**B. Circulating esRAGE and cardiovascular diseases**

Following development of an ELISA system to specifically measure human esRAGE 108, we measured plasma esRAGE level and cross-sectionally examined its association with atherosclerosis in 203 type 2 diabetic and 134 non-diabetic age- and gender-matched subjects 15. esRAGE levels were inversely correlated with carotid and femoral atherosclerosis, as measured as intimal-medial thickness (IMT) by arterial ultrasound. Stepwise regression analyses revealed that plasma esRAGE was the third strongest and an independent factor associated with carotid IMT, following age and systolic blood pressure15. Importantly however, when non-diabetic and diabetic groups were separately

Alzheimer disease have also lower levels of sRAGE in plasma than patients with vascular dementia and controls, suggesting a role for the RAGE axis in this clinical entity as

sRAGE references CAD (non-DM) decreased 106, 153, 154 increased 124 Calcified aortic valve stenosis decreased 155 Carotid atherosclerosis decreased 156 Cerebral ischemia decreased 157 <sup>158</sup> <sup>127</sup> Alzheimer's disease decreased 107 <sup>159</sup> Endothelial dysfunction decreased 160 Diabetes (type 1) increased 122 Diabetes (type 2) increased 123, 124 decreased 120, 121 Hypertension decreased 117 NASH decreased 118 <sup>119</sup> Chronic kidney disease increased 109, 123, 129, 161

Oxidative stress and inflammatory markers positively associated 163, 164

CAD decreased 113, 153 Altzheimer's disease decreased 165 Chronic kidney disease increased 114, 161 <sup>162</sup>

Following development of an ELISA system to specifically measure human esRAGE 108, we measured plasma esRAGE level and cross-sectionally examined its association with atherosclerosis in 203 type 2 diabetic and 134 non-diabetic age- and gender-matched subjects 15. esRAGE levels were inversely correlated with carotid and femoral atherosclerosis, as measured as intimal-medial thickness (IMT) by arterial ultrasound. Stepwise regression analyses revealed that plasma esRAGE was the third strongest and an independent factor associated with carotid IMT, following age and systolic blood pressure15. Importantly however, when non-diabetic and diabetic groups were separately

Table 2. Levels of circulating soluble RAGE in cardiovascular and metabolic diseases.

**B. Circulating esRAGE and cardiovascular diseases** 

Insulin resistance inversely associated 15 Metabolic syndrome decreased 15 Diabetes (type 1) decreased 108, 110 Diabetes (type 2) decreased 15, 113 Hypertension decreased 15 NASH decreased 119 Carotid atherosclerosis decreased 15, 110-112

162

inversely associated 121

no association 109

well 107.

esRAGE

analyzed, inverse correlation between plasma esRAGE level and IMT was significant in non-diabetic population only, suggesting a potential significance of esRAGE in nondiabetic condition. No association of plasma esRAGE with IMT in diabetes was also reported in other study with 110 Caucasian type 2 diabetic subjects 109. Another Japanese research group found an inverse correlation between plasma esRAGE and carotid atherosclerosis in type 1 110 and type 2 diabetic subjects 111. Recently, the same research group also longitudinally examined the predictive significance of plasma esRAGE and sRAGE on progression of carotid atherosclerosis, and found that low circulating esRAGE level as well as sRAGE level was an independent risk factor for the progression of carotid IMT in type 1 diabetic subjects 112. In Chinese type 2 diabetic patients, plasma esRAGE is recently shown to be decreased in angiographically-proved patients with coronary artery disease than those without it 113.

### **C. Low circulating sRAGE as a predictor of cardiovascular diseases**

We also reported an observational cohort study in patients with end-stage renal disease (ESRD) and longitudinally evaluated the effect of plasma esRAGE on cardiovascular mortality 114. The cohort in that study included 206 ESRD subjects, who had been treated by regular hemodialysis for more than 3 months. Even though the plasma esRAGE levels at baseline were higher in ESRD subjects than in those without kidney disease, the subjects in the lowest tertile of plasma esRAGE levels exhibited significantly higher cardiovascular mortality, but not non-cardiovascular mortality. Importantly, even in the subpopulation of non-diabetic subjects alone, low circulating esRAGE level was a predictor of cardiovascular mortality, independent of the other classical risk factors. Thus, low circulating esRAGE or sRAGE level is a potential predictor for atherosclerosis and cardiovascular diseases even in non-diabetic population.

### **D. Circulating sRAGE, esRAGE and metabolic syndrome**

Several components of metabolic syndrome have been shown to be associated with altered plasma sRAGE or esRAGE levels. We first reported that plasma esRAGE levels are already decreased in patients with impaired glucose tolerance as compared with those with normal glucose tolerance (Figure 6A). Moreover, patients with metabolic syndrome showed significantly lower plasma esRAGE than those without it (Figure 6A). Plasma esRAGE levels are inversely correlated with many of the components of metabolic syndrome including body mass index (Figure 6B), blood pressures, fasting plasma glucose, serum triglyceride, and lower HDL-cholesterol levels 15. The majorities of these correlations remained significant even when the non-diabetic or type 2 diabetic subpopulation was extracted for analyses. An inverse correaltion between esRAGE (or sRAGE) and body mass index was also found for control subjects 115, those with type 1 diabetes 116, and those with ESRD 114. Patients with hypertension have been found to have lower plasma sRAGE or esRAGE levels 15, 117. Importantly, our findings also showed that plasma esRAGE was also inversely associated with insulin resistance index, HOMA (Figure 6B), suggesting esRAGE and sRAGE as potential biomarkers for metabolic syndrome and insulin resistance, which could be associated with altered cardiovascular outcomes. Both sRAGE and esRAGE are found to be decreased in patients with liver steatosis 118, 119, which is know to be deeply associated with visceral fat accumulation and insulin resistance.

Fig. 6. Plasma esRAGE levels are decreased in glucose intolerance, metabolic syndrome, obesity and insulin resistance (A) Left panel demonstrates the levels of plasma esRAGE in subjects with normal glucose tolerance (NGT) (n=118), impaired glucose tolerance (IGT) (n=16), and type 2 diabetes (DM) (n=203). Right panel compares the plasma esRAGE levels in subjects with (n-53) or without (n=282) metabolic syndrome (Met) as characterized by modified NCEP criteria. \* p<0.05, ANOVA with multiple comparison (Scheffe's type). (B) Plasma esRAGE levels were inversely associated with body mass index or HOMA insulin resistance index. Logarithm-transformed HOMA index was used for the analyses because of the skewed distribution. Modified from ref 15.

### **E. Circulating sRAGE and esRAGE in diabetes**

The findings regarding plasma levels of the soluble form of RAGE in diabetes are quite confusing. We and other groups have found that plasma esRAGE level is significantly lower in type 1 and type 2 diabetic patients than in non-diabetic controls 15, 110. Plasma sRAGE levels have also been shown to be decreased in diabetic subjects 120, 121, although conflicting findings have also been reported for type 1 122 and type 2 diabetes 123, 124. We examined plasma sRAGE levels by different ELISA system using esRAGE as a standard protein and different sets of antibodies against whole RAGE molecule 125. In our hand, type 2 diabetic subjects without overt nephropathy (0.60 ± 0.28 ng/ml) exhibited significantly (p<0.001, Student's t-test) lower plasma sRAGE level than non-diabetic controls (0.77 ± 0.34 ng/ml) 11. Of note, when diabetic subjects alone were extracted for analyses, a direct association was not observed between plasma soluble RAGE (both sRAGE and esRAGE) levels and the status of glycemic control (i.e. glycated hemoglobin A1c) 15, 109, 116, 120, 126. Thus, these complex findings in diabetic subjects suggest that levels of plasma soluble forms of RAGE are not determined simply by status of glycemic control, and that even plasma esRAGE and sRAGE levels may be under the control of distinct mechanisms. Recent study suggests that sRAGE levels may be significantly influenced by ethnicity 127, which may partially explain controversial findings.

### **F. Circulating sRAGE and esRAGE in CKD**

106 Biomedical Science, Engineering and Technology

Fig. 6. Plasma esRAGE levels are decreased in glucose intolerance, metabolic syndrome, obesity and insulin resistance (A) Left panel demonstrates the levels of plasma esRAGE in subjects with normal glucose tolerance (NGT) (n=118), impaired glucose tolerance (IGT) (n=16), and type 2 diabetes (DM) (n=203). Right panel compares the plasma esRAGE levels in subjects with (n-53) or without (n=282) metabolic syndrome (Met) as characterized by modified NCEP criteria. \* p<0.05, ANOVA with multiple comparison (Scheffe's type). (B) Plasma esRAGE levels were inversely associated with body mass index or HOMA insulin resistance index. Logarithm-transformed HOMA index was used for the analyses because of

The findings regarding plasma levels of the soluble form of RAGE in diabetes are quite confusing. We and other groups have found that plasma esRAGE level is significantly lower in type 1 and type 2 diabetic patients than in non-diabetic controls 15, 110. Plasma sRAGE levels have also been shown to be decreased in diabetic subjects 120, 121, although conflicting findings have also been reported for type 1 122 and type 2 diabetes 123, 124. We examined plasma sRAGE levels by different ELISA system using esRAGE as a standard protein and different sets of antibodies against whole RAGE molecule 125. In our hand, type 2 diabetic subjects without overt nephropathy (0.60 ± 0.28 ng/ml) exhibited significantly (p<0.001, Student's t-test) lower plasma sRAGE level than non-diabetic controls (0.77 ± 0.34 ng/ml) 11. Of note, when diabetic subjects alone were extracted for analyses, a direct association was not observed between plasma soluble RAGE (both sRAGE and esRAGE) levels and the status of glycemic control (i.e. glycated hemoglobin A1c) 15, 109, 116, 120, 126. Thus, these complex findings in diabetic subjects suggest that levels of plasma soluble forms of RAGE are not determined simply by status of glycemic control, and that even plasma esRAGE and sRAGE levels may be under the control of distinct mechanisms. Recent study suggests that sRAGE levels may be significantly influenced by ethnicity 127, which may partially explain

the skewed distribution. Modified from ref 15.

controversial findings.

**E. Circulating sRAGE and esRAGE in diabetes** 

Another important component that can affect plasma sRAGE is the presence of chronic kidney disease. It has been shown that, in peripheral monocytes from subjects with varying severities of CKD, RAGE expression is closely associated with worsening of CKD and is strongly correlated with plasma levels of pentosidine, a marker for AGEs 128. Circulating sRAGE levels have been shown to be increased in patients with decreased renal function, particularly those with ESRD 109, 123, 129. Our observations revealed that plasma esRAGE levels in type 2 diabetic subjects without CKD are lower than non-diabetic controls, which is gradually elevated in accordance with progression of CKD 11. Plasma sRAGE levels in diabetic subjects without CKD also exhibited significantly lower than those of non-diabetic controls 11. Thus, plasma sRAGE and esRAGE are markedly affected by the presence of CKD, which might make the interpretation of the role of soluble RAGE quite complicated 130. It remains to be determined whether the increase in plasma esRAGE in CKD is caused by decreased renal function alone or whether esRAGE levels are upregulated to protect against toxic effects of the RAGE ligands. Successful kidney transplantation resulted in significant decrease in plasma sRAGE 131, implying that the kidneys play a role in sRAGE removal.
