Controversies Over The Role of Vsmcs in Atherosclerotic Cardiovascular Diseases

Abstract

Coronary artery disease (CAD) is by far the most prevalent cardiovascular condition that leads to death in the United States. According to a report filed by the Centers for Disease Control and Prevention (CDC), the disease causes as many as 370,000 deaths annually. Over the years, investigators have been able to identify the major underlying factors that would either directly or indirectly lead to CAD such as atherosclerosis (AS), miRNA regulations, and hypercholesterolemia. In addition, different biomarkers were carefully examined, and cell-specific lineages were traced back to study the proliferation and differentiation of different cell types involved in AS. However, there still had been much controversy and debate over the specific roles of VSMCs. The goal of this review is to rigorously evaluate the current research perspectives on the involvement of VSMCs in AS progression, and how different factors interacting with VSMCs could have contributed to distinct pathological phenotypes.

Introduction

In order to implement and refine any current therapeutic methods to the treatment of coronary artery disease (CAD), it is critical to understand the underlying biochemical and mechanical causes that lead to the initiation and progression of the disease. Atherosclerosis (AS) is a common chronic condition that begins progressing from one’s childhood. AS is usually initiated by hypertension or hypercholesterolemia, which damages the thin endothelium layer of the artery, resulting in an aggregation of white blood cells, especially the macrophages and other macrophage-derived cells, to degrade accumulating low-density-lipoproteins (LDL) and any potential pathogens. The chronic presence of WBCs at the damaged site eventually led to the formation of fibrous plaque, which is composed of multiple lipoproteins and necrotic debris from WBCs, capped by VSMCs and fibrous tissues. Once the plaque has grown to a size that is comparable to the diameter of the arteries, patients are facing the danger of acute fibrous plaque rapture which consequently leads to the notorious coronary artery disease (CAD) due to blood clotting (or thrombosis) and immediate cardiomyocytes death.

VSMCs are involved in various aspects of the disease progression of AS. VSMCs are the dominant producer of extracellular matrix (ECM) in response to atherogenic signals such as INF. By secreting multiple kinds of adhesion molecules including VCAM-1 and ICAM-1, VSMCs also serve as docking sites for monocytes and leukocytes to stabilize the damaged cell layers against apoptosis and promote atherogenesis with the help of platelets. Previously, this process is well-known to be a protective mechanism of damaged vessel walls. However, recent investigations have revealed a different story in which they discovered that the phenotypes and lineages of VSMCs have a major contribution in determining the trajectory of disease progression. A variety of lipoproteins and ligands could induce a drastic change in both the morphology and the functionality of the VSMCs, altering them from inhibiting the AS to promoting it.

Despite the amount of effort being put into elucidating the underlying mechanism of atherosclerosis (AS) and coronary artery disease (CAD), the academic society is still trying to characterize VSMCs’ biochemical and mechanical properties from three major perspectives: 1) fate of the VSMCs, whether they are normal, senescent, or apoptotic; 2) biochemical interactions through epigenetic regulation or ligand-receptor binding; 3) the origin of the VSMCs by lineage-tracing to determine the cell-type specificity of biomarkers. Though not able to discuss the full picture of the situation, this paper will cover the experimental setups, critical results, and potential caveats extensively within these three topics.

Discussion

1. Distinct cell fates

a. Apoptotic VSMCs

Vascular smooth muscle cells (VSMCs) play an important role in the post-injury vascular regeneration process due to its ability to produce a large number of structural proteins, including collagen type I and III, for damaged ECM reconstruction. It was long observed in vitro culture that the presence of VSMCs within monocytes culture would inhibit their apoptosis, which correlates to the phenomenon of accumulation of macrophages at the wounded site, causing chronic inflammation. However, the pathological responses of the presence of apoptotic VSMCs were rarely mentioned in previous studies. Bennett et al. from the University of Cambridge noticed this gap and conducted a series of experiments trying to elucidate the governing biochemical factor behind the anti-apoptotic phenotype of VSMCs. Using transgenic mice with a VSMC-specific toxin-receptor (hDTR) and being atherosclerosis-prone apolipoprotein E-deficient, his group was able to elucidate that the death of VSMCs is insufficient to induce inflammation or any kind of vessel remodeling. However, if apoptotic VSMCs appeared in the established plaque, they would acquire the ability to induce atherosclerotic plaque vulnerability solely. Even though rarely seen in vivo, Bennett et al. further demonstrated that this apoptotic phenotype of VSMCs is powerful enough to trigger undesired clinical outcomes at an earlier stage of AS. Again, hDTR transgenic mice were used as an animal model to study the consequence of VSMC apoptosis by administrating diphtheria toxin (DT), which binds to hDTR and induces cell apoptosis, continuously for 10 to 15 weeks with a high-fat diet, mimicking pathological hypercholesterolemia. It was widely accepted that VSMCs are essential in the process of vascularization. Nevertheless, with only minimal presence of apoptotic VSMCs in the early stage of AS, pathological features such as fibrous cap thinning and necrotic core development can already been observed in the treated mice arteries, indicating that this apoptotic phenotype of VSMCs may be a useful biomarker in predicting disease progression in early stages as well as evaluating the possibility of plaque breakout in late stages.

While Bennett et al. are trying to argue that apoptotic VSMCs have a detrimental effect on the wellness of patients, a set of proteins called NF-E2-related factor 2 (Nrf2)/ Kelch-like ECH-associated protein 1, identified by a group of researchers in Showa University School of Pharmacy, helps to regulate the fate of VSMCs by sensing reactive oxygen species (ROS) that induce oxidative stresses due to vascular injury. Their results showed that Nrf2-induced VSMCs apoptosis may contribute positively to the formation of the neointimal layer and other vascular remodeling events after vascular injury. By down-regulating the anti-apoptotic signaling molecule Keap1 and up-regulating Nrf2 in an event of injury, in vivo models demonstrated significantly faster revascularization with VSMCs apoptosis, preventing the system from experiencing hyperplasia.

While acknowledging the validity of their point of view from both investigators, it was noticed that neither gender nor age of the mice used was specified in the former experiments, and only male mice were used in the latter. Gender plays a critical role in the case of cardiovascular events, and coronary artery disease (CAD) risk profile is particularly associated with females’ reproductive status and menopause transition. In addition, unlike humans, female mice reach their age of sexual maturity within four to six weeks, so that their hormone level, as well as other physiological features, could’ve skewed the experimental results in a great extent. Hence it would be desired to specify the gender and reproductive status of animal models used in any cardiovascular research.

b. Senescent VSMCs

Ras has been identified as a powerful stimulus in diverse cellular events that involve vascularization and angiogenesis. In the case of arterial injury, Ras can be activated to induce vascular smooth muscle cell senescence in the progress of atherogenesis. Minamino et al. presented evidence for not only Ras activation but also downstream signals like ERK signaling, and immunostaining images for both in vitro and in vivo histological samples, demonstrating the co-localization of senescent VSMCs with chronic inflammation in the established atherosclerotic plaque by antibodies against senescence-associated -galactosidase (SA--gal) and -smooth muscle actin. (Minamino Tohru et al., 2003) These pieces of evidence suggest that there might be a heterogeneous population of VSMCs within the established plaque to help reach the homeostatic state of the neointima layer formed by ECM proteins, macrophages and macrophage-derived lipids, and various forms of vascular smooth muscle cells (VSMCs) that are functioning cooperatively yet distinctively. Nevertheless, the biomarkers that used to identify the precise subpopulations of this heterogeneous healing complex may not be specific enough to justify the statement that these antibody-stained populations were exclusively VSMCs. This particular caveat will be discussed in the later sections where other investigators questioned the validity of using those surface proteins as unique biomarkers for VSMCs through the method of embryonic lineage-trancing to study the proliferation as well as differentiation of progenitors of VSMCs.

2. Multiple levels of regulations

a. Transcriptional Regulation: myocardin

Recent efforts to further characterize the phenotypical traits of VSMCs within the atherosclerosis plaque have revealed new pieces of evidence that chronic cholesterol loading of VSMCs drastically alters them to a dysfunctional macrophage-like phenotype. The Fisher group observed in culture plates that VSMCs can undergo phenotype-switching with the presence of cholesterol by staining specific markers for vascular smooth muscle cells (ACTA2) and macrophages (CD68). Their biochemical results were supported by transcriptome profiling and qRT-PCR to quantitatively analyze the expression level of these proteins. With this evidence, they further tested if the external stimulation caused the downregulation of two crucial genetic components for VSMCs contractility: transcription factor myocardin and its co-activator serum-response factor (SRF). Indeed, the Fisher group reported that SRF level was reduced by about 60% to that in the control group, and myocardin level was downregulated to only 25% to that in the control group, indicating a significant loss in VSMCs contractility and mobility to migrate to the wounded site and participate in revascularization of the damaged vessel wall. Though lacking data from in vivo study, which would have been more clinically relevant, this result still profoundly complicates the controversy over the definitive functionality of VSMCs since it’s extremely difficult to directly monitor the physiological condition of a patient in real-time. Ackers-Johnson et al. also reported the inhibitory role of myocardin to the activation of VSMCs, preventing inflammatory response by downregulating the expression levels of multiple cytokines, chemokines, and adhesive molecules for macrophage anchoring. Attenuation of macrophage accumulation was also observed by Ackers-Johnson et al. in ApoE -/- hypercholesterolemic mice models administered with adenoviruses coded with myocardin compare to that of the control group. These results provide insights to further understanding of the physiological regulation of VSMCs on transcriptional levels, yielding potential therapeutic targets for AS and CAD to mitigate the adverse effects from your immune system and lower the risk of AS plaque rupture with CAD disease progression.

Nevertheless, Yuliya et al. and Ackers-Johnson et al. presented several potential therapeutic targets to alleviate hyperplasia and proposed possible interactions between VSMCs and dysfunctional macrophages due to their phenotypical similarities.

b. Post-Transcriptional regulation: miRNA

Choe et al. focused their research on microRNA-regulated VSMC proliferation and differentiation. MicroRNAs are single-stranded non-coding RNA molecules that can bind to the 3’ untranslated region of the mRNA to control the post-transcriptional activity of that particular mRNA. Of major interest, Choe et al. showed that miR-34c plays a crucial role in the process of neointimal hyperplasia both in vitro and in vivo, along with a protein named stem cell factor (SCF). By investigating carefully, these researchers found that miRNA-34c targets SCF, which was reported to induce VSMC proliferation and migration in the early stages of atherosclerosis. Histological staining samples of rat arterial tissue were analyzed under the microscope to study the pathological effects of overexpression of miRNA-34c in vivo, and significant downregulation of SCF is observed with severely attenuated atherogenesis and vascularization. One caveat when comparing these data with those done by other researchers is that Choe et al. used male Sprague-Dawley rats while previous investigators widely utilized ApoE -/- hypercholesterolemic mice models. Still, these results qualitatively illustrated that the VSMC proliferation, migration, and differentiation potencies are regulated by a combination of both genetic and epigenetic factors, and further investigations need to be conducted while considering all these different regulators to accurately characterize the pathogenesis and progression of coronary artery disease.

3. Lineage-tracing: embryological origin-dependence

An even more profound controversy is whether VSMCs are to blame for the deterioration of atherosclerosis due to plaque rupture for several reasons: 1) there’s been no definitive biomarker for VSMC due to antigen loss or transfer by contact during disease progression; 2) VSMCs derived from different embryological origins tend to have distinctive functions and marker expressions; 3) there are other tissues or cell types that express ACTA2 and MYH11 at different stages of differentiation. Cheung et al. presented a possible solution to this problem by artificially inducing different types of VSMCs from human pluripotent stem cells (hPSCs) using growth factors, hormones, or chemicals. After the initial differentiation induction, there are three intermediate lineages: neuroectoderm (NE), lateral plate mesoderm (LM), and paraxial mesoderm (PM). Cheung et al. showed that these origin-specific subtypes of VSMCs require different differential stimuli for fate commitment and distinct biological responses to cytokine stimulations. These results may start a new chapter on researching VSMC characteristics and yield unexpected therapeutic targets due to the change of classifications and the necessity of method refinement.

Conclusion

We conclude that there are some major controversies over the exact functions and phenotypical traits of VSMC during the progression of atherosclerosis and coronary artery disease. Though much effort has been put into elucidating the characteristic similarities and potential interaction between VSMCs with neighboring endothelial cells and accumulating macrophages, there are still plenty of questions left unanswered due to the extreme complexity of biological systems. Evidence of both AS-promoting and AS-inhibiting have been brought up by different researchers trying to argue the specific function of VSMCs at a specific disease stage. However, in the case of understanding a life-threatening condition like coronary heart disease, multiple factors should always be considered to oversee the systematic response.

Finally, we believe that it would be impossible to elucidate the full picture of VSMC-induced CAD without definitive and rigorous characterizations of VSMC interactions with other micro/macromolecules. Therefore, further investigations should be conducted before the selection of any therapeutic targets on human patients.

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