A Complex Extracellular Sphingomyelinase of Pseudomonas aeruginosa Inhibits Angiogenesis by Selective Cytotoxicity to Endothelial Cells

Abstract

The hemolytic phospholipase C (PlcHR) expressed by Pseudomonas aeruginosa is the original member of a Phosphoesterase Superfamily, which includes phosphorylcholine-specific phospholipases C (PC-PLC) produced by frank and opportunistic pathogens. PlcHR, but not all its family members, is also a potent sphingomyelinase (SMase). Data presented herein indicate that picomolar (pM) concentrations of PlcHR are selectively lethal to endothelial cells (EC). An RGD motif of PlcHR contributes to this selectivity. Peptides containing an RGD motif (i.e., GRGDS), but not control peptides (i.e., GDGRS), block the effects of PlcHR on calcium signaling and cytotoxicity to EC. Moreover, RGD variants of PlcHR (e.g., RGE, KGD) are significantly reduced in their binding and toxicity, but retain the enzymatic activity of the wild type PlcHR. PlcHR also inhibits several EC-dependent in vitro assays (i.e., EC migration, EC invasion, and EC tubule formation), which represent key processes involved in angiogenesis (i.e., formation of new blood vessels from existing vasculature). Finally, the impact of PlcHR in an in vivo model of angiogenesis in transgenic zebrafish, and ones treated with an antisense morpholino to knock down a key blood cell regulator, were evaluated because in vitro assays cannot fully represent the complex processes of angiogenesis. As little as 2 ng/embryo of PlcHR was lethal to ∼50% of EGFP-labeled EC at 6 h after injection of embryos at 48 hpf (hours post-fertilization). An active site mutant of PlcHR (Thr178Ala) exhibited 120-fold reduced inhibitory activity in the EC invasion assay, and 20 ng/embryo elicited no detectable inhibitory activity in the zebrafish model. Taken together, these observations are pertinent to the distinctive vasculitis and poor wound healing associated with P. aeruginosa sepsis and suggest that the potent antiangiogenic properties of PlcHR are worthy of further investigation for the treatment of diseases where angiogenesis contributes pathological conditions (e.g., vascularization of tumors, diabetic retinopathy).

Author Summary

Pseudomonas aeruginosa is a major bacterial opportunistic pathogen responsible for acute (e.g., sepsis) and chronic infections (e.g., pulmonary). While it expresses assorted extracellular toxins that in one way or another contribute to pathogenesis, the precise cellular and molecular mechanisms by which these factors act is largely unknown. During septicemia, P. aeruginosa frequently causes vasculitis and thrombosis. Endothelial cells line the entire vascular system of mammals and have sundry key functions in infectious diseases, including thrombosis and inflammation. Endothelial cells are essential to the formation of new blood vessels (angiogenesis) needed for proper wound healing. This report describes in vitro and in vivo experiments demonstrating that an extracellular toxin, PlcHR, at very low (picomolar) concentrations, is highly lethal to endothelial cells and inhibits angiogenesis in vivo. Data herein also suggest that PlcHR is selectively toxic to endothelial cells through its ability to bind a cell receptor(s). These observations may be particularly relevant to the mechanisms by which P. aeruginosa causes vascular lesions and inhibits the healing of wounds during septic infections. Finally, the potent antiangiogenic attribute of PlcHR could be useful in the treatment of noninfectious diseases where angiogenesis contributes their pathogenesis, including the vascularization of tumors and the eye (e.g., diabetic retinopathy).