Ultrastructural and Molecular Analysis of Vascular Smooth Muscle Cells During the Switch from a Physiological to a Pathological Phenotype

Abstract

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.

Keywords: atherosclerosis; autophagy; morphology; phenotype switching; vascular calcification; vascular smooth muscle cells.

Figure 1

Representative confocal microscopy images showing the morphological change in VSMCs during the phenotypic switch. (A) Contractile (CON); (B) Synthetic (SYN), arrows point to focal adhesions; (C) Calcified (CAL) states. Fixed cells were stained with Phalloidin Alexa Fluor 488 (green) and DAPI (blue) to visualize nuclei. Scale bars indicate 20 µm.

Figure 2

Transmission electron microscopy of VSMCs. (A) Representative image of CON VSMCs showing a large oblong nucleus, numerous mitochondria, and a bundle of myofibrils (arrows) in the cytoplasm. (B) Representative image of an SYN VSMC with a lobed nucleus and the cytoplasm displaying few scattered mitochondria and enlarged rough endoplasmic reticulum (RER) cisternae (arrow). m, mitochondrion; n, nucleus. Scale bar, 4 μm.

Figure 3

Transmission electron microscopy of VSMCs. Ultrastructural details of CON (A,C,E) vs. SYN (B,D,F) VMSC cytoplasm. A large number of mitochondria and regular cisternae of RER (arrow) are present in CON cells (A) concerning SYN cells (B), where scanty mitochondria and large roundish cisternae of RER (arrow) are visible. Bundles of myofibrils, sometimes mixed with mitochondria ((C,E) arrows), are present in CON cells. At the same time, they are scarce or absent in SYN cells where autophagic vacuoles and enlarged RER cisternae (arrow) are the main features (B,D,F). In SYN cells, the plasma membrane shows numerous pseudopod-like structures ((F) arrowheads). av, autophagic vacuole; m, mitochondrion; RER, rough endoplasmic reticulum. Scale bars: (A,B,F) 1 μm; (C) 600 nm; (D) 2 μm; (E) 800 nm.

Figure 4

Transmission electron microscopy of VSMCs cultured in calcifying medium for 7 days. Representative images showing scattered calcium microcrystals of different sizes in the cytoplasm ((A) with insert and (B,C)). In (B,C), microcrystals are also evident inside vesicles (arrow) and in the surface protrusions (arrowhead) of the plasma membrane (C). av, autophagic vacuoles. Scale bars, 500 nm; insert, 200 nm.

Figure 5

Intracellular calcium quantification. VSMCs were exposed to a phosphate mixture for 7 days, then lysed, and intracellular calcium levels were measured. Statistical analysis was conducted using a t-test; the bars indicate the mean ± SD. **** p < 0.0001. The data shown represent the averaged results of n = 3 independent experiments.

Figure 6

Evaluation of activation markers in VSMCs. Confocal microscopy images illustrate VSMCs cultured without serum (CON, (A)), with serum (SYN, (B)), and with calcifying media (CAL, (C)) to define the different phenotypes. In (A,B), the cells were fixed and stained with antibodies against α-smooth muscle actin (shown in red in the cytoplasm), galectin-3 (shown in green in the cytoplasm), in C cells stained with antibodies against RUNX-2 (shown in red in the nucleus) and galectin-3 (shown in green in the cytoplasm). DAPI was used for the nuclear visualization (blue). Scale bars: (A,B) 2 μm; (C) 20 µm.

Figure 7

The relative expression levels of α-SMA (A), galectin-3 (B), and RUNX-2 (C) in VSMCs exposed to the different inducing media were assessed. mRNA expression levels were determined using real-time PCR, with normalization to GAPDH. The data presented are means ± SD. Statistical analyses were conducted using one-way ANOVA with Tukey’s post hoc test, with significance levels indicated as *** p < 0.001, **** p < 0.0001. The data shown represent the averaged results of n = 3 independent experiments.