VEGF Protein Levels Research Articles

Itaconic acid induces angiogenesis and suppresses apoptosis via Nrf2/autophagy to prolong the survival of multi-territory perforator flaps

BackgroundPerforator flaps are widely used in hand microsurgery to reconstruct and repair soft tissue injuries. However, ischemia and subsequent ischemia-reperfusion injury may cause distal necrosis of the flap. Itaconic acid (IA) is a modulator of macrophage function, which exerts anti-inflammatory effects in macrophage activation. MethodsThe necrotic area of the flap was detected by measuring the flap temperature with an infrared thermometer. Flap cell apoptosis was detected by TUNEL staining and Western blot analysis of the apoptosis-associated proteins Bcl-2 and Bax. HE staining was used to detect angiogenesis of the skin flaps. CD31 was detected to identify positive vascular expression, and the survival of choke vessels in different areas of the skin flap was assessed by arteriography. In addition, Western blot was performed to quantify the expressions of VEGF, Nrf2, LC3II, SQSTM1, and CTSD. ResultsItaconic acid raises VEGF protein levels in skin flaps and the number of CD31-positive vessels. The skin flaps in the IA treatment group exhibited higher neovascularization and less necrosis than those in the control group. The results of TUNEL staining and Western blot revealed that Itaconic acid attenuated apoptosis in the ischemic area of flap. The combination of itaconic acid and Nrf2 inhibitor ML385 reversed this beneficial effect. The results revealed that itaconic acid attenuated apoptosis, enhanced angiogenesis, and enhanced autophagy. ConclusionIn summary, our findings indicate that itaconic acid might be an effective treatment to reduce flap necrosis. Additionally, this study identified a novel mechanism for the effects of itaconic acid on flap survival.

Mechanism of the HIF-1α/VEGF/VEGFR-2 pathway in the proliferation and apoptosis of human haemangioma endothelial cells.

Haemangiomas (HAs) are prevalent vascular endothelial cell tumours. With respect to the possible involvement of HIF-1α in HAs, we have explored its role in haemangioma endothelial cell (HemEC) proliferation and apoptosis. shRNA HIF-1α and pcDNA3.1 HIF-α were manipulated into HemECs. HIF-α, VEGF, and VEGFR-2 mRNA and protein levels were assessed by qRT-PCR and Western blotting. Cell proliferation and viability, cell cycle and apoptosis, migration and invasion, and ability to form tubular structures were assessed by colony formation assay, CCK-8, flow cytometry, Transwell assay, and tube formation assay. Cell cycle-related protein levels, and VEGF and VEGFR-2 protein interaction were detected by Western blot and immunoprecipitation assays. An Haemangioma nude mouse model was established by subcutaneous injection of HemECs. Ki67 expression was determined by immunohistochemical staining. HIF-1α silencing suppressed HemEC neoplastic behaviour and promoted apoptosis. HIF-1α facilitated VEGF/VEGFR-2 expression and the VEGF had interacted with VEGFR-2 at protein - protein level. HIF-1α silencing arrested HemECs at G0/G1 phase, diminished Cyclin D1 protein level, and elevated p53 protein level. VEGF overexpression partially abrogated the effects of HIF-1α knockdown on inhibiting HemEC malignant behaviours. Inhibiting HIF-1α in nude mice with HAs repressed tumour growth and Ki67-positive cells. Briefly, HIF-1α regulated HemEC cell cycle through VEGF/VEGFR-2, thus promoting cell proliferation and inhibiting apoptosis.

P-631 Alleviating effects of EGCG on ovarian hyperstimulation syndrome and the potential modulating mechanism through the VEGF pathway

Abstract Study question What are the underlying mechanisms of EGCG to alleviate Ovary hyperstimulation syndrome (OHSS)? Summary answer EGCG inhibited VEGF via TGF-β1 classical-SMAD pathway and 67LR-mediated CREB pathway and could reduce the ovarian inflammatory effect and attenuated OHSS progress in rat model. What is known already Ovarian hyperstimulation syndrome (OHSS) is one of the most severe complications of COH during IVF treatment. The pathophysiology of OHSS is characterized by increased capillary permeability, VEGF is an important mediator in OHSS, and serum VEGF levels have been shown to correlate with OHSS severity. (-)-epigallocatechin-3-gallate (EGCG) is the most abundant and biologically active polyphenolic catechin in green tea and has been reported to have multiple effects in humans. Many research indicated that in many pathological processes, EGCG could inhibit VEGF and its receptor expression and have an angiogenesis effect. Herein, EGCG might have a therapeutic effect on OHSS. Study design, size, duration We investigated the role of EGCG in OHSS in vitro and in vivo. The primary human granulosa-lutein(hGL) cells and human granulosa-like tumor (KGN) cell line were cultured and treated with different concentrations of EGCG for 24 hours. Animal OHSS model was established in SD rats by injection of pregnant mare serum gonadotropin (PMSG), and randomly assigned to receive vehicle only or EGCG for 3 days. All experiments were performed 3-8 times for comparisons. Participants/materials, setting, methods The effect of EGCG on KGN and hGL cells was determined by MTT assay. The body weight of rats was measured every day, and the ovary size was measured after removal, the permeability was determined by Evans-blue. The serum estrogen and VEGF level in rats were detected by ELISA. RNA and protein expressions of VEGF, VEGFR-2, TGF-β, and TβR II were detected by qPCR and Western-blotting and immunostaining in vitro and in vivo experiments. Main results and the role of chance Our study demonstrated that administration of EGCG attenuated the development of OHSS in rats, as shown by histological examination and ovarian weight and morphology. The ovary weight was significantly decreased in the EGCG treatment group compared with the OHSS group (147.9 vs 206.5 mg). Additionally, compared to OHSS rats, EGCG treated rats exhibit downregulated ovarian VEGF expression determined by IHC and RT-qPCR. VEGF and E2 protein levels significantly decrease in the serum of the EGCG treatment group. EGCG exerted inhibitory effects on cell growth only in high dose (50uM) and longtime (48h) treatment in KGN and hGL cells. In KGN cells and hGL cells, EGCG significantly reduces the expression of VEGF and TGF-β at the RNA and protein levels. Furthermore, EGCG inhibits TGF-β1-induced VEGF production and secretion in KGN cells by suppressing TGF-β expression and its traditional Smad signaling pathway. EGCG also downregulates VEGF expression through the 67-kDa laminin receptor-mediated PKA-CREB pathway. Limitations, reasons for caution EGCG has been reported to employ a wide range of biological effects. Therefore, its suppressive effects on VEGF and TGF-β signaling pathway might be part of the pharmacological mechanisms to alleviate Ovary hyperstimulation syndrome. Further studies are also needed to explore the therapeutic mechanisms of melatonin from other perspectives. Wider implications of the findings Our findings add mechanistic insight in support of using EGCG as an adjuvant therapy in the management of OHSS. EGCG shows the potential to act as a novel alternative therapeutic drug to treat OHSS. Daily intake of green tea might be beneficial for women under COH to prevent OHSS. Trial registration number Not applicable

Melatonin blunted the angiogenic activity in 3D colon cancer tumoroids by the reduction of endocan

BackgroundComplexity and heterogeneity of the tumor niche are closely associated with the failure of therapeutic protocols. Unfortunately, most data have been obtained from conventional 2D culture systems which are not completely comparable to in vivo microenvironments. Reconstructed 3D cultures composed of multiple cells are valid cell-based tumor models to recapitulate in vivo-like interaction between the cancer cells and stromal cells and the oncostatic properties of therapeutics. Here, we aimed to assess the tumoricidal properties of melatonin on close-to-real colon cancer tumoroids in in vitro conditions.MethodsUsing the hanging drop method, colon cancer tumoroids composed of three cell lines, including adenocarcinoma HT-29 cells, fibroblasts (HFFF2), and endothelial cells (HUVECs) at a ratio of 2: 1: 1, respectively were developed using 2.5% methylcellulose. Tumoroids were exposed to different concentrations of melatonin, from 0.005 to 0.8 mM and 4 to 10 mM, for 48 h. The survival rate was measured by MTT and LDH leakage assays. Protein levels of endocan and VEGF were assessed using western blotting. Using histological examination (H & E) staining, the integrity of cells within the tumoroid parenchyma was monitored.ResultsDespite the reduction of viability rate in lower doses, the structure of tumoroids remained unchanged. In contrast, treatment of tumoroids with higher doses of melatonin, 4 and 10 mM, led to disaggregation of cells and reduction of tumoroid diameter compared to the non-treated control tumoroids (p < 0.05). By increasing melatonin concentration from 4 to 10 mM, the number of necrotic cells increased. Data showed the significant suppression of endocan in melatonin-treated tumoroids related to the non-treated controls (p < 0.05). According to our data, melatonin in higher doses did not alter protein levels of VEGF (p > 0.05).ConclusionsMelatonin can exert its tumoricidal properties on colon cancer tumoroids via the reduction of tumor cell viability and inhibition of the specific pro-angiogenesis factor.

(074) Association of Androgen Receptor Signaling in Human Corpus Cavernosum with Serum Testosterone Levels

Abstract Introduction Testosterone (T) plays an important role in properly developing and maintaining male reproductive tissues. The normal serum testosterone levels can vary between 300 to 1000 ng/dL or 10.4 to 34.7 nmol/L. However, it is unclear whether androgen receptor signaling can vary based on serum T levels or the half-life of testosterone therapy. What remains unknown is whether there is a uniform serum T level above which androgen receptor signaling within the tissue remains similar (saturation hypothesis). Objective We first evaluated whether varying serum levels of T alter androgen receptor (AR) signaling in human penile tissue. Then, we evaluated and compared AR signaling in corpus cavernosum tissue of men treated with short-acting vs. long-acting testosterone. We hypothesized that we would find similar AR signaling in the penile tissue of both eugonadal and hypogonadal men as well as those men receiving short and long-acting testosterone. Methods We obtained human corpus cavernosum (hCC) tissue biopsies during penile surgery (for erectile dysfunction) from 14 men. We recorded their serum T levels on week prior to surgery before 10AM. We used western blot to evaluate adrenergic receptor (AR) and VEGF protein levels. Men without erectile dysfunction undergoing penile surgery for Peyronie's disease were used as controls. Results The median age of participants was 61 (IQR 12) and the median T level was 325ng/dL (IQR 201.45). The results of western blot showed expression of similar levels of VEGF in hCC samples from men with T &amp;gt;200ng/dL. However, men with serum T levels lower than 200 ng/dL had decreased expression of VEGF and AR. Conclusions This finding is significant because it demonstrates that serum testosterone beyond a certain threshold may have a minimal impact on erectile function. If a serum testosterone threshold is identified, this threshold could be a valuable target for future studies on the effects of testosterone on ED. It may alter the treatment threshold for men who have ED as their primary symptom of hypogonadism. This data also suggests the saturation value for penile androgen receptors. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Acerus Pharmaceuticals, Boston Scientific, Coloplast, Endo Pharmaceuticals, Empower Pharmacy, Nestle Health, Olympus, Hims, Inc

MEX3A promotes angiogenesis in colorectal cancer via glycolysis

ABSTRACT As a gastrointestinal malignancy, colorectal cancer (CRC) is a main cause of cancer-related deaths worldwide. Mex-3 RNA-binding family member A (MEX3A) is upregulated in multiple types of tumors and plays a critical role in tumor proliferation and metastasis. However, the function of MEX3A in CRC angiogenesis has not been fully understood. Hence, the aim of this study was to explore the role of MEX3A in CRC angiogenesis and investigate its underlying mechanisms. MEX3A expression in CRC was first investigated by bioinformatics means and then measured by qRT-PCR and Western blot. CCK-8 assay was employed to test cell viability. Angiogenesis assay was used to assess angiogenesis. The protein levels of VEGF, FGF and SDF-1 were evaluated using Western blot. The expression levels of MYC, HK2 and PGK1 were investigated by qRT-PCR. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were determined by Seahorse XP 96. The levels of pyruvate, lactate, citric acid and malate were measured by corresponding kits. Bioinformatics analysis demonstrated high MEX3A expression in CRC tissues and MEX3A enrichment in glycolysis and angiogenesis pathways. Cell assays showed high MEX3A expression in CRC cells and its promoting effects in CRC cell proliferation and glycolysis as well as angiogenesis. Rescue experiment confirmed that glycolysis inhibitor 2-DG could offset the promoting effects of MEX3A on the proliferation, angiogenesis and glycolysis of CRC cells. In conclusion, MEX3A could facilitate CRC angiogenesis by activating the glycolytic pathway, suggesting that MEX3A may be a novel therapeutic target for CRC.

The VEGF protein levels, miR-101-3p, and miR-122-5p are dysregulated in plasma from adolescents with major depression

BackgroundMajor depressive disorder (MDD) is a common and serious psychiatric disorder that affects millions of individuals worldwide. Although the underlying mechanisms are still poorly understood, mounting evidence is indicating a role of growth factors, among them the vascular endothelial growth factor (VEGF or VEGFA). MethodsTo further explore the importance of VEGF, the plasma protein VEGF levels in hospitalized children and adolescents with MDD were investigated before and after antidepressant treatment (10 days on average) and compared to healthy controls. Subsequently, an expression study with 28 microRNAs (miRNAs) targeting VEGF was performed. The study included 39 hospitalized children and adolescents with MDD and 20 healthy controls. ResultsVEGF protein levels were significantly increased in depressed adolescents compared to healthy controls and a short antidepressant treatment significantly decreased the levels in both adolescents and children. In continuation, we found that miR-101-3p was up- and miR-122-5p was down-regulated in depressed adolescents compared to healthy controls and that factors such as age, sex, antidepressant treatment, and haemolysis of the blood samples impact the miRNA results. LimitationsSmall sample size and short treatment period with different pharmacological treatment (fluoxetine, vortioxetine, and venlafaxine). ConclusionThis study substantiates the use of peripheral VEGF as a biomarker of MDD in children and adolescents and highlights the importance of understanding the underlying regulatory mechanisms.

Regulation of VEGFR2 and AKT Signaling by Musashi-2 in Lung Cancer.

Lung cancer is the most frequently diagnosed cancer type and the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) represents most of the diagnoses of lung cancer. Vascular endothelial growth factor receptor-2 (VEGFR2) is a member of the VEGF family of receptor tyrosine kinase proteins, which are expressed on both endothelial and tumor cells, are one of the key proteins contributing to cancer development, and are involved in drug resistance. We previously showed that Musashi-2 (MSI2) RNA-binding protein is associated with NSCLC progression by regulating several signaling pathways relevant to NSCLC. In this study, we performed Reverse Protein Phase Array (RPPA) analysis of murine lung cancer, which suggests that VEGFR2 protein is strongly positively regulated by MSI2. Next, we validated VEGFR2 protein regulation by MSI2 in several human lung adenocarcinoma cell line models. Additionally, we found that MSI2 affected AKT signaling via negative PTEN mRNA translation regulation. In silico prediction analysis suggested that both VEGFR2 and PTEN mRNAs have predicted binding sites for MSI2. We next performed RNA immunoprecipitation coupled with quantitative PCR, which confirmed that MSI2 directly binds to VEGFR2 and PTEN mRNAs, suggesting a direct regulation mechanism. Finally, MSI2 expression positively correlated with VEGFR2 and VEGF-A protein levels in human lung adenocarcinoma samples. We conclude that the MSI2/VEGFR2 axis contributes to lung adenocarcinoma progression and is worth further investigations and therapeutic targeting.

Differential Impact of Intermittent vs. Sustained Hypoxia on HIF-1, VEGF and Proliferation of HepG2 Cells.

Obstructive sleep apnea (OSA) is an emerging risk factor for cancer occurrence and progression, mainly mediated by intermittent hypoxia (IH). Systemic IH, a main landmark of OSA, and local sustained hypoxia (SH), a classical feature at the core of tumors, may act separately or synergistically on tumor cells. Our aim was to compare the respective consequences of intermittent and sustained hypoxia on HIF-1, endothelin-1 and VEGF expression and on cell proliferation and migration in HepG2 liver tumor cells. Wound healing, spheroid expansion, proliferation and migration were evaluated in HepG2 cells following IH or SH exposure. The HIF-1α, endothelin-1 and VEGF protein levels and/or mRNA expression were assessed, as were the effects of HIF-1 (acriflavine), endothelin-1 (macitentan) and VEGF (pazopanib) inhibition. Both SH and IH stimulated wound healing, spheroid expansion and proliferation of HepG2 cells. HIF-1 and VEGF, but not endothelin-1, expression increased with IH exposure but not with SH exposure. Acriflavine prevented the effects of both IH and SH, and pazopanib blocked those of IH but not those of SH. Macitentan had no impact. Thus, IH and SH stimulate hepatic cancer cell proliferation via distinct signaling pathways that may act synergistically in OSA patients with cancer, leading to enhanced tumor progression.

PTTG1 promotes CD34+CD45+ cells to repair the pulmonary vascular barrier via activating the VEGF-bFGF/PI3K/AKT/eNOS signaling pathway in rats with phosgene-induced acute lung injury

Accidental exposure to phosgene can cause acute lung injury (ALI), characterized by uncontrolled inflammation and impaired lung blood-gas barrier. CD34+CD45+ cells with high pituitary tumor transforming gene 1 (PTTG1) expression were identified around rat pulmonary vessels through single-cell RNA sequencing, and have been shown to attenuate P-ALI by promoting lung vascular barrier repair. As a transcription factor closely related to angiogenesis, whether PTTG1 plays a role in CD34+CD45+ cell repairing the pulmonary vascular barrier in rats with P-ALI remains unclear. This study provided compelling evidence that CD34+CD45+ cells possess endothelial differentiation potential. Rats with P-ALI were intratracheally administered with CD34+CD45+ cells transfected with or without PTTG1-overexpressing and sh-PTTG1 lentivirus. It was found that CD34+CD45+ cells reduced the pulmonary vascular permeability and mitigated the lung inflammation, which could be reversed by knocking down PTTG1. Although PTTG1 overexpression enhanced the ability of CD34+CD45+ cells to attenuate P-ALI, no significant difference was found. PTTG1 was found to regulate the endothelial differentiation of CD34+CD45+ cells. In addition, knocking down of PTTG1 significantly reduced the protein levels of VEGF and bFGF, as well as their receptors, which in turn inhibited the activation of the PI3K/AKT/eNOS signaling pathway in CD34+CD45+ cells. Moreover, LY294002 (PI3K inhibitor) treatment inhibited the endothelial differentiation of CD34+CD45+ cells, while SC79 (AKT activator) yielded the opposite effect. These findings suggest that PTTG1 can promote the endothelial differentiation of CD34+CD45+ cells by activating the VEGF-bFGF/PI3K/AKT/eNOS signaling pathway, leading to the repair of the pulmonary vascular barrier in rats with P-ALI.

VEGFA Promotes Skeletal Muscle Regeneration in Aging.

Aging is associated with loss of skeletal muscle regeneration. Differentially regulated vascular endothelial growth factor (VEGF)A with aging may partially underlies this loss of regenerative capacity. To assess the role of VEGFA in muscle regeneration, young (12-14weeks old) and old C57BL/6 mice (24,25 months old) are subjected to cryoinjury in the tibialis anterior (TA) muscle to induce muscle regeneration. The average cross-sectional area (CSA) of regenerating myofibers is 33% smaller in old as compared to young (p<0.01) mice, which correlates with a two-fold loss of muscle VEGFA protein levels (p=0.02). The capillary density in the TA is similar between the two groups. Young VEGFlo mice, with a 50% decrease in systemic VEGFA activity, exhibit a two-fold reduction in the average regenerating fiber CSA following cryoinjury (p< 0.01) in comparison to littermate controls. ML228, a hypoxia signaling activator known to increase VEGFA levels, augments muscle VEGFA levels and increases average CSA of regenerating fibers in both old mice (25% increase, p<0.01) and VEGFlo (20% increase, p<0.01) mice, but not in young or littermate controls. These results suggest that VEGFA may be a therapeutic target in age-related muscle loss.

AGING-RELATED VEGF IMPAIRS MUSCLE REGENERATION

Abstract Purpose Aging is associated with frailty, a parameter that correlates with mortality and loss of muscle mass. The molecular mechanisms behind aging-associated impairment of muscle regeneration remain incompletely understood. We hypothesized VEGF-A with known role in angiogenesis and muscle progenitor differentiation to regulate regeneration in aged skeletal muscle. Methods Young C57BL/6 (10 weeks old) and old C57BL/6 mice (24 months old) were subjected to muscle cryoinjury to induce regeneration. Quantifications of cross-sectional area (CSA) of regenerating myofibers were performed. Tibialis anterior muscle lysates was used for quantifying VEGF-A. To evaluate the role of VEGF in muscle regeneration, a similar experiment was performed on VEGFlo mice with a 75% decrease in VEGF-A activity and littermate controls. ML228, a hypoxia signaling activator that increases VEGF-A levels, was injected into young and old mice as well as VEGFlo and littermate controls. Results Old mice exhibited marked reduction in the VEGF-A protein levels and regenerating myofiber CSA on DPI 10 (1250 vs. 833μm2, p&amp;lt;.001). Similarly, VEGFlo mice exhibited significantly smaller regenerating fiber CSA as compared to littermate controls on DPI 10 (541 vs. 238μm2, p=.0011). Pharmacological augmentation of VEGFA using ML228 increased muscle VEGF levels by 2 folds and skeletal muscle regeneration in both old mice (25% increase in regenerating fiber CSA, p&amp;lt;.01) and VEGFlo (20% increase in regenerating fiber CSA, p&amp;lt;.01) mice, but not young or littermate controls. Conclusions Muscle regeneration declines with aging in correlation with loss of VEGFA levels within skeletal muscle. Supplementation of VEGFA represents a therapeutic target for sarcopenia.

Pumilio RNA-Binding Family Member 1 Plays a Promoting Role on Pancreatic Cancer Angiogenesis.

Our previous studies showed that Pumilio RNA-binding family member 1 (PUM1) gene is abnormally expressed in pancreatic cancer (PC) tissues, and its knockdown suppresses the growth and metastasis of PC cells. Here, we aimed to further investigate its role in angiogenesis. Immunohistochemical assays were carried out to analyze CD31 and PUM1 expression levels in PC tissues and in subcutaneous xenograft tumors. CD31 levels in PC tissues are expressed as microvessel density (MVD). MVD value was positively correlated with PUM1 protein expression. PUM1 was successfully overexpressed or silenced in the PC cell lines. The proliferation, migration, invasion, and tube formation ability of HUVECs were enhanced when cocultured with PC cells overexpressing PUM1. PUM1 overexpression increased extracellular and intracellular VEGFA protein levels in PC cells. Moreover, angiogenesis-related signaling in HUVECs was activated when HUVECs were cocultured with PC cells overexpressing PUM1. Nevertheless, PC cells silenced with PUM1 had the opposite effect. Moreover, subcutaneous xenograft tumors overexpressing PUM1 have the higher expression level of CD31, while subcutaneous xenograft tumors silencing PUM1 have the lower expression level of CD31. In conclusion, PUM1 in PC cells may play a promoting role in PC angiogenesis. PUM1 may be a new regulator of angiogenesis in PC cells.

Development of a method to identify persistent and blanchable redness by skin blotting in mice.

Persistent and blanchable redness (PBR) is not currently included in category I pressure injury (PI), which is defined as non-blanchable redness (NBR). However, PBR progresses to PI in a clinical setting. Therefore, it should be clinically managed as category I PI, and a method to distinctly identify PBR is needed. This study aimed to examine whether PI-related biomarkers can distinguish PRB from transient redness (TR) and NBR using skin blotting. TR, PBR, and NBR models were established by the different conditions of dorsal skin compression. Redness observation and skin blotting were performed, and the skin tissue samples were subjected to histological and molecular biological analyses. The vascular endothelial growth factor (Vegf) b, heat shock protein (Hsp) 90aa1, tumour necrosis factor, interleukin (Il) 1b, and Il6 messenger ribonucleic acid levels were significantly different between the three models. The VEGF-A, VEGF-B, IL-1β, and IL-6 protein levels were different between the three models. Although the results of skin blot examinations were inconsistent with those of the expression analysis of tissue, HSP90α and IL-1β are suggested to be potential markers to distinguish PBR from TR and NBR.

Buyang Huanwu Decoction: A Traditional Chinese Medicine, Promotes Lactate-Induced Angiogenesis in Experimental Intracerebral Hemorrhage.

Identifying the underlying mechanisms and exploring effective therapies for intracerebral hemorrhage (ICH) are urgently needed. Here, we aim to elucidate the potential roles and underlying mechanisms of Buyang Huanwu decoction (BYHWD) in ICH. In the first set of experiments, rats were randomly divided into five groups: Sham, ICH, ICH + sodium oxamate (OXA), ICH + BYHWD, and ICH + BYHWD + OXA. The lactate level around the hematoma was evaluated. PCNA+/vWF+ nuclei were observed. Additionally, an online bioinformatics analysis tool was used to predict the BYHWD druggable targets related to angiogenesis. Then, we validated these predictions. In the second set, exogenous sodium L-lactate (Lac) was infused into the intact brains of rats. Rats were randomly divided into three groups: Sham, Lac, and Lac + YC-1. The numbers of PCNA+/vWF+ nuclei and the expression of HIF-1α and VEGF were evaluated. In the first set of experiments, compared with the ICH group, the BYHWD group exhibited significantly increased numbers of PCNA+/vWF+ nuclei, and neurological dysfunction was markedly improved. Bioinformatics analysis revealed that the improvements caused by BYHWD indicated a role for the HIF-1α pathway. The HIF-1α and VEGF protein levels were upregulated after BYHWD administration. Moreover, we verified that lactate was involved in the predicted mechanisms. In the second set, lactate facilitated angiogenesis and HIF-1α and VEGF expression. Co-infusion with a HIF-1α inhibitor, YC-1, significantly inhibited these effects. Our data suggest that the pharmacological effects of BYHWD involve lactate-induced angiogenesis, these data may provide new evidence for its use in ICH.

The therapeutic role of Jingchuan tablet on ischaemic cerebral stroke via the HIF-1α/EPO/VEGFA signalling pathway

Context Jingchuan tablet (JCT) is a Chinese medicine prescription for treating ischaemic cerebral stroke (ICS). However, its relevant mechanisms remain unclear. Objective To unravel the intrinsic mechanisms of JCT anti-ICS. Materials and methods ‘Hongjingtian’, ‘chuanxiong’, ‘yanhusuo’, ‘bingpian’, ‘cerebral infarction’, ‘cerebral ischemia’ or ‘stroke’ were used as keywords, and then components, targets and underlying mechanisms of JCT anti-ICS were analysed in TCMSP, TTD, DrugBank, STRING and Metascape databases up to June 2020. Male Sprague-Dawley rats under permanent middle cerebral artery occlusion (pMCAO) model, randomly assigned as: model, sham, nimodipine (0.012 g/kg/d) and JCT (0.78, 1.56 and 3.12 g/kg/d) groups, received oral gavage administration for a week. Therapeutic effects were evaluated by detecting the proportion of cerebral infarction, neuronal apoptosis and neurological deficits. Bioactive components were detected by HPLC–MS. Molecular biology and computational docking were used to verify the underlying mechanisms. Results Eighty-one components, 166 targets and HIF-1α/EPO/VEGFA pathway contributed to the anti-ICS effect of JCT. JCT treatment effectively reduced the proportion of cerebral infarction (33.13%), apoptosis rate (14.80%) and neurobehavioural score (2.00). JCT increased the protein levels of HIF-1α (0.84), EPO (0.64) and VEGFA (0.69), respectively (p < 0.05). Gallic acid, salidroside, chlorogenic acid, ethyl gallate, ferulic acid and tetrahydropalmatine detected by HPLC–MS showed good interaction and binding with HIF-1α/EPO/VEGFA. Conclusions Our study demonstrated the mechanisms of JCT anti-ICS associated with the activation of the HIF-1α/EPO/VEGFA pathway, which provided a pharmacological basis for expanding the clinical application and some scientific ideas for further research into the material basis JCT anti-ICS.

The novel prolyl hydroxylase-2 inhibitor caffeic acid upregulates hypoxia inducible factor and protects against hypoxia

Background & aimsHypoxia inducible factor (HIF) is a hypoxia-associated transcription factor that has a protective role against hypoxia-induced damage. Prolyl hydroxylase-2 (PHD2) is a dioxygenase enzyme that specifically hydroxylates HIF targeting it for degradation, therefore, inhibition of the PHD2 enzyme activity acts to upregulate HIF function. This study was to identify novel PHD2 inhibitors. MethodsAn established fluorescence-based PHD2 activity assay was used for inhibitors screening. Western blot and quantitative real-time PCR was used to detect the protein and mRNA levels respectively. Further animal experiment was carried out. ResultsCaffeic acid was screened and identified as a novel PHD2 inhibitor. Caffeic acid treated PC12 and SH-SY5Y neuronal cell lines stabilized endogenous HIF-1α protein levels and consequently increased mRNA levels of its downstream regulated genes VEGF and EPO. Caffeic acid treatment reduced hypoxia-induced cell apoptosis and promoted HIF/BNIP3-mediated mitophagy. Moreover, animal studies indicated that caffeic acid increased the level of HIF-1α protein and mRNA levels of VEGF and EPO in the brain of mice exposed to hypoxia. Conventional brain injury markers including malondialdehyde, lactic acid and lactate dehydrogenase in the caffeic acid treated mice were shown to be reduced to the levels of the control group. ConclusionsThis study suggests that caffeic acid inhibits PHD2 enzyme activity which then activates the hypoxia-associated transcription factor HIF leading to a neuroprotective effect against hypoxia.

Kallistatin prevents ovarian hyperstimulation syndrome by regulating vascular leakage.

Angiogenesis and increased permeability are essential pathological basis for the development of ovarian hyperstimulation syndrome (OHSS). Kallistatin (KS) is an endogenous anti‐inflammatory and anti‐angiogenic factor that participates in a variety of diseases, but its role in OHSS remains unknown. In this study, treating a human ovarian granulosa‐like tumour cell line KGN and human primary granulosa cells (PGCs) with human chorionic gonadotropin (hCG) reduced the expression of KS, but increased the expression of VEGF. Furthermore, we found that KS could attenuate the protein level of VEGF in both KGN cells and human PGCs. More interestingly, we observed that exogenous supplementation of KS significantly inhibited a series of signs of OHSS in mice, including weight gain, ovarian enlargement, increased vascular permeability and up‐regulation of VEGF expression. In addition, KS was proved to be safe on mice ovulation, progression of normal pregnancy and fetus development. Collectively, these findings demonstrated that KS treatment prevented OHSS, at least partially, through down‐regulating VEGF expression. For the first time, these results highlight the potential preventive value of KS in OHSS.

Zinc improves neurological recovery by promoting angiogenesis via the astrocyte-mediated HIF-1α/VEGF signaling pathway in experimental stroke.

BackgroundIschemic stroke is a serious cerebrovascular disease with high morbidity and disability. Zinc accumulation has been shown to play a vital role in neuronal death and blood–brain barrier damage following ischemia in acute stage. However, almost nothing is known about whether zinc is involved in neurological recovery in ischemic prolonged period. This study investigates whether zinc promotes neurological recovery through astrocytes‐induced angiogenesis during ischemic repair phase.MethodsSprague–Dawley rats were subjected to 2 h ischemia/14, 21, and 28 days reperfusion by middle cerebral artery occlusion, then administered ZnCl2 (10 mg/kg) via intraperitoneally daily from 7 days to tissue collection to observe brain tissue morphology, neurological function recovery by cortical width index, Adhesive removal test, and Forelimb placing test. Angiogenesis, astrocyte activation, and HIF‐1α/VEGF pathway were assessed via Western blot, immunofluorescence, and BrdU method in vivo and in vitro.ResultsThe results showed that zinc significantly alleviated brain atrophy and improved neurological function recovery during the cerebral ischemia repair stage. Zinc significantly increased the protein levels of HIF‐1α, VEGF‐A, and VEGF‐R2 in astrocytes, and promoted angiogenesis during cerebral ischemia repair. In vitro and in vivo studies confirmed that zinc promoted angiogenesis via the astrocyte‐mediated HIF‐1α/VEGF signaling pathway.ConclusionsZinc significantly improves neurological function recovery during the cerebral ischemia repair stage, providing new evidence supporting zinc as a potential therapeutic target for ischemic stroke by promoting astrocyte induced angiogenesis.

Fetal hyperhomocysteinemia is associated with placental inflammation and early breakdown of maternal-fetal tolerance in pre-term birth.

Hyperhomocysteinemia (hypHcy) due to impaired folate metabolism is shown to be a risk factor for preterm birth (PTB) and low birth weight (LBW) in mothers. However, the relationship of fetal hypHcy with adverse pregnancy outcomes is under-represented. The present study aims to investigate the association of fetal hypHcy with oxidative stress and placental inflammation that can contribute to an early breakdown of maternal-fetal tolerance in pre-term birth (PTB). Cord blood and placenta tissue were collected from PTB and term infant group. Levels of homocysteine, folic acid, vitamin B12 and oxidative stress markers (MDA, T-AOC, 8-OHdG) were measured in cord blood serum using ELISA and respective standard assay kits. Relative expression of candidate genes (TNF-α, IL-6, IL1-β, VEGF-A, MMP2 and MMP9) was also checked using RT-PCR and immunoblotting/immunohistochemistry. PTB infants showed significantly higher levels of homocysteine (P=.02) and lower levels of vitamin B12 (P=.005) as compared to term infants. We also found that PTB infants with hypHcy had lower T-AOC (P=.003) and higher MDA (P=.04) levels as compared to term infants with normal homocysteine levels. The mRNA and protein levels of TNF-α, VEGF-A, MMP2 and MMP9 were significantly higher in hypHcy PTB infants. Our results show that fetal hypHcy is associated with oxidative stress and an increase in inflammatory markers in the placenta. Thus, in conclusion, our study demonstrates that fetal hypHcy during pregnancy is a potential risk factor that may initiate an early breakdown of uterine quiescence due to activation of inflammatory processes leading to PTB.