Proteins Adsorbed during Intraoperative Hemoadsorption and Their In Vitro Effects on Endothelium

Abstract

(1) Background: Hemoadsorption is a method of blood purification with a wide spectrum of indications. Pre-emptive use of hemoadsorption in patients undergoing heart surgery with cardiopulmonary bypass is considered to reduce the risk of postoperative systemic inflammatory response syndrome. The current study aimed to identify the spectrum of blood proteins adsorbed on the polymer matrix of the CytoSorb hemoadsorption system and to investigate their influence on cultured endothelial cells in vitro. (2) Methods: Adsorbers used for intraoperative hemoadsorption were obtained from patients undergoing on-pump valve surgery in acute endocarditis. Proteins were extracted from the adsorbers, purified, identified with mass-spectrometry and applied to cultured human aortic endothelial cells. (3) Results: A broad range of blood proteins were identified in the material eluted from the CytoSorb adsorber. When added to cultured ECs, these protein extracts caused severe reduction in cell viability and migration. After 24 h exposure, transcriptional changes with up-regulation of multiple metabolic regulators were observed and verified on the protein level. Genes responsible for control of mitosis were significantly down-regulated. (4) Conclusions: In summary, our data reveal that intraoperative hemoadsorption allows broad spectrum removal of a wide range of molecules eliciting endothelial damage.

Keywords: blood purification; endothelium; hemoadsorption; systemic inflammatory response.

Figure 1

Changes in cell viability of HAECs treated with different concentrations of proteins isolated from polymer matrix (CTB-assay) (n = 3). Cells treated with increasing concentrations of eluted proteins (0.125 µg/mL, 0.25 µg/mL, 0.5 µg/mL) for 24 h, have demonstrated a dramatic drop in the rate of metabolism compared to untreated control (** p < 0.001, t-test).

Figure 2

Wound-healing assay in HAECs treated with different concentration of proteins, eluted from the matrix of the hemoadsorber (E) and corresponding concentrations of human serum (S) (n = 3). (a) Representative microphotographs of the HAECs at different time points after scratch. Scratch borders are delineated in red; all microphotographs are taken with 4×magnification. (b) Dynamics of scratch area changes during the 8 h. (c) Percentage of initial scratch area surface after 24 h. Data are presented as mean ± SEM, * p < 0.05 (t-test) compared to the untreated control.

Figure 3

Targets involved into metabolism regulators, up-regulated in HAECs after 24 h treatment. (a) Biological process categories overrepresented in 201 up-regulated genes in HAECs after 24 h treatment with eluted proteins (array data, n = 2). Significance generated with ConsensusPathDB-human (release 34, [26]) is presented on the x-axis as negative logarithmized p-value. (b) Validation of up-regulated protein targets with immunoblotting. Proteins of interest were detected in cell lysates obtained from untreated HAECs (Ctrl), HAECs treated with 0.125 µg/mL of elution (E), 0.125 µg/mL of serum-control (S). Samples from three independent experiments are marked as Exp 1, Exp 2, Exp 3. (c) Signal intensities from protein targets were normalized to signal intensities from β-actin, signals from treated cells are expressed as percent from untreated control. The data are presented as mean values with ± SEM (* p ≤ 0.05; ** p ≤ 0.001; n.s.—no significant difference compared to untreated control). Abbreviations: ASNS−asparagine aminotransferase, PSAT1–phosphoserine aminotransferase, SREBP−sterol regulatory element-binding proteins; tRNA–transfer RNA, MTHFD2−methylene tetrahydrofolate dehydrogenase 2.

Figure 4

Increased expression of PAI-1 in HAECs, treated with proteins eluted from CytoSorb adsorber matrix (n = 3). (a) Immunodetection of tPA and PAI-1 in cell lysates obtained from untreated HAECs (Ctrl), HAECs treated with 0.125 µg/mL of elution €, 0.125 µg/mL of serum (S). Samples from three independent experiments are marked as Exp 1, Exp 2, Exp 3. (b) Signal intensities from PAI-1 and tPA, normalized to β-actin and expressed as fold signal intensity of an untreated control. Data are presented as mean ± SEM (* p ≤ 0.05, n.s.—no significant difference compared to controls).

Figure 5

Targets involved into regulation of the cell cycle, down-regulated in HAECs after 24 h treatment with elution fraction. (a) Biological process categories overrepresented in 119 down-regulated genes in HAECs after 24 h treatment with eluted proteins (array data, n = 2). Significance generated with ConsensusPathDB-human (release 34, [26]) is presented on the x-axis as negative logarithmized p-value. (b) Validation of down-regulated protein targets with immunoblotting (n = 3). Proteins of interest were detected in cell lysates obtained from untreated HAECs (Ctrl), HAECs treated with 0.125 µg/mL of elution (E), 0.125 µg/mL of serum (S). Samples from three independent experiments are marked as Exp 1, Exp 2, Exp 3. KIF20A band is shown with the arrow. (c) Signal intensities from protein targets were normalized to signal intensity from β-actin, signals from treated cells are expressed as percent from untreated control. Data are presented as mean ± SEM. (* p ≤ 0.05; ** p ≤ 0.001).