Disruption of DLL4/NOTCH1 Causes Dysregulated PPARγ/AKT Signaling in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease characterized by vascular remodeling of small pulmonary arteries. Endothelial dysfunction in advanced PAH is associated with proliferation, apoptosis resistance, and endothelial to mesenchymal transition (EndoMT) due to aberrant signaling. DLL4, a cell membrane associated NOTCH ligand, activates NOTCH1 signaling and plays a pivotal role maintaining vascular integrity. Inhibition of DLL4 has been associated with the development of pulmonary hypertension, but the mechanism is incompletely understood. Here we report that BMPR2 silencing in PAECs activated AKT and decreased DLL4 expression. DLL4 loss was also seen in lungs of patients with IPAH and HPAH. Over-expression of DLL4 in PAECs induced BMPR2 promoter activity and exogenous DLL4 increased BMPR2 mRNA through NOTCH1 activation. Furthermore, DLL4/NOTCH1 signaling blocked AKT activation, decreased proliferation and reversed EndoMT in BMPR2-silenced PAECs and ECs from IPAH patients. PPARγ, suppressed by BMPR2 loss, was induced and activated by DLL4/NOTCH1 signaling in both BMPR2-silenced and IPAH PAECs, reversing aberrant phenotypic changes, in part through AKT inhibition. Finally, leniolisib, a well-tolerated oral PI3Kδ/AKT inhibitor, decreased cell proliferation, induced apoptosis and reversed markers of EndoMT in BMPR2-silenced PAECs. Restoring DLL4/NOTCH1/PPARγ signaling and/or suppressing AKT activation may be beneficial in preventing or reversing the pathologic vascular remodeling of PAH.

Immobilized DLL1 has been reported to mimic cell-to-cell interactions and activate NOTCH signaling (1).Therefore, the same approach was adopted to investigate DLL4 signaling and its ability to activate NOTCH signaling in cultured ECs.In experiments studying exogenous DLL4 effects, cells were seeded on plates pre-coated with recombinant DLL4 (0.5 μg/mL in PBS overnight, R&D Systems; Minneapolis, MN).Cells were maintained at 37°C in a humidified incubator with 5% CO2 and 95% air.
Immunofluorescence.Primary PAECs were seeded onto collagen coated 8-well chamber slides (Sigma) at a density of 60,000 cells/well overnight, transfected with BMPR2, CAV1 or control siRNA for 48 h and treated with vehicle (DMSO) or leniolisib (Novartis) for 24 h.Failed donor control or IPAH ECs (50,000 cells/well) were seeded on either BSA or DLL4-coated 8-well chamber slides for 48 h.Cells were then washed 3x with basal medium, fixed with 4% formaldehyde (Polysciences, Inc.) for 20 min at RT, blocked with goat serum (Sigma) plus 0.3% Triton X-100 for 45 min at RT and then washed 3x with PBS supplemented with 0.1% BSA before incubating overnight with primary antibodies.The next day, the slides were washed 3x as above, incubated with secondary antibodies for 1 h, washed 3x as above and mounted using mounting medium with DAPI (4′,6-diamidino-2-phenylindole; Sigma).Cells were imaged using epifluorescence (Leica DMi8 Inverted LED Fluorescence Microscope, Leica, Deerfield, IL) at 20X magnification and analyzed using ImageJ.
For protein lysates, PAECs were plated on either BSA or DLL4 overnight followed by BMPR2 silencing for 24 h.The cells were then transfected with either empty plasmid (CMV) or PPARg (PPARg/ pCMV6-myc-DDK) for 24 h before adding RIPA buffer (ThermoFisher Scientific) supplemented with protease and phosphatase inhibitor cocktail (ThermoFisher Scientific).Additionally, protein lysates were also collected 24 h after over-expressing empty plasmid or DLL4.
RNA Isolation, cDNA Synthesis and Quantitative Real-Time PCR.Total RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions, including DNase I treatment.The iScript cDNA Synthesis Kit (Bio-Rad; Hercules, CA) was used to synthesize cDNA and quantitative real-time PCR (qRT-PCR) analysis was performed using iTaq Universal SYBR Green Supermix with ROX (Bio-Rad) on a Biosystems ViiA TM 7 instrument.Gene expression was normalized to β-actin and delta cycle thresholds were tested for significance.Primer sequences of target genes are listed in Table E4.
Western Blot.Whole cell protein lysates from PAECs, unused failed donor and IPAH cells were lysed with RIPA buffer supplemented with protease and phosphatase inhibitor cocktail.Protein lysates (30 μg) fractionated by 4-12% SDS-PAGE were transferred on nitrocellulose membranes, blocked at RT for 1 h and incubated overnight at 4° C with primary antibody (Table E5).Blots were imaged with ChemiDoc MP Imaging System (BioRad) and densitometry analysis performed with Image Lab software (version 6.1; BioRad).
Cell Proliferation Assays.Primary PAECs were transfected with siCTRL or siBMPR2 for 48 h in T75 flasks, detached with 0.5% trypsin-EDTA and re-seeded (5000 cells/well) on BSA or DLL4 coated 96-well plates.Likewise, failed donor control or IPAH ECs were seeded on BSA or DLL4 coated 96-well plates and cell proliferation over 72 h was quantitated by incorporation of 5-bromo-2-deoxyuridine (BrdU) using a chemiluminescence-based ELISA kit (Sigma).
Cell Apoptosis.Primary PAECs transfected with either BMPR2 or control siRNA for 48 h were replated on collagen-coated 96-well plates at a density of 5000 cell/well overnight.The following day, media was replaced with either complete media or media without serum or growth factors for 24 h.An equal volume of Caspase-Glo 3/7 reagent (Promega) was added to each well for 30 min and luminescence was read with a GloMax® plate reader (Promega).For flow cytometry, primary PAECs were plated on collagen-coated T25 flasks at a density of 220,000 cells/flask.The next day, cells were transfected with either control, BMPR2, JNK1 or CAV1 siRNA for 48 h.Cell media was then replaced with either complete media or media without serum or growth factors and the addition of either vehicle (DMSO) or leniolisib for another 24 h.Cells were harvested, stained with annexin V and propidium iodide (PI) (BD Pharmingen™) and evaluated using either a MACSquant® (Miltenyi) or BD LSRFortessa™ (BD Biosciences) analyzer.Data files were then uploaded and analyzed using FlowJo software version 10.4.2 (Treestar, Ashland, OR).Fluorescence Intensity