Vascular Endothelial Growth Factor A Research Articles

Brown Adipose Tissue Promotes Autologous Fat Grafts Retention Possibly Through Inhibiting Wnt/β-Catenin Pathway.

In plastic surgery, autologous fat grafts (AFG) play an important role because of their abundant supply, biocompatibility, and low rejection rate. However, the lower retention rate of fat grafts limits their widespread use. Brown adipose tissue (BAT) can promote angiogenesis and regulate the level of associated inflammation. This study explored whether BAT has a facilitative effect on fat graft retention. We obtained white adipose tissue (WAT) from c57 mice and combined it with either BAT from c57 mice or phosphate-buffered saline (PBS) as a control. These mixtures were injected subcutaneously into the back of thymus-free nude mice. After 12 weeks, fat grafts were harvested, weighed, and analyzed. We found that the BAT-grafted group had higher mass retention, more mature adipocytes, and higher vascularity than the other group. Further analysis revealed that BAT inhibited M1 macrophages; down-regulated IL-6, IL-1β, and TNF-β; upregulated M2 macrophages and Vascular endothelial growth factor-A (VEGFA); and promoted adipocyte regeneration by inhibiting the Wnt/β-catenin pathway, which together promoted adipose graft retention. The study demonstrated that BAT improved adipose graft retention by promoting angiogenesis, inhibiting tissue inflammation levels and the Wnt/β-catenin pathway. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Retracted] Downregulation of microRNA-206 suppresses clear cell renal carcinoma proliferation and invasion by targeting vascular endothelial growth factorA.

Following the publication of the above paper, it was drawn to the Editor's attention by a concerned reader that there appeared to be several instances of overlapping data panels comparing between the Transwell invasion and migration assay images shown in Figs.2E and 4G, such that data which were intended to show the results from differently performed experiments were apparently derived from a (much) smaller number of original sources. Given the number of cases of overlapping data panels both within and between this pair of figures in the article itself, the Editor of OncologyReports has decided that this paper should be retracted from the Journal on the basis of a lack of confidence in the presented data. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [Oncology Reports 35: 1778-1786, 2016; DOI: 10.3892/or.2015.4538].

Computer Prediction and Experimental Verification of Guizhi Fuling Pill in the Treatment of Benign Prostatic Hyperplasia

Objective: Benign prostatic hyperplasia (BPH) is a prevalent condition among middle-aged and elderly males. The hypoxia-inducible factor-1α (HIF-1α) / vascular endothelial growth factor A (VEGF-A) signaling pathway plays a key role in proliferative disorders. Guizhi Fuling Pill (GFP) provides significant clinical efficacy in addressing BPH and related hyperplastic ailments. This study aims to elucidate the potential involvement of the HIF-1α / VEGF-A signaling pathway in the therapeutic action of GFP against BPH. Methods: Employing network pharmacology and molecular docking techniques, the mechanism underlying GFP against BPH was anticipated. A rat model of BPH was established to validate the primary targets identified via molecular docking. Subcutaneous administration of testosterone propionate was conducted to induce the BPH rat model for experimental validation. Immunohistochemistry was performed to assess the expression of HIF-1α and VEGF-A in prostate tissue across different experimental groups. Results: The network pharmacology analysis unveiled 111 potential constituents of GFP, with key components such as ent-Epicatechin, quercetin, and Mairin exerting influence on BPH. Essential targets encompass ESR1 and HSP90AA1, with signaling pathways like HIF-1, PI3K-AKT, and VEGF prominently associated with cellular apoptosis, angiogenesis, and proliferation. Molecular docking projections underscored the propensity of core components to interact with key targets, exhibiting favorable binding characteristics. In the context of animal experiments, GFP significantly reduced the expression of HIF-1α and VEGF-A in BPH-afflicted rats as compared to the model group. Conclusion: Collectively, this investigation suggests that GFP’s therapeutic potential for BPH could be attributed to its modulation of multifaceted targets, including HIF-1α and VEGF-A.

TSLP is localized in and released from human lung macrophages activated by T2-high and T2-low stimuli: relevance in asthma and COPD

BackgroundMacrophages are the predominant immune cells in the human lung and play a central role in airway inflammation, including asthma and chronic obstructive pulmonary disease (COPD). Thymic stromal lymphopoietin (TSLP), a pleiotropic cytokine mainly expressed by bronchial epithelial cells, plays a key role in asthma and COPD pathobiology. TSLP exists in two variants: the long form (lfTSLP) and a shorter TSLP isoform (sfTSLP). We aimed to localize TSLP in human lung macrophages (HLMs) and investigate the mechanisms of its release from these cells. We also evaluated the effects of the two variants of TSLP on the release of angiogenic factor from HLMs. MethodsWe employed immunofluorescence and Western blot to localize intracellular TSLP in HLMs purified from human lung parenchyma. HLMs were activated by T2-high (IL-4, IL-13) and T2-low (lipopolysaccharide: LPS) immunological stimuli. ResultsTSLP was detected in HLMs and subcellularly localized in the cytoplasm. IL-4 and LPS induced TSLP release from HLMs. Preincubation of macrophages with brefeldin A, known to disrupt the Golgi apparatus, inhibited TSLP release induced by LPS and IL-4. lfTSLP concentration-dependently induced the release of vascular endothelial growth factor-A (VEGF-A), the most potent angiogenic factor, from HLMs. sfTSLP neither activated nor interfered with the activating property of lfTSLP on macrophages. ConclusionsOur results highlight a novel immunologic circuit between HLMs and TSLP. Given the central role of macrophages in airway inflammation, this autocrine loop holds potential translational relevance in understanding innovative aspects of the pathobiology of asthma and chronic inflammatory lung disorders.

Curative effect of the total saponins of Panax japonicus (TSPJ) on type 2 diabetes: Focusing on VEGFA

ObjectiveThe objective of this study was to assess the impact of the total saponins of Panax japonicus (TSPJ) on Type 2 diabetes mellitus (T2DM). ResultsThe intervention of TSPJ was found to have the ability to reverse physiological indicators associated with T2DM, while also enhancing the expression of genes involved in glucose metabolism and intestinal homeostasis. Additionally, alterations in the composition of the gut microbiota were observed. Based on the findings of experimental results and network pharmacology analysis, it is evident that vascular endothelial growth factor A (VEGFA) serves as a prominent shared target between TSPJ and diabetes. The outcomes observed in T2DM mice overexpressing VEGFA align with those observed in T2DM mice treated with TSPJ. ConclusionsTSPJ administration and VEGFA overexpression yield similar effects on T2DM in mice. Thus, in terms of mechanism, by upregulating the expression of VEGFA, TSPJ may ameliorate metabolic imbalance, preserve intestinal homeostasis, and lessen the symptoms of type 2 diabetes. The findings demonstrated the viability of using VEGFA as a type 2 diabetes therapy option and offered important insights into the therapeutic mechanisms by TSPJ in the management of T2DM. To determine the exact mechanisms behind the effects of TSPJ and VEGFA and to assess their potential therapeutic uses, more research efforts are necessary.

AAV-mediated VEGFA overexpression promotes angiogenesis and recovery of locomotor function following spinal cord injury via PI3K/Akt signaling

Spinal cord injury (SCI) is a disorder of the central nervous system resulting from various factors such as trauma, inflammation, tumors, and other etiologies. This condition leads to impairment in motor, sensory, and autonomic functions below the level of injury. Limitations of current therapeutic approaches prompt an investigation into therapeutic angiogenesis through persistent local expression of proangiogenic factors. Here, we investigated whether overexpression of adeno-associated virus (AAV)-mediated vascular endothelial growth factor A (VEGFA) in mouse SCI promoted locomotor function recovery, and whether the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway was mechanistically involved. Three weeks before SCI, AAV-VEGFA was injected at the T10 level to induce VEGFA overexpression. Neurofunctional, histological, and biochemical assessments were done to determine tissue damage and/or recovery of neuromuscular and behavioral impairments. Daily injections of the PI3K/Akt pathway inhibitor LY294002 were made to assess a possible mechanism. AAV-VEGFA overexpression dramatically improved locomotor function and ameliorated pathological injury caused by SCI. Improved motor-evoked potentials in hindlimbs and more spinal CD31-positive microvessels were observed in AAV-VEGFA-overexpressing mice. LY294002 reduced PI3K and Akt phosphorylation levels and attenuated AAV-VEGFA-related improvements. In conclusion, sustained local AAV-mediated VEGFA overexpression in spinal cord can significantly promote angiogenesis and ameliorate locomotor impairment after SCI in a contusion mouse model through activation of the PI3K/Akt signaling pathway.

Maternal Supplementation with Ornithine Promotes Placental Angiogenesis and Improves Intestinal Development of Suckling Piglets.

The blood vessels of the placenta are crucial for fetal growth. Here, lower vessel density and ornithine (Orn) content were observed in placentae for low-birth-weight fetuses versus normal-birth-weight fetuses at day 75 of gestation. Furthermore, the Orn content in placentae decreased from day 75 to 110 of gestation. To investigate the role of Orn in placental angiogenesis, 48 gilts (Bama pig) were allocated into four groups. The gilts in the control group were fed a basal diet (CON group), while those in the experimental groups were fed a basal diet supplemented with 0.05% Orn (0.05% Orn group), 0.10% Orn (0.10% Orn group), and 0.15% Orn (0.15% Orn group), respectively. The results showed that 0.15% Orn and 0.10% Orn groups exhibited increased birth weight of piglets compared with the CON group. Moreover, the 0.15% Orn group was higher than the CON group in the blood vessel densities of placenta. Mechanistically, Orn facilitated placental angiogenesis by regulating vascular endothelial growth factor-A (VEGF-A). Furthermore, maternal supplementation with 0.15% Orn during gestation increased the jejunal and ileal villi height and the concentrations of colonic propionate and butyrate in suckling piglets. Collectively, these results showed that maternal supplementation with Orn promotes placental angiogenesis and improves intestinal development of suckling piglets.

Evaluation of the Levels of Selected Cytokines and Their Possible Influence on the Development of Cardiovascular and Pulmonary Complications in Patients after COVID-19.

Background and Objectives: The aim of this study was to evaluate the levels of selected cytokines and their possible influence on the development of cardiovascular and pulmonary complications in patients hospitalized at the Silesian Centre for Heart Disease in Zabrze after having undergone COVID-19. Materials and methods: The study included 76 randomly selected patients from the SILCOVID-19 database. The median time from symptom onset to the study visit was 102 (86-118) days. The median age of the study group was 53 (44-60) years. Assays of a panel of 30 cytokines were carried out in the serum of patients on a Luminex100 platform using the Milliplex MAP kit from Merck KGaA Germany. Results: There were no statistically significant differences in most of the cytokines analyzed between patients with confirmed or excluded lung lesions or cardiac abnormalities. Additionally, no statistically significant differences in cytokine concentrations according to gender, age, comorbidity of diabetes, renal disease, hypertension, increased risk of thrombotic disease, or psychological disorders were demonstrated. There were high concentrations of cytokines such as platelet-derived growth actor-AA (PDGF-AA), monocyte chemoattractant protein-1 (MCP-1), monokine-induced gamma interferon (MIG), and vascular endothelial growth factor-A (VEGF-A). Conclusions: No direct impact of the dependencies between a panel of cytokines and the incidence of cardiovascular and pulmonary complications in patients hospitalized at the Silesian Centre for Heart Disease in Zabrze after having undergone COVID-19 was demonstrated. The demonstration of high levels of certain cytokines (PDGF-AA, VEGF, MIG, and IP10) that are of significance in the development of many lung diseases, as well as cytokines (MCP-1) that influence the aetiopathogenesis of cardiovascular diseases seems to be highly concerning in COVID-19 survivors. This group of patients should receive further monitoring of these cytokine levels and diagnostic imaging in order to detect more severe abnormalities as early as possible and administer appropriate therapy.

The pathogenicity of vancomycin-resistant Enterococcus faecalis to colon cancer cells

BackgroundThe aim of this study was to investigate the pathogenicity of vancomycin-resistant Enterococcus faecalis (VREs) to human colon cells in vitro.MethodsThree E. faecalis isolates (2 VREs and E. faecalis ATCC 29212) were cocultured with NCM460, HT-29 and HCT116 cells. Changes in cell morphology and bacterial adhesion were assessed at different time points. Interleukin-8 (IL-8) and vascular endothelial growth factor A (VEGFA) expression were measured via RT-qPCR and enzyme-linked immunosorbent assay (ELISA), respectively. Cell migration and human umbilical vein endothelial cells (HUVECs) tube formation assays were used for angiogenesis studies. The activity of PI3K/AKT/mTOR signaling pathway was measured by Western blotting.ResultsThe growth and adhesion of E. faecalis at a multiplicity of infection (MOI) of 1:1 were greater than those at a MOI of 100:1(p < 0.05). Compared to E. faecalis ATCC 29212, VREs showed less invasive effect on NCM460 and HT-29 cells. E. faecalis promoted angiogenesis by secreting IL-8 and VEGFA in colon cells, and the cells infected with VREs produced more than those infected with the standard strain (p < 0.05). Additionally, the PI3K/AKT/mTOR signaling pathway was activated in E. faecalis infected cells, with VREs demonstrating a greater activation compared to E. faecalis ATCC 29212 (p < 0.05).ConclusionVREs contribute to the occurrence and development of CRC by promoting angiogenesis and activating the PI3K/AKT/mTOR signaling pathway.

Anticancer Action of Silver Nanoparticles in SKBR3 Breast Cancer Cells through Promotion of Oxidative Stress and Apoptosis.

Silver nanoparticles (AgNPs) are known as one of the highly utilized NPs owing to their unique characteristics in the field of cancer research. The goal of this research was to explore the oxidative stress, apoptosis, and angiogenesis in SKBR3 breast cancer cells after exposure to AgNPs. The survival rate of SKBR3 cancer cells and MCF-10A normal breast cells was assessed under the effects of different concentrations (0, 32, 64, 128, and 250 μg/ml) by MTT method. The oxidative condition was assessed by measuring reactive oxygen species (ROS) production, total oxidant status (TOS), total antioxidant capacity (TAC), malondialdehyde (MDA), and antioxidant enzyme activity (CAT, GPx, and CAT) using colorimetric-based kits. Flow cytometry and Hoechst 33258 staining were performed to investigate the induction of apoptosis. Furthermore, the expression of Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), and caspase 3 and 7 activity was measured. The cell migration and vascular endothelial growth factor-A (VEGF-A) gene expression, protein kinase B (AKT), phosphatidylinositol 3-kinase (PI3K) were also studied. The MTT results indicated that AgNPs inhibit the SKBR3 cells' viability in a concentration-dependent way. Besides, AgNPs markedly induced oxidative stress via increasing TOS content, MDA production, reduction of TAC, and regulation of antioxidant enzyme level. Additionally, AgNPs promoted apoptosis as revealed by an enhancement in Bax/Bcl-2 expression ratio. Findings also indicated that AgNPs suppress the expression of genes (VEGF-A, AKT, and PI3K) involved in angiogenesis. Altogether, our data revealed that AgNPs initiate oxidative stress and apoptosis in SKBR3 breast cancer cells, dose dependently.

TENAYA and LUCERNE: Two-Year Results from the Phase 3 Neovascular Age-Related Macular Degeneration Trials of Faricimab with Treat-and-Extend Dosing in Year 2

PurposeTo evaluate the 2-year efficacy, durability, and safety of the bispecific antibody, faricimab, which inhibits both angiopoietin-2 and vascular endothelial growth factor-A. DesignTENAYA (NCT03823287) and LUCERNE (NCT03823300) were identically designed, randomized, double-masked, active comparator-controlled phase 3 noninferiority trials across 271 sites worldwide. ParticipantsTreatment-naïve patients with neovascular age-related macular degeneration (nAMD) aged ≥ 50 years. MethodsRandomized (1:1) to intravitreal faricimab 6.0 mg up to every 16 weeks (Q16W) or aflibercept 2.0 mg every 8 weeks (Q8W). Faricimab fixed dosing based on protocol-defined disease activity at weeks 20 and 24 up to week 60, followed up to week 108 by a treat-and-extend−based personalized treatment interval regimen. Main Outcome MeasuresEfficacy analyses included change in best-corrected visual acuity (BCVA) from baseline at 2 years (averaged over weeks 104, 108, and 112) and proportion of patients on Q16W, every 12 weeks (Q12W), and Q8W dosing at week 112 in the intention-to-treat population. Safety analyses included ocular adverse events (AEs) in the study eye through study end at week 112 in patients who received ≥ 1 dose of study treatment. ResultsOf 1326 patients treated across the trials, 1113 (83.9%) completed study treatment (n = 555 faricimab and n = 558 aflibercept). BCVA change from baseline at 2 years was comparable between faricimab and aflibercept in TENAYA (adjusted mean change [95% confidence interval (CI)] +3.7 letters [+2.1 to +5.4] and +3.3 letters [+1.7 to +4.9], respectively; mean difference [95% CI] 0.4 letters [−1.9 to +2.8]) and in LUCERNE (adjusted mean change [95% CI] +5.0 letters [+3.4 to +6.6] and +5.2 [+3.6 to +6.8], respectively; mean difference [95% CI] −0.2 letters [−2.4 to +2.1]). At week 112 in TENAYA and LUCERNE respectively, 59.0% and 66.9% achieved Q16W faricimab dosing, increasing from year 1, and 74.1% and 81.2% achieved ≥ Q12W dosing. Ocular AEs in the study eye were comparable between faricimab and aflibercept in TENAYA (55.0% and 56.5% of patients) and LUCERNE (52.9% and 47.5% of patients) through week 112. ConclusionsTreat-and-extend−based faricimab treatment based on nAMD disease activity maintained vision gains through year 2 with most patients achieving extended dosing intervals.

Reliability, stability during long-term storage, and intra-individual variation of circulating levels of osteopontin, osteoprotegerin, vascular endothelial growth factor-A, and interleukin-17A

BackgroundStudies in many populations have reported associations between circulating cytokine levels and various physiological or pathological conditions. However, the reliability of cytokine measurements in population studies, which measure cytokines in multiple assays over a prolonged period, has not been adequately examined; nor has stability during sample storage or intra-individual variation been assessed.MethodsWe assessed (1) analytical reliability in short- and long-term repeated measurements; (2) stability and analytical reliability during long-term sample storage, and (3) variability within individuals over seasons, of four cytokines—osteopontin (OPN), osteoprotegerin (OPG), vascular endothelial growth factor-A (VEGF-A), and interleukin-17A (IL-17A). Measurements in plasma or serum samples were made with commercial kits according to standard procedures. Estimation was performed by fitting a random or mixed effects linear model on the log scale.ResultsIn repeated assays over a short period, OPN, OPG, and VEGF-A had acceptable reliability, with intra- and inter-assay coefficients of variation (CV) less than 0.11. Reliability of IL-17A was poor, with inter- and intra-assay CV 0.85 and 0.43, respectively. During long-term storage, OPG significantly decayed (− 33% per year; 95% confidence interval [− 54, − 3.7]), but not OPN or VEGF-A (− 0.3% or − 6.3% per year, respectively). Intra- and inter-assay CV over a long period were comparable to that in a short period except for a slight increase in inter-assay CV of VEGF-A. Within-individual variation was small for OPN and VEGF-A, with intra-class correlations (ICC) 0.68 and 0.83, respectively, but large for OPG (ICC 0.11).ConclusionsWe conclude that OPN and VEGF-A can be reliably measured in a large population, that IL-17A is suitable only for small experiments, and that OPG should be assessed with caution due to degradation during storage and intra-individual variation. The overall results of our study illustrate the need for validation under relevant conditions when measuring circulating cytokines in population studies.

Identifying potential Q-markers for quality evaluation of Zhenyuan capsule by integrating chemical analysis, network pharmacology, molecular docking, and molecular dynamics simulations

Quality markers (Q-markers) are of great significance for quality evaluation of herbal medicines. Zhenyuan Capsule (ZYC) is a kind of Chinese patent medicine used to treat cardiovascular diseases. However, reliable and effective Q-markers for ZYC are still lacking. Herein, a UHPLC-Q/Orbitrap-MS/MS was performed to characterise the preliminary chemical profile of ZYC. A total of 86 components were characterised among which 20 constituents were unambiguously identified by reference compounds. Based on network pharmacology, seven major ginsenosides with great importance in the network were identified as Q-markers among which ginsenoside Re with the highest betweenness was screened to inhibit the development of coronary heart disease (CHD) by binding with vascular endothelial growth factor A (VEGFA). Docking and molecular dynamics simulation studies suggested that ginsenoside Re stably bound to VEGFA. Quantitative determination and chemical fingerprinting analysis were performed using HPLC-DAD. The results showed that ginsenosides screened might function as potential Q-markers for ZYC.

A New Generation of Gene Therapies as the Future of Wet AMD Treatment.

Age-related macular degeneration (AMD) is an eye disease and the most common cause of vision loss in the Western World. In its advanced stage, AMD occurs in two clinically distinguished forms, dry and wet, but only wet AMD is treatable. However, the treatment based on repeated injections with vascular endothelial growth factor A (VEGFA) antagonists may at best stop the disease progression and prevent or delay vision loss but without an improvement of visual dysfunction. Moreover, it is a serious mental and financial burden for patients and may be linked with some complications. The recent first success of intravitreal gene therapy with ADVM-022, which transformed retinal cells to continuous production of aflibercept, a VEGF antagonist, after a single injection, has opened a revolutionary perspective in wet AMD treatment. Promising results obtained so far in other ongoing clinical trials support this perspective. In this narrative/hypothesis review, we present basic information on wet AMD pathogenesis and treatment, the concept of gene therapy in retinal diseases, update evidence on completed and ongoing clinical trials with gene therapy for wet AMD, and perspectives on the progress to the clinic of "one and done" therapy for wet AMD to replace a lifetime of injections. Gene editing targeting the VEGFA gene is also presented as another gene therapy strategy to improve wet AMD management.

Exploring the mechanism of curcumin in the treatment of doxorubicin-induced cardiotoxicity based on network pharmacology and molecular docking technology.

Doxorubicin (DOX) is one of the most effective chemotherapeutic agents. However, the nonselective effect leads to serious cardiotoxicity risk in clinical use. Curcumin is a well-known dietary polyphenol that showed a protective effect against the cardiotoxic effect of DOX. This study aimed to assess the role of curcumin in protection against DOX-induced cardiotoxicity. Potential compound and disease targets were obtained from relevant databases, and common targets were screened. Protein-protein interaction (PPI) was used to predict the core targets. Gene ontology (GO) bioprocess analysis and Kyoto encyclopedia of genes and genome enrichment analysis enriched the possible biological processes (BP), cellular components, molecular function, and signaling pathways involved. Finally, the binding of curcumin to target proteins was evaluated through molecular docking. The docking score verified the reliability of the prediction results. In total, 205 curcumin and 700 disease targets were identified. A topological analysis of the PPI network revealed 10 core targets including TP53, tumor necrosis factor-alpha (TNF), AKT1, vascular endothelial growth factor A (VEGFA), prostaglandin-endoperoxide synthase 2 (PTGS2), signal transducer and activator of the transcription 3 (STAT3), HIF1A, MYC, epidermal growth factor receptor (EGFR), and CASP3 (Caspase-3). Furthermore, the enrichment analyses indicated that the effects of curcumin were mediated by genes related to oxidation, inflammation, toxification, cell proliferation, migration, apoptosis, wounding, metabolism, proteolysis, and the signaling pathway of calcium (Ca2+). Molecular docking showed that curcumin could bind with the target proteins with strong molecular force, exhibiting good docking activity. Curcumin has a multi-cardioprotective effect by modulating the core targets' expression in DOX-induced cardiotoxicity. This study elucidated the key target proteins and provided a theoretical basis for further exploring curcumin in the prevention and treatment of DOX-induced cardiotoxicity.

A Water-soluble Form of Dihydroquercetin Reduces LPS-induced Astrogliosis, Vascular Remodeling, and mRNA VEGF-A Levels in the Substantia Nigra of Aged Rats

Background: The age-dependent sporadic form of PD is characterized by the degeneration of dopaminergic (DA) neurons in the Substantia Nigra (SN), gliosis, and vascular changes. Vascular changes may contribute to the onset of the disease and exacerbate the neurodegenerative process, as some vascular changes occur before the onset of neuronal loss. To demonstrate the anti-neuroinflammatory efficacy of a new compound, a water-soluble form of dihydroquercetin (DHQ-WF), we studied the structural changes of microcirculatory vasculature, astroglial GFAP, and vascular endothelial growth factor –A (VEGF-A) mRNA expression in the SN of young and old rats after unilateral nigral treatment by lipopolysaccharide (LPS) and oral administration of DHQ-WF. Materials and methods: The experiments were performed on 18 young (8 weeks - 10 weeks old; 250 g - 320 g) and 18 old (18 months - 19 months old; 390 g - 450 g) male Vistar rats. Young and adult rats from the experimental groups were stereotactically injected with 2 μL LPS solution (LPS from Escherichia coli; 0,01 μL/mL) into one side of the SN. Control young and old rats were similarly injected with 2 μL sterile saline. Half of the animals in both the control and experimental groups (6 animals in each group) received a 2 ml solution containing DHQ-WF at a concentration of 3 mg/ml orally every day. After 8 weeks, brains were harvested and serial cryostat sections were prepared for histochemical (FITC-labeled tomato lectin), immunohistochemical (anti-GFAP Antibody, Cy3 Conjugate) staining, and real-time PCR (mRNA VEGF-A). Results: Eight weeks after LPS injection into the SN, a significant excess of areas occupied by cell bodies and processes of astroglial cells, the density of microcirculatory vessels, and mRNA VEGF-A expression was observed in old animals compared to control old animals and young LPS-treated rats. Oral administration of DHQ-WF to LPS-treated rats resulted in a significant reduction of these parameters in old animals. Conclusion: Injection of LPS into rat SN induces neuroinflammation and vascular angiogenesis, maximally expressed in old animals. Administration of DHQ-WF for 8 weeks significantly reduces these LPS-induced changes. DHQ-WF may be an effective treatment for reducing the effects of neuroinflammation in the aging brain.

Experimental study of tetramethylpyrazine-loaded electroconductive hydrogel on angiogenesis and neuroprotection after spinal cord injury

To explore the mechanisms for repairing spinal cord injury (SCI) with tetramethylpyrazine-loaded electroconductive hydrogel (hereinafter referred to as "TGTP"). A total of 72 female Sprague-Dawley rats were randomly divided into 4 groups: sham operation group (group A), SCI group (group B), SCI+electroconductive hydrogel group (group C), and SCI+TGTP group (group D). Only the vertebral plate was removed in group A, while the remaining groups were subjected to a whole transection model of spinal cord with a 2 mm gap in the lesions. The recovery of hindlimb motor function was evaluated by Basso, Beattie, Bresnahan (BBB) score and modified Rivlin-Tator inclined plate test before operation and at 1, 3, 7, 14, and 28 days after operation, respectively. Animals were sacrificed at 7 days and 28 days after modeling. Neovascularisation was observed by immunofluorescence staining of CD31 and the expression levels of angiopoietin 1 (Ang-1) and Tie-2 were assessed by Western blot assay. At 28 days postoperatively, the expression levels of pro-angiogenic related proteins, including platelet-derived growth factor B (PDGF-B), PDGF receptor β (PDGFR-β), vascular endothelial growth factor A (VEGF-A), and VEGF receptor 2 (VEGFR-2), were also assessed by Western blot. The fibrous scar in the injured area was assessed using Masson staining, while neuronal survival was observed through Nissl staining. Furthermore, LFB staining was utilized to detect myelin distribution and regeneration. Immunofluorescence and Western blot assay were employed to evaluate the expression of neurofilament 200 (NF200). The hindlimb motor function of rats in each group gradually recovered from the 3rd day after operation. The BBB score and climbing angle in group D were significantly higher than those in group B from 3 to 28 days after operation, and significantly higher than those in group C at 14 days and 28 days after operation ( P<0.05). Masson staining showed that the collagen volume fraction in groups B-D were significantly higher than that in group A, and that in group D was significantly lower than that in groups B and C ( P<0.05); a small amount of black conductive particles were scattered at the broken end in group D, and the surrounding collagen fibers were less than those in group C. Nissl and LFB staining showed that the structure of neurons and myelin sheath in the injured area of spinal cord in group D was relatively complete and continuous, and the number of Nissl bodies and the positive area of myelin sheath in group D were significantly better than those in groups B and C ( P<0.05). NF200 immunofluorescence staining and Western blot assay results showed that the relative expression of NF200 protein in group D was significantly higher than that in groups B and C ( P<0.05). CD31 immunofluorescence staining showed that the fluorescence intensity of group D was better than that of groups B and C at 28 days after operation, and tubular or linear neovascularization could be seen. The relative expressions of Ang-1 and Tie-2 proteins in group D were significantly higher than those in groups B and C at 7 and 28 days after operation ( P<0.05). The relative expressions of PDGF-B and PDGFR-β proteins in group D were significantly higher than those in groups B and C, and group B was significantly higher than group C at 28 days after operation ( P<0.05). The relative expressions of VEGF-A and VEGFR2 proteins in group D were higher than those in groups B and C, showing significant difference when compared with group B ( P<0.05), but only the expression of VEGF-A protein was significantly higher than that in group C ( P<0.05). There was significant difference only in VEGFR-2 protein between groups B and C ( P<0.05). TGTP may enhance the revascularization of the injured area and protect the neurons, thus alleviating the injury of spinal cord tissue structure and promoting the recovery of neurological function after SCI in rats.

Expression of VEGFR2 Ligand Binding Domain in Pichia pink™ 4 Cells and Evaluation of Its Interactions with VEGF-A165 Receptor Binding Domain.

Vascular endothelial growth factor A165 (VEGF-A165) and VEGF receptor 2 (KDR) are important mediators of angiogenesis. We aimed to express the soluble KDR ligand-binding domain (sKDR1-3) and evaluate its interaction with the VEGF-A165 receptor-binding domain (VEGFA165-RBD). sKDR1-3 DNA was designed and subcloned into pPinkα-HC plasmid. The cassette was transfected into the Pichia pink™ 4 genome by homologous recombination. We optimized the expression of sKDR1-3 under the induction of different methanol concentrations. VEGFA165-RBD was expressed in E. coli BL21 harboring pET28a( +)─VEGFA165-RBD vector under induction with IPTG with/without lactose. Interaction and biological activity of sKDR1-3 and VEGFA165-RBD were investigated by ELISA and anti-proliferation tests. sKDR1-3 migrated on SDS-PAGE gel as a 35-180kDa protein due to glycosylation. The relative expression level of sKDR1-3 under 1% methanol was higher than 0.5% and 4% methanol induction. IPTG and cysteine were suitable for induction and refolding of VEGFA165-RBD. 25ng sKDR1-3 and 20ng VEGFA165-RBD showed strong binding. sKDR1-3 bound to VEGFA165-RBD and VEGF-A165 with dissociation constants of 0.148 and 0.2nM, respectively. 4-10nM concentrations of sKDR1-3 inhibited the proliferation of HUVE cells induced by 5nM VEGFA165-RBD. In consideration, sKDR1-3 in the nanomolar concentration range, is a promising anticancer drug to inhibit angiogenesis.

The Inhibitory Effect of NSAIDs and Opioids on Spinal Fusion: An Animal Model.

Translational research. To evaluate the relative effects of NSAIDs, opioids, and a combination of the two on spinal fusion inhibition in a rodent model. Nonsteroidal anti-inflammatory drugs (NSAIDs) and opioids are common postoperative analgesic agents. Since NSAIDs inhibit the cyclooxygenase (COX) pathway, they are seldom prescribed following spinal fusion. Opioids may be given instead, but recent evidence suggests opioids also adversely affect spinal fusion quality and success. Eighty male Sprague-Dawley rats underwent L4-5 posterior lumbar fusion and were given one of the following analgesia regimens: saline, morphine (6mg/kg), ketorolac (4mg/kg), or morphine (3mg/kg) and ketorolac (2mg/kg). Serum samples were drawn to evaluate systemic pro-osteoblastic cytokines and vascular endothelial growth factor-A (VEGF-A) levels, which were measured through enzyme-linked immunosorbent assays (ELISA). After six weeks, the rats were sacrificed, and the operated spinal segments underwent manual palpation, microCT, and histologic analysis. Manual palpation scores were significantly diminished in the opioid, NSAID, and multimodal groups when compared with control ( P <0.001). MicroCT fusion scores ( P <0.001) and fusion rates (control: 75% vs . NSAID: 35% vs . opioid: 0% vs . combination: 15%, P <0.001) were significantly diminished in the treatment groups. The bone volume (BV) to tissue volume (TV) ratio (BV/TV) ( P <0.001) and bone mineral density (BMD) ( P <0.001) were all lower in the treatment groups, with the opioid and combined groups having the lowest BMD. Although statistically insignificant ( P <0.09), the concentration of VEGF-A was greater in the control group compared with opioids, NSAIDs, and the combined group. Opioids and NSAIDs, both independently and combined, inhibited spinal fusion and caused inferior bony callus. Administration of opioids resulted in the lowest rate of spinal fusion. We propose this may be due to the inhibition of VEGF-A, which limits angiogenesis to the burgeoning fusion mass.

5-Aza-2'-Deoxycytidine Ameliorates Choroidal Neovascularization by Inhibiting the Wnt/β-Catenin Signaling Pathway.

Choroidal neovascularization (CNV) can constitute the final pathology of many ocular diseases and result in severe vision loss. Studies have demonstrated that DNA methylation is critical in retinal development, aging, and disorders. The current work investigated the effects and underlying mechanism of 5-Aza-2'-deoxycytidine (5-aza-dC), a suppressor of DNA methylation, in the pathological progression of CNV. The DNA methylation profiles of retinal pigment epithelial (RPE)/choroidal complexes in normal and laser-induced CNV mice were assessed by Arraystar Mouse RefSeq Promoter Arrays. The CNV area and blood flow density and intensity were observed by optical coherence tomography angiography, and fluorescence leakage was examined by fundus fluorescein angiography in CNV mice with systemic administration of 5-aza-dC. The effects of 5-aza-dC on the biological functions of bEnd.3 cells were estimated by related assays. Notum gene promoter methylation was measured using bisulfite sequencing PCR. Methyltransferases and Wnt signaling-related genes were detected in animal and cell culture experiments by real-time PCR and immunoblot. Methyltransferases were upregulated, but Notum (a secretion inhibitor of Wnt signaling) was downregulated in the RPE/choroidal complexes of mice with experimental CNV. Intraperitoneal injection of 5-aza-dC inactivated the Wnt pathway and ameliorated the lesion area and the intensity and density of blood flow, as well as the degree of leakage in CNV. In vitro, vascular endothelial growth factor A (VEGFA) stimulation promoted methyltransferases expression and suppressed Notum expression, consequently activating Wnt signaling, whereas exogenous 5-aza-dC reversed VEGFA-induced hyperpermeability, proliferation, migration, and tube formation in bEnd.3 cells via demethylation of Notum promoter. We observed that 5-aza-dC attenuates the growth of CNV by inhibiting the Wnt signaling pathway via promoter demethylation of the Wnt antagonist Notum. These findings provide a theoretical basis for methylation-based treatment with the Notum gene as a potential target for CNV treatment.