The first major finding of this study was a significant association between plasma fibulin-1 levels and PWV in patients with type 2 diabetes and gender- and age matched controls when adjusted for effect-modification from gender and diabetes. In stratified analysis, the association remained significant only in the diabetes group. However, as testing for interaction between diabetes and fibulin-1 was only borderline significant, no firm conclusions regarding differences in the strength of the association between PWV and fibulin-1 in the diabetes group versus the control group can be made in the present study. To the best of our knowledge, we are the first to demonstrate such an association. This suggest that, even though the association was modest, fibulin-1 might be involved in the pathophysiological processes underlying arterial stiffening.
A second major finding was that plasma fibulin-1 levels were lower in patients with type 2 diabetes compared with controls. The finding indicated, that the absolute plasma levels of fibulin-1 could not be interpreted as a simple marker of arterial stiffness, as PWV was higher in the diabetic group compared with the controls. The finding of low plasma fibulin-1 in diabetic patients was unexpected, as a previous study by Cangemi et al  reported markedly higher levels of plasma fibulin-1 in patients with type 2 diabetes than in non-diabetics. Plasma fibulin-1 levels should thus be interpreted in the context of potential confounding factors. Differences in duration of diabetes, gender distribution and anti-diabetic medication in the two studies may account for the observed discrepancies. The diabetes duration was not reported in the Cangemi study , but was probably longer in the older type 2 diabetic patients than in the present study population, and accordingly these patients may have had more severe arterial alterations than our patients. Secondly, we and others  found higher plasma fibulin-1 in non-diabetic females compared with males. The gender distribution (female/male) in the study by Cangemi et al  was 5/31 in the control group, whereas it was 44/46 in the present study. Thus, had more females been represented in the study by Cangemi et al , the plasma fibulin-1 level in the control population might have been higher and the difference between the diabetes and control group less pronounced. Thirdly, metformin-treated patients in our study had significantly lower fibulin-1 levels than patients not receiving metformin. The patients in the study by Cangemi et al  were referred for coronary bypass or carotid atherectomies, and had stopped their metformin treatment before the surgical procedure when the blood samples were drawn. The patients in the present study were in ongoing metformin treatment. As our observations are cross-sectional, causality cannot be inferred, yet if metformin had an effect on plasma fibulin-1 and this effect tapered off as the metformin was metabolized, even the short pause in relation to the surgical procedure may have raised fibulin-1 levels in the diabetes group in the study by Cangemi et al  to untreated values, whereas it remained lowered in our study. Clearly, this hypothesis remains speculative given our study design and requires further testing in an experimental setting. Metformin-treatment could potentially also be associated with changes in PWV or other arterial stiffness indices. A recent study found metformin treatment lowered the augmentation index in patients with non-alcohol fatty liver disease . Yet, regarding PWV, intervention-studies in patients with non-alcoholic fatty liver disease  and with polycystic ovary syndrome [26, 27] have reported conflicting results regarding metformin’s ability to lower PWV, and we did not find any association between metformin treatment and PWV.
A third finding was the lack of association of fibulin-1 with Hba1c and the traditional cardiovascular risk factors cholesterol levels and BP indices including pulse pressure. This is also in contrast with findings in the study by Cangemi et al  and a recent study by Scholze et al in patients with end-stage renal disease , where significant associations were observed between fibulin-1 and fasting plasma glucose, HbA1c, blood pressure and also plasma creatinine. It could thus be hypothesized, that the association of plasma fibulin-1 with HbA1c and BP becomes evident only after a certain disease duration with accumulated damage to the arterial wall caused by longer hyperglycemic and hypertensive exposure, whereas PWV might be a more sensitive indicator of arterial stiffening and hence could be associated with fibulin-1 levels even in the early stages of the disease. Cangemi et al also found fibulin-1 to be associated with carotid compliance, stroke volume/pulse pressure ratio, left atrial volume index and plasma NT-proBNP  and Scholze found fibulin-1 associated with plasma fibrinogen, plasma urea and unadjusted augmentation index , factors not assessed in the present study. We cannot exclude, that adjustment for these parameters might have changed the results of our analysis.
PWV was recorded under standardized examination conditions. The most important parameters to take into account when assessing PWV are age, blood pressure and heart rate . Obesity may affect distance measurement. These parameters were adjusted for in the multivariate analyses.
The present findings extend previous research on the pathophysiological basis of the increased arterial stiffness found in patients with diabetes. Increased brachial-radial PWV in diabetic patients was demonstrated in 1962  and interpreted as an expression of increased diabetic atherosclerosis burden. However, whereas the patchy intimal atherosclerotic lesions may contribute to arterial stiffness, atherosclerosis-independent changes in the ECM of the media layer also play a pivotal role for the increased arterial stiffness seen in diabetic patients. Increased amounts of advanced glycosylation end-products (AGEs) , fibronectin , type IV collagen  and hyaluronan  have been found in the ECM of non-atherosclerotic aortic specimens from diabetic patients compared with non-diabetics. Conversely, decreased content of elastin  and increased content of the elastin-cleaving metalloproteinase matrix metallopeptidase 2  has been reported. These changes all contribute to increase arterial stiffness, yet the pathophysiology remains elusive. First recently, fibulin-1 has been associated with arterial alterations in diabetes patients . It is known that fibulin-1 binds elastin , fibronectin  and proteoglycans , yet, the exact role of fibulin-1 in the arterial wall for arterial stiffness remains to be elucidated. The present findings extend on previous observations by the demonstration of an independent association between plasma fibulin-1 levels and carotid-femoral PWV, the gold standard method for assessment of arterial stiffness , in patients with recently diagnosed type 2 diabetes. However, due to the cross-sectional design, we cannot infer causality of the observed association between plasma fibulin-1 levels and PWV. Neither can the correspondence between arterial wall fibulin-1 and plasma fibulin-1 be assessed in this study.
Application of the mendelian randomization approach  in future studies might provide information regarding the existence of a causal biological association between fibulin-1 and arterial stiffness. This approach is used to make assessment of causality from observational data based on genetic variation [34, 35]. A recent study reported association between a genetic marker for fibulin-2 and hypertension , lending support to the hypothesis that members of the fibulin family could be causally involved in arterial wall changes.
In conclusion, plasma fibulin-1 was independently associated with increased PWV in a sample of well-regulated patients with recently diagnosed type 2 diabetes and gender- and age-matched controls. Fibulin-1 might thus be involved in the pathophysiological processes underlying stiffening of the arterial wall. However, the plasma fibulin-1 level is not a simple marker of the degree of arterial stiffening, as evidenced by lower plasma fibulin-1 concentrations in the diabetes patients. Furthermore, the observed association with metformin treatment and gender confers complexity when making correlative interpretations of data employing plasma fibulin-1 levels.
These observations reflect the complex pathophysiology of arterial stiffening and underline the need for further studies to determine the exact role of fibulin-1 in arterial stiffness and cardiovascular risk.