Quantification of Morphological Modulation, F-Actin Remodeling, and PECAM-1 (CD-31) Redistribution in Endothelial Cells in Response to Fluid-Induced Shear Stress Under Various Flow Conditions

Abstract

Cardiovascular diseases (CVDs) are the number one cause of death globally. Arterial endothelial cell (EC) dysfunction plays a key role in many of these CVDs, such as atherosclerosis. Blood flow-induced wall shear stress (WSS), among many other pathophysiological factors, is known to significantly contribute to EC dysfunction. The present study reports an in vitro investigation of the effect of quantified WSS on ECs, analyzing the EC morphometric parameters and cytoskeletal remodeling. The effects of four different flow cases (low steady laminar (LSL), medium steady laminar (MSL), nonzero-mean sinusoidal laminar (NZMSL), and laminar carotid (LCRD) waveforms) on the EC area, perimeter, shape index (SI), angle of orientation, F-actin bundle remodeling, and platelet endothelial cell adhesion molecule-1 (PECAM-1) localization were studied. For the first time, a flow facility was fully quantified for the uniformity of flow over ECs and for WSS determination (as opposed to relying on analytical equations). The SI and angle of orientation were found to be the most flow-sensitive morphometric parameters. A two-dimensional fast Fourier transform (2D FFT) based image processing technique was applied to analyze the F-actin directionality, and an alignment index (AI) was defined accordingly. Also, a significant peripheral loss of PECAM-1 in ECs subjected to atheroprone cases (LSL and NZMSL) with a high cell surface/cytoplasm stain of this protein is reported, which may shed light on of the mechanosensory role of PECAM-1 in mechanotransduction.

Comparative Evaluation on Impacts of Fibronectin, Heparin–Chitosan, and Albumin Coating of Bacterial Nanocellulose Small-Diameter Vascular Grafts on Endothelialization In Vitro

“…Acridine Orange staining, a fluorescent dye that reports both cytoplasmic RNA and DNA in the nuclei, was used to get a visual impression of cell survival, cell morphology, and its distribution on the BNC surface [ 48 , 49 ]. To determine the shape of the cytoskeleton of the endothelial cells, we additionally stained for F-actin, which is a commonly accepted method for morphological studies on endothelial cells [ 50 , 51 ]. Furthermore, we complemented a CD-31 staining to detect preserved endothelial character of the cells [ 50 ].…”

“…To determine the shape of the cytoskeleton of the endothelial cells, we additionally stained for F-actin, which is a commonly accepted method for morphological studies on endothelial cells [ 50 , 51 ]. Furthermore, we complemented a CD-31 staining to detect preserved endothelial character of the cells [ 50 ]. For cultivated constructs, cell-specific markers were evaluated after fixation in 4% PFA solution for 5 min before staining.…”

Engineering microvascular networks using a KLF2 reporter to probe flow-dependent endothelial cell function

“…Cellular elongation and alignment along the direction of flow is often observed in planar EC preparations exposed to laminar shear stresses 48,49 . In our MVNs, ECs were highly elongated and oriented along the long axis of each vascular branch, even in static culture.…”

Endothelial Mechanosensors for Atheroprone and Atheroprotective Shear Stress Signals

“…Under atheroprotective conditions (11.5±0.9 dynes/cm 2 , 24 h), F-actins aligned with the flow direction, and the cells were full of denser and thicker actin bundles. 52 …”