Plasma Soluble Glycoprotein VI, Platelet Function, Bleeding, and Ischemic Events in Patients Undergoing Elective Percutaneous Coronary Intervention.

BACKGROUND AND AIMS: Glycoprotein VI (GPVI) is the major platelet-specific collagen receptor. GPVI shedding with generation of soluble GPVI (sGPVI) is an endogenous feedback mechanism preventing platelet overstimulation. sGPVI has not been investigated in patients with chronic coronary syndrome (CCS) undergoing percutaneous coronary intervention (PCI), especially regarding its potential value as a predictor of ischemic and bleeding risk. METHODS: Baseline plasma sGPVI levels were available in 318 patients with CCS undergoing PCI. Platelet function was assessed by measuring both adenosine diphosphate (ADP) and collagen-induced platelet aggregation. Co-primary endpoints were a composite of death or myocardial injury at 48 hours after PCI, and Bleeding Academic Research Consortium (BARC) type 1 to 5 bleeding at 30 days. RESULTS: There was no significant correlation between sGPVI and platelet function at baseline or at 48 hours after PCI and loading with antiplatelet drugs. Baseline plasma sGPVI levels were not associated with the ischemic risk: the incidence of the ischemic endpoint was 25.0% in the lower, 22.9% in the middle, and 26.7% in the upper sGPVI tertile (p = 0.82). There was a significant nonlinear relationship between sGPVI and the risk of bleeding: the incidence of the bleeding endpoint was 11.8% in the lower, 12.6% in the middle, and 26.4% in the upper sGPVI tertile (p = 0.006). CONCLUSION: In patients with CCS undergoing PCI, plasma levels of sGPVI did not correlate with ADP- or collagen-induced platelet aggregation. Patients with higher baseline levels of sGPVI may carry an increased risk of bleeding at 30 days after PCI but no excess risk of ischemic events.

The Scavenger Receptor CD68 Regulates Platelet Mediated Oxidized Low-Density Lipoprotein (oxLDL) Deposition in Atherosclerotic Vessels at an Early Stage of Atherosclerosis in LDLR-/-/ApoBec-/- Mice.

BACKGROUND/AIMS: Oxidative modifications of low-density lipoprotein (ox-LDL) play a key role in initial steps of atheroprogression possibly via specific scavenger receptors on inflammatory and endothelial cells. Amongst others, CD68 might play a crucial role in this leading to fatty streak formation. METHODS: Different CD68-Fc fusion proteins were cloned, expressed and tested in vitro for their oxLDL binding properties as a decoy for endogenous oxLDL. Physiological functions were tested in foam cell assays with human monocytes in culture and by binding oxLDL from human blood. The best suited candidate FcIgG2-FL-CD68 was injected twice weekly in LDL receptor and ApoBec deficient mice (LDLR-/-/Apobec-/-), and the oxLDL content was measured in peripheral blood, in different cell types of the spleen and aortic wall by specific oxLDL antibodies using flow cytometry. RESULTS: Different variants of the CD68-Fc bound to copper-oxided LDL (oxLDL), LDL and to a lesser extent HDL with different efficacy in an ELISA based binding assay in vitro. Native oxLDL content in human blood derived from patients with extended atherosclerosis was reduced after passage through a specific protein G column conjugated with the different CD68-Fc fusion proteins. Foam cell formation from human peripheral blood monocyte-platelet co-culture was reduced by the most effective CD68-Fc fusion proteins. oxLDL was not increased in the blood but markedly increased in the vessel wall from LDLR-/-/Apobec-/- mice at an early stage of atherosclerosis. Platelet-like cells in the vessel well contributed most to the increase in tissue oxLDL. FcIgG2-FL-CD68, reduced oxLDL content of aortic vessel wall cells from LDLR-/-/Apobec-/- mice. However a tissue specific reduction on the oxLDL content in peripheral blood, the spleen or cells from the aortic vessel by FcIgG2-FL-CD68 could not be shown. CONCLUSION: Platelets contribute to increased tissue oxLDL in the aortic wall but not in peripheral blood. CD68 seems to play a role in the oxLDL metabolism in the vessel wall at early stages of atherosclerosis. FcIgG2-FL-CD68 could serve as a novel therapeutic option to modify the oxLDL content in the vessel wall.

ADPase CD39 Fused to Glycoprotein VI-Fc Boosts Local Antithrombotic Effects at Vascular Lesions.

BACKGROUND: GPVI (Glycoprotein VI) is the essential platelet collagen receptor in atherothrombosis. Dimeric GPVI-Fc (Revacept) binds to GPVI binding sites on plaque collagen. As expected, it did not increase bleeding in clinical studies. GPVI-Fc is a potent inhibitor of atherosclerotic plaque-induced platelet aggregation at high shear flow, but its inhibition at low shear flow is limited. We sought to increase the platelet inhibitory potential by fusing GPVI-Fc to the ectonucleotidase CD39 (fusion protein GPVI-CD39), which inhibits local ADP accumulation at vascular plaques, and thus to create a lesion-directed dual antiplatelet therapy that is expected to lack systemic bleeding risks. METHODS AND RESULTS: GPVI-CD39 effectively stimulated local ADP degradation and, compared with GPVI-Fc alone, led to significantly increased inhibition of ADP-, collagen-, and human plaque-induced platelet aggregation in Multiplate aggregometry and plaque-induced platelet thrombus formation under arterial flow conditions. GPVI-CD39 did not increase bleeding time in an in vitro assay simulating primary hemostasis. In a mouse model of ferric chloride-induced arterial thrombosis, GPVI-CD39 effectively delayed vascular thrombosis but did not increase tail bleeding time in vivo. CONCLUSIONS: GPVI-CD39 is a novel approach to increase local antithrombotic activity at sites of atherosclerotic plaque rupture or injury. It enhances GPVI-Fc-mediated platelet inhibition and presents a potentially effective and safe molecule for the treatment of acute atherothrombotic events, with a favorable risk-benefit ratio.

Recombinant GPVI-Fc added to single or dual antiplatelet therapy in vitro prevents plaque-induced platelet thrombus formation.

The efficiency of current dual antiplatelet therapy might be further improved by its combination with a glycoprotein (GP) VI-targeting strategy without increasing bleeding. GPVI-Fc, a recombinant dimeric fusion protein binding to plaque collagen and concealing binding sites for platelet GPVI, acts as a lesion-focused antiplatelet drug, and does not increase bleeding in vivo. We investigated, whether GPVI-Fc added in vitro on top of acetylsalicylic acid (ASA), the P2Y12 antagonist ticagrelor, and the fibrinogen receptor antagonist abciximab alone or in combination would increase inhibition of platelet activation by atherosclerotic plaque. Under static conditions, GPVI-Fc inhibited plaque-induced platelet aggregation by 53 %, and increased platelet inhibition by ASA (51 %) and ticagrelor (64 %) to 66 % and 80 %, respectively. Under arterial flow, GPVI-Fc inhibited plaque-induced platelet aggregation by 57 %, and significantly increased platelet inhibition by ASA (28 %) and ticagrelor (47 %) to about 81 % each. The triple combination of GPVI-Fc, ASA and ticagrelor achieved almost complete inhibition of plaque-induced platelet aggregation (93 %). GPVI-Fc alone or in combination with ASA or ticagrelor did not increase closure time measured by the platelet function analyzer (PFA)-200. GPVI-Fc added on top of abciximab, a clinically used anti-fibrinogen receptor antibody which blocks platelet aggregation, strongly inhibited total (81 %) and stable (89 %) platelet adhesion. We conclude that GPVI-Fc added on top of single or dual antiplatelet therapy with ASA and/or a P2Y12 antagonist is likely to improve anti-atherothrombotic protection without increasing bleeding risk. In contrast, the strong inhibition of platelet adhesion by GPVI-Fc in combination with GPIIb/IIIa inhibitors could be harmful.

Cyclic Peptides for Effective Treatment in a Long-Term Model of Graves Disease and Orbitopathy in Female Mice.

A model for human Graves disease in mice was used to compare several treatment approaches. The mice received regular adenovirus (Ad) thyroid-stimulating hormone receptor (TSHR) A subunit immunizations (injections every 4 weeks). The generation of anti-TSHR antibodies, enlarged thyroid sizes (goiter), elevated serum thyroxine levels, retro-orbital fibrosis, and cardiac involvement (tachycardia and hypertrophy) were consistently observed over 9 months. Treatment of established disease in these mice using cyclic peptides that mimic one of the cylindrical loops of the TSHR leucine-rich repeat domain improved or cured all investigated parameters after six consecutive monthly injections. The first significant beneficial effects were observed 3 to 4 months after starting these therapies. In immunologically naïve mice, administration of any of the cyclic peptides did not induce any immune response. In contrast, monthly injections of the full antigenic TSHR A domain as fusion protein with immunoglobulin G crystallizable fragment induced clinical signs of allergy in Ad-TSHR-immunized mice and anti-TSHR antibodies in naïve control mice. In conclusion, cyclic peptides resolved many clinical findings in a mouse model of established Graves disease and orbitopathy. In contrast to blocking TSHR by allosteric modulation, the approach does not incur a direct receptor antagonism, which might offer a favorable side effect profile.

Review of Mouse Models of Graves' Disease and Orbitopathy-Novel Treatment by Induction of Tolerance.

Various approaches have been used to model human Graves' disease in mice, including transfected fibroblasts, and plasmid or adenoviral immunisations with the extracellular A subunit of the human thyrotropin receptor (TSHR). Some of these models were only observed for a short time period or were self-limiting. A long-term model for human Graves' disease was established in mice using continuing immunisations (4-weekly injections) with recombinant adenovirus expressing TSHR. Generation of TSHR binding cAMP-stimulatory antibodies, thyroid enlargement and alterations, elevated serum thyroxin levels, tachycardia and cardiac hypertrophy were maintained for at least 9 months in all Ad-TSHR-immunised mice. Here, we show that these mice suffer from orbitopathy, which was detected by serial orbital sectioning and histomorphometry. Attempts to treat established Graves' disease in preclinical mouse model studies have included small molecule allosteric antagonists and specific antagonist antibodies which were isolated from hypothyroid patients. In addition, novel peptides have been conceived which mimic the cylindrical loops of the TSHR leucine-rich repeat domain, in order to re-establish tolerance toward the antigen. Here, we show preliminary results that one set of these peptides improves or even cures all signs and symptoms of Graves' disease in mice after six consecutive monthly injections. First beneficial effects were observed 3-4 months after starting these therapies. In immunologically naïve mice, administration of the peptides did not induce any immune response.

Rabbit models to study atherosclerosis and its complications - Transgenic vascular protein expression in vivo.

Animal models of atherosclerosis often present limitations of transferability, since important hallmarks of human disease are not completely reproduced in other species. Rabbits have been used in several approaches: 1) inbred strains: Watanabe hereditary hyperlipidemic animals which express a defect in the LDL receptor, 2) transgenic rabbits, which overexpress human lipoproteins, and first knock-out rabbits. 3) native New Zealand white rabbits (NZW) fed with cholesterol-rich diet for at least 8 weeks represent the quickest way to establish arteriosclerosis. Rabbits are arguably the most sensitive animal species to cholesterol overload. Interventions in native or arteriosclerotic arteries are used to induce local thrombus formation, e.g. endothelial denudation or photochemical injury. In contrast to smaller animals, catheterisation of coronary arteries is feasible, whose external ligation serves to induce myocardial infarction. As biological endpoints, arterial vasoreactivity/endothelial dysfunction can be studied in analogy to measurements of brachial artery vasomotion in humans in response to increasing doses of acetylcholine or volume challenges. Tissue fixation allows studying vascular morphology, plaque sizes and thrombi after local interventions. Macrophage and T lymphocyte invasion can be investigated histologically. Positron emission tomography (PET/MRI) offers to measure plaques and content in vivo serially in the same animals. Local virally mediated gene transfer to atherosclerotic rabbit arteries has been established as a rapid and reproducible method to test interesting transgenes. Histological plaque features correspond well to alterations in patients (inflammation and lipid load). Thus, effects of any proteins can be studied directly in the arteriosclerotic disease background - much quicker than after germline transgenesis and cross-breeding.

Prolonged TSH receptor A subunit immunization of female mice leads to a long-term model of Graves' disease, tachycardia, and cardiac hypertrophy.

A transient model for human Graves' disease was successfully established in mice using up to 3 immunizations with recombinant adenovirus expressing the extracellular A-subunit of the human TSH receptor (TSHR) (Ad-TSHR). We studied extension of adenovirally induced TSHR A-subunit immunization in mice by using a novel protocol of long-term 3- and 4-weekly injections. Generation of TSHR binding stimulatory antibodies (capacity to stimulate cAMP activity in TSHR-expressing test cells), goiter, and histological thyroid alterations were maintained for at least 9 months in all Ad-TSHR-immunized mice. In response to injection of 10(10) plaque-forming units of Ad-TSHR, also elevated mean serum T4 levels were observed throughout the study. Moreover, cardiac organ involvement (tachycardia and hypertrophy) were consistently observed in these mice. Higher doses of Ad-TSHR (10(11) plaque-forming units) did not produce consistent elevation of T4 and were not associated with a clear increase in heart rate vs controls, probably because these high doses provoked an immune response-induced tachycardia on their own. In summary, a long-term model of Graves' disease induced by a relatively simple protocol of continuing monthly immunizations should allow to investigate long-term disease mechanisms and may possibly obviate the need for more complicated disease models. Moreover, the clinical outcome predictor of tachycardia and cardiac involvement was reliably detected in the model.

The GPVI-Fc fusion protein Revacept reduces thrombus formation and improves vascular dysfunction in atherosclerosis without any impact on bleeding times.

AIMS: Glycoprotein VI (GPVI) is a key platelet receptor which mediates plaque-induced platelet activation and consecutive atherothrombosis, but GPVI is also involved in platelet-mediated atheroprogression. Therefore, interference in GPVI-mediated platelet activation has the potential to combine short-term and long-term beneficial effects, specificity and safety especially regarding bleeding complications. METHODS AND RESULTS: We investigated the effects of the soluble dimeric GPVI receptor fusion protein, Revacept, an antagonist of collagen-mediated platelet activation, in an animal model of atherosclerosis: twenty week old rabbits, which had been fed on a cholesterol-rich diet for 8 weeks, received Revacept (8 mg/kg) or control twice weekly for 4 weeks. Pharmacokinetics indicated a slight accumulation of the drug in the serum after repeated dosing of Revacept for 3 weeks. A significant improvement of endothelial dysfunction after 0.06 and 0.6 µg/min acetylcholine and a significant decrease of vessel wall thickening were found after Revacept treatment. Accordingly, aortic vessel weight was reduced, and plaque sizes, macrophage and T-cell invasion tended to be reduced in histological evaluations. Bleeding time was determined after tail clipping in mice. Revacept alone or in combination with widely used anti-platelet drugs revealed a high safety margin with no prolongation of bleeding times. CONCLUSION: Repeated doses of Revacept led to a significant improvement of endothelial dysfunction and vascular morphology in atherosclerotic rabbits. Furthermore, no influence of Revacept on bleeding time alone or in combinations with various anti-platelet drugs was found in mice. Thus, the inhibition of collagen-mediated platelet interaction with the atherosclerotic endothelium by Revacept exerts beneficial effects on morphology and vascular function in vivo and seems to have a wide therapeutic window without influencing the bleeding time.

Pharmacological inhibition of platelet-tumor cell cross-talk prevents platelet-induced overexpression of cyclooxygenase-2 in HT29 human colon carcinoma cells.

Cyclooxygenase (COX)-2-derived prostanoids can influence several processes that are linked to carcinogenesis. We aimed to address the hypothesis that platelets contribute to aberrant COX-2 expression in HT29 colon carcinoma cells and to reveal the role of platelet-induced COX-2 on the expression of proteins involved in malignancy and marker genes of epithelial-mesenchymal transition (EMT). Human platelets cocultured with HT29 cells rapidly adhered to cancer cells and induced COX-2 mRNA expression, but not protein synthesis, which required the late release of platelet-derived growth factor and COX-2 mRNA stabilization. Platelet-induced COX-2-dependent prostaglandin E2 (PGE2) synthesis in HT29 cells was involved in the downregulation of p21(WAF1/CIP1) and the upregulation of cyclinB1 since these effects were prevented by rofecoxib (a selective COX-2 inhibitor) and rescued by exogenous PGE2. Galectin-3, which is highly expressed in HT29 cells, is unique among galectins because it contains a collagen-like domain. Thus, we studied the role of galectin-3 and platelet collagen receptors in platelet-induced COX-2 overexpression. Inhibitors of galectin-3 function (ß-lactose, a dominant-negative form of galectin-3, Gal-3C, and anti-galectin-3 antibody M3/38) or collagen receptor-mediated platelet adhesion (revacept, a dimeric platelet collagen receptor GPVI-Fc) prevented aberrant COX-2 expression. Inhibition of platelet-cancer cell interaction by revacept was more effective than rofecoxib in preventing platelet-induced mRNA changes of EMT markers, suggesting that direct cell-cell contact and aberrant COX-2 expression synergistically induced gene expression modifications associated with EMT. In conclusion, our findings provide the rationale for testing blockers of collagen binding sites, such as revacept, and galectin-3 inhibitors in the prevention of colon cancer metastasis in animal models, followed by studies in patients.

Overexpression of ABCG1 protein attenuates arteriosclerosis and endothelial dysfunction in atherosclerotic rabbits.

The ABCG1 protein is centrally involved in reverse cholesterol transport from the vessel wall. Investigation of the effects of ABCG1 overexpression or knockdown in vivo has produced controversial results and strongly depended on the gene intervention model in which it was studied. Therefore, we investigated the effect of local overexpression of human ABCG1 in a novel model of vessel wall-directed adenoviral gene transfer in atherosclerotic rabbits. We conducted local, vascular-specific gene transfer by adenoviral delivery of human ABCG1 (Ad-ABCG1-GFP) in cholesterol-fed atherosclerotic rabbits in vivo. Endothelial overexpression of ABCG1 markedly reduced atheroprogression (plaque size) and almost blunted vascular inflammation, as shown by markedly reduced macrophage and smooth muscle cell invasion into the vascular wall. Also endothelial function, as determined by vascular ultrasound in vivo, was improved in rabbits after gene transfer with Ad-ABCG1-GFP. Therefore, both earlier and later stages of atherosclerosis were improved in this model of somatic gene transfer into the vessel wall. In contrast to results in transgenic mice, over-expression of ABCG1 by somatic gene transfer to the atherosclerotic vessel wall results in a significant improvement of plaque morphology and composition, and of vascular function in vivo.

Effect of the oxLDL binding protein Fc-CD68 on plaque extension and vulnerability in atherosclerosis.

RATIONALE: There is strong evidence that oxidative modification of low-density lipoprotein (oxLDL) plays a critical role in atherogenesis and that oxLDL may profoundly influence the mechanical stability of atherosclerotic plaques. OBJECTIVE: To block oxLDL, we designed, expressed, and tested Fc-CD68, a soluble oxLDL binding protein consisting of human Fc and the extracellular domain of the human oxLDL-binding receptor CD68. METHODS AND RESULTS: Fc-CD68 bound with high specific affinity to oxLDL and strongly bound and colocalized with oxLDL in plaques. To study the effects of repeated administrations of Fc-CD68 on the progression of atherosclerosis and plaque vulnerability, 12- and 16-week old cholesterol-fed ApoE(-/-) mice received either Fc-CD68 (n = 6) or Fc control protein (n = 6 to 8) thrice weekly for 4 weeks. Macroscopic and histological analysis of Sudan red lipid staining showed strong and significant reduction of plaque extension in the aorta and in the aortic root, respectively. Histological analysis of pentachrome- and Sirius-stained sections of the brachiocephalic arteries of 20 week-old ApoE(-/-) mice revealed that Fc-CD68 significantly reduced the occurrence of spontaneous ruptures of established plaques by ≈20%, compared with Fc and drastically increased the collagen content of plaques. Furthermore, in immunostained sections of the brachiocephalic artery and the aortic root, Fc-CD68 reduced the infiltration of plaques with T lymphocytes, and macrophages by ≈50% and 30%, respectively. CONCLUSIONS: The oxLDL binding protein Fc-CD68 attenuates atherosclerosis and strengthens the stability of atherosclerotic plaques.

Inhibition of foam cell formation using a soluble CD68-Fc fusion protein.

The appearance of lipid-rich foam cells is a major feature of vulnerable atherosclerotic plaque formation. The transformation of macrophages into foam cells results from excessive uptake of cholesterol-rich particles by scavenger receptors such as CD68. We cloned a CD68-Fc immunoadhesin, a fusion protein consisting of the extracellular domain of the human CD68 and a human Fc domain, and investigated the function in vitro. Specific binding of CD68-Fc to OxLDL with an affinity of 10 nmol/L was determined by surface plasmon resonance and increased binding to lipid-rich human and ApoE(-/-) mice plaque tissue. This was confirmed both by immunohistochemical staining of CD68-Fc-treated paraffin sections from human plaques and by ELISA-based quantification of CD68-Fc binding to human atherosclerotic plaque extracts. In an in vitro model of macrophage/foam cell formation, CD68-Fc reduced foam cell formation significantly. This was caused both by interference of CD68-Fc with OxLDL uptake into macrophages and platelets and by the inhibition of platelet/OxLDL phagocytosis. Finally, expression of metalloproteinases by macrophages/foam cells was inhibited by CD68-Fc. In conclusion, CD68-Fc seems to be a promising new tool for preventing macrophage/foam cell formation. Thus, CD68-Fc might offer a novel therapeutic strategy for patients with acute coronary syndrome by modulating the generation of vulnerable plaques.

Impact of glycoprotein VI and platelet adhesion on atherosclerosis--a possible role of fibronectin.

Glycoprotein VI (GPVI) mediates binding of platelets to subendothelial collagen during acute arterial thrombosis. GPVI interactions with the activated atherosclerotic vascular endothelium during early atherosclerosis, however, are not well understood. In ApoE-/- mice, platelet adhesion to atherosclerotic arteries was increased, as measured by intravital microscopy. This platelet adhesion was significantly inhibited by IV injection of GPVI-Fc (1 mg/kg body weight). Atherosclerosis in ApoE-/- mice was attenuated both after 7 and 10 weeks of treatment with the anti-GPVI antibody JAQ1 (2 mg/kg body weight i.p. twice weekly). Binding of GPVI-Fc (1 mg/kg IV) occurred to deeper layers, but also to the luminal site of plaques in atherosclerotic rabbits, but not to the vessel wall of healthy littermates. Gene transfer of GPVI-Fc to the carotid vascular wall significantly attenuated athero-progression and endothelial dysfunction in atherosclerotic rabbits in vivo. Specific binding of the soluble GPVI receptor (GPVI-Fc) to fibronectin was found in vitro to coated ELISA plates. Platelet adhesion to fibronectin was significantly inhibited both by GPVI-Fc and by the anti-GPVI antibody 5C4 ex vivo in flow chamber experiments. GPVI plays a role in platelet adhesion to atherosclerotic endothelium in the absence of plaque rupture. Inhibition of GPVI both via GPVI-Fc and anti-GPVI-antibodies results in protection against atherosclerosis in both cholesterol-fed rabbits and ApoE-/- mice. This novel mechanism of GPVI-mediated platelet adhesion-possibly via fibronectin-could relevantly contribute to platelet-triggered atheroprogression.

EMMPRIN (CD147) is a novel receptor for platelet GPVI and mediates platelet rolling via GPVI-EMMPRIN interaction.

The Extracellular Matrix Metalloproteinase Inducer (EMMPRIN, CD147, basigin) is an immunoglobulin-like receptor expressed in various cell types. During cellular interactions homotypic EMMPRIN-EMMPRIN interactions are known to induce the synthesis of matrix metalloproteinases. Recently, we have identified EMMPRIN as a novel receptor on platelets. To our knowledge EMMPRIN has not been shown to serve as adhesion receptor, yet. Here we characterise platelet glycoprotein VI (GPVI) as a novel adhesion receptor for EMMPRIN. Human platelets were prestimulated with ADP and perfused over immobilised recombinant EMMPRIN-Fc or Fc-fragments under arterial shear conditions. ADP-stimulated platelets showed significantly enhanced rolling (but not enhanced firm adhesion) on immobilised EMMPRIN-Fc compared to Fc. Pretreatment of platelets with blocking mAbs anti-EMMPRIN or anti-GPVI leads to a significant reduction of rolling platelets on immobilised EMMPRIN-Fc, whereas pretreatment with blocking mAbs anti-p-selectin, anti-alpha4-integrin or anti-GPIIb/IIIa complex (20 microg/ml each) had no effect. Consistently, chinese hamster ovary (CHO) cells stably transfected with GPVI showed enhanced rolling (but not adhesion) on immobilised EMMPRIN-Fc in comparison to non-transfected CHO cells. Similarly, CHO cells stably transfected with EMMPRIN showed enhanced rolling on immobilised GPVI-Fc (or EMMPRIN-Fc) compared to non transfected CHO-cells. Finally, specific binding of EMMPRIN to GPVI was demonstrated by a modified ELISA and surface plasmon resonance technology with a dissociation constant of 88 nM. Platelet GPVI is a novel receptor for EMMPRIN and can mediate platelet rolling via GPVI-EMMPRIN interaction.

Functional alterations after cardiac sodium-calcium exchanger overexpression in heart failure.

The sodium-calcium exchanger (NCX) is discussed as one of the key proteins involved in heart failure. However, the causal role and the extent to which NCX contributes to contractile dysfunction during heart failure are poorly understood. NCX overexpression was induced by infection with an adenovirus coding for NCX, which coexpressed green fluorescence protein (GFP) (AdNCX) by ex vivo gene transfer to nonfailing and failing rabbit cardiomyocytes. Myocardial gene transfer in rabbits in vivo was achieved by adenoviral delivery via aortic cross-clamping. Peak cell shortening of cardiomyocytes was determined photo-optically. Hemodynamic parameters in vivo were determined by echocardiography (fractional shortening) and tip catheter [maximal first derivative of left ventricular (LV) pressure (dP/dt(max)); maximal negative derivative of LV pressure (-dP/dt(max))]. Peak cell shortening was depressed after NCX gene delivery in isolated nonfailing and in failing cardiomyocytes. In nonfailing rabbits in vivo, basal systolic contractility (fractional shortening and dP/dt(max)) and maximum rate of LV relaxation (-dP/dt(max)) in vivo were largely unaffected after NCX overexpression. However, during heart failure, long-term NCX overexpression over 2 wk significantly improved fractional shortening and dP/dt(max) compared with AdGFP-infected rabbits, both without inotropic stimulation and after beta-adrenergic stimulation with isoproterenol. -dP/dt(max) was also improved after NCX overexpression in the failing rabbits group. These results indicate that short-term effects of NCX overexpression impair contractility of isolated failing and nonfailing rabbit cardiomyocytes. NCX overexpression over 2 wk in vivo does not seem to affect myocardial contractility in nonfailing rabbits. Interestingly, in vivo overexpression of NCX decreased the progression of systolic and diastolic contractile dysfunction and improved beta-adrenoceptor-mediated contractile reserve in heart failure in rabbits in vivo.

Cardiac overexpression of the norepinephrine transporter uptake-1 results in marked improvement of heart failure.

A hyperadrenergic state is one of the key features of human and experimental heart failure. Decreased densities and activities of the presynaptic neuronal norepinephrine (NE) transporter uptake-1 occur both in patients and animal models. It is currently unclear to what extent the reduction of uptake-1 contributes to the deterioration of heart failure. Therefore, we investigated the effects of myocardial overexpression of uptake-1 in both nonfailing rabbit hearts and in an animal model of heart failure. Heart failure was induced in rabbits by rapid ventricular pacing. Adenoviral gene transfer was used to overexpress uptake-1 in the myocardium. Uptake-1 overexpression led to increased NE uptake capacity into the myocardium. In contrast, systemic plasma NE levels in uptake-1-overexpressing failing rabbits (uptake-1-CHF) did not differ from controls. Downregulation of SERCA-2 and beta-adrenergic receptors in the failing myocardium was significantly reversed after uptake-1 overexpression. Uptake-1 overexpression significantly improved left ventricular (LV) diameters (LV end-diastolic diameter: in GCP-overexpressing failing rabbits (GFP-CHF), 17.4+/-0.4 mm; in uptake-1-CHF rabbits, 15.6+/-0.6 mm) and systolic contractility (fractional shortening: GFP-CHF, 20.7+/-0.6%; uptake-1-CHF, 27.3+/-0.7%), as assessed by echocardiography at the end of the heart failure protocol. Intraventricular tip catheter measurements revealed enhanced contractile reserve (dP/dt max with isoproterenol 1.0 microg/kg: GFP-CHF, 6964+/-230 mm Hg/sec; uptake-1-CHF, 7660+/-315 mm Hg/sec) and LV relaxation (dP/dt min with isoproterenol 1.0 microg/kg: GFP-CHF: -3960+/-260 mm Hg/sec; uptake-1-CHF, -4910+/-490 mm Hg/sec). End-diastolic filling pressures (GFP-CHF, 8.5+/-1.2 mm Hg; uptake-1-CHF, 5.6+/-0.7 mm Hg) tended to be lower in uptake-1 overexpressing animals. In summary, local overexpression of uptake-1 in the myocardium results in marked structural and functional improvement of heart failure, thus underlining the importance of uptake-1 as a key protein in heart failure.

Retroviral infection and selection of culture-derived platelets allows study of the effect of transgenes on platelet physiology ex vivo and on thrombus formation in vivo.

UNLABELLED: Background- We recently reported the development of culture-derived (CD) platelets with the aim to express any protein of interest in these platelets. We now report a specific protocol of retroviral infection into the progenitor cells and subsequent selection, which allows to generate large amounts of highly homogenous transgene-expressing CD platelets and to study transgene function rapidly and reliably at large-scale ex vivo and in vivo settings. METHODS AND RESULTS: After retroviral infection and selection, the activation-dependent expression profile of surface markers, aggregation, and granule release were investigated. The function of transgene-expressing CD platelets, the precursor cells of which had been retrovirally infected, compared well to noninfected CD platelets or freshly isolated platelets. Hence, the retroviral infection protocol did not alter platelet physiology. In contrast, adenoviral infection of precursors to CD platelets resulted in marked functional alterations that obviated their use in analytic experiments. Additionally, sufficient amounts of selected CD platelets were generated to warrant intravenous injections into living mice. This approach permitted study of their adhesive profile at endothelial lesions and their effect on thrombus formation in vivo by intravital videofluorescence microscopy. CONCLUSIONS: The novel selection method allowed us to produce recombinant transgene-expressing platelets in sufficient amounts to study genetically modified platelets in vitro and in vivo.

Generation of functional culture-derived platelets from CD34+ progenitor cells to study transgenes in the platelet environment.

The possibility of evaluating the function of transgenes in platelets requires the generation of platelets from nucleated progenitor cells in vitro. In this article, we provide effective culture conditions for generating functional culture-derived (CD) human and mouse platelets from CD34(+) progenitor cells that allow expression of any foreign protein of interest. We have evolved an effective cytokine cocktail (thrombopoietin, stem cell factor, interleukin [IL]-1beta, IL-6) that induces a high yield of CD platelets and optimal shedding from cultivated megakaryocytes generated from CD34(+) progenitor cells. CD platelets showed similar functional and morphological characteristics compared with isolated blood platelets, including surface expression of platelet antigens (CD41, CD42, CD62P), aggregation, release of granule constituents (P-selectin, platelet factor 4, serotonin). Moreover, transmission electron microscopy revealed the presence of typical alpha- and dense granules and dense tubular system in CD platelets. Additionally, we showed that stable transgene expression in CD platelets can be performed through infection of CD34(+) progenitor cells using adenoviral vectors. Thus, we describe a methodology that enables studying functional consequences of transgenes of interest in the natural environment of platelets that may impose substantial impact on potential future platelet research and therapeutic target evaluation. The full text of this article is available online at http://circres.ahajournals.org.

Na+/Ca2+ exchanger overexpression impairs frequency- and ouabain-dependent cell shortening in adult rat cardiomyocytes.

The Na(+)/Ca(2+) exchanger (NCX) may influence cardiac function depending on its predominant mode of action, forward mode or reverse mode, during the contraction-relaxation cycle. The intracellular Na(+) concentration ([Na(+)](i)) and the duration of the action potential as well as the level of NCX protein expression regulate the mode of action of NCX. [Na(+)](i) and NCX expression have been reported to be increased in human heart failure. Nevertheless, the consequences of altered NCX expression in heart failure are still a matter of discussion. We aimed to characterize the influence of NCX expression on intracellular Ca(2+) transport in rat cardiomyocytes by adenoviral-mediated gene transfer. A five- to ninefold (dose dependent) overexpression of NCX protein was achieved after 48 h by somatic gene transfer (Ad.NCX.GFP) versus control (Ad.GFP). NCX activity, determined by Na(+) gradient-dependent (45)Ca(2+)-uptake, was significantly increased. The protein expressions of sarco(endo)plasmic reticulum Ca(2+)-ATPase, phospholamban, and calsequestrin were unaffected by NCX overexpression. Fractional shortening (FS) of isolated cardiomyocytes was significantly increased at low stimulation rates in Ad.NCX.GFP. After a step-wise enhancing frequency of stimulation to 3.0 Hz, FS remained unaffected in Ad.GFP cells but declined in Ad.NCX.GFP cells. The positive inotropic effect of the cardiac glycoside ouabain was less effective in Ad.NCX.GFP cells, whereas the positive inotropic effect of beta-adrenergic stimulation remained unchanged. In conclusion, NCX overexpression results in a reduced cell shortening at higher stimulation frequencies as well as after inhibition of sarcolemmal Na(+)-K(+)-ATPase, i.e., in conditions with enhanced [Na(+)](i). At low stimulation rates, increased NCX expression enhances both intracellular systolic Ca(2+) and contraction amplitude.