Alternatively spliced tissue factor promotes plaque angiogenesis through the activation of hypoxia-inducible factor-1α and vascular endothelial growth factor signaling.

BACKGROUND: Alternatively spliced tissue factor (asTF) is a novel isoform of full-length tissue factor, which exhibits angiogenic activity. Although asTF has been detected in human plaques, it is unknown whether its expression in atherosclerosis causes increased neovascularization and an advanced plaque phenotype. METHODS AND RESULTS: Carotid (n=10) and coronary (n=8) specimens from patients with stable or unstable angina were classified as complicated or uncomplicated on the basis of plaque morphology. Analysis of asTF expression and cell type-specific expression revealed a strong expression and colocalization of asTF with macrophages and neovessels within complicated, but not uncomplicated, human plaques. Our results showed that the angiogenic activity of asTF is mediated via hypoxia-inducible factor-1α upregulation through integrins and activation of phosphatidylinositol-3-kinase/Akt and mitogen-activated protein kinase pathways. Hypoxia-inducible factor-1α upregulation by asTF also was associated with increased vascular endothelial growth factor expression in primary human endothelial cells, and vascular endothelial growth factor-Trap significantly reduced the angiogenic effect of asTF in vivo. Furthermore, asTF gene transfer significantly increased neointima formation and neovascularization after carotid wire injury in ApoE(-/-) mice. CONCLUSIONS: The results of this study provide strong evidence that asTF promotes neointima formation and angiogenesis in an experimental model of accelerated atherosclerosis. Here, we demonstrate that the angiogenic effect of asTF is mediated via the activation of the hypoxia-inducible factor-1/vascular endothelial growth factor signaling. This mechanism may be relevant to neovascularization and the progression and associated complications of human atherosclerosis as suggested by the increased expression of asTF in complicated versus uncomplicated human carotid and coronary plaques.

Novel small leucine-rich repeat protein podocan is a negative regulator of migration and proliferation of smooth muscle cells, modulates neointima formation, and is expressed in human atheroma.

BACKGROUND: Smooth muscle cell (SMC) migration and proliferation critically influence the clinical course of vascular disease. We tested the effect of the novel small leucine-rich repeat protein podocan on SMC migration and proliferation using a podocan-deficient mouse in combination with a model of arterial injury and aortic explant SMC culture. In addition, we examined the effect of overexpression of the human form of podocan on human SMCs and tested for podocan expression in human atherosclerosis. In all these conditions, we concomitantly evaluated the Wnt-TCF (T-cell factor) pathway. METHODS AND RESULTS: Podocan was strongly and selectively expressed in arteries of wild-type mice after injury. Podocan-deficient mice showed increased arterial lesion formation compared with wild-type littermates in response to injury (P<0.05). Also, SMC proliferation was increased in arteries of podocan-deficient mice compared with wild-type (P<0.05). In vitro, migration and proliferation were increased in podocan-deficient SMCs and were normalized by transfection with the wild-type podocan gene (P<0.05). In addition, upregulation of the Wnt-TCF pathway was found in SMCs of podocan-deficient mice both in vitro and in vivo. On the other hand, podocan overexpression in human SMCs significantly reduced SMC migration and proliferation, inhibiting the Wnt-TCF pathway. Podocan and a Wnt-TCF pathway marker were differently expressed in human coronary restenotic versus primary lesions. CONCLUSIONS: Podocan appears to be a potent negative regulator of the migration and proliferation of both murine and human SMCs. The lack of podocan results in excessive arterial repair and prolonged SMC proliferation, which likely is mediated by the Wnt-TCF pathway.

Inactivation of nuclear factor-Y inhibits vascular smooth muscle cell proliferation and neointima formation.

OBJECTIVE: Atherosclerosis and restenosis are multifactorial diseases associated with abnormal vascular smooth muscle cell (VSMC) proliferation. Nuclear factor-Y (NF-Y) plays a major role in transcriptional activation of the CYCLIN B1 gene (CCNB1), a key positive regulator of cell proliferation and neointimal thickening. Here, we investigated the role of NF-Y in occlusive vascular disease. APPROACH AND RESULTS: We performed molecular and expression studies in cultured cells, animal models, and human tissues. We find upregulation of NF-Y and cyclin B1 expression in proliferative regions of murine atherosclerotic plaques and mechanically induced lesions, which correlates with higher binding of NF-Y to target sequences in the CCNB1 promoter. NF-YA expression in neointimal lesions is detected in VSMCs, macrophages, and endothelial cells. Platelet-derived growth factor-BB, a main inductor of VSMC growth and neointima development, induces the recruitment of NF-Y to the CCNB1 promoter and augments both CCNB1 mRNA expression and cell proliferation through extracellular signal-regulated kinase 1/2 and Akt activation in rat and human VSMCs. Moreover, adenovirus-mediated overexpression of a NF-YA-dominant negative mutant inhibits platelet-derived growth factor-BB-induced CCNB1 expression and VSMC proliferation in vitro and neointimal lesion formation in a mouse model of femoral artery injury. We also detect NF-Y expression and DNA-binding activity in human neointimal lesions. CONCLUSIONS: Our results identify NF-Y as a key downstream effector of the platelet-derived growth factor-BB-dependent mitogenic pathway that is activated in experimental and human vasculoproliferative diseases. They also identify NF-Y inhibition as a novel and attractive strategy for the local treatment of neointimal formation induced by vessel denudation.

The complement component C5a is present in human coronary lesions in vivo and induces the expression of MMP-1 and MMP-9 in human macrophages in vitro.

The complement component C5a is formed during activation of the complement cascade and exerts chemotactic and proinflammatory effects. Macrophages, which are localized in the rupture-prone shoulder regions of coronary plaques, are thought to play a major role in plaque destabilization and rupture through the production of matrix metalloproteinases (MMPs). When human monocyte-derived macrophages were stimulated in vitro with C5a, MMP-1 and MMP-9 mRNA levels were significantly increased. Furthermore, C5a up-regulated MMP-1 and MMP-9 antigens and activity, as determined by ELISA and specific activity assays. These effects were blocked by antibodies against the receptor C5aR/CD88. In addition, blocking experiments revealed that MMP-1 expression was mediated by activation of the transcription factor AP-1, and MMP-9 expression was induced by activation of NF-κB and AP-1. Immunohistochemical analysis of human coronary plaques demonstrated the colocalization of C5a, MMP-1, and MMP-9 in vivo. Together, these observations indicate that activation of the complement cascade and formation of C5a may play a role in the onset of acute coronary events by induction of MMPs in atherosclerotic lesions.

Recombinant apolipoprotein A-I Milano rapidly reverses aortic valve stenosis and decreases leaflet inflammation in an experimental rabbit model.

AIMS: Aortic stenosis (AS) is associated with significant morbidity and mortality. Recombinant apolipoprotein A-I Milano (rApoA-I(M)) induces atherosclerotic plaque regression. The aims of this study were to determine the effects of rApoA-I(M) on experimental aortic valve degeneration and its mechanisms of action. METHODS AND RESULTS: New Zealand White rabbits (n = 20) were fed an atherogenic diet for 9 months and then randomized to either placebo or rApoA-I(M). Echocardiography was used to assess the effect of the treatments on AS. Porcine aortic valve myofibroblasts (PAVMF) treated with oxidized low-density lipoprotein served to define the effects of rApoA-I(M) on the expression of monocyte chemoattractant protein-1 (MCP-1), nuclear factor (NF)-kappaB, and alkaline phosphatase (AP). Recombinant apolipoprotein A-I Milano increased aortic valve area (AVA) by 32% (0.25 +/- 0.05 to 0.34 +/- 0.07 cm(2), P < 0.01); whereas AVA remained unchanged in the placebo group (0.24 +/- 0.05 to 0.26 +/- 0.04 cm(2), P = 0.58). Histopathological examination of aortic valves in the rApoA-I(M) animals showed significantly less leaflet thickening, inflammation, and calcification vs. the placebo group. In vitro, rApoA-I(M) significantly inhibited MCP-1, AP, and NF-kappaB and decreased intracellular cholesterol content in PAVMF. CONCLUSION: Recombinant apolipoprotein A-I Milano treatment reverses AS in this experimental rabbit model. The beneficial effects seem to be mediated by enhanced cholesterol removal and by reduced inflammation and calcification.

The electrical substrate of vagal atrial fibrillation as assessed by the signal-averaged electrocardiogram of the P wave.

BACKGROUND: The autonomic nervous system is thought to be involved in the initiation of atrial fibrillation (AF). However, there is a distinct entity of vagal AF characterized by episodes occurring at rest, postprandially, or during sleep. The purpose of this study was to compare intraatrial conduction in patients with vagally mediated AF to those with nonvagal AF, using the signal-averaged electrocardiogram (SAECG) of P wave. METHODS: SAECG of P wave was performed in 58 patients with AF using the Marquette Medical System, and the mean filtered P-wave duration (SAPW) was measured. Nine patients were categorized as having pure vagal AF (Group I), and 42 patients as having nonvagal AF (Group II); the remaining seven patients were excluded from analysis because of incomplete data. RESULTS: The patients in Group I were significantly younger and more likely to have paroxysmal lone AF, as compared to those in Group II. There was no significant difference in left atrial size and left ventricular function in the two groups. The mean SAPW was significantly shorter in Group I when compared to Group II (118 +/- 5 ms vs 149 +/- 39 ms, P < 0.001). Whereas all patients in Group I had a normal SAPW, 79% of patients in Group II had an abnormal SAPW (P < 0.001). A normal SAPW was significantly predictive of vagal AF independent of other co-variables. CONCLUSIONS: (1) Patients with vagal AF are younger, and invariably have paroxysmal lone AF. (2) SAPW is normal and significantly shorter in vagal AF when compared to patients with nonvagal AF. (3) This suggests that those in the vagal AF population have normal intraatrial conduction, which has implications for AF ablation in these patients.

Fenofibrate induces plaque regression in hypercholesterolemic atherosclerotic rabbits: in vivo demonstration by high-resolution MRI.

INTRODUCTION: Fenofibrate has shown to reduce major cardiovascular events and slow angiographic progression of coronary atherosclerosis. The postulated mechanism of action is via the activation of peroxisomal proliferator-activated receptor-alpha (PPAR-alpha), a nuclear transcription factor that controls a variety of cellular functions. We investigated the anti-atherogenic effects of fenofibrate on previously established experimental atherosclerotic lesions. METHOD: Atherosclerotic lesions were induced in the abdominal aorta of New Zealand white (NZW) rabbits (n=19) by a combination of a double-balloon injury and a 9-month hypercholesterolemic diet. The rabbits were randomized into placebo or fenofibrate group. The corresponding treatments were added to the hypercholesterolemic diet. All rabbits underwent MRI examination at randomization and after 6 months of treatment, and were then sacrificed for histopathology. RESULTS: LDL-cholesterol was similarly elevated at randomization and follow-up, and was not significantly modified by fenofibrate therapy. HDL-cholesterol decreased (-27+/-10%, p=0.04) in the placebo and increased (+36.8+/-2%, p=0.04) in the fenofibrate group. MRI showed comparable vessel wall area (VWA) at randomization in both groups. At 15months, a significant increase in VWA was seen in the placebo group (15+/-4%, p=0.007), while fenofibrate treatment was associated with a regression (-11+/-4%, p=0.041) of previously established lesions. Fenofibrate also decreased macrophage and increased smooth muscle cell/collagen content of atherosclerotic lesions. CONCLUSION: MRI measurements can, in conjunction with in vitro histological measurements, contribute to the understanding of the actions of pharmacologic agents in experimental models of atherosclerosis. Fenofibrate significantly regresses atherosclerotic lesions and induced changes in plaque composition associated with a more "stable" phenotype (reduced macrophages and increased SMC). These observations support the potential anti-atherogenic effects of PPAR-alpha agonists.

Decreased reendothelialization and increased neointima formation with endostatin overexpression in a mouse model of arterial injury.

BACKGROUND: Impaired endothelial regeneration contributes to arterial lesion formation. Endostatin is a specific inhibitor of endothelial cell growth and induces endothelial cell apoptosis. We examined the effect of endostatin overexpression on reendothelialization and neointima formation in a mouse model of arterial injury. METHODS AND RESULTS: Mice underwent femoral arterial denudation and received recombinant adenovirus, expressing either murine endostatin (n=19) or control adenoviral vector (n=12), by jugular vein injection. Endostatin gene transfer resulted in high serum levels of endostatin. Strong adenoviral gene expression of beta-galactosidase-expressing control vector was detected in liver tissue and was absent in the injured arterial wall at 1 week. Deposits of endostatin protein were detected along the denuded arterial wall and were not seen in the noninjured contralateral artery at 1 week. Endostatin deposits were also absent in the injured artery of control vector-treated animals. Overexpression of endostatin led to decreased reendothelialization and increased apoptosis of luminal endothelial cells 2 and 4 weeks after arterial injury (P<0.05). In addition, endostatin overexpression resulted in increased neointima formation (P<0.05). Endothelial apoptosis and neointima area correlated positively with endostatin serum levels, whereas the degree of reendothelialization correlated negatively with endostatin serum levels (P<0.05). Furthermore, poor reendothelialization correlated with increased neointima formation (P<0.05). CONCLUSIONS: In summary, decreased reendothelialization and enhanced endothelial apoptosis, in response to endostatin overexpression, were associated with increased neointima formation. These findings demonstrate that high serum levels of endostatin are capable of inhibiting endothelial regeneration and promoting arterial lesion growth in conditions of endothelial injury.

Caspase-3 and tissue factor expression in lipid-rich plaque macrophages: evidence for apoptosis as link between inflammation and atherothrombosis.

BACKGROUND: Macrophages associated with arterial wall lipid deposition contribute to inflammatory processes. Tissue factor (TF) has been implicated in the thrombogenicity of atherosclerotic plaques. Intimal cells undergoing apoptosis have been postulated as a source for TF. However, there is only limited knowledge of cell type, plaque component, and conditions associated with TF expression and apoptosis. We examined the hypothesis that macrophages exposed to conditions of lipid-rich plaque undergo apoptosis and express TF. METHODS AND RESULTS: In human carotid (n=15) and coronary (n=6) atherosclerotic plaques, TF and caspase-3 mRNA and protein expression (evaluated by in situ hybridization and immunohistochemistry) were increased significantly in lipid-rich compared with fibrous plaque components (P<0.01) and correlated with high macrophage content (P<0.05). Double-labeling studies demonstrated colocalization of TF and active caspase-3. In hyperlipidemic mice, expression of TF and active caspase-3 was observed simultaneously and colocalized in neointimal macrophages after arterial injury. In neointima of normolipidemic animals, TF and active caspase-3 were absent after arterial injury. In monocytes cultured in the presence of oxidized LDL, strong induction and colocalization of TF and active caspase-3 were found compared with baseline (P<0.05). Both antigens were significantly decreased after cotreatment with a caspase inhibitor (P<0.05) and were absent in untreated control cells. CONCLUSIONS: The expression of TF as the primary cell-associated activator of the coagulation pathway proves to be closely related to macrophages undergoing apoptosis in conditions of lipid-rich plaque, pointing to a key role of lipid content and inflammatory cell viability in determining plaque thrombogenicity.

Vascular endothelial growth factor regulates reendothelialization and neointima formation in a mouse model of arterial injury.

BACKGROUND: The rate of reendothelialization is critical in neointima formation after arterial injury. Vascular endothelial growth factor (VEGF), a potent endothelial mitogen, has been advocated for accelerating endothelial repair and preventing intimal hyperplasia after percutaneous coronary interventions. However, the precise mechanism of action of VEGF treatment and the physiologic role of endogenous VEGF after arterial injury are not well described. To better understand the role of VEGF in arterial repair, we overexpressed both VEGF and a soluble, chimeric VEGF receptor (VEGF-trap), which binds free VEGF with high affinity, in a mouse model of arterial injury. METHODS AND RESULTS: Four groups of C57BL/6 mice underwent denuding endothelial injury 1 day after systemic injection of recombinant adenovirus expressing (1) VEGF, (2) VEGF-trap, (3) VEGF plus VEGF-trap, or (4) control adenovirus. Circulating levels of adenovirus-encoded proteins were significantly elevated after gene transfer. VEGF overexpression accelerated reendothelialization and increased luminal endothelial cell proliferation 2 weeks after arterial injury (P<0.05), resulting in decreased neointima formation at 4 weeks compared with control (P<0.01). Cotreatment with VEGF-trap completely sequestered free VEGF and abrogated the beneficial effect of VEGF overexpression. Interestingly, sequestration of endogenous VEGF by VEGF-trap overexpression alone also led to delayed reendothelialization at 2 weeks (P<0.01) and increased neointima formation at 4 weeks (P<0.01). CONCLUSIONS: VEGF overexpression accelerated endothelial repair and inhibited neointima formation after arterial injury. Conversely, sequestration of exogenous and/or endogenous VEGF by VEGF-trap delayed reendothelialization and significantly increased neointima size. This demonstrates the therapeutic potential of VEGF but also emphasizes the important physiologic role of endogenous VEGF in vascular repair.

Dendritic cells in neointima formation after rat carotid balloon injury: coordinated expression withanti-apoptotic Bcl-2 and HSP47 in arterial repair.

OBJECTIVES: We sought to evaluate: 1) the contribution of dendritic cells (DCs); and 2) the impact of B-cell lymphoma 2 protein (Bcl-2), a central anti-apoptotic protooncogene, and of heat shock protein 47 (HSP47), indicating subsequent collagen deposition, in neointima formation after angioplasty. BACKGROUND: The origin of neointimal cells and the factors that promote their accumulation are still unclear. Previous studies reported intimal presence of DCs and suggested cells of primarily extravascular origin to contribute to arterial repair. METHODS: Sprague-Dawley rats underwent carotid balloon angioplasty. At different times after angioplasty, tissue sections were analyzed by immunohistochemistry using OX-62 and S100 as DC markers and antibodies against Bcl-2 and HSP47, supplemented by electron microscopic analysis of cell type and apoptosis. RESULTS: Four days after injury, DCs adhered along the internal elastic lamina and demonstrated intense Bcl-2 and HSP47 expression, consistent with low apoptosis. With ongoing neointima enlargement, luminal DCs remained prevalent and were colocalized with Bcl-2 and HSP47, while signaling decreased to basal regions. Media showed no DCs and only low Bcl-2 and HSP47 immunoreactivity. Adventitia transiently revealed a structural separation between day 4 and 7. Whereas the inner layer demonstrated sparse cellularity, apoptosis and no DC, Bcl-2, and HSP47 labeling, the outer layer was characterized by high myofibroblast density with strong Bcl-2 and HSP47 expression but absence of DCs. CONCLUSIONS: We identify DCs as novel components in early neointima formation, promoted by coordinated anti-apoptotic Bcl-2 and HSP47 expression. Despite intense adventitial remodeling, there is no evidence of adventitial cell transmigration.

An increase of VEGF plasma levels is associated with restenosis of drug-eluting stents.

AIMS: Drug-eluting stents (DES) reduce late lumen loss compared to bare metal stents but were not able to eradicate in-stent restenosis (ISR) fully. Vascular endothelial growth factor (VEGF) may inhibit late lumen loss through accelerated reendothelialisation, but may also promote neointima formation by proinflammatory effects. The aim of this study was to evaluate whether endogenous plasma levels of VEGF are associated with development of ISR after implantation of DES. METHODS AND RESULTS: We studied 85 patients who were treated with 159 DES. VEGF plasma levels were determined before and 24 hours after PCI. During the eight-month follow-up period, two patients (2.4%) died of cardiovascular causes and 12 patients (14.5% of patients, 7.6% of stents) developed angiographic ISR. Basal VEGF plasma levels were not different in patients with and without ISR at follow-up. In contrast to patients without ISR, VEGF increased significantly upon PCI in patients with ISR (p<0.005). Patients with a decrease of VEGF after PCI had a restenosis rate of 2.4% compared to a restenosis rate of 26.2% in patients with an increase of VEGF after the procedure (p<0.05). This was independent from clinical and angiographic risk factors. CONCLUSIONS: Basal plasma levels of VEGF are not associated with the development of ISR. However, an increase of VEGF after PCI is associated with a dramatically increased ISR rate after implantation of DES.

Accelerated reendothelialization, increased neovascularization and erythrocyte extravasation after arterial injury in BAMBI-/- mice.

BACKGROUND: Intimal injury rapidly activates TGFß and enhances vascular repair by the growth of endothelial (EC) and vascular smooth muscle cells (VSMC). The response to the TGFß family of growth factors can be modified by BAMBI (BMP, Activin, Membrane Bound Inhibitor) acting as a non-signaling, competitive antagonist of TGFß type I receptors such as ALK 1 and 5. In vivo the effect of BAMBI will depend on its cell-specific expression and of that of the ALK type receptors. We recently reported EC restricted BAMBI expression and genetic elimination of BAMBI resulting in an in vitro and in vivo phenotype characterized by endothelial activation and proliferation involving alternative pathway activation by TGFß through ALK 1. METHODOLOGY/PRINCIPAL FINDINGS: To test the hypothesis that BAMBI modulates arterial response to injury via its effects on endothelial repair and arterial wall neovascularization we used a model of femoral arterial denudation injury in wild type (WT) and BAMBI(-/-) mice. Arterial response was evaluated at 2 and 4 weeks after luminal endothelial denudation of femoral arteries. The BAMBI(-/-) genotype mice showed accelerated luminal endothelial repair at 2 weeks and a highly unusual increase in arterial wall neovascularization compared to WT mice. The exuberant intimal and medial neovessel formation with BAMBI(-/-) genotype was also associated with significant red blood cell extravasation. The bleeding into the neointima at 2 weeks transiently increased it's area in the BAMBI(-/-)genotype despite the faster luminal endothelial repair in this group. Vascular smooth muscle cells were decreased at 2 weeks in BAMBI(-/-) mice, but comparable to wild type at 4 weeks. CONCLUSIONS/SIGNIFICANCE: The absence of BAMBI results in a highly unusual surge in arterial wall neovascularization that surprisingly mimiks features of intra-plaque hemorrhage of advanced atheroma in a mechanical injury model. This suggests important effects of BAMBI on arterial EC homeostasis that need to be further studied in a model of inflammatory atherosclerosis.

Macrophages transmit potent proangiogenic effects of oxLDL in vitro and in vivo involving HIF-1α activation: a novel aspect of angiogenesis in atherosclerosis.

Neovascularization has been linked to the progression and vulnerability of atherosclerotic lesions. Angiogenesis is increased in lipid-rich plaque. Hypoxia-inducible factor alpha (HIF-1α) is a key transcriptional regulator responding to hypoxia and activating genes, which promote angiogenesis, among them vascular endothelial growth factor (VEGF). Oxidized low-density lipoprotein (oxLDL) is generated in lipid-rich plaque by oxidative stress. It triggers an inflammatory response and was traditionally thought to inhibit endothelial cells. New data, however, suggest that oxLDL can activate HIF-1α in monocytes in a hypoxia-independent fashion. We hypothesized that HIF-1α activation in monocyte-macrophages could transmit proangiogenic effects of oxLDL linking hyperlipidemia, inflammation, and angiogenesis in atherosclerosis. First, we examined the effect of oxLDL on HIF-1α and VEGF expression in monocyte-macrophages and on their proangiogenic effect on endothelial cells in vitro in a monocyte-macrophage/endothelial co-culture model. OxLDL strongly induced HIF-1α and VEGF in monocyte-macrophages and significantly increased tube formation in co-cultured endothelial cells. HIF-1α inhibition reversed this effect. Second, we demonstrated a direct proangiogenic effect of oxLDL in an in vivo angiogenesis assay. Again, HIF-1α inhibition abrogated the proangiogenic effect of oxLDL. Third, in a rabbit atherosclerosis model, we studied the effect of dietary lipid lowering on arterial HIF-1α and VEGF expression. The administration of low-lipid diet significantly reduced the expression of both HIF-1α and VEGF, resulting in decreased plaque neovascularization. Our data point to oxLDL as a proangiogenic agent linking hyperlipidemia, inflammation, and angiogenesis in atherosclerosis. This effect is dependent on macrophages and, at least in part, on the induction of the HIF-1α pathway.

Coronary artery disease in aging women: a menopause of endothelial progenitor cells?

The cardiovascular protection provided to women during the reproductive age and the unique angiogenic properties of the female reproductive system provide insights into the complex regulatory network of female sex hormones, angiogenic growth factors, and stem cell regulatory molecules. The intricate and interwoven endometrial physiology of the female menstrual cycle shows that in order to harness the physiologic cardioprotection provided by nature to women of reproductive age, for better cardiovascular therapies in postmenopausal women and the population in general, a coherent and systematic approach is needed.

Carvedilol administration in acute myocardial infarction results in stronger inhibition of early markers of left ventricular remodeling than metoprolol.

BACKGROUND: The structural secuelae of acute myocardial infarction (AMI) is mostly dictated by left ventricular (LV) remodelling, leading to heart failure. Monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play a critical role in LV remodelling. ß-blockers are first line therapy for AMI and heart failure; however, the mechanisms responsible for their benefits remain poorly understood. Different ß-blocker agents have been shown to exert beneficial activities both in AMI and heart failure, however, their role in early remodelling after ischemia/reperfusion is to be fully elucidated. We sought to compare the effect of 2 of the most prescribed ß-blocker agents in early markers of LV remodelling after AMI. METHODS: A reperfused AMI was induced in Yorshire pigs, being randomized to early intravenous carvedilol, metoprolol or placebo. Twenty-four hours after reperfusion markers of early remodelling were addressed in the LV. RESULTS: The early administration of both ß-blockers is able to significantly reduce macrophage infiltration as well as the expression and activity of MCP-1 and MMP-2 compared to placebo. The effects of carvedilol were much stronger than those of metoprolol. Conversely, carvedilol upregulated the expression TIMP-2 to a greater extent than metoprolol. CONCLUSIONS: In an AMI model closely mimicking human pathophysiology, the early administration of carvedilol reduced the expression of markers associated with early LV remodelling to greater extent than metoprolol. These findings may explain the superior clinical benefits exerted by carvedilol in heart failure.

The cardioprotection granted by metoprolol is restricted to its administration prior to coronary reperfusion.

BACKGROUND: Myocardial infarct size is a strong predictor of cardiovascular events. Intravenous metoprolol before coronary reperfusion has been shown to reduce infarct size; however, it is unknown whether oral metoprolol initiated early after reperfusion, as clinical guidelines recommend, is similarly cardioprotective. We compared the extent of myocardial salvage associated with intravenous pre-reperfusion-metoprolol administration in comparison with oral post-reperfusion-metoprolol or placebo. We also studied the effect on suspected markers of reperfusion injury. METHODS: Thirty Yorkshire-pigs underwent a reperfused myocardial infarction, being randomized to pre-reperfusion-metoprolol, post-reperfusion-metoprolol or placebo. Cardiac magnetic resonance imaging was performed in eighteen pigs at day 3 for the quantification of salvaged myocardium. The amounts of at-risk and infarcted myocardium were quantified using T2-weighted and post-contrast delayed enhancement imaging, respectively. Twelve animals were sacrificed after 24h for reperfusion injury analysis. RESULTS: The pre-reperfusion-metoprolol group had significantly larger salvaged myocardium than the post-reperfusion-metoprolol or the placebo groups (31 ± 4%, 13 ± 6%, and 7 ± 3% of myocardium at-risk respectively). Post-mortem analyses suggest lesser myocardial reperfusion injury in the pre-reperfusion-metoprolol in comparison with the other 2 groups (lower neutrophil infiltration, decreased myocardial apoptosis, and higher activation of the salvage-kinase phospho-Akt). Salvaged myocardium and reperfusion injury pair wise comparisons proved there were significant differences between the pre-reperfusion-metoprolol and the other 2 groups, but not among the latter two. CONCLUSIONS: The intravenous administration of metoprolol before coronary reperfusion results in larger myocardial salvage than its oral administration initiated early after reperfusion. If confirmed in the clinical setting, the timing and route of ß-blocker initiation could be revisited.

Reduced acute vascular injury and atherosclerosis in hyperlipidemic mice transgenic for lysozyme.

Hyperlipidemia promotes oxidant stress, inflammation, and atherogenesis in apolipoprotein E-deficient (ApoE((-/-))) mice. Mice transgenic for lysozyme (LZ-Tg) are resistant to acute and chronic oxidative stress and have decreased circulating levels of pro-oxidant advanced glycation end-products (AGEs). Herein we report that TIB-186 macrophages transduced with adenovirus-expressing human LZ (AdV-LZ) containing the AGE-binding domain facilitated AGE uptake and degradation and that AdV-LZ-transduced macrophages and peritoneal macrophages from LZ-Tg mice suppressed the AGE-triggered tumor necrosis factor-alpha response. We assessed atherosclerosis in LZ-Tg mice crossed with ApoE((-/-)) mice (LZ/ApoE((-/-))) and found increased serum LZ levels and decreased AGE and 8-isoprostanes levels, although hyperlipidemia remained similar to ApoE((-/-)) controls. Atherosclerotic plaques and neointimal lesions at the aortic root and descending aorta were markedly decreased (by 40% and 80%, respectively) in LZ/ApoE((-/-)) versus ApoE((-/-)) mice, as were inflammatory infiltrates. The arterial lesions following femoral artery injury in LZ/ApoE((-/-)) mice were suppressed (intimal to media ratio decreased by 50%), as were AGE deposits and vascular smooth muscle cell activation, compared to ApoE((-/-)) mice. Despite hyperlipidemia, development of atheroma and occlusive, inflammatory arterial neointimal lesions in response to injury was suppressed in LZ/ApoE((-/-)) mice. This effect may be due to the antioxidant properties of LZ, which is possibly linked to the AGE-binding domain region of the molecule.

Atherosclerosis regression and TP receptor inhibition: effect of S18886 on plaque size and composition--a magnetic resonance imaging study.

AIMS: Endothelial dysfunction, platelet hyperactivity, and inflammation play a crucial role in atherogenesis. A growing body of evidence suggests that inhibition of the thromboxane A2 (TxA2 or TP) receptor may improve endothelial function and reduce the inflammatory component of atherosclerosis in addition to its demonstrated antiplatelet activity. Consequently, we sought to assess the effect of a novel TP receptor antagonist S18886, on atherosclerotic lesion progression and composition by serial non-invasive magnetic resonance imaging (MRI). METHODS AND RESULTS: S18886 was compared with control in an experimental model of established aortic atherosclerosis in New Zealand White rabbits (n=10). The animals underwent MRI of the abdominal aorta at the time of randomization and at the end of treatment. Subsequently, animals were euthanized and specimens were stained for histopathology and immunohistochemistry with anti-alpha-actin antibodies for vascular smooth muscle cells (VSMC), anti-RAM-11 for macrophages, anti-caspase-3 for apoptotic cells, anti-MMP-1 for metalloproteinases, and anti-endothelin-1 (ET-1) as a marker of endothelial dysfunction. MRI analysis revealed a significant reduction in total vessel area (TVA) and vessel wall area (VWA) in the S18886 group (P<0.05). Immunostaining analysis showed a significant decrease in RAM-11, caspase-3, MMP-1, ET-1 and an increase in alpha-actin in the treated group (P<0.05 vs. control). CONCLUSION: Inhibition of the TP receptor by S18886 causes a regression of advanced atherosclerotic plaques. In addition, the reduction in the markers for macrophages, apoptotic cells, metalloproteinases, and endothelin-1 and the increase in VSMC, suggests that S18886 may not only halt the progression of atherosclerosis, but also transform lesions towards a more stable phenotype. The possibility of combining antithrombotic and antiatherosclerotic activity by means of the administration of TP inhibitors deserves further investigation in a clinical setting.

Evolving concepts in the triad of atherosclerosis, inflammation and thrombosis.

Recent developments into antherothrombosis, the leading cause of morbidity and mortality in Western Society, may help to change our treatment strategy to a more casual approach. The composition of the atherosclerotic plaque, rather than the percent stenosis, appears to be a critical predictor for both risk of plaque rupture and subsequent thrombogenicity. A large lipid core, rich in tissue factor (TF) and inflammatory cells including macrophages, and a thin fibrous cap with compromise of its structural integrity by matrix degrading enzymes, such as metalloproteinases (MMPs), render a lesion susceptible to rupture and subsequent acute thrombosis. Thrombosis may lead to a complete occlusion or, in the case of mural thrombus or intraplaque hemorrhage, to plaque progression. Disruption of a vulnerable or unstable plaque (type IV and Va lesions of the AHA classification) with a subsequent change in plaque geometry and thrombosis may result in an acute coronary syndrome. The high-risk plaque tend to be relatively small, but soft or vulnerable to "passive" disruption because of high lipid content. Inflammatory processes are important components of all stages of atherosclerotic development, including plaque initiation and disruption. As such the early steps in atherosclerotic lesion formation are the over expression of endothelial adhesive protein (i.e. selectins, VCAM and ICAM), chemotactic factors (MCP-1), growth factors (M-CSF), and cytokines (IL-2) that will facilitate the recruitment, internalization and survival of blood-borne inflammatory cells into the vascular wall. Macrophages, following what appears to be a defense mission by protecting the vessel wall from excess lipid accumulation, may eventually undergo apoptosis with release of MMPs and TF. Specific cell recruitment in the vessel wall and build-up of the extracellular matrix are coordinated by a wide variety of stimulators and inhibitors. Active interaction of immune competent cells within the atherosclerotic lesions appears to play a pivotal role in the control of atherosclerotic plaque evolution and, therefore, deserves particular attention from the research community with the ultimate goal of improving preventive and therapeutic medical approaches. Inflammation, thrombosis and atherosclerosis are interdependent and define a triad within the complex pathogenic process of atherothrombosis.