Distraction osteogenesis (DO) is a well-established surgical technique for treating bone defect and limb lengthening. The major drawback of DO is the long treatment period as the external fixator has to be kept in place until consolidation is completed. Calcitonin gene-related peptide (CGRP) has been reported to promote angiogenesis by affecting endothelial progenitor cells (EPCs) in limb ischemia and wound healing. Thus, the goal of this study was to evaluate the angiogenic effect of exogenous CGRP on bone regeneration in a rat DO model. Exogenous CGRP was directly injected into the bone defect after each cycle of distraction in vivo. Micro-computed tomography, biomechanical test and histological analysis were performed to assess the new bone formation. Angiography and immunofluorescence were performed to assess the formation of blood vessels. CD31+CD144+ EPCs in the bone defect were quantified with flow cytometry. In in vitro study, bone marrow stem cells (BMSCs) were used to investigate the effect of CGRP on EPCs production during endothelial differentiation. Our results showed that CGRP significantly promoted bone regeneration and vessel formation after consolidation. CGRP significantly increased the fraction of CD31+CD144+EPCs and the density of capillary density in the bone defect at the end of distraction phase. CGRP increased EPCs population in the endothelial differentiation of BMSCs in vitro by activating PI3K/AKT signaling pathway. Furthermore, differentiated EPCs rapidly assembled into tube-like structures and promoted osteogenic differentiation of BMSCs. In conclusion, CGRP increased EPCs population and promoted blood vessel formation and bone regeneration at the defect region in a DO model.

BACKGROUND: Cardiovascular disease causes >50% of deaths in patients with advanced chronic kidney disease (CKD). Clinical studies suggest that kidney-derived factors contribute to cardiovascular disease development in CKD, independently of comorbidities. However, to date, no kidney-specific humoral risk factor that triggers direct cardiotoxicity has been identified. In this cross-sectional study, we investigate how, in patients with CKD, circulating extracellular vesicles (EVs) facilitate pathological kidney-heart communication, thereby causing cardiotoxicity, impairing cardiac function, and contributing to heart failure progression. METHODS: We investigated the function of EVs from patients with CKD and adenine diet–induced CKD mice on cardiomyocyte and cardiac contractility. microRNA (miRNA) cargo of EVs was identified by small RNA sequencing and quantitative reverse transcription polymerase chain reaction, and their cardiotoxicity was tested by using miRNA mimics. Tissue and cellular origin of CKD-EV-miRNAs were determined from their corresponding primary miRNA expressions in mice. RESULTS: EVs from plasma of patients with CKD, but not from healthy controls, were cardiotoxic; they significantly induced apoptosis both in vitro and in vivo and impaired contractility of adult rat primary cardiomyocytes in vitro. Likewise, EVs from both plasma and kidneys of CKD mice were cardiotoxic. Pharmacologically depleting circulating EVs in CKD mice significantly recovered cardiac function and ameliorated heart failure, improvements that suggest CKD-EVs play a causal role in heart failure pathogenesis. Both human and mouse CKD-EVs were enriched in distinct miRNAs compared with control EVs. CKD-EV-miRNA mimics were cardiotoxic, impairing contractility and downregulating contractile gene expression in human induced pluripotent stem cell–derived cardiomyocytes. It is interesting that levels of endogenous primary miRNAs corresponding to circulating CKD-EV-miRNAs were significantly higher in CKD kidney tissues, specifically in CD45 ^-ve CD31 ^-ve renal cells, but not in CKD hearts, CKD livers, or CKD-peripheral blood mononuclear cells, a result that indicates CKD-EV-miRNAs originate renally. It is remarkable that CKD-EV-miRNA levels correlated with established markers of cardiac injury, thus uncovering the presence of subclinical heart disease and demonstrating heterogeneity in reno-cardiac disease. CONCLUSIONS: Collectively, our human subject and mouse studies show that circulating CKD-EVs, carrying distinct renal-derived miRNAs, mediate the molecular crosstalk that contributes to the pathogenesis of heart failure in CKD. Consequently, CKD-EVs hold promise as diagnostic and prognostic biomarkers for early disease detection and as targets for novel therapeutic interventions in chronic reno-cardiac disease.

Background: A glioma is a tumor originating from glial cells in the central nervous system. Although significant progress has been made in diagnosis and treatment, most high-grade glioma patients are prone to recurrence. Therefore, molecular targeted therapy may become a new direction for adjuvant therapy in glioma. In recent years, many studies have revealed that circular RNA (circRNA) may play an important role in the occurrence and development of many tumors including gliomas. Our previous study found that the expression of hsa_circ_0008922 was up-regulated in glioma tissues upon RNA sequencing. The biological mechanism of circ_0008922 is still unreported in gliomas. Therefore, in this study, we preliminarily outlined the expression of hsa_circ_0008922 in glioma and explored its biological functions. Methods: The expression of hsa_circ_0008922 in forty glioma tissues and four glioma cell lines (A172, U251, SF763 and U87) was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The correlation between hsa_circ_0008922 expression and clinicopathological features of glioma patients was evaluated by Fisher’s exact test. To understand the potential function of hsa_circ_0008922 in glioma, we constructed small interfering RNA (siRNA) to hsa_circ_0008922 to downregulate its expression in glioma cell lines A172 and U251. With these hsa_circ_0008922 downregulated cells, a series of assays were carried out as follows. Cell proliferation was detected by CCK8 assay, migration and invasion were determined by wound healing assay and transwell assay, respectively. Colony formation ability was evaluated by plate clonogenic assay. Moreover, flow cytometry combined with Western blot was performed to analyze apoptosis status and the expression of apoptotic related proteins (caspase 3 and caspase 9). Finally, the possible biological pathways and potential miRNA targets of hsa_circ_0008922 were predicted by bioinformatics. Results: We found that the expression of hsa_circ_0008922 in glioma tissues was 3.4 times higher than that in normal tissues. The expression of has_circ_0008922 was correlated with WHO tumor grade. After down-regulating the expression of hsa_circ_0008922, malignant biological behavior of glioma cells was inhibited, such as cell proliferation, colony formation, migration, and invasion. At the same time, it also induced apoptosis of glioma cells. Predicted analysis by bioinformatics demonstrated that hsa_circ_0008922 may be involved in tumor-related pathways by acting as a molecular sponge for multiple miRNAs (hsa-let-7e-5p, hsa-miR-506-5p, hsa-let-7b-5p, hsa-let-7c-5p and hsa-let-7a-5p). Finally, we integrated our observation to build a circRNA-miRNA-mRNA predictive network.

By virtue of their capability to replicate and regenerate, human stem-cell-derived beta-like cells could be a valuable resource for cellular therapy targeting insulin-dependent diabetes. Here, we present a protocol to generate beta-like cells from human embryonic stem cells (hESCs). We first describe steps for differentiation of beta-like cells from hESCs and CD9-negative beta-like cell enrichment through fluorescence-activated cell sorting. We then detail immunofluorescence, flow cytometry, and glucose-stimulated insulin secretion assay for characterization of human beta-like cells. For complete details on the use and execution of this protocol, please refer to Li et al. (2020).¹.