Connexin 43 (Cx43) is a protein expressed in a variety of mammalian tissues. However, the lack of specific blockers and the absence of known genetic mutants have hampered the investigation of the function of this protein. Cx43-null mice die shortly after birth, thus preventing functional studies in vivo. Here, we report the generation and characterization of a vascular endothelial cell-specific deletion of the Cx43 gene (VEC Cx43 KO) in mice by using the loxP͞Cre system. Using homologous recombination, a mouse line was created carrying loxP sites flanking exon 2 of the Cx43 gene (''floxed'' mice). To produce cell specific deletion of the Cx43 gene, these mice were crossed with animals from a line carrying the Tie 2-Cre transgene. The homozygous VEC Cx43 KO mice survived to maturity. However, they were hypotensive and bradycardic when compared with heterozygous VEC Cx43 KO mice, or to the floxed Cx43 gene mice. The hypotension was associated with marked elevation of plasma nitric oxide (NO) levels as well as elevated plasma angiotensin (Ang) I and II. We hypothesize that endothelial cell Cx43 plays a key role in the formation and͞or action of NO, and that the elevation of Ang II is a secondary event. The specific cellular basis for the hypotension remains to be established, but our findings support the idea that endothelial Cx43 gap junctions are involved in maintaining normal vascular function; moreover, these animals provide the opportunity to determine more clearly the role of endothelial Cx43 in vascular development and homeostasis.

In the vessel wall, endothelial cells are metabolically and electrically coupled to each other and to the adjacent smooth muscle cells by gap junctions composed of connexins. Gap junctions may be formed from combinations of several different connexin proteins, and deletion of one connexin can lead to modification of the expression of another. To reveal a possible interaction between connexin40 (Cx40) and connexin43 (Cx43) in endothelium, we studied their distribution in vessels from C57Bl/6 and Cx40 knockout mice (Cx40-/-) using immunoblots and immunocytochemistry on aortic cross sections and en face whole mounts. En face preparations from C57Bl/6 mice revealed two distinct pools of Cx43, one pericellular and the other intracellular. Cx40 was largely restricted to the periphery of the cells, and in Cx40-/- mice it was, as expected, undetectable. In the Cx40-/- mice, total Cx43 protein was also modestly reduced (immunoblots), but there was a major redistribution of the protein within the cell. The pericellular component of Cx43 was rendered virtually undetectable, and the intracellular compartments were normal or even slightly elevated. Smooth muscle Cx43 was also reduced in the Cx40-/- animals. These findings indicate that the cellular distribution of Cx43 is dependent on the presence of Cx40, and in view of the profound effects on the pericellular pool of the Cx43, the findings suggest that interactions between Cx40 and Cx43 regulate communication between endothelial cells and perhaps between smooth muscle and endothelial cells as well.

Connexin43 is a major component of the gap junctions between pigmented and non-pigmented cells of the double-layered epithelium in the ciliary body of the eye. We directly tested the hypothesis that gap junctions play a crucial role in the production of the aqueous humor by inactivating the GJA1 (connexin43) gene in the pigmented epithelium with cre-loxP technology. To accomplish this, we crossed a line expressing cre recombinase driven by the nestin promoter and a line with floxed connexin43 alleles. Resultant lines exhibited loss of connexin43 from the pigmented epithelium, iris, retinal pigment epithelium and the lens. We observed plasma proteins in the aqueous humor and pathological changes consistent with a loss of intraocular pressure. As the ciliary body is responsible for aqueous humor production, these data support the hypothesis that the gap junctions between pigmented and non-pigmented epithelium are necessary for production of the aqueous humor that is in turn required for the generation of normal intraocular pressure and nourishment of the postnatal lens. The loss of connexin43 expression in the iris correlated with a separation of the posterior pigmented epithelium from the anterior myoepithelium and with meiosis, possibly resulting from a loss of function of the dilator pupillae.

Objective-Vascular disease alters and reduces connexin expression and a reduction in connexin 43 (Cx43) expression diminishes the extent of atherosclerosis observed in a high-cholesterol diet murine model. We hypothesized that connexins might play a role in the smooth muscle cell response to vascular injury. Methods and Results-We therefore studied a line of smooth muscle cell-specific, Cx43 gene knockout mice (SM Cx43 KO) in which the carotid arteries were injured, either by vascular occlusion or by a wire injury. In the SM Cx43 KO mice both types of injury manifested accelerated growth of the neointima and of the adventitia. Isolated vascular smooth muscle cells from the SM Cx43 KO mice grew at a slightly faster rate in culture, and to marginally higher saturation densities than those of control mice, but these changes were not adequate to explain the large changes in the injured vessels. Conclusions-These observations provide direct evidence that smooth muscle Cx43 gap junctions play a multi-faceted role in modulating the in vivo growth response of vascular smooth muscle cells to vascular injury. (Arterioscler Thromb Vasc Biol.