VEGF Levels Research Articles

Analysis of RNA Interference Targeted Against Human Antigen R (HuR) to Reduce Vascular Endothelial Growth Factor (VEGF) Protein Expression in Human Retinal Pigment Epithelial Cells.

VEGF-A or vascular endothelial growth factor-A is an important factor in enabling neovascularization and angiogenesis. VEGF-A is regulated transcriptionally as well as post transcriptionally. Human antigen R (HuR) belonging to the embryonic lethal abnormal vision (ELAV) family is a key regulator promoting stabilization of VEGF-A mRNA. In this research we investigate, whether HuRtargeted RNA interference would enable the reduction of the VEGF-A protein in human retinal pigment epithelial cells (ARPE-19) in-vitro, in normoxic conditions. Three siRNA molecules with sequences complementary to three regions of the HuR mRNA were designed. The three designed siRNA molecules were individually transfected in ARPE-19 cells using Lipofectamine™2000 reagent. Post-transfection (24h, 48h, 72h), downregulation of HuR mRNA was estimated by real-time polymerase reaction, while HuR protein and VEGF-A protein levels were semi-quantitatively determined by western blotting techniques. VEGF-A protein levels were additionally quantified using ELISA techniques. All experiments were done in triplicate. The designed siRNA could successfully downregulate HuR mRNA with concomitant decreases in HuR and VEGF-A protein. The study reveals that HuR downregulation can prominently downregulate VEGF-A, making the protein a target for therapy against pathological angiogenesis conditions such as diabetic retinopathy.

Over-representation analysis of angiogenic factors in immunosuppressive mechanisms in neoplasms and neurological conditions during COVID-19

BackgroundRecent studies emphasized the necessity to identify key (human) biological processes and pathways targeted by the Coronaviridae family of viruses, especially Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronavirus Disease (COVID-19) caused up to 33–55 % death rates in COVID-19 patients with malignant neoplasms and Alzheimer's disease. Given this scenario, we identified biological processes and pathways involved in various diseases which are most likely affected by COVID-19. MethodsThe COVID-19 DisGeNET data set (v4.0) contains the associations between various diseases and human genes known to interact with viruses from Coronaviridae family and were obtained from the IntAct Coronavirus data set annotated with DisGeNET data. We constructed the disease-gene network to identify genes that are involved in various comorbid diseased states. Communities from the disease-gene network were identified using Louvain method and functional enrichment through over-representation analysis methodology was used to discover significant biological processes and pathways shared between COVID-19 and other diseases. ResultThe COVID-19 DisGeNET data set (v4.0) comprised of 828 human genes and 10,473 diseases (including various phenotypes) that together constituted nodes in the disease-gene network. Each of the 70,210 edges connects a human gene with an associated disease. The top 10 genes linked to most number of diseases were VEGFA, BCL2, CTNNB1, ALB, COX2, AGT, HLA-A, HMOX1, FGF2 and COMT. The most vulnerable group of patients thus discovered had comorbid conditions such as carcinomas, malignant neoplasms and Alzheimer's disease. Finally, we identified 15 potentially useful biological processes and pathways for improved therapies. Vascular endothelial growth factor (VEGF) is the key mediator of angiogenesis in cancer. It is widely distributed in the brain and plays a crucial role in brain inflammation regulating the level of angiopoietins. With a degree of 1899, VEGFA was associated with maximum number of diseases in the disease-gene network. Previous studies have indicated that increased levels of VEGFA in the blood results in dyspnea, Pulmonary Edema (PE), Acute Lung Injury (ALI) and Acute Respiratory Distress Syndrome (ARDS). In case of COVID-19 patients with neoplasms and other neurological symptoms, our results indicate VEGFA as a therapeutic target for inflammation suppression. As VEGFs are known to disproportionately affect cancer patients, improving endothelial permeability and vasodilation with anti-VEGF therapy could lead to suppression of inflammation and also improve oxygenation. As an outcome of our study, we make case for clinical investigations towards anti-VEGF therapies for such comorbid conditions affected by COVID-19 for better therapeutic outcomes.

A novel lupene derivative from Thymus capitatus possesses an apoptosis-inducing effect via Let-7 miRNA/Cyclin D1/VEGF cascade in the A549 cell line

Non-small-cell lung carcinoma (NSCLC) is a type of epithelial lung cancer accounting for about 85% of all lung cancers. In our research, a novel lupene derivative namely acetoxy-lup-5(6), 20(29)-diene (ALUP), as well as two known triterpenes; lupeol (LUP) and betulinic acid (BA) were isolated through the chromatographic purification of the 95% ethanolic extract of Thymus capitatus. Identification of the compounds was carried out by physicochemical properties as well as spectral 1D and 2D NMR analysis. The anti-cancer activity of the three triterpenes was assessed on non-small cell lung cancer cell line; A549 using MTT assay and cell cycle analysis using annexin V/propidium iodide. The molecular mechanism underlying anti-apoptotic effects was determined by analyzing Let-7 miRNA and miRNA-21 expression, the mRNA gene expression level of Bax, CASP-8, CD95, Bcl2, KRAS, VEGF, Cyclin D1 using qRT-PCR. Our results revealed that the three isolated compounds ALUP, LUP, and BA caused cell cycle arrest at the G2/M phase with an increase in the apoptosis which may be attributed to their significant effect on raising Bax, CASP-8, and CD95 and reducing the mRNA expression levels of Bcl-2, KRAS, VEGF, and Cyclin D1 compared to control cells. RT-PCR results showed that the ALUP, LUP, and BA significantly downregulated miRNA-21 expression. Meanwhile, the three compounds caused significant overexpression of Let-7 miRNA. This is the first report on the anti-cancer activity of acetoxy-lup-5(6), 20(29)-diene (ALUP) in reducing the proliferation and differentiation of the A549 cell line through inducing apoptosis. Finally, by targeting the Let-7 miRNA/Cyclin D1/VEGF cascade, acetoxy-lup-5(6), 20(29)-diene could be a potential therapeutic agent for lung cancer.

The Potential Mechanism of Curcumin in Treating Oral Squamous Cell Carcinoma Based on Integrated Bioinformatic Analysis.

This study explores the effects of curcumin as a therapeutic agent against oral squamous cell carcinoma (OSCC). We acquired the targets of curcumin from three digital databases, including the Comparative Toxicogenomics Database, Search Tool for Interactions of Chemicals, and SwissTargetPrediction. Then, we identified the differentially expressed genes (DEGs) and the weighted gene coexpression network analysis-based key modules using the expression profiles of GSE23558 to acquire the OSCC-related genes. Additionally, the GeneCards and Online Mendelian Inheritance in Man databases were also used to identify the OSCC-related genes. Finally, curcumin-OSCC interaction genes were obtained by overlapping curcumin targets and OSCC-related genes. The enrichment analysis was performed by the ClusterProfiler algorithm and Metascape, respectively. Then, a protein-protein interaction network was created, and the maximal clique centrality algorithm was used to identify the top 10 hub genes. Besides, we examined the expression levels of hub genes in OSCC using The Cancer Genome Atlas database. 927 DEGs were identified, including 308 upregulated ones and 619 downregulated ones. The cluster one-step network construction function of the WGCNA algorithm recognized a soft-thresholding power of 6, and 9083 genes were acquired. 2591 OSCC-related genes were obtained by overlapping the GSE23558-identified genes and the OSCC-related genes from disease target bases. Finally, we identified 70 candidate drug-disease interaction genes by overlapping the disease-related genes with the curcumin target. The enrichment analysis suggested that response to oxidative stress, epithelial cell proliferation, and AGE/RAGE pathway might involve in the effect of curcumin on OSCC. The topologic study identified the ten hub genes, including VEGFA, AKT1, TNF, HIF1A, EGFR, JUN, STAT3, MMP9, EGF, and MAPK3. A significant difference was observed in VEGFA, AKT1, TNF, HIF1A, EGFR, MMP9, EGF, and MAPK3 expression levels between head and neck squamous cell carcinoma and the normal controls. However, no significant difference was observed in JUN (P = 0.14) and STAT3 (P = 0.054). This study provided an overview and basis for the potential mechanism of curcumin against OSCC. The following experiments should be performed to further understand the effectiveness and safety of curcumin in treating OSCC.

Effect of whole body vibration therapy in the rat model of steroid-induced osteonecrosis of the femoral head.

Background: Steroid-induced Osteonecrosis of the Femoral Head (SIONFH) is a skeletal disease with a high incidence and a poor prognosis. Whole body vibration therapy (WBVT), a new type of physical training, is known to promote bone formation. However, it remains unclear whether WBVT has a therapeutic effect on SIONFH. Materials and methods: Thirty adult male and female Sprague-Dawley (SD) rats were selected and randomly assigned to three experimental groups: the control group, the model group, and the mechanical vibration group, respectively. SIONFH induction was achieved through the combined administration of lipopolysaccharides (LPS) and methylprednisolone sodium succinate for injection (MPS). The femoral head samples underwent hematoxylin and eosin (H&E) staining to visualize tissue structures. Structural parameters of the region of interest (ROI) were compared using Micro-CT analysis. Immunohistochemistry was employed to assess the expression levels of Piezo1, BMP2, RUNX2, HIF-1, VEGF, CD31, while immunofluorescence was used to examine CD31 and Emcn expression levels. Results: The H&E staining results revealed a notable improvement in the ratio of empty lacuna in various groups following WBVT intervention. Immunohistochemical analysis showed that the expression levels of Piezo1, BMP2, RUNX2, HIF-1, VEGF, and CD31 in the WBVT group exhibited significant differences when compared to the Model group (p < 0.05). Additionally, immunofluorescence analysis demonstrated statistically significant differences in CD31 and Emcn expression levels between the WBVT group and the Model group (p < 0.05). Conclusion: WBVT upregulates Piezo1 to promote osteogenic differentiation, potentially by enhancing the HIF-1α/VEGF axis and regulating H-vessel angiogenesis through the activation of the Piezo1 ion channel. This mechanism may lead to improved blood flow supply and enhanced osteogenic differentiation within the femoral head.

Model of toxic fibrosis in Wistar rats: morphological and molecular-genetic parameters of the transition point to cirrhosis

BACKGROUND: To date, when studying the cellular and molecular genetic mechanisms of liver fibrogenesis in experimental rat models, no attention is paid to the transition point of fibrosis to cirrhosis as a separate stage. As this pathology develops, changes in the cell phenotype and gene expression are dynamic in nature, so it is necessary to evaluate them in a long-term temporal dynamics.&#x0D; AIM: The aim of this work is to study the morphological and molecular genetic changes in the liver of Wistar rats during nodular parenchymal rearrangement.&#x0D; METHODS: Fibrosis and cirrhosis of the liver in male Wistar rats was induced with a freshly prepared solution of thioacetamide, which was administered intragastrically through a tube at a dose of 200 mg/kg of body weight 2 times a week for 13 weeks. The level of mRNA of the tweak (tnfsf12), fn14 (tnfrsf12a), ang, vegfa, cxcl12 (sdf), and mmp-9 genes in the liver was detected by real-time polymerase chain reaction. Immunohistochemical study was performed on paraffin sections. -SMA, FAP, CD68, CD206, CX3CR1, CD45 were used as markers. The area of interlobular veins and interlobular arteries was measured (m2). The number of sinusoidal capillaries and interlobular veins was counted.&#x0D; RESULTS: Based on the results obtained, it is possible to establish the transition point of fibrosis to cirrhosis as an independent separate stage of fibrogenesis. This transition was identified at stage F5, and the process itself from F4/F5 to F6.&#x0D; With the growth of fibrous tissue and nodular restructuring of the liver parenchyma, no progression of dystrophic processes and an increase in the zones of necrosis and necrobiosis of hepatocytes were noted. The number of -SMA+ and FAP+ cells in the period F4F5 did not change (p=0.2073 and p=0.3775, respectively). At the same time, significant F6 cirrhosis was accompanied by an increase in their number by 1.5 times (p 0.00001). Differences in the number of CD68+ cells were revealed only at the F4/F5 stage (2.0 times higher than the control, p 0.00001). The number of CD206+, CX3CR1+ and CD45+ cells remained the same. An increase in the number of interlobular veins (p 0.00001) and a decrease in sinusoidal capillaries (p 0.00001) were found compared to the control.&#x0D; The transition to cirrhosis was characterized by changes in the expression levels of tweak, fn14, ang, vegfa, cxcl12, and mmp-9 mRNAs, as well as the presence and strength of the relationship between them. Significant correlations were revealed between the target genes (r=0.50.84; p 0.01).&#x0D; CONCLUSION: The transition point of fibrosis to cirrhosis has special morphological and molecular genetic features that expand knowledge about pathomorphological changes in the liver.

A systems biology approach and in vitro experiment indicated Rapamycin targets key cancer and cell cycle-related genes and miRNAs in triple-negative breast cancer cells.

An anticancer drug known as Rapamycin acts by inhibiting the mammalian target of the Rapamycin pathway. This agent has recently been investigated for its potential therapeutic benefits in sensitizing drug-resistant breast cancer (BC) treatment. The molecular mechanism underlying these effects, however, is still a mystery. Using a systems biology method and in vitro experiment, this study sought to discover essential genes and microRNAs (miRNAs) targeted by Rapamycin in triple-negative BC (TNBC) cells to aid prospective new medications with less adverse effects in BC treatment. We developed the transcription factor-miRNA-gene and protein-protein interaction networks using the freely accessible microarray data sets. FANMOD and MCODE were utilized to identify critical regulatory motifs, clusters, and seeds. Then, functional enrichment analyses were conducted. Using topological analysis and motif detection, the most important genes and miRNAs were discovered. We used quantitative real-time polymerase chain reaction (qRT-PCR) to examine the effect of Rapamycin on the expression of the selected genes and miRNAs to verify our findings. We performed flow cytometry to investigate Rapamycin's impact on cell cycle and apoptosis. Furthermore, wound healing and migration assays were done. Three downregulated (PTGS2, EGFR, VEGFA) and three upregulated (c-MYC, MAPK1, PIK3R1) genes were chosen as candidates for additional experimental verification. There were also three upregulated miRNAs (miR-92a, miR-16, miR-20a) and three downregulated miRNAs (miR-146a, miR-145, miR-27a) among the six selected miRNAs. The qRT-PCR findings in MDA-MB-231 cells indicated that c-MYC, MAPK1, PIK3R1, miR-92a, miR-16, and miR-20a expression levels were considerably elevated following Rapamycin treatment, whereas PTGS2, EGFR, VEGFA, miR-146a, and miR-145 expression levels were dramatically lowered (p < 0.05). These genes are engaged in cancer pathways, transcriptional dysregulation in cancer, and cell cycle, according to the top pathway enrichment findings. Migration and wound healing abilities of the cells declined after Rapamycin treatment, and the number of apoptotic cells increased. We demonstrated that Rapamycin suppresses cell migration and metastasis in the TNBC cell line. In addition, our data indicated that Rapamycin induces apoptosis in this cell line. The discovered vital genes and miRNAs affected by Rapamycin are anticipated to have crucial roles in the pathogenesis of TNBC and its therapeutic resistance.

Immune checkpoint blockade induced shifts in cytokine expression patterns in peripheral blood of head and neck cancer patients are linked to outcome.

Immune-checkpoint blockade (ICB) of programmed-death-1 (PD-1) with pembrolizumab or nivolumab is approved for treating recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC). NadiHN and ADRISK are phase IIB trials investigating in locally advanced (LA) HNSCC having low or high risk of recurrence the potential benefits from adding nivolumab to post-operative radiotherapy or pembrolizumab to cisplatin-based radio-chemotherapy. Along five randomized controlled ICB trials including NadiHN and ADRISK, blood samples were taken before and after starting ICB in n=25 patients. Concentrations of vascular endothelial growth factor A (VEGF), CCL2 (MCP-1), interleukin-6 (IL-6), IL-8, interferon-gamma (IFN-γ), and CXCL10 (IP-10) pre- and post-ICB in EDTA-anticoagulated plasma and serum were compared. We used receiver operating characteristic (ROC) curves to identify optimal cutoff for defining subgroups before analyzing overall survival (OS) applying Kaplan-Meier plots and multivariate Cox regression. We detected huge heterogeneity between cytokine patterns in pre-and post-ICB plasma and serum. We observed high correlation between concentrations of some cytokines. Despite absent systematic OS differences after ICB with pembrolizumab or nivolumab or between LA-HNSCC versus R/M HNSCC patients, we noticed improved outcome of patients having lower IFN-γ concentrations pre- and post-ICB and following ICB reduced concentrations of VEGF, IL-6, and IL-8 but not MCP-1. Contrarily, increases in IL-6, IL-8, and VEGF levels correlated with impaired outcome. Multivariate Cox regression revealed five independent OS predictors among cytokines; using natural logarithms of their hazard ratios to estimate an individual's risk of dying, three cytokine-expression pattern (CEP)-risk groups with no death within mean (95% confidence interval) follow-up of 29.2 (22.1-36.2) months and median OS of 11.3 (8.8-13.8) and 2.9 (0.4-5.4) months were found. Whereas individual pre- or post-ICB cytokine concentrations in serum or plasma alone failed to predict the survivor group, CEP-risk groups may support the identification of individual patients with long-lasting benefit fromICB.

Intralesional Axitinib Injection Mitigates Hypertrophic Scar by Inhibiting Angiogenesis Pathway: A Preliminary Study in a Rabbit Ear Model.

High levels of VEGF and excessive angiogenesis contribute significantly to hypertrophic scar (HS) formation. Our study aimed to preliminarily investigate the effect of axitinib, a selective VEGF receptor tyrosine kinase inhibitor, on angiogenesis of HS and to explore its possible mechanism in a rabbit ear model. Ten male New Zealand white rabbits were used to establish HS models and then randomised to the control and axitinib groups. The scar tissues in the two groups were injected with axitinib or normal saline, and they were evaluated after one month of treatment. Macroscopic scar thickness, vascularity and pliability, as well as histopathological analysis including HE staining and Masson staining and scar elevation index (SEI) between two groups were compared. Immunohistochemical staining of CD31 in two groups was conducted to assess the degree of angiogenesis in HS tissue. The protein expression of protein kinase B (AKT) and ribosomal protein S6 kinase (p70S6K) and their phosphorylation levels in both groups were examined by Western blot analysis. The macroscopic and histological observation showed intralesional axitinib injection significantly reduced scar thickness, vascularity and pliability of HS in the rabbit ear model. The value of SEI in HE assessment was also significantly declined in the axitinib group. Furthermore, immunohistochemical analysis revealed that axitinib suppressed the expression of CD31 in HS tissue, and the mean IOD for blood vessels was significantly lower in the axitinib-treated group. Additionally, axitinib effectively attenuated the protein expression of p70S6K, p-AKT and p-p70S6K by Western blot analysis. Our study suggests that intralesional injection of axitinib can effectively attenuate HS by reducing angiogenesis in the rabbit ear model, and this inhibitory effect may be mediated by suppression of AKT/p70S6K signaling pathway. It indicates that axitinib may be a promising option for the treatment of HS in the future.

Mechanism of procyanidin b2 in the treatment of chronic myeloid leukemia based on integrating network pharmacology and molecular docking.

To study the pharmacological mechanism of PCB2 on chronic myeloid leukemia (CML) by integrating network pharmacological methods systematically. Firstly, the potential target genes of PCB2 were predicted by the pharmacological database and analysis platform (TCMSP and Pharmmapper). Meanwhile, the relevant target genes of CML were collected from GeneCards and DisGene. Pooled data were collected to screen for common target genes. Furthermore, the above intersection genes were imported into the String website to construct a protein-protein interaction (PPI) network, and the Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were further analyzed. Besides, molecular docking was performed to verify the possible binding conformation between PCB2 and candidate targets. Finally, MTT and RT-PCR experiments of K562 cells were performed to verify the above results of network pharmacology. A total of 229 PCB2 target genes were retrieved, among which 186 target genes had interaction with CML. The pharmacological effects of PCB2 on CML were related to some important oncogenes and signaling pathways. The top ten core targets predicted by Network Analysis were as follows: AKT1, EGFR, ESR1, CASP3, SRC, VEGFA, HIF1A, ERBB2, MTOR, and IGF1. Molecular docking studies confirmed that hydrogen bonding was the main interaction force of PCB2 binding targets. According to the molecular docking score, the following three target proteins were most likely to bind to PCB2: VEGFA (-5.5 kcal/mol), SRC (-5.1 kcal/mol), and EGFR (-4.6 kcal/mol). After treatment of PCB2 for 24h, mRNA expression levels of VEGFA and HIF1A decreased significantly in K562 cells. Through integrating network pharmacology combined with molecular docking, the study revealed the potential mechanism of PCB2 anti-chronic myeloid leukemia.

High glucose environment induces NEDD4 deficiency that impairs angiogenesis and diabetic wound healing

BackgroundHealing of diabetic wounds, characterized by impaired angiogenesis, remains a clinical challenge. E3 ligase have been identified as potential therapeutic targets of wound healing. ObjectiveWe assessed the role of E3 ligase NEDD4 in the context of angiogenesis and diabetic wound healing. MethodsThe mRNA expression levels of NEDD4, TSP1 and VEGF were determined by real-time PCR. Western blotting was used to evaluate the protein expression of NEDD4, TSP1 and VEGF. The ubiquitination of TSP1 was evaluated by immunoprecipitation. MTT assay, wound healing assay and tube formation assay were performed to assess the proliferation, migration and angiogenic functions of endothelial cells. The epigenetic modification in the promoter of NEDD4 was confirmed using BSP assay and ChIP-qPCR assay. The role of NEDD4 in wound healing was further verified in diabetic mouse model. ResultsNEDD4 promotes proliferation, migration and tube formation of endothelial cells. It binds to and ubiquitinates TSP1, which lead to TSP1 degradation and thus increased VEGF expression. The inhibitory effect of NEDD4 silencing on the angiogenesis ability of endothelial cells can be restored by TSP1 knockdown. NEDD4 is reduced in diabetic patients, which may due to hypermethylation of NEDD4 promoter mediated via DNMT1 under high glucose condition. Furthermore, inhibition of NEDD4 represses wound healing in diabetic mouse model. ConclusionNEDD4 might promote angiogenesis and wound healing by inhibiting TSP1 via ubiquitination in diabetic patients.

Analysis of age-related differences in hypoxia-related factors in yak brain tissue.

The brain is an important part of the mammalian nervous system, is highly sensitive to hypoxia, and plays an important role in the adaptation of the body to hypoxic environments. This study was conducted to study the distribution and expression of hypoxia-related factors (hypoxia-inducible factor 1α, HIF-1α; erythropoietin, EPO; vascular endothelial growth factor, VEGF; vascular cell adhesion molecule, VCAM) in the cerebellum, cerebrum, medulla oblongata, and corpora quadrigemina in yaks of different ages (4d, 6-months-old and adult). Paraffin sections were obtained from the cerebellum, cerebrum, medulla oblongata, and corpora quadrigemina of healthy yak for 4-day-old, 6-months-old and adult yaks. Histological characteristics were assessed by haematoxylin staining. Immunohistochemical staining was performed to detect the distribution and expression of HIF-1α, EPO, VEGF and VCAM proteins. Immunohistochemical results showed that HIF-1α, EPO, VEGF, and VCAM were expressed in the pyramidal cell layer of the yak cerebrum, and distributed in the cerebellum granulose cell layer, Purkinje cell layer and medulla layer, and were mainly positive in Purkinje cells and medulla. It is expressed in the cell bodies of the medulla oblongata and the quadrimatous neurons. The expression level in the medulla oblongata was higher, indicating may play a crucial role in functional cohesion. The expression of HIF-1α in 4 d cerebellar tissues was higher than that in other age groups, and the expression of HIF-1α in the medulla oblongata increased with age. In addition, the expression levels of EPO and VEGF in the 6-month-old group were slightly higher than those in the other age groups. It is speculated that EPO and VEGF have obvious protective effects on brain tissue in the 6-month-old age group; VCAM showed no significant differences in the cerebrum, cerebellum, medulla oblongata, or corpora quadrigemina of the yaks. This study provides basic data for further exploration of the adaptive mechanism of plateau yak brain tissue.

Analysis of the impact of different schemes of preparation to trabeculectomy on the healing markers on the Tenon fibroblasts cultures

The aim of the study was to assess the influence of different regimes of patient’s preparation before trabeculectomy on the markers of healing process in Tenon’s fibroblast cultures.The studied group consisted of 66 patients with open angle glaucoma undergoing primary trabeculectomy. The patients were divided into 5 groups with different regimes of preparation before the surgery based on application or withdrawal of topical antiglaucoma medications and steroids (G1—patients using antiglaucoma drops until the day of the surgery; G2—patients using antiglaucoma drops until the day of the surgery and additionally dexamethasone for 4 weeks before surgery; G3—patients who stopped using antiglaucoma drops 4 weeks before the surgery and introduced dexamethasone for 4 weeks before surgery; G4—patients who stopped using antiglaucoma drops 4 weeks before the surgery; G5—control group, patients with newly diagnosed glaucoma in whom trabeculectomy was the first treatment option without medical treatment). During trabeculectomy the samples of Tenon’s capsule were obtained. Tenon fibroblasts were isolated directly from the explants to test their proliferation ability and the level of released healing markers. Following factors typical of healing process were evaluated using commercially available ELISA kits: IL 1-β, IL-6, IL-8, VEGF-A, TGF-β1 and MMP-9. Concentrations of IL-1β, IL-6 and TGF-β1 were significantly higher in the group obtaining antiglaucoma drops. Additionally, in this group the fibroblasts revealed the highest proliferation potential, indicating the active healing process. The levels of IL-8, VEGF-A and MMP-9 were similar between the groups. Our study shows that for the best conjunctival anti-inflammatory control, the most influential factor is the withdrawal of antiglaucoma medications.

Myeloid cell-specific deletion of epidermal growth factor receptor aggravates acute cardiac injury.

Myeloid cells, including macrophages, play important roles as first responders to cardiac injury and stress. Epidermal growth factor receptor (EGFR) has been identified as a mediator of macrophage responsiveness to select diseases, though its impact on cardiac function or remodeling following acute ischemic injury is unknown. We aimed to define the role of myeloid cell-specific EGFR in the regulation of cardiac function and remodeling following acute myocardial infarction (MI)-induced injury. Floxed EGFR mice were bred with homozygous LysM-Cre (LMC) transgenic mice to yield myeloid-specific EGFR knockout (mKO) mice. Via echocardiography, immunohistochemistry, RNA sequencing and flow cytometry, the impact of myeloid cell-specific EGFR deletion on cardiac structure and function was assessed at baseline and following injury. Compared with LMC controls, myeloid cell-specific EGFR deletion led to an increase in cardiomyocyte hypertrophy at baseline. Bulk RNASeq analysis of isolated cardiac Cd11b+ myeloid cells revealed substantial changes in mKO cell transcripts at baseline, particularly in relation to predicted decreases in neovascularization. In response to myocardial infarction, mKO mice experienced a hastened decline in cardiac function with isolated cardiac Cd11b+ myeloid cells expressing decreased levels of the pro-reparative mediators Vegfa and Il10, which coincided with enhanced cardiac hypertrophy and decreased capillary density. Overall, loss of EGFR qualitatively alters cardiac resident macrophages that promotes a low level of basal stress and a more rapid decrease in cardiac function along with worsened repair following acute ischemic injury.

Impaired nitric oxide mechanisms underlying lower urinary tract dysfunction in aging rats.

The study aimed to investigate the bladder and urethral activity and nitric oxide (NO)-related molecular changes in aging rats. Rats were divided into two groups: Group Y (young rats; 12 wk) and Group A (aging rats; 15 mo). A 24-h voiding assay was performed, and the urodynamic parameters were evaluated using awake cystometry (CMG) and urethral perfusion pressure (UPP) recordings under urethane anesthesia. The mRNA expression levels of NO-, ischemia-, and inflammation-related markers in urethra and bladder tissues and cGMP levels in the urethra were assessed. Body weight was significantly higher in Group A than in Group Y. Voiding assay results (24 h) were insignificant. In the CMG, the number of non-voiding contractions per voiding cycle and post-void residual volume were significantly higher in Group A than in Group Y; voiding efficiency was significantly lower in Group A than in Group Y. In the UPP recordings, the urethral pressure reduction and high-frequency oscillation (HFO) amplitude were significantly lower in Group A than in Group Y. The mRNA expression levels of Hif-1α, Vegf-a, and Tgf-β1 in the bladder were significantly higher in Group A than in Group Y. The mRNA expression levels of Nos1 and Prkg1 and the cGMP concentrations in the urethra were significantly lower in Group A than in Group Y. Aging rats can be useful models for studying the natural progression of age-related lower urinary tract dysfunctions, for which impaired NO-mediated transmitter function is likely to be an important mechanism.NEW & NOTEWORTHY Aging rats can be useful models for studying the natural progression of age-related lower urinary tract dysfunctions, for which impaired nitric oxide-mediated transmitter function is likely to be an important mechanism.

BaP/BPDE suppressed endothelial cell angiogenesis to induce miscarriage by promoting MARCHF1/GPX4-mediated ferroptosis

Environmental benzo(a)pyrene (BaP) and its ultimate metabolite BPDE (benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide) are universal and inevitable persistent organic pollutants and endocrine disrupting chemicals. Angiogenesis in placental decidua plays a pivotal role in healthy pregnancy. Ferroptosis is a newly identified and iron-dependent cell death mode. However, till now, BaP/BPDE exposure, ferroptosis, defective angiogenesis, and miscarriage have never been correlated; and their regulatory mechanisms have been rarely explored. In this study, we used assays with BPDE-exposed HUVECs (human umbilical vein endothelial cells), decidual tissues and serum samples collected from unexplained recurrent miscarriage and their matched healthy control groups, and placental tissues of BaP-exposed mouse miscarriage model. We found that BaP/BPDE exposure caused ferroptosis and then directly suppressed angiogenesis and eventually induced miscarriage. In mechanism, BaP/BPDE exposure up-regulated free Fe2+ level and promoted lipid peroxidation and also up-regulated MARCHF1 (a novel E3 ligase of GPX4) level to promote the ubiquitination degradation of GPX4, both of which resulted in HUVEC ferroptosis. Furthermore, we also found that GPX4 protein down-regulated the protein levels of VEGFA and ANG-1, two key proteins function for angiogenesis, and thus suppressed HUVEC angiogenesis. In turn, supplement with GPX4 could suppress ferroptosis, recover angiogenesis, and alleviate miscarriage. Moreover, the levels of free Fe2+ and VEGFA in serum might predict the risk of miscarriage. Overall, this study uncovered the crosstalk among BaP/BPDE exposure, ferroptosis, angiogenesis, and miscarriage, discovering novel toxicological effects of BaP/BPDE on human reproductive health. This study also warned the public to avoid exposure to polycyclic aromatic hydrocarbons during pregnancy to effectively prevent adverse pregnancy outcomes.

Pilot Study on the Molecular Pathogenesis of Pyeloureteral Junction Obstruction: Underdevelopment or Fibrosis?

Background and Objectives: Congenital ureteral stenosis is one of the leading causes of impaired urinary drainage and subsequent dilatation of the urinary collecting system, known as hydronephrosis or ureterohydronephrosis. The mechanism that leads to obstruction is not clearly known. Multiple studies in rat models have shown increased angiotensin II and TGFβ levels in obstructed ureteral tissue. The aim of the study is to investigate the expression of fibrosis-related genes in obstructive and normal ureteral tissue. Material and Methods: It is a monocentric pilot study in which nineteen patients were selected prospectively. 17 patients underwent Hynes-Anderson pyeloplasty due to the PUJO; two patients underwent ureteroneocystostomy due to ureterovesical junction obstruction (UVJO); and six patients were chosen for the control group: five underwent nephrectomies due to the kidney tumor and one underwent upper pole heminephrectomy due to the duplex kidney with normal pyeloureteric junctions in all. Tissue RNA was chemically extracted after freezing the biopsy samples in liquid nitrogen, with cDNA synthesis performed immediately after nucleic acid isolation. qPCR was performed to evaluate the relative expression of Tgfb1, Mmp1, Timp1, Pai1, Ctgf, and Vegfa. Expression levels of the Gapdh and Gpi genes (geometric average) were used to calculate the relative expression of the investigated genes. Outliers were removed prior to calculating confidence intervals for the experimental groups, and a Wilcoxon rank-sum test was performed to determine the statistical significance of the differences. Results: Significant differences between healthy and stenotic tissue samples in Ctgf gene expression levels were observed, with the samples from afflicted tissue showing lower expression. No statistical difference in expression levels of Tgfb1, Timp1, Vegfa, Mmp1, and Pai1 was found. Conclusions: These findings suggest that tissue fibrosis, similar to other tissues and organs, is not the leading cause of stenosis, at least at the moment of surgery. Decreased CTGF expression is indicative of the developmental origin of obstruction.

Mechanism of herb pair containing Astragali Radix and Spatholobi Caulis in the treatment of myelosuppression based on network pharmacology and experimental investigation

Ethnopharmacological relevanceThe Astragali Radix and Spatholobi Caulis herb pair (ARSC) is one of the most commonly used herbal combinations for bone marrow suppression. According to traditional Chinese medicine, Astragali Radix strengthens the spleen and replenishes qi, while Spatholobi Caulis is a hematinic agent that promotes blood circulation and enrichment. The compatibility of the two helps the body to tonify the spleen and kidneys and compensate for visceral deficiencies. However, the multi-target mechanism of ARSC in bone marrow suppression has remained largely unknown. Aim of the studyThe aim of this study is to explore the key targets and signaling pathways of the traditional Chinese herbal pair ARSC for the treatment of bone marrow suppression. Materials and methodsThe active components of ARSC and targets for myelosuppression were screened using network databases. Cytoscape 3.8.0 was used to construct compound-target, compound-disease-target and protein-protein interaction (PPI) networks. Go-function and pathway enrichment analyses were performed to explore the potential mechanism. In vivo animal experiments were conducted to verify the molecular mechanisms. ResultsThe 36 active compounds were identified from the ARSC, and a total of 108 genes involved in myelosuppression were screened. VEGFA, IL6, TNF, JUN, STAT3, PTGS2, CASP3 and MMP9 genes were identified as potential drug targets in the PPI network analyzed by CytoHubba. Enrichment analysis indicated that ARSC may treat myelosuppression through various biological processes, such as apoptosis, TNF-α signaling pathway via NF-κB, PI3K/AKT/mTOR signaling pathway, IL6/JAK/STAT3 signaling pathway, P53 signaling pathway and G2/M checkpoint signaling pathway. The results of the experiment showed that the aqueous extract of ARSC significantly alleviated myelosuppression, reduced the apoptosis rate of bone marrow cells, upregulated the mRNA expression levels of TNF-α, IL-6 and VEGF, and promoted NF-κB phosphorylation in myelosuppressed mice. ConclusionsThis study identified the active components and relevant mechanisms of ARSC in the treatment of myelosuppression. Our findings predicted that ARSC could treat bone marrow suppression through multiple components, multiple targets and multiple pathways. Pharmacological experiments showed that ARSC alleviated fluorouracil-induced myelosuppression by reducing the apoptosis rate of bone marrow cells and regulating the TNF-α/NF-κB signaling pathway.

A novel pharmaceutical preparation of Tripterygium wilfordii Hook. f. regulates macrophage polarization to alleviate inflammation in rheumatoid arthritis.

To validate the enhanced therapeutic effect of Tripterygium wilfordii Hook. f. (TWHF) in the treatment of rheumatoid arthritis (RA) by restoring homeostasis of M1/M2 macrophages. This study, using random walk models and network pharmacology, examined the molecular targets and mechanism of TWHF in RA. Based on clinical observations and experiments in arthritis animal models, the effects of TWHF on macrophage polarization, related signal pathways, and targets were examined. Triptolide, a component of TWHF, was used to intervene arthritis rats. Network pharmacological analysis revealed the key RA target genes related to TWHF. TWHF showed a strong correlation with the improvement of inflammatory indicators. TWHF inhibited the factors secreted by M1 macrophages such as IL-1β, IL-6, CXCL8, TNF-α, and VEGF-A, but promoted IL-10 from M2 macrophages. Quantitative liquid-phase chip assay showed that triptolide reduced the levels of TNF-α, CXCL2, and VEGF, while IL-4 and IL-10 were increased in arthritis model. Meanwhile, triptolide inhibited the NF-κB, PI3K/AKT, and p38 MAPK signaling pathways, which in turn improved the RA joint inflammation and fixed immune imbalance. Triptolide downregulate the expression of M1 macrophage-secreted factors that inhibit the overactivation of inflammatory signaling pathways.

Assessment of α9β1 ıntegrın as a new dıagnostıc and therapeutıc target ın Behcet's dısease.

This study aimed to investigate the roles of α9β1 integrin and its ligands in Behçet's disease (BD) by examining serum levels and gene expressions. 15 healthy controls and 30 BD patients (14 active and 16 inactive) were included in the study. Serum levels of ITGA9, ITGB1, TNC, OPN, VCAM-1, VEGF, TSP1, TGM2, Emilin-1, and vWF, were measured by ELISA. Gene expressions of α9β1 (ITGA9 and ITGB1) and its ligands (TNC and SPP1) were evaluated by RT-PCR. Laboratory findings (CRP, ESR, HGB, WBC, RBC, neutrophil, lymphocyte, PLT, RDW, MPV, PCT, and HLA-B51) were obtained from the electronic database. Active BD patients had higher serum levels of α9β1 integrin and its ligands than inactive patients and healthy controls. No significant difference was observed between healthy controls and inactive patients. Gene expressions of ITGB1 and SPP1 were increased in both patient groups compared to healthy controls. ITGA9 and TNC gene expression levels were lower in the active group than in the inactive group. No noticeable differences were found in ITGB1 and SPP1 gene expressions between the patient groups. BD patients exhibited elevated CRP, ESR, WBC, neutrophil, PLT, and PCT levels, while HGB, RBC, and RDW values were lower than healthy controls. Active patients had higher CRP, ESR, WBC, neutrophil, and PLT levels. Significant positive correlations were found between CRP, ESR, WBC, neutrophil, PLT, PCT and serum levels of α9β1 integrin and its ligands. Increased release of α9β1 integrin and its ligands is associated with BD, suggesting their potential as markers for disease severity.