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

Catherine J. Morris, Rebecca J. Kameny, Jason Boehme, Wenhui Gong, Youping He, Terry Zhu, Emin Maltepe, Gary W Raff, Jeffrey R. Fineman, Sanjeev A. Datar

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

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-γ (PPAR-γ), which abrogates production of cellular ROS by NADPH oxidase, is inhibited by Krüppel-like factor 2 (KLF2), a flow-induced transcription factor. We hypothesized that chronically increased pulmonary lymph flow induces a KLF2-mediated decrease in PPAR-γ 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-γ mRNA and protein were significantly decreased. In control LECs exposed to shear forces in vitro, we found similar alterations to KLF2 and PPAR-γ 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-γ, ROS, and bioavailable NO. Conversely, pharmacological inhibition of PPAR-γ 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-γ 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 Krüppel-like factor 2-dependent disrupted peroxisome proliferator-activated receptor-γ signaling that results in the accumulation of reactive oxygen species and decreased bioavailable nitric oxide.

Original languageEnglish (US)
Pages (from-to)H173-H181
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume315
Issue number1
DOIs
StatePublished - Jul 1 2018

Fingerprint

Peroxisome Proliferator-Activated Receptors
Lymph
Endothelial Cells
Lung
Nitric Oxide
NADPH Oxidase
Heart Diseases
Lymphatic Abnormalities
Messenger RNA
Small Interfering RNA
Transfection
Reactive Oxygen Species
Sheep
Proteins
Transcription Factors
Lymph Nodes
Pharmacology

Keywords

  • Bioavailable nitric oxide
  • Krüppel-like factor 2
  • Lymphatic endothelial cell
  • Peroxisome proliferator-activated receptor-γ signaling
  • Pulmonary lymph flow

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine
  • Physiology (medical)

Cite this

KLF2-mediated disruption of PPAR-γ signaling in lymphatic endothelial cells exposed to chronically increased pulmonary lymph flow. / Morris, Catherine J.; Kameny, Rebecca J.; Boehme, Jason; Gong, Wenhui; He, Youping; Zhu, Terry; Maltepe, Emin; Raff, Gary W; Fineman, Jeffrey R.; Datar, Sanjeev A.

In: American Journal of Physiology - Heart and Circulatory Physiology, Vol. 315, No. 1, 01.07.2018, p. H173-H181.

Research output: Contribution to journalArticle

Morris, Catherine J. ; Kameny, Rebecca J. ; Boehme, Jason ; Gong, Wenhui ; He, Youping ; Zhu, Terry ; Maltepe, Emin ; Raff, Gary W ; Fineman, Jeffrey R. ; Datar, Sanjeev A. / KLF2-mediated disruption of PPAR-γ signaling in lymphatic endothelial cells exposed to chronically increased pulmonary lymph flow. In: American Journal of Physiology - Heart and Circulatory Physiology. 2018 ; Vol. 315, No. 1. pp. H173-H181.
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abstract = "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-γ (PPAR-γ), which abrogates production of cellular ROS by NADPH oxidase, is inhibited by Kr{\"u}ppel-like factor 2 (KLF2), a flow-induced transcription factor. We hypothesized that chronically increased pulmonary lymph flow induces a KLF2-mediated decrease in PPAR-γ 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-γ mRNA and protein were significantly decreased. In control LECs exposed to shear forces in vitro, we found similar alterations to KLF2 and PPAR-γ 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-γ, ROS, and bioavailable NO. Conversely, pharmacological inhibition of PPAR-γ 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-γ 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 Kr{\"u}ppel-like factor 2-dependent disrupted peroxisome proliferator-activated receptor-γ signaling that results in the accumulation of reactive oxygen species and decreased bioavailable nitric oxide.",
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AU - Morris, Catherine J.

AU - Kameny, Rebecca J.

AU - Boehme, Jason

AU - Gong, Wenhui

AU - He, Youping

AU - Zhu, Terry

AU - Maltepe, Emin

AU - Raff, Gary W

AU - Fineman, Jeffrey R.

AU - Datar, Sanjeev A.

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N2 - 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-γ (PPAR-γ), which abrogates production of cellular ROS by NADPH oxidase, is inhibited by Krüppel-like factor 2 (KLF2), a flow-induced transcription factor. We hypothesized that chronically increased pulmonary lymph flow induces a KLF2-mediated decrease in PPAR-γ 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-γ mRNA and protein were significantly decreased. In control LECs exposed to shear forces in vitro, we found similar alterations to KLF2 and PPAR-γ 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-γ, ROS, and bioavailable NO. Conversely, pharmacological inhibition of PPAR-γ 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-γ 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 Krüppel-like factor 2-dependent disrupted peroxisome proliferator-activated receptor-γ signaling that results in the accumulation of reactive oxygen species and decreased bioavailable nitric oxide.

AB - 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-γ (PPAR-γ), which abrogates production of cellular ROS by NADPH oxidase, is inhibited by Krüppel-like factor 2 (KLF2), a flow-induced transcription factor. We hypothesized that chronically increased pulmonary lymph flow induces a KLF2-mediated decrease in PPAR-γ 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-γ mRNA and protein were significantly decreased. In control LECs exposed to shear forces in vitro, we found similar alterations to KLF2 and PPAR-γ 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-γ, ROS, and bioavailable NO. Conversely, pharmacological inhibition of PPAR-γ 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-γ 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 Krüppel-like factor 2-dependent disrupted peroxisome proliferator-activated receptor-γ signaling that results in the accumulation of reactive oxygen species and decreased bioavailable nitric oxide.

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KW - Krüppel-like factor 2

KW - Lymphatic endothelial cell

KW - Peroxisome proliferator-activated receptor-γ signaling

KW - Pulmonary lymph flow

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