Browsing by Subject "Cross-linking"

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    Molecular Phenotyping and Mechanisms of Myocardial Fibrosis in Advanced Chronic Kidney Disease
    (Wolters Kluwer, 2023) Narayanan, Gayatri; Halim, Arvin; Hu, Alvin; Avin, Keith G.; Lu, Tzongshi; Zehnder, Daniel; Hato, Takashi; Chen, Neal X.; Moe, Sharon M.; Lim, Kenneth; Medicine, School of Medicine
    Key Points: * Myocardial fibrosis in hearts from patients with CKD is characterized by increased trimeric tensile collagen type I and decreased elastic collagen type III compared with hearts from hypertensive or healthy donors, suggesting a unique fibrotic phenotype. * Myocardial fibrosis in CKD is driven by alterations in extracellular matrix proteostasis, including dysregulation of metalloproteinases and cross-linking enzymes. * CKD-associated mineral stressors uniquely induce a fibronectin-independent mechanism of fibrillogenesis characterized by formation of trimeric collagen compared with proinflammatory/fibrotic cytokines. Background: Myocardial fibrosis is a major life-limiting problem in CKD. Despite this, the molecular phenotype and metabolism of collagen fibrillogenesis in fibrotic hearts of patients with advanced CKD have been largely unstudied. Methods: We analyzed explanted human left ventricular (LV) heart tissues in a three-arm cross-sectional cohort study of deceased donor patients on hemodialysis (HD, n=18), hypertension with preserved renal function (HTN, n=8), and healthy controls (CON, n=17), ex vivo. RNA-seq and protein analysis was performed on human donor hearts and cardiac fibroblasts treated with mineral stressors (high phosphate and high calcium). Further mechanistic studies were performed using primary cardiac fibroblasts, in vitro treated with mineral stressors, proinflammatory and profibrotic cytokines. Results: Of the 43 donor participants, there was no difference in age (P > 0.2), sex (P > 0.8), or body mass index (P > 0.1) between the groups. Hearts from the HD group had extensive fibrosis (P < 0.01). All LV tissues expressed only the trimeric form of collagen type I. HD hearts expressed increased collagen type I (P < 0.03), elevated collagen type I:III ratio (P < 0.05), and decreased MMP1 (P < 0.05) and MMP2 (P < 0.05). RNA-seq revealed no significant differential gene expression of extracellular matrix proteins of interest in HD hearts, but there was significant upregulation of LH2, periostin, α-SMA, and TGF-β1 gene expression in mineral stressor–treated cardiac fibroblasts. Both mineral stressors (P < 0.009) and cytokines (P < 0.03) increased collagen type I:III ratio. Mineral stressors induced trimeric collagen type I, but cytokine treatment induced only dimeric collagen type I in cardiac fibroblasts. Mineral stressors downregulated fibronectin (P < 0.03) and MMP2 zymogen (P < 0.01) but did not significantly affect expression of periostin, MMP1, or cross-linking enzymes. TGF-β upregulated fibronectin (P < 0.01) and periostin (P < 0.02) only. Conclusions: Myocardial fibrosis in advanced CKD hearts is characterized by increased trimeric collagen type I and dysregulated collagen metabolism, and is differentially regulated by components of uremia.
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    β-Aminopropionitrile-Induced Reduction in Enzymatic Crosslinking Causes In Vitro Changes in Collagen Morphology and Molecular Composition
    (PLOS, 2016-11-09) Canelón, Silvia P.; Wallace, Joseph M.; Department of Biomedical Engineering, School of Engineering & Technology
    Type I collagen morphology can be characterized using fibril D-spacing, a metric which describes the periodicity of repeating bands of gap and overlap regions of collagen molecules arranged into collagen fibrils. This fibrillar structure is stabilized by enzymatic crosslinks initiated by lysyl oxidase (LOX), a step which can be disrupted using β-aminopropionitrile (BAPN). Murine in vivo studies have confirmed effects of BAPN on collagen nanostructure and the objective of this study was to evaluate the mechanism of these effects in vitro by measuring D-spacing, evaluating the ratio of mature to immature crosslinks, and quantifying gene expression of type I collagen and LOX. Osteoblasts were cultured in complete media, and differentiated using ascorbic acid, in the presence or absence of 0.25mM BAPN-fumarate. The matrix produced was imaged using atomic force microscopy (AFM) and 2D Fast Fourier transforms were performed to extract D-spacing from individual fibrils. The experiment was repeated for quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Fourier Transform infrared spectroscopy (FTIR) analyses. The D-spacing distribution of collagen produced in the presence of BAPN was shifted toward higher D-spacing values, indicating BAPN affects the morphology of collagen produced in vitro, supporting aforementioned in vivo experiments. In contrast, no difference in gene expression was found for any target gene, suggesting LOX inhibition does not upregulate the LOX gene to compensate for the reduction in aldehyde formation, or regulate expression of genes encoding type I collagen. Finally, the mature to immature crosslink ratio decreased with BAPN treatment and was linked to a reduction in peak percent area of mature crosslink hydroxylysylpyridinoline (HP). In conclusion, in vitro treatment of osteoblasts with low levels of BAPN did not induce changes in genes encoding LOX or type I collagen, but led to an increase in collagen D-spacing as well as a decrease in mature crosslinks.