Donna Lee M. Dinnes

Interactions between meniscal cells and a self assembled biomimetic surface composed of hyaluronic acid, chitosan and meniscal extracellular matrix molecules.

Menisci are one of the most commonly injured parts of the knee with a limited healing potential. This study focuses on fabrication and characterization of biomimetic surfaces for meniscal tissue engineering. To achieve this, a combination of hyaluronic acid/chitosan (HA/CH) mutilayers with covalently immobilized major extracellular matrix (ECM) components of native meniscus, namely collagen I/II (COL.I/II) and chondroitin-6-sulfate (C6S) was employed. Adsorption of the biomolecules was monitored using a quartz crystal microbalance with dissipation (QCM-D) and fourier transform-surface plasmon resonance (FT-SPR). Immobilization of the biomolecules onto HA/CH mutilayers was achieved by sequential adsorption, with optimum binding at a molar ratio of 1.4:1 (COL.I/II: C6S). After adding COL.I/II, the layers became relatively more rigid and large aggregates were evident. The effects of the modified surfaces on cell proliferation, gene expression and proteoglycan production of rat meniscal cells were examined. Quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) analysis showed that primary meniscal cells dedifferentiated rapidly after one passage in monolayer culture. This process could be reversed by culturing the cells on C6S surfaces, as indicated by increases in both collagen II and aggrecan gene expression, as well as proteoglycan production. Cells with abundant lipid vacuoles were evident on all the surfaces over an extended culture period. The results demonstrate the feasibility of C6S surfaces to avoid the dedifferentiation that normally occurs during monolayer expansion of meniscal cells.

Effects of biomimetic surfaces and oxygen tension on redifferentiation of passaged human fibrochondrocytes in 2D and 3D cultures.

Due to its limited healing potential within the inner avascular region, functional repair of the meniscus remains a significant challenge in orthopaedic surgery. Tissue engineering of a meniscus implant using meniscal cells offers the promise of enhancing the reparative process and achieving functional meniscal repair. In this work, using quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) analysis, we show that human fibrochondrocytes rapidly dedifferentiate during monolayer expansion on standard tissue culture flasks, representing a significant limit to clinical use of this cell population for meniscal repair. Previously, we have characterized and described the feasibility of a tailored biomimetic surface (C6S surface) for reversing dedifferentiation of monolayer-expanded rat meniscal cells. The surface is comprised of major meniscal extracellular matrix (ECM) components in the inner region, namely collagen I/II (at a 2:3 ratio) and chondroitin-6-sulfate. We thus have further evaluated the effects of the C6S surface, alongside a number of other tailored surfaces, on cell adhesion, proliferation, matrix synthesis and relevant marker gene expression (collagen I, -II, aggrecan and Sox-9 etc) of passaged human fibrochondrocytes in 2D (coated glass coverslips) and 3D (surface-modified polymeric scaffolds) environments. We show that the C6S surface is permissive for cell adhesion, proliferation and ECM synthesis, as demonstrated using DNA quantification, 1,9-dimethylmethylene blue (DMMB) assay, histology and immunohistochemistry. More importantly, RT-qPCR analyses corroborate the feasibility of the C6S surface for reversing phenotypic changes, especially the downregulation of collagen II, of dedifferentiated human fibrochondrocytes. Furthermore, human fibrochondrocyte redifferentiation was enhanced by hypoxia in the 3D cultures, independent of hypoxia inducible factor (HIF) transcriptional activity and was shown to potentially involve the transcriptional activation of Sox-9.

Expression and stability of two isoforms of ABCG1 in human vascular cells

OBJECTIVE To evaluate the expression of two ABCG1 isoforms that differ in the presence or absence of a 12 amino acid (AA) peptide between the ABC cassette and the transmembrane region, termed ABCG1(+12) and ABCG1(-12), respectively, in human vascular cells and tissues. METHODS AND RESULTS mRNA for both isoforms was expressed in human macrophages, vascular endothelial and smooth muscle cells as well as whole human spleen, lung, liver and brain tissue. However, ABCG1(+12) was not expressed in mouse tissues. 2D gel electrophoresis of ABCG1 protein indicated that both protein isoforms were expressed in human macrophages. Furthermore the half-lives of the two ABCG1 protein isoforms, stably expressed in CHOK1 cells, measured under basal conditions were different, suggesting the presence of a degradation or stabilising signal in or near the 12AA region of ABCG1(+12). CONCLUSION ABCG1(+12) is an isoform of ABCG1 exclusively expressed in human cells at the RNA and protein level. As ABCG1(+12) is not expressed in mice, although mouse models are widely used to elucidate the function of ABCG1, further investigations into the importance of this human ABCG1 isoform are warranted.

Cholesterol accumulation inhibits ER to Golgi transport and protein secretion: studies of apolipoprotein E and VSVGt

Cholesterol excess is typical of various diseases including atherosclerosis. We have investigated whether cholesterol accumulation in the ER (endoplasmic reticulum) can inhibit exit of vesicular cargo and secretion of proteins by studying apoE (apolipoprotein E), a significant glycoprotein in human health and disease. CHO (Chinese hamster ovary) cells expressing human apoE under a cholesterol-independent promoter incubated with cholesterol-cyclodextrin complexes showed increased levels of cellular free and esterified cholesterol, inhibition of SREBP-2 (sterol-regulatory-element-binding protein 2) processing, and a mild induction of ER stress, indicating significant accumulation of cholesterol in the ER. Secretion of apoE was markedly inhibited by cholesterol accumulation, and similar effects were observed in cells enriched with lipoprotein-derived cholesterol and in primary human macrophages. Removal of excess cholesterol by a cyclodextrin vehicle restored apoE secretion, indicating that the transport defect was reversible. That cholesterol impaired protein trafficking was supported by the cellular accumulation of less sialylated apoE glycoforms, and by direct visualization of altered ER to Golgi transport of thermo-reversible VSVG (vesicular stomatitis virus glycoprotein) linked to GFP (green fluorescent protein). We conclude that intracellular accumulation of cholesterol in the ER reversibly inhibits protein transport and secretion. Strategies to correct ER cholesterol may restore homoeostatic processes and intracellular protein transport in conditions characterized by cholesterol excess.

Modulation of collagen II fiber formation in 3-D porous scaffold environments.

Collagen II, a major extracellular matrix component in cartilaginous tissues, undergoes fibrillogenesis under physiological conditions. The present study explored collagen II fiber formation in solution and in two- (coverslip) and three-dimensional (scaffold) environments under different incubation conditions. These conditions include variations in adsorption buffers, the presence of 1-ethyl-3-(3-dimenthylaminopropyl) carbodiimide/N-hydroxysuccinimide crosslinker and the nature of the material surfaces. We extend our observations of collagen II fiber formation in two dimensions to develop an approach for the formation of a fibrillar collagen II network throughout surface-modified polylactide-co-glycolide porous scaffolds. Morphologically, the collagen II network is similar to that present in native articular cartilage. Biological validation of the resultant optimized functional scaffold, using rat bone marrow-derived mesenchymal stem cells, shows appreciable cell infiltration throughout the scaffold with enhanced cell spreading at 24h post-seeding. This economic and versatile approach is thus believed to have significant potential in cartilage tissue engineering applications.

Human macrophage cathepsin B-mediated C-terminal cleavage of apolipoprotein A-I at Ser228 severely impairs antiatherogenic capacity

Apolipoprotein α‐I (apoA‐I) is themajor component of HDLandcentral to the ability of HDLto stimulate ATP‐binding cassette transporter A1 (ABCA1)‐dependent, antiatherogenic export of cholesterol from macrophage foam cells, a key player in the pathology of atherosclerosis. Cell‐mediated modifications of apoA‐I, such as chlorination, nitration, oxidation, and proteolysis, can impair its antiatherogenic function, although it is unknown whether macrophages themselves contribute to such modifications. To investigate this, human monocyte‐derived macrophages (HMDMs) were incubated with human apoA‐I under conditions used to induce cholesterol export. Two‐dimensional gel electrophoresis and Western blot analysis identified that apoA‐I is cleaved (~20–80%) by HMDMs in a time‐dependent manner, generating apoA‐I of lower MW and isoelectric point. Mass spectrometry analysis identified a novel C‐terminal cleavage site of apoA‐I between Ser228‐Phe229. Recombinant apoA‐I truncated at Ser228 demonstrated profound loss of capacity to solubilize lipid and to promoteABCA1‐dependent cholesterol efflux. Protease inhibitors, small interfering RNA knockdown in HMDMs, mass spectrometry analysis, and cathepsin B activity assays identified secreted cathepsin B as responsible for apoA‐I cleavage at Ser228. Importantly, C‐terminal cleavage of apoA‐I was also detected in human carotid plaque. Cleavage at Ser228 is a novel, functionally important post‐translationalmodification of apoA‐Imediated byHMDMsthat limits the antiatherogenic properties of apoA‐I.—Dinnes, D. L.M., White, M. Y., Kockx, M., Traini, M., Hsieh, V., Kim, M.‐J., Hou, L., Jessup, W., Rye, K.‐A., Thaysen‐Andersen, M., Cordwell, S. J., Kritharides, L. Human macrophage cathepsin B‐mediated C‐terminal cleavage of apolipoprotein A‐I at Ser228 severely impairs antiatherogenic capacity. FASEB J. 30, 4239–4255 (2016). www.fasebj.org

HDL heterogeneity and serum efflux capacity.

DOI:10.1097/BOR.0b013e32834b1fb1 There is strong epidemiological evidence of an inverse relationship between HDL-cholesterol (HDL-C) and cardiovascular disease (CVD) and clearly established potentially atheroprotective properties of HDL in animal models and cell culture studies. However, in recent years, some genetic and therapeutic studies have cast doubt on the ability of HDL-C measurement to accurately predict cardiovascular risk and on the therapeutic advantage of raising HDL-C [1]. This is at least in part a consequence of the extreme heterogeneity of HDL particles, which range in diameter from 7–14 nm and contain a diverse array of proteins and lipids [2 && ] in varying proportions and of which HDL-C is only a minor ( 20% mass) component. It is likely that among this diverse population of HDL particles, there will be differences in their biological functionality and atheroprotective activity, and that the development of invitro measures of HDL function will provide potentially more predictive metrics for CVD risk. One of the best-studied candidate atheroprotective functions of HDL is reverse cholesterol transport, whereby cholesterol is removed from peripheral tissues and eventually cleared through biliary excretion. The first step in this process is the removal of cholesterol from cells, referred to as ‘cholesterol efflux’, which has been tested in clinical studies using assays of ‘serum efflux capacity’. These measure the rate at which apoB-depleted sera promote efflux of (H)-cholesterol, usually from murine J774 macrophages preincubated with a cyclic AMP (cAMP) analogue to induce expression of the ATP-binding cassette transporter ABCA1. In this system, ABCA1 is the major defined pathway contributing to cholesterol efflux, although a larger proportion is by other less well-defined pathways and/or diffusional exchange [3]. A seminal study in 2011 demonstrated that serum efflux capacity was inversely related to prevalent CVD and predicted CVD independently of HDL-C [4]. Since then many other studies based on similar assays have appeared, including several in recent months (see Further Reading). One recent study in a large cohort free of CVD at baseline showed that baseline cholesterol efflux capacity was inversely associated with incident CVD over a 9-year follow-up