Stromal-derived Growth Factor Research Articles

AKT in Stromal Fibroblasts Controls Invasion of Epithelial Cells

The tumour microenvironment has an important role in cancer progression and recent reports have proposed that stromal AKT is activated and regulates tumourigenesis and invasion. We have shown, by immuno-fluorescent analysis of oro-pharyngeal cancer biopsies, an increase in AKT activity in tumour associated stromal fibroblasts compared to normal stromal fibroblasts. Using organotypic raft co-cultures, we show that activation of stromal AKT can induce the invasion of keratinocytes expressing the HPV type 16 E6 and E7 proteins, in a Keratinocyte Growth Factor (KGF) dependent manner. By depleting stromal fibroblasts of each of the three AKT isoforms independently, or through using isoform specific inhibitors, we determined that stromal AKT2 is an essential regulator of invasion and show in oro-pharyngeal cancers that AKT2 specific phosphorylation events are also identified in stromal fibroblasts. Depletion of stromal AKT2 inhibits epithelial invasion through activating a protective pathway counteracting KGF mediated invasions. AKT2 depletion in fibroblasts stimulates the cleavage and release of IL1B from stromal fibroblasts resulting in down-regulation of the KGF receptor (fibroblast growth factor receptor 2B (FGFR2B)) expression in the epithelium. We also show that high IL1B is associated with increased overall survival in a cohort of patients with oro-pharyngeal cancers. Our findings demonstrate the importance of stromal derived growth factors and cytokines in regulating the process of tumour cell invasion.

Stromal derived growth factor-1 (CXCL12) modulates synaptic transmission to immature neurons during post-ischemic cerebral repair

In response to ischemic injury, the brain mounts a repair process involving the development of new neurons, oligodendrocytes, and astrocytes. However, the manner in which new neurons integrate into existing brain circuitry is not well understood. Here we observed that during the four weeks after transient middle cerebral artery occlusion (MCAO), doublecortin (DCX)-expressing neural progenitors originating in the subventricular zone (SVZ) were present in the ischemic lesion borderzone, where they received γ-aminobutyric acid (GABA) inputs, a feature that is common to newly developing neurons. The chemokine stromal derived factor-1 (SDF-1 or CXCL12) was enriched in lesional endothelial and microglial cells for up to four weeks after transient MCAO, and application of SDF-1 to acute brain slices enhanced GABAergic inputs to the new neurons. These observations suggest that SDF-1 is in a position to coordinate neovascularization and neurogenesis during the repair process after cerebral ischemia–reperfusion.

Cancer-associated fibroblasts and M2-polarized macrophages synergize during prostate carcinoma progression

Inflammation is now acknowledged as an hallmark of cancer. Cancer-associated fibroblasts (CAFs) force a malignant cross talk with cancer cells, culminating in their epithelial-mesenchymal transition and achievement of stemness traits. Herein, we demonstrate that stromal tumor-associated cells cooperate to favor malignancy of prostate carcinoma (PCa). Indeed, prostate CAFs are active factors of monocyte recruitment toward tumor cells, mainly acting through stromal-derived growth factor-1 delivery and promote their trans-differentiation toward the M2 macrophage phenotype. The relationship between M2 macrophages and CAFs is reciprocal, as M2 macrophages are able to affect mesenchymal-mesenchymal transition of fibroblasts, leading to their enhanced reactivity. On the other side, PCa cells themselves participate in this cross talk through secretion of monocyte chemotactic protein-1, facilitating monocyte recruitment and again macrophage differentiation and M2 polarization. Finally, this complex interplay among cancer cells, CAFs and M2 macrophages, cooperates in increasing tumor cell motility, ultimately fostering cancer cells escaping from primary tumor and metastatic spread, as well as in activation of endothelial cells and their bone marrow-derived precursors to drive de novo angiogenesis. In keeping with our data obtained in vitro, the analysis of patients affected by prostate cancers at different clinical stages revealed a clear increase in the M2/M1 ratio in correlation with clinical values. These data, coupled with the role of CAFs in carcinoma malignancy to elicit expression of stem-like traits, should focus great interest for innovative strategies aimed at the co-targeting of inflammatory cells and fibroblasts to improve therapeutic efficacy.

Ocular neovascularization

Retinal and choroidal vascular diseases constitute the most common causes of moderate and severe vision loss in developed countries. They can be divided into retinal vascular diseases, in which there is leakage and/or neovascularization (NV) from retinal vessels, and subretinal NV, in which new vessels grow into the normally avascular outer retina and subretinal space. The first category of diseases includes diabetic retinopathy, retinal vein occlusions, and retinopathy of prematurity, and the second category includes neovascular age-related macular degeneration (AMD), ocular histoplasmosis, pathologic myopia, and other related diseases. Retinal hypoxia is a key feature of the first category of diseases resulting in elevated levels of hypoxia-inducible factor-1 (HIF-1) which stimulates expression of vascular endothelial growth factor (VEGF), platelet-derived growth factor-B (PDGF-B), placental growth factor, stromal-derived growth factor-1 and their receptors, as well as other hypoxia-regulated gene products such as angiopoietin-2. Although hypoxia has not been demonstrated as part of the second category of diseases, HIF-1 is elevated and thus the same group of hypoxia-regulated gene products plays a role. Clinical trials have shown that VEGF antagonists provide major benefits for patients with subretinal NV due to AMD and even greater benefits are seen by combining antagonists of VEGF and PDGF-B. It is likely that addition of antagonists of other agents listed above will be tested in the future. Other appealing strategies are to directly target HIF-1 or to use gene transfer to express endogenous or engineered anti-angiogenic proteins. While substantial progress has been made, the future looks even brighter for patients with retinal and choroidal vascular diseases.

A Novel Perspective On Hematopoietic Stem/Progenitor Cell Homing - an Expanding Family of Bone Marrow Homing Factors That Can Support SDF-1-Mediated Homing or Even Replace SDF-1

Abstract 1247 Background.Hematopoietic stem progenitor cells (HSPCs) are retained in bone marrow (BM) niches due to the stromal-derived growth factor-1 (SDF-1)–CXCR4 receptor axis and interactions between Very Late Antigen-4 (VLA-4, also known as α 4β1integrin) and its ligand, Vascular Adhesion Molecule-1 (VCAM-1 or CD106). While HSPCs express CXCR4 and VLA-4, their corresponding ligands, SDF-1 and VCAM-1, are expressed by cells in the BM microenvironment (e.g., osteoblasts and fibroblasts). While a role for the SDF-1–CXCR4 axis in the retention of HSPCs in BM under steady-state conditions is undisputed, its role in stem cell homing needs further clarification. For many years, it has been assumed that the chemotactic SDF-1 gradient across the BM–peripheral blood (PB) barrier determines whether cells home from PB into the BM microenvironment. However, this simple explanation has been challenged by several observations supporting the existence of SDF-1–CXCR4-independent homing mechanisms. For example, i) CXCR4−/− fetal liver HSPCs can home to BM in an SDF-1-independent manner, ii) homing of murine HSPCs made refractory to SDF-1 by preincubation and co-injection with a CXCR4 receptor antagonist (AMD3100) is normal or only mildly reduced, iii) HSPCs in which CXCR4 expression is reduced by a SDF-1 intrakine strategy remain able to engraft in lethally irradiated recipients, and, as we recently reported, iv) myeloablative conditioning for transplantation induces a highly proteolytic microenvironment in BM that leads to proteolytic degradation of SDF-1 (Cancer Res. 2010;70:3402, Leukemia. 2012;26:106). Aim of the study.Based on these observations strongly suggesting the involvement of other factors and/or supportive mechanisms in the SDF-1-mediated homing of HSPCs, we became interested in identifying these unknown factors, which support homing of HSPCs when SDF-1 is degraded in the proteolytic microenvironment of BM or even if it is completely absent. Experimental approach.We tested several growth factors, cytokines, bioactive lipids, extracellular nucleotides, and antimicrobial cationic peptides for their potential involvement in homing by employing i) Transwell migration assays and ii) signaling studies in the presence or absence of specific inhibitors. We studied the chemotactic responsiveness of these factors against BM, umbilical cord blood (UCB), and mobilized peripheral blood (mPB) cells. We also focused on the molecular mechanisms responsible for the observed phenomena. Salient observations.Out of >50 different factors tested in addition to SDF-1, only sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), ATP, UTP, and GTP, which are released by cells after BM microenvironment damaged by radio/chemotherapy conditioning for transplantation, were able to chemoattract BM-purified HSPCs. The responsiveness of HSPCs to these factors was inhibited after exposure to pertussis toxin (PTX) and after inhibiting MAPKp42/44 and AKT. Interestingly, in contrast to BM-isolated HSPCs, the chemotactic responsiveness of UCB and mPB HSPCs to these factors was significantly weaker, which suggests desensitization of the corresponding receptors by factors already present in peripheral blood plasma. We also found that BM stroma exposed to myeloablative doses of radio-chemotherapy secretes two antimicrobial cationic peptides, LL-37 and β2-defensin, that, while not direct chemottractants for HSPCs, strongly enhance the responsiveness of HSPCs to an SDF-1 gradient. This phenomenon plays a crucial role in situations in which the SDF-1 homing gradient is impaired by a highly proteolytic BM microenvironment after myeloablative conditioning for transplantation. Conclusions.Since all these direct chemottractants and priming factors are upregulated in BM after myeloblative conditioning for transplantation, a more complex picture of homing emerges that involves several factors that support, and in some situations even replace, the SDF-1–CXCR4 axis. We also conclude that the priming effects by LL-37 and β2-defensin play a critical role in homing of UCB- and mPB-derived HSPCs, which respond robustly to an SDF-1 gradient. Moreover, data in an animal model lend further support to this concept. Disclosures:No relevant conflicts of interest to declare.

A Novel Evidence That PNH Affected Cells Residing in Bone Marrow (BM) Due to Impaired Incorporation of CXCR4 and VLA-4 Into Membrane Lipid Rafts Show Defective SDF-1- and VCAM-1-Mediated Retention in BM What Leads to Their Increased Motility and Impaired Interaction with the BM Stem Cell Niches

AbstractAbstract 1256 Background.Hematopoietic stem progenitor cells (HSPCs) are retained in bone marrow (BM) niches due to the stromal-derived growth factor-1 (SDF-1)–CXCR4 receptor axis and interactions between Very Late Antigen-4 (VLA-4 or a4b1integrin) and its ligand, Vascular Adhesion Molecule-1 (VCAM-1 or CD106). While HSPCs express CXCR4 and VLA-4, their corresponding ligands, SDF-1 and VCAM-1, are expressed by cells in the BM microenvironment (e.g., osteoblasts and fibroblasts). Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon acquired hemolytic anemia that results from the expansion of hematopoietic stem cells with a mutation in one of the enzymes (PIG-A) responsible for glycosylphosphatidylinositol (GPI anchor) biosynthesis, which is a post-translation modification of proteins associated with lipid rafts on the cell membrane surface. Some of these proteins are involved in the resistance of erythrocytes to lysis by the final product of complement cascade (CC) activation, C5b-C9, also known as the membrane attack complex (MAC). Furthermore, as we reported, the CXCR4 receptor, which binds a-chemokine stromal derived factor-1 (SDF-1) is also associated with lipid rafts (Blood 2005;105:40–8) similarly as VLA-4 (a4b1integrin). Recently we demonstrated that the bioactive lipid sphingosine-1-phosphate (S1P), which is a major chemoattractant directing egress of HSPCs from bone marrow (BM) into peripheral blood (PB) during mobilization, is released from lyzed erythrocytes in C5b-C9/MAC-dependent manner (Leukemia 2010;24:976–85). We also demonstrated that PNH affected HSPCs are preferentially mobilized into PB which suggests their defective retention in BM microenvironment (Leukemia 2012;26:1722). Hypothesis.To explain preferential mobilization of PNH patients HSPCs into PB we hypothesized that BM residing HSPCs may have a defective adhesion in BM niches due to defect in lipid raft formation that are required for a proper function of CXCR4-SDF-1 and VLA-4-VCAM-1 retention axes. Experimental strategies.To address this issue we employed for adhesion, chemotaxis and lipid raft formation assays PIG-A mutated human K-562 and TF-1 cells and more importantly BM MNC derived from PNH patients (n=3). PNH affected HSPCs were sorted by FACS as CD34+FLAER− and normal unaffected HSPCs as CD34+FLAER+cell population. Using these cells we studied their i) adhesiveness to immobilized SDF-1 (CXCR4 ligand), VCAM-1 (VLA-4 ligand), fibronectin and BM stroma cells, ii) chemotactic responsiveness in Transwell assays to SDF-1 gradient, iii) ability to incorporate CXCR4 and VLA-4 receptors into membrane lipid rafts and iv) activation of signaling pathways relevant to cell adhesion/migration (MAPKp42/44 and AKT) in response to SDF-1 stimulation. Results.We found that both PIG-A mutated human K-562 and TF-1 cells and more important CD34+FLAER− HSPCs derived from BM of PNH patients in contrast to normal CD34+FLAER+HSPCs show defective adhesion to immobilized SDF-1, VCAM-I, fibronectin and BM stroma cells. This corresponded with weak responsiveness of these cells in chemotactic assays to SDF-1 gradient and weaker phosphorylation of MAPK042/44 and AKT after SDF-1 stimulation. Finally, direct confocal microscopy studies demonstrated defect in incorporation of CXCR4 and VLA-4 into membrane lipid rafts in CD34+FLAER− cells isolated from BM of PNH patients. Conclusions.Based on these observations, we propose a novel view of the pathogenesis of PNH and the expansion of PNH-affected cells in BM microenvironment. Accordingly, the lack of PIG-A protein, which plays an important role in lipid raft formation a proper function of CXCR4 and VLA-4, confers an adhesion defect and an advantage to PNH-affected HSPCs, which become more mobile. These PNH-mutated HSPCs over time may outcompete normal HSPCs for their niches in BM, due to their increased motility, and contribute to the PNH type of hematopoiesis. Defective retention of PNH affected HSPCs and they proper interaction with stem cell niches in BM may lead with time in some of the cases to their malignant transformation. We propose also that drugs that increase BM level of S1P and thus prevent egress of PNH HSPCs from their niches could be employed in PNH treatment along with CC inhibitors (Eculizumab). Disclosures:Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

Inactivation of Rb in stromal fibroblasts promotes epithelial cell invasion.

Stromal-derived growth factors are required for normal epithelial growth but are also implicated in tumour progression. We have observed inactivation of the retinoblastoma protein (Rb), through phosphorylation, in cancer-associated fibroblasts in oro-pharyngeal cancer specimens. Rb is well known for its cell-autonomous effects on cancer initiation and progression; however, cell non-autonomous functions of Rb are not well described. We have identified a cell non-autonomous role of Rb, using three-dimensional cultures, where depletion of Rb in stromal fibroblasts enhances invasive potential of transformed epithelia. In part, this is mediated by upregulation of keratinocyte growth factor (KGF), which is produced by the depleted fibroblasts. KGF drives invasion of epithelial cells through induction of MMP1 expression in an AKT- and Ets2-dependent manner. Our data identify that stromal fibroblasts can alter the invasive behaviour of the epithelium, and we show that altered expression of KGF can mediate these functions.

Nerve Growth Factor Promotes Endothelial Progenitor Cell-Mediated Angiogenic Responses

In response to ischemia, retinal neuronal cells express nerve growth factor (NGF), which can be proangiogenic. Endothelial progenitor cells (EPCs) can participate with the resident vasculature to promote angiogenesis. We postulated that NGF may stimulate CD34⁺ EPCs to convert to an angiogenic phenotype. Human CD34⁺ cells and human retinal endothelial cells (HRECs) were used to examine the effect of NGF on key steps associated with neovascularization. CD34⁺ cells and HRECs were stimulated with NGF (1 to 4 pM) for 24, 48, and 72 hours. Cell migration was measured using a modified Boyden chamber assay. Expression of the receptor for the cytokine stromal derived growth factor 1 (SDF-1), CXCR-4, was assessed by flow cytometry. In vitro angiogenesis was tested using a three-dimensional (3D) extracellular matrix with HRECs/CD34⁺ cell cocultures. NGF receptor activation was assessed by western analysis. NGF promoted proliferation of CD34⁺ cells but not HRECs. Pretreatment of CD34⁺ cells with NGF increased CXCR-4 expression in CD34⁺ cells, resulting in enhanced migration to SDF-1 (P < 0.0001). The enhanced tubule-forming effect of NGF in HRECs was further potentiated by coculture with NGF-pretreated CD34⁺ cells (P < 0.01). The beneficial effect of NGF was blocked (P < 0.0001) by the ERK inhibitor PD98059. In both CD34⁺ and HRECs, NGF increased phosphorylation of neurotrophic tyrosine kinase receptor type 1 (TrkA) receptor by ERK1 activation (P < 0.01). Our in vitro results suggest that NGF released from ischemic nerves in vivo may contribute to the "angiogenic switch" by stimulating the angiogenic behavior of CD34⁺ cells while minimally affecting resident retinal endothelial cells.

Abstract 2831: Studies on the mechanistic basis of the anticancer activity of 2,4-disulfonyl-PBN (OKN-007)

Abstract Sulfatase 2 (Sulf2) is a protein which resides in the extracellular matrix of cancer cells and enzymatically removes sulfate from the 6-O-sulfate esters of heparin. Several tumors have been shown to be enriched in Sulf2 in comparison to normal tissues where it is either absent or present at very low levels. The action of Sulf2 alters growth factor binding to the extracellular matrix of cells and has been shown to be important in the development of many cancers including breast, lung, liver, colon, and pancreas. Sulf2 mobilizes fibroblast growth factors (FGFs), vascular endothelial growth factor (VEGF) and stromal derived growth factor-1 (SDF-1) bound to heparin. We found that OKN-007 acts to weakly inhibit the aryl sulfatase activity of Sulf2 acting on the substrate 4-methylumbelliferyl-sulfate (4-MUS) and to more effectively inhibit the enzymatic activity of Sulf2 when it acts upon its natural substrate the 6-O-sulfate ester of heparin. The Sulf2 enzymatic activity was potently inhibited by suramin, a compound that has 6 phenyl-sulfonyl groups as part of its chemical structure and has been shown to have anti-cancer activity. Utilizing the natural substrate we have shown that other sulfonyl-phenyl compounds act to suppress the activity of Sulf2. We have conducted preliminary studies to see if OKN-007 has anti-cancer activity in a breast cancer mouse xenograph model. Balb/c female ovariectomized Estradiol pellet-implanted nude mice were implanted with MCF-7 breast cancer cells and OKN-007 was administered at 250mg/kg/day in the drinking water. The experiment was terminated at week 5 at which time the data showed that OKN-007 decreased the average tumor volume by 33%. OKN-007 has been shown to have anti-cancer activity in the rat C6 model of glioma. The anti-cancer mechanism of action of OKN-007 is still unknown. Recently we have explored the potential of OKN-007 to alter cancer cell migration in a gradient involving the chemokine SDF-1 and its cellular receptor CXCR4. The CXCL4/SDF-1 gradient is considered to be important in the metastasis of primary cancers. Results with rat C6 glioma cells, U87MG human glioma cells as well as MCF-7 breast cancer cells indicate that OKN-007 at low concentrations inhibits cancer migration in a CXCR4/SDF-1 gradient. These observations need to be explored in more depth utilizing animal models of cancer metastasis. Research supported in part by US Army Concept Grant W81XWH-09-1-0352. Dr. Floyd holds the Merrick Foundation Chair in Aging Research. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2831. doi:1538-7445.AM2012-2831

Abstract 1385: Molecular changes in breast tumors following bevacizumab-based treatment: Final analysis of a randomized neoadjuvant study of bevacizumab or placebo, followed by chemotherapy with or without bevacizumab, in patients with stage II or III breast cancer

Abstract Background: Bevacizumab (bev) has been widely studied in breast cancer (BC), yet no randomized trial in BC has reported in vivo molecular effects of bev on human tumor tissue. We conducted a trial to evaluate the safety, clinical &amp; molecular effects of neoadjuvant chemotherapy plus bev for locally advanced BC. Methods: 90 pts were randomized (2:1:2:1) to 1 of 4 arms: Arm A: TAC (docetaxel, T: 75 mg/m2, doxorubicin, A: 50 mg/m2, cyclophosphamide, C: 500 mg/m2) + low dose bev (7.5 mg/kg); Arm B: TAC + low dose placebo (P); Arm C: TAC + standard dose bev (15 mg/kg); Arm D: TAC + Std-P. A run-in cycle of bev or P was followed by 6 cycles of TAC plus P or bev. Tumor biopsies pre- and 7-10 days post-run-in with bev or P were taken. Unblinding occurred post surgery (Sx): Arms A/C received maintenance bev to complete 52 wks, Arms B/D received no further P. Eligible patients were females with &amp;gt;3 cm, HER2(-) BC. Endpoints included safety and pathologic complete response (pCR) in breast &amp; lymph nodes. To assess the effects of VEGF pathway inhibition on tumor vasculature, PCR analysis using the fluidigm array platform was performed on RNA from the pre- and post-run in samples to evaluate expression of 67 genes known to play a defined role in VEGF signaling. Results: 28 pts were assigned to Arm A, 30 to Arm C, and 32 to Arms B/D. 12 pts came off Tx before surgery (Arm A:2, Arm C:6, Arms B/D:4) and 78 received all Tx, underwent Sx and are evaluable for pCR. Two pts (6%) in Arms B/D, 5 pts (18%) in Arm A and 10 pts (33%) in Arm C had wound healing complications. 17% of pts in Arm C had Gr 3 (N=4) or Gr 4 (N=1) heart failure, none in Arms A/B/D. The pCR rate in evaluable patients was 18% (14/78): 5 (19%) Arm A, 3 (13%) Arm C, 6 (21%) Arms B/D. 45 samples (20 from Arms B/D and 25 from bev arms) were included in the pair-wise analysis to identify 6 genes that were differentially expressed. Bev resulted in a decrease in expression of Dll4, Cox2, Fibronectin (FN_EIIIB), angiopoietin 2 (Angpt2) and ESM1. In addition, upregulation of the cytokine, stromal derived growth factor (SDF1) was observed. Notably, genes such as CD31 or VE-cadherin were not appreciably differentially expressed. Of the genes that were downregulated, Dll4 and Angpt2 represent genes enriched in endothelial tip cells, which guide the migration of newly formed blood vessels. The decrease in these genes likely represents the effect of bev on reducing immature, growing vasculature in the tumor. Conclusions: This trial enabled the clinical and molecular evaluation of breast cancer tumor tissue pre and post-bev. Clinically, bev was associated with more wound healing and heart failure events and similar pCR rates compared to P. Tumor expression analysis for genes in the angiogenesis pathway supports the preclinical hypothesis that bev may primarily target immature tumor vasculature. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1385. doi:1538-7445.AM2012-1385

Abstract 4020: The Novel TrkB Agonist, 7,8-Dihydroxyflavone Enhances Stem Cell Mobilization After Stroke.

Background: Increasing levels of circulating Hematopoietic Stem Cells (HSC)/Hematopoietic Progenitor Cells (HPC), bone marrow derived mononuclear cells that promote repair in areas of injury, have been demonstrated to correlate with improved neurological function following stroke, suggesting a potentially critical role for HSC/HPC’s in limiting stroke injury and/or facilitating stroke recovery. Flavonoids, found in plants and fruit, exert anti-oxidative effects. Recent studies have demonstrated that 7,8 Dihydroxyflavone (DHF) is a potent TrkB agonist mimicking Brain Derived Neurotropic Factor, thus making it a powerful potential tool for treating neurological disorders. Stromal Derived Growth Factor 1-Alpha (SDF1-A) along with its receptor CXCR4 is a potent chemo attractant released by areas of injury. SDF1-A has been shown to mobilize HSC/HPC from the bone marrow to the blood and lead to ‘homing’ of the cells to an area of injury. We investigated the effect of DHF on HSC/HPC function following cerebral ischemia. Methods: Ischemic damage was induced in adult male Long Evans hooded rats (350-400g) with a peri-MCA injection of the vasoconstriction peptide ET-1. The rats were sacrificed at 24 hours post surgery and their bone marrow and blood HSC/HPC enriched using nanoparticles tagged with LIN negative and CD90 markers. Results: Stroked animals showed an increase in bone marrow production of HSC/HPC versus control animals (31.9±7 versus 2±0.5, p&lt;0.05). The mobilization of the HSC/HPC from the bone marrow to the blood was also significantly higher in the stroked animals versus control animals (43±19 versus 3.6±0.3, p&lt;0.05). Following stroke, DHF pre-treated HSC/HPC’s demonstrated significantly improved migration along an SDF-1 gradient compared to controls (129±1.0 versus 108±1.15, p&lt;0.05), despite the fact that DHF alone provided no independent migratory stimulus. Conclusions: The results suggest that DHF may be a viable compound to facilitate HSC/HPC migration post-stroke.

SDF and GABA interact to regulate axophilic migration of GnRH neurons

Stromal derived growth factor (SDF-1) and gamma-aminobutyric acid (GABA) are two extracellular cues that regulate the rate of neuronal migration during development and may act synergistically. The molecular mechanisms of this interaction are still unclear. Gonadotropin releasing hormone-1 (GnRH) neurons are essential for vertebrate reproduction. During development, these neurons emerge from the nasal placode and migrate through the cribriform plate into the brain. Both SDF-1 and GABA have been shown to regulate the rate of GnRH neuronal migration by accelerating and slowing migration, respectively. As such, this system was used to explore the mechanism by which these molecules act to produce coordinated cell movement during development. In the present study, GABA and SDF-1 are shown to exert opposite effects on the speed of cell movement by activating depolarizing or hyperpolarizing signaling pathways, GABA via changes in chloride and SDF-1 via changes in potassium. GABA and SDF-1 were also found to act synergistically to promote linear rather than random movement. The simultaneous activation of these signaling pathways, therefore, results in tight control of cellular speed and improved directionality along the migratory pathway of GnRH neurons.

Elevated Circulating Angiogenic Progenitors and White Blood Cells Are Associated with Hypoxia-Inducible Angiogenic Growth Factors in Children with Sickle Cell Disease

We studied the number and function of angiogenic progenitor cells and growth factors in children aged 5–18 years without acute illness, 43 with Hemoglobin SS and 68 with normal hemoglobin. Hemoglobin SS subjects had at least twice as many mononuclear cell colonies and more circulating progenitor cell than Control subjects. Plasma concentrations of erythropoietin, angiopoietin-2, and stromal-derived growth factor (SDF)-1α were significantly higher in children with Hemoglobin SS compared to Control subjects. In a multivariate analysis model, SDF-1α concentration was found to be associated with both CPC number and total white blood cell count in the Hemoglobin SS group, suggesting that SDF-1α produced by ischemic tissues plays a role in mobilizing these cells in children with Hemoglobin SS. Despite having a higher number of angiogenic progenitor cells, children with Hemoglobin SS had slower migration of cultured mononuclear cells.

A Novel Perspective on Stem Cell Homing – Emerging Interplay Between Bioactive Lipids As Potent Chemoattractants and Cationic Peptides As Underappreciated Modulators of Responsiveness to SDF-1 Gradients

Abstract 2957 Background.Hematopoietic stem progenitor cells (HSPCs) are retained in bone marrow (BM) niches due to the stromal-derived growth factor-1 (SDF-1)–CXCR4 receptor axis and interactions between Very Late Antigen-4 (VLA-4 or a4b1 integrin) and its ligand, Vascular Adhesion Molecule-1 (VCAM-1 or CD106). While HSPCs express CXCR4 and VLA-4, their corresponding ligands, SDF-1 and VCAM-1, are expressed by cells in the BM microenvironment (e.g., osteoblasts and fibroblasts). While a role for the SDF-1–CXCR4 axis in the retention of HSPCs in BM under steady state conditions is undisputed, its role in stem cell homing needs further clarification. For many years, it has been proposed that concentration differences between the chemotactic SDF-1 gradient across the BM–peripheral blood (PB) barrier determines whether cells will home from PB into the BM microenvironment. However, this simple explanation has been challenged by several observations supporting the existence of SDF-1–CXCR4-independent homing mechanisms. For example, i) CXCR4−/– fetal liver HSPCs can home to BM in an SDF-1-independent manner, ii) homing of murine HSPCs made refractory to SDF-1 by preincubation and co-injection with a CXCR4 receptor antagonist (AMD3100) is normal or only mildly reduced, iii) HSPCs in which CXCR4 expression is reduced by a SDF-1 intrakine strategy remain able to engraft in lethally irradiated recipients, and as we recently reported, iv) myeloablative conditioning for transplantation induces a highly proteolytic microenvironment in BM that leads to proteolytic degradation of SDF-1 (Leukemia 2011, doi: 10.1038/leu.2011.185). Aim of the study. Based on these observations that strongly suggest the involvement of other factors and/or supportive mechanisms in the SDF-1-mediated homing of HSPCs we become interested in a role of bioactive lipids (sphingosine-1 phosphate; S1P and ceramide-1 phosphate; C1P) as well as priming phenomenon of SDF-1-CXCR4 axis by cationic antimicrobial peptides (CAMPs) in HSPCs homing. Results. We present evidence that gradients of bioactive lipids, such as S1P and C1P, which are products of membrane lipid metabolism, are involved in stem cell lodgment into BM. In addition, we present another important mechanism that enhances stem cell response to an low SDF-1 concentration gradient, namely, the “priming effect”. This mechanism is based on a phenomenon in which CAMPs such as complement cascade cleavage fragment C3a and stroma fibroblast- and granulocytes-derived antimicrobial peptide LL-37 significantly increase (up to 25 times) the chemotactic responsiveness of HSPCs to very low “tissue concentration-relevant” SDF-1 gradients (1–3 ng/ml). To support this further murine HSPCs primed ex vivo with CAMPs engrafted by 3–5 days faster in lethally irradiated littermates. At the molecular level, this sensitization of the responsiveness to SDF-1 after exposure to CAMPs depends on the incorporation of the CXCR4 receptor into membrane lipid rafts. This phenomenon is specific for SDF-CXCR4 axis as we did not observe that this phenomenon influences S1P- and C1P-mediated in vitro chemotaxis. Overall, these changes of SDF-1, S1P, and C1P chemotactic gradients and the appearance of priming factors in the BM microenvironment and PB plasma are triggered during conditioning for transplantation by radio-chemotherapy by the induction of a proteolytic microenvironment in BM and the activation of the complement cascade (CC). Conclusions. We provide in vivo evidence that C3a and LL-37, which have primarily antimicrobial functions and are harmless to mammalian cells, could be clinically applied to accelerate engraftment as ex vivo priming agent for transplanted human HSPCs. This novel approach based on a short ex vivo incubation of HSPCs in the graft before transplantation by CAMPs (C3a and LL-37) is of particular important in umbilical cord blood transplantations, where the number of available HSPCs in single unit for transplant is limited. This strategy is currently tested in the clinic. Disclosures:No relevant conflicts of interest to declare.

Differential Responsiveness of HSPCs Harvested From Bone Marrow (BM), Mobilized Peripheral Blood (mPB) and Umbilical Cord Blood (UCB) to the BM Homing Factors Stromal-Derived Factor-1 (SDF-1), Sphingosine-1 Phosphate (S1P) and Ceramide-1 Phosphate (C1P) Is Related to the Desensitization of mPB and UCB HSPCs by S1P and C1P Present in mPB and UCB Plasma

Abstract 2958 Background.Hematopoietic stem progenitor cells (HSPCs) are retained in bone marrow (BM) niches in stromal-derived growth factor-1 (SDF-1)–CXCR4 receptor axis-dependent manner. While a role for the SDF-1–CXCR4 axis in the retention of HSPCs in BM under steady state conditions is undisputed, recent evidence confirms that due to induction of proteolytic microenvironment in BM after myeloablative radio-chemotherapy, SDF-1 level in BM decreases (Leukemia 2011, doi: 10.1038/leu.2011.185) which supports the potential role of other non-SDF-1-mediated homing mechanisms. The cumulative evidence indicates that gradients of bioactive lipids, such as sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P), are important homing factors. To support this further we demonstrated recently that S1P and C1P are upregulated in BM after conditioning for transplantation (Leukemia 2011, doi: 10.1038/leu.2011.185). Hypothesis. Based on the fact that SDF-1, S1P and C1P are present at relatively high concentrations in umbilical cord blood (UCB) and mobilized peripheral blood (mPB) plasma, they may desensitize responsiveness of HSPCs to BM homing gradients of SDF-1, S1P and C1P. Experimental approach. Based on this we i) measured concentration of SDF-1, S1P and C1P in BM aspirates, mPB and UCB, ii) evaluated chemotactic responsiveness of BM-, UCB- and mPB-derived HSPCs to homing gradients of SDF-1, S1P and C1P and iii) investigated molecular mechanisms potentially involved in this phenomenon by examining the surface expression of CXCR4 and S1P receptors 1–5 (S1PR1-5) at baseline and following exposure to appropriate ligands. Finally, we modulated the expression of S1PR1-5 on HSPCs and their responsiveness to chemotactic gradients by removal of S1PR1-5 ligands from the culture medium. Results. The highest expression of S1PR1-5 was detected on the surface of BM-derived CD34+/Lin− cells as compared to mPB and UCB counterparts. The downregulation of S1PR1-5 on mPB- and UCB-derived BM CD34+Lin− cells correlated with elevated level of circulating S1P in UCB and mPB plasma. Next, we found that BM-derived HSPCs responded more robustly to S1P and C1P as compared to SDF-1 when these chemoattractants were employed at physiologically relevant concentrations. Addition of S1P to the BM-derived HSPCs or incubation of BM HSPCs in S1P and C1P rich UCB or mPB plasma lead to downregulation of S1PR1-5 and desensitized their responsiveness to S1P and C1P, but not SDF-1 gradient. The expression of S1PR1-5 and responsiveness to S1P gradient by UCB- and mPB-derived HSPCs, however, could be re-established after incubating HSPCs in S1P-free medium. At the same time, since SDF-1 concentration in mPB and UCB is very low, the responsiveness of mPB- and UCB-derived HSPCs to SDF-1 gradient was not affected. Conclusions. We demonstrate, for the first time, significant differences in responsiveness of HSPCs from different sources to homing gradients of bioactive lipids. The relatively high concentration of S1P and C1P in mPB and UCB plasma may potentially desensitize responsiveness of HSPCs to BM homing gradients of bioactive lipids and interfere with their homing. These observations will have substantial clinical implications in HSPCs’ transplantation. Disclosures:No relevant conflicts of interest to declare.

Insulin modulates ischemia-induced endothelial progenitor cell mobilization and neovascularization in diabetic mice

Decreased levels of circulating endothelial progenitor cells (EPCs) predict increased risk of cardiovascular events in diabetic patients. Insulin treatment exerts important cardiovascular protection. Whether and how insulin participates in the EPC regulation of postnatal neovascularization are currently unclear. We employed a mouse hindlimb ischemia model to study EPC mobilization in non-diabetic and streptozotocin-induced diabetic mice. Insulin was administered to diabetic animals postoperatively. To determine the role of EPCs contributing to postnatal vasculogenesis, we used bone marrow-transplanted mice whose bone marrow cells selectively expressed enhanced green fluorescent protein (EGFP). Insulin treatment improved EPC mobilization into peripheral blood, accelerated transcutaneous oxygen pressure restoration and increased capillary density in the ischemic limb associated with partial incorporation of EGFP-positive cells into the capillaries. Insulin treatment restored ischemia-induced release of stromal-derived growth factor 1α and vascular endothelial growth factor (VEGF), and consequently enhanced the activity of Akt and endothelial nitric oxide synthase (eNOS) as well as matrix metalloproteinase-9 in bone marrow. Insulin also augmented tissue-level activation of VEGF/Akt/eNOS pathway. However, all such effects of insulin were completely blocked by combined treatment with a NOS inhibitor. Our data suggested that insulin treatment improved ischemia-induced EPC mobilization and contributed to compensatory neovascularization in diabetic mice through a VEGF/eNOS-related pathway.

An evaluation of bone marrow stromal-derived growth factor-1 and interleukin-8 levels in patients with stage I-III breast cancer with disseminated tumor cells.

10610 Background: Tumor cells that disseminate to the bone marrow (disseminated tumor cells, or DTCs), have been identified in 30% of stage I-III breast cancer (BC) patients and are known to predict outcome. However, the mechanisms involved in DTC metastasis to the bone marrow are unknown. We hypothesized that increases in bone turnover may result in the release of bone growth and “homing” factors into the bone marrow, facilitating BC metastasis to bone marrow and providing a “pre-metastatic niche” for BC cells. The purpose of this study was to determine if bone marrow plasma levels of two well characterized BC-bone “homing” chemokines, stromal-derived growth factor-1 (SDF-1) and interleukin-8 (IL-8), are increased in BC patients with DTCs. Methods: Sixty-five clinical stage I-III BC patients provided informed consent to participate in an IRB-approved study involving collection of bone marrow at the time of surgery for their primary BC. DTCs were assessed using an anti-cytokeratin antibody cocktail following ficoll enrichment and cytospin. Bone marrow plasma SDF-1 and IL -8 levels were quantified using enzyme-linked immunoassays. Results: Mean age was 54 years. Eighty-two percent of patients had T1/T2 tumors, and 38% had lymph node metastases. DTCs were identified in 12/65 (18.5%) of patients. No statistically significant correlations were observed between primary tumor characteristics (ER, PR, HER2, lymph node status, tumor grade) and presence of DTCs. Mean bone marrow plasma SDF-1 levels (1359 ± 379 pg/mL vs. 525 ± 312 pg/mL; p ≤0.001) and IL-8 levels (559 ± 108 pg/mL vs. 62 ± 45 pg/mL; p≤0.001) were significantly higher in DTC-positive vs. DTC-negative patients. Conclusions: Bone marrow plasma SDF-1 and IL-8 levels were significantly increased in patients with DTCs compared to DTC-negative patients. Further studies are needed to determine if patients harboring DTCs have unique microenvironmental factors within the bone marrow that predispose them to tumor cell dissemination.

Abstract 3481: The SDF-1α/PDGF-B pathway plays a critical role in bone marrow cell – derived pericyte differentiation during vasculogenesis of Ewing's sarcoma

Abstract The purpose of this study was to investigate the role of the SDF-1α/PDGF-B pathway in bone marrow cell (BMC) differentiation into pericytes during Ewing's sarcoma growth. Previous findings form our group demonstrated that BMCs migrate to TC71 and A4573 tumors, differentiate into endothelial cells and pericytes and, participate in the development of the tumor vasculature. Vascular endothelial growth factor VEGF165 is the chemotactic stimulus for BMCs. Introducing stromal derived growth factor SDF-1α into VEGF165-inhibited tumors induced chemotaxis of BMCs to a perivascular area where they differentiated into pericytes and up-regulated platelet derived growth factor PDGF-B mRNA levels. These observations prompted us to further investigate the correlation between SDF-1α and PDGF-B and the role of this pathway in BMC differentiation into pericytes. We found that SDF-1α regulates PDGF-B expression through the ELK-1transcription factor and that the SDF-1α/PDGF-B pathway is critical for BMC differentiation into pericytes in vitro. Here, we used AMD3100 to specifically inhibit SDF-1α in both TC71 and A4573 Ewing's tumors and analyzed the effect of SDF-1α inhibition on PDGF-B expression and BMC differentiation into pericytes in vivo. We performed a BM transplant from GFP+ transgenic mice into nude mice in order to track BMC migration to the tumor site. Following engraftment, the mice were injected with either TC71 or A4573 Ewing's sarcoma cells. Once the tumors became palpable, the mice received daily subcutaneous injections of either PBS or AMD3100 for the duration of the experiment. Tumor sections were analyzed by immunohistochemistry (IHC). We found that inhibiting SDF-1α led to smaller vessels with smaller lumens and to a decrease in the microvessel density. We intravenously injected the mice with Hoechst prior to sacrificing and found that AMD3100 decreased tumor blood vessel perfusion. TUNEL staining also showed an increase in tumor apoptosis. To elucidate the effect of SDF-1α on vasculogenesis of Ewing's sarcoma, we stained tumor sections for GFP, CD31, desmin and NG2. Although AMD3100 did not inhibit BMCs from migrating to the tumor site, it decreased BMC differentiation into desmin and NG2 pericytes. We also stained the tumor sections for PDGF-B and confirmed that inhibiting SDF-1α also inhibited PDGF-B. These findings support our previous data that SDF-1α regulates PDGF-B and that this pathway is critical for the differentiation of BMCs into pericytes. All together, our data demonstrates the role of SDF-1α as a signaling molecule which regulates the expression of angiogenic factors such as PDGF-B that are critical for pericyte maturation during tumor growth. Understanding these pathways will allow us to devise new therapeutic approaches for targeting tumor neo-vascularization which may be applied not only to Ewing's sarcoma but to other tumor models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3481. doi:10.1158/1538-7445.AM2011-3481

Improved Biomechanical Properties Of Diabetic And Non-diabetic Skin At Baseline Following Pretreatment With Lentiviral Mediated Overexpression Of Stromal-Derived Growth Factor 1α (SDF-1α)

Improved Biomechanical Properties Of Diabetic And Non-diabetic Skin At Baseline Following Pretreatment With Lentiviral Mediated Overexpression Of Stromal-Derived Growth Factor 1α (SDF-1α)

Estradiol Stimulates Capillary Formation by Human Endothelial Progenitor Cells

Endothelial progenitor cells (EPCs) repair damaged endothelium and promote capillary formation, processes involving receptor tyrosine kinases (RTKs) and heme oxygenase 1 (HO-1). Because estradiol augments vascular repair, we hypothesize that estradiol increases EPC proliferation and capillary formation via RTK activation and induction of HO-1. Physiological concentrations of estradiol (10 nmol/L) increased EPC-induced capillary sprout and lumen formation in matrigel/fibrin/collagen systems. Propyl-pyrazole-triol (PPT; 100 nmol/L; estrogen receptor [ER]-α agonist), but not diarylpropionitrile (ER-β agonist), mimicked the stimulatory effects of estradiol on capillary formation, and methyl-piperidino-pyrazole (ER-α antagonist) abolished the effects of estradiol and PPT. Three different RTK activators (vascular endothelial growth factor, hepatocyte growth factor, and stromal derived growth factor 1) mimicked the capillary-stimulating effects of estradiol and PPT. SU5416 (RTK inhibitor) blocked the stimulatory effects of estradiol and PPT on capillary formation. Estradiol increased HO-1 expression by 2- to 3-fold, an effect blocked by SU5416, and PPT mimicked the effects of estradiol on HO-1. The ability of estradiol to enhance capillary formation, increase expression of HO-1, and augment phosphorylation of extracellular signal–regulated kinase 1/2, Akt, and vascular endothelial growth factor receptor 2 was mimicked by its cell-impermeable analog BSA estradiol. Actinomycin (transcription inhibitor) did not alter the effects of estradiol on RTK activity or vascular endothelial growth factor secretion. We conclude that estradiol via ER-α promotes EPC-mediated capillary formation by a mechanism that involves nongenomic activation of RTKs and HO-1 activation. Estradiol in particular and ER-α agonists in general may promote healing of injured vascular beds by promoting EPC activity leading to more rapid endothelial recovery and capillary formation after injury.