Endothelial progenitor cell biology in disease and tissue regeneration

Endothelial progenitor cells are increasingly being studied in various diseases ranging from ischemia, diabetic retinopathy, and in cancer. The discovery that these cells can be mobilized from their bone marrow niche to sites of inflammation and tumor to induce neovasculogenesis has afforded a novel opportunity to understand the tissue microenvironment and specific cell-cell interactive pathways. This review provides a comprehensive up-to-date understanding of the physiological function and therapeutic utility of these cells. The emphasis is on the systemic factors that modulate their differentiation/mobilization and survival and presents the challenges of its potential therapeutic clinical utility as a diagnostic and prognostic reagent

Synthetic Toll Like Receptor-4 (TLR-4) Agonist Peptides as a Novel Class of Adjuvants

Background: Adjuvants serve as catalysts of the innate immune response by initiating a localized site of inflammation that is mitigated by the interactions between antigens and toll like receptor (TLR) proteins. Currently, the majority of vaccines are formulated with aluminum based adjuvants, which are associated with various side effects. In an effort to develop a new class of adjuvants, agonists of TLR proteins, such as bacterial products, would be natural candidates. Lipopolysaccharide (LPS), a major structural component of gram negative bacteria cell walls, induces the systemic inflammation observed in septic shock by interacting with TLR-4. The use of synthetic peptides of LPS or TLR-4 agonists, which mimic the interaction between TLR-4 and LPS, can potentially regulate cellular signal transduction pathways such that a localized inflammatory response is achieved similar to that generated by adjuvants. Methodology/Principal Findings: We report the identification and activity of several peptides isolated using phage display combinatorial peptide technology, which functionally mimicked LPS. The activity of the LPS-TLR-4 interaction was assessed by NF-kB nuclear translocation analyses in HEK-BLUE TM-4 cells, a cell culture model that expresses only TLR-4, and the murine macrophage cell line, RAW264.7. Furthermore, the LPS peptide mimics were capable of inducing inflammatory cytokine secretion from RAW264.7 cells. Lastly, ELISA analysis of serum from vaccinated BALB/c mice revealed that the LPS peptide mimics act as a functional adjuvant

Estrogen Induced Metastatic Modulators MMP-2 and MMP-9 Are Targets of 3,3′-Diindolylmethane in Thyroid Cancer

Thyroid cancer is the most common endocrine related cancer with increasing incidences during the past five years. Current treatments for thyroid cancer, such as surgery or radioactive iodine therapy, often require patients to be on lifelong thyroid hormone replacement therapy and given the significant recurrence rates of thyroid cancer, new preventive modalities are needed. The present study investigates the property of a natural dietary compound found in cruciferous vegetables, 3,3'-diindolylmethane (DIM), to target the metastatic phenotype of thyroid cancer cells through a functional estrogen receptor.Thyroid cancer cell lines were treated with estrogen and/or DIM and subjected to in vitro adhesion, migration and invasion assays to investigate the anti-metastatic and anti-estrogenic effects of DIM. We observed that DIM inhibits estrogen mediated increase in thyroid cell migration, adhesion and invasion, which is also supported by ER-α downregulation (siRNA) studies. Western blot and zymography analyses provided direct evidence for this DIM mediated inhibition of E(2) enhanced metastasis associated events by virtue of targeting essential proteolytic enzymes, namely MMP-2 and MMP-9.Our data reports for the first time that DIM displays anti-estrogenic like activity by inhibiting estradiol enhanced thyroid cancer cell proliferation and in vitro metastasis associated events, namely adhesion, migration and invasion. Most significantly, MMP-2 and MMP-9, which are known to promote and enhance metastasis, were determined to be targets of DIM. This anti-estrogen like property of DIM may lead to the development of a novel preventive and/or therapeutic dietary supplement for thyroid cancer patients by targeting progression of the disease

LPS peptide mimics lead to NF-κB nuclear translocation.

<p>Nuclear translocation studies of NF-κB were performed by Western blot analysis on both RAW264.7 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030839#pone-0030839-g003" target="_blank">Fig. 3A</a>), HEK-BLUE™-4 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030839#pone-0030839-g003" target="_blank">Fig. 3B</a>), and HEK293 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030839#pone-0030839-g003" target="_blank">Fig. 3C</a>) cells. Cells were stimulated with peptide, either RS01or RS09, at a concentration of 5 µg/ml per 5×10⁴ cells at various time points (15, 30, 60, and 120 min). Nuclear protein fractions were assayed for NF-κB while cytoplasmic proteins fractions were assayed for both NF-κB and IκB-α. HDAC was used as a nuclear protein loading control and actin was used as a cytoplasmic protein loading control. For HEK293 cells, no differences in the levels of cytoplasmic and nuclear NF-kB were observed suggesting that LPS, RS01, and RS09 do not activate the non-TLR-4 expressing HEK293 cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030839#pone-0030839-g003" target="_blank">Fig. 3C</a>).</p

LPS Specific Phage Peptide Sequences.

<p>LPS Specific Phage Peptide Sequences.</p

RAW264.7 secretes inflammatory cytokines in response to RS01 and RS09.

<p>RAW264.7 cells were seeded at a density of 1×10⁶ cells per well of a six well plate followed by addition of either RS01 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030839#pone-0030839-g005" target="_blank">Fig. 5A</a>) or RS09 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030839#pone-0030839-g005" target="_blank">Fig. 5B</a>) and incubated for 24 h after which the culture media was collected. LPS was used as a positive control for both RS01 and RS09 and therefore represented on both <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030839#pone-0030839-g005" target="_blank">Figures 5A and 5B</a>. Culture media was then analyzed for specific cytokine using an antibody array kit. Both RS01 (triangle) and LPS (square) were capable of inducing inflammatory cytokines and chemokines from RAW264.7. Each cytokine is represented as an IDV value which was calculated by comparing the density of each spot with respect to the density of the internal positive controls. The antibody array represents a qualitative comparison of each cytokine and is not qualitative. Each experiment was repeated three times with similar results observed and the standard deviation shown above each sample represents three replicates in one experiment.</p