Recent research indicates that an increase in the steady-state levels of highly reactive dicarbonylic compounds ("carbonyl stress") may lead to the formation of advanced glycation endproducts or AGE. These AGE products could be involved in the etiology of the long-term complications of several human afflictions, such as Diabetes mellitus, ageing, uremia and Alzheimer's disease 12. In diabetic patients with long-standing hyperglycemia, an increase in the generation of AGE products can be partly explained by the process of non-enzymatic glycosylation of proteins. In this reaction, glucose reacts reversibly with the free amino groups of proteins to form unstable Schiff bases, which may undergo an intramolecular rearrangement giving rise to the more stable Amadori product. A further modification of this product originates 3-deoxyglucasone, a reactive dicarbonylic non-protein-bound molecule, which can then form adducts with nearby proteins and irreversibly progress to the formation of a family of compounds collectively known as AGE, many of which are fluorescent and originate protein-proteins crosslinks 2. Most studies have demonstrated that AGE can induce structural and functional alterations of various proteins.

Cell surface receptors for AGE moieties have been detected on different cell types. These proteins appear to contribute to diverse cellular functions, such as the specific recognition and degradation of AGE-modified proteins 3. Depending on the cell type involved, cellular recognition of AGE can be associated with chemotaxis, angiogenesis, oxidative stress, and either cell proliferation or apoptosis. Recent studies have suggested that the nitric oxide synthase (NOS) / nitric oxide (NO) pathway may be involved in the AGE-mediated alterations observed in several cell types, such as retinal vascular endothelial cells and macrophages 45. In addition, NO-mediated vasodilation has been reported to be impaired in diabetes 6.

Collagenous proteins play important roles in the function of several tissues, including bone. They have a long half-life and various free amino groups which are exposed to ambient glucose levels 7. An increase in AGE-modified collagen has been detected in the cortical bone of diabetic and ageing rats 8910. The alterations caused by AGE-modifications of collagen may play a role in the pathogenesis of the osteopenia present in poorly controlled diabetic patients 11. This osteopenia has been attributed to a long-term decrease in the recruitment and bone-forming activity of osteoblasts 12.

In our laboratory we have previously demonstrated that soluble AGE-modified proteins can regulate the growth of mouse MC3T3E1 and rat UMR106 osteoblast-like cells:while initially eliciting an increase in cellular proliferation and differentation, a relatively long-term incubation of these cells with AGE-modified bovine serum albumin (BSA) induces a significant decrease in both parameters 13. We have also described the presence of membrane-associated receptors in both cell lines, which specifically participate in the recognition and degradation of AGE-modified proteins. The expression of these receptors is differentially regulated according to the osteoblastic stage of development 14. Furthermore, we have observed in this system 15 that AGE-BSA can modify the secretion of insulin-like growth factor (IGF)-I, one of the major growth factors involved in bone development 16. In addition, the levels of IGF-binding proteins present in the osteoblastic conditioned media are also regulated by AGE-BSA and the stage of osteoblast development 15.

The aim of the present study was to investigate the effect of an AGE-modified type I collagen matrix obtained in vitro, on the adhesion, spreading, proliferation, differentiation and mineralization of rat osteosarcoma UMR106 and mouse non-transformed MC3T3E1 calvaria-derived cells in culture, throughout their different stages of osteoblastic development. In addition, we also studied the role of reactive oxygen species (ROS) and NOS expression in the possible alterations of osteoblastic growth induced by the glycated collagen matrix.

It has been established that sustained hyperglycemia can cause tissue damage through several mechanisms, including the accelerated formation of AGE on proteins, phospholipids and DNA 17. An important consequence of this non-enzymatic modification involves the recognition of the AGE moieties by specific plasma membrane receptors, which can in turn induce alterations in cellular functions by ROS-mediated mechanisms. The accumulation of AGE occurs on both intracellular and extracellular structures (such as plasma and extracellular matrix proteins [ECM]), altering their bioactivity and associative properties. In particular, the accumulation of AGE on bone type I collagen, which constitutes over 85% of bone ECM proteins, has been previously described in both diabetes and ageing 8910. The integrity of the ECM is essential to maintain the normal function of osteoblasts, the main cell type involved in the formation of bone 18. Thus, AGE-modified ECM may have important consequences on the normal bone-forming activity of osteoblasts, possibly through changes in cell-matrix interactions. Our present study confirms a previous report 10 showing that the formation of AGE on type I collagen can alter osteoblastic growth and inhibit osteoblast differentiation. In addition, we have further extended that study by investigating the effect of AGE-modified collagen on cell adhesion, its relationship to the stage of osteoblastic development, and the possible involvement of oxidative stress and NOS expression as mediators of the action of AGE on osteoblastic cells.

Our results show that UMR106 osteosarcoma cells tend to adhere less to AGE-collagen than to unmodified collagen. However, no differences were found in the case of the non-transformed MC3T3E1 line. These observations seem to indicate that the effect of AGE modification of ECM on cell attachment and growth could depend on cell type. This concept is supported by reports from other investigators who have found either a decrease in cell adhesion induced by AGE collagen in osteosarcoma MG63 cells, fibrosarcoma cells 19 and rat embryo 3Y1 fibroblasts 20, or no effect of AGE on adhesion as in the case of the osteosarcoma sub-clone UMR106-06 21. In this context, the observed differences could be due to different subsets of adhesion molecules involved in the attachment to ECM by different cell types, for example in non-transformed as opposed to tumorigenic cells. It has recently been demonstrated that type I collagen interacts with the α₂β₁ cellular integrin receptor 2223. The sequence Asp-Gly-Glu-Ala (DGEA) present in type I collagen serves as the main recognition site for α₂β₁ integrin, which has been dubbed the collagen-specific receptor. In recent studies, Paul and Bailey 19 found that α₂β₁ is probably involved in the attachment of MG63 osteosarcoma cells to a collagenous matrix, since they were able to inhibit cellular adhesion by an incubation with blocking anti-α₂β₁ antibodies. They also suggested, though did not directly prove, that glycation of type I collagen with methylglyoxal (a potent dicarbonylic AGE precursor) modifies arginine residues present in its Arg-Gly-Asp (RGD) sequence which is common to several other extracellular matrix proteins, and normally is recognized by the α₅β₁ integrin receptor 22. Thus, the RGD sequence is present in the procollagen molecule and could in theory mediate the interaction between bone cells and type I collagen. In actual fact, since anti-α₂ (but not anti-α₅) blocking antibodies inhibit cellular adhesion to a type I collagen substratum, RGD-α₅β₁ interaction does not appear to be normally involved in the cellular attachment to type I collagen 2223. However, it is conceivable that different adhesion sequences of collagen could become either exposed or masked for interaction with several kinds of cellular receptors, as a consequence of molecular alterations of ECM such as AGE modification. Further experiments are in progress in our laboratory to investigate this hypothesis.

In this study we also examined the effect of AGE-modified collagen on osteoblastic proliferation and spreading, differentiation (ALP activity) and mineralization (nodule formation). The effect of AGE on cellular phenotypic expression was found to depend on cell type and/or the stage of osteoblast development. AGE-modified collagen not only decreased ALP activity in phenotypically mature osteoblastic cells, but also diminished nodule formation in post-mitotic osteoblasts in their mineralizing stage of development. AGE-collagen also induced changes in cell proliferation and spreading in a cell-type-specific manner. Thus, after an incubation of 24 hours the glycated matrix significantly inhibited growth and spreading in osteosarcoma UMR106 cells. These observations are in general agreement with the report of Paul and Bailey 19, who found that AGE-collagen decreased the number of matrix-attached MG63 osteosarcoma cells, after an incubation of 20 hours. On the contrary, in the case of non-transformed MC3T3E1 osteoblasts in their proliferating stage of development (24-hour incubation), AGE-modified collagen induced a significant increase in cell growth and spreading. Similarly, in a recent report 10 Katayama found an AGE-collagen-induced increase of DNA synthesis in a primary culture of rat osteoblasts after an incubation of 48 hours. The apparently discordant results obtained in the two cell lines which we studied, may merely be the consequence of dissimilar patterns of intracellular signaling between tumorigenic and non-transformed cells, possibly as a reflection of different receptors involved in their recognition of control or glycated matrices. In post-mitotic mineralizing MC3T3E1 osteoblasts (3 weeks of culture), AGE-collagen was associated with a significant decrease in the number of surviving cells. However, since cells at this stage of development do not divide, the observed effect may be explained by an AGE-induced acceleration of necrosis or apoptosis in the mineralizing cultures. In fact, apoptosis is the endpoint of most recruited osteoblasts in vivo, and it has been shown that the products of different genes are involved in the growth, differentiation and apoptosis of osteoblast-like cells in culture 24.

We have previously described that in UMR106 and MC3T3E1 cells, soluble AGE-BSA initially induces an increase in cellular proliferation and differentiation, while a relatively long-term incubation of these cells with AGE-BSA elicits a significant decrease in both parameters 13. In addition, we have also shown that soluble AGE-modified BSA alters the secretion of IGF-I and IGFBPs-one of the main growth factor systems involved in osteoblastic progression and apoptosis- in a manner that is dependent on the stage of osteoblastic development 15. All of these AGE-BSA induced effects are most probably a consequence of the recognition of soluble AGE structures by their receptors, which we have previously described for both these cell lines 14. On the other hand, the relatively different osteoblastic response to AGE-collagen which we report in the present study, may not only be a consequence of the signal transduction mechanisms induced by the occupancy of AGE-receptors, but also be mediated by integrin or growth factor receptors, which are intimately associated with the process of osteoblast growth and differentiation. In summary, both previous reports and our present study suggest a deleterious effect of AGE-modified proteins on osteoblastic development.

To unravel the mechanisms by which AGE-modified collagen may affect osteoblast development, we studied ROS formation and NOS expression in our culture model. The formation of ROS was inversely correlated with the AGE-collagen-induced changes in cellular proliferation. Thus, an increase in ROS formation was observed in parallel with an inhibition of osteosarcoma UMR106 cell growth. On the contrary, an AGE-mediated increase in MC3T3E1 proliferation was associated with a decrease in ROS formation. ROS have been found to be central to the early stages of the transduction-signaling mechanism initiated as a consequence of the recognition of AGE moieties by their specific receptor RAGE 2526. This signaling involves oxidation of p21^ras by ROS, and subsequent activation of MAP kinases and NF-κB (a nuclear transcription factor). In this sense, the AGE-induced modulation of intracellular ROS levels may also be an important regulator in the expression of certain subsets of genes in osteoblastic cells.

We also assessed the effect of AGE-modified collagen on the expression of NOS. There are a number of studies that have linked an increase in the release of NO with hyperglycemia and diabetes 272829. In UMR106 as well as in MC3T3E1 osteoblasts we have previously shown that the release of NO is very low 30, and that eNOS and iNOS are enhanced after culturing the cells with vanadate, an agent that induces cytotoxicity in both cell lines. The present study provides evidence which suggests that eNOS and iNOS may be involved in the AGE-collagen-induced events observed in osteoblastic cells. Culturing UMR106 and MC3T3E1 osteoblasts for 24 hours on an AGE-modified type I collagen matrix, significantly enhanced the expression of both NOS isoforms. Previous studies have shown that pretreatment of macrophages with AGE-BSA provokes an increase in the levels of LPS- and IFN-γ-induced iNOS mRNA, an effect which is accompanied by an enhanced production of NO 6. However, this effect is only observed in the presence of cytokines: the incubation of macrophages with AGE-BSA or unmodified BSA in the absence of cytokines does not produce any increase in the nitrite levels. To our knowledge, our present findings are the first to document an effect of AGE-modified collagen on the expression of non-cytokine stimulated iNOS in osteoblastic cells. We also detected an even greater increment in the expression of the constitutive isoform eNOS after culturing osteoblast-like cells on an AGE-modified collagenous matrix. These observations are in contrast with a previous report describing an inhibition of eNOS expression in retinal vascular endothelial cells after their incubation for 5 days with soluble glycated albumin 5. The observed differences may be due to the cell system under study, to the time of AGE exposure, or more likely to the cell surface receptors involved in the AGE-modified protein effects and the signaling pathways evoked after their occupation. Nevertheless, the NO/NOS system very probably represents a relevant regulatory mechanism compromised in the cellular consequences of the accumulation of AGE on extracellular matrix molecules.

The present study shows that the non-enzymatic glycosylation of a type I collagen matrix induces alterations on bone cells in culture, which are dependent on (a) cell type and (b) the stage of osteoblastic development. In the osteosarcoma UMR106 cell line, AGE-collagen decreases cellular attachment, spreading, proliferation, and ALP activity. In pre-osteoblastic MC3T3E1 cells, AGE transiently increases cell proliferation and spreading. When these cells attain a mature osteoblastic phenotype, AGE-modified collagen inhibits the expression of ALP, the formation of calcified nodules of mineralization, and the long-term survival of cells. Overall, these results seem to indicate that AGE-collagen induces deleterious effects on osteoblastic growth and function. If these mechanisms were relevant in vivo, the accumulation of AGE on bone extracellular matrix could contribute to the decrease in osteoblastic function which has been described in diabetic patients with osteopenia. Furthermore, we have presented evidence for the involvement of ROS and NOS in the AGE-mediated effects on bone-producing cells.