Impaired Acetylcholine-Induced Endothelium-Dependent Aortic Relaxation by Caveolin-1 in Angiotensin II-Infused Apolipoprotein-E (ApoE?/?) Knockout Mice

Objective

The angiotensin II (AngII)-infused apolipoprotein E-deficient (ApoE^−/−) mouse model is widely used to study atherosclerosis and abdominal aortic aneurysm. An increase in blood pressure has been reported in this model however the underlying mechanism has not been fully explored. In this study, we investigated whether vasomotor dysfunction develops in AngII-infused ApoE^−/− mice and the underlying mechanism involved.

Methods

ApoE^−/− mice were infused with vehicle (distilled water) or AngII subcutaneously for 14 days. Blood pressure and heart rate were measured using the non-invasive tail cuff method. Aortic vascular reactivity and expression of key proteins (endothelial nitric oxide synthase (eNOS), phospho-eNOS and caveolin-1) were assessed using tension myography and Western blotting respectively. Plasma nitric oxide (NO) level was estimated using a colorimetric assay.

Results

AngII infusion caused a time-dependent increase in blood pressure (P<0.001). Aortas from AngII-infused mice were significantly less responsive to acetylcholine-induced endothelium-dependent relaxation when compared to aortas from mice infused with vehicle control (P<0.05). Contractile responses to phenylephrine (P<0.01) and potassium chloride (P<0.001) were significantly enhanced in aortas from AngII-infused mice. eNOS phosphorylation was significantly decreased in the aorta of AngII-infused mice (P<0.05). Aortic caveolin-1 protein expression was significantly increased in AngII-infused mice (P<0.05). Plasma nitrate/nitrite level was significantly reduced in AngII-infused mice (P<0.05). Pharmacological disruption of caveolae using methyl-β-cyclodextrin (MβCD) in isolated aortas from AngII-infused mice caused a significant leftward shift of the acetylcholine-induced relaxation concentration-response curve when compared to vehicle control (P<0.05).

Conclusion

Upregulation of caveolin-1 protein expression and reduced NO bioavailability contributes to aortic endothelial dysfunction in AngII-infused ApoE^−/− mice.     

Vitamin k-antagonists accelerate atherosclerotic calcification and induce a vulnerable plaque phenotype

Background

Vitamin K-antagonists (VKA) are treatment of choice and standard care for patients with venous thrombosis and thromboembolic risk. In experimental animal models as well as humans, VKA have been shown to promote medial elastocalcinosis. As vascular calcification is considered an independent risk factor for plaque instability, we here investigated the effect of VKA on coronary calcification in patients and on calcification of atherosclerotic plaques in the ApoE^−/− model of atherosclerosis.

Methodology/Principal Findings

A total of 266 patients (133 VKA users and 133 gender and Framingham Risk Score matched non-VKA users) underwent 64-slice MDCT to assess the degree of coronary artery disease (CAD). VKA-users developed significantly more calcified coronary plaques as compared to non-VKA users. ApoE^−/− mice (10 weeks) received a Western type diet (WTD) for 12 weeks, after which mice were fed a WTD supplemented with vitamin K₁ (VK₁, 1.5 mg/g) or vitamin K₁ and warfarin (VK₁&W; 1.5 mg/g & 3.0 mg/g) for 1 or 4 weeks, after which mice were sacrificed. Warfarin significantly increased frequency and extent of vascular calcification. Also, plaque calcification comprised microcalcification of the intimal layer. Furthermore, warfarin treatment decreased plaque expression of calcification regulatory protein carboxylated matrix Gla-protein, increased apoptosis and, surprisingly outward plaque remodeling, without affecting overall plaque burden.

Conclusions/Significance

VKA use is associated with coronary artery plaque calcification in patients with suspected CAD and causes changes in plaque morphology with features of plaque vulnerability in ApoE^−/− mice. Our findings underscore the need for alternative anticoagulants that do not interfere with the vitamin K cycle.     

Self-Gated CINE MRI for Combined Contrast-Enhanced Imaging and Wall-Stiffness Measurements of Murine Aortic Atherosclerotic Lesions

Background

High-resolution contrast-enhanced imaging of the murine atherosclerotic vessel wall is difficult due to unpredictable flow artifacts, motion of the thin artery wall and problems with flow suppression in the presence of a circulating contrast agent.

Methods and Results

We applied a 2D-FLASH retrospective-gated CINE MRI method at 9.4T to characterize atherosclerotic plaques and vessel wall distensibility in the aortic arch of aged ApoE^−/− mice after injection of a contrast agent. The method enabled detection of contrast enhancement in atherosclerotic plaques in the aortic arch after I.V. injection of micelles and iron oxides resulting in reproducible plaque enhancement. Both contrast agents were taken up in the plaque, which was confirmed by histology. Additionally, the retrospective-gated CINE method provided images of the aortic wall throughout the cardiac cycle, from which the vessel wall distensibility could be calculated. Reduction in plaque size by statin treatment resulted in lower contrast enhancement and reduced wall stiffness.

Conclusions

The retrospective-gated CINE MRI provides a robust and simple way to detect and quantify contrast enhancement in atherosclerotic plaques in the aortic wall of ApoE^−/− mice. From the same scan, plaque-related changes in stiffness of the aortic wall can be determined. In this mouse model, a correlation between vessel wall stiffness and atherosclerotic lesions was found.     

Toll-like receptor-2 mediates diet and/or pathogen associated atherosclerosis: proteomic findings

Background

Accumulating evidence implicates a fundamental link between the immune system and atherosclerosis. Toll-like receptors are principal sensors of the innate immune system. Here we report an assessment of the role of the TLR2 pathway in atherosclerosis associated with a high-fat diet and/or bacteria in ApoE^+/− mice.

Methods and Results

To explore the role of TLR2 in inflammation- and infection-associated atherosclerosis, 10 week-old ApoE^+/−-TLR2^+/+, ApoE^+/−-TLR2^+/− and ApoE^+/−-TLR2^−/− mice were fed either a high fat diet or a regular chow diet. All mice were inoculated intravenously, once per week for 24 consecutive weeks, with 50 µl live Porphyromonas gingivalis (P.g) (10⁷ CFU) or vehicle (normal saline). Animals were euthanized 24 weeks after the first inoculation. ApoE^+/−-TLR2^+/+ mice showed a significant increase in atheromatous lesions in proximal aorta and aortic tree compared to ApoE^+/−-TLR2^+/− and ApoE^+/−-TLR2^−/− mice for all diet conditions. They also displayed profound changes in plaque composition, as evidenced by increased macrophage infiltration and apoptosis, increased lipid content, and decreased smooth muscle cell mass, all reflecting an unstable plaque phenotype. SAA levels from ApoE^+/−-TLR2^+/+ mice were significantly higher than from ApoE^+/−-TLR2^+/− and ApoE^+/−-TLR2^−/− mice. Serum cytokine analysis revealed increased levels of pro-inflammatory cytokines in ApoE^+/−-TLR2^+/+ mice compared to ApoE^+/−-TLR2^+/− and TLR2^−/− mice, irrespective of diet or bacterial challenge. ApoE^+/−-TLR2^+/+ mice injected weekly for 24 weeks with FSL-1 (a TLR2 agonist) also demonstrated significant increases in atherosclerotic lesions, SAA and serum cytokine levels compared to ApoE^+/−-TLR2^−/− mice under same treatment condition. Finally, mass-spectrometry (MALDI-TOF-MS) of aortic samples analyzed by 2-dimentional gel electrophoresis differential display, identified 6 proteins upregulated greater than 2-fold in ApoE^+/−-TLR2^+/+ mice fed the high fat diet and inoculated with P.g compared to any other group.

Conclusion

Genetic deficiency of TLR2 reduces diet- and/or pathogen-associated atherosclerosis in ApoE^+/− mice, along with differences in plaque composition suggesting greater structural stability while TLR-2 ligand-specific activation triggers atherosclerosis. The present data offers new insights into the pathophysiological pathways involved in atherosclerosis and paves the way for new pharmacological interventions aimed at reducing atherosclerosis.     

VSL#3 Resets Insulin Signaling and Protects against NASH and Atherosclerosis in a Model of Genetic Dyslipidemia and Intestinal Inflammation

Background

Signals generated by the inflammed intestine are thought to contribute to metabolic derangement. The intestinal microbiota contributes to instructing the immune system beyond the intestinal wall and its modulation is a potential target for treating systemic disorders.

Aims

To investigate the pathogenetic role of low grade intestinal inflammation in the development of steatohepatitis and atherosclerosis in a model of genetic dyslipidemia and to test the therapeutic potential of a probiotics intervention in protecting against development of these disorders.

Results

ApoE^−/− mice were randomized to receive vehicle or VSL#3, a mixture of eight probiotics, at the dose of 20×10⁹ colony-forming units/kg/day for three months alone or in combination with 0.2% of dextran sulfate sodium (DSS) in drinking water. Administering DSS to ApoE^−/− mice failed to induce signs and symptoms of colitis but increased intestinal permeability to dextran FITC and, while had no effect on serum lipids, increased the blood levels of markers of liver injury and insulin resistance. DSS administration associated with low level inflammation of intestinal and mesenteric adipose tissues, caused liver histopathology features of steatohepatitis and severe atherosclerotic lesions in the aorta. These changes were prevented by VSL#3 intervention. Specifically, VSL#3 reversed insulin resistance, prevented development of histologic features of mesenteric adipose tissue inflammation, steatohepatitis and reduced the extent of aortic plaques. Conditioned media obtained from cultured probiotics caused the direct transactivation of peroxisome proliferator-activated receptor-γ, Farnesoid-X-receptors and vitamin D receptor.

Conclusions

Low grade intestinal inflammation drives a transition from steatosis to steatohepatitis and worsens the severity of atherosclerosis in a genetic model of dyslipidemia. VSL#3 intervention modulates the expression of nuclear receptors, corrects for insulin resistance in liver and adipose tissues and protects against development of steatohepatitis and atherosclerosis.     

Efficacy and Mechanism of Angiotensin II Receptor Blocker Treatment in Experimental Abdominal Aortic Aneurysms

Background

Despite the importance of the renin-angiotensin (Ang) system in abdominal aortic aneurysm (AAA) pathogenesis, strategies targeting this system to prevent clinical aneurysm progression remain controversial and unproven. We compared the relative efficacy of two Ang II type 1 receptor blockers, telmisartan and irbesartan, in limiting experimental AAAs in distinct mouse models of aneurysm disease.

Methodology/Principal Findings

AAAs were induced using either 1) Ang II subcutaneous infusion (1000 ng/kg/min) for 28 days in male ApoE^−/− mice, or 2) transient intra-aortic porcine pancreatic elastase infusion in male C57BL/6 mice. One week prior to AAA creation, mice started to daily receive irbesartan (50 mg/kg), telmisartan (10 mg/kg), fluvastatin (40 mg/kg), bosentan (100 mg/kg), doxycycline (100 mg/kg) or vehicle alone. Efficacy was determined via serial in vivo aortic diameter measurements, histopathology and gene expression analysis at sacrifice. Aortic aneurysms developed in 67% of Ang II-infused ApoE^−/− mice fed with standard chow and water alone (n = 15), and 40% died of rupture. Strikingly, no telmisartan-treated mouse developed an AAA (n = 14). Both telmisartan and irbesartan limited aneurysm enlargement, medial elastolysis, smooth muscle attenuation, macrophage infiltration, adventitial neocapillary formation, and the expression of proteinases and proinflammatory mediators. Doxycycline, fluvastatin and bosentan did not influence aneurysm progression. Telmisartan was also highly effective in intra-aortic porcine pancreatic elastase infusion-induced AAAs, a second AAA model that did not require exogenous Ang II infusion.

Conclusion/Significance

Telmisartan suppresses experimental aneurysms in a model-independent manner and may prove valuable in limiting clinical disease progression.     

BAFF Receptor mAb Treatment Ameliorates Development and Progression of Atherosclerosis in Hyperlipidemic ApoE?/? Mice

Aims

Option to attenuate atherosclerosis by depleting B2 cells is currently limited to anti-CD20 antibodies which deplete all B-cell subtypes. In the present study we evaluated the capacity of a monoclonal antibody to B cell activating factor-receptor (BAFFR) to selectively deplete atherogenic B2 cells to prevent both development and progression of atherosclerosis in the ApoE^−/− mouse.

Methods and Results

To determine whether the BAFFR antibody prevents atherosclerosis development, we treated ApoE^−/− mice with the antibody while feeding them a high fat diet (HFD) for 8 weeks. Mature CD93⁻ CD19⁺ B2 cells were reduced by treatment, spleen B-cell zones disrupted and spleen CD20 mRNA expression decreased while B1a cells and non-B cells were spared. Atherosclerosis was ameliorated in the hyperlipidemic mice and CD19⁺ B cells, CD4⁺ and CD8⁺ T cells were reduced in atherosclerotic lesions. Expressions of proinflammatory cytokines, IL1β, TNFα, and IFNγ in the lesions were also reduced, while MCP1, MIF and VCAM-1 expressions were unaffected. Plasma immunoglobulins were reduced, but MDA-oxLDL specific antibodies were unaffected. To determine whether anti-BAFFR antibody ameliorates progression of atherosclerosis, we first fed ApoE^−/− mice a HFD for 6 weeks, and then instigated anti-BAFFR antibody treatment for a further 6 week-HFD. CD93⁻ CD19⁺ B2 cells were selectively decreased and atherosclerotic lesions were reduced by this treatment.

Conclusion

Anti-BAFFR monoclonal antibody selectively depletes mature B2 cells while sparing B1a cells, disrupts spleen B-cell zones and ameliorates atherosclerosis development and progression in hyperlipidemic ApoE^−/− mice. Our findings have potential for clinical translation to manage atherosclerosis-based cardiovascular diseases.     

The Bile Acid Sensor FXR Is Required for Immune-Regulatory Activities of TLR-9 in Intestinal Inflammation

Background

Toll like receptors (TLRs) sense the intestinal microbiota and regulate the innate immune response. A dysregulation of TLRs function participates into intestinal inflammation. Farnesoid X Receptor (FXR) is a nuclear receptor and bile acid sensor highly expressed in entero-hepatic tissues. FXR regulates lipid metabolism and innate immunity.

Methodology/Principal Findings

In this study we have investigated whether FXR gene expression/function in the intestine is modulated by TLRs. We found that in human monocytes activation of membrane TLRs (i.e. TLR2, 4, 5 and 6) downregulates, while activation of intracellular TLRs (i.e. TLR3, 7, 8 and 9) upregulates the expression of FXR and its target gene SHP, small heterodimer partner. This effect was TLR9-dependent and TNFα independent. Intestinal inflammation induced in mice by TNBS downregulates the intestinal expression of FXR in a TLR9-dependent manner. Protection against TNBS colitis by CpG, a TLR-9 ligand, was lost in FXR^−/− mice. In contrast, activation of FXR rescued TLR9^−/− and MyD88^−/− mice from colitis. A putative IRF7 response element was detected in the FXR promoter and its functional characterization revealed that IRF7 is recruited on the FXR promoter under TLR9 stimulation.

Conclusions/Significance

Intestinal expression of FXR is selectively modulated by TLR9. In addition to its role in regulating type-I interferons and innate antiviral immunity, IRF-7 a TLR9-dependent factor, regulates the expression of FXR, linking microbiota-sensing receptors to host''s immune and metabolic signaling.     

Pharmacological Activation of the Bile Acid Nuclear Farnesoid X Receptor Is Feasible in Patients with Quiescent Crohn's Colitis

Background

The bile acid-activated nuclear receptor Farnesoid X Receptor (FXR) is critical in maintaining intestinal barrier integrity and preventing bacterial overgrowth. Patients with Crohn''s colitis (CC) exhibit reduced ileal FXR target gene expression. FXR agonists have been shown to ameliorate inflammation in murine colitis models. We here explore the feasibility of pharmacological FXR activation in CC.

Methods

Nine patients with quiescent CC and 12 disease controls were treated with the FXR ligand chenodeoxycholic acid (CDCA; 15 mg/kg/day) for 8 days. Ileal FXR activation was assessed in the fasting state during 6 hrs after the first CDCA dose and on day 8, by quantification of serum levels of fibroblast growth factor (FGF) 19. Since FGF19 induces gallbladder (GB) refilling in murine models, we also determined concurrent GB volumes by ultrasound. On day 8 ileal and cecal biopsies were obtained and FXR target gene expression was determined.

Results

At baseline, FGF19 levels were not different between CC and disease controls. After the first CDCA dose, there were progressive increases of FGF19 levels and GB volumes during the next 6 hours in CC patients and disease controls (FGF19: 576 resp. 537% of basal; GB volumes: 190 resp. 178% of basal) without differences between both groups, and a further increase at day 8. In comparison with a separate untreated control group, CDCA affected FXR target gene expression in both CC and disease controls, without differences between both groups.

Conclusions

Pharmacological activation of FXR is feasible in patients with CC. These data provide a rationale to explore the anti-inflammatory properties of pharmacological activation of FXR in these patients.

Trial Registration

TrialRegister.nl NTR2009     

Decoding the Anti-Trypanosoma cruzi Action of HIV Peptidase Inhibitors Using Epimastigotes as a Model

Background

Aspartic peptidase inhibitors have shown antimicrobial action against distinct microorganisms. Due to an increase in the occurrence of Chagas'' disease/AIDS co-infection, we decided to explore the effects of HIV aspartic peptidase inhibitors (HIV-PIs) on Trypanosoma cruzi, the etiologic agent of Chagas'' disease.

Methodology and Principal Findings

HIV-PIs presented an anti-proliferative action on epimastigotes of T. cruzi clone Dm28c, with IC₅₀ values ranging from 0.6 to 14 µM. The most effective inhibitors, ritonavir, lopinavir and nelfinavir, also had an anti-proliferative effect against different phylogenetic T. cruzi strains. The HIV-PIs induced some morphological alterations in clone Dm28c epimastigotes, as reduced cell size and swollen of the cellular body. Transmission electron microscopy revealed that the flagellar membrane, mitochondrion and reservosomes are the main targets of HIV-PIs in T. cruzi epimastigotes. Curiously, an increase in the epimastigote-into-trypomastigote differentiation process of clone Dm28c was observed, with many of these parasites presenting morphological alterations including the detachment of flagellum from the cell body. The pre-treatment with the most effective HIV-PIs drastically reduced the interaction process between epimastigotes and the invertebrate vector Rhodnius prolixus. It was also noted that HIV-PIs induced an increase in the expression of gp63-like and calpain-related molecules, and decreased the cruzipain expression in epimastigotes as judged by flow cytometry and immunoblotting assays. The hydrolysis of a cathepsin D fluorogenic substrate was inhibited by all HIV-PIs in a dose-dependent manner, showing that the aspartic peptidase could be a possible target to these drugs. Additionally, we verified that ritonavir, lopinavir and nelfinavir reduced drastically the viability of clone Dm28c trypomastigotes, causing many morphological damages.

Conclusions and Significance

The results contribute to understand the possible role of aspartic peptidases in T. cruzi physiology, adding new in vitro insights into the possibility of exploiting the use of HIV-PIs in the clinically relevant forms of the parasite.     

Genetic deletion or TWEAK blocking antibody administration reduce atherosclerosis and enhance plaque stability in mice

Clinical complications associated with atherosclerotic plaques arise from luminal obstruction due to plaque growth or destabilization leading to rupture. Tumour necrosis factor ligand superfamily member 12 (TNFSF12) also known as TNF-related weak inducer of apoptosis (TWEAK) is a proinflammatory cytokine that participates in atherosclerotic plaque development, but its role in plaque stability remains unclear. Using two different approaches, genetic deletion of TNFSF12 and treatment with a TWEAK blocking mAb in atherosclerosis-prone mice, we have analysed the effect of TWEAK inhibition on atherosclerotic plaques progression and stability. Mice lacking both TNFSF12 and Apolipoprotein E (TNFSF12^−/−ApoE^−/−) exhibited a diminished atherosclerotic burden and lesion size in their aorta. Advanced atherosclerotic plaques of TNFSF12^−/−ApoE^−/− or anti-TWEAK treated mice exhibited an increase collagen/lipid and vascular smooth muscle cell/macrophage ratios compared with TNFSF12^+/+ApoE^−/− control mice, reflecting a more stable plaque phenotype. These changes are related with two different mechanisms, reduction of the inflammatory response (chemokines expression and secretion and nuclear factor kappa B activation) and decrease of metalloproteinase activity in atherosclerotic plaques of TNFSF12^−/−ApoE^−/−. A similar phenotype was observed with anti-TWEAK mAb treatment in TNFSF12^+/+ApoE^−/− mice. Brachiocephalic arteries were also examined since they exhibit additional features akin to human atherosclerotic plaques associated with instability and rupture. Features of greater plaque stability including augmented collagen/lipid ratio, reduced macrophage content, and less presence of lateral xanthomas, buried caps, medial erosion, intraplaque haemorrhage and calcium content were present in TNFSF12^−/−ApoE^−/− or anti-TWEAK treatment in TNFSF12^+/+ApoE^−/− mice. Overall, our data indicate that anti-TWEAK treatment has the capacity to diminish proinflamatory response associated with atherosclerotic plaque progression and to alter plaque morphology towards a stable phenotype.     

Activation of farnesoid X receptor prevents atherosclerotic lesion formation in LDLR−/− and apoE−/− mice

The role of farnesoid X receptor (FXR) in the development of atherosclerosis has been unclear. Here, LDL receptor (LDLR^−/−) or apolipoprotein E (apoE^−/−) female or male mice were fed a Western diet and treated with a potent synthetic FXR agonist, WAY-362450. Activation of FXR blocked diet-induced hypertriglyceridemia and elevations of non-HDL cholesterol and produced a near complete inhibition of aortic lesion formation. WAY-362450 also induced small heterodimer partner (SHP) expression and repressed cholesterol 7α-hydroxylase (CYP7A1) and sterol 12 α-hydroxylase (CYP8B1) expression. To determine if SHP was essential for these protective activities, LDLR^−/−SHP^−/− and apoE^−/−SHP^−/− mice were similarly treated with WAY-362450. Surprisingly, a notable sex difference was observed in these mice. In male LDLR^−/−SHP^−/− or apoE^−/−SHP^−/− mice, WAY-362450 still repressed CYP7A1 and CYP8B1 expression by 10-fold and still strongly reduced non-HDL cholesterol levels and aortic lesion area. In contrast, in the female LDLR^−/−SHP^−/− or apoE^−/−SHP^−/− mice, WAY-362450 only slightly repressed CYP7A1 and CYP8B1 expression and did not reduce non-HDL cholesterol or aortic lesion size. WAY-362450 inhibition of hypertriglyceridemia remained intact in LDLR^−/− or apoE^−/− mice lacking SHP of both sexes. These results suggest that activation of FXR protects against atherosclerosis in the mouse, and this protective effect correlates with repression of bile acid synthetic genes, with mechanistic differences between male and female mice.     

Vascular Cellular Adhesion Molecule-1 (VCAM-1) Expression in Mice Retinal Vessels Is Affected by Both Hyperglycemia and Hyperlipidemia

Background

Inflammation has been proposed to be important in the pathogenesis of diabetic retinopathy. An early feature of inflammation is the release of cytokines leading to increased expression of endothelial activation markers such as vascular cellular adhesion molecule-1 (VCAM-1). Here we investigated the impact of diabetes and dyslipidemia on VCAM-1 expression in mouse retinal vessels, as well as the potential role of tumor necrosis factor-α (TNFα).

Methodology/Principal Findings

Expression of VCAM-1 was examined by confocal immunofluorescence microscopy in vessels of wild type (wt), hyperlipidemic (ApoE^−/−) and TNFα deficient (TNFα^−/−, ApoE^−/−/TNFα^−/−) mice. Eight weeks of streptozotocin-induced diabetes resulted in increased VCAM-1 in wt mice, predominantly in small vessels (<10 µm). Diabetic wt mice had higher total retinal TNFα, IL-6 and IL-1β mRNA than controls; as well as higher soluble VCAM-1 (sVCAM-1) in plasma. Lack of TNFα increased higher basal VCAM-1 protein and sVCAM-1, but failed to up-regulate IL-6 and IL-1β mRNA and VCAM-1 protein in response to diabetes. Basal VCAM-1 expression was higher in ApoE^−/− than in wt mice and both VCAM-1 mRNA and protein levels were further increased by high fat diet. These changes correlated to plasma cholesterol, LDL- and HDL-cholesterol, but not to triglycerides levels. Diabetes, despite further increasing plasma cholesterol in ApoE^−/− mice, had no effects on VCAM-1 protein expression or on sVCAM-1. However, it increased ICAM-1 mRNA expression in retinal vessels, which correlated to plasma triglycerides.

Conclusions/Significance

Hyperglycemia triggers an inflammatory response in the retina of normolipidemic mice and up-regulation of VCAM-1 in retinal vessels. Hypercholesterolemia effectively promotes VCAM-1 expression without evident stimulation of inflammation. Diabetes-induced endothelial activation in ApoE^−/− mice seems driven by elevated plasma triglycerides but not by cholesterol. Results also suggest a complex role for TNFα in the regulation of VCAM-1 expression, being protective under basal conditions but pro-inflammatory in response to diabetes.     

Dual Activation of the Bile Acid Nuclear Receptor FXR and G-Protein-Coupled Receptor TGR5 Protects Mice against Atherosclerosis

Bile acid signaling is a critical regulator of glucose and energy metabolism, mainly through the nuclear receptor FXR and the G protein-coupled receptor TGR. The purpose of the present study was to investigate whether dual activation of FXR and TGR5 plays a significant role in the prevention of atherosclerosis progression. To evaluate the effects of bile acid signaling in atherogenesis, ApoE^−/− mice and LDLR^−/− mice were treated with an FXR/TGR5 dual agonist (INT-767). INT-767 treatment drastically reduced serum cholesterol levels. INT-767 treatment significantly reduced atherosclerotic plaque formation in both ApoE^−/− and LDLR^−/− mice. INT-767 decreased the expression of pro-inflammatory cytokines and chemokines in the aortas of ApoE^−/− mice through the inactivation of NF-κB. In addition, J774 macrophages treated with INT-767 had significantly lower levels of active NF-κB, resulting in cytokine production in response to LPS through a PKA dependent mechanism. This study demonstrates that concurrent activation of FXR and TGR5 attenuates atherosclerosis by reducing both circulating lipids and inflammation.     

OX40-OX40L Interaction Promotes Proliferation and Activation of Lymphocytes via NFATc1 in ApoE-Deficient Mice

Background

Our previous studies have shown that OX40-OX40L interaction regulates the expression of nuclear factor of activated T cells c1(NFATc1) in ApoE^−/− mice during atherogenesis. The aim of this study was to investigate whether OX40-OX40L interaction promotes Th cell activation via NFATc1 in ApoE^−/− mice.

Methods and Results

The lymphocytes isolated from spleen of ApoE^−/− mice were cultured with anti-CD3 mAb in the presence or absence of anti-OX40 or anti-OX40L antibodies. The expression of NFATc1 mRNA and protein in isolated lymphocytes were measured by real time PCR (RT-PCR) and flow cytometry (FCM), respectively. The proliferation of lymphocytes was analyzed by MTT method,and the expression of IL-2, IL-4 and IFN-γ in the cultured cells and supernatant were measured by RT-PCR and enzyme-linked immunosorbent assary (ELISA), respectively. After stimulating OX40-OX40L signal pathway, the expression of NFATc1 and the proliferation of leukocytes were significantly increased. Anti-OX40L suppressed the expression of NFATc1 in lymphocytes of ApoE−/− mice. Anti-OX40L or the NFATc1 inhibitor (CsA) markedly suppressed the cell proliferation induced by anti-OX40. Moreover, the expression of IL-2 and IFN-γ was increased in lymphocytes induced by OX40-OX40L interaction. Blocking OX40-OX40L interaction or NFATc1 down-regulated the expression of IL-2 and IFN-γ, but didn’t alter the expression of IL-4 in supernatants.

Conclusion

These results suggest that OX40-OX40L interaction promotes the proliferation and activation of lymphocytes through NFATc1.     

Ablation of TSC2 enhances insulin secretion by increasing the number of mitochondria through activation of mTORC1

Aim

We previously found that chronic tuberous sclerosis protein 2 (TSC2) deletion induces activation of mammalian target of rapamycin Complex 1 (mTORC1) and leads to hypertrophy of pancreatic beta cells from pancreatic beta cell-specific TSC2 knockout (βTSC2^−/−) mice. The present study examines the effects of TSC2 ablation on insulin secretion from pancreatic beta cells.

Methods

Isolated islets from βTSC2^−/− mice and TSC2 knockdown insulin 1 (INS-1) insulinoma cells treated with small interfering ribonucleic acid were used to investigate insulin secretion, ATP content and the expression of mitochondrial genes.

Results

Activation of mTORC1 increased mitochondrial DNA expression, mitochondrial density and ATP production in pancreatic beta cells of βTSC2^−/− mice. In TSC2 knockdown INS-1 cells, mitochondrial DNA expression, mitochondrial density and ATP production were increased compared with those in control INS-1 cells, consistent with the phenotype of βTSC2^−/− mice. TSC2 knockdown INS-1 cells also exhibited augmented insulin secretory response to glucose. Rapamycin inhibited mitochondrial DNA expression and ATP production as well as insulin secretion in response to glucose. Thus, βTSC2^−/− mice exhibit hyperinsulinemia due to an increase in the number of mitochondria as well as enlargement of individual beta cells via activation of mTORC1.

Conclusion

Activation of mTORC1 by TSC2 ablation increases mitochondrial biogenesis and enhances insulin secretion from pancreatic beta cells.     

Glucose-dependent insulinotropic polypeptide prevents the progression of macrophage-driven atherosclerosis in diabetic apolipoprotein E-null mice

Aim

We recently reported that glucose-dependent insulinotropic polypeptide (GIP) prevents the development of atherosclerosis in apolipoprotein E-null (Apoe ^−/−) mice. GIP receptors (GIPRs) are found to be severely down-regulated in diabetic animals. We examined whether GIP can exert anti-atherogenic effects in diabetes.

Methods

Nondiabetic Apoe ^−/− mice, streptozotocin-induced diabetic Apoe ^−/− mice, and db/db mice were administered GIP (25 nmol/kg/day) or saline (vehicle) through osmotic mini-pumps for 4 weeks. The animals were assessed for aortic atherosclerosis and for oxidized low-density lipoprotein-induced foam cell formation in exudate peritoneal macrophages.

Results

Diabetic Apoe ^−/− mice of 21 weeks of age exhibited more advanced atherosclerosis than nondiabetic Apoe ^−/− mice of the same age. GIP infusion in diabetic Apoe ^−/− mice increased plasma total GIP levels by 4-fold without improving plasma insulin, glucose, or lipid profiles. GIP infusion significantly suppressed macrophage-driven atherosclerotic lesions, but this effect was abolished by co-infusions with [Pro³]GIP, a GIPR antagonist. Foam cell formation was stimulated by 3-fold in diabetic Apoe ^−/− mice compared with their nondiabetic counterparts, but this effect was halved by GIP infusion. GIP infusion also attenuated the foam cell formation in db/db mice. In vitro treatment with GIP (1 nM) reduced foam cell formation by 15% in macrophages from diabetic Apoe ^−/− mice, and this attenuating effect was weaker than that attained by the same treatment of macrophages from nondiabetic counterparts (35%). While GIPR expression was reduced by only about a half in macrophages from diabetic mice, it was reduced much more dramatically in pancreatic islets from the same animals. Incubation with high glucose (500 mg/dl) for 9–10 days markedly reduced GIPR expression in pancreatic islet cells, but not in macrophages.

Conclusions

Long-term infusion of GIP conferred significant anti-atherogenic effects in diabetic mice even though the GIPR expression in macrophages was mildly down-regulated in the diabetic state.