Yuanlei Lou

miR-210 activates notch signaling pathway in angiogenesis induced by cerebral ischemia

The compensatory angiogenesis that occurs after cerebral ischemia increases blood flow to the injured area and limits extension of the ischemic penumbra. In this way, it improves the local blood supply. Fostering compensatory angiogenesis is an effective treatment for ischemic cerebrovascular disease. However, angiogenesis in the adult organism is a complex, multi-step process, and the mechanisms underlying the regulation of angiogenesis are not well understood. Although Notch signaling reportedly regulates the vascularization process that occurs in ischemic tissues, little is known about the role of Notch signaling in the regulation of ischemia-induced angiogenesis after ischemic stroke. Recent research has indicated that miR-210, a hypoxia-induced microRNA, plays a crucial role in regulating the biological processes that occur in blood vessel endothelial cells under hypoxic conditions. This study was undertaken to investigate the role of miR-210 in regulating angiogenesis in response to brain ischemia injury and the role of the Notch pathway in the body’s response. We found miR-210 to be significantly up-regulated in adult rat ischemic brain cortexes in which the expression of Notch1 signaling molecules was also increased. Hypoxic models of human umbilical vein endothelial cells (HUVE-12) were used to assess changes in miR-210 and Notch1 expression in endothelial cells. Results were consistent with in vivo findings. To determine the molecular mechanisms behind these phenomena, we transfected HUVE-12 cells with miR-210 recombinant lentiviral vectors. We found that miR-210 overexpression caused up-regulation of Notch1 signaling molecules and induced endothelial cells to migrate and form capillary-like structures on Matrigel. These data suggest that miR-210 is involved in the regulation of angiogenesis in response to ischemic injury to the brain. Up-regulation of miR-210 can activate the Notch signaling pathway, which may contribute to angiogenesis after cerebral ischemia.

Upregulation of MicroRNA-210 Regulates Renal Angiogenesis Mediated by Activation of VEGF Signaling Pathway under Ischemia/Perfusion Injury in vivo and in vitro

Background: MicroRNAs (miRNAs) are endogenous, non-coding, small RNAs that regulate gene expression and function, but little is known about regulation of miRNAs in the kidneys under normal or pathologic conditions. Here, we sought to investigate the potential involvement of miRNAs in renal ischemia/reperfusion (I/R) injury and angiogenesis and to define some of the miRNAs possibly associated with renal angiogenesis. Methods and Results: Male Balb/c mice were subjected to a standard renal I/R. CD31 immunostaining indicated a significant increase of microvessels in the ischemic region. VEGF and VEGFR2 expression were increased in renal I/R at both the mRNA and protein levels which were detected by qRT-PCR and Western blot, respectively. More importantly, 76 microRNAs exhibited more than 2-fold changes using Agilent microRNA microarray, which contains downregulation of 40 miRNAs and upregulation of 36 miRNAs. Upregulation of miR-210 was confirmed by qRT-PCR with prominent changes at 4 and 24 h after reperfusion. Furthermore, overexpression of miR-210 in HUVEC-12 cells enhances VEGF and VEGFR2 expression and promotes angiogenesis on Matrigel in vitro. Conclusion: These findings suggest miR-210 may be involved in targeting the VEGF signaling pathway to regulate angiogenesis after renal I/R injury, which provides novel insights into the angiogenesis mechanism of renal I/R injury.

Bone marrow stromal cells enhance the angiogenesis in ischaemic cortex after stroke: involvement of notch signalling

Angiogenesis takes place after brain ischaemia, and stroke‐induced angiogenesis in ischaemic brain may be associated with improved neurological recovery. Bone MSCs (marrow stromal cells) transplantation can promote this vital angiogenesis in ischaemic zones, but the mechanisms by which MSCs promoting angiogenesis are unclear. The Notch signalling pathway may play an important role in embryonic blood vessels development and tumour angiogenesis, but whether it is also involved in angiogenesis after cerebral ischaemia is uncertain. We therefore investigated the Notch signalling pathway in angiogenesis after stroke. Rats were subjected to MCAo (middle cerebral artery occlusion) and treated intravenously with or without MSCs at 24 h after injury. On day 1, 3 and 7 after treatment with MSCs or PBS, immunofluorescent staining, Western blot and RT‐PCR (reverse transcription–PCR) assays were carried out to evaluate angiogenesis, and expression of VEGF (vascular endothelial growth factor) and Notch signals in the ischaemic cortex. Immunofluorescent showed a significant increase in both new microvessels, VEGF‐positive cells and Notch1‐positive microvessels in the ischaemic cortex in MSCs‐treated group. RT‐PCR indicated that MSC transplantation significantly raised VEGF mRNA and Hes1 mRNA levels in the ischaemic cortex. The data suggest that treatment with MSCs enhances stroke‐induced angiogenesis in ischaemic brain, and that the Notch signalling pathway is involved.

Mesenchymal stem cells regulate the proliferation and differentiation of neural stem cells through Notch signaling

The effects of mesenchymal stem cells (MSCs) on proliferation and cell fate determination of neural stem cells (NSCs) have been investigated. NSCs were co‐cultured with MSCs or NIH3T3 cells using an in vitro transwell system. After 4 days, immunofluorescence staining showed that the number of cells positive for the cell proliferation antigen, ki‐67, in neurospheres in MSCs was greater than in NIH3T3 cells. In some experiments, the top‐layers of MSCs and NIH3T3 cells were removed to induce NSCs differentiation. Seven days after initiating differentiation, the levels of the neuronal marker, NSE, were higher in NSCs in MSCs co‐culture group, and those of glial fibrillary acidic protein (GFAP) were lower, compared with NIH3T3 cells co‐culture group. These were confirmed by immunofluorescence. The role of the Notch signaling pathway analyzed with the specific inhibitor, DAPT, and by examining the expression of Notch‐related genes using RT‐PCR showed that after co‐culturing with MSCs for 24 h, NSCs expressed much higher levels of ki‐67, Notch1, and Hes1 than did NSCs co‐cultured with NIH3T3 cells. Treatment with DAPT decreased ki‐67, Notch1 and Hes1 expression in NCSs, and increased Mash1 expression. The data indicate that the interactions between MSCs and NSCs promote NSCs proliferation and are involved in specifying neuronal fate, mediated in part by Notch signaling.

Effects of low temperatures on proliferation-related signaling pathways in the hippocampus after traumatic brain injury.

To evaluate the influence of low temperatures on the proliferation of neural stem cells (NSCs) and the regulation of their signaling pathways after brain trauma, we examined changes in the expression levels of specific miRNAs and their target genes. We also evaluated NSC proliferation in the hippocampus after brain trauma under low-temperature conditions. We found that the expression profile of miRNAs in the hippocampus after trauma changed at both normal and low temperatures, and the expression of miR-34a decreased significantly lower in rats exposed to low temperatures. There was significant proliferation of endogenous NSCs in the hippocampus after brain trauma at both temperatures, but NSC proliferation was slower at low temperatures. In addition, the expression of Notch1 significantly increased in the hippocampus after brain trauma at both temperatures. However, at low temperatures, the degree of up-regulation of Notch signaling molecules was significantly lower. We conclude that low-temperature environments can inhibit the proliferation of endogenous NSCs in the hippocampus, possibly by alleviating the effects of miR-34a down-regulation and Notch signaling up-regulation induced by traumatic brain injury.

Exposure to lanthanum compound diminishes LPS-induced inflammation-associated gene expression: involvements of PKC and NF-κB signaling pathways

Lanthanum chloride, a rare earth compound, possesses antibacterial and cellular immunity regulating properties. However, the underlying molecular mechanisms remain largely unknown. In this study, we examined the effects of lanthanum chloride on the production of nitric oxide (NO) and tumor necrosis factor-α (TNF-α), the expression of inducible NO synthase (iNOS) and TNF-α in RAW 264.7 cells, a mouse macrophage cell line. We found that the LPS-elicited excessive production of NO and TNF-α in RAW 264.7 cells was inhibited significantly in the presence of lanthanum chloride, and the attenuation of iNOS and TNF-α occurred at mRNA level. Furthermore, the possible signaling components affected by lanthanum chloride in the pathway that lead to LPS-induced iNOS and TNF-α expression were explored. The results indicated the involvements of PKC/Ca2+ and NF-κB in the attenuation of NO and pro-inflammatory cytokine production by lanthanum chloride. Our observations suggest a possible therapeutic application of this agent for treating inflammatory diseases.

Inhibition of Notch signaling facilitates the differentiation of human-induced pluripotent stem cells into neural stem cells

Neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSCs) are becoming an appealing source of cell-based therapies of brain diseases. As such, it is important to understand the molecular mechanisms that regulate the differentiation of iPSCs toward NSCs. It is well known that Notch signaling governs the retention of stem cell features and drives stem cells fate. However, further studies are required to investigate the role of Notch signaling in the NSCs differentiation of iPSCs. In this study, we successfully generated NSCs from human iPSCs using serum-free medium supplemented with retinoic acid (RA) in vitro. We then assessed changes in the expression of Notch signaling-related molecules and some miRNAs (9, 34a, 200b), which exert their regulation by targeting Notch signaling. Moreover, we used a γ-secretase inhibitor (DAPT) to disturb Notch signaling. Data revealed that the levels of the Notch signaling-related molecules decreased, whereas those miRNAs increased, during this differentiation process. Inhibition of Notch signaling accelerated the formation of the neural rosette structures and the expression of NSC and mature neurocyte marker genes. This suggests that Notch signaling negatively regulated the neuralization of human iPSCs, and that this process may be regulated by some miRNAs.

Co-culture with cardiomyocytes induces mesenchymal stem cells to differentiate into cardiomyocyte-like cells and express heart development-associated genes

Co-culture with cardiomyocytes induces mesenchymal stem cells to differentiate into cardiomyocyte-like cells and express heart development-associated genes

Generation of special autosomal dominant polycystic kidney disease iPSCs with the capability of functional kidney-like cell differentiation

BackgroundHuman induced pluripotent stem cells (iPSCs) have been verified as a powerful cell model for the study of pathogenesis in hereditary disease. Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations of PKD or non-PKD genes. The pathogenesis of ADPKD remains unexplored because of the lack of a true human cell model.MethodsSix ADPKD patients and four healthy individuals were recruited as donors of somatic cells from a Chinese ADPKD family without mutations of the PKD genes but carrying SAMSN1 gene deletion. The ADPKD-iPSCs were generated from somatic cells and were induced into kidney-like cells (KLCs) by a novel three-step method involving cytokines and renal epithelium growth medium. Furthermore, we analyzed functional properties of these KLCs by water transportation and albumin absorption assays.ResultsWe successfully generated iPSCs from ADPKD patients and differentiated them into KLCs that showed morphological and functional characteristics of human kidney cells. Further, we also found that ADPKD-iPSC-KLCs had a significantly higher rate of apoptosis and a significantly lower capacity for water transportation and albumin absorption compared to healthy sibling-derived differentiated KLCs. Furthermore, knockdown of SAMSN1 in control iPSCs may attenuate differentiation and/or function of KLCs.ConclusionsThese data show that we have created the first iPSCs established from ADPKD patients without mutations in the PKD genes, and suggest that the deletion mutation of SAMSN1 might be involved in the differentiation and/or function of KLCs. ADPKD-iPSC-KLCs can be used as a versatile model system for the study of kidney disease.

Sodium ferulate induces bone marrow mesenchymal stem cells to differentiate into neural cells by NF-κB signal pathway

Sodium ferulate induces bone marrow mesenchymal stem cells to differentiate into neural cells by NF-κB signal pathway