GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension

Figures

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  Figure 1. GATA6 loss induces SOD2 and other anti-oxidant enzymes deficiency in human and mouse PAECs. (A) An expression of the antioxidant enzymes measured by qPCR in human PAECs (HPAECs) transfected with siGATA6 or control scr siRNA. mRNA levels in scr siRNA transfected cells were set as one. Data are means ± SE, *p < 0.05 vs scr siRNA by Kruskal–Wallis test with post hoc Dunn’s test for multiple comparisons; n = 3–10 (see also Supplemental Table 1). (B) Chromatin immune precipitation (ChiP) assay in HPAECs, n = 8 for SOD2; n = 5 for GPX1. The experiments were repeated three times. Representative gel images and data quantification are shown. Values are means ± SE, **p < 0.01, ***p < 0.001 by Mann Whitney U test. (C,D) Control HPAECs transfected with siGATA6 or scr siRNA (siContr) were assayed by Western blot analysis to detect GATA6 and SOD2. Representative blots are shown. Values are means ± SE of the relative protein levels by densitometry, n = 5, **p < 0.01 by Mann Whitney U test. (E,F) Western blot analysis of whole lung tissue from Gata6 CKO and WT mice. Representative blots are shown. Values are means ± SE of the relative protein levels by densitometry, n = 4, *p < 0.05 by Mann…

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  Figure 2. GATA6 is deficient in both PAEC and PASMC in human PAH lungs. (A) IHC analyses were performed to detect GATA6 (green), CD31 (red) and α-SMA (red) in small PAs from healthy controls (HC) and patients with PAH (SSc-PAH and IPAH) and analysis of nuclear GATA6 in CD31- and SMA-positive cells in small PAs was performed. Left: Images are representative from 3–4 subjects/control, SSc-PAH, and IPAH. Bar equals 50 μm. White arrowheads indicate GATA6-positive cells. Right: Data are in optical density units (OD); Data are means ± SE from 3–4 human subjects per control, 7–8 for PAH (SSc-PAH + IPAH) groups. *p < 0.05 by Mann Whitney U test. (B,C) Immunoblot analysis of early-passage PAEC and PASMC from healthy controls (HC) and subjects with IPAH to detect indicated proteins. Data are means ± SE from n = 5 subjects/group. **p < 0.01 by Mann Whitney U test. (D) Immunoblot analysis of nuclear fractions of PASMC from healthy control (HC) and IPAH subjects. Data are means ± SE from n = 4 subjects/group. *p < 0.05 by Mann Whitney U test. (E) Immunocytochemical analysis of PASMC from healthy control (HC) and IPAH subjects to detect GATA6 (red) and DAPI (blue). Bar equals 50 µm. Representative…

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  Figure 3. GATA6 deficiency is required for increased proliferation of human PAH PAEC and PASMC. (A-D) Human PAH PAECs were transfected with mock ( − ) (A,B), 10 MOI control adenovirus (C,D), or 10 MOI AdGATA6 for 48 h followed by immunoblot analysis (A), cell counts (B), proliferation (Ki67) (C), and apoptosis (Annexin V-FITC Apoptosis Staining/Detection Kit) (D) assays. Data are means ± SE from three (A–C) or four (D) experiments, each performed on the cells from different patient. *p < 0.05 by Mann Whitney U test. (E–H) Immunoblot analysis (E), cell counts (F), proliferation (Ki67) (G), and apoptosis (Annexin V-FITC Apoptosis Staining/Detection Kit) (H) assays of human PAH PASMC transfected with mock ( − ) (E,F), 10 MOI control adenovirus (G,H), or 10 MOI AdGATA6 for 48 h. Data are means ± SE from three (E–G) or four (H) experiments, each performed on the cells from different patient. *p < 0.05 by Mann Whitney U test. (I) RNA-sequencing analysis of adGATA6 and mock-transfected PAH PASMC (3 patients/group). RNA-sequencing data was normalized and clustered. Selected up and down-regulated gene clusters are shown. Genes further studied by immunoblot are boxed. Selected up- and down-regulated…

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  Figure 4. GATA6 expression is induced via BMP10-BMPR2/ALK1 axis. (A–F) HPAECs were transfected with ALK1, BMPR2, Endoglin, or control scr siRNA for 48 h, and then treated with 10 ng/ml BMP10 or vehicle for 6 h for RNA isolation and 24 h for protein isolation. (A,C,E): GATA6 mRNA measured by qPCR. Data are means ± SE; each experiment was repeated at least three times. (B,D,F): GATA6 protein levels were measured by immunoblot analysis. Data are means ± SE, each experiment was repeated at least three times. Representative blots are shown. *p < 0.05, **p < 0.01, ***p < 0.001 by Kruskal–Wallis test with post hoc Dunn’s test for multiple comparisons. (G–I) Human PAH PASMC were treated with 10 ng/ml BMP10 or vehicle ( − ) for 48 h and immunoblot analysis to detect indicated proteins was performed. Data are means ± SE from n = 4 subjects/group. *p < 0.05 by Mann Whitney U test. (J,K) Equal amounts of human PAH HPAEC and PASMC were plated at 6-well plates and treated with 10 ng/ml BMP10 or vehicle ( − ). 48 h later cell counts were performed. Data are means ± SE from n = 3 subjects/group, 3 technical repetitions/subject. *p < 0.05 by Mann Whitney U test. (L,M) HPAECs were transfected with SMAD1…

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  Figure 5. GATA6 deficiency in PAEC and PASMC results in loss of BMP receptors. (A) qPCR of HPAECs transfected with GATA6 or control scr siRNA ( − ) to measure indicated mRNA, each experiment was repeated at least three times. Data are means ± SE, n = 4–7. **p < 0.01, ***p < 0.001 by Mann Whitney U test. (B) Chromatin immune precipitation (ChiP) assay in HPAECs, n = 8–10. Representative gel images and data quantification are shown. Data are means ± SE, ***p < 0.001, ****p < 0.0001 by Mann Whitney U test. (C,D) Immunoblot analysis of control human PASMC transfected with siRNA GATA6 or control scr siRNA for 48 h. Data are means ± SE, 3 subjects/group, *p < 0.05 by Mann Whitney U test. Please see Fig. 2F,G for GATA6 immunoblots. (E,F) Expression of BmpR2, Alk1, ActRIIB, and endoglin measured by qPCR in PAEC (F) and whole lungs (G) from WT and Gata6 CKO mice. Data are means ± SE; E: n = 4–5/group; F: n = 6–11 mice/group. Male and female mice responded similarly. *p < 0.05, **p < 0.01 by Mann Whitney U test ((E,F) BmpR2, Alk1, and ActRIIB) and unpaired τ test (F Endoglin). (G,H) Control HPAECs transfected with siGATA6 or control scr siRNA ( − ) were assayed by immunoblot analysis to detect…

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  Figure 6. Endothelial GATA6 loss promotes GATA6 deficiency and increases growth of PASMC. (A) IHC stainings were performed to detect Gata6, CD31 and α-SMA in WT and Gata6 CKO mice; images are representative from 3 mice/group. Bar = 50 μm. White arrowheads indicate Gata6-positive cells. (B) Schematic representation of experiments (C–F). Media conditioned for 72 h by non-diseased human PAEC (HPAEC) transfected with GATA6 siRNAs or control scr siRNA was added to non-diseased distal human PASMC. (C,D,E) mRNA (C) and immunoblot analyses (D,E) of PASMC after 72 h incubation with HPAEC-conditioned media (CM) were performed. Data are means ± SE from n ≥ 3 repetitions. *p < 0.05, **p < 0.01, ***p < 0.001 by Mann Whitney U test. (F) Cell growth (cell counts) assay of PASMC after 48 h incubation with indicated HPAEC conditioned media (CM). Data are means ± SE, n = 3. *p < 0.05 by Mann Whitney U test. (G,H) PASMC were incubated for 48 h with indicated HPAEC conditioned media (CM) in the presence of 10 nM control IgG, anti-fractalkine antibody (AB), or anti-TGFβ AB. Schematic representation of experiment (G) and cell proliferation analysis (H) are shown. Data are means ± SE, n = 3–4/group. *p < 0.05 by…

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  Figure 7. Treatment with DMF restores expression of the BMP receptors, reverses oxidative stress and pulmonary hypertension in Gata6 CKO mice. (DMF or vehicle were administered daily via i.p. injection for 3 weeks. (A) qPCR analysis of whole lung tissue from WT and Gata6 CKO mice treated with DMF or vehicle to detect expression of indicated BMP receptors. Data are means ± SE, n = 6–12, *p < 0.05. **p < 0.01 by Kruskal–Wallis test followed by Dunn’s multiple comparisons test (BmpR2, ActRIIB, Alk1) and one-way ANOVA followed by post hoc Tukey’s multiple comparison (Endoglin). (B) qPCR analysis of whole lung tissue from WT and Gata6 CKO mice treated with DMF or vehicle to detect expression of the antioxidant enzymes and eNOS. Data are means ± SE, n = 6–17, *p < 0.05., **p < 0.01, ***p < 0.001 by one-way ANOVA followed by post hoc Tukey’s multiple comparisons test (SOD2, GPX1, CAT) and Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test (eNOS). (C,D) mRNA levels of indicated BMP receptors and antioxidant enzymes measured by qPCR in PAEC from WT and Gata6 CKO mice treated with DMF or vehicle. Data are means ± SE, n = 3–6 mice/group, *p < 0.05, **p < 0.01 by Kruskal–Wallis test…

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  Figure 8. Graphical representation of the role of GATA6 in coordinating cross-talk between BMP10 and oxidative stress axis in PAH. GATA6 is an activator of anti-oxidant enzymes and its deficiency in PAEC and PASMC induces oxidative stress and mitochondrial dysfunction. BMP10 induces expression of GATA6 through the ALK1, BMPRII, ENG and ERK pathway. GATA6, in turn, transcriptionally activates BMP receptors in PAEC. Endothelial GATA6 regulates PASMC function via paracrine factors. TGFβ2 secreted by GATA6 deficient PAEC induces PASMC proliferation. Administration of dimethyl fumarate (DMF) to mice with endothelial Gata6 loss restores expression of BMP receptors, resolves oxidative stress, and reverses PH.