Endothelial Connexin Research Articles

Endothelial connexin hemichannels as a novel endothelial regulatory mechanism of vasodilation

The vascular endothelium regulates vasomotor tone in arterioles through intercellular conduction of electrical signals by gap junctions. Endothelial vasodilation is mediated by nitric oxide (NO) and by a hyperpolarizing current initiated in the endothelium. Both signals are transmitted to smooth muscle cells evoking relaxation. Endothelial cells express connexin (Cx) 37,40,43. Cx hemichannels allow the exchange of ions and molecules between intracellular and extracellular compartments that may be associated with endothelial electrical activity. Whether or not Cx hemichannels regulate vasomotor tone is unknown. We evaluated the role of Cx‐hemichannels in the vasodilation process by measuring dye uptake in primary culture of endothelial cells and in isolated mouse mesenteric arterioles. We detected that both acetylcholine (ACh) and bradykinin (BK) increase dye permeability, which was inhibited by connexin mimetic peptides that block Cx43 hemichannels (Gap19). We determined that NO mediates Cx43 hemichannels activity inasmuch as blocking NOS inhibits hemichannel activity. Using biotin switch assay, we observed that ACh induced S‐nitrosylation only in Cx43 at cysteine 271. ACh and BK open endothelial Cx43 hemichannels activity and promote endothelial Ca2+entry from the extracellular space and lead to endothelium hyperpolarization signaling in isolated mesenteric arteries. Blocking Cx43‐hemichannels reduces calcium entry and hyperpolarization. To further assess the physiological role of Cx43 endothelial hemichannels activity, we cannulated isolated mesenteric arterioles and studied ACh‐induced arteriolar relaxation in the presence and absence of connexin mimetic peptides that block Cx43 hemichannels (Gap19) and Cx40 (40Gap 27, a connexin mimetic peptide). Gap19 attenuates ACh‐induced relaxation, but 40Gap27 fails to reduce relaxation. These results indicate that endothelial Cx43 hemichannels participate in arteriolar dilatation. In addition, we replaced cysteine 271 by serine to create a Cx43C271S mouse that failed to cause S‐nitrosylation of Cx43. We detected that this knockin mouse displayed hypertension compared to wild type mice, indicating that Cx43 hemichannels and S‐nitrosylated Cx43 play a role in vascular resistance. Our results suggest that Cx43 hemichannels are a potential therapeutic target in hypertension and vascular disease.

Endothelial Connexins in Developmental and Pathological Angiogenesis.

Connexins (Cxs) constitute a large family of transmembrane proteins that form gap junction channels, which enable the direct transfer of small signaling molecules from cell to cell. In blood vessels, Cx channels allow the endothelial cells (ECs) to respond to external and internal cues as a whole and, thus, contribute to the maintenance of vascular homeostasis. While the role of Cxs has been extensively studied in large arteries, a growing body of evidence suggests that they also play a role in the formation of microvascular networks. Since the formation of new blood vessels requires the coordinated response of ECs to external stimuli, endothelial Cxs may play an important role there. Recent studies in developmental and pathologic models reveal that EC Cxs regulate physiological and pathological angiogenesis through canonical and noncanonical functions, making these proteins potential therapeutic targets for the development of new strategies aimed at a better control of angiogenesis.

Chronic Hypoxia Decreases Endothelial Connexin 40, Attenuates Endothelium-Dependent Hyperpolarization-Mediated Relaxation in Small Distal Pulmonary Arteries, and Leads to Pulmonary Hypertension.

BackgroundAbnormal endothelial function in the lungs is implicated in the development of pulmonary hypertension; however, there is little information about the difference of endothelial function between small distal pulmonary artery (PA) and large proximal PA and their contribution to the development of pulmonary hypertension. Herein, we investigate endothelium‐dependent relaxation in different orders of PAs and examine the molecular mechanisms by which chronic hypoxia attenuates endothelium‐dependent pulmonary vasodilation, leading to pulmonary hypertension.Methods and ResultsEndothelium‐dependent relaxation in large proximal PAs (second order) was primarily caused by releasing NO from the endothelium, whereas endothelium‐dependent hyperpolarization (EDH)–mediated vasodilation was prominent in small distal PAs (fourth–fifth order). Chronic hypoxia abolished EDH‐mediated relaxation in small distal PAs without affecting smooth muscle–dependent relaxation. RNA‐sequencing data revealed that, among genes related to EDH, the levels of Cx37, Cx40, Cx43, and IK were altered in mouse pulmonary endothelial cells isolated from chronically hypoxic mice in comparison to mouse pulmonary endothelial cells from normoxic control mice. The protein levels were significantly lower for connexin 40 (Cx40) and higher for connexin 37 in mouse pulmonary endothelial cells from hypoxic mice than normoxic mice. Cx40 knockout mice exhibited significant attenuation of EDH‐mediated relaxation and marked increase in right ventricular systolic pressure. Interestingly, chronic hypoxia led to a further increase in right ventricular systolic pressure in Cx40 knockout mice without altering EDH‐mediated relaxation. Furthermore, overexpression of Cx40 significantly decreased right ventricular systolic pressure in chronically hypoxic mice.ConclusionsThese data suggest that chronic hypoxia‐induced downregulation of endothelial Cx40 results in impaired EDH‐mediated relaxation in small distal PAs and contributes to the development of pulmonary hypertension.

Endothelial connexins in vascular function.

Gap junctions are essential for intercellular crosstalk in blood and lymphatic vasculature. These clusters of intercellular channels ensure direct communication among endothelial cells and between endothelial and smooth muscle cells, and the synchronization of their behavior along the vascular tree. Gap junction channels are formed by connexins; six connexins form a connexon or hemichannel and the docking of two connexons result in a full gap junction channel allowing for the exchange of ions and small metabolites between neighboring cells. Recent evidence indicates that the intracellular domains of connexins may also function as an interaction platform (interactome) for other proteins, thereby regulating their function. Interestingly, fragments of Cx proteins generated by alternative internal translation were recently described, although their functions in the vascular wall remain to be uncovered. Variations in connexin expression are observed along different types of blood and lymphatic vessels; the most commonly found endothelial connexins are Cx37, Cx40, Cx43 and Cx47. Physiological studies on connexin-knockout mice demonstrated the essential roles of these channel-forming proteins in the coordination of vasomotor activity, endothelial permeability and inflammation, angiogenesis and in the maintenance of fluid balance in the body.

Adenoviral transduction of EGFR into pregnancy-adapted uterine artery endothelial cells remaps growth factor induction of endothelial dysfunction

During pregnancy, uterine vascular vasodilation is enhanced through adapted Ca2+ signaling, facilitated through increased endothelial connexin 43 (Cx43) gap junctional communication (GJC). In preeclampsia (PE), this adaptive response is missing. Of note, the angiogenic factor VEGF can also act via Src and ERK to close Cx43 gap junctions. While VEGFR2 is necessary for such closure, a role VEGFR1 is less clear. We reasoned if VEGFR2 is acting alone, then substituting another growth factor receptor with VEGFR2-like signaling should have the same effect. In uterine artery endothelial cells derived from pregnant sheep (P-UAEC), endogenous EGFR expression is very low. When we used adenovirus to raise EGFR, we also dose-dependently induced EGF-sensitive Cx43 phosphorylation mainly via ERK, and corresponding loss of Ca2+ bursts, but eliminated VEGF effects on phosphorylation of Cx43 or loss of Ca2+ bursting. This surprising observation suggests that while activated EGFR may indeed substitute for VEGFR2, it also sequesters a limited pool of effector molecules needed for VEGFR2 to phosphorylate Cx43. Thus, low endogenous EGFR expression in P-UAEC may be a necessary strategy to allow VEGFR-2 control of GJC, a first step in initiating angiogenesis in healthy pregnancy. Of further note, trophoblasts are rich in EGFR, and we have demonstrated shed PLAP+/EGFR + extracellular vesicles in maternal circulation in first trimester plasma samples using nanoscale high resolution flow cytometry. Collectively our data suggest that placenta derived exosomes positive for EGFR should be further considered as a possible cause of endothelial dysfunction in women with PE.

Syncytiotrophoblast Extracellular Vesicles from Late-Onset Preeclampsia Placentae do not induce a Pro-Inflammatory immune response in THP-1 Macrophages

During pregnancy, uterine vascular vasodilation is enhanced through adapted Ca2+ signaling, facilitated through increased endothelial connexin 43 (Cx43) gap junctional communication (GJC). In preeclampsia (PE), this adaptive response is missing. Of note, the angiogenic factor VEGF can also act via Src and ERK to close Cx43 gap junctions. While VEGFR2 is necessary for such closure, a role VEGFR1 is less clear. We reasoned if VEGFR2 is acting alone, then substituting another growth factor receptor with VEGFR2-like signaling should have the same effect. In uterine artery endothelial cells derived from pregnant sheep (P-UAEC), endogenous EGFR expression is very low. When we used adenovirus to raise EGFR, we also dose-dependently induced EGF-sensitive Cx43 phosphorylation mainly via ERK, and corresponding loss of Ca2+ bursts, but eliminated VEGF effects on phosphorylation of Cx43 or loss of Ca2+ bursting. This surprising observation suggests that while activated EGFR may indeed substitute for VEGFR2, it also sequesters a limited pool of effector molecules needed for VEGFR2 to phosphorylate Cx43. Thus, low endogenous EGFR expression in P-UAEC may be a necessary strategy to allow VEGFR-2 control of GJC, a first step in initiating angiogenesis in healthy pregnancy. Of further note, trophoblasts are rich in EGFR, and we have demonstrated shed PLAP+/EGFR + extracellular vesicles in maternal circulation in first trimester plasma samples using nanoscale high resolution flow cytometry. Collectively our data suggest that placenta derived exosomes positive for EGFR should be further considered as a possible cause of endothelial dysfunction in women with PE.

Single and fractionated ionizing radiation induce alterations in endothelial connexin expression and channel function

Radiotherapy is an effective treatment for most tumor types. However, emerging evidence indicates an increased risk for atherosclerosis after ionizing radiation exposure, initiated by endothelial cell dysfunction. Interestingly, endothelial cells express connexin (Cx) proteins that are reported to exert proatherogenic as well as atheroprotective effects. Furthermore, Cxs form channels, gap junctions and hemichannels, that are involved in bystander signaling that leads to indirect radiation effects in non-exposed cells. We here aimed to investigate the consequences of endothelial cell irradiation on Cx expression and channel function. Telomerase immortalized human Coronary Artery/Microvascular Endothelial cells were exposed to single and fractionated X-rays. Several biological endpoints were investigated at different time points after exposure: Cx gene and protein expression, gap junctional dye coupling and hemichannel function. We demonstrate that single and fractionated irradiation induce upregulation of proatherogenic Cx43 and downregulation of atheroprotective Cx40 gene and protein levels in a dose-dependent manner. Single and fractionated irradiation furthermore increased gap junctional communication and induced hemichannel opening. Our findings indicate alterations in Cx expression that are typically observed in endothelial cells covering atherosclerotic plaques. The observed radiation-induced increase in Cx channel function may promote bystander signaling thereby exacerbating endothelial cell damage and atherogenesis.

The Functional Implications of Endothelial Gap Junctions and Cellular Mechanics in Vascular Angiogenesis.

Angiogenesis—the sprouting and growth of new blood vessels from the existing vasculature—is an important contributor to tumor development, since it facilitates the supply of oxygen and nutrients to cancer cells. Endothelial cells are critically affected during the angiogenic process as their proliferation, motility, and morphology are modulated by pro-angiogenic and environmental factors associated with tumor tissues and cancer cells. Recent in vivo and in vitro studies have revealed that the gap junctions of endothelial cells also participate in the promotion of angiogenesis. Pro-angiogenic factors modulate gap junction function and connexin expression in endothelial cells, whereas endothelial connexins are involved in angiogenic tube formation and in the cell migration of endothelial cells. Several mechanisms, including gap junction function-dependent or -independent pathways, have been proposed. In particular, connexins might have the potential to regulate cell mechanics such as cell morphology, cell migration, and cellular stiffness that are dynamically changed during the angiogenic processes. Here, we review the implication for endothelial gap junctions and cellular mechanics in vascular angiogenesis.

Changes in endothelial connexin 43 expression inversely correlate with microvessel permeability and VE-cadherin expression in endotoxin-challenged lungs.

Endothelial barrier restoration reverses microvessel hyperpermeability and facilitates recovery from lung injury. Because inhibiting connexin 43 (Cx43)-dependent interendothelial communication blunts hyperpermeability in single microvessels, we determined whether endothelial Cx43 levels correlate with changes in microvessel permeability during recovery from lung injury. Toward this, bacterial endotoxin was instilled intratracheally into rat lungs, and at different durations postinstillation the lungs were isolated and blood perfused. Microvessel Cx43 expression was quantified by in situ immunofluorescence and microvessel permeability via a fluorescence method. To supplement the immunofluorescence data, protein levels were determined by immunoblots of lung tissue from endotoxin-instilled rats. Immunofluorescence and immunoblot together revealed that both Cx43 expression and microvessel permeability increased above baseline within a few hours after endotoxin instillation but declined progressively over the next few days. On day 5 postendotoxin, microvessel Cx43 declined to negligible levels, resulting in complete absence of intermicrovessel communication determined by photolytic uncaging of Ca(2+). However, by day 14, both Cx43 expression and microvessel permeability returned to baseline levels. In contrast to Cx43, expression of microvessel vascular endothelial (VE)-cadherin, a critical determinant of vascular barrier integrity, exhibited an inverse trend by initially declining below baseline and then returning to baseline at a longer duration. Knockdown of vascular Cx43 by tail vein injection of Cx43 shRNA increased VE-cadherin expression, suggesting that reduction in Cx43 levels may modulate VE-cadherin levels in lung microvessels. Together, the data suggest that endotoxin challenge initiates interrelated changes in microvessel Cx43, VE-cadherin, and microvessel permeability, with changes in Cx43 temporally leading the other responses.

S35 Bmp9 And Bmp10 Mediate Connexin Expression In Endothelial Cells: Implications For Pah And Hht

Background Germ-line mutations in the bone morphogenetic protein type-II receptor, BMPR-II, underlie 80% of heritable pulmonary arterial hypertension (PAH) cases and approximately 25% of idiopathic PAH cases. PAH may arise due to endothelial dysfunction as mice with BMPR-II deficiency exhibit increased pulmonary vascular permeability. BMP9 is an endothelial quiescence factor and is thought to maintain the integrity of the endothelium. We previously reported that BMPR-II and ALK1 are the key receptors through which BMP9 inhibits the proliferation of human pulmonary artery endothelial cells (hPAECs). We hypothesised that BMPR-II deficiency impacts on endothelial cell connectivity and may contribute to endothelial dysfunction in PAH. Methods Human pulmonary artery endothelial cells were obtained from Lonza and blood outgrowth endothelial cells (BOECs) were isolated from peripheral blood of unaffected controls or PAH patients with identified BMPR-II mutations. Cells were transfected with siRNAs targeting BMPR-II followed by stimulation with BMP9. RNA was extracted and the expression of candidate genes determined by quantitative PCR. Further siRNA studies were performed for ALK1 and endoglin siRNAs. The promotion of gap junction assembly by BMP9 and BMP10 were assessed by immunofluorescence, Western blotting and functionally using parachute assays. Results Screening of candidate BMP9-induced junctional and structural proteins highlighted a subset of endothelial connexins that are BMP9 and BMP10-responsive and dependent on BMPR-II and ALK1. BMP9 and BMP10 increased the expression of the connexins, assessed by Western blotting and immunostaining. In addition, BMP9 and BMP10 significantly increased the transfer of calcein from labelled donor cells to unlabelled acceptor cells, indicating a promotion of endothelial cell connectivity. Conclusion In addition to their roles promoting endothelial quiescence, BMP9 and BMP10 directly promote endothelial cell connectivity by increasing connexin expression and assembly. The central contributions of BMPR-II and ALK1 to this process may implicate impaired endothelial connectivity as a pathological component of PAH and HHT.

Effects of isoprenaline on endothelial connexins and angiogenesis in a human endothelial cell culture system.

Downregulation of endothelial connexins has been shown to result in impaired angiogenesis. Isoprenaline is known to upregulate Cx43 in cardiomyocytes. Effects of isoprenaline on endothelial connexins are unknown. We wanted to investigate whether isoprenaline might induce upregulation of connexins Cx37, Cx40, or Cx43 in human endothelial cells and whether it may promote angiogenesis. Human umbilical vein endothelial cells (HUVECs) were cultured until confluence (5days) and subsequently seeded in Matrigel in vitro angiogenesis assays for 18h. During the entire cell culture and angiogenesis period, cells were treated with vehicle or isoprenaline (100nM). Finally, the resulting angiogenetic network was investigated (immuno)histologically. Moreover, expression of Cx37, Cx40, and Cx43 was determined by Western blot. In addition, we measured functional intercellular gap junction coupling by dye injection using patch clamp technique. Isoprenaline resulted in significantly enhanced expression of endothelial Cx43 and to a lower degree of Cx40 and Cx37. The number of coupling cells was significantly increased. Regarding angiogenesis, we observed significantly enhanced formation of branches and a higher complexity of the tube networks with more branches/length. Isoprenaline increases endothelial connexin expression and intercellular coupling and promotes tube formation.

Endothelial connexin 32 regulates tissue factor expression induced by inflammatory stimulation and direct cell–cell interaction with activated cells

ObjectiveEndothelial cell (EC) interacts with adjacent EC through gap junction, and abnormal expression or function of Cxs is associated with cardiovascular diseases. In patients with endothelial dysfunction, the up-regulation of tissue factor (TF) expression promotes the pathogenic activation of blood coagulation, however the relationship between gap junctions and TF expression in ECs remains uncharacterized. ECs express the gap junction (GJ) proteins connexin32 (Cx32), Cx37, Cx40 and Cx43. We investigated the role of endothelial gap junctions, particularly Cx32, in modulating TF expression during vascular inflammation. Methods and resultsHuman umbilical vein endothelial cells (HUVECs) were stimulated with tumor necrosis factor-α (TNF-α) and TF activity was assessed in the presence of GJ blockers and an inhibitory anti-Cx32 monoclonal antibody. Treatment with GJ blockers and anti-Cx32 monoclonal antibody enhanced the TNF-α−induced TF activity and mRNA expression in HUVECs. TNF-α−activated effector HUVECs or mouse MS-1 cells were co-cultured with non-stimulated acceptor HUVECs and TF expression in acceptor HUVECs was detected. Effector EC induced TF expression in adjacent acceptor HUVECs through direct cell–cell interaction. Cell–cell interaction induced TF expression was reduced by anti-intercellular adhesion molecule-1 (ICAM1) monoclonal antibody. Soluble ICAM1-Fc fusion protein promotes TF expression. GJ blockers and anti-Cx32 monoclonal antibody enhanced TF expression induced by cell–cell interaction and ICAM1-Fc treatment. ConclusionBlockade of endothelial Cx32 increased TF expression induced by TNF-α stimulation and cell–cell interaction which was at least partly dependent upon ICAM1. These results suggest that direct Cx32-mediated interaction modulates TF expression in ECs during vascular inflammation.

Role of endothelial connexin 43 during recovery from lipopolysaccharide‐induced lung injury in rats (LB784)

We showed endothelial Cx43 gap junctions mediate permeability in lung microvessels. However, whether Cx43 gap junctions modulate lung microvessel permeability during recovery from an inflammatory insult is unknown. Toward this, we instilled rats with lipopolysaccharide (LPS; 2mg/kg; IT). Rats were allowed to recover for 1, 5 or 14 days. At each recovery period, the animals’ lungs were isolated and perfused with autologous blood. The pulmonary artery, left atrial and airway pressures were maintained at 10, 3 and 5 cmH2O, respectively. Via a left atrial microcatheter, we loaded microvessels with the Ca2+ indicator, fluo4 AM and Ca2+ cage, NP‐EGTA AM. Uncaging in microvessels of untreated control lungs revealed that, fluo4 fluorescence increased by 39±11% (mean±SEM; n=5 vessels) at 80 µm from the uncaging site compared to that at 0 µm. In contrast, at 5 days post‐LPS treatment the fluorescence increased only 6±2% (P<0.05, n=6). At day 14, fluorescence increase was back at control levels. Further, we determined single microvessel permeability via normalized fluorescence of FITC‐dextran (FDx20) infused into microvessels. The permeability was high at day 1 post LPS treatment (1.9±0.1; n=15 vessels) compared to control (3.6±0.2; P<0.05; n=15). In contrast, permeability recovered by day 5 (3±0.1; n=15) and returned to control levels by day 14. Together the data suggest both interendothelial communication and microvessel permeability were continuously modulated during recovery from LPS‐injury. Thus we speculate, changes in Cx43‐dependent interendothelial communication may underlie decrease in microvessel permeability during recovery from LPS‐injury.Grant Funding Source: '' Supported by NIH HL75503''

Endothelial Cx40 limits myocardial ischaemia/reperfusion injury in mice

Gap junctions are indispensable for the function of heart and blood vessels by providing electrical coupling and direct cell-to-cell transfer of small signalling molecules. Gap junction channels between neighbouring cells are composed of 12 connexins (Cx). Changes in Cx43 expression, localization, and channel properties in cardiomyocytes contribute to infarction and reperfusion injury of the heart. It is increasingly recognized that deleterious consequences of ischaemia/reperfusion (IR) are modulated by the inflammatory response and endothelial function. The role of the endothelial connexins, i.e. Cx40 and Cx37, in cardiac IR injury is, however, not known. Following 30 min ischaemia and 24 h reperfusion, we found a significant increase in myocardial infarct size in mice with endothelial-specific deletion of Cx40 (Cx40del), but not in Cx37-deficient mice. The cardioprotective effect of endothelial Cx40 was associated with a decrease in neutrophil infiltration. Moreover, beneficial effects of endothelial Cx40 were not observed in isolated Langendorff-perfused hearts, suggesting direct involvement of endothelial-leucocyte interactions in the cardiac injury. Single-dose administration of methotrexate, a CD73 activator, reduced infarct size and neutrophil infiltration into the infarcted myocardium in Cx40del but not in control mice. Similar to Cx40del mice, CD73-deficient mice showed increased sensitivity to cardiac IR injury, which could not be conversed by methotrexate. Endothelial Cx40, but not Cx37, is implicated in resistance of the heart to IR injury by activation of the CD73 pathway. Thus, the Cx40-CD73 axis may represent an interesting target for controlling reperfusion damage associated with revascularization in coronary disease.

Local Effects of Pregnancy on Connexin Proteins That Mediate Ca 2+ -Associated Uterine Endothelial NO Synthesis

Uterine artery adaptations during gestation facilitate increases in uterine blood flow and fetal growth. local expression and distribution of uterine artery connexins play roles in mediating in vivo gestational eNOS activation and NO production. We established an ovine model restricting pregnancy to a single uterine horn and measured uterine blood flow, uterine artery shear stress, connexins 37/43, and P(635)eNOS protein levels in uterine artery and systemic artery (omental and renal) endothelium and connexins in vascular smooth muscle. Uterine blood flow and shear stress were locally (unilaterally) and substantially elevated by gestation. During pregnancy, uterine artery endothelial gap junction proteins connexins 37/43 were locally regulated in the gravid horn and elevated 10.3- and 25.6-fold; uterine artery endothelial P(635)eNOS and total eNOS were elevated 3.3- and 2.9-fold; whereas uterine artery vascular smooth muscle connexins 37/43 were locally elevated 12.5- and 5.9-fold, respectively. Less pronounced changes were observed in systemic vasculature except for significant pregnancy-associated increases in omental artery vascular smooth muscle connexin 43 and omental artery endothelial P(635)eNOS and total eNOS. Gap junction blockade using connexin 43, but not connexin 37-specific Gap peptides, abrogated uterine artery endothelial ATP-induced Ca(2+)-mediated NO production. Thus, uterine artery endothelial connexin 43, but not connexin 37, regulates Ca(2+)-mediated NO production required for the vasodilation to accommodate increases in uterine blood flow and shear stress during healthy pregnancies.

Ketone bodies upregulate endothelial connexin 43 (Cx43) gap junctions

Ketosis occurs as a metabolic consequence of negative energy balance in post-calving lactating dairy cows. Metabolism of free fatty acids, released from adipose tissue, generates excessive amounts of acetoacetate (AcAc), β-hydroxybutyrate (BHB), and acetone (Ac) in the liver, which are released into the blood. The effects of ketone bodies on endothelial cells include increased rates of portal vein and liver blood flow and decreased cytokine secretion in response to both bacterial and viral infections. The aim of the current study was to understand the effects of AcAc, BHB and Ac, on expression of connexin 43 (Cx43) and gap junctional intercellular coupling (GJIC) in bovine aortic endothelial cells (BAECs).Confocal microscopy, Western blotting, and real-time quantitative RT-PCR indicated that Cx43 mRNA and protein expression increased after endothelial cell exposure to ketone bodies and that this was accompanied by upregulation of GJIC and cell migration. These effects were most obvious when BAECs were treated with a combination of the three ketones. Ketone bodies were shown to activate ERK and p38 MAPK as early as 3h after treatment and an ERK inhibitor (PD98059) or p38 MAPK inhibitor (SB203580) were found to antagonise the ketone-induced increase in Cx43 protein expression. Thus, ketone bodies up-regulate Cx43 expression and GJIC in BAECs via activation of ERK and p38 MAPK.

Regulation of cardiovascular connexins by mechanical forces and junctions

Connexins form a family of transmembrane proteins that consists of 20 members in humans and 21 members in mice. Six connexins assemble into a connexon that can function as a hemichannel or connexon that can dock to a connexon expressed by a neighbouring cell, thereby forming a gap junction channel. Such intercellular channels synchronize responses in multicellular organisms through direct exchange of ions, small metabolites, and other second messenger molecules between the cytoplasms of adjacent cells. Multiple connexins are expressed in the cardiovascular system. These connexins not only experience the different biomechanical forces within this system, but may also act as effector proteins in co-ordinating responses within groups of cells towards these forces. This review discusses recent insights regarding regulation of cardiovascular connexins by mechanical forces and junctions. It specifically addresses effects of (i) shear stress on endothelial connexins, (ii) hypertension on vascular connexins, and (iii) changes in afterload and the composition of myocardial mechanical junctions on cardiac connexins.

Effect of oxidized LDL on the expression of connexins in cultured human umbilical-vein endothelial cells

The role of connexins in atherogenesis has attracted increasing attention in recent years; however, whether oxidized LDL (low density lipoprotein) is one of the pro-atherosclerotic factors that can influence the expression of endothelial connexins remains unclear. To investigate the effect of oxidized LDL on the gene expression of the gap junction proteins connexin37, connexin40 and connexin43 in cultured HUVEC (human umbilical-vein endothelial cells) in vitro, HUVEC were cultured and divided into groups before being stimulated with various concentrations of oxidized LDL for different times. mRNA expression of connexin37, connexin40 and connexin43 in each group were determined semi-quantitatively by RT-PCR (reverse transcription-PCR), while protein expression of the three connexins was investigated by immunocytochemical staining. Various concentrations of oxidized LDL down-regulated mRNA and protein expression of connexin37, and up-regulated the expression of connexin40 and connexin43. Time-dependent dynamic alterations of the mRNA expression levels of connexin37, connexin40 and connexin43 were also found in HUVEC stimulated by oxidized LDL. We conclude that oxidized LDL can change the expression of connexin genes in cultured HUVEC within a short period, and thus may be involved in the pro-atherosclerotic effect of oxidized LDL.

Cytokines, glucose and angiotensin II (ANG II) and the expression of Connexin (Cx) 37, 40 and 43 in cultured microvascular endothelial cells

Endothelial connexins emerge as molecules that strongly affect the conductive behavior of the endothelial cell layer and are involved in renin release in the kidney. Since many of the cardiovascular disease states are associated with micro‐inflammation and with increased activity of ANG II or with increased glucose, we hypothesized that these could affect expression levels of the connexins 37, 40 and 43. Immortalized microvascular endothelial cells (HMEC‐1) were exposed to Ang II (100 nM), a combination of IL6 (20 IU/ml), IFN (4 ng/ml) and TNFa (20 ng/ml) or to 25 mM Glucose for 4 hours in 50% and 100% confluent HMEC‐1 layers. Then, cells were harvested, RNA extracted and reverse transcribed and subjected to qPCR using pre‐designed primers for Cx37, Cx40 and Cx43 using GAPDH as control. Confluency by itself increased Cx37 expression, but did not affect Cx40 and Cx43 expression. The cytokine mix increased expression of 50% and 100% confluent cells 13‐ and 16‐fold (both P<0.05) for Cx40 and 2‐fold (P<0.05) and 2‐fold (NS) for Cx37. Neither ANG II or high glucose affected Cx expression. The present data indicate that inflammatory factors can affect the gene expression of Cx40 in particular. This might point toward a role for Cx40 in coupling inflammation to microvascular function and to renin release.

Regulation of endothelial connexin40 expression by shear stress

Endothelial connexin (Cx)40 plays an important role in signal propagation along blood vessel walls, modulating vessel diameter and thereby blood flow. Blood flow, in turn, has been shown to alter endothelial Cx40 expression. However, the timing and shear stress dependence of this relationship have remained unclear, as have the signal transduction pathways involved and the functional implications. Therefore, the aim of this study was to quantify the effects of shear stress on endothelial Cx40 expression, to analyze the role of phosphoinositide 3-kinase (PI3K)/Akt signaling involved, and to assess the possible functional consequences for the adaptation of microvascular networks. First-passage human umbilical vein endothelial cells were exposed to defined shear stress conditions and analyzed for Cx40 using real-time RT-PCR and immunoblot analysis. Shear stress caused long-term induction of Cx40 protein expression, with two short-term mRNA peaks at 4 and 16 h, indicating the dynamic nature of the adaptation process. Maximum shear stress-dependent induction was observed at shear levels between 6 and 10 dyn/cm(2). Simulation of this pattern of shear-dependent Cx expression in a vascular adaptation model of a microvascular network led to an improved fit for the simulated results to experimental measurements. Cx40 expression was greatly reduced by inhibiting PI3K or Akt, with PI3K activity being required for basal Cx40 expression and Akt activity taking part in its shear stress-dependent induction.