Morphological characteristics and distribution identification of telocytes in Tibetan sheep testis and epididymis.

Telocytes (TCs) are a type of stromal cell discovered in the various organs of different animals and have many potential functions, including angiogenesis, signalling, and substance transport. However, the TCs have not been detected in the testis or epididymis of Tibetan sheep. This study investigated the position, characteristics, and distribution of TCs in the testis and epididymis of Tibetan sheep using transmission electron microscopy (TEM), toluidine blue staining, immunohistochemistry, and double immunofluorescence to elucidate their possible functions. TEM revealed that TCs were often found near basement membranes and capillaries and were characterised by large nuclei, elongated cytoplasmic protrusions, and many secretory vesicles. We also observed via toluidine staining that TCs were present near basement membrane and interstitial capillaries. Immunohistochemistry and double immunofluorescence revealed the positive expression of CD117, vimentin, platelet derived growth factor receptor α(PDGFRα), PDGFRα + CD117, and PDGFRα + vimentin in TCs. Additionally, we inferred that TCs participates in the formation of the blood-testis and blood-epididymis barriers, as well as in material transport and a stable microenvironment. This study presents the first evidence of the presence of TCs near the basement membrane and blood vessels in the testis and epididymis of Tibetan sheep. These findings provide new insights into the function of TCs in the reproductive systems of plateau animals.

Characterization of Ocular Morphology in Col4a3-/- Mice as a Murine Model for Alport Syndrome.

Purpose: The purpose of this study was to investigate the ocular morphological characteristics of Col4a3-/- mice as a model of Alport syndrome (AS) and the potential pathogenesis. Methods: The expression of collagen IV at 8, 12, and 21 weeks of age was evaluated by immunohistochemistry in wild-type (WT) and Col4a3-/- mice. Hematoxylin and eosin (H&E) staining and thickness measurements were performed to assess the thickness of anterior lens capsule and retina. Ultrastructure analysis of corneal epithelial basement membrane, anterior lens capsule, internal limiting membrane (ILM), and retinal pigment epithelium (RPE) basement membrane was performed using transmission electron microscopy. Finally, Müller cell activation was evaluated by glial fibrillary acidic protein (GFAP) expression. Results: Collagen IV was downregulated in the corneal epithelial basement membrane and ILM of Col4a3-/- mice. The hemidesmosomes of Col4a3-/- mice corneal epithelium became flat and less electron-dense than those of the WT group. Compared with those of the WT mice, the anterior lens capsules of Col4a3-/- mice were thinner. Abnormal structure was detected at the ILM Col4a3-/- mice, and the basal folds of the RPE basement membrane in Col4a3-/- mice were thicker and shorter. The retinas of Col4a3-/- mice were thinner than those of WT mice, especially within 1000 µm away from the optic nerve. GFAP expression enhanced in each age group of Col4a3-/- mice. Conclusions: Our results suggested that Col4a3-/- mice exhibit ocular anomalies similar to patients with AS. Additionally, Müller cells may be involved in AS retinal anomalies. Translational Relevance: This animal model could provide an opportunity to understand the underlying mechanisms of AS ocular disorders and to investigate potential new treatments.

Placental Protein 13 and Syncytiotrophoblast Basement Membrane Ultrastructures in Preeclampsia.

Background and Objectives: Preeclampsia has been linked to an inflammatory response that may be brought on by endothelial cell dysfunction. This paper investigates the pathomechanism of syncytiotrophoblast basement membrane (STBM) damage and Placental Protein 13 (PP13) release, which may have a role in systemic endothelial dysfunction in preeclampsia. Materials and Methods: This comparative cross-sectional study involves 54 preeclampsia patients (27 early-onset preeclampsia and 27 late-onset preeclampsia) and 27 pregnant women with normal blood pressure. An enzyme-linked immunosorbent assay was performed to evaluate maternal blood levels of PP13. Following birth, a portion of the placenta was collected for transmission electron microscope (TEM) and immunohistochemical (IHC) analysis. The data were analyzed using STATA version 15. Results: PP13 expression in the placental syncytiotrophoblast was significantly lower in the early-onset preeclampsia, compared to late-onset preeclampsia and normotensive pregnancy, group (p < 0.001). In contrast, serum PP13 levels were found to be the highest in the early-onset preeclampsia group, although no significant difference were found in mean maternal serum levels of PP13 between the three groups. The decreased PP13 expression in placental syncytiotrophoblast can be attributed to the greater extent of damage in the STBM in early-onset preeclampsia that leads to the release of a larger amount of PP13 into maternal circulation. The hypothesis aligns with the TEM analysis results. Preeclamptic pregnancies showed placental syncytiotrophoblast aponeurosis, whereas normotensive pregnancies did not. Placental lesions and STBM shedding were found to be more pronounced in early-onset preeclampsia compared to late-onset preeclampsia. Conclusions: PP13 and STBM damage may play a role in systemic endothelial dysfunction in preeclampsia.

Brain endothelial GSDMD activation mediates inflammatory BBB breakdown.

The blood-brain barrier (BBB) protects the central nervous system from infections or harmful substances1; its impairment can lead to or exacerbate various diseases of the central nervous system2-4. However, the mechanisms of BBB disruption during infection and inflammatory conditions5,6 remain poorly defined. Here we find that activation of the pore-forming protein GSDMD by the cytosolic lipopolysaccharide (LPS) sensor caspase-11 (refs. 7-9), but not by TLR4-induced cytokines, mediates BBB breakdown in response to circulating LPS or during LPS-induced sepsis. Mice deficient in the LBP-CD14 LPS transfer and internalization pathway10-12 resist BBB disruption. Single-cell RNA-sequencing analysis reveals that brain endothelial cells (bECs), which express high levels of GSDMD, have a prominent response to circulating LPS. LPS acting on bECs primes Casp11 and Cd14 expression and induces GSDMD-mediated plasma membrane permeabilization and pyroptosis in vitro and in mice. Electron microscopy shows that this features ultrastructural changes in the disrupted BBB, including pyroptotic endothelia, abnormal appearance of tight junctions and vasculature detachment from the basement membrane. Comprehensive mouse genetic analyses, combined with a bEC-targeting adeno-associated virus system, establish that GSDMD activation in bECs underlies BBB disruption by LPS. Delivery of active GSDMD into bECs bypasses LPS stimulation and opens the BBB. In CASP4-humanized mice, Gram-negative Klebsiella pneumoniae infection disrupts the BBB; this is blocked by expression of a GSDMD-neutralizing nanobody in bECs. Our findings outline a mechanism for inflammatory BBB breakdown, and suggest potential therapies for diseases of the central nervous system associated with BBB impairment.

Ultrastructural Details of Epiretinal Membrane Foveoschisis.

INTRODUCTION: The aim of this study was to describe differences in the vitreomacular interface (VMI) in idiopathic epiretinal membrane (ERM) foveoschisis compared to macular pseudohole (MPH) and lamellar macular hole (LMH). METHODS: We analysed surgically excised epiretinal material and internal limiting membrane (ILM) specimens obtained from 16 eyes of 16 patients with ERM foveoschisis (6 eyes), MPH (5 eyes), and LMH (5 eyes) during standard pars plana vitrectomy (PPV) with membrane peeling. The three entities were classified according to the newly introduced optical coherence tomography (OCT) terminology. Transmission electron microscopy (TEM) was used to describe the ultrastructural features. RESULTS: We found fibrocellular epiretinal tissues in all samples analysed. However, the cell and collagen composition of the VMI differed between groups. Eyes with ERM foveoschisis were characterized by a higher number of cells, multilayered membranes, and thick strands of vitreous collagen embedding the major cell types of myofibroblasts compared to MPH. Eyes with MPH also showed a predominance of myofibroblasts, but these were located directly on the ILM with no collagen between the cells and the ILM. Eyes with LMH showed a thick, multilayered epiretinal proliferation consisting mainly of non-tractional glial cells, corresponding to hypodense epiretinal proliferation on OCT. Eyes with ERM foveoschisis and MPH were more likely to have incomplete PVD compared to LMH in terms of posterior hyaloid status. DISCUSSION/CONCLUSION: Tractional ERMs in eyes with ERM foveoschisis and MPH differ in their ultrastructure. The main difference is in the amount and topographical distribution of vitreous collagen. However, the epiretinal cell types are predominantly myofibroblasts in both entities. This highlights the importance of distinguishing ERM foveoschisis from both MPH and LMH in terms of pathogenesis and surgical peeling procedures.

Capillary pathology with prominent basement membrane reduplication is the hallmark histopathological feature of scleromyositis.

AIMS: We aim to perform ultrastructural and histopathological analysis of muscle biopsies from a large group of systemic sclerosis (SSc) patients, including some with early/mild SSc features, and examine whether capillary pathology differentiates 'scleromyositis' (SM) from other auto-immune myositis (AIM) subsets. METHODS: Muscle biopsies from a total of 60 SM patients and 43 AIM controls from two independent cohorts were examined by electron microscopy, collagen-4 immunofluorescence (Col4IF) and routine light microscopy. RESULTS: Ultrastructural examination revealed prominent capillary basement membrane (BM) reduplication (4+ layers in >50% of capillaries) in 65% of SM vs 0% of AIM controls (p < 0.001). In SM cases without prominent BM reduplication, capillary dilation was the most distinctive feature, present in 8% of capillaries in SM vs 2% in controls (p = 0.001). Accumulation of ensheathed pericyte processes was another characteristic feature of SM and closely correlated with the degree of BM reduplication (r = 0.833, p < 0.001). On light microscopy, BM marker Col4IF revealed more frequent capillary enlargement in SM than in controls (84% vs 21%, p < 0.001). SM cases were classified as non-inflammatory myopathy (36%), non-specific myositis (33%) or immune-mediated necrotizing myopathy (31%), but despite this histopathological heterogeneity, prominent BM reduplication remained a constant finding. In the 16 SM patients with early/mild SSc features, 63% showed prominent BM reduplication. CONCLUSIONS: These results show that capillary pathology, and in particular prominent capillary BM reduplication, is the hallmark histopathological feature of SM even in patients with early/mild SSc and support the concept of SM as an organ manifestation of SSc and a distinct subset of AIM.

Relationships between Intraocular Pressure, Effective Filtration Area, and Morphological Changes in the Trabecular Meshwork of Steroid-Induced Ocular Hypertensive Mouse Eyes.

We investigated whether an inverse relationship exists between intraocular pressure (IOP) and effective filtration area (EFA) in the trabecular meshwork (TM) in a steroid-induced ocular hypertensive (SIOH) mouse model and the morphological changes associated with the reduction of EFA. C57BL/6 mice (n = 15 per group) received either 0.1% dexamethasone (DEX) or saline eye drops twice daily for five weeks. IOP was measured weekly. Fluorescent tracers were injected into the anterior chamber to label EFA at the endpoint. Injected eyes were fixed and processed for confocal microscopy. EFA in the TM was analyzed. Light and electron microscopy were performed in high- and low-tracer regions of six eyes per group. The mean IOP was ~4 mm Hg higher in DEX-treated than saline-treated control eyes (p < 0.001) at the endpoint. EFA was reduced in DEX-treated eyes compared to controls (p < 0.01) and negatively correlated with IOP (R2 = 0.38, p = 0.002). Reduced thickness of juxtacanalicular tissue (JCT) and increased abnormal extracellular matrix in the JCT were found to be associated with reduced EFA. Our data confirm the inverse relationship between EFA and IOP, suggesting that morphological changes in the JCT contribute to the reduction of EFA, thus elevating IOP in SIOH mouse eyes.

LAMA2 and LOXL4 are candidate FSGS genes.

BACKGROUND: Focal and segmental glomerulosclerosis (FSGS) is a histologic pattern of injury that characterizes a wide spectrum of diseases. Many genetic causes have been identified in FSGS but even in families with comprehensive testing, a significant proportion remain unexplained. METHODS: In a family with adult-onset autosomal dominant FSGS, linkage analysis was performed in 11 family members followed by whole exome sequencing (WES) in 3 affected relatives to identify candidate genes. RESULTS: Pathogenic variants in known nephropathy genes were excluded. Subsequently, linkage analysis was performed and narrowed the disease gene(s) to within 3% of the genome. WES identified 5 heterozygous rare variants, which were sequenced in 11 relatives where DNA was available. Two of these variants, in LAMA2 and LOXL4, remained as candidates after segregation analysis and encode extracellular matrix proteins of the glomerulus. Renal biopsies showed classic segmental sclerosis/hyalinosis lesion on a background of mild mesangial hypercellularity. Examination of basement membranes with electron microscopy showed regions of dense mesangial matrix in one individual and wider glomerular basement membrane (GBM) thickness in two individuals compared to historic control averages. CONCLUSIONS: Based on our findings, we postulate that the additive effect of digenic inheritance of heterozygous variants in LAMA2 and LOXL4 leads to adult-onset FSGS. Limitations to our study includes the absence of functional characterization to support pathogenicity. Alternatively, identification of additional FSGS cases with suspected deleterious variants in LAMA2 and LOXL4 will provide more evidence for disease causality. Thus, our report will be of benefit to the renal community as sequencing in renal disease becomes more widespread.

Scanning electron microscopy of murine skin ultrathin sections and cultured keratinocytes.

Generating high-quality electron microscopy images of the skin and keratinocytes can be challenging. Here we describe a simple protocol for scanning electron microscopy (SEM) of murine skin. The protocol enables characterization of the ultrastructure of the epidermis, dermis, hair follicles, basement membrane, and cell-cell junctions. We detail the specific steps for sample preparation and highlight the critical need for proper orientation of the sample for ultrathin sectioning. We also describe the isolation and preparation of primary keratinocyte monolayers for SEM. For complete details on the use and execution of this protocol, please refer to Biswas et al. (2021).

Characterization of the growth plate-bone interphase region using cryo-FIB SEM 3D volume imaging.

The interphase region at the base of the growth plate includes blood vessels, cells and mineralized tissues. In this region, cartilage is mineralized and replaced with bone. Blood vessel extremities permeate this space providing nutrients, oxygen and signaling factors. All these different components form a complex intertwined 3D structure. Here we use cryo-FIB SEM to elaborate this 3D structure without removing the water. As it is challenging to image mineralized and unmineralized tissues in a hydrated state, we provide technical details of the parameters used. We obtained two FIB SEM image stacks that show that the blood vessels are in intimate contact not only with cells, but in some locations also with mineralized tissues. There are abundant red blood cells at the extremities of the vessels. We also documented large multinucleated cells in contact with mineralized cartilage and possibly also with bone. We observed membrane bound mineralized particles in these cells, as well as in blood serum, but not in the hypertrophic chondrocytes. We confirm that there is an open pathway from the blood vessel extremities to the mineralizing cartilage. Based on the sparsity of the mineralized particles, we conclude that mainly ions in solution are used for mineralizing cartilage and bone, but these are augmented by the supply of mineralized particles.

Mapping the molecular and structural specialization of the skin basement membrane for inter-tissue interactions.

Inter-tissue interaction is fundamental to multicellularity. Although the basement membrane (BM) is located at tissue interfaces, its mode of action in inter-tissue interactions remains poorly understood, mainly because the molecular and structural details of the BM at distinct inter-tissue interfaces remain unclear. By combining quantitative transcriptomics and immunohistochemistry, we systematically identify the cellular origin, molecular identity and tissue distribution of extracellular matrix molecules in mouse hair follicles, and reveal that BM composition and architecture are exquisitely specialized for distinct inter-tissue interactions, including epithelial-fibroblast, epithelial-muscle and epithelial-nerve interactions. The epithelial-fibroblast interface, namely, hair germ-dermal papilla interface, makes asymmetrically organized side-specific heterogeneity in the BM, defined by the newly characterized interface, hook and mesh BMs. One component of these BMs, laminin α5, is required for hair cycle regulation and hair germ-dermal papilla anchoring. Our study highlights the significance of BM heterogeneity in distinct inter-tissue interactions.

Basal lamina characterization in frozen-thawed and long-term grafted human prepubertal ovarian tissue.

RESEARCH QUESTION: Are there differences in the composition and structure of the basal lamina surrounding follicles in prepubertal versus adult human ovarian tissue? DESIGN: Frozen-thawed human ovarian tissue from six prepubertal and seven adult patients was divided into three fragments in each case: two for non-grafted tissue evaluation and one for long-term xenografting to mice. Collagen IV and laminin expression were investigated by immunohistochemistry before and after grafting. The basal lamina was analysed by transmission electron microscopy on frozen-thawed tissue. RESULTS: In frozen-thawed tissue, collagen IV was significantly less expressed around prepubertal follicles than around adult follicles (primordial, P = 0.02; intermediate/growing follicles, P = 0.03), while laminin was significantly more expressed (primordial, P = 0.03; intermediate, P = 0.01). Collagen IV expression was significantly higher around prepubertal primordial follicles in grafted tissue than in non-grafted tissue, reaching similar levels to those in adult tissue. Ultrastructure analysis showed the basal lamina around follicles in prepubertal frozen-thawed tissue to be rather patchy and thinner than around adult follicles (primordial/intermediate, P = 0.001; primary, P = 0.02). CONCLUSIONS: In frozen-thawed tissue, the basal lamina around prepubertal follicles is less mature than around adult follicles, but it becomes similar in both prepubertal and adult subjects after grafting. Grafting could therefore induce maturation of the basal lamina around prepubertal follicles.

EPB41L5 controls podocyte extracellular matrix assembly by adhesome-dependent force transmission.

The integrity of the kidney filtration barrier essentially relies on the balanced interplay of podocytes and the glomerular basement membrane (GBM). Here, we show by analysis of in vitro and in vivo models that a loss of the podocyte-specific FERM-domain protein EPB41L5 results in impaired extracellular matrix (ECM) assembly. By using quantitative proteomics analysis of the secretome and matrisome, we demonstrate a shift in ECM composition characterized by diminished deposition of core GBM components, such as LAMA5. Integrin adhesome proteomics reveals that EPB41L5 recruits PDLIM5 and ACTN4 to integrin adhesion complexes (IACs). Consecutively, EPB41L5 knockout podocytes show insufficient maturation of integrin adhesion sites, which translates into impaired force transmission and ECM assembly. These observations build the framework for a model in which EPB41L5 functions as a cell-type-specific regulator of the podocyte adhesome and controls a localized adaptive module in order to prevent podocyte detachment and thereby ensures GBM integrity.

Age-related ultrastructural neurovascular changes in the female mouse cortex and hippocampus.

Blood-brain barrier (BBB) breakdown occurs in aging and neurodegenerative diseases. Although age-associated alterations have previously been described, most studies focused in male brains; hence, little is known about BBB breakdown in females. This study measured ultrastructural features in the aging female BBB using transmission electron microscopy and 3-dimensional reconstruction of cortical and hippocampal capillaries from 6- and 24-month-old female C57BL/6J mice. Aged cortical capillaries showed more changes than hippocampal capillaries. Specifically, the aged cortex showed thicker basement membrane, higher number and volume of endothelial pseudopods, decreased endothelial mitochondrial number, larger pericyte mitochondria, higher pericyte-endothelial cell contact, and increased tight junction tortuosity compared with young animals. Only increased basement membrane thickness and pericyte mitochondrial volume were observed in the aged hippocampus. Regional comparison revealed significant differences in endothelial pseudopods and tight junctions between the cortex and hippocampus of 24-month-old mice. Therefore, the aging female BBB shows region-specific ultrastructural alterations that may lead to oxidative stress and abnormal capillary blood flow and barrier stability, potentially contributing to cerebrovascular diseases, particularly in postmenopausal women.

Endothelial cell disruption drives increased blood-brain barrier permeability and cerebral edema in the Dahl SS/jr rat model of superimposed preeclampsia.

Preeclampsia is characterized by increases in blood pressure and proteinuria in late pregnancy, and neurological symptoms can appear in the form of headaches, blurred vision, cerebral edema, and, in the most severe cases, seizures (eclampsia). The causes for these cerebral manifestations remain unknown, so the use of animal models that mimic preeclampsia is essential to understanding its pathogenesis. The Dahl salt-sensitive (Dahl SS/jr) rat model develops spontaneous preeclampsia superimposed on chronic hypertension; therefore, we hypothesized that the Dahl SS/jr rat would display cerebrovascular features similar to those seen in human preeclampsia. Furthermore, we predicted that this model would allow for the identification of mechanisms underlying these changes. The pregnant Dahl SS/jr rat displayed increased cerebral edema and blood-brain barrier disruption despite tighter control of cerebral blood flow autoregulation and vascular smooth muscle myogenic tone. Analysis of cerebral endothelial cell morphology revealed increased opening of tight junctions, basement membrane dissolution, and vesicle formation. RNAseq analysis identified that genes related to endothelial cell tight junctions and blood-brain barrier integrity were differentially expressed in cerebral vessels from pregnant Dahl SS/jr compared with healthy pregnant Sprague Dawley rats. Overall, our data reveal new insights into mechanisms involved in the cerebrovascular dysfunction of preeclampsia.NEW & NOTEWORTHY This study uses the Dahl SS/jr rat as a preclinical model of spontaneous superimposed preeclampsia to demonstrate uncoupling of cerebral vascular permeability and blood-brain barrier disruption from cerebral blood flow autoregulatory dysfunction and myogenic tone. Additionally, the data presented in this study lay the foundational framework on which future experiments assessing specific transcellular transport components such as individual transporter protein expression and components of the vesicular transport system (caveolae) can be built to help reveal a potential direct mechanistic insight into the causes of cerebrovascular complications during preeclamptic pregnancies.

Aortic Valve Stenosis and Mitochondrial Dysfunctions: Clinical and Molecular Perspectives.

Calcific aortic stenosis is a disorder that impacts the physiology of heart valves. Fibrocalcific events progress in conjunction with thickening of the valve leaflets. Over the years, these events promote stenosis and obstruction of blood flow. Known and common risk factors are congenital defects, aging and metabolic syndromes linked to high plasma levels of lipoproteins. Inflammation and oxidative stress are the main molecular mediators of the evolution of aortic stenosis in patients and these mediators regulate both the degradation and remodeling processes. Mitochondrial dysfunction and dysregulation of autophagy also contribute to the disease. A better understanding of these cellular impairments might help to develop new ways to treat patients since, at the moment, there is no effective medical treatment to diminish neither the advancement of valve stenosis nor the left ventricular function impairments, and the current approaches are surgical treatment or transcatheter aortic valve replacement with prosthesis.

Low Dose of Genistein Alleviates Mono-(2-Ethylhexyl) Phthalate-Induced Fetal Testis Disorder Based on Organ Culture Model.

Mono-(2-ethylhexyl) phthalate (MEHP) and genistein have been classified as endocrine-disrupting chemicals (EDCs) which interfere with the differentiation and development of the male reproductive system. However, how these two EDCs would affect fetal rat testis development at a low dose was rarely studied. In this study, we established the organ culture system and applied it to evaluate testicular effects following multiple EDC exposure at a low dose. 15.5 days postcoitum fetal rat testes were dissected, cultured, and exposed to vehicle (control), GEN (1 µmol/L, G), MEHP (1 µmol/L, M), or GEN (1 µmol/L)+MEHP (1 µmol/L, G+M). Testicular cell markers, testosterone concentration, redox state, testicular histology, and testicular ultrastructure were evaluated. Our results showed that a low dose of MEHP suppressed the development of Sertoli cells, Leydig cells, and gonocytes by triggering oxidative injuries, which was consistent with the ultrastructural findings. However, coadministration of genistein at a low dose could partially attenuate MEHP-induced fetal testis damage through antioxidative action. Cotreatment of genistein at a low dose may have a promising future on its protecting role for attenuating other EDC-induced reproductive disorders during early life. Based on the results, it can be speculated that dietary intake of isoflavones may make the fetal testis less susceptible to phthalate-induced injury.

Human embryoid bodies as a 3D tissue model of the extracellular matrix and α-dystroglycanopathies.

The basal lamina is a specialized sheet of dense extracellular matrix (ECM) linked to the plasma membrane of specific cell types in their tissue context, which serves as a structural scaffold for organ genesis and maintenance. Disruption of the basal lamina and its functions is central to many disease processes, including cancer metastasis, kidney disease, eye disease, muscular dystrophies and specific types of brain malformation. The latter three pathologies occur in the α-dystroglycanopathies, which are caused by dysfunction of the ECM receptor α-dystroglycan. However, opportunities to study the basal lamina in various human disease tissues are restricted owing to its limited accessibility. Here, we report the generation of embryoid bodies from human induced pluripotent stem cells that model the basal lamina. Embryoid bodies cultured via this protocol mimic pre-gastrulation embryonic development, consisting of an epithelial core surrounded by a basal lamina and a peripheral layer of ECM-secreting endoderm. In α-dystroglycanopathy patient embryoid bodies, electron and fluorescence microscopy reveal ultrastructural basal lamina defects and reduced ECM accumulation. By starting from patient-derived cells, these results establish a method for the in vitro synthesis of patient-specific basal lamina and recapitulate disease-relevant ECM defects seen in the α-dystroglycanopathies. Finally, we apply this system to evaluate an experimental ribitol supplement therapy on genetically diverse α-dystroglycanopathy patient samples.This article has an associated First Person interview with the first author of the paper.

Pericyte abnormalities precede strial capillary basement membrane thickening in Alport mice.

In 129 Sv autosomal Alport mice, the strial capillary basement membranes (SCBMs) progressively thicken between 5 and 9 weeks of age resulting in a hypoxic microenvironment with metabolic stress and induction of pro-inflammatory cytokines and chemokines. These events occur concomitant with a drop in endocochlear potential and a susceptibility to noise-induced hearing loss under conditions that do not permanently affect age/strain-matched littermates. Here we aimed to gain an understanding of events that occur before the onset of SCBM thickening. Alport stria has normal thickness and shows levels of extracellular matrix (ECM) molecules in the SCBMs commensurate with wild-type mice. Hearing thresholds in the 3-week Alport mice do not differ from those of wild-type mice. We performed RNAseq analysis using RNA from stria vascularis isolated from 3-week Alport mice and wild type littermates. Data was processed using Ingenuity Pathway Analysis software and further distilled using manual procedures. RNAseq analysis revealed significant dysregulation of genes involved in cell adhesion, cell migration, formation of protrusions, and both actin and tubulin cytoskeletal dynamics. Overall, the data suggested changes in the cellular architecture of the stria might be apparent. To test this notion, we performed dual immunofluorescence analysis on whole mounts of the stria vascularis from these same animals stained with anti-isolectin gs-ib4 (endothelial cell marker) and anti-desmin (pericyte marker) antibodies. The results showed evidence of pericyte detachment and migration as well as the formation of membrane ruffling on pericytes in z-stacked confocal images from Alport mice compared to wild type littermates. This was confirmed by TEM analysis. Earlier work from our lab showed that endothelin A receptor blockade prevents SCBM thickening and ECM accumulation in the SCBMs. Treating cultured pericytes with endothelin-1 induced actin cytoskeletal rearrangement, increasing the ratio of filamentous to globular actin. Collectively, these findings suggest that the change in type IV collagen composition in the Alport SCBMs results in cellular insult to the pericyte compartment, activating detachment and altered cytoskeletal dynamics. These events precede SCBM thickening and hearing loss in Alport mice, and thus constitute the earliest event so far recognized in Alport strial pathology.

Oriented basement membrane fibrils provide a memory for F-actin planar polarization via the Dystrophin-Dystroglycan complex during tissue elongation.

How extracellular matrix contributes to tissue morphogenesis is still an open question. In the Drosophila ovarian follicle, it has been proposed that after Fat2-dependent planar polarization of the follicle cell basal domain, oriented basement membrane (BM) fibrils and F-actin stress fibers constrain follicle growth, promoting its axial elongation. However, the relationship between BM fibrils and stress fibers and their respective impact on elongation are unclear. We found that Dystroglycan (Dg) and Dystrophin (Dys) are involved in BM fibril deposition. Moreover, they also orient stress fibers, by acting locally and in parallel to Fat2. Importantly, Dg-Dys complex-mediated cell-autonomous control of F-actin fiber orientation relies on the preceding BM fibril deposition, indicating two distinct but interdependent functions. Thus, the Dg-Dys complex works as a crucial organizer of the epithelial basal domain, regulating both F-actin and BM. Furthermore, BM fibrils act as a persistent cue for the orientation of stress fibers that are the main effector of elongation.