ECM510 Sigma-AldrichQCM Chemotaxis Cell Migration Assay, 96-well (8 µm), fluorimetric

Background Recent advances in cell therapies have provided potential opportunities for the treatment of chronic kidney diseases (CKDs). We investigated whether human kidney structures could be preformed in vitro for subsequent implantation in vivo to maximize tissue-forming efficiency. Methods Human renal cells were isolated from unused donor kidneys. Human renal cells were cultured and expanded. Migration was analyzed using growth factors. To form structures, cells were placed in a three-dimensional culture system. Cells were characterized by immunofluorescence, western blots and fluorescence-activated cell sorting using renal cell-specific markers for podocin, proximal and distal tubules and collecting ducts. An albumin uptake assay was used to analyze function. Three-dimensional cultures were implanted into athymic rat kidneys to evaluate survival. Results Human renal cells were effectively expanded in culture and retained their phenotype, migration ability and albumin uptake functions. Human renal cell in three-dimensional culture-formed tubules, which stained positively for proximal, distal tubule and collecting duct markers, and this was confirmed by western blot. Polarity of the tubular cells was determined by the presence of E-cadherin, N-cadherin and Na-K ATPase. Colocalization of labeled albumin and proximal tubule markers proved functionality and specificity of the newly formed tubules. An in vivo study showed that cells survived in the kidney for up to 6 weeks. Conclusions These findings demonstrate that human renal cell grown in three-dimensional culture are able to generate kidney structures in vitro. This system may ultimately be developed into an efficient cell-based therapy for patients with CKD.

Ischemic stroke affecting the adult brain causes increased progenitor proliferation in the subventricular zone (SVZ) and generation of neuroblasts, which migrate into the damaged striatum and differentiate to mature neurons. Meteorin (METRN), a newly discovered neurotrophic factor, is highly expressed in neural progenitor cells and immature neurons during development, suggesting that it may be involved in neurogenesis. Here, we show that METRN promotes migration of neuroblasts from SVZ explants of postnatal rats and stroke-subjected adult rats via a chemokinetic mechanism, and reduces N-methyl-D-asparate-induced apoptotic cell death in SVZ cells in vitro. Stroke induced by middle cerebral artery occlusion upregulates the expression of endogenous METRN in cells with neuronal phenotype in striatum. Recombinant METRN infused into the stroke-damaged brain stimulates cell proliferation in SVZ, promotes neuroblast migration, and increases the number of immature and mature neurons in the ischemic striatum. Our findings identify METRN as a new factor promoting neurogenesis both in vitro and in vivo by multiple mechanisms. Further work will be needed to translate METRN's actions on endogenous neurogenesis into improved recovery after stroke.

The activity of N-hexanoyl-D-erythro-sphingosine, a C6-ceramide against angiogenesis was tested in vitro and in vivo. The effect of ceramide in inhibiting MCF-7 cancer cells was also determined. The aim of this study was to potentiate the effect of ceramide as anti-angiogenic compound that can regulate tumor induced angiogenesis.C6-ceramide inhibited vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cells (HUVEC) tube formation in a dose-dependent manner within 24 hours. Ceramide at concentrations between 12.5 and 25 μM inhibited the viability of MCF-7 cells and reduced VEGF-induced cell migration in 24 hours. At 50 μM, ceramide induced MCF-7 cell death via autophagy as demonstrated by accumulation of MDC in ceramide-treated MCF-7 vacuoles. The expression of VEGF was reduced and the levels of cathepsin D in MCF-7 increased. In vivo, 50 μM ceramide caused a 40% reduction of new vessel formation in the CAM assay within 24 hours. Zebrafish exposed to 100 - 400 μM ceramide had a distinct disruption of blood vessel development at 48 hours post-fertilization. Ceramide-exposed embryos also had primary motoneurons exhibiting abnormal axonal trajectories and ectopic branching. Ceramide induced cell-death was not detected in the zebrafish assay. Collectively, these data indicate that ceramide is a potent anti-angiogenic compound and that the mechanism underlying its anti-angiogenic capabilities does not rely upon the induction of apoptosis.

Annexin II, an abundant phospholipids binding cell surface protein, binds tPA and functions as a regulator of fibrinolysis. Annexin II also mediates angiogenesis and enhances tumor growth and metastasis. However, the mechanism supporting this role is not known. Using human breast cancer model we show that invasive human breast cancer cells (MDA-MB231) synthesize annexin II and tissue plasminogen activator (tPA). In vitro both annexin II and tPA interacts which in turn convert zymogen plasminogen to reactive enzyme plasmin. Cell surface produced plasmin inhibited the migration of MDA-MB231 cells. Silencing of annexin II gene in MDA-MB231 cells abolished tPA binding therefore inhibited tPA dependent plasmin generation. These annexin II suppressed MDA-MB231 cells showed reduced motility. Immunohistochemical analysis of prediagnosed clinical specimens showed abundant secretion of tPA and expression of annexin II on the surface of invasive human breast cancer cells which correlates with neovascularization of the tumor. Taken together, these data indicate that annexin II may regulate localized plasmin generation in breast cancer. This may be an early event switching breast cancer from the prevascular phase to the vascular phase and thus contributing to aggressive cancer with the possibility of metastasis. The data provide a mechanism explaining the role of annexin II in breast cancer progression and suggest that annexin II may be an attractive target for therapeutic strategies aimed to inhibit angiogenesis and breast cancer.

Adipose tissue secretes factors linked to colon cancer risk including leptin. A hallmark of cancer is sustained angiogenesis. While leptin promotes angiogenesis in adipose tissue, it is unknown whether leptin can induce epithelial cells to produce factors that may drive angiogenesis, vascular development and therefore cancer progression. The purpose of this study was to compare the effects of leptin-stimulated colon epithelial cells differing in adenomatous polyposis coli (Apc) genotype (gatekeeper tumor suppressor gene for colon cancer) on angiogenesis. We employed novel colonic epithelial cell lines derived from the Immorto mouse [young adult mouse colon (YAMC)] and the Immorto-Min mouse [Immorto-Min colonic epithelial cell (IMCE)], which carries the Apc Min mutation, to study the effects of leptin-stimulated colon epithelial cells on angiogenesis. We utilized ex vivo rat mesenteric capillary bioassay and human umbilical vein endothelial cell (HUVEC) models to study angiogenesis. IMCE cells stimulated with leptin produced significantly more vascular endothelial growth factor (VEGF) than YAMC (268 +/- 18 versus 124 +/- 8 pg/ml; P < 0.01) cells. Leptin treatment induced dose-dependent increases in VEGF only in IMCE cells. Conditioned media from leptin (50 ng/ml)-treated IMCE cells induced significant capillary formation compared with control, which was blocked by the addition of a neutralizing antibody against VEGF. Conditioned media from leptin-treated IMCE cells also induced HUVEC cell proliferation, chemotaxis, upregulation of adhesion proteins and cell-signaling activation resulting in nuclear factor kappa B nuclear translocation and DNA binding due to VEGF. This is the first study demonstrating that leptin can induce preneoplastic colon epithelial cells to orchestrate VEGF-driven angiogenesis and vascular development, thus providing a specific mechanism and potential target for obesity-associated cancer.

Camptothecin (CPT) was conjugated to the N-terminal of a somatostatin analog (SSA) directly via a carbamate group and a basic N-terminal linking motif, D-Lys-D-Tyr-Lys-D-Tyr-D-Lys. This new CPT-SSA conjugate termed JF-10-81 was evaluated as a receptor-specific delivery system for its anti-invasive and anti-angiogenic activities. It was found that, in addition to blocking migration and invasion of highly invasive prostate cancer PC-3 cells, this conjugate also inhibited in vitro capillary-like tube formation of endothelial cells and in vivo angiogenesis in C57B1/6N female mice. JF-10-81 was found to block PC-3 cell attachment to various extracellular matrix components, mainly to vitronectin, the ligand of cell surface receptors integrin alphaVbeta3 and alphaVbeta5. Additionally, JF-10-81 reduced expression of integrins alphaVbeta3 and alphaVbeta5 on PC-3 cell surfaces, without effects on beta1 or any alphabeta1 heterodimers. This conjugate also inactivated phosphorylation of protein kinase B (PKB/Akt), down-regulated the expression of latent matrix metalloproteinase (MMP) -2 and MMP-9, but had little effect on MMP-3/-10. Meanwhile, membrane type-1 matrix metalloproteinase (MT1-MMP) and the tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) were not detectable in PC-3 cells. alphaVbeta3/alphaVbeta5 and MMP-2/-9 are known to be highly expressed in many tumor cells and play an important role in tumor progression. Our results support that this conjugate could possibly inhibit prostate cancer PC-3 cell invasion through a signaling pathway involving PI3K/Akt, alphaVbeta3/alphaVbeta5 and MMP-2/-9, and this SSA could be used as an efficient vector to deliver CPT or other cytotoxic agents to target sites for cancer therapy.

Patients with tuberous sclerosis complex (TSC) develop hamartomatous tumors showing loss of function of the tumor suppressor TSC1 (hamartin) or TSC2 (tuberin) and increased angiogenesis, fibrosis, and abundant mononuclear phagocytes. To identify soluble factors with potential roles in TSC tumorigenesis, we screened TSC skin tumor-derived cells for altered gene and protein expression. Fibroblast-like cells from 10 angiofibromas and five periungual fibromas produced higher levels of monocyte chemoattractant protein-1 (MCP-1) mRNA and protein than did fibroblasts from the same patient's normal skin. Conditioned medium from angiofibroma cells stimulated chemotaxis of a human monocytic cell line to a greater extent than conditioned medium from TSC fibroblasts, an effect blocked by neutralizing MCP-1-specific antibody. Overexpression of MCP-1 seems to be caused by loss of tuberin function because Eker rat embryonic fibroblasts null for Tsc2 (EEF Tsc2(-/-)) produced 28 times as much MCP-1 protein as did EEF Tsc2(+/+) cells; transient expression of WT but not mutant human TSC2 by EEF Tsc2(-/-) cells inhibited MCP-1 production; and pharmacological inhibition of the Rheb-mTOR pathway, which is hyperactivated after loss of TSC2, decreased MCP-1 production by EEF Tsc2(-/-) cells. Together these findings suggest that MCP-1 is an important paracrine factor for TSC tumorigenesis and may be a new therapeutic target.

Neural stem/progenitor cell (NSPC) migration toward sites of damaged central nervous system (CNS) tissue may represent an adaptive response for the purpose of limiting and/or repairing damage. Little is known of the mechanisms responsible for this migratory response. We constructed a cDNA library of injured mouse forebrain using subtractive suppression hybridization (SSH) to identify genes that were selectively upregulated in the injured hemisphere. We demonstrate that stem cell factor (SCF) mRNA and protein are highly induced in neurons within the zone of injured brain. Additionally, the SCF receptor c-kit is expressed on NSPCs in vitro and in vivo. Finally, we demonstrate that recombinant SCF induces potent NSPC migration in vitro and in vivo through the activation of c-kit on NSPCs. These data suggest that the SCF/c-kit pathway is involved in the migration of NSPCs to sites of brain injury and that SCF may prove useful for inducing progenitor cell recruitment to specific areas of the CNS for cell-based therapeutic strategies.

Current in vitro assays used in assessing tumor motility could be improved by the development of a simple technique that would facilitate studies of the impact of specific genes on pharmacologically altered chemotaxis. We developed a technique that improves on the classic transwell assay by using fluorescence and luminescence to assess chemotaxis. In this transient transfection system, co-transfection of a reporter construct and a gene with an unknown impact on motility are coupled with biochemical assays to quantitate the number of cells that have received a transferred gene, which subsequently crosses the membrane. This assay was found to be less variable than the conventional transwell chamber and is easily adaptable to studies of cell motility or cell invasion. We also demonstrate that this assay can detect the effect of both genetic and pharmacological inhibition of motility alone and in combination. It therefore has the potential to reveal additive or synergistic effects.