Chemoattractants as causative agents, biomarkers and therapeutic targets in vascular pathology

University dissertation from Stockholm : Karolinska Institutet, Department of Medicine

Abstract: Atherosclerosis and restenosis are driven by chronic immune response in the vascular wall. This involves cell migration into and within the vessel wall, cell proliferation as well as accumulation of lipids in the vessel wall. These processes are mediated, among other substances, by molecules belonging to a large family of chemoattractants. The aim of this thesis was to investigate whether several chemoattractants can serve as causative agents, biomarkers and therapeutic targets in atherosclerosis and restenosis. One of the recently discovered chemoattractants, chemokine CXCL16 exists in soluble and transmembrane forms. It ligates chemokine receptor CXCR6 and guides migration of activated Th1 and Tc1 cells. It is identical to scavenger receptor SR-PSOX, which mediates uptake of oxidized low-density lipoprotein (oxLDL) by cells. We detected abundant CXCL16/SR-PSOX and CXCR6 expression in atherosclerotic lesions from human subjects and apolipoprotein-Edeficient (ApoE-/-) mice. In monocytes, CXCL16/SR-PSOX expression was increased by interferon-? (IFN-?), a proatherogenic cytokine abundant in atherosclerotic lesions. This corresponded to increased uptake of oxLDL by monocytes, which was specifically mediated by CXCL16/SR-PSOX. IFN-? injections to ApoE-/- mice induced CXCL16/SR-PSOX expression in atherosclerotic lesions. These data demonstrate a novel role of IFN-? in cellular lipid accumulation through up-regulation of CXCL16/SR-PSOX and suggest that CXCL16/SR-PSOX may serve as a molecular link between lipid metabolism and immune activity in the atherosclerotic lesion. These results also suggested that patients with atherosclerosis may present with altered levels of CXCL16/SR-PSOX in the systemic circulation. Using ELISA we assessed plasma CXCL16/SR-PSOX concentration in stable angina pectoris (SAP) patients, unstable angina pectoris/non-ST-elevation myocardial infarction (UAP/non-STEMI) patients, survivors of a first myocardial infarction (MI) and healthy control subjects. SAP patients exhibited significantly lower median CXCL16/SR-PSOX levels than control subjects, while UAP/non-STEMI patients and post-MI patients tended to have lower CXCL16/SR-PSOX levels compared to controls. At the same time there were no significant correlations between CXCL16/SR-PSOX levels and measures of coronary artery disease (CAD) severity: angiographically determined degree of coronary artery stenosis, plasma lipoprotein fractions, inflammatory cytokines and C-reactive protein. The finding that lower plasma CXCL16/SR-PSOX concentration is associated with CAD might indicate a potential atheroprotective function of CXCL16/SR-PSOX and suggests that it is worth further investigation as a CAD biomarker. CCL11 (eotaxin) is another chemokine, whose systemic levels and genetic variants might be associated with CAD. However, the relationship between CCL11 genetic variants and plasma concentrations in CAD patients has not been studied. We genotyped post-MI patients and healthy controls for a 67G>A single nucleotide polymorphism (SNP) in the CCL11 gene and related the presence of the former to the plasma CCL11 concentrations, measured by ELISA. There were no differences in CCL1 genotype frequencies between patients and controls. Patient G/G carriers had higher circulating CCL11 levels compared both to G/A and A/A patients and G/G controls. At the same time, CCL11 levels did not differ between patients and controls irrespective of the 67G>A SNP variants they carried. No associations were found between plasma CCL11 levels and biochemical indicators of CAD or the degree of coronary artery stenosis in patients. Taken together, we show that despite certain influence of 67G>A SNP on CCL11 plasma levels, neither of them is useful for distinguishing subjects with and without clinical manifestations of coronary atherosclerosis. Leukotriene B4 (LTB4), a potent chemoattractant derived from arachidonic acid, exerts its action by means of specific receptors BLT1 and BLT2. In this study, BLT1 protein was detected in human atherosclerotic plaques, where it colocalized with macrophages, endothelial cells, and smooth muscle cells (SMC). Human coronary artery SMC exposed to either the LTB4 Or the BLT1 partial agonist U75302 exhibited increase of whole-cell ion currents indicating that these cells express functional BLT1 LTB4 induced BLT1-mediated migration and proliferation of SMC in vitro, while treatment with the BLT1 antagonist BIIL284 inhibited balloon injury-induced carotid artery neointimal hyperplasia in rats. In the latter model, intimal SMC exhibited increased expression of BLT1 compared with medial SMC. This could be prevented by transfection with a dominant-negative form Of I?B kinase-?, indicating that BLT1 expression depends on NF-?B. These results show that LTB4 activates functional BLT1 on vascular SMC affecting their chemotaxis and proliferation, and that inhibition of BLT1 signaling reduces neointimal hyperplasia, suggesting this pathway as a possible target for restenosis therapy. In summary, this thesis suggests the novel roles for such chemoattractants as CXCL16/SR-PSOX and LTB4 in the development of atherosclerosis and restenosis, respectively. Moreover, CXCL16/SR-PSOX deserves further investigation as a CAD biomarker, while CCL11 is unlikely to be validated as such. And finally, therapy directed at LTB4/BLT1 axis may be useful for the treatment of post-angioplasty restenosis.

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