Michael Preusch

Periodontal Disease as a Risk Factor for Ischemic Stroke

Background and Purpose— Chronic infectious diseases may increase the risk of stroke. We investigated whether periodontal disease, including periodontitis and gingivitis, is a risk factor for cerebral ischemia. Methods— We performed a case-control study with 303 patients examined within 7 days after acute ischemic stroke or transient ischemic attack, 300 population controls, and 168 hospital controls with nonvascular and noninflammatory neurological diseases. All subjects received a complete clinical and radiographic dental examination. The individual mean clinical attachment loss measured at 4 sites per tooth served as the main indicator for periodontitis. Results— Patients had higher clinical attachment loss than population (P <0.001) and hospital (P =0.010) controls. After adjustment for age, sex, number of teeth, vascular risk factors and diseases, childhood and adult socioeconomic conditions, and lifestyle factors, the risk of cerebral ischemia increased with more severe periodontitis. Subjects with severe periodontitis (mean clinical attachment loss >6 mm) had a 4.3-times-higher (95% confidence interval, 1.85 to 10.2) risk of cerebral ischemia than subjects with mild or without periodontitis (≤3 mm). Severe periodontitis was a risk factor in men but not women and in younger (<60 years) but not older subjects. Periodontitis increased the risk of cerebral ischemia caused by large-artery atherosclerosis, cardioembolism, and cryptogenic etiology. Gingivitis and severe radiologic bone loss were also independently associated with the risk of cerebral ischemia, whereas caries was not. Conclusions— Our study indicates that periodontal disease, a treatable condition, is an independent risk factor for cerebral ischemia in men and younger subjects.

TGF-β1 Limits Plaque Growth, Stabilizes Plaque Structure, and Prevents Aortic Dilation in Apolipoprotein E-Null Mice

Objective—Impairment of transforming growth factor (TGF)-&bgr;1 signaling accelerates atherosclerosis in experimental mice. However, it is uncertain whether increased TGF-&bgr;1 expression would retard atherosclerosis. The role of TGF-&bgr;1 in aneurysm formation is also controversial. We tested whether overexpression of active TGF-&bgr;1 in hyperlipidemic mice affects atherogenesis and aortic dilation. Methods and Results—We generated apolipoprotein E–null mice with transgenes that allow regulated overexpression of active TGF-&bgr;1 in their hearts. Compared to littermate controls, these mice had elevated cardiac and plasma TGF-&bgr;1, less aortic root atherosclerosis (P≤0.002), fewer lesions in the thoracic and abdominal aortae (P≤0.01), less aortic root dilation (P<0.001), and fewer pseudoaneurysms (P=0.02). Mechanistic studies revealed no effect of TGF-&bgr;1 overexpression on plasma lipids or cytokines, or on peripheral lymphoid organ cells. However, aortae of TGF-&bgr;1–overexpressing mice had fewer T-lymphocytes, more collagen, less lipid, lower expression of inflammatory cytokines and matrix metalloproteinase-13, and higher expression of tissue inhibitor of metalloproteinase-2. Conclusions—When overexpressed in the heart and plasma, TGF-&bgr;1 is an antiatherogenic, vasculoprotective cytokine that limits atherosclerosis and prevents aortic dilation. These actions are associated with significant changes in cellularity, collagen and lipid accumulation, and gene expression in the artery wall.

Melagatran Reduces Advanced Atherosclerotic Lesion Size and May Promote Plaque Stability in Apolipoprotein E– Deficient Mice

Objective—Inflammatory mechanisms are involved in atherosclerotic plaque rupture and subsequent thrombin formation. Thrombin not only plays a central role in thrombus formation and platelet activation, but also in the induction of inflammatory processes. We assessed the hypothesis that melagatran, a direct thrombin inhibitor, attenuates plaque progression and promotes stability of advanced atherosclerotic lesions. Methods and Results—Melagatran (500 &mgr;mol/kg/d) or control diet was administered to apolipoprotein E–deficient mice (n=54) with advanced atherosclerotic lesions. Treatment reduced lesion progression in brachiocephalic arteries (P<0.005). Morphometric analysis confirmed that thrombin inhibition promoted plaque stability and resulted in thicker fibrous caps (28.4±14.2 &mgr;m versus 20.8±12.0 &mgr;m; P<0.05), increased media thickness (29.3±9.6 &mgr;m versus 24.4±6.7 &mgr;m; P<0.05), and smaller necrotic cores (73 537±41301 &mgr;m2 versus 126 819±51730 &mgr;m2; P<0.0005). Electro mobility shift assays revealed reduced binding activity of nuclear factor &kgr;B (P<0.05) and activator protein-1 (P<0.05) in aortas of treated mice. Furthermore, immunohistochemistry demonstrated reduced staining for matrix metalloproteinase (MMP)-9 (P<0.05). Melagatran had no significant effect on early lesion formation in C57BL/6J mice. Conclusions—The direct thrombin inhibitor melagatran reduces lesion size and may promote plaque stability in apolipoprotein E–deficient mice, possibly through reduced activation of proinflammatory transcription factors and reduced synthesis of MMP-9.

Sphingosine-1-Phosphate Analogue FTY720 Causes Lymphocyte Redistribution and Hypercholesterolemia in ApoE-Deficient Mice

Objective—Resident immune cells are a hallmark of atherosclerotic lesions. The sphingolipid analogue drug FTY720 mediates retrafficking of immune cells and inhibits their homing to inflammatory sites. We have evaluated the effect of FTY720 on atherogenesis and lipid metabolism. Methods and Results—ApoE−/− mice on a normal laboratory diet received oral FTY720 for 12 weeks, which led to a 2.4-fold increase in serum cholesterol (largely VLDL fraction) and a 1.8-fold increase in hepatic HMGCoA reductase mRNA. FTY720 increased plasma sphingosine-1-phosphate and induced marked peripheral blood lymphopenia. A discoordinate modulation of B, T and monocyte cell numbers was found in peripheral lymphoid organs. Overall depletion of T cells was accompanied by a relative (2-fold) increase in regulatory T cell content paralleled by a similar increase in effector memory T cells (CD4+CD44hiCD62lo) as absolute numbers of both subpopulations remained essentially unchanged. Lymphocyte function was unaltered as indicated by anti-OxLDL antibodies and T cell proliferation. There were no changes in atherosclerotic lesions in early and established atherosclerosis. Conclusions—FTY720 mediated peripheral lymphocyte depletion and retrafficking without altering function and overall balance of pro- and antiatherogenic lymphocyte populations. A net decrease in lymphocyte numbers occurred concomitantly with a more proatherogenic hypercholesterolemia resulting in unaltered atherogenesis.

Evaluation of plaque stability of advanced atherosclerotic lesions in apo E-deficient mice after treatment with the oral factor Xa inhibitor rivaroxaban.

Aim. Thrombin not only plays a central role in thrombus formation and platelet activation, but also in induction of inflammatory processes. Activated factor X (FXa) is traditionally known as an important player in the coagulation cascade responsible for thrombin generation. We assessed the hypothesis that rivaroxaban, a direct FXa inhibitor, attenuates plaque progression and promotes stability of advanced atherosclerotic lesions in an in vivo model. Methods and Results. Rivaroxaban (1 or 5 mg/kg body weight/day) or standard chow diet was administered for 26 weeks to apolipoprotein E-deficient mice (n = 20 per group) with already established atherosclerotic lesions. There was a nonsignificant reduction of lesion progression in the high-concentration group, compared to control mice. FXa inhibition with 5 mg Rivaroxaban/kg/day resulted in increased thickness of the protective fibrous caps (12.3 ± 3.8 μm versus 10.1 ± 2.7 μm; P < .05), as well as in fewer medial erosions and fewer lateral xanthomas, indicating plaque stabilizing properties. Real time-PCR from thoracic aortas revealed that rivaroxaban (5 mg/kg/day) treatment reduced mRNA expression of inflammatory mediators, such of IL-6, TNF-α, MCP-1, and Egr-1 (P < .05). Conclusions. Chronic administration of rivaroxaban does not affect lesion progression but downregulates expression of inflammatory mediators and promotes lesion stability in apolipoprotein E-deficient mice.

Rhizoma Coptidis inhibits LPS-induced MCP-1/CCL2 production in murine macrophages via an AP-1 and NFkappaB-dependent pathway.

Introduction. The Chinese extract Rhizoma coptidis is well known for its anti-inflammatory, antioxidative, antiviral, and antimicrobial activity. The exact mechanisms of action are not fully understood. Methods. We examined the effect of the extract and its main compound, berberine, on LPS-induced inflammatory activity in a murine macrophage cell line. RAW 264.7 cells were stimulated with LPS and incubated with either Rhizoma coptidis extract or berberine. Activation of AP-1 and NFκB was analyzed in nuclear extracts, secretion of MCP-1/CCL2 was measured in supernatants. Results. Incubation with Rhizoma coptidis and berberine strongly inhibited LPS-induced monocyte chemoattractant protein (MCP)-1 production in RAW cells. Activation of the transcription factors AP-1 and NFκB was inhibited by Rhizoma coptidis in a dose- and time-dependent fashion. Conclusions. Rhizoma coptidis extract inhibits LPS-induced MCP-1/CCL2 production in vitro via an AP-1 and NFκB-dependent pathway. Anti-inflammatory action of the extract is mediated mainly by its alkaloid compound berberine.

Association Between Cerebral Ischemia and Cytotoxin-Associated Gene-A–Bearing Strains of Helicobacter pylori

Background and Purpose— Studies on Helicobacter pylori infection and risk of ischemic stroke yielded variable results. Infection with more virulent H. pylori strains, such as cytotoxin-associated gene-A (CagA)–bearing strains, may be of particular relevance for ischemic diseases. We investigated whether H. pylori and CagA seropositivity are independent risk factors for cerebral ischemia or its etiologic subtypes. Methods— We determined IgG antibodies against H. pylori and CagA protein (enzyme immunoassays) in 190 patients with acute cerebral ischemia and in 229 age- and sex-matched control subjects selected randomly from the general population. Results— CagA seropositivity was more common in patients (114/190; 60.0%) than in control subjects (99/229; 43.2%; odds ratio, 1.97; 95% CI, 1.33 to 2.91; P <0.001). This result remained significant after adjustment for age, sex, vascular risk factors and diseases, and childhood and adult social status (odds ratio, 1.84; 95% CI, 1.13 to 3.00; P =0.015). Subgroup analyses yielded similar results in all etiologic stroke subtypes. In contrast, H. pylori seropositivity in general was not associated with increased risk of stroke or its etiologic subtypes. Conclusions— Our results support the hypothesis of an association between infection with CagA-positive H. pylori strains and acute cerebral ischemia.

Interleukin-10 Suppresses Tissue Factor Expression in Lipopolysaccharide-Stimulated Macrophages via Inhibition of Egr-1 and a Serum Response Element/MEK-ERK1/2 Pathway

Atherosclerosis is considered to be an inflammatory disease. Tissue factor (TF), a prothrombotic molecule expressed by various cell types within atherosclerotic plaques, is thought to play an essential role in thrombus formation after atherosclerotic plaque rupture. Recent studies suggest that the antiinflammatory cytokine interleukin-10 (IL-10) has many antiatherosclerotic properties. Therefore, the effects of IL-10 on TF expression in response to inflammation were investigated. Mouse macrophages were stimulated with lipopolysaccharide (LPS) in the presence or absence of IL-10. Pretreatment with IL-10 resulted in a 50% decrease in TF mRNA expression and TF promoter activity. Binding of early growth response gene-1 (Egr-1) to the consensus DNA sequence, a key transcriptional activator of TF expression in response to inflammation, and the expression of Egr-1 mRNA were also inhibited by IL-10. This inhibition was independent of the induction of suppressor of cytokine signaling protein-3 by IL-10. Macrophages that had been transfected with luciferase reporter constructs containing the murine Egr-1 5′-flanking sequence exhibited reduced reporter gene activity in response to LPS stimulation with IL-10 pretreatment. Studies with deletion constructs of the Egr-1 promoter identified the proximal serum response element SRE3 as a potential regulatory site for the IL-10 mediated suppression of Egr-1 expression. Furthermore, activation of the upstream signal-transduction elements, such as mitogen-activated protein kinase kinase (MEK) 1/2, extracellular signal-regulated kinase 1/2, and Elk-1 were also inhibited by IL-10 pretreatment. Taken together, these results demonstrate a pathway for the IL-10 mediated inhibition of TF expression during inflammation and may explain the antiatherosclerotic effects of IL-10.

Oncostatin M is expressed in atherosclerotic lesions: A role for Oncostatin M in the pathogenesis of atherosclerosis

OBJECTIVE Chronic inflammation plays a pivotal role in the development and progression of atherosclerosis. The inflammatory response is mediated by cytokines. The aim of this study was to determine if Oncostatin M (OSM), a monocyte and T-lymphocyte specific cytokine is present in atherosclerotic lesions. We also investigated the roles of signal transducer and activator of transcription (STAT)-1 and STAT-3 in regulating OSM-induced smooth muscle cell (SMC) proliferation, migration and cellular fibronectin (cFN) synthesis. METHODS AND RESULTS Immunostaining of atherosclerotic lesions from human carotid plaques demonstrated the expression of OSM antigen in both macrophages and SMCs. Explanted SMCs from human carotid plaques expressed OSM mRNA and protein as determined by RT-PCR and Western blotting. Using the chow-fed ApoE(-/-) mouse model of atherosclerosis, we observed that OSM was initially expressed in the intima at 20 weeks of age. By 30 weeks, OSM was expressed in both the intima and media. In vitro studies show that OSM promotes SMC proliferation, migration and cFN synthesis. Lentivirus mediated-inhibition of STAT-1 and STAT-3 prevented OSM-induced SMC proliferation, migration and cellular fibronectin synthesis. CONCLUSIONS These findings demonstrate that OSM is expressed in atherosclerotic lesions and may contribute to the progression of atherosclerosis by promoting SMC proliferation, migration and extracellular matrix protein synthesis through the STAT pathway.

Critical Role of Macrophages in Glucocorticoid Driven Vascular Calcification in a Mouse-Model of Atherosclerosis

Objective—Macrophage-derived products are known to play a crucial role during atherogenesis and vascular calcification. Glucocorticoids (GC) are important modulators of immune cell functions, but their specific effects on macrophages behavior during plaque formation are not defined. The present study was therefore designed to investigate the effects of macrophage-specific deletion of the glucocorticoid receptor (GRLysMCre) on atherogenesis and vascular calcification in a hyperlipidemic mouse-model. Methods and Results—Bone marrow was isolated from GRLysMCre mice and wild-type controls (GRflox) and subsequently transplanted into lethally irradiated LDL-receptor–deficient mice. Animals were fed a Western-type diet for 15 or 24 weeks, and atherosclerotic lesions within the aortic sinus were evaluated. At both time points, no significant difference in serum lipid and corticosterone concentrations, atherosclerotic lesion size and macrophage-content within the lesions could be observed. However, GRLysMCre mice showed less calcification as well as a significant reduction of RANKL, BMP2, and Msx2 expression within the vasculature. In vitro studies using conditioned media from macrophages which had been stimulated with dexamethasone demonstrated a dose-dependent increase in calcium deposition by vascular smooth muscle cells. Conclusion—This study demonstrates that macrophage-specific glucocorticoid receptor inactivation reduces vascular calcification without affecting atherosclerotic lesion size in LDL receptor–deficient mice.