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Effect of Collagen Turnover on the Accumulation of Advanced Glycation End Products
Abstract
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
ε-(carboxymethyl)lysine,N
ε-(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.
... Furthermore, AGEs increase the oxidation of low-density lipoprotein (LDL) and promote the development of atherosclerosis [89,90]. Therefore, glycated LDLs are more sensitive to oxidation [90]; they are reduced with difficulty and promote the formation of antibodies that bind AGEs located in vessel walls, which amplify the development of vascular inflammation and atherosclerosis [91]. Glo-1 overexpression has beneficial vascular effects, reduces ROS, and protects against atherogenic LDL formation [92]. ...
... The levels of AGEs (CML, CEL, and pentosides) in skin collagen fibres increase linearly with age [91,149,156]. Verzijl et al. [91] showed that the long turnover time of collagen in the skin is the main reason for the accumulation of AGEs. Collagen is critical not only to the mechanical framework of the skin but also to several cellular processes; it is impaired by glycation in multiple ways [147]. ...
... The levels of AGEs (CML, CEL, and pentosides) in skin collagen fibres increase linearly with age [91,149,156]. Verzijl et al. [91] showed that the long turnover time of collagen in the skin is the main reason for the accumulation of AGEs. Collagen is critical not only to the mechanical framework of the skin but also to several cellular processes; it is impaired by glycation in multiple ways [147]. ...
Ageing is a composite process that involves numerous changes at the cellular, tissue, organ and whole-body levels. These changes result in decreased functioning of the organism and the development of certain conditions, which ultimately lead to an increased risk of death. Advanced glycation end products (AGEs) are a family of compounds with a diverse chemical nature. They are the products of non-enzymatic reactions between reducing sugars and proteins, lipids or nucleic acids and are synthesised in high amounts in both physiological and pathological conditions. Accumulation of these molecules increases the level of damage to tissue/organs structures (immune elements, connective tissue, brain, pancreatic beta cells, nephrons, and muscles), which consequently triggers the development of age-related diseases, such as diabetes mellitus, neurodegeneration, and cardiovascular and kidney disorders. Irrespective of the role of AGEs in the initiation or progression of chronic disorders, a reduction in their levels would certainly provide health benefits. In this review, we provide an overview of the role of AGEs in these areas. Moreover, we provide examples of lifestyle interventions, such as caloric restriction or physical activities, that may modulate AGE formation and accumulation and help to promote healthy ageing.
... The resulting inter-molecular crosslinks alter collagen structure and function by reducing the efficacy of collagenolytic matrix metalloproteinases (MMPs), thereby creating a more compact collagen structure that can disrupt the collagen meshwork in organs such as the skin and omentum [5,[7][8][9][10][11][12]. As a non-enzymatic process, AGEs tend to form on long-lived, low-turnover proteins such as collagen and are present at higher rates in older individuals, as well as diabetic patients who have higher levels of blood glucose [13][14][15][16]. AGE accumulation results in collagen that is more aligned and stiffer than non-crosslinked collagen, which has been shown to increase cancer cell migration [17]. ...
... The staining pattern may reflect AGE modification of collagen as well as collagen-bound cells. Collagen is a long-lived protein with a half-life of 15 years or greater [13]. Of note, many cell types have been shown to be AGE-modified [13,[35][36][37][38]. ...
... Collagen is a long-lived protein with a half-life of 15 years or greater [13]. Of note, many cell types have been shown to be AGE-modified [13,[35][36][37][38]. The use of alternative methods of tissue preparation, involving fixation and permeabilization, may enable better penetration of AGE antibodies through the mesothelial cell layer to access the underlying collagen in the intact tissue context. ...
Ovarian cancer is the sixth leading cause of cancer-related death in women, and both occurrence and mortality are increased in women over the age of 60. There are documented age-related changes in the ovarian cancer microenvironment that have been shown to create a permissive metastatic niche, including the formation of advanced glycation end products, or AGEs, that form crosslinks between collagen molecules. Small molecules that disrupt AGEs, known as AGE breakers, have been examined in other diseases, but their efficacy in ovarian cancer has not been evaluated. The goal of this pilot study is to target age-related changes in the tumor microenvironment with the long-term aim of improving response to therapy in older patients. Here, we show that AGE breakers have the potential to change the omental collagen structure and modulate the peritoneal immune landscape, suggesting a potential use for AGE breakers in the treatment of ovarian cancer.
... Collagen comprises approximately 20-40% of the ECM in the vessel wall [20], and it is primarily responsible for vessel mechanical resistance. Ageing changes the structure of the collagen monomer and reduces protofibril assembly (Fig. 2), resulting in the production of a less dense and more heterogeneous structure in older adults [21]. Total collagen density in vessels increases with ageing, despite a decline in collagen synthesis, as collagen has a long half-life [22]. ...
... Collagen remains in the body for an extended period and is hence prone to ageing-related alterations, such as oxidation, carbonylation, carbamylation, and glycation. Collagen undergoes non-enzymatic modification to form AGEs (Fig. 2), which give rise to cross-linking during ageing [28], forming a network of longer and thicker fibrils [21]. These changes alter the ECM physical and mechanical properties and hence interactions with cells. ...
Each step in angiogenesis is regulated by the extracellular matrix (ECM). Accumulating evidence indicates that ageing-related changes in the ECM driven by cellular senescence lead to a reduction in neovascularisation, reduced microvascular density, and an increased risk of tissue ischaemic injury. These changes can lead to health events that have major negative impacts on quality of life and place a significant financial burden on the healthcare system. Elucidating interactions between the ECM and cells during angiogenesis in the context of ageing is neceary to clarify the mechanisms underlying reduced angiogenesis in older adults. In this review, we summarize ageing-related changes in the composition, structure, and function of the ECM and their relevance for angiogenesis. Then, we explore in detail the mechanisms of interaction between the aged ECM and cells during impaired angiogenesis in the older population for the first time, discussing diseases caused by restricted angiogenesis. We also outline several novel pro-angiogenic therapeutic strategies targeting the ECM that can provide new insights into the choice of appropriate treatments for a variety of age-related diseases. Based on the knowledge gathered from recent reports and journal articles, we provide a better understanding of the mechanisms underlying impaired angiogenesis with age and contribute to the development of effective treatments that will enhance quality of life.
... The extent of ECM damage is likely to be exacerbated by the high concentration of these species (e.g. they comprise >50% of the artery wall by dry mass), the low level and activity of antioxidant defense and repair mechanisms external to cells [22], and the long half-lives of many ECM proteins (~70 years for elastin [23], 60-70 days for some collagens [24]). These factors have been proposed to result in an accumulation of modifications on the ECM of many tissues [25,26]. ...
... 74 years for elastin [23], 60-70 days for some collagens [24]). These factors have been proposed to result, in vivo, in an accumulation of modifications on the ECM of tissues, with this enhanced by disease [25,26]. Consistent with this hypothesis, nitration was detected on a large number of core ECM and ECM-associated proteins in the total proteome dataset (Fig. 2B). ...
Peroxynitrous acid/peroxynitrite (ONOOH/ONOO-) is a powerful oxidizing/nitrating system formed at sites of inflammation, which can modify biological targets, and particularly proteins. Here, we show that multiple proteins from primary human coronary artery smooth muscle cells are nitrated, with LC-MS peptide mass mapping providing data on the sites and extents of changes on cellular and extracellular matrix (ECM) proteins. Evidence is presented for selective and specific nitrations at Tyr and Trp on 11 cellular proteins (out of 3668, including 205 ECM species) in the absence of added reagent ONOOH/ONOO-, with this being consistent with low-level endogenous nitration. A number of these have key roles in cell signaling/sensing and protein turnover. With added ONOOH/ONOO-, more proteins were modified (84 total; with 129 nitrated Tyr and 23 nitrated Trp, with multiple modifications on some proteins), with this occurring at the same and additional sites to endogenous modification. With low concentrations of ONOOH/ONOO- (50 μM) nitration occurs on specific proteins at particular sites, and is not driven by protein or Tyr/Trp abundance, with modifications detected on some low abundance proteins. However, with higher ONOOH/ONOO- concentrations (500 μM), modification is primarily driven by protein abundance. ECM species are major targets and over-represented in the pool of modified proteins, with fibronectin and thrombospondin-1 being particularly heavily modified (12 sites in each case). Both endogenous and exogenous nitration of cell- and ECM-derived species may have significant effects on cell and protein function, and potentially be involved in the development and exacerbation of diseases such as atherosclerosis.
... Due to its avascular nature, it's likely a tear in the AF does not adapt or remodel in the same way other tissue or bone would [8]. Instead, structural changes are potentially irreversible as adult discs have limited healing potential [10]. Research suggests that the collagen turnover time in articular cartilage is approximately 100 years, and it may be even longer in the annulus fibrosus [9,10]. ...
... Instead, structural changes are potentially irreversible as adult discs have limited healing potential [10]. Research suggests that the collagen turnover time in articular cartilage is approximately 100 years, and it may be even longer in the annulus fibrosus [9,10]. Furthermore, injuries that affect the inner annulus or endplate can decompress the nucleus, slowing the "healing" processes which can then result in severe degenerative changes [7]. ...
Many in the scientific community have been debating the extent to which the spine can adapt. While it is widely accepted that vertebral bones can adapt and become stronger/denser with appropriately dosed loading, many unanswered questions remain regarding intervertebral discs (IVDs). Recently, some claim that IVDs can adapt to stress under load and become stronger and more resilient over time based on the findings from a recent study "Imaging of exercise-induced spinal remodeling in elite rowers" by Frenken et al 2022 in the Journal of Science and Medicine in Sport. The Frenken paper investigated the IVD Glycosaminoglycan (GAG) increases in elite rowers over the course of a training period, which was to indicate a positive adaptation to training based on the assumption that GAG is decreased in discs with degeneration. A cause-and-effect relationship does not exist and in fact, is unlikely to exist particularly with a historically high prevalence of disc degeneration in rowers. The Frenken paper supports the notion that the GAG content alters in response to certain stimuli; however, we cannot conclude that a temporary increase in GAG content supports the hypothesis that the IVD adapts positively to load. An alternative postulate is that repetitive biomechanical loading of the intervertebral disc through intra-lumbar flexion (as observed in rowing) does stimulate nutrient transport across the vertebral endplates; however, this biochemical response is of limited benefit and should not be perceived as a positive adaptation to the overall longevity and health of the disc. In this context, the elevation in GAG may be more appropriately interpreted as a maladaptive indicator of undesirable biomechanical intra-lumbar flexion stress that progressively drives cartilaginous endplate sclerosis leading ultimately to the clinically observed accelerated disc degeneration, future back pain symptoms, and reduced GAG concentration in rowers. This contrasting interpretation is consistent with the clinical observation 28 of intra-lumbar flexion biomechanical stress, the transient rise in GAG followed by a reduction that correlates with the radiological findings and back pain symptoms evolving over time in elite rowers.
... Previous studies have reported that aging induces collagen modification via mineralization, accumulation of advanced glycation end products, and depletion of glycosaminoglycans, which affects fiber stability and susceptibility to MMP-mediated degradation [54]. Moreover, collagen modification is known to cause a decrease in the turnover of collagen [55]. Thus, the simultaneous increase in MMP expression and collagen accumulation with aging may be part of the aging process in skeletal muscle. ...
Aging affects several tissues in the body, including skeletal muscle. Multiple types of collagens are localized in the skeletal muscle and contribute to the maintenance of normal muscle structure and function. Since the effects of aging on muscle fibers vary by muscle fiber type, it is expected that the effects of aging on intramuscular collagen might be influenced by muscle fiber type. In this study, we examined the effect of aging on collagen levels in the soleus (slow-twitch muscle) and gastrocnemius (fast-twitch muscle) muscles of 3-, 10-, 24-, and 28-month-old male C57BL/6J mice using molecular and morphological analysis. It was found that aging increased collagen I, III, and VI gene expression and immunoreactivity in both slow- and fast-twitch muscles and collagen IV expression in slow-twitch muscles. However, collagen IV gene expression and immunoreactivity in fast-twitch muscle were unaffected by aging. In contrast, the expression of the collagen synthesis marker heat shock protein 47 in both slow- and fast-twitch muscles decreased with aging, while the expression of collagen degradation markers increased with aging. Overall, these results suggest that collagen gene expression and immunoreactivity are influenced by muscle fiber type and collagen type and that the balance between collagen synthesis and degradation tends to tilt toward degradation with aging.
... Furthermore, AGEs can increase in ammation and oxidative stress in the body, which can further damage tissues and exacerbate diabetes complications (13). AGEs play an important role in the development and progression of T1DM, so they can re ect a patient's blood sugar over a longer period of time (14). It has been found that the generation of AGEs is faster in T1DM patients than the eradication of AGEs, leading to an accumulation of AGEs in the patient's body (15). ...
Background
People with diabetes mellitus are particularly vulnerable to chronic glycation of proteins and tissue damage caused by the buildup of advanced glycation end products (AGEs). A non-invasive optical technique called skin autofluorescence (SAF) can be used to detect the accumulation of AGEs in the skin. There is a significant association between elevated SAF and diabetes and it predicts the development of complications associated with diabetes. This study evaluated the correlation between SAF and both status and development and also SAF's possible clinical utility as a tool for detecting type 1 diabetes mellitus (T1DM) and its complications in children.
Methods
A systematic search was conducted across the following databases: PubMed, MEDLINE, EMBASE, Cochrane Central Register of Control Trials, Science Direct, Scopus, and Web of Science. We used a random effects model for FOX analysis to examined the link between AGEs detected through SAF levels and T1DM in children. The SAF values of children with and without T1DM were compared. Analysis of 95% confidence interval correlation coefficients based on DerSimonian and Laird methods was performed for application of SAF measurements as a noninvasive alternative to T1DM diagnosis and complications.
Results
Three case-control studies and one retrospective cohort study were included in the systematic review and meta-analysis. The AGE Reader® (DiagnOptics Technologies, Groningen, The Netherlands) was used to evaluate the SAF of all included studies. The results showed that there was statistically significant heterogeneity among the four studies (I2 = 82.00% P < 0.05). The random effect model showed that higher SAF levels were positively correlated with T1DM in children [0.20 (0.16, 0.25)], which indicated that in the four studies, the SAF value of T1DM children was higher than that of non-type 1 diabetic children.
Conclusion
Our findings suggest that measuring SAF levels may serve as a non-invasive marker for T1DM in children and its associated complications. Nevertheless, to obtain more robust conclusions and support specific data on T1DM complications, further research with a larger population and a longer follow-up duration is necessary.
... N-linked glycosylation happens in the lumen of the endoplasmic reticulum and Golgi apparatus, and plays a critical role in folding [53]. Protein turn-over has been demonstrated to be the biggest contributing factor to the accumulation of AGEs; this accumulation was shown to be much higher and quicker in cartilage collagen over skin collagen in humans, which corresponds to the faster turn-over rate of skin over cartilage [54]. The higher number of cross-links make the protein matrix of bone stiffer and increase its resistance to proteolysis, which subsequently affects remodelling; individuals with diabetes also display such changes [47]. ...
One of the key questions in forensic cases relates to some form of age inference, whether this is how old a crime scene is, when in time a particular crime was committed, or how old the victim was at the time of the crime. These age-related estimations are currently achieved through morphological methods with varying degrees of accuracy. As a result, biomolecular approaches are considered of great interest, with the relative abundances of several protein markers already recognized for their potential forensic significance; however, one of the greatest advantages of proteomic investigations over genomics ones is the wide range of post-translational modifications (PTMs) that make for a complex but highly dynamic resource of information. Here, we explore the abundance of several PTMs including the glycosylation, deamidation, and oxidation of several key proteins (collagen, fetuin A, biglycan, serum albumin, fibronectin and osteopontin) as being of potential value to the development of an age estimation tool worthy of further evaluation in forensic contexts. We find that glycosylations lowered into adulthood but deamidation and oxidation increased in the same age range.
... Advanced glycation end products (AGEs) are a group of molecules formed nonenzymatically, after the initial attachment of reducing sugars to amino groups of proteins, lipids, or nucleic acids [1]. Long-lived tissues are more prone to AGEs accumulation, and sustain damage due to the formation of cross-links, the modification of proteins, or from inflammation due to their binding to the receptor for AGEs (RAGE) [2][3][4]. Evidences are gathering for their possible involvement in aging [5] and age-related diseases, such as cardiovascular diseases, dementia, and diabetes [6]. ...
Background:
Advanced glycation end products (AGEs) are involved in age-related diseases, but the interaction of gut microbiota with dietary AGEs (dAGEs) and tissue AGEs in the population is unknown.
Objective:
Our objective was to investigate the association of dietary and tissue AGEs with gut microbiota in the population-based Rotterdam Study, using skin AGEs as a marker for tissue accumulation and stool microbiota as a surrogate for gut microbiota.
Design:
Dietary intake of three AGEs (dAGEs), namely carboxymethyl-lysine (CML), N-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MGH1), and carboxyethyl-lysine (CEL), was quantified at baseline from food frequency questionnaires. Following up after a median of 5.7 years, skin AGEs were measured using skin autofluorescence (SAF), and stool microbiota samples were sequenced (16S rRNA) to measure microbial composition (including alpha-diversity, beta-dissimilarity, and taxonomic abundances) as well as predict microbial metabolic pathways. Associations of both dAGEs and SAF with microbial measures were investigated using multiple linear regression models in 1052 and 718 participants, respectively.
Results:
dAGEs and SAF were not associated with either the alpha-diversity or beta-dissimilarity of the stool microbiota. After multiple-testing correction, dAGEs were not associated with any of the 188 genera tested, but were nominally inversely associated with the abundance of Barnesiella, Colidextribacter, Oscillospiraceae UCG-005, and Terrisporobacter, in addition to being positively associated with Coprococcus, Dorea, and Blautia. A higher abundance of Lactobacillus was associated with a higher SAF, along with several nominally significantly associated genera. dAGEs and SAF were nominally associated with several microbial pathways, but none were statistically significant after multiple-testing correction.
Conclusions:
Our findings did not solidify a link between habitual dAGEs, skin AGEs, and overall stool microbiota composition. Nominally significant associations with several genera and functional pathways suggested a potential interaction between gut microbiota and AGE metabolism, but validation is required. Future studies are warranted, to investigate whether gut microbiota modifies the potential impact of dAGEs on health.
... However, there is little current evidence to suggest that articular cartilage late in life is able to regenerate by existing chondrocytes. Furthermore, ECM proteins are known to have an extremely long half-life, such that prevention of ECM degradation is predicted to have a significant impact on cartilage stability 93,94 . This is highlighted by studies which showed that elimination of viable Small Molecule Impact on metabolism Impact on catabolic gene expression Impact on anabolic gene expression FX11 10 Inhibition of aerobic glycolysis Decreased Unknown GSK2837808A 10,34 Inhibition of aerobic glycolysis Minimal Increased 2-Deoxyglucose 42,43 Decreased glycolysis and oxidative phosphorylation Decreased Unknown Galactose 33,56 Increased oxidative phosphorylation, decreased glycolysis Decreased Unknown Dichloroacetate 43,44 Decreased glycolysis, increased oxidative phosphorylation Decreased Unknown 4-Methylumbelliferone 43,58 Decreased glycolysis, increased oxidative phosphorylation, Decreased Unknown Rotenone 9,50 Decreased oxidative phosphorylation Decreased Decreased Antimycin A 9 Decreased oxidative phosphorylation Decreased Decreased Amobarbital 55 Decreased oxidative phosphorylation Decreased Unknown Puerarin 58 Increased mitochondrial biogenesis Decreased Unknown 4-Aminobutyrate 22 Inhibition of Abat and reduced oxidative phosphorylation Decreased Unknown CB-839 18 Decreased glutaminolysis Decreased Unknown Etomoxir 81 Decreased fatty acid oxidation Decreased Unknown Azaserine 91 Inhibition of GFAT-1 mediated glutamine flux into hexosamine pathway Increased Decreased 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 chondrocytes in adult mice protected against OA or that elimination of senescent chondrocytes is also protective against OA 95,96 . ...
Osteoarthritis is a disease that impacts millions around the world, leading to significant financial and medical burden for patients and the healthcare system. However, no effective biomarkers or disease modifying therapeutics exist for the early identification and management of the disease. Inflammation drives chondrocytes to express ECM degrading enzymes and interruption of this pathway is a viable target to prevent degradation of cartilage. It has been demonstrated that inflammation can alter the intracellular metabolism of chondrocytes, a process known as metabolic reprogramming. This metabolic reprogramming is critical for cartilage breakdown by shifting chondrocytes to an ECM-catabolic state and likely as a potential therapeutic target for osteoarthritis. Metabolic modulators hold the potential to reduce chondrocyte inflammatory responses and protect cartilage. In this narrative review, we explore some of the existing examples of interactions between metabolism and inflammatory pathways in chondrocytes. We summarize the impact of inflammatory stimulation on various metabolic pathways and describe several examples by which targeting of metabolism is able to modulate ECM-degrading activity of chondrocytes to protect against cartilage damage.
... On the 10th day of culture, the cells start to secrete ECM and the production was found to be increased in proportion with a period of culture. The fully differentiated chondrocytes secrete ECM which contains type II collagen and aggrecan, which are predominant proteins secreted by the chondrocyte in cartilage [58,59]. The ECM secretion of differentiated chondrocytes contains proteoglycan (GAG), which is stained by Safranin O giving a red/orange colour (Fig. 8d). ...
C. gigantea extract incorporated self-cross-linked alginate dialdehyde-gelatin hydrogel scaffolds for cartilage tissue regeneration in vitro
... A portion of proliferating chondrocytes become hypertrophic, therefore the degradation rate parameter r c 4 should equal the rate parameter for production of hypertrophic chondrocytes, i.e. r c 4 = r hc 1 . The half-lives of type II collagen and proteoglycan are approximately 90 years and 25 years, respectively [98][99][100] . We estimate r co 3 and r pg 3 using (25) . ...
Background and objective:
Osteoarthritis (OA) is a pervasive and debilitating disease, wherein degeneration of cartilage features prominently. Despite extensive research, we do not yet understand the cause or progression of OA. Studies show biochemical, mechanical, and biological factors affect cartilage health. Mechanical loads influence synthesis of biochemical constituents which build and/or break down cartilage, and which in turn affect mechanical loads. OA-associated biochemical profiles activate cellular activity that disrupts homeostasis. To understand the complex interplay among mechanical stimuli, biochemical signaling, and cartilage function requires integrating vast research on experimental mechanics and mechanobiology-a task approachable only with computational models. At present, mechanical models of cartilage generally lack chemo-biological effects, and biochemical models lack coupled mechanics, let alone interactions over time.
Methods:
We establish a first-of-its kind virtual cartilage: a modeling framework that considers time-dependent, chemo-mechano-biologically induced turnover of key constituents resulting from biochemical, mechanical, and/or biological activity. We include the "minimally essential" yet complex chemical and mechanobiological mechanisms. Our 3-D framework integrates a constitutive model for the mechanics of cartilage with a novel model of homeostatic adaptation by chondrocytes to pathological mechanical stimuli, and a new application of anisotropic growth (loss) to simulate degradation clinically observed as cartilage thinning.
Results:
Using a single set of representative parameters, our simulations of immobilizing and overloading successfully captured loss of cartilage quantified experimentally. Simulations of immobilizing, overloading, and injuring cartilage predicted dose-dependent recovery of cartilage when treated with suramin, a proposed therapeutic for OA. The modeling framework prompted us to add growth factors to the suramin treatment, which predicted even better recovery.
Conclusions:
Our flexible framework is a first step toward computational investigations of how cartilage and chondrocytes mechanically and biochemically evolve in degeneration of OA and respond to pharmacological therapies. Our framework will enable future studies to link physical activity and resulting mechanical stimuli to progression of OA and loss of cartilage function, facilitating new fundamental understanding of the complex progression of OA and elucidating new perspectives on causes, treatments, and possible preventions.
... Collagen type I is not the only collagen responsible for the structural properties. Further, the turn-over, i.e. the physiological rebuilding of tissue is important for the accumulation of AGEs since they are only built during the process of aging or with enhanced exposure increased glycation levels (Verzijl et al., 2000). Depending on the glycation level and exposure, the AGEs density might also vary across the crosssection of the fibril and influence the mechanical properties additionally. ...
Cross-links are considered to be a key component of collagen fibrils as they can change the fibrillar behavior in various ways. Advanced-Glycation Endproducts (AGEs), one particular type of cross-links, have been shown to accumulate and impair the mechanical properties of collageneous tissues, whereas enzymatic cross-links (ECLs) are known for stabilizing the structure of the fibril. However, the reasons for whether a given type of cross-link improves or impairs the material properties remain unknown. Here, we use coarse-grained steered molecular models to evaluate the effect of AGEs and ECLs cross-links content on the deformation and failure properties of collagen fibrils. Our simulations show that the collagen fibrils stiffen at high strain levels when the AGEs content exceeds a critical value. In addition, the strength of the fibril increases with AGEs accumulation. By analyzing the forces within the different types of cross-links (AGEs and ECLs) as well as their failure, we demonstrate that a change of deformation mechanism is at the origin of these observations. A high AGEs content reinforces force transfer through AGEs cross-links rather than through friction between sliding tropocollagen molecules. We show that this failure mechanism, which is associated with lower energy dissipation, results in more abrupt failure of the collagen fibril. Our results provide a direct and causal link between increased AGEs content, inhibited intra-fibrillar sliding, increased stiffness, and abrupt fibril fracture. Therefore, they explain the mechanical origin of bone brittleness as commonly observed in elderly and diabetic populations. Our findings contribute to a better understanding of the mechanisms underlying impaired tissue behaviour due to elevated AGEs content and could enable targeted measures regarding the reduction of specific collagen cross-linking levels.
... Historically, OA was seen as a cartilage "wear and tear" phenomenon, but it is now appreciated that its etiology is complex. Systemic inflammation [9], local inflammatory factors and free radicals [10], enzymatic destruction of cartilage extracellular matrix components and subchondral bone [11,12], resident stem cell dysfunction [13,14], production of adipokines by fat [15,16], aging [17,18] and previous joint trauma [19] are all implicated. ...
Background:
Agricultural workers have a higher incidence of osteoarthritis (OA), but the etiology behind this phenomenon is unclear. Calving season, which occurs in mid- to late-winter for ranchers, includes physical conditions that may elevate OA risk. Our primary aim was to determine whether OA biomarkers are elevated at the peak of calving season compared to pre-season, and to compare these data with joint health survey information from the subjects. Our secondary aim was to detect biomarker differences between male and female ranchers.
Methods:
During collection periods before and during calving season, male (n = 28) and female (n = 10) ranchers completed joint health surveys and provided samples of blood, urine, and saliva for biomarker analysis. Statistical analyses examined associations between mean biomarker levels and survey predictors. Ensemble cluster analysis identified groups having unique biomarker profiles.
Results:
The number of calvings performed by each rancher positively correlated with plasma IL-6, serum hyaluronic acid (HA) and urinary CTX-I. Thiobarbituric acid reactive substances (TBARS), a marker of oxidative stress, was significantly higher during calving season than pre-season and was also correlated with ranchers having more months per year of joint pain. We found evidence of sexual dimorphism in the biomarkers among the ranchers, with leptin being elevated and matrix metalloproteinase-3 diminished in female ranchers. The opposite was detected in males. WOMAC score was positively associated with multiple biomarkers: IL-6, IL-2, HA, leptin, C2C, asymmetric dimethylarginine, and CTX-I. These biomarkers represent enzymatic degradation, inflammation, products of joint destruction, and OA severity.
Conclusions:
The positive association between number of calvings performed by each rancher (workload) and both inflammatory and joint tissue catabolism biomarkers establishes that calving season is a risk factor for OA in Montana ranchers. Consistent with the literature, we found important sex differences in OA biomarkers, with female ranchers showing elevated leptin, whereas males showed elevated MMP-3.
... Collagen type I is not the only collagen responsible for the structural properties. Further, the turn-over, i.e. the physiological rebuilding of tissue is important for the accumulation of AGEs since they are only built during the process of aging or with enhanced exposure increased glycation levels (Verzijl et al., 2000). Depending on the glycation level and exposure, the AGEs density might also vary across the crosssection of the fibril and influence the mechanical properties additionally. ...
Cross-links are considered to be a key component of collagen fibrils as they can change the fibrillar behavior in various ways. Advanced-Glycation Endproducts (AGEs), one particular type of cross-links, have been shown to accumulate and impair the mechanical properties of collageneous tissues, whereas enzymatic cross-links (ECLs) are known for stabilizing the structure of the fibril. However, the reasons for whether a given type of cross-link improves or impairs the material properties remain unknown. Here, we use coarse-grained steered molecular models to evaluate the effect of AGEs and ECLs cross-links content on the deformation and failure properties of collagen fibrils. Our simulations show that the collagen fibrils stiffen at high strain levels when the AGEs content exceeds a critical value. In addition, the strength of the fibril increases with AGEs accumulation. By analyzing the forces within the different types of cross-links (AGEs and ECLs) as well as their failure, we demonstrate that a change of deformation mechanism is at the origin of these observations. A high AGEs content reinforces force transfer through AGEs cross-links rather than through friction between sliding tropocollagen molecules. We show that this failure mechanism, which is associated with lower energy dissipation, results in more abrupt failure of the collagen fibril. Our results provide a direct and causal link between increased AGEs content, inhibited intra-fibrillar sliding, increased stiffness, and abrupt fibril fracture. Therefore, they explain the mechanical origin of bone brittleness as commonly observed in elderly and diabetic populations. Our findings contribute to a better understanding of the mechanisms underlying impaired tissue behaviour due to elevated AGEs content and could enable targeted measures regarding the reduction of specific collagen cross-linking levels.
... Collagen and elastin are prevailing proteins in ECM, they have long period of half-life, and, hence, are subjected to pathological non-enzymatic modifications such as glycation, carbonylation, and carbamylation [1]. Advanced glycation end products (AGEs) are considered among the factors contributing to aging, they are detected in different tissues and organs such as kidneys, bones, eyes, skeletal muscles, cartilage, arterial walls, and brain [4][5][6][7]. AGEs are capable of crosslinking and, hence, strengthening collagen structures. In response to the increased stiffness of ECM cells express enzymes remodelling ECM in order to maintain preferable stiffness of the ECM [8]. ...
Extracellular matrix (ECM) is an extracellular tissue structure that, in addition to mechanical support to the cell, is involved in regulation of many cellular processes, including chemical transport, growth, migration, differentiation, and cell senescence. Age-related changes in the structure and composition of the matrix and increase of ECM stiffness with age affect functioning of many tissues and contribute to the development of various pathological conditions. This review considers age-related changes of ECM in various tissues and organs, in particular, effect of ECM changes on aging is discussed.
... Collagen α1(XI protein was detectable at day 0 and day 7 and, in agreement with the mRNA levels, collagen α1(XI) protein reached maximally detectable levels in cell lysates by day 14. The protein detected on day 21 remained high despite the significant decrease in mRNA levels, a result that is not surprising given the long half-life of collagen proteins [24,25]. ...
Cartilage development is a tightly regulated process that involves multiple molecules and signaling pathways. The loss of expression of a single gene can alter cell behavior and disrupt the development of the cartilage. Col11a1 encodes the alpha one chain of the minor fibrillar collagen type XI that is essential in skeletal development. We used an RNAi mediated knockdown approach to investigate the role of Col11a1 expression during chondrogenesis in ATDC5 cells. Col11a1 expression promotes the transition of mesenchymal cells to the chondrogenic phenotype, and reduction of Col11a1 expression interferes with cellular differentiation. Our results indicate that collagen α1(XI) protein is required for chondrocyte cell shape, matrix production, mineralization and gene expression through a mechanism that involves AKT/GSK3β/β-catenin and TCF/LEF activity in ATDC5 cells.
... During development or tissue healing, collagen forms a hierarchical structure wherein tropocollagen is incorporated into fibrils which subsequently form fibers [223]. The formation of this structure proceeds as self-assembly of collagen molecules by various intermolecular cross-links where enzymatic cross-linking plays a crucial role (Fig. 4B) [224].The protein has exceptionally long lifespan that depends on localization within different tissues with the half-life of cartilage collagen estimated at 117 years but skin collagen only at 15 years [225]. Historically, skin collagen glycation has been associated with diabetes as early as 1986 [226]. ...
Diabetes mellitus is the ninth leading cause of mortality worldwide. It is a complex disease that manifests as chronic hyperglycemia. Glucose exposure causes biochemical changes at the proteome level as reflected in accumulation of glycated proteins. A prominent example is hemoglobin A1c (HbA1c), a glycated protein widely accepted as a diabetic indicator. Another emerging biomarker is glycated albumin which has demonstrated utility in situations where HbA1c cannot be used. Other proteins undergo glycation as well thus impacting cellular function, transport and immune response. Accordingly, these glycated counterparts may serve as predictors for diabetic complications and thus warrant further inquiry. Fortunately, modern proteomics has provided unique analytic capability to enable improved and more comprehensive exploration of glycating agents and glycated proteins. This review broadly covers topics from epidemiology of diabetes to modern analytical tools such as mass spectrometry to facilitate a better understanding of diabetes pathophysiology. This serves as an attempt to connect clinically relevant questions with findings of recent proteomic studies to suggest future avenues of diabetes research.
... In addition, higher SAF levels predicted a higher risk of development of type 2 diabetes, and algorithms have been developed to predict individuals with the highest risk of developing type 2 diabetes based on age, BMI, and SAF levels [37]. SAF reflects fluorescent properties present in skin and skin collagen half-life is about 15 years [38]. As a result, SAF is a marker for cumulative accumulation over a much longer period of time than for instance haemoglobin A1c [39]. ...
Background
Skin autofluorescence (SAF) is a non-invasive measure reflecting accumulation of advanced glycation endproducts (AGEs) in the skin. Higher SAF levels are associated with an increased risk of developing type 2 diabetes and cardiovascular disease. An earlier genome-wide association study (GWAS) revealed a strong association between NAT2 variants and SAF. The aim of this study was to calculate SAF heritability and to identify additional genetic variants associated with SAF through genome-wide association studies (GWAS).
Results
In 27,534 participants without diabetes the heritability estimate of lnSAF was 33% ± 2.0% (SE) in a model adjusted for covariates. In meta-GWAS for lnSAF five SNPs, on chromosomes 8, 11, 15 and 16 were associated with lnSAF (P < 5 × 10–8): 1. rs2846707 (Chr11:102,576,358,C > T), which results in a Met30Val missense variant in MMP27 exon 1 (NM_022122.3); 2. rs2470893 (Chr15:75,019,449,C > T), in intergenic region between CYP1A1 and CYP1A2; with attenuation of the SNP-effect when coffee consumption was included as a covariate; 3. rs12931267 (Chr16:89,818,732,C > G) in intron 30 of FANCA and near MC1R; and following conditional analysis 4. rs3764257 (Chr16:89,800,887,C > G) an intronic variant in ZNF276, 17.8 kb upstream from rs12931267; finally, 30 kb downstream from NAT2 5. rs576201050 (Chr8:18,288,053,G > A).
Conclusions
This large meta-GWAS revealed five SNPs at four loci associated with SAF in the non-diabetes population. Further unravelling of the genetic architecture of SAF will help in improving its utility as a tool for screening and early detection of diseases and disease complications.
... Recent studies suggested that collagen fibers lose their parallel arrangement, which appears to be a disorder. Simultaneously, AGE accumulation in tendon tissue increases with age and mainly involves ECM collagen, resulting in abnormal cross-linking of collagen, stiffening of collagen fibers [37], and impaired collagen synthesis in the presence of AGEs cross-linkers, with consequent changes in tendon tissue elasticity [32]. Shinohara et al. [38] found that the deposition of AGEs in the ECM induced nicotinamide adenine dinucleotide phosphate oxidase to produce reactive oxygen species (ROS) through receptor for AGEs. ...
Age-related tendon disorder, a primary motor system disease, is characterized by biological changes in the tendon tissue due to senescence and seriously affects the quality of life of the elderly. The pathogenesis of this disease is not well-understood. Tendon stem/progenitor cells (TSPCs) exhibit multi-differentiation capacity. These cells are important cellular components of the tendon because of their roles in tendon tissue homeostasis, remodeling, and repair. Previous studies revealed alterations in the biological characteristics and tenogenic differentiation potential of TSPCs in senescent tendon tissue, in turn contributing to insufficient differentiation of TSPCs into tenocytes. Poor tendon repair can result in age-related tendinopathies. Therefore, targeting of senescent TSPCs may restore the tenogenic differentiation potential of these cells and achieve homeostasis of the tendon tissue to prevent or treat age-related tendinopathy. In this review, we summarize the biological characteristics of TSPCs and histopathological changes in age-related tendinopathy, as well as the potential mechanisms through which TSPCs contribute to senescence. This information may promote further exploration of innovative treatment strategies to rescue TSPCs from senescence.
... Kidney and skin tissues are rich in long-lived proteins such as collagen and keratin, a matter that may have allowed for the presumed late-acting antiglycation mechanisms of VA to take place and intensify its observed efect there. Collagen, for example, is one of the longest-lived body proteins, with an average half-life extending for years; thus, it is highly susceptible to extensive and advanced glycation [47]. ...
Background:
Glycation is implicated in the pathophysiology of many diseases, including diabetes, cancer, neurodegenerative diseases, and aging. Several natural and synthetic compounds were investigated for their antiglycation activity. We evaluated the antiglycation effect of vanillic acid (VA) using in vitro and in vivo experimental models.
Methods:
In vitro, bovine serum albumin (BSA) (50 mg/ml) was incubated with glucose (50 mM) with or without VA at 1.0-100 mM for 1 week at 37°C, and then, excitation/emission fluorescence was measured at 370/440 nm to determine glycation inhibition. The cytoprotective effect of VA was evaluated using RAW 264.7 cells incubated with or without VA at 7.8-500 μM along with 100-400 μM of methylglyoxal for 48 hours, and cell viability was determined using the MTT assay. Aminoguanidine (AMG) was used as a positive control in both in vitro and cell culture experiments. In vivo, 52 streptozotocin-induced diabetic rats were randomly assigned to 4 groups and treated with 0, 1.5, 4.5, or 15 mg/kg VA for four weeks. Serum fructosamine and blood glycosylated hemoglobin (HbA1c) were then measured, and advanced glycation end-products (AGEs) were detected in the kidneys and the skin of deboned tails using an immunohistochemistry assay.
Results:
VA caused a concentration-dependent effect against BSA glycation (IC50 of 45.53 mM vs. 5.09 mM for AMG). VA enhanced cell viability at all concentrations of VA and methylglyoxal. VA did not affect serum fructosamine or blood HbA1c levels, although it markedly decreased AGEs in the kidney in a dose-dependent manner and decreased AGEs in the skin of deboned tail tissues.
Conclusion:
VA had significant antiglycation activity at cellular and long-term glycation.
... For example, UV radiation absorbed by the collagen protein tyrosine forms a photoaging cross-link called di-tyrosine, which stiffens collagen [31,32] and facilitates wrinkle formation. The half-life of collagen turnover in skin is as long as 14.8 years [33], and when collagen fibers are damaged by UV light, the damaged collagen fibers remain in the dermis for a long time. Since collagen is a protein with a slow metabolic turnover, long-term exposure to sunlight causes accumulation of damage to collagen, resulting in a loss of elasticity. ...
Aminocarbonyl proteins increase with age in the dermal layer. Gene Chip analysis of mRNA expression in human dermal fibroblasts cultured on collagen gels treated with glyceraldehyde as an aminocarbonyl protein and on untreated collagen gels showed a decrease in the amount of aquaporin 1 (AQP1) mRNA. In this study, we clarified the involvement of AQP1 in collagen gel contraction and the production of fibrillin 1 and type I collagen in cultured human dermal fibroblasts. In the experiment, AQP1 siRNA was transfected into cultured human dermal fibroblasts to deplete AQP1, and the cell motility and contractile activity of the collagen gel were assessed. The production of fibrillin 1 and type I collagen was also examined by RT–qPCR and antibody staining. AQP1 depletion decreased the collagen gel contractile activity, and both the amount of mRNA and the antibody staining of fibrillin 1 and type I collagen decreased. Furthermore, the depletion of AQP1 reduced the levels of F-actin and phosphorylated myosin light chain 2, suggesting their involvement in reductions of the motility and collagen gel contractile activity of fibroblasts. These findings suggest that AQP1 is an important biomolecule for cell motility in human dermal fibroblasts and that decreased motility results in decreased expression of extracellular matrix proteins such as fibrillin 1 and type I collagen.
... Collagen is the main structural protein in the dermis, and its glycation induces the cross-linking of proteins. Accumulation of glycated collagen results in a long half-life and slow renewal of proteins in vivo [5]. Moreover, AGEs adversely afect dermal homeostasis [6]. ...
Advanced glycation end products (AGEs) are formed via a nonenzymatic glycosylation reaction called glycation. The formation and accumulation of AGEs increases in skin with age, contributing to the appearance of facial wrinkles and loss of skin elasticity. Therefore, inhibition of AGEs may delay skin aging. The microalgae Parachlorella beijerinckii has been used as a health food supplement for many years and contains carotenoids and vitamins that have antioxidant and anti-inflammatory effects. The aim of this study was to investigate whether Chlorella extract also has antiglycation activity. Antiglycation activity was measured using fluorescent AGEs, Nε-(carboxymethyl) lysine (CML), and Nε-(carboxymethyl) arginine (CMA) from glycated bovine serum albumin and type I collagen in vitro. A gel with a dermis-like structure consisting of collagen and a live fibroblast cell line was glycated with glyoxal. The content of fluorescent AGE, CML, and CMA, and the gel contraction activity were measured. In addition, to investigate the level of inflammation induced by the glycation of the collagen gel, the expression level of the receptor for AGEs and interleukin-8 were examined. Fat-solubleChlorella extract suppressed the formation of fluorescent AGEs, CML, and CMA in both models. These results indicated that Chlorella extract directly inhibited AGE formation. The collagen gel contracted over time during culturing, whereas contraction was inhibited in the glyoxal-treated collagen gel. Chlorella extract remarkably attenuated the glyoxal-induced gel contraction. Moreover, Chlorella extract substantially decreased the fluorescent AGEs, CML, and CMA in the collagen gels with glyoxal. Glyoxal exposure increased the expression levels of interleukin-8 and receptor for AGE proteins in collagen gels, while Chlorella extract inhibited this increase. This study showed that fat-solubleChlorella extract has a direct inhibitory effect on AGEs and decreases receptor expression for AGE-mediated inflammation by reducing AGEs. Chlorella may delay skin aging by inhibiting the formation and accumulation of AGEs.
... Etant donné la difficulté d'éliminer les AGEs notamment sur les protéines à longue demi-vie comme l'élastine et le collagène, ceux-ci s'accumulent au cours de la vie et sont retrouvés en grande quantité dans le derme avec le vieillissement (Thornalley 2008, Xin et al. 2021. Plusieurs AGEs stables s'accumulent avec l'âge au niveau du collagène et de l'élastine de la peau comme le CML, CEL ou encore le pentosidine (Mizutari et al. 1997, Verzijl et al. 2000. Nous savons que le collagène est glyqué par le glucose sur 3 lysines préférentielles alors que les 18 autres résidus lysine sont faiblement glyqués (Reiser et al. 1992). ...
Les personnes diabétiques présentent un risque accru de développer des plaies chroniques, le plus souvent au niveau du pied, pouvant mener jusqu’à l’amputation. Les causes de ce défaut de cicatrisation sont encore mal connues. Il a cependant été montré qu’une des conséquences de l’hyperglycémie est l’accumulation de produits terminaux de glycation, ou AGEs, tel que le Nε-(carboxymethyl)lysine (CML), au niveau de la peau des patients. Notre projet a eu pour objectif de mettre en évidence les effets de la glycation au niveau cutané afin de mieux comprendre les défauts de cicatrisation des diabétiques. Dans un premier temps, notre travail a consisté à étudier le comportement des fibroblastes dermiques humains en réponse à un agent de glycation fort : le glyoxal. Nous avons montré une diminution importante de la prolifération et de la migration cellulaire à la suite d’une accumulation de CML. Il apparaît qu’en présence de glyoxal les fibroblastes produisent correctement le collagène I mais sont incapables de le cliver et/ou de l’assembler correctement. Ces résultats suggèrent que la glycation pourrait retarder la fermeture d’une plaie et induire une fragilité cutanée. Nous avons également pu mettre en évidence une forte accumulation de vésicules lipidiques dans le cytoplasme des fibroblastes en réponse au glyoxal, ce qui traduit un déséquilibre important du métabolisme des lipides comme il est observé chez les diabétiques. Pour aller plus loin, la metformine aux propriétés antiglycantes, pourrait permettre de limiter l’accumulation lipidique. Par la suite, un modèle de peaux 3D avec kératinocytes, fibroblastes et cellules endothéliales a été testé en présence de glyoxal et/ou de metformine. Le glyoxal induit une diminution de la maturité des vaisseaux sanguins, une forte perturbation de la différenciation épidermique avec notamment une diminution de l’épaisseur de l’épiderme et de l’expression de la kératine 1. Dans un modèle 3D de cicatrisation, nous avons montré que la migration des kératinocytes est drastiquement diminuée après traitement au glyoxal. Ces modèles 3D nous ont permis de mettre en évidence que la metformine est capable de rétablir partiellement la différenciation épidermique et d’améliorer la stabilité des vaisseaux sanguins, mais ne permet pas d’augmenter la migration des kératinocytes. Enfin, nous avons testé un pansement contenant de la metformine sur des plaies de compression chroniques induites chez la souris diabétique. Sur ce modèle in vivo nous n’avons cependant pas pu mettre en évidence une amélioration de la cicatrisation en présence de metformine. Ces travaux de thèse ont permis de mettre en évidence que la glycation est à l’origine d’une perturbation forte de différents paramètres essentiels au processus de cicatrisation comme la migration, la prolifération, la différenciation et le remodelage de la matrice extracellulaire. Ces résultats ouvrent d'importantes perspectives pour étudier les complications cutanées liées au diabète et la mise en place d'approches thérapeutiques innovantes comme la metformine qui montre un fort potentiel.
... Under normal, low turnover conditions, chondrocytes resting in the unstressed steady state maintain the synthesis of proteoglycans and other non-collagenous molecules [47]. There is very little turnover of COL type II [48]. Functionally, the middle zone is the first line of resistance to compressive forces [35]. ...
Osteochondral structure reconstruction by tissue engineering, a challenge in regenerative medicine, requires a scaffold that ensures both articular cartilage and subchondral bone remodeling. Functional hydrogels and scaffolds present a strategy for the controlled delivery of signaling molecules (growth factors and therapeutic drugs) and are considered a promising therapeutic approach. Icariin is a pharmacologically-active small molecule of prenylated flavonol glycoside and the main bioactive flavonoid isolated from Epimedium spp. The in vitro and in vivo testing of icariin showed chondrogenic and ostseoinductive effects, comparable to bone morphogenetic proteins, and suggested its use as an alternative to growth factors, representing a low-cost, promising approach for osteochondral regeneration. This paper reviews the complex structure of the osteochondral tissue, underlining the main aspects of osteochondral defects and those specifically occurring in osteoarthritis. The significance of icariin’s structure and the extraction methods were emphasized. Studies revealing the valuable chondrogenic and osteogenic effects of icariin for osteochondral restoration were also reviewed. The review highlighted th recent state-of-the-art related to hydrogels and scaffolds enriched with icariin developed as biocompatible materials for osteochondral regeneration strategies.
... This relatively short-term monitoring has driven the search for other markers encompassing long-term glycemic control [12]. As collagen has a half-life of about 10-15 years, it is an appropriate candidate molecule to monitor a long-term accumulation of AGEs and imposed oxidative stress [13][14][15]. Because of the characteristic fluorescence of AGEs, the collagen-associated AGE-fluorescence can be non-invasively monitored on the skin [16]. ...
Skin autofluorescence (SAF) is a noninvasive method reflecting tissue accumulation of advanced glycation end products (AGEs). We investigated whether, in newly diagnosed children and adolescents with type 1 diabetes (T1D), this surrogate marker of long-term glycemia is associated with markers of the early manifestation phase, residual secretion capacity of the ß-cells, and the occurrence of remission. SAF was measured in 114 children and adolescents (age: 8.0 ± 4.5 years, 44% girls) at the time of T1D diagnosis, and related to HbA1c, C-peptide, diabetic ketoacidosis, and remission. 56 patients were followed up for 1 year. Seventy-four sex- and age-matched healthy individuals served as controls. SAF was higher in the T1D group compared with controls (1.0 ± 0.2 vs. 0.9 ± 0.2, p < 0.001). At the time of diagnosis, SAF correlated with HbA1c (r = 0.285, p = 0.002), was similar in patients with and without ketoacidosis, and was lower in the remitters compared with non-remitters (0.95 ± 0.18 vs. 1.04 ± 0.26, p = 0.027). Unlike HbA1c, SAF was an independent predictor of remission (∆R2 = 0.051, p = 0.004). Former studies consider SAF in diabetic patients as a tool to identify individuals at an increased risk of chronic complications. Here we show that determination of SAF at the time of T1D diagnosis might potentially predict remission, at least in children.
... Remarkably, matrix turnover in mature fibrous tissues is very low, with collagen molecule half-life, in particular, estimated to be anywhere between 95 and 215 years (Verzijl et al., 2000;Sivan et al., 2008). Thus, the permanence of the deposited matrix stresses the importance of its proper establishment during maturation-as ECM integrity is key for the tissue's mechanical role, and endogenous ability to repair and replace the matrix components seemingly limited. ...
The meniscus is an integral load bearing fibrous tissue of the knee joint that derives its mechanical function from the unique geometry and precise organization and composition of its extracellular matrix (ECM). While the importance of the highly specialized ECM is well appreciated in the mature meniscus, how this structural complexity is achieved during development remains poorly understood, and in particular, what interplay exists between the cells that build the matrix and their rapidly evolving microenvironment is unclear. To address these knowledge gaps, we begin by establishing a detailed timeline of the concurrent spatiotemporal changes that occur at both the cellular and matrix level during murine meniscus maturation, through use of Col1-YFP, Col2-CFP, Col10-mCherry fluorescent reporter mice, as well as histological analysis, and region specific high-throughput qPCR. We report that distinct cellular and matrix features defining specific meniscus tissue zones are present at birth, and that regional specialization continues during postnatal growth and maturation, possibly due to onset of load bearing use. Importantly, we define a framework for investigating the reciprocal feedback between cells and their evolving microenvironment—thus laying the foundation for future mechanistic work. Informed by the finding that key structural features of the meniscus matrix are established at birth, the remainder of this thesis addresses how this nascent organization is established. By analyzing key timepoints in knee joint development, we show that the genesis of ordered meniscus matrix is downstream of early cellular patterning characterized by marked fibrillation of the actin cytoskeleton. This suggests that cells and subcellular structures act as a physical template that directs alignment of the deposited fibrous matrix. Through the use of muscular dysgenesis (mdg) and splotch-delayed (Spd) mouse mutants that lack skeletal muscle contraction and joint motion, we further show that this critical cellular re-arrangement prior to meniscus formation does not fully occur without muscle contraction and leads to tissue dissociation—demonstrating that extrinsic forces play an instructive role in the tissue’s formation. Finally, we probe the impact of embryonic cell-mediated physical cues (adhesion, cytoskeletal arrangement) on subsequent meniscus assembly by generating targeted deletion of non-muscle myosin isoforms NM-IIA and NM-IIB (Myh dKO) in meniscus precursor cells during knee development. We demonstrate that cells of Myh dKO animals have defective cellular connectivity and so assemble a disorganized fibrillar matrix at birth, but these deficiencies in matrix alignment are somewhat corrected with postnatal maturation. Together, this work establishes that both cell-generated and extrinsic physical cues are imperative in the establishment of the initial meniscus structure that is built upon and further refined during postnatal growth.
Aging is a natural intrinsic process associated with the loss of fibrous tissue, a slower cell turnover, and a reduction in immune system competence. In the skin, the continuous exposition of environmental factors superimposes extrinsic damage, mainly due to ultraviolet radiation causing photoaging. Although not usually considered a pathogenic event, photoaging affects cutaneous biology, increasing the risk of skin carcinogenesis. At the cellular level, aging is typified by the rise of senescence cells a condition characterized by reduced or absent capacity to proliferate and aberrant hyper-secretory activity. Senescence has a double-edged sword in cancer biology given that senescence prevents the uncontrolled proliferation of damaged cells and favors their clearance by paracrine secretion. Nevertheless, the cumulative insults and the poor clearance of injured cells in the elderly increase cancer incidence. However, there are not conclusive data proving that aged skin represents a permissive milieu for tumor onset. On the other hand, tumor cells are capable of activating resident fibroblasts onto a pro-tumorigenic phenotype resembling those of senescent fibroblasts suggesting that aged fibroblasts might facilitate cancer progression. This review discusses changes that occur during aging that can prime neoplasm or increase the aggressiveness of melanoma and non-melanoma skin cancer.
Neurovascular coupling (NVC), a vital physiological process that rapidly and precisely directs localized blood flow to the most active regions of the brain, is accomplished in part by the vast network of cerebral capillaries acting as a sensory web capable of detecting increases in neuronal activity and orchestrating the dilation of upstream parenchymal arterioles. Here, we report a Col4a1 mutant mouse model of cerebral small vessel disease (cSVD) with age-dependent defects in capillary-to-arteriole dilation, functional hyperemia in the brain, and memory. The fundamental defect in aged mutant animals was the depletion of the minor membrane phospholipid phosphatidylinositol 4,5 bisphosphate (PIP2) in brain capillary endothelial cells, leading to the loss of inwardly rectifying K+ (Kir2.1) channel activity. Blocking phosphatidylinositol-3-kinase (PI3K), an enzyme that diminishes the bioavailability of PIP2 by converting it to phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3), restored Kir2.1 channel activity, capillary-to-arteriole dilation, and functional hyperemia. In longitudinal studies, chronic PI3K inhibition also improved the memory function of aged Col4a1 mutant mice. Our data suggest that PI3K inhibition is a viable therapeutic strategy for treating defective NVC and cognitive impairment associated with cSVD.
Skin ageing results from both the passage of time and from extrinsic forces, predominantly solar ultraviolet irradiation. Intrinsic ageing is characterized by fine wrinkling and homogenous colour on sun‐protected sites whereas extrinsic ageing is characterized by fine and coarse wrinkling, erythema and dyspigmentation on sun‐exposed sites. Two distinct phenotypes of extrinsic ageing have emerged: atrophic and hypertrophic variants. Tools to measure and quantify skin ageing have been developed and offer therapeutic and research applications. Molecular mechanisms distinguishing intrinsic and extrinsic skin ageing are discussed. The implications of skin ageing are cosmetic, medical and social in nature.
Hemidesmosomes are structural protein complexes localized at the interface of tissues with high mechanical demand and shear forces. Beyond tissue anchoring, hemidesmosomes have emerged as force-modulating structures important for translating mechanical cues into biochemical and transcriptional adaptation (i.e. mechanotransduction) across tissues. Here, we discuss the recent insights into the roles of hemidesmosomes in age-related tissue regeneration and aging in C. elegans, mice and humans. We highlight the emerging concept of preserved dynamic mechanoregulation of hemidesmosomes in tissue maintenance and healthy aging.
Aging is a dynamic, time-dependent process characterized by a gradual accumulation of cell damage. Continual functional decline in the intrinsic ability of living organisms to accurately regulate homeostasis leads to increased susceptibility and vulnerability to diseases. Anti-aging research has a long history throughout civilization, with many efforts put forth to understand and prevent the effects of aging. Thus, the major cellular and molecular hallmarks of aging have been identified and multiple strategies aiming to promoting healthy aging and extending the lifespan including lifestyle adjustments, medical treatments, and social programs, have been developed. Here, we use data from the CAS Content Collection to analyze the publication landscape of recent research. We review the advances in knowledge and delineate trends in research advancement on aging factors and attributes, as well as the anti-aging strategies across time, geography, and development pipelines. We also review the current concepts related to the major aging hallmarks on the molecular, cellular, and organismic level, the age-associated diseases, with attention to brain aging and brain health support, as well as the major biochemical processes associated with aging. We further assess the state-of-the-art anti-aging strategies and explore their correlations with age-related diseases. Well-recognized and novel, currently evaluated anti-aging agents have been summarized. Finally, we review clinical applications of anti-aging products with their development pipelines. We hope this review will be helpful for apprehending the current knowledge in the field of aging progression and prevention, in effort to further solve the remaining challenges and fulfil its potential.
Growing clinical evidence suggests a correlation between diabetes and more frequent and severe intervertebral disc failure, partially attributed to accelerated advanced glycation end-products (AGE) accumulation in the annulus fibrosus (AF) through non-enzymatic glycation. However, in vitro glycation (i.e., crosslinking) reportedly improved AF uniaxial tensile mechanical properties, contradicting clinical observations. Thus, this study used a combined experimental-computational approach to evaluate the effect of AGEs on anisotropic AF tensile mechanics, applying the finite element models (FEM) to complement experimental testing and examine difficult-to-measure subtissue-level mechanics. Methylglyoxal-based treatments were applied to induce three physiologically relevant AGE levels in vitro. Models incorporated crosslinks by adapting our previously validated structure-based FEM framework. Experimental results showed that a threefold increase in AGE content resulted in a ∼55% increase in AF circumferential-radial tensile modulus and failure stress and a 40% increase in radial failure stress. Failure strain was unaffected by non-enzymatic glycation. Adapted FEMs accurately predicted experimental AF mechanics with glycation. Model predictions showed that glycation increased stresses in the extrafibrillar matrix under physiologic deformations, which may increase tissue mechanical failure or trigger catabolic remodeling, providing insight into the relationship between AGE accumulation and increased tissue failure. Our findings also added to the existing literature regarding crosslinking structures, indicating that AGEs had a greater effect along the fiber direction, while interlamellar radial crosslinks were improbable in the AF. In summary, the combined approach presented a powerful tool for examining multiscale structure-function relationships with disease progression in fiber-reinforced soft tissues, which is essential for developing effective therapeutic measures. STATEMENT OF SIGNIFICANCE: : Increasing clinical evidence correlates diabetes with premature intervertebral disc failure, likely due to advanced glycation end-products (AGE) accumulation in the annulus fibrosus (AF). However, in vitro glycation reportedly increases AF tensile stiffness and toughness, contradicting clinical observations. Using a combined experimental-computational approach, our work shows that increases in AF bulk tensile mechanical properties with glycation are achieved at the risk of exposing the extrafibrillar matrix to increased stresses under physiologic deformations, which may increase tissue mechanical failure or trigger catabolic remodeling. Computational results indicate that crosslinks along the fiber direction account for 90% of the increased tissue stiffness with glycation, adding to the existing literature. These findings provide insight into the multiscale structure-function relationship between AGE accumulation and tissue failure.
HYBID is a new hyaluronan-degrading enzyme and exists in various cells of the human body. Recently, HYBID was found to over-express in the osteoarthritic chondrocytes and fibroblast-like synoviocytes. According to these researches, high level of HYBID is significantly correlated with cartilage degeneration in joints and hyaluronic acid degradation in synovial fluid. In addition, HYBID can affect inflammatory cytokine secretion, cartilage and synovium fibrosis, synovial hyperplasia via multiple signaling pathways, thereby exacerbating osteoarthritis. Based on the existing research of HYBID in osteoarthritis, HYBID can break the metabolic balance of HA in joints through the degradation ability independent of HYALs/CD44 system and furthermore affect cartilage structure and mechanotransduction of chondrocytes. In particular, in addition to HYBID itself being able to trigger some signaling pathways, we believe that low-molecular-weight hyaluronan produced by excess degradation can also stimulate some disease-promoting signaling pathways by replacing high-molecular-weight hyaluronan in joints. The specific role of HYBID in osteoarthritis is gradually revealed, and the discovery of HYBID raises the new way to treat osteoarthritis. In this review, the expression and basic functions of HYBID in joints were summarized, and reveal potential role of HYBID as a key target in treatment for osteoarthritis.
Collagens serve essential mechanical functions throughout the body, particularly in the connective tissues. In articular cartilage, collagens provide most of the biomechanical properties of the extracellular matrix essential for its function. Collagen plays a very important role in maintaining the mechanical properties of articular cartilage and the stability of the ECM. Noteworthily, many pathogenic factors in the course of osteoarthritis and rheumatoid arthritis, such as mechanical injury, inflammation, and senescence, are involved in the irreversible degradation of collagen, leading to the progressive destruction of cartilage. The degradation of collagen can generate new biochemical markers with the ability to monitor disease progression and facilitate drug development. In addition, collagen can also be used as a biomaterial with excellent properties such as low immunogenicity, biodegradability, biocompatibility, and hydrophilicity. This review not only provides a systematic description of collagen and analyzes the structural characteristics of articular cartilage and the mechanisms of cartilage damage in disease states but also provides a detailed characterization of the biomarkers of collagen production and the role of collagen in cartilage repair, providing ideas and techniques for clinical diagnosis and treatment.
Introduction
Calcium Hydroxylapatite (CaHA) is a common dermal filler used in aesthetic medicine for volumizing and contouring. Understanding mechanisms of actions of CaHA can help improve our understanding of its clinical applications.
Methods
We performed a systematic review to summarize the skin-regeneration related mechanisms of CaHA. Five bibliographic databases were searched for English-language publications that evaluated CaHA in skin regeneration outcomes including neocollagenesis, cell proliferation and growth factors, angiogenesis, vascular dynamic and inflammatory markers, among others. Methodological rigor of included studies was assessed.
Results
Of 2,935 identified citations, 12 studies were included for final analysis. Collagen production was reported by nine studies, cell proliferation by four, elastic fibers and/or elastin by four, and three studies on angiogenesis, while limited studies were available on the other outcomes. Six were clinical/observational studies. Only seven studies had a control group. Overall, studies showed CaHA resulted in increased cell proliferation, increased collagen production and angiogenesis, as well as in higher elastic fiber and elastin formation. Limited and inconclusive evidence was available on the other mechanisms. The majority of the studies had methodological limitations.
Discussion
Current evidence is limited but indicates several mechanisms through which CaHA could lead to skin regeneration, volume enhancement, and contouring.
Systematic review registration
https://doi.org/10.17605/OSF.IO/WY49V .
Osteoarthritis (OA) is the most common age-related joint disease, affecting articular cartilage and other joint structures, causing severe pain and disability. Due to limited understanding of the underlying disease pathogenesis, there are currently no disease modifying drugs for OA. Circadian rhythms are generated by cell-intrinsic timekeeping mechanisms which are known to dampen during ageing, increasing disease risks. In this review, we focus on one emerging area of chondrocyte biology, the circadian clocks. We first provide a historical perspective of circadian clock discoveries and the molecular underpinnings. We will then focus on the expression and functions of circadian clocks in articular cartilage, including their rhythmic target genes and pathways, links to ageing, tissue degeneration and OA, as well as tissue niche-specific entrainment pathways. Further research into cartilage clocks and ageing may have broader implications in the understanding of OA pathogenesis, the standardization of biomarker detection and the development of novel therapeutic routes for the prevention and management of OA and other musculoskeletal diseases.
This article summarizes important molecular mechanisms that drive aging in human skin from the perspective of dermal fibroblasts. The dermis comprises the bulk of the skin and is largely composed of a collagen-rich extracellular matrix (ECM). The dermal ECM provides mechanical strength, resiliency, and an environment that supports the functions of ibroblasts and other types of dermal cells. Fibroblasts produce the dermal ECM and maintain its homeostasis. Fibroblasts attach to the ECM and this attachment controls their morphology and function. During aging, the ECM undergoes gradual degradation that is nitiated by matrix metalloproteinases (MMPs). This degradation alters mechanical forces within the dermal ECM and disrupts he interactions between fibroblasts and the ECM thereby generating an aged fibroblast phenotype. This aged fibroblast phenotype is characterized by collapsed morphology, altered mechanosignaling, induction of CCN1, and activation of transcription factor AP-1, with consequent upregulation of target genes including MMPs and pro-inflammatory mediators. The TGF-beta pathway coordinately regulates ECM production and turnover. Altered mechanical forces, due to ECM fragmentation, down-regulate the type II TGF-beta receptor, thereby reducing ECM production and further increasing ECM breakdown. Thus, dermal aging involves a feed-forward process that reinforces the aged dermal fibroblast phenotype and promotes age-related dermal ECM deterioration. As discussed in the article, the expression of the aged dermal fibroblast phenotype involves both adaptive and cell-autonomous mechanisms.
Cancer cells undergo metabolic reprogramming to rely mostly on aerobic glycolysis (the Warburg effect). The increased glycolytic intake enhances the intracellular levels of reactive sugars and sugar metabolites. These reactive species can covalently modify macromolecules in a process termed glycation. Histones are particularly susceptible to glycation, resulting in substantial alterations to chromatin structure, function, and transcriptional output. Growing evidence suggests a link between dysregulated metabolism of tumors and cancer proliferation through epigenetic changes. This review discusses recent advances in the understanding of histone glycation, its impact on the epigenetic landscape and cellular fate, and its role in cancer. In addition, we investigate the possibility of using histone glycation as biomarkers and targets for anticancer therapeutics.
In an era of global warming knowledge of the effects of solar radiation on humans is of great importance and the latest discoveries in environmental photobiology are presented in this book. The Editor has brought together a wide range of world class contributors to provide the reader with information on the clinical effects of solar radiation, such as inflammation, pigmentation, immune-suppression, cancer and aging, with emphasis on the ethnic or genetic background. The book also offers updates on the biochemical mechanisms involved in the generation of damage to DNA, lipids and proteins and on their removal. Each chapter has been written to provide a "historical" description of the phenomenology followed by the description of the state of the art. In this way, non-specialized and specialised readers alike can be updated in the essential aspects of the field. Key topics include: - Damages from acute versus chronic sun exposure - Skin Color, Melanin, Race/Ethnicity and UV-Induced DNA Damage - The effects of solar radiation on the immune response in humans - Genetic background and UV-induced skin cancer - The photochemistry of indirect damages: Lipid and Protein Damage provoked by UV radiation - DNA repair therapy This title will become an indispensable resource for students and professional at all levels working in fields relating to photochemistry, environmental science, biochemistry and biotechnology.
The pandemic of coronavirus disease 2019 (COVID‑19), caused by a novel severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2), has caused an enormous impact on the global healthcare. SARS-CoV-2 infection primarily targets the respiratory system. Although most individuals testing positive for SARS-CoV-2 present mild or no upper respiratory tract symptoms, patients with severe COVID-19 can rapidly progress to acute respiratory distress syndrome (ARDS). ARDS-related pulmonary fibrosis is a recognized sequelae of COVID-19. Whether post-COVID-19 lung fibrosis is resolvable, persistent, or even becomes progressive as seen in human idiopathic pulmonary fibrosis (IPF) is currently not known and remains a matter of debate. With the emergence of effective vaccines and treatments against COVID-19, it is now important to build our understanding of the long-term sequela of SARS-CoV-2 infection, to identify COVID-19 survivors who are at risk of developing chronic pulmonary fibrosis, and to develop effective anti-fibrotic therapies. The current review aims to summarize the pathogenesis of COVID-19 in the respiratory system and highlights ARDS-related lung fibrosis in severe COVID-19 and the potential mechanisms. It envisions the long-term fibrotic lung complication in COVID-19 survivors, in particular in the aged population. The early identification of patients at risk of developing chronic lung fibrosis and the development of anti-fibrotic therapies are discussed.
The collagen molecular family is the result of nearly one billion years of evolution. It is a unique family of proteins, the majority of which provide general mechanical support to biological tissues. Fibril forming collagens are the most abundant collagens in vertebrate animals and are generally found in positions that resist tensile loading. In animals, cells produce fibril-forming collagen molecules that self-assemble into larger structures known as collagen fibrils. Collagen fibrils are the fundamental, continuous, load-bearing elements in connective tissues, but are often further aggregated into larger load-bearing structures, fascicles in tendon, lamellae in cornea and in intervertebral disk. We know that failure to form fibrillar collagen is embryonic lethal, and excessive collagen formation/growth (fibrosis) or uncontrolled enzymatic remodeling (type II collagen: osteoarthritis) is pathological. Collagen is thus critical to vertebrate viability and instrumental in maintaining efficient mechanical structures. However, despite decades of research, our understanding of collagen matrix formation is not complete, and we know still less about the detailed mechanisms that drive collagen remodeling, growth, and pathology. In this perspective, we examine the known role of mechanical force on the formation and development of collagenous structure. We then discuss a mechanochemical mechanism that has the potential to unify our understanding of collagenous tissue assembly dynamics, which preferentially deposits and grows collagen fibrils directly in the path of mechanical force, where the energetics should be dissuasive and where collagen fibrils are most required. We term this mechanism: Mechanochemical force-structure causality. STATEMENT OF SIGNIFICANCE: Our mechanochemical-force structure causality postulate suggests that collagen molecules are components of mechanochemically-sensitive and dynamically-responsive fibrils. Collagen molecules assemble preferentially in the path of applied strain, can be grown in place by mechanical extension, and are retained in the path of force through strain-stabilization. The mechanisms that drive this behavior operate at the level of the molecules themselves and are encoded into the structure of the biomaterial. The concept might change our understanding of structure formation, enhance our ability to treat injuries, and accelerate the development of therapeutics to prevent pathologies such as fibrosis. We suggest that collagen is a mechanochemically responsive dynamic element designed to provide a substantial "material assist" in the construction of adaptive carriers of mechanical signals.
The basement membrane (BM) constitutes a specialised form of the extracellular matrix (ECM) and plays important roles in many biological processes, such as cell migration, organ and tissue integrity, cell polarity, and the formation of metastases. In metazoans, a canonical BM is formed by only a few conserved structural core proteins: Laminin, Collagen IV, Nidogen and Perlecan. Depending on the tissue’s function and mechanical load, additional matrix proteins interact with, or are incorporated into the BM, resulting in tissue-specific mechanical properties, such as higher stiffness or elasticity, or special resistance to mechanical stress or harmful environmental conditions. In flies, the collagen IV-like protein Pericardin forms an integral constituent of matrices around the heart and tension sensors (chordotonal organs) of the peripheral nervous system. The function and integrity of both organ systems strongly relies on the appropriate establishment of a Pericardin (Prc) matrix and the function of its adapter protein—Lonely heart (Loh). In this review, we provide an overview of the four collagens present in flies, and will discuss our recent work on the formation and function of Pericardin-containing matrices, the role of the adapter protein Lonely heart and the necessity of specialised ECM molecules in tissue architecture and function.
Glycation reactions between proteins and sugars or their metabolites produce advanced glycation end-products, and the glycation of collagen in normal human dermal fibroblasts (NHDF) causes skin spots and freckles. UV exposure induces oxidative stress in NHDF, which overexpresses enzymes that degrade collagen, resulting in dry skin and wrinkling. Herein, we produced a novel polyphenolic decoction of Chaga mushroom extracted with a fermentation medium. Chaga polyphenol decoction (CPD) inhibited the glycation of albumin and collagen gel 3 to 4 times more than 2-aminoguanidine. The antioxidant effects of CPD were investigated using the fluorescence of an intracellular reactive oxygen species (ROS) scavenger, and NHDF exposed to UV-A for 60 min (9.5 J/cm2) after pre-treatment with 190 µg/mL of CPD suppressed ROS scavenger emission by 50 % compared to treatment with phosphate buffer saline. These results suggest that CPD might be a promising glycation inhibitor and ROS scavenger.
Background:
The polycaprolactone-based filler, (PCL-1, Ellansé-S), forms part of the recently growing portfolio of biodegradable collagen-stimulating fillers. It is comprised of a suspension of 25-50 micron diameter microspheres of polycaprolactone (PCL) (30%) in a carboxymethyl cellulose (CMC) gel carrier (70%) and has gained popularity due to its long-term volumizing action.
Objective:
This study outlines a retrospective case series of nine patients injected with the PCL-1, for volume augmentation in the mid-face. Objective volume calculations were performed with the Canfield Vectra 3D Imaging System at two time points post-implantation, with the objective of determining the longevity of the volumizing effect of the bio-stimulating substance.
Results:
A clear increase in volume, between 50-150%, was found in all of the patients at two years, over and above the volume initially injected. All the patients were satisfied with the longevity of the results.
Discussion:
The PCL-based filler is believed to afford immediate volume restoration due to the CMC gel component and a long-term action due to neo-collagenesis, induced by the PCL microspheres. The CMC gel is known to dissipate within 6-8 weeks, only to be replaced by new collagen induced by the PCL particles. Thus soft-tissue formation induced by the PCL particles, ultimately leads to a sustained volumizing effect.
Conclusion:
The PCL-based filler is shown to have a sustained volumizing effects of at least 2 years duration with clear evidence of increase in volume over and above the volume injected, in all of the cases studied. This is indicative of significant neo-collagenesis induced by the PCL microspheres.
Objective
To better understand the challenges of designing therapies to treat damaged vocal fold lamina propria, it is essential to understand the biophysical and pathophysiological mechanisms involved in vocal fold development, maintenance, injury, and aging. This review critically analyses these points to try and direct future efforts and new strategies toward science-based solutions.
Data Sources & Review Methods
MEDLINE, Ovid Embase, and Wed of Science databases were used to identify relevant literature. A scoping review was performed following the preferred reporting items for systematic reviews and meta-analyses extension for scoping reviews checklist.
Results
The layered arrangement of the vocal fold, develops during early childhood and is maintained during adulthood unless injury occurs. The stellate cells of the macular flava are likely to be important in this process. The capacity for vocal fold regeneration and growth is lost during adulthood and repair results in the deposition of fibrous tissue from resident fibroblasts. With advancing age, viscoelastic tissue declines, possibly due to cell senescence. Strategies aimed at replacing fibrous tissue within the vocal folds must either stimulate resident cells or implant new cells to secrete healthy extracellular protein. Injection of basic fibroblast growth factor is the most widely reported therapy that aims to achieve this.
Conclusions
The pathways involved in vocal fold development, maintenance and aging are incompletely understood. Improved understanding has the potential to identify new treatment targets that could potentially overcome loss of vocal fold vibratory tissue.
Diabetes is a rapidly expanding epidemic projected to affect as many as 1 in 3 Americans by 2050. This disease is characterized by devastating complications brought about high glucose and metabolic derangement. The most common of these complications is diabetic bladder dysfunction (DBD) and estimates suggest that 50–80% of patients experience this disorder. Unfortunately, the Epidemiology of Diabetes Interventions and Complications Study suggests that strict glucose control does not decrease ones risk for incontinence, although it does decrease the risk of other complications such as retinopathy, nephropathy and neuropathy. Thus, there is a significant unmet need to better understand DBD in order to develop targeted therapies to alleviate patient suffering. Recently, the research community has come to understand that diabetes produces a systemic state of low-level inflammation known as meta-inflammation and attention has focused on a role for the sterile inflammation-inducing structure known as the NLRP3 inflammasome. In this review, we will examine the evidence that NLRP3 plays a central role in inducing DBD and driving its progression towards an underactive phenotype.
Isolation and structure elucidation of an acid-resistant fluorescent molecule from human extracellular matrix revealed the presence of an imidazo[4,5-b]pyridinium molecule comprising a lysine and an arginine residue cross-linked by a pentose. Structure confirmation was achieved in vitro by the nonenzymatic reaction of ribose with lysine and arginine residues. The cross-link, named pentosidine, could also be synthesized with isomers of ribose, arabinose, xylose, and lyxose as well as by incubating young human collagen with these sugars at 37 ° C. Pentosidine was found in a variety of human tissues including plasma proteins and red blood cells. Its presence in cells grown in culture strongly suggests ribose or ribonucleotide metabolites as precursors. The unexpected discovery of pentose-mediated protein cross-linking raises new questions concerning the aging process.
The aspartic acid in human tooth enamel shows increasing racemization with age. This increase is not seen in the metabolically active protein hemoglobin. The rate constant for the racemization reaction of aspartic acid in human tooth enamel was found to be 8.29 X 10(-4) yr-1. This rate constant suggests that in any protein with a long in vivo lifetime, D-aspartic acid will accumulate with age (about 8% of total aspartic acid in enamel will be the D-enantiomer after 60 years). Thus, racemization may play some role in the aging process affecting metabolically stable tissues in long-lived homeotherms. Aspartic acid racemization in toogh enamel also provides a biochronological tool for assessing the age of living mammals.
A fluorescent compound has been detected in proteins browned during Maillard reactions with glucose in vitro and shown to be identical to pentosidine, a pentose-derived fluorescent cross-link formed between arginine and lysine residues in collagen (Sell, D. R., and Monnier, V. M. (1989) J. Biol. Chem. 264, 21597-21602). Pentosidine was the major fluorophore formed during nonenzymatic browning of ribonuclease and lysozyme by glucose, but accounted for less than 1% of non-disulfide cross-links in protein dimers formed during the reaction. Pentosidine was formed in greatest yields in reactions of pentoses with lysine and arginine in model systems but was also formed from glucose, fructose, ascorbate, Amadori compounds, 3-deoxyglucosone, and other sugars. Pentosidine was not formed from peroxidized polyunsaturated fatty acids or malondialdehyde. Its formation from carbohydrates was inhibited under nitrogen or anaerobic conditions and by aminoguanidine, an inhibitor of advanced glycation and browning reactions. Pentosidine was detected in human lens proteins, where its concentration increased gradually with age, but it did not exceed trace concentrations (less than or equal to 5 mumol/mol lysine), even in the 80-year-old lens. Although its precise carbohydrate source in vivo is uncertain and it is present in only trace concentrations in tissue proteins, pentosidine appears to be a useful biomarker for assessing cumulative damage to proteins by nonenzymatic browning reactions with carbohydrates.
Advanced glycation end-products and glycoxidation products, such as Nepsilon-(carboxymethyl)lysine (CML) and pentosidine, accumulate in long-lived tissue proteins with age and are implicated in the aging of tissue proteins and in the development of pathology in diabetes, atherosclerosis and other diseases. In this paper we describe a new advanced glycation end-product, Nepsilon-(carboxyethyl)lysine (CEL), which is formed during the reaction of methylglyoxal with lysine residues in model compounds and in the proteins RNase and collagen. CEL was also detected in human lens proteins at a concentration similar to that of CML, and increased with age in parallel with the concentration of CML. Although CEL was formed in highest yields during the reaction of methylglyoxal and triose phosphates with lysine and protein, it was also formed in reactions of pentoses, ascorbate and other sugars with lysine and RNase. We propose that levels of CML and CEL and their ratio to one another in tissue proteins and in urine will provide an index of glyoxal and methylglyoxal concentrations in tissues, alterations in glutathione homoeostasis and dicarbonyl metabolism in disease, and sources of advanced glycation end-products in tissue proteins in aging and disease.
The glycoxidation products Nepsilon-(carboxymethyl)lysine and pentosidine increase in skin collagen with age and at an accelerated rate in diabetes. Their age-adjusted concentrations in skin collagen are correlated with the severity of diabetic complications. To determine the relative roles of increased glycation and/or oxidation in the accelerated formation of glycoxidation products in diabetes, we measured levels of amino acid oxidation products, distinct from glycoxidative modifications of amino acids, as independent indicators of oxidative stress and damage to collagen in aging and diabetes. We show that ortho-tyrosine and methionine sulfoxide are formed in concert with Nepsilon-(carboxymethyl)lysine and pentosidine during glycoxidation of collagen in vitro, and that they also increase with age in human skin collagen. The age-adjusted levels of these oxidized amino acids in collagen was the same in diabetic and nondiabetic subjects, arguing that diabetes per se does not cause an increase in oxidative stress or damage to extracellular matrix proteins. These results provide evidence for an age-dependent increase in oxidative damage to collagen and support previous conclusions that the increase in glycoxidation products in skin collagen in diabetes can be explained by the increase in glycemia alone, without invoking a generalized, diabetes-dependent increase in oxidative stress.
A biomechanical failure of the collagen network is postulated in many hypotheses of the development of osteoarthritis with advancing age. Here we investigate the accumulation of non-enzymatic glycation (NEG) products in healthy human articular cartilage, its relation to tissue remodelling and its role in tissue stiffening. Pentosidine levels were low up to age 20 years, and increased linearly after this age. This indicates extensive tissue remodelling at young age, and slow turnover of collagen after maturity has been reached. The slow remodelling is supported by the finding that enzymatic modifications of collagen (hydroxylysine, hydroxylysylpyridinoline, and lysylpyridinoline) were not related to age. The high remodelling is supported by levels of the crosslink lysylpyridinoline (LP) as a function of distance from the articular surface. LP was highest at the surface in mature cartilage (>20 years), whereas in young cartilage (<10 years) the opposite was seen; highest levels were close to the bone. LP levels in cartilage sections at age 14 years are high at the surface and close to the bone, but they are low in the middle region. This indicates that maturation of cartilage in the second decade of life starts in the upper half of the tissue, and occurs last in the tissue close to the bone. The effect of NEG products on instantaneous deformation of cartilage was investigated as a functional of topographical variations in pentosidine levels in vivo and in relation to in vitro induced NEG. Consistently, higher pentosidine levels were associated with a stiffer collagen network. A stiffer and more crosslinked collagen network may become more brittle and more prone to fatigue.
Fragments of the alpha1 C-terminal telopeptide of type I collagen containing the sequence AHDGGR(1209-1214) (CTx) can be measured in urine as an index of bone resorption. We report here that these molecules undergo racemization and isomerization of Asp(1211) in vitro and in vivo, generating a mixture of four isomers: the native peptide form (alphaL), an isomerized form containing a beta-Asp bond (betaL), a racemized form containing a D-Asp residue (alphaD) and an isomerized/racemized form (betaD). To study these reactions at this specific site in collagen, we have employed four immunoassays, each specific for one of the isoforms, and developed HPLC methods for their separation. The kinetics of these reactions were studied in vitro under physiological conditions by incubation of synthetic AHDGGR hexapeptide or mineralized bone collagen. Reactions were found to be strongly shifted towards the beta-Asp forms and slightly in favour of the D-enantiomeric forms. CTx isomers were measured in human urine and in enzymic digests of bovine bone collagen. The results indicated that the extent of racemization and isomerization were correlated with the age and turnover of collagen. The ratios between the native and age-related forms of CTx were elevated in urine from patients with Paget's disease or osteoporosis as compared with that from healthy adults. The alphaL/alphaD CTx ratio had the highest discriminatory power (T-score=23.2; P<0.0001 and T-score=1. 5; P<0.0001 for Paget's disease and osteoporosis respectively). In conclusion, these findings indicate that an assessment of CTx ratios in urine may provide an estimate of bone turnover, aiding in the diagnosis of metabolic bone diseases.
In the time interval datable by radiocarbon, and at the temperatures of most archeological sites, a substantial amount of racemization of aspartic acid takes place. By determination of the amount of racemization of aspartic acid in bones from a particular location which have been dated by the radiocarbon technique, it is possible to calculate the in situ first-order rate constant for interconversion of the L- and D enantiomers of aspartic acid. Once this "calibration" has been calculated, the reaction can be used to date other bones from the deposit that are either too old to be dated by radiocarbon or that are too small for radiocarbon dating. The only assumption required with this approach is that the average temperature experienced by the "calibration" sample is representative of the average temperature experienced by older samples. This "calibration" technique is used herein to date bones from the Olduvai Gorge area in Tanzania, Africa.
Human articular cartilages of various ages were digested with collagenase, and the fluorescence of the digests was measured as a function of age. At acidic pH, all collagenase-treated fractions were found to contain two main fluorophores with fluorescence maxima at 395 and 385 nm (excitation at 295 and 335 nm, respectively). Each fluorophore was isolated from the hydrolysate and its structure was deduced from spectral and chemical data. The 395/295 nm fluorophore was identified as pyridinoline, which is one of the non-reducible cross-linkages in collagen. The 385/335 nm fluorophore was identical to pentosidine, which was isolated from human dura mater and characterized by Sell and Monnier in 1989. Our results showed that the amount of pentosidine per collagen in human articular cartilage increases linearly with age (r = 0.929,p<0.005), while the amount of pyridinoline per collagen remained constant and was not correlated with age (r = 0.20). On the other hand, the amount of pentosidine per pyridinoline increased exponentially during life (r²= 0.839, p<0.05).
Geological samples have been dated on the basis of the degree of racemization of the constituent amino acids by extrapolating rate data from simulated diagenetic studies. The validity of this extrapolation has been questioned, since it is known that, as well as the problem of estimating accurate diagnetic temperatures, factors such as pH and metal ions significantly affect rates of racemization. Results from this study show that further variables affecting the rate of racemization of free amino acids in aqueous solution are ionic strength, the buffer, and buffer concentration. Because of the possible variability in phosphate content in fossil bones, it is significant that racemization increased with increasing concentration of phosphate buffer. Furthermore, since geological matrices vary from sample to sample, and ionic strength may simulate to some extent such matrix changes, it is interesting that, although increasing ionic strength has no effect on racemization rate for a pH of 7.2, at pH 10.0 racemization increases with increasing ionic strength. These data for free amino acids in solution emphasize the necessity to use only the bound amino acid fraction for the dating of fossil specimens. Furthermore, the results suggest that a cautious approach be taken in applying amino acid racemization kinetics of even bound amino acids to geochronology and geothermometry. Concerning the basic mechanism of racemization, our results indicate that steric and proximity effects are equally as important as the inductive strength of the α substituent in determining the relative order of racemization of the amino acids.
N-epsilon-(Carboxymethyl)lysine (CML) is formed on oxidative cleavage of carbohydrate adducts to lysine residues in glycated proteins in vitro [Ahmed et al. (1988) J. Biol. Chem. 263, 8816-8821; Dunn et al. (1990) Biochemistry 29, 10964-10970]. We have shown that, in human lens proteins in vivo, the concentration of fructose-lysine (FL), the Amadori adduct of glucose to lysine, is constant with age, while the concentration of the oxidation product, CML, increases significantly with age [Dunn et al. (1989) Biochemistry 28, 9464-9468]. In this work we extend our studies to the analysis of human skin collagen. The extent of glycation of insoluble skin collagen was greater than that of lens proteins (4-6 mmol of FL/mol of lysine in collagen versus 1-2 mmol of FL/mol of lysine in lens proteins), consistent with the lower concentration of glucose in lens, compared to plasma. In contrast to lens there was a slight but significant age-dependent increase in glycation of skin collagen, 33% between ages 20 and 80. As in lens protein, CML, present at only trace levels in neonatal collagen, increased significantly with age, although the amount of CML in collagen at 80 years of age, approximately 1.5 mmol of CML/mol of lysine, was less than that found in lens protein, approximately 7 mmol of CML/mol of lysine. The concentration of N-epsilon-(carboxymethyl)hydroxylysine (CMhL), the product of oxidation of glycated hydroxylysine, also increased with age in collagen, in parallel with the increase in CML, from trace levels at infancy to approximately 5 mmol of CMhL/mol of hydroxylysine at age 80 . Thus, accumulation of N-(carboxymethyl) amino acids appears to be a general feature of the aging of long-lived proteins by glycation and oxidation reactions.
A sensitive and convenient method for the simultaneous determination of d- and l-aspartic acid in amino acid mixtures is described. The method involves derivatization of the mixture with a chiral fluorogen, followed by high-performance liquid chromatography on a reverse-phase column. The fluorogen used is an adduct of o-phthaldialdehyde with an optically active thiol, N-acetyl-l-cysteine. The sensitivity and accuracy of this method is similar to that using adducts of o-pthaldialdehyde with the achiral thiol, 2-mercaptoethanol. Five picomoles of d-aspartate can be accurately detected in the presence of a 100-fold excess of l-aspartate with a total analysis time (including derivatization) of 10 min.
D-ASPARTIC acid has been shown to accumulate with age in human tooth enamel1 and dentine2 at a rate of about 0.1% yr-1. We have predicted that racemisation should take place in any metabolically stable protein in long-lived mammals and that, as a consequence of racemisation, these proteins will have altered conformations which would probably produce changes in their biological activities or chemical properties3. We have extended these studies to soft tissue proteins, in particular those in the human lens. The proteins in the central portion of the lens are among the most stable in the human body4. Numerous changes in the properties of the lens proteins occur with age and cataract formation-denaturation5, increasing pigmentation6, cross linking5,6 insolubility6-9, and fluorescence10. Pirie11 has suggested that physicochemical processes may be responsible for these changes. We report here the results of D/L enantiomeric analyses of normal human lenses and cataracts: aspartic acid racemisation was seen during ageing and cataract formation.
THE L-amino acids initially present in bone protein undergo slow
racemisation over geological time at a rate which is proportional to
temperature1,2. We have shown3 that at the human
body temperature of ~37°C aspartyl residues in tooth enamel protein
also undergo racemisation at a rate which corresponds to an enrichment
in the D-aspartic acid content of ~0.1% per year. No D-aspartic acid
increase was detected in haemoglobin, a protein with a more rapid
turnover. We concluded that D-aspartyl residues accumulate in the
metabolically stable protein in tooth enamel during the human lifetime
as a result of in situ racemisation. We proposed that the irreversible
first-order rate equation calculated from the enamel results could be
used to deduce the age of any stable protein from a long lived mammal
and thus the age of the organism itself. The error among samples from
old individuals was large, however, probably because attrition and
caries reduce the amount of uncontaminated enamel, and thus tend to
limit the usefulness of tooth enamel for age determinations. In this
report, we show that tooth dentine is a more suitable material, and the
extent of aspartic acid racemisation in this fraction can be used as a
reliable indicator of mammalian age.
The rate of racemization of aspartic acid was measured in young and aged human femoral head cartilage. Normal femoral heads were obtained at postmortem, osteoarthritic specimens at operations for total hip replacement. In order to distinguish between the aspartic acid racemization in collagen from that in proteoglycan (PG), in addition to native tissue, we tested cartilage specimens from which PG had been enzymatically removed. Preliminary results indicate that there is only a very slow collagen turnover in normal adult cartilage. The same is true of residual cartilage from osteoarthritic femoral heads, indicating no rapid repair except where osteophytes are formed. Native, PG-containing cartilage, whether normal or osteoarthritic was found to have unexpectedly high racemization rates.
Recent progress in structure elucidation of products of the advanced Maillard reaction now allows probing specifically for the role of this reaction in the pathogenesis of age- and diabetes-related complications. Pyrraline is a glucose-derived advanced glycation end product against which polyclonal and monoclonal antibodies have been raised. Immunohistochemical localization studies revealed that pyrraline is found predominantly in the sclerosed extracellular matrix of glomerular and arteriolar renal tissues from both diabetic and aged nondiabetic individuals. Pentosidine and carboxymethyllysine are Maillard end products derived from both glucose and ascorbate. In addition, pentosidine can be formed from several other sugars under oxidative conditions, and in vitro studies suggest that a common intermediate involving a pentose is a necessary precursor molecule. The highest levels of these advanced Maillard products are generally found in the extracellular matrix, but these products are also present in lens proteins and in proteins with a fast turnover such as plasma proteins. Diabetes, and especially uremia, greatly catalyzes pentosidine formation. Both conditions are characterized by accelerated cataractogenesis, atherosclerosis, and neuropathy, suggesting that molecular damage by advanced Maillard reaction products may be a common mechanism in their development.
One of the hypotheses trying to explain the process of aging is the idea of glycation of proteins. This reaction, also called the Maillard or browning reaction, may explain age-related symptoms such as cataract, atherosclerosis and modification of collagen-containing tissues. Diabetics, which possess elevated blood sugar levels, show signs of accelerated aging exposing similar complications. The Maillard reaction, which occurs on a large scale in vivo, may play a key role in the initiation of these symptoms.
This study was made to improve the validity of age estimation from teeth using amino acid racemization. The correlation between actual age and the D/L ratio of aspartic acid was investigated by analyzing not only the total amino acid but also its fractionated substances, insoluble collagen and soluble peptide. The coefficient values of correlation between the D/L ratio and actual age in adult lower central incisors were 0.996 (sigma = +/- 1.0 year) for the total amino acid, 0.988 (sigma = +/- 1.8 years) for insoluble collagen, and 0.997 (sigma = +/- 0.9 years) for soluble peptide. The corresponding figures in adult first premolars were 0.991 (sigma = +/- 1.6 years), 0.988 (sigma = +/- 1.9 years), and 0.994 (sigma = +/- 1.4 years), respectively. The reactive velocity of aspartic acid racemization was highest for soluble peptide in both the lower central incisors and first premolars and approximately three times as rapid as that for total amino acid. As a result, age estimated from the analysis of soluble peptide was most accurate. The velocity for insoluble collagen was slightly lower than that for total amino acid. Age estimation was attempted from the teeth of an unknown body. These results suggest that the analyses, not only of total amino acid in dentin but also of its fractionated and extracted substances, can lead to higher reliability in age estimation. Soluble peptide, in particular, has been found to be most effective.
Glycation, oxidation, and nonenzymatic browning of protein have all been implicated in the development of diabetic complications. The initial product of glycation of protein, fructoselysine (FL), undergoes further reactions, yielding a complex mixture of browning products, including the fluorescent lysine-arginine cross-link, pentosidine. Alternatively, FL may be cleaved oxidatively to form N(epsilon)-(carboxymethyl)lysine (CML), while glycated hydroxylysine, an amino-acid unique to collagen, may yield N(epsilon)-(carboxymethyl)hydroxylysine (CMhL). We have measured FL, pentosidine, fluorescence (excitation = 328 nm, emission = 378 nm), CML, and CMhL in insoluble skin collagen from 14 insulin-dependent diabetic patients before and after a 4-mo period of intensive therapy to improve glycemic control. Mean home blood glucose fell from 8.7 +/- 2.5 (mean +/- 1 SD) to 6.8 +/- 1.4 mM (P less than 0.005), and mean glycated hemoglobin (HbA1) from 11.6 +/- 2.3% to 8.3 +/- 1.1% (P less than 0.001). These changes were accompanied by a significant decrease in glycation of skin collagen, from 13.2 +/- 4.3 to 10.6 +/- 2.3 mmol FL/mol lysine (P less than 0.002). However, levels of browning and oxidation products (pentosidine, CML, and CMhL) and fluorescence were unchanged. These results show that the glycation of long-lived proteins can be decreased by improved glycemic control, but suggest that once cumulative damage to collagen by browning and oxidation reactions has occurred, it may not be readily reversed. Thus, in diabetic patients, institution and maintenance of good glycemic control at any time could potentially limit the extent of subsequent long-term damage to proteins by glycation and oxidation reactions.
We undertook an interdisciplinary biomechanical and biochemical study to explore the extent and manner in which the total pool of proteoglycans influences the kinetic and static behavior of bovine articular cartilage in tension. Two biomechanical tests were used: (a) the viscoelastic creep test and (b) a slow constant-rate uniaxial tension test; and two enzymatic proteoglycan extraction procedures were used: (a) chondroitinase ABC treatment and (b) a sequential enzymatic treatment with chondroitinase ABC, trypsin, and Streptomyces hyaluronidase. We found that the viscoelastic creep response of all cartilage specimens may be divided into two distinct phases: an initial phase (less than 15 s), characterized by a rapid increase in strain following load application, and a late phase (15 s less than or equal to t less than 25,000 s), characterized by a more gradual increase in strain. A major finding of this study is that the kinetics of the creep response is greatly influenced by the glycosaminoglycan content of the tissue. For untreated and control specimens, the initial response comprises about 50% of the total strain, while for chondroitinase ABC and sequentially extracted specimens, the initial response comprises up to 83% of the total strain. Furthermore, most untreated and control specimens did not reach equilibrium within the 25,000 s test period, while enzymatically digested specimens often reached equilibrium in less than 100 s. Thus, we conclude that through their physical restraints on collagen, the bulk of proteoglycan present in the tissue acts to retard fibrillar reorganization and alignment under tensile loading, thereby effectively preventing sudden extension of the collagen network. In contrast, the results of our slow constant-rate uniaxial tension experiment show that essentially complete extraction of proteoglycan glycosaminoglycans does not affect the intrinsic tensile stiffness and strength of cartilage specimens or the collagen network in a significant manner. Hence, an important function of the bulk proteoglycans (i.e., the large aggregating type) in cartilage is to retard the rate of stretch and alignment when a tensile load is suddenly applied. This mechanism may be useful in protecting the cartilage collagen network during physiological situations, where sudden impact forces are imposed on a joint.
A simple two-step fluorometric assay of DNA in cartilage explants, utilizing the bisbenzimidazole dye Hoechst 33258, is described. Cartilage explants were prepared for assay by digestion with papain. Aliquots of the digest were mixed with dye solution, and the fluorescence emission measured. The enhancement in fluorescence of dye was specific for DNA, as demonstrated by 97% sensitivity to DNase and resistance to RNase. In addition, little or no interference was caused by non-DNA tissue components, since DNA caused an equal enhancement in fluorescence independent of the presence of papain-digested cartilage. By performing the assay on isolated chondrocytes, the cellular content of DNA was computed to be 7.7 pg per chondrocyte. The assay was stable for at least 2 h and sensitive to as little as 6 ng of DNA or equivalently less than 1000 cells. This procedure offers advantages over other established DNA assays of cartilage and may be especially useful in metabolic studies of cartilage explants.
N epsilon-(Carboxymethyl)lysine (CML) has been identified as a product of oxidation of fructoselysine (FL) in glycated (nonenzymatically glycosylated) proteins in vitro and has also been detected in human tissues and urine [Ahmed et al. (1986) J. Biol. Chem. 261, 4889-4894]. In this study, we compare the amounts of CML and FL in normal human lens proteins, aged 0-79 years, using specific and sensitive assays based on selected ion monitoring gas chromatography-mass spectrometry. Our results indicate that the lens content of FL increases significantly between infancy and about age 5 but that there is only a slight, statistically insignificant increase in FL between age 5 and 80 (mean +/- SD = 1.4 +/- 0.4 mmol of FL/mol of Lys). In contrast, the lens content of the oxidation product, CML, increased linearly with age, ranging from trace levels at infancy up to 8 mmol of CML/mol of lysine at age 79. The ratio of CML to FL also increased linearly from 0.5 to 5 mol of CML/mol of FL between age 1 and 79, respectively. These results indicate that CML, rather than FL, is the major product of glycation detectable in adult human lens protein. The age-dependent accumulation of CML in lens protein indicates that products of both glycation and oxidation accumulate in the lens with age, while the constant rate of accumulation of CML in lens with age argues against an age-dependent decline in free radical defense mechanisms in this tissue.
An age-related accumulation of D-aspartic acid was detected in the white matter of ten normal brains from individuals aged 30 to 80 years. Gray matter showed no systematic increase in D-aspartic acid. The rate constant for D-aspartate formation in the brain is equal to the predicted value calculated for 37 degrees C. Accumulation of the uncommon D-aspartate isomer in myelinated white matter implies that there is little or no turnover of this tissue, and this may have a bearing on dysfunction of the aging brain or on other diseases of myelin.
Osteoarthrosis was induced in one knee joint of dogs by an established surgical procedure. Changes in the articular cartilage in the biosynthesis of collagen and other proteins were sought by radiochemical labelling in vivo, with the following findings. (1) Collagen synthesis was stimulated in all cartilage surfaces of the experimental joints at 2, 8 and 24 weeks after surgery. Systemic labelling with [3H]proline showed that over 10 times more collagen was being deposited per dry weight of experimental cartilage compared with control cartilage in the unoperated knee. (2) Type-II collagen was the radiolabelled product in all samples of experimental cartilage ranging in quality from undamaged to overtly fibrillated, and was the only collagen detected chemically in the matrix of osteoarthrotic cartilage from either dog or human joints. (3) Hydroxylysine glycosylation was examined in the newly synthesized cartilage collagen by labelling dog joints in vivo with [3H]lysine. In experimental knees the new collagen was less glycosylated than in controls. However, no difference in glycosylation of the total collagen in the tissues was observed by chemical analysis. (4) Over half the protein-bound tritium was extracted by 4 M-guanidinium chloride from control cartilage labelled with [3H]proline, compared with one-quarter or less from experimental cartilage. Two-thirds of the extracted tritium separated in the upper fraction on density-gradient centrifugation in CsCl under associative conditions. Much of this ran with a single protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing conditions. The identity of this protein was unknown, although it resembled serum albumin in mobility afte disulphide-bond cleavage.
Over the last 20 years a new chemical method, base on the racemization of aspartic acid, has been developed to be used for the estimation of chronologic age in adult individuals. The method has a good accuracy when used on dental enamel, dentine and cartilage. However, in forensic and archeological cases teeth and cartilage are not always available. Since preliminary studies have shown that there are some age-related changes of the D/L aspartic acid ratio also in bone, this study was carried out to further explore if the method could be used for age estimations of bone. Bone samples from 24 individuals, aged 0.2 to 95.6 years were analysed for the D/L ratios with HPLC-technique. Two different fractions of the bone were examined, an acid-soluble peptide fraction and an acid-insoluble collagen-rich fraction. The analyses showed age-related racemizations in both fractions, although of different rates. The correlation coefficients with age were 0.72 in the peptide fraction, and 0.84 in the collagen-rich fraction. It thus seems as if bone may be used for age estimations when more stable tissues like dentine and cartilage are not available.
Determinations of chronologic age are of great importance in forensic science. At present the aspartic acid racemization method on teeth provides one of the best means in adult individuals. However, if teeth are not available, some other stable tissue has to be used. In this study, the applicability of cartilage from the ribs has been tested. Specimens of rib cartilage were obtained at autopsy from 24 individuals aged 0.2-96 years. An acid-soluble peptide (SP) fraction and an insoluble collagen-rich (IC) fraction were prepared, and the ratio of D/L aspartic acid was determined using the HPLC technique. The correlation coefficient between the D/L ratio and age was r = 0.91 in SP and r = 0.97 in IC. It thus seems as if cartilage from non-weight-bearing areas may be a useful source of tissue for the estimation of chronologic age with the aspartic acid racemization method when teeth are not available.
Products of advanced protein glycosylation (advanced glycation end products, or AGEs) accumulate in tissues as a function of time and sugar concentration. AGEs induce permanent abnormalities in extracellular matrix component function, stimulate cytokine and reactive oxygen species production through AGE-specific receptors, and modify intracellular proteins. Pharmacologic inhibition of AGE formation in long-term diabetic animals prevents diabetic retinopathy, nephropathy, neuropathy, and arterial abnormalities in animal models. Clinical trials in humans are currently in progress.
We investigated whether measurement of aspartic acid racemization in intervertebral discs (IVD) could be used in the postmortem estimation of age at death. The extent of aspartic acid racemization in IVD tissues was found to increase with age. The rate of racemization turned out to be much higher in the nucleus pulposus than in the annulus fibrosus. The relation between age and the D-aspartic acid content in the anterior peripheral annulus fibrosus of IVD was close enough to allow postmortem estimation of age at death based on the extent of aspartic acid racemization in this tissue.
The racemization ration (D/L ratio) of aspartic acid in the dentin (collagen) of deciduous teeth was studied, using dentin of permanent teeth as a reference. D-aspartic acid in deciduous teeth was found to increase almost linearly with aging. The reaction velocity constant of racemization (kyr-1) in deciduous teeth (1.1915 x 10(-3)yr-1) was approximately double the value for permanent teeth (6.2550 x 10(-4)yr-1). The velocity of the racemization reaction was similar between deciduous teeth and permanent teeth in a dry state. The D/L ratio determined using transverse sections of deciduous teeth tended to be high in the crown and low in the root. The reaction velocity of racemization is considered to be affected to some extent by the tooth material, ambient temperature and salivary pH, but most importantly by physiologic resorption of deciduous teeth at the root associated with age.
An improved method for the quantitative derivatization of amino acids with fluorenylmethyl chloroformate (FMOC-Cl) is described. Amino acids are derivatized in borate buffer at pH 11.4 for 40 min at ambient temperature. All amino acids resulted in stable derivatives. In particular, improved derivatization was obtained with the troublesome amino acids His and Tyr: exclusively monosubstituted His and disubstituted Tyr were formed, eluting as free peaks in the chromatogram. These derivatives show a higher fluorescence response than their disubstituted and monosubstituted counterparts, respectively, resulting from other protocols. Under the new conditions, considerable less of the hydrolysis product of FMOC-Cl is seen in the chromatograms. Baseline noise was substantially reduced at a higher emission wavelength (630 nm instead of 313 or 340 nm). With simple precautions, extensive adsorption of the disubstituted derivatives (Lys, Hyl, and Tyr) on plastic or glass surfaces could be prevented. Calibration curves were linear over a 10 to 300 molar ratio of FMOC-Cl to total amino acid. The detection limits are in the femtomole range and the derivatives are stable for more than 48 h, thus permitting automated analysis of multiple samples.
To study the reliabiliity of formulas for calculating mean skin temperature (T
sk), values were computed by 18 different techniques and were compared with the mean of 10,841 skin temperatures measured by infrared thermography. One hundred whole-body infrared thermograms were scanned in ten resting males while changing the air temperature from 40° C to 4° C. Local, regional average and mean skin temperatures were obtained using an image processing system. The agreement frequency, defined as the percentage of the calculated T
sk values which agreed with the corresponding infrared thermographic T
sk within ±0.2° C, ranged for with the various formulas from 7% to 80%. In many sites, the local skin temperature did not coincide with the regional average skin temperature. When the local skin temperatures which showed the highest percentage similarity to the regional average skin temperature within ±0.4° C were applied to the formula, the agreement frequency was markedly improved for all formulas. However, the agreement frequency was not affected by changing the weighting factors from specific constants to individually measured values of regional surface area. By applying the physiologically reliable accuracy range of ±0.2° C in the moderate and ±0.4° C in the cool condition, agreement frequencies of at least 95% were observed in formulas involving seven or more skin temperature measurement sites, including the hand and foot. We conclude that calculation of a reliable mean skin temperature must involve more than seven skin temperature measurement sites regardless of ambient temperature. Optimal sites for skin temperature measurement are proposed for various formulas.
A high-performance liquid chromatographic assay was developed for pyridinium crosslinks and pentosidine in mature collagen of a wide variety of connective tissue hydrolysates by a simple two-step isocratic assay using a reversed-phase column. The crosslinks (including the internal standard pyridoxine) were optimally detected by their native fluorescence by switching wavelengths of the detector during the assay. The method resulted in highly sensitive and accurate measurements, without need for precleaning of the samples: crosslink levels in 200 microm thin slices of the various zones of articular cartilage were easily quantified. The detection limit was as low as 0.4 pmol for the pyridinolines and 0.05 pmol for pentosidine. The intra-assay and inter-assay coefficients of variation were as low as 2% (pyridinolines) and 5% (pentosidine); calibration curves for all compounds were linear over a concentration range larger than two orders of magnitude. With our chromatographic system, the diglycosylated form of hydroxylysylpyridinoline in unhydrolyzed urine was separated as well.
Aggrecan is a key component of the cartilage matrix. During aging, many changes occur in its composition and structure; in particular, there is an increase in the proportion of lower molecular weight monomers and of the "free" binding region. An important question has been whether these changes represent alterations in biosynthesis or whether they are due to the accumulation with age of the partially degraded fragments of the originally synthesized large monomer. In the present work we have used an independent tool, viz., the extent of racemization of aspartic acid to study the molecular "age" of different buoyant density fractions of the aggrecan of human articular cartilage, as well as of isolated free binding region and link protein. By measuring the D/LAsp ratio of the different aggrecan species, we were able to establish directly the relative residence times of these molecules in the cartilage matrix and, in combination with compositional and structural analyses, to define their "history" and calculate some of the kinetics constants characterizing their turnover. The value of the turnover constant for the large monomer in fraction A1D1 is 0.206 per year, which corresponds to a half-life of 3.4 years, while the turnover constant for the free binding region is 0.027 per year, which corresponds to a half-life of 25 years. It is thus clear that the rate of formation and turnover of the large monomer is much more rapid than the final degradation of the free binding region fragments, which explains the accumulation of the latter in cartilage during aging.
Levels of D-aspartic acid (D/L ratio) in cranial non-collagen proteins (acid-soluble peptide fractions) have been reported to increase with age. We isolated total amino acid fractions from the femur and separately isolated acid-insoluble collagen fraction and acid-soluble peptide fractions; then D/L ratios were measured from each fraction by gas chromatography. We evaluated the applicability of their D/L ratios for age estimation based on their correlation coefficient. A sex-related difference was observed in the D/L ratio. In particular, aged females showed a low ratio, suggesting an association with bone disorders. In males, the D/L ratios of acid-soluble peptide fraction showed the highest correlation rate (r = 0.969) with age, and those of total amino acid fraction showed the highest correlation rate (r = 0.633) with age in females. Without separation of male and female, the D/L ratios of total amino acid fraction showed the highest value (r = 0.853). The D/L ratio of acid-soluble peptide fractions differed according to the size of bone powder particles, being higher for larger particle sizes. These results suggest that the application of D/L ratio from total amino acid fraction is the most effective method for estimating age using the human femur. However, care is necessary when studing cadavers that might be females.
There are no reports on rates of amino acid racemization in bones. To investigate the possibility of estimating age by evaluating amino acid residue racemization in human bones, a heating experiment was performed and the rate of aspartic acid racemization was determined using the Arrhenius equation. Assuming an annual mean temperature of 15 degrees C, the rate constant (k) for aspartic acid racemization in bone was calculated, and the racemization rate at 15 degrees C k (y) was 4.1036 x 10(-9)--much lower than that of dentin. These results suggest that it is more difficult to accurately determine age by analyzing aspartic acid residues in bone than in dentin.