Vascular calcification (VC) is a cardiovascular disease characterized by calcium salt deposition in vascular smooth muscle cells (VSMCs). Standard in vitro models used in VC investigations are based on VSMC monocultures under static conditions. Although these platforms are easy to use, the absence of interactions between different cell types and dynamic conditions makes these models insufficient to study key aspects of vascular pathophysiology. The present study aimed to develop a dynamic endothelial cell–VSMC co-culture that better mimics the in vivo vascular microenvironment. A double-flow bioreactor supported cellular interactions and reproduced the blood flow dynamic. VSMC calcification was stimulated with a DMEM high glucose calcification medium supplemented with 1.9 mM NaH₂PO₄/Na₂HPO₄ (1:1) for 7 days. Calcification, cell viability, inflammatory mediators, and molecular markers (SIRT-1, TGFβ1) related to VSMC differentiation were evaluated. Our dynamic model was able to reproduce VSMC calcification and inflammation and evidenced differences in the modulation of effectors involved in the VSMC calcified phenotype compared with standard monocultures, highlighting the importance of the microenvironment in controlling cell behavior. Hence, our platform represents an advanced system to investigate the pathophysiologic mechanisms underlying VC, providing information not available with the standard cell monoculture.

Background: Vascular calcification (VC) is a dynamic, tightly regulated process driven by cellular activity and resembling the mechanisms of bone formation, with specific molecules playing pivotal roles in its progression. We aimed to investigate the involvement of the bone morphogenic proteins (BMP-2, BMP-4, BMPR-1a/1b, and BMPR-2) system in this process. Our study used an advanced in vitro model that simulates the biological environment of the vascular wall, assessing the ability of a phosphate mixture to induce the osteoblastic switch in human coronary artery smooth muscle cells (HCASMCs). Methods: HCASMCs were grown in mono- and co-culture with human coronary artery endothelial cells (HCAECs) in a double-flow bioreactor (LiveBox2 and IVTech), allowing static and dynamic conditions through a peristaltic pump. The VC was stimulated by incubation in a calcifying medium for 7 days. A BMP system Real-Time PCR was performed at the end of each experiment. Results: In monocultures, BMP-2 expression increased in calcified HCASMCs in static (p = 0.01) and dynamic conditions. BMP-4 and the biological receptors were expressed in all the experimental settings, increasing mainly in dynamic flow conditions. In co-cultures, we observed a marked increase in BMP-2 and BMP-4, BMPR-1a (p = 0.04 and p = 0.01, respectively), and BMPR-2 (p = 0.001) in the calcifying setting mostly in dynamic conditions. Conclusions: The increase in BMP-2/4 in co-culture suggests that these genes might promote the switch towards an osteogenic-like phenotype, data also supported by the rise of both BMPR-1a and BMPR-2. Thus, our findings provide insights into the mechanisms by which dynamic co-culture modulates the BMP system activation in an environment mimicking in vivo VC’s cellular and mechanical characteristics.

Background/Objectives: Under physiological conditions, vascular smooth muscle cells (VSMCs) are in a quiescent contractile state, but under pathological conditions, such as atherosclerosis, they change their phenotype to synthetic, characterized by increased proliferation, migration, and production of an extracellular matrix. Furthermore, VSMCs can undergo calcification, switching to an osteoblast-like phenotype, contributing to plaque instability. Methods: In this study, we analyzed the phenotypic changes in VSMCs during the transition from a physiological to a pathological state, a key process in the progression of atherosclerosis, using confocal and transmission electron microscopy, real-time PCR, and intracellular calcium quantification. Results: Confocal and transmission electron microscopy revealed a prominent remodeling of the actin cytoskeleton, increasing autophagic vacuoles in synthetic VSMCs and the deposition of calcium microcrystals in calcified cells. Immunofluorescence analysis revealed differential expression of α-SMA (contractile marker) and galectin-3 (synthetic marker), confirming the phenotypic changes. Real-time PCR further validated these changes, showing upregulation of RUNX-2, a marker of osteogenic transition, in calcified VSMCs. Conclusions: This study highlights the dynamic plasticity of VSMCs and their role in atherosclerosis progression. Understanding the characteristics of these phenotypic transitions can help develop targeted therapies to mitigate vascular calcification and plaque instability, potentially countering cardiovascular disease.

Background/Objectives: Under physiological conditions, vascular smooth muscle cells (VSMCs) are in a quiescent contractile state, but under pathological conditions, such as atherosclerosis, they change their phenotype to synthetic, characterized by increased proliferation, migration, and production of extracellular matrix. Furthermore, VSMCs can undergo calcification, switching to an osteoblast-like phenotype, contributing to plaque instability. Methods: In this study, we analyzed the phenotypic changes of VSMCs during the transition from a physiological to a pathological state, a key process in the progression of atherosclerosis, using confocal and transmission electron micros-copy, Real-time PCR, and intracellular calcium quantification. Results: Confocal and transmission electron microscopy revealed a prominent remodeling of the actin cyto-skeleton, increasing autophagic vacuoles in synthetic VSMCs, and the deposition of calcium microcrystals in calcified cells. Immunofluorescence analysis revealed differ-ential expression of α-SMA (contractile marker) and galectin-3 (synthetic marker), confirming the phenotypic changes. Real-time PCR further validated these changes, showing upregulation of RUNX-2, a marker of osteogenic transition, in calcified VSMCs. Conclusions: This study highlighted the dynamic plasticity of VSMCs and their role in atherosclerosis progression. Understanding the characteristics of these phenotypic transitions can help develop targeted therapies to mitigate vascular calci-fication and plaque instability, potentially countering cardiovascular disease.