Ruud A. Bank

Effect of Collagen Turnover on the Accumulation of Advanced Glycation End Products

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.

Crosslinking by advanced glycation end products increases the stiffness of the collagen network in human articular cartilage: A possible mechanism through which age is a risk factor for osteoarthritis

OBJECTIVE Age is an important risk factor for osteoarthritis (OA). During aging, nonenzymatic glycation results in the accumulation of advanced glycation end products (AGEs) in cartilage collagen. We studied the effect of AGE crosslinking on the stiffness of the collagen network in human articular cartilage. METHODS To increase AGE levels, human adult articular cartilage was incubated with threose. The stiffness of the collagen network was measured as the instantaneous deformation (ID) of the cartilage and as the change in tensile stress in the collagen network as a function of hydration (osmotic stress technique). AGE levels in the collagen network were determined as: Nepsilon-(carboxy[m]ethyl)lysine, pentosidine, amino acid modification (loss of arginine and [hydroxy-]lysine), AGE fluorescence (360/460 nm), and digestibility by bacterial collagenase. RESULTS Incubation of cartilage with threose resulted in a dose-dependent increase in AGEs and a concomitant decrease in ID (r = -0.81, P < 0.001; up to a 40% decrease at 200 mM threose), i.e., increased stiffness, which was confirmed by results from the osmotic stress technique. The decreased ID strongly correlated with AGE levels (e.g., AGE fluorescence r = -0.81, P < 0.0001). Coincubation with arginine or lysine (glycation inhibitors) attenuated the threose-induced decrease in ID (P < 0.05). CONCLUSION Increasing cartilage AGE crosslinking by in vitro incubation with threose resulted in increased stiffness of the collagen network. Increased stiffness by AGE crosslinking may contribute to the age-related failure of the collagen network in human articular cartilage to resist damage. Thus, the age-related accumulation of AGE crosslinks presents a putative molecular mechanism whereby age is a predisposing factor for the development of OA.

Identification of PLOD2 as Telopeptide Lysyl Hydroxylase, an Important Enzyme in Fibrosis

The hallmark of fibrotic processes is an excessive accumulation of collagen. The deposited collagen shows an increase in pyridinoline cross-links, which are derived from hydroxylated lysine residues within the telopeptides. This change in cross-linking is related to irreversible accumulation of collagen in fibrotic tissues. The increase in pyridinoline cross-links is likely to be the result of increased activity of the enzyme responsible for the hydroxylation of the telopeptides (telopeptide lysyl hydroxylase, or TLH). Although the existence of TLH has been postulated, the gene encoding TLH has not been identified. By analyzing the genetic defect of Bruck syndrome, which is characterized by a pyridinoline deficiency in bone collagen, we found two missense mutations in exon 17 of PLOD2, thereby identifying PLOD2 as a putative TLH gene. Subsequently, we investigated fibroblasts derived from fibrotic skin of systemic sclerosis (SSc) patients and found that PLOD2 mRNA is highly increased indeed. Furthermore, increased pyridinoline cross-link levels were found in the matrix deposited by SSc fibroblasts, demonstrating a clear link between mRNA levels of the putative TLH gene (PLOD2) and the hydroxylation of lysine residues within the telopeptides. These data underscore the significance of PLOD2 in fibrotic processes.

Age-related accumulation of Maillard reaction products in human articular cartilage collagen.

Non-enzymic modification of tissue proteins by reducing sugars, the so-called Maillard reaction, is a prominent feature of aging. In articular cartilage, relatively high levels of the advanced glycation end product (AGE) pentosidine accumulate with age. Higher pentosidine levels have been associated with a stiffer collagen network in cartilage. However, even in cartilage, pentosidine levels themselves represent <1 cross-link per 20 collagen molecules, and as such cannot be expected to contribute substantially to the increase in collagen network stiffness. In the present study, we investigated a broad range of Maillard reaction products in cartilage collagen in order to determine whether pentosidine serves as an adequate marker for AGE levels. Not only did the well-characterized AGEs pentosidine, N(epsilon)-(carboxymethyl)lysine, and N(epsilon)-(carboxyethyl)lysine increase with age in cartilage collagen (all P<0.0001), but also general measures of AGE cross-linking, such as browning and fluorescence (both P<0.0001), increased. The levels of these AGEs are all higher in cartilage collagen than in skin collagen. As a functional measure of glycation the digestibility of articular collagen by bacterial collagenase was investigated; digestibility decreased linearly with age, proportional to the extent of glycation. Furthermore, the arginine content and the sum of the hydroxylysine and lysine content of cartilage collagen decrease significantly with age (P<0.0001 and P<0. 01 respectively), possibly due to modification by the Maillard reaction. The observed relationship between glycation and amino acid modification has not been reported previously in vivo. Our present results indicate that extensive accumulation of a variety of Maillard reaction products occurs in cartilage collagen with age. Altogether our results support the hypothesis that glycation contributes to stiffer and more brittle cartilage with advancing age.

The role of collagen in determining bone mechanical properties

The hypothesis of this study was that collagen denaturation would lead to a significant decrease in the toughness of bone, but has little effect on the stiffness of bone. Using a heating model, effects of collagen denaturation on the biomechanical properties of human cadaveric bone were examined. Prior to testing, bone specimens were heat treated at varied temperatures (37–200°C) to induce different degrees of collagen denaturation. Collagen denaturation and mechanical properties of bone were determined using a selective digestion technique and three‐point bending tests, respectively. The densities and weight fractions of the mineral and organic phases in bone also were determined. A repeated measures analysis of variance showed that heating had a significant effect on the biomechanical integrity of bone, corresponding to the degree of collagen denaturation. The results of this study indicate that the toughness and strength of bone decreases significantly with increasing collagen denaturation, whereas the elastic modulus of bone is almost constant irrespective of collagen denaturation. These results suggest that the collagen network plays an important role in the toughness of bone, but has little effect on the stiffness of bone, thereby supporting the hypothesis of this study.

Genetic and Pharmacological Inhibition of Galectin-3 Prevents Cardiac Remodeling by Interfering with Myocardial Fibrogenesis

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

A simplified measurement of degraded collagen in tissues: Application in healthy, fibrillated and osteoarthritic cartilage

Intact triple helical collagen molecules are highly resistant to proteolytic enzymes, whereas degraded (unwound) collagen is easily digested. This fact was exploited to develop a simplified method for the quantification of the amount of degraded collagen in the collagen network of connective tissues. Essentially, the method involves extraction of proteoglycans with 4 M guanidinium chloride, selective digestion of degraded collagen by alpha-chymotrypsin, hydrolysis in 6 M HCl of the released fragments as well as the residual tissue, and then measurement of the amount of hydroxyproline in both pools. Since the digestion of degraded collagen by alpha-chymotrypsin and measurement of hydroxyproline is not restricted to a specific collagen type, this technique can be applied to a wide variety of connective tissues. The method was validated with articular cartilage. Levels of in situ degraded collagen were about four-fold higher in degenerated (fibrillated) cartilage than in its healthy counterpart derived from the same donor. More detailed investigations revealed that the collagen damage in degenerated cartilage is more extensive at the cartilage surface than in the region adjacent to bone. This was not the case in healthy cartilage; identical low values were obtained at the surface and close to the bone. An impaired collagen network has been hypothesized to be the reason for the swelling of cartilage in osteoarthritis (OA). The present paper presents the first experimental evidence to support this hypothesis: more damage to the collagen network (i.e., more degraded collagen molecules within fibrils) is linearly related to more extensive swelling of the OA tissue in hypotonic saline.

Sensitive fluorimetric quantitation of pyridinium and pentosidine crosslinks in biological samples in a single high-performance liquid chromatographic run

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.

The increased swelling and instantaneous deformation of osteoarthritic cartilage is highly correlated with collagen degradation.

OBJECTIVE To provide evidence for the hypothesis that the loss of tensile strength of osteoarthritic (OA) cartilage (resulting in swelling-the hallmark of OA) is due to an impaired collagen network and not to loss or degradation of proteoglycans. METHODS The amount of degraded collagen molecules, the fixed charge density (FCD) on a dry-weight basis, the degree of swelling in saline, and the instantaneous deformation (ID; a test reflecting the tensile stiffness of the collagen network) were measured in full-depth OA femoral condyle samples. In addition, levels of the crosslink hydroxylysylpyridinoline (HP), the amount of degraded collagen molecules, and the degree of swelling were determined in the 3 zones (surface, middle, and deep) of OA cartilage. We also compared the ID of normal and OA cartilage. RESULTS In full-depth OA cartilage, a close relationship was found between swelling and ID. Swelling and ID correlated strongly with the amount of degraded collagen molecules, and were not related to FCD. OA cartilage showed the same zonal pattern in HP levels as normal cartilage (i.e., an increase with depth). No relationship was found between collagen crosslinking and swelling of the surface, middle, and deep zones. In all 3 zones, swelling was proportional to the amount of degraded collagen molecules. Compared with that of normal cartilage, the change in ID of OA cartilage was most pronounced at the surface in a direction parallel to the direction of the collagen fibrils. CONCLUSION The decreased stiffness of the OA collagen network (as measured by swelling and ID) is strongly related to the amount of degraded collagen molecules. The anisotropy in ID parallel and perpendicular to the direction of the fibrils revealed that the impairment of strength resides mainly in, and not between, the fibrils. Proteoglycans play only a minor role in the degeneration of the tensile stiffness of OA cartilage.

Age-related decrease in proteoglycan synthesis of human articular chondrocytes: the role of nonenzymatic glycation

OBJECTIVE To examine the effect of nonenzymatic glycation of cartilage extracellular matrix on the synthetic activity of chondrocytes. METHODS The proteoglycan-synthesis rate (35SO4(2-) incorporation) and levels of advanced nonenzymatic glycation (determined by high-performance liquid chromatography measurement of pentosidine) were evaluated in human articular cartilage from 129 donors, varying in age from 25 to 88 years, and in cartilage with enhanced levels of advanced glycation end-products (AGEs) resulting from incubation with ribose. RESULTS Cartilage showed a strong age-related increase in pentosidine levels (r = 0.97, P < 0.0005) and, concomitantly, a decrease in proteoglycan synthesis (r = -0.98, P < 0.0002). This decrease in proteoglycan synthesis correlated with the increase in pentosidine (r = -0.95, P < 0.02). Moreover, the elevation of pentosidine levels in the in vitro-ribosylated cartilage was proportional with the decrease in proteoglycan synthesis (r = -0.95, P < 0.005). CONCLUSION In both aged and in vitro AGE-enriched cartilage, the rate of proteoglycan synthesis was negatively correlated with the degree of glycation. This suggests that the age-related increase in cartilage AGE levels may be responsible, at least in part, for the age-related decline in the synthetic capacity of cartilage.