Lung heparan sulfates modulate K(fc) during increased vascular pressure: evidence for glycocalyx-mediated mechanotransduction

Abstract

Lung endothelial cells respond to changes in vascular pressure through mechanotransduction pathways that alter barrier function via non-Starling mechanism(s). Components of the endothelial glycocalyx have been shown to participate in mechanotransduction in vitro and in systemic vessels, but the glycocalyx's role in mechanosensing and pulmonary barrier function has not been characterized. Mechanotransduction pathways may represent novel targets for therapeutic intervention during states of elevated pulmonary pressure such as acute heart failure, fluid overload, and mechanical ventilation. Our objective was to assess the effects of increasing vascular pressure on whole lung filtration coefficient (K(fc)) and characterize the role of endothelial heparan sulfates in mediating mechanotransduction and associated increases in K(fc). Isolated perfused rat lung preparation was used to measure K(fc) in response to changes in vascular pressure in combination with superimposed changes in airway pressure. The roles of heparan sulfates, nitric oxide, and reactive oxygen species were investigated. Increases in capillary pressure altered K(fc) in a nonlinear relationship, suggesting non-Starling mechanism(s). nitro-l-arginine methyl ester and heparanase III attenuated the effects of increased capillary pressure on K(fc), demonstrating active mechanotransduction leading to barrier dysfunction. The nitric oxide (NO) donor S-nitrosoglutathione exacerbated pressure-mediated increase in K(fc). Ventilation strategies altered lung NO concentration and the K(fc) response to increases in vascular pressure. This is the first study to demonstrate a role for the glycocalyx in whole lung mechanotransduction and has important implications in understanding the regulation of vascular permeability in the context of vascular pressure, fluid status, and ventilation strategies.

Fig. 1.

Experimental design. A: protocols 1 and 2 = low and standard (ST) tidal volume (Vt) ventilation, respectively, with 2 increases in left atrial pressure (Pla). Protocol 3 = Low Vt ventilation with a single increase in Pla. IG₁ and IG₂ = isogravimetric periods 1 and 2, respectively. Pdo, double-occlusion pressure; K_fc, filtration coefficient. Baseline K_fc1 was determined during step 1 at Pla, 7.5 cmH₂O in protocols 1 and 2. During step 2, Pla₂ was increased to 7.5, 10, 12, 15, or 17 cmH₂O and K_fc2 was measured. The ratio of K_fc2/K_fc1 was used to assess the effect of increasing Pla on whole lung permeability. In protocol 3, lungs were exposed to a single increase in Pla from IG₁ to Pla = 7.5 or 15 cmH₂O. B: pressure-volume curves. Lungs were ventilated with either Low peak inspiratory pressure (PIP) [7–8 cmH₂O, positive end-expiratory pressure (PEEP) = 3 cmH₂O] or ST PIP (10–12 cmH₂O, PEEP = 3 cmH₂O); airflow vs. time was integrated to derive Vt, which was normalized to rat body weight (kg). P_air, air pressure.

Fig. 2.

Effect of Pla on K_fc2/K_fc1. Group numbers represent the 2 values of Pla at which K_fc1 and K_fc2 were derived. All groups had baseline K_fc (K_fc1) assessed at Pla = 7 cmH₂O. The second step in Pla was to 7, 10, 12, 15, or 17 cmH₂O, corresponding to C7-7, C7-10, C7-12, C7-15, C7-17, respectively. The same group numbering system was used in nitro-l-arginine methyl ester (l-NAME)-treated lungs. A: in control lungs (C) during Low Vt, baseline K_fc1 was measured at 7.5 cmH₂O and K_fc2 was measured at 7.5, 10, 12, 15, or 17 cmH₂O. The ratio of K_fc2/K_fc1 vs. Pla was constant at pressures over the range of 7.5 to 12 cmH₂O. K_fc2/K_fc1 increased significantly when Pla₂ = 15 cmH₂O and 17 cmH₂O; n = 8–13/group. B: l-NAME (LN, 200 μM) attenuated the increase in K_fc2/K_fc1 when Pla₂ = 15 and 17 cmH₂O; n = 6–10/group.

Fig. 3.

Attenuation of lung mechanotransduction during Low Vt. Effect of heparanase III (Hep; 150 mIU/ml), l-NAME (200 μM), Mn(III)-tetra(4-benzoic acid) porphyrin chloride (TBAP; 200 μM), and S-nitrosoglutathione (GSNO; 500 μM) on K_fc2/K_fc1 when Pla = 15 cmH₂O. Baseline K_fc1 ratio (C7–7) is shown for comparison. Heparanase III and l-NAME significantly reduced the increase in K_fc2 when Pla = 15 cmH₂O. TBAP had no effect on pressure-induced increase in K_fc2/K_fc1. GSNO increased mean K_fc2/K_fc1 to 9.06+3.26 vs. untreated lungs at Pla = 15 cmH₂O (P < 0.0001). Experiments were performed during Low Vt ventilation; n = 8–9/group.

Fig. 4.

Zonal characteristics (ZC) during increase in Pla. A: ZC was calculated at each Pla for control lungs and l-NAME-treated lungs at Low Vt. ZC increased significantly between Pla = 7.5 and 10 cmH₂O but was not statistically changed when Pla > 12 cmH₂O. l-NAME had no effect on ZC relative to controls at any given Pla. B: heparanase III (HepIII) and l-NAME had no effect on ZC at Pla = 15 cmH₂O relative to untreated lungs. ZC analysis suggests recruitment was not influencing the affects of treatments (heparanase and l-NAME) on K_fc during elevated vascular pressure. Open circles represent outliers.

Fig. 5.

Interstitial volume after elevated Pla during Low Vt. Retained lung weight after measurement of K_fc2 was used as a marker of interstitial volume (ΔVi) and normalized to the predicted lung weight (PLW). Heparanase III and l-NAME significantly reduced ΔVi/PLW, consistent with a reduction in vascular permeability.

Fig. 6.

Effect of Vt on K_fc. A: K_fc1 was identical during Low Vt vs. ST Vt. B: at ST Vt, increasing Pla₂ to 15 cmH₂O significantly increased the K_fc2/K_fc1 ratio compared with baseline (Pla = 7.5 cmH₂O). Heparanase III (Hep7–15) significantly reduced the K_fc2/K_fc1 ratio at Pla = 15 cmH₂O; inactive heparanase [Hep7–15 + diethyl pyrocarbonate (DEPC)] had no effect on K_fc2. l-NAME reduced K_fc2 by almost 50% during ST Vt and Pla = 15 cmH₂O, but the decrease did not reach statistical significance. GSNO significantly increased K_fc2 when Pla = 15 cmH₂O. C: increases in Pla to 17 cmH₂O result in marked pulmonary edema that was significantly reduced by heparanase (Hep7–17) and l-NAME (LN7–17).

Fig. 7.

ZC after elevated Pla with ST Vt. A: ZC was calculated at Pla = 15 cmH₂O for control lungs (C7–15), heparanase III-treated (Hep7–15), and l-NAME-treated lungs (LN7–15). There was no difference in ZC between groups. Baseline ZC at Pla = 7.5 cmH₂O (C7–7) is shown for comparison. B: ZC shown for controls (C7–7) and when Pla = 17 cmH₂O (C7–17); heparanase and l-NAME had no effect on ZC at Pla = 17 cmH₂O.

Fig. 8.

Interstitial volume after elevated Pla during ST Vt. Retained lung weight after measurement of K_fc2 was used as a marker of interstitial volume and normalized to the predicted lung weight. A: during ST Vt, heparanase III (Hep7–15) and l-NAME (LN7–15) had no effect on ΔVi/PLW compared with control lungs (C7–15). B: during ST Vt and Pla = 17 cmH₂O, lung water increased significantly and was attenuated by both heparanase and l-NAME.

Fig. 9.

Pressure-conditioning: single pressure step protocol. Lungs exposed to a single increase in Pla from isogravimetric conditions to 15 cmH₂O (C0–15 cmH₂O) had a K_fc significantly less than the K_fc derived from the double pressure step protocol at Pla₂ = 15 cmH₂O. Heparanase III and l-NAME reduced the single-step K_fc by 20–50%, but this reduction did not reach statistical significance.

Fig. 10.

Immunohistochemistry of heparan sulfates. Lungs were immune-stained with anti-heparan sulfate antibody (HSS-1) for heparan sulfates. A: control lungs show significant vascular staining for heparan sulfates. B: heparanase abolishes all heparan sulfate staining, demonstrating removal of cell surface heparan sulfates. C: control lungs stained with 3G10, which recognizes the neoepitope created by heparanase. Note absence of neoepitope staining. D: heparanase treatment results in robust staining for neoepitope formation demonstrating activity of the enzyme.

Fig. 11.

Immunohistochemistry for nitrotyrosine. A: control lungs at Pla = 7.5 cmH₂O showed patchy staining. B: when lungs were exposed to Pla = 15 cmH₂O, marked nitrotyrosine staining was evident. C: heparanase III treatment prior to increasing Pla attenuated nitrotyrosine staining.

Fig. 12.

Lung nitrate/nitrite (NOx) concentrations. NOx was measured in lung tissue following increased Pla. A: relationship of NOx concentration vs. K_fc2/K_fc1 ratio for pooled Low Vt (LTV) and ST Vt (STV) data. Inset shows close up of relationship at low NOx concentrations; note linear correlation of Low Vt group vs. NOx and left shift of Low Vt vs. ST Vt group. B: during Low Vt ventilation, an increase in Pla from 7 to 15 cmH₂O results in a 4-fold increase in tissue NOx. GSNO further increased tissue NOx levels when Pla = 15 cmH₂O. Heparanase treatment at low Pla had no effect on NOx (Hep7–7) but significantly reduced NOx concentrations at Pla = 15 cmH₂O, (Hep7–15). C: during ST Vt increases in Pla to 15 cmH₂O result in large increases in NOx to 0.70; GSNO at Pla = 15 cmH₂O further increased NOx to 2.60. When Pla = 17 cmH₂O, NOx increased to 8.41; heparanase significantly reduced the NOx to 0.54, when Pla = 15 cmH₂O (n = 4–6). m1–m4, fit parameters. Chisq, chi squared.

Fig. 13.

Schematic of hypothesized role of glycocalyx in lung vascular mechanotransduction. Left: during static conditions, the glycocalyx maintains barrier function over the intercellular junction. Right: during increased vascular pressure, the increased hydraulic flow through the glycocalyx deforms or stresses the glycosaminoglycan (GAG) fibers, which in turn activates endothelial nitric oxide synthase (eNOS) and leads to barrier dysfunction. ΔPc, change in capillary pressure; Q, flow; ZO-1 and ZO-2, zonula occludens-1 and -2; vin, vinculin, VE-Cad, vascular endothelial cadherin; ECM, extracellular matrix.