New soluble angiopoietin analog of Hepta-ANG1 prevents pathological vascular leakage.

Vascular leak is a key driver of organ injury in diseases, and strategies that reduce enhanced permeability and vascular inflammation are promising therapeutic targets. Activation of the angiopoietin-1 (ANG1)-Tie2 tyrosine kinase signaling pathway is an important regulator of vascular quiescence. Here we describe the design and construction of a new soluble ANG1 mimetic that is a potent activator of endothelial Tie2 in vitro and in vivo. Using a chimeric fusion strategy, we replaced the extracellular matrix (ECM) binding and oligomerization domain of ANG1 with a heptameric scaffold derived from the C-terminus of serum complement protein C4-binding protein α. We refer to this new fusion protein biologic as Hepta-ANG1, which forms a stable heptamer and induces Tie2 phosphorylation in cultured cells, and in the lung following intravenous injection of mice. Injection of Hepta-ANG1 ameliorates vascular endothelial growth factor- and lipopolysaccharide-induced vascular leakage, in keeping with the known functions of Angpt1-Tie2 in maintaining quiescent vascular stability. The new Hepta-ANG1 fusion is easy to produce and displays remarkable stability with high multimericity that can potently activate Tie2. It could be a new candidate ANG1 mimetic therapy for treatments of inflammatory vascular leak, such as acute respiratory distress syndrome and sepsis.

CD34+ enriched cell products intended for autologous transendocardial CD34+ cell transplantation release significant amounts of angiopoietin-1.

OBJECTIVES: Cell-based therapy has emerged as a promising strategy for the treatment of patients with heart failure. Increasing evidence supports the hypothesis that paracrine mechanisms mediated by soluble factors released by the cells play a predominate role in reparative processes. The aim of our study was to analyze which cytokines are released by CD34+ enriched cell products intended for autologous transendocardial CD34+ cell transplantation in patients with cardiomyopathy. MATERIAL AND METHODS: The peripheral blood CD34+ cells from 12 patients were mobilized with granulocyte colony-stimulating factor, collected via apheresis and enriched by immunoselection. RESULTS: In CD34+ enriched cell population, hematopoietic, but not mesenchymal or endothelial, progenitors were detected. Except for angiopoietin-1, other measured cytokines (FGF1, FGF2, VEGF, PDGF, IL-6, HGH, SDF-1α/CXCL12, NRG1) were not released by CD34+ cells. The average concentration of angiopoietin-1 released by 5×106 CD34+ cells grown in neutral DMEM medium was 213.6±130.0pg/mL (range: 74-448pg/mL). Angiopoietin-1 secretion correlated well with CD34+ cell's capacity for generating colonies derived from hematopoietic progenitors (Pearson's correlation=0.964; P<0.001). CONCLUSION: Our study presents angiopoietin-1 as an interesting candidate and suggests future studies to explore how its release by CD34+ cells might impact the success of autologous CD34+ cell transplantation.

Angiopoietin-1 regulates microvascular reactivity and protects the microcirculation during acute endothelial dysfunction: role of eNOS and VE-cadherin.

The growth factor angiopoietin-1 (Ang-1) plays an essential role in angiogenesis and vascular homeostasis. Nevertheless, the role of Ang-1 in regulating vascular tone and blood flow is largely unexplored. Endothelial nitric oxide synthase (eNOS) and the junctional protein VE-cadherin are part of the complex signalling cascade initiated by Ang-1 in endothelial cells. In this study, we aimed to investigate the mechanisms underlying acute effects of Ang-1 on microvascular reactivity, permeability and blood flow, and hypothesise that eNOS and VE-cadherin underpin Ang-1 mediated vascular effects that are independent of angiogenesis and proliferation. Myography of isolated microarterioles from male C3H/HeN mice (7-10 weeks) was employed to measure vascular reactivity in vitro. Microcirculatory function in vivo was evaluated by intravital microscopy and Doppler fluximetry in dorsal window chambers. Ang-1 and its stable variant MAT.Ang-1 induced a concentration-dependent vasodilation of arterioles in vitro, which was blocked with nitric oxide (NO) synthesis inhibitor l-NAME. In vivo, MAT.Ang-1 restored to control levels l-NAME induced peripheral vasoconstriction, decreased blood flow and microvascular hyperpermeability. Tissue protein expression of VE-cadherin was reduced by NOS inhibition and restored to control levels by MAT.Ang-1, whilst VE-cadherin phosphorylation was increased by l-NAME and subsequently reduced by MAT.Ang-1 administration. Moreover, MAT.Ang-1 alone did not modulate systemic levels of angiogenetic factors. Our novel findings report that Ang-1 induces arteriolar vasodilation via release of NO, suggesting that Ang-1 is an important regulator of microvascular tone. As MAT.Ang-1 ameliorates detrimental effects on the microcirculation induced by inhibition of NO synthesis and stabilizes the endothelial barrier function through VE-cadherin, we propose that this Ang-1 variant may serve as a novel therapeutic agent to protect the microcirculation against endothelial dysfunction.