<p>Smooth muscle cells from pig aortic media were grown in tissue culture, in medium containing 10% calf serum. Lipid biosynthesis from radioactive substrates 1-¹⁴C-acetate, U-¹⁴C-D-glucose, 1-¹⁴C-oleic acid and ³²P-phosphoric acid was measured. In addition, the influence of various sera, including pig serum, normolipemic human serum (NLHS), and hyperlipemic human serum (HLHS) on lipid biosynthesis from acetate and phosphoric acid was studied</p> <p>Compared to calf serum, all three test sera caused a stimulation of lipid synthesis in the lipid classes, phospholipid (PL), free fatty acids (FFA), triglycerides (TG) and cholesterol esters (CE), and an inhibition of cholesterol plus diglyceride (S + DG) synthesis. The extent of stimulation was least for pig serum and greatest for HLHS; the inhibition of S + DG was greatest for HLHS and least for pig serum. It was noted that the HLHS stimulation of CE synthesis was proportionately greater than the stimulation of the other lipid classes and that the HLHS inhibition of S + DG was significantly greater than that seen with the other test sera.</p> <p>The morphology of cultured aortic smooth muscle cells grown in 10% calf serum and 10% HLHS was examined by means of scanning and transmission electronmicroscopy. It was observed that HLHS caused degenerative alterations in the morphology of the cultured smooth muscle cells, such as an abundance of lipid droplets and cellular debris. The implications of these results in relation to the development of atherosclerosis are discussed.</p> / Thesis / Master of Science (MS)

Platelet-derived growth factor D-chain (PDGF-D) is the newest member of the PDGF family of mitogens and chemo-attractants; it is expressed in a wide variety of cell types, including vascular smooth muscle cells (SMCs). The molecular mechanisms regulating PDGF-D transcription are unknown. Here I investigated the effects of angiotensin II (ATIl) and IL-1 beta on the transcription of PDGF-D and changes in vascular SMCs phenotype. Primer extension analysis mapped a single transcriptional start site to the ccAG CGC motif of PDGF-D promoter. Several potential transcription factor binding sites such as SpI, Ets-1, NF-??B, IRF-1, p53, Smad4 and AP1 were located in the proximal 1168bp of the PDGF-D promoter. ATII-inducible Ets-1 and PDGF-D gene expression is mediated via H202. IL-I beta supresses PDGF-D promoter activity, mRNA and protein expression in SMCs through NF-??B p65, IRF-1 and HDAC1, which form complex in the PDGF-D promoter. This study provides the first direct link between NF-KB and the PDGF-D promoter, IRF-1 with any member of the PDGF family and a new example of HDAC mediated inhibition of gene expression. In summary, this study investigates for the first time the mechanisms mediating the transcriptional regulation of PDGF-D in vascular SMCs. This provides valuable insights into the molecular control of vascular phenotype, and opens up potential opportunities for therapeutic intervention.

Immunopathological disorders are no longer defined by dysregulated T-helper (Th) type 1/ Th2 responses but account for modulatory cell types such as regulatory and Th17 cells. The newly defined Th17 subset is an effector memory subtype which regulates mucosal host defense responses. A distinctive feature of interleukin (IL)-17 is its ability to invoke neutrophilic responses and to synergize cytokine responses in proximal structural cells. This effect is most evident for proinflammatory cytokines and neutrophil-mobilizing chemokines which are under the regulatory control of the canonical, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. The uniqueness of the IL-17A receptor (IL-17RA) signal transduction pathway however has been a limiting factor in uncovering IL-17-mediated effector functions since the receptor bears little homology to other known receptors and contains a unique cytoplasmic consensus binding motif. Hence, the composition, dynamics and subunit interactions of the IL-17R complex have become an emerging area of research where novel recruitment motifs and adaptor proteins are actively being explored. Our study sought to uncover the signal transduction and molecular mechanisms mediating the initiation and amplification responses induced by IL-17. We hypothesize that (i) IL-17 represents a key cytokine which initiates inflammatory responses by acting on proximal structural cells to rapidly release neutrophil-mobilizing chemokines and myeloid growth factors and that (ii) IL-17 directly promotes survival responses of immune effector cells. Genomic analysis of stimulated human airway smooth muscle cells support the proinflammatory nature of IL-17 as NF-κB associated genes and chemokines were most significantly upregulated within 2 hours. However, IL-17 induced a modest fold increase in gene expression levels whereby only 4 genes achieved greater than 2 fold increases. This, along with the observation that IL-17 enhanced IL-1β-mediated CXCL8 expression via transcriptional promoter activation levels and post-transcriptional mRNA stabilization mechanisms suggests that IL-17 cooperatively functions with secondary cytokines to mediate inflammatory responses. Despite activating the p38-mitogen-activated protein kinase (MAPK) signaling pathway in peripheral blood neutrophils, IL-17 did not directly affect the apoptotic capacity of these cells but unexpectedly antagonized the survival response mediated by the granulocyte-macrophage colony stimulating factor (GM-CSF). Collectively, our results suggest that IL-17 is a potent synergistic cytokine which signals via the MAPK-NF-κB pathway to indirectly recruit neutrophils via CXC-chemokines produced by non-hematopoietic cells and that IL-17 may potentially dampen inflammatory responses by directly antagonizing inflammatory effector cells.