Pulmonary arterial hypertension (PAH) is a vascular remodeling disease characterized by persistent elevation of pulmonary arterial (PA) pressure and premature death. PA smooth muscle cells (PASMCs) from PAH patients present a cancer-like hyperproliferative and apoptosis-resistant phenotype contributing to obliteration of the vascular lumen. Although prevention of DNA damage-induced cell death appears to be an adaptive mechanism used by PAH-PASMCs to growth, the molecular players engaged to orchestrate the repair of DNA lesion remain largely unknown. Cancer cells presenting elevated levels of DNA damage/replication stress (RS) rely on activation of the CHK1 pathway to restrain the accumulation of deleterious levels of DNA damage.
We hypothesize that PAH-PASMCs have developed an orchestrated response mediated by CHK1 to overcome RS/DNA damage, allowing their survival and proliferation.
Methods and Results:
Using Western blot (WB), we showed that, compared to control cells, isolated PAH-PASMCs display elevated levels of DNA damage/RS (pRPA32 and γH2AX, p<0.05) and exhibit constitutive activation of the ATR/CHK1 pathway. Consistently, CHK1 was overexpressed (Immunofluorescence) in distal PAs from PAH patients as well as in three animal models of PAH; the monocrotaline (MCT) rat, the Fawn-Hooded rat (FHR) and the simian immunodeficiency virus (SIV)-infected macaque models. In vitro, we demonstrated that reduced miR-424 expression (qPCR, p<0.05) accounts for CHK1 up-regulation in PAH-PASMCs. Pharmacological (MK-8776) and molecular (siCHK1) inhibition of CHK1 exacerbates the levels of RS and DNA damage. These effects were associated with diminished PAH-PASMCs proliferation (Ki67 labeling, p<0,01) and resistance to apoptosis (TUNEL labeling, p<0.5). In vivo, we provide evidence that MK-8776 significantly improves established PAH by decreasing the mean PA pressure (right heart catheterization) and PA medial wall thickness (EVG staining) in the MCT and FHR models.
CHK1 promotes vascular remodeling in PAH by mitigating DNA damage and represents a new promising therapeutic target.