Soluble LDL-receptor is induced by TNF-α and inhibits hepatocytic clearance of LDL-cholesterol.

Defective LDL-C clearance and hence its elevation in the circulation is an established risk factor for cardiovascular diseases (CVDs) such as myocardial infarction (MI). A soluble LDL-receptor (sLDL-R) has been detected in human plasma which correlates strongly with circulating LDL-C and classical conditions that promote chronic inflammation. However, the mechanistic interplay between sLDL-R, inflammation, and CVDs remains to be investigated. Here, we report that stimulation of HepG2 cells with TNF-α induces the release of sLDL-R into culture supernatants. In addition, TNF-α induces gene expression of peptidases ADAM-17 and MMP-14 in HepG2 cells, and inhibiting these peptidases using TMI 1 significantly reduces the TNF-α induced sLDL-R release. We found that a soluble form of recombinant LDL-R (100 nM) can strongly bind to LDL-C and form a stable complex (KD = E-12). Moreover, incubation of HepG2 cells with this recombinant LDL-R resulted in reduced LDL-C uptake in a dose-dependent manner. In a nested case-control study, we found that baseline sLDL-R in plasma is positively correlated with plasma total cholesterol level. Furthermore, a twofold increase in plasma sLDL-R was associated with a 55% increase in the risk of future MI [AOR = 1.55 (95% CI = 1.10-2.18)]. Nevertheless, mediation analyses revealed that a significant proportion of the association is mediated by elevation in plasma cholesterol level (indirect effect ß = 0.21 (95% CI = 0.07-0.38). Collectively, our study shows that sLDL-R is induced by a pro-inflammatory cytokine TNF-α via membrane shedding. Furthermore, an increase in sLDL-R could inhibit hepatic clearance of LDL-C increasing its half-life in the circulation and contributing to the pathogenesis of MI. KEY MESSAGES: TNF-α causes shedding of hepatocytic LDL-R through induction of ADAM-17 and MMP-14. sLDL-R binds strongly to LDL-C and inhibits its uptake by hepatocytic cells. Plasma sLDL-R is positively correlated with TNF-α and cholesterol. Plasma sLDL-R is an independent predictor of myocardial infarction (MI). Plasma cholesterol mediates the association between sLDL-R and MI.

Interleukin-6 trans-signaling induced laminin switch contributes to reduced trans-endothelial migration of granulocytic cells.

BACKGROUND AND AIMS: Laminins are essential components of the endothelial basement membrane, which predominantly contains LN421 and LN521 isoforms. Regulation of laminin expression under pathophysiological conditions is largely unknown. In this study, we aimed to investigate the role of IL-6 in regulating endothelial laminin profile and characterize the impact of altered laminin composition on the phenotype, inflammatory response, and function of endothelial cells (ECs). METHODS: HUVECs and HAECs were used for in vitro experiments. Trans-well migration experiments were performed using leukocytes isolated from peripheral blood of healthy donors. The BiKE cohort was used to assess expression of laminins in atherosclerotic plaques and healthy vessels. Gene and protein expression was analyzed using Microarray/qPCR and proximity extension assay, ELISA, immunostaining or immunoblotting techniques, respectively. RESULTS: Stimulation of ECs with IL-6+sIL-6R, but not IL-6 alone, reduces expression of laminin α4 (LAMA4) and increases laminin α5 (LAMA5) expression at the mRNA and protein levels. In addition, IL-6+sIL-6R stimulation of ECs differentially regulates the release of several proteins including CXCL8 and CXCL10, which collectively were predicted to inhibit granulocyte transmigration. Experimentally, we demonstrated that granulocyte migration is inhibited across ECs pre-treated with IL-6+sIL-6R. In addition, granulocyte migration across ECs cultured on LN521 was significantly lower compared to LN421. In human atherosclerotic plaques, expression of endothelial LAMA4 and LAMA5 is significantly lower compared to control vessels. Moreover, LAMA5-to-LAMA4 expression ratio was negatively correlated with granulocytic cell markers (CD177 and myeloperoxidase (MPO)) and positively correlated with T-lymphocyte marker CD3. CONCLUSIONS: We showed that expression of endothelial laminin alpha chains is regulated by IL-6 trans-signaling and contributes to inhibition of trans-endothelial migration of granulocytic cells. Further, expression of laminin alpha chains is altered in human atherosclerotic plaques and is related to intra-plaque abundance of leukocyte subpopulations.

Activation of the JAK/STAT3 and PI3K/AKT pathways are crucial for IL-6 trans-signaling-mediated pro-inflammatory response in human vascular endothelial cells.

BACKGROUND: IL-6 classic signaling is linked to anti-inflammatory functions while the trans-signaling is associated with pro-inflammatory responses. Classic signaling is induced via membrane-bound IL-6 receptor (IL-6R) whereas trans-signaling requires prior binding of IL-6 to the soluble IL-6R. In both cases, association with the signal transducing gp130 receptor is compulsory. However, differences in the downstream signaling mechanisms of IL-6 classic- versus trans-signaling remains largely elusive. METHODS: In this study, we used flow cytometry, quantitative PCR, ELISA and immuno-blotting techniques to investigate IL-6 classic and trans-signaling mechanisms in Human Umbilical Vein Endothelial Cells (HUVECs). RESULTS: We show that both IL-6R and gp130 are expressed on the surface of human vascular endothelial cells, and that the expression is affected by pro-inflammatory stimuli. In contrast to IL-6 classic signaling, IL-6 trans-signaling induces the release of the pro-inflammatory chemokine Monocyte Chemoattractant Protein-1 (MCP-1) from human vascular endothelial cells. In addition, we reveal that the classic signaling induces activation of the JAK/STAT3 pathway while trans-signaling also activates the PI3K/AKT and the MEK/ERK pathways. Furthermore, we demonstrate that MCP-1 induction by IL-6 trans-signaling requires simultaneous activation of the JAK/STAT3 and PI3K/AKT pathways. CONCLUSIONS: Collectively, our study reports molecular differences in IL-6 classic- and trans-signaling in human vascular endothelial cells; and elucidates the pathways which mediate MCP-1 induction by IL-6 trans-signaling.

Expression and characterization of αvß5 integrin on intestinal macrophages.

Macrophages play a crucial role in maintaining homeostasis in the intestine, but the underlying mechanisms have not yet been elucidated fully. Here, we show for the first time that mature intestinal macrophages in mouse intestine express high levels of αvß5 integrin, which acts as a receptor for the uptake of apoptotic cells and can activate molecules involved in several aspects of tissue homeostasis such as angiogenesis and remodeling of the ECM. αvß5 is not expressed by other immune cells in the intestine, is already present on intestinal macrophages soon after birth, and its expression is not dependent on the microbiota. In adults, αvß5 is induced during the differentiation of monocytes in response to the local environment and it confers intestinal macrophages with the ability to promote engulfment of apoptotic cells via engagement of the bridging molecule milk fat globule EGF-like molecule 8. In the absence of αvß5, there are fewer monocytes in the mucosa and mature intestinal macrophages have decreased expression of metalloproteases and IL 10. Mice lacking αvß5 on haematopoietic cells show increased susceptibility to chemical colitis and we conclude that αvß5 contributes to the tissue repair by regulating the homeostatic properties of intestinal macrophages.