PAH is an obliterative vascular disease characterized by an increased proliferation and apoptosis resistance of the vascular wall cells, including pulmonary arterial smooth muscle cells (PASMCs). Interestingly, these cells are exposed to a sustained stressful environment known to cause fatal DNA damage. In contrary, PAH-PASMCs survive and proliferate in this environment, suggesting that PAH-PASMC have developed adaptive mechanisms. Our proteomic studies showed that the pyrophosphatase MuT homolog-1 (MTH1) is among the highly overexpressed proteins in PAH-PASMCs. MTH1 hydrolyses 8- oxo-2′-deoxyguanosine riphosphate (8OHdG) and prevents its incorporation in the DNA, a highly mutagenic damage and DNA strand break inducer. We hypothesized that the increased expression of MTH1 in PAH-PASMC is a selective advantage to adapt to the stressful environment, a protection permitting PAH-PASMCs to survive and therefore contributing to vascular remodeling.
Methods and Results:
We confirmed by western blot MTH1 overexpression (2-fold increase) in lungs (p<0.01), distal pulmonary arteries (PA)(p<0.01) and isolated PASMCs (p<0.05) from PAH patients (n=9) compared to control donors (n=9). Similarly, MTH1 was increased in lung and distal PAs (p<0.05) of PAH animal models (monocrotaline, n=5, and Sugen/Hypoxia, n=5). In addition, we found that MTH1 inhibition by siRNA and S-Crizotinib in PAH-PASMCs was associated with an increase in both nuclear and mitochondrial 8OHdG levels (Immunofluorescence n=4, p<0,01), resulting in DNA damage (Comet Assay, mitochondrial PCR, n=3 p<0,05), decreased mitochondrial function (Seahorse, n=3, p<0,05) and the inhibition of proliferation/apoptosis resistance (Ki67/TUNEL, n=4 p<0,01).
In vitro, MTH1 is essential to PAH-PASMC survival. Its inhibition reduces cell proliferation and apoptosis resistance associated with genome instability and loss of mitochondrial function. Thus, MTH1 represents a new interesting target in PAH and our current in vivo preliminary studies comfort the promising therapeutic potential of the (S)-Crizotinib in PAH treatments.