Decellularization of human umbilical arteries / Descelularización de las arterias umbilicales humanas

SUMMARY: Arterial obstruction in small diameter (<6 mm) vessels are many times treated with grafts, however autologous aren't always available and synthetic have a high rate of complications. Decellularization of umbilical arteries may provide a solution, but the ideal method is debatable. We compare effectiveness between SDS and Triton X-100. Umbilical cords obtained from full term pregnancies with normal development and no evident complications in the newborn, were micro-dissected within 12 h and stored in phosphate buffered saline without freezing. Arteries were then processed for decellularization using 0.1 % and 1 % SDS, and 1 % Triton X100 protocols. Evaluation of cellular and nuclear material, collagen fibers, elastic fibers, and glycosoaminoglycans of the extracellular matrix (ECM) were evaluated as well as morphometric analysis under histological and immunohistochemical techniques. Triton X-100 was ineffective, preserving nuclear remains identified by immunofluorescence, had the most notable damage to elastic fibers, and decrease in collagen. SDS effectively eliminated the nuclei and had a less decrease in elastic fibers and collagen. Laminin was preserved in all groups. No significant differences were identified in luminal diameters; however the middle layer decreased due to decellularization of muscle cells. In conclusion, 0.1 % SDS decellularization was the most effective in eliminating cells and preserving the main components of the ECM.

RESUMEN: La obstrucción arterial en vasos de pequeño diámetro (<6 mm) se trata muchas veces con injertos, sin embargo, los autólogos no siempre están disponibles y los sintéticos tienen una alta tasa de complicaciones. La descelularización de las arterias umbilicales puede proporcionar una solución, pero el método ideal es discutible. Comparamos la efectividad entre los métodos SDS y Triton X-100. Cordones umbilicales obtenidos a partir de embarazos a término con evolución normal y sin complicaciones evidentes del recién nacido, se microdiseccionaron en 12 horas y se almacenaron en solución salina con fosfato sin congelación. Las arterias se procesaron luego para la descelularización usando los protocolos de SDS al 0,1 % y 1 %, y Triton X-100 al 1 %. Se realizó la evaluación de material celular y nuclear, fibras de colágeno, fibras elásticas y glucosoaminoglicanos de la matriz extracelular (MEC), así como el análisis morfométrico bajo técnicas histológicas e inmunohistoquímicas. Triton X-100 fue ineficaz, conservando los restos nucleares identificados por inmunofluorescencia, tuvo el daño más notable a las fibras elásticas y la disminución del colágeno. SDS efectivamente eliminó los núcleos y tuvo una disminución menor en las fibras elásticas y el colágeno. Laminina fue preservado en todos los grupos. No se identificaron diferencias significativas en los diámetros luminales; sin embargo, la capa media disminuyó debido a la descelularización de las células musculares. la descelularización con SDS al 0,1 % fue la más efectiva para eliminar células y preservar los principales componentes de la MEC.

Human umbilical artery smooth muscle exhibits a 2-APB-sensitive capacitative contractile response evoked by vasoactive substances and expresses mRNAs for STIM, Orai and TRPC channels

After depletion of intracellular Ca2+ stores the capacitative response triggers an extracellular Ca2+ influx through store-operated channels (SOCs) which refills these stores. Our objective was to explore if human umbilical artery smooth muscle presented this response and if it was involved in the mechanism of serotonin- and histamine-induced contractions. Intracellular Ca2+ depletion by a Ca2+-free extracellular solution followed by Ca2+ readdition produced a contraction in artery rings which was inhibited by the blocker of Orai and TRPC channels 2-aminoethoxydiphenyl borate (2-APB), suggesting a capacitative response. In presence of 2-APB the magnitude of a second paired contraction by serotonin or histamine was significantly less than a first one, likely because 2-APB inhibited store refilling by capacitative Ca2+ entry. 2-APB inhibition of sarcoplasmic reticulum Ca2+ release was excluded because this blocker did not affect serotonin force development in a Ca2+-free solution. The PCR technique showed the presence of mRNAs for STIM proteins (1 and 2), for Orai proteins (1, 2 and 3) and for TRPC channels (subtypes 1, 3, 4 and 6) in the smooth muscle of the human umbilical artery. Hence, this artery presents a capacitative contractile response triggered by stimulation with physiological vasoconstrictors and expresses mRNAs for proteins and channels previously identified as SOCs.

Endothelial nitric oxide synthase traffic inducer in the umbilical vessels of the patients with pre-eclampsia / 华中科技大学学报（医学）（英德文版）

The expression of endothelial nitric oxide synthase traffic inducer (NOSTRIN) was examined in the umbilical vessels of the patients with pre-eclampsia (PE) to explore its possible role in the pathogenesis of PE. The NOSTRIN mRNA in umbilical tissues was determined by RT-PCR. The eNOS activity in umbilical vessels was spectrophotometrically detected. NO2-/NO3-, the stable metabolic end products of NO, was measured by using nitrate reductase. RT-PCR showed that the expression level of NOSTRIN was significantly higher in women with PE than in the normal group (P<0.01). The activity of eNOS was significantly decreased in PE group [(12.83+/-3.61) U/mg] than in normal group [(21.72+/-3.83) U/mg] (P<0.01). The level of NO2-/NO3- in PE patients (27.53+/-7.48) micromol/mg was significantly lower than that of normal group (54.27+/-9.53) micromol/mg (P<0.01). The significant negative correlation existed between the expression of NOSTRIN and the activity of eNOS in umbilical vessels of women with PE (r=-0.58, P<0.01). It was concluded that the level of NOSTRIN expression was increased in umbilical vessel of women with PE, indicating that it may be involved in the pathogenesis of PE.