Jag2 expression is upregulated in hypoxia-treated rats and PASMCs.

(A) Visualization of Jag2 expression in the GSE85618 dataset. (B) Jag2 mRNA and protein (C) expression by qRT-PCR, immunohistochemistry, and western blotting in lung tissue of normal and hypoxia-treated rats. N = 6 rats per group. (D) Immunofluorescence double staining of α-SMA and Jag2 in lung tissues of normal and hypoxia-treated rats. Scale bar, 10 μm. (E) Jag2 mRNA and protein expression by qRT-PCR, immunofluorescence (F), and western blotting (G) in normal and hypoxia-treated PASMCs. Each group N = 3, and three biological replicates per group for cellular experiments. Scale bar, 25 μm. Data shown are mean ± SD; ***P < 0.001.</p

Inhibition of Jag2 effectively ameliorates hypoxia-induced PH in rats.

(A) Fluorescence microscopy images of lung tissue four weeks after intratracheal AAV.1Jag2 instillation, where green fluorescence represents the expression and localization of AAV1 in lung tissue. DAPI stains the nuclei blue. Scale bar=10μm. (B) Representative immunoblot images and quantitative analysis of Jag2 protein expression in the control and AAV1.Jag2 groups. (C) Waveform diagram and quantitative analysis of RVSP (mmHg) in the indicated groups. (D) Representativemicroscopic images of distal pulmonary vascular stained with H&E, immunostained for α-SMA, and Masson trichrome in control, HPH, and HPH+Jag2i rats. Scale bar=10μm. (E) Fulton index RV/(LV+S) in the three groups. (F) The relative medial thickness expressed as a ratio of (total vascular area ‐ lumen area) to total vascular area (media/CSA). N=6 rats per group. Data shown are mean ± SD; **P < 0.01, ***P < 0.001.</p

Primers for real-time PCR (rat).

Notch pathway has played a significant role in the pathophysiology of pulmonary hypertension (PH). However, the role of Jagged 2 (Jag2), one ligand of Notch, remains to be elucidated.Therefore, determining the contribution of Jag2 to PH and its impact on pulmonary artery smooth muscle cells (PASMCs) was the aim of this investigation. Adeno-associated virus-mediated Jag2 inhibition was used to explore the role of Jag2 in peripheral pulmonary vascular remodeling assessed in a rat model of chronic hypoxia (10% O2, 4 weeks) induced pulmonary hypertension. In vitro, the effect of Jag2 silencing on hypoxia (1% O2, 24h) induced rat PASMCs was determined. Group differences were assessed using a 2-sided unpaired Student’s t-test for two groups and one-way ANOVA for multiple groups. Jag2 upregulation was first confirmed in rats with sustained hypoxia-induced PH using publicly available gene expression data, experimental PH rat models and hypoxia induced rat PASMCs. Jag2 deficiency decreased oxidative stress injury, peripheral pulmonary vascular remodeling (0.276±0.020 vs. 0.451±0.033 μm, PPJag2 knockdown decreased proliferation (1.227±0.051 vs. 1.45±0.07, P = 0.012), increased apoptosis (16.733%±0.724% vs. 6.56%±0.668%, P</div

Heat map and volcano plot of the GSE85618 dataset.

(A) A visual representation in the form of a heat map to highlight the top 40 genes that show significant differences in expression levels within the GSE85618 dataset. Each column in the heat map corresponds to a particular sample, while each row represents the expression level of a specific gene. (B) volcano plot of the GSE85618 dataset showing the fold change (x-axis) differentially expressed genes. (TIF)</p

Absence of Jag2 alleviates hypoxia-induced oxidative stress injury in rats.

(A)Comparison of DHE staining and quantitative analysis in rat lung tissue samples treated with Control, HPH, and HPH+Jag2i. (B) Comparison of lung SOD, MPO, and MDA activity in the different groups. (C) Representative immunoblot images and quantitative analysis of Nrf2 and HO-1 protein expression inControl, HPH, and HPH+Jag2i rats. N=6 rats per group. The values expressed are mean ± SD. *P< 0.05, **P <0.01, ***P < 0.001.</p

Immunoblot images of α-SMA, PCNA and Sirt1.

(A) Representative immunoblot images and quantitative analysis of α-SMA and PCNA protein expression in Control, HPH, and HPH+ Jag2i rats. N=6 rats per group. (B) Representative western blots and quantitative analysis of Sirt1 protein expression in Control and Si-Sirt1 groups. (C) Representative western blots and quantitative analysis of Sirt1 protein expression in Control, Hypoxia and Hy+Si-Jag2 groups. *P (TIF)</p

Jag2 deficiency prevents proliferation and promotes apoptosis in hypoxia-treated PASMCs.

(A) PASMCs were transfected with Si-NC or Si-Jag2 and analyzed by qRT-PCR and western blotting (B). (C) PASMC viability in control, hypoxia, and hypoxia+Si-Jag2 groups was detected by the CCK8 assay. (D) EdU analysis of PASMCs in the indicated groups. (E) Flow cytometry and quantitative analysis of apoptosis in the three groups. (F) Representative western blots and quantitative analysis of Bax/Bcl2 in the three groups. *P<0.05, **P<0.01, ***P<0.001, each group N = 3. Three biological replicates per group for cellular experiments.</p

Jag2 regulates mitochondrial injury in hypoxia-induced PASMCs via Sirt1 signaling.

(A)Comparison of Sirt1-6 mRNA expression between control, hypoxia, and hypoxia+Si-Jag2 groups by qRT‒PCR. (B) Representative western blots and quantitative analysis (C) of Sirt1 protein expression in hypoxia+Si-Jag2 and hypoxia+Si-Jag2+Si-Sirt1 groups. (D) JC-1 staining and quantitative analysis (E) of the intracellular mitochondrial membrane potential in the indicated groups. (F) Intracellular mitochondrial superoxide levels of the two groups were tested using the MitoSOX assays and flow cytometry. (G) Representative western blots and quantitative analysis (H) of Coxiv and Tom20 in the two groups. *P<0.05, **P<0.01, ***P<0.001, each group N = 3. Three biological replicates per group for cellular experiments.</p

Raw image of western blots.

Notch pathway has played a significant role in the pathophysiology of pulmonary hypertension (PH). However, the role of Jagged 2 (Jag2), one ligand of Notch, remains to be elucidated.Therefore, determining the contribution of Jag2 to PH and its impact on pulmonary artery smooth muscle cells (PASMCs) was the aim of this investigation. Adeno-associated virus-mediated Jag2 inhibition was used to explore the role of Jag2 in peripheral pulmonary vascular remodeling assessed in a rat model of chronic hypoxia (10% O2, 4 weeks) induced pulmonary hypertension. In vitro, the effect of Jag2 silencing on hypoxia (1% O2, 24h) induced rat PASMCs was determined. Group differences were assessed using a 2-sided unpaired Student’s t-test for two groups and one-way ANOVA for multiple groups. Jag2 upregulation was first confirmed in rats with sustained hypoxia-induced PH using publicly available gene expression data, experimental PH rat models and hypoxia induced rat PASMCs. Jag2 deficiency decreased oxidative stress injury, peripheral pulmonary vascular remodeling (0.276±0.020 vs. 0.451±0.033 μm, PPJag2 knockdown decreased proliferation (1.227±0.051 vs. 1.45±0.07, P = 0.012), increased apoptosis (16.733%±0.724% vs. 6.56%±0.668%, P</div

Jag2 knockdown ameliorates mitochondrial dysfunction induced by hypoxia.

(A) JC-1 staining of the intracellular mitochondrial membrane potential of control, hypoxia, and hypoxia+Si-Jag2 groups. JC-1 aggregates produce red fluorescence; JC-1 monomer produces green fluorescence. An increase in the relative ratio of red to green fluorescence indicates mitochondrial depolarization. (B) Intracellular mitochondrial superoxide levels in the three groups were tested using the MitoSOX assay and flow cytometry. (C) Representative western blots and quantitative analysis (D) of Coxiv and Tom20 in three groups. *P<0.05, **P<0.01, ***P<0.001, each group N = 3. Three biological replicates per group for cellular experiments.</p