Biomedical subjects

Irene E Kochevar

Publications and source records attributed to Irene E Kochevar.

15 recordsLinked to original sources

PubMed2006

7-Dehydrocholesterol enhances ultraviolet A-induced oxidative stress in keratinocytes: roles of NADPH oxidase, mitochondria, and lipid rafts.

Long wavelength solar UVA radiation stimulates formation of reactive oxygen species (ROS) and prostaglandin E(2) (PGE(2)), which are involved in skin photosensitivity and tumor promotion. High levels of 7-dehydrocholesterol (7-DHC), the precursor to cholesterol, cause exaggerated photosensitivity to UVA in patients with Smith-Lemli-Opitz syndrome (SLOS). Partially replacing cholesterol with 7-DHC in keratinocytes rapidly (<5 min) increased UVA-induced ROS, intracellular calcium, phospholipase A(2) activity, PGE(2), and NADPH oxidase activity. UVA-induced ROS and PGE(2) production were inhibited in these cells by depleting the Nox1 subunit of NADPH oxidase using siRNA or using a mitochondrial radical quencher, MitoQ. Partial replacement of cholesterol with 7-DHC also disrupted membrane lipid raft domains, although depletion of cholesterol, which also disrupts lipid rafts, did not affect UVA-induced increases in ROS and PGE(2). Phospholipid liposomes containing 7-DHC were more rapidly oxidized by a free radical mechanism than those containing cholesterol. These results indicate that 7-DHC enhances rapid UVA-induced ROS and PGE(2) formation by enhancing free radical-mediated membrane lipid oxidation and suggests that this mechanism might underlie the UVA photosensitivity in SLOS.

Calcium↗

PubMed2005

Ultraviolet A induces apoptosis via reactive oxygen species in a model for Smith-Lemli-Opitz syndrome.

Solar ultraviolet A (UVA) radiation induces many responses in skin including oxidative stress, DNA damage, inflammation, and skin cancer. Smith-Lemli-Opitz syndrome (SLO-S) patients show dramatically enhanced immediate (5 min) and extended (24-48 h) skin inflammation in response to low UVA doses compared to normal skin. Mutations in Delta7-dehydrocholesterol reductase, which converts 7-dehydrocholesterol to cholesterol, produces high levels of 7-dehydrocholesterol in SLO-S patient's serum. Since 7-dehydrocholesterol is more rapidly oxidized than cholesterol, we hypothesized that 7-dehydrocholesterol enhances UVA-induced oxidative stress leading to keratinocyte death and inflammation. When keratinocytes containing high 7-dehydrocholesterol and low cholesterol were exposed to UVA (10 J/cm2), eightfold greater reactive oxygen species (ROS) were produced than in normal keratinocytes after 15 min. UVA induced 7-dehydrocholesterol concentration-dependent cell death at 24 h. These responses were inhibited by antioxidants, reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor (diphenyleneiodonium) and a mitochondria-specific radical quencher. Cell death was characterized by activation of caspases-3, -8, and -9 and by phosphatidylserine translocation. Studies using antioxidants and specific caspase inhibitors indicated that activation of caspase-8, but not caspase-9, mediates ROS-dependent caspase-3 activation and suggested that ROS from NADPH oxidase activate caspase-8. These results support a ROS-mediated apoptotic mechanism for the enhanced UVA-induced inflammation in SLO-S patients.

Antioxidants↗

PubMed2005

Involvement of UVB-induced reactive oxygen species in TGF-beta biosynthesis and activation in keratinocytes.

TGF-beta produced by keratinocytes in response to UVB (290-320 nm) is a potential mediator for effects of acute and chronic solar radiation on skin. This study was designed to determine whether reactive oxygen species (ROS) mediate UVB-induced TGF-beta biosynthesis in keratinocytes and the subsequent activation of the latent TGF-beta complex. UVB irradiation elevated both total (latent plus active) and active TGF-beta in the keratinocyte supernatants, with a greater increase in the active form. UVB irradiation induced up to a 30% increase in ROS, and the ROS were detected up to 90 min after irradiation. NAC and Trolox, cytoplasmic ROS scavengers, abolished the UVB-induced TGF-beta and intracellular ROS, suggesting that UVB-induced ROS are involved in TGF-beta regulation. Inhibitors of NADPH oxidase activity, DPI and apocynin, decreased UVB-induced ROS. The increase in NADPH oxidase activity was mediated by EGFR activation. UVB-induced ROS also activated latent TGF-beta complex by stimulating MMP-2 and -9 activities. In summary, physiological doses of UVB increase intracellular ROS, which upregulate TGF-beta biosynthesis and activation of TGF-beta through increased activity of MMPs.

ErbB Receptors↗

PubMed2005

Evaluation of photochemical tissue bonding for closure of skin incisions and excisions.

BACKGROUND AND OBJECTIVES: Photochemical tissue bonding (PTB) is a new non-thermal technique for tissue repair involving application of a photochemically active dye and irradiation with visible light. The objective was to compare PTB with standard sutures and the tissue adhesive, octyl cyanoacrylate, for closure of skin incisions and excisions. STUDY DESIGN/MATERIALS AND METHODS: Incisions and excisions made on the flanks of a Hanford mini-pig were secured with subcutaneous sutures. Superficial closure methods were 3-0 monofilament sutures, PTB (Rose Bengal and green light), tissue adhesive and the combination of tissue adhesive then PTB. Wounds were evaluated 2, 4, and 6 weeks postoperatively for cosmetic outcomes and histology. RESULTS: Cosmetic outcomes and histological scar width of incisions and excisions did not differ among the treatment groups at 2, 4, and 6 weeks. CONCLUSION: PTB is as effective as standard sutures for wound closure in porcine skin in terms of cosmetic outcomes and safety.

Animals↗

PubMed2005

Photochemical repair of Achilles tendon rupture in a rat model.

BACKGROUND: Photochemical tissue bonding (PTB) is an emerging technique for bonding or sealing tissue surfaces that requires light and a photoactive dye for its effect. The potential of PTB for tendon repair was assessed in a rat model. MATERIALS AND METHODS: The optical properties of bovine tendon were determined ex vivo to gauge the depth of light penetration as a function of wavelength and dosimetry parameters were established for PTB repair of ruptured tendon. PTB was then tested in vivo to repair transected tendons in Sprague-Dawley rats. Repair strengths were measured using a strain gauge up to 14 days post treatment. RESULTS: The effective penetration depth in tendon was estimated to be 0.68 mm at 514 nm. Following PTB treatment of mechanically ruptured tendon, significant bonding was dependent on the presence of both light and dye and attained a plateau strength at a fluence of 125 J/cm2. In a subsequent in vivo study to investigate PTB for repair of transected rat Achilles tendon, the ultimate stress required to break the repaired tendon was measured immediately after irradiation and at 7 and 14 days post-repair. Results showed that the difference in the ultimate stress between control and PTB treatment groups was statistically significant immediately after treatment and at 7 days (p = 0.04) but not 14 days (p = 0.75) post-repair. CONCLUSIONS: PTB provides a benefit to tendon repair at early stages in repair and is worthy of further investigation as a potential surgical adjunct for tendon repair in orthopedic surgeries.

Achilles Tendon↗

PubMed2004

Singlet oxygen signaling: from intimate to global.

Reactive oxidizing species (ROS), such as hydrogen peroxide, nitric oxide, superoxide anion, and singlet oxygen, can damage cells as well as initiate responses such as new gene expression. The cell response evoked is strongly dependent on several factors. The subcellular location for formation of an ROS may be especially important for a highly reactive ROS, because it diffuses only a very short distance before reacting with a cellular molecule. How does a short-lived, locally acting ROS trigger responses at distant subcellular sites? This issue is discussed in light of a study of gene expression initiated by singlet oxygen, a ROS that exists for less than 100 ns in cells. Singlet oxygen is generated after a photosensitizer absorbs light energy and transfers the energy to molecular oxygen. To assess the role of singlet oxygen in light-induced gene expression in Arabidopsis, a mutant was used that accumulates the photosensitizer protochlorphyllide in chloroplasts. Three mechanisms are discussed that may connect the site of singlet oxygen formation to the signal transduction components: (i) direct oxidation of a signaling component by singlet oxygen, (ii) formation of oxidation products near the sites of singlet oxygen formation that diffuse to and react with signaling components, and (iii) alteration of the redox balance of the cell to a more oxidized state such that a greater proportion of a signaling pathway component is oxidized.

Animals↗

PubMed2004

Photochemical keratodesmos as an adjunct to sutures for bonding penetrating keratoplasty corneal incisions.

PURPOSE: To evaluate the benefit of photochemical keratodesmos (PKD) in acute wound closure of penetrating keratoplasty (PKP) corneal incisions in vivo. SETTING: Massachusetts General Hospital and Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA. METHODS: Penetrating keratoplasty incisions were performed in both eyes of 6 New Zealand white rabbits, followed by application of 1.5 mM rose bengal dye at the wound edges. The incision in 1 eye of each rabbit was irradiated with neodymium:YAG laser light at 532 nm and fluence of 40 J/cm2; the contralateral control eye was untreated. Intraocular pressure at which fluid leaked at the edges (IOPL) was determined immediately after surgery. RESULTS: The mean IOPL was 410 mm Hg +/- 70 (SD) in the PKD-treated eyes and 250 +/- 150 mm Hg in the unirradiated eyes. The difference was statistically significant by paired t test (P<.05). CONCLUSIONS: Photochemical keratodesmos may be a useful adjunct to sutures for approximating PKP corneal incisions in the immediate postoperative period. This process does not induce high temperature, and thus denaturation can be avoided and structural integrity restored.

Animals↗

PubMed2004

Photochemical keratodesmos for bonding corneal incisions.

PURPOSE: To evaluate the immediate and long-term effectiveness of a dye-plus-laser irradiation treatment (photochemical keratodesmos [PKD]) for sealing corneal incisions. METHODS: Incisions (3.5 mm) in rabbit corneas were treated on the incision walls with rose bengal dye followed by exposure to 514-nm laser radiation. PKD was evaluated in three groups (n = 3-6) using laser fluences of 115, 153, or 192 J/cm(2) (180-, 240-, and 300- second exposures, respectively) compared with an untreated group (n = 8). The intraocular pressure at which leakage occurred (IOP(L)) during infusion of saline into the anterior chamber was determined. In a long-term study, treated and control corneas were observed weekly for 10 weeks for the appearance of neovascularization, anterior chamber inflammation, iridocorneal adhesion, corneal melting, and scarring. RESULTS: Immediately after treatment, the IOP(L) increased with increasing laser fluence, producing IOPs of 230 +/- 90, 370 +/- 120, and more than 500 mm Hg at 115, 153, and 192 J/cm(2), respectively, compared with 40 +/- 20 mm Hg in control eyes (P < 0.005). No reduction in the IOP(L) was observed up to 14 days after surgery. Corneal melting in PKD-treated or control eyes was not observed in the 10-week healing study. Neovascularization, which peaked at 4 weeks but resolved by 8 weeks, was detected around the incision in both PKD-treated and control eyes. CONCLUSIONS: Immediate and lasting sealing of corneal incisions was obtained in eyes treated with PKD, using short irradiation times. These results suggest that PKD has potential for improved corneal tissue bonding.

Animals↗

PubMed2004

Effects of UVR and UVR-induced cytokines on production of extracellular matrix proteins and proteases by dermal fibroblasts cultured in collagen gels%.

Synthesis of extracellular matrix (ECM) proteins and their degradation by matrix metalloproteinases (MMP) are part of the dermal remodeling resulting from chronic exposure of skin to ultraviolet radiation (UVR). We have compared two alternative mechanisms for these responses, namely, a direct mechanism in which UV-B or UV-A is absorbed by fibroblasts and an indirect mechanism in which cytokines, produced in skin in response to UVR, stimulate production of the ECM proteins and MMP. These studies were carried out on human dermal fibroblasts grown in contracted, free-floating 9 day old collagen gels as a dermal equivalent. Synthesis of tropoelastin, collagen, fibrillin, MMP-1, -2, -3 and -9 and tissue inhibitors of metalloproteinases (TIMP)-1 and -2 were measured. Tropoelastin, collagen and fibrillin levels were stable between days 4 and 10, and MMP and TIMP decreased by day 10. Neither UV-B (2.5-50 mJ/cm2) nor UV-A (2-12 J/cm2) altered synthesis of ECM proteins, but UV-A increased MMP-1 and -3 production. Tropoelastin synthesis increased in response to transforming growth factor-beta1 (5 ng/mL) treatment. Both interleukin-1beta and tumor necrosis factor-alpha (10 ng/mL) decreased fibrillin messenger RNA levels but increased MMP-1, -3 and -9 synthesis markedly. Collagen synthesis was not modulated by UV-B, UV-A or cytokine treatment. These results indicate that certain cytokines may have greater effects on production of ECM proteins and MMP than absorption of UV-B and UV-A by fibroblasts grown in dermal equivalents and suggest that the former pathway may play a role in the dermal remodeling in photoaged skin.

Cell Culture Techniques↗

PubMed2003

Downregulation of epidermal growth factor receptor signaling by singlet oxygen through activation of caspase-3 and protein phosphatases.

Downregulation of survival signaling pathways contributes to the cytotoxicity of reactive oxygen species (ROS) and may underlie certain therapies for hyperproliferative diseases. We have investigated the role of singlet oxygen, an ROS formed by photosensitization, in the regulation of survival signaling via the epidermal growth factor receptor (EGFR). Exposure of human keratinocytes to singlet oxygen resulted in rapid loss of EGFR, which was not blocked by either inhibition of receptor internalization or by interrupting the major proteolytic pathways (proteasome, lysosome or calpain). However, pretreatment with a caspase-3 inhibitor, DEVD-FMK, inhibited EGFR degradation. Caspase-3 cleavage was detected as early as 5 min after singlet oxygen treatment, and recombinant active caspase-3 completely cleaved EGFR in a keratinocyte membrane fraction. The singlet oxygen-induced loss of EGFR was accompanied by dephosphorylation of EGFR as well as of Akt and extracellular signal-regulated kinase 1/2 (ERK)1/2. Singlet oxygen-induced protein dephosphorylation was not dependent on activation of caspase-3. In contrast, inhibition of protein phosphatases (PPs) with okadaic acid completely blocked dephosphorylation of EGFR, ERK1/2 and Akt as well as degradation of EGFR. These results indicate that the oxidative stress produced by singlet oxygen rapidly disrupts EGFR-mediated signaling by decreasing both the protein level and its phosphorylation. These responses depended on intertwined activation of caspase-3 and PPs.

Apoptosis↗

PubMed2003

Influence of hydration on dihydroxyacetone-induced pigmentation of stratum corneum.

Dihydroxyacetone, the browning ingredient in sunless tanning formulations, reacts with amino acids in the outer stratum corneum to form a mixture of high molecular weight pigments. Our initial observations indicated that high hydration of dihydroxyacetone-treated skin completely inhibited development of pigmentation. To investigate the mechanism underlying this effect, studies were carried out in isolated murine epidermis, polyvinyl alcohol/lysine films, and lysine in glycerol/water solvent. Murine epidermis treated with dihydroxyacetone showed a biphasic dependence on relative humidity: maximum pigmentation developed at 84% relative humidity and minimum pigmentation at 0% and 100% relative humidity. Filaggrin proteolysis, which shows a similar dependence on relative humidity and provides free amino acids in the outer stratum corneum, did not account for the relative humidity dependence of dihydroxyacetone pigmentation. A similar biphasic pigmentation response was obtained when polyvinyl alcohol film containing lysine was treated with dihydroxyacetone and incubated at various relative humidities, indicating that the structure of the stratum corneum was not a major factor. To remove the influence of the matrix, the reaction of dihydroxyacetone with lysine was followed at varying concentrations of water in mixed glycerol/buffer solvent. Again, greater pigment formation was found at an intermediate level of water (6% vol/vol) and little pigmentation at 0% and 100% water content. These results are consistent with a requirement for water at low relative humidity, which facilitates formation of free amine groups needed for the initial reaction with dihydroxyacetone, and with inhibition of the dehydration reactions by water through the law of mass action at high relative humidity.

Amino Acids↗

PubMed2003

Ultraviolet A radiation induces rapid apoptosis of human leukemia cells by Fas ligand-independent activation of the Fas death pathways.

Endogenous cellular chromophores absorb ultraviolet A radiation (UVA, 290-320 nm), the major UV component of terrestrial solar radiation, leading to the formation of reactive oxidizing species that initiate apoptosis, gene expression and mutagenesis. UVA-induced apoptosis of T helper cells is believed to underlie the UVA phototherapy for atopic dermatitis and other T cell-mediated inflammatory skin diseases. We have evaluated the involvement of the Fas-Fas ligand (FasL) pathway in rapid UVA-induced apoptosis in human leukemia HL-60 cells. UVA-induced apoptosis was not inhibited by pretreatment with a neutralizing anti-Fas antibody, although the same UVA treatment initiated cleavage of caspase-8 and subsequent processing of Bid and caspase-3-like proteases. Inhibition of caspase-8 by Lle-Glu (OMe)-Thr-Asp(OMe)-fluoromethyl ketone completely blocked caspase-3 cleavage and apoptosis in UVA-treated cells, suggesting that apoptosis was initiated by the Fas pathway. This inference was supported by demonstrating that immunoprecipitates obtained from UVA-treated cells using anti-Fas antibody contained caspase-8 and Fas-associating protein with death domain (FADD). In addition, Fas clustering in response to UVA treatment was observed by immunofluorescence microscopy. These data support a mechanism for rapid, UVA-induced apoptosis in HL-60 cells involving initial formation of the Fas-FADD-caspase-8 death complex in an FasL-independent manner.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Singlet oxygen-induced activation of Akt/protein kinase B is independent of growth factor receptors.

Singlet oxygen (1O2)-induced cytotoxicity is believed to be responsible for responses to photodynamic therapy and for apoptosis of T helper cells after UV-A treatment. Other cytotoxic oxidants, such as hydrogen peroxide and peroxynitrite have been shown to stimulate cell survival signaling pathways in addition to causing cell death. Both these oxidants stimulate the Akt/protein kinase B survival signaling pathway through activation of membrane tyrosine kinase growth factor receptors. We evaluated the ability of 1O2 to activate the Akt/protein kinase B pathway in NIH 3T3 cells and examined potential activation pathways. Exposure of fibroblasts to 1O2 elicited a strong and sustained phosphorylation of Akt, which occurred concurrently with phosphorylation of p38 kinase, a proapoptotic signal. Inhibition of phosphatidylinositol-3-OH kinase (PI3-K) completely blocked Akt phosphorylation. Significantly, cell death induced by 1O2 was enhanced by inhibition of PI3-K, suggesting that activation of Akt by 1O2 may contribute to fibroblast survival under this form of oxidative stress. 1O2 treatment did not induce phosphorylation of platelet-derived growth factor receptor (PDGFR) or activate SH-PTP2, a substrate of growth factor receptors, suggesting that PDGFR was not activated. In addition, specific inhibition of PDGFR did not affect Akt phosphorylation elicited by 1O2. Activation of neither focal adhesion kinase (FAK) nor Ras protein, both of which mediate responses to reactive oxygen species, appeared to be pathways for the 1O2-induced activation of the PI3-K-Akt survival pathway. Thus, activation of Akt by 1O2 is mediated by PI3-K and contributes to a survival response that counteracts cell death after 1O2-induced injury. However, unlike the response to other oxidants, activation of the PI3-K-Akt by 1O2 does not involve activation of growth factor receptors, FAK or Ras protein.

Animals↗

PubMed2002

Enhancement of porcine skin graft adherence using a light-activated process.

BACKGROUND: Skin grafts are widely used in plastic surgery, and burn and ulcer wound management. Rapid and sustained adherence, the ability to resist shear stress, and a void-free surface-to-surface contact are critical to the success of graft survival. Mechanical and adhesive fixation aids are currently used to achieve graft adherence and they are not free of problems. Photochemical tissue bonding (PTB) is an emerging laser technique with numerous applications in surgical specialties. In the current study, PTB was investigated as a means to bond and enhance the adherence of skin grafts. METHODS: In this study, ex vivo porcine skin grafts treated with a photosensitizing dye, rose bengal (RB), were approximated dermis-to-dermis and irradiated with visible light from an argon laser at 514 nm. The adherence of the skin grafts was measured immediately after irradiation. Dose-response relationships between the light and the dye with adherence of the grafts were established. The surface temperature of the skin under irradiation was monitored and the viability of the skin cells in the grafts was also measured. RESULTS: Results showed that the skin graft adherence was RB dose-dependent in a statistically significant manner with the concentration of RB reaching a plateau value of 0.1% (w/v) of RB. Graft adhesion also increased with laser fluence up to 504 J/cm(2) in the presence of 0.1% RB. No fluence dependence was observed in the absence of RB. Thermogram results showed that the maximal surface temperature during irradiation was less than 40 degrees C. Histological investigation and trypan blue exclusion assays demonstrated that skin grafts retained cell viability and collagen organization after PTB. CONCLUSIONS: This ex vivo study demonstrates that PTB using argon laser irradiation and RB enhances skin graft adherence by forming dermal-dermal bonding. The increase in adherence is a function of the concentration of RB and the laser fluence. The results also suggest that the PTB is a potentially safe procedure because it is nonthermal in nature and does not significantly affect the skin cell viability.

Animals↗

PubMedDate not supplied

Spatially resolved cellular responses to singlet oxygen.

Singlet oxygen (1O2) is unique amongst reactive oxygen species formed in cells in that it is an excited state molecule with an inherent upper lifetime of 4 micros in water. Whether the lifetime of 1O2 in cells is shortened by reactions with cellular molecules or reaches the inherent maximum value is still unclear. However, even with the maximum lifetime, the diffusion radius is only approximately 220 nm during three lifetimes (approximately 5% 1O2 remaining), much shorter than cellular dimensions indicating that the primary reactions of 1O2 will be subcellularly localized near the site of 1O2 formation. This fact has raised the question of whether spatially resolved cellular responses to 1O2 occur, i.e. whether responses can be initiated by generation and reaction of 1O2 at a particular subcellular location that would not have been produced by 1O2 generation at other subcellular sites. In this paper, we discuss examples of spatially resolved responses initiated by 1O2 as a function of distance from the site of generation of 1O2. Three levels are recognized, namely, a molecular level where the primary oxidation product directly modifies the behavior of a cell, an organelle level where the initial photo-oxidation products initiate mechanisms that are unique to the organelle and the cellular level where mediators diffuse from their site of formation to the target molecules that initiate the response. These examples indicate that, indeed, spatially resolved responses to 'O2 occur in cells.

Humans↗