Giannoula Klement

Various conventional chemotherapeutic drugs can block angiogenesis or even kill activated, dividing endothelial cells. Such effects may contribute to the antitumor efficacy of chemotherapy in vivo and may delay or prevent the acquisition of drug-resistance by cancer cells. We have implemented a treatment regimen that augments the potential antivascular effects of chemotherapy, that is devoid of obvious toxic side effects, and that obstructs the development of drug resistance by tumor cells. Xenografts of 2 independent neuroblastoma cell lines were subjected to either continuous treatment with low doses of vinblastine, a monoclonal neutralizing antibody (DC101) targeting the flk-1/KDR (type 2) receptor for VEGF, or both agents together. The rationale for this combination was that any antivascular effects of the low-dose chemotherapy would be selectively enhanced in cells of newly formed vessels when survival signals mediated by VEGF are blocked. Both DC101 and low-dose vinblastine treatment individually resulted in significant but transient xenograft regression, diminished tumor vascularity, and direct inhibition of angiogenesis. Remarkably, the combination therapy resulted in full and sustained regressions of large established tumors, without an ensuing increase in host toxicity or any signs of acquired drug resistance during the course of treatment, which lasted for >6 months. This article may have been published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

Platelets, in addition to their function in hemostasis, play an important role in wound healing and tumor growth. Because platelets contain angiogenesis stimulators and inhibitors, the mechanisms by which platelets regulate angiogenesis remain unclear. As platelets adhere to activated endothelium, their action can enhance or inhibit local angiogenesis. We therefore suspected a higher organization of angiogenesis regulators in platelets. Using double immunofluorescence and immunoelectron microscopy, we show that pro- and antiangiogenic proteins are separated in distinct subpopulations of alpha-granules in platelets and megakaryocytes. Double immunofluorescence labeling of vascular endothelial growth factor (VEGF) (an angiogenesis stimulator) and endostatin (an angiogenesis inhibitor), or for thrombospondin-1 and basic fibroblast growth factor, confirms the segregation of stimulators and inhibitors into separate and distinct alpha-granules. These observations motivated the hypothesis that distinct populations of alpha-granules could undergo selective release. The treatment of human platelets with a selective PAR4 agonist (AYPGKF-NH(2)) resulted in release of endostatin-containing granules, but not VEGF-containing granules, whereas the selective PAR1 agonist (TFLLR-NH(2)) liberated VEGF, but not endostatin-containing granules. In conclusion, the separate packaging of angiogenesis regulators into pharmacologically and morphologically distinct populations of alpha-granules in megakaryocytes and platelets may provide a mechanism by which platelets can locally stimulate or inhibit angiogenesis.

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.

Platelet microparticles are a normal constituent of circulating blood. Several studies have demonstrated positive correlations between thrombotic states and platelet microparticle levels. Yet little is known about the processes by which platelet microparticles are generated in vivo. We now characterize microparticles derived directly from megakaryocytes. Video microscopy of live mouse megakaryocytes demonstrated that microparticles form as submicron beads along the lengths of slender, unbranched micropodia. These microparticles are CD41(+), CD42b(+), and express surface phosphatidylserine. Megakaryocyte microparticle generation is resistant to inhibition of microtubule assembly, which is critical to platelet formation, and augmented by inhibition of actin polymerization. To determine whether circulating microparticles are derived primarily from activated platelets or megakaryocytes, we identified markers that distinguish between these 2 populations. CD62P and LAMP-1 were found only on mouse microparticles from activated platelets. In contrast, full-length filamin A was found in megakaryocyte-derived microparticles, but not microparticles from activated platelets. Circulating microparticles isolated from mice were CD62P(-), LAMP-1(-) and expressed full-length filamin A, indicating a megakaryocytic origin. Similarly, circulating microparticles isolated from healthy volunteers were CD62P(-) and expressed full-length filamin A. Cultured human megakaryocytes elaborated microparticles that were CD41(+), CD42b(+), and express surface phosphatidylserine. These results indicate that direct production by megakaryocytes represents a physiologic means to generate circulating platelet microparticles.