Vascular Smooth Muscle Cell Development and Cardiovascular Malformations : Cardiology Discovery

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Vascular Smooth Muscle Cell Development and Cardiovascular Malformations

Qi, Lihua1; Kong, Wei2,3,4; Fu, Yi2,3,4,∗

Editor(s): Fu, Xiaoxia; Xu, Tianyu

Author Information
Cardiology Discovery 1(4):p 259-268, December 2021. | DOI: 10.1097/CD9.0000000000000035
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The cardiovascular system is the earliest system formed in the embryo and it plays a critical role in vertebrate development and homeostasis.[1,2] The progenitors of smooth muscle cells (SMCs) that form the vascular system during embryogenesis have a wide variety of lineages, including the neural crest (NC), secondary heart field (SHF), somatic, and splanchnic mesoderm.[3] Defects in patterning and morphogenesis related to the various vascular developmental programs can give rise to various congenital cardiovascular diseases.[4–8] Similarly, SMC-specific gene knockout in mice leads to various disruptions and cardiovascular defects, further demonstrating the key role of SMCs in cardiovascular development. Elucidating the mechanisms underlying vascular SMC (VSMC) development will not only advance our knowledge of vascular wall biology but also help to identify genes that cause vascular wall diseases and congenital vascular defects when they are mutated in humans.

In conditions such as arteriosclerosis and neointimal hyperplasia, intimal SMC aggregation is a key event.[2] The long-standing dogma is that the majority of the SMCs in the tunica intima come from the tunica media, and once SMCs in the tunica media switch from a contractile state to a secretory state, they become migratory in nature and travel to the tunica intima, where they proliferate and form neointimal lesions.[2] However, this view has been questioned by Holifield et al[9] who put forward the idea that the mature SMCs in the tunica media of injured carotid arteries in dogs do not undergo phenotypic alterations. In other words, VSMC phenotypic alterations alone do not fully explain the diversity of SMC responses to common risk factors that lead to vessel injury and remodeling.

Recent evidence suggests that SMCs in different regions of the vascular system originate from different progenitors. This may explain the varied biological behavior of SMCs, which may directly contribute to region-specific vascular diseases. Hence, to better understand the diversities of VSMCs, we must trace their lineages back to their progenitor cells. The aim of this study was to review the diverse progenitor cells of VSMCs and reveal the roles of the diverse progenitor cells in cardiovascular development, abnormal vascular smooth muscle development, and the associated cardiovascular malformations.

A lineage perspective of VSMCs development

Vascular system development involves 2 distinct mechanisms, namely, vasculogenesis and angiogenesis.[10] Vasculogenesis occurs when mesoderm-derived angioblasts differentiate to form the primary vascular endothelial plexus. Angiogenesis, on the other hand, is the formation of a secondary vascular network that buds off from an existing vessel.[10] Recruitment of a variety of progenitors that differentiate into VSMCs is crucial for angiogenesis. Distinct vessels, or even distinct regions of the same vessel, consist of SMC populations of diverse origins, each with its unique lineage and developmental history.[11] Hence, the vascular smooth muscle is regarded as a mosaic tissue. This mosaic pattern makes the SMC differentiation programs complex. We discuss all aspects of SMC differentiation in detail in the sections below [Figure 1].

Figure 1:
A lineage perspective of vascular smooth muscle development. Different color represents different embryonic origins for SMCs as indicated in the key. The NC (yellow) contributes SMCs to the ascending and arch portions of the aorta, the ductus arteriosus (higher magnification of the ductus arteriosus indicated in the circle), the innominate, and the right and left common carotid arteries. The SHF (blue) gives rise to the SMCs in the base of the aorta and pulmonary tract. Both NC and SHF contribute to the wall of the pulmonary trunk and left/right pulmonary artery (white). The somites cells (red) built up the wall of the descending thoracic artery. The proepicardium (wathet blue) contributes to SMCs in the coronary artery. Pleural mesothelial cells (brown) undergo mesothelial-to-mesenchymal transition and differentiate into SMCs in lung vessels. Splanchnic mesoderm cells (green) give rise to SMCs of the abdominal aorta. Nephrogenic stromal cells (purple) give rise to SMCs of the renal vessel. Peritoneal mesothelial cells (orange) undergo mesothelial-to-mesenchymal transition and differentiate into SMCs in gut vessels. SMC: Smooth muscle cell.

Lineage tracing analysis of vertebrate embryos has revealed that VSMCs originate from a minimum of 7 discrete progenitor cells.[12] Among them, SHF- and cardiac NC-derived SMCs play major essential roles in vessel generation. The cardiac NC contributes to SMCs that end up in the aortic arch, ascending aorta, ductus arteriosus, right subclavian, brachiocephalic, and right and left common carotid arteries.[6] The SHF-derived progenitor cells migrate to the aortic sac and contribute to SMCs in the aortic base and pulmonary trunk.[13] Additionally, pro-epicardium-derived cells form the VSMCs in the coronary artery.[14] Somites are responsible for the VSMCs in the descending thoracic aorta.[15] Mesothelial cells form VSMCs in the gut and lung vessels via mesothelial-to-mesenchymal transition.[16] Nephrogenic stromal cells, derived from the metanephric mesenchyme, develop into the VSMCs of the renal vessels.[17] Embryonic stem cells also play an irreplaceable role in smooth muscle development.[18] Lastly, there is some controversy regarding whether endothelial cells can transdifferentiate into VSMCs. In an emerging report, the endothelium was shown to form hematopoietic stem cells, which in turn, differentiate to form SMCs and embryonic endothelium, while Azzoni et al[19] demonstrated that the early extra-embryonic endothelium can develop into mesoangioblasts expressing SMC markers. Even more interestingly, a recent single-cell RNA sequencing analysis of the developing cardiac outflow tract (OFT) revealed that the VSMCs at the base of the great arteries exhibited convergent development, with intermediate cell subpopulations undergoing either mesenchymal-to-VSMC transition or myocardial-to-VSMC transdifferentiation.[20] This study has provided great insight into the development of VSMCs and further reflected the obvious heterogeneity of the origins of VSMCs.

Cardiac NC derived SMCs

The NC is a strip of cells that lines the dorsal region of the neural tube in vertebrate embryos. It is divided into the cranial NC, which facilitates craniofacial and cranial ganglia development, and the trunk NC, which aids in the formation of the peripheral nervous system.[6] The caudal region of cranial NC, which lies between the level of the midotic placode to the third somite is termed cardiac NC and is crucial for cardiovascular development. Cardiac NC cells migrate collectively or individually along specific routes and eventually differentiate into incredibly multiple arrays of cell types, including VSMCs.[21–23] In the vascular system, cardiac NC cells have 2 identifiable fates. Some cardiac NC cells, around the pharyngeal arch arteries (PAAs), differentiate into VSMCs that make up the aortic arch, ascending aorta, ductus arteriosus, and brachiocephalic and right and left common carotid arteries.[6] The remaining cardiac NC cells migrate to the OFT and aid in the development of the aorta and pulmonary trunk septation.[6] The fate and role of the cardiac NC are particularly well established in various animal models. For instance, Jiang et al[24] discovered that the cardiac NC cell in mice supplies SMCs for the aortic arch, ascending aorta, ductus arteriosus, right subclavian, brachiocephalic, and right and left common carotid arteries. Bergwerff et al,[25] on the other hand, found that NC cells in quail-chick chimeras provide SMCs for the branchial arch arteries.

There are 2 potential paths: dorsolateral and ventromedial.[25] The dorsolateral path is the path used in the abovementioned cases. It remains unclear whether the NC cells originating from the ventral part of the neural tube give rise to SMCs. Ali et al[26] reported that a multi-potential cell population originating from the ventral region of the neural tube can migrate to the craniofacial mesenchyme to form SMCs of the craniofacial arteries and great vessels. However, Boot et al[27,28] reported that the ventral neural tube-derived cells do not contribute to the heart and OFT system in chick-quail chimeras. Nevertheless, Bergwerff et al[25] challenged the view of Boot et al, reporting that the ventral NC cells, although not required for aortic arch development, are crucial for re-patterning and formation of the smooth muscle of the arterial tunica media. Furthermore, ventral NC cells were reported to migrate to the pharyngeal arch to differentiate into SMCs of the PAAs.[29] However, whether the ventral NC cells are involved in the development of SMCs requires experimental verification.

SHF-derived SMCs

Although cardiac NC cell-derived SMCs participate in the formation of the ascending aorta, this is not the case in the aortic base or pulmonary trunk. The SMCs in these locations are of unknown origin. The SHF is located in the splanchnic mesoderm below the foregut floor. Research initially suggested that the SHF-derived cells only provide myocardium to the cardiac arterial pole, thereby elongating the OFT.[30] However, this view was challenged by findings that SHF-derived SMCs participate in the development of the pulmonary trunk and aorta in OFT.[13] Subsequently, several mouse studies confirmed that the aortic base and pulmonary trunk require the SHF. Waldo et al[30] labeled SHF-derived cells by lineage tracer dye microinjection and traced the fate of the labeled cells. They discovered that the SHF-derived cells followed 2 unique migration paths to the cardiac arterial pole. One population traveled to the OFT and formed cardiomyocytes, whereas the other population migrated to the aortic sac to form SMCs that make up the aortic base and pulmonary trunk. Similarly, Hu et al[31] showed that SHF-derived cells differentiated into VSMCs at the base of the pulmonary trunk and ascending aorta.

The mammalian vascular formation is a complex dynamic process. Complex interactions among numerous lineages are required to achieve VSMC development. For instance, cardiac NC- and SHF-derived SMCs can simultaneously contribute to the tunica media of the ascending aorta. Cardiac NC-derived cells tend to populate the tunica media of the posterior region and the inner medial aspect of the anterior region of the ascending aorta. SHF-derived cells tend to populate the outer medial aspect of the posterior and anterior regions of the ascending aorta. The cardiac NC cells and SHF-derived cells form the tunica media of the distal and proximal portion of the aorta and pulmonary trunk, respectively.

Somites and dorsal aorta SMC development

Soon after gastrulation, the paraxial mesoderm gives rise to somites that reside on opposite sides of the neural tube. The somites are initially divided into the ventral sclerotome and the dorsal dermomyotome, and they contribute to multiple tissues and organs. As the dorsal aorta forms close to the somites, many researchers believed that somites contribute to the vascular cells of the dorsal aorta. In 2006, Pouget et al[15] confirmed that somite-derived cells travel to the dorsal aorta to form the smooth muscle in the tunica media in chick-quail chimeras. Thereafter, Scaal and Wiegreffe[32] reported that SMCs derived from the sclerotome, identified as secondary SMCs, migrate ventrally to replace primary SMCs. Moreover, Esner et al[33] reported that some of the dorsal aorta SMCs might arise from the myotome. Thus, smooth muscle tissues that build up the wall of the descending thoracic aorta are “segmentally” derived from various somites, with each somite producing different SMC progenitors.

Coronary artery SMC development

Coronary vessel development is brought about by a step-by-step angiogenic program, involving the initial formation of an immature vessel plexus that is then remodeled into a mature vascular bed. During the remodeling stage, SMCs of different origins migrate into the coronary vessels to form one or more SMC layers surrounding the endothelium, contributing to the coronary vessel walls. There are 3 known origins of coronary artery SMCs, comprising the pro-epicardium, endothelium, and endocardium-derived cardiac cushion mesenchyme.[34] The pro-epicardium is a cluster of coronary vasculature progenitor cells outside of the initial primitive heart tube (overlaying the septum transversum and sinus venosus). Transplanted pro-epicardium in quail-chick chimeras contributed to the formation of donor-derived coronary artery SMCs.[35] Thus, it was thought for a long time that all the SMCs of the coronary vasculature arise from the pro-epicardium. Lineage tracing confirmed this view that pro-epicardium-derived cells travel to the myocardium to form coronary SMCs. In the past few years, it has been found that post-natal coronary vessels were derived from initial embryonic coronary vessels. Recently, additional progenitor cells (besides pro-epicardium-derived cells) were reported to have the ability to form the post-natal coronary arteries. Tian et al[36] used genetic lineage tracing to confirm that neonatal endocardial cells generated a distinct compartment of the coronary circulation in a very short period after birth. Recently, a third source of coronary smooth muscle was discovered. Chen et al[37] reported that endocardium-derived cardiac cushion mesenchyme can differentiate into coronary SMCs and pericytes. Endocardial cells can undergo endothelial-to-mesenchymal transdifferentiation into primitive mesenchymal progenitors, expressing platelet-derived growth factor receptors (PDGFRs), such as PDGFRα and PDGFRβ.[38] These findings indicate that, besides the contributions from the epicardium, the endothelium and endocardium-derived cardiac cushion mesenchyme also contribute to the smooth muscle layer of coronary vessels. Furthermore, the cardiac NC is essential for building up the smooth muscle wall of the coronary vascular tree. Thus, coronary artery SMCs originate from multiple precursors in the developing embryo.

Moreover, these different sources of SMCs can compensate for each other in cases of mutation and injury. For example, in cases of defective epicardial differentiation, a non-epicardial source of coronary smooth muscle was observed.[39] Additionally, epicardium-specific deletion of Pdgfrβ or recombining binding protein suppressor of hairless (Rbpj; which can act as a component of the Notch signaling pathway) triggered a severely delayed development of coronary artery smooth muscle, but this delay did not occur in full Pdgfrβ-knockout mice.[39] This was presumably because the Pdgfrβ+ cushion mesenchyme compensated for it.[39]

Another interesting observation is that cardiac pericytes serve as intermediate progenitors of coronary artery SMCs, as indicated by lineage tracing of a single labeled epicardial cell.[40] The coronary vascular plexus, covered with pericytes, receives arterialization signals and differentiates into smooth muscle. This process is regulated by Notch signaling.[41] Importantly, Red-Horse et al[42] and Pérez-Pomares et al[43] found that some mouse coronary artery SMCs originate from the sinus venosus; the venous endothelial cells travel to the heart muscle, where they form a vascular plexus, which is then remodeled into arteries, veins, and capillaries.[42,44] All these findings provide insights into the detailed development of coronary SMCs.

SMCs originating from mesothelial cells

It has long been known that the pro-epicardial mesothelium produces coronary SMCs, but whether other mesothelia form SMCs was unknown. Recent evidence suggests that pleural mesothelial cells that are positive for Wilms tumor-1 which is expressed in most mesothelial cells, can undergo mesothelial-to-mesenchymal transition and differentiate into SMCs in the lung vessels. Wilm et al[16] found that peritoneum cells differentiate into intestinal blood SMCs by marking mesenteric vessels (Wilms tumour-1+) and associated branches in the gut wall. However, the mesothelial origin of VSMCs is not limited to the epicardium and peritoneum. Pérez-Pomares et al[43] found that mesothelial cells form the ventral wall SMCs of the avian dorsal aorta. Xu et al[45] found that nephrogenic stromal cells derived from the metanephric mesenchyme form SMCs of renal vessels.

VSMCs originating from stem/progenitor cells

Embryonic stem cells (ESCs) can also form SMCs in vitro and in vivo. Fetal liver kinase 1 (FLK1), as a vascular endothelial growth factor receptor, is among the earliest-induced vascular markers.[46] FLK1-positive mouse ESCs cultured on type IV collagen differentiated into SMCs, which were dependent on multiple stimuli, namely, growth factors, extracellular matrix (ECM), and cytokines. FLK1-positive ESCs injected into chick embryos differentiated into SMCs of yolk sac blood vessels. Collagen IV has also been shown to stimulate ESCs to form SMCs.[46] Furthermore, NADPH oxidase (NOX4) was shown to drive ESC differentiation into nuclear factor erythroid 2-related factor 3 (NRF3), which is also related to the migration of SMC precursors during early embryo development. Pepe et al[47] further confirmed that NRF3 is crucial for ESC differentiation into SMCs.

Bone marrow-derived cells can also form SMCs involved in angiogenesis.[47] The bone marrow has 2 discrete stem cell populations, namely, mesenchymal stem cells (MSCs) and hematopoietic stem cells. MSCs originating from adipose tissue have been shown to differentiate into SMCs.[48,49] Williams et al[50,51] isolated endothelium from adipose tissue and transplanted it onto a graft surface, and sub-endothelial SMCs were observed. Wang et al[52] found that adipose-derived stem cells differentiate into SMCs by regulating transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-4 expression. Gu et al[18] found that human umbilical cord-derived MSCs express massive amounts of SMC markers under TGF-β1 induction. MiR-503 and MiR-222-5p also regulate MSC differentiation into SMCs.[18,53] Collectively, these data show that stem/progenitor cells are important sources of SMCs. However, the key molecular mechanisms controlling stem cell differentiation into SMCs are not well understood.

VSMCs originating from endothelial cell transdifferentiation

Endothelial cells are thought by some researchers to give rise to VSMCs. However, this is a controversial issue. Seifert et al[54] reported that some aortic endothelial cells in quail embryos will transdifferentiate into primordial VSMCs. This preliminary result suggests that endothelial cells are capable of differentiating into sub-endothelial SMCs. In vitro experimentation also confirmed that adult bovine aortic endothelial cells can transform into smooth muscle actin (SMA)-positive cells by activating TGF-β1. The TGF-β–SMADd2/3-Slug signaling pathway is essential for endothelial-to-mesenchymal transition.[55] Tabarsi et al[56] found SMCs formed from endothelial cells in cases of intimal thickening in pulmonary hypertension. Overall, these studies confirmed that endothelial cell populations can give rise to SMCs, which has intriguing implications for SMC development.

Major signaling pathways involved in VSMC development

Vascular development is a highly organized process that relies on precise signaling. Multiple essential signaling axes are involved in smooth muscle formation. Here, we briefly summarize key signaling axes particularly required for smooth muscle development [Figure 2].

Figure 2:
Major signal pathways involved in VSMC development. BMP: Bone morphogenetic protein; OFT: Outflow tract; SHH: Sonic hedgehog; TGF: Transforming growth factor; VSMC: Vascular smooth muscle cell.

TGF-β signaling

The TGF-β pathway is crucial for normal vessel formation, as it controls matrix synthesis, SMC differentiation, and vascular morphogenesis. Mice with deletions of the TGF-β isoforms (TGF-β1, -β2, and -β3) and their receptors exhibited early embryonic lethality, reduced angiogenesis, abnormal capillary tube formation, and SMC hypoplasia.[57] The roles of TGF-β and the receptors in SMC development have been demonstrated in detail by gene deletion experiments. TGF-β2 deletion results in perinatal death due to abnormalities in OFT and heart vessel growth and remodeling.[58] Boezio et al[59] generated zebrafish lacking the TGF-β receptor ALK5 and found a distinct dilation of the OFT, with SMC disorganization, an increased number of endothelial cells, and ECM disorganization. In turn, endothelial-specific ALK5 overexpression in Alk5−/− zebrafish protected against these defects.[59] Smooth muscle protein 22-alpha (SM22α)-specific TGF-β receptor II-knockout embryos exhibited embryonic lethality due to multiple defects, including OFT anomalies, right dorsal aortic persistence, and distal aortic dilation.[59] These findings reveal that endothelial TGF-β signaling regulates OFT SMC development. Recent research has also shown that BMP receptors, as members of the TGF-β superfamily of receptors, are required for VSMC differentiation and development. Aberrant BMP signaling disturbs the development of lung arterial SMCs and leads to pulmonary arterial hypertension.[60] El-Bizri et al[61] found that VSMC-specific deletion of the BMP type-1A receptor results in embryonic lethality owing to massive vascular damage. Smad4 is a principle regulator of the TGF-β/BMP network, and it is highly expressed in VSMCs during vessel formation. SMC-specific Smad4 deletion leads to embryonic lethality due to vascular defects such as reduced VSMC proliferation, differentiation, migration, spreading, and attachment.[62,63] Taken together, the findings indicate that TGF-β signaling is crucial for the development of embryonic VSMCs.

Notch signaling

The Notch signaling network is a highly conserved essential pathway for the formation of multiple organs in vertebrates. Numerous reports have demonstrated the essential function of the Notch signaling network in angiogenesis and vasculogenesis during mammalian embryo development, including in SMC development.[64] To date, 4 Notch receptors have been found to be involved in mammalian VSMC development. Notch1-knockout mice exhibited embryonic lethality due to vascular developmental defects.[65–67] Baeten et al[68] and Wang et al[69] reported that Notch2 and Notch3 cooperate with each other to regulate vascular smooth muscle development. Takeshita et al[70] found that embryos with Notch1 mutations or Notch1/Notch4 double mutations exhibited massive dysregulation of angiogenic vascular remodeling. SMC-specific Notch2 deletion gives rise to pulmonary arteries and aorta narrow due to SMC defects. Baeten et al[68] found that Notch2 and Notch3 silencing in VSMCs leads to patent ductus arteriosus. Domenga et al[71] found that Notch3 is required to produce functional arteries by regulating VSMC differentiation and maturation in small resistance arteries. They found that artery-specific Notch3-knockout mice had a thinner VSMC coat, vessel enlargement, and altered VSMC shape and size in small resistance arteries.[71] Moreover, Ghosh et al[72] demonstrated that Notch3 signaling is fundamental for arterial specification of VSMCs. They found that Notch3 was selectively expressed in pulmonary artery VSMCs, and it was continuously activated from the late fetal period to the early post-natal period. Notch3 was also required to maintain the smooth muscle gene expression profile and the morphological characteristics of the pulmonary artery after birth.[72] Our lab recently found that cartilage oligomeric matrix protein interacts with the activated Notch1 signaling pathway to drive ESC differentiation into VSMCs.[73]

Notch signaling is crucial for the NC precursors to differentiate into proper OFT patterns and in aortic arch artery formation. Suppressing Notch activity in these cells in mice causes malformation of the smooth muscle layer of the aortic arteries, resulting in congenital heart defects, namely, pulmonary artery stenosis and aortic arch patterning defects. Ex vivo research involving neural tube explants from mutant embryos shows that mutant NC derivatives lost their differentiation ability in SMCs.[74–76] These findings suggest that Notch signaling positively regulates cardiac NC cell differentiation into SMCs.

Notch signaling is also needed for SHF-derived cells to differentiate into SMCs. Moreover, inhibiting the Notch axis in the SHF severely dysregulates the cardiac NC, leading to defective migration to the OFT and subsequent aortic arch artery (AAA) anomalies.[13]

Jagged-1 is an important Notch ligand, with NC cell-specific Jagged-1 deletion impairing SMC differentiation and aortic arch remodeling.[7,77] SMC-specific inactivation of Jagged-1 in mice (using an SM22a-Cre driver line) led to patent ductus arteriosus, along with early post-natal mortality. Moreover, these mice displayed a common characteristic, namely, a remarkable dysregulation in contractile SMC differentiation in the ductus arteriosus wall and adjoining descending aorta. Additionally, these mice exhibited strong downregulation of contractile proteins, such as SMA, SM22a, and smooth muscle myosin heavy chain.[78]

It is well known that epicardium-derived cells give rise to coronary vessel SMCs. Consistently, complete suppression of Notch signaling in epicardium-derived cells markedly alters the coronary vessel morphology, as epicardium-derived cells cannot form SMCs in the absence of Notch signaling.[79] Alternately, conditional Notch signaling stimulation in epicardium-derived cells causes premature differentiation of SMCs.[79] To some extent, Notch activation appears to be dependent on TGF-β. TGF-β upregulates the Notch ligand Jagged-1 along with the SMC markers actin alpha 2 (ACTA2), calponin 1 (CNN1), and myocardin (MYOCD). Jagged-1 knockdown disrupted the effects of TGF-β, which involved partial downregulation of ACTA2 and CNN1 and complete downregulation of MYOCD.[80] This suggests that Jagged-1 is essential for the TGF-β-driven expression of SMC markers.[81] Additionally, the Notch axis mediates TGF-β-regulated human ESC differentiation into SMCs.[81] Notch signaling upregulates SMC markers in human ESCs. Collectively, these results suggest that the Notch signaling pathway plays a major role in regulating embryonic vascular morphogenesis and remodeling. The 4 Notch receptors exhibit overlapping roles in VSMC development.

Wnt signaling

The highly conserved Wnt signaling axes regulate cell differentiation, apoptosis, and orientation. Deficiency in the Wnt receptor Frizzled-5 is embryonically lethal, as it severely impairs yolk sac angiogenesis.[82] Wnt3a promotes SMC differentiation by upregulating SM22-α in vitro.[83] Wnt7b is essential for pulmonary vascular smooth muscle integrity. Wnt7b and β-catenin are crucial for Pdgfrα and Pdgfrβ expression and pulmonary SMC precursor proliferation. Wnt signaling increased SMC precursor proliferation as well as Pdgfrα and Pdgfrβ expression. Additionally, Pdgfr expression is, in part, directly regulated by the ECM protein tenascin C (TNC), with the Wnt/TNC/PDGFR network being essential for the development of SMC.[84]

Hedgehog signaling

Hedgehog signaling is a powerful morphogen mediating angiogenesis during embryonic development. Recent studies have shown that during the formation of the aorta and coronary vessels in the yolk sac, Indian hedgehog and sonic hedgehog (Shh) mediate embryonic vascular smooth muscle development.[85] Lawson et al[86] were the first to report the role of Shh in normal embryonic angiogenesis.[86] They reported that notochord-based Shh signaling promotes aortic development in the zebrafish embryo. Vokes et al[87] also reported that Shh signaling has an essential role in dorsal aortic development in avian and mouse embryos. Lavine and Ornitz[88] revealed that Shh migrates from the cardiac epicardial layer during mouse embryonic development to promote coronary vessel formation in the sub-epicardial space by upregulating VEGFs and Angiotensin II.

Cardiovascular malformations related to abnormal VSMC development

The first step of vascular wall formation is the development of a primary vascular network. In this process, vascular endothelial cells begin to invest the vessel wall to form a nascent capillary-like vascular network. After the establishment of the primary vascular system, various progenitor cells migrate, proliferate, and differentiate into VSMCs and further build up the embryonic vascular system. Dysregulation of the precursor cells (direct or indirect), including dysregulation of the molecular pathways that control VSMC migration, proliferation, and differentiation, can cause common congenital cardiovascular diseases. In particular, mutation of particular genes that regulate VSMC development can cause vascular morphology defects.

OFT malformation

OFT deformity is the most common deformity among congenital heart diseases. Cardiac NC cells travel to pharyngeal arches 3 and 4 and continue to the OFT to take part in the separation of the OFT into the pulmonary trunk and ascending aorta.[89] Defective OFT smooth muscle development leads to severe malformations, including abnormal OFT septation (such as persistent truncus arteriosus), abnormal patterning of the AAAs and great arteries (such as the interrupted aorta and double aortic arch), and tetralogy of Fallot.[23,89]

These malformations have been verified in various cardiac NC ablation models. In avian embryos, dorsal neural tube removal resulted in persistent truncus arteriosus.[90–92] In chicks, NC ablation also led to persistent truncus arteriosus, impaired patterning of the great vessels, and OFT misalignments.[8,93] Furthermore, in mice, knockout of Notch2 under the Pax3 promoter decreased cardiac NC-derived SMC proliferation, leading to pulmonary artery stenosis, impaired aortic arch patterning, and ventricular septal damage.[76] Knockout of the transcription factor Myocd under the Wnt1 promoter in mice led to patent ductus arteriosus due to failure of cardiac NC cell differentiation into SMCs.[94]Bmp6 and Bmp7 inactivation in mice disturbed cardiac NC cell migration, leading to OFT, cardiac valve, and septation defects.[95] Also in mice, knockout of Alk2 (a type I receptor in the BMP signaling pathway) impaired cardiac NC cell migration to the OFT and reduced SMC formation around the AAAs, leading to aberrant aortic arch maturation and persistent truncus arteriosus.[96]

SHF-derived cells are crucial for extending the OFT and proper alignment of the aorta and pulmonary trunk over the left and right ventricles, as these cells add myocardial tissue to the OFT. SHF ablation results in pulmonary atresia and tetralogy of Fallot.[97] Many recent studies have demonstrated that SHF-derived cells form SMCs at the definitive arterial pole.[30,98–100] Abnormal SHF-derived cell migration causes arterial pole misalignment, leading to malformations such as overriding aorta and double outlet right ventricle.[101] Ward et al[97] ablated the SHF in chick embryos and found that this led to disrupted OFT elongation and alignment, causing tetralogy of Fallot and pulmonary atresia.

Many studies have demonstrated a link between the SHF (which is necessary for extending the OFT, resulting in a properly aligned aorta and pulmonary trunk over the left and right ventricle) and the cardiac NC (which is necessary for OFT septation and normal cardiac looping) during OFT formation. NC ablation restricts SHF-derived cell proliferation, suggesting that the NC presence in the pharynx is crucial for the normal generation of SHF-derived cells.[23] In cardiac NC-ablated embryos, SHF-derived cells do not elongate the tube due to impeding myocardial cells add to the OFT.[62] The shortened OFT leads to abnormal cardiac looping.[23] The cardiac NC cell-related cardiovascular formation defects can be divided into 2 types: direct (due to zero structural contribution by cardiac NC cells, which is caused by defective or deficient NC formation) and indirect (due to lack of cardiac NC cells, causing abnormal signaling and/or tissue interactions). In addition to the cardiac NC affecting SHF-derived cells, cardiac NC cell migration also depends to some degree on the SHF.[23] NC ablation led to defective SHF-derived myocardium formation on the heart tube, and the abnormally shortened OFT resulted in arterial pole misalignment, causing defects such as the overriding aorta and double outlet right ventricle.[21,100] Other studies reported that cardiac NC ablation did not disrupt the addition of SHF-derived SMCs to the arterial pole, but the loss of cardiac NC interactions led the SHF-derived cells to undergo massive proliferation in the pharynx floor, which disturbed the intersection between the vascular smooth muscle and myocardium, disrupting arterial pole patterning.[21] Collectively, these findings suggest that the SHF-derived cells and cardiac NC cells are mutually necessary for normal OFT development.[21,22,30]

This raises a confounding research question: how does the NC regulate SHF development, as their cell populations are not in direct contact? Recent studies suggest that signaling is a critical bridge between the NC and SHF.[102] Fibroblast growth factor (FGF8) has been shown to influence the proliferation, differentiation, and migration of NC cells and SHF-derived cells in a context-dependent manner. NC ablation upregulates FGF8 signaling in the pharynx, which occurs at the same time that SHF-derived myocardium is added to the heart tube.[93,102] In contrast, blocking FGF signaling using an FGF receptor 1 blocker rescued SHF development, OFT alignment, and cardiac looping.[102] Hence, although the SHF-derived cells and cardiac NC cells are discrete cell populations (with differing roles in OFT development), they are interdependent during critical stages of OFT development in the pharynx.[89] Developmental impairment of 1 cell population can affect both.[89]

AAA malformation

The aortic arch and its primary branches are generated from 3 paried PAAs, numbered 3, 4, and 6. Defective PAA formation or remodeling results in various congenital diseases, such as interruption of the aortic arch (IAA), right-sided aortic arch (RAA), and aberrant origin of the right subclavian artery (A-RSA).

Many signaling molecules regulate PAA formation and remodeling, including T-box transcription factor 1 (TBX1), Notch receptors, and endothelins.[75,103,104] Knockout of Hrt1/Hey1 (which encodes a downstream transcription factor in the Notch axis) impairs VSMC differentiation in the fourth PAA and leads to congenital vascular abnormalities, namely, RAA, IAA, and A-RSA.[7] Dominant-negative mutation of mastermind-like (MAML) gene blocks Notch signaling, inhibits smooth muscle addition to these arteries, and thereby impairs PAA remodeling.[105] Mutations related to the TGF-β superfamily network, namely, the BMP axis (BMP6, BMP7), in cardiac NC cells in mice led to defective PAAs.[95,106] Thus, cardiac NC cells clearly travel to the developing heart to form VSMCs and aid in PAA remodeling, and this process is regulated by diverse signaling pathways.

Coronary artery malformation

It is well known that pro-epicardial cells travel over the primitive tubular heart and form the VSMCs of the cardiac coronary arteries. During the transition of pro-epicardial cells into VSMCs of the coronary arteries, there are specific regulatory steps that guide cell fates and migratory paths. The cells must undergo an epithelial-to-mesenchymal transition to leave the surface and migrate into the deeper layers of the myocardium to form the smooth muscle of the cardiac stroma coronary artery. Specifically deletion of either Pdgfrα or Pdgfrβ interrupts the ability of epicardial-derived cells to produce SMCs.[38] Additionally, in mice with both α4 integrin and vascular cell adhesion molecule-1 knockout, the epicardium does not form coronary arteries.[107,108] Moreover, Cx43α1 gap junctions also regulate the deployment of the pro-epicardial cells, with Cx43α1 knockout resulting in abnormal coronary artery patterning and abnormal deployment of pro-epicardium-derived VSMCs.[109]

Summary and perspectives

For many years, the structural and functional diversity of VSMCs were deemed to be due to the plasticity of SMC responses to various mechanical and biologic variations in the immediate environment. Nonetheless, recent reports indicate that the diversity of the SMCs in adult blood vessels reflects their varied developmental histories. Although progress has been made in our understanding of the origins and differentiation of SMCs in a large number of vessels, certain questions still need to be addressed in the future.

First, most research on the origins and differentiation of SMCs relies on lineage tracing in developing embryos, but the developmental fates of cells in different species are varied. Differences induced using different species or promoters make exploring the origins of SMCs challenging. For example, information about the role of the cardiac NC in cardiovascular formation was obtained from cardiac NC ablation and labeling studies in mice and quail-chick chimeras, but differences were observed between these models. There are significant differences in the timing of events related to the function of the cardiac NC in OFT septation. One difference is that cardiac NC cells appear in the mouse distal OFT 2.5 days earlier than the NC-derived cells enter the OFT in the chicks.[6] Another difference is the route followed by the cardiac NC cells into the OFT cushions. In quail-chick chimeras, these cells enter the OFT cushions sub-endocardially or sub-myocardially. However, in mice, they only enter sub-endocardially. These migration routes differ due to species-specific differences in the timing of key events during cardiovascular formation along with species- and species ablation system-specific structural differences.

Species also vary in terms of the locations of arteries and veins, which indicates the outcomes of differences in developmental fates. In humans, the larger coronary arteries and veins have multiple layers. The coronary veins are located sub-epicardially while coronary arteries are intramyocardial. In contrast, in rodents, the arteries are intramyocardial (deep within the myocardium), and the veins are sub-epicardial (on the surface). In mice, coronary artery segments proximal to the aorta have multiple layers, whereas the more distal ones have a single layer. Thus far, the states and cell types regarding the developing VSMCs need to be systematically investigated. Additional examination is necessary to elucidate the specific functions of the vascular progenitors using more varying advanced methods and in multiple species using multiple tools. Greater precision in lineage mapping will require development of mouse models that allow more than 1 cell type in a single tissue to be traced. Moreover, additional new models to examine cell heterogeneity and vascular cell development are urgently required. Nevertheless, recently, single-cell RNA sequencing has allowed systematic and unbiased delineation of heterogeneous cells, elucidating the cell types and states involved in OFT formation. This has laid the foundation for additional studies on the origins of SMCs at the single-cell level.[20]

Second, because of the dynamic nature of blood vessel development and the spatiotemporal-specific embryonic smooth muscle development, the molecular mechanisms underlying the differentiation of different progenitors into SMCs have yet to be clarified. For example, the precise molecular mechanisms underlying how aortic SMCs are derived from the cardiac NC, SHF, cardiac mesoderm, and somatic mesoderm are unknown.[12] The boundaries between smooth muscle from discrete embryonic origins have been predicted to signify areas of specific aortic dissection vulnerability.[21] Therefore, future research should clarify the spatiotemporal mechanisms underlying the development of VSMCs of different origins, deepening the understanding of the VSMC development mechanism, and elucidating the cause of aortic dissection. One useful observation regarding identifying the mechanisms underlying the development of region-specific SMCs is that aortic smooth muscle tissues with different embryonic origins have unique histological characteristics, so the technology could be developed based on this to label the smooth muscle borders. Although pluripotent stem cells, including ESCs and induced pluripotent stem cells, have been used to attempt to achieve this, the detailed intermediate VSMC development process remains unclear and further investigation is required. Spatial transcriptomics is a novel technology that (1) allows all gene activity in a single tissue section to be assessed and (2) allows the spatially resolved expression to be displayed in relation to the tissue section. As this technology allows gene expression patterns to be investigated, it will help us to gain a better understanding of biological processes and disease.[11,110] In the future, connecting the complementary vascular and stem cell biology disciplines will likely bring about rapid advances in our understanding of cardiovascular health and the ability to prevent and treat cardiovascular disease. In summary, the recent progress in the understanding of the diverse origins of VSMCs has greatly expanded our view of the function of VSMCs. This not only represents fundamental progress in biological research but also provides a foundation to interrogate the developmental pathways operating during vascular injury and disease.



Conflicts of interest


Editor note: Wei Kong is an Editorial Board Member of Cardiology Discovery. The article was subject to the journal's standard procedures, with peer review handled independently by this editor and her research groups.


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Vascular smooth muscle; Development; Diverse progenitor; Signaling pathway; Cardiovascular malformation

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