Glucosepane is likely the most important form of persistent cross-linking in aging human tissue. There is some remaining uncertainty, but it appears that the vast majority of cross-links in old tissues are based on glucosepane. Cross-links are the consequence of advanced glycation end-products (AGEs), sugary metabolic waste that can bond with the structural molecules of the extracellular matrix. Where two such molecules are linked together by a single AGE (a “cross-link”), it reduces their ability to move relative to one another. The presence of many persistent cross-links thus degrades the structural properties of that tissue. This is particularly true of elasticity, vital to the correct function of skin and, more importantly, blood vessels. Cross-linking is likely an important contribution to arterial stiffening, and the hypertension and cardiovascular disease that follows as a consequence.
The solution to this aspect of aging is to find a way to periodically remove cross-links. That effort has been hampered by the fact that the important cross-links in humans and laboratory species such as mice are completely different. That was well demonstrated by the high profile failure of the cross-link breaker compound alagebrium to perform in humans in the same way that it does in rats. Further, the tools required to work with important human AGEs such as glucosepane have been lacking. Without necessary line items such as animal models, a cheap method of synthesizing glucosepane, and antibodies specific to glucosepane, scientists avoided this part of the field in favor of easier programs of research. Fortunately the SENS Research Foundation started to fund efforts to solve this tooling problem some years ago, and, once started and shown to be productive, that line of work has continued.
Today’s paper reports on the development of specific antibodies for glucosepane by the same group that first produced a robust, low-cost method of glucosepane synthesis. Antibodies that are highly specific to the molecules under study are needed for any rigorous program of development, as without them many assays of cells and tissues become questionable or impossible. This paper is an important step forward, just as much so as the synthesis of glucosepane. This part of the field of cross-link study is being opened up, and the more researchers to participate, the sooner we’ll see successful trials of cross-link breaking drugs capable of removing glucosepane from the human body. There is at present one startup biotech company working towards that goal, and in a better world there would be a dozen, a mirror of the developing senolytics industry.
Glucosepane is among the most abundant AGEs found in human tissues. It is formed from lysine, arginine, and glucose, and it is over an order of magnitude more abundant than any other AGE crosslink in extracellular matrix (ECM). Notably, glucosepane levels have been shown to correlate with various disease states, including diabetic retinopathy, microalbuminuria, and neuropathy. While the exact mechanisms behind glucosepane-mediated dysfunction remain unclear, it is believed to impair the functional and mechanical properties of proteins in the ECM and interfere with proteolytic degradation of collagen.
To date, the primary method for identifying glucosepane in tissues has required exhaustive enzymatic degradation followed by high pressure liquid chromatography-mass spectrometry (LC/MS). Although these protocols have proven effective in quantifying glucosepane in bulk tissue extracts, they are labor-intensive and the degradation process destroys the tissue architecture, making it difficult to examine the localization of glucosepane.
In recent years, anti-AGE antibodies have emerged as useful tools for studying AGEs and have the advantage of being compatible with the evaluation of intact tissues, enabling immunohistochemical staining and imaging procedures. Several anti-AGE antibodies have been produced by immunization of animals with AGEs generated either from total synthesis or through in vitro glycation methods. Such methods involve the incubation of an immunogenic carrier protein, such as BSA, with glucose or other reactive sugar metabolites. Reaction conditions that generate glucosepane are known also to generate a range of AGE by-products, including carboxymethyllysine. These in vitro preparation methods are unlikely to produce antibodies that are specific for glucosepane, although no such studies have been reported.
To avoid this expected complication, we decided to synthesize homogeneous, synthetic glucosepane immunogens. Herein, we describe the development and characterization of the first antibodies known to selectively recognize glucosepane. To this end, we have created a synthetic glucosepane immunogen that closely resembles glucosepane found in vivo and used it to generate a polyclonal antibody serum that recognizes glucosepane both in vitro and in ex vivo tissue samples. We have demonstrated that the antibodies can bind to glucosepane with high degrees of specificity and sensitivity through ELISA studies and have employed these antibodies in immunohistochemical experiments.
Interestingly, these latter studies demonstrate that glucosepane accumulates within sub-components of the retina, specifically the retinal pigment epithelium (RPE), Bruch’s membrane, and choroid, which are anatomic areas highly affected by AMD and diabetic retinopathy.