Discovery of novel amide derivatives against VEGFR-2/tubulin with potent antitumor and antiangiogenic activity.

Dual-target agents have more advantages than drug combinations for cancer treatment. Here, we designed and synthesized a series of novel VEGFR-2/tubulin dual-target inhibitors through a molecular hybridization strategy, and the activities of all the synthesized compounds were tested against tubulin and VEGFR-2. Among which, compound 19 exhibited strong potency against tubulin and VEGFR-2, with IC50 values of 0.76 ± 0.11 µM and 15.33 ± 2.12 nM, respectively. Additionally, compound 19 not only had significant antiproliferative effects on a series of human cancer cell lines, especially MGC-803 cells (IC50 = 0.005 ± 0.001 µM) but also overcame drug resistance in Taxol-resistant MGC-803 cells, with an RI of 1.8. Further studies showed that compound 19 could induce tumor cell apoptosis by reducing the mitochondrial membrane potential, increasing the level of ROS, facilitating the induction of G2/M phase arrest, and inhibiting the migration and invasion of tumor cells in a dose-dependent manner. In addition, compound 19 also exhibits potent antiangiogenic effects by blocking the VEGFR-2/PI3K/AKT pathway and inhibiting the tubule formation, invasion, and migration of HUVECs. More importantly, compound 19 demonstrated favorable pharmacokinetic profiles, robust in vivo antitumor efficacy, and satisfactory safety profiles. Overall, compound 19 can be used as a lead compound for the development of tubulin/VEGFR-2 dual-target inhibitors.

Increase of PCSK9 expression in diabetes promotes VEGFR2 ubiquitination to inhibit endothelial function and skin wound healing.

Diabetic foot ulcers (DFUs) are a serious vascular disease. Currently, no effective methods are available for treating DFUs. Pro-protein convertase subtilisin/kexin type 9 (PCSK9) regulates lipid levels to promote atherosclerosis. However, the role of PCSK9 in DFUs remains unclear. In this study, we found that the expression of PCSK9 in endothelial cells (ECs) increased significantly under high glucose (HG) stimulation and in diabetic plasma and vessels. Specifically, PCSK9 promotes the E3 ubiquitin-protein ligase NEDD4 binding to vascular endothelial growth factor receptor 2 (VEGFR2), which led to the ubiquitination of VEGFR2, resulting in its degradation and downregulation in ECs. Furthermore, PCSK9 suppresses the expression and activation of AKT, endothelial nitric oxide synthase (eNOS), and ERK1/2, leading to decreased nitric oxide (NO) production and increased superoxide anion (O2._) generation, which impairs vascular endothelial function and angiogenesis. Importantly, using evolocumab to limit the increase in PCSK9 expression blocked the HG-induced inhibition of NO production and the increase in O2._ production, as well as inhibited the phosphorylation and expression of AKT, eNOS, and ERK1/2. Moreover, evolocumab improved vascular endothelial function and angiogenesis, and promoted wound healing in diabetes. Our findings suggest that targeting PCSK9 is a novel therapeutic approach for treating DFUs.

ALK5/VEGFR2 dual inhibitor TU2218 alone or in combination with immune checkpoint inhibitors enhances immune-mediated antitumor effects.

Transforming growth factor ß (TGFß) is present in blood of patients who do not respond to anti-programmed cell death (ligand) 1 [PD-(L)1] treatment, and through synergy with vascular endothelial growth factor (VEGF), it helps to create an environment that promotes tumor immune evasion and immune tolerance. Therefore, simultaneous inhibition of TGFß/VEGF is more effective than targeting TGFß alone. In this study, the dual inhibitory mechanism of TU2218 was identified through in vitro analysis mimicking the tumor microenvironment, and its antitumor effects were analyzed using mouse syngeneic tumor models. TU2218 directly restored the activity of damaged cytotoxic T lymphocytes (CTLs) and natural killer cells inhibited by TGFß and suppressed the activity and viability of regulatory T cells. The inactivation of endothelial cells induced by VEGF stimulation was completely ameliorated by TU2218, an effect not observed with vactosertib, which inhibits only TGFß signaling. The combination of TU2218 and anti-PD1 therapy had a significantly greater antitumor effect than either drug alone in the poorly immunogenic B16F10 syngeneic tumor model. The mechanism of tumor reduction was confirmed by flow cytometry, which showed upregulated VCAM-1 expression in vascular cells and increased influx of CD8 + CTLs into the tumor. As another strategy, combination of anti-CTLA4 therapy and TU2218 resulted in high complete regression (CR) rates in CT26 and WEHI-164 tumor models. In particular, immunological memory generated by the combination of anti-CTLA4 and TU2218 in the CT26 model prevented the development of tumors after additional tumor cell transplantation, suggesting that the TU2218-based combination has therapeutic potential in immunotherapy.

Successful pazopanib treatment of undifferentiated pleomorphic sarcoma with coamplification of PDGFRA, VEGFR2 and KIT: A case report.

Undifferentiated pleomorphic sarcoma (UPS) is a high-grade, aggressive soft tissue sarcoma (STS) with a poor prognosis, and no definitive or effective treatment is currently available for it. Pazopanib, an orally available multiple tyrosine kinase inhibitor, has been approved for the treatment of advanced STS. The present study documents the case of a 51-year-old man with advanced UPS with coamplification of platelet-derived growth factor receptor A (PDGFRA), vascular endothelial growth factor receptor 2 (VEGFR2) and stem cell factor receptor (KIT) genes. The patient exhibited a marked and sustained response to pazopanib. The patient presented with a retroperitoneal tumour with pancreatic head lymph node metastasis, and bone metastases in the second/fifth thoracic vertebrae and left femur. Based on the histological analysis of the retroperitoneal tumour and femoral mass, the patient was diagnosed with UPS. Palliative radiation therapy was administered to the left femur and second/fifth thoracic vertebrae to prevent fractures. After radiation therapy, the patient achieved a partial response after eight courses of doxorubicin. A comprehensive genomic profiling analysis (FoundationOne® CDx) revealed coamplification of PDGFRA, VEGFR2 and KIT genes. Hence, pazopanib was initiated as a second-line treatment. Notably, the retroperitoneal tumour shrank, and no new lesions developed for 3 years after the initiation of pazopanib treatment. This response suggests that the coamplification of PDGFRA, VEGFR2 and KIT may predict favourable outcomes in response to pazopanib.

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. Sunitinib was rationally modified to spirocyclopropyloxindoline carboxamides and their in vitro cytotoxic profiling were evaluated. The molecular modelling studies enabled the screening of designed analogues and identified possible interactions within the type III allosteric inhibitor binding site of VEGFR-2. The biological screening of compounds 15a-y, revealed the ability of compound 15w to inhibit the cell growth in MCF-7 cells 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. VEGFR-2 inhibition was validated through HUVEC tube formation inhibition assay. 15w also inhibited cell migration in wound healing assay. The qualitative assessment of apoptosis induction by 15w in MCF-7 cells was evaluated through AO/EB and DAPI staining studies, whereas apoptotic quantification and cell cycle analysis were performed through FACS analysis. The current study strives to sequentially optimize the structural attributes of 3-alkenyl oxindole core to surpass the existing challenges of well-known VEGFR-2 inhibitors and compound 15w was identified to be a prominent lead towards the development of clinical drug candidates.

Anti-proliferation evaluation of new derivatives of indole-6-carboxylate ester as receptor tyrosine kinase inhibitors.

Aim: The main goal was to create two new groups of indole derivatives, hydrazine-1-carbothioamide (4a and 4b) and oxadiazole (5, and 6a-e) that target EGFR (4a, 4b, 5) or VEGFR-2 (6a-e). Materials & methods: The new derivatives were characterized using various spectroscopic techniques. Docking studies were used to investigate the binding patterns to EGFR/VEGFR-2, and the anti-proliferative properties were tested in vitro. Results: Compounds 4a (targeting EGFR) and 6c (targeting VEGFR-2) were the most effective cytotoxic agents, arresting cancer cells in the G2/M phase and inducing the extrinsic apoptosis pathway. Conclusion: The results of this study show that compounds 4a and 6c are promising cytotoxic compounds that inhibit the tyrosine kinase activity of EGFR and VEGFR-2, respectively.

[Box: see text].

Semaxanib, a VEGF inhibitor, suppresses melanogenesis by modulating CRTC3 independently of VEGF signaling.

BACKGROUND: Dysregulation of melanogenesis contributes to the development of skin hyperpigmentation diseases, which poses a treatment challenge. Following the establishment of CRTC3 screening methods to explore small molecules inhibiting melanogenesis for the topical treatment of hyperpigmentation diseases, we identified a candidate molecule, semaxanib. OBJECTIVE: To explore the antimelanogenic effects of semaxanib, a vascular endothelial growth factor receptor (VEGFR) 2 inhibitor, for potential applications in hyperpigmentation management and to unravel the role of VEGF signaling in melanocyte biology by investigating mechanism of action of semaxanib. METHODS: Mouse-derived spontaneously immortalized melanocytes, B16F10, and normal human primary epidermal melanocytes cells were treated with semaxanib, and cellular responses were assessed using cell viability assays and melanin content measurements. Molecular mechanisms were investigated using transcriptional activity assays, reverse-transcription polymerase chain reaction, and immunoblotting analysis. In vivo studies were conducted using an epidermis-humanized transgenic mouse model and ex vivo human skin tissues. RESULTS: Semaxanib ameliorated melanin content in cultured melanocytes by downregulating the expression of melanogenesis-associated genes by suppressing the CRTC3/microphthalmia-associated transcription factors. Topical application of semaxanib reduced melanin accumulation in the ultraviolet B-stimulated ex vivo human epidermis and tail of K14-stem cell factor transgenic mice. Mechanistically, the antimelanogenic effect induced by semaxanib was associated with SIK2-CRTC3-MITF rather than VEGF signaling in melanocytes. CONCLUSION: Semaxanib emerges as a promising candidate for the development of therapeutics for hyperpigmentation, potentially working independently of VEGF signaling in human melanocytes.

Tetrahydroisoquinoline reduces angiogenesis by interacting myeloma cells with HUVECs mediated by extracellular vesicles.

Multiple myeloma (MM) is a neoplastic condition resulting from the uncontrolled expansion of B-cell-derived plasma cells. The importance of angiogenesis in MM development has also been demonstrated. Extracellular vesicles (EVs) have vital functions in interactions between neighboring cells, such as angiogenesis. The objective of this in vitro study was to examine the transfection and angiogenesis effects of MM-EVs on endothelial cells (ECs) upon treatment with Tetrahydroisoquinoline (THIQ) as a bioactive organic compound derivative from isoquinoline. Following treatment of multiple myeloma cells (U266) with THIQ, MM-EVs were harvested and transmigrated to human umbilical vein endothelial cells (HUVEC) in a co-culture model. EVs transmigration was traced by flow cytometry. Correspondingly, the expression of angiogenic genes and/or proteins in U266 cells and HUVECs was measured by RT-PCR and ELISA methods. Likewise, the proliferation and migration of HUVECs treated with THIQ-treated MM-EVs were visualized and estimated by performing both tube formation and scratch wound healing methods. Surprisingly, the anti-angiogenic effect of THIQ-treated MM-EVs was evident by the decreased expression of CD34, VEGFR2, and IL-6 at the mRNA and/or protein levels after internalization of MM-EVs in HUVEC. Finally, tube formation and scratch wound healing experiments showed inhibition of HUVEC cell proliferation and migration by THIQ-treated MM-EVs compared to control MM-EVs. MM-EVs derived from THIQ-treated myeloma cells (U266) inhibited angiogenesis in HUVECs. This phenomenon is coordinated by the internalized THIQ-treated MM-EVs in HUVECs, and ultimately the reduction of angiogenic factors and inhibition of tube formation and scratch wound healing.

Hepatic arterial infusion chemotherapy plus camrelizumab and apatinib for advanced hepatocellular carcinoma.

BACKGROUND AND AIMS: There is limited information on combination of hepatic arterial infusion chemotherapy (HAIC) and systemic therapy for advanced hepatocellular carcinoma (Ad-HCC). We aim to compare the efficacy and safety of HAIC plus camrelizumab (a PD-1 inhibitor) and apatinib (an VEGFR-2 inhibitor) versus camrelizumab and apatinib for Ad-HCC. METHODS: From April 2019 to October 2022, 416 patients with Ad-HCC who received either HAIC plus camrelizumab and apatinib (TRIPLET protocol, n = 207) or camrelizumab and apatinib (C-A protocol, n = 209) were reviewed retrospectively. The propensity score matching (PSM) was used to reduce selective bias. Overall survival (OS) and progression-free survival (PFS) were compared using the Kaplan-Meier method with the log-rank test. Cox regression analyses of independent prognostic factors were evaluated. RESULTS: After PSM 1:1, 109 patients were assigned to two groups. The median OS of not reached in the TRIPLET group was significantly longer than that of 19.9 months in the C-A group (p < 0.001), while in the TRIPLET group, the median PFS of 11.5 months was significantly longer than that of 9.6 months in the C-A group (p < 0.001). Multivariate analyses showed that the factors significantly affected the OS were CTP grade, tumor number > 3, and TRIPLET treatment (p < 0.001). Grade 3/4 adverse events occurred at a rate of 82.1% vs. 71.3% in TRIPLET and C-A groups, respectively. CONCLUSION: The TRIPLET protocol has promising survival benefits in the management of patients with Ad-HCC, with acceptable safety. TRAIL REGISTRATION: The study has been retrospectively registered at Chinese Clinical Trial Registry ( https://www.chictr.org.cn/ , ChiCTR2300075828).

Mtnr1b deletion disrupts placental angiogenesis through the VEGF signaling pathway leading to fetal growth restriction.

The placenta, as a "transit station" between mother and fetus, has functions delivering nutrients, excreting metabolic wastes and secreting hormones. A healthy placenta is essential for fetal growth and development while the melatonergic system seems to play a critical physiological role in this organ since melatonin, its synthetic enzymes and receptors are present in the placenta. In current study, Mtnr1a and Mtnr1b knockout mice were constructed to explore the potential roles of melatonergic system played on the placental function and intrauterine growth retardation (IUGR). The result showed that Mtnr1a knockout had little effect on placental function while Mtnr1b knockout reduced placental efficiency and increased IUGR. Considering the extremely high incidence of IURG in sows, the pregnant sows were treated with melatonin. This treatment reduced the incidence of IUGR. All the evidence suggests that the intact melatonergic system in placenta is required for its function. Mechanistical studies uncovered that Mtnr1b knockout increased placental oxidative stress and apoptosis but reduced the angiogenesis. The RNA sequencing combined with histochemistry study identified the reduced angiogenesis and placental vascular density in Mtnr1b knockout mice. These alterations were mediated by the disrupted STAT3/VEGFR2/PI3K/AKT pathway, i.e., Mtnr1b knockout reduced the phosphorylation of STAT3 which is the promotor of VEGFR2. The downregulated VEGFR2 and its downstream elements of PI3K and AKT expressions, then, jeopardizes the angiogenesis and placental development.

Design, synthesis and cytotoxic evaluation of new thieno[2,3-d]pyrimidine analogues as VEGFR-2/AKT dual inhibitors, apoptosis and autophagy inducers.

Novel thieno[2,3-d]pyrimidine analogues were designed, synthesized and evaluated for anti-proliferative activity against HepG-2, PC-3 and MCF-7 cancer cell lines. In addition, WI-38 normal cell line was used to explore the safety of all the tested compounds. Compounds 2 (IC50 = 4.29 µM HePG-2, 10.84 µM MCF-7), 6 (IC50 = 14.86 µM HePG-2, 8.04 µM PC-3 and 12.90 µM MCF-7) and 17 (IC50 = 9.98 µM HePG-2, 33.66 µM PC-3 and 14.62 µM MCF-7) were the most promising candidates on the tested cancer cells with high selective toxicity-sparing normal cells. A further mechanistic evaluation revealed promising kinase inhibitory activity, where compound 2 inhibited VEGFR-2 and AKT at IC50 = 0.161 and 1.06 µM, respectively, Furthermore, derivative 6 inhibited VEGFR-2 and AKT at IC50 = 0.487 and 0.364 µM, respectively, while compound 17 showed IC50 = 0.164 and 0.452 µM, respectively. Moreover, compounds 2, 6 resulted in G1 phase cell cycle arrest while candidate 17 arrest cell cycle at G2/M phase. Similar to the apoptosis results, compound 17 showed the highest autophagic induction among the evaluated derivatives. Finally, docking studies were conducted to assess the binding patterns of these active derivatives. The results showed that the binding patterns inside the active sites of both the VEGFR-2 and AKT-1 (allosteric pocket) crystal structures were identical to the reference ligands.

Exploitation of enhanced prime editing for blocking aberrant angiogenesis.

INTRODUCTION: Aberrant angiogenesis plays an important part in the development of a variety of human diseases including proliferative diabetic retinopathy, with which there are still numerous patients remaining a therapeutically challenging condition. Prime editing (PE) is a versatile gene editing approach, which offers a novel opportunity to genetically correct challenging disorders. OBJECTIVES: The goal of this study was to create a dominant-negative (DN) vascular endothelial growth factor receptor (VEGFR) 2 by editing genomic DNA with an advanced PE system to block aberrant retinal angiogenesis in a mouse model of oxygen-induced retinopathy. METHODS: An advanced PE system (referred to as PE6x) was established within two lentiviral vectors, with one carrying an enhanced PE guide RNA and a canonical Cas9 nickase fused with an optimized reversal transcriptase, and the other conveying a nicking guide RNA and a DN-MLH1 to improve PE efficiency. Dual non-integrating lentiviruses (NILVs) produced with the two lentiviral PE6x vectors were then employed to create a mutation of VEGFR2 T17967A by editing the Mus musculus VEGFR2 locus in vitro and in vivo, leading to generation of a premature stop codon (TAG, K796stop) to produce DN-VEGFR2, to interfere with the wild type VEGFR2 which is essential for angiogenesis. RESULTS: NILVs targeting VEGFR2 delivered into cultured murine vascular endothelial cells led to 51.06 % VEGFR2 T17967A in the genome analyzed by next generation sequencing and the production of DN-VEGFR2, which was found to hamper VEGF-induced VEGFR2 phosphorylation, as demonstrated by Western blot analysis. Intravitreally injection of the dual NILVs into postnatal day 12 mice in a model of oxygen-induced retinopathy, led to production of retinal DN-VEGFR2 in postnatal day 17 mice which blocked retinal VEGFR2 expression and activation as well as abnormal retinal angiogenesis without interfering with retinal structure and function, as assessed by electroretinography, optical coherence tomography, fundus fluorescein angiography and histology. CONCLUSION: DN-VEGFR2 resulted from editing genomic VEGFR2 using the PE6x system can be harnessed to treat intraocular pathological angiogenesis.

Screening and Anti-angiogenesis Activity of Chiloscyllium plagiosum Anti-human VEGFR2 Single-domain Antibody.

Recently, the incidence of malignant tumors is on the rise and searching for new treatments on it has become the research priority. Blocking the vascular endothelial growth factor (VEGF) and its receptor (VEGFR) is one of the treatment strategies that used in the development of specific anti-angiogenic drugs. The deficiencies in tissue penetration and affinity maturation become the weakness of these drugs in anti-tumors applications. The single heavy chain antibody found in Chiloscyllium plagiosum, which has a low molecular weight and superior tissue penetration of variable region (VNARs), was considered to have the high antigen binding activity and stability. This type of antibody has a simple structure that can be prokaryoticaly expressed, which makes it easily to produce new antiangiogenic target drugs. Specific anti-IgNAR rabbit multiple antibodies have been used to assess the level of VNARs in sharks and have shown a significant enrichment of IgNAR after triple immunization. An anti-VEGFR2 phage library was used for the targeted VNARs screening, and five candidate VNARs sequences were subsequently obtained by phage screening, followed by combined screening with the transcriptome library, and analysis of conserved regions along with 3D modelling matched the VNAR profile. ELISA and cell-based assays showed that two of the VNARs, VNAR-A6 and VNAR-E3, had a superior antigen affinity and anti-angiogenic activity thereby being able to inhibit human Umbilical Vein Endothelial Cells proliferation and migration. The anti-VEGFR2 VNARs derived from the immunized Chiloscyllium plagiosum and screened by phage library, which provide the new research ideas and specific approaches for the development of new drugs. The anti-VEGFR2 VNARs are capable for blocking the VEGF-VEGFR pathway, which of these may contribute to expanding the use of anti-angiogenic drugs.

VEGF as a Key Actor in Recurrent Respiratory Papillomatosis: A Narrative Review.

Recurrent respiratory papillomatosis (RRP) is a benign disease of the upper aerodigestive tract caused by human papillomavirus (HPV) types 6 and 11. The clinical course is unpredictable and some patients, especially younger children, experience a high rate of recurrence with a significant impact on their quality of life. The molecular mechanisms of HPV infection in keratinocytes have been extensively studied throughout the years, with particular regard to its role in causing malignant tumors, like cervical cancer and head and neck carcinomas. A minor but not negligible amount of the literature has investigated the molecular landscape of RRP patients, and some papers have studied the role of angiogenesis (the growth of blood vessels from pre-existing vasculature) in this disease. A central role in this process is played by vascular endothelial growth factor (VEGF), which activates different signaling cascades on multiple levels. The increased knowledge has led to the introduction of the VEGF inhibitor bevacizumab in recent years as an adjuvant treatment in some patients, with good results. This review summarizes the current evidence about the role of VEGF in the pathophysiology of RRP, the molecular pathways activated by binding with its receptors, and the current and future roles of anti-angiogenic treatment.

Unveiling the potential of isatin-grafted phenyl-1,2,3-triazole derivatives as dual VEGFR-2/STAT-3 inhibitors: Design, synthesis and biological assessments.

The use of VEGFR-2 inhibitors as a stand-alone treatment has proven to be ineffective in clinical trials due to the robustness of cellular response loops that lead to treatment resistance when only targeting VEGFR-2. The over-activation of the signal transducer/activator of transcription 3 (STAT-3) is expected to significantly impact treatment failure and resistance to VEGFR-2 inhibitors. In this study, we propose the concept of combined inhibition of VEGFR-2 and STAT-3 to combat induced STAT-3-mediated resistance to VEGFR-2 inhibition therapy. To explore this, we synthesized new isatin-grafted phenyl-1,2,3-triazole derivatives "6a-n" and "9a-f". Screening on PANC1 and PC3 cancer cell lines revealed that compounds 6b, 6 k, 9c, and 9f exhibited sub-micromolar ranges. The most promising molecules, 6b, 6 k, 9c, and 9f, demonstrated the highest inhibition when tested as dual inhibitors on VEGFR-2 (with IC50 range 53-82 nM, respectively) and STAT-3 (with IC50 range 5.63-10.25 nM). In particular, triazole 9f showed the best results towards both targets. Inspired by these findings, we investigated whether 9f has the ability to trigger apoptosis in prostate cancer PC3 cells via the assessment of the expression levels of the apoptotic markers Caspase-8, Bcl-2, Bax, and Caspase-9. Treatment of the PC3 cells with compound 9f significantly inhibited the protein expression levels of VEGFR-2 and STAT-3 kinases compared to the control.

The Impact of the VEGF/VEGFR2/PI3K/AKT Signaling Axis on the Proliferation and Migration Abilities of Human Dental Pulp Stem Cells.

The potential therapeutic benefits of human dental pulp stem cells (HDPSCs) in dental regenerative medicine have been demonstrated. However, little is known about the molecular mechanisms regulating the biological characteristics of HDPSCs. The experiment aims to explore whether VEGF activates signaling pathways such as FAK, PI3K, Akt, and p38 in HDPSCs, and to investigate the molecular mechanisms by which VEGF influences proliferation and migration of HDPSCs. Normal and inflamed human dental pulp (HDP) samples were collected, and the levels of VEGF in HDP were assessed. HDPSCs were cultured and purified. HDPSCs were stimulated with lipopolysaccharide (LPS) at gradient concentrations, and real-time quantitative polymerase chain reaction (qPCR) was used to assess changes in VEGF mRNA. Gradient concentrations of VEGF were used to stimulate HDPSCs, and cell migration ability was evaluated through scratch assays and Transwell chamber experiments. Phosphorylation levels of FAK, AKT, and P38 were assessed using Western blotting. Inhibitors of VEGFR2, FAK, AKT, P38, and VEGF were separately applied to HDPSCs, and cell migration ability and phosphorylation levels of FAK, AKT, and P38 were determined. The results indicated significant differences in VEGF levels between normal and inflamed HDP tissues, with levels in the inflamed state reaching 435% of normal levels (normal: 87.91 ng/mL, inflamed: 382.76 ng/mL, P < 0.05). LPS stimulation of HDPSCs showed a significant increase in VEGF mRNA expression with increasing LPS concentrations (LPS concentrations of 0.01, 0.1, 1, and 10 µg/mL resulted in VEGF mRNA expressions of 181.2%, 274.2%, 345.8%, and 460.9%, respectively, P < 0.05). VEGF treatment significantly enhanced the migration ability of HDPSCs in Transwell chamber experiments, with migration rates increasing with VEGF concentrations (VEGF concentrations of 0, 1, 10, 20, 50, and 100 ng/mL resulted in migration rates of 8.41%, 9.34%, 21.33%, 28.41%, 42.87%, and 63.15%, respectively, P < 0.05). Inhibitors of VEGFR2, FAK, AKT, P38, and combined VEGF stimulation demonstrated significant migration inhibition, with migration rates decreasing to 8.31%, 12.64%, 13.43%, 18.32%, and 74.17%, respectively. The migration rate with combined VEGF stimulation showed a significant difference (P < 0.05). The analysis of phosphorylation levels revealed that VEGF stimulation significantly activated phosphorylation of FAK, AKT, and P38, with phosphorylation levels increasing with VEGF concentrations (P < 0.05). The VEGF/VEGFR2 signaling axis regulated the migration ability of HDPSCs through the FAK/PI3K/AKT and P38MAPK pathways. This finding highlighted not only the crucial role of VEGF in injury repair of HDPSCs but also provided important clues for a comprehensive understanding of the potential applications of this signaling axis in dental regenerative medicine.

Molecular docking aided machine learning for the identification of potential VEGFR inhibitors against renal cell carcinoma.

Renal cell carcinoma is a highly vascular tumor associated with vascular endothelial growth factor (VEGF) expression. The Vascular Endothelial Growth Factor -2 (VEGF-2) and its receptor was identified as a potential anti-cancer target, and it plays a crucial role in physiology as well as pathology. Inhibition of angiogenesis via blocking the signaling pathway is considered an attractive target. In the present study, 150 FDA-approved drugs have been screened using the concept of drug repurposing against VEGFR-2 by employing the molecular docking, molecular dynamics, grouping data with Machine Learning algorithms, and density functional theory (DFT) approaches. The identified compounds such as Pazopanib, Atogepant, Drosperinone, Revefenacin and Zanubrutinib shown the binding energy - 7.0 to - 9.5 kcal/mol against VEGF receptor in the molecular docking studies and have been observed as stable in the molecular dynamic simulations performed for the period of 500 ns. The MM/GBSA analysis shows that the value ranging from - 44.816 to - 82.582 kcal/mol. Harnessing the machine learning approaches revealed that clustering with K = 10 exhibits the relevance through high binding energy and satisfactory logP values, setting them apart from compounds in distinct clusters. Therefore, the identified compounds are found to be potential to inhibit the VEGFR-2 and the present study will be a benchmark to validate the compounds experimentally.

Targeted Delivery of Diphtheria Toxin into VEGFR1/VEGFR2 Overexpressing Cells Induces Anti-angiogenesis Activity.

BACKGROUND: Vascular Endothelial Growth Factor Receptors (VEGFR1 and VEGFR2) are tyrosine kinase receptors expressed on endothelial cells and tumor vessels and play an important role in angiogenesis. In this study, three repeats of VEGFR1 and VEGFR2 binding peptide (VGB3) were genetically fused to the truncated diphtheria toxin (TDT), and its in vitro activity was evaluated. METHODS: The recombinant construct (TDT-triVGB3) was expressed in bacteria cells and purified with nickel affinity chromatography. The binding capacity and affinity of TDT-triVGB3 were evaluated using the enzyme-linked immunosorbent assay. The inhibitory activity of TDT-triVGB3 on viability, migration, and tube formation of human endothelial cells was evaluated using MTT, migration, and tube formation assays. RESULTS: TDT-triVGB3 selectively detected VEGFR1 and VEGFR2 with high affinity in an enzyme- linked immunosorbent assay and significantly inhibited viability, migration, and tube formation of human endothelial cells. CONCLUSION: The developed TDT-triVGB3 is potentially a novel agent for targeting VEGFR1/ VEGFR2 over-expressing cancer cells.