Notch3 signaling promotes the development of pulmonary arterial hypertension

Abstract

Notch receptor signaling is implicated in controlling smooth muscle cell proliferation and in maintaining smooth muscle cells in an undifferentiated state. Pulmonary arterial hypertension is characterized by excessive vascular resistance, smooth muscle cell proliferation in small pulmonary arteries, leading to elevation of pulmonary vascular resistance, right ventricular failure and death. Here we show that human pulmonary hypertension is characterized by overexpression of NOTCH3 in small pulmonary artery smooth muscle cells and that the severity of disease in humans and rodents correlates with the amount of NOTCH3 protein in the lung. We further show that mice with homozygous deletion of Notch3 do not develop pulmonary hypertension in response to hypoxic stimulation and that pulmonary hypertension can be successfully treated in mice by administration of N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a gamma-secretase inhibitor that blocks activation of Notch3 in smooth muscle cells. We show a mechanistic link from NOTCH3 receptor signaling through the Hairy and enhancer of Split-5 (HES-5) protein to smooth muscle cell proliferation and a shift to an undifferentiated smooth muscle cell phenotype. These results suggest that the NOTCH3-HES-5 signaling pathway is crucial for the development of pulmonary arterial hypertension and provide a target pathway for therapeutic intervention.

Figure 1

Notch3 is a marker for PAH and PH disease severity. (a) Northern blot analysis of Notch3 relative to 18S RNA from human lungs with idiopathic PAH (three subjects - left panel), mice with hypoxia-induced PH (three animals - middle panel), and rats with monocrotaline-induced PH (three animals - right panel) compared to control lungs. (b) Upper panel: Western blot analysis of Notch3 ICD relative to Gapdh in the same lung tissue as in A. Lower panel: Relative expression values obtained by densitometry of Notch3 ICD protein normalized to Gapdh (n = 20 for each group). (c) Northern blot analysis of Notch3 from organs of mice exposed to hypoxia (H) or normoxia (C) for 6 weeks (left panel), Western blot analysis of Notch3 ICD in the same organs (right panel, top). Relative expression values obtained by densitometry of Notch3 protein normalized to Gapdh (n = 4 per group) (right panel, bottom). (d) Western blot analysis of NOTCH3 ICD and HES5 relative to GAPDH in the lungs of subjects with varying severity of PAH and control individuals. PVR = pulmonary vascular resistance (left panel, top). Western blot analysis of Notch3 ICD and Hes5 relative to Gapdh from mouse lungs during development of hypoxia-induced PH, or rat lungs during development of monocrotaline-induced PH, compared to control animals (middle and right panel, top). Lower panel: Relative expression values obtained by densitometry of Notch3 ICD or Hes5 protein normalized to Gapdh (n = 1 for each PVR listed; n = 20 animals for each timepoint). (e) Northern blot analysis of total RNA from the lungs of mice and rats as in D.

Figure 2

Notch3 and Hes5 expression are specific to sPASMCs in the lung. (a) Notch 3 (red) and α-SM-actin (green) immunofluorescence staining in small pulmonary arteries from humans (top panels) and mice (bottom panels) with and without PAH/PH. Nuclei are counterstained with DAPI (blue). Notch3 staining is confined to sPASMCs and predominates in vessels from PAH/PH lung tissue. Scale bar = 50 μm. (b) Hes5 (red) and α-SM-actin (green) immunofluorescence staining in small pulmonary arteries 75–100 μm in diameter in pulmonary hypertensive and normotensive human (top panels) and mouse (bottom panels) lung tissue. Nuclei are counterstained with DAPI (blue). Hes5 staining is confined to sPASMCs and predominates in vessels from PAH/PH lung tissue. Scale bar = 50 μm. (c) qRT-PCR analysis of total RNA from subcultured sPASMCs derived from the lungs of ten individuals with and ten individuals without PAH (three subcultures per subject). NOTCH3 and HES5 values are normalized to 18S rRNA control. (d) Upper panel: Western blot analysis of NOTCH3 and HES5 in subcultured sPASMCs derived from the lungs of three human subjects with and three human subjects without PAH. Lower panel: Relative expression values obtained by densitometric analysis of NOTCH3 and HES5 normalized to GAPDH (n = ten PAH subjects’ subcultures, n = ten non-PAH subjects’ subcultures).

Figure 3

Notch3 increases vSMC proliferative capacity in vitro. (a) Left panel: Western blot demonstrating increase in NOTCH3 ICD and HES5 in three sPASMC subcultures (from different individuals) infected with Adeno-Notch3 ICD compared to the same subcultures infected with Adeno-lacZ. Right panel: Relative expression values obtained by densitometry of Notch3 ICD and HES5 protein normalized to GAPDH (n = three subjects’ subcultures per group). (b) Stimulation of human sPASMC proliferation by constitutive Notch3 ICD expression. Left panel: Averaged change in cell number after Adeno-Notch3 ICD or Adeno-lacZ transduction. (ten human sPASMC subcultures, 12 viral infections per subculture). Right panel: ³[H] leucine incorporation in the same cells as in left panel. (c) Effects of HES5 siRNA and its scrambled control (30 nM). Left panel: qRT-PCR analysis of total RNA from Adeno-Notch3 ICD-infected human sPASMCs (n = three independent subcultures from ten individuals tested) and control vector-infected SMCs (Adeno-lacZ; n = three aliquots of same subcultures tested) treated with scrambled or HES5 siRNA. Values are normalized to 18S rRNA control. Right panel, top: Western blot analysis of protein from sPASMCs treated with HES5 or scrambled RNA after either Adeno-lacZ or Adeno-Notch3 ICD transfection. Right panel, bottom: Relative expression values obtained by densitometry of HES5 protein normalized to TUBULIN (n = three independent experiments for each human subculture, with ten human subcultures per group). (d) Left panel: Growth curve of sPASMCs from ten human lungs without PAH (three subcultures per subject), treated with either scrambled or HES5 siRNA, followed by transfection with either Adeno-Notch3 ICD or Adeno-lacZ. *P < 0.01 compared to control groups. Right panel: ³[H] leucine incorporation for the same cells as in left panel. *P < 0.01 compared to control groups.

Figure 4

Faster growth rates and lower levels of contractile vSMC markers in sPASMCs from PAH humans compared with non-PAH subjects are dependent on HES5. (a) qRT-PCR analysis of genes associated with vSMC differentiation in sPASMCs (n = ten subjects per group, three subcultures per individual) from people with and without PAH. Average expression values are normalized to 18S rRNA. (b) Left panel: Growth curves of sPASMCs isolated from human lungs (n = ten individuals per group, three subcultures per individual) with and without PAH. *P < 0.01 versus controls. Right panel: ³[H] leucine incorporation for the same cells as in the left panel. *P < 0.01 versus controls. (c) Effects of HES5 and scrambled siRNA (30 nM). Left panel: qRT-PCR analysis of total RNA from PAH and non-PAH sPASMCs (n = three independent subcultures from ten humans per group) treated with HES5 or scrambled siRNA. Values are normalized to 18S rRNA. Right panel, top: Western blot analysis of protein derived from the same cells in the left panel. Right panel, bottom: Relative expression values obtained by densitometry of HES5 protein normalized to TUBULIN (n = three independent experiments for each human subculture with ten human subcultures per group). (d) Left panel: Growth curve of sPASMCs isolated from the PAH and non-PAH lungs (three subcultures per human subject) treated with either HES5 or scrambled siRNA. *P < 0.01 compared to control groups. Right panel: ³[H] leucine incorporation for the same cells as in left panel. *P < 0.01 compared to control groups. (e) Effect of selective knockdown of HES5 on qRT-PCR measurement of MHC, SMOOTHELIN, α-SM-ACTIN, and CALPONIN mRNA in sPASMCs from humans with and without PAH (n = ten human subjects per group, three subcultures/human). Data were normalized to 18S rRNA levels and represent means from three independent experiments.

Figure 5

Notch3^−/− mice are resistant to the development of hypoxic PH. (a) Upper panel: Western blot analysis of Notch3 ICD and Hes5 in Notch3^−/− and Notch3^+/+ lung tissues. Lower panel: Relative expression values obtained by densitometry of Notch3 ICD or Hes5 protein normalized to Gapdh (n = 20 per group). (b) Averaged SBP and RVSP in Notch3^−/− mice as well as Notch3^+/+ and Notch3^+/− littermates at serial timepoints under hypoxic conditions of 10% oxygen (ten readings per animal over one hour, 20 animals per group at each timepoint). (c) Hematoxylin and eosin-stained sections (rows one and three) and immunohistochemical analysis of PCNA (rows two and four) of small pulmonary arteries from lungs of Notch3^−/− and Notch3^+/+ mice after 4 and 6 weeks of hypoxia. Dark nuclei are PCNA positive. Results are representative sections from at least ten animals per group for each timepoint. Scale bar = 25 μm. (d) Percentage of sPASMCs that are PCNA-positive in Notch3^−/− and Notch3^+/+ mice (n = ten for animals/group, ten lung sections/animal) at timepoints during hypoxia. (e) Pulmonary angiograms of Notch3^−/− and Notch3^+/+ animals after 6 weeks of hypoxia. (f) Ratio of the weight of right ventricle (RV), to that of left ventricle plus septum (LV + S), as an index of RV hypertrophy in Notch3^−/−, Notch3^+/+ mice, and Notch3^+/− mice (for all groups, n = 20) after 6 weeks of hypoxia.

Figure 6

DAPT treatment reverses the development of hypoxic PH in mice. Animals were exposed to 2 weeks of 10% oxygen, followed by 4 weeks of 10% oxygen and treatment with subcutaneous DAPT or placebo (DMSO). (a) Left panel: Western blot analysis of Notch3 ICD and Hes5 in the lungs of mice receiving DAPT as a function of time. Right panel: Relative expression values obtained by densitometry of Notch3 ICD and Hes5 protein normalized to Gapdh (n = ten for each timepoint in each group). (b) Hes5 (red) and α-SM-actin (green) immunofluorescence staining in small pulmonary arteries from hypoxic mice treated for 4 weeks with DAPT or DMSO. Nuclei are counterstained with DAPI. Scale bar = 25 μm. (c) Averaged RVSP (left panel) and SBP (right panel) in mice under hypoxic conditions (ten readings per animal, 20 animals per group at each timepoint). *P < 0.01 versus DMSO control. (d) Left panel: Hematoxylin and eosin-stained sections (rows one and three) and immunohistochemical analysis of PCNA (rows two and four) of small pulmonary arteries from the lungs of mice after treatment with DAPT or placebo (days 15–42) and 6 weeks of hypoxia. Dark nuclei are PCNA positive. Results are representative sections from five animals per group per timepoint. Scale bar = 25 μm. Right panel: Percentage of sPASMCs that are PCNA-positive in DAPT- and placebo-treated mice in hypoxia. (n = ten animals per timepoint for each group, ten sections/animal). (e) Left panel: TUNEL staining (green) with DAPI nuclear staining (blue) in small pulmonary arteries from the same animals lungs as in D. Results are representative sections from five animals per group per timepoint. Scale bar = 25 μm. Right panel: Percentage of sPASMCs that are TUNEL-positive in DAPT- and placebo-treated mice in hypoxia. (n = ten animals per timepoint for each group, ten sections/animal). (f) Pulmonary angiograms of DAPT-and placebo-treated animals after 6 weeks of hypoxia. (g) Ratio of the weight of right ventricle (RV), to that of left ventricle plus septum (LV + S), as an index of RV hypertrophy in DAPT- (n = 20) versus placebo-treated (n = 20) mice after 6 weeks of hypoxia.