A scalable approach to topographically mediated antimicrobial surfaces based on diamond.

Bio-inspired Topographically Mediated Surfaces (TMSs) based on high aspect ratio nanostructures have recently been attracting significant attention due to their pronounced antimicrobial properties by mechanically disrupting cellular processes. However, scalability of such surfaces is often greatly limited, as most of them rely on micro/nanoscale fabrication techniques. In this report, a cost-effective, scalable, and versatile approach of utilizing diamond nanotechnology for producing TMSs, and using them for limiting the spread of emerging infectious diseases, is introduced. Specifically, diamond-based nanostructured coatings are synthesized in a single-step fabrication process with a densely packed, needle- or spike-like morphology. The antimicrobial proprieties of the diamond nanospike surface are qualitatively and quantitatively analyzed and compared to other surfaces including copper, silicon, and even other diamond surfaces without the nanostructuring. This surface is found to have superior biocidal activity, which is confirmed via scanning electron microscopy images showing definite and widespread destruction of E. coli cells on the diamond nanospike surface. Consistent antimicrobial behavior is also observed on a sample prepared seven years prior to testing date.

Inhibition of hypoxia inducible factor-1α downregulates the expression of epithelial to mesenchymal transition early marker proteins without undermining cell survival in hypoxic lens epithelial cells.

PURPOSE: The purpose of this study was to identify potential therapeutic strategies to slow down or prevent the expression of early-onset epithelial to mesenchymal transition (EMT) marker proteins (fibronectin and alpha smooth muscle actin, α-SMA) without sacrificing the synthesis and accumulation of the prosurvival protein vascular endothelial growth factor (VEGF) in cultured virally transformed human lens epithelial (HLE) cells. METHODS: HLE-B3 cells, maintained in a continuous hypoxic environment (1% oxygen), were treated with SB216763, a specific inhibitor of glycogen synthase kinase-3ß (GSK-3ß) catalytic activity. Western blot analysis was employed to detect the cytoplasmic and nuclear levels of ß-catenin, as well as the total lysate content of fibronectin and α-SMA. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of VEGF in cell culture medium. A hypoxia-inducible factor-1α (HIF-1α) translation inhibitor and an HIF-2α translation inhibitor were independently employed to evaluate the effect of hypoxia inducible factor inhibition on EMT marker protein and VEGF expression. XAV932 was used to assess the suppression of nuclear ß-catenin and its downstream effect on EMT marker proteins and VEGF expression. RESULTS: SB216763-treated HLE-B3 cells caused marked inhibition of GSK-3ß activity prompting a significant increase in the translocation of cytoplasmic ß-catenin to the nucleus. The enhancement of nuclear ß-catenin looked as if it positively correlated with a significant increase in the basal expression of VEGF as well as increased expression of fibronectin and α-SMA. In conjunction with SB216763, coadministration of an HIF-1α translation inhibitor, but not an HIF-2α translation inhibitor, markedly suppressed the expression of fibronectin and α-SMA without affecting VEGF levels. Treatment with XAV932 significantly reduced the level of nuclear ß-catenin, but the levels of neither the EMT marker proteins nor VEGF were changed. CONCLUSIONS: Recently, we reported that nuclear ß-catenin, but not HIF-2α, regulates the expression of fibronectin and α-SMA in atmospheric oxygen. In marked contrast, data from the hypoxic condition clearly establish that nuclear ß-catenin plays little apparent role in the expression of EMT marker proteins. Instead, the loss of HIF-1α (but not HIF-2α) decreases the expression of the EMT marker proteins without sacrificing the levels of the prosurvival protein VEGF. These findings support the development of a potentially relevant therapeutic strategy to undermine the progression of normal cells to the mesenchymal phenotype in the naturally hypoxic lens without subverting cell viability.

Lenticular cytoprotection, part 2: link between glycogen synthase kinase-3ß, epithelial to mesenchymal transition, and mitochondrial depolarization.

PURPOSE: The inhibition of GSK-3ß blocks mitochondrial membrane permeability transition (mMPT) for HLE-B3 cells in atmospheric oxygen. GSK-3ß, as part of a multifactorial complex, also regulates nuclear levels of ß-catenin, a known coordinator of cell survival and adhesion. The purpose of these studies was to demonstrate a novel, but likely disadvantageous, link between ß-catenin's influence on the expression of the pro-survival protein, vascular endothelial growth factor (VEGF), resulting in enhanced lens epithelial cell mitochondrial protection against depolarization and nuclear ß-catenin as an inducer of epithelial to mesenchymal transition (EMT). METHODS: Virally transformed human lens epithelial cells (HLE-B3) were treated with SB216763, a specific inhibitor of GSK-3ß catalytic activity and XAV939, a specific ß-catenin inhibitor that bars the translocation of ß-catenin from cytoplasm to the nucleus. Western blot analysis was employed to detect the levels of cytoplasmic and nuclear ß-catenin and phospho-ß-catenin, pBcl-2 and the EMT proteins, α-smooth muscle actin (α-SMA), and fibronectin. ELISA was used to measure the levels of VEGF in cell culture supernatants. JC-1 analysis was performed to analyze the influence of either SB216763 or XAV939 on mitochondrial depolarization. RESULTS: Cultured lens epithelial cells maintained in hypoxia (1% oxygen) and subsequently reintroduced into atmospheric oxygen and treated with the GSK-3ß inhibitor SB216763 illustrated a marked inhibition of phosphorylation of glycogen synthase (downstream substrate of GSK-3ß) and significant increase in nuclear translocation of ß-catenin. The augmented nuclear ß-catenin levels positively correlated with increased expression of α-SMA and fibronectin, both marker proteins indicative of EMT. The enhanced nuclear ß-catenin activity also elicited increased VEGF and pBcl-2 expression, resulting in increased resistance to mitochondrial depolarization. Treatment of the cells with the ß-catenin inhibitor XAV939 resulted in decreased expression of nuclear ß-catenin, VEGF levels, pBcl-2, and EMT proteins, as well as increased mitochondrial depolarization. CONCLUSIONS: The data support a model whereby the onset of epithelial to mesenchymal transition may circuitously benefit from the enhanced synthesis of VEGF by setting up a potentially harmful situation whereby the resulting mesenchymal cell population may be more resistant to mitochondrial depolarization than the lens epithelial cell population from which it originated. These findings support the potential therapeutic relevance of developing strategies to undermine the progression of normal cells to mesenchymal transition without subverting cell viability.

Lenticular cytoprotection. Part 1: the role of hypoxia inducible factors-1α and -2α and vascular endothelial growth factor in lens epithelial cell survival in hypoxia.

PURPOSE: The prosurvival signaling cascades that mediate the unique ability of human lens epithelial cells to survive in their naturally hypoxic environment are not well defined. Hypoxia induces the synthesis of the hypoxia inducible factor HIF-1α that in turn, plays a crucial role in modulating a downstream survival scheme, where vascular endothelial growth factor (VEGF) also plays a major role. To date, no published reports in the lens literature attest to the expression and functionality of HIF-2α and the role it might play in regulating VEGF expression. The aim of this study was to identify the functional expression of the hypoxia inducible factors HIF-1α and HIF-2α and establish their role in regulating VEGF expression. Furthermore, we demonstrate a link between sustained VEGF expression and the ability of the hypoxic human lens epithelial cell to thrive in low oxygen conditions and resist mitochondrial membrane permeability transition (also referred to as lenticular cytoprotection). METHODS: Hypoxia inducible factor translation inhibitors were used to demonstrate the role of HIF-1α and HIF-2α and the simultaneous expression of both hypoxic inducible factors to determine their role in regulating VEGF expression. Axitinib, which inhibits lenticular cell autophosphorylation of its VEGF receptor, was employed to demonstrate a role for the VEGF-VEGFR2 receptor complex in regulating Bcl-2 expression. Specific antisera and western blot analysis were used to detect the protein levels of HIF-1α and HIF-2α, as well as the proapoptotic protein, BAX and the prosurvival protein, Bcl-2. VEGF levels were analyzed with enzyme-linked immunosorbent assay (ELISA). The potentiometric dye, 5,5',6,6'-tetrachloro1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine iodide, was used to determine the effect of the inhibitors on mitochondrial membrane permeability transition. RESULTS: Cultured human lens epithelial cells (HLE-B3) maintained under hypoxic condition (1% oxygen) displayed consistent accumulation of VEGF throughout the 72 h incubation period. Using hypoxia inducible factor translation inhibitors targeting HIF-1α or HIF-2α, the specific inhibition of each protein did not diminish VEGF synthesis. The combined inhibition of HIF-1α and HIF-2α expression, using a double hypoxia inducible factor translation inhibitor, markedly decreased the level of VEGF. The inhibition of VEGF synthesis was associated with a profound deficiency in the level of the prosurvival protein, Bcl-2. Axitinib also prevented the VEGF-mediated expression of Bcl-2. The loss of VEGF coupled with the decrease in intracellular Bcl-2 correlated with marked mitochondrial depolarization, an early predictor of cellular apoptosis. CONCLUSIONS: Our data support a model in which the sustained synthesis of VEGF in human lens epithelial cells, maintained under hypoxic condition, is regulated by a compensatory inter-relationship between HIF-1α and HIF-2α. VEGF acts as a prosurvival factor in hypoxic lens epithelial cells by maintaining consistent expression of the prosurvival protein Bcl-2, which likely prevents the translocation of cytosolic BAX to the outer mitochondrial membrane, thus preventing the initiation of mitochondrial depolarization.

Lenticular mitoprotection. Part B: GSK-3ß and regulation of mitochondrial permeability transition for lens epithelial cells in atmospheric oxygen.

PURPOSE: Loss of integrity of either the inner or outer mitochondrial membrane results in the dissipation of the mitochondrial electrochemical gradient that leads to mitochondrial membrane permeability transition (mMPT). This study emphasizes the role of glycogen synthase kinase 3beta (GSK-3ß) in maintaining mitochondrial membrane potential, thus preventing mitochondrial depolarization (hereafter termed mitoprotection). Using 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763), an inhibitor of GSK-3ß, and drawing a distinction between it and 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (UO126), an inhibitor of extracellular-signal-regulated kinase (ERK) phosphorylation, the means by which GSK-3ß influences mitoprotection in cultured human lens epithelial (HLE-B3) cells and normal, secondary cultures of bovine lens epithelial cells, maintained in atmospheric oxygen, was investigated. METHODS: Virally transfected human lens epithelial cells (HLE-B3) and normal cultures of bovine lens epithelial cells were exposed to acute hypoxic conditions (about 1% O2) followed by exposure to atmospheric oxygen (about 21% O2). Specific antisera and western blot analysis was used to examine the state of phosphorylation of ERK and GSK-3ß, as well as the phosphorylation of a downstream substrate of GSK-3ß, glycogen synthase (GS, useful in monitoring GSK-3ß activity). The potentiometric dye, 1H-benzimidazolium-5,6-dichloro-2-[3-(5,6-dichloro-1,3-diethyl-1,3-dihydro-2H-benzimidazol-2-ylidene)-1-propenyl]-1,3-diethyl-iodide (JC-1), was used to monitor mitochondrial depolarization upon exposure of inhibitor treatment relative to the control cells (mock inhibition) in atmospheric oxygen. Caspase-3 activation was scrutinized to determine whether mitochondrial depolarization inevitably leads to apoptosis. RESULTS: Treatment of HLE-B3 cells with SB216763 (12 µM) inactivated GSK-3ß activity as verified by the enzyme's inability to phosphorylate its substrate, GS. SB216763-treated cells were not depolarized relative to the control cells as demonstrated with JC-1 fluorescent dye analysis. The HLE-B3 cells treated with UO126, which similarly blocked phosphorylation of GS, were nevertheless prone to mMPT relative to the control cells. Western blot analysis determined that Bcl-2-associated X (BAX) levels were unchanged for SB216763-treated or UO126-treated HLE-B3 cells when compared to their respective control cells. However, unlike the SB216763-treated cells, the UO126-treated cells showed a marked absence of Bcl-2, as well as phosphorylated Bcl-2 relative to the controls. UO126 treatment of bovine lens epithelial cells showed similar results with pBcl-2 levels, while the Bcl-2 content appeared unchanged relative to the control cells. HLE-B3 and normal bovine lens cell cultures showed susceptibility to mMPT associated with the loss of pBcl-2 by UO126 treatment. CONCLUSIONS: MITOCHONDRIAL DEPOLARIZATION MAY OCCUR BY ONE OF TWO KEY OCCURRENCES: interruption of the electrochemical gradient across the inner mitochondrial membrane resulting in mMPT or by disruption of the integrity of the inner or outer mitochondrial membrane. The latter scenario is generally tightly regulated by members of the Bcl-2 family of proteins. Inhibition of GSK-3ß activity by SB216763 blocks mMPT by preventing the opening of the mitochondrial permeability transition pore. UO126, likewise, inhibits GSK-3ß activity, but unlike SB216763, inhibition of ERK phosphorylation induces the loss of intracellular pBcl-2 levels under conditions where intracellular BAX levels remain constant. These results suggest that the lenticular mitoprotection normally afforded by the inactivation of GSK-3ß activity may, however, be bypassed by a loss of pBcl-2, an anti-apoptotic member of the Bcl-2 family. Bcl-2 prevents the translocation of BAX to the mitochondrial outer membrane inhibiting depolarization by disrupting the normal electrochemical gradient leading to mMPT.

Lenticular mitoprotection. Part A: Monitoring mitochondrial depolarization with JC-1 and artifactual fluorescence by the glycogen synthase kinase-3ß inhibitor, SB216763.

PURPOSE: Dissipation of the electrochemical gradient across the inner mitochondrial membrane results in mitochondrial membrane permeability transition (mMPT), a potential early marker for the onset of apoptosis. In this study, we demonstrate a role for glycogen synthase kinase-3ß (GSK-3ß) in regulating mMPT. Using direct inhibition of GSK-3ß with the GSK-3ß inhibitor SB216763, mitochondria may be prevented from depolarizing (hereafter referred to as mitoprotection). Cells treated with SB216763 showed an artifact of fluorescence similar to the green emission spectrum of the JC-1 dye. We demonstrate the novel use of spectral deconvolution to negate the interfering contributing fluorescence by SB216763, thus allowing an unfettered analysis of the JC-1 dye to determine the mitochondrial membrane potential. METHODS: Secondary cultures of virally transfected human lens epithelial cells (HLE-B3) were exposed to acute hypoxic conditions (approximately 1% O2) followed by exposure to atmospheric oxygen (approximately 21% O2). The fluorescent dye JC-1 was used to monitor the extent of mitochondrial depolarization upon exposure of inhibitor treatment relative to the control cells (mock inhibition) in atmospheric oxygen. Annexin V-fluorescein isothiocyanate/propidium iodide staining was implemented to determine cell viability. RESULTS: Treatment of HLE-B3 cells with SB216763 (12 µM), when challenged by oxidative stress, suppressed mitochondrial depolarization relative to control cells as demonstrated with JC-1 fluorescent dye analysis. Neither the control nor the SB216763-treated HLE-B3 cells tested positive with annexin V-fluorescein isothiocyanate/propidium iodide staining under the conditions of the experiment. CONCLUSIONS: Inhibition of GSK-3ß activity by SB216763 blocked mMPT relative to the slow but consistent depolarization observed with the control cells. We conclude that inhibition of GSK-3ß activity by the GSK-3ß inhibitor SB216763 provides positive protection against mitochondrial depolarization.