CHAPTER 7: BIOLOGY OF THE VESSEL WALL

The pathogenesis of common cardiovascular diseases, including atherosclerosis and hypertension, involves diverse and complex pathways. Significant advances in our understanding of the underlying pathobiologies at a molecular and cellular level are increasingly leading to the development of new therapeutic strategies targeted at primary etiologies rather than secondary manifestations of vascular disease. Thus, it is essential for both inquisitive scientists and clinicians who are taking care of patients with cardiovascular disease to understand the normal physiology, signaling pathways, and function of the different cell types that constitute the layers of the vascular wall, in addition to the complex interactions between the vasculature, the heart and the entire human organism (Fig. 7–1). This chapter will provide relevant basic knowledge about the underlying physiology and function of each component of the vasculature and how disease conditions alter this carefully regulated system.

Early Vessel Formation—Vasculogenesis

Vasculogenesis is the earliest form of vessel generation to occur; in fact, it is one of the earliest biologic programs in the developing embryo. Vasculogenesis has been recognized and studied for at least a century.¹ It is initiated soon after gastrulation (when the single-layered blastula is reorganized to form the ectoderm, mesoderm, and endoderm) by the aggregation of mesodermal progenitor cells into clusters referred to as blood islands, which are the earliest vascular structures. These clusters then undergo partial lineage commitment, with the outer cells becoming endothelial precursor cells (referred to as angioblasts) and the central cells becoming hematopoietic progenitor cells. Blood islands soon coalesce and fuse, and a central lumen emerges. These primitive vessels undergo progressive remodeling and maturation, and eventually a primitive vasculature emerges (Fig. 7–2).

Vascular endothelial growth factor (VEGF) and its receptor VEGF receptor-2 (VEGFR-2) are critical players during vasculogenesis, with VEGFR-2 knockout leading to embryonic death caused by a severe defect in vasculogenesis in mouse models.² With respect to the key cells participating in vasculogenesis, it was once believed that a bipotent hemangioblast cell capable of differentiation into endothelial and hematopoietic cells was responsible for blood island formation and vasculogenesis. This dogma has now been challenged. Contemporary cell reprogramming studies have shown that during commitment to a hematopoietic fate, primitive cells may express endothelial markers in a transitory manner, but that they do not exist in a truly bipotent state.³ Indeed, mounting evidence has now quite convincingly shown that hemogenic endothelial cells are responsible for definitive hematopoietic cells in the embryo^4,5,6,7 and that their hemogenic ability is reliant on a specific microenvironmental vascular niche.⁷ Although this may appear to be a semantic point, the presumptive existence of a hemangioblast was the central rationale for the many cell therapy studies conducted in the past two decades seeking to use bone marrow (BM)-derived cells to generate new vessels in the adult.^8,9,10 The fact that these clinical studies failed to demonstrate efficacy for new vessel formation using BM-derived cell therapy⁸ may be attributable to this likely incorrect assumption regarding the existence of a bipotent hemangioblast.

An additional important point with respect to vasculogenesis is the fact that it implies de novo vessel formation in the setting of blood islands and the concurrent deployment of hematopoietic progenitor cells. Although the potential for de novo new vessel formation in the adult continues to be debated, what is almost certain is that the classic developmental vasculogenesis program, including blood islands and hemogenic cells, does not arise in the adult.⁹

Angiogenesis

An ongoing source of confusion in vascular biology is that “angioblasts” are cells that are operative during vasculogenesis, whereas the process of angiogenesis does not involve angioblasts but rather new vessel formation from preexisting vessels. Specifically, angiogenesis is a well-described biologic program in development and in the adult. It can be defined as the formation of new capillaries from preexisting vessels by the sprouting or splitting of these preexisting vessels by transcapillary pillars or posts of extracellular matrix and/or cells (see Fig. 7–2).¹⁰ To facilitate this capillary sprouting, endothelial cells possess a well-developed capacity for migration, which is under the control of VEGF and other cytokines.¹¹ Although angiogenesis occurs in the adult, it remains unclear if progenitor cell deployment is a requirement during angiogenesis. A contribution from BM-derived or circulating cells now appears less likely, but it remains possible that endothelial or other progenitor populations resident in the local vessel wall may participate in this process.^12,13

Arteriogenesis

Arteriogenesis is yet another mechanism of vessel formation, although in the strictest sense it is not truly a mechanism of de novo vessel formation but merely vessel remodeling. Arteriogenesis is defined as the formation of larger diameter arteries from preexisting capillaries or arterioarteriolar anastomoses, typically initiated by altered blood flow and shear stress, and involving vessel dilation and remodeling (see Fig. 7–2).¹⁰ Recent exacting studies using mouse models permitting fate tracking of endothelial cells have demonstrated that arteriogenesis is the main mechanism of collateral vessel formation in the heart after myocardial infarction, which involves the remodeling and growth of preexisting arteries.¹⁴

The precise scenarios in the adult where vasculogenesis, angiogenesis, and arteriogenesis are the dominant vascular processes for new vessel formation remain to be clearly defined. As stated, we and others remain guarded about the existence of vasculogenesis in the adult.⁹ With respect to angiogenesis, there are certain situations where this is clearly the dominant process, such as new vessel ingrowth into tumor tissue,¹⁵ or capillary ingrowth into an advanced atherosclerotic plaque.¹⁶ Conversely, where a preexisting vascular network exists and likely when the distances involved are greater—such as following the occlusion of a major epicardial coronary artery—arteriogenesis is likely to be the most important process.¹⁴

Endothelial Cells

The endothelium is strategically positioned as a critical interface between the blood and tissues (Fig. 7–3). Not surprisingly, endothelial cells are pivotal to maintaining vascular homeostasis, and multiple vascular diseases arise from endothelial dysfunction, including atherosclerosis and hypertension. The metabolic state of the endothelial cell is closely regulated by surrounding vascular and nonvascular parenchymal cells, all of which are supported by underlying capillary networks. The metabolic and functional state of the endothelium is influenced by multiple factors, including the degree of oxygenation,¹⁷ physiological forces such as shear stress, metabolic changes, inflammatory responses, and the local cytokine milieu.¹¹

Among its many functions, the endothelium is of particular importance for maintaining and protecting the integrity and function of the vascular wall, with specific roles. These include (1) functioning as a metabolic tissue that actively secretes vasoactive factors governing vascular tone; (2) acting as an anticoagulant and antithrombotic surface; (3) serving as a barrier to most circulating blood constituents; (4) regulating the transendothelial passage of specific molecules, proteins, and cells across this barrier, and (5) participating in the inflammatory response via active leukocyte recruitment and facilitation of leukocyte margination from the lumen into the vessel wall and adjacent tissues.

Permeability

The endothelium regulates vascular permeability at a macrovascular and microvascular level, with transendothelial fluid and macromolecule transport being differentially regulated depending on vessel size. In the microcirculation, endothelial permeability is a key factor that facilitates the delivery of nutrients to tissues as well as the exchange of by-products from various metabolic pathways. Larger caliber vessels typically function as vascular conduits, and in these vessels the endothelium generally acts more as a barrier. The endothelium also serves as a barrier to the underlying vascular smooth muscle; dynamic regulation allows for selective exposure of the underlying vascular wall and smooth muscle cells to mitogenic, thrombotic, and vasoactive agents that have specific downstream effects. As we discuss below, the principal mechanisms whereby molecules or substances can pass across the endothelium are transendothelial vesicular transport in caveolae and modulation of intercellular contacts.

Endothelial cell contact is maintained through adherens and tight junctions. Vascular endothelial (VE) cadherin is a cell junctional protein within adherens junctions and is important for interendothelial cell contacts. VE-cadherin interacts with the actin cytoskeleton, plakoglobin, and catenins to promote cell adhesion.¹⁸ Various tight junctions are formed between endothelial cells that include interaction between occludins, claudins, and junctional adhesion molecule-1. These interendothelial cell interactions are carefully regulated and important for mediating permeability changes to allow passage of macromolecules and extravasation of immune cells, and they are also of importance for other vascular functions such as control of angiogenesis. Vessel size and vascular bed location are related to the integrity of these interendothelial contacts. An important example of this is tight junctions in different vascular beds, with those forming the blood–brain barrier being among the most developed in the entire vascular system. Vasoactive agents, including VEGF, histamine, and prostaglandins, are active in capillaries and postcapillary venules and act to regulate permeability.¹⁹ Tight junctions within arteries are less permeable to solute flux but are regulated by similar vasoactive agonists.

As an alternate means of transendothelial passage, vesicular transport is important for the transfer of water-soluble macromolecules from the luminal to abluminal surface of the endothelium. Caveolae vesicles that contain the structural protein caveolin and are formed from the plasma membrane—are important for this process.²⁰ Caveolae are located at sites where other kinases, G proteins, docking proteins, and related receptors are present; these molecules are important for signal transduction in the endothelium.²¹

Endothelial cell contraction is another aspect of endothelial barrier function. Contraction occurs in response to a variety of stimuli, including thrombin, histamine, and ionomycin, which leads to changes in cell shape that open intercellular gap junctions. This signaling cascade and the subsequent contractile response are thought to be the underlying mechanisms for tissue edema that occurs in response to histamine and bradykinin. The main intracellular pathways involved include activation of protein kinase C; myosin light-chain phosphorylation; tyrosine kinase activation; and stimulation of Rho, a small G protein.²²

As well as functioning as a physical permeability barrier, it should be noted that the endothelium also modulates various signaling pathways and secretes factors that control permeability and are discussed in this section. Therefore, the endothelium has both a direct and an indirect role in the control of vascular permeability.

Hemostasis

At rest under normal hemodynamic conditions, the endothelium presents an anti-thrombotic surface that inhibits the adhesion of platelets and activation of the coagulation cascade (Fig. 7–4). However, the endothelium can serve as a functional antithrombotic, thrombolytic or intermediate interface. Anticoagulants regulated by the endothelium include those that inhibit platelet aggregation, such as prostacyclin and nitric oxide²³; antithrombin III²⁴; heparin-like molecules; and thrombomodulin, which activates endothelial protein C.²⁵ On the other hand, conditions of inflammation or cellular stress, such as those produced by air pollution, can induce an activated endothelial state, which is characterized by the production of procoagulants such as tissue factor,²⁶ factor VIII, factor Va, and plasminogen activator inhibitor 1. In particular, tissue factor production and release is regulated by multiple proinflammatory and proatherogenic pathways, with tissue factor being closely linked to complex atherosclerotic lesions.²⁷ Recently, an alternatively spliced tissue factor isoform was described, which is produced by activated endothelial cells and macrophages, and associated with angiogenesis, neointimal formation, and advanced atherosclerosis.²⁸ Interestingly, although it was long believed that factor VIII was produced by hepatocytes, it is now understood that endothelial cells are the major and potentially even the exclusive source of plasma factor VIII.²⁹ The underlying factors and regulatory mechanisms that govern endothelial cell hemostasis continue to be active areas of scientific research.

Inflammation

Endothelial cells also modulate and contribute to inflammatory processes. Endothelial secretion of chemotactic molecules and upregulated expression of adhesion molecules function to recruit leukocytes to sites of inflammation. Infiltrating macrophages and T lymphocytes, in addition to cytokines and arachidonic acid metabolites, also stimulate secretion of these molecules from the endothelium. The vascular inflammatory response is predominantly mediated by chemokines, classified as CXC and CC ligands dependent on positioning of the first two cysteines in the amino acid sequence. These chemokines interact with G protein receptors classified as CXCR and CCR.^30,31

Induction of the differential expression of the different adhesion molecules mediates the accumulation of various leukocyte classes at sites of inflammation. Many proinflammatory mediators are thought to contribute to the endothelial–leukocyte interaction, with the complete endothelial inflammatory response program being orchestrated by a multitude of molecular players. Among these, L- and P-selectins along with vascular cell adhesion molecule (VCAM)-1 are important for the initial capture of local leukocytes from blood flow to the site of active inflammation. Leukocyte rolling is then initiated by E- and P-selectins.³² Interactions of beta2-integrins, platelet endothelial cell adhesion molecule (PECAM) and CAM-1 with leukocytes leads to adhesion. At the sites of atherosclerotic plaques the surface expression of VCAM-1 and interactions between monocyte chemotactic protein-1 (MCP-1) and the monocyte receptor CCR2 facilitates leukocyte recruitment. This response is also mediated by interleukin (IL)-8 and its receptor CXCR2, as well as fractalkine and its receptor CX3CL1. As well as inflammation, these sequential steps of capture, adhesion, and migration are also relevant in tumor biology, when malignant cells undergo contact-initiated interactions with endothelial cells to facilitate transendothelial migration and cancer cell extravasation.³³

Vascular Tone

Endothelial cells secrete an array of vasoactive metabolites that are important regulators of various vascular and systemic physiologic functions, including vascular tone. In turn, these factors play a key role in controlling both systemic blood pressure and regional blood flow to specific organs or tissue beds. These major vasoactive factors, summarized in Table 7–1, include potent relaxing factors such as nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factors, as well as constricting factors such as endothelin. This section will describe the most prominent regulatory molecules.

Nitric Oxide

Furchgott and Zawadzki³⁴ first described an endothelium-derived relaxing factor (EDRF) in the context of a dilatory response in aortic rings in response to acetylcholine; EDRF was subsequently determined to be nitric oxide (NO).³⁵ Central to NO biology, nitric oxide synthase (NOS) oxidizes guanidine nitrogens of L-arginine, leading to the formation of citrulline and NO. NOS has three known isoforms in mammals that were first identified in various tissues: brain (nNOS, neuronal NOS or NOS type I), macrophages (iNOS, inducible NOS or NOS type II), and endothelial cells (eNOS, endothelial NOS or NOS type III). These three isoforms have binding sites for nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide, and flavin mononucleotide, in addition to a calcium-calmodulin binding site. Tetrahydrobiopterin is an important cofactor for NO synthases participating in electron transfer from the heme group of the enzyme to the L-arginine and is required for NO production by all three NO synthase isoforms.³⁶ Under normal conditions, these enzymes couple oxidation of the amino acid substrate L-arginine with the reduction of molecular oxygen to form NO and L-citrulline. Under conditions of oxidative stress, the electron transfer is shifted to oxygen, which results in the formation of a superoxide anion, termed uncoupling of NOS; this is thought to be important in various cardiovascular disease states.³⁶

The release of NO from NOS is regulated by many factors, including acetylcholine, norepinephrine, bradykinin, thrombin, ATP, vasopressin, platelet-derived factors such as serotonin and histamine, fatty acids, ionophores, and hemodynamic factors such as shear stress. NO crosses the smooth muscle cell membrane and binds the heme moiety of soluble guanylate cyclase, which leads to formation of cyclic guanosine monophosphate (cGMP); cGMP reduces intracellular calcium concentrations, with subsequent dephosphorylation of myosin light chain and smooth muscle relaxation.^37,38 Nitroglycerin is a drug that exerts its vasodilatory effects through its conversion to NO.

Increased intracellular calcium levels act to activate eNOS by binding of calcium and calmodulin, with eNOS activity also regulated through enzyme phosphorylation. Different patterns of shear stress enhance eNOS expression, including high levels of shear and vasoprotective unidirectional laminar shear stress. Conversely, low shear stress and oscillatory shear stress are associated with reduced eNOS and are considered atherogenic.³⁹ Recently, it was established that laminar shear stress (LSS) activates a NOX2/p47phox complex to activate eNOS phosphorylation and NO production, while conversely, oscillatory shear stress activates a NOX1/NOXO1 complex to uncouple eNOS.³⁹

Increased shear stress leads to an acute release of NO from the endothelium, with this response only dependent on calcium in the first few seconds and the continued activation of eNOS in response to shear stress being maintained by serine phosphorylation.⁴⁰ Physical activity also increases eNOS expression in endothelial cells, thought to be reflective of the increased shear stress secondary to high cardiac output during sustained exercise.⁴¹ Reduced eNOS expression has been shown in the presence of oxidized low-density lipoprotein (LDL) cholesterol, glycated LDL, hypoxia, and inflammation (tumor necrosis factor [TNF]-alpha).^42,43,44 Hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (“statins”) have been shown to stabilize eNOS messenger RNA, which leads to elevated eNOS levels. This is thought to be one of the underlying mechanisms that contribute to the pleiotropic beneficial clinical effects of these agents. As an unexpected paradigm shift, it was recently also shown that eNOS is expressed by circulating erythrocytes, with cross-bone marrow transplant studies demonstrating an important role for erythrocyte eNOS in blood pressure regulation.⁴⁵

Endothelium-derived Hyperpolarizing Factors

Initial studies following the discovery of NO demonstrated that vascular smooth muscle cells could become hyperpolarized and that this was dependent on the presence of endothelium-derived factors following challenge with vasodilators. This was determined to be mediated by hyperpolarizing factors separate from NO.⁴⁶ Two further endothelium-derived hyperpolarizing factors (EDHFs) were then identified: 14,15-epoxyeicosatrienoic acid (14,15-EET) and hydrogen peroxide. The molecule 14,15-EET is a cytochrome P450 metabolite of arachidonic acid.⁴⁷ This epoxide and its metabolite 14,15-dihydroxyeicosatrienoic acid are both released from the endothelium, reach the adjacent vascular smooth muscle membrane, and open calcium-activated potassium channels. The subsequent efflux of potassium leads to hyperpolarization and closing of voltage-dependent calcium channels, smooth muscle cell relaxation, and vasodilatation.⁴⁸ 14,15-EET also has direct beneficial effects on the endothelium, and has been shown to inhibit endothelial senescence via the mTORC2/Akt signaling pathway.⁴⁹ Hydrogen peroxide is also released by endothelial cells and mediates similar vasodilatory effects.^50,51,52 It has been noted that with decreasing caliber of vessels from large conduits to small arterioles, the role of NO is less dominant and EDHF-dependent vasodilatation is more prominent.⁵³ Interestingly, there is a regulatory relationship between NO and EDHF. EETs can induce NO production in various tissues, whereas some of the effects of EETs are regulated by the activation of NOS and increased NO production⁵⁴ As another example of the interplay among these pathways, excessive superoxide production leads to oxidative degradation of NO with subsequent loss of NO mediated vasodilation. Hydrogen peroxide is a by-product of superoxide production and can alternatively serve as an EDHF, which can maintain small-vessel vasodilatation.

Prostacyclin

Prostacyclin, or prostaglandin I2 (PGI2), is a prostanoid produced from cyclooxygenase (COX)-1 and COX-2 enzymatic activity on arachidonic acid. PGI2 is released from endothelial cells and exerts powerful vasodilatory effects by relaxation of vascular smooth muscle cells, which is mediated by an increase in intracellular cyclic adenosine monophosphate (cAMP) levels. Recent data suggest that hydrogen peroxide generation affects endothelial PGI2 production, with COX-1, and not COX-2, the main source of endothelial PGI2 under conditions of mild oxidative stress.⁵² Prostacyclin also has an antithrombotic role and suppresses platelet activation and aggregation, as well as reducing growth factor release from macrophages and endothelial cells.⁵⁵ These various effects are thought to contribute to the adverse side-effect profile of COX-2 inhibitors, although these factors alone cannot fully account for the observed adverse effects of the COX-2 inhibitors.^52,56 Prostacyclin synthesis is stimulated predominantly by bradykinin (most potent), substance P, epidermal growth factor, platelet-derived growth factor (PDGF), and adenine nucleotides.⁵⁵ In eNOS knockout mice, prostacyclin was demonstrated to compensate for loss of NO.⁵⁷ Epoprostenol (a synthetic prostacyclin), iloprost (a chemically stable analog of prostacyclin), and treprostinil (a tricyclic benzidine analog of epoprostenol) have efficacy in the clinical treatment of pulmonary hypertension.⁵⁸

Other Prostaglandins

In addition to prostacyclin, under pathologic conditions the endothelium can produce other prostaglandins with vasoconstrictive properties, including prostaglandin H2 (PGH2) and thromboxane. PGH2 is a product of COX-1 and COX-2 activity, and thromboxane is a product of the action of thromboxane synthase on PGH2.⁵⁹ Furthermore, hormones such as acetylcholine and endothelin-1 stimulate the release of endothelium-derived constricting factors (EDCFs) that act on the thromboxane or other receptors in models of hypertension. Depending on the model or clinical disease state, thromboxane A2, PGH2, prostaglandin F2α, prostaglandin E2, and paradoxically PGI2 can all act as EDCFs.⁶⁰

Another cytochrome P450 metabolite of arachidonic acid with vasoconstrictive properties is 20-hydroxyeicosatetraenoic acid (20-HETE).⁶¹ This metabolite depolarizes the vascular smooth muscle membrane through the inhibition of calcium activated potassium channels and subsequent promotion of vasoconstriction. Angiotensin II, endothelin, and catecholamines stimulate 20-HETE production whereas NO inhibits its production.⁶¹ Production of 20-HETE is increased in many common diseases, including hypertension, diabetes, and kidney disease.

Angiotensin-Converting Enzyme

Endothelial cells, notably in the pulmonary vasculature, synthesize and express angiotensin-converting enzyme (ACE) on their cell surface. The main function of ACE is converting angiotensin I to the potent vasoconstrictor angiotensin II and degrading bradykinin. Interestingly, at the molecular level, it has been demonstrated that with ligand binding of ACE inhibitor, ACE can directly signal through its cytoplasmic tail, which leads to downstream changes in gene expression.⁶² The relevance of this in different cardiovascular diseases is not yet well known. Vascular and cardiac cells contain most components of the renin/angiotensin system,⁶³ which supports the important contribution of locally produced angiotensin II to cardiovascular function. The local production of angiotensin II is also supportive evidence of the effectiveness of ACE inhibitors and angiotensin receptor antagonists when total circulating levels of renin or angiotensin II are not increased.

ACE-II is a carboxypeptidase enzyme that functions to cleave an amino acid from angiotensin I or angiotensin II. Its overall effect is a reduction in angiotensin II with subsequent increase in the metabolite angiotensin I to VII that has vasodilatory effects. The interplay between ACE-I and ACE-II regulates angiotensin II levels and vasomotor tone⁶⁴

Endothelins

Endothelins (ETs) encompass a family of peptides that are synthesized and secreted by many cell types, including endothelial cells. Three ETs (ET-1, ET-2, and ET-3) are described, each being comprised of 18 amino acids. Initial synthesis starts with preproendothelin, which is processed and results in “big endothelin.” Big endothelin is released and converted to its active form by ET-converting enzyme. Synthesis is stimulated by angiotensin II, hypoxia, oxidized LDL, inflammatory cytokines, and low shear stress.⁶⁵ ET mediates its effects on the vasculature through three described receptors: ET-A, ET-B, and ET-C. These receptors have specificity for different endothelins and lead to activation of different downstream cell signaling pathways. The ET-A receptor is expressed predominantly in vascular smooth muscle, and the ET-B receptor is found on endothelial cells. Activation of the ET-A receptor stimulates potent vasoconstriction, whereas activation of ET-B stimulates the release of NO and vasodilatation.⁶⁶ ET-1 stimulates a slow, intense and sustained contraction thought to be mediated by activation of the phosphoinositide/protein kinase C pathway and opening of voltage-dependent L-type calcium channels.⁶⁷ Of note, low, subthreshold concentrations of ET-1 enhance vasoconstriction mediated by other metabolites, including serotonin, alpha-adrenergic agonists, and angiotensin II, also thought to be mediated via the protein kinase C pathway.

ET-1 is a growth factor for smooth muscle cells⁶⁸ as well as a chemoattractant for monocytes. In vitro, angiotensin II has been shown to stimulate the production of ET-1 in smooth muscle cells. In vivo it has been shown that the hypertensive effects of angiotensin II are mediated through ET. These data suggest that there is interplay between the endothelin and angiotensin pathways in the pathogenesis of diseases such as hypertension, diabetes, and heart failure. Several ET receptor antagonists are now approved for clinical use in pulmonary hypertension, including bosentan, ambrisentan, and macitentan. Bosentan is a mixed ET-A and ET-B receptor antagonist, whereas the newer agents ambrisentan and macitentan are selective for ET-A; therefore, they have theoretical benefits in terms of preserving NO activity and vasodilator functions mediated through ET-B receptors while antagonizing vasoconstriction and smooth muscle cell proliferation mediated by ET-A receptors.^58,69

Hemodynamic Influences

Endothelial cells use mechanosensing to transduce the frictional force from blood flow (fluid shear stress) into biochemical and cellular signals; in this fashion, circulatory hemodynamics, including stretch, strain, and shear stress, are able to modulate many endothelial functions.⁷⁰ Stretch of the vessel wall and shear stress have been shown to independently affect endothelial cell function and/or morphology. In vitro data have demonstrated that endothelial cell stretch leads to change in endothelial cell shape,⁷¹ activation of intracellular signaling, increase in intracellular calcium and superoxide levels,⁷² and cell proliferation.⁷³ Exposure of endothelial cells to shear stress leads to cytoskeletal changes with reorganization and alignment of the actin filaments and microtubules as well as cellular realignment in the direction of flow. Shear stress also alters the function of the endothelium, including activation of potassium currents; secretion of vasoactive and growth factors such as the production of various microRNAs, NO, endothelin, prostacyclin, and basic fibroblast growth factors; elevation of tissue factor expression; increased uptake of LDL; and enhanced tissue plasminogen activator secretion.^70,74

Hemodynamics vary in different vascular beds. Low shear stress vascular areas such as branch points and curvatures demonstrate predilections for the formation of atherosclerotic lesions.⁷⁵ Oscillations of flow have been demonstrated in the proximal carotid arteries, carotid bulbs, and distal aorta.⁷⁶ Oscillatory shear stress leads to increased endothelial production of reactive oxygen species (ROS), increased expression of adhesion molecules and simulation of monocyte adhesion.^39,77

The sensing and transduction of mechanical signals by endothelial cells is mediated by multiple factors, including extracellular glycosaminoglycans, selectin molecules, and PECAM-1. The coexpression of PECAM-1 and VE-cadherin has been shown to promote shear responsiveness to surrounding cells that are not normally affected by shear stress.⁷⁸ In addition, mechanosensors activate integrins that modify cytoskeletal properties and downstream signaling.^79,80 In turn, flow sensitive ion channels and selective G proteins act to modulate mechanotransduction.^81,82 Caveolae contain many signaling molecules, including G proteins, which are significant in downstream signaling pathway activation in response to shear stress.⁸³

Endothelial-to-Mesenchymal Transition

Endothelial-to-mesenchymal transition (EndMT) is a pervasive biologic program whereby endothelial cells undergo a series of molecular and structural rearrangements and change their phenotype to that of a mesenchymal cell (Fig. 7–5).⁸⁴ EndMT is known to play a fundamental role in cardiac development⁸⁴ and has now been implicated in several cardiovascular disease states, including atherosclerosis,⁸⁵ vein graft remodeling,⁸⁶ cerebral cavernous malformations,⁸⁷ cardiac fibrosis,⁸⁸ vascular calcification,⁸⁹ and pulmonary hypertension.⁹⁰ Essentially, under the influence of TGF-β, fibroblast growth factor (FGF), and other signaling pathways, the pathological milieu that arises under these various pathologic states promotes this endothelial phenotypic switch, with some studies finding that EndMT gives rise to fibroblasts.⁸⁸ In contrast, in different disease models, we and others have identified that EndMT may give rise to vascular smooth muscle cells (VSMCs)⁸⁶ or even other mesenchymal cell types, such as osteoprogenitor cells.⁸⁹ Importantly, EndMT is a specific subtype of the broader epithelial-to-mesenchymal transition (EMT) program.⁸⁴ EMT has been particularly well described in renal fibrosis, with current studies indicating that the cellular transition that occurs is only partial, such that EMT gives rise to fibroblast-like cells but not fully mature fibroblasts.^91,92 We identified a similar phenomenon with EndMT during vein graft remodeling, where EndMT gave rise to immature VSMCs that did not express the full array of mature smooth muscle cell contractile proteins.⁸⁶ As a common theme of these studies, EndMT has typically been associated with disease progression, and there appear to be multiple therapeutic opportunities for blocking this program in an effort to decrease disease severity.

Vascular Smooth Muscle Cell Contraction

Contraction and relaxation of the vascular wall is mediated by changes in medial VSMC tone, which as already mentioned can be affected by various signaling molecules, hormones, and other factors. As with the endothelium, similar biochemical pathways are activated through vasoactive agonists, with the ultimate smooth muscle cell contractile response determined also by the vascular microenvironment and macroenvironment.

Initial stimulation by vasoactive agonists leads to hydrolysis of phosphoinositides by phospholipase C, which results in generation of inositol triphosphate (IP3) and diacylglycerol (DAG).⁹³ IP3 conducts downstream signaling by binding an IP3 receptor on intracellular organelles with calcium stores that can be easily mobilized into the cytoplasm; release of calcium granules initiates downstream signaling that culminates in VSMC contraction.⁹⁴ IP3 activity is modulated by binding of IP3 to its receptor in addition to phosphorylation and binding of ATP and NADH.⁹⁵ Intracellular calcium stores are maintained by influx through L-type calcium voltage channels and transient receptor potential.⁹⁶ Calcium-mediated downstream signaling is disrupted with reuptake of calcium into the endoplasmic reticulum through sarcoplasmic reticulum calcium-ATPase and capture of calcium via calcium-ATPase on the plasma membrane.⁹⁷ Activation of phospholipase C also leads to generation of DAG, an activator of protein kinase C, an important calcium- and phospholipid-dependent enzyme that enhances contraction with elevated levels of intracellular calcium.⁹⁸ Metabolism of DAG produces glycerol, fatty acids, and ultimately leukotrienes and eicosanoids that also modulate VSMC tone.

Different vasoactive agonists induce differing magnitudes and lengths of contraction. There are two phases of smooth muscle contraction: an initial rapid component and a more sustained phase of contraction. The initial phase of contraction is dependent on the formation of actin-myosin cross-bridges in response to elevated levels of intracellular calcium; sustained contraction is present after the initial acute increase in intracellular calcium. Phasic contraction of smooth muscle is thought to be mediated by a sliding filament mechanism. Development of tension is regulated by myosin light-chain kinase (MLCK)-mediated phosphorylation of the myosin light chain (MLC) (Fig. 7–6). Hormonal stimulation leads to increased intracellular calcium, calmodulin binding, and association with MLCK and its transitioning to an active form. MLCK phosphorylates MLC, with subsequent actin activation of magnesium-ATPase and cross-bridge formation. With sequestration of calcium, there is a dissociation of calcium from calmodulin and subsequent deactivation of MLCK. Dephosphorylation of myosin via myosin phosphatase leads to cessation of cross-bridge action. Of note, during sustained contraction, there is a low calcium concentration and low-energy consumption state, which suggests there is a low cycling state. This state of slow, tonic contraction that achieves high levels of force with low energy expenditure is referred to as the latch state or latch-bridge state. This latch phenomenon is generally thought to be largely caused by the dephosphorylation of myosin during its attachment to actin. However, unphosphorylated myosin can bind to actin and, although it may not be actively cycling, it likely contributes to force maintenance.⁹⁹ In turn, caldesmon enhances the binding force of unphosphorylated myosin to actin potentially contributing to the latch state, while extracellular signal-regulated kinase (ERK) phosphorylation of caldesmon decreases this binding force to very low levels.¹⁰⁰

Contractility is also modulated by the small-molecular-weight guanosine triphosphatase (GTPase) Rho. The active Rho form (GTP bound) activates Rho kinase, which subsequently inhibits myosin phosphatase type 1.¹⁰¹ This leads to a sustained MLC phosphorylation and sensitization of the contractile apparatus to calcium. Increased levels of Rho have been noted in hypertension, making it a therapeutic target.¹⁰² Rho kinase inhibitors have been shown to lower vascular resistance and blood pressure in animal models and may have efficacy as novel antihypertensive agents.^103,104

VSMC contractile signaling is counteracted by relaxation stimuli, such as intracellular cyclic nucleotides, including cyclic AMP and GMP. NO binds to guanylate cyclase in VSMCs, leading to a reduction in intracellular calcium and reduction of MLC phosphorylation. Cyclic nucleotides are degraded by 3′,5′-cyclic nucleotide phosphodiesterases (PDEs).¹⁰⁵ These are the target of multiple therapeutic agents, including PDE type 5 inhibitors such as sildenafil, that lead to increased cGMP levels. On the basis of their vasodilatory effects in the pulmonary circulation, PDE type 5 inhibitors are now widely used in the treatment of primary pulmonary hypertension.⁵⁸

Regulation of Vascular Smooth Muscle Cell Growth and Hypertrophy

In a nonpathological state, the VSMC is relatively refractory to growth stimuli and exists in a quiescent, differentiated, “contractile” state.⁹⁷ VSMC growth and proliferation is inhibited by a range of factors, including NO, prostacyclin, heparan sulfate, and transforming growth factor beta (TGF-β).^106,107,108 Although, depending on the context and strength of stimulus, TGF-β activity may also promote proliferation and phenotypic changes. VSMCs are maintained in a differentiated contractile state through signaling mediated by cyclic GMP-dependent protein kinase G. VSMCs are partially shielded from circulating factors through the protective endothelium. Disruption of the endothelial barrier leads to exposure of the VSMCs to promitogenic factors such as PDGF and thrombin, which leads to a mitogenic response and switch to the “synthetic” phenotype; repair of the endothelium inhibits further proliferation.

In pathological states, the vascular environment favors VSMC switching to the “synthetic” phenotype. Synthetic VSMCs are noted for their downregulation of contractile proteins/filaments, increased proliferation, and migratory capacity.⁹⁷ They are classically implicated in neointimal hyperplasia (see later section). Alternatively, degradation of the ECM also leads to a synthetic VSMC phenotype.¹⁰⁹ This is mediated through metalloproteinases released by activated cells within the vascular wall or infiltrated inflammatory cells. Agents that promote phenotypic switching from the quiescent contractile to the active synthetic state may be secreted by intrinsic vascular cells or invading immune cells. PDGF is a potent promitogenic agent involved in VSMC switching to the synthetic state; it is composed of two peptide chains (A and B) and can be secreted in multiple forms (heterodimer AB, or homodimer AA or BB). Various growth hormones regulate the release of PDGF from endothelial cells, including TGF-β, FGF, TNF, and thrombin.¹¹⁰ PDGF promotes growth through suppression of the contractile phenotype¹¹¹ and activation of signaling pathways described above for vasoconstrictive agonists: hydrolysis of phosphoinositide, mobilization of calcium in addition to influx, and activation of protein kinase C. Insulin growth factor 1 (IGF-1) is another growth factor that facilitates movement of cells through the growth cycle and enhances PDGF-mediated mitogenic effects on VSMCs.¹¹² In turn, PDGF regulates IGF-1 production by endothelial cells.¹¹³ Smooth muscle proliferation is also modulated by IL-1, FGF, and endothelin. The inflammatory cytokine IL-1 has many vascular effects, including effects on mitogenesis.¹¹⁴ FGF is a potent agonist of the VSMC mitogenic response, which is particularly notable during neointimal hyperplasia.¹¹⁵ FGF is found within the subendothelial matrix, and its release is triggered by heparin and proteinases.¹¹⁶ Smooth muscle cell release of FGF is thought to be a key response to arterial injury. Endothelin-1 may also induce VSMC growth through binding at the ET-A receptor and downstream elevation in intracellular calcium, activation of protein kinase C, and increase in ROS production. Endothelin-1 production is modulated via hyperinsulinemia,¹¹⁷ shear stress and pressure,¹¹⁸ and angiotensin II.¹¹⁹

In terms of downstream intracellular signaling, VSMC stimulation by the previously mentioned factors leads to recruitment of different protein complexes that subsequently activate multiple cellular signaling cascades. Following activation, many growth factor receptors dimerize and self-phosphorylate tyrosine residues. Different protein complexes either bind directly to tyrosine kinase (phospholipase C-γ, the proto-oncogene c-Src, phosphatidylinositol 3-kinase) or through linker proteins Grb and Shc (pyk-2 paxillin). Grb and Shc link receptors to Ras (ubiquitous GTPase), which initiates a serine/threonine kinase signaling cascade involving mitogen-activated protein kinases (MAPKs), with the final response being VSMC growth (Fig. 7–7). Another kinase important to this response is c-Abl, an Src substrate that activates Rac, with subsequent effects on cytoskeletal rearrangement.¹²⁰ These protein complexes are also activated through G protein–coupled receptors.^121,122 This is thought to be the mechanism of action for the observed growth-promoting properties of vasoconstrictive agonists such as angiotensin II, thrombin, and ET-1. PDGF and other classic growth factors promote cell-cycle progression and proliferation through activation of cell-cycle proteins and inhibition of cell-cycle inhibitors. Hypertrophy-enhancing agents such as angiotensin have a more modest effect on cell-cycle proteins such as cyclins and enhance a cell cycle inhibitor.¹²³

Another regulator of the growth pathway is ROS. Various growth-promoting agonists stimulate NADPH oxidases Nox1 and Nox5,^124,125 with production of hydrogen peroxide and superoxide, which can give rise to other ROS.^126,127 Hydrogen peroxide activates mitogen signaling molecules such as Src, Ras, p38MAPK, and Akt/protein kinase B, thus promoting entry into the cell cycle.^{128,129,130,131,132} Hydrogen peroxide also inactivates protein tyrosine phosphatases, leading to prolonged phosphorylation of key signaling molecules.¹³³

Notably, VSMC growth may occur through hyperplasia and/or hypertrophy. Classic growth factors stimulate hyperplasia, which is associated with the switch to an active synthetic phenotype. On the other hand, hypertrophy is a response to chronic stimulation with vasoconstrictor agonists. Hypertrophy is secondary to an increase in smooth muscle cell mass with increase in protein synthesis and no accompanying replication of DNA. For example, this can be seen in response to angiotensin II and thrombin^134,135 or as a pathologic response to hypertension in large vessels.

Noncoding RNAs (ncRNAs) are a group of various types of RNA moieties that, in general, are not translated into proteins. As a relatively recent but very major paradigm shift, ncRNAs are being increasingly recognized as important regulators of gene and protein expression. The subtypes of ncRNAs and their mechanisms of action are comprehensively described in Chapter 6 and Figs. 6–7, 6–8, 6–9. In the vasculature, our knowledge of ncRNAs is in its infancy—but growing rapidly. Already, it is clear that ncRNAs are major players in the full spectrum of vascular biology, including all anatomical layers considered in this chapter (eg, endothelium, smooth muscle cells, intima, media, adventitia). Furthermore, various ncRNAs have been identified that are stable outside the cellular milieu in the blood. Specifically, circulating microRNAs (miRNA, a very short ncRNA moiety of ~22 nucleotides) exist in a stable extracellular form, and strong associations have been identified between the levels of specific miRNAs and various cardiovascular diseases, such as acute myocardial infarction, coronary artery disease, heart failure, stroke, and hypertension.¹³⁶ Mechanistically, it is also being quickly appreciated that ncRNAs play a major role in the biology of the vessel wall. However, at the present time, our knowledge of these ncRNA pathways is obviously quite incomplete and appears more as an isolated potpourri of effects, rather than an integrated understanding. Nevertheless, the fact that certain ncRNA moieties can exist in the extracellular compartment has significant implications, because it means they can act as messengers between cells or even between organs within the body, akin to circulating hormones or cytokines.¹³⁷ Many hundreds or even thousands of ncRNAs are likely implicated in the biology of the vessel wall. The study of ncRNAs currently represents one of the areas most intensely researched in vascular biology. A summary of several recent studies describing a complete pathway of action of specific vascular miRNAs is presented in Fig. 7–8.

ECM is a major component of the vascular wall. It functions as a medium for transport of nutrients; a receptacle for secreted substances from surrounding cellular structures; and a mediator of migration and proliferation of VSMCs, endothelial cells, and monocytes. The ECM has various components, each of which has distinct roles in maintaining the integrity and function of the vasculature, summarized in Table 7–2. The interactions between the different components of the ECM and vascular cells comprise a complex, large signaling network involving numerous signaling receptors. These signaling networks ultimately affect various components of vascular cell development and biology, including response to hemodynamics, organ development, hypertrophy, angiogenesis, and development of atherosclerotic plaques.

The degradation and reformation of the ECM is an important component of multiple processes that maintain vascular wall function. Vascular cell hypertrophy, proliferation, and migration are dependent on initial degradation of the ECM. One of the early events in angiogenesis includes the degradation of the ECM to allow for capillary formation. Enzymes that selectively digest individual components of the ECM are known as metalloproteinases (MMPs); these are secreted by inflammatory and vascular cells (including endothelial cells, VSMCs, fibroblasts, and resident macrophages). In addition, these same cell populations also produce counterregulatory tissue inhibitors of metalloproteinases (TIMPs), which promote ECM stability.

MMPs belong to one of two classes: secreted or membrane-spanning (with an active site outside the cell). Secreted MMPs are divided into several groups: type IV collagenases (also referred to as gelatinases), stromelysins, and interstitial collagenases. Secreted MMPs are produced as inactive zymogens that can be activated by plasmin. Cytokines also regulate MMP and TIMP activity at a transcriptional and post-transcriptional level. The membrane-spanning MMPs are made up of disintegrin and metalloproteinase domain proteins. These are multifunctional proteins that contribute to cell adhesion as well as degrading matrix components to release paracrine factors, such as monocyte colony stimulating factor, TNF-alpha, Fas ligand, and Notch.¹³⁸

MMP secretion and expression are regulated at multiple levels, with inflammation being a major global pro-MMP stimulus. Cytokine stimulation has been shown to increase expression and secretion of MMPs by VSMCs (MMP-1, MMP-9, MMP-3, MMP-2 zymogen), which facilitates degradation of major matrix components.^139,140,141 In microvascular and venous endothelial cells, MMP-3 and MMP-9 expression is also induced by cytokines.¹⁴² The overall net effect of cytokines in the vascular wall appears to be a shift in balance between MMPs and TIMPs, with an end result of ECM degradation and remodeling. ROS have been shown to stimulate activation and expression of MMP-9.^143,144 This is thought to be significant in vascular pathologies that have increased levels of oxidative stress, such as atherosclerosis and hypertension. Importantly, TIMPs and MMPs play a pivotal role in the pathology of atherosclerotic plaques and aneurysms.^141,145 MMPs are highly expressed in the shoulder regions of atherosclerotic plaques¹⁴⁶ and predipose to plaque rupture. Although produced by several cell types, as noted, in atherosclerosis, activated macrophages are especially notable for their production of MMPs and ROS.^143,146 Abdominal aneurysms also have an abundance of MMP activity.^141,147 Both vascular pathologies are mediated by oxidative stress, inflammation, and altered hemodynamics, which also likely contribute to the expression and activation of MMPs. Of note, expression of MMPs has also been shown in the aneurysms of patients with Marfan syndrome.¹⁴⁸

The pathobiology of most vascular diseases is notable for a complex interplay of all the previously mentioned cellular players and molecular pathways. Here we study several key disease states to further explore these relationships and their role in promoting disease.

Atherosclerosis and Endothelial Dysfunction

In many vascular diseases, there is notable endothelial dysfunction, which may manifest as loss of vasodilator response, prothrombotic events, initiation of leukocyte adhesion, and/or stimulation of VSMC migration and proliferation. However, this process is especially important in atherosclerosis, with endothelial dysfunction being clinically detectable before the macroscopic presence of formed atherosclerotic plaques. Among the many reports to demonstrate an association between endothelial dysfunction and vascular disease are several seminal clinical studies from the 1990s that demonstrated associations between endothelium-dependent arterial vasoreactivity and various cardiovascular risk states, such as diabetes,¹⁴⁹ cigarette smoking,¹⁵⁰ and positive family history.¹⁵¹ The fact that endothelium-dependent arterial vasoreactivity is an early event in atherosclerosis has been reinforced by a multitude of basic mechanistic studies. One of the hallmarks of impaired endothelium-dependent arterial vasoreactivity is impaired eNOS activation, with resulting decreased NO production.¹⁵² Destabilization of eNOS mRNA under conditions of endothelial cell stress is an additional mechanism that leads to decreased NO production.¹⁵³ Inhibition of Rho GTPase through statins prevents this destabilization, which adds to the mechanisms through which HMG CoA reductase inhibitors mediate vasculoprotective effects.¹⁵⁴

Recruitment of monocytes and macrophages into the vessel wall is another aspect of endothelial dysfunction.¹⁵⁵ LDL modification occurs when the subintimal space becomes more oxidized. Modified LDL then activates various toll-like receptors (TLRs), including TLR-4, with subsequent induction of proinflammatory gene expression and infiltration of macrophages. TLR-4 antagonism reduces infiltration of monocytes/macrophages and expression of IL-6 and MMP-9 in atherosclerotic lesions of diabetic mice.¹⁵⁶ Adhesion molecule expression is amplified through secretion of inflammatory cytokines by activated macrophages and vascular cells. In humans, polymorphisms of the fractalkine receptor CX3CR1 have been demonstrated to alter adhesive and chemoattractant properties, influencing the likelihood of developing atherosclerosis and acute coronary syndromes.¹⁵⁷

Intimal proliferation in atherosclerosis is secondary to migration and hyperplasia of VSMCs and myofibroblasts, in addition to ECM accumulation. Growth factors, including PDGF, FGF, and IGF-1, are thought to contribute to proliferation, which is also influenced by less well understood factors, such as disruption of the integrity of the internal elastic lamellae.¹⁵⁸ These signaling pathways also become dysfunctional in the atherosclerotic endothelium, which can further enhance plaque progression.⁸⁵

Hypertension

Hypertension is characterized by dysfunction of the endothelium and VSMCs. In chronic disease, there is impairment in endothelium-dependent relaxation, as well as increased endothelium-dependent constrictor activity, which is influenced by endothelial peroxisome proliferator-activated receptor gamma (PPARγ) and systemic angiotensin signaling.¹⁵⁹ Vessel wall mass is increased, with acute hypertension leading to thickening of the medial and adventitial layers and with significant adventitial hyperplasia.¹⁶⁰ VSMCs are an integral aspect of this response, with both smooth muscle hyperplasia and hypertrophy being more or less prominent depending on arterial size and muscularity.¹⁶¹ Interestingly, nestin is an intermediate filament protein in smooth muscle cells that participates in the regulation of hypertension-mediated hyperplasia,¹⁶¹ which is also governed by the cAMP-PKA-EGFR-CREB/ERK pathway.¹⁶² However, the smooth muscle cell hypertrophic response to hypertension appears to be controlled by alternate signaling pathways.¹⁶¹ Phenotypic VSMC modulation also occurs in response to hypertension, with altered smooth muscle contractile protein expression.¹⁶³

Neointimal Hyperplasia

Neointimal hyperplasia is a hyperproliferative VSMC response that arises in the weeks to months following acute arterial injury, although it also contributes to other pathologies, such as pulmonary arterial remodeling in pulmonary hypertension and during vein graft remodeling on exposure of the donor segment of venous vessel to systemic arterial pressures. From the 1980s to the 2000s, intense efforts were devoted to understanding neointimal hyperplasia because it is the pathological process responsible for restenosis that arises following percutaneous coronary intervention (PCI; balloon angioplasty and stenting). In the setting of PCI, acute arterial injury arises from the physical trauma inflicted on the host vasculature by ballooning and stenting, which includes endothelial denudation, overstretch injury, and disruption of the elastic lamellae.

The cardinal feature of neointimal hyperplasia is VSMC proliferation. As stated, resting VSMCs exist in the “contractile state,” expressing high levels of contractile proteins and reduced levels of inflammatory markers. On arterial injury, VSMCs undergo a phenotypic switch from the contractile to synthetic state, with reduced expression of contractile proteins and enhanced matrix and collagen expression. It is these synthetic VSMCs that largely comprise the neointima.¹⁶⁴ Neointimal formation occurs on the luminal side of the innermost aspect of the arterial elastic laminae, this being the intimal layer. Importantly, in healthy vessels, the intima is only one to two cells thick and comprised of endothelial cells and sometimes a subendothelial pericyte cell layer.¹³ Exuberant VSMC proliferation with collagen and matrix production in this intimal layer, in this context called the neointima, may lead to significant neointimal formation with luminal narrowing.¹⁶⁵ In the context of clinical PCI, this may culminate in coronary stenosis or even complete coronary artery occlusion.

Interestingly, on acute arterial injury, a series of very early events arise that appear to dictate the subsequent hyperplastic response. One of the earliest events after acute arterial injury is rapid expression of MCP-1, RANTES, and other inflammatory cytokines by medial VSMCs.¹⁶⁶ This surge in expression of inflammatory and chemotactic cytokines triggers an early influx of inflammatory cells, including monocytes and T cells, which then orchestrate the injury repair and hyperplastic response.¹⁶⁶ However, many medial VSMCs succumb to the intense cellular and oxidative stresses and undergo cell apoptosis and death. Next, days to weeks after the initial injury, lineage tracking studies have shown that some of the surviving medial VSMCs are able to proliferate and give rise to new synthetic VSMCs that both repopulate the media and also give rise to the neointima.^165,167 The extent of neointimal hyperplasia is regulated by typical signaling proteins known to regulate VSMCs, such as PDGF ligand, TGF-β, FGF, stromal cell-derived factor 1 (SDF-1), and RANTES,^166,168 with higher order regulation from a range of factors, including p21^Cip1, p27, and p53.^168,169 Other factors that may dictate the extent of neointimal formation include the severity of the initial injury, whether or not the elastic laminae are fully disrupted and there is exposure of the underlying adventitia,¹⁵⁸ whether there is concurrent atherosclerosis, and how rapidly the endothelial layer is reestablished.¹⁷⁰

Neointimal hyperplasia was a major clinical concern in the era of stand-alone balloon angioplasty and then the bare metal stent era, where clinically significant restenosis arose in 30% to 40% of lesions that had previously undergone intervention. However, in the contemporary era of drug-eluting stents (DES), clinically significant restenosis arises in only 5% to 10% of treated lesions.^171,172 Unlike bare metal stents, DES are loaded with specific drugs, often related to chemotherapeutic agents such as everolimus or sirolimus, with the primary effect of reducing VSMC hyperplasia. However, inhibition of reendothelialization was a detrimental off-target effect that was associated with stent thrombosis. Thankfully, with improved stent delivery platforms with thinner struts and better optimized drugs with improved release kinetics, the delayed endothelial healing seen with first-generation DES (Cypher and Taxus) has largely been overcome.^171,173 In turn, it is believed that this earlier reendothelialization that occurs with second-generation DES helps to further slow the intimal hyperplastic response.¹⁷⁰ Thus, it is pleasing that the clinical impact of neointimal hyperplasia has been dramatically reduced in recent years, attesting also to the vital importance of the well-conducted translational research that facilitated this progress.

During vein graft remodeling, when segments of vein must adapt to systemic arterial pressures, such as in coronary artery bypass graft surgery or arteriovenous fistula formation, neointimal hyperplasia also arises. Interestingly, unlike acute injury where the endothelium is denuded, the pathobiology of intimal hyperplasia in vein graft remodeling appears to involve EndMT, with endothelial cells switching phenotype and giving rise to synthetic VSMCs under the governance of TGF-β pathway signaling.⁸⁶

Diabetes

Diabetes has profound effects on the biology of the vessel wall, including the endothelium and VSMCs.^174,175 In the endothelium, diabetes increases the production of ROS and reduces available NO. This leads to impaired endothelium-dependent vasodilatation. However, additional mechanisms of diabetes-associated endothelial dysfunction are multifold. Vascular expression of proinflammatory genes is elevated in diabetes.¹⁷⁶ Diabetes and the metabolic syndrome also cause a range of detrimental changes in blood clotting, including increased clotting factor formation, low protein C levels, increased tendency for platelet aggregation, altered plasminogen activator inhibitor-1 levels, increased thrombin formation, hypofibrinolysis, and a prothrombotic fibrin clot phenotype.^177,178,179 The production of advanced glycated end products acting on endothelium, macrophages, and VSMCs is another final common pathway in diabetes that also increases the rate of progression of vascular disease. Histopathologically, in larger conduit vessels these events contribute to the formation of atherosclerotic lesions, but in resistance arteries there is arteriopathy, characterized by wall thickening, luminal narrowing, inflammation, and remodeling. This is seen in the associated vascular manifestations of diabetes, including retinopathy, nephropathy, and ischemia of peripheral tissues.¹⁷⁴ An overview of the key pathologic diabetic factors affecting the vasculature is shown in Fig. 7–9.

The vascular wall contains several populations of resident vascular progenitor cells that shall be briefly reviewed below. As an important note, the cardiovascular community has devoted intense efforts into defining the origins and role of endogenous stem cells in the heart, and despite these efforts, numerous controversies and unanswered questions remain. The progenitor populations occupying the vascular wall are even less well understood, and we expect that significant advances will be made on this front in the near future.

Endothelial Progenitor Cells

Although there has been intense debate regarding the existence of circulating endothelial progenitor cells (EPCs),¹⁰ presumed to have arisen from the bone marrow, an overlooked potential location for EPCs is the vessel wall itself. Indeed, in vitro, most vessel-derived endothelial populations may be passaged several times, attesting to their proliferative capacity. Whether all endothelial cells possess inherent capacity for a degree of expansion and proliferation, or whether there is a discrete EPC niche in the vessel wall, remains to be rigorously defined. However, Ingram et al previously demonstrated that the vessel wall contains a complete hierarchy of EPCs, which can be defined both their clonogenic and proliferative potential.¹⁸⁰ Subsequently, Naito et al went on to define an EPC population that resides among other non-EPCs of the vessel wall. These resident progenitor endothelial cells comprise approximately 1% of all endothelial cells, are not derived from the bone marrow, and reside predominantly in veins and capillaries.¹⁸¹

Pericytes and Mesenchymal Stem Cells

Increasingly, data are emerging to suggest that pericytes represent a type of mesenchymal stem (also called stromal) cell (MSC).^13,182 Although there is little question now that pericytes fulfill the formal criteria to be termed MSCs, including differentiation into adipocytes, chondrocytes, and osteocytes,¹⁸² their role in vascular biology remains to be fully defined. Specifically, it is very well described that the investment of endothelial cells by pericytes is fundamental for the stabilization and maturation of new vessels.¹⁸³ Neither has the dependency of this vessel-stabilizing effect on MSC characteristics been defined, nor has the possibility that MSCs may contribute in other ways to vascular biology.

Medial Vascular Progenitor Cells

It has long been suspected that VSMC progenitor cells reside in the medial layer, although the presence of any VSMC progenitor must be reconciled against the intrinsic capacity of VSMCs for proliferation and phenotypic switching. Recently, Tang et al¹⁸⁴ described the existence of a progenitor population residing in the outer aspect of the medial layer and that gave rise to VSMCs. However, these findings subsequently generated significant controversy¹⁸⁵ and remain to be replicated.

Adventitial Progenitor Cells

Perhaps the most convincing data for the existence of resident vascular progenitor cells is from the adventitia. A series of studies from Majetsky et al showed that a murine stem cell antigen-1 (Sca-1+) population resides in the adventitia and that it was potent in vitro for giving rise to smooth muscle cells and endothelial cells.¹⁸⁶ Using indirect methods, it was suggested that Sca-1+ progenitor cells can contribute to atherosclerotic plaque formation.¹⁸⁷ More recent data have emerged using Gli-1 as a marker of adventitial MSC-like cells.¹⁸⁸ Gli-1+ adventitial progenitor cells coexpress Sca-1 and can give rise to myofibroblasts that contribute substantially to organ fibrosis (Fig. 7–10). Accordingly, ablation of Gli-1+ cells substantially reduces organ fibrosis.¹⁸⁸ Interestingly, additional studies have also suggested that the adult adventitia may also harbor a resident macrophage progenitor cell population.¹⁸⁹ Whether or not resident adventitial progenitor cells can be harnessed for therapeutic applications requires further study.

Major clinical treatment advances that have impacted patient care have been made in the last decades, which owe their success to a core understanding of the biology of the vessel wall. Notable among these are major reductions in post-PCI restenosis by attenuating neointimal hyperplasia,¹⁷¹ tailoring of antiplatelet therapies after PCI based on an understanding of reendothelialization and its kinetics after PCI,¹⁹⁰ anti-VEGF therapies that block angiogenesis and thereby exert antitumor effects,¹⁹¹ and the expanded arsenal of vasodilator therapies that are now available for use in patients with pulmonary hypertension.⁵⁸ Yet much still remains to be understood, including the details of EndMT and how it can be manipulated for therapeutic gain, resident vascular progenitor cells and their role in health and disease, and the complex interactions at the systemic level between the vasculature, the heart and the entire human organism (see Fig. 7–1). By understanding these and other aspects of vessel biology, untold therapeutic opportunities are likely to be disclosed. In sum, the importance of a thorough understanding of the vessel wall in health and disease cannot be gainsaid, and we foresee redoubled efforts in this field in the coming years.

Chapter 7 is dedicated in loving memory of Cyril Kovacic (1937-2016), who passed away during the final stages of drafting this chapter. We acknowledge the authors of the most recent previous versions of this chapter, Drs. Kathy K. Griendling, David G. Harrison, and R. Wayne Alexander, for their inspirational contribution. Jason Kovacic receives research support from the National Institutes of Health (K08HL111330 and R01HL130423), the Foundation Leducq (Transatlantic Network of Excellence), the American Heart Association (14SFRN20840000), and Astra Zeneca.