Lysyl oxidase-mediated crosslinking in granulation tissue collagen in two models of hyperglycemia.

Enzymatically mediated crosslinks and nonenzymatic glycation were quantified in granulation tissue collagen in two models of hyperglycemia, diabetes and galactosemia, that have opposite effects on collagen solubility. The effects of castration, which alters collagen solubility, was also investigated. Collagen from both diabetic and galactosemic rats had significantly increased levels of dihydroxylysinonorleucine (DHLNL), a difunctional reducible crosslink. Galactosemic rats had significantly decreased levels of hydroxypyridinium, a trifunctional product of DHLNL and hydroxylysine, relative to control values, while diabetic rats had normal levels. Values for all other detectable crosslinks in collagen from hyperglycemic rats were indistinguishable from control values. Nonenzymatic glycation was increased in both groups of hyperglycemic rats. In diabetic rats, but not in galactosemic rats, nonenzymatic glycation was strongly correlated with DHLNL content. Castration had no effect on crosslink content of collagen from diabetic or galactosemic rats. This study demonstrates that (1) collagen crosslinking is abnormal in granulation tissue collagen in both experimental diabetes and galactosemia, (2) these changes are similar to those observed in skin collagen from insulin-dependent diabetic subjects and (3) the crosslinking abnormalities are not correlated with alterations in collagen solubility. We conclude that hyperglycemia-associated increases in immature crosslinks cannot account for altered collagen solubility, although impaired maturation of such crosslinks may be partially responsible for the lathyrogenic effect of galactosemia.     

Lysyl oxidase-mediated crosslinking in granulation tissue collagen in two models of hyperglycemia

Enzymatically mediated crosslinks and nonenzymatic glycation were quantified in granulation tissue collagen in two models of hyperglycemia, diabetes and galactosemia, that have opposite effects on collagen solubility. The effects of castration, which alters collagen solubility, was also investigated. Collagen from both diabetic and galactosenic rats had significantly increased levels of dihydroxylysinonorleucine (DHLNL), a difunctional reducible crosslink. Galactosemic rats had significantly decreased levels of hydroxypyridinium, a trifunctional product of DHLNL and hydroxylyse, relative to control values, while diabetic rats had normal levels. Values for all other detectable crosslinks in collagen from hyperglycemic rats were indistinguishable from control values. Nonezymatic glycation was increased in both groups of hyperglycemic rats. In diabetic rats, but not in galactosemic rats, nonenzymatic glycation was strongly correlated DHLNL content. Castration had no effect on crosslink content of collagen from diabetic or galactosemic rats. This study demonstrates that (1) collagen crosslinking is abnormal in granulation tissue collagen in both experimental diabetes and galactosemia, (2) these changes are similar to those observed in skin collagen from insulin-dependent diabetic subjects and (3) the crosslinking abnormalities are not correlated with alterations in collagen solubility. We conclude that hyperglycemia-associated increases in immature crosslinks cannot acount for altered collagen solubility, although impaired maturation of such crosslinks may be partially responsible for the lathyrogenic effect of galactosemia.     

AGE-breakers cleave model compounds,but do not break Maillard crosslinks in skin and tail collagen from diabetic rats

Advanced glycation end products (AGE), formed by nonenzymatic Maillard reactions between carbohydrate and protein, contribute to the increase in chemical modification and crosslinking of tissue proteins with age. Acceleration of AGE formation in collagen during hyperglycemia, with resultant effects on vascular elasticity and basement membrane permeability, is implicated in the pathogenesis of diabetic complications. AGE-breakers, such as N-phenacylthiazolium (PTB) and N-phenacyl-4,5-dimethylthiazolium (PMT) halides, have been proposed as therapeutic agents for reversing the increase in protein crosslinking in aging and diabetes. We have confirmed that these compounds, as well as the AGE-inhibitor pyridoxamine (PM), cleave the model AGE crosslink, phenylpropanedione, and have studied the effects of these compounds in reversing the increased crosslinking of skin and tail collagen isolated from diabetic rats. Crosslinking of skin collagen, measured as the half-time for solubilization of collagen by pepsin in 0.5M acetic acid, was increased approximately 5-fold in diabetic, compared to nondiabetic rats. Crosslinking of tail tendon collagen, measured as insolubility in 0.05 N acetic acid, was increased approximately 10-fold. Collagen preparations were incubated in the presence or absence of AGE-breakers or PM in phosphate buffer, pH 7.4, for 24h at 37 degrees C. These treatments did not decrease the half-time for solubilization of diabetic skin collagen by pepsin or increase the acid solubility of diabetic tail tendon collagen. We conclude that, although AGE-breakers and PM cleave model crosslinks, they do not significantly cleave AGE crosslinks formed in vivo in skin collagen of diabetic rats.     

A comparison of the PAS-reaction in the dermis of diabetic and non-diabetic humans

Dermopathy is a part of the diabetic syndrome that decreases the solubility of dermal collagen. In the present study, the PAS-reactive component of collagen has been analysed in diabetic and non-diabetic human dermis by photographic densitometry. The PAS-reaction was significantly lower in diabetics than non-diabetics, and age seemed to be of no consequence. The results are interpreted to indicate a decrease in collagen-associated sugar residues in diabetics.     

Effect of cadmium on collagen content and solubility in rat bone

The toxic action of cadmium in the bone tissue is known, but its mechanisms are still unexplained. We examined whether Cd influences collagen content and its solubility in the femoral bone of three-week-old female rats exposed to 5 or 50 mg Cd/l in drinking water. Non-cross linked collagen was extracted with 0.5 M acetic acid, and two acid-insoluble collagen fractions were extracted with pepsin and 4.0 M guanidine hydrochloride, respectively. SDS/PAGE showed the presence of two collagen types, I and V, in all three extracted fractions. Exposure of rats to Cd for 6 months increased the amount of acid-soluble collagens type I and V and decreased the level of acid-insoluble collagens. The amount of total collagen extracted from the bones of rats exposed to 50 mg Cd/l was reduced by about 14% as compared to control and those intoxicated with 5 mg Cd/l. The solubility of type I bone collagen (determined as the percentage of acetic-soluble fraction of total collagen) was increased 2.9- and 3.0-fold in rats intoxicated with 5 and 50 mg Cd/l, respectively. Similarly, the solubility of type V collagen was increased 2.3- and 2.7-fold, respectively. Our results indicate that Cd treatment affects bone collagen by decreasing its content and increasing its solubility.     

The effect of experimental diabetes on the molecular characteristics of soluble rat-tail tendon collagen

Acid soluble rat-tail tendon collagen was prepared from animals rendered diabetic by treatment with either streptozotocin or alloxan and from matched controls. In comparison to the normal, the diabetic collagens consistently demonstrated decreased solubility of reconstituted fibrils, marked increase in intrinsic viscosity and a decreased ratio of alpha to beta components. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gels revealed a marked decrease in migration of alpha1, alpha2, and beta components from both types of diabetic collagen. These data indicate that diabetic collagens are larger than normal and are capable of higher degrees of polymerization due to increased intra- and inter-molecular interactions. These changes could explain, in part, the altered response of diabetic connective tissues to inflammation and trauma.     

Changes in the cross-linking of collagen from rat tail tendons due to diabetes

The acid solubility of Type I collagen from rat tail tendons decreases due to diabetes. This finding has been taken as evidence that collagen from diabetics may be more cross-linked than normal. We compared CNBr peptide maps prepared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for [3H] NaBH4-reduced tail tendons from streptozotocin-diabetic rats with maps from age-matched control rats. At least through 30 weeks of diabetes, the distribution of mass of both cross-linked and uncross-linked CNBr peptides was identical in diabetic and control tendons. Therefore, the number of cross-linked peptides did not increase due to diabetes. We analyzed the 3H-cross-linking compounds present on the CNBr peptides and found that the 3H content of peptides cross-linked in control tendons through the bivalent, reduced cross-links hydroxylysinonorleucine and lysinonorleucine was diminished on corresponding peptides from diabetic tendons as a function of duration of diabetes. The cross-linked peptides, however, persisted. Therefore, we conclude that a larger fraction of these bivalent cross-links is found in an unknown, non-reducible form in tendons from diabetic compared with control rats. This resembles a phenomenon normally associated with maturation and/or aging where the non-reducible form of the cross-links is acid-stable. An increase in the fraction of the cross-links that is non-reducible and acid-stable would explain, at least in part, the decrease in acid solubility of the collagen. Non-enzymatic glycation (NEG) was not very specific, since most CNBr peptides bound some glucose. However, peptides from the alpha 2-chain seemed to be preferential targets for NEG. While NEG clearly increased due to diabetes, we found no evidence that increased NEG led to an increased number of cross-links in tail tendon collagen from streptozotocin diabetic rats.     

4-Thiaproline reduces heart lipid peroxidation and collagen accumulation in the diabetic db/db mouse

Summary Collagen accumulation is a main pathological feature of diabetic cardiomyopathy. The underlying mechanisms seem to be increased cross linking by reactive carbonyles. The purpose of the study was to decrease the collagen content of total ventricular tissue by the oral administration of thiaproline, which could reduce collagen due to its functions as a proline analogue, blocking collagen production and as a free oxygen radical scavenger, blocking reactive carbonyles and oxygen species and subsequently collagen cross linking.Thiaproline was administered to genetically diabetic db/db mice and compared to untreated animals. Total ventricular collagen as expressed by hydroxyproline was significantly lower in the treated group (means 0.23 micromoles/10 tissue in the treated vs 0.35 micromoles/100 mg tissue in the untreated group, p < 0.001). Significantly more collagen could be eluted in the treated group (p < 0.001) and carboxymethyllysine was significantly reduced in the treated group (p < 0.001). Di-tyrosine and glycemic control did not differ between the groups. Glutathione was significantly increased in the TP treated experimental group (p < 0.001) and lipid peroxidation products were significantly decreased (means 0.221 absorbance in the treated group versus 0.321 absorbance in the untreated diabetic group) correlating with total ventricular collagen content (r = 0.87, p < 0.01).We conclude that thiaproline reduced total ventricular collagen content by inhibiting collagen cross linking as reflected by increased solubility of collagen and expressed by higher elution quantity of collagen. Thiaproline, and/or its metabolites induced increase of heart glutathione which may well have been scavenging reactive carbonyles derived from lipid peroxidation and advanced stage nonenzymatic glycosylation as shown by decreased total ventricular carboxy-methyllysine and lipid peroxidation products paralleling reduced heart collagen content.It remains to be shown that the successful reduction of heart collagen by thiaproline is paralleled by improved functional properties.     

Cis 3 hydroxyproline reduces glomerular basement membrane thickness and collagen type IV synthesis in diabetic rats

Summary Glomerular basement membrane thickening is thought to be due to increased collagen synthesis and abnormal cross linking. Based upon the observation that the incorporation of distinct proline analogues leads to increased degradation of the newly abnormally formed collagen we administered cis 3 hydroxyproline orally to streptozotocin diabetic rats.Measuring glomerular basement membrane thickness we found in the treated group significantly lower values. The solubility of collagen in the treated group was significantly increased, indicating the mechanism of action of the proline analogue. The collagen content of kidneys in the treated group was reduced as well correlating with the basement membrane thickness. Provided the absence of toxicity of cis 3 hydroxyproline its pharmaceutical use for the inhibition of basement membrane proliferation seems promising.     

Nonenzymatic glycation of collagen in aging and diabetes

Considerable progress has been made in our understanding of nonenzymatic glycation of collagen, and the relationship between glycation of collagen and changes in connective tissue associated with aging and diabetes. Recent studies surveyed in this review suggest the following conclusions: 1. Collagen content of early glycation products does not appear to increase throughout the life span in normal human subjects, although small increases may occur that are linked to glycemic changes. These products are increased, relative to age-matched controls, in experimental diabetes and in diabetes mellitus in collagen from virtually all tissues analyzed. 2. Collagen content of browning products increases with aging and appears to be higher in diabetic subjects than in age-matched controls. Rates of accumulation may be accelerated in subpopulations of diabetic subjects at high risk for developing complications. 3. Increases in early glycation products do not appear to be associated with alterations in collagen solubility, thermal rupture time, or mechanical strength, nor is there an association with most diabetic complications. Alterations in these products may, however, affect conformation, ligand binding, lysyl oxidase-mediated cross-linking, and interactions between collagen and other macromolecules in the extracellular matrix. 4. Increased content of browning products is associated with many physicochemical changes in collagen as well as with long-term complications in diabetes mellitus. 5. Regulatory mechanisms have been identified in vivo that may serve to control or limit the formation of glycation products. 7. Pharmacologic agents have been identified that may be able to reduce collagen content of late glycation products. Despite the progress that has been made in this field, many areas of uncertainty and controversy exist. For example, there is not yet a consensus that the browning products associated with collagen exclusively comprise advanced Maillard products derived from nonenzymatically glycated residues. There is evidence that oxidative reactions involving lipids also play a role in generating fluorophores and chromophores that may alter properties of collagen. Thus, in the extracellular matrix collagen may be continuously modified by at least three very different processes: Maillard reactions, interactions with oxidizing lipids, and enzymatically mediated cross-linking. The interrelationships between these and possibly other posttranslational modifications remain a poorly understood area of great complexity.     

Protective effect of Withania somnifera (Solanaceae) on collagen glycation and cross-linking

Modification of collagen such as non-enzymatic glycation and cross-linking plays an important role in diabetic complications and age-related diseases. We evaluate the effect of Withania somnifera on glucose-mediated collagen glycation and cross-linking in vitro. Extent of glycation, viscosity, collagen-linked fluorescence and pepsin solubility were assessed in different experimental procedures to investigate the effect of W. somnifera. Tail tendons obtained from rats (Rattus norvegicus) weighing 250-275 g were incubated with 50 mM glucose and 100 mg of metformin or Withania root powder or ethanolic extract of Withania under physiological conditions of temperature and pH for 30 days. Formation of advanced glycation end products (AGE) was measured by fluorescent method whereas the cross-linking of collagen was assessed by pepsin digestion and viscosity measurements. Tendon collagen incubated with glucose showed an increase in glycation, AGE and cross-linking of collagen. The collagen incubated with W. somnifera and metformin ameliorates these modifications. The ethanolic extract of Withania showed more prominent effect than Withania root powder. The activity of ethanolic extract of Withania is comparable to metformin, a known antiglycating agent. In conclusion, Withania could have therapeutic role in the prevention of glycation induced pathogenesis in diabetes mellitus and aging.     

Are the changes in collagen caused by chronical X-irradiation similar to aging?

It has been shown that in the skin of chronically irradiated rats the proportion of collagen type III as compared to collagen type I is increased; on the other hand, no changes in the overall proportion to collagen were observed in the skin. It appears that the increased proportion of collagen type III in chronically irradiated rats is responsible for the decreased solubility of cutaneous collagen in these animals. Concomitantly, indirect evidence was accumulated for the presence of an additional cross-link in type III collagen, present only when irradiated animals served as the collagen source. This cross-link is located subterminally as long as it is not removed by limited pepsin digestion. It was concluded that the physiological decrease in solubility and the decrease in solubility observed in chronically irradiated animals have a different molecular background.     

Type II collagen of lamprey

The major collagen in lamprey notochord is type II, as determined by its amino acid composition and solubility properties. This collagen has a distribution of charged residues indistinguishable from higher vertebrate Type II collagens as judged by its SLS banding pattern. Lamprey type II collagen has a higher thermal stability than lamprey skin collagen, in contrast to the identical melting temperatures for these types in mammals. A minor collagen in lamprey notochord has solubility properties, amino acid composition, and electrophoretic mobility similar to that of 1 alpha, 2 alpha, 3 alpha collagen in human cartilage.     

Comparative analysis of collagens solubilized from human foetal, and normal and osteoarthritic adult articular cartilage, with emphasis on type VI collagen

The different collagen types were extracted sequentially, by 4 M guanidinium chloride and pepsin, from human foetal and normal and osteoarthritic adult articular cartilage. They were characterized by electrophoresis and immunoblotting. Most of the collagenous proteins present in articular cartilage from young human foetuses were solubilized: almost 40% of the total collagen was extracted in the native form with 4 M guanidinium chloride. Type VI collagen was detected in this fraction as high-molecular-mass chains (185-220 kDa) and a low-molecular-mass chain (140 kDa). Type II, IX and XI collagens were also present, but were extracted more extensively by pepsin digestion. Comparative analysis of normal and osteoarthritic cartilage from adults reveals some major differences: an increase in the solubility of the collagen and modifications of soluble collagen types in osteoarthritic cartilage. Furthermore, type VI collagen was present at a higher concentration in guanidinium chloride extracts of osteoarthritic cartilage than those of normal tissue. This finding was corroborated by electron microscopic observations of the same samples: abundant (100 nm) periodic fibrils were observed in the disorganized pericellular capsule of cloned cells in osteoarthritic cartilage. In normal tissues the pericellular zone was more compact and contained only a few such banded fibrils. The differences in the collagen types solubilized from normal and osteoarthritic cartilage, although corresponding to a minor proportion of the total collagen, demonstrate that important modifications in chondrocyte metabolism and in the collagenous network do occur in degenerated cartilage.     

Glycation end-product cross-link breaker reduces collagen and improves cardiac function in aging diabetic heart

Aging and diabetes mellitus (DM) both affect the structure and function of the myocardium, resulting in increased collagen in the heart and reduced cardiac function. As part of this process, hyperglycemia is a stimulus for the production of advanced glycation end products (AGEs), which covalently modify proteins and impair cell function. The goals of this study were first to examine the combined effects of aging and DM on hemodynamics and collagen types in the myocardium in 12 dogs, 9-12 yr old, and second to examine the effects of the AGE cross-link breaker phenyl-4,5-dimethylthazolium chloride (ALT-711) on myocardial collagen protein content, aortic stiffness, and left ventricular (LV) function in the aged diabetic heart. The alloxan model of DM was utilized to study the effects of DM on the aging heart. DM induced in the aging heart decreased LV systolic function (LV ejection fraction fell by 25%), increased aortic stiffness, and increased collagen type I and type III protein content. ALT-711 restored LV ejection fraction, reduced aortic stiffness and LV mass with no reduction in blood glucose level (199 +/- 17 mg/dl), and reversed the upregulation of collagen type I and type III. Myocardial LV collagen solubility (%) increased significantly after treatment with ALT-711. These data suggest that an AGE cross-link breaker may have a therapeutic role in aged patients with DM.     

Increased glycosylation of glomerular basement membrane collagen in diabetes

Basement membrane was purified from glomeruli isolated from normal and streptozotocin-diabetic rats. After extraction of non-collagen protein with 8M urea, the extent of glycosylation in glomerular basement membrane collagen was determined with a specific colorimetric reaction that detects carbohydrate in ketoamine linkage with proteins. The level of glycosylation of glomerular basement membrane collagen purified from diabetic rats was significantly greater than that in non-diabetic animals. Increased basement membrane glycosylation may alter structure-function relationships of the capillary filtration barrier.     

Increased type-III/type-I collagen ratios in diabetic human conjunctival biopsies

The ratio of type-III to type-I collagen is measured in human conjunctival biopsies from control and diabetic subjects. The tissue is digested by CNBr and the resulting peptides are quantified by SDS polyacrylamide gel electrophoresis. The peptides used are alpha 1-(I)CB7 and alpha 1-(III)CB8. In control population, of type-III collagen slightly increases with age. In two diabetic populations, (juvenile onset diabetes and maturity onset diabetes), the percentage of type-III collagen is significantly higher than in age-matched control groups. These data plus those previously obtained on genetically diabetic mice indicate that diabetes mellitus affects the expression of interstitial collagen phenotype. Preliminary results on prediabetic subjects suggest the role of genetic factors in such alterations.     

The influence of 1-hydroxyethane-1,1-diphosphonate and dichloromethanediphosphonate on lysine hydroxylation and cross-link formation in rat bone, cartilage and skin collagen.

The effects in vivo of dichloromethanediphosphonate and 1-hydroxyethane 1,1-diphosphonate on collagen solubility, hydroxylation of lysine and proline and on the formation of collagen intermolecular cross-links were studied by using rat bone, cartilage and skin tissues. Dichloromethanediphosphonate decreased bone collagen solubility both in acetic acid and after pepsin treatment. Although none of the diphosphonates had any effect on the hydroxylation of proline, dichloromethane-diphosphonate, but not 1-hydroxyethane-1,1-diphosphonate, increased the number of hydroxylysine residues in the alpha-chains of bone, skin and cartilage collagen. The stimulatory effect was dose-dependent. The dichloromethanediphosphonate-mediated increase in hydroxylysine residues in bone and cartilage was manifested in an increase of dihydroxylysinonorleucine, the cross-link that is formed by the condensation of two hydroxylysine residues. The cross-link hydroxylysinonorleucine, a condensation product of hydroxylysine and lysine, on the other hand, was decreased. The total number of intermolecular cross-links was not changed by the diphosphonate.     

Changes in collagen cross-linking and lysyl oxidase by estrogen.

Dermal collagen solubility and lysyl oxidase activity of bones were measured in DDD mice of advancing age. Insoluble fractions of the dermal collagen increased more rapidly in females than in males after 5 weeks of age. Activity of the lysyl oxidase extracted from bones was higher in females than in males after 4 weeks of age. After sexual maturation, such sex differences were always observed in skin as well as in bone tissue. In other experimental animals, dermal collagen solubility was markedly decreased by estrogen treatment and lysyl oxidase was remarkably activated by estrogen in both skin and bone. Thus it is clear that estrogen stimulates the enzyme activity and accelerates the maturation of collagen and elastin in extracellular space.