The Emerging Role of Pericyte-Derived Extracellular Vesicles in Vascular and Neurological Health.

Pericytes (PCs), as a central component of the neurovascular unit, contribute to the regenerative potential of the central nervous system (CNS) and peripheral nervous system (PNS) by virtue of their role in blood flow regulation, angiogenesis, maintenance of the BBB, neurogenesis, and neuroprotection. Emerging evidence indicates that PCs also have a role in mediating cell-to-cell communication through the secretion of extracellular vesicles (EVs). Extracellular vesicles are cell-derived, micro- to nano-sized vesicles that transport cell constituents such as proteins, nucleic acids, and lipids from a parent originating cell to a recipient cell. PC-derived EVs (PC-EVs) play a crucial homeostatic role in neurovascular disease, as they promote angiogenesis, maintain the integrity of the blood-tissue barrier, and provide neuroprotection. The cargo carried by PC-EVs includes growth factors such as endothelial growth factor (VEGF), connecting tissue growth factors (CTGFs), fibroblast growth factors, angiopoietin 1, and neurotrophic growth factors such as brain-derived neurotrophic growth factor (BDNF), neuron growth factor (NGF), and glial-derived neurotrophic factor (GDNF), as well as cytokines such as interleukin (IL)-6, IL-8, IL-10, and MCP-1. The PC-EVs also carry miRNA and circular RNA linked to neurovascular health and the progression of several vascular and neuronal diseases. Therapeutic strategies employing PC-EVs have potential in the treatment of vascular and neurodegenerative diseases. This review discusses current research on the characteristic features of EVs secreted by PCs and their role in neuronal and vascular health and disease.

Inflammation up-regulates cochlear expression of TRPV1 to potentiate drug-induced hearing loss.

Aminoglycoside antibiotics are essential for treating life-threatening bacterial infections, despite the risk of lifelong hearing loss. Infections induce inflammation and up-regulate expression of candidate aminoglycoside-permeant cation channels, including transient receptor potential vanilloid-1 (TRPV1). Heterologous expression of TRPV1 facilitated cellular uptake of (fluorescently tagged) gentamicin that was enhanced by agonists, and diminished by antagonists, of TRPV1. Cochlear TRPV1 was immunolocalized near the apical membranes of sensory hair cells, adjacent supporting cells, and marginal cells in the stria vascularis. Exposure to immunostimulatory lipopolysaccharides, to simulate of bacterial infections, increased cochlear expression of TRPV1 and hair cell uptake of gentamicin. Lipopolysaccharide exposure exacerbated aminoglycoside-induced auditory threshold shifts and loss of cochlear hair cells in wild-type, but not in heterozygous Trpv1+/- or Trpv1 knockout, mice. Thus, TRPV1 facilitates cochlear uptake of aminoglycosides, and bacteriogenic stimulation upregulates TRPV1 expression to exacerbate cochleotoxicity. Furthermore, loss-of-function polymorphisms in Trpv1 can protect against immunogenic exacerbation of aminoglycoside-induced cochleotoxicity.

Vascular regeneration in adult mouse cochlea stimulated by VEGF-A165 and driven by NG2-derived cells ex vivo.

Can damaged or degenerated vessels be regenerated in the ear? The question is clinically important, as disruption of cochlear blood flow is seen in a wide variety of hearing disorders, including in loud sound-induced hearing loss (endothelial injury), ageing-related hearing loss (lost vascular density), and genetic hearing loss (e.g., Norrie disease: strial avascularization). Progression in cochlear blood flow (CBF) pathology can parallel progression in hair cell and hearing loss. However, neither new vessel growth in the ear, nor the role of angiogenesis in hearing, have been investigated. In this study, we used an established ex vivo tissue explant model in conjunction with a matrigel matrix model to demonstrate for the first time that new vessels can be generated by activating a vascular endothelial growth factor (VEGF-A) signal. Most intriguingly, we found that the pattern of the newly formed vessels resembles the natural 'mesh pattern' of in situ strial vessels, with both lumen and expression of tight junctions. Sphigosine-1-phosphate (S1P) in synergy with VEGF-A control new vessel size and growth. Using transgenic neural/glial antigen 2 (NG2) fluorescent reporter mice, we have furthermore discovered that the progenitors of "de novo" strial vessels are NG2-derived cells. Taken together, our data demonstrates that damaged strial microvessels can be regenerated by reprogramming NG2-derived angiogenic cells. Restoration of the functional vasculature may be critical for recovery of vascular dysfunction related hearing loss.

Microvascular networks in the area of the auditory peripheral nervous system.

Using transgenic fluorescent reporter mice in combination with an established tissue clearing method, we detail heretofore optically opaque regions of the spiral lamina and spiral limbus where the auditory peripheral nervous system is located and provide insight into changes in cochlear vascular density with ageing. We found a relatively dense and branched vascular network in young adults, but a less dense and thinned network in aged adults. Significant reduction in vascular density starts early at the age of 180 days in the region of the spiral limbus (SL) and continues into old age at 540 days. Loss of vascular volume in the region of spiral ganglion neurons (SGN) is delayed until the age of 540 days. In addition, we observed that two vascular accessory cells are closely associated with the microvascular system: perivascular resident macrophages and pericytes. Morphologically, perivascular resident macrophages undergo drastic changes from postnatal P7 to young adult (P30). In postnatal animals, most perivascular resident macrophages exhibit a spherical or nodular shape. In young adult mice, the majority of perivascular resident macrophages are elongated and display an orientation parallel to the vessels. In our imaging, some of the perivascular resident macrophages are caught in the act of transmigrating from the blood circulation. Pericytes also display morphological heterogeneity. In the P7 mice, pericytes are prominent on the capillary walls, relatively large and punctate, and less uniform. In contrast, pericytes in the P30 mice are relatively flat and uniform, and less densely distributed on the vascular network. With triple fluorescence labeling, we did not find obvious physical connection between the two systems, unlike neuronal-vascular coupling found in brain. However, using a fluorescent (FITC-conjugated dextran) tracer and the enzymatic tracer horseradish peroxidase (HRP), we observed robust neurovascular exchange, likely through transcytotic transport, evidenced by multiple vesicles present in the endothelial cells. Taken together, our data demonstrate the effectiveness of tissue-clearing methods as an aid in imaging the vascular architecture of the SL and SGNs in whole mounted mouse cochlear preparations. Structure is indicative of function. The finding of differences in vascular structure in postnatal and young adult mice may correspond with variation in hearing refinement after birth and indicate the status of functional activity. The decrease in capillary network density in the older animals may reflect the decreased energy demand from peripheral neural activity. The finding of active transcytotic transport from blood to neurons opens a potential therapeutic avenue for delivery of various growth factors and gene vectors into the inner ear to target SGNs.

Simplified, automated methods for assessing pixel intensities of fluorescently-tagged drugs in cells.

Assessing the cytoplasmic uptake of fluorescently-tagged drugs in heterogeneous cell types currently involves time-consuming manual segmentation of confocal microscopy images. We developed a set of methods that incorporate map algebra techniques to facilitate and expedite image segmentation, particularly of the parenchyma of intermediate cells in the stria vascularis of the inner ear. Map algebra is used to apply a convolution kernel to pixel neighborhoods to create a masking image to select pixels in the original image for further operations. Here, we describe the utility of integrated intensity-based, percentile-based, and local autocorrelation-based methods to automate segmentation of images into putative morphological regions for pixel intensity analysis. Integrated intensity-based methods are variants of watershed segmentation tools that determine morphological boundaries from rates of change in integrated pixel intensity. Percentile- and local autocorrelation-based methods evolved out of the process of developing map algebra- and integrated intensity-based tools. We identified several simplifications that are surprisingly effective for image segmentation and pixel intensity analysis. These methods were empirically validated on three levels: first, the algorithms were developed based on iterations of inspected results; second, algorithms were tested for various types of robustness; and third, developed algorithms were validated against results from manually-segmented images. We conclude the key to automated segmentation is supervision of output data.

Culture media-based selection of endothelial cells, pericytes, and perivascular-resident macrophage-like melanocytes from the young mouse vestibular system.

The vestibular blood-labyrinth barrier (BLB) is comprised of perivascular-resident macrophage-like melanocytes (PVM/Ms) and pericytes (PCs), in addition to endothelial cells (ECs) and basement membrane (BM), and bears strong resemblance to the cochlear BLB in the stria vascularis. Over the past few decades, in vitro cell-based models have been widely used in blood-brain barrier (BBB) and blood-retina barrier (BRB) research, and have proved to be powerful tools for studying cell-cell interactions in their respective organs. Study of both the vestibular and strial BLB has been limited by the unavailability of primary culture cells from these barriers. To better understand how barrier component cells interact in the vestibular system to control BLB function, we developed a novel culture medium-based method for obtaining EC, PC, and PVM/M primary cells from tiny explants of the semicircular canal, sacculus, utriculus, and ampullae tissue of young mouse ears at post-natal age 8-12 d. Each phenotype is grown in a specific culture medium which selectively supports the phenotype in a mixed population of vestibular cell types. The unwanted phenotypes do not survive passaging. The protocol does not require additional equipment or special enzyme treatment. The harvesting process takes less than 2 h. Primary cell types are generated within 7-10 d. The primary culture ECs, PCs, and PVM/M shave consistent phenotypes more than 90% pure after two passages (∼ 3 weeks). The highly purified primary cell lines can be used for studying cell-cell interactions, barrier permeability, and angiogenesis.

Local mechanisms for loud sound-enhanced aminoglycoside entry into outer hair cells.

Loud sound exposure exacerbates aminoglycoside ototoxicity, increasing the risk of permanent hearing loss and degrading the quality of life in affected individuals. We previously reported that loud sound exposure induces temporary threshold shifts (TTS) and enhances uptake of aminoglycosides, like gentamicin, by cochlear outer hair cells (OHCs). Here, we explore mechanisms by which loud sound exposure and TTS could increase aminoglycoside uptake by OHCs that may underlie this form of ototoxic synergy. Mice were exposed to loud sound levels to induce TTS, and received fluorescently-tagged gentamicin (GTTR) for 30 min prior to fixation. The degree of TTS was assessed by comparing auditory brainstem responses (ABRs) before and after loud sound exposure. The number of tip links, which gate the GTTR-permeant mechanoelectrical transducer (MET) channels, was determined in OHC bundles, with or without exposure to loud sound, using scanning electron microscopy. We found wide-band noise (WBN) levels that induce TTS also enhance OHC uptake of GTTR compared to OHCs in control cochleae. In cochlear regions with TTS, the increase in OHC uptake of GTTR was significantly greater than in adjacent pillar cells. In control mice, we identified stereociliary tip links at ~50% of potential positions in OHC bundles. However, the number of OHC tip links was significantly reduced in mice that received WBN at levels capable of inducing TTS. These data suggest that GTTR uptake by OHCs during TTS occurs by increased permeation of surviving, mechanically-gated MET channels, and/or non-MET aminoglycoside-permeant channels activated following loud sound exposure. Loss of tip links would hyperpolarize hair cells and potentially increase drug uptake via aminoglycoside-permeant channels expressed by hair cells. The effect of TTS on aminoglycoside-permeant channel kinetics will shed new light on the mechanisms of loud sound-enhanced aminoglycoside uptake, and consequently on ototoxic synergy.

Uptake of fluorescent gentamicin by peripheral vestibular cells after systemic administration.

OBJECTIVE: In addition to cochleotoxicity, systemic aminoglycoside pharmacotherapy causes vestibulotoxicity resulting in imbalance and visual dysfunction. The underlying trafficking routes of systemically-administered aminoglycosides from the vasculature to the vestibular sensory hair cells are largely unknown. We investigated the trafficking of systemically-administered gentamicin into the peripheral vestibular system in C56Bl/6 mice using fluorescence-tagged gentamicin (gentamicin-Texas-Red, GTTR) imaged by scanning laser confocal microscopy to determine the cellular distribution and intensity of GTTR fluorescence in the three semicircular canal cristae, utricular, and saccular maculae at 5 time points over 4 hours. RESULTS: Low intensity GTTR fluorescence was detected at 0.5 hours as both discrete puncta and diffuse cytoplasmic fluorescence. The intensity of cytoplasmic fluorescence peaked at 3 hours, while punctate fluorescence was plateaued after 3 hours. At 0.5 and 1 hour, higher levels of diffuse GTTR fluorescence were present in transitional cells compared to hair cells and supporting cells. Sensory hair cells typically exhibited only diffuse cytoplasmic fluorescence at all time-points up to 4 hours in this study. In contrast, non-sensory cells rapidly exhibited both intense fluorescent puncta and weaker, diffuse fluorescence throughout the cytosol. The numbers and size of fluorescent puncta in dark cells and transitional cells increased over time. There is no preferential GTTR uptake by the five peripheral vestibular organs' sensory cells. Control vestibular tissues exposed to Dulbecco's phosphate-buffered saline or hydrolyzed Texas Red had negligible fluorescence. CONCLUSIONS: All peripheral vestibular cells rapidly take up systemically-administered GTTR, reaching peak intensity 3 hours after injection. Sensory hair cells exhibited only diffuse fluorescence, while non-sensory cells displayed both diffuse and punctate fluorescence. Transitional cells may act as a primary pathway for trafficking of systemic GTTR from the vasculature to endolymph prior to entering hair cells.

Isolation and culture of endothelial cells, pericytes and perivascular resident macrophage-like melanocytes from the young mouse ear.

This protocol describes a growth medium-based approach for obtaining cochlear endothelial cells (ECs), pericytes (PCs) and perivascular resident macrophage-like melanocytes (PVM/Ms) from the stria vascularis of mice aged between P10 and P15 (P, postnatal day). The procedure does not involve mechanical or enzymatic digestion of the sample tissue. Explants of stria vascularis, 'mini-chips', are selectively cultured in growth medium, and primary cell lines are obtained in 7-10 d. The method is simple and reliable, and it provides high-quality ECs, PVM/Ms and PCs with a purity >90% after two passages. This protocol is suitable for producing primary culture cells from organs and tissues of small volume and high anatomical complexity, such as the inner ear capillaries. The highly purified primary cell lines enable cell culture-based in vitro modeling of cell-cell interactions, barrier control function and drug action.

Endothelial cell, pericyte, and perivascular resident macrophage-type melanocyte interactions regulate cochlear intrastrial fluid-blood barrier permeability.

The integrity of the fluid-blood barrier in the stria vascularis is critical for maintaining inner ear homeostasis, especially for sustaining the endocochlear potential, an essential driving force for hearing function. However, the mechanisms that control intrastrial fluid-blood barrier permeability remain largely unknown. At the cellular level, the intrastrial fluid-blood barrier comprises cochlear microvascular endothelial cells connected to each other by tight junctions (TJs), an underlying basement membrane, and a second line of support consisting of cochlear pericytes and perivascular resident macrophage-type melanocytes. In this study, we use a newly established primary cell culture-based in vitro model to show that endothelial cells, pericytes, and perivascular resident macrophage-type melanocytes interact to control intrastrial fluid-blood barrier permeability. When the endothelial cell monolayer was treated with pericyte--or perivascular resident macrophage-type melanocyte--conditioned media, the permeability of the endothelial cell monolayer was significantly reduced relative to an untreated endothelial cell monolayer. Further study has shown the pericytes and perivascular resident macrophage-type melanocytes to regulate TJ expression in the endothelial cell monolayer. The new cell culture-based in vitro model offers a unique opportunity to obtain information on the organ-specific characteristics of the cochlear blood/tissue barrier. Our finding demonstrates the importance of signaling among pericytes, endothelial cells, and perivascular resident macrophage-type melanocytes to the integrity of the intrastrial fluid-blood barrier.

Bone marrow cell recruitment mediated by inducible nitric oxide synthase/stromal cell-derived factor-1alpha signaling repairs the acoustically damaged cochlear blood-labyrinth barrier.

Using a mouse model with noise-induced cochlear blood-labyrinth-barrier (CBLB) injury, we examined the effects of inducible nitric oxide synthase (iNOS) on the recruitment of bone marrow-derived cells (BMDCs) to the CBLB after acoustic injury. Lethally irradiated C57BL/6J and B6.129P2-Nos2(tm1Lau)/J mice were transplanted with GFP(+)-BMDCs from C57Bl/6-Tg (UBC GFP) mice. Four weeks after transplantation, we assessed the population of GFP(+)-BMDCs in the CBLB. Only small numbers of GFP(+)-BMDCs were found to infiltrate the area of the CBLB in the control recipient mice. However, robust GFP(+)-BMDC migration occurred in the area of the CBLB within the injured cochlea during the first week following acoustic trauma, and further BMDC accumulation was seen by 2 weeks posttrauma. After 4 weeks, the BMDCs were integrated into vessels. Local iNOS from perivascular resident macrophages was found to be important for BMDC infiltration, since mice deficient in iNOS (Inos(-/-)) and mice with iNOS that had been inhibited by 1400W displayed reduced BMDC infiltration. Stromal cell-derived factor-1α (SDF-1α) and its chemokine receptor 4 (CXCR4) were required for the iNOS-triggered recruitment. BMDC recruitment was significantly reduced by the inhibition of SDF-1α activity. Inhibition of the iNOS/SDF-1α signaling pathway reduced vascular repair as observed by reduced vascular density. Our study revealed an intrinsic signaling pathway of iNOS that mediates SDF-1α to promote GFP(+)-BMDC infiltration/targeting in cochlear vascular repair.

Competitive antagonism of fluorescent gentamicin uptake in the cochlea.

Aminoglycosides enter inner ear hair cells via apical endocytosis, or mechanoelectrical transduction channels, implying that, in vivo, aminoglycosides enter hair cells from endolymph prior to exerting their cytotoxic effect. If so, circulating aminoglycosides likely cross the strial blood-labyrinth barrier and enter marginal cells prior to clearance into endolymph. We characterized the competitive antagonism of unconjugated aminoglycosides on the uptake of fluorescent gentamicin (GTTR) in the stria vascularis and kidney cells at an early time point. In mice, uptake of GTTR by kidney proximal tubule cells was competitively antagonized by gentamicin at all doses, but only weakly by kanamycin (mimicking in vitro data). GTTR fluorescence was approximately 100-fold greater in proximal tubule cells than in the stria vascularis. Furthermore, only high molar ratios of aminoglycosides significantly reduced strial uptake of GTTR. Thus, gentamicin antagonism of GTTR uptake is more efficacious in proximal tubules than in the stria vascularis. Competitive antagonism of GTTR uptake is indicative of specific cell-regulatable uptake mechanisms (e.g., ion channels, transporters) in the kidney. Strial uptake mechanisms have lower specific affinity for gentamicin, and/or density (compared to the kidney), yet may be critical to transport gentamicin across the strial blood-labyrinth barrier into marginal cells.