Moderate- and High-Intensity Exercise Improves Lipoprotein Profile and Cholesterol Efflux Capacity in Healthy Young Men.

Background Exercise is associated with a reduced risk of cardiovascular disease. Increased high-density lipoprotein cholesterol (HDL-C) levels are thought to contribute to these benefits, but much of the research in this area has been limited by lack of well-controlled subject selection and exercise interventions. We sought to study the effect of moderate and high-intensity exercise on HDL function, lipid/lipoprotein profile, and other cardiometabolic parameters in a homogeneous population where exercise, daily routine, sleep patterns, and living conditions were carefully controlled. Methods and Results Male Army recruits (n=115, age 22±0.3 years) completed a 12-week moderate-intensity exercise program. A subset of 51 subsequently completed a 15-week high-intensity exercise program. Fitness increased and body fat decreased after moderate- and high-intensity exercise (P<0.001). Moderate-intensity exercise increased HDL-C and apolipoprotein A-I levels (6.6%, 11.6% respectively), and decreased low-density lipoprotein cholesterol and apolipoprotein B levels (7.2%, 4.9% respectively) (all P<0.01). HDL-C and apolipoprotein A-I levels further increased by 8.2% (P<0.001) and 6.3% (P<0.05) after high-intensity exercise. Moderate-intensity exercise increased ABCA-1 (ATP-binding cassette transporter A1) mediated cholesterol efflux by 13.5% (P<0.001), which was sustained after high-intensity exercise. In a selected subset the ability of HDLs to inhibit ICAM-1 (intercellular adhesion molecule-1) expression decreased after the high (P<0.001) but not the moderate-intensity exercise program. Conclusions When controlling for exercise patterns, diet, and sleep, moderate-intensity exercise improved HDL function, lipid/lipoprotein profile, fitness, and body composition. A sequential moderate followed by high-intensity exercise program showed sustained or incremental benefits in these parameters. Improved HDL function may be part of the mechanism by which exercise reduces cardiovascular disease risk.

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

Apolipoprotein A-I (apoA-I) is the major component of HDL and central to the ability of HDL to 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 promote ABCA1-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-translational modification of apoA-I mediated by HMDMs that 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.

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.

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.

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.

Influence of biodegradable and non-biodegradable material surfaces on the differentiation of human monocyte-derived macrophages.

Monocyte-derived macrophages (MDM) and multinucleated foreign body giant cells (FBGC) are the primary cell types that remain at the cell-material interface of polyurethane (PU)-based medical devices as a result of chronic inflammatory responses. In vitro studies have demonstrated that MDM possess degradative potential toward PU, which can result in device failure. Because most studies have followed the degradation potential, morphology, and function of these cells only once fully differentiated, the current study investigated the influence of a non-degradable control tissue culture-grade polystyrene (TCPS) surface relative to two degradable model polycarbonate-urethanes (PCNU), of different chemistry, on various parameters of MDM morphology and function during a 14-day differentiation time course. The differentiation of human monocytes isolated from whole blood on PCNU materials resulted in increased cell attachment, decreased multinucleation, and significant decreases in cell spreading when compared with cells differentiated on TCPS. Actin-stained podosome-like cell adhesion structures were increased in PCNU-adherent cells, accompanied by an alteration in beta-actin and vinculin protein expression. The expression of the CD68 macrophage marker was reduced when cells were adherent to the PCNU materials and compared with TCPS, suggesting altered cell activation by the degradable relative to non-degradable materials. The degradative potential of these cells was altered by the material surface they were exposed to as measured by esterase activity and protein expression of monocyte-specific esterase. This was also supported by physical material breakdown evident in scanning electron microscopy images that illustrated holes in the PCNU films generated by the presence of differentiating MDM. It was concluded from these studies that PCNU materials significantly alter the function and morphology of differentiating MDM. This must be taken into consideration when studying cell-material interactions because these cells will receive cues from their immediate environment (including the biomaterial) upon differentiation, thereby affecting their resulting phenotype.

Intracellular phospholipase A2 expression and location in human macrophages: influence of synthetic material surface chemistry.

Phospholipase A(2) (PLA(2)) enzymes participate in a potent inflammatory pathway through the liberation of arachidonic acid upon hydrolysis of membrane glycerophospholipids. The presence of implanted polycarbonate-urethane (PCNU) materials, used in several medical applications, has the ability to influence inflammatory responses of human macrophages that are recruited to a tissue-material interface; however, the specific inflammatory pathways that are activated upon macrophage attachment to PCNU are largely unknown. Previous studies suggested the participation of PLA(2) pathways in material degradation with the use of chemical inhibitors, such as aristolochic acid (ARIST), however not accurately defining the specific PLA(2) enzymes involved. The current study aimed to establish specific groups of PLA(2) involved in the macrophage foreign body response to PCNU. ARIST was assessed for specific effects on secretory PLA(2) (sPLA(2)) protein expression and non-specific effects on key proteins, beta-actin and monocyte-specific esterase, implicated in the macrophage attack on PCNU materials. Macrophage attachment to PCNU materials induced increased intracellular expression of cytosolic PLA(2) (cPLA(2)), but not sPLA(2), relative to tissue culture polystyrene (TCPS) as detected by immunoblot analysis, demonstrating an early and delayed stimulation during the time course of increased cPLA(2) protein expression. Laser scanning confocal microscopy images indicated a change in location of cPLA(2) in macrophages adherent to PCNU surfaces compared to TCPS. This study has illustrated changes in macrophage cPLA(2) expression in response to cell-attachment to PCNU surfaces, demonstrating that the macrophage foreign body response to biomaterials induces a potent inflammatory pathway, which may lead to tissue damage near the site of material implantation.

Material surfaces affect the protein expression patterns of human macrophages: A proteomics approach.

Monocyte-derived macrophages (MDM) are key inflammatory cells and are central to the foreign body response to implant materials. MDM have been shown to exhibit changes in actin cytoskeleton, multinucleation, cell size, and function in response to small alterations in polycarbonate-urethane (PCNU) surface chemistry. Although PCNU chemistry has an influence on de novo protein synthesis, no assessments of the protein expression profiles of MDM have yet been reported. The rapid emerging field of expression proteomics facilitates the study of changes in cellular protein profiles in response to their microenvironment. The current study applied proteomic techniques, 2-dimensional electrophoresis (2-DE) combined with MALDI-ToF (matrix assisted laser desorption ionization-time of flight) mass spectrometry, to determine differences in MDM protein expression influenced by PCNU. Results indicated that MDM responded to material chemistry by modulation of structural proteins (i.e. actin, vimentin, and tubulin). Additionally, intracellular protein modulation which requires proteins responsible for trafficking (i.e. chaperone proteins) and protein structure modification (i.e. bond rearrangement and protein folding) were also altered. This study demonstrated for the first time that a proteomics approach was able to detect protein expression profile changes in MDM cultured on different material surfaces, forming the basis for utilizing further quantitative proteomics techniques that could assist in elucidation of the mechanisms involved in MDM-material interaction.

The human macrophage response during differentiation and biodegradation on polycarbonate-based polyurethanes: dependence on hard segment chemistry.

Human monocytes, isolated from whole blood, were seeded onto tissue culture grade polystyrene (PS) and three polycarbonate-based polyurethanes (PCNUs) (synthesized with either 1,6-hexane diisocyanate (HDI) or 4,4'-methylene bis-phenyl diisocyanate (MDI), poly(1,6-hexyl 1,2-ethyl carbonate) diol (PCN) and 1,4-butanediol (BD) in different stoichiometric ratios (HDI:PCN:BD 4:3:1 or 3:2:1 and MDI:PCN:BD 3:2:1) (referred to as HDI431, HDI321 and MDI321, respectively). Following their differentiation to monocyte-derived macrophages (MDMs) the cells were trypsinized and reseeded onto each of the PCNUs synthesized with either 14C-HDI or 14C-BD and degradation was measured by radiolabel release (RR). When the differentiation surface was MDI321, there was more RR from 14C-HDI431 than from any other surface (p < 0.0001) whereas the amount of esterase (identified by immunoblotting) as well as the esterase activity was the greatest in MDM differentiated on PS, reseeded on 14C-HDI431 (p < 0.0001). The effect of potential degradation products (methylene dianiline (MDA) and BD) from the PCNUs was carried out to determine possible links between products and substrate-induced activation of MDM. MDA was found to inhibit RR 60% from MDM seeded on 14C-MDI321B (p < 0.0001), approximately 20% from 14C-HDI431 (p = 0.002) and no effect from 14C-HDI321B. MDA inhibited esterase activity 30% from MDM only on 14C-MDI321B (p = 0.003), but no effect on esterase activity was observed for the other two polymers. BD had no inhibitory effect on RR from any PCNU, but did inhibit esterase activity in MDM on 14C-HDI431 (p = 0.025). This study indicates that the degradation of a specific material is a multi-factorial process, dictated by its susceptibility to hydrolysis, the effect of specific products generated during this course of action, and perhaps not as well appreciated, the material's inherent ability to influence enzyme synthesis and release.

Phospholipase A2 pathway association with macrophage-mediated polycarbonate-urethane biodegradation.

Activation of the phospholipase A2 (PLA2) pathway is a key cell signaling event in the inflammatory response. The PLA2 family consists of a group of enzymes that hydrolyze membrane phospholipids, resulting in the liberation of arachidonic acid (AA), a precursor to pro-inflammatory molecules. Given the well-documented activating role of biomaterials in the inflammatory response to medical implants, the present study investigated the link between PLA2 and polycarbonate-based polyurethane (PCNU) biodegradation, and the effect that material surface had on PLA2 activation in the U937 cell line. PCNUs were synthesized with poly(1,6-hexyl 1,2-ethyl carbonate)diol, 1,4-butanediol and one of two diisocyanates (hexane 1,6-diisocyanate or 4,4'-methylene bisphenyl diisocyanate) in varying stoichiometries and incubated with adherent U937 cells. PLA2 inhibiting agents resulted in significantly decreased PCNU biodegradation (p < 0.05). Moreover, when activation of PLA2 was assessed (3H-AA release), significantly more 3H-AA was released from PCNU-adherent U937 cells than polystyrene-adherent U937 cells (p < 0.05) which was significantly decreased in the presence of PLA2 inhibitors. The pattern of inhibition of U937 cell-mediated biodegradation and 3H-AA release that was modulated by PCNU surface differences, suggests a role for secretory PLA2 along with cytosolic PLA2. Understanding PCNU activation of intracellular pathways, such as PLA2, will allow the design of materials optimized for their intended use.