Mechanotransduction via endothelial adhesion molecule CD31 initiates transmigration and reveals a role for VEGFR2 in diapedesis.

Engagement of platelet endothelial cell adhesion molecule 1 (PECAM, PECAM-1, CD31) on the leukocyte pseudopod with PECAM at the endothelial cell border initiates transendothelial migration (TEM, diapedesis). We show, using fluorescence lifetime imaging microscopy (FLIM), that physical traction on endothelial PECAM during TEM initiated the endothelial signaling pathway. In this role, endothelial PECAM acted as part of a mechanotransduction complex with VE-cadherin and vascular endothelial growth factor receptor 2 (VEGFR2), and this predicted that VEGFR2 was required for efficient TEM. We show that TEM required both VEGFR2 and the ability of its Y1175 to be phosphorylated, but not VEGF or VEGFR2 endogenous kinase activity. Using inducible endothelial-specific VEGFR2-deficient mice, we show in three mouse models of inflammation that the absence of endothelial VEGFR2 significantly (by ≥75%) reduced neutrophil extravasation by selectively blocking diapedesis. These findings provide a more complete understanding of the process of transmigration and identify several potential anti-inflammatory targets.

AMG232 inhibits angiogenesis in glioma through the p53-RBM4-VEGFR2 pathway.

AMG232 effectively inhibits cancers with wild-type p53 (also known as TP53) by reactivating p53, but whether it inhibits glioma angiogenesis remains unclear. This study confirms that AMG232 inhibits the proliferation of glioma endothelial cells (GECs) in a dose-dependent manner and inhibits the angiogenesis of GECs. p53 and RNA-binding motif protein 4 (RBM4) were expressed at low levels in GECs, while MDM2 and vascular endothelial growth factor receptor 2 (VEGFR2, also known as KDR) were highly expressed. In vitro and in vivo experiments confirmed that AMG232 upregulated p53 and RBM4, and downregulated MDM2 and VEGFR2 by blocking the MDM2-p53 interaction. Both p53 silencing and RBM4 silencing significantly upregulated the expression of VEGFR2, promoted the proliferation, migration and tube formation of GECs, and reversed the effects of AMG232 on downregulating VEGFR2 and inhibiting the angiogenesis of GECs. AMG232 increased RBM4 expression by upregulating p53, and p53 bound to RBM4 and promoted its transcription. RBM4 bound to and shortened the half-life of VEGFR2, promoting its degradation. Finally, AMG232 produced a significant decrease in new vessels and hemoglobin content in vivo. This study proves that AMG232 inhibits glioma angiogenesis by blocking the MDM2-p53 interaction, in which the p53-RBM4-VEGFR2 pathway plays an important role.

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.

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.

Vasorin promotes endothelial differentiation of glioma stem cells via stimulating the transcription of VEGFR2.

Gliomas are highly vascularized malignancies, but current anti-angiogenic treatments have not demonstrated practical improvements in patient survival. Studies have suggested that glioma-derived endothelial cell (GdEC) formed by glioma stem cell (GSC) differentiation may contribute to the failure of this treatment. However, the molecular mechanisms involved in GSC endothelial differentiation remain poorly understood. We previously reported that vasorin (VASN) is highly expressed in glioma and promotes angiogenesis. Here, we show that VASN expression positively correlates with GdEC signatures in glioma patients. VASN promotes the endothelial differentiation capacity of GSC in vitro and participates in the formation of GSC-derived vessels in vivo. Mechanistically, vascular endothelial growth factor receptor 2 (VEGFR2) is a critical factor that mediates the regulation of VASN on GSC endothelial differentiation. Separation of cell chromatin fractionation and chromatin immunoprecipitation-sequencing analysis show that VASN interacts with Notch1 and co-translocates into the cell nuclei, where VASN binds to the VEGFR2 gene promoter to stimulate its transcription during the progression of GSC differentiation into GdEC. Together, these findings elucidate the role and mechanisms of VASN in promoting the endothelial differentiation of GSC and suggest VASN as a potential target for anti-angiogenic therapy based on intervention in GdEC formation in gliomas.

DAB2IP associates with hereditary angioedema: Insights into the role of VEGF signaling in HAE pathophysiology.

BACKGROUND: In the recent years, there was an important improvement in the understanding of the pathogenesis of hereditary angioedema (HAE). Notwithstanding, in a large portion of patients with unknown mutation (HAE-UNK) the genetic cause remains to be identified. OBJECTIVES: To identify new genetic targets associated with HAE, a large Argentine family with HAE-UNK spanning 3 generations was studied. METHODS: Whole exome sequencing was performed on affected family members to identify potential genetic variants associated with HAE-UNK. In silico analyses and experimental studies were applied to assess the role of the identified gene variant. RESULTS: A missense variant (p.D239N) in DAB2IP was identified. The variant occurred in the C2-domain, the region interacting with vascular endothelial growth factor receptor 2 (VEGFR2). It was found to be rare, and predicted to have a detrimental effect on the functionality of DAB2IP. Protein structure modeling predicted changes in the mutant p.D239N protein structure, impacting protein stability. The p.D239N variant affected the subcellular localization of VEGFR2. Cells transfected with the DAB2IP-239N transcript exhibited an intracellular distribution, and VEGFR2 remained associated with the cell membrane. The altered localization pattern indicated reduced colocalization of the mutant protein with VEGFR2, suggesting a diminished ability of VEGFR2 binding. CONCLUSIONS: The study identified a novel missense variant (p.D239N) in DAB2IP in a family with HAE-UNK and highlighted the role of dysregulated VEGF-mediated signaling in altered endothelial permeability. DAB2IP loss-of-function pathogenic variants lead to the impairment of the endothelial VEGF/VEGFR2 ligand system and represent a new pathophysiologic cause of HAE-UNK.

Dual drug-loaded polymeric mixed micelles for ovarian cancer: Approach to enhanced therapeutic efficacy of albendazole and paclitaxel.

Chemotherapy resistance remains a significant challenge in treating ovarian cancer effectively. This study addresses this issue by utilizing a dual drug-loaded nanomicelle system comprising albendazole (ABZ) and paclitaxel (PTX), encapsulated in a novel carrier matrix of D-tocopheryl polyethylene glycol 1000 succinate vitamin E (TPGS), soluplus and folic acid. Our objective was to develop and optimize this nanoparticulate delivery system using solvent evaporation techniques to enhance the therapeutic efficacy against ovarian cancer. The formulation process involved pre-formulation, formulation, optimization, and comprehensive characterization of the micelles. Optimization was conducted through a 32 factorial design, focusing on the effects of polymer ratios on particle size, zeta potential, polydispersity index (PDI) and entrapment efficiency (%EE). The optimal formulation demonstrated improved dilution stability, as indicated by a critical micelle concentration (CMC) of 0.0015 mg/mL for the TPGS-folic acid conjugate (TPGS-FOL). Extensive characterization included differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR), and Fourier-transform infrared spectroscopy (FTIR). The release profile exhibited an initial burst followed by sustained release over 90 h. The cytotoxic potential of the formulated micelles was superior to that of the drugs alone, as assessed by MTT assays on SKOV3 ovarian cell lines. Additionally, in vivo studies confirmed the presence of both drugs in plasma and tumour tissues, suggesting effective targeting and penetration. In conclusion, the developed TPGS-Fol-based nanomicelles for co-delivering ABZ and PTX show promising results in overcoming drug resistance, enhancing solubility, sustaining drug release, and improving therapeutic outcomes in ovarian cancer treatment.

Efficacy of ramucirumab combination chemotherapy as second-line treatment in patients with advanced adenocarcinoma of the stomach or gastroesophageal junction after exposure to checkpoint inhibitors and chemotherapy as first-line therapy.

FOLFOX plus nivolumab represents a standard of care for first-line therapy of advanced gastroesophageal cancer (aGEC) with positive PD-L1 expression. The efficacy of second-line VEGFR-2 inhibition with ramucirumab (RAM) plus chemotherapy after progression to immunochemotherapy remains unclear. Medical records of patients with aGEC enrolled in the randomized phase II AIO-STO-0417 trial after treatment failure to first-line FOLFOX plus nivolumab and ipilimumab were retrospectively analyzed. Patients were divided into two groups based on second-line therapy: RAM plus chemotherapy (RAM group) or treatment without RAM (control group). Eighty three patients were included. In the overall population, progression-free survival (PFS) in the RAM group was superior to the control (4.5 vs 2.9 months). Responders (CR/PR) to first-line immunochemotherapy receiving RAM containing second-line therapy had prolonged OS from start of first-line therapy (28.9 vs 16.5 months), as well as second-line OS (9.6 vs 7.5 months), PFS (5.6 vs 2.9 months) and DCR (53% vs 29%) compared to the control. PD-L1 CPS ≥1 was 42% and 44% for the RAM and the control, respectively. Patients with CPS ≥1 in the RAM group showed better tumor control (ORR 25% vs 10%) and improved survival (total OS 11.5 vs 8.0 months; second-line OS 6.5 vs 3.9 months; PFS 4.5 vs 1.6 months) compared to the control. Prior exposure to first-line FOLFOX plus dual checkpoint inhibition followed by RAM plus chemotherapy shows favorable response and survival rates especially in patients with initial response and positive PD-L1 expression and has the potential to advance the treatment paradigm in aGEC.

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.

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.

VEGFR affects miR-3200-3p-mediated regulatory T cell senescence in tumour-derived exosomes in non-small cell lung cancer.

Numerous studies have demonstrated that regulatory T (Treg) cells play an important role in the tumour microenvironment (TME). The aim of this study was to investigate whether VEGFR2 affects the expression of miR-3200-3p in exosomes secreted by tumour cells, thereby influencing Treg senescence in the TME. The results showed that VEGFR2 expression level was the highest in Calu-1 cells, and after transfection with si-VEGFR2, the exosomes secreted from Calu-1 cells were extracted and characterised with no significant difference from the exosomes of the untransfected group, but the expression of miR-3200-3p in the exosomes of the transfected si-VEGFR2 group was elevated. The Cell Counting Kit-8 (CCK-8) and flow cytometry (FCM) results suggested that exosomes highly expressing miR-3200-3p could inhibit Treg cell viability and promote apoptosis levels when treated with Treg cells. Detection of the senescence-associated proteins p16 INK4A and MMP3 by western blot (WB) revealed that exosomes highly expressing miR-3200-3p were able to elevate their protein expression levels. Tumour xenograft experiments demonstrated that exosomes with high miR-3200-3p expression promoted Treg cell senescence and inhibited subcutaneous tumour growth in nude mice. Dual-luciferase reporter assays and RNA pull-down assays showed that miR-3200-3p could be linked with DDB1. Overexpression of DDB1 reverses changes in DCAF1/GSTP1/ROS protein expression caused by exosomes with high miR-3200-3p expression. In conclusion, inhibition of VEGFR2 expression in tumour cells promotes the expression of miR-3200-3p in exosomes secreted by tumour cells. miR-3200-3p enters the TME through exosomes and acts on DDB1 in Treg cells to promote senescence of Treg cells to inhibit tumour progression.

Suppression of retinal neovascularization by intravitreal injection of cryptotanshinone.

Neovascular eye diseases, including proliferative diabetic retinopathy and retinopathy of prematurity, is a major cause of blindness. Laser ablation and intravitreal anti-VEGF injection have shown their limitations in treatment of retinal neovascularization. Identification of a new therapeutic strategies is in urgent need. Our study aims to assess the effects of Cryptotanshinone (CPT), a natural compound derived from Salvia miltiorrhiza Bunge, in retina neovascularization and explore its potential mechanism. Our study demonstrated that CPT did not cause retina tissue toxicity at the tested concentrations. Intravitreal injections of CPT reduced pathological angiogenesis and promoted physical angiogenesis in oxygen-induced retinopathy (OIR) model. CPT improve visual function in OIR mice and reduced cell apoptosis. Moreover, we also revealed that CPT diminishes the expression of inflammatory cytokines in the OIR retina. In vitro, the administration of CPT effectively inhibited endothelial cells proliferation, migration, sprouting, and tube formation induced by the stimulation of human retinal vascular endothelial cells (HRVECs) with VEGF165. Mechanistically, CPT blocking the phosphorylation of VEGFR2 and downstream targeting pathway. After all, the findings demonstrated that CPT exhibits potent anti-angiogenic and anti-inflammatory effects in OIR mice, and it has therapeutic potential for the treatment of neovascular retinal diseases.

Polyene phosphatidylcholine promotes tibial fracture healing in rats by stimulating angiogenesis dominated by the VEGFA/VEGFR2 signaling pathway.

One of the factors that predispose to fractures is liver damage. Interestingly, fractures are sometimes accompanied by abnormal liver function. Polyene phosphatidylcholine (PPC) is an important liver repair drug. We wondered if PPC had a role in promoting fracture healing. A rat model of tibial fracture was developed using the modified Einhorn model method. X-rays were used to detect the progression of fracture healing. Progress of ossification and angiogenesis at the fracture site were analyzed by Safranin O/fast green staining and CD31 immunohistochemistry. To investigate whether PPC has a direct angiogenesis effect, HUVECs were used. We performed MTT, wound healing, Transwell migration, and tube formation assays. Finally, RT-qPCR and Western blot analysis were used to study the underlying mechanism. The results showed that PPC significantly shortened the apparent recovery time of mobility in rats. PPC treatment significantly promoted the formation of cartilage callus, endochondral ossification, and angiogenesis at the fracture site. In vitro, PPC promoted the proliferative viability of HUVECs, their ability to heal wounds, and their ability to penetrate membranes in the Transwell apparatus and increased the tube formation of cells. The transcription of VEGFA, VEGFR2, PLCγ, RAS, ERK1/2 and MEK1/2 was significantly up regulated by PPC. Further, the protein level results demonstrated a significant increase in the expression of VEGFA, VEGFR2, MEK1/2, and ERK1/2 proteins. In conclusion, our findings suggest that PPC promotes angiogenesis by activating the VEGFA/VEGFR2 and downstream signaling pathway, thereby accelerating fracture healing.

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.

Tuning spacer length improves the functionality of the nanobody-based VEGFR2 CAR T cell.

BACKGROUND: The chimeric antigen receptor-expressing T (CAR-T) cells for cancer immunotherapy have obtained considerable clinical importance. CAR T cells need an optimized intracellular signaling domain to get appropriately activated and also for the proper antigen recognition, the length and composition of the extracellular spacer are critical factors. RESULTS: We constructed two third-generation nanobody-based VEGFR2-CARs containing either IgG1 hinge-CH2-CH3 region or hinge-only as long or short extracellular spacers, respectively. Both CARs also contained intracellular activating domains of CD28, OX40, and CD3ζ. The T cells from healthy individuals were transduced efficiently with the two CARs, and showed increased secretion of IL-2 and IFN-γ cytokines, and also CD69 and CD25 activation markers along with cytolytic activity after encountering VEGFR2+ cells. The VEGFR2-CAR T cells harboring the long spacer showed higher cytokine release and CD69 and CD25 expression in addition to a more efficient cytolytic effect on VEGFR2+ target cells. CONCLUSIONS: The results demonstrated that the third-generation anti-VEGFR2 nanobody-based CAR T cell with a long spacer had a superior function and potentially could be a better candidate for solid tumor treatment.

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.

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.

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 (variable new antigen receptor, 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 C. 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.

Inhibition of soluble epoxide hydrolase enhances the dentin-pulp complex regeneration mediated by crosstalk between vascular endothelial cells and dental pulp stem cells.

BACKGROUND: Revascularization and restoration of normal pulp-dentin complex are important for tissue-engineered pulp regeneration. Recently, a unique periodontal tip-like endothelial cells subtype (POTCs) specialized to dentinogenesis was identified. We have confirmed that TPPU, a soluble epoxide hydrolase (sEH) inhibitor targeting epoxyeicosatrienoic acids (EETs) metabolism, promotes bone growth and regeneration by angiogenesis and osteogenesis coupling. We hypothesized that TPPU could also promote revascularization and induce POTCs to contribute to pulp-dentin complex regeneration. Here, we in vitro and in vivo characterized the potential effect of TPPU on the coupling of angiogenesis and odontogenesis and investigated the relevant mechanism, providing new ideas for pulp-dentin regeneration by targeting sEH. METHODS: In vitro effects of TPPU on the proliferation, migration, and angiogenesis of dental pulp stem cells (DPSCs), human umbilical vein endothelial cells (HUVECs) and cocultured DPSCs and HUVECs were detected using cell counting kit 8 (CCK8) assay, wound healing, transwell, tube formation and RT-qPCR. In vivo, Matrigel plug assay was performed to outline the roles of TPPU in revascularization and survival of grafts. Then we characterized the VEGFR2 + POTCs around odontoblast layer in the molar of pups from C57BL/6 female mice gavaged with TPPU. Finally, the root segments with DPSCs mixed with Matrigel were implanted subcutaneously in BALB/c nude mice treated with TPPU and the root grafts were isolated for histological staining. RESULTS: In vitro, TPPU significantly promoted the migration and tube formation capability of cocultured DPSCs and HUVECs. ALP and ARS staining and RT-qPCR showed that TPPU promoted the osteogenic and odontogenic differentiation of cultured cells, treatment with an anti-TGF-ß blocking antibody abrogated this effect. Knockdown of HIF-1α in HUVECs significantly reversed the effect of TPPU on the expression of angiogenesis, osteogenesis and odontogenesis-related genes in cocultured cells. Matrigel plug assay showed that TPPU increased VEGF/VEGFR2-expressed cells in transplanted grafts. TPPU contributed to angiogenic-odontogenic coupling featured by increased VEGFR2 + POTCs and odontoblast maturation during early dentinogenesis in molar of newborn pups from C57BL/6 female mice gavaged with TPPU. TPPU induced more dental pulp-like tissue with more vessels and collagen fibers in transplanted root segment. CONCLUSIONS: TPPU promotes revascularization of dental pulp regeneration by enhancing migration and angiogenesis of HUVECs, and improves odontogenic differentiation of DPSCs by TGF-ß. TPPU boosts the angiogenic-odontogenic coupling by enhancing VEGFR2 + POTCs meditated odontoblast maturation partly via upregulating HIF-1α, which contributes to increasing pulp-dentin complex for tissue-engineered pulp regeneration.

Tanshinone IIA attenuates osteoarthritis via inhibiting aberrant angiogenesis in subchondral bone.

Excessive angiogenesis in subchondral bone is a pathological feature of osteoarthritis (OA). Tanshinone IIA (TIIA), an active compound found in Salvia miltiorrhiza, demonstrates significant anti-angiogenic properties. However, the effect of TIIA on abnormal subchondral angiogenesis in OA is still unclear. This study aims to investigate the mechanism of TIIA in modulating subchondral bone angiogenesis during OA and assess its therapeutic potential in OA. Our findings demonstrate that TIIA attenuated articular cartilage degeneration, normalized subchondral bone remodeling, and effectively suppressed aberrant angiogenesis within subchondral bone in monosodium iodoacetate (MIA)-induced OA mice. Additionally, the angiogenesis capacity of primary CD31hiEmcnhi endothelial cells was observed to be significantly reduced after treatment with TIIA in vitro. Mechanically, TIIA diminished the proportion of hypertrophic chondrocytes, ultimately leading to a substantial reduction in the secretion of vascular endothelial growth factor A (VEGFA). The supernatant of hypertrophic chondrocytes promoted the tube formation of CD31hiEMCNhi endothelial cells, whereas TIIA inhibited this process. Furthermore, TIIA effectively suppressed the expression of vascular endothelial growth factor receptor 2 (VEGFR2) along with its downstream MAPK pathway in CD31hiEmcnhi endothelial cells. In conclusion, our data indicated that TIIA could effectively inhibit the abnormal angiogenesis in subchondral bone during the progression of OA by suppressing the VEGFA/VEFGR2/MAPK pathway. These findings significantly contribute to our understanding of the abnormal angiogenesis in OA and offer a promising therapeutic target for OA treatment.