Apoptosis Of HemECs Research Articles

Revealing the mechanism of 755-nm long-pulsed alexandrite laser in inhibiting infantile hemangioma endothelial cells through transcriptome sequencing

Laser therapy has shown promising outcomes in treating infantile hemangiomas. However, the molecular mechanisms underlying laser treatment for IH remain incompletely elucidated. This study aimed to unravel the molecular mechanisms of laser therapy in IH treatment. We evaluated the inhibitory effects of laser treatment on the proliferation and promotion of apoptosis in human hemangioma endothelial cells (HemECs) through cell counting kit-8 (CCK-8) assay, Hoechst 33342 staining, and flow cytometric analysis. Transcriptome sequencing analysis of HemECs following laser treatment revealed a significant decrease in the expression level of the GSTM5 gene. The qRT-PCR and western blot analysis also showed that GSTM5 expression in HemECs was downregulated compared to human umbilical vein endothelial cells (HUVECs), and concomitantly, the p62-Nrf2 pathway was suppressed. Using siRNA to downregulate GSTM5 expression, we observed that inhibiting GSTM5 expression could restrain cell proliferation, elevate intracellular ROS levels, and induce apoptosis in HemECs. Furthermore, upon inhibition of the p62-Nrf2 pathway using p62-specific siRNA, a significant decrease in GSTM5 expression and an elevation in intracellular ROS levels were noted in laser-treated HemECs. These findings suggested that laser treatment may operate by inhibiting the p62-Nrf2 pathway, thereby downregulating GSTM5 expression, elevating ROS levels, and consequently inducing apoptosis in HemECs.

Osmundacetone Inhibits Angiogenesis of Infantile Hemangiomas through Inducing Caspases and Reducing VEGFR2/MMP9.

This study aims to explore the potential of Osmundacetone (OSC) as a new treatment for infantile hemangiomas (IH), the most common benign tumors in infancy. Currently, propranolol serves as the primary treatment for IH, but its effectiveness is limited, and it poses challenges of drug resistance and side effects. Therefore, there is a pressing need to identify alternative therapies for IH. The effects of OSC on the proliferation and apoptosis of HemECs (endothelial cells from hemangiomas) were assessed using CCK-8 assay, colony formation assay, HOCHEST 33342 staining, and flow cytometry. Western blot analysis was performed to investigate OSC's influence on Caspases and angiogenesis-related proteins. Animal models were established using HemECs and BALB/c mice, and histological and immunohistochemical staining were conducted to evaluate the impact of OSC on mouse hemangiomas, VEGFR2, and MMP9 expression. OSC treatment significantly reduced HemECs' viability and colony-forming ability, while promoting apoptosis, as indicated by increased HOCHEST 33342 staining. OSC upregulated the protein expression of Bax, PARP, Caspase9, Caspase3, AIF, Cyto C, FADD, and Caspase8 in HemECs. In animal models, OSC treatment effectively reduced hemangioma size and improved histopathological changes. OSC also suppressed VEGFR2 and MMP9 expression while elevating Caspase3 levels in mouse hemangiomas. OSC demonstrated promising results in inhibiting HemECs' proliferation, inducing apoptosis, and ameliorating pathological changes in hemangiomas in mice. Moreover, it influenced the expression of crucial caspases and angiogenesis-related proteins. These findings suggest that OSC holds potential as a novel drug for clinical treatment of IH.

MicroRNA‑203a‑3p suppresses endothelial cell proliferation and invasion, and promotes apoptosis in hemangioma by inactivating the VEGF‑mediated PI3K/AKT pathway.

Our previous study demonstrated that microRNA-203a-3p (miR-203a-3p) was involved in the regulation of long non-coding RNA MEG8-mediated the progression of hemangioma, which is a benign tumor characterized by endothelial hyperplasia in the blood vessels and primarily occurring in infants and females. Therefore, the present study aimed to further investigate the effects of miR-203a-3p on endothelial cell proliferation, invasion and apoptosis, as well as its underlying mechanism in hemangioma. Human hemangioma endothelial cells (HemECs) were first transfected with either miR-203a-3p mimics or a miR-203a-3p inhibitor. Subsequently, vascular endothelial growth factor A (VEGFA) was overexpressed in these cells. Cell proliferation (by Cell Counting Kit-8 assay), apoptosis (by TUNEL assay), invasion (by Transwell assay) and PI3K/AKT signaling (by western blot) were assessed following transfection of these cells. Notably, transfection with miR-203a-3p mimics caused a reduction in cell proliferation, invasion and in the phosphorylation levels of PI3K and AKT, and promoted cell apoptosis in HemECs. By contrast, transfection with the miR-203a-3p inhibitor exerted the opposite effects compared with those of the miR-203a-3p mimics. miR-203a-3p was revealed to directly suppress VEGFA expression in HemECs. VEGFA overexpression alone increased cell proliferation and invasion, but decreased apoptosis. Furthermore, VEGFA co-transfection reversed the effects mediated by miR-203a-3p mimics transfection in HemECs. Mechanistically, miR-203a-3p was demonstrated to inactivate the PI3K/AKT pathway, whereas VEGFA overexpression produced the opposite effect. VEGFA co-transfection also attenuated the miR-203a-3p mimics-induced inactivation of PI3K/AKT signaling in HemECs. In conclusion, these data suggested that miR-203a-3p may inhibit endothelial cell proliferation and invasion, and promote apoptosis by inactivating VEGFA and PI3K/AKT signaling in hemangioma. These findings also implicated miR-203 as a possible treatment option for this disease.

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.

Cellular proliferation of Hemangioma endothelial cells (HemECs) under targeted regulation of LncRNA MALAT1 via miR-494-3p/PTEN Axis

The current study is aimed to explore the regulation of lncRNA MALAT1 in human HemECs functions. In our study, relative expressions of MALAT1, miR-494-3p and PTEN in HemECs (HemEC) were determined using qRT-PCR methods. MTT assays were used to measure cell viability. The rate of cell apoptosis was assessed using caspase-3 assay. Transfections were performed to mediate lncRNA MALAT1 and miR-494-3p expression in HemEc cells. Also, Bioinformatic analysis and Luciferase reporter were used to predict and validate the bindings between MALAT1 and PTEN, and PTEN and miR-494-3-p. MALAT1 was highly expressed in HemECs. Cell proliferation increased significantly due to MALAT1 overexpression in HemECs while MALAT1 overexpression significantly reduced cell apoptosis in HemECs. On the other hand, contradictory results were observed due to the reduction of MALAT1 in HemEC. We also found that MALAT1 interacts with miR-494-3p/PTEN to mediate cellular functions. Collectively, the results showed that the MALAT1 expression was negatively associated with miR-494-3p and positively matched the PTEN expression. In addition, MALAT1 acted as a ceRNA by binding with miR-494-3p to up-regulate PTEN in HemECs. Our study showed that MALAT1 accelerates the proliferation of HemEC cells by controlling the miR-494-3p/PTEN axis promoting new insight into IH treatment.

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.

Long noncoding RNA LINC00342 promotes growth of infantile hemangioma by sponging miR-3619-5p from HDGF.

Infantile hemangiomas (IH) are a type of benign vascular neoplasm that may cause permanent scarring. Hemangioma-derived endothelial cells (HemECs) are commonly used as an in vitro model to study IH. Long noncoding RNA is a type of RNA transcript longer than 200 nucleotides that does not encode any protein. LINC00342 was discovered to regulate proliferation and apoptosis in nonsmall cell lung cancer. However, the role of LINC00342 in IH has never been reported before. Expressions of LINC00342 and miR-3619-5p were detected in proliferating versus normal skin tissues. Colony formation and Cell-Couting Kit 8 assays were carried out to study the effects on cell proliferation after knockdown and overexpression of LINC00342, respectively. Meanwhile caspase-3 activity and nucleosomal fragmentation assay were applied to detect cell apoptosis. Micro-RNA binding sites on LINC00342 and hepatoma-derived growth factor (HDGF) were predicted and confirmed via dual-luciferase reporter assay. Biotin RNA pulldown assay was used to verify the direct binding between RNA molecules. LINC00342 enhanced proliferation and inhibited apoptosis in HemECs. MiR-3619-5p targeted both LINC00342 and HDGF, where LINC00342 sponged miR-3619-5p and positively regulated HDGF. HDGF knockdown rescued the effects of LINC00342 on HemECs. The LINC00342-miR-3619-5p-HDGF signaling pathway could regulate cell proliferation and apoptosis in HemECs.NEW & NOTEWORTHY The role of LINC00342 in infantile hemangiomas has not yet been elucidated. This paper highlights the regulatory role of LINC00342 in cell proliferation and apoptosis in hemangioma-derived endothelial cells and the underlying molecular mechanisms. The findings would provide potential target for treatment of infantile hemangiomas.

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.

LncRNA OIP5-AS1 Promotes the Proliferation of Hemangioma Vascular Endothelial Cells via Regulating miR-195-5p/NOB1 Axis.

Background: Hemangioma, which is a benign vascular neoplasm, has been demonstrated to be the most common tumor of infancy, resulting from aberrant proliferation of endothelial cells and pericytes.Objective: The aim of this study was to determine the role of lncRNA OIP5-AS1 on the proliferation and metastasis of human hemangioma endothelial cells (HemECs).Methods: In this study, we examined the expression of OIP5-AS1 and miR-195-5p in hemangiomas patients. The effects of knockdown or ectopic expression of OIP5-AS1 on the viability, proliferation and apoptosis of HemECs were explored. Rescue assays were performed to explore the effects of OIP5-AS1/miR-195-5p/NOB1 axis on biological behaviors in the HemECs.Results: It was found that OIP5-AS1 was overexpressed in both hemangioma tissues and HemECs cells. OIP5-AS1 knockdown suppressed the proliferation, migration, and invasion of HemECs cell, while overexpression of OIP5-AS1 had opposite effects LncRNA OIP5-AS1 acted as a ceRNA through binding with miR-195-5p to upregulate NOB1 in HemECs.Conclusion: Taken together, lncRNA OIP5-AS1 acted as a ceRNA to drive proliferation, migration and invasion of HemECs through regulating miR-195-5p/NOB1 axis.

Curcumin-induced apoptosis of hemangioma endothelial cells by downregulating expression of vascular endothelial growth factor and angiogenin 2

Objective To investigate the effect of curcumin on vascular endothelial growth factor (VEGF) and angiogenin (Ang)-2 in hemangioma endothelial cells (HemECs) and the apoptosis mechanism. Methods HemECs were separated and cultured by enzyme digestion, and were treated with 0, 6.25, 12.50, 25.00, 50.00 and 100.00 μmol/L of curcumin. Cell counting kit-8 (CCK-8) was used to detect the cell proliferation toxicity of HemECs. TdT-mediated dUTP nick end labeling (TUNEL) method was used to detect the apoptosis of HemECs. Real-time reverse transcriptase-polymerase chain reaction(RT-qPCR) was used to detect the mRNA expression of VEGF, Ang-1, and Ang-2. The cell immunofluorescence was used to detect the protein expression of vascular endothelial growth factor receptor (VEGFR)1, VEGFR2, endothelium-specific tyrosine kinase receptor (Tie)1 and Tie2. Results The survival rate of cells treated with 25.00 or 50.00 μmol/L curcumin for 48 h was significantly lower than that of cells treated with 25.00 or 50.00 μmol/L curcumin for 24 h [(62.34±10.08)% vs. (79.25±8.17)% and (15.23±4.01)% vs. (26.10±6.05)%; t=3.192, P=0.010 and t=3.668, P=0.004]. The number of TUNEL positive cells treated with 25.00 μmol/L curcumin for 48 h (16.00±2.80) was significantly greater than that after treatment with 0 μmol/L curcumin (5.00±1.40; t=8.607, P=0.000). The VEGF mRNA relative expression in HemECs treated with 25.00 μmol/L curcumin for 24 h (0.34±0.09) was significantly lower than that after treatment with 0 μmol/L curcumin (1.00±0.12; t=8.193, P=0.001). The Ang-2 mRNA relative expression in HemECs treated with 25.00 μmol/L curcumin for 24 h (0.45±0.07) was significantly lower than that after treatment with 0 μmol/L curcumin (0.99±0.12; t=8.193, P=0.001). The absorbance values of VEGFR2 and Tie2 protein expression in HemECs treated with 25 μmol/L curcumin for 24 h (0.30±0.05 and 0.30±0.04) were significantly lower than those after treatment with 0 μmol/L curcumin (0.48±0.09, 0.52±0.07; t=4.300, P=0.002; t=6.602, P=0.000). Conclusion Curcumin can inhibit the growth of HemECs by inducing apoptosis. The underlying mechanism may be closely related with down-regulations of VEGF and Ang-2. Key words: Hemangioma endothelial cells; Apoptosis; Vascular endothelial growth factor; Angiogenin 2

Curcumin induces apoptosis and inhibits proliferation in infantile hemangioma endothelial cells via downregulation of MCL-1 and HIF-1α.

Background:Curcumin has been used as an alternative medicine for the treatment of infantile hemangiomas (IHs); however, the mechanism underlying the effectiveness of curcumin in IHs remains largely unclear.Methods:In this study, we isolated primary human hemangioma endothelial cells (HemECs) from fresh surgical specimens of 3 patients. We treated HemECs by curcumin and investigated the alterations in proliferative and apoptotic signaling pathways with cell counting kit-8, flow cytometry, western blotting, immunofluorescence, and real-time polymerase chain reaction.Results and Conclusion:We found that curcumin potently inhibited proliferation in HemECs, achieving low-micromolar IC50 (the half maximal inhibitory concentration) value. We also observed that treatment with curcumin induced apoptosis in HemECs, as evidenced by positively Annexin-V-FITC staining, caspase-3 activation, and cleavage of poly(adenosine diphosphate-ribose) polymerase (PARP) in the treated cells. Moreover, we showed that curcumin suppressed the expression of antiapoptotic protein myeloid cell leukemia-1 (MCL-1), hypoxia-inducible factor 1α (HIF-1α), and vascular endothelial growth factor (VEGF).Altogether, our study suggests that the effectiveness of curcumin in IHs may be associated with its potent antiproliferative and apoptotic activities in HemECs.

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.