27 research outputs found

    Human macrophage foam cells degrade atherosclerotic plaques through cathepsin K mediated processes

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    <p>Abstract</p> <p>Background</p> <p>Proteolytic degradation of Type I Collagen by proteases may play an important role in remodeling of atherosclerotic plaques, contributing to increased risk of plaque rupture.</p> <p>The aim of the current study was to investigate whether human macrophage foam cells degrade the extracellular matrix (ECM) of atherosclerotic plaques by cathepsin K mediated processes.</p> <p>Methods</p> <p>We 1) cultured human macrophages on ECM and measured cathepsin K generated fragments of type I collagen (C-terminal fragments of Type I collagen (CTX-I) 2) investigated the presence of CTX-I in human coronary arteries and 3) finally investigated the clinical potential by measuring circulating CTX-I in women with and without radiographic evidence of aortic calcified atherosclerosis.</p> <p>Results</p> <p>Immune-histochemistry of early and advanced lesions of coronary arteries demonstrated co-localization of Cathepsin-K and CTX-I in areas of intimal hyperplasia and in shoulder regions of advanced plaques. Treatment of human monocytes with M-CSF or M-CSF+LDL generated macrophages and foam cells producing CTX-I when cultured on type I collagen enriched matrix. Circulating levels of CTX-I were not significantly different in women with aortic calcifications compared to those without.</p> <p>Conclusions</p> <p>Human macrophage foam cells degrade the atherosclerotic plaques though cathepsin K mediated processes, resulting in increase in levels of CTX-I. Serum CTX-I was not elevated in women with aortic calcification, likely due to the contribution of CTX-I from osteoclastic bone resorption which involves Cathepsin-K. The human macrophage model system may be used to identify important pathway leading to excessive proteolytic plaque remodeling and plaque rupture.</p

    Lysophosphatidic acid enhances stromal cell-directed angiogenesis.

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    Ischemic diseases such as peripheral vascular disease (PVD) affect more than 15% of the general population and in severe cases result in ulcers, necrosis, and limb loss. While the therapeutic delivery of growth factors to promote angiogenesis has been widely investigated, large-scale implementation is limited by strategies to effectively deliver costly recombinant proteins. Multipotent adipose-derived stromal cells (ASC) and progenitor cells from other tissue compartments secrete bioactive concentrations of angiogenic molecules, making cell-based strategies for in situ delivery of angiogenic cytokines an exciting alternative to the use of recombinant proteins. Here, we show that the phospholipid lysophosphatidic acid (LPA) synergistically improves the proangiogenic effects of ASC in ischemia. We found that LPA upregulates angiogenic growth factor production by ASC under two- and three-dimensional in vitro models of serum deprivation and hypoxia (SD/H), and that these factors significantly enhance endothelial cell migration. The concurrent delivery of LPA and ASC in fibrin gels significantly improves vascularization in a murine critical hindlimb ischemia model compared to LPA or ASC alone, thus exhibiting the translational potential of this method. Furthermore, these results are achieved using an inexpensive lipid molecule, which is orders-of-magnitude less costly than recombinant growth factors that are under investigation for similar use. Our results demonstrate a novel strategy for enhancing cell-based strategies for therapeutic angiogenesis, with significant applications for treating ischemic diseases

    LPA enhances the proangiogenic effects of ASC under ischemia <i>in</i><i>vitro</i>.

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    <p>Expression of (<b>a</b>) <i>VEGF</i> and (<b>b</b>) <i>FGF2</i> are upregulated by SD/H and further enhanced with the addition of 25 μM LPA. In both cases, the addition of Ki16425 abrogates this effect (<i>n</i> = 4). Data represents combined gene expression from three unique donors. *<i>p</i> < 0.05 vs. control, **<i>p</i> < 0.01 vs. control, ***<i>p</i> < 0.001 vs. control.</p

    LPA receptor expression in human ASC is dependent on oxygen microenvironment.

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    <p>(<b>a</b>) <i>LPAR1</i> expression is unchanged by SD/H, but (<b>b</b>) <i>LPAR2</i> expression is significantly higher in SD/H. <i>LPAR3</i> expression is undetectable in either condition, but SD/H also increases expression of (<b>c</b>) <i>LPAR4</i> and (<b>d</b>) <i>LPAR5</i> (<i>n</i> = 4). **<i>p</i> < 0.01 vs. control, ***<i>p</i> < 0.001 vs. control.</p

    ASC treated with LPA promote endothelial cell migration.

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    <p>(<b>a</b>) Medium conditioned by ASC in the presence of LPA under SD/H is significantly more chemoattractive to ECFC than medium from non-treated or inhibitor-treated ASC. The addition of LPA or Ki16425 to unconditioned medium had no effect on ECFC migration (<i>n</i> = 5). (<b>b</b>) LPA promotes VEGF secretion from ASC entrapped in 3D fibrin gels under SD/H compared to cells treated with no LPA or Ki16425. (<i>n</i> = 6). *<i>p</i> < 0.05 vs. control, **<i>p</i> < 0.01 vs. control, ***<i>p</i> < 0.001 vs. control (<b>c</b>) LPA also increases production of other angiogenic and inflammatory cytokines. Data are presented as average subtracted background fluorescence intensity normalized to positive controls. </p

    Co-delivery of LPA with ASC in fibrin gels significantly improves angiogenesis in a murine model of critical limb ischemia.

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    <p>Representative H&E stained sections from the quadriceps of (<b>a</b>) normal hindlimbs and (<b>b</b>) ischemic limbs treated with fibrin gels containing 25 μM LPA, (<b>c</b>) ASC, or (<b>d</b>) LPA and ASC show the presence of more large vessels in limbs treated with cells and LPA together. Similarly, CD31 staining reveals the formation of larger intramuscular blood vessels in limbs receiving (<b>h</b>) both LPA and ASC, while (<b>e</b>) normal tissue and defects receiving (<b>f</b>) LPA and (<b>g</b>) ASC alone have fewer and smaller vessels. (<b>i</b>) Blood vessel quantification from H&E stained sections confirms these results (<i>n=8</i>). Scale bars represent 100 μm; arrows indicative of vessels with defined lumens and erythrocytes. *<i>p</i> < 0.05 vs. other groups. (<b>j</b>) Hindlimbs were visually assessed for severity of toe and foot necrosis in each treatment group. </p

    Mesenchymal Stem Cell Seeding of Porcine Small Intestinal Submucosal Extracellular Matrix for Cardiovascular Applications

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    <div><p>In this study, we investigate the translational potential of a novel combined construct using an FDA-approved decellularized porcine small intestinal submucosa extracellular matrix (SIS-ECM) seeded with human or porcine mesenchymal stem cells (MSCs) for cardiovascular indications. With the emerging success of individual component in various clinical applications, the combination of SIS-ECM with MSCs could provide additional therapeutic potential compared to individual components alone for cardiovascular repair. We tested the <i>in vitro</i> effects of MSC-seeding on SIS-ECM on resultant construct structure/function properties and MSC phenotypes. Additionally, we evaluated the ability of porcine MSCs to modulate recipient graft-specific response towards SIS-ECM in a porcine cardiac patch <i>in vivo</i> model. Specifically, we determined: 1) <i>in vitro</i> loading-capacity of human MSCs on SIS-ECM, 2) effect of cell seeding on SIS-ECM structure, compositions and mechanical properties, 3) effect of SIS-ECM seeding on human MSC phenotypes and differentiation potential, and 4) optimal orientation and dose of porcine MSCs seeded SIS-ECM for an <i>in vivo</i> cardiac application. In this study, histological structure, biochemical compositions and mechanical properties of the FDA-approved SIS-ECM biomaterial were retained following MSCs repopulation <i>in vitro</i>. Similarly, the cellular phenotypes and differentiation potential of MSCs were preserved following seeding on SIS-ECM. In a porcine <i>in vivo</i> patch study, the presence of porcine MSCs on SIS-ECM significantly reduced adaptive T cell response regardless of cell dose and orientation compared to SIS-ECM alone. These findings substantiate the clinical translational potential of combined SIS-ECM seeded with MSCs as a promising therapeutic candidate for cardiac applications.</p></div
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