Delayed Microvascular Shear Adaptation in Pulmonary Arterial Hypertension. Role of Platelet Endothelial Cell Adhesion Molecule-1 Cleavage

Abstract

Rationale: Altered pulmonary hemodynamics and fluid flow-induced high shear stress (HSS) are characteristic hallmarks in the pathogenesis of pulmonary arterial hypertension (PAH). However, the contribution of HSS to cellular and vascular alterations in PAH is unclear.

Objectives: We hypothesize that failing shear adaptation is an essential part of the endothelial dysfunction in all forms of PAH and tested whether microvascular endothelial cells (MVECs) or pulmonary arterial endothelial cells (PAECs) from lungs of patients with PAH adapt to HSS and if the shear defect partakes in vascular remodeling in vivo.

Methods: PAH MVEC (n = 7) and PAH PAEC (n = 3) morphology, function, protein, and gene expressions were compared with control MVEC (n = 8) under static culture conditions and after 24, 72, and 120 hours of HSS.

Measurements and main results: PAH MVEC showed a significantly delayed morphological shear adaptation (P = 0.03) and evidence of cell injury at sites of nonuniform shear profiles that are critical loci for vascular remodeling in PAH. In clear contrast, PAEC isolated from the same PAH lungs showed no impairments. PAH MVEC gene expression and transcriptional shear activation were not altered but showed significant decreased protein levels (P = 0.02) and disturbed interendothelial localization of the shear sensor platelet endothelial cell adhesion molecule-1 (PECAM-1). The decreased PECAM-1 levels were caused by caspase-mediated cytoplasmic cleavage but not increased cell apoptosis. Caspase blockade stabilized PECAM-1 levels, restored endothelial shear responsiveness in vitro, and attenuated occlusive vascular remodeling in chronically hypoxic Sugen5416-treated rats modeling severe PAH.

Conclusions: Delayed shear adaptation, which promotes shear-induced endothelial injury, is a newly identified dysfunction specific to the microvascular endothelium in PAH. The shear response is normalized on stabilization of PECAM-1, which reverses intimal remodeling in vivo.

Keywords: endothelial cell; microcirculation; molecular biology; pulmonary arterial hypertension; shear stress.

Figures

Figure 1.

Morphological shear adaptation of pulmonary arterial hypertension (PAH) microvascular endothelial cells (MVECs) is delayed. Representative phase-contrast images (scale bar, 400 µm) of control MVEC, PAH MVEC, and PAH pulmonary arterial endothelial cells (PAECs) after exposure to high shear stress for 24, 72, and 120 hours. Arrow indicates direction of flow. Inlays show 1.5× magnification (scale bar, 200 µm). To the right, quantifications of shear adaptation (control, n = 8; PAH, n = 7; PAEC, n = 3; one-way analysis of variance) presented as number of not shear-adapted cells. Nr. = number.

Figure 2.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) protein expression and interendothelial localization are disturbed in pulmonary arterial hypertension (PAH) microvascular endothelial cells (MVECs). (A) Protein expression of the shear sensors vascular endothelial (VE)-cadherin, PECAM-1, and vascular endothelial growth factor receptor 2 (VEGFR2) in static control versus PAH MVEC (control, n = 6; PAH, n = 7; unpaired Student's t test). Representative Western blots are shown. Data normalized to total ERK1/2. (B) Relative (rel.) mRNA expression of control and PAH MVEC under static culture conditions and after 72 hours of high shear stress (control, n = 3; PAH, n = 3; two-way analysis of variance). (C) Representative PECAM-1 and VE-cadherin immunostaining of control and PAH MVEC under static culture conditions, and (D) 72 hours after high shear stress (scale bars, 50 µm). Arrowheads highlight areas of low peripheral PECAM-1. Nuclei were counterstained with Hoechst. Image intensities are not equal. Arrow represents direction of flow. a.u. = arbitrary units.

Figure 3.

The delayed pulmonary arterial hypertension (PAH) microvascular endothelial cell (MVEC) shear adaptation facilitates endothelial injury at sites of nonuniform flow profiles. Representative side-by-side comparison of control and PAH MVEC shear adaptation 48 hours after application of uniform (laminar, inner branch) and nonuniform (bifurcation, outer branch) shear profiles (scale bars, 400 µm). White areas indicate sites of severe cell loss. Arrows present general direction of flow. Platelet endothelial cell adhesion molecule-1 (green) and nuclei (blue) were stained (scale bars, 50 µm). Arrowheads indicate interendothelial gaps. Owing to the channel geometry, some autofluorescence and light scattering is recognizable.

Figure 4.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) silencing resembles delayed shear adaptation, whereas stabilization of PECAM-1 normalizes shear responsiveness in all forms of pulmonary arterial hypertension (PAH). (A) Representative phase-contrast images (scale bar, 400 μm) of control microvascular endothelial cell (MVEC) treated with either scrambled RNA (scMVEC) or small interfering RNA against PECAM-1 (siPECAM-1) at 72 hours after high shear stress (HSS). Inlays show 1.5× magnifications (scale bar, 200 μm). Arrow indicates direction of flow. To the right, representative Western blots for silencing efficiency. Blot intensities were quantified (italic characters). ERK1/2 was loading control. (B) Representative immunostaining (scale bar, 20 μm) of active caspases (green) in control and PAH MVEC. Cells were partly treated with the caspase inhibitor Z-Asp-2,6-dichlorobenzoyloxymethylketone (Z-Asp). Nuclei (blue) were counterstained with Hoechst. (C) Representative terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling (scale bar, 20 μm). (D) Representative phase-contrast images of PAH MVEC with and without Z-Asp treatment after 72 hours of HSS. Arrow indicates direction of flow. To the right, representative Western blots for full-length PECAM-1 (130 kD) and its truncated cytoplasmic fragment (28 kD). ERK1/2 and glyceraldehyde phosphate dehydrogenase (GAPDH) were loading controls. (E) Quantification of non–shear-adapted cell fractions after 24, 72, and 120 hours of HSS (control, n = 3; PAH, n = 3; unpaired Student's t test).

Figure 5.

Caspase inhibition stabilizes platelet endothelial cell adhesion molecule-1 (PECAM-1) levels and attenuates intimal thickening in Sugen5416 and hypoxia (SuHx)-treated rats. (A) Study design. SuHx rats were treated with vehicle or Z-Asp-2,6-dichlorobenzoyloxymethylketone (Z-Asp) after the hypoxic period, and control animals, held under normoxic conditions, were vehicle treated (control, n = 5; SuHx, n = 8; Z-Asp, n = 12; one-way analysis of variance or unpaired Student's t test). (B) Relative protein expression of active caspase 3 as a general marker for proapoptotic signaling, and (C) C-terminal PECAM-1 in whole lung lysates. Representative blots are shown with β-actin as loading control. (D) Representative immunohistochemical staining for von Willebrand factor (green), α-smooth muscle actin (red), and nuclei (blue) on small peripheral lung vessels (outer diameter [OD] ≤ 60 µm; scale bar, 20 µm). To the right, corresponding quantification of vessels characterized by occlusive lesions. (E) Representative elastic van Gieson staining (scale bar, 20 µm) and associated quantifications of intimal and medial wall thickness. a.u. = arbitrary units; Cath = open chest right ventricular catherization; Ctrl = control; Echo = echocardiography; rel. = relative.

Figure 6.

Microvascular endothelial cell (MVEC) dysfunction contributes to pulmonary arterial hypertension (PAH) via defective shear responses and consequent shear-induced injury. The defective endothelial response to shear is an intrinsic dysfunction of the microvascular endothelium, wherefore high shear stress might be both a trigger and a maintenance factor for the vascular remodeling in PAH. We propose exuberant proapoptotic signaling and caspase-mediated cytoplasmic cleavage of platelet endothelial cell adhesion molecule-1 (PECAM-1) as cause for the disturbed signaling and delayed shear response. The endothelial shear adaptation can be normalized by prevention of PECAM-1 cleavage through caspase blockage with Z-Asp-2,6-dichlorobenzoyloxymethylketone (Z-Asp). Binding sites of the used PECAM-1 antibodies are depicted in blue.