Expression Levels Of VEGF Research Articles

VR-10 polypeptide interacts with CD36 to induce cell apoptosis and autophagy in choroid-retinal endothelial cells: Identification of VR-10 as putative novel therapeutic agent for choroid neovascularization (CNV) treatment

Choroid neovascularization (CNV) is important adverse pathological changes that contributes to the aggravation of hypoxic-ischemic eye diseases, and our preliminary work evidences that the thrombospondin-1 (TSP-1) synthetic polypeptide VR-10 may be the candidate therapeutic agent for the treatment of CNV, but its detailed effects and molecular mechanisms are not fully delineated. In this study, the CNV models in BN rats were established by using the laser photocoagulation method, which were further subjected to VR-10 peptide treatment. The RNA-seq and bioinformatics analysis suggested that VR-10 peptide significantly altered the expression patterns of genes in the rat ocular tissues, and the changed genes were especially enriched in the CD36-associated signal pathways. Next, by performing the Real-Time qPCR and Western Blot analysis, we expectedly found that VR-10 upregulated the anti-angiogenesis biomarker (PEDF) and downregulated pro-angiogenesis biomarkers (VEGF, HIF-1 and IL-17) in rat tissues. In addition, we evidenced that VR-10 downregulated CDK2, CDK4, CDK6, Cyclin D1 and Cyclin D2 to induce cell cycle arrest, upregulated cleaved Caspase-3, Bax and downregulated Bcl-2 to promote cell apoptosis, and increased LC3B-II/I ratio and facilitate p62 degradation to promote cell autophagy in RF/6A cells, which were all reversed by knocking down CD36. Moreover, VR-10 upregulated PEDF, and decreased the expression levels of VEGF, HIF-1 and IL-17 to block angiogenesis of RF/6A cells in a CD36-dependent manner. Taken together, VR-10 peptide interacts with its receptor CD36 to regulate the biological functions of RF/6A cells, and these data suggest that VR-10 peptide may be the putative therapeutic drug for the treatment of CNV in clinic.

Aconiti lateralis radix praeparata total alkaloids exert anti-RA effects by regulating NF-κB and JAK/STAT signaling pathways and promoting apoptosis.

Aconiti Lateralis Radix Praeparata (“Fuzi” in Chinese) is one of the traditional herbs widely used to intervene rheumatoid arthritis (RA), while Fuzi total alkaloids (FTAs) are the main bioactive components. However, the treatment targets and specific mechanisms of FTAs against RA have not been fully elucidated. The purpose of the present study was to confirm the anti-rheumatism effects of FTAs and reveal its potential molecular mechanisms. In TNF-α-induced MH7A cells model, we found that FTAs showed inhibitory effects on proliferation. While, FTAs significantly decreased the expression levels of IL-1β, IL-6, MMP-1, MMP-3, PGE2, TGF-β, and VEGF. FTAs also enhanced the progress of apoptosis and arrested the cell cycle at G0/G1 phase to prevent excessive cell proliferation. In addition, FTAs inhibited the hyperactivity of NF-κB and JAK/STAT signaling pathways, and regulated the cascade reaction of mitochondrial apoptosis signaling pathway. The results suggested that FTAs exerted anti-inflammatory effects by inhibiting NF-κB and JAK/STAT signaling pathways, promoted apoptosis by stimulating mitochondrial apoptosis signaling pathway, and inhibited cell proliferation by modulating cell cycle progression.

Artificial Microvesicles: New Perspective on Healing Tendon Wounds

Tendons have a limited capacity to repair both naturally and following clinical interventions. Damaged tissue often presents with structural and functional differences, adversely affecting animal performance, mobility, health, and welfare. Advances in cell therapies have started to overcome some of these issues, however complications such as the formation of ectopic bone remain a complication of this technique. Regenerative medicine is therefore looking toward future therapies such as the introduction of microvesicles (MVs) derived from stem cells (SCs). The aim of the present study was to assess the characteristics of artificially derived MVs, from equine mesenchymal stem cells (MSCs), when delivered to rat tendon cells in vitro and damaged tendons in vivo. The initial stages of extracting MVs from equine MSCs and identifying and characterizing the cultured tendon stem/progenitor cells (TSCs) from rat Achilles tendons were undertaken successfully. The horse MSCs and the rat tendon cells were both capable of differentiating in 3 directions: adipogenic, osteogenic, and chondrogenic pathways. The artificially derived equine MVs successfully fused with the TSC membranes, and no cytotoxic or cytostimulating effects were observed. In addition, co-cultivation of TSCs with MVs led to stimulation of cell proliferation and migration, and cytokine VEGF and fractalkine expression levels were significantly increased. These experiments are the first to show that artificially derived MVs exhibited regeneration-stimulating effects in vitro, and that fusion of cytoplasmic membranes from diploid cell lines originating from different species was possible. The experiment in vivo demonstrated the influence of MVs on synthesis of collagen I and III types in damaged tendons of rats. Explorations in vivo showed accelerated regeneration of injured tendons after introduction of the MVs into damaged areas. The results from the studies performed indicated obvious positive modifying effects following the administration of MVs. This represents the initial successful step required prior to translating this regenerative medicine technique into clinical trials, such as for tendon repair in injured horses.

Fenugreek Extract-Loaded Polycaprolacton/Cellulose Acetate Nanofibrous Wound Dressings for Transplantation of Unrestricted Somatic Stem Cells: An In Vitro and In Vivo Evaluation

Complex pathophysiology of diabetic wounds causes a delayed wound healing response. Advanced wound dressing materials that deliver biochemical cues are of particular interest in wound healing research. Here, we developed a dual-function delivery vehicle for drug and cell delivery applications to treat diabetic wounds. The delivery system was developed via electrospinning of polycaprolacton/cellulose acetate solution containing fenugreek extract. The produced delivery vehicle was characterized using microstructural studies, cell viability assay, cytoprotection assay, cell migration assay, In Vitro anti-inflammatory assay, free radical scavenging assay, tensile strength studies, swelling studies, and protein adsorption test. Scaffolds were then seeded with 30000 unrestricted somatic stem cells and transplanted into the rat model of excisional diabetic wound. Wound healing assay showed that the co-delivery of fenugreek extract and unrestricted somatic stem cells led to a substantial improvement in the healing activity of electrospun dressings, as evidenced by higher wound contraction, epithelial thickness, and collagen deposition in this group compared with other experimental groups. Gene expression analysis showed that dual-function delivery system could increase the expression level of VEGF, b-FGF, and collagen type II genes. Furthermore, the tissue expression level of IL-1β and glutathione peroxidase genes was significantly reduced in this group compared with other groups. This study shows that the developed system may be considered as a potential treatment modality for diabetic wounds in the clinic.

Analysis of the absorbed constituents and mechanism of liquidambaris fructus extract on hepatocellular carcinoma.

Background: Hepatocellular carcinoma (HCC) refers to one of the top 10 cancers in terms of morbidity and mortality globally, seriously influencing people’s lives. First recorded in Compendium of Materia Medica, liquidambaris fructus (LF) generates definite anti-liver tumor effect. However, its effective substances and mechanism remain to be elucidated. Methods: Serum pharmacochemistry and UPLC-QTOF-MS technologies were employed to explore the plasma of rats after intragastric administration of liquidambaris fructus extract (LFE) in order to find the active ingredients. Subsequently, DEN-induced rat liver cancer model was established with the purpose of investigating the anti-tumor activity of LFE from physiological, pathological and biochemical aspects. Finally, non-target metabonomics combined with q-PCR and Western blot methods were adopted for revealing the mechanism. Results: Totally 11 prototype blood transfused ingredients, including imperatorin and phellopterin were detected. LFE presents excellent impact on enhancing the quality of life, prolonging the life cycle, reducing inflammatory reaction, protecting hepatocytes, improving body immunity and killing liver tumor cells. Altogether 82 endogenous differential metabolites were found in metabonomics, suggesting that LFE can treat HCC by acting on key targets of PTEN/PI3K/Akt pathway and fatty acid metabolism. Further research also verified that LFE can upregulate the relative expression levels of PTEN, PDCD4, Caspase 9, Caspase 3, Bax and Bad as well as lower the relative expression levels of PI3K, AKT, VEGFA and Bcl-2. Conclusion: This study revealed the pharmacodynamic material basis of LFE in the treatment of HCC, and from the perspective of metabolomics proved that the effects of inhibiting the growth of tumor cells, promoting tumor cell apoptosis, reducing inflammatory reaction, protecting hepatocytes, improving the survival state of tumor rats, and prolonging the life cycle are related to its impact on PTEN/PI3K/Akt, fatty acid metabolism and other key signal pathways.

Microporous structures on mineralized collagen mediate osteogenesis by modulating the osteo-immune response of macrophages.

It is a new hot pot in tissue engineering and regenerative medicine to study the effects of physicochemical properties of implanted biomaterials on regulating macrophage polarization to promote bone regeneration. In this study, we designed and fabricated mineralized collagen (MC) with different microporous structures via in vitro biomimetic mineralization method. The microporous structures, mechanical properties, shore hardness and water contact angle measurements were tested. Live/dead cell staining, CCK-8 assay, phalloidine staining, staining of focal adhesions were used to detect cell behavior. ELISA, qRT-PCR, ALP, and alizarin red staining (ARS) were performed to appraise osteogenic differentiation and investigated macrophage response and their subsequent effects on the osteogenic differentiation. The results showed that RAW264.7 and MC3T3-E1 cells were able to survive on the MC. MC with the microporous structure of approximately 84 μm and 70%–80% porosity could promote M2 macrophage polarization and increase the expression level of TGF-β and VEGF. Moreover, the gene expression of the osteogenic markers ALP, COL-1, and OCN increased. Therefore, MC with different microporous structures mediated osteoimmunomodulation in bone regeneration. These data will provide a new idea of biomaterials inducing bone repair and direct the optimal design of novel immune biomaterials, development, and rational usage.

Kuoxin Decoction promotes lymphangiogenesis in zebrafish and in vitro based on network analysis.

Background: Inadequate lymphangiogenesis is closely related to the occurrence of many kinds of diseases, and one of the important treatments is to promote lymphangiogenesis. Kuoxin Decoction (KXF) is an herbal formula from traditional Chinese medicine used to treat dilated cardiomyopathy (DCM), which is associated with lymphangiogenesis deficiency. In this study, we comprehensively verified whether KXF promotes lymphangiogenesis in zebrafish and in vitro based on network analysis. Methods: We performed virtual screening of the active compounds of KXF and potential targets regarding DCM based on network analysis. Tg (Flila: EGFP; Gata1: DsRed) transgenic zebrafish embryos were treated with different concentrations of KXF for 48 h with or without the pretreatment of MAZ51 for 6 h, followed by morphological observation of the lymphatic vessels and an assessment of lymphopoiesis. RT-qPCR was employed to identify VEGF-C, VEGF-A, PROX1, and LYVE-1 mRNA expression levels in different groups. After the treatment of lymphatic endothelial cells (LECs) with different concentrations of salvianolic acid B (SAB, the active ingredient of KXF), their proliferation, migration, and protein expression of VEGF-C and VEGFR-3 were compared by CCK-8 assay, wound healing assay, and western blot. Results: A total of 106 active compounds were identified constituting KXF, and 58 target genes of KXF for DCM were identified. There were 132 pathways generated from KEGG enrichment, including 5 signaling pathways related to lymphangiogenesis. Zebrafish experiments confirmed that KXF promoted lymphangiogenesis and increased VEGF-C and VEGF-A mRNA expression levels in zebrafish with or without MAZ51-induced thoracic duct injury. In LECs, SAB promoted proliferation and migration, and it could upregulate the protein expression of VEGF-C and VEGFR-3 in LECs after injury. Conclusion: The results of network analysis showed that KXF could regulate lymphangiogenesis through VEGF-C and VEGF-A, and experiments with zebrafish confirmed that KXF could promote lymphangiogenesis. Cell experiments confirmed that SAB could promote the proliferation and migration of LECs and upregulate the protein expression of VEGF-C and VEGFR-3. These results suggest that KXF promotes lymphangiogenesis by a mechanism related to the upregulation of VEGF-C/VEGFR-3, and the main component exerting this effect may be SAB.

Inhibitory effect of low‑intensity pulsed ultrasound on the fibrosis of the infrapatellar fat pad through the regulation of HIF‑1α in a carrageenan‑induced knee osteoarthritis rat model.

Fibrotic changes in the infrapatellar fat pad (IFP) are involved in the pathogenesis of knee osteoarthritis (KOA). HIF-1α is a transcription factor that is activated during hypoxia and is suggested to play a role in fibrosis in various organs. However, its participation in the fibrotic changes in IFP remains unclear. Therefore, we investigated the role of HIF-1α in IFP fibrosis using a carrageenan-induced KOA rat model and evaluated the potential of low-intensity pulsed ultrasound (LIPUS) as a novel treatment for KOA. A rat model was prepared by intra-articular injection of 0.5% carrageenan (50 µl) using 8-week-old male Wistar rats. Fibrosis of the IFP was evaluated histologically by hematoxylin and eosin and Sirius Red staining at 1 and 2 weeks after intra-articular injection. The mRNA expression levels of HIF-1α and fibrosis-related molecules, CTGF and VEGF, were analyzed using reverse transcription-quantitative PCR, and the DNA binding activity of HIF-1α was assessed using a binding assay. In addition, the effect of irradiation with LIPUS on the fibrosis of IFP was verified. Histological studies demonstrated a significant increase in the fibrosis of IFP 1 and 2 weeks after intra-articular injection of carrageenan, accompanied by overexpression of CTGF and VEGF, which was followed by upregulation of transcriptional activation of HIF-1α. Moreover, intervention with LIPUS for 2 weeks after injection of carrageenan attenuated fibrosis of IFP, accompanied by a significant reduction in the transcriptional activation of HIF-1α and decreased the gene expression levels of HIF-1α, CTGF, and VEGF. The present study demonstrated that activation of HIF-1α promoted fibrosis of IFP in carrageenan-induced arthritis in rats and that intervention with LIPUS decreased the activity of HIF-1α and inhibited fibrosis. These results suggest that LIPUS may serve as a novel approach for the treatment of KOA, through its modulation of HIF-1α.

Effect of Netrin-1 on VEGFA Expression in T-ALL Cells and Its Related Mechanism

ELISA assays were applied to detect the levels of Netrin-1 and VEGFA in the bone marrow (BM) samples from children in the T-ALL and control group. The level of Netrin-1 and VEGFA were compared between control children and patients, and the liner correlation between Netrin-1 and VEGFA was analyzed. The T-ALL cells Jurkat and Molt-4 were culture in vitro, and the cells were treated with different concentration of Netrin-1 (0, 25, 50, 100 ng/ml) for 24 h, quantitative RT-PCR (qRT-PCR) and Western blot were used to detect the VEGFA expression in Jurkat, Molt-4 cells. The expression of Netrin-1 receptors in T-ALL cells was detected by qRT-PCR and the interaction between Netrin-1 and receptor in each cells was detected by co-IP. Furthermore, Western blot was used to detect the phosphorylation level of key prateins of AKT signal transduction pathway including Akt and mTOR in T-ALL cells treated with Netrin-1 (100 ng/ml). The expression of VEGFA and phosphorylation of AKT pathway transducers were detected by Western blot, after T-ALL cells treated with Netrin-1 (100 ng/ml) combined with inhibitors specific to Akt or mTOR. The expression level of Netrin-1 and VEGFA in T-ALL patients BM samples were both signi-ficantly higher than that of control group. And the expression level of Netrin-1 was positively correlated with that of VEGFA（r2=0974). With the increase of Netrin-1 concentration, the expression level of VEGFA also increased(P<0.05）. Netrin-1 interacted with its receptor, integrin-β4 at the Netrin-1 concentration of 100 ng/ml. Further, the treatment of Netrin-1 could increase the phosphorylation of Akt and mTOR, which were the key transducers of AKT pathway. After treatment of T-ALL cells with Netrin-1 (100 ng/mL) and Akt inhibitor, the expression of VEGFA and phosphorylation of Akt or mTOR decreased. When the cells were treated with Netrin-1(100 ng/ml) and mTOR inbititor, the phosphorylation level of mTOR and the expression of VEGFA decreased, the phosphorylation level of Akt increased. The expression of Netrin-1 and VEGFA in bone marrow of childred with T-ALL were abnormal, and there was a linear relationship between them. Netrin-1 can interact with its receptor, integrin-β4 and activate AKT transduction pathway to elevate the expression of VEGFA in T-ALL cells.

Electro-acupuncture Promotes Angiogenesis via Exosomal miR-210 in the Hypoxia-induced HUVECs Mediated HIF-1α/VEGF/Notch 1 Signal Pathway.

Acupuncture has been wildly applied for cerebral ischemia treatment in China for thousands of years, while the specific mechanism remains uncertain. Recently, many studies have shown that acupuncture promotes angiogenesis after ischemia occurs. Here, we examined the effect of electro-acupuncture (EA) exosomes on angiogenesis in hypoxia-induced human umbilical vein endothelial cells (HUVECs). To investigate whether EA exosomal miR-210 promotes angiogenesis in the hypoxiainduced HUVECs via the HIF-1α/VEGF/Notch 1 signal pathway. The middle cerebral artery occlusion (MCAO) model was established and treated with EA therapy. Then, exosomes were identified and isolated from rats' plasma in the MCAO+EA group by transmission electron microscopy (TEM), surface markers expressions, and PKH26 reagent. MiR- 210 mimic, miR-210 inhibitor, and HIF-1α were transfected. Flow cytometry, CCK-8 assay, and Transwell assay were conducted to assess the migration, apoptosis, and proliferation of each group of cells. Western blot and quantitative PCR were performed to detect the CD34, HIF-1α, VEGF, Notch 1, and miR-210 expression levels in each group. MiR-210 was significantly upregulated in exosomes of the MCAO plasma, and further enhanced by EA therapy. EA-EXOs and miR-210 mimic inhibited cell apoptosis, promoted cell proliferation and cell migration in hypoxia-induced HUVECs. However, the miR-210 inhibitor reversed the proliferation and migration number induced by EA-EXOs. Besides, EA-EXOs and miR- 210 mimic further enhanced those HIF-1α, VEGF, and Notch 1 levels compared to the hypoxia treatment only. Silencing HIF-1α or miR-210 reversed the high expressions of those three angiogenic factors induced by hypoxia and EA-EXO. qPCR showed similar trends with their relative mRNAs. To analyze these associations quantificationally, Spearman's rank correlation coefficient was calculated. As revealed by results, the expression of proteins and mRNA were highly correlative with each other. These results indicated that EA-EXO miR-210 promotes angiogenesis in hypoxia conditions via HIF-1α/VEGF/Notch 1 signal pathway.

Kisspeptin treatment improves fetal-placental development and blocks placental oxidative damage caused by maternal hypothyroidism in an experimental rat model.

Maternal hypothyroidism is associated with fetal growth restriction, placental dysfunction, and reduced kisspeptin/Kiss1R at the maternal-fetal interface. Kisspeptin affects trophoblastic migration and has antioxidant and immunomodulatory activities. This study aimed to evaluate the therapeutic potential of kisspeptin in the fetal-placental dysfunction of hypothyroid Wistar rats. Hypothyroidism was induced by daily administration of propylthiouracil. Kisspeptin-10 (Kp-10) treatment was performed every other day or daily beginning on day 8 of gestation. Feto-placental development, placental histomorphometry, and expression levels of growth factors (VEGF, PLGF, IGF1, IGF2, and GLUT1), hormonal (Dio2) and inflammatory mediators (TNFα, IL10, and IL6), markers of hypoxia (HIF1α) and oxidative damage (8-OHdG), antioxidant enzymes (SOD1, Cat, and GPx1), and endoplasmic reticulum stress mediators (ATF4, GRP78, and CHOP) were evaluated on day 18 of gestation. Daily treatment with Kp-10 increased free T3 and T4 levels and improved fetal weight. Both treatments reestablished the glycogen cell population in the junctional zone. Daily treatment with Kp-10 increased the gene expression levels of Plgf, Igf1, and Glut1 in the placenta of hypothyroid animals, in addition to blocking the increase in 8-OHdG and increasing protein and/or mRNA expression levels of SOD1, Cat, and GPx1. Daily treatment with Kp-10 did not alter the higher protein expression levels of VEGF, HIF1α, IL10, GRP78, and CHOP caused by hypothyroidism in the junctional zone compared to control, nor the lower expression of Dio2 caused by hypothyroidism. However, in the labyrinth zone, this treatment restored the expression of VEGF and IL10 and reduced the GRP78 and CHOP immunostaining. These findings demonstrate that daily treatment with Kp-10 improves fetal development and placental morphology in hypothyroid rats, blocks placental oxidative damage, and increases the expression of growth factors and antioxidant enzymes in the placenta.

Reducing VEGFB expression regulates the balance of glucose and lipid metabolism in mice via VEGFR1.

In recent years, studies have demonstrated that vascular endothelial growth factor B (VEGFB) can affect the metabolism of fatty acids and glucose, and it is expected to become a target for the diagnosis and treatment of metabolic diseases such as obesity and diabetes. At present, the specific mechanism that VEGFB regulates lipid and glucose metabolism balance is not completely understood. The present study used systemic VEGFB gene-knockout mice to investigate the effects of downregulation of the VEGFB gene on lipid metabolism and insulin secretion, and to explore the mechanism of the VEGFB pathway involved in the regulation of glucose and lipid metabolism. The morphological changes in the liver and pancreas of mice after VEGFB gene deletion were observed under a light microscope and a scanning electron microscope, and the effects of VEGFB gene deletion on lipid metabolism and blood glucose balance were detected by a serological technique. The detection indexes included total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol. Simultaneously, fasting blood glucose, glycosylated hemoglobin A1c (HbA1c), fasting insulin and glucagon were measured. Insulin sensitivity was assessed by using the insulin tolerance tests and glucose tolerance tests, and function of β-cell islets was evaluated by using the insulin resistance index (HOMA-IR) and pancreatic β-cell secretion index (HOMA-β). Τhe protein expression changes of vascular endothelial growth factor receptor 1 (VEGFR1) and vascular endothelial growth factor receptor 2 (VEGFR2) in mouse islets were detected by western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) after the VEGFB gene was knocked down to analyze the mechanism of VEGFB that may be involved in glucose and lipid metabolism. It was observed that after VEGFB was knocked down, mouse hepatocytes exhibited steatosis and increased secretory vesicles in islet cells. The lipid metabolism indexes such as TG, TC and LDL increased significantly; however, the levels of FBS, postprandial blood glucose and HbA1c decreased, whereas the glucose tolerance increased. Serum insulin secretion increased and HOMA-IR decreased since VEGFB was knocked down. Western blotting and RT-qPCR results revealed that the expression levels of VEGFR1 and neuropilin-1 decreased after the VEGFB gene was knocked down, while the expression levels of VEGFA and VEGFR2 increased. The absence of VEGFB may be involved in the regulation of glucose and lipid metabolism in mice by activating the VEGFA/VEGFR2 signaling pathway. VEGFB is expected to become a new target for the treatment of metabolic diseases such as obesity and diabetes. At present, the mechanism of VEGFB involved in regulating lipid metabolism and glucose metabolism is not completely clear. It was identified that downregulating VEGFB improved lipid metabolism and insulin resistance. The role of VEGFB/VEGFR1 pathway and other family members in regulating glucose and lipid metabolism was detected, which provided a theoretical and experimental basis for VEGFB to affect the regulation of glucose and lipid metabolism balance.

Gastric cancer-derived mesenchymal stem cells regulate the M2 polarization of macrophages within gastric cancer microenvironment via JAK2/STAT3 signaling pathway

Objective: To investigate the role and mechanism of tumor-derived mesenchymal stem cells in regulating the M2 polarization of macrophages within gastric cancer microenvironment. Methods: Gastric cancer tissues and the adjacent non-cancerous tissues were collected from patients underwent gastric cancer resection in the First People's Hospital of Lianyungang during 2018. In our study, THP-1-differentiated macrophages were co-cultured with gastric cancer-derived mesenchymal stem cells (GC-MSCs). Then, the M2 subtype-related gene, the markers expressed on cell surface and the cytokine profile were analyzed by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), flow cytometry and Luminex liquid chip, respectively. The key cytokines mediating the inducing effect of GC-MSCs on macrophage polarization into the M2 subtype were detected and screened by Luminex liquid chip, which were further confirmed by the neutralizing antibody test. The expressions of macrophage proteins involved in M2 polarization-related signaling pathways under the different co-culture conditions of GC-MSCs were detected by western blot. Results: In Mac+ GC-MSC-culture medium (CM) group, the expression levels of Ym-1 and Fizz-1 (1.53±0.32 and 13.22±1.05, respectively), which are markers for M2 subtype, were both significantly higher than those of Mac group (1.00±0.05 and 1.21±0.38, respectively, P<0.05). The level of iNOS in Mac+ GC-MSC-CM group (0.60±0.41) was significantly lower than that of Mac group (1.06±0.38, P=0.023). In Mac+ GC-MSC-Transwell (TW) group, the expression levels of Ym-1 and Fizz-1 (1.47±0.09 and 13.16±2.77, respectively) were both significantly higher than those of Mac group (1.00±0.05 and 1.21±0.38, respectively, P<0.05). The level of iNOS in Mac+ GC-MSC-CM group (0.56±0.03) was significantly lower than that of Mac group (1.06±0.38, P=0.026). The ratios of CD163(+) /CD204(+) cells in Mac+ GC-MSC-CM and Mac+ GC-MSC-TW groups (3.80% and 4.40%, respectively) were both remarkably higher than that of Mac group (0.60%, P<0.05). The expression levels of IL-10, IL-6, MCP-1 and VEGF in Mac+ GC-MSC-CM group were (592.60±87.52), (1 346.80±64.70), (11 256.00±29.03) and (1 463.90±66.67) pg/ml, respectively, which were significantly higher than those of Mac group [(41.03±2.59), (17.35±1.79), (5 213.30±523.71) and (267.12±12.06) pg/ml, respectively, P<0.05]. The levels of TNF-α, IP-10, RANTES and MIP-1α were (95.57±9.34), (410.48±40.68), (6 967.30±1.29) and (1 538.70±283.04) pg/ml, which were significantly lower than those of Mac group [(138.01±24.31, (1 298.60±310.50), (14 631.00±4.21) and (6 633.20±1.47) pg/ml, respectively, P<0.05]. The levels of IL-6 and IL-8 in GC-MSCs [(11 185.02±2.82) and (12 718.03±370.17) pg/ml, respectively] were both strikingly higher than those of MSCs from adjacent non-cancerous gastric cancer tissues [(270.71±59.38) and (106.04±32.84) pg/ml, repectively, P<0.05]. The ratios of CD86(+) cells in Mac+ IL-6-blocked-GC-MSC-CM and Mac+ IL-8-blocked-GC-MSC-CM groups (28.80% and 31.40%, respectively) were both higher than that of Mac+ GC-MSC-CM group (24.70%). Compared to Mac+ GC-MSC-CM group (13.70%), the ratios of CD204(+) cells in Mac+ IL-6-blocked-GC-MSC-CM and Mac+ IL-8-blocked-GC-MSC-CM groups (9.90% and 8.70%, separately) were reduced. The expression levels of p-JAK2 and p-STAT3, which are proteins of macrophage M2 polarization-related signaling pathway, in Mac+ GC-MSC-CM group (0.86±0.01 and 1.08±0.01, respectively) were significantly higher than those of Mac group (0.50±0.01 and 0.82±0.01, respectively, P<0.05). The expression levels of p-JAK2 in Mac+ IL-6-blocked-GC-MSC-CM group (0.47±0.02) were significantly lower those that of Mac+ GC-MSC-CM group (0.86±0.01, P<0.05). The expression levels of p-JAK2 and p-STAT3 in Mac+ IL-8-blocked-GC-MSC-CM group (0.50±0.01 and 0.85±0.01, respectively) were both significantly lower than those of Mac+ GC-MSC-CM group (0.86±0.01 and 1.08±0.01, P<0.05). The expression levels of p-JAK2 and p-STAT3 in Mac+ IL-6/IL-8-blocked-GC-MSC-CM group (0.37±0.01 and 0.65±0.01, respectively) were both significantly lower than those of Mac+ GC-MSC-CM group (0.86±0.01 and 1.08±0.01, P<0.05). Conclusion: GC-MSCs promote the activation of JAK2/STAT3 signaling pathway in macrophages via high secretions of IL-6 and IL-8, which subsequently induce the macrophage polarization into a pro-tumor M2 subtype within gastric cancer microenvironment.

Adenosine-Prefabricated Adipose Tissue Improves Fat Graft Survival by Promoting VEGF-Dependent Angiogenesis.

Angiogenesis plays an important role in determining the fat graft survival. However, clinical preconditioning techniques that target angiogenesis during fat grafting have not been established so far. Adenosine has emerged as a regulator of angiogenesis under hypoxic conditions; therefore, the aim of this study was to investigate the effects and underlying mechanisms of adenosine prefabrication on fat graft survival. In the first animal study, a total of 32 mice were transplanted with fat prefabricated with vehicle (Control, N = 16) or adenosine (Adenosine, N = 16). In the second animal study, 24 mice were divided into three groups based on the type of fat graft: Control (N = 8), Adenosine (N = 8), and Axitinib (cotreatment of adenosine with axitinib, N = 8). At 1- and 4-weeks post-transplantation, grafts were evaluated by histopathological and biochemical assessment. Adenosine-induced vascular endothelial growth factor (VEGF) production and angiogenesis were determined using cell cultures. The retention volumes of fat grafts in the adenosine group were significantly increased until 4weeks. Fat grafts from the adenosine group exhibited greater structural integrity, reduced fibrosis, and increased blood vessels. The expression levels of angiogenesis-related genes, Vegfa, Vegfr1, Vegfr2, and Vwf, were elevated in the adenosine group. Furthermore, adenosine upregulated VEGF production in preadipocytes, thereby enhancing the migration of endothelial cells. Treatment with the axitinib, VEGF receptor inhibitor, abrogated the adenosine-induced angiogenesis in the fat grafts. Adenosine prefabrication in fat improved the graft survival by enhancing angiogenesis through the VEGF/VEGFR axis in the preadipocytes and endothelial cells. Therefore, this method may be used as a novel strategy to increase the retention rate in fat grafts.

Epigenetic restoration and activation of ERβ: an inspiring approach for treatment of triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer. TNBC lacks targeted therapy receptors, rendering endocrine and HER2-targeted therapies ineffective. TNBC is typically treated with cytotoxic chemotherapy followed by surgery. Targeting epigenetic modifications could potentially be a new effective TNBC target therapy. The aim of this study is to examine the effects of epigenetic drugs, decitabine as DNA methyltransferase inhibitor (DNMTI) and vorinostat as histone deacetylase inhibitor (HDACI), and the ERβ agonist DPN on ERα and ERβ re-expressions in the MDA-MB-231 cells as a model of TNBC.MethodsUsing MTT assay, the IC50 of decitabine, vorinostat, and DPN on MDA-MB-231 cells were determined. The effects of all drugs alone or in combinations on MDA-MB-231 cells were evaluated. qRT-PCR was used to determine ERα & ERβ gene expression. Caspase-3 activity and the protein expression levels of VEGF, Cyclin D1, and IGF-1 were assessed.ResultsBoth ERα and ERβ mRNA were re-expressed in different high levels in all treated groups, especially in the triple therapy group compared with control. Significantly, the triple drugs therapy showed the lowest levels of VEGF, Cyclin D1, and IGF-1 and the highest level of Caspase-3 activity, indicating a possible antitumor effect of ERβ activation through decreasing proliferation and angiogenesis and increasing apoptosis in MDA-MB-231 cells.ConclusionsThe antiproliferative effect of ERβ could be retained when co-expressed with Erα using a powerful epigenetic combination of Decitabine and vorinostat with DPN.

Newly Synthesized Benzimidazoles Inhibit Vascular Endothelial Growth Factor and Matrix Metalloproteinase-2 and -9 Levels in Prostate Cancer Cells.

Investigating the effects of newly synthesized agents on various molecular mechanisms to understand their mechanism of action is an important step of pre-clinical screening. Benzimidazoles are composed of a unique fused benzene and imidazole ring and have attracted great attention due to their broad bioactivities, including antitumor. In the current study, we reported the synthesis of novel benzimidazole derivatives and investigated the possible cytotoxic and anti-angiogenic effects on human prostate cancer and umbilical vein endothelial cells (HUVECs). MTT assay was used to assess cell viability. A scratch assay was conducted to monitor the migration of cells. mRNA expression levels of VEGF, MMP-2, and MMP-9 were evaluated using qPCR. Changes in protein levels were evaluated by western blotting. Compound G1, having a chlorine moiety, showed a potent cytotoxic activity on both prostate cancer cells and HUVECs, and inhibited cell migration via decreasing the mRNA and protein levels of key angiogenesis-related molecules such as VEGF, MMP-2, and MMP-9. These results suggest that newly synthesized G1 may be a novel anti-angiogenic agent for prostate cancer treatment.

Identification of potential biomarkers and small‑molecule compounds related to intracerebral hemorrhage with bioinformatics analysis.

This study aimed to further explore the underlying molecular mechanism of intracerebral hemorrhage (ICH), gene expression profile GSE24265, containing perihematomal tissues, contralateral grey and white matters were retrieved and analyzed. The data was hierarchically clustered and the differentially expressed genes (DEGs) were screened. Functional analysis and protein interaction analysis of DEG hubs were performed, and the miRNA‑transcription factor (TF)‑target network was built. In addition, the candidate small-molecule compounds that might reverse the expression of an ICH‑linked gene were identified by CMap. This method revealed a total of 408 DEGs. Five modules including chemokinerelated, antigen immune-related, pathogen infection, cell reaction, and positive regulation of tyrosine phosphorylation and MAPK cascade were identified. The expression levels of CCL5, CXCL8, ICAM1, IL-1B, IL-6, VCAM1, and VEGFA were correlated with ICH among the top 10 hub genes obtained in the protein-protein interaction (PPI) network. A total of 237 miRNA‑TF‑target regulatory relationships were obtained, including 6 TFs, 11 miRNAs and 105 target genes. Finally, the CMap database identified Prestwick-1083, xamoterol, ifosfamide, methyldopate, nifurtimox, propranolol, and methoxamine as potential therapeutic agents for ICH while doxorubicin, menadione and azacitidine may increase its pathogenicity. Furthermore, CCL5, CXCL8 and VEGFA may be novel candidate susceptibility genes for ICH. Some small-molecule drugs, including xamoterol may be used for the treatment of ICH.

Extract of Marsdenia tenacissima (Roxb.) Moon [Apocynaceae] Suppresses Hepatocellular Carcinoma by Inhibiting Angiogenesis.

The extract of Marsdenia tenacissima (Roxb.) Moon [Apocynaceae] (MTE) has shown a significant anti-cancer effect on hepatocellular carcinoma (HCC), but its mechanism remains unclear. In this study, we used transcriptomics methods to investigate the underlying mechanism of MTE against HCC. Both MHCC97H and HepG2 cell lines were treated with MTE. The cell viability and migration were measured using the cell counting kit-8 assay and transwell assay. RNA-sequencing was used to identify differentially expressed genes (DEGs) between HepG2 cells treated with and without MTE. The expression levels of selected DEGs—vascular endothelial growth factor-A (VEGFA), platelet-derived growth factor receptor-β (PDGFRB), and von Willebrand factor (VWF)—were verified by RT-PCR and Western blot. The effect of conditioned medium from HCC cells with MTE treatment (CM-MTE) on blood vessels was observed by tube formation assay of HUVECs and chick chorioallantoic membrane (CAM) assay. A mouse model of HCC patient-derived tumor xenograft (PDX) was established and treated with MTE. The effect of MTE on the growth and angiogenesis of HCC-PDX was analyzed. The results demonstrated that MTE inhibited the viability and migration of HCC cells. RNA-seq showed that MTE treatment downregulated multiple genes associated with metabolism and angiogenesis. The expression levels of VEGFA, VWF, PDGFB, and PDGFRB in HCC cells were significantly suppressed by MTE. Meanwhile, MTE effectively inhibited the tube-forming capability of HUVECs and the angiogenesis of chick CAM. In vivo experiments revealed that the extract reduced tumor volume, inhibited the proliferation of HCC cells, and expanded the necrotic area of the tumor. Immunohistochemical results showed that the expression levels of CD31, PDGFB, VEGF, VWF, and PDGFRB in the HCC-PDX tumor tissues were all downregulated by MTE in a dose-dependent manner. Taken together, MTE could inhibit angiogenesis by repressing the expression of VEGF, VWF, PDGF, and PDGFRB in HCC cells, a mechanism that may enable MTE to counter HCC development.

Mechanism of ischemic brain injury repair by endothelial progenitor cell‑derived exosomes.

Ischemic stroke is a refractory disease that seriously endangers human health and life. The main treatment aim of stroke is to alleviate brain injury. The present study aimed to investigate the effects and mechanisms of endothelial progenitor cell (EPC)‑derived exosomes in repairing ischemic brain injury. Sprague‑Dawley rat models of cerebral ischemia‑reperfusion (IR) injury were established by middle cerebral artery occlusion. The IR model rats were then treated with PBS, EPC or exosomes; untreated and Sham rats were used as control. EPCs were obtained from tibias and femurs, and exosomes were isolated from the EPCs and characterized. To measure brain injury, 2,3,5‑triphenyltetrazolium chloride staining was used to measure the infarct area, neurological deficit was scored, hematoxylin and eosin staining was used to examine pathological changes and TUNEL staining was used to quantify apoptosis. Immunofluorescence staining and reverse transcription‑quantitative PCR were used to determine CD31 and VEGF protein and mRNA expressions, respectively, and western blot analysis was used out to measure the protein expression levels of Wnt3a, GSK‑3β and phosphorylated (p)‑GSK‑3β. Compared with rats in the Control and Sham groups, in IR model rats the nerve fibers were slightly necrotic and swollen and the number of nerve cells was reduced. Following EPC treatment, the brain tissue exhibited mild liquefaction and degeneration in the small focus area with mild edema in the stroma. The numbers of nerve cells decreased, and the distribution of nerve cells was not very uniform; proliferation of glial cells was observed. Following treatment with exosomes, the distribution of nerve cells was more uniform with less degeneration and necrosis; the proliferation of glial cells was remarkable. Compared with the Control group, the infarct size, neurological defect score, percentage of apoptotic cells, expression of CD31, VEGF, Wnt3a, and p‑GSK‑3β were significantly higher in the IR model (P<0.05). After EPC and exosome treatments, the infarct size, neurological defect score, percentage of apoptotic cells, expression of Wnt3a, and p‑GSK‑3β were significantly reduced (P<0.05), whereas the mRNA and protein expression levels of CD31 and VEGF were significantly increased (P<0.05). Results from the present study demonstrated that EPC‑derived exosomes may alleviate ischemic injury by inhibiting apoptosis and promoting angiogenesis. These findings suggested that exosomes may have a protective role for nerve cells and may be a potentially effective option for treating stroke. However, human clinical studies are needed to validate these findings from animals.

Correlation between the Expression of VEGF and Ki67 and Lymph Node Metastasis in Non-small-Cell Lung Cancer: A Systematic Review and Meta-Analysis.

Background Lymph node metastasis is the most common and important way of metastasis in NSCLC and is also the most important factor affecting lung cancer stage and prognosis. It is very important to analyze the relationship between the expression of vascular endothelial growth factor (VEGF) and Ki67 and lymph node metastasis (LNM) in non-small-cell lung cancer (NSCLC). Methods We searched the PubMed, EMBASE, and Cochrane Library and conducted meta-analyses using the R meta-package. Relative risk (RR) with a 95% confidence interval (95% CI) was the main indicator. Results Totally, 18 studies were considered eligible, with 4521 patients, including 1518 LNM-positive patients and 3033 LNM-negative patients. The incidence of LNM in Ki67-negative patients was lower than that in Ki67-positive patients (RR = 0.66, 95% CI: 0.44, 0.98). The incidence of LNM in VEGF-A-negative patients was lower than that in VEGF-A-positive patients (RR = 0.64, 95% CI: 0.49, 0.83). The incidence of LNM in VEGF-C negative patients was lower than that in VEGF-C positive patients (RR = 0.68, 95% CI: 0.53, 0.88). The incidence of LNM in VEGF-D negative and positive patients were of no significant differences (RR = 0.84, 95% CI: 0.61, 1.14). Conclusion The high expression of Ki67, VEGF-A, and VEGF-C significantly increases the risk of lymph node metastasis in NSCLC, while the VEGF-D expression has no correlation with lymph node metastasis. The expression levels of Ki67, VEGF-A, and VEGF-C show a good potential for lymph node metastasis prediction.