Hisaka Kawate

The GPCR modulator protein RAMP2 is essential for angiogenesis and vascular integrity

Adrenomedullin (AM) is a peptide involved both in the pathogenesis of cardiovascular diseases and in circulatory homeostasis. The high-affinity AM receptor is composed of receptor activity-modifying protein 2 or 3 (RAMP2 or -3) and the GPCR calcitonin receptor-like receptor. Testing our hypothesis that RAMP2 is a key determinant of the effects of AM on the vasculature, we generated and analyzed mice lacking RAMP2. Similar to AM-/- embryos, RAMP2-/- embryos died in utero at midgestation due to vascular fragility that led to severe edema and hemorrhage. Vascular ECs in RAMP2-/- embryos were severely deformed and detached from the basement membrane. In addition, the abnormally thin arterial walls of these mice had a severe disruption of their typically multilayer structure. Expression of tight junction, adherence junction, and basement membrane molecules by ECs was diminished in RAMP2-/- embryos, leading to paracellular leakage and likely contributing to the severe edema observed. In adult RAMP2+/- mice, reduced RAMP2 expression led to vascular hyperpermeability and impaired neovascularization. Conversely, ECs overexpressing RAMP2 had enhanced capillary formation, firmer tight junctions, and reduced vascular permeability. Our findings in human cells and in mice demonstrate that RAMP2 is a key determinant of the effects of AM on the vasculature and is essential for angiogenesis and vascular integrity.

Vascular Endothelial Adrenomedullin-RAMP2 System Is Essential for Vascular Integrity and Organ Homeostasis

Background— Revealing the mechanisms underlying the functional integrity of the vascular system could make available novel therapeutic approaches. We previously showed that knocking out the widely expressed peptide adrenomedullin (AM) or receptor activity-modifying protein 2 (RAMP2), an AM-receptor accessory protein, causes vascular abnormalities and is embryonically lethal. Our aim was to investigate the function of the vascular AM-RAMP2 system directly. Methods and Results— We generated endothelial cell–specific RAMP2 and AM knockout mice (E-RAMP2−/− and E-AM−/−). Most E-RAMP2−/− mice died perinatally. In surviving adults, vasculitis occurred spontaneously. With aging, E-RAMP2−/− mice showed severe organ fibrosis with marked oxidative stress and accelerated vascular senescence. Later, liver cirrhosis, cardiac fibrosis, and hydronephrosis developed. We next used a line of drug-inducible E-RAMP2−/− mice (DI-E-RAMP2−/−) to induce RAMP2 deletion in adults, which enabled us to analyze the initial causes of the aforementioned vascular and organ damage. Early after the induction, pronounced edema with enhanced vascular leakage occurred. In vitro analysis revealed the vascular leakage to be caused by actin disarrangement and detachment of endothelial cells. We found that the AM-RAMP2 system regulates the Rac1-GTP/RhoA-GTP ratio and cortical actin formation and that a defect in this system causes the disruption of actin formation, leading to vascular and organ damage at the chronic stage after the gene deletion. Conclusions— Our findings show that the AM-RAMP2 system is a key determinant of vascular integrity and homeostasis from prenatal stages through adulthood. Furthermore, our models demonstrate how endothelial cells regulate vascular integrity and how their dysregulation leads to organ damage.

Adrenomedullin in sinusoidal endothelial cells play protective roles against cold injury of liver.

Donor organ damage caused by cold preservation is a major problem affecting liver transplantation. Cold preservation most easily damages liver sinusoidal endothelial cells (LSECs), and information about the molecules modulating LSECs function can provide the basis for new therapeutic strategies. Adrenomedullin (AM) is a peptide known to possess anti-apoptotic and anti-inflammatory properties. AM is abundant in vascular endothelial cells, but levels are comparatively low in liver, and little is known about its function there. In this study, we demonstrated both AM and its receptors are expressed in LSECs. AM treatment reduced LSECs loss and apoptosis under cold treatment. AM also downregulated cold-induced expression of TNFalpha, IL1beta, IL6, ICAM1 and VCAM1. AM reduced apoptosis and expression of ICAM1 and VCAM1 in an in vivo liver model subjected to cold storage. Conversely, apoptosis was exacerbated in livers from AM and RAMP2 (AM receptor activity-modifying protein) knockout mice. These results suggest that AM expressed in LSECs exerts a protective effect against cold-organ damage through modulation of apoptosis and inflammation.

A non-inheritable maternal Cas9-based multiple-gene editing system in mice.

The CRISPR/Cas9 system is capable of editing multiple genes through one-step zygote injection. The preexisting method is largely based on the co-injection of Cas9 DNA (or mRNA) and guide RNAs (gRNAs); however, it is unclear how many genes can be simultaneously edited by this method, and a reliable means to generate transgenic (Tg) animals with multiple gene editing has yet to be developed. Here, we employed non-inheritable maternal Cas9 (maCas9) protein derived from Tg mice with systemic Cas9 overexpression (Cas9 mice). The maCas9 protein in zygotes derived from mating or in vitro fertilization of Tg/+ oocytes and +/+ sperm could successfully edit the target genome. The efficiency of such maCas9-based genome editing was comparable to that of zygote microinjection–based genome editing widely used at present. Furthermore, we demonstrated a novel approach to create “Cas9 transgene-free” gene-modified mice using non-Tg (+/+) zygotes carrying maCas9. The maCas9 protein in mouse zygotes edited nine target loci simultaneously after injection with nine different gRNAs alone. Cas9 mouse-derived zygotes have the potential to facilitate the creation of genetically modified animals carrying the Cas9 transgene, enabling repeatable genome engineering and the production of Cas9 transgene-free mice.

Novel Regulation of Cardiac Metabolism and Homeostasis by the Adrenomedullin-Receptor Activity-Modifying Protein 2 System

Adrenomedullin (AM) was identified as a vasodilating and hypotensive peptide mainly produced by the cardiovascular system. The AM receptor calcitonin receptor-like receptor associates with receptor activity-modifying protein (RAMP), one of the subtypes of regulatory proteins. Among knockout mice (−/−) of RAMPs, only RAMP2−/− is embryonically lethal with cardiovascular abnormalities that are the same as AM−/−. This suggests that the AM-RAMP2 system is particularly important for the cardiovascular system. Although AM and RAMP2 are highly expressed in the heart from embryo to adulthood, their analysis has been limited by the embryonic lethality of AM−/− and RAMP2−/−. For this study, we generated inducible cardiac myocyte-specific RAMP2−/− (C-RAMP2−/−). C-RAMP2−/− exhibited dilated cardiomyopathy-like heart failure with cardiac dilatation and myofibril disruption. C-RAMP2−/− hearts also showed changes in mitochondrial structure and downregulation of mitochondria-related genes involved in oxidative phosphorylation, &bgr;-oxidation, and reactive oxygen species regulation. Furthermore, the heart failure was preceded by changes in peroxisome proliferator-activated receptor-&ggr; coactivator 1&agr; (PGC-1&agr;), a master regulator of mitochondrial biogenesis. Metabolome and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) imaging analyses revealed early downregulation of cardiolipin, a mitochondrial membrane-specific lipid. Furthermore, primary-cultured cardiac myocytes from C-RAMP2−/− showed reduced mitochondrial membrane potential and enhanced reactive oxygen species production in a RAMP2 deletion–dependent manner. C-RAMP2−/− showed downregulated activation of cAMP response element binding protein (CREB), one of the main regulators of mitochondria-related genes. These data demonstrate that the AM-RAMP2 system is essential for cardiac metabolism and homeostasis. The AM-RAMP2 system is a promising therapeutic target of heart failure.

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.

Functional differentiation of RAMP2 and RAMP3 in their regulation of the vascular system

Adrenomedullin (AM) is a vasoactive peptide that possesses various bioactivities. AM receptors are dimers consisting of CLR with one of two accessory proteins, RAMP2 or RAMP3. The functional difference between CLR/RAMP2 and CLR/RAMP3 and the relationship between the two receptors remain unclear. To address these issues, we generated RAMP2 and RAMP3 knockout (-/-) mice and have been studying their physiological activities in the vascular system. AM-/- and RAMP2-/- mice die in utero due to blood vessel abnormalities, which is indicative of their essential roles in vascular development. In contrast, RAMP3-/- mice were born normally without any major abnormalities. In adult RAMP3-/- mice, postnatal angiogenesis was normal, but lymphangiography using indocyanine green (ICG) showed delayed drainage of subcutaneous lymphatic vessels. Moreover, chyle transport by intestinal lymphatics was delayed in RAMP3-/- mice, which also showed more severe interstitial edema than wild-type mice in a tail lymphedema model, with characteristic dilatation of lymphatic capillaries and accumulation of inflammatory cells. In scratch-wound assays, migration of isolated RAMP3-/- lymphatic endothelial cells was delayed as compared to wild-type cells, and AM administration failed to enhance the re-endothelialization. The delay in re-endothelialization was due to a primary migration defect rather than a decrease in proliferation. These results suggest that RAMP3 regulates drainage through lymphatic vessels, and that the AM-RAMP3 system could be a novel therapeutic target for controlling postoperative lymphedema.

Endogenous CGRP protects against neointimal hyperplasia following wire-induced vascular injury.

Neointimal hyperplasia is the primary lesion underlying atherosclerosis and restenosis after percutaneous coronary intervention. Calcitonin gene-related peptide (CGRP) is produced by alternative splicing of the primary transcript of the calcitonin/CGRP gene. Originally identified as a strongly vasodilatory neuropeptide, CGRP is now known to be a pleiotropic peptide widely distributed in various organs and tissues. Our aim was to investigate the possibility that CGRP acts as an endogenous vasoprotective molecule. We compared the effect of CGRP deficiency on neointimal formation after wire-induced vascular injury in wild-type and CGRP knockout (CGRP-/-) mice. We found that neointimal formation after vascular injury was markedly enhanced in CGRP-/- mice, which also showed a higher degree of oxidative stress, as indicated by reduced expression of nitric oxide synthase, increased expression of p47phox, and elevated levels of 4HNE, as well as greater infiltration of macrophages. In addition, CGRP-deficiency led to increased vascular smooth muscle cell (VSMC) proliferation within the neointima. By contrast, bone marrow-derived cells had little or no effect on neointimal formation in CGRP-/-mice. In vitro analysis showed that CGRP-treatment suppressed VSMC proliferation, migration, and ERK1/2 activity. These results clearly demonstrate that endogenous CGRP suppresses the oxidative stress and VSMC proliferation induced by vascular injury. As a vasoprotective molecule, CGRP could be an important therapeutic target in cardiovascular disease.

Endogenous α‐calcitonin gene‐related peptide mitigates liver fibrosis in chronic hepatitis induced by repeated administration of concanavalin A

Background: α‐Calcitonin gene‐related peptide (αCGRP) is a 37‐amino acid pleiotropic peptide that we previously showed to exert a hepatoprotective effect during concanavalin A (Con A)‐induced acute hepatitis. In the present study, we used αCGRP−/− mice to further investigate the antifibrogenic and hepatoprotective effects of endogenous αCGRP in Con A‐induced chronic hepatitis.

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.