Pre-Analytical Modification of Serum miRNAs: Diagnostic Reliability of Serum miRNAs in Hemolytic Diseases.

Circulating microRNAs (miRNAs) are useful biomarkers of hemolysis. Since blood cells are the main origins of circulating miRNAs, we evaluated blood cell-related pre-analytical modification of the miRNA signatures during blood drawing and serum processing. The levels of miRNA before and after ex vivo blood drawing were analyzed with the reverse transcriptase-based polymerase chain reaction method. Furthermore, the changes of miRNA signatures caused by different time-lag between blood drawing and serum preparation by 24 h were evaluated. Finally, we compared the miRNA levels between leftover samples and samples of hemolytic diseases. Blood drawing procedure induced increments of red blood cell (RBC)-related miRNAs (miR-451a, miR-486) about 2-fold. One hour standing of blood samples before serum separation induced almost the same increases in RBC-related miRNAs. To test the clinical usefulness of miR-451a as a biomarker of hemolytic diseases, we analyzed miRNAs of samples from 10 normal subjects, 30 leftover samples in the clinical laboratory, and 20 samples from patients with hemolytic diseases. Serum miR-451a significantly increased in patients with hemolytic anemia more than the levels of pre-analytical modification. In conclusion, the pre-analytical modification of serum miRNAs did not disturb the usefulness of RBC-derived miRNAs as biomarkers of hemolytic diseases.

Oral administration of nicorandil enhances the survival of ischemic skin flaps in rats.

Nicorandil has an anti-apoptotic effect on ischemic myocardium through the activation of ATP-sensitive potassium (K(ATP)) channel. We tested the hypothesis that oral administration of nicorandil had a protective effect on ischemic skin flaps. A cranially based skin flap measuring 3x7 cm in full thickness was made on the back of rats. The rats were divided into a control group and 8 nicorandil groups (group 1-8) according to different doses and timings of administration. On day 7 at 5 cm, groups 1 to 6 (10 or 30 mg/kg twice per day for 3 days starting at 24 h before, 0.5 h before or 0.5 h after the operation) showed significantly higher blood perfusion change rate (73.3+/-2.9%-79.1+/-4.1% vs. 25.9+/-8.6%, P<0.01), and significantly higher survival rate (68.8+/-4.8-75.2+/-8.2% vs. 47.0+/-2.8%, P<0.05) than the control group. Many more surviving blood vessels were also observed in these groups. In contrast, no significant effects were found either in group 7 (30 mg/kg twice per day for 3 days starting 24 h after the operation) or group 8 (30 mg/kg once at 0.5 h after the operation). We did not find an angiogenic effect of nicorandil in vitro. Therefore, our results confirmed that the oral administration of nicorandil could protect tissues from necrosis in ischemic skin flaps. In addition, its protective effect depends on the time of first administration and the duration.

Cold preservation of islets in UW solution--with special reference to apoptosis.

BACKGROUND: Apoptosis progresses in cultured islets. Little is known with regard to apoptosis under cold preservation. We examined viability and function of islets in University of Wisconsin (UW) solution. MATERIALS AND METHODS: Isolated rat islets were cultured overnight (overnight group) and further treated with 7-day culture in RPMI 1640 medium at 37 degrees C (culture group) or 7-day preservation in UW solution at 4 degrees C (preservation group). They were evaluated by glucose-stimulated insulin secretion test. Apoptosis was examined by TdT-mediated dUTP-biotin nick end-labeling (TUNEL) assay. Expression of caspase mRNA and the ratio of Bax to Bcl-2 were evaluated by reverse-transcriptase polymerase chain reaction (RT-PCR). RESULTS: Islet recovery after 7 days was significantly lower in culture group than in preservation group (44.0 +/- 3.7% versus 75.0 +/- 4.9%, P < 0.05). The stimulation index in the culture group was significantly lower than in the overnight group (2.1 +/- 0.2 versus 4.1 +/- 0.4, P < 0.05). The apoptotic index in the culture group was significantly higher than both in the overnight group and in the preservation group (38.0 +/- 3.0% versus 10.8 +/- 2.0 and 27.0 +/- 4.0%, P < 0.05). Caspase 3, 8, and 9 mRNA in the culture group expressed more than in the other groups. Bax/Bcl-2 in the culture group was significantly lower than in the overnight group (3.2 +/- 0.66 versus 8.1 +/- 0.95, P < 0.05), suggesting that apoptosis had been already destined early after isolation. CONCLUSIONS: The preservation group showed better recovery and function than the culture group. Apoptosis contributed to islet loss under culture and it was significantly suppressed under cold preservation.

Immunohistochemical analysis of bcl-2, Bax and Bak expression in thyroid glands from patients with Graves' disease.

In order to clarify the role of apoptosis and the expression of Bcl-2 family proteins in the pathology of Graves' disease (GD), we evaluated the apoptosis by in situ end-labeling of fragmented DNA and the expression of Bcl-2, Bax and Bak by immunohistochemistry in thyroid tissues from 20 patients with GD and in normal thyroid tissues from 6 patients with follicular adenoma (N). Apoptotic nuclei were found in thyrocytes and in germinal center of lymphoid follicles. Bcl-2 was strongly expressed in both GD and N thyrocytes. Bax was not expressed in either GD or N thyrocytes. Bak was expressed in thyrocytes from 5 of 20 patients with GD, while it was detected in all N thyrocytes. In lymphoid follicles Bcl-2 was expressed in the mantle zone, while Bax and Bak were both expressed in the germinal center. The percentage of apoptotic nuclei in GD thyrocytes was low (0~3.6%), and negatively correlated with the weight of the thyroid glands resected (rs = -0.43, P<0.05). It was greater in Bak-positive GD thyrocytes than in Bak-negative ones (mean +/- SD; 1.7 +/- 0.7% vs. 0.7 +/- 0.9%, P<0.05). These findings suggest that the differential expression of Bcl-2 family proteins in both thyrocytes and lymphoid follicles may be involved in the pathology of GD.

Palmitate-induced apoptosis of microvascular endothelial cells and pericytes.

BACKGROUND: Recent observations in the EURODIAB Complications Study demonstrated that markers of insulin resistance are strong risk factors for retinopathy incidence in patients with diabetes. However, the molecular mechanism underlying this remains to be elucidated. In this study, we investigated the influence of palmitate, a major saturated free fatty acid in plasma, on the apoptotic cell death of cultured microvascular endothelial cells (EC) and retinal pericytes. MATERIALS AND METHODS: The intracellular formation of reactive oxygen species (ROS) was detected using the fluorescent probe CM-H(2)DCFDA. DNA synthesis was determined by measuring [(3) H]-thymidine incorporation into cells. DNA fragmentations of EC were quantitatively analyzed in an enzyme-linked immunosorbent assay, and DNA laddering was evaluated on agarose gel electrophoresis. RESULTS: Palmitate increased ROS generation in microvascular EC. Furthermore, palmitate significantly inhibited DNA synthesis and induced apoptotic cell death in EC, which were completely prevented by an antioxidant, N-acetylcysteine. Palmitate up-regulated pericyte mRNA levels of a receptor for advanced glycation end products (AGE), and thereby potentiated the apoptotic effects of AGE on pericytes. CONCLUSIONS: The results suggest that palmitate could induce apoptotic cell death in microvascular EC and pericytes through the overgeneration of intracellular ROS, and thus be involved in the development of diabetic retinopathy.