Effects And Micro RNA Mechanisms Of 1α,25-Dihydroxyvitamin D3 On Pulmonary Arterial Hypertension

Chapter 1 1α, 25-Dihydroxyvitamin D3 prevents differentiation of human lung fibroblasts via micro RNA-27 b targeting vitamin D receptor Objective:1. To determine the role of 1,25(OH)2D3 in regulating differentiation of fibroblasts induced by TGF-β1. 2. To examine the role of mi R-27 b in regulating differentiation of fibroblasts. 3. To investigate target gene and the relevant mechanisms of mi R-27 b. Methods: 1. Cell medical intervention: the cells were divided into three groups: control group,TGF-β1 group and TGF-β1+ VD group. MRC5 cells were cultured to about 70-80%confluence and serum-starved for 24 h, and then they were treated for 48 h in 2% FBSmedium with ethanol vehicle or 1,25(OH)2D3 in the absence or presence of rh TGF-β1.TGF-β1+ VD group were treated with 1,25(OH)2D3(100 n M) and rh TGF-β1(10ng/ml). TGF-β1 group were treated with ethanol vehicle and rh TGF-β1(10 ng/ml).Control group were treated with ethanol and PBS vehicles. 2. Total RNA and total protein of cells were extracted. The levels of α-SMA and VDRwere determined by QRT-PCR, immunofluorescence and western blotting analysis,normalized to β-actin expression. 3. Mi RNAs of cells were extracted. The expression of mi R-27 b was assessed byQRT-PCR and normalized to U6 expression. 4. To determine whether mi R-27 b regulates the differentiation phenotype of thepulmonary fibroblasts, we transfected human lung fibroblasts with scramble, mi R-27bmimic or mi R-27 b inhibitor and evaluated α-SMA levels in these cells. We furtherinvestigated whether or not mi R-27 b regulates VDR gene expression. MRC5 cellswere transfected with scramble, mi R-27 b mimic or mi R-27 b inhibitor and VDR levelswere evaluated in these cells. 5. We sought to further substantiate that mi R-27 b targets VDR 3’UTR directly.Luciferase reporter constructs were used, incorporating a wild-type or mutant 3’UTRof VDR in which the sequence corresponding to the seed region was altered. Thereporter vectors were then co-transfected into 293 A cells with scramble, mi R-27bmimic or mi R-27 b mimic/mi R-27 b inhibitor and the luciferase activity of these cellswere detected. Results: 1. Effects of 1,25(OH)2D3 on differentiation and VDR protein expression of human lungfibroblasts induced by TGF-β1: TGF-β1 significantly up-regulated α-SMA expressionsat the m RNA and protein levels in MRC5 cells, but 1,25(OH)2D3 could markedlyinhibit this effect. Furthermore, TGF-β significantly decreased the protein expressionof VDR, and treatment of TGF-β-stimulated fibroblasts with 1,25(OH)2D3 effectivelyup-regulated VDR protein level. However, the levels of the VDR transcripts did notchange. 2. Effects of 1,25(OH)2D3 on TGF-β1-induced mi R-27 b expression in human lungfibroblasts: mi R-27 b expression levels were found to be significantly higher in MRC5cells induced by TGF-β1. However, treatment of TGF-β-stimulated fibroblasts with1,25(OH)2D3 effectively decreased mi R-27 b expression.3. mi R-27 b regulates differentiation and VDR expression of human lung fibroblasts.Overexpression of mi R-27 b markedly increased the baseline levels of the α-SMAtranscripts and α-SMA proteins in lung fibroblasts. The mi R-27 b inhibitor decreasedthe α-SMA levels in lung fibroblasts. Furthermore, mi R-27 b inhibitor attenuatedTGF-β1-induced α-SMA expression at both m RNA and protein levels in lungfibroblasts. The reduced expression of α-SMA protein in 1,25(OH)2D3-treated cellswas attenuated by mi R-27 b mimic.mi R-27 b mimic reduced VDR protein levels in lung fibroblasts. The mi R-27 b inhibitorincreased the VDR protein levels in these cells. However, mi R-27 b had no effect on thelevels of the VDR transcripts. Furthermore, the reduced expression of VDR protein inTGF-β1-treated cells was attenuated by mi R-27 b inhibitor. The mi R-27 b mimicresulted in an decrease of VDR protein expression in lung fibroblasts treated with1,25(OH)2D3. Likewise, mi R-27 b had no effect on the levels of the VDR transcripts. 4. Effects of mi R-27 b on targeting VDR 3’UTR: mi R-27 b mimic significantly decreasedluciferase activity and introduction of the mi R-27 b inhibitor increased luciferaseactivity. We then mutated mi R-27 b target site to confirm that mi R-27 b was binding tothis sequence. Interestingly, the effects of mi R-27 b mimic or mi R-27 b inhibitor wereessentially abolished. Conclusion:1. 1,25(OH)2D3(100 n M) inhibits differentiation and down-regulates mi R-27 b expression in human lung fibroblasts induced by TGF-β1. 2. mi R-27 b overexpressing decreased expression of VDR protein and increased expression of α-SMA while reducing levels of mi R-27 b had opposing effects. 3. 1,25(OH)2D3 inhibits lung fibroblasts differentiation induced by TGF-β1 via mi R-27 b targeting VDR 3’UTR. Chapter 2 1α, 25-Dihydroxyvitamin D3 protects rats from monocrotaline-induced pulmonary arterial hypertension Objective:1. To observe the effects of 1,25(OH)2D3 on pulmonary vessels in rat model of X monocrotaline-induced pulmonary arterial hypertension. 2. To study the effects of 1,25(OH)2D3 on TGF-β1 expression in lung tissue of monocrotaline-induced rat model. Methods: 1. Laboratory animals and groups: 32 male Sprague-Dawley rats were randomly dividednto four groups: control group(n=8), VD group(n=8), MCT model group(n=8) andMCT+ VD group(n=8), respectively.2. The establishment of rat model of monocrotaline-induced pulmonary arterialhypertension and the effect of 1,25(OH)2D3 on MCT-induced rat: MCT group andMCT+VD group were subcutaneously injected with monocrotaline at a dose of60mg/kg body weight once only. Control group and VD group received subcutaneousinjection of saline vehicle. Simultaneously, VD group and MCT+VD group wereintraperitoneally injected with 1,25(OH)2D3 by 0.5ug/100 g body weight twice a week,for 3 weeks. Control group and MCT group were intraperitoneally treated with salinevehicle. 3. On the 22 nd day, mean pulmonary arterial pressures(m PAP) were measured and rathearts were isolated. The right ventricle(RV) was separated from the left ventricle(LV)and septum(S), and weighed. The weight ratio of the right ventricle to the leftventricle plus the septum(RV/(LV +S) ratio) was calculated as an index of rightventricular hypertrophy(RVHI). 4. The lung tissues were fixed in 4% paraformaldehyde, embedded in paraffin, andsectioned. After hematoxylin and eosin(HE) staining was performed, these sectionswere examined using light microscopy. Morphometric analysis was performed in thepulmonary small artery. Medial wall thickness and wall area were calculated with thefollowing formula: medial thickness(WT %)= medial wall thickness/external diameter×100%; wall area(WA %)= medial wall cross-sectional area /external cross-sectionalarea ×100%. 5. TGF-β1 expressions in lung tissues of rats were determined by QRT-PCR and westernblotting analysis, normalized to β-actin expression. Results: 1. Effects of 1,25(OH)2D3 on m PAP, RVHI and morphometric index of MCT inducedrats: MCT at a dose of 60mg/kg body weight was subcutaneously injected for 3 weeks.The m PAP, RVHI, WT % and WA % of MCT induced rats obviously increasedcompared to control group, but 1,25(OH)2D3 could markedly inhibited these effects. 2. Effects of 1,25(OH)2D3 on pulmonary small artery morphology in MCT induced rats:sections of lung tissue in rats stained by HE showed that the pulmonary arteriole wallsin the Control group and VD group were thin and single layered under lightmicroscopy. Infiltration of inflammatory cells did not exist in pulmonary perivascularspaces. Conspicuous pulmonary vascular remodeling could be observed in MCT group,characterized by pulmonary arteriole media muscularization, significant thickening andperivascular inflammatory cells infiltration. After 1,25(OH)2D3 treatment, medialhypertrophy and perivascular inflammatory cells infiltration decreased. 3. Effects of 1,25(OH)2D3 on TGF-β1 expression in lung tissue of MCT-induced rats:TGF-β1 protein expression increased in lung tissue of MCT-induced rats.1,25(OH)2D3 could reduce TGF-β1 protein level. However, the levels of the TGF-β1transcripts did not change. Conclusion:1. Subcutaneously injection of MCT at a dose of 60mg/kg body weight once only for 3 weeks successfully established rat model of MCT-induced pulmonary arterial hypertension, increased in pulmonary artery pressure and pulmonary vascular inflammation and promoted pulmonary vascular remodeling. 2. 1,25(OH)2D3 prevents development of MCT-induced pulmonary arterial hypertension in rats and improves pulmonary hemodynamics, pulmonary vascular remodeling and right ventricular hypertrophy. 3. 1,25(OH)2D3 could reduce TGF-β1 protein level of lung tissue in MCT-induced rats, therefore inhibits pulmonary vascular remodeling. Chapter 3 micro RNA mechanisms of 1α, 25-Dihydroxyvitamin D3 on monocrotaline-induced pulmonary artery hypertension Objective:1. To determine the role of 1,25(OH)2D3 in α-SMA, VDR, COL1A1 and OPN expressions of lung tissue in MCT-induced rats. 2. To study the effects of 1,25(OH)2D3 on mi R-27 b expression of lung tissue in MCT-induced rats. Methods: 1. The establishment of rat model of monocrotaline-induced pulmonary arterialhypertension and the effects of 1,25(OH)2D3 on MCT-induced rat. 2. On the 22 nd day, the lung tissues were collected. Some lung tissue specimens werefixed in OCT diluted in phosphate buffered saline(PBS)(1:1), quick-frozen inisopentane on dry ice and sectioned. Total RNA, total protein and mi RNAs of lungtissues were extracted.3. The levels of α-SMA, VDR, COL1A1 and OPN in lung tissues of rats weredetermined by QRT-PCR and western blotting analysis, normalized to β-actinexpression. 4. The expressions of mi R-27 b in lung tissues of rats were assessed by QRT-PCR andnormalized to U6 expression. Results: 1. Effects of 1,25(OH)2D3 on α-SMA, COL1A1 and OPN expressions in lung tissue ofMCT-induced rats: α-SMA, COL1A1 and OPN expressions at the m RNA and proteinlevels increased in lung tissue of MCT-induced rats. 1,25(OH)2D3 could reduceα-SMA, COL1A1 and OPN expressions. 2. Effects of 1,25(OH)2D3 on VDR expression in lung tissue of MCT-induced rats: VDRprotein expression decreased in lung tissue of MCT-induced rats. 1,25(OH)2D3 couldup-regulate VDR protein level. However, the levels of the VDR transcripts did notchange. 3. Effects of 1,25(OH)2D3 on mi R-27 b expression in lung tissue of MCT-induced rats:mi R-27 b expression increased in lung tissue of MCT-induced rats. 1,25(OH)2D3 couldreduce mi R-27 b expression. Conclusion:1. Subcutaneously injection of MCT at a dose of 60mg/kg body weight once only for 3 weeks induced pulmonary vascular remodeling and pulmonary vascular inflammation in rats. The myofibroblasts in lung tissue of MCT-induced rats increased. α-SMA, COL1A1 and OPN expressions up-regulated in lung tissue of MCT-induced rats. 2. 1,25(OH)2D3 reduced α-SMA, COL1A1 and OPN expressions via VDR in lung tissue of MCT-induced rats. 3. mi R-27 b might regulate α-SMA protein levels of lung tissue in MCT-induced rats.