Soluble Endoglin Stimulates Inflammatory and Angiogenic Responses in Microglia That Are Associated with Endothelial Dysfunction

Increased soluble endoglin (sENG) were observed in human brain arteriovenous malformations (bAVMs), and overexpression of sENG with vascular endothelial growth factor (VEGF)-A induced dysplastic vessel formation in mouse brain. However, the underlying mechanism of sENG-induced vascular malformations is not clear. While evidence suggests the role of sENG as a pro-inflammatory modulator, increased microglial accumulation and inflammations were observed in bAVMs. Therefore, we hypothesized that microglia mediate sENG-induced inflammation and endothelial cell (EC) dysfunction in bAVMs. In this study, we confirmed that sENG with VEGF-A overexpression induced dysplastic vessel formation. Remarkably, there were increased microglial activation around dysplastic vessels with expression of NLRP3, inflammasome marker. We found that sENG increased the gene expression of VEGF-A, pro-inflammatory cytokines/inflammasome mediators (TNF-α, IL-6, NLRP3, ASC, Caspase-1, and IL-1β), and proteolytic enzyme (MMP-9) in BV2 microglia. The conditioned media from sENG-treated BV2 (BV2-sENG-CM) significantly increased angiogenic factors (Notch-1 and TGFβ) and pERK1/2 in ECs while it decreased IL-17RD, an anti-angiogenic mediator. Finally, the BV2-sENG-CM significantly increased EC migration and tube formation. Together, our study demonstrates that sENG provokes microglia to release angiogenic/inflammatory responses which may be involved in EC dysfunction. Our study suggests the contribution of microglia in the pathology of sENG-associated vascular malformations.

Ion Conductance-Based Perfusability Assay of Vascular Vessel Models in Microfluidic Devices

We present a novel methodology based on ion conductance to evaluate the perfusability of vascular vessels in microfluidic devices without microscopic imaging. The devices consisted of five channels, with the center channel filled with fibrin/collagen gel containing human umbilical vein endothelial cells (HUVECs). Fibroblasts were cultured in the other channels to improve the vascular network formation. To form vessel structures bridging the center channel, HUVEC monolayers were prepared on both side walls of the gel. During the culture, the HUVECs migrated from the monolayer and connected to the HUVECs in the gel, and vascular vessels formed, resulting in successful perfusion between the channels after culturing for 3–5 d. To evaluate perfusion without microscopic imaging, Ag/AgCl wires were inserted into the channels, and ion currents were obtained to measure the ion conductance between the channels separated by the HUVEC monolayers. As the HUVEC monolayers blocked the ion current flow, the ion currents were low before vessel formation. In contrast, ion currents increased after vessel formation because of creation of ion current paths. Thus, the observed ion currents were correlated with the perfusability of the vessels, indicating that they can be used as indicators of perfusion during vessel formation in microfluidic devices. The developed methodology will be used for drug screening using organs-on-a-chip containing vascular vessels.

Chronic Alcohol Reduces Bone Formation Through Inhibiting Proliferation and Promoting Aging of Endothelial Cells in Type-H Vessels

Background: Chronic alcohol is one of the leading risk factors for male osteoporosis . Angiogenesis and osteogenesis coupled by type-H vessels coordinate the biological process of bone homeostasis to prevent osteopenia. It is unknown whether alcohol inhibits type-H-vessel-dependent bone formation. Aims: This study aimed to determine whether alcohol hampers proliferation and promotes aging of endothelial cells of type-H vessels, and whether alcohol inhibits the differentiation of bone marrow-mesenchymal stem cells (BM-MSCs) into osteoblasts through reducing the number and secretion of endothelial cells in type-H vessels. Materials and Methods: Two-month-old mice fed with alcohol liquid diet (28% of calories) or normal liquid diet for two months. The tibias were isolated and detected with X-ray and micro-CT. Paraffin-embedded or frozen tibial sections were prepared and used for immunohistochemical or immunofluorescence staining respectively in vivo . Human Umbilical Vein Endothelial Cells (HUVECs) were treated with different-concentrated alcohol for 12 hours. The conditioned medium of the above HUVECs cells was collected to culture human BM-MSCs, which were induced to differentiate into osteoblasts in vitro . Results: The alcoholic diet retarded the bone growth and lead to osteoporosis, impaired bone formation of osteoblasts, and decreased CD31 hi EMCN hi type-H-vessel formation through inhibiting proliferation and promoting aging of endothelial cells in mice. Alcohol treatment obviously increased the expression of p16, while significantly decreased the expression of Bmi-1, CDK6, Cyclin D, E2F1 and BMP2 compared to vehicle. Alcohol inhibited the differentiation of BM-MSCs into osteoblasts through reducing the BMP2 secretion of endothelial cells in type-H vessels. Conclusions: Alcoholic diet impaired CD31 hi EMCN hi type-H-vessel formation through inhibiting proliferation and promoting aging of endothelial cells via Bmi-1/p16 signaling, and inhibited the differentiation of BM-MSCs into osteoblasts through reducing the BMP2 secretion of endothelial cells in type-H vessels. It provides a basis for developing a new treatment strategy targeting aging endothelial cells of type-H-vessel to prevent alcoholic osteopenia.

Cyclic pulsation stress promotes bone formation of tissue engineered laminae through the F-actin/YAP-1/β-Catenin signaling axis

Mechanical loads are fundamental regulators of bone formation and remodeling. However, the molecular regulation of mechanotransduction during vertebral laminae regeneration remains poorly understood. Here, we found that cerebrospinal fluid pulsation (CSFP) stress—cyclic pulsation stress—could promote the osteogenic and angiogenic abilities of rat mesenchymal stromal cells (MSC), thereby promoting tissue-engineered laminae’s bone and blood vessel formation. In the process, F-actin relayed CSFP stress to promote the nuclear translocation of YAP1, which then decreased the degradation and promoted the nuclear translocation of β-Catenin. In turn, the nuclear translocation of β-Catenin promoted the osteogenic differentiation and angiogenic abilities of MSC, thereby promoting tissue-engineered laminae’s bone and blood vessel formation. Thus, we conclude that CSFP promotes the osteogenesis and angiogenesis of tissue-engineered laminae through the F-actin/YAP-1/β-Catenin signaling axis. This study advances our understanding of vertebral laminae regeneration and provides potential therapeutic approaches for spinal degeneration after spinal laminectomy.

snoRNA23 enhances the progression of hepatocellular carcinoma via regulation of the Wnt/?-catenin pathway

Small nucleolar non-coding RNA(snoRA)23 is upregulated in human pancreatic ductal adenocarcinoma. However, to the best of our knowledge, the role of snoRA23 in hepatocellular carcinoma progression has not been determined. MTT and colony formation assays were used to assess the cell viability and proliferation of HCC cells with snoRA23 knocked down, respectively, and a lymphatic vessel formation assay was used to determine tube formation ability of Human dermal lymphatic endothelial cells treated with conditioned media from HCC cell cultures. The results showed that snoRA23 knockdown attenuated cell viability, colony formation,and lymphatic vessel formation in HCC cells. snoRA23 was correlated with the prolonged overall survival of patients with HCC. Additionally, snoRA23 knockdown downregulated the Wnt/?-catenin signaling pathway by decreasing Wnt3a expression and ?-catenin levels.?-methylacyl-CoA racemase (AMACR) levels were notably decreased by snoRA23 depletion. Finally, it was confirmed that AMACR overexpression partially rescued snoRA23-modulated HCC tumorigenesis. The results of the present study provide further insight into the role of non-coding RNAs in the development and progression of HCC.

Pro-Angiogenic and Osteogenic Effects of Adipose Tissue-Derived Pericytes Synergistically Enhanced by Nel-like Protein-1

An important objective of vascularized tissue regeneration is to develop agents for osteonecrosis. We aimed to identify the pro-angiogenic and osteogenic efficacy of adipose tissue-derived (AD) pericytes combined with Nel-like protein-1 (NELL-1) to investigate the therapeutic effects on osteonecrosis. Tube formation and cell migration were assessed to determine the pro-angiogenic efficacy. Vessel formation was evaluated in vivo using the chorioallantoic membrane assay. A mouse model with a 2.5 mm necrotic bone fragment in the femoral shaft was used as a substitute for osteonecrosis in humans. Bone formation was assessed radiographically (plain radiographs, three-dimensional images, and quantitative analyses), and histomorphometric analyses were performed. To identify factors related to the effects of NELL-1, analysis using microarrays, qRT-PCR, and Western blotting was performed. The results for pro-angiogenic efficacy evaluation identified synergistic effects of pericytes and NELL-1 on tube formation, cell migration, and vessel formation. For osteogenic efficacy analysis, the mouse model for osteonecrosis was treated in combination with pericytes and NELL-1, and the results showed maximum bone formation using radiographic images and quantitative analyses, compared with other treatment groups and showed robust bone and vessel formation using histomorphometric analysis. We identified an association between FGF2 and the effects of NELL-1 using array-based analysis. Thus, combinatorial therapy using AD pericytes and NELL-1 may have potential as a novel treatment for osteonecrosis.

Nuclear SUN1 stabilizes endothelial cell junctions to regulate blood vessel formation

Endothelial cells line all blood vessels and coordinate blood vessel formation and the blood-tissue barrier via endothelial cell-cell junctions. The nucleus also regulates endothelial cell behaviors, but the mechanisms are poorly understood. Here we show that nuclear-localized SUN1, a LINC complex component that connects the nucleus to the cytoskeleton, regulates endothelial cell-cell junction communication and blood vessel formation. Loss of murine endothelial Sun1 impaired blood vessel formation and destabilized junctions. At the cellular level, SUN1 stabilized endothelial cell-cell junctions and promoted barrier function. Abnormal SUN1-depleted junctions resembled those seen with loss of microtubules, and they were accompanied by impaired microtubule dynamics and actomyosin hypercontractility. Angiogenic sprouts formed but retracted in SUN1-depleted endothelial cells, and vessels of zebrafish lacking SUN1 had abnormal extension and were defective in forming connections. Thus, endothelial SUN1 regulates peripheral cell-cell junctions from the nucleus, likely via microtubule-based interactions, and this long-range regulation is important for blood vessel formation and barrier function.