Paracrine Shear-Stress-Dependent Signaling from Endothelial Cells Affects Downstream Endothelial Function and Inflammation

Abstract

Cardiovascular diseases (CVDs), mainly ischemic heart disease (IHD) and stroke, are the leading cause of global mortality and major contributors to disability worldwide. Despite their heterogeneity, almost all CVDs share a common feature: the endothelial dysfunction. This is defined as a loss of functionality in terms of anti-inflammatory, anti-thrombotic and vasodilatory abilities of endothelial cells (ECs). Endothelial function is greatly ensured by the mechanotransduction of shear forces, namely, endothelial wall shear stress (WSS). Low WSS is associated with endothelial dysfunction, representing the primary cause of atherosclerotic plaque formation and an important factor in plaque progression and remodeling. In this work, the role of factors released by ECs subjected to different magnitudes of shear stress driving the functionality of downstream endothelium has been evaluated. By means of a microfluidic system, HUVEC monolayers have been subjected to shear stress and the conditioned media collected to be used for the subsequent static culture. The results demonstrate that conditioned media retrieved from low shear stress experimental conditions (LSS-CM) induce the downregulation of endothelial nitric oxide synthase (eNOS) expression while upregulating peripheral blood mononuclear cell (PBMC) adhesion by means of higher levels of adhesion molecules such as E-selectin and ICAM-1. Moreover, LSS-CM demonstrated a significant angiogenic ability comparable to the inflammatory control media (TNFα-CM); thus, it is likely related to tissue suffering. We can therefore suggest that ECs stimulated at low shear stress (LSS) magnitudes are possibly involved in the paracrine induction of peripheral endothelial dysfunction, opening interesting insights into the pathogenetic mechanisms of coronary microvascular dysfunction.

Keywords: endothelial dysfunction; inflammatory response in endothelial cells; mechanotransduction; microvascular dysfunction; shear stress.

Figure 1

(A) F-actin fluorescence analysis on HUVECs cultured in static and dynamic conditions. Nuclei are stained in blue, while actin filaments are stained in red. White arrow indicates the flow direction. (B) Quantitative analysis of the angles subtended by the cell’s major axis and the flow direction (p < 0.001). Scalebar: 200 μm.

Figure 2

PECAM-1 (CD31) staining in HUVECs cultured in static and dynamic conditions. Represented in blue are the cells’ nuclei, while in red are PECAM-1 proteins. White arrow indicates the flow direction. Scalebar: 100 μm.

Figure 3

Western blot analysis of eNOS (A) and E-selectin (B) in HUVECs cultured under shear stress. (C,D) represent the normalization data of eNOS and E-selectin, respectively (* p < 0.05 vs. control).

Figure 4

PBMC adhesion assay. (A) Leukocyte adhesion on Y-shaped PPFCs; notably, there is a higher adhesion rate in branch segments (indicated in red) compared to straight segments (indicated in green). (B) HUVEC monolayer visualized in phase contrast and fluorescent adherent PBMCs. (C) Quantification of adhesion rate by fluorescence (*** p < 0.005). Scalebar: 200 μm.

Figure 5

(A) PBMC adhesion assay on straight segments of PPFC. (B) Quantitative fluorescence analysis. Scalebar: 200 μm.

Figure 6

MTT assay on HUVECs subjected to control (CTR), TNFα- and LSS-conditioned medium (CM) for 24 and 72 h. (* p < 0.05; *** p < 0.005).

Figure 7

Western blot analysis of HUVEC cell lysates after 24 h of stimulation with TNFα-, LSS- and HSS-conditioned media (-CMs). (A,B) show the expression of eNOS and E-selectin, respectively, while (C,D) represent the normalization data by using tubulin expression level (* p < 0.05 and *** p < 0.005).

Figure 8

ICAM-1 mRNA expression in HUVECs cultured for 24 h with TNFα-, LSS- and HSS-conditioned media (-CMs). ICAM-1 mRNA expression is represented relative to CTR-CM (2^−ΔΔCt).

Figure 9

PBMC adhesion assay on HUVEC monolayer cultured for 24 h with TNFα-, LSS- and HSS-conditioned media (-CMs). (A) Immunofluorescence imaging of representative fields of the different conditions. (B) Adherent PBMC absolute count on HUVEC monolayers. (C) Fluorescence quantification of the adherent PBMCs (** p < 0.01 and *** p < 0.005). Scalebar: 200 μm.

Figure 10

Tube formation assay on Matrigel. Representative bright field images of the same central area were taken at different time points per each condition. Scalebar: 500 μm.

Figure 11

Computer reconstruction obtained in ImageJ Angiogenesis Analyzer plug-in of master segments and junctions observed in HUVECs seeded on Matrigel and stimulated for 8 h with different CMs.

Figure 12

Angiogenesis analysis of tube formation assay at different time points. (* p < 0.05, ** p < 0.01).

Figure 13

(A) Phase contrast (scalebar: 500 μm) and (B) tubulin fluorescence images (scalebar: 200 μm) of tube formation at 24 h performed with HUVECs in presence of different conditioned media.

Figure 14

Angiogenesis analysis of tube formation images at 24 h. (* p < 0.05).

Figure 15

pro-MMP-2 expression evaluated by zymography. On the right, quantitative analysis of the absolute expression of pro-MMP-2 in different conditioned media (CMs) is shown. (* p < 0.05, *** p < 0.005).

Figure 16

Schematic representation of the results obtained. Endothelial cells (ECs) exposed to LSS are characterized by decreased eNOS levels, an increase in adhesion molecules and the cytoplasmatic localization of PECAM-1 (cPECAM-1), while ECs challenged with HSS display high eNOS level and limited expression of adhesion molecules and PECAM expression at adherens junctions (mPECAM-1). Tube formation is significantly increased in ECs cultured in LSS-CM demonstrating the presence of angiogenic factors in the conditioned media. The image on the right represents a proposed mechanism correlating coronary microvascular dysfunction (CMD) to epicardial coronary artery disease (CAD) associated with low shear stress regions. Coronary arteries are rich in regions experiencing low shear stress during the cardiac cycle, which are often inner curvatures or bifurcations. ECs at these regions are then exposed to low shear forces for a prolonged period before atherogenesis. During this period, it is possible that ECs could release various factors affecting nearby cells as well as downstream microvascular endothelial cells ultimately promoting CMD. In fact, CMD was found to precede in time epicardial CAD, and this is likely to be related to hemodynamic changes in the microvascular compartment affecting upstream blood flow (smart.servier.com).