Podcasts about cx43

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.09.527850v1?rss=1 Authors: Fadjukov, J., Wienbar, S., Milicevic, N., Hakanen, S., Vihinen-Ranta, M., Ihalainen, T. O., Schwartz, G. W., Nymark, S. Abstract: Retinal pigment epithelium (RPE) at the back of the eye is a monolayer of cells with an extensive network of gap junctions that contributes to retinal health in a multitude of ways. One of those roles is the phagocytosis of photoreceptor outer segments. This renewal is under circadian regulation and peaks after light onset. Connexin 43 (Cx43) is the most predominantly expressed gap junction protein in RPE. In this study, we examine how gap junctions and specifically, Cx43 phosphorylation, contribute to phagocytosis in both human embryonic stem cell derived RPE and mouse RPE monolayers. We show that both Rac1 and CDK5 have differences in protein localization at different points in phagocytosis, and that by using their effectors, the capability of RPE for phagocytosis changes. CDK5 has not yet been reported in RPE tissue, and here we show that it likely regulates Cx43 localization and resulting electrical coupling. We find that gap junctions in RPE are temporally highly dynamic during phagocytosis and that regulation of gap junctions via phosphorylation is likely critical for maintaining eye health. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.02.514878v1?rss=1 Authors: Matthaeus, C., Juettner, R., Gotthardt, M., Rathjen, F. G. Abstract: The IgCAM coxsackie-adenovirus receptor (CAR) is essential for embryonic heart development and for electrical conduction in the mature heart. However, it is not well-understood how CAR exerts these effects at the cellular level. To address this question, we analysed the spontaneous beating of cultured embryonic hearts and cardiomyocytes from wildtype and CAR knockout (KO) embryos. Surprisingly, in the absence of CAR, cultured cardiomyocytes showed an increased frequency of beating and calcium cycling. Increased beating of heart organ cultures was also induced by application of reagents that bind to the extracellular region of CAR, such as the adenovirus fiber knob. However, the calcium cycling machinery, including calcium extrusion via SERCA2 and NCX, was not disrupted in CAR KO cells. In contrast, CAR KO cardiomyocytes displayed an increase in the size, but decrease in total number, of membrane-localized Cx43 clusters. This was accompanied by improved cell-cell coupling between CAR KO cells, as demonstrated by increased intercellular dye diffusion. Our data indicate that CAR may modulate the localization and oligomerization of Cx43 at the plasma membrane, which could in turn influence electrical propagation between cardiomyocytes via gap junctions. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.22.216598v1?rss=1 Authors: Mozafari, S., Deboux, C., Laterza, C., Ehrlich, M., Kuhlmann, T., Martino, G., Baron-Van Evercooren, A. Abstract: Oligodendrocytes are extensively coupled to astrocytes, a phenomenon ensuring glial homeostasis and maintenance of CNS myelin. Molecular disruption of this communication occurs in demyelinating diseases such as multiple sclerosis. Less is known about the vulnerability and reconstruction of the panglial network during adult demyelination-remyelination. Here, we took advantage of LPC-induced demyelination to investigate the expression dynamics of the oligodendrocyte specific connexin 47 (Cx47) and whether this dynamic could be modulated by grafted iPSC-neural progeny. Our data show that deconstruction of the panglial network following demyelination is larger in size than demyelination. Loss of Cx47 expression is timely rescued during remyelination and accelerated by the grafted neural precursors. Moreover, mouse and human iPS-derived oligodendrocytes express Cx47, which co-labels with astrocyte Cx43, indicating their integration into the panglial network. These data suggest that full lesion repair following transplantation occurs by panglial reconstruction in addition to remyelination. Targeting panglial elements by cell therapy or pharmacological compounds may help accelerating or stabilizing re/myelination in myelin disorders. Copy rights belong to original authors. Visit the link for more info

Eine effektive Regulation der Gewebedurchblutung erfordert eine Koordination der Reaktion einzelner Gefäßzellen bzw. verschiedener Gefäßabschnitte. Der zur Koordination erforderliche interzelluläre Signalaustausch kann zumindest teilweise über Gap Junction-Kanäle erfolgen, die als interzelluläre Verbindungen den Austausch von elektrischen und chemischen Signalstoffen zwischen benachbarten Zellen ermöglichen. Dieser Austausch kann über die Modulation der Permeabilität von Gap Junction-Kanälen reguliert werden. Aus Untersuchungen an Modellzellen (HeLA-Zellen) war bereits bekannt dass NO eine solche Modulatorwirkung ausübt, wenn die Gap Junctions nur Connexin 37 (Cx37) enthalten während kein Effekt von NO auf Gap Junctions zu beobachtet war, wenn Gap Junctions aus Cx43 oder Cx40 gebildet wurden. Da Endothelzellen normalerweise alle drei Connexine exprimieren, sollte in der vorliegenden Arbeit untersucht werden, inwieweit NO in diesen Zellen überhaupt eine nachweisbare Wirkung auf die Gap Junction Permeabilität und damit auf den Signalaustausch entfaltet. Als Modell des Signalaustauschs wurde die Ausbreitung von Calciumwellen jeweils zwischen Endothelzellen oder glatten Muskelzellen allein oder zwischen beiden Zelltypen untersucht. Nach Auslösung von interzellulären Calciumwellen als Folge einer mechanischen Stimulation von einzelnen Zellen konnte zunächst gezeigt werden, dass die interzelluläre Ausbreitung von Calcium unter den gewählten Versuchsbedingungen über Gap Junctions-erfolgte. Im Gegensatz zum Modellsystem der HeLa Zellen, in denen nur Cx37 exprimiert war, zeigte NO in den Endothelzellen (humane Nabelschnur, alle drei Connexine exprimiert) abgesehen von einer geringradigen Verzögerung keinen Hemmeffekt auf die Gap Junction-abhängige Ausbreitung von Calcium-Signalen. Wurde jedoch Cx43 durch Behandlung mit siRNA herunterreguliert, führte NO auch in den Endothelzellen zu einer Hemmung der interzellulären Calciumwellenausbreitung. Auch in intakten Endothelzellen, die mit glatten Muskelzellen kokultiviert wurden, ließ sich bei genauerer Analyse ein Hemmeffekt von NO nachweisen. Dieser war jedoch auf die Zellbereiche beschränkt, in denen Endothelzellen und glatte Muskelzellen unmittelbar benachbart waren (myoendotheliale Junctions). In diesen myoendo-thelialen Gap Junctions, fanden wir auf der Endothelseite immunhistochemisch überwiegend Cx37 exprimiert. Aufgrund dieser präferentiellen Lokalisation von Cx37 scheint daher NO eine besondere Rolle bei der Modulation des Calciumaustauschs (und potentiell auch anderer Signalmoleküle wie IP3 oder cyclische Nukleotide) zu spielen. Die Kontrolle des Calciumaustauschs könnte funktionell eine calciumabhängige glattmuskuläre Kontraktion bei Endothelstimulation verhindern und somit die endothelabhängige Dilatation verstärken. Diese bisher unbekannte NO-Wirkung auf Cx37-exprimierende Gap Junctions könnte einen weiteren Mechanismus der Gefäßtonusregulation darstellen.

Background: Gap junctional calcium signal propagation (transfer of calcium or a calcium releasing messenger via gap junctions) between vascular cells has been shown to be involved in the control of vascular tone. We have shown before that nitric oxide (NO) inhibits gap junctional communication in HeLa cells exclusively expressing connexin 37 (HeLa-Cx37) but not in HeLa-Cx40 or HeLa-Cx43. Here we studied the effect of NO on the gap junctional calcium signal propagation in endothelial cells which, in addition to Cx37, also express Cx40 and Cx43. Furthermore, we analyzed the impact of NO on intermuscle and on myoendothelial gap junction-dependent calcium signal propagation. Since specific effects of NO at one of these three junctional areas (interendothelial/myoendothelial/ intermuscle) may depend on a differential membrane localization of the connexins, we also studied the distribution of the vascular connexins in small resistance arteries. Results: In endothelial (HUVEC) or smooth muscle cells (HUVSMC) alone, NO did not affect gap junctional Ca2+ signal propagation as assessed by analyzing the spread of Ca2+ signals after mechanical stimulation of a single cell. In contrast, at myoendothelial junctions, it decreased Ca2+ signal propagation in both directions by about 60% (co-cultures of HUVEC and HUVSMC). This resulted in a longer maintenance of calcium elevation at the endothelial side and a faster calcium signal propagation at the smooth muscle side, respectively. Immunohistochemical stainings (confocal and two-photon-microscopy) of cells in co-cultures or of small arteries revealed that Cx37 expression was relatively higher in endothelial cells adjoining smooth muscle (culture) or in potential areas of myoendothelial junctions (arteries). Accordingly, Cx37 - in contrast to Cx40 - was not only expressed on the endothelial surface of small arteries but also in deeper layers (corresponding to the internal elastic lamina IEL). Holes of the IEL where myoendothelial contacts can only occur, stained significantly more frequently for Cx37 and Cx43 than for Cx40 (endothelium) or Cx45 (smooth muscle). Conclusion: NO modulates the calcium signal propagation specifically between endothelial and smooth muscle cells. The effect is due to an augmented distribution of Cx37 towards myoendothelial contact areas and potentially counteracts endothelial Ca2+ signal loss from endothelial to smooth muscle cells. This targeted effect of NO may optimize calcium dependent endothelial vasomotor function.

Vaskuläre Erkrankungen sind als Ursache für Mortalität und Morbidität führend in westlichen Industrieländern. Es gibt zunehmend Hinweise darauf, dass oxLDL eine herausragende Rolle bei der Atheroskleroseinduktion bzw. -progression zukommt. Die initiale Wirkung von im Blut zirkulierendem oxLDL findet auf der Ebene der Interaktion mit dem vaskulären Endothel statt und resultiert in der endothelialen Dysfunktion. Da die Zellfunktion durch die Integrität der Endothelzellschicht bzw. deren interzelluläre Kommunikation mitbestimmt ist, wäre es denkbar, dass für die oxLDL-induzierten Veränderungen im Endothel u. a. die Beeinflussung der Zell-Zell-Kommunikation via Gap Junctions eine Rolle spielt. Bislang war jedoch wenig darüber bekannt, welchen Einfluss oxLDL auf die interzelluläre Kommunikation über Gap Junctions in Endothelzellen ausübt. Außerdem war es nicht geklärt, inwiefern diese Veränderungen in der Zell-Zell-Kommunikation die Induktion und den Schweregrad der oxLDL-induzierten Apoptose beeinflussen. Ziele der Studie waren daher i) zu analysieren, ob und über welche Mechanismen oxLDL einen Einfluss auf die interzelluläre Kommunikation über Gap Junctions in Endothelzellen ausübt, ii) zu untersuchen, welche Bedeutung der interzellulären Kommunikation über Gap Junctions bzw. einzelnen Connexinen bei der Induktion der Apoptose zukommt. Mittels der Dye-Transfer-Methode nach Farbstoffinjektion in eine einzelne Zelle konnten wir zeigen, dass oxLDL eine signifikante Steigerung der interzellulären Kommunikation über Gap Junctions in HUVEC induziert. Dieser Effekt ist dosisabhängig: er zeigte sich nur bei geringen oxLDL-Konzentrationen (15 bzw. 26 μg/ml) und wurde bei weiterer Erhöhung der Konzentration bis auf 100 μg/ml wiederum aufgehoben. Die durch oxLDL verstärkte Zell-Zell-Kommunikation wurde in Endothelzellen durch einen cAMP/PKA abhängigen Mechanismus vermittelt, wobei die cAMP-Freisetzung durch ein Cyclooxygenaseprodukt, wahrscheinlich Prostacyclin, getriggert wurde. Mittels immunhistochemischer Färbungen für Cx37 und Cx43 konnten wir nicht bestätigen, dass die oxLDL-induzierte Verstärkung der Zell-Zell-Kommunikation infolge einer Hochregulation der Connexin-Expression auftritt. Im zweiten Teil der Studie wurde der Einfluss von oxLDL auf die Apoptoseinduktion analysiert. Die Apoptose wurde mittels der Annexin V - Propidium Iodid Färbung bzw. durch Nachweis des Mitochondrienmembranpotentials durchflusszytometrisch erfasst. OxLDL verursachte einen signifikanten Anstieg der Apoptoserate in HUVEC. Zur Aufklärung der Rolle bestimmter Connexine wurden weitere Experimenten in Cx-transfizierten HeLa-Zellen durchgeführt. In diesen Zellen erhöhen einzelne Connexine die Apoptoserate in unterschiedlichem Ausmaß: Cx43 > Cx40 > Cx37. Um zu prüfen, ob die bloße Anwesenheit der Connexine dafür von Bedeutung war oder ob von Connexinen gebildete Gap Junctions dafür von Bedeutung waren, wurden weitere Experimente durchgeführt. Dafür wurden in einem neuen Versuchsansatz Zellen in Suspension (keine Zell-Zell-Kontakte) sowie adhärente Zellen im Monolayer (bestehende Zell-Zell- Kontakte) einer proapoptotischen Stimulation durch Streptonigrin unterzogen. Die Zellen in Suspension wiesen erst zu einem deutlich späteren Zeitpunkt apoptotische Veränderungen auf. Das deutet auf eine Beteiligung der Gap Junctions bei der Apoptoseinduktion hin. Diese Interpretation wurde durch Befunde einer weiteren Versuchsreihe bestätigt. Bei Inkubation von apoptotischen Cx43-positiven Zellen mit intakten Zellen wurde die Apoptoserate der Letzteren nur dann signifikant erhöht, wenn diese ebenfalls Connexin 43 exprimierten und funktionelle Gap Junctions mit den bereits apoptotischen Zellen de novo bilden konnten. Somit demonstriert diese Arbeit, dass Gap Junctions eine wichtige Rolle bei der Apoptoseinduktion spielen. In nachfolgenden Studien soll in Atherosklerose-Modellen überprüft werden, ob und inwiefern die hier beschriebenen Mechanismen auch unter den In-Vivo-Bedingungen bei den oxLDL-assoziierten Gefäß/Endothelschäden eine Rolle spielen.

In the present study, the testes of 32 bovine embryos with different crown-rump length (2.5- 90 cm CRL) and of 15 sexually mature bulls (Deutsches Fleckvieh) were investigated using light- and electron microscope as well as glycohistochemical and immunohistochemical methods. The gestation period was divided into 3 stages; early, mid, and late gestation. Developmental changes in the testicular morphogenesis were therefore analyzed in details during these phases. Generally, embryonic development of bovine testis involves the same mechanism described in other mammals. At the first stage of this study (2.5 cm CRL/43 dpc), the anlage of the testes protruded to the coelomic cavity as paired bean-shaped structures on either side of the dorsal mesentery medial to the mesonephros. It consists of primitive testicular cords, interstitium, and rete testis blastema. Proceeding with fetal age, these basic testicular structures are further differentiated. The tunica albuginea is separated into two layers: an outer fibrous layer (tunica fibrosa) with some mesenchymal cells, numerous fibroblast, and much fibrous content and an inner cellular layer with several blood vessels (tunica vasculosa). The testicular cords are surrounded by a marked basal lamina and peritubular cells and lined by two types of cells: a large number of dark polygonal cells with irregular nuclei, pre-Sertoli cells and small number of large light round cells with relatively round nuclei, the prespermatogonia. The average number of the germ cells per cross section of cord increases, particularly form 3.5 to 14 cm CRL, resulting in a germ cell maximum at the end of this stage (14 cm CRL). Although most of the germ cells are located toward the periphery of the cord, some are also found in the center. Pre-Sertoli cells form a complete layer at the periphery of the cords. Generally, these cells are irregular in shape and numerous but considerably smaller than the germ cells. Unlike prespermatogonia, mitotic figures are seen in pre-Sertoli cells during the whole embryonic life. As a consequence of the expansion in the interstitium, the seminiferous cords are progressively separated from each other. The testicular interstitium is rapidly differentiated and is composed of several islets or clusters of polygonal Leydig cells, peritubular flattened cells surrounding the testicular cords, connective tissue cells, and numerous blood vessels. In the present study, fetal Leydig cells were first recognized at 3.5 cm CRL. Thereafter, the average number of these cells is rapidly increased to attain their maximum with the end of the first gestation period (14 cm CRL). This generation of Leydig cells however dedifferentiates progressively with developmental age. A continuous system of basal lamina joins the testicular cords with rete strands from 10 cm CRL and onwards. This system establishes the first connection between these two testicular components via ill-developed uncanalized straight tubules (tubuli recti). Rete testis channels are lined by simple layer of cuboidal epithelium with round nuclei occupying most of the cytoplasm and enclosed by well-defined basal lamina. The adult bovine testis is enclosed by a connective tissue capsule, tunica albuginea, composed predominantly of collagen fibers and few elastic fibers. Most of the testicular parenchyma is made up of the convoluted seminiferous tubules (tubuli seminiferi contorti), two-ended convoluted loops, with both ends opening into the rete testis via specialized terminal segments. The seminiferous tubules of sexually mature bulls are enclosed by a distinct lamina propria and are lined by two cell populations, non-proliferating Sertoli cells and highly proliferating spermatogenic cells. The bovine lamina propria consists of basal lamina, collagen and elastic fibers, and 3-5 layers of partially overlapping myofibroblasts. Additionally, fibrocytes, collagen fibrils, and fibroblasts-like cells form the outermost border of the tubulus. Sertoli cells are easily identifiable elements of the seminiferous epithelium. Adult Sertoli cells are large irregularly shaped cells with their broad bases resting on the basal lamina while the remaining cytoplasmic processes extend upward to the tubular lumen. They are characterized by round or oval euchromatin-rich nuclei situating in the basal portion near the basal lamina of the seminiferous tubules. Adult bovine germ cells are present in four morphologically different groups, i.e., spermatogonia, spermatocytes, spermatids, and spermatozoa. The seminiferous cycle stages are identified using changes in the germ cell nuclei as well as location and shape of spermatids. According to this method, eight stages are defined in the seminiferous epithelium of bovine. The interstitial or intertubular tissue of adult bovine testis consists of Leydig cells, macrophages, scattered lymphocytes and plasma cells, and contains numerous blood and lymph vessels. Not all Leydig cells have contact to blood or lymph capillaries. The excurrent duct system of the adult bovine testis consists of terminal segment of the convoluted seminiferous tubules, straight tubules, and rete testis. The terminal segment can be further subdivided into a proximal (transitional) region, middle portion, and distal part (terminal plug). The proximal region is lined by typical Sertoli cells while the last two parts are lined by modified Sertoli cells. The tubulus rectus of adult bovine testis is composed of three morphologically different regions: a proximal cup-shaped region, a middle narrow stalk, and a distal festooned portion. The rete testis is a complicated centrally positioned meshwork of intercommunicating channels that lies within the mediastinum testis parallel to the long axis of epididymis. The simple cuboidal epithelium of straight tubules and rete testis is shown to contain some lymphocytes and macrophages. The cellular distribution of glycoconjugates within the fetal and adult bovine testis was investigated using thirteen (ConA, PSA, LCA, PNA, GSA-I, ECA, DBA, SBA, HPA, VVA, WGA, UEA-I, LTA) different fluorescein isothiocyanate (FITC) conjugated lectins. In fetal testes, detection of sugar moieties by lectins was carried out on Bouin õ s-fixed paraffin-embedded sections while in adult it was performed on both Bouin õ s-fixed paraffin-embedded and acetone-fixed frozen sections. Only five lectins (PSA, PNA, GSA-I, DBA, WGA) showed a positive reaction in the embryonic testes. PNA, GSA-I, DBA, and WGA were detected in the germ cells whereas PSA, DBA and WGA labeled the fetal Leydig cells. None of the lectins used was observed in the pre-Sertoli cells. Further on, some lectins were seen in tunica albuginea (PSA, PNA, GSA-I, WGA), basal lamina of testicular cords (PSA, WGA), interstitial blood vessels (PSA, GSA-I, WGA), mediastinum testis (PSA, PNA, WGA) and rete testis epithelium (PNA). In adult animals, spermatogonia and spermatocytes were positively stained with PSA, LCA, DBA, SBA, and VVA. All the lectins investigated except that of the fucose-binding lectin (UEA-I and LTA) were definitely detected in the acrosome of round and elongated spermatids. These results indicate a role for carbohydrates in spermiogenesis. Apical Sertoli cells processes and Leydig cells were weakly stained with PSA and LCA as well. DBA binding sites were also seen in the Leydig cells. Immunohistochemical studies were performed using the Avidin-Biotin-Peroxidase Complex (ABC) method for localization of fibroblast growth factor-1 (FGF-1), fibroblast growth factor-2 (FGF-2), S-100, laminin, alpha-smooth muscle actin (á -SMA), vascular endothelial growth factor (VEGF), connexin 43 (Cx43), CD4, CD8, CD68, angiotensin-converting enzyme (ACE), and galactosyltransferase (GalTase) in the bovine testis. The expression of FGF-1 and FGF-2 was further investigated in the adult bovine testis using in situ hybridization and PCR. Immunohistochemically, FGF-1 was seen in the Sertoli cells, Leydig cells, endothelium of the blood vessels, and epithelium of straight tubules and rete testis of fetal and adult testis. It was additionally detected in spermatogonia and spermatids of sexual mature animals. FGF-2 exhibited a striking positive reaction in fetal (from 6 to 30 cm CRL) and adult Leydig cells. Moreover, it showed marked reaction in the endothelium of blood vessels and in the epithelium of tubulus rectus and rete testis. FGF-2 was also localized in some spermatogonia, and myofibroblasts. By means of in situ hybridization, FGF-1 and FGF-2 mRNA were found in Leydig and Sertoli cells as well as in the modified Sertoli cells of the terminal segment. FGF-1 transcripts were additionally recognized in the straight tubules and rete testis epithelium. Distinct S100 immunostaining was observed in the Sertoli cells, endothelium of blood vessels and in the rete testis epithelium of fetal and adult testis. Laminin was localized to the basal lamina of seminiferous tubules, blood vessels, myofibroblasts, and rete testis. Although á -SMA was detected in smooth muscle cells of the blood vessels, no immunoreactivity was seen in the peritubular cells during the whole gestation period. The myofibroblasts surrounding the seminiferous tubules and rete testis showed intense positive reaction for á -SMA in the adult testis. VEGF was detected in the acrosomes of the elongating spermatids. Connexin 43 was localized to gap junctions between Leydig cells in the fetal and adult life as well as to the seminiferous epithelium apical to spermatogonia and basal to spermatocytes, a position correlating with Sertoli-Sertoli cell junctions. The detection of cells positive for CD4, CD8, CD68 within the adult testis interstitium clearly indicate the presence of lymphocytes and macrophages within this testicular compartment. GalTase showed striking positive reaction in the Golgi complex of Sertoli cells, Leydig cells, and some spermatocytes as well as at the cell membrane of elongating spermatids and in the simple cuboidal epithelium of rete testis. ACE positive reaction was found in the prespermatogonia (only at 6-10 cm CRL) and in fetal and adult testicular blood vessels. The functional significance of these immunocytochemically-demonstrated proteins is discussed.