Chronic hypertension induces cardiac remodeling, including left ventricular hypertrophy and fibrosis, through a combination of both hemodynamic and humoral factors. In previous studies, we showed that the heptapeptide ANG-(1-7) prevented mitogen-stimulated growth of cardiac myocytes in vitro, through a reduction in the activity of the MAPKs ERK1 and ERK2. In this study, saline- or ANG II-infused rats were treated with ANG-(1-7) to determine whether the heptapeptide reduces myocyte hypertrophy in vivo and to identify the signaling pathways involved in the process. ANG II infusion into normotensive rats elevated systolic blood pressure >50 mmHg, in association with increased myocyte cross-sectional area, ventricular atrial natriuretic peptide mRNA, and ventricular brain natriuretric peptide mRNA. Although infusion with ANG-(1-7) had no effect on the ANG II-stimulated elevation in blood pressure, the heptapeptide hormone significantly reduced the ANG II-mediated increase in myocyte cross-sectional area, interstitial fibrosis, and natriuretic peptide mRNAs. ANG II increased phospho-ERK1 and phospho-ERK2, whereas cotreatment with ANG-(1-7) reduced the phosphorylation of both MAPKs. Neither ANG II nor ANG-(1-7) altered the ERK1/2 MAPK kinase MEK1/2. However, ANG-(1-7) infusion, with or without ANG II, increased the MAPK phosphatase dual-specificity phosphatase (DUSP)-1; in contrast, treatment with ANG II had no effect on DUSP-1, suggesting that ANG-(1-7) upregulates DUSP-1 to reduce ANG II-stimulated ERK activation. These results indicate that ANG-(1-7) attenuates cardiac remodeling associated with a chronic elevation in blood pressure and upregulation of a MAPK phosphatase and may be cardioprotective in patients with hypertension.

Rationale There is growing evidence that the myocardium responds to injury by recruiting c-kit+ cardiac progenitor cells to the damage tissue. Even though the ability of exogenously introducing c-kit+ cells to injured myocardium has been established, the capability of recruiting these cells through modulation of local signaling pathways by gene transfer has not been tested. Objective To determine whether stem cell factor gene transfer mediates cardiac regeneration in a rat myocardial infarction model, through survival and recruitment of c-kit+ progenitors and cell-cycle activation in cardiomyocytes, and explore the mechanisms involved. Methods and Results Infarct size, cardiac function, cardiac progenitor cells recruitment, fibrosis, and cardiomyocyte cell-cycle activation were measured at different time points in controls (n=10) and upon stem cell factor gene transfer (n=13) after myocardial infarction. We found a regenerative response because of stem cell factor overexpression characterized by an enhancement in cardiac hemodynamic function: an improvement in survival; a reduction in fibrosis, infarct size and apoptosis; an increase in cardiac c-kit+ progenitor cells recruitment to the injured area; an increase in cardiomyocyte cell-cycle activation; and Wnt/β-catenin pathway induction. Conclusions Stem cell factor gene transfer induces c-kit+ stem/progenitor cell expansion in situ and cardiomyocyte proliferation, which may represent a new therapeutic strategy to reverse adverse remodeling after myocardial infarction.

We have previously shown that the endocannabinoid anandamide and its metabolically stable analog (R)-methanandamide produce vasorelaxation in rabbit aortic ring preparations in an endothelium-dependent manner that could not be mimicked by other CB 1 cannabinoid receptor agonists (Am J Physiol 282: H2046 -H2054, 2002). Here, we show that (R)-methanandamide and abnormal cannabidiol stimulated nitric oxide (NO) production in rabbit aortic endothelial cells (RAEC) in a dosedependent manner but that other CB 1 and CB 2 receptor agonists, such as cis-3R- [2-hydroxy-4-(1,1- Results from this study suggest that in RAEC, (R)-methanandamide acts on a novel non-CB 1 and non-CB 2 anandamide receptor and signals through G i and phosphatidylinositol 3-kinase, leading to Akt activation, eNOS phosphorylation, and NO production.

Previous studies showed that angiotensin-(1-7) [Ang-(1-7)] attenuates cardiac remodeling by reducing both interstitial and perivascular fibrosis. Although a high affinity binding site for Ang-(1-7) was identified on cardiac fibroblasts, the molecular mechanisms activated by the heptapeptide hormone were not identified. We isolated cardiac fibroblasts from neonatal rat hearts to investigate signaling pathways activated by Ang-(1-7) that participate in fibroblast proliferation. Ang-(1-7) reduced 3H-thymidine, -leucine and –proline incorporation into cardiac fibroblasts stimulated with serum or the mitogen endothelin-1 (ET-1), demonstrating that the heptapeptide hormone decreases DNA, protein and collagen synthesis. The reduction in DNA synthesis by Ang-(1-7) was blocked by the AT(1-7) receptor antagonist [D-Ala7]-Ang-(1-7), showing specificity of the response. Treatment of cardiac fibroblasts with Ang-(1-7) reduced the Ang II- or ET-1-stimulated increase in phospho-ERK1 and –ERK2. In contrast, Ang-(1-7) increased dual-specificity phosphatase DUSP1 immunoreactivity and mRNA, suggesting that the heptapeptide hormone increases DUSP1 to reduce MAP kinase phosphorylation and activity. Incubation of cardiac fibroblasts with ET-1 increased cyclooxygenase 2 (COX-2) and prostaglandin synthase (PGES) mRNAs, while Ang-(1-7) blocked the increase in both enzymes, suggesting that the heptapeptide hormone alters the concentration and the balance between the proliferative and anti-proliferative prostaglandins. Collectively, these results indicate that Ang-(1-7) participates in maintaining cardiac homeostasis by reducing proliferation and collagen production by cardiac fibroblasts in association with up-regulation of DUSP1 to reduce MAP kinase activities and attenuation of the synthesis of mitogenic prostaglandins. Increased Ang-(1-7) or agents that enhance production of the heptapeptide hormone may prevent abnormal fibrosis that occurs during cardiac pathologies.