Dynasore Modulates Store-Operated Calcium Entry and Mitochondrial Calcium Release in Corneal Epithelial Cells.

Dysregulation of calcium homeostasis can precipitate a cascade of pathological events that lead to tissue damage and cell death. Dynasore is a small molecule that inhibits endocytosis by targeting classic dynamins. In a previous study, we showed that dynasore can protect human corneal epithelial cells from damage due to tert-butyl hydroperoxide (tBHP) exposure by restoring cellular calcium (Ca2+) homeostasis. Here we report results of a follow-up study aimed at identifying the source of the damaging Ca2+. Store-operated Ca2+ entry (SOCE) is a cellular mechanism to restore intracellular calcium stores from the extracellular milieu. We found that dynasore effectively blocks SOCE in cells treated with thapsigargin (TG), a small molecule that inhibits pumping of Ca2+ into the endoplasmic reticulum (ER). Unlike dynasore however, SOCE inhibitor YM-58483 did not interfere with the cytosolic Ca2+ overload caused by tBHP exposure. We also found that dynasore effectively blocks Ca2+ release from internal sources. The inefficacy of inhibitors of ER Ca2+ channels suggested that this compartment was not the source of the Ca2+ surge caused by tBHP exposure. However, using a Ca2+-measuring organelle-entrapped protein indicator (CEPIA) reporter targeted to mitochondria, we found that dynasore can block mitochondrial Ca2+ release due to tBHP exposure. Our results suggest that dynasore exerts multiple effects on cellular Ca2+ homeostasis, with inhibition of mitochondrial Ca2+ release playing a key role in protection of corneal epithelial cells against oxidative stress due to tBHP exposure.

Differential Efficacy of Small Molecules Dynasore and Mdivi-1 for the Treatment of Dry Eye Epitheliopathy or as a Countermeasure for Nitrogen Mustard Exposure of the Ocular Surface.

The ocular surface comprises the wet mucosal epithelia of the cornea and conjunctiva, the associated glands, and the overlying tear film. Epitheliopathy is the common pathologic outcome when the ocular surface is subjected to oxidative stress. Whether different stresses act via the same or different mechanisms is not known. Dynasore and dyngo-4a, small molecules developed to inhibit the GTPase activity of classic dynamins DNM1, DNM2, and DNM3, but not mdivi-1, a specific inhibitor of DNM1L, protect corneal epithelial cells exposed to the oxidant tert-butyl hydroperoxide (tBHP). Here we report that, while dyngo-4a is the more potent inhibitor of endocytosis, dynasore is the better cytoprotectant. Dynasore also protects corneal epithelial cells against exposure to high salt in an in vitro model of dysfunctional tears in dry eye. We now validate this finding in vivo, demonstrating that dynasore protects against epitheliopathy in a mouse model of dry eye. Knockdown of classic dynamin DNM2 was also cytoprotective against tBHP exposure, suggesting that dynasore's effect is at least partially on target. Like tBHP and high salt, exposure of corneal epithelial cells to nitrogen mustard upregulated the unfolded protein response and inflammatory markers, but dynasore did not protect against nitrogen mustard exposure. In contrast, mdivi-1 was cytoprotective. Interestingly, mdivi-1 did not inhibit the nitrogen mustard-induced expression of inflammatory cytokines. We conclude that exposure to tBHP or nitrogen mustard, two different oxidative stress agents, cause corneal epitheliopathy via different pathologic pathways. SIGNIFICANCE STATEMENT: Results presented in this paper, for the first time, implicate the dynamin DNM2 in ocular surface epitheliopathy. The findings suggest that dynasore could serve as a new topical treatment for dry eye epitheliopathy and that mdivi-1 could serve as a medical countermeasure for epitheliopathy due to nitrogen mustard exposure, with potentially increased efficacy when combined with anti-inflammatory agents and/or UPR modulators.

Roles unveiled for membrane-associated mucins at the ocular surface using a Muc4 knockout mouse model.

Membrane-associated mucins (MAMs) are proposed to play critical roles at the ocular surface; however, in vivo evidence has been lacking. Here we investigate these roles by phenotyping of a Muc4 KO mouse. Histochemical analysis for expression of the beta-galactosidase transgene replacing Muc4 revealed a spiraling ribbon pattern across the corneal epithelium, consistent with centripetal cell migration from the limbus. Depletion of Muc4 compromised transcellular barrier function, as evidenced by an increase in rose bengal staining. In addition, the corneal surface was less smooth, consistent with disruption of tear film stability. While surface cells presented with well-developed microprojections, an increase in the number of cells with fewer microprojections was observed. Moreover, an increase in skin-type keratin K10 and a decrease in transcription factor Pax6 was observed, suggesting an incipient transdifferentiation. Despite this, no evidence of inflammatory dry eye disease was apparent. In addition, Muc4 had no effect on signaling by toll-like receptor Tlr4, unlike reports for MUC1 and MUC16. Results of this study provide the first in vivo evidence for the role of MAMs in transcellular barrier function, tear film stability, apical epithelial cell architecture, and epithelial mucosal differentiation at the ocular surface.

Dynasore Protects Corneal Epithelial Cells Subjected to Hyperosmolar Stress in an In Vitro Model of Dry Eye Epitheliopathy.

Epitheliopathy at the ocular surface is a defining sign of dry eye disease, a common disorder that affects 10% to 30% of the world's population. Hyperosmolarity of the tear film is one of the main drivers of pathology, with subsequent endoplasmic reticulum (ER) stress, the resulting unfolded protein response (UPR), and caspase-3 activation implicated in the pathway to programmed cell death. Dynasore, is a small molecule inhibitor of dynamin GTPases that has shown therapeutic effects in a variety of disease models involving oxidative stress. Recently we showed that dynasore protects corneal epithelial cells exposed to the oxidant tBHP, by selective reduction in expression of CHOP, a marker of the UPR PERK branch. Here we investigated the capacity of dynasore to protect corneal epithelial cells subjected to hyperosmotic stress (HOS). Similar to dynasore's capacity to protect against tBHP exposure, dynasore inhibits the cell death pathway triggered by HOS, protecting against ER stress and maintaining a homeostatic level of UPR activity. However, unlike with tBHP exposure, UPR activation due to HOS is independent of PERK and mostly driven by the UPR IRE1 branch. Our results demonstrate the role of the UPR in HOS-driven damage, and the potential of dynasore as a treatment to prevent dry eye epitheliopathy.

Recombinant Human Clusterin Seals Damage to the Ocular Surface Barrier in a Mouse Model of Ophthalmic Preservative-Induced Epitheliopathy.

There is a significant unmet need for therapeutics to treat ocular surface barrier damage, also called epitheliopathy, due to dry eye and related diseases. We recently reported that the natural tear glycoprotein CLU (clusterin), a molecular chaperone and matrix metalloproteinase inhibitor, seals and heals epitheliopathy in mice subjected to desiccating stress in a model of aqueous-deficient/evaporative dry eye. Here we investigated CLU sealing using a second model with features of ophthalmic preservative-induced dry eye. The ocular surface was stressed by topical application of the ophthalmic preservative benzalkonium chloride (BAC). Then eyes were treated with CLU and sealing was evaluated immediately by quantification of clinical dye uptake. A commercial recombinant form of human CLU (rhCLU), as well as an rhCLU form produced in our laboratory, designed to be compatible with U.S. Food and Drug Administration guidelines on current Good Manufacturing Practices (cGMP), were as effective as natural plasma-derived human CLU (pCLU) in sealing the damaged ocular surface barrier. In contrast, two other proteins found in tears: TIMP1 and LCN1 (tear lipocalin), exhibited no sealing activity. The efficacy and selectivity of rhCLU for sealing of the damaged ocular surface epithelial barrier suggests that it could be of therapeutic value in treating BAC-induced epitheliopathy and related diseases.

Clusterin, other extracellular chaperones, and eye disease.

Proteostasis refers to all the processes that maintain the correct expression level, location, folding and turnover of proteins, essential to organismal survival. Both inside cells and in body fluids, molecular chaperones play key roles in maintaining proteostasis. In this article, we focus on clusterin, the first-recognized extracellular mammalian chaperone, and its role in diseases of the eye. Clusterin binds to and inhibits the aggregation of proteins that are misfolded due to mutations or stresses, clears these aggregating proteins from extracellular spaces, and facilitates their degradation. Clusterin exhibits three main homeostatic activities: proteostasis, cytoprotection, and anti-inflammation. The so-called "protein misfolding diseases" are caused by aggregation of misfolded proteins that accumulate pathologically as deposits in tissues; we discuss several such diseases that occur in the eye. Clusterin is typically found in these deposits, which is interpreted to mean that its capacity as a molecular chaperone to maintain proteostasis is overwhelmed in the disease state. Nevertheless, the role of clusterin in diseases involving such deposits needs to be better defined before therapeutic approaches can be entertained. A more straightforward case can be made for therapeutic use of clusterin based on its proteostatic role as a proteinase inhibitor, as well as its cytoprotective and anti-inflammatory properties. It is likely that clusterin works together in this way with other extracellular chaperones to protect the eye from disease, and we discuss several examples. We end this article by predicting future steps that may lead to development of clusterin as a biological drug.

Transcriptome Analysis of Pterygium and Pinguecula Reveals Evidence of Genomic Instability Associated with Chronic Inflammation.

Solar damage due to ultraviolet radiation (UVR) is implicated in the development of two proliferative lesions of the ocular surface: pterygium and pinguecula. Pterygium and pinguecula specimens were collected, along with adjacent healthy conjunctiva specimens. RNA was extracted and sequenced. Pairwise comparisons were made of differentially expressed genes (DEGs). Computational methods were used for analysis. Transcripts from 18,630 genes were identified. Comparison of two subgroups of pterygium specimens uncovered evidence of genomic instability associated with inflammation and the immune response; these changes were also observed in pinguecula, but to a lesser extent. Among the top DEGs were four genes encoding tumor suppressors that were downregulated in pterygium: C10orf90, RARRES1, DMBT1 and SCGB3A1; C10orf90 and RARRES1 were also downregulated in pinguecula. Ingenuity Pathway Analysis overwhelmingly linked DEGs to cancer for both lesions; however, both lesions are clearly still benign, as evidenced by the expression of other genes indicating their well-differentiated and non-invasive character. Pathways for epithelial cell proliferation were identified that distinguish the two lesions, as well as genes encoding specific pathway components. Upregulated DEGs common to both lesions, including KRT9 and TRPV3, provide a further insight into pathophysiology. Our findings suggest that pterygium and pinguecula, while benign lesions, are both on the pathological pathway towards neoplastic transformation.

Biosynthetic scaffolds for partial meniscal loss: A systematic review from animal models to clinical practice.

Acute or degenerative meniscus tears are the most common knee lesions. Meniscectomy provides symptomatic relief and functional recovery only in the short- to mid-term follow-up but significantly increases the risk of osteoarthritis. For this reason, preserving the meniscus is key, although it remains a challenge. Allograft transplants present many disadvantages, so during the last 20 years preclinical and clinical research focused on developing and investigating meniscal scaffolds. The aim of this systematic review was to collect and evaluate all the available evidence on biosynthetic scaffolds for meniscus regeneration both in vivo and in clinical studies. Three databases were searched: 46 in vivo preclinical studies and 30 clinical ones were found. Sixteen natural, 15 synthetic, and 15 hybrid scaffolds were studied in vivo. Among them, only 2 were translated into clinic: the Collagen Meniscus Implant, used in 11 studies, and the polyurethane-based scaffold Actifit®, applied in 19 studies. Although positive outcomes were described in the short- to mid-term, the number of concurrent procedures and the lack of randomized trials are the major limitations of the available clinical literature. Few in vivo studies also combined the use of cells or growth factors, but these augmentation strategies have not been applied in the clinical practice yet. Current solutions offer a significant but incomplete clinical improvement, and the regeneration potential is still unsatisfactory. Building upon the overall positive results of these "old" technologies to address partial meniscal loss, further innovation is urgently needed in this field to provide patients better joint sparing treatment options.

Membrane-associated mucins of the human ocular surface in health and disease.

Mucins are a family of high molecular weight, heavily-glycosylated proteins produced by wet epithelial tissues, including the ocular surface epithelia. Densely-packed O-linked glycan chains added post-translationally confer the biophysical properties of hydration, lubrication, anti-adhesion and repulsion. Membrane-associated mucins (MAMs) are the distinguishing components of the mucosal glycocalyx. At the ocular surface, MAMs maintain wetness, lubricate the blink, stabilize the tear film, and create a physical barrier to the outside world. In addition, it is increasingly appreciated that MAMs function as cell surface receptors that transduce information from the outside to the inside of the cell. Recently, our team published a comprehensive review/perspectives article for molecular scientists on ocular surface MAMs, including previously unpublished data and analyses on two new genes MUC21 and MUC22, as well as new MAM functions and biological roles, comparing human and mouse (PMID: 31493487). The current article is a refocus for the audience of The Ocular Surface. First, we update the gene and protein information in a more concise form, and include a new section on glycosylation. Next, we discuss biological roles, with some new sections and further updating from our previous review. Finally, we provide a new chapter on MAM involvement in ocular surface disease. We end this with discussion of an emerging mechanism responsible for damage to the epithelia and their mucosal glycocalyces: the unfolded protein response (UPR). The UPR offers a novel target for therapeutic intervention.

Prospects on the Potential In Vitro Regenerative Features of Mechanically Treated-Adipose Tissue for Osteoarthritis Care.

Gathering a better grasp on the adipose stromal vascular fraction (SVF) is demanding among clinicians for osteoarthritis (OA) care because of its promising but multifaceted clinical outcomes. The aim of this preclinical in vitro study was to test whether the mechanical approach with Hy-Tissue SVF system, a class IIa CE marked device of adipose tissue micro-fragmentation, influences the biological features and functions of SVF. We compared mechanical generated-SVF (mSVF) with the enzymatic generated-SVF (eSVF) by testing cell survival, phenotype, differentiation, and paracrine properties using ELISA assays. Both adipose SVF showed 80% viable cells and enrichment for CD-44 marker. The mSVF product preserved the functions of cell populations within the adipose tissue; however, it displayed lowered nucleated cell recovery and CFU-F than eSVF. As for multipotency, mSVF and eSVF showed similar differentiation commitment for osteochondral lineages. Both adipose SVF exhibited an increased release of VEGF, HGF, IGF-1 and PDGF-bb, involved in pathways mediating osteochondral repair and cell migration. Both mSVF and eSVF also displayed high release for the anti-inflammatory cytokine IL-10. After in vitro culture, supernatants from both mSVF and eSVF groups showed a low release of cytokines except for IL-10, thereby giving evidence of functional changes after culture expansion. In this study, mSVF showed active cell populations in the adipose tissue comparable to eSVF with excellent survival, differentiation and paracrine properties under a new mechanical adipose tissue micro-fragmentation system; thereby suggesting its potential use as a minimally invasive technique for OA treatment.

Therapeutic Potential of the Molecular Chaperone and Matrix Metalloproteinase Inhibitor Clusterin for Dry Eye.

Evidence is presented herein supporting the potential of the natural homeostatic glycoprotein CLU (clusterin) as a novel therapeutic for the treatment of dry eye. This idea began with the demonstration that matrix metalloproteinase MMP9 is required for damage to the ocular surface in mouse dry eye. Damage was characterized by degradation of OCLN (occludin), a known substrate of MMP9 and a key component of the paracellular barrier. Following up on this finding, a yeast two-hybrid screen was conducted using MMP9 as the bait to identify other proteins involved. CLU emerged as a strong interacting protein that inhibits the enzymatic activity of MMP9. Previously characterized as a molecular chaperone, CLU is expressed prominently by epithelia at fluid-tissue interfaces and secreted into bodily fluids, where it protects cells and tissues against damaging stress. It was demonstrated that CLU also protects the ocular surface in mouse dry eye when applied topically to replace the natural protein depleted from the dysfunctional tears. CLU is similarly depleted from tears in human dry eye. The most novel and interesting finding was that CLU binds selectively to the damaged ocular surface. In this position, CLU protects against epithelial cell death and barrier proteolysis, and dampens the autoimmune response, while the apical epithelial cell layer is renewed. When present at high enough concentration, CLU also blocks staining by vital dyes used clinically to diagnose dry eye. None of the current therapeutics have this combination of properties to "protect, seal, and heal". Future work will be directed towards human clinical trials to investigate the therapeutic promise of CLU.

Dynasore protects ocular surface mucosal epithelia subjected to oxidative stress by maintaining UPR and calcium homeostasis.

The mucosal epithelia of the ocular surface protect against external threats to the eye. Using a model of human stratified corneal epithelial cells with mucosal differentiation, we previously demonstrated that a small molecule inhibitor of dynamin GTPases, dynasore, prevents damage to cells and their transcellular barriers when subjected to oxidative stress. Investigating mechanisms, we now report the novel finding that dynasore acts by maintaining Ca+2 homeostasis, thereby inhibiting the PERK branch of the unfolded protein response (UPR) that promotes cell death. Dynasore was found to protect mitochondria by preventing mitochondrial permeability transition pore opening (mPTP), but, unlike reports using other systems, this was not mediated by dynamin family member DRP1. Necrostatin-1, an inhibitor of RIPK1 and lytic forms of programmed cell death, also inhibited mPTP opening and further protected the plasma membrane barrier. Significantly, necrostatin-1 did not protect the mucosal barrier. Oxidative stress increased mRNA for sXBP1, a marker of the IRE1 branch of the UPR, and CHOP, a marker of the PERK branch. It also stimulated phosphorylation of eIF2α, the upstream regulator of CHOP, as well as an increase in intracellular Ca2+. Dynasore selectively inhibited the increase in PERK branch markers, and also prevented the increase intracellular Ca2+ in response to oxidative stress. The increase in PERK branch markers were also inhibited when cells were treated with the cell permeable Ca2+ chelator, BAPTA-AM. To our knowledge, this is the first time that dynasore has been shown to have an effect on the UPR and suggests therapeutic applications.

Histological, Histomorphometrical, and Biomechanical Studies of Bone-Implanted Medical Devices: Hard Resin Embedding.

The growing incidence of degenerative musculoskeletal disorders as well as lifestyle changes has led to an increase in the surgical procedures involving implanted medical devices in orthopedics. When studying implant/tissue interface in hard materials (i.e., metals or dense plastics) and/or in large bone segments, the hard plastic embedding of the intact undecalcified tissue envelope with the implant in situ is needed. The aim of this work is to describe the advances and the possibilities of high-temperature methyl methacrylate (MMA) embedding for the histological, histomorphometrical, and biomechanical assessment of bone-implanted medical devices. Unlike routine techniques, undecalcified bone processing histology, using high-temperature MMA, requires a complex and precise sample processing methodology and the availability of sophisticated equipment and software for both sample preparation and analyses. MMA embedding permits the evaluation of biological responses to the presence of implanted medical devices without implant removal, allowing simultaneous qualitative and quantitative histological evaluation, both static and dynamic histomorphometry, and biomechanical analyses not possible with tissue decalcification. MMA embedding, despite being a demanding procedure, is still preferred to other kinds of resin-based embedding because of its peculiar characteristics, which allow the study of samples of big dimensions also implanted with hard materials without reducing the sample or removing the material. Dynamic measurements are allowed together with biomechanical investigations at the bone-biomaterial interface, obtaining a comprehensive and precise evaluation of the safety and effectiveness of medical devices for orthopedic regenerative, reconstructive, and reparative surgery.

Spinal fusion procedures in the adult and young population: a systematic review on allogenic bone and synthetic grafts when compared to autologous bone.

This systematic review aims to compare clinical evidences related to autologous iliac crest bone graft (ICBG) and non-ICBG (local bone) with allografts and synthetic grafts for spinal fusion procedures in adult and young patients. A systematic search was carried out in three databases (PubMed, Scopus, Web of Science, Cochrane Central Register of Controlled Trials) to identify clinical studies in the last 10 years. The initial search retrieved 1085 studies, of which 24 were recognized eligible for the review. Twelve studies (4 RCTs, 5 prospective, 3 retrospective) were focused on lumbar spine, 9 (2 RCTs, 2 prospective, 4 retrospective, 1 case-series) on cervical spine and 3 (1 RCT, 2 retrospective) on spinal fusion procedures in young patients. Calcium phosphate ceramics, allografts, bioglasses, composites and polymers have been clinically investigated as substitutes of autologous bone in spinal fusion procedures. Of the 24 studies included in this review, only 1 RCT on cervical spine was classified with high level of evidence (Class I) and showed low risk of bias. This RCT demonstrated the safety and efficacy of the proposed treatment, a composite bone substitute, that results in similar and on some metrics superior outcomes compared with local autograft bone. Almost all other studies showed moderately or, more often, high incidence of bias (Class III), thus preventing ultimate conclusion on the hypothesized beneficial effects of allografts and synthetic grafts. This review suggests that users of allografts and synthetic grafting should carefully consider the scientific evidence concerning efficacy and safety of these bone substitutes, in order to select the best option for patient undergoing spinal fusion procedures.

Relevance of humanized three-dimensional tumor tissue models: a descriptive systematic literature review.

Despite numerous advances in tumor screening, diagnosis, and treatment, to date, tumors remain one of the leading causes of death, principally due to metastasis and the physiological damage produced by tumor growth. Among the main limits related to the study of tumor physiology there is the complex and heterogeneity nature of its environment and the absence of relevant, simple and inexpensive models able to mimic the biological processes occurring in patients allowing the correct clinical translation of results. To enhance the understanding of the mechanisms of tumors and to develop and evaluate new therapeutic approaches the set-up of advanced and alternative models is mandatory. One of the more translational approaches seems to be the use of humanized three-dimensional (3D) tissue culture. This model allows to accurately mimic tumor morphology and biology, maintaining the native microenvironment without any manipulation. However, little is still known on the real clinical relevance of these models for the study of tumor mechanisms and for the screening of new therapy. The aim of this descriptive systematic literature review was to evaluate and summarize the current knowledge on human 3D tumor tissue culture models. We reviewed the strategies employed by researchers to set-up these systems, also considering the different approaches and culture conditions used. All these aspects greatly contribute to the existing knowledge on tumors, providing a specific link to clinical scenarios and making the humanized 3D tumor tissue models a more attractive tool both for researchers and clinicians.

A Rationale for the Use of Clotted Vertebral Bone Marrow to Aid Tissue Regeneration Following Spinal Surgery.

Vertebral body bone marrow aspirate (V-BMA), easily accessible simultaneously with the preparation of the site for pedicle screw insertion during spinal procedures, is becoming an increasingly used cell therapy approach in spinal surgery. However, the main drawbacks for V-BMA use are the lack of a standardized procedure and of a structural texture with the possibility of diffusion away from the implant site. The aim of this study was to evaluate, characterize and compare the biological characteristics of MSCs from clotted V-BMA and MSCs from whole and concentrate V-BMAs. MSCs from clotted V-BMA showed the highest cell viability and growth factors expression (TGF-ß, VEGF-A, FGF2), the greatest colony forming unit (CFU) potency, cellular homogeneity, ability to differentiate towards the osteogenic (COL1AI, TNFRSF11B, BGLAP) and chondrogenic phenotype (SOX9) and the lowest ability to differentiate toward the adipogenic lineage (ADIPOQ) in comparison to all the other culture conditions. Additionally, results revealed that MSCs, differently isolated, expressed different level of HOX and TALE signatures and that PBX1 and MEIS3 were down-regulated in MSCs from clotted V-BMA in comparison to concentrated one. The study demonstrated for the first time that the cellular source inside the clotted V-BMA showed the best biological properties, representing an alternative and advanced cell therapy approach for patients undergoing spinal surgery.

Membrane-associated mucins of the ocular surface: New genes, new protein functions and new biological roles in human and mouse.

The mucosal glycocalyx of the ocular surface constitutes the point of interaction between the tear film and the apical epithelial cells. Membrane-associated mucins (MAMs) are the defining molecules of the glycocalyx in all mucosal epithelia. Long recognized for their biophysical properties of hydration, lubrication, anti-adhesion and repulsion, MAMs maintain the wet ocular surface, lubricate the blink, stabilize the tear film and create a physical barrier to the outside world. However, it is increasingly appreciated that MAMs also function as cell surface receptors that transduce information from the outside to the inside of the cell. A number of excellent review articles have provided perspective on the field as it has progressed since 1987, when molecular cloning of the first MAM was reported. The current article provides an update for the ocular surface, placing it into the broad context of findings made in other organ systems, and including new genes, new protein functions and new biological roles. We discuss the epithelial tissue-equivalent with mucosal differentiation, the key model system making these advances possible. In addition, we make the first systematic comparison of MAMs in human and mouse, establishing the basis for using knockout mice for investigations with the complexity of an in vivo system. Lastly, we discuss findings from human genetics/genomics, which are providing clues to new MAM roles previously unimagined. Taken together, this information allows us to generate hypotheses for the next stage of investigation to expand our knowledge of MAM function in intracellular signaling and roles unique to the ocular surface.

Deregulated miRNAs in osteoporosis: effects in bone metastasis.

Starting from their role exerted on osteoblast and osteoclast differentiation and activity pathways, microRNAs (miRNAs) have been recently identified as regulators of different processes in bone homeostasis. For this purpose, in a recent review, we highlighted, as deregulated miRNAs could be involved in different bone diseases such as osteoporosis. In addition, recent studies supported the concept that osteoporosis-induced bone alterations might offer a receptive site for cancer cells to form bone metastases, However, to date, no data on specific-shared miRNAs between osteoporosis and bone metastases have been considered and described to clarify the evidence of this link. The main goal of this review is to underline as deregulated miRNAs in osteoporosis may have specific roles in the development of bone metastases. The review showed that several circulating osteoporotic miRNAs could facilitate tumor progression and bone-metastasis formation in several tumor types, i.e., breast cancer, prostate cancer, non-small-cell lung cancer, esophageal squamous cell carcinoma, and multiple myeloma. In detail, serum up-regulation of pro-osteoporotic miRNAs, as well as serum down-regulation of anti-osteoporotic miRNAs are common features of all these tumors and are able to promote bone metastasis. These results are of key importance and could help researcher and clinicians to establish new therapeutic strategies connected with deregulation of circulating miRNAs and able to interfere with pathogenic processes of osteoporosis, tumor progressions, and bone-metastasis formation.

GPR158 in the Visual System: Homeostatic Role in Regulation of Intraocular Pressure.

Purpose: GPR158 is a newly characterized family C G-protein-coupled receptor, previously identified in functional screens linked with biological stress, including one for susceptibility to ocular hypertension/glaucoma induced by glucocorticoid stress hormones. In this study, we investigated GPR158 function in the visual system. Methods: Gene expression and protein immunolocalization analyses were performed in mouse and human brain and eye to identify tissues where GPR158 might function. Gene expression was perturbed in mice, and in cultures of human trabecular meshwork cells of the aqueous outflow pathway, to investigate function and mechanism. Results:GPR158 is highly expressed in the brain, and in this study, we show prominent expression specifically in the visual center of the cerebral cortex. Expression was also observed in the eye, including photoreceptors, ganglion cells, and trabecular meshwork. Protein was also localized to the outer plexiform layer of the neural retina. Gpr158 deficiency in knockout (KO) mice conferred short-term protection against the intraocular pressure increase that occurred with aging, but this was reversed over time. Most strikingly, the pressure lowering effect of the acute stress hormone, epinephrine, was negated in KO mice. In contrast, no disruption of the electroretinogram was observed. Gene overexpression in cell cultures enhanced cAMP production in response to epinephrine, suggesting a mechanism for intraocular pressure regulation. Overexpression also increased survival of cells subjected to oxidative stress linked to ocular hypertension, associated with TP53 pathway activation. Conclusions: These findings implicate GPR158 as a homeostatic regulator of intraocular pressure and suggest GPR158 could be a pharmacological target for managing ocular hypertension.