The insulin-like growth factor system and adenocarcinoma of the colon

The insulin-like growth factor (IGF) system is important in normal growth and development. However, it is also known to be involved with malignant transformation and cellular proliferation. IGF binding proteins modulate the biological activity of IGF-I, either potentiating or inhibiting its activity, as well as determining how much enters the circulation at any one time. IGF binding protein-4 (IGFBP-4), for example is believed to be inhibitory to the effects of IGF-I. This thesis shows that the colon cancer cell lines Colo 205, HT29 and WiDR proliferate in response to IGF-I, and that IGFBP-4 at high concentrations inhibits their growth. However, it was found that with lower concentrationsof IGFBP-4, proliferation in HT29 and WiDR cells increased. Nevertheless in two cell lines, IGFBP-4 partially negated the proliferative effects of IGF-I. An antibody against IGFBP-4 was used to show that endogenous IGFBP-4 plays an important role in modifying cell growth. In order to start in vivo experiments which required considerable quantities of IGFBP-4, this protein was produced in an expression system and purified using an immunoaffinity column method. The rhIGFBP-4 thus produced was shown to be functional and to inhibit colorectal cancer cell growth in vitro. A nude mouse model of colon cancer was produced and the expression of components of the IGF system in this model determined using PCR. Experiments were performed using conditioned medium from Colo 205 cells to investigate IGFBP-4 protease activity. This thesis shows that manipulation of the IGF system is a potential target for further research into treatment for adenocarcinoma of the colon

Effects of control and primed periodontal ligament stem cells on endothelial cell behavior <i>in vitro</i>.

<p>(A) CM of control PDLSCs or cells treated with DFX or FGF-2 alone does not induce HMEC-1 proliferation as determined by MTT (n = 8). (B) PDLSCs induce endothelial cell migration in the transwell migration assay. Both untreated and treated PDLSCs are able to induce HMEC-1 migration, but there is no difference in chemotactic potential between control and primed PDLSCs. Negative control: culture medium containing 0.1% FBS. Positive control: culture medium containing 10% FBS. FGF control: culture medium conta0ining 10 ng/mL FGF-2. D+F control: culture medium containing 100 μM DFX and 10 ng/mL FGF-2. Scale bar = 300 μm. Data are expressed as mean ± SEM. ** = p-value ≤0.01; *** = p-value ≤ 0.001 compared to either FGF-2 control or D+F control.</p

Characterization of human periodontal ligament stem cells.

<p>PDLSCs display mesenchymal-like cell morphology comprising spindle-shaped and polygonal cell types (A). Successful adipogenic, chondrogenic and osteogenic induction (n = 3) is confirmed by histochemical staining of respectively lipid droplets with Oil red O (B), aggrecan (C) and the presence of calcified matrix by means of Alizarin Red S staining (D). Furthermore, flow cytometric-analysis showed the expression of MSC-markers CD44, CD73, CD90 and CD105 (E).</p

Exposure to DFX leads to decreased cell numbers of endothelial cells after 72 hours.

<p>Cell numbers were monitored via Trypan Blue Exclusion, only 72 hours of DFX treatment resulted in a decreased cell count (n = 3). Control: culture medium containing 0.1% FBS. The conditions included in this experiment only contained the different priming agents and were lacking factors secreted by PDLSCs. DFX = deferoxamine; FGF-2 = fibroblast growth factor-2; PDLSC = periodontal ligament stem cells. Data are expressed as mean ± SEM and were analyzed by Two-Way ANOVA. *** = p-value ≤0.001.</p

Angiogenic Capacity of Periodontal Ligament Stem Cells Pretreated with Deferoxamine and/or Fibroblast Growth Factor-2

<div><p>Periodontal ligament stem cells (PDLSCs) represent a good source of multipotent cells for cell-based therapies in regenerative medicine. The success rate of these treatments is severely dependent on the establishment of adequate vasculature in order to provide oxygen and nutrients to the transplanted cells. Pharmacological preconditioning of stem cells has been proposed as a promising method to augment their therapeutic efficacy. In this study, the aim was to improve the intrinsic angiogenic properties of PDLSCs by <i>in vitro</i> pretreatment with deferoxamine (DFX; 100μM), fibroblast growth factor-2 (FGF-2; 10ng/mL) or both substances combined. An antibody array revealed the differential expression of several proteins, including vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). ELISA data confirmed a 1.5 to 1.8-fold increase in VEGF for all tested conditions. Moreover, 48 hours after the removal of DFX, VEGF levels remained elevated (1.8-fold) compared to control conditions. FGF-2 and combination treatment resulted in a 5.4 to 13.1-fold increase in PlGF secretion, whereas DFX treatment had no effect. Furthermore, both PDLSCs as pretreated PDLSCs induced endothelial migration. Despite the significant elevated VEGF levels of pretreated PDLSCs, the induced endothelial migration was not higher by pretreated PDLSCs. We find that the observed induced endothelial cell motility was not dependent on VEGF, since blocking the VEGFR1-3 with Axitinib (0.5nM) did not inhibit endothelial motility towards PDLSCs. Taken together, this study provides evidence that preconditioning with DFX and/or FGF-2 significantly improves the angiogenic secretome of PDLSCs, in particular VEGF and PlGF secretion. However, our data suggest that VEGF is not the only player when it comes to influencing endothelial behavior by the PDLSCs.</p></div

The effect of DFX, FGF-2 or combination treatment of the MSC marker expression as demonstrated by flow cytometry or on cell proliferation (measured with Trypan Blue Exclusion assay).

<p>(A) Pretreatment does not influence MSC marker expression of PDLSC proliferation (n = 6). (B) Cell proliferation was monitored during treatment by means of Trypan Blue Exclusion assay (n = 5).</p

Periodontal ligament stem cells do not elicit endothelial cell migration via VEGF.

<p>Conditioned medium of untreated and treated PDLSCs induces endothelial cell migration. In the presence of 0.5 nM Axitinib (AX), a VEGFR1-3 inhibitor, endothelial cells continue to migrate towards factors secreted by PDLSCs. Negative control: culture medium containing 0.1% FBS. Positive control: culture medium containing 10% FBS. FGF control: culture medium containing 10ng/mL FGF-2. D+F control: culture medium containing 100 μM DFX and 10 ng/mL FGF-2. Scale bar = 300μm. Data are expressed as mean ± SEM. ** = p-value ≤0.01; *** = p-value ≤ 0.001 compared to either FGF control or D+F control.</p

Treatment with DFX and/or FGF-2 increases VEGF and PlGF secretion of PDSLCs.

<p>PDLSCs were pretreated either with 100 μM DFX for 24 hours, with 10 ng/mL FGF-2 for 72 hours or with a combination of both substances. (A-D) Antibody array of the CM of PDLSCs pretreated with DFX (n = 1), FGF-2 and the combination treatment (n = 2). Graphs A-C show pixel density of the differentially expressed proteins. As determined by ELISA (n = 5), DFX treatment resulted in a 1.8 fold increase in VEGF secretion (E), whereas FGF-2 treatment increased VEGF secretion 1.5 fold (F). Finally, a combination of both agents led to a 2.7 fold increase in VEGF secretion (G). ELISA of PlGF showed that not DFX (H) but treatment with FGF-2 (I) and FGF-2 combined with DFX (J) significantly upregulated PlGF secretion. Data are expressed as mean ± SEM and analyzed with Two-Way-ANOVA, ** = p ≤ 0.01; *** = p ≤ 0.001.</p

Schematic overview of PDLSC pretreatment procedures.

<p>After overnight incubation standard PDLSCs culture medium was switched to standard medium containing only 0.1% FBS, supplemented with either 100μM DFX or 10 ng/mL FGF-2. CM of DFX-treated cells was collected after 24 hours whereas CM of FGF-2-treated cells was collected after 72 hours. In the case of combination treatment, DFX was added 48 hours after the start of FGF-2 pretreatment and CM was collected 24 hours later. Subsequently cells were washed and priming media were replaced by standard culture medium containing 0.1% FBS. This wash-out medium was collected after 8, 24 and 48 hours.</p

Chorioallantoic membrane (CAM) assay.

<p>At day 9 of embryonic development, matrigel droplets with or without 50,000 hDPSC were applied onto the CAM of a chicken embryo (black arrow, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071104#pone-0071104-g005" target="_blank">Figure 5a</a>). 72 hours later, pictures were taken (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071104#pone-0071104-g005" target="_blank">figure 5c–e</a>). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071104#pone-0071104-g005" target="_blank">Figure 5c</a> is a control matrigel droplet, while <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071104#pone-0071104-g005" target="_blank">Figure 5d</a> is a droplet containing 50,000 hDPSC. White scale bars represent 1 mm. To assess angiogenesis, two circles (with a radius of 1.5 and 2 mm, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071104#pone-0071104-g005" target="_blank">figure 5e</a>), were digitally positioned around the matrigel droplets and the blood vessels intersecting these circles were counted double blind. Graph 5b: hDPSC were able to significantly increase the number of capillaries intersecting both circles. This assay was repeated 4 times with hDPSC of 4 different donors to gain a database of at least 27 individual eggs. Values are represented as means ± S.E.M.</p