Thioflavin T fluorescence in human serum: Correlations with vascular health and cardiovascular risk factors
Introduction
Amyloid deposition is implicated in neurodegenerative diseases [1]. Amyloid fibrils display a characteristic morphology, increased β-structure relative to the non-fibrillar form, and the ability to interact with, and alter the spectral properties of the dye thioflavin T (ThT). Amyloid deposits are also associated with atherosclerosis [2], with histological analysis showing amyloid in the aortic media of 97%, and in the aortic intima of 35% of people over 50 years of age [3]. Immunohistochemical studies of human coronary artery plaque demonstrate accumulation of apolipoproteins, including apolipoprotein (apo)A-I, apoB, apoC-II and apoE, which co-localise with serum amyloid P, a marker of amyloid structures [4].
Plaque amyloid deposits may contribute to age-related, diminished vascular elasticity. Loss of arterial elasticity, which can be measured non-invasively by pulse-wave analysis, occurs in several high cardiovascular risk conditions including diabetes, coronary artery disease, rheumatoid arthritis (RA), systemic lupus erythematosis (SLE), and renal disease [5], [6], [7], [8]. Amyloid structures composed of the β-amyloid peptide or apoC-II activate macrophages [9], [10], which may represent an early event in atherosclerosis. We have shown that oxidised cholesterol metabolites found in human atheroma promote apoC-II amyloid fibril formation, suggesting that oxidative modification of the lipid component could promote amyloid deposition [11]. In addition, the direct oxidative modification of low-density lipoproteins (LDL), generates amyloid-like properties, including the ability to interact with ThT [12]. These observations suggest that amyloid-like structures may influence several processes associated with atherosclerosis.
Amyloid-like structures also result from modification of human serum albumin (HSA) by incubation with glucose [13]. Protein modification by glucose generates advanced glycation end-products (AGEs), which are a structurally heterogeneous group of compounds produced via the non-enzymatic reaction of reducing carbohydrates with amino groups of proteins, followed by a series of oxidation, dehydration and rearrangement reactions [14]. AGE accumulation in diabetes is considered a key trigger of endothelial dysfunction leading to vascular damage [15], [16], [17]. A family of receptors for AGEs has been identified in macrophages and vascular cells, and may play an essential role in inflammation and atherosclerosis progression [2], [16], [17]. We hypothesise that the covalent modification of proteins, in blood or the sub-endothelial space, generates amyloid-like structures that trigger early events in atherosclerosis. Accordingly, we examined the properties of the major ThT binding species in human sera and their association with known cardiovascular risk markers.
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Subjects
The study was approved by the St. Vincent's Hospital Human Research Ethics Committee, methodologies conformed to the standards set by the Declaration of Helsinki, and each subject gave written, informed consent. Human subjects were recruited from St. Vincent's Hospital Clinics and assessed by the clinician investigators (A.J.J., M.W., A.M.W., T.G., E.R., A.L., and L.C.). Recruited subjects had no known diabetes, and were not receiving hypoglycaemic drugs or therapeutic insulin. Absence of
Serum ThT fluorescence shows natural variation between subjects
Subject demographics are shown in Table 1. ThT fluorescence in sera from 35 controls, 36 RA, and 34 SLE subjects showed 8.75-fold variation from the lowest to highest values, with average values (± SD) of 0.97 ± 0.26, 1.12 ± 0.45, and 0.74 ± 0.23 for the control, RA, and SLE sets respectively. The average ThT fluorescence value over all 105 samples was 0.95 ± 0.44 units (Table 1).
Serum ThT fluorescence tracks with vascular dysfunction and cardiovascular risk factors
All subjects were ranked and grouped into tertiles, on the basis of their serum ThT fluorescence measurements. Measurements
Discussion
ThT has been widely used to diagnose tissue amyloid deposits and to follow amyloid fibril formation from purified proteins and peptides in vitro. In the presence of amyloid fibrils and amyloid-like structures, ThT has distinct fluorescence excitation and emission spectra relative to the free dye. This fluorescence change is generally considered specific to amyloid-like structures, and is not induced upon binding to native protein monomers or amorphous aggregates [23]. We report, for the first
Conclusion
The correlation of total serum ThT measurements with vascular dysfunction and other vascular risk factors suggests that ThT fluorescence in serum may represent a novel, inexpensive, and simple measurement for assessment of cardiovascular risk. We have shown that ThT fluorescence detects glycated proteins and AGEs. These serum components, which have firm links with cardiovascular disease progression [16], [17], are likely to comprise a major contribution to serum ThT fluorescence levels. Both
Acknowledgments
The authors thank Dr Michael Bailey for assistance with fluorescence spectroscopy. This work was supported by the ARC (DP0449510) and NH&MRC (350229), an Australian NHF Clinical Research Fellowship (A.J.J.), an NH&MRC Neil Hamilton Fairley Fellowship (A.W.), and a University of Melbourne ECR Grant (A.S.J.).
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