Vascular smooth muscle cells (VSMCs) play important roles not only in the physiological functions of the blood vessels, such as vasoconstriction, vasodilatation and extracellular matrix production, but also in the pathogenesis of vascular diseases, particularly atherosclerosis and hypertension. VSMCs are mostly of mesodermal origin, although some are of neuroectodermal origin, for example, VSMCs present in the aorta and in blood vessels arising from the aortic arch. VSMCs of neuroectodermal origin are implicated in defects of cardiovascular morphogenesis, such as bicuspid aortic valve, coarctation of the aorta, patent ductus arteriosus and tetralogy of Fallot. The origin, location in the vascular tree, gender, species, strain and age influence the phenotype of VSMCs and their propensity to migration and growth. In a healthy adult organism, VSMCs have a quiescent and differentiated contractile phenotype characterized by early markers (e.g., SM α-actin, SM22-α), intermediate markers (h-caldesmon, calponin) and late markers (SM myosins, smoothelin) of VSMC differentiation. However, after blood vessel injury, surgery or explantation in vitro, VSMCs undergo a phenotypic modulation to synthetic phenotype, which endows them with high activity in migration, growth and proteosynthesis. These features can lead to stenosis or to obliteration of the vascular lumen and impaired blood supply to various tissues and organs.
- blood vessels
- smooth muscle cells
- contractile phenotype
- synthetic phenotype
- phenotypic modulation
- vascular diseases
- developmental pathology
Vascular smooth muscle cells (VSMCs) are an important component of blood vessels. The cells are located in the medium part of a blood vessel, that is, tunica media, where they are oriented in a circle around the vascular lumen and form numerous layers. In medium vessels, there are up to 40 layers of VSMCs, and in large vessels, there are up to 60 layers. As the medium part of the blood vessel wall, the tunica media is located between the tunica intima and tunica adventitia, and is separated from these parts by the lamina elastica interna and the lamina elastica externa, respectively. The tunica intima (also referred to as tunica interna) contains a semipermeable monolayer of endothelial cells and forms the luminal part of a blood vessel, contacting the blood. The tunica adventitia, that is, the exterior part of a blood vessel (also referred to as tunica externa), contains fibroblasts, nerves and, in bigger vessels, also small blood vessels supplying the vascular wall, called vasa vasorum. In addition, the tunica adventitia anchors blood vessels to the adjacent tissues (Figure 1) . This structure is similar in arteries and in veins; in veins, the tunica media is usually thinner, due to the lower blood pressure in the venous bed, and the tunica intima in some veins contains valves in order to keep blood flowing in a single direction (Figure 2) . The smallest blood vessels are capillaries, which lack the three classical layers of a blood vessel wall. They consist only of a fine tubular structure built of endothelial cells, which is surrounded by pericytes, which are somewhat like VSMCs and are phenotypically similar to VSMCs (Figure 3) .
VSMCs play important roles in the physiological functioning of blood vessels and in pathological changes in them. In healthy blood vessels of an adult organism, VSMCs ensure that the blood vessels contract and relax, and in this way, they make a marked contribution to the regulation of blood circulation. In healthy adult blood vessels, VSMCs are in a quiescent nonproliferative phenotype, referred to as contractile phenotype. This phenotype is characterized by abundant contractile fibers containing VSMC-specific contractile proteins, such as α-isoform of actin and the SM-1 and SM-2 myosin heavy chain isoforms, and other specific proteins associated with the contractile apparatus [4, 5, 6, 7, 8]. Under pathological conditions accompanying the onset and development of vascular diseases, VSMCs undergo a process referred to as phenotypic modulation, that is, they switch from the contractile phenotype to the synthetic phenotype, characterized by a loss of contractile filaments and associated molecules, and by increased formation of organelles associated with proteosynthesis (Figure 4) . VSMCs of synthetic phenotype are active in migration and growth. This can lead to intimal thickening, formation of atherosclerotic plaques, thickening of the blood vessel wall during hypertension, and finally to stenosis or full obliteration of the vascular lumen [4, 5, 6, 9, 10, 11]. Similar changes in VSMCs also occur after vascular surgery and when VSMCs are cultivated in vitro, particularly under conventional static conditions and in standard serum-supplemented media [6, 7, 12].
This chapter summarizes the most important knowledge on the role of VSMCs in the physiological behavior and in pathological alterations of blood vessels, on the contractile and synthetic phenotype of VSMC, and on the implication of these cells in developmental disorders of the cardiovascular system, atherosclerosis and hypertension. This chapter includes the author’s own experience in her studies of gender-related differences in the migration and proliferation of VSMCs, and in studying the role of VSMCs in vascular remodeling during hypoxic pulmonary hypertension.
2. Vascular smooth muscle cell origin and its role in vascular pathology
2.1. Sources of VSMCs
The VSMCs arise from two main sources: the mesoderm and the neuroectoderm, that is, the ectoderm of the neural crest. Most of the VSMCs in the vascular tree are of mesodermal origin, and are formed mainly from mesenchyme, that is, a type of connective tissue found mostly during the development of an embryo ; or from mesothelium via the epithelial-to-mesenchymal transition .
VSMCs differentiated from neural crest cells reside in the cardiac outflow tract, the ascending aorta, the aortic arch, the proximal thoracic aorta, the brachiocephalic trunks, the common carotid arteries, the internal and external carotid arteries and subclavian arteries, and also in the blood vessels of the facial structures and the forebrain [15, 16, 17]. The pericytes in regions supplied by these vessels are also of neuroectodermal origin . Neural crest cells also differentiate into adventitial fibroblasts. However, the endothelial cells of all the vessels are of mesodermal origin [15, 19]. In addition, the coronary and pulmonary arteries and the descending aorta remain devoid of neuroectodermal VSMCs and contain only mesodermal VSMCs, similarly as the remaining vessels in the body .
The factors that regulate the differentiation of neural crest cells into VSMCs include the Notch and Hippo signaling pathways, fibronectin, transforming growth factor-β (TGF-β), Smad2, and myocardin-related transcription factor B.
The Notch plays a critical, cell-autonomous role in the differentiation of neural crest precursors into smooth muscle cells both in vitro and in vivo. Mutations in components of the Notch signaling pathway result in defects of the cardiac outflow tract . For proper Notch signaling, Hippo signaling is required. Neural crest-specific deletion of the Hippo effectors Yap and Taz produces neural crest precursors that migrate normally, but fails to differentiate into VSMCs . In addition, Notch signaling is regulated by fibronectin 1 (Fn1), which is synthesized by the neural crest cells (NCCs) and mediates the morphogenesis of the aortic arch artery. The Fn1 signals are delivered into NCCs through integrin α5β1 adhesion receptors and lead to the differentiation of NCCs into VSMCs .
TGF-β plays a controversial role in VSMC differentiation. On the one hand, TGF-β induces the differentiation of VSMCs from NCCs. This differentiation is mediated by Smad2, a transcription factor which is required for TGF-β-induced nuclear translocation of myocardin-related transcription factor B (MRTFB). MRTFB enhanced the binding of Smad2 to a promoter of the expression of genes encoding differentiation markers of VSMCs [24, 25]. On the other hand, in mature VSMCs of neural crest origin, TGF-β increased the synthesis of DNA, which is known to be associated with a loss of differentiation markers in VSMCs. At the same time, TGF-β inhibited the growth of VSMCs of mesodermal origin. This dual effect of TGF-β was explained by the different composition of TGF-β receptors in VSMCs of different origin. In neuroectodermal VSMCs, subunit II is non-glycosylated, while in mesodermal VSMCs, subunit II of this receptor is fully glycosylated .
2.2. The role of VSMC origin in developmental pathology
Proper differentiation of neural crest cells into VSMCs is required for normal cardiovascular morphogenesis. If this differentiation is defective, various cardiovascular disorders can occur, for example, bicuspid aortic valve, coarctation of the aorta, patent ductus arteriosus, tetralogy of Fallot, aneurysm of the thoracic aorta, and intracranial aneurysm.
Bicuspid aortic valve (BAV) is associated with the decreased expression of MYH11, the gene encoding the myosin heavy chain in VSMCs of neural crest origin, impaired contraction of these cells and decreased TGF-β signaling based on phosphorylation of SMAD2. In addition, patients with BAV are at higher risk of developing aneurysms of the thoracic aorta than patients with tricuspid aortic valve .
The major role in the pathogenesis of coarctation of the aorta in humans is attributed to deregulation of the Forkhead Box C1 (FOXC1) transcription factor or its downstream genes . FOXC1 is also involved in the pathogenesis of ocular diseases, particularly glaucoma. FOXC1 dysfunction causes disruptions in basement membrane integrity and lower resistance of cells to cell death in response to oxidative stress [29, 30]. In addition, patients with BAV and coarctation of the aorta are more prone to developing an intracranial aneurysm .
Patent ductus arteriosus, that is, a temporary fetal vessel that bypasses the lungs by shunting the aortic arch to the pulmonary artery, can result from insufficient proliferation, differentiation and contractility of a specific smooth muscle subpopulation that shares a common neural crest precursor with cardiovascular melanocytes .
Tetralogy of Fallot (TOF) is a serious congenital disorder characterized by a ventricular septal defect, overriding aorta, right ventricular outflow tract obstruction (i.e., pulmonary stenosis) and right ventricular hypertrophy. TOF has a polygenic origin, being caused by a combination of deleterious mutations in genes essential for apoptosis and cell growth, for the assembly of the sarcomere, and also for the neural crest and the secondary heart field, that is, the cellular basis of the right ventricle and its outflow tract. Numerous genetic abnormalities are associated with TOF. They include mutation of the gene encoding myosin binding protein C3 (MYBPC3) , mutations and polymorphism of the gene encoding vascular endothelial growth factor (VEGF), which regulates vasculogenesis and angiogenesis [34, 35], mutation of the gene encoding Neuropilin1 (NRP1), a membrane co-receptor of VEGF , and mutation of the gene encoding Jagged1 (JAG1, also designated as CD339), that is, a cell surface protein interacting with receptors in the mammalian Notch signaling pathway , and also trisomy of chromosome 21, that is, Down’s syndrome .
3. Vascular smooth muscle cells in healthy adult vessels
In healthy adult blood vessels, the vast majority of VSMCs are in a quiescent non-proliferative phenotype. This phenotype is usually defined as contractile, while the phenotype of proliferating VSMCs has been widely referred to as synthetic. However, the population of quiescent nonproliferating VSMCs is highly heterogeneous, varying dynamically from primarily contractile cells to synthetic cells specialized in extracellular matrix production . In addition, the blood vessel wall contains abundant stem/progenitor cells, which are largely responsible for VSMC accumulation in the intima during vascular remodeling, such as neointimal hyperplasia and arteriosclerosis [10, 39, 40, 41]).
The degree of VSMC differentiation can be determined by the level of expression of specific markers of VSMC differentiation at mRNA and protein level. Markers of VSMC differentiation have been divided into early, mid-term and late, according their appearance during embryonic development , during restoration of differentiated phenotype of VSMC  or during differentiation of stem cells toward VSMCs [43, 44]. For example, during the early stage of differentiation of embryonic stem cell-derived embryoid bodies, SM α-actin is the first to be detected, followed by myocardin, SM22-α and smooth muscle myosin heavy chain (SM-MHC). The expression of SM-α actin, myocardin, SM22-α and SM-MHC was found to begin on day 0, 8, 11, 13, respectively, during early embryonic vascular development .
Early markers of VSMC differentiation include SM α-actin, myocardin and SM22-α [7, 43, 45]. Mid-term markers are h-caldesmon and SM-calponin [46, 47, 48, 49, 50], although these markers have been designated in other studies as early markers . Late markers are desmin, meta-vinculin, SM-1 and SM-2 isoforms of myosin heavy chain, and smoothelin [7, 43, 44]. Markers of VSMC differentiation, stained by immunofluorescence, are demonstrated in Figure 5 [8, 51, 52, 53, 54, 55, 56, 57].
SM α-actin is the earliest known marker of VSMC differentiation, but its expression alone does not provide definitive evidence for a smooth muscle lineage . SM α-actin is located in thin filaments of VSMCs, together with tropomyosin, troponin and calponin. Alpha-actin is the predominant isoform in the VSMCs of a healthy adult vessel. Other actin isoforms in VSMCs include β- and γ-actin, but they are minority components in adult physiological VSMCs, being localized predominantly in immature VSMCs during development or in dedifferentiated VSMCs in diseased vessels [4, 5, 9, 58].
Myocardin is a transcription factor essential for VSMC-specific differentiation. It is a transcriptional coactivator of the serum response factor involved in cell cycle regulation, apoptosis, cell growth and cell differentiation. Myocardin induces the expression of SM α-actin, SM22-α, calponin and SM-MHC. Mice lacking myocardin die during embryogenesis from a lack of differentiated VSMCs. During supraphysiological mechanical load, for example during hypertension and prolonged stretching of VSMCs in vitro, myocardin is translocated from the nuclei to the cytoplasm and is degraded by the proteasome, which led to phenotypic modulation of VSMCs toward the synthetic and proliferative phenotype [9, 59, 60, 61, 62].
SM22-α is an actin-binding protein of the calponin family that is involved in calcium-independent smooth muscle contraction [45, 63]. SM22-α interacts directly and co-localizes with F-actin, and it therefore participates in the organization of the actin cytoskeleton in differentiated VSMCs. SM22-α facilitates the assembly of actin filaments into bundles, enhances the contractility and the mobility of VSMCs, and maintains the contractile phenotype in VSMCs . Disruption of SM22-α is involved in osteochondrogenesis in arterial diseases and also in vascular inflammation [45, 65]. SM22-α attenuated vascular inflammation by suppressing the IKK-IκBα-NF-κB signaling cascades .
Caldesmon is a cytoskeletal protein interacting with actin, tropomyosin, myosin, calmodulin and phospholipids. Due to alternative splicing of one gene, calmodulin occurs in two isoforms, namely high molecular weight (89–93 kDa) caldesmon isoforms (h-caldesmon), and low molecular weight (59–63 kDa) caldesmon isoforms (l-caldesmon). H-caldesmon is present in adult and fully differentiated smooth muscle cells, while l-caldesmon is found in non-muscle cells and in de-differentiated smooth muscle cells. H-caldesmon is also a marker of tumors, for example of soft tissue tumors of the skin. All isoforms are potent inhibitors of the actin-tropomyosin activated myosin MgATPase. Smooth muscle caldesmon, together with tropomyosin, is a mediating factor for Ca2+-dependent inhibition of smooth muscle contraction [66, 67].
Calponin is another actin filament-associated regulatory protein expressed in smooth muscle cells and in many types of nonmuscle cells. It occurs in three isoforms, that is, calponin 1, 2 and 3, encoded with three homologous genes, CNN1, CNN2 and CNN3, respectively. All three isoforms inhibit actin-activated myosin ATPase and stabilize the actin cytoskeleton. Calponin 1 is specifically expressed in smooth muscle cells and plays a role in fine-tuning smooth muscle contractility. Similarly as in caldesmon, the interaction of calponin with actin inhibits actomyosin MgATPase activity. Calponin 2 is expressed both in smooth muscle cells and in nonmuscle cells, and it regulates multiple actin cytoskeleton-based functions. Calponin 3 participates in actin cytoskeleton-based activities in embryonic development, myogenesis and neuronal plasticity [68, 69, 70]. Another important role of calponin is its tumor-suppressing effect. The levels of calponin 1 and 2 have been found to be decreased in tumor cells, and transfection of these cells with gene encoding calponin 1 reduced their growth and malignancy. The level of calponin 2 in the serum of patients can also be used as a biomarker of tumor diseases, for example, breast cancer [70, 71].
Desmin, together with vimentin, forms intermediate filaments in VSMCs. During the development and specialization of cells toward smooth muscle cells, desmin replaces vimentin as the predominant component of intermediate filaments. Desmin is upregulated during differentiation of VSMCs from stem cells, for example, human bone marrow-derived mesenchymal stem cells (MSCs)  and also from embryonic mesothelial cells via epithelial-to-mesenchymal transition . Desmin and smooth muscle myosin were expressed together in the cells, and their acquisition appeared indicative of the terminal differentiation of smooth muscle . However, the amount of desmin was found to be much lower in VSMCs than in smooth muscle cells of the digestive, respiratory, and urogenital tract, and also much lower than the amount of vimentin in VSMCs . For example, rat aortic smooth muscle cells contain 51% of vimentin alone-positive cells, 48% with both vimentin and desmin and 1% with desmin alone .
Meta-vinculin is a high-molecular form of vinculin, that is, a protein of the focal adhesion plaques associated with integrin adhesion receptors in cells . Meta-vinculin and vinculin are co-localized in focal adhesion plaques . Together with SM-MHC, meta-vinculin is considered as a marker of well-differentiated contractile VSMCs [76, 77]). During phenotypic modulation of VSMCs, for example, in venous grafts used as aortocoronary bypasses , in human coronary arteries affected by arteriosclerosis  or during cultivation of VSMCs , the content of meta-vinculin in VSMCs decreases, and meta-vinculin can fully disappear from cells . In cell culture conditions, the content of meta-vinculin in VSMCs can be reestablished when the cells reach confluence . During hypoxic pulmonary hypertension induced in newborn calves, proliferation occurred almost exclusively in the meta-vinculin-negative VSMC population rather than in the VSMC population expressing meta-vinculin .
SM-1 and SM-2 isoforms of myosin heavy chain (SM-MHC) are located in thick filaments of fully differentiated VSMCs. In intact adult rat thoracic aorta, the ratio of SM-1:SM-2 is 80:20 both for mRNA and for protein . VSMCs also contain the non-muscle (NM) isoform of myosin heavy chain, which is present, for example, in fibroblasts, macrophages, lymphocytes and platelets [81, 82]. The expression of myosin heavy chain isoforms in VSMCs is highly variable. The same VSMC cell can contain all three isoforms of myosin heavy chain or only SM myosins [7, 83]. This leads to a functional diversity of VSMCs, which have a fast contractile gene program, giving rise to a phasic smooth muscle phenotype, or a slow contractile gene program, giving rise to a tonic smooth muscle phenotype . During vascular diseases, such as atherosclerosis , after vascular surgery, for example, coronary angioplasty  or after explantation of VSMCs in vitro , the expression of SM-1 myosin, and particularly of SM-2 myosin, decreases rapidly, while the content of NM myosin increases.
Smoothelin (SMTN) is a cytoskeletal protein present exclusively in contractile smooth muscle cells. The SMTN family of proteins consists of two isoforms of SMTN, namely SMTN-A, SMTN-B, and the SMTN-like protein 1 (SMTNL1). The SMTN-A isoform is located predominantly in visceral smooth muscle cells, while the SMTN-B isoform is located in vascular smooth muscle cells, and SMTNL1 is located in both visceral and vascular smooth muscle cells. SMTN-A and SMTNL1 are associated with the contractile apparatus in VSMCs, and their main function is to increase the contraction potential of VSMCs, to contribute to vascular adaptations in various physiological and pathological conditions, such as pregnancy, exercise training or hypertension and to contribute to the maintenance of the contractile phenotype [86, 87].
4. Vascular smooth muscle cells in vascular pathology
Typical examples of vascular diseases accompanied by vascular remodeling are atherosclerosis and hypertension. These two diseases have several common features, mainly thickening of the vessel wall and narrowing of the vascular lumen, which is due, among other reasons, to the activation of migration, growth and proteosynthesis in VSMCs.
The onset of atherosclerosis is usually associated with mechanical or biochemical damage to the endothelial cell layer. Mechanical damage occurs during hypertension or during vascular surgery. Biochemical damage is due to the presence of various harmful compounds in the blood, for example, reactive oxygen species (ROS), nicotine or products of nonenzymatic glycation, and also due to elevated concentrations of originally physiological biomolecules, such as glucose, cholesterol or homocysteine. Biochemically damaged endothelium becomes thrombogenic, permeable and immunogenic. Platelets adhering to the endothelium then release platelet-derived growth factor (PDGF), a potent mitogen and chemoattractant for VSMCs [88, 89]. The production of PDGF-like molecules and other growth factors, for example VEGF, is also activated in the VSMCs themselves . Other examples of growth factors that can penetrate into the vascular wall from the blood through the damaged endothelial barrier, and that can stimulate the proliferation of VSMCs, include epidermal growth factor (EGF), fibroblast growth factor (FGF) , nerve growth factor (NGF) , insulin-like growth factor-1 (IGF-1)  and TGF–β . In addition to growth factors, other blood-borne molecules that can penetrate into the blood vessel wall can change the composition of its extracellular matrix (ECM) and can influence the behavior of VSMCs, include albumin, globulins including immunoglobulins, hemoglobin, lipoproteins, fibrinogen, fibrin, thrombin and thrombospondin [94, 95, 96, 97, 98].
Damaged endothelium is also prone to inflammatory activation. Endothelial cells increase their expression of adhesion molecules of immunoglobulin and selectin families, namely intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelial-leucocyte adhesion molecule-1 (ELAM-1) [6, 11]. These molecules, present on the membrane of endothelial cells, bind the cells of the immune system, namely leucocytes, lymphocytes, monocytes, macrophages and mast cells. These cells are then stimulated by chemotactic factors to migrate inside the vascular wall. The chemotactic factors include interleukins (e.g. interleukin-1), tumor necrosis factors (TNFs, e.g. TNF-α) and monocyte chemoattractant protein-1 (MCP-1), and are produced in VSMCs, endothelial cells and inflammatory cells [11, 99]. In addition, immunoglobulin adhesion molecules, such as VCAM-1, can be expressed directly by VSMCs . Another potent source of inflammatory cells penetrating inside the vascular wall is tunica adventitia, particularly its vasa vasorum [40, 101]. As concerns infiltration of the vascular wall with inflammatory cells, atherosclerosis has been considered as a specific type of inflammation [4, 102, 103]. In addition, the inflammatory cells produce proteolytic enzymes, such as chymase, tryptase and metalloproteases (MMPs, e.g. MMP-2, MMP-9 and MMP-13), which degrade the ECM and liberate the VSMCs from their proliferation control exerted by ECM [11, 104, 105]. The inflammatory cells also produce ROS, which can cause cell death in high concentrations. In low concentrations, however, they can stimulate phenotypic modulation and proliferation of VSMCs directly or indirectly by damage to the vascular ECM [11, 106]. Macrophages, like VSMCs, can proliferate within the damaged vascular wall [4, 107] and can also store lipids and form so-called foam cells (Figure 6) .
Under all these pathological conditions, VSMCs in the damaged vascular wall undergo phenotypic modulation, migrate to the tunica intima and proliferate. The proliferation of VSMCs can be very massive and can lead to considerable stenosis or to full obliteration of the vascular lumen. For this reason, early investigators of atherogenesis compared the proliferation of VSMCs to the growth of tumor cells and considered atherosclerotic plaques as “benign tumors of VSMCs” [108, 109].
During phenotypic modulation, the late markers of VSMC differentiation are the first to be lost, while the early markers can persist even in migrating and proliferating cells. For example, smoothelin-B is the first smooth muscle cell marker that disappears when vascular tissues are compromised, for example, in atherosclerosis or restenosis . Smoothelin was not detected in primary or long-term smooth muscle cell cultures, which simulate the pathological conditions in damaged blood vessels in vivo . Similarly, in cultured VSMCs derived from the rat thoracic aorta and grown in 10% serum for 3–5 days to sub-confluence, the expression of SM-1 myosin at mRNA level decreased by 30% and the expression of SM-2 myosin at mRNA level decreased by 80%. At the protein level, SM-1 myosin was detectable at a reduced level in confluent cells, whereas SM-2 myosin was absent in confluent cells. Cultivation of confluent cells in serum-free media, that is under conditions used for restoration of the contractile phenotype, had little or no effect on SM-1 or SM-2 myosins at the mRNA level. In contrast, the level of SM α-actin, the earliest marker of VSMC differentiation, decreased both at the mRNA level and at the protein level. However, it did not fully disappear in these cells, and growth arrest by serum withdrawal or by high cell population density led to renewed SM α-actin expression in these cells . In addition, phenotypically modulated VSMCs synthesize a spectrum of ECM molecules, which is altered quantitatively and qualitatively. For example, these VSMCs synthesize higher amounts of collagen type I, III and V, elastin and glycosaminoglycan, particularly chondroitin sulfate A/C and dermatan sulfate [6, 110]. In addition, phenotypically-modulated VSMCs synthesize ECM molecules, which are typical for osteoblasts and are involved in matrix calcification, for example, osteopontin and osteonectin [111, 112]. These VSMCs also showed increased expression and DNA-binding activity of a transcription factor named Core binding factor alpha1 (Cbfa1) , expression of Runt-related transcription factor-2 (Runx2) , and increased expression and activity of alkaline phosphatase, an enzyme involved in matrix mineralization .
Two types of hypertension should be distinguished, namely systemic hypertension and pulmonary hypertension. Systemic hypertension can be a cause or a consequence of atherosclerosis, while pulmonary hypertension does not depend on the systemic blood pressure and has specific causes of its own, mainly pulmonary hypoxia.
Both types of hypertension include phenotypic modulation and proliferation of VSMCs, that is hyperplasia, which means an increasing number of VSMCs. In addition, systemic hypertension is characterized by the hypertrophy of VSMCs, that is an increase in VSMC volume. This hypertrophy is often associated with polyploidy, mainly tetraploidy and, to a lesser extent, also octoploidy. The polyploidization of VSMCs has been attributed to so-called “incomplete growth stimulation,” which is sufficient only for DNA synthesis and the onset of mitosis, but not for subsequent karyokinesis and cytokinesis. Incomplete growth stimulation has been attributed to an increased level of contractile agonists, such as angiotensin II, arginine vasopressin (also known as antidiuretic hormone), adrenaline, bradykinin and serotonin, which act as weak mitogens for VSMCs [114, 115]). Incomplete mitosis without karyokinesis and cytokinesis is referred to as endomitosis . If only cytokinesis is absent, binucleated or multinucleated, VSMCs are formed. If only DNA synthesis occurs, without complete mitosis, the process of cell polyploidization is referred to as endoreduplication. The increase in volume and ploidy in VSMCs has often been referred to as “specific hypertrophy,” and has been considered by some investigators as a physiological response of VSMCs to mechanical loading during blood circulation, that is, as a certain type of VSMC differentiation. Polyploid VSMCs are more effective in synthesis of contractile proteins and mechanically resistant ECM proteins, and occur even under physiological conditions. For example, the aorta of healthy young rats contains 8–10% of polyploid VSMCs. However, during hypertension and with increasing age, the number of polyploid VSMCs can reach several tens % [114, 116, 117]. For this reason, the polyploidy of VSMCs has been proposed as a biomarker of senescence . In addition, contractile proteins, such as actin and myosin, are not synthesized in proper isoforms typical for differentiated VSMCs. In other words, polyploidy of VSMCs appears to be associated with decreased-to-absent expression of muscle-specific proteins . Accordingly, the maximal force per cross-sectional area generated by the hypertrophic smooth muscle in aorta from hypertensive rats was lower than in normal rat aorta . In addition, increased synthesis of ECM proteins (e.g., collagen III, fibronectin) can lead to increased stiffness of the blood vessel wall, which further worsens the hypertension .
The main cause of pulmonary hypertension is alveolar hypoxia, which is due to a lower concentration of oxygen in the atmosphere, for example, at high altitudes  or in experimental isobaric or hypobaric hypoxic chambers [122, 123, 124]. Alveolar hypoxia also occurs during obstruction of airways, for example, during bronchial asthma [125, 126] and chronic obstructive pulmonary disease (including pulmonary emphysema) [121, 127, 128], during interstitial fibrosis, which hampers the diffusion of oxygen from the alveoles to the capillaries [121, 128], during sterile and microbial inflammations in lungs [124, 126], during thromboembolism in pulmonary arteries  and also during extrapulmonary diseases, such as liver diseases  and cardiac diseases ).
Pulmonary hypertension leads to phenotypic modulation and hyperplasia of VSMCs [121, 132, 133], but usually not to VSMC polyploidization. Surprisingly, the number of tetraploid VSMCs in pulmonary arteries even decreased, as revealed by flow cytometry of pulmonary arterial medial cells obtained from calves exposed to hypoxia in a hypobaric hypoxic chamber . Tetraploid VSMCs were found in the pulmonary arteries of Eker rats, that is an animal model of somatic mutations in the tuberous sclerosis complex-2 (TSC2) gene .
Other factors associated with pulmonary hypertension include vasoconstriction [121, 131], damage to VSMCs by ROS , synthesis of ECM , degradation of ECM proteins by proteases [104, 105, 123], infiltration of the vascular wall with immunocompetent cells, particularly mast cells [104, 105], and inflammatory activation of VSMCs [132, 133, 135]. In addition, not only VSMCs in the tunica media, but also fibroblasts in the tunica adventitia migrate, proliferate and undergo inflammatory activation, and they therefore contribute to vascular remodeling [133, 136].
4.3. Other factors influencing the phenotype and proliferation of VSMCs
The propensity of VSMCs to phenotypic modulation and activation of migration and proliferation can also be influenced by their origin, their location in the vascular system, species, strain, breeding conditions, age and gender.
As mentioned earlier, VSMCs are of neuroectodermal and mesodermal origin. These two types of VSMCs respond in a different manner to various factors playing roles in the pathogenesis of vascular diseases acquired in adulthood, for example, atherosclerosis. For example, the tunica media of the aortic arch composed of VSMCs of neural crest origin calcified significantly earlier than the tunica media of the descending aorta composed of mesoderm-derived VSMCs . Fluid shear stress, another factor contributing to the development of vascular diseases, inhibited the proliferation of mesodermal VSMCs but induced the proliferation of neuroectodermal VSMCs by increasing the expression of cyclin D1 (which mediates cell cycle progression from the G1 phase to the DNA-replicative S phase), by downregulating the cell cycle inhibitor p21 and by activating the Akt pathway in a manner dependent on phosphoinositide 3-kinase . In addition, mesodermal VSMCs derived from avian embryonic vessels expressed about 10 times more SM α-actin and tropoelastin than neuroectodermal VSMCs .
An example of regional differences in VSMC growth is the higher incidence of polyploid VSMCs in the aorta and in other big elastic arteries than in smaller muscular arteries, for example, mesenteric arteries [114, 115]. A possible explanation is the lower mechanical load of big arteries, leading to relatively little damage to the endothelial barrier and lower permeability of this barrier for mitogens from blood. Another explanation is that elastin keeps the VSMCs in a contractile quiescent state, and these VSMCs are therefore less responsive to growth stimulation and undergo incomplete mitosis [140, 141]. Other examples include higher resistance of human VSMCs from the internal mammary artery to dedifferentiation and induction of migration and proliferation in comparison with VSMCs from other arteries, particularly VSMCs from coronary arteries , and a higher propensity of human arterial VSMCs than of venous VSMCs to form atherosclerotic lesions . In spite of this, phenotypic alterations to venous VSMCs appear to be critical for the development of primary varicose veins. The VSMCs of varicose veins showed a lower expression of desmuslin, an intermediate filament protein, which resulted in decreased expression of SM α-actin, SM-MHC and smoothelin, disassembly of actin stress fibers and increased levels of collagen synthesis and MMP-2 expression . Phenotypic modulation, migration and proliferation of VSMC intima also occurs in vein grafts implanted in arterial position, for example, as an aortocoronary bypass. The VSMCs in vein grafts decreased their expression of myocardin, SM-1 and SM-2 myosins  and meta-vinculin . Phenotypic modulation and proliferation of VSMCs in vein grafts can be attenuated by a perivascular drug delivery system releasing sirolimus and preventing distension of the vein grafts [145, 146], and also by transduction of VSMCs with microRNA-145-encoding plasmids .
However, it should be pointed out that, in addition to regional differences in the VSMC phenotype and growth, the VSMC population of the same vessel is highly heterogeneous, containing a wide spectrum of VSMCs varying from primarily contractile phenotype to synthetic cells specialized in extracellular matrix production, and also less-differentiated progenitor cells [10, 38, 39, 40, 41].
Species-specific differences have been found, for example, in the expression of isoforms of arginase, that is an enzyme that stimulates VSMC proliferation and collagen deposition, and thus implicated in the vascular damage during atherosclerosis and during systemic and pulmonary hypertension. Specifically, rat VSMCs expressed isoform I of arginase, while human VSMCs expressed only arginase II . Another example is a species-specific difference in VSMC-endothelial cell interaction. In co-culture with endothelial cells, the proliferation of VSMCs derived from human aorta was inhibited, while the proliferation of bovine aortic VSMCs was stimulated .
Strain-specific differences in VSMC growth have been studied mainly in rats. It was found that polyploidization of aortic VSMCs was the highest in the Wistar-Kyoto (WKY) strain of rats (i.e., normotensive inbred rats related to spontaneously hypertensive rats, SHR); it was intermediate in SHR (genetically hypertensive rat), and it was lowest in Sprague-Dawley rats (i.e., normotensive outbred rats) and in Fischer rats (i.e., normotensive inbred rats). Nonarterial cells (venous VSMCs and lung cells) from WKY and SHR remained essentially diploid, suggesting that polyploidization is also tissue-specific . At the same time, the proliferation, that is hyperplasia, of VSMCs derived from the aorta of SHR rats was markedly higher than that found in aortic VSMCs from WKY rats . The propensity of VSMCs to migration and proliferation can also be influenced by breeding conditions. In our earlier study, the migration and proliferation of cultured aortic VSMC derived from Wistar rats raised under conventional conditions was higher than in cells from Wistar rats raised under specific pathogen-free (SPF) conditions .
The phenotype and growth of VSMCs can also be influenced by the age of the organism. The proliferation activity of fetal and neonatal VSMCs is higher, and their differentiation status is physiologically lower, than in adult VSMCs [152, 153]. In adult organisms, increased age is associated mainly with negative factors affecting VSMCs, for example, oxidative damage, DNA damage (including telomere attrition), mitochondrial dysfunction, apoptosis, pro-inflammatory secretory phenotype associated with the loss of VSMC differentiation markers, such as SM α-actin and SM22-α, increased expression of transcription factors Msx2 and Runx2 and of bone morphogenetic protein-2, that is markers of osteoblast transition of VSMCs, and increased sensitivity of β-adrenoceptors, which are implicated in the inhibition of cellular proliferation [154, 155, 156, 157]. These changes in VSMC behavior caused by donor age can be further magnified by proliferative aging of VSMCs during their cultivation in vitro .
Gender plays an important role in the propensity of VSMCs to migration and proliferation. It is generally known that estrogens decrease the proliferative activity of VSMCs, while androgens increase it. This is considered as the main cause of the higher incidence of cardiovascular diseases in male organisms. However, VSMCs from males proliferated faster even without the actual presence of sex hormones in the cell culture medium. For example, VSMCs isolated from the thoracic aorta of adult male rats migrated earlier from the explants and proliferated faster than their female counterparts [158, 159]. These differences were enhanced in a serum-free medium  and after repeated passaging of VSMCs , and were also apparent in newborn rats  and in both WKY rats and SHR rats . These differences have been explained by prenatal synthesis of androgens initiated by the expression of specific genes in the SRY locus on the Y chromosome. Among others, androgens increase the sensitivity of VSMCs to adrenergic hormones, which persists throughout life, and also without the actual presence of physiological levels of androgens [163, 164].
Vascular smooth muscle cells (VSMCs) are physiological and the most numerous component of the arterial and venous wall, and they ensure vasoconstriction and vasodilatation and other functions, such as synthesis of extracellular matrix. However, VSMCs are also implicated in vascular disorders, such as defects of cardiovascular morphogenesis, atherosclerosis, and systemic and pulmonary hypertension. The VSMCs in healthy adult blood vessels are in the quiescent contractile state, characterized by specific markers of VSMC differentiation namely SM α-actin, myocardin and SM22-α (early markers), h-caldesmon and SM calponin (intermediate markers) and desmin, meta-vinculin, SM-1 and SM-2 isoforms of myosin heavy chain and smoothelin (late markers). However, in pathologically changed blood vessels, VSMCs lose their differentiation markers, activate migration and proliferation and increase proteosynthesis. This VSMC behavior can lead to remodeling of the vascular wall, including stenosis and obliteration of the vascular lumen.
This study was supported by the Czech Health Research Council, Ministry of Health of the Czech Republic (project No. 15-33018A). Further support was provided by BIOCEV – Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University (CZ.1.05/1.1.00.02.0109), a European Regional Development Fund project. Mr. Robin Healey (Czech Technical University in Prague) is gratefully acknowledged for his language revision of the manuscript.
Conflict of interest
The authors declare no conflict of interest.