Enhancer of Zeste Homolog 2 Induces Pulmonary Artery Smooth Muscle Cell Proliferation

Pulmonary Arterial Hypertension (PAH) is a progressively devastating disease characterized by excessive proliferation of the Pulmonary Arterial Smooth Muscle Cells (PASMCs). Studies suggest that PAH and cancers share an apoptosis-resistant state featuring excessive cell proliferation. The proliferation of cancer cells is mediated by increased expression of Enhancer of Zeste Homolog 2 (EZH2), a mammalian histone methyltransferase that contributes to the epigenetic silencing of target genes. However, the role of EZH2 in PAH has not been studied. In this study, it is hypothesized that EZH2 could play a role in the proliferation of PASMCs.In the present study, the expression patterns of EZH2 were investigated in normal and hypertensive mouse PASMCs. The effects of EZH2 overexpression on the proliferation of human PASMCs were tested. PASMCs were transfected with EZH2 or GFP using nucleofector system. After transfection, the cells were incubated for 48 hours at 37°C. Proliferation and cell cycle analysis were performed using flow cytometry. Apoptosis of PASMCs was determined using annexin V staining and cell migration was tested by wound healing assay.EZH2 protein expression in mouse PASMCs were correlated with an increase in right ventricular systolic pressure and Right Ventricular Hypertrophy (RVH). The overexpression of EZH2 in human PASMCs enhances proliferation, migration, and decrease in the rate of apoptosis when compared to GFP-transfected cells. In the G2/M phase of the EZH2 transfected cells, there was a 3.5 fold increase in proliferation, while there was a significant decrease in the rate of apoptosis of PASMCs, when compared to control.These findings suggest that EZH2 plays a role in the migration and proliferation of PASMCs, which is a major hallmark in PAH. It also suggests that EZH2 could play a role in the development of PAH and can serve as a potential target for new therapies for PAH

EZH2 overexpression reduces apoptosis in HPASMCs.

<p>HPASMCs cells were transfected with a plasmid expressing EZH2 or with GFP using 4D-Nucleofector system. Apoptosis was measured in transfected cells by using Annexin V staining. Propidium iodide (PI) incorporation was measured by flow cytometry to assess the mode of cell death. Relative fluorescence units represent the intensity of annexin V incorporation and PI incorporation was quantified. The noted experiments are representative of a minimum of four similar evaluations. ^*<i>P</i><0.05 compared with control.</p

Increased expression of EZH2 in PASMCs was associated with PAH progression in hypoxia-induced PAH mouse model.

<p>Five age and sex-matched C57BL/6 mice were exposed to room air (21% FiO2) or to hypoxia (10% FiO2) at sea level. Averaged (A) RVSP and (B) RV/LV+S in mice at serial time points (0, 3 and 6 weeks) under hypoxic conditions of 10% oxygen (ten readings per mouse over 1 h, 20 mice per group at each time point). (C) EZH2 protein expression by western blot at serial time points under hypoxic conditions of 10% oxygen. All experiments were repeated at least 3 independent times ^*<i>P</i><0.05 compared with 0 hrs.</p

EZH2 overexpression modulates HPASMC phenotype.

<p>The HPASMCs were transfected with EZH2 or control plasmid and expression levels of calponin, a SMC phenotypic marker was assessed for A) mRNA by real-time PCR and B) protein levels by Western blot. All experiments were repeated at least 3 times and graph plotted is an average of three independent experiments. P<0.005 compared to controls. C) EZh2 increases binding to calponin-1 promoter in HPASMCs. DNA fragments interacting with H3K27me3 were pulled down by anti H3K27me3 or normal rabbit antibody. The presence of Calponin promoter DNA was checked by PCR using calponin promoter specific primers (upper panel). MYOD1 promoter segment binding to H3K27me3 was used as a positive control (lower panel). Calponin promoter region was amplified in EZH2 transfected cells and immunoprecipitated with anti-H3K27me3 but not in vector transfected cells (lanes 2 and 5). The immnoprecipitation with normal rabbit IgG did not yield any PCR amplification (lanes 3 and 6).</p

EZH2 overexpression induces proliferation of HPASMCs.

<p>HPASMCs were transfected with a plasmid expressing EZH2 or control plasmids using 4D-Nucleofector system. A) EZH2 protein expression was analyzed by western blot (upper panel) and mRNA expression was analyzed by real-time PCR (lower panel) to determine the transfection efficiency. B) The transfected cells were fixed and labeled with PI after 48 hrs and analyzed for cell cycle by flow cytometry. Percentage number of cells in S and G2/M phase of the cell cycle is plotted (upper panel) and representative histograms from the cell cycle analysis of EZH2 and control GFP plasmid transfected cells are shown (lower panel). C) Real-time PCR analysis of cell proliferation markers expressed as normalized fold expression is shown. All experiments were repeated at least 3 times and graphs shown are average of three independent experiments.</p

EZH2 expression induced in proliferating HPASMCs.

<p>HPASMCs were serum starved (1% FBS treated) or 10% serum treated for 24 h, and then (A) EZH2 expression was detected by qRT–PCR. (<b>B</b>) EZH2 protein expression was analyzed in serum-starved or 10% serum-treated HPASMCs by Western blotting. All experiments were repeated at least 3 independent times. ^*<i>P</i><0.05 compared with 5% FBS.</p

EZH2 overexpression markedly increases cell migration in HPASMCs as measured by wound healing assay.

<p>The HPASMCs were transfected with EZH2 and GFP plasmid or only with GFP plasmid (Control). An artificial wound was created using a 1 ml pipette on a confluent monolayer of cells. A) Images were taken at 0 and 24 hrs after wound. B) Migration was quantified visually as the number of cells migrating in to the gap and average of three experiments is plotted.</p