Heterocellular interaction enhances recruitment of alpha and beta-catenins and ZO-2 into functional gap-junction complexes and induces gap junction-dependant differentiation of mammary epithelial cells

Gap junctions (GJ) are required for mammary epithelial differentiation. Using epithelial (SCp2) and myoepithelial-like (SCg6) mouse-derived mammary cells, the role of heterocellular interaction in assembly of GJ complexes and functional differentiation (β-casein expression) was evaluated. Heterocellular interaction is critical for β-casein expression, independent of exogenous basement membrane or cell anchoring substrata. Functional differentiation of SCp2, co-cultured with SCg6, is more sensitive to GJ inhibition relative to homocellular SCp2 cultures differentiated by exogenous basement membrane. Connexin (Cx)32 and Cx43 levels were not regulated across culture conditions; however, GJ functionality was enhanced under differentiation-permissive conditions. Immunoprecipitation studies demonstrated association of junctional complex components (α-catenin, β-catenin and ZO−2) with Cx32 and Cx43, in differentiation conditions, and additionally with Cx30 in heterocellular cultures. Although β-catenin did not shuttle between cadherin and GJ complexes, increased association between connexins and β-catenin in heterocellular cultures was observed. This was concomitant with reduced nuclear β-catenin, suggesting that differentiation in heterocellular cultures involves sequestration of β-catenin in GJ complexes.     

Electrotonic myofibroblast-to-myocyte coupling increases propensity to reentrant arrhythmias in two-dimensional cardiac monolayers

In pathological conditions such as ischemic cardiomyopathy and heart failure, differentiation of fibroblasts into myofibroblasts may result in myocyte-fibroblast electrical coupling via gap junctions. We hypothesized that myofibroblast proliferation and increased heterocellular coupling significantly alter two-dimensional cardiac wave propagation and reentry dynamics. Co-cultures of myocytes and myofibroblasts from neonatal rat ventricles were optically mapped using a voltage-sensitive dye during pacing and sustained reentry. The myofibroblast/myocyte ratio was changed systematically, and junctional coupling of the myofibroblasts was reduced or increased using silencing RNAi or adenoviral overexpression of Cx43, respectively. Numerical simulations in two-dimensional models were used to quantify the effects of heterocellular coupling on conduction velocity (CV) and reentry dynamics. In both simulations and experiments, reentry frequency and CV diminished with larger myofibroblast/myocyte area ratios; complexity of propagation increased, resulting in wave fractionation and reentry multiplication. The relationship between CV and coupling was biphasic: an initial decrease in CV was followed by an increase as heterocellular coupling increased. Low heterocellular coupling resulted in fragmented and wavy wavefronts; at high coupling wavefronts became smoother. Heterocellular coupling alters conduction velocity, reentry stability, and complexity of wave propagation. The results provide novel insight into the mechanisms whereby electrical myocyte-myofibroblast interactions modify wave propagation and the propensity to reentrant arrhythmias.     

Cardiac telocytes — their junctions and functional implications

Telocytes (TCs) form a cardiac network of interstitial cells. Our previous studies have shown that TCs are involved in heterocellular contacts with cardiomyocytes and cardiac stem/progenitor cells. In addition, TCs frequently establish 'stromal synapses' with several types of immunoreactive cells in various organs ( www.telocytes.com ). Using electron microscopy (EM) and electron microscope tomography (ET), we further investigated the interstitial cell network of TCs and found that TCs form 'atypical' junctions with virtually all types of cells in the human heart. EM and ET showed different junction types connecting TCs in a network (puncta adhaerentia minima, processus adhaerentes and manubria adhaerentia). The connections between TCs and cardiomyocytes are 'dot' junctions with nanocontacts or asymmetric junctions. Junctions between stem cells and TCs are either 'stromal synapses' or adhaerens junctions. An unexpected finding was that TCs have direct cell-cell (nano)contacts with Schwann cells, endothelial cells and pericytes. Therefore, ultrastructural analysis proved that the cardiac TC network could integrate the overall 'information' from vascular system (endothelial cells and pericytes), nervous system (Schwann cells), immune system (macrophages, mast cells), interstitium (fibroblasts, extracellular matrix), stem cells/progenitors and working cardiomyocytes. Generally, heterocellular contacts occur by means of minute junctions (point contacts, nanocontacts and planar contacts) and the mean intermembrane distance is within the macromolecular interaction range (10-30 nm). In conclusion, TCs make a network in the myocardial interstitium, which is involved in the long-distance intercellular signaling coordination. This integrated interstitial system appears to be composed of large homotropic zones (TC-TC junctions) and limited (distinct) heterotropic zones (heterocellular junctions of TCs).     

Hormonal modulation of gap junctions in rat ovarian follicles

Summary Homocellular gap junctions between granulosa cells and between theca interna cells, and heterocellular gap junctions between granulosa cells and oocytes persist in rat ovarian follicles for as long as 90 days following hypophysectomy. Gonadotrophic and/or steroid hormones are therefore not required for the maintenance of gap junctions between these cells during early follicular growth. However, replacement therapy with estrogen and human chorionic gonadotrophin results in amplification of gap junctions in granulosa and theca interna cells respectively. Within 24 h following hormonal stimulation, growth of gap junctions is characterized by the appearance of formation plaques as observed in freeze-fracture replicas and by the association of microfilamentous material located subadjacent to gap junction membrane observable in thin-sectioned cells.     

IGFBP6 regulates vascular smooth muscle cell proliferation and morphology via cyclin E–CDK2

Despite the high prevalence of varicose veins, the underlying pathogenesis of this disease remains unclear. The present study aims to explore the role of insulin-like growth factor binding protein 6 (IGFBP6) in vascular smooth muscle cells (VSMCs). Using a protein array approach, we identified several differentially expressed proteins between varicose great saphenous veins and normal great saphenous veins. Bioinformatic analysis showed that IGFBP6 was closely related to cell proliferation. Further validation confirmed that IGFBP6 was one of the most highly expressed proteins in varicose vein tissue. Knocking down IGFBP6 in VSMCs significantly attenuated cell proliferation and induced the S phase arrest during the cell cycle. Further experiments demonstrated that IGFBP6 knockdown increased cyclin E ubiquitination, which reduced expression of cyclin E and phosphorylation of CDK2. Furthermore, IGFBP6 knockdown arrested centrosome replication, which subsequently influenced VSMC morphology. Ultimately, IGFBP6 was validated to be involved in VSMC proliferation in varicose vein tissues. The present study reveals that IGFBP6 is closely correlated with VSMC biological function and provides unprecedented insights into the underlying pathogenesis of varicose veins.     

Gap junctions between oocyte and follicle cells in Acerentomon sp. (Insecta,Protura)

Each oocyte in the ovary of Acerentomon is surrounded by a layer of follicle cells (FC) and possesses a group of specialized, so-called chorion-producing cells (CPC). The FCs lying immediately under the CPCs form processes which make contact with the oocyte. Gap junctions occur at the points of contact between the oolemma and the membrane of the processes. A possible role of the heterocellular gap junctions in Acerentomon ovary is the coordination of development of the oocyte and CPCs.     

Troglitazone has no effect on K(ATP) channel opener induced-relaxations in rat aorta and in human saphenous veins from patients with type 2 diabetes

Troglitazone, a thiazolidinedione derivative, is an oral antidiabetic agent that enhances insulin sensitivity in insulin-resistant states. K(ATP) channels, on the other hand, have important roles protecting cardiovascular system in ischemic and/or hypoxic states. They are also important in the control of vascular tone, and therefore of blood pressure. We tested whether troglitazone can directly affect vascular K(ATP) channel opener-induced relaxations in vitro. 1, 10 or 100 microM troglitazone incubations for 30 min did not alter cromakalim (a K(ATP) channel opener)--induced relaxations in endothelium-denuded aortas from rat, saphenous veins from type 2 diabetic and nondiabetic patients. In addition, we compared the sensitivity to cromakalim in diabetic saphenous veins with that of nondiabetic veins. The concentration-response curve for cromakalim was shifted to the right in diabetic vein. pD2 values for cromakalim were 6.85+/-0.08 vs. 6.61+/-0.04 (p<0.05) in nondiabetic (n:10) and diabetic (n:7) veins respectively. % maximum response of cromakalim was also significantly decreased by 24+/-3% in diabetic veins. However, responsiveness of veins to phenylephrine or sodium nitroprusside were similar in both groups. The results obtained may be clinically useful 1. suggesting that in ischaemic and/or hypoxic insults troglitazone may not worsen vascular dilatation, through K(ATP) channel, in diabetic patients who are more prone to these conditions than healthy people, 2. providing an evidence that diabetes causes an impaired dilatation of human saphenous vein through K(ATP) channels. This may partly be related with diabetes-induced vascular complications, such as vasospasm and even hypertension. Accordingly, since saphenous veins are used as conduit vessels in coronary by-pass graft surgery, the results also suggest that the defective dilatation through K(ATP) channels may play a role on the performance of saphenous vein grafts in type 2 diabetes.     

Venous Distensibility in Patients with Varicose Veins

Forearm veins were studied to determine whether patients with primary varicosity of the saphenous veins had a generalized abnormality of the venous system. Distensibility of the superficial forearm and hand veins was measured in 25 patients with varicosity of the saphenous veins, and in 25 control subjects. Patients with saphenous varicosity had a significantly greater distensibility of the undistorted forearm veins than control subjects. Hysteresis of distensibility curves was more prnounced in patients with varicosity than in control subjects; mean hysteresis index was 0.65 ± 0.06 versus 0.28 ± 0.02 in controls. These investigations suggest that an increased distensibility of the venous system is the predisposing factor in the development of varicose veins.     

Signaling across myoendothelial gap junctions--fact or fiction?

Gap junctions interconnect vascular cells homocellularly, thereby allowing the spread of signals along the vessel wall, which serve to coordinate vessel behavior. In addition, gap junctions provide heterocellular coupling between endothelial and vascular smooth muscle cells, creating so-called myoendothelial gap junctions (MEGJs). Endothelial cells control vascular tone by the release of factors that relax vascular smooth muscle. Endothelial factors include nitric oxide, prostaglandins, and an additional dilator principle, which acts by smooth muscle hyperpolarization and is therefore named endothelium-derived hyperpolarizing factor (EDHF). Whether this principle indeed relies on a factor or on intact MEGJs, which allow direct current transfer from endothelial to smooth muscle cells, has recently been questioned. Careful studies revealed the presence of vascular cell projections that make contact through the internal elastic lamina, exhibit the typical GJ morphology, and express connexins in many vessels. The functional study of the physiological role of MEGJs is confined by the difficulty of selectively blocking these channels. However, in different vessels studied in vitro, the dilation related to EDHF was sensitive to experimental interventions that block MEGJs more or less specifically. Additionally, bidirectional electrical coupling between endothelial and smooth muscle cells was demonstrated in isolated small vessels. In marked contrast, similar approaches used in conjunction with intravital microscopy, which allows examination of vascular behavior in the intact animal, did not verify electrical or dye-coupling in different models investigated. The discrepancy between in vitro and in vivo investigations may be due to size and origin of the vessels studied using these distinct experimental approaches. Additionally, MEGJ coupling is possibly tightly controlled in vivo by yet unknown mechanisms that prevent unrestricted direct signaling between endothelial and smooth muscle cells.     

Myofibroblasts cause heterogeneous Cx43 reduction and are unlikely to be coupled to myocytes in the healing canine infarct

Following myocardial infarction (MI) inflammatory responses transform cardiac fibroblasts to myofibroblasts, which in vitro studies show form heterocellular gap junctions with cardiac myocytes via Connexin43 (Cx43). The ability to form heterocellular junctions in the intact heart and the impact of these junctions on propagation is unclear. We used a canine model of MI and characterized the distribution and quantity of myofibroblasts in surviving epicardial cells [epicardial border zone (EBZ)]. We found a significant increase in myofibroblasts within the EBZ and no gap junction plaques between myofibroblasts and myocytes. Because myofibroblasts produce IL-1β, which downregulates Cx43, we asked whether myofibroblast proliferation causes loss of Cx43 near myofibroblast clusters. In vitro studies showed that IL-1β caused loss of Cx43 and reduced coupling. Western blot showed a significant increase of IL-1β in the EBZ, and immunohistochemistry showed a loss of Cx43 in regions of myofibroblasts in the intact heart. Additionally, dye studies in intact heart showed no coupling between myocytes and myofibroblasts. To quantify the effect of myofibroblasts on propagation we used a two-dimensional subcellular computer model of the EBZ, which showed that heterogeneities in myofibroblast density lead to conduction abnormalities. In conclusion, an increase of myofibroblasts in the infarcted heart causes heterogeneous Cx43 levels, possibly as a result of the release of IL-1β and decreased cell-cell communication, which leads to conduction abnormalities following MI.     

Heterocellular cultures of pulmonary alveolar epithelial cells grown on laminin-5 supplemented matrix

The pulmonary alveolar epithelium consists of alveolar type I (AT1) and alveolar type II (AT2) cells. Interactions between these two cell types are necessary for alveolar homeostasis and remodeling. These interactions have been difficult to study in vitro because current cell culture models of the alveolar epithelium do not provide a heterocellular population of AT1 and AT2 cells for an extended period of time in culture. In this study, a new method for obtaining heterocellular cultures of AT1- and AT2-like alveolar epithelial cells maintained for 7 d on a rat tail collagen-fibronectin matrix supplemented with laminin-5 is described. These cultures contain cells that appear by their morphology to be either AT1 cells (larger flattened cells without lamellar bodies) or AT2 cells (smaller cuboidal cells with lamellar bodies). AT1-like cells stain for the type I cell marker aquaporin-5, whereas AT2-like cells stain for the type II cell markers surfactant protein C or prosurfactant protein C. AT1/AT2 cell ratios, cell morphology, and cell phenotype-specific staining patterns seen in 7-d-old heterocellular cultures are similar to those seen in alveoli in situ. This culture system, in which a mixed population of phenotypically distinct alveolar epithelial cells are maintained, may facilitate in vitro studies that are more representative of AT1-AT2 cell interactions that occur in vivo.     

Functional comparison of the human isolated femoral artery, internal mammary artery, gastroepiploic artery, and saphenous vein

Human femoral, internal mammary, and gastroepiploic arteries and saphenous veins are used as bypass grafts for coronary surgery or for reconstruction in arterial occlusive disease. We have characterized the contractile responses of these vessels to various agents that are liberated during cardiac or vascular surgery. In organ baths, U46619 (a stable thromboxane A2 mimetic), norepinephrine, endothelin-1, angiotensin II, and KCl caused concentration-dependent contractions in all vessels tested. Leukotriene C4 did not induce any contraction in the arteries, whereas a contraction was obtained in the saphenous vein rings. U46619 induced the most powerful contraction in all vessels tested. The pD2 values for each agent did not differ among the different vessels. When responses were expressed as a percentage of KCl-induced contraction, the contraction of endothelin-1 (151+/-5%) and leukotriene C4 (43+/-5%) was more significant on saphenous veins than on arteries. In conclusion, thromboxane A2 appears to be the most potent endogenous constricting agent on different human vascular beds. Our second finding is that saphenous veins are more sensitive to contract to leukotriene C4 and endothelin-1 than arteries. These properties may influence early and (or) long-term vein graft patency.     

Endothelial control of vascular tone by nitric oxide and gap junctions: a haemodynamic perspective

Local haemodynamic forces acting on the endothelium modulate vascular tone through mechanisms that normalize intimal shear stress. This flow-dependent diameter response contributes to the optimization of circulatory function and is mediated via shear stress-induced release of NO, vasodilator prostanoids and a putative endothelium-derived hyperpolarizing factor or EDHF. There is growing evidence that NO/prostanoid independent relaxations involve direct heterocellular signalling between endothelial and smooth muscle cells via gap junctions.     

Unusual features of intercellular junctions in the larvacean Oikopleura dioica

Summary In the pelagic larvacean Oikopleura dioica, the epithelium lining the alimentary tract consists of ciliated and unciliated cell types. The ciliated cells also exhibit an apical border of long microvilli. Between the microvilli, the cellular membrane often projects deeply down into the cytoplasm; the membranes of these invaginations and those of apicolateral interdigitations may be associated with one another by tight junctions. Some of these junctions may be autocellular. The tight junctions are seen by freeze-fracture to be very simple in construction, composed of a single row of intramembranous particles, which may be fused into a P-face ridge. There is a dense cytoplasmic fuzz associated with these tight junctions which may extend into adjoining zonula adhaerens-like regions. The invaginations of the apical membranes are, in addition, associated by gap junctions which may also be autocellular. More conventional homocellular and heterocellular tight and gap junctions occur along the lateral borders of ciliated cells and between ciliated and unciliated cells. These gap junctions possess a reduced intercellular cleft and typical P-face connexons arranged in macular plaques, with complementary E-face pits. Both cell types exhibit extensive stacks of basal and lateral interdigitations. The tight junctions found here are unusual in that they are associated with a dense cytoplasmic fuzz which is normally more characteristic of zonulae adhaerentes.     

The determination of the collagen and elastin amount in the human varicose vein by the computer morphometric method

The results of the works dealing with alterations of the connective tissue in varicose vein wall are not ambiguous, so the exact cause of the vein dilatation has still not been established. We were determining the collagen and elastin amounts in human varicose vein wall in comparison with non-dilated long saphenous vein by the light microscopy and computer morphometric method. We have found the lesser amount of collagen in varicose veins than in non-dilated veins, the amounts of the elastin in both the varicose and non-varicose veins were without the statistical significance.     

Coupled heterocellular arrays in the brain

Gap junctions are transcellular pathways that enable a dynamic metabolic coupling and a selective exchange of biological signaling mediators. Throughout the course of the brain development these intercellular channels are assembled into regionally and temporally defined patterns. The present review summarizes the possibilities of heterocellular gap junctional pairing in the brain parenchyma, involving glial cells, neurons and neural precursors as well as it highlights on the meaningfulness of these coupled arrays to the concept of brain functional compartments.     

Intercellular Ca2+ signalling: the artery wall

The fact that many cells in the cardiovascular system are coupled via gap junctions enables direct intercellular Ca2+ signalling. Ca2+ ions and/or inositol trisphosphate (IP3) can pass through these aqueous pores. Cell-cell coupling can occur between cells of the same type, or via special junctions between adjacent cells of different types. Homocellular coupling acts to amplify and prolong Ca2+ signals, whereas heterocellular coupling allows complex interactions between the cells, including the modulation of their respective responses. This review will focus on the interactions between cells that form the blood vessel wall, illustrating how cell-cell communication defines important physiological functions.     

Arterial Shear Stress Reduces Eph-B4 Expression in Adult Human Veins

Vein graft adaptation to the arterial environment is characterized by loss of venous identity, with reduced Ephrin type-B receptor 4 (Eph-B4) expression but without increased Ephrin-B2 expression. We examined changes of vessel identity of human saphenous veins in a flow circuit in which shear stress could be precisely controlled. Medium circulated at arterial or venous magnitudes of laminar shear stress for 24 hours; histologic, protein, and RNA analyses of vein segments were performed. Vein endothelium remained viable and functional, with platelet endothelial cell adhesion molecule (PECAM)-expressing cells on the luminal surface. Venous Eph-B4 expression diminished (p = .002), Ephrin-B2 expression was not induced (p = .268), and expression of osteopontin (p = .002) was increased with exposure to arterial magnitudes of shear stress. Similar changes were not found in veins placed under venous flow or static conditions. These data show that human saphenous veins remain viable during ex vivo application of shear stress in a bioreactor, without loss of the venous endothelium. Arterial magnitudes of shear stress cause loss of venous identity without gain of arterial identity in human veins perfused ex vivo. Shear stress alone, without immunologic or hormonal influence, is capable of inducing changes in vessel identity and, specifically, loss of venous identity.     

Selecting a treatment for primary varicose veins.

The treatment of varicose veins includes injection/compression sclerotherapy and surgical stripping or ligation or both. Surgery appears to be favoured when the saphenous system is involved or when the patient is 35 to 64 years old or presents with ankle edema or flare. On the other hand, sclerotherapy has been found to be more effective in patients with dilated superficial veins or incompetent perforating veins in the lower legs and to be more acceptable and less expensive than surgical treatment.     

Effluxes of 3,4-Dihydroxyphenylalanine, 3,4-Dihydroxyphenylglycol, and Norepinephrine from Four Blood Vessels During Basal Conditions and During Nerve Stimulation

Abstract: Effluxes of 3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenyglycol, and norepinephrine from four superfused canine blood vessels (saphenous and portal veins and mesenteric and pulmonary arteries) were studied under basal conditions and during nerve stimulation. From quantification of the compounds a series of indices of activities at neuroeffector junctions are proposed. These are (a) basal overflow of 3,4-dihydroxyphenylglycol as an index of vesicular-cytoplasmic translocation of norepinephrine, (b) the increase in 3,4-dihydroxyphenyglycol overflow attributable to nerve stimulation as an index of neuronal reuptake of norepinephrine released by stimulation, (c) the sum of the increases in overflows of norepinephrine and 3,4-dihydroxyphenylglycol attributable to nerve stimulation as an index of evoked release of norepinephrine, and (d) the efflux of 3,4-dihydroxyphenylalanine as an index of the activity of tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of norepinephrine. There were clear differences between these indices in the vessels. Correlation coefficients of the indices among vessels indicated that a high tissue norepinephrine level was associated with high biosynthetic capacity and high vesicular-cytoplasmic exchange but not with high release. There was no evidence suggesting feedback inhibition of synthesis by neuroplasmic norepinephrine—whether arising from vesicular-cytoplasmic translocation or from reuptake from the junctional cleft. The major value of these indices will probably be in determining the intergrated effects of pharmacologic agents at neuroeffector junctions in different blood vessels.