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Background— Endothelial dysfunction is the initiating event of atherosclerosis. The expression of connexin40 (Cx40), an endothelial gap junction protein, is decreased during atherogenesis. In the present report, we sought to determine whether Cx40 contributes to the development of the disease.Methods and Results— Mice with ubiquitous deletion of Cx40 are hypertensive, a risk factor for atherosclerosis. Consequently, we generated atherosclerosis-susceptible mice with endothelial-specific deletion of Cx40 (Cx40del mice). Cx40del mice were indeed not hypertensive. The progression of atherosclerosis was increased in Cx40del mice after 5 and 10 weeks of a high-cholesterol diet, and spontaneous lesions were observed in the aortic sinuses of young mice without such a diet. These lesions showed monocyte infiltration into the intima, increased expression of vascular cell adhesion molecule-1, and decreased expression of the ecto-enzyme CD73 in the endothelium. The proinflammatory phenotype of Cx40del mice was confirmed in another model of induced leukocyte recruitment from the lung microcirculation. Endothelial CD73 is known to induce antiadhesion signaling via the production of adenosine. We found that reducing Cx40 expression in vitro with small interfering RNA or antisense decreased CD73 expression and activity and increased leukocyte adhesion to mouse endothelial cells. These effects were reversed by an adenosine receptor agonist.Conclusions— Cx40-mediated gap junctional communication contributes to a quiescent nonactivated endothelium by propagating adenosine-evoked antiinflammatory signals between endothelial cells. Alteration in this mechanism by targeting Cx40 promotes leukocyte adhesion to the endothelium, thus accelerating atherosclerosis.

Pax-1, a member of a murine multigene family, belongs to the paired box-containing class of developmental control genes first identified in Drosophila. The Pax-1 gene encodes a sequence-specific DNA-binding protein with transcriptional activating properties and has been found to be mutated in the autosomal recessive mutation undulated (un) on mouse chromosome 2 with vertebral anomalies along the entire rostrocaudal axis. By radioactive in situ hybridization (ISH) using a fragment from the murine Pax-1 paired box that is almost identical to the respective sequences from the cognate human gene HuP48 and fluorescence in situ hybridization (FISH) using a complete mouse Pax-1 cDNA, we have assigned the human homologue of murine Pax-1, the PAX1 locus, to chromosome 20p. The map position of PAX1 after FISH (FL-pter value of 0.34 +/- 0.04) corresponds to band p11.2. These results confirm the exceptional homology between human chromosome 20 and the distal segment of mouse chromosome 2, extending from bands F to G, and add PAX1 to the group of genes on 20p like PTPA, PRNP, SCG1, BMP2A, which are located in proximity on both chromosomes.

A murine multigene family has been identified that shares a conserved sequence motif, the paired box, with developmental control and tissue-specific genes of Drosophila. To date five murine paired box-containing genes (Pax genes) have been described and one, Pax-1, has been associated with the developmental mutant phenotype undulated. Here we describe the paired boxes of three novel Pax genes, Pax-4, Pax-5, and Pax-6. Comparison of the eight murine paired domains of the mouse, the five Drosophila paired domains, and the three human paired domains shows that they fall into six distinct classes: class I comprises Pox meso, Pax-1, and HuP48; class II paired, gooseberry-proximal, gooseberry-distal, Pax-3, Pax-7, HuP1, and HuP2; class III Pax-2, Pax-5, and Pax-8; class IV Pax-4; class V Pox neuro; and class VI Pax-6. Pax-1 and the human gene HuP48 have identical paired domains, as do Pax-3 and HuP2 as well as Pax-7 and HuP1, and are likely to represent homologous genes in mouse and man. Identical intron-exon structure and extensive sequence homology of their paired boxes suggest that several Pax genes represent paralogs. The chromosomal location of all novel Pax genes and of Pax-3 and Pax-7 has been determined and reveals that they are not clustered.

undulated (un) homozygous mice exhibit vertebral malformations along the entire rostro-caudal axis. Pax 1, a murine paired box-containing gene, is expressed in ventral sclerotome cells and later in intervertebral disks along the entire vertebral column. We localized the Pax 1 gene on chromosome 2 between beta 2-microglobulin and the agouti locus to an area where un maps. DNA analysis of the un mutant revealed a point mutation in a highly conserved part of the paired box of Pax 1, leading to a Gly-Ser replacement. The chromosomal location and the mutation in the paired box of un mice in conjunction with Pax 1 gene expression in wild-type mice implicate a causative role of Pax 1 in generation of the vertebral column.

U. R. Deutsch