This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.In contrast with the full term ductus, constriction of the immature ductus frequently fails to cause luminal obliteration and only moderately increases diffusion distances for oxygen and nutrients within its muscle media. This is associated with less cell death and less remodeling and leads to persistent ductus luminal blood flow after birth. In addition to the absence of anatomic remodeling, the immature newborn ductus progressively loses its ability to constrict during the first days after birth. The explanation for the impaired contractility of the persistently patent immature newborn ductus is currently unknown. During the past year we examined ductus from full term and immature fetal and newborn baboons to determine how constriction of the ductus arteriosus affects the concentrations of oxygen, glucose, glycogen, and ATP in the ductus wall in vivo; and, to determine how these changes relate to the appearance of cell death in the vessel's wall and to the ability of the ductus to constrict after birth. We determined cell death (using TUNEL staining), energy status (using enzyme-linked bioluminescence imaging) and tissue hypoxia (using HIF1 alpha and VEGF mRNA expression as a surrogate marker) in frozen ductus obtained from baboons: 185d fetus (n= 11), 1-2 d old full term newborn (n=10), 125 d fetus (n=14), 125 d (6 d PRN) newborn (with open ductus, n=16), 125 d (6 d PRN) newborn (with closed ductus, n=10). In the full term baboon ductus, the expression of our two surrogate markers of hypoxia (HIF1alpha and VEGF) increased and the average concentrations of ATP, glucose and glycogen decreased following postnatal ductus constriction. The concentrations of ATP, glucose, and glycogen were significantly related to the degree of ductus constriction. In the full term baboon ductus there was also a significant relationship between the degree of ductus constriction and the logarithmic transformation of the number of TUNEL-positive cells (our indicator of cell death) (area of ductus lumen versus TUNEL log10: r=-0.92, p15% TUNEL-positive cells) only occurred when the ductus was closed and there was no Doppler flow through its lumen. In the premature ductus, ATP concentrations were directly related to tissue glucose concentrations but not to glycogen concentrations. Similarly, cell death (TUNEL staining) was related to tissue glucose concentrations but not to glycogen concentrations in the immature ductus. This is consistent with our previous in vitro studies that showed that the primary substrate utilized during glycolysis is glucose, not glycogen. In addition, during tight constriction and tissue hypoxia, the immature ductus actually develops regions where glycogen concentrations were actually greater than those observed in the fetus. We have previously shown that ductus rings, incubated in vitro under hypoxic conditions, develop both regions of glycogen depletion as well as regions of glycogen surplus: interestingly, the regions of glycogen surplus are more commonly seen in the immature than the mature ductus. We hypothesize that this adaptive mechanism could add to the ability ,of the immature ductus to tolerate episodes of hypoxia and nutrient shortage making it more resistant to developing postnatal cell death and permanent closure. Although the changes in HIF1alphaNEGF expression and ATP concentrations were significantly related to the degree of ductus constriction in the premature ductus, we were surprised to find that both HIF1alphaNEGF expression and ATP concentrations were still significantly altered after birth even when the immature ductus remained open and continued to have persistent luminal blood flow. We used ductus arteriosus from preterm lamb fetuses to determine if the drop in ductus ATP concentrations, that occur after birth in preterm newborns with an open ductus, is sufficient to alter ductus contractility. We examined the contractile behavior of immature sheep ductus in vitro exposed to 80% 02 and two different concentrations of glucose. At low glucose concentrations, ATP concentrations were not maintained and fell to 31 % of the starting (0 hr) fetal ductus levels. This is similar to the in vivo drop in ATP concentration observed in the immature newborn baboon ductus that remains open after birth. In contrast, in the rings exposed to normal glucose concentrations, ATP concentrations were stable during the 24 hr incubation, There was no difference between the mo experimental groups in the incidence of cell death (TUNEL staining) or remodeling during the in vitro incubation (24 hours). Ductus with normal concentrations of glucose and ATP contracted when exposed to 80% oxygen for 24 hours; in contrast, ductus with low glucose and low ATP failed to contract when exposed to 80% oxygen.
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