Effect of serum concentration on adhesion of monocytic THP-1 cells onto cultured EC monolayer and EC-SMC co-culture

Background:The adhesion of monocytes to the endothelium following accumulation of low-density lipoprotein (LDL) in subendothelial spaces is an important step in the development of intimal hyperplasia in arterially implanted vein grafts and atherosclerosis in both animals and humans. However, it is not well known how serum factors affect the adhesion of monocytes. Methods: We have studied the effect of fetal calf serum (FCS), which we considered a source of LDL, on the adhesion of monocytes to endothelial cells (ECs) by using human monocytic THP-1 cells and both a monolayer of cultured bovine aortic endothelial cells (EC monoculture) and a co-culture with bovine aortic smooth muscle cells (EC-SMC co-culture). Results: It was found that the addition of FCS to the medium greatly affected the adhesion of THP-1 cells, and the higher the concentration of FCS in the medium, the greater the adhesion of THP-1 cells to endothelial cells. Adhesion of THP-1 cells to an EC-SMC co-culture was approximately twofold greater than that to an EC monoculture, and after adhering to endothelial cells, many THP-1 cells trans-migrated into the layer of smooth muscle cells. Conclusion: The results suggest that the elevation of the LDL (cholesterol) level in blood provides a favorable condition for the development of intimal hyperplasia and atherosclerosis by promoting the adhesion of monocytes to the endothelium and their subsequent migration into subendothelial spaces.

Platelet-mediated adhesion facilitates leukocyte sequestration in hypoxia-reoxygenated microvessels

Leukocyte transendothelial migration and sequestration are two distinct outcomes following leukocyte adhesion to endotheli- um during ischemia-reperfusion injury, in which platelets may play a pivotal role. In the present study, we established an in vitro hypoxia-reoxygenation model to mimic ischemia-reperfusion injury and found platelet pre-incubation significantly in- creased leukocyte adhesion to endothelial cells after hyoxia-reoxygenation （over 67%）. Blockade of endothelial-cell-expressed adhesion molecules inhibited leukocyte direct adhesion to endothelial cells, while platelet-mediated leukocyte adhesion was suppressed by blockade of platelet-expressed adhesion molecules. Further experiments revealed platelets acted as a bridge to mediate leukocyte adhesion, and platelet-mediated adhesion was the predominant pattern in the presence of platelets. However, platelet pre-incubation significantly suppressed leukocyte transendothelial migration after hypoxia-reoxygenation （over 31%）, which could be aggravated by blockade of endothelial-cell-expressed adhesion molecules, but alleviated by blockade of plate- let-expressed adhesion molecules. This would indicate that platelet-mediated adhesion disrupted leukocyte transendothelial migration. An in vivo meseuteric ischemia-reperfusion model demonstrated leukocyte transfusion alone caused mild leukocyte adhesion to reperfused vessels and subsequent leukocyte infiltration, while simultaneous leukocyte and platelet transfusion led to massive leukocyte adhesion and sequestration within reperfused microvessels. Our studies revealed platelets enhanced leu- kocyte adhesion to endothelial cells, but suppressed leukocyte transendothelial migration. Overall, this leads to leukocyte se- questration in hypoxia-reoxygenated microvessels.

Endothelial Notch activation promotes neutrophil transmigration via downregulating endomucin to aggravate hepatic ischemia/reperfusion injury

Inflammatory leukocytes infiltration is orchestrated by mechanisms involving chemokines,selectins,addressins and other adhesion molecules derived from endothelial cells(ECs),but how they respond to inflammatory cues and coordinate leukocyte transmigration remain elusive.In this study,using hepatic ischemia/reperfusion injury(HIRI)as a model,we identified that endothelial Notch activation was rapidly and dynamically induced in liver sinusoidal endothelial cells(LSECs)in acute inflammation.In mice with EC-specific Notch activation(NICeCA),HIRI induced exacerbated liver damage.Consistently,endothelial Notch activation enhanced neutrophil infiltration and tumor necrosis factor(TNF)-αexpression in HIRI.Transcriptome analysis and further qRT-PCR as well as immunofluorescence indicated that endomucin(EMCN),a negative regulator of leukocyte adhesion,was downregulated in LSECs from NICeCA mice.EMCN was downregulated during HIRI in wild-type mice and in vitro cultured ECs insulted by hypoxia/re-oxygenation injury.Notch activation in ECs led to increased neutrophil adhesion and transendothelial migration,which was abrogated by EMCN overexpression in vitro.In mice deficient of RBPj,the integrative transcription factor of canonical Notch signaling,although overwhelming sinusoidal malformation aggravated HIRI,the expression of EMCN was upregulated;and pharmaceutical Notch blockade in vitro also upregulated EMCN and inhibited transendothelial migration of neutrophils.The Notch activation-exaggerated HIRI was compromised by blocking LFA-1,which mediated leukocyte adherence by associating with EMCN.Therefore,endothelial Notch signaling controls neutrophil transmigration via EMCN to modulate acute inflammation in HIRI.