The Role of Vascular Smooth Muscle Cells in Arterial Remodeling: Focus on Calcification-Related Processes

Arterial remodeling refers to the structural and functional changes of the vessel wall that occur in response to disease, injury, or aging. Vascular smooth muscle cells (VSMC) play a pivotal role in regulating the remodeling processes of the vessel wall. Phenotypic switching of VSMC involves oxidative stress-induced extracellular vesicle release, driving calcification processes. The VSMC phenotype is relevant to plaque initiation, development and stability, whereas, in the media, the VSMC phenotype is important in maintaining tissue elasticity, wall stress homeostasis and vessel stiffness. Clinically, assessment of arterial remodeling is a challenge; particularly distinguishing intimal and medial involvement, and their contributions to vessel wall remodeling. The limitations pertain to imaging resolution and sensitivity, so methodological development is focused on improving those. Moreover, the integration of data across the microscopic (i.e., cell-tissue) and macroscopic (i.e., vessel-system) scale for correct interpretation is innately challenging, because of the multiple biophysical and biochemical factors involved. In the present review, we describe the arterial remodeling processes that govern arterial stiffening, atherosclerosis and calcification, with a particular focus on VSMC phenotypic switching. Additionally, we review clinically applicable methodologies to assess arterial remodeling and the latest developments in these, seeking to unravel the ubiquitous corroborator of vascular pathology that calcification appears to be.

2.1. Arterial Remodeling Arterial remodeling reflects the adaptation of the vessel wall to biochemical and biomechanical stimuli [ 9 , 12 ]. In the vasculature, two types of remodeling can be distinguished: outward and inward remodeling, with respective hypertrophy (thickening) or hypotrophy (thinning) of the vessel wall. Large conduit vessels do not have the ability to constrict in response to stress, and therefore show hypertrophic remodeling. Atherosclerosis is characterized by an increase in vessel diameter, with the thickening of both the media and intima, and classically termed outward hypertrophic remodeling [ 28 ]. Aneurysm formation is characterized by an increase in vessel diameter, with a thinning of the vessel wall, and termed outward hypothropic remodeling [ 29 ] ( Figure 2 ). Inward remodeling is less frequently seen and is observed in more muscular peripheral arteries, probably reflecting the sustained vasoconstriction of vessels [ 30 ]. Under low laminar flow, platelet derived growth factors (PDGF) and transforming growth factor-β (TGF-β) promote inward remodeling by increasing VSMC proliferation and collagen deposition [ 31 ]. Inward remodeling also contributes to atherosclerosis, but does not lead to an increased vessel size ( Figure 2 ). Atherosclerosis development involves many cellular processes, like the recruitment of inflammatory cells by chemotaxis and infiltration [ 32 ]. VSMCs are connected to a fenestrated network of elastin and collagen fibers. The capacity of the vessel wall to elastically distend is important to accommodate the volume ejected with each heartbeat and to limit peripheral pressure pulsations. The active tone and spatial arrangement of VSMCs may influence the mechanical load on the ECM components and, therefore, modulate vessel diameter and stiffness [ 18 ]. Chronic exposure to high blood pressure increases tensile stress [ 9 ] to which VSMCs respond by proliferation, resulting in hyperplasia and thickening of the vascular wall. Concomitant activity of MMPs facilitates the structural breakdown of elastin ECM, and synthetic VSMCs produce collagen ECM, attempting to preserve stiffness homeostasis [ 33 ]. High blood pressure thus aggravates age-related stiffening of arteries [ 34 , 35 , 36 ]. Additional ECM disturbances, such as the presence of calcium crystals, have a further impact on the stiffening of the medial layer [ 37 , 38 ]. During aging, it is generally accepted that the number of cells in the vasculature decreases, although the causes of this finding remain to be established [ 34 ]. It has been hypothesized that VSMCs become senescent and that cell division rates decrease [ 39 ]. The recent literature ignores vessel wall cellularity and often refers to cellular processes, such as apoptosis, inflammation, calcification and epigenetic effects, all playing a part in vessel wall aging [ 20 ]. Additionally, with aging, collagen content in major arterial vasculature increases, whereas elastin content decreases and the number of VSMCs declines [ 40 ]. As a consequence, remaining VSMCs are embedded in a collagen-enriched ECM with fewer cellular focal adhesions [ 41 , 42 , 43 ]. Within arterial vessels, differences exist in the content of elastin to VSMC ratios [ 44 ]. Large arteries close to the heart contain more elastin and are therefore called “elastic” arteries. It is particularly elastic arteries that stiffen with age [ 45 ]. Large artery stiffening results in decreased arterial compliance, especially in those aged over 60 years [ 19 , 46 , 47 ]. Peripheral vessels contain more VSMCs relative to elastin and are termed “muscular” arteries. In muscular arteries, the relative elastin content increases with age, most likely caused by the decline in the number of VSMCs and decreased collagen content [ 44 ]. It should be noted that the absolute amount of ECM proteins in the vasculature decreases with age, but that fat and extracellular material, such as calcium crystals, increase [ 48 ]. Taken together, the number of VSMCs within the vasculature strongly correlates with vascular stiffening and the arterial remodeling processes [ 34 ]. Endothelial function plays an important role in arterial remodeling. Blood flow and normal wall shear stress stimulate ECs to produce NO ( Figure 3 ). NO induces the relaxation of local VSMCs, which leads to dilation of the vessel wall. Endothelium-dependent vasodilation decreases with age, which appears to be associated (perhaps causally or bi-directionally) with a quiescent state of VSMCs and is clearly implicated to be associated with hypertension and CVD [ 49 , 50 ]. Under pathological conditions, ECs are known to produce cytokines and growth factors [ 51 ], which induces VSMCs phenotype switching from contractile to a more synthetic phenotype. An increased wall shear stress increases endothelial derived NO release, further decreasing VSMC proliferation and increasing VSMC apoptosis [ 52 ], resulting in outward remodeling and stif

Platelets have shown to affect VSMC inflammatory phenotypes and injury responses [ 63 ]. Additionally, platelet EVs induce an inflammatory response in VSMC in vitro [ 64 ]. Platelet factor 4 (PF4) has a central role in the stimulation of VSMC-mediated cytokine release, which is primarily affected by increased krüppel-like factor 4 (KLF-4) transcription. Experimental atherosclerosis models indicated that PF4 is proatherogenic and it has been found to penetrate deep into the vessel wall, underlining the importance of platelets aside from their effect on the endothelium [ 65 ].

The ECM in which VSMCs are embedded also affects VSMC phenotype. VSMC phenotype is modulated via integrin receptors that are present on ECM proteins [ 87 ]. ECM consists of structural proteins, such as collagen, elastin and proteoglycans. For instance, the proteoglycan heparin promotes VSMC contractility. In vitro, heparin treatment of VSMCs induces a contractile phenotype and slows down its proliferation [ 69 , 88 ]. Collagen has pleiotropic effects on VSMC phenotype, depending on the type of collagen. Collagen type-I and fibronectin induce a synthetic VSMC phenotype [ 89 , 90 ] with proliferation [ 91 , 92 ]. On the contrary, collagen type-IV and laminin promote a contractile VSMC phenotype [ 89 , 93 ]. Intact elastin is associated with a contractile VSMC phenotype. The loss of elastin, by a genetic mutation, is associated with increased hypertrophy and hyperplasia of VSMCs [ 94 , 95 ]. Additionally, elastin remodeling, by the modulation of cathepsins and MMPs, promotes VSMC phenotypic switching that induces calcification [ 96 , 97 ]. Not only composition, but also the organization of structural fibers, determines VSMC phenotype. Culturing VSMCs in 3D compared to 2D increases contractile expression markers and induces TGF-β expression [ 98 , 99 ]. Culturing VSMCs on scaffold templates controls spatial organization and morphological response of VSMCs, indicating that VSMCs react to structural environmental changes to retain vessel function [ 100 ].

KLF4 is an important transcriptional regulator defining VSMC phenotype during development [ 103 ] and after vascular injury [ 104 ]. KLF4 expression is increased in lesions of ApoE -/- mice on a Western type diet [ 105 ]. Additionally, there is increased binding of KLF4 to α SMA and SM22 α promotors upon vascular injury in mouse carotid arteries [ 104 ]. The loss of KLF4 has favorable effects, inhibiting plaque pathogenesis and reducing plaque vulnerability [ 106 ]. KLF4 is critical in the regulation of phenotypic switching of VSMCs, both in vitro and in vascular injury models. KLF4 expression results in profound activation of pluripotency genes such as Oct4 and Sox2, indicating that VSMCs can reactivate its pluripotency network in response to vascular stress or damage [ 5 ]. Moreover, KLF4 activates > 800 pro-inflammatory VSMC genes, of which many are atherosclerosis relevant [ 106 ]. Additionally, KLF4 knock-out in an atherosclerosis animal model revealed the switch of VSMCs toward a synthetic phenotype, while suppressing the macrophage-like form [ 106 ].

Several pathologies are associated with VSMC phenotype switching [ 12 ]. Synthetic VSMCs produce EVs [ 2 , 122 , 123 ], that have been found in both the intimal and medial layers of the vessel wall [ 124 ]. VSMCs are known to release EVs upon phenotypic switching towards synthetic or osteogenic phenotype. EVs derived from VSMCs share similarities with EVs from osteoblasts, having calcium-binding capacities and osteoblast-like ECM production [ 2 , 80 ]. Recently, it was shown that not all EVs promote calcification [ 125 ]. This implies that EV content is variable and dependent on VSMC-mediated biogenesis. A specific subclass of EVs are exosomes, which express tetraspanins (CD9, CD63 and CD81) and differ in expression pattern, and, hence, mineralizing capacity [ 125 ]. Furthermore, calcifying conditions in vitro increase the multi-vesicular body, forming enzyme sphingomyelin phosphodiesterase 3 (SMPD3) and subsequent exosome genesis [ 80 ]. Consequently, the inhibition of SMPD3 ablates the generation of exosomes and their subsequent calcification. Additionally, sortilin has been suggested as a key player in the VSMC-mediated calcifying EVs genesis and release. Sortilin is a sorting receptor that directs target proteins to a designated location via the secretory or endocytic compartment [ 126 ]. Recent findings have identified that sortilin promotes vascular calcification via the trafficking and loading of tissue-nonspecific alkaline phosphatase into EVs [ 127 ]. Moreover, sortilin co-localized with calcification in human calcified vessels [ 127 ]. Differences between mineralizing and non-mineralizing EVs eventually determine calcification of the ECM in proximity of VSMCs. Hence, better insight into EV composition, such as lipid content and RNA and protein profile, might provide new insights into the mechanism by which VSMC derived EVs contribute to vascular calcification and result in novel targets for treatment. In response to calcified ECM, neighboring ECs and VSMCs react and induce the production of osteogenic factors, such as bone morphogenetic protein 2 (BMP-2) and 4 (BMP-4) [ 128 , 129 ]. Besides this expression of bone-associated proteins, the number of VSMCs decreases with age, and an increase of collagen-to-elastin ratio further increases the stiffening of the vessel wall [ 130 ]. In hypertension, calcium handling is disturbed, which is associated with an increased activation of L-type calcium channels and sensitivity of (hypertensive) patients toward calcium channel blockers [ 131 ]. Increased intracellular calcium induces the activation of receptors coupled to phospholipase C, leading to the generation of second messengers that trigger cytokines, ROS and miRNAs, and also cellular derived EVs [ 132 , 133 ]. Additionally, increased intracellular calcium activates the contractile machinery of VSMCs, leading to hyper-contractility. Excessive intracellular calcium rapidly disintegrates both mitochondria and structural components of VSMCs, and results in calcium depositions within elastic fibers [ 130 ]. Consequently, elasticity of the vasculature decreases, further contributing to increased blood pressure [ 24 , 134 ] and VSMC stiffening [ 135 , 136 ]. Specific calcium antagonists, blocking L-type voltage-dependent calcium channels, reduce calcium internalization and normalize blood pressure [ 137 , 138 , 139 ]. Of note, these calcium channel blockers also prevent calcification of the vasculature [ 140 ].

Atherosclerosis is characterized by intimal plaque build-up and often referred to as a thickening of the vessel wall, ultimately narrowing the lumen of the vessel. Atherosclerosis is considered an inflammatory disease, starting with endothelial stress and subsequent monocyte infiltration [ 150 , 151 ]. Clinically, plaques may rupture, resulting in stroke or myocardial infarction [ 104 ]. Besides the classical view that systemic factors are causative for atherosclerosis, recent data suggest that local vascular processes also initiate atherogenesis [ 104 ]. Lineage tracing studies revealed that many of the cells present in atherosclerotic plaques are derived from VSMC precursors [ 30 ]. Additionally, it is now recognized that atherosclerotic plaque macrophages have lineage traces of VSMCs, indicating that cells in the intima originate from the vascular media, and not necessarily from the circulating mesenchymal cells that infiltrate the vessel wall at sites of injury [ 152 ]. Arterial remodeling is considered key to the development of atherosclerosis [ 148 , 153 ]. Arterial remodeling of the vessel wall can be initiated via many processes, including oxidative stress [ 2 ], proliferation [ 58 ], VSMC phenotype switching [ 154 ], cell infiltration, apoptosis and calcification [ 155 , 156 , 157 ].

Fibrosis of different organs contributes to up to 50% of worldwide mortality [ 181 , 182 ]. In the vasculature, myofibroblasts produce ECM proteins such as collagen and fibronectin [ 12 ]. Myofibroblasts are derived from MSC-like cells that reside in the vessel wall. The role of these MSC-like cells in the vessel wall is not fully understood, and is believed to play an important role in structural vessel repair. In vascular fibrosis, excessive collagen deposition leads to a reduced compliance of the vessel, which indicates increased arterial stiffness. Furthermore, high blood pressure induces structural changes including hypertrophy of the vessel wall [ 183 , 184 ] and cellular [ 185 ] and ECM remodeling [ 186 ]. Lineage tracing studies have shown that resident Gli1 + MSC-like cells are a major contributor of vascular fibrosis in arterial remodeling [ 187 ]. Acute vessel wall injury revealed that a significant number of Gli1 + cells were detected in the media and neointima, and that they express VSMC markers such as α SMA and CNN1. Additionally, some 50% of the newly formed VSMCs were derived from Gli + progenitors, indicating the involvement of adventitial cells in arterial remodeling [ 188 ]. Likewise, angiotensin II induced hypertension was caused by an exuberant production of collagen in the adventitial layer [ 189 ]. VSMCs that migrate towards the adventitia start expressing stem cell markers Sca-1 and CD34 [ 186 ]. These “adventitial stem cells” change towards fibroblasts and deposit collagen and other ECM, contributing to adventitial fibrosis [ 190 , 191 ]. During atherosclerosis development, fate-tracing studies in mice have shown that MSC-like cells migrate into the media and neo-intima. Also, MSC-like Gli1 + cells were a major source of osteoblasts-like cells, significantly contributing to the process of vascular calcification in both vascular media and intima. The genetic ablation of Gli + cells before injury dramatically reduced the severity of vascular calcification [ 192 ]. These data indicate that fibrotic processes are regulated by myofibroblasts and MSC-like cells and that these are important in the development of atherosclerosis and vascular calcification.

Arterial stiffness can be determined by measuring local vessel diameter and distension by ultrasound or MRI [ 198 ]. The measured data can be used to calculate the local distensibility coefficient (DC) or compliance. PWV, as well as DC, are well known to be pressure dependent and require blood pressure adjustment [ 199 ]. Adjustment is valid when considering group analyses, but lacks applicability in the use of individual cases [ 32 , 200 ]. PWV is largely dependent on the elastic properties of the large arteries. Therefore, PWV should be interpreted with care, since it only reflects the stiffness of the aorta. In conduit arteries, the relative amount of VSMCs are limited and, thus, the contribution of ECM to arterial stiffening may be expected to be more pronounced. In peripheral, more muscular, arteries, age-related stiffening is not as straightforward [ 201 , 202 ]. PWV in the lower extremities can change significantly, which may explain why no evident relation between PWV and age has been reported [ 203 ]. Additionally, hypertrophy of VSMCs is commonly observed in hypertension [ 204 ], with the potential to (partially) determine PWV [ 205 ]. Thus, PWV is an important clinical marker for risk assessment, as it correlates with cardiovascular risk, mortality, and organ damage of the heart, kidney and brains [ 206 , 207 ]. Techniques for measuring PWV are developing. cfPWV is most commonly used for aortic stiffness, but is susceptible to local wave reflections and not suitable for obtaining information on carotid arteries [ 208 ]. Ultrasound ultrafast imaging techniques are being explored to directly measure the propagation of the pulse wave, with limited success for the common carotid artery [ 209 ]. Elastography is a promising technique, as it measures the propagation of shear waves in the tissue [ 210 ]. The propagation of the shear wave is directly proportional to the elastic modulus of the tissue [ 16 ]. PWV can also be measured using MRI, which also allows for segmentation. Its drawbacks are costs and long scanning times [ 33 ]. Currently, the available PWV toolbox can only be applied to large and medium-sized arteries, but techniques are developing for smaller vessels. At present, the interpretation of stiffness measurements in terms of arterial wall constitutive properties remains a challenge [ 187 ]. Medial calcifications are known to co-localize with elastin fragmentation spots, but the mechanical contribution of the calcific deposits to vessel stiffness remains to be established; the elastin degradation alone could be sufficient to explain stiffening [ 211 ].

Calcification imaging in the clinic is mostly based on CT, which is an angiography technique based on X-ray radiation and makes use of computer processing to create a detailed 3D image. Calcification scores, such as the coronary artery calcium (CAC) score, can be derived from CT scans and, in recent decades, this has been used as a measure of atherosclerotic burden. The Agatson score is the most frequently used method to calculate CAC. The Agatston score is defined as a weighed sum of the calcification area, with density given as Hounsfield Units [ 226 ]. CAC represents an accurate measure to assess atherosclerotic burden [ 227 , 228 ]. Additionally, CAC correlates with traditional risk factors for atherosclerosis, such as hypertension, hypercholesterolemia and diabetes [ 227 , 228 ]. CAC also associates with cardiovascular morbidities and mortalities, including cardiac death and stroke [ 229 ]. CAC is regarded as the best clinical predictor for the accumulation and progression of vascular calcification over time [ 230 , 231 , 232 ]. CAC can be used to identify high-risk individuals that need immediate medical attention or intervention [ 233 ]. Interventions to slow down or even regress calcification, using the supplementation of vitamin K, are currently being conducted [ 234 ]. High dietary intake of vitamin K is associated with a lower risk of coronary heart disease [ 235 , 236 , 237 ] and supplementation with vitamin K significantly delays the progression of calcification [ 238 ]. Conversely, statin treatment is known for its beneficial cardiovascular effects, however, it is also shown to increase CAC score [ 239 ]. This indicates that type of calcification, as well as the volume or density of calcification, determines whether it is plaque-stabilizing or plaque-destabilizing. Indeed, CAC volume is positively and independently associated with CVD, whereas CAC density was inversely correlated with CVD risk [ 240 ]. Measuring CAC as a clinical marker for CVD has some limitations. Firstly, CAC is limited to coronary arteries, which do not represent the calcification of other vessels of the arterial system. CAC does not represent total plaque size, as histopathological studies showed that the area of calcium deposition was much smaller than total plaque volume [ 241 , 242 ]. Additionally, CAC score poorly correlated with luminal narrowing and severity [ 217 , 243 , 244 ]. Secondly, distinction between medial and intimal calcification is not possible with current resolutions of CT. Intimal and medial calcification are different pathologies, with different risk factors for developing CVD [ 245 ]. Additionally, microcalcification (< 15 μm) cannot be assessed, as the resolution of CT is some 200 μm [ 246 ]. Thirdly, evidence suggests that atherosclerotic plaques that have denser calcification have smaller lipid cores, with increased stability of plaques, resulting in lower CVD risk [ 247 ]. Greater CAC volume, however, is associated with a higher risk of CVD [ 248 ]. This indicates that future research should integrate density and volume into CAC scoring. Finally, CAC involves exposure to radiation, which limits the use of calcium scores as a follow-up on atherosclerosis progression [ 249 ].

Arterial remodeling is defined as the adaptation of the vessel wall away from a normal homeostatic state towards pathologic development, triggered by chronic exposure to stress signals. Different arterial remodeling processes have been described, such as inflammation, oxidative stress, lipid accumulation, and degradation of the ECM. Many of these are regulated by VSMCs and affect vessel wall morphology and properties. VSMCs exhibit cellular plasticity and, upon stress stimuli, they may differentiate towards synthetic, macrophage-like and osteogenic VSMCs. As a consequence, VSMCs initiate and support remodeling processes involving ECM synthesis and cell proliferation, migration and contraction, which aid in maintaining tissue functionality. Disruption of VSMC phenotypic plasticity has many pathological consequences, including the development of hypertension, atherosclerosis, aneurysm formation, intimal and medial calcification, and vascular fibrosis, ultimately leading to increased CVD morbidity and mortality. Early detection of vascular remodeling is key in the prevention and prognosis of CVD. Clinically, assessment of calcification-mediated remodeling remains difficult. Assessment tools include measurements of pulse wave velocity, distensibility coefficient and intima-media thickness. These techniques may capture changes in dynamic and geometric vessel wall properties, but cannot directly monitor the underlying arterial remodeling processes. The addition of techniques, such as 18 F-NaF PET and CT imaging may provide further mechanistic insight into vascular calcification development and other VSMC-mediated processes. Combining expertise in vascular biology, clinical vascular expertise and multi-scale/multi-modality imaging should advance the understanding and identification of critical determinants of vascular remodeling, ultimately resulting in tailor-made diagnoses and treatments.