KLF2-mediated disruption of PPAR-γ signaling in lymphatic endothelial cells exposed to chronically increased pulmonary lymph flow

Lymphatic abnormalities associated with congenital heart disease are well described, yet the underlying mechanisms remain poorly understood. Using a clinically relevant ovine model of congenital heart disease with increased pulmonary blood flow, we have previously demonstrated that lymphatic endothelial cells (LECs) exposed in vivo to chronically increased pulmonary lymph flow accumulate ROS and have decreased bioavailable nitric oxide (NO). Peroxisome proliferator-activated receptor-gamma (PPAR-gamma), which abrogates production of cellular ROS by NADPH oxidase, is inhibited by Kruppel-like factor 2 (KLF2), a flow-induced transcription factor. We hypothesized that chronically increased pulmonary lymph flow induces a KLF2-mediated decrease in PPAR-gamma and an accumulation of cellular ROS, contributing to decreased bioavailable NO in LECs. To better understand the mechanisms that transduce the abnormal mechanical forces associated with chronically increased pulmonary lymph flow, LECs were isolated from the efferent vessel of the caudal mediastinal lymph node of control (n = 5) and shunt (n = 5) lambs. KLF2 mRNA and protein were significantly increased in shunt compared with control LECs, and PPAR-gamma mRNA and protein were significantly decreased. In control LECs exposed to shear forces in vitro, we found similar alterations to KLF2 and PPAR-gamma expression. In shunt LECs. NADPH oxidase subunit expression was increased, and bioavailable NO was significantly lower. Transfection of shunt LECs with KLF2 siRNA normalized PPAR-gamma, ROS, and bioavailable NO. Conversely, pharmacological inhibition of PPAR-gamma in control LECs increased ROS equivalent to levels in shunt LECs at baseline. Taken together, these data suggest that one mechanism by which NO-mediated lymphatic function is disrupted after chronic exposure to increased pulmonary lymph flow is through altered KLF2-dependent PPAR-gamma signaling, resulting in increased NADPH oxidase activity, accumulation of ROS, and decreased bioavailable NO. NEW & NOTEWORTHY Lymphatic endothelial cells, when exposed in vivo to chronically elevated pulmonary lymph flow in a model of congenital heart disease with increased pulmonary blood flow, demonstrate Kruppel-like factor 2-dependent disrupted peroxisome proliferator-activated receptor-gamma signaling that results in the accumulation of reactive oxygen species and decreased bioavailable nitric oxide.