Mechanisms and Consequences of oxLDL Induced Endothelial Cell Stiffening

Endothelial biomechanics is emerging as a key player in the regulation of vascular endothelial cells. Here, we address the mechanism of endothelial stiffening induced by oxidized-LDL (oxLDL), a potential determinant of cardiovascular disease. We also investigate the role of oxLDL-induced endothelial stiffening in lumen formation. Endothelial stiffness was estimated by analyzing progressive membrane deformation using Micropipette Aspiration. We show that oxLDL induces RhoA activation and that oxLDL-induced endothelial stiffening is abrogated by inhibition of RhoA or its downstream target ROCK. 7-ketocholesterol, a major oxysterol in oxLDL increases endothelial cell stiffening, contractility and network formation. OxLDL-induced increase in endothelial force generation estimated in a gel contraction assay also required RhoA/ROCK activity. OxLDL also facilitates formation of endothelial branching networks in 3D collagen gels in vitro and induces increased formation of functional blood vessels in a Matrigel plug assay in vivo. Both effects are RhoA and ROCK dependent. Importantly, loading cells with cholesterol prevented oxLDL-induced RhoA activation and the downstream signaling cascade and reversed oxLDL-induced lumen formation. The observed effects on oxLDL-induced endothelial stiffening and its association with increased endothelial angiogenic activity in vitro and formation of functional vessels in vivo suggest a potential role for endothelial biomechanics in cardiovascular disease

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

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

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

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

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

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

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