Göran Bondjers

Interferon-γ Induces Secretory Group IIA Phospholipase A2 in Human Arterial Smooth Muscle Cells INVOLVEMENT OF CELL DIFFERENTIATION, STAT-3 ACTIVATION, AND MODULATION BY OTHER CYTOKINES

Increased expression of secretory non-pancreatic phospholipase A2 (sPLA2-IIA) could be part of the inflammatory reaction in atherosclerosis. However, the factors controlling sPLA2-IIA production in human vascular cells are unknown. We investigated regulation of sPLA2-IIA expression and secretion by human arterial smooth muscle cells in culture (HASMC). SPLA2-IIA was induced after 3–14 days of culture in non-proliferating conditions. SPLA2-IIA was co-expressed with heavy caldesmon, a cytoskeleton protein, and p27, a G1 cyclin inhibitor, proteins characteristically expressed by differentiated cells. Further incubation with 50–500 units/ml of interferon (IFN)-γ significantly increased sPLA2-IIA mRNA and secretion. IFN-γ-induced sPLA2-IIA was found to be active in cell media and associated with cell membrane proteoglycans. IFN-γ induced sPLA2-IIA expression was antagonized by tumor necrosis factor (TNF)-α and interleukin (IL)-10. TNF-α added individually induced a significant but transient (4 h) increase in sPLA2-IIA secretion. IL-10 by itself did not affect sPLA2-IIA expression and secretion. IFN-γ-stimulated sPLA2-IIA transcription involved STAT-3 protein. Interestingly, IL-6 but not IFN-γ up-regulated the sPLA2-IIA expression in HepG2 cells, thus sPLA2-IIA induction by IFN-γ response appears to be cell specific. In summary, conditions leading to cell differentiation induced sPLA2-IIA expression in HASMC and further exposure to IFN-γ can up-regulate sPLA2-IIA transcription and secretion. This IFN-γ stimulatory effect can be modulated by other cytokines.

Hypoxia increases 25-hydroxycholesterol-induced interleukin-8 protein secretion in human macrophages

Interleukin-8 (IL-8) is a chemotactic factor for T-lymphocytes and smooth muscle cells and may therefore have an important effect in atherogenesis. It is secreted from oxysterol-containing foam cells which have been found in hypoxic zones in atherosclerotic plaques. The aim of this study was to investigate the effect of hypoxia on the secretion of IL-8 by oxysterol-stimulated macrophages. Hypoxia enhances 25-hydroxycholesterol (25-OH-chol)-induced IL-8 secretion in human monocyte-derived macrophages. The effect is most pronounced when macrophages are incubated with low concentrations of 25-OH-chol. Both 25-OH-chol and hypoxia increases the intracellular level of the signalling molecule hydrogen peroxide (H(2)O(2)). This event coincided with an enhanced binding of the transcription factor c-jun to the IL-8 gene promoter and an increased IL-8 mRNA expression in hypoxic macrophages. These observations suggest that similar intracellular signalling pathways are used for both 25-OH-chol-induced IL-8 expression and hypoxia-induced IL-8 expression. Thus, hypoxia in atherosclerotic plaques may increase the secretion of IL-8 from oxysterol-containing foam cells, which subsequently may accelerate the progression of atherosclerosis.

25-hydroxycholesterol induces lipopolysaccharide-tolerance and decreases a lipopolysaccharide-induced TNF-α secretion in macrophages

Several different oxysterols are formed when LDL is oxidized. The role of oxysterols in the inflammatory process in the atherosclerotic plaque is not totally elucidated. In this study we have investigated the effect of four different oxysterols on an LPS-induced TNF-alpha secretion in human macrophages. Cultured human macrophages were incubated with 7-keto-, 7beta-hydroxy-, 27-hydroxy- and 25-hydroxycholesterol for 24 h before exposure to endotoxin (LPS) for 3 h. All oxysterols, except 7-ketocholesterol, significantly decreased an LPS-induced TNF-alpha secretion. The most pronounced effect was obtained with 25-hydroxycholesterol, where the TNF-alpha secretion was reduced to 8%. This decreased effect was also found on the TNF-alpha mRNA level. The decreased LPS-induced TNF-alpha secretion coincided with an increased binding of the transcription factors Sp1 and Sp3 to the TNF-alpha promoter. In vitro studies of the TNF-alpha promoter suggests possible interactions between Sp1 and Sp3 and the NF-kappaB transcription factor complex that might affect the transcriptional initiation.

PROCESSING OF PDGF GENE PRODUCTS DETERMINES INTERACTIONS WITH GLYCOSAMINOGLYCANS

The platelet derived growth factor (PDGF), a mitogen for mesenchymal cells, may be bound to and inhibited by heparin and other glycosaminoglycans. PDGF is a homo‐ or heterodimer of A‐ and B‐chains. They occur as short (A109 and B110) and long (A125 and B160) isoforms. The latter contain basic carboxyl‐terminal extensions. Dimeric A125 binds to heparin through its basic extension in a two‐step reaction. The mechanism involves a conformational change and is consistent with a Monod–Wyman–Changeux allosteric model. Previous indirect experiments suggested that three critical amino acids (basic R111, K116 and polar T125) might be involved. Here, direct binding experiments using dimeric full‐length mutants in surface plasmon resonanse analysis showed that all three critical amino acids in an R(X)4K(X)8T‐motif contributed in a concerted manner to the high affinity binding. Mutations of these amino acids to alanine resulted in large thermodynamic changes, loss of the allosteric mechanism and order(s) of magnitude lower binding affinity. The binding mechanism and affinity of long dimeric rB were similar to the mutants. Short dimeric rA109 and rB110 showed 100 times lower binding affinity than rA125. Consequently, interactions with glycosaminoglycans in tissues varies between PDGF isoforms and may influence their local accumulation and activity. Copyright

Expression of PDGF receptors and ligand-induced migration of partially differentiated human monocyte-derived macrophages: Influence of IFN-γ and TGF-β

In the early atherosclerotic lesion, monocytes accumulate at sites of inflammation and endothelial injury. Platelet-derived growth factor (PDGF), produced for example by macrophages, is a chemoattractant for smooth muscle cells and possibly also for macrophages. During early differentiation into macrophages, human monocytes (early hMDM) showed lower expression of PDGF alpha-receptor (PDGF-Ralpha) than beta-receptor (PDGF-Rbeta) mRNA. Early hMDM showed increased random motility (chemokinesis) in the presence of PDGF of the long (BB(L)) but not short (BB(S)) B-chain homodimer. Neither PDGF-AA(S) nor PDGF-AA(L) affected early hMDM motility. Since increased cytokine levels accompany inflammation, the influence of interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta) on PDGF-R expression and migratory response were studied. Only PDGF-Ralpha mRNA was highly upregulated by IFN-gamma. TGF-beta only had minor effects on receptor mRNAs. Upregulation of PDGF-Ralpha levels by IFN-gamma was accompanied by significantly increased migration (chemotaxis) towards PDGF-AA(L) only. Consequently, IFN-gamma modulates PDGF-Rs expression in early hMDM and, subsequently, the chemotactic activity of PDGF-AA(L) on IFN-gamma-stimulated early hMDM. This suggests that PDGF-AA(L) may be involved in attracting activated monocytes to sites of inflammation and injury.

The Molecular Basis for Lipoprotein Interaction with Vascular Tissue

Local and focal retention of lipoproteins rather than increased influx appears to be the basis for lipoprotein deposition in arterial tissue during atherogenesis. Binding of low density lipoproteins to arterial chondroitin sulphate rich proteoglycans is mediated by specific hydrophilic peptide sequences in apoB, characterized by a high frequency of basic amino acids. One of these sequences is considered to be involved in the interaction between the lipoproteins and the LDL receptor. Consequently, it might be postulated that the receptor-mediated cellular uptake of LDL could be inhibited in the presence of an excess of arterial proteoglycans. However, LDL which has been precipitated by proteoglycan and subsequently resolubilized is taken up more avidly than native LDL both in macrophages and in smooth muscle cells. This appears to be due to a selection by the proteoglycans of a more reactive fraction of LDL. This fraction has a smaller size and less surface phospholipids. As smaller LDL particles also have a higher transfer rate into the arterial tissue they may be particularly atherogenic. Low density lipoproteins appear to be taken up with higher affinity by arterial macrophages than by smooth muscle cells. The selective transfer of the LDL to macrophages can be inhibited by alpha-tocopherol suggesting that oxidative modification may be involved in this process. In fact, binding of LDL to arterial proteoglycans also appears to increase the susceptibility of the lipoproteins to oxidative modification, as well as the susceptibility to proteolytic degradation. Thus, the interaction of LDL with proteoglycans might be involved in several of the key elements of the atherogenic process.