Smooth muscle Cxcl12 contributions to vascular remodeling in flow and hypoxia-induced pulmonary hypertension

Figures

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  Figure 1. Left pneumonectomy combined with hypoxia results in exacerbated PH and RVH.A, the illustration shows the hypothesis that increased blood flow and Hx result in severe PH. B, schematics showing experimental designs. C, right ventricular systolic pressure (RVSP) measurements were conducted for control (n = 6), LP (n = 11), Hx (n = 8), and LP/Hx (n = 12) mice. D, Fulton Index (FI) was calculated for right ventricle hypertrophy (RVH) in control (n = 10), LP (n = 13), Hx (n = 9), and LP/Hx (n = 10) mice. E, cross-sections of hearts were stained with H&E, demonstrating a thickened RV wall (red arrowheads) in LP/Hx mice. F, right ventricle (RV) echocardiographic measurements were conducted in control (n = 9–10), LP (n = 7–8), Hx (n = 9–10), and LP/Hx (n = 7–10) mice. G, representative echocardiogram images of the RV in end-diastole were captured with the RV outlined in red dashedlines. H, left ventricle echocardiographic measurements were conducted in control (n = 5), LP (n = 7), Hx (n = 5–6), and LP/Hx (n = 7) mice. I, RVSP and FI measurements in control (n = 6–10), LP POD 90 (n = 7–8), and LP/Hx recovery mice (n = 3–4). Each dot represents an individual mouse. Statistical analysis was…

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  Figure 2. LP/Hx results in exacerbated vascular remodeling and accumulation of Sma + cells on distal arterioles. A, tissue clearing and deep whole lung lobe staining were performed on experimental mouse lungs. Sma is in red. Scale bar represents 1000 μm. B, precision cut lung slices (PCLSs) from control, LP, Hx, and LP/Hx mice showing increased accumulation of Sma (white) on the arterioles of LP/Hx mice. Scale bar represents 1000 μm. C, graph shows the percentage of distal arterioles (small: <20 μm, medium: 20–50 μm, large: >50 μm) with >50% Sma coverage. N = 3 for each experimental group. Each dot represents one PCLS inspected from a unique animal. D, PCLSs from experimental animals stained for Cd31 (endothelium, green), Sma (SMCs, white), Pdgfrβ (pericytes, red), and DAPI (blue). Scale bar represents 100 μm. Yellow boxes demonstrate increased magnification. Error bars demonstrate mean ± standard error. Statistical analysis was performed with one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01 ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 indicates statistical significance.

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  Figure 3. Fate mapping using Acta2-CreERT2::R26-mTmG mice suggests SMC origin cells contribute to increased muscularization on distal arterioles.A, fate mapping experiments were conducted for Acta2-mTmG mice in control, LP, Hx, and LP/Hx mice. IF staining was performed for Sma (red) and DAPI (blue). Notice both the colocalization of GFP + cells and Sma staining in LP and LP/Hx mice and the presence of GFP-, Sma + cells increased in the distal vasculature of LP/Hx mice compared to Hx and LP (white arrow). Scale bar represents 100 μm. B, precision cut lung slices (PCLS) from Acta2-mTmG experimental mice suggest the accumulation of endogenous GFP (green) in LP/Hx mice. The white boxes indicate the magnified areas in panels to the right (C, D), which are used for quantification. Scale bar represents 1000 μm. C, quantifications of endogenous GFP coverage in Acta2-mTmG control, LP, Hx, and LP/Hx mice. Each dot represents one vessel inspected from a PCLS. For each condition, six vessels were inspected from three (n = 3) biological samples. D, mean intensity was quantified for GFP in control, LP, Hx, and LP/Hx mice. N = 3 for each group. Each dot represents the average of six vessels inspected…

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  Figure 4. Pulmonary overcirculation combined with hypoxia leads to upregulation of Cxcl12 and an increase in contractility/proliferative cell markers in SMCs.A, real-time qPCR (qRT-PCR) of isolated SMCs (Cd146+) from control, LP, Hx, and LP/Hx mice for Cxcl12. The graph shows the relative expression of Cxcl12 compared to the housekeeping gene B2m. N = 3 for each experimental group. B, Precision cut lung slices (PCLS) from Acta2-CreERT2::R26-mTmG mice showing increased accumulation of Cxcl12 (white) in GFP + cells (SMCs) on distal arterioles (yellow arrows) in control, LP, Hx, and LP/Hx mice. Scale bar represents 100 μm. C, qRT-PCR of isolated SMCs (Cd146+) from control, LP, Hx, and LP/Hx mice for secretory/contractility (Acta2, Tagln, Myh11) and proliferative (Ccnd1, Pcna) cell markers. The graph shows the relative expression of each gene over the housekeeping gene B2m. N = 3 for each experimental group. Each dot represents a unique sample. Statistical analysis was performed with one-way ANOVA. Error bars demonstrate mean ± standard error. ∗p < 0.05, ∗∗p < 0.01 indicates statistical significance.

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  Figure 5. Healthy human pulmonary artery smooth muscle cells exposed to laminar shear stress result in CXCL12 upregulation.A, schematic image shows the proposed flow and cell culture experiments. B, IF staining was performed on healthy human pulmonary artery smooth muscle cells (hPASMCs) for SMA (green) and CXCL12 (red) with and without exposure to laminar shear stress. DAPI (blue). Scale bar represents 50 μm. C, graphs show immunofluorescence (IF) quantifications of CXCL12 in hPASMCs exposed to laminar shear stress and controls including the percent of overall cells expressing CXCL12 and mean intensity of CXCL12. N = 3 biological samples with three images quantified from each sample. Each symbol represents an image taken from a biological sample. Statistical analysis was performed with the Mann-Whitney test. Error bars demonstrate mean ± standard error. ∗p < 0.05, ∗∗∗p < 0.001 indicates statistical significance.

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  Figure 6. CXCL12 is upregulated in remodeled vessels in patients with PAH-CHD.A, staining of lung tissue from nondiseased patients and patients with PAH-CHD (VSD) for CXCL12 (red), SMA (white), CD31 (green), and DAPI (blue). Yellow arrows highlight CXCL12 accumulation in remodeled distal vasculature. Scale bar represents 100 μm. B, the vessel area and mean intensity of SMA immunofluorescence (IF) staining in human lung samples were quantified for patients with PAH-CHD and nondiseased controls. N = 3 for each group. Each symbol represents an individual calculation of one vessel from a biological sample. For each sample, three vessels were inspected and quantified. C, the vessel area and mean intensity of CXCL12 IF staining in lung samples from patients with PAH-CHD and nondiseased controls. N = 3 for each group. Each symbol represents a calculation of one vessel inspected from a biological sample. For each sample, three vessels were inspected and quantified. D, schematic illustration shows the role of Cxcl12 and the response of SMCs, ECs, and PCs to increased blood flow and Hx. Statistical analysis was performed with the Mann-Whitney test. Error bars demonstrate mean ± standard error. ∗∗p <…