Shock Wave Therapy Promotes Cardiomyocyte Autophagy and Survival during Hypoxia.

BACKGROUND: Autophagy plays an important role in cardiovascular disease. Controversy still exists regarding the effect of autophagy on ischemic/hypoxic myocardium. Cardiac shock wave therapy (CSWT) is an effective alternative treatment for refractory ischemic heart disease. Whether CSWT can regulate cardiomyocyte autophagy under hypoxic conditions is not clear. We established a myocardial hypoxia model using the H9c2 cell line and performed shock waves (SWs) treatment to evaluate the effect of SW on autophagy. METHODS: The H9c2 cells were incubated under hypoxic conditions, and SW treatment was then performed at energies of 0.02, 0.05, or 0.10 mJ/mm2. The cell viability and intracellular ATP level were examined. Western blot analysis was used to assess the expression of LC3B, AMPK, mTOR, Beclin-1, Sirt1, and HIF-1α. Autophagic vacuoles were visualized by monodansylcadaverine staining. RESULTS: After the 24-hour hypoxic period, cardiomyocyte viability and ATP levels were decreased and autophagy was significantly increased in H9c2 cells. SW treatment with an energy of 0.05 mJ/mm2 significantly increased the cellular viability, ATP level, LC3B-II/I, and number of autophagic vacuoles. In addition, phosphorylated AMPK and Sirt1 were increased and phosphorylated mTOR and HIF-1α were decreased after SW treatment. CONCLUSION: SW treatment can potentially promote cardiomyocyte autophagy during hypoxia and protect cardiomyocyte function by regulating the AMPK/mTOR pathway.

Blue light emitting diode induces apoptosis in lymphoid cells by stimulating autophagy.

The present study was performed to examine the induction of apoptotic cell death and autophagy by blue LED irradiation, and the contribution of autophagy to apoptosis in B cell lymphoma A20 and RAMOS cells exposed to blue LED. Irradiation with blue LED reduced cell viability and induced apoptotic cell death, as indicated by exposure of phosphatidylserine on the plasma outside membrane and fragmentation of DNA. Furthermore, the mitochondrial membrane potential increased, and apoptotic proteins (PARP, caspase 3, Bax, and bcl-2) were observed. In addition, the level of intracellular superoxide anion (O2(-)) gradually increased. Interestingly the formation of autophagosomes and level of LC3-II were increased in blue LED-irradiated A20 and RAMOS cells, but inhibited after pretreatment with 3-methyladenine (3-MA), widely used as an autophagy inhibitor. Inhibition of the autophagic process by pretreatment with 3-MA blocked blue LED irradiation-induced caspase-3 activation. Moreover, a significant reduction of both the early and late phases of apoptosis after transfection with ATG5 and beclin 1 siRNAs was shown by the annexin V/PI staining, indicating a crucial role of autophagy in blue LED-induced apoptosis in cells. Additionally, the survival rate of mice irradiated with blue LED after injection with A20 cells increased compared to the control group. Our data demonstrate that blue LED irradiation induces apoptosis via the mitochondrial-mediated pathway, in conjunction with autophagy. Further studies are needed to elucidate the precise mechanism of blue LED-induced immune cell death.

Cytosolic Delivery of Liposomal Vaccines by Means of the Concomitant Photosensitization of Phagosomes.

One of the greatest pharmaceutical challenges in vaccinology is the delivery of antigens to the cytosol of antigen-presenting cells (APCs) in order to allow for the stimulation of major histocompatibility complex (MHC) class I-restricted CD8(+) T-cell responses, which may act on intracellular infections or cancer. Recently, we described a novel method for cytotoxic T-lymphocyte (CTL) vaccination by combining antigens with a photosensitizer and light for cytosolic antigen delivery. The goal of the current project was to test this immunization method with particle-based formulations. Liposomes were prepared from dipalmitoylphosphatidylcholine and cholesterol, and the antigen ovalbumin (OVA) or the photosensitizer tetraphenyl chlorine disulfonate (TPCS2a) was separately encapsulated. C57BL/6 mice were immunized intradermally with OVA liposomes or a combination of OVA and TPCS2a liposomes, and light was applied the next day for activation of the photosensitizer resulting in cytosolic release of antigen from phagosomes. Immune responses were tested both after a prime only regime and after a prime-boost scheme with a repeat immunization 2 weeks post priming. Antigen-specific CD8(+) T-cell responses and antibody responses were analyzed ex vivo by flow cytometry and ELISA methods. The physicochemical stability of liposomes upon storage and light exposure was analyzed in vitro. Immunization with both TPCS2a- and OVA-containing liposomes greatly improved CD8(+) T-cell responses as compared to immunization without TPCS2a and as measured by proliferation in vivo and cytokine secretion ex vivo. In contrast, OVA-specific antibody responses (IgG1 and IgG2c) were reduced after immunization with TPCS2a-containing liposomes. The liposomal formulation protected the photosensitizer from light-induced inactivation during storage. In conclusion, the photosensitizer TPCS2a was successfully formulated in liposomes and enabled a shift from MHC class II to MHC class I antigen processing and presentation for stimulation of strong CD8(+) T-cell responses. Therefore, photosensitive particulate vaccines may have the potential to add to current vaccine practice a new method of vaccination that, as opposed to current vaccines, can stimulate strong CD8(+) T-cell responses.

Effect of 808 nm Diode Laser on Swimming Behavior, Food Vacuole Formation and Endogenous ATP Production of Paramecium primaurelia (Protozoa).

Photobiomodulation (PBM) has been used in clinical practice for more than 40 years. To clarify the mechanisms of action of PBM at cellular and organism levels, we investigated its effect on Paramecium primaurelia (Protozoa) irradiated by an 808 nm infrared diode laser with a flat-top handpiece (1 W in CW). Our results led to the conclusion that: (1) the 808 nm laser stimulates the P. primaurelia without a thermal effect, (2) the laser effect is demonstrated by an increase in swimming speed and in food vacuole formation, (3) the laser treatment affects endogenous adenosine triphosphate (ATP) production in a positive way, (4) the effects of irradiation dose suggest an optimum exposure time of 50 s (64 J cm(-2) of fluence) to stimulate the Paramecium cells; irradiation of 25 s shows no effect or only mild effects and irradiation up to 100 s does not increase the effect observed with 50 s of treatment, (5) the increment of endogenous ATP concentration highlights the positive photobiomodulating effect of the 808 nm laser and the optimal irradiation conditions by the flat-top handpiece.

Resveratrol couples apoptosis with autophagy in UVB-irradiated HaCaT cells.

UVB radiation causes about 90% of non-melanoma skin cancers by damaging DNA either directly or indirectly by increasing levels of reactive oxygen species (ROS). Skin, chronically exposed to both endogenous and environmental pro-oxidant agents, contains a well-organised system of chemical and enzymatic antioxidants. However, increased or prolonged free radical action can overwhelm ROS defence mechanisms, contributing to the development of cutaneous diseases. Thus, new strategies for skin protection comprise the use of food antioxidants to counteract oxidative stress. Resveratrol, a phytoalexin from grape, has gained a great interest for its ability to influence several biological mechanisms like redox balance, cell proliferation, signal transduction pathways, immune and inflammatory response. Therefore, the potential of resveratrol to modify skin cell response to UVB exposure could turn out to be a useful option to protect skin from sunlight-induced degenerative diseases. To investigate into this matter, HaCaT cells, a largely used model for human skin keratinocytes, were treated with 25 or 100 µM resveratrol for 2 and 24 hours prior to UVB irradiation (10 to 100 mJ/cm(2)). Cell viability and molecular markers of proliferation, oxidative stress, apoptosis, and autophagy were analyzed. In HaCaT cells resveratrol pretreatment: reduces UVB-induced ROS formation, enhances the detrimental effect of UVB on HaCaT cell vitality, increases UVB-induced caspase 8, PARP cleavage, and induces autophagy. These findings suggest that resveratrol could exert photochemopreventive effects by enhancing UVB-induced apoptosis and by inducing autophagy, thus reducing the odds that damaged cells could escape programmed cell death and initiate malignant transformation.

Suppression of low-dose hyper-radiosensitivity in human lung cancer cell line A549 by radiation-induced autophagy.

This study explored the role of radiation-induced autophagy in low-dose hyperradiosensitivity (HRS) in the human lung cancer cell line A549. A549 cells, either treated with an autophagic inhibitor 3-methyladenine (3-MA), or with a vehicle control, were irradiated at different low doses (≤0.5 Gy). The generation of autophagy was examined by laser scanning confocal microscopy. Western blotting was used to detect the expression of microtubule-associated protein l light chain 3B II (LC3B-II). Flow cytometry (FCM) and clonogenic assays were used to measure the fraction of surviving cells at the low irradiation doses. Our results showed that there was a greater inhibition of autophagic activity, but a higher degree of low-dose HRS in A549 cells treated with 3-MA than in control group. Our data demonstrated that radiation-induced autophagy is correlated with HRS in A549 cells, and is probably one of the mechanisms underlying HRS.

Poly(ADP-ribose) polymerase-1 regulates the mechanism of irradiation-induced CNE-2 human nasopharyngeal carcinoma cell autophagy and inhibition of autophagy contributes to the radiation sensitization of CNE-2 cells.

The aim of the present study was to investigate the role of autophagy in response to ionizing radiation (IR) in CNE-2 human nasopharyngeal carcinoma cells and to demonstrate the function of poly(ADP-ribose) polymerase-1 (PARP-1) in the regulation of IR-induced autophagy. Microtubule-associated protein 1 light chain 3 (LC3) and poly(ADP-ribose) (PAR) were assessed using western blotting. Ultrastructural analysis was performed using transmission electron microscopy (TEM). The percentage of apoptotic cells was assessed by flow cytometry. The MTT method was used to detect cell viability of CNE-2 cells at different time points after IR. Clonogenic survival assays were used to evaluate the radiosensitivity of nasopharyngeal carcinoma cells treated with IR and IR combined with autophagy inhibitor (chloroquine phosphate), with autophagy inducer (rapamycin) or with PARP-1 inhibitor 3-amino benzamide (3AB). IR induced a massive accumulation of autophagosomes detected by TEM and intensified the conversion of cytosolic LC3-I to LC3-II. PARP-1 activation was accompanied by strong upregulation of PAR and LC3-II expression in CNE-2 cells. Compared with radiation alone, chloroquine phosphate (CDP) or 3AB combined with IR significantly decreased cell viability, as well as the autophagic ratio and LC3-II protein levels. Inhibition of autophagy increased radiation-induced apoptosis; rapamycin (RAPA) significantly decreased cell viability as well, but RAPA increased the autophagic ratio and LC3-II protein levels; induction of autophagy increased radiation-induced apoptosis. To conclude, PARP-1 regulates IR-induced autophagy, and PARP-1 inhibitor contributes to the radiation sensitization of CNE-2 cells. Blockade of autophagy with CDP enhanced the cytotoxicity of radiotherapy in CNE-2 cells. This suggests that inhibition of autophagy or PARP-1 may be used as an adjuvant therapy to treat nasopharyngeal carcinoma.

ATG7 deficiency suppresses apoptosis and cell death induced by lysosomal photodamage.

Photodynamic therapy (PDT) involves photosensitizing agents that, in the presence of oxygen and light, initiate formation of cytotoxic reactive oxygen species (ROS). PDT commonly induces both apoptosis and autophagy. Previous studies with murine hepatoma 1c1c7 cells indicated that loss of autophagy-related protein 7 (ATG7) inhibited autophagy and enhanced the cytotoxicity of photosensitizers that mediate photodamage to mitochondria or the endoplasmic reticulum. In this study, we examined two photosensitizing agents that target lysosomes: the chlorin NPe6 and the palladium bacteriopheophorbide WST11. Irradiation of wild-type 1c1c7 cultures loaded with either photosensitizer induced apoptosis and autophagy, with a blockage of autophagic flux. An ATG7- or ATG5-deficiency suppressed the induction of autophagy in PDT protocols using either photosensitizer. Whereas ATG5-deficient cells were quantitatively similar to wild-type cultures in their response to NPe6 and WST11 PDT, an ATG7-deficiency suppressed the apoptotic response (as monitored by analyses of chromatin condensation and procaspase-3/7 activation) and increased the LD(50) light dose by > 5-fold (as monitored by colony-forming assays). An ATG7-deficiency did not prevent immediate lysosomal photodamage, as indicated by loss of the lysosomal pH gradient. However, unlike wild-type and ATG5-deficient cells, the lysosomes of ATG7-deficient cells recovered this gradient within 4 h of irradiation, and never underwent permeabilization (monitored as release of endocytosed 10-kDa dextran polymers). We propose that the efficacy of lysosomal photosensitizers is in part due to both promotion of autophagic stress and suppression of autophagic prosurvival functions. In addition, an effect of ATG7 unrelated to autophagy appears to modulate lysosomal photodamage.

Dual functions of autophagy in the response of breast tumor cells to radiation: cytoprotective autophagy with radiation alone and cytotoxic autophagy in radiosensitization by vitamin D 3.

In MCF-7 breast tumor cells, ionizing radiation promoted autophagy that was cytoprotective; pharmacological or genetic interference with autophagy induced by radiation resulted in growth suppression and/or cell killing (primarily by apoptosis). The hormonally active form of vitamin D, 1,25D 3, also promoted autophagy in irradiated MCF-7 cells, sensitized the cells to radiation and suppressed the proliferative recovery that occurs after radiation alone. 1,25D 3 enhanced radiosensitivity and promoted autophagy in MCF-7 cells that overexpress Her-2/neu as well as in p53 mutant Hs578t breast tumor cells. In contrast, 1,25D 3 failed to alter radiosensitivity or promote autophagy in the BT474 breast tumor cell line with low-level expression of the vitamin D receptor. Enhancement of MCF-7 cell sensitivity to radiation by 1,25D 3 was not attenuated by a genetic block to autophagy due largely to the promotion of apoptosis via the collateral suppression of protective autophagy. However, MCF-7 cells were protected from the combination of 1,25D 3 with radiation using a concentration of chloroquine that produced minimal sensitization to radiation alone. The current studies are consistent with the premise that while autophagy mediates a cytoprotective function in irradiated breast tumor cells, promotion of autophagy can also confer radiosensitivity by vitamin D (1,25D 3). As both cytoprotective and cytotoxic autophagy can apparently be expressed in the same experimental system in response to radiation, this type of model could be utilized to distinguish biochemical, molecular and/or functional differences in these dual functions of autophagy.

Resveratrol-mediated downregulation of Rictor attenuates autophagic process and suppresses UV-induced skin carcinogenesis.

Macroautophagy is a cellular response to various environmental stresses that ensures lysosomal degradation of long-lived and damaged proteins and cellular organelles. It occurs through the formation of an autophagosome, which then fuses with a lysosome to form an autolysosome. Depending on the cellular context, autophagy may promote cancer cell survival or it may serve as a mechanism of tumor suppression. Herein, we show that resveratrol, a natural phytoalexin, induces premature senescence in human A431 SCC cells, and that resveratrol-induced premature senescence is associated with a blockade of autolysosome formation, as assessed by the absence of colocalization of LC3 and Lamp-2, markers for autophagosomes and lysosomes, respectively. Further, we show that resveratrol downregulates the level of Rictor, a component of mTORC2, leading to decreased RhoA-GTPase and altered actin cytoskeleton organization. Exogenous overexpression of Rictor restores RhoA-GTPase activity and actin cytoskeleton network, and decreases resveratrol-induced senescence-associated ß-gal activity, indicating a direct role of Rictor in senescence induction. Rictor is overexpressed in UV-induced murine SCCs, whereas its expression is diminished by oral administration of resveratrol. These data indicate that resveratrol attenuates autophagic process via Rictor, and suggest that downregulation of Rictor may be a mechanism of tumor suppression associated with premature senescence.

Intracellular targeting specificity of novel phthalocyanines assessed in a host-parasite model for developing potential photodynamic medicine.

Photodynamic therapy, unlikely to elicit drug-resistance, deserves attention as a strategy to counter this outstanding problem common to the chemotherapy of all diseases. Previously, we have broadened the applicability of this modality to photodynamic vaccination by exploiting the unusual properties of the trypanosomatid protozoa, Leishmania, i.e., their innate ability of homing to the phagolysosomes of the antigen-presenting cells and their selective photolysis therein, using transgenic mutants endogenously inducible for porphyrin accumulation. Here, we extended the utility of this host-parasite model for in vitro photodynamic therapy and vaccination by exploring exogenously supplied photosensitizers. Seventeen novel phthalocyanines (Pcs) were screened in vitro for their photolytic activity against cultured Leishmania. Pcs rendered cationic and soluble (csPcs) for cellular uptake were phototoxic to both parasite and host cells, i.e., macrophages and dendritic cells. The csPcs that targeted to mitochondria were more photolytic than those restricted to the endocytic compartments. Treatment of infected cells with endocytic csPcs resulted in their accumulation in Leishmania-containing phagolysosomes, indicative of reaching their target for photodynamic therapy, although their parasite versus host specificity is limited to a narrow range of csPc concentrations. In contrast, Leishmania pre-loaded with csPc were selectively photolyzed intracellularly, leaving host cells viable. Pre-illumination of such csPc-loaded Leishmania did not hinder their infectivity, but ensured their intracellular lysis. Ovalbumin (OVA) so delivered by photo-inactivated OVA transfectants to mouse macrophages and dendritic cells were co-presented with MHC Class I molecules by these antigen presenting cells to activate OVA epitope-specific CD8+T cells. The in vitro evidence presented here demonstrates for the first time not only the potential of endocytic csPcs for effective photodynamic therapy against Leishmania but also their utility in photo-inactivation of Leishmania to produce a safe carrier to express and deliver a defined antigen with enhanced cell-mediated immunity.

Circadian-clock driven cone-like photoreceptor phagocytosis in the neural retina leucine zipper gene knockout mouse.

PURPOSE: Whereas much information is available on rod outer segment phagocytosis by the retinal pigmented epithelium (RPE), corresponding data for cones are quite limited, especially in laboratory models of normal rats and mice with very low cone numbers. To characterize the light and circadian control of cone photoreceptor phagocytosis in mice, we capitalized on the blue cone-like phenotype of neural retina leucine zipper gene (Nrl) null mice (Nrl(-/-)). METHODS: Nrl(-/-) mice were maintained under standard cyclic light (12h:12h light-dark [LD] cycle; light=300 lux) for one month, then divided into two groups: 1) continued maintenance in LD (36 mice); or 2) transferred to constant darkness (DD; 21 mice) for 36 h. Animals were sacrificed every 3 h over 24 h, and their eyes were rapidly enucleated and fixed. Cryosections were stained using specific cone short-wavelength opsin antibodies. Phagosome numbers in the RPE were quantified with a morphometric system. We monitored the expression of c-mer proto-oncogene tyrosine kinase (MerTK) in wild-type and knockout mice using a specific MerTK antibody. RESULTS: In LD, cone phagocytosis showed a statistically significant peak of activity 1 h after light onset, 2-3 fold higher than at other times. In constant darkness, the temporal phagocytic profile resembled that of LD (significant peak at 1 h of subjective day), but the number of phagosomes was decreased at all time points. Immunostaining of MerTK in wild-type and Nrl(-/-) mice showed expression at the apical surface of the RPE. CONCLUSIONS: Cone-like outer segment phagocytosis in Nrl(-/-) mice shows a similar profile to that of rods in normal mice and other species. These data are the first to quantify blue cone-like photoreceptor phagocytosis under different lighting conditions in mice, and suggest this model may constitute a valuable system for investigating circadian regulation of cone function.

Processes of blue light-induced damage to retinal pigment epithelial cells lacking phagosomes.

PURPOSE: To experimentally clarify the processes of the changes induced by blue light directly on the retinal pigment epithelium (RPE) before the formation of phagosomes or the accumulation of lipofuscin. METHODS: We developed a new experimental method in which primary cultured cells of very young pigmented rats were exposed to several intensities and durations of blue light (wavelength = 440+/-10 nm). RESULTS: At 1.0 mW/cm2, the damage was limited to mitochondria. At 2.0 mW/cm2, the cytoplasm exhibited large whorls of membrane or whorled inclusions, which were consistent with autophagic vacuoles. At 4.0 mW/cm2, the RPE cells showed lysis of the cytoplasm and a nucleus that was consistent with necrosis. CONCLUSIONS: Our results suggested that damage induced by blue light to cultured RPE cells may originate in the mitochondria and end in necrosis. The type of cell death induced in the RPE by blue light seems to be determined mainly by the intensity of the light, but is also related to the duration of exposure.

Retinal light damage: protective effect of alpha-tocopherol.

We studied histologically the protective effect of alpha-tocopherol to retinal light damage. After 3-week-old albino rats were fed with an alpha-tocopherol deficient or supplemented diet and kept in a 12-hour dim light/12-hour dark environment for 8 weeks, each animal was exposed to intense light (2500 lux) for 1, 3, 6, 12, 24, and 72 hours. The eyes were enucleated and prepared for transmission electron microscopy study and image analysis of phagosomes. Before light exposure, the alpha-tocopherol content of the neural retina of the deficient and supplemented groups was 0.3 microgram and 23.34 micrograms, respectively. After 1- and 3-hour exposures, morphological changes in the retinal pigment epithelium and photoreceptor membranes were more extensive in the deficient group than in the supplemented group. After a 24-hour exposure, pyknotic photoreceptor nuclei were more numerous in the deficient group than the supplemented group. After 3-, 6-, and 12-hour exposures, large phagosomes were more numerous in the deficient group than in the supplemented group. These findings suggest that alpha-tocopherol can protect the retina from light injury for up to 24 hours of exposure.

Solar pruning of retinal rods in albino rainbow trout.

Morphology of the central retina and scotopic visual sensitivity were compared in juvenile albino and normally pigmented rainbow trout living under natural and reduced daylight. Outdoor albinos avoided exposing their eyes to direct sunlight, whereas normals were indifferent to it. After 4 months outdoors (approximately 10,000 lux in albinos, approximately 100,000 lux in normals), albinos had severely truncated or missing rod outer segments (ROS) and some missing rod ellipsoids, but normal numbers of photoreceptor nuclei and fully intact cones. Albino estimated ROS volume was only 7.1% of normal in July, but increased to 20% by the following February, mainly via an increase in numbers of ROS. However, in albinos moved indoors October 7 and exposed to 10-30 lux ambient daylight, both the number and length of ROS increased significantly, with estimated ROS volume reaching 95% of normal by 34 days. Albinos generally had more phagosomes (approximately 3 x normal) and more macrophages (approximately 2 x normal) in their outer retina. An optomotor reflex was used to define the effect of ROS volume on the ability to respond visually during dark adaptation. In July, albinos and normals from outdoor raceways (3 months) or indoor raceways (35 days) showed equal sensitivity after first being placed in darkness, but after 1 h in darkness, outdoor albinos with 6% of normal ROS volume were 2.0 log units less sensitive than indoor or outdoor normals, whereas indoor albinos with 53% of normal ROS volume were only 0.7 log units less sensitive. This verifies that most rod cell bodies of albino trout can persist without functional ROS in indirect sunlight, and can regrow functional outer segments in dim daylight. This finding is distinct from the extensive retinal light damage observed in albino rats exposed to more moderate cyclic light, in which entire rod cells degenerate early on.