Extracellular Vesicles From Auditory Cells as Nanocarriers for Anti-inflammatory Drugs and Pro-resolving Mediators.

Drug- and noise-related hearing loss are both associated with inflammatory responses in the inner ear. We propose that intracochlear delivery of a combination of pro-resolving mediators, specialized proteins and lipids that accelerate the return to homeostasis by modifying the immune response rather than by inhibiting inflammation, might have a profound effect on the prevention of sensorineural hearing loss. However, intracochlear delivery of such agents requires a reliable and effective method to convey them, fully active, directly to the target cells. The present study provides evidence that extracellular vesicles (EVs) from auditory HEI-OC1 cells may incorporate significant quantities of anti-inflammatory drugs, pro-resolving mediators and their polyunsaturated fatty acid precursors as cargo, and potentially could work as carriers for their intracochlear delivery. EVs generated by HEI-OC1 cells were divided by size into two fractions, small (≤150 nm diameter) and large (>150 nm diameter), and loaded with aspirin, lipoxin A4, resolvin D1, and the polyunsaturated fatty acids (PUFA) arachidonic, eicosapentaenoic, docosahexanoic, and linoleic. Bottom-up proteomics revealed a differential distribution of selected proteins between small and large vesicles. Only 17.4% of these proteins were present in both fractions, whereas 61.5% were unique to smaller vesicles and only 3.7% were exclusively found in the larger ones. Importantly, the pro-resolving protein mediators Annexin A1 and Galectins 1 and 3 were only detected in small vesicles. Lipidomic studies, on the other hand, showed that small vesicles contained higher levels of eicosanoids than large ones and, although all of them incorporated the drugs and molecules investigated, small vesicles were more efficiently loaded with PUFA and the large ones with aspirin, LXA4 and resolvin D1. Importantly, our data indicate that the vesicles contain all necessary enzymatic components for the de novo generation of eicosanoids from fatty acid precursors, including pro-inflammatory agents, suggesting that their cargo should be carefully tailored to avoid interference with their therapeutic purpose. Altogether, these results support the idea that both small and large EVs from auditory HEI-OC1 cells could be used as nanocarriers for anti-inflammatory drugs and pro-resolving mediators.

Preliminary Characterization of Extracellular Vesicles From Auditory HEI-OC1 Cells.

OBJECTIVES: Isolate, purify, and characterize extracellular vesicles (EVs) obtained from auditory HEI-OC1 cells, and evaluate their suitability for intracochlear transport and delivery of pharmacological drugs and/or pro-resolution mediators of acute inflammatory processes. METHODS: HEI-OC1 EVs were isolated and purified using the exoEasy Maxi Kit, and their size was evaluated by nanoparticle tracking techniques. Bottom-up proteomics of the EVs, either freshly obtained or stored for up to 4 months at -20°C, was performed by LC-ESI-MS/MS. LC-ESI-MS/MS-MRM was used to measure the loading of dexamethasone inside EVs following co-incubation at room temperature for 1 hour with and without 5 minutes sonication. RESULTS: Routinely, we were able to obtain purified fractions of >2 × 109 EVs/mL, with diameters varying between 50 and 800 nm. Bottom-up proteomics showed that among the most abundant EVs proteins, 19.2% were cytoplasmic, 17.2% were membrane localized, 12.3% were cytosolic, and 14.6% were nucleolar. No significant differences between fresh and stored EVs were detected. Importantly, co-incubation of HEI-OC1 EVs (1 × 108 EVs/mL) with dexamethasone (10 mM) resulted in the incorporation of 10.1 ± 1.9 nM dexamethasone per milliliter of EVs suspension. CONCLUSIONS: Altogether, the results suggest that EVs from HEI-OC1 cells could be advantageously used as biological nanocarriers for the delivery of specific molecules and pharmacological drugs into the inner ear.

Resolution of Cochlear Inflammation: Novel Target for Preventing or Ameliorating Drug-, Noise- and Age-related Hearing Loss.

A significant number of studies support the idea that inflammatory responses are intimately associated with drug-, noise- and age-related hearing loss (DRHL, NRHL and ARHL). Consequently, several clinical strategies aimed at reducing auditory dysfunction by preventing inflammation are currently under intense scrutiny. Inflammation, however, is a normal adaptive response aimed at restoring tissue functionality and homeostasis after infection, tissue injury and even stress under sterile conditions, and suppressing it could have unintended negative consequences. Therefore, an appropriate approach to prevent or ameliorate DRHL, NRHL and ARHL should involve improving the resolution of the inflammatory process in the cochlea rather than inhibiting this phenomenon. The resolution of inflammation is not a passive response but rather an active, highly controlled and coordinated process. Inflammation by itself produces specialized pro-resolving mediators with critical functions, including essential fatty acid derivatives (lipoxins, resolvins, protectins and maresins), proteins and peptides such as annexin A1 and galectins, purines (adenosine), gaseous mediators (NO, H2S and CO), as well as neuromodulators like acetylcholine and netrin-1. In this review article, we describe recent advances in the understanding of the resolution phase of inflammation and highlight therapeutic strategies that might be useful in preventing inflammation-induced cochlear damage. In particular, we emphasize beneficial approaches that have been tested in pre-clinical models of inflammatory responses induced by recognized ototoxic drugs such as cisplatin and aminoglycoside antibiotics. Since these studies suggest that improving the resolution process could be useful for the prevention of inflammation-associated diseases in humans, we discuss the potential application of similar strategies to prevent or mitigate DRHL, NRHL and ARHL.

Working with Auditory HEI-OC1 Cells.

HEI-OC1 is one of the few mouse auditory cell lines available for research purposes. Originally proposed as an in vitro system for screening of ototoxic drugs, these cells have been used to investigate drug-activated apoptotic pathways, autophagy, senescence, mechanism of cell protection, inflammatory responses, cell differentiation, genetic and epigenetic effects of pharmacological drugs, effects of hypoxia, oxidative and endoplasmic reticulum stress, and expression of molecular channels and receptors. Among other several important markers of cochlear hair cells, HEI-OC1 cells endogenously express prestin, the paradigmatic motor protein of outer hair cells. Thus, they can be very useful to elucidate novel functional aspects of this important auditory protein. HEI-OC1 cells are very robust, and their culture usually does not present big complications. However, they require some special conditions such as avoiding the use of common anti-bacterial cocktails containing streptomycin or other antibiotics as well as incubation at 33 °C to stimulate cell proliferation and incubation at 39 °C to trigger cell differentiation. Here, we describe how to culture HEI-OC1 cells and how to use them in some typical assays, such as cell proliferation, viability, death, autophagy and senescence, as well as how to perform patch-clamp and non-linear capacitance measurements.

HEI-OC1 cells as a model for investigating drug cytotoxicity.

The House Ear Institute-Organ of Corti 1 (HEI-OC1) is one of the few, and arguable the most used, mouse auditory cell line available for research purposes. Originally proposed as an in vitro system for screening of ototoxic drugs, it has been used to investigate, among other topics, apoptotic pathways, autophagy and senescence, mechanism of cell protection, inflammatory responses, cell differentiation, effects of hypoxia, oxidative and endoplasmic reticulum stress, and expression of molecular channels and receptors. However, the use of different techniques with different goals resulted in apparent contradictions on the actual response of these cells to some specific treatments. We have now performed studies to characterize the actual response of HEI-OC1 cells to a battery of commonly used pharmacological drugs. We evaluated cell toxicity, apoptosis, viability, proliferation, senescence and autophagy in response to APAP (acetaminophen), cisplatin, dexamethasone, gentamicin, penicillin, neomycin, streptomycin, and tobramycin, at five different doses and two time-points (24 and 48 h), by flow cytometry techniques and caspase 3/7, MTT, Cytotoxicity, BrdU, Beclin1, LC3 and SA-ß-galactosidase assays. We also used HEK-293 and HeLa cells to compare some of the responses of these cells to those of HEI-OC1. Our results indicate that every cell line responds to the each drug in a different way, with HEI-OC1 cells showing a distinctive sensitivity to at least one of the mechanisms under study. Altogether, our results suggest that the HEI-OC1 might be a useful model to investigate biological responses associated with auditory cells, including auditory sensory cells, but a careful approach would be necessary at the time of evaluating drug effects.

HEI-OC1 cells as a model for investigating prestin function.

The House Ear Institute-Organ of Corti 1 (HEI-OC1) is a mouse auditory cell line that endogenously express, among other several markers of cochlear hair cells, the motor protein prestin (SLC26A5). Since its discovery fifteen years ago, and because of the difficulties associated with working with outer hair cells, prestin studies have been performed mostly by expressing it exogenously in non-specific systems such as HEK293 and TSA201, embryonic kidney cells from human origin, or Chinese Hamster Ovary (CHO) cells. Here, we report flow cytometry and confocal laser scanning microscopy studies on the pattern of prestin expression, as well as nonlinear capacitance (NLC) and whole cell-patch clamping studies on prestin motor function, in HEI-OC1 cells cultured at permissive and non-permissive conditions. Our results indicate that both total prestin expression and plasma membrane localization increase in a time-dependent manner when HEI-OC1 cells differentiate under non-permissive culture conditions. In addition, we demonstrate that HEI-OC1 cells have a robust NLC associated to prestin motor function, which decreases when the density of prestin molecules present at the plasma membrane increases. Altogether, our results show that the response of endogenously expressed prestin in HEI-OC1 cells is different from the response of prestin expressed exogenously in non-auditory cells, and suggest that the HEI-OC1 cell line may be an important additional tool for investigating prestin function.

Acetaminophen and NAPQI are toxic to auditory cells via oxidative and endoplasmic reticulum stress-dependent pathways.

Pain relievers containing N-acetyl-para-aminophenol, also called APAP, acetaminophen or paracetamol, in combination with opioid narcotics are top-selling pharmaceuticals in the U.S. Individuals who abuse these drugs for as little as sixty days can develop tinnitus and progressive bilateral sensorineural hearing loss. Recently published studies indicate that APAP and its metabolic product N-acetyl-p-benzoquinoneimine (NAPQI) are the primary ototoxic agents in this type of pain relievers. However, the mechanisms underlying the deleterious effects of these drugs on auditory cells remain to be fully characterized. In this study, we report cellular, genomic, and proteomic experiments revealing that cytotoxicity by APAP and NAPQI involves two different pathways in Immortomouse-derived HEI-OC1 cells, implicating ROS overproduction, alterations in ER morphology, redistribution of intra-cisternal chaperones, activation of the eIF2α-CHOP pathway, as well as changes in ER stress and protein folding response markers. Thus, both oxidative and ER stress are part of the cellular and molecular mechanisms that contribute to the cytotoxic effects of APAP and NAPQI in these cells. We suggest that these in vitro findings should be taken into consideration when designing pharmacological strategies aimed at preventing the toxic effects of these drugs on the auditory system.

In vitro assessment of antiretroviral drugs demonstrates potential for ototoxicity.

Several studies have reported an increased incidence of auditory dysfunction among HIV/AIDS patients. We used auditory HEI-OC1 cells in cell viability, flow cytometry and caspases 3/7-activation studies to investigate the potential ototoxicity of fourteen HIV antiretroviral agents: Abacavir, AZT, Delavirdine, Didenosine, Efavirenz, Emtricitabine, Indinavir, Lamivudine, Nefinavir, Nevirapine, Tenofovir, Ritonavir, Stavudine and Zalcitabine, as well as combinations of these agents as used in the common anti-HIV cocktails Atripla™, Combivir™, Epzicom™, Trizivir™, and Truvada™. Our results suggested that most of the single assayed anti-HIV drugs are toxic for HEI-OC1 auditory cells. The cocktails, on the other hand, decreased auditory cells' viability with high significance, with the following severity gradient: Epzicom âˆ¼ Trizivir >> Atripla âˆ¼ Combivir > Truvada. Interestingly, our results suggest that Trizivir- and Epzicom-induced cell death would be mediated by a caspase-independent mechanism. l-Carnitine, a natural micronutrient known to protect HEI-OC1 cells against some ototoxic drugs as well as to decrease neuropathies associated with anti-HIV treatments, increased viability of cells treated with Lamivudine and Tenofovir as well as with the cocktail Atripla, but had only minor effects on cells treated with other drugs and drug combinations. Altogether, these results suggest that some frequently used anti-HIV agents could have deleterious effects on patients' hearing, and provide arguments in favor of additional studies aimed at elucidating the potential ototoxicity of current as well as future anti-HIV drugs.

Acetaminophen ototoxicity after acetaminophen/hydrocodone abuse: evidence from two parallel in vitro mouse models.

OBJECTIVE: Acetaminophen/hydrocodone, a commonly used analgesic preparation, has been linked to rapidly progressing sensorineural hearing loss in human patients. The cellular and molecular mechanisms underlying the ototoxic effects of this drug combination are currently unknown, but are usually associated with high doses of hydrocodone. This study was aimed at identifying the specific agent responsible for hearing loss from toxic killing of cochlear sensory cells. STUDY DESIGN: Dose-response study. SETTING: University laboratory and private research facility. SUBJECTS AND METHODS: Math1 green fluorescent protein neonatal mouse cochlear cultures as well as a mouse auditory cell line (HEI-OC1) were exposed in vitro to different concentrations of acetaminophen, hydromorphone (the active metabolite of hydrocodone), and the micronutrient L-carnitine, either alone or combined. Using fluorescent and light microscopy, we quantified the sensory hair cells from a 600-microm basal segment before and after treatment. Acetaminophen/hydrocodone-induced apoptosis of HEI-OC1 was evaluated by caspase 3-activation studies. Statistically significant cell survival was determined with Student t test and analysis of variance. RESULTS: Cell death was associated mainly with exposure to acetaminophen, was slightly potentiated when combined with hydromorphone, and was partially prevented by L-carnitine. Exposure to hydrocodone or hydromorphone alone failed to kill either cochlear hair cells or HEI-OC1 cells. CONCLUSION: Our findings point to acetaminophen, rather than hydrocodone, as the primary cytotoxic agent. Hydrocodone, however, may work synergistically with acetaminophen, increasing the damage to auditory cells. These findings are an important first step toward understanding the mechanism of acetaminophen/hydrocodone ototoxicity and may lead to future treatment strategies for hearing loss from ototoxic medications.

Pivotal role of Harakiri in the induction and prevention of gentamicin-induced hearing loss.

Gentamicin is a widely used ototoxic agent. In this study, we shed light on the mechanisms underlying gentamicin-induced hearing loss. More importantly, we demonstrate in vivo and in vitro the effectiveness of a strategy for preventing drug-induced hearing loss using l-carnitine (LCAR), a safe micronutrient that plays a key role in energy metabolism and detoxification [Rebouche, C. J. & Seim, H. (1998) Annu. Rev. Nutr. 18, 39-61]. We show that LCAR prevents changes in hearing threshold and cochlear damage in newborn guinea pigs exposed to gentamicin in utero. Mechanistically, gentamicin-induced apoptosis of auditory cells is mediated by the extracellular signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinase (MAPK) pathway through up-regulation of the proapoptotic factor Harakiri (Hrk). Most important, small interfering RNA (siRNA) experiments demonstrate that Hrk up-regulation is crucial for gentamicin-induced apoptosis. LCAR, in contrast, prevents both gentamicin-induced Hrk up-regulation and apoptosis acting by means of c-Jun N-terminal kinase (JNK). Together, these results outline pathways for gentamicin-induced hearing loss and its prevention and assign a key role to Hrk in these processes. Thus, our data offer a conceptual framework for designing clinical trials using a safe micronutrient, LCAR, as a simple preventive strategy for iatrogenically induced ototoxicity.

ROCK-dependent and ROCK-independent control of cochlear outer hair cell electromotility.

Outer hair cell electromotility is crucial for the proper function of the cochlear amplifier, the active process that enhances sensitivity and frequency discrimination of the mammalian ear. Previous work (Kalinec, F., Zhang, M., Urrutia, R., and Kalinec, G. (2000) J. Biol. Chem. 275, 28000-28005) has suggested a role for Rho GTPases in the regulation of outer hair cell electromotility, although the signaling pathways mediated by these enzymes remain to be established. Here we have investigated the cellular and molecular mechanisms underlying the homeostatic regulation of the electromotile response of guinea pig outer hair cells. Our findings defined a ROCK-mediated signaling cascade that continuously modulates outer hair cell electromotility by selectively targeting the cytoskeleton. A distinct ROCK-independent pathway functions as a fast resetting mechanism for this system. Neither pathway affects the function of prestin, the unique molecular motor of outer hair cells. These results extend our understanding of a basic mechanism of both normal human hearing and deafness, revealing the key role of the cytoskeleton in the regulation of outer hair cell electromotility and suggesting ROCK as a molecular target for modulating the function of the cochlear amplifier.

A cochlear cell line as an in vitro system for drug ototoxicity screening.

Aminoglycoside antibiotics, loop diuretics, antineoplastic agents and other commonly used pharmacological drugs are ototoxic. Understanding of the cellular and molecular mechanisms underlying drug ototoxicity, however, has been hampered by the limited availability of inner ear tissues and drug side effects on laboratory animals. Immortalized cell lines derived from the auditory sensory organ, sensitive to ototoxic drugs and growing in environments that can be systematically manipulated, would facilitate the research directed at elucidating these mechanisms. Such immortalized cell lines could also be used to discover novel therapeutic agents for preventing drug-induced sensorineural hearing loss. Here, we report a conditionally immortalized organ of Corti-derived epithelial cell line, which shows evidence of activation of apoptosis when exposed to known ototoxic drugs. This cell line may be an excellent in vitro system to investigate the cellular and molecular mechanisms involved in ototoxicity and for screening of the potential ototoxicity or otoprotective properties of new pharmacological drugs.

Cisplatin-induced apoptosis in auditory cells: role of death receptor and mitochondrial pathways.

Cisplatin, a commonly used chemotherapeutic agent, has a major limitation due to its ototoxicity. Previous studies have shown that cisplatin induces apoptosis in auditory sensory cells, but the underlying mechanisms remain to be elucidated. In this study, cisplatin was found to induce apoptosis in a cochlear cell line, in a dose- and duration-dependent manner. Specific caspase assays revealed an early (6 h) but transient increase in caspase 8 activity, and a delayed (12 h) increase in caspase 9 activity. The enhanced caspase 8 activity was preceded by upregulation of p53 expression, and coincided with cleavage of Bid to its truncated form. This was followed temporally by activation and mitochondrial translocation of Bax, induction of mitochondrial permeability transition, release of cytochrome c into the cytosol, activation of caspase 9, and entry into the execution phase of apoptosis. Our results indicate the involvement of both the death receptor mechanisms as well as mitochondrial pathways in cisplatin-induced apoptosis of auditory cells in an in vitro model system.