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Cardiovascular diseases are among the leading causes of death worldwide and particularly in the developed World. The search for new therapeutic approaches for improving the functions of the damaged heart is therefore a critical endeavour. Myocardial infarction, which can lead to heart failure, is associated with irreversible loss of functional cardiomyocytes. The loss of cardiomyocytes poses a major difficulty for treating the damaged heart since terminally differentiated cardiomyocytes have very limited regeneration potential. Currently, the only effective treatment for severe heart failure is heart transplantation but this option is limited by the acute shortage of donor hearts. The high incidence of heart diseases and the scarcity donor hearts underline the urgent need to find alternative therapeutic approaches for treating cardiovascular diseases. Pluripotent embryonic stem (ES) cells can differentiate into functional cardiomyocytes. Therefore the engraftment of ES cell-derived functional cardiomyocytes or cardiac progenitor cells into the damaged heart to regenerate healthy myocardial tissues may be used to treat damaged hearts. Stem cell-based therapy therefore holds a great potential as a very attractive alternative to heart transplant for treating heart failure and other cardiovascular diseases. A major obstacle to the realisation of stem cell-based therapy is the lack of donor cells and this in turn is due to the fact that, currently, the molecular mechanisms or the regulatory signal transduction mechanisms that are responsible for mediating ES cell differentiation into cardiomyocytes are not well understood. Overcoming this huge scientific challenge is absolutely necessary before the use of stem cell-derived cardiomyocytes to treat the damaged heart can become a reality. Therefore the aim of this thesis was to investigate the signal transduction pathways that are involved in the differentiation of stem cells into cardiomyocytes. The first objective was the establishment and use of cardiomyocyte differentiation models using H9c2 cells and P19 stem cells to accomplish the specific objectives of the thesis. The specific objectives of the thesis were, the investigation of the roles of (i) nitric oxide (ii) protein kinase C (PKC), (iii) p38 mitogen-activated protein kinase (p38 MAPK) (vi) phosphoinositide 3-kinase (PI3K) and (vi) nuclear factor-kappa B (NF-kB) signalling pathways in the differentiation of stem cells to cardiomyocytes and, more importantly, to identify where possible any points of convergence and potential cross-talk between pathways that may be critical for differentiation to occur. P19 cells were routinely cultured in alpha minimal essential medium (α-MEM) supplemented with 100 units/ml penicillin /100 μg/ml streptomycin and 10% foetal bovine serum (FBS). P19 cell differentiation was initiated by culturing the cells in microbiological plates in medium containing 0.8 % DMSO to form embryoid bodies (EB). This was followed by transfer of EBs to cell culture grade dishes after four days. H9c2 cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% FBS. Differentiation was initiated by incubating the cells in medium containing 1% FBS. In both models, when drugs were employed, they were added to cells for one hour prior to initiating differentiation. Cell monolayers were monitored daily over a period of 12 or 14 days. H9c2 cells were monitored for morphological changes and P19 cells were monitored for beating cardiomyocytes. Lysates were generated in parallel for western blot analysis of changes in cardiac myosin heavy chain (MHC), ventricular myosin chain light chain 1(MLC-1v) or troponin I (cTnI) using specific monoclonal antibodies. H9c2 cells cultured in 1% serum underwent differentiation as shown by the timedependent formation of myotubes, accompanied by a parallel increase in expression of both MHC and MLC-1v. These changes were however not apparent until 4 to 6 days after growth arrest and increased with time, reaching a peak at day 12 to 14. P19 stem cells cultured in DMSO containing medium differentiated as shown by the timedependent appearance of beating cardiomyocytes and this was accompanied by the expression of cTnI. The differentiation of both P19 stem cells and H9c2 into cardiomyocytes was blocked by the PI3K inhibitor LY294002, PKC inhibitor BIM-I and the p38 MAPK inhibitor SB2035800. However when LY294002, BIM-I or SB2035800 were added after the initiation of DMSO-induced P19 stem cell differentiation, each inhibitor failed to block the cell differentiation into beating cardiomyocytes. The NF-kB activation inhibitor, CAPE, blocked H9c2 cell differentiation into cardiomyocytes. Fast nitric oxide releasing donors (SIN-1 and NOC-5) markedly delayed the onset of differentiation of H9c2 cells into cardiomyocytes while slow nitric oxide releasing donors (SNAP and NOC-18) were less effective in delaying the onset of differentiation or long term differentiation of H9c2 cells into cardiomyocytes. Akt (protein kinase B) is the key downstream target of PI3K. Our cross-talk data also showed that PKC inhibition and p38 MAPK inhibition respectively enhanced and reduced the activation of Akt, as determined by the phosphorylation of Akt at serine residue 473. In conclusion, PKC, PI3K, p38 MAPK and NF-kB are relevant for the differentiation of stem cells into cardiomyocytes. Our data also show that the PKC, PI3K and p38 MAPK signalling pathways are activated as very early events during the differentiation of stem cells into cardiomyocytes. Our data also suggest that PKC may negatively regulate Akt activation while p38 MAPK inhibition inhibits Akt activation. Our fast NO releasing donor data suggest that nitric oxide may negatively regulate H9c2 cell differentiation.

Abstract: Approximately 70% of patients infected with hepatitis C virus (HCV) develop chronic infection, which has been reported to be due to impaired specific T cell responses. Myeloid dendritic cells (mDCs) are potent antigen-presenting cells that regulate T cell responses, however their role during chronic hepatitis C (CHC) is not fully understood. My hypothesis was that the activity of mDCs to regulate T-cell response against HCV might be changed during CHC infection, and this change might contribute to the impaired T-cell responses that lead to the persistence of HCV infection. The objectives of my thesis were to compare the immunogenic activity (which stimulates T-cell proliferation), tolerogenic activity (which kills T cells), and apoptosis of mDCs from CHC patients and healthy donors. In this thesis, I found that mDCs from CHC patients expressed lower level of activating molecules, HLA-DR and CD86, compared to mDCs from healthy volunteers. When mDCs were cocultured with T cells, there were fewer T cells proliferating in the patient group than in the healthy group. This result indicated that the ability of mDCs to stimulate T cell proliferation was impaired in CHC patients. Moreover, mDCs from CHC patients underwent spontaneous apoptosis at a higher rate than mDCs from healthy donors. Nuclear factor-kappa B (NF-κB) activity, which is critical for mDC function and prevention of apoptosis, was diminished in mDCs from CHC patients. I further studied the tolerogenic activity of mDCs during CHC infection. mDCs from CHC patients expressed up-regulated levels of Fas ligand and the ligand 2 of PD-1 compared to mDCs from healthy volunteers. mDCs from CHC patients can kill T cells, while mDCs from healthy volunteers could not. This result indicated that the tolerogenic activity of mDCs was up-regulated in CHC patients. In conclusion, mDCs from CHC patients demonstrated functional changes with increased apoptosis, and diminished NF-κB activity. mDCs from CHC patients have impaired immunogenic activity to stimulate T-cell proliferation, and have up-regulated tolerogenic activity to kill T cells. These changes might be additional novel mechanisms of immune evasion by HCV, and contribute to the impaired specific T-cell responses observed in CHC patients.

Streptococci are the dominant oral commensal organisms. They, along with putative periodontal pathogens, normally colonize multiple oral tissue sites. An emerging paradigm indicates that the host can distinguish between pathogenic and nonpathogenic stimuli, and commensal bacteria could modulate expression of host genes to contribute to mucosal tolerance. Here, I hypothesized that an oral commensal Streptococcus cristatus can attenuate epithelial proinflammatory response to Fusobacterium nucleatum via regulation of Toll-Like Receptor (TLR) signaling, as a mechanism for oral mucosal tolerance to polymicrobial infection. I first demonstrated that S. cristatus itself did not provoke IL-8 production in epithelial cells, and it was able to inhibit IL-8 responses to several putative oral pathogens including F. nucleatum. The inhibitory effect of S. cristatus on IL-8 was independent of its viability and its coaggregation with F. nucleatum, was not related to soluble bacterial products, and appeared to require bacterial contact with epithelial cells. Similar effects were seen with several other species of oral streptococci. Next, I performed pathway-specific microarray analysis to specifically monitor host gene modulation by S. cristatus on a broad scale. I found that S. cristatus altered the F. nucleatum–induced expression of a number of proinflammatory cytokine genes. Profiling of TLR signaling related genes revealed that S. cristatus most significantly impacted the downstream pathways, especially NF-κB, rather than altering TLRs and their adaptors and interacting proteins. Lastly, I examined the underlying mechanism(s) involved in the modulating effect of S. cristatus by looking specifically at its impact on the nuclear factor kappaB (NF-κB) pathway under the Toll-like receptor signaling background. I found that the IL-8 suppression by S. cristatus was coincident with the inhibition of NF-κB activation and IκB-α degradation. Pre-incubation with TLR2 and TLR4 antibodies, however, did not affect the epithelial response to either species alone or in combination. I thus conclude that the oral commensal S. cristatus is not only tolerated by the host, but also able to modulate host inflammatory response to pathogenic species through inhibiting IκB-NF-κB signaling module. The anti-inflammatory effect of S. cristatus might represent a regulatory mechanism present at the epithelial surface to tolerate polymicrobial colonization.

Septic shock is a major cause of deaths resulting from uncontrolled inflammation and circulatory failure. Recent studies suggest that the vascular isoform of ATP-sensitive K+ (KATP) channels is an important contributor to septic susceptibility. To understand the molecular mechanisms for channel regulation during sepsis, we performed studies in isolated endothelium-denuded mesenteric rings. Lipopolysaccharides (LPS) induced vascular relaxation and hyporeactivity to phenylephrine. The LPS-treated aortic smooth muscle cells displayed hyperpolarization and augmentation of KATP channel activity. Both were due to an up-regulation of Kir6.1 and SUR2B surface expression. The up-regulation relied on transcriptional and translational mechanisms, in which nuclear factor-¦ÊB (NF-¦ÊB) and Protein kinase A (PKA) played a critical role. Oxidative stress occurs during sepsis and may act as another regulatory mechanism affecting KATP channel activity and vascular contractility. We found that micromolar concentrations of H2O2 impaired the pinacidil-induced vasodilation. The effect attributed to the suppression of KATP channel activity, which can be fully produced by reactivity oxidants. Unlike the Kir6.1/SUR2B channel, the Kir6.2/SUR2B channel was insensitive to 1mM H2O2, indicating that the modulation sites are located in Kir6.1. Site-directed mutational analysis showed that three cysteine residues located in N-terminus and the core region of Kir6.1 were likely to mediate the redox-dependent channel modulation. Arginine vasopressin (AVP) is a vasoconstrictor that is successfully applied to manage sepsis. However, the downstream target of AVP is uncertain. Our studies show that AVP-induced vasoconstriction depended on V1a receptor, Protein kinase C (PKC) and KATP channel. Additionally, AVP decreased Kir6.1/SUR2B channel activity through V1a receptor. The inhibitory effect was caused by a suppression of the channel open state probability. The channel inhibition was mediated by phosphorylation of the channel protein by PKC. The widespread involvement of the vascular KATP channel in vascular responses to endotoxemia strongly suggests that the temporospatial control of channel activity may constitute an important intervention to vascular tone, blood pressure and organ-tissue perfusion in septic shock. Such a control appears feasible by targeting several modulatory mechanisms of intracellular signaling, Kir6.1/SUR2B expression, redox state and channel protein phosphorylation as demonstrated in this dissertation.

Stress activates the hypothalamic-pituitary-adrenal (HPA) axis resulting in increased serum glucocorticoids (GCs); GCs are used clinically for their potent anti-inflammatory actions. Thus, because the stress-induced increase in GC is anti-inflammatory, stress predictably suppresses the inflammatory phase of wound healing and slows closure of cutaneous wounds. Previous studies from this laboratory suggest that the steroid hormone dehydroepiandrosterone (DHEA) and its metabolites androstenediol (AED) and androstenetriol (AET) counter-regulate the immune-suppressive functions of GCs. Thus we hypothesized that pharmacological treatment of animals with AED would diminish the health adverse effects of elevated GCs. It is further hypothesized that by counterbalancing the GC-mediated transcriptional regulation of cytokine gene expression through interactions with the transcriptional activator NF-κB, AED will restore inflammation and improve wound healing in stressed animals. A murine model of cutaneous wound healing was used. Male CD-1 mice underwent daily cycles of restraint stress (RST), which began three days prior to wounding. Animals were treated with AED at 3 timepoints, while control animals received vehicle treatment. Photoplanimetry was utilized to assess healing kinetics of the wounds. Wounds were harvested at various times and processed for real time PCR analysis, or the TransAM™ assay. The results show that RST delayed wound closure and altered the kinetics of inflammatory gene expression compared to control animals and did so by modulating NF-κB activation. Treatment with AED prevented the anti-inflammatory effects of RST and augmented NF-κB driven gene expression above control levels. Preliminary data suggest the effects of AED are manifest at the level of transcription initiation complex formation, and future studies will test this hypothesis. In conclusion, the data indicate that AED may be a viable pharmacological approach to functionally antagonize the effects of stress and presumably glucocorticoids, thereby improving wound healing.