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

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

To successfully develop a functional tissue-engineered vascular patch, recapitulating the hierarchical structure of vessel is critical to mimic mechanical properties. Here, we use a cell sheet engineering strategy with micropatterning technique to control structural organization of bovine aortic vascular smooth muscle cell (VSMC) sheets. Actin filament staining and image analysis showed clear cellular alignment of VSMC sheets cultured on patterned substrates. Viability of harvested VSMC sheets was confirmed by Live/Dead® cell viability assay after 24 and 48 hours of transfer. VSMC sheets stacked to generate bilayer VSMC patches exhibited strong inter-layer bonding as shown by lap shear test. Uniaxial tensile testing of monolayer VSMC sheets and bilayer VSMC patches displayed nonlinear, anisotropic stress-stretch response similar to the biomechanical characteristic of a native arterial wall. Collagen content and structure were characterized to determine the effects of patterning and stacking on extracellular matrix of VSMC sheets. Using finite-element modeling to simulate uniaxial tensile testing of bilayer VSMC patches, we found the stress-stretch response of bilayer patterned VSMC patches under uniaxial tension to be predicted using an anisotropic hyperelastic constitutive model. Thus, our cell sheet harvesting system combined with biomechanical modeling is a promising approach to generate building blocks for tissue-engineered vascular patches with structure and mechanical behavior mimicking native tissue.

Every 37 seconds, someone in the United States dies from cardiovascular disease. Vascular calcification is one of the underlying causes of these fatal events. Medial calcification develops following arteriosclerosis, or hardening of the arteries. Medial calcification is characterized by the deposition of hydroxyapatite in the medial layer of the arteries after normal vascular smooth muscle cells undergo a phenotypic switch to resemble osteoblast-like cells. It is hypothesized that this switch is caused by the wingless related (WNT)-Signaling pathway. The WNT-Signaling pathway, upon activation, causes the upregulation of osteogenic markers for the development of osteoblast-like cells. Current treatments alleviate consequences of calcification but do not address the disease. Due to a lack of cures for calcification, a novel therapy for this disease is overdue. By studying human aortic smooth muscle cells and confirming the role of WNT-Signaling as it relates to calcification, a possible therapeutic target for calcification can be identified.

Cardiovascular disease (CVD) is the leading cause of death in the United States, and hypertension has been recognized as a major contributor to its manifestation and progression. Vascular smooth muscle cells control the tone and elasticity of vessels and their dysfunction in hypertension contributes to arterial remodeling and subsequent end organ damage. Evidence has indicated that the Endoplasmic Reticulum (ER) Unfolded Protein Response (UPR) may be compromised in hypertension, while the contribution of protein aggregate formation (a main driver of UPR activation) is undefined. Glucose regulated protein-78 (GRP78), a residential ER chaperone, acts to aid in the proper folding of nascent peptides during translation, while also acting as the primary signal transducer for UPR. We hypothesize overexpression of GRP78 can protect against Angiotensin II induced protein aggregation in vascular smooth muscle cells (VSMCs) to reduce pathological ER UPR signaling and hypertensive vascular remodeling. To test this hypothesis, we investigated protein aggregate induction by Ang II stimulation as well as ER UPR activation, and if overexpression of the ER-resident chaperone glucose regulated protein 78 (GRP78) could protect against these as well as VSMC remodeling markers: hypertrophy, collagen production and inflammation. Utilizing pre-amyloid oligomer (PAO) immunofluorescence staining to identify Ang II induction of amyloid in VSMCs, we found amyloid accumulation was maximal at 48h post stimulation, which could be prevented with adenoviral overexpression of GRP78. Ang II significantly induced ER stress markers p-IRE1α, p-PERK and cleaved ATF6 in VSMCs. Overexpression of GRP78 was able to attenuate these ER stress responders. Interestingly, shotgun proteomic analysis of triton X-100 insoluble aggregate fractions revealed proteostasis machinery enriched in Ang II treated VSMC aggregates (HSP70, VCP, CryAB), which were attenuated with GR78 overexpression. To investigate pathological VSMC remodeling markers, we found that Ang II induced VSMC collagen production, immune cell adhesion, VCAM-1 expression, and hypertrophy (via protein synthesis) which was attenuated by GRP78 overexpression. Utilizing a VSMC-promoter derived GRP78 overexpression mouse model (GRP78TG SM22α Cre-/- or GRP78TG SM22α Cre+/-), we investigated the effect of ER stress inhibition on Ang II induced vascular remodeling. Importantly, hypertrophy and fibrosis in the aorta and the cardiac vasculature were assessed by Masson’s Trichrome and Sirius red staining and found to be increased in Cre-/- mice, while Cre+/- were significantly protected. These effects were accompanied with a significant reduction in Ang II-induced aortic amyloid burden (PAO) and ER UPR signaling. Blood pressure was monitored via tail cuff which revealed GRP78 Cre+/- mice were not protected against Ang II induced hypertension. Overall, these findings indicate that VSMC protein aggregation activates the ER stress response and contribute to hypertensive vascular remodeling. Furthermore, therapeutically targeting this mechanism via overexpression of GRP78 may elude new pharmacological interventions for arterial stiffness, while addressing fundamental questions about the mechanisms involved. / Biomedical Sciences

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13515号 / 論医博第2265号 / 新制||医||1061(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 湊谷 謙司, 教授 石見 拓, 教授 江木 盛時 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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