Platelets secrete stromal cell-derived factor 1alpha and recruit bone marrow-derived progenitor cells to arterial thrombi in vivo.

The accumulation of smooth muscle and endothelial cells is essential for remodeling and repair of injured blood vessel walls. Bone marrow-derived progenitor cells have been implicated in vascular repair and remodeling; however, the mechanisms underlying their recruitment to the site of injury remain elusive. Here, using real-time in vivo fluorescence microscopy, we show that platelets provide the critical signal that recruits CD34+ bone marrow cells and c-Kit+ Sca-1+ Lin- bone marrow-derived progenitor cells to sites of vascular injury. Correspondingly, specific inhibition of platelet adhesion virtually abrogated the accumulation of both CD34+ and c-Kit+ Sca-1+ Lin- bone marrow-derived progenitor cells at sites of endothelial disruption. Binding of bone marrow cells to platelets involves both P-selectin and GPIIb integrin on platelets. Unexpectedly, we found that activated platelets secrete the chemokine SDF-1alpha, thereby supporting further primary adhesion and migration of progenitor cells. These findings establish the platelet as a major player in the initiation of vascular remodeling, a process of fundamental importance for vascular repair and pathological remodeling after vascular injury.

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.

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.

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.

Soluble glycoprotein VI dimer inhibits platelet adhesion and aggregation to the injured vessel wall in vivo.

Platelet-collagen interactions play a fundamental role in the process of arterial thrombosis. The major platelet collagen receptor is the glycoprotein VI (GPVI). Here, we determined the effects of a soluble dimeric form of GPVI on platelet adhesion in vitro and in vivo. We fused the extracellular domain of GPVI with the human immunoglobulin Fc domain. The soluble dimeric form of GPVI (GPVI-Fc) specifically bound to immobilized collagen. Binding of GPVI-Fc to collagen was inhibited competitively by soluble GPVI-Fc, but not control Fc lacking the external GPVI domain. GPVI-Fc inhibited the adhesion of CHO cells that stably express human GPVI and of platelets on collagen and attenuated thrombus formation under shear conditions in vitro. To test the effects of GPVI-Fc in vivo, arterial thrombosis was induced in the mouse carotid artery, and platelet-vessel wall interactions were visualized by intravital fluorescence microscopy. Infusion of GPVI-Fc but not of control Fc virtually abolished stable arrest and aggregation of platelets following vascular injury. Importantly, GPVI-Fc but not control Fc, was detected at areas of vascular injury. These findings further substantiate the critical role of the collagen receptor GPVI in the initiation of thrombus formation at sites of vascular injury and identify soluble GPVI as a promising antithrombotic strategy.

Antigen presenting cells transfected with LMP2a RNA induce CD4+ LMP2a-specific cytotoxic T lymphocytes which kill via a Fas-independent mechanism.

Recent reports have demonstrated that EBV can be used as a target of specific CTL-based treatments in severe chronic EBV, immunoblastic B cell lymphoma and Hodgkin's disease (HD). Based upon the promising results form these in vivo studies, it has been suggested that an antigen-specific CTL-based immunotherapy may be of benefit in treating EBV-associated tumors such as HD and nasopharyngeal carcinoma (NPC) which express the potentially immunogenic antigens, LMP1 and LMP2a. Recent work form our group has demonstrated that LMP2a-specific CTLs may be generated in vitro using autologous antigen presenting cells which have been transfected with polyadenylated LMP2a RNA in the presence of a cationic lipid. In this study, we demonstrate that the presence of the lipid enhances dendritic cell (DC) transfection efficiency and appears to protect the intracellular LMP2a RNA from degradation by cellular RNAses. Significantly, these improvements resulted in the transfected DCs having a superior ability to stimulate autologous T cell proliferation. These LMP2a + DCs were used to stimulate LMP2a-specific effector cells which were predominantly a mixture of cytotoxic and helper CD4+ T cells. The molecular mechanisms whereby these CD4+ T cells lyzed their LMP2a-expressing targets was investigated and we show that, although expressing Fas ligand on their surface, LMP2a-specific CD4+ effector cells kill their targets using the Ca2+-dependent perforin/granzyme pathway which is the same mechanism used by CD8+ CTLs.