Zhi-Feng Deng

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.

Pre-conditioned mesenchymal stem cells: a better way for cell-based therapy

Ischemic heart disease is the major cause of death globally, and a recently developed stem cell transplantation is a promising therapy for myocardial infarction. Mesenchymal stem cells (MSCs) exist in a wide range of tissues, and their differentiation potential and immunoregulatory capacity make them a more optimal candidate for regenerative medicine. However, the poor survival and low differentiation efficiency of the donor cells in the infarcted myocardium challenged therapeutic efficacy of MSC transplantation. To this end, many researchers have focused on improving the microenvironments of MSCs before and after transplantation and on trying to figure out the mechanisms. A recent study by Boopathy and colleagues reported the pro-cardiovascular differentiation effect of oxidative stress on cultured MSCs and the underlying signal pathways, leading to the notion that MSCs pre-conditioned with oxidative reagents promote cardiac differentiation efficiency of MSCs and may result in better clinical effect for ischemic heart diseases.

tRNA-Derived Small Non-Coding RNAs in Response to Ischemia Inhibit Angiogenesis

Ischemic injuries will lead to necrotic tissue damage, and post-ischemia angiogenesis plays critical roles in blood flow restoration and tissue recovery. Recently, several types of small RNAs have been reported to be involved in this process. In this study, we first generated a rat brain ischemic model to investigate the involvement of new types of small RNAs in ischemia. We utilized deep sequencing and bioinformatics analyses to demonstrate that the level of small RNA fragments derived from tRNAs strikingly increased in the ischemic rat brain. Among these sequences, tRNAVal- and tRNAGly-derived small RNAs account for the most abundant segments. The up-regulation of tRNAVal- and tRNAGly-derived fragments was verified through northern blot and quantitative PCR analyses. The levels of these two fragments also increased in a mouse hindlimb ischemia model and cellular hypoxia model. Importantly, up-regulation of the tRNAVal- and tRNAGly-derived fragments in endothelial cells inhibited cell proliferation, migration and tube formation. Furthermore, we showed that these small RNAs are generated by angiogenin cleavage. Our results indicate that tRNA-derived fragments are involved in tissue ischemia, and we demonstrate for the first time that tRNAVal- and tRNAGly-derived fragments inhibit angiogenesis by modulating the function of endothelial cells.

Downregulation of the Long Non-Coding RNA Meg3 Promotes Angiogenesis After Ischemic Brain Injury by Activating Notch Signaling.

Angiogenesis after ischemic brain injury contributes to the restoration of blood supply in the ischemic zone. Strategies to improve angiogenesis may facilitate the function recovery after stroke. Recent researches have demonstrated that dysfunction of long non-coding RNAs are associated with angiogenesis. We have previously reported that long non-coding RNAs (lncRNAs) are aberrantly expressed in ischemic stroke. However, little is known about long non-coding RNAs and theirs role in angiogenesis after stroke. In this study, we identified a rat lncRNAs, Meg3, and found that Meg3 was significantly decreased after ischemic stroke. Overexpression of Meg3 suppressed functional recovery and decreased capillary density after ischemic stroke. Downregulation of Meg3 ameliorated brain lesion and increased angiogenesis after ischemic stroke. Silencing of Meg3 resulted in a proangiogenic effect evidenced by increased endothelial cell migration, proliferation, sprouting, and tube formation. Mechanistically, we showed that Meg3 negatively regulated notch pathway both in vivo and in vitro. Inhibition of notch signaling in endothelial cells reversed the proangiogenic effect induced by Meg3 downregulation. This study revealed the function of Meg3 in ischemic stroke and elucidated its mechanism in angiogenesis after ischemic stroke.

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.

Repression of SIRT1 Promotes the Differentiation of Mouse Induced Pluripotent Stem Cells into Neural Stem Cells

The use of transplanting functional neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSCs) has increased for the treatment of brain diseases. As such, it is important to understand the molecular mechanisms that promote NSCs differentiation of iPSCs for future NSC-based therapies. Sirtuin 1 (SIRT1), a NAD+-dependent protein deacetylase, has attracted significant attention over the past decade due to its prominent role in processes including organ development, longevity, and cancer. However, it remains unclear whether SIRT1 plays a role in the differentiation of mouse iPSCs toward NSCs. In this study, we produced NSCs from mouse iPSCs using serum-free medium supplemented with retinoic acid. We then assessed changes in the expression of SIRT1 and microRNA-34a, which regulates SIRT1 expression. Moreover, we used a SIRT1 inhibitor to investigate the role of SIRT1 in NSCs differentiation of iPSCs. Data revealed that the expression of SIRT1 decreased, whereas miRNAs-34a increased, during this process. In addition, the inhibition of SIRT1 enhanced the generation of NSCs and mature neurocytes. This suggests that SIRT1 negatively regulated the differentiation of mouse iPSCs into NSCs, and that this process may be regulated by miRNA-34a.

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.

Role of Phosphorylated HDAC4 in Stroke-Induced Angiogenesis

Acetylation or deacetylation of chromatin proteins and transcription factors is part of a complex signaling system that is involved in the control of neurological disorders. Recent studies have demonstrated that histone deacetylases (HDACs) exert protective effects in attenuating neuronal injury after ischemic insults. Class IIa HDAC4 is highly expressed in the brain, and neuronal activity depends on the nucleocytoplasmic shuttling of HDAC4. However, little is known about HDAC4 and its roles in ischemic stroke. In this study, we report that phosphorylation of HDAC4 was remarkably upregulated after stroke and blockade of HDAC4 phosphorylation with GÖ6976 repressed stroke-induced angiogenesis. Phosphorylation of HDAC4 was also increased in endothelial cells hypoxia model and suppression of HDAC4 phosphorylation inhibited the tube formation and migration of endothelial cells in vitro. Furthermore, in addition to the inhibition of angiogenesis, blockade of HDAC4 phosphorylation suppressed the expression of genes downstream of HIF-VEGF signaling in vitro and in vivo. These data indicate that phosphorylated HDAC4 may serve as an important regulator in stroke-induced angiogenesis. The protective mechanism of phosphorylated HDAC4 is associated with HIF-VEGF signaling, implicating a novel therapeutic target in stroke.

Extracellular Vesicles Secreted by Human Urine-Derived Stem Cells Promote Ischemia Repair in a Mouse Model of Hind-Limb Ischemia

Background/Aims: Our previous studies have shown that human urine-derived stem cells (USCs) have great potential as a cell source for cytotherapy and tissue engineering and that extracellular vesicles (EVs) secreted by USCs (USCs-EVs) can prevent diabetes-induced kidney injury in an animal model. The present study was designed to evaluate the effects of USCs-EVs on ischemia repair. Methods: USCs-EVs were isolated and purified by a battery of centrifugation and filtration steps. The USCs-EVs were then characterized by transmission electron microscopy, western blot and tunable resistive pulse sensing techniques. After intramuscularly transplanting USCs-EVs into an ischemic mouse hind-limb, we observed the therapeutic effects of USCs-EVs on perfusion by laser doppler perfusion imaging, angiogenesis and muscle regeneration by histology and immunohistochemistry techniques over 21 days. We subsequently tested whether USCs-EVs can induce the proliferation of a human microvascular endothelial cell line HMEC-1 and a mouse myoblast cell line C2C12 by cell counting kit 8 assay in vitro. Meanwhile, the potential growth factors in the USCs-EVs and supernatants of the USCs cultures were detected by enzyme-linked immunosorbent assay. Results: The USCs-EVs were spherical vesicles with a diameter of 30–150 nm and expressed exosomal markers, such as CD9, CD63 and Tsg101. Ischemic limb perfusion and function were markedly increased in the hind-limb ischemia (HLI) model after USCs-EVs administration. Moreover, angiogenesis and muscle regeneration levels were significantly higher in the USCs-EVs treatment group than in the PBS group. The in vitro experiments showed that USCs-EVs facilitated HMEC-1 and C2C12 cell proliferation in a dose-dependent manner. Conclusions: These results revealed for the first time that USCs-EVs efficiently attenuate severe hind-limb ischemic injury and represent a novel therapy for HLI.

Systematic Analysis of RNA Regulatory Network in Rat Brain after Ischemic Stroke

Although extensive studies have identified large number of microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) in ischemic stroke, the RNA regulation network response to focal ischemia remains poorly understood. In this study, we simultaneously interrogate the expression profiles of lncRNAs, miRNAs, and mRNAs changes during focal ischemia induced by transient middle cerebral artery occlusion. A set of 1924 novel lncRNAs were identified and may involve brain injury and DNA repair as revealed by coexpression network analysis. Furthermore, many short interspersed elements (SINE) mediated lncRNA:mRNA duplexes were identified, implying that lncRNAs mediate Staufen1-mediated mRNA decay (SMD) which may play a role during focal ischemia. Moreover, based on the competitive endogenous RNA (ceRNA) hypothesis, a stroke regulatory ceRNA network which reveals functional lncRNA:miRNA:mRNA interactions was revealed in ischemic stroke. In brief, this work reports a large number of novel lncRNAs responding to focal ischemia and constructs a systematic RNA regulation network which highlighted the role of ncRNAs in ischemic stroke.