Vascular Endothelial Growth Factor mRNA Expression Research Articles

Adipose-Derived Stem Cell Transplantation Inhibits Vascular Inflammatory Responses and Endothelial Dysfunction in Rats with Atherosclerosis.

PurposeThis study aimed to investigate the effect of adipose-derived stem cell (ADSC) transplantation on atherosclerosis (AS) and its underlying mechanisms.Materials and MethodsIn our study, rat AS model was established, and ADSCs were isolated and cultured. Atherosclerotic plaque and pathological symptoms of thoracic aorta were measured by Oil Red O staining and Hematoxylin-Eosin staining, respectively. Total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were measured by an automatic biochemical analyzer. Expressions of vascular endothelial growth factor (VEGF), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), aortic endothelin-1 (ET-1), interleukin-6 (IL-6), c-reactive protein (CRP), and tumor necrosis factor α (TNF-α) were measured by enzyme linked immunosorbent assay, VEGF, VCAM-1, ICAM-1, ET-1, respectively, and NF-κB p65 mRNA expressions were detected by quantitative real-time polymerase chain reaction. Protein expressions of VEGF, VCAM-1, ICAM-1, ET-1, NF-κB p65, p-NF-κB p65, and IκBα were measured by western blot. Moreover, NF-κB p65 expression was measured by immunofluorescence staining.ResultsADSC transplantation alleviated the pathological symptoms of aortic AS. ADSC transplantation decreased the levels of TC, TG, and LDL-C and increased serum HDL-C level. Meanwhile, ADSC transplantation decreased the levels of IL-6, CRP, and TNF-α in AS rats. Moreover, the expressions of VEGF, ET-1, VCAM-1, and ICAM-1 were decreased by ADSC transplantation. ADSC transplantation inhibited phosphorylation of NF-κB p65 and promoted IκBα expression in AS rats.ConclusionOur study demonstrated that ADSC transplantation could inhibit vascular inflammatory responses and endothelial dysfunction by suppressing NF-κB pathway in AS rats.

TNF-alpha increases angiogenic potential in a co-culture system of dental pulp cells and endothelial cells.

We recently demonstrated that a co-culture system of human umbilical vein endothelial cells (HUVECs) and human dental pulp stem cells (hDPSCs) could enhance angiogenesis ability in vitro. However, whether tumor necrosis factor α (TNF-α) could promote blood vessel formation during pulp regeneration remained unknown. The aim of this study was to investigate the effects of TNF-α on the formation of endothelial tubules and vascular networks in a co-culture system of hDPSCs and HUVECs. hDPSCs were co-cultured with HUVECs at a ratio of 1:5. The Matrigel assay was performed to detect the total tubule branching lengths and numbers of branches, and the Cell-Counting Kit 8 assay was performed to examine the effect of TNF-α on cell proliferation. Real-time polymerase chain reactions and western blot were used to detect vascular endothelial growth factor (VEGF) mRNA and protein expression. The Matrigel assay showed significantly greater total branching lengths and numbers of branches formed in the experimental groups treated with different concentrations of TNF-α compared with the control group. The decomposition times of the tubule structures were also significantly prolonged (P < 0.05). Treatment with 50 ng/ml TNF-α did not significantly change the proliferation of co-cultured cells, but it significantly increased the VEGF mRNA and protein expression levels (p < 0.05). In addition, the migration abilities of HUVECs and hDPSCs increased after co-culture with TNF-α (p < 0.05). TNF-α enhanced angiogenic ability in vitro in the co-culture system of hDPSCs and HUVECs.

Establishment of a rat model of dimethylbenzanthracene-induced vulvar squamous intraepithelial lesions

To establish a SD rat model of vulvar squamous intraepithelial lesions. Seventy female SD rats were randomized into 4 groups, namely the blank control group (n=10), mechanical irritation group (n=10), acetone solution group (n=10), and mechanical irritation with DMBA acetone solution group (n=40, model group), and the corresponding treatments were administered 3 times a week for 14 weeks. The changes of the vulvar skin of the rats were observed regularly until the 18th week. The expression of mutant p53 (mtp53) and vascular endothelial growth factor (VEGF) proteins were detected using immunohistochemistry and Western blotting, and the expressions of mtp53 and VEGF mRNA were detected with qRT- PCR in the blank control group and model group. No significant differences were found in the morphological or histopathological changes of the skin among the blank control group, mechanical irritation group and acetone solution group. In the model group, low-grade squamous intraepithelial lesions (LSIL) occurred in 28 rats (70%) and high-grade squamous intraepithelial lesions (HSIL) in 11 rats (27.5%) at 14 weeks, with a success rate of 97.5% in inducing vulvar squamous intraepithelial lesions. Compared with the blank control group, the rats in the model group showed significantly increased expressions of mtp53 and VEGF at both the protein level (P &lt; 0.05) and the mRNA level (P &lt; 0.05). DMBA in acetone solution combined with mechanical irritation can induce vulvar squamous intraepithelial lesions in female SD rats.

Corin is a key regulator of endochondral ossification and bone development via modulation of vascular endothelial growth factor A expression.

Corin has been studied extensively within the vascular system and is known to regulate blood pressure. We have shown that corin is one of the most highly upregulated genes during osteogenic differentiation of human adipose-derived stem cells (hASCs). This study tested the hypothesis that, through modulation of angiogenic signalling pathways, corin is a critical regulator of osteogenic differentiation and endochondral ossification. In vitro, corin expression in hASC was suppressed via siRNA knockdown and vascular endothelial growth factor A (VEGF-A) expression was quantified via reverse transcription polymerase chain reaction. In vivo, a murine corin knockout model (female, 10weeks) was used to determine the effect of corin deficiency on long bone development. Wild-type and corin knockout long bones were compared via haematoxylin and eosin staining to assess tissue characteristics and cellular organization, three-point bending to assess mechanical characteristics, and immunohistochemistry to visualize VEGF-A expression patterns. Corin knockdown significantly (p<0.05) increased VEGF-A mRNA expression during osteogenic differentiation. In vivo, corin knockout reduced tibial growth plate thickness (p<0.01) and severely diminished the hypertrophic region. Corin knockout femurs had significantly increased stiffness (p<0.01) and maximum loads (p<0.01) but reduced postyield deflections (p<0.01). In corin knockout mice, VEGF-A expression was increased near the growth plate but was reduced throughout the tibial shaft and distal head of the tibiae. This is the first study to show that corin is a key regulator of bone development by modulation of VEGF-A expression. Further elucidation of this mechanism will aid in the development of optimized bone tissue engineering and regenerative medicine therapies.

Amelioration of diabetic nephropathy in db/db mice treated with tibetan medicine formula Siwei Jianghuang Decoction Powder extract

Siwei Jianghuang Decoction Powder (SWJH) documented originally in the Four Medical Tantras-Blue Glaze exhibited beneficial effects on diabetic nephropathy (DN) via combined synergistically action of multiple formula components including Curcumae longae Rhizoma, Berberidis dictyophyllae Cortex, Phyllanthi Fructus and Tribuli Fructus. This study investigated the effects of SWJH on DN in db/db mice and possible underlying mechanisms. The ten weeks old db/db mice treated with SWJH by intra-gastric administration once a day for 8 weeks. After 8 weeks, body weight, water and food intake of mice were recorded. The level of fasting blood glucose (FBG) was measured. Serum creatinine (Scr), blood urea nitrogen (BUN), urine microalbumin (UMAlb), serum uric acid (UA) and urinary albumin excretion (UAE) were detected. An enzyme-linked immunosorbent assay was performed to test serum vascular endothelial growth factor (VEGF) and transforming growth factor-β1 (TGF-β1). Real-time PCR and Western blot analysis were used to test mRNA and protein expression of hypoxia inducible factor-1α (HIF-1α), VEGF and TGF-β1 in kidney tissue. SWJH treatment significantly reduced the levels of FBG, Scr, BUN, UMAlb, UA and UAE and retarded renal fibrosis. SWJH treatment further significantly reduced serum TGF-β1 level and downregulated the expression of HIF-1α, VEGF and TGF-β1 at both mRNA and protein levels. Principal component analysis and partial least squares regression and hierarchical cluster analysis demonstrated that SWJH treatment significantly ameliorated renal damage in DN mice. These consequences suggested that SWJH formulations were effective in the treatment of DN through regulating the HIF-1α, VEGF and TGF-β1 overexpression.

Ameliorative Effects of α-Tocopherol and/or Coenzyme Q10 on Phenytoin-Induced Cognitive Impairment in Rats: Role of VEGF and BDNF-TrkB-CREB Pathway.

Phenytoin is one of the most well-known antiepileptic drugs that cause cognitive impairment which is closely related to cAMP response element-binding protein (CREB) brain-derived neurotrophic factor (BDNF) signaling pathway. Moreover, vascular endothelial growth factor (VEGF), an endothelial growth factor, has a documented role in neurogenesis and neuronal survival and cognitive impairment. Therefore, this study aimed to investigate the influence of powerful antioxidants: α-Toc and CoQ10 alone or combined in the preservation of brain tissues and the maintenance of memory formation in phenytoin-induced cognitive impairment in rats. The following behavioral test novel object recognition and elevated plus maze were assessed after 14days of treatment. Moreover, VEGF, BDNF, TrkB, and CREB gene expression levels in the hippocampus and prefrontal cortex were estimated using RT-PCR. Both α-Toc and CoQ10 alone or combined with phenytoin showed improvement in behavioral tests compared to phenytoin. Mechanistically, α-Toc and/or CoQ10 decreases the VEGF mRNA expression, while increases BDNF-TrKB-CREB mRNA levels in hippocampus and cortex of phenytoin-treated rats. Collectively, α-Toc and/or CoQ10 alleviated the phenytoin-induced cognitive impairment through suppressing oxidative damage. The underlying molecular mechanism of the treating compounds is related to the VEGF and enhancing BDNF-TrkB-CREB signaling pathway. Our study indicated the usefulness α-Toc or CoQ10 as an adjuvant to antiepileptic drugs with an advantage of preventing cognitive impairment and oxidative stress.

Bisphenol-A impairs cellular function and alters DNA methylation of stress pathway genes in first trimester trophoblast cells

Humans are exposed to Bisphenol A (BPA) from the consumer products and plastic substances. However, impacts of low levels of BPA exposure on placental developmental processes such as first trimester trophoblast cell growth, angiogenesis and epigenetic modifications are not well studied. Low concentration of BPA (1 nM) affected cell proliferation of human placental first trimester trophoblasts using a model cell, HTR8/SVneo. BPA abolished both basal- and vascular endothelial growth factor (VEGF)-stimulated tube formation in these cells. BPA significantly down regulated mRNA expression of VEGF, proliferating cell nuclear antigen, intercellular adhesion molecule 1 with concomitant upregulation of 11-β-hydroxysteroid dehydrogenase 2 mRNA and protein expression in HTR8/SVneo cells. BPA also lowered CpG methylation of gene promoter associated with metabolic and oxidative stress. This study demonstrated that BPA at 1 nM not only affected cellular growth, development and angiogenic activities but also affected DNA methylation of stress response and down-regulation of angiogenic growth factors in first trimester trophoblast cells.

A molecular basis for the synergy between 17‑allylamino‑17‑demethoxy geldanamycin with Capecitabine and Irinotecan in human colorectal cancer cells through VEFG and MMP-9 gene expression

Anti-proliferative, anti-metastatic and anti-angiogenic effects of 17‑allylamino‑17‑demethoxy geldanamycin (17-AAG) were studied alone and in combination with Capecitabine (Cap) and/or Irinotecan (IR) on HT-29 human colorectal carcinoma cells. Expression of MMP-9 (matrix metalloproteinase‑9) and VEGF (vascular endothelial growth factor) mRNA was analyzed by real-time PCR method. The study was further followed by wound scratch assay for migration assessment. Nitric oxide content, Malondialdehyde generation and total anti-oxidant capacity were also assessed. Results showed significant differences between mono- and double therapy (p < 0.05). Combination of 17-AAG with IR or Cap resulted in synergistic effect (Combination Index < 1). Among double combination groups only Cap/17-AAG showed significant differences in MMP-9 and VEGF genes expression and wound healing assay. Moreover, a significant decrease of wound area in our triple combination group was obtained, indicating the antagonistic effect. IR/17-AAG and IR/Cap double combination groups resulted in down-regulation of MMP-9 and VEGF mRNA expression, respectively. Significant generation of MDA and decrease in TAC values have been observed in all our tested groups, however, the IR/17-AAG combination was the only group that could elevate NO concentration, significantly. Our findings demonstrated potent anti-angiogenesis and anti-metastatic effects for 17-AAG when it is provided in double combination especially with Cap, suggesting a new protocol in colorectal cancer combination therapy. These findings may indicate that down-regulation of VEGF and MMP-9 genes is directly related to angiogenesis and metastasis.

A Mesenchymal stem cell line (B10) increases angiogenesis in a rat MCAO model

A human mesenchymal stem cell line (B10) transplantation has been shown to improve ischemia-induced neurological deficits in animal stroke models. To understand the underlying mechanism, we have investigated the effects of B10 transplantation on cerebral angiogenesis in a rat middle cerebral artery occlusion (MCAO) model. B10 cells were transplanted intravenously 24 h after MCAO. Immunofluorescence staining results showed that compared to PBS-groups, vWF positive vessel and endoglin positive new vessels were increased in B10-transplanted MCAO groups in the lesion areas. The mRNA of angiogenesis factors including placental growth factor and hypoxia inducible factor (HIF)-1α were increased 3 days after MCAO in the core and IBZ areas of B10-transplanted group. Angiopoetin1 mRNA was increased only in the IBZ. Western blotting results showed that HIF-1α and vascular endothelial growth factor (VEGF) proteins were increased in B10-transplanted group. Both HIF-1α and VEGF were expressed in macrophage/microglia in the core area. In the IBZ, however, HIF-1α was expressed both in astrocytes and macrophage/microglia, while VEGF was expressed only in macrophage/microglia. Moreover, TGFβ protein levels were found to be increased in B10-transplanted group in the core and IBZ regions. Cell culture experiments using a human microglia cell line (HMO6) and B10 showed that IL-1β induced VEGF mRNA expression in both cell types. IL-1β was found to be highly expressed in B10 cells, and its co-culture with HMO6 further increased that in B10. Co-culture increased VEGF mRNA in both B10 and HMO6. In the rat brains, IL-1β was expressed in macrophage/microglia and transplanted-B10 cells in the core. IL-1β positive cell number was increased slightly, but significantly in B10-transplanted rats. To explore further, IL-1β expression was silenced in B10 cells by transfecting mRNA specific siRNA, and then transplanted in MCAO rats. Immunostaining result showed that endoglin positive area was decreased in IL-1β-silenced B10 transplanted groups compared to nonsilenced-B10 transplanted groups. Interestingly, vessel-like structure appeared as early as 3 days after MCAO in IL-1β-silenced B10-transplanted group. Thus our results demonstrated that B10 cells increased angiogenesis in MCAO rat model, through the regulation of HIF-1α and VEGF expression, where IL-1β might play a role.

Effect of Berberine on Angiogenesis and HIF-1&alpha; / VEGF Signal Transduction Pathway in Rats with Cerebral Ischemia - Reperfusion Injury.

To investigate the effect of berberine on angiogenesis and signal transduction pathway of hypoxia-inducible growth factor-1&alpha; (HIF-1&alpha;) / vascular endothelial growth factor (VEGF) in rats with cerebral ischemia-reperfusion injury. An experimental study. Luoyang Central Hospital Affiliated with Zhengzhou University, China, from 2016 to 2017. Forty-five rats were randomly divided into control group, model group and berberine group, 15 rats in each group. The model of rat with cerebral ischemia-reperfusion injury was duplicated by suture method. Neurological score was evaluated before, after operation, and 7 days after administration. Microvessel density (MVD) of cerebral ischemia- reperfusion cortex was detected by immunohistochemistry. HIF-1&alpha; mRNA and VEGF mRNA of cerebral ischemia- reperfusion cortex were detected by RT-PCR. HIF-1&alpha;, and VEGF protein expression of cerebral ischemia-reperfusion cortex was detected by Western Blotting. After operation and 7 days of administration, the neurological scores of the control group, model group, and berberine group were different (all p<0.001). After 7 days of administration, neurological score of berberine group was lower than that of model group (p <0.001), MVD, HIF-1&alpha; mRNA and VEGF mRNA expression. HIF-1&alpha; and VEGF protein expression levels were lower in model group than berberine group (all p <0.001). Berberine can promote angiogenesis in the rat with cerebral ischemia-reperfusion injury. Its mechanism may be activation of HIF-1&alpha; / VEGF signal transduction pathway.

Expression of Rap1, guanosine triphosphate Rap1, vascular endothelial growth factor and β-catenin in experimental choroidal neovascularization

Objective To observe the expression of Rap1, guanosine triphosphate-Rap1 (GTP-Rap1), vascular endothelial growth factor (VEGF) and β-catenin in experimental choroidal neovascularization (CNV). Methods Forty-two brown Norwegian rats were randomly divided into a blank control group (7 rats) and a model group (35 rats). Both eyes were enrolled. The CNV model was established by holmium ion laser photocoagulation in the model group. At 3, 7, 14, 21, and 28 days after photocoagulation, fluorescein fundus angiography (FFA) and choroidal vascular smear were performed to observe the degree of fluorescein leakage and CNV area in rats; Western blot and real-time quantitative polymerase chain reaction (RT-PCR) were used to detect the expression of Rap1, GTP-Rap1, VEGF, β-catenin and mRNA in CNV. Results The results of FFA examination showed that a large disc-shaped fluorescein leaked in the photo-condensation spot 14 days after photocoagulation. Laser confocal microscopy showed that compared with 7 days after photocoagulation, CNV area increased at 14, 21, 28 days after photocoagulation, and the difference were statistically significant (t=3.725, 5.532, 3.605;P<0.05). Western blot showed that there was no significant difference in the relative expression of Rap1 protein in CNV at different time points after photocoagulation between the two groups (P=0.156). Compared with the blank control group, the relative expression of GTP-Rap1 protein was significantly decreased, the relative expression of VEGF and β-catenin protein were significantly increased in the model group (P=0.000). The results of RT-PCR showed that there was no significant difference in the relative expression of Rap1 mRNA at different time points after photocoagulation between the two groups (P=0.645), but there were significant difference in the relative expression of β-catenin mRNA (P=0.000). At 7, 14, 21 and 28 days after photocoagulation, there were significant difference in the relative expression of GTP-Rap1 and VEGF mRNA between the two groups (P=0.000). Conclusions The expression of GTP-Rap1 in experimental CNV is significantly lower than that in normal rats. Key words: Choroidal neovascularization/treatment; rap1 GTP-binding proteins; Vascular endothelial growth factors; beta Catenin

Effect of different doses of Astragalus membranaceus on levels of vascular endothelial growth factor and basic fibroblast growth factor in serum and lung tissues of rats with pulmonary embolism

Objective To evaluate the effect of different doses of Astragalus membranaceus on the levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in serum and lung tissues of rats with pulmonary embolism. Methods Seventy-six clean-grade healthy male Sprague-Dawley rats, aged 8-9 weeks, weighing 140-170 g, were assigned to control group (group C, n=11) and experimental group (group E, n=65) by a random number table method.The rats with pulmonary embolism in group E were further divided into 4 subgroups using a random number table method: pulmonary embolism group (group P), low-dose Astragalus membranaceus group (group H1), median-dose Astragalus membranaceus group (group H2) and high-dose Astragalus membranaceus group (group H3). The model of pulmonary embolism was established by injecting autologous blood clots into the right jugular vein.At 1 h and 1, 2, 3, 4, 5 and 6 days after successful establishment of the model, Astragalus membranaceus 20, 40 and 60 g/kg were injected intraperitoneally in H1-3 groups, respectively, while the equal volume of normal saline was given instead in group P. The chest was opened after anesthesia on day 7, and blood samples were collected from cardiac chambers for determination of concentrations of serum VEGF and bFGF by enzyme-linked immunosorbent assay.The pulmonary specimens were obtained from the upper lobe of right lungs for determination of the expression of VEGF and bFGF mRNA (using real-time polymerase chain reaction). Results Compared with group C, the concentrations of serum VEGF and bFGF were significantly increased, and the expression of VEGF and bFGF mRNA in lung tissues was up-regulated in the other four groups (P<0.05). Compared with group P, the serum bFGF concentration was significantly increased, and the expression of VEGF and bFGF mRNA in lung tissues was up-regulated in H1-3 groups (P<0.05). Compared with group H1, the serum bFGF concentration was significantly increased, the expression of VEGF mRNA and bFGF mRNA in lung tissues was up-regulated in H2 and H3 groups (P<0.05). Compared with group H2, the expression of VEGF and bFGF mRNA in lung tissues was significantly up-regulated in group H3 (P<0.05). Conclusion Astragalus membranaceus can up-regulate the expression of VEGF and bFGF in lung tissues in a dose-dependent manner, thus improving pulmonary embolism in rats. Key words: Astragalus membranaceus; Pulmonary embolism; Vascular endothelial growth factors; Fibroblast growth factor 2

Electroacupuncture for myocardial ischemia injury in rats via AMPK-HDAC5-HIF-1α signaling

To explore the impact of electroacupuncture (EA) on the AMPKα-HDAC5-HIF-1α signaling in the heart of the rats with myocardial ischemia (MI) via detecting the expressions of AMP-activated protein kinase α (AMPKα), histone deacetylase 5 (HDAC5), hypoxia inducible factor (HIF-1α) and vascular endothelial growth factor (VEGF). Thirty-six healthy male SD rats were randomized into a sham operation group (6 rats), a sham + EA group (6 rats), a model group (12 rats) and an EA group (12 rats). We ligated the left anterior descending artery (LAD) for MI model, and exposed the heart of rats after opening the chest without ligation for the rats in the sham operation gorup and the sham + EA group. On the 2nd day after LAD ligation, EA was applied at "Neiguan" (PC 6) with 2 Hz/15 Hz and 1.5-2 mA for 30 min in the EA group and sham+EA group, once a day for 4 days. The same fixation was used in the sham operation group and the model group, without EA. Myocardial infarction area was observed by TTC staining and serum cardiac troponin T (cTnT) was detected by radioimmunoassay. The expression of VEGF mRNA was detected by real time PCR. The protein expressions of AMPKα, HDAC5, HIF-1α and VEGF were detected by western blot. Compared with the sham operation group, 4 days after LAD ligation, the myocardial infarction was obvious and the expression of serum cTnT increased in the model group (P<0.01); and the proterin expression of HIF-1α in myocardial tissue ascended (P<0.01); the expression of VEGF mRNA decreased (P<0.05); the changes of the protein expressions of AMPKα、HDAC5、VEGF were not obvious (all P>0.05). After EA for 4 days, the myocardial infarction area and cTnT expression decreased in the EA group (both P<0.01); the VEGF mRNA and protein expressions and AMPKα, HDAC5, HIF-1α protein expressions increased (P<0.05, P<0.01). EA could regulate the AMPKα-HDAC5-HIF-1α signaling in myocardial tissue, which may activate VEGF expression for angiogenesis signaling, reduce myocardial infarction area so as to achieve cardioprotective effect.

Involvement of vascular endothelial growth factor (VEGF) and mitogen-activated protein kinases (MAPK) in the mechanism of neuroleptic drugs.

Recent evidence suggests that the mitogen activated protein kinase (MAPK)-associated signaling pathway in the frontal cortical areas demonstrates abnormal activity in cases of schizophrenia. Moreover, schizophrenia patients often display alterations in the regional cellular energy metabolism and blood flow of the brain; these are shown to parallel changes in angiogenesis primarily mediated by vascular endothelial growth factor (VEGF). The present study examines the differential effects of time-dependent treatment with haloperidol, olanzapine and amisulpride (20μM) on VEGF and MAPK mRNA expression and VEGF level, using the T98 cell line as an example of nerve cells. For the purposes of comparison, the effect of neuroprotective pituitary adenylate cyclase-activating polypeptide (PACAP) on the expression of VEGF mRNA and secretion were also evaluated in this cell model. RT-PCR analysis revealed that all the tested neuroleptics increased VEGF mRNA expression after 72-h incubation; however, only haloperidol and olanzapine also increased the level of VEGF detected by ELISA, and they demonstrated significantly stronger effects than PACAP. Haloperidol and olanzapine, but not amisulpride, decreased MAPK14 mRNA expression in T98G cells after 72-h incubation. The obtained results suggest that haloperidol and olanzapine can trigger the MAPK and VEGF signaling pathway, which may contribute to their neuroprotective mechanism of action.

MiR‑20b negatively regulates VEGF expression by targeting STAT3 in H22 hepatocellular carcinoma cells.

Vascular endothelial growth factor (VEGF) promotes angiogenesis during tumor growth, and its expression involves multiple signaling pathways and transcription factors. In the present study, transforming growth factor (TGF)‑β1 promoted upregulation of VEGF and downregulation of microRNA (miR)‑20b expression in mouse H22 hepatocellular carcinoma cells. miR‑20b negatively regulated both constitutive VEGF expression and TGF‑β1‑induced VEGF expression. The miRanda algorithm predicted that a binding site of the miR‑20b GCAAUCUGGGCACUUU sequence was present in the signal transducer and activator of transcription (STAT)3 3'‑untranslated region. Following transfection of miR‑20b mimics into H22 cells, expression of STAT3 protein was downregulated. A dual‑luciferase activity assay revealed that miR‑20b directly targeted STAT3 to regulate its expression, and that interference with STAT3 expression significantly downregulated VEGF mRNA and protein expression. Interference with STAT3 expression resulted in increased VEGF expression in H22 cells overexpressing miR‑20b, but expression was lower than that in quiescent H22 cells. This indicated that STAT3 was involved in the negative regulation of VEGF expression in H22 cells by miR‑20b. The data demonstrated that miR‑20b negatively regulated VEGF expression by directly targeting STAT3 in H22 cells.

Adiponectin enhances biological functions of vascular endothelial progenitor cells through the mTOR-STAT3 signaling pathway.

Adiponectin (APN), an adipose tissue-excreted adipokine, plays protective roles in metabolic and cardiovascular diseases. In this study, the effects and mechanisms of APN on biological functions of rat vascular endothelial progenitor cells (VEPCs) were investigated in vitro. After administrating APN in rat VEPCs, the proliferation was measured by methyl thiazolyl tetrazolium (MTT) method, the apoptotic rate was test by Flow cytometry assay, mRNA expression of B-cell lymphoma-2 (Bcl-2) and vascular endothelial growth factor (VEGF) was determined by real-time reverse transcriptase polymerase chain reaction (RT-PCR), and protein expression of mechanistic target of rapamycin (mTOR), signal transducer and activator of transcription 3 (STAT3) and phospho-STAT3 (pSTAT3) was analyzed by Western blot. It was suggested that APN promoted the optical density (OD) value of VEPCs, enhanced mRNA expression of Bcl-2 and VEGF, and inhibited cell apoptotic rate. Furthermore, protein expression of pSTAT3 was also increased in the presence of APN. Moreover, APN changed-proliferation, apoptosis and VEGF expression of VEPCs were partially suppressed after blocking the mTOR-STAT3 signaling pathway by the mTOR inhibitor XL388. It was indicated that APN promoted biological functions of VEPCs through targeting the mTOR-STAT3 signaling pathway.

STROBE: The correlation of Cyr61, CTGF, and VEGF with polymyositis/dermatomyositis.

This study aims to explore the roles of cysteine-rich protein 61 (Cyr61/CCN1), connective tissue growth factor (CTGF/CCN2) and vascular endothelial growth factor (VEGF) in the vascular process of polymyositis (PM)/dermatomyositis (DM).Real-time quantitative polymerase chain reaction was used to determine the mRNA expression of Cyr61, CTGF, and VEGF in muscle tissues of initially treated PM/DM patients and controls. Enzyme-linked immunosorbent assay (ELISA) was used to determine the serum levels of Cyr61, CTGF, and VEGF of initially treated PM/DM patients before and after treatment. Data were statistically analyzed using statistical software SPSS 17.0.The mRNA expression levels of Cyr61, CTGF, and VEGF in muscle tissues were higher in the PM and DM groups than in the control group (P < .05). Differences in the mRNA expression levels of Cyr61, CTGF, and VEGF in muscle tissues between the PM and DM groups were not statistically significant (P > .05). Before treatment, the serum levels of Cyr61, CTGF, and VEGF were higher in the PM and DM groups than in the control group (P < .05). Furthermore, in the PM and DM groups, the expression levels of Cyr61, CTGF, and VEGF in serum at 6 months after treatment were lower than those before treatment (P < .05).Cyr61, CTGF, and VEGF are involved in the pathogenesis of PM/DM. These may be involved in the pathogenesis mainly by affecting the formation of blood vessels and promoting inflammatory response. This suggests that microvascular lesions play an important role in the immune pathogenesis of inflammatory myopathy PM/DM.

Expression and clinical significance of HIF-1, VEGF and MMP-2 in patients with pathological scar

Objective To investigate the expression levels and significance of hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) in pathological scar. Methods 73 patients with pathological scar from March 2015 to February 2017 in our hospital were selected, including 42 cases in hypertrophic scar group and 31 cases in keloid group. 20 cases of non pathological scar patients and 20 cases of normal plastic surgery were selected as control group. mRNA and protein expression levels of HIF-1, VEGF and MMP-2 in skin tissue of each group were detected by real-time fluorescence quantitative and Western blot, and the clinical significance was analyzed. Results Compared with non pathological scar tissue and normal skin tissue, the mRNA and protein expression levels of HIF-1α, VEGF and MMP-2 in pathological scar tissue were significantly increased, and the differences were statistically significant (P 0.05); the mRNA and protein expression levels of MMP-2 in keloid group were significantly lower than those in hypertrophic scar group (P<0.05). Pearson correlation analysis showed that there was a significant positive correlation between HIF-1α and VEGF, MMP-2 in pathological scar tissues (r=0.623, 0.507, P<0.05). Conclusions The expressions of HIF-1α, VEGF and MMP-2 in pathological scar tissue is obviously increased, which is closely related to the formation of pathological scar. Key words: Cicatrix/PA; Hypoxia-inducible factor 1, alpha subunit/ME; Vascular endothelial growth factors/ME; Matrix metalloproteinase 2/ME

Increased expression of IL-33 in rosacea skin and UVB-irradiated and LL-37-treated HaCaT cells.

Rosacea is one of the most common dermatoses of adults. Although the detailed pathophysiology remains unknown, it is thought that rosacea is caused by a consistently aberrant, innate immune response, and that LL-37 plays an important role. However, involvement of the inflammatory cytokine IL-33 has not yet been studied. We explored the role played by IL-33 in the pathophysiology of rosacea. First, we immunohistochemically evaluated the expression of IL-33 and its receptor (ST2) in rosacea skin. Second, we exposed HaCaT cells to ultraviolet B (UVB) irradiation in the presence or absence of LL-37 and measured the expression of proinflammatory cytokines including IL-33. We also analysed VEGF (vascular endothelial growth factor) mRNA expression and protein release after costimulation of HaCaT cells by LL-37 and IL-33. Immunohistochemically, IL-33 expression was enhanced in the skin of rosacea patients, especially with erythematotelangiectatic subtype. In vitro, UVB and LL-37 synergistically increased mRNAs expression of proinflammatory cytokines, especially IL-33 and IL-1β. IL-33 protein release was also synergistically increased by LL-37 and UVB treatment. LL-37 and IL-33 stimulated VEGF mRNA expression and VEGF release from HaCaT cells. Our findings suggest that rosacea skin with abundant LL-37 may robustly produce and release IL-33 when exposed to UV radiation. IL-33 may participate in the angiogenesis and vasodilation of rosacea skin by enhancing VEGF release.

Impact of G-CSF on expressions of Egr-1 and VEGF in acute ischemic cerebral injury.

The aim of the present study was to investigate the protective effect of granulocyte colony-stimulating factor (G-CSF) on acute ischemic cerebral injury, and its mechanism through the impact of G-CSF on early growth response-1 (Egr-1) and vascular endothelial growth factor (VEGF) expressions. Male Sprague-Dawley (SD) rats were divided them into three groups, i.e., the sham, model and G-CSF groups to measure the effect of G-CSF on the volume of cerebral infarction and level of lactate dehydrogenase (LDH) in rats. Hematoxylin and eosin (H&E) staining method was performed for histopathological examination. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis were used to detect the mRNA and protein expressions of Egr-1 and VEGF in different groups. Furthermore, Statistical Product and Service Solutions (SPSS) 17.0 software was applied to detect the differences in the expression of Egr-1 and VEGF between the two groups. Compared with the sham group, we found that the volume of cerebral infarction and LDH content in the model group were significantly elevated. By contrast, in the model group, those indicators in the G-CSF group were obviously decreased. H&E staining results also showed that G-CSF could decrease the necrotic area in cerebral infarction and the incidence of inflammation, and sustain the integrity of the molecular structure. Immunofluorescence staining results revealed that the protein expressions of Egr-1 and VEGF in the model group were all significantly decreased, while those in the G-CSF group were remarkably elevated. RT-PCR and western blot analysis revealed that the mRNA and protein expressions of Egr-1 and VEGF in the model group were decreased obviously, but those in the G-CSF group were elevated significantly, and the differences between the two groups showed statistical significance (P<0.05). G-CSF manifests a significant protective effect on the acute ischemic cerebral injury, which may be realized through its effect on the expressions of Egr-1 and VEGF.