Enhancing mechanical properties of tissue‐engineered constructs via lysyl oxidase crosslinking activity

Elbjeirami, Wafa M.; Yonter, Edward O.; Starcher, Barry; West, Jennifer L.

doi:10.1002/jbm.a.10021

Cited by 143 publications

(84 citation statements)

References 32 publications

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“…This is not surprising given in vitro data that LOX treatment of isolated collagen gels resulted in a significant increase in GЈ (12,22). In vivo studies of rat heart and aorta also showed that increased stiffness was secondary to cross-linking by members of the LOX family rather than to increased collagen deposition (4,30,37).…”

Section: Discussionmentioning

confidence: 83%

“…We hypothesized that early changes in GЈ resulted from increased collagen cross-linking, which has been shown in in vitro as well as in vivo studies to increase the GЈ of collagen in the absence of increased deposition. Lysyl oxidase (LOX)-mediated cross-linking activity in particular increases collagen stiffness and is known to be upregulated early after liver injury (4,12,22,30,37). We therefore treated a second cohort of rats with the LOX inhibitor BAPN over a period of 2 wk.…”

Section: Study Design and Histologymentioning

confidence: 99%

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Increased stiffness of the rat liver precedes matrix deposition: implications for fibrosis

Georges¹,

Hui²,

Gombos³

et al. 2007

American Journal of Physiology-Gastrointestinal and Liver Physi

506

401

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Liver fibrosis, the response to chronic liver injury, results from the activation of mesenchymal cells to fibrogenic myofibroblasts. We have recently shown that two key myofibroblast precursor populations, hepatic stellate cells and portal fibroblasts, undergo activation in culture in response to increasing substrate stiffness. We therefore hypothesized that alterations in liver stiffness precede myofibroblast activation and fibrosis in vivo as well. To test this hypothesis, we induced fibrosis in rats by twice weekly injections of carbon tetrachloride (CCl4) and then killed the animals at various time points ranging from 3 to 70 days after the initiation of injury. The shear storage modulus of the whole liver was measured on fresh tissue; fixed and frozen tissue from the same livers was used to quantify fibrosis. We observed that liver stiffness increased immediately and continued to increase, leveling out by day 28. Fibrosis, measured histologically by trichrome staining as well as by quantitative sirius red staining, increased with time, although these increases were delayed relative to changes in stiffness. There was no direct correlation between stiffness and fibrosis at early or late time points. Treatment of a second cohort of rats with the lysyl oxidase inhibitor, β-aminopropionitrile (BAPN), partially prevented early increases in liver stiffness. We concluded that increases in liver stiffness precede fibrosis and potentially myofibroblast activation. Liver stiffness appears to result from matrix cross-linking and possibly other unknown variables in addition to matrix quantity. We suggest that increased liver stiffness may play an important role in initiating the early stages of fibrosis.

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Section: Discussionmentioning

confidence: 83%

Section: Study Design and Histologymentioning

confidence: 99%

Increased stiffness of the rat liver precedes matrix deposition: implications for fibrosis

Georges¹,

Hui²,

Gombos³

et al. 2007

American Journal of Physiology-Gastrointestinal and Liver Physi

506

401

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“…There are five isoforms: LOX and the LOX-like enzymes LOXL1, LOXL2, LOXL3, and LOXL4, all encoded by separate genes and with overlapping, but distinct, functions and substrate specificity. Multiple studies have shown that LOX-mediated cross-linking significantly increases ECM stiffness (8,15,20,29).The LOXs play an important role in liver fibrosis. LOX expression in the liver, as determined by immunohistochemistry, is upregulated as early as 24 h after bile duct ligation (BDL) and continues to increase through at least 72 h, preceding increases in ␣-SMA (7).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Hepatic stellate cells and portal fibroblasts are the major cellular sources of collagens and lysyl oxidases in normal liver and early after injury

Perepelyuk

Terajima²,

Wang

et al. 2013

American Journal of Physiology-Gastrointestinal and Liver Physi

156

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Perepelyuk M, Terajima M, Wang AY, Georges PC, Janmey PA, Yamauchi M, Wells RG. Hepatic stellate cells and portal fibroblasts are the major cellular sources of collagens and lysyl oxidases in normal liver and early after injury. Am J Physiol Gastrointest Liver Physiol 304: G605-G614, 2013. First published January 17, 2013 doi:10.1152/ajpgi.00222.2012.-Liver fibrosis is characterized by excessive deposition of extracellular matrix proteins by myofibroblasts derived from hepatic stellate cells and portal fibroblasts. Activation of these precursors to myofibroblasts requires matrix stiffness, which results in part from increased collagen crosslinking mediated by lysyl oxidase (LOX) family proteins. The aims of this study were to characterize the mechanical changes of early fibrosis, to identify the cells responsible for LOX production in early injury, and to determine which cells in normal liver produce collagens and elastins, which serve as substrates for LOXs early after injury. Hepatocytes and liver nonparenchymal cells were isolated from normal and early-injured liver and examined immediately for expression of LOXs and matrix proteins. We found that stellate cells and portal fibroblasts were the major cellular sources of fibrillar collagens and LOXs in normal liver and early after injury (1 day after bile duct ligation and 2 and 7 days after CCl 4 injury). Activity assays using stellate cells and portal fibroblasts in culture demonstrated significant increases in LOX family enzymatic activity as cells became myofibroblastic. LOX family-mediated deoxypyridinoline and pyridinoline cross-links increased after CCl 4-mediated injury. There was a significant association between liver stiffness (as quantified by the shear storage modulus G=) and deoxypyridinoline levels; increased deoxypyridinoline levels were also coincident with significantly increased elastic resistance to large strain deformations, consistent with increased cross-linking of the extracellular matrix. These data suggest a model in which the liver is primed to respond quickly to injury, activating potential mechanical feed-forward mechanisms.collagen cross-linking; liver fibrosis; pyridinoline; deoxypyridinoline; extracellular matrix PATHOLOGICAL FIBROSIS is characterized by excessive accumulation of extracellular matrix (ECM) proteins, most notably fibrillar collagens. The majority of ECM in organ fibrosis is deposited by myofibroblasts, proliferative and motile cells characterized by expression of ␣-smooth muscle actin (␣-SMA), which migrate to the site of injury or differentiate from preexisting cells in the organ. In the liver, the major myofibroblast precursor cells are hepatic stellate cells and portal fibroblasts.Myofibroblast differentiation from precursor cells requires mechanical tension. This has been shown in vitro for general fibroblast-to-myofibroblast activation (14), as well as for hepatic stellate cell and portal fibroblast activation to myofibroblasts in the liver (19,36) and suggests that increases in liver stiffness precede myofi...

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“…Because collagen I is the most common protein found in vertebrate animals and is structurally highly conserved, it is generally well tolerated for in vivo studies, and multiple cell types readily adhere to this substrate. In addition, the elastic moduli of a collagen I gel can be readily manipulated by varying collagen orientation, fibril crosslinking, concentration, or even biochemical modification or mutation (Christner et al, 2006;Girton et al, 1999;Martin et al, 1996;Roeder et al, 2002), thereby increasing its biological versatility (Elbjeirami et al, 2003;Grinnell, 2003). The magnitude and directional orientation of externally imposed tension can also be easily manipulated with collagen preparations.…”

Section: D Organotypic Model Systemsmentioning

confidence: 99%

Demystifying the Effects of a Three‐Dimensional Microenvironment in Tissue Morphogenesis

Johnson

Leight

Weaver

2007

Methods in Cell Biology

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Tissue morphogenesis and homeostasis are dependent on a complex dialogue between multiple cell types and chemical and physical cues in the surrounding microenvironment. The emergence of engineered three-dimensional (3D) tissue constructs and the development of tractable methods to recapitulate the native tissue microenvironment ex vivo has led to a deeper understanding of tissuespecific behavior. However, much remains unclear about how the microenvironment and aberrations therein directly affect tissue morphogenesis and behavior. Elucidating the role of the microenvironment in directing tissue-specific behavior will aid in the development of surrogate tissues and tractable approaches to diagnose and treat chronic-debilitating diseases such as cancer and atherosclerosis. Toward this goal, 3D organotypic models have been developed to clarify the mechanisms of epithelial morphogenesis and the subsequent maintenance of tissue homeostasis. Here we describe the application of these 3D culture models to illustrate how the microenvironment plays a critical role in regulating mammary tissue function and signaling, and discuss the rationale for applying precisely defined organotypic culture assays to study epithelial cell behavior. Experimental methods are provided to generate and manipulate 3D organotypic cultures to study the effect of matrix stiffness and matrix dimensionality on epithelial tissue morphology and signaling. We end by discussing technical limitations of currently available systems and by presenting opportunities for improvement.

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Enhancing mechanical properties of tissue‐engineered constructs via lysyl oxidase crosslinking activity

Cited by 143 publications

References 32 publications

Increased stiffness of the rat liver precedes matrix deposition: implications for fibrosis

Increased stiffness of the rat liver precedes matrix deposition: implications for fibrosis

Hepatic stellate cells and portal fibroblasts are the major cellular sources of collagens and lysyl oxidases in normal liver and early after injury

Demystifying the Effects of a Three‐Dimensional Microenvironment in Tissue Morphogenesis

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