Brain calcification is a common occurrence in the aging process, with >20% of individuals over the age of 65 showing hardened plaques in the basal ganglia. Loss of the vitamin D receptor (VDR) in transgenic mice leads to formation of calcified plaques in the basal ganglia and thalamus within the mice. Vitamin D signals through two known vitamin D responsive proteins, protein disulfide isomerase A3 (PDIA3) and VDR. In vitro, vitamin D has been demonstrated to suppress calcification in osteoblast-like cells. Here, we aim to elucidate which of either PDIA3 or VDR transduce vitamin D mediated suppression of calcification in vitro. PDIA3 or VDR were selectively knocked out in human osteosarcoma (SaOs) cells using CRISPR-Cas9 technology to generate PDIA3 KO or VDR KO cells. Knockout for PDIA3 or VDR was confirmed by RT-qPCR assay or western blot analysis. The calcification of SaOs-2 cells was induced with treatment of β-glycerophosphate along with ascorbic acid allowing for determination of whether loss of PDIA3 or VDR would lead to altered calcium deposition. Cells null for PDIA3 but not VDR grew at a significantly slower rate than wild-type (WT) cells. Intriguingly, PDIA3 and VDR KO cells displayed significantly more calcification relative to WT control cells. Calcitriol or the synthetic analogue EB1089 suppressed calcification in vitro in WT and PDIA3 KO but not VDR KO cells as measured by alizarin red staining. These data suggest VDR is critical for mediating vitamin D’s inhibition of calcification in vitro, and that PDIA3 has a role in suppressing calcification. This study provides novel insights into vitamin D signaling and provides a foundation for further study and understanding of vitamin D related pathologies.

MicroRNA-9 (miR-9) modulates gene expression and demonstrates high structural conservation and wide expression in the central nervous system. Bioinformatics analysis predicts almost 100 ion channels, membrane transporters and receptors, including genes linked to primary familial brain calcification (PFBC), as possible miR-9-5p targets. PFBC is a neurodegenerative disorder, characterized by bilateral and symmetrical calcifications in the brain, associated with motor and behavioral disturbances. In this work, we seek to study the influence of miR-9-5p in regulating genes involved in PFBC, in an osteogenic differentiation model with SaOs-2 cells. During the induced calcification process, solute carrier family 20 member 2 (SLC20A2) and platelet-derived growth factor receptor beta (PDGFRB) were downregulated, while platelet-derived growth factor beta (PDGFB) showed no significant changes. Significantly decreased levels of SLC20A2 and PDGFRB were caused by the presence of miR-9-5p, while PDGFB showed no regulation. We confirmed the findings using an miR-9-5p inhibitor and also probed the cells in electrophysiological analysis to assess whether such microRNA might affect a broader range of ion channels, membrane transporters and receptors. Our electrophysiological data show that an increase of the miR-9-5p in SaOs-2 cells decreased the density and amplitude of the output ionic currents, indicating that it may influence the activity, and perhaps the expression, of some ionic channels. Additional investigations should determine whether such an effect is specific to miR-9-5p, and whether it could be used, together with the miR-9-5p inhibitor, as a therapeutic or diagnostic tool.

BACKGROUND: Calcification is a natural process of bone formation or osteogenesis. However, calcium is able to be deposited abnormally in soft tissues such as the aorta, adipose tissue and liver, causing these to harden. Abnormal calcification in arteries is a common factor contributing to high blood pressure and, further, many severe cardiovascular diseases such as atherosclerosis and coronary disease. In liver and adipose tissue, calcification always takes place accompanied by excess extracellular matrix (ECM) accumulation which is called fibrosis, contributing to cirrhosis and metabolic disorders including insulin resistance. In addition, it is documented that severe calcification in adipose tissues is able to cause damage to the micro-vascular system, and calcification in perivascular adipose tissue (PVAT) is a key effector of arterial stiffness. Dystrophic calcification, one of the most common types of abnormal calcification, usually occurs as a reaction to tissue damage such as obesity-induced inflammation. Increasing numbers of studies indicate that abnormal calcification is the result of re-differentiation towards osteogenesis which occurs in the nascent resident cells under the stimulation of multiple factors. The BMP/Smad signaling pathway is commonly known to participate in bone formation and is implicated in mineralization as well as local induction of inflammation. Importantly, BMP/Smad signaling as an inducer of the osteochondrogenic phenotype in vascular calcification is fully appreciated. However, the molecular events of dystrophic calcification triggered by obesity-induced chronic inflammation still remain unclear. Our previous studies have identified that imbalance with increased activity of neutrophil elastase (NE), a Ser protease mainly released by neutrophils during inflammation, and decreased serum levels of the NE inhibitor α1-antitrypsin A1AT, contributes to the development of obesity-related metabolic complications including insulin resistance, fatty liver and chronic inflammation. This study explored the effects of NE on abnormal calcification in soft tissues, which may be mediated by BMP/Smad signaling pathway, and, furthermore, the molecular mechanism by which NE activates the BMP/Smad signaling pathway. METHODS: Wild-type mice were fed with either a high-fat high-fructose diet (HFHFD), a high-fat diet (HFD) alone or a normal chow diet (NCD), and NE-knockdown mice were fed with a HFHFD. Adipose tissue and liver were extracted from all mice. H&E staining and immunofluorescence staining (IF) detected the inflammation condition. Alizarin staining and von kossa staining were used to detect calcium deposits. 3,3′-Diaminobenzidine (DAB) staining was used to examine active phospho-Smad1/5 signaling. Regarding nascent resident cells which have potential ability of osteogenic re-differentiation, 3t3l1 fibroblast and human hepatic stellate cell (hHSC) were cultured in dishes and 6-well plates with coverslips. In our previous research, mouse aortic smooth muscle cells (mASMC) seeded in 6-well plates grew in an osteogenic medium (10mM β-glycerophosphate and 10mM Calcium chloride) in the presence or absence of NE (10nM). Calcium deposits were detected by Alizarin staining. 3t3l1 and hHSC was treated with NE (20nM, 30nM, 40nM), BMP2, TGFβ1 or NE combined with BMP2, TGFβ1 or NE inhibitor GW311616A (Axon). Further, we used specific chemical inhibitors, LDN-193189, BMP-ALK2/3 inhibitor, SB525334, TGFβ-ALK5 inhibitor, and I-191, PAR2 antagonist to investigate the molecular mechanism of NE’s effects on Smad signaling pathways. Cells in dishes were harvested, and the proteins were measured by western blot. Coverslips in 6-well plates were used for immunofluorescence. RESULTS: The most severe calcification was found in the adipose tissue of HFHFD fed wild-type mice and moderate calcification took place in the HFD mouse group while NCD mice rarely had calcium deposits. NE-knockdown significantly prevented calcium deposits in adipose tissue compared with HFHFD wild-type mice. Consistently, we found increased phospho-Smad1/5 (p-Smad1/5) signaling in the adipose tissues of mice on the HFHFD and HFD mice while p-Smad1/5 was prevented in the NE-knockout group. Furthermore, NE enhanced calcium deposits in mASMC cultured in osteogenic medium. NE significantly activated p-Smad1/5 signaling in hHSC in the dose-effect relationship and contributes to an additive effect on p-Smad1/5 in the presence of BMP2 or TGFβ1. Although p-Smad1/5 was only slightly aroused by NE in 3t3l1 fibroblast, NE was able to promote p-Smad1/5 activation tremendously and specifically in the presence of BMP2 or TGFβ1 but not p-Smad2/3 which is the main downstream signaling of TGFβ1. Chemical inhibition of ALK2/3, not ALK5 or PAR2, was able to completely block NE’s effects in hSHC on p-Smad1/5 activation. In addition, the cleavage of osteoblast-cadherin or CDH11 (OB-cadherin) was observed in hHSC, which may indicate a lower beta-catenin abundance in the hHSC cells which were treated with NE. CONCLUSION: NE has the potential to contribute to abnormal calcification in soft tissues including the liver, adipose tissue and aorta via activating canonical ALK2/3-BMP-Smad1/5 signaling pathway in the mesenchymal stem cell/MSC-lineage cells. In addition, NE is likely to break cell-cell adhesion which may contribute to cell proliferation and re-differentiation towards osteogenesis and fibrosis. / 2024-01-28T00:00:00Z

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18146号 / 医博第3866号 / 新制||医||1002(附属図書館) / 31004 / 京都大学大学院医学研究科医学専攻 / (主査)教授 鈴木 茂彦, 教授 妻木 範行, 教授 戸口田 淳也 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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

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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.

Osteoarthritis is the most common form of arthritis. Genetic models have been developed to determine if and how a targeted gene may influence cartilage degenerative changes. The itga1-null mouse model has an inhibited integrin α1B1 through a ubiquitous integrin α1 subunit knockout, which leads to fibrosis in articular cartilage through excessive signalling from transforming growth factor beta (TGFB). Depleting this TGFB signalling is proposed to have a protective effect on cartilage. This project is part of a foregoing study where a cartilage-specific knockout of TGFB receptor type II (TBRII) was used to deplete TGFB signalling in articular cartilage of the itga1-null mice to reduce the severity of cartilage degradation. This project continues the analysis of the genetic model into bone architecture at the knee. Mouse hindlimbs were scanned at a 13μm resolution using micro-computed tomography and segmented into 3D datasets containing calcified tissues and bone of the knee and surroundings. Quantification methods for trabecular bone parameters (bone volume fraction, trabecular separation, and trabecular thickness) and ectopic calcification of soft tissues were developed. Loss of trabecular bone around the involved joint is a hallmark of post-traumatic osteoarthritis. However, the results from this study showed no significant changes in trabecular bone of itga1-null mouse knees despite observing severe osteoarthritic changes in the adjacent cartilage. There were no significant effects in peri-articular trabecular bone when the TBRII knockout in cartilage was activated, but there were significant increases in ectopic calcifications of the menisci and collateral ligaments. These ectopic calcifications were also seen in tamoxifen control mice, suggesting that tamoxifen, along with TBRII depletion in cartilage, had a role in increased abnormal calcifications. Although integrin α1B1 inhibition appears to have an important role in cartilage degeneration, it does not appear to influence the bony changes that normally accompany post-traumatic arthritis. / Thesis / Master of Applied Science (MASc) / Osteoarthritis is a common joint disorder, associated mainly with cartilage degradation. Some genes have been identified that cause or prevent osteoarthritis. A previous study used two of these genes in a genetic mouse model to explore how osteoarthritis may develop. Removing the integrin α1 subunit from mice caused osteoarthritic changes in the cartilage of the mouse knee. When the transforming growth factor beta gene was removed from the cartilage, these changes were less severe. This project continued the study by exploring changes in bone around the mouse knee. We quantified bone changes around the mouse knee using high-resolution micro-computed tomography scans. Contrary to common findings in post-traumatic osteoarthritis, we found that there were no significant changes in the bone around the knees even where severe cartilage changes had been identified. However, there were significant increases in calcifications of soft tissues including the meniscus and ligaments around the knee.

La calcification de la valve aortique (CVA) est une maladie cardiovasculaire de plus en plus répandue, particulièrement en Amérique du Nord. Elle cause le rétrécissement de la valve aortique et le seul traitement actuellement disponible est le remplacement chirurgical. Des études menées par le Dr Patrick Mathieu (Institut de Cardiologie et de Pneumologie de Québec) ont montré qu’une surexpression d’une ectonucléotide pyrophosphatase/phosphodiestérase de type 1 (ENPP1) est à l’origine de cette sténose. Une solution à cette maladie serait donc de trouver un inhibiteur d’ENPP1. Inspirées des travaux du groupe de Pfizer visant ENPP1 pour le traitement de la chondrocalcinose articulaire et l’ostéoarthrite, quelques familles d’inhibiteurs de type quinazoline-4-pipéridine sulfamides (QPS) ont été synthétisés et testées in vitro. Une étude en modélisation moléculaire sur le site potentiel de liaison des inhibiteurs sur ENPP1 est en cours, en collaboration avec le Pr Patrick Lagüe (Université Laval, Département de biochimie, microbiologie et bio-informatique) et son équipe pour optimiser le design de la structure des composés. Les composés d’une des familles, les QPS-pyrimidine, ont été testés in vitro sur quelques lignées cellulaires cancéreuses (HT-1080, HT-29, M21 et MCF-7) pour mesurer leur activité antiproliférative. Ces composés ont une inhibition de croissance médiane (IC50) de l’ordre du micromolaire et représentent donc un point de départ intéressant pour la mise au point de nouveaux traitements anticancéreux. / The calcification of the aortic valve (CAV) is a cardiovascular disease increasingly widespread, particularly in North America. It causes narrowing of the aortic valve and currently available only treatment is surgical replacement. Studies by Dr. Patrick Mathieu (Institute of Cardiology and Pneumology of Quebec) showed that overexpression of an ectonucleotide pyrophosphatase/phosphodiesterase type 1 (ENPP1) is the origin of the stenosis. A solution to this disease would be to find an inhibitor of ENPP1. Inspired by Pfizer’s group works on ENPP1 for the treatment of osteoarthritis and chondrocalcinosis, some members of the quinazoline-4-piperidine sulfonamides (QPS) inhibitor family were synthesized and tested in vitro. A study in molecular modelling on the potential binding site inhibitor on ENPP1 is underway in collaboration with Prof. Patrick Lagüe (Université Laval, Department of biochemistry, microbiology and bioinformatics) and his team to optimize the design of compounds structure. The compounds of one family, the QPS-pyrimidine, were tested in vitro on some cancer cell lines (HT-1080, HT-29, M21 and MCF-7) to measure their antiproliferative activity. These compounds have a median growth inhibition (GI50) in the micromolar range and thus represent an interesting starting point for the development of new cancer treatments.

Background and Objectives: Mitral stenosis with extensive mitral annular calcification (MAC) remains surgically challenging in respect to clinical outcome. Prolonged surgery time with imminent ventricular rupture and systolic anterior motion can be considered as a complex of causal factors. The aim of our alternative hybrid approach was to reduce the risk of annual rupture and paravalvular leaks and to avoid obstruction of the outflow tract. A review of the current literature was also carried out. Materials and Methods: Six female patients (mean age 76 9 years) with severe mitral valve stenosis and severely calcified annulus underwent an open implantation of an Edwards Sapien 3 prosthesis on cardiopulmonary bypass. Our hybrid approach involved resection of the anterior mitral leaflet, placement of anchor sutures and the deployment of a balloon expanded prosthesis under visual control. Concomitant procedures were carried out in three patients. Results: The mean duration of cross-clamping was 95 31 min and cardiopulmonary bypass was 137 60 min. The perioperative TEE showed in three patients an inconspicuous, heart valve-typical gradient on all implanted prostheses and a clinically irrelevant paravalvular leakage occurred in the anterior annulus. In the left ventricular outflow tract, mild to moderately elevated gradients were recorded. No adverse cerebrovascular events and pacemaker implantations were observed. All but one patient survived to discharge. Survival at one year was 83.3%. Conclusions: This “off label” implantation of the Edwards Sapien 3 prosthesis may be considered as a suitable bail-out approach for patients at high-risk for mitral valve surgery or deemed inoperable due to extensive MAC.