HUVEC Tube Formation Research Articles

AKT, a Key Transmitter of HIF-1α and AR Signaling Pathways, Has a Critical Role in the Apigetrin-Mediated Anti-Cancer Effects in Prostate Cancer Cells

Apigetrin is a flavonoid glycoside phytochemical that is derived from various herbs and exhibits several beneficial biological activities, including anti-oxidant, anti-inflammatory, anti-obesity, and anti-cancer effects. In the present study, we elucidated the anti-cancer effect and targeting mechanism of apigetrin in LNCaP and PC-3 cells through various experiments, including cell viability by CELLOMAXTM Viability Assay kit, cell migration by scratch wound assays, and 2D-and 3D- cell growth assay. Apigetrin inhibited the viability, migration, proliferation, and growth of cells in long-term 2D- and 3D- cultures cell growth. A high dose of apigetrin induced apoptosis, as evidenced by increased cleavage of poly ADP-ribose polymerase (PARP) and caspase-3 (c-cas3) in both LNCaP and PC-3 cells. Furthermore, apigetrin inhibited AR, PSA, HIF-1α, and VEGF expression in LNCaP and PC-3 cells. Apigetrin also suppressed the hypoxia-induced HIF-1α expression in these cells. Furthermore, apigetrin reduced hypoxia-induced VEGF secretion in the culture medium and inhibited hypoxia-induced tube formation of HUVECs. Silencing of AKT revealed that the anti-cancer activity of apigetrin is mediated via AKT. Thus, our data suggest that apigetrin exerts anti-cancer effects by inhibiting AKT, a central key of HIF-1α and AR signaling, in early-and late-stage prostate cancer cells.

The circ_FAM53B-miR-183-5p-CCDC6 axis modulates the malignant behaviors of papillary thyroid carcinoma cells.

Papillary thyroid carcinoma (PTC) is a common thyroid malignancy. Circular RNAs (circRNAs) have been implicated in the development of PTC. Here, we explored the function and mechanism of circRNA family with sequence similarity 53, member B (circ_FAM53B) in PTC pathogenesis. Circ_FAM53B, microRNA (miR)-183-5p and coiled-coil domain containing 6 (CCDC6) levels were gauged by quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blotting. The direct relationship between miR-183-5p and circ_FAM53B or CCDC6 was verified by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Our data showed that circ_FAM53B expression was reduced in PTC tissues and cells. Circ_FAM53B expression restrained proliferation, migration, and invasion and triggered apoptosis of PTC cells, as well as hindered HUVEC tube formation. Circ_FAM53B repressed miR-183-5p expression. MiR-183-5p re-expression reversed the effects of circ_FAM53B on cell behaviors. MiR-183-5p targeted and inhibited CCDC6, and circ_FAM53B upregulated CCDC6 through miR-183-5p competition. MiR-183-5p knockdown repressed cell proliferation, migration, invasion, and tube formation and facilitated apoptosis by upregulating CCDC6. Furthermore, circ_FAM53B reduced tumor growth in vivo. Collectively, our findings suggest that circ_FAM53B affects PTC cell biological behaviors via the miR-183-5p-CCDC6 axis.

Total alkaloid fraction of Leonurus japonicus Houtt. Promotes angiogenesis and wound healing through SRC/MEK/ERK signaling pathway

Ethnopharmacological relevanceLeonurus japonicus Houtt., also known as motherwort, is a traditional Chinese medicine that was first identified in Shennong Bencao Jing, the first and essential pharmacy monograph in China. L. japonicus has been regarded as a good gynecological medicine since ancient times. It has been widely used in clinical settings for treatment of gynecological diseases and postnatal rehabilitation with good efficacy and low adverse effects. Aim of the studyThe main purpose of this study was to determine the angiogenic and wound healing effects of total alkaloid fraction from L. japonicus Houtt. (TALH) in vivo and in vitro. In addition, the main bioactive components of total alkaloids were to be identified and analyzed in this study. Materials and methodsFirst, the UHPLC/Q-TOF-MS method was used to identify and quantify the major components in the TALH extract. The wound healing activity was evaluated in vivo using a rat full-thickness cutaneous wound model. Histological study of wound healing in rat model was performed via immunohistochemistry and immunofluorescence. Cell proliferation was determined by MTT assay. Wound healing and transwell assays were used for detection of cell migration. The effect on tube formation was determined by tube formation assay in HUVECs. Western blot and RT-PCR were used to detect the expressions of relative proteins and genes respectively. Knock-down of SRC by siRNA was done to verify the crucial role of SRC in promotion of angiogenesis induced by TALH. ResultsSeven characteristic peaks were recognized in the UHPLC/Q-TOF-MS spectrum, while four of the main components were quantified. The wound model in rats showed that treatment of TALH promoted wound healing by stimulating cellular proliferation and collagen deposition. In vitro experiments showed that co-treatment of TALH and VEGF increased cell proliferation, migration and tube formation in HUVECs. Mechanistic studies suggested that the co-treatment increased gene expressions of SRC, MEK1/2 and ERK1/2, as well as the phosphorylation levels of these proteins. Furthermore, the effect of co-treatment was attenuated after SRC knockdown, suggesting that SRC plays an important role in angiogenesis and wound healing induced by TALH and VEGF co-treatment. ConclusionOur results showed that TALH was one of the main active components of L. japonicus that promoted angiogenesis and wound healing by regulating the SRC/MEK/ERK pathway. Our study provided scientific basis for better clinical application of L. japonicas.

Ligustilide Inhibits Tumor Angiogenesis by Downregulating VEGFA Secretion from Cancer-Associated Fibroblasts in Prostate Cancer via TLR4.

Simple SummaryInhibiting the production of vascular endothelial growth factor A (VEGFA) can inhibit angiogenesis, thereby inhibiting tumor growth. We found that ligustilide can inhibit the secretion of VEGFA from prostate cancer-related fibroblasts (CAFs), and this signaling pathway is related to Toll-like receptor 4 (TLR4)-ERK/JNK/p38. Using the above receptors or signals pathway molecule blockers can block the effect of ligustilide to downregulate the secretion of VEGFA from CAFs. The concentration of ligustilide used in this study does not directly inhibit the growth of CAFs, but changes its function, and this study is different from the direct blocking effect of the existing VEGFA antibodies, but cuts off the source of VEGFA, which is expected to become a novel therapeutic strategy for oncology.CAFs secrete VEGFA in the tumor microenvironment to induce angiogenesis and promote tumor growth. The downregulation of VEGFA secretion from CAFs helps block angiogenesis and exerts an anti-tumor effect. In vivo experiments showed that the angiogenesis of the tumor-bearing mice in the ligustilide group was significantly reduced. The results of MTT, tube formation, Transwell and scratch experiments showed that ligustilide did not affect the proliferation of HUVECs in a certain concentration range (<60 μM), but it inhibited the proliferation, tube formation and migration of HUVECs induced by CAFs. At this concentration, ligustilide did not inhibit CAF proliferation. The qPCR and WB results revealed that ligustilide downregulated the level of VEGFA in CAFs via the TLR4-ERK/JNK/p38 signaling pathway, and the effect was attenuated by blockers of the above molecules. Ligustilide also downregulated the autocrine VEGFA of HUVECs induced by CAFs, which inhibited angiogenesis more effectively. In addition, ligustilide inhibited glycolysis and HIF-1 expression in CAFs. Overall, ligustilide downregulated the VEGFA level in CAFs via the TLR4-ERK/JNK/p38 signaling pathway and inhibited the promotion of angiogenesis. This study provides a new strategy for the anti-tumor effect of natural active molecules, namely, blockade of angiogenesis, and provides a new candidate molecule for blocking angiogenesis in the tumor microenvironment.

Scutellaria barbata D.Don (SBD) extracts suppressed tumor growth, metastasis and angiogenesis in Prostate cancer via PI3K/Akt pathway

BackgroundScutellaria barbata D.Don (SBD) is derived from the dried whole plant of Labiate which has been widely used to treat patients with multiple cancer. It was previously reported that the ethanol extract of SBD is able to promote apoptosis, and inhibit cell proliferation and angiogenesis in cancer.Materials and methodsCCK8, Edu assays and colony formation assay were performed to assess the effect of SBD on PCa cell growth. Effect of SBD on apoptosis and cell cycle was detected by flow cytometry. Transwell and wounding healing assay were conducted to detect the invasion and migration activities of PCa cells. Western blot was employed to detect the protein expression. 2RRV1 mouse xenograft model was established to detect the effect of SBD on prostate cancer. Angiogenesis was analysed by coculturing PCa cell lines and HUVECs.ResultsThe results showed that SBD induced a significant decrease in cell viability and clonogenic growth in a dose-dependent manner. SBD induced cell apoptosis and cell cycle G2/M phase arrest by inactivating PI3K/AKT signalling pathway. Treatment with SBD also significantly decreased the cell migration and invasion via phenotypic inversion of EMT that was characterized by the increased expression of E-cadherin and Vimentin, and decreased expression of N-cadherin, which could be partially attributed to inhibiting PI3K/AKT signalling pathway. Subsequently, using AKT inhibitor MK2206, we concluded that PI3K/AKT are also involved in cell apoptosis and metastasis of PCa cells stimulated by SBD. Apart from its direct effects on PCa cells, SBD also exhibited anti-angiogenic properties. SBD alone or conditioned media from SBD-treated PCa cells reduced HUVEC tube formation on Matrigel without affecting HUVEC viability. Furthermore, 22RV1 xenograft C57BL/6 mice treated with SBD in vivo showed a significant inhibitory in tumour size and tumour weight without toxicity. In addition, administration with medium- or high-dose of SBD significantly inhibited the cell proliferation and enhanced the damage to tumour tissues.ConclusionsCollectively, our in vitro and in vivo findings suggest that SBD has the potential to develop into a safe and potent alternative therapy for PCa patients.

Smoking Impairs Hematoma Formation and Dysregulates Angiogenesis as the First Steps of Fracture Healing.

Bone fracture healing is an overly complex process in which inflammation, osteogenesis, and angiogenesis are tightly coupled, and delayed fracture repair is a very common health risk. One of the major causes of delayed healing is the formation of insufficient vasculature. Precise regulation of blood vessels in bone and their interplay with especially osteogenic processes has become an emerging topic within the last years; nevertheless, regulation of angiogenesis in (early) diseased fracture repair is still widely unknown. Here, we aim to develop an in vitro model for the analysis of early fracture healing which also enables the analysis of angiogenesis as a main influencing factor. As smoking is one of the main risk factors for bone fractures and developing a delay in healing, we model smoking and non-smoking conditions in vitro to analyze diverging reactions. Human in vitro fracture hematomas mimicking smokers’ and non-smokers’ hematomas were produced and analyzed regarding cell viability, inflammation, osteogenic and chondrogenic differentiation, and angiogenic potential. We could show that smokers’ blood hematomas were viable and comparable to non-smokers. Smokers’ hematomas showed an increase in inflammation and a decrease in osteogenic and chondrogenic differentiation potential. When analyzing angiogenesis, we could show that the smokers’ hematomas secrete factors that drastically reduced HUVEC proliferation and tube formation. With an angiogenesis array and gene expression analysis, we could identify the main influencing factors: Anpgt1/2, Tie2, and VEGFR2/3. In conclusion, our model is suitable to mimic smoking conditions in vitro showing that smoking negatively impacts early vascularization of newly formed tissue.

A novel risk score model based on five angiogenesis-related long non-coding RNAs for bladder urothelial carcinoma

BackgroundTumour angiogenesis is an independent risk factor for bladder urothelial carcinoma (BUC) progression, but viable and promising antiangiogenic targets are understudied. Emerging evidence suggests that long non-coding RNAs (lncRNAs) play prominent role in the tumour microenvironment and tumour angiogenesis.MethodsThe clinical data of BUC patients were obtained from TCGA database and clinical specimens of 138 BUC patients. Univariate and multivariate COX regression analyses were used to identify survival-related ARLNRs (sARLNRs) from The Molecular Signatures Database v4.0. Fisher’s exact probability method was used to detect the correlations between sARLNRs levels and clinicopathological characteristics. A chain of experiments including FACS, qPCR, immunohistochemistry, tube formation, migration and invasion assays, combining with co-culture models, were utilized to validate the clinical significance and angiogenetic correlation of sARLNRs.ResultsFive sARLNRs were employed to establish an angiogenesis-related risk score model, by which patients in the low-risk group obtained better overall survival than those in the high-risk group. The expression of AC005625.1 and AC008760.1 was significantly related to ECs percentage, tumour size and muscle invasion status. Besides, AC005625.1 and AC008760.1 expressed lower in BUC cell lines and tumour tissues than that in normal urothelial cells and adjacent normal tissues, with much lower levels in more advanced T stages. A prominently higher proportion of ECs was detected in tumour tissues with lower expression of AC005625.1 and AC008760.1. In the co-culture models, we found that knockdown of AC005625.1 and AC008760.1 in BUC cells increased the tube formation, migration and invasion abilities of HUVEC. The expression levels of CD31, VEGF-A, VIMENTIN and N-CADHERIN were also enhanced in HUVEC cells co-cultured with siR-AC005625.1 and siR-AC008760.1-treated T24 cells.ConclusionIn the study, we identify five sARLNRs and validate their clinical significance, angiogenesis correlation and prognosis-predictive values in BUC. These findings may provide a new perspective and some promising antiangiogenic targets for clinical diagnosis and treatment strategies of BUC.

Decidualization-derived cAMP promotes decidual NK cells to be angiogenic phenotype.

During early pregnancy, a large number of CD56bright natural killer cells (NKs) are accumulated in the decidua; unlike peripheral and cord blood NK cells (pNK and cNK), these decidual NK cells (dNK) display a great capacity to secrete a series of angiogenic/vascular factors, which are essential for placental development. However, the mechanism underlying the formation of dNK cells with an angiogenic phenotype remains unclear. First, we compared the difference between dNK and cNK/pNK cells in terms of the expression of CD56 and VEGF, and the regulation of the tube formation. The effect of cAMP on the differentiation of NK cells was evaluated by its analog and inhibitor stimulation. We further analyzed the differences in the phenotype of dNK cells and the expression of VEGF in dNK cells from normal pregnancies and miscarriages. Different from cNK and pNK, dNK cells displayed high expression of CD56 and VEGF. And dNK cells showed a higher capacity of inducing tube formation of HUVEC by trophoblast. Meanwhile, we observed thatcAMP-analogue increased the percentage of CD56bright NK population in cNK cells with up-regulated VEGF secretion and tube formation of HUVEC by trophoblast, which could be inhibited by pretreatment with VEGFR neutralizing antibody. Similar changes occurred when co-culturing cNK cells with DSCs but not ESCs. Interestingly, the inhibitor of cAMP signaling (Metadoxine, META) could significantly inhibit the upregulation of VEGF in cNK cells by DSCs. Furthermore, DSCs could secret much more cAMP than ESCs. Notably, decreased CD56bright NK population and VEGF secretion by dNK were related to pregnancy loss. These findings suggest that dNK cells display an angiogenic phenotype that can be induced by decidualized cAMP signaling. Our study indicates the significance of decidualization-derived cAMP in regulating angiogenesis of decidual NKs and reveals complex crosstalk between different cell types in a critical period during early pregnancy.

Design, synthesis and biological evaluation of novel FAK inhibitors with better selectivity over IR than TAE226

In this study, 28 novel focal adhesion kinase (FAK) inhibitors were designed and synthesized based on FAK inhibitor TAE226. Compound 18b displayed good inhibition of FAK (IC50 = 45 nM) with at least 22 fold of selectivity over insulin receptor (IR, IC50 > 1000 nM) and exhibited potent anticancer activity against Hela, HCT116 and MDA-MB-231 cell lines with IC50 values of 0.27, 0.19 and 0.26 μM respectively, compared to TAE226 (2.68, 0.64 and 4.19 μM respectively). 18b also inhibited the clone formation and migration of HCT-116 cells, tube formation of HUVECs, and stimulated cell cycle arrest in the G2/M phase, inducing apoptosis by promoting ROS production. The FAK-Src-ERK signaling pathway was inhibited by 18b in a dose-dependent manner. Moreover, 18b showed adequate oral bioavailability of 16.37% and 75.90% tumor growth inhibition in the HCT116 xenograft model was observed. These results indicate that 18b is a promising selective inhibitor of FAK.

Effect of Neiyi Prescription of QIU on autophagy and angiogenic ability of endometriosis via the PPARγ/NF-κB signaling pathway.

Neiyi Prescription of QIU (NYPQ) is a traditional Chinese medicine prescription for the treatment of endometriosis (EMS). Here, we aimed to examine the effects and mechanisms of NYPQ on angiogenic ability in EMS. EMS rats were established with estradiol valerate and autologous transplantation. EMS rats were intraperitoneally injected with chloroquine (CQ, 40mg/kg), rapamycin (RAPA, 1mg/kg), and monoclonal antibody VEGF (anti-VEGF, 3mg/g/d) or administered 5, 10, 20mg/g/d NYPQ decoction through oral gavage for 4weeks, respectively. By the before and end of the treatment period, the volume of the endometriotic lesions was measured. The pathological morphology, angiogenesis, and the number of autophagosomes of the endometriotic lesion were observed by hematoxylin and eosin staining, immunohistochemistry, and transmission electron microscope, respectively. The cell viability, apoptosis, and angiogenesis of HUVECs were detected by MTT, flow cytometry, and lumen formation experiment, respectively. The expression levels of VEGF, autophagy-/apoptosis-/PPARγ/NF-κB- pathway-related proteins in endometrium tissues or HUVECs were detected by western blot assays. The autophagy agonist rapamycin reduced the lesion size, the microvessel density, and VEGF expression, and promoted the production of autophagosomes and the expression of autophagy-related proteins, while the autophagy inhibitor chloroquine had the opposite effects. In vivo, NYPQ could dose-dependently reduce lesion volume and microvessel density, ameliorate histopathological features and promote autophagosome production of ectopic endometrium. Moreover, serum-containing NYPQ could significantly inhibit the cell viability and tube formation of HUVECs and elevate HUVECs apoptosis. Besides, NYPQ significantly reduced VEGF and promoted autophagy-/apoptosis-related protein expressions. Also, NYPQ might promote autophagy and inhibit angiogenesis by activating the PPARγ/NF-κB pathway. Collectively, these findings indicate that NYPQ has therapeutic potential in experimentally induced peritoneal endometriosis, and its mechanism may be related to the activation of the PPARγ/NF-κB signaling pathway.

A novel alginate from Sargassum seaweed promotes diabetic wound healing by regulating oxidative stress and angiogenesis

Diabetic skin ulcer is one of the most severe complications in diabetes, however, current therapeutic approaches are not effective enough. Agents modulating oxidative stress, inflammation, and angiogenesis are quite promising for alleviation of diabetic skin ulcers. In this study, a novel Sargassum kjellmanianum-derived polysaccharide (SARP) was prepared. SARP was an alginate with Mw of 45.4 kDa, consisting of 76.56% mannuronic acid, 18.89% guluronic acid, and 4.55% glucuronic acid. SARP could attenuate oxidative stress-induced cell damage via activating nuclear factor erythroid 2-related factor 2 (Nrf2). SARP also promoted the migration and tube formation of HUVECs, which was related to the increased vascular endothelial growth factor (VEGF) expression. In diabetic wound model, SARP (iv, 200 mg/kg) administration increased angiogenesis, alleviated oxidative stress, ameliorated diabetes-related aberrations, and thereby accelerated diabetic wound healing. These findings identified SARP had potential to be developed as a drug candidate for diabetic skin ulcers.

Bioactivity and miRNome Profiling of Native Extracellular Vesicles in Human Induced Pluripotent Stem Cell-Cardiomyocyte Differentiation.

Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell‐cardiomyocyte (hiPSC‐CM) differentiation and maturation, i.e., from hiPSC (hiPSC‐EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC‐EV compared to EV from committed cell populations. hiPSC‐EV also significantly increase cell cycle activity of hiPSC‐CM. Global miRNA expression profiles, obtained by small RNA‐seq analysis, corroborate an EV‐miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC‐EV are enriched in pluripotency‐associated miRNA with higher in vitro pro‐angiogenic and pro‐proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC‐EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications.

Intratumoral heterogeneity of MYC drives medulloblastoma metastasis and angiogenesis.

Intratumoral heterogeneity is crucially involved in metastasis, resistance to therapy, and cancer relapse. Amplifications of the proto-oncogene MYC display notable heterogeneity at the single-cell level and are associated with a particularly dismal prognosis in high-risk medulloblastomas (MBs). The aim of this study was to establish the relevance of interclonal cross-talk between MYC-driven and non-MYC-driven MB cells. We used fluorescence in situ hybridization, single-cell transcriptomics, and immunohistochemistry, in vitro isogenic cell models, non-targeted proteomics, mass spectrometry-based metabolite quantification, HUVECs tube formation assay, and orthotopic in vivo experiments to investigate interclonal cross-talk in MB. We found that the release of lactate dehydrogenase A (LDHA) from MYC-driven cells facilitates metastatic seeding and outgrowth, while secretion of dickkopf WNT signaling pathway inhibitor 3 from non-MYC-driven cells promotes tumor angiogenesis. This tumor-supporting interaction between both subclones was abrogated by targeting the secretome through pharmacological and genetic inhibition of LDHA, which significantly suppressed tumor cell migration. Our study reveals the functional relevance of clonal diversity and highlights the therapeutic potential of targeting the secretome to interrupt interclonal communication and progression in high-risk MB.

H2O2-responsive VEGF/NGF gene co-delivery nano-system achieves stable vascularization in ischemic hindlimbs

Peripheral vascular disease (PVD) is a common clinical manifestation of atherosclerosis. Vascular endothelial growth factor (VEGF) gene therapy is a promising approach for PVD treatment. However, due to single-gene therapy limitations and high H2O2 pathological microenvironment, VEGF gene therapy are not as expectations and its clinical application are limited. Synergistic effects of Nerve factors and vascular factors in angiogenesis have attracted attention in recent years. In this study, VEGF and nerve growth factor (NGF) genes co-delivery nanoparticles (VEGF/NGF-NPs) were prepared by using H2O2 responsive 6s-PLGA-Po-PEG as a carrier. 6s-PLGA-Po-PEG could react with H2O2 specifically due to the internal peroxalate bond. Angiogenic effects of VEGF/NGF-NPs has been evaluated in cells and hindlimb ischemia mice model. Results showed that VEGF/NGF-NPs promoted VEGF and NGF co-expression simultaneously, eliminated excessive H2O2, strengthened reactions between SH-SY5Ys and HUVECs, and finally enhanced migration, tube formation, proliferation and H2O2 damage resistance of HUVECs. VEGF/NGF-NPs also recovered blood perfusion, promoted the expression of VEGF, NGF, eNOS and NO, and enhanced vascular coverage of pericytes. Treatment effects of VEGF/NGF-NPs may related to VEGF/eNOS/NO pathway. Altogether, VEGF/NGF-NPs eliminated excessive H2O2 while achieving gene co-delivery, and promoted stable angiogenesis. It’s a promising way for PVD treatment by using VEGF/NGF-NPs.Graphical

Fraxetin protects rat brains from the cerebral stroke via promoting angiogenesis and activating PI3K/Akt pathway

Objective Fraxetin has antioxidant, anti-inflammation and neuroprotective functions, however, its role in ischemic stroke is still vague. Herein, this study delves into the underlying mechanism. Methods Ischemia and reperfusion operation were performed to establish the cerebral stroke rat models. The brain functions were evaluated with neurological score. The brain infarcted volume in fraxetin group was measured by 2,3,5-triphenyltetrazolium chloride staining. The blood-brain barrier permeability, CD34 enrichment, and the brain water content were measured by Evans blue staining, immunofluorescence staining, and wet-dry method, respectively. Real-time quantitative polymerase chain reaction (RT-qPCR) and western blot (WB) were applied to examine the levels of angiogenesis- and PI3K/Akt pathway-related factors. MTT and tube formation assays were used to measure the viability and tube formation of HUVECs. Results Fraxetin decreased the brain injury-related neurological score, brain infarction, and cerebral edema and maintained blood-brain barrier permeability, whereas it promoted the angiogenesis in ischemia-damaged brain via enhancing CD34 enrichment, the expressions of VEGF, Ang-1, Tie-2, and CD-31, viability of HUVECs, as well as activating the phosphorylation of PI3K and Akt. Importantly, wortmannin (a specific PI3K inhibitor) impeded the fraxetin-induced cell viability, angiogenesis, and phosphorylation of Akt and PI3K in HUVECs. Conclusions Fraxetin has protective effects on the brain ischemia-reperfusion injury and promotes angiogenesis for cerebral repair via phosphorylation of PI3K and Akt.

Inhibition of FGFR2 enhances chemosensitivity to gemcitabine in cholangiocarcinoma through the AKT/mTOR and EMT signaling pathways

AimTo investigate the oncogenic role of FGFR2 in carcinogenesis in cholangiocarcinoma (CCA) cells. In addition, the feasibility of using FGFR inhibitors in combination with standard chemotherapy was also explored for the chemosensitizing effect in CCA cells. Main methodsFive CCA cell lines were used to screen FGFR2 expression by Western immunoblotting. Two CCA cell lines, KKU-100 and KKU-213A, were knocked down of the FGFR2 gene using siRNA. Cell viability was assessed by the MTS cell proliferation assay. Reproductive cell death was assessed by clonogenic assay. The effects on cell migration and invasion were analyzed by the Transwell chamber method. Cell cycle analysis was performed by flow cytometry. Cell angiogenesis was assessed by HUVEC tube formation and human angiogenesis antibody array analysis. Proteins associated with proliferative and metastatic properties were evaluated by Western blotting. Key findingsKnockdown of FGFR2 suppressed cell growth and colony formation in CCA cells in association with G2/M cell cycle arrest and downregulation of STAT3, cyclin A and cyclin B1. Silencing FGFR2 enhanced the suppressive effect of gemcitabine (Gem) on cell migration and invasion. The combination of infigratinib, an FGFR inhibitor, and Gem, interrupted cell growth, migration, and invasion via downregulation of FGFR/AKT/mTOR pathways and the EMT-associated proteins vimentin and slug. Moreover, the combination also suppressed tube formation together with decreased expression of the proangiogenic factor VEGF. SignificanceInhibition of FGFRs by infigratinib enhanced the antitumor effect of Gem in CCA cells through downregulation of the FGFR/AKT/mTOR, FGFR/STAT3 and EMT signaling pathways.

Exosomal miR-205-5p enhances angiogenesis and nasopharyngeal carcinoma metastasis by targeting desmocollin-2

The aim of this study was to investigate whether and how exosomal miR-205-5p regulated angiogenesis and nasopharyngeal carcinoma (NPC) metastasis. We found that up-regulated serum exosomal miR-205-5p levels were associated with NPC progression and worse overall survival of NPC patients. miR-205-5p over-expression significantly increased tube formation, wound healing, migration and invasion of NPC cells, and lung metastasis of NPC tumors, whereas miR-205-5p inhibition had opposite effects. Exosomal miR-205-5p from NPC cells promoted the migration, tube formation, and microvessel density (MVD) of HUVECs in vitro and in vivo. Furthermore, bioinformatics-, luciferase reporter-, and biotinylated miR-205-5p-based pull-down assays indicated that miR-205-5p directly bound to the 3′ UTR of desmocollin-2 (DSC2). Exosomal miR-205-5p targeted DSC2 to enhance the EGFR/ERK signaling and MMP2/MMP9 expression, promoting angiogenesis and NPC metastasis, which was abrogated by DSC2 over-expression. Finally, the levels of miR-205-5p transcripts were positively correlated with MVD but negatively with DSC2 expression in NPC tissues, and patients with miR-205high/DSC2low NPC had worse overall survival. In conclusion, exosomal miR-205-5p promotes angiogenesis and NPC metastasis by targeting DSC2 to enhance EGFR/ERK signaling and MMP expression. This exosomal/miR-205-5p/EGFR/ERK axis may be a new therapeutic target for intervention of NPC metastasis.

Angiogenesis Driven by the CEBPD-hsa-miR-429-VEGFA Signaling Axis Promotes Urothelial Carcinoma Progression.

Background and Purpose: This research aimed to excavate the alternative mechanism of CEBPD on tumor growth and explore the biological significance of the CEBPD/hsa-miR-429/VEGFA axis on angiogenesis in urothelial carcinoma (UC). Methods: Quantitative RT-PCR, immunoblotting assay and tube formation examined the effect of hsa-miR-429 mimic or/and inhibitor on VEGFA expression and angiogenesis in CEBPD-overexpressing UC-derived cells. The association between CEBPD, hsa-miR-429, VEGFA and microvascular density (MVD) and clinical outcome were evaluated in 296 patients with UBUC and 340 patients with UTUC, respectively. Results: The increase in the transcript and protein of VEGFA as well as HUVECs tube formation was diminished upon the treatment of hsa-miR-429 mimic in CEBPD-overexpressing BFTC909 and TCCSUP. Nevertheless, the inhibited regulation of hsa-miR-429 mimic on the expression of VEGFA and ability of HUVECs tube formation was rescued by the combined incubation with hsa-miR-429 inhibitor in these two UC-derived cell lines. Furthermore, the clinical correlations showed that the higher level of VEGFA or MVD has a positive correlation with the expression of CEBPD and a negative relation to hsa-miR-429 and leads to tumor aggressiveness with worse disease-specific, metastasis-free survival in UBUC and UTUC cohorts. Conclusions: We decipher the oncogenic mechanism of CEBPD on angiogenesis through the hsa-miR-429 inhibition to stabilize the expression of VEGFA in UC. The novel research unveiled the modulation of the CEBPD/hsa-miR-429/VEGFA axis on the progression of UC and could be accessible to theranostic biomarkers.

Endoglin Modulates TGFβR2 Induced VEGF and Proinflammatory Cytokine Axis Mediated Angiogenesis in Prolonged DEHP-Exposed Breast Cancer Cells.

Angiogenesis is the process of vascular network development and plays a crucial role in cancer growth, progression, and metastasis. Phthalates are a class of environmental pollutants that have detrimental effects on human health and are reported to increase cancer risk. However, the interplay between phthalate exposure and angiogenesis has not been investigated thoroughly. In this study, we investigated the effect of prolonged di (2-ethylhexyl) phthalate (DEHP) treatment on the angiogenic potential of triple-negative breast cancer. MDA-MB-231 cells were exposed to physiological concentrations of DEHP for more than three months. Prolonged DEHP exposure induced angiogenesis in breast cancer cells. Endoglin (ENG)/CD105 is a membrane glycoprotein and an auxiliary receptor of the TGFβ receptor complex. In endothelial cells, ENG is highly expressed and it is a prerequisite for developmental angiogenesis. A literature review highlights endoglin as a well-known mesenchymal stem cell marker responsible for vascular development and angiogenesis. NGS analysis showed that endoglin overexpression in DEHP-exposed MDA-MB-231 cells correlated with tumor development and growth. An in vivo zebrafish xenograft assay showed that VEGFA induced sprouting of the subintestinal vein (SIV) in embryos injected with DEHP-exposed cells. Endoglin knockdown reduced SIV sprouting and VEGFA expression in zebrafish embryos. An in vitro HUVEC tube formation assay showed that endoglin depletion reversed DEHP-induced VEGF-mediated HUVEC tube formation in coculture. DEHP-induced endoglin activated TGFβ/SMAD3/VEGF and MAPK/p38 signaling in MDA-MB-231 cells. A cytokine angiogenesis antibody array showed induced expression of the inflammatory cytokines IL1α, IL1β, IL6, and IL8, along with GMCSF and VEGF. Endoglin knockdown reversed DEHP-induced activation of the TGFβ/SMAD3/VEGF signaling axis, MAPK/p38 signaling, and cytokine regulation, limiting angiogenesis potential both in vivo and in vitro. Targeting endoglin might serve as a potential alternative treatment to control angiogenesis, leading to metastasis and limiting cancer progression.

Membrane progesterone receptor \u03b1 (mPR\u03b1) enhances hypoxia-induced vascular endothelial growth factor secretion and angiogenesis in lung adenocarcinoma through STAT3 signaling

Lung cancer remains a huge challenge to public health because of its high incidence and mortality, and lung adenocarcinoma (LUAD) is the main subtype of lung cancer. Hypoxia-induced vascular endothelial growth factor (VEGF) release and angiogenesis have been regarded as critical events in LUAD carcinogenesis. In the present study, membrane progesterone receptor α (mPRα) is deregulated within LUAD tissue samples; increased mPRα contributes to a higher microvessel density (MVD) in LUAD tissues. mPRα knockdown in A549 and PC-9 cells significantly inhibited STAT3 phosphorylation, as well as HIF1α and VEGF protein levels, decreasing cancer cell migration and invasion. The in vivo xenograft model further confirmed that mPRα enhanced the aggressiveness of LUAD cells. Furthermore, mPRα knockdown significantly inhibited hypoxia-induced upregulation in HIF1α and VEGF levels, as well as LUAD cell migration and invasion. Under the hypoxic condition, conditioned medium (CM) derived from mPRα knockdown A549 cells, namely si-mPRα-CM, significantly inhibited HUVEC migration and tube formation and decreased VEGF level in the culture medium. In contrast, CM derived from mPRα-overexpressing A549 cells, namely mPRα-CM, further enhanced HUVEC migration and tube formation and increased VEGF level under hypoxia, which was partially reversed by STAT3 inhibitor Stattic. In conclusion, in LUAD cells, highly expressed mPRα enhances the activation of cAMP/JAK/STAT3 signaling and increases HIF1α-induced VEGF secretion into the tumor microenvironment, promoting HUVEC migration and tube formation under hypoxia.