Vascular Endothelial Growth Factor mRNA Levels Research Articles

Exosomes Derived from Mesenchymal Stem Cells Increase the Viability of Damaged Endometrial Cells via the miR-99b-5p/PCSK9 Axis.

The aim of this article was to investigate whether exosomes derived from bone marrow mesenchymal stem cells repair damaged endometrial stromal cells (EnSCs) through the miR-99b-5p/PCSK9 axis. Exosomes derived from bone marrow mesenchymal stem cells (BMSC-exos) were isolated by ultracentrifugation and characterized using transmission electron microscopy and nanoflow cytometry. A mifepristone-induced EnSC injury model was established in vitro, and the uptake of BMSC-exos was assessed. EnSCs were divided into three groups: the normal group (ctrl), EnSC injury group (model), and BMSC-exo treatment group. The effects of BMSC-exos on EnSC proliferation, apoptosis, and vascular endothelial growth factor (VEGF) expression were assessed by coculturing MSC-exos with endometrial cells. Furthermore, high-throughput sequencing was used to identify differentially expressed genes (DEGs). Through bioinformatics analysis, reverse transcription-quantitative polymerase chain reaction, western blotting, the CCK8 assay, immunohistochemistry, and dual-luciferase experiments, the potential mechanism by which BMSC-exos-derived miRNAs repair EnSC injury was studied. BMSC-exos expressed the marker proteins CD9 and CD63. Laser confocal microscopy showed that BMSC-exos could enter damaged EnSCs. In the BMSC-exos-EnSC coculture group compared with the model group, BMSC-exos significantly increased the proliferation of damaged EnSCs and inhibited cell apoptosis in a dose-dependent manner. The expression levels of Caspase-3, Caspase-9, Bax, and VEGF mRNA were significantly downregulated in the BMSC-exos-EnSC coculture group, whereas Bcl-2 expression was upregulated. We identified 28 overlapping DEGs between the model and ctrl groups and between the BMSC-exo and model groups. Transfection with miR-99b-5p mimics significantly decreased PCSK9 gene expression and inhibited the expression of the autophagy-related proteins Beclin-1 and LC3-II/I and apoptosis, thereby promoting EnSC proliferation. Transfection with a miR-99b-5p inhibitor showed the opposite effects. Beclin-1, LC3-II/I, and PCSK9 expression in the thin endometrium was significantly increased. miR-99b-5p promoted cell proliferation by targeting PCSK9. BMSC-exos promoted endometrial proliferation, and miR-99b-5p inhibited cell apoptosis and promoted EnSC proliferation by targeting PCSK9, providing a new target for the treatment of thin endometrium.

MiR-34a ameliorates arterial blood flow in rats with lower limb arteriosclerosis obliterans via Sirt1 signaling pathway.

In this study, we investigated the impact of microRNA-34a (miR-34a) on lower limb arteriosclerosis obliterans in rats through the Sirtuin 1 (Sirt1) signaling pathway. Thirty-six Sprague-Dawley rats were divided into normal, model, and miR-34a mimics groups. Rats in the normal group were raised normally, while the model group underwent lower limb arteriosclerosis obliterans induction and received saline injections. The miR-34a mimics group also underwent arteriosclerosis obliterans modeling but received miR-34a mimics injections. Immunohistochemistry revealed significantly increased vascular endothelial growth factor (VEGF) expression in both model and miR-34a mimics groups compared to the normal group, with the miR-34a mimics group showing higher levels. Western blotting indicated elevated Sirt1 protein expression in both non-normal groups, with the miR-34a mimics group exhibiting significantly higher levels. Quantitative polymerase chain reaction (qPCR) demonstrated higher levels of miR-34a, VEGF mRNA, and Sirt1 mRNA in the model group compared to the normal group, but significantly lower levels than the miR-34a mimics group. Enzyme-linked immunosorbent assay (ELISA) showed increased VEGF content in the model group compared to the normal group but decreased compared to the miR-34a mimics group. Hemorrheological detection revealed a reduced PU index in both non-normal groups compared to the normal group, with a significant increase in the miR-34a mimics group compared to the model group. Overall, miR-34a upregulation enhanced VEGF expression in rat blood vessels, ameliorating arterial blood flow in lower limb arteriosclerosis obliterans through the Sirt1 signaling pathway.

Mechanism of ultrafiltration extract of Angelicae Sinensis Radix and Hedysari Radix regulates HIF-1α signaling pathway mediated by renal hypoxia to ameliorate renal fibrosis in DKD rats

This study explored the mechanism of the ultrafiltration extract of Angelicae Sinensis Radix and Hedysari Radix in ameliorating renal fibrosis in the rat model of diabetic kidney disease(DKD) based on the expression of hypoxia-inducible factor-1α(HIF-1α)/vascular endothelial growth factor(VEGF) and HIF-1α/platelet-derived growth factor(PDGF)/platelet-derived growth factor receptor(PDGFR) signaling pathways in the DKD rats. After 1 week of adaptive feeding, 50 male SPF-grade Wistar rats were randomized into a blank group(n=7) and a modeling group. After 24 h of fasting, the rats in the modeling group were subjected to intraperitoneal injection of streptozocin and fed with a high-sugar and high-fat diet to establish a DKD model. After modeling, the rats were randomly assigned into model(n=7), low-dose ultrafiltration extract(n=7), medium-dose ultrafiltration extract(n=7), irbesartan(n=8), and high-dose ultrafiltration extract(n=8) groups. After intervention by corresponding drugs for 12 weeks, the general conditions of the rats were observed. The body weights and blood glucose levels of the rats were measured weekly, and the 24 h urinary protein(24hUP) was measured at the 6th and 12th weeks of drug administration. After the last drug administration, the renal function indicators were determined. Masson staining was employed to observe the pathological changes of the renal tissue. The expression of prolyl hydroxylase domain 2(PHD2) and HIF-1α in the renal tissue was detected by immunohistochemistry(IHC). Real-time qPCR was employed to determine the mRNA levels of PHD2, VEGF, PDGF, and PDGFR in the renal tissue. Western blot was employed to determine the protein levels of HIF-1α, VEGF, PDGF, and PDGFR in the renal tissue. The results showed that compared with the model group, drug administration lowered the levels of glycosylated serum protein(GSP), aerum creatinine(Scr), and blood urea nitrogen(BUN) in a dose-dependent manner(P<0.05 or P<0.01) and mitigated the pathological changes in the renal tissue. Furthermore, drug administration up-regulated mRNA level of PHD2(P<0.05 or P<0.01), down-regulated the mRNA levels of VEGF, PDGF, and PDGFR(P<0.05 or P<0.01) and the protein levels of HIF-1α, VEGF, PDGF, and PDGFR(P<0.01) in the renal tissue, and increased the rate of PHD2-positive cells(P<0.01). In conclusion, the ultrafiltration extract of Angelicae Sinensis Radix and Hedysari Radix effectively alleviated the renal fibrosis in DKD rats by inhibiting the expression of key proteins in the HIF-1α signaling pathway mediated by renal hypoxia and reducing extracellular matrix(ECM) deposition.

Minoxidil delivered via a stem cell membrane delivery controlled release system promotes hair growth in C57BL/6J mice.

Objective: Umbilical cord-derived mesenchymal stem cell membrane-loaded minoxidil (MXD) nanoparticles (STCM-MXD-NPs) were prepared to investigate their effects on hair growth in C57BL/6J mice. Methods: STCM-MXD-NPs were obtained by freeze-thawing and differential centrifugation, and their effects on hair growth were evaluated using C57BL/6J mice. The mRNA and protein expression levels of vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) were detected by real-time polymerase chain reaction and enzyme-linked immunosorbent assays, respectively. Protein expression levels of marker of proliferation Ki-67 (MKI67) and β-catenin (CTNNB) in skin tissue were detected by immunohistochemistry. Results: STCM-MXD-NPs improved MXD solubility. They released the drug slowly, increasing its transdermal properties, accumulation in the skin, and content in the hair bulb tissues with a better efficacy than that of ordinary MXD. Moreover, STCM-MXD-NPs significantly upregulated the mRNA and protein levels of VEGF and IGF-1 and promoted the protein expression of MKI67 and CTNNB in mouse skin tissues, promoting mouse hair growth. Conclusion: Stem cell membrane-loaded MXD nanoparticles with slow-release properties increased MXD accumulation in the skin by improving its transdermal properties, increasing VEGF, IGF-1, MKI67, and CTNNB expression levels and promoting hair growth in C57BL/6J mice.

Assessment of plasma soluble Tie-2 level to distinguish moyamoya disease from atherosclerotic cerebrovascular disease and predict postoperative neovascularization.

Moyamoya disease (MMD) is a chronic steno-occlusive cerebrovascular disease and features the formation of hazy collateral vessels at the base of the brain. Angiopoietin (Ang)-1 and -2, their receptor Tie-2, and vascular endothelial growth factor (VEGF) that regulate angiogenesis might be important in MMD pathophysiology and postoperative collateral formation. The goal of this study was to determine whether levels of these angiogenic factors could predict collateralization in patients with MMD. A total of 196 patients with MMD and 57 with atherosclerotic cerebrovascular disease serving as controls were enrolled. Ang-1, Ang-2, Tie-2, and VEGF mRNA levels were analyzed in middle cerebral artery (MCA) arterial wall specimens by using real-time quantitative polymerase chain reaction. MCA and peripheral plasma concentrations of Ang-1, Ang-2, soluble Tie-2 (sTie-2), and VEGF were examined by enzyme-linked immunosorbent assay. Cerebral arteriography was performed 6 months after bypass surgery to assess the postoperative collateralization. In MCA specimens, patients with MMD exhibited higher expression levels of Ang-1 and Ang-2 but lowered VEGF expression. The patients with MMD had significantly higher concentrations of Ang-1 and Ang-2 but lower levels of VEGF in MCA plasma. Peripheral plasma concentrations of these growth factors were not changed. MCA and peripheral plasma sTie-2 levels were both reduced in patients with MMD. The 6-month follow-up showed that patients with good collateral formation had lower sTie-2 levels in both MCA and peripheral plasma. Furthermore, the Suzuki stage and peripheral plasma sTie-2 level were significantly correlated with good postoperative collateral formation on multivariate analysis. Ang-1, Ang-2, Tie-2, and VEGF are involved in MMD pathogenesis. The peripheral plasma level of sTie-2 can differentiate MMD from atherosclerotic cerebrovascular disease and serve as a novel biomarker to predict postoperative collateral formation.

Celecoxib/Cyclodextrin Eye Drop Microsuspensions: Evaluation of In Vitro Cytotoxicity and Anti-VEGF Efficacy for Retinal Diseases.

Celecoxib (CCB), a cyclooxygenase-2 inhibitor, is capable of reducing oxidative stress and vascular endothelial growth factor (VEGF) expression in retinal cells and has been shown to be effective in the treatment of diabetic retinopathy and age-related macular degeneration. However, the ocular bioavailability of CCB is hampered due to its very low aqueous solubility. In a previous study, we developed 0.5% (w/v) aqueous CCB eye drop microsuspensions (MS) containing randomly methylated β-cyclodextrin (RMβCD) or γ-cyclodextrin (γCD) and hyaluronic acid (HA) as ternary CCB/CD/HA nanoaggregates. Both formulations exhibited good physicochemical properties. Therefore, we further investigated their cytotoxicity and efficacy in a human retina cell line in this study. At a CCB concentration of 1000 μg/mL, both CCB/RMβCD and CCB/γCD eye drop MS showed low hemolysis activity (11.1 ± 0.3% or 4.9 ± 0.2%, respectively). They revealed no signs of causing irritation and were nontoxic to retinal pigment epithelial cells. Moreover, the CCB eye drop MS exhibited significant anti-VEGF activity by reducing VEGF mRNA and protein levels compared to CCB suspended in phosphate buffer saline. The ex vivo transscleral diffusion demonstrated that a high quantity of CCB (112.47 ± 37.27 μg/mL) from CCB/γCD eye drop MS was deposited in the porcine sclera. Our new findings suggest that CCB/CD eye drop MS could be safely delivered to the ocular tissues and demonstrate promising eye drop formulations for retinal disease treatment.

Vascular endothelial growth factor (VEGF) -A, -C and VE-cadherin as potential biomarkers in early breast cancer patients

BackgroundVascular endothelial growth factor (VEGF) -A and -C act as multifunctional molecules and growth factors, while VE-cadherin (cadherin 5, CDH5) is the endothelial junction protein. AimTo assess the relationship between intratumoral VEGF -A, -C and CDH5 levels and clinical outcome, in primary, early-stage, breast cancer patients. Patients and methodsThe study included 69 node-negative (N0) breast cancer patients, all of whom had not received any prior hormonal or chemotherapeutic systemic therapy that would affect the course of disease. The median follow-up period was 144 months. Intratumoral mRNA levels of VEGF -A, -C and CDH5 were determined by RT-qPCR. Prognostic performance was evaluated by Cox proportional hazards regression, Kaplan-Meier analysis, as well as by the multivariable approach based on the least absolute shrinkage and selection operator (LASSO) logit regression. Classification of patients into the low and high subgroups was performed using the outcome-oriented cut-off point categorization approach. ResultsOf the measured mRNAs, only CDH5 mRNA (t = −2.17; p = 0.04) and VEGF-C mRNA (t = −2.41; p = 0.03) showed significant differences between values in patient subgroups with distant metastasis and those without recurrences, respectively. These t-test results were in agreement with the Cox regression by which CDH5 mRNA reached the most pronounced hazard ratio (HR=2.07; p = 0.05), followed by VEGF-C mRNA (HR=1.59; p = 0.005). HR values above 1.0 indicate that high levels of either CDH5 or VEGF-C mRNAs associated with a higher risk of poor clinical outcome. Distant recurrence incidence was 26% for the CDH5high and 3% for the CDH5low subgroup (Kaplan–Meier analysis). Distant recurrence incidence was 23% for the VEGF-Chigh and 0% for VEGF-Clow subgroup. The independent prognostic value of VEGF-C mRNA was confirmed by LASSO regression. ConclusionIntratumoral VEGF-A levels did not associate with disease outcome in primary, early-stage, breast cancer patients, whilst raised levels of either CDH5 or VEGF-C prognosticated a high risk of distant metastasis.

Effect of a 630 nm light on vasculogenic mimicry in A549 lung adenocarcinoma cells in vitro

ObjectiveThe objective of this study was to investigate the effect of photodynamic therapy (PDT) on the formation of vasculogenic mimicry (VM) in the human lung adenocarcinoma A549 cell line in vitro. MethodsThe participants were divided into a blank control group, a photosensitizer group, a light group, and a PDT group. Cells from each group were cultured in three dimensions using Matrigel, and vasculogenic mimicry generation was observed microscopically. Periodic Acid-Schiff (PAS) staining was used to verify the vasculogenic mimicry structure. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was used to detect the expression levels of cellular osteopontin (OPN) and vascular endothelial growth factor (VEGF) mRNA. Western blotting was used to detect the expression levels of cellular OPN and VEGF protein. ResultsA549 cells cultured on Matrigel for about six hours revealed VM on PAS staining, and the number of formations was significantly reduced in the PDT group compared with other groups (P < 0.05). The RT-PCR results showed that the PDT group downregulated OPN and VEGF mRNA expression compared with each control group (P < 0.05). Western blot results showed that OPN and VEGF protein expression was downregulated in the PDT group compared with each control group (P < 0.05). The results of RT-PCR showed that the expression of OPN and VEGF mRNA was downregulated in the PDT group compared with each control group (P < 0.05). The results of Western blotting showed that the expression of OPN and VEGF was downregulated in the protein PDT group compared with each control group (P < 0.001). ConclusionPhotodynamic therapy significantly inhibited the formation of vasculogenic mimicry in human lung adenocarcinoma A549 cells in vitro and downregulated the expression of OPN, VEGF mRNA, and protein levels.

Differential expression of angiogenesis-related genes in goat uterus and corpus luteum during pregnancy.

Vascularization and the control of luteal and endometrial development are regulated by hypoxia-inducible factors (HIFs) and vascular endothelial growth factor (VEGF) during pregnancy. In this study, the mRNA and protein expression levels of HIFs (HIF1A, HIF2A and HIF3A) and VEGF in goat uterine and ovarian tissues during various stages of pregnancy were evaluated. A total of 42 Hair goats were used and were allocated into six groups, namely embryo-positive (G1), early pregnancy (G2), mid-term pregnancy (G3), late pregnancy (G4), oocyte-positive group (G5) and diestrus group (G6). The mRNA expression of the examined genes was evaluated by RT-qPCR, and protein expression was evaluated by immunohistochemistry (IHC). In caruncles, HIF1A mRNA expression was greater in G1, G2 and G4 than in G3 (p < .05). HIF1A and HIF2A expression was greater in G1 than in G5 (p < .05). In cotyledons, HIF1A, HIF2A and HIF3A mRNA expression was greater in G2 and G3 compared to G4 (p < .05). In luteal tissue, HIF1A mRNA expression was greater in G1 and G2 than in G3 and G4 (p < .05). In the immunohistochemical examination, HIF1A, HIF2A, HIF3A and VEGF immunoreactions were detected in uterine and luteal tissues. Findings suggest that HIFs and VEGF are involved in the regulation of ovarian functions as well as the processes of implantation and placentation.

Correlation Analysis of Vascular Endothelial Growth Factor Level with Clinicopathological Features and Prognosis in Patients with Diabetic Nephropathy: A Biopsy-based Study.

Globally, Type 2 diabetes mellitus (T2DM) is one of the most prevalent chronic diseases, which poses a great potential threat to the human body. Diabetic nephropathy (DN), a very common complication in T2DM, is also the main trigger for end-stage renal disease. A thorough understanding of the pathogenesis is the key as well as the breakthrough for future diagnosis and treatment of DN. This investigation aims to provide more in-depth and accurate guidance for future follow-up research by analyzing the role of vascular endothelial growth factor (VEGF) in the kidney tissue of DN patients. Seventy-nine patients with suspected DN who underwent renal needle biopsy in our hospital from January 2015 to June 2019 were selected as the research participants. After the biopsy, 36 cases were confirmed as DN, and the other 43 were T2DM with primary glomerulonephritis. Determination of VEGF mRNA and protein expression in renal tissue employed PCR and Western blot, and the connection between VEGF mRNA level and clinical pathology (such as renal function, inflammatory factors and pathological manifestations) was discussed. The disease recurrence in DN patients was recorded through the 3-year prognostic followed up, and the related influencing factors were analyzed. In kidney tissue, VEGF mRNA level and protein expression were notably higher in DN patients than in diabetic patients (P<0.05). Pearson correlation coefficient analysis identified that VEGF mRNA and protein had a positive connection with blood urea nitrogen (BUN), serum creatinine (Scr), 24-hoururinetotalprotein(24hUTP) and C-reactive protein (CRP) (P<0.05). Among the various clinicopathological features of DN patients, age, BMI, sex, family history, smoking, drinking, exercise habits, clinical presentations and pathological changes had no significant relationship with VEGF level (P>0.05), but the course of the disease, fasting blood glucose (FBG), glycosylated hemoglobin (HBALC) and pathological stages of nephropathy had a close connection with VEGF level (P<0.05). Prognostic follow-up revealed that VEGF mRNA was noticeably higher in patients with recurrence than in those without (P<0.05). When VEGF mRNA >5.20 in kidney tissue, the sensitivity and specificity for predicting the 3-year recurrence were 85.71% and 84.00% respectively (P<0.05). Finally, Logistic regression analysis identified the independence of FBG, HBALC and VEGF levels as the influencing factors for the prognostic recurrence of DN (P<0.05).VEGF expression in kidney tissue of DN patients is closely linked to renal function and increases as the disease progresses, which is an independent risk factor associated with the prognostic recurrence of DN, with great potential significance for future DN diagnosis and treatment.

The expression analyses of GSK3B, VEGF, ANG1, and ANG2 in human brain microvascular endothelial cells treated with the synthetic cannabinoid XLR-11

The endocannabinoid system receptors, cannabinoid receptors type-1 (CBR-1) and −2 (CBR-2), are implicated in several behavioral and cognitive processes. Many studies have indicated a correlation between cannabinoid receptors and angiogenesis. The current study aims to reveal the possible molecular signaling involved in brain angiogenesis induced by the activation of CBR-1 and CBR-2. We investigated whether the synthetic cannabinoid XLR-11, an agonist of CBR-1 and CBR-2, influences the mRNA and protein expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (ANG1) and −2 (ANG2) in human brain microvascular endothelial cells (hBMVEs). Furthermore, we determined the phosphorylation of glycogen synthase kinase 3 beta (GSK3B) expression. Treatment of hBMVEs cells with XLR-11 elevated the mRNA levels of VEGF, ANG1, and ANG2. The secretion of these proangiogenic factors was increased in the media. Furthermore, the intracellular expression of VEGF, ANG1, ANG2, and GSK3B was significantly increased. This current research provides a new possible approach by targeting the cannabinoid receptors to control and regulate brain angiogenesis for treating a variety of angiogenesis-related diseases. This could be achived by using different agonists or antagonists of the cannabinoid receptors based on the nature of the diseases.

A cell-permeable peptide inhibitor of p55PIK signaling alleviates suture-induced corneal neovascularization and inflammation

To prepare an ophthalmic solution with a cell-permeable TAT peptide (TAT-N24) as the main cell-permeable peptide inhibitor of p55PIK signaling and observe its therapeutic effect on suture-induced corneal neovascularization (CNV) in rats. Sprague-Dawley rats were used to establish a corneal suture (CS) model of CNV. The vehicle and 0.9% TAT-N24 ophthalmic solution was topically administered. CNV induction was assessed on the basis of the clinical performance of each group. Hematoxylin-eosin staining was used to observe pathological changes, and immunohistochemical staining and confocal immunofluorescence were used to determine the localization of factors associated with corneal tissue. The mRNA expression levels of hypoxia-inducible factor (HIF-1α), vascular endothelial growth factor (VEGF-A), nuclear transcription factor κB (NF-κB p65), tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), and interleukin (IL)-6 were determined using real-time quantitative polymerase chain reaction. Western blotting was performed to detect the protein expression levels of HIF-1α and NF-κB p65. TAT-N24 slowed CNV production and reduced the expression of HIF-1α and inflammatory factors in CS models. The mRNA levels of HIF-1α, VEGF-A, NF-kB, TNF-α, IL-1β, and IL-6 significantly decreased. Moreover, the protein levels of HIF-1α and NF-κB p65 were significantly decreased. TAT-N24 can treat CNV and ocular inflammation by inhibiting the HIF-1α/NF-κB signaling pathway in CS. In the early treatment of corneal foreign body trauma, topical application of TAT-N24 can not only reduce the inflammatory response but also inhibit corneal neovascularization.

Mechanism of vascular endothelial cell-derived exosomes modified with vascular endothelial growth factor in steroid-induced femoral head necrosis

Steroid-induced avascular necrosis of the femoral head (SANFH) is an intractable orthopedic disease. This study investigated the regulatory effect and molecular mechanism of vascular endothelial cell (VEC)-derived exosomes (Exos) modified with vascular endothelial growth factor (VEGF) in osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in SANFH. VECs were cultured in vitro and transfected with adenovirus Adv-VEGF plasmids. Exos were extracted and identified. In vitro/vivo SANFH models were established and treated with VEGF-modified VEC-Exos (VEGF-VEC-Exos). The internalization of Exos by BMSCs, proliferation and osteogenic and adipogenic differentiation of BMSCs were determined by the uptake test, cell counting kit-8 (CCK-8) assay, alizarin red staining, and oil red O staining. Meanwhile, the mRNA level of VEGF, the appearance of the femoral head, and histological analysis were assessed by reverse transcription quantitative polymerase chain reaction and hematoxylin-eosin staining. Moreover, the protein levels of VEGF, osteogenic markers, adipogenic markers, and mitogen-activated protein kinase (MAPK)/extracellular regulated protein kinases (ERK) pathway-related indicators were examined by Western blotting, along with evaluation of the VEGF levels in femur tissues by immunohistochemistry. Glucocorticoid (GC) induced adipogenic differentiation of BMSCs and inhibited osteogenic differentiation. VEGF-VEC-Exos accelerated the osteogenic differentiation of GC-induced BMSCs and inhibited adipogenic differentiation. VEGF-VEC-Exos activated the MAPK/ERK pathway in GC-induced BMSCs. VEGF-VEC-Exos promoted osteoblast differentiation and suppressed adipogenic differentiation of BMSCs by activating the MAPK/ERK pathway. VEGF-VEC-Exos accelerated bone formation and restrained adipogenesis in SANFH rats. VEGF-VEC-Exos carried VEGF into BMSCs and motivated the MAPK/ERK pathway, thereby promoting osteoblast differentiation of BMSCs in SANFH, inhibiting adipogenic differentiation, and alleviating SANFH.

Protective Effect of NO2-OA on Oxidative Stress, Gliosis, and Pro-Angiogenic Response in Müller Glial Cells.

Inflammation and oxidative and nitrosative stress are involved in the pathogenesis of proliferative retinopathies (PR). In PR, a loss of balance between pro-angiogenic and anti-angiogenic factors favors the secretion of vascular endothelial growth factor (VEGF). This vascular change results in alterations in the blood-retinal barrier, with extravasation of plasma proteins such as α2-macroglobulin (α2M) and gliosis in Müller glial cells (MGCs, such as MIO-M1). It is well known that MGCs play important roles in healthy and sick retinas, including in PR. Nitro-fatty acids are electrophilic lipid mediators with anti-inflammatory and cytoprotective properties. Our aim was to investigate whether nitro-oleic acid (NO2-OA) is beneficial against oxidative stress, gliosis, and the pro-angiogenic response in MGCs. Pure synthetic NO2-OA increased HO-1 expression in a time- and concentration-dependent manner, which was abrogated by the Nrf2 inhibitor trigonelline. In response to phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS), NO2-OA prevented the ROS increase and reduced the gliosis induced by α2M. Finally, when hypoxic MGCs were incubated with NO2-OA, the increase in VEGF mRNA expression was not affected, but under hypoxia and inflammation (IL-1β), NO2-OA significantly reduced VEGF mRNA levels. Furthermore, NO2-OA inhibited endothelial cell (BAEC) tubulogenesis. Our results highlight NO2-OA's protective effect on oxidative damage, gliosis; and the exacerbated pro-angiogenic response in MGCs.

Synergistic therapeutic effects of intracerebral transplantation of human modified bone marrow-derived stromal cells (SB623) and voluntary exercise with running wheel in a rat model of ischemic stroke

BackgroundMesenchymal stromal cell (MSC) transplantation therapy is a promising therapy for stroke patients. In parallel, rehabilitation with physical exercise could ameliorate stroke-induced neurological impairment. In this study, we aimed to clarify whether combination therapy of intracerebral transplantation of human modified bone marrow-derived MSCs, SB623 cells, and voluntary exercise with running wheel (RW) could exert synergistic therapeutic effects on a rat model of ischemic stroke.MethodsWistar rats received right transient middle cerebral artery occlusion (MCAO). Voluntary exercise (Ex) groups were trained in a cage with RW from day 7 before MCAO. SB623 cells (4.0 × 105 cells/5 μl) were stereotactically injected into the right striatum at day 1 after MCAO. Behavioral tests were performed at day 1, 7, and 14 after MCAO using the modified Neurological Severity Score (mNSS) and cylinder test. Rats were euthanized at day 15 after MCAO for mRNA level evaluation of ischemic infarct area, endogenous neurogenesis, angiogenesis, and expression of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF). The rats were randomly assigned to one of the four groups: vehicle, Ex, SB623, and SB623 + Ex groups.ResultsSB623 + Ex group achieved significant neurological recovery in mNSS compared to the vehicle group (p < 0.05). The cerebral infarct area of SB623 + Ex group was significantly decreased compared to those in all other groups (p < 0.05). The number of BrdU/Doublecortin (Dcx) double-positive cells in the subventricular zone (SVZ) and the dentate gyrus (DG), the laminin-positive area in the ischemic boundary zone (IBZ), and the mRNA level of BDNF and VEGF in SB623 + Ex group were significantly increased compared to those in all other groups (p < 0.05).ConclusionsThis study suggests that combination therapy of intracerebral transplantation SB623 cells and voluntary exercise with RW achieves robust neurological recovery and synergistically promotes endogenous neurogenesis and angiogenesis after cerebral ischemia, possibly through a mechanism involving the up-regulation of BDNF and VEGF.

Effect of hypoxia on HIF-1α/MDR1/VEGF expression in gastric cancer cells treated with 5-fluorouracil.

Fluorouracil chemotherapeutic drugs are the classic treatment drugs of gastric cancer. But the problem of drug resistance severely limits their clinical application. This study aims to investigate whether hypoxia microenvironment affects gastric cancer resistance to 5-fluorouracil (5-FU) and discuss the changes of gene and proteins directly related to drug resistance under hypoxia condition. Gastric cancer cells were treated with 5-FU in hypoxia/normoxic environment, and were divided into a Normoxic+5-FU group and a Hypoxia+5-FU group. The apoptosis assay was conducted by flow cytometry Annexin V/PI double staining. The real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression level of hypoxia inducible factor-1α (HIF-1α), multidrug resistance (MDR1) gene, P-glycoprotein (P-gp), and vascular endothelial growth factor (VEGF) which were related to 5-FU drug-resistance. We analyzed the effect of hypoxia on the treatment of gastric cancer with 5-FU. Compared with the Normoxic+5-FU group, the apoptosis of gastric cancer cells treated with 5-FU in the Hypoxia+5-FU group was significantly reduced (P<0.05), and the expression of apoptosis promoter protein caspase 8 was also decreased. Compared with the the Normoxic+5-FU group, HIF-1α mRNA expression in the Hypoxia+5-FU group was significantly increased (P<0.05), and the mRNA and protein expression levels of MDR1, P-gp and VEGF were also significantly increased (all P<0.05). The increased expression of MDR1, P-gp and VEGF had the same trend with the expression of HIF-1α. Hypoxia is a direct influencing factor in gastric cancer resistance to 5-FU chemotherapy. Improvement of the local hypoxia microenvironment of gastric cancer may be a new idea for overcoming the resistance to 5-FU in gastric cancer.

TNF-α and IL-1β Promote Renal Podocyte Injury in T2DM Rats by Decreasing Glomerular VEGF/eNOS Expression Levels and Altering Hemodynamic Parameters.

Diabetic nephropathy (DN) is a serious microvascular complication in those with type 2 diabetes mellitus (T2DM). Evidence confirms that serum tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the T2DM stage are proposed as prognostic markers for DN development, but it is unclear how they affect renal podocyte-associated nephrin and WT-1 expression. In the presence of podocyte injury, glomerular vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS) and hemodynamic parameters are dysregulated. The current research aimed to clarify the relationship of TNF-α and IL-1β with podocyte injury by altering VEGF/eNOS expression and hemodynamic parameters. A high-fat diet/streptozotocin-induced DN rat model was established. Serum TNF-α and IL-1β levels were tracked in the pre-T2DM, T2DM and DN stages. In the DN stage, the mRNA and protein expression levels of renal TNF-α, IL-1β, VEGF, eNOS, nephrin and WT-1 were studied. Renal hemodynamic parameters, including peak systolic velocity, end-diastolic flow velocity and mean velocity were measured with a color Doppler ultrasound technique. Compared to those in the normal control (CTL) group, serum TNF-α and IL-1β levels increased significantly in the pre-T2DM stage (obesity, insulin resistance and hyperlipidemia), T2DM stage (hyperglycemia) and DN stage (abnormal renal functions) (all: P < 0.05) in the DN group. Serum TNF-α and IL-1β levels in the T2DM stage were significantly higher than those in the pre-T2DM stage (two: P < 0.05). Compared to the CTL group, renal nephrin, WT-1, TNF-α, IL-1β, eNOS and VEGF expression and hemodynamic parameters in the DN stage all showed significant differences separately (all: P < 0.05). Increased serum and renal TNF-α and IL-1β levels played important roles in reducing renal nephrin and WT-1 expression levels, which may be related to the fact that the former affected renal VEGF/eNOS expression and blood flow parameters in the DN rats.

Vascular Endothelial Growth Factor and Erythropoietin Show Different Expression Patterns in the Early and Late Hypoxia Preconditioning Phases and May Correlate with DNA Methylation Status in the Mouse Hippocampus.

Liu, Na, Yanbo Zhang, Pu Zhang, Kerui Gong, Chunyang Zhang, Kai Sun, and Guo Shao. Vascular endothelial growth factor and erythropoietin show different expression patterns in the early and late hypoxia preconditioning phases and may correlate with DNA methylation status in the mouse hippocampus. High Alt Med Biol. 23:361-368, 2022. Background: Vascular endothelial growth factor (VEGF) and erythropoietin (EPO) have been proven to participate in neuroprotection induced by hypoxia preconditioning (HPC), and they can be regulated by hypoxia-inducible factor 1 (HIF-1). It has been reported that DNA methylation can affect VEGF and EPO expression. This study aimed to explore the expression of VEGF and EPO in the early phase and late phase of HPC and whether their expression was affected by DNA methylation. Method: Acute repeated HPC mice were used as the animal model, and detection of molecular changes was performed immediately (early phase) and 1 day (late phase) after HPC treatment. The mRNA and protein expression levels of VEGF, EPO, and DNA methyltransferases (DNMTs) in the hippocampi were measured by real-time polymerase chain reaction and western blotting, respectively. The activity of DNMTs and global methylation levels were analyzed by enzyme-linked immunosorbent assay. DNA methylation levels of VEGF and EPO promoters, which were catalyzed by DNMTs, were determined by bisulfite-modified DNA sequencing. Results: The expression of VEGF was increased in the early phase and late phase of HPC (p < 0.05), whereas the expression of EPO was unchanged in the early phase (p > 0.05) of HPC and was increased in the late phase (p < 0.05). VEGF and EPO expression were negatively correlated with the DNA methylation levels of their promoters. DNMT3A and DNMT3B were decreased in the early phase and late phase (p < 0.05), whereas DNMT1 was unchanged in the early phase and late phase (p > 0.05). Conclusions: Our data demonstrated that DNMTs affect VEGF and EPO expression by regulating the DNA methylation levels of the promoters of VEGF and EPO.

Changes in arterial blood vessels and VEGF and Ang-1 expression in pregnant and non-pregnant yak uterine caruncle.

We investigated the structural features of arterial blood vessels in yak uterine caruncle and the effects of the expression of vascular regulation-related factors on angiogenesis in pregnant and non-pregnant yak uterus. Three-dimensional specimens of the uterine artery of non-pregnant and pregnant yaks were produced to observe and measure the distribution characteristics and number of arterial vessels in the uterus and caruncle in the two periods. The uterine caruncle structure was observed and analysed by haematoxylin-eosin staining. The expression features of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) in the uterine caruncle were detected with immunohistochemistry, quantitative real-time PCR (qRT-PCR) and western blotting. The length and number of blood vessels in the caruncle were increased, the degree of curvature was decreased, and the folding was more complicated during pregnancy as compared with that during non-pregnancy. The immunohistochemical results demonstrated that VEGF and Ang-1 were mainly expressed strongly in the mucosal epithelial cytoplasm. The glandular lumen of the uterine gland, lymphocytes and the media and adventitia of blood vessels are widely distributed, and they are all positive. VEGF and Ang-1 mRNA and protein levels were highest in pregnancy, followed by that in the luteal phase and in the follicular phase, and three stages were significantly different (p < .05). These findings provide an anatomical reference and theoretical basis for improving the diagnosis and treatment of yak reproductive disorders and other diseases in high-altitude and low-oxygen environments.

NF90 interacts with components of RISC and modulates association of Ago2 with mRNA

BackgroundNuclear factor 90 (NF90) is a double-stranded RNA-binding protein involved in a multitude of different cellular mechanisms such as transcription, translation, viral infection, and mRNA stability. Recent data suggest that NF90 might influence the abundance of target mRNAs in the cytoplasm through miRNA- and Argonaute 2 (Ago2)-dependent activity.ResultsHere, we identified the interactome of NF90 in the cytoplasm, which revealed several components of the RNA-induced silencing complex (RISC) and associated factors. Co-immunoprecipitation analysis confirmed the interaction of NF90 with the RISC-associated RNA helicase, Moloney leukemia virus 10 (MOV10), and other proteins involved in RISC-mediated silencing, including Ago2. Furthermore, NF90 association with MOV10 and Ago2 was found to be RNA-dependent. Glycerol gradient sedimentation of NF90 immune complexes indicates that these proteins occur in the same protein complex. At target RNAs predicted to bind both NF90 and MOV10 in their 3′ UTRs, NF90 association was increased upon loss of MOV10 and vice versa. Interestingly, loss of NF90 led to an increase in association of Ago2 as well as a decrease in the abundance of the target mRNA. Similarly, during hypoxia, the binding of Ago2 to vascular endothelial growth factor (VEGF) mRNA increased after loss of NF90, while the level of VEGF mRNA decreased.ConclusionsThese findings reveal that, in the cytoplasm, NF90 can associate with components of RISC such as Ago2 and MOV10. In addition, the data indicate that NF90 and MOV10 may compete for the binding of common target mRNAs, suggesting a role for NF90 in the regulation of RISC-mediated silencing by stabilizing target mRNAs, such as VEGF, during cancer-induced hypoxia.