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      Possible Cytotoxic Effect of the Expression of a Connexin 43-LacZ Fusion Gene in Cells of the Vascular Wall



      Journal of Vascular Research

      S. Karger AG

      Dominant negative construct, Gap junction, Cell growth, Transgenic mice

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          Connexin 43 (Cx43) gap junctions are hypothesized to play a key role in many aspects of vascular function. In an effort to evaluate the importance of connexins in vascular function we took advantage of the fact that a Cx43-LacZ fusion protein has been reported to effectively reduce dye transfer in NIH 3T3 fibroblasts by acting as a dominant negative construct. We explored the use of this dominant negative construct in cultured vascular smooth muscle (VSM) cells and in transgenic mice. We examined the viability of cultured VSM cells expressing the Cx43-LacZ fusion protein under the control of a cytomegalovirus promoter. We also selectively expressed the dominant negative construct in the endothelial cells of transgenic mice under the control of a Tie 2 promoter. Transient transfection of cultured VSM cells led to good initial expression of the Cx43-LacZ fusion protein as evidenced by X-gal staining. Following 10 days of G418 selection, 300 cell clones were examined. None expressed the fusion protein, based on X-gal staining and Western blot analysis, but all contained the transgene, based on PCR analysis. The fusion protein was expressed in a few isolated cells, suggesting that cell division was inhibited by the fusion protein. In agreement with this finding was the fact that expression of the Cx43-LacZ fusion protein was not observed in any of seven Tie 2-Cx43-LacZ transgenic mouse lines. Moreover, a very low yield of mice carrying the transgene was observed (7/136; 5.1%). Analysis of 65 embryos at embryonic day 11.5 showed similar results. These data strongly suggest that the expression of the Cx43-LacZ fusion protein prevents the formation of both stable clones and transgenic animals. This may be due to a cytotoxic effect of the dominant negative construct or to the fact that successful cell propagation is not possible if gap junctional transmission is completely blocked.

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          Most cited references 3

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          Uniform vascular-endothelial-cell-specific gene expression in both embryonic and adult transgenic mice.

          TIE2 is a vascular endothelial-specific receptor tyrosine kinase essential for the regulation of vascular network formation and remodeling. Previously, we have shown that the 1.2-kb 5' flanking region of the TIE2 promoter is capable of directing beta-galactosidase reporter gene expression specifically into a subset of endothelial cells (ECs) of transgenic mouse embryos. However, transgene activity was restricted to early embryonic stages and not detectable in adult mice. Herein we describe the identification and characterization of an autonomous endothelial-specific enhancer in the first intron of the mouse TIE2 gene. Furthermore, combination of the TIE2 promoter with an intron fragment containing this enhancer allows it to target reporter gene expression specifically and uniformly to virtually all vascular ECs throughout embryogenesis and adulthood. To our knowledge, this is the first time that an in vivo expression system has been assembled by which heterologous genes can be targeted exclusively to the ECs of the entire vasculature. This should be a valuable tool to address the function of genes during physiological and pathological processes of vascular ECs in vivo. Furthermore, we were able to identify a short region critical for enhancer function in vivo that contains putative binding sites for Ets-like transcription factors. This should, therefore, allow us to determine the molecular mechanisms underlying the vascular-EC-specific expression of the TIE2 gene.
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            Formation of heteromeric gap junction channels by connexins 40 and 43 in vascular smooth muscle cells.

             Le D. He,  J. Burt,  S Taffet (1999)
            Connexin (Cx) 43 and Cx40 are coexpressed in several tissues, including cardiac atrial and ventricular myocytes and vascular smooth muscle. It has been shown that these Cxs form homomeric/homotypic channels with distinct permeability and gating properties but do not form functional homomeric/heterotypic channels. If these Cxs were to form heteromeric channels, they could display functional properties not well predicted by the homomeric forms. We assessed this possibility by using A7r5 cells, an embryonic rat aortic smooth muscle cell line that coexpresses Cxs 43 and 40. Connexons (hemichannels), which were isolated from these cells by density centrifugation and immunoprecipitated with antibody against Cx43, contained Cx40. Similarly, antibody against Cx40 coimmunoprecipitated Cx43 from the same connexon fraction but only Cx40 from Cx (monomer) fractions. These results indicate that heteromeric connexons are formed by these Cxs in the A7r5 cells. The gap junction channels formed in the A7r5 cells display many unitary conductances distinct from homomeric/homotypic Cx43 or Cx40 channels. Voltage-dependent gating parameters in the A7r5 cells are also quite variable compared with cells that express only Cx40 or Cx43. These data indicate that Cxs 43 and 40 form functional heteromeric channels with unique gating and conductance properties.
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              Blockade of connexin 43 expression by stable transfection of antisense cDNA in cultured vascular smooth muscle cells.

              Gap junctional communication is involved in embryogenesis, cell growth control, and coordinated contraction of cardiac myocytes. It has been hypothesized that gap junctions coordinate responses of vascular cells to constrictor or dilator stimulation. Three connexin (Cx) proteins, 37, 40, and 43, are found in the vasculature. Cx43 gap junctions are widely distributed along the vascular tree, although a precise physiologic role in vascular function is unknown because of a lack of specific functional inhibitors and of suitable animal models. To investigate the role of Cx43 in intercellular communication among vascular smooth muscle (VSM) cells, we selectively modified the expression of the Cx43 gene using antisense cDNA stable transfections in culture. Results show that in cells stably transfected with antisense Cx43 cDNA, gene expression of Cx43 could be reduced to 20% of that observed in vector-transfected cells. In spite of the mRNA and protein reduction, the antisense Cx43 cDNA-transfected cells did not show a significant reduction in dye transfer or a difference in cell growth rate as compared with control. These results suggest either that the residual amount of Cx43 protein is sufficient for dye transfer and growth control or that the dye transfer in these cells can be mediated by Cx40 or other connexin proteins. Therefore, more potent approaches, such as dominant negative and gene knockout, are required to fully block gap junctional communication in VSM cells.

                Author and article information

                J Vasc Res
                Journal of Vascular Research
                S. Karger AG
                June 2001
                25 May 2001
                : 38
                : 3
                : 203-211
                Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Va., USA
                51048 J Vasc Res 2001;38:203–211
                © 2001 S. Karger AG, Basel

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                Page count
                Figures: 5, Tables: 1, References: 22, Pages: 9
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