Vascular calciﬁcation is a signiﬁcant contributor to cardiovascular risk in chronic kidney disease (CKD) patients, and its extent and severity has been correlated with mortality in several studies. Hyperphosphatemia predisposes these patients to early and progressive vascular calcification: it appears to be involved in a number of mechanisms that trigger and promote the progression of this active and cell-mediated pathological process, in which vascular smooth muscle cells (VSMCs) residing in the tunica media of blood vessels are the main cell type actively involved. We developed an in vitro model to elucidate the molecular and cellular mechanisms involved in the pathogenesis of vascular calcification: in particular, we challenged rat VSMCs for 7-15 days with high Pi (inorganic phosphorous) with the purpose to reproduce in vitro the same pathological process that occurs in CKD patients in vivo. We investigated the high Pi-induced calcium deposition and the modulation of different cellular biological processes (apoptosis, autophagy and VSMC osteoblastic differentiation) through molecular biology, proteomic and immunohistochemistry analysis. First of all, we studied the modulation of osteonectin (SPARC), a major non collagenous protein of bone matrix that is associated, generally, with remodeling of tissues, mineralization and pathological responses to injury. Since there are controversial results regarding its role during the process of vascular calcification, we investigated osteonectin expression both in vitro and ex-vivo, and the results suggest a pro-calcifying role of this protein in the process of vascular calcification. Then, we developed an experimental strategy to delay the progression of calcium deposition in our in vitro model. We studied the potential effect of repeated and short time suspension of high Pi treatment (process that we called “Wash Out”) on the progression of calcium deposition, trying to reproduce the same temporal decrease of Pi levels that occurs in CKD patients treated with haemodialysis. Surprisingly, we discovered that it is sufficient a temporary total absence or partial decrease in Pi concentration under a so called “trigger threshold” during the process of calcification to obtain a substantial inhibition of calcium deposition. The molecular and cellular pathways involved in this protective action are apoptosis, VSMC osteoblastic differentiation and autophagy: the formers are partially inhibited, while the latter is incremented after the “Wash Out” treatment. Finally, we investigated the mechanism of action of two drugs CKD patients are treated with in the attempt to reduce hyperphosphatemia and to contrast secondary hyperparathyroidism, respectively: Lanthanum Chloride (LaCl3) and Calindol. We demonstrated that these compounds significantly delay the progression of high Pi-induced VSMC calcium deposition, but with different mechanisms of action: both of them delay VSMC osteoblastic transformation, and, in particular, lanthanum chloride preserves VSMC lineage markers expression and partially prevents VSMC apoptosis, whereas calindol increases the expression of an anti-calcifying protein. These in vitro discoveries can suggest that vascular calcification is a multifactorial process that involves different cellular and molecular pathways, and that it is of relevant importance to control as more as possible phosphate levels in CKD patients, even with diet, because high-Pi is the most dangerous key regulator of vascular calcification in end stage renal disease pathology.
MOLECULAR AND CELLULAR MECHANISMS OF VASCULAR CALCIFICATION: PATHOGENESIS AND TREATMENT / F. Elli ; relatore: M. G. Cozzolino ; correlatore: P. Ciceri. - Milano : Università degli studi di Milano. Università degli Studi di Milano, 2014 Dec 11. ((27. ciclo, Anno Accademico 2014.
|Titolo:||MOLECULAR AND CELLULAR MECHANISMS OF VASCULAR CALCIFICATION: PATHOGENESIS AND TREATMENT|
|Supervisori e coordinatori interni:||COZZOLINO, MARIO GENNARO|
|Data di pubblicazione:||11-dic-2014|
|Parole Chiave:||Vascular calcification, Chronic Kidney Disease, Vascular Smooth Muscle Cells|
|Settore Scientifico Disciplinare:||Settore MED/14 - Nefrologia|
|Citazione:||MOLECULAR AND CELLULAR MECHANISMS OF VASCULAR CALCIFICATION: PATHOGENESIS AND TREATMENT / F. Elli ; relatore: M. G. Cozzolino ; correlatore: P. Ciceri. - Milano : Università degli studi di Milano. Università degli Studi di Milano, 2014 Dec 11. ((27. ciclo, Anno Accademico 2014.|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.13130/elli-francesca_phd2014-12-11|
|Appare nelle tipologie:||Tesi di dottorato|