Biomedical subjects

Stanislav I Tomarev

Publications and source records attributed to Stanislav I Tomarev.

14 recordsLinked to original sources

PubMed2006

Optimedin induces expression of N-cadherin and stimulates aggregation of NGF-stimulated PC12 cells.

Optimedin, also known as olfactomedin 3, belongs to a family of olfactomedin domain-containing proteins. It is expressed in neural tissues and Pax6 is involved in the regulation of its promoter. To study possible effects of optimedin on the differentiation of neural cells, we produced stably transfected PC12 cell lines expressing optimedin under a tetracycline-inducible promoter. Cells expressing high levels of optimedin showed higher growth rates and stronger adhesion to the collagen extracellular matrix as compared with control PC12 cells. After stimulation with nerve growth factor (NGF), optimedin-expressing cells demonstrated elevated levels of N-cadherin, beta-catenin, alpha-catenin and occludin as compared with stimulated, control PC12 cells. Expression of optimedin induced Ca(2+)-dependent aggregation of NGF-stimulated PC12 cells and this aggregation was blocked by the expression of N-cadherin siRNA. Expression of optimedin also changed the organization of the actin cytoskeleton and inhibited neurite outgrowth in NGF-stimulated PC12 cells. We suggest that expression of optimedin stimulates the formation of adherent and tight junctions on the cell surface and this may play an important role in the differentiation of the brain and retina through the modulation of cytoskeleton organization, cell-cell adhesion and migration.

Animals↗

PubMed2006

Dual origin of avian lymphatics.

The earliest signs of the lymphatic vascular system are the lymph sacs, which develop adjacent to specific embryonic veins. It has been suggested that sprouts from the lymph sacs form the complete lymphatic vascular system. We have studied the origin of the jugular lymph sacs (JLS), the dermal lymphatics and the lymph hearts of avian embryos. In day 6.5 embryos, the JLS is an endothelial-lined sinusoidal structure. The lymphatic endothelial cells (LECs) stain (in the quail) positive for QH1 antibody and soybean agglutinin. As early as day 4, the anlagen of the JLS can be recognized by their Prox1 expression. Prox1 is found in the jugular section of the cardinal veins, and in scattered cells located in the dermatomes along the cranio-caudal axis and in the splanchnopleura. In the quail, such cells are positive for Prox1 and QH1. In the jugular region, the veins co-express the angiopoietin receptor Tie2. Quail-chick-chimera studies show that the peripheral parts of the JLS form by integration of cells from the paraxial mesoderm. Intra-venous application of DiI-conjugated acetylated low-density lipoprotein into day 4 embryos suggests a venous origin of the deep parts of the JLS. Superficial lymphatics are directly derived from the dermatomes, as shown by dermatome grafting. The lymph hearts in the lumbo-sacral region develop from a plexus of Prox1-positive lymphatic capillaries. Both LECs and muscle cells of the lymph hearts are of somitic origin. In sum, avian lymphatics are of dual origin. The deep parts of the lymph sacs are derived from adjacent veins, the superficial parts of the JLS and the dermal lymphatics from local lymphangioblasts.

Animals↗

PubMed2006

Mutated mouse and human myocilins have similar properties and do not block general secretory pathway.

PURPOSE: The present study compared properties of wild-type and mutated mouse and human myocilin (Myoc) proteins as a prerequisite for development of a mouse model of glaucoma. METHODS: cDNA encoding full-length mouse Myoc was cloned into the p3XFLAG-CMV-14 vector. Tyr423His and Ile463Ser mutations were introduced into the mouse Myoc protein by in vitro mutagenesis. Intracellular localization and secretion of wild-type and mutated mouse Myoc proteins were studied in immunostaining and Western blotting experiments, respectively, after transfection into COS-7 cells. RESULTS: Similar to human MYOC, wild-type and mutated mouse Myoc demonstrated vesicular staining in transfected cells. However, while wild-type human and mouse Myoc were preferentially located in both the endoplasmic reticulum and Golgi, mutated human and mouse Myoc were located mainly in the endoplasmic reticulum and were excluded from Golgi. Similar to mutations in human MYOC, mutations in mouse Myoc dramatically reduced its secretion from transfected cells. Secretion of mutated Myoc was partially restored by culturing cells at 30 degrees C instead of 37 degrees C. The presence of mutated human MYOC prevented secretion of wild-type mouse Myoc but did not dramatically affect secretion of alkaline phosphatase, thrombospondin, Timp3 or olfactomedin-1. CONCLUSIONS: Properties of the mouse Myoc protein are similar to those of the human MYOC. The presence of mutated mouse or human Myoc does not block a general secretory pathway. Expression of mutated Myoc in the eye in mice may mimic human glaucoma and lead to development of a genetic mouse model of glaucoma.

Amino Acid Sequence↗

PubMed2004

Mafs, Prox1, and Pax6 can regulate chicken betaB1-crystallin gene expression.

During lens fiber cell differentiation, the regulation of crystallin gene expression is coupled with dramatic morphological changes. Here we report that Mafs, Prox1, and Pax6, which are essential transcription factors for normal lens development, bind to three functionally important cis elements, PL1, PL2, and OL2, in the chicken betaB1-crystallin promoter and may cooperatively direct the transcription of this lens fiber cell preferred gene. Gel shift assays demonstrated that Mafs bind to the MARE-like sequences in the PL1 and PL2 elements, whereas Prox1, a sequence-specific DNA-binding protein like its Drosophila homolog Prospero, interacts with the OL2 element. Furthermore, Pax6, a known repressor of the chicken betaB1-crystallin promoter, binds to all three of these cis elements. In transfection assays, Mafs and Prox1 activated the chicken betaB1-crystallin promoter; however, their transactivation ability was repressed when co-transfected with Pax6. Taken together with the known spatiotemporal expression patterns of Mafs, Prox1, and Pax6 in the developing lens, we propose that Pax6 occupies and represses the chicken betaB1-crystallin promoter in lens epithelial cells, and is displaced by Prox1 and Mafs, which activate the promoter, in differentiating cortical fiber cells.

Animals↗

PubMed2004

Genetically increasing Myoc expression supports a necessary pathologic role of abnormal proteins in glaucoma.

Despite the importance of MYOC for glaucoma, the protein's normal function(s) and the pathogenic mechanism(s) of MYOC mutations are not clear. Elevated intraocular pressure (IOP) and glaucoma are sometimes induced by corticosteroids, and corticosteroid use can result in substantially increased MYOC expression. It has been suggested, therefore, that steroid-induced MYOC protein levels cause steroid-induced glaucoma and that protein level-increasing mutations in MYOC contribute to glaucoma not associated with steroid use. A causative role of elevated MYOC levels in steroid-induced glaucoma is controversial, however, and it is not clear if elevated MYOC levels can result in IOP elevation. To directly test if increased levels of MYOC can cause IOP elevation and glaucoma, we generated bacterial artificial chromosome transgenic mice that overexpress Myoc at a level similar to that induced by corticosteroid use. These mice do not develop elevated IOP or glaucoma. Our present findings, along with the absence of glaucoma in mice completely lacking MYOC, show that changing the level of MYOC is not pathogenic (from absent to approximately 15 times normal). These findings suggest that noncoding sequence variants are unlikely to influence glaucoma and that disease pathogenesis in primary open-angle glaucoma patients is dependent upon the expression of abnormal mutant proteins. This work does not support a causative role for increased MYOC levels or the MYOC gene in steroid-induced glaucoma.

Adrenal Cortex Hormones↗

PubMed2004

Microarray analysis of changes in mRNA levels in the rat retina after experimental elevation of intraocular pressure.

PURPOSE: The goal of this study was to identify altered patterns of retinal mRNA expression after experimental elevation of intraocular pressure (IOP) in a rat glaucoma model. METHODS: Brown Norway rats (N = 16) received unilateral episcleral vein injection of hypertonic saline to elevate IOP. IOP was monitored daily by handheld tonometer, and retinas were collected 8 days and 5 weeks after surgery. Comparison of mRNA levels between experimental and fellow retinas was made using gene microarrays (rat U34A rat arrays; Affymetrix, Santa Clara, CA). Semiquantitative RT-PCR was used to confirm selected results from array analysis and to compare with alterations after optic nerve transection. RESULTS: IOP elevation for 5 weeks resulted in reproducible changes in levels of 81 mRNAs. Of these, 74 increased, whereas only 7 decreased. The expression levels of 27 of these same messages were changed after 8 days of IOP elevation. In addition, four other genes demonstrated altered expression after the shorter period of elevated IOP exposure. Approximately half of the mRNAs with altered expression were associated with either neuroinflammatory responses or apoptosis. For 25 of the selected functionally relevant messages altered by array analysis, the alterations were confirmed by semiquantitative RT-PCR. The levels of 24 of 25 selected messages were also changed after optic nerve transection. CONCLUSIONS: The activation of glia and the complement system after IOP elevation, which is similar to that described in several neurodegenerative diseases and after optic nerve transection, suggests that this rat glaucoma model could be used to evaluate the neuroprotective potential of therapeutic agents that target these processes.

Animals↗

PubMed2004

Gene expression profile of the rat eye iridocorneal angle: NEIBank expressed sequence tag analysis.

PURPOSE: To characterize gene expression pattern in the combined tissues of the rat iridocorneal angle by expressed sequence tag (EST) analysis, as part of the NEIBank project. METHODS: RNA was extracted from dissected tissues of the rat iridocorneal angle (iris, ciliary body, trabecular meshwork, and Schlemm's canal) and used to construct unamplified, non-normalized cDNA libraries in the pSPORT1 vector. Approximately 5000 clones were sequenced from the 5'-end. Clones were clustered and identified using the GRIST software, a procedure based on BLAST comparisons. Complete sequences of several novel cDNAs showing eye-preferred expression patterns were obtained. The expression patterns of several genes have been investigated by Northern blot and in situ hybridization, as well as by RT-PCR. RESULTS: After analysis and removal of non-mRNA sequences, 2195 independent clusters, potentially representing individual eye angle-expressed clones were obtained. The expression profile of the combined rat eye angle tissues was more similar to that of the human iris than to human trabecular meshwork. Several cDNAs encoding transcription factors essential for normal eye development and function including Pax-6, Six3, c-Maf, Maf1, Sox-4, Foxc1, Rx, and Ldb2 were present among sequenced clones. A number of tested cDNAs showed eye-preferred expression patterns. Myocilin, which is abundant in human eye angle tissues, was not observed in the rat collection; however, transcripts for three other olfactomedin-domain proteins were seen. Latrotoxin receptor (CL1AA) and optimedin were shown to be expressed in the iris and ciliary body, as well as in the ganglion and inner nuclear cell layers of the retina, whereas the rat orthologue of the human HNOEL-iso gene was expressed in the iris and sclera and less actively in the trabecular meshwork, retina, and optic nerve. CONCLUSIONS: The iridocorneal libraries are a good source of novel uncharacterized genes and molecular markers for the tissues of the eye angle. Although myocilin is not abundantly expressed in rat eye angle, other olfactomedin-containing genes are expressed there and may play important roles in normal eye function and disease.

Amino Acid Sequence↗

PubMed2004

Pdlim2, a novel PDZ-LIM domain protein, interacts with alpha-actinins and filamin A.

PURPOSE: To characterize properties of Pdlim2, a novel PDZ and LIM domain-containing protein. METHODS: cDNA encoding Pdlim2 was identified in a cDNA library of transcripts expressed in the tissues of the rat eye irido-corneal angle. The expression pattern of the Pdlim2 gene was studied by Northern blot analysis and in situ hybridization. Proteins interacting with Pdlim2 were identified by pull-down assay and mass spectrometry. Intracellular localization of Pdlim2 was investigated by confocal microscopy. RESULTS: Rat Pdlim2 protein belongs to the ALP subfamily of proteins containing the PDZ domain in the N-terminal portion and the LIM domain in the C-terminal portion of the protein. The Pdlim2 gene was specifically expressed in the corneal epithelial cells, but not in the corneal stroma and endothelium nor in other ocular tissues. Pdlim2 was also expressed in the lung. In rat corneal and lung extracts, alpha-actinin-1, alpha-actinin-4, filamin A, and myosin heavy polypeptide 9 were co-immunoprecipitated with Pdlim2. Myosin VI was co-immunoprecipitated with Pdlim2 from corneal but not lung extracts. alpha-Actinins were the most abundant among immunoprecipitated proteins. Direct interaction of Pdlim2 with alpha-actinins and filamin was confirmed using pull-down assays and gel overlay assay with purified proteins. Pdlim2 and alpha-actinins were co-localized mainly to stress fibers after transfection into COS-7 cells. In transfected COS-7 cells, complexes of Pdlim2 and alpha-actinin-1 were preferentially located along the basal aspect. CONCLUSIONS: These results suggest that Pdlim2, like other ALP subfamily members, may act as an adapter that directs other proteins to the cytoskeleton.

Actinin↗

PubMed2003

Three different fates of cells migrating from somites into the limb bud.

Cells from the ventrolateral dermomyotomal lips at limb levels undergo epithelio-mesenchymal transition and migrate as individual and undifferentiated cells into the limb buds. The cells give rise to myocytes and blood vascular endothelial cells (BECs) in the limb. Using vascular endothelial growth factor receptor-3 (VEGFR-3) as a marker, it has also been shown that the somites contribute to endothelial cells of lymphatic vessels in the limbs, but it is unknown where the lymphangiogenic precursors are located within the somite. In this study we used the transcription factor Prox1 as a lymphatic marker and investigated whether cells in the dorso-lateral quarter of the somite differentiate into lymphatic endothelial cells (LECs) of the limbs. To label the migrating cells, the dorso-lateral part of an epithelial brachial somite was grafted homotopically from quail into chick embryos at HH stages 13-14. The chick hosts were incubated until day 10-11 of development. The quail cell nuclei were identified with QCPN (anti-quail) antibodies. Cell differentiation was analysed by immunohistochemical staining with QH1, anti-desmin and anti-Prox1 antibodies, and by in situ hybridisation with Prox1 probes. Our results confirm that quail cell nuclei are incorporated into the myotubes of the limb muscles. Quail cells are found in the endothelium of limb blood vessels and lymphatics, predominantly the dermal lymphatics. This indicates that superficial lymphatics develop independently from the deep ones and shows that cells migrating from the lateral somitic edge into the limb buds differentiate into three cell populations: myocytes, BECs and LECs.

Animals↗

PubMed2003

Lymphangioblasts in embryonic lymphangiogenesis.

BACKGROUND: The origin of the lymphatic endothelium, either from the venous system or mesenchymal lymphangioblasts, presents as a persistent controversy. Recently, highly specific markers of the lymphatic endothelium have been found, enabling us to reinvestigate the embryonic origin of the lymphatics. METHODS AND RESULTS: The homeobox transcription factor, Prox1, is expressed in lymphatic, but not in blood vascular, endothelial cells throughout murine and avian development and in adult human tissues. Here we show expression of scattered Prox1-positive cells in the dermatome of 4-day-old chick embryos by in situ hybridization and immunostaining. These cells obviously form the Prox1-positive lymphatic networks in the skin of the body wall and the limbs during further embryonic development. The Prox1 protein is localized in the nuclei of the lymphatic endothelial cells (LECs). CONCLUSIONS: The results strongly suggest that the superficial lymphatics develop independently from the deep ones, and are derived from mesenchymal lymphangioblasts rather than veins. Our results argue against the unique origin of lymphatics from veins and suggest a heterogenous origin of LECs. The results are discussed in the context of historical data.

Animals↗

PubMed2003

Gene expression profile of the human trabecular meshwork: NEIBank sequence tag analysis.

PURPOSE: To characterize the gene expression pattern in the human trabecular meshwork (TM) and identify candidate genes for glaucoma by expressed sequence tag (EST) analysis as part of the NEIBank project. METHODS: RNA was extracted from dissected human TM and used to construct unamplified, un-normalized cDNA libraries in the pSPORT1 vector. More than 4000 clones were sequenced from the 5' end. Clones were clustered and identified using GRIST software. In addition, the expression patterns of genes encoding olfactomedin-domain proteins were analyzed by RT-PCR. RESULTS: After non-mRNA contaminants were removed, 3459 independent TM-expressed clones were obtained. These were grouped in 1888 clusters, potentially representing individual expressed genes. Transcripts for the myocilin gene, a locus for inherited glaucoma, formed the third most abundant cluster in the TM collection, and several other genes implicated in glaucoma (PITX2, CYP1B1, and optineurin) were also represented. One abundant TM transcript was from the gene for the angiopoietin-like factor CTD6, which is located at on the long arm of chromosome 1, area 36.2-36.1 in the region of the glaucoma locus GLC3B, whereas other transcripts were from genes close to known glaucoma loci. The TM collection contains cDNAs for genes that are preferentially expressed in the lymphatic endothelium (matrix Gla protein, apolipoprotein D precursor, and selenoprotein P precursor). In addition to EST profiling, RT- PCR was used to detect transcripts of the olfactomedin-domain proteins latrotoxin receptor Lec3 and optimedin in the TM. CONCLUSIONS: The TM libraries are a good source of molecular markers for TM and candidate genes for glaucoma. The abundance of myocilin cDNAs corresponds to the critical role of this gene in glaucoma and contrasts with libraries derived from cultured tissue. The expression profile raises the possibility that cells of the TM and Schlemm's canal may be more similar to lymphatic, rather than blood vascular endothelium.

Aged↗

PubMed2002

The transcription factor Prox1 is a marker for lymphatic endothelial cells in normal and diseased human tissues.

Detection of lymphatic endothelal cells (LECs) has been problematic because of the lack of specific markers. The homeobox transcription factor Prox1 is expressed in LECs of murine and avian embryos. We have studied expression of Prox1 in human tissues with immunofluorescence. In 19-wk-old human fetuses, Prox1 and vascular endothelial growth factor receptor-3 (VEGFR-3) are coexpressed in LECs of lymphatic trunks and lymphatic capillaries. Prox1 is located in the nucleus, and its expression is mutually exclusive with that of the blood vascular marker PAL-E. Prox1 is a constitutive marker of LECs and is found in tissues of healthy adults and lymphedema patients. Blood vascular endothelial cells (BECs) of hemangiomas express CD31 and CD34, but not Prox1. A subset of these cells is positive for VEGFR-3. Lymphatics in the periphery of hemangiomas express Prox1 and CD31, but not CD34. In lymphangiomas, LECs express Prox1, CD31, and VEGFR-3, but rarely CD34. In the stroma, spindle-shaped CD34-positive cells are present. We show that Prox1 is a reliable marker for LECs in normal and pathologic human tissues, coexpressed with VEGFR-3 and CD31. VEGFR-3 and CD34 are less reliable markers for LECs and BECs, respectively, because exceptions from their normal expression patterns are found in pathologic tissues.

Antigens, CD34↗

PubMed2002

Optimedin: a novel olfactomedin-related protein that interacts with myocilin.

Mutations in the MYOC gene may lead to juvenile open-angle glaucoma with high intraocular pressure, and are detected in about 4% of people with adult onset glaucoma. Most of these mutations are found in the third exon of the gene encoding the olfactomedin-like domain located at the C terminus of the protein. Another olfactomedin-related protein, known as noelin or pancortin, is involved in the generation of neural crest cells. Here we describe the identification of a novel olfactomedin-related gene, named optimedin, located on chromosome 1p21 in humans. Optimedin and noelin are both expressed in brain and retina. However, unlike noelin, rat optimedin is also highly expressed in the epithelial cells of the iris and the ciliary body in close proximity to the sites of Myoc expression. In the human eye, optimedin is expressed in the retina and the trabecular meshwork. Both optimedin and myocilin are localized in Golgi and are secreted proteins. The presence of mutant myocilin interferes with secretion of optimedin in transfected cells. Optimedin and myocilin interact with each other in vitro as judged by the GST pulldown, co-immunoprecipitation and far-western binding assays. The C-terminal olfactomedin domains are essential for interaction between optimedin and myocilin, while the N-terminal domains of both proteins are involved in the formation of protein homodimers. We suggest that optimedin may be a candidate gene for disorders involving the anterior segment of the eye and the retina.

Amino Acid Sequence↗

PubMed2002

Prox1 is differentially localized during lens development.

Prox1, the vertebrate cognate of Drosophila Prospero, is a homeodomain protein essential for the development of the lens, liver and lymphatic system. While it is well established that the subcellular distribution of Prospero changes during development, this had not been demonstrated for Prox1. Here, high-resolution confocal microscopy demonstrated that Prox1 protein is predominately cytoplasmic in the lens placode as well as the lens epithelium and germinative zone throughout development. However during fiber cell differentiation, Prox1 protein redistributes to cell nuclei. Finally, as lens fiber cells condense their chromatin in response to lens denucleation, Prox1 remains in the nucleus but does not appear to interact with DNA. Thus, it appears that the function of Prox1, like that of its Drosophila cognate Prospero, is at least partially controlled by changes in its subcellular distribution during development.

Animals↗