Cytokine Adsorber Use during DCD Heart Perfusion Counteracts Coronary Microvascular Dysfunction

Abstract

Microvascular dysfunction (MVD) in cardiac allografts is associated with an impaired endothelial function in the coronary microvasculature. Ischemia/reperfusion injury (IRI) deteriorates endothelial function. Hearts donated after circulatory death (DCD) are exposed to warm ischemia before initiating ex vivo blood perfusion (BP). The impact of cytokine adsorption during BP to prevent MVD in DCD hearts is unknown. In a porcine DCD model, we assessed the microvascular function of hearts after BP with (DCD-BP^CytoS, n = 5) or without (DCD-BP, n = 5) cytokine adsorption (CytoSorb^®). Microvascular autoregulation was assessed by increasing the coronary perfusion pressure, while myocardial microcirculation was measured by Laser-Doppler-Perfusion (LDP). We analyzed the immunoreactivity of arteriolar oxidative stress markers nitrotyrosine and 4-hydroxy-2-nonenal (HNE), endothelial injury indicating cell adhesion molecules CD54, CD106 and CD31, and eNOS. We profiled the concentration of 13 cytokines in the perfusate. The expression of 84 genes was determined and analyzed using machine learning and decision trees. Non-DCD hearts served as a control for the gene expression analysis. Compared to DCD-BP, relative LDP was improved in the DCD-BP^CytoS group (1.51 ± 0.17 vs. 1.08 ± 0.17). Several pro- and anti-inflammatory cytokines were reduced in the DCD-BP^CytoS group. The expression of eNOS significantly increased, and the expression of nitrotyrosine, HNE, CD54, CD106, and CD31, markers of endothelial injury, majorly decreased in the DCD-BP^CytoS group. Three genes allowed exact differentiation between groups; regulation of HIF1A enabled differentiation between perfusion (DCD-BP, DCD-BP^CytoS) and non-perfusion groups. CAV1 allowed differentiation between BP and BP^CytoS. The use of a cytokine adsorption device during BP counteracts preload-dependent MVD and preserves the microvascular endothelium by preventing oxidative stress and IRI of coronary arterioles of DCD hearts.

Keywords: cytokine adsorption; cytokines; donation after circulatory death; heart transplantation; hemoadsorption; machine perfusion; microvascular dysfunction; oxidative stress.

Figure 1

Workflow. BP: blood perfusion. DCD: donation after circulatory death. BP^CytoS: blood perfusion with an integrated cytokine adsorption device.

Figure 2

Microvascular function. (A) Pressure-Microcirculation-Matching. (B) Pressure-Flow-Matchig. (C) Preload-Microcirculation-Matching. (D) Myocardial workload * p < 0.05, *¹ p = 0.053, *² p = 0.067 and ** p < 0.001 compared to DCD-BP. LDP in DCD hearts is only shown as a reference. BP: blood perfusion. BP^CytoS: blood perfusion with an integrated cytokine adsorption device. DCD: donation after circulatory death. DP: developed pressure. LDP: laser Doppler perfusion. LV: left-ventricular.

Figure 3

Perfusion pressure. Light grey: DCD. Grey: DCD-BP. Dark grey: DCD-BP^CytoS. LV: left ventricular. MP: machine perfusion. BP: blood perfusion. DCD: donation after circulatory death. BP^CytoS: blood perfusion with an integrated cytokine adsorption device.

Figure 4

Cytokine profile. IL: interleukine. INF: interferone. TNF: tumor necrosis factor. MP: machine perfusion. BP: blood perfusion. DCD: donation after circulatory death. BP^CytoS: blood perfusion with an integrated cytokine adsorption device.

Figure 5

Representative photomicrographs of immunohistochemical staining of endothelial oxidative stress (A,B). Semiquantitative analysis of arteriolar immunoreactivity (C,D). BP: blood perfusion. CD: cluster of differentiation. DCD: donation after circulatory death. BP^CytoS: blood perfusion with an integrated cytokine adsorption device. NT: nitrotyrosine. HNE: hydrxynonenal.

Figure 6

Representative photomicrographs of immunohistochemical staining of injury markers (A–D). Semiquantitative analysis of arteriolar immunoreactivity (E–H). ** p < 0.001 compared to DCD. ^## p < 0.001 compared to DCD-BP. BP: blood perfusion. CD: cluster of differentiation. DCD: donation after circulatory death. BP^CytoS: blood perfusion with an integrated cytokine adsorption device. eNOS: endothelial nitric oxide synthase.

Figure 7

(A) Volcano plots for regulated genes of the group DCD, (B) DCD-BP, and (C) DCD-BP^CytoS compared to Control. (D) Principal component analysis on the Borruta preselected variables, (E) Decision tree to show key regulated genes, which allow differentiation between groups. BP: blood perfusion. BP^CytoS or Cy: blood perfusion with an integrated cytokine adsorption device. Crtl: control. DCD: donation after circulatory death.