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京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22641号 / 医博第4624号 / 新制||医||1044(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 湊谷 謙司, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23380号 / 医博第4749号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 木村 剛, 教授 YOUSSEFIAN Shohab / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Vascular smooth muscle cells display a certain level of phenotype plasticity. Under specific conditions fully differentiated cells are able to undergo dedifferentiation and to restart growth and proliferation. An organ culture method is a useful technique for the analysis of dedifferentiation of vascular smooth muscle cells, because it provides an opportunity for studying the changes in cell phenotype. The aim of this study was to investigate the basic contractile characteristics in rat femoral arteries cultured for different time periods (from one to three days). In addition, the effects of fetal bovine serum (FBS), that contains various growth factors and other biological active molecules, on contractile function were studied. We also tried to attenuate cell dedifferentiation by lowering the calcium influx, because calcium is an important second messenger participating in cell growth and proliferation. To achieve this goal we used cultivation with nifedipine, a voltage-dependent calcium channel inhibitor. The cultivation without FBS slightly decreased arterial contractility, whereas the cultivation with FBS decreased arterial contractility considerably. The major change in contractility of arteries cultivated with FBS occurred approximately within 24 hours of cultivation. The cultivation with...

Cardiovascular disease is the leading cause of human death worldwide. Currently, the prevalence of cardiovascular disease and health care costs associated with its onset continue to increase in both developed and developing societies. Concordant with the need to improve preventative measures is the imperative to develop more effective and efficient remedies for incident cardiovascular pathologies. Increased aortic stiffness with aging has recently emerged as an early, independent, and consistent physiological predictor of cardiovascular disease and represents an attractive target for possible therapeutic options. The success of any biomedical strategy in this regard is incumbent upon comprehension of biological processes and mechanical properties attributable to constituent components within the aortic wall. This dissertation tested the hypothesis that aging-induced changes to smooth muscle maintenance of biomechanical homeostasis within the aorta lead to undesirable increases in stiffness, correlative with increased risk of negative cardiovascular outcomes. Conventionally, mechanical studies and models have identified extracellular matrix as the primary determinant of changes in stiffness, but new research presented here shows that this may not be true. In viable ex vivo preparations of aortic tissue, roughly half of the maximal elastic modulus results from alpha-agonist activation of smooth muscle cells. Investigation of the biochemical interactions that characterize this effect revealed a link between aging and decreased expression of Src, a kinase involved in numerous signaling pathways governing cellular growth and survival, as well as defective regulation of focal adhesions between the smooth muscle cells and extracellular matrix. These findings were integrated into a model of aortic contractility and stiffness that establishes an aging-impaired regulatory complex comprising focal adhesions and non-muscle actin cytoskeleton in vascular smooth muscle cells. A better understanding of the mechanisms underlying this model may motivate the design of potential therapeutics, deliverable to previously overlooked target sites within aortic smooth muscle, and ultimately novel treatments for aging-induced cardiovascular disease. / 2017-10-27T00:00:00Z

Airway hyperresponsiveness (AHR) is a hallmark of asthma where constriction of airway smooth muscle (ASM) causes excessive airway narrowing. Asthmatics, unlike healthy subjects, cannot prevent or reverse this narrowing by stretching their airways with a deep inspiration (DI). Since stretching of isolated ASM causes dramatic reductions in force generation and asthmatics tend to have stiffer airways, researchers hypothesize that reduced ASM stretching during breathing and DIs results in hyperreactive airways. However, counterintuitively, excised measurement on intact airways show narrowing is minimally reversed by pressure oscillations simulating breathing and DIs. We hypothesized that AHR does not result from reduced capacity to stretch the airways; furthermore, each constituent of the airway wall experiences different strain magnitude during breathing and DIs. To test this, we used an intact airway system which controls transmural pressure (Ptm) to simulate breathing while measuring luminal diameter in response to ASM agonists. An ultrasound system and automated segmentation algorithm were implemented to quantify and compare the ability of Ptm fluctuations to reverse and prevent narrowing in larger (diameter=5.72±0.52mm) relative to smaller airways (diameter=2.92±0.29mm). We found the ability of Ptm oscillations to reverse airway narrowing was proportional to strain imposed on the airway wall. Further, tidal-like breathing Ptm oscillations (5-15cmH2O) after constriction imposed 196% more strain in smaller compared to larger airways (14.6% vs. 5.58%), resulting in 76% greater reversal of narrowing (41.2% vs. 23.4%). However, Ptm oscillations applied before and during constriction resulted in the same steady-state diameter as when Ptm oscillations were applied only after constriction. To better understand these results, we optimized an ultrasound elastography technique utilizing finite element-based image registration to estimate spatial distributions of displacements, strains, and material properties throughout an airway wall during breathing and bronchoconstriction. This required we formulate and solve an inverse elasticity problem to reconstruct the distribution of nonlinear material properties. Strains and material properties were radially and longitudinally heterogeneous, and patterns and magnitudes changed significantly after induced narrowing. Taken together, these data show AHR likely does not emerge due to reduced straining of airways prior to challenge, but remodeling that stiffens airway walls might serve to sustain constriction during an asthmatic-like attack.

BACKGROUND: Sirtuin-1 (SirT1) is a NAD+-dependent deacetylase essential for maintaining the structure and function of the vasculature. Reduced SirT1 expression and activity has been correlated with the development of vascular diseases, mainly attributed to loss of SirT1’s anti-oxidant and anti-inflammatory beneficial effects. We previously found that deletion of vascular smooth muscle (VSM) SirT1 in mice is associated with increased matrix metalloproteinases (MMPs) and the subsequent development of aortic dissections or ruptures in response to the hypertensive peptide angiotensin II. Based on these previous findings, we hypothesize that loss of SirT1 activity is involved in the pathogenesis of AA. SirT1 is a stress response gene, its deacetylase activity can be impaired by excessive oxidative stress. We postulate that mutating three cysteine residues in SirT1’s catalytic domain can prevent its inactivation by oxidative insults and protect against AA and other vascular diseases. OBJECTIVES: assess the role of SirT1 in a genetic mouse model of Marfan Syndrome that develops AA; (2) Determine design and optimize an enzyme-based colorimetric ELISA to determine SirT1 activity in mouse VSM cells and aortas; (3) Produce an adeno-associated virus (AAV) expressing an oxidant-resistant triple mutant SirT1 in VSM cells that has the potential to mitigate the downstream outcomes derived from alterations in SirT1 activity, such as MMPs activation and development of AA in mgR-/- mice. METHODS: mgR-/- and littermate mgR+/+ (WT) mice aortas and VSM cells were cultured in conditioned medium and the activity of released MMPs was determined by in-gel zymography. For the development of the SirT1 activity assay, we designed a multi-step sandwich ELISA that captures a biotin- and FLAG-tagged acetylated p53 peptide, used as SirT1 deacetylase substrate. Amounts of acetylated and total p53 peptide were sequentially detected with antibodies and colorimetric substrates as index of SirT1 deacetylase activity. AAVs expressing a control or triple mutant SirT1 (3M) were produced in HEK293T cells; VSM cells were then infected with control or 3M AAV and SirT1 protein expression levels were measured by Western Blot. RESULTS: MMPs activity is increased in aortas and VSMC of mgR-/- mice; the first stage of optimization of the SirT1 activity assay successfully defined the assay conditions and experimental design, and it is ready to be optimized with mgR-/- cell and tissue samples; our novel control and SirT1 triple mutant AAVs were produced and successfully overexpressed in VSM cells. / 2024-03-14T00:00:00Z

Thromboxane A2 (TXA2) and ET-1 have been known to play important roles in modulating vascular contraction and growth. The present study was undertaken to examine the effect of TXA2 on the induction of endothelin-1 (ET-1) mRNA and protein levels in smooth muscle cells derived from rat heart. U-46619, a stable TXA2 mimetic, superinduced preproET-1 mRNA in the presence of cycloheximide in these cells. This effect could be blocked by SQ-29548, a TXA2/prostaglandin H2 receptor antagonist and by actinomycin D, an RNA synthesis inhibitor. In addition, H7, a protein kinase C inhibitor, could abolish the induction. Transient transfection experiment revealed that the elevated ET-1 mRNA level after U-46619 treatment was a result of the activation of ET-1 gene activity. The elevated ET-1 message level was accompanied by increased ET-1 release into the cultured medium. These results show that the short-lived TXA2 can induce potent and long-lived ET-1. These findings support a potential role for ET-1 in the pathogenesis of coronary atherosclerosis and hypertension evoked by TXA2.

Objective: To determine if cannabinoid receptor 2 (CB2) plays a role in atherosclerosis, we investigated the effects of systemic CB2 gene deletion on hyperlipidemia-induced atherogenesis in low density lipoprotein receptor-deficient (Ldlr-/-) mice. Methods and results: Ldlr-/- and CB2/Ldlr double knockout (CB2-/-Ldlr-/-) mice were fed an atherogenic diet for 8 and 12 weeks. Morphometric analysis revealed no significant difference between the atherosclerotic lesion area in the proximal aortas of Ldlr-/- and CB2-/-Ldlr-/- mice after 8 or 12 weeks on the atherogenic diet. The macrophage and smooth muscle cell (SMC) content, as revealed by immunohistochemical staining, did not differ significantly between Ldlr-/- and CB2-/-Ldlr-/- lesions after 8 weeks. However, after 12 weeks, CB2-/-Ldlr-/- lesions displayed greater macrophage content (86.6±4.1 versus 75.2±7.5%, P<0.05) and SMC content (11.1±5.1 versus 4.2±2.4%, P<0.05) compared to controls. Lesional apoptosis, as determined by in situ TUNEL analysis, was reduced ∼50% in CB2-/-Ldlr-/- lesions after 12 weeks. CB2-/-Ldlr-/- lesions displayed significantly reduced collagen content and increased elastin fiber fragmentation after 12 weeks, which was associated with an ∼57% increase in matrix metalloproteinase 9 (MMP) levels. In vitro, CB2-/- macrophages secreted ∼1.8-fold more MMP9 activity than CB2+/+ macrophages. Conclusions: CB2 receptor deficiency affects atherogenesis in Ldlr-null mice by increasing lesional macrophage and SMC content, reducing lesional apoptosis and altering extracellular matrix components, in part, by upregulating MMP9. These results suggest that pharmacological manipulation of CB2 receptors might exert multiple and complex effects on atherogenesis and plaque stability.