Fibulin-1 is a marker for arterial extracellular matrix alterations in type 2 diabetes.

BACKGROUND: Extracellular matrix alterations are important elements in the arterial changes seen in diabetes, being associated with increased vascular stiffness and the development of cardiovascular diseases. However, no biomarkers for diabetes-related arterial changes have been defined. METHODS: Mammary artery specimens from 17 men with type 2 diabetes and 18 nondiabetic individuals were used for microarray expression profiling, quantitative real-time PCR, immunoassay, and immunohistochemical analyses. A derived candidate marker, fibulin-1, which is an elastin-associated matrix molecule, was measured immunochemically in plasma from (a) 70 patients scheduled for vascular surgery, (b) 305 patients with type 2 diabetes examined with carotid ultrasonography and echocardiography, and (c) 308 patients with type 2 diabetes, followed for 15 years. RESULTS: The most upregulated transcript in nonatherosclerotic arterial tissue from patients with type 2 diabetes encoded the extracellular matrix protein, fibulin-1. Higher concentrations of fibulin-1-protein were present in artery extracts from patients with diabetes than extracts from individuals without diabetes, and increased fibulin-1 immunostaining was apparent around the external elastic lamina of diabetic arteries. Patients with diabetes displayed increased plasma concentrations of fibulin-1 (P = 0.006). Plasma fibulin-1 concentrations correlated with hemoglobin A(1c) (P < 0.001), arterial stiffness indices including pulse pressure (P < 0.001), and carotid compliance (P = 0.004), as well as plasma N-terminal pro-B-type natriuretic peptide concentrations (P < 0.001) and were predictive of 15-year mortality (P = 0.013). CONCLUSIONS: Fibulin-1 accumulates in the arterial wall and in plasma of patients with type 2 diabetes, and appears to be a factor associated with arterial extracellular matrix changes in type 2 diabetes.

Total and isoform-specific quantitative assessment of circulating fibulin-1 using selected reaction monitoring MS and time-resolved immunofluorometry.

PURPOSE: Targeted proteomics using SRM-MS combined with stable-isotope dilution has emerged as a promising quantitative technique for the study of circulating protein biomarkers. The purpose of this study was to develop and characterize robust quantitative assays for the emerging cardiovascular biomarker fibulin-1 and its circulating isoforms in human plasma. EXPERIMENTAL DESIGN: We used bioinformatics analysis to predict total and isoform-specific tryptic peptides for absolute quantitation using SRM-MS. Fibulin-1 was quantitated in plasma by nanoflow-LC-SRM-MS in undepleted plasma and time-resolved immunofluorometric assay (TRIFMA). Both methods were validated and compared to a commercial ELISA (CircuLex). Molecular size determination was performed under native conditions by SEC analysis coupled to SRM-MS and TRIFMA. RESULTS: Absolute quantitation of total fibulin-1, isoforms -1C, and -1D was performed by SRM-MS. Fibulin-1C was the most abundant isoform in plasma. Circulating fibulin-1 isoforms were homo -or hetero multimeric complexes (range 318-364 kDa). Good correlation was obtained between SRM-MS and TRIFMA but not CircuLex. CONCLUSIONS AND CLINICAL RELEVANCE: For biomarker studies using smaller cohorts, SRM-MS provides an alternative measure of total and specific fibulin-1 isoforms in undepleted plasma. For larger cohorts TRIFMA provides a faster platform for fibulin-1 quantitation in plasma. While the correlation between these methods was acceptable, low correlation was obtained between the commercial CircuLex assay and SRM-MS or TRIFMA.

Fibulin-1C, C1 Esterase Inhibitor and Glucose Regulated Protein 75 Interact with the CREC Proteins, Calumenin and Reticulocalbin.

Affinity purification, immunoprecipitation, gel electrophoresis and mass spectrometry were used to identify fibulin-1C, C1 esterase inhibitor and glucose regulated protein 75, grp75, as binding partners of the CREC proteins, calumenin and reticulocalbin. Surface plasmon resonance was used to verify the interaction of all three proteins with each of the CREC proteins. Fibulin-1C interacts with calumenin and reticulocalbin with an estimated dissociation constant around 50-60 nM. The interaction, at least for reticulocalbin, was not dependent upon the presence of Ca2+. C1 esterase inhibitor interacted with both proteins with an estimated dissociation constant at 1 µM for reticulocalbin and 150 nM for calumenin. The interaction, at least for calumenin, was dependent upon the presence of Ca2+ with strong interaction at 3.5 mM while no detectable interaction could be found at 0.1 mM. Grp75 binds with an affinity of approximately 3-7 nM with reticulocalbin as well as with calumenin. These interactions suggest functional participation of the CREC proteins in chaperone activity, cell proliferation and transformation, cellular aging, haemostasis and thrombosis as well as modulation of the complement system in fighting bacterial infection.

Fibulins and their role in cardiovascular biology and disease.

Fibulins are a group of extracellular matrix proteins of which many are present in high amounts in the cardiovascular system. They share common biochemical properties and are often found in relation to basement membranes or elastic fibers. Observations in humans with specific mutations in fibulin genes, together with results from genetically engineered mice and data from human cardiovascular tissue suggest that the fibulin family of proteins play important functional roles in the cardiovascular system. Moreover, fibulin-1 circulates in high concentrations in plasma and may function as a cardiovascular disease marker.

Metformin, but not rosiglitazone, attenuates the increasing plasma levels of a new cardiovascular marker, fibulin-1, in patients with type 2 diabetes.

OBJECTIVE The extracellular matrix protein fibulin-1 is upregulated in the arterial wall in type 2 diabetes (T2D) and circulates in increased concentrations in diabetes. Metformin is an antidiabetic drug with beneficial cardiovascular disease effects in diabetes. We hypothesized that metformin would influence the increased level of plasma fibulin-1 in diabetes. RESEARCH DESIGN AND METHODS After a 4-week run-in period, 371 eligible patients with T2D were randomized to treatment groups in a factorial design including insulin alone (control), +metformin, +rosiglitazone, or +both metformin and rosiglitazone. Plasma fibulin-1 was analyzed at the beginning of the study and after 18 and 24 months. RESULTS Plasma fibulin-1 increased in all groups throughout the 2-year period; however, the increase was strongly attenuated among patients treated with metformin. A highly significant difference was observed when the mean change in plasma fibulin-1 was compared between metformin- and non-metformin-treated individuals both at 18 and 24 months of treatment, but rosiglitazone had no effect. Metformin and rosiglitazone alone reduced the HbA1c levels to comparable levels and in combination even further. CONCLUSIONS Metformin attenuates the increase in plasma fibulin-1 concentrations in T2D, independently of glycemic effects. Changes in fibulin-1 may reflect an important element in diabetic arteriopathy that can be influenced by metformin.