Vascular Endothelial Growth Factor a Promotes Chronic Itch via VEGFA-VEGFR2-PI3K-TRPV1 Axis in Allergic Contact Dermatitis.

Introduction: Allergic contact dermatitis (ACD), a prevalent skin disorder affecting up to 20% of the population, triggers significant discomfort and health implications. Our research investigates the pivotal role of Vascular Endothelial Growth Factor A (VEGFA) in chronic itching associated with ACD. Methods: Bioinformatics methods were utilized to identify differentially expressed genes (DEGs) between ACD models and patients. In vivo models of chronic pruritus in mice induced by 2,4-dinitrofluorobenzene (DNFB) were employed. Mice were administered subcutaneously with a VEGFA inhibitor, sFlt1, and compared to a control group. Real-time RT-PCR, Western blot, and immunohistochemical staining were performed to evaluate VEGFA expression and the impact of sFlt1 on itching behavior. Results: The analysis revealed that VEGFA is significantly upregulated in ACD skin, primarily expressed by keratinocytes. Administration of the VEGFA inhibitor sFlt1 in the ACD mouse model led to a substantial reduction in scratching behavior, indicating that VEGFA may mediate pruritus through the VEGFA-VEGFR2-PI3K-TRPV1 signaling pathway. Discussion: These findings suggest that VEGFA plays a crucial role in ACD-associated pruritus and may serve as a potential therapeutic target. However, further research is required to validate these findings and to explore additional molecular pathways involved in the pruritic response in ACD.

The expression level of VEGFR2 regulates mechanotransduction, tumor growth and metastasis of high grade serous ovarian cancer cells.

Recent data shows that alterations in the expression and/or activation of the vascular endothelial growth factor receptor 2 (VEGFR2) in high grade serous ovarian cancer (HGSOC) modulate tumor progression. However, controversial results have been obtained, showing that in some cases VEGFR2 inhibition can promote tumorigenesis and metastasis. Thus, it is urgent to better define the role of the VEGF/VEGFR2 system to understand/predict the effects of its inhibitors administered as anti-angiogenic in HGSOC. Here, we modulated the expression levels of VEGFR2 and analyzed the effects in two cellular models of HGSOC. VEGFR2 silencing (or its pharmacological inhibition) promote the growth and invasive potential of OVCAR3 cells in vitro and in vivo. Consistent with this, the low levels of VEGFR2 in OV7 cells are associated with more pronounced proliferative and motile phenotypes when compared to OVCAR3 cells, and VEGFR2 overexpression in OV7 cells inhibits cell growth. In vitro data confirmed that VEGFR2 silencing in OVCAR3 cells favors the acquisition of an invasive phenotype by loosening cell-ECM contacts, reducing the size and the signaling of focal adhesion contacts (FAs). This is translated into a reduced FAK activity at FAs, ECM-dependent alterations of mechanical forces through FAs and YAP nuclear translocation. Together, the data show that low expression, silencing or inhibition of VEGFR2 in HGSOC cells alter mechanotransduction and lead to the acquisition of a pro-proliferative/invasive phenotype which explains the need for a more cautious use of anti-VEGFR2 drugs in ovarian cancer.

Apatinib has anti-tumor effects and induces autophagy in lung cancer cells with high expression of VEGFR-2.

Objectives: This study investigated the inhibitory effect of apatinib on lung cancer cells with high expression of vascular endothelial growth factor-2 (VEGFR-2) and on inducing cellular autophagy and drug resistance. Materials and Methods: The expression of VEGFR-2 was detected using western blotting and RT-PCR. Cell proliferation was measured using the CCK8 and colony formation assays. The cell apoptosis rate was determined using flow cytometry and tunnel assay. Cellular autophagy was detected by measuring the expression of LC3-II using Western blotting and cellular immunofluorescence. The inhibitory effect of apatinib on lung cancer cells and transplanted tumors was observed after treatment with the autophagy inhibitor chloroquine. Results: Apatinib dose-dependently inhibited the proliferation of H1975 and H446 cells; it induced apoptosis via the PARP and caspase-3 pathways in H1975 and H446 cells and effectively inhibited the growth of transplanted tumors. Apatinib induced autophagy in a dose-dependent manner in H1975 and H446 cells. The inhibitory effect of apatinib on cells and the promotion of apoptosis were significantly enhanced after treatment with chloroquine. Immunohistochemistry showed that combining apatinib with chloroquine could reduce the expression of CD31 and Ki67 and increase the expression of caspase-3. Conclusion: Apatinib inhibits proliferation and induces apoptosis in H1975 and H1446 lung cancer cells with high VEGFR2 expression and autophagy in H1975 and H446 cells.

Computer-Aided Design of VEGFR-2 Inhibitors as Anticancer Agents: A Review.

Due to its intricate molecular and structural characteristics, vascular endothelial growth factor receptor 2 (VEGFR-2) is essential for the development of new blood vessels in various pathological processes and conditions, especially in cancers. VEGFR-2 inhibitors have demonstrated significant anticancer effects by blocking many signaling pathways linked to tumor growth, metastasis, and angiogenesis. Several small compounds, including the well-tolerated sunitinib and sorafenib, have been approved as VEGFR-2 inhibitors. However, the widespread side effects linked to these VEGFR-2 inhibitors-hypertension, epistaxis, proteinuria, and upper respiratory infection-motivate researchers to search for new VEGFR-2 inhibitors with better pharmacokinetic profiles. The key molecular interactions required for the interaction of the small molecules with the protein target to produce the desired pharmacological effects are identified using computer-aided drug design (CADD) methods such as pharmacophore and QSAR modeling, structure-based virtual screening, molecular docking, molecular dynamics (MD) simulation coupled with MM/PB(GB)SA, and other computational strategies. This review discusses the applications of these methods for VEGFR-2 inhibitor design. Future VEGFR-2 inhibitor designs may be influenced by this review, which focuses on the current trends of using multiple screening layers to design better inhibitors.

Synthesis and in vitro exploration of the 8-carbo substituted 5-methoxyflavones as anti-breast and anti-lung cancer agents targeting protein kinases (VEGFR-2 & EGFR).

The 8-aryl-, 8-styryl- and 8-arylethynyl substituted 5-methoxyflavones were synthesized and characterized using a combination of spectroscopic techniques. Single crystal X-ray diffraction (XRD) study on a representative compound 3h shows an inverted dimer linked by fused ten and six-membered ring motifs involving intermolecular CO⋯HC and CH⋯OC hydrogen bonds. Compounds 3b, 3c, 3d, 4a and 4b exhibited strong activity against the human breast (MCF-7) cancer cell line (IC50 = 13.68 ± 0.72, 16.91 ± 0.40, 13.63 ± 0.36, 14.66 ± 0.47 and 12.26 ± 0.45 µM, respectively) and lung (A549) cancer cell line (IC50 = 15.38 ± 0.33, 10.00 ± 0.28, 12.38 ± 0.30, 12.84 ± 0.33 and 8.47 ± 0.30 µM, respectively) compared to quercetin (IC50 = 40.61 ± 1.07 and 58.17 ± 0.50 µM, respectively). Compounds 3b, 3c and 4b exhibited dual inhibitory effect against the vascular endothelial growth factor receptor-2 (VEGFR-2) and the epidermal growth factor receptor (EGFR) tyrosine kinase phosphorylation. Molecular docking revealed that strong alignment with the enzyme backbone is achieved mostly by hydrophobic (π-π, and π-H) contacts and by hydrogen bonding interaction with the residues in the active sites of VEGFR-2 and EGFR. The test compounds possess favorable drug-likeness properties, supporting their potential as promising therapeutic candidates.

Zinc Finger Protein 536 is a Potential Prognostic Biomarker that Promotes Neuroblastoma Progression via VEGFR2-PI3K-AKT Pathway.

BACKGROUND: Neuroblastoma (NB) is a well-known pediatric malignancy intertwined with neurodevelopment. Previously implicated in neuronal differentiation, Zinc Finger Protein 536 (ZNF536) has emerged as a promising prognostic and immune-related biomarker in our pan-cancer analysis. METHODS: Single-cell RNA transcriptome sequencing, bulk transcriptome analysis, and immuno-histochemistry were used to assess ZNF536 expression and its association with prognosis. Cell proliferation, migration, invasion, and differentiation in ZNF536-knockdown NB cell lines were detected to evaluate the effect of ZNF536 on tumor cells. Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), a potential target of ZNF536, and its downstream PI3K/AKT signaling cascade were investigated using transcriptome sequencing, CUT&Tag, quantitative real-time PCR (qRT-PCR), and Western blotting. The role of ZNF536 in tumorigenesis and the potential regulation axis was evaluated in vivo using a BALB/c nude mouse xenograft tumor model. RESULTS: ZNF536 mRNA and protein expression were significantly higher in NB patients with poor prognosis. In vitro, ZNF536 knockdown curtailed proliferation, migration, and invasion of NB cells while fostering differentiation. ZNF536 regulated VEGFR2 expression, thus activating the PI3K-AKT pathway. In vivo, ZNF536 knockdown reduced tumor growth and proliferation via the VEGFR2-PI3K-AKT pathway. CONCLUSION: ZNF536 resulted as a novel prognostic biomarker in NB, promoting oncogenesis through VEGFR2-PI3K-AKT signaling axis modulation, suggesting its therapeutic potential in managing NB progression.

Combined inhibition of MET and VEGF enhances therapeutic efficacy of EGFR TKIs in EGFR-mutant non-small cell lung cancer with concomitant aberrant MET activation.

BACKGROUND: Aberrant activation of mesenchymal epithelial transition (MET) has been considered to mediate primary and acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC). However, mechanisms underlying this process are not wholly clear and the effective therapeutic strategy remains to be determined. METHODS: The gefitinib-resistant NSCLC cell lines were induced by concentration increase method in vitro. Western blot and qPCR were used to investigate the relationship between MET and vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) signaling pathway. Double luciferase reporter gene and co-immunoprecipitation were used to further reveal the regulation mechanism between MET and VEGF/VEGFR2. The effect of combined inhibition of MET and VEGF/VEGFR2 signaling pathway on the therapeutic sensitivity of EGFR-TKI in gefitinib resistant cell lines with MET aberration was verified ex vivo and in vivo. RESULTS: We successfully obtained two gefitinib-resistant NSCLC cell lines with EGFR mutation and abnormal activation of MET. We observed that MET formed a positive feedback loop with the VEGF/VEGFR2 signaling, leading to persistent downstream signaling activation. Specifically, MET up-regulated VEGFR2 expression in a MAPK/ERK/ETS1-dependent manner, while VEGF promoted physical interaction between VEGFR2 and MET, thereby facilitating MET phosphorylation. A MET inhibitor, crizotinib, combined with an anti-VEGF antibody, bevacizumab, enhanced the sensitivity of NSCLC cells to gefitinib and synergistically inhibited the activation of downstream signaling in vitro. Dual inhibition of MET and VEGF combined with EGFR TKIs markedly restrained tumor growth in both human NSCLC xenograft models and in an EGFR/MET co-altered case. CONCLUSIONS: Our work reveals a positive feedback loop between MET and VEGF/VEGFR2, resulting in continuous downstream signal activation. Combined inhibition of MET and VEGF/VEGFR2 signaling pathway may be beneficial for reversing EGFR TKIs resistance.

Design, synthesis and biological evaluation of indazole derivatives as VEGFR-2 kinase inhibitors with anti-angiogenic properties.

The strategy of inhibiting angiogenesis, specifically by targeting vascular endothelial growth factor receptor 2 (VEGFR-2), has been proven effective in tumor treatment. In this study, we designed several VEGFR-2 kinase inhibitors based on an indazole scaffold. Among them, the most potent compound, 30, inhibits VEGFR-2 (IC50 = 1.24 nM) with subtle selectivity over other kinases. It demonstrates significant inhibitory activity against HUVEC angiogenesis and inhibits cell migration in a dose-dependent manner. Additionally, it exhibits low acute toxicity in mice. In vivo studies, compound 30 demonstrates favorable pharmacokinetic profiles. It suppresses tumor angiogenesis in the zebrafish subintestinal vessel model, indicating that it may be a potential angiogenesis inhibitor for further development.

Design, synthesis, and biological investigations of new pyrazole derivatives as VEGFR2/CDK-2 inhibitors targeting liver cancer.

New Series of N-Manniche bases 3,4 (a-c) and 5,6 (a-b) were synthesized through the reaction of benzaldehyde and amine with 3-methyl-4-(aryldiazenyl)-1H-pyrazol-5-ol derivatives 2(a-c), they were fully characterized by FT-IR, (1H, 13C) NMR data in addition to their mass spectra. The Structural Activity Relationship of the target compounds were examined for their cytotoxicity. Some newly synthesized compounds showed promising antiproliferation properties when tested against HepG2 cancer cells. Compounds 4a, 5a, and 6b showed potent cytotoxicity against HepG2 with IC50 values of 4.4, 3.46 and 2.52 µM compared to Sorafenib (IC50 = 2.051 µM) and Roscovitine (IC50 = 4.18 µM). Furthermore, they were safe against the THLE2 cells with higher IC50 values. Compound 6b exhibited promising dual VEGFR2/CDK-2 inhibition activities; it had an IC50 value of 0.2 µM with VEGFR2 inhibition of 93.2%, and it had an IC50 value of 0.458 µM with CDK-2 inhibition of 88.7%. In comparison to the untreated control group (0.95%), compounds 5a (38.32%) and 6b (42.9%) considerably increased the cell population in total apoptosis. In addition, compounds 5a and 6b arrested the cell population at G0-G1 and S phases, respectively. Molecular docking experiments confirmed the virtual binding mechanism of the most active drugs, which were found to have good binding affinities with both receptor active sites.

A Recombinant Lentiviral Vegfr2-Silencing Vector Attenuates Roxarsone-Promoted Growth of Rat Vascular Endothelial Cells and Angiogenesis in Matrigel Plug and B16F10 Xenograft Models.

Roxarsone (ROX) is widely used as a feed addictive for livestock and poultry. ROX promotes angiogenesis, which can lead to health problems, and it is necessary to identify methods to counter this angiogenic effect of ROX. The VEGF/VEGFR2 signaling pathway is involved in the growth and reconstruction of new blood vessels during angiogenesis. In this study, a recombinant lentiviral vector encoding Vegfr2 shRNA was transfected into rat vascular endothelial cells and used in mouse matrigel plug and melanoma xenograft models to investigate its potential to regulate ROX-induced angiogenesis and tumor growth. Treating endothelial cells with ROX increased cell proliferation, migration, and a tube-like structure of growth relative to the control group. The addition of the lentiviral Vegfr2-silencing vector significantly attenuated the effects of ROX on endothelial cells. The hemoglobin content of mouse matrigel plugs treated with ROX was increased significantly. This effect was dramatically attenuated by the co-administration of shRNA targeting Vegfr2. The volume, weight and CD34 staining of the melanoma xenograft tumors increased by ROX were also attenuated by Vegfr2 silence. These results indicate that the down-regulation of VEGFR2 protein plays an inhibitory role in the ROX-promoted angiogenesis in vivo and in vitro. These data support the targeting of Vegfr2 gene as an effective means to treat ROX-induced angiogenesis and tumor growth.

Novel quinazolin-4-one based derivatives bearing 1,2,3-triazole and glycoside moieties as potential cytotoxic agents through dual EGFR and VEGFR-2 inhibitory activity.

The toxicity that was caused by the developed medications for anticancer treatment is, unfortunately, an earnest problem stemming from the various involved targets, and accordingly, intense research for overcoming such a phenomenon remains indispensable. In the current inquiry, an innovative category of substituted quinazoline-based glycosides incorporating a core of 1,2,3-triazole and attached to distinct acetylated likewise deprotected sugar segments are created and produced synthetically. The resulted 1,2,3-triazolyl-glycosides products were investigated for their ability to cause cytotoxicity to several human cancer cell lines. The quinazoline based glycosyl-1,2,3-triazoles 10-13 with free hydroxy sugar moiety revealed excellent potency against (IC50 range = 5.70-8.10 µM, IC50 Doxorubicin = 5.6 ± 0.30 µM, IC50 Erlotinib = 4.3 ± 0.1 µM). against MCF-7 cancer cell line. In addition, the derived glycosides incorporating quinazolinone and triazole core 6-13 with acetylated and deprotected sugar parts showed excellent and superior potency against HCT-116 (IC50 range = 2.90-6.40 µM). The potent products were revealed as safe cytotoxic agents as indicated by their studied safety profiles. Additional research of promising candidates inhibitory analysis performed against EGFR and VEGFR-2. The hydroxylated glycosides incorporating triazole and quinazoline system 11 and 13 with N-methyl substitution of quinazolinone, gave excellent potency against EGFR (IC50 = 0.35 ± 0.11 and 0.31 ± 0.06 µM, correspondingly) since glycoside 13 revealed comparable IC50 (3.20 ± 0.15 µM) to sorafenib against VEGFR-2. For more understanding of its action mode, it was analyzed how the 1,2,3-triazolyl glycoside 13 made an effect on the apoptosis induction and the arrest of the cell cycle. It was revealed that it had the ability to stop HCT-116 cells in their cell cycle's G1 stage. Moreover, the influence of quinazolinone-1,2,3-triazole-glycoside 13 upon p53, Bax, and Bcl-2 levels in HCT-116 units was also studied for future approaches toward its behavior. Additionally, the latter derivative may trigger apoptosis, as indicated by a significant increase in apoptotic cells. Furthermore, molecular docking was simulated to make an obvious validation and comprehension acquirement of the binding's characteristics also attractions among the most forceful compounds side by side with their aimed enzymes.

AB042. Combined anti-PD-L1 and anti-VEGFR2 therapy promotes the antitumor immune response in glioblastoma multiforme by reprogramming tumor microenvironment.

BACKGROUND: Inhibitors of programmed cell death ligand 1 (PD-L1) and vascular endothelial growth factor receptor 2 (VEGFR2) are commonly used in the clinic, but they are beneficial for only a minority of glioblastoma multiforme (GBM) patients. GBM has significant immunosuppressive properties, and there are many immunosuppressive cells and dysfunctional effector T-cell in the tumor microenvironment (TME), which is one of the important reasons for the failure of clinical treatment of GBM. P21-activated kinase 4 (PAK4) is a threonine protein kinase, and as a pivotal immune suppressor in the TME. PAK4 knockdown attenuates vascular abnormalities and promotes T-cell infiltration. METHODS: Using RNA sequencing (RNA-seq) technology, western blotting, and immunofluorescence, we identified changes in genes expression following VEGFR2 knockdown. The impact of anti-PD-L1 and anti-VEGFR2 on GBM cells apoptosis was assessed using coculture assays, western blotting, and flow cytometry. Additionally, the therapeutic efficacy of anti-PD-L1 and anti-VEGFR2 therapy was evaluated through in vivo experiments, immunohistochemistry, and immunofluorescence. RESULTS: Our studies revealed that VEGFR2 binds and phosphorylates signal transducer and activator of transcription 3 (p-STAT3), thereby regulating the expression of PAK4. Anti-PD-L1 and anti-VEGFR2 therapy can increase the secretion of interferon-gamma (IFN-γ), granzyme B, and perforin by immune cells and promoting the cytotoxic effects of cytotoxic cluster of differentiation 8 (CD8)+ T cells, and overexpression of PAK4 could reverse this effect. We also demonstrated that combination therapy with anti-PD-L1 and anti-VEGFR2 agents prevents tumor growth in an intracranial tumor model. CONCLUSIONS: Our results support that anti-VEGFR2 therapy can downregulate PAK4, reprogram the TME by increasing CD8+ T cells infiltration and activation, and enhance the therapeutic effect of anti-PD-L1 therapy on GBM cells.

Tibial transverse transport combined with platelet-rich plasma sustained-release microspheres activates the VEGFA/VEGFR2 pathway to promote microcirculatory reconstruction in diabetic foot ulcer.

This study proposes to investigate the therapeutic efficacy and mechanism of combining tibial transverse transport (TTT) with platelet-rich plasma (PRP) for diabetic foot ulcer (DFU). The diabetic rabbit model was constructed with Streptozotocin, which was intervened with TTT and PRP. PRP injection combined with TTT significantly promoted vascularisation and enhanced CD31, VEGFA, and VEGFR2 expressions compared to traditional TTT. However, the VEGFR2 inhibitor suppressed these phenomena. In the in vitro injury model, PRP reversed the diminished human umbilical vein endothelial cells (HUVECs) function and vascularisation caused by high-glucose damage. Additionally, PRP reduced inflammation and oxidative stress (approximately 47% ROS level) and enhanced VEGFA and VEGFR2 expression in HUVECs. However, the knockdown of VEGFR2 reversed the effect of PRP. In conclusion, TTT combined with intraosseous flap injection of PRP sustained-release microspheres activated the VEGFA/VEGFR2 pathway to promote microcirculatory reconstruction in DFU. These findings may provide new potential therapeutic strategies for DFU.

Anti-proliferative 2,3-dihydro-1,3,4-thiadiazoles targeting VEGFR-2: Design, synthesis, in vitro, and in silico studies.

In this study, we present the design, synthesis, and evaluation of six new thiadiazole derivatives designed as VEGFR-2 inhibitors. The most promising compound, 18b, demonstrated promising inhibitory activity against VEGFR-2, with an IC50 value of 0.165 µg/mL. The in vitro assessments on MCF-7 and HepG2 cell lines revealed the superior anti-proliferative effects of compound 18b, exhibiting IC50 values of 0.06 and 0.17 µM, respectively. Further investigations into the cell cycle distribution of compound 18b on MCF-7 cells exhibited a cell cycle arrest at the S phase (52.96 %) and significantly reducing the percentage of cells in the G0-G1 and G2/M phases. Additionally, compound 18b demonstrated a remarkable pro-apoptotic effect, with 45.29 % total apoptosis, characterized by both early and late apoptosis, and minimal necrosis. These findings were corroborated by RT-PCR analysis, revealing a significant downregulation of the anti-apoptotic gene Bcl2 and upregulation of the pro-apoptotic gene BAX in compound 18b-treated cells compared to control MCF-7 cells. Moreover, in silico studies involving molecular docking, Density Functional Theory (DFT) calculations, Molecular Dynamics (MD) simulations, MM-GBSA, Principle Component Analysis of Trajectories (PCAT), in addition to Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) predictions underscored the molecular interactions, energetics, and pharmacokinetic properties of compound 18b and the other derivatives further supporting its potential. Our integrated approach, combining in vitro experimens with in silico predictions provides valuable insights into the therapeutic potential of compound 18b as a robust VEGFR-2 inhibitor and lays the groundwork for future optimization.

Fucoxanthin Attenuates Angiogenesis by Blocking the VEGFR2-Mediated Signaling Pathway through Binding the Vascular Endothelial Growth Factor.

Fucoxanthin, a dietary carotenoid, is predominantly found in edible brown algae and is commonly consumed worldwide. Fucoxanthin has been shown to possess beneficial health activities such as antidiabetic, anti-inflammatory, antimutagenic, and antiobesity; however, the effects of fucoxanthin on VEGF-mediated angiogenesis and its possible binding with VEGF are unknown. Here, different lines of evidence supported the suppressive roles of fucoxanthin in VEGF-mediated angiogenesis. In human umbilical vein endothelial cells, fucoxanthin remarkedly suppressed VEGF-mediated cell proliferative, migration, and invasive abilities, as well as tube formation, without cytotoxicity. In addition, fucoxanthin inhibited the subintestinal vessel formation of zebrafish in vivo. In signaling cascades, fucoxanthin was proposed to interact with VEGF, thus attenuating VEGF's functions in activating the VEGF receptor and its related downstream signaling, i.e., phosphorylations of MEK and Erk. Fucoxanthin also significantly blocked VEGF-triggered ROS formation. Furthermore, the outcomes of applying fucoxanthin in cancer cells were identified, which included (i) inhibiting VEGF-mediated cell proliferation and migration and (ii) inhibiting NF-κB translocation via limiting MMP2 expression. These lines of investigations supported the antiangiogenic roles of fucoxanthin, as well as reviewing its signaling mechanisms, in blocking the VEGF-triggered responses. The results would benefit the potential development of fucoxanthin for the prevention and treatment of angiogenesis-related diseases.

3D physiologically-informed deep learning for drug discovery of a novel vascular endothelial growth factor receptor-2 (VEGFR2).

Angiogenesis is an essential process in tumorigenesis, tumor invasion, and metastasis, and is an intriguing pathway for drug discovery. Targeting vascular endothelial growth factor receptor 2 (VEGFR2) to inhibit tumor angiogenic pathways has been widely explored and adopted in clinical practice. However, most drugs, such as the Food and Drug Administration -approved drug axitinib (ATC code: L01EK01), have considerable side effects and limited tolerability. Therefore, there is an urgent need for the development of novel VEGFR2 inhibitors. In this study, we propose a novel strategy to design potential candidates targeting VEGFR2 using three-dimensional (3D) deep learning and structural modeling methods. A geometric-enhanced molecular representation learning method (GEM) model employing a graph neural network (GNN) as its underlying predictive algorithm was used to predict the activity of the candidates. In the structural modeling method, flexible docking was performed to screen data with high affinity and explore the mechanism of the inhibitors. Small -molecule compounds with consistently improved properties were identified based on the intersection of the scores obtained from both methods. Candidates identified using the GEM-GNN model were selected for in silico modeling using molecular dynamics simulations to further validate their efficacy. The GEM-GNN model enabled the identification of candidate compounds with potentially more favorable properties than the existing drug, axitinib, while achieving higher efficacy.

Vascular endothelial growth factor receptor 2 as a potential host target for the inhibition of enterovirus replication.

Because host kinases are key regulators of multiple signaling pathways in response to viral infections, we previously screened a kinase inhibitor library using rhabdomyosarcoma cells and human intestinal organoids in parallel to identify potent inhibitors against EV-A71 infection. We found that Rho-associated coiled-coil-containing protein kinase (Rock) inhibitor efficiently suppressed the EV-A71 replication and further revealed Rock1 as a novel EV-A71 host factor. In this study, subsequent analysis found that a variety of vascular endothelial growth factor receptor (VEGFR) inhibitors also had potent antiviral effects. Among the hits, Pazopanib, with a selectivity index as high as 254, which was even higher than that of Pirodavir, a potent broad-spectrum picornavirus inhibitor targeting viral capsid protein VP1, was selected for further analysis. We demonstrated that Pazopanib not only efficiently suppressed the replication of EV-A71 in a dose-dependent manner, but also exhibited broad-spectrum anti-enterovirus activity. Mechanistically, Pazopanib probably induces alterations in host cells, thereby impeding viral genome replication and transcription. Notably, VEGFR2 knockdown and overexpression suppressed and facilitated EV-A71 replication, respectively, indicating that VEGFR2 is a novel host dependency factor for EV-A71 replication. Transcriptome analysis further proved that VEGFR2 potentially plays a crucial role in combating EV-A71 infection through the TSAd-Src-PI3K-Akt pathway. These findings expand the range of potential antiviral candidates of anti-enterovirus therapeutics and suggest that VEGFR2 may be a key host factor involved in EV-A71 replication, making it a potential target for the development of anti-enterovirus therapeutics. IMPORTANCE: As the first clinical case was identified in the United States, EV-A71, a significant neurotropic enterovirus, has been a common cause of hand, foot, and mouth disease (HFMD) in infants and young children. Developing an effective antiviral agent for EV-A71 and other human enteroviruses is crucial, as these viral pathogens consistently cause outbreaks in humans. In this study, we demonstrated that multiple inhibitors against VEGFRs effectively reduced EV-A71 replication, with Pazopanib emerging as the top candidate. Furthermore, Pazopanib also attenuated the replication of other enteroviruses, including CVA10, CVB1, EV-D70, and HRV-A, displaying broad-spectrum anti-enterovirus activity. Given that Pazopanib targets various VEGFRs, we narrowed the focus to VEGFR2 using knockdown and overexpression experiments. Transcriptomic analysis suggests that Pazopanib's potential downstream targets involve the TSAd-Src-PI3K-Akt pathway. Our work may contribute to identifying targets for antiviral inhibitors and advancing treatments for human enterovirus infections.

Urolithin B inhibits the differentiation of M1 macrophages and relieves the inflammation around the implants under osteoporosis via down-regulating the phosphorylation of VEGFR2.

The inflammation causes the destroyed osseointegration at the implant-bone interface, significantly increasing the probability of implant loosening in osteoporotic patients. Currently, inhibiting the differentiation of M1 macrophages and the inflammatory response could be a solution to stabilize the microenvironment of implants. Interestingly, some natural products have anti-inflammatory and anti-polarization effects, which could be a promising candidate for stabilizing the implants' microenvironment in osteoporotic patients. This research aims to explore the inhibitory effect of Urolithin B(UB) on macrophage M1 polarization, which ameliorates inflammation, thus alleviating implant instability. We established an osteoporosis mouse model of implant loosening. The mouse tissues were taken out for morphological analysis, staining analysis, and bone metabolic index analysis. In in vitro experiments, RAW264.7 cells were polarized to M1 macrophages using lipopolysaccharide (LPS) and analyzed by immunofluorescence (IF) staining, Western blot (WB), and flow cytometry. The CSP100 plus chip experiments were used to explore the potential mechanisms behind the inhibiting effects of UB. Through observation of these experiments, UB can improve the osseointegration between the implants and femurs in osteoporotic mice and enhance the stability of implants. The UB can inhibit the differentiation of M1 macrophages and local inflammation via inhibiting the phosphorylation of VEGFR2, which can be further proved by the weakened inhibited effects of UB in macrophages with lentivirus-induced overexpression of VEGFR2. Overall, UB can specifically inhibit the activation of VEGFR2, alleviate local inflammation, and improve the stability of implants in osteoporotic mice.

Quercetin inhibits endothelial & hepatocellular carcinoma cell crosstalk via reducing extracellular vesicle-mediated VEGFR2 mRNA transfer.

This study aims to investigate the regulatory effects of quercetin extracellular vesicles (EVs)-mediated expression of vascular endothelial growth factor receptor 2 (VEGFR2) in hepatocellular carcinoma (HCC)-derived circulating tumor cells (CTCs) and the underlying mechanisms. CTCs were isolated from patients with pathologically diagnosed HCC, with VEGFR2 expression visualized by fluorescence in situ hybridization (FISH). The human HCC cell line Huh-7 and SK-HEP-1 were used for in vitro studies to assess EVs uptake, VEGFR2 mRNA transfer, invasion, migration, cancer stem cell (CSC) properties, and VEGF secretion. Results showed that VEGFR2 mRNA was commonly expressed in HCC-CTCs, with a higher incidence in biphenotypic CTCs. Its expression was limited in HCC cell lines, but present in certain liver cells. In vitro experiments confirmed that VEGFR2 mRNA could be transferred to HCC cells via EVs from primary tumor endothelial cells (PTECs), which was impaired by quercetin treatment. Quercetin significantly reduced VEGFR2 mRNA and protein expression in HCC cells, weakened their invasive and metastatic capacities, and diminished VEGFR2-mediated CSC properties. In vivo, quercetin reduced VEGF secretion, impaired angiogenesis, slowed tumor growth, and decreased the number and proportion of VEGFR2-positive CTCs. In summary, VEGFR2 mRNA is present in HCC-CTCs, potentially sourced from PTECs-derived EVs. Quercetin effectively inhibits VEGFR2 expression, impacting HCC cell invasion, metastasis, and CSC characteristics. Besides, it reduces VEGFR2-positive CTCs in vivo. These effects support its therapeutic potential in HCC treatment by targeting the angiogenesis and tumor dissemination pathway.

Design, Development of Pyrazole-Linked Spirocyclopropyl Oxindole-Carboxamides as Potential Cytotoxic Agents and Type III Allosteric VEGFR-2 Inhibitors.

Tumor progression depends on angiogenesis, which is stimulated by growth factors like VEGF, targeting VEGFR kinase with small molecules is an effective anti-angiogenic therapeutic approach. The rational modification of sunitinib (VEGFR-2 inhibitor) to spirocyclopropyloxindoline carboxamides have been performed and their in vitro cytotoxic profiling was evaluated. The molecular modelling studies enabled the screening of designed analogues and identifying the possible interactions within the type III allosteric inhibitor binding site of VEGFR-2. The biological screening of synthesized compounds 15 a-y, revealed the ability of compound 15 w to inhibit the cell growth in MCF-7 cell line with IC50 value of 3.87±0.19 µM and alongside inhibition of VEGFR-2 kinase at a IC50 concentration of 4.34±0.13 µM was observed. Also, VEGFR-2 inhibition was validated through HUVEC tube formation inhibition assay. The qualitative assessment of apoptosis induction by 15 w in MCF-7 cells was evaluated through staining studies such as AO/EB and DAPI staining, whereas quantification of apoptosis and cell cycle analysis were performed through FACS analysis. The metastatic ability of the cancer cells was evaluated through inhibition of cell migration by a scratch wound healing assay. The current study strives to sequentially optimize the structural attributes of the 3-alkenyl oxindole core to surpass the existing challenges of well-known VEGFR-2 inhibitors. The findings observed from this study highlights that compound 15 w to be a prominent lead towards the development of clinical drug candidates.