Original Article Statins Ameliorate Endothelial Barrier Permeability Changes in the Cerebral Tissue of Streptozotocin-Induced Diabetic Rats Arshag D. Mooradian, 1 Michael J. Haas, 1 Oksana Batejko, 1 Meri Hovsepyan, 1 and Stephen S. Feman 2 Statins may have favorable effects on endothelial barrier function. The effect of rosuvastatin and simvastatin ther- apy (10 mg/kg) for 5 weeks on blood-brain barrier (BBB), blood-retinal barrier (BRB), and cardiac muscle permeabil- ity of streptozotocin-induced diabetic rats was studied. The size-selective permeability of different vascular beds to a group of fluorescein isothiocyanate dextrans of vary- ing molecular weights was measured. The volume of distri- bution of 250-, 70-, and 40-kDa dextrans in the cerebral tissue of diabetic rats were significantly increased. The volume of distribution of these dextrans in cerebral tissue was normalized by both statins. Diabetes did not signifi- cantly alter the BRB, but both statins decreased the volume of distribution of 70- and 40-kDa dextrans in the retina. The volume of distribution of 40 kDa in cardiac muscle was increased in diabetes, and this change was prevented with statin treatment. Treatment with rosuvastatin and meval- onate (150 mg/kg in drinking water for 5 weeks) did not alter the volume of distribution measurements. We con- cluded that 1) diabetes in rats is associated with significant changes in the BBB permeability; 2) statin treatment im- proves the endothelial barrier function in cerebral tissue, retina, and cardiac muscle; and 3) this statin effect could not be attributed to HMGCoA reductase inhibition. Diabetes 54:2977–2982, 2005 O ne of the sentinel features of atherosclerosis is endothelial cell dysfunction that manifests it- self in a variety of ways including poor nitric oxide production, poor vasodilatory response, and increased adhesiveness to leukocytes (1). Another potential endothelial dysfunction commonly observed in diabetes is altered permeability to macromolecules. Diabetes in humans and in animal models has been found to cause significant alterations in endothelial per- meability in various vascular beds (2–5). Potential mech- anisms underlying the diabetes-related changes in the blood-brain barrier (BBB) include altered expression of key structural and enzymatic proteins, alterations in the lipid composition and fluidity of the membranes, alter- ations in the neurotransmitter activity, and increased oxidative damage of the endothelial cells (2,6). Another likely contributor to these changes is the activation of protein kinase C that is shown to play an important role in increased permeability of the peripheral and cerebral circulation (7,8). Finally, recent studies have shown that inactivation of the rho-GTPase has a critical role in endo- thelial barrier function (9,10). Statins are known to have many pleiotropic effects (11). However, the effect of statins on the functional integrity of the microvasculature of diabetic animals has not been well studied. Statins are known to alter endothelial cell func- tion, smooth muscle cell migration and proliferation, and some aspects of vascular inflammation (11). In addition, statins have been shown to improve endothelial barrier permeability in the aorta of Watanabe hyperlipidimic rabbits (12). However, the effect of statins on endothelial barrier permeability in various tissues of diabetic animals has not been comprehensively evaluated. To address this question, the permeability of the BBB, blood-retinal bar- rier (BRB), and cardiac muscle to a group of dextrans with varying sizes was studied in diabetic rats treated with statins. RESEARCH DESIGN AND METHODS Male Fischer 344 rats at 4 months of age were obtained from Harlan Industries (Indianapolis, IN). A group of rats was rendered diabetic with a single intraperitoneal injection of 1.3% streptozotocin in citrate buffer (35 mg/kg) as described previously (13,14). These rats can be kept without insulin therapy for up to 12 weeks. The diabetic state was confirmed by repeated measures of glucosuria and by plasma glucose concentrations in excess of 300 mg/dl at the day of the experiment. The following groups of rats were studied: control (vehicle-injected group) (n = 10), diabetic rats on regular diet and ad libitum tap water for drinking (n = 10), and diabetic rats fed rat diet along with either rosuvastatin (n = 10) or simvastatin (n = 10) at 10-mg/kg dose in the drinking water for a total of 5 weeks. Daily water consumption was monitored to adjust the dose of statins delivered daily. In order to determine whether suppression of HMGCoA reductase activity by statins is involved in modulating endothelial cell barrier integrity, an additional group of diabetic rats (n = 10) treated with rosuvastatin (10 mg/kg in the drinking water) and mevalonate (150 mg/kg in the drinking water for 5 weeks) was studied. In vivo endothelial permeability measurements. The permeability of endothelial cells in cerebral, retinal, and cardiac tissue was estimated by measurements of the volume of distribution of fluorescein isothiocyanate (FITC)-labeled dextrans of various molecular weights (15). The rats were injected through the tail vein with 0.2 ml 0.9% saline solution containing 200 mg/ml of dextrans molecular weight 4,000, 10,000, 20,000, 40,000, 70,000, and 250,000. The FITC dextrans were purchased from Sigma (St. Louis, MO). Five hours after injection with FITC dextrans the animals were killed. Brain (cerebrum) and heart (left ventricular) (100 mg each wet weight) and 50 mg of retina were rinsed in buffer and disrupted mechanically with a polytron From the 1 Division of Endocrinology, Diabetes, and Metabolism, Department of Internal Medicine, Saint Louis University School of Medicine, St. Louis, Missouri; and the 2 Department of Ophthalmology, Saint Louis University School of Medicine, St. Louis, Missouri. Address correspondence and reprint requests to Arshag D. Mooradian, MD, Division of Endocrinology, Saint Louis University Medical School, 1402 South Grand Blvd., St. Louis, MO 63104. E-mail: mooradad@slu.edu. Received for publication 5 May 2005 and accepted in revised form 12 July 2005. BBB, blood-brain barrier; BRB, blood-retinal barrier; FITC, fluorescein isothiocyanate. © 2005 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. DIABETES, VOL. 54, OCTOBER 2005 2977