Angiogenesis-related Factors Research Articles

Inhibitory Effects of Pectic Polysaccharide Isolated from Diospyros kaki Leaves on Tumor Cell Angiogenesis via VEGF and MMP-9 Regulation.

Persimmon leaves are an attractive source of phytochemicals with potential health benefits. However, there are only a few reports on the chemical properties and biological activity of the polysaccharide fractions (PLE-I–III) of persimmon leaves. We evaluated the angiogenesis-inhibiting ability of pectic-polysaccharides. The molecular weight of PLEs was determined using a high-performance size-exclusion chromatography system. Tube formation assay of human umbilical vein endothelial cells (HUVECs) was performed using Matrigel-coated 96-well plates. Matrix metalloproteinase (MMP-9), vascular endothelial growth factor (VEGF), PI3K, Akt, and p38 phosphorylation levels were determined using Western blotting; VEGF and MMP-9 transcript levels were measured using SYBR Green qRT-PCR. PLE-I–III significantly inhibited HUVEC tube formation at 12.5 and 25 μg/mL. Among them, PLE-II showed the strongest anti-tube formation activity, and the mRNA/protein expression of angiogenesis-related factors (VEGF/MMP-9) was significantly reduced by PLE-II. PLE-II also suppressed the phosphorylation of PI3K/AKT and p38, JNK, and NF-κB p65 in HUVECs. These results suggest that the polysaccharide PLE-II isolated from persimmon leaves inhibited VEGF and MMP-9 expression in HUVECs via regulation of PI3K/AKT, p38, JNK, and NF-κB p65 signaling pathways.

Exosomes from microRNA-126 overexpressing mesenchymal stem cells promote angiogenesis by targeting the PIK3R2-mediated PI3K/Akt signalling pathway.

microRNA‐126 (miR‐126), an endothelial‐specific miRNA, is associated with vascular homeostasis and angiogenesis. However, the efficiency of miR‐126‐based treatment is partially compromised due to the low efficiency of miRNA delivery in vivo. Lately, exosomes have emerged as a natural tool for therapeutic molecule delivery. Herein, we investigated whether exosomes derived from bone marrow mesenchymal stem cells (BMMSCs) can be utilized to deliver miR‐126 to promote angiogenesis. Exosomes were isolated from BMMSCs overexpressed with miR‐126 (Exo‐miR‐126) by ultracentrifugation. In vitro study, Exo‐miR‐126 treatment promoted the proliferation, migration and angiogenesis of human umbilical vein endothelial cells (HUVECs). Furthermore, the gene/protein expression of angiogenesis‐related vascular endothelial growth factor (VEGF) and angiotensin‐1 (Ang‐1) were up‐regulated after incubation with Exo‐miR‐126. Additionally, the expression level of phosphoinositol‐3 kinase regulatory subunit 2 (PIK3R2) showed an inverse correlation with miR‐126 in HUVECs. Particularly, the Exo‐miR‐126 treatment contributed to enhanced angiogenesis of HUVECs by targeting PIK3R2 to activate the PI3K/Akt signalling pathway. Similarly, Exo‐miR‐126 administration profoundly increased the number of newly formed capillaries in wound sites and accelerated the wound healing in vivo. The results demonstrate that exosomes derived from BMMSCs combined with miR‐126 may be a promising strategy to promote angiogenesis.

Hypoxia-inducible factor-1α mediates and regulates angiogenesis-related factors expression in hepatocellular carcinoma

Objective: To investigate the expression of hypoxia-inducible factor (HIF)-1α, vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) during the progression of liver diseases and the molecular mechanism of HIF-1α in regulating the expression of angiogenic factors in hepatocellular carcinoma (HCC). Methods: Serum samples from hospitalized patients with liver cancer, liver cirrhosis, and chronic hepatitis were collected, and healthy people were used as controls. Mouse models of hepatocarcinogenesis were used to detect the dynamic expression of HIF-1α, VEGF and Ang-2. Enzyme-linked immunosorbent assay was used to quantitatively analyze the serum levels of HIF-1α, VEGF and Ang-2 in patients with liver disease and mice. HIF-1α-specific miRNA expression plasmids was constructed to transfect HepG2 human HCC cells. HIF-1α mRNA transcriptional interference effects were analyzed on biological behavior, VEGF and Ang-2 expression, and epithelial mesenchymal transformation (EMT) in human HCC cell line. The sample means of multiple groups were compared by analysis of variance and q test and the sample rate was compared by χ (2) test. Results: In patients with chronic liver disease, the serum expression of HIF-1α, VEGF and Ang-2 in the liver cancer group (145.6 ± 32.6) μg/L, (458.9 ± 125.3) μg/L and (42.9 ± 5.1) μg/L was significantly higher than the liver cirrhosis (P < 0.001) (79.5 ± 28.4) μg/L, (206.8 ± 56.8) μg/L and (26.2 ± 6.1) μg/L and chronic hepatitis group (60.1 ± 18.8) μg/L, (178.1 ± 85.4) μg/L and (21.8 ± 6.9) μg/L. In addition, HIF-1α was positively correlated with VEGF (r = 0.937, P < 0.001), HIF-1α and Ang-2 (r = 0.933, P < 0.001), and VEGF and Ang-2 (r = 0.910, P < 0.001). Mouse models of hepatocarcinogenesis confirmed that HIF-1α, VEGF and Ang-2 had progressively increased during the process of malignant transformation from normal hepatocytes, hepatocyte degeneration, and precancerous lesions to canceration. HIF-1α miRNA intervention plasmid had transformed HepG2 cells. Compared with the blank group, HIF-1α mRNA, HIF-1α, VEGF and Ang-2 were decreased by 88.1%, 59.8%, 54.0% and 36.0% at 72h, respectively. The expression level of EMT-related protein Snail (0.26 ± 0.02 and 0.67 ± 0.09, q = 6.75, P < 0.003), VIM (0.27±0.08 and 0.73±0.04, t = 10.35, P < 0.001) and Twist (0.24 ± 0.07 and 0.73 ± 0.02, q = 12.08, P < 0.001) was significantly reduced, but the expression level of E-cadherin (0.76 ± 0.08 and 0.27 ± 0.09, q = 7.05, P < 0.002) was significantly increased. Conclusion: HIF-1α mediates and regulates angiogenesis-related factors such as VEGF and Ang-2 expression in hepatocellular carcinoma. Furthermore, HIF-1α transcriptional interference can significantly affect the biological characteristics and EMT transformation of hepatocellular carcinoma cells.

Human mesenchymal stromal/stem cells recruit resident pericytes and induce blood vessels maturation to repair experimental spinal cord injury in rats

Angiogenesis is considered to mediate the beneficial effects of mesenchymal cell therapy in spinal cord injury. After a moderate balloon-compression injury in rats, injections of either human adipose tissue-derived stromal/stem cells (hADSCs) or their conditioned culture media (CM-hADSC) elicited angiogenesis around the lesion site. Both therapies increased vascular density, but the presence of hADSCs in the tissue was required for the full maturation of new blood vessels. Only animals that received hADSC significantly improved their open field locomotion, assessed by the BBB score. Animals that received CM-hADSC only, presented haemorrhagic areas and lack pericytes. Proteomic analyses of human angiogenesis-related factors produced by hADSCs showed that both pro- and anti-angiogenic factors were produced by hADSCs in vitro, but only those related to vessel maturation were detectable in vivo. hADSCs produced PDGF-AA only after insertion into the injured spinal cord. hADSCs attracted resident pericytes expressing NG2, α-SMA, PDGF-Rβ and nestin to the lesion, potentially contributing to blood vessel maturation. We conclude that the presence of hADSCs in the injured spinal cord is essential for tissue repair.

LncRNA H19 Promotes Cell Proliferation, Migration, and Angiogenesis of Glioma by Regulating Wnt5a/β-Catenin Pathway via Targeting miR-342

Glioma is the most common malignant brain tumor and long non-coding RNAs (lncRNAs) have been reported to play an important role in the growth and angiogenesis of glioma. However, the potential mechanisms of lncRNA H19 in glioma remain unclear. In the present study, the effects of lncRNA H19 on glioma cell proliferation, migration, and angiogenesis were evaluated. The expression levels of H19, miR-342, and Wnt5a in glioma tissues and cells were detected by RT-qPCR or Western blotting. Dual luciferase reporter assay confirmed the interaction between H19, miR-342, and Wnt5a. Cell proliferation, migration, and angiogenesis were analyzed by colony formation, transwell, and tube formation assays, respectively. IHC was performed to test the angiogenesis-related factor CD31. H19 and Wnt5a expression were remarkably upregulated in glioma tissues and cells, whereas miR-342 expression was downregulated. Moreover, functional analysis confirmed that knockdown of H19 or overexpression of miR-342 suppressed glioma cell proliferation, migration, and angiogenesis in vitro. Besides, H19 was found to directly target miR-342 to promote Wnt5a expression and activate β-catenin pathway in glioma cells. Moreover, suppression of miR-342 or overexpression of Wnt5a reversed the inhibitory effect of sh-H19 on glioma growth and metastasis. Additionally, we verified that H19 promoted glioma cell proliferation, migration, and angiogenesis via miR-342/Wnt5a/β-catenin axis in vivo. H19 regulates glioma cell growth and metastasis through miR-342 to mediate Wnt5a/β-catenin signaling pathway, which provides new therapeutic targets for glioma treatment.

Formononetin inhibits inflammation and promotes gastric mucosal angiogenesis in gastric ulcer rats through regulating NF-κB signaling pathway

To investigate the effects of formononetin on rats with gastric ulcer and further to explore its possible mechanism. Rats were randomly divided into sham operation group (Sham), model group (Model), omeprazole control group (Omeprazole) and formononetin in different dose groups (FOR-L, FOR-M, FOR-H). Rats model with gastric ulcer were established by 100% glacial acetic acid. Hematoxylin–eosin (H&E) staining was used to observe the pathological morphology of gastric mucosa. Immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) were used to detect the level of inflammatory and angiogenesis related factors. The expressions of nuclear factor kappa-B (NF-κB) signaling pathway-related proteins were detected by western blot. Formononetin and omeprazole could ameliorate the pathological morphology of gastric mucosa in gastric ulcer rats. Compared with Model group, the levels of tumor necrosis factor (TNF)-α, Interleukin (IL)-1β, IL-6, myeloperoxidase (MPO), human endothelin (ET)-1 and p-P65 protein in formononetin treatment and omeprazole groups were significantly decreased (p < 0.05). Moreover, formononetin could increase the content of vascular endothelial growth factor (VEGF), nitric oxide (NO) and the levels of CD34, tight junction proteins (ZO-1 and occludin) and p-IκBα in a dose-dependent manner. Formononetin can ameliorate gastric ulcer in rats by inhibiting inflammation and promoting gastric mucosal angiogenesis, and its mechanism maybe related to NF-κB signaling pathway.

Enhanced angiogenic effects of RGD, GHK peptides and copper (II) compositions in synthetic cryogel ECM model

Synthetic oligopeptides are a promising alternative to natural full-length growth factors and extracellular matrix (ECM) proteins in tissue regeneration and therapeutic angiogenesis applications. In this work, angiogenic properties of dual and triple compositions containing RGD, GHK peptides and copper (II) ions (Cu2+) were for the first time studied. To reveal specific in vitro effects of these compositions in three-dimensional scaffold, adamantyl group bearing peptides, namely Ada-Ahx-GGRGD (1) and Ada-Ahx-GGGHK (2), were effectively immobilized in bioinert pHEMA macroporous cryogel via host-guest β-cyclodextrin-adamantane interaction. The cryogels were additionally functionalized with Cu2+ via the formation of GHK-Cu complex. Angiogenic responses of HUVECs grown within the cryogel ECM model were analyzed. The results demonstrate that the combination of RGD with GHK and further with Cu2+ dramatically increases cell proliferation, differentiation, and production of a series of angiogenesis related cytokines and growth factors. Furthermore, the level of glutathione, a key cellular antioxidant and redox regulator, was altered in relation to the angiogenic effects. These results are of particular interest for establishing the role of multiple peptide signals on regeneration related processes and for developing improved tissue engineering materials.

Overexpression of NDRG2 promotes the therapeutic effect of pazopanib on ovarian cancer

Objectives Ovarian cancer is the second commonly seen cancer in the US, patients with ovarian cancer are commonly diagnosed in the advanced stage. Pazopanib is an inhibitor of multiple tyrosine kinases and has been approved in treatment for carcinoma by FDA. N-myc downstream-regulated gene 2 (NDRG2) has been regarded as a cancer suppressor gene and presented an inhibition effect in cancer proliferation, invasion, and migration. Design: NDRG2 was overexpressed or inhibited in SKOV-3 cells, then experiments were performed to detect the apoptosis of cells. The expression or secretion of pro-cancer molecules was detected. And the expression of apoptosis-related proteins and the ASK1/JNK1 signaling pathway was detected. Methods The NDRG2 overexpression and inhibition model was firstly constructed in SKOV-3 cells, the apoptotic cells were detected using flow cytometry. The expression of cellular metastasis genes was detected using the qPCR method. The angiogenesis factors was detected using the ELISA method. Expression of each target protein was detected using western blotting analysis. Results NDRG2 overexpression and inhibition model were constructed in the SKOV-3 cell line, overexpression of NDRG2 enhanced the effect of pazopanib on inhibition of the expression of metastasis-related molecules and angiogenesis-related factors. The apoptosis process of cells was also enhanced after overexpression of NDRG2, and these effects were regulated by the activation of the ASK1/JNK1 signaling pathway. Limitations: The effect of NDRG2 in animal models and more cell lines needs to be explored in further study. Conclusions NDRG2 might be a therapeutic target in treatment for ovarian cancer.

Sequential paracrine mechanisms are necessary for the therapeutic benefits of stem cell therapy.

Stem cell injections are an attractive therapeutic tool. It has been demonstrated that injected stem cells promote tissue repair and regeneration via paracrine mechanisms. However, the effects of injected stem cells continue for far longer than they are present. We hypothesized that the effects of injected stem cells are prolonged because of a sequential paracrine relay mechanism. Conditioned media was collected from mesenchymal stem cells (MSCs) after 24 h. This media was then added to RAW264.7. Media was collected from the macrophages after 24 h and was then added to endothelial cells (ECs). This conditioned macrophage media, but not control media, promoted wound healing and induced EC differentiation. Similar results were observed with primary macrophages. To identify the active paracrine factors released by macrophages in response to stimulation by MSC conditioned media we used an antibody array, identifying increased expression of the angiogenesis-related proteins stromal cell-derived factor 1 (SDF1) and plasminogen activator inhibitor-1 (PAI-1). Knockdown of either protein inhibited the ability of conditioned media derived from MSC paracrine factor-stimulated macrophages to induce EC differentiation both in vitro and in vivo. Conditioned media derived from postnatal day 7 (P7) mouse macrophages induced EC differentiation. Moreover, SDF1 and PAI-1 levels were >120 higher in P7 macrophages compared with adult macrophages, suggesting that MSC paracrine factors promote adult macrophages to adopt a juvenile phenotype. These results indicate that MSC paracrine factors induce macrophages to secrete SDF1 and PAI-1, in-turn inducing endothelial cells to differentiate. Identification of a sequential paracrine mechanism opens new therapeutic avenues for stem cell therapy.

Bone marrow mesenchymal stem cells-induced exosomal microRNA-486-3p protects against diabetic retinopathy through TLR4/NF-κB axis repression.

Diabetic retinopathy (DR) is a chronic disease causing health and economic burdens on individuals and society. Thus, this study is conducted to figure out the mechanisms of bone marrow mesenchymal stem cells (BMSCs)-induced exosomal microRNA-486-3p (miR-486-3p) in DR. The putative miR-486-3p binding sites to 3'untranslated region of Toll-like receptor 4 (TLR4) was verified by luciferase reporter assay. High glucose (HG)-treated Muller cells were transfected with miR-486-3p or TLR4-related oligonucleotides and plasmids to explore theirs functions in DR. Additionally, HG-treated Muller cells were co-cultured with BMSC-derived exosomes, exosomes collected from BMSCs that had been transfected with miR-486-3p or TLR4-related oligonucleotides and plasmids to explore their functions in DR. MiR-486-3p, TLR4 and nuclear factor-kappaB (NF-κB) expression, angiogenesis-related factors, oxidative stress factors, viability and apoptosis in HG-treated Muller cells were detected by RT-qPCR, western blot analysis, ELISA, MTT assay and flow cytometry, respectively. MiR-486-3p was poorly expressed while TLR4 and NF-κB were highly expressed in HG-treated Muller cells. TLR4 was a target of miR-486-3p. Upregulating miR-486-3p or down-regulating TLR4 inhibited oxidative stress, inflammation and apoptosis, and promoted proliferation of HG-treated Muller cells. Meanwhile, BMSC-derived exosomes inhibited oxidative stress, inflammation and apoptosis, and promoted proliferation of HG-treated Muller cells. Restoring miR-486-3p further enhanced, while up-regulating TLR4 reversed, the improvement of exosomes treatment. Our study highlights that up-regulation of miR-486-3p induced by BMSC-derived exosomes played a protective role in DR mice via TLR4/NF-κB axis repression.

The three-phase enriched environment paradigm promotes neurovascular restorative and prevents learning impairment after ischemic stroke in rats

Enriched environment (EE) with a complex combination of sensorimotor, cognitive and social stimulations has been shown to enhance brain plasticity and improve recovery of functions in animal models of stroke. The present study extended these findings by assessing whether the three-phase EE intervention paradigm would improve neurovascular remodeling following ischemic stroke.Male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion (MCAO). A three-phase EE intervention paradigm was designed in terms of the different periods of cerebral ischemia by periodically rearranging the EE cage. Morris water maze (MWM) tests were performed to evaluate the learning and memory function. Multimodal MRI was applied to examine alterations to brain structures, intracranial vessels, and cerebral perfusion on the 31st day after MCAO. The changes of capillaries ultrastructure were examined by transmission electron microscope. Double-immunofluorescent staining was used to evaluate neurogenesis and angiogenesis. The expression of angiogenesis-related factors and neurovascular remodeling related signaling pathways including Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/glycogen synthase kinase-3 (GSK-3)/β-catenin and the axon guidance molecules were detected by Western blot analysis.MRI measurements revealed that EE treatment significantly increased survival volume of cortex and striatum, improved cerebral blood flow (CBF), amplified anterior azygos cerebral artery (azACA), ipsilateral internal carotid artery (ICA) and anterior communicating artery (AComA) vessel signal compared with standard housed rats (IS). Consistent with these findings, EE reduced ischemic BBB damage of capillary, enhanced endogenous angiogenesis and modified the expression of VEGF, Ang-1 or Ang-2 in ischemic rats. Additionally, this proangiogenic effect was consistent with the increased progenitor cell proliferation and neuronal differentiation in the peri-infarct cortex and striatum after EE intervention. Specifically, EE intervention paradigm markedly increased expression of phosphorylated PI3K, AKT and GSK-3, but reduced phosphorylated β-catenin. Moreover, the axon guidance proteins expression level was significant higher in EE group. In parallel to these findings, EE significantly enhanced recovery of lost spatial learning memory function in MCAO rats without affecting infarct size.Together, MRI findings along with histological results strongly supported that the three-phase EE paradigm benefited neurovascular reorganization and thereby improved poststroke cognitive function. Moreover, our findings suggest that this type of EE paradigm induced neurogenesis and angiogenesis, at least in part, via regulating PI3K/AKT/GSK-3/β-catenin signaling pathway and activation of the intrinsic axonal guidance molecules in animal models of ischemic stroke.

Preconditioning Effect of High-Intensity Interval Training (HIIT) and Berberine Supplementation on the Gene Expression of Angiogenesis Regulators and Caspase-3 Protein in the Rats with Myocardial Ischemia-Reperfusion (IR) Injury.

Objective It has been shown that angiogenesis is a desirable treatment for patients with ischemic heart disease. We set out to investigate the impact of high-intensity interval training (HIIT) and berberine supplementation on the gene expression of angiogenesis-related factors and caspase-3 protein in rats suffering from myocardial ischemic-reperfusion injury. Methods Fifty rats were divided into the following groups: (1) trained, (2) berberine supplemented, (3) combined, and (4) IR. Each cohort underwent five sessions of HIIT per week for a duration of 8 weeks followed by induction of ischemia. Seven days after completion of reperfusion, changes in the gene expression of angiogenesis-related factors and caspase-3 protein were evaluated in the heart tissue. Results We observed a significant difference between four groups in the transcript levels of vascular endothelial cell growth factor (VEGF), fibroblast growth factor-2 (FGF2), and thrombospondin-1(TSP-1) (p ≤ 0.05). However, the difference in endostatin (ENDO) levels was not significant among the groups despite a discernible reduction (p ≥ 0.05). Moreover, caspase-3 protein and infarct size were significantly reduced in the intervention groups (p ≤ 0.05), and cardiac function increased in response to these interventions. Conclusion The treatments exert their effect, likely, by reducing caspase-3 protein and increasing the expression of angiogenesis-promoting factors, concomitant with a reduction in inhibitors of the process.

104P Angiogenesis-related factors and clinical outcomes in combination therapy with paclitaxel (PTX), ramucirumab (RAM) plus nivolumab (Nivo) for advanced gastric cancer (AGC)

104P Angiogenesis-related factors and clinical outcomes in combination therapy with paclitaxel (PTX), ramucirumab (RAM) plus nivolumab (Nivo) for advanced gastric cancer (AGC)

Anti-angiogenic effect of tripterygium glycosides tablets in animal models of rheumatoid arthritis: A systematic review and meta-analysis

To explore and summarize the beneficial effects of a traditional Chinese medicine preparation, Tripterygium glycosides tablets (TGT), in rheumatoid arthritis (RA) animal models of neovascularization, and to provide a reference for future clinical applications and research on its pharmacologic mechanism. We searched the databases PubMed, Embase, Web of Science, Chinese National Knowledge Infrastructure, VIP, Wan Fang and SinoMed (China Biomedical Document Service System) to identify studies of TGT with outcome indicators of angiogenesis-related factors that were published before April 2020. Subgroup analysis and meta-regression were performed for dosage and duration of TGT. Statistical tests and subgroup analysis were conducted using RevMan 5.3, and meta-regression and sensitivity analysis were conducted using STATA/SE 15.0. Fourteen studies of TGT in RA rats were included in this analysis. Treatment with TGT significantly reduces synovial microvessel density and the expression of vascular endothelial growth factor (VEGF), VEGF receptor 2, hypoxia inducible factor α, c-Fos, c-Jun, angiopoietin-1 and angiopoietin-2 compared with control groups (P < .05). Subgroup analysis did not show a significant association of the mRNA levels of VEGF in synovium, assessed using quantitative real-time PCR, with duration or dosage of TGT. Meta-regression analysis also indicated that the effects of dosage and duration were not significantly associated with differences in VEGF mRNA levels. Sensitivity analysis on VEGF mRNA levels did not fundamentally change the results. TGT can reduce synovial neovascularization by decreasing synovial microvessel density and expression of VEGF, VEGF receptor 2, hypoxia-inducible factor α, c-Fos, c-Jun, Ang-1 and Ang-2, thereby suppressing pannus formation and bone destruction in rat models of RA. Additional well-designed studies are required to confirm these findings.

Thymosin‑β�4 induces angiogenesis in critical limb ischemia mice via regulating Notch/NF‑κB pathway

Thymosin-β 4 (Tβ4) has been reported to exert a pro-angogenic effect on endothelial cells. However, little is known on the role and underlying mechanisms of Tβ4 on critical limb ischemia (CLI). The present study aimed there-fore to investigate the mechanisms and pro-angiogenic effects of Tβ4 in CLI mice. Tβ4 overexpression lentiviral vector was first transfected into HUVEC and CLI mice model, and inhibitors of Notch pathway (DAPT) and NF-κB pathway (BMS) were also applied to HUVEC and CLI mice. Subsequently, MTT, tube formation and wound healing assays were used to determine the cell viability, angiogenesis and migratory ablity of HUVEC, respectively. Western blotting, reverse transcription, quantitative PCR, immunofluorescence and immunohistochemistry were used to detect the expression of the angiogenesis-related factors angiopoietin-2 (Ang2), TEK receptor tyrosine kinase 2 (tie2), vascular endothelial growth factor A (VEGFA), CD31 and α-smooth muscle actin (α-SMA) and the Notch/NF-κB pathways-related factors NOTCH1 intracellular domain (N1ICD), Notch receptor 3 (Notch3), NF-κB and p65 in HUVEC or CLI mice muscle tissues. The results demonstrated that Tβ4 not only enhanced the cell viability, angiogenesis and migratory ability of HUVEC but also promoted the expression of Ang2, tie2, VEGFA, N1ICD, Notch3, NF-κB, and phosphorylated (p)-p65 in HUVEC. In addition, Tβ4 promoted the expression of CD31, α-SMA Ang2, tie2, VEGFA, N1ICD and p-p65 in CLI mice muscle tissues. Treatment with DAPT and BMS had opposite effects of Tβ4, whereas Tβ4 reversed the effect of DAPT and BMS. The findings from the present study suggested that Tβ4 may promote angiogenesis in CLI mice via regulation of Notch/NF-κB pathways.

Therapeutic effect of integrin-linked kinase gene-modified bone marrow-derived mesenchymal stem cells for streptozotocin-induced diabetic cystopathy in a rat model

BackgroundDiabetic cystopathy (DCP) is a chronic complication of diabetes mainly within the submucosal and muscular layers of the bladder due to the hyperglycemia-induced ischemia. As no effective therapies are currently available, the administration of optimized mesenchymal stem cells (MSCs) provides a potential treatment of DCP. Thus far, new strategy, such as genetic modification of MSCs, has been developed and has shown promising outcomes of various disorders.MethodsThis study was conducted using integrin-linked kinase (ILK) gene-modified bone marrow-derived stem cells (BMSCs) for streptozotocin (STZ)-induced diabetic cystopathy in a rat model. In total, 68 male Sprague-Dawley rats were randomized into five groups: sham control (control group, n = 10); DCP model alone (DM group, n = 10); DCP rats intravenously treated with BMSCs (BMSC group, n = 16); DCP rats accepted adenoviral vector-infected BMSCs (Ad-null-BMSC group, n = 16) and DCP rats accepted ILK adenoviral vector-infected BMSCs (Ad-ILK-BMSC group, n = 16). Diabetic rats accepted cell transplantation in the experimental group (2 rats per group) were sacrificed for the bladder tissue on the third day, 7th day, and 14th day of treatment respectively ahead of schedule. At 4 weeks after treatment, all rats in five groups accepted urodynamic studies to evaluate bladder function and were sacrificed for bladder tissue.ResultsOur data showed that the underactive bladder function was significantly improved in DCP rats intravenously treated with ILK gene-modified BMSCs compared to those in the DM, BMSCs, and Ad-null-BMSC group. Meanwhile, we found that gene-modified BMSC treatment significantly promoted the activation of the AKT/GSK-3β pathway by increasing phosphorylation and led to the enhancement of survival. In addition, the expression levels of angiogenesis-related protein vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and stromal cell-derived factor-1 (SDF-1) were significantly higher in the Ad-ILK-BMSC group than that in the DM, BMSCs, and Ad-null-BMSC group as assessed by enzyme-linked immunosorbent assay and western blot. As two indicators of vascular endothelial cell markers, the expression of von Willebrand factor (vWF) and CD31 by western blot and immunofluorescent staining revealed that the percentage of the vascular area of the bladder tissue significantly increased in Ad-ILK-BMSC group compared with the BMSCs and Ad-null-BMSC group on the 14th day of treatment. Histological and immunohistochemical staining (hematoxylin and eosin (HE), vWF, Ki67, and TUNNEL) on the bladder tissue revealed statistically different results between groups.ConclusionILK gene-modified BMSCs restored the bladder function and histological construction via promoting the process of angiogenesis and protecting cells from high glucose-associated apoptosis in STZ-induced DCP rat model, which provides a potential for the treatment of patients with DCP.

Radiofrequency irradiation attenuates angiogenesis and inflammation in UVB-induced rosacea in mouse skin.

Rosacea is a skin inflammatory condition accompanied by cutaneous signs such as oedema, flushing, erythema, telangiectasia and pustules. Generally, rosacea is triggered by ultraviolet B (UVB) exposure. When exposed to UVB, skin epidermis thickens and produces elevated levels of pro-inflammatory cytokines, especially keratinocyte-related VEGF, a potent angiogenic factor. The upregulations of VEGF expression and its secretion promote the formation of new blood vessels and exacerbates rosacea. In this study, radiofrequency (RF) irradiation reduced keratinocyte proliferation in the epidermal layer, the expressions of pro-inflammatory cytokines, angiogenesis-related inflammatory factors and VEGF in our UVB-induced model of rosacea in vitro and in vivo. RF irradiation attenuated VEGF-induced angiogenesis-associated processes such as tube formation, cell migration and endothelial cell proliferation. Notably, blood vessel densities in the skins of UVB-treated mice and rosacea patients were significantly decreased by RF irradiation. These results provide experimental and molecular evidence regarding the effectiveness of RF irradiation for the treatment of rosacea.

Sphingosine kinase 1/sphingosine 1-phosphate/sphingosine 1-phosphate receptor 1 pathway: A novel target of geniposide to inhibit angiogenesis

ObjectiveRheumatoid arthritis (RA) is a common inflammatory autoimmune disease characterized by the formation of joint synovitis and pannus. Sphingosine 1-phosphate (S1P) is an important mediator related to angiogenesis, inflammation and autoimmunity. As Geniposide (GE) has potent immuno-modulation function, we investigated the effects on the dynamic balance of angiogenesis-related factors and Sphingosine kinase 1 (SphK1)-S1P-S1P receptor 1 (S1PR1) signal transduction in adjuvant-induced arthritis (AA) rats. MethodThe model evaluation was performed from paw swelling degree, arthritis index and movement score. The immunohistochemistry and enzyme-linked immunosorbent assay were used to study the microvascular density (MVD) and pro/anti-angiogenic factors levels. The cell viability was examined by cell counting kit-8 assay. SphK1, S1PR1 mRNA and protein levels in fibroblast-like synoviocytes (FLSs) were detected by quantitative real-time polymerase chain reaction and Western blotting. ResultsThe results showed that GE can apparently suppressed the inflammatory pathological status. The arthritis index, paw swelling and MVD of AA rats were decreased with dose dependence (⁎P < 0.05, ⁎⁎P < 0.01). In addition, GE can reduce the secretion of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1), promote the secretion of endostatin (ES) and inhibit excessive proliferation of FLSs (⁎P < 0.05, ⁎⁎P < 0.01). Importantly, GE can significantly inhibit the activity of SphK1, the level of S1P and the expression of SphK1 and S1PR1 in FLSs (⁎P < 0.05, ⁎⁎P < 0.01). ConclusionIt indicated that GE reduces the activity of SphK1 by restoring the dynamic balance between pro/anti-angiogenic factors, thereby interfering with SphK1-S1P-S1PR1 signal transduction, reducing the formation of synovial microvessels and exerting anti-angiogenesis effect of RA.

Novel ECM Patch Combines Poly(vinyl alcohol), Human Fibroblast-Derived Matrix, and Mesenchymal Stem Cells for Advanced Wound Healing.

Decellularized extracellular matrix (ECM)-based scaffold has been a very useful resource for effective tissue regeneration. In this study, we report a novel ECM patch that physically combines human fibroblast-derived matrix (hFDM) and poly(vinyl alcohol) (PVA) hydrogel. hFDM was obtained after decellularization of in vitro cultured human fibroblasts. We investigated the basic characteristics of hFDM alone using immunofluorescence (fibronectin, collagen type I) and angiogenesis-related factor analysis. Successful incorporation of hFDM with PVA produced an hFDM/PVA patch, which showed excellent cytocompatibility with human mesenchymal stem cells (hMSCs), as assessed via cell adhesion, viability, and proliferation. Moreover, in vitro scratch assay using human dermal fibroblasts showed a significant improvement of cell migration when treated with the paracrine factors originated from the hMSC-incorporated hFDM. To evaluate the therapeutic effect on wound healing, hMSCs were seeded on the hFDM/PVA patch and they were then transplanted into a mouse full-thickness wound model. Among four experimental groups (control, PVA, hFDM/PVA, hMSC/hFDM/PVA), we found that hMSC/hFDM/PVA patch accelerated the wound closure with time. More notably, histology and immunofluorescence demonstrated that compared to the other interventions tested, hMSC/hFDM/PVA patch could lead to significantly advanced tissue regeneration, as confirmed via nearly normal epidermis thickness, skin adnexa regeneration (hair follicle), mature collagen deposition, and neovascularization. Additionally, cell tracking of prelabeled hMSCs suggests the in vivo retention of transplanted cells in the wound region after the transplantation of hMSC/hFDM/PVA patch. Taken together, our engineered ECM patch supports a strong regenerative potential toward advanced wound healing.

Triiodothyronine potentiates angiogenesis-related factor expression through PI3K/AKT signaling pathway in human osteoarthritic osteoblasts.

Objective(s):Previous study has indicated that triiodothyronine (T3) facilitated cartilage degeneration in osteoarthritis (OA). This study aimed to investigate the effects of T3 on angiogenesis-related factor expression in human osteoblasts of OA subchondral bone.Materials and Methods:The subchondral bone specimens were obtained from OA patients and healthy participants. The expressions of VEGF, HIF-1α, AKT, and phosphorylated AKT was detected by immunohistochemistry, Western blotting, and RT-qPCR in OA. Angiogenesis-related factor expression in OA osteoblasts was measured by treating different concentrations of T3. The hypoxia model and PX-478 (HIF-1α inhibitor) were employed to confirm the regulative role of HIF-1α for VEGF expression. The level of VEGF secretion was examined in osteoblasts supernatant using ELISA. Results:Immunohistochemistry showed strong staining of VEGF and HIF-1α in OA subchondral bone. The expression of VEGF, HIF-1α, and p-AKT in OA osteoblasts was higher than that of normal osteoblasts at protein and mRNA levels. The physiological concentration of T3 (10-7 M) in OA osteoblasts up-regulated the expression of VEGF, HIF-1α, and p-AKT after 24 hr and 48 hr culture, while a higher dose of T3 displayed the adverse effects. Additionally, VEGF and p-AKT expression was down-regulated when PX-478 inhibited HIF-1α protein. Conclusion:Our results suggested that local T3 could effectively increase angiogenesis-related factor expression by PI3K/AKT signaling pathway, and HIF-1α regulated the VEGF expression in OA osteoblasts.