Synthetic Antiangiogenic Vascular Endothelial Growth Factor-A Splice Variant Revascularizes Ischemic Muscle in Peripheral Artery Disease.

BACKGROUND: Alternative splicing in the eighth exon C-terminus of VEGF-A (vascular endothelial growth factor-A) results in the formation of proangiogenic VEGF165a and antiangiogenic VEGF165b isoforms. The only known difference between these 2 isoform families is a 6-amino acid switch from CDKPRR (in VEGF165a) to SLTRKD (in VEGF165b). We have recently shown that VEGF165b can induce VEGFR2-activation but fails to induce VEGFR1 (VEGF receptor 1)-activation. The molecular mechanisms that regulate VEGF165b's ability toward differential VEGFR2 versus VEGFR1 activation/inhibition are not yet clear. METHODS AND RESULTS: Hypoxia serum starvation was used as an in vitro peripheral artery disease model. Unilateral single ligation of the femoral artery was used as a preclinical peripheral artery disease model. VEGFR1 activating ligands have 2 arginine (RR) residues in their eighth exon C-terminus, that were replaced by lysine-aspartic acid (KD) in VEGF165b. A synthetic anti-angiogenic VEGF165b splice variant in which the KD residues were switched to RR (VEGF165bKD→RR) activated both VEGFR1- and VEGFR2-signaling pathways to induce ischemic-endothelial cell angiogenic capacity in vitro and enhance perfusion recovery in a severe experimental-peripheral artery disease model significantly higher than VEGF165a. Phosphoproteome arrays showed that the therapeutic efficacy of VEGF165bKD→RR over VEGF165a is due to its ability to induce P38-activation in ischemic endothelial cells. CONCLUSIONS: Our data shows that the KD residues regulate VEGF165b's VEGFR1 inhibitory property but not VEGFR2. Switching these KD residues to RR resulted in the formation of a synthetic/recombinant VEGF165bKD→RR isoform that has the ability to activate both VEGFR1- and VEGFR2-signaling and induce ischemic-endothelial cell angiogenic and proliferative capacity that matched the angiogenic requirement necessary to achieve perfusion recovery in a severe experimental-peripheral artery disease model.

Combination of arsenic trioxide and apatinib synergistically inhibits small cell lung cancer by down-regulating VEGFR2/mTOR and Akt/c-Myc signaling pathway via GRB10.

BACKGROUND: Small cell lung carcinoma (SCLC) is characterized by -poor prognosis, -high predilection for -metastasis, -proliferation, and -absence of newer therapeutic options. Elucidation of newer pathways characterizing the disease may allow for development of targeted therapies and consequently favorable outcomes. METHODS: The current study explored the combinatorial action of arsenic trioxide (ATO) and apatinib (APA) in vitro and in vivo. In vitro models were tested using -H446 and -H196 SCLC cell lines. The ability of drugs to reduce -metastasis, -cell proliferation, and -migration were assessed. Using bioinformatic analysis, differentially expressed genes were determined. Gene regulation was assessed using gene knock down models and confirmed using Western blots. The in vivo models were used to confirm the resolution of pathognomic features in the presence of the drugs. Growth factor receptor bound protein (GRB) 10 expression levels of human small cell lung cancer tissues and adjacent tissues were detected by IHC. RESULTS: In combination, ATO and APA were found to significantly reduce -cell proliferation, -migration, and -metastasis in both the cell lines. Cell proliferation was found to be inhibited by activation of Caspase-3, -7 pathway. In the presence of drugs, it was found that expression of GRB10 was stabilized. The silencing of GRB10 was found to negatively regulate the VEGFR2/Akt/mTOR and Akt/GSK-3ß/c-Myc signaling pathway. Concurrently, absence of metastasis and reduction of tumor volume were confirmed in vivo. The immunohistochemical results confirmed that the expression level of GRB10 in adjacent tissues was significantly higher than that in human small cell lung cancer tissues. CONCLUSIONS: Synergistically, ATO and APA have a more significant impact on inhibiting cell proliferation than each drug independently. ATO and APA may be mediating its action through the stabilization of GRB10 thus acting as a tumor suppressor. We thus, preliminarily report the impact of GRB10 stability as a target for SCLC treatment.

Molecular Analysis of Renal/Adrenal Angiosarcomas Reveals High Frequency of Recurrent Genetic Alterations.

Angiosarcomas of the kidney and adrenal gland are rare, highly aggressive vascular neoplasms. Their genomic profile has not been systematically studied to date. We report the clinicopathologic and molecular features of six angiosarcomas centered in the kidney/adrenal gland. All patients were male adults, ranging from 58 to 77 years of age. Tumor sizes ranged from 2.5 to 22.5 cm. Half of the cases demonstrated hot spot mutations in the KDR gene, while one-third demonstrated mutations in the PIK3CA gene; both of these gene alterations being previously described, preferentially in breast angiosarcomas. In addition, two cases each demonstrated BRIP1 gene amplification, CTNNB1 and ETV6 mutations, which have not been previously reported in angiosarcoma. Notably, molecular studies were critical in establishing the correct diagnoses in three cases: one was an epithelioid angiosarcoma originally misdiagnosed as metastatic adenocarcinoma to the adrenal gland, the second was a vasoformative angiosarcoma that mimicked hemangioma, and the third was a collision tumor between a high-grade angiosarcoma and a chromophobe renal cell carcinoma which was originally diagnosed as a sarcomatoid renal cell carcinoma. In summary, angiosarcomas of the kidney and adrenal gland have a high frequency of recurrent genetic alterations, some of them being shared with other angiosarcoma subtypes, while other appear to be novel. In particular, activating hot spot KDR and PIK3CA mutations represent potential therapeutic targets for these highly aggressive cancers.

The KDR Gene rs2071559 and the VEGF Gene rs6921438 May Be Associated with Diabetic Nephropathy in Caucasians with Type 2 Diabetes Mellitus.

The aim of our study was to investigate an association between polymorphisms of either the VEGF (vascular endothelial growth factor) gene (rs6921438) or the KDR (kinase insert domain receptor) gene (rs2071559, rs2305948) and DN (diabetic nephropathy) in Caucasians with T2DM (type 2 diabetes mellitus). The second aim was to investigate the effect of either the VEGF gene (rs6921438) or the KDR gene (rs2071559, rs2305948) on the immune expression of either VEGF or KDR in the renal tissues of T2DM subjects (to test the functional significance of tested polymorphisms). The study included 897 Caucasians with T2DM for at least ten years (344 patients with DN and 553 patients without DN). Each subject was genotyped and analyzed for KDR (rs1617640, rs2305948) and VEGF (rs6921438) polymorphisms. Kidney tissue samples taken from 15 subjects with T2DM (autopsy material) were immunohistochemically stained for the expression of VEGF and KDR. We found that the rs2071559 KDR gene was associated with an increased risk of DN. In addition, the GG genotype of the rs6921438 VEGF gene had a protective effect. We found a significantly higher numerical area density of VEGF-positive cells in T2DM subjects with the A allele of the rs6921438-VEGF compared to the homozygotes for wild type G allele (7.0 ± 2.4/0.1 mm2 vs. 1.24 ± 0.5/0.1 mm2, respectively; p < 0.001). Moreover, a significantly higher numerical area density of KDR-positive cells was found in T2DM subjects with the C allele of rs2071559 (CC + CT genotypes) compared to the homozygotes for wild type T allele (9.7± 3.2/0.1 mm2 vs. 1.14 ± 0.5/0.1 mm2, respectively; p < 0.001) To conclude, our study showed that the presence of the C allele of the rs2071559 KDR gene was associated with a higher risk of DN, while the G allele of the rs6921438-VEGF conferred protection against DN in Slovenian T2DM subjects.

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

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

Dual effects of targeting neuropilin-1 in lenvatinib-resistant hepatocellular carcinoma: inhibition of tumor growth and angiogenesis.

In the era of immunotherapy, lenvatinib (LEN) still holds an important position in the sequential treatment of advanced hepatocellular carcinoma (HCC). However, the sustained therapeutic effect of LEN is not sufficient, and there is a need to address the development of resistance. Neuropilin-1 (NRP1) is known to act as a coreceptor for epidermal growth factor receptor (EGFR), Met, and vascular endothelial growth factor receptor 2 (VEGFR2), which have been reported to be involved in LEN resistance. In this study, we used cell culture and in vivo xenograft models to evaluate the contribution of NRP1 in the acquisition of LEN resistance in HCC as well as the potential of NRP1 as a therapeutic target. LEN resistance increased EGF/EGFR and hepatocyte growth factor (HGF)/Met signaling in liver cancer cells and VEGFA/VEGFR2 and HGF/Met signaling in vascular endothelial cells, thereby promoting cell proliferation, cell migration, and angiogenesis. We found that activation of NRP1 is essential for the enhancement of these signaling. In addition, NRP1 inhibition combined with LEN therapy synergistically improved the antitumor effects against LEN-resistant HCC, indicating that NRP1 is an attractive therapeutic target.NEW & NOTEWORTHY We demonstrated that neuropilin-1 (NRP1) was an essential coreceptor mediating the activation of multiple signaling pathways in the acquisition of resistance to lenvatinib (LEN) in HCC. The addition of NRP1 inhibition to LEN had a synergistic antitumor effect on LEN-resistant HCC in culture and in vivo xenograft models.

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.

Prime Editing of Vascular Endothelial Growth Factor Receptor 2 Attenuates Angiogenesis In Vitro.

Vascular endothelial growth factor receptor (VEGFR)-2 is a key switch for angiogenesis, which is observed in various human diseases. In this study, a novel system for advanced prime editing (PE), termed PE6h, is developed, consisting of dual lentiviral vectors: (1) a clustered regularly interspaced palindromic repeat-associated protein 9 (H840A) nickase fused with reverse transcriptase and an enhanced PE guide RNA and (2) a dominant negative (DN) MutL homolog 1 gene with nicking guide RNA. PE6h was used to edit VEGFR2 (c.18315T>A, 50.8%) to generate a premature stop codon (TAG from AAG), resulting in the production of DN-VEGFR2 (787 aa) in human retinal microvascular endothelial cells (HRECs). DN-VEGFR2 impeded VEGF-induced phosphorylation of VEGFR2, Akt, and extracellular signal-regulated kinase-1/2 and tube formation in PE6h-edited HRECs in vitro. Overall, our results highlight the potential of PE6h to inhibit angiogenesis in vivo.

Mutation in the Kinase Domain Alters the VEGFR2 Membrane Dynamics.

BACKGROUND: Recently, the substitution R1051Q in VEGFR2 has been described as a cancer-associated "gain of function" mutation. VEGFR2R1051Q phosphorylation is ligand-independent and enhances the activation of intracellular pathways and cell growth both in vitro and in vivo. In cancer, this mutation is found in heterozygosity, suggesting that an interaction between VEGFR2R1051Q and VEGFR2WT may occur and could explain, at least in part, how VEGFR2R1051Q acts to promote VEGFR2 signaling. Despite this, the biochemical/biophysical mechanism of the activation of VEGFR2R1051Q remains poorly understood. On these bases, the aim of our study is to address how VEGFR2R1051Q influences the biophysical behavior (dimerization and membrane dynamics) of the co-expressed VEGFR2WT. METHODS: We employed quantitative FLIM/FRET and FRAP imaging techniques using CHO cells co-transfected with the two forms of VEGFR2 to mimic heterozygosity. RESULTS: Membrane protein biotinylation reveals that VEGFR2WT is more exposed on the cell membrane with respect to VEGFR2R1051Q. The imaging analyses show the ability of VEGFR2WT to form heterodimers with VEGFR2R1051Q and this interaction alters its membrane dynamics. Indeed, when the co-expression of VEGFR2WT/VEGFR2R1051Q occurs, VEGFR2WT shows reduced lateral motility and a minor pool of mobile fraction. CONCLUSIONS: This study demonstrates that active VEGFR2R1051Q can affect the membrane behavior of the VEGFR2WT.

Clinical course and vascular endothelial growth factor signaling system expression in maxillary angiosarcoma: A case report.

Maxillary angiosarcoma, an aggressive tumor derived from vascular endothelial cells, is very rare. Recently, antivascular endothelial growth factor (VEGF) therapies have attracted considerable attention. We describe the clinical course of a patient with maxillary angiosarcoma and discuss the expression of VEGF signaling molecules assessed via immunohistological analysis. An 81-year-old man presented with an aggressive tumor in the left maxillary sinus. Biopsy revealed atypical nuclear cell proliferation, and the tumor was suspected to be a sarcoma. The maxillary malignancy was treated using a multidisciplinary approach with a combination of surgery, radiotherapy, and regional chemotherapy. Examination of the specimen obtained in the first surgery revealed maxillary angiosarcoma, found to be positive for CD31, while negative for CD34, D2-40, and factor Ⅷ. Although no pathological residual tumor was observed after the planned wide surgery, cervical lymph node and distant metastases occurred. The patient died 24 months after the first surgery. Staining revealed VEGF receptor (VEGFR) 1, VEGFR2, phosphorylated Ak strain transforming, mitogen-activated protein kinase, and signal transducer and activator of transcription 3 positivity. Although our findings do not indicate that anti-VEGF therapy is beneficial for treating maxillary angiosarcomas, we found that VEGFR signaling pathways were activated in maxillary angiosarcomas similar to angiosarcomas originating at other sites. Herein, we report a case of maxillary angiosarcoma, focused on VEGFR and signaling pathway activation. To our knowledge, this is the first report to describe VEGFR system immunostaining findings in maxillary angiosarcoma.

Endothelial cell heterogeneity in colorectal cancer: tip cells drive angiogenesis.

This study aims to uncover the heterogeneity of endothelial cells (ECs) in colorectal cancer (CRC) and their crucial role in angiogenesis, with a special focus on tip cells. Using single-cell RNA sequencing to profile ECs, our data suggests that CRC ECs predominantly exhibit enhanced angiogenesis and decreased antigen presentation, a shift in phenotype largely steered by tip cells. We also observed that an increase in the density and proportion of tip cells correlates with CRC occurrence, progression, and poorer patient prognosis. Furthermore, we identified endothelial cell-specific molecule 1 (ESM1), specifically expressed in tip cells, sustains a VEGFA-KDR-ESM1 positive feedback loop, promoting angiogenesis and CRC proliferation and migration. We also found the enrichment of KDR in tip cells and spotlight a unique long-tail effect in VEGFA expression: while VEGFA is primarily expressed by epithelial cells, the highest level of VEGFA expression is found in individual myeloid cells. Moreover, we observed that effective PD-1 blockade immunotherapy significantly reduced tip cells, disrupting the VEGFA-KDR-ESM1 positive feedback loop in the process. Our investigation into the heterogeneity of ECs in CRC at a single-cell level offers important insights that may contribute to the development of more effective immunotherapies targeting tip cells in CRC.

Sinensetin Inhibits Angiogenesis in Lung Adenocarcinoma via the miR-374c-5p/VEGF-A/VEGFR-2/AKT Axis.

Sinensetin is a product isolated from Orthosiphon aristatus, and its antitumor activities have been well established. This study focused on the role and mechanism of sinensetin in lung adenocarcinoma (LUAD). LUAD cells were treated with various concentrations of sinensetin. The proliferation, migration, invasion, and angiogenesis of LUAD cells were detected using colony formation, transwell, and tube formation assays, respectively. The protein levels of VEGF-A, VEGFR-2, and phosphorylated AKT (ser473) were measured by western blotting. The targeted relationship between VEGF-A and miR-374c-5p was verified by luciferase reporter assay. BALB/c nude mice inoculated with A549 cells were treated with sinensetin (40 mg/kg/day) by gavage for 21 days to investigate the effect of sinensetin on tumor growth and angiogenesis in vivo. We found that sinensetin reduced proliferation, migration, invasion, angiogenesis, and cancer stem characteristics of LUAD cells. Sinensetin also suppressed LUAD tumor growth and angiogenesis in vivo. Sinensetin downregulated VEGF-A expression in LUAD cells by enhancing miR-374c-5p expression. MiR-374c-5p inhibited the VEGF-A/VEGFR-2/AKT pathway in LUAD cells. The antitumor effect of sinensetin was reversed by overexpression of VEGF-A or inhibition of miR-374c-5p. Overall, sinensetin upregulates miR-374c-5p to inhibit the VEGF-A/VEGFR-2/AKT pathway, thereby exerting antitumor effect on LUAD.

Immunomic longitudinal profiling of the NeoPembrOv trial identifies drivers of immunoresistance in high-grade ovarian carcinoma.

PD-1/PD-L1 blockade has so far shown limited survival benefit for high-grade ovarian carcinomas. By using paired samples from the NeoPembrOv randomized phase II trial (NCT03275506), for which primary outcomes are published, and by combining RNA-seq and multiplexed immunofluorescence staining, we explore the impact of NeoAdjuvant ChemoTherapy (NACT) ± Pembrolizumab (P) on the tumor environment, and identify parameters that correlated with response to immunotherapy as a pre-planned exploratory analysis. Indeed, i) combination therapy results in a significant increase in intraepithelial CD8+PD-1+ T cells, ii) combining endothelial and monocyte gene signatures with the CD8B/FOXP3 expression ratio is predictive of response to NACT + P with an area under the curve of 0.93 (95% CI 0.85-1.00) and iii) high CD8B/FOXP3 and high CD8B/ENTPD1 ratios are significantly associated with positive response to NACT + P, while KDR and VEGFR2 expression are associated with resistance. These results indicate that targeting regulatory T cells and endothelial cells, especially VEGFR2+ endothelial cells, could overcome immune resistance of ovarian cancers.

MEG3-Mediated Oral Squamous-Cell-Carcinoma-Derived Exosomal miR-421 Activates Angiogenesis by Targeting HS2ST1 in Vascular Endothelial Cells.

Exosomal microRNAs (miRNAs) from cancer cells play a key role in mediating the oral squamous cell carcinoma (OSCC) microenvironment. The objective of this study was to investigate how the long non-coding RNA (lncRNA) MEG3 affects OSCC angiogenesis through exosomal miR-421. Global miRNA microarray analysis and quantitative real-time PCR (qRT-PCR) were performed to determine the level of miRNAs in OSCC cell-derived exosomes. Cell migration, invasion, tube formation, immunohistochemistry, and hemoglobin concentrations were used to study the effects of exosomal miR-421 in angiogenesis. Western blotting was used to determine the expression level of HS2ST1 and VEGFR2-related downstream proteins. MiRNA array and qRT-PCR identified the upregulation of miR-421 in OSCC cell-derived exosomes. Furthermore, exosomal miR-421 can be taken up by human umbilical vein endothelial cells (HUVECs) and then target HS2ST1 through VEGF-mediated ERK and AKT phosphorylation, thereby promoting HUVEC migration, invasion, and tube formation. Additionally, forced expression of the lncRNA MEG3 in OSCC cells reduced exosomal miR-421 levels and then increased HS2ST1 expression, thereby reducing the VEGF/VEGFR2 pathway in HUVECs. Our results demonstrate a novel mechanism by which lncRNA MEG3 can act as a tumor suppressor and regulate endothelial angiogenesis through the exosomal miR-421/HS2ST1 axis, which provides a potential therapeutic strategy for OSCC angiogenesis.

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.

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.

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.

Exosomal circPVT1 promotes angiogenesis in laryngeal cancer by activating the Rap1b-VEGFR2 signaling pathway.

Laryngeal cancer (LC) is the second most common head and neck cancer and has a decreasing 5-year survival rate worldwide. Circular RNAs (circRNAs) regulate cancer development in diverse ways based on their distinct biogenesis mechanisms and expansive regulatory roles. However, currently, there is little research on how exosomal circRNAs are involved in the development of LC. Here, we demonstrated that circPVT1, a circRNA derived from the well-studied long noncoding RNA PVT1, is correlated with disease progression in LC and promotes angiogenesis both in vivo and in vitro. Mechanistically, circPVT1 is loaded into LC cell-secreted exosomes and taken up by vascular epithelium cells. By sponging miR-30c-5p, exosomal circPVT1 promotes Rap1b expression, which dramatically enhances vascular endothelial growth factor receptor 2 and the phosphatidylinositol 3-kinase (PI3K)/AKT pathway activation, ultimately resulting in the induction of angiogenesis. Furthermore, our xenograft models demonstrated that the combination of short hairpin RNA-circPVT1 and cetuximab showed high efficacy in inhibiting tumor growth and angiogenesis. Collectively, these findings uncover a novel mechanism of exosomal circRNA-mediated angiogenesis modulation and provide a preclinical rationale for testing this analogous combination in patients with LC.

HNRNPA2B1 stabilizes NFATC3 levels to potentiate its combined actions with FOSL1 to mediate vasculogenic mimicry in GBM cells.

BACKGROUND: Vasculogenic mimicry (VM) is an enigmatic physiological feature that influences blood supply within glioblastoma (GBM) tumors for their sustained growth. Previous studies identify NFATC3, FOSL1 and HNRNPA2B1 as significant mediators of VEGFR2, a key player in vasculogenesis, and their molecular relationships may be crucial for VM in GBM. AIMS: The aim of this study was to understand how NFATC3, FOSL1 and HNRNPA2B1 collectively influence VM in GBM. METHODS: We have investigated the underlying gene regulatory mechanisms for VM in GBM cell lines U251 and U373 in vitro and in vivo. In vitro cell-based assays were performed to explore the role of NFATC3, FOSL1 and HNRNPA2B1 in GBM cell proliferation, VM and migration, in the context of RNA interference (RNAi)-mediated knockdown alongside corresponding controls. Western blotting and qRT-PCR assays were used to examine VEGFR2 expression levels. CO-IP was employed to detect protein-protein interactions, ChIP was used to detect DNA-protein complexes, and RIP was used to detect RNA-protein complexes. Histochemical staining was used to detect VM tube formation in vivo. RESULTS: Focusing on NFATC3, FOSL1 and HNRNPA2B1, we found each was significantly upregulated in GBM and positively correlated with VM-like cellular behaviors in U251 and U373 cell lines. Knockdown of NFATC3, FOSL1 or HNRNPA2B1 each resulted in decreased levels of VEGFR2, a key growth factor gene that drives VM, as well as the inhibition of proliferation, cell migration and extracorporeal VM activity. Chromatin immunoprecipitation (ChIP) studies and luciferase reporter gene assays revealed that NFATC3 binds to the promoter region of VEGFR2 to enhance VEGFR2 gene expression. Notably, FOSL1 interacts with NFATC3 as a co-factor to potentiate the DNA-binding capacity of NFATC3, resulting in enhanced VM-like cellular behaviors. Also, level of NFATC3 protein in cells was enhanced through HNRNPA2B1 binding of NFATC3 mRNA. Furthermore, RNAi-mediated silencing of NFATC3, FOSL1 and HNRNPA2B1 in GBM cells reduced their capacity for tumor formation and VM-like behaviors in vivo. CONCLUSION: Taken together, our findings identify NFATC3 as an important mediator of GBM tumor growth through its molecular and epistatic interactions with HNRNPA2B1 and FOSL1 to influence VEGFR2 expression and VM-like cellular behaviors.

High expression of DEC2 distinguishes chondroblastic osteosarcoma and promotes tumour growth by activating the VEGFC/VEGFR2 signalling pathway.

Osteosarcoma (OS) is the most common primary malignant bone tumour in children and young adults. Account for 80% of all OS cases, conventional OS are characterized by the presence of osteoblastic, chondroblastic and fibroblastic cell types. Despite this heterogeneity, therapeutic treatment and prognosis of OS are essentially the same for all OS subtypes. Here, we report that DEC2, a transcriptional repressor, is expressed at higher levels in chondroblastic OS compared with osteoblastic OS. This difference suggests that DEC2 is disproportionately involved in the progression of chondroblastic OS, and thus, DEC2 may represent a possible molecular target for treating this type of OS. In the human chondroblastic-like OS cell line MNNG/HOS, we found that overexpression of DEC2 affects the proliferation of the cells by activating the VEGFC/VEGFR2 signalling pathway. Enhanced expression of DEC2 increased VEGFR2 expression, as well as increased the phosphorylation levels at sites Y951 and Y1175 of VEGFR2. On the one hand, activation of VEGFR2Y1175 enhanced cell proliferation through VEGFR2Y1175-PLCγ1-PKC-SPHK-MEK-ERK signalling. On the other hand, activation of VEGFR2Y951 decreased mitochondria-dependent apoptosis rate through VEGFR2Y951-VARP-PI3K-AKT signalling. Activation of these two signalling pathways resulted in enhanced progression of chondroblastic OS. In conclusion, DEC2 plays a pivotal role in cell proliferation and apoptosis-resistance in chondroblastic OS via the VEGFC/VEGFR2 signalling pathway. These findings lay the groundwork for developing focused treatments that target specific types of OS.