©Anders Wetterholm


NAVIGATE HOMEPARTICIPANTSRESEARCHMANAGEMENTPUBLICATIONSPOSITIONS

RESEARCH

From genomics to mechanisms and treatment of CAD
Gene and protein expression in human atherosclerosis

Understanding the regulation and physiological role of proprotein convertase subtilisin/kexin type 9 (PCSK9)
Thyroid hormone (T3) in lipid metabolism

Functional studies on PDGFD in vivo and in vitro
Understanding the role of HIF signaling in atherosclerosis
Vascular inflammation, endothelial dysfunction and insulin resistance
Leukotrienes in AAA formation

 


 

From genomics to mechanisms and treatment of CAD
Recent application of molecular genomics in genome-wide association (GWA) studies has identified an increasing number of genomic regions that are firmly associated with CAD. However, the identity of the culprit genes and the biological mechanisms underlying their associations with CAD are poorly known. We hypothesized that functional characterisation of genes, proteins and pathways implicated by GWA studies will help elucidating mechanisms predisposing to CAD and identify a new generation of targets for prediction, prevention and treatment. In the past two years, a total of 21 novel CAD loci have been identified by us and our international collaborators. For three of these loci, the culprit gene has already been identified by a combination of bioinformatics, expression analysis in human target tissues and locus fine-mapping, and generation of gene-targeted mouse models has started to provide phenotypic characterisation of the novel genes. In parallel, disease-associated pathways are being elucidated in cell systems and target tissues from mouse models. We will also apply targeted intervention, by inhibitory monoclonal antibodies and silencing RNA, in model systems as a further means of determining biological mechanisms. Small molecule inhibitors will be identified for promising target molecules. Furthermore, we have started to examine the clinical utility of genetic variants and plasma proteins encoded by the CAD risk genes as novel biomarkers and as components of novel risk scores in longitudinal cohort studies. Using a different approach, we have initiated whole genome resequencing of large families with distinct forms of monogenic hyperlipidemias, and already identified potentially causal mutations in novel genes related to familial hypercholesterolemia, hypertriglyceridemia and hyperalphalipoproteinemia. An example of an initial, proof-of-concept intervention study conducted in humans is the study of arginase inhibition demonstrating improvement in endothelial function in patients with CAD, particularly in patients with type-2 diabetes (T2D).

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Gene and protein expression in human atherosclerosis
We are currently exploring the transcriptome and proteome of human atherosclerosis. For this purpose, we have built a large tissue biobank, BiKE, with plaque RNA and histological samples combined with detailed clinical data from 600 patients undergoing carotid endarterectomies due to risk of imminent stroke, which constitutes the largest biobank of its kind in the world today. The complete plaque transcriptome has been analysed in 150 patients by using Affymetrix global expression arrays. Our data show enhanced mRNA expression of a set of proinflammatory genes including those for TNF superfamily members, several chemokines, interferon-induced genes, and the enzymatic machinery involved in biosynthesis of leukotrienes and prostaglandins. Furthermore, we have developed in silico tools to evaluate the clinical utility of transcriptome and proteome analyses. Analysis of lipid mediator profiles in patient samples have also been used for correlations with high-resolution ultrasound (measuring lipid content) and gene expression profiles of target cascade enzymes. In addition, due to the availability of DNA from all patients, it has been possible to assess the effect of specific gene polymorphisms on gene expression levels in pathological tissue. This will provide insights into the molecular pathogenesis of atherosclerosis and help formulating novel hypotheses and identify new therapy targets.
In a parallel approach, based on gene expression profiling in multiple vascular biopsies from patients undergoing coronary artery bypass grafting (CABG) for symptomatic CAD and in endothelial cell culture systems, we have identified a core set of candidate endothelial genes, which may be implicated in plaque angiogenesis or leukocyte transmigration activity. We now analyze the biology of these genes, including several known and orphan G-protein coupled receptors (GPCR), and putative transcriptional regulators. Our primary approach is to generate knockouts, and study their phenotypes alone, or in combination with mouse models for atherosclerosis. To date, we have generated knockout mice of three GPCRs and one GPCR-co-receptor. Of these, three (GPR116, Ramp2 and S1P1) display strong spontaneous cardiovascular phenotypes and one so far lacks abnormalities. We are now in the process of breeding these mutations into atherosclerosis-prone backgrounds.

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Understanding the regulation and physiological role of proprotein convertase subtilisin/kexin type 9 (PCSK9)
PCSK9, a protein that reduces the number of low-density lipoprotein (LDL) receptors, constitutes an interesting pharmacological target. Since the physiological role of PCSK9 is unclear, we considered it important to investigate how PCSK9 is regulated in model systems and in man. We have found that estrogen, thyroid hormone and growth hormone all reduce circulating PCSK9, in line with the notion that in man, the cholesterol lowering effects of these hormones are mediated by a reduction of PCSK9. Interestingly, a diet supplemented with polyunsaturated fatty acids (PUFA) reduces PCSK9, thereby explaining why LDL-cholesterol is reduced when our diet is supplemented with PUFA. Our data also suggest that PCSK9 stabilizes plasma LDL-cholesterol during the diurnal phases of the day as well as during fasting. Analysis of circulating PCSK9 levels in four cohorts of healthy, middle-aged Swedish men and women (n=5722) revealed that PCSK9 levels vary over a ~ 50-fold range in healthy subjects and that common and low frequency genetic variants in the PCSK9 gene locus influence the pronounced inter-individual variation in circulating PCSK9 levels.

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Thyroid hormone (T3) in lipid metabolism
Since cholesterol metabolism is under extensive hormonal control, novel aspects of hormonal regulation have been addressed by the Center, with the emphasis placed on thyroid hormone (T3). Results obtained so far show that T3 stimulates bile acid synthesis in man while it lowers PCSK9 (see above). Furthermore, reverse cholesterol transport including biliary cholesterol secretion is stimulated by T3 in rodents. T3 also promotes hepatic cholesterol secretion. Treatment of human subjects with the liver-specific T3 receptor agonist eprotirome drastically reduces LDL cholesterol, triglycerides and Lp(a), both as monotherapy and as add-on to treatment with the cholesterol absorption inhibitor ezetimibe. These data indicate that selective stimulation of the thyroid hormone receptor may be a promising therapeutic avenue.
Vascular immunology of atherosclerosis
Using mouse models of atherosclerosis, we have identified a peptide sequence of the LDL protein, apoB-100, as an immunodominant epitope that drives proatherogenic T-cell responses, cloned the T-cell receptor recognizing this epitope, and made transgenic mice that overexpress this particular T-cell receptor. Ongoing studies are determining the impact of this T-cell response on lipid metabolism and atherosclerosis. Furthermore, we have developed vaccination strategies that trigger atheroprotective rather than proatherogenic immune responses, identified a peptide sequence in apoB that triggers innate immunity, and determined the role in atherosclerosis of innate signal transduction pathways. Recent work has shown that regulatory T-cells modulate lipoprotein metabolism leading to hyperlipidemia when regulatory T-cells are eradicated.
In longitudinal cohort studies, we have found that low levels of IgM antibodies directed against phosphorylcholine (anti-PC) are associated with increased risk of CAD and ischemic stroke. Also, low levels of anti-PC provide independent prognostic information after an acute coronary syndrome event. Anti-PC has anti-inflammatory properties and seems to inhibit the uptake of oxLDL by the macrophage scavenger receptor CD36. Similarly, we have obtained evidence of an involvement of autoantibodies against the native and MDA-modified apoB-100 peptide 210 in early atherosclerosis, plaque formation, vascular remodeling and clinical CAD.

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Functional studies on PDGFD in vivo and in vitro
In 2010, the PDGFD gene was identified as a novel susceptibility gene for CAD by groups in the Theme Center and other members of the Coronary Artery Disease (C4D) Genetics Consortium. To identify the underlying mechanisms for the CAD association, PDGFD-associated pathways are now being subjected to in-depth functional characterization using both in vitro and in vivo models as well as by analyzing microarray expresion data from human atheromatous plaques and vascular tissues derived from the BiKE and ASAP biobanks. Most importantly, a combined Apoe-/- and Pdgfd-/- (double knock-out (KO)) mouse has been generated to assess the role of PDGF-D in atherogenesis, and a rat carotid artery balloon injury model is employed to determine the role of PDGF-D during vascular remodeling after injury. Thirdly, PDGFD transgenic mice are used to explore molecular mechanisms in pulmonary hypertension. A mouse model of inducible pulmonary hypertension is also used to study pericyte-deficient PDGF-B retention motif knockouts.
Human cytomegalovirus as a trigger of the acute coronary syndrome
Having optimised staining protocols to detect CMV proteins in vascular tissue specimens; we found CMV protein expression in 93% of aortic aneurysms and in 58% of atherosclerotic plaques. In atherosclerotic plaques, viral proteins were detected primarily in the shoulder region of the plaques where rupture often occurs. Increased levels of a particular T-cell phenotype, the CD28-null T-cells, in the blood of patients with evolving myocardial infarction (MI) constitutes indirect clinical evidence that CMV reactivation precedes plaque rupture and MI. Interestingly, CD4-positive CD28-negative T-cells only exist in CMV-infected individuals. We showed that these T-cells are mainly directed against CMV and provide high inflammatory activity as they contain perforin and produce high levels of IFN-gamma and TNF-alpha upon stimulation. We are currently testing the hypothesis that defective CMV particles (called dense bodies), produced during CMV infection and carrying mainly CMV RNA and microRNA, mediate the expansion of the CD28-negative T-cells. These particles are anticipated to trigger a unique response in plasmacytoid dendritic cells, which are known to be crucial in the expansion of CD28-negative T-cells.
The endothelin B receptor (EDNRB) is known to trigger smooth muscle cell contraction, and ariginase counteracts this process through production of nitric oxide (NO) that will relax the vessel. For unknown reasons ENDRB expression is upregulated in human atherosclerosis lesions. It has also been demonstrated that increased arginase 2 (Arg2) expression is associated with low NO production, promotes aberrant vessel wall remodelling and neointima formation, and that Arg2 is upregulated in human atherosclerosis lesions. We found that CMV highly upregulates Arg2 and EDNRB expression in vitro, both in endothelial cells and smooth muscle cells. In atherosclerotic plaques, we found that primarily CMV-infected smooth muscle cells exhibited increased EDNRB and Arg 2 expression, which implies that CMV has a potential role in modifying factors relevant to vasospasm.

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Understanding the role of HIF signaling in atherosclerosis
Endothelial hypoxia could be an initiating event in atherosclerosis. To elucidate the role of HIF-1alpha in atherogenesis we have generated a range of mouse models where HIF-1alpha, HIF-2alpha, VEGF-A, or VHL have been incapacitated by tissue-specific gene disruption in myeloid cells, endothelial cells or the myocardium. The HIF genetic models are now being examined for their effect on the development of atherosclerotic lesions in combined mouse models with atherosclerosis-prone mice (e.g. LDL-receptor- and apolipoprotein E-deficient mice). The mouse models are also used in DNA array-supported gene expression profiling experiments to screen for cardiovascular target genes that are dysregulated by HIF-1alpha during both generation and progression of atherosclerotic lesions. The observations made in the mouse models will be validated in the BiKE collection of human atherosclerotic plaques (see above).
Role of a dominant negative regulator of hypoxia-inducible signalling in pulmonary hypertension
We have discovered that alternative splicing of the HIF-3alpha locus generates a product that encodes a potent dominant negative regulator of hypoxia signalling. This negative regulator, designated NEPAS, is involved in negative feedback loop regulation of ET-1 expression, and disruption of its gene in mouse models results in enlargement of the right ventricle and impaired lung remodelling, i.e. a syndrome of pulmonary hypertension. In mechanistic studies, we are now exploring why only a selected network of HIF target genes are subject to negative regulation by NEPAS and wish to identify mechanisms determining recruitment of NEPAS to these specific target promoters. These studies should yield important information on the mode of regulation of genes by HIF and NEPAS during embryonic development and in the immediate postnatal phase (the perturbation of which is leading to pulmonary hypertension, cardiac hypertrophy and heart failure). Observations made in the mouse models will be validated in the BiKE collection of human atherosclerotic plaques (see above).
Understanding the role of eicosanoids in atherosclerosis
Eicosanoids, especially the leukotrienes, have been in the focus of attention as candidate inflammatory mediators in human atherosclerosis. We are using the BiKE collection of specimens obtained by carotid endarterectomy to assess expression profiles of enzymes and receptors in the eicosanoid cascade(s). In the past two years, we have shown that thromboxane synthase expression is increased in human atherosclerotic plaque and increased in lesions of patients with recent symptoms of thrombotic events. Furthermore, we have explored the expression of all 6 human lipoxygenase (LO) genes in human plaques. We found that 15-LO type 2 is the only LO, besides 5-LO, that is significantly upregulated, with higher levels in symptomatic than asymptomatic plaques, suggesting a role for this enzyme in the “healing response” of the vessel wall. We have also established LC-MS platforms for analysis of oxylipins (fully established), sphingolipids (partially established) and nitro fatty acids (under development) that are used to monitor lipid mediator profiles in patient samples, and subjected the data to network analysis in a “systems biology” approach.

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Vascular inflammation, endothelial dysfunction and insulin resistance
Insulin resistance plays a major role in the pathogenesis of T2D and is an important risk factor for cardiovascular disease. Endothelial dysfunction, characterized by reduced bioactivity of NO and increased activity of the vasoconstrictor and pro-inflammatory peptide ET-1, is present in insulin-resistant states and is an important factor promoting the development of cardiovascular complications in patients with insulin resistance. The effect of ET-1 on endothelial function and glucose uptake has been investigated in subjects with insulin resistance, and the molecular mechanism behind the effect of ET-1 on glucose uptake has been further examined in cell culture experiments. Subsequently, clinical studies with ET receptor antagonists have been conducted in patients with endothelial dysfunction, insulin resistance and T2D. Intra-arterial dual ETA/ETB receptor blockade enhanced basal and insulin-stimulated forearm glucose uptake in insulin resistant subjects (n=11). ET-1 directly impaired glucose uptake in skeletal muscle cells via a receptor-dependent mechanism. The effect of exogenous ET-1 on basal forearm glucose uptake was studied in subjects with insulin resistance (n=9) and in cultured human skeletal muscle cells. Intra-arterial ET-1 infusion not only induced vascular dysfunction, but also acutely impaired forearm glucose uptake in individuals with insulin resistance and in skeletal muscle cells from T2D subjects. The mechanism seems to be related to signaling downstream of IRS1 Ser636.
Arginase inhibition: novel therapuetic strategy in atherosclerosis and myocardial ischemia-reperfusion
Arginase expression and activity has been shown to be increased in arteries from patients with CAD as well as in experimental models of myocardial ischemia. Arginase inhibition attenuates myocardial ischemia-reperfusion injury as revealed by reduction in infarct size in experimental animal models. The mechanism behind this effect is related to increased production of NO due to increased availability of the common substrate L-arginine.

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Leukotrienes in AAA formation
Contrary to what one would expect, we have found that the spasmogenic and immune modulating cysteinyl-leukotrienes (cys-LT), rather than LTB4, are implicated in human AAA. The enzyme catalyzing the committed step in cys-LT biosynthesis, i.e., LTC4S, is upregulated both in the vessel wall and thrombus. Furthermore, LTD4 triggers the release of MMP2 from the diseased vessel wall and this response can be counteracted by a CysLT1 antagonist, which is a drug used in the clinical management of asthma, thus suggesting a therapeutic potential of anti-leukotrienes in AAA. Functional studies in vivo are now being conducted in calcium chloride and elastase-induced mouse/rat models.

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