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Development of effective treatment against vascular calcification : a major clinical issue in an ageing population
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Abstract
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| This thesis focusses on finding novel therapeutic targets and therapies for arterial media calcification, a major cardiovascular complication in elderly and patients with chronic kidney disease (CKD), diabetes and osteoporosis. Arterial media calcification refers to the deposition of calciumphosphate crystals (i.e. hydroxyapatite) in the medial layer of the arterial wall favoring arterial stiffness, hypertension and left ventricular hypertrophy ultimately leading to heart failure, impaired coronary perfusion and cardiac stroke. Moreover arterial calcification is associated with increased mortality in CKD patients. In general, the arterial media calcification process encompasses both active and passive processes involving a phenotypic switch of vascular smooth muscle cells and endothelial cells towards bone-like cells and mesenchymal stem-like cells (progenitor cells of the osteoblast lineage), and precipitation of calcium and phosphate crystals in the arterial wall respectively. Furthermore, arterial media calcification is linked to a decrease in circulating calcification inhibitors (i.e. pyrophosphate, matrix gla-protein, fetuinA, sclerostin and osteoprotegerin) and an increase in circulating calcification stimulators (i.e. phosphate, calcium, uremic toxins, inflammation, hyperglycemia and vascular ageing). Calcification in the medial layer of the arterial wall highly resembles physiological bone mineralization, making it extremely difficult to develop safe and efficient treatments against arterial media calcification without affecting physiological bone mineralization; i.e. the so-called calcification paradox. This is of particular relevance to CKD patients, since they may suffer from either high bone turnover disease (i.e. osteitis fibrosa) or low bone turnover disease (i.e. osteomalacia, adynamic bone). Therefore, it is indispensable to evaluate novel compounds concomitantly against arterial media calcification and potential deleterious effect on bone in different animal models of arterial media calcification including the non-CKD warfarininduced arterial media calcification rat model and the CKD induced arterial media calcification rat models such as the adenine rat model. The first research objective of this thesis investigated the involvement of protein-bound uremic toxins, indoxyl sulfate and p-cresyl sulfate, in the arte- Summary 208 rial media calcification process. During CKD, both indoxyl sulfate and p-cresyl sulfate accumulate in the blood circulation inducing deleterious effects on surrounding organs such as the kidney, bone and vasculature. Due to conflicting clinical data on whether or not these uremic toxins contribute to cardiovascular mortality in CKD patients, we exposed CKD rats to vehicle, indoxyl sulfate (150 mg/kg per day) or p-cresyl sulfate (150 mg/kg per day) for either 4 days (short-term exposure) or 7 weeks (long-term exposure) to study early and late vascular alterations by toxin exposure. Results showed that both protein-bound uremic toxins, at concentrations similar to those found in CKD patients, significantly increased calcification in the aorta and peripheral vessels. Furthermore, by performing a proteomic analysis of arterial samples, an association between calcification events, acute-phase response signaling and coagulation and glucometabolic signaling pathways was found. Additional metabolic linkage to these pathways revealed that indoxyl sulfate and p-cresyl sulfate exposure engendered a pro-diabetic state as evidenced by elevated resting glucose and reduced GLUT1 expression. Inflammation and coagulation signaling pathways were also activated in the non-calcified aortic tissue of CKD animals exposed to short-term uremic toxins exposure indicating that these signaling pathways are causally implicated in toxin-induced arterial calcification. In conclusion, this objective revealed that in a context of CKD, both indoxyl sulfate and p-cresyl sulfate directly promote vascular calcification via activation of inflammation and coagulation pathways and were strongly associated with impaired glucose homeostasis. Further studies are needed to investigate whether agonist or antagonists of these pathways are capable of influencing the arterial media calcification process. In the second research objective, studies that target the hydroxyapatite crystal growth in the arterial wall were performed through extracellular nucleotide mediated effects. Both interventions with the purinergic independent (i.e. TNAP inhibition) and dependent (i.e. P2X receptor activation) pathways were explored. The enzyme TNAP regulates the breakdown of the calcification inhibitor pyrophosphate (a metabolic degradation product of nucleotides) into the calcification stimulator inorganic phosphate. Pyrophosphate prevents further incorporation of inorganic phosphate into the hydroxyapatite crystals. Because the expression of TNAP is increased in calcified arteries as well as its genetic overexpression in endothelial cells and vascular smooth muscle cells led to the development of arterial media calcification, selective inhibitors against TNAP enzymatic activity were developed. We investigated the prophylactic effect of the pharmacological TNAP inhibitor SBI-425 on arterial calcification and bone metabolism in both a non-CKD (warfarin induced arterial calcification rat model) and CKD related context (adenine induced arterial Summary 209 calcification rat model). Daily administration of the TNAP inhibitor SBI-425 (10 mg/kg/day) to warfarin exposed rats for 7 weeks significantly reduced arterial calcification. Unexpectedly, as TNAP activity is a hundred times higher in the bones versus arteries, administration of SBI-425 resulted in decreased bone formation rate and mineral apposition rate, and increased osteoid maturation time and this without significant changes in osteoclast- and eroded perimeter. This was an important finding as CKD patients already suffer from a compromised bone status and thus therefore it was imperative to investigate whether TNAP-inhibitor SBI-425, either or not in combination with pyrophosphate supplementation, was able to inhibit arterial media calcification in a CKD context without aggravating bone metabolism. In this study, administration of a high phosphate diet for two weeks followed by a 0,75% adenine diet for four weeks induced the development of a stable chronic renal failure as indicated by hyperphosphatemia, hypocalcemia and high serum creatinine levels. Also, the presence of CKD induced arterial media calcification and bone metabolic defects. However, treating these CKD rats with pyrophosphate (120?mol/kg/ day) alone or in combination with TNAP inhibitor SBI-425 (10mg/kg/day) reduced the presence of arterial media calcification, which was not the case for SBI-425 treatment alone. Moreover, SBI-425 alone and in combination with pyrophosphate increased osteoid area pointing to a less efficient bone mineralization. Then again, mRNA expression profile of the aortic tissue revealed that in a context of CKD elevated NPP3 gene expression, rather than TNAP, might be responsible for the stimulation of the arterial calcification process. Based on these findings, evaluating higher dosages of SBI-425 to block CKD related arterial calcification has limited relevance and purpose. Future studies must focus on (i) determining whether other phosphatases such as NPP3 are more relevant in the calcification process during CKD and (ii) focus on whether higher dosages of pyrophosphate administration are able to halt the progression of pre-existing arterial calcifications in a CKD setting as well as establishing the optimal route of administration including intraperitoneal injections versus oral administration. In vitro studies have shown that the synthetic ATP analogue and P2X receptor agonist ?,?-meATP is a potent inhibitor of vascular smooth muscle cell calcification. For this reason, we have studied the effect of ATP analogue ?,?-meATP on the development of arterial media calcification in a non-CKD warfarin rat model. Interestingly, daily intra-peritoneal injections with ?,?meATP (2 mg/kg/day) significantly reduced the calcifications in the aorta and peripheral vessels which was further reinforced by a significant reduction in aortic Von Kossa positive area % versus vehicle group. However, ?,?-meATP did not alter the mRNA expression of osteo/chondrogenic marker genes, Summary 210 calcium-phosphate metabolism nor did it influence apoptotic lesions in the arterial wall. The exact mechanism by which ?,?-meATP mediates its inhibitory effects on arterial media calcification remains inconclusive, however it could be that ?,?-meATP interferes with the formation of calcium-phosphate crystals via its breakdown product methylene bisphosphonate. Unfortunately, ?,?-meATP induced characteristics of adynamic bone disease, as indicated by a significant lower amount of osteoid tissue and a decrease in bone formation rate, questioning the safety and efficiency of ATP analogue ?,?-meATP in a CKD context. Future studies must elucidate the ability of ?,?-meATP and its metabolite methylene bisphosphonate to prevent the development of arterial media calcification in rats with chronic renal failure. To conclude, developing efficient and safe therapies against arterial media calcification remains challenging as the studies performed in the frame of this thesis report that compounds (i.e. TNAP-inhibitor SBI-425 and ?,?meATP) inhibit the development of calcifications in the arterial wall however, also compromise physiological bone mineralization. Therefore, exploring new therapeutic targets related to inflammation, coagulation and/or glucose homeostasis, investigating the dissimilarities between the calcification process in the arteries and bone, as well as trying to selectively target compounds such as ?,?-meATP to the blood vessels by using i.e. nanoparticle conjugated to antibody drug delivery system might all be promising ways to obtain safe and efficient anti-arterial calcification therapies. |
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Language
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English
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Publication
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Antwerpen
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Universiteit Antwerpen, Faculteit Farmaceutische, Biomedische en Diergeneeskundige Wetenschappen, Departement Biomedische Wetenschappen
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2021
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Volume/pages
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221 p.
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Note
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d'Haese, Patrick C. [Supervisor]
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Verhulst, Anja [Supervisor]
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Neven, Ellen [Supervisor]
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Full text (open access)
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