Immune Responses Aginst Aldehyde Modified Extracellular Matrix in Atherosclerosis

Atherosclerosis is a chronic inflammatory disease and the leading cause of myocardial infarction and stroke. Accumulation, aggregation and subsequent oxidation of LDL in the arterial wall are considered as key events in the development of atherosclerosis. The oxidation of LDL generates reactive aldehydes, including malondialdehyde (MDA) that modifies ApoB in LDL. Immune responses against MDA-modified epitopes on ApoB have been shown to be linked to atherosclerotic disease. This thesis is focused on the possibility that LDL oxidation result in the release of MDA that modified surrounding extracellular matrix (ECM) proteins. These modifications may subsequently target immune responses against the plaque ECM and influence the atherosclerotic process. Our studies provide evidence for the presence of MDA-modifications on ECM proteins during atherosclerosis. We also show that antibodies against several MDA-modified ECM proteins are present in human plasma. A prospective clinical study showed that subjects that later suffered from acute cardiovascular events had significantly lower IgG and IgM antibody levels against MDA-modified fibronectin. These epidemiological results indicate that immune responses against MDA-modified fibronectin may have protective effects in atherosclerotic disease. To investigate the functional role of immune responses against modified matrix proteins in atherosclerosis, we immunized Apoe-/- mice with two different MDA-modified matrix proteins to which we had found antibodies in human plasma. MDA-modified fibronectin immunization significantly decreased the atherosclerotic plaque development in both aorta and in subvalvular lesions, while MDA-modified laminin resulted in increased plaque development. Finally we show that injection of Alum, an adjuvant commonly used in vaccines, results in LDL oxidation and aldehyde generation at the injection site

Plasma S100A8/A9 Correlates With Blood Neutrophil Counts, Traditional Risk Factors, and Cardiovascular Disease in Middle-Aged Healthy Individuals.

The S100 alarmins A8, A9, and A8/A9, secreted by activated neutrophils and monocytes/macrophages, are involved in the pathogenesis of various inflammatory diseases. S100A8/A9 has previously been linked to atherogenesis and cardiovascular (CV) disease. We investigated whether S100A8, A9, and A8/A9 correlate with carotid artery disease and CV risk in apparently healthy individuals

Plasma fibronectin deficiency impedes atherosclerosis progression and fibrous cap formation

Atherosclerotic lesions are asymmetric focal thickenings of the intima of arteries that consist of lipids, various cell types and extracellular matrix (ECM). These lesions lead to vascular occlusion representing the most common cause of death in the Western world. The main cause of vascular occlusion is rupture of atheromatous lesions followed by thrombus formation. Fibronectin (FN) is one of the earliest ECM proteins deposited at atherosclerosis-prone sites and was suggested to promote atherosclerotic lesion formation. Here, we report that atherosclerosis-prone apolipoprotein E-null mice lacking hepatocyte-derived plasma FN (pFN) fed with a pro-atherogenic diet display dramatically reduced FN depositions at atherosclerosis-prone areas, which results in significantly smaller and fewer atherosclerotic plaques. However, the atherosclerotic lesions from pFN-deficient mice lacked vascular smooth muscle cells and failed to develop a fibrous cap. Thus, our results demonstrate that while FN worsens the course of atherosclerosis by increasing the atherogenic plaque area, it promotes the formation of the protective fibrous cap, which in humans prevents plaques rupture and vascular occlusion

Increased aldehyde-modification of collagen type IV in symptomatic plaques - A possible cause of endothelial dysfunction.

Subendothelial LDL-adhesion and its subsequent oxidation are considered as key events in the development of atherosclerotic lesions. During oxidation of LDL, reactive aldehydes such as malondialdehyde (MDA) are formed, which modify apolipoprotein B100. However, the possibility that these reactive aldehydes could leak out of the LDL-particle and modify surrounding extracellular matrix proteins has been largely unexplored. We have investigated if aldehyde-modification of collagen type IV, one of the major basement membrane components, in plaques is associated with cardiovascular events

Increased Inflammation in Atherosclerotic Lesions of Diabetic Akita-LDLr

Background. Diabetes is associated with increased cardiovascular disease, but the underlying cellular and molecular mechanisms are poorly understood. One proposed mechanism is that diabetes aggravates atherosclerosis by enhancing plaque inflammation. The Akita mouse has recently been adopted as a relevant model for microvascular complications of diabetes. Here we investigate the development of atherosclerosis and inflammation in vessels of Akita mice on LDLr−/− background. Methods and Results. Akita-LDLr−/− and LDLr−/− mice were fed high-fat diet from 6 to 24 weeks of age. Blood glucose levels were higher in both male and female Akita-LDLr−/− mice (137% and 70%, resp.). Male Akita-LDLr−/− mice had markedly increased plasma cholesterol and triglyceride levels, a three-fold increase in atherosclerosis, and enhanced accumulation of macrophages and T-cells in plaques. In contrast, female Akita-LDLr−/− mice demonstrated a modest 29% increase in plasma cholesterol and no significant increase in triglycerides, atherosclerosis, or inflammatory cells in lesions. Male Akita-LDLr−/− mice had increased levels of plasma IL-1β compared to nondiabetic mice, whereas no such difference was seen between female diabetic and nondiabetic mice. Conclusion. Akita-LDLr−/− mice display considerable gender differences in the development of diabetic atherosclerosis. In addition, the increased atherosclerosis in male Akita-LDLr−/− mice is associated with an increase in inflammatory cells in lesions

A constitutive endogenous osteopontin production is important for macrophage function and differentiation.

Macrophages are involved in the pathological process underlying atherosclerosis and constitutively express the multifunctional protein osteopontin which has important exogenous effects on these cells. However, the effect of the endogenous osteopontin expression on macrophage function has been sparsely studied. To shed light on the importance of the endogenous osteopontin expression, RAW 264.7 macrophage-like cells were silenced in osteopontin expression using RNAi. The cells were analysed for basic functions including attachment, migration, apoptosis and for the expression of macrophage differentiation markers and cytokines. The macrophages with silenced osteopontin expression showed impaired migration and an increased rate of serum starvation-induced apoptosis as compared to osteopontin-producing control cells. Furthermore, the cells with silence osteopontin expression had an altered phenotype with monocyte-like characteristics, including decreased expression of macrophage scavenger receptor A type 1. The altered phenotype of these cells could not be reversed by presence of extracellular osteopontin. In addition the cells with silenced osteopontin expression had a lower expression of IL-12 mRNA and the anti-apoptotic Flip mRNA. We conclude that a constitutive endogenous osteopontin production is important for proper basic functions of macrophages and our study indicates that the constitutive osteopontin production is involved in maintaining macrophages in a differentiated phenotype

The functional impact of G protein-coupled receptor 142 (Gpr142) on pancreatic β-cell in rodent

We have recently shown that the G protein-coupled receptor 142 (GPR142) is expressed in both rodent and human pancreatic β-cells. Herein, we investigated the cellular distribution of GPR142 within islets and the effects of selective agonists of GPR142 on glucose-stimulated insulin secretion (GSIS) in the mouse islets and INS-1832/13 cells. Double-immunostaining revealed that GPR142 immunoreactivity in islets mainly occurs in insulin-positive cells. Potentiation of GSIS by GPR142 activation was accompanied by increased cAMP content in INS-1832/13 cells. PKA/Epac inhibition markedly suppressed the effect of GPR142 activation on insulin release. Gpr142 knockdown (Gpr142-KD) in islets was accompanied by elevated release of MCP-1, IFNγ, and TNFα during culture period and abolished the modulatory effect of GPR142 activation on the GSIS. Gpr142-KD had no effect on Ffar1, Ffar2, or Ffar3 mRNA while reducing Gpr56 and increasing Tlr5 and Tlr7 mRNA expression. Gpr142-KD was associated with an increased expression of Chrebp, Txnip, RhoA, and mitochondrial Vdac1 concomitant with a reduced Pdx1, Pax6, and mitochondrial Vdac2 mRNA levels. Long-term exposure of INS-1832/13 cells to hyperglycemia reduced Gpr142 and Vdac2 while increased Chrebp, Txnip, and Vdac1 mRNA expression. GPR142 agonists or Bt 2 -cAMP counteracted this effect. Glucotoxicity-induced decrease of cell viability in Gpr142-KD INS-1 cells was not affected by GPR142-agonists while Bt 2 -cAMP prevented it. The results show the importance of Gpr142 in the maintenance of pancreatic β-cell function in rodents and that GPR142 agonists potentiate GSIS by an action, which most likely is due to increased cellular generation of second messenger molecule cAMP

Autoantibodies against basement membrane collagen type IV are associated with myocardial infarction

Collagen type IV is the major constituent of basement membranes underlying endothelial cells and is important for endothelial cell attachment and function. Autoantibodies against native collagen type IV have been found in various autoimmune diseases. Oxidation of LDL in the vascular wall results in the formation of reactive aldehydes, which could modify surrounding matrix proteins. Like oxidized LDL, these modified matrix proteins are likely to induce immune responses. We examined whether autoantibodies against native or aldehyde-modified collagen type IV are associated with myocardial infarction. IgM and IgG against native and aldehyde-modified collagen type IV were measured by ELISA in serum from 387 survivors of a first myocardial infarction and 387 age- and sex-matched controls. Post-infarction patients had significantly increased levels of IgM against native collagen type IV, and IgG against native collagen type IV was present at detectable level in 17% of patients as opposed to 7% of controls (p < 0.001). Controlling for major cardiovascular risk factors demonstrated that the presence of IgG against native collagen type IV was associated with myocardial infarction (OR 2.9 (1.6–5.4), p = 0.001). Similarly, subjects in the highest quartile of IgM against native collagen type IV had increased risk of having suffered myocardial infarction (OR 3.11 (1.8–5.4), p < 0.001) after adjusting for cardiovascular risk factors. In contrast, IgG against aldehyde-modified collagen type IV was decreased in myocardial infarction patients, but this association was not independent of established cardiovascular risk factors. Autoantibodies against collagen type IV are associated with myocardial infarction independently of traditional cardiovascular risk factors

Inhibitory effect of UDP-glucose on cAMP generation and insulin secretion

Type-2 diabetes (T2D) is a global disease caused by the inability of pancreatic β-cells to secrete adequate insulin. However, the molecular mechanisms underlying the failure of β-cells to respond to glucose in T2D remains unknown. Here, we investigated the relative contribution of UDP-glucose (UDP-G), a P2Y14-specific agonist, in the regulation of insulin release using human isolated pancreatic islets and INS-1 cells. P2Y14 was expressed in both human and rodent pancreatic β-cells. Dose-dependent activation of P2Y14 by UDP-G suppressed glucose-stimulated insulin secretion (GSIS) and knockdown of P2Y14 abolished the UDP-G effect. 12-h pretreatment of human islets with pertussis-toxin (PTX) improved GSIS and prevented the inhibitory effect of UDP-G on GSIS. UDP-G on GSIS suppression was associated with suppression of cAMP in INS-1 cells. UDP-G decreased the reductive capacity of nondiabetic human islets cultured at 5 mm glucose for 72 h and exacerbated the negative effect of 20 mm glucose on the cell viability during culture period. T2D donor islets displayed a lower reductive capacity when cultured at 5 mm glucose for 72 h that was further decreased in the presence of 20 mm glucose and UDP-G. Presence of a nonmetabolizable cAMP analog during culture period counteracted the effect of glucose and UDP-G. Islet cultures at 20 mm glucose increased apoptosis, which was further amplified when UDP-G was present. UDP-G modulated glucose-induced proliferation of INS-1 cells. The data provide intriguing evidence for P2Y14 and UDP-G's role in the regulation of pancreatic β-cell function