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.

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 enhances reporter gene expression after transfection of vascular cells in vitro.

BACKGROUND: Restenosis after percutaneous coronary intervention remains a serious clinical problem. Progress in local gene therapy to prevent restenosis has been hindered by concerns over the safety and efficacy of viral vectors and the limited efficiency of nonviral techniques. This study investigates the use of adjunctive ultrasound to enhance nonviral gene delivery. METHODS AND RESULTS: Cultured porcine vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) were transfected with naked or liposome-complexed luciferase reporter plasmid for 3 hours. Ultrasound exposure (USE) for 60 seconds at 1 MHz, 0.4 W/cm2, 30 minutes into this transfection period enhanced luciferase activity 48 hours later by 7.5-fold and 2. 4-fold, respectively. Luciferase activity after lipofection of ECs was similarly enhanced 3.3-fold by adjunctive USE. USE had no effect on cell viability, although it inhibited VSMC but not EC proliferation. CONCLUSIONS: Adjunctive USE was associated with enhanced transgene expression in VSMCs and ECs and reduced VSMC but not EC proliferation in vitro, which suggests that ultrasound-assisted local gene therapy has potential as an antirestenotic therapy.

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.

Substance P potentiates the algogenic effects of intraarterial infusion of adenosine.

OBJECTIVES: This study investigated whether substance P potentiates the muscular and cardiac pain caused by the intraarterial infusion of adenosine, an autocoid known to induce muscular and cardiac ischemic-like pain in humans. BACKGROUND: Substance P is involved in the generation of neurogenic inflammation and causes cutaneous hyperalgesia. Because substance P is present in perivascular nerves it might also cause muscular and cardiac hyperalgesia. To test this hypothesis its effects on adenosine-induced muscular and cardiac pain were investigated in humans. METHODS: A randomized, crossover study of the algogenic effects of the intrailiac infusion of increasing scalar doses (from 125 to 2,000 micrograms/min) of adenosine or substance P (11.2 pmol/min) for 3 min, followed by the simultaneous infusion of substance P plus the same doses of adenosine, was carried out in nine patients with no evidence of peripheral vascular disease. A similar protocol was carried out by infusing increasing scalar doses of adenosine (from 50 to 800 micrograms/min) or substance P (11.2 pmol/min) for 3 min, followed by the simultaneous infusion of substance P plus the same doses of adenosine, into the left coronary artery of eight patients with angina. Pain severity, assessed by a visual analog scale, is presented as median. The remaining data are presented as mean value +/- 1 SD. RESULTS: All patients experienced pain during both adenosine and substance P plus adenosine infusion; no patient experienced pain during the infusion of substance P alone. During intrailiac infusion, all patients experienced pain in the right leg that occurred earlier (207 +/- 152 vs. 321 +/- 154 s, p < 0.05) and was greater (47 vs. 30 mm, p < 0.05) during the simultaneous infusion of substance P plus adenosine than during the infusion of adenosine. Similarly, during intracoronary infusion, all patients experienced chest pain that occurred earlier (409 +/- 242 vs. 596 +/- 210 s, p < 0.05) and was greater (51 vs. 33 mm, p < 0.05) during the simultaneous infusion of substance P plus adenosine than during infusion of adenosine. No patient exhibited electrocardiographic signs of ischemia. CONCLUSIONS: Substance P does not cause muscular or cardiac pain, but it provokes muscular and cardiac hyperalgesia.

Substance P for evaluation of coronary endothelial function after cardiac transplantation.

The endothelium-dependent vasodilator substance P dilates normal and diseased coronary vessels in humans in vivo and produces a maximal response similar to that seen with intracoronary isosorbide dinitrate. Twelve cardiac transplant recipients underwent intracoronary infusion of substance P after routine annual investigations. All patients were well, with no evidence of rejection and with angiographically normal coronary arteries. Substance P was infused at 2 ml/min for 2 min into the coronary artery, starting at a dose of 1.4 pmol/min and increasing by doubling increments, and followed by isosorbide dinitrate (1 mg/min) infused over 2 min. Coronary artery diameter was measured in 23 vessel segments from 12 transplant recipients. The following doses were infused: saline solution (1 ml/min), substance P (0.7 [three patients], 1.4, 2.8, 5.6, 11.2, 22.4 pmol/min) and isosorbide dinitrate (1 mg/min). The mean percent increase in diameter (+/- SEM) in response to increasing doses of substance P was as follows: 0, 6.5 +/- 2.9%, 10.9 +/- 2.9%, 12.1 +/- 2.9%, 16.5 +/- 2.6%, 19.2 +/- 3.1% and 25.8 +/- 2.2%, respectively. Half maximal dilation was produced with 1.4 to 2.8 pmol/min of substance P; the maximal response (mean percent diameter change) was 22 +/- 2.5%. This was not significantly different from that achieved with isosorbide dinitrate. It is concluded that coronary endothelial function as assessed by response to substance P is preserved in cardiac transplant recipients with angiographically normal coronary arteries. Substance P may be a suitable agent for testing endothelial function in these patients.

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.

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.

Alterations in c-fos expression, cell proliferation and apoptosis in pressure distended human saphenous vein.

OBJECTIVES: Saphenous vein graft failures, resulting from thrombosis and the abnormal proliferation, migration and apoptosis of vascular smooth muscle cells (VSMC) are major limitations of coronary artery bypass surgery. We investigated whether surgical trauma of human saphenous vein induces the early response gene c-fos and causes alterations in rates of proliferation and apoptosis. METHODS: Surgically prepared human vein consisted of distended (at 350 mmHg for 2 min) or non-distended segments of vein maintained in serum free RPMI at 37 degrees C and 5% CO2 for various time intervals. c-fos expression was detected by Northern analysis. Cell proliferation and apoptosis were determined by [3H]thymidine incorporation combined with proliferation cell nuclear antigen (PCNA) immunostaining and TUNEL, respectively. Labelling indices for proliferation and apoptosis were correlated with vessel was thicknesses. RESULTS: Control, freshly isolated vein expressed no c-fos. Surgically prepared vein synthesized c-fos 1 h following harvesting. There was a significant increase in c-fos in distended compared to non-distended vein. c-Fos protein increased in surgically prepared vein 24 h after harvesting. There was a significant increase in vascular cell proliferation in the non-distended compared to the distended vein: mean (S.E.M.) 1279 (218) vs. 863 (155) dpm/microgram DNA, P < 0.05, n = 6. In addition, the apoptotic index was significantly lower in the media of non-distended vs. distended vein 0.82 (0.2) vs. 5.5 (1.5), P < 0.05, n = 5. CONCLUSIONS: These findings demonstrate that surgical preparation of human saphenous vein increases expression of c-fos mRNA and apoptosis and reduces proliferation when compared with non-distended vein. These changes may influence the failure of saphenous vein grafts.

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.

Sensory neuropeptide effects in human skin.

1 Neuropeptides released from sensory nerves may account for cutaneous flare and wheal following local trauma. In 28 normal subjects we have studied the effects of four sensory neuropeptides given by intradermal injection on the forearm or back. 2 All peptides caused a flare distant from the site of injection, presumably due to an axon reflex. Substance P (SP) was the most potent (geometric mean dose causing 50% of maximum flare, 4.2 pmol). Neurokinin A (NKA) was the next most potent with neurokinin B (NKB) and calcitonin gene-related peptide (CGRP) the least. The distant flare response to SP, NKA and NKB was maximal at 5 min and disappeared within 2 h. 3 CGRP caused a local erythema over the site of injection at doses above 0.5 pmol which at higher doses lasted for up to 12 h. 4 SP, NKA and NKB caused wheals at doses above 5 pmol with SP and NKB being the most potent. CGRP (up to 250 pmol) did not consistently cause wheal formation. There was no significant effect of coinjection of CGRP upon the response to SP although there was a tendency for an enhancement of the wheal response. 5 The H1-histamine antagonist terfenadine (60 mg orally) significantly inhibited the wheal and distant flare response to histamine (5 nmol) and NKA, but not that caused by NKB. The distant flare of CGRP was also reduced but the local erythema was unaltered. 6. Aspirin (600 mg orally) significantly inhibited the distant flare response to SP, NKA and CGRP, but not that caused by NKB or histamine; the local erythema induced by CGRP was unaffected by aspirin. Aspirin also inhibited the wheal formed by NKA but not the wheal induced by the other substances. 7. These results suggest that tachykinins cause a distant flare response partially via the release of histamine and cyclo-oxygenase products, but cause a wheal by a direct effect on the skin microvasculature. The order of potency SP > NKB > NKA suggests that an SPp or NK, receptor is involved in the wheal response. CGRP by contrast has a direct vasodilator effect which is very prolonged.

Action of calcitonin gene-related peptide upon bovine vascular endothelial and smooth muscle cells grown in isolation and co-culture.

1. Bovine aortic endothelial cells (BAE) and smooth muscle cells (BASM) were grown separately and in co-culture. 2. Calcitonin gene-related peptide (CGRP) caused dose-dependent activation of adenylate cyclase in each cell type when grown in isolation. The concentration of CGRP causing half-maximal activation in BAE and BASM was 200 nM and 310 nM, respectively. 3. In cells grown in co-culture exposure to bradykinin produced dose-dependent elevations in cyclic GMP content which were maximal 1 min after application of the agonist. 4. CGRP (1 nM-1 microM) did not stimulate a rise in cyclic GMP in co-cultures. 5. Displaceable CGRP binding was identified throughout the wall of the bovine aorta. 6. We conclude that CGRP receptors coupled to adenylate cyclase are present on BAE and BASM, but there is no coupling of these receptors to the release of any agent (such as endothelium-derived relaxing factor) that activates guanylate cyclase.

Potent vasoactive properties of endothelin 1 in human skin.

The effect of the endothelial cell-derived peptide endothelin 1 was investigated in human skin. Intradermal injection of endothelin 1 (1-100 pmol) caused a dose-dependent area of pallor that was associated with a significant reduction in basal skin blood flow, measured by laser-Doppler blood flowmeter (with 1 pmol endothelin, P = 0.012, analysis of variance). The coadministration of endothelin 1 (1-100 pmol) with the neuropeptide vasodilator calcitonin gene-related peptide (CGRP) inhibited the vasodilator response to CGRP (10 pmol) by up to 82.7 +/- 9.2% (with 100 pmol endothelin, P less than 0.001). The response of the prostanoid vasodilator prostaglandin E2 (10 pmol) was inhibited by endothelin in a similar manner. In addition to the vasoconstrictor effects, endothelin 1 produced a dose-dependent flare that surrounded the area of pallor, and this was associated with a significant increase in blood flow (P less than 0.05) within the flare area. The H1 antagonist terfenadine (120 mg po) significantly reduced the flare area associated with endothelin 1: flare 5 min after intradermal endothelin (10 pmol, placebo treated), 668 +/- 405 mm2; terfenadine treated, 201 +/- 257 mm2 (P less than 0.05). The flare was also significantly attenuated when endothelin (10 pmol) was injected into local anesthetic-treated skin. Thus intradermal injection of endothelin in humans causes long-lasting vasoconstriction at the site of injection and a surrounding flare. Results suggest that the flare component is partially histamine dependent and the result of an axon reflex. This study demonstrates the potent activity of endothelin in human skin. It is possible that endothelin could be relevant to the local response of skin to injury.

Sodium cromoglycate: evidence of tachykinin antagonist activity in the human skin.

The mechanism of action of the antiasthmatic drug sodium cromoglycate (SCG) is unclear. One possibility is that SCG antagonizes the effects of the tachykinin substance P (SP), an agent known to cause airway edema. However, when SP is inhaled by humans, it has no demonstrable effect on airway function; therefore, the possibility that SCG prevents SP-induced changes in microvascular permeability was examined in human skin in vivo where potent edema-producing effects are seen. SCG (5-500 nmol) caused significant (P < 0.05) dose-dependent inhibition of SP-induced edema (wheal) formation when coadministered by intradermal injection. There was no effect on the nonreceptor-mediated flare response. SCG also significantly (P < 0.05) inhibited the wheal response to the related tachykinin neurokinin B but had no inhibitory effect on the cutaneous responses to histamine and prostaglandin E2. In addition, SCG (0.1-10 mM) caused dose-dependent inhibition of binding of SP labeled with 125I-labeled Bolton-Hunter to a number of tissues known to contain SP binding sites, as assessed by autoradiography. These concentrations were equivalent to the final concentrations of SCG found to inhibit the wheal response in the skin. The possibility that SCG interacted with SP was investigated both by gel filtration and high-performance liquid chromatography. No strong interaction was demonstrated with an 8,000 M excess of SCG under both hydrophobic and hydrophilic conditions. These results raise the possibility that SCG may have tachykinin antagonist properties.

Intravascular stents: a new technique for tissue processing for histology, immunohistochemistry, and transmission electron microscopy.

BACKGROUND: Study of the vascular response to stent implantation has been hampered by difficulties in sectioning metal and tissue without distortion of the tissue stent interface. The metal is often removed before histochemical processing, causing a loss of arterial architecture. Histological and immunohistochemical sections should be 5 microns with an intact tissue stent interface. OBJECTIVES: To identify the most suitable cutting and grinding equipment, embedding resin, and slides for producing thin sections of stented arteries with the stent wires in situ for histological, immunohistochemical, and transmission electron microscopic (TEM) analyses. METHODS: 20 balloon stainless steel stents were implanted in the coronary arteries of 10 pigs. Twenty eight days later the stented arterial segments were excised, formalin fixed, embedded in five different resins (Epon 812, LR white, T9100, T8100, and JB4), and sectioned with two different high speed saws and a grinder for histological, immunohistochemical, and TEM analyses. Five stented human arteries were obtained at necropsy and processed using the best of the reported methods. RESULTS: The Isomet precision saw and grinder/polisher unit reliably produced 5 microns sections with most embedding resins; minimum section thickness with the horizontal saw was 400 microns. Resin T8100, a glycol methacrylate, enabled satisfactory sectioning, grinding, and histological (toluidine blue, haematoxylin and eosin, and trichromatic and polychromatic stains) and immunohistochemical analyses (alpha smooth muscle actin, von Willebrand factor, vimentin, proliferating cell nuclear antigen, and CD68 (mac 387)). T9100 and T8100 embedded stented sections were suitable for ultrastructural examination with TEM. Stented human arterial sections showed preserved arterial architecture with the struts in situ. CONCLUSION: This study identified the optimal methods for embedding, sawing, grinding, and slide mounting of stented arteries to achieve 5 microns sections with an intact tissue metal interface, excellent surface qualities, histological and immunohistochemical staining properties, and suitability for TEM examination. The technique is applicable to experimental and clinical specimens.

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.

Delivery of antisense oligonucleotides to the vascular wall.

Antisense oligonucleotides are short segments of synthetic DNA designed to contain sequences of bases complementary to the DNA or RNA of a particular target gene of interest. By binding to the target, the antisense oligonucleotides can prevent translation of the gene into protein via different mechanisms including destruction of the AS-ODN-nucleic acid hybrid by RNAse H, steric hindrance of the ribosome causing interference of protein elongation, or blockage of the initiation of protein translation (1). Figure 1 shows the possible mechanisms of action of AS-ODNs. Fig. 1. Possible mechanisms of action of antisense oligonucleotides. 1, Aptameric or specific binding to transcription factors; 2, triplex formation via binding to doublestranded DNA resulting in steric inhibition of transcription of DNA into RNA; 3, specific binding to splice junctions or poly A signals inhibits mRNA maturation; 4, inhibition of transport from the nucleus; 5, specific binding to mRNA causing inhibition of translation via steric hindrance of ribosomal complexes; 6, duplex formation causing RNAse H mediated cleavage of mRNA; 7, aptameric binding to protein causing inhibition of protein function.

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.

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.