Sci. Aging Knowl. Environ., 26 January 2005
Vol. 2005, Issue 4, p. pe3
[DOI: 10.1126/sageke.2005.4.pe3]


Apoptotic Killing of Fibroblasts by Matrix-Bound Advanced Glycation Endproducts

Mark E. Obrenovich, and Vincent M. Monnier

The authors are in the Department of Pathology at Case Western Reserve University, Cleveland, OH 44106, USA. E-mail: vmm3{at} (V.M.M.)

Key Words: extracellular matrix • skin • advanced glycation endproduct • RAGE • apoptosis • diabetes • cancer


Aging of the extracellular matrix (ECM) is characterized by the accumulation of postsynthetic modifications due to glycation (the so-called Maillard Reaction). During this reaction, protein molecules become modified through the nonenzymatic adduction of sugar residues onto proteins. Such adducts, commonly known as glycation adducts, can be identified in the blood (for example, as glycated hemoglobin A1c) or in the urine. Through a subsequent complex series of reactions, these adducts yield a variety of reaction products known as advanced glycation endproducts (AGEs). Other processes, like oxidation, can contribute to the specific accumulation of these AGEs in long-lived tissues, such as skin. In this regard, mammalian skin contains insoluble collagen-rich ECM that accumulates AGEs at a rate that increases with age in nearly all mammalian species investigated so far (1) and is dramatically higher in individuals with diseases such as diabetes and end-stage renal disease as compared with healthy individuals (2, 3). Both aging and diabetes are also associated with problems of wound healing and the occurrence of higher than normal rates of fibroblast apoptosis.

Now, new evidence from Alikhani, Graves, and colleagues at Boston University School of Dental Medicine implicates carboxymethyl-lysine (CML), a major AGE contained in the ECM in skin, in dermal fibroblast death by apoptosis (4).

CML Induces Fibroblast Apoptosis

Using chemically modified collagen preparations that were specifically enriched in CML, the authors demonstrated an ability to induce apoptosis in fibroblasts both in vivo and in vitro. Apoptosis, or programmed cell death, can be instigated through several routes, including a cytoplasmic pathway that involves tumor necrosis factor (TNF) and a mitochondrial pathway that is activated when damaged mitochondria release cytochrome C. Each pathway involves the activation of specific caspases (cysteine proteases that generally exist in the cell in inactive form and are activated upon cleavage) (see "More Than a Sum of Our Cells", "Membranes of Death", and "Ironing Out Cell Death").

Alikhani et al. found that AGE-induced apoptosis occurred in vivo and in vitro directly through the activation of the effector caspase 3, which was activated via both the caspase 8-dependent cytoplasmic pathways and the caspase 9-dependent mitochondrial pathway. Subsequent in vivo experiments established that injection of CML-rich but not unmodified collagen into mouse connective tissue induced fibroblast apoptosis that was decreased by 50 to 80% by pretreatment with caspase 8 and 9 inhibitors. Because CML is a known ligand for the receptor for advanced glycation end product (RAGE; see below) (5), the authors investigated whether this activity was receptor mediated. In support of this idea, they found that antibodies that bind to the extracellular domain of RAGE and inhibit the interaction of CML-modified proteins with this receptor effectively blocked apoptosis. This finding is important in that it shows the apoptosis is mediated through RAGE signaling. Further, using their cell culture system and DNA microarrays, they found that the CML-collagen preparation enhanced the global expression of proapoptotic mRNAs, as well as mRNAs encoding several classes of molecules that include ligands, receptors, adaptor molecules, and mitochondrial proteins.

Importantly, the pattern of expression of apoptotic markers was not identical with the pattern of apoptotic genes induced by TNF{alpha}, which was explored in a parallel experiment. This finding indicates that contamination during sample preparation by bacterial endotoxin, which can induce apoptosis via TNF{alpha}, is an unlikely culprit. Moreover, TNF{alpha} alone might have originated via RAGE activation in macrophages, which produce numerous other factors and cytokines that are associated with cell death. Likewise, a major redox-sensitive signal transduction pathway that involves NADPH oxidase and the transcription factor NF-{kappa}B would also have led to the production of NF-{kappa}B-regulated cytokines, including TNF{alpha} (6). Thus, the differential pattern of expression implies that TNF{alpha}-independent pathways may be involved in the induction of apoptosis by CML-collagen.

Maillard Reaction Products Modify Tissue Matrices during Aging

The evidence generated by Alikhani et al. joins a growing number of observations that implicate several products of the Maillard reaction in structural and biological alterations that occur in aging and age-related diseases (7) (see Obrenovich Perspective). In skin, these changes include (i) decreased collagen synthesis and elasticity, (ii) increased protein cross-linking, (iii) decreased digestibility (by proteases) and turnover rate, and (iv) impaired tensile properties and altered surface charges, all of which may in part explain why skin becomes thin, prone to shear-stress forces, and impaired in its wound-healing capacity in old age.

To date, more than 15 AGE structures have been characterized in skin collagen. AGE structures such as CML, fructosyl-lysine (glycated lysine), and AGE cross-links such as glucosepane are among the main modifications found in aging collagen (8). The biologically active CML is formed nonenzymatically from the oxidation of residues glycated by glucose or any other sugar, as well as from the reaction of carbonyl groups in oxoaldehydes, such as glyoxal and glycolaldehyde, and ascorbic acid oxidation products (see Monnier Perspective). However, importantly, CML can also be generated via lipid peroxidation, which is why it is also called an advanced lipoxidation product (ALE) (9), as well as by inflammation via oxidation of serine resides by the myeloperoxidase system (10).

Biological Effects of AGEs and CML-Rich Proteins: The Role of RAGE

Strictly defined, the acronym "AGE" applies only to stable endproducts from the reaction of reducing sugars, such as glucose, and amines. However, it has become synonymous with advanced Maillard reaction endproducts, a broader descriptor that nowadays includes a larger number of reactive carbonyl compounds from various metabolic sources (7). In the context of biological experiments, however, many investigators have been using the term "AGE" to indicate proteins that were incubated long-term with glucose in phosphate buffer under oxidizing conditions, leading to preparations rich in CML and a host of other products, some of which were recently characterized (11). Such preparations are known to have broad biological properties, including the ability to activate or cause the release of (i) inflammatory cytokines and factors such as TNF{alpha}, IL-1, IL-6, and TGF{beta}; (ii) cell adhesion molecules such as VCAM-1, ICAM-1, and E-selectin; and (iii) metalloproteinases such as MMP2, MMP3, and MMP13, to name just a few. Many more effects depending on the cell type and system under investigation have been reported [for recent reviews, see (12, 13)].

Some of the AGE-induced effects are mediated, in part, by AGE ligands that bind several members of scavenger and nonscavenger receptor families (classes of receptors that either remove, or signal the presence of, offending substrates). The best characterized is RAGE, which is a cell-surface receptor of the immunoglobulin family present in a large number of cells, including vascular endothelial cells, smooth muscle cells, macrophages, monocytes, and neuronal cells. The implication of RAGE in a host of diabetes and age-related inflammatory conditions in vivo is supported by the following findings: (i) infusion of soluble RAGE (which inhibits RAGE signaling by acting as a decoy receptor) to mice suppresses the progression of atherosclerosis, diabetic nephropathy, and other diabetes-related abnormalities; (ii) transgenic mice that overproduce RAGE exhibit exacerbation of indices of nephropathy and retinopathy; (iii) RAGE knockout animals are protected from the lethal effects of sepsis; and (iv) transgenic mice that express a defective form of RAGE display impaired nerve regeneration (14-16) (see also "Provoking RAGE", "Suppressed RAGE", and "Once More, With Feeling").

Although to our knowledge no aberrant phenotype has yet been described for RAGE knockout mice under nonstressed conditions, RAGE appears to have the attributes of a pleiotropic gene that is beneficial in some but deleterious in other circumstances, apparently especially those that are associated with chronic stimulation. In that regard, the implications of RAGE activation are broad, extending to diabetes, atherosclerosis, Alzheimer's disease, arthritis, periodontitis, and most likely all inflammatory states. Its relevance for cancer is discussed below.

AGEs Lead to Increases in Apoptosis

The decision of whether a cell survives, proliferates, or dies rests within a complex network and amid "cross-talk" between pathways for survival, adhesion, or proliferative signals and those signals that ultimately favor death or apoptosis. A dichotomy is readily evident in the complex interplay between the various known signaling pathways and their intermediates. Diagrammed in Fig. 1 are general pathways and signal transduction intermediates, which have been selected to illustrate the point that, ultimately, growth and even oncogenic events or cell death depends upon these pathways or factors that tip the net balance in favor of one event or the other. In that regard, RAGE was shown to transduce signaling via the Ras-Raf-MEK/ERK pathway in smooth muscle cells (17).

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Fig. 1. Select signaling pathways and the intersection between apoptosis and cell-cycle activation. AGE-RAGE was found to activate the Ras-Raf-MEK/ERK pathway in endothelial cells. [Reproduced from (29) with permission from]

AGEs have been found to be proapoptotic for cultured retinal pericytes, umbilical cord and corneal endothelial cells, neuronal cells, and renal mesangial cells (18-21). The results of Alikhani et al. are important in that they demonstrate that matrix-linked and not only soluble AGEs can mediate apoptosis, and that they do so by inducing expression of proapoptotic cytokines (21-23). Because fibroblasts play important roles in the maintenance and healing of dermal connective tissue, the accumulation of AGEs in skin may have a detrimental effect, in part, through promoting fibroblast apoptosis. In that regard, enhanced apoptosis has been linked to many of the detrimental effects of aging.


What are the implications of Alikhani and coauthors' observations? Cell death is likely innocuous if the cell can be replaced. However, in the retina, CML-triggered death of endothelial cells (which line the microvasculature) or pericytes (which are located next to the endothelial cells) might contribute to the microvascular disease that is associated with hyperglycemia. In the kidney, for example, podocytes (cells that control glomerular permeability to macromolecules), might become permeable to the serum protein albumin if undergoing apoptosis caused by contact with a highly modified CML-rich basement membrane. One condition in which the CML-apoptosis paradigm might be particularly important is the sun-exposed, inflamed skin, in which the 5- to 10-fold reported increase in CML abundance relative to that in unexposed skin (24) might have profound biological effects. In this case, apoptosis might be deleterious by impeding wound healing but beneficial by removing precancerous cells.

In many other instances, however, the consequences of cells sitting on CML-rich matrix might be more devastating than those from apoptosis if cells survive in a state of chronic stimulation, thereby sending deleterious signals to neighboring cells. In the retina, for example, pigment epithelial cells might undergo phenotypic changes when sitting on the CML-rich Bruch's membrane (an ECM layer) and react by forming drusen, molecular debris that accumulates near the retina and is associated with age-related macular degeneration (25, 26) (see "The Eyes Have It" and "Tarnished Vision"), impairing thereby the microarchitecture of the retina. Conceivably, the persistent existence of such cells would make their therapeutic replacement by progenitor stem cells very difficult.

Although many more examples of deleterious CML-cell interaction can be cited or conceptualized, one intriguing thought is that the accumulation of CML in the aging ECM might also have beneficial properties. CML formation prevents age-related matrix cross-linking by blocking reactive lysyl residues and might thus decrease the progression of tissue stiffening. In addition, the CML-rich old matrix might represent an ultimate anticancer mechanism by sending RAGE-dependent proapoptotic signals to metastatic cells entering into contact with the modified matrix (see "Trash Talk"). Indeed, downregulation of RAGE has been associated with increased rates of lung cancer growth (27). However, RAGE signaling has also been associated with enhanced metastasis (28), raising the question of how RAGE exactly influences apoptotic versus proliferative signaling. In that regard, it is unfortunate that Alikhani and colleagues, while carefully documenting changes in apoptotic gene expression after treatment with CML-modified collagen, did not report on the status of proliferative gene expression.

In summary, the discovery of apoptotic signals in a model of senescent ECM consisting of CML-modified collagen and fibroblasts is likely to have major implications for our understanding of cell-matrix interaction in aging and age-related diseases. It is possible that a self-reinforcing vicious cycle would result from the chemical aging and accumulation of CML-rich ECM and abnormal signals stemming from the cells embedded in such modified matrix, as depicted in Fig. 2. If the concept in this figure is correct, inhibition of AGE accumulation in aging and age-related diseases would represent the most powerful intervention against the aging of the ECM.

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Fig. 2. Conceptual integration of vicious self-reinforcing cycles of chemical and biological aging of the ECM, leading to either apoptosis or perpetuation of a chronic inflammatory state.


January 26, 2005
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Citation: M. E. Obrenovich, V. M. Monnier, Apoptotic Killing of Fibroblasts by Matrix-Bound Advanced Glycation Endproducts. Sci. Aging Knowl. Environ. 2005 (4), pe3 (2005).

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