Paraoxonases-2 and -3 Are Important Defense Enzymes against Pseudomonas aeruginosa Virulence Factors due to Their Anti-Oxidative and Anti-Inflammatory Properties

The pathogen Pseudomonas aeruginosa causes serious damage in immunocompromised patients by secretion of various virulence factors, among them the quorum sensing N-(3-oxododecanoyl)-L-homoserine lactone (3OC12) and the redox-active pyocyanin (PCN). Paraoxonase-2 (PON2) may protect against P. aeruginosa infections, as it efficiently inactivates 3OC12 and diminishes PCN-induced oxidative stress. This defense could be circumvented because 3OC12 mediates intracellular Ca2+-rise in host cells, which causes rapid inactivation and degradation of PON2. Importantly, we recently found that the PON2 paralogue PON3 prevents mitochondrial radical formation. Here we investigated its role as additional potential defense mechanism against P. aeruginosa infections. Our studies demonstrate that PON3 diminished PCN-induced oxidative stress. Moreover, it showed clear anti-inflammatory potential by protecting against NF-κB activation and IL-8 release. The latter similarly applied to PON2. Furthermore, we observed a Ca2+-mediated inactivation and degradation of PON3, again in accordance with previous findings for PON2. Our results suggest that the anti-oxidative and anti-inflammatory functions of PON2 and PON3 are an important part of our innate defense system against P. aeruginosa infections. Furthermore, we conclude that P. aeruginosa circumvents PON3 protection by the same pathway as for PON2. This may help identifying underlying mechanisms in order to sustain the protection afforded by these enzymes

Fibroblast growth factors are required for efficient tumor angiogenesis

Although the function of vascular endothelial growth factor in the induction of tumor angiogenesis is well understood, the role of a second group of angiogenic factors, the fibroblast growth factors (FGFs), remains elusive. We used a recombinant adenovirus expressing soluble FGF receptor (AdsFGFR) to interfere with FGF function in tumor angiogenesis. AdsFGFR repressed endothelial cell proliferation in vitro and inhibited tumor angiogenesis in an ex vivo bioassay, in which endothelial cells were cocultured with angiogenic tumor biopsies in a collagen gel. Moreover, AdsFGFR repressed tumor angiogenesis and hence tumor growth in vivo in allograft transplantation experiments. Whereas adenoviral expression of a soluble form of VEGF receptor 1 (AdsFlt) predominantly affected the initiation of tumor angiogenesis, soluble FGF receptor (sFGFR) appeared to impair the maintenance of tumor angiogenesis. The combination of sFGFR and soluble Flt exhibited a synergistic effect in the repression of tumor growth. Finally, i.v. injection of AdsFGFR resulted in a dramatic repression of tumor growth in a transgenic mouse model of pancreatic β cell carcinogenesis. Similar to control infections with AdsFlt, tumor-associated vessel density was decreased, indicating that the expression of sFGFR impaired tumor angiogenesis. These data indicate that FGFs play a critical role in tumor angiogenesis

Fibroblast growth factors are required for efficient tumor angiogenesis

Although the function of vascular endothelial growth factor in the induction of tumor angiogenesis is well understood, the role of a second group of angiogenic factors, the fibroblast growth factors (FGFs), remains elusive. We used a recombinant adenovirus expressing soluble FGF receptor (AdsFGFR) to interfere with FGF function in tumor angiogenesis. AdsFGFR repressed endothelial cell proliferation in vitro and inhibited tumor angiogenesis in an ex vivo bioassay, in which endothelial cells were cocultured with angiogenic tumor biopsies in a collagen gel. Moreover, AdsFGFR repressed tumor angiogenesis and hence tumor growth in vivo in allograft transplantation experiments. Whereas adenoviral expression of a soluble form of VEGF receptor 1 (AdsFlt) predominantly affected the initiation of tumor angiogenesis, soluble FGF receptor (sFGFR) appeared to impair the maintenance of tumor angiogenesis. The combination of sFGFR and soluble Flt exhibited a synergistic effect in the repression of tumor growth. Finally, i.v. injection of AdsFGFR resulted in a dramatic repression of tumor growth in a transgenic mouse model of pancreatic β cell carcinogenesis. Similar to control infections with AdsFlt, tumor-associated vessel density was decreased, indicating that the expression of sFGFR impaired tumor angiogenesis. These data indicate that FGFs play a critical role in tumor angiogenesis

One Enzyme, Two Functions: PON2 PREVENTS MITOCHONDRIAL SUPEROXIDE FORMATION AND APOPTOSIS INDEPENDENT FROM ITS LACTONASE ACTIVITY*

The human enzyme paraoxonase-2 (PON2) has two functions, an enzymatic lactonase activity and the reduction of intracellular oxidative stress. As a lactonase, it dominantly hydrolyzes bacterial signaling molecule 3OC12 and may contribute to the defense against pathogenic Pseudomonas aeruginosa. By its anti-oxidative effect, PON2 reduces cellular oxidative damage and influences redox signaling, which promotes cell survival. This may be appreciated but also deleterious given that high PON2 levels reduce atherosclerosis but may stabilize tumor cells. Here we addressed the unknown mechanisms and linkage of PON2 enzymatic and anti-oxidative function. We demonstrate that PON2 indirectly but specifically reduced superoxide release from the inner mitochondrial membrane, irrespective whether resulting from complex I or complex III of the electron transport chain. PON2 left O2˙̄ dismutase activities and cytochrome c expression unaltered, and it did not oxidize O2˙̄ but rather prevented its formation, which implies that PON2 acts by modulating quinones. To analyze linkage to hydrolytic activity, we introduced several point mutations and show that residues His114 and His133 are essential for PON2 activity. Further, we mapped its glycosylation sites and provide evidence that glycosylation, but not a native polymorphism Ser/Cys311, was critical to its activity. Importantly, none of these mutations altered the anti-oxidative/anti-apoptotic function of PON2, demonstrating unrelated activities of the same protein. Collectively, our study provides detailed mechanistic insight into the functions of PON2, which is important for its role in innate immunity, atherosclerosis, and cancer