Multiple Intravenous Bolus Dosing and Invasive Hemodynamic Assessment in a Hypoxia-Induced Mouse Pulmonary Artery Hypertension Model.

Pulmonary arterial hypertension (PAH) is a progressive life-threatening disease, primarily affecting small pulmonary arterioles of the lung. Currently, there is no cure for PAH. It is important to discover new compounds that can be used to treat PAH. The mouse hypoxia-induced PAH model is a widely used model for PAH research. This model recapitulates human clinical manifestations of PAH Group 3 disease and is an important research tool to evaluate the effectiveness of new experimental therapies for PAH. Research using this model often requires the administration of compounds in mice. For a compound that needs to be given directly into the bloodstream, optimizing intravenous (IV) administration is a key part of the experimental procedures. Ideally, the IV injection system should permit multiple injections over a set time course. Although the mouse hypoxia-induced PAH model is very popular in many laboratories, it is technically challenging to perform multiple IV bolus dosing and invasive hemodynamic assessment in this model. In this protocol, we present step-by-step instructions on how to carry out multiple IV bolus dosing via mouse jugular vein and perform arterial and right ventricle catheterization for hemodynamic assessment in mouse hypoxia-induced PAH model.

Point of care, bone marrow mononuclear cell therapy in ischemic heart failure patients personalized for cell potency: 12-month feasibility results from CardiAMP heart failure roll-in cohort.

AIM: Heart failure following myocardial infarction (MI) is a potentially lethal problem with a staggering incidence. The CardiAMP Heart Failure trial represents the first attempt to personalize marrow-derived cell-based therapy to individuals with cell characteristics associated with beneficial responses in prior trials. Before the initiation of the randomized pivotal trial, an open-label "roll-in cohort" was completed to ensure the feasibility of the protocol's procedures. METHODS: Patients with chronic post-MI heart failure (NYHA class II-III) receiving stable, guideline-directed medical therapy with a left ventricular ejection fraction between 20 and 40% were eligible. Two weeks prior to treatment, a ~ 5 mL bone marrow aspiration was performed to examine "cell potency". On treatment day, a 60 mL bone marrow aspiration, bone marrow mononuclear cell (BM MNC) enrichment and transendocardial injection of 200 million BM MNC's was performed in a single, point of care encounter. Patients were then followed to assess clinical outcomes. RESULTS: The cell potency small volume bone marrow aspirate, the 60 mL bone marrow aspirate, and transendocardial injections were well tolerated in 10 patients enrolled. There were no serious adverse events related to bone marrow aspiration or cell delivery. Improvement in 6-min walk distance was observed at 6 months (+47.8 m, P = 0.01) and trended to improvement at 12 months (+46.4, P = 0.06). Similarly, trends to improved NYHA heart failure functional class, quality of life, left ventricular ejection fraction and recruitment of previously akinetic left ventricular wall segments were observed. CONCLUSION: All CardiAMP HF protocol procedures were feasible and well tolerated. Favorable functional, echo and quality of life trends suggest this approach may offer promise for patients with post MI heart failure. The randomized CardiAMP Heart Failure pivotal trial is underway to confirm the efficacy of this approach. CLINICAL TRIAL REGISTRATION: https://clinicaltrials.gov/ct2/show/NCT02438306.

The CardiAMP Heart Failure trial: A randomized controlled pivotal trial of high-dose autologous bone marrow mononuclear cells using the CardiAMP cell therapy system in patients with post-myocardial infarction heart failure: Trial rationale and study design.

BACKGROUND: Heart failure following myocardial infarction is a common, disabling, and deadly condition. Direct injection of autologous bone marrow mononuclear cells into the myocardium may result in improved functional recovery, relieve symptoms, and improve other cardiovascular outcomes. METHODS: CardiAMP-HF is a randomized, double-blind, sham-controlled, pivotal trial designed to investigate the safety and efficacy of autologous bone marrow mononuclear cells treatment for patients with medically refractory and symptomatic ischemic cardiomyopathy. The primary end point is change in 6-minute walk distance adjusted for major adverse cardiovascular events at 12 months following treatment. Particularly novel aspects of this trial include a cell potency assay to screen subjects who have bone marrow cell characteristics that suggest a favorable response to treatment, a point-of-care treatment method, a high target dose of 200 million cells, and an efficient transcatheter intramyocardial delivery method that is associated with high cell retention. CONCLUSIONS: This novel approach may lead to a new treatment for those with ischemic heart disease suffering from medically refractory heart failure.

Cgnl1, an endothelial junction complex protein, regulates GTPase mediated angiogenesis.

AIMS: The formation of cell-cell and cell-extra cellular matrix (ECM) contacts by endothelial cells (ECs) is crucial for the stability and integrity of a vascular network. We previously identified cingulin-like 1 (Cgnl1) in a transcriptomic screen for new angiogenic modulators. Here we aim to study the function of the cell-cell junction associated protein Cgnl1 during vessel formation. METHODS AND RESULTS: Unlike family member cingulin, Cgnl1 expression is enriched in ECs during vascular growth. Cgnl1 is important for the formation of multicellular tubule structures, as shown in vitro using loss-of function assays in a 3D matrix co-culture system that uses primary human ECs and supporting mural cells. Further studies revealed that Cgnl1 regulates vascular growth by promoting Ve-cadherin association with the actin cytoskeleton, thereby stabilizing adherens junctions. Cgnl1 also regulates focal adhesion assembly in response to ECM contact, promoting vinculin and paxillin recruitment and focal adhesion kinase signalling. In vivo, we demonstrate in a postnatal retinal vascular development model in mice that Cgnl1 function is crucial for sustaining neovascular growth and stability. CONCLUSIONS: Our data demonstrate a functional relevance for Cgnl1 as a defining factor in new vessel formation both in vitro and in vivo.

THSD1 preserves vascular integrity and protects against intraplaque haemorrhaging in ApoE-/- mice.

AIMS: Impairment of the endothelial barrier leads to microvascular breakdown in cardiovascular disease and is involved in intraplaque haemorrhaging and the progression of advanced atherosclerotic lesions that are vulnerable to rupture. The exact mechanism that regulates vascular integrity requires further definition. Using a microarray screen for angiogenesis-associated genes during murine embryogenesis, we identified thrombospondin type I domain 1 (THSD1) as a new putative angiopotent factor with unknown biological function. We sought to characterize the role of THSD1 in endothelial cells during vascular development and cardiovascular disease. METHODS AND RESULTS: Functional knockdown of Thsd1 in zebrafish embryos and in a murine retina vascularization model induced severe haemorrhaging without affecting neovascular growth. In human carotid endarterectomy specimens, THSD1 expression by endothelial cells was detected in advanced atherosclerotic lesions with intraplaque haemorrhaging, but was absent in stable lesions, implying involvement of THSD1 in neovascular bleeding. In vitro, stimulation with pro-atherogenic factors (3% O2 and TNFα) decreased THSD1 expression in human endothelial cells, whereas stimulation with an anti-atherogenic factor (IL10) showed opposite effect. Therapeutic evaluation in a murine advanced atherosclerosis model showed that Thsd1 overexpression decreased plaque vulnerability by attenuating intraplaque vascular leakage, subsequently reducing macrophage accumulation and necrotic core size. Mechanistic studies in human endothelial cells demonstrated that THSD1 activates FAK-PI3K, leading to Rac1-mediated actin cytoskeleton regulation of adherens junctions and focal adhesion assembly. CONCLUSION: THSD1 is a new regulator of endothelial barrier function during vascular development and protects intraplaque microvessels against haemorrhaging in advanced atherosclerotic lesions.

Sox7 controls arterial specification in conjunction with hey2 and efnb2 function.

SoxF family members have been linked to arterio-venous specification events and human pathological conditions, but in contrast to Sox17 and Sox18, a detailed in vivo analysis of a Sox7 mutant model is still lacking. In this study we generated zebrafish sox7 mutants to understand the role of Sox7 during vascular development. By in vivo imaging of transgenic zebrafish lines we show that sox7 mutants display a short circulatory loop around the heart as a result of aberrant connections between the lateral dorsal aorta (LDA) and either the venous primary head sinus (PHS) or the common cardinal vein (CCV). In situ hybridization and live observations in flt4:mCitrine transgenic embryos revealed increased expression levels of flt4 in arterial endothelial cells at the exact location of the aberrant vascular connections in sox7 mutants. An identical circulatory short loop could also be observed in newly generated mutants for hey2 and efnb2. By genetically modulating levels of sox7, hey2 and efnb2 we demonstrate a genetic interaction of sox7 with hey2 and efnb2. The specific spatially confined effect of loss of Sox7 function can be rescued by overexpressing the Notch intracellular domain (NICD) in arterial cells of sox7 mutants, placing Sox7 upstream of Notch in this aspect of arterial development. Hence, sox7 levels are crucial in arterial specification in conjunction with hey2 and efnb2 function, with mutants in all three genes displaying shunt formation and an arterial block.

Cardiac function in a long-term follow-up study of moderate and severe porcine model of chronic myocardial infarction.

BACKGROUND: Novel therapies need to be evaluated in a relevant large animal model that mimics the clinical course and treatment in a reasonable time frame. To reliably assess therapeutic efficacy, knowledge regarding the translational model and the course of disease is needed. METHODS: Landrace pigs were subjected to a transient occlusion of the proximal left circumflex artery (LCx) (n = 6) or mid-left anterior descending artery (LAD) (n = 6) for 150 min. Cardiac function was evaluated before by 2D echocardiography or 3D echocardiography and pressure-volume loop analysis. At 12 weeks of follow-up the heart was excised for histological analysis and infarct size calculations. RESULTS: Directly following AMI, LVEF was severely reduced compared to baseline in the LAD group (-17.1 ± 1.6%, P = 0.009) compared to only a moderate reduction in the LCx group (-5.9 ± 1.5%, P = 0.02) and this effect remained unchanged during 12 weeks of follow-up. CONCLUSION: Two models of chronic MI, representative for different patient groups, can reproducibly be created through clinically relevant ischemia-reperfusion of the mid-LAD and proximal LCx.

Similar effect of autologous and allogeneic cell therapy for ischemic heart disease: systematic review and meta-analysis of large animal studies.

RATIONALE: In regenerative therapy for ischemic heart disease, use of both autologous and allogeneic stem cells has been investigated. Autologous cell can be applied without immunosuppression, but availability is restricted, and cells have been exposed to risk factors and aging. Allogeneic cell therapy enables preoperative production of potent cell lines and immediate availability of cell products, allowing off-the-shelf therapy. It is unknown which cell source is preferred with regard to improving cardiac function. OBJECTIVE: We performed a meta-analysis of preclinical data of cell therapy for ischemic heart disease. METHODS AND RESULTS: We conducted a systematic literature search to identify publications describing controlled preclinical trials of unmodified stem cell therapy in large animal models of myocardial ischemia. Data from 82 studies involving 1415 animals showed a significant improvement in mean left ventricular ejection fraction in treated compared with control animals (8.3%, 95% confidence interval, 7.1-9.5; P<0.001). Meta-regression revealed a similar difference in left ventricular ejection fraction in autologous (8.8%, 95% confidence interval, 7.3-10.3; n=981) and allogeneic (7.3%, 95% confidence interval, 4.4-10.2, n=331; P=0.3) cell therapies. CONCLUSIONS: Autologous and allogeneic cell therapy for ischemic heart disease show a similar improvement in left ventricular ejection fraction in large animal models of myocardial ischemia, compared with placebo. These results are important for the design of future clinical trials.

Intracoronary infusion of encapsulated glucagon-like peptide-1-eluting mesenchymal stem cells preserves left ventricular function in a porcine model of acute myocardial infarction.

BACKGROUND: Engraftment and survival of stem cells in the infarcted myocardium remain problematic in cell-based therapy for cardiovascular disease. To overcome these issues, encapsulated mesenchymal stem cells (eMSCs) were developed that were transfected to produce glucagon-like peptide-1, an incretin hormone with known cardioprotective effects, alongside MSC endogenous paracrine factors. This study was designed to investigate the efficacy of different doses of intracoronary infusion of eMSC in a porcine model of acute myocardial infarction (AMI). METHODS AND RESULTS: One hundred pigs were subjected to a moderate AMI (posterolateral AMI; n=50) or a severe AMI (anterior AMI; n=50), whereupon surviving animals (n=36 moderate, n=33 severe) were randomized to receive either intracoronary infusion of 3 incremental doses of eMSC or Ringers' lactate control. Cardiac function was assessed using invasive hemodynamics, echocardiography, and histological analysis. A trend was observed in the moderate AMI model, whereas in the severe AMI model, left ventricular ejection fraction improved by +9.3% (P=0.004) in the best responding eMSC group, because of a preservation of left ventricular end-systolic volume. Arteriolar density increased 3-fold in the infarct area (8.4±0.9/mm(2) in controls versus 22.2±2.6/mm(2) in eMSC group; P<0.001). Although not statistically significant, capillary density was 30% higher in the border zone (908.1±99.7/mm(2) in control versus 1209.0±64.6/mm(2) in eMSC group; P=ns). CONCLUSIONS: eMSCs enable sustained local delivery of cardioprotective proteins to the heart, thereby enhancing angiogenesis and preserving contractile function in an animal AMI model.

Effect of shear stress alteration on atherosclerotic plaque vulnerability in cholesterol-fed rabbits.

Previously, we created an experimental murine model for the induction of vulnerable plaque (VP). Although this murine model offers the opportunity to study the different molecular biological pathways that regulate plaque destabilization, the size of the animals severely limits the use of the model for in vivo diagnostics and percutaneous interventions. This study aimed to create a VP model in the rabbit, based on the murine model, to aid the assessment and development of novel diagnostic and interventional tools. New Zealand white rabbits were fed on a 2% cholesterol diet. After 1 week, a shear stress-altering device was implanted around the right carotid artery. Twelve weeks after cast placement, the carotid artery was isolated and processed for (immuno-)histological analysis to evaluate the presence of a VP phenotype. Atherosclerotic plaques with high lipid and macrophage content, low vascular smooth muscle cell content and intimal neovascularization were located upstream and downstream of the cast. The plaques lacked a significant necrotic core. In conclusion, we were able to create atherosclerotic plaques with a phenotype beyond that of a fatty streak, with a high percentage of lipids and macrophages, a thick cap with some vascular smooth muscle cells and neovascularization. However, as there was only a small necrotic core, the overall phenotype seems less vulnerable as compared to the thin fibrous cap atheroma in patients.

Intracoronary stem cell infusion after acute myocardial infarction: a meta-analysis and update on clinical trials.

BACKGROUND: Several cell-based therapies for adjunctive treatment of acute myocardial infarction have been investigated in multiple clinical trials, but the benefits still remain controversial. This meta-analysis aims to evaluate the efficacy of bone marrow-derived mononuclear cell (BMMNC) therapy in patients with acute myocardial infarction, but also explores the effect of newer generations of stem cells. METHODS AND RESULTS: A random-effects meta-analysis was performed on randomized controlled trials investigating the effects of stem cell therapy in patients with acute myocardial infarction that were published between January 2002 and September 2013. The defined end points were left ventricular (LV) ejection fraction, LV end-systolic and end-diastolic volumes, infarct size, and major adverse cardiac and cerebrovascular event rates. Also, several subgroup analyses were performed on BMMNC trials. Overall, combining the results of 22 randomized controlled trials (RCTs), LV ejection fraction increased by +2.10% (95% confidence interval [CI], 0.68-3.52; P=0.004) in the BMMNC group as compared with controls, evoked by a preservation of LV end-systolic volume (-4.05 mL; 95% CI, -6.91 to -1.18; P=0.006) and a reduction in infarct size (-2.69%; 95% CI, -4.83 to -0.56; P=0.01). However, there is no effect on cardiac function, volumes, or infarct size, when only RCTs (n=9) that used MRI-derived end points were analyzed. Moreover, no beneficial effect could be detected on major adverse cardiac and cerebrovascular event rates after BMMNC infusion after a median follow-up duration of 6 months. CONCLUSIONS: Intracoronary infusion of BMMNC is safe, but does not enhance cardiac function on MRI-derived parameters, nor does it improve clinical outcome. New and possibly more potent stem cells are emerging in the field, but their clinical efficacy still needs to be defined in future trials.

Divergence of zebrafish and mouse lymphatic cell fate specification pathways.

In mammals, the homeodomain transcription factor Prox1 acts as the central regulator of lymphatic cell fate. Its restricted expression in a subset of cardinal vein cells leads to a switch towards lymphatic specification and hence represents a prerequisite for the initiation of lymphangiogenesis. Murine Prox1-null embryos lack lymphatic structures, and sustained expression of Prox1 is indispensable for the maintenance of lymphatic cell fate even at adult stages, highlighting the unique importance of this gene for the lymphatic lineage. Whether this pre-eminent role of Prox1 within the lymphatic vasculature is conserved in other vertebrate classes has remained unresolved, mainly owing to the lack of availability of loss-of-function mutants. Here, we re-examine the role of Prox1a in zebrafish lymphangiogenesis. First, using a transgenic reporter line, we show that prox1a is initially expressed in different endothelial compartments, becoming restricted to lymphatic endothelial cells only at later stages. Second, using targeted mutagenesis, we show that Prox1a is dispensable for lymphatic specification and subsequent lymphangiogenesis in zebrafish. In line with this result, we found that the functionally related transcription factors Coup-TFII and Sox18 are also dispensable for lymphangiogenesis. Together, these findings suggest that lymphatic commitment in zebrafish and mice is controlled in fundamentally different ways.

Absence of chemokine (C-x-C motif) ligand 10 diminishes perfusion recovery after local arterial occlusion in mice.

OBJECTIVE: In arteriogenesis, pre-existing anastomoses undergo enlargement to restore blood flow in ischemic tissues. Chemokine (C-X-C motif) ligand 10 (CXCL10) is secreted after Toll-like receptor activation. Toll-like receptors are involved in arteriogenesis; however, the role of CXCL10 is still unclear. In this study, we investigated the role for CXCL10 in a murine hindlimb ischemia model. APPROACH AND RESULTS: Unilateral femoral artery ligation was performed in wild-type (WT) and CXCL10(-/-) knockout (KO) mice and perfusion recovery was measured using laser-Doppler perfusion analysis. Perfusion recovery was significantly lower in KO mice compared with WT at days 4 and 7 after surgery (KO versus WT: 28±5% versus 81±13% at day 4; P=0.003 and 57±12% versus 107±8% at day 7; P=0.003). Vessel measurements of α-smooth muscle actin-positive vessels revealed increasing numbers in time after surgery, which was significantly higher in WT when compared with that in KO. Furthermore, α-smooth muscle actin-positive vessels were significantly larger in WT when compared with those in KO at day 7 (wall thickness, P<0.001; lumen area, P=0.003). Local inflammation was assessed in hindlimb muscles, but this did not differ between WT and KO. Chimerization experiments analyzing perfusion recovery and histology revealed an equal contribution for bone marrow-derived and circulating CXCL10. Migration assays showed a stimulating role for both intrinsic and extrinsic CXCL10 in vascular smooth muscle cell migration. CONCLUSIONS: CXCL10 plays a causal role in arteriogenesis. Bone marrow-derived CXCL10 and tissue-derived CXCL10 play a critical role in accelerating perfusion recovery after arterial occlusion in mice probably by promoting vascular smooth muscle cell recruitment and maturation of pre-existing anastomoses.

Efficiency of statin treatment on EPC recruitment depends on baseline EPC titer and does not improve angiographic outcome in coronary artery disease patients treated with the Genous stent.

The objective of this study was to assess the effect of high-dose atorvastatin treatment on endothelial progenitor cell (EPC) recruitment and angiographic and clinical outcome in coronary artery disease (CAD) patients treated with the Genous EPC-capturing stent. The HEALING IIB study was a multicenter, open-label, prospective trial that enrolled 100 patients. Patients were started on 80 mg atorvastatin qd, at least 2 weeks before the index procedure and continued for at least 4 weeks after the index procedure. Eighty-seven patients were included in this analysis. EPC levels significantly increased as early as 2 weeks after the start of atorvastatin. Remarkably, among this group, 31 patients proved to be nonresponders to atorvastatin treatment (i.e., no increase in EPC levels), while 56 patients were responders (i.e., rise in EPC count between week -2 and 0). Compared to responders, nonresponders had a significantly higher baseline EPC count (76 ± 10 vs. 41 ± 5, p < 0.01) with a lower late luminal loss (LLL) at 6- and 18-month follow-up (FU) (0.61 ± 0.07 vs. 0.88 ± 0.08, p < 0.05, and 0.50 ± 0.08 vs. 0.82 ± 0.08, p < 0.01 respectively). Furthermore, baseline EPC count was inversely correlated with LLL at 6-month FU (R = -0.42, p < 0.001). Patients with a higher EPC count at baseline showed no increase in EPC recruitment in response to statin treatment but had favorable LLL at 6- and 18-month FU, whereas patients with lower EPC count were responsive to statin therapy, but EPCs might be less functional as they had higher LLL at 6- and 18-month FU. These data imply that although statin treatment can enhance EPC titer in patients with low baseline levels, there is no indication for a possible beneficial clinical effect with EPC capture stents.

Clinical study using adipose-derived mesenchymal-like stem cells in acute myocardial infarction and heart failure.

Adipose tissue represents an abundant, accessible source of regenerative cells that can be easily obtained in sufficient amount for therapy. Adipose-derived regenerative cells (ADRC) are comprised of leukocytes, smooth muscles, endothelial cells, and mesenchymal stem cells. In contrast to bone-marrow-derived MSC, the abundance of adipose tissue in patients and the higher frequency per unit mass of regenerative cells allow for the isolation of cells in therapeutic meaningful amounts in less than 2h after donor tissue acquisition.Harvest of adipose tissue can thus follow primary PCI, allowing efficient treatment within 24h. This obviates the need for extensive cell culturing in GMP clean room facilities and makes ADSCs a promising and practical autologous cell source. In the following chapter, we will describe the liposuction procedure for stem cell harvest, two cell delivery techniques, and pressure/volume loop analysis for the follow-up of our patients enrolled in the clinical studies.

Intracoronary infusion of allogeneic mesenchymal precursor cells directly after experimental acute myocardial infarction reduces infarct size, abrogates adverse remodeling, and improves cardiac function.

RATIONALE: Mesenchymal precursor cells (MPCs) are a specific Stro-3+ subpopulation of mesenchymal stem cells isolated from bone marrow. MPCs exert extensive cardioprotective effects, and are considered to be immune privileged. OBJECTIVE: This study assessed the safety, feasibility, and efficacy of intracoronary delivery of allogeneic MPCs directly after acute myocardial infarction in sheep. METHODS AND RESULTS: Initially, intracoronary delivery conditions were optimized in 20 sheep. These conditions were applied in a randomized study of 68 sheep with an anterior acute myocardial infarction. Coronary flow was monitored during MPC infusion, and cardiac function was assessed using invasive hemodynamics and echocardiography at baseline and during 8 weeks follow-up. Coronary flow remained within thrombolysis in myocardial infarction III definitions in all sheep during MPC infusion. Global left ventricular ejection fraction as measured by pressure-volume loop analysis deteriorated in controls to 40.7±2.6% after 8 weeks. In contrast, MPC treatment improved cardiac function to 52.8±0.7%. Echocardiography revealed significant improvement of both global and regional cardiac functions. Infarct size decreased by 40% in treated sheep, whereas infarct and border zone thickness were enhanced. Left ventricular adverse remodeling was abrogated by MPC therapy, resulting in a marked reduction of left ventricular volumes. Blood vessel density increased by >50% in the infarct and border areas. Compensatory cardiomyocyte hypertrophy was reduced in border and remote segments, accompanied by reduced collagen deposition and apoptosis. No microinfarctions in remote myocardial segments or histological abnormalities in unrelated organs were found. CONCLUSIONS: Intracoronary infusion of allogeneic MPCs is safe, feasible, and markedly effective in a large animal model of acute myocardial infarction.

Oxidative stress and pathological changes after coronary artery interventions.

Oxidative stress greatly influences the pathogenesis of various cardiovascular disorders. Coronary interventions, including balloon angioplasty and coronary stent implantation, are associated with increased vascular levels of reactive oxygen species in conjunction with altered endothelial cell and smooth muscle cell function. These alterations potentially lead to restenosis, thrombosis, or endothelial dysfunction in the treated artery. Therefore, the understanding of the pathophysiological role of reactive oxygen species (ROS) generated during or after coronary interventions, or both, is essential to improve the success rate of these procedures. Superoxide O2(·-) anions, whether derived from uncoupled endothelial nitric oxide synthase, nicotinamide adenine dinucleotide phosphate oxidase, xanthine oxidase, or mitochondria, are among the most harmful ROS. O2(·-) can scavenge nitric oxide, modify proteins and nucleotides, and induce proinflammatory signaling, which may lead to greater ROS production. Current innovations in stent technologies, including biodegradable stents, nitric oxide donor-coated stents, and a new generation of drug-eluting stents, therefore address persistent oxidative stress and reduced nitric oxide bioavailability after percutaneous coronary interventions. This review discusses the molecular mechanisms of ROS generation after coronary interventions, the related pathological events-including restenosis, endothelial dysfunction, and stent thrombosis-and possible therapeutic ways forward.

Biological mechanisms of microvessel formation in advanced atherosclerosis: the big five.

Advanced atherosclerotic lesions prone to rupture are characterized by a distinct histomorphology and pathobiology that became in recent years, increasingly related to the process of intraplaque neovascularization. Molecular mechanisms that regulate angiogenesis and that are active in the plaque region may destabilize advanced lesions by promoting microvessel growth and thus providing an entry route for inflammatory cells secondary to the luminal endothelium. In addition, angiogenic factors can also define intraplaque microvessel integrity and endothelial barrier function, determining the prevalence of intraplaque hemorrhaging. Here, we aim to compose a hypothetical model for angiogenic regulation of vulnerable plaque development, based on the evidence of clinical correlation and experimental functional studies that are provided for five of the most well-described angiogenic pathways in the current literature.