The effects of stenting on coronary endothelium from a molecular biological view: Time for improvement?

Coronary artery stenting following balloon angioplasty represents the gold standard in revascularization of coronary artery stenoses. However, stent deployment as well as percutaneous transluminal coronary angioplasty (PTCA) alone causes severe injury of vascular endothelium. The damaged endothelium is intrinsically repaired by locally derived endothelial cells and by circulating endothelial progenitor cells from the blood, leading to re‐population of the denuded regions within several weeks to months. However, the process of re‐endothelialization is often incomplete or dysfunctional, promoting in‐stent thrombosis and restenosis. The molecular and biomechanical mechanisms that influence the process of re‐endothelialization in stented segments are incompletely understood. Once the endothelium is restored, endothelial function might still be impaired. Several strategies have been followed to improve endothelial function after coronary stenting. In this review, the effects of stenting on coronary endothelium are outlined and current and future strategies to improve endothelial function after stent deployment are discussed.     

Contribution of recipient-derived cells in allograft neointima formation and the response to stent implantation

Allograft coronary disease is the dominant cause of increased risk of death after cardiac transplantation. While the percutaneous insertion of stents is the most efficacious revascularization strategy for allograft coronary disease there is a high incidence of stent renarrowing. We developed a novel rabbit model of sex-mismatched allograft vascular disease as well as the response to stent implantation. In situ hybridization for the Y-chromosome was employed to detect male cells in the neointima of stented allograft, and the population of recipient derived neointimal cells was measured by quantitative polymerase chain reaction and characterized by immunohistochemistry. To demonstrate the participation of circulatory derived cells in stent neointima formation we infused ex vivo labeled peripheral blood mononuclear cells into native rabbit carotid arteries immediately after stenting. Fourteen days after stenting the neointima area was 58% greater in the stented vs. non-stented allograft segments (p = 0.02). Male cells were detected in the neointima of stented female-to-male allografts. Recipient-derived cells constituted 72.1+/-5.7% and 81.5+/-4.2% of neointimal cell population in the non-stented and stented segments, respectively and the corresponding proliferation rates were only 2.7+/-0.5% and 2.3+/-0.2%. Some of the recipient-derived neointimal cells were of endothelial lineage. The ex vivo tagged cells constituted 9.0+/-0.4% of the cells per high power field in the stent neointima 14 days after stenting. These experiments provide important quantitative data regarding the degree to which host-derived blood-borne cells contribute to neointima formation in allograft vasculopathy and the early response to stent implantation.     

Effect of balloon pre-dilation on performance of self-expandable nitinol stent in femoropopliteal artery

Balloon pre-dilation is usually performed before implantation of a nitinol stent in a femoropopliteal artery in a case of severe blockage or calcified plaque. However, its effect on performance of the nitinol stent in a diseased femoropopliteal artery has not been studied yet. This study compares the outcomes of stenting with pre-dilation and without it by modelling the entire processes of stent deployment. Fatigue deformation of the implanted stent is also modelled under diastolic–systolic blood pressure, repetitive bending, torsion, axial compression and their combination. Reduced level of stress in the stent occurs after stenting with pre-dilation, but causing the increased damage in the media layer, i.e. the middle layer of the arterial wall. Generally, pre-dilation increases the risk of nitinol stent’s fatigue failure. Additionally, the development of in-stent restenosis is predicted based on the stenting-induced tissue damage in the media layer, and no severe mechanical irritation is induced to the media layer by pre-dilation, stent deployment or fatigue loading.

     

Lipids: potential regulators of nitric oxide generation

The endothelium is a dynamic organ that secretes several biologically active substances and plays a major functional role in the health of an organism in both physiological and pathological conditions. For instance, the endothelium is involved in control of the exchange of plasma and tissue biomolecules, regulation of vessel tone, inflammation, lipid metabolism, vessel growth and remodeling, and modulation of coagulation and fibrinolysis. The endothelium generates nitric oxide, which is a key regulator of vasodilation and plays important roles in preventing, or in some cases promoting, numerous cardiovascular diseases. Several recent studies have examined the interplay between lipids and nitric oxide generation, especially in relation to atherosclerosis. The endothelium is continuously exposed to circulating lipids in the form of lipoproteins and protein carriers that may have a direct impact on nitric oxide synthesis and function. The purpose of this review is to illustrate some of the recent findings that link lipids (plasma and cellular) to nitric oxide generation (see Fig. 1).     

Coronary Injury Score Correlates with Proliferating Cells and Alpha-Smooth Muscle Actin Expression in Stented Porcine Coronary Arteries

Neointimal formation and cell proliferation resulting into in-stent restenosis is a major pathophysiological event following the deployment of stents in the coronary arteries. In this study, we assessed the degree of injury, based on damage to internal elastic lamina, media, external elastic lamina, and adventitia following the intravascular stenting, and its relationship with the degree of smooth muscle cell proliferation. We examined the smooth muscle cell proliferation and their phenotype at different levels of stent injury in the coronary arteries of domestic swine fed a normal swine diet. Five weeks after stent implantation, swine with and without stents were euthanized and coronaries were excised. Arteries were embedded in methyl methacrylate and sections were stained with H&E, trichrome, and Movat’s pentachrome. The expression of Ki67, α-smooth muscle actin (SMA), vimentin, and HMGB1 was evaluated by immunofluorescence. There was a positive correlation between percent area stenosis and injury score. The distribution of SMA and vimentin was correlated with the degree of arterial injury such that arteries that had an injury score >2 did not have immunoreactivity to SMA in the neointimal cells near the stent struts, but these neointimal cells were positive for vimentin, suggesting a change in the smooth muscle cell phenotype. The Ki67 and HMGB1 immunoreactivity was highly correlated with the fragmentation of the IEL and injury in the tunica media. Thus, the extent of coronary arterial injury during interventional procedure will dictate the degree of neointimal hyperplasia, in-stent restenosis, and smooth muscle cell phenotype.     

药物涂层球囊概述及其在外周动脉疾病治疗中的应用进展

经皮经腔血管成形术(PTA)已广泛用于外周动脉疾病(PAD)的治疗。然而,该技术存在血管壁弹性回缩和内膜增生等不足。PTA术后植入金属裸支架(BMS)虽然可以减少血管壁弹性回缩,但由此引起的支架内再狭窄(ISR)又成为治疗中的一个突出问题。药物洗脱支架(DES)被用来解决狭窄问题,但晚期支架内血栓形成(LST)、内皮化延迟和必须长期抗血小板治疗等问题也随之而来。在这样的背景下,药物涂层球囊(DCB)获得了快速发展。DCB作为非支架方案,可将所携载的活性药物转移至病变段血管壁,对ISR或原发病变均有较好的治疗效果。本文简要介绍了DCB的发展历史,并通过实验室研究、动物实验和临床试验,从机制上阐述涂层技术、涂层药物、赋形剂等对DCB功效和安全性的影响以及DCB在PAD治疗中的应用进展。     

Rescue of arterial function by angiotensin-(1-7): towards improvement of endothelial function by drug-eluting stents

Purpose: To explore the hypothesis that stent placement decreases dilator function of various arteries outside the stented segment and that angiotensin- (1-7) improves this function, and to assess the contribution of dilator signal compounds. A further objective was to test the hypothesis that on-stent delivery of Ang-(1-7) reduces neointima formation and improves endothelial function. Methods: Abdominal aortic stenting or sham operation was performed in the rat four weeks after stenting and treatment with intravenous saline or Ang-(1-7) infusion (24 µg/kg/h); vasomotor function in isolated thoracic aorta and brachial and iliac artery was measured in organ baths. Furthermore, Ang-(1-7)-eluting stents were designed and placed in rat abdominal aorta. Neointima formation and aortic function were tested after four weeks. Results: Relaxation of the thoracic aorta to metacholine was decreased after stenting compared with shams due to a decrease in nitric oxide-mediated response (67% reduction in maximal NO-dependent response). Ang-(1-7) restored the response mainly through increased prostaglandin- and possibly also endothelial-derived hyperpolarising factor-mediated relaxation. Relaxation in the brachial artery decreased after stenting (maximal response dropped by 50%), whilst contractions to phenylephrine increased. Ang-(1-7) normalised vasomotor function. Iliac artery function remained unaltered after stenting but Ang-(1-7) increased maximal relaxations by 65%. Delivery of Ang-(1-7) by means of a drug-eluting stent improved endothelial function. Conclusion: Stenting differentially affects dilator and contractile function in various arterial beds. Ang-(1-7) both improves dilator function and normalises contractile function. Delivery of protective peptides such as Ang-(1-7) from the stent is a new therapy option that merits further development and exploration. (Neth Heart J 2008;16:293-8.)     

In silico prediction of the mechanobiological response of arterial tissue: application to angioplasty and stenting

One way to restore physiological blood flow to occluded arteries involves the deformation of plaque using an intravascular balloon and preventing elastic recoil using a stent. Angioplasty and stent implantation cause unphysiological loading of the arterial tissue, which may lead to tissue in-growth and reblockage; termed "restenosis." In this paper, a computational methodology for predicting the time-course of restenosis is presented. Stress-induced damage, computed using a remaining life approach, stimulates inflammation (production of matrix degrading factors and growth stimuli). This, in turn, induces a change in smooth muscle cell phenotype from contractile (as exists in the quiescent tissue) to synthetic (as exists in the growing tissue). In this paper, smooth muscle cell activity (migration, proliferation, and differentiation) is simulated in a lattice using a stochastic approach to model individual cell activity. The inflammation equations are examined under simplified loading cases. The mechanobiological parameters of the model were estimated by calibrating the model response to the results of a balloon angioplasty study in humans. The simulation method was then used to simulate restenosis in a two dimensional model of a stented artery. Cell activity predictions were similar to those observed during neointimal hyperplasia, culminating in the growth of restenosis. Similar to experiment, the amount of neointima produced increased with the degree of expansion of the stent, and this relationship was found to be highly dependant on the prescribed inflammatory response. It was found that the duration of inflammation affected the amount of restenosis produced, and that this effect was most pronounced with large stent expansions. In conclusion, the paper shows that the arterial tissue response to mechanical stimulation can be predicted using a stochastic cell modeling approach, and that the simulation captures features of restenosis development observed with real stents. The modeling approach is proposed for application in three dimensional models of cardiovascular stenting procedures.     

The involvement of vascular endothelial growth factor and flt-1 in the process of neointimal proliferation in pig coronary arteries following stent implantation

To clarify the role of vascular endothelial growth factor (VEGF) in the process of restenosis, a Palmaz-Schatz stent was implanted in the left anterior descending coronary artery of male pigs at 2 weeks after balloon injury (balloon/artery ratio 1.2:1). The animals were euthanized at 1, 2, and 4 weeks after stenting, and western blot and immunohistochemical analysis were performed using VEGF, fms-like tyrosine kinase (flt)-1, and platelet-derived growth factor (PDGF) antibodies. The expressions of VEGF and flt-1 protein in the neointima were observed as early as 1 week after stenting and remained for up to 4 weeks, while re-endothelialization was complete at 2 weeks. These protein expressions were demonstrated in proliferated smooth muscle cells throughout the entire period after stenting and, in addition, they were observed in the macrophages and endothelial cells of microvessels around stent struts at 4 weeks. The expression pattern of VEGF corresponded with that of PDGF, a growth factor well-known to induce neointimal proliferation. The cell proliferative activity, measured by the proliferating cell nuclear antigen index, around the struts remained high until 4 weeks after stenting, while that in the other areas declined at 4 weeks. These results suggest that VEGF is involved in the process of restenosis not only through its angiogenic properties and induction of monocyte chemotaxis, but also by a synergistic effect with PDGF.     

Pathology of in-stent restenosis

The process of in-stent restenosis parallels wound healing responses. Stent deployment results in early thrombus deposition and acute inflammation, granulation tissue development, and ultimately smooth muscle cell proliferation and extracellular matrix synthesis. The severity of arterial injury during stent placement correlates with increased inflammation and late neointimal growth. These pathological findings provide useful targets for therapies aimed at reducing the incidence of in-stent restenosis.     

Rac1 regulates cardiovascular development and postnatal function of endothelium

Rac1 is a member of the small Rho GTPase family, which controls actin cytoskeleton and focal adhesion dynamics in cellular protrusions. While Rac1 therefore contributes to regulation of endothelial cell-cell and cell-matrix interactions, a detailed understanding of its role in endothelium function is lacking. Recently, the role of Rac1 in development and postnatal regulation of the cardiovascular system has been investigated in murine models lacking Rac1 specifically in endothelium. Homozygous endothelial deletion was lethal, primarily due to defects in angiogenesis. Rac1-deficient endothelial cells were unable to form cellular protrusions/lamellipodia, leading to impaired cell-cell and cell-matrix interactions, and resulting in dysfunctional adhesion, motility, permeability and capillary morphogenesis. Development was normal in the heterozygous model, however a hypertensive phenotype was observed as a result of reduced nitric oxide signalling. Nitric oxide synthase activity was regulated by Rac1 at multiple levels; expression, mRNA stability and uptake of the nitric oxide synthase substrate L-arginine. Therefore, Rac1 activity is essential in regulating developmental and postnatal angiogenesis and cardiovascular function, by controlling nitric oxide production, and formation of endothelial cell protrusions.Key words: Rac1, angiogenesis, endothelial, motility, lamellipodia, nitric oxide, nitric oxide synthase     

Inflammation and in-stent restenosis: the role of serum markers and stent characteristics in carotid artery stenting

Background

Carotid angioplasty and stenting (CAS) may currently be recommended especially in younger patients with a high-grade carotid artery stenosis. However, evidence is accumulating that in-stent restenosis (ISR) could be an important factor endangering the long-term efficacy of CAS. The aim of this study was to investigate the influence of inflammatory serum markers and procedure-related factors on ISR as diagnosed with duplex sonography.

Methods

We analyzed 210 CAS procedures in 194 patients which were done at a single university hospital between May 2003 and June 2010. Periprocedural C-reactive protein (CRP) and leukocyte count as well as stent design and geometry, and other periprocedural factors were analyzed with respect to the occurrence of an ISR as diagnosed with serial carotid duplex ultrasound investigations during clinical long-term follow-up.

Results

Over a median of 33.4 months follow-up (IQR: 14.9–53.7) of 210 procedures (mean age of 67.9±9.7 years, 71.9% male, 71.0% symptomatic) an ISR of ≥70% was detected in 5.7% after a median of 8.6 months (IQR: 3.4–17.3). After multiple regression analysis, leukocyte count after CAS-intervention (odds ratio (OR): 1.31, 95% confidence interval (CI): 1.02–1.69; p = 0.036), as well as stent length and width were associated with the development of an ISR during follow-up (OR: 1.25, 95% CI: 1.05–1.65, p = 0.022 and OR: 0.28, 95% CI: 0.09–0.84, p = 0.010).

Conclusions

The majority of ISR during long-term follow-up after CAS occur within the first year. ISR is associated with periinterventional inflammation markers and influenced by certain stent characteristics such as stent length and width. Our findings support the assumption that stent geometry leading to vessel injury as well as periprocedural inflammation during CAS plays a pivotal role in the development of carotid artery ISR.     

Biodegradable stents as a platform to drug loading

Despite technical and mechanical improvement in coronary stents the incidence of restenosis caused by in-stent neointimal hyperplasia remains high. Oral administration of numerous pharmacological agents has failed to reduce restenosis after coronary stenting in humans, possibly owing to insufficient local drug concentration. Therefore, drug-eluting stents were developed as a vehicle for local drug administration. The authors developed a new drug-eluting polymer stent that is made of poly-l-lactic acid polymer mixed with tranilast, an anti-allergic drug that inhibits the migration and proliferation of vascular smooth muscle cells induced by platelet-derived growth factor and transforming growth factor->1. Polymer stents might be superior to polymer-coated metallic stents as local drug delivery stents in terms of biodegradation and the amount of loaded drug. Drug-mixed polymer stents can be loaded with a larger amount of drug than can drug-coated metallic stents because the polymer stent struts can contain the drug. Clinical application is required to assess the safety and efficacy of drug-eluting polymer stents against stent restenosis.     

Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. The feasibility of using magnetic forces to capture and retain endothelial outgrowth cells (EOC) labeled with super paramagnetic iron oxide nanoparticles (SPION) has been shown previously. But this technique requires the development of a mechanically functional stent from a magnetic and biocompatible material followed by in-vitro and in-vivo testing to prove rapid endothelialization. We developed a weakly ferromagnetic stent from 2205 duplex stainless steel using computer aided design (CAD) and its design was further refined using finite element analysis (FEA). The final design of the stent exhibited a principal strain below the fracture limit of the material during mechanical crimping and expansion. One hundred stents were manufactured and a subset of them was used for mechanical testing, retained magnetic field measurements, in-vitro cell capture studies, and in-vivo implantation studies. Ten stents were tested for deployment to verify if they sustained crimping and expansion cycle without failure. Another 10 stents were magnetized using a strong neodymium magnet and their retained magnetic field was measured. The stents showed that the retained magnetism was sufficient to capture SPION-labeled EOC in our in-vitro studies. SPION-labeled EOC capture and retention was verified in large animal models by implanting 1 magnetized stent and 1 non-magnetized control stent in each of 4 pigs. The stented arteries were explanted after 7 days and analyzed histologically. The weakly magnetic stents developed in this study were capable of attracting and retaining SPION-labeled endothelial cells which can promote rapid healing.     

Ultrastructural localization of nitric oxide synthase and endothelin in coronary and pulmonary arteries of newborn rats

This is the first report on the ultrastructural distribution of nitric oxide synthase and endothelin immunoreactivities in the coronary and pulmonary arteries of newborn Wistar rats. The distribution of nitric oxide synthase and endothelin was investigated using pre-embedding peroxidase-antiperoxidase immunocytochemistry. In both arteries examined, positive labelling for nitric oxide synthase was localized both in the endothelium and smooth muscle, whereas positive labelling for endothelin was localized in the endothelium exclusively. In the coronary artery, approximately 80% and 55% of the endothelial cells examined were positive for nitric oxide synthase and endothelin, respectively, whereas in the pulmonary artery, 77% and 60% of the endothelial cells were positive for nitric oxide synthase and endothelin, respectively. These findings indicate that nitric oxide synthase and endothelin are colocalized in some of the endothelial cells of the newborn rat. In the endothelium, nitric oxide synthase and endothelin immunoreactivities were distributed throughout the cell cytoplasm and in association with the membranes of intracellular organelles. In smooth muscle, a relationship of nitric oxide synthase immunoreactivity to endoplasmic reticulum was observed in the pulmonary artery. In summary, in the newborn rat, endothelial cells of the coronary and pulmonary artery are rich in nitric oxide synthase (neuronal isoform) and endothelin, and it is suggested therefore that they may be substantially involved in vasomotor control of the cardiac and pulmonary circulation during early stages of postnatal development.     

Stent expansion in curved vessel and their interactions: a finite element analysis

Coronary restenosis after angioplasty has been reduced by stenting procedure, but in-stent restenosis (ISR) has not been eliminated yet, especially in tortuous vessels. In this paper, we proposed a finite element method (FEM) to study the expansion of a stent in a curved vessel (the CV model) and their interactions. A model of the same stent in a straight vessel (the SV model) was also studied and mechanical parameters of both models were researched and compared, including final lumen area, tissue prolapse between stent struts and stress distribution. Results show that in the CV model, the vessel was straightened by stenting and a hinge effect can be observed at extremes of the stent. The maximum tissue prolapse of the CV model was more severe (0.079 mm) than the SV model (0.048 mm); and the minimum lumen area of the CV was decreased (6.10 mm(2)), compared to that of the SV model (6.28 mm(2)). Tissue stresses of the highest level were concentrated in the inner curvature of the CV model. The simulations offered some explanations for the clinical results of ISR in curved vessels and gave design suggestions of the stent and balloon for tortuous vessels. This FEM provides a tool to study mechanisms of stents in curved vessels and can improve new stent designs especially for tortuous vessels.     

Rac1 regulates cardiovascular development and postnatal function of endothelium

Rac1 is a member of the small Rho GTPase family, which controls actin cytoskeleton and focal adhesion dynamics in cellular protrusions. While Rac1 therefore contributes to regulation of endothelial cell-cell and cell-matrix interactions, a detailed understanding of its role in endothelium function is lacking. Recently, the role of Rac1 in development and postnatal regulation of the cardiovascular system has been investigated in murine models lacking Rac1 specifically in endothelium. Homozygous endothelial deletion was lethal, primarily due to defects in angiogenesis. Rac1-deficient endothelial cells were unable to form cellular protrusions/lamellipodia, leading to impaired cell-cell and cell-matrix interactions, and resulting in dysfunctional adhesion, motility, permeability and capillary morphogenesis. Development was normal in the heterozygous model, however a hypertensive phenotype was observed as a result of reduced nitric oxide signalling. Nitric oxide synthase activity was regulated by Rac1 at multiple levels; expression, mRNA stability and uptake of the nitric oxide synthase substrate L-arginine. Therefore, Rac1 activity is essential in regulating developmental and postnatal angiogenesis and cardiovascular function, by controlling nitric oxide production, and formation of endothelial cell protrusions.     

Role of Pin1 in neointima formation: Down-regulation of Nrf2-dependent heme oxygenase-1 expression by Pin1

Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to intima formation after stenting and balloon angioplasty. Pin1, a peptidyl prolyl isomerase recognizing phosphorylated Ser/Thr–Pro, isomerizes the peptide bond. Because Pin1 overexpression is associated with transformation and the uncontrolled cell growth of tumors, we hypothesized that Pin1 functions as a chronic stimulator of VSMC proliferation. Pin1-positive smooth muscle cells were seen in the neointimal region of the femoral artery after guidewire injury. Exposure of VSMCS to platelet-derived growth factor (PDGF) increased Pin1 expression in a concentration-dependent manner. Basal cell growth rate and cyclin D1 expression were enhanced in Pin1-overexpressing VSMCs (Pin1-VSMCs). Moreover, PDGF-induced production of reactive oxygen species (ROS) in Pin1-VSMCs was higher than in control VSMCs. In Pin1-VSMCs, heme oxygenase-1 (HO-1) induction in response to nitric oxide donor was suppressed compared to control VSMCs. Nuclear translocation of nuclear factor E2-related factor-2 (Nrf2) was also diminished in Pin1-VSMCs. In contrast, the activity of the inducible minimal antioxidant response element (ARE) was potentiated in Pin1-null mouse embryonic fibroblasts (MEFs), compared to Pin1-wild-type MEFs. Moreover, Nrf2 ubiquitination was stimulated by Pin1 overexpression. Intraperitoneal injection of juglone (a Pin1 inhibitor) for 3 weeks (1 mg/kg, two times a week) significantly suppressed neointimal formation induced by wire injury. In conclusion, Pin1 induction during neointimal formation may be associated with ROS-mediated VSMC proliferation via down-regulation of Nrf2/ARE-dependent HO-1 expression. Pin1 may be a novel therapeutic target for several vascular diseases including atherosclerosis and stenosis.     

Unraveling the biological significance of nitric oxide

Independent investigations into the biochemical changes and cytostatic properties induced in immunostimulated macrophages and studies involving the identity and mechanism of action of endothelium-derived relaxing factor led to the finding of a new metabolic pathway which converts L-arginine to nitric oxide and citrulline. The pathway has since been reported in a number of additional cell types including cells in the central nervous system (CNS). In the endothelium and CNS nitric oxide is acting as a signaling agent with the evidence supporting activation of the enzyme guanylate cyclase in the target cell. Nitric oxide is toxic and evidence supports a cytostatic/cytotoxic function as the primary action of macrophage-derived nitric oxide.