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Introduction : Recent studies demonstrate the vasculoprotective effects of cell-based therapies to inhibit limit restenosis. Delivery of autologous late outgrowth endothelial progenitor cells (eEPC) result in early reendothelialization and inhibition of neointimal hyperplasia. Additional anti-inflammatory treatment may further reduce neointima generation. Methods and Results : CD34+ cells were isolated using immunomagnetic beads and cultured in endothelial cell medium to obtain eEPC. Early passage cells were transduced ex vivo by a retroviral vector expression of IL-1ra (eEPC IL-1ra) or empty vector (eEPC pLXSN) and expanded up to 46 population doublings. Expression levels were confirmed by RT-PCR. Athymic nude rats underwent carotid balloon and wire injury immediately followed by local delivery of eEPCs, eEPC IL-1ra, eEPC pLXSN or buffer for 20 minutes (n=22). Elastica van Giesson staining revealed a decrease in the intima/media ratio from 1.53 ± 0.11 to 1.13 ± 0.09 (mean ± SEM) after transplantation of eEPC (p=0.009). However, no additional reduction in neointima generation was observed after transplantation of eEPC IL-1ra. Endothelial-cell specific staining demonstrated an enhancement of reendothelialisation by eEPCs 24 hours after vascular injury. After 2 weeks a reduction of Ki67+ proliferating cells from 37.0 ± 8.5 Ki67+ cells in the control group to 11.67 ± 2.85 Ki67+ cells after eEPC transplantation (P=0.03) was found. Yet, no additional anti-proliferative effects were observed after transplantation of eEPC IL-1ra. Thus, enhancement of reendothelialization through local delivery of eEPCs may be sufficient for the inhibition of neointima generation since additional anti-inflammatory treatment using eEPC IL-1ra had no beneficial effect. Conclusion : Local delivery of blood-derived expanded endothelial progenitor cells after vascular injury contributes to endothelial regeneration, inhibits neointimal smooth muscle cell proliferation and reduces neointima generation. Autologous cell transplantation after vascular injury may be a feasible strategy for promotion of reendotheli-alisation and improvement of vascular regeneration.

Background: Recent studies suggest that endothelial progenitor cells (EPC) from bone marrow or peripheral blood improve myocardial function in experimental myocardial infarction (MI). Since applications for cell therapy are limited by the number of available cells, expansion of EPC may facilitate its therapeutic use in ischemic disease. The aim of this study was to expand late outgrowth EPC from peripheral blood from patients with acute myocardial infarction, characterize them and investigate their therapeutic effect in experimental MI. Methods and Results: Venous blood samples were obtained from patients with acute MI (n=51), stable angina (sAP, n=57) and healthy controls (H,n=47). CD34+ cells were isolated using immunomagnetic beads (Miltenyi Biotec). CD34+ cells cultured on fibronectin in endothelial cell medium formed colonies after 1–2 weeks and were further expanded for up to 3 months to generate late outgrowth EPC (eEPC). Expansion was observed up to 2.9x10′9 (MI), 11x10′9 (sAP) and 7x10′9 cells (H) with a mean culture duration of 61 days. Expanded cells showed an endothelial morphology and expressed endothelial surface markers (CD31, VEGF-R2, CD105). Intramyocardial transplantion of 1x10′6 eEPC in experimental myocardial infarction in athymic nude rats revealed improvement in echocardiographic ejection fraction after 2 weeks. This was associated with enhanced vessel density after 1 week and increased mRNA expression of HGF. No differences in infarct size were observed. Similarly in a chronic model of myocardial infarction (eEPC transplantation 1 week after MI) myocardial function significantly improved after 5 weeks in comparison to the control group. Conclusion: Expansion of eEPC from circulating CD34+ cells in patients with coronary artery disease is feasible and improves myocardial function after local transplantation in acute and chronic myocardial infarction. The large number of generated eEPC may prove benefical for therapeutic use and this advantage may prevail time-consuming expansion procedures.

Mantle cell lymphoma (MCL) is a distinctive non-Hodgkin's lymphoma subtype, characterized by overexpression of cyclin D1 as a consequence of the chromosomal translocation t(11;14)(q13;q32). MCL remains an incurable disease, combining the unfavourable clinical features of aggressive and indolent lymphomas. The blastic variant of MCL, which is often associated with additional cytogenetic alterations, has an even worse prognosis and new treatment options are clearly needed. The present study investigated the effect of a specific proteasome inhibitor, lactacystin, on cell cycle progression and apoptosis in two lymphoma cell lines harbouring the t(11;14)(q13;q32) and additional cytogenetic alterations, including p53 mutation (NCEB) and p16 deletion (Granta 519). Granta cells were more susceptible to inhibition of the proteasome with respect to inhibition of proliferation and apoptosis induction. No changes were observed in the expression levels of the G1 regulatory molecules cyclin D1 and cdk4, but cell cycle arrest and apoptosis induction was accompanied by accumulation of the cdk inhibitor p21 in both cell lines. Increased p53 expression was only observed in Granta cells with wild-type p53. Cleavage of procaspase-3 and -9 was observed but cleavage of procaspase-8 was not involved in apoptosis induction. The proapoptotic effect of lactacystin was reversed by pretreatment with the pancaspase inhibitor zVAD.fmk. Lactacystin was also effective in inducing apoptosis in lymphoma cells from MCL patients. We conclude that inhibition of the proteasome might be a promising therapeutic approach for this incurable disease.

SummaryTumor cell survival critically depends on heterotypic communication with benign cells in the microenvironment. Here, we describe a survival signaling pathway activated in stromal cells by contact to B cells from patients with chronic lymphocytic leukemia (CLL). The expression of protein kinase C (PKC)-βII and the subsequent activation of NF-κB in bone marrow stromal cells are prerequisites to support the survival of malignant B cells. PKC-β knockout mice are insusceptible to CLL transplantations, underscoring the in vivo significance of the PKC-βII-NF-κB signaling pathway in the tumor microenvironment. Upregulated stromal PKC-βII in biopsies from patients with CLL, acute lymphoblastic leukemia, and mantle cell lymphoma suggests that this pathway may commonly be activated in a variety of hematological malignancies.