Expression Levels Of Vascular Endothelial Growth Factor A Research Articles

LncRNA XIST promotes neovascularization in diabetic retinopathy by regulating miR-101-3p/VEGFA.

This study sought to investigate the regulation of long noncoding RNA (lncRNA) XIST on the microRNA (miR)-101-3p/vascular endothelial growth factor A (VEGFA) axis in neovascularization in diabetic retinopathy (DR). Serum of patients with DR was extracted for the analysis of XIST, miR-101-3p, and VEGFA expression levels. High glucose (HG)-insulted HRMECs and DR model rats were treated with lentiviral vectors. MTT, transwell, and tube formation assays were performed to evaluate cell viability, migration, and angiogenesis, and ELISA was conducted to detect the levels of inflammatory cytokines. Dual-luciferase reporter, RIP, and RNA pull-down experiments were used to validate the relationships among XIST, miR-101-3p, and VEGFA. XIST and VEGFA were upregulated and miR-101-3p was downregulated in serum from patients with DR. XIST knockdown inhibited proliferation, migration, vessel tube formation, and inflammatory responsein HG-treated HRMECs, whereas the above effects were nullified by miR-101-3p inhibition or VEGFA overexpression. miR-101-3p could bind to XIST and VEGFA. XIST promoted DR development in rats by regulating the miR-101-3p/VEGFA axis. LncRNA XIST promotes VEGFA expression by downregulating miR-101-3p, thereby stimulating angiogenesis and inflammatory response in DR.

Blocking the ATR-SerRS-VEGFA pathway targets angiogenesis for UV-induced cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the second most prevalent form of skin cancer, with an escalating incidence rate and a notable potential (up to 5%) for metastasis. Ultraviolet radiation (UVA and UVB) exposure is the primary risk factor for cSCC carcinogenesis, with literature suggesting ultraviolet radiation (UVR) promotes vascular endothelial growth factor A (VEGFA)expression. This study aims to investigate UVR-induced upregulation of VEGFA and explore combination therapeutic strategies. The skin squamous cell carcinoma cell line A431 was exposed to specific durations of ultraviolet radiation. The effect of emodin on ATR/SerRS/VEGFA pathway was observed. The cell masses were also transplanted subcutaneously into mice (n = 8). ATR inhibitor combined with emodin was used to observe the growth and angiogenesis of the xenografts. The results showed that UV treatment significantly enhanced the phosphorylation of SerRS and the expression level of VEGFA in A431 cells (p < 0.05). Treatment with emodin significantly inhibited this expression (p < 0.05), and the combination of emodin and ATR inhibitor further enhanced the inhibitory effect (p < 0.05). This phenomenon was further confirmed in the xenograft model, which showed that the combination of ATR inhibitor and emodin significantly inhibited the expression of VEGFA to inhibit angiogenesis (p < 0.05), thus showing an inhibitory effect on cSCC. This study innovatively reveals the molecular mechanism of UV-induced angiogenesis in cSCC and confirms SerRS as a novel target to inhibit cSCC angiogenesis and progression in vitro and in vivo studies.

Downregulation of lncRNA MIR17HG reduced tumorigenicity and Treg-mediated immune escape of non-small-cell lung cancer cells through targeting the miR-17-5p/RUNX3 axis.

Long noncoding RNA MIR17HG was involved with the progression of non-small-cell lung cancer (NSCLC), but specific mechanisms of MIR17HG-mediated immune escape of NSCLC cells were still unknown. The present study investigated the function of MIR17HG on regulatory T cell (Treg)-mediated immune escape and the underlying mechanisms in NSCLC. Expression of MIR17HG and miR-17-5p in NSCLC tissue samples were detected using quantitative real-time PCR (qRT-PCR). A549 and H1299 cells were transfected with sh-MIR17HG, miR-17-5p inhibitor, or sh-MIR17HG + miR-17-5p inhibitor, followed by cocultured with Tregs. Cell proliferation was measured using 5-ethynyl-20-deoxyuridine (Edu) staining assay and cell counting kit-8 (CCK-8) assay. Flow cytometry was used for determining positive numbers of FOXP3+CD4+/CD25+/CD8+ Tregs. Through subcutaneous injection with transfected A549 cells, a xenograft nude mouse model was established. Weights and volumes of xenograft tumors were evaluated. Additionally, the expressions of immune-related factors includingtransforming growth factor beta (TGF-β), vascular endothelial growth factor A (VEGF-A), interleukin-10 (IL-10), IL-4, and interferon-gamma (IFN-γ) in cultured cells, were evaluated by enzyme-linked immunosorbent assay and western blot analysis. Then, miR-17-5p was decreased and MIR17HG was enhanced in both NSCLC tissues and cell lines. MIR17HG knockdown significantly suppressed cell proliferation, tumorigenicity, and immune capacity of Tregs in A549 and H1299 cells, whereas sh-MIR17HG significantly reduced expression levels of VEGF-A, TGF-β, IL-4, and IL-10 but promoted the IFN-γ level in vitro and in vivo. Moreover, downregulation of miR-17-5p significantly reversed the effects of sh-MIR17HG. Additionally, we identified that runt- related transcription factor 3 (RUNX3) was a target of miR-17-5p, and sh-MIR17HG and miR-17-5p mimics downregulated RUNX3 expression. In conclusion, downregulation of MIR17HG suppresses tumorigenicity and Treg-mediated immune escape in NSCLC through downregulating the miR-17-5p/RUNX3 axis, indicating that this axis contains potential biomarkers for NSCLC.

Mirna-383-5p Functions as an Anti-oncogene in Glioma through the Akt/mTOR Signaling Pathway by Targeting VEGFA.

Previously, we have screened 59 differentially expressed miRNAs and 419 mRNAs in the glioblastoma samples that have been compared to the peritumoral tissues using bioinformatics analyses, which included miRNA-383-5p and vascular endothelial growth factor A (VEGFA). miRNA-383-5p and VEGFA/Akt/mTOR pathway play important regulatory roles in the malignant biological behavior of glioma. Glioma cell lines, U87 and U251, were collected for in vitro experiments. miRNA-383-5p and VEGFA expression levels were detected with qRT-PCR and WB. The protein expressions of Akt, mTOR, and VEGFR in U87 and U251 were detected with WB. The effect of miRNA-383-5p on the VEGFA activity was verified by dual-luciferase reporter assay. CCK-8 was used to examine the U87 and U251 cells' inhibition. Flow cytometry and transwell assays were used to detect cell apoptosis and invasion, respectively. Our research data indicated overexpression of miRNA-383-5p to suppress malignant biological behavior, which was manifested as promoting the apoptosis of U87 and U251 cells and inhibiting invasion, proliferation, and metastasis. VEGFA is one of the downstream target genes of miRNA-383- 5p. miRNA-383-5p could inhibit the expression of VEGFA and Akt/mTOR signaling pathways. Overexpression of VEGFA can reverse the inhibitory effect of miRNA-383-5p and reactivate the Akt/mTOR signaling pathway. Our results indicate that miRNA-383-5p functions as an anti-oncogene by inhibiting the VEGFA/Akt/mTOR signaling pathway in glioma cells. These data provide potential therapeutic targets for glioblastoma.

Camptothecin enhances the anti-tumor effect of low-dose apatinib combined with PD-1 inhibitor on hepatocellular carcinoma

Apatinib has been shown to apply to a variety of solid tumors, including advanced hepatocellular carcinoma. Preclinical and preliminary clinical results confirmed the synergistic antitumor effects of apatinib in combination with anti-programmed death-1 (PD-1) inhibitors. In this study, we investigated camptothecin (CPT) enhances the anti-tumor effect of low-dose apatinib combined with PD-1 inhibitor on hepatocellular carcinoma. CPT combined with a PD-1 inhibitor enhances the anti-tumor effects of low-dose apatinib in hepatocellular carcinoma which was evaluated in making use of the H22 mouse model (n = 32), which was divided into four groups. Immunohistochemical staining and western blotting were used to detect nuclear factor erythroid 2-related factor 2 (Nrf2) as well as sequestosome 1 (p62), vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor receptor 2 (VEGFR2), PD-1, and programmed cell death ligand 1 (PD-L1). The results showed that the average size of the tumor of the combination group (Group D) was significantly less than that of the apatinib + PD-1 inhibitor group (Group C). The expression levels of Nrf2, p62, VEGFA, VEGFR2, PD-1, and PD-L1 in the apatinib + PD-1 inhibitor group(Group C) were lower than those in the control group (Group A) (P < 0.05). The expression levels of these genes in the apatinib + PD-1 inhibitor group (Group C) were significantly lower in the combination group (Group D) (P < 0.05). There was no obvious difference in body weight and liver and kidney functions between the four groups of mice. In conclusion, CPT improves the anti-tumor effect of low-dose apatinib combined with PD-1 inhibitor on hepatocellular carcinoma

Decreased FGF23 inhibits placental angiogenesis via the ERK1/2-EGR-1 signaling pathway in preeclampsia

PurposeThis study aimed to investigate the expression of fibroblast growth factor 23 (FGF23) in pregnant women with preeclampsia and elucidate its role in promoting placental angiogenesis through the ERK1/2-EGR-1 signaling pathway. MethodsSerum FGF23 levels were measured by ELISA in healthy pregnant women and patients with preeclampsia during the first, second, and third trimesters of pregnancy. Wound healing, Transwell, and tube formation assays were performed to investigate the effects of FGF23 on cell migration, invasion and tube formation. The expression of vascular endothelial growth factor A (VEGF-A) and its upstream signaling molecules, p-ERK, and EGR-1, in placental tissues was detected by RT-qPCR and western blotting. Additionally, the effect of FGF23 on VEGF-A, p-ERK, and EGR-1 expression was further explored in vitro. ResultsSerum FGF23 levels increased with gestational age. During the third trimester, the control group exhibited a more pronounced increase in FGF23 levels than the preeclampsia group. Administering exogenous FGF23 promoted trophoblast cell migration, invasion and enhanced tube formation in vascular endothelial cells. The expression levels of VEGF-A, p-ERK, and EGR-1 in the placental tissues were significantly lower in the preeclampsia group than in the control group. In vitro experiments confirmed that FGF23 up-regulated VEGF-A expression through the p-ERK/EGR-1 signaling pathway. ConclusionThe serum level of FGF23 decreased in pregnant women with preeclampsia, inhibiting the ERK1/2-EGR-1 pathway and resulting in decreased expression of VEGF-A, thereby inhibiting placental angiogenesis. This could be a potential mechanism involved in the progression of preeclampsia.

Tetramethylpyrazine regulates angiogenesis of endothelial cells in cerebral ischemic stroke injury via SIRT1/VEGFA signaling pathway

This study aims to investigate whether tetramethylpyrazine(TMP) can stimulate angiogenesis in cerebral microvascular endothelial cells and alleviate cerebral ischemic stroke(CIS) and to explore the underlying mechanisms. In the animal study, adult Sprague-Dawley rats(n=15) were assigned into sham surgery(sham), middle cerebral artery occlusion/reperfusion(MCAO/R), and MCAO/R+TMP(intraperitoneal injection of 20 mg·kg~(-1)) groups. The neurological function was evaluated by the Z-Longa method. The cerebral infarction volume was detected by TTC staining. Enzyme-linked immunosorbent assay(ELISA) was employed to detect the expression of vascular endothelial growth factor(VEGF), angiopoietin(Ang), and platelet-derived growth factor(PDGF). Immunofluorescence staining was employed to detect Ki67 and the expression of vascular endothelial growth factor A(VEGFA) and slient information regulator 1(SIRT1). Western blot was employed to determine the expression levels of VEGFA, SIRT1, angiopoietin-2(Ang-2), and platelet-derived growth factor B(PDGFB). In the cell study, mouse brain-derived endothelial cells(Bend.3) were cultured, and the optimal concentration of TMP was determined. Then, VEGF, Ang, and PDGF were detected by ELISA after the addition of cabozantinib. Western blot was employed to measure the expression of VEGFA, Ang-2, and PDGFB. Immunofluorescence staining was used to detect CD31, CD34, and Ki67, and the proliferation, migration, and tube formation ability of Bend.3 cells were observed in vitro. Western blot and immunofluorescence staining were performed to measure the expression of SIRT1 and VEGFA after addition of the SIRT1-specific inhibitor selisistat(EX-527). The results showed that compared with the sham group, the MCAO/R group had severe neurological function damage, increased infarction volume, up-regulated expression of VEGF, VEGFA, Ang, Ang-2, PDGF, and PDGFB, and down-regulated expression of Ki67 and SIRT1(P&lt;0.01). Compared with the MCAO/R group, the MCAO/R+TMP group presented alleviated neurological function damage, reduced infarction volume, and activated expression of VEGF, VEGFA, Ang, Ang-2, PDGF, PDGFB, Ki67, and SIRT1(P&lt;0.01). The cell experiments showed that compared with the normal group, Bend.3 cells were activated by oxygen glucose deprivation/reoxygenation(OGD/R) treatment(P&lt;0.05, P&lt;0.01). Compared with the OGD/R group, the OGD/R+TMP group upregulated the expression levels of VEGF, VEGFA, Ang, Ang-2, PDGF, PDGFB, SIRT1, Ki67, CD31, and CD34, enhanced the angiogenic ability of Bend.3 cells without being inhibited by BMS or EX-527(P&lt;0.05, P&lt;0.01, P&lt;0.001). The results suggest that TMP can activate the SIRT1/VEGFA signaling pathway to stimulate angiogenesis and alleviate CIS injury.

In Vitro Investigation of Gelatin/Polycaprolactone Nanofibers in Modulating Human Gingival Mesenchymal Stromal Cells.

The aesthetic constancy and functional stability of periodontium largely depend on the presence of healthy mucogingival tissue. Soft tissue management is crucial to the success of periodontal surgery. Recently, synthetic substitute materials have been proposed to be used for soft tissue augmentation, but the tissue compatibility of these materials needs to be further investigated. This study aims to assess the in vitro responses of human gingival mesenchymal stromal cells (hG-MSCs) cultured on a Gelatin/Polycaprolactone prototype (GPP) and volume-stable collagen matrix (VSCM). hG-MSCs were cultured onto the GPP, VSCM, or plastic for 3, 7, and 14 days. The proliferation and/or viability were measured by cell counting kit-8 assay and resazurin-based toxicity assay. Cell morphology and adhesion were evaluated by microscopy. The gene expression of collagen type I, alpha1 (COL1A1), α-smooth muscle actin (α-SMA), fibroblast growth factor (FGF-2), vascular endothelial growth factor A (VEGF-A), transforming growth factor beta-1 (TGF-β1), focal adhesion kinase (FAK), integrin beta-1 (ITG-β1), and interleukin 8 (IL-8) was investigated by RT-qPCR. The levels of VEGF-A, TGF-β1, and IL-8 proteins in conditioned media were tested by ELISA. GPP improved both cell proliferation and viability compared to VSCM. The cells grown on GPP exhibited a distinct morphology and attachment performance. COL1A1, α-SMA, VEGF-A, FGF-2, and FAK were positively modulated in hG-MSCs on GPP at different investigation times. GPP increased the gene expression of TGF-β1 but had no effect on protein production. The level of ITG-β1 had no significant changes in cells seeded on GPP at 7 days. At 3 days, notable differences in VEGF-A, TGF-β1, and α-SMA expression levels were observed between cells seeded on GPP and those on VSCM. Meanwhile, GPP showed higher COL1A1 expression compared to VSCM after 14 days, whereas VSCM demonstrated a more significant upregulation in the production of IL-8. Taken together, our data suggest that GPP electrospun nanofibers have great potential as substitutes for soft tissue regeneration in successful periodontal surgery.

Expression of interleukin-37, vascular endothelial growth factor A, and transforming growth factor-β1 and their correlation with T cells in children with primary immune thrombocytopenia

To investigate the expression of interleukin-37 (IL-37), vascular endothelial growth factor A (VEGFA), and transforming growth factor-β1 (TGF-β1) in children with primary immune thrombocytopenia (ITP) and their correlation with T cells. A retrospective analysis was conducted on 45 children with ITP (ITP group) who were admitted to Handan Central Hospital from January 2020 to April 2022, and 30 healthy children who underwent physical examination during the same period were included as the healthy control group. The mRNA expression levels of IL-37, VEGFA, and TGF-β1 and the levels of regulatory T cells (Treg) and helper T cells 17 (Th17) were measured before and after treatment, and the correlation between the mRNA expression levels of IL-37, VEGFA, and TGF-β1 and the levels of Treg, Th17, and Treg/Th17 ratio were analyzed. Compared with the healthy control group, the ITP group had a significantly higher mRNA expression level of IL-37 and a significantly higher level of Th17 before and after treatment, as well as significantly lower mRNA expression levels of VEGFA and TGF-β1 and significantly lower levels of Treg and Treg/Th17 ratio (P<0.05). After treatment, the ITP group had significant reductions in the mRNA expression level of IL-37 and the level of Th17 and significant increases in the mRNA expression levels of VEGFA and TGF-β1 and the levels of Treg and Treg/Th17 ratio (P<0.05). Correlation analysis showed that in the ITP group, the mRNA expression levels of IL-37 and TGF-β1 were negatively correlated with the levels of Treg and Treg/Th17 ratio (P<0.05) and were positively correlated with the level of Th17 (P<0.05) before and after treatment; the mRNA expression level of VEGFA was positively correlated with the levels of Treg and Treg/Th17 ratio (P<0.05) and was negatively correlated with the Th17 level (P<0.05) before and after treatment. Abnormal expression levels of IL-37, VEGFA, and TGF-β1 may be observed in children with ITP, which is significantly associated with the imbalance of Treg/Th17 ratio. It is speculated that the cytokines such as IL-37, VEGFA, and TGF-β1 may be involved in the development and progression of ITP or may become important potential targets for the treatment of children with ITP. Citation:Chinese Journal of Contemporary Pediatrics, 2023, 25(11): 1131-1136.

Catalpol Prevents Glomerular Angiogenesis Induced by Advanced Glycation End Products via Inhibiting Galectin-3.

The main characteristics of diabetic nephropathy (DN) at the early stage are abnormal angiogenesis of glomerular endothelial cells (GECs) and macrophage infiltration. Galectin-3 plays a pivotal role in the pathogenesis of DN via binding with its ligand, advanced glycation end products (AGEs). Catalpol, an iridoid glucoside extracted from Rehmannia glutinosa, has been found to ameliorate vascular inflammation, reduce endothelial permeability, and protect against endothelial damage in diabetic milieu. However, little is known about whether catalpol could exert an anti-angiogenesis and anti-inflammation effect induced by AGEs. Mouse GECs (mGECs) and RAW 264.7 macrophages were treated with different concentrations of AGEs (0, 50, 100, 200 and 400 µg/mL) for different time (0, 6, 12, 24 and 48 h) to determine the optimal concentration of AGEs and treatment time. Cells were treated with catalpol (10 µmol/L), GB1107 (1 µmol/L, galectin-3 inhibitor), PX-478 (50 µmol/L, HIF-1α inhibitor), adenovirus-green fluorescent protein (Ad-GFP) [3×107 plaque-forming unit (PFU)/mL] or Ad-galectin-3-GFP (2×108 PFU/mL), which was followed by incubation with 50 µg/mL AGEs. The levels of galectin-3, vascular endothelial growth factor A (VEGFA) and pro-angiogenic factors angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), tunica interna endothelial cell kinase-2 (Tie-2) were detected by enzymelinked immunosorbent assay (ELISA). Cell counting kit-8 (CCK-8) assay was used to evaluate the proliferation of these cells. The expression levels of galectin-3, vascular endothelial growth factor receptor 1 (VEGFR1), VEGFR2, and hypoxia-inducible factor-1α (HIF-1α) in mGECs and those of galectin-3 and HIF-1α in RAW 264.7 macrophages were detected by Western blotting and immunofluorescence (IF) staining. The rat DN model was established. Catalpol (100 mg/kg) or GB1107 (10 mg/kg) was administered intragastrically once a day for 12 weeks. Ad-galectin-3-GFP (6×107 PFU/mL, 0.5 mL) or Ad-GFP (6×106 PFU/mL, 0.5 mL) was injected into the tail vein of rats 48 h before the sacrifice of the animals. The expression of galectin-3, VEGFR1, VEGFR2, and HIF-1α in renal cortices was analyzed by Western blotting. The expression of galectin-3, F4/80 (a macrophage biomarker), and CD34 (an endothelium biomarker) in renal cortices was detected by IF staining, and collagen accumulation by Masson staining. The expression levels of galectin-3 and VEGFA were significantly higher in mGECs and RAW 264.7 macrophages treated with 50 µg/mL AGEs for 48 h than those in untreated cells. Catalpol and GB1107 could block the AGEs-induced proliferation of mGECs and RAW 264.7 macrophages. Over-expression of galectin-3 was found to reduce the inhibitory effect of catalpol on the proliferation of cells. Catalpol could significantly decrease the levels of Ang-1, Ang-2 and Tie-2 released by AGEs-treated mGECs, which could be reversed by over-expression of galectin-3. Catalpol could significantly inhibit AGEs-induced expression of galectin-3, HIF-1α, VEGFR1, and VEGFR2 in mGECs. The inhibitory effect of catalpol on galectin-3 in AGEs-treated mGECs was impaired by PX-478. Moreover, catalpol attenuated the AGEs-activated HIF-1α/galectin-3 pathway in RAW 264.7 macrophages, which was weakened by PX-478. Additionally, catalpol significantly inhibited the expression of galectin-3, macrophage infiltration, collagen accumulation, and angiogenesis in the kidney of diabetic rats. Over-expression of galectin-3 could antagonize these inhibitory effects of catalpol. Catalpol prevented the angiogenesis of mGECs and macrophage proliferation via inhibiting galectin-3. It could prevent the progression of diabetes-induced renal damage.

Effect of diosgenin on mTOR/FASN/HIF-1α/VEGFA expression in rats with non-alcoholic fatty liver disease

The present study aimed to investigate the effect of diosgenin on mammalian target of rapamycin(mTOR), fatty acid synthase(FASN), hypoxia inducible factor-1α(HIF-1α), and vascular endothelial growth factor A(VEGFA) expression in liver tissues of rats with non-alcoholic fatty liver disease(NAFLD) and explore the mechanism of diosgenin on lipogenesis and inflammation in NAFLD. Forty male SD rats were divided into a normal group(n=8) fed on the normal diet and an experimental group(n=32) fed on the high-fat diet(HFD) for the induction of the NAFLD model. After modeling, the rats in the experimental group were randomly divided into an HFD group, a low-dose diosgenin group(150 mg·kg~(-1)·d~(-1)), a high-dose diosgenin group(300 mg·kg~(-1)·d~(-1)), and a simvastatin group(4 mg·kg~(-1)·d~(-1)), with eight rats in each group. The drugs were continuously given by gavage for eight weeks. The levels of triglyceride(TG), total cholesterol(TC), low-density lipoprotein cholesterol(LDL-C), alanine transaminase(ALT), and aspartate transaminase(AST) in the serum were detected by the biochemical method. The content of TG and TC in the liver was detected by the enzyme method. Enzyme-linked immunosorbent assay(ELISA) was used to measure interleukin 1β(IL-1β) and tumor necrosis factor α(TNF-α) in the serum. Lipid accumulation in the liver was detected by oil red O staining. Pathological changes of liver tissues were detected by hematoxylin-eosin(HE) staining. The mRNA and protein expression levels of mTOR, FASN, HIF-1α, and VEGFA in the liver of rats were detected by real-time fluorescence-based quantitative polymerase chain reaction(PCR) and Western blot, respectively. Compared with the normal group, the HFD group showed elevated body weight and levels of TG, TC, LDL-C, ALT, AST, IL-1β, and TNF-α(P&lt;0.01), increased lipid accumulation in the liver(P&lt;0.01), obvious liver steatosis, up-regulated mRNA expression levels of mTOR, FASN, HIF-1α, and VEGFA(P&lt;0.01), and increased protein expression levels of p-mTOR, FASN, HIF-1α, and VEGFA(P&lt;0.01). Compared with the HFD group, the groups with drug treatment showed lowered body weight and levels of TG, TC, LDL-C, ALT, AST, IL-1β, and TNF-α(P&lt;0.05, P&lt;0.01), reduced lipid accumulation in the liver(P&lt;0.01), improved liver steatosis, decreased mRNA expression levels of mTOR, FASN, HIF-1α, and VEGFA(P&lt;0.05, P&lt;0.01), and declining protein expression levels of p-mTOR, FASN, HIF-1α, and VEGFA(P&lt;0.01). The therapeutic effect of the high-dose diosgenin group was superior to that of the low-dose diosgenin group and the simvastatin group. Diosgenin may reduce liver lipid synthesis and inflammation and potentiate by down-regulating the mTOR, FASN, HIF-1α, and VEGFA expression, playing an active role in preventing and treating NAFLD.

Enterococcus faecalis promotes the progression of colorectal cancer via its metabolite: biliverdin

BackgroundEnterococcus faecalis (Efa) has been shown to be a “driver bacteria” in the occurrence and development of colorectal cancer (CRC). This study aims to explore the effect of specific metabolites of Efa on CRC.MethodsThe pro-tumor effects of Efa were assessed in colonic epithelial cells. The tumor-stimulating molecule produced by Efa was identified using liquid chromatography mass spectrometry (LC-MS). The proliferative effect of metabolites on CRC cells in vitro was assayed as well. The concentration of vascular endothelial growth factor A (VEGFA) and interleukin-8 (IL-8) was determined using enzyme-linked immunosorbent assay (ELISA). Tubular formation assay of human umbilical vein endothelial cells (HUVEC) and cell migration assay were applied to study angiogenesis. Additionally, western blot analysis was used to investigate key regulatory proteins involved in the angiogenesis pathway. Tumor growth was assessed using mouse models with two CRC cells and human colon cancer organoid.ResultsCo-incubation with the conditioned medium of Efa increased the proliferation of cultured CRC cells. Biliverdin (BV) was determined as the key metabolite produced by Efa using LC-MS screening. BV promoted colony formation and cell proliferation and inhibited cell cycle arrest of cultured CRC cells. BV significantly increased the expression level of IL-8 and VEGFA by regulating the PI3K/AKT/mTOR signaling pathway, leading to the acceleration of angiogenesis in CRC. The up-regulation of proliferation and angiogenesis by BV were also confirmed in mice.ConclusionIn conclusion, BV, as the tumor-stimulating metabolite of Efa, generates proliferative and angiogenic effects on CRC, which is mainly mediated by the activation of PI3K/AKT/mTOR.

Down-Regulation of miR-93 Negatively Correlates with Overexpression of VEGFA and MMP3 in Endometriosis: A Cross-Sectional Study.

Endometriosis is identified as presence of the endometrium outside the uterine cavity. Retrograde menstruation contributes to the endometrial tissue implantation and the establishment of endometriotic lesions at ectopic sites. It has been suggested that the endometriotic lesions are rich in angiogenic growth factors, while they have an essential role in survival and invasion of these cells. We investigated regulation of microRNA-93 (miR-93) and its involvement with vascular endothelial growth factor A (VEGFA) and matrix metalloproteinase (MMP) 3 expression in women with endometriosis. This was a cross-sectional study at Central Surgical Installation, Dr. Cipto Mangunkusumo General Hospital, Jakarta, Indonesia, between October 2020 and November 2021. Eutopic and ectopic endometrial tissues were collected from 30 subjects with laparoscopically-confirmed endometriotic women. Normal endometrial cells of non-endometriosis women served as controls. Total RNA was isolated from all samples and a quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to analyze the expression of miR-93, VEGFA and MMP. There was no significant difference in the expression levels of VEGFA (2.14 ± 0.50, P=0.719) and MMP3 (2.99 ± 0.42, P=0.583) between endometriotic lesions of endometriosis women and the healthy endometrium. Expression of miR-93 was significantly lower in the eutopic endometrium (16.7 fold) and ectopic endometriotic lesion (20 fold) compared to the normal endometrium (P<0.001). Furthermore, we also observed a significant correlation between miR-93, VEGFA expression in eutopic endometrium obtained from women with endometriosis (r=-0.544, P=0.029). Expression of the miR-93 was also negatively correlated with MMP3 expression in both eutopic (r=-0.412, P=0.01) and ectopic (r=-0.539, P=0.03) endometrial cells of women with endometriosis. VEGFA and MMP3 expression levels trended to be increased in both eutopic and ectopic endometrial tissues of endometriosis women, while down-regulation of miR-93 might be involved in the alteration of VEGFA and MMP3 in endometriosis.

Biejiajian pill inhibits progression of hepatocellular carcinoma by downregulating PDGFRβ signaling in cancer-associated fibroblasts

Ethnopharmacological relevanceBiejiajian pill (BJJP) is a canonical formula that is clinically used to treat chronic liver disease, especially to decrease the incidence of hepatocellular carcinoma (HCC). However, the mechanisms underlying the prevention of HCC progression by BJJP remain unclear. Aim of the studyThis study aimed to determine whether BJJP inhibits HCC progression by downregulating platelet-derived growth factor receptor beta (PDGFRβ) signaling in cancer-associated fibroblasts (CAFs) in a mouse model of diethylnitrosamine (DEN)/carbon tetrachloride (CCl4)-induced HCC. Materials and methodsC57BL/6 male mice were intraperitoneally injected with DEN 2 weeks after birth, followed by repeated injections of CCl4 weekly from 6 weeks of age onwards, to recapitulate features of HCC. At week 14, BJJP was orally administered to mice. The effects of BJJP on HCC progression were evaluated using histology, immunohistochemistry, and serum biochemical marker levels. Transcriptome analysis, molecular docking, quantitative real-time PCR, and Western blot were used to study the genes targeted by BJJP and the associated signaling pathway. The effects of BJJP on PDGFRβ signaling in CAFs and the underlying mechanism were demonstrated. ResultsBJJP treatment significantly suppressed carcinogenesis and cancer progression, and it ameliorated liver inflammation in mice with HCC. A total of 176 genes, including PDGFRβ, were significantly downregulated after BJJP treatment and five components of BJJP with high binding affinity to PDGFRβ were identified. BJJP inhibited the phosphorylation of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), and glycogen synthase kinase 3 beta (GSK3β) by suppressing PDGFRβ expression in CAFs, and it also downregulated the expression of the downstream proteins hepatocyte growth factor (HGF) and vascular endothelial growth factor A (VEGF-A). Furthermore, BJJP-containing serum consistently reduced PDGFRβ, HGF, and VEGF-A expression levels in HSC-derived CAFs in vitro. Importantly, PDGF-BB induced PDGFRβ activation in CAFs and both BJJP and sunitinib (a kinase inhibitor) inhibited PDGF-BB/PDGFRβ signaling. ConclusionBJJP inhibits the progression of HCC through suppressing VEGF-A and HGF expression in CAFs by downregulating PDGFRβ signaling.

Systematic Analysis of CXC Chemokine-Vascular Endothelial Growth Factor A Network in Colonic Adenocarcinoma from the Perspective of Angiogenesis.

Background Tumor angiogenesis plays a vital role in tumorigenesis, proliferation, and metastasis. Recently, vascular endothelial growth factor A (VEGFA) and CXC chemokines have been shown to play vital roles in angiogenesis. Exploring the expression level, gene regulatory network, prognostic value, and target prediction of the CXC chemokine-VEGFA network in colon adenocarcinoma (COAD) is crucial from the perspective of tumor angiogenesis. Methods In this study, we analyzed gene expression and regulation, prognostic value, target prediction, and immune infiltrates related to the CXC chemokine-VEGFA network in patients with COAD using multiple databases (cBioPortal, UALCAN, Human Protein Atlas, GeneMANIA, GEPIA, TIMER (version 2.0), TRRUST (version 2), LinkedOmics, and Metascape). Results Our results showed that CXCL1/2/3/5/6/8/11/16/17 and VEGFA were markedly overexpressed, while CXCL12/13/14 were underexpressed in patients with COAD. Moreover, genetic alterations in the CXC chemokine-VEGFA network found at varying rates in patients with COAD were as follows: CXCL1/2/17 (2.1%), CXCL3/16 (2.6%), CXCL5/14 (2.4%), CXCL6 (3%), CXCL8 (0.8%), CXCL11/13 (1.9%), CXCL12 (0.6%), and VEGFA (1.3%). Promoter methylation of CXCL1/2/3/11/13/17 was considerably lower in patients with COAD, whereas methylation of CXCL5/6/12/14 and VEGFA was considerably higher. Furthermore, CXCL9/10/11 and VEGFA expression was notably correlated with the pathological stages of COAD. In addition, patients with COAD with high CXCL8/11/14 or low VEGFA expression levels survived longer than patients with dissimilar expression levels. CXC chemokines and VEGFA form a complex regulatory network through coexpression, colocalization, and genetic interactions. Moreover, many transcription factor targets of the CXC chemokine-VEGFA network in patients with COAD were identified: RELA, NFKB1, ZFP36, XBP1, HDAC2, SP1, ATF4, EP300, BRCA1, ESR1, HIF1A, EGR1, STAT3, and JUN. We further identified the top three miRNAs involved in regulating each CXC chemokine within the network: miR-518C, miR-369-3P, and miR-448 regulated CXCL1; miR-518C, miR-218, and miR-493 regulated CXCL2; miR-448, miR-369-3P, and miR-221 regulated CXCL3; miR-423 regulated CXCL13; miR-378, miR-381, and miR-210 regulated CXCL14; miR-369-3P, miR-382, and miR-208 regulated CXCL17; miR-486 and miR-199A regulated VEGFA. Furthermore, the CXC chemokine-VEGFA network in patients with COAD was notably associated with immune infiltration. Conclusions This study revealed that the CXC chemokine-VEGFA network might act as a prognostic biomarker for patients with COAD. Moreover, our study provides new therapeutic targets for COAD, serving as a reference for further research in the future.

METTL3 dual regulation of the stability of LINC00662 and VEGFA RNAs promotes colorectal cancer angiogenesis

PurposeThe angiogenesis is among the primary factors that affect tumor recurrence and distant organ metastasis in colorectal cancer (CRC). N6-methyladenosine (m6A) modification is one of the most common chemical modifications in eukaryotic mRNA, especially at the post-transcriptional level. Methyltransferase-like 3 (METTL3) promoting angiogenesis in a variety of tumors has been reported. However, the mechanism of how METTL3 dual-regulates the stability of long non-coding RNAs (lncRNAs) and vascular-related factor RNAs to affect angiogenesis in CRC is unclear.Methods64 paired CRC and adjacent normal tissues were collected. In vitro, quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC), actinomycin assay, methylated RNA immunoprecipitation (MeRIP) experiment,3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) and colony formation assay were performed. The functions were also studied in zebrafish model animals in vivo.ResultsWe found that the vascular endothelial growth factor A(VEGFA), METTL3 and LINC00662 RNAs were highly expressed in CRC, and that METTL3 was significantly positively correlated with LINC00662 and VEGFA. The protein expression levels of CD31, CD34, VEGFA, m6A and METTL3 were all significantly increased in the CRC tissues. The angiogenesis experiments both in vivo and in vitro found that METTL3 and LINC00662 promoted angiogenesis in CRC. The actinomycin assay indicated that METTL3 maintained the stability of LINC00662 and VEGFA RNAs. In addition, the MeRIP experiment confirmed that the LINC00662 and VEGFA RNAs had METTL3-enriched sites.ConclusionThese findings suggest that METTL3 and LINC00662 may both serve as diagnostic and prognostic predictive biomarkers for CRC and potential targets for anti-vascular therapy.

The association of umbilical coiling and angiogenesis markers: Impact assessment of gestational diabetes

IntroductionThe purpose of this study was to determine the association between the postnatal umbilical coiling index (pUCI) and vascular endothelial growth factor A (VEGFA) and its receptor (VEGFR2) in parturients with and without gestational diabetes mellitus (GDM). MethodsWithin 24 h following birth, the umbilical cord and pUCI of 29 newborns with GDM and 28 neonates with non-GDM parturients were prospectively examined. Real-time PCR tests were used to determine the expression levels of the VEGFA and VEGFR2 genes, measured from the umbilical cord. The Mann-Whitney and Chi-squared tests were used to compare continuous and discrete variables with and without GDM. ResultsThe median (IQR) of maternal age was 30 (26-34) years. There were no differences in demographic features between GDM and non-GDM parturients. While there was a marginal difference in VEGFA expression levels between the GDM and non-GDM groups (P-values = 0.07), no difference was detected for VEGFR2 (P-values = 0.75). Comparing hyper- and hypocoiling cords revealed a small difference in VEGFA levels (P-values = 0.05), but no change in VEGFR2 (P-values = 0.50). Furthermore, in both GDM and non-GDM parturients, down-regulated VEGFA was the general rule among abnormal pUCIs. DiscussionThe GDM and coiling state both are associated with the amount of VEGFA expression, but neither is related to VEGFR2. Furthermore, regardless of whether the patient has GDM or not, the abnormal coiling pattern appears to be related to the VEGFA down-regulation.

LncRNA-UCA1 regulates lung adenocarcinoma progression through competitive binding to miR-383

ABSTRACT The present study aimed to assess the role of the long non-coding RNA-urothelial cancer associated 1 (lncRNA-UCA1)/microRNA (miR)-383/vascular endothelial growth factor A (VEGFA) axis in regulating lung adenocarcinoma physiology through in vivo and in vitro experiments. The expression profile of lncRNA-UCA1 was analyzed by genome-wide analysis from GSE146459. The cell counting Kit-8, colony formation, wound healing and transwell assays were performed to evaluate the effects of lncRNA-UCA1 in vitro. In addition, luciferase reporter assays were performed to confirm the binding site. The expression levels of miR-383 and VEGFA in tumor cells were measured using reverse transcription-quantitative PCR. HCC-78 was also transfected with miR-383 mimics, inhibitors and siRNA-VEGFA before their viability was also assessed. Xenograft models were established in nude mice to investigate the tumor characteristics in vivo. The expression of lncRNA-UCA1 was significantly increased in tumor tissues and cells compared with adjacent tissues or HBE cells. Silencing lncRNA-UCA1 expression in cells resulted in a reduction in lung cancer cell viability. In addition, lncRNA-UCA1 silencing increased the expression of miR-383. Inhibiting miR-383 expression increased HCC-78 proliferation, migration and invasion, whilst reducing their apoptosis. miR-383 was shown to specifically target VEGFA to inhibit its expression at both the protein and mRNA levels. VEGFA knockdown resulted in a reduction in all aforementioned aspects of HCC-78 cell activity. In addition, inhibiting miR-383 expression led to larger tumor sizes in vivo. To conclude, the results of the study suggest that lncRNA-UCA1 can regulate the expression of miR-383 and, in turn, VEGFA.

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.

The Effect of Eugenol Treatment on Diabetic Cardiomyopathy in Streptozotocin-Induced Diabetic Rats

Cardiovascular diseases account for most of the morbidity and mortality associated with diabetes. Diabetic cardiomyopathy (DCM) is associated with heart failure in diabetic patients without relation to other cardiovascular diseases such as hypertension or coronary artery disorders. Eugenol is a phenolic compound extracted from the clove tree and exhibits effective mitigation of hyperglycemic conditions in diabetic animals. Thus, in the current study, we aimed to explore the effect of eugenol treatment on rats with DCM. The experimental animals included 30 Sprague Dawley male rats which are divided into three experimental groups (10 rats each) as the following: the non-diabetic control group (ND), diabetic group (D), and a treated-diabetic group (20mg/kg/day of eugenol) (D+E). Diabetes was induced by streptozotocin (STZ) injection (60 mg/ kg). After 6 weeks, blood samples and left ventricles were collected for analysis. Serum glucose levels, heart weight/body weight ratio, and the myocardial mRNA expression of transforming growth factor β1 (TGF-β1), tumor necrosis factor-α (TNF-α), caspase 3 (casp3), vascular endothelial growth factor-A (VEGF-A), and collagen IV were evaluated. Furthermore, the myocardial superoxide dismutase (SOD) activity was measured. Diabetic rats showed a significant appearance of hyperglycemia and increased expression of myocardial TNF-α, TGF-β1, caspase 3, and VEGF-A compared to the control group (P &lt; 0.05), and a tendency to increase collagen IV (P &lt; 0.1). On the other hand, the eugenol treatment mitigates diabetic-associated hyperglycemia and the increased mRNA expression levels of myocardial TGF-β1, VEGF-A, caspase 3, and TNF-α (P &lt; 0.05). In addition, the overexpression of collagen IV was inhibited, and the myocardial SOD activity was improved in the diabetic rats treated with eugenol. The study provided evidence that eugenol may have a potential therapeutic effect in the experimental models of DCM by reducing the expression of pro-inflammatory, pro-fibrotic, angiogenic, and pro-apoptotic factors (TNF-α, TGF-β, collagen IV, VEGF-A, and caspase 3 respectively). It is recommended for further studies investigate the exact molecular processes by which eugenol may ameliorate the DCM phenotype.