Failure of postnatal ductus arteriosus closure in prostaglandin transporter-deficient mice.

BACKGROUND: Prostaglandin E(2) (PGE(2)) plays a major role both in maintaining patency of the fetal ductus arteriosus and in closure of the ductus arteriosus after birth. The rate-limiting step in PGE(2) signal termination is PGE(2) uptake by the transporter PGT. METHODS AND RESULTS: To determine the role of PGT in ductus arteriosus closure, we used a gene-targeting strategy to produce mice in which PGT exon 1 was flanked by loxP sites. Successful targeting was obtained because neither mice hypomorphic at the PGT allele (PGT Neo/Neo) nor global PGT knockout mice (PGT(-/-)) exhibited PGT protein expression; moreover, embryonic fibroblasts isolated from targeted mice failed to exhibit carrier-mediated PGE(2) uptake. Although born in a normal mendelian ratio, no PGT(-/-) mice survived past postnatal day 1, and no PGT Neo/Neo mice survived past postnatal day 2. Necropsy revealed patent ductus arteriosus with normal intimal thickening but dilated cardiac chambers. Both PGT Neo/Neo and PGT(-/-) mice could be rescued through the postnatal period by giving the mother indomethacin before birth. Rescued mice grew normally and had no abnormalities by gross and microscopic postmortem analyses. In accordance with the known role of PGT in metabolizing PGE(2), rescued adult PGT(-/-) mice had lower plasma PGE(2) metabolite levels and higher urinary PGE(2) excretion rates than wild-type mice. CONCLUSIONS: PGT plays a critical role in closure of the ductus arteriosus after birth by ensuring a reduction in local and/or circulating PGE(2) concentrations.

Small-molecule cyclic alpha V beta 3 antagonists inhibit sickle red cell adhesion to vascular endothelium and vasoocclusion.

Abnormal adhesion of sickle red blood cells (SS RBCs) to vascular endothelium may play an important role in vasoocclusion in sickle cell disease. Accruing evidence shows that endothelial alpha V beta 3-integrin has an important role in SS RBC adhesion because of its ability to bind several adhesive proteins implicated in this interaction. In the present studies, we tested therapeutic efficacy of small-molecule cyclic pentapeptides for their ability to block alpha V beta 3-mediated SS RBC adhesion by using two well-established assay systems, i.e., cultured human umbilical vein endothelial cells (HUVEC) and artificially perfused mesocecum vasculature of the rat under flow conditions. We tested the efficacy of two RGD-containing cyclic pentapeptides, i.e., cRGDFV (EMD 66203) and cRGDF-ACHA (alpha-amino cyclohexyl carboxylic acid) (EMD 270179), based on their known ability to bind alpha V beta 3. An inactive peptide, EMD 135981 (cR beta-ADFV) was used as control. Cyclization and the introduction of D-Phe (F) results in a marked increase in the ability of cyclic peptides to selectively bind alpha V beta 3 receptors. In the mesocecum vasculature, both EMD 66203 and EMD 270179 ameliorated platelet-activating factor-induced enhanced SS RBC adhesion, postcapillary blockage, and significantly improved hemodynamic behavior. Infusion of a fluorescent derivative of EMD 66203 resulted in colocalization of the antagonist with vascular endothelium. Also, pretreatment of HUVEC with either alpha V beta 3 antagonist resulted in a significant decrease in SS RBC adhesion. Because of their metabolic stability, the use of these cyclic alpha V beta 3 antagonists may constitute a novel therapeutic strategy to block SS RBC adhesion and associated vasoocclusion under flow conditions.

Prostaglandin signaling in the renal collecting duct: release, reuptake, and oxidation in the same cell.

Prostaglandins mediate autacrine and paracrine signaling over short distances. We used the renal collecting duct as a model system to test the hypothesis that local control of prostaglandin signaling is achieved by expressing inactivation in the same cell as synthesis. Immunocytochemical studies demonstrated that renal collecting ducts in situ express the prostaglandin (PG) synthesis enzyme, cyclooxygenase-1 (COX-1), as well as both components of prostaglandin metabolic inactivation, i.e. the prostaglandin uptake carrier prostaglandin transporter (PGT) and the enzyme 15-hydroxyprostaglandin dehydrogenase. We characterized this system further using the collecting duct cell line Madin-Darby canine kidney (MDCK), which retains COX-2 and prostaglandin dehydrogenase expression but which has lost PGT expression. When we reintroduced PGT, it was correctly sorted to the apical membrane where it altered the sidedness of prostaglandin E2 (PGE2) release, a process we call "vectorial release via sided reuptake." Importantly, although COX-2 and prostaglandin dehydrogenase are expressed in the same MDCK cell, they must be compartmentalized because even in the presence of excess dehydrogenase newly synthesized PGE2 is released largely un-oxidized. However, when PGE2 undergoes first release and then PGT-mediated reuptake, significant oxidation takes place, suggesting that PGT imports PGE2 into the prostaglandin dehydrogenase compartment. Our data are consistent with a new model that offers significant new mechanisms for the fine control of eicosanoid signaling.

Effect of fetal hemoglobin on microvascular regulation in sickle transgenic-knockout mice.

In sickle cell disease, intravascular sickling and attendant flow abnormalities underlie the chronic inflammation and vascular endothelial abnormalities. However, the relationship between sickling and vascular tone is not well understood. We hypothesized that sickling-induced vaso-occlusive events and attendant oxidative stress will affect microvascular regulatory mechanisms. In the present studies, we have examined whether microvascular abnormalities expressed in sickle transgenic-knockout Berkeley (BERK) mice (which express exclusively human alpha- and beta(S)-globins with <1% gamma-globin levels) are amenable to correction with increased levels of antisickling fetal hemoglobin (HbF). In BERK mice, sickling, increased oxidative stress, and hemolytic anemia are accompanied by vasodilation, compensatory increases in eNOS and COX-2, and attenuated vascular responses to NO-mediated vasoactive stimuli and norepinephrine. The hypotension and vasodilation (required for adequate oxygen delivery in the face of chronic anemia) are mediated by non-NO vasodilators (i.e., prostacyclin) as evidenced by induction of COX-2. In BERK mice, the resistance to NO-mediated vasodilators is associated with increased oxidative stress and hemolytic rate, and in BERK + gamma mice (expressing 20% HbF), an improved response to these stimuli is associated with reduced oxidative stress and hemolytic rate. Furthermore, BERK + gamma mice show normalization of vessel diameters, and eNOS and COX-2 expression. These results demonstrate a strong relationship between sickling and microvascular function in sickle cell disease.