Functional Impairment of Endothelial Colony Forming Cells (ECFC) in Patients with Severe Atherosclerotic Cardiovascular Disease (ASCVD)

Abstract

Endothelial dysfunction is a key factor in atherosclerosis. However, the link between endothelial repair and severity of atherosclerotic cardiovascular disease (ASCVD) is unclear. This study investigates the relationship between ASCVD, markers of inflammation, and circulating endothelial progenitor cells, namely hematopoietic cells with paracrine angiogenic activity and endothelial colony forming cells (ECFC). Two hundred and forty-three subjects from the TELARTA study were classified according to the presence of clinical atherosclerotic disease. ASCVD severity was assessed by the number of involved vascular territories. Flow cytometry was used to numerate circulating progenitor cells (PC) expressing CD34 and those co-expressing CD45, CD34, and KDR. Peripheral blood mononuclear cells ex vivo culture methods were used to determine ECFC and Colony Forming Unit- endothelial cells (CFU-EC). The ECFC subpopulation was analyzed for proliferation, senescence, and vasculogenic properties. Plasma levels of IL-6 and VEGF-A were measured using Cytokine Array. Despite an increased number of circulating precursors in ASCVD patients, ASCVD impaired the colony forming capacity and the angiogenic properties of ECFC in a severity-dependent manner. Alteration of ECFC was associated with increased senescent phenotype and IL-6 levels. Our study demonstrates a decrease in ECFC repair capacity according to ASCVD severity in an inflammatory and senescence-associated secretory phenotype context.

Keywords: atherosclerosis; endothelial progenitor cells; senescence.

Figure 1

ECFC formation according to ACSVD presence and severity. PBMC from 112 patients were seeded onto gelatin-coated wells and observed every 2 days as described in Materials and Methods. (A,B) Colony progeny obtention (in%) in the presence or absence of ASCVD (A) or according to the extent of ASCVD (B). (C,D) Number of ECFC colonies formed by PBMC according to the presence or absence of ASCVD (C) or the extent of ASCVD (D). Data are presented as median (IQR) and percentages (%). ASCVD indicates atherosclerotic cardiovascular disease. p of comparisons are obtained from the Mann–Whitney or Khi2-test as appropriate.

Figure 2

ECFC proliferative capacity and senescence according to ASCVD presence and severity. ECFC were used at passage 3 as described in Materials and Methods. (A) Proliferation assayed by BrdU incorporation. Results were expressed in rlu/s of luminescence measurements. Data represent mean ± SD of 55 independent experiments performed in triplicate. (B) Representative images from ECFC according to the extent of ASCVD (magnification 20×). (C) The percentage of senescent cells was determined as the number of cells expressing SA-β-galactosidase (blue cells) relative to the total number of cells in each field. Histograms represent mean ± SD of 55 independent experiments performed in triplicate. ASCVD indicates atherosclerotic cardiovascular disease. p of comparisons are obtained from the Mann–Whitney test.

Figure 3

ECFC vasculogenic potential according to the presence and severity of ASCVD. ECFC were used at passage 3 as described in Materials and Methods. (A) Representative experiment of capillary-like sprout formation from 3D in vitro angiogenesis assay with collagen gel-embedded spheroids obtained with ECFC from patients according to the extent of ASCVD (number of ASCVD) (magnification 20×). (B–D) Quantification of the number of branching points (B), the number of sprouts (C), and the cumulative length (D) per spheroids according to the absence or presence of ASCVD. For each experiment, sprouts from 20 spheroids were counted. Data represent means ± SD of 55 independent experiments. ASCVD indicates atherosclerotic cardiovascular disease. p of comparisons are obtained from the Mann–Whitney test.

Figure 4

Relation between senescence and angiogenic functions of ECFC. (A) Senescence according to the number of sprouts per spheroid. (B) Senescence according to the number of branching points per spheroid. (C) Senescence according to the cumulative length per spheroid. R² and p are from Pearson correlation.

Figure 5

Relations between ECFC proliferative capacity or senescence and clinical characteristics. (A,B) Carotid thickness (mm) according to the proliferative capacity and the senescence of ECFC. (C,D) IL-6 level according to the proliferative capacity and the senescence of ECFC. (E,F) VEGF level according to the proliferative capacity and the senescence of ECFC. R² and p are from Pearson correlation. IL-6 indicates Interleukin 6 and VEGF indicates vascular endothelium growth factor.

Figure 6

Relation between VEGF level and angiogenic function of ECFC. (A) VEGF level according to the number of sprouts per spheroid (B) VEGF level according to the number of branching points per spheroid (C) VEGF level according to the cumulative length per spheroid. R² and p are from Pearson correlation. VEGF indicates vascular endothelium growth factor.