Modeling elastin-associated vasculopathy with patient induced pluripotent stem cells and tissue engineering

Abstract

Elastin-associated vasculopathies are life-threatening conditions of blood vessel dysfunction. The extracellular matrix protein elastin endows the recoil and compliance required for physiologic arterial function, while disruption of function can lead to aberrant vascular smooth muscle cell proliferation manifesting through stenosis, aneurysm, or vessel dissection. Although research efforts have been informative, they remain incomplete as no viable therapies exist outside of a heart transplant. Induced pluripotent stem cell technology may be uniquely suited to address current obstacles as these present a replenishable supply of patient-specific material with which to study disease. The following review will cover the cutting edge in vascular smooth muscle cell modeling of elastin-associated vasculopathy, and aid in the development of human disease modeling and drug screening approaches to identify potential treatments. Vascular proliferative disease can affect up to 50% of the population throughout the world, making this a relevant and critical area of research for therapeutic development.

Introduction

Elastin is one of many extracellular matrix proteins secreted by cells, along with proteoglycans, hyaluronan, and collagen fibers, and components of elastin have been found in VSMCs, endothelial cells, chondrocytes, and fibroblasts [1]. The role of elastin in these cell types is to provide compliance in response to cell stretching and to facilitate the recoil essential to blood vessel physiology. Even in an in vitro monolayer, tropoelastin monomers have displayed the inherent physical property of elastic extensibility [2]. Age-related inflammation via the continued activity of matrix metalloproteinase-12, also known as elastase, progressively degrades elastin fibers, resulting in stiffer tissues and impaired wound healing [3]. As elastin fibers are formed primarily during the early neonatal and late fetal stages of life, with formation rapidly declined throughout adulthood, loss of elastin leads to progressive dysfunction in blood vessels [4, 5]. Degradation of elastin can lead to aberrant vascular proliferation, leading to the development of vasculopathies including thoracic or abdominal aortic aneurysm and dissection and intimal hyperplasia.

Elastin polymers are initially formed by cells through the secretion of the 60–75 kDa monomer tropoelastin, a soluble protein which becomes subsequently crosslinked outside the cell by the enzyme lysyl oxidase. As illustrated in Fig. 1, these soluble monomers first require a scaffold onto which they can be deposited, with the main scaffold proteins in VSMCs being fibrillin-1 and fibrillin-2 [6, 7]. These fibrillins are cysteine-rich glycoproteins thought to provide additional flexibility to the elastin polymer. Fibrillins are also known to interact with cell surface receptors, such as integrins and proteoglycans, and are thought to thus mediate signal transduction [8, 9]. Once assembled, the adaptor protein fibulin-5 is thought to further aid in the maturation of elastin scaffolds in the presence of integrins [10].

Fig. 1.

Schematic illustration of biogenesis of elastin fiber via assembly of tropoelastin monomers. Tropoelastin is translated and binds to its molecular chaperone (such as EBP or FKBP65) in the rough endoplasmic reticulum. The tropoelastin–chaperone complex is transported through the Golgi and secreted via vehicle to the cell membrane. Extracellularly, the secreted tropoelastin is oxidized by lysyl oxidase, detached from the molecular chaperone, attached to fibulin5 which binds to integrin α_vβ₃, and further associated with the fibrillin-1 and other attached tropoelastin monomers to form polymers of elastic fibers

As an extracellular protein, elastin must interact with additional factors on the cellular surface to transmit signals to the internal environment of the cell. Elastin is believed to act as a negative regulator of VSMC proliferation and a positive regulator of VSMC maturation through affecting the polymerization state of smooth muscle actin (SMA). The ratio of monomeric (globular, G-actin) SMA to polymeric (filamentous, F-actin) is thought to affect the proliferation of VSMCs. In elastin-deficient VSMCs during in vitro culture, smooth muscle actin filament assembly is significantly reduced and cells display a hyperproliferative phenotype [11]. Addition of recombinant tropoelastin to elastin null VSMCs elevated levels of active Rho-A, rescued F-actin formation, and inhibited proliferation and migration in a dose-dependent manner [12].

The functionality of elastin fibers and relevant signaling pathways have been characterized through the implementation of “gold standard” in vitro molecular biology approaches using primary VSMCs acquired from patients as well as in vivo physiologic experiments with various knockout mouse models. Proposed mechanisms for elastin signaling derived through these approaches include direct binding of elastin to integrins or G-protein coupled receptors (GPCR), or indirect signaling through the activity of elastin adaptor molecules. Studies on the interaction of the elastin C-terminal RKRK motif with integrin α_vβ₃ [13–16] or the internal tropoelastin VGVAPG domain with adaptor GPCRs, glycosaminoglycans, or the molecular chaperone elastin-binding protein [12, 13, 17, 18] have been detailed elsewhere. Additional intracellular signaling events between the transcriptional coactivator of the serum response factor (SRF), myocardin-related transcription factor (MRTF), and actin polymerization have been proposed [19, 20], with TGF-β perhaps augmenting these effects [21].

Although these gold standard approaches have yielded many insights into elastin biology, these approaches are inherently limited in scope. Primary VSMCs have limited accessibility as patient samples are available only following heart transplant or expiration. It has also been found that following repeated culturing, primary VSMC begin to lose some properties of smooth muscle [22], and exhibit an absence of extracellular elastin fiber formation in cell culture models. This is reflected through the comparably minimal studies of elastin biology conducted with primary VSMCs. Similarly, mouse models, though informative as an in vivo surrogate, possess inherent physiologic differences to humans, particularly with regard to the cardiovascular system. Furthermore, the genetic phenotypes underlying elastin complex pathologies (e.g., Eln^+/−, Fbn1^+/−, Fbln5^+/−) are not faithfully recapitulated in mouse models [10, 23–26] and thus models may not accurately reflect human patients. Therefore, employing a novel, broadly applicable approach which faithfully reflects human elastin pathologies would be beneficial towards the advancement of the field.

Induced pluripotent stem cell platform

iPSC modeling

To further the study of VSMC proliferation pathologies, there is a requirement for an appropriate model system. A desirable alternative modeling system comes through the advent of induced pluripotent stem cells (iPSCs) (Fig. 2). The seminal discovery of using the Yamanaka reprogramming factors to take fully differentiated cells and induce a state of pluripotency has revealed a novel methodology for an array of disease modeling approaches [27]. Using iPSCs as a modeling system is preferable to primary cell lines because the supply is readily accessible from patients in a non-invasive manner, and the cells can self-renew following the induction of pluripotency, effectively making the supply endless. iPSCs are also advantageous compared to mouse models, as the iPSCs come from a human source, avoiding confounding cross-species artifacts. The findings from iPSC research are additionally readily transferable to a clinical setting, as the cells being treated originate from an affected patient.

Fig. 2.

Schematic illustration of current approaches to modeling elastin-associated diseases via induced pluripotent stem cells and tissue engineering. The generation of induced pluripotent stem cells (iPSCs) can be mediated by introduction of exogenous reprogramming factors (e.g., OCT4, SOX2, c-MYC and KLF4; the Yamanaka factors) into donor’s somatic cells (e.g., dermal fibroblasts or peripheral blood mononucleocytes). Subsequently, the iPSCs can be specifically differentiated into the desired cell types such as vascular smooth muscle cells. Multiple applications can be achieved using these iPSC-derived vascular smooth muscle cells, including establishment of a 2D culture-based disease model, 3D engineered tissue ring model for studying the basic pathological mechanisms, and generation of tissue-engineered blood vessel (TEBV) as a vascular graft for clinical purpose

2D modeling

There are several applications of iPSC technology to study VSMC proliferation in disease. The first is the basic modeling of biology in a two-dimensional culture plate (Fig. 2). Differentiation protocols have been determined for VSMCs in particular, though as the iPSCs are pluripotent, these techniques can be and have been extrapolated to a variety of cell types [28]. Indeed, such protocols have been implemented to model SVAS, WBS, Marfan syndrome, and Down syndrome in two-dimensional culture systems effectively [11, 29, 30]. A two-dimensional system using iPSCs provides a unique and highly useful platform for a detailed mechanistic study of elastin-associated vasculopathy.

3D modeling

In addition to the above, a newer approach has been to model iPSC-derived VSMCs cultured in a three-dimensional system (Fig. 2). Using ring-shaped agarose molds, VSMCs can self-assemble into tissue culture rings which maintain the typical markers for smooth muscle: SMA, MYH11, SM22α [31]. These iPSC-derived tissue culture rings similarly maintain the functionality of VSMCs, displayed through contractility and stretch versus strain analyses compared to surgically prepared primary tissue rings [31]. Such a three-dimensional model provides additional insight into how VSMCs interact as a network of cells, more closely matching the in vivo environment of blood vessels. Furthermore, the 3D vascular tissue ring approach has successfully been implemented to model the vascular hyperproliferation and loss of contractility observed in patients with a haploinsufficiency of elastin [31]. In this way, tissue rings create a unique platform for both disease modeling and drug screening in vitro under more physiologic conditions.

Tissue-engineered blood vessels

A final modeling approach is a further extrapolation of the three-dimensional iPSC-derived tissue culture rings. The vascular cells can further be applied to fabricate tissue-engineered blood vessels (TEBVs). TEBVs are formed through the seeding or mixing of iPSC-derived VSMCs around a biodegradable fibrous or hydrogel-like scaffold (Fig. 2) [32, 33]. As the TEBV cells grow, they can be further conditioned both with externally provided growth media and through a controlled internal pulsatile flow, mimicking the flow of blood through a developing vessel. Once mature, these TEBVs have shown to replicate the circulatory force requirements of natural blood vessels, and contain secreted extracellular matrix proteins essential to blood vessel biology [34]. It has been reported that iPSC-based TEBVs can be generated via a number of approaches. TEBVs have been produced by seeding the iPSC-VSMCs onto a polyglycolic acid scaffold [35, 36] which both histologically resemble native vessels and were implantable into a rat model [35]. Moreover, vascular cells can be mixed with a collagen gel to initially cast the tubule shape structure, and subsequent TEBVs can be further advanced via the addition of an endothelial layer in the lumen [32]. Furthermore, using these approaches, pathologic phenotypes such as Hutchinson–Gilford progeria syndrome were successfully reproduced in the TEBV platform [37]. In this way, TEBVs provide a useful model for disease modeling as well as a potential future form of therapy preferable to a stent, either as a decellularized general use product or as a patient-specific vessel, as the TEBV is naturally produced and would be allogenic or syngeneic.

Fundamental questions

An analysis of the current state of the field of elastin biology and VSMC proliferation reveals several important questions which need to be addressed in future research and modeling approaches. These fundamental questions are more suitably addressed through the iPSC platform than the aforementioned gold standard approaches. First, how does extracellular elastin communicate with the nucleus? Why does aberrant signaling in elastinopathies lead to changes in VSMC proliferation? Data are conflicting on any precise mechanism of action, and current modeling approaches are insufficient to address this concern, as extracellular elastin polymer has not been producible in vitro in cultured VSMCs, and mouse models are not suitable for a detailed mechanistic study. Second, how is the post-transcriptional regulation of elastin controlled so as to produce the appropriate balance of elastin isoforms needed for the cell at any given time? Elastin is notoriously difficult to work with experimentally due to the variety of mRNA transcripts produced to create different isoforms of tropoelastin. Though tropoelastin mRNA has been identified as a target for miR-29a, the mechanism of how this governs appropriate isoform expression remains unclear and should be addressed [38]. Third, how does the lineage specificity of VSMCs alter the signal transduction process? In the media of large elastin arteries, there are two distinct subtypes of VSMCs. One originates from the cardiac neural crest, and is thus ectoderm, while the other is from the local mesenchyme, and is thus mesodermal. These different VSMC subtypes elicit different responses upon stimulation by some peptide growth factors, notably TGF-β [39]. It is thus necessary for future experimental approaches to consider lineage differentiation alongside a mechanistic assessment.

How does extracellular elastin communicate with the nucleus?

Biomechanical sensing

The connection between the forces provided to the cell surface and corresponding changes to the cellular internal environment is an essential process in all cells and is of particular importance for arterial responses to changes in blood flow. The endothelial cells of the intima are the initial point of contact between the circulation and the artery, and so these cells possess several mechanisms to sense fluid shear stress from the lumen. These include stretch-activated ion channels, integrins, cell surface receptors, and glycoproteins [40]. These signals can then be transduced by the endothelial cells through the process of mechanotransduction and propagated to the extracellular matrix and VSMCs of the medial artery [40]. Mechanotransduction acts as a feedback network which links the sensory inputs to operational outputs, such as cytoskeletal or cell surface receptor rearrangements, leading to global changes in cellular morphology and transcriptional regulation [41]. Mechanotransduction is an extremely fast process, occurring on the order of microseconds, several orders of magnitude faster than the speed of signaling through soluble factors [42]. Thus, mechanotransduction processes are of high importance to cells and systems which directly experience and respond to variable forces, such as the VSMCs of arteries. Proliferative adaptations of the arterial cell wall in response to stress have been reviewed elsewhere [43]. Another key factor in biomechanical sensing of arteries is the extracellular matrix, which not only can bind, integrate and control the presentation of growth factors and ligands to cells, but also provides a protective buffer to shield the arterial cells from the high stress of blood flow. In the aorta, VSMCs sense a force of 3–5 kPa, despite the 100–200 kPa of pressure imposed on the arterial wall following systolic ejection [43]. Thus, the maintenance of the extracellular matrix is essential to normal biomechanical sensing in arteries, and is achieved through the dynamic activity of deposition and secretion by endothelial cells and VSMCs and degradation by matrix metalloproteinases.

Maladaptation can occur when the biomechanical sensory apparatus of VSMCs is dysfunctional. In VSMCs the two principal resultant pathologies are stenosis, or the hyperproliferation of VSMCs to thicken the arterial medial layer and occlude the lumen, and aneurysms, a bowing out of the arterial wall in response to the pressure demand of blood flow as a result of wall thinning. Studies using the gold standard approaches have been informative, finding this irregular sensing could result from mutations in the internal cellular network, such as SMA, MYH11, or myosin light chain kinase, whose signaling and mechanics have been well-described [44–46]. Additionally, such maladaptive responses to improper biomechanical sensing could arise from deficiencies in extracellular matrix components of VSMCs, namely in elastin [24, 47–49], fibrillin-1 [21, 23], or fibulin-5 [10, 25, 26]. However, these gold standard approaches are insufficient to address the mechanistic “how” between the signaling events that connect to extracellular environment to the nucleus. The iPSC platform is well-suited to investigate this pathway, as pathology-specific VSMCs can be compared to control VSMCs with regard to extracellular environment, tension sensing, and downstream activation of factors leading to the proliferative or contractile response.

Modeling elastin complex communication with the nucleus with iPSCs

As noted above, tropoelastin monomers in vitro have been shown to display an inherent elastic extensibility [2], which allows differential tension to be sensed across membrane-bound integrins [50]. As integrins form the fundamental connection between the extracellular elastin fibers and the intracellular actomyosin machinery, it is probable that the defective elastin complexes prevalent in the aforementioned vascular pathologies can be differentially sensed through the mechanotransduction machinery, generating an aberrant proliferative or contractile response. Stiffness can be directly measured via transfection of a fluorescent talin tension sensor [50], which can be readily employed over a variety of elastin complex deficiencies through the iPSC-VSMC platform. Using patient-derived iPSC-VSMCs at the single cell level in vitro reflects the in vivo phenotype as in both cases a more forceful stretch on the actomyosin machinery—arising from the interaction between integrins and a stiffer, less elastic matrix—leads to enhanced FAK signaling and cellular proliferation [51, 52]. Downstream effectors on this signaling cascade could readily be studied through standard molecular biology approaches using this platform, providing insights unavailable from conventional techniques. The iPSC-VSMC platform thus provides an extensive and more physiologic setting to study basic elastin biology.

iPSCs as a drug screening platform

In addition to effective disease modeling, the iPSC platform is well-suited for high-throughput drug screening. As elastin-associated vasculopathies often show defective smooth muscle actin filament formation, and thus an impaired contractility [11, 12, 31], rescue of these phenotypic sequelae following drug exposure can be assessed, either through a direct measurement of contraction in response to exogenous vasoconstrictor addition, or through the presence of smooth muscle actin filaments via immunofluorescence. Likewise, the aberrant proliferation commonly seen in these pathologies can be monitored through a cell cycle marker such as Ki67, or one of de novo proliferation such as EdU. Finally, levels of elastin deposition and fiber formation can be assessed using a high-throughput immunofluorescence approach in the absence of cell permeabilization, so as to exclusively visualize extracellular elastin.

In addition, functionality of drug-treated iPSC-VSMCs can be assessed using the 3D vascular tissue rings [31] and TEBVs [37, 53]. Through measurements of stress versus strain, vessel burst pressure, and ECM tension, phenotypically rescued VSMCs can be validated at the functional level, an essential prerequisite for clinical application. Furthermore, the 3D vascular tissue ring format, once generated under automation, could itself be applied directly as a high-throughput drug screening platform with the above assays.

Elastin fiber production in iPSCs

An obstacle in the field of iPSC modeling of elastin-associated vasculopathy has been the effective development of elastin fibers in vitro. Such development is essential for the accurate study of disease mechanisms as well as in multi-dimensional models of disease structures. This remains an active area of investigation throughout the elastin-associated vasculopathy field, and several methods have been implemented to improve extracellular deposition of elastin and subsequent fiber formation in in vitro and in vivo systems. Employing a coculture system of bovine VSMCs with human dermal fibroblasts has been shown to enhance elastin deposition in fibrin gel-based engineered vascular grafts [35]. Antagonism of miR-29a in vitro increased extracellular elastin deposits in both elastin haploinsufficient models and three-dimensional constructs [38]. In engineered artery models, use of biaxial—circumferential and longitudinal—stretching during growth improved mechanical properties of the arteries as well as maturation of elastin fibers [54, 55]. In models of pulmonary arterial hypertension, another elastin-associated vasculopathy, elastin fiber assembly appeared dependent on signaling by bone morphogenetic protein receptor 2 and by TGF-β, and manipulation of these signaling pathways was shown to increase fiber formation [56]. Additional extracellular matrix proteins to those involved in elastin fibers have been implicated as negatively correlated with elastin protein formation. These include the proteoglycan decorin [56] and the chondroitin sulfate proteoglycan versican [57]. Furthermore, exposure of dermal fibroblast cells to the polyphenol tannic acid similarly displayed enhanced secretion and stability of elastin fibers in vitro [58]. Finally, a cell-free model leveraging a fast-degrading elastomer has shown to activate host vascular remodeling mechanisms which could similarly improve elastin fiber maturation [59]. Due to the availability and expandability of iPSCs with which to study disease pathologies, application of these approaches to augment elastin fiber production in vitro is much more feasible than with the gold standard approaches. As such, techniques can readily be employed in 2D and 3D platforms for disease mechanistic studies as well as tissue engineering.

Efficient endogenous expression of elastin polymers in TEBVs provides a way to couple these techniques in a physiologically relevant setting for tissue engineering and future drug screening. The overexpression of elastin cDNA through a doxycycline-inducible promoter, optimization of culture conditions to include pro-elastin factors like TGF-β, IGF, and retinoic acid, and the application of biaxial stretching are all potential methods of enhance expression [53, 55, 60], while implementation of a miR-29a inhibitor during the growth process of TEBVs displayed enhanced deposition of elastin, improving vessel compliance [38].

How is the post-transcriptional regulation of elastin controlled so as to produce the appropriate balance of elastin isoforms?

The production of elastin by VSMCs is regulated at the transcriptional level, the post-transcriptional level, and at the post-translational level. Gold standard approaches using primary VSMCs have been informative at the transcriptional regulatory level [61–64] and at the post-translational level as detailed above. Post-transcriptionally, gold standard approaches have been less informative, as at least 11 different splice isoforms of tropoelastin are produced, increasing the difficulty of molecular biology approaches. As the ratio of tropoelastin isoforms synthesized changes through the fetal, neonatal, and adult periods, isoform expression likely plays an essential role in proper assembly of elastin fibers [4]. Indeed, it has been suggested that the cessation of continued production of tropoelastin protein is under post-transcriptional regulation, rather than controlled at the transcriptional level [5]. It is known that tropoelastin mRNA stability can be affected by transforming growth factor beta (TGF-β) activity and by vitamin D3 [65–67]. More directly, microRNA-29a (miR-29a) has been shown to lead to degradation of tropoelastin mRNA [38], while inhibition of miR-29a in human cells was shown to increase the expression of elastin and upregulate protein levels in elastin haploinsufficient patients, though a precise mechanism remains unclear.

It would be useful, then, to assess the role of elastin splice variant expression in the context of vascular pathologies. This is much more feasibly performed in an iPSC platform than with primary cells, due to the ability to readily obtain large quantities of patient-specific cells to perform the relevant molecular biology experiments on, or with an animal model where cross-species differences and inherent differences in cardiovascular physiology could confound results. Differential isoform expression in these pathological models can be assessed using RNA-sequencing and exon arrays, which have previously been implemented to quantify myoblast isoform switching during cellular differentiation [68]. Notably, as miR-29a antagonism has been shown to upregulate elastin protein expression, analysis of the 3′-UTR landscape of elastin isoform mRNA, and specifically an analysis of alternative polyadenylation of this region and 3′UTR length, indicators of potential binding sites for miRNA, could provide highly informative toward addressing this question [69–71]. Both detailed descriptions of experimental designs of this type, and more general commentary on the implications of tissue specific of temporal-dependent expression of mRNA isoforms have been discussed elsewhere [70–75]. Moreover, use of both 3D vascular tissue ring and TEBV formats will allow the study of elastin isoform expression in vitro under more physiological conditions. Though still a complex problem, the iPSC platform provides a robust foundation of patient-specific cells to readily perform isoform specific overexpression or antagonism to begin to address the role differential isoform expression may play in vascular elastinopathies.

How does the lineage specificity of VSMCs alter the signal transduction process?

The VSMCs which comprise the aorta arise from different developmental lineages. In the aortic segments proximal to the heart, i.e., the ascending aorta, these neural crest-derived VSMCs are the predominant, and essentially exclusive, type of VSMC, while the more distal regions of the same arteries are of the paraxial mesoderm, and the aortic root is derived from the lateral plate mesoderm [39]. Importantly, in the elastin-associated vasculopathies SVAS and thoracic aortic aneurysm and dissection, the aberrant proliferation of vascular smooth muscle cells is restricted to the proximal, neural crest-derived VSMCs, indicating this subtype may undergo unique signal transduction relative to the mesenchymal VSMCs [48].

The importance of lineage specificity for VSMCs has been discussed as essential for studying subtypes of VSMC pathologies such as SVAS [11] and Marfan syndrome [30], though the gold standard approaches are limited in this area. Primary VSMCs of each unique subtype would be difficult to sufficiently acquire, and would be complicated by the loss of the VSMC phenotype in culture. Though mouse models can aid in visualizing more global physiologic effects on the arterial regions arising from differing lineage, an in-depth mechanistic analysis of signaling pathways is not feasible with this model either. The iPSC platform, however, is highly suited to address the role or importance of lineage specificity on vascular pathology as pluripotent, patient-specific, progenitors can be directed to differentiate into VSMCs arising from varying lineage. Indeed, robust, detailed protocols have been developed for differentiating vast quantities of neural crest, lateral plate mesoderm, and paraxial mesoderm-derived VSMCs for research purposes [29, 30, 76]. The iPSC model thus takes the advantages provided by an in vitro platform to investigate the role of lineage specificity on signal transduction, without the limitations of finite accessibility and expandability. Further, 3D iPSC-derived tissue culture rings or TEBVs could be created using lineage-specific VSMCs from patients with VSMC proliferation pathologies as a useful and accessible platform for drug screening in addition to a mechanistic study, following the parameters discussed above.

Conclusion

Aberrant proliferation of VSMCs is associated with an array of progressive disease phenotypes and can result in stenosis, aneurysm, dissection, heart failure, or atherosclerosis. These conditions have no available and effective current therapy outside of a heart transplant or vessel bypass, though insights into the underlying causes are under active investigation. Inappropriate biomechanical sensing in the arterial wall can lead to maladaptation pathways being activated, leading to an abnormal state of VSMC proliferation. As the principal effector of arterial compliance in the extracellular matrix, a likely candidate is the elastin polymer, or its substituent adaptor proteins fibrillin-1 and fibulin-5. Indeed, mutations in these proteins have been found to be in strong correlation with VSMC proliferation disorders [11, 23, 26, 30, 49]. To effectively study VSMC proliferation, an appropriate model system must be developed to address deficiencies in the field. Though primary cell cultures and mouse modeling have provided useful insights previously, implementation of induced pluripotent stem cell biology is a valuable and highly complementary approach. iPSC technology can be used to acquire patient-specific VSMCs in a minimally invasive manner, create a source of self-renewing cells which can be differentiated into lineage specific VSMCs, and model VSMC biology in a two-dimensional environment for detailed mechanistic studies, a three-dimensional environment for studying cellular network interactions and as a platform for drug screening, and through TEBVs for future clinical applications.

Abbreviation

VSMC

Vascular smooth muscle cell