Biomedical subjects

D T Shima

Publications and source records attributed to D T Shima.

24 records · Page 2Linked to original sources

PubMed1995

Alterations in gene expression associated with changes in the state of endothelial differentiation.

The endothelium maintains a developmental plasticity which allows rapid phenotypic change in response to extracellular signals during normal processes, such as corpus luteum formation and wound healing, and in pathologic processes, such as tumor angiogenesis. Endothelial cells (EC) in culture have been very useful for investigating various aspects of endothelial growth and behavior. In spite of documented similarities between EC in vitro and the endothelium in vivo, many characteristics of the vessel endothelium are lost when the cells are placed into culture. We have undertaken to identify differences in gene expression between differentiated vessel endothelium and dedifferentiated EC. We utilized a new technique called differential display which compares polymerase chain reaction (PCR)-amplified mRNA from two (or more) cell populations. Endothelium scraped directly from freshly obtained aortas, and demonstrated to be free of contaminants, were used as the source of differentiated RNA, whereas proliferating, primary explanted EC grown for five days in the presence of basic fibroblast growth factor (bFGF) provided a pool of 'dedifferentiated' RNA. Using differential display, we have observed numerous reproducible differences in gene expression. To confirm that the expression differences visualized by differential display represented actual differences in gene expression, we isolated vessel-specific and culture-specific cDNA tags for additional analysis. Three cDNA tags specific to vessel endothelium were cloned and sequenced, and compared to nucleotide and protein databases. Two of the clones (A1 and 2.5) displayed no significant sequence similarity, whereas a third clone (A2) is nearly identical to a human expressed sequence tag (EST) and has significant sequence similarities to a plant and Xenopus ubiquitin-like protein. Northern and/or in situ hybridization analysis of the A1 and A2 genes confirmed their restricted expression to the vessel endothelium. The expression of A1 by the endothelium in vivo is not simply a function of growth state, as cultured cells did not express A1 even when grown to postconfluence. One other cDNA fragment, selected as a culture-induced gene, was identified by sequence analysis as the bovine homologue of laminin B1, and Northern analysis confirmed that expression was induced upon culturing of EC. Use of differential display to study endothelial gene expression will allow us to investigate the molecular mechanisms that underlie initiation and maintenance of endothelial differentiation.

Animals↗

PubMed1995

Hypoxic induction of endothelial cell growth factors in retinal cells: identification and characterization of vascular endothelial growth factor (VEGF) as the mitogen.

BACKGROUND: New vessel growth is often associated with ischemia, and hypoxic tissue has been identified as a potential source of angiogenic factors. In particular, ischemia is associated with the development of neovascularization in a number of ocular pathologies. For this reason, we have studied the induction of endothelial cell mitogens by hypoxia in retinal cells. MATERIALS AND METHODS: Human retinal pigment epithelium (hRPE) were grown under normoxic and hypoxic conditions and examined for the production of endothelial mitogens. Northern analysis, biosynthetic labeling and immunoprecipitation, and ELISA were used to assess the levels of vascular endothelial growth factor/vascular permeability factor (VEGF) and basic fibroblast growth factor (bFGF), two endothelial cell mitogens and potent angiogenic factors. Soluble receptors for VEGF were employed as competitive inhibitors to determine the contribution of the growth factor to the hypoxia-stimulated mitogen production. RESULTS: Following 6-24 hr of hypoxia, confluent and growing cultures of hRPE increase their levels of VEGF mRNA and protein synthesis. Biosynthetic labeling studies and RT-PCR analysis indicate that the cells secrete VEGF121 and VEGF165, the soluble forms of the angiogenic factor. In contrast, hRPE cultured under hypoxic conditions show reduced steady-state levels of basic fibroblast growth factor (bFGF) mRNA and decreased bFGF protein synthesis. Unlike VEGF, bFGF is not found in conditioned media of hRPE following 24 hr of hypoxia. Using a soluble high-affinity VEGF receptor as a competitive inhibitor of VEGF, we demonstrate that a VEGF-like activity is the sole hypoxia-inducible endothelial mitogen produced by cultured hRPE. CONCLUSIONS: From this comparison we conclude that hRPE do not respond to hypoxia with a general, nonspecific increase in the overall levels of growth factors, as is seen during cell wounding responses or serum stimulation. The physiological relevance of data from this in vitro model are affirmed by separate studies in an animal model of retinal ischemia-induced ocular neovascularization (1) in which retina-derived VEGF levels have been shown to correlate spatio-temporally with the onset of angiogenesis. Taken together, these data support the hypothesis that the induction of VEGF by hypoxia mediates the rapid, initial angiogenic response to retinal ischemia.

Alternative Splicing↗

PubMed1994

Vascular endothelial growth factor/vascular permeability factor is temporally and spatially correlated with ocular angiogenesis in a primate model.

Ischemia often precedes neovascularization. In ocular neovascularization, such as occurs in diabetic retinopathy, a diffusible angiogenic factor has been postulated to be produced by ischemic retina and to lead to neovascularization of the retina, optic nerve, or iris. However, no angiogenic factor has been conclusively identified that satisfies this hypothesis. Vascular endothelial growth factor/vascular permeability factor, hereafter referred to as VEGF, is a likely candidate for an ocular angiogenic factor because it is a secreted mitogen, specific for endothelial cells, and is upregulated by hypoxia. We investigated the association of VEGF with the development of experimental iris neovascularization in the cynomolgus monkey. Following the production of retinal ischemia by laser occlusion of all branch retinal veins, VEGF was increased in the aqueous fluid, and the aqueous VEGF levels changed synchronously and proportionally with the severity of iris neovascularization. Northern analysis and in situ hybridization revealed that VEGF messenger RNA is upregulated in the ischemic retina. These observations support the hypothesis that ocular neovascularization is regulated by a diffusible factor and identify VEGF as a likely candidate for a retina-derived vascular permeability and angiogenesis factor in vivo.

Animals↗

PubMed1993

Synthesis and secretion of vascular permeability factor/vascular endothelial growth factor by human retinal pigment epithelial cells.

The presence of the secreted angiogenic molecule, vascular permeability factor/vascular endothelial growth factor, was investigated in human retinal pigment epithelial cells. Northern and in situ hybridization analysis of cultured cells identified vascular permeability factor/vascular endothelial growth factor mRNA. Western analysis of cell lysates identified a 42 kD protein that comigrated with human recombinant vascular permeability factor/vascular endothelial growth factor. Immunoassay measurements detected vascular permeability factor/vascular endothelial growth factor protein in cell lysates and conditioned media in vitro and in cell lysates isolated directly from post-mortem eyes. These data demonstrate that human retinal pigment epithelial cells can synthesize the secreted angiogenic peptide vascular permeability factor/vascular endothelial growth factor in vitro and in situ. The production and secretion of this factor by human retinal pigment epithelial cells may be important in the pathogenesis of ocular neovascularization.

Antisense Elements (Genetics)↗

PubMed1992

Monoclonal antibody ST1 identifies an antigen that is abundant in the axolotl and newt limb stump but is absent from the undifferentiated regenerate.

Monoclonal antibodies (mAb) utilized in regeneration studies to date identify antigens that are up-regulated in the blastema. We obtained a monoclonal antibody, designated ST1 (Stump 1), that is reactive to an extracellular matrix (ECM) antigen exhibiting the opposite distribution; ST1 is an abundant antigen of the limb stump soft tissues but is absent from within the blastema. The border between abundance and absence of mAb ST1 reactivity was sharp and extended as a concavity into the stump. This distinct dichotomy led to further studies relevant to understanding how this extracellular matrix antigen is modulated during regeneration. mAb ST1 reactivity decreased in the internal tissues at the distal end of the limb prior to blastema formation and remained absent until the onset of differentiation. The initial decrease in mAb ST1 reactivity was dependent on the combined effects of injury and the wound epithelium but was nerve independent. At blastema stages of regeneration, the distribution of tenascin, ascertained by mAb MT1 reactivity, closely matched the area without reactivity to mAb ST1. The spatial and temporal distribution of the ST1 antigen in unamputated limbs and during regeneration did not correspond to any previously described ECM component.

Ambystoma mexicanum↗

PubMedDate not supplied

Segregation of COPI-rich and anterograde-cargo-rich domains in endoplasmic-reticulum-to-Golgi transport complexes.

Membrane traffic between the endoplasmic reticulum (ER) and the Golgi complex is regulated by two vesicular coat complexes, COPII and COPI. COPII has been implicated in the selective packaging of anterograde cargo into coated transport vesicles budding from the ER [1]. In mammalian cells, these vesicles coalesce to form tubulo-vesicular transport complexes (TCs), which shuttle anterograde cargo from the ER to the Golgi complex [2] [3] [4]. In contrast, COPI-coated vesicles are proposed to mediate recycling of proteins from the Golgi complex to the ER [1] [5] [6] [7]. The binding of COPI to COPII-coated TCs [3] [8] [9], however, has led to the proposal that COPI binds to TCs and specifically packages recycling proteins into retrograde vesicles for return to the ER [3] [9]. To test this hypothesis, we tracked fluorescently tagged COPI and anterograde-transport markers simultaneously in living cells. COPI predominated on TCs shuttling anterograde cargo to the Golgi complex and was rarely observed on structures moving in directions consistent with retrograde transport. Furthermore, a progressive segregation of COPI-rich domains and anterograde-cargo-rich domains was observed in the TCs. This segregation and the directed motility of COPI-containing TCs were inhibited by antibodies that blocked COPI function. These observations, which are consistent with previous biochemical data [2] [9], suggest a role for COPI within TCs en route to the Golgi complex. By sequestering retrograde cargo in the anterograde-directed TCs, COPI couples the sorting of ER recycling proteins [10] to the transport of anterograde cargo.

Animals↗