Leukocyte Bim deficiency does not impact atherogenesis in ldlr -/- mice, despite a pronounced induction of autoimmune inflammation

Proapoptotic Bcl-2 family member Bim is particularly relevant for deletion of autoreactive and activated T and B cells, implicating Bim in autoimmunity. As atherosclerosis is a chronic inflammatory process with features of autoimmune disease, we investigated the impact of hematopoietic Bim deficiency on plaque formation and parameters of plaque stability. Bim−/− or wild type bone marrow transplanted ldlr−/− mice were fed a Western type diet (WTD) for 5 or 10 weeks, after which they were immunophenotyped and atherosclerotic lesions were analyzed. Bim−/− transplanted mice displayed splenomegaly and overt lymphocytosis. CD4+ and CD8+ T cells were more activated (increased CD69 and CD71 expression, increased interferon gamma production). B cells were elevated by 147%, with a shift towards the pro-atherogenic IgG-producing B2 cell phenotype, resulting in a doubling of anti-oxLDL IgG1 antibody titers in serum of bim−/− mice. Bim−/− mice displayed massive intraplaque accumulation of Ig complexes and of lesional T cells, although this did not translate in changes in plaque size or stability features (apoptotic cell and macrophage content). The surprising lack in plaque phenotype despite the profound pro-atherogenic immune effects may be attributable to the sharp reduction of serum cholesterol levels in WTD fed bim−/− mice

Modulation of Mouse Coagulation Gene Transcription following Acute In Vivo Delivery of Synthetic Small Interfering RNAs Targeting HNF4α and C/EBPα

Hepatocyte nuclear factor 4α (HNF4α) and CCAAT/enhancer-binding protein α (C/EBPα) are important for the transcriptional control of coagulation factors. To determine in vivo the direct role of HNF4α and C/EBPα in control of genes encoding coagulation factors, a synthetic small interfering (si)RNA approach was used that enabled strong reduction of mouse hepatic HNF4α and C/EBPα under conditions that minimized target-related secondary effects. For both HNF4α and C/EBPα, intravenous injection of specific synthetic siRNAs (siHNF4α and siC/EBPα) resulted in more than 75% reduction in their liver transcript and protein levels 2 days post-injection. For siHNF4α, this coincided with marked and significantly reduced transcript levels of the coagulation genes Hrg, Proz, Serpina5, F11, F12, F13b, Serpinf2, F5, and F9 (in order of magnitude of effect) as compared to levels in control siRNA injected animals. Significant decreases in HNF4α target gene mRNA levels were also observed at 5 days post-siRNA injection, despite a limited level of HNF4α knockdown at this time point. Compared to HNF4α, C/EBPα knockdown had a modest impact on genes encoding coagulation factors. A strong reduction in C/EBPα transcript and protein levels resulted in significantly affected transcript levels of the control genes Pck1 and Fasn and a modest downregulation for coagulation genes Fba, Fbg and F5. F5 and F11 were the sole coagulation genes that were significantly affected upon prolonged (5 day) C/EBPα knockdown. We conclude that in the mouse, HNF4α has a direct and essential regulatory role for multiple hepatic coagulation genes, while a role for C/EBPα is more restricted. In addition, this study demonstrates that synthetic siRNA provides a simple and fast means for determining liver transcription factor involvement in vivo

The immediate and late effects of thyroid hormone (triiodothyronine) on murine coagulation gene transcription

Thyroid dysfunction is associated with changes in coagulation. The aim of our study was to gain more insight into the role of thyroid hormone in coagulation control. C57Black/6J mice received a low-iodine diet and drinking water supplemented with perchlorate to suppress endogenous triiodothyronine (T3) and thyroxine (T4) production. Under these conditions, the impact of exogenous T3 on plasma coagulation, and hepatic and vessel-wall-associated coagulation gene transcription was studied in a short- (4 hours) and long-term (14 days) setting. Comparing euthyroid conditions (normal mice), with hypothyroidism (conditions of a shortage of thyroid hormone) and those with replacement by incremental doses of T3, dosages of 0 and 0.5 μg T3/mouse/day were selected to study the impact of T3 on coagulation gene transcription. Under these conditions, a single injection of T3 injection increased strongly hepatic transcript levels of the well-characterized T3-responsive genes deiodinase type 1 (Dio1) and Spot14 within 4 hours. This coincided with significantly reduced mRNA levels of Fgg, Serpinc1, Proc, Proz, and Serpin10, and the reduction of the latter three persisted upon daily treatment with T3 for 14 days. Prolonged T3 treatment induced a significant down-regulation in factor (F) 2, F9 and F10 transcript levels, while F11 and F12 levels increased. Activity levels in plasma largely paralleled these mRNA changes. Thbd transcript levels in the lung (vessel-wall-associated coagulation) were significantly up-regulated after a single T3 injection, and persisted upon prolonged T3 exposure. Two-week T3 administration also resulted in increased Vwf and Tfpi mRNA levels, whereas Tf levels decreased. These data showed that T3 has specific effects on coagulation, with Fgg, Serpinc1, Proc, Proz, Serpin10 and Thbd responding rapidly, making these likely direct thyroid hormone receptor targets. F2, F9, F10, F11, F12, Vwf, Tf and Tfpi are late responding genes and probably indirectly modulated by T

Atherothrombosis model by silencing of protein C in APOE*3-Leiden.CETP transgenic mice

Murine atherosclerosis models are key for investigation of atherosclerosis pathophysiology and drug development. However, they do not feature spontaneous atherothrombosis as a final stage of atherosclerosis. Transgenic mice expressing both the human mutant apolipoprotein E form APOE*3-Leiden and human cholesteryl ester transfer protein (CETP), i.e. APOE*3-Leiden.CETP mice, feature a moderate hyperlipoproteinemia and atherosclerosis phenotype. In contrast to apolipoprotein E deficient (Apoeˉ(/)ˉ) mice, APOE*3-Leiden.CETP mice respond well to lipid-lowering and anti-atherosclerotic drugs. The aim of the study was to investigate whether silencing of anticoagulant Protein C (Proc) allows APOE*3-Leiden.CETP mice to feature thrombosis as a final stage of atherosclerosis. Female APOE*3-Leiden.CETP mice were fed a Western-type diet to induce advanced atherosclerosis, followed by an injection with a small interfering RNA targeting Proc (siProc). Presence of atherosclerosis and atherothrombosis was determined by histologic analysis of the aortic root. Atherosclerosis severity in the aortic root area of APOE*3-Leiden.CETP mice varied from type “0” (no lesions) to type “V” lesions (advanced and complex lesions). Atherothrombosis following siProc injection was observed for 4 out of 21 APOE*3-Leiden.CETP mice (19% incidence). The atherothrombosis presented as large, organized, fibrin- and leukocyte-rich thrombi on top of advanced (type “V”) atherosclerotic plaques in the aortic root. This atherothrombosis was comparable in appearance and incidence as previously reported for Apoeˉ(/)ˉ mice with a more severe atherosclerosis (19% incidence). APOE*3-Leiden.CETP mice with modest hyperlipidemia and atherosclerosis can develop atherothrombosis upon transient Proc-silencing. This further extends the use of these mice as a test model for lipid-lowering and anti-atherosclerotic drugs

Hepatic transcript levels and plasma levels of procoagulant coagulation factors.

<p>Hepatic transcript levels (A and C) and plasma levels (B) of procoagulant coagulation factors in mice treated with 0 μg T₃ (white bars) or 0.5 μg T₃ (black bars). Panel A shows the T₃-induced changes in hepatic transcript levels 4 hours after a single T₃ injection. Panels B and C show plasma levels and T₃-induced changes in hepatic transcripts for 14 days, respectively. Data are presented as mean with the error bar representing the calculated maximum expression level (panels A and C) or mean±SEM (standard error of the mean) (panel B) of n = 12 mice per group, with the vehicle-treated group set as a reference. Relative expression levels (A and C) were compared using the comparative threshold cycle method with ß-actin as internal control. *p<0.05, **p<0.01, and ***p<0.001 as compared to vehicle-treated mice. Fg-γ: Fibrinogen-γ plasma levels.</p

Predilection of Low Protein C-induced Spontaneous Atherothrombosis for the Right Coronary Sinus in Apolipoprotein E deficient mice

Silencing of anticoagulant protein C using RNA interference (siProc) evokes low incident but spontaneous atherothrombosis in the aortic root of apolipoprotein E–deficient (Apoe−/−) mice. The aims of the current study were (1) to analyze if plaque characteristics or circulating factors could be linked to atherothrombosis susceptibility, (2) to increase the incidence of atherothrombosis by transiently increasing blood pressure, and (3) to direct atherothrombosis to an additional predefined vascular site by applying a semi-constrictive collar around the carotid artery. siProc-driven spontaneous atherothrombosis in the aortic root of Apoe−/− mice was reproduced and occurred at an incidence of 23% (9 out of 39 mice), while the incidence of collar-induced atherothrombosis in the carotid artery was 2.6% (1 out of 39 mice). Treatment with phenylephrine, to transiently increase blood pressure, did not increase atherothrombosis in the aortic root of the Apoe−/− mice nor in the carotid arteries with collars. Plaques in the aortic root with an associated thrombus were lower in collagen and macrophage content, and mice with atherothrombosis had significantly more circulating platelets. Plasma protein C, white blood cell counts, total cholesterol, fibrinogen, serum amyloid A, and IL-6 were not different amongst siProc treated mice with or without thrombosis. Remarkably, our data revealed that thrombus formation preferably occurred on plaques in the right coronary sinus of the aortic root. In conclusion, there is a predilection of low protein C-induced spontaneous atherothrombosis in Apoe−/− mice for the right coronary sinus, a process that is associated with an increase in platelets and plaques lower in collagen and macrophage content

Tumor necrosis factor-alpha promotes atherosclerotic lesion progression in APOE*3-Leiden transgenic mice

Tumor necrosis factor-alpha (TNFalpha) is a pleiotropic cytokine exerting both inflammatory and cell death modulatory activity, and is thought to play a role in the pathogenesis of atherosclerosis. Studies in mice indicated that TNFalpha affects atherosclerosis minimally or not under conditions that allow fatty streak formation. Here, we examined the possible role of TNFalpha in advanced and complex atherosclerotic lesions. To induce atherosclerosis, TNFalpha-deficient (Tnf-/-) APOE*3-Leiden and control APOE*3-Leiden only mice were fed a cholesterol-rich diet. Comparable levels of plasma cholesterol and triglycerides and the systemic inflammatory parameters, serum amyloid A and soluble intercellular adhesion molecule-1 were found in APOE*3-LeidenTnf-/- and control mice. Although absence of TNFalpha did not affect the quantitative area of atherosclerosis, APOE*3-LeidenTnf-/- mice had a higher relative number of early lesions (46.1% vs. 21.4%) and a lower relative number of advanced lesions (53.9% vs. 78.6%, P=0.04). In addition, the advanced lesions in APOE*3-LeidenTnf-/- mice showed less necrosis (9.9+/-12.1% vs. 23.4+/-19.3% of total lesion area, P=0.04) and an increase in apoptosis (1.5+/-1.5% vs. 0.4+/-0.6% of total nuclei, P=0.03). Our data indicate that TNFalpha stimulates the formation of lesions towards an advanced phenotype, with more lesion necrosis and a lower incidence of apoptosi