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

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

Upregulated pro-LOX expression in PASMC isolated from patients with idiopathic pulmonary arterial hypertension (IPAH) is associated with increased stiffness.

<p>A: Western blot analysis on pro-LOX in PASMC from normal subjects (Normal) and IPAH patients (IPAH). B: Time courses of the membrane deformation in normal PASMC (open circles) and IPAH-PASMC (closed circles) cultured on uncoated cover slips. Microaspiration was used to determine the membrane deformation which is inversely related to membrane stiffness. <i>P</i><0.01 between the two curves (using two-way ANOVA). C: Time courses of the membrane deformation in normal PASMC and IPAH-PASMC cultured on collagen-coated cover slips. <i>P</i><0.01 between the two curves (using two-way ANOVA). D: Time courses of the membrane deformation in IPAH-PASMC treated with (IPAH+βAPN) or without (IPAH) the LOX inhibitor βAPN. <i>P</i><0.01 between the two curves (using two-way ANOVA).</p

The protein expression level of CTR1, pro-LOX and HIF-1α is increased in whole-lung tissues of mice with HPH.

<p>A–C: Western blot analysis of mouse CTR1 (A), pro-LOX (B) and HIF-1α (C) in total and membrane proteins extracted from whole-lung lung tissues of normoxic control mice (Nor, n = 5) and chronically hypoxic (Nor, n = 5) mice. Proteins from Nor and Hyp mouse lungs were solubilized in 3% DDM/1× RIPA buffer and utilized for Western blot analysis using antibodies specific for mouse CTR1, pro-LOX, and HIF-1α. β-actin or β-tubulin was used as a loading control. D: Summarized data (mean±SE) showing protein expression levels of CTR1, pro-LOX and HIF-1α in lungs tissues isolated from Nor and Hyp mice. The band intensity was quantitated with ImageJ software, normalized with respect to the loading control, and then shown relative to control (% of Nor). **<i>P</i><0.01 vs. Nor.</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

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

Activity of lysyl oxidase (LOX) is increased in whole-lung tissues of chronically hypoxic mice and in culture media from human PASMC exposed to hypoxia or treated with CoCl₂.

<p>Enzymatic activity of LOX was determined using Fluorimetric Lysyl Oxidase Assay Kit by monitoring LOX-catalyzed H₂O₂ release from the fluorescent substrate in HRP-coupled reaction. A: LOX activity in lung tissue homogenates from normoxic (Nor, room air for 5 weeks, n = 5) and hypoxic (Hyp, 10% O₂ for 5 weeks, n = 5) mice. **<i>P</i><0.01 vs. Nor. B: LOX activity in culture media collected from human PASMC after 24, 48 and 72 hrs of exposure to normoxia (Nor, 21% O₂) or hypoxia (Hyp, 3% O₂). *<i>P</i><0.05, **<i>P</i><0.01 vs. Nor. C: LOX activity in culture media collected from PASMC treated with vehicle (Cont) or CoCl₂ (100 µM for 48 hrs). **<i>P</i><0.01 vs. Cont. Each bar graph displays the Cu-dependent activity of LOX determined by subtracting values obtained in the presence of BCS (a Cu chelator) from values in the absence of BCS. LOX activity is expressed in nanomoles of H₂O₂ released from the cells and normalized to the amount of total protein in each sample. Data are shown as mean±SE.</p

PASMC migration is dependent on Cu and inhibited by knockdown of CTR1.

<p>Cell migration was determined by the modified Boyden chamber assay. Cells were plated on top of the porous (8-µm pore) membrane. After 48 hrs, the membrane was fixed and stained using Diff-Quick and the migrated cells in randomly chosen fields were counted at 200× magnification. A: Representative images (<i>a</i>) showing human PASMC cultured under normoxic (Nor) or hypoxic (Hyp) conditions in the absence (Control) or presence (BCS) of 200 µM BCS (a Cu chelator). Summarized data (mean±SE) showing migrated cell counts (<i>b</i>) in normoxic (Nor) and hypoxic (Hyp) PASMC treated with (BCS) or without (Control) BCS. **<i>P</i><0.01 vs. Control. The Cu-dependent cell migration (<i>c</i>) was determined by the percent changes in migrated cell counts between Control and BCS-treated cells in Nor and Hyp. Data are shown as mean±SE. *<i>P</i><0.05 vs. Nor. B: Representative images (<i>a</i>) showing human PASMC treated with scrambled siRNA (Control-siRNA) or CTR1-siRNA under Nor or Hyp conditions. Summarized data (mean±SE) showing migrated cell counts (<i>b</i>) in Nor and Hyp PASMC treated with Control-siRNA (solid bars) or CTR1-siRNA (open bars). **<i>P</i><0.01 vs. Control. The Cu-dependent cell migration (<i>c</i>) was determined by the percent changes in migrated cell counts between PASMC treated with Control-siRNA and CTR1-siRNA in normoxia (Nor) and hypoxia (Hyp). Data are shown as mean±SE. *<i>P</i><0.05 vs. Nor.</p