Vascular Endothelial Growth Factor Receptor Inhibitor Research Articles

(E)-N-(3-(1-(2-(4-(2,2,2-Trifluoroacetamido)benzoyl)hydrazono)ethyl)phenyl)nicotinamide: A Novel Pyridine Derivative for Inhibiting Vascular Endothelial Growth Factor Receptor-2: Synthesis, Computational, and Anticancer Studies.

(E)-N-(3-(1-(2-(4-(2,2,2-Trifluoroacetamido)benzoyl)hydrazono)ethyl)phenyl)nicotinamide (compound 10) was designed as an antiangiogenic VEGFR-2 inhibitor with the essential pharmacophoric structural properties to interact with the catalytic pocket of VEGFR-2. The designed derivative was synthesized, and its structure was confirmed through Ms, elemental, 1H, and 13C spectral data. The potentiality of the designed pyridine derivative to bind with and inhibit the vascular endothelial growth factor receptor-2 (VEGFR-2) enzyme was indicated by molecular docking assessments. In addition, six molecular dynamic (MD) experiments proved its correct binding with VEGFR-2 over 100 ns. Additionally, the molecular mechanics energies, combined with the generalized born and surface area (MM-GBSA) analysis, identified the precise binding with optimum energy. To explore the stability and reactivity of the designed pyridine derivative, density functional theory (DFT) calculations, including electrostatic potential maps and total electron density, were carried out. Additionally, the absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis demonstrated its general likeness and its safety. The designed compound was synthesized to evaluate its effects against VEGFR-2 protein, cancer, and normal cells. The in vitro results were concordant with the in silico results, because the new pyridine derivative (compound 10) displayed VEGFR-2 inhibition with an IC50 value of 65 nM and displayed potent cytotoxic properties against hepatic (HepG2) and breast (MCF-7) cancer cell lines with IC50 values of 21.00 and 26.10 μM, respectively; additionally, it exhibited high selectivity indices against the normal cell lines (W-38) of 1.55 and 1.25, respectively. The obtained results present compound 10 as a new lead VEGFR-2 inhibitor for further biological investigation and chemical modifications.

Novel pyrazolo[3,4-d]pyrimidines as potential anticancer agents: Synthesis, VEGFR-2 inhibition, and mechanisms of action

Novel pyrazolo[3,4-d] pyrimidine derivatives bearing carbon-aryl(heteryl)idene moieties were synthesized via a condensation reaction of 5-aminopyrazoles and cyclic lactams. The preparation of the target compounds employed bioisosterism, where a pyrazole ring was a major replacement. Fifteen target compounds were investigated for their antiproliferative activity on five human cancer cell lines; derivative (E)− 1-methyl-9-(3,4,5-trimethoxybenzylidene)− 6,7,8,9-tetrahydropyrazolo[3,4-d]pyrido[1,2-a]pyrimidin-4(1H)-one (10k) showed the highest activity (IC50 value (0.03–1.6 μM), on selected cell lines. Results of an in vivo experiment on an HT-29 xenograft nude mouse model also confirmed that 10k inhibited tumor growth. The proposed anticancer mechanism of 10k in HT-29 and HCT-116 cells was that 10k caused G2/M phase arrest in cancer cells and decreased the mitochondrial membrane potential (Δψmt). Additional studies were conducted on HUVEC, where 10k significantly inhibited HUVEC cell migration, adhesion, and tube formation activity. Molecular modeling studies revealed that 10k forms hydrogen bonds with cys-919 of vascular endothelial growth factor receptor 2 (VEGFR-2) and inhibit VEGFR-2 kinase activity. Moreover, tubulin polymerization assay results showed that 10k formed hydrogen bonds with Asn-101 and Gln-11 of tubulin. Furthermore, it could change the aberration of microtubule arrangements in HUVEC and inhibit tubulin polymerization. These results indicate that the main anticancer activity of 10k may be mediated by anti-vascular effects and inhibition of tubulin polymerization in pre-clinical trials.

Effect of triamcinolone acetonide on retinal inflammation and angiogenesis induced by pericyte depletion in mouse

Triamcinolone acetonide (TA) has been shown to improve morphological and functional outcome in diabetic macular edema (DME) patients. However, the functional mechanism of TA has not been elucidated yet. In this study we investigated the detailed functional mechanism of TA using culture cells and retinopathy mouse models in which retinal inflammation and abnormal angiogenesis were induced by pericyte depletion. TA significantly prevented retinal hemorrhage, edema and partially improved abnormal angiogenesis. TA decreased retinal vascular endothelial growth factor (VEGF) concentration, presumably by preventing recruitment of macrophages into retina and TA also inhibited expression of inflammatory cytokines in retina. TA inhibited proliferation/migration of vascular endothelial cells and vessel sprouting. No direct inhibition of VEGF receptor 2 (VEGFR2) autophosphorylation was observed by TA. These results suggested that TA improved inflammatory retinal events which were induced in pericyte-deleted mice by mainly decreasing macrophage-derived VEGF and expression of inflammatory cytokines followed by attenuation of vascular permeability and proliferation/migration of endothelial cells. Furthermore, in these processes, translocation of glucocorticoid receptor (GR) was partially involved.

Repurposing Lenvatinib as A Potential Therapeutic Agent against Thyroid Eye Disease by Suppressing Adipogenesis in Orbital Adipose Tissues.

Thyroid eye disease (TED) is the most common orbital disease in adults. Targeting expanded orbital adipose tissue (OAT) removed by surgery has therapeutic potential. However, drugs targeting OAT are unavailable because of the lack of deciphering features of OAT. Here, we aimed to investigate the mechanism underlying OAT expansion and identify a drug targeting OAT in TED. We found an increasing number of adipocytes with smaller size in TED-derived OATs as compared with controls, indicating that hyperplasia rather than hypertrophy contributed to OAT enlargement in TED. Typically smaller-sized adipocytes in TED patient-derived OATs were noted to localize surrounding vessels. RNA sequencing revealed enriched vascular endothelial growth factor receptor (VEGFR) genes in adipocytes differentiated from preadipocytes of TED-derived stromal vascular fraction (SVF). Similarly, OATs in patients with TED also expressed a higher level of VEGFR-1 and -2. We induced adipogenesis in TED-derived SVF with or without Lenvatinib, an FDA-approved small-molecule VEGFR inhibitor. Lenvatinib significantly suppressed lipid accumulation in a dose-dependent manner. In conclusion, our study revealed the potential anti-adipogenic effect of Lenvatinib on the OAT of TED-affected patients. In addition to proposing a drug for TED treatment, this study shows the therapeutic potential of anti-adipogenesis drugs targeting the VEGF pathway.

Synthesis,characterization and biological activities of nitrogen-containing Combretastatin A-4 derivatives

A series of new combretastatin A4 derivatives (6a-6c, 9a-12c) containing nitrogen atoms and nitrogen heterocycles were synthesized by condensation reaction, reduction reaction, hydrolysis reaction and Crutius rearrangement, and the structures were confirmed by 1H NMR, 13C NMR and Mass spectrum. The antitumor cytotoxicity of a series of newly synthesized compounds were evaluated in vitro toward the cell line of hepatoma cell with erianin as positive control. Amoung all the compound, 6b exhibit the most antitumor activity against HepG2 (Human hepatoellular carcinomas) cell line with the inhibition rate 93.85%, and the other compounds also showed moderate to potent antitumor activity. To understand the interaction of series with active binding site of receptor, docking study was performed with VEGFR-2 (Vascular Endothelial Growth Factor Receptor 2) kinase domain in complex with axitinib using Sybyl. The compound with the highest total score was 9c, reaching 8.92, which was higher than that of the positive control erianin This research highlights the therapeutic potential of novel VEGFR-2 inhibitors in treating cancers and provides a promising strategy for drug discovery. Graphical abstract

Focused ultrasound stimulation of the inflammatory reflex at the spleen ameliorates pulmonary arterial hypertension in rodents

Abstract Background Inflammation is a major contributor in pulmonary arterial hypertension (PAH) pathogenesis. Non-invasive, focused ultrasound stimulation (FUS) of the spleen activates the neuroimmune inflammatory reflex (IR) and suppresses systemic inflammation [1]. Purpose We aimed to explore whether daily FUS of the spleen improves haemodynamics and biomarkers by modulating the IR, in a rat model of PAH. Methods Sprague-Dawley rats (n=12) were injected s.c. with Sugen5416 (VEGF receptor inhibitor) and then, were placed in a hypoxic chamber (FiO2=10%) for 21 days, followed by 14 days of re-exposure to normoxia (FiO2=10%). At day 21, rats were randomized to either FUS (n=6) or sham-stimulation of the spleen (n=6). Each FUS- or sham-stimulation session consisted of 12 minutes. After 14 days of treatment, in a terminal experiment, right ventricular systolic pressure (RVSP) and arterial pressure were measured invasively, as well as biomarkers, in each animal (Figure 1A). Results FUS significantly reduced RVSP compared with the sham-stimulation (Mean±SEM, 50.83±3.57 mmHg vs 72.50±5.81; p=0.009), resulting in a 30% relative reduction (Figure 1B). Mean systemic arterial pressure was similar in the 2 groups (Mean±SEM, 79.83±3.73 mmHg vs. 87.00±2.95; p=0.137) (Figure 1C), as was the change in heart rate between day 1 and day 14 of treatment period (−19.53±5.36% vs. −16.79±3.1, p=0.61) (Figure 1D). Consistently, plasma brain natriuretic peptide (BNP) levels are reduced in the FUS group (119.45±19.93 ng/μl vs. 319.39±91.85; p=0.019), indicative of reduced myocardial wall stress in the FUS group (Figure 1E). Conclusion Non-invasive FUS of the spleen reduced RVSP by ∼30% and BNP without significantly affecting systemic pressure or heart rate, in rats with severe PAH. Non-invasive FUS, by modulating the IR, may exert an anti-inflammatory effect in PAH. FUS of the spleen is noninvasive, safe, widely available and easy to perform, and should be further explored as a possible therapeutic option in PAH. Funding Acknowledgement Type of funding sources: Private hospital(s). Main funding source(s): Northwell Health

Engineering placenta-like organoids containing endogenous vascular cells from human-induced pluripotent stem cells.

The placenta is an essential organ that maintains the health of both the fetus and its mother. Understanding the development of human placenta has been hindered by the limitations of existing animal models and monolayer cell cultures. Models that can recapitulate the essential aspects of human placental multicellular components and vasculature are still lacking. Herein, we presented a new strategy to establish placenta-like organoids with vascular-like structures from human-induced pluripotent stem cells in a defined three-dimensional (3D) culture system. The resulting placenta-like tissue resembles first-trimester human placental development in terms of complex placental components and secretory function. The multicellular tissue was characterized by the inclusion of trophoblasts (cytotrophoblasts, syncytiotrophoblasts, extravillous trophoblasts, and other endogenous vascular cells), which were identified by immunofluorescence, flow cytometry analyses, real-time quantitative reverse transcription polymerase chain reaction and single-cell RNA-seq. Moreover, the 3D tissue was able to secrete the placenta-specific hormone human chorionic gonadotropin β (hCG-β)and vascular endothelial growth factor A (VEGFA). The tissue responded to the inflammatory factor tumor necrosis factor-α (TNF-α)and VEGF receptor inhibitors. This new model system can represent the major features of placental cellular components, and function, which have not been realized in 2D monolayer cultures. The developed tissue system might open new avenues for studying normal early human placental development and its disease states.

Combination of apatinib with apo-IDO1 inhibitor for the treatment of colorectal cancer

Colorectal cancer (CRC) is the third most common cancer in the world. Recently, many clinical studies have demonstrated the therapeutic potential of immune checkpoint therapy combined with inhibitors of vascular endothelial growth factor receptor 2 (VEGFR2) in colon cancer. Compound B37, identified in our previous experiment, is an apo-form indoleamine-2,3-dioxygenase 1 inhibitor (apo-IDO1 inhibitor), which has been shown to significantly suppress tumor growth combined with an anti-PD1 antibody. We speculated whether this apo-IDO inhibitor (B37) combined with a VEGFR2 inhibitor (apatinib) would further improve its anti-tumor activity. Therefore, a syngeneic mouse colon cancer model (mouse colon cancer cell line CT26) was established to investigate the anti-tumor activity of B37 combined with apatinib. As expected, the combination of B37 and apatinib (VEGFR2 inhibitor) improved the therapeutic effect compared with apo-IDO1 inhibitor and apatinib monotherapy, as shown by the reduced growth of transplanted tumors, weakened proliferation, and increased apoptosis of cancer cells. Specifically, there was a 24.8% reduction in tumor volume using apatinib and 31.3% reduction using B37. The combination-treated group showed remarkable inhibition of tumor growth (52.2%). For tumor weight, there was a 29.2% reduction in the apatinib-treated group and 35.0% reduction in the B37-treated group. The combination-treated group showed a 56.3% reduction. Moreover, the combination therapy reprogrammed the immune microenvironment by increasing infiltration of CD4+ and CD8+ T cells, decreasing the ratio of regulatory T cells, and promoting the killing ability of T cells manifested by elevated expression of IFN-γ and granzyme B in the combination-treated group. Our study indicates that the combination of apo-IDO1 inhibitor and apatinib is a promising strategy for CRC therapy.

Paeoniflorin binds to VEGFR2 to restore autophagy and inhibit apoptosis for podocyte protection in diabetic kidney disease through PI3K-AKT signaling pathway

Ethnopharmacological relevancePaeoniflorin (PF) was found to exhibit renal protection from diabetic kidney disease (DKD) in previous trials, but its specific mechanism remains to be elucidated. Aim of the studyThis study furtherly explored the specific mechanism of PF in protect podocyte injury in DKD. Materials and methodsWe observed the effects of PF on renal tissue and podocytes in DKD by constructing the vitro and vivo models after measuring the pharmacokinetic characteristics of PF. Target proteins of PF were found through target prediction, and verified by molecular docking, CESTA, and SPR, and then furtherly explored the downstream regulation mechanism related to podocyte autophagy and apoptosis by network prediction and co-immunoprecipitation. Finally, by using the target protein inhibitor in vivo and knocking down the target protein gene in vitro, it was verified that PF played a role in regulating autophagy and apoptosis through the target protein in diabetic nephropathy. ResultsThis study found that in STZ-induced mice model, PF could improve the renal biochemical and pathological damage and podocyte injure (p < 0.05), upregulate autophagy activity (p < 0.05), but inhibit apoptosis (p < 0.01). Vascular endothelial growth factor receptor 2 (VEGFR2), predicted as the target of PF, directly bind with PF reflected by molecular docking and surface plasmon resonance detection. Animal studies demonstrated that VEGFR2 inhibitors have a protective effect similar to that of PF on DKD. Network prediction and co-immunoprecipitation further confirmed that VEGFR2 was able to bind PIK3CA to regulate PI3K-AKT signaling pathway. Furthermore, PF downregulated the phosphorylation of PI3K and AKT (p < 0.05). In vitro, similarly to autophagy inhibitors, PF was also found to improve podocyte markers (p < 0.05) and autophagy activity (p < 0.05), decrease caspase 3 protein (p < 0.05) and further inhibited VEGFR2-PI3K-AKT activity (p < 0.05). Finally, the results of VEGFR2 knockdown were similar to the effect of PF in HG-stimulated podocytes. ConclusionIn conclusion, PF restores autophagy and inhibits apoptosis by targeting the VEGFR2-mediated PI3K-AKT pathway to improve renal injury in DKD, that provided a theoretical basis for PF treatment in DKD.

The crosstalk between lung cancer cells and platelets promotes tumor angiogenesis in vivo and in vitro.

We previously showed that the crosstalk of H1975 cells and platelets (PLTs) may promote tumor angiogenesis. This study aimed to determine whether other lung cell lines (LC) interacting with PLTs could affect tumor angiogenesis through in vivo and in vitro experiments. Cell Counting Kit-8, EdU cell proliferation, wound healing, Transwell invasion, F-actin staining, tube formation, ELISA and western blot assays were performed to investigate the properties and the expression levels of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), p-VEGFR2, PI3K, p-PI3K, Akt, p-Akt and eNOS in supernatants or HUVECs. Then, using mouse models, immunohistochemistry was applied to detect the expression levels of CD31 and VEGF. Compared with single-cultured HUVECs (EC) or incubation with either LC supernatant (EC + LC) or activated PLT supernatant (EC + PLT), incubation with SN_LCP (supernatant derived from LC cocultured with PLT, named the EC + LC + PLT group) improved the viability, proliferation, migration, invasion, and tube formation activities of HUVECs and the expression of F-actin, VEGF, VEGFR2, p-VEGFR2, p-PI3K, p-Akt and eNOS in HUVECs. Higher expression levels of CD31 and VEGF were found in the LLC + PLT (mouse model inoculated with Lewis lung cancer (LLC) cells cocultured with PLTs) group than in the LLC (mouse model inoculated with LLC cells alone) group. However, the increased angiogenic properties of HUVECs were inhibited by apatinib, an inhibitor of VEGFR2. Lung carcinoma cells interacting with PLTs may play a key role in lung carcinoma angiogenesis through the VEGF/VEGFR2 signaling pathway.

Cardiovascular adverse events induced by immune checkpoint inhibitors: A real world study from 2018 to 2022.

BackgroundThe reported rate of cardiovascular adverse events (CAE) caused by immune checkpoint inhibitors (ICI) is low but potentially fatal. Assess the risk of CAE in cancer patients and compare the incidence of CAE between Chinese developed ICIs and imported ICIs.MethodsA retrospective analysis was performed on cancer patients treated with ICI for at least four cycles in the Second Affiliated Hospital of Dalian Medical University from January 2018 to March 2022. Baseline characteristics, physiological and biochemical values, electrocardiographic and echocardiographic findings were compared between patients with and without CAE.ResultsAmong 495 patients treated with ICIs, CAEs occurred in 64 patients (12.93%). The median time to the event was 105 days (61–202). The patients with low neutrophil-to-lymphocyte ratio (L-NLR) were significantly associated with the risk of developing CAE (hazard ratio HR 3.64, 95% confidence ratio CI 1.86–7.15, P = 0.000). Patients with higher comorbidity burden significantly increased the risk of developing CAE (HR 1.30, 95% CI 1.05–1.61, P = 0.014). Those who received a combination of ICI and vascular endothelial growth factor receptor (VEGFR) inhibitors (HR 2.57, 95% CI 1.37–4.84, P = 0.003) or thoracic radiation therapy (HR 32.93, 95% CI 8.81–123.14, P = 0.000) were at a significantly increased risk of developing CAE. Compared to baseline values, creatine kinase is -oenzymes (CK-MB) (95% CI -9.73 to -2.20, P = 0.003) and cardiac troponin I (cTnI) (95% CI -1.06 to -0.06, P = 0.028) were elevated, and the QTc interval prolonged (95% CI -27.07 to -6.49, P = 0.002). Using nivolumab as a control, there was no difference in CAE risk among the eight ICIs investigated. However, the results of the propensity matching showed that programmed death-ligand 1 (PD-L1) inhibitors had lower CAE occurrence compared with programmed cell death protein 1 (PD-1) inhibitors (adjusted HR = 0.38, P = 0.045).ConclusionPatients who received concurrent VEGFR inhibitors and ICIs had a history of thoracic radiation therapy, L-NLR, and higher comorbidity burden had an increased risk of CAEs. Elevated cTnI, CK-MB, and QTc, can be used to monitor CAEs. There was no significant difference in CAE risks between Chinese domestic and imported ICIs. PD-L1 inhibitors had lower CAE occurrence than PD-1 inhibitors.

Artemisinin derivative FO-ARS-123 as a novel VEGFR2 inhibitor suppresses angiogenesis, cell migration, and invasion

Anti-angiogenesis targeting vascular endothelial growth factor receptor 2 (VEGFR2) has been considered an important strategy for cancer therapy. VEGFR2 inhibitors targeting tumor angiogenic pathways have been widely used in clinical cancer treatment. However, inherent or acquired resistance to anti-angiogenic drugs may occur and thus limit their clinical application. New VEGFR2 inhibitors are still highly demanded. The aim of this study was to investigate VEGFR2-targeted artemisinin (ARS)-type compounds for cancer treatment. Here, we reported the ARS derivative FO-ARS-123 as a novel VEGFR2 inhibitor, which displayed potent binding activity with VEGFR2 in in silico by molecular docking (pKi, 0.40 ± 0.31 nM) and in vitro by microscale thermophoresis (Kd, 1.325 ± 0.055 μM). In addition, compound FO-ARS-123 displayed a strong inhibition on cell proliferation of a broad range of cancer cells as well as suppressed cell migration and invasion. Remarkably, FO-ARS-123 exerted profound anti-angiogenesis effects in the in vitro tube formation assay and in vivo CAM assay. These results suggest that FO-ARS-123 might be a novel and promising anti-angiogenesis agent for cancer treatment.

A case report of pre-eclampsia-like endothelial injury in the kidney of an 85-year-old man treated with ibrutinib

BackgroundGlomerular endotheliosis is the pathognomonic glomerular lesion in pre-eclampsia that has also been described in those taking tyrosine kinase inhibitors for cancer treatment. Ibrutinib is a Bruton’s tyrosine kinase inhibitor used to treat chronic lymphocytic leukemia (CLL). We report the first known case of glomerular endotheliosis on kidney biopsy in a patient on ibrutinib monotherapy.Case presentationThe patient presented with acute on chronic kidney disease, proteinuria, low C3 and C4 and a high rheumatoid factor titer. A kidney biopsy was performed to confirm a preliminary diagnosis of membranoproliferative glomerulonephritis (MPGN), the most common glomerular disease in patients with CLL. Unexpectedly, the kidney biopsy showed pre-eclampsia-like lesions on light and electron microscopy: occlusion of glomerular peripheral capillary lumens by swollen reactive endothelial cells. Findings of glomerulonephritis were not seen, and there were no specific glomerular immune deposits by immunofluorescence or electron microscopy.ConclusionsCLL is known to cause glomerular lesions, mainly MPGN. There is increasing evidence that ibrutinib, a major treatment for CLL, can cause kidney disease, but the precise pathology is not characterized. We present a patient with CLL on ibrutinib with signs of glomerular endotheliosis. Based on the absence of CLL-induced kidney pathologies typically seen on the kidney biopsy and the non-selectivity of ibrutinib, we attributed the glomerular endotheliosis to ibrutinib. In pre-eclampsia, increased soluble fms-like tyrosine kinase 1 (sFlt1) levels induce endothelial dysfunction by decreasing vascular endothelial growth factor (VEGF). Ibrutinib has been demonstrated to have non-selective tyrosine kinase inhibition, including inhibition of VEGF receptor (VEGFR) and epidermal growth factor receptor (EGFR). VEGFR and EGFR inhibitors have recently been described in the literature to cause hypertension, proteinuria, and glomerular endotheliosis. Kidney biopsy should be performed in CLL patients on ibrutinib that present with acute kidney injury (AKI) or proteinuria to determine whether the clinical picture is attributable to the disease itself or a complication of the therapy.

Novel artemisinin derivative FO8643 with anti-angiogenic activity inhibits growth and migration of cancer cells via VEGFR2 signaling

The vascular endothelial growth factor receptor 2 (VEGFR2) is widely recognized as a key effector in angiogenesis and cancer progression and has been considered a critical target for the development of anti-cancer drugs. Artemisinin (ARS) and its derivatives exert profound efficacy in treating not only malaria but also cancer. As a novel ARS-type compound, FO8643 caused significant suppression of the growth of a panel of cancer cells, including both solid and hematologic malignancies. In CCRF-CEM leukemia cells, FO8643 dramatically inhibited cell proliferation coupled with increased apoptosis and cell cycle arrest. Additionally, FO8643 restrained cell migration in the 2D wound healing assay as well as in a 3D spheroid model of human hepatocellular carcinoma HUH-7 cells. Importantly, SwissTargetPrediction predicted VEGFR2 as an underlying target for FO8643. Molecular docking simulation further indicated that FO8643 formed hydrogen bonds and hydrophobic interactions within the VEGFR2 kinase domain. Moreover, FO8643 directly inhibited VEGFR2 kinase activity and its downstream action including MAPK and PI3K/Akt signaling pathways in HUH-7 cells. Encouragingly, FO8643 decreased angiogenesis in the chorioallantoic membrane assay in vivo. Collectively, FO8643 is a novel ARS-type compound exerting potential VEGFR2 inhibition. FO8643 may be a viable drug candidate in cancer therapy.

Adenosine-Prefabricated Adipose Tissue Improves Fat Graft Survival by Promoting VEGF-Dependent Angiogenesis.

Angiogenesis plays an important role in determining the fat graft survival. However, clinical preconditioning techniques that target angiogenesis during fat grafting have not been established so far. Adenosine has emerged as a regulator of angiogenesis under hypoxic conditions; therefore, the aim of this study was to investigate the effects and underlying mechanisms of adenosine prefabrication on fat graft survival. In the first animal study, a total of 32 mice were transplanted with fat prefabricated with vehicle (Control, N = 16) or adenosine (Adenosine, N = 16). In the second animal study, 24 mice were divided into three groups based on the type of fat graft: Control (N = 8), Adenosine (N = 8), and Axitinib (cotreatment of adenosine with axitinib, N = 8). At 1- and 4-weeks post-transplantation, grafts were evaluated by histopathological and biochemical assessment. Adenosine-induced vascular endothelial growth factor (VEGF) production and angiogenesis were determined using cell cultures. The retention volumes of fat grafts in the adenosine group were significantly increased until 4weeks. Fat grafts from the adenosine group exhibited greater structural integrity, reduced fibrosis, and increased blood vessels. The expression levels of angiogenesis-related genes, Vegfa, Vegfr1, Vegfr2, and Vwf, were elevated in the adenosine group. Furthermore, adenosine upregulated VEGF production in preadipocytes, thereby enhancing the migration of endothelial cells. Treatment with the axitinib, VEGF receptor inhibitor, abrogated the adenosine-induced angiogenesis in the fat grafts. Adenosine prefabrication in fat improved the graft survival by enhancing angiogenesis through the VEGF/VEGFR axis in the preadipocytes and endothelial cells. Therefore, this method may be used as a novel strategy to increase the retention rate in fat grafts.

VEGF‑mediated tumour growth and EMT in 2D and 3D cell culture models of hepatocellular carcinoma

The purpose of the present study was to evaluate the effects of vascular endothelial growth factor (VEGF) on tumorigenic properties in two-dimensional (2D) and three-dimensional (3D) cultures of hepatoma cells. The proliferation and invasion of hepatoma cells was assessed using wound healing, chemotaxis Transwell, invasion, tube-forming and hanging drop assays in both 2D and 3D cultures. The expression levels of epithelial-mesenchymal transition (EMT) and stemness markers were analysed using reverse transcription-quantitative PCR (RT-qPCR) for mRNA expression and immunofluorescence assay for protein expression. To validate the role of VEGF in tumour growth, a VEGF receptor (VEGFR) inhibitor (sorafenib) was used. The results demonstrated that the hepatoma cells formed 3D spheroids that differed in size and density in the absence and presence of the growth factor, VEGF. In all spheroids, invasion and angiogenesis were more aggressive in 3D cultures in comparison to 2D conditions following treatment with VEGF. Mechanistically, the VEGF-mediated increase in the levels of EMT markers, including Vimentin, N-cadherin 2 (Cadherin 2) and Thy-1 Cell Surface Antigen was observed in the 2D and 3D cultures. Sorafenib treatment for 24 h culminated in a marked reduction in cell migration, cell-cell adhesion, spheroid compaction and EMT gene expression in 3D models as compared to the 2D models. On the whole, the findings of the present study suggested that as compared to the 2D cell cultures, 3D cell cultures model may be used as a more realistic model for the study of tumour growth and invasion in the presence of angiogenic factors, as well as for tumour inhibitor screening.

PRMT4-mediated arginine methylation promotes tyrosine phosphorylation of VEGFR-2 and regulates filopodia protrusions

SummaryThrough tightly controlled multilayer mechanisms, vascular endothelial growth factor receptor-2 (VEGFR-2) activation and its downstream signal transduction govern vasculogenesis and pathological angiogenesis, such as tumor angiogenesis. Therefore, it is critical to understand the molecular mechanisms governing VEGFR-2 signal transduction. We report that protein arginine methyltransferase 4 (PRMT4) via its highly conserved EVH1 and PH domain-like N-terminal domain binds to VEGFR-2 and mediates methylation of the juxtamembrane arginine 817 (R817) on VEGFR-2. Methylation of R817 selectively increases phosphorylation of tyrosine 820 (Y820). Phosphorylation of Y820 facilitates the c-Src binding with VEGFR-2 via Src homology domain 2 (SH2). Interfering with the methylation of R817 or phosphorylation of Y820 inhibits VEGFR-2-induced filopodia protrusions, a process that is critical for the core angiogenic responses of VEGFR-2. Methylation of R817 is an important previously unrecognized mechanism of the angiogenic signaling of VEGFR-2, with implications for the development of novel-targeted VEGFR-2 inhibitors.

CD8+ T-cell Responses Are Boosted by Dual PD-1/VEGFR2 Blockade after EGFR Inhibition in Egfr-Mutant Lung Cancer.

Epidermal growth factor receptor (EGFR) is the most frequently mutated driver oncogene in nonsmoking-related, non-small cell lung cancer (NSCLC). EGFR-mutant NSCLC has a noninflamed tumor microenvironment (TME), with low infiltration by CD8+ T cells and, thus, immune-checkpoint inhibitors, such as antiprogrammed cell death-1 (anti-PD-1), have weak antitumor effects. Here, we showed that CD8+ T-cell responses were induced by an EGFR-tyrosine kinase inhibitor (TKI) in syngeneic Egfr-mutant NSCLC tumors, which was further pronounced by the sequential dual blockade of PD-1 and vascular endothelial growth factor receptor 2 (VEGFR2). However, the simultaneous triple blockade had no such effect. The PD-1/VEGFR2 dual blockade did not exert tumor-inhibitory effects without pretreatment with the EGFR-TKI, suggesting that the treatment schedule is crucial for the efficacy of the dual blockade therapy. Pretreatment with EGFR-TKI increased the CD8+ T-cell/regulatory T-cell (Treg) ratio, while also increasing the expression of immunosuppressive chemokines and chemokine receptors, as well as increasing the number of M2-like macrophages, in the TME. Discontinuing EGFR-TKI treatment reversed the transient increase of immunosuppressive factors in the TME. The subsequent PD-1/VEGFR2 inhibition maintained increased numbers of infiltrating CD8+ T cells and CD11c+ dendritic cells. Depletion of CD8+ T cells in vivo abolished tumor growth inhibition by EGFR-TKI alone and the sequential triple therapy, suggesting that EGFR inhibition is a prerequisite for the induction of CD8+ T-cell responses. Our findings could aid in developing an alternative immunotherapy strategy in patients with cancers that have driver mutations and a noninflamed TME.

Tivozanib for the treatment of advanced renal cell carcinoma

Introduction Renal cell carcinoma (RCC) accounts for 2.4% of cancers, with clear cell kidney cancer being the most common histologic subtype. Despite recent therapeutic advances, the prognosis for patients with advanced disease remains poor, with a 5-year survival rate of only 13.9% reported in the United States. Tivozanib is a novel inhibitor of vascular endothelial growth factor (VEGF) receptor (VEGFR) -1, -2, and -3 and has recently been approved by the US FDA for use in patients with relapsed or recurrent advanced RCC following 2 or more prior systemic therapies. Areas covered Here, the authors provide a review focusing on the development of tivozanib, compare tivozanib to other agents in the VEGFR inhibitor class, detail how tivozanib fits into current treatment landscape, and describe ongoing studies involving this agent. Expert opinion Given tivozanib’s excellent safety profile and demonstrated clinical benefit in patients with pretreated disease, tivozanib is likely to be heavily utilized in the later line setting. The ongoing TINIVO-2 study may further impact the treatment landscape by evaluating the use of tivozanib plus nivolumab in patients who have progressed on an immune checkpoint inhibitor.

RCC Real-World Data: Prognostic Factors and Risk Stratification in the Immunotherapy Era.

Simple SummaryNowadays, most metastatic renal cell carcinoma (mRCC) patients are candidates for immunotherapy. Risk stratification is based on the IMDC model that was developed in an earlier era of vascular endothelial growth factor receptor inhibitors (VEGFRi). An updated, more accurate model is needed. This paper suggests an updated risk-stratification model based on five factors that are strongly correlated with overall survival in a real-world cohort of patients with mRCC treated with checkpoint inhibitors in any line of treatment during the course of their disease. Compared with the commonly used IMDC criteria, in this cohort, our model was better able to predict survival.Immunotherapy has transformed the landscape of treatment in metastatic renal cell carcinoma (mRCC) in the last decade. Currently, prognostic risk stratification is based on the model developed in the era of vascular endothelial growth factor receptor inhibitors (VEGFRi) by Heng in 2009. Our study aims to find the most relevant risk criteria for mRCC patients treated with checkpoint inhibitors (CPI). In a retrospective cohort study, laboratory, pathology, demographic, and clinical data were retrieved from electronic medical records of consecutive mRCC patients treated with CPI in a tertiary center between 2015 and 2020. An unbiased multivariate analysis was performed to define predictive variables with a bootstrap validation step. We analyzed data on 127 patients with a median follow-up of 60 months. The median overall survival (OS) since the diagnosis of metastatic disease was 57 months. The response rate for CPI was 39%. Five risk factors were correlated with worse OS: intact primary kidney tumor (HR 2.33, p = 0.012), liver metastasis (HR 3.33, p = 0.001), <one year to treatment start (HR 1.98, p = 0.029), elevated platelets (HR 3.06, p = 0.015), and Karnofsky performance status <80% (HR = 3.42, p = 0.001). The model received a C-index of 70.7 compared with a score of 62.0 for the Heng’s model. When dividing patients into “low-risk” (0–1 risk factors) and “high-risk” (2–5 risk factors), there was good separation between the groups, with an HR of 5.9 (p < 0.0001). This study presents a new prognostic model for mRCC in the immunotherapy era with improved accuracy. Further research is needed to validate this model in larger cohorts.