Transient receptor potential vanilloid 4-mediated disruption of the alveolar septal barrier: a novel mechanism of acute lung injury

Abstract

Disruption of the alveolar septal barrier leads to acute lung injury, patchy alveolar flooding, and hypoxemia. Although calcium entry into endothelial cells is critical for loss of barrier integrity, the cation channels involved in this process have not been identified. We hypothesized that activation of the vanilloid transient receptor potential channel TRPV4 disrupts the alveolar septal barrier. Expression of TRPV4 was confirmed via immunohistochemistry in the alveolar septal wall in human, rat, and mouse lung. In isolated rat lung, the TRPV4 activators 4alpha-phorbol-12,13-didecanoate and 5,6- or 14,15-epoxyeicosatrienoic acid, as well as thapsigargin, a known activator of calcium entry via store-operated channels, all increased lung endothelial permeability as assessed by measurement of the filtration coefficient, in a dose- and calcium-entry dependent manner. The TRPV antagonist ruthenium red blocked the permeability response to the TRPV4 agonists, but not to thapsigargin. Light and electron microscopy of rat and mouse lung revealed that TRPV4 agonists preferentially produced blebs or breaks in the endothelial and epithelial layers of the alveolar septal wall, whereas thapsigargin disrupted interendothelial junctions in extraalveolar vessels. The permeability response to 4alpha-phorbol-12,13-didecanoate was absent in TRPV4(-/-) mice, whereas the response to thapsigargin remained unchanged. Collectively, these findings implicate TRPV4 in disruption of the alveolar septal barrier and suggest its participation in the pathogenesis of acute lung injury.

Figure 1

Activation of TRPV4 expressed in lung increases endothelial permeability. In human (A), rat (B), and mouse (C) lung, TRPV4 was expressed in the alveolar septal compartment (left panels) and in bronchial epithelium (not shown). TRPV4 expression in vascular smooth muscle in extra-alveolar vessels (right panels) was observed in human and rat lung, whereas little was seen in mouse lung. Western blotting (D) showed similar TRPV4 expression in rat microvascular (MV) and pulmonary artery (PA) endothelium (TRPV4/β-actin band density was 1.1 in both groups). However, TRPV4 was not consistently expressed in endothelium of extra-alveolar vessels in intact lung (A–C). The TRPV4 agonist 4αPDD increased the filtration coefficient K_f in isolated rat lung (p=0.021) in a dose-dependent fashion (p=0.002); *p<0.05 vs 1 or 5 μmol/L. The TRPV1 agonist 4α-phorbol-12,13-didecanoate-20-homovanillate (4αPDDHV) had no effect (p=0.201, paired t-test).

Figure 2

Activation of TRPV4 or store-operated channels increases permeability in a Ca²⁺ entry-dependent fashion. In low (0.02 mmol/L) extracellular [Ca²⁺] (striped bars), K_f was measured at baseline (BL) and 45 min after treatment with the TRPV4 agonists 4αPDD or EETs or thapsigargin which activates store-operated channels. A final K_f was measured 15 min after Ca²⁺ add-back (2.2 mmol/L, closed bars). Vehicle (A) or the TRPV antagonist ruthenium red (1 μmol/L, panel B) was added 15 min prior to treatment. Ca²⁺ entry was required for the permeability response to all agonists (A), yet only the response to 4αPDD or EETs was blocked by ruthenium red (B). P values for ANOVA are shown above each group; post hoc tests identified specific differences: *p<0.05 vs. BL, ^#p<0.05 vs agonist in low Ca²⁺.

Figure 3

Microscopic assessment of perivascular cuffing in extra-alveolar vessels. Perivascular cuffs were infrequently observed in control rat lungs (A). Perivascular cuffing induced by 4αPDD (B), 14,15-EET (C), or thapsigargin (D) was heterogeneous, and when cuffing appeared (arrow), cuff volume fraction was no different between groups (see Table 2). Extra-alveolar vessels often appeared no different control. Scale bars are 100 μm. PA, pulmonary arteriole; Br, bronchiole; L, lymphatic.

Figure 4

Assessment of endothelial ultrastructure in rat lung. Endothelial cell integrity was assessed using transmission electron microscopy. Random blocks were selected from glutaraldehyde-fixed lung one hr after treatment with vehicle or drug, and measurement of K_f to document endothelial permeability. Representative micrographs from extra-alveolar vessels (A–D) and septal capillaries (E–H) are shown. Endothelial ultrastructure was retained in control lung (A and E), though occasional blebs or breaks in septal capillaries were observed. 4αPDD (B and F) and 14,15-EET (C and G) rarely altered junctional morphology. However, both agonists resulted in endothelial breaks and blebs (asterisk) in the septal capillary endothelium (F and G), as well as blebs in the alveolar epithelium (inset in panel G). Thapsigargin resulted in development of gaps at junctions between endothelial cells in extra-alveolar vessels (arrowhead, D), but had no impact in the septal compartment (H). Scale bars (A–H) are 2 μm; scale bar for inset in G is 500 nm.

Figure 5

Activation of TRPV4 increases endothelial permeability in mouse lung. A. Using primers designed to amplify across the pore-loop region of TRPV4, the predicted 612 bp product was observed in wild type and heterozygote mice, but not in null animals. B. In isolated mouse lung, we showed that the TRPV4 agonist 4αPDD (10 μmol/L) increased endothelial permeability (K_f) only in TRPV4+/+ mice (p=0.036, paired t-test). In contrast, activation of store-operated channels via thapsigargin (150 nmol/L) increased permeability in both wild type and null mice (p=0.032 and 0.008, respectively, paired t-test). These results confirm the role of TRPV4 in mediating acute lung injury. *p<0.05 vs. Baseline (paired t-test).