Faculty Administrative Relations Committee report, 2016-2017

Annual report for a committee of the University of Tennessee at Chattanooga Faculty Senate

Microrna Regulation On The Expression Of CD38 And Other Asthma Related Genes In Human Airway Smooth Muscle Cells

University of Minnesota Ph.D. dissertation. April-2015. Major: Veterinary Medicine. Advisor: Mathur Kannan. 1 computer file (PDF); x, 200 pages.CD38 is a multifunctional enzyme that regulates intracellular calcium ([Ca++]i ) homeostasis. It is expressed in airway smooth muscle (ASM) cells where it elevates [Ca++]i through its enzymatic product cyclic ADPribose (cADPR) and increases ASM contractility. Increased expression of CD38 in the ASM cells derived from the asthmatic patients (AS-HASM) and attenuated airway hyperresponsiveness to contractile stimuli shown by CD38-/- mice implicate the importance of CD38 in asthma. The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is considered to be an important mediator for airway pathology in asthma. The reason for the differential expression of TNF-alpha-induced-CD38 in AS-HASM cells, does not involve transcriptional regulation of CD38 which is through signaling pathways mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K), and transcription factors nuclear factor kappa-B (NF-kB) and AP-I. Thus I hypothesized that post-transcriptional regulation of CD38 by microRNAs account for the differential expression of TNF-alpha induced -CD38 in AS-HASM cells. Among the several potential microRNAs predicted for CD38 by microRNA target-predicting algorithms, I selected miR-140-3p and miR-708 for further studies, as these showed differential expression in the AS-HASM cells compared to those from healthy subjects. Overexpression of these either microRNAs in ASM cells inhibited the TNF-alpha induced expression of CD38 at messenger RNA (mRNA) and protein levels. Luciferase-reporter assays with a mutated 3'UTR of the CD38 transcript confirmed the specific target sites for both microRNAs. Transcript stability assays revealed that mRNA degradation is not the mechanism underlying regulation by microRNAs. Examination of the expression and activation levels of proteins in the upstream signaling pathways of CD38 revealed that miR-140-3p, by inactivating p38 MAPK and NF-kB, and miR-708, by inactivating c-Jun N-terminal kinase (JNK) MAPK and Akt by elevating the expression of their phosphatases MKP-1 and PTEN respectively, control the expression of CD38 indirectly. Further, we found that miR-708 downregulates the expression of many chemokines and inhibits the serum induced proliferation of human ASM cells. We conclude that both microRNAs have therapeutic potential in controlling asthma related symptoms through regulating the expression of CD38 and chemokines and controlling the proliferation of human ASM cells

Phagocytosis of mast cell granules results in decreased macrophage superoxide production

The mechanism by which phagocytosed mast cell granules (MCGs) inhibit macrophage superoxide production has not been defined. In this study, rat peritoneal macrophages were co-incubated with either isolated intact MCGs or MCG-sonicate, and their respiratory burst capacity and morphology were studied. Co-incubation of macrophages with either intact MCGs or MCG-sonicate resulted in a dose-dependent inhibition of superoxide- mediated cytochrome c reduction. This inhibitory effect was evident within 5 min of incubation and with MCG-sonicate was completely reversed when macrophages were washed prior to activation with PMA. In the case of intact MCGs, the inhibitory effect was only partially reversed by washing after a prolonged co-incubation time. Electron microscopic analyses revealed that MCGs were rapidly phagocytosed by macrophages and were subsequently disintegrated within the phagolysosomes. Assay of MCGs for superoxide dismutase (SOD) revealed the presence of significant activity of this enzyme. A comparison of normal macrophages and those containing phagocytosed MCGs did not reveal a significant difference in total SOD activity. It is speculated that, although there was no significant increase in total SOD activity in macrophages containing phagocytosed MCGs, the phagocytosed MCGs might cause a transient increase in SOD activity within the phagolysosomes. This transient rise in SOD results in scavenging of the newly generated superoxide. Alternatively, MCG inhibition of NADPH oxidase would explain the reported observations

Deranged aortic intima-media thickness, plasma triglycerides and granulopoiesis in Sl/Sl(d) mice.

Studies were carried out to evaluate the impact of a high-fat dietary regimen on aortic wall thickness, peripheral blood leukocyte profile, and plasma cholesterol and triglyceride levels in the mast cell-deficient Sl/Sl(d) mouse. The results demonstrated that the mean aortic wall thickness of Sl/Sl(d) mice was significantly higher than their normal littermates, and were increased in both genotypes after a 17-day high-fat regimen. In comparison with normal littermates, Sl/Sl(d) genotypes had elevated levels of plasma triglycerides with normal levels of plasma cholesterol, and the high-fat diet markedly lowered the triglyceride levels. Total peripheral blood leukocytes, the monocyte and granulocyte counts, and hemoglobin levels were significantly lower in Sl/Sl(d) mice, although the number of lymphocytes, eosinophils and basophils were the same in both genotypes. Interestingly, the high-fat diet regimen elevated leukocyte counts and the number of monocytes and granulocytes in Sl/Sl(d) mice

Poloxamer 407-induced atherosclerosis in mice appears to be due to lipid derangements and not due to its direct effects on endothelial cells and macrophages.

Coronary heart disease secondary to atherosclerosis is still the leading cause of death in the US. Animal models used for elucidating the pathogenesis of this disease primarily involve rabbits and pigs. Previous studies from this laboratory have demonstrated intraperitoneal injections of poloxamer 407 (P-407) in both male and female mice will lead to hyperlipidemia and atherosclerosis, suggesting the use of this polymer to develop a mouse model of atherosclerosis. In order to understand the mechanism of P-407-induced hyperlipidemia and vascular lesion formation, we evaluated the direct effects of P-407 on endothelial cell and macrophage functions in vitro, and its in vivo effects on the oxidation of circulating lipids following long-term (4 month) administration. Our results demonstrated that incubation of P-407 with human umbilical vein endothelial cells in culture did not influence either cell proliferation or interleukin-6 and interleukin-8 production over a concentration range of 0-40 microM. In addition, nitric oxide production by macrophages was not affected by P-407 over a concentration range of 0-20 microM. Finally, we demonstrated that while P-407 could not induce the oxidation of LDL-C in vitro, long-term (4 month) administration of P-407 in mice resulted in elevated levels of oxidized lipids in the plasma. Thus, it is suggested that the formation of atherosclerotic lesions in this mouse model of atherosclerosis does not result from either direct stimulation of endothelial cells or macrophage activation by P-407. Instead, these data would support the premise that oxidation of lipids (perhaps low-density lipoprotein cholesterol) by an indirect mechanism following injection of P-407 may represent one of the mechanisms responsible for atheroma formation

Effect of mast cell granules on the gene expression of nitric oxide synthase and tumour necrosis factor-alpha in macrophages.

Previous studies have shown that mast cell granules (MCG) inhibit numerous macrophage functions including tumour cytotoxicity, superoxide and nitric oxide (NO) production, and FCgamma2a receptor-mediated phagocytosis. In this study, the effect of MCG on macrophage TNF alpha and nitric oxide synthase (iNOS) mRNA expression, and the production and fate of TNF alpha were examined. Upon activation with LPS+IFN gamma, macrophages expressed both TNF alpha and iNOS mRNA and produced both TNF alpha and NO. Co-incubation of LPS+IFN gamma-activated macrophages with MCG resulted in dose-dependent inhibition of iNOS mRNA expression. TNF alpha production in the activated macrophages was decreased by MCG, which was associated with a reduction in TNF alpha mRNA expression. MCG were also capable of degrading both macrophage-generated and recombinant TNF alpha. The direct effect of MCG on TNF alpha was partially reversed by a mixture of protease inhibitors. These results demonstrate that MCG decrease the production of NO and TNF alpha by inhibiting macrophage iNOS and TNF alpha gene expression. Furthermore, MCG post-transcriptionally alter TNF alpha levels via proteolytic degradation

Constitutive Expression of Functionally Active Protease-Activated Receptors 1 and 2 in Human Conjunctival Epithelial Cells

Protease-activated receptors (PARs) are G-protein-coupled receptors which initiate inflammatory responses when activated by specific serine proteases. This study was conducted to examine whether human conjunctival epithelial cells (HCECs) express functionally active PAR1 and PAR2 using Chang conjunctival epithelial cells as in vitro model. We performed RT-PCR and immunofluorescence analyses to determine the expression of PAR1 and PAR2, and monitored the production of IL-6 after activating HCECs with PAR1 activating agents (thrombin or TFLLRN) or PAR2 activating agents (tryptase, trypsin, or SLIGKV). The results show that HCECs constitutively express PAR1 and PAR2 mRNA and proteins, and produce significant amounts of IL-6 when incubated with specific PAR-activating enzymes or agonist peptides. Thrombin- and tryptase-induced HCEC activation was blocked by PAR1 and PAR2 neutralizing antibodies, respectively, and by specific enzyme inhibitors. The constitutive expression of PAR1 and PAR2, and their activation by thrombin and tryptase, respectively, may have important implications in ocular inflammation