Matrigel-coated stents reduce intimal thickening in a large animal vascular stent model.

BACKGROUND: Restenosis within vascular stents is primarily due to intimal thickening secondary to intimal hyperplasia (IH) which occurs maximally around stent struts. Dedifferentiation of vascular smooth muscle cells (VSMC) with subsequent migration and proliferation is believed to be a key event in IH formation. Matrigel (basement membrane protein) has been shown to inhibit dedifferentiation of VSMC in vitro. Our aim was to test the in vivo effect of Matrigel on IH formation using a novel sheep vascular stent model. METHODS: Twenty vascular stents were implanted in the renal arteries of ten sheep. The left renal artery of each sheep was used to deploy uncoated stent and the right renal artery was used to deploy Matrigel-coated stent. Five sheep were analysed at four weeks and five at eight weeks after stent implantation. The sheep were sacrificed at the end of the study periods and the stented renal artery segments were examined by histology. Luminal, intimal and medial areas were determined using computer-assisted morphometric analysis. RESULTS: All stent sites were widely patent without thrombosis. No luminal stenosis was seen angiographically. IH was quantified from histology cross-sections and expressed as an intima to media (I/M) ratio. The ratio was significantly reduced in the matrigel-coated sites at eight weeks (uncoated 0.49+/-0.23; Matrigel-coated 0.32+/-0.12; p value <0.05). CONCLUSIONS: The sheep renal artery vascular stent model is feasible for the study of stent biology. IH was reduced by Matrigel-coated stents.

Detection of dissection and remodeling of atherosclerotic lesions in rabbits after balloon angioplasty by magnetic-resonance imaging.

OBJECTIVE: To assess the usefulness of magnetic-resonance imaging (MRI) for monitoring acute changes after angioplasty of preexisting lesions in rabbits with basal lesions similar to those observed in humans. METHODS: A combination of Fogarty balloon injury (1 week after initiation of diet) and a mildly hypercholesterolemic diet (0.2% cholesterol and 5% peanut oil) was used to promote the rapid formation of atherosclerotic lesions in 16 New Zealand white rabbits. After 5 months of the diet, angioplasty was performed on these lesions with a Grüntzig catheter in both iliac arteries and the abdominal aorta. MRI was used to monitor the initial formation of lesions after 3 and 5 months of the diet, and 2 days, 2 weeks, and 1 and 2 months after angioplasty. RESULTS: The combination of early Fogarty injury and mildly hypercholesterolemic diet induced fibroproliferative lesions similar to type Vb atherosclerotic lesions seen in humans. Angioplasty induced deep dissections at the shoulders of lesions in the majority of animals. These dissections often extended into the media. The cellular, proliferative response after angioplasty was localized and limited to sites of dissection. A significant increase in area of arterial wall was observed after angioplasty at sites of dissection without any loss of lumen. In contrast, proximal and distal to the sites of injury, there was no change in wall area but a transient reduction in lumen area. CONCLUSIONS: Comparison of MRI results with histology confirmed that changes in the wall and lumen, including small linear dissections in the lesions and arterial remodeling, are detectable by MRI.

Magnetic resonance imaging to study lesions of atherosclerosis in the hyperlipidemic rabbit aorta.

We have developed a high resolution magnetic resonance (MR) imaging technique to serially assess lesions of atherosclerosis in a rabbit model. A volume phased array coil was designed and used to image the abdominal aortas of six atherosclerotic rabbits and two age-, sex-, and weight-matched controls. Lesions of atherosclerosis were induced by a combination of repeat balloon injury and a hyperlipidemic diet. All animals were imaged on at least two occasions 9-16 months after initiation of atherosclerosis. In addition, animals were imaged immediately after sacrifice. Anatomic dissection and histology were performed to verify the MR findings. The volume phased array coil improves the image signal-to-noise ratio over existing extremity coils and resulted in higher resolution images of the abdominal aorta. Proton density-weighted images acquired with 2D/3D fast spin-echo are the most useful sequence to outline the vessel wall and to differentiate wall from lumen and background. Progressive wall thickening and lumen stenosis were observed in the serial images of the diseased rabbits. Wall thickness and lumen area derived noninvasively from the in vivo MR images correlate with postmortem MR images and sections of aorta examined by dissection microscopy and histology. Spin-echo and fast spin-echo imaging with a phased array body coil can be used to accurately assess plaque dimensions, and potentially can be used to image intraplaque features and to monitor lesion progression or regression. It should also be possible to adapt these techniques to assess human disease, especially for peripheral vascular problems.

Platelet-derived growth factor. Distinct signal transduction pathways associated with migration versus proliferation.

Figure 2 summarizes our current interpretation of data concerning signals from the activated PDGF receptor involved in directed migration and proliferation of human arterial SMC. Binding of PDGF (PDGF-BB or PDGF-AA) causes PDGF-receptor dimerization, tyrosine autophosphorylation, and subsequent binding of several molecules containing SH2 domains to the activated receptor. Binding and activation of PLC gamma by the PDGF receptor leads to PIP2 hydrolysis, resulting in generation of diacylglycerol (DAG) and IP3. Subsequently, intracellular levels of calcium are elevated as a result of IP3-mediated calcium release from intracellular compartments. The decreased levels of PIP2 and increased levels of calcium both favor actin-filament disassembly by inducing capping of actin-filament barbed ends and actin-monomer sequestration. A localized, and transient, actin-filament disassembly enables the cell to extend filopodia towards PDGF, thereby enabling chemotaxis to take place. At a later time and/or in a different compartment, actin-filament assembly is promoted by PDGF by a mechanism that is not completely understood, but that may involve small GTP-binding proteins, such as Rho, and formation of DAG. Migration on collagen requires functional alpha 2 beta 1 integrins, which may either constitute a permissive state required for a cell to migrate, or which may be actively involved in intracellular signals leading to migration. PDGF-induced DNA synthesis and proliferation involves activation of Ras, MAP kinase kinase, and MAP kinase. Cross-talk between PKA signaling and tyrosine-kinase receptor signaling results in PKA inhibition of the MAP kinase cascade, probably at the level of Raf. Activation of PI 3-kinase, or a PI 3-kinase-like enzyme, is also likely to contribute to the mitogenic effects of PDGF in these cells (Bornfeldt, unpublished observation). What determines if a SMC will migrate and/or proliferate in response to PDGF? Results are starting to emerge that show regulation of expression of molecules involved in intracellular signaling with different phenotypic states of SMC. For example, expression of PLC gamma is very low in intact vascular wall (where SMC show a "contractile phenotype"), and induced when SMC are converted to a "synthetic phenotype" in culture. Proliferation and expression of MAP kinase, but not calcium signaling, appear to be regulated by the extracellular matrix, and the profile of integrin expression is different in SMC in culture compared to SMC in the vascular wall. Thus, the relation between expression of signaling molecules involved in migration and signaling molecules involved in proliferation, as well as cross-talk between different signal-transduction pathways, may determine the net effect of PDGF.

The adhesive and migratory effects of osteopontin are mediated via distinct cell surface integrins. Role of alpha v beta 3 in smooth muscle cell migration to osteopontin in vitro.

Osteopontin is an arginine-glycine-aspartate containing acidic glycoprotein postulated to mediate adhesion, migration, and biomineralization in diverse tissues. The mechanisms explaining this multifunctionality are not well understood, although it is known that one osteopontin receptor is the alpha v beta 3 integrin. In this work, we studied human smooth muscle cells varying in alpha v beta 3 levels to identify additional osteopontin receptors. We report that, in addition to alpha v beta 3, both alpha v beta 5 and alpha v beta 1 are osteopontin receptors. Moreover, the presence or absence of alpha v beta 3 on the cell surface altered the adhesive and migratory responses of smooth muscle cells to osteopontin. Adhesion of alpha v beta 3-deficient cell populations to osteopontin was only half that of cells containing alpha v beta 3, and migration toward an osteopontin gradient in the Boyden chamber was dependent on cell surface alpha v beta 3. Although alpha v beta 3-deficient smooth muscle cells were unable to migrate to osteopontin, they did migrate significantly in response to vitronectin and fibronectin. These findings represent the first description of alpha v beta 5 and alpha v beta 1 as osteopontin receptors and suggest that, while adhesion to osteopontin is supported by integrins containing beta 1, beta 3, and beta 5, migration in response to osteopontin appears to depend on alpha v beta 3. Thus, interaction with distinct receptors is one mechanism by which osteopontin may initiate multiple functions.

Serial magnetic resonance imaging of experimental atherosclerosis detects lesion fine structure, progression and complications in vivo.

A major problem in the study of lesions of atherosclerosis is the difficulty of imaging noninvasively the lesions and following their progression in vivo. To address this problem, we have developed advanced magnetic resonance techniques to noninvasively and serially image advanced lesions of atherosclerosis in the rabbit abdominal aorta. Both lumen and wall were imaged with high resolution. Progression of disease, resulting in increase in lesion mass, decrease in arterial lumen, or stenosis, and intralesion complications, can be detected. Images acquired in vivo correlate with the fine structure of the lesions of atherosclerosis, including the fibrous cap, necrotic core, and lesion fissures, as verified by gross examination, dissection microscopy, and histology. The ability to noninvasively identify the features of atherosclerotic plaques, has significant implications for determining risks and benefits associated with different therapeutic approaches.

Dynamic expression of alpha 1 beta 1 and alpha 2 beta 1 integrin receptors by human vascular smooth muscle cells. Alpha 2 beta 1 integrin is required for chemotaxis across type I collagen-coated membranes.

Vascular smooth muscle cells (SMCs) in the media of normal arteries express alpha 1 beta 1 integrin with no detectable alpha 2 beta 1 as determined by immunocytochemistry. In contrast, immunoprecipitation of integrins expressed by human SMCs cultured from medial explants shows strong expression of alpha 2 beta 1 and no expression of alpha 1 beta 1. The apparent reciprocal expression of these two collagen and laminin receptors was confirmed by flow cytometric analysis of fluorescent labeled cells. Freshly isolated SMCs had detectable alpha 1, alpha 3, alpha 5, and alpha v subunits with low levels of detectable beta 3 and no detectable alpha 2. Cultured SMCs expressed alpha 2, alpha 3, alpha 5 and alpha v subunits with little or no alpha 1 or beta 3. Neither alpha 4 nor alpha 6 were detectable in freshly isolated or cultured cells. Expression of alpha 2 beta 1 receptors by cultured SMCs appears to be required for the migration of these cells on type I collagen. Migration of cultured SMCs across a type I collagen-coated membrane toward two different chemotactic stimuli, platelet-derived growth factor-BB (1 nmol/L) and insulin-like growth factor-(1 nmol/L), was Mg2+ dependent and inhibited by preincubation of cells with an affinity-purified polyclonal anti-alpha 2 beta 1 antibody or by monoclonal antibodies directed against the individual alpha 2 or beta 1 subunits. Attachment to type 1 collagen membranes was not affected by antibodies under conditions where migration was significantly impeded. The combined data show that SMC expression of alpha 1 beta 1 and alpha 2 beta 1 integrin receptors for collagen and laminin is dynamic and reciprocal and may be important with respect to SMC migration on type I collagen. These findings are potentially important in understanding the pathophysiology of vascular diseases, for example, atherosclerosis and restenosis following balloon angioplasty, where SMC migration is a contributing factor.

Characterization of GMP-140 (P-selectin) as a circulating plasma protein.

GMP-140 is a 140-kD granule membrane protein, found in the alpha granules of platelets and the Weibel-Palade bodies of endothelial cells, that is surface expressed on cell activation and mediates neutrophil attachment. Cloning data for GMP-140 from an endothelial library predict a soluble form of the protein, the transcription message for which is also found in platelets. In this study, we report the detection by enzyme-linked immunosorbent assay of soluble GMP-140 in plasma centrifuged for 3 h at 100,000 g (to remove platelet microparticles) and confirm its identity by purification from plasma. Plasma concentrations were found to be 0.251 +/- 0.043 micrograms/ml (means +/- SD, n = 10) in normal male controls and 0.175 +/- 0.063 micrograms/ml (means +/- SD, n = 10) in normal female controls. The purified protein had an identical molecular mass (nonreduced) to platelet membrane GMP-140 (approximately 3 kD lower, reduced) and was immunoblotted by polyclonal anti-GMP-140, and the anti-GMP-140 monoclonal antibodies AK4 and AK6. Analytical gel filtration studies indicated that the plasma GMP-140 eluted as a monomer whereas detergent-free, platelet membrane GMP-140 eluted as a tetramer consistent with plasma GMP-140 lacking a transmembrane domain. Purified plasma GMP-140 bound to the same neutrophil receptor as the membrane-bound form, and when immobilized on plastic, bound neutrophils equivalently to immobilized platelet membrane GMP-140. Since it has been shown that fluid-phase GMP-140 is antiinflammatory and downregulates CD18-dependent neutrophil adhesion and respiratory burst, its presence in plasma may be of major importance in preventing the inadvertent activation of neutrophils in the circulation.

GMP-140 binding to neutrophils is inhibited by sulfated glycans.

GMP-140 is a 140-kDa granule membrane glycoprotein localized to the alpha-granules of platelets and the Weibel-Palade bodies of endothelial cells. Expression of GMP-140 on the activated cell surface has been shown to mediate the adhesion of thrombin-activated platelets to neutrophils and monocytes and the transient adhesion of neutrophils to endothelium. In contrast, fluid-phase GMP-140 strongly inhibits the CD18-dependent adhesion of tumor necrosis factor alpha-activated neutrophils to endothelium suggesting that GMP-140 can also serve an anti-adhesive function. In the present report, it is demonstrated that fluid-phase GMP-140 which exists predominantly as a tetramer binds to a single class of high affinity receptor on neutrophils and HL60 cells. Binding of 125I-labeled GMP-140 to neutrophils and HL60 cells and the rosetting of neutrophils and HL60 cells by thrombin-activated platelets were inhibited by EDTA, excess unlabeled fluid-phase GMP-140, Fab fragments of an affinity-purified rabbit anti-GMP-140 antibody, and by the murine anti-GMP-140 monoclonal antibody, AK 4. Both neutrophil and HL60 GMP-140 binding and platelet rosetting were strongly inhibited by heparin, fucoidin, and dextran sulfate 500,000, were partially inhibited by dextran sulfate 5,000 and lambda- and kappa-carrageenan, but were not inhibited by chondroitins 4- and 6-sulfate. Since this sulfated glycan specificity is identical to that previously reported by us for GMP-140, the present results suggest that the sulfated glycan binding site and the neutrophil receptor binding site on GMP-140 are either identical or proximal.

Adhesion protein GMP140 inhibits superoxide anion release by human neutrophils.

The respiratory burst of blood neutrophils has a critical role in the destruction of microorganisms and tissue damage in inflammation. Neutrophils adhere in a dose-dependent fashion to granule membrane protein 140 (GMP140), a member of the LEC-CAM (lectin/epidermal growth factor/complement-binding domain cell adhesion molecule) family of adhesion proteins when it is immobilized onto plastic surfaces. Adherence to GMP140 was associated with less superoxide anion generation than adherence to other surfaces, an effect that is especially remarkable after activation of neutrophils with tumor necrosis factor alpha, an agent that on other surfaces promotes adhesion and spreading. However, on GMP140 the cells fail to spread and instead remain rounded and refractile. Neutrophils adhering to GMP140 were also deficient in superoxide anion generation to formylmethionylleucylphenylalanine. Furthermore, fluid-phase GMP140 also inhibited the superoxide generation by neutrophils stimulated by tumor necrosis factor alpha. The effect of GMP140 was reversible by washing and was inhibited by anti-GMP140 Fab antibody. GMP140 appears to be a natural antiinflammatory molecule that may prevent the inappropriate activation of neutrophils in the circulation.

Proarrhythmic effects of antiarrhythmic drugs.

Drugs that are described as antiarrhythmic drugs may actually aggravate arrhythmia in several ways and these are termed proarrhythmic effects. The most common type of proarrhythmia is a paradoxical increase in the frequency of episodes of the target arrhythmia. This type of effect had not been suspected until recently and has not been widely publicized. It is a phenomenon common to all antiarrhythmic drugs when they are used to treat arrhythmias based on a re-entrant mechanism (the most common mechanism of clinical arrhythmias). Different drugs vary in their tendency to produce this type of proarrhythmic response. These differences are explicable in terms of the relative effects of the drugs on refractoriness and conduction times in the re-entrant circuit. Proarrhythmic effects are most important in the treatment of ventricular tachycardias because recurrences are often fatal. Proarrhythmic effects on ventricular tachycardia can now be predicted at electrophysiological study before commencement of long-term therapy, and potentially dangerous treatment can be avoided. The key to proper treatment to proarrhythmia is to recognize that it is a drug-induced problem and to withdraw the offending drug.

Prevention of activated neutrophil adhesion to endothelium by soluble adhesion protein GMP140.

Neutrophils and monocytes, but not lymphocytes, adhered strongly to plastic surfaces coated with GMP140, a protein of endothelial cells and platelets. This adhesion of neutrophils was mediated by GMP140 and not by the CD18 integrin complex. By contrast, GMP140 in solution inhibited the CD18-dependent adhesion of tumor necrosis factor-alpha-activated neutrophils to plastic surfaces and resting endothelium, but not of resting neutrophils to tumor necrosis factor-alpha-activated endothelium. Thus, the binding of a soluble form of an adhesion protein selectively inhibited another set of adhesive events. Soluble GMP140 may be important in maintaining the nonadhesiveness of neutrophils in the circulation and may serve to limit inflammatory reactions.

Characterization of human platelet GMP-140 as a heparin-binding protein.

Human platelet GMP-140 has been identified as a heparin-binding protein. Purified platelet GMP-140 bound to Heparin-Sepharose CL-6B and was eluted by approximately 0.5 M sodium chloride. Radioiodinated GMP-140 bound specifically and saturably to heparin immobilized on Matrex-Pel 102 beads. Binding of radioiodinated GMP-140 to heparin-Matrex-Pel 102 beads was divalent cation-independent and was strongly inhibited by excess fluid phase GMP-140 and heparin and by other sulfated glycans such as fucoidin and dextran-sulfate. Binding was not inhibited by chondroitins 4- and 6-sulfate or mannose 6-phosphate.

Comparison of immediate versus day to day variability of ventricular tachycardia induction by programmed stimulation.

The use of programmed stimulation to assess long-term oral antiarrhythmic drug efficacy for ventricular tachycardia is complicated by the fact that mode of ventricular tachycardia induction varies from day to day in the absence of drug therapy. The purpose of this prospective study was to assess whether mode of ventricular tachycardia induction is more reproducible within one study than from day to day. Thirty-nine consecutive patients with documented sustained ventricular tachyarrhythmias secondary to coronary artery disease underwent three inductions of ventricular tachycardia at 15 min intervals in the absence of drug therapy. A stimulation protocol in which the only major variable was the number of extrastimuli required for tachycardia induction was used. Subsequent day to day variability in mode of tachycardia induction was also assessed in the same patients at two further drug-free inductions at intervals of 5 +/- 2 days. The number of extrastimuli required for tachycardia induction was significantly more reproducible at the immediate repeat studies than from day to day (69% of patients versus 31%, p less than 0.01). From these data, probability tables were derived that show the likelihood that changes in inducibility at subsequent tachycardia inductions are due to chance. The QRS configuration of induced ventricular tachycardia was also more reproducible at the immediate studies (64% versus 26%, p less than 0.01). Basic electrophysiologic and stimulation variables differed over a significantly wider range from day to day than at the immediate studies.(ABSTRACT TRUNCATED AT 250 WORDS)