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Recombinant COMP-Ang1, a chimera of angiopoietin-1 (Ang1) and a short coiled-coil domain of cartilage oligomeric matrix protein (COMP), is under consideration as a therapeutic agent capable of inducing the homing of cells with increased angiogenesis. However, the potentials of COMP-Ang1 to stimulate migration of mesenchymal stem cells (MSCs) and the associated mechanisms are not completely understood. We examined the potential of COMP-Ang1 on bone marrow (BM)-MSCs, human periodontal ligament stem cells (PDLSCs), and calvarial osteoblasts. COMP-Ang1 augmented Tie-2 induction at protein and mRNA levels and increased proliferation and expression of runt-related transcription factor 2 (Runx2), osterix , and CXCR4 in BMMSCs, but not in osteoblasts. The COMP-Ang1-mediated increases were inhibited by Tie-2 knockdown and by treating inhibitors of phosphoinositide 3-kinase (PI3K), LY294002, or p38 mitogen-activated protein kinase (MAPK), SB203580. Phosphorylation of p38 MAPK and Akt was prevented by siRNA-mediated silencing of Tie-2 . COMP-Ang1 also induced in vitro migration of BMMSCs and PDLSCs. The induced migration was suppressed by Tie-2 knockdown and by CXCR4-specific peptide antagonist or LY294002, but not by SB203580. Furthermore, COMP-Ang1 stimulated the migration of PDLSCs into calvarial defect site of rats. Collectively, our results demonstrate that COMP-Ang1-stimulated proliferation, differentiation, and migration of progenitor cells may involve the Tie-2-mediated activation of p38 MAPK and PI3K/Akt pathways. [ABSTRACT FROM AUTHOR]

Abstract: Angiopoietins 1 and 2, ligands for the receptor kinase Tie-2, have been proposed to play critical but opposing roles in vascular development. Since signaling by Tie-2 is likely affected by other endothelial cell receptors such as Flk-1, the receptor for VEGF, and cell–cell adhesion receptors PECAM1 and VE-cad, we explored their interactions in a 3D model of vasculogenesis. When murine embryoid bodies (EBs) were treated with VEGF in Matrigel in the presence or absence of Ang-1 or Ang-2 for eight days, Ang-1 abrogated vascular sprouting for treatments started at days 0 or 3. In contrast, Ang-2 greatly accelerated vascular sprouting compared to untreated EBs. These results were confirmed in a second model system where VEGF treated HUVECs were grown in Matrigel in the presence or absence of Ang-1 or Ang-2. Since vascular sprouting must be precisely controlled in the developing embryo, it is likely that cell–cell adhesion molecules play a role in sensing the density of vascular sprouts. In this respect, we have shown that PECAM1 and CEACAM1 play essential roles in vascular sprouting. We now show that PECAM1 is associated with Tie-2, becomes phosphorylated on its ITIMs, and recruits the inhibitory phosphatases SHP-1 and SHP-2. In addition, PECAM1 is associated with VE-cad and may similarly regulate its signaling via recruitment of SHP-1/2. [Copyright &y& Elsevier]

Abstract: Introduction: Endothelial activation leading to vascular barrier breakdown plays an essential role in the pathophysiology of multiple-organ dysfunction syndrome (MODS) in sepsis. Increasing evidence suggests that the function of the vessel-protective factor Angiopoietin-1 (Ang-1), a ligand of the endothelial-specific Tie2 receptor, is inhibited by its antagonist Angiopoietin-2 (Ang-2) during sepsis. In order to reverse the effects of the sepsis-induced suppression of Ang-1 and elevation of Ang-2 we aimed to investigate whether an intravenous injection of recombinant human (rh) Ang-1 protects against MODS in murine sepsis. Methods: Polymicrobiological abdominal sepsis was induced by cecal ligation and puncture (CLP). Mice were treated with either 1μg of intravenous rhAng-1 or control buffer immediately after CLP induction and every 8h thereafter. Sham-operated animals served as time-matched controls. Results: Compared to buffer-treated controls, rhAng-1 treated septic mice showed significant improvements in several hematologic and biochemical indicators of MODS. Moreover, rhAng-1 stabilized endothelial barrier function, as evidenced by inhibition of protein leakage from lung capillaries into the alveolar compartment. Histological analysis revealed that rhAng-1 treatment attenuated leukocyte infiltration in lungs and kidneys of septic mice, probably due to reduced endothelial adhesion molecule expression in rhAng-1 treated mice. Finally, the protective effects of rhAng-1 treatment were reflected by an improved survival time in a lethal CLP model. Conclusions: In a clinically relevant murine sepsis model, intravenous rhAng-1 treatment alone is sufficient to significantly improve a variety of sepsis-associated organ dysfunctions and survival time, most likely by preserving endothelial barrier function. Further studies are needed to pave the road for clinical application of this therapy concept. [Copyright &y& Elsevier]

Abstract: Molecular mechanisms regulating the remodeling of the lymphatic vasculature from an immature plexus of vessels to a hierarchal network of initial and collecting lymphatics are not well understood. One gene thought to be important for this process is Angiopoietin-2 (Ang-2). Ang2−/− mice have previously been reported to exhibit an abnormal lymphatic phenotype but the precise nature of the lymphatic defects and the underlying mechanisms have yet to be defined. Here we demonstrate by whole-mount immunofluorescence staining of ear skin and mesentery that lymphatic vessels in Ang2−/− mice fail to mature and do not exhibit a collecting vessel phenotype. Furthermore, dermal lymphatic vessels in Ang2−/− pups prematurely recruit smooth muscle cells and do not undergo proper postnatal remodeling. In contrast, Ang2 knock-out Ang1 knock-in mice do develop a hierarchal lymphatic vasculature, suggesting that activation of Tie-2 is required for normal lymphatic development. Taken together, this work pinpoints a specific lymphatic defect of Ang2−/− mice and further defines the sequential steps in lymphatic vessel remodeling. [Copyright &y& Elsevier]

Abstract: There are limited and conflicting studies investigating the role of the angiopoietin family in human thyroid cancer development and progression. We have investigated cytokines angiopoietin-1, -2 and their receptor (Tie-2), known to be involved in angiogenesis, in the serum of 52 thyroid cancer patients (21 cases of papillary cancers, PTC; 8 follicular cancers, FTC; 12 medullary cancers, MTC and 11 anaplastic cancers, ATC), using ELISA assays. The control consisted of 27 healthy volunteers. Statistically significant lower concentrations of Ang-1 were found in patients with thyroid cancers as compared with the control (p <0.003). The levels of Ang-2 and Tie-2 did not differ significantly between thyroid cancer patients and control. We have also compared the results of Ang-1, Ang-2, and Tie-2 determinations obtained in different histopathological subgroups of cancer patients. These results revealed lower Ang-1 concentrations in ATC (p <0.05), MTC (p <0.02), FTC (p <0.01) and in PTC patients (p <0.05) than control. We have also observed lower Ang-2 concentration in PTC patients (p <0.03) and Tie-2 in FTC patients (p <0.02 ) in comparison to controls. In conclusion, the Angs/Tie-2 system dysfunction may play an important role in thyroid cancerogenesis and decreased concentration of Ang-1 in serum can be a useful additional biomarker for the presence of thyroid cancers. [Copyright &y& Elsevier]

Phenotypically and functionally different types of microvascular endothelial cells (MVECs) derived from the developing corpus luteum were isolated and characterized by our group. We investigated whether these cytokeratin-positive (CK+) and cytokeratin-negative (CK−) MVECs differed in the expression of angiogenic factors and their regulation under hypoxia. Using quantitative RT-PCR, VEGF and its receptors, Flk-1 and Flt-1, were detected in CK− MVECs. The mRNA expression of Flk-1 mRNA was 100 times as high as that of Flt-1 mRNA. CK+ MVECs expressed VEGF and Flt-1 mRNA, but were devoid of Flk-1 transcripts. No Ang-1 mRNA was demonstrated in either cell type, and Ang-2 mRNA was found only in CK− MVECs. Tie-2 mRNA was detected in both MVEC types, but levels were 150 times as high in CK− MVECs as in CK+ MVECs. mRNA of hypoxia-inducible factors Hif-1α and Hif-1β was expressed in both MVEC types. After hypoxia, neither VEGF, nor Flk-1, nor Tie-2 mRNA expression was altered in either MVEC type. Flt-1 expression and Ang-2 mRNA expression were significantly increased at about 2.5-fold (P < 0.05) in CK− MVECs, but not in CK+ MVECs. Our study demonstrates the varying expression and regulation of angiogenesis-related factors and receptors in phenotypically different MVEC types. [Copyright &y& Elsevier]

Spontaneously hypertensive stroke-prone rats (SHRSP), a model for genetic stroke susceptibility, suffer spontaneous stroke and enhanced injury after experimental stroke, in part due to abnormal cerebrovascular development. We hypothesized that angiopoietin system genes in SHRSP may follow unique patterns of expression after experimentally induced stroke. SHRSP, hypertensive control rats (SHR), and normotensive controls (WKY) were subjected to experimental middle cerebral artery occlusion, and brain RNA was analyzed for expression of angiogenic genes. Expression of angiopoietin-2 increased after stroke in all rat strains and was significantly enhanced in SHRSP compared with control strains. In addition, expression of angiopoietin-1 and the angiopoietin receptor dropped markedly after stroke in SHRSP animals, but was not different after ischemia in SHR and WKY strains. Thus, the SHRSP brain elaborates a unique and specific pattern of angiopoietin system gene expression after stroke which may underlie stroke susceptibility of these rats. [Copyright &y& Elsevier]