Endothelial Dysfunction Following Enhanced TMEM16A Activity in Human Pulmonary Arteries

Figures

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  Figure 1. TMEM16A defines calcium-activated chloride current in pulmonary arterial endothelial cells (PAECs). (a) Western blot showing TMEM16A expression in donor PAECs, pulmonary arterial smooth muscle cells (PASMCs) and human lung homogenate (hLH) (N = 2–3 patients). Vinculin served as loading control. (b) Representative whole-cell, Ca2+-activated Cl− current (IClCa) traces (left) and normalized current-voltage (I-V) relationships measured with voltage clamp in PAECs showing the effect of benzbromarone (Bbr) (right). (c) Calculated Bbr-sensitive current in donor PAECs and PASMCs. Figures were generated with n = 5–13 cells from healthy donors. (d,e) Representative isometric tension measurements and quantification showing endothelial contribution of Bbr effectiveness on U46619 pre-constricted mouse pulmonary arteries with either endothelium removed (no EC; d) or incubation with 300 µm L-NAME (e) (n = 4–7). ** p < 0.01, *** p < 0.001, ANOVA with Bonferroni post-hoc test, data are presented as mean ± s.e.m.

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  Figure 2. TMEM16A accounts for increased Ca2+-activated Cl− current in IPAH PAECs. Immunofluorescence staining of (a) 3D precision cut lung slices (PCLS), (b) lung sections and (c) PAECs obtained from healthy donor lungs and patients suffering from IPAH (BP = antibody blocking peptide, scale bar = 50 µm for PCLS, 50 µm for PAECs and 50 µm for lung sections). (d) The effect of Bbr on representative whole-cell IClCa traces (left) and normalized current-voltage relationships (right) measured with voltage clamp in donor and IPAH PAECs (VehI/BbrI = IPAH PAECs perfused with vehicle or Bbr; VehD/BbrD = donor PAECs perfused with vehicle or Bbr). (e) Comparison of the Ca2+-activated Cl− current density of donor and IPAH PAECs at 120 mV. Figures were generated with n = 12–13 cells from at least N = 4 patient samples. # p < 0.01, *** p < 0.0001, ANOVA with Bonferroni post-hoc test, data are presented as mean ± s.e.m.

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  Figure 3. Upregulation of TMEM16A in human PAECs. (a) Immunofluorescence staining of TMEM16A in PAECs infected with CtrlAd or Ano1Ad (BP = antibody blocking peptide; scale bar = 20 µm). (b) Representative whole-cell IClCa traces (left) and normalized current-voltage relationships (right) measured with voltage-clamp showing the effect of Bbr in donor PAECs transfected with CtrlAd. (c) Representative whole-cell IClCa traces (left) and normalized current-voltage relationships (right) measured with voltage-clamp showing the effect of Bbr in donor PAECs transfected with Ano1Ad and overexpressing TMEM16A. (d) Consecutive, calculated Bbr-sensitive current comparing primary PAECs infected with CtrlAd or Ano1Ad. Figures were generated with 8–13 cells from N = 2 healthy donors, data are presented as mean ± s.e.m.

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  Figure 4. TMEM16A-mediated membrane depolarization disrupts Ca2+ dynamics of human PAECs. (a) Fluorometric measurements indicating shift in relative resting membrane potential (Em) of donor PAECs infected with CtrlAd or Ano1Ad using DiBAC4(3) dye. (b) Representative traces depict changes in intracellular Ca2+ detected in PAECs transfected with CtrlAd or Ano1Ad. (c–e) The effect of TMEM16A overexpression on cytosolic baseline Ca2+ concentration ([Ca2+]i), store depletion and Ca2+ influx using Fura-2 in donor PAECs infected with CtrlAd or Ano1Ad (BHQ = butylhydroquinone). Figures were generated with 80-116 cells from N = 3 healthy donors. * p < 0.05, *** p < 0.001, paired (a) and unpaired t-tests (c–e).

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  Figure 5. Enhanced TMEM16A mediates metabolic changes of PAECs. (a–b) Seahorse mitochondrial stress test profiles of TMEM16A-overexpressing primary PAECs showing the ratio of oxygen consumption rate (OCR) to extracellular acidification rate (ECAR) OCR/ECAR. (c) Proliferation of human PAECs overexpressing TMEM16A measured with thymidine incorporation (n = 5). (d,e) Cas3/Cas7 apoptosis assay and cell-cycle analysis of human PAECs overexpressing TMEM16A. (STS = staurosporin). (f) Western blots of PAECs infected with TMEM16A-overexpressing Ano1Ad or control CtrlAd with quantifications of PCNA, cleaved PARP/PARP and Cyclin D1. Figures were generated with 13 separate sets of experiments. * p < 0.05, ratio-paired (a) or paired t-test.

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  Figure 6. Increased TMEM16A activity causes angiogenic dysfunction. (a) Representative pictures of PAECs wound healing assay, expressing either Ano1Ad or control CtrlAd, taken after 24, 36 and 48 h (scale bar = 200 µm). (b) Quantification of the wound healing assay. (c) Matrigel tube-formation assay with representative bright-field (BF) and mCherry fluorescence pictures of Ano1Ad or CtrlAd-infected PAECs (scale bar = 200 µm). (d) Quantification showing the number of branching points in comparison to CtrlAd. Figures were generated with 4 separate sets of experiments with triplicate in each group. ** p < 0.01, *** p < 0.001, (b) ANOVA with Bonferroni post-hoc test, (d) paired t-test.

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  Figure 7. Elevated TMEM16A activity alters ERK1/2 signaling. (a) Western blots showing protein levels of p38, Akt and SAPK/JNK obtained from of PAECs infected with either Ano1Ad or control CtrlAd. Figures were generated with 6 samples. (b) Western blots showing ERK1/2 pathway activation in PAECs infected with either Ano1Ad or CtrlAd with quantification. Figures were generated with 6 samples. * p < 0.05, ratio-paired t-test.

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  Figure 8. Elevated TMEM16A activity disturbs eNOS activation: (a) Noninduced nitric oxide levels and ACh-induced nitric oxide production of control (CtrlAd) and TMEM16A-overexpressing (Ano1Ad) human PAECs. Figures were generated with 8 sets of experiments with quadruplicate in each group and normalized to protein content. (b) Western blots showing ACh-induced changes in eNOS phosphorylation of CtrlAd and Ano1Ad-infected donor PAECs with quantification following the eNOS phosphorylation pattern at activatory Ser1177 and inhibitory Thr495 sites after 5, 15 and 30 min of ACh stimulation. (c) Quantification of basal, noninduced level of eNOS phosphorylation at Ser1177 and Thr495 as well as phosphorylation of Ser1177 15 min after ACh stimulation. Figures were generated with 6 samples. * p < 0.05, *** p < 0.001, ratio-paired t-test.

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  Figure 9. Enhanced TMEM16A results in reduced ACh-induced vasorelaxation of human donor pulmonary arteries. (a) Western blot of healthy donor pulmonary artery infected with either CtrlAd or Ano1Ad with quantification of TMEM16A overexpression (right). (b) Immunofluorescence staining of donor 3D, precision-cut lung slices (PCLSs) infected either with CtrlAd or Ano1Ad (scale bar = Width = 1257.93 µm, Height = 1257.93 µm, Depth = 82.00 µm for CtrlAd and 247.00 µm for Ano1Ad). (c) Representative isometric tension measurements showing ACh-mediated vasorelaxation of U46619 pre-constricted donor pulmonary arteries (PAs) infected with either CtrlAd or Ano1Ad (left) and quantification of the results (right) showing 2–3 PAs from the same donor in both groups. (d) The effect of TMEM16A overexpression on 30 µM ACh-induced vasorelaxation of 300 nM U46619 preconstricted healthy donor pulmonary arteries. Figure was generated with 7 pairs of vessels taken from N = 3 donors. Unpaired t-test.