Aldosterone and not plasminogen activator inhibitor-1 is a critical mediator of early angiotensin II/N-G-nitro-L-arginine methyl ester-induced myocardial injury
ABSTRACT Angiotensin II (Ang II) increases levels of aldosterone and plasminogen activator inhibitor-1 (PAI-1). Both aldosterone and PAI-1 seem to promote cardiovascular (CV) injury. Our objective was to determine the roles of PAI-1 and aldosterone in the development of myocardial and renal damage in a model with high Ang II and low nitric oxide (NO) availability, a pattern seen in patients with heart failure, diabetes mellitus, and arteriosclerosis.
Mice on a moderately high sodium diet were treated with the NO synthase inhibitor NG-nitro-l-arginine methyl ester (L-NAME) for 14 days plus Ang II during days 8 through 14. The roles of aldosterone and PAI-1 in the development of CV injury were assessed using the mineralocorticoid receptor antagonist spironolactone (0, 1.5, 15, and 50 mg x 100 g(-1) x day(-1)) and PAI-1-deficient mice (PAI-1-/-). Ang II/L-NAME-treated mice showed glomerular ischemia, proteinuria, and necrosis of myocytes and vascular smooth muscle cells with an associated mixed inflammatory response, deposition of loose collagen, and neovascularization. Compared with saline-drinking mice, Ang II/L-NAME-treated mice had significantly increased heart to body weight (HW/BW) ratios, cardiac and renal damage assessed by histological examination, PAI-1 immunoreactivity, and proteinuria. Spironolactone treatment decreased PAI-1 immunoreactivity and reduced in a dose-dependent fashion cardiac and renal damage. PAI-1-/- animals had a similar degree of CV injury as PAI-1+/+ animals.
Mineralocorticoid receptor antagonism, but not PAI-1 deficiency, protected mice from developing Ang II/L-NAME-mediated myocardial and vascular injury and proteinuria, suggesting that aldosterone, but not PAI-1, plays a key role in the development of early Ang II/L-NAME-induced cardiovascular injury.
- SourceAvailable from: Gail K Adler
[Show abstract] [Hide abstract]
- "For example, E2 treatment increased cardiac AT 1 R levels and activation of either ERs or AT 1 R can increase PKC-ERK pathways and PAI-1 (Alexaki VI et al. 2006; Smith LH et al. 2004). In our study we used cardiac PAI-1 levels as a marker of early cardiovascular injury as previously described (Oestreicher EM et al. 2003). While E2 treatment increased PAI-1 and other mediators of cardiovascular injury, the increase in these factors were not associated with a detectable increase in cardiac injury histopathology, possibly due to relative insensitivity of this method and to the relatively short duration of treatment. "
ABSTRACT: We tested the hypothesis that 17beta-estradiol (E(2)) has dual effects on the heart, increasing levels of proteins thought to have beneficial cardiovascular effects (e.g. endothelial nitric oxide (NO) synthase (eNOS)) as well as those thought to have detrimental cardiovascular effects (e.g. type 1 angiotensin II (AngII) receptor (AT(1)R)). Ovariectomized Wistar rats consuming a high-sodium diet received one of four treatments (n=7 per group): group 1, placebo pellets; group 2, E(2) (0 x 5 mg/pellet, 21-day release); group 3, NOS inhibitor, N(omega)-nitro-L-arginine-methyl-ester (L-NAME; 40 mg/kg per day for 14 days) plus Ang II (0 x 225 mg/kg per day on days 11-14); group 4, E(2) plus L-NAME/Ang II. E(2) increased cardiac levels of estrogen receptors ESR1 and ESR2, an ESR-associated membrane protein caveolin-3, eNOS, and phosphorylated (p)eNOS, thus, exerting potentially beneficial cardiovascular effects on NO. However, E(2) also increased cardiac levels of proteins associated with cardiovascular injury and inflammation including, AT(1)R, protein kinase C delta (PRKCD), phosphorylated PRKC, and phosphorylated extracellular signal regulated kinase (pMAPK)3/1, plasminogen activator inhibitor-1 (PAI-1), osteopontin and ED-1, a monocyte/macrophage-specific protein. E(2) treatment led to similar protein changes in the hearts of L-NAME/Ang II-treated rats except that the increase in peNOS was prevented, and L-NAME/Ang II and E(2) had additive effects in increasing cardiac PRKCD and PAI-1. Thus, the highest levels of cardiac PAI-1 and PRKCD occurred in L-NAME/Ang II-treated rats receiving E(2). In summary, E(2) treatment increased cardiac expression of AT(1)R as well as the expression of pro-inflammatory and prothrombotic factors.Journal of Endocrinology 11/2008; 200(1):75-84. DOI:10.1677/JOE-08-0199
[Show abstract] [Hide abstract]
- "We chose to further test the NO pathway using the more sensitive RT-PCR technique and using larger mouse tissues such as the heart. We have previously shown that chronic HS diet in WT mice or in rats is not associated with cardiac tissue damage (Turchin et al., 2006; Rocha et al., 2000; Martinez et al., 2002; Oestreicher et al., 2003). We hypothesized that Cav-1 deficiency during HS diet may affect eNOS expression/activity not only in the blood vessels, but also in the heart. "
ABSTRACT: Changes in dietary sodium intake are associated with changes in vascular volume and reactivity that may be mediated, in part, by alterations in endothelial nitric oxide synthase (eNOS) activity. Caveolin-1 (Cav-1), a transmembrane anchoring protein in the plasma membrane caveolae, binds eNOS and limits its translocation and activation. To test the hypothesis that endothelial Cav-1 participates in the dietary sodium-mediated effects on vascular function, we assessed vascular responses and nitric oxide (NO)-mediated mechanisms of vascular relaxation in Cav-1 knockout mice (Cav-1-/-) and wild-type control mice (WT; Cav-1+/+) placed on a high-salt (HS; 4% NaCl) or low-salt (LS; 0.08% NaCl) diet for 16 days. After the systolic blood pressure was measured, the thoracic aorta was isolated for measurement of vascular reactivity and NO production, and the heart was used for measurement of eNOS expression and/or activity. The blood pressure was elevated in HS mice treated with NG-nitro-l-arginine methyl ester and more so in Cav-1-/- than WT mice and was significantly reduced during the LS diet. Phenylephrine caused vascular contraction that was significantly reduced in Cav-1-/- (maximum 0.25 +/- 0.06 g/mg) compared with WT (0.75 +/- 0.22 g/mg) on the HS diet, and the differences were eliminated with the LS diet. Also, vascular contraction in response to membrane depolarization by high KCl (96 mM) was reduced in Cav-1-/- (0.27 +/- 0.05 g/mg) compared with WT mice (0.53 +/- 0.12 g/mg) on the HS diet, suggesting that the reduced vascular contraction is not limited to a particular receptor. Acetylcholine (10(-5) M) caused aortic relaxation in WT mice on HS (23.6 +/- 3.5%) and LS (23.7 +/- 5.5%) that was enhanced in Cav-1-/- HS (72.6 +/- 6.1%) and more so in Cav-1-/- LS mice (93.6 +/- 3.5%). RT-PCR analysis indicated increased eNOS mRNA expression in the aorta and heart, and Western blots indicated increased total eNOS and phosphorylated eNOS in the heart of Cav-1-/- compared with WT mice on the HS diet, and the genotypic differences were less apparent during the LS diet. Thus Cav-1 deficiency during the HS diet is associated with decreased vasoconstriction, increased vascular relaxation, and increased eNOS expression and activity, and these effects are altered during the LS diet. The data support the hypothesis that endothelial Cav-1, likely through an effect on eNOS activity, plays a prominent role in the regulation of vascular function during substantial changes in dietary sodium intake.AJP Heart and Circulatory Physiology 04/2008; 294(3):H1258-65. DOI:10.1152/ajpheart.01014.2007
- [Show abstract] [Hide abstract]
ABSTRACT: Matrixmetalloproteinasen beeinflussen die Entstehung, die Progression und die Komplikationen der Atherosklerose. Sie spielen eine wichtige Rolle beim Abbau der vaskulären Basalmembran und der Extrazellularmatrix. Vermutlich sind sie über diese Mechanismen und über die Beeinflussung der Endothelzellbarriere wesentlich an der Pathogenese der Atherosklerose beteiligt. Im ersten Teil der Arbeit beschäftigten wir uns mit der Frage, ob MMPs durch Endothelzellen gebildet werden können. Bei unseren Versuchen konnten wir die Präsenz von MMP-2 und MMP-9 im Überstand von humanen Nabelschnurvenenendothelzellen (HUVECs) nachweisen. Somit sind menschliche Endothelzellen direkt an der lokalen Produktion von MMPs beteiligt. Im zweiten Teil der Arbeit wurde untersucht, wie MMP-2 in den Nabelschnurendothelien gebildet und reguliert wird. MMP-2 wird in Endothelzellen zeitabhängig sezerniert. Bradykinin führt dosisabhängig zu einer gesteigerten pro-MMP-2 Sekretion. Bei der Stimulation mit PMA und Interleukin 1-beta wird demgegenüber vermehrt intermediäres und aktives MMP-2 im Überstand detektiert. Nach der Vorbehandlung der Zellen mit dem Proteinsynthesehemmer Cycloheximid war nach 24 Stunden nahezu kein pro-MMP-2 mehr im Überstand der Endothelzellkulturen nachweisbar. Das Tubulusgift Colchicin führte zu einer signifikanten Verringerung der freigesetzten pro-MMP-2 Menge. Es ließ sich jedoch noch eine geringe Menge an intermediären und aktiven MMP-2 nachweisen. In weiteren Experimenten sollte untersucht werden, ob die Komponenten des Renin-Angiotensin-Aldosteron Systems, welche als wichtige Faktoren in der Pathogenese der Atherosklerose diskutiert werden, die endotheliale Synthese und Freisetzung von MMP-2 regulieren. Dabei zeigte sich, dass Aldosteron einen signifikanten stimulatorischen Effekt auf die pro-MMP-2 Menge im Überstand der HUVECs besitzt. Im Gegensatz dazu ließ sich bei der Stimulation der Endothelzellen mit Angiotensin II nur eine tendenziell vorhandene Erhöhung der Angiotensin II induzierten MMP-2 Bildung nachweisen. Zusammenfassend ist von einer Beeinflussung der endothelialen MMP-Bildung durch Komponenten des RAAS auszugehen. Die geringe Stimulierbarkeit der endothelialen MMP und die hohe interexperimentelle Variabilität der MMP-Bildung lassen jedoch keine abschließende Bewertung zu. In Zukunft wird es sicherlich notwendig sein, weitere Studien (evtl. mit molekularbiologischen Ansätzen) zur näheren Charakterisierung des Zusammenhanges zwischen RAAS und der MMP-Bildung in HUVECs durchzuführen. Matrixmetalloproteinases play an important role in the formation, progression and the complications of atherosclerosis. Likely, Matrixmetalloproteinases are involved in the pathogensis of atherosclerosis by influencing the endothelial barrier by decomposing the basalmembrane and the extracellular matrix. It´s questionable whether endothelial cells produce MMPs and whether the renin-angiotensin-aldosterone system, which is presumed to play a role in the pathogenesis of atherosclerosis, could influence this production. In the first step of this study we detected the gelatinaes MMP-2 and MMP-9 in conditioned medium of HUVECs (human umbilical vein endothelial cells). Zymographic analysis revealed gelatinolytic activity at 72 (pro-MMP-2), 64 (intermediate MMP-2) and 62 kD (active MMP-2). The treatment of HUVECs with bradykinin led to an increased secretion of pro-MMP-2. When we stimulated HUVECs with PMA as well as IL-1β, there was an increased appearence of intermediate and active MMP-2 in the conditioned medium. After the incubation with Cycloheximide, a proteinsynthesis inhibitor, little pro-MMP-2 was found. That means, that most of pro-MMP-2 must have been syntheticized de-novo and that only a few Proteinases were already ready for secretion in vesicels. The transport of these vesicles can be hindered with Colchicin. In the next step we investigated the influence of the renin-angiotensin-aldosterone-system upon the endothelial MMP-2. Aldosterone led to an increased appearence of pro-MMP-2 in the conditioned medium. In contrast to that we found only a tendency of increased pro-MMP-2 in the angiotensin II conditioned medium. In summary we proceed from the assumption that the RAA-System could influence the endothelial MMP-Production. However the low ability to stimulate the endothelial MMP and the high interexperimentel variability don´t allow a final valuation. In the future it will be necessary to carry out more studies to characterize the correlation between MMPs and the renin-angiotensin-aldosterone system.