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Biochemistry Research International
Volume 2012 (2012), Article ID 685267, 11 pages
Hematopoietic Stem Cell Mobilization and Homing after Transplantation: The Role of MMP-2, MMP-9, and MT1-MMP
1Research & Development, Canadian Blood Services, 8249 114th Street NW, Edmonton, AB, Canada T6G 2R8
2Department of Medicine, University of Alberta, Canadian Blood Services Building, 8249 114th Street NW, Edmonton, AB, Canada T6G 2R8
Received 2 September 2011; Accepted 2 December 2011
Academic Editor: Yong Li
Copyright © 2012 Neeta Shirvaikar et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Hematopoietic stem/progenitor cells (HSPCs) are used in clinical transplantation to restore hematopoietic function. Here we review the role of the soluble matrix metalloproteinases MMP-2 and MMP-9, and membrane type (MT)1-MMP in modulating processes critical to successful transplantation of HSPC, such as mobilization and homing. Growth factors and cytokines which are employed as mobilizing agents upregulate MMP-2 and MMP-9. Recently we demonstrated that MT1-MMP enhances HSPC migration across reconstituted basement membrane, activates proMMP-2, and contributes to a highly proteolytic bone marrow microenvironment that facilitates egress of HSPC. On the other hand, we reported that molecules secreted during HSPC mobilization and collection, such as hyaluronic acid and thrombin, increase MT1-MMP expression in cord blood HSPC and enhance (prime) their homing-related responses. We suggest that modulation of MMP-2, MMP-9, and MT1-MMP expression has potential for development of new therapies for more efficient mobilization, homing, and engraftment of HSPC, which could lead to improved transplantation outcomes.
1. Hematopoietic Stem/Progenitor Cells (HSPCs) Transplantation
HSPC transplantation is a clinical procedure in which HSPCs capable of reconstituting normal bone marrow (BM) function are administered intravenously to a patient who has undergone preparative regimens including chemotherapy and/or irradiation. Approximately 60,000 autologous and allogeneic HSPC transplants are performed annually worldwide to treat various cancers and diseases of the blood and immune system [1]. During steady-state homeostasis, approximately 0.06% of BM HSPCs circulate continuously in the peripheral blood (PB) [2], but this number can be increased with the use of chemotherapeutic drugs (e.g., cyclophosphamide) and/or growth factors and cytokines (e.g., granulocyte colony-stimulating factor (G-CSF)) that "mobilize" HSPC from BM into the PB [3]. Currently mobilized (m)PB HSPC collection has almost replaced BM harvest for autologous and most allogeneic transplantations because it is relatively easy to collect by apheresis in an outpatient setting and because engraftment after transplantation is faster. G-CSF is the most commonly used mobilizing agent in the clinic with regimens using 10 μg/kg/day for five days when used alone, or 10 to 14 days when used in combination with chemotherapeutic agents [3]. Randomized trials of mPB transplantation have shown that neutrophil and platelet engraftment generally occurs at a median of 9–14 days compared to 21 days with BM [4]. This has been attributed to the higher number of HSPC collected and transplanted. A limitation of mPB HSPC transplantation is that patients' responses to G-CSF vary: 5–10% of allogeneic normal donors mobilize poorly and up to 40% of autologous patients fail to mobilize depending on their disease and the intensity/number of prior chemotherapy regimens [5]. Hence, elucidation of the molecular mechanisms of HSPC mobilization could lead to more efficient mobilizing agents and development of better protocols.
An alternative source of HSPC is cord blood (CB) obtained at the time of childbirth, after the umbilical cord has been detached from the newborn. Since the first CB transplant in 1988, more than 30,000 CB transplants have been performed worldwide in pediatric and adult patients [1, 6]. CB has several advantages over BM and mPB as source of HSPC for transplantation. CB contains lower numbers of more immature, immunocompetent T cells and thus requires less stringent HLA matching; this means that a mismatch at one or two loci can be tolerated without significant increase in graft versus host disease (GvHD) or decrease in overall survival [6, 7]. However, the main limitation of CB transplantation is the low CD34+ cell dose available in one CB unit which is generally insufficient to support engraftment in adult patients. Retrospective analyses of outcomes of CB and BM transplantation in adults have reported delayed neutrophil engraftment: 27 days with CB versus 18 days with BM and platelet engraftment: 60 days with CB versus 29 days with BM [7]. Currently efforts are being made to increase CB cell dose in order to speed up engraftment and hematopoietic recovery. Strategies to use more than one CB unit [7] or to expand CB CD34+ cells ex vivo [8], however have met with limited success. A more comprehensive knowledge of CB HSPC biology and the mechanisms of their homing is expected to improve CB transplantation outcomes.
2. Mobilization of HSPC
During homeostasis, continuous traffic of HSPC between the BM and PB contributes to normal hematopoiesis. The ability to enhance these physiological processes and, for example, enforce egress (mobilization) of HSPC from the BM to circulation has been invaluable in clinical transplantation [9]. Several cytokines (G-CSF, granulocyte macrophage-CSF, Flt-3 ligand, interleukin (IL)-8, and stem cell factor (SCF)/kit-ligand) and chemokines (stromal cell-derived factor (SDF)-1 and GROβ) can trigger mobilization in varying degrees [3, 10]. We recently demonstrated that G-CSF also increases plasma hepatocyte growth factor (HGF) levels in mobilized patients and expression of its receptor c-Met in HSPC and myeloid cells, suggesting that GCSF-mediated HSPC mobilization occurs in part through the HGF/c-Met axis [11].
Another important axis that influences mobilization is the SDF-1/CXCR4 axis. This axis is essential for retention of HSPC in the BM, and perturbation of the SDF-1 gradient in the BM as well as a decrease in the responsiveness of HSPC to SDF-1 leads to mobilization [12].
The proteases carboxypeptidase M (CPM) [13] and CD26 [14] cleave the C-terminus of SDF-1 resulting in attenuated chemotactic responses of HSPC; moreover, G-CSF-induced mobilization is impaired in CD26-deficient mice [14]. Desensitization of CXCR4 by the urokinase-mediated plasminogen activation system during G-CSF-induced mobilization has been demonstrated [15]. In murine studies, SDF-1 concentration in the BM decreased following G-CSF administration and correlated with mobilization, suggesting that a physiological drop in SDF-1 level in the BM is a critical step in HSPC mobilization [12, 16]. A decrease in BM SDF-1 levels has been reported to coincide with a peak of proteolytic activity of neutrophil elastase (NE), cathepsin G (CG), and matrix metalloproteinase (MMP)-9 [17]. Both SDF-1 and CXCR4 are targets of degradation by NE, CG, MMP-9, MMP-2, and membrane type (MT)1-MMP [18–20] and can be inactivated by proteolytic cleavage. The roles of these MMPs will be discussed in detail in subsequent sections.
Recent studies have demonstrated that thrombolytic agents such as microplasmin, tenecteplase, and recombinant tissue plasminogen activator enhance G-CSF-induced mobilization in murine models [21]. Adhesion molecules also play an important role in the retention of HSPC in the BM. Very late antigen (VLA)-4 is expressed by HSPC and its ligand, vascular cell adhesion molecule (VCAM)-1, is constitutively expressed by endothelial and stromal cells [22]. Disruption of the VCAM-1/VLA-4 axis with a small molecule inhibitor of VLA-4 resulted in the mobilization of more HSC over basal levels [23]. CD44, a polymorphic integral membrane glycoprotein, binds to several extracellular matrix (ECM) components such as hyaluronic acid (HA), fibronectin, and collagen and mice treated with anti-CD44 antibody or lacking CD44 exhibit impaired mobilization in response to G-CSF [24].
Components of innate immunity also participate in G-CSF-induced HSPC mobilization [25]. During G-CSF-induced mobilization, the complement cascade (CC) is activated by the classical pathway. However, in the early steps of the CC, C1q, C3, and its cleavage fragments C3a and desArgC3a increase retention of HSPC in BM while in the later steps of the CC, C5, and its cleavage fragments C5a and desArgC5a enhance HSPC mobilization and their egress into PB [26–28].
3. Homing of HSPC
The success of clinical HSPC transplantation relies on the inherent ability of transplanted HSPC to home efficiently to the BM niche and engraft. Interactions between HSPC and their niches that are disrupted during mobilization need to be reestablished during their homing to the BM and its repopulation. Previously it was believed that mobilization and homing were mirror images of each other; however, emerging evidence now suggests that this is not the case, although both processes involve many of the same adhesive and chemotactic interactions. The homing of HSPC to BM is a rapid process occurring within hours after their transplantation and is a prerequisite for their repopulation and engraftment [29]. Homing involves three consecutive steps: extravasation from PB through the BM endothelium, migration through stroma, and lodgement into niches.
The extravasation of circulating HSPC within the BM requires a set of molecular interactions that mediates the recognition of circulating HSPC by the endothelium of BM sinusoids [30]. The mechanisms of HSPC extravasation are similar to those of leukocytes into inflamed tissues in that they are mediated by adhesion molecules. The BM endothelium constitutively expressing P-selectin, E-selectin, and VCAM-1 mediates rolling and tethering of HSPC to the blood vessel wall prior to their extravasation [31]. HSPC expressing CD44 and VLA-4 receptors interact with their cognate ligands on the endothelial surface. The coordinated action of these adhesion molecules is triggered by SDF-1 presented at the surface of endothelial cells. SDF-1 mediates activation of lymphocyte function-associated antigen-1, VLA-4 and VLA-5, converting the rolling of HSPC into stable arrest on the endothelium [32].
SDF-1—CXCR4 signalling is critical in the regulation of HSPC homing, engraftment, and retention in the BM. Blockade of CXCR4 was shown to inhibit HSPC homing and engraftment, whereas overexpression of CXCR4 by CB and mPB CD34+ cells leads to increased SDF-1 induced in vitro migration and homing in NOD/SCID mouse models [33, 34]. We recently showed that mPB CD34+ cells that had higher responsiveness to SDF-1 had high CXCR4 expression and could compensate for a lower CD34+ cell dose in achieving faster hematopoietic engraftment after transplantation [35]. SDF-1 binding to CXCR4 activates phosphatidylinositol 3-kinase (PI3K), the phospholipase C-γ (PLC-γ)/protein kinase C (PKC) cascade, and p44/42 mitogen-activated protein kinase (MAPK) [36]. SDF-1—CXCR4 signalling also activates the atypical PKC subtype PKCζ which mediates cell polarization, adhesion, MMP-9 secretion, and chemotaxis of CD34+ cells [37]. Interactions mediated by the Rho family GTPases (Rac, Rho, and Cdc42) have also been implicated in HSPC homing and engraftment. Following engraftment, deletion of Rac-1 and Rac-2 GTPases led to massive mobilization of HSPC from BM. Knocking out Rac-1 significantly reduced migration towards SDF-1 and attenuated the homing of murine HSC to the endosteum, which is essential for long-term HSC repopulation [38]. We previously demonstrated that Rac-1 colocalization with CXCR4 in membrane lipid rafts of HSPC promotes their in vivo homing in a murine model [39]. Moreover, in vitro treatment of HSPC with supernatants of leukapheresis products (SLPs) or their components, such as C3a or platelet-derived microvesicles (PMVs), modulates the SDF-1—CXCR4 axis and speed up in vivo homing in murine models [39, 40].
Proteases regulate HSPC migration and tissue localization and have been shown to also play important functions in HSPC homing. The roles of proteolytic enzymes particularly MMP-2, MMP-9, and MT1-MMP in this process will be discussed in detail in subsequent sections.
4. Matrix Metalloproteinases
MMPs belong to a family of Zn2+-binding, Ca2+-dependent endopeptidases whose essential function is proteolysis of the ECM, a process that is required in several cellular processes [41]. Currently, 24 human MMPs have been identified that have structural similarities but vary in their expression profiles and substrate specificities. MMPs are classified based on substrate recognition into stromelysins (MMP-3, MMP-10, and MMP-11), matrilysins (MMP-7, MMP-26), gelatinases (MMP-2 and MMP-9), and collagenases (MMP-1, MMP-8, MMP-13, and MMP-14) [42]. Apart from ECM molecules, MMPs act on a whole array of substrates including other proteinases and MMPs, proteinase inhibitors, growth factors, cytokines, chemokines, cell surface receptors, and cell adhesion molecules and regulate many processes such as cell migration, proliferation, apoptosis, angiogenesis, tumor expansion, and metastasis [42–44]. The expression and function of MMPs are regulated at different levels. Generally expressed at low levels, MMPs are upregulated during tissue remodeling, inflammation, wound healing, and cancer progression [45, 46]. They are synthesized as latent enzymes that are either secreted or membrane-anchored. Six MT-MMPs have been identified so far, of which four, MT1-/MMP-14, MT2-/MMP-15, MT3-/MMP-16, and MT5-/MMP-24, have a transmembrane domain while the other two, MT4-/MMP-17 and MT6-/MMP-25, have a glycosylphosphatidylinositol domain [42]. Their membrane anchoring allows them to carry out pericellular proteolysis. MMPs are activated by the proteolytic release of the N-terminal propeptide domain. Once active, they can be inhibited by endogenous tissue inhibitors of metalloproteinases (TIMPs), the reversion-inducing cysteine-rich protein with Kazal motifs (RECK), and tissue factor pathway inhibitor-2 as well as by plasma inhibitor (α2-macroglobulin) [47, 48]. Therefore, a balance between MMPs and their inhibitors is important to ECM remodeling in the tissue and in HSPC migration.
The gelatinases MMP-2 and MMP-9 have been extensively studied in cancer and other diseases. MMP-2 is secreted by fibroblasts, endothelial cells, epithelial cells, and transformed cells whereas MMP-9 is produced predominantly by leukocytes [49]. MMP-2 and MMP-9 are required for physiological processes such as ECM remodeling during growth and development, inflammation, wound healing, angiogenesis, and leukocyte mobilization [41, 46]. They are also involved in pathological processes such as cancer, inflammation, and neural and vascular degenerative diseases [45, 46]. Although MMP-2 and MMP-9 are secreted by cells in the developing embryo, mice deficient in these gelatinases are viable. However, mice deficient in MMP-2 exhibit defects in developmental angiogenesis whereas mice deficient in MMP-9 show delayed vascularization and ossification resulting in moderate skeletal abnormalities [49]. MMP-2 and MMP-9 are similar in many respects, but differ in their glycosylation pattern, activation, and substrate specificity. The 92 kDa MMP-9 has two glycosylation sites in the prodomain and the catalytic domain whereas the 72 kDa MMP-2 is a nonglycosylated protein [42]. MMP-2 activation is a cell surface event mediated by the formation of a ternary complex containing MT1-MMP, TIMP-2, and proMMP-2 [42]. The N-terminal domain of TIMP-2 binds to MT1-MMP whereas the C-terminal domain binds the hemopexin domain of proMMP-2. An adjacent MT1-MMP free of TIMP-2 subsequently activates proMMP-2 by cleaving its propeptide domain. On the other hand, MMP-9 activation is mediated by a proteolytic cascade involving MMP-3, MMP-2 and MMP-13 [42]. MMP-3 is activated by plasmin generated from plasminogen by urokinase-type plasminogen activator (uPA) bound to its receptor on the cell surface [42]. Similarly, MMP-2 activates proMMP-13 which then activates proMMP-9 [50]. MMP-2 and MMP-9 share similar proteolytic activities and degrade a number of ECM molecules such as gelatin, collagen types IV, V, and XI, and laminin. In addition MMP-2 also degrades collagen types I, II and III. Both also cleave several non-ECM molecules such as SDF-1, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, plasminogen, and uPA, among many others [42–44].
MT1-MMP also plays important roles in both physiological and pathological processes. Mice deficient in MT1-MMP exhibit craniofacial dysmorphism, dwarfism, osteopenia, fibrosis of soft tissues, arthritis, and premature death, emphasizing the function of MT1-MMP in ECM remodelling during development [51]. On the other hand, elevated MT1-MMP expression has been observed in a wide variety of cancers including lung, breast, cervical, brain, liver, head, and neck, indicating MT1-MMP's role in tumor progression and metastasis [45]. In addition, it has been implicated in angiogenesis, bone development, atherosclerosis, inflammation, and wound healing [44] by virtue of its ability to degrade several ECM macromolecules including collagens, laminins, fibronectin, aggregan, and fibronectin and to activate proMMP-2 and proMMP-13 [42, 44, 52]. MT1-MMP cleaves CD44, processes αv integrin to its mature form, and degrades tissue transglutaminase, thus modifying the immediate cell environment and affecting cellular functions in a variety of ways [44]. MT1-MMP has a domain structure composed of a propeptide region, catalytic domain, hinge region, hemopexin-like domain, and a type I transmembrane domain with a cytoplasmic tail at the C-terminus which anchors it to the cell surface [42]. MT1-MMP is also expressed in its latent form and cleavage of the propeptide region by furin or related protein convertases renders it active. Activation of MT1-MMP takes place in the trans-Golgi network complex during secretion, and the enzyme is expressed in its active form at the cell surface [53]. Active MT1-MMP is inhibited by RECK [54], TIMP-2, TIMP-3 and TIMP-4 but not by TIMP-1 [48]; however, TIMP-2 has a dual role. On the one hand it inhibits MT1-MMP and on the other promotes activation of proMMP-2 [47]. At the cell surface, the 60 kDa active MT1-MMP molecule undergoes self-proteolysis by removal of its catalytic domain which results in an inactive 44 kDa species [55]. A high level of truncated MT1-MMP coincides with high proMMP-2 activation. In migrating tumor cells, MT1-MMP localizes predominantly in the lamellipodia through its interaction with CD44 [56]. It has been reported that this interaction and the consequent shedding of CD44 stimulate cell motility [57]. The hemopexin domain of MT1-MMP interacts with the stem region of CD44 which interacts with F-actin through its cytoplasmic domain. Binding of MT1-MMP with CD44 indirectly links the proteinase to the cytoskeleton and thereby enables its localization to the lamellipodia [58]. MT1-MMP can be internalized by both clathrin-dependent and caveolae-dependent pathways, and recycled back to the surface, and the internalization process is essential for the enzyme to promote cell migration [58]. The redistribution of MT1-MMP to sites of degradation, such as the lamellipodia of endothelial cells and the invadopodia of tumor cells, is highly complex and involves a dynamic interplay between endocytic and exocytic processes [59]. In most cell types surface expression of MT1-MMP is low due to rapid endocytosis resulting in intracellular accumulation of the proteinase in the early and late endosomes. This dynamic regulation suggests that the spatiotemporal recruitment of MT1-MMP to specialized domains plays a critical role in its invasive properties [60]. Various growth factors, chemokines, and inflammatory mediators have been reported to modulate MT1-MMP expression in malignant and normal cells. In the fibrosarcoma cell line HT1080, Rac-1 modulation of MT1-MMP and its processing to its 44 kDa form correlated with proMMP-2 activation [61]. Furthermore, Rac-1 has been demonstrated to promote hemophilic complex formation of MT1-MMP, recruitment to the lamellipodia-rich cell surface, and subsequent proMMP-2 activation [62]. In Lewis lung carcinoma cell line, type 1 insulin growth factor-1 increases invasiveness of these cells through increased MT1-MMP expression via the PI3K/AKT/mTOR pathway [63]. The SDF-1−CXCR4 axis promotes melanoma cell invasion and metastasis by upregulating MT1-MMP through Rac-1 and RhoA-GTPases [64]. Moreover, through activation of Rac-1 and its downstream effector ERK1/2, SDF-1 intracellularly upregulates MT1-MMP; however, when these cells were in contact with Matrigel, a PI3K-dependent transient redistribution of MT1-MMP to the cell surface was observed [65]. In endothelial cells, VEGF-mediated upregulation of MT1-MMP at the mRNA level occurs through the MAPK/JNK pathway, whereas protein expression is regulated by PI3K [66, 67]. MT1-MMP clustering on the cell surface is dependent on cortical actin polymerization which is regulated by PI3K, and this clustering of MT1-MMP has been suggested to be more important than its internalization [59]. In mesenchymal stromal cells (MSCs), we reported that MT1-MMP mediates their chemotactic migration towards SDF-1 and HGF [68]. Increased chemoinvasion of MSC through upregulation of MT1-MMP by cytokines TGF-β, TNF-α, and IL-1 was also reported [69]. In hematopoietic cells, MT1-MMP has been demonstrated to mediate trans-endothelial migration of monocytes, and the interaction of monocytes with fibronectin and endothelial ligands, such as VCAM-1 and intracellular cell adhesion molecule (ICAM)-1, increased MT1-MMP clustering and localization into membrane protrusions or lamellipodia [70].
5. MMPs in HSPC Mobilization
MMPs have been traditionally considered to facilitate cell migration by breaching basement membrane barriers comprised of ECM proteins. The mobilizing agent G-CSF induces neutrophil proliferation, activation, and degranulation with the subsequent release of the serine proteases NE, CG, and MMP-9 into the BM, making it a highly proteolytic microenvironment [17, 71]. MMP-9 has been reported to be elevated in plasma after mobilization with G-CSF or IL-8 [71, 72]. Mobilization is thought to occur predominantly through the action of neutrophils and release of granulocytes from the BM always precedes mobilization of HSPC [73]. However, HSPC themselves contribute to this process by secreting MMP-2 and MMP-9 [74]. We have reported that while BM HSPCs in steady-state do not secrete MMPs, upon stimulation with G-CSF, among other growth factors and cytokines, HSPCs secrete both MMP-2 and MMP-9 leading to their enhanced migration through reconstituted basement membrane (Matrigel) [74]. Both mature leukocytes as well as immature CD34+ cells from in vivo G-CSF-mobilized PB highly express MT1-MMP compared to their steady-state (ss)BM and ssPB counterparts. Furthermore, cell surface expression of MT1-MMP in in vivo mobilized polymorphonuclear cells (PMNs) and monocytes was 8 and 12 times higher than in their respective ssPB counterparts. G-CSF exerts its effects on other signalling axes that also result in upregulation of MMP-9 and MT1-MMP. For example, we observed that HGF increases in the plasma of mobilized patients and that G-CSF, through induction of HGF, upregulates MMP-9 and MT1-MMP expression in BM PMN [11]. In addition, activation of the CC by G-CSF triggers a series of reactions generating various bioactive peptides including the C5 cleavage fragments (C5a and desArgC5a) which have been shown to play an important role in mobilization [25, 75]. In particular, we recently demonstrated that C5a increases MMP-9 and MT1-MMP expression in PMN and mononuclear cells (MNCs), contributing to a microenvironment that is conducive to the egress of HSPC from the BM [28]. MT1-MMP causes pericellular degradation by processing several ECM components (such as gelatin, fibronectin, laminin, vitronectin, and fibrillar collagens) [41, 43, 52]. This is further substantiated by observation that G-CSF-induced migration of MNC and HSPC through Matrigel (containing collagen type IV, laminin, entactin) is MT1-MMP dependent. MT1-MMP expressed by HSPC activates proMMP-2 secreted by BM stromal cells [76, 77]. Active MMP-2 could subsequently initiate a cascade of activation of other MMPs including MMP-9 and MMP-13, which not only degrade the ECM but also disrupt adhesive interactions between HSPC and their niches. One of the most potent of these interactions is between SDF-1, produced by stromal cells, including endothelial cells, and osteoblasts, and its receptor CXCR4, expressed by HSPC. Recently, we demonstrated that C5a, apart from upregulating MT1-MMP, decreases CXCR4 expression in PMN, which disrupts their chemotaxis towards SDF-1. Moreover, this impaired chemoattraction is partially restored by the potent MT1-MMP inhibitor EGCG (which also inhibits proMMP-2 activation), suggesting that MT1-MMP contributes towards reduced retention of HSPC in the BM microenvironment [28].
In addition to matrix remodeling, MT1-MMP cleaves adhesion molecules, such as CD44 and integrins, and chemokines such as SDF-1. Reduced retention of HSPC in the BM during G-CSF-induced mobilization is also due to a decrease in active SDF-1 in the BM which coincides with peak proteolytic activity [12, 16]. SDF-1 can be cleaved and inactivated by several proteases which are activated during mobilization such as NE, CG, MMP-2 and MMP-9, CD26, CPM, and MT1-MMP [13, 14, 17–20]. MT1-MMP cleavage of SDF-1 results in loss of binding to CXCR4 and reduced chemoattraction for CD34+ cells [18]. Therefore, during G-CSF-induced mobilization, upregulation of MT1-MMP expression in HSPC has consequences that could lead to their reduced retention in the BM and resultant egress into PB.
A low SDF-1 level in the BM during cytokine-induced mobilization has been suggested to result from suppression of SDF-1-producing osteoblasts, and although the mechanism of this phenomenon is not completely understood [78], it has been suggested to occur in light of an observed increase in osteoclast activity [79]. Active bone remodeling takes place during G-CSF mobilization, and increased cathepsin K and MMP-9 secretion by osteoclasts has been implicated in high bone turnover [79, 80]. Similarly, MT1-MMP also plays an important role in bone remodeling as shown by the fact that MT1-MMP−/− mice exhibit severe skeletal defects [51]. MT1-MMP is also known to be expressed by osteoclasts and recently it was demonstrated that MT1-MMP was necessary for macrophage fusion during multinucleated osteoclast formation and differentiation [81]. Accordingly, 8-day-old MT1-MMP−/− mice exhibited impaired osteoclast function, which did not, however, result in increased bone mass since osteoblast function is also compromised in these mice [51, 81]. These results suggest that MT1-MMP could have a dual role in bone development. Nevertheless, the role of MT1-MMP in bone remodeling during G-CSF-induced mobilization requires further investigation.
It was postulated that MT1-MMP expression in HSPC is regulated by the endogenous inhibitor RECK and that high MT1-MMP and low RECK levels in HSPC resulted in the egress of BM progenitors into circulation [54, 77]. In a recent publication, we provided evidence that MT1-MMP expression on the surface of HSPC is regulated by its incorporation into membrane lipid rafts, and that both MT1-MMP expression and proMMP-2 activation are PI3K-dependent [76]. Moreover, in co-cultures of fibroblasts with BM CD34+ cells, proMMP-2 is not activated; however, pre-incubation of CD34+ cells with G-CSF highly upregulated MT1-MMP, which was then able to activate proMMP-2 in similar co-cultures. Consistent with this, in co-cultures of stromal cells with mPB CD34+ cells that had been transfected with MT1-MMP siRNA, active MMP-2 was not detectable [76]. The MT1-MMP activation of proMMP-2 in the BM microenvironment is important because active MMP-2 not only initiates the activation of other MMPs that play a role in matrix remodeling, but also inactivates SDF-1 and CXCR4 and adhesion molecules, processes which facilitate egress of HSPC from BM niches across the ECM and subendothelial membranes.
MT1-MMP promotes cell motility by pericellular ECM degradation [41, 52, 53]. In this respect we demonstrated the role of MT1-MMP in the migration of CD34+ cells and MNC across reconstituted basement membrane as specific inhibition of MT1-MMP by siRNA significantly abrogated their migration [76]. On the other hand, upregulation of MT1-MMP expression in HSPC by G-CSF could lead to their reduced retention in the BM and enhanced egress into PB [77]. The definitive proof of this would involve studies using MT1-MMP−/− mice, but such mice have severe developmental abnormalities resulting in their early death [51]; hence in vivo mobilization experiments in MT1-MMP knockout mice are difficult. However, these experiments have been carried out in chimeric mouse models [77].
The initial cell response to cytokine or chemokine stimulation is the reorganization of the actin cytoskeleton during which several signalling pathways are activated. MT1-MMP has been shown to be modulated by PI3K, MAPK, and Rho family GTPases depending on the cell type and stimulant [64, 82]. We demonstrated that although both PI3K and MAPK are activated in hematopoietic cells upon G-CSF stimulation, MT1-MMP expression and proMMP-2 activation are only PI3K dependent. Furthermore, inhibition of the PI3K-AKT axis also inhibits cell polarization, co-localization of MT1-MMP with F-actin, and trans-Matrigel migration of mPB CD34+ cells [76]. Murine HSPC studies have shown that cytokine-mediated lipid raft clustering activated the AKT-FOXO signalling pathway, which is essential for entry into cell cycle, and inhibition of lipid raft formation by MβCD led to repression of this pathway and hibernation-like state of HSPC [83]. The recruitment of the regulatory PI3K subunit p85 to lipid rafts after cytokine stimulation is required for activation of the downstream effectors of the PI3K-AKT axis and is dependent on lipid raft integrity [84]. Consistent with these findings, we showed that G-CSF-induced PI3K activation in lipid rafts leads to reorganization of the actin cytoskeleton, recruitment of MT1-MMP into lipid rafts, and proMMP-2 activation [76], which increases the degradation of basement membrane and interstitial matrix in the BM microenvironment and eventually the egress of HSPC into circulation as shown in Figure 1(a). Mobilizing signals such as G-CSF expand the number of myeloid cells in the BM, which secrete proteolytic enzymes (including MMP-2 and MMP-9) that disrupt interactions that retain the HSPC in the BM (e.g., SDF-1/CXCR4, VCAM-1/VLA-4, ECM). Permeabilization of the endothelial barrier occurs with granulocytes "paving the way" for HSPC. Chemoattractants in the blood (e.g., bioactive lipids such as sphingosine 1-phosphate, HGF) further facilitate egress into PB of HSPC [73] (Figure 1(a)).
6. MMPs in HSPC Homing
MMPs degrade various ECM molecules and facilitate HSPC transmigration across basement membrane barriers. SDF-1 and other growth factors induce the secretion of MMP-2 and MMP-9, thus facilitating in vitro migration of HSPC across reconstituted basement membrane towards SDF-1 [73]. Incubation of CB-derived HSPC with SCF induces MMP-2 and MMP-9 secretion and homing in NOD/SCID mice [85]. We demonstrated that MT1-MMP mediates migration of CB CD34+ cells and megakaryocytic progenitors towards an SDF-1 gradient [86]. Recently another group reported, using a chimeric mouse model, that engraftment levels of MT1-MMP−/− c-Kit+ cells were significantly lower than those of wild-type cells, and inhibition of MT1-MMP by monoclonal antibody attenuated homing of human HSPC in a NOD/SCID mouse model [77].
We have demonstrated that HSPC primed by SLP or their components (fibrinogen, fibronectin, complement C1q, complement C3a, PMV, HA, thrombin) respond better to an SDF-1 gradient [26, 39, 40, 87, 88]. Furthermore, murine HSPC that have been primed with C3a, PMV, or SLP before transplantation engrafted faster in lethally irradiated mice [39, 40, 87]. This priming effect was due to increased incorporation of CXCR4 and Rac-1 GTPase into membrane lipid rafts and to increased MMP-2 and MMP-9 secretion, indicating that the SDF-1—CXCR4 axis cooperates with MMPs during HSPC homing [39].
We recently investigated the effects of two priming molecules, HA and thrombin, on the modulation of MT1-MMP expression and its activity [88]. First, we found that HA and thrombin upregulated MT1-MMP expression in CB HSPC. Secondly, HA- and thrombin-primed chemoinvasion of HSPC towards a low SDF-1 gradient was MT1-MMP dependent. It has been established that the SDF-1—CXCR4 axis promotes the chemotaxis not only of normal but also of tumor cells [70, 89]. For example, the coordinated interaction of CXCR4 and MT1-MMP is required for melanoma cell metastasis to lungs [65]. CXCR4 was required for the initial phases of melanoma cell chemotaxis and their arrival in the lungs, whereas MT1-MMP was necessary for subsequent invasion and dissemination of the tumor. We can therefore speculate that a coordinated interaction between CXCR4 and MT1-MMP is also required by HSPC for their homing to the BM. MT1-MMP expressed in CB HSPC (upregulated by HA and thrombin), activates proMMP-2 secreted by endothelial cells. Active MMP-2 could thereby participate in the extravasation process and facilitate homing by activating other MMPs and degrading ECM barriers [73]. In this respect, MT1-MMP has been shown to facilitate trans-endothelial migration of monocytes through clustering of MT1-MMP at the lamellipodia upon contact with activated endothelial cells or the immobilized endothelial ligands VCAM-1 and ICAM-1 [70]. Priming of homing-related responses of CB HSPC showed that HA and thrombin activated the PI3K-AKT signalling axis and Rac-1 GTPase [88]. MT1-MMP expression and proMMP-2 activation were dependent on both these signalling pathways. We demonstrated that intracellular crosstalk between these pathways leads to signal amplification of a low SDF-1 gradient, leading to enhanced MT1-MMP expression on the cell surface of CB HSPC and increase chemoinvasion towards SDF-1 [88]. Thus, agents such as HA and thrombin that positively regulate the SDF-1—CXCR4 axis may also prime the homing-related responses of HSPC by upregulating MT1-MMP. HSPCs then attach/tether to and extravasate the sinusoid endothelium. MT1-MMP promotes activation of proMMP-2 that degrades ECM barriers. HSPCs are chemoattracted to their BM niches due to amplified chemotactic response towards SDF-1 produced by osteoblasts and stromal cells (Figure 1(b)).
7. Therapeutic Strategies to Improve Transplantation Outcomes
Accumulating evidence indicates an important role for MMP-2, MMP-9, and MT1-MMP in HSPC mobilization and homing. In particular, modulation of MT1-MMP could become a potential target for development of therapeutic strategies that could improve transplantation outcomes. First, critical to optimizing clinical mobilizing regimens is an understanding of the molecular mechanisms that regulate HSPC mobilization [73, 90, 91]. Such studies have already led to the development of new mobilizing agents that resulted in a rapid collection of more HSPC for transplantation. For example, recent use of Plerixafor (AMD3100) which reversibly binds CXCR4 and disrupts SDF-1—CXCR4 interactions has demonstrated that a combination of AMD3100 and G-CSF results in greater mobilization efficacy compared to G-CSF alone [92, 93].
The main problem with G-CSF-induced mobilization is the variable kinetics of mobilization as shown by the significant number of patients/donors who either mobilize poorly or fail to mobilize. Recent findings indicate that positive regulation of MT1-MMP increases migration/mobilization of HSPC, and hence the development of mobilizing agents that increase MT1-MMP expression could enhance mobilization efficiency. For example, cytokines such as HGF that upregulate MT1-MMP expression in HSPC [11] could also synergize with G-CSF and increase mobilization efficiencies in patients who mobilize poorly with G-CSF alone. This could be tested in a clinical setting following confirmatory results from murine models. On the other hand, potential inhibitors such as green tea polyphenol EGCG, which inhibits MT1-MMP expression and proMMP-2 activation, and statins, which like methyl-β-cyclodextrin disrupt lipid raft formation, could inhibit MT1-MMP incorporation into lipid rafts and thereby negatively affect HSPC mobilization. This could result in lowering the number of HSPC collected, and therefore we suggest that overcoming these inhibitory effects could improve transplantation outcomes.
Secondly, a successful transplantation outcome also depends on the ability of a large number of intravenously injected HSPC to rapidly find their way to the BM. This has led to the design of strategies to increase the number of HSPC available by ex vivo expansion of HSPC before transplantation. Another strategy is to enhance the homing potential of a limited number of HSPC by their ex vivo exposure to agents such as C3a and PMV that prime their chemotactic responses by positively regulating the SDF-1—CXCR4 axis, as we previously showed in murine models [40, 87]. Interestingly, this strategy of ex vivo exposure of CB HSPC to C3a is already being evaluated in clinical trial [94]. Therefore we suggest that other priming agents that enhance the responsiveness of CB HSPC towards SDF-1 and upregulate MMPs expression could be used for ex vivo short-term treatment/priming of CB HSPC and be evaluated in clinical trials.
Lastly, a better understanding of the molecular mechanisms of HSPC migration is not only beneficial for designing novel therapeutic strategies discussed above but could also be applied to enhancement of the homing properties of other types of stem cells such as MSC which share common migration mechanisms.
Conflict of Interests
The authors declare no potential conflicts of interest.
The authors' research on "Mechanisms of HSPC Mobilization and Homing" has been funded by an Operating Grant (XE00025) from the Canadian Blood Services (CBS) Research and Development/Canadian Institutes for Health Research Blood Utilization and Conservation Partnership Initiative to A. Janowska-Wieczorek.
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There was no telling at first what William Price meant by "big news" when he called the Houston Press on the afternoon of February 2 from his doctor's office, where he had gone when his blood pressure shot sky high after the day's events. To Price, a librarian at Kashmere Gardens Elementary, big news could be anything from rumors of the embezzlement of thousands of dollars in donated money to the principal's removing the telephone from the teacher's lounge. For months, Price had been telling the Press about what he called serious mismanagement at Kashmere Gardens, including cheating on the Texas Assessment of Academic Skills.
If some of Price's stories seemed outlandish, the story Price told that afternoon topped them all: Police officers had come to the school and searched his briefcase. They told him the principal had reported that Price was carrying a gun and intended to shoot her. When the police found no gun, Northeast District Superintendent E.O. Curtis, who was on the scene, asked Price to come to the district office. There, he was relieved of his duties with pay.
The memorandum Curtis gave Price said the librarian was accused of "calling students in your class 'niggers;' re-enacting the Jasper, Texas incident by having a child lie down on the floor with both feet in a chair while you slowly pulled; telling your students that you hated Blacks; telling your students that Ms. Jefferson, the principal, does not like you and you don't like her, etc." No mention was made of a gun.
In the view of local TV stations, Price was right about his big news. The tale of the racist librarian led Channel 2's "Live at Five" and ran on several stations on two consecutive days. But barely a peep was heard from parents of the children who were allegedly subjected to such abuse. The Houston Chronicle quoted the Kashmere Gardens PTA president quoting "a group of parents" quoting their children quoting Price as saying, "This is what happened to a man in Jasper, Texas, and this is what should happen to all [racial slur]" after he "grabbed a fourth-grade student by his feet Monday and dragged him across the library floor."
The district issued a statement saying, "HISD absolutely will not tolerate this type of behavior, and if this allegation is true, this person's employment with HISD will be terminated." Other than that, district spokesperson Terry Abbott declined to comment until an investigation is completed.
Except for one specialist who works with disabled children, Price is the only white teacher at Kashmere Gardens Elementary. He is hyperbolic -- visit his home a few times and he'll say you've been there "dozens of times." He also adjusts details to suit his needs. Curtis's repeatedly asking Price to clean off his desk before leaving campus is later recast as Curtis's refusing to let Price clean off his desk before leaving campus, an explanation for his inability to produce certain promised documents. Yet his exaggeration tends to the theatrical, not the cruel. When Good Morning America supposedly queried him about a possible appearance after the local news coverage, Price asked an acquaintance, deadpan, if a tuxedo wouldn't be "too much" to wear on the show.
"I ain't never heard him say nothing toward a black person. No black person -- no kids, no adults, since I've been working there with him," says Julia Keller, a black mother of twins who volunteered in the Kashmere Gardens library every day. Keller says Price asked Jefferson to give her a school lunch every day. When the school wouldn't, he bought it for her himself. She says he also purchased crayons, paper, pencils and other supplies for the students -- and a microwave and coffee maker for the teachers -- with his own money. The idea that Price would call students "niggers" doesn't add up for Keller.
"I feel this way: If he's the only white person in that school, and he's been there four years, how is he going to have racism toward a black person?" she asks. "That's what I don't understand. That's stupid."
So what did happen in the library on Monday, February 1, the first day of Black History Month? Price's story seems typically theatrical: He gave a fourth-grade class a lesson on civil rights, showing a movie about onetime slave Jane Pittman and explaining concepts such as racism and segregation. Students brought up the subject of the murder in Jasper, where a black man, James Byrd Jr., was dragged behind a truck and decapitated. The kids wanted to know why people do things like that to other people. They wanted to know what "decapitated" meant. They wanted to know why Byrd didn't just untie himself from the truck. Price says he asked a volunteer from the class to demonstrate by lying down and putting his feet up on a table, which he told the class to pretend was a moving truck. Then he asked Jeremy if the boy could reach up to his feet to untie an imaginary rope. He couldn't.
There was another teacher in the room when all this was going on, but according to Houston Federation of Teachers president Gayle Fallon, all teachers at the school have been instructed not to discuss the incident with anyone. "There are a number of teachers telling us, 'This charge isn't real,' " Fallon says.
Price says he referenced the "N word" as an example of racism but never actually said it. He says he also referenced the "H word" as the counterpart used to denigrate whites.
The following day, he was greeted by the HISD police. Department Captain Jeffery McMillin confirms that two officers did visit the school because of reports that Price had a gun, but the officers did not find a weapon. How that resulted in Price's suspension due to allegations of racism, McMillin could not say: "I don't know where the two connect."
Somehow, though, Officer R.E. Peters wrote up both the gun and the alleged racial harassment. His police report lists five complainants, all Kashmere Gardens parents. Each either failed to return calls from the Press or refused to discuss the allegations of racism, but one, Priscilla Wilson, did say Jefferson called her to the school that Tuesday, and another, Cynthia Catina, would say only that she reported the incident to the school herself.
Judging by the evidence, it's no wonder Jefferson would want to get rid of Price. For one thing, Price had his share of run-ins with his previous principal, Joyce Andrews at Bastian Elementary; in fact, the whole faculty was divided into two camps over Andrews. In 1994 Price was involuntarily transferred from Bastian after having a heart attack on campus; shortly afterward Andrews was removed from the school in an HISD "intervention."
At Kashmere Gardens, Price had been waging war with Jefferson for months over the way she runs the school. He claims his every offer to help the school, by calling in favors from contacts at Southwestern Bell and other businesses to running a reading competition, was rejected by Jefferson. "All that threatened her," Price says.
Frustrated, Price says, he started to fight back. Among Price's milder allegations: Jefferson refused to buy supplies for students, kept Price's orders for library books sitting on her desk for weeks and locked new audio-visual and computer equipment in a closet where no one could access it. In fact, only two weeks before the HISD police escorted him of campus, Price gave Jefferson a seven-page letter detailing his complaints. "Why when I mention our wonderful test scores in public people look at me and smile like I should be in an institution?" Price wrote. "Do they know something I don't know?"
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Price says he made attempts to inform the district of allegations of cheating and mismanagement at the school. Since June, he says, he has contacted Curtis five times. "I told him something was going to happen to me" he says, adding that Curtis simply made note of his complaints. When the Press asked if Curtis would respond to Price's statements, spokesman Abbott said there would be no comment.
Price also points out that Jefferson has tried to pin charges of racism on him before. In October, when two HISD investigators visited Kashmere as part of their probe of cheating on the TAAS, Price says assistant principal Vivian Davis accused him of calling them. Shortly afterward, he says, Jefferson told him parents had complained about him. Price claims Jefferson also called children in one fifth-grade class to her office and tried to tape-record their saying Price had made racist comments.
Price's union representative, Carol Galloway, put an end to those accusations, though Galloway says she didn't know about the tape recording. "It was a parental complaint, but it wasn't about racism. They didn't present it within policy, that's all."
The race-related allegations, along with the added reports about the Jasper incident, apparently rematerialized after the gun search proved fruitless. Now, with the Jasper tie-in, the story has been getting nibbles from national reporters. Although Price agreed to be interviewed by several TV stations, he laments the resulting effect on his life; he says he's been recognized at restaurants, the grocery store and the copy shop. "Six million people now think I'm a racist," says Price. "People asking me if I said those things; people asking me why did I drag that kid -- it tears my heart out.

subset: commoncrawl


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17 Comments
creepy but beautiful! voted!
Cool concept and great design. +1
vintage creepy, awesome treatment of the pic, would try tio blend in morte the skulls in my opninion, but the colors are perfect and the placemnet, even if its to boxxy gives the sensation of a tarot card wich as you said was the whole emotion and feel you were going for, nice piece, love it, great first sub, voted, keep em coming my friend….peace..
Pretty weird in a nice sort of way.
thanks so much for looking at my first sub, you guys rule!
creepy good, although the box design kinda dilutes the impact a bit. +1
Cool vintage look…I agree the skulls could be integrated/blended more, but awesome look to the piece none the less.
You have a very creepy vintage style artwork..
I love it…
voyager9 said: creepy good, although the box design kinda dilutes the impact a bit. +1
Maybe you can leave the edges torn or burned, but not straight?
In any case, +1. Like everyone has said it's "creepy" and good :)
i'm not liking how it's a whole rectangle thing. If the design blended with the shirt more, it'd look a lot better.
Still, it's very creative, and amazing as it is. Voted ;).
Ok…honey! I figured it out. I had to sign up before I could vote. So I voted! Good luck!!! xoxo J
Love it!!
I think it could work with other t-shirt colours too like DarkChocolate. You have my vote
nice illustration +1
Nice vintage design ! +1
nice work!


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OGDEN, Utah – According to the U.S. State Department 300,000 children in the United States are at risk of becoming victims of sex trafficking. In light of those figures Weber State University's Nontraditional Student Center will be hosting the "Not For Sale" Campaign from March 28-April 1.
One of the week's highlights will be a panel discussion, "Protecting Your Family," held on March 29 at 12:30 p.m. in WSU's Shepherd Union Building. The discussion will feature speakers from Utah Health and Human Rights and Child Rescue who work closely with victims of sex trafficking. They will expand on the "how-tos" of protecting self and family from falling prey to sexual exploitation.
Also scheduled are screenings of the movie "Taken," a fictional story depicting sex trafficking in Europe. On Monday and Wednesday students and the community will join in a "March For Victims" to protest sex trafficking. The 30-minute march through campus will begin in the Shepherd Union Atrium and end at the Stewart Bell Tower Plaza.
All events are free and open to the public. The campaign is sponsored by WSU's Nontraditional Student Center and Women's Studies, Feminist United Network and Stand.
Week's scheduled events include:
Monday (March 28): 6 p.m. Protest March, "March For Victims" – Shepherd Union
Tuesday (March 29): 12:30 p.m. Panel Discussion, "Protecting Your Family" –
Shepherd Union Room 312
7:30 p.m. Screening of film "Taken" – Shepherd Union Wildcat
Wednesday (March 30): 10:30 p.m. Protest March, "March For Victims" – Shepherd
Union Atrium
2:30 p.m. Screening of film "Taken" – Shepherd Union Wildcat
Friday (April 1): 1:30 p.m. Book discussion, "Not For Sale: The Return of the
Global Slave Trade & How We Can Fight It" – Shepherd Union
Room 232
Visit weber.edu/wsutoday for more news about Weber State University.
- cw -
- Contact:
Sarah Stanton, Nontraditional Student Center
801-626-7501 · email@example.com
Colette M. Williamson, office of Media Relations
801-626-7212 · email@example.com


text:
Privacy guaranteed - Your email is not shared with anyone.
Separate names with a comma.
Discussion in 'General Glocking' started by DaBigBR, Jul 18, 2006.
Try to guess which finger I'm holding up. I'll give you 3 guesses but you'll only need one.
Probably your thumb. You can take it out of your *** now.
Well I missed waking up to your's back there. Just trying recapture the feeling.
I think he means August 1st, but I may be mistaken. I know in our computers dates are given YYYY/MM/DD
I just got fitted for my duty belt today. Soon as I get it issued to me, I'll post the pics.
Thanks for the explanation. I hope you stay safe!
Here's my setup. I like having my back as clear as possible, saves me a lot of pain.
Left to right:
G&G ASP Cuff case w/ ASP Chain cuffs
Open top Double Mag case with 2 G22 Mag's (Soon to to be for G21 SF)
Loop for Baton/Stinger LED
Safariland Tactical Taser Holster w/ Taser and Taser Cartridge holder
Motorola XTS5000 In Metal holder w/ 2-wire earpiece and mic
Streamlight Stinger LED in Safariland holder
SL-20 ring
ASP in swivel scabbard
G22 in Safariland Raptor Lev III (Soon to be replaced by G21SF in same style holster)
MK-4 Pepper Spray in case
G&G ASP Cuff case w/ ASP Link cuffs
all on Safariland Duty Belt
Glock 36 worn on vest under right arm (for weak hand drawing)
whats that thing on the bottom?
Do you mean the belt keepers with the key keeper?
now that you named it... didnt notice its a line of beltkeepers
Above is the original belt that I passed out of our academy with 13 years ago. Nothing more than the .38, a dump pouch baton ring and handcuffs. I went all wild and got a double split six and 2 x comp2 speedloaders on it and was considered a "Tackleberry" hehe.
The current belt (below) is in standard configuration, with the addition os a leatherman and a torch ring.
You guys carry torches???
I kill me.
Need a hand?
Figure it out. Every vehicle has a torch. Every copper known to man carries one in the dark yet a torch ring is not standard!
Back to the top.
I added a body mic back to mine, unchanged other than that.
No spare mags 4teecal? What state are you in? Did Victoria go to Glocks yet?
I have a lot of stuff, pic doesn't show my car-mic holder.
Is it just me, or do you carry two OC cans, one in front of the taser upside down and one rightside up in front of your gun?
The one next to the mag holder is the OC can, the one next to my Taser is a flashlight.


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Love Live! Reoccupies the Yamanote Line
- Categories: Anime, News
- Date: Dec 20, 2014 16:22 JST
- Tags: Idol, Image Gallery, Itasha, Love Live!, Marketing, Otaku, Trains
Cheerful idol anime Love Live! has apparently achieved the awe-inspiring milestone of 10 million players for their cutesy rhythm game "School Idol Festival", and so is bringing the idols back to the trains of the Yamanote line to yet again to spread word of this achievement and hopefully hoover up even more players.
Users who have ridden the railway line rushed to twitter to share pictures of the now "transformed" trains:
The ads during the first Yamanote advertisement campaign:


text:
Artist description:
just a wand in the the grass
Canvas Draw Created Feb 18, 2012 640 x 460px Unless noted Copyright 2012 soraxfangirl.
We encourage you to submit a comment and let the artist know what you think of this drawing. Please log in or join to post a comment.
Post a comment
soraxfangirl Feb 23, 2012
Thanks i used the grass brush.
jellybean2011 Feb 18, 2012
what a clever idea and wonderful grass 5h fav
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DSC – TVR Sagaris Progress
Posted on August 10th, 2008 in GTR2 – 2 Comments
The Belgian Dutch Skinning Team has been making some progress with the latest addition to their Dutch Supercar Challenge mod for GTR2.
Guntha is turning a street-legal TVR Sagaris into a proper racing car. The car already sports a rear wing, the newest previews can be found below.


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Steve Webber has worked in the Real Estate Industry for over 35 years. He has been an agent, broker, manager, and real estate company owner. He was co-owner of one of the largest Coldwell Banker Company Franchises, with offices in Utah, Colorado and Nevada. He also was director of Sales at Promontory, a luxury golf and ski community in Park City for three years. He has been involved in thousands of real estate transactions. His experience in real estate gives his clients an advantage whether they are buying, selling, building or developing real estate. He holds the GRI, CRS and CRB professional designations. Steve is married to his wife Jill and they have four children. They live in St. George in Entrada.
As a sales professional working under the Sotheby's International Realty brand, Steve specializes in luxury and second homes, golf communities, and investment properties. He uses his experience and love for the Southern Utah area to help his clients discover the property that meets their unique needs. Steve's affiliation with Sotheby's International Realty gives his selling clients local, national, and international exposure when marketing their home.


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Ivomec Injection with Ivermectin
By Merial. 1% Ivermectin injectable solution to rid cattle of internal and external parasites including G.I. tract worms, lungworms, grubs, lice, and mange mites. Safe for calves, breeding bulls, and cows. Not for use in female dairy cattle of breeding age. Not to be administered by injection in horses. Each ml of Ivomec contains 10 mg of ivermectin, sufficient to treat 110 lb of body weight (maximum 10 ml per injection site). Give subcutaneously. Store at room temperature. Do not use if frozen. 50 ml pack. Not for use in small animals. Needles and syringes are available separately.
Please click on "More Information" for dosage instructions.
Ivomec Injection: Give subcutaneously only, to reduce risk of potentially fatal clostridial infection of the injection site. Animals should be appropriately restrained to achieve the proper route of administration. Use of a 16-gauge 1/2" to 3/4" needle is suggested. Inject under the loose skin in front of or behind the shoulder.


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Infinity by Journey – Classic Rock Pick of the Week
Three studio albums, and not much success.
A change was needed.
Time to find a strong lead singer, and that new lead singer was Steve Perry.
Robert Fleischmann was briefly the lead singer, but their were some differences and issues that just didn't work out.
'Infinity' was released by Columbia Records in 1978, and is the most successful Journey album to date.
Surprisingly though, the album only reached number 21 on the Billboard Top 200.
Roy Thomas Baker, who also worked with Queen, produced the album.
Baker had a successful method of stacking harmonies.
He would have the vocalists sing each harmony part in unison.
The effect made three or four voices sound like more.
You can hear this on 'Anytime' and 'Feeling that Way.'
Three singles came out of 'Infinity' – 'Wheel In The Sky', Feeling That Way/Anytime' and 'Lights.'
A great album from an amazing group.
That's Classic Rock.
Here's a cool video of 'Feeling That Way/Anytime."


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Thousands attend vigil at Jonathan Law High School in Milford for slain student Maren Sanchez
MILFORD - Thousands of people attended a vigil Monday night in Milford for Maren Sanchez, the student who was killed at Jonathan Law High School last Friday.
Many people at the vigil wore purple, Sanchez's favorite color, and carried purple flowers. Gov. Dannel Malloy was among the attendees.
Sanchez was involved in the school's production of "Little Shop of Horrors" and her drama teacher says the play will be performed in her memory.
Police say Sanchez, 16, was pushed down a stairwell and stabbed with a knife in the neck and torso by a male classmate. Students say the suspect attacked Sanchez after she declined his invitation to the junior prom, which was to be held that night, but was postponed after the attack. Police say they're investigating the prom angle.
Police have declined to identify the suspect, but he has been identified in media reports as 16-year-old Chris Plaskon, a junior at the school.
The junior prom has not been rescheduled yet, but the Autumn Rose Florist in Milford says that when it is, it will give out free corsages and boutonnieres made with purple flowers in honor of Sanchez.
A wake for Sanchez will be held Thursday at the Cody-White Funeral Home on Broad Street in Milford. The wake will be open to the public and will be held from 2 p.m. to 7 p.m. No funeral service is planned, but a memorial fund has been set up in her name.


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The Arizona Board of Regents voted Thursday to approve contract extensions for UA athletic director Greg Byrne, baseball coach Andy Lopez and women's golf coach Laura Ianello, and Steve Walker — the leader of the Wildcats' new sand volleyball program — received his first deal as a head coach.
The Board approved all contracts unanimously and without discussion during its meeting in Flagstaff.
Byrne received a two-year extension through 2018 at his current salary of $440,000 per year. The Wildcats' social media-savvy athletic director could be a hot name to replace retiring or older bosses at other programs; Thursday's extension, and a $500,000 retention bonus due to Byrne on Sept. 30, 2016, could be a deterrent to schools looking to hire him away.
Lopez, who makes a base salary of $160,000 per year, had his contract extended by one year through 2018. Lopez can make an extra $50,000 per year if he reaches performance bonuses.
Walker, a former UA volleyball assistant who was tabbed to run the sand volleyball program this year, will make $70,000 per year as part of a deal that runs through 2016.
Ianello received a raise of $6,041, bringing her base salary to $75,000 per year. Her new contract runs through 2016.
Byrne said Thursday that he appreciated the support of UA president Ann Weaver Hart and the board of regents.
"I think we've got a great group of coaches, student-athletes and staff, and I think we're collectively moving our program forward to have sustained and long-term success," he said.
Ianello, the golf coach, "has done a great job of continuing our wonderful tradition of success in women's golf," Byrne said.
"We want her to be our coach for a long time," he said.
The sand volleyball program is "certainly exciting," Byrne said.
Lopez, who won a College World Series championship in 2012, ranks among the UA's longest-tenured and most popular coaches.
"I love Andy Lopez,"
Byrne said. "If I could have my son play college baseball for any coach in the country, it would be him. … This is the way of showing the belief and support we have in him."


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Ever heard of a 'bagel head' injection? No, neither had we, but the bizarre beauty trend has taken Japan by storm.
If you're wondering how this works, it basically leaves a bagel-shaped circle on the forehead, hence the name (suddenly, bagels are no longer our favourite snack).
National Geographic's Taboo show has filmed the procedure, which starts with a saline injection in the forehead. The bump is then creating by pressing into the centre of the saline blob and voila, you've got your bagel.
Once the body has absorbed the saline, the bagel disappears. However, permanent or not, would you try this latest trend? Watch the video below (warning: do not watch if you have a fear of needles) and let us know your thoughts in a comment.
See more weird beauty treatments in the gallery below:


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Editor's Note: ESPN Insider has teamed with Blue Ribbon College Basketball Yearbook to provide a comprehensive look at all Division I teams. To order the complete 2006-07 edition of Blue Ribbon College Basketball Yearbook, visit www.blueribbonyearbook.com or call 1-866-805-BALL (2255).
(Information in this team report is as of October 1.)
It seems like forever since Valparaiso was in the NCAA Tournament. Or even the Mid-Continent championship game.
Records show it has been only two years. But with back-to-back MCC Tournament disappointments, the Crusaders have fallen on hard times, at least in comparison to the glory days of yesteryear, 2004. Injuries, for the second straight season, decimated Valparaiso's roster and sent the program into the second tier of conference teams after a long run at or near the top.
With Shawn Huff (7.5 ppg, 4.0 rpg) and Jimmie Miles (4.9 ppg, 1.7) hampered and limited to only 34 games combined, Homer Drew's crew struggled to find stability in 2005-06.
"That really changed our program the rest of the year," Drew said. "Their injuries really hit us hard. We never could get over the hump."
The season unraveled shortly after the turn of the new year. Huff missed the final 14 games with a broken foot and Miles missed most of January because of illness and injuries.
"We were 11-4 and had just won six in a row and were playing very, very well," said Drew, who had considered it his most talented team in almost a decade.
Four straight losses later, Valparaiso's hyped season came crashing down. The Crusaders even lost second-half leads of 22 points and 15 points to IUPUI and UMKC, respectively.
Valparaiso stumbled to a .500 conference record (eight conference wins were its lowest since 1991-92) before being blown out by eventual champion Oral Roberts in the MCC Tournament semifinals. The 19-point loss was Valparaiso's worst in the league tournament since Drew's first season in 1988-89.
Now, without the five leading scorers back on the nation's 13th-highest scoring team (79.6 points per game), Drew has got to wonder where the offense is going to come from.


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Artist description:
well as usual, i started off having no idea what i was doing >.< and here we are this weird colourful scene came out. Whats your thoughts on it?
Flash Draw Created May 19, 2009 470 x 338px Unless noted Copyright 2009 Random-ness-ness.
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Very cool, love this! ^^
its awesome random i love it 5hs
Wow! You have a great imagination! It kinda looks like a mountain/waterfall scape, with a canyon in the middle. Very interesting. The colours work well together.
when i look at it i think rocks,mountains,waterfall? great work!!! i like this kind stuff makes brain move :D


text:
Crystal Palace v Leicester City
Crystal Palace midfielder Mile Jedinak is nursing a sore back and his fitness will be assessed ahead of the visit of leaders Leicester.
Connor Wickham, James McArthur and Marouane Chamakh all remain sidelined by injuries.
Leicester's Andy King is a doubt with the sickness bug that caused him to miss Monday's win against Newcastle.
Fellow midfielder Matty James is not yet ready to a return from a cruciate knee ligament injury.
Jonathan Pearce: "Exactly a year ago Leicester were still bottom. But their 4-3 defeat at Spurs sparked the miracle. They won seven of nine games to stay up.
"Now, in one of the greatest football stories ever told, they can see the silverware shimmering. But they have to remain steadfast. If a remarkable year is bracketed with defeat, the wolves will be at the Foxes' door.
"Palace shouldn't beat them. They're the only side in the country without a 2016 league win. Their home form is shocking.
"But they know that they could be in a real relegation fight by the time their FA Cup semi-final comes around. They need a win. I've a sneaky feeling they'll get it."
Crystal Palace manager Alan Pardewbacks his side to derail Leicester's title bid, as they did to Liverpool and Manchester City in the past two seasons: "It has been a banana-skin, this place, in the past and it's typical of Crystal Palace
"Our history is littered with upsets against the odds, that's what this club is about. Not one pundit thinks we'll win, so that's actually when Palace are good.
"It's been almost the perfect season for them (Leicester) but it doesn't always come all the way through, that perfect run. Maybe their blips are still to come."
Leicester City manager Claudio Ranieri: "We have a tight-knit, wonderful dressing room and a team that works. However, we know we haven't done anything yet.
"Everyone is rooting for us to win the Premier League. It's incredible that a small club are punching above their weight like this in the big-money era.
"Now we're going to fight to qualify for Europe. And if we stay up there, we'll try to get into the Champions League and then go for the title. But we have to take it one step at a time."
I don't see Leicester winning all their remaining games and I just have a sneaking suspicion they will have a little hiccup here.
Crystal Palace remain on an awful run in the league but I watched them against Reading in the FA Cup last weekend and I think they can cause Leicester some problems.
Prediction: 2-1
Lawro's full predictions v BBC athletics commentator Steve Cram
- Think you can do better than Lawro? Predict the score for this match and the rest of this round's Premier League fixtures in our Predictor game.
- Leicester beat Crystal Palace for the first time in five matches in the reverse fixture last October, with Jamie Vardy scoring the only goal.
Crystal Palace
- Crystal Palace have gone 12 Premier League matches without a win (D4, L8).
- Alan Pardew's side have suffered five straight defeats at Selhurst Park, and only Aston Villa have a worse overall home record this season.
- They are the only club in the top four divisions without a league victory in 2016.
- Palace have failed to keep a clean sheet in their last 10 league matches, conceding 23 goals.
Leicester City
- The league leaders have won six of their last eight matches, with their only defeat in 12 coming at Arsenal last month (W7, D4, L1).
- Leicester have won 12 games by a single goal this season, more than any other Premier League side.
- Claudio Ranieri's team have the best away record in the top flight, picking up 31 points so far.
- The Foxes have scored in a league-high 27 of their 30 games this season.
- N'Golo Kante has made an unrivalled 128 tackles in the division this season.
- Leicester were top of the table with eight matches to play in 1962-63, but won only once in the remainder of the season and finished fourth.
- Jamie Vardy has scored 19 league goals this season. The last Leicester player to score 20 times in a top-flight season was Gary Lineker in 1984-85.