Effect of vitamin D on aortic remodeling in streptozotocin-induced diabetes

The experiments were performed in 30 male Wistar rats (RccHan:WIST, age 4 months) obtained from Harlan Laboratories (Harlan Laboratories, Inc., The Netherlands). The animals were kept in a room with controlled temperature (21 ± 2°C) and lighting (12:12-h light–dark cycle) with free access to food pellets and tap water. All experimental procedures were approved by the Estonian National Board of Animal Experiments and were conducted in accordance with the European Communities Directive (86/609/EEC).

Rats were randomly assigned to three groups of equal size: control group, diabetic group, and cholecalciferol-treated diabetic group. Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ) 50 mg/kg (Sigma-Aldrich, St. Louis, MO, USA) freshly dissolved in 0.9% NaCl solution. Blood samples were taken 48 h later from the tail vein and glucose levels were measured with a glucometer (Glucocard X-meter, Arkray Inc., Japan). Rats with glucose levels >15 mmol/L were considered diabetic. One animal died 2 days following STZ injection and another animal did not develop hyperglycemia. Immediately after confirmation of diabetes, one diabetic group of animals was submitted to supplementation with cholecalciferol (Sigma-Aldrich, St. Louis, MO, USA) 12.5 μg (500 IU) kg^-1 body weight, dissolved in 0.3 ml olive oil administered orally. This dose was expected to be below that which causes hypercalcemia and soft tissue calcification since animal models of arterial wall calcification require the administration of much higher doses of vitamin D [20]. Cholecalciferol was chosen over calcitriol, the hormonal form of vitamin D, to further reduce the risk of soft tissue calcification. Cholecalciferol was administered every other day by gavage for a period of 10 weeks. Weekly, body weight was monitored and glycosuria was assessed with reagent strips (Combur Test, Roche, Germany) to exclude ketosis.

After 10 weeks, the animals were anesthetised with a mixture of fentanyl (0.07 mg/kg, Gedeon-Richter Plc., Hungary), midazolam (5 mg/kg, Roche Pharma AG, Germany), and ketamine (75 mg/kg Vetoquinol Biowet Sp. z.o.o., Poland) administered subcutaneously. The optimal concentrations of the anesthetic substances were determined in pilot experiments. After induction of anesthesia, animals were placed on a heating pad and body temperature was maintained at 37°C. A 2.5 F high-fidelity, dual pressure sensor catheter with 50 mm separation between sensors (SPC-721, Millar Instruments Inc., TX, USA) was introduced via the femoral artery into the descending aortic trunk so that the distal sensor was positioned at the beginning of the descending aorta and the resulting position of the proximal sensor was just proximal to the aortic bifurcation. Mean arterial pressure (MAP) was determined from measurements made by the proximal pressure transducer. Arterial pressure was increased and decreased by infusion of phenylephrine (50 μg/min) and nitroglycerine (30 μg/min), respectively, via a catheter inserted into the femoral vein. Measurements of PWV were performed similarly to what has been described by other investigators [21, 22]. Briefly, pulse pressure waves were recorded simultaneously at the two aortic sites and PWV was calculated by dividing the propagation distance by propagation time using an automated foot-to-foot method. The foot of the pressure wave was defined by the peak of the second time derivative of pressure during each pulse. As the distance between the sensors is fixed at 50 mm, this calculation provides a highly accurate measurement of PWV. Data were acquired at a sampling rate of 2 kHz (PowerLab, ADInstruments, Australia) and feature extraction and calculations made with custom scripts in Spike2 v.6. software (Cambridge Electronic Design, UK). PWV was plotted against MAP at 5 mmHg increments to characterise PWV over a wide range of MAP from 50 to 200 mmHg.

After the haemodynamic measurements were completed, blood samples were taken from the tail vein for assessment of glucose levels. The rats were euthanised by drawing blood by cardiac puncture; part was used to measure glycated haemoglobin (HbA1c) level and the remaining portion was centrifuged at 3000 rpm for 15 minutes to obtain serum. Serum 25-hydroxyvitamin D [25(OH)D] level was measured using a radioimmune assay (25-Hydroxyvitamin D, ¹²⁵I Ria Kit, Diasorin Corporation, USA). ADMA was determined from serum samples by an enzyme-linked immunoassay using a commercial kit (DLD Diagnostika, Germany). Calcium concentration in serum was determined by a colorimetric test (Calcium liquicolor, HUMAN Gesellschaft für Biochemica und Diagnostica mbH, Germany).

The aortic samples for histological analysis were fixed in 10% formalin for 12 hours and embedded in paraffin with vacuum infiltration processor (Tissue-Tek® VIP^TM 5 Jr, Sakura, USA). Specimens were cut with microtome Ergostar HM 200 (Microm, Germany) at four-μm thickness sections and stained using the hematoxylin-eosin, resorcin-fuchsin, and van Gieson methods for examination by light microscopy (Olympus BX50, Japan).

Estimation of the internal diameter of the aorta was performed by measuring two inner diameters at right angle for each cross-section of the thoracic aorta. At least eight different cross-sections of the aorta were analysed for each rat.

Thickness of the medial layer of the aorta was determined in thoracic aorta cross-sections by ten consecutive measurements in a systematic manner to evaluate all segments of the circumference of the aorta. At least six different cross-sections of aorta were analysed for each rat.

The staining intensities of the elastic fibers in the media and collagen fibers in the media and adventitia were evaluated on a subjective scale ranging from 0 to 3 (0 – no staining of fibers, 1 – poor staining of fibers, 2 – moderate staining of fibers, 3 – intensive staining of fibers). The evaluations were performed by two independent observers in a blinded fashion; the scores were summed and used for statistical analysis.

Results are expressed as means ± standard deviation (SD). Differences between the groups were evaluated using the one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc analysis for multiple comparisons of group means. Semi-quantitative data were compared by the Kruskal-Wallis one-way ANOVA followed by Mann–Whitney U test. Differences were considered to be statistically significant when p was <0.05. All statistical comparisons were performed with Statistica software (version 8; StatSoft, USA).

The results are presented in Table 1. The initial body weights were similar in control and diabetic groups. The final body weights in the diabetic groups were significantly lower than in the control group. There were no differences between the diabetic treated and untreated groups in body weight after the treatment period. The levels of blood glucose and HbA1c were found to be significantly increased in the diabetic rats and were not affected by the vitamin D supplementation. Serum 25(OH)D level was significantly decreased in the untreated diabetic group, compared to the control group. Administration of vitamin D resulted in a significantly higher serum 25(OH)D level than that in the control group. Serum levels of ADMA were significantly lower in the control group compared with both diabetic groups. Vitamin D supplementation did not prevent the elevation of serum ADMA concentration. Serum calcium levels were similar between all groups, indicating that the administered dose of cholecalciferol remained within safe limits without increasing the risk of soft tissue calcification and possibly contributing to aortic stiffening.

Before administration of the vasoactive substances, resting systolic blood pressure (SBP), diastolic blood pressure (DBP), pulse pressure (PP), mean arterial pressure (MAP), and heart rate (HR) were not statistically different between all groups (Table 2). Intravenous infusion of phenylephrine increased MAP to 200 mmHg, followed by infusion of nitroglycerine, which decreased MAP to 50 mmHg. The diabetic rats had a significantly higher PWV compared to the control rats across a supraphysiological range of MAP (170–200 mmHg), but not at a lower MAP range. The non-linear PWV-MAP curve for the diabetic treated group was similar to that of diabetic untreated group, indicating that aortic stiffness was similar at every given level of MAP (Figure 1). Although all three groups received similar doses of phenylephrine, MAP above 170 mmHg was not achieved in diabetic treated rats. The reason for this effect is unknown, but may possibly include a lower sensitivity to phenylephrine.

Aortae of the control group showed a regular vascular morphology, while several alterations were noted in the structure of aortae of rats in the untreated and treated diabetic groups. More pronounced changes were found in the aortae of the untreated diabetic group, where focal irregular arrangement of elastic fibers was noted together with decreased staining intensity of elastic fibers and increased internal diameter of the aorta (Table 3, Figures 2 and 3). Changes in the medial thickness and in collagen staining were not statistically significant compared to the control group (Table 3). Milder changes of the aortic wall, particularly regarding medial elastic fibers with no focal disarrangements were noted in the diabetic treated group (Table 3, Figure 3). Untreated diabetes was also associated with reduced ratio of elastin to collagen that was prevented by vitamin D supplementation (Table 3). Both in untreated and treated diabetic groups no focal thickenings or other significant alterations of the intimal layer were found.