Proliferation Of HemECs Research Articles

Inhibition of infantile hemangioma growth and promotion of apoptosis via VEGF/PI3K/Akt axis by 755-nm long-pulse alexandrite laser

BackgroundInfantile hemangioma (IH) is a common vascular tumor in female infants, which can lead to aesthetic issues and facial scarring. This study aimed to investigate the inhibitory effects and underlying mechanisms of 755 nm long-pulsed alexandrite laser on IH. MethodsHemangioma endothelial cells (HemECs) were exposed to 755 nm long-pulsed alexandrite laser to evaluate its impact on cell proliferation and apoptosis. A patient-derived xenograft model was established to assess the inhibitory effects of laser treatment on IH in vivo. ResultsIn vitro, 755 nm long-pulsed alexandrite laser effectively suppressed the proliferation of HemECs and induced cell apoptosis. Laser treatment significantly inhibited the volume and weight of tumors, accompanied by significant downregulation of vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor receptor 2 (VEGFR2), phosphatidylinositol 3-kinase (PI3K), and protein kinase B (Akt) expression levels in both hemangioma cells and tumors. Additionally, laser treatment resulted in the conversion of VEGFA165a to VEGFA165b. TUNEL staining demonstrated increased apoptosis in tumor cells after laser treatment, along with upregulation of cleaved caspase-3 and Bax, and downregulation of Bcl-2. ConclusionIn addition to the principle of selective photothermal decomposition, modulation of the VEGF/PI3K/Akt axis may serve as a potential mechanism for IH treatment using a long pulse-width 755 nm laser. This sheds valuable light on the molecular mechanisms underlying IH pathogenesis and potential therapeutic targets while providing a theoretical basis for the safe and efficient management of proliferative IH using laser therapy.

Apolipoprotein A-I Binding Protein Inhibits the Formation of Infantile Hemangioma through Cholesterol-Regulated Hypoxia-Inducible Factor 1α Activation

Infantile hemangioma (IH) is the most frequent vascular tumor of infancy with unclear pathogenesis, of which disordered angiogenesis is considered to involve IH formation. We previously found that apolipoprotein A-I binding protein (AIBP)-also known as NAXE (NAD (P)HX epimerase)-a regulator of cholesterol metabolism, plays a critical role in the pathological angiogenesis of mammals. Here, we found that AIBP had much lower expression levels in both IH patients' tissues and hemangioma endothelial cells (HemECs), compared to adjacent normal tissues and human dermal vascular endothelial cells (HDMECs), respectively. Knockout of NAXE by CRISPR-Cas9 in HemECs enhanced tube formation and migration, and NAXE overexpression impaired tube formation and migration of HemECs. Interestingly, AIBP suppressed the proliferation of HemECs in hypoxia. We then found that reduced expression of AIBP correlated with increased Hif1α levels in IH patients' tissues and HemECs. Further mechanistic investigation demonstrated that AIBP disrupted Hif1α signaling via cholesterol metabolism under hypoxia. Notably, AIBP significantly inhibited the development of IH in immunodeficient mice. Furthermore, using the validated mouse endothelial cell (EOMA) and NAXE-/- mouse models, we demonstrated that both endogenous AIBP from tumors and AIBP in the tumor microenvironment limit the formation of hemangioma. These findings suggested that AIBP was a player in the pathogenesis of IH and could be a potential pharmacological target for treating IH.

Hemangioma Endothelial Cells and Hemangioma Stem Cells in Infantile Hemangioma.

Hemangioma is one of the most common benign tumors in infants and young children. The 2 most important cells in the course of infantile hemangioma (IH) are hemangioma stem cells (HemSCs) and hemangioma endothelial cells (HemECs). Infantile hemangioma is characterized by massive proliferation of HemECs, but current studies indicate that HemSCs play an important role in pathogenesis of IH. This review aimed to identify molecules that influence HemSC differentiation and HemEC proliferation and apoptosis to help clarify the pathogenesis of IH and provide novel drug targets for the treatment of IH. Relevant basic science studies related to IH were identified by searching Google Scholar, Embase, PubMed, MEDLINE, and peer-reviewed journal articles. Hemangioma stem cells can differentiate into HemECs, pericytes, and adipocytes. In the proliferating phase of IH, HemSCs mainly differentiate into HemECs and pericytes to promote angiogenesis. In the regressive phase, they mainly differentiate into adipocytes. Therefore, increasing the proportion of HemSCs differentiating into adipocytes, inhibiting the proliferation of HemECs, and promoting the apoptosis of HemECs can facilitate the regression of IH.

Propranolol inhibits the angiogenic capacity of hemangioma endothelia via blocking β-adrenoceptor in mast cell.

Propranolol, a non-selective blocker of the β-adrenoceptor (AR), is a first-line treatment for infantile hemangioma (IH). Mast cells have been implicated in the pathophysiology of propranolol-treated hemangioma. However, the function of mast cells remains unclear. HMC-1s (Human mast cell line) having been treated with propranolol for 24 h were centrifuged, washed with PBS twice, and maintained in cell culture medium for another 24 h. The supernatants with propranolol which were named as propranolol-treated HMC-1s supernatants were obtained. The expression of cytokines and mediators was examined among HMC-1s dealt with propranolol. HemECs (hemangioma endothelial cells) were co-cultured with propranolol-treated HMC-1s supernatants, and their proliferation and apoptosis were investigated. The autophagic-related protein was examined in HemECs using immunoblot. In propranolol-treated HMC-1s, the expressions of ADRB1 (β1-AR) and ADRB2 (β2-AR) were reduced by 70% and 60%, respectively, and that of cytokines and mediators were reduced. The proliferation was decreased, but apoptosis and autophagy were induced in HemECs treated with propranolol-treated HMC-1s supernatants. However, propranolol can work well in shRNA-ADRB1 or shRNA-ADRB2 transfected HMC-1s. Propranolol inhibit the proliferation of HemECs and promote their apoptosis and autophagy through acting on both β1 and β2 adrenoceptor in mast cell. Treated with propranolol, β1, and β2 adrenoceptor on human mast cell expression was reduced significantly. After hemangioma endothelial cell treated with the supernatants from propranolol-treated human mast cell, its proliferation was decreased, but apoptosis and autophagy were significantly induced. Propranolol can work well in shRNA-ADRB1 or shRNA-ADRB2 transfected HMC-1s. Mast cells may have a role in the action of propranolol in infantile hemangioma through both β1 and β2 adrenoceptors to inhibit the angiogenic capacity of hemangioma endothelial cells.

Effects of miR-125b-5p on the proliferation and apoptosis of human hemangioma endothelial cells HemES and its mechanism

Objective: This article mainly studies the effects of miR-125b-5p on the proliferation and apoptosis of human hemangioma endothelial cells HemECs. Methods: RT-qPCR was used to detect the expressions of miR-125b-5p and MCL-1 mRNA in HemECs and collateral cells of human hemangioma endothelial cells. HemECs were selected and divided into control group, miR-NC group, miR-125b-5p mimic group, miR-125b-5p inhibitor group, pc-MCL-1 group, miR-125b-5p+ pc-MCL-1 group, 9 multiple holes in each group. . HemECs were transfected with 100 nmol · L-1 of miR-NC, miR-125b-5p mimic, miR-125b-5p inhibitor, pc-MCL-1 plasmids separately or in combination. MTT method was used to detect the proliferation of HemECs. The apoptosis of HemECs was detected by flow cytometry. Dual luciferase report was used to to detect targeting relationship. The relative expression levels of Ki67, PCNA, cleaved caspase-3, Bax, Bcl-2, p-p70s6k/ p70s6k, p-AKT/AKT, and p-mTOR/mTOR proteins were detected by Western blot. Results: By comparing the expression levels of miR-125b-5p in hemangioma tissues and cells, HemECs cell lines with obvious down-regulation effects were selected for follow-up experiments. Compared with the control group, the proliferation of HemECs and the expressions of Ki67 and PCNA in the miR-125b-5p mimic group were decreased significantly (P＜0.01). The apoptotic rate of HemECs and the expression levels of cleaved Caspase-3 and Bax were increased significantly, while the expression of Bcl-2 was decreased significantly (P＜0.01). The expression levels of p-AKT/AKT, p-mTOR/mTOR and p-p70S6K/p70S6K were down-regulated significantly (P ＜0.01); the proliferation of HemECs and the expressions of Ki67 and PCNA in the miR-125b-5p inhibitor group were increased significantly (P ＜0.01); the apoptosis rate and the expressions of cleaved Caspase-3 and Bax were decreased significantly, and the expression of Bcl-2 was increased (P＜0.05, P＜0.01). miR-125b-5p targeted down-regulation of MCL-1. Compared with miR-125b-5p mimic group, the proliferation of HemECs and the expressions of Ki67 and PCNA in miR-125b-5p+ pc-MCL-1 group were increased significantly (P＜0.01), the apoptosis rate of HemECs and the expressions of cleaved Caspase-3 and Bax were decreased significantly, while the expression of Bcl-2 was increased (P＜0.01). The expressions of p-AKT/AKT, p-mTOR/mTOR, and p-p70S6K/p70S6K was also increased significantly (P＜0.01). Conclusion: miR-125b-5p inhibits the proliferation of human hemangioma endothelial cells and induces apoptosis. The mechanism may be related to the targeted down-regulation of MCL-1 expression and inhibition of AKT/mTOR pathway activation.

Silencing microRNA-210 in Hypoxia-Induced HUVEC-Derived Extracellular Vesicles Inhibits Hemangioma

Background: Hemangioma (Hem) is a benign tumor commonly seen in infancy with a relative high morbidity. Human umbilical vein endothelial cell (HUVEC)-derived extracellular vesicles (EVs) are actively participated in Hem. Therefore, this study is designed to figure out the underlying mechanism of HUVEC-derived EVs in Hem. Methods: Initially, EVs were separated from HUVECs and identified. HUVEC-derived EVs in normoxia or hypoxia were then cultivated with Hem endothelial cells (HemECs) to test the proliferation, apoptosis, and migration of HemECs. Microarray analysis was performed to select microRNAs (miRs) with differential expression. miR-210 in hypoxia-induced HUVECs was silenced, and the relevant EVs were extracted and then co-cultured with HemECs to perform biological effect experiments. Then, the target relation between miR-210 and homeobox A9 (HOXA9) was identified by the dual luciferase reporter gene assay and RNA immunoprecipitation assay. Moreover, xenograft transplantation was also applied to confirm the in vitro experiments. Results: Hypoxia-induced HUVECs promoted release of EVs, which were absorbed by HemECs. Hypoxia-induced HUVEC-EVs promoted HemEC proliferation and migration and inhibited apoptosis. miR-210 from the hypoxia-induced HUVEC-EVs was highly expressed and promoted HemEC growth. Silencing miR-210 expression in the hypoxia-induced HUVEC-EVs suppresses Hem development in vivo. In addition, miR-210 targeted HOXA9. Conclusion: Silencing miR-210 in HUVEC-derived EVs could suppress Hem by targeting HOXA9. This investigation may provide novel insights for Hem treatment.

Down-regulation of miR-556-3p inhibits hemangioma cell proliferation and promotes apoptosis by targeting VEGFC

The purpose of this study was to determine the effect of microRNA (miR)-556-3p on cell proliferation and apoptosis of hemangioma-derived endothelial cells (HemECs). qRT-PCR was used to compare the expressions of miR-556-3p in HemECs and normal cells. The target gene was identified using dual-luciferase reporter assay. Cell proliferation was measured with MTT assay, while western blotting was used to assay VEGFC expression levels. Apoptosis was assayed with FITC Annexin V Apoptosis assay kit. miR-556-3p was overexpressed in HemECs. Transfection with miR-556-3p inhibitor resulted in decreased proliferation of HemECs (p<0.05). The expression of the target gene of miR-556-3pi.e. VEGFC was upregulated when miR-556-3p inhibitor was transfected. The transfection also resulted in increased apoptosis. In HemECs, miR-556-3p is overexpressed and VEGFC expression is low. Thus, miR-556-3p or VEGFC might be potential targets for treatment of angiosarcoma.

Propranolol inhibits proliferation and induces apoptosis of hemangioma-derived endothelial cells via Akt pathway by down-regulating Ang-2 expression

Hemangioma is one of the commonest benign vascular tumors among children. Propranolol is the first-line therapeutic drug for hemangioma. However, the effects and mechanisms of propranolol in hemangioma have not been thoroughly elaborated. In this study, the effects and mechanisms of propranolol were explored using hemangioma-derived endothelial cells (HemECs). The expression of GLUT1 were determined by immunofluorescence staining. qRT-PCR assay was conducted to detect the mRNA expressions of angiopoietin-2 (Ang-2) and Tie-2. Western blot assay was carried out to measure the protein levels of Ang-2, Tie-2, protein kinase-B (Akt) and phospholyrated-Akt (p-Akt). Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8) assay and Western blot of Ki67 protein level. Cell apoptosis was measured by flow cytometry analysis and Western blot of Bax and Bcl-2 levels. We found that propranolol inhibited proliferation and induced apoptosis in human umbilical vein endothelial cells (HUVECs) and HemECs. Moreover, propranolol inhibited the expressions of Ang-2 and Tie-2 in HUVECs and HemECs. Functional analysis revealed that Ang-2 attenuated the effects of propranolol on HemEC proliferation and apoptosis. Mechanistical analysis showed that propranolol inhibited the Akt pathway by regulating Ang-2 expression in HemECs. Futhermore, inhibition of the Akt pathway attenuated the effects of Ang-2 on proliferation and apoptosis in HemECs. In conclusion, propranolol inhibited proliferation and induced apoptosis of HemECs via Akt pathway by down-regulating Ang-2 expression, which contributes to our understanding on the pathogenesis of hemangioma and promotes the development of therapeutic approaches for hemangioma.

Lycium barbarum polysaccharide induced apoptosis and inhibited proliferation in infantile hemangioma endothelial cells via down-regulation of PI3K/AKT signaling pathway.

Lycium barbarum polysaccharide (LBP) has a variety of pharmacological and biological activities such as anti-inflammatory, antioxidation, anti-apoptosis, immune regulation and other pharmacological effects; however, the effect of LBP on infantile hemangioma (IH) was less reported. Primary human hemangioma endothelial cells (HemECs) were isolated from fresh surgical specimens of patients. HemECs was treated with LBP and the changes in proliferative and apoptotic signaling pathways were investigated by performing cell counting kit-8, cloning formation experiment, in vitro angiogenesis experiment, flow cytometry, Western blot, immunofluorescence, HE stain and real-time quantitative polymerase chain reaction. We found that LBP potently inhibited the proliferation of HemECs and achieved a low-micromolar IC50 (45 and 40 μg/ml, the half maximal inhibitory concentration) value and less angiogenesis, however, the IC50 had no effect on human umbilical vein endothelial cells (HUVECs) viability. LBP treatment induced apoptosis in HemECs, which was supported by positive Annexin-V-FITC staining, the activation of cleaved caspase-3 and Bcl-2-associated X protein (Bax) and the inhibition of B-cell lymphoma/leukemia-2 (Bcl-2). Moreover, the result demonstrated that LBP suppressed the expressions of proliferating cell nuclear antigen (PCNA), Ki67, vascular endothelial growth factor (VEGF), VEGFR2 and phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signal pathway. PI3K-specific agonist (IGF-1) had promotive effects on HemECs proliferation, which was reversed by LBP. Our study suggests that the effectiveness of LBP in IHs may be associated with its potent anti-proliferative and apoptotic activities in HemECs. Thus, our findings may provide an effective medicine for IHs treatment.

Propranolol inhibits proliferation and invasion of hemangioma-derived endothelial cells by suppressing the DLL4/Notch1/Akt pathway

Infantile hemangioma (IH) is one of the most common benign vascular tumors of infancy. Propranolol has been recently introduced for the treatment of IH. However, the mechanism of protective effect has not been fully understood. In this study, hemangioma-derived endothelial cells (HemECs) were isolated and treated with propranolol. The cell viability was measured by MTT assay, and the cell cycle arrest was detected using flow cytometry. Cell invasion was determined using transwell assay. The expressions of matrix metalloproteinase (MMP)-2, MMP-9, Delta-like 4 (DLL4), Notch1, Akt, p-Akt, and vascular endothelial growth factor (VEGF) were detected using western blot. HemECs were incubated with recombinant human DLL4 (rhDLL4) to investigate the role of DLL4/Notch1 in the effect of propranolol. The results showed that propranolol inhibited cell viability of HemECs in a time-dependent manner. Propranolol suppressed cell proliferation of HemECs by arresting cell progression at G0/G1 phase. Propranolol inhibited the invasion ability of HemECs and reduced the expression levels of MMP-2 and MMP-9 in HemECs. Besides, propranolol treatment blocked the DLL4/Notch1 and Akt signaling and inhibited VEGF expression in HemECs. Treatment with rhDLL4 activated the Akt signaling and attenuated the effect of propranolol on HemECs. Our data indicated that propranolol inhibited the cell proliferation and invasion of HemECs. The effect was possibly involved in the DLL4/Notch1/Akt signaling pathway.

MicroRNA-143 acts as a suppressor of hemangioma growth by targeting Bcl-2

Infantile hemangioma is the most common vascular tumor affecting infants, which is associated with clonal expansion of endothelial cells. The aim of this study is to determine the role of microRNA (miR)-143 in the growth and survival of hemangioma-derived endothelial cells (HemECs). We examined the expression of miR-143 in patients with proliferating-phase (n=10) and involuting-phase (n=8) hemangiomas. The effects of ectopic expression of miR-143 on the viability, proliferation, cell cycle distribution, and apoptosis of HemECs were explored. We also identified the target gene(s) that was involved in the activity of miR-143. It was found that proliferating hemangiomas had significantly (P<0.05) lower levels of miR-143 than involuting counterparts. Reexpression of miR-143 significantly reduced the viability and proliferation of HemECs, while knockdown of miR-143 led to an increase in the proliferation of HemECs. Moreover, overexpression of miR-143 arrested HemECs at the G0/G1 phase and promoted caspase-3-dependent apoptosis. At the molecular level, miR-143 overexpression significantly promoted the expression of p21 and p53 and reduced the expression of cyclin D1, CDK2, CDK4, and Bcl-2. Silencing of Bcl-2 phenocopied the effect of miR-143 overexpression on the proliferation and apoptosis of HemECs. Furthermore, co-expression of Bcl-2 reversed the growth-suppressive effect of miR-143 on HemECs. Taken together, miR-143 acts as a suppressor in the growth of HemECs, at least partially, through downregulation of Bcl-2. Reexpression of miR-143 may represent a potential therapeutic strategy for the treatment of proliferating hemangiomas.

Sildenafil suppresses the proliferation and enhances the apoptosis of hemangioma endothelial cells.

Treatment of infantile hemangiomas (IH) with propranolol was first reported in 2008. Regressions of lymphatic malformations combined with pulmonary hypertension was first reported in 2012 after three children received treatment with oral sildenafil, which serves as an antagonist of phosphodiesterase isoform-5 (PDE-5). A marked expression of endothelial cells in the cytoplasm of IH tissues was obtained in our previous study. Therefore, the present study hypothesized that the antagonist of PDE-5, sildenafil, may lead to the regression of hemangiomas. To assess this hypothesis, the proliferation and apoptosis of specimen-derived hemangioma endothelial cells (HemECs) was determined in vitro by an MTT assay and flow cytometry, respectively, following treatment with sildenafil. The potential mechanisms underlying the mRNA and protein expression levels of inhibitor of differentiation 1 (Id-1) were determined by reverse transcription-quantitative polymerase chain reaction and western blotting. The results demonstrated that 5 µM sildenafil suppressed the proliferation of HemECs and significantly enhanced the rate of apoptosis after 24 h. Additionally, the mRNA and protein expression levels of Id-1 were downregulated following treatment with sildenafil. Therefore, the present study concluded that PDE-5 may be a potential therapeutic target for hemangiomas and Id-1 may serve a vital role in the associated signaling transduction pathways.

Rapamycin inhibits the proliferation of endothelial cells in hemangioma by blocking the mTOR-FABP4 pathway

FABP4 is widely expressed in both normal and pathologic tissues. It promotes cell proliferation, survival and migration of endothelial cells, and therefore, angiogenesis. However, the role of FABP4 in hemangioma or hemangioma endothelial cells (HemECs) has not been explored. In this study, we investigated whether FABP4 directly regulates the proliferation of HemECs. The expression of cell cycle checkpoint genes was analyzed with the microarray data of human dermal microvascular endothelial cells (HDVECs) and infantile hemangioma endothelial cells. Real-time RT-PCR and western blotting were used to examine the expression of FABP4 in HemECs. Next, the FABP4 expression was inhibited in HemECs using siRNA or rapamycin and upregulated using retroviral transduction of HemECs to assess its influence on proliferation of HemECs. The microarray data showed that cell cycle checkpoint genes were upregulated in HemECs. Moreover, HemECs showed significantly higher proliferation rates than HDVECs. The expression of FABP4 and mTOR was increased in the HemECs. While FABP4 knockdown reduced the BrdU incorporation and cell number of HemECs as expected, cell proliferation was accelerated by FABP4 over-expression. Moreover, rapamycin (10nM) inhibited mTOR-FABP4 signaling and HemEC proliferation. Taken together, these results indicated that mTOR signaling pathway-activated FABP4 directly regulates the proliferation of endothelial cells in hemangioma. Rapamycin and inhibitors of FABP4 have therapeutic potential for treating infantile hemangiomas.

Effects of propranolol on the proliferation and apoptosis of hemangioma-derived endothelial cells

Background/PurposePropranolol, a non-selective beta-blocker, has recently been introduced as a novel treatment modality for proliferating hemangiomas. However, the mechanism of action of this therapy is unknown. In this study, we investigated propranolol in the etiology of hemangiomas that are associated with the proliferation and apoptosis of hemangioma-derived endothelial cells (HemECs). MethodsHemECs were isolated from freshly resected hemangioma specimens. We studied propranolol-treated HemECs in vitro. We measured the effect of propranolol on HemEC viability using the Cell Counting Kit-8 (CCK-8) assay and proliferation and apoptosis using a BrdU labeling assay, annexin-V-fluorescein isothiocyanate/propidium iodide flow cytometry, and Hoechst 33342 fluorescent staining. We explored the potential mechanisms of propranolol-induced HemEC dysfunction using western blot analysis, a caspase assay kit, and real-time quantitative PCR. ResultsWe observed that propranolol had inhibitory effects on the viability and proliferation of HemECs. HemEC apoptosis significantly increased with 100μM propranolol treatment. Vascular endothelial growth factor (VEGF) expression was down-regulated by propranolol in a dose-dependent manner. We also demonstrated activation of the caspase cascade, including caspase-9 and caspase-3 of the intrinsic pathway, and an increased p53 gene expression and Bax/Bcl-xL ratio in HemECs treated with 100μM propranolol. ConclusionsWe obtained novel data that suggests propranolol could inhibit HemEC proliferation and induce apoptosis. The effects were likely mediated through the suppression of VEGF expression, activation of caspase-9 and caspase-3, up-regulation of the pro-apoptotic genes p53 and Bax and down-regulation of the anti-apoptotic gene Bcl-xL.