Heterogeneity in vascular smooth muscle cell embryonic origin in relation to adult structure, physiology, and disease

Abstract

Regional differences in vascular physiology and disease response exist throughout the vascular tree. While these differences in physiology and disease correspond to regional vascular environmental conditions, there is also compelling evidence that the embryonic origins of the smooth muscle inherent to the vessels may play a role. Here we review what is known regarding the role of embryonic origin of vascular smooth muscle cells during vascular development. The focus of this review is to highlight the heterogeneity in the origins of vascular smooth muscle cells and the resulting regional physiologies of the vessels. Our goal is to stimulate future investigation into this area and provide a better understanding of vascular organogenesis and disease.

Introduction

Life requires a constant ebb and flow of nutrients and waste to and from cells. Unicellular organisms can simply take in nutrients and release cellular wastes across the phospholipid membrane. With the evolution of complex multi-cellular organisms, a simple absorptive/secretive interaction with the environment is often insufficient to facilitate basic metabolic needs. These needs become particularly apparent during development. A developing vertebrate embryo can only subsist for a finite time without a vasculature. One of the earliest vertebrate organ systems to develop is the vascular system to allow for continued rapid maturation of complex multicellular organisms. Failure of the cardiovascular system to develop results in early termination of the embryo.

While continual maturation occurs postnatally, the majority of patterning for the cardiovascular system is established before and during the prenatal period. Multiple lines of evidence support the hypothesis that embryonic origin plays an important role in vascular biology. In reviewing the genesis and structure of the vascular system, we will highlight the importance of understanding the implications of the origin and differentiation of these cellular elements on the physiology of an adult organ system.

Current surgical treatments of cardiovascular ailments are relatively crude. Surgical interventions repair structural problems but little or no thought is given to replicating the underlying cellular characteristics of the vessel in the mended region. When artificial vascular grafts are used, the grafts, while tubular, often fail to replicate specialized regional properties inherent to the vessel. While these interventions work to fix the problem at hand, underlying biological issues have not been addressed. By understanding the implications of development and underlying biology of the cardiovascular system we suggest that better-informed strategies will formulate better interventions for the treatment of cardiovascular diseases.

Vascular Structure

The cardiovascular system is generally organized in three basic layers: an outer stromal or connective tissue-rich layer, a middle muscular layer, and an inner single cell protective layer. In the vasculature, these layers are referred to as the tunica adventitia, tunica media, and tunica intima. Like the epicardium, the adventitia is a connective tissue-rich protective layer. In the largest vessels such as the aorta, the adventitia contains the vasa vasorum which is the source of oxygenated blood for the media of these large vessels (Kawabe and Hasebe, 2014). While rich in connective tissue, the adventitia of the major vessels also contains a Sca1⁺ cell population capable of giving rise to VSMCs (Passman et al., 2008). While the embryonic origin of these cells is unknown, they appear in the perivascular space between the aortic arch and pulmonary trunk between 15.5 and 18.5 days post coitum (dpc) in mice. The adventitia has a critical role in vascular maintenance and repair.

The tunica media is the main contractile layer of the vascular system containing vascular smooth muscle cells (VSMCs) and extracellular matrix that is arranged in concentric layers of muscle alternating with elastin or matrix. In the smallest of vessels including specific capillary and post-capillary venules, VSMCs are replaced with pericytes. These cells have characteristics of both smooth muscle and connective tissue cells but their lineal relationship to either population is not currently understood. The VSMCs of the media provide tonic contractions that allow for the perpetuation of blood flow (Owens et al., 2004). Those same VSMCs deposit the elastin and collagen contributing to the extracellular matrix of the media (Owens et al., 2004). This extracellular matrix provides the elasticity critical to proper vascular function (Rosenquist et al., 1990; Ruckman et al., 1994; Thieszen et al., 1996).

VSMCs are a very plastic population of cells with the ability to modulate their phenotype based on environmental cues (Owens et al., 2004). Depending on these cues, VSMCs can exhibit a differentiated contractile phenotype or a synthetic proliferative phenotype. These phenotypic switching cues can come from within the tunica media, from cell to cell contact, or from autocrine factors (Owens, 1995). The tunica intima as well as the adventitia can also have a powerful impact on the phenotype switching of VSMCs. Growth factors, contractile agonists, NO, and modulation of vascular permeability all influence phenotype switching. The VSMCs also integrate mechanical signals from the surrounding tissue and extracellular matrix when establishing phenotypic plasticity (Owens, 1995). The contractile and synthetic phenotypes are extremes on either end of a spectrum. VSMCs fluidly exist anywhere along the continuum of this spectrum. The embryonic origins of VSMCs varies based on the vessel in question and will be further examined in the following section.

While there is no definitive marker for VSMCs, these cells do express different markers depending on their differentiation or phenotypic state. Alpha-smooth muscle actin (αSMA) is an early marker of VSMC commitment (Owens, 1995). It is also expressed during development by other myogenic lineages. SM22 is a marker of early differentiated VSMCs but it is also widely expressed in other developing myogenic lineages (Li et al., 1996). Smooth muscle myosin heavy chain (smMHC) is a marker of mature contractile VSMCs (Owens, 1995; Manabe and Owens, 2001). Calponin is a member of the calmodulin family of proteins and is another late stage marker of VSMCs (Gimona et al., 1990). All of these markers are highly expressed in contractile VSMCs but are down-regulated in synthetic VSMCs (Owens, 1995; Owens et al., 2004). An isoform of non-muscular myosin heavy chain, NM-B MHC, is highly expressed in embryonic VSMCs but has also been identified as one of the best markers of synthetic VSMCs (Kuro-o et al., 1991; Rensen et al., 2007). An array of molecular markers must be used to identify VSMCs from other mesenchymal cells. Identification of regionally specific VSMC markers would further facilitate in vivo and in vitro studies of VSMC characteristics.

The intimal layer is a single cell layer of endothelial cells that can modulate the activity of the VSMCs based on interactions with flowing blood. The endothelial cells of the intima sit on a basal lamina with an internal elastic lamina, which together provides both stability and flexibility to the endothelial layer. Since they are in direct contact with the lumen of the vessel, endothelial cells are poised to play a critical role in tissue homeostasis (Chien et al., 1998; Cines et al., 1998; Traub and Berk, 1998; Arnal et al., 1999; Andersen and Stender, 2000; Baldwin and Thurston, 2001; Woo et al., 2011; Bazigou and Makinen, 2013). There are known regional variations in endothelial cell function (Petzelbauer et al., 1993; Aird, 2007a; Aird, 2007b; Yano et al., 2007; Zhang et al., 2008; Burridge and Friedman, 2010; Aird, 2012), however it is not clear to what extent these are due to regional differences in endothelial cell embryonic origin or the overlying VSMC population.

Vascular Development

In mammals, the development of a healthy cardiovascular system depends upon the recruitment and coordinated differentiation of cells from diverse embryonic origins. Vasculogenesis begins with specification of angioblasts (Vokes and Krieg, 2002; Sato et al., 2008; Ren et al., 2010). Derived from the mesoderm, angioblasts form an endothelial plexus throughout the developing embryo and the yolk sac (Schmidt et al., 2007). The vasculature begins forming by the condensation of this endothelial plexus. Cell-cell junctions form and endothelial cells rearrange to generate patent lumens (Houser et al., 1961; Jin et al., 2005). These primitive vessels remodel, branch, and recruit mural cells (fibroblasts and VSMCs).

VSMC progenitors arise from distinct embryonic sources including splanchnic mesoderm (Wilm et al., 2005), somitic mesoderm (Esner et al., 2006; Wasteson et al., 2008), neural crest (NC) (Le Lievre and Le Douarin, 1975; Jiang et al., 2000), mesothelia, (Que et al., 2008) and other embryonic cell types (Gittenberger-de Groot et al., 1999; Rinkevich et al., 2012). While the embryonic origins of many VSMC populations is known, the exact nature of the VSMC precursor remains elusive. A Flk1-postive cell population is capable of producing both endothelial and VSM cell populations in vitro and contributed to the vascular network when injected into chick embryos (Yamashita et al., 2000). Follow-up studies looking at the developmental role of Flk-1 found that Flk-1 lineage cells give rise to endothelial cells, cardiac muscle, and skeletal muscle, but found no evidence of a lineage label in smooth muscle cells (Motoike et al., 2003). It is therefore possible that subpopulations within the mesoderm possess the ability to differentiate into VSMC however the exact pathways involved remain unknown.

In the aorta, splanchnic mesodermal cells are first recruited and differentiate into VSMCs. Before the splanchnic mesoderm cells completely encircle the dorsal aorta, the cells are displaced by somitic mesodermal cells. Differentiation of these somitic mesodermal cells begins in the ventral anterior end of the vessel. Differentiation then proceeds around the circumference of the vessel (ventral to dorsal), and down the length of the aorta toward the diaphragm (anterior to posterior).

Meanwhile, the outflow tract of the aorta begins as the aortic sac feeding a series of six paired pharyngeal arch arteries surrounded by the pharyngeal arch mesoderm. The cardiac NC migrates down the pharyngeal arches to invade the aortic sac. A subset of the cardiac NC participates in septation of the truncus arteriosus into the aortic arch and the pulmonary trunk (Kirby, Gale et al. 1983). The rest of the cardiac NC remains in the pharyngeal arch arteries and becomes the VSMCs of the aortic arch and the arteries of the head and neck (Le Lievre and Le Douarin, 1975). The border that forms between the NC-derived VSMCs of the ascending aortic arch (aAo) and mesoderm-derived VSMCs of the descending aorta (dAo) is maintained throughout development and into adulthood (Le Lievre and Le Douarin, 1975; Jiang et al., 2000). Arch arteries one, two and five bilaterally resorb while arch artery three become sections of the vessels of the head and neck. Arch artery four on the left unilaterally becomes the aortic arch while the right side resorbs. Arch artery six contributes to the pulmonary trunk and ductus arteriosus.

Once cells encircle the aorta and differentiate into VSMCs, they undergo a closely regulated process of layer formation within the media. Different regions of the vasculature consistently form different numbers of layers in the media. Lumen diameter is the best predictor for the number of layers that exist in the media (Wolinsky and Glagov, 1964; Wolinsky and Glagov, 1967). However, studies of transgenic mice that manipulate animal’s mass or vascular matrix components demonstrate that there are regional mechanisms that can compensate for increased vascular demands without changing the number of vascular smooth muscle layers (Dilley and Schwartz, 1989; Faury et al., 2003; Wagenseil and Mecham, 2009; Hoglund and Majesky, 2012).

Much of the initial patterning of the vasculature occurs rapidly and before the heart even begins beating. Once the heart beats, perfusing vessels with blood, the vasculature begins growing and remodeling through angiogenesis (Blatnik et al., 2005; Chen and Tzima, 2009; Culver and Dickinson, 2010). Hemodynamic force is one of the most powerful forces in vascular remodeling and is required for complete vascular and embryonic development.

Heterogeneity of Vascular Physiology and Disease

Heterogeneity of blood vessels is critical to cardiovascular function. Different regions of the vasculature require different physical properties in order to meet distinct physiological requirements. The outflow tract of the heart is an elastic artery that contains multiple elastic lamina and smooth muscle cell layers able to withstand the pulsatile force produced by the beating heart (Shirwany and Zou, 2010). While the descending aorta is also considered an elastic artery, the extracellular components differ greatly from the ascending aorta and the blood velocity has decreased substantially compared to the aortic arch (Gadson et al., 1993; Ruckman et al., 1994). As the blood moves further out in the vasculature tree, the vessels become more muscular and contain fewer elastic lamina (Figure 2). These vessels must maintain vascular tone in order to deliver blood to their target tissues and organs (Shirwany and Zou, 2010). The capillary bed contains pericytes that function to regulate capillary blood flow and permeability. The venous system, while patterned similarly to the arteriole system, has distinct differences. In its return to the heart, blood relies on a series of valves in the veins to keep moving against gravity and at a lower pressure than in the arteries, particularly in the limbs (Meissner et al., 2007; Bazigou and Makinen, 2013). While there are pericyte cells associated with veins, they do not play a significant contractile role as they do in muscular arteries. Rather, veins rely in part on skeletal muscle contractions to act like peripheral muscle pumps to return blood to the heart (Alimi et al., 1994). There is a large amount of variation in structure and function along the vascular tree.

Figure 2.

Structural differences between elastic and muscular arteries

The media of elastic arteries, such as the aorta, consist of multiple organized layers of VSMCs interspersed with layers of elastin. Muscular arteries, such as the mesenteric artery, have fewer elastic lamina within the media.

Differences in vascular structure are also reflected within the three lamina. For example, regional variation in endothelial cells of the tunica intima regulates vessel permeability (Katora and Hollis, 1976), while great variability in connective tissue elements has been noted along the entirety of the vascular tree (Gadson et al., 1993; Ruckman et al., 1994). Significant heterogeneity in structure and function has been noted for the tunica media (van Meurs-van Woezik et al., 1983; Stenmark and Mecham, 1997), whose major cellular components are VSMCs or pericytes. In the large conducting arteries of the cardiac outflow tract, this layer contains significant amounts of elastin contributing to the distensible properties that are essential for reacting to blood volume and pressure changes during the cardiac cycle (Fawcett et al., 1994). Conversely, medium-sized muscular arteries (e.g. coronary and mesenteric vessels) contain less elastin but are enriched in VSMCs critical for regulation of vessel diameter and distribution of blood to specific tissues of the body. Capillaries consist of an endothelium wrapped by a single pericyte. In addition to differences in their basic morphology, these vessels have regionally distinct expression of matrix and signaling pathway components often linked to the tunica media (Subbiah et al., 1981; Flaim et al., 1985; Sufka et al., 1990; Gadson et al., 1993; Ruckman et al., 1994; Thieszen et al., 1996; Gadson et al., 1997; Andersen and Stender, 2000; Ko et al., 2001; Leroux-Berger et al., 2011; Cheung et al., 2012; Reslan et al., 2013; Trigueros-Motos et al., 2013).

Not only do the physiologies of these vascular regions differ, but the regions of the vasculature are differentially susceptible to vascular disease. First, atherosclerosis does not plague the entire vasculature equally. Branch points and places with higher turbulent flow, including the aortic arch, develop plaques more frequently than other regions (VanderLaan et al., 2004; Van Assche et al., 2011). Even when these atherosclerotic-prone vessels are transplanted into a region that does not develop plaques, atherosclerosis still occurs (Haimovici and Maier, 1971). Secondly, under calcifying conditions similar to those seen in cases of kidney failure, the ascending aorta calcifies much more rapidly compared to the descending aorta (Leroux-Berger et al., 2011). In fact, VSMCs isolated from the ascending aorta retain this propensity to calcify, linking this condition to VSMC biology and possibly reflective of their embryonic origin. These data support our hypothesis that VSMCs from different regions of the vasculature are intrinsically different and have the potential to contribute to disease.

This begs the question, “what determines these differences in physiological and pathological states?” These characteristics could be dependent on environmental factors or embryonic origins of the cells that constitute the vessels. We propose that a combination of vascular environment and embryonic origin account for regional variation in physiology and disease in the cardiovascular system (Figure 3).

Figure 3. Both vascular environment and embryonic origin of the vascular components play a role in establishing regionally specific vascular physiology and disease.

There is abundant evidence that embryonic origin of VSCMs plays an important role in vascular biology. Differences in VSMC characteristics such as gene expression have been attributed to their embryonic origin (Zhang et al., 2012). Studies also suggest that when vessels prone to atherosclerosis are placed in a vascular region that does not typically develop atherosclerosis, they retain their predisposition to disease (Haimovici and Maier, 1971). Additionally, differentiation of VSMCs from embryonic stem cells through NC- or mesoderm-lineages provide evidence that VSMC characteristics are programmed based largely on embryonic origin (Cheung et al., 2012). The proximal aorta is particularly interesting because its VSMCs arise from two distinct embryonic origins: NC and somitic mesoderm (Jiang et al., 2000; Wasteson et al., 2008). This juxtaposition of VSMCs from different embryonic origins in the aorta affords the opportunity to test the hypothesis that embryonic origin of VSMCs relates to the vascular function of distinct regions within a single vessel. While distinct differences have been described in the regions of the embryonic aorta, these differences converge in the adult aorta (Pfaltzgraff et al., 2014).

These studies suggest that individual VSMC characteristics are strongly determined by embryonic origin. However, definitive evidence that embryonic origin dictates vascular phenotype has not been provided (Gittenberger-de Groot et al., 1999).

Congenital cardiovascular defects also occur at the boundaries between vascular regions of different embryonic origins. Two examples of these conditions are coarctation of the aorta and interruption of the aortic arch (Jacobs et al., 1995; Tanous et al., 2009). Building a better understanding of the embryonic origin and patterning of the vasculature could lead to greater comprehension of how to prevent or effectively repair these conditions. Current surgical means of repair, while correcting the structural defect, fail to address the underlying patterning problems. Many patients who have had coarctation repairs as older children or adults develop hypertension without re-coarctation suggesting that there is an underlying cause of the structural defect has not been addressed by surgery (O'Sullivan et al., 2002). This is additional evidence that regional differences in vascular biology need to be understood to be able to properly address vascular diseases.

Future Directions

Role of embryonic origin and environment in VSMC biology

By exploring the molecular and physiological characteristics of the regions of the aorta, we have been able to describe the convergence of regional embryonic vascular characteristics in the adult. What is still unclear, however, is the process through which this convergence occurs. Is there a single stimulus that triggers convergence or is it a gradual postnatal change? Some possible environmental triggers for this convergence are changes in oxygen tension and the closing of the ductus arteriosus.

Further studies examining different vessels with VSMCs from the same embryonic origin would give insight both into environmental influences and the role of sub-regional embryonic origin. Some such studies have been performed comparing two vessels each with NC VSMC origin, the ductus arteriosus and the aAo (Shelton et al., 2014). Subdivisions within a single embryonic origin could have very different vascular phenotypes. For example, Hox codes could provide additional position information about vascular origin. In fact, Hox expression has been directly linked to vascular phenotypes (Pruett et al., 2008; Pruett et al., 2012; Trigueros-Motos et al., 2013). While we have demonstrated a regional embryonic phenotype that correlates with embryonic origin, perhaps subdomains of the aorta can be identified based on a combination of Hox code expression as well as embryonic origins.

Impact of VSMC origins on endothelial cell function

One of the major questions that remains unanswered is to what extent VSMC origin influences the biology of the underlying endothelial cells. We can image a number of different assays which might shed light in the area. In vitro matrix culture of endothelial cells is known to result in tube formation and is used as an assay of angiogenesis (Ponce, 2009). Perhaps co-culture of endothelial cells with VSMCs from different embryonic origins would produce differences in the angiogenic properties of the endothelial cells. Another similar assay would be to use a microfluidic device that would allow for the co-culture of VSMCs and endothelial cells under flow conditions in vitro. These simplified vessels could then be exposed to different flow and pulsatile conditions and assayed to determine any influence of the VSMC population on endothelial nitric oxide activity. Finally, a more definitive study would be to injury a vessel in vivo, and transplant various VMSCs from diverse origins over the injured vessel (Shelton and Bader, 2012). In this way, the media of the vessel would repopulate with the transplanted VSMCs and the vessel could later be harvested and studied for endothelial physiology, morphology, and molecular signatures.

Role of embryonic VSMC phenotypes on vascular physiology and disease

If embryonic origin has consequences for physiology and disease in the adult, then we might expect to see some embryonic characteristics reemerge in diseased adult vessels. One possible direction would be to describe the vascular characteristics of the different regions of the aorta from an apoE^−/− mouse (Meir and Leitersdorf, 2004). The apoE^−/− mouse readily develops atherosclerosis and could be used as a model of vascular disease to test if regions of the aorta take on their embryonic characteristics under vascular stress. One study focusing on the role of Hox codes in atherosclerosis heterogeneity found that HoxA9 plays an important role in establishing athero-resistance in the dAo (Trigueros-Motos et al., 2013). While this study focused on genes that did not change between WT and apoE^−/− conditions, it would be of interest to determine whether the embryonic expression pattern reemerges in times of vascular stress. Another example of a genetic model for vascular stress would be the diabetes-prone, leptin-deficient (ob/ob) mouse model (Drel et al., 2006). Leptin-deficient mice provide a model for diabetes and obesity, which causes vascular stress. Based on the hypothesis that the embryonic profile reemerges in stressed or diseased tissue, one would predict that the aAo and dAo would have distinct characteristics in an adult suffering from vascular stress.

Summary

The cardiovascular system is a carefully orchestrated mosaic of cell types derived from diverse developmental origins. It is not hard to imagine then, how embryonic origin could play a large role in physiology and disease in the adult. A better understanding of the similarities and differences between these different vascular regions can help us understand how to prevent and treat these vascular diseases in a more effective and efficient manner that actually addresses the underlying biology of the system.

Figure 1. Basic structure of the cardiovascular system consists of three layers.

In the vasculature, the first layer is the tunica intima (e, blue). This layer is adjacent to the lumen of the vessel and contains endothelial cells. The middle layer is the tunica media (m, red). This layer consists of VSMCs and extra-cellular matrix. The outer layer is the tunica adventitia and is composed of matrix, fibroblasts, and vascular progenitor cells (a, green).