D. Rudenko

Nocturnal hemodialysis is associated with restoration of early-outgrowth endothelial progenitor-like cell function.

BACKGROUND AND OBJECTIVES Angiogenesis is a key response to tissue ischemia that may be impaired by uremia. Although early-outgrowth endothelial progenitor-like cells promote angiogenesis in the setting of normal renal function, cells from uremic patients are dysfunctional. When compared with conventional hemodialysis, it was hypothesized that nocturnal hemodialysis would improve the in vivo angiogenic activity of these cells in a well described model of ischemic vascular disease. DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENTS Early-outgrowth endothelial progenitor-like cells were cultured from healthy controls (n = 5) and age- and gender-matched conventional hemodialysis (12 h/wk, n = 10) and nocturnal hemodialysis (30 to 50 h/wk, n = 9) patients. Cells (5 × 10(5)) or saline were injected into the ischemic hindlimb of athymic nude rats 1 day after left common iliac artery ligation. RESULTS Although conventional dialysis cell injection had no effect versus saline, nocturnal hemodialysis and healthy control cell injection significantly improved ischemic hindlimb perfusion and capillary density. Nocturnal hemodialysis cell injection was also associated with significant increases in endogenous angiopoietin 1 expression in the ischemic hindlimb compared with saline and conventional dialysis cell injection. CONCLUSIONS In contrast to a conventional dialytic regimen, nocturnal hemodialysis is associated with a significantly improved ability of early-outgrowth endothelial progenitor-like cells to promote angiogenesis and thus restore perfusion in a model of ischemic vascular disease.

Novel regulatory role of neuropilin-1 in endothelial-to-mesenchymal transition and fibrosis in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an intense fibrotic reaction termed tumor desmoplasia, which is in part responsible for its aggressiveness. Endothelial cells have been shown to display cellular plasticity in the form of endothelial-to-mesenchymal transition (EndMT) that serves as an important source of fibroblasts in pathological disorders, including cancer. Angiogenic co-receptor, neuropilin-1 (NRP-1) actively binds TGFβ1, the primary mediator of EndMT and is involved in oncogenic processes like epithelial-to-mesenchymal transition (EMT). NRP-1 and TGFβ1 signaling have been shown to be aberrantly up-regulated in PDAC. We report herein a positive correlation between NRP-1 levels, EndMT and fibrosis in human PDAC xenografts. Loss of NRP-1 in HUVECs limited TGFβ1-induced EndMT as demonstrated by gain of endothelial and loss of mesenchymal markers, while maintaining endothelial cell architecture. Knockdown of NRP-1 down-regulated TGFβ canonical signaling (pSMAD2) and associated pro-fibrotic genes. Overexpression of NRP-1 exacerbated TGFβ1-induced EndMT and up-regulated TGFβ signaling and expression of pro-fibrotic genes. In vivo, loss of NRP-1 attenuated tumor perfusion and size, accompanied by reduction in EndMT and fibrosis. This study defines a previously unrecognized role of NRP-1 in regulating TGFβ1-induced EndMT and fibrosis, and advocates NRP-1 as a therapeutic target to reduce tumor fibrosis and PDAC progression.

Perlecan Heparan Sulfate Proteoglycan Is a Critical Determinant of Angiogenesis in Response to Mouse Hind-Limb Ischemia

BACKGROUND Perlecan is a heparan sulfate proteoglycan (HSPG) constituent of the extracellular matrix with roles in cell growth, differentiation, and angiogenesis. The role of the HS side chains in regulating in vivo angiogenesis after hind-limb ischemia is unknown. METHODS Heparan sulfate (HS)-deficient perlecan (Hspg2(Δ3/Δ3)) mice (n = 35), containing normal perlecan core protein but deficient in HS side chains, and wild-type (n = 33) littermates underwent surgical induction of hind-limb ischemia. Laser Doppler perfusion imaging (LDPI) and contrast-enhanced ultrasonography (CEU) provided serial assessment of hind-limb perfusion. Harvested muscles underwent immunostaining for endothelial cell density (CD31), real-time reverse transcription polymerase chain reaction RT-PCR for vascular endothelial growth factor (VEGF) mRNA expression and western blot analysis for VEGF and fibroblast growth factor (FGF)2 protein expression at days 2 and 28. RESULTS Serial LDPI showed significantly greater perfusion recovery in ischemic limbs of wild-type compared with Hspg2(Δ3/Δ3) mice. CEU showed that normalized microvascular perfusion was increased in wild-type compared with Hspg2(Δ3/Δ3) mice at day 28 (0.67 ± 0.12 vs 0.26 ± 0.08; P = 0.001). CD31-positive cell counts were significantly higher in wild-type compared with Hspg2(Δ3/Δ3) mice on day 28 (122 ± 30 cells vs 84 ± 34 cells per high-power field [HPF]; P < 0.05). Endogenous VEGF mRNA expression (P < 0.05) and VEGF protein expression (P < 0.002) were significantly decreased in the ischemic limbs of Hspg2(Δ3/Δ3) mice compared with wild-type mice at day 2 and day 28, respectively. FGF2 protein expression showed no significant differences. CONCLUSIONS These results suggest that the HS side chains in perlecan are important mediators of the angiogenic response to ischemia through a mechanism that involves upregulation of VEGF expression.

EXPLORING THE BENEFICIAL EFFECTS OF S100A6 OVEREXPRESSION IN ACUTE INFARCTION-REPERFUSION: PREVENTION OF FIBROSIS, ENHANCED ANGIOGENESIS AND ATTENUATION OF MYOCARDIAL HYPERTROPHY

BACKGROUND: An important issue limiting cell therapy in the treatment of infarcted heart is the extensive loss of transplanted cells. Manipulation of the cells prior to the transplantation has been proposed in order to improve their biologic and functional properties through enhancement of cell survival, homing, retention, differentiation or proliferation. METHODS: The objective was to optimize human stem cells using a novel strategy of preconditioning with oxytocin (OT) and their preparation for clinical applications with improved viability and therapeutic function. Human mesenchymal stem cells (hMSC) were treated with OT from 0.1 nM to 1 mM (range of physiological and pharmacological doses) with either short bursts or continuously to determine the conditions with highest responsiveness. OT signalling pathways were investigated according to phosphorylation of proteins or activation of specific effectors. The LIVE/DEAD assay kit assessed cellular viability. Multilineage differentiation was confirmed by in vitro assays for tri-potentiality based on adipogenesis (oil red staining) and osteogenesis (alizarin staining). Phenotype and surface markers were evaluated by standard fluorescence-activated cell sorting techniques to validate changes in protein expression following OT treatment at different time points/conditions. RESULTS: OT receptor mRNAwas found in hMSC by RT-PCR and proteins byWestern blotting.OT rapidly (5-15minutes) and significantly increased phosphorylation of PI3K/AKT (1,6 fold) and ERK1/2 (3,0 folds) all P<0.05, both pathways involved in cell survival. OT improved survival of hMSC exposed to hypoxia and serum starvation culture conditions mimicking myocardial infarct (P<0.05). OT treatment did not alter differentiation potential as demonstrated by adipogenesis and osteogenesis. In fact, results showed that continuous OT treatment promoted adipogenic differentiation (p<0.05). Similarly, OT continuous treatment demonstrated favorable effects on osteogenesis.Multipotent hMSCwere negative for: CD14,CD34, CD45 andCD133; and positive for: CD44, CD73, CD29, CD166 and CD105; OT treatment did not affect the phenotype of hMSC up to the second passage. OT treated hMSC maintained their growth capacity for cell expansion in vitro. CONCLUSION: Human MSC express OT-receptor functioning with a rapid and strong response to OT exposure as demonstrated by activation of survival signaling pathways, enhancing the capacity of stem cells to resist ischemic conditions. Besides having prosurvival and cytoprotective effects, OT preconditioning supports the cells to maintain their stemness and promote their differentiation and proliferation potential for post-engraftment function. Ex vivo cellular preconditioning with OT represents an attractive strategy to improve hMSC survival and reparative potential without the need for genetic manipulation. FRQS, HSF, Montreal University

526 Targeted Delivery of MicroRNA-126 Improves Perfusion in Chronic Hindlimb Ischemia

were treated with a MMP inhibitor, doxycycline, post-surgery. Doxycycline prevented the reduction in plaque size in the AVF group (p 0.05). Plaque MMP-9 expression was increased in AVF vs. control and sham (10.8% 1.4, 3.9% 0.7, 4.6% 1.1, respectively; p 0.0001). MMP-3 expression was increased in AVF vs. control (9.5% 1.5, 5.0% 0.8, respectively; p 0.05). MMP-2 expression was decreased in AVF vs. sham (5.7% 0.5, 11.3% 1.6, respectively; p 0.05). In addition, we overexpressed the tissue inhibitor of metalloproteinase (TIMP-1) in mice before surgery. The increased Timp-1 prevented the reduction in plaque size in the AVF group (p 0.05). All groups had comparable body weight, blood pressure, lipid levels and hematological parameters. There was also no difference in smooth muscle cell and collagen content between groups. To further understand the role of MMPs, we devised an endothelial cell (EC)-macrophage co-culture system where the ECs were exposed to high (15 dynes/cm) or low (0.5 dynes/cm) shear stress. Macrophage migration exposed to EC effluent was performed using a scratch assay. There was a 2.6 fold increase in migration in the high flow condition compared to the low (p 0.05). When the cells were exposed to the MMP inhibitor drug, GM6001, migration was decreased 2 fold (p 0.05). CONCLUSION: Our findings suggest that MMPs play a role in shear stress inducedplaque regression. Shear stress acting on ECs may influence the cells within the plaque by increasing MMP expression allowing for better macrophage mobility and egress which are characteristics of regressing plaques.