Intimal Cushion Formation Research Articles

Effect of Long-term Administration of Prostaglandin E1 on Morphologic Changes in Ductus Arteriosus

To improve survival of patients with hypoplastic left heart syndrome, combination therapy with bilateral pulmonary artery banding and prostaglandin E1 (PGE1)-mediated ductal patency was developed as an alternative for high-risk neonates in Japan. However, the effect of long-term PGE1 administration on ductus arteriosus remains unclear. Synchrotron radiation-based X-ray phase-contrast tomography (XPCT) enables clear visualization of soft tissues at an approximate spatial resolution of 12.5 μm. We aimed to investigate morphologic changes in ductus arteriosus after long-term PGE1 infusion using XPCT. Seventeen ductus arteriosus tissue samples from patients with hypoplastic left heart syndrome were obtained during the Norwood procedure. The median duration of lipo-prostaglandin E1 (lipo-PGE1) administration was 48 days (range, 3 to 123). Structural analysis of ductus arteriosus was performed and compared with conventional histologic analysis. The XPCT was successfully applied to quantitative measurements of ductal media. Significant correlation was found between the duration of lipo-PGE1 infusion and mass density of ductal media (R= 0.723, P= .001). The duration of lipo-PGE1 administration was positively correlated with elastic fiber staining (R= 0.799, P < .001) and negatively correlated with smooth muscle formation (R=-0.83, P < .001). No significant increase in intimal cushion formation was found after long-term lipo-PGE1 administration. Expression of ductus arteriosus dominant PGE2-receptor EP4 almost disappeared in specimens when lipo-PGE1 was administered over 3 days. Disorganized elastogenesis and little intimal cushion formation after long-term lipo-PGE1 administration suggest that ductus arteriosus remodeled to the elastic artery phenotype. Because EP4 was downregulated and ductus arteriosus exhibited elastic characteristics, the dosage of lipo-PGE1 might be decreased after a definite administration period.

CCN3 secreted by prostaglandin E2 inhibits intimal cushion formation in the rat ductus arteriosus

The ductus arteriosus (DA), an essential fetal shunt between the pulmonary trunk and the descending aorta, changes its structure during development. Our previous studies have demonstrated that prostaglandin E2 (PGE2)-EP4 signaling promotes intimal cushion formation (ICF) by activating the migration of DA smooth muscle cells via the secretion of hyaluronan. We hypothesized that, in addition to hyaluronan, PGE2 may secrete other proteins that also regulate vascular remodeling in the DA. In order to detect PGE2 stimulation-secreted proteins, we found that CCN3 protein was increased in the culture supernatant in the presence of PGE2 in a dose-dependent manner by nano-flow liquid chromatography coupled with tandem mass spectrometry analysis and enzyme-linked immunosorbent assay. Quantitative RT-PCR analysis revealed that PGE2 stimulation tended to increase the expression levels of CCN3 mRNA in DA smooth muscle cells. Immunohistochemical analysis revealed that CCN3 was highly localized in the entire smooth muscle layers and the endothelium of the DA. Furthermore, exogenous CCN3 inhibited PGE2-induced ICF in the ex vivo DA tissues. These results suggest that CCN3 is a secreted protein of the DA smooth muscle cells induced by PGE2 to suppress ICF of the DA. The present study indicates that CCN3 could be a novel negative regulator of ICF in the DA to fine-tune the PGE2-mediated DA remodeling.

DNA microarray profiling identified a new role of growth hormone in vascular remodeling of rat ductus arteriosus

The ductus arteriosus (DA), a fetal arterial connection between the pulmonary artery and the aorta, has a character distinct from the adjacent arteries. We compared the transcriptional profiles of the DA and the aorta of Wistar rat fetuses on embryonic day 19 (preterm) and day 21 (near-term) using DNA microarray analyses. We found that 39 genes were expressed 2.5-fold greater in the DA than in the aorta. Growth hormone (GH) receptor (GHR) exhibited the most significant difference in expression. Then, we found that GH significantly promoted migration of DA smooth muscle cells (SMCs), thus enhancing the intimal cushion formation of the DA explants. GH also regulated the expression of cytoskeletal genes in DA SMCs, which may retain a synthetic phenotype in the smooth muscle-specific cytoskeletal genes. Thus, the present study revealed that GH-GHR signal played a role in the vascular remodeling of the DA.

Prostaglandin E2-activated Epac Promotes Neointimal Formation of the Rat Ductus Arteriosus by a Process Distinct from That of cAMP-dependent Protein Kinase A

We have demonstrated that chronic stimulation of the prostaglandin E2-cAMP-dependent protein kinase A (PKA) signal pathway plays a critical role in intimal cushion formation in perinatal ductus arteriosus (DA) through promoting synthesis of hyaluronan. We hypothesized that Epac, a newly identified effector of cAMP, may play a role in intimal cushion formation (ICF) in the DA distinct from that of PKA. In the present study, we found that the levels of Epac1 and Epac2 mRNAs were significantly up-regulated in the rat DA during the perinatal period. A specific EP4 agonist, ONO-AE1-329, increased Rap1 activity in the presence of a PKA inhibitor, PKI-(14-22)-amide, in DA smooth muscle cells. 8-pCPT-2'-O-Me-cAMP (O-Me-cAMP), a cAMP analog selective to Epac activator, promoted migration of DA smooth muscle cells (SMC) in a dose-dependent manner. Adenovirus-mediated Epac1 or Epac2 gene transfer further enhanced O-Me-cAMP-induced cell migration, although the effect of Epac1 overexpression on cell migration was stronger than that of Epac2. In addition, transfection of small interfering RNAs for Epac1, but not Epac2, significantly inhibited serum-mediated migration of DA SMCs. In the presence of O-Me-cAMP, actin stress fibers were well organized with enhanced focal adhesion, and cell shape was widely expanded. Adenovirus-mediated Epac1, but not Epac2 gene transfer, induced prominent ICF in the rat DA explants when compared with those with green fluorescent protein gene transfer. The thickness of intimal cushion became significantly greater (1.98-fold) in Epac1-overexpressed DA. O-Me-cAMP did not change hyaluronan production, although it decreased proliferation of DA SMCs. The present study demonstrated that Epac, especially Epac1, plays an important role in promoting SMC migration and thereby ICF in the rat DA.

Fate of the “opened” arterial duct: Lessons learned from bilateral pulmonary artery banding for hypoplastic left heart syndrome under the continuous infusion of prostaglandin E1

Fate of the “opened” arterial duct: Lessons learned from bilateral pulmonary artery banding for hypoplastic left heart syndrome under the continuous infusion of prostaglandin E1

Chronic activation of the prostaglandin receptor EP4 promotes hyaluronan-mediated neointimal formation in the ductus arteriosus

PGE, a potent vasodilator, plays a primary role in maintaining the patency of the ductus arteriosus (DA). Genetic disruption of the PGE-specific receptor EP4, however, paradoxically results in fatal patent DA (PDA) in mice. Here we demonstrate that EP4-mediated signals promote DA closure by hyaluronic acid-mediated (HA-mediated) intimal cushion formation (ICF). Chronic EP4 stimulation by ONO-AE1-329, a selective EP4 agonist, significantly enhanced migration and HA production in rat DA smooth muscle cells. When HA production was inhibited, EP4-mediated migration was negated. Activation of EP4, adenylyl cyclase, and PKA all increased HA production and the level of HA synthase 2 (HAS2) transcripts. In immature rat DA explants, ICF was promoted by EP4/PKA stimuli. Furthermore, adenovirus-mediated Has2 gene transfer was sufficient to induce ICF in EP4-disrupted DA explants in which the intimal cushion had not formed. Accordingly, signals through EP4 have 2 essential roles in DA development, namely, vascular dilation and ICF. The latter would lead to luminal narrowing, helping adhesive occlusion and permanent closure of the vascular lumen. Our results imply that HA induction serves as an alternative therapeutic strategy for the treatment of PDA to the current one, i.e., inhibition of PGE signaling by cyclooxygenase inhibitors, which might delay PGE-mediated ICF in immature infants.

Isolated ductus arteriosus aneurysm in the fetus and infant: a multi-institutional experience

OBJECTIVESThe purpose of this study was to describe the clinical characteristics and outcome and to elucidate the pathogenesis of ductus arteriosus aneurysm (DAA).BACKGROUNDDuctus arteriosus aneurysm is a rare lesion that can be associated with severe complications including thromboembolism, rupture and death.METHODWe reviewed the clinical records, diagnostic imaging studies and available histology of 24 cases of DAA, diagnosed postnatally (PD) in 15 and antenatally (AD) in 9 encountered in five institutions.RESULTSOf PD cases, 13 presented at <2 months, and all AD cases were detected incidentally after 33 weeks of gestation during a late trimester fetal ultrasound study. Of the 24, only 4 had DAA-related symptoms and 6 had associated syndromes: Marfan, Smith-Lemli-Opitz, trisomies 21 and 13 and one possible Ehlers-Danlos. Three had complications related to the DAA: thrombus extension into the pulmonary artery, spontaneous rupture, and asymptomatic cerebral infarction. Six underwent uncomplicated DAA resection for ductal patency, DAA size or extension of thrombus. In the four examined, there was histologic evidence of reduced intimal cushions in two and abnormal elastin expression in two. Five of the 24 died, with only one death due to DAA. Of 19 survivors, all but one remain clinically asymptomatic at a median follow-up of 35 months; however, two have developed other cardiac lesions that suggest Marfan syndrome. A review of 200 consecutive third trimester fetal ultrasounds suggests an incidence of DAA of 1.5%.CONCLUSIONSDuctus arteriosus aneurysm likely develops in the third trimester perhaps due to abnormal intimal cushion formation or elastin expression. Although it can be associated with syndromes and severe complications, many affected infants have a benign course. Given the potential for development of other cardiac lesions associated with connective tissue disease, follow-up is warranted.

Nitric oxide mediates LC-3-dependent regulation of fibronectin in ductus arteriosus intimal cushion formation.

Ductus arteriosus intimal cushion formation is characterized by fibronectin-dependent smooth muscle cell (SMC) migration. Enhanced fibronectin synthesis in ductus SMC is regulated by the interaction of LC-3, a microtubule-associated protein, with an AU-rich element (ARE) in the 3'-untranslated region of fibronectin mRNA, facilitating its recruitment to polyribosomes for translation. Since nitric oxide (NO) is implicated in posttranscriptional gene regulation and is produced in the ductus, we investigated its mechanistic role in LC-3-mediated fibronectin synthesis. NO production was sevenfold higher in ductus vs. aortic SMC (P<0.005) associated with increased neuronal NO synthase (nNOS) expression. The NOS inhibitor L-NMMA decreased fibronectin synthesis by approximately 45-50% (P<0.05), whereas the NO donor, SNAP, increased ductus fibronectin synthesis approximately onefold (P<0.05); neither agent altered fibronectin mRNA levels. Immunoblotting revealed that SNAP increased and L-NMMA reduced a membrane-associated phosphorylated form of LC-3. RNA gel mobility shift assays confirmed that NO enhanced LC-3 binding to the fibronectin mRNA ARE. Our studies indicate a tissue-specific program in the ductus arteriosus whereby elevated nNOS expression and NO production regulate the posttranscriptional increase in fibronectin synthesis required for SMC motility.

Gene transfer in utero biologically engineers a patent ductus arteriosus in lambs by arresting fibronectin-dependent neointimal formation.

Closure of the ductus arteriosus requires prenatal formation of intimal cushions, which occlude the vessel lumen at birth. Survival of newborns with severe congenital heart defects, however, depends on ductal patency. We used a gene transfer approach to create a patent ductus arteriosus by targeting the fibronectin-dependent smooth muscle cell migration required for intimal cushion formation. Fetal lamb ductus arteriosus was transfected in utero with hemagglutinating virus of Japan liposomes containing plasmid encoding 'decoy' RNA to sequester the fibronectin mRNA binding protein. Fibronectin translation was inhibited and intimal cushion formation was prevented. We thus established the essential role of fibronectin-dependent smooth muscle cell migration in intimal cushion formation in the intact animal and the feasibility of incorporating biological engineering in the management of congenital heart disease.

Cell-extracellular matrix interactions in the ductus arteriosus and perinatal pulmonary circulation

Our studies and those of others have shown that changes in the extracellular matrix have profound effects on vascular remodeling. In the ductus, increased production of endothelial hyaluronan and smooth muscle cell chondroitin sulfate and fibronectin and impaired elastin fiber assembly are features critical to smooth muscle cell migration into the subendothelium and intimal cushion formation. There is a developmentally orchestrated process that involves post-transcriptional mechanisms of gene regulation. Closure of the ductus arteriosus is associated with further changes in matrix expression and programmed cell death. The changes in the extracellular matrix that induce neointimal formation are also observed in pathological conditions in pulmonary and coronary arteries. Plasticity of the pulmonary circulation in the perinatal period also involves matrix regulation, and processes that prevent the normal decrease in pulmonary vascular resistance will result in impaired matrix regulation, and in the development of structural changes in the pulmonary arteries, including abnormal smooth muscle cell differentiation, hypertrophy and proliferation, which sustain the elevation in pulmonary artery pressure.

Regulation of fetal lamb ductus arteriosus smooth muscle cell migration by indomethacin and dexamethasone.

Postnatal closure of the ductus arteriosus (DA) requires the development, during late gestation, of "intimal cushions." These structures, which partially occlude the vessel lumen, are characterized by smooth muscle cell (SMC) migration into an expanded subendothelium. DA SMC migration is dependent upon increased fibronectin (FN) production. We hypothesized that indomethacin (INDO) or dexamethasone, which could influence FN production, may affect SMC migration and DA intimal cushion formation. SMC harvested from the DA and aorta of 100-d fetal lambs were seeded onto three-dimensional collagen gels. Migration into the gels was quantitatively assessed by phase-contrast microscopy. INDO retarded DA SMC migration (p < 0.05), but it had no effect on aorta SMC migration. Synthesis of FN was measured after [35S]-methionine radiolabeling of cells, gelatin sepharose extraction of conditioned media, and resolution by SDS-PAGE. Despite the decrease in migration, INDO did not affect FN synthesis in DA SMC, whereas dexamethasone, a stabilizer of FN mRNA that increased DA FN synthesis by 44%, had no further effect on SMC migration. We then investigated whether INDO might be influencing SMC migration by decreasing collagenase production or altering cell shape through changes in F-actin polymerization. Collagenase activity, assessed by zymography using collagen types I and IV, was similar in control and treated DA and aorta cells. Image analysis of the actin cytoskeleton after rhodamine-phalloidin staining of DA SMC, however, revealed significant shortening of INDO-treated DA SMC relative to control, consistent with the observed reduction in migration.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in distribution of elastin and elastin receptor during intimal cushion formation in the ductus arteriosus

Changes in distribution of elastin and elastin receptor were studied during maturation of the ductus arteriosus. In this vessel intimal thickening is a normal process. It starts with separation of endothelial cells from the internal elastic lamina by the formation of a wide subendothelial region, followed by an increase in number of radially orientated cells in the inner media and subendothelial region. For the first time the nature of these originally inner media cells could be definitely determined as being derived from smooth muscle cells. Areas rich in these modified smooth muscle cells contained less elastin as compared with regions rich in circularly orientated smooth muscle cells. The internal elastic lamina had disappeared underneath intimal cushions of the canine ductus arteriosus, whereas in the fetal human ductus arteriosus, splitting of the internal elastic lamina was observed. Elastin was not present underneath separated endothelial cells, which suggests that these cells do not synthesize elastin, at least not after separation. Both smooth muscle cells and endothelial cells demonstrated the presence of the elastin receptor. Changes in its distribution were not observed. The temporal and spatial association between the altering distribution of elastin and the absence of normal cushion formation in the persistent ductus arteriosus suggests a role of the elastin receptor in the process of intimal thickening.

Ultrastructural and immunohistochemical changes of the extracellular matrix during intimal cushion formation in the ductus arteriosus of the dog

The changes of the intima during subendothelial edema formation were studied by ultrastructural and immunohistochemical methods in the ductus arteriosus (DA) of the dog. Subendothelial edema formation is the first stage in the development of intimal cushions in the DA. Development of intimal cushions is a physiological process preceding normal spontaneous closure after birth. The material consisted of normal canine DA and DA from a strain of dogs with hereditary persistence of the DA (PDA). In the normal DA intimal thickening starts with separation of the endothelial cells from the internal elastic lamina by a widened subendothelial region (SR). Initially this SR is, at the ultrastructural level, composed of granular and amorphous material. Collagen fibrils and elastin are not detected. During the formation of the SR a shedding of the basal lamina underneath the endothelial cells is observed. In the PDA the endothelial cells remain attached to the internal elastic lamina. The topography of the extracellular matrix components collagen type I, III, IV, fibronectin and laminin were studied immunohistochemically. These are important factors in the adherence of the endothelial cells to the underlying intimal layers. Laminin and collagen type I are diffusely present before but absent after detachment of the endothelial cells. Collagen type 111, barely detectable before detachment, becomes visible underneath the detached cells. No changes are observed in the distribution of collagen type IV and fibronectin. Comparison of the normal DA with the various types of the PDA strain and controls allowed the conclusion that the observed changes in the extracellular matrix components were confined to those parts of the vessel wall that showed development of intimal thickening. The observed alterations both at the ultrastructural and immunohistochemical level do not explain the initiation of the process of endothelial cell detachment, which have been shown in a previous study to be related to an increase in hyaluronic acid.

Qualitative and quantitative differences in protein synthesis comparing fetal lamb ductus arteriosus endothelium and smooth muscle with cells from adjacent vascular sites

During late gestation, intimal cushions form in the ductus arteriosus (DA) and these cause the vessel to close when it constricts in the postnatal period. The formation of intimal cushions suggests highly specialized functions of DA endothelial and smooth muscle cells. To investigate these properties, we established, from fetal lambs on Day 138 of a 148-day term gestation, primary cell cultures of DA endothelium and smooth muscle and compared them to cells derived from the adjacent pulmonary artery and aorta. Purity of the endothelial cell cultures from each vascular site was assessed by the contact inhibited “cobblestone” monolayer phenotype, by positive immunofluorescence for factor VIII and by angiotensin converting enzyme activity. Purity of smooth muscle cell cultures at each vascular site was assessed by the “hills and valleys” phenotype and by positive immunofluorescence with a smooth muscle actin specific monoclonal antibody. Endothelial and smooth muscle cells had different growth curves, ultrastructural features, and protein profiles on single and two-dimensional SDS-polyacrylamide gel electrophoresis (PAGE), but vascular sites were similar. To further determine whether differences related to DA origin were indeed present, endothelial and smooth muscle cells from all three vascular sites were incubated with the radiolabeled amino acids [ 14C]leucine, [ 14C]proline, and [ 14C]valine and the proteins in both the cells and the conditioned medium were analyzed by autoradiography after SDS-PAGE. A dense band corresponding to a 42-kDa protein was observed in valine-labeled DA endothelial cells and conditioned medium and a 52-kDa protein was observed in the conditioned medium of leucine-labeled DA smooth muscle cells only. Further isolation and characterization of these endothelial and smooth muscle proteins will be necessary to determine whether they are related to the mechanism of intimal cushion formation in the late gestation DA or are present abnormally in association with the intimal proliferation observed in pulmonary and systemic vascular disease.