Simultaneous intravital imaging of macrophage and neutrophil behaviour during inflammation using a novel transgenic zebrafish.

The zebrafish is an outstanding model for intravital imaging of inflammation due to its optical clarity and the ability to express fluorescently labelled specific cell types by transgenesis. However, although several transgenic labelling myeloid cells exist, none allow distinction of macrophages from neutrophils. This prevents simultaneous imaging and examination of the individual contributions of these important leukocyte subtypes during inflammation. We therefore used Bacterial Artificial Chromosome (BAC) recombineering to generate a transgenic Tg(fms:GAL4.VP16)i186 , in which expression of the hybrid transcription factor Gal4-VP16 is driven by the fms (CSF1R) promoter. This was then crossed to a second transgenic expressing a mCherry-nitroreductase fusion protein under the control of the Gal4 binding site (the UAS promoter), allowing intravital imaging of mCherry-labelled macrophages. Further crossing this compound transgenic with the neutrophil transgenic Tg(mpx:GFP)i114 allowed clear distinction between macrophages and neutrophils and simultaneous imaging of their recruitment and behaviour during inflammation. Compared with neutrophils, macrophages migrate significantly more slowly to an inflammatory stimulus. Neutrophil number at a site of tissue injury peaked around 6 hours post injury before resolving, while macrophage recruitment increased until at least 48 hours. We show that macrophages were effectively ablated by addition of the prodrug metronidazole, with no effect on neutrophil number. Crossing with Tg(Fli1:GFP)y1 transgenic fish enabled intravital imaging of macrophage interaction with endothelium for the first time, revealing that endothelial contact is associated with faster macrophage migration. Tg(fms:GAL4.VP16)i186 thus provides a powerful tool for intravital imaging and functional manipulation of macrophage behaviour during inflammation.

Translational mini-review series on immunology of vascular disease: inflammation, infections and Toll-like receptors in cardiovascular disease.

Cardiovascular disease, in which atherosclerosis is the major underlying cause, is currently the largest cause of death in the world. Atherosclerosis is an inflammatory disease characterized by the formation of arterial lesions over a period of several decades at sites of endothelial cell dysfunction. These lesions are composed of endothelial cells, vascular smooth muscle cells, monocytes/macrophages and T lymphocytes (CD4(+)). As the lesions progress some can become unstable and prone to disruption, resulting in thrombus formation and possibly a myocardial infarction or stroke depending upon the location. Although the exact triggers for plaque disruption remain unknown, much recent evidence has shown a link between the incidence of myocardial infarction and stroke and a recent respiratory tract infection. Interestingly, many reports have also shown a link between a family of pattern recognition receptors, the Toll-like receptors, and the progression of atherosclerosis, suggesting that infections may play a role in both the progression of atherosclerosis and in inducing the more severe complications associated with the disease.

P2X receptor characterization and IL-1/IL-1Ra release from human endothelial cells.

BACKGROUND AND PURPOSE: The pro-inflammatory cytokine, interleukin-1beta (IL-1beta), has been implicated in the pathogenesis of atherosclerosis, potentially via its release from vascular endothelium. Endothelial cells (EC) synthesize IL-1beta in response to inflammatory stimuli, but the demonstration and mechanism of release of IL-1 from ECs remains unclear. In activated monocytes, efficient release of bioactive IL-1beta occurred via activation of ATP-gated P2X(7) receptors (P2X(7)Rs). Activation of P2X(7)R in ECs from human umbilical vein (HUVECs) released IL-1 receptor antagonist (IL-1Ra). The purpose of this study was to provide a quantitative investigation of P2XR expression and function, in parallel with IL-1beta and IL-1Ra synthesis, processing and release, in HUVECs under pro-inflammatory conditions. EXPERIMENTAL APPROACH: Quantitative RT-PCR, immunoblotting, ELISA, flow cytometry, and whole-cell patch clamp recordings were used to determine protein expression and receptor function. IL-8-luciferase-reporter was used as an IL-1 sensitive bioassay. KEY RESULTS: HUVECs expressed P2X(4)R and P2X(7)R subtypes and both were significantly up-regulated under inflammatory conditions. P2X(7)R currents were increased 3-fold by inflammatory stimuli, whereas no P2X(4)R-mediated currents were detected. Caspase-1, but not IL-1beta, was present intracellularly under basal conditions; inflammatory stimuli activated the synthesis of intracellular pro-IL-1beta and increased caspase-1 levels. Activation of P2X(7)Rs resulted in low-level release of bioactive IL-1beta and simultaneous release of IL-1Ra. The net biological effect of release was anti-inflammatory. CONCLUSIONS AND IMPLICATIONS: Endothelial P2X(7)Rs induced secretion of both pro- and anti-inflammatory IL-1 receptor ligands, the balance of which may provide a means for altering the inflammatory state of the arterial vessel wall.

Serum lipids in the GENECARD study of coronary artery disease identify quantitative trait loci and phenotypic subsets on chromosomes 3q and 5q.

Coronary artery disease (CAD) and dyslipidemia have strong genetic components. Heterogeneity complicates evaluating genetics of complex diseases such as CAD; incorporating disease-related phenotypes may help reduce heterogeneity. We hypothesized that incorporating lipoproteins in a study of CAD would increase the power to map genes, narrow linkage peaks, identify phenotypic subsets, and elucidate the contribution of established risk factors to genetic results. We performed ordered subset analysis (OSA) and quantitative trait linkage (QTL) using serum lipoproteins and microsatellite markers in 346 families with early-onset CAD. OSA defined homogeneous subsets and calculated lod scores across a chromosome after ranking families by mean lipoprotein values. QTL used variance components analysis. We found significantly increased linkage to chromosome 3q13 (LOD 5.10, p = 0.008) in families with higher HDL cholesterol, lower LDL and total cholesterol, lower triglycerides, and fewer CAD risk factors, possibly due to a concentrated non-lipoprotein-related genetic effect. OSA identified linkage on chromosome 5q34 in families with higher cholesterol, possibly representing a hereditary lipoprotein phenotype. Multiple QTLs were identified, with the strongest for: total cholesterol on chromosome 5q14 (LOD 4.3); LDL on 20p12 (LOD 3.97); HDL on 3p14 (LOD 1.65); triglycerides on 18q22 (LOD 1.43); and HDL/TC ratio on 3q27-28 (LOD 2.06). Our findings suggest the presence of etiologic heterogeneity in families with early-onset CAD, potentially due to differential effects of lipoprotein phenotypes. Candidate genes are under investigation.

Regulation of expression and signalling modulator function of mammalian tribbles is cell-type specific.

The constant need to respond to changes in the environment is a common feature for all life forms. During evolution, a number of intracellular signal processing systems have evolved to fulfill this requirement. One of the most ancient such systems is the mitogen activated protein kinase (MAPK) signalling network, shared by all eukaryotes. Activation of MAPKs is key to regulation of mitosis and in cellular responses to stress or hormones, for instance. In addition, activity of this signalling system is essential during embryonic development. However, many aspects of MAPK mediated responses are strongly cell-type specific. A family of proteins, called tribbles have recently been described as novel regulators of MAPK function. Our group has previously shown that alterations in tribbles levels lead to profound changes in the activation of the various MAPKs. However, little is known about the cell-type specific aspects of regulation of tribbles expression. Here, we report that expression of all three members of the human tribbles family is dynamically controlled in response to inflammatory stimulation. This regulation, however, is strongly cell-type dependent. Our observations suggest regulation of tribbles expression may play an important role in the cell-type specific cellular responses, mediated by the MAPK network.

Ultrasound-mediated delivery of TIMP-3 plasmid DNA into saphenous vein leads to increased lumen size in a porcine interposition graft model.

Progressive saphenous vein graft (SVG) narrowing and occlusion remains a major limitation of coronary artery bypass grafting and is an important target for gene therapy. Ex vivo adenoviral gene transfer of tissue inhibitor of metalloproteinase 3 (TIMP-3) reduces adverse SVG remodelling postarterialization, but concerns remain over the use of viral vectors in patients. Ultrasound exposure (USE) in the presence of echocontrast microbubbles (ECM) substantially enhances nonviral gene delivery. We investigated the effects of ultrasound-enhanced gene delivery (UEGD) of TIMP-3 plasmid on vascular remodelling in porcine SVG. Maximal luciferase activity (3000-fold versus naked plasmid alone) and TIMP-3 transgene expression in porcine vascular smooth muscle cells in vitro was achieved using USE at 1 MHz, 1.8 mechanical index (MI), 6% duty cycle (DC) in the presence of 50% (v/v) BR14 ECM (Bracco). These conditions were therefore utilized for subsequent studies in vivo. Yorkshire White pigs received carotid interposition SVG that were untransfected or had undergone ex vivo UEGD of lacZ (control) or TIMP-3 plasmids. At 28 d postgrafting, lumen and total vessel area were significantly greater in the TIMP-3 group (10.1+/-1.2 and 25.5+/-2.2 mm2, respectively) compared to untransfected (6.34+/-0.5 and 20.8+/-1.9 mm2) or lacZ-transfected (6.1+/-0.7 and 19.7+/-1.2 mm2) controls (P<0.01). These data indicate that nonviral TIMP-3 plasmid delivery by USE achieves significant biological effects in a clinically relevant model of SV grafting, and is the first study to demonstrate the potential for therapeutic UEGD to prevent SVG failure.

A potential pharmacogenomic strategy for anticoagulant treatment in non-ST elevation acute coronary syndromes: the role of interleukin-1 receptor antagonist genotype.

OBJECTIVES: Our aim was to determine a pharmacogenomic approach to heparin use in non-ST elevation acute coronary syndromes, specifically the impact of interleukin (IL)-1 receptor antagonist polymorphisms upon von Willebrand factor (vWF) responses to unfractionated heparin (UFH) and low molecular weight heparin (LMWH). BACKGROUND: In acute coronary syndromes (ACS), identification of specific biological or genetic targets to direct pharmacological treatment remains a challenge. vWF has been shown to predict future cardiovascular risk and the response to anticoagulant treatments during non-ST elevation ACS. IL-1 receptor antagonist (IL-1RN) polymorphisms predict the change in vWF between 24 and 48 h (Delta vWF) during non-ST elevation ACS. METHODS: We genotyped at the IL-1 locus, 67 patients with non-ST elevation ACS who received either LMWH or UFH, and measured vWF levels at 24 and 48 h. RESULTS: LMWH was superior to UFH in reducing the rise in vWF between 24 and 48 h in the cohort as a whole. However, when patients were stratified by IL-1RN genotype, LMWH was superior to UFH in reducing Delta vWF only in allele *2 carriers (0.51 iU mL(-1) vs. 1.37, P < 0.01), but not in non-carriers (- 0.03 iU mL(-1) vs. 0.15, P = NS). CONCLUSION: IL-1RN genotype may be a useful marker to identify patients that benefit from LMWH in non-ST elevation ACS.

Response of very small (2 mm) porcine coronary arteries to balloon angioplasty and stent implantation.

OBJECTIVE: To investigate the response of very small coronary arteries to stent deployment and balloon angioplasty. SETTING: Normal porcine coronary arteries. METHODS: 24 pigs underwent intervention to two main coronary arteries, in segments 2.0 mm in diameter, with balloons whose diameter was 2.5 mm at standard pressure. Twelve arteries received a BiodivYsio small vessel (SV) stent; 12 an NIR SV stent; 12 standard BiodivYsio stent, and 12 balloon only. The arteries were harvested at 28 days, fixed, embedded in plastic, and cut and ground in cross section. The injury score and histomorphometry were assessed. RESULTS: The BiodivYsio SV stent was associated with 20% less injury (p = 0.16), a 30% larger lumen (p = 0.13), and a 25% smaller neointima (p = 0.03) than the NIR SV stent, despite identical oversize. The standard BiodivYsio stent exhibited less recoil but 29% greater injury (p = 0.01), 59% more neointima (p = 0.00), and 18% less lumen (p = 0.27) than the BiodivYsio SV. Of all interventions, balloon only was associated with little injury, little neointima, major vessel shrinkage, and the largest lumen. CONCLUSION: Despite uniform oversize dilatation, both injury and response varied widely in very small porcine coronary arteries, depending on whether a stent or balloon was used, the stent design, and the number and/or thickness of struts. The response to different stent designs is considerable and is related to the degree of injury.

Drug eluting stents: maximising benefit and minimising cost.

A policy of selective implantation of drug eluting stents, in a minority of lesions most likely to benefit, seems to be a rational way to employ this new and currently costly technology.

Coronary artery stretch versus deep injury in the development of in-stent neointima.

OBJECTIVE: To investigate the relative importance of stent induced arterial stretch and deep injury to the development of in-stent neointima. SETTING: Normal porcine coronary arteries METHODS: 30 BiodivYsio stents (Biocompatibles) were deployed at a stent to artery ratio of 1.25:1 (a moderate injury) and harvested at 28 days. Multiple serial cross sections were analysed morphometrically and the neointimal areas were correlated with the type and degree of injury. RESULTS: Arterial stretch occurred in 78% of struts (77% of sections) and produced moderate neointimal growth (neointimal area 1.93 (0.13) mm2). Deep injury (rupture of the internal elastic lamina) occurred in 20% of struts (23% of sections) and produced a 1.7-fold increase in neointimal area (3.33 (0.41) mm2) compared with stretch only (p = 0.0002). With even deeper injury (rupture of the external elastic lamina), there was a 2.6-fold increase in neointimal area (5.01 (0.48) mm2) compared with stretch only (p = 0.02). A new injury score, incorporating both stretch and deep injury, correlated with neointimal area (r = 0.60, p < 0.001). CONCLUSIONS: Stretch of the coronary artery in a stent is common, and a major contributor to neointima formation, even in the absence of deep injury. Deep injury is, however, a more potent stimulus to neointima formation than stretch. Greater degrees of stretch are associated with thicker neointima. Where neither deep injury nor stretch are seen, the stent has no effect upon the development of neointima.

Critical review of unstable angina and non-ST elevation myocardial infarction.

Within the coronary vasculature the progression of a stable atherosclerotic plaque into a vulnerable and ultimately unstable lesion leads to a cascade of events culminating in the clinical presentation of unstable angina or acute myocardial infarction. In recent years studies have provided new insights in to the pathology and natural history, stimulating advances in diagnosis, treatment, and management. The review discusses the progress made including the role of inflammation, cardiac biomarkers, antiplatelet therapy, and percutaneous intervention. Current issues of debate and future directions are also addressed.

Localization of c-Myb and induction of apoptosis by antisense oligonucleotide c-Myb after angioplasty of porcine coronary arteries.

Previous studies have shown that inhibition of the proto-oncogene c-myb inhibits neointimal formation in various animal models. However, the temporal and spatial expression of c-Myb in the vessel wall after injury is not known, and the mechanism of action of antisense oligonucleotide (AS-ODN-c-myb) inhibition remains unclear. One potential effect of cell cycle dysregulation by inhibition of c-myb is an increase in the rates of apoptosis. In this study, c-Myb expression after percutaneous transluminal coronary angioplasty (PTCA) injury and induction of apoptosis after AS-ODN-c-myb treatment were determined. Immunohistochemistry and cellular phenotyping were used to localize c-Myb expression in porcine coronary arteries at various time intervals after PTCA. In vitro, the effects of AS-ODN-c-myb on the apoptosis of porcine vascular smooth muscle cells (PVSMCs) and endothelial cells were determined by using a cell-death ELISA and time-lapse video microscopy. In vivo, local delivery of AS-ODN-c-myb was performed after PTCA of pig coronary arteries, and apoptosis was quantified at 6 hours. c-Myb is induced in pig coronary arteries after angioplasty, with maximal expression in inflammatory cells at 18 hours and in vascular smooth muscle cells at 3 to 7 days. In vitro, AS-ODN-c-myb enhanced PVSMCs (6.8+/-0.8% [P=<0.001] versus 0.5% serum) but not endothelial cell apoptosis (1.4+/-0.5% [P=NS] versus 0.5% serum). In vivo, 6 hours after porcine coronary angioplasty and delivery of AS-ODN-c-myb, the proportion of apoptotic cells within the media was 4.2+/-0.8% (PTCA alone), 2.3+/-0.2% (PTCA+vehicle), and 9.0+/-1.1% (PTCA+AS-ODN-c-myb; P<0.05 versus PTCA alone and P<0.01 versus PTCA+saline). c-Myb is expressed after PTCA of pig coronary arteries, and AS-ODN-c-myb induces apoptosis of PVSMCs in vitro and medial cells in vivo.

Interleukin 1 receptor antagonist gene polymorphism and restenosis after coronary angioplasty.

BACKGROUND: Percutaneous transluminal coronary angioplasty (PTCA) is limited by the recurrence of luminal stenosis, which occurs in up to 50% of procedures. It has been shown that patient specific factors, perhaps genes, contribute to this process. OBJECTIVE: To determine whether completion of healing after PTCA is part of an acute self limiting inflammatory process and whether polymorphism at important inflammatory gene loci might determine susceptibility to restenosis after PTCA. DESIGN: DNA samples were collected from 171 patients attending for elective PTCA in Sheffield (S) and Leicester (L), who were scheduled to undergo follow up angiography (at four months (L) or six months (S)) as part of other restenosis studies. At follow up angiography, the patients were separated into restenosers (> 50% luminal narrowing) and non-restenosers (< 50% luminal narrowing). Four DNA polymorphisms within interleukin 1 (IL-1) related loci (IL-1A (-889), IL-1B (-511), IL-1B (+3954), and IL-1RN intron 2 VNTR (variable number tandem repeat)) were genotyped using methods based on polymerase chain reaction. Significance was assessed by chi(2) analysis of the relevant contingency table, and the magnitude of effect was estimated by calculating odds ratios. The Mantel-Haenszel (MH) test was applied to summarise data across the two populations. RESULTS: Allele 2 at IL-1RN (IL-1RN*2) was significantly over represented in the non-restenoser group (L+S, 34% v 23% in restenosers). Furthermore, IL-1RN*2 homozygosity was increased in the non-restenoser population compared with the restenosers (MH test: p = 0.0196 (L+S); p = 0.031 (L+S, single vessel disease only), and the effect seemed to be restricted to the single vessel disease subpopulation. For other polymorphism within IL-1 related loci no significant associations were found with either restenosis or non-restenosis. CONCLUSIONS: IL-1RN*2 may be associated with protection from restenosis after PTCA for individuals with single vessel disease. As this polymorphism has functional significance, this finding suggests that alteration in an individual's inflammatory predisposition may modulate the blood vessel response to injury.

TGFbeta is active, and correlates with activators of TGFbeta, following porcine coronary angioplasty.

OBJECTIVE: Restenosis following angioplasty involves processes that may be influenced by local production of cytokines. We investigated the expression of active and total transforming growth factor beta (TGFbeta) following porcine coronary angioplasty (PTCA), and have correlated this with the expression of potential in vivo activators of TGFbeta: mannose-6-phosphate/insulin-like growth factor-II (M6P/IGF-II) receptor and thrombospondin-1. METHODS: Oversized porcine PTCA was performed and the arteries excised after selected intervals. Levels of in situ active and total (active plus latent) TGFbeta were determined using a modified plasminogen activator-inhibitor/luciferase bioassay. RESULTS: Levels of active TGFbeta significantly increased 2 h to 7 days after angioplasty, compared to non-injured controls. Levels returned to baseline by 28 days. Active TGFbeta in tissues adjacent to the injured artery did not change. Total TGFbeta was significantly higher than controls 2-6 h after injury. M6P/IGF-II receptor mRNA was upregulated between 6 h and 3 days after injury, with protein detectable at 3-28 days. Thrombospondin-1 was detected between 1 h and 14 days. CONCLUSIONS: We conclude that balloon injury causes an early rapid increase in levels of active TGFbeta, that correlates with the expression of TGFbeta activators. Thus, TGFbeta is a good potential target for anti-restenotic therapies.

Phosphorylcholine-coated stents in porcine coronary arteries: in vivo assessment of biocompatibility.

AIMS: To examine the angiographic (quantitative coronary angiography), morphometric, light microscopic (LM) (i.e., histology and immunohistochemical staining) and electron microscopic (EM) findings after implantation of phosphorylcholine (PC)-coated compared to uncoated stents in porcine coronary arteries. METHODS: Forty (25 PC-coated, 15 uncoated) divYsio stents were implanted into the coronary arteries of 20 pigs. Quantitative coronary angiography (QCA) was performed pre-stent and post-implantation in fifteen pigs, at 28 days. Two pigs were killed at 5 days (LM and scanning EM), one pig at 14 days (scanning EM) and 17 pigs at 28 days (LM, scanning EM, transmission EM). At 28 days, thirty-two of 34 stented segments excised were formalin-fixed, of which 30 were embedded in resin and sectioned for morphometry and LM. Remaining stents were examined by TEM and SEM. RESULTS: No angiographically occlusive thrombosis occurred in any of the stents. LM at 5 days showed endothelialization of PC-coated and uncoated stents, which was also confirmed by scanning EM at 14 days. At 28 days, QCA and morphometry showed no significant differences between PC-coated and uncoated stents. A few inflammatory cells were seen in both stent types at 5 days but there was no inflammatory or additional tissue reaction to PC-coated compared to uncoated stents at 28 days. CONCLUSIONS: The divYsio stents, with or without PC coating, performed equally well in terms of acute patency, 28-day QCA and morphometry. The PC coating allows a stent to endothelialize normally and is not associated with specific histological changes. The PC coating on the divYsio stent appears biocompatible.

Effect of selective or combined inhibition of integrins alpha(IIb)beta(3) and alpha(v)beta(3) on thrombosis and neointima after oversized porcine coronary angioplasty.

BACKGROUND: Thrombosis and neointima formation limit the efficacy of coronary angioplasty (PTCA). Clinical trials have implicated the adhesion molecules integrin alpha(IIb)beta(3) and integrin alpha(v)beta(3) in these processes. The roles of these molecules in vascular smooth muscle cell adhesion, platelet aggregation, and the thrombotic and neointimal response to oversize porcine PTCA was investigated by use of a selective alpha(IIb)beta(3) antagonist (lamifiban), a selective alpha(v)beta(3) antagonist (VO514), and a combined alpha(IIb)beta(3)/alpha(v)beta(3) antagonist (G3580). METHODS AND RESULTS: In vitro, both alpha(v)beta(3) inhibitors caused dose-dependent inhibition of porcine vascular smooth muscle cell adhesion to vitronectin but not to collagen type IV, fibronectin, or laminin, whereas selective alpha(IIb)beta(3) inhibition had no effect. Intravenous infusions of either alpha(IIb)beta(3) inhibitor in swine profoundly inhibited ex vivo platelet aggregation to ADP, whereas selective alpha(v)beta(3) inhibition had no effect. In a porcine PTCA model, intravenous infusions of the integrin antagonists were administered for 14 days after oversized balloon angioplasty injury. After PTCA, there was regional upregulation of integrin alpha(v)beta(3) in the developing neointima, as assessed by immunohistochemistry. Six hours after PTCA, obstruction of lumen by thrombus was reduced significantly by alpha(IIb)beta(3) inhibition compared with either control or alpha(v)beta(3) inhibition (mean control, 18.7%; VO514, 18.5%; lamifiban, 6.4%; G3580, 7.9%). Twenty-eight days after PTCA, there was a significant reduction of neointima with inhibitors of either integrin (mean intima/media ratio: control, 3.08; VO514, 1.33; lamifiban, 0.97; G3580, 1.32). CONCLUSIONS: We conclude that both integrin alpha(IIb)beta(3) and integrin alpha(v)beta(3) participate in neointima development after experimental angioplasty.

Opportunities for the treatment of inflammation in cardiovascular disease.

Atherosclerosis, and the clinical presentation of atherosclerosis, both have their basic pathogenesis in inflammatory mechanisms. The use of mouse models of atherosclerosis has emphasised the importance of inflammation in atherogenesis and the use of serum markers of inflammation in epidemiological studies has shown the importance of inflammatory status in determining the presentation of atherosclerotic disease. Therapeutic opportunities will arise from the manipulation of these inflammatory mechanisms. Proof of this principle has been shown with the use of aspirin and statin drugs as well as the emerging roles for peroxisome proliferator-activated receptor (PPAR) agonists. It is likely that both refinement of existing anti-inflammatory agents and the identification of new inflammatory mechanisms will afford real opportunities for the treatment of atherosclerotic cardiovascular disease.