Collagen molecules in articular cartilage have an exceptionally long lifetime, which makes them susceptible to the accumulation of advanced glycation end products (AGEs). In fact, in comparison to other collagen-rich tissues, articular cartilage contains relatively high amounts of the AGE pentosidine. To test the hypothesis that this higher AGE accumulation is primarily the result of the slow turnover of cartilage collagen, AGE levels in cartilage and skin collagen were compared with the degree of racemization of aspartic acid (% D-Asp, a measure of the residence time of a protein). AGE (N ⑀ -(carboxymethyl)lysine, N ⑀ -(carboxyethyl)lysine, and pentosidine) and % D-Asp concentrations increased linearly with age in both cartilage and skin collagen (p < 0.0001). The rate of increase in AGEs was greater in cartilage collagen than in skin collagen (p < 0.0001). % D-Asp was also higher in cartilage collagen than in skin collagen (p < 0.0001), indicating that cartilage collagen has a longer residence time in the tissue, and thus a slower turnover, than skin collagen. In both types of collagen, AGE concentrations increased linearly with % D-Asp (p < 0.0005). Interestingly, the slopes of the curves of AGEs versus % D-Asp, i.e. the rates of accumulation of AGEs corrected for turnover, were identical for cartilage and skin collagen. The present study thus provides the first experimental evidence that protein turnover is a major determinant in AGE accumulation in different collagen types. From the age-related increases in % D-Asp the half-life of cartilage collagen was calculated to be 117 years and that of skin collagen 15 years, thereby providing the first reasonable estimates of the half-lives of these collagens.Nonenzymatic glycation is a post-translational modification of proteins in vivo, which is initiated by the spontaneous reaction of sugars with lysine residues in proteins and eventually results in the formation of advanced glycation end products (AGEs), 1 such as N ⑀ -(carboxymethyl)lysine (CML), N ⑀ -(carboxyethyl)lysine (CEL), and pentosidine (1-3). Because AGEs are irreversible chemical modifications of protein, they accumulate with age in long lived proteins such as lens crystallins and tissue collagens (1, 3-9). Because collagen molecules in articular cartilage have an exceptionally long lifetime (Ͼ100 years) (10, 11), they are highly susceptible to the accumulation of AGEs. Indeed, in comparison to other collagen-rich tissues (such as skin), articular cartilage contains relatively high amounts of pentosidine (3, 12). Although differences in AGE levels between different proteins have been attributed to differences in protein turnover rates (3,(12)(13)(14), no quantitative evidence to support this assumption is available.To compare protein turnover rates, information on the residence time of a protein in tissue can be obtained from the racemization of aspartic acid. Amino acids are incorporated into peptides and proteins as the L-enantiomers. During aging, racemization slowly converts the L-form into a race...

Chronic organ injury leads to fibrosis and eventually organ failure. Fibrosis is characterized by excessive synthesis, remodeling, and contraction of extracellular matrix produced by myofibroblasts. Myofibroblasts are the key cells in the pathophysiology of fibrotic disorders and their differentiation can be triggered by multiple stimuli. To develop anti-fibrotic therapies, it is of paramount importance to understand the molecular basis of the signaling pathways contributing to the activation and maintenance of myofibroblasts. Several signal transduction pathways, such as transforming growth factor (TGF)-β, Wingless/Int (WNT), and more recently yes-associated protein 1 (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) signaling, have been linked to the pathophysiology of fibrosis. Activation of the TGF-β1-induced SMAD complex results in the upregulation of genes important for myofibroblast function. Similarly, WNT-stabilized β-catenin translocates to the nucleus and initiates transcription of its target genes. YAP and TAZ are two transcriptional co-activators from the Hippo signaling pathway that also rely on nuclear translocation for their functioning. These three signal transduction pathways have little molecular similarity but do share one principle: the cytosolic/nuclear regulation of its transcriptional activators. Past research on these pathways often focused on the isolated cascades without taking other signaling pathways into account. Recent developments show that parts of these pathways converge into an intricate network that governs the activation and maintenance of the myofibroblast phenotype. In this review, we discuss the current understanding on the signal integration between the TGF-β, WNT, and YAP/TAZ pathways in the development of organ fibrosis. Taking a network-wide view on signal transduction will provide a better understanding on the complex and versatile processes that underlie the pathophysiology of fibrotic disorders.

Background-Galectin-3 has been implicated in the development of organ fibrosis. It is unknown whether it is a relevant therapeutic target in cardiac remodeling and heart failure. Methods and Results-Galectin-3 knock-out and wild-type mice were subjected to angiotensin II infusion (2.5 µg/kg for 14 days) or transverse aortic constriction for 28 days to provoke cardiac remodeling. The efficacy of the galectin-3 inhibitor N-acetyllactosamine was evaluated in TGR(mREN2)27 (REN2) rats and in wild-type mice with the aim of reversing established cardiac remodeling after transverse aortic constriction. In wild-type mice, angiotensin II and transverse aortic constriction perturbations caused left-ventricular (LV) hypertrophy, decreased fractional shortening, and increased LV end-diastolic pressure and fibrosis (P<0.05 versus control wild type). Galectin-3 knock-out mice also developed LV hypertrophy but without LV dysfunction and fibrosis (P=NS). In REN2 rats, pharmacological inhibition of galectin-3 attenuated LV dysfunction and fibrosis. To elucidate the beneficial effects of galectin-3 inhibition on myocardial fibrogenesis, cultured fibroblasts were treated with galectin-3 in the absence or presence of galectin-3 inhibitor. Inhibition of galectin-3 was associated with a downregulation in collagen production (collagen I and III), collagen processing, cleavage, cross-linking, and deposition. Similar results were observed in REN2 rats. Inhibition of galectin-3 also attenuated the progression of cardiac remodeling in a long-term transverse aortic constriction mouse model. Conclusions-Genetic disruption and pharmacological inhibition of galectin-3 attenuates cardiac fibrosis, LV dysfunction, and subsequent heart failure development. Drugs binding to galectin-3 may be potential therapeutic candidates for the prevention or reversal of heart failure with extensive fibrosis. (Circ Heart Fail. 2013;6:107-117.)

The hallmark of fibrotic processes is an excessive accumulation of collagen. The deposited collagen shows an increase in pyridinoline cross-links, which are derived from hydroxylated lysine residues within the telopeptides. This change in cross-linking is related to irreversible accumulation of collagen in fibrotic tissues. The increase in pyridinoline cross-links is likely to be the result of increased activity of the enzyme responsible for the hydroxylation of the telopeptides (telopeptide lysyl hydroxylase, or TLH). Although the existence of TLH has been postulated, the gene encoding TLH has not been identified. By analyzing the genetic defect of Bruck syndrome, which is characterized by a pyridinoline deficiency in bone collagen, we found two missense mutations in exon 17 of PLOD2, thereby identifying PLOD2 as a putative TLH gene. Subsequently, we investigated fibroblasts derived from fibrotic skin of systemic sclerosis (SSc) patients and found that PLOD2 mRNA is highly increased indeed. Furthermore, increased pyridinoline cross-link levels were found in the matrix deposited by SSc fibroblasts, demonstrating a clear link between mRNA levels of the putative TLH gene (PLOD2) and the hydroxylation of lysine residues within the telopeptides. These data underscore the significance of PLOD2 in fibrotic processes.The biosynthesis of collagen molecules involves several intracellular post-translational modifications followed by excretion and extracellular aggregation of the collagen molecules into fibrils, which are subsequently stabilized by intermolecular cross-links (1, 2). Two related routes are responsible for the formation of these collagen cross-links, namely the allysine route, in which a lysine residue in the telopeptide is converted by lysyl oxidase into the aldehyde allysine, and the hydroxyallysine route, in which a hydroxylysine residue in the telopeptide is converted into the aldehyde hydroxyallysine. Subsequently, the allysine or the hydroxyallysine reacts with a Lys or Hyl residue in the triple helix to form di-, tri-, or tetrafunctional cross-links (3-6). The mature cross-links hydroxylysylpyridinoline or lysylpyridinoline are formed via the hydroxyallysine route and occur in a variety of connective tissues such as bone, tendon, ligaments, and cartilage (7). In contrast, collagen in the skin is mainly cross-linked via the allysine route. Interestingly, in fibrotic skin (lipodermatosclerosis, keloid) and organ fibrosis (lung, liver), which is characterized by an excessive accumulation of collagen, an increase in crosslinks derived from the hydroxyallysine route is found (8 -15). It has been shown that the amount of hydroxyallysine-derived cross-links is related to the irreversible accumulation of collagen in fibrotic tissues, indicating that collagen containing hydroxyallysine-derived cross-links is more difficult to degrade than collagen containing allysine-derived cross-links (10 -12, 14, 15). Inhibition of the formation of hydroxyallysine-derived cross-links in fibrosis is therefore likely to resu...