Emodin suppresses alkali burn-induced corneal inflammation and neovascularization by the vascular endothelial growth factor receptor 2 signaling pathway.

OBJECTIVE: To investigate the effects of emodin on alkali burn-induced corneal inflammation and neovascularization. METHODS: The ability of emodin to target vascular endothelial growth factor receptor 2 (VEGFR2) was predicted by molecular docking. The effects of emodin on the invasion, migration, and proliferation of human umbilical vein endothelial cells (HUVEC) were determined by cell counting kit-8, Transwell, and tube formation assays. Analysis of apoptosis was performed by flow cytometry. CD31 levels were examined by immunofluorescence. The abundance and phosphorylation state of VEGFR2, protein kinase B (Akt), signal transducer and activator of transcription 3 (STAT3), and P38 were examined by immunoblot analysis. Corneal alkali burn was performed on 40 mice. Animals were divided randomly into two groups, and the alkali-burned eyes were then treated with drops of either 10 µM emodin or phosphate buffered saline (PBS) four times a day. Slit-lamp microscopy was used to evaluate inflammation and corneal neovascularization (CNV) in all eyes on Days 0, 7, 10, and 14. The mice were killed humanely 14 d after the alkali burn, and their corneas were removed and preserved at －80 ℃ until histological study or protein extraction. RESULTS: Molecular docking confirmed that emodin was able to target VEGFR2. The findings revealed that emodin decreased the invasion, migration, angiogenesis, and proliferation of HUVEC in a dose-dependent manner. In mice, emodin suppressed corneal inflammatory cell infiltration and inhibited the development of corneal neovascularization induced by alkali burn. Compared to those of the PBS-treated group, lower VEGFR2 expression and CD31 levels were found in the emodin-treated group. Emodin dramatically decreased the expression of VEGFR2, p-VEGFR2, p-Akt, p-STAT3, and p-P38 in VEGF-treated HUVEC. CONCLUSION: This study provides a new avenue for evaluating the molecular mechanisms underlying corneal inflammation and neovascularization. Emodin might be a promising new therapeutic option for corneal alkali burns.

The pathological roles and potential mechanisms of vascular endothelial growth factor receptor-3 in gastric cancer.

OBJECTIVE: To investigate the roles and underlying mechanisms of vascular endothelial growth factor receptor-3 (VEGFR-3) in gastric cancer (GC). METHODS: VEGFR-3 gene expression profiles in human gastric adenocarcinoma (GAC) tissues were analysed using The Cancer Genome Atlas database. Human GC cell lines and were used for in vitro studies. Mouse models of GC and distant metastasis were used for in vivo studies. Silencing of VEGFR-3 gene expression was achieved using small interfering RNA. RESULTS: VEGFR-3 gene expression was significantly elevated in GAC tissues and GC cells. Higher VEGFR-3 expression was positively correlated with more advanced stages and a greater number of metastatic lymph nodes. In vitro studies in GC cells showed that knockdown of VEGFR-3 gene expression significantly suppressed cell proliferation and migration, but promoted apoptosis. In vivo investigations revealed that silencing of VEGFR-3 gene expression exhibited significant inhibition on tumour growth and metastasis. Further mechanistic studies showed that VEGFR-3 exerted its pathological roles by affecting the key molecules in the apoptotic and epithelial-mesenchymal transition pathways. CONCLUSION: The molecular pathways associated with VEGFR-3-mediated pathological effects could be targets in the development of novel approaches for the diagnosis, prognosis and treatment of GC.

Molecular basis of VEGFR1 autoinhibition at the plasma membrane.

Ligand-independent activation of VEGFRs is a hallmark of diabetes and several cancers. Like EGFR, VEGFR2 is activated spontaneously at high receptor concentrations. VEGFR1, on the other hand, remains constitutively inactive in the unligated state, making it an exception among VEGFRs. Ligand stimulation transiently phosphorylates VEGFR1 and induces weak kinase activation in endothelial cells. Recent studies, however, suggest that VEGFR1 signaling is indispensable in regulating various physiological or pathological events. The reason why VEGFR1 is regulated differently from other VEGFRs remains unknown. Here, we elucidate a mechanism of juxtamembrane inhibition that shifts the equilibrium of VEGFR1 towards the inactive state, rendering it an inefficient kinase. The juxtamembrane inhibition of VEGFR1 suppresses its basal phosphorylation even at high receptor concentrations and transiently stabilizes tyrosine phosphorylation after ligand stimulation. We conclude that a subtle imbalance in phosphatase activation or removing juxtamembrane inhibition is sufficient to induce ligand-independent activation of VEGFR1 and sustain tyrosine phosphorylation.

NRP1 interacts with endoglin and VEGFR2 to modulate VEGF signaling and endothelial cell sprouting.

Endothelial cells express neuropilin 1 (NRP1), endoglin (ENG) and vascular endothelial growth factor receptor 2 (VEGFR2), which regulate VEGF-A-mediated vascular development and angiogenesis. However, the link between complex formation among these receptors with VEGF-A-induced signaling and biology is yet unclear. Here, we quantify surface receptor interactions by IgG-mediated immobilization of one receptor, and fluorescence recovery after photobleaching (FRAP) measurements of the mobility of another coexpressed receptor. We observe stable ENG/NRP1, ENG/VEGFR2, and NRP1/VEGFR2 complexes, which are enhanced by VEGF-A. ENG augments NRP1/VEGFR2 interactions, suggesting formation of tripartite complexes bridged by ENG. Effects on signaling are measured in murine embryonic endothelial cells expressing (MEEC+/+) or lacking (MEEC-/-) ENG, along with NRP1 and/or ENG overexpression or knockdown. We find that optimal VEGF-A-mediated phosphorylation of VEGFR2 and Erk1/2 requires ENG and NRP1. ENG or NRP1 increase VEGF-A-induced sprouting, becoming optimal in cells expressing all three receptors, and both processes are inhibited by a MEK1/2 inhibitor. We propose a model where the maximal potency of VEGF-A involves a tripartite complex where ENG bridges VEGFR2 and NRP1, providing an attractive therapeutic target for modulation of VEGF-A signaling and biological responses.

The Relation of VEGFA, VEGFR2, VEGI, and HIF1A Genetic Variants and Their Serum Protein Levels with Breast Cancer in Egyptian Patients.

Breast cancer is the most common type of cancer in Egyptian females. Polymorphisms in the angiogenesis pathway have been implicated previously in cancer risk and prognosis. The aim of the current study was to determine whether certain polymorphisms in the genes of vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor receptor 2 (VEGFR2), vascular endothelial growth inhibitor (VEGI), and hypoxia-inducible factor-1α (HIF1A) associated with breast cancer development. The study included 154 breast cancer patients and 132 apparently healthy age-matched females as a control group. VEGFA rs25648 genotyping was performed using (ARMS) PCR technique; while VEGFR2 rs2071559, VEGI rs6478106, and HIF-1α rs11549465 were genotyped by the PCR-RFLP method. Serum levels of VEGF, VEGFR2, VEGI, and HIF1A proteins in breast cancer patients and controls were measured by ELISA. There was a significant association between the VEGFA rs25648 C allele and breast cancer risk (OR 2.5, 95% CI 1.7-3.6, p < 0.001). VEGFA rs25648 C/C genotype was statistically significantly higher in breast cancer patients vs. control (p < 0.001). Participants with the T/T and T/C VEGFR2 rs2071559 genotypes had 5.46 and 5 higher odds, respectively, of having breast cancer than those with the C/C genotype. For the VEGI rs6478106 polymorphism, there was a higher proportion of C allele in breast cancer patients vs. control (p = 0.003). Moreover, the C/C genotype of VEGI rs6478106 was statistically significantly higher in breast cancer patients vs. control (p = 0.001). There was no significant difference in genotypes and allele frequencies of HIF1A rs11549465 polymorphism between breast cancer cases and control individuals (p > 0.05). Serum levels of VEGFA, VEGI, and HIF1A were considerably greater in women with breast cancer than in the control (p < 0.001). In conclusion, the genetic variants VEGFA rs25648, VEGFR2 rs2071559, and VEGI rs6478106 revealed a significant association with increased breast cancer risk in Egyptian patients.

Association of KDR (rs2071559, rs1870377), CFH (rs1061170, rs1410996) genes variants and serum levels with pituitary adenoma.

INTRODUCTION: Pituitary adenomas (PA) are slow-growing, benign tumors that usually do not metastasize to other body organs. Although they are referred to as benign, tumor growth can eventually put pressure on nearby structures, spread to surrounding tissues, and cause symptoms. The exact cause of PA is unknown, and the pathogenesis is multifactorial. METHODS: Our study included PA patients and healthy volunteers. Genomic DNA was extracted using the DNA salting-out method. All participants were genotyped for the KDR rs2071559, rs1870377, CFH rs1061170, and rs1410996 polymorphisms. Serum levels of KDR and CFH were examined using the ELISA method. RESULTS: The results of the present study showed that KDR rs2071559 A allele was associated with the occurrence of PA, hormonally active PA, invasive PA, and PA without recurrence development. KDR rs1870377 increased the probability of invasive PA and PA recurrence. CFH rs1061170 C allele was associated with hormonally active PA and the T allele was associated with non-invasive PA development. CONCLUSION: KDR rs2071559, rs1870377, and CFH rs1061170 could be potential biomarkers associated with PA.

Association analysis of Vascular Endothelial Growth Factor-A (VEGF-A) polymorphism in rheumatoid arthritis using computational approaches.

Rheumatoid arthritis (RA), is marked by joint inflammation leading to pannus formation which results in cartilage destruction promoting bone erosion. The pathological hallmark of RA includes synovial hyperplasia and synovial angiogenesis. Active tissue neovascularization is observed in RA. Vascular endothelial Growth factor A (VEGFA), an endothelial cell-specific proangiogenic molecule is triggered by hypoxic cells and its levels are upregulated in RA. The aim of this study was to investigate functional and pathogenic VEGFA variants and to identify the impact of point mutation in VEGFA's interaction with VEGFR2 and how these polymorphisms affect the susceptibility and severity of RA. We investigated impact of these point mutations on the stability of VEGFA using various computational tools. These mutations were further identified by conservational profile as they are highly involved as structural and functional mutations. Furthermore, these selected variants were modelled and docked against targeted domain regions IGD2 and IGD3 of VEGFR2. Further molecular dynamic simulations were performed using Gromacs. Out of 168 nsSNPS, 19 were highlighted as highly pathogenic using insilico prediction tools. InterPro and ConSurf revealed domains and conserved variants respectively. After stability analysis, we concluded that almost all the mutations were responsible for decreasing the protein stability. HOPE predicted that all the selected damaging nsSNPs were present in the domain which is essential for the functioning of VEGFA protein. Constructed Ramachandran plot and ERRAT validated the quality of all the models. Based on the interactions predicted by STRING database, we performed Protein-Protein docking between VEGFA and VEGFR2. We found few conserved interactions and new polar contacts among wild-type and mutants with VEGFR2. From the simulations, we concluded that mutant R108Q was the most stabilizing mutant among all others whereas R82Q, C86Y, and R108W complexed with VEGFR2 were comparatively less stabilizing as compared to the wild type. This study provides insight into pathogenic nsSNPs that can affect VEGFA protein structure and function. These high-risk variants must be taken into consideration for genetic screening of patients suffering from RA.

In vivo antiangiogenic effect of nimbolide, trans-chalcone and piperine for use against glioblastoma.

BACKGROUND: Angiogenesis is an important hallmark of Glioblastoma (GBM) marked by elevated vascular endothelial growth factor-A (VEGF-A) and its receptor 2 (VEGFR-2). As previously reported nimbolide (NBL), trans-chalcone (TC) and piperine (PPR) possess promising antiangiogenic activity in several cancers however, their comparative efficacy and mechanism of antiangiogenic activity in GBM against VEGFR-2 has not been elucidated. METHODS: 2D and 3D spheroids cultures of U87 (Uppsala 87 Malignant Glioma) were used for evaluation of non-cytotxoic dose for anti-angiogenic activity. The antiangiogenic effect was investigated by the GBM U87 cell line bearing chick CAM model. Excised U87 xenografts were histologically examined for blood vascular density by histochemistry. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the presence of avian and human VEGF-A and VEGFR-2 mRNA transcripts. RESULTS: Using 2D and 3D spheroid models, the non-cytotoxic dose of NBL, TC and PPR was ≤ 11 µM. We found NBL, TC and PPR inhibit U87-induced neoangiogenesis in a dose-dependent manner in the CAM stand-alone model as well as in CAM U87 xenograft model. The results also indicate that these natural compounds inhibit the expression of notable angiogenic factors, VEGF-A and VEGFR-2. A positive correlation was found between blood vascular density and VEGF-A as well as VEGFR-2 transcripts. CONCLUSION: Taken together, NBL, TC and PPR can suppress U87-induced neoangiogenesis via a reduction in VEGF-A and its receptor VEGFR-2 transcript expression at noncytotoxic concentrations. These phytochemicals showed their utility as adjuvants to GBM therapy, with Piperine demonstrating superior effectiveness among them all.

Synthesis, molecular modeling Insights, and anticancer assessment of novel polyfunctionalized Pyridine congeners.

The present study describes synthesizing a novel series of polyfunctionalized pyridine congeners 1-18 and assessed for cytotoxic efficacies versus HCT-116, MCF-7, and HepG-2 among one non-cancerous BJ-1 human normal cell. Most compounds were precisely potent anticancer candidate drugs. The molecular impact of the most active compounds 9, 10, 11, 13, 15, and 17 was evaluated after MCF-7 treatment. The gene expression of pro- and ant-apoptosis markers P53, Bax, Caspase-3 and Bcl-2 as well as VEGFR-2 and HER2 were determined. Compounds 13 and 15 induced upregulation of pro-apoptosis of P53, Bax, Caspase-3 and downregulation of anti-apoptosis Bcl-2 gene. However, compound 15 showed higher effect compared to 13 and respective control. Moreover, a slight reduction in HER2 gene expression was detected due to compound 15 treatment, while VEGFR-2 gene was upregulated. In agreement, the immunoblotting analysis showed higher accumulation of P53, Bax, Caspase-3 proteins and of decrease the Bcl-2 protein levels. Furthermore, docking studies united with molecular dynamic simulation exposed compounds 13 and 15 fitting in the middle of the active site at the interface linking the ATP binding site and the allosteric hydrophobic binding pocket. Finally, we performed Petra/Osiris/ Molinspiration (POM) analysis for the newly synthesized compounds. The evaluation of primary in silico parameters revealed significant differences among individual polyfunctionalized pyridine compounds, highlighting the most promising candidates. These preliminary results may help in coordinating and initiating other research projects focused on polyfunctionalized pyridine compounds, especially those with predicted bioactivity, low toxicity, optimal ADME parameters, and promising perspectives.

Endothelial VEGFR2-PLCγ signaling regulates vascular permeability and antitumor immunity through eNOS/Src.

Endothelial phospholipase Cγ (PLCγ) is essential for vascular development; however, its role in healthy, mature, or pathological vessels is unexplored. Here, we show that PLCγ was prominently expressed in vessels of several human cancer forms, notably in renal cell carcinoma (RCC). High PLCγ expression in clear cell RCC correlated with angiogenic activity and poor prognosis, while low expression correlated with immune cell activation. PLCγ was induced downstream of vascular endothelial growth factor receptor 2 (VEGFR2) phosphosite Y1173 (pY1173). Heterozygous Vegfr2Y1173F/+ mice or mice lacking endothelial PLCγ (Plcg1iECKO) exhibited a stabilized endothelial barrier and diminished vascular leakage. Barrier stabilization was accompanied by decreased expression of immunosuppressive cytokines, reduced infiltration of B cells, helper T cells and regulatory T cells, and improved response to chemo- and immunotherapy. Mechanistically, pY1173/PLCγ signaling induced Ca2+/protein kinase C-dependent activation of endothelial nitric oxide synthase (eNOS), required for tyrosine nitration and activation of Src. Src-induced phosphorylation of VE-cadherin at Y685 was accompanied by disintegration of endothelial junctions. This pY1173/PLCγ/eNOS/Src pathway was detected in both healthy and tumor vessels in Vegfr2Y1173F/+ mice, which displayed decreased activation of PLCγ and eNOS and suppressed vascular leakage. Thus, we believe that we have identified a clinically relevant endothelial PLCγ pathway downstream of VEGFR2 pY1173, which destabilizes the endothelial barrier and results in loss of antitumor immunity.

Association of single nucleotide variants in VEGFA and KDR with the risk and angiogenic features of diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype and dependent on angiogenesis (AG), whose main effectors are VEGFA and VEGFR2. Functional single nucleotide variants (SNVs) are described in VEGFA and KDR genes. However, it still unknown whether VEGFA - 2578C/A, -2489C/T, -1154G/A, -634G/C, -460C/T and KDR-604T/C, -271G/A, +1192G/A and +1719A/T SNVs act on DLBCL risk and angiogenic features. Genomic DNA from 168 DLBCL patients and 205 controls was used for SNV genotyping. Angiogenesis was immunohistochemically assessed in tumor biopsies, with reactions for VEGFA, VEGFR2, and CD34. VEGFA -1154GG genotype were associated with 1.6-fold higher DLBCL risk. KDR + 1192GG plus KDR + 1719 TT and KDR + 1192GG plus VEGFA - 2578CC combined genotypes are associated with 2.19- and 2.04-fold higher risks of DLBCL, respectively. VEGFA - 634GG or GC genotypes are associated with increased microvessel density and VEGFA levels. No relationship was observed between SNVs and cell-of-origin classification of DLBCL, but higher VEGFA and VEGFR2 were seen in non-germinal center tumors.

PDGFRA, KIT, and KDR Gene Amplification in Glioblastoma: Heterogeneity and Clinical Significance.

Glioblastoma (GBM) is the most frequent tumor of the central nervous system, and its heterogeneity is a challenge in treatment. This study examined tumoral heterogeneity involving PDGFRA, KIT, and KDR gene amplification (GA) in 4q12 and its association with clinical parameters. Specimens from 22 GBM cases with GA for the 4q12 amplicon detected by FISH were investigated for homogeneous or heterogeneous coamplification patterns, diffuse or focal distribution of cells harboring GA throughout tumor sections, and pattern of clustering of fluorescence signals. Sixteen cases had homogenously amplification for all three genes (45.5%), for PDGFRA and KDR (22.7%), or only for PDGFRA (4.6%); six cases had heterogeneous GA patterns, with subpopulations including GA for all three genes and for two genes - PDGFRA and KDR (13.6%), or GA for all three and for only one gene - PDGFRA (9.1%) or KIT (4.6%). In 6 tumors (27.3%), GA was observed in focal tumor areas, while in the remaining 16 tumors (72.7%) it was diffusely distributed throughout the pathological specimen. Amplification was universally expressed as double minutes and homogenously stained regions. Coamplification of all three genes PDGFRA, KIT, and KDR, age ≥ 60 years, and total tumor resection were statistically associated with poor prognosis. FISH proved effective for detailed interpretation of molecular heterogeneity. The study uncovered an even more diverse range of amplification patterns involving the 4q12 oncogenes in GBM than previously described, thus highlighting a complex tumoral heterogeneity to be considered when devising more effective therapies.

Adverse effect of VEGFR-2 (rs1870377) polymorphism on the clinical course of COVID-19 in females and males in an age-dependent manner.

The COVID-19 pandemic has affected people worldwide with varying clinical presentations ranging from mild to severe or fatal, and studies have found that age, gender, and some comorbidities can influence the severity of the disease. It would be valuable to have genetic markers that might help predict the likely outcome of infection. For this objective, genes encoding VEGFR-2 (rs1870377), CCR5Δ32 (rs333), and TLR3 (rs5743313) were analyzed for polymorphisms in the peripheral blood of 160 COVID-19 patients before COVID-19 vaccine was available in Türkiye. We observed that possession of the VEGFR-2 rs1870377 mutant allele increased the risk of severe/moderate disease in females and subjects ≥65 years of age, but was protective in males <65 years of age. Other significant results were that the CCR5Δ32 allele was protective against severe disease in subjects ≥65 years of age, while TLR3 rs5743313 polymorphism was found to be protective against severe/moderate illness in males <65 years of age. The VEGFR-2 rs1870377 mutant allele was a risk factor for severe/moderate disease, particularly in females over the age of 65. These findings suggest that genetic polymorphisms have an age- and sex-dependent influence on the severity of COVID-19, and the VEGFR-2 rs1870377 mutant allele could be a potential predictor of disease severity.

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.

Endothelial-specific loss of IKKß disrupts pulmonary endothelial angiogenesis and impairs postnatal lung growth.

Pulmonary angiogenesis drives alveolarization, but the transcriptional regulators directing pulmonary angiogenesis remain poorly defined. Global, pharmacological inhibition of nuclear factor-kappa B (NF-κB) impairs pulmonary angiogenesis and alveolarization. However, establishing a definitive role for NF-κB in pulmonary vascular development has been hindered by embryonic lethality induced by constitutive deletion of NF-κB family members. We created a mouse model allowing inducible deletion of the NF-κB activator, IKKß, in endothelial cells (ECs) and assessed the effect on lung structure, endothelial angiogenic function, and the lung transcriptome. Embryonic deletion of IKKß permitted lung vascular development but resulted in a disorganized vascular plexus, while postnatal deletion significantly decreased radial alveolar counts, vascular density, and proliferation of both endothelial and nonendothelial lung cells. Loss of IKKß impaired survival, proliferation, migration, and angiogenesis in primary lung ECs in vitro, in association with decreased expression of VEGFR2 and activation of downstream effectors. Loss of endothelial IKKß in vivo induced broad changes in the lung transcriptome with downregulation of genes related to mitotic cell cycle, extracellular matrix (ECM)-receptor interaction, and vascular development, and the upregulation of genes related to inflammation. Computational deconvolution suggested that loss of endothelial IKKß decreased general capillary, aerocyte capillary, and alveolar type I cell abundance. Taken together, these data definitively establish an essential role for endogenous endothelial IKKß signaling during alveolarization. A deeper understanding of the mechanisms directing this developmental, physiological activation of IKKß in the lung vasculature may provide novel targets for the development of strategies to enhance beneficial proangiogenic signaling in lung development and disease.NEW & NOTEWORTHY This study highlights the cell-specific complexity of nuclear factor kappa B signaling in the developing lung by demonstrating that inducible loss of IKKß in endothelial cells impairs alveolarization, disrupts EC angiogenic function, and broadly represses genes important for vascular development.

The E2 ubiquitin-conjugating enzymes UBE2D1 and UBE2D2 regulate VEGFR2 dynamics and endothelial function.

Vascular endothelial growth factor receptor 2 (VEGFR2, encoded by KDR) regulates endothelial function and angiogenesis. VEGFR2 undergoes ubiquitination that programs this receptor for trafficking and proteolysis, but the ubiquitin-modifying enzymes involved are ill-defined. Herein, we used a reverse genetics screen for the human E2 family of ubiquitin-conjugating enzymes to identify gene products that regulate VEGFR2 ubiquitination and proteolysis. We found that depletion of either UBE2D1 or UBE2D2 in endothelial cells caused a rise in steady-state VEGFR2 levels. This rise in plasma membrane VEGFR2 levels impacted on VEGF-A-stimulated signalling, with increased activation of canonical MAPK, phospholipase Cγ1 and Akt pathways. Analysis of biosynthetic VEGFR2 is consistent with a role for UBE2D enzymes in influencing plasma membrane VEGFR2 levels. Cell-surface-specific biotinylation and recycling studies showed an increase in VEGFR2 recycling to the plasma membrane upon reduction in UBE2D levels. Depletion of either UBE2D1 or UBE2D2 stimulated endothelial tubulogenesis, which is consistent with increased VEGFR2 plasma membrane levels promoting the cellular response to exogenous VEGF-A. Our studies identify a key role for UBE2D1 and UBE2D2 in regulating VEGFR2 function in angiogenesis.

Exposure to Valproic acid (VPA) resulted in alterations in the expression of angiogenic genes (NRP-1, VEGFA, VEGFR-2 and sFlt1) and histological modifications in the placenta of mice (Mus musculus).

Valproic acid (VPA), an anti-epileptic drug (AED), has been reported to exhibit anti-angiogenic properties. This study aimed to examine the impact of VPA on the expression of NRP-1 and additional angiogenic factors, as well as angiogenesis, in mouse placenta. Pregnant mice were divided into four groups: control (K), solvent control (KP), VPA treatment at a dose of 400 mg/kg body weight (BW) (P1), and VPA treatment at a dose of 600 mg/kg BW (P2). The mice were subjected to daily treatment via gavage from embryonic day (E) 9 to E14 and E9 to E16. Histological analysis was performed to evaluate Microvascular Density (MVD) and percentage of the placental labyrinth area. In addition, a comparative analysis of Neuropilin-1 (NRP-1), vascular endothelial growth factor (VEGFA), vascular endothelial growth factor receptor (VEGFR-2), and soluble (sFlt1) expression was conducted in relation to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The results of the MVD analysis and percentage of labyrinth area in the E14 and E16 placentas indicated that the treated groups were significantly lower than the control group. The relative expression levels of NRP-1, VEGFA, and VEGFR-2 in the treated groups were lower than those in the control group at E14 and E16. Meanwhile, the relative expression of sFlt1 in the treated groups at E16 was significantly higher than in the control group. Changes in the relative expression of these genes inhibit angiogenesis regulation in the mouse placenta, as evidenced by reduced MVD and a smaller percentage of the labyrinth area.

Association of 410L, 1016I and 1534C kdr mutations with pyrethroid resistance in Aedes aegypti from Ouagadougou, Burkina Faso, and development of a one-step multiplex PCR method for the simultaneous detection of 1534C and 1016I kdr mutations.

BACKGROUND: Since 2000, Burkina Faso has experienced regular dengue cases and outbreaks, making dengue an increasingly important health concern for the country. Previous studies in Burkina Faso reported that resistance of Aedes aegypti to pyrethroid insecticides was associated with the F1534C and V1016I kdr mutations. The current study reports high resistance of Ae. aegypti populations to pyrethroid insecticides, likely supported by mutations in the voltage-gated sodium channel, here evidenced by genotyping the kdr SNPs V410L, V1016I and F1534C. We also describe a new multiplex PCR-based diagnostic of F1534C and V1016I kdr SNPs. METHODS: Larvae of Ae. aegypti were collected from three health districts of Ouagadougou in 2018. The resistance status of Ae. aegypti to permethrin (15 µg/ml) and deltamethrin (10 µg/ml) was tested using bottles and to malathion (5%) using WHO tube tests. All bioassays used 1-h exposure and mortality recorded 24 h post-exposure. Bioassay results were interpreted according to WHO thresholds for resistance diagnosis. The kdr mutations were screened using AS-PCR and TaqMan methods in exposed and non-exposed Aedes mosquitoes. RESULTS: Females from all health districts were resistant to permethrin and deltamethrin (< 20% mortality) but were fully susceptible to 5% malathion. The F1534C and V1016I kdr mutations were successfully detected using a newly developed multiplex PCR in perfect agreement with TaqMan method. The 1534C/1016I/410L haplotype was correlated with permethrin resistance but not with deltamethrin resistance; however, the test power was limited by a low frequency of dead individuals in deltamethrin exposure. CONCLUSIONS: Resistance to pyrethroid insecticides is associated with kdr mutant haplotypes, while the absence of substantial resistance to malathion suggests that it remains a viable option for dengue vector control in Ouagadougou.

Evaluation of the evolutionary genetics and population structure of Culex pipiens pallens in Shandong province, China based on knockdown resistance (kdr) mutations and the mtDNA-COI gene.

BACKGROUND: Mosquitoes are important vectors for a range of diseases, contributing to high rates of morbidity and mortality in the human population. Culex pipiens pallens is dominant species of Culex mosquito in northern China and a major vector for both West Nile virus and Bancroftian filariasis. Insecticide application were largely applied to control the mosquito-mediated spread of these diseases, contributing to increasing rates of resistance in the mosquito population. The voltage-gated sodium channel (Vgsc) gene is the target site of pyrethroids, and mutations in this gene cause knockdown resistance (kdr). While these kdr mutations are known to be critical to pyrethroid resistance, their evolutionary origins remain poorly understood. Clarifying the origins of these mutations is potential to guide further vector control and disease prevention efforts. Accordingly, the present study was designed to study the evolutionary genetics of kdr mutations and their association with the population structure of Cx. p. pallens in Shandong province, China. METHODS: Adult Culex females were collected from Shandong province and subjected to morphological identification under a dissection microscope. Genomic DNA were extracted from the collected mosquitoes, the Vgsc gene were amplified via PCR and sequenced to assess kdr allele frequencies, intron polymorphisms, and kdr codon evolution. In addition, population genetic diversity and related population characteristics were assessed by amplifying and sequencing the mitochondrial cytochrome C oxidase I (COI) gene. RESULTS: Totally, 263 Cx. p. pallens specimens were used for DNA barcoding and sequencing analyses to assess kdr allele frequencies in nine Culex populations. The kdr codon L1014 in the Vgsc gene identified two non-synonymous mutations (L1014F and L1014S) in the analyzed population. These mutations were present in the eastern hilly area and west plain region of Shandong Province. However, only L1014F mutation was detected in the southern mountainous area and Dongying city of Shandong Province, where the mutation frequency was low. Compared to other cities, population in Qingdao revealed significant genetic differentiation. Spatial kdr mutation patterns are likely attributable to some combination of prolonged insecticide-mediated selection coupled with the genetic isolation of these mosquito populations. CONCLUSIONS: These data suggest that multiple kdr alleles associated with insecticide resistance are present within the Cx. p. pallens populations of Shandong Province, China. The geographical distributions of kdr mutations in this province are likely that the result of prolonged and extensive insecticide application in agricultural contexts together with frequent mosquito population migrations. In contrast, the low-frequency kdr mutation detected in central Shandong Province populations may originate from the limited selection pressure in this area and the relative genetic isolation. Overall, the study compares the genetic patterns revealed by a functional gene with a neutral marker and demonstrates the combined impact of demographic and selection factors on population structure.

Polymorphic Variants of Genes Encoding Angiogenesis-Related Factors in Infertile Women with Recurrent Implantation Failure.

Recurrent implantation failure (RIF) is a global health issue affecting a significant number of infertile women who undergo in vitro fertilization (IVF) cycles. Extensive vasculogenesis and angiogenesis occur in both maternal and fetal placental tissues, and vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) family molecules and their receptors are potent angiogenic mediators in the placenta. Five single nucleotide polymorphisms (SNPs) in the genes encoding angiogenesis-related factors were selected and genotyped in 247 women who had undergone the ART procedure and 120 healthy controls. Genotyping was conducted by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). A variant of the kinase insertion domain receptor (KDR) gene (rs2071559) was associated with an increased risk of infertility after adjusting for age and BMI (OR = 0.64; 95% CI: 0.45-0.91, p = 0.013 in a log-additive model). Vascular endothelial growth factor A (VEGFA) rs699947 was associated with an increased risk of recurrent implantation failures under a dominant (OR = 2.34; 95% CI: 1.11-4.94, padj. = 0.022) and a log-additive model (OR = 0.65; 95% CI 0.43-0.99, padj. = 0.038). Variants of the KDR gene (rs1870377, rs2071559) in the whole group were in linkage equilibrium (D' = 0.25, r2 = 0.025). Gene-gene interaction analysis showed the strongest interactions between the KDR gene SNPs rs2071559-rs1870377 (p = 0.004) and KDR rs1870377-VEGFA rs699947 (p = 0.030). Our study revealed that the KDR gene rs2071559 variant may be associated with infertility and rs699947 VEGFA with an increased risk of recurrent implantation failures in infertile ART treated Polish women.