Adrenomedullin-RAMP2 and -RAMP3 Systems Regulate Cardiac Homeostasis during Cardiovascular Stress.

Adrenomedullin (AM) is a peptide hormone with multiple physiological functions, which are regulated by its receptor activity-modifying proteins, RAMP2 and RAMP3. We previously reported that AM or RAMP2 knockout (KO) (AM-/-, RAMP2-/-) is embryonically lethal in mice, whereas RAMP3-/- mice are apparently normal. AM, RAMP2, and RAMP3 are all highly expressed in the heart; however, their functions there are not fully understood. Here, we analyzed the pathophysiological functions of the AM-RAMP2 and AM-RAMP3 systems in hearts subjected to cardiovascular stress. Cardiomyocyte-specific RAMP2-/- (C-RAMP2-/-) and RAMP3-/- showed no apparent heart failure at base line. After 1 week of transverse aortic constriction (TAC), however, C-RAMP2-/- exhibited significant cardiac hypertrophy, decreased ejection fraction, and increased fibrosis compared with wild-type mice. Both dP/dtmax and dP/dtmin were significantly reduced in C-RAMP2-/-, indicating reduced ventricular contractility and relaxation. Exposing C-RAMP2-/- cardiomyocytes to isoproterenol enhanced their hypertrophy and oxidative stress compared with wild-type cells. C-RAMP2-/- cardiomyocytes also contained fewer viable mitochondria and showed reduced mitochondrial membrane potential and respiratory capacity. RAMP3-/- also showed reduced systolic function and enhanced fibrosis after TAC, but those only became apparent after 4 weeks. A reduction in cardiac lymphatic vessels was the characteristic feature in RAMP3-/-. These observations indicate the AM-RAMP2 system is necessary for early adaptation to cardiovascular stress through regulation of cardiac mitochondria. AM-RAMP3 is necessary for later adaptation through regulation of lymphatic vessels. The AM-RAMP2 and AM-RAMP3 systems thus play separate critical roles in the maintenance of cardiovascular homeostasis against cardiovascular stress.

Vasoprotective Activities of the Adrenomedullin-RAMP2 System in Endothelial Cells.

Neointimal hyperplasia is the primary lesion underlying atherosclerosis and restenosis after coronary intervention. We previously described the essential angiogenic function of the adrenomedullin (AM)-receptor activity-modifying protein (RAMP) 2 system. In the present study, we assessed the vasoprotective actions of the endogenous AM-RAMP2 system using a wire-induced vascular injury model. We found that neointima formation and vascular smooth muscle cell proliferation were enhanced in RAMP2+/- male mice. The injured vessels from RAMP2+/- mice showed greater macrophage infiltration, inflammatory cytokine expression, and oxidative stress than vessels from wild-type mice and less re-endothelialization. After endothelial cell-specific RAMP2 deletion in drug-inducible endothelial cell-specific RAMP2-/- (DI-E-RAMP2-/-) male mice, we observed markedly greater neointima formation than in control mice. In addition, neointima formation after vessel injury was enhanced in mice receiving bone marrow transplants from RAMP2+/- or DI-E-RAMP2-/- mice, indicating that bone marrow-derived cells contributed to the enhanced neointima formation. Finally, we found that the AM-RAMP2 system augmented proliferation and migration of endothelial progenitor cells. These results demonstrate that the AM-RAMP2 system exerts crucial vasoprotective effects after vascular injury and could be a therapeutic target for the treatment of vascular diseases.

Adrenomedullin-RAMP2 System in Vascular Endothelial Cells.

Vascular endothelial cells play key roles in maintaining vascular and organ homeostasis. Adrenomedullin (AM), originally identified as a vasodilating peptide, is now recognized to be a pleiotropic molecule involved in both circulatory homeostasis and the pathogenesis of cardiovascular diseases. We have reported that knockout mice deficient in AM or receptor activity-modifying protein 2 (RAMP2), an AM-receptor accessory protein, show vascular endothelial cell deformities that are embryonically lethal. To directly clarify the pathophysiological functions of the vascular AM-RAMP2 system, we generated vascular endothelial cell-specific RAMP2 knockout mice. Using these mice, we found that the AM-RAMP2 system is a key determinant of vascular integrity and homeostasis from prenatal stages through adulthood. This review highlights the functions of AM-RAMP2 in vascular endothelial cells.

Calcitonin gene-related peptide (CGRP) receptors are important to maintain cerebrovascular reactivity in chronic hypertension.

Cerebral blood flow autoregulation (CA) shifts to higher blood pressures in chronic hypertensive patients, which increases their risk for brain damage. Although cerebral vascular smooth muscle cells express the potent vasodilatatory peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) and their receptors (calcitonin receptor-like receptor (Calclr), receptor-modifying proteins (RAMP) 1 and 2), their contribution to CA during chronic hypertension is poorly understood. Here we report that chronic (10 weeks) hypertensive (one-kidney-one-clip-method) mice overexpressing the Calclr in smooth muscle cells (CLR-tg), which increases the natural sensitivity of the brain vasculature to CGRP and AM show significantly better blood pressure drop-induced cerebrovascular reactivity than wt controls. Compared to sham mice, this was paralleled by increased cerebral CGRP-binding sites (receptor autoradiography), significantly in CLR-tg but not wt mice. AM-binding sites remained unchanged. Whereas hypertension did not alter RAMP-1 expression (droplet digital (dd) PCR) in either mouse line, RAMP-2 expression dropped significantly in both mouse lines by about 65%. Moreover, in wt only Calclr expression was reduced by about 70% parallel to an increase of smooth muscle actin (Acta2) expression. Thus, chronic hypertension induces a stoichiometric shift between CGRP and AM receptors in favor of the CGRP receptor. However, the parallel reduction of Calclr expression observed in wt mice but not CLR-tg mice appears to be a key mechanism in chronic hypertension impairing cerebrovascular reactivity.

Adrenomedullin-RAMP2 system is crucially involved in retinal angiogenesis.

Adrenomedullin (ADM) is an endogenous peptide first identified as a strong vasodilating molecule. We previously showed that in mice, homozygous knockout of ADM (ADM(-/-)) or its receptor regulating protein, RAMP2 (RAMP2(-/-)), is embryonically lethal due to abnormal vascular development, thereby demonstrating the importance of ADM and its receptor signaling to vascular development. ADM expression in the retina is strongly induced by ischemia; however, its role in retinal pathophysiology remains unknown. Here, we analyzed oxygen-induced retinopathy (OIR) using heterozygous ADM and RAMP2 knockout mice models (ADM(+/-) or RAMP2(+/-), respectively). In addition, we analyzed the role of the ADM-RAMP2 system during earlier stages of retinal angiogenesis using an inducible endothelial cell-specific RAMP2 knockout mouse line (DI-E-RAMP2(-/-)). Finally, we assessed the ability of antibody-induced ADM blockade to control pathological retinal angiogenesis in OIR. In OIR, neovascular tufts, avascular zones, and hypoxic areas were all smaller in ADM(+/-) retinas compared with wild-type mice. ADM(+/-) retinas also exhibited reduced levels of VEGF and eNOS expression. DI-E-RAMP2(-/-) showed abnormal retinal vascular patterns in the early stages of development. However, ADM enhanced the proliferation and migration of retinal endothelial cells. Finally, we found intravitreal injection of anti-ADM antibody reduced pathological retinal angiogenesis. In conclusion, the ADM-RAMP2 system is crucially involved in retinal angiogenesis. ADM and its receptor system are potential therapeutic targets for controlling pathological retinal angiogenesis.