Vigdis Bjerkeli

Myocardial expression of CC- and CXC-chemokines and their receptors in human end-stage heart failure

OBJECTIVES Chemokines regulate several biological processes, such as chemotaxis, collagen turnover, angiogenesis and apoptosis. Based on the persistent immune activation with elevated circulating levels of chemokines in patients with congestive heart failure (CHF), we have hypothesised a pathogenic role for chemokines in the development of CHF. The objective of this study was to examine mRNA levels and cellular localisation of chemokines and chemokine receptors in human CHF. METHODS We examined explanted hearts from ten patients with end-stage heart failure (all chambers) and in ten organ donors using an RNase protection assays and immunohistochemical techniques. RESULTS Our main findings were: (i) expression of eight chemokine and nine chemokine receptor genes in both failing and nonfailing myocardium, (ii) particularly high mRNA levels of monocyte chemoattractant protein (MCP)-1 and CXC-chemokine receptor 4 (CXCR4), in both chronic failing and nonfailing myocardium, (iii) decreased mRNA levels of MCP-1 and interleukin (IL)-8 in the failing left ventricles compared to failing left atria, (iv) decreased chemokine (e.g., MCP-1 and IL-8) and increased chemokine receptor (e.g., CCR2, CXCR1) mRNA levels in failing left ventricles and failing left atria compared to corresponding chambers in the nonfailing hearts and (v) immunolocalisation of MCP-1, IL-8 and CXCR4 to cardiomyocytes. CONCLUSION The present study demonstrates for the first time chemokine and chemokine receptor gene expression and protein localisation in the human myocardium, introducing a new family of mediators with potentially important effects on the myocardium. The observation of chemokine dysregulation in human end-stage heart failure may represent a previously unknown mechanism involved in progression of chronic heart failure.

Myocardial gene expression of leukaemia inhibitory factor, interleukin-6 and glycoprotein 130 in end-stage human heart failure.

Background Studies in different animal models and plasma analyses in humans suggest that members of the interleukin‐6 (IL‐6) cytokine family may be involved in the pathogenesis of congestive heart failure (CHF). Accordingly, we have examined IL‐6‐related cytokines in chronic CHF in humans by analysing gene and protein expression in myocardium derived from patients with end‐stage heart failure and donor hearts.

Gene expression analysis of peripheral T cells in a subgroup of common variable immunodeficiency shows predominance of CCR7– effector‐memory T cells

Common variable immunodeficiency (CVID) represents a heterogeneous group of antibody deficiency syndromes, characterized by defective antibody production in which T cell deficiency may play a pathogenic role. A subgroup of CVID patients has impaired in vitro T cell proliferation. Using microarray analyses of T cells from these patients, we found a gene expression pattern different from healthy controls and patients with X‐linked agammaglobulinaemia. The profile of the differentially expressed genes suggests enhanced cytotoxic effector functions, antigen experienced or chronically activated T cells and a predominance of CCR7– T cells. Further experiments using flow cytometry revealed a striking predominance of CCR7– T cells in a subgroup of CVID patients, and an association with impaired T cell proliferation. Our observations indicate that a predominance of CCR7– T cells with effector‐memory cell features and with reduced proliferative capacity may characterize a subgroup of CVID.

Raised serum levels of interleukin-18 is associated with disease progression and may contribute to virological treatment failure in HIV-1-infected patients

To gain further insight into the possible role of interleukin (IL)‐18 in HIV‐1 infection we examined serum levels of IL‐18 in various clinical and immunological stages of HIV‐1 infection during cross‐sectional (n = 41) and longitudinal testing (n = 20) and during HAART (n = 21, 24 months follow‐up). Our main findings were that HIV‐1‐infected patients had significantly raised IL‐18 levels comparing healthy controls, particularly in those with advanced disease, that while HAART induced a marked decline in IL‐18, virological treatment failure was associated with persistently raised IL‐18 levels during such therapy and that our in vitro experiments showed an IL‐18‐mediated up‐regulation of the HIV‐1 coreceptor CXCR4 and the pro‐apoptotic mediator TRAIL in PBMC from HIV‐1‐infected patients receiving HAART. HIV‐1 infection appears to be characterized by persistently raised IL‐18 levels and during HAART, such a pattern was associated with virological treatment failure, possibly contributing to immunodeficiency and HIV‐1 replication in these patients.

Effects of interferon-α on gene expression of chemokines and members of the tumour necrosis factor superfamily in HIV-infected patients

We examined the effect of interferon (IFN)‐α on the expression of 375 genes relevant to inflammatory and immunological reactions in peripheral blood mononuclear cells (PBMC) from HIV‐infected patients by cDNA expression array and real‐time quantitative RT‐PCR. Our main findings were: (i) IFN‐α induced up‐regulation of several genes in the tumour necrosis factor (TNF) superfamily including the ligands APRIL, FasL, TNF‐α and TRAIL, with particularly enhancing effects on the latter in HIV‐infected patients. (ii) While IFN‐α markedly up‐regulated the expression of anti‐angionetic ELR– CXC‐chemokines (e.g. MIG and IP‐10), it suppressed the expression of angiogenic ELR+ CXC‐chemokines (e.g. GRO‐α, IL‐8 and ENA‐78), with similar patterns in both patients and controls. (iii) IFN‐α induced a marked increase in gene expression of the HIV co‐receptor CCR5 in both patients and controls. We suggest that these effects may contribute to both the therapeutic and toxic effects of IFN‐α. Moreover, our findings underscore that the biological effects of IFN‐α in HIV infection are complex and that the clinical net effects of IFN‐α treatment may be difficult to predict. However, the potent enhancing effect of IFN‐α on several pro‐apoptotic genes in the TNF superfamily and the enhancing effect on CCR5 expression suggest a possible pathogenic role of IFN‐α in the progression of HIV‐related immunodeficiency and suggests caution in the therapeutic use of IFN‐α in HIV‐infected individuals.

Interleukin 23 Levels Are Increased in Carotid Atherosclerosis Possible Role for the Interleukin 23/Interleukin 17 Axis

Background and Purpose— Interleukin (IL)-23 is a cytokine in the IL-12 family, mainly produced by antigen-presenting cells with a central role in inflammation. We hypothesize that IL-23 is also important in atherogenesis and investigate this in a population with carotid atherosclerosis. Methods— Plasma levels of IL-23 were measured in patients with carotid artery stenosis and in healthy controls. The mRNA levels of IL-23 and its receptor, IL-23R, were measured in atherosclerotic plaques, nonatherosclerotic vessels, peripheral blood mononuclear cells, and plasmacytoid dendritic cells. Results— Our findings were as follows: (1) patients with carotid atherosclerosis (n=177) had significantly raised plasma levels of IL-23 when compared with healthy controls (n=24) with particularly high levels in those with the most recent symptoms. (2) mRNA levels of IL-23 and IL-23R were markedly increased in carotid plaques (n=68) when compared with nonatherosclerotic vessels (n=8–10). Immunostaining showed colocalization to plaque macrophages. (3) Patients with carotid atherosclerosis had increased mRNA levels of both IL-23 and IL-23R in plasmacytoid dendritic cells, but not in peripheral blood mononuclear cells. (4) IL-23 increased IL-17 release in monocytes and particularly in peripheral blood mononuclear cells from patients with carotid atherosclerosis, but not in cells from healthy controls. (5) IL-23 gave a prominent tumor necrosis factor release in monocytes from patients with carotid atherosclerosis but not in cells from healthy controls. (6) High plasma levels of IL-23 were associated with increased mortality during follow-up. Conclusions— We have shown an association between IL-23 and disease progression in patients with carotid atherosclerosis, potentially involving IL-17-related mechanisms.

The Carnitine-butyrobetaine-trimethylamine-N-oxide pathway and its association with cardiovascular mortality in patients with carotid atherosclerosis

BACKGROUND AND PURPOSE γ-butyrobetaine (γBB) is a metabolite from dietary Carnitine, involved in the gut microbiota-dependent conversion from Carnitine to the pro-atherogenic metabolite trimethylamine-N-oxide (TMAO). Orally ingested γBB has a pro-atherogenic effect in experimental studies, but γBB has not been studied in relation to atherosclerosis in humans. The aim of this study was to evaluate associations between serum levels of γBB, TMAO and their common precursors Carnitine and trimethyllysine (TML) and carotid atherosclerosis and adverse outcome. METHODS Serum γBB, Carnitine, TML and TMAO were quantified by high performance liquid chromatography in patients with carotid artery atherosclerosis (n = 264) and healthy controls (n = 62). RESULTS Serum γBB (p = 0.024) and Carnitine (p = 0.001), but not TMAO or TML, were increased in patients with carotid atherosclerosis. Higher levels of γBB and TML, but not TMAO or Carnitine were independently associated with cardiovascular death also after adjustment for age and eGFR (adjusted HR [95%] 3.3 [1.9-9.1], p = 0.047 and 6.0 [1.8-20.34], p = 0.026, respectively). CONCLUSIONS Patients with carotid atherosclerosis had increased serum levels of γBB, and elevated levels of γBB and its precursor TML were associated with cardiovascular mortality. Long-term clinical studies of γBB, as a cardiovascular risk marker, and safety studies regarding dietary supplementation of γBB, are warranted.

Matrix Metalloproteinase 7 Is Associated with Symptomatic Lesions and Adverse Events in Patients with Carotid Atherosclerosis

Background Atherosclerosis is a major cause of cerebrovascular disease. Matrix metalloproteinases (MMPs) play an important role in matrix degradation within the atherosclerotic lesion leading to plaque destabilization and ischemic stroke. We hypothesized that MMP-7 could be involved in this process. Methods Plasma levels of MMP-7 were measured in 182 consecutive patients with moderate (50–69%) or severe (≥70%) internal carotid artery stenosis, and in 23 healthy controls. The mRNA levels of MMP-7 were measured in atherosclerotic carotid plaques with different symptomatology, and based on its localization to macrophages, the in vitro regulation of MMP-7 in primary monocytes was examined. Results Our major findings were (i) Patients with carotid atherosclerosis had markedly increased plasma levels of MMP-7 compared to healthy controls, with particularly high levels in patients with recent symptoms (i.e., within the last 2 months). (ii) A similar pattern was found within carotid plaques with markedly higher mRNA levels of MMP-7 than in non-atherosclerotic vessels. Particularly high protein levels of MMP-7 levels were found in those with the most recent symptoms. (iii) Immunhistochemistry showed that MMP-7 was localized to macrophages, and in vitro studies in primary monocytes showed that the inflammatory cytokine tumor necrosis factor-α in combination with hypoxia and oxidized LDL markedly increased MMP-7 expression. (iv) During the follow-up of patients with carotid atherosclerosis, high plasma levels of MMP-7 were independently associated with total mortality. Conclusion Our findings suggest that MMP-7 could contribute to plaque instability in carotid atherosclerosis, potentially involving macrophage-related mechanisms.

The Antiatherogenic Function of HDL Is Impaired in Hyperhomocysteinemic Subjects

High plasma homocysteine concentrations have been associated with increased risk of cardiovascular disease, whereas plasma HDL concentration is inversely correlated to such disorders. We hypothesized that hyperhomocysteinemic subjects may have dysfunctional HDL. We therefore investigated the ability of serum from hyperhomocysteinemic male and female subjects (n = 10) and control subjects (n = 10) to induce cholesterol efflux and to inhibit release of inflammatory mediators from human umbilical vein endothelial cell. We found that serum from hyperhomocysteinemic subjects had impaired ability to induce cholesterol efflux from lipid-loaded macrophages compared with healthy controls. HDL from those with markedly raised homocysteine concentrations had a reduced antiinflammatory effect in tumor necrosis factor-alpha-activated endothelial cells with an attenuated suppressive effect on interleukin-6 growth-related oncogene-alpha release. Also, the activity of paraoxonase in serum, a multifunctional enzyme with antioxidative effects in relation to the function of HDL, was significantly reduced in hyperhomocysteinemic subjects, in particular those with markedly raised homocysteine concentration. Our findings suggest that hyperhomocysteinemic individuals have dysfunctional HDL particles with attenuated antiatherogenic activity and may represent a novel explanation for the increased risk of cardiovascular events in these individuals.

High Levels of S100A12 Are Associated With Recent Plaque Symptomatology in Patients With Carotid Atherosclerosis

Background and Purpose— Atherosclerosis is a progressive chronic disease, in which inflammation plays a key role. The calcium-binding proteins calgranulins including S100A8, S100A9, and S100A12 are involved in many cellular activities and pathological processes including inflammation. We therefore hypothesized that calgranulins may be markers of plaque instability in patients with carotid atherosclerosis. Methods— Plasma levels of S100A8/A9 and S100A10 were measured in 159 consecutive patients with high-grade carotid stenosis and in 22 healthy control subjects. The mRNA levels of calgranulins were also measured within the atherosclerotic carotid plaques, and their regulation was analyzed in vitro in monocytes. Results— Our main findings were: (1) plasma levels of S100A12 were significantly higher in patients with carotid atherosclerosis compared with healthy control subjects with the highest levels in patients with the most recent symptoms (ie, within 2 months); (2) plasma levels of S100A8/S100A9 showed a modest increase in patients with symptoms in the previous 2 to 6 months but not in the other patients; (3) mRNA levels of S100A8, S100A9, and S100A12 showed increased expression in atherosclerotic carotid plaques from patients with the most recent symptoms compared with the remaining patients; (4) in THP-1 monocytes, activation of Toll-like receptors 2 and 4 increased mRNA levels of S100A8, S100A9, and S10012 and interleukin-1&bgr;, interferon &ggr;, and releasate from thrombin-activated platelets significantly enhanced the expression of S100A12. Conclusions— Our findings support a link between calgranulins and atherogenesis and suggest that these mediators, and in particular S100A12, may be related to plaque instability.