Vascular Endothelial Growth Factor mRNA Expression Research Articles

MiR-600 suppresses HeLa cell proliferation by inhibiting hypoxia-inducible factor-1α signaling pathway

To determine whether miR-600 suppresses the proliferation of HeLa cells by inhibiting hypoxia-inducible factor-1α (HIF-1α) signaling pathway and its effect on expressions of cyclin D1 and vascular endothelial growth factor (VEGF). HeLa cells were transfected with miR-600 mimic and plasmid-HIF-1α, either alone or in combination, to up-regulate miR-600 and HIF-1α expressions in the cells. Six hours after the transfection, the cell viability was assessed using MTT assay, and the mRNA and protein expressions of VEGF, cyclin D1, and HIF-1α were analyzed with qPCR and Western blotting. The viability of HeLa cells showed no obvious changes 6 h after transfection with miR-600 mimic or Plasmid-HIF-1α. At 24 h and 48 h, the cells transfected with miR-600 mimic showed a time-dependent reduction of cell viability, while the cells transfected with Plasmid-HIF-1α alone and with both miR-600 mimic and Plasmid-HIF-1α showed increased cell viability. The cell viabilities in Plasmid-HIF-1α group were significantly higher than those in miR-600 mimic+Plasmid-HIF-1α group at 24 h and 48 h. Six hours after transfection with miR-600 mimic, the cells exhibited significantly decreased expressions of VEGF, cyclin D1, and HIF-1α, which were all significantly up-regulated in Plasmid-HIF-1α group and miR-600 mimic+Plasmid-HIF-1α group. VEGF, cyclin D1, and HIF-1α expressions were significant higher in Plasmid-HIF-1α group than in miR-600 mimic+ Plasmid-HIF-1α group. miR-600 suppresses the proliferation of HeLa cells and down-regulate the expressions of cyclin D1 and VEGF by inhibiting HIF-1α signaling pathway.

Human Red Blood Cells Modulate Cytokine Expression in Monocytes/Macrophages Under Anoxic Conditions.

In the bone marrow (BM) hematopoietic niche, the oxygen tension is usually very low. Such condition affects stem and progenitor cell proliferation and differentiation and, at cellular level regulates hematopoietic growth factors, chemokines and adhesion molecules expression. In turn, these molecules affect the proliferation and maturation of other cellular components of the niche. Due to the complexity of the system we started the in vitro investigations of the IL-6, IL-8, TNFα cytokines expression and the vascular endothelial growth factor (VEGF), considered key mediators of the hematopoietic niche, in human macrophages and macrophage cell line. Since in the niche the oxygen availability is mediated by red blood cells (RBCs), we have influenced the anoxic cell cultures by the administration of oxygenated or deoxygenated RBCs (deoxy RBCs). The results reported in this brief paper show that the presence of RBCs up-regulates IL-8 mRNA while IL-6 and VEGF mRNA expression appears down-regulated. This does not occur when deoxy RBCs are used. Moreover, it appears that the administration of RBCs leads to an increase of TNFα expression levels in MonoMac 6 (MM6). Interestingly, the modulation of these factors likely occurs in a hypoxia-inducible factor-1α (HIF-1α) independent manner. Considering the role of oxygen in the hematopoietic niche further studies should explore these preliminary observations in more details.

Loss of sphingosine 1-phosphate receptor 3 gene function impairs injury-induced stromal angiogenesis in mouse cornea

AbstractSphingosine 1-phosphate (S1P) is a bioactive sphingolipid generated through sphingosine kinase1 (SPK1)-mediated phosphorylation of sphingosine. We show here that injury-induced S1P upregulation increases corneal neovascularization through stimulating S1PR3, a cognate receptor. since this response was suppressed in S1PR3-knockout mice. Furthermore, Cayman10444, a selective S1PR3 inhibitor, reduced this response in WT mice. Such reductions in neovascularization were associated with reduced vascular endothelial growth factor A (VEGF-A) mRNA expression levels in WT TKE2 corneal epithelial cells and macrophages treated with CAY10444 as well as macrophages isolated from S1PR3 KO mice. S1P increased tube-like vessel formation in human vascular endothelial cells (HUVEC) and human retinal microvascular endothelial cells (HRMECs) cells expressing S1PR3. In S1PR3 KO mice, TGFβ1-induced increases in αSMA gene expression levels were suppressed relative to those in the WT counterparts. In S1PR3 deficient macrophages, VEGF-A expression levels were lower than in WT macrophages. Transforming growth factor β1(TGFβ1) upregulated SPK1 expression levels in ocular fibroblasts and TKE2 corneal epithelial cells. CAY10444 blocked S1P-induced increases in VEGF-A mRNA expression levels in TKE2 corneal epithelial cells. Endogenous S1P signaling upregulated VEGF-A and VE-cadherin mRNA expression levels in HUVEC. Unlike in TKE2 cells, SIS3 failed to block TGFβ1-induced VEGF-A upregulation in ocular fibroblasts. Taken together, these results indicate that injury-induced TGFβ1 upregulation increases S1P generation through increases in SPK1 activity. The rise in S1P formation stimulates the S1PR3-linked signaling pathway, which in turn increases VEGF-A expression levels and angiogenesis in mouse corneas.Injury-induced transforming growth factor β1 increases sphingosine 1-phosphate (S1P) generation by upregulating in sphingosine kinase 1 activity. The high levels of S1P formation stimulate the S1PR3-linked signaling pathway, which in turn increases vascular endothelial growth factor-A expression levels and angiogenesis in mouse corneas.

Effects of bioactive glass on proliferation, differentiation and angiogenesis of human umbilical vein endothelial cells

OBJECTIVE To investigate the effects of phytic acid derived bioactive P2O5-SiO2-CaO gel-glasses (PSC) on the proliferation, differentiation and angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro. METHODS HUVECs were cultured in PSC extracts, which were prepared with endothelial cell medium (ECM) at a gradient concentration of 0.01, 0.1, 1 and 2 g/L. Cells cultured in ECM were used as the control. The effect of PSC on HUVECs proliferation was assessed on the 1st, 3rd, 5th, 7th and 10th days with (4, 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide assay (MTT), and the optimum PSC concentration for HUVECs proliferation was used in the following experiments. The subsequent experiments were divided into two groups. The experimental group used PSC extracts to culture HUVECs (PSC group) and the control group used ECM to culture HUVECs (ECM group). Gene expression of angiogenic factors, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), was detected on the 2nd, 4th and 7th days by real-time reverse transcription-polymerase chain reaction (real-time RT-PCR). The morphology and number of tubules formation were observed at the 4th and 10th hours. Image J software was used for counting and quantitative analysis. RESULTS The results of MTT assay showed that 0.1 g/L PSC group had the most significant effect on promoting HUVECs proliferation. The optical density values of 0.1 g/L PSC group on the 5th and 7th days were significantly higher than those of the other PSC groups and the control group (P < 0.05). The result of real-time RT-PCR showed that 0.1 g/L PSC extract up-regulated the mRNA expression of VEGF and bFGF significantly (P < 0.05). On the 4th day, the gene expressions of VEGF and bFGF in PSC group were 1.59 and 1.45 times higher than those in ECM group respectively, and on the 7th day, the gene levels of VEGF and bFGF in PSC group were 1.98 and 1.37 times higher than those in ECM group respectively. The tubule formation assay showed that the maturity and density of the tubules in 0.1 g/L PSC group was much better than that in the ECM group at the 10th hour. The quantitative analysis by Image J indicated that the tubules number in PSC group (29.63±2.29) was higher than in the ECM group (20.13±2.36), with statistical significance (P < 0.05). CONCLUSION PSC showed significant promoting effects on HUVECs' proliferation, differentiation and angiogenesis in vitro.

Effects of hyperbaric oxygen therapy on vascular endothelial growth factor protein and mRNA in crush injury patients: A randomized controlled trial study

IntroductionLimb injury is a major health concern that imposes a direct danger to both life and limb viability. At Kandou Hospital Manado, hyperbaric oxygen (HBO) therapy has been performed as an adjunctive treatment for crush injury although no study has yet directly compared outcomes of HBO versus conventional therapy. Documentation of the association between HBO therapy and the healing process of crush injury is necessary to reduce the gaps in the literature and to establish an evidence-based clinical use of HBO therapy. MethodsIn this study, we assessed the changes in the vascular endothelial growth factor (VEGF) serum levels and VEGF mRNA expression as biomarkers of wound healing using ELISA and qRT-PC at four different measurement times: at baseline, after receiving initial treatment (debridement and limb-salvage surgeries), 2 h after the first session HBOT, and after a full 10 HBOT sessions. A randomized controlled trial (RCT) was used to enroll patients subjected to crush injury who were admitted to the Surgical Emergency Department (ER) of Kandou Hospital Manado, Indonesia. ResultsVEGF serum levels increased significantly in patients suffering from crush injury who received HBO therapy versus the control group. The increased VEGF serum is expected to result in an acceleration time of wound healing and a reduction in amputation rate. ConclusionThere was a significant difference between crush injury patients who received conservative therapy versus those receiving HBO therapies; thus, there was an increased likelihood of an accelerated wound healing and a reduction in the risk of amputation.

Triiodothyronine stimulates steroid and VEGF production in murine Leydig cells via cAMP-PKA pathway.

Thyroid hormones affect testicular development as well as functions like spermatogenesis and steroidogenesis, thereby influencing male fertility. Our group earlier showed that the stimulatory role of the thyroid hormone, T3 , on the production of vascular endothelial growth factor (VEGF) by murine Leydig cells is mediated by steroids and hypoxia-inducible factor-1 (HIF-1α). The current study further defines the signalling pathway(s) utilised by T3 to stimulate the production of steroids, VEGF and HIF-1α in mouse Leydig tumour cell line (MLTC-1). Specific inhibitors for different signalling molecules were used to study the role of cyclic AMP (cAMP), and its downstream mediators. Expression of VEGF and HIF-1α mRNA were measured by quantitative RT-PCR; VEGF secretion by ELISA; steroid secretion by radioimmunoassay and HIF-1α protein levels by western blotting. Inhibitors of adenylate cyclase (AC), protein kinase A (PKA), sarcoma kinase (SrcK), phosphoinositide 3-kinase (PI3K) and MAP kinase kinase (MEK1/2) abolished the T3 -induced increase in VEGF mRNA and protein levels. The same signalling molecules also mediated the increased production of steroids and HIF-1α protein in response to T3 . Therefore, it was concluded that T3 stimulates steroid secretion and HIF-1α protein in MLTC-1 cells through the AC-cAMP-PKA-PI3K-MEK pathway, which in turn stimulate VEGF production.

Knockdown of miR-203a-3p alleviates the development of bronchopulmonary dysplasia partly via the up-regulation of vascular endothelial growth factor A.

Bronchopulmonary dysplasia (BPD) is characterized by impaired vascular and alveolar development, and the underlying molecular mechanisms have remained elusive. MicroRNAs are important players in various biological functions including the pathogenesis of BPD. The present study aimed to examine the expression of miR-203a-3p in the peripheral blood of BPD patients and elucidate the mechanisms underlying miR-203a-3p-mediated progression of BPD. We examined the expression of miR-203a-3p in the peripheral blood of BPD patients and found that miR-203a-3p was up-regulated in the patients. Additionally, the mRNA expression levels of vascular endothelial growth factor A (VEGFA) and hypoxia-inducible factor-1alpha were down-regulated in the BPD patients. Further in vitro studies showed that miR-203a-3p suppressed the expression of VEGFA in RLE-6TN cells by targeting the VEGFA 3' untranslated region. Overexpression of miR-203a-3p inhibited the viability of RLE-6TN cells and induced cell apoptosis, whereas the knockdown of miR-203a-3p exerted opposite effects. VEGFA treatment significantly attenuated the increase in the RLE-6TN cell apoptotic rates induced by miR-203a-3p overexpression; while VEGFA knockdown significantly increased the cell apoptotic rates of RLE-6TN cells, which was partially reversed by the treatment with miR-203a-3p inhibitor. Furthermore, miR-203a-3p was up-regulated, whereas VEGFA was down-regulated in the lung tissues of BPD rats, and sequestration of the expression of miR-203a-3p prevented hyperoxia-induced lung damage, increased VEGFA mRNA and protein expression levels, and promoted the protein expression of ERK, PI3K, and p38 in the lung tissues of BDP rats. In summary, the findings of our study indicate that miR-203a-3p knockdown alleviates hyperoxia-induced lung tissue damage in the BPD rat model, and its effect may be associated with the up-regulation of VEGF.

Targeted Temperature Management Suppresses Hypoxia-Inducible Factor-1α and Vascular Endothelial Growth Factor Expression in a Pig Model of Cardiac Arrest.

BackgroundThe hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF)/VEGF receptor subtype 2 (VEGFR-2) pathway has been implicated in ischemia/reperfusion injury. The aim of this study was to clarify whether whole-body hypothermic targeted temperature management (HTTM) inhibits the HIF-1α/VEGF/VEGFR-2 pathway in a swine model of cardiac arrest (CA) and cardiopulmonary resuscitation (CPR).MethodsTwenty-four domestic male Beijing Landrace pigs were used in this study. CA was electrically induced with ventricular fibrillation and left untreated for 8 min. Return of spontaneous circulation (ROSC) was achieved in 16 pigs, which were randomly assigned either to normothermia at 38 °C or to HTTM at 33 °C (each group: n = 8). HTTM was intravascularly induced immediately after ROSC. The core temperature was reduced to 33 °C and maintained for 12 h after ROSC. The serum levels of HIF-1α, VEGF, VEGFR-2, and neuron-specific enolase (NSE) were measured with enzyme immunoassay kits 0.5, 6, 12, and 24 h after ROSC. The expression of HIF-1α, VEGF, and VEGFR-2 in cerebral cortical tissue was measured by RT-PCR and Western blot analysis 24 h after ROSC. Neurological deficit scores and brain cortical tissue water content were evaluated 24 h after ROSC.ResultsThe serum levels of HIF-1α, VEGF, and VEGFR-2 were significantly increased under normothermia within 24 h after ROSC. However, these increases were significantly reduced by HTTM. HTTM also decreased cerebral cortical HIF-1α, VEGF, and VEGFR-2 mRNA and protein expression 24 h after ROSC (all p < 0.05). HTTM pigs had better neurological outcomes and less brain edema than normothermic pigs.ConclusionThe HIF-1α/VEGF/VEGFR-2 system is activated following CA and CPR. HTTM protects against cerebral injury after ROSC, which may be part of the mechanism by which it inhibits the expression of components of the HIF-1α/VEGF/VEGFR-2 signaling pathway.

Effects of silencing epididymal vascular endothelial growth factor (VEGF) expression on hyaluronidase (HYD) activity in arsenic poisoning rats through downregulating VEGF receptor 2 (VEGFR2)

ABSTRACT RNA interference (RNAi) was used to investigate the role of epididymal vascular endothelial growth factor (VEGF) gene expression on sperm hyaluronidase (HYD) in a rat model of arsenic poisoning and to identify a new gene therapy target for male infertility caused by arsenic poisoning. The Rat model of chronic arsenic poisoning was established. And we found that positive expression of VEGF and VEGF receptor 2 (VEGFR2) was observed by Immunohistochemical staining in the epididymal tissues of arsenic-exposed rats. Subsequently, VEGF-shRNA-1, VEGF-shRNA-2 and VEGF shRNA-3 expression vectors containing epididymal VEGF-shRNA lentivirus were constructed and injected into the bilateral epididymis of each group of rats (Control group, NC-shRNA negative infection group, VEGF-shRNA-1 group, VEGF-shRNA-2 group, VEGF-shRNA-3 group) (n = 10 per group). Compared with the negative infection group and the normal control group, the expression of VEGF and VEGFR2 mRNA and protein levels were significantly decreased following epididymal infection. In addition, the HYD activity was all significantly lower than that in the normal control group and the negative infection group. Taken together, epididymal VEGF gene silencing may inhibit the activity of sperm HYD through downregulating VEGFR2.

Effects of electroacupuncture on proangiogenesis process and protein turnover in a mouse model of sarcopenia

To observe the effect of electroacupuncture(EA) on proangiogenesis process and protein turn-over in a mouse model of sarcopenia, so as to explore its potential molecular mechanism anti-aging. Fourteen 30-week-old male SAMP8 mice were randomly divided into a model group (n=7) and an EA group (n=7). Seven anti-rapidly aging SAMR1 mice of the same age were used as the control group (n=7). EA (1 mA, 4 Hz) was applied to bilateral "Zusanli"(ST36) and "Yanglingquan"(GB34) for 20 minutes each time once a day, 6 times a week for 4 weeks. The exhausted running platform was used to test the sports function. Gastrocnemius muscle mass and relative ratio of gastrocnemius muscle mass to body mass were measured. HE staining and transmission electron microscope were used to observe the morphology, and the cross-sectional area of gastrocnemius muscle was calculated. Relative protein expressions of protein kinase B (AKT) , phosphorylated (p) -AKT, mammalian target of rapamycin (mTOR) , p-mTOR, p70 ribosomal protein S6 kinase (p70S6K) , p-p70S6K,hypoxia inducible factor-1α (HIF-1α) and relative mRNA expressions of HIF-1α, vascular endothelial growth factor A (VEGF-A) , muscle RING finger-1 (MuRF-1) and muscle atrophy F-box (MAFbx) were detected by Western blot and real-time fluorescence quantitative PCR, seperatively. Compared with the control group, the running time and distance, body mass and gastrocnemius mass, and the ratio of gastrocnemius mass to body mass decreased(P<0.01, P<0.05), cross-sectional area of gastrocnemius, related protein expression of p-AKT,p-mTOR, p-p70S6K and HIF-1α, mRNA expression of HIF-1α and VEGF-A decreased (P<0.01), while mRNA expression of MuRF1 and MAFbx increased (P<0.01) in the model group. Following EA intervention, the running time and distance, body mass and gastrocnemius mass and the ratio of gastrocnemius mass to body mass increased (P<0.05), cross-sectional area of gastrocnemius, related protein expression of p-AKT,p-mTOR, p-p70S6K and HIF-1α, mRNA expression of HIF-1α and VEGF-A were significantly up-regulated (P<0.01), mRNA expression of MuRF1 and MAFbx down-regulated (P<0.01, P<0.05) in the EA group compared with the model group. EA may delay the aging muscle atrophy in mice by regulating the gastrocnemius muscle's proangiogenesis process and protein turnover.

Influence of porcine urinary bladder matrix and porcine acellular dermal matrix on wound healing of full-thickness skin defect in diabetic mice

Influence of porcine urinary bladder matrix and porcine acellular dermal matrix on wound healing of full-thickness skin defect in diabetic mice

Association of JMJD3, MMP-2 and VEGF expressions with clinicopathological features of invasive ductal breast carcinoma

To examine the expressions of JMJD3, matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) in invasive ductal breast carcinoma, their association with the clinicopathological features of the patients and the effect of JMJD3 overexpression on proliferation and MMP-2 and VEGF expressions in breast cancer cells. The protein and mRNA expressions of JMJD3, MMP-2, and VEGF in invasive ductal breast carcinoma and paired adjacent tissues were detected by immunohistochemistry and RT-PCR, respectively, and their correlation with the clinicopathological characteristics of the patients was analyzed. Kaplan-Meier survival analysis was used to evaluate the correlation of JMJD3, MMP-2 and VEGF expression levels with the survival of the patients. In breast cancer MDA-MB-231 cells transfected with a JMJD3-expression plasmid, the expression of Ki67 was examined immunohistochemically, the cell proliferation was assessed with CCK8 assay, and the mRNA expressions of MMP-2 and VEGF were detected with RT-PCR. Breast cancer tissues had significantly lower JMJD3 expression and higher MMP-2 and VEGF expressions at both the mRNA and protein levels than the adjacent tissue (P < 0.05). The positivity rates of JMJD3, MMP-2 and VEGF in breast cancer tissues were significantly correlated with tumor diameter, differentiation, TNM stage, lymph node metastasis, and molecular subtypes (P < 0.05). KaplanMeier analysis showed that JMJD3 expression level was positively while MMP-2 and VEGF were inversely correlated with the disease-free survival time of the patients (P < 0.05). Cox regression analysis identified JMJD3, MMP-2, VEGF and tumor differentiation as independent prognostic factors of breast cancer. Spearman correlation analysis suggested a negative correlation of JMJD3 with MMP2 (r=-0.569, P < 0.05) and VEGF (r=-0.533, P < 0.05) and a positive correlation between MMP2 and VEGF (r=0.923, P < 0.05). In MDA-MB-231 cells, overexpression of JMJD3 inhibited the proliferation of MDA-MB-231 cells and the expression of MMP-2 and VEGF. The expressions of JMJD3, MMP-2 and VEGF in invasive ductal breast carcinoma are closely correlated to tumor proliferation, invasion, metastasis and prognosis and can be used for prognostic evaluation of breast cancer.

Weichang'an Formula Inhibits Tumor Growth in Combination with Bevacizumab in a Murine Model of Colon Cancer-Making up for the Deficiency of Bevacizumab by inhibiting VEGFR-1.

Aim: Angiogenesis plays an important role in the initiation, development, and metastasis of malignant tumors. Antiangiogenic drugs combined with immune therapy are considered to have a synergistic effect on anti-tumor strategy. Weichang’an formula (WCAF) is a prescription of traditional Chinese medicine (TCM) based on pharmaceutical screening and clinical experience. The aim of this study is to examine the effect of WCAF and its combined action with Bevacizumab (BEV) in colorectal cancer, and to identify the possible mechanism of action. Methods: A human colon cancer cell (HCT 116) subcutaneous xenograft model was established in BALB/c-nu/nu mice. Tumor-bearing mice were randomized into each of four groups: control, WCAF treated, BEV treated, and WCAF plus BEV treated. Apoptosis was detected by TUNEL assay. Western blot was used to assess the protein levels of Leptin-R, STAT3, p-STAT3, BCL-2, and VEGFR-1. Immunohistochemistry was used to detect the micro-vessel density (MVD) and AKT1. Leptin and Vascular endothelial growth factor A (VEGF-A) mRNA expression were detected by Real-time PCR (RT-PCR). A network pharmacology study and validation assay were carried out to find the underlying molecular targets of WCAF related to immune regulation. Results: Compared with the control group, WCAF reduced tumor weight and volume, as well as promoted tumor cell apoptosis. WCAF treatment decreased the mRNA expression of Leptin and VEGF-A, while the protein levels of CD31, LEP-R, VEGFR-1, STAT3, and p-STAT3 were decreased in tumor tissues. In addition, VEGFR-1 protein expression was decreased in the WCAF group and the WCAF plus BEV group but not in the BEV group. The combination of WCAF and BEV demonstrated a partial additive anti-tumor effect in vivo. The pharmacological network also found there are 26 WCAF target proteins related to cancer immune and 12 cancer immune related pathways. The AKT1 protein expression in the WCAF and WCAF + BEV groups were significantly lower than the that in the control group (p < 0.01). Conclusion: WCAF can inhibit tumor growth and promote apoptosis and inhibit tumor angiogenesis in subcutaneous xenografts of human colon cancer HCT-116 in nude mice. WCAF also makes up for the deficiency of BEV by inhibiting VEGFR-1. The VEGFR-1 expression between the combination group and BEV alone achieved statistically significant difference (p < 0.01). Combined with BEV, WCAF showed a partial additive anti-tumor effect. The mechanism may be related to Leptin/STAT3 signal transduction, VEGF-A, VEGFR-1 and WCAF target proteins related to cancer immune such as leptin and AKT1.

Effects of Axial Compression and Distraction on Vascular Bud and VEGFA Expression in the Vertebral Endplate of an Ex Vivo Rabbit Spinal Motion Segment Culture Model.

An ex vivo study of the rabbit's vertebral endplate. The aim of this study was to assess the effect of axial compression and distraction on vascular buds and vascular endothelial growth factor (VEGFA) expression of the vertebral endplate (VEP). The abnormal load can lead to intervertebral disc degeneration (IDD), whereas axial distraction can delay this process. The effects of different mechanical loads on the intervertebral disc (IVD) have been hypothesized to be related to changes in the vascular buds of the VEP; moreover, the process that might involve the vascular endothelial growth factor (VEGF) within the VEP. Rabbit spinal segments (n = 40) were harvested and randomly classified into four groups: Control group, no stress was applied; Group A, a constant compressive load applied; Group B, compression load removed for a fixed time daily on a continuous basis, and substituted with a distraction load for 30 minutes; and Group C, compression removed for 30 minutes for a fixed period daily on a continuous basis. Tissue specimens were collected before the culture (day 0) and on day 14 post-culture of each group for analysis of IVDs' morphology, and protein and mRNA expression of Aggrecan, COL2al, VEGFA, and vascular endothelial growth factor receptor 2 of the VEPs. Application of axial distraction and dynamic load compression significantly delayed time- and constant compression-mediated VEP changes and IDD. Moreover, the degree of degeneration was associated with loss of vascular buds, as well as the downregulation of VEGFA and its receptor. The regulation of vascular buds and VEGF expression in the VEP represents one of the mechanisms of axial distraction and dynamic loading.Level of Evidence: N/A.

A role for phosphodiesterase type 5 inhibitors in remodelling the urinary bladder after radiation exposure.

Minimizing the toxicity of radiotherapy is challenging. We investigated the effects of a phosphodiesterase type-5 inhibitor (PDE5I) on the urinary bladder after pelvic radiotherapy. Eight rats were assigned to each group (group 1: control; group 2: radiation; group 3: radiation plus PDE5I). Radiation dose was 10 Gy/one fraction. Udenafil (20 mg/kg, daily for 4 weeks) was administered in group 3. Cystometry was performed 4 weeks after treatment, followed by real-time PCR for PDE5, vascular endothelial growth factor (VEGF), and endothelial nitric oxide synthase (eNOS) mRNA, western blotting for PDE5, cyclic GMP-dependent protein kinase (PRKG), VEGF164, Akt, eNOS and NADPH oxidase (NOX)-2 proteins, and immunohistochemistry for eNOS. The expression of both VEGF mRNA and eNOS mRNA was higher in group 3 than in group 2. VEGF and eNOS protein expression improved with PDE5I treatment. Akt protein phosphorylation was higher in group 3 than in group 2, but NOX-2 protein expression was lower in group 3 than in group 2. Immunohistochemistry showed that the mean density of arterioles expressing eNOS was higher in group 3 than in group 2. Cystometry revealed that the intercontraction interval was remarkably longer in group 3 than in group 2 but that the maximal voiding pressure was higher in group 2 than in group 3. Daily treatment with a PDE5I after radiotherapy may prevent bladder storage dysfunction, potentially due to its effects on vasodilation and angiogenesis and through minimizing tissue oxidative damage by means of the VEGF/Akt/eNOS pathway.

Activation of the VEGF-A/ERK/PLA2 Axis Mediates Early Retinal Endothelial Cell Damage Induced by High Glucose: New Insight from an In Vitro Model of Diabetic Retinopathy.

Early blood retinal barrier (BRB) dysfunction induced by hyperglycemia was related to increased pro-inflammatory activity of phospholipase A2 (PLA2) and the upregulation of vascular endothelial growth factor A (VEGF-A). Here, we tested the role of VEGF-A in high glucose (HG)-induced damage of human retinal endothelial cells (HRECs) mediated by Ca++-dependent (cPLA2) and Ca++-independent (iPLA2) PLA2s. HRECs were treated with normal glucose (5 mM, NG) or high glucose (25 mM, HG) for 48 h with or without the VEGF-trap Aflibercept (Afl, 40 µg/mL), the cPLA2 inhibitor arachidonoyl trifluoromethyl ketone (AACOCF3; 15 µM), the iPLA2 inhibitor bromoenol lactone (BEL; 5 µM), or VEGF-A (80 ng/mL). Both Afl and AACOCF3 prevented HG-induced damage (MTT and LDH release), impairment of angiogenic potential (tube-formation), and expression of VEGF-A mRNA. Furthermore, Afl counteracted HG-induced increase of phospho-ERK and phospho-cPLA2 (immunoblot). VEGF-A in HG-medium increased glucose toxicity, through upregulation of phospho-ERK, phospho-cPLA2, and iPLA2 (about 55%, 45%, and 50%, respectively); immunocytochemistry confirmed the activation of these proteins. cPLA2 knockdown by siRNA entirely prevented cell damage induced by HG or by HG plus VEGF-A, while iPLA2 knockdown produced a milder protective effect. These data indicate that VEGF-A mediates the early glucose-induced damage in retinal endothelium through the involvement of ERK1/2/PLA2 axis activation.

Effect of mild hypothermia on behaviors of rats with intracerebral hemorrhage and the possible mechanism

To explore the effect of mild hypothermia on inflammatory response and angiogenesis in brain tissues of rats with intracerebral hemorrhage (ICH) and its possible mechanism for improving behavioral deficits of the rats After ICH. A total of 120 healthy male SD rats were randomly divided into sham operation group, ICH group and mild hypothermia group. Rat models of ICH were established in the latter two groups by stereotactic injection of autogenous blood in the brain, and the rats in the sham operation group received injection of normal saline in the same manner. At 15 min after modeling, the rats in hypothermia group were subjected to mild hypothermia (30-32 ℃) for 8 h followed by rewarming (37-38 ℃); the body temperature was maintained at 37-38 ℃ in the other two groups. At 2, 4, 7, 14 and 21 days after the treatment, Longa scoring, balance beam scoring and Berderson scoring were used to evaluate the behavioral deficits of the rats. Immunohistochemical staining was used to detect the protein expressions of tumor necrosis factor-α (TNF-α) and nuclear factor-κB (NF-κB) in the brain tissue of the rats, and the mRNA expressions of α subunit of hypoxia-inducible factor 1 (HIF1-α) and vascular endothelial growth factor (VEGF) were detected using RT- PCR. At 2, 4, 7, 14 and 21 days after the treatment, the behavioral scores of the rats were significantly higher in ICH group and mild induced hypothermia group than in the sham operation group (P < 0.05 or 0.01). The protein expressions of TNF-α and NF-κB and mRNA expressions of HIF1-α and VEGF were significantly higher in ICH group and mild hypothermia group than in the sham operation group (P < 0.01). The behavioral scores were significantly lower in mild hypothermia group than in ICH group (P < 0.05), and the protein expressions of TNF-α and NF-κB were lower and the mRNA expressions of HIF1- α and VEGF were higher in mild hypothermia group than in ICH group (P < 0.05 or 0.01). Mild hypothermia can improve behavioral deficits in rats with ICH possibly by antagonizing brain inflammation and promoting angiogenesis.

Effects of ultrasound on vascular endothelial growth factor in cartilage, synovial fluid, and synovium in rabbit knee osteoarthritis.

Ultrasound is commonly used to treat knee osteoarthritis (KOA), which has unique advantages with regard to relieving pain and inflammation as well as delaying cartilage degeneration, but the underlying mechanisms are less clear. The study aimed to investigate the therapeutic effects of ultrasound on vascular endothelial growth factor (VEGF) expression in cartilage, the synovium, and synovial fluid (SF) in a rabbit model of KOA. Twenty-four New Zealand rabbits were randomly divided into ultrasound (group A), sham ultrasound (group B) and no-ACLT control groups (group C). Six weeks after undergoing anterior cruciate ligament transection (ACLT), group A was treated with ultrasound and group B was treated with sham ultrasound. Two weeks thereafter, the morphology of the synovium and cartilage were observed. Cartilage and synovium were scored using the Mankin scale and Krenn V scores, respectively. VEGF expression in the cartilage, SF, and synovium of ACLT knee joints was analyzed via immunohistochemistry, western blotting, and RT-PCR. Cartilage degeneration and synovitis were the most severe in group B and the least severe in group C. Similarly, Mankin scores and Krenn V scores were highest in group B and lowest in group C (p<0.05). There were also significant differences in the VEGF IOD of cartilage or synovium, VEGF protein content in SF, and VEGF mRNA expression in cartilage or SF (p<0.05). Ultrasound can relieve synovitis and delay cartilage degradation, and the mechanisms of ultrasound for the treatment of KOA may involve inhibition of the expression of VEGF in the synovium, SF, and cartilage.

MiR-126 facilitates apoptosis of retinal ganglion cells in glaucoma rats via VEGF-Notch signaling pathway.

The aim of this study was to explore the effect of micro ribonucleic acid (miR)-126 on the apoptosis of retinal ganglion cells in glaucoma rats via the vascular endothelial growth factor (VEGF)-Notch signaling pathway. A total of 36 Sprague-Dawley (SD) rats were randomly divided into three groups, including normal group (n=12), model group (n=12) and miR-126 antagomir group (n=12). Rats in normal group did not receive any treatment. In model group and miR-126 antagomir group, the rats were used to establish glaucoma models and intervened with normal saline and miR-126 antagomir, respectively. Intraocular pressure was detected at the completion of modeling and the last intervention, at 7 days after which samples were taken. Western blotting was adopted to detect the relative protein expressions of Notch1 and Notch2. The content of VEGF was examined via enzyme-linked immunosorbent assay (ELISA). Quantitative polymerase chain reaction (qPCR) was carried out to detect the messenger RNA (mRNA) expressions of VEGF, Notch1 and Notch2. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to detect cell apoptosis. After modeling, intraocular pressure in model group and miR-126 antagomir group was significantly higher than that in normal group (p<0.05). At the end of the intervention, intraocular pressure in miR-126 antagomir group was notably lower than that in model group (p<0.05). VEGF content in model group and miR-126 antagomir group was notably higher than that in normal group (p<0.05). However, it was markedly lower in miR-126 antagomir group than model group (p<0.05). Model group exhibited remarkably higher protein expressions of Notch1 and Notch2 than normal group (p<0.05). However, the protein expressions of Notch1 and Notch2 in miR-126 antagomir group were evidently reduced (p<0.05). Besides, the mRNA expressions of VEGF, Notch1 and Notch2 in model group were significantly higher than those in normal group (p<0.05). However, they were significantly lower in miR-126 antagomir group than those in model group (p<0.05). Furthermore, the apoptosis rate in model group was distinctly higher than that in normal group (p<0.05). However, it was notably lower in miR-126 antagomir group than model group (p<0.05). MiR-126 facilitates the apoptosis of retinal ganglion cells in glaucoma rats by promoting the VEGF-Notch signaling pathway.

A Comparative Study: Gene Expression VEGF, FLT-1and KDR And level of PLGF In Preeclampsia And Normal Pregnancy.

involved The vascular endothelial growth factor (VEGF) family of angiogenic growth factors in the pathophysiology of these inconveniences . aims This study to examine Maternal serum levels of placental growth factor (PLGF) and Placental mRNA expression of VEGF, fms-like tyrosine kinase 1( FLT-1) and kinase insert domain receptor (KDR) in normotensive control (NC) women (n = 24) and women with preeclampsia (PE) (n = 50) [25 severe PE (SPE) and 25 Mild PE (MPE)]. We conducted a cross-sectional study in 50 preeclamptic women: 25women with mild PE, 25 patients with severe PE. While 24 normotensive pregnant women matched for age as a control group. we measured by real time PCR (RTPCR) Placental mRNA expression of VEGF FLT-1and KDR . and we measured Maternal serum levels of PLGF by ELISA. there was statistically significant difference between the three study groups as regards all 4 studied laboratory variables. KDR, FLT-1 and PLGF were all statistically significantly higher in control group as compared to either of the two PE groups. The mean VFGF expression was statistically higher in cases of severe PE than Mild PE, both were higher than control group (p < 0.0005).