Mainz, Univ., Diss., 2001.

No description available.

No description available.

Malachite green(MG), an N-methylated diaminotriphenylmethane dye, has been widely used as an antifungal agent in aquaculture. Malachite green is reduced to and persists as leucomalachite green(LMG) in the tisssues of fish. Concern over MG and LMG are due to the potential for consumer exposure, suggestive evidence of tumor promotion in rodent liver, and suspicion of carcinogenicity based on structure-activity relationships. Several hepatotxicants and liver carcinogens have been shown to alter cell-cell signaling by interference with gap junction intercellular communication (GJIC).This study wanted to determine if disruption of cell-cell interactions occurs in rat liver epithelial cells in response to MG and LMG treatment. Rat liver epithelial cells(WB) were treated with LMG(0~6£gg/ml) or MG.(0~5£gg/ml) for one hour and gap junction was analyzed using the scrape- loading/dye transfer assay. The viability and proliferation of rat liver epithelial cells treated with LMG or MG were determined by MTT and colony forming efficiency. In addition, expression and intracellular localization of connexin43, E-cadherin,£]-catenin,£\-tubulin, ZO-1 and occludin were determined by immunoblot and immunostain analysis. A clear decrease in the distance of dye transfer was evident following treatment with MG(0~5£gg/ml) or LMG(0~6£gg/ml). Treatment with LMG and MG at different concentrations resulted in a decrease in cell viability and cell proliferation. Preincubation of cells with protein kinase C(PKC) inhibitor decreased the inhibition of GJIC by 5ug/ml MG and the specific MEK 1 inhibitor decreased substantially the inhibition of GJIC by 5£gg/ml MG and 5£gg/ml LMG. On the other hand, the specific PI3 kinase inhibitor decreased the inhibition of GJIC only by 3£g/mlLMG and we treated WB cells with EDTA to chelate extracellular calcium ion. The decrease of free calcium ion caused the expression of GJIC. At the transcriptional level, 10£gg/ml LMG and 10£gg/ml MG after treatment for one hour caused no change in the level of connexin43 mRNA. At the translational level, the different concentrations of MG or LMG after treatment for one hour or 24 hours caused a decrease in the level of the concentrations of connexin43 protein, E-cadherin protein,£]-catenin protein,£\-tubulin protein, ZO-1 protein and changed the distribution of occludin and ZO-1. Therefore, these data speculated the hypothesis that disruption of cell-cell signaling by interference with GJIC may contribute to LMG and MG toxicity, carcinogenicity and apoptosis.

Hypoglycemia is a severe side effect of insulin overdose in the diabetic population and can result in various neurological sequalae including seizures, coma, and brain death. There is still a limited understanding of the generation and propagation of hypoglycemic seizures, which may exacerbate hypoglycemia-induced neuronal damage. Moreover, glucose reperfusion after a period of transient hypoglycemia has been shown to cause neuronal hyperexcitability which can have further damaging effects. Gap junctional communication can be involved in the spread of hypoglycemic injury in two ways: 1) by providing a cytoplasmic continuity in which seizures can easily propagate and 2) by engaging the astrocytic network in metabolic compensation as well as enhancing astrocytic buffering of K+. This study aims to investigate the role that gap junctions play during brain energy deprivation. Results from these experiments show that gap junction blockade can have a neuroprotective role during hypoglycemia and glucose reperfusion.

国立情報学研究所で電子化したコンテンツを使用している。

国立情報学研究所で電子化したコンテンツを使用している。 / 国立情報学研究所で電子化したコンテンツを使用している。

No description available.

In reconstructive and plastic surgery, there exists a growing demand of adequate tissue implants, since currently available strategies for autologous transplantation are limited by complications including transplant failure and donor site morbidity. By developing in vitro and in vivo autologous substitutes for defective tissue sites, adipose tissue engineering can address these challenges, although there are several obstacles to overcome. One of the major limitations is the sufficient vascularization of in vitro engineered large constructs that remains crucial and demanding for functional tissues. Decellularized jejunal segments may represent a suitable scaffolding system with preexisting capillary structures that can be repopulated with human microvascular endothelial cells (hMVECs), and a luminal matrix applicable for the adipogenic differentiation of human adipose-derived stem cells (hASCs). Hence, co-culture of these cells in jejunal segments, utilizing a custom-made bioreactor system, was characterized in terms of vascularization and adipose tissue development. Substantial adipogenesis of hASCs was demonstrated within the jejunal lumen in contrast to non-induced controls, and the increase of key adipogenic markers was verified over time upon induction. The development of major extracellular matrix components of mature adipose tissue, such as laminin and collagen IV, was shown within the scaffold in induced samples. Successful reseeding of the vascular network with hMVECs was demonstrated in long-term culture and co-localization of vascular structures and adipogenically differentiated hASCs was observed. Therefore, these results represent a novel approach for in vitro engineering of vascularized adipose tissue constructs that warrants further investigations in preclinical studies. Another still existing obstacle in adipose tissue engineering is the insufficient knowledge about the applied cells, for instance the understanding of how cells can be optimally expanded and differentiated for successful engineering of tissue transplants. Even though hASCs can be easily isolated from liposuction of abdominal fat depots, yielding low donor site morbidity, huge numbers of cells are required to entirely seed complex and large 3D matrices or scaffolds. Thus, cells need to be large-scale expanded in vitro on the premise of not losing their differentiation capacity caused by replicative aging. Accordingly, an improved differentiation of hASCs in adipose tissue engineering approaches remains still desirable since most engineered constructs exhibit an inhomogeneous differentiation pattern. For mesenchymal stem cells (MSCs), it has been shown that growth factor application can lead to a significant improvement of both proliferation and differentiation capacity. Especially basic fibroblast growth factor (bFGF) represents a potent mitogen for MSCs, while maintaining or even promoting their osteogenic, chondrogenic and adipogenic differentiation potential. As there are currently different contradictory information present in literature about the applied bFGF concentration and the explicit effect of bFGF on ASC differentiation, here, the effect of bFGF on hASC proliferation and differentiation capacity was investigated at different concentrations and time points in 2D culture. Preculture of hASCs with bFGF prior to adipogenic induction showed a remarkable effect, whereas administration of bFGF during culture did not improve adipogenic differentiation capacity. Furthermore, the observations indicated as mode of action an impact of this preculture on cell proliferation capacity, resulting in increased cellular density at the time of adipogenic induction. The difference in cell density at this time point appeared to be pivotal for increased adipogenic capacity of the cells, which was confirmed in a further experiment employing different seeding densities. Interestingly, furthermore, the obtained results suggested a cell-cell contact-mediated mechanism positively influencing adipogenic differentiation. As a consequence, subsequently, studies were conducted focusing on intercellular communication of these cells, which has hardly been investigated to date. Despite the multitude of literature on the differentiation capacity of ASCs, little is reported about the physiological properties contributing to and controlling the process of lineage differentiation. Direct intercellular communication between adjacent cells via gap junctions has been shown to modulate differentiation processes in other cell types, with connexin 43 (Cx43) being the most abundant isoform of the gap junction-forming connexins. Thus, in the present study we focused on the expression of Cx43 and gap junctional intercellular communication (GJIC) in hASCs, and its significance for adipogenic differentiation of these cells. Cx43 expression in hASCs was demonstrated histologically and on the gene and protein expression level and was shown to be greatly positively influenced by cell seeding density. Functionality of gap junctions was proven by dye transfer analysis in growth medium. Adipogenic differentiation of hASCs was shown to be also distinctly elevated at higher cell seeding densities. Inhibition of GJIC by 18α-glycyrrhetinic acid significantly compromised adipogenic differentiation, as demonstrated by histology, triglyceride quantification, and adipogenic marker gene expression. Flow cytometry analysis showed a lower proportion of cells undergoing adipogenesis when GJIC was inhibited, further indicating the importance of GJIC in the differentiation process. Altogether, these results demonstrate the impact of direct cell-cell communication via gap junctions on the adipogenic differentiation process of hASCs and may contribute to further integrate direct intercellular crosstalk in rationales for tissue engineering approaches. / In der rekonstruktiven und plastischen Chirurgie besteht ein wachsender Bedarf an adäquaten Gewebetransplantaten, da die derzeit verfügbaren Strategien für autologe Transplantationen von Geweben durch Komplikationen wie beispielsweise Transplantatversagen sowie Morbiditäten an der Entnahmestelle beeinträchtigt werden. Das Tissue Engineering kann dieser Problematik jedoch durch die Entwicklung von in vitro und in vivo gezüchtetem, autologen Gewebeersatz für defekte Gewebestellen begegnen, wobei es dabei noch mehrere Hindernisse zu überwinden gilt. Eine der größten Limitationen ist die ausreichende Vaskularisierung der in vitro hergestellten, großen Konstrukte, welche für die Funktion des Gewebes entscheidend ist. Hierfür können dezellularisierte, jejunale Segmente ein geeignetes Gerüstsystem darstellen, deren bereits vorhandene Kapillarstrukturen mit humanen, mikrovaskulären Endothelzellen (hMVECs) und deren luminale Matrix mit humanen Stammzellen aus dem Fettgewebe (hASCs), mit anschließender adipogen Differenzierung, besiedelt werden können. Im Rahmen der vorliegenden Arbeit wurden diese Konstrukte mit Hilfe eines maßgeschneiderten Bioreaktorsystems kultiviert und die Kokultur der Zellen in der jejunalen Matrix hinsichtlich der Fettgewebeentwicklung untersucht. Im Gegensatz zu nicht-induzierten Kontrollen wurde nach adipogener Induktion innerhalb des jejunalen Lumens eine substanzielle Fettgewebebildung der hASCs, sowie ein Anstieg wichtiger adipogener Marker im zeitlichen Verlauf nachgewiesen. Die Bildung wesentlicher extrazellulärer Matrixkomponenten des reifen Fettgewebes, wie beispielsweise Laminin und Kollagen IV, wurde innerhalb der Matrix bei induzierten Proben ebenso beobachtet. Die erfolgreiche Neubesiedlung des Gefäßnetzes mit hMVECs konnte in der Langzeitkultur gezeigt und eine Kolokalisation von Gefäßstrukturen und differenzierten hASCs beobachtet werden. Somit stellen diese Ergebnisse einen vielversprechenden, neuen Ansatz für die in vitro Entwicklung von vaskularisierten Fettgewebekonstrukten dar, welcher jedoch noch weitere Untersuchungen in präklinischen Studien erfordert. Eine weitere Limitation in der Entwicklung von Fettgewebe ist das unzureichende Wissen über die verwendeten Zellen – so zum Beispiel wie Zellen optimal expandiert und differenziert werden können, um einen Gewebeersatz erfolgreich herzustellen. Auch wenn hASCs leicht aus abdominalen Liposuktionen, welche zu einer relativ geringen Morbidität an der Entnahmestelle führen, isoliert werden können, ist eine sehr große Anzahl an Zellen erforderlich, um komplexe und große 3D-Matrizes vollständig mit Zellen zu besiedeln. So müssen Zellen in vitro im großen Maßstab expandiert werden, wobei auf die Erhaltung ihrer Differenzierungskapazität und die Vermeidung des replikativen Alterns geachtet werden muss. Da viele der entwickelten Konstrukte des Weiteren ein inhomogenes Differenzierungsmuster aufweisen, ist eine Verbesserung der adipogenen Differenzierung von ASCs im Rahmen von Tissue Engineering Ansätzen wünschenswert. Für mesenchymale Stammzellen (MSCs) wurde bereits gezeigt, dass die Anwendung von Wachstumsfaktoren zu einer deutlichen Verbesserung der Proliferations- und Differenzierungskapazität führen kann. Insbesondere der Wachstumsfaktor bFGF (basic fibroblast growth factor) stellt ein starkes Mitogen für MSCs dar, wobei er das osteogene, chondrogene und adipogene Differenzierungspotenzial der Zellen aufrechterhält und sogar fördert. Da es in der Literatur derzeit unterschiedliche und teilweise widersprüchliche Informationen über die verwendeten bFGF Konzentrationen und den expliziten Effekt von bFGF auf die Differenzierung von ASCs gibt, wurde der Effekt von bFGF auf die Proliferations- und Differenzierungsfähigkeit mit unterschiedlichen Konzentrationen und zu unterschiedlichen Zeitpunkten in der 2D Kultur untersucht. Die Vorkultur der hASCs mit bFGF vor der adipogenen Induktion hatte einen beachtlichen Effekt auf die Differenzierung, während die Verabreichung von bFGF während der Kultur, die adipogene Differenzierungsfähigkeit der Zellen nicht verbesserte. Darüber hinaus zeigten die Ergebnisse einen Einfluss der Vorkultur auf die Zellproliferation, was zu einer erhöhten Zelldichte zum Zeitpunkt der adipogenen Induktion führte. Der Unterschied in der Zelldichte zu diesem Zeitpunkt schien entscheidend für die gesteigerte Differenzierungskapazität der Zellen zu sein, was sich in einem weiteren Experiment mit unterschiedlichen Aussaatdichten bestätigte. Interessanterweise deuteten die Ergebnisse außerdem darauf hin, dass ein Zell-Zell-Kontakt-vermittelter Mechanismus die adipogene Differenzierung positiv beeinflusst. Daher wurden anschließend Untersuchungen zur interzellulären Kommunikation dieser Zellen durchgeführt, welche bisher kaum erforscht wurde. Trotz der Vielzahl an Literatur über die Differenzierungsfähigkeit von ASCs ist wenig über die physiologischen Prozesse bekannt, die zur Differenzierung in verschiedene Zelltypen beitragen und diese kontrollieren. So wurde gezeigt, dass die direkte interzelluläre Kommunikation zwischen benachbarten Zellen über Gap Junctions Differenzierungsprozesse moduliert. Connexin 43 (Cx43) stellt dabei die häufigste Isoform der Gap Junction-bildenden Connexine dar. Im Rahmen dieser Arbeit wurde die Expression von Cx43 und die interzelluläre Kommunikation durch Gap Junctions (gap junctional intercellular communication; GJIC) in hASCs, sowie ihre Bedeutung für die adipogene Differenzierung untersucht. Die Cx43 Expression in hASCs wurde histologisch und auf Gen- und Proteinexpressionsebene nachgewiesen und wurde durch die Zellaussaatdichte nachweislich stark beeinflusst. Die Funktionalität der Gap Junctions konnte mit Hilfe eines Assays zur Übertragung von Farbstoffen untersucht werden. Es zeigte sich hierbei eine zelldichteabhängige, adipogene Differenzierungkapazität der hASCs. Die Hemmung der GJIC durch 18α-Glycyrrhetinsäure beeinträchtigte die adipogene Differenzierung deutlich, wie sich durch die Histologie, die Triglyceridquantifizierung und die adipogene Markergenexpression beobachten ließ. Bei Hemmung der GJIC zeigte sich mit Hilfe der Durchflusszytometrie, dass weniger Zellen adipogen differenzieren konnten, was die Bedeutung von GJIC im Differenzierungsprozess hervorhebt. Zusammenfassend veranschaulichen diese Ergebnisse den Einfluss direkter Zell-Zell-Kommunikation über Gap Junctions auf den adipogenen Differenzierungsprozess von hASCs und könnten somit in Zukunft dazu beitragen, direkte interzelluläre Kommunikation in Tissue Engineering Ansätze zu integrieren.

Adenoviruses are non-enveloped, dsDNA tumor viruses responsible for a breadth of pathogenesis including acute respiratory disease and viral myocarditis. Gap junctions, which are formed by connexin proteins, directly couple the cytoplasms of apposed cells enabling immunological, metabolic, and electrical intercellular communication. The gap junction protein connexin43 (Cx43; gene name – GJA1) is the most widely expressed human connexin protein and is the predominant connexin in the working myocardium. Given the immunological role for Cx43 gap junctions, we hypothesized that gap junctions would be targeted during adenoviral infection. We find reduced Cx43 protein due to suppression of GJA1 transcription dependent upon β-catenin during adenoviral infection, with viral protein E4orf1 sufficient to induce β-catenin phosphorylation. Loss of gap junction function occurs prior to reduced Cx43 protein levels with Ad5 infection rapidly inducing Cx43 phosphorylation at residues previously demonstrated to alter gap junction conductance. Direct Cx43 interaction with ZO-1 plays a critical role in gap junction regulation. We find loss of Cx43/ZO-1 complexing during Ad5 infection by co-immunoprecipitation, with complementary studies in human induced pluripotent stem cell derived-cardiomyocytes revealing Cx43 gap junction remodeling concomitant with reduced ZO-1 complexing. These findings demonstrate specific targeting of gap junction function by Ad5 leading to disruptions in intercellular communication which would contribute to dangerous pathological states including arrhythmias in infected hearts. Intercellular junction proteins belonging to classically defined unique junctions exhibit extensive cross-talk and interdependency for expression and localization. We find reduced connexin43 (Cx43) phosphorylation at a known internalization motif, leading us to hypothesize that gap junctions are maintained during adenoviral infection in order to stabilize intercellular junctions and adenoviral receptors therein. Utilizing immunofluorescence confocal microscopy, we demonstrate that Cx43 reductions are primarily cytosolic with Cx43 preservation at the plasma membrane. Click-IT chemistry, a non-radioactive pulse-chase technique, reveals that Cx43 ½ life is extended during adenoviral infection. In order to test if remaining Cx43 exists in de facto gap junctions (i.e. not undocked or cytosolic connexons) we utilized 1 % Triton X-100 solubility fractionation and find Cx43 is indeed primarily junctional during adenoviral infection. Having demonstrated increases in junctional Cx43, we next asked how tightly coupled cells were during adenoviral infection and by ECIS measurements of electrical resistance we demonstrate a transient increase in mechanical coupling during infection. Our future aims are to uncover changes in Coxsackievirus and adenovirus receptor (CAR) protein localization to determine if adenoviral-induced changes to subcellular architecture predisposes neighboring cells to infection and enhances viral spread. These findings will add to the existing model of adenoviral infection and more broadly, contribute to the therapeutic design of adenoviral vectors for cancer and gene therapy. / Doctor of Philosophy / The human heart will beat more than 3 billion times during the average lifetime. This is accomplished by billions of individual heart muscle cells, called cardiomyocytes, contracting in synchrony. Cardiomyocytes require direct cell to cell communication in order to receive the proper cues and work in concert. Outside of the heart, including the lining of the lungs which acts as a first line of defense against invading pathogens, direct cell to cell communication is important for mounting proper immune responses. A primary means by which cells communicate directly with neighboring cells is through gap junctions which are formed of proteins called connexins. Six connexin proteins form a channel in the cell surface that binds to a similar channel on an apposing cell to create a continuous gap junction channel, coupling the cell interiors directly. The most widely expressed human connexin, and the most abundant connexin in the heart, is connexin43 (Cx43; gene name – GJA1). Adenoviruses are pathogens commonly associated with respiratory illnesses in addition to more serious diseases including viral myocarditis, or infection of the heart. Given that Cx43 gap junctions enable direct intercellular communication important in initiating immune responses, we hypothesized that adenovirus would target Cx43 and gap junctions during infection. We find reduced Cx43 protein in cells infected with human adenovirus, and revel that the expression of the GJA1 gene is suppressed. We next focused on potential signaling pathways that are changed during adenoviral infection. β-catenin is a factor with several cellular roles including regulating expression of specific genes including GJA1 (Cx43). We demonstrate β-catenin is activated during adenoviral infection and that this is necessary for reducing Cx43 transcripts. A pathway that activates β-catenin in this manner is the PI3K/Akt signaling axis, which has previously been shown to be turned on during adenovirus infection by a viral protein called E4orf1. We find the adenoviral protein E4orf1 is sufficient to induce β-catenin activation revealing a potential therapeutic target for future studies. We next determined that direct cell to cell communication through gap junctions is reduced before loss of the gap junction protein Cx43 during infection. Gap junctions are modified by the cell to change their ability to couple cells independently of protein levels alone and we find gap junction modifications consistent with altered communication ability. Furthermore, the gap junction protein Cx43 interacts with the cellular skeleton protein Zonula Occludens-1 (ZO-1) during movement into and out of gap junction clusters. We determined alterations in Cx43/ZO-1 interactions consistent with gap junction remodeling. In complimentary studies we find the same gap junction remodeling in cardiomyocytes revealing arrhythmogenic potential during acute adenoviral infection in human heart cells. Localized with gap junctions are several other junction proteins including the Coxsackievirus and adenovirus receptor (CAR) which is critical in cardiac development and also the primary receptor for species C adenoviruses (used in our studies). CAR expression has been demonstrated to alter Cx43 levels and indeed, many junctional proteins influence the expression and/or localization of other junctional proteins. Interestingly, despite reduced Cx43 levels and reduced gap junction function (cell to cell communication), we detected decreases in a gap junction modification that is associated with gap junction degradation, suggesting that new gap junction protein Cx43 is not being made but already synthesized Cx43 is degraded more slowly. We hypothesized Cx43 is maintained during adenoviral infection in order to recruit other junctional components, principally CAR, on uninfected neighbor cells to predispose them to infection. We observed using microscopy that Cx43 reductions are primarily inside the cell but Cx43 is preserved on the cell surface and at junctions between cells. We next asked if the protein is being degraded more slowly and find Cx43 exists for longer in infected cells signifying that it is being degraded more slowly. Utilizing a fractionation technique to separate gap junction connexin from connexon that is non-junctional or inside the cell, we detect an increase in junctional Cx43, revealing maintenance of Cx43 gap junction structures. Having now identified adenoviral-mediated maintenance of Cx43 gap junction structures, we next wanted to test for changing in mechanical coupling (i.e. how tightly are the cells connected to one another) where we demonstrate an increase in mechanical coupling during adenoviral infection. Our future directions are to determine if this increase in Cx43 gap junction maintenance and mechanical coupling is concomitant with changes in CAR expression/localization on uninfected neighboring cells and if altered, does this predispose uninfected neighbors of infected cells to infection.