Huixia Lu

Cold Exposure Promotes Atherosclerotic Plaque Growth and Instability via UCP1-Dependent Lipolysis

Summary Molecular mechanisms underlying the cold-associated high cardiovascular risk remain unknown. Here, we show that the cold-triggered food-intake-independent lipolysis significantly increased plasma levels of small low-density lipoprotein (LDL) remnants, leading to accelerated development of atherosclerotic lesions in mice. In two genetic mouse knockout models (apolipoprotein E−/− [ApoE−/−] and LDL receptor−/− [Ldlr−/−] mice), persistent cold exposure stimulated atherosclerotic plaque growth by increasing lipid deposition. Furthermore, marked increase of inflammatory cells and plaque-associated microvessels were detected in the cold-acclimated ApoE−/− and Ldlr−/− mice, leading to plaque instability. Deletion of uncoupling protein 1 (UCP1), a key mitochondrial protein involved in thermogenesis in brown adipose tissue (BAT), in the ApoE−/− strain completely protected mice from the cold-induced atherosclerotic lesions. Cold acclimation markedly reduced plasma levels of adiponectin, and systemic delivery of adiponectin protected ApoE−/− mice from plaque development. These findings provide mechanistic insights on low-temperature-associated cardiovascular risks.

Aortic adventitial angiogenesis and lymphangiogenesis promote intimal inflammation and hyperplasia

INTRODUCTION Adventitial inflammation is known to influence neointimal formation and vascular remodeling. The present study was aimed to clarify the relationship between neointima hyperplasia and adventitial angiogenesis and lymphangiogenesis after balloon-induced aortic endothelial injury. METHODS Seventy male Wistar rats were randomly divided into six interventional groups and one control group. The intimal area/medial area ratio (I/M ratio), the adventitial macrophage index, and the number of adventitial microvessels (Ad-MV) and lymphatic vessels (Ad-LV) in the aorta were measured, and the mRNA expressions of VEGF-A, VEGFR-1, VEGF-C, VEGFR-3, PDGF-B, and PDGFR-beta in the aortic wall were quantified by real-time RT-PCR. RESULTS Compared with the control group, the I/M ratio, macrophage index, Ad-MV, Ad-LV, and the mRNA expressions of VEGF-A, VEGFR-1, VEGF-C, VEGFR-3, PDGF-B, and PDGFR-beta in interventional groups increased significantly after balloon-induced injury. I/M ratio showed significant correlations with Ad-MV and Ad-LV after balloon intervention. Multiple linear regression analysis indicated that Ad-MV and Ad-LV were independent factors of intimal hyperplasia. CONCLUSION Adventitial angiogenesis and lymphangiogenesis are induced by intimal inflammation after balloon injury, and these neogenetic vessels in turn promote intimal inflammation and hyperplasia probably via delivery and activation of inflammatory cells.

Role of Toll-Like Receptor 4 on Lupus Lung Injury and Atherosclerosis in LPS-Challenge ApoE−/− Mice

To investigate the pathologic mechanisms of toll-like receptor 4 (TLR4) in lung injury and atherosclerosis, ApoE−/− or wild-type mice were intraperitoneally administered saline, lipopolysaccharides (LPS), or LPS plus TAK-242 (TLR4 inhibitor), respectively, twice a week for 4 weeks. Serum autoantibody of antinuclear antibody (ANA), anti-double-stranded DNA (anti-dsDNA), and cytokines of interferon-gamma (IFN-γ), tumor necrosis factor (TNF-α), and interleukin-1 (IL-1β) were assessed by ELISA. Hematoxylin and eosin (HE) and Perls stains for lung pathomorphology as well as HE staining for atherosclerosis were employed. TLR4 in macrophages was detected by double immunofluorescent staining. While protein expressions of TLR4, nuclear factor-kappa B p65 (NF-κB p65), and B cell activating factor belonging to the TNF family (BAFF) were examined by immunohistochemistry. We found that serum autoantibody (ANA and anti-dsDNA), cytokines (IFN-γ, TNF-α, IL-1β), lung inflammation, and intima-media thickness in brachiocephalic artery were obviously increased after LPS challenge in both genotypes, but to a lesser extent in wild-type strains. And those alterations were alleviated by coadministration of LPS and TAK-242. Mechanistically, upregulation of TLR4, NF-κb, and BAFF was involved. We concluded that TLR4/NF-κb/BAFF in macrophages might be a possible common autoimmune pathway that caused lung injury and atherosclerosis. TLR4 signal will be a therapeutic target in atherosclerosis and immune-mediated lung injury.

PEDF improves atherosclerotic plaque stability by inhibiting macrophage inflammation response

BACKGROUND Atherosclerosis is a vascular disease with plaque formation and growth. Instable plaque with chronic inflammation is closely related to adverse cardiac outcomes. Pigment epithelium-derived factor (PEDF) is an endogenous multifunctional cytokine that possesses the ability of anti-inflammation. The aim of this study is to detect whether PEDF has protective effect on the stability of atherosclerotic plaque and to explore whether the effect of anti-inflammation involved. METHODS AND RESULTS ApoE-/- mice fed with high fat diet and RAW264.7 cells were used to evaluate anti-inflammatory activities of PEDF both in vivo and in vitro. PEDF overexpression improved atherosclerotic plaque stability in ApoE-/- mice. The expression of inflammatory factors (interleukin-1β [IL-1β], interleukin-6 [IL-6], tumor necrosis factor-α [TNF-α], monocyte chemotactic protein-1 [MCP-1] and matrix metalloproteinase [MMP-9]) was significantly decreased with PEDF overexpression in vivo and in vitro. The anti-inflammation effect of PEDF was attenuated by PPAR-γ specific antagonist GW9662. In addition, PEDF significantly decreased the expression of phosphorylated ERK-MAPK, p38-MAPK and JNK-MAPK. GW9662 partly reversed the PEDF-mediated depression of phosphorylated ERK- and p38-MAPK but has no significant effect on JNK-MAPK. CONCLUSIONS PEDF has protective effect on increasing AS plaque stability through ameliorating macrophage inflammation. PPAR-γ and downstream MAPKs were involved in the mechanism.

Apolipoprotein E-knockout mice show increased titers of serum anti-nuclear and anti-dsDNA antibodies.

Apolipoprotein E-knockout (ApoE(-/-)) mice, atherosclerosis-prone mice, show an autoimmune response, but the pathogenesis is not fully understood. We investigated the pathogenesis in female and male ApoE(-/-) mice. The spleens of all ApoE(-/-) and C57BL/6 (B6) mice were weighed. The serum IgG level and titers of anti-nuclear antibody (ANA) and anti-double-stranded DNA (anti-dsDNA) antibody were assayed by ELISA. Apoptosis of spleen tissue was evaluated by TUNEL. TLR4 level in spleen tissue was tested by immunohistochemistry and Western blot analysis. Levels of MyD88, p38, phosphorylated p38 (pp38), interferon regulatory factor 3 (IRF3) and Bcl-2-associated X protein (Bax) in spleen tissue were detected by Western blot analysis. We also survey the changes of serum autoantibodies, spleen weight, splenocyte apoptosis and the expressions of TLR4, MyD88, pp38, IRF3 and Bax in spleen tissue in male ApoE(-/-) mice after 4weeks of lipopolysaccharide (LPS), Toll-like receptor 4 ligand, administration. ApoE(-/-) mice showed splenomegaly and significantly increased serum level of IgG and titers of ANA and anti-dsDNA antibody as compared with B6 mice. Splenocyte apoptosis and the expression of TLR4, MyD88, pp38, IRF3 and Bax in spleen tissue were significantly lower in ApoE(-/-) than B6 mice. The expression of TLR4, MyD88, IRF3, pp38, and Bax differed by sex in ApoE(-/-) spleen tissue. The down-regulation of TLR4 signal molecules induced by LPS led to decreased expression of Bax and increased serum titers of ANA and anti-dsDNA antibody. Therefore, the TLR4 signal pathway may participate in maintaining the balance of splenocyte apoptosis and autoantibody production in ApoE(-/-) mice.

Apolipoprotein E-knockout mice on high-fat diet show autoimmune injury on kidney and aorta

BACKGROUND Apolipoprotein E-knockout (ApoE(-/-)) mice is a classic model of atherosclerosis. We have found that ApoE(-/-) mice showed splenomegaly, higher titers of serum anti-nuclear antibody (ANA) and anti-dsDNA antibody compared with C57B6/L (B6) mice. However, whether ApoE(-/-) mice show autoimmune injury remains unclear. METHODS AND RESULTS Six females and six males in each group, ApoE(-/)(-), Fas(-/-) and B6 mice, were used in this study. The titers of serum ANA, anti-dsDNA antibody and creatinine and urine protein were measured by ELISA after 4 months of high-fat diet. The spleen weight and the glomerular area were determined. The expressions of IgG, C3 and macrophage in kidney and atherosclerotic plaque were detected by immunostaining followed by morphometric analysis. Similar to the characteristics of Fas(-/-) mice, a model of systemic lupus erythematosus (SLE), ApoE(-/-) mice, especially female, displayed significant increases of spleen weight and glomerular area when compared to B6 mice. Also, elevated titers of serum ANA, anti-dsDNA antibody and creatinine and urine protein. Moreover, the expressions of IgG, C3 and macrophage in glomeruli and aortic plaques were found in ApoE(-/-) mice. In addition, the IgG and C3 expressions in glomeruli and plaques significantly increased (or a trend of increase) in female ApoE(-/-) mice compared with males. CONCLUSIONS Apolipoprotein E-knockout mice on high-fat diet show autoimmune injury on kidney and aorta.

Lymphangiogenesis promotes inflammation and neointimal hyperplasia after adventitia removal in the rat carotid artery

Lymphatic vessels exist in adventitia in the atherosclerotic coronary artery and play an important role in the inflammatory and immune response. After adventitia removal, the carotid wall of rat model showed significantly increased ratio of intimal to medial area (I/M ratio), the number of adventitial lymphatic vessels (Ad-LV) and microvessels (Ad-MV), and macrophage index and expression of VEGF-C, VEGFR-3, PDGF-B and PDGFR-beta. The I/M ratio was significantly correlated with Ad-LV and macrophage index but not Ad-MV. These results suggest that adventitial lymphangiogenesis is stimulated by growth factors released by inflammatory cells in vasculature after adventitia removal, and these neogenetic lymph vessels in turn promote intimal inflammation and hyperplasia, probably via delivery and activation of inflammatory cells.

Targeting blood thrombogenicity precipitates atherothrombotic events in a mouse model of plaque destabilization

Although some features of plaque instability can be observed in genetically modified mouse models, atherothrombosis induction in mice has been attested to be difficult. We sought to test the hypothesis that alterations in blood thrombogenicity might have an essential role in the development of atherothrombosis in ApoE−/− mice. In a mouse model of plaque destabilization established in our laboratory, we targeted blood thrombogenicity by systemically overexpressing murine prothrombin via adenovirus-mediated gene transfer. Systemic overexpression of prothrombin increased blood thrombogenicity, and remarkably, precipitated atherothrombotic events in 70% of the animals. The affected plaques displayed features of culprit lesions as seen in human coronary arteries, including fibrous cap disruption, luminal thrombosis, and plaque hemorrhage. Treatment with aspirin and clopidogrel substantially reduced the incidence of atherothrombosis in this model. Mechanistically, increased inflammation, apoptosis and upregulation of metalloproteinases contributed to the development of plaque destabilization and atherothrombosis. As conclusions, targeting blood thrombogenicity in mice can faithfully reproduce the process of atherothrombosis as occurring in human coronary vessels. Our results suggest that blood-plaque interactions are critical in the development of atherothrombosis in mice, substantiating the argument that changes in blood coagulation status may have a determinant role in the onset of acute coronary syndrome.

Tongxinluo mitigates atherogenesis by regulating angiogenic factors and inhibiting vasa vasorum neovascularization in apolipoprotein E-deficient mice.

Vasa vasorum (VV) neovascularization contributes to atherogenesis and its expansion and distribution is correlated with intraplaque expression of angiogenic factors. The present study investigated the roles of Tongxinluo (TXL), a traditional Chinese medication, on VV proliferation and atherogenesis. In vitro, TXL pre-treatment reversed the tumor necrosis factor-a (TNF-a) induced expression of vascular endothelial growth factor A (VEGF-A) and angiopoietin-1 (ANGPT-1) but not ANGPT-2, leading to increased ratio of ANGPT-1 to ANGPT-2. Consistently, TXL treatment (at a dosage of 0.38, 0.75, 1.5 g/kg/d, respectively) decreased the expression of VEGF-A while increased that of ANGPT-1 in early atherosclerotic lesions of apolipoprotein E deficient (apoE−/−) mice. On aortic ring assay, microvessels sprouting from aortas were significantly inhibited in TXL-treated mice. Moreover, VV neovascularization in plaques was markedly reduced with TXL treatment. Histological and morphological analysis demonstrated that TXL treatment reduced plaque burden, plaque size and changed the plaque composition. These data suggest that TXL inhibits early atherogenesis through regulating angiogenic factor expression and inhibiting VV proliferation in atherosclerotic plaque. Our study shed new light on the anti-atherosclerotic effect of TXL.

Apolipoprotein E deficiency and high‑fat diet cooperate to trigger lipidosis and inflammation in the lung via the toll‑like receptor 4 pathway

Apolipoprotein E deficiency (ApoE−/−) combined with a high-fat Western-type diet (WD) is known to activate the toll-like receptor (TLR4) pathway and promote atherosclerosis. However, to date, the pathogenic effects of these conditions on the lung have not been extensively studied. Therefore, the present study examined the effects of ApoE−/− and a WD on lung injury and investigated the underlying mechanisms. ApoE−/− and wild-type mice were fed a WD or normal chow diet for 4, 12 and 24 weeks. Lung inflammation, lung cholesterol content and cytokines profiles in bronchoalveolar lavage fluid (BALF) were determined. TLR4 and its main downstream molecules were analyzed with western blot analysis. In addition, the role of the TLR4 pathway was further validated using TLR4-targeted gene silencing. The results showed that ApoE−/− mice developed lung lipidosis following 12 weeks of receiving a WD, as evidenced by an increased lung cholesterol content. Moreover, dependent on the time period of receiving the diet, those mice exhibited pulmonary inflammation, which was manifested by initial leukocyte recruitment (at 4 weeks), by increased alveolar septal thickness and mean linear intercept as well as elevated production of inflammation mediators (at 12 weeks), and by granuloma formation (at 24 weeks). The expression levels of TLR4, myeloid differentiation primary response 88 (MyD88) and nuclear factor kappa B were markedly upregulated in ApoE−/− WD mice at week 12. However, these effects were ameliorated by shRNA-mediated knockdown of TLR4. By contrast, ApoE−/− ND or wild-type WD mice exhibited low-grade or no inflammation and mild lipidosis. The levels of TLR4 and MyD88 in those mice showed only minor changes. In conclusion, ApoE deficiency acts synergistically with a WD to trigger lung lipidosis and inflammation at least in part via TLR4 signaling.