Loss of cystic fibrosis transmembrane conductance regulator impairs lung endothelial cell barrier function and increases susceptibility to microvascular damage from cigarette smoke

Abstract

Abnormal lung microvascular endothelial vascular barrier function may contribute to pulmonary inflammation, such as that occurring during inhalation of cigarette smoke (CS). Cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel expressed in both epithelial and endothelial cells, regulates the organization of tight junctions between epithelial cells and has also been implicated in the transport of sphingosine-1 phosphate (S1P), a vascular barrier-enhancing sphingolipid. Because CS has been shown to affect CFTR function, we hypothesized that CFTR function contributes to lung endothelial cell barrier and that CFTR dysfunction worsens CS-induced injury. CFTR inhibitors GlyH-101 or CFTRinh172 caused a dose-dependent increase in pulmonary or bronchial endothelial monolayer permeability, which peaked after 4 hours. CFTR inhibition was associated with both intercellular gaps and actin stress fiber formation compared with vehicle-treated cells. Increasing endothelial S1P, either by exogenous treatment or by inhibition of its degradation, significantly improved the barrier function in CFTR-inhibited monolayers. Both cultured lung endothelia and the lung microcirculation visualized in vivo with intravital two-photon imaging of transgenic mice deficient in CFTR showed that CFTR dysfunction increased susceptibility to CS-induced permeability. These results suggested that CFTR function might be required for lung endothelial barrier, including adherence junction stability. Loss of CFTR function, especially concomitant to CS exposure, might promote lung inflammation by increasing endothelial cell permeability, which could be ameliorated by S1P.

Keywords: COPD; S1P; ceramides; cystic fibrosis; sphingolipids.

Figure 1

Dose-dependent increase in lung endothelial monolayer permeability (decreased transendothelial normalized resistance) in response to treatment with cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTR(inh)-172 in human pulmonary artery endothelial cells (HPAECs; A; n = 3–5; P < .05 vs. control) and CFTR inhibitor GlyH-101 in rat lung microvascular endothelial cells (RLMVECs; B; n = 1) and in endothelial cells isolated and purified from sheep bronchial artery (SBA) with CFTR(inh)-172 (C; n = 5). Asterisk indicates P < .05 vs. control, shown at 3.5 hours. DMSO, dimethyl sulfoxide.

Figure 2

Fluorescence microscopy of rat lung microvascular endothelial cells (RLMVECs) treated with specific cystic fibrosis transmembrane conductance regulator (CFTR) inhibitors CFTR(inh)-172 or GlyH-101 (not pictured) demonstrated internalization of β-catenin from the cell periphery (yellow arrows vs. arrowheads; A); and increased actin stress fibers (yellow arrows; B) compared with vehicle-treated cells. Images were acquired by epifluorescence (A) and confocal (B) microscopy of CFTR-inhibited versus vehicle-treated endothelial cells after staining for nuclei (4′,6-diamidino-2-phenylindole, blue; A and B) and β-catenin (red; A) or actin (TR-phalloidin, red; B). Scale bar in A = 70 μm, and insets are magnified by 100%.

Figure 3

A, Exogenous sphingosine-1 phosphate (S1P; 1 μM) attenuated the decrease in transendothelial resistance (TER) in response to cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor GlyH-101 (10 μM) in rat lung microvascular endothelial cells (RLMVECs). B, Increase in endogenous S1P, stimulated by administration of 4-deoxypyridoxine (4DP; 5 mM), also attenuated the decrease in TER in response to CFTR inhibitor GlyH-101 (10 μM) in RLMVECs, shown at 2-hour timepoint, P < .01 GlyH + H₂O versus every other condition, n = 1–4. DMSO, dimethyl sulfoxide.

Figure 4

A, Cystic fibrosis transmembrane conductance regulator (CFTR) inhibition with Gly-H (5 μM) or CFTR-inh172 (10 μM) worsened the time-dependent decrease in transendothelial resistance (TER) in response to cigarette smoke (CS) extract (CS, 5%) in rat lung microvascular endothelial cells (RLMVECs). This was compared with ambient air extract used as control (AC). B, Increase in endogenous sphingosine-1 phosphate, stimulated by administration of 4-deoxypyridoxine (4DP), attenuated the decrease in TER in response to CS (5%) in RLMVECs (n = 2–6).

Figure 5

Effect of cigarette smoke extract (CSE) on the lung microcirculation captured in real time in the pulmonary microvasculature of a living wild-type (WT; A) and cystic fibrosis transmembrane conductance regulator (CFTR)–deficient (B) mouse. Three-dimensional reconstruction of fluorescein isothiocyanate– labeled vessels (green) surrounding alveoli (dark regions) and Rho-G6-labeled neutrophils (orange) imaged via intravital 2-photon microscopy before (AI and BI) and after (A and BII–IV) intravenous administration of CSE (100 μL of 20% CSE). Nuclei were stained with intravenous Hoechst (blue). Note increasing neutrophil trafficking and plasma extravasation (asterisks) into airspaces after CSE administration in the CFTR-deficient (B) but not WT (A) mouse and compared to a CFTR-deficient mouse not receiving CSE (C).