The Rejuvenative Impact of Anisotropic Topographies on Senescent Endothelial Cells

Objectives: Thrombogenicity of foreign material surfaces is a major adverse event of ventricular assist device (VAD) therapy. Endothelialization of the luminal surface on micro engineered substrates is a promising strategy but is limited by replicative capacity of endothelial cells (ECs). We aim to study the impact of anisotropic topographies on the endothelialization performance of senescent ECs.

Methods: Fully characterized human umbilical cord vein ECs (HUVECs) from three different donors underwent in vitro expansion until replicative senescence (RS) was achieved. RS was determined by SA-B Gal positive staining and reduced relative telomere length determined using monochrome multiplex quantitative PCR. Moreover, we compared and quantify the adherens junction organization and the overall cell architecture of non-senescent and senescent HUVEC monolayers seeded on a flat and a micro-structured surface with anisotropic topography (depth, line width, and pitch of 1 µm) under static conditions and 1.4 Pa laminar flow. Differences in the population means were analyzed by two-way ANOVA.

Result: Senescent HUVECs seized to proliferate after 8 weeks, 70 ± 4% of cultured cells were positive for SA-β-Gal after 17 population doublings (PDs). The relative telomere length decreased from 1.2 ± 0.2 after 3 PDs to 0.6 ± 0.3 (p = 0.0018) after 17 PDs. RS induced a decline in the integrity of adherens junctions. VE-cadherin (VEC) connectivity index (CI) decreased from 0.7 ± 0.1, to 0.4 ± 0.1 (p = 0.02) after 17 PDs. Senescent endothelial cell monolayers lost their ability to elongate and orient their actin filaments parallel to the flow direction (cells orientation after 3 PDs under flow 11 ± 1.3 degrees, cells orientation after 17 PDs under flow 44 ± 2.1 degrees, p = 0.04). Under static conditions, monolayers cultured on micro structured surfaces improved the integrity of adherens junctions as shown by the increase of the VEC CI from 0.7 ± 0.1 to 0.9 ± 0.1 (p = 0.04) in young ECs (3 PDs) and from 0.4 ± 0.1 to 0.7 ± 0.1 (p = 0.01) in senescent ECs (17 PDs). Elongation and orientation of both young and senescent ECs improved on micro structured surfaces (PDL 3: 6 ± 0.7 degrees on the micro structured surface, on the flat surface 39 ± 6.5 degrees, p = 0.04, PDL 17: 9 ± 1.3 degrees on the micro structured surface, on the flat surface 46 ± 4.2 degrees, p = 0.03).

Conclusion: RS impairs the formation of a stable EC monolayer. Cultivation on micro structured topography improves contact and adhesion between neighboring senescent ECs suggesting micro structured surfaces as a potential strategy to improve VAD endothelialization.

Publication History

Article published online:
19 February 2021