Vascular Endothelial Growth Factor mRNA Research Articles

Prognostic significance of VHL, HIF1A, HIF2A, VEGFA and p53 expression in patients with clear‑cell renal cell carcinoma treated with sunitinib as first‑line treatment

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell cancer, characterized by the highest mortality rate among other RCC subtypes due to the occurrence of metastasis and drug resistance following surgery. The Von Hippel-Lindau tumor suppressor (VHL)-hypoxia-inducible factor 1 subunit α (HIF1A)/hypoxia-inducible factor 2α (HIF2A)-vascular endothelial growth factor A (VEGFA) protein axis is involved in the development and progression of ccRCC, whereas sunitinib, a tyrosine kinase inhibitor, blocks the binding of VEGFA to its receptor. The aim of the present study was to examine the possible association of the gene expression of VHL, HIF1A, HIF2A, VEGFA and tumor protein P53 (P53) in cancer tissue with the outcome of ccRCC patients who were treated with sunitinib as first-line therapy following nephrec-tomy. A total of 36 ccRCC patients were enrolled, 11 of whom were administered sunitinib post-operatively. Tumor and control samples were collected, and mRNA and protein levels were assessed by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. High mRNA and protein expression levels of HIF2A and VEGFA were found to be associated with shorter overall survival (OS) and progression-free survival (PFS) rates, as well as with unfavorable risk factors of cancer recurrence and mortality. Resistance to sunitinib was also observed; the OS and PFS rates were shorter (median OS and PFS: 12 and 6 months, respectively, vs. undetermined). Sunitinib resistance was associated with high HIF2A and VEGFA protein levels (b=0.57 and b=0.69 for OS and PFS, respectively; P<0.001). Taken together, the findings of this study suggest that the protein levels of HIF2A and VEGFA in tumor tissue may serve as independent prognostic factors in ccRCC. ccRCC patients with increased intratumoral HIF2A and VEGFA protein levels, and unaltered VHL protein levels, are not likely to benefit from sunitinib treatment following nephrectomy; however, this hypothesis requires verification by large-scale replication studies.

Semaphorin 7a participants in pterygium by regulating vascular endothelial growth factor.

To investigate the relationship between semaphorin 7a expression and cell proliferation and migration in pterygium fibroblasts. Twenty-six patients with surgically diagnosed pterygium were enrolled, including 15 cases of primary pterygium and 11 cases of recurrent pterygium. In addition, 12 cases of normal conjunctival tissue were collected. The expression of semaphorin 7a in normal conjunctival tissue, primary pterygium and recurrent pterygium was detected by real-time polymerase chain reaction. Recurrent pterygium fibroblasts were isolated and cultured, and the expression of semaphorin 7a was silenced by small interfering RNA (siRNA) interference technique. Furthermore, the effects of si-semaphorin 7a interference on the mRNA and protein levels of β1-integrin, vascular endothelial growth factor A (VEGFA) and vascular endothelial growth factor receptor (VEGFR), and on fibroblast proliferation were analyzed. Transwell assay was used to detect the effect of semaphorin 7a interference on fibroblast migration. Semaphorin 7a was highly expressed in the primary pterygium and recurrent pterygium samples than that of the normal conjunctival tissue. Compared with the primary pterygium, the expression of semaphoring 7a in the recurrent pterygium samples was significantly increased (P<0.05). The mRNA and protein expression levels of β1-integrin, VEGFA and VEGFR were decreased after si-semaphorin 7a transfection, and as well as the cell proliferation and migration. Semaphorin 7a might play important roles in the pathogenesis of pterygium by affecting the expression of β1-integrin, VEGFA and VEGFR.

Electroacupuncture on ST36 and GB39 Acupoints Inhibits Synovial Angiogenesis via Downregulating HIF-1α/VEGF Expression in a Rat Model of Adjuvant Arthritis.

Introduction. The hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) play a key role in synovial angiogenesis in rheumatoid arthritis (RA). Therefore, this study aimed to test the hypothesis that electroacupuncture (EA) may inhibit RA synovial angiogenesis via HIF-1α/VEGF expression. Methods. Sprague-Dawley rats were randomly distributed to 4 groups: control, adjuvant arthritis (AA), AA+electroacupuncture (AA+EA), and AA+sham EA groups. AA model was induced by injection of Freund's complete adjuvant in bilateral hind footpad. 3 days after injection, EA was delivered to the acupoints Zusanli (ST 36) and Xuanzhong (GB 39) once every two days for a total of 8 times in the AA+EA group, while sham EA treatment was applied in the AA+sham EA group. The arthritis score, paw volume, and H&E staining for each animal were measured. CD34 expression in synovial tissue of ankle joint was observed by immunohistochemistry. HIF-1α and VEGF mRNA and protein levels in synovial tissue were determined by real-time quantitative PCR and Western blot, respectively. Results. Compared with rats in AA group, EA stimulation significantly decreased arthritis scores, paw volume, and pathological damage of synovial tissues. Moreover, EA markedly suppressed the synovial angiogenesis of AA rats, as evidenced by reduced CD34 positive expression. Furthermore, EA significantly reduced HIF-1α and VEGF mRNA and protein levels in synovial of AA rats. Finally, the CD34 expression in synovial tissue was positively correlated with HIF-1α and VEGF protein levels. Conclusion. EA on ST36 and GB39 acupoints can effectively inhibit synovial angiogenesis in the AA rat model via downregulating HIF-1α/VEGF expression.

Inhibitory Effect of Semen Litchi Drug Serum on the Proliferation of Human Hepatoma HepG2 Cells and Expression of VEGF and MMP-9.

To investigate inhibitory effect of semen litchi drug serum on proliferation of human hepatoma HepG2 cells and its effect on the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9). An experimental study. College of Pharmacy, Guangxi University of Chinese Medicine, China, from June 2017 to January 2018. Semen litchi drug serum with concentrations of 0 mg/kg (control group), 3 g/kg (low dose group), 6 g/kg (medium dose group) and 12 g/kg (high dose group) was used to act on HepG2 cells at the logarithmic phase. Inhibitory effect of semen litchi drug serum on cell growth, expression of VEGF and MMP-9 mRNA and protein was detected. Inhibitory effect of semen litchi drug serum on the proliferation of HepG2 cells significantly increased with the increase of drug concentration, which was dose-time dependent. Expression levels of VEGF and MMP-9 mRNA in HepG2 cells after 48 hours of treatment by semen litchi low-dose group, medium-dose group, and high-dose group were lower than those in control group (all p <0.001). After acting on HepG2 cells for 48 hours, relative expressions of VEGF and MMP-9 protein in semen litchi low-dose group, medium-dose group, and high-dose group were lower than those in control group (all p<0.001). Semen litchi drug serum can inhibit proliferation of hepatoma cells in vitro. The anti-hepatoma effect of semen litchi drug serum may be exerted through down-regulating the expression of VEGF and MMP-9 and inhibiting angiogenesis of hepatocellular carcinoma.

Exosomes secreted by endothelial progenitor cells improve the bioactivity of pulmonary microvascular endothelial cells exposed to hyperoxia in vitro.

Paracrine factors secreted by endothelial progenitor cells (EPCs) are suggested to be responsible, in part, for the improved microvascular development in bronchopulmonary dysplasia (BPD) models. This study aims to investigate the potential role of exosomes derived from EPCs (EPC-EXOs), a component of paracrine secretion, in angiogenesis by mediating the activity of PMVECs exposed to hyperoxia. EPCs were isolated from bone marrow of rats. EPC-EXOs were isolated by ExoQuick-TC kits from the conditioned media of EPCs. The PMVECs were divided into three groups, including the normal group, the hyperoxia group (exposed to 85% O2) and the EPC-EXOs treatment group (exposed to 85% O2 and EPC-EXOs with the concentration of 100 µg/mL). The activities of proliferation, migration and tube formation of PMVECs were detected at the endpoint. The mRNA and protein expression levels of VEGF, VEGFR2 and eNOS in different groups were detected by real-time quantitative PCR and western blot. We found EPC-EXOs exhibited a cup or biconcave morphology, with the size ranging from 30 to 150 nm, and positive for the characteristic exosomal surface marker proteins, CD63 and TSG101. Comparing to the control group, Hyperoxic stress impaired the proliferation, migration, and tubule formation of PMVECs, and decreased the expression of endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), and vascular endothelial growth factor receptor 2 (VEGFR-2) of PMVECs. Comparing to the hyperoxia group, EPC-EXOs treatment enhanced the bioactivity of PMVECs in vitro, and increased the expression of eNOS, VEGF and VEGFR2. Our data demonstrate EPCs secrete exosomes that have independent angiogenic activity in vitro. This may help explain in part the protective effects of EPCs on hyperoxic injury in the developing lung vasculature and may represent a promising therapeutic strategy for BPD.

Effects of Obesity and Acute Resistance Exercise on Skeletal Muscle Intercellular Communication Pathways

Obesity (OB) disrupts cellular communication consistent with lower skeletal muscle capillarization. Exosomes, small microvesicles, transport and deliver mRNA, miRNA, and proteins in an endocrine manner and are released by muscle during aerobic exercise. The effects of resistance exercise (REx) on exosome biogenesis is unknown. PURPOSE: Investigate if resistance exercise increases skeletal muscle exosome biogenesis pathways and if this response is impaired in obesity. METHODS: Lean (LN) and obese (OB) (n=8/group) sedentary men and women performed 3 sets of 8-12 repetitions/set of acute, single leg knee extension resistance exercise at 80% of 1-RM. Vastus lateralis biopsies were obtained at rest and at 15 min, and 3 hr post-exercise. Muscle mRNA, protein expression, fiber typing, and capillary staining were measured. RESULTS: The gene expression of the exosome biogenesis components hepatocyte growth factor-regulated tyrosine kinase (HGS) and vacuolar protein sorting mutant (VPS4a) were lower in OB than LN at rest (~25%) and at 15 min post- (~20%), but not 3 hr post-exercise. Expression of exosome surface markers apoptotic linked gene-2 interacting protein X (Alix) was lower (OB ~35% and LN ~20% 15min post-exercise) and tumor susceptibility gene-101 (TSG-101) was higher (OB ~50% and LN ~40% 3hr post-exercise) in response to REx in both groups. Acute resistance exercise increased vascular endothelial growth factor (VEGF) mRNA similarly in LN and OB. Interestingly, anti-angiogenic thrombospondin-1 (TSP-1) mRNA was increased by acute REx only in OB (~230% 3hr post-exercise). miR-130a (angiogenesis), miR-206 (myoblast to myotube differentiation) and miR-503 (repressor of cell proliferation) were increased in OB at rest and following exercise. Type II fiber size was greater and capillary density was lower in OB. CONCLUSION: Obesity alters skeletal muscle exosome biogenesis, angiogenic, and muscle differentiation pathways possibly contributing to greater muscle fiber size and lower muscle capillarization. Resistance exercise alters skeletal muscle exosome marker expression similarly in both lean and obese.

Ureaplasma species modulate cell adhesion molecules and growth factors in human brain microvascular endothelial cells

Ureaplasma species (spp.) are considered commensals of the adult urogenital tract, but may cause chorioamnionitis and preterm birth as well as sepsis and meningitis in neonates. Pathomechanisms in Ureaplasma-driven neuroinflammation are largely unknown. This study addressed mRNA and protein expression of intercellular and vascular cell adhesion molecules (ICAM-1, VCAM-1), granulocyte-colony stimulating factor (G-CSF), and vascular endothelial growth factor (VEGF) in native or lipopolysaccharide (LPS) co-stimulated human brain microvascular endothelial cells (HBMEC) exposed to Ureaplasma (U.) urealyticum or U. parvum. Ureaplasma spp. reduced G-CSF mRNA (p < 0.05) and protein expression (p < 0.01) and increased VEGF mRNA levels (p < 0.01) in native HBMEC. Upon co-stimulation, Ureaplasma isolates enhanced LPS-evoked VEGF and ICAM-1 mRNA expression (p < 0.05), but mitigated G-CSF and VCAM-1 mRNA responses (p < 0.05). In line with previous findings, our results indicate an ability of Ureaplasma spp. to compromise blood-brain barrier integrity, mitigate immune defense, and subdue neuroprotective mechanisms. This may facilitate intracerebral inflammation, allow chronic infections, and promote brain injury. More pronounced effects in co-stimulated cells may indicate an immunomodulatory capacity of Ureaplasma spp.

Vascular endothelial growth factor is up-regulated by leukemia inhibitory factor and interleukin-6 in human endometriotic stromal cells

Objective: To compare the expression of leukemia inhibitory factor (LIF), interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) in tissue and fluid samples from patients with endometriosis, and investigate whether LIF and IL-6 regulate VEGF in human endometriotic stromal cells (ESC). Methods: The levels of VEGF, LIF, IL-6 in serum, peritoneal fluid of patients with and without endometriosis were measured by ELISA. The mRNA of these three factors in the ectopic and eutopic endometrial tissue and stromal cells were measured by real-time PCR. ESC derived from ovarian endometriomas were cultured using the method of primary cell culture with LIF and IL-6, and the level of VEGF mRNA and protein were measured by the method of real-time PCR and ELISA respectively. Results: VEGF and IL-6 concentration were 1.2 and 1.3 times higher in the serum of patients with endometriosis than in the control group [(94±19) versus (78±17) ng/L; (45±14) versus (35±9) ng/L; all P<0.05]. VEGF and IL-6 concentration were 1.2 and 1.4 times higher in the peritoneal fluid of patients with endometriosis than in the control group [(110±25) versus (91±21) ng/L; (69±20) versus (49±15) ng/L; all P<0.05]. VEGF and IL-6 concentrations in peritoneal fluid of patients with endometriosis were 1.2 and 1.5 times higher than in serum (all P<0.01). VEGF, LIF and IL-6 mRNA expression were 2.2, 8.6, 44.7 times higher in ESC compared with the matching eutopic endometrial stromal cells (all P<0.01). LIF and IL-6 mRNA were 2.0 and 64.8 times higher in ectopic endometrial tissue than the matching eutopic endometrial tissue (all P<0.05).ESC cultured with LIF, IL-6 and LIF+IL-6 induce VEGF protein secretion [(106±18), (124±30), (140±27) ng/L] by 1.3, 1.5 and 1.7 times (all P<0.05). Conclusion: Overexpression of LIF and IL-6 may synergistically contribute to upregulation of VEGF in ESC and promote development of endometriosis.

The Effect and Mechanism of Vascular Endothelial Growth Fac-tor (VEGF) on Tumor Angiogenesis in Gallbladder Carcinoma

To investigate the effect of vascular endothelial growth factor (VEGF) on tumor angiogenesis in gallbladder carcinoma. Fifty one patients with gallbladder carcinoma were enrolled as observation group. Thirty healthy people were included as control group. Chemically synthesized siRNA sequences targeting VEGF was transfected with VEGF-siRNA. A blank group (group B), a negative control group (transfected with independent sequence, group C), and an inhibition group (transfected with VEGF siRNA, group D) were established. Physiological saline was set as group A. The expression of VEGF was detected by qRT-PCR. The expression of VEGF protein was detected by Western blot. MVD was used to measure microvessel density. CCK-8, Transwell and flow cytometry were used to detect cell proliferation, invasion and apoptosis. The tumor volume of nude mice and VEGF mRNA expression in group D was significantly smaller than that in group B and C (P<0.05). The MVD density in group B and C was significantly higher than that in group D (P<0.01). The proliferation of cells was detected from the 3rd day, and the proliferation of cells in the blank and negative control groups was faster than that of the inhibition group (P<0.05). The apoptosis rate of the blank group and the negative control group was lower than that of the inhibition group (P<0.001). VEGF is highly expressed in serum of patients with cholangiocarcinoma, it promotes angiogenesis, proliferation and invasion of gallbladder cancer cells, and inhibits apoptosis of tumor cells.

Exosomes from Microglia Attenuate Photoreceptor Injury and Neovascularization in an Animal Model of Retinopathy of Prematurity

The role of microglia in the pathophysiology of ischemic retinal diseases has been studied extensively. Exosomes from microglial cells exert protective effects during several nervous system diseases, but their roles in hypoxia-induced retinopathy remain unclear. In our study, exosomes derived from microglial cells were injected into the vitreous body of mice with oxygen-induced retinopathy (OIR). Results showed that exosome-treated OIR mice exhibited smaller avascular areas and fewer neovascular tufts in addition to decreased vascular endothelial growth factor (VEGF) and transforming growth factor β (TGF-β) expression. Moreover, photoreceptor apoptosis was suppressed by exosome injection. Mechanistically, exosomes from microglial cells were incorporated into photoreceptors in vitro and inhibited the inositol-requiring enzyme 1α (IRE1α)-X-box binding protein 1 (XBP1) cascade, which contributes to hypoxia-induced photoreceptor apoptosis. Furthermore, the exosomes also downregulated the mRNA and protein levels of VEGF and TGF-β in hypoxia-exposed photoreceptors. A microRNA assay showed that microRNA-24-3p (miR-24-3p) levels were extremely high in exosomes from microglial cells, suggesting that this could be the key molecule that inhibits the hypoxia-induced expression of IRE1α in photoreceptors. These findings delineate a novel exosome-mediated mechanism of microglial cell-photoreceptor crosstalk that facilitates normal angiogenesis and visual function in OIR mice; thus, our results also suggest a potential therapeutic approach for retinopathy of prematurity.

Hormonal regulation of vascular endothelial growth factor A (VEGFA) gene expression in granulosa and theca cells of cattle1.

Vascular endothelial growth factor A (VEGFA) stimulates angiogenesis and is associated with increased vascularity in ovarian follicles of cattle. The objectives of this study were to investigate the developmental and hormonal regulation of VEGFA expression in ovarian granulosa and theca cells (TC) of cattle. Bovine ovaries were collected from a local slaughterhouse and granulosa cells (GC) and TC were collected from small (SM; 1 to 5 mm) and large (LG; 8 to 20 mm) follicles. Cells were collected fresh or cultured in serum-free medium and treated with various factors that regulate angiogenesis and follicular development. RNA was collected for analysis of VEGFA mRNA abundance via quantitative PCR. In SM-follicle GC (SMGC), prostaglandin E2 (PGE2) and FSH decreased (P < 0.05) VEGFA mRNA abundance by 30 to 46%, whereas in LG-follicle GC (LGGC), PGE2 and FSH were without effect (P > 0.10). In SMGC, dihydrotestosterone (DHT), sonic hedgehog (SHH), and growth differentiation factor-9 (GDF9) decreased (P < 0.05) VEGFA expression by 30 to 40%. Fibroblast growth factor-9 (FGF9) and estradiol (E2) were without effect (P > 0.10) on VEGFA mRNA in both SMGC and LGGC, whereas progesterone increased (P < 0.05) VEGFA mRNA in LGGC but had no effect in LGTC. Bone morphogenetic protein-4 (BMP4), LH, and FGF9 increased (P < 0.05) abundance of VEGFA mRNA by 1.5- to 1.9-fold in LGTC. Insulin-like growth factor-1 (IGF1) was without effect (P > 0.10) on VEGFA mRNA in both TC and GC. An E2F transcription factor inhibitor, HLM0064741 (E2Fi), dramatically (i.e., 8- to 13-fold) stimulated (P < 0.01) the expression of VEGFA mRNA expression in both SMGC and LGTC. Abundance of VEGFA mRNA was greater (P < 0.05) in LGGC and SMGC than in LGTC. Also, SMTC had greater (P < 0.05) abundance of VEGFA mRNA than LGTC. In conclusion, VEGFA mRNA abundance was greater in GC than TC, and VEGFA expression decreased in TC during follicle development. Some treatments either suppressed, stimulated, or had no effect on VEGFA expression depending on the cell type. The inhibition of E2F transcription factors had the greatest stimulatory effect of all treatments evaluated, and thus, E2Fs may play an important role in regulating angiogenesis during follicle growth in cattle.

Inhibitory effect of Tanshinone IIA on the proliferation of human retinal pigment epithelial cells in hypoxia and its mechanism

Objective To investigate the effect of Tanshinone ⅡA on the proliferation and the signaling pathway of human retinal pigment epithelial (RPE) cells in hypoxia. Methods CoCl2 (150 μmol/L) was used to simulate hypoxic condition and the ARPE-19 cells cultured in vitro were divided into blank control group, hypoxia control group, Tanshinone ⅡA group and hypoxia-inducible factor-1α (HIF-1α) inhibitor group.The different doses of Tanshinone ⅡA were used to treat ARPE-19 for 24, 48 and 72 hours, respectively.The inhibitory rate of cell proliferation of different groups were detected by MTT after 24, 48 and 72 hours of administration cultured, and the apoptosis rate and the cell cycle distribution of cells in the hypoxia were analyzed by flow cytometry.Real-time PCR and Western blot were used to detect the expressions of mRNA and protein of HIF-1α and vascular endothelial growth factor (VEGF). Results MTT assay showed that Tanshinone ⅡA could inhibit the proliferation of ARPE-19 cells in a dose- and time-dependent manner, and the proliferation inhibitory rate gradually increased in the 1, 5 and 10 mg/L Tanshinone ⅡA groups, with significant differences between any two groups (all at P 0.05). Conclusions Tanshinone ⅡA can inhibit the proliferation of RPE cells, induce apoptosis by arresting cells at G0/G1 phase.The mechanism is related to the suppression of HIF-1α/VEGF signaling pathway. Key words: Tanshinone ⅡA; Retinal pigment epithelial cells; Hypoxia-inducible factor-1α; Vascular endothelial growth factor; Proliferation

Correlation of MiR-152 expression with VEGF expression in placental tissue of preeclampsia rat and its influence on apoptosis of trophoblast cells.

To detect the expression of micro ribonucleic acid (miR)-152 in the placental tissues of preeclampsia (PE) rats and its correlation with the expression of vascular endothelial growth factor (VEGF), and to investigate the influence of miR-152 on the apoptosis of trophoblast cells. A Sprague Dawley (SD) rat model of PE was established, the total RNA and total protein in the placental tissues were extracted, and reverse transcription-polymerase chain reaction (RT-PCR) was applied to determine the expression level of miR-152 in the placental tissues. The rats were divided into miR-152 high expression group (High group) and miR-152 low expression group (Low group) according to the expression level of miR-152. RT-PCR, immunohistochemistry and Western blotting assay were performed to detect the expressions of VEGF messenger RNA (mRNA) and protein, respectively, in the placental tissues of the two groups of rats. Meanwhile, the BeWo trophoblast cell lines were used in in-vitro experiment, which were divided into Control group and miR-152 mimic group. 10 μL miR-152 mimic were added into each well plate in miR-152 mimic group, and 24 h later, TUNEL staining and flow cytometry were utilized to assess the cell apoptosis in both groups. At the same time, the expressions of B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax) in the two groups of cells were measured using Western blotting assay. The expression level of miR-152 in the placentas of PE rats was increased markedly compared with that of normal rats. The expression levels of VEGF mRNA and protein in the rat placenta in High group were notably higher than those in Low group. In vitro experiment results indicated that miR-152 mimic could promote the apoptosis of BeWo trophoblast cells, up-regulate the pro-apoptotic gene Bax and inhibit the anti-apoptotic gene Bcl-2 simultaneously. The expression of miR-152 is increased in the placental tissues of PE rats, and it is positively correlated with VEGF. In addition, the increased miR-152 expression can promote the apoptosis of trophoblast cells.

The Role of the Local Bone Marrow Renin-Angiotensin System in Multiple Myeloma

Objective:Angiotensin II promotes growth and angiogenesis via type 1 receptors (AGTR1) in certain tumors. In this study, we examine the bone marrow AGTR1 expression in multiple myeloma (MM) and its relationship with the regulation of angiogenesis and prognostic factors.Materials and Methods:Bone marrow AGTR1 mRNA levels of 39 MM patients and 15 healthy controls were analyzed with quantitative RT-PCR. Immunohistochemical staining of the tissue vascular endothelial growth factor (VEGF), CD34, and factor VIIIrAg (fVIIIrAg) was used to assess bone marrow angiogenesis.Results:Bone marrow samples of the patients showed increased VEGF, fVIIIrAg, and CD34 staining and higher AGTR1 expression levels when compared to controls. Patients with severe-diffuse bone marrow infiltration showed higher bone marrow VEGF, fVIIIrAg, CD34, and AGTR1 mRNA levels when compared to other patients.Conclusion:AGTR1 expression was found positively correlated with plasma β2-microglobulin level and patients with increased AGTR1 expression showed increased bone marrow CD34 levels.

Opposite effects of demethylating treatment on VEGFA and HIF1A expression in MDA-MB-231 breast cancer cell line in hypoxic microenvironment

Hypoxic microenvironment in breast tumors has been associated with tumor aggressiveness, therapy resistance, and worse prognosis. HIF-1α (hypoxia inducible factor-1α) plays important roles in hypoxia by regulating a set of genes, such as VEGFA (vascular endothelial growth factor), which is related to angiogenesis. Studies have shown that DNA methylation acts on the gene control of HIF1A in hypoxia. However, little is known about the HIF1A transcriptional regulation in aggressive breast cancer MDA-MB-231 cell line. Therefore, the present study analyzed the effect of a demethylating agent on the transcription levels of HIF1A, and its target gene VEGFA, in the MDA-MB-231 cell line, in hypoxia. Herein, we demonstrated that 5′-aza-2′-deoxycitidine treatment in hypoxia reverted and raised HIF1A mRNA levels, but decreased VEGFA mRNA levels. Additionally, in both normoxia and hypoxia, HIF-1α protein levels increased in response to the demethylating treatment. The results of the current study indicated that HIF1A could be regulated by DNA methylation in normoxia and hypoxia. Furthermore, our results indicated a distinct regulation of VEGFA transcripts, independent of HIF-1α, in 5′-aza-dC-treated MDA-MB-231 cells in hypoxia.

Association of vascular endothelial growth factor (VEGF) Gene polymorphisms and expression with the risk of endometriosis: a case-control study.

Endometriosis is a polygenic and multifactorial gynecology situation which might be associated with angiogenesis. In the current study we assess the role of vascular endothelial growth factor (VEGF) - 2578 A/C, and + 936 C/T polymorphisms in susceptibility to endometriosis and checking the expression of VEGF mRNA in eutopic tissue of endometrium with and without endometriosis. The study was comprised of 300 patients who underwent laparascopic or laparotomy surgery with 100 cases who had confirmed histological diagnosis of endometriosis, and 200 controls with no histological diagnosis of disease. The genotyping of VEGF polymorphisms was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique and the gene expression in tissue was determined using Real-Time PCR assay. There was no important difference of allele distribution of the - 2578 A/C (P = 0.7) and + 936 C/T (P = 0.5) polymorphisms among endometriosis cases and controls. Study of VEGF expression during the menstrual cycle, showed that endometrial tissue in cases group expressed more VEGF mRNA at the secretory phase compared to the proliferative phase (P = 0.03). Our results suggest that - 2578 A/C and + 936 C/T polymorphisms of VEGF did not seem to have impact on endometriosis predisposition in our study population. Also we did not find any link between VEGF mRNA expression and risk of endometriosis.

Mesenchymal Stem Cells: a Promising Therapeutic Tool for Acute Kidney Injury.

Acute kidney injury (AKI) is a rapid loss of renal function. It has high mortality rates. Still, renal replacement therapy is considered the best solution for recovering AKI. This opens a line of thought to develop an alternative therapy for it without complications. Mesenchymal stem cells are considered a new therapy for treating kidney diseases. The aim of this work was to address the anti-apoptotic, antioxidative, and pro-angiogenic effects of adipose tissue-derived MSCs (AD-MSCs) and bone marrow-MSCs (BM-MSCs) for treating AKI. Adult male Wistar rats were assigned into nine groups (n = 10): (1) the control group; (2) the AKI group, receiving cisplatin; (3) the AKI group treated with AD-MSCs (1 × 106); (4) the AKI group treated with AD-MSCs (2 × 106); (5) the AKI group treated with AD-MSCs (4 × 106); (6) the AKI group treated with losartan; (7) the AKI group treated with BM-MSCs (1 × 106); (8) the AKI group treated with BM-MSCs (2 × 106); and (9) the AKI group treated with BM-MSCs (4 × 106). The results showed a significant rise in creatinine, urea, and cystatin C (cys C) levels and upregulation of p38 mRNA, whereas a significant decline in NAD(P)H quinone oxidoreductase 1 (NQO-1) protein and downregulation of B-cell lymphoma-2 (Bcl-2) mRNA and vascular endothelial growth factor (VEGF) mRNA were recorded in AKI. MSCs could improve renal functions manifested by decreased urea, creatinine, and cys C levels; downregulation of p38; and upregulation of Bcl-2 and VEGF. Moreover, MSC therapy could induce NQO-1 in the treated rats relative to the untreated rats. So, cell-based therapy can reduce AKI through the antioxidative, anti-apoptotic, and pro-angiogenic properties of MSCs. Therefore, the findings received in this attempt create a fertile base for the setup of cell therapy in patients with AKI.

Higher melatonin in the follicle fluid and MT2 expression in the granulosa cells contribute to the OHSS occurrence

BackgroundOvarian hyperstimulation syndrome (OHSS) is a common and severe complication for patients undergoing IVF/ICSI-ET. Melatonin widely participates in the regulation of female reproductive endocrine activity. However, whether melatonin participates in the progression of OHSS is largely unknown. This study aims to identify the predictive value of follicular fluid (FF) melatonin for OHSS establishment and the underlying mechanism.MethodsAll participants of this case-control study were enrolled at the Reproductive Medicine Center located in the First Affiliated Hospital of Zhengzhou University in China from January to October in 2017. Quantitative real-time PCR and western blot were used to examine the mRNA and protein levels. Primary granulosa cells were extracted and cultured for in vitro studies. Melatonin concentration was measured by ELISA. Logistic analysis and receiver-operating characteristic (ROC) curves were used to evaluate the predicting value of melatonin on OHSS occurrence.Main outcome measuresThe expression level of melatonin receptor 2 (MT2), P450 aromatase cytochrome (aromatase), vascular endothelial growth factor (VEGF), and inducible nitric oxide synthase (iNOS) mRNA in human primary granulosa cells. The concentration of melatonin in FF. The predicting value of melatonin on OHSS and the cut-off value of the prediction.ResultsFF melatonin concentrations were significantly higher in patients with OHSS compared to non-OHSS group (35.94 ± 10.18 ng/mL vs 23.93 ± 10.94 ng/mL, p<0.001). The expression of MT2 mRNA (p = 0.0459) and protein in granulosa cells was also significantly higher in the OHSS group. When using a cut-off level of 27.52 ng/ml, the sensitivity and specificity of FF melatonin to predict OHSS was 84.6 and 74.0%, respectively (p < 0.0001). We also found that melatonin could up-regulates aromatase mRNA, VEGF mRNA expression and down-regulates iNOS mRNA expression in the granulosa cells.ConclusionOHSS patients have higher melatonin in the FF as well as higher MT2 expression in the granulosa cells. The melatonin in FF might be used as an effective predictor for the occurrence of OHSS.

BMAL1 Links Bevacizumab Resistance in Colorectal Cancer to Circadian Rhythm and Heme Receptor REVERBA

Heme sensor Nuclear Receptor Subfamily 1 Group D Member 1 (NR1D1/REVERBA) and its target gene Aryl Hydrocarbon Receptor Nuclear Translocator-Like (ARNTL/BMAL1) regulate circadian rhythm and angiogenesis. Given their involvement in the interplay between clock disruptions (e.g. by mutations or sleep deprival), cancer risk and vascular biology, we hypothesized that these transcription factors contribute to resistance against anti-angiogenic therapy with vascular endothelial growth factor (VEGFA) neutralizing antibody (bevacizumab, abbrev. Beva) in colorectal cancer (CRC). REVERBA bound to a predicted -672 bp Retinoic Acid Receptor-Related Orphan Receptor Alpha (RORA)-responsive element (RORE) adjacent to a BMAL1 DNA-binding motif (E-box) in the proximal promoter of the human VEGFA gene. REVERBA, indirectly and together with BMAL1, increased VEGFA mRNA and protein expression. Conversely, REVERBA siRNA and REVERBA-antagonist hemin decreased VEGFA synthesis. Beva treatment inhibited CRC growth in a C57BL/6J Apc min/+ genetically modified mouse model (GEMM) and BALB/c nu/nu mouse HCT116 xenografts, without attenuating BMAL1 levels or microvessel density (MVD). In CRC patient tumors (n=74), high BMAL1 protein expression was associated with clinical non-response to combination chemotherapy with Beva (*p=0.0061) and reduced progression-free survival (PFS) [*p=0.0223, Hazard Ratio (HR)=1.69]. In addition, single nucleotide polymorphisms (SNPs) in the BMAL1 (ARNTL) gene correlated with shorter overall survival (OS) [rs11022780, *p=0.014, HR=1.61] and PFS (rs7396943, rs7938307, rs2279287) in patients undergoing combination chemotherapy with Beva. Thus, BMAL1 is associated with Beva resistance in CRC. Since the REVERBA-BMAL1-VEGFA axis can be pharmacologically targeted, interference with this signaling pathway could circumvent resistance to anti-angiogenic therapy in CRC. Funding Statement: This study was supported by grants to EB from the Deutsche Krebshilfe (#108287 and #111086). FE received a fellowship from the “Studienstiftung des deutschen Volkes”. JB was supported by the Translational Physician Scientist (TraPS) Program of the Medical Faculty Mannheim of the Heidelberg University. WW was awarded a fellowship from the Chinese Scholarship Council (#201408080116). ME was supported by the State of Baden-Wurttemberg for “Center of Geriatric Biology and Oncology (ZOBEL) – Perspektivforderung” and “Biology of Frailty - Sonderlinie Medizin”. ATB and ME received funds from the European Commission Seventh Framework Programme (Contract No. 278981 [ANGIOPREDICT]) and are supported by the European Commission Horizon 2020 Programme (Contract No. 754923 [COLOSSUS]). ATB is further supported by Science Foundation Ireland under grant 13/CDA/2183 ”Coloforetell”. HJL was partly funded by the National Cancer Institute (grant number P30CA014089), the Gloria Borges WunderGlo Foundation -The Wunder Project, the Dhont Family Foundation, the San Pedro Peninsula Cancer Guild, the Daniel Butler Research Fund, and the Call to Cure Research Fund Declaration of Interests: The authors declare no conflicts of interest. The reagent mBeva was obtained on the basis of a material transfer agreement (MTA#10082012) from Roche Diagnostics GmbH (Penzberg, Germany). HJL has received clinical trial financial support from Merck Serono and Roche and honoraria for advisory board membership and lectures from Bayer, Boehringer Ingelheim, Genentech, Merck Serono and Roche Ethics Approval Statement: Xenograft studies conformed to the 2010/63/EU guidelines and were approved by the Dept. of Health and Children, Dublin, Ireland (B100/3654) and University College Dublin Animal Research Ethics Committee (P12-27). Therapy studies were conducted in accordance with ethical guidelines (Declaration of Helsinki) and approved by the local ethics committees for each participating site. All patients provided written informed consent for the analysis of molecular correlates.

Qishen Yiqi Dropping Pills Ameliorates Doxorubicin-induced Cardiotoxicity in Mice via Enhancement of Cardiac Angiogenesis.

BackgroundQishen Yiqi Dropping Pills (QYDP) is a Chinese traditional medicine that has been applied to treat coronary heart disease and ischemic heart failure in China. However, few studies have explored whether QYDP exerted an effect on doxorubicin (Doxo)-induced cardiotoxicity. Hence, in this study we investigated the effect of QYDP on cardiotoxicity induced by doxorubicin (Doxo) and its potential mechanism.Material/MethodsMale C57BL/6 mice (20–25 g, 8–10 weeks old) were randomly assigned to 4 groups: Control group, QYDP group, Doxo group, and QYDP+Doxo group. The mice were intraperitoneal injected with Doxo weekly for 4 weeks to mimic the chronic toxicity. Four weeks after Doxo injection, echocardiography was applied to evaluate the left ventricular (LV) function, and the structure of the cardiac muscle fibers was analyzed with anti-actinin-2 antibody staining by immunofluorescence. Moreover, TUNEL staining and western blot analysis of Bax protein, Bcl-2 protein, and cleaved caspase-3 protein expression levels were conducted to explore whether QYDP exerted effect on cardiac apoptosis. In addition, Masson trichrome staining and western blot analysis of α-SMA protein expression levels were used to evaluate whether QYDP exerted an effect on cardiac fibrosis. Western blots and quantitative real-time polymerase chain reaction were applied to detect the vascular endothelial growth factor (VEGF) protein and mRNA levels in the myocardial tissue, and anti-CD31 antibody staining by immunohistochemistry was employed to explore whether QYDP exerted an effect on cardiac angiogenesis.ResultsQYDP effectively attenuated cardiac dysfunction and cardiac muscle fibers disruption in Doxo treated mice. Moreover, QYDP reduced myocardial apoptosis and myocardial fibrosis in Doxo treated mice, accompanied with elevated protein levels of VEGF and enhancement of myocardial microvessel density.ConclusionsQYDP could protect against Doxo-induced cardiotoxicity, which may be closely associated with enhanced cardiac angiogenesis. Hence, QYDP could be a promising alternative for the treatment of Doxo-induced cardiotoxicity.