Tissue factor and PAR1 promote microbiota-induced intestinal vascular remodelling.

The gut microbiota is a complex ecosystem that has coevolved with host physiology. Colonization of germ-free (GF) mice with a microbiota promotes increased vessel density in the small intestine, but little is known about the mechanisms involved. Tissue factor (TF) is the membrane receptor that initiates the extrinsic coagulation pathway, and it promotes developmental and tumour angiogenesis. Here we show that the gut microbiota promotes TF glycosylation associated with localization of TF on the cell surface, the activation of coagulation proteases, and phosphorylation of the TF cytoplasmic domain in the small intestine. Anti-TF treatment of colonized GF mice decreased microbiota-induced vascular remodelling and expression of the proangiogenic factor angiopoietin-1 (Ang-1) in the small intestine. Mice with a genetic deletion of the TF cytoplasmic domain or with hypomorphic TF (F3) alleles had a decreased intestinal vessel density. Coagulation proteases downstream of TF activate protease-activated receptor (PAR) signalling implicated in angiogenesis. Vessel density and phosphorylation of the cytoplasmic domain of TF were decreased in small intestine from PAR1-deficient (F2r(-/-)) but not PAR2-deficient (F2rl1(-/-)) mice, and inhibition of thrombin showed that thrombin-PAR1 signalling was upstream of TF phosphorylation. Thus, the microbiota-induced extravascular TF-PAR1 signalling loop is a novel pathway that may be modulated to influence vascular remodelling in the small intestine.

Hypoxia increases macrophage motility, possibly by decreasing the heparan sulfate proteoglycan biosynthesis.

Macrophages are recruited and retained in hypoxic sites in atherosclerotic lesions and tumors. Furthermore, macrophages are suggested to be a major source of HSPG synthesis in atherosclerotic lesions. HSPG are, among other things, known to regulate cell motility, cell adhesion, and receptor interaction. The aim of this study was to investigate the effect of hypoxia on HSPG expression and macrophage motility. We also explored the potential regulation of HSPG by the transcription factor HIF-1alpha. The nondirected cell motility was increased in HMDM after 24 h exposure to hypoxia (0.5% O(2)) compared with normal cell culture condition (21% O(2)). Enzymatic degradation of HS GAG further increased the motility of the HMDM in hypoxia, indicating a role of reduced cell-associated HSPG in the increased HMDM motility. HMDM exposed to 24 h of hypoxia had lower mRNA expressions of syndecan-1 and -4 compared with cells exposed to normal cell culture conditions. Protein levels of syndecan-1 were also decreased significantly in response to hypoxia, and cells subjected to hypoxia had lower mRNA expression for key enzymes involved in HS biosynthesis. In addition, hypoxia was found to reduce the relative content of HS GAG. Transfecting THP-1 cells with siHIF-1alpha indicated that this transcription factor was not involved in the hypoxia-induced modifications of HSPG expression. Given the documented multiple functions of HSPG in macrophage behavior, the hypoxia-induced modifications of HSPG may be of relevance for the development of atherosclerotic lesions and tumor progression.

IFNgamma regulates PDGF-receptor alpha expression in macrophages, THP-1 cells, and arterial smooth muscle cells.

The recruitment of monocyte-derived macrophages (MDMs) and arterial smooth muscle cells (ASMCs) contributes to inflammation and development of intimal hyperplasia during atherosclerosis. Platelet-derived growth factor (PDGF) is a potent mitogen for SMC, signalling through PDGF-receptor subunits alpha (Ralpha) and beta (Rbeta). We have previously found that interferon gamma (IFNgamma) upregulates PDGF-Ralpha mRNA expression in human MDM (hMDM) which causes an increased migration towards PDGF. In the present study, we found that IFNgamma mediated an upregulation of PDGF-Ralpha mRNA also in THP-1 cells. The induction of PDGF-Ralpha in both hMDM and THP-1 cells was caused by STAT1 binding to the PDGF-Ralpha promoter. In human ASMCs, IFNgamma again stimulated a transient STAT1-binding to the PDGF-Ralpha promoter. However, this was not followed by an upregulation of PDGF-Ralpha mRNA. IFNgamma-stimulation resulted in augmented expression of PDGF-Ralpha protein in differentiated hMDM. Early hMDM only expressed an immature and not fully glycosylated form of the PDGF-Ralpha protein. In contrast, THP-1 cells did not synthesize PDGF-Ralpha protein, implying further posttranscriptional inhibition. Our results contribute to a better understanding of the complex regulation of PDGF-Ralpha expression and how proinflammatory factors may contribute to PDGF-related hyperplasia in vascular diseases.

Fatty acids cause alterations of human arterial smooth muscle cell proteoglycans that increase the affinity for low-density lipoprotein.

OBJECTIVE: The dyslipidemia of insulin resistance, with high levels of albumin-bound fatty acids, is a strong cardiovascular disease risk. Human arterial smooth muscle cell (hASMC) matrix proteoglycans (PGs) contribute to the retention of apoB lipoproteins in the intima, a possible key step in atherogenesis. We investigated the effects of high NEFA levels on the PGs secreted by hASMCs and whether these effects might alter the PG affinity for low-density lipoprotein. METHODS AND RESULTS: hASMC exposed for 72 hours to high concentrations (800 micromol/L) of linoleate (LO) or palmitate upregulated the core protein mRNAs of the major PGs, as measured by quantitative PCR. Insulin (1 nmol/L) and the PPARgamma agonist rosiglitazone (10 micromol/L) blocked these effects. In addition, high LO increased the mRNA levels of enzymes required for glycosaminoglycan (GAG) synthesis. Exposure to NEFA increased the chondroitin sulfate:heparan sulfate ratio and the negative charge of the PGs. Because of these changes, the GAGs secreted by LO-treated cells had a higher affinity for human low-density lipoprotein than GAGs from control cells. Insulin and rosiglitazone inhibited this increase in affinity. CONCLUSIONS: The response of hASMC to NEFA could induce extracellular matrix alterations favoring apoB lipoprotein deposition and atherogenesis.

Characterisation and application of antibodies specific for the long platelet-derived growth factor A and B chains.

The platelet-derived growth factor (PDGF) family comprises important mitogens for mesenchymal cells. The active dimeric form of PDGF consists of four structurally related A, B, C, and D chains. All PDGF-variants bind to PDGF-receptors. The A and B chains occur with and without basic C-terminal amino acid extensions as long (A(L) and B(L)) and short (A(S) and B(S)) isoforms. PDGF-A and -B form homo- or heterodimers. The biological relevance of short and long isoforms is unknown, although it may relate to different affinities for glycosaminoglycans of the cell glycocalix and intercellular matrix. Commercially available anti-PDGF-A and anti-PDGF-B antibodies cannot discriminate between the short and the long isoforms. Thus, to investigate the function of the long and short isoforms, we raised antibodies specific for the long A and B chain isoforms. The antibodies were affinity-purified and their properties analysed by surface plasmon resonance. Inhibition studies with different PDGF homodimers and dot-blot studies proved their high specificity for the respective isoforms. Both antibodies recognised the target PDGF homodimers complexed to the glycocalix of human arterial smooth muscle cells and human monocyte-derived macrophages. By using these specific antibodies, we were able to confirm at the protein level the synthesis of PDGF-A and -B during differentiation of human monocyte-derived macrophages and to demonstrate the presence of the PDGF-A(L) and PDGF-B(L) isoforms in human arterial tissue.

Fatty acids induce increased granulocyte macrophage-colony stimulating factor secretion through protein kinase C-activation in THP-1 macrophages.

Insulin resistance and type 2 diabetes are associated with elevated circulating levels of nonesterified FA (NEFA) and lipoprotein remnants. The dyslipidemia is an important contributor to the excess arterial disease associated with insulin resistance and type 2 diabetes, but the mechanisms involved are elusive. In the present study we examined the effect of NEFA on macrophages. For this purpose, we utilized human macrophages, prepared by treating THP-1 monocytes with phorbol ester. We found that albumin-bound NEFA at physiological levels increase the secretion of granulocyte macrophage-colony stimulating factor (GM-CSF) by the THP-1 macrophages in a dose-dependent manner. The effect was registered as an increase in mRNA, and the amount of GM-CSF secreted correlated with the accumulation of TAG and DAG in the cell. The NEFA-induced rise in GM-CSF appeared to be mediated by activation of protein kinase C, probably acting on extracellular signal-regulated kinases 1 and 2 and being calcium dependent. We speculate that increased secretion of GM-CSF by resident macrophages in the intima exposed chronically to high levels of NEFA, such as those present in insulin resistance, may contribute to a proatherogenic response of arterial cells.