Generation of a novel ex-vivo model to study re-endothelialization

Figures & data

Figure 1. (A) Schematic of the denudation setup. (B) The structure of native artery with intact EC layer from H&E and immunofluorescent (IF) staining. (C) The structure of denuded artery with EC free area. Scale bars 500 µm and 200 μm (for H&E); 100 μm and 25 μm (for IF staining).

Figure 2. (A,B) Representative staining of CD31 and αSMA on denudation protocol using trypLE Select and trypsin-EDTA at RT and 37 °C with its Respective incubation period. (C,D) Quantification of EC coverage post denudation protocol at RT and 37 °C respectively. ** p < 0.01 and * p < 0.05 compared to native artery using two-way ANOVA. (N = 4; Scale bar: 100 μm).

Figure 3. (A,B) Representative image of Alcian blue staining from denudation using trypLE Select and trypsin-EDTA enzyme at RT and 37 °C. (C,D) Intensity analysis of Alcian blue at RT and 37 °C respectively. ** p < 0.01 compared to native tissue using two-way ANOVA. (N = 4, scale bar: 200 μm).

Figure 4. (A) Intima-media thickness of denuded hUA at day 0 and after cultured for 7 days (B). (C) Result shows non-significant increase of intima-media thickness at day 7 compared to day 0. L = lumen (paired t-test, N = 5, scale bar 200 μm) (D) immunophenotype of isolated cells is CD31⁺CD146⁺vWF⁺VEGFR2⁺αSMA^-. (Scale bar: 100 μm).

Table 1. Comparison of chemical and mechanical endothelial denudation.

Injury type	Chemical denudation	Mechanical denudation
Approach	Proteolytic enzyme, EDTA, urea, detergent	Flexible wire, balloon catheter, forceps, needle
Endothelial removal	Yes	Yes
SMC injury	Can be controlled	Yes
Isolation of EC	Proteolytic enzyme: yes	No
Modelling difficulties	Ex-vivo: moderate Animal model: moderate	Ex-vivo: easy Animal model: difficult
Advantages	Specifically removes EC Less invasive Reduce variability	Mimic pathophysiological condition (e.g.: balloon catheter injury) Rapid and efficient
Disadvantages	Specific and tissue dependency Does not mimic pathophysiological condition	Invasive Variability in the severity of injury

Data availability statement

The data that support the findings of this study are available from the corresponding author, [NS], upon reasonable request.