Modulation of angiotensin II and norepinephrine-induced plasminogen activator inhibitor-1 expression by AT1a receptor deficiency
2007; Elsevier BV; Volume: 72; Issue: 1 Linguagem: Inglês
10.1038/sj.ki.5002268
ISSN1523-1755
AutoresNancy J. Brown, Jennifer Bradford, Z. Wang, William Lea, Lijun Ma, Ji Ma, Douglas E. Vaughan, Agnes B. Fogo,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoAngiotensin (Ang) II stimulates plasminogen activator inhibitor-1 (PAI-1) expression in many cell types by mechanisms that are cell-type specific. We measured effects of Ang II or norepinephrine on PAI-1 expression in wild type (WT) and Ang type-1a receptor knockout mice (AT1a-/-) in the presence or absence of the non-specific AT1 antagonist losartan. Ang II and norepinephrine increased systolic blood pressure equally, whereas losartan decreased the pressor response of the former but not the latter in WT mice. In AT1a-/- mice, baseline systolic blood pressure was lower with no effect of Ang II, norepinephrine, or losartan. Ang II stimulated PAI-1 expression in the heart, aorta, and kidney and markedly in the liver of WT mice. In AT1a-/- mice, Ang II-stimulated PAI-1 was significantly attenuated compared with the WT in the heart and aorta but significantly enhanced in the kidney. Losartan decreased the induction in the aorta and liver of WT, and in the kidney and liver of AT1a-/- mice. Norepinephrine increased PAI-1 expression in WT heart and aorta, and in AT1a-/- heart, kidney, and liver with no effect of losartan. Renal PAI-1 expression correlated with AT1b receptor mRNA. We conclude that Ang II stimulates PAI-1 expression in part through the AT1b receptor in the kidney and liver. Further, norepinephrine induces PAI-1 expression in vivo with AT1a receptor deficiency modulating the effect. Angiotensin (Ang) II stimulates plasminogen activator inhibitor-1 (PAI-1) expression in many cell types by mechanisms that are cell-type specific. We measured effects of Ang II or norepinephrine on PAI-1 expression in wild type (WT) and Ang type-1a receptor knockout mice (AT1a-/-) in the presence or absence of the non-specific AT1 antagonist losartan. Ang II and norepinephrine increased systolic blood pressure equally, whereas losartan decreased the pressor response of the former but not the latter in WT mice. In AT1a-/- mice, baseline systolic blood pressure was lower with no effect of Ang II, norepinephrine, or losartan. Ang II stimulated PAI-1 expression in the heart, aorta, and kidney and markedly in the liver of WT mice. In AT1a-/- mice, Ang II-stimulated PAI-1 was significantly attenuated compared with the WT in the heart and aorta but significantly enhanced in the kidney. Losartan decreased the induction in the aorta and liver of WT, and in the kidney and liver of AT1a-/- mice. Norepinephrine increased PAI-1 expression in WT heart and aorta, and in AT1a-/- heart, kidney, and liver with no effect of losartan. Renal PAI-1 expression correlated with AT1b receptor mRNA. We conclude that Ang II stimulates PAI-1 expression in part through the AT1b receptor in the kidney and liver. Further, norepinephrine induces PAI-1 expression in vivo with AT1a receptor deficiency modulating the effect. Activation of the renin–angiotensin–aldosterone system causes vascular toxicity in part through effects of angiotensin (Ang) II to induce the expression of plasminogen activator inhibitor-1 (PAI-1),1Vaughan D.E. Lazos S.A. Tong K. Angiotensin II regulates the expression of plasminogen activator inhibitor-1 in cultured endothelial cells.J Clin Invest. 1995; 95: 995-1001Crossref PubMed Scopus (505) Google Scholar,2Feener E.P. Northrup J.M. Aiello L.P. King G.L. Angiotensin II induces plasminogen activator inhibitor-1 and -2 expression in vascular endothelial and smooth muscle cells.J Clin Invest. 1995; 95: 1353-1362Crossref PubMed Scopus (254) Google Scholar a major physiological inhibitor of fibrinolysis, in vitro and in vivo.3Saksela O. Rifkin D.B. Cell-associated plasminogen activation:regulation and physiologic functions.Annu Rev Cell Biol. 1988; 4: 93-126Crossref PubMed Scopus (706) Google Scholar Increased PAI-1 has been implicated in the pathogenesis of coronary arterial thrombosis and myocardial infarction in mouse models4Eren M. Painter C.A. Atkinson J.B. et al.Age-dependent spontaneous coronary arterial thrombosis in transgenic mice that express a stable form of human plasminogen activator inhibitor-1.Circulation. 2002; 106: 491-496Crossref PubMed Scopus (151) Google Scholar and with increased risk of atherosclerotic thrombotic events in studies in humans.5Thogersen A.M. Jansson J.H. Boman K. et al.High plasminogen activator inhibitor and tissue plasminogen activator levels in plasma precede a first acute myocardial infarction in both men and women: evidence for the fibrinolytic system as an independent primary risk factor.Circulation. 1998; 98: 2241-2247Crossref PubMed Scopus (566) Google Scholar,6Collet J.P. Montalescot G. Vicaut E. et al.Acute release of plasminogen activator inhibitor-1 in ST-segment elevation myocardial infarction predicts mortality.Circulation. 2003; 108: 391-394Crossref PubMed Scopus (137) Google Scholar In addition, by preventing the activation of matrix metalloproteinases and the degradation of extracellular proteins by plasmin, PAI-1 promotes fibrosis.7Eitzman D.T. McCoy R.D. Zheng X. et al.Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene.J Clin Invest. 1996; 97: 232-237Crossref PubMed Scopus (511) Google Scholar, 8Kaikita K. Fogo A.B. Ma L.-J. et al.Plasminogen activator inhibitor-1 deficiency prevents hypertension and vascular fibrosis in response to chronic nitric oxide synthase inhibition.Circulation. 2001; 104: 839-844Crossref PubMed Scopus (147) Google Scholar, 9Huang Y. Haraguchi M. Lawrence D.A. et al.A mutant, noninhibitory plasminogen activator inhibitor type 1 decreases matrix accumulation in experimental glomerulonephritis.J Clin Invest. 2003; 112: 379-388Crossref PubMed Scopus (125) Google Scholar, 10Ingelfinger J.R. Forestalling fibrosis.N Engl J Med. 2003; 349: 2265-2266Crossref PubMed Scopus (17) Google Scholar In the kidney, increased PAI-1 expression contributes to the pathogenesis of both glomerular injury and tubulointerstitial fibrosis.9Huang Y. Haraguchi M. Lawrence D.A. et al.A mutant, noninhibitory plasminogen activator inhibitor type 1 decreases matrix accumulation in experimental glomerulonephritis.J Clin Invest. 2003; 112: 379-388Crossref PubMed Scopus (125) Google Scholar, 11Oda T. Jung Y.O. Kim H.S. et al.PAI-1 deficiency attenuates the fibrogenic response to ureteral obstruction.Kidney Int. 2001; 60: 587-596Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 12Ma J. Weisberg A. Griffin J.P. et al.Plasminogen activator inhibitor-1 deficiency protects against aldosterone-induced glomerular injury.Kidney Int. 2006; 69: 1064-1072Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar Ang II stimulates PAI-1 expression in vitro in a variety of cell types including adipocytes, astrocytes, endothelial cells, vascular smooth muscle cells (VSMCs), proximal tubular epithelial cells, and mesangial cells.1Vaughan D.E. Lazos S.A. Tong K. Angiotensin II regulates the expression of plasminogen activator inhibitor-1 in cultured endothelial cells.J Clin Invest. 1995; 95: 995-1001Crossref PubMed Scopus (505) Google Scholar, 2Feener E.P. Northrup J.M. Aiello L.P. King G.L. Angiotensin II induces plasminogen activator inhibitor-1 and -2 expression in vascular endothelial and smooth muscle cells.J Clin Invest. 1995; 95: 1353-1362Crossref PubMed Scopus (254) Google Scholar, 13Rydzewski B. Zelezna B. Tang W. et al.Angiotensin II stimulation of plasminogen activator inhibitor-1 gene expression in astroglial cells from the brain.Endocrinology. 1992; 130: 1255-1262Crossref PubMed Scopus (45) Google Scholar, 14Gesualdo L. Ranieri E. Monno R. et al.Angiotensin IV stimulates plasminogen activator inhibitor-1 expression in proximal tubular epithelial cells.Kidney Int. 1999; 56: 461-470Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 15Skurk T. Lee Y.M. Hauner H. Angiotensin II and its metabolites stimulate PAI-1 protein release from human adipocytes in primary culture.Hypertension. 2001; 37: 1336-1340Crossref PubMed Scopus (114) Google Scholar The mechanism through which Ang II stimulates PAI-1 expression depends on the cell type. For example, in endothelial cells and proximal tubular epithelial cells, the effect of Ang II on PAI-1 expression appears to be at least partially mediated through its hexapeptide metabolite angiotensin-3–8 (Ang IV).14Gesualdo L. Ranieri E. Monno R. et al.Angiotensin IV stimulates plasminogen activator inhibitor-1 expression in proximal tubular epithelial cells.Kidney Int. 1999; 56: 461-470Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 16Kerins D.M. Hao Q. Vaughan D.E. Angiotensin induction of PAI-1 expression in endothelial cells is mediated by the hexapeptide angiotensin IV.J Clin Invest. 1995; 96: 2515-2520Crossref PubMed Scopus (300) Google Scholar, 17Mehta J.L. Li D.Y. Yang H. Raizada M.K. Angiotensin II and IV stimulate expression and release of plasminogen activator inhibitor-1 in cultured human coronary artery endothelial cells.J Cardiovasc Pharmacol. 2002; 39: 789-794Crossref PubMed Scopus (40) Google Scholar Thus, administration of pharmacological agents that block the conversion of Ang II to Ang IV prevents the effect of Ang II on PAI-1 expression. Similarly, Ang II-induced endothelial PAI-1 expression is blocked by AT4 receptor antagonists, but not by selective AT1 or AT2 receptor antagonists.16Kerins D.M. Hao Q. Vaughan D.E. Angiotensin induction of PAI-1 expression in endothelial cells is mediated by the hexapeptide angiotensin IV.J Clin Invest. 1995; 96: 2515-2520Crossref PubMed Scopus (300) Google Scholar In contrast, AT1 receptor antagonism decreases PAI-1 expression in astrocytes, VSMCs, and adipocytes.2Feener E.P. Northrup J.M. Aiello L.P. King G.L. Angiotensin II induces plasminogen activator inhibitor-1 and -2 expression in vascular endothelial and smooth muscle cells.J Clin Invest. 1995; 95: 1353-1362Crossref PubMed Scopus (254) Google Scholar, 13Rydzewski B. Zelezna B. Tang W. et al.Angiotensin II stimulation of plasminogen activator inhibitor-1 gene expression in astroglial cells from the brain.Endocrinology. 1992; 130: 1255-1262Crossref PubMed Scopus (45) Google Scholar, 15Skurk T. Lee Y.M. Hauner H. Angiotensin II and its metabolites stimulate PAI-1 protein release from human adipocytes in primary culture.Hypertension. 2001; 37: 1336-1340Crossref PubMed Scopus (114) Google Scholar Studies in the rat provide conflicting data as to whether Ang II increases cardiovascular and renal PAI-1 expression in vivo through the AT1 receptor or via conversion of Ang II to Ang IV and activation of the AT4 receptor. We have reported previously that AT1 receptor antagonism abolished acute Ang II-induced hepatic, renal, aortic, and cardiac PAI-1 expression.18Nakamura S. Nakamura I. Ma L. et al.Plasminogen activator inhibitor-1 expression is regulated by the angiotensin type 1 receptor in vivo.Kidney Int. 2000; 58: 251-259Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar Likewise, AT1 receptor antagonism decreased Ang II-induced aortic and cardiac PAI-1 expression in Sprague–Dawley rats.19Chen H.C. Bouchie J.L. Perez A.S. et al.Role of the angiotensin AT(1) receptor in rat aortic and cardiac PAI-1 gene expression.Arterioscler Thromb Vasc Biol. 2000; 20: 2297-2302Crossref PubMed Google Scholar On the other hand, 2-week infusion of either Ang II or Ang IV significantly increased cardiac PAI-1 expression, whereas only Ang II increased renal PAI-1 expression.20Abrahamsen C.T. Pullen M.A. Schnackenberg C.G. et al.Effects of angiotensins II and IV on blood pressure, renal function, and PAI-1 expression in the heart and kidney of the rat.Pharmacology. 2002; 66: 26-30Crossref PubMed Scopus (17) Google Scholar However, none of these studies delineated the role of specific AT1 receptor subtypes in Ang II-stimulated PAI-1 expression. We therefore tested the hypothesis that Ang II induces PAI-1 expression via the AT1a receptor in vivo. To do this, we compared the effect of a 4-h infusion of Ang II or an equivalent pressor dose of norepinephrine on cardiac, aortic, renal, and hepatic PAI-1 expression in wild type (WT) mice and in mice lacking the AT1a receptor (AT1a-/-), in the presence and absence of the non-specific AT1 receptor antagonist losartan. Figure 1 illustrates the effect of Ang II or norepinephrine on systolic blood pressure (SBP) in WT and AT1a-/- mice. Ang II (mean SBP over 4 h 125.9±5.2 mm Hg, P=0.004 versus saline) or norepinephrine (mean SBP 127.0±4.4 mm Hg, P=0.002) significantly increased pressure compared with normal saline (mean SBP 103.0±5.5 mm Hg) in the WT mice. The pressor effects of Ang II and norepinephrine were similar (P=0.877). Pre-treatment with losartan significantly decreased baseline SBP (Figure 1, P=0.002) and attenuated the SBP response to Ang II (mean SBP 108.8±7.4 mm Hg, P=0.030 versus Ang II alone during first 3 h, P=0.530 versus saline) in WT mice. There was no effect of losartan on the pressor response to norepinephrine (mean SBP during norepinephrine and losartan 125.3±7.4 mm Hg). Baseline SBP was significantly lower in AT1a-/- mice compared to WT (Figure 1, P<0.001). Moreover, there was no effect of either Ang II (P=0.778) or norepinephrine (P=0.648) on SBP in AT1a-/- mice. Pre-treatment with losartan abolished the difference in baseline SBP between WT and AT1a-/- mice (P=0.890); there was no effect of losartan on SBP during Ang II or norepinephrine in AT1a-/- mice. Table 1 shows the effect of treatment on plasma aldosterone concentrations in WT and AT1a-/- mice. Ang II only numerically tended to increase aldosterone in WT mice. However, aldosterone concentrations were significantly higher in Ang II-infused WT mice compared with Ang II-infused AT1a-/- mice. In addition, losartan significantly decreased aldosterone concentrations in Ang II- and norepinephrine-infused WT mice, but did not significantly affect aldosterone concentrations in AT1a-/- mice.Table 1Effect of treatment on plasma aldosterone concentrationsWild typeAT1a-/-Vehicle512.9±273.4246.5±54.9Ang II616.4±73.8352.9±80.6*Ang II+losartan158.6±43.7†,‡199.8±10.7Norepinephrine595.7± 81.3431.2±107.1Norepinephrine+losartan124.6±18.1†,‡294.2±96.8Plasma aldosterone concentrations are given in pg/ml.*P<0.05 versus wild type Ang II, †P<0.05 versus wild type Ang II, ‡P<0.05 versus wild type norepinephrine. Open table in a new tab Plasma aldosterone concentrations are given in pg/ml. *P<0.05 versus wild type Ang II, †P<0.05 versus wild type Ang II, ‡P<0.05 versus wild type norepinephrine. Ang II increased PAI-1 expression in heart (9.9-fold, P<0.001 versus saline), aorta (4.6-fold, P<0.05), kidney (2.5-fold, P<0.05), and liver (97-fold, P<0.001), respectively, of WT mice (Figure 2). Losartan attenuated Ang II-induced PAI-1 expression in the aorta (1.8-fold, NS versus saline) and liver (9.8-fold, P<0.01 versus Ang II alone). In AT1a-/- mice, Ang II-induced PAI-1 expression was diminished in the heart (P<0.05 versus WT Ang II) and aorta (P<0.05 versus WT Ang II), equivalent in the liver (43-fold versus WT saline), but enhanced in kidney (9.1-fold versus WT saline, P<0.05 versus WT Ang II) compared with WT mice. Losartan attenuated Ang II-induced PAI-1 expression in the kidney (1.6-fold versus WT saline, P<0.05 versus Ang II alone) and liver (11.9-fold, P<0.05 versus Ang II alone) of AT1a-/- mice. To determine whether the effect of Ang II on PAI-1 expression was pressor-dependent, we assessed whether norepinephrine increased PAI-1 expression in WT or AT1a-/- mice. Norepinephrine increased PAI-1 expression in the heart (15.6-fold) and aorta (7.4-fold) but not the kidney or liver of WT mice. Even though norepinephrine did not increase blood pressure, norepinephrine increased PAI-1 expression in heart (8.2-fold versus WT saline, P<0.001), kidney (7.7-fold, P<0.01), and liver (50.9-fold, P<0.001) in AT1a-/- mice. Losartan did not affect norepinephrine induced PAI-1 expression. To determine whether norepinephrine induced PAI-1 mRNA expression via an effect on TGF-β expression, we measured TGF-β mRNA expression in the heart, kidney, and liver (Table 2). Norepinephrine did not increase TGF-β expression in any organ in WT or AT1a-/- mice. TGF-β expression was similar in the heart of WT and AT1a-/- mice. TGF-β expression was decreased in the liver of AT1a-/- mice compared with WT mice, regardless of treatment. On the other hand, renal TGF-β expression was increased in saline-treated and norepinephrine+losartan-treated AT1a-/- mice compared with WT mice. Renal TGF-β mRNA expression correlated with renal PAI-1 expression in AT1a-/- mice (r=0.783, P<0.001), but not in WT mice (r=0.194, P=0.441). However, there was no relationship between relative renal pSmad2 protein, a marker of TGF-β signaling, and renal PAI-1 expression (P=0.582).Table 2Effect of treatment on TGF-β mRNA expressionRelative mRNA expressionPSmad2/α-tubulin proteinWild typeAT1aP-valueWild typeAT1aP-valueHeart Saline1.05±0.200.89±0.18NS Ang II0.80±0.100.57±0.20NS Ang II+losartan0.76±0.080.45±0.08*NS NE1.12±0.240.79±0.24NS NE+losartan0.83±0.190.83±0.19NSKidney Saline1.00±0.061.69±0.410.0080.82±0.220.45±0.06NS Ang II0.79±0.110.86±0.18†NS0.85±0.140.59±0.10NS Ang II+losartan1.29±0.070.75±0.06†0.0260.71±0.131.02±0.23*NS NE0.85±0.171.16±0.07*NS0.81±0.130.66±0.12NS NE+losartan0.52±0.06*1.09±0.16*0.0200.94±0.110.77±0.11NSLiver Saline1.00±0.080.16±0.050.012 Ang II0.88±0.140.31±0.120.050 Ang II+losartan0.83±0.090.17±0.070.043 NE0.73±0.320.14±0.020.030 NE+losartan0.73±0.390.11±0.020.057NS, not significant.*P<0.05 versus saline, †P≤0.001 versus saline. Open table in a new tab NS, not significant. *P<0.05 versus saline, †P≤0.001 versus saline. Ang II also significantly increased circulating active plasma PAI-1 antigen in WT and AT1a-/- mice and losartan prevented this effect (Figure 3). Norepinephrine significantly increased interleukin (IL)-6 and active PAI-1 antigen in AT1a-/- mice, but not in WT mice. Circulating active plasma PAI-1 antigen concentrations correlated with renal (r=0.904, P<0.001) and hepatic (r=0.731, P<0.001) PAI-1 expression. In addition, IL-6 (r=0.632, P=0.006) and active PAI-1 antigen (r=0.666, P=0.003) correlated with plasma aldosterone concentrations in AT1a-/- mice, but not in WT mice. After controlling for losartan treatment, PAI-1 mRNA expression in the kidney of AT1a-/- mice also correlated significantly with circulating aldosterone (r=0.433, P=0.04). There was no correlation between renal PAI-1 expression and aldosterone in WT mice. There was no correlation between cardiac, hepatic, or aortic PAI-1 mRNA expression and aldosterone in either strain. To determine whether differences in patterns of PAI-1 mRNA expression in the kidney and liver of WT and AT1a-/- mice resulted from differences in expression of the AT1b receptor, we measured AT1a and AT1b receptor mRNA expression. In WT mice, Ang II decreased AT1a receptor expression in the kidney and liver, even during losartan (Table 3). Norepinephrine alone also decreased AT1a receptor expression in WT mice. As expected, AT1a receptor mRNA expression was reduced to artifactual levels in the AT1a-/- mice. Renal and hepatic AT1b receptor expression was statistically similar in WT and AT1a-/- mice. However, the relationship between AT1b receptor and PAI-1 mRNA expression differed in the kidneys of WT and AT1a-/- mice. Thus, while renal PAI-1 mRNA expression correlated with AT1b receptor expression in both WT (r=0.767, P<0.001) and AT1a-/- (r=0.514, P=0.020) mice, the slope of the relationship was significantly steeper in the AT1a-/- mice (slope 6.8 versus 0.5, P<0.001). Hepatic PAI-1 expression did not correlate with AT1a or AT1b receptor expression in WT or AT1a-/- mice.Table 3Effect of treatment on AT1a and AT1b receptor mRNA expressionRelative mRNA expressionWild typeAT1aP-valueKidney AT1a receptor Saline0.83±0.080.05±0.01<0.001 Ang II0.48±0.10*0.04±0.020.006 Ang II+losartan0.60±0.08†0.04±0.000.002 NE0.60±0.04†0.07±0.02<0.001 NE+losartan1.20±0.04*0.05±0.01<0.001 AT1b receptor Saline1.13±0.361.92±1.22NS Ang II3.34±1.30‡,§,∥,¶1.04±0.46NS Ang II+losartan1.26±0.200.62±0.110.029 NE0.48±0.060.61±0.07NS NE+losartan0.59±0.180.74±0.15NSLiver AT1a receptor Saline1.03±0.070.02±0.01<0.001 Ang II0.93±0.140.01±0.010.003 Ang II+losartan0.57±0.14‡0.07±0.060.03 NE0.55±0.09†0.01±0.000.001 NE+losartan0.62±0.190.00±0.000.03 AT1b receptor Saline1.57±1.040.52±0.05NS Ang II0.31±0.100.60±0.53NS Ang II+losartan0.57±0.120.30±0.06NS NE2.89±2.750.23±0.03NS NE+losartan12.85±12.820.26±0.11NSNE, norepinephrine; NS, not significant.*P<0.001 versus saline, †P<0.01 versus saline, ‡P<0.05 versus saline, §P<0.05 versus Ang II+losartan, ∥P<0.001 versus NE, ¶P<0.005 versus NE+losartan. Open table in a new tab NE, norepinephrine; NS, not significant. *P<0.001 versus saline, †P<0.01 versus saline, ‡P<0.05 versus saline, §P<0.05 versus Ang II+losartan, ∥P<0.001 versus NE, ¶P<0.005 versus NE+losartan. Figures 4 and 5 show the effect of saline, Ang II, and norepinephrine on PAI-1 mRNA signal in the heart and aorta from WT and AT1a-/- mice. Hybridization with control sense probes showed no specific signal. There was minimal expression of PAI-1 mRNA in cardiac myocytes and aorta by in situ hybridization in saline-infused WT or AT1a-/- mice. In Ang II-treated WT mice, PAI-1 mRNA signal was increased (2–3+) in the myocytes of the left ventricle and septum as well as in aortic smooth muscle cells (2–3+) and aortic endothelium (1+). In contrast, in AT1a-/- mice, Ang II infusion induced a variable, but overall modest (1–3+) increase in PAI-1 expression in the endocardium, but not in ventricular myocytes. Only trace PAI-1 mRNA signal was observed in aortae from Ang II-infused AT1a-/- mice. Infusion of norepinephrine induced a strong (3–4+) increase in PAI-1 mRNA signal in the myocytes of the left ventricle, a 1+ increase in signal in the myocytes of the right ventricle, and a 1–2+ increase in PAI-1 mRNA signal in the endocardium of hearts from WT mice; there was also a 1–2+ increase in PAI-1 mRNA signal in the vascular smooth muscle and endothelium of the aorta. Norepinephrine induced a 2+ increase in PAI-1 mRNA signal in the endocardium of AT1a-/- mice, with focal areas of increased PAI-1 mRNA signal in the left ventricle, with only trace signal in the aorta.Figure 5Effect of saline, Ang II, and norepinephrine on PAI-1 expression by in situ hybridization in aorta (original magnification × 400). There was minimal PAI-1 expression in the aorta in saline-infused WT mice (a) or AT1a-/- mice (e). Ang II stimulated a marked increase in PAI-1 expression in aortic VSMCs and a modest increase in aortic endothelium in WT mice (b). This was attenuated by pretreatment with losartan (c). Norepinephrine induced strong PAI-1 expression in aortic vascular smooth muscle and endothelium of WT mice (d). Only trace PAI-1 aortic signal was observed in Ang II-treated (f) or norepinephrine-treated (g) AT1a-/- mice.View Large Image Figure ViewerDownload (PPT) Figure 6 shows the effect of saline, Ang II, and norepinephrine on PAI-1 mRNA expression in the kidney from WT and AT1a-/- mice. During saline infusion, there was strong PAI-1 expression in the glomeruli of AT1a-/-, but not WT, mice. Ang II induced mild diffuse expression of PAI-1 in the kidney of both WT and AT1a-/- mice with 2+ increase in signal in the mesangium of AT1a-/- animals. Norepinephrine induced strong signal in the main renal artery and its major branches in both WT and AT1a-/-, whereas norepinephrine also increased PAI-1 focally in peritubular capillaries in the AT1a-/- animals. Following saline, the liver (not shown) of both WT and AT1a-/- mice showed similar very low-level diffuse PAI-1 signal. Ang II and norepinephrine induced intense signal in centrilobular hepatocytes in both WT and AT1a-/- mice. To test the hypothesis that Ang II stimulates PAI-1 expression via the AT1a receptor in vivo, we studied the effect of genetic AT1a deficiency and pharmacological AT1 receptor blockade on PAI-1 expression. The data suggest that Ang II regulates PAI-1 expression in the heart and aorta primarily via the AT1a receptor, whereas the AT1b receptor contributes significantly to Ang II-stimulated PAI-1 expression in the kidney and liver. In addition, this study provides the first evidence that norepinephrine regulates tissue PAI-1 expression in vivo. AT1a receptor deficiency was associated with decreased basal PAI-1 expression in the heart and aorta, but not in the kidney or liver. These data concur with the data of Tsujino et al.,21Tsujino T. Naito Y. Kawasaki D. et al.Circadian expression of plasminogen activator inhibitor-1 in angiotensin II type 1a receptor knockout mice.Clin Exp Hypertens. 2005; 27: 159-168Crossref PubMed Scopus (11) Google Scholar who reported that the effect of AT1a deficiency on circadian PAI-1 expression differs in an organ-specific fashion. In that study, the investigators found depressed trough PAI-1 expression in the heart and a lack of circadian variation in PAI-1 expression in the aorta of AT1a-/- mice, whereas hepatic PAI-1 expression continued to exhibit circadian rhythm and there was no difference in PAI-1 expression in the kidney of AT1a-/- versus WT mice. Previous studies using pharmacological receptor antagonists have consistently demonstrated that Ang II induces PAI-1 expression in vitro in cardiomyocytes22Chen H.C. Bouchie J.L. Perez A.S. et al.Role of the angiotensin AT(1) receptor in rat aortic and cardiac PAI-1 gene expression.Arterioscler Thromb Vasc Biol. 2000; 20: 2297-2302Crossref PubMed Scopus (46) Google Scholar and VSMCs22Chen H.C. Bouchie J.L. Perez A.S. et al.Role of the angiotensin AT(1) receptor in rat aortic and cardiac PAI-1 gene expression.Arterioscler Thromb Vasc Biol. 2000; 20: 2297-2302Crossref PubMed Scopus (46) Google Scholar,23Papakonstantinou E. Roth M. Kokkas B. et al.Losartan inhibits the angiotensin II-induced modifications on fibrinolysis and matrix deposition by primary human vascular smooth muscle cells.J Cardiovasc Pharmacol. 2001; 38: 715-728Crossref PubMed Scopus (38) Google Scholar and in vivo18Nakamura S. Nakamura I. Ma L. et al.Plasminogen activator inhibitor-1 expression is regulated by the angiotensin type 1 receptor in vivo.Kidney Int. 2000; 58: 251-259Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 20Abrahamsen C.T. Pullen M.A. Schnackenberg C.G. et al.Effects of angiotensins II and IV on blood pressure, renal function, and PAI-1 expression in the heart and kidney of the rat.Pharmacology. 2002; 66: 26-30Crossref PubMed Scopus (17) Google Scholar, 22Chen H.C. Bouchie J.L. Perez A.S. et al.Role of the angiotensin AT(1) receptor in rat aortic and cardiac PAI-1 gene expression.Arterioscler Thromb Vasc Biol. 2000; 20: 2297-2302Crossref PubMed Scopus (46) Google Scholar in the heart and aorta via its AT1 receptor. A recent study in cultured VSMCs from AT1a-/- suggests that Ang IV also induces PAI-1 expression in VSMCs. In this study, Ang II-induced cardiac and aortic PAI-1 expression was dramatically attenuated in AT1a-/- mice, indicating that the AT1a receptor primarily mediates increased cardiac and aortic PAI-1 expression in this in vivo model.24Esteban V. Ruperez M. Sanchez-Lopez E. et al.Angiotensin IV activates the nuclear transcription factor-kappaB and related proinflammatory genes in vascular smooth muscle cells.Circ Res. 2005; 96: 965-973Crossref PubMed Scopus (85) Google Scholar In contrast, the finding in kidney and liver that Ang II-induced PAI-1 expression was increased or preserved in AT1a-/- mice and that pharmacological AT1 receptor antagonism abolished this expression indicates that the AT1b receptor contributes to the regulation of PAI-1 expression in these organs. Indeed, the relationship between PAI-1 and AT1b receptor mRNA expression was enhanced in AT1a-/- compared with WT mice. In addition, the localization of PAI-1 signal to peritubular capillaries in the norepinephrine-treated AT1a-/- mice is in proximity to the tubular localization of AT1b receptors by immunohistochemistry.25Hoffmann A. Cool D.R. Characterization of two polyclonal peptide antibodies that recognize the carboxy terminus of angiotensin II AT1A and AT1B receptors.Clin Exp Pharmacol Physiol. 2005; 32: 936-943Crossref PubMed Scopus (4) Google Scholar These findings are compatible with prior data indicating that non-specific pharmacological AT1 receptor antagonism18Nakamura S. Nakamura I. Ma L. et al.Plasminogen activator inhibitor-1 expression is regulated by the angiotensin type 1 receptor in vivo.Kidney Int. 2000; 58: 251-259Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar decreases PAI-1 expression in these organs. We also report that norepinephrine-induced PAI-1 expression in the heart and aorta of WT mice and the heart of AT1a-/- mice. Of note, AT1a deficiency unmasked an effect of norepinephrine on PAI-1 expression in the kidney and liver, while at the same time the pressor response to norepinephrine was blunted. This study did not specifically address the mechanism through which norepinephrine increases PAI-1 expression. Norepinephrine increases cardiomyocyte expression of TGF-β in male Sprague–Dawley rats26Briest W. Homagk L. Rassler B. et al.Norepinephrine-induced changes in cardiac transforming growth factor-beta isoform expression pattern of female and male rats.Hypertension. 2004; 44: 410-418Crossref PubMed Scopus (45) Google Scholar and enhances the effect of TGF-β on PAI-1 expression in cardiac fibroblasts;27Akiyama-Uchida Y. Ashizawa N. Ohtsuru A. et al.Norepinephrine enhances fibrosis mediated by TGF-beta in cardiac fibroblasts.Hypertension. 2002
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