Pressure is proinflammatory in lung venular capillaries

Abstract

Endothelial responses may contribute importantly to the pathology of high vascular pressure. In lung venular capillaries, we determined endothelial [Ca(2+)](i) by the fura-2 ratioing method and fusion pore formation by quantifying the fluorescence of FM1-43. Pressure elevation increased endothelial [Ca(2+)](i). Concomitantly evoked exocytotic events were evident in a novel spatial-temporal pattern of fusion pore formation. Fusion pores formed predominantly at vascular branch points and colocalized with the expression of P-selectin. Blockade of mechanogated Ca(2+) channels inhibited these responses, identifying entry of external Ca(2+) as the critical triggering mechanism. These endothelial responses point to a proinflammatory effect of high vascular pressure that may be relevant in the pathogenesis of pressure-induced lung disease.

Figure 1

Endothelial [Ca²⁺]_i in lung venular capillaries. (a) Images are of the 340:380 ratio color-coded for [Ca²⁺]_i. Images were obtained at P_LA of 5 cmH₂O (left) and after 30 minutes of P_LA increase to 20 cmH₂O (right). Endothelial [Ca²⁺]_i is given for a single cell (arrowheads). Replicated in 9 capillaries. (b) Group data are shown as 5-minute averages at P_LA of 5 cmH₂O and after 30 minutes of P_LA increase to 20 cmH₂O. Infusions of gadolinium (n = 4) or Ca²⁺-free dextran (n = 4) were started 10 minutes before pressure elevation. *P < 0.05 vs. P_LA of 5 cmH₂O; ^#P < 0.05 vs. control (n = 9).

Figure 2

Fusion pore formation in lung venular capillaries. (a) Images of a single capillary taken at different P_LA. P_LA of 5 cmH₂O. Images are before (top) and after (bottom) pressure elevation was obtained after 5 minutes of FM1-43 infusion. P_LA of 20 cmH₂O. Images were obtained at different time points after start of dye infusion. Color code shows fluorescence intensities. Capillary branch point (arrowheads) and midsegmental (arrow) locations are marked. Vessel margins are depicted by line sketches. Replicated in 8 capillaries. (b) After 30 minutes at P_LA of 20 cmH₂O, FM1-43 was infused into a lung venular capillary (horizontal bar) and then washed out by the blood flow (arrow). Shown is capillary fluorescence per unit surface area. Replicated in 8 capillaries. (c) Capillary fluorescence was quantified in pixels 5 × 5 μm², at midsegmental and branch point locations after 5 minutes of FM1-43 infusion. Data were obtained at P_LA of 5 cmH₂O and after 30 minutes of P_LA increase to 20 cmH₂O in control (n = 8) and gadolinium-treated (n = 4) lungs. Gadolinium did not increase fluorescence above control at P_LA of 5 cmH₂O (not shown). *P < 0.05 vs. P_LA of 5 cmH₂O.

Figure 3

P-selectin expression in lung venular capillaries. (a) Images show indirect immunofluorescence of P-selectin at P_LA of 5 cmH₂O (left) and after 30 minutes of P_LA increase to 20 cmH₂O (right). Vessel margins are depicted by line sketch. Color code shows fluorescence intensities at branch point (arrowheads) and midsegmental (arrows) locations. Replicated in 8 capillaries. (b) P-selectin expression was quantified as the product of the mean fluorescence intensity and fluorescent area. The x-axis indicates period elapsed after a 1-minute washout of unbound FITC-IgG with blood flow. Data were obtained at P_LA of 5 cmH₂O (open symbols) and after 30 minutes of P_LA increase to 20 cmH₂O (filled symbols). Plots are control (circles; n = 8), 4°C (squares; n = 3), and gadolinium treatment (triangles; n = 4). Decay of P-selectin fluorescence was determined from the linear regression slope (P < 0.01 for all regressions).

Figure 4

[Ca²⁺]_i dependence of fusion pore formation and P-selectin expression in lung venular capillaries. (a) Images of a pressure-stressed lung capillary at a branch point. After 30 minutes at P_LA of 20 cmH₂O, FM1-43 was infused for 5 minutes, and the capillary was imaged at a specific, landmarked location (top). Subsequently, FM1-43 fluorescence was photobleached by exposure to unfiltered mercury lamp illumination, and the capillary was stained for P-selectin expression and then reimaged at the identical location (bottom). Line sketches depict vessel margins. Replicated in 6 capillaries. (b) Single vessels were consecutively stained with FM1-43 for fusion pore detection and for indirect immunofluorescence of P-selectin at P_LA of 5 and 20 cmH₂O. Graph shows data from pixel-by-pixel analyses (pixel size: 5 × 5 μm²). Line was drawn by linear regression (r_s = 0.781; P < 0.001; n = 11). (c) Endothelial [Ca²⁺]_i was correlated with expressions of fusion pores and P-selectin. Data were obtained at P_LA of 5 and 20 cmH₂O. After determination of endothelial [Ca²⁺]_i, fura-2 fluorescence was photobleached, and vessels were stained with FM1-43 for fusion pore fluorescence (open circles; n = 4) or for immunofluorescence of P-selectin (filled circles; n = 6). FM1-43 and P-selectin fluorescences were quantified in 5 × 5 μm² pixels at sites of [Ca²⁺]_i determination. Lines were drawn by linear regressions for FM1-43 (r_s = 0.949; P < 0.001) and P-selectin (r_s = 0.826; P < 0.001) against [Ca²⁺]_i.