IntroductionHuman mesenchymal stem cells (hMSCs) from bone marrow are characterized by their ability of self-renewal paired with the capacity to differentiate into diverse mesodermal cell types such as osteoblasts, chondrocytes, and adipocytes. 1,2 Moreover, hMSCs were shown to give rise to cells beyond the germ layers with visceral mesoderm, neuroectoderm, or endoderm characteristics. [2][3][4] Additional functions have been reported for hMSCs in providing cytokine and growth factor support for the expansion of hematopoetic 5 and embryonic stem cells, 6 or by playing an immunomodulatory role. 7 One of the most remarkable but least understood findings is the ability of hMSCs to migrate from bone marrow or peripheral blood into damaged tissues. Transplantation experiments in animals and patients demonstrated that mesenchymal stem cells migrate to sites of injury, where they enhance wound healing, 8 support tissue regeneration following myocardial infarction, 9 home to and promote the restoration of bone marrow microenvironment after damage by myeloablative chemotherapy, 10 or help to overcome the molecular defect in children with osteogenesis imperfecta. 11 Another interesting observation is that systemically delivered hMSCs are mobilized to and integrate into tumor tissue. 12 Taken together, these exciting features have rendered hMSCs a promising tool for tissue engineering 13 as well as multiple cell and gene therapy strategies. [14][15][16] Detailed studies have demonstrated that homing of hematopoetic stem cells from blood into bone marrow or their mobilization from bone marrow into blood and tissues is mainly controlled by cytokines/chemokines, adhesion molecules, and proteolytic enzymes. [17][18][19] However, little is known about the molecular mechanisms regulating cell movement and relocalization in hMSCs.A key requirement for cells to reach distant target sites is the ability to traverse the protein fibers of the extracellular matrix (ECM) which is present between cells of all tissue types. 20 Basement membranes represent a specialized form of the ECM that separate epithelium or endothelium from stroma by a dense layer of ECM. To overcome these matrix barriers, migrating cells require specific proteolytic enzymes. Besides some serine-and cysteineproteinases, in particular the matrix metalloproteinases (MMPs) consisting of more than 24 zinc-dependent endopeptidases, are capable of degrading ECM components. Consequently, MMPs are found to be involved in various physiologic and pathologic processes. 21 The 2 gelatinases, MMP-2 and MMP-9, preferentially cleave denatured collagens (gelatin), laminin, and collagen type IV as the major constituent of basement membranes. 20,21 Biosynthesis and activity of the gelatinases are associated with the invasive capacity of various cell types such as leukocytes, endothelial cells, and metastasizing tumor cells. 22-24 MMP-2 and MMP-9 are secreted from the cells as latent zymogens which are rapidly complexed by their specific endogenous inhibitors, the tissue inhibitor of ...

The tissue inhibitors of metalloproteinases (TIMPs) are well recognized for their role in extracellular matrix remodeling by controlling the activity of matrix metalloproteinases (MMPs). Independent of MMP inhibition, TIMPs act as signaling molecules with cytokine-like activities thereby influencing various biological processes including cell growth, apoptosis, differentiation, angiogenesis, and oncogenesis. Recent studies on TIMP-1's cytokine functions have identified complex regulatory networks involving a specific surface receptor and subsequent signaling pathways including miRNA-mediated posttranscriptional regulation of gene expression that ultimately control the fate and behavior of the cells. The present review summarizes the current knowledge on TIMP-1 as a cytokine modulator of cell functions, outlines recent progress in defining molecular pathways that transmit TIMP-1 signals from the cell periphery into the nucleus, and discusses TIMP-1's role as a cytokine in the pathophysiology of cancer and other human diseases.

Background The CXCL12/CXCR4 chemokine ligand/receptor axis controls (progenitor) cell homeostasis and trafficking. So far, an atheroprotective role of CXCL12/CXCR4 has only been implied through pharmacological intervention, particularly as the somatic deletion of the CXCR4 gene in mice is embryonically lethal. Moreover, cell-specific effects of CXCR4 in the arterial wall and underlying mechanisms remain elusive, prompting us to investigate the relevance of CXCR4 in vascular cell types for atheroprotection. Methods We examined the role of vascular CXCR4 in atherosclerosis and plaque composition by inducing an endothelial cell (EC, BmxCreERT2-driven)-specific or smooth muscle cell (SMC, SmmhcCreERT2- or TaglnCre-driven)-specific-deficiency of CXCR4 in an apolipoprotein E-deficient mouse model. To identify underlying mechanisms for effects of CXCR4, we studied endothelial permeability, intravital leukocyte adhesion, involvement of the Akt/WNT/β-catenin signaling pathway and relevant phosphatases in VE-cadherin expression and function, vascular tone in aortic rings, cholesterol efflux from macrophages, and expression of SMC phenotypic markers. Finally, we analyzed associations of common genetic variants at the CXCR4 locus with the risk for coronary heart disease, along with CXCR4 transcript expression in human atherosclerotic plaques. Results The cell-specific deletion of CXCR4 in arterial ECs (n=12-15) or SMCs (n=13-24) markedly increased atherosclerotic lesion formation in hyperlipidemic mice. Endothelial barrier function was promoted by CXCL12/CXCR4, which triggered Akt/WNT/β-catenin-signaling to drive VE-cadherin expression and stabilized junctional VE-cadherin complexes through associated phosphatases. Conversely, endothelial CXCR4-deficiency caused arterial leakage and inflammatory leukocyte recruitment during atherogenesis. In arterial SMCs, CXCR4 sustained normal vascular reactivity and contractile responses, whereas CXCR4-deficiency favored a synthetic phenotype, the occurrence of macrophage-like SMCs in the lesions, and impaired cholesterol efflux. Regression analyses in humans (n=259,796) identified the C-allele at rs2322864 within the CXCR4 locus to be associated with increased risk for coronary heart disease. In line, C/C risk genotype carriers showed reduced CXCR4 expression in carotid artery plaques (n=188), which was furthermore associated with symptomatic disease. Conclusions Our data clearly establish that vascular CXCR4 limits atherosclerosis by maintaining arterial integrity, preserving endothelial barrier function, and a normal contractile SMC phenotype. Enhancing these beneficial functions of arterial CXCR4 by selective modulators might open novel therapeutic options in atherosclerosis.

Human mesenchymal stem cells (hMSCs) exhibit the potential to contribute to a wide variety of endogenous organ tissue repair. However, the signals governing hMSC mobilization out of the bone marrow, release into the bloodstream, and migration/invasion into the target tissue are largely unknown. Since canonical Wnt signaling regulates not only tumor but also various stem cell attributes, we hypothesized that this signal transduction pathway might also be involved in governing the transmigration of hMSCs through human extracellular matrix (ECM). Stimulation of hMSCs with recombinant Wnt3a or LiCl resulted in the accumulation of the transcriptional activator ␤-catenin, its translocation into the nucleus, and the upregulation of typical Wnt target genes such as cyclin D1 and membrane-type matrix metalloproteinase-1 (MT1-MMP). Moreover, both stimuli significantly enhanced hMSC proliferation up to 40%. In addition, an increase of more than twofold in the ability of hMSCs to transmigrate through Transwell filters coated with human ECM was observed. In a reverse approach, Wnt signaling in hMSCs was inhibited by knocking down the expression of either ␤-catenin or low-density lipoprotein receptor-related protein 5 using RNA interference technology. These inhibition strategies resulted in downregulation of the Wnt target genes cyclin D1 and MT1-MMP, in a reduced proliferation rate, and in a strikingly diminished invasion capacity (64% and 52%). Taken together, this study provides for the first time decisive evidence that canonical Wnt signaling is critically involved in the regulation of the proliferation, as well as of the migration/invasion capacity of hMSCs, representing essential stem cell features indispensable during tissue regeneration processes.

Tissue inhibitor of metalloproteinases 1 (TIMP-1) is a matrix metalloproteinase (MMP)-independent regulator of growth and apoptosis in various cell types. The receptors and signaling pathways that are involved in the growth factor activities of TIMP-1, however, remain controversial. RNA interference of TIMP-1 has revealed that endogenous TIMP-1 suppresses the proliferation, metabolic activity, and osteogenic differentiation capacity of human mesenchymal stem cells (hMSCs). The knockdown of TIMP-1 in hMSCs activated the Wnt/β-catenin signaling pathway as indicated by the increased stability and nuclear localization of β-catenin in TIMP-1-deficient hMSCs. Moreover, TIMP-1 knockdown cells exhibited enhanced β-catenin transcriptional activity, determined by Wnt/β-catenin target gene expression analysis and a luciferase-based β-cateninactivated reporter assay. An analysis of a mutant form of TIMP-1 that cannot inhibit MMP indicated that the effect of TIMP-1 on β-catenin signaling is MMP independent. Furthermore, the binding of CD63 to TIMP-1 on the surface of hMSCs is essential for the TIMP-1-mediated effects on Wnt/β-catenin signaling. An array analysis of microRNAs (miRNAs) and transfection studies with specific miRNA inhibitors and mimics showed that let-7f miRNA is crucial for the regulation of β-catenin activity and osteogenic differentiation by TIMP-1. Let-7f was up-regulated in TIMP-1-depleted hMSCs and demonstrably reduced axin 2, an antagonist of β-catenin stability. Our results demonstrate that TIMP-1 is a direct regulator of hMSC functions and reveal a regulatory network in which let-7f modulates Wnt/β-catenin activity.plasticity | osteogenesis | canonical Wnt signaling

The regulative network conducting adult stem cells in endogenous tissue repair is of prime interest for understanding organ regeneration as well as preventing degenerative and malignant diseases. One major signal transduction pathway which is involved in the control of these (patho)physiological processes is the Wnt pathway. Recent results obtained in our laboratories showed for the first time that canonical Wnt signaling is critically involved in the control of the migration/invasion behaviour of human mesenchymal stem cells (hMSC). In the first part of this review, we describe that the regenerative state is closely linked to the activation of the Wnt pathway. Central hallmarks of activated stem cells are recapitulated in a similar way also in cancer metastasis, where the acquisition of an invasive cancer stem cell phenotype is associated with the induction of Wnt-mediated epithelial to mesenchymal transition (EMT). In the second part, the influence of proinflammatory cytokines such as transforming growth factor (TGF-)beta1, interleukin (Il-)1beta, and tumor necrosis factor (TNF-)alpha is discussed with regard to the invasive characteristics of hMSC. In this context, special attention has been paid on the role of matrix metalloproteinases (MMPs), such as MMP-2, MMP-9 and membrane type 1 (MT1)-MMP, as well as on the tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2. Putative cross-talks between different signal transduction pathways that may amplify the invasive capacity of this stem cell population are also discussed. Finally, the consequences towards future drug-mediated therapeutical modifications of Wnt signaling in stem cells and tumor cells are highlighted.

Mesenchymal stem cells (MSCs) can contribute to tissue repair by actively migrating to sites of tissue injury. However, the cellular and molecular mechanisms of MSC recruitment are largely unknown. The nuclear factor (NF)-kappaB pathway plays a pivotal role in regulating genes that influence cell migration, cell differentiation, inflammation, and proliferation. One of the major cytokines released at sites of injury is tumor necrosis factor-alpha (TNF-alpha), which is known to be a key regulator of the NF-kappaB pathway. Therefore, we hypothesized that TNF-alpha may lead to MSC invasion and proliferation by activation of the NF-kappaB pathway. TNF-receptor 1 and 2, NF-kappaB (p65), and IkappaB kinase 2 (IKK-2) are expressed in human MSCs (hMSCs). Stimulation of hMSCs with TNF-alpha caused a p65 translocation from the cytoplasm to nucleoplasm but did not change the expression profile of MSC markers. TNF-alpha strongly augmented the migration of hMSCs through the human extracellular matrix. Using lentiviral gene transfer, overexpressing a dominant-negative mutant of IKK-2 (dn-IKK-2) significantly blocked this effect. NF-kappaB target genes associated with migration (vascular cell adhesion molecule-1, CD44, and matrix metalloproteinase 9) were upregulated by TNF-alpha stimulation and blocked by dn-IKK-2. Moreover, using the bromodeoxyuridine assay, we showed that the inhibition of the NF-kappaB pathway caused a significant reduction in the basal proliferation rate. TNF-alpha stimulated the proliferation of hMSCs, whereas overexpression of dn-IKK-2 significantly blocked this effect. TNF-alpha led to the upregulated expression of the proliferation-associated gene cyclin D1. In conclusion, we demonstrated that the NF-kappaB pathway components, p65 and IKK-2, are expressed in hMSCs. Our data provide evidence that this signal transduction pathway is implicated in TNF-alpha-mediated invasion and proliferation of hMSCs. Therefore, hMSC recruitment to sites of tissue injury may, at least in part, be regulated by the NF-kappaB signal transduction pathway.

MicroRNAs (miRNAs) are versatile regulators of gene expression with profound implications for human disease including atherosclerosis, but whether they can exert posttranslational functions to control cell adaptation and whether such noncanonical features harbor pathophysiological relevance is unknown. Here, we show that miR-126-5p sustains endothelial integrity in the context of high shear stress and autophagy. Bound to argonaute-2 (Ago2), miR-126-5p forms a complex with Mex3a, which occurs on the surface of autophagic vesicles and guides its transport into the nucleus. Mutational studies and biophysical measurements demonstrate that Mex3a binds to the central U- and G-rich regions of miR-126-5p with nanomolar affinity via its two K homology domains. In the nucleus, miR-126-5p dissociates from Ago2 and binds to caspase-3 in an aptamer-like fashion with its seed sequence, preventing dimerization of the caspase and inhibiting its activity to limit apoptosis. The antiapoptotic effect of miR-126-5p outside of the RNA-induced silencing complex is important for endothelial integrity under conditions of high shear stress promoting autophagy: ablation of Mex3a or ATG5 in vivo attenuates nuclear import of miR-126-5p, aggravates endothelial apoptosis, and exacerbates atherosclerosis. In human plaques, we found reduced nuclear miR-126-5p and active caspase-3 in areas of disturbed flow. The direct inhibition of caspase-3 by nuclear miR-126-5p reveals a noncanonical mechanism by which miRNAs can modulate protein function.