Vascular calcifications (VC) are abnormal mineral depositions in the vascular tree and are frequently observed in patients with atherosclerosis, chronic kidney disease (CKD), and diabetes [1,2]. VC affects more than 70% of the population over 70 years, leading to different cardiovascular complications, thus increasing morbidity and mortality. Currently, medical treatments are not available; preventive actions based on diet and integration with natural bioproducts, like quercetin, curcumin, resveratrol, and magnesium, have been reported to inhibit the disease process in experimental models [3]. An in-depth understanding of the molecular mechanisms of vascular calcification represents the key to developing effective therapeutic strategies to treat VC. A valuable approach for better understanding underlying mechanisms is represented by in-vitro cell model systems that recapitulate the cellular characteristics and their hemodynamic environment. In particular, these systems may allow to evaluate also the role of active compounds in the treatment of vascular calcification. This work aims to develop an advanced in-vitro model that can recapitulate the complex biological environment of the vascular wall to increase the effectiveness of in-vitro tests of active compounds in the reduction and/or slow down of vascular calcification before proceeding to clinical studies. To best mimic the environment of systemic vasculature, Human Coronary Artery Endothelial cells (HCAECs) and Human Coronary Artery Smooth Muscle cells (HCASMCs) were tested using a double-flow bioreactor (LiveBox2, IVTech Srl, Massarosa, Italy) composed by two chambers that can be perfused by independent flows thus allowing static and/or dynamic conditions. HCAECs (45.000 cells) were seeded in each upper chamber of the LiveBox2 and cultured for seven days in supplemented Endothelial Cell GM MW2. HCASMCs (30.000 cells) were seeded in each bottom chamber of the LB2 and cultured in supplemented Medium 231; the two compartments are separated by a polyester porous membrane (0.45 ?m pore size). Upper chambers were subjected to a 250 ul/min flow rate for the dynamic condition. For the induction of HCASMCs calcification, Medium 231 was replaced with DMEM HG supplemented with 1,9 mM phosphates solution (NaH2PO4/Na2HPO4) for seven days. After the incubation period, HCAECs and HCASMCs viability was tested with CellTiter-Blue Cell Viability Fluorometric Assay. For Calcium quantification, HCASMCs were subjected to HCl and overnight freezing to optimize the lysis, and Sigma-Aldrich Calcium Colorimetric Assay Kit was used following manufacturer instructions. Cell samples were collected, and Real Time-PCR of specific genes was performed. Data analysis was conducted using the Student's t-test or ANOVA with a significance level of ? = 0.05. The analysis of cell viability demonstrated that calcifying media significantly decreased HCASMCs viability in static conditions. The presence of HCAECs and the dynamic flow seem to attenuate the antiproliferative effect of the phosphates solution on the HCASMCs. Regarding HCAECs' viability, we found no significant differences during calcifying conditions with respect to control, both in static and dynamic conditions. The positive effect of HCAECs and the dynamic flow was also highlighted in calcium amount, which is considerably reduced for static conditions and when endothelial cells are present. To assess the ability of phosphates mixture to induce the osteoblastic switch of HCASMCs, a key event in vascular calcification, the Real Time-PCR was performed and showed, with respect to control, a significant increase of: 1) BMP2 and BMPR1a receptor, as well as of BMPR1b, also if no significantly involved in vascular calcification; 2) IL-1?, a potent proinflammatory cytokine playing diverse roles in the pathogenesis of atherosclerotic vascular calcification; 3) CX43, the most abundant gap junction protein involved in osteoblastic differentiation; 4) mTOR1, which is involved in the maintenance of cellular homeostasis.
A dynamic in-vitro model of smooth muscle and endothelial cells cultures for vascular calcification study
Ceccherini E;Persiani E;Gisone I;Del Ry S;Guiducci L;Cabiati M;Morales MA;Cecchettini A;Vozzi F
2022
Abstract
Vascular calcifications (VC) are abnormal mineral depositions in the vascular tree and are frequently observed in patients with atherosclerosis, chronic kidney disease (CKD), and diabetes [1,2]. VC affects more than 70% of the population over 70 years, leading to different cardiovascular complications, thus increasing morbidity and mortality. Currently, medical treatments are not available; preventive actions based on diet and integration with natural bioproducts, like quercetin, curcumin, resveratrol, and magnesium, have been reported to inhibit the disease process in experimental models [3]. An in-depth understanding of the molecular mechanisms of vascular calcification represents the key to developing effective therapeutic strategies to treat VC. A valuable approach for better understanding underlying mechanisms is represented by in-vitro cell model systems that recapitulate the cellular characteristics and their hemodynamic environment. In particular, these systems may allow to evaluate also the role of active compounds in the treatment of vascular calcification. This work aims to develop an advanced in-vitro model that can recapitulate the complex biological environment of the vascular wall to increase the effectiveness of in-vitro tests of active compounds in the reduction and/or slow down of vascular calcification before proceeding to clinical studies. To best mimic the environment of systemic vasculature, Human Coronary Artery Endothelial cells (HCAECs) and Human Coronary Artery Smooth Muscle cells (HCASMCs) were tested using a double-flow bioreactor (LiveBox2, IVTech Srl, Massarosa, Italy) composed by two chambers that can be perfused by independent flows thus allowing static and/or dynamic conditions. HCAECs (45.000 cells) were seeded in each upper chamber of the LiveBox2 and cultured for seven days in supplemented Endothelial Cell GM MW2. HCASMCs (30.000 cells) were seeded in each bottom chamber of the LB2 and cultured in supplemented Medium 231; the two compartments are separated by a polyester porous membrane (0.45 ?m pore size). Upper chambers were subjected to a 250 ul/min flow rate for the dynamic condition. For the induction of HCASMCs calcification, Medium 231 was replaced with DMEM HG supplemented with 1,9 mM phosphates solution (NaH2PO4/Na2HPO4) for seven days. After the incubation period, HCAECs and HCASMCs viability was tested with CellTiter-Blue Cell Viability Fluorometric Assay. For Calcium quantification, HCASMCs were subjected to HCl and overnight freezing to optimize the lysis, and Sigma-Aldrich Calcium Colorimetric Assay Kit was used following manufacturer instructions. Cell samples were collected, and Real Time-PCR of specific genes was performed. Data analysis was conducted using the Student's t-test or ANOVA with a significance level of ? = 0.05. The analysis of cell viability demonstrated that calcifying media significantly decreased HCASMCs viability in static conditions. The presence of HCAECs and the dynamic flow seem to attenuate the antiproliferative effect of the phosphates solution on the HCASMCs. Regarding HCAECs' viability, we found no significant differences during calcifying conditions with respect to control, both in static and dynamic conditions. The positive effect of HCAECs and the dynamic flow was also highlighted in calcium amount, which is considerably reduced for static conditions and when endothelial cells are present. To assess the ability of phosphates mixture to induce the osteoblastic switch of HCASMCs, a key event in vascular calcification, the Real Time-PCR was performed and showed, with respect to control, a significant increase of: 1) BMP2 and BMPR1a receptor, as well as of BMPR1b, also if no significantly involved in vascular calcification; 2) IL-1?, a potent proinflammatory cytokine playing diverse roles in the pathogenesis of atherosclerotic vascular calcification; 3) CX43, the most abundant gap junction protein involved in osteoblastic differentiation; 4) mTOR1, which is involved in the maintenance of cellular homeostasis.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
