Glioblastoma multiforme (GBM) is an aggressive central nervous system (CNS) cancer characterized by enhanced tumor cell motility, pernicious invasion into the normal brain, extensive tumor-induced angiogenesis, and adaptive resistance to current therapeutic paradigms. One of the difficulties associated with GBM is the ability of the tumor cells to infiltrate normal CNS tissue. Neurosurgeons can remove the primary tumor mass, but peripheral cells that are inaccessible will ultimately result in a secondary lesion that can lead to death. The matrix metalloproteinases (MMP) are well known for their abilities to facilitate processes of cellular motility and invasion through their clearance of extracellular matrix (ECM). A specific member of this family, MMP-1, is not observed in normal brain, yet its expression is a common characteristic of GBM. The various causes of MMP-1 expression, and its consequences in GBM cells are unknown. As such, functional studies were conducted related to the induction of MMP-1 expression via another molecule intrinsic to GBM, nitric oxide (NO). The exposure of GBM cell lines to nanomolar concentrations of NO produced significant inductions of MMP-1 expression and GBM cell motility. The specific removal of MMP-1 with siRNA elicited an abrogation of NO-stimulated motility, suggesting a pathological contribution by this enzyme. Furthermore, recent accumulating evidence suggests that MMP-1 contributes to tumor cell survival and related angiogenesis in other cancer settings. To investigate these capabilities in GBM, cell lines were stably engineered to have either MMP-1 over-expression or knock-down. Both tumor formation and size were significantly reduced with MMP-1 knock-down and significantly increased with over-expression. In a model of GBM cell-induced angiogenesis, the presence of MMP-1 contributed to an angiogenic phenotype. Further angiogenesis studies revealed a significant recruitment of host endothelium to the tumor interstitium in vivo. Proteomic studies suggest that one mechanism by which MMP-1 could influence angiogenesis is through the easement of the anti-angiogenic tissue inhibitor of metalloproteinases-4 (TIMP-4), since the removal of MMP-1 elicits a significant increase in TIMP-4 detection. Altogether, these functional data present MMP-1 as a promising target for future therapeutic investigation, because it is unique to the GBM environment and contributes to key overlapping GBM pathologies.

The inflammatory nature of the tumor microenvironment provides a cytokine and chemokine rich proliferative environment. Much of the responsibility of this environment is due to the production of Reactive Oxygen Species (ROS). These studies examined the proliferative rich tumor environment from a new perspective of Nitric Oxide Synthase (NOS) dysregulation. NOS’s have the ability to become uncoupled and generate superoxide in lieu of nitric oxide (NO). A requirement of NOS for the production of NO is the cofactor tetrahydrobiopterin (BH4) and when it is missing NOS becomes uncoupled and turns into a peroxynitrite synthase. Here I demonstrate that NOS is uncoupled in tumor cells due to depleted BH4 levels. This uncoupling leads to decreased NO signaling and increased pro-inflammatory, pro-survival, signaling as a result of the increased generation of ROS/RNS from uncoupled NOS activity. I was able to recouple NOS through exogenous BH4 both in vitro and in vivo, reducing ROS/RNS and reestablishing NO signaling through cGMP protein associated kinase. Reduction of ROS/RNS resulted in the reduced activity of two major constitutively active transcription factors in breast cancer cells, NFκB and STAT3. In MCF-7 and MDA231 cells I found that increased NO-dependent PKG signaling led to tumor cell toxicity mediated by downregulation of β-catenin. Downregulation of β-catenin led to increased protein levels of p21 in MCF-7 and p27 in MDA 231cells, ultimately resulting in cell death. These results suggest that there is potential for BH4 as a therapeutic agent since exogenous dietary BH4 ameliorates chemically induced colitis, and reduced azoxymethane (AOM) induced colon and spontaneously developing mammary carcinogenesis.

The purpose of this study was to identify the effects of a hemoglobin-based oxygen carrier, HBOC-201, on the cardiovascular system. Systemic cardiovascular parameters of mean arterial pressure (MAP), pulse pressure, heart rate, and oxygen saturation, as well as vascular resistance, were examined. A murine model of the cardiovascular system and microvasculature was employed. Sprague-Dawley rats (male; 230-530g; N = 13) were anaesthetised and surgically prepared for intravital microscopy of the spinotrapezius muscle. Increasing doses of HBOC-201 (2 mg/kg, 22 mg/kg, 230 mg/kg, and 780 mg/kg) and an iso-oncotic volume control were administered to assess for a dose-response relationship. MAP displayed a significant increase from baseline for both treatment groups, with no significant difference between the two. Arteriolar diameter displayed no changes from baseline, or between treatment groups or across doses. Based on these results, the noted changes in MAP were due to hypervolemia, and not a property of HBOC-201, itself.

Cell damage represents a major pathomechanism in many diseases of high clinical interest, such as myocardial infarction (MI), where it plays an important role in ischemia-reperfusion (I/R) injury. Considerable progress has been made towards identifying physiological and pharmacological agents that play a key role in myocardial preconditioning against I/R injury and also elucidating the molecular changes leading to such protection.Second messengers in cellular signaling pathways, such as cGMP have been well implicated as key players in ischemic and pharmacological preconditioning (PC) of the heart. Phosphodiesterase type 5 (PDE-5) is an enzyme that specifically hydrolyzes cGMP thereby decreasing its tissue concentration. Sildenafil is a potent selective inhibitor of PDE-5 and therefore allows the accumulation of cGMP in several tissues shown to express PDE-5, including pulmonary and coronary arteries. We initially hypothesized that vasodilation induced by sildenafil may release several endogenous mediators including adenosine, bradykinin or nitric oxide (NO), that may trigger a signaling cascade leading to protection against I/R injury. Our results show that sildenafil, at a clinically relevant dose, induced powerful acute and delayed cardioprotection against I/R injury in an in vivo rabbit model via opening of mitoKATP channels. The acute cardioprotective effect of sildenafil was dependent on activation of protein kinase C in rabbits. Moreover, we observed that sildenafil induced delayed PC by NO produced through activation of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in the mouse heart. The expression of iNOS/eNOS was regulated by ERK phosphorylation and the delayed protection against I/R was blocked by PD98059, a selective ERK inhibitor. Furthermore, sildenafil-induced delayed protection was abolished in the intact heart as well as adult myocytes derived from adenosine A1 receptor knock-out mice suggesting an essential role of A1 receptor in protection. Taken together, these studies suggest that sildenafil is a powerful tool to reduce I/R injury in the animal models. Future clinical studies with relatively safe and effective PDE-5 inhibitors may have an enourmous impact on the use of these compounds in reducing I/R injury in the heart and other organs.

Recent studies have shown that the potent phosphodiesterase-5 (PDE-5) inhibitor, sildenafil citrate, induces a powerful cardioprotective effect against ischemia-reperfusion (I/R) injury in rabbit and mouse hearts. However, the effect of this drug in inducing long-term protection against I/R injury remains unknown. The goal of this study was to identify the duration of the protective window of sildenafil citrate as well as vardenafil, a more potent PDE-5 inhibitor. Rabbits were treated with sildenafil (0.7 mg/kg, iv), vardenafil (0.143 mg/kg), or an equivalent volume of saline. After 24 hrs, 48 hrs, 96 hrs, or 7 days of sildenafil treatment, the hearts were subjected to I/R. In the vardenafil groups, the hearts were subjected to I/R at 24 hrs and 7 days after administration of the drug. To evaluate the role of nitric oxide (NO) in cardioprotection, a non-selective blocker of nitric oxide synthase, L-NAME (15 mg/kg, iv) was administered 10 minutes prior to I/R. The results show significant reductions in infarct size in hearts treated with sildenafil and vardenafil as compared to the corresponding saline controls at all time points. The protective effects of sildenafil and vardenafil were abrogated in animals treated with L-NAME. L-NAME had no effect on infarct size in saline treated control rabbits. These data suggest that both sildenafil and vardenafil induce a long-term protective effect against myocardial infarction which is mediated via a NO-dependent pathway. These studies are important in exploiting the clinical potential of PDE-5 inhibitors in terms of protection against ischemia/reperfusion injury in patients with coronary artery disease.

In the US today, blood transfusion is safer than ever. Nevertheless, the century-old quest for a suitable blood substitute persists. The elimination of unwanted side effects, especially transfusion-transmitted diseases, the problems and high cost factor involved in collecting and storing human blood, the pending worldwide shortages, and the need for compatibility testing are the driving forces contributing towards the development of blood substitutes. The leading research is focusing on hemoglobin-based oxygen carriers (HBOCs), which are limited in clinical application due to the pressor effect they induce. In this study, the mechanisms through which HBOCs affect mean arterial pressure (MAP), arteriolar diameter, and nitric oxide levels in the microcirculation were investigated, using Oxyglobin (HBOC-301), a third generation glutaraldehyde-polymerized bovine hemoglobin. The spinotrapezius muscle of female Sprague-Dawley rats was exteriorized for microcirculatory observations. HBOC in doses of 0.1, 1.0, 10.0, and 100.0 μM i.v., LNAME (30 mg/kg, i.v.), and papaverine (100 μM, topically) were given to the rat. Heparinized saline (0.1 ml and 0.5 ml, i.v.) served as control. MAP was monitored continuously through a cannula in the right carotid artery. Images of the feed, arcade and transverse arterioles were captured using a Zeiss Axioplan microscope, equipped with a digital camera, and imaging software. All doses of HBOC produced an overall vasoconstriction of the arterioles leading to an elevated MAP. Following L-NAME pretreatment, HBOC administration alone and with papaverine produced no significant elevation in MAP, indicating that the increase in resistance required basal amounts of nitric oxide (NO). This study concludes that the constriction of the arterioles correlated with the level of hypertension, and that these effects occur in a dose-dependent manner as a consequence of NO scavenging.

Prior studies have demonstrated the effect of diazoxide in protecting against apoptosis via mitochondrial KATP channel opening in vitro. The current investigations are designed to determine if sildenafil, a phosphodiesterase-5 inhibitor and known mitochondrial KATP channel opener, would protect against chronic doxorubicin cardiomyopathy both in vivo and in vitro.Male ICR mice were randomized to 1 of 4 treatments: saline, sildenafil (0.7 mg/kg IP), doxorubicin (5 mg/kg IP), and sildenafil (0.7 mg/kg IP)+doxorubicin. Apoptosis was determined using the terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling and in situ oligo ligation methods. Desmin distribution was determined via immunofluorescence. Bcl-2 was analyzed by Western blot. Left ventricular function was measured in Langendorff mode. Electrocardiographical analysis measured changes indicative of doxorubicin cardiotoxicity (ST-prolongation). In vitro studies using adult ventricular cardiomyocytes were exposed to doxorubicin (1 μM), sildenafil (1 μM) with or without NG-nitro-L-arginine methyl ester (L-NAME; 100 μM), or 5-hydroxydecanoate (5-HD; 100 μM) 1 hour before doxorubicin and incubated for 18 hours. Doxorubicin-treated mice demonstrated increased apoptosis and desmin disruption, which was attenuated in the sildenafil+doxorubicin group. Bcl-2 decreased in the doxorubicin group but was maintained at basal levels in the sildenafil+doxorubicin group. Left ventricular developed pressure and rate pressure product were significantly depressed in the doxorubicin group but attenuated in the sildenafil+doxorubicin group. ST-interval significantly increased in the doxorubicin group over 8 weeks. In the sildenafil+doxorubicin group, ST-interval remained unchanged from baseline. Doxorubicin significantly increased apoptosis, caspase-3 activation, and disruption of mitochondrial membrane potential in vitro,. In contrast, sildenafil significantly protected against doxorubicin cardiotoxicity; however, protection was abolished by both L-NAME and 5-HD. Cell viability studies using spectrophotometer and flow cytometric techniques demonstrated that sildenafil did not affect the antitumor efficacy of doxorubicin in PC-3 cells in vitro. In fact, flow cytometry data indicate that sildenafil, when combined with doxorubicin, was synergistic in the antineoplastic action of doxorubicin. Prophylactic treatment with sildenafil prevented apoptosis and left ventricular dysfunction in a chronic model of doxorubicin-induced cardiomyopathy. Moreover, these studies provide relevant clinical data on the safety and efficacy of sildenafil, leading the way for clinical trials in humans receiving doxorubicin chemotherapy.

The American Cancer Society estimates more than 141,000 new cases of and about 50,000 deaths from colorectal cancer every year. Treatment options include surgery, radiation therapy and targeted therapies such as anti-angiogenics. However, no therapies address the key driving factor of colorectal cancer: inflammation. It is well known that chronic inflammatory conditions such as Crohn’s Disease, ulcerative colitis, diabetes, obesity and cigarette smoking all elevate the risk of developing colorectal cancer. One of the hallmarks of chronic inflammation is the elevated levels of reactive oxygen/nitrogen species (ROS/RNS). A primary source of these ROS/RNS is uncoupled Nitric Oxide Synthase (NOS). Under non-inflammatory conditions NOS generates Nitric Oxide. However, in an inflammatory environment, such as the oxidative tumor microenvironment, NOS’s cofactor tetrahydrobiopterin (BH4) is oxidized to dihydrobiopterin (BH2). NOS bound to BH2 is said to be uncoupled and produces superoxide O2-and peroxynitrite (ONOO-). Previous work in our and other’s labs have shown that increased production of ROS/RNS leads to the activation of pro-inflammatory/proliferative molecules such as NFκB, Stat3, β-Catenin and Akt. NOS can be re-coupled by supplementing cells and animals with BH4 or its precursor Sepiapterin (SP). Herein we show that recoupling NOS with SP in HCT116, Caco-2 and HT29 cells, decreased tumor cell proliferation, increased β-Catenin degradation and decreased Akt activity. We also see increased tumor cell death measured by in vitro clonogenic assay, as well as decreased metabolic uptake in Azoxymethane/Dextran Sodium Sulfate (AOM/DSS) induced colorectal cancer in vivo measured by [18F]-fluorodeoxyglucose ([18F]-FDG) positron emitted topography (PET) imaging. We believe by recoupling NOS both in vivo and in vitro we are modulating Wnt signaling via Akt and GSK-3β. Lastly, we conducted studies to determine a mechanistic explanation of how tumor cells maintain a decreased BH4:BH2 ratio.

Sand flies (order Diptera) are vectors of Leishmania parasites (Trypanosomatida), which are inoculated into the host skin together with the vector saliva. Sand fly saliva plays the important role in the Leishmania transmission; in naive host it supresses the host immune response assisting Leishmania to establish the infection, while in repeatedly bitten host it elicits a protective immune response. The submitted thesis focuses on the effect of sand fly saliva on macrophages, the key cells in the infection control. In the first part of the thesis we established a laboratory model L. major - P. papatasi - Balb/c to describe the protective effect of saliva immunization on Leishmania infection development. Immunized mice were protected against Leishmania infection which was reflected in the ear lesion size, parasite load in the ear dermis and draining lymph nodes but also in cytokine production. On the contrary, produced lower amount of nitric oxide, while arginase activity was comparable with nonimmunized group. The IgG antibodies against saliva served as a marker of exposure to sandflies while IgG antibodies against Leishmania antigens served as a marker of infection severity. The experiments were aimed on the possibility of cross-protectivity in Balb/c mice against L. major between closely related...

Doctor of Philosophy / Department of Anatomy and Physiology / David C. Poole / The overall aim of this dissertation is to better understand the mechanisms determining skeletal muscle oxygen delivery-utilization matching in health and disease. Emphasis is directed toward the role of nitric oxide (NO) bioavailability in modulating muscle microvascular oxygenation (PO2mv; the sole driving force for blood-myocyte oxygen flux) during transitions in metabolic demand. The first investigation of this dissertation (Chapter 2) demonstrates that alterations in NO bioavailability have a major impact on skeletal muscle PO2mv kinetics following both the onset and cessation of contractions. Specifically, increased NO levels (via the NO donor sodium nitroprusside; SNP) elevates whereas reduced NO levels (non-specific NOS inhibition with NG-nitro-L-arginine methyl ester; L-NAME) diminishes muscle PO2mv at the onset and during recovery from contractions in the spinotrapezius muscle of healthy young rats. Consistent with these results, inhibition of the neuronal NO synthase isoform (S-methyl-L-thiocitrulline; SMTC; Chapter 3) reveals alterations in NO-mediated regulation of skeletal muscle PO2mv with advanced age that likely contribute to exercise intolerance in this population. In Chapter 4 we observed that pronounced oxidative stress is implicated in these pathological responses seen in aged and diseased states. Transient elevations in the oxidant hydrogen peroxide to levels seen in the early stages of senescence and cardiovascular diseases promote detrimental effects on skeletal muscle contractile function (i.e., augmented oxygen cost of force production). Chapter 5 demonstrates that endurance exercise training improves skeletal muscle microvascular oxygenation (i.e., greater PO2mv and slower PO2mv kinetics) across the metabolic transient partly via enhanced NO-mediated function in healthy young individuals. These data carry important clinical implications given that exercise training may ameliorate NO-mediated function, muscle microvascular oxygenation deficits and consequently exercise intolerance in aged and diseased populations. In conclusion, alterations in NO bioavailability have a major impact on the dynamic balance between skeletal muscle oxygen delivery and utilization (i.e., PO2mv kinetics) in health and disease. While advanced age or the predations of disease impair considerably skeletal muscle microvascular oxygenation, exercise training-induced adaptations on the oxygen transport system constitute a non-pharmacological therapeutic intervention potentially capable of mitigating these microcirculatory deficits.