Knockdown of VEGFR2 inhibits proliferation and induces apoptosis in hemangioma-derived endothelial cells

Abstract

Angiogenesis is a process of development and growth of new capillary blood vessels from pre-existing vessels. Angiogenic growth factors play important roles in the development and maintenance of some malignancies, of which vascular endothelial growth factor (VEGF)/VEGFR2 interactions are involved in proliferation, migration, and survival of many cancer cells. The aim of this study was to investigate the function of VEGFR2 in human hemangiomas (HAs). Using immunohistochemistry assay, we examined the expression levels of VEGF, VEGFR2, Ki-67, glucose transporter-1 (Glut-1), phosphorylated protein kinase B (p-AKT) and p-ERK in different phases of human HAs. Positive expression of VEGF, VEGFR2, Ki-67, Glut-1, p-AKT and p-ERK was significantly increased in proliferating phase HAs, while decreased in involuting phase HAs (P=0.001; P=0.003). In contrast, cell apoptotic indexes were decreased in proliferating phase HAs, but increased in involuting phase HAs (P<0.01). Furthermore, we used small hairpin RNA (shRNA)-mediated VEGFR2 knockdown in primary HA-derived endothelial cells (HemECs) to understand the role of VEGF/VEGFR2 signaling. Knockdown of VEGFR2 by Lv-shVEGFR2 inhibited cell viability and induced apoptosis in primary HemECs companied with decreased expression of p-AKT, p-ERK, p-p38MAPK and Ki-67 and increased expression of caspase-3 (CAS-3); Overexpression of VEGFR2 promoted cell viability and blocked apoptosis in Lv-VEGFR2-transfected HemECs. Taken together, our findings demonstrate that, increased expression of VEGFR2 is involved in the development of primary HemECs possibly through regulation of the AKT and ERK pathways, suggesting that VEGFR2 may be a potential therapeutic target for HAs.

lncRNA MEG3 Suppresses the Tumorigenesis of Hemangioma by Sponging miR-494 and Regulating PTEN/ PI3K/AKT Pathway

“…The characteristics of HDEC were listed as follows: the nucleus of HDEC was very clear, the cells showed long spindle and polygonal, and the "vessel cavity" structure could be formed. The positive expression of PCNA and VEGF was confirmed to be increased in proliferating phase HAs but decreased in involuting phase HAs as previously reported [5,6,23]. HDEC and CRL-2586 EOMA cell lines were cultured in Dulbecco's Modified Eagle's medium (DMEM, Sigma, Aldrich, USA) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS, Sigma, Aldrich, USA), 100U/ml of penicillin and 100μg/ml of streptomycin (Invitrogen, Carlsbad, CA, USA), and were placed in a humidified atmosphere containing 5% CO2 at 37°C.…”

“…Low expression of MEG3 acts as an independent factor for predicting poor prognosis in cancer patients [12,15,25,26], and the downregulation of MEG3 is attributed to its promoter hypermethylation in cancer [27][28][29], and the negative modulation by DNMT1 in glioma [30]. MEG3 and VEGF have the differential expression levels in HAs [5,18]. In the present study, we found that, MEG3 expression was decreased and harbored the negative association with VEGF expression in proliferating phase HAs rather than in the involuting phase HAs, suggesting that MEG3 might be a negative regulator of HAs proliferation.…”

“…Our previous studies showed that VEGF expression can be used to differentiate the proliferating and involuting phase HAs by immunohistochemistry analysis [5]. Therefore, the expression levels of MEG3 and VEGF were estimated in different phase HAs (n=15) by qRT-PCR analysis, which indicated that MEG3 expression was downregulated (Fig.…”

“…Cell morphological observation were performed as previously reported [4][5][6]23]. The characteristics of HDEC were listed as follows: the nucleus of HDEC was very clear, the cells showed long spindle and polygonal, and the "vessel cavity" structure could be formed.…”

“…The somatic mutations, such as von Hippel-Lindau (VHL) contribute to the development of HAs [3]. Our previous studies showed that insulin like growth factor 2 (IGF2)/IGF2R and vascular endothelial growth factor receptor 2 (VEGFR2) pathways promote HAs cell proliferation and reduce cell apoptosis [4,5] but pharmacological inhibition of hypoxia inducible factor-1 (HIF-1) results in the growth inhibition in HAs cells [6]. However, little knowledge is known about the molecular mechanisms underlying HAs growth.…”