Sema3a promotes osteogenic differentiation of BMSCs under 2Gy radiation by reducing ROS and inflammatory factors

This is the data of the article: <b>Sema3a promot</b><b>es</b><b> osteogenic differentiation of BMSC</b><b>s</b><b> </b><b>under 2Gy radiation </b><b>by</b><b> reducing </b><b>ROS</b><b> and inflammatory factors</b><p><b></b></p

Sema3a promotes osteogenic differentiation of BMSC under 2Gy radiation by reducing ROS and inflammatory factors

This is the data of our research (<b>Sema3a promot</b><b>es</b><b> osteogenic differentiation of BMSC</b><b>s</b><b> </b><b>under 2Gy radiation </b><b>by</b><b> reducing </b><b>ROS</b><b> and inflammatory factors</b><p><b></b></p>)<br

Smooth muscle cell-specific FoxM1 controls hypoxia-induced pulmonary hypertension

Rationale: Forkhead box M1 (FoxM1) is a transcription factor that promotes cell proliferation by regulating a broad spectrum of genes that participate in cell cycle regulation, such as Cyclin B, CDC25B, and Aurora B Kinase. We have shown that hypoxia, a well-known stimulus for pulmonary hypertension (PH), induces FoxM1 in pulmonary artery smooth muscle cells (PASMC) in a HIF-dependent pathway, resulting in PASMC proliferation, while the suppression of FoxM1 prevents hypoxia-induced PASMC proliferation. However, the implications of FoxM1 in the development of PH remain less known. Methods: We determined FoxM1 levels in the lung samples of idiopathic PAH (pulmonary arterial hypertension) (IPAH) patients and hypoxia-induced PH mice. We generated constitutive and inducible smooth muscle cell (SMC)-specific FoxM1 knockdown or knockout mice as well as FoxM1 transgenic mice which overexpress FoxM1, and exposed them to hypoxia (10 % O2, 90% N2) or normoxia (Room air, 21 % oxygen) for four weeks, and measured PH indices. We also isolated mouse PASMC (mPASMC) and mouse embryonic fibroblasts (MEF) from these mice to examine the cell proliferation and expression levels of SMC contractile proteins. Results: We showed that in hypertensive human lungs or mouse lungs, FoxM1 levels were elevated. Constitutive knockout of FoxM1 in mouse SMC caused early lethality, whereas constitutive knockdown of FoxM1 in mouse SMC prevented hypoxia-induced PH and PASMC proliferation. Inducible knockout of FoxM1 in SMC reversed hypoxia-induced pulmonary artery wall remodeling in existing PH. Overexpression of FoxM1 enhanced hypoxia-induced pulmonary artery wall remodeling and right ventricular hypertrophy in mice. Alteration of FoxM1 status did not affect hypoxia-induced hypoxia-inducible factor (HIF) activity in mice. Knockout of FoxM1 decreased PASMC proliferation and induced expression of SMC contractile proteins and TGF-β/Smad3 signaling. Conclusions: Our studies provide clear evidence that altered FoxM1 expression in PASMC contributes to PH and uncover a correlation between Smad3-dependent signaling in FoxM1-mediated proliferation and de-differentiation of PASMC

Smooth muscle cell-specific FoxM1 controls hypoxia-induced pulmonary hypertension

RATIONALE: Forkhead box M1 (FoxM1) is a transcription factor that promotes cell proliferation by regulating a broad spectrum of genes that participate in cell cycle regulation, such as Cyclin B, CDC25B, and Aurora B Kinase. We have shown that hypoxia, a well-known stimulus for pulmonary hypertension (PH), induces FoxM1 in pulmonary artery smooth muscle cells (PASMC) in a HIF-dependent pathway, resulting in PASMC proliferation, while the suppression of FoxM1 prevents hypoxia-induced PASMC proliferation. However, the implications of FoxM1 in the development of PH remain less known. METHODS:We determined FoxM1 levels in the lung samples of idiopathic PAH (pulmonary arterial hypertension) (IPAH) patients and hypoxia-induced PH mice. We generated constitutive and inducible smooth muscle cell (SMC)-specific FoxM1 knockdown or knockout mice as well as FoxM1 transgenic mice which overexpress FoxM1, and exposed them to hypoxia (10% O2, 90% N2) or normoxia (Room air, 21% oxygen) for four weeks, and measured PH indices. We also isolated mouse PASMC (mPASMC) and mouse embryonic fibroblasts (MEF) from these mice to examine the cell proliferation and expression levels of SMC contractile proteins. RESULTS:We showed that in hypertensive human lungs or mouse lungs, FoxM1 levels were elevated. Constitutive knockout of FoxM1 in mouse SMC caused early lethality, whereas constitutive knockdown of FoxM1 in mouse SMC prevented hypoxia-induced PH and PASMC proliferation. Inducible knockout of FoxM1 in SMC reversed hypoxia-induced pulmonary artery wall remodeling in existing PH. Overexpression of FoxM1 enhanced hypoxia-induced pulmonary artery wall remodeling and right ventricular hypertrophy in mice. Alteration of FoxM1 status did not affect hypoxia-induced hypoxia-inducible factor (HIF) activity in mice. Knockout of FoxM1 decreased PASMC proliferation and induced expression of SMC contractile proteins and TGF-β/Smad3 signaling. CONCLUSIONS: Our studies provide clear evidence that altered FoxM1 expression in PASMC contributes to PH and uncover a correlation between Smad3-dependent signaling in FoxM1-mediated proliferation and de-differentiation of PASMC

Downregulation of HIF-1α by siRNA significantly attenuates mRNA expression of CTR1 in hypoxic PASMC.

<p>A: Real-time RT-PCR analysis on HIF-1α (<i>a</i>), HIF-2α (<i>b</i>) and CTR1 (<i>c</i>) in hypoxic PASMC treated with (50–200 pmol) or without (0 pmol) siRNA specifically targeting HIF-1α, HIF-2α and CTR1, respectively. Data are shown in mean±SE. ***<i>P</i><0.01 vs. control hypoxic cells (0-pmol siRNA). B: Real-time RT-PCR analysis on human CTR1 (<i>a</i>), ATP7A (<i>b</i>), HIF-1α (<i>c</i>) and HIF-2α (<i>d</i>) in hypoxic PASMC treated with 100-pmol scrambled siRNA (Control-siRNA, open bars), HIF-1α-siRNA (solid bars), HIF-2α-siRNA (light grey bars), and CTR1-siRNA (dark grey bars), respectively. ***<i>P</i><0.01 vs. hypoxic cells treated with scrambled siRNA (Control-siRNA).</p

Chelation of Cu and knockdown of CTR1 both decrease Bcl-2 expression in human PASMC during hypoxia.

<p>A: Western blot analysis on PCNA (a marker for cell proliferation) and Bcl-2 (an anti-apoptotic protein) in PASMC transfected with scrambled siRNA (Cont) or human CTR1-siRNA (siRNA) and PASMC treated with the Cu chelator BCS. B: Summarized data (mean±SE) showing PCNA (left panel) and Bcl-2 (right panel) protein levels in PASMC transfected with Control-siRNA or hCTR1-siRNA and PASMC treated with BCS. *<i>P</i><0.05 vs. Control-siRNA.</p

The mRNA expression level of Cu transporters (CTR1 and ATP7A) and lysyl oxidase (LOX) is increased in whole-lung and pulmonary artery (PA) tissues of mice with chronically hypoxia-induced pulmonary hypertension (HPH).

<p>Whole lung tissues and isolated PA tissue from normoxic (Nor, 21% O₂) and hypoxic (Hyp, 10% O₂ for 5 weeks) mice were homogenized and their mRNA transcripts evaluated by RT-PCR utilizing primers specific for ATOX1, ATP7A, CCS, CTR1, LOX, GAPDH or 18s rRNA (internal controls). A: RT-PCR products from whole-lung tissues were separated on 2% agarose gels (upper panel) and the band intensities quantitated by ImageJ, normalized to intensity of GAPDH, and graphed relative to Nor (n = 4 Nor mouse lungs; n = 8 Hyp mouse lungs). B: PA dissected from Nor and Hyp mice were used for RNA extraction (n = 5) and analyzed by quantitative PCR. Real-time PCR reaction was set with primers specific for the indicated genes. The cycle threshold C(t) values were normalized to 18s rRNA to obtain ΔC(t)_, quantified relative to normoxic control for each of the indicated genes (ΔΔC(t)), and graphed as % of normoxic control. C: Representative records of right ventricular pressure (RVP, left panel) and summarized data (mean±SE) showing RV systolic pressure (RVSP) in Nor (n = 6) and Hyp (n = 13) mice. D: Representative records (left panel) and summarized data (right panel, mean±SE) of right ventricular contractility (RV-±dp/dt_max) in Nor and Hyp mice. E: Summarized data (mean±SE) showing the ratio of right ventricle (RV) weight to left ventricle (LV) and septum (S) weight [RV/(LV+S)] in Nor (n = 7) and Hyp (n = 7) mice. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 vs. Nor.</p

Inhibition of LOX downregulates PCNA (a marker for cell proliferation) and Bcl–2 (an anti-apoptotic protein) in human PASMC during hypoxia.

<p>A: Western blot analysis on PCNA and Bcl–2 in hypoxic PASMC treated with (+) or without (-) the irreversible LOX inhibitor βAPN (for 48 hrs). B: Summarized data (mean±SE) showing PCNA (left panel) and Bcl–2 (right panel) protein levels in control PASMC (open bars) and PASMC treated with βAPN (solid bars). **<i>P</i><0.01 vs. Control.</p

Hypoxia-mediated upregulation of mRNA expression of Cu transporters (CTR1, ATP7A) and lysyl oxidase (LOX) is associated with an increase in Cu transportation in human pulmonary arterial smooth muscle cells (PASMC).

<p>A: Real-time RT-PCR analysis on ATP7A, CTR1, and LOX (left panel) and ⁶⁴Cu uptake (mean±SE) in human PASMC exposed to normoxia (Nor) and hypoxia (Hyp, 3% O₂ for 48 hrs, n = 3; right panel). B: Real-time RT-PCR analysis on ATP7A, CTR1, and LOX (left panel) and ⁶⁴Cu uptake (mean±SE, right panel) in human PASMC treated with vehicle (Cont) and CoCl₂ (100 µM for 48 hrs, n = 3; right pane). Lactate dehydrogenase (LDH) and erythropoietin (EPO) were used as positive controls. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 vs. Hyp or CoCl₂.</p

PASMC motility is dependent on Cu and the Cu-dependent PASMC motility is augmented by hypoxia.

<p>Cell motility was determined by a scratch wound assay over a period of 12: Representative images showing normoxic (Nor) and hypoxic (Hyp) PASMC immediately (0 hr) or 12 hrs after scratch with a sterile pipette in the absence (Control) or presence (BCS) of 200 µM BCS (a Cu chelator). B: Representative images showing control (-CoCl₂) and 100-µm CoCl₂-treated PASMC immediately (0 hr) or 12 hrs after scratch in the absence (Control) or presence (BCS) of BCS. C: Summarized data (mean±SE) showing gap closure (<i>a</i>) measured at 12 hr in Nor and Hyp PASMC treated with (BCS) or without (Control) BCS. **<i>P</i><0.01 vs. Control. The Cu-dependent cell motility (<i>b</i>) was determined by the percent changes in gap closure between Control and BCS-treated PASMC under Nor and Hyp conditions. *<i>P</i><0.05 vs. Nor. D: Summarized data (mean±SE) showing gap closure (<i>a</i>) measured at 12 hr in PASMC treated with (+CoCl₂) or without (-CoCl₂) CoCl₂ in the absence (Control) or presence (BCS) of BCS. *<i>P</i><0.05 vs. Control. The Cu-dependent cell motility (<i>b</i>) was determined by the percent changes in gap closure between Control and BCS-treated PASMC in the absence (-) or presence (+) of CoCl₂.</p