The neoplastic production of the insulin-like growth factor binding protein (IGFBP)-2 often correlates with tumor malignancy and aggressiveness. Since IGFBP-2 contains an RGD motif in its C-terminus, it was hypothesized that this protein may act independently of IGF on tumor cells through integrins. To investigate this, integrin binding, intracellular signaling and the impact of IGFBP-2 on cell adhesion and proliferation were examined in two tumor cell lines. In tracer displacement studies, up to 30% of the added 125 I-hIGFBP-2 specifically bound to the cells. Bound 125 I-hIGFBP-2 was reversibly displaced by IGFBP-2, IGFBP-1 and RGD-(Gly-Arg-Asp)-containing peptides, but not by IGFBP-3, -4, -5, -6 and RGE-(Gly-Arg-Glu)-containing peptides. Blocking with antibodies directed against different integrins and with fibronectin demonstrated that IGFBP-2 cell surface binding is specific for 5 1-integrin. Incubation of IGFBP-2 with equimolar quantities of IGF-I and IGF-II annihilated RGD-specific binding. IGFBP-2 binding at the cell surface led to dephosphorylation of the focal adhesion-kinase (FAK) of up to 37% (P<0·01), and of the p42/44 MAP-kinases of up to 40% (P<0·01). In addition, IGFBP-2 promoted de-adhesion of the cells dose-dependently by up to 30% (P<0·05), and reduced proliferation by 24% (P<0·01). Since one of the cell lines used does not express a functional IGF-I receptor, these data demonstrate that IGFBP-2 can act in an IGF-independent manner, at least in part by an interaction with 5 1-integrin.

IGF binding proteins (IGFBPs) modulate IGF cellular bioavailability and may directly regulate tumor growth and invasion. We have previously shown that IGFBP-2 binds and localizes IGF-I to the pericellular matrix and have provided some evidence suggesting that the heparin binding domain (HBD) or the arginine-glycine-aspartic acid (RGD) integrin binding motif may be involved in these interactions. However, the precise mechanisms involved remain to be elucidated. We therefore mutated the HBD or RGD sequence of IGFBP-2 and investigated consequent effects on extracellular matrix (ECM) binding, IGF-induced proliferation, and migration of neuroblastoma cells. IGFBP-2 and its arginine-glycine-glutamic acid (RGE) mutant similarly bound ECM components, whereas binding of mutant HBD-IGFBP-2 to each of the ECM substrates was markedly reduced by 70-80% (P < 0.05). IGF-I (100 ng/ml) increased incorporation of 3H-thymidine in neuroblastoma SK-N-SHEP cells by approximately 30%, an effect blunted by exogenously added native or either mutant IGFBP-2. Overexpression of IGFBP-2 and its RGE mutant potently promoted SHEP cell proliferation (5-fold), whereas SHEP cell proliferation was negligible when HBD-IGFBP-2 was overexpressed. Addition or overexpression of IGFBP-2 and its RGE mutant potently (P < 0.05) enhanced SHEP cell migration/invasion through the ECM. However, overexpression of the HBD-IGFBP-2 mutant potently inhibited (50-60%) SHEP cell invasion through ECM. Thus, IGFBP-2, which binds to the ECM, enhances proliferation and metastatic behavior of neuroblastoma cells, functions that directly or indirectly use the HBD but not the integrin binding sequence. Our novel findings thus point to a key role for the HBD of IGFBP-2 in the control and regulation of neuroblastoma growth and invasion.

Background-Bartonella species are the only known bacterial pathogens causing vasculoproliferative disorders in humans (bacillary angiomatosis [BA]). Cellular and bacterial pathogenetic mechanisms underlying the induction of BA are largely unknown. Methods and Results-Activation of hypoxia-inducible factor-1 (HIF-1), the key transcription factor involved in angiogenesis, was detected in Bartonella henselae-infected host cells in vitro by immunofluorescence, Western blotting, electrophoretic mobility shift, and reporter gene assays and by immunohistochemistry in BA tissue lesions in vivo. Gene microarray analysis revealed that a B henselae infection resulted in the activation of genes typical for the cellular response to hypoxia. HIF-1 was essential for B henselae-induced expression of vascular endothelial growth factor as shown by inhibition with the use of HIF-1-specific short-interfering RNA. Moreover, infection with B henselae resulted in increased oxygen consumption, cellular hypoxia, and decreased ATP levels in host cells. Infection with a pilus-negative variant of B henselae did not lead to cellular hypoxia or activation of HIF-1 or vascular endothelial growth factor secretion, suggesting a crucial role of this bacterial surface protein in the angiogenic reprogramming of the host cells. Key Words: angiomatosis, bacillary Ⅲ Bartonella henselae Ⅲ angiogenesis Ⅲ HIF-1 protein Ⅲ hypoxia A ngiogenesis is a multistep process resulting in the formation of new blood vessels from preexisting vasculature. Newly formed vessels supply oxygen and nutrients to growing tumors and are necessary for tumor progression and metastasis. 1 Angiogenesis is also a component of various cardiovascular and inflammatory diseases. 2 Hypoxiainducible factor-1 (HIF-1) is a key transcription factor for the induction of angiogenic growth factors that adjust the vascular oxygen supply to tissue metabolic demands. 3,4 Of the many genes induced by HIF-1, vascular endothelial growth factor (VEGF) plays a critical role in triggering angiogenesis as the major hypoxia-inducible mitogen for endothelial cells. 5 Interestingly, human herpesvirus-8 (HHV-8) and several Bartonella species induce angiogenesis in humans. causes the vasculoproliferative disorder Kaposi's sarcoma, 6 which has a high frequency among immunocompromised patients, such as those infected with HIV. HHV-8 -infected cells express VEGF on HIF-1 activation, 7 and this mechanism was implicated in endothelial cell proliferation in the lesions. 8 Bartonella henselae and B quintana are the etiologic agents of bacillary angiomatosis (BA) and bacillary peliosis (BP), which are histologically characterized as lobulated proliferation of mainly capillary-sized vessels and predominantly affect HIV patients. 9 These slow-growing bacteria are facultative intracellular pathogens that, like HHV-8, also induce VEGF in host cells in vitro and in BA or BP lesions of patients. 10 Endothelial cells are one presumed habitat of Bartonella. 11 Dissecting the angioproliferative strategies used Received...

IntroductionHuman multipotent mesenchymal stromal cells (MSCs) can be readily expanded from bone marrow aspirates by ex vivo culture and characterized by immunophenotype and plasticity. 1 These cells maintain plasticity for classical mesenchymal tissues and others to a lesser extent. 2 Moreover, expanded MSCs display immunomodulatory properties (ie, inhibition of antigen presentation, maturation of dendritic cells, cytotoxicity of natural killer [NK] cells, and proliferation of peripheral blood mononuclear cells [PBMCs]) in response to polyclonal stimulation or in alloresponses. [3][4][5] These immunoregulatory features of MSCs led to their clinical application in pediatric and adult patients. [6][7][8] However, the identity of soluble factors necessary for the inhibition exerted by MSCs remains unclear. Particularly, the type of mitogens used for stimulation of PBMCs may have consequences for the predominant inhibitory pathway engaged by MSC-derived factors. 9 Among others, interferon-␥ (IFN␥) has been proposed as a prime candidate for initiation of MSC-mediated immune regulation. 9-12 A wellknown IFN␥-inducible gene in this context is indoleamine 2,3-dioxygenase (IDO). 13 IDO initiates degradation of the essential amino acid tryptophan and, in concert with other enzymes, might possibly give rise to metabolites of tryptophan, which inhibit PBMC activation. Blocking this enzyme in cocultures of MSCs and HLA-mismatched PBMCs almost completely abrogated the inhibitory effects of MSCs on the proliferating allogeneic PBMCs. 11,12 However, other researchers could not find an effect of IDO in their experimental system. 14 Moreover, IFN␥ itself has also been shown to turn MSCs into antigen-presenting cells. 15 Some antiproliferative effects are mediated by insulin-like growth factor-binding proteins (IGFBPs), in particular IGFBP3. 16 This family of proteins has been involved in growth inhibition in several experimental settings relevant for the system studied here. 17,18 In order to address the relevance of IFN␥-induced IDO expression for the inhibitory function of human MSCs in alloresponses, we analyzed human IFN␥ receptor 1 (R1)-deficient MSCs. Materials and methodsThese studies were approved by the Institutional Review Board (IRB) of the University Hospital Tübingen, Germany. Cell cultureAfter informed consent was obtained in accordance with the Declaration of Helsinki, cryoconserved bone marrow aspirates from a boy with a frameshift mutation in the IFN␥-R1 subunit of the IFN␥ receptor were thawed. Control specimens of MSCs were derived from excess material of standard bone marrow biopsies in children treated for leukemia (IRB approval 241/2005V). MSC cultures were established as reported earlier. 19 The deletion of a T at position 523 in the IFN␥-R1 subunit was verified by conventional sequence analysis of a reverse transcriptase-polymerase chain reaction (RT-PCR) product (data not shown). Plasticity assays and flow cytometry were carried out as described elsewhere (data not shown). 20 Proliferation assaysHLA-mis...