Arterial Pulsatility Augments Microcirculatory Perfusion and Maintains the Endothelial Integrity during Extracorporeal Membrane Oxygenation via hsa_circ_0007367 Upregulation in a Canine Model with Cardiac Arrest

Abstract

Background: The impairment of microcirculation is associated with the unfavorable outcome for extracorporeal membrane oxygenation (ECMO) patients. Studies revealed that pulsatile modification improves hemodynamics and attenuates inflammation during ECMO support. However, whether flow pattern impacts microcirculation and endothelial integrity is rarely documented. The objective of this work was to explore how pulsatility affects microcirculation during ECMO.

Methods: Canine animal models with cardiac arrest were supported by ECMO, with the i-Cor system used to generate nonpulsatile or pulsatile flow. The sublingual microcirculation parameters were examined using the CytoCam microscope system. The expression of hsa_circ_0007367, a circular RNA, was measured during ECMO support. In vitro validation was performed in pulmonary vascular endothelial cells (PMVECs) exposed to pulsatile or nonpulsatile flow, and the expressions of hsa_circ_0007367, endothelial tight junction markers, endothelial adhesive molecules, endothelial nitric oxide synthases (eNOS), and NF-κB signaling activity were analyzed.

Results: The pulsatile modification of ECMO enhanced microcirculatory perfusion, attenuated pulmonary inflammation, and stabilized endothelial integrity in animal models; meanwhile, the expression of hsa_circ_0007367 was significantly upregulated both in animals and PMVECs exposed to pulsatile flow. In particular, upregulation of hsa_circ_0007367 stabilized the expressions of endothelial tight junction markers zonula occludens- (ZO-) 1 and occludin, followed by modulating the endothelial nitric oxide synthases (eNOS) activity and inhibiting the NF-κB signaling pathway.

Conclusion: The modification of pulsatility contributes to microcirculatory perfusion and endothelial integrity during ECMO. The expression of hsa_circ_0007367 plays a pivotal role in this protective mechanism.

Figure 1

Pulsatility improves the microcirculatory parameters in on-ECMO animal models with cardiac arrest. (a) Animals were exposed to nonpulsatile or pulsatile circuits, and PVD were measured at different time points; (b) animals were exposed to nonpulsatile or pulsatile circuits, and MFI were measured at different time points. ^∗Significantly different between groups, p < 0.05; ^∗∗significantly different between groups, p < 0.01. Abbreviations: ECMO: extracorporeal membrane oxygenation; PVD: perfused vessel density; MFI: microvascular flow index.

Figure 2

Pulsatility attenuates lung injury and upregulates the expression of hsa_circ_0007367 (circUBAP2), which is a microcirculation-related circRNA. (a) Representative images of lung tissues with hematoxylin and eosin staining in nonpulsatile group animals after 6 hours of ECMO; (b) representative images of lung tissues with hematoxylin and eosin staining in pulsatile group animals after 6 hours of ECMO; (c) comparison of the circUBAP2 expressions in lung tissues between these two groups; (d) correlation analysis between the expression of circUBAP2 and the PVD value; (e) correlation analysis between the expression of circUBAP2 and the MFI value. ^∗∗Significantly different between groups, p < 0.01. Abbreviations: PVD: perfused vessel density; MFI: microvascular flow index.

Figure 3

Pulsatility maintains the expressions of ZO-1 and occludin. (a) Immunofluorescent analysis of the ZO-1 distribution in PMVECs exposed to nonpulsatile or pulsatile flow for 6 hours; (b) immunofluorescent analysis of the occludin distribution in PMVECs exposed to nonpulsatile or pulsatile flow for 6 hours; (c) the expression of ZO-1 in PMVECs exposed to nonpulsatile or pulsatile flow for 6 hours using qPCR; (d) the expression of occludin in PMVECs exposed to nonpulsatile or pulsatile flow for 6 hours using qPCR; (e) the expressions of ZO-1 and occludin in PMVECs exposed to nonpulsatile or pulsatile flow for 6 hours using western blot assay. ^∗∗Significantly different between groups, p < 0.01. Abbreviations: ZO-1: zonula occludens-1; DAPI: 4′,6-diamidino-2-phenylindole; PMVEC: pulmonary microvascular endothelial cell.

Figure 4

hsa_circ_0007367 (UBAP2) preserves the expressions of ZO-1 and occludin. (a) The expression of circUBAP2 in PMVECs treated with nonpulsatile or pulsatile flow for 6 hours; (b) the expressions of ZO-1 and occludin in PMVECs treated with circUBAP2 knockdown or scrambled siRNA using western blot assay; (c) immunofluorescent analysis of the ZO-1 distribution in PMVECs treated with circUBAP2 silencing or scramble siRNA; (d) immunofluorescent analysis of the occludin distribution in PMVECs treated with circUBAP2 silencing or scrambled siRNA. ^∗∗Significantly different between groups, p < 0.01. Abbreviations: ZO-1: zonula occludens-1; DAPI: 4′,6-diamidino-2-phenylindole; PMVEC: pulmonary microvascular endothelial cell.

Figure 5

hsa_circ_0007367 (UBAP2) is needed for the pulsatility-mediated ZO-1 and occludin upregulation. (a) The distribution of ZO-1 in PMVECs treated with control siRNA or siRNA targeting circUBAP2, followed by 6-hour exposure to pulsatile flow; (b) the distribution of occludin in PMVECs treated with control siRNA or siRNA targeting circUBAP2, followed by 6-hour exposure to pulsatile flow; (c) the expressions of ZO-1 and occludin detected with western blotting in PMVECs treated with control siRNA or si-circUBAP2, followed by 6-hour exposure to pulsatile flow or nonpulsatile flow; (d) the expression of ZO-1 mRNA in PMVECs treated with control siRNA or si-circUBAP2, followed by 6-hour exposure to different flow patterns; (e) the expression of occludin mRNA in PMVECs treated with control siRNA or si-circUBAP2, followed by 6-hour exposure to different flow patterns. ^∗p < 0.05; ^∗∗p < 0.01. Abbreviations: ZO-1: zonula occludens-1; DAPI: 4′,6-diamidino-2-phenylindole; si: small interfering RNA; Ctrl: control; PMVEC: pulmonary microvascular endothelial cell.

Figure 6

hsa_circ_0007367 (UBAP2) is required for the suppression of endothelial adhesion and inflammation mediated by pulsatile flow. (a) The expressions of endothelial adhesive molecules, NF-κB, and eNOS proteins using western blotting in PMVECs treated with control siRNA or si-circUBAP2, followed by 6-hour exposure to pulsatile flow or nonpulsatile flow; (b) the expression of NF-κB mRNA in PMVECs treated with different siRNAs and different flow patterns; (c) the expression of eNOS mRNA in PMVECs treated with different siRNAs and different flow patterns. ^∗p < 0.05; ^∗∗p < 0.01. Abbreviations: ZO-1: zonula occludens-1; DAPI: 4′,6-diamidino-2-phenylindole; si: small interfering RNA; Ctrl: control; eNOS: endothelial nitric oxide synthases; PMVEC: pulmonary microvascular endothelial cell.