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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...

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.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21029号 / 医科博第90号 / 新制||医科||6(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 山下 潤, 教授 湊谷 謙司, 教授 原田 浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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 leads in morbidity and mortality in Western societies with no known cure. NADPH oxidase (Nox) contributes to atherosclerosis through the indirect activation of macrophages leading to the internalization of oxidized low density lipoproteins (oxLDL). Chronic inflammation in activated macrophages contributes to atherosclerosis. Because macrophages are positioned at the cross-roads of lipid metabolism in vessel walls, they are important in the cellular pathology of atherosclerosis. Components of the endocannabinoid (eCB) system are vital to atherosclerotic development, since the eCB system has been found to play an important role in the amelioration of atherosclerosis. The eCB system has several components, including the G-protein-coupled cannabinoid receptors (CB1 and CB2); their endogenous ligands, 2-arachidonoylglycerol (2-AG) and anandamide (AEA); and biosynthetic enzymes that produce and degrading these compounds. CB2 signaling has been shown to upregulate immunoprotective and anti-oxidative pathways, whereas CB1 signaling has opposite effects. We hypothesized a mechanistic link between scavenger receptor activation and Nox activity, which leads to enhanced 2-AG biosynthesis via a signaling pathway that activates diacylglycerol lipase beta (DAGLB). Activation of CB2-mediated signaling by enhanced “eCB tone” can potentially reduce oxidative stress in macrophages. The released 2-AG is subsequently catabolized hydrolytic enzymes, leading to enhanced 2-AGbiosynthesis via activated DAGLB. We first proved that macrophage treated with oxLDL can activate Nox and increase reactive oxygen species production. We used human and mouse macrophages to demonstrate cause and effect. Secondly, we demonstrated that increased levels of superoxide causes enhanced 2-AG biosynthesis within the macrophage, and that upregulation in eCB production is an adaptive response to oxidative stress. Finally, we identified and quantified the serine hydrolases found in smooth muscle cells (SMCs) using an activity-based protein profiling (ABPP)-MudPIT approach that our laboratory has previously done using human macrophages. Additionally, the catabolism of 2-AG by primary SMCs was explored to demonstrate SMCs can hydrolyze 2-AG to its metabolites arachidonic acid and glycerol by the known hydrolytic enzymes. We demonstrated that enhancing endocannabinoid tone within the vessel wall is a valuable strategy to reduce the occurrence of inflammation that leads to atherosclerosis.

In 2018, 1 in every 10 infants born in the United States was born preterm. The majority of neonatal deaths and nearly a third of infant deaths that occur are linked to preterm birth. Preterm birth is initiated when the quiescent state of the uterus ends prematurely, leading to contractions and parturition beginning as early as 32 weeks, though the origins are not well understood. Tocolytics are pharmaceuticals utilized to postpone preterm labor, but currently only manage to prolong pregnancy for up to 48 hours and have not proven effective in completely preventing preterm delivery. To enable research and discovery of therapeutics with potential to better address preterm birth, the capability to study isolated cell processes of pregnant uterine tissue in vitro is needed. Our development of an in vitro model of the myometrium utilizing uterine myocytes - uterine smooth muscle cells (uSMCs) responsible for contractions - provides a platform to examine the cellular mechanisms of late-stage pregnancy potentially involved in preterm birth. In this thesis, we discuss the optimized culture of uterine SMCs on a flexible polydimethylsiloxane (PDMS) substrate functionalized using a cationic solution, Poly-L-lysine (PLL), followed by extracellular matrix (ECM) protein coating. Using the model we developed, we then exposed this elastic substrate with uterine SMCs to different strain rates in order to investigate the impact of mechanical strain parameters on uterine SMC hypertrophy in the uterus during late-stage pregnancy. It was found that PLL and ECM protein coatings significantly impact cell morphology and density in unstrained substrates. It was also observed that when exposed to strain conditions, strain significantly increased hypertrophic morphological traits in select conditions. These results indicate that both surface and mechanical properties of in vitro systems impact uterine SMC phenotype, offering further understanding of cellular pathways involved in the uterus under mechanical load. / 2024-05-31T00:00:00Z

yes / Type 2 diabetes mellitus prevalence is growing globally, and the leading cause of mortality in these patients is cardiovascular disease. Epigenetic mechanisms such as microRNAs (miRs) and DNA methylation may contribute to complications of type 2 diabetes mellitus. We discovered an aberrant type 2 diabetes mellitus–smooth muscle cell phenotype driven by persistent up-regulation of miR-145. This study aimed to determine whether elevated expression was due to changes in methylation at the miR-145 promoter. Smooth muscle cells were cultured from saphenous veins of 22 non-diabetic and 22 type 2 diabetes mellitus donors. DNA was extracted, bisulphite treated and pyrosequencing used to interrogate methylation at 11 CpG sites within the miR-145 promoter. Inter-patient variation was high irrespective of type 2 diabetes mellitus. Differential methylation trends were apparent between non-diabetic and type 2 diabetes mellitus–smooth muscle cells at most sites but were not statistically significant. Methylation at CpGs −112 and −106 was consistently lower than all other sites explored in non-diabetic and type 2 diabetes mellitus–smooth muscle cells. Finally, miR-145 expression per se was not correlated with methylation levels observed at any site. The persistent up-regulation of miR- 145 observed in type 2 diabetes mellitus–smooth muscle cells is not related to methylation at the miR-145 promoter. Crucially, miR-145 methylation is highly variable between patients, serving as a cautionary note for future studies of this region in primary human cell types.

Yes / Abdominal aortic aneurysm (AAA) is a silent, progressive disease with high mortality and increasing prevalence with aging. Smooth muscle cell (SMC) dysfunction contributes to gradual dilatation and eventual rupture of the aorta. Here we studied phenotypic characteristics in SMC cultured from end-stage human AAA (5cm) and cells cultured from a porcine carotid artery (PCA) model of early and end-stage aneurysm. Human AAA-SMC presented a secretory phenotype and expressed elevated levels of differentiation marker miR-145 (2.2-fold, P<.001) and senescence marker SIRT-1 (1.3-fold, P<.05), features not recapitulated in aneurysmal PCA-SMC. Human and end-stage porcine aneurysmal cells were frequently multi-nucleated (3.9-fold, P<.001 and 1.8-fold, P<.01 respectively, versus control cells) and displayed aberrant nuclear morphology. Human AAA-SMC exhibited higher levels of the DNA damage marker H2AX (3.9-fold, P<.01 vs. control SMC). These features did not correlate with patients’ chronological age; and are therefore potential markers for pathological premature vascular aging. Early-stage PCA-SMC (control and aneurysmal) were indistinguishable from one another across all parameters. The principal limitation of human studies is tissue availability only at end-stage disease. Refinement of a porcine bioreactor model would facilitate study of temporal modulation of SMC behaviour during aneurysm development and potentially identify therapeutic targets to limit AAA progression. / Supported in part by a grant from the Leeds Teaching Hospitals Charitable Foundation (9R11/8002)