The main findings of this study can be summarized as follows: (1) oral AST-120 reduced the plasma levels of AGEs, probably, via absorption of dietary AGEs; (2) AST-120 therapy decreased circulating and tissue levels of AGEs and resulted in improvements in neovascularization and blood flow recovery in a hind-limb ischemia model in diabetic mice; and (3) the polarity of macrophages in the ischemic tissues was predominantly pro-inflammatory M1 phenotype in diabetic mice, and AST-120 therapy reversed this polarity into the pro-angiogenic M2 phenotype.
In patients with diabetes, neovascularization is insufficient to overcome the loss of blood flow that occurs due to arterial narrowing or occlusion . Angiogenesis—defined as the sprouting of new blood vessels from pre-existing vascular structures—is a physiological reaction to tissue ischemia . This process starts with the degradation of non-fibrillar collagens in the basement membrane, followed by migration and proliferation of pre-existing vascular endothelial cells or circulating endothelial progenitor cells (EPCs) . Angiogenesis is initiated by hypoxia and inflammation, and it involves angiogenic, anti-angiogenic, and maturation factors . Previous studies have found that alterations in VEGF expression, attenuation of monocyte migratory ability, and impaired EPC mobilization contribute to impairments in neovascularization in diabetes [21, 22]. Formation of AGEs is one of the major mechanisms responsible for vascular damage in diabetes . In RAGE knockout mice, collateral growth and blood flow recovery are reduced when compared with WT mice . Blockade of the formation of AGEs by aminoguanidine also helps in the restoration of perfusion in ischemic tissues . Our data are, broadly, in line with these findings; however, they also provide novel insights into the pathological mechanisms and possible therapeutic approaches.
Many agents have been developed to attenuate the damage induced by AGEs, including inhibitors and breakers of AGEs, antioxidants, natural substances, and anti-inflammatory molecules . Diet-derived AGEs are essential sources of AGEs, and the removal of exogenous AGEs is a potential approach to reduce serum levels of AGEs. It has been demonstrated in healthy subjects that dietary restriction alone could reduce serum levels of AGEs by 30–40% [26, 27]. In patients with diabetes or renal failure, restriction of dietary AGEs could attenuate tissue injury related to AGEs [28, 29]. AST-120 is an oral adsorbent that can bind to many low-molecular-weight (100–10,000 kDa) compounds. It has been used to reduce a variety of uremic toxins in patients with CKD, with concomitant improvements in the intimal-medial thickness and flow-mediated dilatation [30, 31]. AST-120 could completely adsorb CML, a well-recognized food-derived AGE, and reduce serum levels of AGEs in patients with CKD . For the first time, we demonstrated that AST-120 could also reduce serum levels of AGEs in diabetic mice with normal renal functions. Circulating AGEs were reduced by approximately 67% in diabetic mice treated with AST-120, resulting in a serum level equal to that in non-diabetic mice. Although the adsorptive property of AST-120 was not examined in gastric or intestinal fluids, in vitro data suggest that AST-120 could adsorb AGEs in food and, subsequently, decrease the serum levels of AGEs. The evidence collectively suggests that adsorption of exogenous AGEs may be a potential therapeutic approach to attenuate ischemic injury in diabetes. Further studies are required to clarify the optimal dose of AST-120 and the degree of reduction in the levels of AGEs required to produce clinical benefits.
Few studies have addressed the underlying mechanisms via which AGEs attenuate neovascularization. Tamarat et al. demonstrated that the formation of AGEs reduced degradation of the extracellular matrix degradation and, subsequently, abrogated the angiogenic process in diabetic mice . Tanii et al. demonstrated that AGEs disturb the recruitment and functions of pericytes via the platelet-derived growth factor-BB/protein kinase C axis, which regulates the maturation of capillary vessels . Our findings demonstrated that the deleterious effects of AGEs on neovascularization might be related to inflammatory activation in ischemic tissues through the binding of AGEs with RAGE. Macrophages are the principal cells that participate in the inflammatory and angiogenic processes following tissue ischemia. We demonstrated a significant increase in the macrophage infiltration in ischemic tissues in diabetic mice, which was reversed by reducing the AGEs with AST-120 therapy. The infiltration by macrophages was accompanied by increases in pro-inflammatory cytokines and decreases in pro-angiogenic cytokines. The effects of AGEs on macrophages may be mediated through the binding to RAGE, as demonstrated by the co-expression of RAGE and F4/80 positive cells in ischemic tissues. AST-120 decreased not only the circulating AGEs but also the AGE-positive cells, RAGE-positive cells, and RAGE as well as F4/80 double-positive cells in ischemic tissues. The detailed mechanism of enhanced activation of macrophages by AGEs, mainly via the RAGE/NF-κB pathway was demonstrated by Jin et al. .
In addition to inflammatory activation, macrophages respond to environmental signals and transform into different functional phenotypes, ranging from pro-inflammatory (classic M1 activation) to pro-angiogenic phenotypes (alternative M2 activation) [34, 35]. The pro-angiogenic property is not demonstrated in all subsets of macrophages. M2 macrophages promote angiogenesis by producing pro-angiogenic cytokines and growth factors. In diabetic mice, we found the pro-inflammatory M1 phenotype to be the predominant one in ischemic tissues. In non-diabetic mice, the predominant macrophages were of the pro-angiogenic M2 phenotype. A comparable change in pro-inflammatory cytokines (TNF-α, IL-6, MCP-1, and Cxcl6) and pro-angiogenic cytokine (VEGF) expression supports the role of macrophage polarization in the angiogenic process. AST-120 reversed the M1/M2 polarization, resulting in subsequent improvements in neovascularization. The effects of AGEs on macrophage polarity were demonstrated in in vitro studies as well. Either AGEs or high-glucose conditions enhance the transformation of macrophages into M1 phenotype. Administration of AST-120 could reverse the change in polarity.
The impact of macrophage polarization on the angiogenic process was investigated in previous studies. Angiogenic growth factors and cytokines are highly expressed in M2 rather than M1 macrophages, and M2 macrophages promote tube formation by these secretory factors in vitro. [36, 37]. Neutralizing these growth factors could impair the M2-induced angiogenesis . In animal models, the infusion of anti-inflammatory M2 macrophages but not pro-inflammatory M1 macrophages promotes angiogenesis . Collectively, AGE-RAGE related alternations in macrophage polarization might be a probable mechanism underlying the microangiopathy in diabetes.
Some limitations of this study must be acknowledged. First, AGEs are recognized as some of the most potent toxins in diabetes that can be reduced by AST-120. Although mice without CKD were used in this experiment, we could not exclude the possibility that the benefits were derived from reductions in other toxins. Second, the causal relationship between AGEs and neovascularization and detailed mechanical pathways involved could not be proven directly in this animal model.