Endothelial regrowth after arterial injury: from vascular repair to therapeutics

Time for primary review 32 days. Interventional strategies for patients with coronary artery disease such as percutaneous transluminal coronary angioplasty or coronary stent implantation invariably result in a marked degree of vascular injury (deendothelialization, mechanical damage to the medial and adventitial layers) [1–3]. The loss of the endothelial monolayer is associated with a variety of deleterious consequences such as thrombus formation, neointimal thickening, and abnormal responses to endothelium-dependent agonists [3–5]. These effects may contribute to each of the known limitations of these techniques (vessel occlusion, restenosis, coronary spasm). The objective of the present review will be to present applied reendothelialization as a possible treatment for patients with coronary artery disease undergoing mechanical revascularization. Our aim will be twofold: first, to summarize current knowledge on the beneficial effect of endothelial regrowth after arterial injury; second, to discuss the potential role of growth factors or other compounds to accelerate reendothelialization. Occupying a strategically important location between circulating blood and tissues and having the ability to respond to changes in its physical, chemical, and humoral environment by the production of bioactive substances, the normal endothelium controls the tone and the proliferative state of the underlying vascular smooth muscle cells (VSMCs) and maintains a non-adhesive luminal surface. Modulation of VSMC tone is mediated by the synthesis and release of endothelium-derived relaxing and contracting factors. The proliferative state of VSMCs is controlled through these endothelium-derived factors and through the secretion of matrix proteins. Anticoagulant, fibrinolytic, and antithrombotic properties of the endothelium contribute to the maintenance of the fluidity of the blood. Prostacyclin (PGI2), a potent vasorelaxant and platelet-inhibitory metabolite of arachidonic acid was the first endothelium-derived vasoactive factor discovered [6]. After the seminal observation of Furchgott and Zawadski in 1980 [7]that an intact endothelial layer was required for acetylcholine-induced vasorelaxation, further studies demonstrated that a labile, diffusible, nonprostanoid substance called endothelium-derived relaxing factor (EDRF) mediates endothelium-dependent vasorelaxation [8]. Based on the striking similarities between nitric oxide (NO) and EDRF it was postulated that EDRF and NO were identical [9]. Measurement of NO production by endothelial cells provided direct evidence for this hypothesis [10]. The endothelial cell (EC) synthesizes EDRF-NO from the terminal guanido nitrogen atom(s) of l-arginine [11]. The enzyme (NO-synthase) responsible for this reaction has been isolated and characterized [12]. Several isoforms of NO-synthase were identified: two isoforms, present in endothelial (eNOS) and neuronal cells (nNOS), which require calcium and calmodulin, and a form (iNOS) which is Ca2+-independent and can be observed in immunoactivated cells, including ECs and VSMCs, after treatment with endotoxin, tumor necrosis factor (TNF) or other cytokines [13]. The arginine-NO signaling pathway appears thus to be ubiquitous, and may contribute to a variety of biological functions [13]. In addition to EDRF-NO, the existence of other nonprostanoid relaxing mediators [14], including an endothelium-derived hyperpolarizing factor (EDHF) [15], has been proposed. It has been postulated that EDHF may be an endogenous activator of potassium channels in VSMCs [15]. The endothelium-derived contracting factors mediating endothelium-dependent vasoconstriction belong to two categories: (a) vasoconstrictor metabolites of arachidonic acid (TXA2, endoperoxides) and (b) a potent peptidergic vasoconstrictor substance named endothelin-1 [16–18]. Endothelin-1 acts on vascular smooth muscle through endothelin-A receptors and is the most potent endogenous vasoconstrictor yet identified. Endothelin-1 is not stored in endothelial cell but m-RNA expression, protein synthesis and secretion are induced within minutes by appropriate stimuli such as angiotensin II, catecholamines, growth factors (basic FGF) or thrombin [18]. The production of endothelin-1 is inhibited by nitric oxide and prostacyclin [19, 20]. In addition, angiotensin II (Ang II) may also be regarded as an endothelium-derived contracting factor: angiotensin converting enzyme (ACE) has been localized in the ECs of capillaries and large arteries and vein throughout the vasculature [21], while bovine aortic cells in culture express renin and are able to synthesize Ang II intracellularly [22, 23]. In addition, the production of superoxide anion, elicited in ECs through an NADH-dependent pathway [24], may also be considered, by its effects on EDRF-NO, as a contracting agent. Indeed EDRF is rapidly inactivated by superoxides [25, 26]while superoxide dismutase reverses this effect [27, 28]. The endothelium produces a number of molecules that stimulate VSMC proliferation (e.g., endothelin, platelet derived growth factor,…) [29, 30]and is also responsible for the modulation of compounds such as angiotensin that have been implicated in growth-related metabolism [31, 32]. Because VSMCs in the mature, healthy blood vessel wall exhibit minimal proliferation activity [33, 34], the actions of these growth promoters must be countered by factors that inhibit such metabolism. The endothelium is the likely source of such molecules because, when damaged, VSMC proliferation is an almost immediate response [35, 36]. This observation suggests that the intact endothelium produces both growth promoters and inhibitors in a balanced, coordinate manner. It has been shown for some time that ECs are a source of heparin and heparin sulfates, which do have growth-inhibitory properties. More recently, it has also been suggested that EDRF-NO also acts as a powerful growth modulator. Growth inhibition of VSMCs, when in coculture with ECs, was primarily ascribed to the actions of endothelium-derived heparin/heparan sulfate proteoglycans [37–39]. Addition of heparin or heparan sulfate glycosaminoglycans to tissue culture medium inhibits VSMC growth [38–40]. Infusion of heparin reduces neointimal proliferation in balloon abraded arteries [41]. In vitro and in vivo studies suggest that heparin may prevent VSMC entry into the S phase of the cell cycle [42, 43]. The intracellular increase in cyclic GMP, a part of the signal transduction of EDRF-NO, and NO donors depress the incorporation of 3H-thymidine in DNA in cultured VSMCs [44]. The physiologic relevance of this effect was corroborated by results of co-culture experiments in which the inhibitory effect of ECs on VSMC growth was increased when cultured cells were incubated with l-arginine, the substrate for EDRF-NO synthesis, while this effect was decreased when cells were incubated with nitro-l-arginine, a l-arginine analog blocking the NO synthase [45]. These observations were confirmed by studies performed in vivo in models of balloon injury [46, 47]and of atherosclerosis [48, 49]. In these studies, l-arginine inhibited neointimal formation and this effect was reversed by l-NAME (N-nitro-l-arginine-methyl-ester), an other l-arginine analog blocking NO synthase. The normal properties of the endothelium include the maintenance of a non-adhesive luminal surface and anticoagulant, fibrinolytic, and antithrombotic actions. The maintenance of normal blood fluidity and the preservation of a non-thrombogenic surface is characterized by several EC membrane regulatory mechanisms. ECs synthesize heparin-like glycosaminoglycans that bind antithrombin III and catalyze the inactivation of coagulation proteases such as thrombin and factor X [50], providing potent antithrombin activity of the vascular surface and protecting against thrombus formation. They also constitutively synthesize thrombomodulin, an intrinsic membrane receptor that binds thrombin, and converts it into a potent activator of protein C. Activated protein C acts as an anticoagulant in the presence of protein S, inactivating factors Va and VIIa [51], and as stimulus for local fibrinolysis by binding endothelial plasminogen activator inhibitor (PAI). Intravascular fibrinolysis is initiated at the vessel surface by the synthesis and release by ECs of tissue plasminogen activator (t-PA), a highly specific serine protease that cleaves plasminogen and converts it into regulatory by endothelial synthesis and release of plasminogen activator an important role in the and of the inhibits platelet EDRF-NO inhibits platelet and have of EDRF (NO) and can to inhibit platelet arterial injury is associated with EC loss the underlying vessel wall which is by a layer of and [29, factors and for VSMCs, such as which are in the vessel wall After balloon reendothelialization of the vessel from of intact endothelium to the endothelial injury also and of some of the cells, VSMCs, of the underlying this to the of This is associated with a from a to a [29, to VSMC proliferation and and synthesis of matrix which are responsible for has also been in studies have shown that in the VSMCs in an matrix It was both in models and in that in addition to the two the response to balloon injury is associated with a in the of the artery at the a two of vessel after balloon angioplasty have been which is deleterious in of and may be the of vessel to a and which is beneficial in of and to for neointimal formation. In the and demonstrated that vascular was the in at vessel has been confirmed as a of after balloon angioplasty in The biological of this are In the ECs rapidly the cycle to endothelial and can be initiated by loss of or growth factors by ECs, VSMCs and circulating cells from the of the and from the of arteries This within the first after arterial and or The of endothelial regrowth from the is to models and for in the the endothelial is in the and in the the most important factors the to be the of the and the number of within the while reendothelialization can be after of in with ECs have been to be of regrowth after reendothelialization the endothelium abnormal In to normal ECs, ECs as a with with blood and are and with the In is on reendothelialization percutaneous transluminal coronary angioplasty in in after that balloon injury is associated with a loss of the EC In addition, of at suggests that is reendothelialization at the within of balloon angioplasty while reendothelialization between and is after The factors the response of ECs after balloon injury have not been It is likely that the release of growth factors for ECs a role in reendothelialization. In the vessel Growth a growth factor for both ECs and VSMCs is and stored by ECs and VSMCs injury both a release of stored from cells and an increased of from ECs and VSMCs The effect of on cells is by the of in the matrix by the increased of receptors in ECs balloon injury The role of endogenous in endothelial after arterial injury is by the of and protein with proliferative ECs at the of reendothelialization The observation that blood of increase after balloon angioplasty in suggests that this factor also be in Growth is a growth factor for ECs It is in the vessel wall by VSMCs and its binding are to ECs The observation for the observation that has effects on role for in endothelial has also been suggested by the of in the vessel wall balloon injury in by the release of the protein after balloon angioplasty in balloon injury is associated with EC loss the underlying vessel wall which is by a layer of and [29, This may to thrombus formation, when is vessel wall In the thrombus was observed in and of the within and after balloon injury of the artery In this platelet and thrombus decreased when the endothelial may to vessel a role in the formation of neointimal Indeed in a of balloon the of against was associated with a in neointimal In addition, thrombin has potent effects on the growth of VSMCs antithrombin inhibit the of thrombus at the by a matrix for growth may the of The role of the endothelium in the underlying growth has been by the results of experiments models of endothelial injury [35, The loss of the endothelial in a large with or minimal medial as performed by a of or a is to the formation of a within [35, The role of reendothelialization in this was suggested by endothelial injury on a to cells is associated with endothelial regrowth within and formation In addition, in models with a of the that first have a degree of [35, the of proliferation of VSMCs in the is in in The of this effect some of the properties of the endothelium such as the of a VSMCs against circulating or platelet factors and the release of growth inhibitory molecules have to that the is not the of the degree of injury to the with EC loss and reendothelialization within may also in arteries growth may the is is to to that the endothelium is in vascular after balloon its potential role has been vascular after balloon injury is an that may or may not of vascular cells may in the vascular the endothelium is to a part Indeed the endothelium is strategically to as a cell and humoral as as an for responses In this the by and that a in vessel induced by a in blood is on an intact endothelium the in a of NO a role in the of the vessel in response to increased blood with this ECs to endothelial after arterial It has been that the of the abnormal for after the reendothelialization has been These studies that vessel elicited through endothelium agonists endothelial In some of these experiments receptor mediated was in both and were for this include in arterial or the of balloon In an response of arterial to angioplasty has also been a that is with of the After balloon of properties to a production of and EDRF-NO the of VSMCs to relaxing or contracting factors is This suggests that the presence of endothelium may the of arterial at of as and EDRF-NO to inhibit platelet and production thrombus formation. the production of EDRF-NO, a potent inhibitor of VSMC in endothelium role in neointimal formation. The observation that is a between the of endothelial and the degree of is with this hypothesis these that the endothelium after balloon angioplasty may be with to its inhibitory effects on vascular thrombus formation and the or the abnormal of the endothelium after arterial injury may to a variety of deleterious consequences such as increased neointimal thickening, or of vasorelaxation in response to endothelium-dependent a a treatment that the of a endothelial layer applied reendothelialization is likely to be associated with beneficial effects at the injury the possible strategies to increase endothelial regrowth in vivo after arterial the of EC growth factors has been the most and are important growth factors for ECs in vitro is specific for ECs, are also growth factors for other cell such for VSMCs The in vivo of these growth factors has been associated with an increased of in tissues and have been in vivo in an to increase endothelial regrowth after arterial The first that the in vivo of was associated with a increase in EC on arteries was performed by In this the evidence for the effect of on EC in and further demonstrated that EC regrowth be within in a of balloon by of the of responses on the endothelium of and of the increase in the was in ECs from arteries in ECs from suggested by the the of ECs of arteries to may be by a number of receptors when cell is It is of to that may reendothelialization of arteries when at in this In a of balloon of the have observed a increase in endothelial regrowth after of of a for the beneficial effect of on endothelial regrowth is not to of of also of vascular endothelium in vivo these studies the beneficial effect of on EC growth in it was of to the of the that in response to the of the growth the effect of of on responses to endothelium-dependent agonists after vascular injury In this as of of was associated with a degree of reendothelialization. after responses not between the and the In the endothelium-dependent acetylcholine-induced of the was that of the The by which the to are not but the endothelium-dependent responses observed after treatment are not to endothelial studies performed in arteries have demonstrated abnormal endothelium-dependent responses in the of reendothelialization this suggests that in addition to its effect on EC also some of ECs and normal responses to endothelium-dependent In of this hypothesis are the results of a a beneficial effect of of on the responses of in the In this abnormal endothelium-dependent responses have been in the of important changes at the endothelial these abnormal responses are to be to a synthesis or an of EDRF [48, The in endothelium-dependent responses in this suggests that in addition to an effect on ECs, may also to changes at the endothelial of of from to after balloon on endothelium-dependent at The response to observed in arteries of was by treatment with In addition, the response to in was to the response observed in arteries from the of the The effect of on neointimal after arterial injury is of for in a a increase in neointimal of a for VSMCs in vitro in the observed an inhibition of neointimal to after artery balloon and was increase in in the by a degree of neointimal was observed in and after these studies suggest that the effect of on neointimal may be the of a between and inhibitory on VSMC studies the that functions of the endothelium such as production of NO are to the of luminal by neointimal [46, reendothelialization may thus neointimal formation. the other which is a potent growth factor for VSMCs neointimal formation. such as the the of the and the may between is growth factors by of the that its binding are present on ECs, but not other cell the effects of are to ECs by the hypothesis that a direct of to the surface of a balloon artery accelerate reendothelialization. In this was after balloon injury of the and the of reendothelialization was in the the neointimal was to a degree in arteries with In addition, demonstrated a of cells in the of This result was by the that in the EC proliferation after balloon injury in a thus appears to be as potent as in endothelial its for ECs may a potential the inhibition of VSMC growth by the endothelium will not be by direct results were observed by other local or of from not effect of local on endothelial proliferation and endothelial regrowth in a of balloon This may be by in and of More recently, the effects of direct of after angioplasty in a have been In this balloon injury and with the acid of was observed as as and for at increase in the of was observed after reendothelialization by while the of reendothelialization in arteries was at and at In this a of the consequences of reendothelialization was arteries of normal endothelium-dependent responses within while arteries demonstrated in endothelium-dependent responses at arteries neointimal and a luminal in with in studies that endothelial growth factors have the potential to accelerate endothelial regrowth in to endothelial and to with It is important to that to are on a potential effect of endothelial growth factors on vascular of is as an treatment for patients with coronary or artery studies have as the first mechanical to after balloon angioplasty a after coronary between and have been of after stent implantation has been to growth to VSMC proliferation and thrombus formation are to of arterial stent a in has been in with the of balloon by changes in antithrombotic of these and have been to the of an intact endothelial monolayer and results have been the of stent reendothelialization. In the has been as after for it was observed that arteries to and in and coronary has been as In a of in was observed in within implantation In the of coronary at was observed in of after implantation have of EC growth factors may accelerate the of an intact endothelial monolayer after stent to this stent in a Our in a of balloon injury by stent in arteries as suggested to and after endothelial of the arterial is experiments performed in the and local of protein or local of the for demonstrated of the vessel by This was associated with an of EC and a in thrombus formation These effects on endothelial and thrombus formation were associated with a in growth within the The potential for to growth was further by a by In this a of stent performed balloon was associated with a of ECs and an stent and a in of local protein on stent of stent after local of or of the surface from the of the stent is observed in thrombus is the from and of the stent in the and with blood cells and thrombus of surface from of stent the are from and of the stent with endothelial cell layer the stent cells to be in of in and the of the results suggest that may an to prevent both thrombus formation and growth at the of stent of EC growth other treatment strategies have been to increase endothelial regrowth endothelial after arterial injury in These strategies include of and endothelial cell The arterial vessel wall the of the renin angiotensin renin and enzyme (ACE) are present in inhibitors are known to inhibit neointimal in models of balloon This effect has been to the inhibition of the of angiotensin to angiotensin II or to inhibition of with of NO release More it has also been demonstrated in a of balloon inhibitors may endothelial and effects on endothelial were observed in other models in coronary arteries effect of inhibition on endothelial regrowth the two of and endothelial observed in inhibition was associated with endothelial regrowth in vivo in a of balloon injury This with in vitro studies that that inhibitors proliferation and of ECs are several potential by which inhibition may accelerate endothelial inhibition of the of angiotensin to angiotensin that angiotensin II inhibits proliferation of growth coronary ECs in a manner. and that increased of bovine aortic endothelial cells in vitro and that angiotensin II this may to inhibition of associated with of NO it has been suggested that NO may proliferation and of ECs in vitro studies that the renin angiotensin may endothelial inhibition also endothelial its effects and the associated changes in blood and wall studies are to the of the effects of inhibition on endothelial In addition to a effect on endothelial demonstrated that inhibition a effect on EC These suggest that inhibitors may also have effects on EC from or Indeed the observed of the of the ECs with in the of observed in with inhibitors has been associated with of ECs This observation is in with the that inhibition the properties of the of inhibition on endothelial of after of artery in a and an inhibitor In controls the is to the of the artery the of the artery In inhibitor reendothelialization is but with a the of the The and of inhibition on endothelial may a role in the beneficial effects of inhibition on as neointimal is to the presence of a endothelium in vivo studies, inhibitors in models of balloon arterial injury have inhibitory effects on neointimal These results suggest that a of the direct effect of angiotensin II on VSMC growth to of may also neointimal by endothelial The inhibitory of on the of atherosclerosis has been suggested by an of and The of in ECs a direct effect of on vascular This is further by the that proliferation and of ECs in vitro a effect of on endothelial regrowth was in a of balloon injury by two In these studies, the effects of on the endothelium were both and associated with a in The by which endothelial in vivo is has been shown to direct effects on ECs potential role for has been by the that in cultured VSMCs It is also of to that the effect with on reendothelialization is of the that with of or The of local of ECs to accelerate endothelial a endothelial cell was first in This was applied to balloon arteries In these were a a the endothelial cell endothelial of the vessel after balloon injury This has also been shown to platelet it to prevent This suggests that EC may not be to after balloon injury; of endothelial to the of cells to arterial or the for a to the cells may this have to that the that endothelial cells may be isolated from blood in are to the luminal surface of artery the to of cell in to vascular of this has also been applied to to and to of the This two of to with ECs to of the The of in ECs applied to was by More recently, in vivo studies that ECs with a tissue plasminogen activator in an to local activity were from the stent to the effects of local on EC to the underlying substrate this is specific for the in these experiments or it to techniques of applied to to be of the is which form of protein or is the most appropriate to endothelial suggest in the vessel wall a of the local of heparin binding may be an The of such a are its and to the of is to the of for molecules that do not in the vessel wall a which is the for most of The of is the protein production at the it is that the in to the The is the to transduction to an the molecules by the cells have to be In addition, to the of or in the it is to that a phase has been initiated by in In this the and of local of of a for are in patients undergoing balloon angioplasty of and are performed at on the effect of this on endothelial and will be the that circulating endothelial cell are in endothelial after angioplasty the for the of strategies to endothelial after In the normal endothelium an important role in vascular its has deleterious increased neointimal formation, and abnormal to rapidly a endothelial layer may thus be beneficial after arterial