Regulation of the micromechanical properties of pulmonary endothelium by S1P and thrombin: role of cortactin

Abstract

Disruption of pulmonary endothelial cell (EC) barrier function is a critical pathophysiologic event in highly morbid inflammatory conditions such as sepsis and acute respiratory disease stress syndrome. Actin cytoskeleton, an essential regulator of endothelial permeability, is a dynamic structure whose stimuli-induced rearrangement is linked to barrier modulation. Here, we used atomic force microscopy to characterize structural and mechanical changes in the F-actin cytoskeleton of cultured human pulmonary artery EC in response to both barrier-enhancing (induced by sphingosine 1-phosphate (S1P)) and barrier-disrupting (induced by thrombin) conditions. Atomic force microscopy elasticity measurements show differential effects: for the barrier protecting molecule S1P, the elastic modulus was elevated significantly on the periphery; for the barrier-disrupting molecule thrombin, on the other hand, it was elevated significantly in the central region of the cell. The force and elasticity maps correlate with F-actin rearrangements as identified by immunofluorescence analysis. Significantly, reduced expression (via siRNA) of cortactin, an actin-binding protein essential to EC barrier regulation, resulted in a shift in the S1P-mediated elasticity pattern to more closely resemble control, unstimulated endothelium.

FIGURE 1

Correlation between elasticity and morphology for human pulmonary EC. AFM images, (A) height and (B) deflection, showing the morphology of human pulmonary EC grown to confluence. Network of actin fibers and cell processes are visible, particularly in the deflection image. Elasticity maps (C) were obtained to find the ratio, E_C/E_n, between elastic moduli at the cell periphery (E_C) and cell nucleus (E_n). (D) The crosses in the elasticity map indicate the peripheral region of the analyzed cell, and the nucleus region is enclosed. Subsequently, the average value of all data points inside each region was determined to find E_C/E_n. In this particular case, a value E_C/E_n = 1.05 ± 0.02 was found. Elasticity maps were obtained by transforming (E) force versus distance curves into (F) indentation curves and fitting the approach portion of the curves (black solid lines) with the Hertz model (red solid lines). The retraction curve (E) is displayed with dashed lines. At forces used during contact mode imaging (∼1 nN), the cell deformation (penetration depth of the tip) was typically between 50 nm and 200 nm.

FIGURE 2

S1P and thrombin induce differential effects on EC cytoskeletal structure. Cultured HPAEC were treated with vehicle (control: images A, D, and G), S1P (1 μM: images B, E, and H), or thrombin (1 unit/ml: images C, F, and I) for 5 min and then fixed as described in Materials and Methods. Separate samples were then analyzed by AFM (top row; images A, B, and C) or standard immunofluorescence for F-actin (middle row; images D, E, and F) and cortactin (bottom row; images G, H, and I) distribution. Vehicle-treated EC exhibited disorganized F-actin staining and scattered peripheral and cytoplasmic cortactin staining (D and G), whereas S1P induced peripheral F-actin (small arrows, E) and cortactin redistribution (open arrows, H) as previously described (15). In contrast, thrombin produced intercellular gap formation (arrows, F and I), F-actin stress fiber formation (small arrows, F), and loss of peripheral cortactin staining (I). All AFM images are displayed in deflection mode and have a size of 80 × 80 μm². The scale, displayed as white bar in F-actin control image = 10 μm, is the same for all fluorescence images.

FIGURE 3

S1P and thrombin induce differential effects on EC barrier function. HPAEC were plated in monolayer on gold microelectrodes and grown to confluence to measure TER as described in Materials and Methods. After 30 min of baseline measurements, S1P (1 μM) or thrombin (1 unit/ml) were added at time 0 to individual wells. S1P (solid squares, solid line) induced rapid and sustained increased barrier function (indicated by increased resistance across the EC monolayer), whereas thrombin (open squares, dashed line) rapidly and potently decreased EC barrier function (indicated by decreased resistance). Representative TER tracings normalized to starting resistances are shown. Experiments were independently performed dozens of times and are consistent with previously published results (13).

FIGURE 4

Elasticity changes in human pulmonary EC induced by barrier-disrupting (thrombin) and barrier-enhancing agents (S1P). Deflection and height AFM images are displayed alongside elasticity maps for comparison between morphological and mechanical properties. (A–C) Untreated cells used as control, (D–F) cells treated with thrombin and (G–I) cells treated with S1P. Data were acquired using the same cantilever with 0.06 N/m spring constant in PBS buffer at room temperature, as described in Materials and Methods.

FIGURE 5

Differential effects of S1P and thrombin on distribution of endothelial elasticity. (A–C) Cross sections for multiple cells (four per condition) illustrating differences in elasticity at the cell's periphery (edges) and nucleus (central region) for (A) control, (B) thrombin-treated, and (C) S1P-treated cells. The scale in the x axis is common to all panels. In each panel, the y axis is the same for all cross sections.

FIGURE 6

Depletion of cortactin protein expression does not attenuate S1P-induced cortical F-actin rearrangement. HPAEC were treated with control or cortactin siRNA as described in Materials and Methods. (A) Western blot of whole-cell lysates demonstrated depletion of cortactin protein expression by ∼90% in the cortactin siRNA-treated EC. (B) Control and cortactin siRNA-treated HPAEC were stimulated with vehicle (control) or S1P (1 μM) for 5 min and then fixed and immunostained for F-actin. S1P rapidly induced a dramatic increase in peripheral F-actin in both types of siRNA-treated EC. Scale for all images: white bar in Control siRNA control image = 10 μm.

FIGURE 7

Depletion of cortactin alters morphology and elastic properties for S1P-stimulated cells. (A–C) Cortactin levels were depleted using cortactin-SiRNA, and (D–F) cells were treated with control-SiRNA; thus, cortactin levels were not suppressed. (C) The elastic modulus at the cells periphery, E_c, decreased noticeably with respect to that at the cell's nucleus, E_n, for EC having depleted cortactin content, but (F) E_c remained slightly increased compared to E_n for cells treated with control RNA. Shown is a representative sample of multiple analyzed cells.