A Novel Therapeutic Agent for Rescuing the Adverse Effects of Advanced Glycation End-products in the Intervertebral Disk

Abstract

Introduction Long-lived structural proteins such as collagen and aggrecan, particularly in tissues with low turnover, such as the intervertebral disk, are susceptible to accumulation of advanced glycation end-products (AGEs).1 The increase in AGEs, along with other changes in the matrix, has been implicated in the degeneration of the IVD.1,2 The changes induced by increased AGEs include specific deteriorations in the elastic and viscoelastic mechanical behaviors of the IVD tissues.3 We aim to mechanistically investigate the relationship between AGEs and the IVD tissue material behavior. We hypothesize that it may be possible to rescue the deleterious effects of AGEs using a novel AGEs-breaker4 and restore the tissue mechanical function. To investigate this hypothesis, we sought out to (1) investigate the effects of tissue mechanical behavior by inducing the formation of AGEs in vitro; and (2) reduce the amount of AGEs and restore the tissue mechanical behavior from both in vitro inductions and in vivo accumulations of the AGEs using a novel AGEs breaker. Materials and Methods A total of 11 fresh frozen lumbar spines (T12/L1 - L5/S1) were collected (5M/6F) with a mean age of 82.5 years ± 10.2. The cross-sections of the lumbar disk were visually categorized into Thompson grades 1 to 55 and corroborated with fluoroscopic imaging. The grade 1 (“Control”) and grade 5 (“Degenerate”) disks were used for this study (total 27 disks). The disks were separated into annulus fibrosus (AF) and nucleus pulposus (NP) tissues. The Control disks were tested mechanically and then divided to undergo 0, 2, 4, 8, and 10 days of ribosylation in 0.6 M ribose at 37°C.6 The treated control disks, along with the degenerated disks, were then tested mechanically. The resulting force-deformation curves, accounting for the probe geometry, were used to compute elastic modulus and tan δ. Following mechanical measurements, the 0-day and 4-day treated control NP tissues were incubated in 0.15 M of a thiazolium salt known to cleave AGEs products4 for 4 days. The degenerate NP tissues were also incubated for 4 days in the thiazolium salt solution. Following the second incubation, all tissues were mechanically assessed again, and then subjected to acid hydrolysis by 6N hydrochloric acid (16 hours, 110C). The collagen cross-linking by AGEs, a sugar-derived posttranslational modification of amino acids, was quantified by measuring the autofluorescence of the acid hydrolysates at 370 nm emission and 440 nm excitation and divided by collagen content. The collagen content was measured from the acid-hydrolysates using a chloramine T absorbance assay that measured the hydroxyproline, an amino acid on the collagen protein that constitutes approximately 14% of collagen by mass7. Results The in vitro ribosylation of the Control AF and NP tissues resulted in a significant increase in the advanced glycation end-products (AGEs). Correspondingly, the changes in AGEs resulted in changes in the elastic ( p < 0.001; ANOVA) and viscoelastic ( p < 0.001; ANOVA) behavior of the tissues (Fig 1). The elastic behavior of the AF tissue was more affected by the increase of AGEs, whereas the NP viscoelastic behavior sustained greater changes compared to AGEs. Treatment by thiazolium salts resulted in significant reductions in AGEs in the Grade 1, Grade 1 + Ribosylation, and Grade 5 NP disk tissues (Fig. 2). Correspondingly, these changes in AGEs also resulted in changes in both the indentation modulus and tan d of the thiazolium-treated tissues. Conclusion Although disk degeneration is coupled with changes in cell behavior, matrix composition, and tissue-level mechanics of the IVD, we show here that modulating the levels of the AGEs can produce specific changes in elastic and viscoelastic properties differentially in the AF and NP tissues that are similar to that of degeneration.3,8 Furthermore, reduction in AGEs restores the tissue functional mechanical behavior comparable to a healthy disk. Many questions remain such as the biological response towards AGE-breakers and viable delivery methods. The results here, however, suggest that modulating the levels of AGEs may be a feasible strategy towards restoring tissue mechanical function of the IVD. I confirm having declared any potential conflict of interest for all authors listed on this abstract No Disclosure of Interest None declared Sivan, et al, Biochem J 2006;299:29–35. Pokharna HK and FM Phillips. Spine 23(15): 1645–1648 Morse, et al. Trans Ortho Res Soc 2012 Vasan, et al. Ar biochem biophys 419(1): 89–96 Thompson JP et al. Spine 1990;15 Tang SY, et al. Bone 40(4):1144–1151 Woessner JF. Ar biochem biophysics 93:440–447 Jazini, et al. Spine 2011. DOI: 10.1097/BRS.0b013e31822ce81f