Microinjection of clustered regulatory interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-related RNA and DNA into fertilized eggs is a novel approach for creating gene-modified mice. Blastocysts obtained just before implantation may be appropriate for testing the fidelity of CRIPSR/Cas9-mediated genome editing because they can be individually handled in vitro and obtained 3 days after microinjection, thus allowing researchers to check mutations rapidly. However, it is not known whether indel mutations caused by the CRISPR/Cas9 system can be reproducibly detected in embryos. In this study, we assessed the detection of CRISPR/Cas9-induced mutations in embryos. T7 endonuclease I was more effective than Surveyor nuclease for detecting mutations in annealed fragments derived from 2 plasmids, which contained nearly identical sequences. Mouse fertilized eggs were microinjected with CRISPR/Cas9-related RNA/DNA to examine whether non-homologous end joining-mediated knockout and homologous recombination-mediated knockin occurred in the endogenous receptor (G protein-coupled) activity modifying protein 2 (Ramp2) gene. Individual blastocysts were lysed to obtain crude DNA solutions, which were used for polymerase chain reaction (PCR) assays. T7 endonuclease I-based PCR and sequencing analysis demonstrated that 25–100% of the embryos were knockout embryos and 7–57% of the embryos were knockin embryos. Our results also established that crude DNA from a single blastocyst was an appropriate template for Whole genome amplification and subsequent assessment by PCR and the T7 endonuclease I-based assay. The single blastocyst-based assay was useful for determining whether CRISPR/Cas9-mediated genome editing worked in murine embryos.

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.

Aims Controlling vascular integrity is expected to be a novel therapeutic target of cancers as well as cardiovascular diseases. Adrenomedullin (AM) and its receptor-modulating protein, RAMP2, have been identified as essential mediators of cardiovascular homeostasis. In this study, we used inducible vascular endothelial cell-specific RAMP2 knockout (DI-E-RAMP2^−/−) mice to clarify the contribution made by the endogenous AM-RAMP2 system to angiogenesis and metastasis. Methods and results Subcutaneously transplanted sarcoma or melanoma cells showed less growth and angiogenesis in DI-E-RAMP2^−/− than in control mice. On the other hand, after the transplantation of B16BL6 melanoma cells into hindlimb footpads, spontaneous metastasis to the lung was enhanced in DI-E-RAMP2^−/− mice. Early after RAMP2 gene deletion, DI-E-RAMP2^−/− mice showed enhanced vascular permeability, endothelial–mesenchymal transition (EndMT)-like change, and systemic oedema. Within the lungs of DI-E-RAMP2^−/− mice, pulmonary endothelial cells were deformed, and inflammatory cells infiltrated the vessel walls and expressed the chemotactic factors S100A8/9 and SAA3, which attract tumour cells and mediate the formation of a pre-metastatic niche. Conversely, the overexpression of RAMP2 suppressed tumour cell adhesion to endothelial cells, tumour metastasis, and improved survival. Conclusion These findings indicate that the AM-RAMP2 system regulates vascular integrity, whereas RAMP2 deletion promotes vascular permeability and EndMT-like change within primary lesions and formation of pre-metastatic niches in distant organs by destabilizing the vascular structure and inducing inflammation. Vascular integrity regulated by the AM-RAMP2 system could thus be a hopeful therapeutic target for suppressing tumour metastasis.

Background: Calcitonin gene-related peptide (CGRP) is a 37-amino acid peptide and produced by alternative splicing of the transcript of the calcitonin/CGRP gene. Originally identified as a strong vasodilatory and hypotensive peptide, CGRP is now known to be a pleiotropic molecule distributed in various organs, including the brain. Method: In this study, we used CGRP knockout mice (CGRP−/−) to examine the actions of endogenous CGRP during cerebral ischemia. To induce acute and chronic cerebral ischemia, mice were subjected to middle cerebral artery occlusion (MCAO) and bilateral common carotid artery stenosis (BCAS). Results: In the cerebral cortex of wild-type mice, CGRP expression was upregulated after acute infarction. In CGRP−/− subjected to MCAO or BCAS, recovery of cerebral blood flow was slower and exhibited more extensive neuronal cell death. Expression of the inflammatory cytokines was higher in CGRP−/− than wild type in the acute phase of ischemia. Pathological analysis during the chronic phase revealed more extensive neuronal cell loss and demyelination and higher levels of oxidative stress in CGRP−/− than wild-type. CGRP−/− also showed less compensatory capillary growth. In an eight-arm radial maze test, CGRP−/− exhibited poorer reference memory than wild-type. On the other hand, CGRP administration promoted cerebral blood flow recovery after cerebral ischemia. We also found that CGRP directly inhibited the cell death of primary cortical neurons. Conclusion: These results indicate endogenous CGRP is protective against ischemia-induced neuronal cell injury. CGRP could, thus, be a novel candidate for use in the treatment of both cerebral ischemia and progression of cognitive decline.

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.

Non-alcoholic steatohepatitis (NASH) is a severe form of fatty liver disease that is defined by the presence of inflammation and fibrosis, ultimately leading to cirrhosis and hepatocellular carcinoma. Treatment with human placental extract (HPE) reportedly ameliorates the hepatic injury. We evaluated the effect of HPE treatment in a mouse model of NASH. In the methione- and choline-deficient (MCD) diet-induced liver injury model, fibrosis started from regions adjacent to the sinusoids. We administered the MCD diet with high-salt loading (8% NaCl in the drinking water) to mice deficient in the vasoprotective molecule RAMP2 for 5 weeks, with or without HPE. In both the HPE and control groups, fibrosis was seen in regions adjacent to the sinusoids, but the fibrosis was less pronounced in the HPE-treated mice. Levels of TNF-α and MMP9 expression were also significantly reduced in HPE-treated mice, and oxidative stress was suppressed in the perivascular region. In addition, HPE dose-dependently increased survival of cultured endothelial cells exposed to 100 μM H2O2, and it upregulated expression of eNOS and the anti-apoptotic factors bcl-2 and bcl-xL. From these observations, we conclude that HPE ameliorates NASH-associated pathologies by suppressing inflammation, oxidative stress and fibrosis. These beneficially effects of HPE are in part attributable to its protective effects on liver sinusoidal endothelial cells. HPE could thus be an attractive therapeutic candidate with which to suppress progression from simple fatty liver to NASH.