The proteasome regulates collagen-induced platelet aggregation via nuclear-factor-kappa-B (NFĸB) activation.

INTRODUCTION: Platelets possess critical hemostatic functions in the system of thrombosis and hemostasis, which can be affected by a multitude of external factors. Previous research has shown that platelets have the capacity to synthesize proteins de novo and more recently a multicatalytic protein complex, the proteasome, has been discovered in platelets. Due to its vital function for cellular integrity, the proteasome has become a therapeutic target for anti-proliferative drug therapies in cancer. Clinically thrombocytopenia is a frequent side-effect, but the aggregatory function of platelets also appears to be affected. Little is known however about underlying regulatory mechanisms and functional aspects of proteasome inhibition on platelets. Our study aims to investigate the role of the proteasome in regulating collagen-induced platelet aggregation and its interaction with NFkB in this context. MATERIAL AND METHODS: Using fluorescence activity assays, platelet aggregometry and immunoblotting, we investigate regulatory interactions of the proteasome and Nuclear-factor-kappa-B (NFkB) in collagen-induced platelet aggregation. RESULTS: We show that collagen induces proteasome activation in platelets and collagen-induced platelet aggregation can be reduced with proteasome inhibition by the specific inhibitor epoxomicin. This effect does not depend on Rho-kinase/ROCK activation or thromboxane release, but rather depends on NFkB activation. Inhibition of the proteasome prevented cleavage of NFκB-inhibitor protein IκBα and decreased NFκB activity after collagen stimulation. Inhibition of the NFκB-pathway in return reduced collagen-induced platelet proteasome activity and cleavage of proteasome substrates. CONCLUSIONS: This work offers novel explanations how the proteasome influences collagen-dependent platelet aggregation by involving non-genomic functions of NFkB.

The tyrosine phosphatase SHP-1 regulates hypoxia inducible factor-1α (HIF-1α) protein levels in endothelial cells under hypoxia.

INTRODUCTION: The tyrosine phosphatase SHP-1 negatively influences endothelial function, such as VEGF signaling and reactive oxygen species (ROS) formation, and has been shown to influence angiogenesis during tissue ischemia. In ischemic tissues, hypoxia induced angiogenesis is crucial for restoring oxygen supply. However, the exact mechanism how SHP-1 affects endothelial function during ischemia or hypoxia remains unclear. We performed in vitro endothelial cell culture experiments to characterize the role of SHP-1 during hypoxia. RESULTS: SHP-1 knock-down by specific antisense oligodesoxynucleotides (AS-Odn) increased cell growth as well as VEGF synthesis and secretion during 24 hours of hypoxia compared to control AS-Odn. This was prevented by HIF-1α inhibition (echinomycin and apigenin). SHP-1 knock-down as well as overexpression of a catalytically inactive SHP-1 (SHP-1 CS) further enhanced HIF-1α protein levels, whereas overexpression of a constitutively active SHP-1 (SHP-1 E74A) resulted in decreased HIF-1α levels during hypoxia, compared to wildtype SHP-1. Proteasome inhibition (MG132) returned HIF-1α levels to control or wildtype levels respectively in these cells. SHP-1 silencing did not alter HIF-1α mRNA levels. Finally, under hypoxic conditions SHP-1 knock-down enhanced intracellular endothelial reactive oxygen species (ROS) formation, as measured by oxidation of H2-DCF and DHE fluorescence. CONCLUSIONS: SHP-1 decreases half-life of HIF-1α under hypoxic conditions resulting in decreased cell growth due to diminished VEGF synthesis and secretion. The regulatory effect of SHP-1 on HIF-1α stability may be mediated by inhibition of endothelial ROS formation stabilizing HIF-1α protein. These findings highlight the importance of SHP-1 in hypoxic signaling and its potential as therapeutic target in ischemic diseases.

Hepatitis C virus induced endothelial inflammatory response depends on the functional expression of TNFα receptor subtype 2.

In hepatitis C virus (HCV) infection, morbidity and mortality often result from extrahepatic disease manifestations. We provide evidence for a role of receptors of the innate immune system in virally induced inflammation of the endothelium in vitro and in vivo. Corresponding to the in vitro finding of an HCV-dependent induction of proinflammatory mediators in endothelial cells, mice treated with poly (I:C) exhibit a significant reduction in leukocyte rolling velocity, an increase in leukocyte adhesion to the vessel wall and an increased extravasation of leukocytes. HCV directly promotes activation, adhesion and infiltration of inflammatory cells into the vessel wall by activation of endothelial viral receptors. Poly (I:C) induces the expression of TLR3 in vivo and hereby allows for amplification of all of the aforementioned responses upon viral infection. Proinflammatory effects of viral RNA are specifically mediated by TLR3 and significantly enhanced by tumor necrosis factor alpha (TNFα). HCV-RNA induces the endothelial expression of TNFα and TNFα receptor subtype 2 and we provide evidence that leucocyte adhesion and transmigration in response to activation of viral RNA receptors seem to depend on expression of functional TNFR2. Our results demonstrate that endothelial cells actively participate in immune mediated vascular inflammation caused by viral infections.

SHP-2 regulates growth factor dependent vascular signalling and function.

Cellular responses to the environment are mediated by intracellular signalling pathways monitoring several essential cellular processes, such as proliferation, migration, differentiation and survival. Cellular dysfunction is caused by dysregulation of intracelleular signalling pathways and may ultimately result in pathophysiological conditions. The non- transmembrane protein tyrosine phosphatase SHP-2 has been shown to be important for the control of cellular behaviour. It influences the activity of several growth factor and cytokine dependent signalling pathways by association with growth factor receptors, cell surface adhesion molecules and adaptor molecules such as Gab-1, Grb2 and IRS-1. Upon FGF-2, EGF and insulin stimulation SHP-2 regulates MAPK pathway activation. In addition, SHP-2 is involved in the regulation of cell survival by influencing the PI3-K/Akt pathway upon EGF, IGF and PDGF stimulation. Due to these properties, SHP-2 function has recently gained more interest in vascular processes, such as in the differentiation of cardiac progenitor cells and angiogenic events. Indeed, SHP-2 was shown to positively regulate endothelial cell motility and angiogenesis in vitro and in vivo as well as controlling intracellular pH of endothelial and vascular smooth muscle cells. On the other hand, SHP-2 was also demonstrated to be responsible for down regulation of VEGF receptor 2 activation upon dopamin and collagen stimulation. Finally, mutations in the Ptpn11 gene (encoding SHP-2) underlie the developmental disorders Noonan syndrome and Leopard syndrome characterized by congenital heart disease and hematologic abnormalities. Different mutations in this gene also result in myeloid and lymphoid malignancies. This article summarizes the role of SHP-2 in signalling pathways relevant for vascular biology and associated disorders.

The endothelial tyrosine phosphatase SHP-1 plays an important role for vascular haemostasis in TNFα -induced inflammation in vivo.

INTRODUCTION: Inflammation and endothelium-derived superoxides are important pathomechanisms in atherothrombotic diseases. We could previously show that the tyrosine phosphatase SHP-1 acts as a negative regulator in endothelial superoxide production. In this study we investigated the influence of SHP-1 on platelet-endothelium interaction and arterial thrombosis in TNFα -induced endothelial inflammation in vivo. METHODS: Arteriolar thrombosis and platelet rolling in vivo were investigated in C57BL/6 mice using intravital microscopy in the dorsal skinfold chamber microcirculation model. RESULTS: Inhibition of SHP-1 by the specific pharmacological inhibitor sodium stibogluconate did not significantly enhance platelet-endothelium interaction in vivo under physiological conditions but led to an augmented fraction of rolling platelets in TNFα -induced systemic inflammation. Accordingly, ferric-chloride-induced arteriolar thrombus formation, which was already increased by SHP-1 inhibition, was further enhanced in the setting of TNFα -induced inflammation. Platelet aggregation in vitro as well as ex vivo was not influenced by SHP-1-inhibition. In cultured endothelial cells, sodium stibogluconate increased TNFα -induced surface expression of p-selectin and von Willebrand factor. Additionally, TNFα increased SHP-1 activity and protein expression. CONCLUSIONS: The endothelial tyrosine phosphatase SHP-1 plays an important role for vascular hemostasis in vivo, which is crucial in TNF α -induced endothelial inflammation where it may serve as an autoinhibitory molecule to prevent excess inflammatory response and thrombus formation.

Prothrombotic effects of tumor necrosis factor alpha in vivo are amplified by the absence of TNF-alpha receptor subtype 1 and require TNF-alpha receptor subtype 2.

INTRODUCTION: Elevated serum levels of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) correlate with an increased risk for atherothrombotic events and TNFα is known to induce prothrombotic molecules in endothelial cells. Based on the preexisting evidence for the impact of TNFα in the pathogenesis of autoimmune disorders and their known association with an acquired hypercoagulability, we investigated the effects of TNFα and the role of the TNF receptor subtypes TNFR1 and TNFR2 for arteriolar thrombosis in vivo. METHODS: Arteriolar thrombosis and platelet-rolling in vivo were investigated in wildtype, TNFR1-/-, TNFR2-/- and TNFR1-/R2-/- C57BL/6 mice using intravital microscopy in the dorsal skinfold chamber microcirculation model. In vitro, expression of prothrombotic molecules was assessed in human endothelial cells by real-time PCR and flow cytometry. RESULTS: In wildtype mice, stimulation with TNFα significantly accelerated thrombotic vessel occlusion in vivo upon ferric chloride injury. Arteriolar thrombosis was much more pronounced in TNFR1-/- animals, where TNFα additionally led to increased platelet-endothelium-interaction. TNFα dependent prothrombotic effects were not observed in TNFR2-/- and TNFR1-/R2- mice. In vitro, stimulation of human platelet rich plasma with TNFα did not influence aggregation properties. In human endothelial cells, TNFα induced superoxide production, p-selectin, tissue factor and PAI-1, and suppressed thrombomodulin, resulting in an accelerated endothelial dependent blood clotting in vitro. Additionally, TNFα caused the release of soluble mediators by endothelial cells which induced prothrombotic and suppressed anticoagulant genes comparable to direct TNFα effects. CONCLUSIONS: TNFα accelerates thrombus formation in an in vivo model of arteriolar thrombosis. Its prothrombotic effects in vivo require TNFR2 and are partly compensated by TNFR1. In vitro studies indicate endothelial mechanisms to be responsible for prothrombotic TNFα effects. Our results support a more selective therapeutic approach in anticytokine therapy favouring TNFR2 specific antagonists.

Hydrogen sulfide-releasing aspirin derivative ACS14 exerts strong antithrombotic effects in vitro and in vivo.

OBJECTIVE: Hydrogen sulfide (H(2)S)-releasing NSAIDs exert potent anti-inflammatory effects beyond classical cyclooxygenase inhibition. Here, we compared the platelet inhibitory effects of the H(2)S-releasing aspirin derivative ACS14 with its mother compound aspirin to analyze additional effects on platelets. METHODS AND RESULTS: In platelets of mice fed with ACS14 for 6 days (50 mg/kg per day), not only arachidonic acid-induced platelet aggregation but also ADP-dependent aggregation was decreased, an effect that was not observed with an equimolar dose of aspirin (23 mg/kg per day). ACS14 led to a significantly longer arterial occlusion time after light-dye-induced endothelial injury as well as decreased thrombus formation after ferric chloride-induced injury in the carotid artery. Bleeding time was not prolonged compared with animals treated with equimolar doses of aspirin. In vitro, in human whole blood, ACS14 (25-500 µmol/L) inhibited arachidonic acid-induced platelet aggregation, but compared with aspirin additionally reduced thrombin receptor-activating peptide-, ADP-, and collagen-dependent aggregation. In washed human platelets, ACS14 (500 µmol/L) attenuated αIIbß3 integrin activation and fibrinogen binding and increased intracellular cAMP levels and cAMP-dependent vasodilator-stimulated phosphoprotein (VASP) phosphorylation. CONCLUSIONS: The H(2)S-releasing aspirin derivative ACS14 exerts strong antiaggregatory effects by impairing the activation of the fibrinogen receptor by mechanisms involving increased intracellular cyclic nucleotides. These additional antithrombotic properties result in a more efficient inhibition of thrombus formation in vivo as achieved with aspirin alone.

Response of VEGF to activation of viral receptors and TNFα in human mesangial cells.

Vascular endothelial growth factor (VEGF) plays an important role in glomerular homeostasis as well as in the pathogenesis of kidney diseases as glomerulonephritis (GN) and diabetic nephropathy. Mesangial cells (MC), which are an integral part of the functional glomerular filtration barrier in that providing structural support, can behave like inflammatory cells and produce mediators as chemokines and growth factors; they are known to express viral receptors, with TLR3 having been attributed relevance in viral disease-associated GN. Experiments were performed on human MC in cell culture. Stimulation experiments were performed with poly (I:C) and hepatitis C RNA from patients with hepatitis C infection. We hereby show a TLR3-mediated upregulation of VEGF and its receptor subtype 2 (VEGF-R2) in human MC upon activation of viral receptors by poly (I:C) and hepatitis C virus. The increase in VEGF expression levels is further enhanced by tumor necrosis factor alpha (TNFα) which also induces the cytokines IL-6 and IL-8 as well as the chemokines MCP-1 and RANTES. These effects are potentiated by preincubation of MC with poly (I:C), just as the induction of the viral receptors TLR3, RIG-1, and MDA5 themselves. Moreover, MCP-1 itself is able to significantly increase mesangial VEGF expression. Therefore, with VEGF and VEGF-R2 being induced upon viral receptor activation in human MC, a novel role of TLR3 in mediating glomerular damage in virally induced or aggravated GN is inferred. TNFα and MCP-1 are seemingly important in amplifying VEGF effects in the setting of virally induced inflammation, with TNFα being also able to induce other mediators of glomerular pathology in GN.

ARNO regulates VEGF-dependent tissue responses by stabilizing endothelial VEGFR-2 surface expression.

AIMS: The vascular endothelial growth factor (VEGF) stimulates angiogenesis by induction of vessel permeability, proliferation, and migration of endothelial cells, an important process in ischaemic diseases. ADP-ribosylation factor (ARF) nucleotide-binding site opener (ARNO) (cytohesin-2) is a guanine exchange factor important for cellular signalling through ARF GTPases. However, a role for ARNO in VEGF-dependent endothelial processes has so far not been documented. Therefore, we investigated whether ARNO has a role in VEGF-dependent activation of endothelial cells and thus vessel permeability. METHODS AND RESULTS: ARNO expression was observed in endothelial cells in vitro by RT-PCR, western blotting, and immunofluorescence as well as ex vivo by immunohistochemical staining of mouse aorta. Treatment with the cytohesin inhibitor SecinH3 or with an ARNO siRNA prevented VEGF-dependent Akt activation, assessed by detection of phosphorylated Akt, and proliferation of endothelial cells in vitro, measured by methylthiazoletetrazolium (MTT) reduction. In addition, ARNO suppression reduced VEGF-induced permeability in vessels of the mouse (C57BL/6) cremaster muscle in vivo, as measured by extravasation of fluorescein isothiocyanate (FITC)-dextran. Moreover, ARNO knock-down accelerated ligand-induced reduction in vascular endothelial growth factor receptor-2 (VEGFR-2) surface expression, internalization, and degradation, as assessed by flow cytometry and western blotting, respectively. CONCLUSION: Our findings indicate an important and novel role for endothelial ARNO in VEGF-dependent initiation of angiogenesis by regulation of VEGFR-2 internalization in endothelial cells, resulting in the activation of the Akt pathway, vessel permeability, and ultimately endothelial proliferation. Thus, ARNO may be a new essential player in endothelial signalling and angiogenesis.

Late and very late catch-up after 90Sr/90Y beta-irradiation for the treatment of coronary in-stent restenosis.

Since late vessel failure has been speculated as a significant limitation of vascular brachytherapy (VBT), we conducted a prospective clinical evaluation at 6, 12, 24, 36 and 60 months follow-up after irradiation with (90)Sr/(90)Y for in-stent restenosis (ISR) regardless of the patient's symptomatic status. Complete five-year follow-up is reported for 104 consecutive patients. The cumulative rate of death was 13.5% (6 months: 0.96%; 12 months: 2.88%; 24 months: 4.81%; 36 months: 7.69%), of acute myocardial infarction 4.81% (2.88%; 4.81%; 4.81%; 4.81%), of late thrombotic occlusion 4.81% (3.85%; 4.81%; 4.81%; 4.81%), of target lesion revascularization (TLR) 27.9% (8.65%; 12.5%; 17.3%; 21.2%), of target vessel revascularization (TVR) 43.3% (12.5%; 19.2%; 22.1%; 29.8%), and of all major adverse cardiovascular events (MACE) 61.5% (16.3%; 26.9%; 31.7%; 42.3%), respectively. Considered that the annual incidence of TVR after the first year following drug-eluting stenting for in-stent restenosis has been reported as approximately 3% per year, an incidence of 5.8% per year following VBT of our study population clearly indicates a more pronounced, delayed and, even in the fifth year after the index procedure, ongoing restenotic process following beta-irradiation of in-stent restenotic lesions associated with clinically relevant adverse cardiovascular events.

Bioactivation of dermal scaffolds with a non-viral copolymer-protected gene vector.

The use of scaffolds in skin tissue engineering is accompanied with low regeneration rates and high risk of infection. In this study, we activated an FDA-approved collagen scaffold for dermal regeneration by incorporation of copolymer-protected gene vectors (COPROGs) to induce a temporary release of VEGF. In vitro results show that the presence of COPROGs did not affect the distribution, attachment, proliferation and viability of cells in the scaffold. A transient release of VEGF was observed for up to 3 weeks. Moreover a high amount of VEGF was also found in the cells and associated with the scaffold. In a full skin defect model in nude mice, VEGF levels were significantly increased compared to controls in VEGF gene activated scaffolds 14 d after implantation, but not in skin from the wound edge. Results showed an increased amount of non-adherent cells, especially erythrocytes, and von Willebrandt factor (vWF) and a yellow red appearance of gene activated scaffolds in relation to controls. This suggests the presence of leaky vessels. In this work we show that the bioactivation of collagen scaffolds with COPROGs presents a new technology that allows a local release of therapeutic proteins thus enhancing the regenerative potential in vivo.

CD34+CD140b+ cells and circulating CXCL12 correlate with the angiographically assessed severity of cardiac allograft vasculopathy.

AIMS: We sought to determine whether circulating vascular progenitor cells, such as endothelial progenitor cells (EPCs) or smooth muscle progenitor cells (SPCs), were associated with the severity of cardiac allograft vasculopathy (CAV). METHODS AND RESULTS: CD34(+)CD140b(+) SPCs and CD34(+)KDR(+) EPCs were measured in the peripheral circulation of 187 adult heart transplant recipients by flow cytometry. Cardiac allograft vasculopathy was quantified by angiography using a CAV-specific scoring system. Cardiac allograft vasculopathy was present in 84 patients (44.7%) and was classified as mild in 59 and severe in 25 cases. Circulating SPCs were more frequently detectable in CAV patients than in patients without CAV. The number of CD34(+)CD140b(+) cells showed a stepwise increase in patients with moderate and severe CAV. Smooth muscle progenitor cell counts were higher in patients with coronary stent implant compared with unstented patients with CAV. In contrast, peripheral CD34(+)KDR(+) EPC counts were not changed in CAV patients. Plasma CXCL12 levels correlated with the degree of CAV and SPC counts. None of the different immunosuppressive drug regimes was related to the SPC count or the CXCL12 levels. A multivariate regression analysis revealed that the SPC count was independently associated with the presence of CAV. CONCLUSION: Circulating SPCs, but not EPCs, and plasma CXCL12 concentrations are elevated in CAV patients, indicating that they play prominent roles in transplant arteriosclerosis.

Suppression of DNA-PKcs enhances FGF-2 dependent human endothelial cell proliferation via negative regulation of Akt.

Angiogenesis initiation is crucially dependent on endothelial proliferation and can be stimulated by the fibroblast growth factor 2 (FGF-2). The DNA dependent protein kinase (DNA-PK), long known for its importance in repairing DNA double strand breaks, belongs to the phosphatidylinositol-3 kinase (PI3-K) super family and has recently been identified as one of the enzymes phosphorylating and activating Akt. Due to its similarity with PI3-K, we hypothesized that DNA-PK may have similar effects on endothelial angiogenic processes and signalling. We used primary endothelial cells (HUVEC and PAEC) and human microvascular endothelial cells (HMEC) to study the role of DNA-PK in endothelial proliferation and signalling. DNA-PKcs suppression with the compound NU7026 or with siRNA induced basal endothelial cell proliferation as well as enhanced FGF-2 dependent proliferation. This was associated with an increase in phosphorylated Akt. Tube formation was not affected by DNA-PKcs clearly showing that the role of DNA-PK in endothelial processes differs from that of PI3-K. Our findings indicate DNA-PK as an important enzyme maintaining the quiescent endothelial phenotype by actively inhibiting Akt thus restraining endothelial cell proliferation preventing excessive growth.

An optimized flow cytometry protocol for analysis of angiogenic monocytes and endothelial progenitor cells in peripheral blood.

Circulating adult CD34(+)VEGFR2(+) endothelial progenitor cells (EPCs) have been shown to differentiate into endothelial cells, thus contributing to vascular homeostasis. Furthermore, a subset of circulating CD14(+) monocytes coexpresses CD16 together with the angiopoietin receptor Tie2 and has been functionally implicated in tumor angiogenesis. However, clinically applicable protocols for flow cytometric quantification of EPCs and Tie2(+) monocytes in peripheral blood and a consensus on reference values remain elusive. The number of Tie2(+)CD14(+)CD16(mid) angiogenic monocytes and CD34(+)VEGFR2(+)CD45(low/-) EPCs was assessed in the peripheral venous blood of patients with stable coronary artery disease by three-color flow cytometry using specific monoclonal antibodies conjugated to PerCP, PE, PE-Cy7, APC, and APC-Cy7. Scatter multigating with exclusion of dead cells was performed to dissect complex mononuclear cell populations. This analysis was further refined by matching bright fluorochromes (PE-Cy7, PE, APC) with dimly expressed markers (CD34, VEGFR2, Tie2), by automatic compensation for minimizing fluorescence spillover and by using fluorescence-minus-one (FMO) controls to determine positive/negative boundaries. Presuming a Gaussian distribution, we obtained average values (mean +/- SD) of 1.45 +/- 1.29% for Tie2(+)CD14(+)CD16(mid) monocytes (n = 11, range: 0.12-3.64%) and 0.019 +/- 0.013% for CD34(+)VEGFR2(+)CD45(low/-) EPCs (n = 17, range: 0.003-0.042%). The intra- and inter-assay variability was 1.6% and 4.5%, respectively. We have optimized a fast and sensitive assay for the flow cytometric quantification of circulating angiogenic monocytes and EPCs in cardiovascular medicine. This protocol may represent a basis for standardized analysis and monitoring of these cell subsets to define their normal range and prognostic/diagnostic value in clinical use.

Novel role of toll-like receptor 3, RIG-I and MDA5 in poly (I:C) RNA-induced mesothelial inflammation.

Viral inflammation and infection of mesothelial cells (MC) are a major problem in several organ systems including pleura, pericardium and peritoneum. Toll-like receptors (TLRs) are an essential part of the innate immune system for early recognition of pathogen-associated molecular patterns. TLRs recognise molecular patterns associated with microbial pathogens and induce an immune response. TLR3 recognises dsRNA of viral origin as exemplified by poly (I:C) RNA, a synthetic analogue of viral dsRNA. The helicases RIG-I and MDA5 may also act as sensors of viral infections. MC exhibit an expression of TLR3, RIG-I and MDA5. Poly (I:C) RNA stimulation resulted in an up-regulation of proinflammatory cytokines and chemokines as well as type I interferons. This novel finding of functional expression of viral sensors on human MC may indicate a novel link between viral infections and mesothelial inflammation and indicates a pathophysiologic role of viral receptors in these processes.

Inhibition of the tyrosine phosphatase SHP-2 suppresses angiogenesis in vitro and in vivo.

Endothelial cell survival is indispensable to maintain endothelial integrity and initiate new vessel formation. We investigated the role of SHP-2 in endothelial cell survival and angiogenesis in vitro as well as in vivo. SHP-2 function in cultured human umbilical vein and human dermal microvascular endothelial cells was inhibited by either silencing the protein expression with antisense-oligodesoxynucleotides or treatment with a pharmacological inhibitor (PtpI IV). SHP-2 inhibition impaired capillary-like structure formation (p < 0.01; n = 8) in vitro as well as new vessel growth ex vivo(p < 0.05; n = 10) and in vivo in the chicken chorioallantoic membrane (p < 0.01, n = 4). Additionally, SHP-2 knock-down abrogated fibroblast growth factor 2 (FGF-2)-dependent endothelial proliferation measured by MTT reduction (p < 0.01; n = 12). The inhibitory effect of SHP-2 knock-down on vessel growth was mediated by increased endothelial apoptosis (annexin V staining, p < 0.05, n = 9), which was associated with reduced FGF-2-induced phosphorylation of phosphatidylinositol 3-kinase (PI3-K), Akt and extracellular regulated kinase 1/2 (ERK1/2) and involved diminished ERK1/2 phosphorylation after PI3-K inhibition (n = 3). These results suggest that SHP-2 regulates endothelial cell survival through PI3-K-Akt and mitogen-activated protein kinase pathways thereby strongly affecting new vessel formation. Thus, SHP-2 exhibits a pivotal role in angiogenesis and may represent an interesting target for therapeutic approaches controlling vessel growth.

Stereospecific and redox-sensitive increase in monocyte adhesion to endothelial cells by homocysteine.

OBJECTIVE: Previous studies have shown that elevated homocysteine (Hcy) levels promote the development of atherosclerotic lesions in atherosclerosis-prone animal models. There is evidence that oxidant stress contributes to Hcy's deleterious effects on the vasculature. The accumulation and adhesion of monocytes to the vascular endothelium is a critical event in the development of atherosclerosis. We investigated the effects of Hcy on the interaction between human endothelial cells (EC) (EC line EA.hy 926 and primary human umbilical vein EC [HUVEC]) and the monocytic cell line THP-1, and the impact of vascular oxidant stress and redox-sensitive signaling pathways on these events. METHODS AND RESULTS: L-Hcy, but not D-Hcy, increases the production of reactive oxygen species inside EC, enhances nuclear factor(NF)-kappaB activation, and stimulates intercellular adhesion molecule-1 (ICAM-1) RNA transcription and cell surface expression. This leads to a time- and dose-dependent increase in monocyte adhesion to ECs. Pretreatment of ECs with superoxide scavengers (MnTBAP and Tiron) or with an inhibitor of NF-kappaB activation abolished Hcy-induced monocyte adhesion, ICAM-1 expression, and nuclear translocation of NF-kappaB. CONCLUSIONS: These findings suggest that reactive oxygen species produced under hyperhomocysteinemic conditions may induce a proinflammatory situation in the vessel wall that initiates and promotes atherosclerotic lesion development.

Three-year clinical follow-up after strontium-90/yttrium-90 beta-irradiation for the treatment of in-stent coronary restenosis.

Because late vessel failure has been speculated as a possible limitation of vascular brachytherapy, we conducted a prospective clinical evaluation at 6, 12, 24, and 36 months of follow-up after irradiation with strontium-90/yttrium-90 for in-stent restenosis, regardless of the patient's symptomatic status. We report complete 3-year follow-up data for 106 consecutive patients. The cumulative rate of death at 6, 12, 24, and 36 months was 0.9%, 0.9%, 0.9%, and 1.9% respectively. The corresponding rates for acute ST-elevation myocardial infarction were 2.8%, 4.7%, 4.7%, and 4.7%, respectively. The cumulative rate of late thrombotic occlusion at 6, 12, 24, and 36 months was 3.8%, 4.7%, 4.7%, and 4.7%, respectively. The corresponding rates of target lesion revascularization and target vessel revascularization were 8.5% and 12.3% (p = 0.046), 14.2% (p = 0.157) and 18.0% (p = 0.046), 12.3% and 18.9% (p = 0.008), and 21.7% (p = 0.083) and 29.2% (p = 0.005), respectively. The cumulative rate of all major adverse cardiovascular events at 6, 12, 24, and 36 months was 16.1%, 24.5% (p = 0.003), 27.4% (p = 0.083), and 35.8% (p = 0.003), respectively. In conclusion, these results indicate a delayed and, even in the third year after the index procedure, continued restenotic process after beta irradiation of in-stent restenotic lesions.