Immunohistomorphology of pancreatic islet microvasculature and the immunophenotypic analysis of CEPC in adult diabetic rats / Inmunohistomorfología de microvasculatura de islotes pancreatic y el análisis inmunofenotípico de CEPC en ratas diabéticas adultas

SUMMARY: Hyperglycaemia is one of the main causes for the endothelial cell (EC) damage in diabetic patients. Even though circulating endothelial progenitor cells (CEPC) could be used as a prognosis for microvascular complications, there is very little information on the islet microvasculature. We analysed by immunohistochemistry and by flow cytometric immunophenotyping, the expression of CD34 on EC and the expressions of CD31, CD34, CD45 and CD133 on CEPC in Streptozotocin (STZ)-induced diabetic rats. Peripheral blood and tissue specimens were obtained from rats of different treatment regimens: STZ treatment, control saline (NS) and sodium citrate (CB) treatments. Blood cells were exposed to flow cytometric immunophenotyping for CD133, CD31, CD34, CD45 and CD133. While tissues from the pancreas, liver and kidney were routinely processed and stained immunohistochemically for CD34. There was a tendency of an increased in CD45-/CD133+/CD31+/CD34+ cells (0.04 ± 0.11 %) in diabetic rats compared to the controls (CB: 0.03 ± 0.04 %; Saline: 0.01 ± 0.03 %). But there was no significant statistical difference between them. The expression pattern of CD34 on the EC in the organs' vascular beds including arterioles, venules, capillaries and sinusoids was extremely heterogeneous across and within treatment regimens. The ECs in the sinusoids of the liver presented similar CD34 expression patterns across different treatment regimens, while the expression of CD34 on the ECs of sinusoidal capillaries in the pancreas vary with the treatment regimen. We conclude that the degree of endothelial cell damage is not uniform across organs' vascular beds in the rat, contrary to mice and humans. Furthermore, the sinusoids in the pancreas and the kidney may have the same degree of endothelial damage when exposed to the same deleterious causes.

RESUMEN: La hiperglucemia es una de las principales causas del daño de las células endoteliales (EC) en pacientes diabéticos. A pesar de que las células progenitoras endoteliales circulantes (CEPC) podrían utilizarse como pronóstico de las complicaciones microvasculares, hay muy poca información sobre la microvasculatura de los islotes. Se analizaron por inmunohistoquímica y por inmunofenotipificación citométrica de flujo, la expresión de CD34 en EC y las expresiones de CD31, CD34, CD45 y CD133 en CEPC en ratas diabéticas inducidas por estreptozotocina (STZ). Se obtuvieron muestras de sangre y tejidos periféricos a partir de ratas de diferentes regímenes de tratamiento: tratamiento con STZ, solución salina control (NS) y citrato de sodio (CB). Las células sanguíneas fueron expuestas a inmunofenotipado por citometría de flujo para CD133, CD31, CD34, CD45 y CD133. Mientras que los tejidos del páncreas, el hígado y el riñón fueron rutinariamente procesados y teñidos inmunohistoquímicamente para CD34. Se observó una tendencia a un aumento en las células CD45- / CD133 + / CD31 + / CD34 + (0,04 ± 0,11 %) en ratas diabéticas en comparación con los controles (CB: 0,03 ± 0,04 %; Salino: 0,01 ± 0,03 %). Pero no hubo diferencias estadísticamente significativas entre ellos. El patrón de expresión de CD34 en la EC en los lechos vasculares de los órganos incluyendo arteriolas, vénulas, capilares y sinusoides fue extremadamente heterogéneo a través de y dentro de los regímenes de tratamiento. Las EC en los sinusoides del hígado presentaron patrones de expresión de CD34 similares a través de diferentes regímenes de tratamiento, mientras que la expresión de CD34 en las CE de capilares sinusoidales en el páncreas varía con el régimen de tratamiento. Concluimos que el grado de daño de las células endoteliales no es uniforme en los lechos vasculares de los órganos en la rata, en comparación de los ratones y los seres humanos. Además, los sinusoides en el páncreas y el riñón pueden tener el mismo grado de daño endotelial cuando se exponen a las mismas causas deletéreas.

Ultrastructural changes of pancreatic islets microcirculation in nonobese diabetic (NOD) mice.

The objective of this study was to investigate the ultrastructural changes of vascular pancreatic islets using a transmission electron microscopic technique. The major ultrastructural changes of microvessel in NOD mice are indicated by the swelling and vacuolization of the endothelial cell. Swollen cells are the first noticeable lesion of the cell response in reversible degeneration that is caused by the failure of homeostatic control. Loss in endothelial cell homeostasis is primarily a marker of endothelial dysfunction that plays a key role in the pathogenesis of diabetic vascular disease by losing the control of vascular tone. Diabetes also associates with an increased generation of oxygen-derived free radicals that may impair vasodilatation through the inactivation of vasodilators. In conclusion, consistent with a hypothesis that loss of the modulatory role of the endothelium may be a critical and initiating factor in the development of diabetic vascular disease, the ultrastructural changes in this study may indicate the first sign of endothelial dysfunction. This dysfunction correlates to the relationship between diabetes and reversible lesions of vessels in NOD mice, making for a better understanding of the pathophysiology of diabetic vascular disease to set the stage for further investigation to restore endothelial dysfunction in diabetes.

Effective glycemic control achieved by transplanting non-viral cationic liposome-mediated VEGF-transfected islets in streptozotocin-induced diabetic mice

Hypoxic damage is one of the major causes of islet graft failure and VEGF is known to play a crucial role in revascularization. To address the effectiveness of a cationic lipid reagent as a VEGF gene carrier, and the beneficial effect of VEGF-transfected islets on glycemic control, we used effectene lipid reagent in a transfection experiment using mouse islets. Transfection efficiencies were highest for 4 microgram/microliter cDNA and 25 microliter effectene and cell viabilities were also satisfactory under this condition, and the overproduction of VEGF mRNA and protein were confirmed from conditioned cells. A minimal number of VEGF-transfected islets (100 IEQ/animal) were transplanted into streptozotocin (STZ)-induced diabetic mice. Hyperglycemia was not controlled in the islet transplantation (IT)-alone group (0/8) (non- diabetic glucose mice number/total recipient mice number) or in the IT-pJDK control vector group (0/8). However, hyperglycemia was completely abrogated in the IT-pJDK-VEGF transduced group (8/8), and viable islets and increased VEGF-transfected grafts vascularization were observed in renal capsules.