Comparison of Human and Experimental Pulmonary Veno-Occlusive Disease

Abstract

Pulmonary veno-occlusive disease (PVOD) occurs in humans either as heritable form (hPVOD) due to biallelic inactivating mutations of EIF2AK4 (encoding GCN2), or as a sporadic form at older age (sPVOD). The chemotherapeutic agent Mitomycin C is a potent inducer of PVOD in humans and in rats (MMC-PVOD). Here we compared human hPVOD and sPVOD, and MMC-PVOD pathophysiology at the histological, cellular and molecular levels to unravel common altered pathomechanisms. MMC-exposure in rats was primarily associated with arterial and microvessels remodeling and secondarily followed by venous remodeling, when PVOD became symptomatic. In all forms of PVOD tested, there were convergent GCN2-dependent but eIF2α-independent pulmonary protein overexpression of heme oxygenase 1 (HO-1) and CCAAT-enhancer-binding protein (C/EBP) homologous protein (CHOP), two downstream effectors of GCN2 signaling and endoplasmic reticulum (ER) stress. In human PVOD samples, CHOP immunohistochemical staining mainly labeled endothelial cells in remodeled veins and arteries. Strong HO-1 staining was observed only within capillary hemangiomatosis foci, where intense microvascular proliferation occurs. HO-1 and CHOP stainings were not observed in control and pulmonary arterial hypertension lung tissues, supporting the specificity for CHOP and HO-1 involvement in PVOD pathobiology. In vivo loss of GCN2 (EIF2AK4 mutations carriers and Eif2ak4-/-rats) or in vitro GCN2 inhibition in cultured pulmonary artery endothelial cells (PAECs) using pharmacological and siRNA approaches demonstrated that GCN2 lossof-function negatively regulates BMP-dependent SMAD1/5/9 signaling. Exogenous BMP9 was still able to reverse GCN2 inhibition-induced PAECs proliferation. In conclusion, we identified CHOP and HO-1 inhibition, and BMP9 as potential therapeutic options for PVOD.

Proteomic Analysis of KCNK3 Loss of Expression Identified Dysregulated Pathways in Pulmonary Vascular Cells

“…Among these proteins, we selected HMOX1 (or HO-1) and IFIT3 for confirmation by the semi-quantitative Western blot approach to confirm these findings due to the importance of HMOX1 and EIF2AK2 (or PKR) in the pulmonary field [ 12 ] and due to the novelty of IFIT3 in the pulmonary hypertension field.…”

“…EIF2 is crucial for protein synthesis and the initiator binding of tRNA to the ribosome. EIF2 signaling is known to play a critical role in the vascular remodeling and proliferation of PASMCs in chronic hypoxia-induced pulmonary hypertension [ 19 ], as well as in pulmonary vein occlusive disease [ 12 ]. EIF2 signaling is a part of the integrated stress response (ISR).…”

“…ISR is mediated by several ISR kinases (GCN2, PERK, HRI), and the protein kinase R (PKR)—also named EIF2AK2—is known to phosphorylate the α-subunit of eIF2α, leading to the activation of activating transcription factor 4 (ATF4). ATF4 is known to induce the expression of other transcription factors, including HO-1 [ 12 ]. Here, we found that EIF2AK2 is reduced in hPASMCs and hPAECs transfected with siKCNK3.…”

“…Here, we found that HO-1 was overexpressed in hPASMCs and hPAECs transfected with siKCNK3. HO-1 plays a crucial role in the survival of vascular smooth muscle [ 23 ], and could play major role in many human diseases, including pulmonary veino-occlusive disease [ 12 , 22 ]. HO-1 promotes cancer development, since HO-1 overexpression is observed in different types of cancers promoting cell survival [ 24 ], while the inhibition of HO-1 reduces cell viability [ 25 ].…”

“…Total proteins from hPASMCs and hPAECs or rat lung-tissue samples were prepared as described previously [ 12 ]. Anti-HO-1 (Abcam; ab13248) was used at 1/1000, anti-IFIT3 (Clinisciences, Nanterre, France; sc-393396) was used at 1/1000, and anti-β-Actin was used for normalization.…”