The trans-differentiation of injury-activated fibroblasts to myofibroblasts is a process that provides contractile strength for wound closure. Persistent myofibroblast differentiation, however, is associated with fibrotic pathologies such as organ fibrosis, vascular remodeling, and atherosclerotic plaque formation. Myofibroblasts acquire a contractile phenotype with biochemical properties characteristic of both smooth muscle cells and stromal fibroblasts. The cyto-contractile protein, smooth muscle α-actin (SMαA) is a biomarker of myofibroblast differentiation. Expression of the SMαA gene, ACTA2, is regulated by cis-acting elements and transcription factors that activate or repress the ACTA2 promoter. Purine-rich element binding proteins A (Purα) and B (Purβ) are sequence-specific, single-stranded DNA (ssDNA)/RNA-binding proteins that act as transcriptional repressors of ACTA2 expression. Both Pur proteins interact with the purine-rich strand of a cryptic muscle-CAT (MCAT) enhancer motif in 5'-flanking region of the ACTA2 promoter. Despite significant sequence homology with Purα, Purβ was identified as the dominant repressor of ACTA2 expression in mouse embryonic fibroblasts and vascular smooth muscle cells by virtue of gain-of function and loss-of-function analyses in cultured cells. Biophysical studies indicated that Purβ reversibly self-associates in solution to form a homodimer. Quantitative DNA-binding assays revealed that Purβ interacts with the purine-rich strand of the ACTA2 MCAT motif via a cooperative, multisite binding mechanism to form a high-affinity 2:1 Purβ-ssDNA complex. In this dissertation, a combination of computational, biochemical, and cell-based approaches were employed to elucidate the molecular basis of Purβ repressor interaction with the ACTA2 gene. Limited proteolysis of recombinant mouse Purβ in the presence and absence of the purine-rich strand of the ACTA2 MCAT element led to the identification of a core ssDNA-binding region that retains the ability to dimerize in solution. Knockdown of endogenous Purβ in mouse embryonic fibroblasts via RNA interference induced SMαA expression and conversion to a myofibroblast-like phenotype. To map the specific structural domains in the core region of Purβ that account for its unique ACTA2 repressor and ssDNA-binding functions, computational homology models of the Purβ monomer and dimer were generated based on the x-ray crystal structure of an intramolecular subdomain of Drosophila melanogaster Purα. Empirical biochemical and cell-based analyses of rationally-designed Purβ truncation proteins revealed that the assembled Purβ homodimer is composed of three separate purine-rich ssDNA-binding subdomains. Evaluation of the effects of anionic detergent and high-salt on the binding of Purβ to ssDNA implicated the involvement of hydrophobic and electrostatic interactions in mediating high-affinity nucleoprotein complex formation. This inference was validated by site-directed mutagenesis experiments, which identified several basic amino acid residues required for the ACTA2 repressor activity of Purβ. Collectively, the findings described herein establish the structural and chemical basis for the cooperative interaction of Purβ with the ACTA2 MCAT enhancer and for Purβ-dependent suppression of myofibroblast differentiation.

KCl is traditionally used as a stimulus to examine the role of increases in cytosolic Ca2+ on the regulation of smooth muscle contraction. KCl bypasses GPCR activation, thereby avoiding activation of additional cell signaling systems, such as phospholipase C and PKC that are inherent to Gaq and Ga12/13 stimulation. GPCR activation causes Ca2+ sensitization (greater force for a given increase in Ca2+) by a ROCK- and PKC-dependent inhibition of myosin light chain (MLC) phosphatase. Recent studies have demonstrated that KCl can also produce Ca2+ sensitization, implying that Ca2+ itself may induce Ca2+ sensitization. To test the hypothesis that Ca2+ can induce Ca2+ sensitization, we permeabilized rabbit femoral artery rings with beta-escin and subjected the tissues to a Ca2+ concentration response curve in the absence (control) and presence of selective inhibitors of ROCK and PKC, the general ser/thr kinase inhibitor, staurosporine, and the MLCK inhibitor, wortmannin. For a comparison, tissues permeabilized with beta-escin were also contracted with the alpha-adrenergic agonist, phenylephrine (PE), and exposed to the same complement of inhibitors. Interestingly, Ca2+-induced contraction was inhibited by a PKC inhibitor in beta-escin-permeabilized tissue. Wortmannin and staurosporine nearly abolished Ca2+-induced contraction. The ROCK inhibitor, H-1152, significantly reduced Ca2+- and PE-induced contractions in beta-escin permeabilized tissues. Western blots showed significant decreases in basal MYPT1-pThr853 by ROCK inhibitors and reduction of MLC-p by ROCK and PKC inhibitors. These data support our hypothesis that elevations in Ca2+ or constitutive ROCK activity may cause ROCK-dependent Ca2+ sensitization. Moreover, data also suggest the possibility of PKC-directed activation of the RhoA/ROCK cascade. Once the mechanism for smooth muscle Ca2+ sensitization is understood, advanced treatments for vascular hyper-contraction disorders, such as vasospasm and hypertension, may be possible.

Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family of secreted proteins, which include in addition to BDNF, nerve growth factor (NGF) and neurotrophin 3-6 (NT-3-6). BDNF mediates its functions by activating two cell surface receptors, pan-neurotrophin receptor (P75NTR) and tropomyosin-related kinase B (TrkB) and their downstream intracellular cascades. BDNF is best known for its role in neuronal survival, regulation of neuronal differentiation, migration and activity-dependent synaptic plasticity. However, BDNF is widely expressed in non-neuronal tissues as well. The localization and the function of BDNF in intestinal smooth muscle cells (SMCs) are not well defined. Thus, the main purpose of the present study was the identification and characterization of BDNF in intestinal SMCs. Using xviii biochemical and molecular techniques, we have demonstrated in this study that BDNF is synthesized and released in rabbit intestinal longitudinal SMCs cultures. Furthermore, gut neuropeptides, Pituitary Adenylate Cyclase Activating Peptide (PACAP) and substance P (SP) increased BDNF expression and release in SMCs cultures after 24 hrs and 48 hrs incubation. We have also shown that intracellular Ca2+ levels are essential for SP stimulation of BDNF expression and secretion. Lastly, we have demonstrated that exogenous BDNF enhanced carbachol (CCh)-induced contraction of isolated longitudinal muscle strips, and this was inhibited by preincubation with TrkB inhibitor K252a and PLC inhibitor U73122 sugesting that BDNF sensitize longitudinal SMCs to CCh by activating PLC pathway, which is normally absent in those muscle cells. These results provide new insight into the mechanisms of neurotrophin (BDNF) modulation of gut function, which may lead to new therapeutic avenues for treatment of gastrointestinal disorders, and explain some of the pathological changes associated with inflammation such as hypercontractility associated with gut infection or IBD.

The Melatonin, a close derivative of serotonin, is involved in physiological regulation of circadian rhythms. In the gastrointestinal (GI) system, melatonin exhibits endocrine, paracrine and autocrine actions and is implicated in the regulation of GI motility. Generally, melatonin actions oppose the stimulatory actions of serotonin on motility. However, it is not known whether melatonin can also act directly on GI smooth muscle cells. The aim of the present study was to determine the expression of melatonin receptors in smooth muscle and identify their signaling pathways. Muscle cells were isolated from rabbit distal stomach by enzymatic digestion, filtration and centrifugation and cultured in DMEM-10. Expression of melatonin receptors, MT1 and MT2, was determined by RT-PCR and Western blot. G protein activity was measured by melatonin-induced increase in Gα binding to [35S]GTPγS. Phosphoinositide (PI)-specific phospholipase C (PLC-) activity was measured by ion-exchange chromatography. Cytosolic Ca2+ was measured in fura-2 loaded cells and muscle contraction was measured by scanning micrometry. In cultured gastric smooth muscle cells MT1 was detected by RT-PCR and western blot. Melatonin activated Gαq, but not Gαs, Gαi1, Gαi2, or Gαi3. Consistent with activation of Gαq, melatonin stimulated PLC-β activity (PI hydrolysis), increased cytosolic Ca2+, and elicited muscle contraction. Stimulation of PLC-β activity was blocked by the expression of Gq minigene and contraction was blocked by the PLC-β inhibitor, U73122. We conclude that gastric smooth muscle cells express receptors for melatonin (MT1) coupled to Gq. The receptors mediate stimulation of PLC- activity and increase in cytosolic Ca2+, and elicit muscle contraction.

Gastrointestinal (GI) smooth muscle possesses distinct regional and functional properties that distinguish it from other types of visceral and vascular smooth muscle. On the basis of electrical properties and contractile phenotype, GI smooth muscles have been classified into phasic (non-sphinteric) and tonic (sphinteric) smooth muscles. The biochemical basis of phasic and tonic phenotypes of smooth muscle is not clear and is the major question of inquiry of the present study. Phosphorylation of Ser19 on the 20 kDa myosin light chain (MLC) is essential for acto-myosin interaction and contraction in both phasic and tonic muscles. The levels of MLC20 phosphorylation are regulated by Ca2+/calmodulin-dependent MLC kinase (MLCK) and MLC phosphatase (MLCP), and the activity of these enzymes are in turn regulated by various signaling molecules whose expression and activity are important in determining the strength and duration of their activity. The signaling proteins are AMP kinase (MLCK activity), Rho kinase, zipper-interacting protein kinase (ZIPK), CPI-17 and telokin (MLCP activity), phosphodiesterase 5 (PDE5) and multi-drug resistance protein 5 (MRP5). The overarching goal of the dissertation is to identify the differences in the signaling pathways that regulate MLCK and MLCP activities, and thus MLC20 phosphorylation and muscle function. Using biochemical, molecular and functional approaches, and antrum (distal stomach) and fundus (proximal stomach) of rabbit stomach as models of phasic and tonic smooth muscles, respectively, the present study characterized important differences in the signaling pathways that highly correlate with the contractile phenotype. These include: 1) tissue-specific expression of contractile proteins such as myosin heavy chain isoforms, actin, caldesmon, calponin, - and β-tropomyosin, smoothelin-A and -B; 2) higher expression of AMPK, selective feedback inhibition of MLCK activity via AMPK-mediated phosphorylation, and higher expression of telokin and activation of MLCP correlate with the rapid cyclical contractile function in phasic muscle; 3) higher expression and activation of Rho kinase/ZIPK/MYPT1 and PKC/CPI-17 pathways, preferential inhibition of MLCP activity, and sustained phosphorylation of MLC20 correlate with the sustained contraction in tonic muscle; and 4) rapid termination of cGMP signal and muscle relaxation by preferential degradation and efflux of cGMP via higher expression of PDE5 and MRP5, respectively, correlate with the brief relaxation and rapid restoration of contraction in tonic muscle. It is anticipated that these findings could be important in providing the underlying mechanisms involved in the pathophysiology of smooth muscle function and new insights for the development of therapeutic agents that should act on smooth muscle in the gut to treat motility disorders as well as in other regions such as airways and vascular smooth muscle where similar intracellular mechanisms may prevail.

Development of novel ill vitro and ill vivo models for determining primary events in HLRCC tumourigenesis Linda O'Flaherty, Mansfield College Thesis submitted for degree of Doctor of Philosophy Nuffield Department of Clinical Medicine, University of Oxford Hilary Term 2012 Germline mutations of fumarate hydratase (FR), encoding an enzyme of the tricarboxylic acid (TCA) cycle, predispose affected individuals to hereditary leiomyomatosis and renal cell cancer (HLRCC). FH-deficient cells and tissues have been shown to accumulate fumarate, exhibit S-(2-succinyl) cysteine (2SC) protein modifications and to constitutively express hypoxia-inducible factor alpha (HIF -1 a and -20.), under nonnoxic conditions. This thesis presents a phenotypic characterisation of FhI-I- mouse embryonic fibroblasts (MEFs), generated from previously reported conditional Fhl knockout mice, as a new in vitro system for investigating and identifying biochemical and metabolic pathways that are dysregulated as a result of FhI inactivation. These cell lines reproduced the aforementioned phenotypes, in addition to an observed shift from oxidative phosphorylation (OXPHOS) to glycolytic metabolism. Re-expression of either full length, mitochondrial-targeted FH (FhI-I- +FH) or cytoplasmic FH (Fhrl- +FHl'1MTS) in FhI-deficient MEFs was sufficient to reduce intracellular fumarate and to correct for the dysregulation of the Hif pathway. These results were of particular interest as they demonstrated that nonnoxic stabilisation of Hif-Ia occurs independently of the persistent mitochondrial defect observed in Fhrl- +FHl'1MTS MEFs. These findings were corroborated in vivo following the development of transgenic mouse models, ubiquitously expressing either FH or FHl'1MTS in mice with targeted inactivation of FhI in renal tubular cells. Surprisingly, the cytoplasmic-restricted FH (FHl'1MTS) transgene was just as efficient as the transgenic mice expressing mitochondrial- targeted FH at rescuing the cystic phenotype associated with Fh I-deficiency in the kidneys. As the function of cytoplasmic FH has remained poorly understood, these results go some way to extricating a role for this isofonn of FH. The results of this thesis demonstrate that these novel in vitro and in vivo models, used either alone or in combination, are a versatile and robust paradigm for studying altered cell metabolism in not only HLRCC but other diseases associated with metabolic dysregulation.

Tissue engineered blood vessels (TEBVs) have great potential as tools for disease modeling and drug screening. However, existing methods for fabricating TEBVs create homogenous tissue tubes, which may not be conducive to modeling focal vascular diseases such as intimal hyperplasia or aneurysm. In contrast, our lab has a unique modular system for fabricating TEBVs. Smooth muscle cells (SMCs) are seeded into an annular agarose mold, where they aggregate into vascular tissue rings, which can be stacked and fused into small diameter TEBVs. Our goal is to create a platform technology that may be used for fabricating focal vascular disease models, such as intimal hyperplasia. Because tubes are fabricated from individual ring units, each ring can potentially be customized, enabling the creation of focal changes or regions of disease along the tube length. In these studies, we first demonstrated our ability to modulate cell phenotype within individual SMC ring units using incorporated growth factor-loaded degradable gelatin microspheres. Next, we evaluated fusion of ring subunits to form composite tissue tubes, and demonstrated that cells retain their spatial positioning within individual rings during fusion. By incorporating electrospun polycaprolactone cannulation cuffs at each end, tubes were mounted on bioreactors after only 7 days of fusion to impart luminal medium flow for 7 days at a physiological shear stress of 12 dyne/cm2. We then created focal heterogeneities along the tube length by fusing microsphere-containing rings in the central region of the tube between rings without microspheres. In the future, microspheres may be used to deliver growth factors to this localized region of microsphere incorporation and induce disease phenotypes. Due to the challenges of working with primary human SMCs, we next evaluated human mesenchymal stem cells (hMSCs) as an alternative cell source to generate vascular SMCs. We evaluated the effects of microsphere-mediated platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and transforming growth factor beta-1 (TGF-β1) delivery on ring thickness, proliferation, and contractile protein expression over a 14 day period. Finally, we created a structurally distinct region of smooth muscle within tissue tubes by fusing human aortic SMCs in a central region between hMSC rings. In summary, we developed a platform technology for creating modular tubular tissues that may be further developed into an in vitro intimal hyperplasia model. It may also be modified to model other focal vascular diseases, such as aneurysm, or to create other types of multi-tissue tubular structures, such as trachea.

MicroRNAs play an important role in biological regulation. Recently miR-34a has been reported to regulate tumour cell cycle progression and apoptosis. However, the functional role of miR-34a in smooth muscle cell (SMC) differentiation from stem cells is yet unclear. Main objectives of this PhD project are to determine the functional role of miR-34a and its target genes in SMC differentiation and underlying mechanisms. Mouse embryonic stem (ES) cells were seeded on collagen coated flasks in differentiation medium to allow SMC differentiation. Upon analysis, miR-34a was significantly up-regulated during SMC differentiation. Results demonstrated that overexpression of miR-34a significantly promoted SMC-specific gene expression, while knockdown of miR-34a inhibited expression of SMC specific gene. Enforced expression and knockdown of miR-34a in differentiating ES cells up-regulated and down-regulated, respectively, several SMC transcription factors in a similar manner. It was also found that miR-34a overexpression in stem cells promoted SMC differentiation in vivo. Furthermore, deacetylase sirtuin 1 (Sirt1) was identified as one of the top targets of miR-34a. Surprisingly, Sirt1 was demonstrated to be positively regulated by miR-34a during SMC differentiation in a cellular context and RNA sequence dependent manner. VIII Mechanistically, the data suggested that miR-34a promoted differentiating stem cells arrest at G0/G1 phase, and a significant decreased incorporation of miR-34a and SirT1 RNA into Ago2-RISC complex was observed upon SMC differentiation. The results demonstrated that Sirt1 acted as a transcriptional activator in the regulation of SMC gene during ES cell differentiation. Finally, H3K9 tri-methylation around the promoter regions of the SMαA and SM22α genes was also found to be significantly inhibited by SirT1 overexpression. These findings suggest that miR-34a plays an important role in SMC differentiation from ES cells. Meanwhile, Sirt1 can be regulated by miR-34a through an unexpected pathway and it was identified as a functional modulating target in miR-34a mediated SMC differentiation.

Orientador: José Ricardo Paciência Rodrigues / Banca: Afonso Mazario / Banca: Henrique Benedito Brenelli / Banca: Cleverson Teixeira / Banca: Gilberto Uemura / Resumo: Estudos de expressão gênica têm identificado vários grupos moleculares de carcinoma de mama, com diferentes comportamentos clínico e biológico. A correlação entre "cDNA microarray" e imunoistoquímica(IQ) com marcadores para citoceratinas, Her2/neu, receptor de estrógeno(RE) e de células basais mioepiteliais (1A4, S-100 e p63), identificaram cinco grupos: (1) luminal A (RE+; Her2/neu-), (2) luminal B (RE+; Her2/neu+), (3) superexpressão de Her2/neu (RE-; Her2/neu+), (4) tipo basal (RE-; Her2/neu-; Ck 5/6 +) e (5) nenhum destes ("null"). Os de tipo luminal expressam citoceratinas de padrão luminal (Ck8/18) e os de tipo basal expressam citoceratinas 5/6 e 14 ou marcadores de células basais mioepiteliais. Avaliamos a expressão de Ck5/6, Ck8/18 e 1A4 em material de citoinclusão, comparando-a ao espécime cirúrgico. Material e Métodos: Foram selecionados 62 casos, seqüenciais, de carcinoma de mama diagnosticados por PAAF, com citoinclusão e espécime cirúrgico. Cortes de citoinclusão e do espécime cirúrgico foram imunocorados para Ck 5/6, Ck 8/18 e 1A4. Resultados e Conclusão: Os valores, em porcentagem, de sensibilidade, especificidade, valor preditivo positivo(VPP), valor preditivo negativo(VPN) e acurácia foram, respectivamente: Ck5/6 (77, 100, 100, 92 e 94); Ck8/18 ( 98, 66, 96, 80 e 95) e 1A4 ( 92, 96, 85, 98 e 95). Portanto, a identificação de Ck5/6, Ck8/18 e 1A4 por IQ em material de citoinclusão é método confiável, com resultados muito próximos aos obtidos no espécime cirúrgico e pode contribuir para a classificação dos carcinomas mamários de expressão luminal e basal, fornecendo informações importantes que possam orientar na escolha do tratamento, bem como na avaliação de fatores prognósticos e preditivos. A importância da obtenção de dados morfológicos e imunoistoquímicos sobre os carcinomas mamários através do material... (Rewsumo completo, clicar acesso eletrônico abaixo) / Abstract: Genetic expression studies have identified many molecular groups of breast carcinoma, with different clinical and biological behavior. The correlation between cDNA microarray and immunohistochemistry (IHC) with markers for cytokeratin, Her2/neu, estrogen receptor (ER) and of basal myoepithelial cells (1A4, S-100 e p63), identified five groups: (1) luminal A (ER+; Her2/neu-), (2) luminal B (ER+; Her2/neu+), (3) overexpression of Her2/neu (ER- ; Her2/neu+), (4) basal-like (ER- ; Her2/neu-; Ck 5/6 +) and (5) none of them (null). The luminal-like express cytokeratines of luminal pattern (Ck8/18) and the basal-like express cytokeratines 5/6 and 14 or markers of myoepithelial basal cells. We have evaluated the expression of Ck5/6, Ck8/18 and 1A4 in cell block comparing it to the surgical specimen. Material and Methods: 43 62 cases have been selected, sequencial, of breast carcinoma diagnosed through fine needle aspiration (FNA), with cell block and surgical specimen. Cuts of cell block and from the surgical specimen were immunostained for Ck 5/6, Ck 8/18 and 1A4. The value, in percentage, of sensibility, specificity, positive predictive value, negative predictive value, and accuracy were respectively: Ck5/6 (77, 100, 100, 92 e 94); Ck8/18 (98, 66, 96, 80 e 95) e 1A4 ( 92, 96, 85, 98 e 95). Therefore, the identification of CK5/6, 8/18 and 1A4 for IHC in cell block is a reliable method, with results very close to the ones obtained in the surgical specimen, and it can contribute to the sub classification of the breast carcinomas of luminal and basal expression, providing important information, which can orientate the treatment... (Complete abstract click electronic access below) / Doutor

Pulmonary arterial hypertension (PAH) is a disease in which pulmonary vascular resistance increases. The cell membrane of pulmonary artery smooth muscle cells (PASMC) in PAH patients is depolarised, resulting in disrupted Ca2+ signalling leading to smooth muscle constriction and PASMC proliferation and migration. In rat pulmonary artery (PA) smooth muscle the KV7 K+ channels, encoded by the KCNQ genes, have been proposed to contribute to the resting K+ current, promoting low resting tone by maintaining a negative membrane potential and low intracellular Ca2+. KV7 channel activating drugs have the potential to counteract the dysfunctional signalling during PAH by causing hyperpolarisation. This study set out to determine if the KV7 channels are expressed in human PA and if so whether they can alter vascular tone, PASMC proliferation and/or migration due to their ability to reduce intracellular Ca2+ indirectly. The effects of KV7 K+ channel modulators on human PA tone were measured using myography, while KCNQ gene expression was examined with quantitative PCR. Markers of proliferation (5-bromo-2'-deoxy-uridine (BrdU) and Ki67 antigen), were used to measure PASMC proliferation, while migration was assessed using the scratch-wound assay. Human PASMCs express all KCNQ genes, except KCNQ2. The KV7 channel blockers XE991, linopirdine and (-)chromanol 293B, constricted PAs. The KV7 channel activators retigabine and zinc pyrithione (ZnPy) relaxed PAs pre-constricted with agonists. The retigabine response was enhanced in PAs constricted with Bay K 8644, abolished in ionomycin constricted PAs and reduced in the presence of 90 mM K+, suggesting inhibition of voltage-gated Ca2+ influx. Similar experiments on rat PAs suggest that only part of the ZnPy-induced relaxation can be attributed to KV7 channel activation. The KCNQ5 gene remained in cultured PASMCs while no KV7 channel modulator altered proliferation or migration. Thus KV7.5 channels could possibly be a marker of differentiated PASMCs and/or be involved in the regulation of cell phenotype. The results imply that KV7 channels play a role in regulating PA tone and Ca2+ signalling in PA smooth. It is concluded that although KCNQ5 transcripts are preserved in proliferating PASMC, it is unlikely they play a role in PASMC proliferation or migration. In summary, KV7 channel activators may be useful in the treatment of PAH since they can prevent vasoconstriction.