Elevated levels of plasma von Willebrand factor and the risk of macro- and microvascular disease in type 2 diabetic patients with microalbuminuria

Peter Gæde, Pernille Vedel, Hans-Henrik Parving and Oluf Pedersen

Steno Diabetes Center, Gentofte, Denmark



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. The purpose of this study was to examine the concept suggesting that microalbuminuria in combination with high levels of plasma von Willebrand factor is a stronger predictor for cardiovascular disease and microvascular complications than microalbuminuria alone in type 2 diabetic patients.

Methods. One hundred and sixty patients with type 2 diabetes mellitus and persistent microalbuminuria were followed for an average of 3.8 (SD 0.3) years. 70% of the patients were treated with angiotensin converting enzyme (ACE)-inhibitors. Patients in this subanalysis were divided into two groups according to baseline plasma von Willebrand factor levels below or above the median. The main outcome was cardiovascular disease (cardiovascular mortality, non-fatal stroke, non-fatal myocardial infarction, coronary artery bypass graft and revascularization or amputation of legs), progression to diabetic nephropathy or progression in diabetic retinopathy.

Results. At baseline the two groups were comparable for HbA1c, fasting levels of s-total-cholesterol, s-HDL-cholesterol and s-triglycerides, systolic and diastolic blood pressure, gender, known diabetes duration, smoking habits, previous cardiovascular disease and antihypertensive therapy as well as retinopathy. Odds ratio for cardiovascular disease was 1.11 (95% CI 0.45–2.73, P=0.82) (multiple logistic regression), odds ratio for progression to nephropathy was 1.08 (0.41–2.85, P=0.87) and odds ratio for progression in retinopathy was 0.96 (0.46–2.00, P=0.92), all with plasma von Willebrand factor levels above the median.

Conclusions. Our results do not support the suggestion that the combination of high plasma levels of von Willebrand factor and microalbuminuria is a stronger predictor for cardiovascular disease, progression to diabetic nephropathy or progression in diabetic retinopathy than microalbuminuria alone in patients with type 2 diabetes and persistent microalbuminuria.

Keywords: cardiovascular disease; endothelial dysfunction; microalbuminuria; microvascular complications; type 2 diabetes mellitus; von Willebrand factor



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Microalbuminuria is a strong predictor for the development of both micro- and macrovascular disease in patients with type 1 as well as type 2 diabetes mellitus [1,2]. In both types of diabetes, microalbuminuria clusters with hypertension, severe retinopathy and atherosclerotic cardiovascular disease. Furthermore, microalbuminuria is associated with vascular endothelial dysfunction, which could underlie this clustering [3,4]. However, there is important heterogeneity in this association in both types of diabetes. In a cross-sectional kidney biopsy study in microalbuminuric type 2 diabetic patients, elevated plasma levels of von Willebrand factor (vWF) as a marker of generalized endothelial dysfunction was only seen in patients with typical or atypical patterns of diabetic glomerulopathy, but not in patients with normal histology [5]. In a prospective study in type 2 diabetic patients it was found that elevated levels of urinary albumin excretion rate was associated with an increased risk of new cardiovascular events only in the presence of elevated levels of plasma vWF [4]. The observed heterogeneity has led to the hypothesis that only microalbuminuria with generalized endothelial damage confers an increased risk of cardiovascular disease and renal failure, thereby introducing the terms ‘malignant’ and ‘benign’ microalbuminuria [6]. The aim of the present prospective study was to examine whether microalbuminuria associated with the presence of endothelial dysfunction as indicated by high plasma levels of vWF is a stronger predictor for cardiovascular disease and progression in diabetic microangiopathy than microalbuminuria alone in type 2 diabetic patients suffering from persistent microalbuminuria.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The present study is a sub-study of the Steno type 2 study reported previously [7]. In short, 160 patients (age 45–65 years) with type 2 diabetes mellitus and persistent microalbuminuria (urinary albumin excretion rate 30–300 mg/24 h) were followed in an open, parallel trial. With concealed randomization, patients were randomized to either: (i) intensive multifactorial intervention comprising behaviour modification and polypharmacological therapy targeting several modifiable risk factors such as diet, exercise, smoking habits, hyperglycaemia, hypertension, dyslipidaemia and microalbuminuria at Steno Diabetes Center, Copenhagen, Denmark (n=80); or (ii) to standard multifactorial intervention at their general practitioners according to guidelines from the Danish Medical Association. The primary endpoint was progression to diabetic nephropathy. Patients in the intensively treated group were seen at least every third month during an average follow-up period of 3.8 years. Intensive multifactorial intervention was associated with risk reductions in the progression rate to nephropathy (odds ratio 0.27 (95% CI 0.10–0.75)), progression in retinopathy (odds ratio 0.45 (0.22–0.93)) and progression in autonomic neuropathy (odds ratio 0.31 (0.12–0.78) as compared to standard intervention [7]. A significant risk reduction was also seen with intensive therapy for a combined endpoint for macrovascular disease, mainly due to a lower progression rate in peripheral artery disease.

Patients
All 160 participating patients were recruited from the Steno Diabetes Center during 1992–93. Microalbuminuria was defined as a urinary albumin excretion rate (AER) of 30–300 mg per 24 h in four of six 24 h urine samples. Diabetes was defined by 1985 WHO criteria. Exclusion criteria were age older than 65 or younger than 40 years; a stimulated serum C-peptide concentration less than 600 pmol/l 6 min after intravenous injection of 1 mg of glucagon; pancreatic insufficiency or diabetes secondary to pancreatitis; alcohol abuse; non-diabetic kidney disease; malignancy; or life-threatening disease with death probable within 4 years. Informed consent was obtained from all participants. The protocol was in accordance with the Helsinki declaration and was approved by the ethics committee of Copenhagen County.

In the present post-hoc analysis, patients were divided into two groups according to baseline plasma vWF below the median or above the median in the cohort as originally proposed by Stehouwer et al. [4].

Assessments
Endpoint examinations were planned and performed at baseline and after 4 years of follow-up unless otherwise indicated. Blood pressure was measured twice after 20 min rest in the supine position with a Hawksley random-zero sphygmomanometer. Every second year, AER was measured in three consecutive 24 h urine collections. Retinal photographs of two 45–50 degree fields (macula-temporal and disc-nasal) were taken in both eyes through dilated pupils. The photographs were graded by two independent ophthalmologists, masked to the original treatment allocation, according to the EURODIAB six-level grading scale [8]. The resting 12-lead electrocardiogram was classified by the Minnesota code [9] by two independent, masked graders. Cause of death was taken from the death certificate. Cardiovascular death was defined as codes 390–459 according to the International Classification of Diseases, 9th edn. WHO criteria were used to define acute myocardial infarction and stroke. Procedures were those noted in hospital discharge letters.

Assays
Unless otherwise stated all blood samples were taken at 0800 after an overnight fast. Patients did not take their drugs in the morning of the day of blood sampling. HbA1c was measured by ion-exchange high-performance liquid chromatography (Bio-Rad VARIANT, California, USA) and the non-diabetic reference range in our laboratory was 4.1–6.4%. Serum total cholesterol and serum HDL-cholesterol concentrations were measured by chromatography. Triglycerides were measured with colorimetry. AER was measured by ELISA [10]. Serum creatinine was measured with routine methods. Plasma vWF was measured by ELISA, as described previously [11].

Endpoints
Cardiovascular disease was as in the original study by Stehouwer et al. [4] a combined endpoint in this case consisting of cardiovascular mortality, non-fatal stroke, non-fatal myocardial infarction, coronary artery bypass graft and revascularization or amputation of legs. Diabetic nephropathy was diagnosed if median AER was greater than 300 mg per 24 h in at least one of the 2-yearly examinations. Progression in diabetic retinopathy was an increase of at least one level in any eye or development of new diabetic retinopathy.

Statistical analysis
Comparison of groups at baseline was by one-way analysis of variance or Kruskal–Wallis test whenever appropriate for numerical variables. {chi}2-test was used to compare categorical variables. Changes in variables during follow-up were compared using Student's t-test or Mann–Whitney test. For endpoints, odds ratios with 95% confidence intervals were calculated from multiple logistic regression analysis with HbA1c, age, gender, known diabetes duration, smoking habits, original treatment allocation (intensive therapy or standard therapy) and the endpoint of interest at baseline as covariates. Thus, adjustments were made for imbalances between groups at baseline and during follow up in this post-hoc analysis.

Since treatment with angiotensin converting enzyme (ACE)-inhibitors is a potential confounder of plasma vWF levels [12], odds ratios for endpoints were also adjusted for ACE-inhibitor treatment during follow-up in another regression model. Treatment with insulin may also affect circulating levels of vWF [13], therefore a third regression model was adjusted for insulin treatment during follow-up.

To test whether a linear interaction exists between levels of plasma vWF and any of the three endpoints, plasma vWF concentration were entered in the multiple logistic regression model instead of the grouped variable. A non-linear association between plasma vWF levels and endpoints was examined with polynomial regression by entering both plasma vWF levels and squared plasma vWF levels in the model.

P-values less than 0.05 were considered statistically significant.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The range of fasting plasma levels of vWF at baseline was from 0.64 to 5.35 U/ml with a median value of 1.50 U/ml in the cohort. As shown in Table 1Go, the two groups below and above the median of vWF were comparable at baseline for the listed clinical and biochemical variables. The number of patients from the original intensive therapy group and standard therapy group were equally divided into the two new groups. The only significant difference between the group with plasma vWF below the median and the group above the median was in the drug treatment for hyperglycaemia since more patients with high levels of plasma vWF were treated with insulin.


View this table:
[in this window]
[in a new window]
 
Table 1. Baseline characteristics of patients with plasma von Willebrand levels below the median (‘benign’ microalbuminuria) and von Willebrand factor above the median (‘malignant’ microalbuminuria) in 160 type 2 diabetic patients with microalbuminuria

 
Five patients withdrew from the study during the mean follow-up period of 3.8 (SD 0.3) years. These patients did not differ from the rest of the cohort in clinical and biochemical variables at baseline (data not shown).

As seen from Table 2Go, the two groups with plasma vWF below and above the median, respectively, did not differ significantly in the changes seen in clinical and biochemical variables during follow-up, although weight gain tended to be greater in the group with plasma vWF below the median.


View this table:
[in this window]
[in a new window]
 
Table 2. Changes at the end of the intervention trial in clinical and biochemical variables and in the number of patients treated with insulin and ACE-inhibitors

 
During the follow-up period, 13 patients in the group with plasma vWF below the median experienced a cardiovascular disease event as compared to 16 patients in the vWF above the median group (odds ratio 1.11 (95% CI 0.45–2.73), P=0.82) (Table 3Go). In the present study, patients from the original intensive therapy group and the standard therapy group have been pooled in each of the two new subgroups according to plasma vWF below or above the median. This approach did not change the odds ratio for a CHD event with plasma vWF above the median significantly (odds ratio 1.10; 0.28–4.24) in the original intensive therapy group compared to 1.15 (0.32–4.14) in the original standard therapy group.


View this table:
[in this window]
[in a new window]
 
Table 3. Risk of cardiovascular disease event, progression to diabetic nephropathy, and progression in diabetic retinopathy during 3.8 years of follow-up in microalbuminuric type 2 diabetic patients divided into two groups according to plasma von Willebrand factor (vWF) levels below or above the median*

 
Testing for a linear or non-linear association with plasma vWF levels and coronary heart disease events did not show any association (P=0.45 and P=0.71, respectively). As seen from Table 3Go, there was no association with levels of plasma vWF above the median and progression to diabetic nephropathy defined as a median AER above 300 mg per 24 h in three consecutive 24-h urine samples. This was also the case with the test for a linear or a non-linear association (P=0.99 and P=0.88, respectively). Finally, no relation with plasma levels of von Willebrand factor and progression in retinopathy was seen (P=0.63 for a linear association and P=0.70 for a non-linear association).

As shown in Table 3Go, adjustments for treatment with ACE-inhibitors or insulin during follow-up did not change the odds ratios for endpoints with elevated plasma vWF significantly.



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The aim of our study was to examine whether microalbuminuria in combination with high levels of plasma vWF is a stronger predictor for cardiovascular disease, progression to diabetic nephropathy and progression in diabetic retinopathy than microalbuminuria alone in type 2 diabetic patients. We did not find such an association in the present study population for any of the endpoints over a 3.8 year follow-up period. Nor did we find that patients with plasma vWF levels above the median had a higher baseline prevalence of cardiovascular disease or diabetic retinopathy. Since only a fraction of the type 2 diabetic patients in the present study had a kidney biopsy performed we cannot decide whether the previously reported cross-sectional association between high levels of vWF and typical diabetic kidney lesions as well as atypical lesions apply to our study population [5]. However, levels of AER and serum creatinine did not differ between the two groups at baseline.

In the cross-sectional kidney biopsy study, all microalbuminuric patients with typical diabetic glomerulopathy had diabetic retinopathy and proliferative retinopathy was only present in these patients. However, patients with atypical patterns of renal injury or normal renal structure were equally divided into a group having simplex background retinopathy and a group without diabetic retinopathy [5]. Both typical and atypical renal lesions were associated with worse metabolic control, but furthermore, plasma vWF levels in patients with proliferative retinopathy were significantly higher than in patients without retinopathy [5,14]. In our prospective study we did not see any significant associations with elevated levels of plasma vWF and progression in diabetic retinopathy.

All patients in the intensively treated group and 47% of the patients in the control group in the Steno Type 2 Study were treated with ACE-inhibitors. ACE inhibition has been shown to counteract endothelial activation in hypertensive patients with impaired glucose tolerance [12], and recently treatment with the ACE-inhibitor ramipril has been shown to decrease mortality in high-risk patients, including patients with diabetes [15]. A subanalysis in diabetic patients showed a significant decrease of 16% with 10 mg ramipril daily on a composite endpoint of diabetic complications compared to placebo.

It is noteworthy that, in the original study proposing the term ‘malignant’ microalbuminuria, only a few of the patients received treatment with ACE-inhibitors [4]. Another epidemiological study which found an association between elevated levels of vWF and cardiovascular mortality in both diabetic and non-diabetic subjects, does not give any information about medical treatment [16]. Finally, a recent study from the same group which found the same association in type 2 diabetic patients with microalbuminuria has excluded patients treated with ACE-inhibitors [17].

It should be noted that there was a perfect match of patients from the intensively treated group and in patients treated with ACE-inhibitors during follow-up between the ‘malignant’ and ‘benign’ microalbuminuria group in the present study. Furthermore, adjustment for treatment with ACE-inhibitors during follow-up did not give any significant association between elevated plasma vWF levels and any of the endpoints. However, statistical adjustment may not obviously in all cases sufficiently compensate for biological complexity.

The number of cardiovascular events as defined previously was 29 in the 160 type 2 diabetic patients with persistent microalbuminuria included in the present study giving an odds ratio of 1.11 (95% CI 0.45–2.73) with elevated plasma vWF. In comparison, 11 events in 28 patients were observed in the original study by Stehouwer et al. [4], which gave a relative risk with elevated levels of plasma vWF of 3.66 (1.3–11.9). Assuming an equal difference in risk for cardiovascular disease, in the present study the power would be 85% of detecting this difference at a 5% significance level. Because of the small difference in the number of events between the groups with plasma vWF below and above the median, respectively, the actual power is, however, much lower. Although an 11% difference in the risk for a CVD event with elevated plasma vWF could prove clinically important, it should be noted that the 95% confidence intervals are much narrower in the present study than in the study by Stehouwer et al. [4]. We did not have sufficient power to detect an association between elevated levels of vWF and cardiovascular mortality as have been found in other studies [16,17], since only five patients died from cardiovascular causes during follow-up.

It should again be mentioned that the design of our study is a post-hoc analysis and as such is a limitation of our results. The question of selection bias is also important, however inclusion criteria in the Steno type 2 study was simply type 2 diabetes and microalbuminuria, and we therefore do not believe that selection bias is a major problem in this case.

In conclusion, the results of this study with a mean follow-up time of 3.8 years do not support the hypothesis that phenotypic heterogeneity in type 2 diabetic patients with microalbuminuria and, in most cases, concomitant treatment with ACE-inhibitors can be explained by differences in plasma vWF levels. Thus, according to our results, there is no case for ‘benign’ or ‘malignant’ microalbuminuria as suggested [6]. However, further large-scale, long-term, prospective studies are required to validate this conclusion.



   Notes
 
Correspondence and offprint requests to: Peter Gæde, Steno Diabetes Center, Niels Steensens Vej 2, DK-2820 Gentofte, Denmark. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Borch-Johnsen K, Kreiner S. Proteinuria: value as a predictor of cardiovascular mortality in insulin-dependent diabetes mellitus. BMJ1987; 294: 1651–1654[ISI][Medline]
  2. Mogensen CE. Microalbuminuria predicts clinical proteinuria and early mortality in maturity-onset diabetes mellitus. N Engl J Med1984; 310: 356–360[Abstract]
  3. Stehouwer CDA. Von Willebrand factor, dysfunction of the vascular endothelium, and the development of renal and vascular complications in diabetes. In: Mogensen CE, ed. The Kidney and Hypertension in Diabetes Mellitus. 3rd edn. Kluwer, Boston, 1997; 155–163
  4. Stehouwer CDA, Nauta JJP, Zeldenrust GC, Hackeng WHL, Donker AJM, Den Ottolander GJH. Urinary albumin excretion, cardiovascular disease and endothelial dysfunction in non-insulin dependent diabetes mellitus. Lancet1992; 340: 319–323[ISI][Medline]
  5. Fioretto P, Stehouwer CD, Mauer M et al. Heterogeneous nature of microalbuminuria in NIDDM: studies of endothelial function and renal structure. Diabetologia1998; 41: 233–236[ISI][Medline]
  6. Stehouwer CD, Yudkin JS, Fioretto P, Nosadini R. How heterogeneous is microalbuminuria in diabetes mellitus? The case for ‘benign’ and ‘malignant’ microalbuminuria. Nephrol Dial Transplant1998; 13: 2751–2754[Free Full Text]
  7. Gæde P, Vedel P, Parving H-H, Pedersen O. Intensified multifactorial intervention in patients with type 2 diabetes mellitus and microalbuminuria: the Steno type 2 randomised study. Lancet1999; 353: 617–622[ISI][Medline]
  8. Aldington SJ, Kohner EM, Meuer S, Klein R, Sjølie A-K. Methodology for retinal photography and assessment of diabetic retinopathy: the EURODIAB IDDM Complications Study. Diabetologia1995; 38: 437–444[ISI][Medline]
  9. Blackburn H, Keys A, Simonson E, Rautaharju P, Punsar S. The electrocardiogram in population studies: a classification system. Circulation1960; 21: 1160–1175[ISI]
  10. Feldt-Rasmussen B, Dinesen B, Deckert M. Enzyme immunoassay: an improved determination of urinary albumin in diabetics with incipient nephropathy. Scan J Clin Lab Invest1985; 45: 539–544[ISI][Medline]
  11. Ingerslev J. A sensitive ELISA for von Willebrand factor (Vwf:Ag). Scand J Clin Lab Invest1987; 47: 143–149[ISI][Medline]
  12. Ferri C, Desideri G, Baldoncini R et al. Early activation of vascular endothelium in non-obese, nondiabetic essential hypertensive patients with multiple metabolic abnormalities. Diabetes1998; 47: 660–667[Abstract]
  13. Heywood DM, Mansfield MW, Grant PJ. Levels of von Willebrand factor, insulin resistance syndrome and a common vWF gene polymorphism in non-insulin-dependent (type 2) diabetes mellitus. Diab Med1996; 13: 720–725[ISI][Medline]
  14. Fioretto P, Mauer M, Brocco E et al. Patterns of renal injury in NIDDM patients with microalbuminuria. Diabetologia1996; 39: 1569–1576[ISI][Medline]
  15. The Heart Outcomes Prevention Evaluation Study Investigators. Effects of an angiotensin-converting-enzyme inhibitor, ramipril, on cardiovascular events in high-risk patients. N Engl J Med2000; 342: 145–153[Abstract/Free Full Text]
  16. Jager A, van Hinsberg VWM, Kostense PJ et al. Von Willebrand factor, C-reactive protein and 5-year mortality in diabetic and nondiabetic subjects: the Hoorn Study. Arterioscler Thromb Vasc Biol1999; 19: 3071–3078[Abstract/Free Full Text]
  17. Jager A, van Hinsberg VWM, Kostense PJ et al. Prognostic implications of retinopathy and a high plasma von Willebrand factor concentration in type 2 diabetic subjects with microalbuminuria. Nephrol Dial Transplant2001; 16: 529–536[Abstract/Free Full Text]
Received for publication: 21.12.00
Revision received 31. 5.01.