Rapamycin Inhibits Proliferation of Hemangioma Endothelial Cells by Reducing HIF-1-Dependent Expression of VEGF

Hemangiomas are tumors formed by hyper-proliferation of vascular endothelial cells. This is caused by elevated vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we show that elevated VEGF levels produced by hemangioma endothelial cells are reduced by the mTOR inhibitor rapamycin. mTOR activates p70S6K, which controls translation of mRNA to generate proteins such as hypoxia inducible factor-1 (HIF-1). VEGF is a known HIF-1 target gene, and our data show that VEGF levels in hemangioma endothelial cells are reduced by HIF-1α siRNA. Over-expression of HIF-1α increases VEGF levels and endothelial cell proliferation. Furthermore, both rapamycin and HIF-1α siRNA reduce proliferation of hemangioma endothelial cells. These data suggest that mTOR and HIF-1 contribute to hemangioma endothelial cell proliferation by stimulating an autocrine loop of VEGF signaling. Furthermore, mTOR and HIF-1 may be therapeutic targets for the treatment of hemangiomas.     

microRNA-151-5p在肾血管性高血压大鼠血管内皮细胞中的表达及意义

目的:研究microRNA-151-5p(miR-151-5p)在两肾一夹(2K1C)肾血管性高血压大鼠中的表达,并为miR-151-5p参与调节血管内皮细胞功能提供理论依据。方法:建立2K1C大鼠模型,获得胸主动脉血管内皮,采用荧光定量PCR技术检测血管内皮细胞中miR-151-5p的表达,通过数据库及生物信息学软件预测miR-151-5p的靶基因,并对靶基因进行GO富集和KEGG pathway分析。结果:与假手术组大鼠相比较,实验组大鼠胸主动脉血管内皮细胞miR-151-5p的表达量显著升高(P0.05)。GO分析显示miR-151-5p的靶基因参与蛋白加工水解、Notch受体加工等多种生物学功能(P0.01);KEGG pathway分析显示miR-151-5p的靶基因参与Notch信号通路、血管平滑肌收缩、代谢途径、细菌感染和RNA转运等信号通路。结论:2K1C大鼠血管内皮细胞中miR-151-5p表达升高,可能通过对其靶基因APH1A的调控参与血管内皮细胞功能调节。     

p38 MAPK mediates gamma-irradiation-induced endothelial cell apoptosis, and vascular endothelial growth factor protects endothelial cells through the phosphoinositide 3-kinase-Akt-Bcl-2 pathway

Therapeutic radiation is widely used in cancer treatments. The success of radiation therapy depends not only on the radiosensitivity of tumor cells but also on the radiosensitivity of endothelial cells lining the tumor vasculature. Vascular endothelial growth factor (VEGF) plays a critical role in protecting endothelial cells against a number of antitumor agents including ionizing radiation. Strategies designed to overcome the survival advantage afforded to endothelial cells by VEGF might aid in enhancing the efficacy of radiation therapy. In this report we examined the signaling cascade(s) involved in VEGF-mediated protection of endothelial cells against gamma-irradiation. gamma-Irradiation-induced apoptosis of human dermal microvascular endothelial cells (HDMECs) was predominantly mediated through the p38 MAPK pathway as an inhibitor of p38 MAPK (PD169316), and dominant negative mutants of p38 MAPK could significantly enhance HDMEC survival against gamma-irradiation. Inhibition of the PI3K and MAPK pathways markedly up-regulated gamma-irradiation-mediated p38 MAPK activation resulting in enhanced HDMEC apoptosis. In contrast, VEGF-treated HDMECs were protected from gamma-irradiation-induced apoptosis predominantly through the PI3K/Akt pathway. Bcl-2 expression was markedly elevated in VEGF-treated HDMECs, and it was significantly inhibited by the PI3K inhibitor LY294002. HDMECs exposed to irradiation showed a significant decrease in Bcl-2 expression. In contrast, VEGF-stimulated HDMECs, when irradiated, maintained higher levels of Bcl-2 expression. Taken together our results suggest that gamma-irradiation induces endothelial cell apoptosis predominantly via the activation of p38 MAPK, and VEGF protects endothelial cells against gamma-irradiation predominantly via the PI3K-Akt-Bcl-2 signaling pathway.     

miR-221 is required for endothelial tip cell behaviors during vascular development

Angiogenesis requires coordination of distinct cell behaviors between tip and stalk cells. Although this process is governed by regulatory interactions between the vascular endothelial growth factor (Vegf) and Notch signaling pathways, little is known about the potential role of microRNAs. Through deep sequencing and functional screening in zebrafish, we find that miR-221 is essential for angiogenesis. miR-221 knockdown phenocopied defects associated with loss of the tip cell-expressed Flt4 receptor. Furthermore, miR-221 was required for tip cell proliferation and migration, as well as tip cell potential in mosaic blood vessels. miR-221 knockdown also prevented "hyper-angiogenesis" defects associated with Notch deficiency and miR-221 expression was inhibited by Notch signaling. Finally, miR-221 promoted tip cell behavior through repression of two targets: cyclin dependent kinase inhibitor 1b (cdkn1b) and phosphoinositide-3-kinase regulatory subunit 1 (pik3r1). These results identify miR-221 as an important regulatory node through which tip cell migration and proliferation are controlled during angiogenesis.     

Notch activation induces endothelial cell cycle arrest and participates in contact inhibition: role of p21Cip1 repression

Although previous studies demonstrate that appropriate Notch signaling is required during angiogenesis and in vascular homeostasis, the mechanisms by which Notch regulates vascular function remain to be elucidated. Here, we show that activation of the Notch pathway by the ligand Jagged1 reduces the proliferation of endothelial cells. Notch activation inhibits proliferation of endothelial cells in a cell-autonomous manner by inhibiting phosphorylation of the retinoblastoma protein (Rb). During cell cycle entry, p21Cip1 is upregulated in endothelial cells. Activated Notch inhibits mitogen-induced upregulation of p21Cip1 and delays cyclin D-cdk4-mediated Rb phosphorylation. Notch-dependent repression of p21Cip1 prevents nuclear localization of cyclin D and cdk4. The necessity of p21Cip1 for nuclear translocation of cyclin D-cdk4 and S-phase entry in endothelial cells was demonstrated by targeted downregulation of p21Cip1 by using RNA interference. We further demonstrate that when endothelial cells reach confluence, Notch is activated and p21Cip1 is downregulated. Inhibition of the Notch pathway at confluence prevents p21Cip1 downregulation and induces Rb phosphorylation. We suggest that Notch activation contributes to contact inhibition of endothelial cells, in part through repression of p21Cip1 expression.     

Notch信号通路在血管损伤修复中的作用

Notch信号通路是进化中高度保守的信号转导通路,其调控细胞增殖、分化和凋亡的功能涉及几乎所有组织和器官。血管损伤后,Notch信号通路分子表达改变,引起内皮细胞（endothelial cell,EC）和血管平滑肌细胞（vascular smooth muscle cell,VSMC）表型改变,其增殖、迁移、抗凋亡等能力也随之变化,从而参与血管的损伤修复。Notch信号通路能够促进EC和VSMC增殖以及VSMC迁移至内膜,并提高其存活能力,凶此能够促进新生内膜的形成。     

Down-regulation of mir-424 contributes to the abnormal angiogenesis via MEK1 and cyclin E1 in senile hemangioma: its implications to therapy

Background

Senile hemangioma, so-called cherry angioma, is known as the most common vascular anomalies specifically seen in the aged skin. The pathogenesis of its abnormal angiogenesis is still unclear.

Methodology/Principal Findings

In this study, we found that senile hemangioma consisted of clusters of proliferated small vascular channels in upper dermis, indicating that this tumor is categorized as a vascular tumor. We then investigated the mechanism of endothelial proliferation in senile hemangioma, focusing on microRNA (miRNA). miRNA PCR array analysis revealed the mir-424 level in senile hemangioma was lower than in other vascular anomalies. Protein expression of MEK1 and cyclin E1, the predicted target genes of mir-424, was increased in senile hemangioma compared to normal skin or other anomalies, but their mRNA levels were not. The inhibition of mir-424 in normal human dermal microvascular ECs (HDMECs) using specific inhibitor in vitro resulted in the increase of protein expression of MEK1 or cyclin E1, while mRNA levels were not affected by the inhibitor. Specific inhibitor of mir-424 also induced the cell proliferation of HDMECs significantly, while the cell number was decreased by the transfection of siRNA for MEK1 or cyclin E1.

Conclusions/Significance

Taken together, decreased mir-424 expression and increased levels of MEK1 or cyclin E1 in senile hemangioma may cause abnormal cell proliferation in the tumor. Senile hemangioma may be the good model for cutaneous angiogenesis. Investigation of senile hemangioma and the regulatory mechanisms of angiogenesis by miRNA in the aged skin may lead to new treatments using miRNA by the transfection into senile hemangioma.     

miR-125b-5p对人血管瘤内皮细胞HemES增殖和凋亡的影响及其机制

目的:研究miR-125b-5p对人血管瘤内皮细胞HemECs增殖、凋亡的影响.方法:RT-qPCR检测人血管瘤内皮细胞HemECs及其旁系组织细胞中miR-125b-5p与MCL-1 mRNA的表达;选取HemECs细胞分为对照组、miR-NC 组、miR-125b-5p mimic 组、miR-125b-5p in...     

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.     

Notch Signaling Change in Pulmonary Vascular Remodeling in Rats with Pulmonary Hypertension and Its Implication for Therapeutic Intervention

Pulmonary hypertension (PH) is a fatal disease that lacks an effective therapy. Notch signaling pathway plays a crucial role in the angiogenesis and vascular remodeling. However, its roles in vascular remodeling in PH have not been well studied. In the current study, using hypoxia-induced PH model in rat, we examined the expression of Notch and its downstream factors. Then, we used vessel strip culture system and γ-secretase inhibitor DAPT, a Notch signaling inhibitor to determine the effect of Notch signaling in vascular remodeling and its potential therapeutic value. Our results indicated that Notch 1–4 were detected in the lung tissue with variable levels in different cell types such as smooth muscle cells and endothelial cells of pulmonary artery, bronchia, and alveoli. In addition, following the PH induction, all of Notch1, Notch3, Notch4 receptor, and downstream factor, HERP1 in pulmonary arteries, mRNA expressions were increased with a peak at 1–2 weeks. Furthermore, the vessel wall thickness from rats with hypoxia treatment increased after cultured for 8 days, which could be decreased approximately 30% by DAPT, accompanied with significant increase of expression level of apoptotic factors (caspase-3 and Bax) and transformation of vascular smooth muscle cell (VSMC) phenotype from synthetic towards contractile. In conclusion, the current study suggested Notch pathway plays an important role in pulmonary vascular remodeling in PH and targeting Notch signaling pathway could be a valuable approach to design new therapy for PH.     

miR-29c-3p Increases Cell Viability and Suppresses Apoptosis by Regulating the TNFAIP1/NF-κB Signaling Pathway via TNFAIP1 in Aβ-Treated Neuroblastoma Cells

Alzheimer’s disease (AD) is the most common cause of dementia among older people in worldwide. miR-29c-3p was reported to play a role in AD development. However, the detail function of miR-29c-3p in AD remains unclear. The aim of this research is to analyze the functional mechanism of miR-29c-3p in AD. The RNA levels of miR-29c-3p and Tumor necrosis factor-α-inducible protein-1 (TNFAIP1) were detected by Quantitative real time polymerase chain (qRT-PCR) reaction. Western blot assay was carried out to examine the protein levels of TNFAIP1, Bax, B-cell lymphoma-2 (Bcl-2), Cleaved caspase 3, and Nuclear factor-k-gene binding (NF-κB). The interaction between miR-29c-3p and TNFAIP1 was predicted by online tool TargrtScan and verified using the dual luciferase reporter assay and RNA immunoprecipitation RIP (RIP) assay. Besides, cell proliferation and apoptosis rate were determined by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometry analysis, respectively. Aβ treatment decreased miR-29c-3p expression and increased TNFAIP1 expression. Overexpression of miR-29c-3p mitigated the effects of Aβ on proliferation and apoptosis. Similarly, knockdown of TNFAIP1 also reversed the effects of Aβ on cell progression. Interestingly, miR-29c-3p suppressed the expression of TNFAIP1 via binding to 3′UTR of TNFAIP1 mRNA. As expected, overexpression of TNFAIP1 reversed the effects of miR-29c-3p on Aβ-mediated cell progression. Besides, we also confirmed that miR-29c-3p affected Aβ-mediated cell progression by regulating TNFAIP1/NF-κB signaling pathway. In conclusion, our findings confirmed that miR-29c-3p attenuated Aβ-induced neurotoxicity through regulation of NF-κB signaling pathway by directly targeting TNFAIP1, providing the potential value for the treatment of AD patients.

     

miR-126 enhances the sensitivity of non-small cell lung cancer cells to anticancer agents by targeting vascular endothelial growth factor A

Increasing evidence suggests that hsa-miR-126 (miR-126) is down-regulated in non-small cell lung cancer (NSCLC) cell lines and the restoration of miR-126 impairs tumor cell proliferation, migration, invasion, and survival by targeting specific molecules. Here, we reported for the first time that miR-126 was involved in regulating the response of NSCLC cells to cancer chemotherapy. After transfected A549 cells with miR-126 mimic or inhibitor, we found that an elevated level of miR-126 was significantly associated with a decreased half maximal inhibitory concentration of adriamycin (ADM) and vincristine, an increased accumulation of ADM, down-regulation of vascular endothelial growth factor A (VEGFA) and multidrug resistance-associated protein 1 (MRP1), and inactivation of the Akt signaling pathway. Furthermore, enhanced expression of miR-126 suppressed the growth of A549 xenograft and inhibited the expression of VEGFA and MRP1. miR-126 could efficiently down-regulate VEGFA expression through the interaction with the VEGFA 3'-untranslated region, whereas restoration of VEGFA could partially attenuate the suppression of MRP1 by miR-126. However, LY294002, an inhibitor of the PI3K/Akt signaling pathway, diminished this effect, suggesting that enhanced expression of miR-126 increased the sensitivity of NSCLC cells to anticancer agents through negative regulation of a VEGF/PI3K/Akt/MRP1 signaling pathway.     

Cocoa procyanidins inhibit proliferation and angiogenic signals in human dermal microvascular endothelial cells following stimulation by low-level H2O2

Procyanidins extracted from cocoa play a role in the defense against oxidative stress, as well as in vascular and immune functions. We previously reported that pentameric procyanidins isolated from cocoa inhibit the expression of the tyrosine kinase ErbB2 gene, thus slowing the growth of cultured human aortic endothelial cells. We herein investigate the further consequences of such inhibition by cocoa procyanidins, particularly regarding the protein level in phosphorylation patterns and the effects on the proliferation of human dermal microvascular endothelial cells (HDMECs) following angiogenic stimulation with low-level H2O2. We report herein that both the pentameric and octameric procyanidin fractions of cocoa inhibit the proliferation of HDMECs, whereas the pentameric fraction modulates the activity of several crucial proteins in angiogenic signaling by altering their tyrosine phosphorylation. Similar to aortic endothelial cells, the pentameric procyanidin fraction down-regulates the expression of ErbB2 tyrosine kinase in HDMECs. In conclusion, we report evidence suggesting that polyphenols may influence endothelial growth signaling, thus affecting angiogenesis in vitro. If these observations are applicable in vivo, they suggest a beneficial effect for cells overexpressing ErbB2, such as in specific neoplasias     

MicroRNA-194 reduces inflammatory response and human dermal microvascular endothelial cells permeability through suppression of TGF-β/SMAD pathway by inhibiting THBS1 in chronic idiopathic urticaria

Chronic idiopathic urticaria (CIU) is a polyetiological dermatologic disease. Reports have stated that some microRNAs (miRNAs) have their roles to play in inflammatory response. In this present study, we aim to investigate whether miR-194 has an effect on attenuating inflammatory response and human dermal microvascular endothelial cells (HDMECs) permeability of CIU mast cells through TGF-β/SMAD pathway by binding to thrombospondin 1 (THBS1). The Gene Expression Omnibus database was used to obtain the CIU-related microarray data, and then the analysis of differentially expressed genes was conducted and the miRNA regulated by THBS1 was predicted. After transfection of different mimic, inhibitor, or small interfering RNA, the effect of miR-194 on inflammatory reaction, mast cell degranulation, histamine release rate, HDMECs permeability, and the expression of THBS1, interferon γ (IFN-γ), TGF-β, Smad3, and interleukin 4 (IL-4) were detected. THBS1 was verified to be the miR-194 target. After transfected with overexpressed miR-194 and si-THBS1, the degranulation rate, histamine release rate, and HDMECs permeability were significantly reduced, while the expression of IFN-γ was higher, and the expression of THBS1, TGF-β, Smad3, IL-4 was significantly lower, accompanied with alleviated inflammatory reaction. Our study provides evidence that miR-194 negatively modulates THBS1 and inhibits the activation of TGF-β/SMAD pathway, thereby alleviating the inflammatory response and HDMECs permeability of mast cells in CIU.     

NOTCH1 regulates the proliferation and migration of bladder cancer cells by cooperating with long non-coding RNA HCG18 and microRNA-34c-5p

In recent years, the NOTCH signaling pathway has been gradually studied in human malignancies. Inactivation of the NOTCH signaling pathway was uncovered to be correlated with the carcinogenesis of bladder cancer (BCa). Nevertheless, the specific molecular mechanism of NOTCH1 (one of the core factors of the NOTCH signaling pathway) is not well elucidated in BCa. This study focused on the mechanism by which NOTCH1 affects the biological behaviors of BCa cells. According to the experimental results of quantitative real-time polymerase chain reaction, NOTCH1 was dysregulated in BCa tissues and cell lines. The prognostic value of NOTCH1 for the patients with BCa was determined using the Kaplan-Meier method. Mechanism investigations revealed that NOTCH1 is a target of miR-34c-5p in BCa. Furthermore, microarray analysis was used to find the dysregulated long noncoding RNAs (lncRNA), which can bind with miR-34c-5p. Mechanism experiments further demonstrated the rationality of the HCG18-miR-34c-5p-NOTCH1 pathway. Functional assays were then applied to validate the inhibitory influences of NOTCH1 on the proliferation and migration of BCa cells. Furthermore, the inhibitory effects of NOTCH1 could be affected by miR-34c-5p or lncRNA HCG18. All findings in this study revealed that NOTCH1 suppresses the BCa progression by cooperating with lncRNA HCG18 and miR-34c-5p.