Unraveling endothelin-1 induced hypercontractility of human pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension

Figures

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  Fig 1. Detection of ET-1 induced contraction in HPASMC by electrical impedance. Electrical impedance was measured as described in the methods section. Triplicate wells were used with or without (CON) 1 nM, 10 nM or 100 nM ET-1 (A). The line graphs represent the average normalized cell index across triplicate wells and the error bars are the standard deviation at each time point. The bar graphs represent the average negative peak for each triplicate and the error bars are the standard deviation at that time point. Triplicate wells treated with vehicle (CON), 1 uM BQ123 plus 10 nM ET-1, 10 nM ET-1 or 10 nM of sarafotoxin (SF6C) were measured as well (B). This figure illustrates a non-PAH HPASMC (Control-1) strain that is representative of results obtained from various HPASMC strains. * p< 0.01 vs CON; ** p< 0.001 vs CON.

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  Fig 2. Dot plot comparing ET-1 induced contraction magnitudes in non-PAH and PAH HPASMC. Contraction magnitudes were calculated for non-PAH (n = 5) and PAH (n = 5) HPASMC by taking the maximum negative peak value of each sample and dividing by one of the non-PAH samples giving a relative cell index value. Vertical dots indicate relative cell index for each non-PAH (triangle) and PAH (circle) HPASMC sample. The horizontal line represents the median of the group. A Welch’s two sample t-test assuming unequal variance was performed between the non-PAH and PAH groups; p-value = 0.055.

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  Fig 3. Effect of vascular effectors, bradykinin and angiotensin, on HPASMC contraction. A representative HPASMC sample (Control-5) showing the average normalized cell index of triplicate wells treated without (CON) or with 100 nM bradykinin (BK) or 100 nM angiotensin II (AT) (A). Bar graph where bar heights represent average relative cell index at negative peak for n = 4 HPASMC strains. The error bars represent standard deviation. Relative cell index at negative peak was calculated by dividing the minimum cell index of the treatment group (AT, BK or ET-1) by the control group (CON) at that same time point. This was done for each HPASMC cell strain. Then, an average and standard deviation were calculated across n = 4 biological replicates. * p< 0.01 vs CON.

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  Fig 4. LIMK effect on ET-1 promoted contraction in HPASMC. Representative western blot of non-PAH (Control-1) and PAH (PAH-3) HPASMC proteins probed for p-cofilin, total cofilin and beta actin after pretreatment with or without 10 uM LIMi3 or 10 uM Y27632 and treatment with or without ET-1. Each sample had 22 ug of protein loaded per well on two separate gels which later were probed for p-cofilin and beta actin or total cofilin and beta actin (A). A representative HPASMC sample (Control-1) showing the average normalized cell index of triplicate wells pretreated with or without LIMKi3 1 h before treatment without (CON) or with ET-1 (B, C). Another representative HPASMC (PAH-1) showing the result of pretreated with MMCPP targeted against cofilin (Cofilin MMCPP) for one h before addition of ET-1 (D, E). Bar graphs represent average relative cell index at negative peak for n = 4 HPASMC strains and the error bars are the standard deviation (C, E). NS = not significant vs ET-1.

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  Fig 5. Effect of ROCK siRNA knockdown on the ET-1 induced contraction in non-PAH and PAH HPASMC. Western blots showing protein levels of ROCK1 and ROCK2 after siRNA knockdown with ROCK1, ROCK2 or control siRNA in non-PAH (Control-1) and PAH (PAH-1) HPASMC. Thirty ug of total protein was loaded for non-PAH samples and 15 ug of total protein was loaded for the PAH samples (A). Representative impedance experiments showing the average of triplicate wells which were pretreated with negative control (CON) siRNA, ROCK1 siRNA or ROCK2 siRNA and then treated with or without ET-1. The line graph represents the average normalized cell index across triplicate wells and the error bars are the standard deviation (B, D). These values at the negative peak of the contraction are illustrated with bar graphs (C, E). ROCK1 KD, ROCK1 knockdown with ROCK1 siRNA; ROCK2 KD, ROCK2 knockdown with ROCK2 siRNA; NS = not significant vs siRNA CON + ET-1; *p< 0.01 vs siRNA CON + ET-1.

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  Fig 6. Effect of ROCK inhibitor on the ET-1 induced contraction in non-PAH and PAH HPASMC. Representative experiments showing triplicate wells which were pretreated without or with 1, 10 uM or 25 uM Y27632 (ROCKin) for 1 h and then with or without ET-1 in non-PAH (A) and PAH (B) HPASMC. These values at the negative peak of the cell index are illustrated with bar graphs with error bars representing standard deviations. #p< 0.05 vs CON.

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  Fig 7. Effect of caldesmon, MEK and p90RSK inhibition on ET-1 induced cell contraction. Representative experiments showing bar graphs with standard deviation error bars for caldesmon activity inhibition with a caldesmon MMCPP (CaD MMCPP) or negative control MMCPP (Neg MMCPP) (A), MEK inhibition with U0126 (U) (B) and p90RSK inhibition with BI-D1870 (BI) (C). All inhibitors were pre-incubated with the cells for 1 h before addition of ET-1. #p< 0.05 vs CON; &p< 0.05 vs ET-1.

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  Fig 8. The effect of calcium channel blockers on ET-1 and bradykinin induced cell contraction. Representative experiments showing bar graphs with standard deviation error bars for the effects of 10 uM nifedipine (Nif), 10 uM thapsigargin (thaps) (A, C), or mibefradil (Mib) (B, D) on ET-1 (A, B) or bradykinin (BK) (C, D) induced cell contraction. All inhibitors were pre-incubated with the cells for 1 h before addition of ET-1 or BK. #p< 0.05 vs CON.