The fibrinolysis inhibitor α2-antiplasmin restricts lymphatic remodelling and metastasis in a mouse model of cancer.

Remodelling of lymphatic vessels in tumours facilitates metastasis to lymph nodes. The growth factors VEGF-C and VEGF-D are well known inducers of lymphatic remodelling and metastasis in cancer. They are initially produced as full-length proteins requiring proteolytic processing in order to bind VEGF receptors with high affinity and thereby promote lymphatic remodelling. The fibrinolytic protease plasmin promotes processing of VEGF-C and VEGF-D in vitro, but its role in processing them in cancer was unknown. Here we explore plasmin's role in proteolytically activating VEGF-D in vivo, and promoting lymphatic remodelling and metastasis in cancer, by co-expressing the plasmin inhibitor α2-antiplasmin with VEGF-D in a mouse tumour model. We show that α2-antiplasmin restricts activation of VEGF-D, enlargement of intra-tumoural lymphatics and occurrence of lymph node metastasis. Our findings indicate that the fibrinolytic system influences lymphatic remodelling in tumours which is consistent with previous clinicopathological observations correlating fibrinolytic components with cancer metastasis.

Multiple ecto-nucleoside triphosphate diphosphohydrolases facilitate intracellular replication of Legionella pneumophila.

Legionella pneumophila is an opportunistic pathogen that replicates within alveolar macrophages resulting in the onset of severe atypical pneumonia. Previously we have identified Lpg1905, a eukaryotic-type ecto-NTPDase (nucleoside triphosphate diphosphohydrolase) from L. pneumophila that was required for optimal intracellular replication and virulence in a mouse lung infection model. In the present study, we characterized the activity of a second eukaryotic-type NTPDase, Lpg0971, from L. pneumophila. We observed that recombinant Lpg0971 hydrolysed only ATP and exhibited divalent cation preference for manganese (II) ions. Similar to lpg1905, an lpg0971 mutant carrying the plasmid pMIP was attenuated in a mouse lung infection model and impaired for replication in human macrophages and amoebae. Increased trafficking of the LCV (Legionella-containing vacuole) to a LAMP-1 (lysosome-associated membrane protein-1)-positive compartment was observed for both the lpg1905 and lpg0971 mutants carrying pMIP. Complementation with either lpg1905 or lpg0971 restored intracellular replication, suggesting that a minimum level of ATPase activity was required for this function. A double lpg1905/0971 mutant was not more impaired for intracellular replication than the single mutants and complementation of the double mutant with lpg0971, but not lpg1905, restored intracellular replication. This suggested that although the NTPDases have overlapping activities they have distinct functions. Unlike many eukaryotic-type proteins from L. pneumophila, neither Lpg1905 nor Lpg0971 were translocated into the host cell by the Dot/Icm (defective in organelle trafficking/intracellular multiplication) type IV secretion system. Overall our data suggest that the ability of L. pneumophila to replicate in eukaryotic cells relies in part on the ability of the pathogen to hydrolyse ATP within an intracellular compartment.