Heat shock protein 90 inhibitors protect and restore pulmonary endothelial barrier function

Abstract

Heat shock protein 90 (hsp90) inhibitors inactivate and/or degrade various client proteins, including many involved in inflammation. Increased vascular permeability is a hallmark of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Thus, we tested the hypothesis that hsp90 inhibitors may prevent and/or restore endothelial cell (EC) permeability after injury. Exposure of confluent bovine pulmonary arterial endothelial cell (BPAEC) monolayer to TGF-beta1, thrombin, bacterial lipopolysaccharide (LPS), or vascular endothelial growth factor (VEGF) increased BPAEC permeability, as revealed by decreased transendothelial electrical resistance (TER). Treatment of injured endothelium with hsp90 inhibitors completely restored TER of BPAEC. Similarly, preincubation of BPAEC with hsp90 inhibitors prevented the decline in TER induced by the exposure to thrombin, LPS, VEGF, or TGF-beta1. In addition, hsp90 inhibitors restored the EC barrier function after PMA or nocodazole-induced hyperpermeability. These effects of the hsp90 inhibitors were associated with the restoration of TGF-beta1- or nocodazole-induced decrease in VE-cadherin and beta-catenin expression at EC junctions. The protective effect of hsp90 inhibitors on TGF-beta1-induced hyperpermeability was critically dependent upon preservation of F-actin cytoskeleton and was associated with the inhibition of agonist-induced myosin light chain (MLC) and myosin phosphatase target subunit 1 (MYPT1) phosphorylation, F-actin stress fibers formation, microtubule disassembly, increase in hsp27 phosphorylation, and association of hsp90 with hsp27, but independent of p38MAPK activity. We conclude that hsp90 inhibitors exert barrier protective effects on BPAEC, at least in part, via inhibition of hsp27-mediated, agonist-induced cytoskeletal rearrangement, and therefore may have useful therapeutic value in ALI, ARDS, and other pulmonary inflammatory disease.

Figure 1.

Effects of TGF-β1 and radicicol on endothelial monolayer integrity. (a–d) bovine pulmonary arterial endothelial cells (BPAEC) were cultured in 35-mm dishes for 10 days to allow a confluent monolayer to form. Cells were then exposed for 18 hours to (a) vehicle (0.1% DMSO); (b) TGF-β1 (50 ng/ml); (c) TGF-β1 (50 ng/ml) and RA (1μg/ml), given at the same time; or (d) RA (1μg/ml) alone. Endothelial cell (EC) detachment induced by TGF-β1 (b) is shown by arrows. Phase contrast: original magnification ×100. (e–h) Under similar culture conditions, EC were treated with 10 ng/ml TGF-β1 for 6 hours and stained with silver nitrate to reveal borders between adjacent EC; treatments: (e) vehicle (0.1% DMSO); (f) TGF-β1 (10 ng/ml); (g) TGF-β1 (10 ng/ml) and RA (1μg/ml), given at the same time; or (h) TGF-β1 (10 ng/ml), alone (phase contrast). Original magnification ×320.

Figure 2.

Effects of TGF-β1 and radicicol on BPAEC cytoskeletal rearrangement. BPAEC grown on glass coverslips for 10 days were treated with (a, e, h, l) vehicle (0.1% DMSO), (b, f, i, m) RA (1 μg/ml) for 6 hours, (c, g, j, n) TGF-β1 (10 ng/ml, 6 h), or (d, h, k, o) TGF-β1 plus RA (given at the same time, 6 h). Cells were then fixed and double-stained for VE-cadherin (a–d), β-catenin (e–h), F-actin (h–k), and β-tubulin (l–o) as described in Materials and Methods. Bars = 20 μm.

Figure 3.

Effects of TGF-β1 and hsp90 inhibitors on BPAEC transendothelial resistance (TER). BPAEC were plated on gold microelectrodes and TER was measured as described in Materials and Methods. (a) EC were treated with either vehicle (0.1% DMSO) or TGF-β1, at the time indicated by arrow, and TER was monitored for 18 hours. (b) EC were treated with vehicle or one of three hsp-90 inhibitors at the time indicated by arrow. (c) Cells were pre-treated with vehicle or one of three hsp90 inhibitors for 3 hours (RA, 17-AAG, 17-DMAG; all 1 μg/ml) followed by TGF-β1 (10 ng/m, time indicated by arrow) and TER was monitored for 18 hours. (d) Cells were exposed to TGF-β1, as above (time indicated by first arrow), followed by hsp90 inhibitor treatment, 2 to 3 hours after injury, at the nadir of the TER response (time indicated by the second arrow), and TER was monitored for 18 hours. Data are means ± SE (n = 4 for all groups).

Figure 4.

Effects of TGF-β1 and radicicol on MLC and MYPT1 phosphorylation. Confluent BPAEC monolayers were incubated with vehicle (0.1% DMSO), TGF-β1 (10 ng/ml) alone or together with RA (1 μg/ml) for the indicated periods of time. (a) MLC or (b) MYPT1 phosphorylation was monitored by Western blotting with di-phospo-MLC and phospho-MYPT–specific antibodies, respectively, as described in Materials and Methods. Quantitative analysis of MLC and MYPT1 phosphorylation is expressed as the ratio of phosphorylated to total protein. (c, d) EC were treated with RA alone for 6 hours. Data are means ± SE (n = 5 for all groups). *P ≤ 0.05 compared with vehicle; ^#P ≤ 0.05 compared with corresponding TGF-β1 alone treatment.

Figure 5.

Effect of radicicol on TGF-β1–induced p38 MAPK activation. BPAEC were treated with (a) vehicle (0.1% DMSO), TGF-β1 (10 ng/ml), TGF-β1 plus RA (1 μg/ml), given together, or (b) RA alone for the indicated periods of time. Phosphorylated p38MAPK expression was detected by Western analysis using antibodies against phospho-p38MAPK as described in Materials and Methods. Phosphorylation of proteins was quantified as the ratio of phospho-p38MAPK to total protein. Data are means ± SE (n = 4 for all groups). *P ≤ 0.05 compared with vehicle.

Figure 6.

Effects of TGF-β1 and radicicol on hsp27 phosphorylation and hsp90/hsp27 complex formation. BPAEC were treated with vehicle (0.1% DMSO), TGF-β1 (10 ng/ml), TGF-β1 plus RA (1 μg/ml), given together, or RA alone for 6 hours. (a) The level of phosphorylated hsp27 was detected by immunoblotting using antibody directed against phospho-hsp27, as described in Materials and Methods. Hsp27 phosphorylation was quantified as the ratio of phospho-hsp27 to total hsp27. (b) Cell lysates were immunoprecipitated with antibody against hsp27 as described in Materials and Methods. Immunoprecipitates were separated by SDS-PAGE and immunoblotted with antibodies against hsp90 or hsp27. The levels of hsp90 associated with hsp27 were quantified as ratio of hsp90 to hsp27. Data are means ± SE (n = 4 for all groups). *P < 0.05.

Figure 7.

Role of microtubules and actin microfilaments on the barrier-protective effect of radicicol. (A) BPAEC were plated on gold microelectrodes and TER was measured as described in Materials and Methods. (a) Cells were pretreated with RA (1 μg/ml) for 3 hours (first arrow) followed by cytochalasin B (5 μg/ml; blue line) indicated by the second arrow; or EC were first treated with cytochalasin B (indicated by the second arrow) and at the nadir of TER response they received RA (1 μg/ml), indicated by the third arrow. Control groups were treated with vehicle (0.1% DMSO) or cytochalasin B alone. TER was monitored for 18 hours. (b) Cells were treated as above but instead of the actin microfilament disrupter, cytochalasin B, microtubules were disassembled by nocodazole (5 μM), indicated by the second arrow. Data are means ± SE (n = 4 for all groups). (B) BPAEC grown on glass coverslips were treated (a, d): with vehicle (0.1% DMSO), (b, e): nocodazole (5 μM), or (c, f): nocodazole plus RA (1 μg/ml), given together, for 6 hours. Cells were then fixed and double-stained for VE-cadherin (a–c) and β-catenin (d–f) as described in Materials and Methods. Bars = 20 μm.

Figure 8.

Effects of radicicol on EC barrier disruption induced by receptor- and non–receptor-mediated agonists. BPAEC were plated on gold microelectrodes and TER was measured as described in Materials and Methods. Cells were pretreated with RA (1 μg/ml, indicated by the first arrow) for 3 hours and then exposed to (a) thrombin (100 nM), (b) VEGF (50 ng/ml), (c) LPS (1,000 EU/ml), or (d) PMA (100 nM), as indicated by the second arrow. To examine the effect of RA on restoration of injured EC, RA was added at the nadir of the TER response (indicated by the third arrow). Control groups were treated with vehicle (0.1% DMSO) or with agonists alone. TER was monitored for 18 hours. Data are means ± SE (n = 4).