Matrigel cytometry: a novel method for quantifying angiogenesis in vivo.

Many of the current in vivo methods to evaluate angiogenesis are poorly quantifiable. Recently, the Matrigel plug assay has become the method of choice in many studies involving in vivo testing for angiogenesis. When known angiogenic factors are mixed with Matrigel and injected subcutaneously into mice, endothelial cells migrate into the gel plug. These endothelial cells form vessel-like structures, a process that mimics the formation of capillary networks. Here, we present a modification of the traditional Matrigel assay with improved method to quantify the amount of endothelial cells that incorporate into the plug. The removed plugs were subjected to a mild protease treatment, yielding intact cells. The liberated cells were then stained using an endothelial cell-specific markers, and counted by flow cytometry. This novel combination of FACS analysis with the traditional Matrigel assay improves the ability to quantify in vivo angiogenesis, and for the first time enables to determine the number of migrating and proliferating endothelial cells which reflects the angiogenesis rate.

Transglutaminase in Plasmodium parasites: activity and putative role in oocysts and blood stages.

Transglutaminase was identified in malaria parasites by immunofluorescence microscopy using alpha-transglutaminase antiserum. Functional enzyme was demonstrated in vivo and in vitro using labeled polyamines that become incorporated into protein substrates through TGase activity. In Plasmodium falciparum intraerythrocytic parasites, transglutaminase activity was stage-dependent: it was weak in ring-forms but much stronger in trophozoites and schizonts. High levels of activity were detected in P. gallinaceum zygotes and ookinetes and in capsules of oocysts developing on mosquito midguts. Unlike most known transglutaminases, the enzymatic activity in Plasmodium was Ca(2+)-independent. Furthermore, levels of activity were similar at 37 and 26 degrees C. Parasite transglutaminase may be responsible for the modification of erythrocytic cytoskeleton in infected cells and it may facilitate the construction of oocyst capsules by cross-linking mosquito-derived basement membrane components with Plasmodium-derived proteins.

Interaction of Plasmodium gallinaceum ookinetes and oocysts with extracellular matrix proteins.

Plasmodium ookinetes are elongate, motile and invasive while inside the mosquito gut but promptly metamorphose into spherical immobile oocysts upon coming in contact with the basement membrane surrounding the midgut. There they begin a prolonged growth period characterized by massive DNA synthesis for the production of sporozoites. Living Plasmodium gallinaceum ookinetes attached avidly to the murine extracellular matrix proteins, laminin and collagen type IV. In ELISA-type assays, the main ookinete surface protein, Pgs28 was implicated as a mediator of parasite attachment to these basement membrane constituents. Laminin and collagen IV adhered to ookinete and oocyst lysates spotted onto nitrocellulose membranes. Receptor-ligand blot assays demonstrated that Pgs28 and an oocyst-specific antigen recognized by the mAb 10D6 interact with murine collagen IV and laminin. 10D6 antigen was also recognized by monospecific antiserum against the human epidermal growth factor receptor. Mosquito-derived laminin was incorporated into oocyst capsules of P. gallinaceum growing in Aedes aegypti. We hypothesize that contact with the mosquito basement membrane triggers the transformation of ookinetes into oocysts. Coalescence of basement membrane proteins onto the capsules masks developing oocysts from the mosquito's immune system and facilitates their prolonged extracellular development in the mosquito body cavity.