Background: Disturbances in metabolic homeostasis, resulting in cardiovascular disease, are the major cause of morbidity and mortality in patients with chronic kidney disease (CKD). Mineral deposition in the vascular smooth muscle cell (VSMC)-rich medial layer of the vessel wall is a prominent driver of mortality in these patients. Vascular calcification (VC) is now recognized as an important predictor and independent risk factor for all-cause and cardiovascular mortality in patients with advanced CKD. Prolonged impairment in kidney functions, characteristic of chronic CKD, results in marked changes in blood biochemistry, namely elevated circulating levels of inorganic phosphate (Pi) and calcium. These changes in circulating mineral content trigger osteogenic transdifferentiation and apoptosis of the VSMCs, subsequently leading to VC. In addition to kidney-related metabolic changes, chronic inflammation and liver disease are also associated with increased VC. T-cell death-associated gene 51 (TDAG51) and growth differentiation factor 10 (GDF10) have been previously reported to contribute to metabolic regulation in conditions of atherogenic VC, osteogenesis, and adipogenesis. However their role in medial VC and associated morbidities remains unknown. Methods and Results: Using a combination of in vitro, ex vivo and in vivo models findings presented in this thesis demonstrate that TDAG51 is an important modulator of VC, and as such is upregulated by conditions of hyperphosphatemia. I show that VSMCs from TDAG51-/- mice exhibit reduced expression and activity of key driver of osteogenic transdifferentiation, Runt-related transcription factor 2 (Runx2). To explain these observations, I demonstrate reduced expression of type III sodium-dependent Pi transporter, Pit-1, as well as intracellular Pi in these cells. Additionally, GDF10, an established inhibitor of osteogenesis, was identified to be significantly upregulated in TDAG51-/- VSMCs and mice. In line with these observations, knockdown of human homologue of TDAG51, pleckstrin homology-like domain, family A, member 1, resulted in increased expression of GDF10. Consistent with the anti-osteogenic role of GDF10, treatment with recombinant human GDF10 reduced Pi-mediated hydroxyapatite deposition, Runx2 expression and activity in primary mouse VSMCs. Interestingly, GDF10-/- mice develop severe adiposity, hepatic lipid accumulation, injury and inflammation. To explain this phenotype, a marked increase in the expression and activity of established driver of adipogenesis, peroxisome-proliferator-activated receptor γ (PPAR γ) in the livers of GDF10-/- mice was demonstrated. Complementary experiments in cultured hepatocytes demonstrate that treatment with recombinant human GDF10 attenuates nuclear PPAR γ expression and subsequent lipid accumulation, inflammation and fibrosis in these cells. Conclusion: This work highlights TDAG51 as an important regulator of Pi-mediated VC through downregulation of Pit-1, Runx2, and GDF10. Additionally, GDF10 has been described as a novel systemic inhibitor and a potential diagnostic marker for VC. Lastly, this work further characterizes GDF10 as an adipokine important in the regulation of hepatic lipid levels, which can indirectly affect vascular health. / Thesis / Doctor of Philosophy (Medical Science)

Medial calcification refers to mineral deposition in the middle layer of arteries. This mineralization is common in chronic kidney disease patients and causes an increased chance of cardiovascular complications. Calcitriol, the active form of vitamin D, is often administered to these patients to treat an associated condition, secondary hyperparathyroidism. Unfortunately, calcitriol treatment may promote vascular calcification due to increasing serum calcium and phosphate. We examined the effects of calcitriol supplementation on vascular smooth muscle cell (VSMC) calcification, through atomic absorption, scanning electron microscopy, and western blot analysis. Additionally, we examined the effects of the combinations of calcitriol, fibroblast growth factor 23 (FGF-23), and klotho. We determined that calcitriol supplementation alone increased calcification but was not associated with a transition towards an osteoblast-like phenotype. On the other hand, the combination of calcitriol and FGF-23 caused a decrease in calcification, but this decrease was attenuated with the further addition of klotho.

Vascular calcification (VC) is accelerated in patients with chronic kidney disease (CKD), resulting in increased risk of cardiovascular disease and mortality. Although the consequences of VC are associated with elevated pulse wave velocity (PWV) and left ventricular hypertrophy (LVH), the temporal impact on blood pressure changes is unknown. Mineral imbalance in CKD greatly contributes to the development of VC, and elevated serum phosphate is a major risk factor. Magnesium, which plays an important role in bone regulation, has been recently shown to be a modifier of VC, but whether magnesium inhibits calcification in CKD is unknown. A modified adenine model of CKD was developed in rats, characterized by mineral imbalance and progressive VC. During the development of VC, pulse pressure increased, which was driven by a drop in diastolic blood pressure, rather than systolic hypertension. Continuous pressure recordings in conscious rats using radiotelemetry revealed a significant increase in systolic variability associated with development of VC. Regional VC was associated with regional changes in the hemodynamic profile of the CKD rats. For example, only thoracic aortic calcification was associated with elevated PWV and pulse pressure. In contrast, the presence of abdominal and thoracic calcification differentially affected proximal and distal arterial pressure wave forms. CKD animals exhibited LVH, which was further increased by the presence of VC. In addition, fibroblast growth factor 23, which regulates renal excretion of phosphate, was elevated in CKD animals at every time point and was associated with LVH independently from VC. Development of VC was characterized in an in vitro organ model. Phosphate elevation in vitro caused VC in aortas. In vitro, magnesium supplementation inhibited initiation and progression of VC. CKD animals given a magnesium diet also demonstrated attenuated development of VC. In patients with stage 3-5 CKD (excluding dialysis), dietary phosphate was associated with the progression of coronary artery calcification even after adjusting for use of phosphate binders, total dietary energy and total dietary protein. Given the serious negative outcomes associated with development of VC, these findings fill key gaps in knowledge regarding the detection, management, prevention and treatment of VC in CKD. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2013-12-01 15:12:54.388

Vascular calcification is the formation of mineralised tissue within the walls of arteries. The pathology has many similarities to embryonic bone formation and involves the osteogenic differentiation of vascular smooth muscle cells (VSMCs) and matrix mineralisation. Recent studies have demonstrated that the bioactive sphingolipids, ceramide and sphingosine-1-phosphate (S1P), regulate embryonic bone formation. Ceramide can be generated by lysosomal acid sphingomyelinase (L-SMase) and neutral sphingomyelinase (N-SMase), and be converted to sphingosine by acid ceramidase (ACDase) and subsequently to S1P by sphingosine kinases (SK1 & SK2). This study tested the hypothesis that ceramide and S1P also regulate VSMC matrix mineralisation. VSMCs were cultured in the presence of 3 mM β-glycerophosphate (BGP) to induce osteogenic differentiation and matrix mineralisation. During VSMC mineralisation there were decreases in the activities of L-SMase and N-SMase and increases in the levels of C18 and C20 ceramide. S1P levels also increased during mineralisation as did SK1 and SK2 mRNA and SK activity. These results demonstrate that ceramide and S1P have the potential to regulate VSMC mineralisation. The exogenous addition of C2 ceramide decreased the rate of VSMC matrix mineralisation. Consistent with this, when VSMCs were cultured with 3 mM BGP and the joint L-SMase and ACDase inhibitor, desipramine, total ceramide levels increased and no matrix mineralisation was detected. These findings suggest that ceramide is an inhibitor of VSMCs matrix mineralisation. It was also noted in the presence of 3 mM BGP and desipramine that the mineralisation-associated increase in S1P was inhibited. In agreement with this, when exogenous S1P was added to the VSMCs an increase in matrix mineralisation was observed. Thus, S1P acts as a promoter of matrix mineralisation. To determine how S1P was promoting matrix mineralisation the signalling roles of the ezrin, radixin and moesin (ERM) proteins were investigated. The short-term stimulation of VSMCs with S1P led to the phosphorylation of the ERM proteins and over the mineralisation time-course, when S1P levels increased, the levels of ERM phosphorylation also increased. When VSMCs were cultured in the presence of 3 mM BGP and the inhibitor of ezrin phosphorylation, NSC668394, a decrease in matrix mineralisation was observed. No increases in ERM phosphorylation were seen in the presence of desipramine during the mineralisation time-course Therefore, S1P may be increasing matrix mineralisation through promoting the phosphorylation of the ERM proteins. This work has demonstrated that ceramide inhibits and S1P promotes VSMC matrix mineralisation in vitro. Additionally, this work identifies activation of ERM proteins, downstream of S1P, as a novel signalling pathway promoting matrix mineralisation. Characterisation of novel regulators of VSMC matrix mineralisation in vitro gives insight into the complex mechanisms contributing to vascular calcification in vivo and will aid in identification of novel therapeutic targets.

Premature death from cardiovascular disease is especially high in patients with chronic kidney disease (CKD). Vascular calcification is a potent risk factor for developing cardiac-related morbidity and mortality and is especially prominent within the CKD population. Deficiencies in serum levels of fetuin-A as well as inadequate production of matrix Gla protein (MGP) correlate inversely with the extent of vascular calcification and time spent on dialysis. Fetuin-A is a well-known systematic regulator of bone metabolism and MGP is a local antagonist of bone forming proteins. To meet the clinical need of at-risk patients prone to cardiac-related mortality. We propose a targeted protein therapy to treat arteriosclerotic arteries. The focus of this thesis was to characterize the binding interactions of fetuin-A with calcium mineral in a simulated body fluid and to study the in vitro effects of a fetuin vitamin K2 co-therapy on the prevention of calcification of vascular smooth muscle cells.

Vascular calcification is prevalent in ageing, in atherosclerosis, and especially in patients with Chronic Kidney Disease (CKD), with associated increased morbidity and mortality. The phenotypic transition of Vascular Smooth Muscle Cells (VSMCs) into osteoblastic/chondrogenic-like cells is critical for the development of calcification in CKD patients. Osteocytes, terminally differentiated osteoblasts, have recently emerged as major regulators of calcification in bone. Recently, osteocytelike cells have been observed in human peripheral arteries with medial vascular calcification. However, it remains undetermined as to whether VSMCs can undergo osteocytic differentiation within a calcifying environment and the functional role of osteocyte formation in the development of medial vascular calcification. Initial studies have characterised the ectonucleotide pyrophosphatase/phosphodiesterase 1 knockout (Enpp1-/-) mouse as a valid model of medial vascular calcification, which is employed throughout this thesis. This thesis has compared VSMCs to osteoblasts undergoing osteocytic differentiation in vitro. VSMC in vitro calcification was accompanied by up-regulated expression of osteocyte markers, including Sost, E11, Dmp1, Phex, Mepe and Fgf23. Immunohistochemistry confirmed the appearance of sclerostin and E11 in calcified aortae from the Enpp1-/- mouse. Further studies have identified a direct inhibitory role for the osteocyte specific gene FGF23 in modulating vascular calcification. The inhibitory effect of FGF23 on VSMC calcification was mediated through the MAPK/ERK signalling pathway. This thesis has also determined the role of BMP9, a highly osteogenic bone morphogenic protein, in vascular calcification, which induces VSMC calcification through a Smad signalling mechanism. Furthermore, VSMC expression of the osteocytic marker Sost was markedly increased following BMP9 treatment. Intriguingly, BMP9 was markedly elevated in serum from dialysis patients and a significant correlation was observed between dialysis time and BMP9 concentration in patients receiving haemodialysis. The work described herein has demonstrated that vascular calcification is associated with an osteocyte phenotype, and reports a direct inhibitory effect of the osteocyte specific gene FGF23 on vascular calcification. Furthermore, this thesis has shown that BMP9 induces the expression of the osteocytic marker Sost in VSMCs, and appears to play a critical role in vascular calcification.

Obesity is a major risk factor for insulin resistance, type 2 diabetes, cardiovascular disease (CVD), and vascular calcification. Vascular calcification is correlated with advanced CVD and a significant predictor of cardiovascular events. Obese individuals tend to have increased levels of circulating leptin, an adipocytokine that is a significant independent predictor of cardiovascular disease. We have shown that daily intraperitoneal injections of exogenous leptin (125 μg/mouse/d) can promote vascular calcification in an ApoE-/- mouse model of atherosclerosis. This increase in calcification is associated with an increase in the expression of several osteoblast-specific markers and is independent of any affect on atherosclerotic lesion size. Our studies suggest that leptin mediates the osteogenic differentiation of vascular smooth muscle cells (VSMCs) to promote vascular calcification via a pathway involving the inhibition of glycogen synthase kinase (GSK)-3 activity. Other studies have suggested that endoplasmic reticulum (ER) stress-induced GSK-3 activity promotes the development of atherosclerosis. Therefore, we hypothesized that during the progression of vascular disease, GSK-3 functions as a checkpoint for VSMCs at which cells can commit to: i) de-differentiation, thereby contributing to atherosclerosis, or ii) osteogenic differentiation, thereby contributing to vascular calcification. We investigated the effects of modulating GSK-3 activity on the differentiation of VSMCs in vitro. We found that many of the molecular tools that are typically used to modulate ER stress can promote the expression of osteoblast-specific markers and the osteogenic differentiation of MOVAS cells. However, because many of these interventions affect multiple pathways in MOVAS cells, the specific role of the ER stress – GSK-3 pathway is difficult to discern. Future studies are required to determine the effects of direct modulation of GSK-3 on vascular calcification and to delineate the mechanisms/effects of various ER stressors in the osteogenic differentiation of VSMCs. / Thesis / Doctor of Philosophy (Medical Science)

Chronic kidney disease (CKD) is a worldwide health problem with rising incidence and high cardiovascular mortality. CKD compromises cardiovascular function, in part, characterized by vascular calcification (VC), elevated pulse wave velocity (PWV) and pulse pressure (PP). Through manipulation of dietary adenine, we produced a model characterized by graded severity of CKD, VC and hyperphosphatemia. To our knowledge, we are the first to explore the relationship between aortic calcium content and changes in circulatory function in rodents with CKD. Fourteen-week old Sprague-Dawley rats received a diet containing an adenine concentration (0.25-0.75%) plus high-normal dietary phosphate (1%), for up to 10 weeks. Circulatory changes were determined by arterial radiotelemetry (n=6) and by assessment of aortic pulse wave velocity (PWV, n=32). VC was assessed using the calcium-O-cresophthalein-complexone assay. At sacrifice, kidney function (creatinine (µmol/L)) was worst in the group with VC (251.3±60.2 µmol/L), compared to non-calcifying CKD (200.3±68.8 µmol/L) or control (50.0±16.2 µmol/L). PWV (cm/s) adjusted for blood pressure (BP) was markedly elevated in animals with VC (3.23±0.33 log(cm/s)) versus non-calcifying CKD (2.85± 0.12 log(cm/s)) or control (2.96±0.08 log(cm/s)). Arterial pressure radiotelemetry revealed that there was an increase in pulse pressure (38±4.7 mmHg to 58 ±15.2 mmHg) during the development of VC. Systolic pressure remained relatively stable throughout (129±8.7 mmHg), diastolic pressure fell during weeks 9 and 10 of the study (91±6.0 mmHg down to 74±9.1 mmHg), a fall that almost fully accounted for the changes in pulse pressure. The calcifying CKD animals also exhibited left ventricular hypertrophy (LVH) compared to CKD or control animals (2.32±0.3 vs 2.03±0.2, 1.80±0.1 g/kg respectively). Manipulating dietary adenine produces a graded severity of CKD with calcification which impact circulatory changes (PP and PWV). These altered circulatory functions are likely to be key factors in the enhanced LVH. This model appears to be a useful for the study of CKD-associated VC. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2009-12-16 15:10:49.384

Cardiovascular disease is a significant health crisis, representing 32% of deaths worldwide in 2019. Vascular calcification (VC), a major contributor to cardiovascular disease, is a regulated biomineralization process whose exact mechanisms are unknown. Additionally, vascular smooth muscle cells (VSMCs) significantly contribute to VC by undergoing a phenotypic switch and differentiating into osteoblast-like cells. When factors like hypertension cause disturbed laminar flow in the body’s vasculature, the mechanical stress promotes the phenotypic switch and calcification of VSMCs via mechanotransduction. VC is also induced by the Wnt pathway, which is activated via mineral imbalance and mechanical stimulation. However, the exact mechanisms behind mechanotransduction in relation to VC, Wnt, and VSMC differentiation are unknown. If uncovered, knowledge of the mechanisms may be used to create effective treatments for VC.

Cardiovascular disease is among the leading causes of death in the US. It stems from the pathological buildup of plaque within the vasculature known as vascular calcification. Medial calcification, or arteriosclerosis is the buildup of plaque within the medial layer of the arteries resulting in artery wall stiffening and reduction of blood flow. Evidence suggests that the vascular smooth muscles cells (VSMCs) that line the medial layer of the arteries, undergo a phenotypic switch to osteoblast-like cells to deposit calcium while in this pathological state. The Wnt/BETA-catenin pathway could potentially play a role in the phenotypic modulation. Inhibition of the Wnt signaling pathway could be a promising approach to combat vascular calcification. Sclerostin (SOST) has been shown to be upregulated during arteriosclerosis in a manner that is indicative of the possible therapeutic potential of the protein. Therefore, we propose to confirm the role of Wnt signaling in vascular calcification and investigate the effects of SOST treatment on vascular calcification.