Fig 1: CD248 regulates PDGF-BB-induced signal pathways and promotes a PDGF induced phenotype switch of PASMCs. A, After hPASMCs were treatment with PDGF-BB (20 ng/ml) for 0 or 30 min, CD248 (green) and PDGFR-ß (red) expression was detected by confocal microscopy. The cell nucleus was stained with DAPI (blue). B, Co-immunoprecipitation using CD248 antibody can pull down PDGFR-ß in hPASMCs. C, Coimmunoprecipitation using PDGFR-ß antibody can pull down CD248 in hPASMCs. IP: immunoprecipitant; Input: whole cell lysate. D, hPASMCs were transfected with siCD248 or siCRTL for 24 h before treatment with PDGF-BB (20 ng/mL) for another 30 min; left, p-PDGFR-ß, p-ERK ½, and p-NF-?B p65 protein levels were measured; right, relative expression of p-PDGFR-ß, p-ERK 1/2 and p-NF-?B p65; E, Left, p-ERK 1/2 and p-NF-?B p65 protein levels were measured after PDGF-BB induction of hPASMCs treated with ontuxizumab or Isotype IgG (10 µg/mL); right, relative expression of p-ERK 1/2 and p-NF-?B p65; F, hPASMCs were transfected as indicated, cultured, and administered with PDGF-BB (20 ng/mL), and the mRNA levels of proliferative and migration-related markers were detected by real-time PCR; G, hPASMCs were administered with PDGF-BB and treated with ontuxizumab or Isotype IgG (10 µg/mL), and the mRNA levels of proliferative and migration-related markers were detected by real-time PCR; H and I, hPASMCs were transfected as in (F), (H) the mRNA levels of synthetic and (I) contractile related markers were detected by real-time PCR; J and K, hPASMCs were treated as in (G), (J) the mRNA levels of synthetic and (K) contractile-related markers were detected by real-time PCR. The results are expressed as mean ± SD (n = 3 for each group), * P < .05, ** P < .01; one-way ANOVA with Tukey's multiple comparisons test or two-way ANOVA analysis
Fig 2: Disruption of CD248 attenuates MCT-induced pulmonary vascular remodeling and PAH. A, Schematic of CD248 knockdown in rats with MCT-injection; B and C, (B) RVSP; and (C) RVHI in lenti-shCD248 or lenti-Negative Control (NC) rats 4 weeks after MCT injection (n = 6 rats per group); D, H&E staining and immunofluorescent staining of pulmonary arteries (PAs) (<100 µm in diameter) stained with aSMA (green) and DAPI (blue). Scale bar, 50 µm; E, quantitative analysis of PA medial wall thickness for images in (D) (n = 3 rats per group, 10 PAs per rat); F, Percentage of non-muscularized vessels, partially muscularized vessels, and fully muscularized vessels from MCT-treated rats (n = 5 rats per group); G, left, representative immunofluorescent staining of PAs labeled with Ki67 in red. PASMCs were labeled using a-SMA staining (green), right, represent the ratio of cells positive for Ki67 in PAs. Arrows indicate positive cells; H, left, WB analysis of CD248, p-NF-?B p65, and NOX4 in lung tissues from MCT-treated rats; right, relative expression of CD248, p-NF-?B p65, and NOX4 (n = 3 biological replicates for each group). The results are expressed as mean ± SD, * P < .05, ** P < .01; one-way ANOVA with Tukey's multiple comparisons test, or two-way ANOVA analysis. Scale bar: 50 µm
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