Comparing Redox and Intracellular Signalling Responses to Cold Plasma in Wound Healing and Cancer.

Cold plasma (CP) is an ionised gas containing excited molecules and ions, radicals, and free electrons, and which emits electric fields and UV radiation. CP is potently antimicrobial, and can be applied safely to biological tissue, birthing the field of plasma medicine. Reactive oxygen and nitrogen species (RONS) produced by CP affect biological processes directly or indirectly via the modification of cellular lipids, proteins, DNA, and intracellular signalling pathways. CP can be applied at lower levels for oxidative eustress to activate cell proliferation, motility, migration, and antioxidant production in normal cells, mainly potentiated by the unfolded protein response, the nuclear factor-erythroid factor 2-related factor 2 (Nrf2)-activated antioxidant response element, and the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway, which also activates nuclear factor-kappa B (NFκB). At higher CP exposures, inactivation, apoptosis, and autophagy of malignant cells can occur via the degradation of the PI3K/Akt and mitogen-activated protein kinase (MAPK)-dependent and -independent activation of the master tumour suppressor p53, leading to caspase-mediated cell death. These opposing responses validate a hormesis approach to plasma medicine. Clinical applications of CP are becoming increasingly realised in wound healing, while clinical effectiveness in tumours is currently coming to light. This review will outline advances in plasma medicine and compare the main redox and intracellular signalling responses to CP in wound healing and cancer.

In vitro and in vivo evaluation of diethyldithiocarbamate with copper ions and its liposomal formulation for the treatment of Staphylococcus aureus and Staphylococcus epidermidis biofilms.

Surgical site infections (SSIs) are mainly caused by Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) biofilms. Biofilms are aggregates of bacteria embedded in a self-produced matrix that offers protection against antibiotics and promotes the spread of antibiotic-resistance in bacteria. Consequently, antibiotic treatment frequently fails, resulting in the need for alternative therapies. The present study describes the in vitro efficacy of the Cu(DDC)2 complex (2:1 M ratio of diethyldithiocarbamate (DDC-) and Cu2+) with additional Cu2+ against S. aureus and S. epidermidis biofilms in models mimicking SSIs and in vitro antibacterial activity of a liposomal Cu(DDC)2 + Cu2+ formulation. The in vitro activity on S. aureus and S. epidermidis biofilms grown on two hernia mesh materials and in a wound model was determined by colony forming unit (CFU) counting. Cu2+-liposomes and Cu(DDC)2-liposomes were prepared, and their antibacterial activity was assessed in vitro using the alamarBlue assay and CFU counting and in vivo using a Galleria mellonella infection model. The combination of 35 µM DDC- and 128 µM Cu2+ inhibited S. aureus and S. epidermidis biofilms on meshes and in a wound infection model. Cu(DDC)2-liposomes + free Cu2+ displayed similar antibiofilm activity to free Cu(DDC)2 + Cu2+, and significantly increased the survival of S. epidermidis-infected larvae. Whilst Cu(DDC)2 + Cu2+ showed substantial antibiofilm activity in vitro against clinically relevant biofilms, its application in mammalian in vivo models is limited by solubility. The liposomal Cu(DDC)2 + Cu2+ formulation showed antibiofilm activity in vitro and antibacterial activity and low toxicity in G. mellonella, making it a suitable water-soluble formulation for future application on infected wounds in animal trials.

The combination of diethyldithiocarbamate and copper ions is active against Staphylococcus aureus and Staphylococcus epidermidis biofilms in vitro and in vivo.

Staphylococcus aureus and Staphylococcus epidermidis are associated with life-threatening infections. Despite the best medical care, these infections frequently occur due to antibiotic resistance and the formation of biofilms of these two bacteria (i.e., clusters of bacteria embedded in a matrix). As a consequence, there is an urgent need for effective anti-biofilm treatments. Here, we describe the antibacterial properties of a combination treatment of diethyldithiocarbamate (DDC) and copper ions (Cu2+) and their low toxicity in vitro and in vivo. The antibacterial activity of DDC and Cu2+ was assessed in vitro against both planktonic and biofilm cultures of S. aureus and S. epidermidis using viability assays, microscopy, and attachment assays. Cytotoxicity of DDC and Cu2+ (DDC-Cu2+) was determined using a human fibroblast cell line. In vivo antimicrobial activity and toxicity were monitored in Galleria mellonella larvae. DDC-Cu2+ concentrations of 8 µg/ml DDC and 32 µg/ml Cu2+ resulted in over 80% MRSA and S. epidermidis biofilm killing, showed synergistic and additive effects in both planktonic and biofilm cultures of S. aureus and S. epidermidis, and synergized multiple antibiotics. DDC-Cu2+ inhibited MRSA and S. epidermidis attachment and biofilm formation in the xCELLigence and Bioflux systems. In vitro and in vivo toxicity of DDC, Cu2+ and DDC-Cu2+ resulted in > 70% fibroblast viability and > 90% G. mellonella survival. Treatment with DDC-Cu2+ significantly increased the survival of infected larvae (87% survival of infected, treated larvae vs. 47% survival of infected, untreated larvae, p < 0.001). Therefore, DDC-Cu2+ is a promising new antimicrobial with activity against planktonic and biofilm cultures of S. epidermidis and S. aureus and low cytotoxicity in vitro. This gives us high confidence to progress to mammalian animal studies, testing the antimicrobial efficacy and safety of DDC-Cu2+.

Zinc Homeostasis Alters Zinc Transporter Protein Expression in Vascular Endothelial and Smooth Muscle Cells.

INTRODUCTION: Zinc is an important essential micronutrient with anti-oxidative and anti-inflammatory properties in humans. The role of zinc in signalling has been characterized in the nervous, endocrine, gastrointestinal, renal and reproductive systems. Relatively little is known regarding its role in the vascular system, but the role of zinc homeostasis in augmenting vascular health and vasorelaxation is emerging. Zinc transport proteins are integral to the protective function of zinc, but knowledge of their expression in vascular endothelial and smooth muscle cells is lacking. METHODOLOGY: Human coronary artery endothelial cells and pulmonary artery smooth muscle cells were assessed for gene expression (RT-PCR) of SLC39A (ZIP), SLC30A (ZnT) and metallothionein (MT) families of Zn transporters and storage proteins. Protein expression (fluorescence confocal microscopy) was then analysed for the proteins of interest that changed mRNA expression: ZIP2, ZIP12, ZnT1, ZnT2 and MT1/2. RESULTS: Endothelial and smooth muscle cell mRNA expression of ZnT1, ZnT2 and MT1 was significantly downregulated by low and high Zn conditions, while ZIP2 and ZIP12 expression was induced by Zn depletion with the Zn chelator, TPEN. Changes in gene expression were consistent with protein expression levels for ZIP2, ZIP12 and MT1, where ZIP2 was localized to intracellular bodies and ZIP12 to lamellipodia. CONCLUSION: Vascular endothelial and smooth muscle cells actively regulate specific Zn transport and metallothionein gene and protein expressions to achieve Zn homeostasis.

NFκB Inhibition Mitigates Serum Amyloid A-Induced Pro-Atherogenic Responses in Endothelial Cells and Leukocyte Adhesion and Adverse Changes to Endothelium Function in Isolated Aorta.

The acute phase protein serum amyloid A (SAA) is associated with endothelial dysfunction and early-stage atherogenesis. Stimulation of vascular cells with SAA increases gene expression of pro-inflammation cytokines and tissue factor (TF). Activation of the transcription factor, nuclear factor kappa-B (NFκB), may be central to SAA-mediated endothelial cell inflammation, dysfunction and pro-thrombotic responses, while targeting NFκB with a pharmacologic inhibitor, BAY11-7082, may mitigate SAA activity. Human carotid artery endothelial cells (HCtAEC) were pre-incubated (1.5 h) with 10 µM BAY11-7082 or vehicle (control) followed by SAA (10 µg/mL; 4.5 h). Under these conditions gene expression for TF and Tumor Necrosis Factor (TNF) increased in SAA-treated HCtAEC and pre-treatment with BAY11-7082 significantly (TNF) and marginally (TF) reduced mRNA expression. Intracellular TNF and interleukin 6 (IL-6) protein also increased in HCtAEC supplemented with SAA and this expression was inhibited by BAY11-7082. Supplemented BAY11-7082 also significantly decreased SAA-mediated leukocyte adhesion to apolipoprotein E-deficient mouse aorta in ex vivo vascular flow studies. In vascular function studies, isolated aortic rings pre-treated with BAY11-7082 prior to incubation with SAA showed improved endothelium-dependent vasorelaxation and increased vascular cyclic guanosine monophosphate (cGMP) content. Together these data suggest that inhibition of NFκB activation may protect endothelial function by inhibiting the pro-inflammatory and pro-thrombotic activities of SAA.

Low-density lipoprotein modified by myeloperoxidase oxidants induces endothelial dysfunction.

Low-density lipoprotein (LDL) modified by hypochlorous acid (HOCl) produced by myeloperoxidase (MPO) is present in atherosclerotic lesions, where it is implicated in the propagation of inflammation and acceleration of lesion development by multiple pathways, including the induction of endothelial dysfunction. Thiocyanate (SCN-) ions are utilised by MPO to produce the oxidant hypothiocyanous acid (HOSCN), which reacts with LDL in a different manner to HOCl. Whilst the reactivity of HOCl-modified LDL has been previously studied, the role of HOSCN in the modification of LDL in vivo is poorly defined, although emerging evidence suggests that these particles have distinct biological properties. This is important because elevated plasma SCN- is linked with both the propagation and prevention of atherosclerosis. In this study, we demonstrate that both HOSCN- and HOCl-modified LDL inhibit endothelium-mediated vasorelaxation ex vivo in rat aortic ring segments. In vitro experiments with human coronary artery endothelial cells show that HOSCN-modified LDL decreases in the production of nitric oxide (NO•) and induces the loss of endothelial nitric oxide synthase (eNOS) activity. This occurs to a similar extent to that seen with HOCl-modified LDL. In each case, these effects are related to eNOS uncoupling, rather than altered expression, phosphorylation or cellular localisation. Together, these data provide new insights into role of MPO and LDL modification in the induction of endothelial dysfunction, which has implications for both the therapeutic use of SCN- within the setting of atherosclerosis and for smokers, who have elevated plasma levels of SCN-, and are more at risk of developing cardiovascular disease.