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  • br Materials and Methods br

    2020-05-20


    Materials and Methods
    Results
    Discussion HSCs have been identified as major collagen-producing all trans retinoic acid in the injured liver (1), and activation of HSCs has become the central link for liver fibrosis (2). The factors that regulate the biological behavior of HSCs include soluble regulatory factors and nonsoluble regulatory factors, the latter mainly refer to ECM itself. The interaction between HSCs and ECM alters the ability of HSCs to create ECM, which means ECM can directly regulate the activation of HSCs. A previous study has shown that HSCs have no active proliferative activity in the matrix culture simulating the environment of Disse space and produce only small amounts of ECM (13). In the plastic culture dish without any packets, HSCs show active proliferation and may gradually transform into myofibroblasts with activation state, generating a large amount of collagen (13). HSCs produce different ECM components when cultured in different types of single collagen matrix. This ECM-HSC interaction was previously attributed to integrin receptors that can upregulate MMP-2 (14), but there is no evidence that the integrin receptors can mediate activation of HSCs. The discovery of DDR has great significance for explaining the phenomenon that HSCs have different biological activities under different conditions of ECM. DDRs, a novel subfamily of receptor tyrosine kinases, were discovered during the search for tyrosine kinase proteins expressed in human malignancies 5, 6. DDRs can modulate cell responses such as adhesion, migration, differentiation, survival and proliferation, in response to the changes in the amount and configuration of collagen in microenvironment. In in vitro primary culture, activated HSCs induce DDR2 expression, whereas HSCs in period of quiescence restored by matrix gel decreased DDR2 expression (15). Type I collagen can upregulate DDR2 mRNA expression of HSCs and promote HSC tyrosinephosphorylation (15). HSCs with DDR2 overexpression showed stronger cell proliferation and migration activity (15). As a result, a positive feedback mechanism between collagen and HSCs can be formed: collagen induces HSCs activation and DDR2 expression, and HSCs with the overexpression of DDR2 produce more collagen by proliferation and activation. The in vitro results are in line with our in vivo study on liver fibrosis, indicating DDR2 may regulate the biological activation of HSCs, leading to liver fibrosis in response to the changes in collagen contents after alcohol-induced liver injury.
    Introduction Diabetes mellitus is characterized by impaired glucose metabolism and a doubled risk of bone fracture [1]. The pathogenesis of diabetic bone fragility can be broadly divided into cellular and extracellular matrix (ECM) aspects. Cellular complications include diminished osteoblast differentiation [2], increased osteoblast apoptosis [3], and increased differentiation of mesenchymal stromal cells into adipocytes at the expense of osteoblasts [4], [5]. Together, these cellular events down-regulate bone formation in diabetes, and promote diabetic osteopenia. In addition to cellular abnormalities, alterations in the organic extracellular matrix of bone are typical in diabetes. Collagen is the most abundant protein in bone organic matrix, and it undergoes intra- and extracellular post-translational modifications in order to help form a functional extracellular matrix. To stabilize collagen fibrils, lysyl oxidase catalyzes the oxidative deamination of lysine or hydroxylysine residues in the collagen telopeptide regions resulting in the formation of peptidyl aldehydes. Lysine or hydroxylysine-derived aldehydes spontaneously react to form intra- and intermolecular cross-links between collagen molecules [6]. Lysyl oxidase-dependent collagen cross-linking is essential for bone strength [7]. Elevated levels of glucose in diabetes result in non-enzymatic modifications of extra- and intra-cellular proteins. Aldoses, ketoses and oxidized lipids react with basic residues of proteins, and initiate a complex non-enzymatic cascade of modifications resulting in heterogeneous products collectively known as advanced glycation end products (AGEs). Some AGEs interfere with the function of proteins by causing non-enzymatic cross-linking. Long-lived proteins such as type I collagen accumulate higher levels of AGEs than short-lived proteins. Non-enzymatic AGE modification and non-enzymatic cross-linking of collagen molecules interfere with the normal structure of collagen fibrils, which reduces bone strength in diabetes [8].