Disrupted NOS signaling in lymphatic endothelial cells exposed to chronically increased pulmonary lymph flow

Abstract

Associated abnormalities of the lymphatic circulation are well described in congenital heart disease. However, their mechanisms remain poorly elucidated. Using a clinically relevant ovine model of a congenital cardiac defect with chronically increased pulmonary blood flow (shunt), we previously demonstrated that exposure to chronically elevated pulmonary lymph flow is associated with: 1) decreased bioavailable nitric oxide (NO) in pulmonary lymph; and 2) attenuated endothelium-dependent relaxation of thoracic duct rings, suggesting disrupted lymphatic endothelial NO signaling in shunt lambs. To further elucidate the mechanisms responsible for this altered NO signaling, primary lymphatic endothelial cells (LECs) were isolated from the efferent lymphatic of the caudal mediastinal node in 4-wk-old control and shunt lambs. We found that shunt LECs (n = 3) had decreased bioavailable NO and decreased endothelial nitric oxide synthase (eNOS) mRNA and protein expression compared with control LECs (n = 3). eNOS activity was also low in shunt LECs, but, interestingly, inducible nitric oxide synthase (iNOS) expression and activity were increased in shunt LECs, as were total cellular nitration, including eNOS-specific nitration, and accumulation of reactive oxygen species (ROS). Pharmacological inhibition of iNOS reduced ROS in shunt LECs to levels measured in control LECs. These data support the conclusion that NOS signaling is disrupted in the lymphatic endothelium of lambs exposed to chronically increased pulmonary blood and lymph flow and may contribute to decreased pulmonary lymphatic bioavailable NO.

Keywords: nitric oxide signaling; nitric oxide synthase.

Fig. 1.

Lymphatic endothelial cells (LECs) isolated from the efferent lymphatic vessel of the caudal mediastinal lymph node of control and shunt lambs. A–D: representative micrographs of immunofluorescence staining for nuclei with 4′,6-diamidino-2-phenylindole (DAPI, blue) (A and B) or the lymphatic-specific marker Prox1 (red) (C and D). E: Western blot demonstrates that LECs, but not pulmonary artery smooth muscle cells (SMC) isolated from control (n = 2, 2) and shunt (n = 2, 2) lambs each express the lymphatic-specific markers Prox1 and LYVE-1. F and G: flow cytometric analysis of a representative LEC isolate, gated on nucleated cells (DAPI positive). LECs stained with the appropriate isotype control (gray) are compared with LECs stained with LYVE-1 (green) or Prox1 (red). Greater than 98% of nucleated cells are LYVE-1 and Prox1 positive.

Fig. 2.

Bioavailable NO (NO_x) and endothelial nitric oxide synthase (eNOS) expression is decreased in shunt LECs, but inducible nitric oxide synthase (iNOS) expression is increased. A: NO_x levels in control (40.9 ± 6.3 μM) and shunt (6.1 ± 2.7 μM) LECs, *P < 0.001. B: eNOS mRNA expression is decreased 4-fold in shunt LECs, *P < 0.05. Relative RNA expression quantified by quantitative real-time PCR (qPCR) and normalized to control. Data are shown as means ± SE. C: eNOS protein expression is decreased 3-fold in shunt LECs, *P < 0.05. D: iNOS protein expression is increased 1.8-fold in shunt LECs, *P < 0.01. E: neuronal nitric oxide synthase (nNOS) is not expressed in control or shunt LECs; +, positive control (tissue homogenate from right ventricle) for nNOS. Protein levels in control and shunt LECs quantified by Western blot. For presentation graphically, densitometry in each lane has been normalized to β-actin and to control. For all experiments, n = 3 control, 3 shunt.

Fig. 3.

Nitric oxide synthase (NOS) activity in LECs, as a percentage of arginine converted to citrulline. A: total NOS activity (in the presence of Ca²⁺) is increased in shunt LECs, 40.7 ± 24% vs. 17.6 ± 8% in control LECs, as measured by an in vitro maximum enzyme reaction rate assay. NOS activity is abrogated in both control (0.8 ± 3%) and shunt (4.9 ± 2.7%) LECs in the presence of the nonspecific inhibitor N^G-nitro-l-arginine (l-NNA). B: iNOS activity (NOS activity in the absence of Ca²⁺) is similarly increased in shunt LECs, 41.2 ± 28% vs. 16.7 ± 7% in control LECs, and l-NNA also inhibits iNOS activity in control (1.5 ± 3.7%) and shunt (2.9 ± 2.6%) LECs. For NOS activity assays, n = 3 control and 3 shunt and were tested in triplicate, *P < 0.05. NOS activity assays with l-NNA were tested in duplicate, control LECs vs. control LECs + inhibitor (†) and shunt LECs vs. shunt LECs + inhibitor (§), P < 0.05 for each.

Fig. 4.

eNOS protein-protein interactions and Ser¹¹⁷⁷ phosphorylation in LECs. Following eNOS immunoprecipitation (IP) in control and shunt LECs, Western blots were performed for eNOS [immunoblot (IB)], 90-kDa heat shock protein (HSP90), caveolin-1, calmodulin, and phospho-eNOS-serine-1177. Normalized to control LECs, the steric inhibitor caveolin-1 associated with eNOS is decreased in shunt LECs (0.47 ± 0.11); conversely, the relative amount of the chaperone HSP90 associated with eNOS increases in shunt LECs (1.53 ± 0.17), and the relative amount of calmodulin associated with eNOS increases in shunt LECs to 1.49 ± 0.25. The relative amount of phosphorylated Ser¹¹⁷⁷ decreases in shunt LECs (0.43 ± 0.07). Data are shown as mean ratios [caveolin-1, HSP90, calmodulin, or phospho (p)-eNOS-Ser¹¹⁷⁷/eNOS] ± SD and normalized to control. For all experiments, n = 3 control, 3 shunt, with *P < 0.05.

Fig. 5.

Nitration level in LECs. A: overall nitration is increased 2.4-fold in shunt LECs compared with control LECs, *P < 0.05. Nitrotyrosine (3-NT) levels in control and shunt LEC lysates, quantified by Western. For presentation graphically, densitometry in each lane has been normalized to β-actin and to control. B: nitration of eNOS is increased 1.4-fold in shunt LECs compared with control LECs, *P < 0.05. Following IP with an antibody against eNOS, the membrane was probed by IB for eNOS and 3-NT. Data are shown as a mean ratio of 3-NT-eNOS/eNOS ± SD. For all experiments, n = 3 control, 3 shunt.

Fig. 6.

Measurement of reactive oxygen species (ROS) in LECs. Micrographs (×10) of control (A) and shunt (B) LECs. Nuclei are stained with DAPI (blue), and activated (CellROX) fluorophore (red), marks ROS. C: ROS accumulation is increased 2.6-fold in shunt LECs (+CellROX) compared with control LECs (+CellROX), *P < 0.05. ROS was quantified using a fluorescence microplate reader; for each assay, n = 3 control and 3 shunt and were tested in duplicate; data are presented as mean signal intensities ± SD normalized to control. Note that, in the absence of CellROX, the signal is negligible in both control and shunt LECs (−CellROX). D: pharmacological inhibition of iNOS with the selective inhibitor N-{[3-(aminomethyl)phenyl]methyl}ethanimidamide dihydrochloride (1400W) results in a 2.5-fold reduction in ROS levels in shunt LECs (§P < 0.05) and is equivalent to ROS levels measured in control LECs. Note that iNOS inhibition does not affect ROS levels in control LECs; n = 3 control and 3 shunt and were tested in quadruplicate in the absence or presence of 20 μM 1400W for 6 h before addition of CellROX. E: pharmacological inhibition using the nonselective NOS inhibitor 2-(2-aminoethyl)isothiourea dihydrobromide (AET) results in 1.6-fold decreased accumulation of ROS in shunt LECs (§P < 0.05) and 2.9-fold decreased ROS in control LECs (†P < 0.05). For this assay, n = 2 control and 2 shunt and were tested in duplicate in the absence or presence of 100 μM AET for 24 h before addition of CellROX. All data are presented as mean signal intensities ± SD normalized to control.