Receptor activity-modifying protein 1 regulates the phenotypic expression of BMSCs via the Hippo/Yap pathway.

Receptor activity-modifying protein 1 (RAMP1) might be a critical regulator during bone wound healing. However, the roles and mechanisms of RAMP1 in osteogenesis remain unclear. Here, we aimed to elucidate the role of RAMP1 and explore the effects of Yes-associated protein 1 (Yap1), an effector of the Hippo/Yap pathway, in this process. We used a RAMP1 overexpression lentiviral system in bone marrow mesenchymal stem cells (BMSCs), which enhanced RAMP1 expression in an effective, appropriate, and sustained manner. Alkaline phosphatase (ALP) activity assays and alizarin red staining showed that RAMP1 promoted osteogenic differentiation of BMSCs after calcitonin gene-related peptide (CGRP) treatment (10 -8 mol/L). Moreover, real-time polymerase chain reaction and Western blot analysis indicated that RAMP1 upregulated the expression of osteogenic phenotypic markers (ALP, runt-related transcription factor 2, osteopontin; p < 0.05). To further uncover the mechanism of RAMP1 in osteogenic differentiation, we used verteporfin (10 -7 mol/L) to block Yap1. Notably, verteporfin impaired RAMP1-induced osteogenesis. Taken together, our findings confirmed that RAMP1 is a key mediator of bone regeneration and indicate that RAMP1 promotes CGRP-induced osteogenic differentiation of BMSCs via regulation of the Hippo/Yap pathway.

The influence of receptor activity-modifying protein-1 overexpression on angiogenesis in mouse brain capillary endothelial cells.

Receptor activity-modifying protein-1 (RAMP1) is highly expressed in the heart and vasculature, indicating that it might be related to the vascular system. However, the effects of RAMP1 on angiogenesis and the intrinsic mechanisms underlying this process remain unclear. Here, we verified that RAMP1 is a critical regulator of angiogenesis in a mouse brain capillary endothelial cell line (bEnd.3). We first constructed a RAMP1 overexpression lentiviral vector system and stably transfected bEnd.3 cells. We further showed that RAMP1 overexpression could lead to bEnd.3 migration and capillary tube formation in Matrigel without exogenous calcitonin gene-related peptide (CGRP) treatment. At the same time, RAMP1 overexpression had little effect on proliferation. More importantly, vascular endothelial growth factor (VEGF) and CGRP expression levels were not significantly higher in RAMP1-overexpressing cells than in control cells (P > 0.05), indicating that RAMP1 did not function through upregulating VEGF or CGRP expression in bEnd.3 cells. Strikingly, RAMP1 transfection increased adrenomedullin 2 (AM2) expression levels ( P < 0.05). Taken together, these data contribute to a better understanding of the molecular mechanisms of RAMP1 in angiogenesis.

CGRP regulates the dysfunction of peri-implant angiogenesis and osseointegration in streptozotocin-induced diabetic rats.

Diabetes is a chronic systematic disease which results in neuropathy and dysfunctional bone metabolism and microcirculation. Calcitonin gene related peptide (CGRP) is an important neuropeptide that is involved in bone formation and vascular response. This study aimed to elucidate the role of CGRP in diabetic peri-implant angiogenesis and osteogenesis, which is yet to be reported. In vivo, we injected streptozotocin into SD rats to establish an experimental diabetes model. We then implanted 1 mm × 5 mm Ti implants into rat tibiae and injected lentivirus into the bone marrow cavity to overexpress or silence the peri-implant CGRP expression. We also applied overexpression lentivirus and silencing short hair RNA (shRNA) in rat bone marrow mesenchymal stem cells (BMSCs) to investigate the biological effects of CGRP in vitro. Through the investigation of diabetic neurons, blood, and peri-implant bone, we could observe that diabetes led to decreased synthesis and expression of CGRP, and high CGRP expression were only seen in peri-implant tissues in the early-to-middle phase of diabetic bone integration. Microfil perfusion followed by micro-CT analysis showed that the overexpression of CGRP enhanced peri-implant angiogenesis via increased vessel volume and thickness. Regarding osteogenesis, CGRP was found to improve the impaired osseointegration, as observed through micro-CT reconstruction and H&E staining. Similarly, overCGRP alleviated the hyperglycemia-triggered decrease in mineralization, and rescued ALP activity and the mRNA and protein expression of VEGF-A, ALP, and OPN. CGRP also attenuated the high glucose-induced production of reactive oxygen species (ROS). Our results demonstrate the potential promotive role of CGRP in early-to-middle phase of osseointegration, as CGRP could regulate the diabetes-induced dysfunctions in peri-implant angiogenesis and osseointegration. Our study provides a new insight into the diabetic peri-implant vasculature and the potential positive effect of CGRP on diabetic peri-implant vessels and bone.

The protective role of calcitonin gene-related peptide (CGRP) in high-glucose-induced oxidative injury in rat aorta endothelial cells.

Endothelial dysfunction is considered to be an initial indicator in diabetes-induced macrovascular complications. Evidence has shown that CGRP is an important neuropeptide active in vascular system, especially in vasorelaxation. This study aimed to investigate the role of CGRP in high-glucose-induced endothelial dysfunction in rat aorta endothelial cells (RAECs). Quantitative-real time PCR and western blots were used to determine the efficiency of overexpression and interference of CGRP. After incubation with normal glucose (5.5 mM) or high glucose (33 mM), the cell viability and cell apoptosis were tested. Afterwards, the Nitric Oxide (NO) production, the mRNA expression of inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS) and angiotensin II (Ang II) and the level of reactive oxygen species (ROS) were determined. The involvement of ERK1/2-NOX4 was determined through western blots and the translocation of p47phox was also observed via cell immunofluorescence. CGRP alleviated the high-glucose-induced cell apoptosis while CGRP did not have an obvious impact on cell viability. Meanwhile, CGRP increased the NO production as well as the eNOS mRNA expression and reversely decreased the stimulated expression of iNOS and Ang II by high glucose. In addition, CGRP attenuated the high-glucose-stimulated intracellular ROS production by ERK1/2-NOX4 and the translocation of p47phox. These results indicated the protective role of CGRP in high-glucose-induced oxidative injury in RAECs possibly through inhibiting ERK1/2-NOX4. Our findings might help to further understand the potential role and possible mechanism of CGRP in endothelial dysfunction caused by high glucose.

Overexpression of calcitonin gene-related peptide protects mouse cerebral microvascular endothelial cells from high-glucose-induced damage via ERK/HIF-1/VEGF signaling.

In the diabetic brain, hyperglycemia damages the cerebrovasculature and impairs neurovascular crosstalk. Calcitonin gene-related peptide (CGRP) is an important neuropeptide that is active in the vascular system. In this study, we aimed to investigate whether CGRP is involved in the high-glucose-induced damage in mouse cerebral microvascular endothelial (b.END3) cells and the possible mechanism in vitro. The overexpression of CGRP by lentiviral transduction inhibited cell apoptosis but not proliferation. In contrast to the promoting of angiogenesis and migration under normal glucose, CGRP inhibited hyperglycemia-induced tube formation but had no effect on migration. Calcitonin gene-related peptide partly reduced the increased level of intracellular reactive oxygen species (ROS) and altered nitric oxide synthase mRNA expression. Furthermore, CGRP suppressed the increased HIF-1α/VEGF-A expression and the phosphorylation of ERK1/2 in hyperglycemia. The ERK inhibitor U0126 showed similar inhibition of cell apoptosis, tube formation and HIF-1α/VEGF expression as that exhibited by lenti-CGRP. These findings demonstrate the protective role of CGRP overexpression against high-glucose-induced cerebrovascular changes in b.END3 cells, possibly through the inhibition of ERK/HIF-1/VEGF signaling.