Endothelial hyperpermeability in severe pulmonary arterial hypertension: role of store-operated calcium entry

Abstract

Here, we tested the hypothesis that animals with severe pulmonary arterial hypertension (PAH) display increased sensitivity to vascular permeability induced by activation of store-operated calcium entry. To test this hypothesis, wild-type and transient receptor potential channel 4 (TRPC4) knockout Fischer 344 rats were given a single injection of Semaxanib (SU5416; 20 mg/kg) followed by 3 wk of exposure to hypoxia (10% oxygen) and a return to normoxia (21% oxygen) for an additional 2-3 wk. This Semaxanib/hypoxia/normoxia (i.e., SU5416/hypoxia/normoxia) treatment caused PAH, as evidenced by development of right ventricular hypertrophy, pulmonary artery medial hypertrophy, and occlusive lesions within precapillary arterioles. Pulmonary artery pressure was increased fivefold in Semaxanib/hypoxia/normoxia-treated animals compared with untreated, Semaxanib-treated, and hypoxia-treated controls, determined by isolated perfused lung studies. Thapsigargin induced a dose-dependent increase in permeability that was dependent on TRPC4 in the normotensive perfused lung. This increase in permeability was accentuated in PAH lungs but not in Semaxanib- or hypoxia-treated lungs. Fluid accumulated in large perivascular cuffs, and although alveolar fluid accumulation was not seen in histological sections, Evans blue dye conjugated to albumin was present in bronchoalveolar lavage fluid of hypertensive but not normotensive lungs. Thus PAH is accompanied by a TRPC4-dependent increase in the sensitivity to edemagenic agents that activate store-operated calcium entry.

Keywords: Semaxanib; Sugen 5416; TRPC4; calcium channels; canonical transient receptor potential 4; edema; filtration coefficient.

Fig. 1.

Thapsigargin induces a dose-dependent increase in filtration coefficient (K_f) that requires transient receptor potential channel 4 (TRPC4) expression. A: after an isogravimetric state was established, a baseline K_f was measured and thapsigargin at the indicated concentration was added to the reservoir and allowed to recirculate for 15 min before a second K_f was measured. Thapsigargin induced a dose-dependent increase in permeability in lungs from wild-type (TRPC^+/+) and TRPC4 knockout (TRPC4^−/−) rats, estimated by K_f. Data are expressed as mean ± SE; n = 6–10 per concentration. *P < 0.05; **P < 0.01; ****P < 0.0001, significant difference using one-way ANOVA with Bonferroni's post hoc test. B: concentration-response curves generated from data obtained in Fig. 1A reveal the half maximal concentration for increased permeability in TRPC4^+/+ lungs is ∼49 nM, similar to that previously reported in Sprague-Dawley rat lungs (11). Within the range of concentrations tested, the apparent half-maximal concentration is right shifted to at least 220 nM in TRPC4^−/− lungs.

Fig. 2.

The TRPC4-dependent increase in permeability corresponds with formation of perivascular fluid cuffs and not with alveolar flooding. A: lungs were isolated and perfused as described in materials and methods. Baseline perfusion pressures were <10 cmH₂O and the Fulton index (RV/LV + S) was <0.30 in TRPC4^+/+ and TRPC4^−/− lungs, characteristic features of a normotensive circulation. B: thapsigargin increased K_f ∼2-fold in TRPC4^+/+ lungs, and the increase in permeability was significantly reduced in TRPC4^−/− lungs. *P < 0.05 using unpaired t-test. C: thapsigargin promoted fluid accumulation in perivascular cuffs, especially in TRPC4^+/+ lungs but did not cause alveolar flooding. Arrows identify perivascular cuffs and L denotes a dilated lymphatic channel.

Fig. 3.

Hyperpermeability response to thapsigargin is revealed in animals with severe pulmonary arterial hypertension. A: lungs from Semaxanib/hypoxia/normoxia-treated animals were isolated and perfused as described in materials and methods. Baseline pulmonary perfusion pressure was ∼50 cmH₂O and the Fulton index was ∼0.75 in TRPC4^+/+, TRPC4^+/−, and TRPC4^−/− lungs; there were no differences among groups in either of measurements. The dashed line reflects average values reported in normotensive TRPC4^+/+ lungs from Fig. 1 for comparison. B: thapsigargin (150 nM) increased K_f ∼10-fold in TRPC4^+/+ lungs. This hyperpermeability response to thapsigargin was not abolished in TRPC4^−/− lungs at the 150-nM concentration. C: thapsigargin induced extensive perivascular fluid cuffs, especially in TRPC4^+/+ lungs (arrows). Arteries and arterioles were remodeled, with evidence for medial hypertrophy, shown by arrowheads. Complex obliterative lesions were resolved in small precapillary arterioles (*), consistent with previous reports in the F344 rat (5). Dilated lymphatics were seen (L). As previously reported (5), complex lesions were less severe in TRPC4^−/− than in TRPC4^+/+ lungs. D: thapsigargin (150 nM) promotes accumulation of Evans blue dye conjugated to albumin in the bronchoalveolar lavage of pulmonary arterial hypertensive (PAH) but not normotensive lungs. PAH increases sensitivity to the thapsigargin (150 nM)-induced increase in permeability (left), consistent with the data shown in Figs. 2 and 3. This hyperpermeability response corresponds with the appearance of Evans blue dye in the bronchoalveolar lavage fluid, an effect that is not seen in normotensive lungs (middle). Two representative lung images are shown from the normotensive and PAH animals (right). The left PAH lung represents the low responder reported in the accompanying graph, whereas the right PAH lung represents one of the high responders. BL, baseline; TG, thapsigargin. *P < 0.05 using unpaired t-test.

Fig. 4.

Three-week hypoxia exposure is not sufficient to sustain either the pulmonary arterial hypertension or the hyperpermeability response to thapsigargin. A: animals were exposed to hypoxia (10% oxygen) for 3 wk and then returned to normoxia for an additional 2 wk. Pulmonary perfusion pressures were ∼10 cmH₂O and Fulton indexes were ∼0.30 in both TRPC4^+/+ and TRPC4^−/− lungs. The dashed line reflects average responses reported in normotensive TRPC4^+/+ lungs from Fig. 1 for comparison. P = ns using unpaired t-test. B: thapsigargin (75 and 150 nM) increased K_f ∼2-fold in TRPC4^+/+ lungs. This effect was abolished in TRPC4^−/− lungs at 75 nM (P < 0.05 using unpaired t-test), but not at 150 nM, thapsigargin. C: thapsigargin induced perivascular cuffing without evidence of alveolar flooding, especially apparent in lungs from TRPC4^+/+ rats (arrows). Despite the normal pulmonary artery pressures seen in A, pulmonary artery and arteriole media were remodeled, characteristic of the hypertrophy and hyperplasia that accompanies chronic hypoxia exposure (arrowhead).

Fig. 5.

Semaxanib inoculation is not sufficient to induce pulmonary arterial hypertension or cause a hyperpermeability response to thapsigargin. A: F344 rats received a single subcutaneous Semaxanib injection, and were then maintained under normoxia for an additional five wk. Lungs were isolated and perfused as described in materials and methods. Pulmonary perfusion pressures were ∼10 cmH₂O, and the Fulton index was ∼0.30 in both TRPC4^+/+ and TRPC4^−/− rats. The dashed line reflects average responses reported in normotensive TRPC4^+/+ lungs from Fig. 1 for comparison. P = ns using unpaired t-test. B: thapsigargin increased K_f ∼2-fold at 75 and 150 nM concentrations in both TRPC4^+/+ and TRPC4^−/− lungs. P = ns using unpaired t-test. C: extensive perivascular cuffs were noted in both TRPC4^+/+ and TRPC4^−/− lungs (arrows), while fluid was not seen in alveoli.