Heterocellular Contact at the Myoendothelial Junction Influences Gap Junction Organization

Abstract

Abstract-Heterocellular communication between vascular smooth muscle cells (VSMC) and endothelial cells (EC) at the myoendothelial junction (MEJ) is a critical part of control of the arteriolar wall. We have developed an in vitro model of the MEJ composed of primary cultures of murine EC and VSMC. Immunoctytochemistry and immunoblots demonstrated Cx37 and Cx43 in both cell types, whereas Cx40 was found only in EC. Cx37 was excluded from the MEJ in both EC and VSMC. Connexin composition as well as functionality of the gap junctions at the MEJ was assessed by measuring diffusional transfer of biocytin and Cy3. Using connexin-specific blockers and manipulations of expression of individual connexin proteins, we confirmed that Cx37 is not a part of EC-VSMC coupling, and we demonstrated that heterotypic gap junctions are functional at the MEJ. We speculate that specific gap junction organization may be a vital component of EC-VSMC contact at the MEJ. One form of communication between endothelial cells (EC) and vascular smooth muscle cells (VSMC) appears to be via extensions of the 2 cell types that pass through the internal elastic lamina and make contacts called myoendothelial junctions (MEJ). [3][4][5] Gap junctions at the points of cell-cell contact in the MEJ form pathways for flow of second messengers or electrical current between the two cells, 4,6 -8 and this communication is critical in unifying their function.Although the physiological importance of the MEJ is well known, research on this structure is limited because of the anatomical location of MEJ, limited access to both cell types in vivo, bioavailablity of inhibitors in vivo, and isolation of sufficient quantities of protein from both cell types. 2 In view of these complexities, an in vitro model would be highly desirable. We, therefore, developed such a model from primary cultures of mouse EC and VSMC. We used immunoblots to identify the connexins expressed and immunocytochemistry to identify connexin proteins trafficked to the MEJ. In addition, we measured movement of diffusible solutes through the gap junctions at the MEJ and used a combination of gap junctional blockers including connexin mimetic peptides. These data allowed us to ascertain the functional and spatial organization of connexins at the MEJ. Materials and MethodsAn expanded Materials and Methods section is available in the online supplement at http://circres.ahajournals.org. AnimalsAll procedures and protocols in this study were approved by the University of Virginia Animal Care and Use Committee. All mice (including Cx37 Ϫ/Ϫ and Cx40 Ϫ/Ϫ ; kind gifts of D.L. Paul 9,10 ) were on a C57Bl/6 background. Cell CultureIsolation of mouse EC was based on methods described by Srinivasan et al 11 and Shi et al,12 with modifications. EC were used between passages 2 and 4. Isolation of VSMC was based on the method of Owens et al, 13 with modifications. VSMC were used between passages 2 and 3.A vascular cell coculture was created by inverting a FBS precoated polyester Transwell insert (Corning) and...

Oxidized Phospholipids Alter Vascular Connexin Expression, Phosphorylation, and Heterocellular Communication

“…We compared these in vivo findings with responses of a vascular cell coculture model. 17 In this model, OxPAPC treatment led to major alterations in connexin protein and to phosphorylation of Cx43, an effect that has been shown to inhibit gap junction permeability. 18 We conclude that changes in connexin expression, heterocellular communication between EC and VSMC, and phosphorylation of Cx43 induced by OxPAPC may be important pathological developments during the course of atherosclerotic disease.…”

“…17 Primary cultures of mouse VSMC were plated on the underside of a Transwell insert with a pore diameter of 0.4 m. On VSMC confluence, the Transwell insert was inverted, and primary cultures of EC were plated on the top side of the Transwell insert for at least 2 days after confluence before being used. When lipids were added to the vascular cell coculture, they were added in 7.5% FBS for 8 hours.…”

“…For immunoblot analysis, the vascular cell cocultures were snap-frozen with liquid nitrogen, and EC and VSMC were removed with Laemmli sample buffer (Bio-Rad) and run on a 12.5% SDS-PAGE. Antibodies used for identification of connexins have previously been shown to be specific 17,22 and include anti-Cx37 (ADI; Zymed), anti-Cx40 (ADI; Zymed), anti-Cx43 (Sigma [intracellular loop for immunoblot; C-terminal tail for immunohistochemistry]), and anti-tyrosine 265-specific Cx43 (Santa Cruz Biotechnology). Antibodies raised against Cx45 were not considered specific (eg, Theis et al 23 ).…”

“…As previously described, EC were loaded with biocytin (Molecular Probes), and biocytin transfer to VSMC was assessed by cutting transverse sections of the vascular cell coculture and exposing the biocytin with fluorophore-conjugated streptavidin. 17 The pixel intensity in the XZ direction was measured every 1 m down the length of the Transwell pore. The gap junction inhibitor 18 ␣-glycyrrhetinic acid (18 ␣-GA; 50 mol/L; Sigma) was used as previously described.…”

“…The gap junction inhibitor 18 ␣-glycyrrhetinic acid (18 ␣-GA; 50 mol/L; Sigma) was used as previously described. 17 …”