level of TIMP-2 at week 1 and week 5. This evidence suggests that elevated oxidative stress after Ovx is responsible for reduction in MMP-2 activity, mainly affecting the conversion of proMMP-2 to active-form MMP-2, and not protein synthesis. This also implies that estrogen can improve MMP-2 function through its antioxidant capacity.

NO produced by iNOS has been implicated in the modulation of MMP-2 activation. After previous studies, we reported that after Ovx, eNOS appeared dysfunctional with lack of cofactor BH₄,³² and induction of iNOS protein expression presented at 2 weeks after surgery, which may contribute to the compensation for eNOS dysfunction by producing NO. Long-term treatment with AG reduced NO levels in Ovx rats.¹⁹ Several studies have established the ability of NO to regulate vessel thickness and cellular composition. Models of chronic NO synthase inhibition have shown a decrease in lumen diameter, an increase in vascular smooth muscle cell and endothelial cell proliferation, and thickening of the adventitial tissue.^13,14 Conversely, inhibition of NO synthase by L-NAME can augment activity of MMP-2 in the myocardium.³³ Peroxynitrite, the product of superoxide anion and NO, has been demonstrated to suppress the activation of proMMP-2.³⁴ Therefore, despite oxidative stress elevated in the E₂-deficient environment, treatment with AG reduced NO production attenuating the formation of peroxynitrite in Ovx rats that may contribute to preventing MMP-2 dysfunction. In addition, AG is a prototype therapeutic agent for the prevention of formation of advanced glycation endproducts (AGE). AGE-crosslinking of long-lived structural proteins, such as collagen and elastin, has been correlated with the severity of diabetic complications as well as in certain pathophysiological states commonly seen in aging.³⁵ AGE-crosslinking causes proteins that are normally flexible to become rigid. The cells, tissues, and blood vessels become stiff and increasingly dysfunctional. One of the consequences of AGE-crosslinking of collagen is decreased susceptibility to proteolytic and chemical degradation, leading to collagen accumulation. Therefore, preventing AGE-formation by AG may also contribute to reverse collagen accumulation caused by Ovx.

Although the mechanisms responsible for increases in blood pressure in postmenopausal women are complex and multifaceted, E₂ deficiency-induced impaired MMP-2 activation and collagen accumulation in blood vessels is a possible mechanism for postmenopausal hypertension. Recently, it has been suggested that early wave reflections from a short arterial tree to the heart and increased aortic rigidity contribute to isolated systolic hypertension in women. Postmenopausal development of central conduit artery stiffening superimposed on the shorter arterial tree may lead to hemodynamic changes in older women.³⁶ Therefore, the interplay of MMP-2, MT1-MMP, and TIMP-2 expression in large arteries shows new insight into the pathophysiology of blood vessels in E₂-deficient conditions. Further studies should be conducted to evaluate the regulatory pattern in small arteries.

Disclosure

The authors declared no conflict of interest.

References

1. Tayebjee MH, MacFadyen RJ, Lip GYH. Extracellular matrix biology: a new frontier in linking the pathology and therapy of hypertension. J Hypertens 2003;21:2211–2218. | Article | PubMed |
2. Jacob MP. Extracellular matrix remodeling and matrix metalloproteinases in the vascular wall during aging and in pathological conditions. Biomed Pharmacother 2003;57:195–202. | Article | PubMed |
3. Murphy G, Docherty AJP. The matrix metalloproteinases and their inhibitors in connective tissue remodeling. Am J Respir Cell Mol Biol 1992;7:120–125. | PubMed | ISI | ChemPort |
4. Woessner JF Jr. The family of matrix metalloproteinases. Ann NY Acad Sci 1994;732:11–21. | Article | PubMed | ChemPort |
5. Donnelly R, Collinson DJ, Manning G. Hypertension, matrix metalloproteinases and target organ damage. J Hypertens 2003;21:1627–1630. | Article | PubMed |
6. Ward RV, Hembry RM, Reynolds JJ, Murphy G. The purification of TIMP2 from its 72 kDa pro-gelatinase complex. Biochem J 1991;278:179–187. | PubMed | ISI | ChemPort |
7. Ellerbroek SM, Wu YI, Overall CM, Stack MS. Functional interplay between type 1 collagen and cell surface matrix metalloproteinase activity. J Biol Chem 2001;276:24833–24842. | Article | PubMed | ISI | ChemPort |
8. Zervoudaki A, Economou E, Pitsavos C, Vasiliadou K, Aggeli C, Tsioufis K, Toutouza M, Stefanadis C, Toutouzas P. The effects of Ca²⁺ channel antagonists on plasma concentrations of matrix metalloproteinase-2 and -9 in essential hypertension. Am J Hypertens 2004;17:273–276. | Article | PubMed |
9. Spiers JP, Kelso EJ, Siah WF, Edge G, Song G, McDermott BJ, Hennessy M. Alterations in vascular matrix metalloproteinase due to ageing and chronic hypertension: effects of endothelin receptor blockade. J Hypertens 2005;23:1717–1724. | Article | PubMed |
10. Chesler N, Ku D, Galis ZS. Transmural pressure induces matrix degrading activity in porcine arteries ex vivo. Am J Physiol 1999;277:H2002–H2009. | PubMed | ChemPort |
11. Zhang Y, Stewart KG, Davidge ST. Estrogen replacement reduces age-associated remodeling in rat mesenteric arteries. Hypertension 2000;36:970–974. | PubMed |
12. Nelson K, Melendez JA. The powerhouse takes control of the cell: the role of mitochondria in signal transduction. Free Radic Biol Med 2004;37:768–784. | Article | PubMed | ISI | ChemPort |
13. Steudel W, Scherrer-Crosbie M, Bloch KD, Weimann J, Huang PL, Jones RC, Picard MH, Zapol WM. Sustained pulmonary hypertension and right ventricular hypertrophy after chronic hypoxia in mice with congenital deficiency of nitric oxide synthase 3. J Clin Invest 1998;101:2468–2477. | Article | PubMed | ISI | ChemPort |
14. Arribas SM, Gonzalez C, Graham D, Dominiczak AF, McGrath JC. Cellular changes induced by chronic nitric oxide inhibition in intact rat basilar arteries revealed by confocal microscopy. J Hypertens 1997;15:1685–1693. | Article | PubMed | ChemPort |
15. Mitchell JB, DeGraff W, Kaufman D, Krishna MC, Samuni A, Finkelstein E, Ahn MS, Hahn SM, Gamson J, Russo A. Inhibition of oxygen-dependent radiation-induced damage by nitroxide superoxide dismutase mimic, tempol. Arch Biochem Biophys 1991;289:62–70. | Article | PubMed | ISI | ChemPort |
16. Cuzzocrea S, McDonald MC, Mazzon E, Siriwardena D, Costantino G, Fulia F, Cucinotta G, Gitto E, Cordaro S, Barberi I, De Sarro A, Caputi AP, Thiemermann C. Effects of tempol, a membrane-permeable radical scavenger, in a gerbil model of brain injury. Brain Res 2000;875:96–106. | Article | PubMed | ISI | ChemPort |
17. Prockop DJ, Kivirikko KI. Collagens: molecular biology, diseases, and potentials for therapy. Annu Rev Biochem 1995;64:403–434. | Article | PubMed | ISI | ChemPort |
18. Sternlicht MD, Werb Z. How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol 2001;17:463–516. | Article | PubMed | ISI | ChemPort |
19. Lee YM, Cheng PY, Hong SF, Chen SY, Lam KK, Sheu JR, Yen MH. Oxidative stress induces vascular heme oxygenase-1 expression in ovariectomized rats. Free Radic Biol Med 2005;39:108–117. | Article | PubMed |
20. Itoh Y, Seiki M. MT1-MMP: a potent modifier of pericellular microenvironment. J Cell Physiol 2006;206:1–8. | Article | PubMed | ISI | ChemPort |
21. Garcia PM, Gimenez J, Bonacasa B, Carbonell LF, Miguel SG, Quesada T, Hernandez I. Estrogen exerts a beneficial effect on coronary vascular remodeling in the early stages of hypertension in spontaneously hypertensive rats. Menopause 2005;12:453–459. | Article | PubMed |
22. Wingrove CS, Garr E, Godsland IF, Stevenson JC. 17 beta-oestradil enhances release of matrix metalloproteinase-2 from human vascular smooth muscle cells. Biochim Biophys Acta 1998;1406:169–174. | PubMed |
23. Guccione M, Silbiger S, Lei J, Neugarten J. Estradiol upregulates mesangial cell MMP-2 activity via the transcription factor AP-2. Am J Physiol-Renal Physiol 2002;282:F164–F169. | PubMed |
24. Dubey RK, Jackson EK. Estrogen-induced cardiorenal protection: potential cellular, biochemical, and molecular mechanisms. Am J Physiol-Renal Physiol 2001;280:F365–F388. | PubMed |
25. Shwaery GT, Vita JA, Keaney FR Jr. Antioxidant protection of LDL by physiological concentratons of 17 beta-estradiol. Requirement for estradiol modification. Circulation 1997;95:1378–1385. | PubMed | ChemPort |
26. Dubey RK, Tyurina YY, Tyurin VA, Gillespie DG, Branch RA, Jackson EK, Kagan VE. Estrogen and tamoxifen metabolites protect smooth muscle cell membrane phospholipids against peroxidation and inhibit cell growth. Circ Res 1999;84:229–239. | PubMed | ISI | ChemPort |
27. Dantas AP, Tostes RC, Fortes ZB, Costa SG, Nigro D, Carvalho MH. In vivo evidence for antioxidant potential of estrogen in microvessels of female spontaneously hypertensive rats. Hypertension 2002;39:405–411. | Article | PubMed |
28. Wassmann S, Baumer AT, Strehlow K, van Eickels M, Grohe C, Ahlbory K, Rosen R, Bohm M, Nickenig G. Endothelial dysfunction and oxidative stress during estrogen deficiency in spontaneously hypertensive rats. Circulation 2001;103:435–441. | PubMed | ISI | ChemPort |
29. Lacort M, Leal AM, Liza M, Martin C, Martinez R, Ruiz-Larrea MB. Protective effect of estrogens and catecholestrogens against peroxidative membrane damage in vitro. Lipids 1995;30:141–146. | Article | PubMed |
30. Ruiz-Larrea MB, Martin C, Martinez R, Navarro R, Lacort M, Miller NJ. Antioxidant activities of estrogens against aqueous and lipophilic radicals; differences between phenol and catechol estrogens. Chem Phys Lipids 2000;105:179–188. | Article | PubMed | ChemPort |
31. Signorelli SS, Neri S, Sciacchitano S, Pino LD, Costa MP, Pennisi G, Ierna D, Caschetto S. Duration of menopause and behavior of malondialdehyde, lipids, lipoproteins and carotid wall artery intima-media thickness. Maturitas 2001;39:39–42. | Article | PubMed | ISI | ChemPort |
32. Lam KK, Lee YM, Hsiao G, Chen SY, Yen MH. Estrogen therapy replenishes vascular tetrahydrobiopterin and reduces oxidative stress in ovariectomized rats. Menopause 2006;13:294–302. | Article | PubMed |
33. Matsunaga T, Weihrauch DW, Moniz MC, Tessmer J, Warltier DC, Chilian WM. Angiostatin inhibits coronary angiogenesis during impaired production of nitric oxide. Circulation 2002;105:2185–2191. | Article | PubMed | ISI | ChemPort |
34. Owens MW, Milligan SA, Jourd'heuil D, Grisham MB. Effects of reactive metabolites of oxygen and nitrogen on gelatinase A activity. Am J Physiol-Lung Cell Mol Physiol 1997;273:L445–L450.
35. Thornalley PJ. Use of aminoguanidine (Pimagedine) to prevent the formation of advanced glycation endproducts. Arch Biochem Biophys 2003;419:31–40. | Article | PubMed | ISI | ChemPort |
36. Safar ME, Smulyan H. Hypertension in women. Am J Hypertens 2004;17:82–87. | Article | PubMed |

Acknowledgments

This work was supported in part by research grants from the National Science Council (NSC 94-2320-B-016-039 to Y.-M.L.), Taipei, Taiwan.