Note:

Endothelial cells (ECs) and smooth muscle cell (SMC) play a key part during development of fibrosis in the intima being partly responsible for synthesis of matrix metalloproteinase (MMPs) and various regulators and substrates of these enzymes. Omega-3 (n-3) polyunsaturated fatty acids (PUFA) consumption, mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), has beneficial effects on atherosclerosis but its effect on the development of fibrosis is relatively unknown. <i>Objective:</i> Determine the effects of EPA and DHA, alone or in combination, on fibrosis-related factors in aortic SMCs (AoSMCs) and human umbilical vein ECs (HUVECs) and human aortic ECs (HAECs). <i>Results:</i> Treatment of cells with/without 10 μM DHA, EPA, oleic acid (OA) or vehicle control (VC) altered expression of MMPs, regulators and substrates of MMPs and inflammatory cytokines. EPA increased the α-actin:β-actin ratio indicative of a more contractile SMC phenotype and gelatinase (MMP-2 and -9) activity in HUVECs. In aortic cells, EPA and DHA decreased uPAR mRNA and protein expressions. DHA, EPA and DHA: EPA (at 3:1 and 1:1) decreased SMC migration, this did not involve uPA/plasmin activity. <i>Conclusion:</i> EPA and DHA could decrease inflammatory cytokines and the fibrogenic environment in atherosclerotic lesions by decreasing MMP expression and activity. These fatty acids may also reduce SMC migration and proliferation, independently of uPA/plasmin activity, potentially reducing SMC build up in the intima. This could possibly prevent and/or show plaque progression and increase the stability of advanced plaques.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 199-207). Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 70-92).

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 189-204). Also available on the Internet.

The excitation-contraction-relaxation cycle of the guinea pig ileum longitudinal smooth muscle was studied in muscles contracted by a muscarinic agent, cis-2-methyl-4-dimethylaminomethyl-1,3-dioxolane methiodide (CD) and by 60 mM KC1. Aspects of the cycle were investigated by analyzing the active transport enzyme activities in the sarcolemma, the tissue Ca depots which could release Ca for contraction and the sensitivity of the contractile responses to extracellular ion changes. Essentially net changes of intracellular Ca, Mg, Na and K content during contractions were measured by a modified 'La method'. The tissues were washed for 30 min in 160 mM Tris-HCl solution (pH 7.4) containing 10 mM LaCl₃ at 4°C in order to seal the intracellular ions in the cell and displace extracellular ions. A method to loosen the 'intercellular cementing' substance by reducing the tissue Ca and Mg was developed as an adjunct to the preparation of a sarcolemmal enriched microsomal fraction. The method reduced the tenacity of the tissue and made the tissue easy to disrupt by a mild homogenizing procedure. The method also appeared to aid the extraction of contractile proteins. The microsomal fraction was not detectably contaminated by mitochondria and was enriched with vesicles of sarcolemma, probably originating from the muscle caveolae. The sarcolemma enriched microsomal fraction had a Ca-ATPase activity that was progressively stimulated by 10⁻⁷ to 2.4 x 10⁻⁴ M free Ca²⁺ , did not require Mg and was inhibited by La. The microsomal Ca-ATPase activity was not due to contamination by actomyosin. The actomyosin Ca-r-ATPase in the soluble fraction had a higher affinity than the microsomal Ca-ATPase for Ca and for La. The microsomal Ca-ATPase activity was postulated to be associated with an active Ca pump thought to he located in the cayeolae. The microsomal fraction had a Mg-dependent ATPase that could Be stimulated by Na, but K and ouabain had very little additional effect. The addition of an activating factor in the soluble fraction conferred some K and ouabain sensitivity to the Mg-dependent Na-ATPase, which indicated that a Na,K-ATPase was present in this tissue. Low doses of ouabain contracted the longitudinal ileum but the responses were not antagonized by raising the external K concentration five fold, as would be expected if ouabain acted by inhibiting the Na,K-ATPase. However, the ouabain response was rapidly lost when extracellular Ca was removed from the medium and the decline of the response followed the same time course as the loss of extracellular Ca. The peak of the ouabain contraction coincided with significant increases of intracellular Ca and Na, but K loss was not apparent until relaxation ensued. The results suggested that ouabain has an early direct effect on membrane permeability before it inhibited the Na,K-ATPase. CD (2 x 10⁻⁷ M) and 60 mM KCl induced phasic and tonic contractions of the longitudinal muscle of the ileum. The phasic contraction declined from 100% to 7% over 10 min when Ca was omitted from the physiological medium. This decline followed the time course of the loss of extracellular Ca. This, together with the fact that low concentrations of LaCl₃ inhibited the phasic component, indicated that Ca bound to the outer aspect of the cell was responsible for the phasic component. The tonic component was lost more rapidly than the phasic component when the Ca was removed from the Tyrode's solution. The tonic component seemed activated by free Ca mobilized from the extracellular space. The extracellular origin of the Ca for contraction was consistent with the observed small net gain of intracellular Ca that occurred during the phasic and tonic contractions. The minimal volume of the sarcoplasmic reticulum and the abundance of caveolae was also consistent with the high sensitivity of the tissue to extracellular Ca concentrations. The intracellular Ca gained during contraction wa,s extruded within 30 sec after the CD or 60 mM KCl were washed out of the tissue bath, Following washout of CD, the muscle was quiescent for the 20 to 30 min 'equilibration' phase. Spontaneous activity was absent during this phase and tension was below baseline. After a maximal CD contraction, a second response to CD or to 60 mM KCl induced during the 'equilibration' phase had an altered or desensitized biphasic appearance. Responses of the muscle to CD for 10 min were accompanied by a cytoplasmic loss of K. After washout of CD, the K was regained slowly over 20 to 30 min. Stimulation of the tissue by 60 mM KCl did not cause a loss of K from the muscle nor did it cause desensitization of the muscle. Higher extracellular K concentrations decreased the time required after CD contractions for the return of spontaneous activity and prevented muscle desensitization to repeated doses of CD, probably by accelerating the return of intracellular K levels to normal. It was proposed that during contraction, elevated intracellular Ca activated K channels, thereby increasing K permeability and causing the 'after-hyperpolarization' and subsequent desensitization which follows muscarinic induced contractions. / Pharmaceutical Sciences, Faculty of / Graduate

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Indiana University-Purdue University Indianapolis (IUPUI) / There are many clinical diseases involving abnormal differentiation of smooth muscle, such as atherosclerosis, hypertension and asthma. In these diseases, one important pathological process is the disruption of the balance between differentiation and proliferation of smooth muscle cells. Serum Response Factor (SRF) has been shown to be a key regulator of smooth muscle differentiation, proliferation and migration through its interaction with various accessory proteins. Myocardin Related Transcrition Factors (MRTFs) are important co-activators of SRF that induce smooth muscle differentiation. Elucidating the mechanism of how MRTFs and SRF discriminate between genes required to regulate smooth muscle differentiation and those regulating proliferation will be a significant step toward finding a cure for these diseases. We hypothesized that SWI/SNF ATPdependent chromatin remodeling complexes, containing Brg1 and Brm, may play a role in this process. Results from western blotting and quantitative reverse transcription - polymerase chain reaction (qRT-PCR) analysis demonstrated that expression of dominant negative Brg1 or knockdown of Brg1 with silence ribonucleic acid (siRNA) attenuated expression of SRF/MRTF dependent smooth muscle-specific genes in primary cultures of smooth muscle cells. Immunoprecipitation assays revealed that Brg1, SRF and MRTFs form a complex in vivo and that Brg1 directly binds MRTFs, but not SRF, in vitro. Results from chromatin immunoprecipitation assays demonstrated that dominant negative Brg1 significantly attenuated SRF binding and the ability of MRTFs to increase SRF binding to the promoters of smooth muscle-specific genes, but not proliferation-related early response genes. The above data suggest that Brg1/Brm containing SWI/SNF complexes play a critical role in differentially regulating expression of SRF/MRTF-dependent genes through controlling the accessibility of SRF/MRTF to their target gene promoters. To examine the role of SWI/SNF in smooth muscle cells in vivo, we have generated mice harboring a smooth muscle-specific knockout of Brg1. Preliminary analysis of these mice revealed defects in gastrointestinal (GI) development, including a significantly shorter gut in Brg1 knockout mice. These data suggest that Brg1-containing SWI/SNF complexes play an important role in the development of the GI tract.