Patient-derived Endothelial Cells Research Articles

S141 BMP9 is required for LPS-mediated neutrophil recruitment to PAH-patient derived blood outgrowth endothelial cells with BMPR-II mutations

IntroductionHeterozygous mutations in the gene encoding bone morphogenetic protein (BMP) receptor II (BMPR-II) are present in >70% of patients with heritable pulmonary arterial hypertension (hPAH) and 15–26% of idiopathic PAH...

Inhibitory Effect of Natural Anti-Inflammatory Compounds on Cytokines Released by Chronic Venous Disease Patient-Derived Endothelial Cells

Large vein endothelium plays important roles in clinical diseases such as chronic venous disease (CVD) and thrombosis; thus to characterize CVD vein endothelial cells (VEC) has a strategic role in identifying specific therapeutic targets. On these bases we evaluated the effect of the natural anti-inflammatory compounds α-Lipoic acid and Ginkgoselect phytosome on cytokines/chemokines released by CVD patient-derived VEC. For this purpose, we characterized the levels of a panel of cytokines/chemokines (n = 31) in CVD patients' plasma compared to healthy controls and their release by VEC purified from the same patients, in unstimulated and TNF-α stimulated conditions. Among the cytokines/chemokines released by VEC, which recapitulated the systemic profile (IL-8, TNF-α, GM-CSF, INF-α2, G-CSF, MIP-1β, VEGF, EGF, Eotaxin, MCP-1, CXCL10, PDGF, and RANTES), we identified those targeted by ex vivo treatment with α-Lipoic acid and/or Ginkgoselect phytosome (GM-CSF, G-CSF, CXCL10, PDGF, and RANTES). Finally, by investigating the intracellular pathways involved in promoting the VEC release of cytokines/chemokines, which are targeted by natural anti-inflammatory compounds, we documented that α-Lipoic acid significantly counteracted TNF-α-induced NF-κB and p38/MAPK activation while the effects of Ginkgo biloba appeared to be predominantly mediated by Akt. Our data provide new insights into the molecular mechanisms of CVD pathogenesis, highlighting new potential therapeutic targets.

Coagulation on Endothelial Cells: The Underexposed Part of Virchow’s Triad

The process of thrombin generation involves numerous plasma proteases and cofactors. Interaction with the vessel wall, in particular endothelial cells (ECs), influences this process but data on this interaction is limited. We evaluated thrombin generation on EA.hy926, human coronary arterial ECs (HCAECs) and patient-derived human venous ECs (HVECs) by means of a modified calibrated automated thrombogram (CAT) method and especially looked into contribution of the intrinsic and extrinsic pathways. Thrombin generation was measured in presence of confluent ECs with normal pooled and factor XII-deficient (FXII-deficient) platelet-poor plasma, with/without active site inhibited factor VIIa (ASIS) to block the extrinsic pathway and corn trypsin inhibitor for blocking contact activation (intrinsic pathway). Fetal bovine serum (FBS) was removed from culture conditions as FXIIa from the serum retained on ECs apparently, thereby inducing strong contact activation. In serum-free conditions, EA.hy926 and patient-derived HVECs induced thrombin generation mainly via the contact activation pathway with minor influence of ASIS on peak height and very low thrombin generation curves in FXII-deficient plasma. HVECs derived from coronary arterial bypass graft (CABG) patients showed increased thrombin generation compared to control patients, which could be ascribed to increased contact activation. Contribution of the extrinsic pathway on patient-derived ECs was limited. We conclude that the CAT method in combination with serum-free cultured ECs offers a valuable high-throughput method to evaluate endothelial influences on thrombin generation, which appears to involve predominantly contact activation on ECs. Contact activation-mediated thrombin generation was increased on ECs from CABG patients compared to controls.

Identification of Novel Angiogenic Proteins in Multiple Myeloma Patient-Derived Endothelial Cells Using Proteome-Wide Analysis.

Abstract 2825Poster Board II-801Novel technologies are needed to identify new and more efficient biomarkers and improved molecular targets for accurate diagnosis and treatment of multiple myeloma (MM). Proteomics, i.e., the study of proteins and protein pathways involved in disease, is a new dimension in preclinical and clinical development. Preliminary studies in global protein expression of bone marrow endothelial cells (ECs) of patients with MM (MMECs) and ECs from monoclonal gammopathy of undetermined significance (MGUS-MGECs) vs. human umbilical vein endothelial cells (HUVECs), has shown at least 20 proteins differentially expressed in MMECs. The association of monodimensional electrophoresis with MALDI-MS analysis allowed us to identify a series of proteins that are shown to be important biomarkers to differentiate MMECs vs. MGECs and HUVECs, and distinguish different stages in MM progression, i.e., diagnosis, relapse, and refractory disease. These proteins are known to play important roles in cellular functions such as glycolysis (α-enolase and glyceraldehyde-3-phosphate dehydrogenase), cell-cycle regulation (14-3-3 zeta/delta protein, mitotic checkpoint serine/threonine-protein kinase BUB1), apoptosis (annexin V), angiogenesis and metastasis (vimentin, α-filamin) and they might contribute to the adverse evolution of the disease. Preliminary results suggest that the increased expression of α-filamin in ECs from an active desease, refractory desease and stable desease, the upregulation of vimentin in ECs from an active desease and down regulation of annexin VI in ECs from a refractory desease is related to the cell overangiogenic potential, indicate a common machinery involved with the structural organization of the cytoskeleton and with the connection of matrix and cell–cell external signals with the intracellular signaling pathways. On the other hand, changes in the expression of structural proteins (Filamin, vimentin, alpha-actinin), could account, at least in part, for the different morphologies displayed by migrating endothelial cells. In order to understand the role of these proteins in the angiogenic switch, the molecular targets potentially useful in the antiangiogenic treatment of MM were silenced by siRNA experiments and their potential involvement in migration and proliferation was investigated by proliferation, cell adhesion and invasion essays. This study has delineated the functional profile of the proteome of ECs in patients with MM in order to understand some of the mechanisms involved in neovascularisation and progression of MM and to identify new targets useful in the treatment of these patients. Because angiogenesis and lymphangiogenesis actively contribute to cancer progression, future studies to establish the role of these angiogenic proteins in disease may suggest potential new targets for tissue-specific therapies. Disclosures:No relevant conflicts of interest to declare.

Zoledronic acid affects over-angiogenic phenotype of endothelial cells in patients with multiple myeloma

Therapeutic doses of zoledronic acid markedly inhibit in vitro proliferation, chemotaxis, and capillarogenesis of bone marrow endothelial cells of patients with multiple myeloma. Zoledronic acid also induces a sizeable reduction of angiogenesis in the in vivo chorioallantoic membrane assay. These effects are partly sustained by gene and protein inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in an autocrine loop. Mevastatin, a specific inhibitor of the mevalonate pathway, reverts the zoledronic acid antiangiogenic effect, indicating that the drug halts this pathway. Our results provide evidence of a direct antiangiogenic activity of zoledronic acid on multiple myeloma patient-derived endothelial cells due to at least four different mechanisms identified either in vitro or in vivo. Tentatively, we suggest that the zoledronic acid antitumoral activity in multiple myeloma is also sustained by antiangiogenesis, which would partly account for its therapeutic efficacy in multiple myeloma.

Vasculogenic mimicry by bone marrow macrophages in patients with multiple myeloma

Bone marrow macrophages of patients with active and nonactive multiple myeloma (MM), monoclonal gammopathies of undetermined significance (MGUS) and benign anemia (controls) were stimulated for 7 days with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), and analysed for the expression of endothelial cell (EC) markers by reverse transcription (RT)-PCR, real-time RT-PCR, western blot and immunofluorescence. Their vasculogenic ability was investigated in vitro in a Matrigel assay and in vivo on bone marrow biopsies through dual immunofluorescence and confocal laser microscopy. Active MM macrophages exposed to VEGF and bFGF acquired EC markers and formed capillary-like structures mimicking paired bone marrow ECs (multiple myeloma patient-derived endothelial cells, MMECs), with major responsiveness compared to macrophages from nonactive MM, MGUS or controls. Bone marrow biopsies of active MM harbored 'mosaic' vessels, being formed by MMECs, EC-like macrophages and macrophages themselves. These figures were rare in nonactive MM and absent in MGUS or controls. Our data indicate that macrophages contribute to build neovessels in active MM through vasculogenic mimicry, and this ability proceeds parallel to progression of the plasma cell tumors. Macrophages may be a target for the MM antivascular treatment.

Chronic Vascular Inflammation in Patients With Type 2 Diabetes

Chronic vascular inflammation may play a role in the development of macrovascular complications in diabetic patients. In this study, we examine the association of endothelial expression of two inflammatory mediators, receptor for advanced glycation end product (RAGE) and monocyte chemoattractant protein-1 (MCP-1), with type 2 diabetes using novel endothelial biopsy and RT-PCR techniques. Endothelial samples are obtained from the aorta of 12 patients with type 2 diabetes and 23 control subjects who underwent cardiac catheterization for chest pain syndrome or heart transplant follow-up. Endothelial cells are purified using magnetic beads with adsorbed CD146 antibody and subjected to RT-PCR analysis of RAGE and MCP-1 transcripts. The association of RAGE and MCP-1 expression with type 2 diabetes is assessed with chi(2) test and confirmed with in vitro experiments on human aorta endothelial cells. RT-PCR reveals gene expression patterns in patient-derived endothelial cells. Strong associations are observed between induction of RAGE mRNA and diabetes (P < 0.01) and between induction of RAGE and MCP-1 transcripts (P < 0.05). Treatment of cultured human aortic endothelial cells with S100b induces the expression of MCP-1 and RAGE transcripts. Endothelial cells can be harvested during cardiac catheterization and can be characterized with respect to molecular phenotypes under the influence of both genetic and environmental factors. Induction of RAGE and MCP-1 transcripts in patients with diabetes supports a role of chronic vascular inflammation in macrovascular complications.