GAL3 Regulates Smooth Muscle Cell Survival in PAH via its NWGR Motif

Pulmonary arterial hypertension (PAH) is a complex and fatal disorder characterized by an unrelenting increase in pulmonary arterial pressure. Excessive proliferation of pulmonary artery smooth muscle cells (PASMC) leads to increased vascular resistance and eventually right ventricular failure and death. Our laboratory has identified a key role for Galectin‐3 (GAL‐3 or Lgals3 ) in driving PASMC proliferation and elevated pulmonary pressures. However, the mechanisms by which GAL‐3 alters PASMC behavior remains poorly understood. GAL‐3 contains a BH1 like domain that has an NWGR sequence with high homology to the anti‐apoptotic protein, BCL2. Our central hypothesis is GAL‐3 upregulation plays an important role in driving PASMC proliferation through repression of apoptosis via the NWGR domain. In order to test the role of this domain we have made an adenoviral vector encoding the GAL‐3 G182A mutation and have shown that it expresses equally to a WT GAL‐3. We have also made a GAL‐3 KO rat and have isolated GAL‐3 PASMCs from these animals (RPASMC). Our in vitro studies show that overexpression of GAL‐3 G182A cause a decrease in viability using flow cytometry upon treatment overnight with 0.5% serum containing media(p<.001). However there was no significant difference in survival(p=.840) when cells were conditioned in normal media. Proliferation experiments similarly showed no significant difference between GAL‐3 WT and G182A in KO human aortic endothelial cells(HAEC) and human PASMC (p>.05). When exposed to apoptotic stimuli (10ng/mL TNFα and 1μg/ml Cycloheximide) GAL‐3 G182A overexpressing GAL‐3 KO RPASMCs had significantly (p<.001) decreased vitality as compared to GAL‐3 WT overexpressing cells. Overall our data suggests that the NWGR domain of GAL‐3 is important in regulating PASMC survivability, and our future work will investigate the role of GAL‐3 mediated apoptosis in animal models of PAH. Support or Funding Information F31HL147428 R01HL125926‐03