Coarctation of the aorta: nonsurgical treatment using stent implantation.

INTRODUCTION: Coarctation of the aorta (CoA) accounts for 5%-8% of all congenital heart defects. If left untreated, most patients with significant CoA will have varying degrees of morbidity (e.g. hypertension, stroke, collateral formation and ventricular hypertrophy), possibly even mortality. Traditionally, treatment for this condition is surgical. Herein, we report stenting during catheterisation as an alternative nonsurgical treatment option for patients with CoA, and present the treatment outcomes of patients who underwent this treatment option. METHODS: We retrospectively reviewed four patients (2 men and 2 women; age range 20-41 years) who underwent CoA stenting under general anaesthesia for the treatment of native CoA or restenosis of CoA at our institution. Three patients had a 40-mm Palmaz stent inserted, while one had a 39-mm Cheatham-Platinum covered stent inserted. Angiography and measurement of pressure gradients were performed before and after stent implantation to ensure good treatment outcomes. RESULTS: The patients' treatment outcomes were good, with a significant reduction in pressure gradients across the narrowed segments. Angiography showed relief of CoA. The patients were followed up for 1-3 years, during which no complications were noted. CONCLUSION: This is the first reported series in Singapore on the nonsurgical treatment of CoAs in adult patients using stents during interventional cardiac catheterisation. This less invasive procedure may lead to a new paradigm shift with regard to the treatment of CoA.

Retinoic acid biosynthetic enzyme ALDH1 localizes in a subset of retinoid-dependent tissues during xenopus development.

Control of retinoic acid synthesis in vertebrate organisms is undoubtedly important for regulating the numerous retinoid signaling events which occur during development. The mechanisms which accomplish this task involve enzymes such as class I aldehyde dehydrogenase (ALDH1), which has recently been found to be conserved from amphibians to mammals and which functions as a retinoic acid biosynthetic enzyme in vivo. Here we have found that Xenopus ALDH1 mRNA and protein is expressed in a subset of retinoid-dependent tissues which develop shortly after neurulation during the tail bud stages. ALDH1 mRNA was first clearly detectable by in situ hybridization in stage 28 tail bud embryos localized in the olfactory placode and pronephros, and at stage 35 mRNA was also detected in the pronephric duct. Antibodies were generated against Xenopus ALDH1, and immunohistochemistry was used to demonstrate that ALDH1 protein accumulates in the olfactory placode, pronephros, and dorsal retina at stage 28, and additionally in the lens placode and pronephric duct at stage 35. Neither ALDH1 mRNA nor protein was detected in the posterior region of Xenopus embryos during the tail bud stages. In contrast to neurula stage embryos in which retinoic acid is distributed in an anteroposterior gradient with the high end posteriorly, we found that tail bud stage embryos have retinoic acid present in significant levels in both the head and trunk regions, but with no detection in the posterior region. These findings are consistent with ALDH1 contributing to retinoic acid synthesis needed for development of certain head structures (olfactory placodes, dorsal retina, lens placode) and certain trunk structures (pronephros and pronephric duct). Dev Dyn 1999;215:264-272.

Stimulation of premature retinoic acid synthesis in Xenopus embryos following premature expression of aldehyde dehydrogenase ALDH1.

In order for nuclear retinoic acid receptors to mediate retinoid signaling, the ligand retinoic acid must first be produced from its vitamin A precursor retinal. Biochemical studies have shown that retinal can be metabolized in vitro to retinoic acid by members of the aldehyde dehydrogenase enzyme family, including ALDH1. Here we describe the first direct evidence that ALDH1 plays a physiological role in retinoic acid synthesis by analysis of retinoid signaling in Xenopus embryos, which have plentiful stores of maternally derived retinal. The Xenopus ALDH1 gene was cloned and shown to be highly conserved with chick and mammalian homologs. Xenopus ALDH1 was not expressed at blastula and gastrula stages, but was expressed at the neurula stage. We used a retinoic acid bioassay to demonstrate that retinoic acid is normally undetectable in embryos from fertilization to the initial gastrula stage, but that a tremendous increase in retinoic acid occurs during neurulation when ALDH1 is first expressed. Overexpression of ALDH1 by injection of Xenopus embryos with mRNAs encoding the mouse, chick or Xenopus ALDH1 homologs induced high levels of retinoic acid detection during the blastula stage. Thus, premature expression of ALDH1 stimulates premature synthesis of retinoic acid. These findings reveal an important conserved role for ALDH1 in retinoic acid synthesis in vivo, and demonstrate that conversion of retinoids from the aldehyde form to the carboxylic acid form is a crucial regulatory step in retinoid signaling.

Alcohol dehydrogenases in Xenopus development: conserved expression of ADH1 and ADH4 in epithelial retinoid target tissues.

Mammalian alcohol dehydrogenases ADH1 (class I ADH) and ADH4 (class IV ADH) function as retinol dehydrogenases contributing to the synthesis of retinoic acid, the active form of vitamin A involved in growth and development. Xenopus laevis ADH1 and ADH4 genes were isolated using polymerase chain reaction primers corresponding to conserved motifs of vertebrate ADHs. The predicted amino acid sequence of Xenopus ADH1 was clearly found to be an ortholog of ADH1 from the related amphibian Rana perezi. Phylogenetic tree analysis of the Xenopus ADH4 sequence suggested this enzyme is likely to be an ADH4 ortholog, and this classification was more confidently made when based also on the unique expression patterns of Xenopus ADH1 and ADH4 in several retinoid-responsive epithelial tissues. Northern blot analysis of Xenopus adult tissues indicated nonoverlapping patterns of ADH expression, with ADH1 mRNA found in small intestine, large intestine, liver, and mesonephros and ADH4 mRNA found in esophagus, stomach, and skin. These nonoverlapping tissue-specific patterns are identical to those previously observed for mouse ADH1 and ADH4, thus providing further evidence that Xenopus ADH1 and ADH4 are orthologs of mouse ADH1 and ADH4, respectively. During Xenopus embryonic development ADH1 mRNA was first detectable by Northern blot analysis at stage 35, whereas ADH4 mRNA was undetectable through stage 47. Whole-mount in situ hybridization indicated that ADH1 expression was first localized in the pronephros during Xenopus embryogenesis, thus conserved with mouse embryonic ADH1 which is first expressed in the mesonephros. ADH4 expression was not detected in Xenopus embryos by whole-mount in situ hybridization but was localized to the gastric mucosa of the adult stomach, a property shared by mouse ADH4. Conserved expression of ADH1 and ADH4 in retinoid-responsive epithelial tissues of amphibians and mammals argue that these enzymes may perform essential retinoid signaling functions during development of the pronephros, mesonephros, liver, and lower digestive tract in the case of ADH1 and in the skin and upper digestive tract in the case of ADH4.

Retinoic acid and alcohol/retinol dehydrogenase in the mouse adrenal gland: a potential endocrine source of retinoic acid during development.

Retinoid signaling requires the conversion of retinol to retinoic acid by a two-step process, the first of which can be catalyzed in vitro by class I and class IV alcohol dehydrogenases (ADH). These enzymes may participate in local retinoic acid synthesis in some target tissues, although other studies suggest retinoic acid may also be supplied to tissues via the bloodstream, much like an endocrine hormone. Here we have analyzed the expression of these two ADHs as well as retinoic acid production in the adrenal gland, an organ known to be an endocrine source of other hormones. In situ hybridization revealed high levels of both class I and class IV ADH messenger RNAs in adrenal glands of 16.5-day mouse embryos and adults. Class I ADH protein was immunohistochemically detected in embryonic and adult adrenal glands, the latter primarily in the zona fasiculata of the cortex. Abundant class IV ADH protein was detected in the embryonic adrenal as well as in the zona glomerulosa and zona fasiculata of the adult adrenal cortex. Interestingly, class IV ADH protein was found in only a subset of adult cortical cells arranged in radial columns, thus providing further evidence for centripetal cell migration during adrenocortical differentiation. Using a retinoic acid bioassay, adrenal glands from 16.5 day embryos were found to have significantly higher levels of retinoic acid than embryonic liver. The adult adrenal was found to have approximately 15.5 pmol/g of retinoic acid, whereas the adult liver had 24.8 pmol/g, and brain, heart, and spleen each had less than 1.0 pmol/g. Because previous findings indicate that the adrenal gland is not a retinoid target tissue, our detection of both alcohol/retinol dehydrogenases and significant amounts of retinoic acid in this organ suggests that it functions as a potential endocrine source of this hormone during mouse development.

Class IV alcohol/retinol dehydrogenase localization in epidermal basal layer: potential site of retinoic acid synthesis during skin development.

Vitamin A (retinol) plays a signaling role in the development of skin and other epithelial tissues. This is accomplished by a two-step metabolic pathway in which the rate-limiting step is oxidation of retinol to retinal, followed by oxidation of retinal to retinoic acid, which serves as the active ligand to activate nuclear retinoic acid receptors. Previous studies in mouse skin have shown that retinol oxidation is catalyzed by a cytosolic retinol dehydrogenase that may be a member of the alcohol dehydrogenase (ADH) enzyme family. Analysis of the ADH family has shown that class IV ADH is the most efficient isozyme for retinol oxidation but that other isozymes can catalyze this reaction. Here we have examined mouse skin for the expression of genes encoding class I ADH and class IV ADH, the only ADH isozymes in this species able to function as retinol dehydrogenases in vitro. In situ hybridization analysis of mouse skin revealed that class I ADH mRNA was absent, whereas class IV ADH mRNA was abundant and localized in the epidermal basal layer, providing evidence that the skin retinol dehydrogenase previously identified was class IV ADH. Immunohistochemical studies indicated that class I ADH protein was absent in the mouse skin, but class IV ADH protein was detected primarily in the basal layer of the epidermis, with less detection in the spinous layer and no detection in the cornified layer. This apparent down-regulation of class IV ADH expression during keratinocyte terminal differentiation provides evidence that the basal layer of the epidermis may be the primary site of local retinoic acid synthesis needed for retinoid signaling in the skin.

Initiation of retinoid signaling in primitive streak mouse embryos: spatiotemporal expression patterns of receptors and metabolic enzymes for ligand synthesis.

The requirement of vitamin A (retinol) for successful completion of vertebrate embryogenesis is well established. Retinoid signaling involves a two-step metabolic event in which retinol is first converted to retinal, and then retinal is converted to the active ligand retinoic acid, which modulates the transcriptional activity of a nuclear retinoic acid receptor (RAR). During mouse embryogenesis, retinoic acid is not detected at 6.5 days of embryonic development (E6.5) when gastrulation first initiates, but it is detected at E7.5 and later. This suggests that retinoid signaling during embryogenesis may be initiated during the primitive streak stage. Here we have used whole-mount in situ hybridization to examine E6.5-E8.5 mouse embryos for expression of RAR alpha, RAR beta, RAR gamma, and two enzymes, class IV alcohol dehydrogenase (ADH-IV) and class I aldehyde dehydrogenase (ALDH-I), which have been shown to have retinol and retinal dehydrogenase activities, respectively. At E6.5, RAR alpha mRNA was expressed ubiquitously in embryonic and extraembryonic tissues, RAR gamma mRNA was detected throughout all embryonic tissues, but mRNAs for RAR beta, ADH-IV, and ALDH-I were not detected. By E7.5, RAR alpha mRNA was still ubiquitous, RAR beta mRNA was now observed in presumptive hindbrain ectoderm and adjacent mesenchyme, RAR gamma mRNA was still observed in all embryonic tissues, and ADH-IV as well as ALDH-I mRNAs were now both expressed in primitive streak mesoderm. In E8.5 embryos, RAR alpha mRNA was still ubiquitous, RAR beta mRNA was present in the caudal hindbrain as well as the closed neural tube and foregut, RAR gamma mRNA was widespread but most prevalent in caudal embryonic tissues, and mRNAs for both ADH-IV and ALDH-I were expressed in cranial mesenchyme, somites, and paraxial mesoderm. Thus, ADH-IV and ALDH-1, two metabolic enzymes able to convert retinol to retinoic acid, are both initially expressed in primitive streak mesoderm at E7.5 when retinoic acid is first detectable. On the other hand, RAR alpha and RAR gamma expression is widespread and present at E6.5 prior to retinoic acid detection. These results suggest that upregulation of ADH-IV and ALDH-I gene expression in primitive streak mesoderm may lead to retinoic acid synthesis and initiation of retinoid signaling during mouse embryogenesis.

Localization of class I and class IV alcohol dehydrogenases in mouse testis and epididymis: potential retinol dehydrogenases for endogenous retinoic acid synthesis.

The vitamin A metabolite retinoic acid plays an essential signaling role in spermatogenesis by acting as a ligand for nuclear retinoic acid receptors. However, little is known about the regulation of retinoic acid synthesis from vitamin A (retinol). Here we have examined mouse testis and epididymis for the presence of endogenous retinoic acid and for the expression of genes encoding class I and class IV alcohol dehydrogenases (ADH), both of which catalyze retinol oxidation, the rate-limiting step in the conversion of retinol to retinoic acid. Using a bioassay we found that mouse testis and epididymis both have significant levels of retinoic acid ranging from 7 to 8 pmol/g, an amount known to be sufficient to optimally activate retinoic acid receptors. In situ hybridization analysis of mouse testis revealed that class I ADH mRNA was localized in Sertoli cells and Leydig cells, while class IV ADH mRNA was confined to late spermatids. In the epididymis, class I ADH mRNA was detected in both principal and basal cells, whereas class IV ADH mRNA was limited to basal cells. Immunohistochemical analyses of testis indicated that class I ADH protein was localized in Sertoli and Leydig cells, whereas class IV ADH protein was observed only in late spermatids. Class I ADH protein was localized in principal and basal cells of the cauda epididymidis but only in basal cells of the caput epididymidis. Class IV ADH protein was limited to basal cells along the entire length of the epididymis. These results support a role for ADHs during spermatogenesis, potentially as retinol dehydrogenases catalyzing local retinoic acid synthesis in the testis and epididymis.

Expression patterns of class I and class IV alcohol dehydrogenase genes in developing epithelia suggest a role for alcohol dehydrogenase in local retinoic acid synthesis.

Vitamin A (retinol) regulates embryonic development and adult epithelial function via metabolism to retinoic acid, a pleiotrophic regulator of gene expression. Retinoic acid is synthesized locally and functions in an autocrine or paracrine fashion, but the enzymes involved remain obscure. Alcohol dehydrogenase (ADH) isozymes capable of metabolizing retinol include class I and class IV ADHs, with class III ADH unable to perform this function. ADHs also metabolize ethanol, and high levels of ethanol inhibit retinol metabolism, suggesting a possible mode of action for some of the medical complications of alcoholism. To explore whether any ADH isozymes are linked to retinoic acid synthesis, herein we have examined the expression patterns of all known classes of ADH in mouse embryonic and adult tissues, and also measured retinoic acid levels. Using in situ hybridization, class I ADH mRNA was localized in the embryo to the epithelia of the genitourinary tract, intestinal tract, adrenal gland, liver, conjunctival sac, epidermis, nasal epithelium, and lung, plus in the adult to epithelia within the testis, epididymis, uterus, kidney, intestine, adrenal cortex, and liver. Class IV ADH mRNA was localized in the embryo to the adrenal gland and nasal epithelium, plus in the adult to the epithelia of the esophagus, stomach, testis, epididymis, epidermis, and adrenal cortex. Class III ADH mRNA, in contrast, was present at low levels and not highly localized in the embryonic and adult tissues examined. We detected significant retinoic acid levels in the fetal kidney, fetal/adult intestine and adrenal gland, as well as the adult liver, lung, testis, epididymis, and uterus--all sites of class I and/or class IV ADH gene expression. These findings indicate that the expression patterns of class I ADH and class IV ADH, but not class III ADH, are consistent with a function in local retinoic acid synthesis needed for the development and maintenance of many specialized epithelial tissues.

Ethanol inhibition of retinoic acid synthesis as a potential mechanism for fetal alcohol syndrome.

Retinoic acid (RA) is known to act as a signaling molecule during embryonic development, but little is known about the regulation of RA synthesis from retinol. The rate-limiting step in RA synthesis is the oxidation of retinol, a reaction that can be catalyzed by alcohol dehydrogenase (ADH). Ethanol is also a substrate for ADH, and high levels of ethanol inhibit ADH-catalyzed retinol oxidation. This has prompted us to hypothesize that ethanol-induced defects observed in fetal alcohol syndrome involve ethanol inhibition of ADH-catalyzed RA synthesis. Here, we have examined the effect of ethanol on RA levels in cultured mouse embryos by using a bioassay. Treatment with 100 mM ethanol, but no 10 mM, led to a significant decrease in RA detection in 7.5-day-old embryos. Using whole-mount in situ hybridization, we detected mRNA for class IV ADH, but not ethanol-active cytochrome P450 2E1, in 7.5- and 8.5-day-old embryos, indicating that an ADH-linked pathway exists at these stages for metabolizing retinol and ethanol. Thus, the observed ethanol-induced reduction in RA may be caused by ethanol inhibition of retinol oxidation catalyzed by class IV ADH. In our postulated mechanism for fetal alcohol syndrome, this enzyme may well play a crucial role.

Retinoic acid synthesis in mouse embryos during gastrulation and craniofacial development linked to class IV alcohol dehydrogenase gene expression.

Endogenous retinoic acid (RA) has been observed in vertebrate embryos as early as gastrulation, but the mechanism controlling spatiotemporal synthesis of this important regulatory molecule remains unknown. Some members of the alcohol dehydrogenase (ADH) family catalyze retinol oxidation, the rate-limiting step in RA synthesis. Here we have examined mouse embryos for the presence of endogenous RA and expression of ADH genes. RA was not detected in egg cylinder stage embryos but was detected in late primitive streak stage embryos. Detection of class IV ADH mRNA, but not class I or class III, coincided with the onset of RA synthesis, being absent in egg cylinder embryos but present in the posterior mesoderm of late primitive streak embryos. During neurulation, RA and class IV ADH mRNA were colocalized in the craniofacial region, trunk, and forelimb bud. Class IV ADH mRNA was detected in cranial neural crest cells and craniofacial mesenchyme as well as trunk and forelimb bud mesenchyme. The spatiotemporal expression pattern and enzymatic properties of class IV ADH are thus consistent with a crucial function in RA synthesis during embryogenesis. In addition, the finding of endogenous RA and class IV ADH mRNA in the craniofacial region has implications for the mechanism of fetal alcohol syndrome.

Bovine oxytocin transgenes in mice. Hypothalamic expression, physiological regulation, and interactions with the vasopressin gene.

To gain insights into the molecular mechanisms that restrict the expression of the oxytocin gene to anatomically defined groups of neurons in the hypothalamus, we generated transgenic mice bearing bovine oxytocin genomic fragments. Appropriate neuron-specific and physiological regulation was observed in mice bearing transgene bOT3.5, which consists of the oxytocin structural gene flanked by 0.6 kilobase pair (kbp) of upstream and 1.9 kbp of downstream sequences. bOT3.5 is expressed in oxytocin magnocellular neurons in the mouse supraoptic nucleus and paraventricular nucleus, but transgene RNAs are excluded from vasopressin neurons. Replacement of the drinking diet of the transgenic mice with 2% (w/v) NaCl for 7 days significantly increased the abundance of bovine oxytocin transcripts in the supraoptic nucleus, but not in the paraventricular nucleus, in parallel with the endogenous mouse oxytocin RNA. Surprisingly, mimicry of the endogenous oxytocin gene expression pattern was lost with larger transgenes. Addition of 0.7 kbp of contiguous downstream sequences (transgene bOT) or linkage to the bovine vasopressin gene (transgene VP-B/bOT3.5) repressed hypothalamic expression. No mice were derived bearing transgene bOT6.4, which consists of the oxytocin structural gene flanked by 3 kbp of upstream and 2.6 kbp of downstream sequences, suggesting that the presence of this DNA is detrimental to normal embryonic development. These data suggest that while bOT3.5 contains sufficient cis-acting sequences to mediate expression to particular subsets of hypothalamic neurons, the overall regulation of the oxytocin gene is governed by multiple interacting enhancers and repressors.

Cloning of the mouse class IV alcohol dehydrogenase (retinol dehydrogenase) cDNA and tissue-specific expression patterns of the murine ADH gene family.

Humans possess five classes of alcohol dehydrogenase (ADH), including forms able to oxidize ethanol or formaldehyde as part of a defense mechanism, as well as forms acting as retinol dehydrogenases in the synthesis of the regulatory ligand retinoic acid. However, the mouse has previously been shown to possess only three forms of ADH. Hybridization analysis of mouse genomic DNA using cDNA probes specific for each of the five classes of human ADH has now indicated that mouse DNA cross-hybridizes to only classes I, III, and IV. With human class II or class V ADH cDNA probes, hybridization to mouse genomic DNA was very weak or undetectable, suggesting either a lack of these genes in the mouse or a high degree of mutational divergence relative to the human genes. cDNAs for murine ADH classes I and III have previously been cloned, and we now report the cloning of a full-length mouse class IV ADH cDNA. In Northern blot analyses, mouse class IV ADH mRNA was abundant in the stomach, eye, skin, and ovary, thus correlating with the expression pattern for the mouse Adh-3 gene previously determined by enzyme analysis. In situ hybridization studies on mouse stomach indicated that class IV ADH transcripts were abundant in the mucosal epithelium but absent from the muscular layer. Comparison of the expression patterns for all three mouse ADH genes indicated that class III was expressed ubiquitously, whereas classes I and IV were differentially expressed in an overlapping set of tissues that all contain a large component of epithelial cells. This expression pattern is consistent with the ability of classes I and IV to oxidize retinol for the synthesis of retinoic acid known to regulate epithelial cell differentiation. The results presented here indicate that the mouse has a simpler ADH gene family than the human but has conserved class IV ADH previously shown to be a very active retinol dehydrogenase in humans.

Over-expression of oxytocin in the testes of a transgenic mouse model.

The bovine oxytocin gene has been expressed in the testes of two independent transgenic mouse lines. Hybridization and RNase protection analysis showed that the oxytocin transgene was transcribed from the normal functional promoter in the Sertoli cells of the seminiferous tubules in a developmentally regulated manner. Immunohistochemistry indicated that both oxytocin and neurophysin epitopes were expressed together in the Sertoli cells at stages I-V and X-XII of the cycle of the seminiferous epithelium. Furthermore, analysis with high-performance liquid chromatography showed that there was a tenfold increase in the amount of amidated oxytocin present in testicular extracts from the transgenic mice. However, there appeared to be no detectable effect of this overproduction of hormone on testicular morphology or fertility parameters. A significant decrease by 50% was detected only in the levels of intratesticular testosterone and dihydrotestosterone. The results point to a local paracrine role for oxytocin in the modulation of Leydig cell function.

Neuron-specific expression and physiological regulation of bovine vasopressin transgenes in mice.

We have used transgenic mice to analyse the regulation of the bovine vasopressin (BVP) gene. We find that the restriction of BVP gene expression to anatomically and functionally distinct hypothalamic neuronal groups is achieved, in part, by selective repression. The expression of a 1.25 kb BVP proximal promoter, which on its own confers general expression of a reporter to most peripheral and brain tissues, was limited by sequences in the BVP structural gene to neural cells in the adrenal medulla and brain. Transgene expression in the hypothalamus was shown to be regulated by the physiological stimulus of dehydration in parallel with the endogenous gene. The expression of a larger 13.4 kb BVP transgene, containing 9 kb of 5' upstream sequence, the VP structural gene and 1.5 kb 3' of the transcription unit, was even more restricted and resembles that of the endogenous mouse gene. Hypothalamic expression of the 13.4 kb BVP transgene was regulated appropriately in response to an osmotic challenge.

Neuropeptide gene expression in transgenic animals.

Transgenic animal techniques offer today's neuroscientist the ability to experimentally manipulate neurosecretory systems with a precision undreamt of by our predecessors. The range of techniques now available, building as it does on our growing knowledge of physiological systems at the inter- and intercellular level, allows us to critically define molecular lesions and ask about their consequences to the whole organism. Neuroscientist should grasp the opportunities afforded by these recent developments.