In twenty patients with type 2 diabetes with no history of stroke, P-NT-proBNP was strongly associated with volumes of white matter hyperintensities. Furthermore, white matter hyperintensities were higher and brain parenchyma fraction was lower in our patients, and in particular patients with CAD, compared to age and sex matched controls, however not when adjusting for conventional risk factors.
Plasma NT-proBNP and white matter hypertensities
Our main finding that elevated P-NT-proBNP levels were associated with presence and severity of WMH has to our best knowledge not been described before. The association was independent of all tested CV risk factors and particular age and systolic blood pressure, which are considered the main predictors of WMH. Noteworthy, the association between P-NT-proBNP and WMH was strengthened and not attenuated by the addition of CV risk factors and in particular CIMT, and this may suggest that the cause is not related to an age systemic atherosclerosis but indeed organ heart and brain specific, therefore linking the heart with the brain. In humans the left ventricle is the main source of P-NT-proBNP with increased myocyte stretch or myocardial ischemia. It therefore seemed unlikely, based on physiology that elevated P-NT-proBNP from the heart per se directly promoted WMH or cerebrovascular disease in the present study. In contrast, heart disease such as heart failure or presence of atrial fibrillation that are known determinants of higher P-NT-proBNP levels and associated with increased risk of strokes, could be a common driver of both elevated P-NT-proBNP and WMH . Our patients, however, were without acute illness and had normal ejection fraction without atrial fibrillation or valvular disease, and patients also had lower levels of P-NT-proBNP than seen in congestive heart failure . Furthermore, we performed concomitant analyses with measures of subclinical heart disease, and the association between P-NT-proBNP and WMH remained significant after adjustment for LVEF, CCS and asymptomatic CAD. Along that line when patients with CAD were excluded, the association between P-NT-proBNP and WMH actually persisted. This is particular important, since the differences in P-NT-proBNP levels between patients with and without CAD were large and this could drive a false association between P-NT-proBNP and WMH. Of note, we only included patients with or without previously screen detected asymptomatic CAD in order to include patients with a wide risk profile. Accordingly, our present study suggests that the cause of the association between P-NT-proBNP and WMH is not explained by a common typical CV or heart disease driver, therefore possibly directly linking P-NT-proBNP and WMH. Along that line, recent studies have documented that plasma brain natiurectic peptide (BNP) itself increases the risk of stroke . WMH are commonly found on MRI of elderly individuals with frequencies ranging from 11-21% at age 60 to 94% at age 82 . Although often referred to as 'incidental', WMH have been associated with decline in cognitive function in independently living elderly populations and with a two-fold increased risk of dementia or stroke . Furthermore, WMH are regarded as expressions of chronic hypoperfusion and small vessel cerebral disease. Small vessel cerebral disease is the most prevalent asymptomatic neurological disease, its incidence reported to be 6- to 10-fold that of symptomatic stroke [9, 10]. Presence of WMH has been reported to predict a three-fold increased risk of stroke and two-fold increased risk of mortality in the general population . In the recent Framingham Offspring Stroke Study, the presence of WMH predicted CV mortality and this was independent of risk factors and strokes or dementia . Following the discussion of the mechanism underlying the relationship between P-NT-proBNP and WMH, the reverse could also be true; WMH induced elevated P-NT-proBNP levels through abnormal neuroendocrine signals to the cardiac ventricles and thereby functional asymptomatic heart failure . Accordingly, the chronic cerebral hypoperfusion seen in patients with WMH could indirectly promote NT-proBNP realise from the heart. Our measurement of cardiac autonomic neuropathy (heart rate variability), however, did not change the association between P-NT-proBNP and WMH in the present study. Increased P-NT-proBNP levels could, however, also reflect other counter actions to the damages associated with WMH and accordingly mediated through other pathways. Along that line, other studies have suggested that BNP include several actions in addition to vasodilation and promotion of natriuresis and diuresis such as inhibition of the sympathetic nervous system and inhibition of several hormone systems, including the renin-angiotensin-aldosterone-system (RAAS), endothelins, cytokines, and vasopressin [18, 19].
Finally, the correlation between P-NT-proBNP and WMH could also be brain to brain instead of heart to brain. Along that line, damage or hypoperfusion of the brain (WMH) might directly induce realise of NT-proBNP from the brain . Brain natriuretic peptide (BNP) was actually first described in the porcine brain and accordingly later localized in the hypothalamus, thalamus, cerebellum, pons and cortex of the human brain . Few studies have investigated BNP and NT-proBNP from the brain in disease. Specifically, in rats status epilepticus induced increase in plasma ANP (8), and occlusion of the middle cerebral artery stimulates BNP mRNA expression in rat brain tissues . In humans, the BNP gene promoter region contains a hypoxia-inducible factor-1 binding site, which activates BNP expression , and in patients with subarachnoid hemorrhage plasma BNP concentrations were higher than controls. Some studies have also shown that P-NT-proBNP is increased in patients with acute ischemic stroke [6, 7, 23–25] but these studies may not have convincingly demonstrated that this association is independent from heart disease. In contrast, Tomati et al. examined all included patients with acute stroke with echocardiography in order to exclude the presence of heart disease and hereby investigate if BNP levels were elevated in patients with acute stroke, independently of heart disease. The study demonstrated that plasma BNP levels were indeed associated with the severity of cerebrovascular disease and particular volume of brain infarct size. One human CT study also recently suggested that the ischemic brain tissue per se release NT-proBNP into the circulation . Importantly, all our patients were without acute illnesses; since acute disease such as stroke might be a condition that per se with sympathetic and catecholamines activity, cause stress of the heart and therefore BNP secretion. In contrast, our patients with CAD had higher WMH and lower BPF, however no longer after adjustment for age and sex (Table 1). Our study also demonstrated that WMH is higher and BPF lower in our patients compared to age-matched controls, where 69% had no signs of WMH.
All our patients had microalbuminuria and several studies have demonstrated that microalbuminuria identifies a subgroup of patients with increased morbidity and mortality from CV disease but recent studies also suggest that P-NT-proBNP can further stratify the group into patients with high or low risk for CV events [1, 3, 5]. Specifically, we have previously identified P-NT-proBNP as a powerful predictor of all cause and cardiovascular mortality that included strokes in patients with type 2 diabetes . In that study, 80% of patients in the upper P-NT-proBNP tertile died, compared to 30% of patients in the lower tertile during 15 years of follow-up. The cause of poor prognosis in patients with elevated P-NT-proBNP is not known. We therefore performed the above described cross-sectional study of 200 patients with microalbuminuria and in this study, which examined patients for subclinical CAD and CV disease, we demonstrated no independent correlations between P-NT-proBNP and measurements of peripheral systolic blood pressure, CIMT, CCS, MPI, CAG or echo abnormalities [11–13]. P-NT-proBNP levels, however, have been shown by others to correlate with CIMT , CCS , MPI , CAG  and echo abnormalities  in other populations. Along that line, all our patients received intensive multifactorial intervention at Steno Diabetes Center for years before entering the study and this intervention aimed at CV prevention includes improved glycemic, lipid, and blood pressure control, as well as antithrombotic therapy and lifestyle modification according to international guidelines . Intensive multifactorial treatment according to the Steno-2 trail reduces CV disease and mortality with 50% . Of note, the medical treatment in our large cohort included statins (95% of patients), aspirin (92%), and RAAS-blocking agents (98%) and yielded mean total cholesterol levels of 3.7 mM, arterial blood pressures of 133/75 mmHg, and haemoglobinA1c levels of 53 mmol/mol (7.9%), respectively. We have therefore speculated that the intensive multifactorial medical treatment may be responsible for a weakening of the associations currently found between P-NT-proBNP levels and the previously described measurements of subclinical CAD and CV disease [11–13]. The Steno-2 trial showed that P-NT-proBNP levels might still hold prognostic capability despite multifactorial treatment . Along that line, the association between P-NT-proBNP and WMH in the present study could suggest that the residual risk seen in these patients could be cerebrovascular and that this risk is revealed with increased levels of P-NT-proBNP. If P-NT-proBNP in general can be used as an independent biomarker in the detection of subclinical WMH or cerebrovascular disease is, however, not known. Along that line, our present study is small and included a relative selected population of type 2 diabetic patients. For an example, the intensive medical therapy prescribed in our patients but also the selection of high risk CV patients with microalbuminuria and/or asymptomatic CAD. Along that line, the confounders on P-NT-proBNP levels could be even more pronounced in complicated CV patients, and therefore more than we were able to adjust for.
Today, it is generally accepted that the P-NT-proBNP assay may be useful in the screening, diagnosis and perhaps monitoring of patients with heart failure. In the general population, BNP inversely correlates with hyperinsulinemia but in diabetic patients P-NT-proBNP levels are increased compared to controls, and in addition BNP levels are altered by several factors in stroke patients [34–36]. P-NT-proBNP is a prognostic marker of CV disease in several populations including diabetic patients, and perhaps also a diagnostic tool for LV dysfunction in diabetic patients [5, 31]. As described above, recent studies have demonstrated that BNP might also be important in patients with stroke. Specifically, Saritas et al., showed that plasma BNP levels could serve as a measurement of the severity and clinical progression in stroke patients . Furthermore, the Framingham Offspring Study demonstrated that BNP offered improvement in the accuracy of stroke risk . This could also be true in diabetic patients but is not formally proven . Finally, it is important to note that few studies have investigated if WMH or other cerebrovascular disease in these patients on intensive medical therapy could serve a CV preventive treatment target. In one study, more aggressive antihypertensive treatment was associated with reduced progression in WMH but if this reduction is associated with reduced risk is not known . Accordingly, the clinical impact of the presence of WMH and the true meaning of NT-proBNP and WMH associations need to be investigated in larger and longitudinal studies.
Strengths and limitations
Our detection of brain structure in the present study was performed with 3D MRI scan, which is a well established, recognized and accurate strong non-invasive modality for these measurements. The MRI is sensitive with a three-dimensional quantification and low covariability . Most studies that include MRI measurements in patients with diabetes are from subpopulations  and fewer studies report volumetric measurements as in the present study [41, 42]. Although our study only included twenty patients with diabetes, we demonstrated a broad variation of WMH and brain volumes among our patients that were selected among 200 patients with or without asymptomatic CAD in order to include patients with a wide-ranging CV risk profile. The several multivariate analyses we performed as described in various combination of 2–3 CV risk factors in addition to age and sex, due to the relative low sample size, should obviously be interpreted with caution and we did not test for the multiple testing in these analyses.