Shear Stress and Cyclic Circumferential Stretch, But Not Pressure, Alter Connexin43 Expression in Endothelial Cells

Hemodynamic forces play a critical role in atherogenesis, as evidenced by the focal pattern of development of atherosclerotic lesions. Whereas disturbed flow in the branches and curved regions of large arteries is proatherogenic, laminar flow in the straight parts of vessels is atheroprotective. In addition, hypertension and age-related changes in arterial stiffness are important risk factors of the disease. Hemodynamic forces induce various changes in the structure and function of vascular endothelium, many of which reflect alterations in gene expression. Endothelial cells are linked by gap junctions, which facilitate the propagation of electrical and chemical signals along the vascular wall. Using an in vitro perfusion system, we investigated the effects of pulsed unidirectional and oscillatory flows in combination with different levels of hydrostatic pressure and circumferential stretch on the expression of Cx43 in endothelial cells. Our results show that shear stress and circumferential stretch, but not pressure, modulate the expression of Cx43. In view of the distribution of this protein along the vascular tree, our findings provide new insights into the role of mechanical forces on gap junctional communication in regions prone to the development of atherosclerosis.     

Regulation of connexin 43 gene expression by cyclical mechanical stretch in neonatal rat cardiomyocytes

Myocardial cells respond to changes in the mechanical forces imposed on them with changes in myocardial tension in the short term and with structural remodeling in the long term. Since these responses involve intercellular communication, we have investigated regulation of the gap junction proteins, connexin 43 (Cx43), connexin 40 (Cx40) and connexin 37 (Cx37), by cyclical mechanical stretch. Results were compared with parallel experiments on c-fos and GAPDH. Twenty percent stretch of cultured rat cardiomyocytes caused a 3-fold increase in Cx43 mRNA levels by 2 h. c-fos mRNA levels increased after 30 min of stretch, whereas Cx40, Cx37, and GADPH mRNA did not change. Protein levels of Cx43 increased by 4 h and remained elevated for 16 h. New protein synthesis was not a requirement for the stretch-induced rise in Cx43 expression, since mRNA levels were unaffected by treatment with cycloheximide. In addition, mechanical stretch induced alkalization of cardiomyocytes that was antagonized by inhibiting Na-H exchanger (NHE). Gap junction potential (Gj) was concomitantly elevated. Chemical closure of Cx channels by insulin was followed by inhibition of NHE. In conclusion, cyclical mechanical stretch caused increased expression of the gap junction protein Cx43 in cardiomyocytes and also the Gj. The augmentation of Cx43 mRNA expression and its functional status were associated with activation of NHE.     

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.     

Shear stress-induced upregulation of connexin 43 expression in endothelial cells on upstream surfaces of rat cardiac valves

Endothelial expression of the gap junction proteins, connexin (Cx) 37, Cx40, and Cx43, varies within the vascular network. While previous studies suggest that shear stress may upregulate Cx43, it is not well understood if shear stress affects the expression of all endothelial connexins and to what extent. Endothelial cells on the upstream and downstream surfaces of cardiac valves are subjected to considerably different intensities of shear stress. We therefore reasoned that we could determine the extent hemodynamic forces affect the expression of Cx37, Cx40, and Cx43 by comparing their immunohistochemical distribution on the upstream and downstream surfaces of rat cardiac valves. We found 70- to 200-fold greater expression of Cx43 in the endothelial cells on the upstream than on the downstream surfaces. However, Cx37 was expressed almost equally in the endothelial cells on upstream and downstream surfaces, and Cx40, a major connexin in most vascular endothelial cells, was not detected on either surface. In addition to the heterogeneity in Cx43 expression, endothelial cells on the upstream surface were 35% to 65% smaller than those on the corresponding downstream surface. These results suggest that shear stress may affect endothelial cell size and Cx43 expression but not Cx37 expression.     

Flow regulates intercellular communication in HAEC by assembling functional Cx40 and Cx37 gap junctional channels

Direct cell-to-cell transfer of ions and small signaling molecules via gap junctions plays a key role in vessel wall homeostasis. Vascular endothelial gap junctional channels are formed by the connexin (Cx) proteins Cx37, Cx40, and Cx43. The mechanisms regulating connexin expression and assembly into functional channels have not been fully identified. We investigated the dynamic regulation of endothelial gap junctional intercellular communication (GJIC) by fluid flow and the participation of each vascular connexin in functional human endothelial gap junctions in vitro. Human aortic endothelial cells (HAEC) were exposed for 5, 16, and 24 h to physiological flows in a parallel-plate flow chamber. Connexin protein expression and localization were evaluated by immunocytochemistry, and functional GJIC was evaluated by dye injection. Connexin-mimetic peptide inhibitors were used to assess the specific connexin composition of functional channels. HAEC monolayers in culture exhibited baseline functional communication at a striking low level despite abundant expression of Cx43 and Cx40 localized at cell-to-cell appositions. Upon exposure to flow, GJIC by dye spread demonstrated a significant time-dependent increase from baseline levels, reaching 7.5-fold in 24 h. Inhibition studies revealed that this response was mediated primarily by Cx40, with lesser contributions of the other two vascular connexins assembled into functional homotypic and/or heterotypic channels. This is the first study to demonstrate that flow simultaneously and differentially regulates expression of the Cx37, Cx40, and Cx43 proteins and their involvement in the augmentation of intercellular communication by dye transfer in human endothelial cells in vitro.     

Levels of gap junction proteins in coronary arterioles and aorta of hamsters exposed to the cold and during hibernation and arousal.

There are marked changes in vascular dynamics during prolonged periods in the cold, entrance into hibernation, and arousal to euthermy. Cell-to-cell communication through gap junction channels plays a pivotal role in the control of vasomotor function. Multiple gap junction proteins are expressed in blood vessels, including connexins 37 (Cx37), 40 (Cx40), 43 (Cx43), and 45 (Cx45). Using immunolabeling techniques combined with confocal microscopy, we quantitated the levels of these connexins in coronary arterioles and the thoracic aorta of the golden hamster in four physiological conditions: normal control animals at euthermy; cold-exposed animals (before entrance into hibernation); during hibernation; and after 2-hr arousal from hibernation. In all groups, Cx37 was localized between endothelial cells of the aorta and Cx40 was observed between endothelial cells of coronary arterioles and the aorta. Cx43 was confined to smooth muscle cells of the aorta. Labeling for Cx45 was detected in the endothelium of the ascending aorta. The expression of Cx37 was significantly reduced in cold-exposed, hibernating, and aroused animals. Immunolabeling for Cx40 was increased in the coronary arteriolar endothelium of the cold-exposed group compared with normal controls, hibernating, and aroused animals, perhaps to facilitate intercellular communication during the prolonged circulatory changes to vascular dynamics required to maintain core temperature during cold adaptation. Cx40 expression was unchanged in the aorta. Cx43 immunoexpression in the aorta remained constant under all conditions examined. These changes in connexin expression did not occur during the rapid circulatory changes associated with arousal from hibernation.     

Shear stress-induced upregulation of connexin 43 expression in endothelial cells on upstream surfaces of rat cardiac valves

Endothelial expression of the gap junction proteins, connexin (Cx) 37, Cx40, and Cx43, varies within the vascular network. While previous studies suggest that shear stress may upregulate Cx43, it is not well understood if shear stress affects the expression of all endothelial connexins and to what extent. Endothelial cells on the upstream and downstream surfaces of cardiac valves are subjected to considerably different intensities of shear stress. We therefore reasoned that we could determine the extent hemodynamic forces affect the expression of Cx37, Cx40, and Cx43 by comparing their immunohistochemical distribution on the upstream and downstream surfaces of rat cardiac valves. We found 70- to 200-fold greater expression of Cx43 in the endothelial cells on the upstream than on the downstream surfaces. However, Cx37 was expressed almost equally in the endothelial cells on upstream and downstream surfaces, and Cx40, a major connexin in most vascular endothelial cells, was not detected on either surface. In addition to the heterogeneity in Cx43 expression, endothelial cells on the upstream surface were 35% to 65% smaller than those on the corresponding downstream surface. These results suggest that shear stress may affect endothelial cell size and Cx43 expression but not Cx37 expression.     

Expression of connexin 37, 40 and 43 in rat mesenteric arterioles and resistance arteries

Connexins are the protein constituents of gap junctions which mediate intercellular communication in most tissues. In arterioles gap junctions appear to be important for conduction of vasomotor responses along the vessel. Studies of the expression pattern of connexin isoforms in the microcirculation are sparse. We investigated the expression of the three major vascular connexins in mesenteric arterioles (diameter <50 micro m) from male Sprague-Dawley rats, since conducted vasomotor responses have been described in these vessels. The findings were compared with those obtained from upstream small resistance arteries. Indirect immunofluorescence techniques were used on whole mounts of mesenteric arterioles and on frozen sections of resistance arteries (diameter approximately 300 micro m). Mesenteric arterioles expressed Cx40 and Cx43 in the endothelial layer, and Cx37 was found in most but not all vessels. Connexins were not demonstrated in the media. In resistance arteries endothelial cells expressed Cx37, Cx40 and Cx43. Ultrastructural studies of mesenteric arterioles confirmed that gap junction plaques between endothelial cells are present, whereas myoendothelial, or smooth muscle cell gap junctions could not be demonstrated. The findings suggest that smooth muscle cells in mesenteric arterioles may not be well coupled and favour that conducted vasomotor responses in these vessels are propagated through the endothelial cell layer.     

Gap junction communication between uterine stromal cells plays a critical role in pregnancy-associated neovascularization and embryo survival

In the uterus, the formation of new maternal blood vessels in the stromal compartment at the time of embryonic implantation is critical for the establishment and maintenance of pregnancy. Although uterine angiogenesis is known to be influenced by the steroid hormones estrogen (E) and progesterone (P), the underlying molecular pathways remain poorly understood. Here, we report that the expression of connexin 43 (Cx43), a major gap junction protein, is markedly enhanced in response to E in uterine stromal cells surrounding the implanted embryo during the early phases of pregnancy. Conditional deletion of the Cx43 gene in these stromal cells and the consequent disruption of their gap junctions led to a striking impairment in the development of new blood vessels within the stromal compartment, resulting in the arrest of embryo growth and early pregnancy loss. Further analysis of this phenotypical defect revealed that loss of Cx43 expression resulted in aberrant differentiation of uterine stromal cells and impaired production of several key angiogenic factors, including the vascular endothelial growth factor (Vegf). Ablation of CX43 expression in human endometrial stromal cells in vitro led to similar findings. Collectively, these results uncovered a unique link between steroid hormone-regulated cell-cell communication within the pregnant uterus and the development of an elaborate vascular network that supports embryonic growth. Our study presents the first evidence that Cx43-type gap junctions play a critical and conserved role in modulating stromal differentiation, and regulate the consequent production of crucial paracrine signals that control uterine neovascularization during implantation.     

Functional expression of Ca²⁺ dependent mammalian transmembrane gap junction protein Cx43 in slime mold Dictyostelium discoideum

In this paper, we expressed murine gap junction protein Cx43 in Dictyostelium discoideum by introducing the specific vector pDXA. In the first step, the successful expression of Cx43 and Cx43-eGFP was verified by (a) Western blot (anti-Cx43, anti-GFP), (b) fluorescence microscopy (eGFP-Cx43 co-expression, Cx43 immunostaining), and (c) flow cytometry analysis (eGFP-Cx43 co-expression). Although the fluorescence signals from cells expressing Cx43-eGFP detected by fluorescence microscopy seem relatively low, analysis by flow cytometry demonstrated that more than 60% of cells expressed Cx43-eGFP. In order to evaluate the function of expressed Cx43 in D. discoideum, we examined the hemi-channel function of Cx43. In this series of experiments, the passive uptake of carboxyfluorescein was monitored using flow cytometric analysis. A significant number of the transfected cells showed a prominent dye uptake in the absence of Ca(2+). The dye uptake by transfected cells in the presence of Ca(2+) was even lower than the non-specific dye uptake by non-transformed Ax3 orf+ cells, confirming that Cx43 expressed in D. discoideum retains its Ca(2+)-dependent, specific gating function. The expression of gap junction proteins expressed in slime molds opens a possibility to the biological significance of intercellular communications in development and maintenance of multicellular organisms.     

Connexin45 interacts with zonula occludens-1 and connexin43 in osteoblastic cells

The relative expression of connexin43 and connexin45 modulates gap junctional communication and production of bone matrix proteins in osteoblastic cells. It is likely that changes in gap junction permeability are determined by the interaction between these two proteins. Cx43 interacts with ZO-1, which may be involved in trafficking of Cx43 or facilitating interactions between Cx43 and other proteins. In this study we sought to identify proteins that associate with Cx45 by coprecipitation in non-denaturing conditions. Cx45 was isolated with a 220-kDa protein that we identified as ZO-1. Under the same conditions, Cx43 also was isolated with anti-Cx45 antiserum from Cx45-transfected ROS cells (ROS/Cx45 cells). Cx43 antiserum could also coprecipitate ZO-1 in the transfected and untransfected ROS cells. Double label immunofluorescence studies showed that ZO-1, Cx43, and Cx45 colocalized at appositional membranes in ROS/Cx45 cells suggesting that all three proteins are normally associated in the cells. Additionally, we found that in vitro translated ZO-1 binds to the carboxyl-terminal of Cx45 indicating that there is a direct interaction between the carboxyl-terminal of Cx45 and ZO-1. These studies demonstrate that ZO-1 interacts with Cx45 as well as with Cx43, and suggest that the interaction of connexins with ZO-1 may play a role in regulating the composition of the gap junction and may modulate connexin-connexin interactions.     

Cyclic stretch downregulates arterial vascular connexin43 protein expression: an ex vivo study

Vascular cells may communicate through gap junctions that are formed by connexin (Cx) proteins. We investigated differential regulation of arterial gap junctions by steady and cyclic stretch and the underlying mechanotransduction pathways. Ex vivo culture of rabbit thoracic aortas was used to investigate regulation of Cx43 by cyclic stretch. After culturing for 6 or 24 h, Cx43 protein levels were quantified using Western blot. Cultures under a pulsatile pressure (mean 80 mmHg, pulse 30 mmHg) decreased Cx43 protein at both 6 and 24 h as compared with cultures under a steady pressure (80 mmHg). The regulation of Cx43 protein was mediated by pulsatile pressure-induced cyclic stretch, not by cyclic stress. Protein levels of active and total Src were also decreased by cyclic stretch at 24 h. The Src- specific inhibitor PP1 in steady culture only or in both steady and pulsatile culture conditions eliminated the difference in Cx43 protein levels between the two culture conditions. Addition of reactive oxygen species inhibitor apocynin to the pulsatile culture abolished the differences in Src and Cx43 protein levels between the two cultures. Thus, Src and reactive oxygen species appear to play a role in cyclic stretch-mediated regulation of Cx43 protein. These results are likely to have important implications in cardiovascular physiology and pathophysiology under conditions wherein significant alterations in the level of cyclic stretch are present.     

Endothelial connexin32 enhances angiogenesis by positively regulating tube formation and cell migration

The gap junction proteins connexin32 (Cx32), Cx37, Cx40, and Cx43 are expressed in endothelial cells, and regulate vascular functions involving inflammation, vasculogenesis and vascular remodeling. Aberrant Cxs expression promotes the development of atherosclerosis which is modulated by angiogenesis; however the role played by endothelial Cxs in angiogenesis remains unclear. In this study, we determined the effects of endothelial Cxs, particularly Cx32, on angiogenesis. EA.hy926 cells that had been transfected to overexpress Cx32 significantly increased capillary length and the number on branches compared to Cx-transfectant cells over-expressing Cx37, Cx40, and Cx43 or mock-treated cells. Treatment via intracellular transfer of anti-Cx32 antibody suppressed tube formation of human umbilical vein endothelial cells (HUVECs) compared to controls. In vitro wound healing assays revealed that Cx32-transfectant cells significantly increased the repaired area while anti-Cx32 antibody-treated HUVECs reduced it. Ex vivo aorta ring assays and in vivo matrigel plaque assays showed that Cx32-deficient mice impaired both vascular sprouting from the aorta and cell migration into the implanted matrigel. Therefore endothelial Cx32 facilitates tube formation, wound healing, vascular sprouting, and cell migration. Our results suggest that endothelial Cx32 positively regulates angiogenesis by enhancing endothelial cell tube formation and cell migration.     

Connexin43 deficiency causes dysregulation of coronary vasculogenesis

The connexin43 knockout (Cx43alpha1 KO) mouse dies at birth from outflow obstruction associated with infundibular pouches. To elucidate the origin of the infundibular pouches, we used microarray analysis to investigate gene expression changes in the pouch tissue. We found elevated expression of many genes encoding markers for vascular smooth muscle (VSM), endothelial cells, and fibroblasts, cell types that are epicardially derived and essential for coronary vasculogenesis. This was accompanied by increased expression of VEGF and genes in the TGFbeta and VEGF/Notch/Eph cell-signaling pathways known to regulate vasculogenesis/angiogenesis. Using immunohistochemistry and a VSM lacZ reporter gene, we confirmed an abundance of ectopic VSM and endothelial cells in the infundibular pouch and in some regions of the right ventricle forming secondary pouches. This was associated with distinct thinning of the compact myocardium. TUNEL labeling showed increased apoptosis in the pouch tissue, in agreement with the finding of altered expression of many apoptotic genes. Defects in vascular remodeling were indicated by a marked reduction in the branching complexity of the distal coronary arteries. In the near term KO mouse, we also observed a profusion of large coronary vascular plexuses subepicardially. This was associated with elevated epicardial expression of VEGF and abnormal epicardial cell morphology. Together, these observations indicate that dysregulated coronary vasculogenesis plays a pivotal role in formation of the infundibular pouches and suggests an essential role for Cx43alpha1 gap junctions in coronary vasculogenesis and vascular remodeling.     

N-cadherin and Cx43alpha1 gap junctions modulates mouse neural crest cell motility via distinct pathways

Our previous studies showed an essential role for connexin 43 or alpha1 connexin (Cx43alpha1) gap junctions in the modulation of neural crest cell motility. Cx43alpha1 gap junctions and N-cadherin containing adherens junctions are expressed in migrating cardiac neural crest cells. Analysis of the N-cadherin knockout (KO) mouse model revealed that N-cadherin is essential for gap junction mediated dye coupling but not for expression of Cx43alpha1 gap junctions in neural crest cells. Time lapse videomicroscopy and motion analysis showed that the motility of N-cadherin KO neural crest cells were altered, but the motility changes differed compared to Cx43alpha1 KO neural crest cells. These observations suggest that the role of N-cadherin in cell motility is not simply mediated via the modulation of Cx43alpha1 mediated cell-cell communication. This was confirmed by a parallel analysis of wnt-1 deficient neural crest cells, which also showed a reduction in dye coupling, and yet no change in cell motility. Analysis of p120 catenin (p120ctn), an Amardillo family protein known to play a role in cell motility, showed that it is colocalized with N-cadherin and Cx43alpha1 in migrating neural crest cells. This subcellular distribution was altered in the N-cadherin and Cx43alpha1 KO neural crest cells. Given these results, we propose that N-cadherin and Cx43alpha1 may modulate neural crest cell motility by engaging in a dynamic cross-talk with the cell's locomotory apparatus through p120ctn signaling.