Vascular Endothelial Growth Factor Receptor Type Research Articles

Dysregulated VEGF/VEGFR-2 Signaling and Plexogenic Lesions in the Embryonic Lungs of Chickens Predisposed to Pulmonary Arterial Hypertension.

Plexiform lesions are a hallmark of pulmonary arterial hypertension (PAH) in humans and are proposed to stem from dysfunctional angioblasts. Broiler chickens (Gallus gallus) are highly susceptible to PAH, with plexiform-like lesions observed in newly hatched individuals. Here, we reported the emergence of plexiform-like lesions in the embryonic lungs of broiler chickens. Lung samples were collected from broiler chickens at embryonic day 20 (E20), hatch, and one-day-old, with PAH-resistant layer chickens as controls. Plexiform lesions consisting of CD133+/vascular endothelial growth factor receptor type-2 (VEGFR-2)+ angioblasts were exclusively observed in broiler embryos and sporadically in layer embryos. Distinct gene profiles of angiogenic factors were observed between the two strains, with impaired VEGF-A/VEGFR-2 signaling correlating with lesion development and reduced arteriogenesis. Pharmaceutical inhibition of VEGFR-2 resulted in enhanced lesion development in layer embryos. Moreover, broiler embryonic lungs displayed increased activation of HIF-1α and nuclear factor erythroid 2-related factor 2 (Nrf2), indicating a hypoxic state. Remarkably, we found a negative correlation between lung Nrf2 activation and VEGF-A and VEGFR-2 expression. In vitro studies indicated that Nrf2 overactivation restricted VEGF signaling in endothelial progenitor cells. The findings from broiler embryos suggest an association between plexiform lesion development and impaired VEGF system due to aberrant activation of Nrf2.

VEGFR2 targeted microbubble-based ultrasound molecular imaging improving the diagnostic sensitivity of microinvasive cervical cancer

BackgroundThe current diagnostic methods of microinvasive cervical cancer lesions are imaging diagnosis and pathological evaluation. Pathological evaluation is invasive and imaging approaches are of extremely low diagnostic performance. There is a paucity of effective and noninvasive imaging approaches for these extremely early cervical cancer during clinical practice. In recent years, ultrasound molecular imaging (USMI) with vascular endothelial growth factor receptor type 2 (VEGFR2) targeted microbubble (MBVEGFR2) has been reported to improve the early diagnosis rates of breast cancer (including ductal carcinoma in situ), pancreatic cancer and hepatic micrometastases. Herein, we aimed to assess the feasibility of MBVEGFR2-based USMI in extremely early cervical cancer detection to provide an accurate imaging modality for microinvasive cervical cancer (International Federation of Gynecology and Obstetrics (FIGO) Stage IA1 and IA2).ResultsWe found MBVEGFR2-based USMI could successfully distinguish extremely early lesions in diameter < 3 mm from surrounding normal tissues (all P < 0.05), and the sensitivity gradually decreased along with increasing tumor diameter. Moreover, normalized intensity difference (NID) values showed a good linear correlation with microvessel density (MVD) (R2 = 0.75). In addition, all tumors could not be identified from surrounding muscles in subtracted ultrasound images when mice were administered MBCon.ConclusionsOverall, MBVEGFR2-based USMI has huge potential for clinical application for the early detection of microinvasive cervical cancer (FIGO Stage IA1 and IA2), providing the foothold for future studies on the imaging screening of this patient population.

Inhibition of VEGFR-2 by SU5416 increases neonatally glutamate-induced neuronal damage in the cerebral motor cortex and hippocampus.

Vascular endothelial growth factor (VEGF) exerts neuroprotective or proinflammatory effects, depending on what VEGF forms (A-E), receptor types (VEGFR1-3), and intracellular signaling pathways are involved. Neonatal monosodium glutamate (MSG) treatment triggers neuronal death by excitotoxicity, which is commonly involved in different neurological disorders, including neurodegenerative diseases. This study was designed to evaluate the effects of VEGFR-2 inhibition on neuronal damage triggered by excitotoxicity in the cerebral motor cortex (CMC) and hippocampus (Hp) after neonatal MSG treatment. MSG was administered at a dose of 4 g/kg of body weight (b.w.) subcutaneously on postnatal days (PD) 1, 3, 5, and 7, whereas the VEGFR-2 inhibitor SU5416 was administered at a dose of 10 mg/kg b.w. subcutaneously on PD 5 and 7, 30 min before the MSG treatment. Neuronal damage was assessed using hematoxylin and eosin staining, fluoro-Jade staining, and TUNEL assay. Additionally, western blot assays for some proteins of the VEGF-A/VEGFR-2 signaling pathway (VEGF-A, VEGFR-2, PI3K, Akt, and iNOS) were carried out. All assays were performed on PD 6, 8, 10, and 14. Inhibition of VEGFR-2 signaling by SU5416 increases the neuronal damage induced by neonatal MSG treatment in both the CMC and Hp. Moreover, neonatal MSG treatment increased the expression levels of the studied VEGF-A/VEGFR-2 signaling pathway proteins, particularly in the CMC. We conclude that VEGF-A/VEGFR-2 signaling pathway activation could be part of the neuroprotective mechanisms that attempt to compensate for neuronal damage induced by neonatal MSG treatment and possibly also in other conditions involving excitotoxicity.

Mechanism of Qingre Huoxue Fang treatment on inhibiting angiogenesis of rheumatoid arthritis based on network pharmacology and in vitro experiments.

This study aimed to explore the mechanism of Qingre Huoxue Fang (QRHXF) treatment on anti-angiogenesis in rheumatoid arthritis (RA) based on network pharmacology and in vitro experiments. We used the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Therapeutic Target (TTD) database to extract the active components of QRHXF and potential targets for regulating angiogenesis. First, we used Cytoscape bioinformatics software to construct the network of QRHXF-angiogenesis and screened the potential targets. Then, we performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis on the potential core targets. In addition, enzyme-linked immune assay and Western blot were used for in vitro validation and to verify the effects of different concentrations of QRHXF on the expression levels of the vascular endothelial growth factor receptor type 1 (VEGFR-1) and VEGFR-2 cytokines and phosphoinositide 3-kinase (PI3k) and Ak strain transforming (Akt) proteins in human umbilical vein endothelial cells (HUVECs). In results, we screened 179 core QRHXF antiangiogenic targets, including vascular endothelial growth factor (VEGF) cytokines. Enrichment analysis showed that the targets were enriched in 56 core signaling pathways, including PI3k and Akt. In vitro experiments showed that the migration distance and square, adhesion optical density (OD) values, and the number of branch points in tube formation significantly decreased in the QRHXF group compared with the induced group (P<0.01). Notably, the serum levels of VEGFR-1 and VEGFR-2 were lower compared with the induced group (P<0.05 or P<0.01). In addition, the expressions of PI3K and p-Akt proteins were decreased in the middle- and high doses groups (P<0.01). This study's results suggest that the downstream mechanism of QRHXF anti-angiogenesis might inhibit the PI3K-Akt signalling pathway and downregulate VEGF-1 and VEGF-2.

Mechanism of Qingre Huoxue Fang treatment on inhibiting angiogenesis of rheumatoid arthritis based on network pharmacology and in vitro experiments.

This study aimed to explore the mechanism of Qingre Huoxue Fang (QRHXF) treatment on anti-angiogenesis in rheumatoid arthritis (RA) based on network pharmacology and in vitro experiments. We used the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Therapeutic Target (TTD) database to extract the active components of QRHXF and potential targets for regulating angiogenesis. First, we used Cytoscape bioinformatics software to construct the network of QRHXF-angiogenesis and screened the potential targets. Then, we performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis on the potential core targets. In addition, enzyme-linked immune assay and Western blot were used for in vitro validation and to verify the effects of different concentrations of QRHXF on the expression levels of the vascular endothelial growth factor receptor type 1 (VEGFR-1) and VEGFR-2 cytokines and phosphoinositide 3-kinase (PI3k) and Ak strain transforming (Akt) proteins in human umbilical vein endothelial cells (HUVECs). In results, we screened 179 core QRHXF antiangiogenic targets, including vascular endothelial growth factor (VEGF) cytokines. Enrichment analysis showed that the targets were enriched in 56 core signaling pathways, including PI3k and Akt. In vitro experiments showed that the migration distance and square, adhesion optical density (OD) values, and the number of branch points in tube formation significantly decreased in the QRHXF group compared with the induced group (P<0.01). Notably, the serum levels of VEGFR-1 and VEGFR-2 were lower compared with the induced group (P<0.05 or P<0.01). In addition, the expressions of PI3K and p-Akt proteins were decreased in the middle- and high doses groups (P<0.01). This study's results suggest that the downstream mechanism of QRHXF anti-angiogenesis might inhibit the PI3K-Akt signalling pathway and downregulate VEGF-1 and VEGF-2.

VEGFR2 insufficiency enhances phosphotoxicity and undermines Klotho's protection against peritubular capillary rarefaction and kidney fibrosis.

Vascular endothelial growth factor (VEGF) and its cognate receptor (VEGFR2) system are crucial for cell functions associated with angiogenesis and vasculogenesis. Klotho contributes to vascular health maintenance in the kidney and other organs in mammals, but it is unknown whether renoprotection by Klotho is dependent on VEGF/VEGFR2 signaling. We used heterozygous VEGFR2-haploinsufficient (VEGFR2+/-) mice resulting from heterozygous knockin of green fluorescent protein in the locus of fetal liver kinase 1 encoding VEGFR2 to test the interplay of Klotho, phosphate, and VEGFR2 in kidney function, the vasculature, and fibrosis. VEGFR2+/- mice displayed downregulated VEGF/VEGFR2 signaling in the kidney, lower density of peritubular capillaries, and accelerated kidney fibrosis, all of which were also found in the homozygous Klotho hypomorphic mice. High dietary phosphate induced higher plasma phosphate, greater peritubular capillary rarefaction, and more kidney fibrosis in VEGFR2+/- mice compared with wild-type mice. Genetic overexpression of Klotho significantly attenuated the elevated plasma phosphate, kidney dysfunction, peritubular capillary rarefaction, and kidney fibrosis induced by a high-phosphate diet in wild-type mice but only modestly ameliorated these changes in the VEGFR2+/- background. In cultured endothelial cells, VEGFR2 inhibition reduced free VEGFR2 but enhanced its costaining of an endothelial marker (CD31) and exacerbated phosphotoxicity. Klotho protein maintained VEGFR2 expression and attenuated high phosphate-induced cell injury, which was reduced by VEGFR2 inhibition. In conclusion, normal VEGFR2 function is required for vascular integrity and for Klotho to exert vascular protective and antifibrotic actions in the kidney partially through the regulation of VEGFR2 function.NEW & NOTEWORTHY This research paper studied the interplay of vascular endothelial growth factor receptor type 2 (VEGFR2), high dietary phosphate, and Klotho, an antiaging protein, in peritubular structure and kidney fibrosis. Klotho protein was shown to maintain VEGFR2 expression in the kidney and reduce high phosphate-induced cell injury. However, Klotho cytoprotection was attenuated by VEGFR2 inhibition. Thus, normal VEGFR2 function is required for vascular integrity and Klotho to exert vascular protective and antifibrotic actions in the kidney.

Transient Exposure of Endothelial Cells to Doxorubicin Leads to Long-Lasting Vascular Endothelial Growth Factor Receptor 2 Downregulation.

Doxorubicin (Dox) is an effective antineoplastic drug with serious cardiotoxic side effects that persist after drug withdrawal and can lead to heart failure. Dysregulation of vascular endothelium has been linked to the development of Dox-induced cardiotoxicity, but it is unclear whether and how transient exposure to Dox leads to long-term downregulation of Endothelial Vascular Endothelial Growth Factor Receptor type2 (VEGFR2), essential for endothelial cells function. Using an in vitro model devised to study the long-lasting effects of brief endothelial cells exposure to Dox, we show that Dox leads to sustained protein synthesis inhibition and VEGFR2 downregulation. Transient Dox treatment led to the development of long-term senescence associated with a reduction in VEGFR2 levels that persisted days after drug withdrawal. By analyzing VEGFR2 turnover, we ruled out that its downregulation was depended on Dox-induced autophagy. Conversely, Dox induced p53 expression, reduced mTOR-dependent translation, and inhibited global protein synthesis. Our data contribute to a mechanistic basis to the permanent damage caused to endothelial cells by short-term Dox treatment.

Ocimum sanctum Linn. Extract Improves Cognitive Deficits in Olfactory Bulbectomized Mice via the Enhancement of Central Cholinergic Systems and VEGF Expression.

This study aimed to clarify the antidementia effects of ethanolic extract of Ocimum sanctum Linn. (OS) and its underlying mechanisms using olfactory bulbectomized (OBX) mice. OBX mice were treated daily with OS or a reference drug, donepezil (DNP). Spatial and nonspatial working memory performance was measured using a modified Y maze test and a novel object recognition test, respectively. Brain tissues of the animals were subjected to histochemical and neurochemical analysis. OS treatment attenuated OBX-induced impairment of spatial and nonspatial working memories. OBX induced degeneration of septal cholinergic neurons, enlargement of the lateral ventricles, and suppression of hippocampal neurogenesis. OS and DNP treatment also depressed these histological damages. OS administration reduced ex vivo activity of acetylcholinesterase in the brain. OBX diminished the expression levels of genes coding vascular endothelial growth factor (VEGF) and VEGF receptor type 2 (VEGFR2). Treatment with OS and DNP reversed OBX-induced decrease in VEGF gene and protein expression levels without affecting the expression of the VEGFR2 gene. These results demonstrate that the administration of OS can lessen the cognitive deficits and neurohistological damages of OBX and that these actions are, at least in part, mediated by the enhancement of central cholinergic systems and VEGF expression.

Cyclooxygenase inhibition attenuates brain angiogenesis and independently decreases mouse survival under hypoxia.

Although cyclooxygenase (COX) role in cancer angiogenesis has been studied, little is known about its role in brain angioplasticity. In the present study, we chronically infused mice with ketorolac, a non‐specific COX inhibitor that does not cross the blood–brain barrier (BBB), under normoxia or 50% isobaric hypoxia (10% O2 by volume). Ketorolac increased mortality rate under hypoxia in a dose‐dependent manner. Using in vivo multiphoton microscopy, we demonstrated that chronic COX inhibition completely attenuated brain angiogenic response to hypoxia. Alterations in a number of angiogenic factors that were reported to be COX‐dependent in other models were assayed at 24‐hr and 10‐day hypoxia. Intriguingly, hypoxia‐inducible factor 1 was unaffected under COX inhibition, and vascular endothelial growth factor receptor type 2 (VEGFR2) and C‐X‐C chemokine receptor type 4 (CXCR4) were significantly but slightly decreased. However, a number of mitogen‐activated protein kinases (MAPKs) were significantly reduced upon COX inhibition. We conclude that additional, angiogenic factor‐independent mechanism might contribute to COX role in brain angioplasticity, probably including mitogenic COX effect on endothelium. Our data indicate that COX activity is critical for systemic adaptation to chronic hypoxia, and BBB COX is essential for hypoxia‐induced brain angioplasticity. These data also indicate a potential risk for using COX inhibitors under hypoxia conditions in clinics. Further studies are required to elucidate a complete mechanism for brain long‐term angiogenesis regulation through COX activity.

Detection and quantification of VEGFR-1 in the nuclei of tumor cells by a new flow cytometry-based method

Flow cytometry using fluorescent antibodies (FC) is the method of choice for the quantitation of proteins expressed at the surface or inside the cell, but, however, does not allow to selectively measure nuclear expression. We therefore sought to develop a method for the extraction of intact cell nuclei, which can be used for their subsequent immunofluorescent analysis by FC. The studied protein was vascular endothelial growth factor-receptor-type 1 (VEGFR-1) which is important in tumor survival and metastasis. Two human cell lines, A431 (epidermoid carcinoma of skin with low invasive and metastatic potential) and BRO (highly aggressive amelanotic melanoma), were used as examples for tumor cells, and normal human fibroblasts PHF served as a control line. The quality of the extracted nuclei was assessed by their intactness and purity from cytoplasm. The high content of the nuclear markers (PCNA = proliferating cell nuclear antigen, lamin A/C) in the extracted nuclei with almost complete absence of the cytoplasmic β-tubulin demonstrated that the protocol can be used to obtain a pure suspension of single intact cell nuclei. The measurement of the nuclear VEGFR-1 content revealed that it was present only in tumor cell nuclei and that in more malignant BRO cells the receptor content was 1.75 times higher than in A431 (p = 0.014). Thus, the developed method of extraction of cell nuclei for subsequent FC analysis is suitable for the quantitative evaluation of protein content in the native nucleus.

Are the Cognitive Alterations Present in Children Born From Preeclamptic Pregnancies the Result of Impaired Angiogenesis? Focus on the Potential Role of the VEGF Family.

Evidence from clinical studies has proposed that children born from preeclamptic women have a higher risk of suffering neurological, psychological, or behavioral alterations. However, to date, the mechanisms behind these outcomes are poorly understood. Here, we speculate that the neurodevelopmental alterations in the children of preeclamptic pregnancies result from impaired angiogenesis. The pro-angiogenic factors vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) are key regulators of both vascular and neurological development, and it has been widely demonstrated that umbilical blood of preeclamptic pregnancies contains high levels of soluble VEGF receptor type 1 (sFlt-1), a decoy receptor of VEGF. As a consequence, this anti-angiogenic state could lead to long-lasting neurological outcomes. In this non-systematic review, we propose that alterations in the circulating concentrations of VEGF, PlGF, and sFlt-1 in preeclamptic pregnancies will affect both fetal cerebrovascular function and neurodevelopment, which in turn may cause cognitive alterations in post-natal life.

Abstract 3919: Targeting urease to human VEGFR2 elicits antitumor activity in triple-negative breast cancer models

Abstract Introduction: Vascular endothelial growth factor receptor type 2 (VEGFR2) expression is one of the most prominent biomarkers of the tumor-associated neovasculature. Moreover, VEGFR2 can be aberrantly expressed on the surface of tumor cells. We have previously described the development of V21-DOS47, an immunoconjugate composed of the VHH-portion of a camelid single domain anti-VEGFR2 antibody (V21H4) and jack bean urease, which converts endogenous urea into ammonia and hydroxyl ions. V21-DOS47 is the second in a class of antibody-urease drugs. The first, L-DOS47, is currently in clinical testing for non-small cell lung cancer. In this study, we identified tumor cells with VEGFR2 expression, tested V21-DOS47 binding to tumor cells, and investigated in vivo activity in both immunocompetent and immunodeficient mice. Methods: Flow cytometry experiments were performed with biotinylated V21H4 antibody and anti-biotin fluorochrome-conjugated secondary antibody. Detection of urease by flow cytometry was performed with an anti-urease antibody followed by incubation with a fluorochrome-conjugated secondary antibody. Western blotting was used for the assessment of protein expression. For in vivo experiments, VEGFR2-overexpressing derivatives of human MDA-MB-231 and murine 4T1 breast cancer cell lines were generated. Balb/c or nude mice were inoculated with tumor cells: 2.5 x 105 4T1 or 1x106 MDA-MB-231, respectively, on Day 0 of each experiment. Intravenous injections of V21-DOS47 at a dose of 10 µg/kg were started on Day 3 and continued on Days: 5, 7, 9 and 11. Results: After screening human tumor cell lines for VEGFR2 expression by RT-PCR and Western blotting, we determined that two triple-negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) expressed the highest levels of VEGFR2. Binding of V21H4 to the cell surface of both cell lines was confirmed by flow cytometry. In vivo studies were conducted using both immunodeficient (with human MDA-MB-231-hVEGFR2 cells) and immunocompetent (with murine 4T1-hVEGFR2 cells) mice. In the syngeneic/immunocompetent model of Balb/c mice implanted with 4T1-hVEGFR2 cells, but not wild-type 4T1 cells, the antitumor effect of V21-DOS47 was significant and long-lasting. However, the results obtained from the initial experiment in the MDA-MB-231-hVEGFR2 model suggest that the antitumor effect of V21-DOS47 is transient in immunodeficient mice. Conclusions: Our data show successful targeting of the DOS47 platform to human VEGFR2 expressed on tumor cells. Our in vivo data indicate that the antitumor activity of V21-DOS47 is enhanced in immunocompetent mice, which suggests that the immune system is a significant component of the antitumor activity of the V21-DOS47 immunoconjugate. Citation Format: Angelika Muchowicz, Anna Bujak, Beata Pyrzynska, Justyna Karolczak, Abdessamad Zerrouqi, Magdalena Ozga, Lukasz Szewczyk, Baomin Tian, Wah Wong, Marni Uger, Katarzyna Poplawska, Dorota Gierej, Pawel Wisniewski, Heman Chao, Magdalena Winiarska, Radoslaw Zagozdzon. Targeting urease to human VEGFR2 elicits antitumor activity in triple-negative breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3919.

Ultrasound molecular imaging of breast cancer in MCF-7 orthotopic mice using gold nanoshelled poly(lactic-co-glycolic acid) nanocapsules: a novel dual-targeted ultrasound contrast agent.

BackgroundThe development of nanoscale molecularly targeted ultrasound contrast agents (UCAs) with high affinity and specificity is critical for ultrasound molecular imaging in the early detection of breast cancer.PurposeTo prospectively evaluate ultrasound molecular imaging with dual-targeted gold nanoshelled poly(lactide-co-glycolic acid) nanocapsules carrying vascular endothelial growth factor receptor type 2 (VEGFR2) and p53 antibodies (DNCs) in MCF-7 orthotopic mice model.MethodsDNCs were fabricated with an inner PLGA and outer gold nanoshell spherical structure. Its targeting capabilities were evaluated by confocal laser scanning microscopy (CLSM) and flow cytometry (FCM) in vitro. Contrast-enhanced ultrasound imaging (CEUS) with DNCs was evaluated qualitatively and quantitatively in vitro and in MCF-7 orthotopic mice model by two different systems. The biodistribution of NCs in mice was preliminary investigated. Differences were calculated by using analysis of variance.ResultsDNCs showed a well-defined spherical morphology with an average diameter of 276.90±110.50 nm. In vitro, DNCs exhibited high target specificities (79.01±5.63% vs. 2.11±1.07%, P<0.01; 75.54±6.58% vs. 5.21±3.12%, P<0.01) in VEGFR2- and p53-positive cells compared with control cells. In vivo, CEUS displayed a significantly higher video intensity in two systems using DNCs in comparison with non-targeted PLGA@Au NCs and single-targeted NCs. Biodistribution studies revealed that more DNCs in breast cancer tissue could be detected in mice than in other NCs (P<0.05).ConclusionDNCs were demonstrated to be novel dual-targeted UCAs and may have potential applications in early non-invasive visualization of breast cancer.

Glutamate Neonatal Excitotoxicity Modifies VEGF-A, VEGF-B, VEGFR-1 and VEGFR-2 Protein Expression Profiles During Postnatal Development of the Cerebral Cortex and Hippocampus of Male Rats.

Vascular endothelial growth factor (VEGF) exerts both neuroprotective and proinflammatory effects in the brain, depending on the VEGF (A-E) and VEGF receptor (VEGFR1-3) types involved. Neonatal monosodium glutamate (MSG) treatment triggers an excitotoxic degenerative process associated with several neuropathological conditions, and VEGF messenger RNA (mRNA) expression is increased at postnatal day (PD) 14 in rat hippocampus (Hp) following the treatment. The aim of this work was to establish the changes in immunoreactivity to VEGF-A, VEGF-B, VEGFR-1 and VEGFR-2 proteins induced by neonatal MSG treatment (4g/kg, subcutaneous, at PD1, 3, 5 and 7) in the cerebral motor cortex (CMC) and Hp. Samples collected from PD2 to PD60 from control and MSG-treated male Wistar rats were assessed by western blotting for each protein. Considering that immunoreactivity measured by western blotting is related to the protein expression level, we found that each protein in each cerebral region has a specific expression profile throughout the studied ages, and all profiles were differentially modified by MSG. Specifically, neonatal MSG treatment significantly increased the immunoreactivity to the following: (1) VEGF-A at PD8-PD10 in the CMC and at PD6-PD8 in the Hp; (2) VEGF-B at PD2, PD6 and PD10 in the CMC and at PD8-PD9 in the Hp; and (3) VEGFR-2 at PD6-PD8 in the CMC and at PD21-PD60 in the Hp. Also, MSG significantly reduced the immunoreactivity to the following: (1) VEGF-B at PD8-PD9 and PD45-PD60 in the CMC; and (2) VEGFR-1 at PD4-PD6 and PD14-PD21 in the CMC and at PD4, PD9-PD10 and PD60 in the Hp. Our results indicate that VEGF-mediated signalling is involved in the excitotoxic process triggered by neonatal MSG treatment and should be further characterized.

ERK signaling is required for VEGF-A/VEGFR2-induced differentiation of porcine adipose-derived mesenchymal stem cells into endothelial cells

BackgroundCell-based therapy that can rejuvenate the endothelium with stimulated adipose-derived mesenchymal stem cells (AMSCs) is a promising therapeutic strategy for the re-endothelialization of denuded arteries at the stenting site. Previously, we have shown that silencing of MMP-2 and MMP-14 inhibits vascular endothelial growth factor receptor type 2 (VEGFR2) cleavage, and induces differentiation of AMSCs toward the endothelial cell (EC) lineage. In this study, we examined the underlying signaling pathways that regulate differentiation of AMSCs to ECs in vitro through VEGFR2.MethodsAMSCs were isolated from porcine abdominal adipose tissue. The isolated AMSCs were characterized by positive expression of CD29, CD44, and CD90 and negative expression of CD11b and CD45. The isolated MSCs were transfected with siRNA to silence MMP-2, MMP-14, and angiotensin receptor 2 (ATR2). Cells were suspended either in endothelial basal media (EBM) or endothelial growth media (EGM) with various treatments. Flow cytometry was performed to examine the expression of EC markers, and western blot analysis was performed to examine the expression and activity of various kinases. Scratch assay was performed to examine the cell migration. Data were analyzed by ANOVA using PRISM GraphPad.ResultsAfter 10 days of stimulation for EC differentiation, the morphology of AMSCs changed to a morphology similar to that of ECs. Silencing MMP-2 and MMP-14 resulted in significant decrease in the number of migrated cells compared with the EGM-only group. ATR2 siRNA transfection did not affect the migration and differentiation of AMSCs to ECs. Stimulation of AMSCs for EC differentiation with or without MMP-2 or MMP-14 siRNA resulted in significant increase in p-ERK, and significant decrease in p-JNK. There was no significant change in p-p38 in all three groups compared with the EBM group. ERK inhibition resulted in significant decrease in the expression of EC markers in the EGM, EGM + MMP-2 siRNA, and EGM + MMP-14 siRNA groups. The VEGFR2 kinase inhibitor induced a dose-dependent inhibition of ERK.ConclusionThe ERK signaling pathway is critical for VEGF-A/VEGFR2-induced differentiation of AMSCs into ECs. These findings provide new insights into the role of the ERK signaling pathway in AMSC differentiation to ECs for potential clinical use in cardiovascular diseases.

MMP-2 and MMP-14 Silencing Inhibits VEGFR2 Cleavage and Induces the Differentiation of Porcine Adipose-Derived Mesenchymal Stem Cells to Endothelial Cells.

The molecular mechanisms that control the ability of adipose‐derived mesenchymal stem cells (AMSCs) to remodel three‐dimensional extracellular matrix barriers during differentiation are not clearly understood. Herein, we studied the expression of matrix metalloproteinases (MMPs) during the differentiation of AMSCs to endothelial cells (ECs) in vitro. MSCs were isolated from porcine abdominal adipose tissue, and characterized by immunopositivity to CD44, CD90, CD105, and immunonegativity to CD14 and CD45. Plasticity of AMSCs was confirmed by multilineage differentiation. The mRNA transcripts for MMPs and Tissue Inhibitor of Metalloproteinases (TIMPs), and protein expression of EC markers were analyzed. The enzyme activity and protein expression were analyzed by gelatin zymography, enzyme‐linked immunosorbent assay (ELISA), and Western blot. The differentiation of AMSCs to ECs was confirmed by mRNA and protein expressions of the endothelial markers. The mRNA transcripts for MMP‐2 and MMP‐14 were significantly increased during the differentiation of MSCs into ECs. Findings revealed an elevated MMP‐14 and MMP‐2 expression, and MMP2 enzyme activity. Silencing of MMP‐2 and MMP‐14 significantly increased the expression of EC markers, formation of capillary tubes, and acetylated‐low‐density lipoprotein uptake, and decreased the cleavage of vascular endothelial growth factor receptor type 2 (VEGFR2). Inhibition of VEGFR2 significantly decreased the expression of EC markers. These novel findings demonstrate that the upregulation of MMP2 and MMP14 has an inhibitory effect on the differentiation of AMSCs to ECs, and silencing these MMPs inhibit the cleavage of VEGFR2 and stimulate the differentiation of AMSCs to ECs. These findings provide a potential mechanism for the regulatory role of MMP‐2 and MMP‐14 in the re‐endothelialization of coronary arteries following intervention. Stem Cells Translational Medicine 2017;6:1385–1398

Vasohibin-1 Is a Poor Prognostic Factor of Ovarian Carcinoma.

Vasohibin-1 (VASH1) is an identified negative feedback inhibitor of angiogenesis induced by vascular endothelial growth factor (VEGF) in vascular endothelial cells (ECs). Expression of VASH1 has been reported not only in ECs of normal tissue, but also in ECs surrounding malignant tumors. In malignant tumors, VASH1 is also gaining attention as a prognosis prediction marker. The aim of this study is to investigate the correlation between VASH1 expression and vascular-related factors and various clinicopathological outcomes in clinical cases of ovarian carcinoma. We retrospectively analyzed clinical records of 58 patients with ovarian carcinoma. The expression patterns of VASH1 and other vascular-related factors (CD31 as markers of microvessel density (MVD), VEGF receptor type 2 (VEGFR2), D2-40 as markers of lymphovessel density), and Ki67 (as proliferation markers of cancer cells) were examined immunohistochemically. We studied the correlation between immunohistochemical expression and overall survival. VASH1 expression pattern significantly differed between Federation of Obstetrics and Gynecology (FIGO) Stages. Numbers of VASH1-positive vessels had a significant positive correlation with MVD (Speaman's correlation coefficient (ρ) was 0.51, p < 0.001), VEGFR2-positive vessels (ρ = 0.61, p < 0.001), and percentage of Ki67 (ρ = 0.28, p = 0.034). The Cox univariable analyses revealed that the group of high VASH1 expression (> 14.6 vessels per mm2) at Stages I-III is a prognostic factor (HR = 3.3, 95%CI = 0.4-8.4; p = 0.013). Our results indicate that VASH1 expression in ovarian carcinoma is significantly associated with vascular-related factors and Ki67 expression. We propose that VASH1 is a prognostic marker in ovarian carcinoma.

Vascular Endothelial Growth Factor Receptor Type 1 Signaling Prevents Delayed Wound Healing in Diabetes by Attenuating the Production of IL-1β by Recruited Macrophages

The persistence of proinflammatory macrophages, which are recruited to the granulation tissue, impairs the healing of diabetic wounds. Herein, we examined the role of vascular endothelial growth factor receptor type 1 (VEGFR1) signaling in streptozotocin (STZ)-induced diabetic wound healing. Angiogenesis, lymphangiogenesis, and the healing of full-thickness skin wounds were impaired in STZ-treated wild-type (WT) mice compared with vehicle-treated WT mice, with attenuated recruitment of VEGFR1-positive macrophages expressing vascular endothelial growth factor (VEGF)-A, VEGF-C, and VEGF-D to the wound granulation tissue. These phenomena were even more prevalent in STZ-treated VEGFR1 tyrosine kinase knockout mice (VEGFR1 TK(-/-) mice). STZ-treated WT mice, but not STZ-treated VEGFR1 TK(-/-) mice, showed accelerated wound healing when treated with placenta growth factor. Compared with that of STZ-treated WT mice, the wound granulation tissue of STZ-treated VEGFR1 TK(-/-) mice contained more VEGFR1-positive cells expressing IL-1β [a classic (M1) activated macrophage marker] and fewer VEGFR1-positive cells expressing the mannose receptor [CD206; an alternatively activated (M2) macrophage marker]. Treatment of STZ-treated VEGFR1 TK(-/-) mice with an IL-1β-neutralizing antibody restored impaired wound healing and angiogenesis/lymphangiogenesis and induced macrophages in the wound granulation tissue to switch to an M2 phenotype. Taken together, these results suggest that VEGFR1 signaling plays a role in regulating the balance between macrophage phenotypes in STZ-induced diabetic wounds, prevents impaired diabetic wound healing, and promotes angiogenesis/lymphangiogenesis.

Expression of Vascular Endothelial Growth Factor Has a Regulatory Role in Gingival Venules in Experimental Diabetes.

In the present study, the possible localization and role of vascular endothelial growth factor receptor type 2 (VEGFR2) in the regulation of gingival venules in a rat model of experimental diabetes are examined. Six weeks after streptozotocin premedication, Wistar male rats presenting blood sugar levels >20 mmol/L were selected for investigation. The VEGFR2 antagonist ZM323881 [5-((7-benzyloxyquinazolin-4-yl)amino)-4-fluoro-2-methylphenol-hydrochloride] (20 μg/mL) was dripped onto the gingiva between the mandibular incisors. Changes in diameter of the selected gingival venule were measured by vital microscopy combined with digital photography at specified times. Immunohistochemical staining was used to localize VEGFR2. For controls, the same protocol was used on animals with normal blood sugar levels and healthy gingiva. There was a significant difference between the baseline venule diameter of the diabetic and the control groups (47 ± 1 and 28 ± 2 μm, respectively). After 15, 30, and 60 minutes of local application of ZM323881, significant vasoconstriction was observed in the venules of diabetic rats compared with the baseline (81.4% ± 4.6%, 81.8% ± 4.4%, and 80.6% ± 5.1%, respectively). The control group showed no change in the venule diameter. The immunohistochemical analysis showed significantly increased VEGFR2 expression in the mast cells along the venules in the diabetic group, whereas mast cells were rarely found in the control group. The findings suggest that VEGF expression is increased in gingiva in experimentally induced diabetes. After VEGFR2 activation, the mast cell-derived vasodilatory and inflammatory mediators may contribute markedly to the concomitant changes in the microcirculation.

Antitumoral Effect of Sunitinib-eluting Beads in the Rabbit VX2 Tumor Model.

Purpose To measure plasmatic sunitinib concentration (PSC) and intratumoral sunitinib concentration (ITSC) after transcatheter arterial chemoembolization (TACE) with two different sizes of sunitinib-eluting beads (SEBs) in rabbits with VX2 hepatic allografts and to investigate treatment effects on vascular endothelial growth factor receptor type 2 (VEGFR2) phosphorylation, tumor volume, and histopathologic changes. Materials and Methods The protocol was approved by the French Ethics Committee for Animal Experiments (Comité d'Ethique en Expérimentation Animale du Centre INRA de Jouy-en-Josas et AgroParisTech, or COMETHEA, approval no. 11/028). Two experiments were performed. In the first, seven animals received 0.05 mL of 100-300-μm SEBs (1.5 mg of sunitinib) in the hepatic artery, and six animals received saline injections. In the second, eight animals received 0.05 mL of 70-150-μm SEBs (1.5 mg of sunitinib), seven received 0.05 mL of 70-150-μm unloaded beads, and seven received oral sunitinib (6 mg every day). Tumor size was monitored with ultrasonography. PSC, ITSC, and phosphorylation of VEGFR2 were assessed on days 1 and 14. After the animals were sacrificed, histopathologic analysis was performed. The Kruskal-Wallis test, Mann-Whitney U test, and Fisher exact test were used to look for statistically significant differences between groups. Results Maximum PSC after TACE with 100-300-μm SEBs was 0.002 μg/mL on day 1. ITSC was 17.8 μg/g on day 1 and 0.16 μg/g on day 14. After TACE with 70-150-μm SEBs, ITSC was 40.4 μg/g on day 1 and 27.4 μg/g on day 14. Phosphorylation of VEGFR2 was inhibited until day 14 after TACE with both sizes of SEBs. The size of VX2 tumors treated with 70-150-μm SEB TACE increased less (-2%) than that of tumors treated with unloaded beads (+42%) and oral sunitinib (6 mg every day; +1853%; P = .044). Conclusion SEB TACE resulted in minimal PSC, high ITSC, and sustained VEGFR2 phosphorylation inhibition until day 14. (©) RSNA, 2016.