Biomedical subjects

K Addicks

Publications and source records attributed to K Addicks.

At least 55 records · Page 3Linked to original sources

PubMed1999

Perlecan maintains the integrity of cartilage and some basement membranes.

Perlecan is a heparan sulfate proteoglycan that is expressed in all basement membranes (BMs), in cartilage, and several other mesenchymal tissues during development. Perlecan binds growth factors and interacts with various extracellular matrix proteins and cell adhesion molecules. Homozygous mice with a null mutation in the perlecan gene exhibit normal formation of BMs. However, BMs deteriorate in regions with increased mechanical stress such as the contracting myocardium and the expanding brain vesicles showing that perlecan is crucial for maintaining BM integrity. As a consequence, small clefts are formed in the cardiac muscle leading to blood leakage into the pericardial cavity and an arrest of heart function. The defects in the BM separating the brain from the adjacent mesenchyme caused invasion of brain tissue into the overlaying ectoderm leading to abnormal expansion of neuroepithelium, neuronal ectopias, and exencephaly. Finally, homozygotes developed a severe defect in cartilage, a tissue that lacks BMs. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and elevated expression of cartilage extracellular matrix genes, suggesting that perlecan protects cartilage extracellular matrix from degradation.

Animals↗

PubMed1999

Nitric oxide synthase expression and role during cardiomyogenesis.

OBJECTIVE: The aim of the present study was the investigation of the expression of NOS during cardiomyogenesis and its functional role. DESIGN: The qualitative and quantitative expression of NOS isoforms during different stages of cardiac development was evaluated using immunocytochemistry and dot blots, respectively. The functional relevance of NOS expression during cardiomyogenesis was investigated using the in vitro ES cell-differentiation model and selective pharmacological agents. RESULTS: On day 7.5 of embryonic development (E7.5) none of the NOS isoforms were expressed in the embryo, whereas the inducible (iNOS), as well as the endothelial (eNOS) isoforms were detected in the extraembryonic parts. In contrast, starting from E9.5 rat and murine embryos displayed prominent iNOS and eNOS expression. This was correlated with high expression of soluble guanylylcyclase (sGC) as well as high cyclic GMP (cGMP) content. During further development after E14.5 both, iNOS as well as eNOS, started to be downregulated and shortly prior to birth reduced staining for eNOS was found, whereas iNOS was hardly detectable. We further investigated whether NO plays a role for cardiomyogenesis, using in vitro ES cell-derived cardiomyocytes differentiating within embryoid bodies (EBs). The NOS expression pattern in these cells paralleled the one detected in vivo. We demonstrate that continuous incubation of EBs with the NOS inhibitors L-NMMA (2-10 mM) or L-NA (2-10 mM) for 4 to 9 days after plating resulted in a pronounced differentiation arrest of cardiomyocytes, whereas this effect could be reversed by coapplication of the NO-donor spermine-NONOate (10 microM). CONCLUSIONS: Both, iNOS and eNOS isoforms are prominently expressed during early stages of cardiomyogenesis. Around E14.5 NOS expression starts to decline. Moreover, the NO-generation is required for cardiomyogenesis since NOS inhibitors prevent the maturation of terminally differentiated cardiomyocytes using the ES cell system.

Animals↗

PubMed1999

Expression of inducible nitric oxide synthase (iNOS/NOS II) in the cochlea of guinea pigs after intratympanical endotoxin-treatment.

Since NO is believed to be involved in cochlear physiology, presence of the constitutive isoforms of nitric oxide synthase (NOS), and the target enzyme of NO, soluble guanylyl cyclase (sGC) in structures of the mammalian cochlea have been demonstrated. To date, no reports have been published regarding the detection of the inducible isoform (NOS II) in the cochlea. In order to show the capability of iNOS expression in cochlear tissue, a mixture of proinflammatory bacterial lipopolysaccharides (LPS) and tumor necrosis factor alpha (TNF-alpha) was injected into the tympanic cavity of guinea pigs, vs. saline-solution as control. Paraffin sections of LPS/TNF-alpha treated and saline-treated cochleae (6 h) were examined immunohistochemically with specific antibodies to neuronal, endothelial and inducible NOS and to sGC. Initiated expression of iNOS in the cochlea was observed in the wall of blood vessels of the spiral ligament (SL) and the modiolus, in supporting cells of the organ of Corti, in the limbus, in nerve fibers and in a part of the perikarya of the spiral ganglion after LPS/TNFalpha-treatment. iNOS was not detected in saline-treated control tissue. Expression of both constitutive NOS-isoforms (endothelial and neuronal NOS) and of sGC showed no significant differences in both experimental groups. Endothelial eNOS and neuronal bNOS were detected co-localized in ganglion cells, in nerve fibers, in cells of the SL and in supporting cells of the organ of Corti, but not in sensory cells. Strong labeling for bNOS became evident in the endosteum of the cochlea, while in the endothelium of blood vessels and in the epithelium of the limbus only eNOS could be labeled. sGC could be detected in SL, in supporting and sensory cells of the organ of Corti, in nerve fibers, ganglion cells, in the wall of blood vessels and in the limbus-epithelium. While small amounts of NO, generated by bNOS and eNOS, seem to support the cochlear blood flow and auditory function as well as neurotransmission, high amounts of iNOS-generated NO could have dysregulative and neurotoxic effects on the inner ear during bacterial and viral infections of the middle and inner ear.

Animals↗

PubMed1999

Expression of inducible nitric oxide-synthase in the vestibular system of hydropic guinea pigs.

Immunohistochemical investigations of the guinea pig vestibular system, using a specific antibody to the inducible isoform of NO-synthase (iNOS/NOS II), have been performed 3 weeks after surgical closure of the right endolymphatic duct (n = 7). Endolymphatic hydrops (ELH) of the right temporal bone became evident by excavation of the Reissner's membrane in all seven animals. Those animals revealed iNOS-expression in ganglion cells, in the wall of blood vessels and in nerve fibers of the right vestibular system, while the corresponding left temporal bones and temporal bones of non-operated controls (n = 6) as well as of sham-operated animals (n = 3) did not show any iNOS-positive structures. iNOS-generated NO could be involved in the pathophysiology of vestibular dysfunction in Meniere's disease.

Animals↗

PubMed1999

Inhibition of phosphatidylinositol-3-kinase blocks development of functional embryonic cardiomyocytes.

Culturing murine embryonic stem (ES) cells within embryoid bodies (EBs) has been reported to reproduce cardiomyocyte development from primitive precursor cells to highly specialized phenotypes of cardiac tissue. We show here that the specific inhibitor of phosphatidylinositol-3-kinase (PI-3-kinase), LY294002, blocks the growth and induces apoptosis as well as necrosis of D3 ES cells within early EBs. Treatment of EBs from day 3 to day 7 with 50 microM LY294002 resulted in a massive loss of alpha-actinin-stained cardiomyocytes after plating the EBs for additional 7 days. In parallel we observed a strong decrease in the number of EBs containing area(s) with beating cardiomyocytes. The specific action of the PI-3-kinase inhibitor on development of cardiomyocytes was demonstrated by the observation that formation of endothelial cells was not affected in the same EBs. Our results provide the first evidence that signal transduction via the PI-3-kinase pathway is essential for mammalian early cardiomyocyte development.

Animals↗

PubMed1999

Immunolocalization of inducible and constitutive nitric oxide synthases in human bladder cancer.

OBJECTIVES: Nitric oxide (NO) is synthesized by the enzyme family of NO synthases (NOS) and plays an important role in tumor growth and angiogenesis. NO generation by inducible NOS (iNOS) also influences the cytotoxicity of macrophages and tumor-induced immunosuppression. Before now, the expression of iNOS and constitutive NOS in bladder carcinoma tissue had not been determined. METHODS: Bladder carcinoma tissue specimens were procured from 18 patients (mean age 69.7 years) undergoing transurethral resection. In every patient, tumor biopsies were compared with biopsies of benign bladder regions. Histochemical NADPH-diaphorase staining and NOS immunohistochemistry were performed on all tissue specimens. RESULTS: Positive NADPH-diaphorase staining was detected in all sections from bladder carcinoma tissue. NOS immunohistochemistry showed a different pattern. The malignant epithelial cells were highly iNOS positive. Specimens of bladder mucosa outside of the malignant regions showed only a weak positive iNOS immunostaining. The endothelial cells of abundant precapillary vessels in the stroma of bladder tumors showed a highly positive endothelial NOS (eNOS) immunostaining compared with the stroma of nonmalignant bladder tissue. Neuronal NOS immunoreactivity was only found in nitrinergic fibers in the fibromuscular stroma. CONCLUSIONS: Bladder carcinoma tissue had a high iNOS content; benign tissue did not. NO generation from iNOS in the malignant epithelium and from eNOS in tumor stroma may play different roles in tumor angiogenesis and tumor-induced immunosuppression.

Aged↗

PubMed1999

Localization of the NO/cGMP-pathway in the cochlea of guinea pigs.

The presence of nitric oxide synthase (NOS) in substructures of the cochlea of guinea pigs is an issue of current focus. Moreover, information concerning the localization of cells effected by the NO/cGMP-pathway are rare. Paraffin sections of guinea pig cochlea were incubated with specific antibodies to the three known NOS isoforms, soluble guanylyl cyclase (sGC) and cyclic guanosine-monophosphate (cGMP), the second messenger system of NO. While detection of inducible iNOS failed in all cochlear structures, expression of endothelial eNOS was found in the spiral ligament, in the stria vascularis, in cells of the organ of Corti, in nerve fibers and in some perikaryia of the spiral ganglion. The cochlear nerve showed an accentuated affinity for immunostaining in distal, basal segments of the cochlea. Neuronal bNOS was found predominantly in the endosteum of the modiolus and cochlea and was less intensively present in all perikaryia of the spiral ganglion and in the spiral ligament. Supporting cells of the organ of Corti and cells in the limbus spiralis displayed only modest immunostaining, while bNOS was not found in outer and inner hair cells. NOS detection was accompanied by immunoreactivity to sGC and to cGMP. The presence of NOS and its second messenger system gives evidence for a possible involvement in neurotransmission, regulation of the cochlear amplifier and in homeostasis.

Animals↗

PubMed1999

Stage specific glycosylation pattern for lactoseries carbohydrates in the developing chick retina.

Based on the idea of differentiation-related changes in the glycosylation pattern of neurons, the expression of two cell surface oligosaccharide epitopes, N-acetyl-lactosamine (NALA), and its sulpho-glucuronyl derivative (HNK-1), was studied, by immunohistochemistry and Western blot experiments, in the developing chick retina beginning on day 2 of incubation (E2) until day 18 post-hatching. NALA was detectable on neuroepithelial cells as soon as the primary optic vesicles formed, and this pattern continued until E3. During subsequent retinal development NALA expression became progressively restricted in concert with the appearance of postmitotic neurons as revealed by neurite outgrowth, and with the formation of synaptic contacts until it disappeared at the end of the incubation period. The pattern of NALA expression was the inverse of HNK-1 which was detected for the first time at E3 on postmitotic ganglion cells accumulating at the vitreal surface. The number of HNK-1+ cells steadily increased until around E10, when the entire neural epithelium was labelled. Synchronously to synaptogenesis, most neurons lost their HNK-1 immunoreactivity. At the time of hatching the adult-like pattern was found, characterised by subpopulations of labelled horizontal, bipolar, amacrine, and ganglion cells. Immunoblot experiments demonstrated transient NALA glycosylation of protein bands, partially identical in their apparent molecular weight to those proteins with HNK-1 glycosylation. The observed temporospatial changes in the glycosylation patterns of distinct proteins during retinal development suggest NALA as a suitable marker for neuronal proliferation, and HNK-1 for differentiation and establishment of final synaptic configuration.

Age Factors↗

PubMed1999

Selective expression of the NOS II isoform during mouse vestibulocochlear receptorgenesis.

Based on in vitro studies, nitric oxide (NO) is reported to be involved in initial neuronal differentiation. In order to compare this finding with the situation in vivo, we have looked for the expression of the three NO synthase isoforms in the developing mouse vestibulocochlear system. From these isoforms only the inducible NOS II is expressed during inner ear development. Examination of a series of embryonic and early postnatal animals, up to postnatal day 6, reveals a maturation-dependent, monophasic expression of this isoform. Initial expression is observed by day 10 of gestation in nerve cells of the vestibolocochlear ganglion and on their fibres. By day 14 of gestation, these afferent fibres penetrate the epithelium of the prospective receptor fields making contact with early, differentiating immunoreactive cochlear hair cells and receptor cells of the macula and crista ampullaris. This receptor-cell-derived immunoreactivity vanished in differentiated sensory hair cells by postnatal day 6, when both the constitutive isoforms and subsequent activated members of the down stream second messenger cascade (guanylate cyclase/cGMP) of the adult mouse were not then detectable. The strict phasic expression of NOS-II, independent of the second messenger system mentioned above, implies that there is a unique role for the inducible NOS isoform in nerve cell differentiation, independent of the NO/guanylate cyclase/cGMP pathway.

Aging↗

PubMed1999

Regulation of the L-type Ca2+ channel during cardiomyogenesis: switch from NO to adenylyl cyclase-mediated inhibition.

In adult mammalian cardiomyocytes, stimulation of muscarinic receptors counterbalances the beta-adrenoceptor-mediated increase in myocardial contractility and heart rate by decreasing the L-type Ca2+ current (ICa) (1, 2). This effect is mediated via inhibition of adenylyl cyclase and subsequent reduction of cAMP-dependent phosphorylation of voltage-dependent L-type Ca2+ channels (3). Little is known, however, about the nature and origin of this pivotal inhibitory pathway. Using embryonic stem cells as an in vitro model of cardiomyogenesis, we found that muscarinic agonists depress ICa by 58 +/-3% (n=34) in early stage cardiomyocytes lacking functional beta-adrenoceptors. The cholinergic inhibition is mediated by the nitric oxide (NO)/cGMP system since it was abolished by application of NOS inhibitors (L-NMA, L-NAME), an inhibitor of the soluble guanylyl cyclase (ODQ), and a selective phosphodiesterase type II antagonist (EHNA). The NO/cGMP-mediated ICa depression was dependent on a reduction of cAMP/protein kinase A (PKA) levels since application of the catalytic subunit of PKA or of the PKA inhibitor PK) prevented the carbachol effect. In late development stage cells, as reported for ventricular cardiomyocytes (2, 4), muscarinic agonists had no effect on basal ICa but antagonized beta-adrenoceptor-stimulated ICa by 43 +/-4% (n=16). This switch in signaling pathways during development is associated with distinct changes in expression of the two NO-producing isoenzymes, eNOS and iNOS, respectively. These findings indicate a fundamental role for NO as a signaling molecule during early embryonic development and demonstrate a switch in the signaling cascades governing ICa regulation.

Adenine↗

PubMed1999

Nitric oxide decreases microvascular permeability in bradykinin stimulated and nonstimulated conditions.

This study examined the occurrence of endothelial nitric oxide (NO)-synthase (NOS-III) in terminal mesenteric vessels and the involvement of NO in microvascular permeability. Possible effects were studied in bradykinin (BK)-induced and basal conditions. NOS expression was investigated by using NOS-III immunohistochemistry and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry on the light- and electron-microscopic levels. Permeability was examined in dissected mesenteries of male rats weighing 250-300 g. Tissue treatment was performed with BK (100 nM), sodium nitroprusside (SNP, 1 and 10 microM), L-nitroarginine (L-NA, 300 microM), BK and L-NA, BK and SNP, L-NA and SNP, as well as with BK, SNP (10 microM), and the guanylylcyclase inhibitor ODQ (10 microM), and BK and ODQ alone. Pharmacologically induced permeability changes were studied with fluorescein isothiocyanate (FITC)-dextran 70 kDa as a tracer for macromolecular transport. Video images were analyzed with computer determination of integrated optical density (IOI). Results were statistically verified by analysis of variance and t test. Microvascular permeability was increased by 168% after BK treatment and was enhanced by NO-synthesis inhibition with L-NA by 607%. However, the NO donor SNP led to a reduced tracer extravasation to 105 and 58%, respectively, an effect blocked by ODQ. Under basal conditions without prior BK induction, L-NA also causes an increase of IOI by 25%, whereas coapplication with SNP resulted in only a 10% increase of permeability. These results point out that NO has a modulatory role for microvascular permeability by supporting the barrier function of the endothelial lining in stimulated and nonstimulated conditions.

Animals↗

PubMed1999

Establishment of ionic channels and signalling cascades in the embryonic stem cell-derived primitive endoderm and cardiovascular system.

The first organ system to be established in early embryogenesis is the cardiovascular system which develops upon interaction with hypoblastic cells of the primitive endoderm. Here we focus on recent work on embryoid bodies derived from pluripotent embryonic stem (ES) cells. Ca(2+) oscillations and Ca(2+) signalling pathways during the differentiation of primitive endodermal cell layers are reported. Furthermore, the development-dependent expression of ion channels and the buildup of signalling cascades involved in the modulation of voltage-dependent L-type Ca(2+) channels during early cardiomyogenesis and the formation of functional vascular structures in the process of vasculogenesis and angiogenesis are reviewed. We also report on the use of green fluorescent protein reporter gene expression under the control of cardiac-specific promoters, e.g. the human cardiac alpha-actin promoter, which enables the identification and in vivo characterization of cardiomyocytes at very early stages of cardiomyogenesis.

Animals↗

PubMed1998

Functional characteristics of ES cell-derived cardiac precursor cells identified by tissue-specific expression of the green fluorescent protein.

In contrast to terminally differentiated cardiomyocytes, relatively little is known about the characteristics of mammalian cardiac cells before the initiation of spontaneous contractions (precursor cells). Functional studies on these cells have so far been impossible because murine embryos of the corresponding stage are very small, and cardiac precursor cells cannot be identified because of the lack of cross striation and spontaneous contractions. In the present study, we have used the murine embryonic stem (ES, D3 cell line) cell system for the in vitro differentiation of cardiomyocytes. To identify the cardiac precursor cells, we have generated stably transfected ES cells with a vector containing the gene of the green fluorescent protein (GFP) under control of the cardiac alpha-actin promoter. First, fluorescent areas in ES cell-derived cell aggregates (embryoid bodies [EBs]) were detected 2 d before the initiation of contractions. Since Ca2+ homeostasis plays a key role in cardiac function, we investigated how Ca2+ channels and Ca2+ release sites were built up in these GFP-labeled cardiac precursor cells and early stage cardiomyocytes. Patch clamp and Ca2+ imaging experiments proved the functional expression of the L-type Ca2+ current (ICa) starting from day 7 of EB development. On day 7, using 10 mM Ca2+ as charge carrier, ICa was expressed at very low densities 4 pA/pF. The biophysical and pharmacological properties of ICa proved similar to terminally differentiated cardiomyocytes. In cardiac precursor cells, ICa was found to be already under control of cAMP-dependent phosphorylation since intracellular infusion of the catalytic subunit of protein kinase A resulted in a 1.7-fold stimulation. The adenylyl cyclase activator forskolin was without effect. IP3-sensitive intracellular Ca2+ stores and Ca2+-ATPases are present during all stages of differentiation in both GFP-positive and GFP-negative cells. Functional ryanodine-sensitive Ca2+ stores, detected by caffeine-induced Ca2+ release, appeared in most GFP-positive cells 1-2 d after ICa. Coexpression of both ICa and ryanodine-sensitive Ca2+ stores at day 10 of development coincided with the beginning of spontaneous contractions in most EBs. Thus, the functional expression of voltage-dependent L-type Ca2+ channel (VDCC) is a hallmark of early cardiomyogenesis, whereas IP3 receptors and sarcoplasmic Ca2+-ATPases are expressed before the initiation of cardiomyogenesis. Interestingly, the functional expression of ryanodine receptors/sensitive stores is delayed as compared with VDCC.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1998

Cardiac specific expression of the green fluorescent protein during early murine embryonic development.

We demonstrate the establishment of transgenic mice, where the expression of the green fluorescent protein (GFP) is under control of the human cardiac alpha-actin promoter. These mice display cardiac specific GFP expression already during early embryonic development. Prominent GFP fluorescence was observed at the earliest stage of the murine heart anlage (E8). Cardiomyocytes of different developmental stages proved GFP positive, but the intensity varied between cells. We further show that contractions of single GFP positive cardiomyocytes can be monitored within the intact embryo. At later stages of embryonic development, the skeletal musculature was also GFP positive, in line with the known expression pattern of cardiac alpha-actin. The tissue specific labeling of organs is a powerful new tool for embryological as well as functional investigations in vivo.

Actins↗

PubMed1998

Nitric oxide synthase in the vestibulocochlear system of mice.

The exact distribution of nitric oxide-synthases (NOS) and the NO-target enzyme soluble guanylyl cyclase (sGC) in the cochlea and vestibular organ is an issue of current discussion. The existence of NOS-isoforms in the cochlea of the guinea pig has been described recently, while information about the vestibular system are still rare and non-satisfying. In order to gain more information, immunostaining was performed, using specific antibodies to NOS I-III and to sGC, on paraffin sections of complete temporal bones from mice. NOS III could be detected in cochlea and vestibular ganglion cells, in nerve fibres, in outer hair cells of the cochlear and in the sensory epithelium of the maculae. Also, the spiral ligament and the limbus epithelium was positive to NOS III. NOS I was found in the sensory epithelium of the maculae and cristae ampullares, outer and inner hair cells of the cochlea, in nerve fibres and in ganglion cells. In contrast to that NOS II could not be detected at all. Furthermore, a strong NOS I immunoreaction was displayed on the endosteum of the bone, while the periosteum was lacking of NOS. NOS detection was accompanied by immunoreactivity to sGC. The findings imply that NOS I and III-generated NO is involved in neurotransmission and other regulative processes in the vestibulocochlear system.

Animals↗

PubMed1998

Postnatally induced inactivation of gp130 in mice results in neurological, cardiac, hematopoietic, immunological, hepatic, and pulmonary defects.

The pleiotrophic but overlapping functions of the cytokine family that includes interleukin (IL)-6, IL-11, leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, and cardiotrophin 1 are mediated by the cytokine receptor subunit gp130 as the common signal transducer. Although mice lacking individual members of this family display only mild phenotypes, animals lacking gp130 are not viable. To assess the collective role of this cytokine family, we inducibly inactivated gp130 via Cre-loxP-mediated recombination in vivo. Such conditional mutant mice exhibited neurological, cardiac, hematopoietic, immunological, hepatic, and pulmonary defects, demonstrating the widespread importance of gp130-dependent cytokines.

Animals↗

PubMed1998

Localisation of the nitric oxide (NO)/cGMP-pathway in the vestibular system of guinea pigs.

The exact distribution of nitric oxide-synthases (NOS) in the vestibular system has not been described satisfying yet. Immunostaining, using specific antibodies to the three known NOS-isoforms, to cyclic guanosine monophosphate (cGMP) and soluble guanylyl-cyclase (sGC), the second messenger system of nitric oxide (NO), was performed on paraffin sections of temporal bone from guinea pigs. eNOS could be detected in vestibular ganglion cells and in nerve fibres, including the calyces, surrounding the type 1 hair cells (HC). bNOS was found in the sensory epithelium, ganglion cells and in bone, while iNOS could not be found. NOS-detection was accompanied by reactivity to sGC and to cGMP. This finding implies that b- and eNOS-generated NO is involved in regulative processes in neurotransmission and regulation of blood flow.

Animals↗

PubMed1998

Beta1 integrin deficiency impairs migration and differentiation of mouse embryonic stem cell derived neurons.

Cell-matrix interaction plays an important role during neuronal development, which is demonstrated by comparing wild type (D3)- and beta1 integrin-deficient (G201) embryonic stem cell derived neurons. In D3 preparations complex networks of functionally coupled neurons with bi- and multipolar morphologies develop. In contrast, neuronal differentiation is retarded in G201 derived neurons, recognised by limited migration and restricted morphological differentiation. Furthermore, beta1 integrin deficiency causes a delay in expression of major neurotransmitters like GABA and glutamate as well as of synaptophysin. These findings indicate a prominent role of beta1 integrin for both morphological and chemical differentiation.

Animals↗