Bijoy Chellan

S100/Calgranulin-Mediated Inflammation Accelerates Left Ventricular Hypertrophy and Aortic Valve Sclerosis in Chronic Kidney Disease in a Receptor for Advanced Glycation End Products–Dependent Manner

Objective—S100A12 and fibroblast growth factor 23 are biomarkers of cardiovascular morbidity and mortality in patients with chronic kidney disease (CKD). We tested the hypothesis that human S100/calgranulin would accelerate cardiovascular disease in mice subjected to CKD. Approach and Results—A bacterial artificial chromosome of the human S100/calgranulin gene cluster containing the genes and regulatory elements for S100A8, S100A9, and S100A12 was expressed in C57BL/6J mouse (hBAC-S100) to generate a novel humanized mouse model. CKD was induced by ureteral ligation, and hBAC-S100 mice and wild-type mice were studied after 10 weeks of chronic uremia. hBAC-S100 mice with CKD showed increased fibroblast growth factor 23 in the hearts, left ventricular hypertrophy, diastolic dysfunction, focal cartilaginous metaplasia, and calcification of the mitral and aortic valve annulus together with aortic valve sclerosis. This phenotype was not observed in wild-type mice with CKD or in hBAC-S100 mice lacking the receptor for advanced glycation end products with CKD, suggesting that the inflammatory milieu mediated by S100/receptor for advanced glycation end products promotes pathological cardiac hypertrophy in CKD. In vitro, inflammatory stimuli including interleukin-6, tumor necrosis factor-&agr;, lipopolysaccarides, or serum from hBAC-S100 mice upregulated fibroblast growth factor 23 mRNA and protein in primary murine neonatal and adult cardiac fibroblasts. Conclusions—Myeloid-derived human S100/calgranulin is associated with the development of cardiac hypertrophy and ectopic cardiac calcification in a receptor for advanced glycation end products–dependent manner in a mouse model of CKD. We speculate that fibroblast growth factor 23 produced by cardiac fibroblasts in response to cytokines may act in a paracrine manner to accelerate left ventricular hypertrophy and diastolic dysfunction in hBAC-S100 mice with CKD.

Enzymatically Modified Low-Density Lipoprotein Promotes Foam Cell Formation in Smooth Muscle Cells via Macropinocytosis and Enhances Receptor-Mediated Uptake of Oxidized Low-Density Lipoprotein

Objective— Enzyme-modified nonoxidized low-density lipoprotein (ELDL) is present in human atherosclerotic lesions. Our objective is to understand the mechanisms of ELDL uptake and its effects on vascular smooth muscle cells (SMC). Approach and Results— Transformation of murine aortic SMCs into foam cells in response to ELDL was analyzed. ELDL, but not acetylated or oxidized LDL, was potent in inducing SMC foam cell formation. Inhibitors of macropinocytosis (LY294002, wortmannin, amiloride) attenuated ELDL uptake. In contrast, inhibitors of receptor-mediated endocytosis (dynasore, sucrose) and inhibitor of caveolae-/lipid raft–mediated endocytosis (filipin) had no effect on ELDL uptake in SMC, suggesting that macropinocytosis is the main mechanism of ELDL uptake by SMC. Receptor for advanced glycation end products (RAGE) is not obligatory for ELDL-induced SMC foam cell formation, but primes SMC for the uptake of oxidized LDL in a RAGE-dependent manner. ELDL increased intracellular reactive oxygen species, cytosolic calcium, and expression of lectin-like oxidized LDL receptor-1 in wild-type SMC but not in RAGE−/− SMC. The macropinocytotic uptake of ELDL is regulated predominantly by intracellular calcium because ELDL uptake was completely inhibited by pretreatment with the calcium channel inhibitor lacidipine in wild-type and RAGE−/− SMC. This is in contrast to pretreatment with PI3 kinase inhibitors which completely prevented ELDL uptake in RAGE−/− SMC, but only partially in wild-type SMC. Conclusions— ELDL is highly potent in inducing foam cells in murine SMC. ELDL endocytosis is mediated by calcium-dependent macropinocytosis. Priming SMC with ELDL enhances the uptake of oxidized LDL.

IL-22 is induced by S100/calgranulin and impairs cholesterol efflux in macrophages by downregulating ABCG1

S100A8/9 and S100A12 are emerging biomarkers for disease activity of autoimmune and cardiovascular diseases. We demonstrated previously that S100A12 accelerates atherosclerosis accompanied by large cholesterol deposits in atherosclerotic lesions of apoE-null mice. The objective of this study was to ascertain whether S100/calgranulin influences cholesterol homeostasis in macrophages. Peritoneal macrophages from transgenic mice expressing human S100A8/9 and S100A12 in myeloid cells [human bacterial artificial chromosome (hBAC)/S100] have increased lipid content and reduced ABCG1 expression and [3H]cholesterol efflux compared with WT littermates. This was associated with a 6-fold increase in plasma interleukin (IL)-22 and increased IL-22 mRNA in splenic T cells. These findings are mediated by the receptor for advanced glycation endproducts (RAGE), because hBAC/S100 mice lacking RAGE had normal IL-22 expression and normal cholesterol efflux. In vitro, recombinant IL-22 reduced ABCG1 expression and [3H]cholesterol efflux in THP-1 macrophages, while recombinant S100A12 had no effect on ABCG1 expression. In conclusion, S100/calgranulin has no direct effect on cholesterol efflux in macrophages, but rather promotes the secretion of IL-22, which then directly reduces cholesterol efflux in macrophages by decreasing the expression of ABCG1.

Differing rates of cholesterol absorption among inbred mouse strains yield differing levels of HDL-cholesterol.

Inbred strains of mice with differing susceptibilities to atherosclerosis possess widely varying plasma HDL levels. Cholesterol absorption and lipoprotein formation were compared between atherosclerosis-susceptible, low-HDL C57BL6/J mice and atherosclerosis-resistant, high-HDL FVBN/J mice. [3H]cholesterol and triglyceride appeared in the plasma of FVB mice gavaged with cholesterol in olive oil at a much higher rate than in C57 mice. The plasma cholesterol was found almost entirely as HDL-cholesterol in both strains. Inhibition of lipoprotein catabolism with Tyloxapol revealed that the difference in the rate of [3H]cholesterol appearance in the plasma was due entirely to a greater rate of chylomicron secretion from the intestine of the FVB mice. Lipid absorption into the 2nd quarter of the small intestine is greater in the FVB mice and indicates that this region may contain the factors that give rise to the differences in absorption observed between the two mouse strains. Additionally, ad libitum feeding prior to cholesterol gavage accentuates the absorption rate differences compared with fasting. The resultant remodeling of the increased levels of chylomicron in the plasma may contribute to increased plasma HDL. Intestinal gene expression analysis reveals several genes that may play a role in these differences, including microsomal triglyceride transfer protein and ABCG8.

Alginic Acid Cell Entrapment: A Novel Method for Measuring In Vivo Macrophage Cholesterol Homeostasis

Macrophage conversion to atherosclerotic foam cells is partly due to the balance of uptake and efflux of cholesterol. Cholesterol efflux from cells by HDL and its apoproteins for subsequent hepatic elimination is known as reverse cholesterol transport. Numerous methods have been developed to measure in vivo macrophage cholesterol efflux. Most methods do not allow for macrophage recovery for analysis of changes in cellular cholesterol status. We describe a novel method for measuring cellular cholesterol balance using the in vivo entrapment of macrophages in alginate, which retains incorporated cells while being permeable to lipoproteins. Recipient mice were injected subcutaneously with CaCl2 forming a bubble into which a macrophage/alginate suspension was injected, entrapping the macrophages. Cells were recovered after 24 h. Cellular free and esterified cholesterol mass were determined enzymatically and normalized to cellular protein. Both normal and cholesterol loaded macrophages undergo measureable changes in cell cholesterol when injected into WT and apoA-I-, LDL-receptor-, or apoE-deficient mice. Cellular cholesterol balance is dependent on initial cellular cholesterol status, macrophage cholesterol transporter expression, and apolipoprotein deficiency. Alginate entrapment allows for the in vivo measurement of macrophage cholesterol homeostasis and is a novel platform for investigating the role of genetics and therapeutic interventions in atherogenesis.

LIGHT/TNFSR14 Can Regulate Hepatic Lipase Expression by Hepatocytes Independent of T Cells and Kupffer Cells

LIGHT/TNFSF14 is a costimulatory molecule expressed on activated T cells for activation and maintenance of T cell homeostasis. LIGHT over expressed in T cells also down regulates hepatic lipase levels in mice through lymphotoxin beta receptor (LTβR) signaling. It is unclear whether LIGHT regulates hepatic lipase directly by interacting with LTβR expressing cells in the liver or indirectly by activation of T cells, and whether Kupffer cells, a major cell populations in the liver that expresses the LTβR, are required. Here we report that LIGHT expression via an adenoviral vector (Ad-LIGHT) is sufficient to down regulate hepatic lipase expression in mice. Depletion of Kupffer cells using clodronate liposomes had no effect on LIGHT-mediated down regulation of hepatic lipase. LIGHT-mediated regulation of hepatic lipase is also independent of LIGHT expression by T cells or activation of T cells. This is demonstrated by the decreased hepatic lipase expression in the liver of Ad-LIGHT infected recombination activating gene deficient mice that lack mature T cells and by the Ad-LIGHT infection of primary hepatocytes. Hepatic lipase expression was not responsive to LIGHT when mice lacking LTβR globally or only on hepatocytes were infected with Ad-LIGHT. Therefore, our data argues that interaction of LIGHT with LTβR on hepatocytes, but not Kupffer cells, is sufficient to down regulate hepatic lipase expression and that this effect can be independent of LIGHT’s costimulatory function.

Enzyme-modified non-oxidized LDL (ELDL) induces human coronary artery smooth muscle cell transformation to a migratory and osteoblast-like phenotype

Enzyme modified non-oxidative LDL (ELDL) is effectively taken up by vascular smooth muscle cells (SMC) and mediates transition into foam cells and produces phenotypic changes in SMC function. Our data show that incubation of human coronary artery SMC (HCASMC) with low concentration of ELDL (10 μg/ml) results in significantly enhanced foam cell formation compared to oxidized LDL (200 μg/ml; p < 0.01) or native LDL (200 μg/ml; p < 0.01). Bioinformatic network analysis identified activation of p38 MAPK, NFkB, ERK as top canonical pathways relevant for biological processes linked to cell migration and osteoblastic differentiation in ELDL-treated cells. Functional studies confirmed increased migration of HCASMC upon stimulation with ELDL (10 μg/ml) or Angiopoietin like protein 4, (ANGPTL4, 5 μg/ml), and gain in osteoblastic gene profile with significant increase in mRNA levels for DMP-1, ALPL, RUNX2, OPN/SPP1, osterix/SP7, BMP and reduction in mRNA for MGP and ENPP1. Enhanced calcification of HCASMC by ELDL was demonstrated by Alizarin Red staining. In summary, ELDL is highly potent in inducing foam cells in HCASMC and mediates a phenotypic switch with enhanced migration and osteoblastic gene profile. These results point to the potential of ELDL to induce migratory and osteoblastic effects in human smooth muscle cells with potential implications for migration and calcification of SMCs in human atherosclerosis.