VEGF Protein Expression Research Articles

Extracellular Vesicle-Derived microRNAs of Human Wharton's Jelly Mesenchymal Stromal Cells May Activate Endogenous VEGF-A to Promote Angiogenesis.

Despite low levels of vascular endothelial growth factor (VEGF)-A, the secretome of human Wharton’s jelly (WJ) mesenchymal stromal cells (MSCs) effectively promoted proangiogenic responses in vitro, which were impaired upon the depletion of small (~140 nm) extracellular vesicles (EVs). The isolated EVs shared the low VEGF-A profile of the secretome and expressed five microRNAs, which were upregulated compared to fetal dermal MSC-derived EVs. These upregulated microRNAs exclusively targeted the VEGF-A gene within 54 Gene Ontology (GO) biological processes, 18 of which are associated with angiogenesis. Moreover, 15 microRNAs of WJ-MSC-derived EVs were highly expressed (Ct value ≤ 26) and exclusively targeted the thrombospondin 1 (THBS1) gene within 75 GO biological processes, 30 of which are associated with the regulation of tissue repair. The relationship between predicted microRNA target genes and WJ-MSC-derived EVs was shown by treating human umbilical-vein endothelial cells (HUVECs) with appropriate doses of EVs. The exposure of HUVECs to EVs for 72 h significantly enhanced the release of VEGF-A and THBS1 protein expression compared to untreated control cells. Finally, WJ-MSC-derived EVs stimulated in vitro tube formation along with the migration and proliferation of HUVECs. Our findings can contribute to a better understanding of the molecular mechanisms underlying the proangiogenic responses induced by human umbilical cord-derived MSCs, suggesting a key regulatory role for microRNAs delivered by EVs.

A synergy of estradiol with leptin modulates the long non-coding RNA NEAT1/ mmu-miR-204-5p/IGF1 axis in the uterus of high-fat-diet-induced obese ovariectomized mice

Obesity increases the risk of developing cancers for both males and females. This study investigated potential crosstalk between estradiol and leptin signaling pathways within the endometrium of high-fat-diet-induced obese ovariectomized mice to gain insight into possible links between obesity and endometrial cancer. We administered 17-β estradiol (0.2 μg/mouse subcutaneously) and/or recombinant mouse leptin (1 μg/g Bwt intraperitoneally.,) for 20 h to high-fat-diet-induced obese ovariectomized mice. The uterine tissues of experimental animals after treatments were studied by histological, immunohistochemical, quantitative real-time PCR (gene/miRNAs), and methylation-specific PCR analyses. Quantitative real-time PCR analysis revealed significantly increased expression of Cyclin d1, Esr1, Igf1, Igfbp2, Vegf, Oct4, and Pgr after estradiol and leptin co-treatment. Methylation-specific PCR results indicated that the hormonal dependent transcriptional regulation of Vegf, Igf1, and Pgr is independent of promoter methylation. The decreased expression of mmu- miR-204-5p after estradiol and leptin treatments correlated with the increased expression of long non-coding RNA Neat1. Insilico analysis confirmed the interaction of Neat1 and mmu- miR-204-5p and gene targets of mmu-miR-204-5p, including Igf1 were analyzed in this study. Immunohistochemical analyses revealed subcellular localization and increased expression of ESR, VEGF, phospho-Estrogen Receptor-α (pTyr537), and LEPR proteins following estradiol and leptin exposure. Overall, the data from our in vivo studies suggest the regulation of Neat1-mmu-miR-204−5p- Igf1 axis and associated gene expression changes in uterine tissue after estradiol and leptin co-treatment. In humans, long-term exposure to estradiol and leptin can alter endometrial homeostasis through the NEAT1-miR-204−5p-Igf1 axis and favor carcinogenic pathways, which provide mechanistic insight into the obesity-associated endometrial cancer.

Engineered resveratrol-loaded fibrous scaffolds promotes functional cardiac repair and regeneration through Thioredoxin-1 mediated VEGF pathway

Despite recent advancements, mortality due to coronary heart disease (CHD) remains high. Recently, the use of tissue-engineered grafts and scaffolds has emerged as a candidate for supporting the myocardium after an ischemic event. Resveratrol is a naturally occurring plant-based non-flavonoid polyphenolic compound found in many natural foods, including grapes and red wine. We embedded resveratrol in a polycaprolactone (PCL) scaffold and evaluated the cardio-therapeutic effects in a murine model of myocardial infarction (MI), with animals being grouped into Sham (S), Myocardial Infarction (MI), MI + PCL, and MI + PCL-Resveratrol (MI + PCL-R). After 4 and 8 weeks, echocardiography was performed to assess ejection fraction (EF) and fractional shortening (FS), which was followed by immunohistochemistry and immunofluorescence analysis at 8 weeks. The MI + PCL-R group showed a significant improvement in EF and FS compared with the MI + PCL group at 4 and 8-weeks post-surgery. PCL-R scaffolds treated hearts revealed decreased inflammatory cell infiltration, improved collagen extracellular matrix (ECM) secretion and blood vessel network formation following MI. The immunofluorescence analysis revealed resveratrol-loaded scaffolds promote increased expression of cTnT, Cx-43, Trx-1, and VEGF proteins. This study reports resveratrol-mediated rescue of ischemic myocardium when delivered through a biodegradable polymeric scaffold system after MI.

LncRNA MIR205HG regulates melanomagenesis via the miR-299-3p/VEGFA axis.

In this study, we investigated the role of lncRNA MIR205HG in melanomagenesis. Quantitative real-time PCR (qRT-PCR) analysis showed that MIR205HG levels were significantly upregulated in melanoma cell lines compared to normal human melanocytes. Similarly, MIR205HG levels were significantly higher melanoma tissues than adjacent normal skin tissues (n=30). CCK-8 and flow cytometry assays showed that MIR205HG knockdown significantly decreased the viability of melanoma cells. Dual luciferase reporter and RNA pull-down assays confirmed that MIR205HG directly binds to microRNA (miR)-299-3p. Targetscan analysis and dual luciferase reporter assays showed that miR-299-3p directly binds to the 3’UTR of VEGFA mRNA. Wound healing and transwell invasion assays showed that MIR205HG knockdown decreased in vitro migration and invasiveness of melanoma cells, and these effects were reversed by treatment with miR-299-3p inhibitor. MIR205HG-silenced melanoma cells showed increased miR-299-3p expression and lower levels of both VEGFA mRNA and protein. Tumor volumes were significantly smaller in nude mice xenografted with MIR205HG knockdown melanoma cells than the controls. These results demonstrate that MIR205HG supports melanoma growth via the miR-299-3p/VEGFA axis. This makes MIR205HG a potential therapeutic target for the treatment of melanoma.

Desmoplastic Small Round Cell Tumor: A Review of Main Molecular Abnormalities and Emerging Therapy.

Simple SummaryDesmoplastic small round cell tumor is a rare neoplasm with extremely aggressive behavior. Despite the multimodal treatment for newly diagnosed patients with chemotherapy, cytoreductive surgery and radiation, the cure rate is still low. For relapsed or progressive disease, there is limited data regarding second and third-line therapies. Novel agents have shown only modest activity. Recent molecular changes have been identified in this disease and this opens opportunities to be explored in future clinical trials.Desmoplastic small round cell tumor (DSRCT) is an extremely rare, aggressive sarcoma affecting adolescents and young adults with male predominance. Generally, it originates from the serosal surface of the abdominal cavity. The hallmark characteristic of DSRCT is the EWSR1–WT1 gene fusion. This translocation up-regulates the expression of PDGFRα, VEGF and other proteins related to tumor and vascular cell proliferation. Current management of DSRCT includes a combination of chemotherapy, radiation and aggressive cytoreductive surgery plus intra-peritoneal hyperthermic chemotherapy (HIPEC). Despite advances in multimodal therapy, outcomes remain poor since the majority of patients present disease recurrence and die within three years. The dismal survival makes DSRCT an orphan disease with an urgent need for new drugs. The treatment of advanced and recurrent disease with tyrosine kinase inhibitors, such as pazopanib, sunitinib, and mTOR inhibitors was evaluated by small trials. Recent studies using comprehensive molecular profiling of DSRCT identified potential therapeutic targets. In this review, we aim to describe the current studies conducted to better understand DSRCT biology and to explore the new therapeutic strategies under investigation in preclinical models and in early phase clinical trials.

Oral Contraceptive Steroids Promote Papillary Thyroid Cancer Metastasis by Targeting Angiogenesis and Epithelial-Mesenchymal Transition.

Thyroid cancer is the most prevalent type of endocrine malignancy with the highest incidence rate among women under 45 years old. Ethinylestradiol (EE) and levonorgestrel (LNG) are two steroid components of low-dose oral contraceptives used all over the world. In this study, we aimed to examine the possible effects of the combination of these two steroids on metastasis and angiogenic factors in BCPAP papillary thyroid cancer (PTC) cell line. After treatment of BCPAP cells with the combination of 20 nM EE and 90 nM LNG, mRNA expression levels of long noncoding RNAs HOTAIR and MALAT1, angiogenic and antiangiogenic gene markers VEGFA and THBS1, and epithelial-mesenchymal transition (EMT) biomarkers CDH1, CDH2, FN1, and VIM were analyzed by real-time PCR. Additionally, the protein expression of VEGFA was semiquantified by Western blotting. Results showed that the combination of LNG and EE significantly elevated the level of VEGFA protein and mRNA expression of VEGFA, MALAT1, HOTAIR, CDH2, FN1, and VIM genes while decreased CDH1 gene expression but had no marked effect on the expression of THBS1 gene in comparison with the control group. Also, our results suggest that LNG and EE may increase the metastatic and migratory properties of BCPAP cells via modulating angiogenic and EMT biomarkers. These data may highlight the potential of exogenous steroids in the advancement of PTC tumors.

Regulation mechanism of miR-494-3p on endometrial receptivity in mice via PI3K/AKT/mTOR pathway

Successful implantation requires endometrial receptivity. To investigate the mechanisms of miR-494-3p on endometrial receptivity, GnRHa's superovulation scheme was designed to reduce endometrial receptivity, and the pregnant mice were injected with miR-494-3p antagomir. The regulatory role of miR-494-3p was identified by RT-qPCR, uterine blastocyst count, scanning electron microscopy, hematoxylin-eosin (HE) staining, and Western blot. Results indicated that miR-494-3p antagomir increased uterine blastocysts numbers, promoted the pinocytosis expressions, and increased endometrial thickness. Besides, miR-494-3p antagomir significantly increased leukemia inhibitory factor (LIF), Ang-2 and VEGF protein expressions, and up-regulated p-AKT/AKT and p-mTOR/mTOR protein ratios in endometrium. Luciferase assay confirmed that LIF was a potential target of miR-494-3p. Subsequently, human endometrial epithelial cells (hEECs) were transfected with miR-494-3p inhibitor and PI3K inhibitor (LY294002). The role of miR-494-3p was identified by RT-qPCR, CCK-8 assay, transwell assay and flow cytometry. Results indicated that miR-494-3p inhibitor significantly increased proliferation and invasion, and significantly inhibited apoptosis in hEECs, while LY294002 reversed its biological function. Overall, these results suggested that miR-494-3p is the key regulator of endometrial receptivity in mice, regulating this complex process through the PI3K/AKT/mTOR pathway. Understanding the role of miR-494-3p in endometrial receptivity is of great significance for exploring new targets for the diagnosis and treatment of early pregnancy failure, and improving the success rates of artificial reproduction.

Human antigen R promotes angiogenesis of endothelial cells cultured with adipose stem cells derived exosomes via overexpression of vascular endothelial growth factor in vitro

ABSTRACT Recent studies showed that exosomes obtained from adipose-derived stem cells (ADSCs) could improve the angiogenesis of fat grafts via overexpression of vascular endothelial growth factor (VEGF). Human antigen R (HuR) promotes the expression of VEGF in many cancers, but the effect of HuR in normal endothelial cells in the presence of ADSC-derived exosomes remains unclear. We aimed to investigate the effect of HuR on the expression of VEGF and angiogenesis of human umbilical vein endothelial cells (HUVECs) cultured with ADSCs-derived exosomes. The HuR-overexpressed HUVECs (HuR-HUVECs) were cocultured with ADSCs-derived exosomes. qRT-PCR and Western blotting were performed to examine the stability and expression of VEGF-A mRNA and protein. The proliferation, migration, and proangiogenic capacity of HuR-HUVECs were evaluated using cell counting kit-8 (CCK-8), scratch wound healing, and Matrigel tube formation assay. qRT-PCR showed that HuR-HUVECs had higher expression and slower attenuation of VEGF-A mRNA. Western blotting confirmed higher expression of VEGF-A in HuR-HUVECs. CCK-8, scratch wound healing, and Matrigel tube formation assay demonstrated an increased proangiogenic effect in HuR-HUVECs. HuR promotes angiogenesis of HUVECs cocultured with ADSCs-derived exosomes via stabilization and overexpression of VEGF in vitro. The HuR/VEGF pathway is an important regulatory mechanism of angiogenesis in endothelial cells.

Chitosan and gelatin biopolymer supplemented with mesenchymal stem cells (Velgraft®) enhanced wound healing in goats (Capra hircus): Involvement of VEGF, TGF and CD31

AimCell-based therapy has emerged as promising strategy for chronic and impaired wounds treatment. Current research is focused on developing biomaterial systems that act as a niche for mesenchymal stem cells (MSCs) to promote wound healing through paracrine molecular cascading. This study was aimed to evaluate the wound healing potential of Velgraft, a ready-to-use biodegradable artificial skin substitute, on excision wound in goats. Materials and methodsTwelve male goats were randomized divided in to three groups of four animals each. After infliction of surgical wound, Velgraft and Soframycin were applied on wounds of the animals of Groups II and III while Group I (sham operated) served as control. Wound diameters were measured at pre-defined time-points for determination of progressive wound healing up to 28 days. Skin sections were stained using Hematoxylin and eosin (H&E) for examining the histoarchitectural changes, Masson trichome staining for ascertaining collagen synthesis and immunohistochemistry for expression of CD31, VEGF and TGF-β1 proteins to determine post-treatment angiogenesis in the inflicted wounds. ResultsVelgraft application appreciably enhanced wound closure by day 21 which was confirmed through restoration of the normal skin architecture as evident based on histopathological examination and characterized by complete regeneration of epidermal layers, collagen fibers, blood capillaries and hair follicular formation. Stimulation of angiogenesis markers was also observed at different time-points post-Velgraft application; which is suggestive of the improved angiogenesis and vasculogenesis. ConclusionVelgraft facilitates wound healing by augmenting early wound closure, enhancing collagen synthesis and deposition, trichosis development and promoting revascularization and epidermal layers restoration.

Healing Effects of Curcumin Nanoparticles in Deep Tissue Injury Mouse Model.

Chronic inflammation and lack of angiogenesis are the important pathological mechanisms in Deep Tissue Injury (DTI). Curcumin is a well-known anti-inflammatory and antioxidant agent. However, curcumin is unstable under acidic and alkaline conditions and can be rapidly metabolized and excreted in the bile, which shortens its bioactivity and efficacy. This study aimed to prepare curcumin-loaded poly (lactic-co-glycolic acid) nanoparticles (CPNPs) and to elucidate the protective effects and underlying mechanisms of wound healing in DTI models. CPNPs were evaluated for particle size, biocompatibility, in vitro drug release and their effect on in vivo wound healing. The results of in vivo wound closure analysis revealed that CPNP treatments significantly improved wound contraction rates (p<0.01) at a faster rate than the other three treatment groups. H&E staining revealed that CPNP treatments resulted in complete epithelialization and thick granulation tissue formation. In contrast, control groups resulted in a lack of compact epithelialization and persistence of inflammatory cells within the wound sites. Quantitative real-time PCR analysis showed that treatment with CPNPs suppressed IL-6 and TNF-α mRNA expression, and up-regulated TGF-β, VEGF-A and IL-10 mRNA expression. Western blot analysis showed up-regulated protein expression of TGF-β, VEGF-A and phosphorylated-STAT3. Our results showed that CPNPs enhanced wound healing in DTI models through modulation of the JAK2/STAT3 signalling pathway and subsequent upregulation of pro-healing factors.

Effects of HIF-1α on Spermatogenesis of Varicocele Rats by Regulating VEGF/PI3K/Akt Signaling Pathway.

Hypoxia-inducible factor-1α (HIF-1α) participates in the regulation of spermatogenic function in rats with varicocele (VC), and the PI3K/Akt pathway plays an important role in it. In the present research, we applied the CRISPR/Cas9 gene editing technique to silence the HIF-1α gene of VC rat testis, to explore the effect of HIF-1α on apoptosis of spermatogenic cells in VC rats through the PI3K/Akt pathway. Sprague Dawley rats were randomly assigned to four groups, including the normal rat group (group N), VC model group (group V), VC + HIF-1α-lentivirus group (group H), and VC + luciferase-lentivirus group (group L). Apoptosis of spermatogenic cells in rat testis was tested by TUNEL Kit. The morphologic changes of seminiferous tubules were viewed by a light microscope. Expressions of VEGF, Akt, p-Akt, p70S6K, and p-p70S6K were detected by means of Western blot, immunofluorescence, or immunohistochemistry methods. One-way ANOVA was applied to analyze the diverseness between groups. Compared with group N, the distribution of germ cells was disordered, apoptosis of spermatogenic cells increased significantly, and the expression of VEGF, p-Akt, and p-p70S6K was also increased in group V. Compared with group V, the damage of seminiferous epithelium in group H was improved, and the arrangement of the seminiferous epithelium was almost orderly. Apoptosis of spermatogenic cells decreased significantly, and the expression of VEGF, p-Akt, and p-p70S6K protein was decreased (P < 0.05). There was no significant difference between group N and group H (P > 0.05).In conclusion, HIF-1α is regulated by hypoxia in rats with varicocele to regulate its downstream gene VEGF which regulates spermatogenesis, and the PI3K/Akt signaling pathway plays a regulatory role in this process.

Correlation between the Expression of Angiogenic Factors and Stem Cell Markers in Human Uveal Melanoma.

Uveal melanoma (UM) is the most common malignant tumor of the eye with extremely high metastatic potential. UM tumor cells can disseminate only hematogenously, thus, angiogenic signals have a particular role in the prognosis of the disease. Although the presence of cancer stem cells (CSCs) in densely vascularized UMs has been reported previously, their role in the process of hematogenous spread of UM has not been studied. In this study, we investigated the regulation of angiogenesis in UM in correlation with the presence of CSCs. Seventy UM samples were collected to analyze the expression of CSC markers and angiogenic factors. The expression of CSC markers was studied by RT-PCR, Western blotting techniques and IHC-TMA technique. RT-PCR showed high expression of CSC markers, particularly nestin, FZD6 and SOX10 and somewhat lower expression of NGFR. The protein expression of FZD6, HIF-1α and VEGFA was further evaluated in 52 UM samples by the IHC-TMA technique. We report here for the first time a significant correlation between FZD6 and VEGFA expression in UM samples. The observed correlation between FZD6 and VEGFA suggests the presence of CSCs in UM that are associated with the vascularization process.

Tumor-associated macrophage, angiogenesis and lymphangiogenesis markers predict prognosis of non-small cell lung cancer patients

BackgroundThe tumor microenvironment (TME) is a critical player in tumor progression, metastasis and therapy outcomes. Tumor-associated macrophages (TAMs) are a well-recognized core element of the TME and generally characterized as M2-like macrophages. TAMs are believed to contribute to tumor progression, but the mechanism behind this remains unclear. We aimed to investigate the clinical, angiogenic, and lymphangiogenic significance of TAMs in non-small cell lung cancer (NSCLC).MethodsUtilizing combined immunohistochemistry and digital image analysis, we assessed CD68, CD163, VEGF-A, and VEGF-C expression in 349 patients with NSCLC. Subsequently, the potential association between M2 TAMs and angiogenic VEGF-A and/or lymphangiogenic VEGF-C was evaluated for its prognostic value. Furthermore, the effects of M2 TAMs on angiogenesis and lymphangiogenesis were explored via an in vitro co-culture system.ResultsCD68 and CD163 expression were found to directly correlate with VEGF-A and/or VEGF-C expression (all p < 0.001). Furthermore, elevated M2 ratio (CD163+/CD68+) was significantly associated with poor overall survival (p = 0.023). Dual expression of M2 ratiohigh and VEGF-Chigh (M2 ratiohighVEGF-Chigh) was correlated with worse overall survival (p = 0.033). Multivariate analysis revealed that M2 ratiohigh [HR (95% CI) = 1.53 (1.01–2.33), p = 0.046] and combined M2 ratiohighVEGF-Chigh expression [HR (95% CI) = 2.01 (1.28–3.16), p = 0.003] were independent predictors of poor overall survival. Notably, we confirmed that M2 macrophages significantly enhanced the protein and mRNA expression of both VEGF-A and VEGF-C, while M1 macrophages induced only mRNA expression of VEGF-A in A549 cells.ConclusionsThis study suggests that TAMs are significantly associated with angiogenesis and lymphangiogenesis, contributing to the progression of NSCLC. Furthermore, elevated M2 ratio, similar to combined high M2 ratio and high VEGF-C expression, is a strong indicator of poor prognosis in patients with NSCLC, providing insight for future TAM-based immunotherapy strategies.

Metabolite secretions of Lactobacillus plantarum YYC-3 may inhibit colon cancer cell metastasis by suppressing the VEGF-MMP2/9 signaling pathway

BackgroundColorectal cancer (CRC) is a major clinical challenge, and the gut microbiome plays important roles in the occurrence and metastasis of CRC. Lactobacillus and their metabolites are thought to be able to suppress the growth of CRC cells. However, the antimetastatic mechanism of Lactobacillus or their metabolites toward CRC cells is not clear. Therefore, the aim of this study was to assess the inhibitory mechanism of cell-free supernatants (CFSs) of L. rhamnosus GG, L. casei M3, and L. plantarum YYC-3 on metastasis of CRC cells.ResultsYYC-3 CFS showed the highest inhibitory effect on CRC cell growth, invasion and migration, and inhibited MMP2, MMP9, and VEGFA gene and protein expression, and protein secretion. Furthermore, it suppressed the activities of MMPs by gelatin zymography. Moreover, the effective compounds in these CFSs were analyzed by Q Exactive Focus liquid chromatography–mass spectrometry.ConclusionsOur results showed that metabolite secretions of YYC-3 may inhibited cell metastasis by downregulating the VEGF/MMPs signaling pathway. These data suggest that treatment of CRC cells with metabolites from L. plantarum YYC-3 may reduce colon cancer metastasis.

Thalidomide Inhibits Angiogenesis via Downregulation of VEGF and Angiopoietin-2 in Crohn's Disease.

Immune-mediated angiogenesis is important in the pathogenesis of inflammatory bowel disease and targeted treatment could alleviate the disease. Thalidomide is an effective drug in inflammatory bowel disease, which might be related to its multiple role in anti-inflammatory, immunoregulatory, and anti-angiogenesis. This study is to investigate the effect of thalidomide on angiogenesis in tissues from patients and in vitro cells. Angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), VEGF, and CD31 expressions in intestinal mucosa from pediatric CD patients before and after thalidomide treatment were measured by immunohistochemistry. Western blotting and polymerase chain reaction were performed to characterize the change of angiogenic factors before and after treatment in remission. Human umbilical vein endothelial cells (HUVECs) treated by thalidomide were used to examine its effect on endothelial cell proliferation and migration and capillary-like structures. Results showed that VEGF and Ang-2 levels were significantly greater in CD patients over controls. Thalidomide produced a significant reduction in protein expression of Ang-2 and VEGF, along with a decrease in mRNA expression of Ang-2. While, Ang-1 level did not show a statistically significant change. Thalidomide significantly inhibited cell proliferation in a dose-dependent manner. It also suppressed VEGF- and Ang-2-induced cell migration and capillary-like tube formation in HUVECs. Therefore, our study suggests that VEGF and Ang-2 levels are up-regulated in pediatric CD patients. It also indicated that thalidomide can be able to deactivate endothelium by the downregulation effect on angiogenic factors by targeting VEGF and Ang-2.

Abstract 13469: Novel Role of Extracellular SOD-derived H 2 O 2 Signaling in Exercise-induced Vascular Adaptation in Type 2 Diabetes

Background: Exercise training promotes vascular adaptation (restoration of endothelial function and angiogenesis) in type2 diabetes (T2D). Since eNOS uncoupling/O 2 - are increased in T2D, exercise may promote vascular adaptation via mechanism other than eNOS-NO axis. Extracellular superoxide dismutase (ecSOD) is a secreted copper (Cu) containing SOD that catalyzes the dismutation of O 2 - to H 2 O 2 and its full activity requires Cu transporter ATP7A. We reported that ATP7A-ecSOD pathway is reduced in T2D and that ecSOD-derived H 2 O 2 promotes VEGFR2 signaling and angiogenesis in endothelial cells. However, role of ATP7A-ecSOD axis and H 2 O 2 signaling in vascular adaptation to exercise in T2D have not been reported. Oxidation of Cysteine residues of targets proteins to generate cysteine sulfenic acid (Cys-OH) is a key initial event in H 2 O 2 -mediated signaling. Results: Here we show that ATP7A protein (49%), ecSOD activity (51%) and extracellular H 2 O 2 levels in blood vessels and skeletal muscles were significantly decreased in high fat diet-induced T2D mice compared to control C57Bl6 mice, which were rescued by volunteer wheel exercise (2 weeks) or in T2D/ATP7A overexpressing mice. In parallel, exercise training restored impaired endothelium-dependent relaxation (EDR) of resistant arteries and angiogenesis (CD31+ capillary, 2.4-fold) in skeletal muscle of T2D mice, which were inhibited by exogenous catalase or in T2D/ecSOD KO mice, but not by L-NAME, suggesting that ecSOD-derived H 2 O 2 , but not eNOS/NO, mediates exercise-induced beneficial effects. Mechanistically, exercise significantly increased Cys-OH formation of PKG1α (2.3-fold), which was shown to induce EDR in H 2 O 2 -, but not NO/cGMP-, dependent manner, in T2D vascular tissues as well as Cys-OH formation of AMPK (3.6-fold) and AMPK downstream angiogenic PGC1α and VEGF protein expression in T2D skeletal muscles. Of note, these exercise-induced Cys oxidation of PKG1α and AMPK were not observed in control or T2D/ecSOD KO mice. Conclusion: Exercise-induced ATP7A-ecSOD axis-mediated out-side in H 2 O 2 signaling plays an important role in promoting vascular adaptation in T2D via Cys oxidation of redox-sensitive kinases required for endothelial function and angiogenesis in skeletal muscles.

Abstract 15238: Cardiomyocyte Specific Pellino1 Knockout Leads to Increased Oxidative Stress, Reduced Anti-apoptotic Signaling and Cardiac Dysfunction in a Murine Myocardial Infarction Model

Introduction: Ischemic heart disease is the leading cause of death in adults in the US. We have shown that overexpression of E3 ligase Pellino1 (Peli1) increased vessel density, triggered anti-apoptotic signaling, and preserved cardiac function in the infarcted myocardium. In the present study, we aimed to explore whether the loss of Peli1 function in cardiomyocytes has any effect on cardiac function using a genetically-modified murine myocardial infarction (MI) model. Methods: Wild type (WT) and cardiomyocyte-specific Peli1 knockout (CP1KO -/- ) mice were divided into 4 groups [wild-type-sham (WTS), CP1KO -/- sham (CP1KO -/- S), WTMI and CP1KO -/- MI] to undergo either left anterior descending artery ligation or sham surgery. Phosphorylation of Mitogen-Activated Protein Kinase-Activated Protein Kinase-2 (p-MK2) and expression of VEGF, Bcl2, and cIAP2 were quantified by Western blot analysis. Cardiac function by two-dimensional echocardiography was performed at 30 days post-op to assess ejection fraction (EF), fractional shortening (FS), left ventricular internal diameter in both systole and diastole (LVIDs and LVIDd). Results: We observed no significant change in cardiac functions between WTS and CP1KO-/-S group. However, in MI groups, CP1KO -/- mice showed significant loss of systolic functions, represented by both EF (26.60%±2.340 vs. 33.46%±1.84; p&lt;0.029, n=13) and FS (12.43%± 1.19 vs. 15.93%±0.98; n=13, p&lt;0.03) when compared to WTMI group. Western blot analysis of heart tissue 24-hour post-MI showed a significant reduction in the expression of p-MK2 (p=0.0045) in CP1KO -/- group compared to WT mice. Similarly, expression of VEGF (p=0.0227), BCl-2 (p=0.0073) and cIAP2 (p=0.0483) was also found to be significantly downregulated in CP1KO -/- group compared to WTMI group at 4-day post-MI. We also analyzed oxidative stress marker, 3-NT, which was significantly upregulated (p=0.0001) in CP1KO mice compared to WTMI. Conclusion: Loss of Peli1 in cardiomyocyte results in the impairment of cardiac function along with increased oxidative stress, reduced angiogenic and survival protein expression, which suggests Peli1 plays an important role in the cardiomyocyte signaling during myocardial infarction.

Synergistic interactions of PlGF and VEGF contribute to blood-retinal barrier breakdown through canonical NFκB activation

To investigate the role of placental growth factor/vascular endothelial growth factor (PlGF-VEGF) heterodimers are involved in the blood-retinal barrier (BRB) breakdown and the associated mechanism, human retinal endothelial cells (HRECs) were treated with recombinant human (rh)PlGF-VEGF heterodimers and rhPlGF and studied in normal and high-glucose conditions. HREC barrier function was evaluated by the measurement of trans-endothelial electrical resistance (TEER). Adeno-Associated Virus Type 5 (AAV5) vectors overexpressed PlGF in the retina by intravitreal injection into the C57BL6 mouse eye. AAV5-GFP vector and naïve animals were used as controls. Immunofluorescence (IF) and western blots examined the protein expression of PlGF-VEGF heterodimers, VEGF, PlGF, NFκB, p-IκBα, ZO-1, and VE-cadherin in HREC and mouse retina. PlGF-VEGF heterodimers were detected predominantly in the HREC cell nuclei based on IF and cytoplasmic and nuclear fractionation experiments. High glucose treatment increased PlGF-VEGF nuclear abundance. Dot immunoblotting demonstrated a strong affinity of the 5D11D4 antibody to PlGF-VEGF heterodimers. rhPlGF-VEGF disrupted the barrier function of HREC, which was prevented by the neutralization of PlGF-VEGF by the 5D11D4 antibody. Stimulation of HRECs with rhPlGF also led to an increase in the nuclear signals for PlGF-VEGF, p-IκBα, and colocalization of NFκB p65 and PlGF-VEGF in the nuclei. The selective IKK2 inhibitor IMD0354 disrupted the nuclear colocalization. Treatment with IMD0354 restored the barrier function of HREC, as indicated by the ZO-1 and VE-cadherin expression. In the mouse retinas, PlGF overexpression by AAV5 vector reduced ZO-1 expression and increased abundance of pIκBα. PIGF/VEGF heterodimers mediate BRB breakdown potentially through the canonical NFκB activation.

Effects of salvianolate lyophilized injection combined with Xueshuantong injection in regulation of BBB function in a co-culture model of endothelial cells and pericytes

The combined use of two or more different drugs can better promote nerve recovery and its prognosis for treatment of stroke. The salvianolate lyophilized injection (SLI) and Xueshuantong Injection (XST) are two standardized Chinese medicine injections which have been widely used in the treatment of cerebrovascular diseases. Salvianolic acid B (Sal B) and Notoginsenoside R1 (NR1) is respectively one of the active constituents of SLI and XST, which have certain effects on stroke. In this study, we established a co-culture of endothelial cells and pericytes for oxygen-glucose deprivation/reperfusion (OGD/R) injury model to study the effects of SLI and Sal B or XST and NR1 alone, or with their combinations (1S1X) in regulation of BBB function. The results showed that compared with the OGD/R group, treatment with SLI, XST and SalB and NR1 can significantly increase the TEER, reduce the permeability of Na-Flu, enhance the expression of tight junctions (TJs) between cells, and stabilize the basement membrane (BM) composition. In addition, the combination of 1S1X is superior to the XST or SLI alone in enhancing the TJs between cells and stabilizing the BM. And the active components SalB and NR1 can play a strong role in these two aspects, even with the whole effects. Furthermore, the study showed that XST, Sal B and NR1 increases in Ang-1and Tie2, while decrease in Ang-2 and VEGF protein expressions. Overall, these findings suggest that SLI combined with XST (1X1S) has protective effects on co-culture of endothelial cells and pericytes after OGD/R. Moreover, its protective effect might be associated with increase of TJs and BMs through activation of Ang/Tie-2 system signaling pathway.

Nanodelivery of Resveratrol-Loaded PLGA Nanoparticles for Age-Related Macular Degeneration.

Age-related macular degeneration, precisely neovascular form, is the leading cause of vision loss and the key treatment includes intravitreal injections of anti-vascular endothelial growth factor (anti-VEGF) agents. A method to increase local concentration of drug at posterior segment of the eye and to reduce the frequency of intravitreal injections is an unmet need. Resveratrol, a naturally occurring antioxidant and anti-inflammatory polyphenol, was loaded in PLGA polymeric nanoparticles to study their sustained release property and effectiveness in reducing expression of VEGF protein in vitro. Nanoparticles were characterized using FTIR, DSC, size, encapsulation efficiency, TEM, and in vitro drug release studies. Using MTT assay, the cytotoxicity of formulation was evaluated on ARPE-19 cells. The cellular uptake and VEGF expression levels were also evaluated in in vitro settings. The optimized formulation had a particle size of 102.7nm with - 47.30mV of zeta potential. Entrapment efficiency was found to be 65.21%. The cell viability results suggested compatibility of developed formulation. Cellular uptake and VEGF expression levels for the formulated nanoparticles specified that the developed formulation showed potential cellular uptake and had displayed anti-angiogenic property by inhibiting VEGF expression in vitro. The results showed successful development of resveratrol-loaded nanoparticles which may be used for neovascular AMD treatment alone or in combination with anti-VEGF agents.