| The intrastrial fluid-blood barrier separates the stria vascularis (SV) from peripheral circulation. The integrity of the barrier is critical for maintaining inner ear homeostasis, especially for sustaining the endocochlear potential (EP), an essential driving force for hearing function. Disruption of the barrier is closely associated with a number of hearing disorders, including autoimmune inner ear disease, noise-induced hearing loss, age related hearing loss, and several genetically linked diseases. Despite the importance of the intrastrial fluid-blood barrier, little is understood about regulation of the barrier and the mechanisms that control its permeability.In the classic view, the intrastrial fluid-blood barrier comprises basement membrane and endothelial cells (ECs) that connect to each other with tight junctions to form a diffusion barrier that prevents most blood-borne substances from entering the ear. In a recent study, we found that the intrastrial fluid-blood barrier also includes a large number of pericytes and perivascular-resident macrophage-like melanocytes (PVM/Ms).The PVM/Ms are in close contact with vessels through cytoplasmic processes. The structural complexity of PVM/Ms’ capillary morphology suggests functional complexity. The cell types of the intrastrial fluid-blood barrier have yet to be characterized functionally, and the role of PVM/Ms in the intrastrial fluid-blood barrier is particularly elusive. In an earlier study, we characterized the PVM/Ms as resident macrophages, because the cells are positive for several macrophage surface molecules, including F4/80, CD68, and CDllb. In this study, we further characterized the PVM/Ms as expressing several melanocyte markers. Overall, our results show that PVM/Ms are a hybrid cell type and differ from the classical macrophage. Given their morphological similarities to astrocytes and glial cells, we speculate the PVM/Ms might have a functional role similar to that of astrocytes in the blood-brain barrier and glial cells in the blood-retina barrier. Astrocytes and glial cells are known to have essential roles in regulation of barrier integrity and tissue-oriented functions. Barriers without these cells lose tight-junction proteins, become leaky, and are associated with tissue edema. In our study, we tested whether communication between ECs and PVM/Ms is essential for the barrier function and, more specifically, whether such signaling controls microvessel permeability. The hypotheses were tested in both in vitro and in vivo models. Primary endothelial and PVM/M cell lines from mouse cochlea were established for in vitro tests, and the results were validated in vivo by ablating PVM/Ms. A transgenic mouse model with a diphtheria toxin (DT) receptor under control of a human integrin aM promoter (CD11b) enabled transient depletion of the PVM/Ms.Our results demonstrate that PVM/Ms’ regulation of intrastrial fluid-blood barrier integrity, mediated by pigment epithelialderived factor (PEDF), affects the expression of tight junctionassociated proteins. The experiment provides evidence that signaling between PVM/Ms and ECs regulates intrastrial fluid-blood barrier integrity and that regulation of permeability is instrumental in maintaining a normal EP and hearing threshold.Part I:The Characteristics of perivascular-resident macrophage-like melanocytes in the stria vascularies in the intrastrial fluid-blood barrierObjective:To analyze the characteristics of perivascular-resident macrophage-like melanocytes and the structural complexity of PVM/Ms’ capillary morphology.Methods:1. Stria vascularies of mice were viewed in a Philips EM 100 transmission electron microscope.2. Indentified the characteristics of perivascular-resident macrophage-like melanocytes by Immunohistochemistry and Fluorescence Microscopy.3. Got perivascular-resident macrophage-like melanocytes from in vivo and in vitro models.4. Indentified the characteristics of perivascular-resident macrophage-like melanocytes by RT-PCR.Results:1. Confocal images and 3D reconstructed confocal images of the SV show a large population of PVM/Ms sandwiched between marginaland basal cell layers of the SV. The PVM/Ms are highly invested on the abluminal surface of capillaries and are structurally intertwined with ECs with dendritic processes that also interface with the capillary wall.2. The PVM/Ms contain melanin pigment granules in the cytoplasm and express melanocyte marker proteins, including cytosolic GSTa4, GST, and Kir4.1. mRNA for Gst, Gpf480, and Kir4.1 are detected by RT-PCR in isolated and purified PVM/Ms.Conclusion:1. The PVM/Ms are in close contact with vessels through cytoplasmic processes. The structural complexity of PVM/Ms’ capillary morphology suggests functional complexity.2. PVM/Ms are a hybrid cell type and differ from the classical macrophage. They are identified here as melanocyte-like macrophages.Part II:PVM/Ms control the integrity of the intrastrial fluid-blood barrier in the SV by affecting the expression of tight- and adherens-junction proteinsObjective:To explore PVM/Ms control the integrity of the intrastrial fluid-blood barrier in the SV by affecting the expression of tight- and adherens-junction proteins.Methods:1. Established ECs and PVM/Ms cell lines from mice strail vascularies.2. The permeability of the endothelial barrier was measured directly in a co-culture model with an EC and PVM/Ms.3. Analyzed EP and ABR of the mice in which a transgene encoding a DT receptor was used for transient depletion of PVM/Ms.4. The permeability of intrastrial fluid-blood barrier of mice was measured by different ways in which a transgene encoding a DT receptor was used for transient depletion of PVM/Ms.5. we speculated that PVM/Ms control barrier permeability by affecting global expression of tight junction-associated proteins. Then we provided evidence by in vivo and in vitro models.Results:1. ECs monolayers were highly permeable in the absence of PVM/Ms.2. Transgenic ablation of PVM/Ms results in significant leakage from vessels and in hearing loss.3. PVM/Ms control barrier permeability by affecting the expression of tight junction-associated proteins,ZO-l,Occludin and Ve-cadherin.Conclusion:1. We describes a growth medium-based approach for obtaining cochlear endothelial cells (ECs) and perivascular resident macrophage-like melanocytes (PVM/Ms) from the stria vascularis of mice. The highly purified primary cell lines enable cell culture-based in vitro modeling of cell-cell interactions, barrier control function and drug action.2. PVM/M regulates the intrastrial fluid-blood barrier integrity by affecting the expression of tight junction associated proteins, that is important for EP and hearing.Part III:PEDF is an essential signaling molecule for PVM/Ms regulating the integrity of the intrastrial fluid-blood barrierObjective:To explore the signaling molecule for PVM/Ms regulating the integrity of the intrastrial fluid-blood barrier by using a siRNA-targeting vector to suppress Pedf expression.Methods:1. Indentified the expression of PEDF and PEDFR in PVM/Ms and ECs by immunohistochemistry and ELASA.2. Established the in vivo and in vitro models by an siRNA-targeting vector to suppress Pedf expression.3.In vivo and in vitro models, we tested expression of ZO-1,Ooccludin, and Ve-cadherin by RT-PCR and western-blotting before and after transfection.Results:1. PEDF is expressed in primary cultured PVM/Ms, whereas PEDF receptor (PEDFR) is expressed in primary cultured ECs. And we can see the same result in the whole-mount tissue of strial vascularies of mice.2. QRT-PCR analysis of Pedf transcripts from the in vitro samples showed the siRNA suppressed the Pedf gene in PVM/Ms by 80% and a 60% down-regulation of Pedf gene expression in vivo by siRNA.3. Consistent with the down-regulation of ZO-1, occludin, and ve-cadherin seen in vitro, down-regulation of Pedf gene expression by siRNA resulted in dramatically decreased protein expression in capillaries as compared with a vector control group.Conclusions:1. PEDF can regulate the expression of ZO-1, Ooccludin, and Ve-cadherin.2. PEDF signaling between PVM/Ms and ECs is an important mediator of the effect PVM/Ms on expression of tight- and adherens-junction proteins. |
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