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
Anatomy of a medium-large sized artery in the adult. A. Four-color confocal microscope image of a mouse femoral artery. Staining was with 4′,6-diamidino-2-phenylindole (DAPI) shown in blue to mark nuclei, anti-SM22α to mark smooth muscle cells in red, and CD31 to mark endothelial cells in white. The internal and external elastic laminae (shown in green) were not stained, but were imaged using autofluoresence emitted when excited with a 488-nm laser line. B. Schematic representation of adjacent confocal image identifying vascular anatomic structures/layers. Reproduced with permission from Kovacic JC and Boehm M. Resident vascular progenitor cells: an emerging role for non-terminally differentiated vessel-resident cells in vascular biology, Stem Cell Res. 2009 Jan;2(1):2-15.13
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.
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, which are vesicles that contain the structural protein caveolin that 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, which 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.
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.
Pathways of thrombosis and thrombolysis. Under normal conditions, the endothelium is antithrombotic. Antithrombin III (AT III) binds thrombin and serves to clear thrombin from the circulation. Prostacyclin (prostaglandin I2 [PGI2]) inhibits platelet aggregation, and thrombomodulin (TM) activates protein C, which inhibits plasminogen activator inhibitor 1 (PAI-1) and interacts with protein S to inactivate activated factors V and VIII, thus limiting thrombosis. Because PAI-1 inhibits the tissue plasminogen activator (PA)–catalyzed conversion of plasminogen to plasmin, PAI-1 inhibition leads to accumulation of plasmin and fibrinolysis. With stimulation with inflammatory cytokines, there is increased expression of tissue factor on the endothelial cell surface. Tissue factor participates in the activation of factor X which, in turn, promotes assembly of the prothrombinase complex, producing thrombin. Under these conditions, endothelial cells thus amplify the thrombotic response. Used with permission from Bernard Lassègue, PhD.
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 with 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
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.
TABLE 7–1.Major Vasoactive Factors Produced by the Endothelium |Favorite Table|Download (.pdf) TABLE 7–1. Major Vasoactive Factors Produced by the Endothelium
|Main Vasoactive Factors ||Action |
|Prostacyclin, nitric oxide, endothelial derived hyperpolarizing factor ||Vasodilation |
|Endothelin, prostanoids ||Vasoconstriction |
|Factor VIII antigen, von Willebrand factor, tissue factor, thrombomodulin, and tissue plasminogen activator ||Key regulators of the coagulation/fibrinolytic system |
|Collagen, elastin, glycosaminoglycans, fibronectin ||Maintaining the structural components of the extracellular matrix |
|Matrix metalloproteinases, tissue inhibitors of metalloproteinases ||Regulation of extracellular matrix, degradation and remodeling |
|Heparans and growth factors ||Smooth muscle cell proliferation |
|Lipoprotein lipase, low-density lipoprotein (LDL) receptor, scavenger LDL receptor ||Lipid metabolism |
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 type I), macrophages (iNOS, inducible NOS type II), and endothelial cells (eNOS, endothelial 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 a 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 it 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, 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
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.
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 (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
Endothelial cells use mechanosensing to transduce the frictional force from blood flow (fluid shear stress) into biochemical and cellular signals, and 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 (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.
Key aspects of the molecular and cellular changes that occur with endothelial-to-mesenchymal transition. BM (black line), basement membrane; DDR2, discoidin domain receptor 2; EMT, epithelial-to-mesenchymal transition; FSP1, fibroblast specific protein 1; MMPs, matrix metalloproteinases; αSMA, alpha-smooth muscle actin. Reproduced with permission from Kovacic JC, Mercader N, Torres M, et al: Epithelial-to-mesenchymal and endothelial-to-mesenchymal transition: from cardiovascular development to disease, Circulation. 2012 Apr 10;125(14):1795-1808.84