Human Fetal Heart Research Articles

MicroRNA microarray expression profiling in human myocardial infarction.

MicroRNAs (miRNAs), small non-coding RNA molecules, are negative regulators of gene expression. Recent studies have indicated their role in various forms of cardiovascular disease. In spite of the number of miRNA microarray analyses performed, little is known about the genome-wide miRNA expression pattern in human myocardial infarction (MI). Using miRNA microarrays and bioinformatic analysis, miRNA expression was analyzed on human MI and foetal hearts compared to healthy adult hearts, to determine whether there is any similar expression pattern between MI and foetal hearts, and to identified miRNAs that have not previously been described as dysregulated in cardiovascular diseases. Of 719 miRNAs analyzed, ∼ 50% were expressed in human hearts, 77 miRNAs were absent from all tested tissues and 57 were confidently dysregulated in at least one tested group. Some expression patterns appeared to be similar in MI and foetal hearts. Bioinformatic analysis revealed 10 miRNAs as dysregulated in MI not yet related to cardiovascular disease, and 5 miRNAs previously described only in animal models of cardiovascular diseases. Finally, qRT-PCR analysis confirmed dysregulation of 7 miRNAs, miR-150, miR-186, miR-210, miR-451, and muscle-specific, miR-1 and miR-133a/b; all of these are believed to be involved in various physiological and pathological processes.

MORPHOMETRIC STUDY OF THE VENTRICULAR SEGMENT OF THE HUMAN FETAL HEART BETWEEN 13 AND 20 WEEKS' GESTATION

Our objective was to determine the normal dimensions of the ventricular segment of the human fetal heart between 13 and 20 weeks' gestation. Study Design: 103 hearts obtained by necropsy were dissected and measurements of different portions of ventricles were determined under stereoscopic magnification. In each ventricle were measured anteroposterior and lateral diameters, inlet and outlet length, and thickness of walls at different levels. Our results showed the cardiac apex was constituted by the left ventricle in 68.9% of the hearts. Both ventricles showed linear growth during this period of fetal development. Ranges in median values of external and internal ventricular measurements were determined. The left ventricular wall was thicker than the right, and the right ventricular cavity was larger. This study provides morphometric reference information concerning the dimensions and growth of both ventricles of the fetal heart, which may be useful in pediatric cardiac surgery and echocardiography.

Human connexin31.9, unlike its orthologous protein connexin30.2 in the mouse, is not detectable in the human cardiac conduction system

In the human heart connexin(Cx)40, Cx43 and Cx45-containing gap junctional channels electrically couple cardiomyocytes, forming a functional syncytium. In the mouse heart, additionally, Cx30.2-containing gap junctions have been detected in the atrioventricular node where they are implicated, together with Cx45, in impulse delay. However, whether the human ortholog of Cx30.2, Cx31.9, is expressed in the human heart has not previously been investigated. We therefore generated Cx31.9 specific antibodies to test for the expression of Cx31.9 in the human heart. These antibodies recognized the Cx31.9 protein in HeLaCx31.9 transfectants by immunofluorescence and immunoblot analyses. However, we did not find punctate Cx31.9 specific immunofluorescence signals in the working myocardium or in the impulse generation and conduction system of adult or fetal human heart. Complementary immunoblot analyses did not reveal Cx31.9 protein in the adult atrial or ventricular myocardium. We conclude that the Cx31.9 protein, unlike its counterpart in the mouse, is not expressed in detectable quantities and is thus unlikely to contribute to the impulse generation and conduction system or the working myocardium of the human heart.

Abstract 1456: Microrna-499 Regulates (cardio)myocyte Differentiation in Human-Derived Cardiomyocyte Progenitor Cells

To improve regeneration of the injured myocardium, it is necessary to enhance the intrinsic capacity of the heart to regenerate itself and/or replace the damaged tissue by cell transplantation. One of the potential sources for cell replacement therapy are heart-derived progenitor cells. Cardiomyocyte progenitor cells (CMPCs) are obtained from human fetal hearts and adult human biopsies, easily expanded in culture, and efficiently differentiated into beating cardiomyocytes. Recently, several studies have demonstrated that microRNAs (miRNAs) are important for transcriptional regulation during angiogenesis, heart development, and maintenance of stem cell populations by translational repression. We hypothesize that, since miRNAs regulate stem cell maintenance and differentiation, miRNAs are involved in differentiation of human cardiomyocyte progenitor cells in vitro . Methods and results: Human fetal CMPCs were isolated, cultured, and efficiently differentiated into beating cardiomyocytes. miRNA expression was profiled in proliferating and differentiated CMPCs. We observed that miRNA-499 was highly upregulated upon differentiation (>100 fold, microarray and qRT-PCR). Since tissue distribution of miRNA-499 was unknown, several mouse tissues were analyzed. qRT-PCR demonstrated that miRNA-499 is highly enriched in cardiac tissue. This was confirmed via in situ-hybridization, miRNA-499 was localized in cardiomyocytes in human fetal and adult hearts. Transient transfection of miRNA- 499 in undifferentiated CMPCs reduced their proliferation rate by 15% (MTT assay and FACS analysis). Moreover, upon miR-499 transfection spontaneous beating was observed earlier and expression levels of cardiac troponin T, alpha-cardiac actinin, and Mlc-2v was increased. This enhanced cardiomyocyte differentiation occurred via repression of Sox6. Conclusion: Our results demonstrate a strong regulatory role of miRNAs in human CMPC proliferation and differentiation into cardiomyocytes. By modulating miRNA-499 expression levels, the differentiation of CMPCs is affected, resulting in enhanced cardiomyogenesis.

Abstract 1451: Hyperpolarization Induces Differentiation Of Human Cardiomyocyte Progenitor Cells

Background: Recently, we have isolated cardiomyocyte progenitor cells (hCMPCs) from human fetal and adult hearts. These cells differentiate into spontaneously beating cardiomyocytes when stimulated with 5-azacytidine. Subsequent stimulation by TGFβ enhances differentiation efficiency to nearly 100%. The underlying molecular mechanisms mediating this cardiomyogenic differentiation are not understood. In skeletal myoblasts, hyperpolarization-mediated activation of calcineurin signaling is crucial for myogenic differentiation. In hCMPCs, whole-cell patch clamp recordings showed a hyperpolarized membrane potential after stimulation with TGFβ or BMP. We hypothesized that hyperpolarization and calcineurin signaling regulate cardiomyogenic differentiation of hCMPCs after TGFβ stimulation. Methods & Results: To test whether hyperpolarization initiates cardiomyogenic differentiation, hyperpolarization was induced by 1) co-culture of hCMPCs with HEK 293 cells overexpressing a Kir2.1GFP fusion protein (KWGF cells) or 2) culture of hCMPCs overnight in medium containing low potassium concentrations. During co-culture, Lucifer Yellow dye injection in KWGF cells spread to neighboring hCMPCs, indicating cellular coupling. This resulted in stable hyperpolarization in hCMPCs, which could be blocked by addition of the gap junction inhibitor halothane. After two weeks, qPCR analysis revealed increased expression of cardiac sarcomeric genes in the hCMPCs in a dose-dependent manner. Induction of hyperpolarization by culturing hCMPCs with low potassium concentrations also resulted in increased expression of cardiac genes and the formation of spontaneously beating cells. Immunofluorescence staining revealed striated patterns of troponin I and α-actinin. Interestingly, hyperpolarization also increased intracellular calcium levels in hCMPCs, as measured by ratiometric imaging of indo-1 fluorescence, and, subsequently, a time-dependent increase in NFAT-Luciferase reporter activity, indicating activation of the calcineurin pathway. Conclusion: TGFβ and/or BMP-mediated hyperpolarization of hCMPCs induces calcineurin-mediated cardiomyogenic differentiation.

Characterization of Recombinant Human Prolyl 3-Hydroxylase Isoenzyme 2, an Enzyme Modifying the Basement Membrane Collagen IV

The single 3-hydroxyproline residue in the collagen I polypeptides is essential for proper fibril formation and bone development as its deficiency leads to recessive osteogenesis imperfecta. The vertebrate prolyl 3-hydroxylase (P3H) family consists of three members, P3H1 being responsible for the hydroxylation of collagen I. We expressed human P3H2 as an active recombinant protein in insect cells. Most of the recombinant polypeptide was insoluble, but small amounts were also present in the soluble fraction. P3H1 forms a complex with the cartilage-associated protein (CRTAP) that is required for prolyl 3-hydroxylation of fibrillar collagens. However, coexpression with CRTAP did not enhance the solubility or activity of the recombinant P3H2. A novel assay for P3H activity was developed based on that used for collagen prolyl 4-hydroxylases (C-P4H) and lysyl hydroxylases (LH). A large amount of P3H activity was found in the P3H2 samples with (Gly-Pro-4Hyp)5 as a substrate. The Km and Ki values of P3H2 for 2-oxoglutarate and its certain analogues resembled those of the LHs rather than the C-P4Hs. Unlike P3H1, P3H2 was strongly expressed in tissues rich in basement membranes, such as the kidney. P3H2 hydroxylated more effectively two synthetic peptides corresponding to sequences that are hydroxylated in collagen IV than a peptide corresponding to the 3-hydroxylation site in collagen I. These findings suggest that P3H2 is responsible for the hydroxylation of collagen IV, which has the highest 3-hydroxyproline content of all collagens. It is thus possible that P3H2 mutations may lead to a disease with changes in basement membranes.

Anti-La (SS-B) but not anti-Ro52 (SS-A) antibodies cross-react with laminin--a role in the pathogenesis of congenital heart block?

Cross-reactions between maternally derived autoantibodies and fetal cardiac antigens have been postulated to play a role in the pathogenesis of congenital heart block (CHB). We have explored the cross-reactivity of autoantibodies to the small ribonuclear autoantigens, La/SS-B and Ro/SS-A, with laminin, the major component of cardiac sarcolemmal membrane using affinity-purified antibodies from patients with Sjögren's syndrome (SS). Anti-La antibodies purified from eight of 10 patients cross-reacted significantly with mouse laminin by ELISA. In contrast, purified antibodies to Ro52 from the same 10 patients showed little or no binding to laminin. Laminin inhibited up to 70% binding of anti-La antibodies to La antigen, and La inhibited up to 65% binding of anti-La antibodies to laminin. The cross-reaction was further examined on cryosections of 10 human fetal hearts aged from 8.7 to 14.9 weeks of gestation, two normal adult hearts, and one pathological adult heart with a diagnosis of dilated cardiomyopathy. Anti-Ro52 antibodies did not bind to the surface of cardiac cells. However, anti-La antibodies from seven of 10 patients tested bound to the surface of fetal myocytes from hearts aged 9.4 to 14.9 weeks of gestation, and also to the myocytes from the pathological adult heart but not to normal adult hearts. Preincubation with La antigen abolished the binding of anti-La antibodies to the surface of adult heart myocytes with dilated cardiomyopathy, and pre-incubation with mouse laminin could partially block this binding. These results suggest that molecular mimicry between laminin and La, but not Ro52, may act as a target for specific maternal autoantibodies, and contribute to the pathogenesis of CHB at a critical stage during fetal cardiac development.

Progenitor cells isolated from the human heart: a potential cell source for regenerative therapy.

In recent years, resident cardiac progenitor cells have been identified in, and isolated from the rodent heart. These cells show the potential to form cardiomyocytes, smooth muscle cells, and endothelial cells in vitro and in vivo and could potentially be used as a source for cardiac repair. However, previously described cardiac progenitor cell populations show immature development and need co-culture with neonatal rat cardiomyocytes in order to differentiate in vitro. Here we describe the localisation, isolation, characterisation, and differentiation of cardiomyocyte progenitor cells (CMPCs) isolated from the human heart. hCMPCs were identified in human hearts based on Sca-1 expression. These cells were isolated, and FACS, RT-PCR and immunocytochemistry were used to determine their baseline characteristics. Cardiomyogenic differentiation was induced by stimulation with 5-azacytidine. hCMPCs were localised within the atria, atrioventricular region, and epicardial layer of the foetal and adult human heart. In vitro, hCMPCs could be induced to differentiate into cardiomyocytes and formed spontaneously beating aggregates, without the need for co-culture with neonatal cardiomyocytes. The human heart harbours a pool of resident cardiomyocyte progenitor cells, which can be expanded and differentiated in vitro. These cells may provide a suitable source for cardiac regeneration cell therapy. (Neth Heart J 2008;16:163-9.).

Accessory Atrioventricular Pathways

By definition, accessory atrioventricular pathways are aberrant muscle bundles that connect the atrium to a ventricle outside of the regular atrioventricular conduction system. Clinically, they may manifest as substrates for ventricular preexcitation. The first accessory pathway in a patient who suffered from Wolff-Parkinson-White syndrome was described in 1943 by Wood, Wolferth, and Geckler.1 Shortly after, Ohnell created a reconstruction of an accessory pathway that very elegantly showed the close proximity of the pathway to the fibrous attachment of the mitral valve and its relationship with the sulcus coronarius (Figure, A).2 Subsequent histological studies have demonstrated unequivocally that these pathways are the anatomic substrates for the classical Wolff-Parkinson-White variety of preexcitation. Figure. A, Ohnell’s depiction of an accessory bundle in the left atrioventricular groove.2 Reprinted with permission from Blackwell Publishing. B, Histological section stained with Masson’s trichrome (fibrous tissue in green and myocardium in red) to show an accessory left-sided pathway (arrow) skirting the mitral annulus. C, Histological section showing a broad right-sided accessory pathway (arrow) that is formed by a myocardial pouch extending from the right atrial appendage to the ventricle and ultimately traced to a small right ventricular vein. This pathway is some distance from the fibrous annulus of the tricuspid valve. *Valvar annulus. Article p 1508 Accessory atrioventricular pathways are found most often in the parietal atrioventricular junctional areas, including the paraseptal areas. They breach the insulation provided by the fibrofatty tissues of the atrioventricular groove (sulcus tissue) and the hingelines (fibrous annulus) of the valves. They are rarely found in the area of fibrous continuity between the aortic and mitral valves because in this area, there is usually a wide gap between the atrial myocardium and ventricular myocardium to accommodate the aortic outflow tract. On the left parietal side, the accessory pathways tend …

Magnetocardiography for fetal arrhythmias

The failing adult heart is known to revert to fetal patterns of contractile protein expression during pathologic remodeling, yet very little is known about the electrophysiology or electromechanics of the living human fetal heart during normal prenatal development or during stress. This owes largely to the inability to easily record the fetal electrocardiogram (fECG). While fetal echocardiography provides mechanical assessment of heart rhythm, it does not provide details of the electrophysiology of the fetal heart. These details, along with the genetics of fetal and neonatal demise, are emerging as potential leading factors in defining prenatal sudden death risk. While echocardiography has been utilized extensively for 20 years in prenatal high risk screening, it has not had the desired outcome in reducing overall fetal sudden death to the extent that sudden death has been reduced in the infant, child, and adult. Late gestation unexplained fetal death costs 26,000 fetal lives yearly in the United States and over 4 million fetal lives each year worldwide. Fetal Magnetocardiography (fMCG) opens a new window in fetal healthcare assessment. Fetal cardiac deaths may be preventable because the diseases that lead to these deaths are often treatable, especially if the sophisticated modern ICU monitoring and management techniques could be translated to the field of prenatal care.

Spectral analysis of fetal heart rate variability for fetal surveillance: review of the literature

Cardiotocography has a poor diagnostic value in detecting fetal acidosis. Spectral analysis of fetal heart rate variability can be used to monitor the fetal autonomic nervous system. To determine the value of spectral analysis for fetal surveillance. A systematic search was performed in the electronic databases CENTRAL (the Cochrane Library; 2007, Issue 3), PUBMED and EMBASE up to May 2007. Articles that described spectral analysis of human fetal heart rate variability and compared the energy in spectral bands with blood-gas values obtained by funipuncture or from the umbilical cord immediately postpartum were included. Six studies met the inclusion criteria. The included studies were heterogeneous, various methods of spectral analysis and different frequency bands were used and the outcome measures varied. Five out of six studies showed a decrease in spectral energy in the low frequency (LF) band in case of fetal distress. An extremely low LF power had a sensitivity of 97.5% and a specificity of 86.1% to detect fetal distress. Spectral analysis could be a promising method for fetal surveillance. Larger prospective studies are needed to determine the exact diagnostic value of spectral analysis. For further research, standardisation of spectral analysis is recommended. Studies should focus on real time monitoring.

Human fetal heart rate variability-characteristics of autonomic regulation in the third trimester of gestation

To describe developmental aspects of the sympatho-vagal heart rate regulation in the human fetus by applying numerics to visual descriptions of fetal heart rate patterns throughout the third trimester of pregnancy. The focus is to determine potential benefits of this alternative means of assessing the maturation of the fetal autonomic nervous system by analysis of fetal heart rate variability (fHRV). The magnetocardiograms of 103 normal fetuses between 24+1 and 41+6 weeks of gestation were studied. Fetal heart beat intervals were determined with a temporal precision of 1 ms. The levels of fetal activity were estimated according to characteristic heart rate patterns (I-III) prior to 32, between 32-35 and beyond 35 (groups 1-3) completed weeks. Mean heart rate (mHR), standard deviation of normal-to-normal beat intervals (SDNN) and root mean square of successive differences of normal beats (RMSSD) served as fHRV indices, mean permutation entropy (PE_Mean) as complexity measure. SDNN/RMSSD was introduced as a potential marker for sympatho-vagal balance. Low activity fHRP (I) were characterized by significantly lower level fHRV indices and higher PE_Mean when compared to fHRP II. We found that SDNN/RMSSD decreases with gestation in fHRP I, which suggests increasing vagal influence. In fHRP III (assigned to active awake fetuses only after 32 weeks), highest level SDNN and mHR are associated with a dramatically reduced complexity. fHRV indices cluster characteristically with the activity levels. We conclude that a combined analysis of fHRV, based on SDNN/RMSSD and PE_Mean, and fHRP is advantageous in the assessment of maturation of the fetal autonomic nervous system.

Innervation of pulmonary veins: Morphologic pattern and pathways of nerves in the human fetus

The aim of the study was to determine the anatomy of intrinsic nerves supplying human pulmonary veins (PVs). Twenty-two hearts of human fetuses with full sets of PVs were examined using a histochemical method for acetylcholinesterase in order to stain transmurally intrinsic neural structures on non-sectioned PVs for subsequent stereomicroscopic examination. Findings of the study demonstrate that epicardiac nerve extensions from both the dorsal right atrial and the middle dorsal subplexuses reached the right superior as well as the right inferior PVs, whereas the left superior PV was supplied by nerve extensions from the left dorsal subplexus. The left and middle dorsal subplexuses contributed nerves to the left inferior PV. The ganglia related topographically to PVs were patchy in distribution. On the left and right superior PVs, 38+/-6 and 31+/-3 ganglia were found, respectively, whereas 46+/-7 and 38+/-7 ganglia were identified on the left and right inferior PVs. The size of ganglia was similar for all four veins, ranging in area from 0.004+/-0.0003 to 0.007+/-0.0004 mm(2). The total area of ganglia distributed on a given PV was similar, ranging from 0.15+/-0.0003 to 0.25+/-0.0004 mm(2). The present findings demonstrate that the richest ganglion sites supplying intrinsic nerves to the human PVs are located on the posterior sides of both inferior and the left superior PVs and, therefore, these sites may be considered primary targets for focal pulmonary vein ablation in catheter-based therapy of atrial fibrillation.

Historical Perspectives

When neonatology was emerging as a clinical discipline in the late 1950s and early 1960s, it had the advantage of a strong foundation of basic research in fetal and neonatal physiology. In particular, the new clinical enterprise (it was not yet a subspecialty) focused on cardiorespiratory pathophysiology. This foundation came from a few distinguished research laboratories studying fetal and neonatal physiology and the processes of the transition from fetal to neonatal life. The preeminent laboratory in the field was the Nuffield Institute for Medical Research at Oxford, directed by Geoffrey S. Dawes. Geoffrey Dawes became the director of the Institute in 1948. He received his medical degree in 1943, and in the following 5 years, he gained recognition as a prominent investigator in pharmacology and physiology. The very short interval between receiving his degree and being named director of an important research institute indicates how quickly the academic leadership recognized his exceptional qualities. As the new director, Dawes set the primary focus of research for the Nuffield Institute as developmental physiology. In this role, the Nuffield became the successor to the path of research started by Sir Joseph Barcroft at Cambridge early in the twentieth century. Dawes and his coinvestigators concentrated particularly on the preparations for and mechanisms of adaptation to extrauterine life. A central focus of the research was the adaptive mechanisms in the circulatory and respiratory systems, including placental function, establishment of lung function, and changes in the pulmonary and systemic circulations. Although most of the work examined cardiorespiratory physiology and its neurohumoral regulation, researchers also studied fetal growth and metabolism, and their studies included the responses to stresses such as hypoxia and hemorrhage. One of the hallmarks of the research of this highly productive group was the elegance of their experimental designs. The clinical implications of this …

Altered expression of mitochondrial and extracellular matrix genes in the heart of human fetuses with chromosome 21 trisomy

BackgroundThe Down syndrome phenotype has been attributed to overexpression of chromosome 21 (Hsa21) genes. However, the expression profile of Hsa21 genes in trisomic human subjects as well as their effects on genes located on different chromosomes are largely unknown. Using oligonucleotide microarrays we compared the gene expression profiles of hearts of human fetuses with and without Hsa21 trisomy.ResultsApproximately half of the 15,000 genes examined (87 of the 168 genes on Hsa21) were expressed in the heart at 18–22 weeks of gestation. Hsa21 gene expression was globally upregulated 1.5 fold in trisomic samples. However, not all genes were equally dysregulated and 25 genes were not upregulated at all. Genes located on other chromosomes were also significantly dysregulated. Functional class scoring and gene set enrichment analyses of 473 genes, differentially expressed between trisomic and non-trisomic hearts, revealed downregulation of genes encoding mitochondrial enzymes and upregulation of genes encoding extracellular matrix proteins. There were no significant differences between trisomic fetuses with and without heart defects.ConclusionWe conclude that dosage-dependent upregulation of Hsa21 genes causes dysregulation of the genes responsible for mitochondrial function and for the extracellular matrix organization in the fetal heart of trisomic subjects. These alterations might be harbingers of the heart defects associated with Hsa21 trisomy, which could be based on elusive mechanisms involving genetic variability, environmental factors and/or stochastic events.

Pluripotent stem cells exhibiting similar characteristics can be isolated from human fetal bone marrow, heart, liver, muscle, lung, derma, kidney, and fat

Previously, we reported that a cell population derived from human fetal bone marrow (BM), termed here Flk1(+)CD34(-) postembryonic pluripotent stem cells (PPSCs) that have the characteristics of mesenchymal stem cells (MSCs), could differentiate into ectodermal, endodermal and mesodermal cell types at the single cell level in vitro, and that these cells could also differentiate into the epithelium of liver, lung, gut, as well as the hematopoietic and endothelial lineages after transplantion into irradiated non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. In this study, we further isolated pluripotent stem cells from human fetal heart, liver, muscle, lung, derma, kidney, and fat and then analyzed the characteristics and function of these stem cells. It was found that the phenotype of the culture-expanded pluripotent stem cells from different fetal tissues was similar to BM-derived Flk1(+)CD34(-) PPSCs, i.e. Flk1 and CD44 positive, GlyA, CD34, CD45, class I-HLA and HLA-DR negative. Morphologically, these cells were fibroblast-like and the doubling time was about 30 h. More importantly, culture-expanded pluripotent stem cells from all these fetal tissues were able to differentiate into cells with morphologic and phenotypic characteristics of adipocytes, osteocytes, neurons, glial cells and hepatocytes. These pluripotent stem cells with characteristics similar to fetal BM-derived Flk1(+)CD34(-) PPSCs can be selected and cultured from tissues other than the BM. This phenomenon may help explain the "stem cell plasticity" found in multiple human tissues. In addition, as fetal BM-derived Flk1 + CD34(-) PPSCs, these pluripotent stem cells from different fetal tissues had the capacity for self-renewal and multi-lineage differentiation even after being expanded for more than 40 population doublings in vitro. Thus, they may be an ideal source of stem cells for treatment of inherited or degenerative diseases.

Homocysteine, hRIP3 and congenital cardiovascular malformations

Elevated serum homocysteine (Hcys) levels have been suggested to contribute to congenital cardiovascular malformations, neural tube defects, and cardiovascular diseases. To investigate the mechanisms resulting in cardiovascular diseases and birth defects, Kuang-Hueih Chen et al. identified and characterized a novel gene, named rHCY2, whose expression was markedly up-regulated when Hcys was elevated in rat. In vivo, rHCY2 gene could induce chicken embryonic cells apoptosis and embryonic malformations. Its N-terminal kinase domain is apparently similar to human receptor-interacting serine-threonine kinase 3 (hRIP3). In view of this, we hypothesize that a link between the teratogenic effects of Hcys and hRIP3 is theoretically plausible. However, given the lack of data on the topic, it remains to be seen whether an elevated serum Hcys level will increase the expression of hRIP3. Using normal and abnormal human fetal hearts and cultured normal human fetal cardiomyocytes, we show that congenital cardiovascular malformations are associated with the overexpression of hRIP3, and evidence is found for a certain association between overexpression of hRIP3 and homocysteine-induced congenital cardiovascular malformations. Folic acid and anti-hRIP3 antibodies seem to favor maintenance of the shape and ultrastructure of cultured human fetal cardiomyocytes.

CDNA cloning, characterization and expression analysis ofDTX2, a human WWE and RING-finger gene, in human embryos

The WWE domain is a conserved globular domain in several proteins and predicted to mediate specificprotein-protein interactions in ubiquitin and ADP ribose conjugation systems. The RING domain is a conserved and specialized zinc-finger motif with 40-60 residues binding to two zinc atoms, which is also probably involved in mediating protein-protein interactions. Here, from human fetal heart cDNA library, we identified DTX2, a human WWE & RING-finger gene, with high similarity with its homologues. Evaluation of full-length cDNA obtained by RACE indicated it encodes a protein composed of two WWE domains and a RING-finger region. The DTX2 gene located in human chromosome 7q11.23 spanning approximately 44.3 kb on the genome and the deduced protein is 622 amino acids. Northern analysis revealed DTX2 was expressed in the 18-week, 22.5-week human embryo hearts and adult hearts, especially with high levels in the 18-week and adult hearts. Taken together, these results indicate that DTX2 is a gene encoding a WWE-RING-finger protein and involved in regulating heart development and heart functions.

Primary and secondary transcriptional effects in the developing human Down syndrome brain and heart

Microarray analysis of transcript levels in fetal cerebellum and heart tissues of Down syndrome patients showed a disruption only of chromosome 21 gene expression.

Inhibitory Effect of Monoclonal Antibodies Against CXCR3-B NH2-Terminus on the Proliferation of U937 Cells.

The role of CXCR3 in mediating the angiostatic activity of IFN-inducible ELR−CXC chemokines has been further defined with the finding that CXCR3 exists as two alternatively spliced variants, termed CXCR3A and CXCR3B respectively. The chemokine CXCL4/PF4 shares several activities with CXCL9, CXCL10, and CXCL11, but only binds to its specific receptor CXCR3B for the angiostatic activity. CXCR3B, a novel chemokine receptor, is 51 amino acid residues longer at the NH2-terminus than CXCR3A. Unlike activation of CXCR3A, which is pertussis toxin-sensitive and activates cellular proliferation, activation of CXCR3B is pertussis toxin-resistant and leads to inhibit cellular proliferation. Thus, the 51 amino acid residues at CXCR3B NH2-terminus are very important for the CXCL4/PF4 binding and for the CXCR3B inhibitory effect. In the present studies, a GST-tagged fusion protein of CXCR3B (a.a 1-51) was used as the immunogen for generation of monoclonal antibodies (mAbs) against CXCR3B (a.a. 1–51), and three peptides derived from CXCR3B NH2-terminus (a.a. 1–19, a.a. 17–35, and a.a. 33–51) for epitope mapping of the established mAbs. Nine hybrodoma cell lines secreting mAbs against CXCR3B were established, and we found that two mAbs reacted specifically with the CXCR3B a.a. 1–19, and one antibody with the CXCR3B a.a. 33–51. Three of these antibodies could also bind to CXCR3B on the surface of human intestinal epithelial Caco2 cells, and CXCR3B recognized by the antibodies were localized in the vascular endothelial cells of human fetal heart tissues. The data from Cell proliferation assay showed that three mAbs could inhibit the growth of U937 cells, which were found to express CXCR3B as well. We conclude that the novel panel of CXCR3B mAbs could be useful tools for the CXCR3B functional studies. Meanwhile, CXCR3B may be not only the inhibitory target on endothelial cells, but also the new directly reactive site of CXCR3B-positive tumor cells such as promonocytic U937 cells.