Abstract-The transition from stable to rupture-prone and ruptured atherosclerotic plaques involves many processes, including an altered balance between inflammation and fibrosis. An important mediator of both is transforming growth factor (TGF)-␤, and a pivotal role for TGF-␤ in atherogenesis has been postulated. Here, we determine the in vivo effects of TGF-␤ inhibition on plaque progression and phenotype in atherosclerosis. Recombinant soluble TGF-␤ receptor II (TGF␤RII:Fc), which inhibits TGF-␤ signaling, was injected in apolipoprotein E-deficient mice for 12 weeks (50 g, twice a week intraperitoneally) as early treatment (treatment age 5 to 17 weeks) and delayed treatment (age 17 to 29 weeks). In the early treatment group, inhibition of TGF-␤ signaling treatment resulted in a prominent increase in CD3-and CD45-positive cells in atherosclerotic lesions. Most profound effects were found in the delayed treatment group. Plaque area decreased 37.5% after TGF␤RII:Fc treatment. Moreover, plaque morphology changed into an inflammatory phenotype that was low in fibrosis: lipid cores were 64.6% larger, and inflammatory cell content had increased 2.7-fold. The amount of fibrosis decreased 49.6%, and intraplaque hemorrhages and iron and fibrin deposition were observed frequently. TGF␤RII:Fc treatment did not result in systemic effects. These results reveal a pivotal role for TGF-␤ in the maintenance of the balance between inflammation and fibrosis in atherosclerotic plaques.

Background-Cathepsin K (catK), a lysosomal cysteine protease, was identified in a gene-profiling experiment that compared human early plaques, advanced stable plaques, and advanced atherosclerotic plaques containing a thrombus, where it was highly upregulated in advanced stable plaques. Methods and Results-To assess the function of catK in atherosclerosis, catK Ϫ/Ϫ /apolipoprotein (apo) E Ϫ/Ϫ mice were generated. At 26 weeks of age, plaque area in the catK Ϫ/Ϫ /apoE Ϫ/Ϫ mice was reduced (41.8%) owing to a decrease in the number of advanced lesions as well as a decrease in individual advanced plaque area. This suggests an important role for catK in atherosclerosis progression. Advanced plaques of catK Ϫ/Ϫ /apoE Ϫ/Ϫ mice showed an increase in collagen content. Medial elastin fibers were less prone to rupture than those of apoE Ϫ/Ϫ mice. Although the relative macrophage content did not differ, individual macrophage size increased. In vitro studies of bone marrow derived-macrophages confirmed this observation. Scavenger receptor-mediated uptake (particularly by CD36) of modified LDL increased in the absence of catK, resulting in an increased macrophage size because of increased cellular storage of cholesterol esters, thereby enlarging the lysosomes.

The hematopoietic deficiency of Hck and Fgr led to attenuated atherosclerotic plaque formation by abrogating endothelial adhesion and transmigration; paradoxically, it also promoted plaque instability by causing monocyte subset imbalance and subendothelial accumulation, raising a note of caution regarding src kinase-targeted intervention in plaque inflammation.

SummaryAlthough many epidemiological studies have shown an association between hyperfibrinogenemia and atherosclerosis, it is not established whether elevated fibrinogen has an etiological role in the pathogenesis or is only a reflection of the ongoing disease.We have studied the contribution of fibrinogen to the development of atherosclerosis in atherosclerosis-prone ApoE*3-Leiden mice that have been cross-bred with transgenic mice overexpressing fibrinogen. Genetic compound offspring were used to evaluate the progression of atherosclerotic lesions after being fed an atherogenic diet for 7 weeks. It was observed that the lesion area of the plaques as well as the severity of the lesions in the aortic valve was comparable in control single transgenic ApoE*3-Leiden mice and in double transgenic apoE*3-Leiden mice overexpressing fibrinogen. No thrombus or fibrin deposition was observed in atherosclerotic lesions in either group of mice.These results indicate that elevated plasma fibrinogen concentrations in ApoE*3-Leiden transgenic mice do not affect the progression of diet-induced atherosclerotic lesions.

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Background: Membrane cholesterol is known to modulate a variety of cell signaling pathways and functions. While cholesterol depletion by High-Density Lipoproteins (HDL) has potent antiinflammatory effects in various cell types, its effect on inflammatory responses in macrophages remain ill defined. Methods & Results: Pre-incubation of human and murine macrophages in vitro with human reconstituted (apolipoproteinA-I/phosphatidylcholine) or native HDL significantly decreased LPS-induced anti-inflammatory IL-10 production, while the opposite was observed for the pro-inflammatory mediators IL-12 and TNF. We show that these effects are mediated by passive cholesterol depletion and lipid raft disruption, without involvement of ABCA1, ABCG1, SR-B1 or CD36. These pro-inflammatory effects are confirmed in vivo in peritoneal macrophages from ApoA-I transgenic mice, which have high circulating HDL levels. In line, innate responses required for clearance of P. aeruginosa bacterial infection in lung were compromised in mice with low HDL levels. Native and reconstituted HDL enhances Toll Like Receptor-induced signaling by activating protein kinase C (PKC), since inhibition of PKC ablated the observed HDL effects. Using microarray analysis and macrophages from NF-kB luciferase mice, we observed that HDL induces NF-kB activation. Western blot and ChIP-PCR analyses showed that in particular the p65 subunit was activated. Using specific knock-out mice for the upstream activation pathways, we show that the observed HDL effects are independent of the upstream kinases IKK, NIK and CKII. Furthermore, using STAT1 knock-out mice we observed that also STAT1 is involved in the pro-inflammatory HDL effects on IL-10 and IL-12 secretion. On the other hand, using pharmacological inhibitors, we show that HDL enhances ADAM protease activity, thereby mediating TNF release. Conclusion and Clinical Relevance: HDL exerts pro-inflammatory effects on macrophages via passive cholesterol depletion by activation of PKC, NF-kB and STAT1. These pro-inflammatory activities on macrophages could at least partly underlie the disappointing therapeutic potential of HDL raising therapy in current cardiovascular clinical trials.

This study explored whether bacterial endotoxins, in the form of lipopolysaccharides (LPS), could have an injurious effect on the biliary tract in conjunction with ischemia. A total of 64 rats were randomly assigned to 4 groups: sham operation (sham group), 1 mg/kg LPS intraperitoneal (LPS group), hepatic ischemia/reperfusion (IR; IR group), and IR combined with LPS (IR+LPS group). Following 1 or 6 hours of reperfusion, serum liver tests, bile duct histology, immunofluorescence microscopy (zonula occludens-1 [ZO-1]), bile composition (bile salts, phospholipids, lactate dehydrogenase), hepatic gene expression (bile salt transporters and inflammatory mediators), as well as serum and biliary cytokine concentrations were quantified and compared between the study groups. In addition, the integrity of the blood biliary barrier (BBB) was assayed in vivo using horseradish peroxidase (HRP). LPS administration induced severe small bile duct injury following 6 hours of reperfusion. Furthermore, total bile salts and bilirubin concentrations in serum were increased in the LPS groups compared with sham controls (LPS, + 3.3-fold and +1.9-fold; IR+LPS, + 3.8-fold and +1.7-fold, respectively). The BBB was impaired in the LPS groups as evidenced by elevated levels of HRP in bile (+4.9-fold), and decreased expression of claudin 1 (-6.7-fold) and claudin 3 (-3.6-fold). LPS was found to be a potent inducer of small bile duct injury following hepatic ischemia and 6 hours of reperfusion. This injury was associated with increased permeability of the BBB and impaired hepatic bile salt clearance. Liver Transplantation 23 194-206 2017 AASLD.