Expression Levels Of VEGF Research Articles

Thymoquinone has a synergistic effect with PHD inhibitors to ameliorate ischemic brain damage in mice

BackgroundA blockage in a blood vessel can cause reduced blood flow to the brain, which eventually leads to the death of surrounding tissue. Several studies have attempted to develop an effective intervention to reverse this process and improve the health status of affected individuals. Due to its indirect effect on cellular functions and metabolism, the hypoxia-inducible factor (HIF-1α) protein has been proposed as a promising transcription factor in the treatment of stroke. PurposeThe current study aims to explore the relation between HIF-1 α and thymoquinone (TQ) in the attenuation of ischemic brain damage and the possible mechanism of this relation to reduce cell death. MethodsFor this purpose, dimethyloxallyl glycine (DMOG), 8 mg/kg, Acriflavine (ACF), 1.5 mg/kg, and both combined with TQ (5 mg/kg) were assessed. Male C57 mice were used to establish an ischemic stroke model by using endothelin-1 (ET-1) (400 pmole/μl) intra- cranial injection. The ultrastructure alterations of neuronal soma, axons, and mitochondria after stroke and treatment were well addressed. Besides, the expression levels of VEGF, HIF-1α, Nrf2, and HO-1 were evaluated. Meanwhile, apoptosis and autophagy-related proteins were also investigated. ResultsTreatment of ischemic stroke by TQ can activate the HIF-1α pathway and its downstream genes such as VEGF, TrkB, and PI3K, which in turn enhance angiogenesis and neurogenesis. Our study revealed that TQ has the same effect as DMOG to activate HIF-1 α and can improve motor dysfunction after ischemic stroke. Further, we demonstrated that both TQ and DMOG effectively attenuate the organelle's damage following ischemic stroke, which was confirmed by the cryogenic transmission electron microscope. The synergistic effect of TQ and DMOG may lead to a chemo-modulation action in the autophagy process after stroke onset and this result is validated by the western blot and rt-qPCR techniques. ConclusionOur finding revealed the potential role of TQ as a HIF-1 α activator to reduce cell death, modulate autophagy and decrease the infarct volume after ischemic stroke onset. The neuroprotective effect of TQ is achieved by decreasing the inflammation and increasing angiogenesis as well as neurogenesis via induction of the HIF-1α-VEGF/Nrf2-HO-1-TrkB-PI3K pathway.

Exosomes Derived from Bone Marrow Mesenchymal Stem Cells Promote Angiogenesis in Ischemic Stroke Mice via Upregulation of MiR-21-5p.

Exosomes derived from bone mesenchymal stem cells (BMSC-Exos) are one of the main factors responsible for the therapeutic effects of BMSCs. The study aimed to investigate whether BMSC-Exos could promote angiogenesis in ischemic stroke mice via miR-21-5p. In ischemic stroke mice, the therapeutic effects of BMSC-Exos were evaluated by neurological functions and infarct volume. Microvessel density was detected by BrdU/vWF immunofluorescence staining. In in vitro experiments, the proangiogenic effects of BMSC-Exos were assessed via proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). The miR-21-5p inhibitor was transfected into BMSCs using Lipofectamine 2000. miR-21-5p expression was detected by qRT-PCR. The expression levels of VEGF, VEGFR2, Ang-1, and Tie-2 were determined by Western blot. BMSC-Exos significantly improved neurological functions and reduced infarct volume, upregulated microvessel density, and miR-21-5p expression after cerebral ischemia. In vitro assays revealed that BMSC-Exos enhanced HUVECs functions including proliferation, migration, and tube formation. BMSC-Exos increased the expression levels of VEGF, VEGFR2, Ang-1, and Tie-2. However, the proangiogenic effects of BMSC-Exos on HUVECs were reversed by the miR-21-5p inhibitor. These results suggest that BMSC-Exos could promote angiogenesis via miR-21-5p upregulation, making them an attractive treatment strategy for stroke recovery.

Protective effect of royal jelly on fluoride-induced nephrotoxicity in rats via the some protein biomarkers signalling pathways: a new approach for kidney damage

Introduction Protective effect of royal jelly (RJ) on fluoride-induced nephrotoxicity was investigated in this study. Methods 42 healthy male Wistar rats (n = 42, 8 weeks of age) were divided equally into 6 groups with 7 rats in each; (1) Group-1: Controls fed with standard diet; (2) Group-2: RJ [100 mg/kg] bw (body weight), by oral gavage; (3) Group-3: Fluoride [50 mg/kg] bw, in drinking water; (4) Group-4: Fluoride [100 mg/kg] bw, in drinking water; (5) Group-5: RJ [100 mg/kg] bw, by oral gavage + Fluoride [50 mg/kg] bw, in drinking water; (6) Group-6: RJ [100 mg/kg] bw, by oral gavage + Fluoride [100 mg/kg] bw, in drinking water. After 8 weeks, all rats were decapitated and their kidney tissues were removed for further analysis. The protein expression levels of caspase-3, caspase-6, caspase-9, Bcl-2, Bax, VEGF, GSK-3, BDNF, COX-2 and TNF-α proteins in kidney tissue were analysed by western blotting technique Results RJ increased Bcl-2, COX-2, GSK-3, TNF-α and VEGF protein levels and a decreased caspase-3, caspase −6, caspase-9, Bax and BDNF protein levels in fluoride-treated rats. Conclusion RJ application may have a promising therapeutical potential in the treatment of many diseases in the future by reducing kidney damage.

Effect of estrogen application timing on adipose tissue revascularization and immune regulation in rats after autologous fat transplantation

Purpose: To study the effect of timing of estrogen application on adipose tissue revascularization and immune regulation in rats given autologous fat transplantation.Methods: Ninety (90) healthy rats were selected for use in this study. The rats were randomly divided into a study group (n = 45) and control group (n = 45). After ovariectomy, the study group was given estrogen replacement therapy before autologous fat transplantation, while the control group was given estrogen replacement therapy after autologous fat transplantation. The rats were observed for 6 weeks after transplantation. Micro-vessel density, wet weight of transplanted fat, VEGF expression level, levels of M1 and M2 in macrophages, and macrophage infiltration rate were determined 40 days postautologous fat transplantation.Results: Forty days after autologous fat transplantation, microvessel density, wet weight of transplanted fat, expression level of VEGF and levels of M1 and M2 in macrophages were significantly higher in study group rats than in controls (p < 0.05). There was 80 % macrophage infiltration in the study group rats, while the corresponding control value (61 %) was significantly lower (p < 0.05).Conclusion: Estrogen treatment before autologous fat transplantation in rats is helpful for the revascularization of adipose tissue, enhances vascular regeneration, promotes the survival of adipose tissue after transplantation, and activates immune cells. It also promotes the production of immune factors, and improves immunoregulation in adipose tissue. Thus, this therapeutic strategy may be useful in clinical practice, but further clinical trials are required.

Mechanism of Shiliu Buxue Syrup for anemia using integrated metabolomics and network pharmacology

For many years, Shiliu Buxue Syrup (SLBXS) has been used in the treatment of anemia in Xinjiang, China. However, the potential therapeutic mechanism of SLBXS in the treatment of anemia remains unclear. We qualitatively analyzed the ingredients of SLBXS and predicted the underlying mechanisms by network pharmacology. A mice model of anemia was established by subcutaneous injection of 1-Acetyl-2-phenylhydrazine (APH). Spleen metabolomics was performed to screen potential biomarkers and pathways related to anemia. Furthermore, core targets of crucial pathways were experimentally validated. Finally, molecular docking was used for predicting interactions between compositions and targets. Network pharmacology indicated that the 230 SLBXS ingredients may affect 141 target proteins to regulate the PI3K/AKT and HIF-1 signaling pathways. Metabolomics revealed that SLBXS could mediate 30 biomarkers, such as phosphoric acid, l-pyroglutamic acid, alpha-Tocopherol, 1-stearoyl-rac-glycerol, and dihydroxyacetone phosphate, to regulate drug metabolism-other enzymes, glutathione metabolism, glycolysis or gluconeogenesis, nicotinate and nicotinamide metabolism, nitrogen metabolism, and purine metabolism. Western blot indicated that SLBXS can regulate the protein expression levels of AKT1, Bcl2, Caspase3, HIF-1α, VEGF-A, and NOS2. The molecular docking revealed that most of the compositions had a good binding ability to the core targets. Based on these findings, we speculate that SLBXS treats anemia mainly by modulating the PI3K/AKT and HIF-1 pathways and glutathione and glycolytic metabolisms.

Effect of low energy shock wave on testicular microenvironment homeostasis in rats

ObjectiveTo further investigate whether two sets of low-energy extracorporeal shock waves (LESWs) impulse parameters, i.e., 0.02 mJ/mm2 for 500 impulses and 0.04 mJ/mm2 for 500 impulses, which have been shown to directly affect the testes, can promote testicular spermatogenesis or positively regulate homeostasis of the testicular microenvironment. Methods(1) Twenty-four experimental rats were randomly divided into a 0.02 mJ/mm2 500 impulses group (L1 group), a 0.04 mJ/mm2 500 impulses group (M1 group), a sham intervention group (S group) and a blank control group (N group). The experiment period was 8 weeks. (2) Apoptosis of the spermatogenic cells in the left testicle was detected by the TUNEL method, VEGF and eNOs protein expression was detected by immunohistochemistry, and histomorphological changes were observed in PAS-stained sections. Moreover, the morphologies of the spermatogenic tubules and testicular stroma were quantitatively analyzed by stereological analysis. The right testicle was used for Western blot detection of the protein expression levels of Bax, Cytochrome C, Caspase-3, Bcl-2, VEGF and eNOs. ResultsCompared with the other three groups, the rate of M1 testicular germ cell apoptosis induced by shock treatment was higher, the expression levels of proapoptotic proteins increased significantly while that of the antiapoptotic protein was lower, and the suppression of cell proliferation correlated with the protein expression levels. Additionally, with respect to the absolute volume of the seminiferous tubules, the absolute interstitial testicular volume notably increased, producing a series of biological effects working against testicular sperm production and function. However, there was no significant difference between the L1 group and the N and S groups. ConclusionsLESWs treatment with impulse parameters of 0.02 mJ/mm2 for 500 impulses showed a better protective effect on testicular spermatic function in rats and has a positive regulatory biological effect.

Nicaraven-loaded electrospun wound dressings promote diabetic wound healing via proangiogenic and immunomodulatory functions: a preclinical investigation.

The current study developed a biopolymer-based wound dressing by electrospinning of Nicaraven-loaded collagen solution. Firstly, collagen was dissolved in acetic acid, and then Nicaraven was added to the polymeric solution at three different concentrations of 2 w/w%, 4 w/w%, and 6 w/w%. The resulting solution was then electrospun. Various experiments were performed to characterize the produced wound dressings. In vitro studies showed that Nicaraven-loaded scaffolds were not toxic against L929 fibroblast cells and protected them against oxidative stress. Wound healing potential of different formulations of Nicaraven-loaded collagen wound dressings was studied in a rat model of the excisional diabetic wound. The study showed that the collagen/4% Nicaraven and collagen/6% Nicaraven wound dressings exhibited a significantly higher percentage of wound closure, the thickness of the epithelium, and collagen deposition compared with collagen/2% Nicaraven, collagen-only, and sterile gauze groups. Gene expression study showed that the developed wound dressings reduced the tissue expression levels of glutathione peroxidase, NFKβ, and matrix metalloproteinase 9 (MMP9) genes. In addition, in the wounds treated with collagen/4% Nicaraven and collagen/6% Nicaraven scaffolds, wound healing was associated with a higher tissue expression level of b-FGF, VEGF, and collagen type I genes. Overall, wound healing activity of collagen/4% Nicaraven and collagen/6% Nicaraven wound dressings was not significantly different. This study implies that collagen wound dressings incorporated with 4% and 6% Nicaraven can be considered a potential candidate to treat diabetic wounds in the clinic.

Irbesartan protects against type 2 diabetic nephropathy by regulating miR-93/VEGF and its downstream effectors

Purpose: To investigate the protective effect of irbesartan against type 2 diabetic nephropathy (T2DN), and the mechanism involved.Methods: Wistar rats (n = 48) were used in this study. Diabetes mellitus (DM) was established in the rats via injection of streptozotocin (STZ). Thereafter, the DM rats were randomly divided into diabetic nephropathy (DN) group and irbesartan group, with 16 rats in each group. Rats in the control group (n = 16) received normal saline in place of irbesartan.Results: Total cholesterol (TC), triglycerides (TGs), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C), as well as protein expression levels of VEGF, FN, collagen IV in DN group were significantly higher in irbesartan group than the corresponding levels in normal control group, while miR-93 protein expression level was significantly lower than that in normal control group (p < 0.05). However, the expression level of mir-93 was significantly higher in irbesartan group than in DN group, while levels of TC, TGs, HDL-C, LDL-C and VEGF, as well as protein levels of FN and collagen IV protein were significantly lower than those in DN group (p < 0.05). There was no obvious change in the renal tissues of the normal control group. In contrast, in the DN group, glomerularcapillary loop hypertrophy, narrow glomerular cavity, thick basement membrane, mesangial matrix, and vacuolar degeneration in renal tubular epithelial cells, were evident. Compared with DN group, the pathological changes in the irbesartan group were significantly mitigated (p < 0.05).Conclusion: Irbesartan protects DM rats against type 2 diabetic nephropathy by regulating mir-93/VEGF and its downstream effector molecules. This provides some ideas for the development of new drugs for the prevention of type 2 diabetic nephropathy.

Sildenafil-Induced Revascularization of Rat Hindlimb Involves Arteriogenesis through PI3K/AKT and eNOS Activation.

Hypoxia and inflammation play a major role in revascularization following ischemia. Sildenafil inhibits phosphodiesterase-5, increases intracellular cGMP and induces revascularization through a pathway which remains incompletely understood. Thus, we investigated the effect of sildenafil on post-ischemic revascularization. The left femoral artery was ligated in control and sildenafil-treated (25 mg/kg per day) rats. Vascular density was evaluated and expressed as the left/right leg (L/R) ratio. In control rats, L/R ratio was 33 ± 2% and 54 ± 9%, at 7- and 21-days post-ligation, respectively, and was significantly increased in sildenafil-treated rats to 47 ± 4% and 128 ± 11%, respectively. A neutralizing anti-VEGF antibody significantly decreased vascular density (by 0.48-fold) in control without effect in sildenafil-treated animals. Blood flow and arteriolar density followed the same pattern. In the ischemic leg, HIF-1α and VEGF expression levels increased in control, but not in sildenafil–treated rats, suggesting that sildenafil did not induce angiogenesis. PI3-kinase, Akt and eNOS increased after 7 days, with down-regulation after 21 days. Sildenafil induced outward remodeling or arteriogenesis in mesenteric resistance arteries in association with eNOS protein activation. We conclude that sildenafil treatment increased tissue blood flow and arteriogenesis independently of VEGF, but in association with PI3-kinase, Akt and eNOS activation.

Report of Exosomes Isolated from a Human Uterine Leiomyoma Cell Line and Their Impact on Endometrial Vascular Endothelial Cells.

Uterine leiomyomas are the most common pelvic tumor in women of reproductive age; they cause irregular heavy menstrual bleeding leading to anemia and subsequent negative effects on quality of life. Exosomes have arisen as main players of disease progression in several illnesses, including a range of benign and malignant conditions; however, their role in leiomyomas’ pathophysiology remains unknown. We investigated the effect of exosomes derived from human uterine leiomyoma tumor cells (HULM) and human myometrial cells (UTSM) on the behavior of human endometrial microvascular endothelial cells (HEMEC). HULM- and UTSM-derived exosomes were isolated and cocultured with HEMECs. Then, cell proliferation, mRNA expression, tube formation assay, and RNA-seq were performed. Treatment of HEMEC with HULM-derived exosomes increased cell proliferation by 60% compared to control untreated cells, upregulated C-MYC and VEGFA expression levels, and increased tube formation, length, and branching (markers of angiogenesis). Profiling of miRNA revealed that 84 miRNAs were significantly downregulated and 71 were upregulated in HULM-derived exosomes compared to UTSM-derived exosomes. These findings suggest that HULM-derived exosomes might have effects on HEMEC function, containing factors that enhance endometrial proliferation and angiogenesis, which may contribute to heavy menstrual bleeding. Further research on exosomes in uterine leiomyoma may identify possible novel biomarkers for treatment.

An electrospun PGS/PU fibrous scaffold to support and promote endothelial differentiation of mesenchymal stem cells under dynamic culture condition

Shear stress is one of the dominant mechanical stimuli present in a vessel wall and an important regulator of endothelial cells functions. It has been shown that shear stress can derive endothelial differentiation of mesenchymal stem cells (MSCs). Engineering a vascular basement membrane thus requires a scaffold that is not only biocompatible but also able to withstand shear stress and support endothelial differentiation of MSCs in the presence of shear stress. In this study, we fabricated an electrospun poly(glycerol sebacate) (PGS)/polyurethane (PU) fibrous scaffold and characterized its physical properties, mechanical properties, and platelet adhesion and hemolysis induction. The developed PGS/PU indicated desirable hydrophilicity (∼41°), an elastic modulus (∼27 MPa) close to that of the Saphenous vein, and no significant level of hemolysis (∼0.03%) and platelet adhesion. The PGS/PU was next seeded with MSCs and implemented in a custom-made bioreactor for applying shear stress (magnitude: ∼15 dyn/cm2, duration: 24 h). Investigating the endothelial-specific gene expression in the presence of shear stress revealed that expression levels of VEGF R-2, VWF, and PCAM-1 significantly increased for the cells seeded on the PGS/PU scaffold compared to those seeded on a glass coverslip. It is thereby concluded that the developed PGS/PU fibrous scaffold can support and significantly promote the endothelial differentiation of MSCs in the presence of shear stress without the need for any growth factors. The findings of this study could provide insights into engineering a functional vascular basement membrane in vitro for vascular tissue engineering applications.

The Combined Use of Platelet-Rich Plasma Clot Releasate and Allogeneic Human Umbilical Cord Mesenchymal Stem Cells Rescue Glucocorticoid-Induced Osteonecrosis of the Femoral Head.

Glucocorticoid-induced osteonecrosis of the femoral head (ONFH) is a refractory disease. The treatment options for ONFH, especially nonsurgical ones, merit further investigation. To evaluate the combinatorial therapeutic effects of platelet-rich plasma clot releasate (PRCR) and umbilical cord mesenchymal stem cells (UC-MSCs) on glucocorticoid-induced ONFH, a dexamethasone (DEX)-treated cell model and a high-dose methylprednisolone (MPS)-treated rat model were established. Cell counting kit-8 (CCK-8) assay was performed in vitro to determine the optimum dosage of PRCR for UC-MSC viability. The effects of PRCR, UC-MSCs, and PRCR + UC-MSCs on cell viability, apoptosis, migration, and differentiation capacities of DEX-treated bone marrow mesenchymal stem cells (BMSCs) and human umbilical vein endothelial cell (HUVECs) were explored via Transwell assays. Western blotting was conducted to evaluate the expression levels of RUNX2, VEGF, caspase-3, and Bcl-2 in the coculture systems. Ultrasound-guided intra-articular PRCR, UC-MSCs, and PRCR + UC-MSC injections were performed on the ONFH model rats. Microcomputed tomography, histological and immunohistochemical analyses, tartrate-resistant acid phosphatase (TRAP) staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were used to assess the therapeutic effects of PRCR and UC-MSCs on bone loss and necrosis induced by high-dose MPS. Results of this study revealed that the in vitro application of PRCR, UC-MSCs, and PRCR + UC-MSCs reversed the impaired proliferation and migration capacities and resisted apoptosis of BMSCs and HUVECs induced by DEX. Moreover, the PRCR and UC-MSC application significantly improved the alkaline phosphatase (ALP) and alizarin red (ALR) staining of BMSCs and tube formation capacity of HUVECs and promoted the protein expression of RUNX2 in BMSCs and VEGF in HUVECs. Similarly, in the ONFH rat model, the intra-articular injection of UC-MSCs and PRCR improved the subchondral bone mass parameters; promoted the expression of ALP, RUNX2, and VEGF; suppressed osteoclast overactivity; and resisted cell apoptosis. The combination of PRCR and UC-MSCs shows promising therapeutic effects in treating glucocorticoid-induced ONFH. The current study provides important information on intra-articular therapy, paving the way for the clinical management of ONFH in the future.

Abstract 335: Macrophage Interleukin-1 Beta Dependent Angiogenesis During Wound Healing

Angiogenesis during wound healing is thought to require an inflammation suppressed state. However, there is growing evidence that early infiltrating inflammatory macrophages may play an important role in setting the stage for angiogenesis. Here we sought to understand the relationship between the potent inflammatory cytokine, IL-1β, and vascular endothelial growth factor-A (VEFG-A) in the context of two experimental models of angiogenesis-dependent healing: skin punch biopsy and hind limb ischemia. We established a macrophage-specific IL-1 β -deletion model ( mIL-1 β ) to study the relationship between IL-1β and VEGF-A in the context of these models. Using a punch biopsy model, wounds were quantified, showing remarked expansion of the wounds in mIL-1 β -deleted mice on day 2 of the healing process, resulting in slower wound closure. Analysis from mIL-1 β -deleted mice confirmed decreased macrophage expression of IL-1β and VEGF-A early at day two post-wounding by punch biopsy. In our second approach, we used femoral artery ligation to demonstrate hind limb ischemia. We hypothesized that mIL-1 β -deleted mice would exhibit reduced blood flow recovery due to impaired angiogenesis. Analysis from mIL-1 β -deleted mice indicates reduced IL-1β and VEGF-A expression. In establishing a pathway of IL-1β-dependent macrophage VEGF-A upregulation in the acute injury state, we examined transcriptional factors STAT3 and NF-κB. Constitutively active STAT3 and IKK-2 (NF-κB activator) appear to increase VEGF-A expression, while inhibition of STAT3 and NF-κB exhibits a dose-dependent reduction of VEGF-A expression. In summary, two models of angiogenesis-dependent healing from injury indicate that macrophage VEGF-A expression is dependent on IL-1β expression during early inflammation. Strategies to alleviate impaired wound healing where VEGF-A levels may not be sufficient could require upregulation of the downstream effectors of IL-1β to restore macrophage VEGF-A expression levels required for adequate angiogenesis.

Thioredoxin-1 Ameliorates Oxygen-Induced Retinopathy in Newborn Mice through Modulation of Proinflammatory and Angiogenic Factors.

Oxygen-induced retinopathy (OIR) is an animal model for retinopathy of prematurity, which is a leading cause of blindness in children. Thioredoxin-1 (TRX) is a small redox protein that has cytoprotective and anti-inflammatory properties in response to oxidative stress. The purpose of this study was to determine the effect of TRX on OIR in newborn mice. From postnatal day 7, C57BL/6 wild type (WT) and TRX transgenic (TRX-Tg) mice were exposed to either 21% or 75% oxygen for 5 days. Avascular and neovascular regions of the retinas were investigated using fluorescence immunostaining. Fluorescein isothiocyanate-dextran and Hoechst staining were used to measure retinal vascular leakage. mRNA expression levels of proinflammatory and angiogenic factors were analyzed using quantitative polymerase chain reaction. Retinal histological changes were detected using immunohistochemistry. In room air, the WT mice developed well-organized retinas. In contrast, exposing WT newborn mice to hyperoxia hampered retinal development, increasing the retinal avascular and neovascular areas. After hyperoxia exposure, TRX-Tg mice had enhanced retinal avascularization compared with WT mice. TRX-Tg mice had lower retinal neovascularization and retinal permeability during recovery from hyperoxia compared with WT mice. In the early stages after hyperoxia exposure, VEGF-A and CXCL-2 expression levels decreased, while IL-6 expression levels increased in WT newborn mice. Conversely, no differences in gene expressions were observed in the TRX-Tg mouse retina. IGF-1 and Angpt1 levels did not decrease during recovery from hyperoxia in TRX-Tg newborn mice. As a result, overexpression of TRX improves OIR in newborn mice by modulating proinflammatory and angiogenic factors.

A novel risk score model based on five angiogenesis-related long non-coding RNAs for bladder urothelial carcinoma

BackgroundTumour angiogenesis is an independent risk factor for bladder urothelial carcinoma (BUC) progression, but viable and promising antiangiogenic targets are understudied. Emerging evidence suggests that long non-coding RNAs (lncRNAs) play prominent role in the tumour microenvironment and tumour angiogenesis.MethodsThe clinical data of BUC patients were obtained from TCGA database and clinical specimens of 138 BUC patients. Univariate and multivariate COX regression analyses were used to identify survival-related ARLNRs (sARLNRs) from The Molecular Signatures Database v4.0. Fisher’s exact probability method was used to detect the correlations between sARLNRs levels and clinicopathological characteristics. A chain of experiments including FACS, qPCR, immunohistochemistry, tube formation, migration and invasion assays, combining with co-culture models, were utilized to validate the clinical significance and angiogenetic correlation of sARLNRs.ResultsFive sARLNRs were employed to establish an angiogenesis-related risk score model, by which patients in the low-risk group obtained better overall survival than those in the high-risk group. The expression of AC005625.1 and AC008760.1 was significantly related to ECs percentage, tumour size and muscle invasion status. Besides, AC005625.1 and AC008760.1 expressed lower in BUC cell lines and tumour tissues than that in normal urothelial cells and adjacent normal tissues, with much lower levels in more advanced T stages. A prominently higher proportion of ECs was detected in tumour tissues with lower expression of AC005625.1 and AC008760.1. In the co-culture models, we found that knockdown of AC005625.1 and AC008760.1 in BUC cells increased the tube formation, migration and invasion abilities of HUVEC. The expression levels of CD31, VEGF-A, VIMENTIN and N-CADHERIN were also enhanced in HUVEC cells co-cultured with siR-AC005625.1 and siR-AC008760.1-treated T24 cells.ConclusionIn the study, we identify five sARLNRs and validate their clinical significance, angiogenesis correlation and prognosis-predictive values in BUC. These findings may provide a new perspective and some promising antiangiogenic targets for clinical diagnosis and treatment strategies of BUC.

HIF-2α-targeted interventional chemoembolization multifunctional microspheres for effective elimination of hepatocellular carcinoma

Transcatheter arterial chemoembolization (TACE) is widely used for the treatment of advanced hepatocellular carcinoma (HCC). However, the long-term hypoxic microenvironment caused by TACE seriously affects the therapeutic effect of TACE. HIF-2α plays a crucial role on the chronic hypoxia process, which might be an ideal target for TACE therapy. Herein, a multifunctional polyvinyl alcohol (PVA)/hyaluronic acid (HA)-based microsphere (PT/DOX-MS) co-loaded with doxorubicin (DOX) and PT-2385, an effective HIF-2α inhibitor, was developed for enhanced TACE treatment efficacy. In vitro and in vivo studies revealed that PT/DOX-MS had a superior ability to treat HCC by blocking the tumor cells in G2/M phase, prompting cell apoptosis, and inhibiting tumor angiogenesis. The antitumor mechanisms of PT/DOX-MS were possibly due to that the introduction of PT-2385 could effectively inhibit the expression level of HIF-2α in hypoxic HCC cells, thereby down-regulating the expression levels of Cyclin D1, VEGF and TGF-α. In addition, the combination of DOX and PT-2385 could jointly inhibit VEGF expression, which was another reason accounting for the combined anti-cancer effect of PT/DOX-MS. Overall, our study demonstrated that PT/DOX-MS is a promising embolic agent for enhanced HCC treatment via the combined effect of hypoxia microenvironment improvement, chemotherapy, and embolization.

Effect of Neiyi Prescription of QIU on autophagy and angiogenic ability of endometriosis via the PPARγ/NF-κB signaling pathway.

Neiyi Prescription of QIU (NYPQ) is a traditional Chinese medicine prescription for the treatment of endometriosis (EMS). Here, we aimed to examine the effects and mechanisms of NYPQ on angiogenic ability in EMS. EMS rats were established with estradiol valerate and autologous transplantation. EMS rats were intraperitoneally injected with chloroquine (CQ, 40mg/kg), rapamycin (RAPA, 1mg/kg), and monoclonal antibody VEGF (anti-VEGF, 3mg/g/d) or administered 5, 10, 20mg/g/d NYPQ decoction through oral gavage for 4weeks, respectively. By the before and end of the treatment period, the volume of the endometriotic lesions was measured. The pathological morphology, angiogenesis, and the number of autophagosomes of the endometriotic lesion were observed by hematoxylin and eosin staining, immunohistochemistry, and transmission electron microscope, respectively. The cell viability, apoptosis, and angiogenesis of HUVECs were detected by MTT, flow cytometry, and lumen formation experiment, respectively. The expression levels of VEGF, autophagy-/apoptosis-/PPARγ/NF-κB- pathway-related proteins in endometrium tissues or HUVECs were detected by western blot assays. The autophagy agonist rapamycin reduced the lesion size, the microvessel density, and VEGF expression, and promoted the production of autophagosomes and the expression of autophagy-related proteins, while the autophagy inhibitor chloroquine had the opposite effects. In vivo, NYPQ could dose-dependently reduce lesion volume and microvessel density, ameliorate histopathological features and promote autophagosome production of ectopic endometrium. Moreover, serum-containing NYPQ could significantly inhibit the cell viability and tube formation of HUVECs and elevate HUVECs apoptosis. Besides, NYPQ significantly reduced VEGF and promoted autophagy-/apoptosis-related protein expressions. Also, NYPQ might promote autophagy and inhibit angiogenesis by activating the PPARγ/NF-κB pathway. Collectively, these findings indicate that NYPQ has therapeutic potential in experimentally induced peritoneal endometriosis, and its mechanism may be related to the activation of the PPARγ/NF-κB signaling pathway.

Recombinant human erythropoietin accelerated the proliferation of non-small cell lung cancer cell lines and reduced the expression of VEGF, HIF-1α, and PD-L1 under a simulated hypoxic environment in vitro.

BackgroundAs erythropoietin (EPO) has been used to treat anemia in cancer patients, negative controversy has continued. Unfortunately, its effects on non‐small‐cell lung carcinoma (NSCLC) cell lines are uncertain and the phenomenon of inducing immune escape of tumor cells remains to be explored. This study aimed to provide an important basis for the application of exogenous EPO in the treatment of tumor‐associated anemia.MethodsCells were cultured in 1% O2, 5% CO2, and 94% N2 to simulate a hypoxic environment of the tumor. A549 cell line (lower expression EPOR) and NCI‐H838 cell line (higher expression EPOR) were treated with 2 and 8 U/ml recombinant human EPO (rhEPO). CCK‐8 method was used to determine the logarithmic growth phase of the cells and to detect cell proliferation. The expression levels of VEGF, HIF‐1α, and PD‐L1 were determined by western blot. One‐way ANOVA was used for statistical analysis between groups, with p < 0.05 indicating a significant difference.ResultsHypoxia itself could decrease the survival rate of NSCLC cells. Under the hypoxic condition, rhEPO induced tumor cells proliferation, especially in the NCI‐H838 cell line, where 2 U/ml rhEPO increased the total number of surviving cells (Hypoxia + rhEPO 2 U/ml vs. Hypoxia, p < 0.05). Western blot analysis showed that hypoxia upregulated the expression of VEGF, HIF‐1α, and PD‐L1 in NSCLC cell lines (Normoxia vs. Hypoxia, p < 0.05), but may not be dependent on the expression levels of EPOR. RhEPO decreased the expression levels of VEGF and HIF‐1α. In the A549 cell line, it depended on the concentration of rhEPO and was particularly obvious in HIF‐1α (Hypoxia vs. Hypoxia + rhEPO 2 U/ml vs. Hypoxia + rhEPO 8 U/ml, p < 0.05). A low concentration of rhEPO may not reduce VEGF expression. In the NCI‐H838 cell line, the effect of rhEPO on VEGF was more obvious, but it may be independent of rhEPO concentrations. The downregulation of PD‐L1 expression by rhEPO was only presented in the A549 cell line and required higher rhEPO concentrations (Hypoxia + rhEPO 8 U/ml vs. Hypoxia&Hypoxia + rhEPO 2 U/ml, p < 0.05).ConclusionThe effect of prolonged high concentrations of rhEPO under hypoxic conditions resulted in accelerated cells proliferation of non‐small‐cell lung cancer and was independent of EPOR expression levels on the cell lines surface. Hypoxia resulted in increased expression of VEGF, HIF‐1α, and PD‐L1 on the NSCLC cell lines. Under normoxic conditions, rhEPO did not affect the expression of VEGF, HIF‐1α, and PD‐L1; but under hypoxic conditions, the application of rhEPO reduced the expression of VEGF, HIF‐1α, and PD‐L1, producing an impact on the biological behavior of tumor cells.

Effects of miR-363 on the Biological Activities of Eutopic Endometrial Stromal Cells in Endometriosis.

EMs is a kind of benign disease with certain malignant behaviors. The adhesion, invasive growth, and angiogenesis of ectopic endometrial cells are the pathological basis of EMs occurrence, but its etiology and pathogenesis have not been completely illustrated yet. In our research, we aim to investigate the role of miR-363 in the pathogenesis of endometriosis. Real-time quantitative PCR was used to detect the expression of miR-363 before and after ESC/NSC transfection. CCK-8, flow cytometry, and transwell assay were used to detect the effect of the miR-363 expression on cell proliferation, apoptosis, and invasion. The effects of the miR-363 expression on the contents of Fas/APO-1 and ICAM-1 in cell culture supernatant were detected by ELISA. qRT-PCR and WB assay were used to detect the effects of the miR-363 expression on the mRNA and protein expression levels of ICAM-1, MMP-7, and VEGF in ESC. The increased expression of miR-363 could inhibit the proliferation and invasion of ESC, promote apoptosis, and inhibit the secretion of FAS/APO-1 and ICAM-1. The knockdown expression of miR-363 promoted proliferation and invasion of NSC, inhibited apoptosis, and promoted secretion of FAS/APO-1 and ICAM-1. VCAM-1, VEGF, and MMP-7 were detected in ESCs before transfection. The protein expression level was higher than that of NSCs. Compared with pretransfection, the protein levels of VCAM-1, VEGF, and MMP-7 in the M-363 group were significantly downregulated. The downregulated expression of miR-363 was associated with a stronger cell proliferation ability, a lower cell apoptosis rate, and a stronger ESC. Migration is associated with invasiveness, proliferation, angiogenesis, and immune escape. The low expression of miR-363 promotes endogenesis through posttranscriptional regulation of target genes VCAM-1, MMP-7, and VEGF. The differential expression of miR-363 between ESC and NSC may be an important factor in the many biological differences between ESC and NSC.

Effects of Placenta-Derived Human Amniotic Epithelial Cells on the Wound Healing Process and TGF-β Induced Scar Formation in Murine Ischemic-Reperfusion Injury Model.

Pressure ulcers (PUs), a result of ischemic reperfusion (IR) injuries, are prevalent skin problems which show refractoriness against standard therapeutic approaches. Besides, scar formation is a critical complication of ulcers that affects functionality and the skin's cosmetic aspect. The current study aimed to investigate the effects of placenta-derived human amniotic epithelial cells (hAECs), as important agents of regenerative medicine and stem cell therapy, on accelerating the healing of IR ulcers in mice. We also evaluated the effects of these cells on reducing the TGFβ-induced scar formation. Male Balb/c mice at the age of 6-8weeks were subjected to three IR cycles. Afterward, the mice were divided into three experimental groups (n = 6 per group), including the control group, vehicle group, and hAECs treatment group. Mice of the treatment group received 100μL of fresh hAECs 1 × 106 cell/ml suspension in PBS. Afterward, mice were assessed by histological, stereological, molecular, and western blotting techniques at 3, 7, 14, and 21days after wounding. The histological and stereological results showed the most diminutive scar formation and better healing in the hAECs treated group compared to control group. Furthermore, our results demonstrated that the expression level of Col1A1 on days 3, 14, and 21 in the hAECs treated group was significantly lower than control. Additionally, injection of hAECs significantly reduced the expression level of Col3A1 on days 3, 7, and 21 while increased Col3A1 on the day 14. Otherwise, in the hAECs treated group, the expression levels of VEGFA on days 7 and 14 were higher, which showed that hAECs could promote angiogenesis and wound healing. Also, cell therapy significantly lowered the protein levels of TGF-β1 on day 14, while the protein level of TGF-β3 on day 14 was significantly higher. This data could demonstrate the role of hAECs in scar reduction in IR wounds. These results suggest that hAECs can promote re-epithelialization and wound closure in an animal model of PU. They also reduced scar formation during wound healing by reducing the expression of TGF-β1/ TGF-β3 ratio.