The relationship between endothelial progenitor cell populations and epicardial and microvascular coronary disease-a cellular, angiographic and physiologic study

Abstract

Background: Endothelial progenitor cells (EPCs) are implicated in protection against vascular disease. However, studies using angiography alone have reported conflicting results when relating EPCs to epicardial coronary artery disease (CAD) severity. Moreover, the relationship between different EPC types and the coronary microcirculation is unknown. We therefore investigated the relationship between EPC populations and coronary epicardial and microvascular disease.

Methods: Thirty-three patients with a spectrum of isolated left anterior descending artery disease were studied. The coronary epicardial and microcirculation were physiologically interrogated by measurement of fractional flow reserve (FFR), index of microvascular resistance (IMR) and coronary flow reserve (CFR). Two distinct EPC populations (early EPC and late outgrowth endothelial cells [OECs]) were isolated from these patients and studied ex vivo.

Results: There was a significant inverse relationship between circulating OEC levels and epicardial CAD severity, as assessed by FFR and angiography (r=0.371, p=0.04; r=-0.358, p=0.04; respectively). More severe epicardial CAD was associated with impaired OEC migration and tubulogenesis (r=0.59, p=0.005; r=0.589, p=0.004; respectively). Patients with significant epicardial CAD (FFR<0.75) had lower OEC levels and function compared to those without hemodynamically significant stenoses (p<0.05). In contrast, no such relationship was seen for early EPC number and function, nor was there a relationship between IMR and EPCs. There was a significant relationship between CFR and OEC function.

Conclusions: EPC populations differ in regards to their associations with CAD severity. The number and function of OECs, but not early EPCs, correlated significantly with epicardial CAD severity. There was no relationship between EPCs and severity of coronary microvascular disease.

Figure 1. Higher circulating OEC levels and function are associated with less severe epicardial CAD, as assessed by FFR.

(A) Association between OEC levels and FFR. (B) Association between OEC migration and FFR. (C) Association between OEC tubulogenesis and FFR. Correlations were assessed using the Spearman rank test. Images on the right panel show (A) OEC colony; scale bar, 100 µm, (B) OECs migrating onto transwell membrane, identified by co-staining with Ulex europaeus lectin (green) and DAPI (blue); scale bar, 50 µm, (C) network formation on Matrigel by OECs; scale bar, 100 µm.

Figure 2. Circulating OEC levels and function according to significant (FFR<0.75) and non-significant (FFR≥0.75) LAD disease.

Patients with significant LAD disease (FFR<0.75) had; (A) lower OEC levels (p<0.05), (B) impaired OEC migration (p<0.01), and (C) OEC tubulogenesis (p<0.01) compared with those with non-significant disease. The comparison between groups was assessed using the Student's t test. Data are expressed as mean±SEM. Sig, significant.

Figure 3. Higher circulating OEC levels and function are associated with less severe epicardial CAD, as assessed by angiography.

(A) Association between angiographic LAD stenosis severity and OEC levels. (B) Association between LAD stenosis and OEC migration. (C) Association between LAD stenosis and OEC tubulogenesis. Correlations were assessed using the Spearman rank test.

Figure 4. Circulating early EPC levels do not correlate with severity of epicardial CAD.

(A) Association between early EPC levels and FFR. (B) Association between early EPC levels and stenosis severity. (C) Image example of early EPCs, identified by co-staining with Dil-Ac-LDL (red) and Ulex europaeus lectin (green); scale bar, 50 µm. Correlations were assessed using the Spearman rank test. Dil-Ac-LDL, 1,1′dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanide-labeled acetylated low-density lipoprotein.

Figure 5. Flow cytometry-derived EPC definitions.

(A) Cells isolated from whole blood by density gradient separation were gated by forward and side scatter to select mononuclear cells by excluding erythrocytes, granulocytes and cell debris. (B) The mononuclear-gated cells that stained positive for CD34-FITC were analyzed for the presence or absence of CD45-PC5. (C) Mononuclear cells were also analyzed for co-staining of CD34-FITC and KDR-PE. MNC, mononuclear cell; CD34-FITC, fluorescein isothiocyanate-conjugated CD34; CD45-PC5, phycoerythrin-Cy5-conjugated CD45; KDR-PE, phycoerythrin-conjugated KDR.

Figure 6. There is no correlation between circulating EPC levels and coronary microvascular integrity, as assessed by IMR.

(A) Association between early EPC levels and IMR. (B) Association between OEC levels and IMR. Correlations were assessed using the Spearman rank test.

Figure 7. The relationship between circulating OEC level and function and CFR.

(A) Association between OEC levels and CFR. (B) Association between OEC migration and CFR. (C) Association between OEC tubulogenesis and CFR. Correlations were assessed using the Spearman rank test.