Ang 1

Mouse lung tissues were homogenized with an ice-cold lysis buffer. The homogenates were centrifuged at 12,000 rpm for 15 minutes at 4°C and the total protein concentrations were determined using a BCA protein assay kit (Pierce Co, IL). An aliquot (30 μg) of the protein lysate was separated on a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane by electrophoresis. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline and incubated with the following primary antibodies overnight: neural glial antigen (NG) 2, fibroblast specific protein (FSP) -1 (1:1000, abcam), PHD2, HIF-1α and HIF-2α (1:1000, Novus Bio, CO), Notch3, β-myosin heavy chain (MHC), transforming growth factor (TGF)-β and Ang-1 (1:1000, Sigma, MO). The membranes were then washed and incubated for 2 hours with an anti-rabbit or anti-mouse secondary antibody conjugated with horseradish peroxidase (1:5000, Santa Cruz, CA). Densitometric analysis of the bands were carried out using image acquisition and analysis software (TINA 2.0).

The lung, HCASMC and MLEC were harvested and homogenized in 300 μl of lysis buffer and total protein concentrations were determined using a BCA protein assay kit (Pierce Co, IL). Fifteen microgram of protein were subjected to SDS-PAGE on 10% polyacrylamide gels and transferred to a PVDF membrane. The blots were probed with Sirt3 and Tie-2 (1:1000, Cell Signaling, MA), NG2 (1:1000, abcam), PHD2 and HIF-2α (1:1000, Novus Bio, CO), Notch3 and Ang-2 (1:1000, Santa Cruz, CA) and Ang-1 (1:1000, Sigma, MO) antibodies. The membranes were then washed and incubated with a secondary antibody coupled to horseradish peroxidase and densitometric analysis was carried out using image acquisition and analysis software (TINA 2.0).

The concentration of VEGF, ANG-1, TGFβ, IGF-1, and HGF in CM was determined in duplicate wells by sandwich ELISA, following the manufacturer’s guidelines for each kit (VEGF: Sigma-Aldrich; ANG-1, TGFβ, IGF-1, HGF: LifeSpan BioSciences, Inc., WA).

Subconfluent cultures were fixed using 4% paraformaldehyde at room temperature (RT) for 10 min, followed by permeabilization with 0.01% Triton ×100 for 4 min. Cells were stained with primary antibodies—YKL-40 (sc-393590), VEGF-A (sc-152), and VEGF-B (sc-1876) from Santa Cruz Biotech, Olig 2 (ab42453), MAP2 (ab11267), ANG1 (ab8451), and ANG2 (ab8452) from Abcam, GFAP (mab 360) from Millipore, and nestin (N5413-R) from Sigma, for 2 h at RT. Subsequently, cells were stained with Alexa Fluor-conjugated secondary antibodies (Invitrogen). For Western blotting, cell monolayers were washed thrice with 1× PBS and harvested using trypsin. Cell pellets were lysed in MPER containing 1× protease inhibitor cocktail (Thermo Scientific). A total of 40 µg of cell lysate was loaded in each experiment, and electrophoresed samples were transferred onto PVDF membrane (Pall Life Science). Membranes were probed using respective primary antibodies (CD44, HPA005787, dilution 1:1,000, Sigma; α-tubulin, T9026, dilution 1:5,000, Sigma; and VEGF-A, VEGF-B, ANG1, and ANG2 at 1:1000 dilution) overnight. Membranes were washed thrice in 1× PBST and probed with secondary antibody (antimouse HRP, 616520, dilution 1:5,000; antigoat HRP, 611620, dilution 1:1,000; and antirabbit HRP, 656120, dilution 1:1,000; Invitrogen) for 2 h at RT. Blots were developed using ECL substrate (Thermo Scientific).

The hearts were harvested and homogenized in lysis buffer for Western blot analysis. Following immunoblotting, the membranes were immunoblotted with VEGF, Ang-2, Tie-2, NFκbp65, ICAM-1, MyD88, IRK-4, TNFα and TLR4 (1∶1000, Santa Cruz, CA), PHD1, PHD2, PHD3 and HIF-1α (Novus Bio, CO) and Ang-1 (1∶1000, Sigma, MO) antibodies. The membranes were then washed and incubated with a secondary antibody coupled to horseradish peroxidase and densitometric analysis was carried out using image acquisition and analysis software (TINA 2.0). Cardiac hypertrophic gene β-myosin heavy chain (β-MHC) and ANP expression was examined by western blot analysis. Heart tissue sections were stained with H&E (Haematoxylin and Eosin, Sigma, MO). Cardiomyocyte size (area) (40X) was measured by using NIH image analysis.