NOTCH3 and Pulmonary Arterial Hypertension

Figure 1. NOTCH3 is activated by the binding of the JAGGED-1 ligand, stimulating the proteolytic S2 cleavage by ADAM of the NOTCH3 extracellular domain (ECD) and intracellular domain (ICD). Then, S3 cleavage by γ-secretase liberates the ICD into the cytoplasm. The ICD translocates to the nucleus and binds to the RBP-J transcription complex and functions as a transcriptional enhancer of Hairy/Enhancer of Split (HES) and Hairy/Enhancer of Split-related (HRT/HEY) genes.

Figure 2. (A) Left: Western blot analysis of NOTCH3 ICD and HES-5 relative to GAPDH in the lungs of subjects with varying severities of PAH and control individuals. ICD, intracellular domain. PVR, pulmonary vascular resistance. Right: Relative expression values obtained by densitometry of NOTCH3 ICD or HES-5 protein normalized to GAPDH (n = 1 for each PVR listed). (B) Left: Western blot analysis of Notch3 ICD and Hes-5 relative to Gapdh from mouse lungs during the development of hypoxia-induced pulmonary hypertension, compared to normoxic animals. Right: Relative expression values obtained by densitometry of Notch3 ICD or Hes-5 proteins normalized to Gapdh (n = 20 animals for each time point). RVSP, right ventricular systolic pressure (mmHg). (C) Left: Western blot analysis of Notch3 ICD and Hes-5 relative to Gapdh from rat lungs during the development of Sugen-induced pulmonary hypertension, compared to control animals (For (B–D), four measurements of RSVP were taken over a 10-min period and averaged). Right: Relative expression values obtained by densitometry of Notch3 ICD or Hes-5 protein normalized to Gapdh (n = 20 animals for each time point). (D) Left: Western blot analysis of Notch3…

Figure 3. (A) Left: Western blot analysis of NOTCH ligands relative to GAPDH in subcultured sPASMCs from PAH and non-PAH individuals. Right: Relative expression values obtained by densitometry of NOTCH3 ligand proteins normalized to GAPDH (n = 10 sPASMC subcultures per group). (B) Left: NOTCH3 ligand (red) and α-smooth muscle actin (ACTA2; green) immunofluorescence staining of human PAH and non-PAH-subcultured sPASMCs. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 25 μm. Right: Relative fluorescence values obtained by densitometry of NOTCH3 ligands normalized to background readings (n = 10 representative cells for each NOTCH3 ligand). (C) Left: Western blot analysis of NOTCH ligands relative to GAPDH in lung tissue from three different individuals. Right: Relative expression values obtained by densitometry of NOTCH3 ligand proteins normalized to GAPDH (n = 20 lung samples from 20 patients in each group). Figure modified and adapted from previous publication [8].

Figure 4. Rats were intraperitoneally injected with Sugen (20 mg/kg) followed by 3 weeks of hypoxia and 2 weeks of normoxia to induce pulmonary hypertension (PH). Subsequently, animals were treated with subcutaneous anti-NOTCH3 Ab 28042 (40 mg/kg) or placebo (PBS) under normoxic conditions for 10 weeks. (A) Western blot analysis of Jag-1, Notch3 ICD, and Hes-5 in the lungs of Sugen-hypoxia rats treated with anti-NOTCH3 Ab 28042 or PBS, normalized to Gapdh. (B) H&E-stained sections of small pulmonary arteries from lungs of rats at the beginning of treatment and during treatment with anti-NOTCH3 Ab 28042 or PBS. Results are representative sections from five rats per group at each time point. Scale bar, 25 μm. (C) Top: Representative continuous-wave Doppler signals measured from the right ventricular outflow tract before and during rat treatment with anti-NOTCH3 Ab 28042 compared to controls. Main pulmonary artery blood flow in PBS-treated rats showed shortened acceleration to peak velocity and midsystolic notching (yellow arrows), indicative of elevated pulmonary vascular resistance. Bottom: Graph of pulmonary artery acceleration time (PAAT; three measurements per animal per timepoint) in…