Abstract 15621: Checkpoint Kinases Important Players in Lung Fibrogenesis and Pulmonary Vascular Remodeling: A Translational Study

Abstract

Introduction: Pulmonary fibrosis (PF) is an important commonality in pathogenesis of interstitial lung diseases, and pulmonary hypertension (PH) is a well-recognized severe complication of PF. Both significantly impact patients’ survival and functional status, but without validated treatments. In the lung, parenchymal and vascular remodeling share pathomechanisms, mainly including excessive proliferation and resistance to apoptosis of fibroblasts (LFs) and pulmonary arterial smooth muscle cells (PASMCs). In cancer, sustained cell proliferation is ensured, in part, by a fine tuning of cell cycle and DNA repair machinery, of which checkpoint kinases (CHK1 and CHK2) are critical regulators. They are upregulated in cancer and their inhibition are currently tested in clinical trials. However, these pathways have never been explored in PF-PH. We thus hypothesized that CHK1/2 are upregulated in PF and contribute to both fibrotic and vascular lesions in PF-PH patients. Methods and Results: Increased DNA damage (γH2Ax and p(S4/S8)-RPA32) as well as augmented expression and activity of CHK1/2 was observed by WB in lung and isolated LFs from PF patients compared to controls (p<0.05, n=8~15 per group) with marked expression in fibroblastic foci. Similarly, increased levels of CHK1/2 and DNA damage were detected by WB and IF in remodeled pulmonary arteries (PA) of PF patients with or without PH. Similar findings were noted in mice exposed to bleomycin. In isolated PF-LFs and PASMCs, dual inhibition of CHK1/2 using MK-8776 and LY2606368 significantly reduced expression of TCTP and RAD51 (two factors required for efficient DNA repair), leading to exacerbation of DNA damage and resulted in reduced cell proliferation (Ki67 labeling, WB PCNA) and resistance to apoptosis (Annexin V assay, WB Survivin). Furthermore, inhibition of CHK1/2 mitigated the hyper-activated state of PF-LFs, as illustrated by reduced expression of FN, CTGF, and pSTAT3 (WB). Similar results were observed in control LFs exposed to TGF-β1; all p<0.05. Conclusions: Our data provide compelling evidence that CHK1/2 are involved in lung fibrogenesis and PA remodeling in PF. Current experiments aim to determine whether CHK1/2 inhibitors elicit beneficial effects in animal models of PF-PH.