IntroductionBone marrow-derived and circulating hematopoietic progenitor cells have been shown to contribute to cardiovascular tissue regeneration. 1,2 CD34 ϩ hematopoietic progenitor cells were widely and successfully used in different transplantation models, including myocardial regeneration. 3 A subset of circulating CD34 ϩ cellsexpressing markers such as CD133 and vascular endothelial growth factor (VEGF) receptor-2 (flk1)-are endothelial progenitor cells (EPCs) with the ability to differentiate into mature endothelial cells, thus contributing to re-endothelialization and neovascularization. 4 In contrast, the ability of hematopoietic cells to transdifferentiate into functional cardiomyocytes is controversial. [5][6][7][8] Moreover, enhanced angiogenesis in response to progenitor cells may be influenced mainly by the release of growth factors rather than by cellular differentiation. 9 CCN1 (formerly known as CYR61) is a cysteine-rich heparinbinding protein that is encoded by an immediate early gene and belongs to the novel CCN gene family (connective tissue growth factor [CTGF], cysteine-rich angiogenic protein 61 [CYR61], and nephroblastoma overexpressed [Nov]). 10 The expression of CCN1 is rapidly induced in response to growth factors. 11 CCN1 is expressed by all types of vascular cells, is associated with the extracellular matrix (ECM) and mediates cell adhesion, migration, proliferation and neovascularization through cell type-specific binding mainly to ␣ 6 ␤ 1 , ␣ M ␤ 2 , and ␣ V ␤ 3 integrins. 10,[12][13][14] It is noteworthy that CCN1-deficient mice suffer embryonic death as a result of placental vascular insufficiency and compromised vessel integrity because of impaired vessel bifurcations and impaired VEGF-C expression, establishing CCN1 as a novel and essential regulator of vascular development. 15 In addition, it has been shown recently that CCN1 is critically involved in cardiac development. 16 Besides its ability to promote tumor growth, 17 we recently reported that enhanced CCN1 expression is associated with several cardiovascular pathophysiologic settings, such as atherosclerotic plaque formation, 18 enhanced vascular mechanical stretch, 19 cardiac pressure overload, and ischemic cardiomyopathy. 20 Recent evidence suggested that CCN1 expression is induced in various cardiovascular disorders, suggesting a role for CCN1 in cellular and tissue self-renewal, potentially involving circulating progenitor cells. Because the impact of CCN1 on progenitor cells is largely unknown, we used circulating human bone marrow-derived CD34 ϩ cells to study CCN1 effects on migration, release of growth factors, endothelial proliferation and neovascularization, which are important for cardiovascular regeneration. Materials and methods ReagentsAll chemicals were obtained from Sigma (Taufkirchen, Germany) unless otherwise specified. All cell culture plates were from TTP (Trasadingen, Switzerland). RGD peptides (Arg-Gly-Asp) and GRGDSP peptides (GlyArg-Gly-Asp-Ser-Pro) were from Bachem (Weil am Rhein, Germany). Human ...

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We have investigated the role of tyrosine phosphorylation of the cyclin-dependent kinase (cdk) inhibitor p27 Kip1 using the acute promyelocytic leukemia cell line NB4 together with granulocyte colonystimulating factor (G-CSF). Short-term G-CSF stimulation resulted in a rapid tyrosine dephosphorylation of p27 Kip1 accompanied by a change in its binding preferences to cdks. On G-CSF stimulation, p27 Kip1 dissociated from cdk4 and associated with cdk2. Binding assays with recombinant p27 Kip1 confirmed that tyrosine-phosphorylated p27 Kip1 preferentially bound to cdk4, whereas unphosphorylated protein preferentially associated with cdk2. In addition, studies with p27 Kip1 point mutations revealed a decisive role of Tyr88 and Tyr89 in binding to cdk4. Furthermore, phosphorylation of Tyr88 and Tyr89 was accompanied by strong nuclear translocation of p27 Kip1 .Taken together, this report provides the first evidence that tyrosine phosphorylation of p27 Kip1 plays a crucial role in binding to cdks and its subcellular localization. Moreover, both effects are mediated by application of G-CSF. (Blood.