Loss of adenomatous poliposis coli-α3 integrin interaction promotes endothelial apoptosis in mice and humans

Abstract

Rationale: Pulmonary hypertension (PH) is characterized by progressive elevation in pulmonary pressure and loss of small pulmonary arteries. As bone morphogenetic proteins promote pulmonary angiogenesis by recruiting the Wnt/β-catenin pathway, we proposed that β-catenin activation could reduce loss and induce regeneration of small pulmonary arteries (PAs) and attenuate PH.

Objective: This study aims to establish the role of β-catenin in protecting the pulmonary endothelium and stimulating compensatory angiogenesis after injury.

Methods and results: To assess the impact of β-catenin activation on chronic hypoxia-induced PH, we used the adenomatous polyposis coli (Apc(Min/+)) mouse, where reduced APC causes constitutive β-catenin elevation. Surprisingly, hypoxic Apc(Min/+) mice displayed greater PH and small PA loss compared with control C57Bl6J littermates. PA endothelial cells isolated from Apc(Min/+) demonstrated reduced survival and angiogenic responses along with a profound reduction in adhesion to laminin. The mechanism involved failure of APC to interact with the cytoplasmic domain of the α3 integrin, to stabilize focal adhesions and activate integrin-linked kinase-1 and phospho Akt. We found that PA endothelial cells from lungs of patients with idiopathic PH have reduced APC expression, decreased adhesion to laminin, and impaired vascular tube formation. These defects were corrected in the cultured cells by transfection of APC.

Conclusions: We show that APC is integral to PA endothelial cells adhesion and survival and is reduced in PA endothelial cells from PH patient lungs. The data suggest that decreased APC may be a cause of increased risk or severity of PH in genetically susceptible individuals.

Figure 1. Apc^Min/+ mice in chronic hypoxia demonstrate increased right ventricular systolic pressure (RVSP) and RV hypertrophy and small vessel loss compared to wild type C57 littermate controls

Measurements of (A) RVSP, (B) right ventricular weight relative to that of left ventricle and septum (RV/LV+S), (C) muscularization of peripheral arteries at alveolar wall and duct level and (D) number of peripheral alveolar duct and wall arteries per 100 alveoli in mice exposed to room air (Normoxia), three weeks of 10% O₂ (Hypoxia) and three weeks of recovery in room air (Recovery) as described in the Methods. Representative images of muscularized pulmonary arteries (C) and vessel number (D) are shown above the corresponding measurements. Bars represent mean ±SEM from experiments involving 10 animals per group. **P<0.001, ***P<0.0001 vs. normoxia, #P<0.01, ##P<0.001 vs. C57, one way ANOVA with Bonferroni’s post-test. Scale bar=25µm (C) and 100µm (D).

Figure 2. Apc^Min/+ mvPAECs demonstrate reduced survival and growth

(A) Representative western immunoblots for βC and APC in lysates from C57 littermate and Apc^Min/+ mvPAECs. Densitometric values are shown relative to α-tubulin. ***P<0.0001, unpaired t-test. (B) Apoptosis was measured by the Caspase 3/7 assay in C57 and Apc^Min/+ mvPAECs exposed to a range of serum concentrations (0–10%) under either normoxia (left panel) or hypoxia (right panel). After 24 hours, lysates were analyzed for luciferase activity (LU) as described in the Methods. Camptothecin was used as a positive control. (C) Proliferation was assesed by cell count assays in C57 and Apc^Min/+ mvPAECs exposed to a range of VEGF concentrations (0–50ng/ml) under either normoxia (left panel) or hypoxia (right panel). Cell numbers were measured 72 hours after the addition of VEGF as described in the Methods. Bars represent mean ±SEM from N=3 experiments. *P<0.01, **P<0.001, ***P<0.0001 vs. C57 at 10% FBS (apoptosis assay) or baseline (VEGF proliferation assay); #P<0.01, ##P<0.001, ###P<0.0001 vs. C57, one way ANOVA with Bonferroni’s post-test.

Figure 3. Apc^Min/+ mvPAECs demonstrate reduced adhesion to matrigel resulting in decreased tube formation

(A) Adhesion assay was performed by counting the number of C57 littermate and or Apc^Min/+ mvPAECs attached to a matrigel coated surface 30 minutes after seeding. Bars represent mean ±SEM from N=3 experiments. ***P<0.0001, unpaired t-test. (B) Representative photomicrographs of tube formation assays in matrigel at six hours. Scale bar=150µm. Tube number was analyzed as described in the Methods. Bars represent mean ±SEM from N=3 experiments. **P<0.001, unpaired t-test. (C) Representative immunofluorescence microscopy photomicrographs demonstrate abundant focal adhesions in controls (C-E) but not in Apc^Min/+ mvPAECs (F-H) as judged by vinculin (red) at the tips of actin (green) fibers at the cell periphery. Scale bar=30µm.

Figure 4. APC deficiency in mvPAECs reduces adhesion to laminin in a βC independent manner

(A) Representative western immunoblots for APC and βC in human mvPAECs lysates treated with scrambled (SC) or APC siRNA. Densitometric values are shown relative to α-tubulin. Bars represent mean ±SEM of n=3. ***P<0.0001, unpaired t-test. (B, C, D) Adhesion of APC and SC siRNA treated human mvPAEC on increasing amounts of fibronectin (FN, panel B), collagen IV (CIV, panel C), and laminin (LN, panel D). (E) Adhesion to all three substrates (10µg per well) was measured in cells co-transfected with APC siRNA and scrambled or βC siRNA. The average number of cells was calculated by counting the total number of cells in six random fields per well (200x magnification). Bars represent mean ±SEM from N=3 experiments. *P<0.01, **P<0.001, ***P<0.0001 vs. uncoated, #P<0.01,##P<0.001, ###P<0.0001 vs. SC, one way ANOVA with Bonferroni’s post-test.

Figure 5. Laminin induces APC dependent ILK-1 and Akt activation

(A) Immunoprecipitation with α3 integrin antibody and immunoblotting with APC antibody in whole cell lysates of human mvPAECs following adhesion to CIV, LN and FN. (B) ILK-1 kinase assay performed in scrambled and APC siRNA treated human mvPAECs seeded on CIV, LN and FN coated surfaces. Phosphorylation of the GSK3β substrate was used as a measure of ILK kinase activity as decribed in the Methods. ILK-1 activity was measured by densitometry against total ILK-1 levels. Total GSK3β levels in whole lysates are also shown. **P<0.001, one way ANOVA with Bonferroni’s post-test Bars represent mean ±SEM from N=3 experiments. (C) Akt phosphorylation in whole cell lysates recovered from scrambled or APC siRNA treated human mvPAEC seeded on CIV, LN and FN coated surfaces. Densitometric values are shown relative to total Akt. Bars represent mean ±SEM from N=3 experiments. ***P<0.0001, scrambled vs. corresponding APC siRNA, one way ANOVA with Bonferroni’s post-test.

Figure 6. The α3 cytoplasmic tail regulates matrix (ECM) binding specificity and requires preservation of the QPSXXE motif

(A) Diagram illustrating the WT and mutant α3 and α4 integrin constructs. Details of the their construction can be found in the Methods. (B and C) Adhesion to CIV, LN and FN was measured in human mvPAECs transfected with the mutant α4 chimera containing the α3 cytoplasmic tail (α4Δ3) (B) or the corresponding α3 chimera containing the α4 cytoplasic tail (α3Δ4) (C) following SC or APC siRNA treatment. (D) Adhesion to CIV, LN and FN in mvPAECs transfected with either vector or mutant α3 integrin construct containing mutations in the amino acids within the QPSXXE motif (Δα3) as described in the Methods. Bars represent mean ±SEM from N=3 experiments. ***P<0.0001 versus CIV, one way ANOVA with Bonferroni’s post-test.

Figure 7. Microvascular PAECs from IPAH patients demonstrate adhesion defects and reduced APC protein expression

(A) Tube formation in matrigel, (B) immunohistochemistry for APC and CD31 in patient microvessels, (C) adhesion to CIV, LN and FN, (D) ILK-1 activation assay and (E) WB for APC, ILK-1, α3 and β1 integrin in IPAH versus healthy donor mvPAECs. Lysates from mvPAECs were isolated from five healthy donors and five IPAH patients were used for western immunoblotting. Line separating first three samples from samples 4+5 in (E) was placed to indicate that samples were run in different gels. (D) Transfection of a WT APC expression vector versus empty plasmid and tube formation in IPAH mvPAECs. Bars represent mean ±SEM from N=5 experiments or patients. ***P<0.0001, unpaired t-test in A and F. ***P<0.0001 one way ANOVA with Bonferroni’s post-test versus all other groups in C and D. ***P<0.0001 versus corresponding control in E. Scale bar=150 µm in A and F. Scale bar=25µm in B.

Figure 8. Proposed model for involvement of APC in laminin adhesion and pAkt mediated mvPAEC survival

(A) Upon binding laminin, α3β1 integrin complex recruits APC to the α3 cytoplasmic tail (B). Once there, APC facilitates ILK-1 activation, formation of focal adhesion complexes by recruitment of vinculin, paxillin and actin, followed by activation of pAkt leading to cell survival (C).