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Plasma Levels of Cellular Fibronectin in Diabetes

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Plasma Levels of Cellular Fibronectin in Diabetes Pathophysiology/Complications ORIGINAL ARTICLE Plasma Levels of Cellular Fibronectin in Diabetes SUZAN D.J.M. KANTERS, MD, PHD RINI C.J.M. FRIJNS, MD contain an extra type III structural domain JAN-DIRK BANGA, MD, PHD JAAP J. BEUTLER, MD, PHD called ED-A (3,4). Usually, Ͻ1–2% of total ALE ALGRA, MD, PHD ROB FIJNHEER, MD, PHD fibronectins in plasma consists of this cellu- lar fibronectin (5). Elevated plasma levels of cellular fibro- nectin have been reported in patients with rheumatoid vasculitis, in preeclamptic OBJECTIVE — Cellular fibronectin is an endothelium-derived protein involved in suben- women, in patients with collagen vascular dothelial matrix assembly. Elevated plasma levels of cellular fibronectin therefore reflect loss disorders, in acute trauma, in sepsis syn- of endothelial cell polarization or injury to blood vessels. Consequently, elevated plasma lev- drome, and in thrombotic thrombocy- els of circulating cellular fibronectin have been described in clinical syndromes with vascular damage, although not in diabetes or atherosclerosis. topenic purpura (5–9). These observations suggest that the intravascular accumulation RESEARCH DESIGN AND METHODS — We determined fibronectin levels in 52 of cellular fibronectin reflects injury to blood patients with type 1 diabetes, 50 patients with type 2 diabetes, 54 patients with a history of vessels. Vessel wall damage with character- ischemic stroke, 23 patients with renal artery stenosis, and 64 healthy subjects. istic endothelial extracellular matrix changes is also found in subjects with diabetes. The RESULTS — Circulating cellular fibronectin was significantly elevated in patients with dia- accumulation of proteins in the suben- betes (4.3 ± 2.8 µg/ml) compared with patients with ischemic stroke (2.0 ± 0.9 µg/ml), patients dothelial matrix in patients with diabetes is with renovascular hypertension (1.7 ± 1.1 µg/ml), and healthy subjects (1.4 ± 0.6 µg/ml). visible by light microscopy as periodic acid- Patients with diabetes and at least one cardiovascular risk factor had an almost 2.5-fold increase Schiff–positive material, consisting of in cellular fibronectin compared with diabetic subjects without such a risk factor. In multi- variate regression analysis, higher triglycerides, current or past cigarette smoking, and higher laminin, type IV collagen, and fibronectin urinary albumin excretion were independently associated with an increase in circulating cel- (10). Plasma levels of circulating cellular lular fibronectin in diabetes. fibronectin could therefore reflect matrix changes and hence vessel wall damage in CONCLUSIONS — These results suggest that circulating cellular fibronectin may be a patients with diabetes. marker protein for endothelial cell activation, especially in diabetes. Prospective studies are In the present study, we investigated needed to explore this possibility. whether the plasma level of cellular fibro- nectin reflects the vascular changes in Diabetes Care 24:323–327, 2001 patients with diabetes, compared with healthy nondiabetic individuals or nondia- betic patients with a history of ischemic hange in endothelial function is con- on cell surfaces. They promote cell-cell and stroke and nondiabetic patients with renal sidered an early pivotal step in the cell-matrix interactions and thus play a role artery stenosis. Furthermore, we studied Cdevelopment of arterial disease. Endo- in tissue construction and reconstruction relations between various cardiovascular risk thelial function can be assessed either by (1). A single gene encodes for various dis- factors and circulating cellular fibronectin in vascular function tests using invasive or tinct fibronectin moieties, with differences in diabetic individuals. noninvasive techniques or by determination primary structure resulting from alternative of plasma concentrations of specific endo- splicing of the primary mRNA transcript RESEARCH DESIGN AND thelial proteins, such as von Willebrand fac- (2). The predominant fibronectin in plasma METHODS tor (vWF). Circulating cellular fibronectin is secreted by hepatocytes and lacks extra may be another useful marker of endo- domain (ED) segments. The fibronectins Patient characteristics thelial activation. found in subendothelial and connective tis- Diabetic patients, renovascular hyperten- Fibronectins are large glycoproteins sue matrices are produced locally by sive patients, and control subjects were found in plasma, in extracellular matrix, and endothelial cells and fibroblasts and often recruited from the outpatient clinic, and ischemic stroke patients were recruited From the Department of Internal Medicine (S.D.J.M.K., J.-D.B.), the Julius Center for Patient-Oriented Research from the clinic of the University Medical (A.A.), the Department of Neurology (A.A., R.C.J.M.F.), the Department of Nephrology and Hypertension Center Utrecht. The study protocol was (J.J.B.), and the Department of Hematology (R.F.), University Medical Center, Utrecht, the Netherlands. Address correspondence and reprint requests to Jan-Dirk Banga, MD, PhD, Department of Internal Med- approved by the ethics committee of the icine, G02.228, University Medical Center Utrecht, P.O. Box 85500, 3508 GA, Utrecht, the Netherlands. hospital and all patients gave informed con- E-mail: [email protected]. sent. We investigated the following groups Received for publication 10 July 2000 and accepted in revised form 27 October 2000. of patients: Abbreviations: BSA, bovine serum albumin; ED, extra domain; MoAb, monoclonal antibody; PBS, phos- phate-buffered saline; vWF, von Willebrand factor. Group 1. We studied 102 patients with dia- A table elsewhere in this issue shows conventional and Système International (SI) units and conversion betes: 52 with type 1 and 50 with type 2 factors for many substances. diabetes, aged 19–73 years, with a duration DIABETES CARE, VOLUME 24, NUMBER 2, FEBRUARY 2001 323 Circulating cellular fibronectin in diabetes Table 1—Demographic, clinical, and bio- ischemic stroke and from 30 nondiabetic was detected by addition of peroxidase-con- chemical characteristics of 102 patients with patients with a previous (Ͼ1 week) tran- jugated anti-human fibronectin antibodies diabetes sient ischemic attack or minor ischemic (0.6 µg/ml) (Dako, Glostrup, Denmark). stroke associated with a moderate-to-severe Binding of the antibody was detected by stenosis of the internal carotid artery (13). adding 0.25 µg/ml O-phenylene-diamine in Age (years) 48.8 ± 14.6 These 54 ischemic stroke patients were 0.1 mol/l citrate-phosphate buffer (pH 5) Men 62 32–88 years of age, and 70% were men. with 2.5 mmol/l H O . The reaction was Type 1 diabetes 51 2 2 The diagnosis of acute ischemic stroke was stopped by adding 3 mol/l sulfuric acid. Diabetes duration (years) 13.5 ± 9.8 established by history, clinical examination, Absorbance at 490 nm was read on a HbA (%) 8.3 ± 1.3 1c and cerebral computed tomography or microplate reader. As a standard, cellular Total cholesterol (mmol/l) 5.3 ± 0.9 magnetic resonance imaging. Some 38% of fibronectin was purified from cultured HDL cholesterol (mmol/l) 1.2 ± 0.4 the patients with an acute ischemic stroke human fetal lung fibroblasts (5,9). ED-A Triglycerides (mmol/l) 1.7 ± 1.0 and 47% of the patients with a sympto- fibronectin was isolated from the medium of LDL cholesterol (mmol/l) 3.3 ± 0.8 matic stenosis of the internal carotid artery GM-1380 human fetal lung fibroblast grown BMI (kg/m2) 26.4 ± 4.2 had a history of hypertension. in Dulbecco’s modified Eagle’s medium con- Treatment for hypertension 25 Group 3. We included 23 nondiabetic taining 10% fibronectin-depleted fetal calf Current or past cigarette 64 hypertensive patients with angiographic serum by gelatin-affinity chromatography smoking evidence of Ͼ50% atherosclerotic stenosis (5). MoAb 3E2 was raised using fibronectin History of vascular disease of a renal artery (renovascular and athero- antigen released by cultures of human breast Angina pectoris 11 sclerotic hypertensive patients) in our study cancer cell lines (15,16). The interassay coef- Myocardial infarction 10 (14). They were 45–71 years of age, had a ficient of variation of a standard (1.0 µg cel- Stroke 2 mean arterial pressure in the range of lular fibronectin per milliliter) measured in Intermittent claudication 4 97–153 mmHg (mean 125), and 57% were 12 microtiter plates was 8.1%, and the intra- Micro- or macroalbuminuria* 26 men. Their duration of hypertension was in assay coefficient of variation was 4.0% (12 Data are means ± SD or %. *Urinary albumin excre- the range of 0.5–36 years (mean 9). Of samples). An amount of 0.4 µg/ml in plasma tion Ͼ30 mg/24 h. these patients, 10 also had evidence of could be detected in the linear range of the coronary artery disease, 4 had proven ath- standard curve. MoAb 3E2 is specific for the erosclerosis of the carotid arteries, and 11 ED-A domain, and the critical residues were of diabetes of 0.4–47 years, and 62% were had evidence of atherosclerosis in the aorta, recently identified (17). men. Demographic, clinical, and biochem- or in femoral or iliac arteries. ical characteristics of these patients are Group 4. Healthy control subjects num- Statistical analysis shown in Table 1. Of these patients, 73 bered 64 individuals, were 19–79 years of Data are presented as means ± SD. Differ- were included in a lipid-lowering study, age, and 42% were men. They were ences between groups of patients and con- and 29 participated in a study on the effect asymptomatic for vascular disease and trol subjects were described by 95% CIs of long-term physical exercise on glycemic were not being administered cardiovascu- based on the t test for independent sam- control (11,12). Their baseline data were lar medication. ples. These differences were adjusted for used for the current study. Inclusion criteria age by linear regression analysis. for the lipid-lowering study were as fol- Blood sampling and laboratory Linear regression analysis was used to lows: type 1 or type 2 diabetes, LDL cho- investigations evaluate the determinants of circulating cel- lesterol Ͼ2.6 mmol/l or triglycerides Ͼ1.7 Blood was sampled by an evacuated tube lular fibronectin in diabetes.
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