David Cervi

Platelets actively sequester angiogenesis regulators

Clinical trials with antiangiogenic agents have not been able to validate plasma or serum levels of angiogenesis regulators as reliable markers of cancer presence or therapeutic response. We recently reported that platelets contain numerous proteins that regulate angiogenesis. We now show that accumulation of angiogenesis regulators in platelets of animals bearing malignant tumors exceeds significantly their concentration in plasma or serum, as well as their levels in platelets from non-tumor-bearing animals. This process is selective, as platelets do not take up a proportional amount of other plasma proteins (eg, albumin), even though these may be present at higher concentrations. We also find that VEGF-enriched Matrigel pellets implanted subcutaneously into mice or the minute quantities of VEGF secreted by microscopic subcutaneous tumors (0.5-1 mm(3)) result in an elevation of VEGF levels in platelets, without any changes in its plasma levels. The profile of other angiogenesis regulatory proteins (eg, platelet-derived growth factor, basic fibroblast growth factor) sequestered by platelets also reflects the presence of tumors in vivo before they can be macroscopically evident. The ability of platelets to selectively take up angiogenesis regulators in cancer-bearing hosts may have implications for the diagnosis and management of many angiogenesis-related diseases and provide a guide for antiangiogenic therapies.

Tumors Stimulate Platelet Delivery of Angiogenic Factors in Vivo : An Unexpected Benefit

The interaction between platelets and the tumor microenvironment results in the modulation of angiogenesis, although the mechanisms governing this regulation remain unclear. This study explores the differences in the communication between wounded tissues and healthy, tumor-conditioned, and frozen platelets. Platelet-rich plasma obtained from healthy (PRP) or tumor-bearing (TPRP) mice was applied to dorsal, full-thickness wounds on diabetic mice. Wound healing was evaluated using macroscopic criteria and a staging system based on angiogenesis and stromal cell proliferation. Proteomic analysis was used to compare the levels of angiogenic proteins contained in the platelet preparations. TPRP-treated wounds reached 90% wound closure 5.6 to 9.5 days earlier than PRP-treated and nontreated wounds, respectively. TPRP induced a fourfold increase in stromal cell proliferation compared with nontreated wounds, and a 2.5-fold increase compared with PRP-treated wounds. TPRP induced the highest stimulation of angiogenesis with a fourfold increase compared with nontreated controls. On day 21, wounds treated with TPRP showed a typical architecture with thick collagen bundles. Although the levels of angiogenesis regulators detected via SELDI-ToF were similar between the PRP and TPRP treatment regimens, the enhanced healing capacity of TPRP suggests improved platelet delivery as indicated by frozen TPRP preparations that had lost most of their pro-angiogenic drive. In conclusion, these results show that intact tumor-conditioned platelets display an improved ability to deliver angiogenesis regulators to wounded tissues.

Vascular Endothelial Growth Factor—A Positive and Negative Regulator of Tumor Growth

Over the past decade, the well-documented role of vascular endothelial growth factor (VEGF) in tumor angiogenesis has led it to become one of the leading therapeutic targets for the treatment of cancer. Emerging evidence from genetically modified animal models, however, suggests that elevated levels of VEGF, or a proangiogenic phenotype, may impede, rather than promote, early tumor development and progression. For example, hypermorph VEGF transgenic mice display delayed progression of a retroviral-induced murine leukemia, and knockdown of VEGF expression within the myeloid compartment accelerates tumor progression. Several mechanisms have been proposed to explain this paradox, whereby VEGF induces changes within the hematopoietic compartment and tumor microenvironment through recruitment of tumor inhibitory monocytic cells and the negative regulation of tumor angiogenesis. Thus, it is apparent that the levels of VEGF expression in both tumor and nontumor tissues, as well as the context and timing of its modulation relative to cancer induction, play an important role in determining the effects of VEGF expression on tumorigenicity. In light of these recent findings, the various mechanisms underlying the negative role of VEGF during early tumor development, progression, and metastasis will be discussed.

Direct transcriptional regulation of MDM2 by Fli-1

The Ets transcription factor, Fli-1, has been shown to play a pivotal role in the induction and progression of Friend Murine Leukemia Virus (F-MuLV)-induced erythroleukemia, with its overexpression leading to erythroblast survival, proliferation, and inhibition of terminal differentiation. P53 inactivation is an additional genetic alteration that occurs in late-stage leukemic progression associated with in vivo and in vitro immortalization. Since p53 protein expression levels are low, to undetectable, in primary erythroleukemic cells that express elevated levels of Fli-1, we investigated the potential regulation of p53 by Fli-1. We assessed whether the overexpression of Fli-1 could partially regulate p53 via modulation of its well-established regulator, MDM2. In this paper, we demonstrate that the promoter of MDM2 contains a consensus binding site for Fli-1 that is bound by this transcription factor in vitro and in vivo, resulting in MDM2 transcriptional regulation. We further substantiate these observations in vivo by demonstrating a positive correlation in the expression of Fli-1 and MDM2, and a negative correlation with p53 in leukemic tissues obtained from mice with Friend Disease. These observations depict a significant function of Fli-1 overexpression in the indirect control of p53, evidently capable of leading to an increasingly aggressive erythroleukemic clone in vivo.

Phosphorylation status of c-Kit and Epo receptors, and the presence of wild-type p53 confer in vitro resistance of murine erythroleukemic cells to Celecoxib

It is well established that selective COX-2 inhibitors exhibit potent effects against progression of select solid tumours. However, their effects on liquid tumours have not been fully established. By taking advantage of murine Friend Disease we have shown a strong antileukemic effect of celecoxib by determining novel in vitro targets. Western blot analyses revealed the expression of COX-2 in a panel of Friend Virus-transformed, splenic-derived primary erythroleukemic blasts and established cell lines generated in our laboratory. We have shown that celecoxib at concentrations as low as 20 μM significantly suppresses proliferation of the selected murine erythroleukemia cell line HB60-5. The greatest proliferative inhibition was seen at 40 μM of celecoxib, resulting in apoptosis. Our results also demonstrate that treatment of the established murine erythroleukemia cell line HB60-5 with celecoxib results in suppression of c-Kit and erythropoietin receptor (Epo-R) phosphorylation resulting in apoptosis, likely through decreased levels of survival factors. However, upon overexpression of c-Kit alone in these cells a significant increase in survival and twofold increase in proliferation in the presence of celecoxib were observed (P<0.05). Finally, since responsiveness of our murine erythroleukemia cell lines to celecoxib is above the reported physiologically achievable levels in vivo, we have provided in vitro evidence to suggest that reduced sensitivity of erythroleukemic cells to lower doses of celecoxib may be a consequence of the loss of wild-type p53. These findings are pivotal in addressing potential discrepancies associated with sensitivity of murine erythroleukemic cells to celecoxib in vitro versus in vivo.