Purinergic P2Y2 Receptor-Induced Activation of Endothelial TRPV4 Channels Mediates Lung Ischemia-Reperfusion Injury

Abstract

Lung ischemia-reperfusion injury (IRI), characterized by inflammation, vascular permeability, and lung edema, is the major cause of primary graft dysfunction after lung transplantation. We recently reported that endothelial cell (EC) TRPV4 channels play a central role in lung edema and dysfunction after IR. However, the cellular mechanisms for lung IR-induced activation of endothelial TRPV4 channels are unknown. In a left-lung hilar ligation model of IRI in mice, we found that lung IR increases the efflux of extracellular ATP (eATP) through pannexin 1 (Panx1) channels at the EC membrane. Elevated eATP activated elementary Ca²⁺ influx signals through endothelial TRPV4 channels through purinergic P2Y2 receptor (P2Y2R) signaling. P2Y2R-dependent activation of TRPV4 channels was also observed in human and mouse pulmonary microvascular endothelium in ex vivo and in vitro surrogate models of lung IR. Endothelium-specific deletion of P2Y2R, TRPV4, and Panx1 in mice had substantial protective effects against lung IR-induced activation of endothelial TRPV4 channels, lung edema, inflammation, and dysfunction. These results identify endothelial P2Y2R as a novel mediator of lung edema, inflammation, and dysfunction after IR, and show that disruption of endothelial Panx1-P2Y2R-TRPV4 signaling pathway could represent a promising therapeutic strategy for preventing lung IRI after transplantation.

Keywords: Lung ischemia-reperfusion injury; Pannexin 1; TRP channels; hypoxia-reoxygenation; purinergic receptor signaling; vascular endothelial barrier.

Figure 1.. Lung IR increases TRPV4 channel activity in the pulmonary endothelium.

(A) Left-lung hilar-ligation model of IRI (see Methods). Mice in the IR group underwent left lung hilar ligation for 1 hour followed by reperfusion for 2 hours. Control mice underwent sham surgery with no hilar ligation. (B) Left, representative images of left lungs from TRPV4^fl/fl sham, TRPV4^fl/fl IR, and TRPV4_EC^−/− IR mice showing gross injury after IR and protection in TRPV4_EC^−/− mice. Center, image showing the ordering system used for dissecting out small PAs (4^th-order, dashed area) from left lungs for analysis of overall Ca²⁺ signals and TRPV4 sparklet activity. Right, grayscale image showing approximately 15 ECs from a field of view in a PA loaded with Fluo-4 AM. Red outline shows one EC. (C) Representative fractional fluorescence (F/F₀) traces showing baseline Ca²⁺ signaling activity in the pulmonary endothelium from TRPV4^fl/fl sham, TRPV4^fl/fl IR, TRPV4_EC^−/− sham, and TRPV4_EC^−/− IR mice. (D) Total baseline Ca²⁺ signaling activity, represented as sites per cell (top) and events per cell (bottom), in PAs from TRPV4^fl/fl sham, TRPV4^fl/fl IR, TRPV4_EC^−/− sham, and TRPV4_EC^−/− IR mice, with and without the TRPV4 inhibitor GSK219 (100 nM) (n = 5–6/group, *P < 0.05, **P < 0.01; ns, not significant; two-way ANOVA). (E) Representative F/F₀ traces reflecting TRPV4 sparklet activity in the presence of cyclopiazonic acid (CPA, 20 μM), used to eliminate intracellular Ca²⁺ release signals, in the pulmonary endothelium from TRPV4^fl/fl sham, TRPV4^fl/fl IR, TRPV4_EC^−/− sham, and TRPV4_EC^−/− IR mice. Dotted lines represent quantal levels (single-channel amplitudes) determined from all-point histograms.(11) (F) TRPV4 sparklet activity per site (left; NP_O per site, where N is the number of channels and P_O is the open-state probability) and TRPV4 sparklet sites per cell (right) in the presence of CPA in the pulmonary endothelium from TRPV4^fl/fl sham, TRPV4^fl/fl IR, TRPV4_EC^−/− sham, and TRPV4_EC^−/− IR mice (n = 5–6/group; *P < 0.05, ***P < 0.001; ns, not significant; two-way ANOVA). (G) TRPV4 mRNA levels in freshly isolated pulmonary endothelium from TRPV4^fl/fl mice, expressed relative to sham (n = 4) and IR (n = 5) (ns, not significant; unpaired t-test). (H) Baseline endothelial Ca²⁺ signaling activity in small PAs from TRPV4^fl/fl mice in the presence of apyrase (10 U/mL), an eATP-diphosphohydrolase, presented as sites per cell (left) and events per cell (right) (n = 5–6/group; ***P < 0.001; two-way ANOVA). (I) Endothelial TRPV4 sparklet sites per cell and sparklet activity in small PAs from TRPV4^fl/fl mice in the presence of CPA in pulmonary endothelium from TRPV4^fl/fl sham, TRPV4^fl/fl IR, and TRPV4_EC^−/− IR mice (n = 5–6/group; ***P < 0.001; two-way ANOVA). (J) Diagram depicting induction of TRPV4 Ca²⁺ channel activity by eATP after lung IR, leading to elevated intracellular Ca²⁺.

Figure 2.. Lung IR increases TRPV4 sparklet activity via P2Y2R signaling in the pulmonary endothelium.

Ca²⁺ signals were recorded from the endothelium of Fluo-4-loaded small PAs. (A) Total Ca²⁺ signaling activity (sites per cell and events per cell) and (B) TRPV4 sparklet activity (sparklet sites per cell and sparklet activity per site) in ECs from small PAs of C57BL6/J mice in the absence and presence of the P2Y2R inhibitor, AR-C 118925XX (ARC, 10 μM) (n = 5–6/group; ***P < 0.001; ns, not significant; two-way ANOVA). (C) Representative images of left lungs from P2Y2R^fl/fl sham, P2Y2R^fl/fl IR, and P2Y2R_EC^−/− IR mice showing gross lung injury after IR and protection in P2Y2R_EC^−/− mice. (D) Representative F/F₀ traces showing baseline Ca²⁺ signaling activity in the pulmonary endothelium. (E) Dot plot showing total baseline Ca²⁺ signaling activity for P2Y2R^fl/fl sham, P2Y2R^fl/fl IR, P2Y2R_EC^−/− sham, and P2Y2R_EC^−/− IR mice, with and without the TRPV4 inhibitor GSK219 (100 nM) (n = 5–6/group; **P < 0.01, ***P < 0.001; ns, not significant; two-way ANOVA). (F) Representative TRPV4 sparklet traces and (G) analysis of sparklet activity in the pulmonary endothelium from P2Y2R^fl/fl sham, P2Y2R^fl/fl IR, P2Y2R_EC^−/− sham, and P2Y2R_EC^−/− IR mice (n = 5–6/group; **P < 0.01, ***P < 0.001; ns, not significant; two-way ANOVA). (H) TRPV4 mRNA levels in freshly isolated pulmonary ECs from P2Y2R^fl/fl mice (sham, n = 4; IR, n = 5; unpaired t-test). Data are expressed relative to the sham group. (I) Diagram depicting eATP-induced activation of endothelial P2Y2R signaling and Ca²⁺ influx through TRPV4 channels after lung IR.

Figure 3.. Endothelium-specific P2Y2R deletion decreases IR-induced neutrophil infiltration and lung edema and improves lung function after IR.

(A) Partial pressure of arterial oxygen (PaO₂), (B) lung compliance, and (C) lung edema (wet/dry weight ratio), measured in P2Y2R^fl/fl sham, P2Y2R^fl/fl IR, P2Y2R_EC^−/− sham, and P2Y2R_EC^−/− IR mice (n = 5–6/group; ***P < 0.001; ns, not significant; two-way ANOVA). (D) Representative immunostaining images showing neutrophil infiltration (stained pink with alkaline phosphatase). Scale bar: 50 μm. Quantification of the number of neutrophils per highpowered field (HPF; right) from P2Y2R^fl/fl sham, P2Y2R^fl/fl IR, P2Y2R_EC^−/− sham, and P2Y2R_EC^−/− IR mice (n = 5–6/group; ***P < 0.001; two-way ANOVA). (E) Concentrations of the proinflammatory cytokines CXCL1/KC, CXCL2/MCP-1, IL-6, and TNFα in left-lung BAL fluid from P2Y2R^fl/fl sham, P2Y2R^fl/fl IR, P2Y2R_EC^−/− sham, and P2Y2R_EC^−/− IR mice (n = 5–6/group; ***P < 0.001; ns, not significant; two-way ANOVA).

Figure 4.. Exposure of small PAs to acute HR induces endothelial TRPV4 sparklet activity through P2Y2R stimulation.

En face small PAs were exposed to 1 hour hypoxia followed by 10 minutes reoxygenation (HR) or normoxia (N) for 70 minutes. The final 10 minutes included incubation with the Ca²⁺ indicator, Fluo-4 (10 μM), for each group. (A) HR model and (B) analysis of baseline Ca²⁺ signaling activity and (C) TRPV4 sparklet activity after normoxia or HR in the pulmonary endothelium of small PAs from TRPV4^fl/fl and TRPV4_EC^−/− mice, in the presence or absence of the TRPV4 inhibitor, GSK219 (100 nM) (n = 5–6/group; ***P < 0.001; ns, not significant; two-way ANOVA). (D) Total baseline Ca²⁺ signaling activity and (E) TRPV4 sparklet activity after normoxia or HR in the pulmonary endothelium of small PAs from P2Y2R^fl/fl and P2Y2R_EC^−/− IR mice, in the presence and absence of the TRPV4 inhibitor GSK219 (n = 5–6/group; *P < 0.05, ***P < 0.001; ns, not significant; two-way ANOVA).

Figure 5.. Exposure to acute HR increases TRPV4 channel activity in human PMVECs.

(A) Top left, an image showing a lung wedge sample obtained during lung transplantation surgery. Top right, CD31 immunostaining showing ECs in small PAs isolated from human lungs (left). Bottom, greyscale images showing ECs from fluo-4-loaded, freshly isolated small PAs from human lungs. Small PAs were exposed to normoxia or HR. (B) F/F₀ traces showing baseline endothelial Ca²⁺ signaling activity in small PAs from human lungs (right). (C) Analyzed data showing total baseline endothelial Ca²⁺ signaling activity in the absence and presence of GSK219 (100 nM) in small PAs from human lungs (n = 5/group, ***P < 0.001, two-way ANOVA). (D) Analyzed data showing total baseline endothelial Ca²⁺ signaling activity in the absence and presence of ARC (10 μM) in small PAs from human lungs (n = 5/group, ***P < 0.001, two-way ANOVA). (E) Top, representative whole-cell patch-clamp traces of TRPV4 currents, defined as currents in the presence of the TRPV4 agonist GSK101 (10 nM) minus those in the presence of GSK101 + the TRPV4 antagonist GSK219 (100 nM), in human PMVECs. Outward currents through TRPV4 channels were recorded using −100 mV to +100 mV voltage ramps. Experiments were performed in the presence of ruthenium red (RuR; 1 μM) to prevent Ca²⁺ influx through TRPV4 channels and subsequent Ca²⁺-dependent activation of K⁺ channels. Bottom, averaged outward currents in human PMVECs at +100 mV under basal conditions, in the presence of GSK101 (10 nM) or GSK101 + GSK219 (100 nM), and GSK219-sensitive currents (GSK101 minus [GSK101 + GSK219]) (*P < 0.05, **P < 0.01, ***P < 0.001 vs. Basal; two-way ANOVA). (F) Calbryte 520-loaded human PMVECs (top) and representative traces (bottom) and analyzed data (G) showing total baseline Ca²⁺ signaling activity in the absence and presence of ARC (10 μM) in human PMVECs exposed to normoxia or HR. (H) Representative traces and (I) analyzed data showing TRPV4 sparklet activity in the absence and presence of ARC (10 μM) in human PMVECs (n = 5–7/group, ***P < 0.001; two-way ANOVA).

Figure 6.. Panx1-effluxed eATP mediates IR-induced activation of endothelial P2Y2R–TRPV4 signaling.

(A) Representative images of left lungs from Panx1^fl/fl sham, Panx1^fl/fl IR, and Panx1_EC^−/− IR mice showing gross injury after IR and protection in Panx1_EC^−/− mice. (B) Bioluminescence measurements of eATP (nM) released from small PAs of Panx1^fl/fl sham, Panx1^fl/fl IR, Panx1_EC^−/− sham, and Panx1_EC^−/− IR mice (n = 6/group; ***P < 0.001; ns, not significant; two-way ANOVA). (C) Panx1 mRNA levels in freshly isolated pulmonary ECs from Panx1^fl/fl sham (n = 4) and Panx1_EC^fl/fl IR (n = 5) mice (ns, not significant; unpaired t-test). Data are expressed relative to the sham group. (D) Total baseline Ca²⁺ signaling activity and (E) TRPV4 sparklet activity in the pulmonary endothelium from Panx1^fl/fl sham, Panx1^fl/fl IR, Panx1_EC^−/− IR, and Panx1_EC^−/− IR mice in the absence and presence of the TRPV4 inhibitor GSK219 (100 nM) (n = 5–6/group, *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant; two-way ANOVA). (F) Total baseline Ca²⁺ signaling activity and (G) TRPV4 sparklet activity in the pulmonary endothelium from Panx1^fl/fl sham and Panx1_EC^−/− IR mice in the absence and presence of GSK219 (100 nM) (n = 5–6/group, *P < 0.05, *P < 0.01, ***P < 0.001; ns, not significant; two-way ANOVA).

Figure 7.. Outline of lung IR-induced Panx1–eATP–P2Y2R–TRPV4 signaling in pulmonary ECs.

eATP, effluxed through endothelial Panx1 channels, activates the endothelial P2Y2R, which, in turn, increases Ca²⁺ influx via activation of endothelial TRPV4 channels to mediate endothelial barrier dysfunction, neutrophil infiltration into the alveolar space, lung edema, and dysfunction after lung IR.