Blue light treatment of Pseudomonas aeruginosa: Strong bactericidal activity, synergism with antibiotics and inactivation of virulence factors.

Pseudomonas aeruginosa is among the most common pathogens responsible for both acute and chronic infections of high incidence and severity. Additionally, P. aeruginosa resistance to conventional antimicrobials has increased rapidly over the past decade. Therefore, it is crucial to explore new therapeutic options, particularly options that specifically target the pathogenic mechanisms of this microbe. The ability of a pathogenic bacterium to cause disease is dependent upon the production of agents termed 'virulence factors', and approaches to mitigate these agents have gained increasing attention as new antibacterial strategies. Although blue light irradiation is a promising alternative approach, only limited and preliminary studies have described its effect on virulence factors. The current study aimed to investigate the effects of lethal and sub-lethal doses of blue light treatment (BLT) on P. aeruginosa virulence factors. We analyzed the inhibitory effects of blue light irradiation on the production/activity of several virulence factors. Lethal BLT inhibited the activity of pyocyanin, staphylolysin, pseudolysin and other proteases, but sub-lethal BLT did not affect the production/expression of proteases, phospholipases, and flagella- or type IV pili-associated motility. Moreover, a eukaryotic cytotoxicity test confirmed the decreased toxicity of blue light-treated extracellular P. aeruginosa fractions. Finally, the increased antimicrobial susceptibility of P. aeruginosa treated with sequential doses of sub-lethal BLT was demonstrated with a checkerboard test. Thus, this work provides evidence-based proof of the susceptibility of drug-resistant P. aeruginosa to BLT-mediated killing, accompanied by virulence factor reduction, and describes the synergy between antibiotics and sub-lethal BLT.

Radiation induced-tubulogenesis in endothelial cells is antagonized by the antiangiogenic properties of green tea polyphenol (-) epigallocatechin-3-gallate.

Radiation therapy is a widely-used option for the treatment of a variety of solid tumors. Although effective, ionizing radiation (IR) may give rise to various side effects, including secondary tumors. In agreement with this, recent reports have demonstrated increased invasive potential in different tumor-derived cell lines following radiation treatment. Many of the molecular effects of IR specifically on the endothelial cells involved in tumor neo-vascularization remain unknown. In this study, we found that low sublethal single doses of IR applied to human umbilical vein endothelial cells stimulated cell migration and in vitro tubulogenesis. This correlated with an increase in membrane type-1 matrix metalloproteinase (MT1-MMP) protein expression, a crucial enzyme that promotes endothelial cell migration and tube formation, and of caveolin-1, a protein that regulates tube formation. Cell adhesion was also promoted by IR, reflected in increased gene expression levels of cell surface beta(3) integrin. Pretreatment of the cells with epigallocatechin-3-gallate (EGCg), a green tea catechin that possesses anti-angiogenic properties, prevented most of the IR-induced cellular and molecular events. These observations suggest that current protocols involving radiation therapy for the treatment of cancer can paradoxically promote angiogenesis, but can be improved by combination with anti-angiogenic molecules such as EGCg to target those tumor-derived endothelial cells that escaped IR-induced apoptosis.

Irradiation of rat mesangial cells alters the expression of gene products associated with the development of renal fibrosis.

To determine the ability of radiation to modulate mesangial cell expression of various molecules involved in promoting extracellular matrix (ECM) accumulation [fibronectin, plasminogen activator-inhibitor 1 (Pai1), and tissue inhibitor of metalloproteinase-2 (Timp2)] and degradation (Tgfb, plasminogen activators u-PA or t-PA, matrix metalloproteinases Mmp2 and Mmp9), primary cultures of rat mesangial cells (passage number 6-11) were placed in serum-free medium 24 h prior to irradiation with single doses of 0.5-20 Gy (137)Cs gamma rays. After irradiation, cells were maintained in serum-free medium for a further 48 h. Irradiation of quiescent mesangial cells resulted in significant (P < 0.05) time- and dose-dependent increases in Fn and Pai1 mRNA and/or immunoreactive protein. Despite an increase in Tgfb1 mRNA, there was little evidence for an increase in total Tgfb protein. Indeed, active levels remained unaltered after irradiation. Irradiation led to differential changes in MMP expression; active Mmp2 levels increased, while Mmp9 levels appeared unaltered. In addition, secretion of plasminogen activators into the medium was unchanged after irradiation, while secretion of Timp2 increased. We conclude that irradiating mesangial cells leads to altered production of various molecules involved in accumulation and degradation of extracellular matrix.

Pathophysiology of premature skin aging induced by ultraviolet light.

BACKGROUND: Long-term exposure to ultraviolet irradiation from sunlight causes premature skin aging (photoaging), characterized in part by wrinkles, altered pigmentation, and loss of skin tone. Photoaged skin displays prominent alterations in the collagenous extracellular matrix of connective tissue. We investigated the role of matrix-degrading metalloproteinases, a family of proteolytic enzymes, as mediators of collagen damage in photoaging. METHODS: We studied 59 whites (33 men and 26 women, ranging in age from 21 to 58 years) with light-to-moderate skin pigmentation, none of whom had current or prior skin disease. Only some of the participants were included in each of the studies. We irradiated their buttock skin with fluorescent ultraviolet lights under standard conditions and obtained skin samples from irradiated and nonirradiated areas by keratome or punch biopsy. In some studies, tretinoin and its vehicle were applied to skin under occlusion 48 hours before ultraviolet irradiation. The expression of matrix metalloproteinases was determined by in situ hybridization, immunohistology, and in situ zymography. Irradiation-induced degradation of skin collagen was measured by radioimmunoassay of soluble cross-linked telopeptides. The protein level of tissue inhibitor of matrix metalloproteinases type 1 was determined by Western blot analysis. RESULTS: A single exposure to ultraviolet irradiation increased the expression of three matrix metalloproteinases -- collagenase, a 92-kd gelatinase, and stromelysin -- in skin connective tissue and outer skin layers, as compared with nonirradiated skin. The degradation of endogenous type I collagen fibrils was increased by 58 percent in irradiated skin, as compared with nonirradiated skin. Collagenase and gelatinase activity remained maximally elevated (4.4 and 2.3 times, respectively) for seven days with four exposures to ultraviolet irradiation, delivered at two-day intervals, as compared with base-line levels. Pretreatment of skin with tretinoin (all-trans-retinoic acid) inhibited the induction of matrix metalloproteinase proteins and activity (by 70 to 80 percent) in both connective tissue and outer layers of irradiated skin. Ultraviolet irradiation also induced tissue inhibitor of matrix metalloproteinases-1, which regulates the enzyme. Induction of the inhibitor was not affected by tretinoin. CONCLUSIONS: Multiple exposures to ultraviolet irradiation lead to sustained elevations of matrix metalloproteinases that degrade skin collagen and may contribute to photoaging. Treatment with topical tretinoin inhibits irradiation-induced matrix metalloproteinases but not their endogenous inhibitor.

UVC activation of the HeLa cell membrane "TGF alpha ase," a metalloenzyme.

We have investigated the effect of UVC irradiation on "TGF alpha ase" activity using both intact HeLa cells and isolated membrane fragments with an assay based on the previously described nonapeptide substrate method [Brown et al. (1992): J Cell Biochem 48:411-423]. This method allows recognition of cleavage at the scissile bond cognate with that of the TGF alpha N-terminal cleavage site from its membrane-bound precursor. The level of ectoendopeptidase (including "TGF alpha ase") activity observed on intact cells was lower than that of ectoaminopeptidases. Addition of foetal bovine serum (FBS) enhanced aminopeptidase and dipeptidyl peptidase activity but inhibited "TGF alpha ase" activity, while phorbol 12-myristate 13-acetate (PMA) had no significant effect on the ectopeptidases monitored, expect for "TGF alpha ase," which was also inhibited, in contradistinction to their effects in other cell systems. Sublethal UVC irradiation (10 Jm-2) of the cultures resulted in activation of the ectoaminopeptidase and ectoendopeptidases which was maximal 16 and 20-24 h after irradiation, respectively. The addition of FBS to these irradiated cells appeared to reduce the increase in endopeptidase products, due in part to increased aminopeptidase activity but also to the direct inhibitory effect of FBS on the "TGF alpha ase." The activation of these proteases by UVC, even at high fluences (500 Jm-2), was not observed within the first 30 min after the cells were irradiated. Purified plasma membrane fragments were prepared from suspension cultures of HeLa cells and displayed high levels of "TGF alpha ase" activity. The rate of "TGF alpha ase" activity using 140 nM peptide substrate (P9) was 5.6 pmol/min/mg membrane protein, which was elevated to 13.7 pmol/min/mg membrane protein, 20 h after the cells had been irradiated with 10 Jm-2 UVC. Inhibition studies indicate that the plasma membrane "TGF alpha ase is a metalloenzyme as it was inhibited by EDTA, EGTA, and 1,10-phenanthroline but not by elastase or serine protease inhibitors. "TGF alpha ase" activity on intact cells was shown to be inhibited by 1,10-phenanthroline, which further supports this suggestion. Treatment of the membranes with Triton X-100 resulted in a loss of "TGF alpha ase" activity, raising the possibility that this enzyme may require a cofactor to be fully functional. We show that in purified membrane preparations of HeLa cells there is evidence for the presence of a "TGF alpha ase" which can be activated by UV irradiation, which differs from the putative "TGF alpha ase" described in various other cell lines, and which does not seem dependent on protein kinase C (PKC) activity.

Molecular basis of sun-induced premature skin ageing and retinoid antagonism.

Damage to skin collagen and elastin (extracellular matrix) is the hallmark of long-term exposure to solar ultraviolet irradiation, and is believed to be responsible for the wrinkled appearance of sun-exposed skin. We report here that matrix-degrading metalloproteinase messenger RNAs, proteins and activities are induced in human skin in vivo within hours of exposure to ultraviolet-B irradiation (UVB). Induction of metalloproteinase proteins and activities occurred at UVB doses well below those that cause skin reddening. Within minutes, low-dose UVB upregulated the transcription factors AP-1 and NF-kappa B, which are known to be stimulators of metalloproteinase genes. All-trans retinoic acid, which transrepresses AP-1 (ref. 8), applied before irradiation with UVB, substantially reduced AP-1 and metalloproteinase induction. We propose that elevated metalloproteinases, resulting from activation of AP-1 and NF-kappa B by low-dose solar irradiation, degrade collagen and elastin in skin. Such damage, if imperfectly repaired, would result in solar scars, which through accumulation from a lifetime of repeated low-dose sunlight exposure could cause premature skin ageing (photoageing).

Induction of tissue-type plasminogen activator and 72-kDa type-IV collagenase by ionizing radiation in rat astrocytes.

Radiation-induced damage in the central nervous system (CNS) is believed to be targeted to glial or endothelial cells or both, although the pathophysiology of the process is still poorly understood. In this study, we irradiated rat astrocytes with single doses of X-rays and then estimated the levels of tissue plasminogen activator (tPA) and collagenase in serum-free medium and cell extracts at different times. Fibrin zymography revealed increased levels of intracellular tPA activity at 12 hr after irradiation. Gelatin zymography showed continuously increasing levels of extracellular 72-kDa type-IV collagenase after irradiation. Quantitative enzymatic activities by densitometry showed a 3- to 4-fold elevation in the level of the intracellular tPA activity at 12 hr and a 5- to 6-fold increase in the level of the extracellular 72-kDa type-IV collagenase activity at 48 hr. An ELISA with specific antibodies for tPA and 72-kDa type-IV collagenase indicated a 5-fold increase in the level of tPA at 12 hr and a more-than-7-fold increase in the level of 72-kDa type-IV collagenase at 48 hr. This study adds considerable credibility to the proposed role of plasminogen activators and type-IV collagenase in the development of CNS damage after radiotherapy for brain tumors.

Irreversible photolabeling of active site of neutral endopeptidase-24.11 "enkephalinase" by azidothiorphan and [14C]-azidothiorphan.

Azidothiorphan and its [14C]-labeled analogue have been developed as photoaffinity ligands for the active site of the neutral endopeptidase 24.11. In in vitro assays azidothiorphan inhibits the endopeptidase activity with a Ki of 0.75 nM. After ultraviolet irradiation the inhibitor binds irreversibly to the enzyme, and many factors suggest that the photolabeling occurs at the active site. The binding is accompanied by a loss of enzymatic activity, and the inclusion of the competitive inhibitor thiorphan protects the endopeptidase from this inactivation. In addition the binding of another competitive inhibitor [3H]N-[(R,S)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl]-glycine to the active site of endopeptidase-24.11 is inhibited after irradiation with azidothiorphan. Experiments with [14C]-azidothiorphan have shown that very little nonspecific binding of inhibitor to enzyme occurs and the the labeled probe remains bound under denaturing conditions. Azidothiorphan has also been found to produce a long-lasting naloxone-reversible analgesia after intracerebroventricular administration. The results show that azidothiorphan should prove useful both for structural studies and for investigations on the synthesis and turnover of the neutral endopeptidase-24.11.