Decellularized porcine conjunctiva as an alternative substrate for tissue-engineered epithelialized conjunctiva.

PURPOSE: The long-term success of visual rehabilitation in patients with severe conjunctival scarring is reliant on the reconstruction of the conjunctiva with a suitable substitute. The purpose of this study is the development and investigation of a re-epithelialized conjunctival substitute based on porcine decellularized conjunctiva (PDC). METHODS: PDC was re-epithelialized either with pre-expanded human conjunctival epithelial cells (PDC + HCEC) or with a human conjunctival explant placed directly on PDC (PDC + HCEx). Histology and immunohistochemistry were performed to evaluate epithelial thickness, proliferation (Ki67), apoptosis (Caspase 3), goblet cells (MUC5AC), and progenitor cells (CK15, ΔNp63, ABCG2). The superior construct (PDC + HCEx) was transplanted into a conjunctival defect of a rabbit (n = 6). Lissamine green staining verified the epithelialization in vivo. Orbital tissue was exenterated on day 10 and processed for histological and immunohistochemical analysis to examine the engrafted PDC + HCEx. A human-specific antibody was used to detect the transplanted cells. RESULTS: From day-14 in vitro onward, a significantly thicker epithelium and greater number of cells expressing Ki67, CK15, ΔNp63, and ABCG2 were noted for PDC + HCEx versus PDC + HCEC. MUC5AC-positive cells were found only in PDC + HCEx. The PDC + HCEx-grafted rabbit conjunctivas were lissamine-negative during the evaluation period, indicating epithelial integrity. Engrafted PDC + HCEx showed preserved progenitor cell properties and an increased number of goblet cells comparable to those of native conjunctiva. CONCLUSION: Placing and culturing a human conjunctival explant directly on PDC (PDC + HCEx) enables the generation of a stable, stratified, goblet cell-rich construct that could provide a promising alternative conjunctival substitute for patients with extensive conjunctival stem and goblet cell loss.

Decellularized human corneal stromal cell sheet as a novel matrix for ocular surface reconstruction.

The shortage of donor corneas as well as the limitations of tissue substitutes leads to the necessity to develop alternative materials for ocular surface reconstruction. Corneal surface substitutes must fulfill specific requirements such as high transparency, low immunogenicity, and mechanical stability combined with elasticity. This in vitro study evaluates a decellularized matrix secreted from human corneal fibroblasts (HCF) as an alternative material for ocular surface reconstruction. HCF from human donors were cultivated with the supplementation of vitamin C to form a stable and thick matrix. Furthermore, due to enhanced cultivation time, a three-dimensional like multilayered construct which partly mimics the complex structure of the corneal stroma could be generated. The formed human cell-based matrices (so-called cell sheets [CS]) were subsequently decellularized. The complete cell removal, collagen content, ultrastructure, and cell toxicity of the decellularized CS (DCS) as well as biomechanical properties were analyzed. Surgical feasibility was tested on enucleated porcine eyes. After decellularization and sterilization, a transparent, thick, cell free, and sterile tissue substitute resulted, which allowed expansion of limbal epithelial stem cells with no signs of cytotoxicity, and good surgical feasibility. DCS seem to be a promising new corneal tissue substitute derived from human cells without the limitation of donor material; however, future in vivo studies are necessary to further elucidate its potential for ocular surface reconstruction.

Treatment of diabetic retinopathy through neuropeptide Y-mediated enhancement of neurovascular microenvironment.

Diabetic retinopathy (DR) is one of the most severe clinical manifestations of diabetes mellitus and a major cause of blindness. DR is principally a microvascular disease, although the pathogenesis also involves metabolic reactive intermediates which induce neuronal and glial activation resulting in disruption of the neurovascular unit and regulation of the microvasculature. However, the impact of neural/glial activation in DR remains controversial, notwithstanding our understanding as to when neural/glial activation occurs in the course of disease. The objective of this study was to determine a potential protective role of neuropeptide Y (NPY) using an established model of DR permissive to N-methyl-D-aspartate (NMDA)-induced excitotoxic apoptosis of retinal ganglion cells (RGC) and vascular endothelial growth factor (VEGF)-induced vascular leakage. In vitro evaluation using primary retinal endothelial cells demonstrates that NPY promotes vascular integrity, demonstrated by maintained tight junction protein expression and reduced permeability in response to VEGF treatment. Furthermore, ex vivo assessment of retinal tissue explants shows that NPY can protect RGC from excitotoxic-induced apoptosis. In vivo clinical imaging and ex vivo tissue analysis in the diabetic model permitted assessment of NPY treatment in relation to neural and endothelial changes. The neuroprotective effects of NPY were confirmed by attenuating NMDA-induced retinal neural apoptosis and able to maintain inner retinal vascular integrity. These findings could have important clinical implications and offer novel therapeutic approaches for the treatment in the early stages of DR.

MSC Transplantation Improves Lacrimal Gland Regeneration after Surgically Induced Dry Eye Disease in Mice.

Dry eye disease (DED) is a multifactorial disease characterized by a disrupted tear film homeostasis and inflammation leading to visual impairments and pain in patients. Aqueous-deficient dry eye (ADDE) causes the most severe progressions and depends mainly on the loss of functional lacrimal gland (LG) tissue. Despite a high prevalence, therapies remain palliative. Therefore, it is of great interest to develop new approaches to curatively treat ADDE. Mesenchymal stem/stromal cells (MSC) have been shown to induce tissue regeneration and cease inflammation. Moreover, an increasing amount of MSC was found in the regenerating LG of mice. Therefore, this study investigated the therapeutic effect of MSC transplantation on damaged LGs using duct ligation induced ADDE in mice. Due to the transplantation of sex-mismatched and eGFP-expressing MSC, MSC could be identified and detected until day 21. MSC transplantation significantly improved LG regeneration, as the amount of vital acinar structures was significantly increased above the intrinsic regeneration capacity of control. Additionally, MSC transplantation modulated the immune reaction as macrophage infiltration was delayed and TNFα expression decreased, accompanied by an increased IL-6 expression. Thus, the application of MSC appears to be a promising therapeutic approach to induce LG regeneration in patients suffering from severe DED/ADDE.

Analysis of lacrimal gland derived mesenchymal stem cell secretome and its impact on epithelial cell survival.

In situ regeneration of lacrimal gland (LG) tissue would be a promising approach to curatively treat dry eye disease (DED). Mesenchymal stem cells (MSC) exhibit therapeutic effects in a variety of pathological conditions and our group recently reported that their number increases in regenerating mouse LG. Since the therapeutic effects are suggested to arise from secreted trophic factors, the application of MSC-secreted proteins seems to be a promising approach to induce/enhance LG regeneration. Therefore, this study aims to optimize the isolation of murine LG-MSC and analyze their secretome to investigate its potential for LG epithelial cell survival in vitro. For optimization, LG-MSC were isolated by an explant technique or cell sorting and their secretome was investigated under normal and inflammatory conditions. Results showed that the secretome of MSC had beneficial effects on the viability of ethanol-damaged LG epithelial cells. Additional, Lipocalin-2, prosaposin, ras GTPase-activating protein-binding protein 1 (Rac1) and signal transducer and activator of transcription 1 (STAT1), proteins that were up-regulated under inflammatory conditions, further improved the cell survival of ethanol-damaged LG epithelial cells. Interestingly, recovery of cell viability was highest, when the cells were incubated with STAT1. Summarizing, this study identified promising proteins for further studies on LG regeneration.

Müller Cells Stabilize Microvasculature through Hypoxic Preconditioning.

BACKGROUND/AIMS: Hypoxia of the retina is a common pathogenic drive leading to vision loss as a result of tissue ischemia, increased vascular permeability and ultimately retinal neovascularisation. Here we tested the hypothesis that Müller cells stabilize the neurovascular unit, microvasculature by suppression of HIF-1α activation as a result of hypoxic preconditioning. METHODS: Tube Formation Assay and In vitro Vascular Permeability Image Assay were used to analyze angiogenesis and vascular integrity. Seahorse XF Cell Mito Stress Test was used to measure mitochondrial respiration. Gene and protein expression were examined by qRTPCR, ELISA and western blot. RESULTS: Hypoxic insult induces a significant induction of proangiogenic factors including vascular endothelial growth factor (VEGF) and angiopoietinlike 4 (ANGPTL-4) resulting in angiogenesis and increased vascular permeability of vascular endothelial cells. Hypoxic preconditioning of a human retinal Müller glia cell line significantly attenuates HIF-1α activation through the inhibition of mTOR and concomitant induction of aerobic glycolysis, stabilizing endothelial cells. CONCLUSION: Hypoxic preconditioning of Müller cells confers a robust protection to endothelial cells, through the suppression of HIF1α activation and its downstream regulation of VEGF and ANGPTL-4.

Evaluation of Plastic-Compressed Collagen for Conjunctival Repair in a Rabbit Model.

IMPACT STATEMENT: Conjunctival integrity is crucial for a healthy ocular surface and visual acuity. In severe cases of inflammatory surface disorders or after trauma, thermal or chemical burns as well as after ocular surgery, a surgical reconstruction using conjunctival substitutes is required. Due to limitations of currently used substitutes, such as the amniotic membrane, there is a need for the development of new scaffolds of consistent quality for conjunctival reconstruction. This study explored the biocompatibility and surgical usability of plastic-compressed collagen as an alternative conjunctival substitute in a rabbit model.

Generation and characterisation of decellularised human corneal limbus.

PURPOSE: Limbal epithelial stem cells (LESC) reside in a niche in the corneo-scleral transition zone. Deficiency leads to pain, corneal opacity, and eventually blindness. LESC transplantation of ex-vivo expanded human LESC on a carrier such as human amniotic membrane is a current treatment option. We evaluated decellularised human limbus (DHL) as a potential carrier matrix for the transplantation of LESC. METHODS: Human corneas were obtained from the local eye bank. The limbal tissue was decellularised by sodium desoxychelate and DNase solution and sterilised by γ-irradiation. Native limbus- and DHL-surface structures were assessed by scanning electron microscopy and collagen ultrastructure using transmission electron microscopy. Presence and preservation of limbal basement membrane proteins in native limbus and DHL were analysed immunohistochemically. Absence of DNA after decellularisation was assessed by Feulgen staining and DNA quantification. Presence of immune cells was explored by CD45 staining, and potential cytotoxicity was tested using a cell viability assay. RESULTS: In the DHL, the DNA content was reduced from 1.5 ± 0.3 µg/mg to 0.15 ± 0.01 µg/mg; the three-dimensional structure and the arrangement of the collagen fibrils were preserved. Main basement membrane proteins such as collagen IV, laminin, and fibronectin were still present after decellularisation and γ-irradiation. CD45-expressing cells were evident neither in the native limbus nor in the DHL. DHL did not convey cytotoxicity. CONCLUSIONS: The extracellular matrix (ECM) of the limbus provides a tissue specific morphology and three-dimensionality consisting of particular ECM proteins. It therefore represents a substantial component of the stem cell niche. The DHL provides a specific limbal niche surrounding, and might serve as an easily producible carrier matrix for LESC transplantation.

snRPN controls the ability of neurons to regenerate axons.

BACKGROUND: Retinal ganglion cells (RGCs) of mammals lose the ability to regenerate injured axons during postnatal maturation, but little is known about the underlying molecular mechanisms. OBJECTIVE: It remains of particular importance to understand the mechanisms of axonal regeneration to develop new therapeutic approaches for nerve injuries. METHODS: Retinas from newborn to adult monkeys (Callithrix jacchus)1 were obtained immediately after death and cultured in vitro. Growths of axons were monitored using microscopy and time-lapse video cinematography. Immunohistochemistry, Western blotting, qRT-PCR, and genomics were performed to characterize molecules associated with axonal regeneration and growth. A genomic screen was performed by using retinal explants versus native and non-regenerative explants obtained from eye cadavers on the day of birth, and hybridizing the mRNA with cross-reacting cDNA on conventional human microarrays. Followed the genomic screen, siRNA experiments were conducted to identify the functional involvement of identified candidates. RESULTS: Neuron-specific human ribonucleoprotein N (snRPN) was found to be a potential regulator of impaired axonal regeneration during neuronal maturation in these animals. In particular, up-regulation of snRPN was observed during retinal maturation, coinciding with a decline in regenerative ability. Axon regeneration was reactivated in snRPN-knockout retinal ex vivo explants of adult monkey. CONCLUSION: These results suggest that coordinated snRPN-driven activities within the neuron-specific ribonucleoprotein complex regulate the regenerative ability of RGCs in primates, thereby highlighting a potential new role for snRPN within neurons and the possibility of novel postinjury therapies.

Decellularised conjunctiva for ocular surface reconstruction.

Conjunctival reconstruction is an integral component of ocular surface restoration. Decellularised tissues are frequently used clinically for tissue engineering. This study identifies porcine decellularised conjunctiva (PDC) and human decellularised conjunctiva (HDC) as promising substitutes for conjunctival reconstruction. PDC and HDC were nearly DNA-free, structurally intact and showed no cytotoxic effects in vitro, which was confirmed by DNA quantification, histology, transmission electron microscopy, collagen quantification and cytotoxicity assay. Comparing the biomechanical properties to amniotic membrane (AM), the most frequently applied matrix for ocular surface reconstruction today, the decellularised conjunctiva was more extensible and elastic but exhibited less tensile strength. The in vivo application in a rabbit model proofed significantly enhanced transplant stability and less suture losses comparing PDC and HDC to AM while none of the matrices induced considerable inflammation. Ten days after implantation, all PDC, 4 of 6 HDC but none of the AM transplants were completely integrated into the recipient conjunctiva with a partially multi-layered epithelium. Altogether, decellularised conjunctivas of porcine and human origin were superior to AM for conjunctival reconstruction after xenogeneic application in vivo. STATEMENT OF SIGNIFICANCE: Conjunctival integrity is essential for a healthy ocular surface and clear vision. Its reconstruction is required in case of immunological diseases, after trauma, chemical or thermal burns or surgery involving the conjunctiva. Due to limitations of currently used substitute tissues such as amniotic membrane, there is a need for the development of new matrices for conjunctival reconstruction. Decellularised tissues are frequently applied clinically for tissue engineering. The present study identifies porcine and human decellularised conjunctiva as biocompatible and well tolerated scaffolds with superior integration into the recipient conjunctiva compared to amniotic membrane. Decellularised conjunctiva depicts a promising substitute for conjunctival reconstruction in ophthalmology.

Evaluation of Decellularized Porcine Jejunum as a Matrix for Lacrimal Gland Reconstruction In Vitro for Treatment of Dry Eye Syndrome.

Purpose: Dry eye syndrome (DES) can cause blindness in severe cases, but mainly palliative treatments exist. A tissue-engineered lacrimal gland (LG) could provide a curative treatment. We aimed to evaluate decellularized porcine jejunum (SIS-Muc) as a scaffold for porcine LG epithelial cells. Methods: To evaluate SIS-Muc as a potential scaffold, basement membrane proteins in SIS-Muc and native LG were compared (immunohistochemistry [IHC]). Porcine LG epithelial cells cultured on plastic were characterized (immunocytochemistry), and their culture supernatant was compared with porcine tears (proteomics). Epithelial cells were then seeded onto SIS-Muc in either a static (cell crown) or dynamic culture (within a perfusion chamber) and metabolic (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and secretory capacities (ß-hexosaminidase assay), protein expression (IHC), and ultrastructure transmission electron microscopy (TEM) compared in each. Results: Collagen IV and laminin were found in both native LG and SIS-Muc. When cultured on plastic, LG epithelial cells expressed pan-cytokeratin, Rab3D, HexA, and produced mucins, but lysozyme and lactoferrin expression was nearly absent. Some porcine tear proteins (lipocalin-2 and lactoferrin) were found in LG epithelial cell culture supernatants. When LG cells were cultured on SIS-Muc, metabolic and ß-hexosaminidase activities were greater in dynamic cultures than static cultures (P < 0.05). In both static and dynamic cultures, cells expressed pan-cytokeratin, Rab3D, lysozyme, and lactoferrin and produced mucins, and TEM revealed cell polarization at the apical surface and cell-cell and cell-scaffold contacts. Conclusions: SIS-Muc is a suitable scaffold for LG cell expansion and may be useful toward reconstruction of LG tissue to provide a curative treatment for DES. Dynamic culture enhances cell metabolic and functional activities.

The interplay between histone deacetylases and rho kinases is important for cancer and neurodegeneration.

Rho associated coiled-coil containing kinases (ROCKs) respond to defined extra- and intracellular stimuli to control cell migration, cell proliferation, and apoptosis. Histone deacetylases (HDACs) are epigenetic modifiers that regulate nuclear and cytoplasmic signaling through the deacetylation of histones and non-histone proteins. ROCK and HDAC functions are important compounds of basic and applied research interests. Recent evidence suggests a physiologically important interplay between HDACs and ROCKs in various cells and organisms. Here we summarize the crosstalk between these enzymatic families and its implications for cancer and neurodegeneration.

Analysis of Histone Deacetylase-Dependent Effects on Cell Migration Using the Stripe Assay.

For normal embryonic development/morphogenesis, cell migration and homing are well-orchestrated and important events requiring specific cellular mechanisms. In diseases such as cancer deregulated cell migration represents a major problem. Therefore, numerous efforts are under way to understand the molecular mechanisms of tumor cell migration and to generate more efficient tumor therapies. Cell migration assays are one of the most commonly used functional assays. The wound-healing assay or the Boyden chamber assay are variations of these assays. Nearly all of them are two-dimensional assays and the cells can only migrate on one substrate at a time. This is in contrast to the in vivo situation where the cells are faced simultaneously with different surfaces and interact with different cell types. To approach this in vivo situation we used a modified version of the stripe assay designed by Bonhoeffer and colleagues to examine mechanisms of axonal guidance. The design of this assay allows cells to decide between two different substrates offered at the same time. Utilizing alternating neuronal substrates for migration analyses we can partially mimic the complex in vivo situation for brain tumor cells. Here we describe the detailed protocol to perform a modified version of the stripe assay in order to observe substrate-dependent migration effects in vitro, to analyze the effect of Rho-dependent kinases (ROCKS), of histone deacetylases (HDACs) and of other molecules on glioma cells.

Development of Causative Treatment Strategies for Lacrimal Gland Insufficiency by Tissue Engineering and Cell Therapy. Part 2: Reconstruction of Lacrimal Gland Tissue: What Has Been Achieved So Far and What Are the Remaining Challenges?

The lacrimal gland is located in the upper temporal compartment of the orbita, and along with the ocular surface, eye lids, and sensory and motor nerves forms the lacrimal functional unit (LFU). The LFU is responsible for producing, distributing, and maintaining the tear film in order to maintain a smooth, moist, and regular ocular surface epithelium such that appropriate refractive properties are achieved and the eyeball is protected against dust, debris, and pathogens. If the main lacrimal gland is impaired (due to either disease or injury), this balance is disrupted, and severe quantitative dry eye syndrome (DES) can develop. DES treatments remain palliative, with the most commonly used therapies being based on tear substitution, tear retention, and control of inflammation on the ocular surface. Causative treatments such as salivary gland transplantation have shown to reduce symptoms in very severe cases, however can cause problems on the ocular surface due to different properties of saliva and tears. Therefore, causative approaches for treating DES by regeneration or reconstruction of lacrimal gland tissue depending on disease severity seem highly appealing. This article reviews current approaches for in vitro reconstruction of lacrimal gland tissue. Finally, the limitations that must be overcome before a new, tissue-engineered therapy may be delivered to clinic will be discussed.

Development of Causative Treatment Strategies for Lacrimal Gland Insufficiency by Tissue Engineering and Cell Therapy. Part 1: Regeneration of Lacrimal Gland Tissue: Can We Stimulate Lacrimal Gland Renewal In Vivo?

Severe dry eye syndrome (DES) is a complex disease that is commonly caused by inflammatory and degenerative changes in the lacrimal gland, and can result in severe pain and disruption to visual acuity. In healthy subjects, the ocular surface is continually lubricated by the tear film that ensures that the ocular surface remains moist and free of debris, enabling normal vision. The lacrimal fluid, mid-layer of the tear film, is mainly produced by the lacrimal gland and if this is dysfunctional for any reason, severe DES can develop. Currently, only palliative treatments for DES exist that aim to either replace or retain tears and/or minimize inflammation. A curative approach that aims to trigger the regeneration of existing lacrimal gland tissue in situ may, therefore, be very beneficial to DES patients. This article reviews the different approaches that have been explored toward lacrimal gland regeneration. Progress to date in vitro, in vivo, and in man is described with a focus on clinical feasibility and efficacy. Promising candidates for drug-dependent treatment of DES are growth factors and cytokines, such as hepatocyte growth factor (HGF) and tumor necrosis factor α-stimulated gene 6 protein (TSG-6). Only a few studies have evaluated gene therapy for lacrimal gland deficiencies, but with promising results. However gene therapy carries a variety of risks regarding carcinogenesis and therefore a treatment in the near future using this approach seems to be unlikely. Cell therapies utilizing mesenchymal stem cells (MSCs) seem to be more applicable than those using human amniotic membrane (hAM) epithelial cells or induced pluripotent stem (iPS) cells, since MSCs combine the favorable traits of both (multipotency, capability to stimulate regeneration immunomodulatory and non-immunogenic properties).

Engineering of a Secretory Active Three-Dimensional Lacrimal Gland Construct on the Basis of Decellularized Lacrimal Gland Tissue.

Lacrimal gland (LG) insufficiency is a main cause for severe dry eye leading to pain, visual impairment, and eventually loss of sight. Engineering of transplantable LG tissue with secretory capacity is a desirable goal. In this study, a three-dimensional decellularized LG (DC-LG) scaffold with preserved LG morphology was generated by treatment with 1% sodium deoxycholate and DNase solution using porcine LG tissue. To address clinical applicability, the primary in vitro culture of secretory active LG cells from a small tissue biopsy of 1.5 mm diameter was introduced and compared with an established isolation method by enzymatic digestion. Cells from both isolation methods depicted an epithelial phenotype, maintained their secretory capacity for up to 30 days, and exhibited progenitor cell capacity as measured by aldehyde dehydrogenase-1 activity, side population assay, and colony-forming units. Cells from passage 0 were reseeded into the DC-LG and secretory active cells migrated into the tissue. The cells resembled an LG-like morphology and the constructs showed secretory activity. These results demonstrate the possibility of engineering a secretory competent, three-dimensional LG construct using LG cells expanded from a small tissue biopsy and DC-LG as a matrix that provides the native structure and physiological niche for these cells.

The Influence of Oxygen on the Proliferative Capacity and Differentiation Potential of Lacrimal Gland-Derived Mesenchymal Stem Cells.

PURPOSE: The application of lacrimal gland-derived mesenchymal stem cells (LG-MSC) for the regeneration of lacrimal gland tissue could result in a novel therapy for dry-eye syndrome. To optimize the culture conditions, the purpose of this study was to evaluate the influence of low oxygen on phenotype, differentiation potential, proliferative, and regenerative capacity of murine LG-MSC. METHODS: Murine LG-MSC were cultured in 21% and 5% oxygen and characterized by flow cytometry, cell sorter assisted proliferation-, and colony forming unit-assays. Reactive oxygen species (ROS) levels as well as lineage differentiation were evaluated. The effect of conditioned medium of LG-MSC from both oxygen conditions (CM MSC 21%, respectively, CM MSC 5%) on lacrimal gland epithelial cells (LG-EC) was examined in wound healing and proliferation assays. RESULTS: Cells under both culture conditions revealed differentiation potential and presented a MSC-specific flow cytometric phenotype. In 5% oxygen, cells yielded less ROS, showed a stable morphology, higher colony forming potential, and an increased proliferation capacity. Five percent oxygen significantly increased the number of CD44+ LG-MSC. Furthermore, CM MSC 5% significantly enhanced migration and proliferation in LG-EC. CONCLUSIONS: In vitro expansion in low oxygen preserves the proliferation capacity and differentiation potential of LG-MSC and increases the effects of conditioned medium on migration and proliferation in LG-EC. Therefore, expansion in low oxygen seems to be an excellent method, to obtain vital MSC. Also, an increased number of LG-MSC expressing CD44 was observed under low oxygen, which might be a valuable marker to identify a potent MSC subpopulation.

Opposing signaling of ROCK1 and ROCK2 determines the switching of substrate specificity and the mode of migration of glioblastoma cells.

Despite current advances in therapy, the prognosis of patients with glioblastoma has not improved sufficiently in recent decades. This is due mainly to the highly invasive capacity of glioma cells. Little is known about the mechanisms underlying this particular characteristic. While the Rho-kinase (ROCK)-dependent signaling pathways involved in glioma migration have yet to be determined, they show promise as one of the candidates in targeted glioblastoma therapy. There are two ROCK isoforms: ROCK1, which is upregulated in glioblastoma tissue compared to normal brain tissue, and ROCK2, which is also expressed in normal brain tissue. Blockage of both of these ROCK isoforms with pharmacologic inhibitors regulates the migration process. We examined the activities of ROCK1 and ROCK2 using knockdown cell lines and the newly developed stripe assay. Selective knockdown of either ROCK1 or ROCK2 exerted antidromic effects on glioma migration: while ROCK1 deletion altered the substrate-dependent migration, deletion of ROCK2 did not. Furthermore, ROCK1 knockdown reduced cell proliferation, whereas ROCK2 knockdown enhanced it. Along the signaling pathways, key regulators of the ROCK pathway are differentially affected by ROCK1 and ROCK2. These data suggest that the balanced activation of ROCKs is responsible for the substrate-specific migration and the proliferation of glioblastoma cells.

Macula-less rat and macula-bearing monkey retinas exhibit common lifelong proteomic changes.

The visual consequences of age-related alterations in the neural retina have been well documented, but little is known about their molecular bases. We performed a comparative proteomic analysis of the retinas in marmosets and rats to identify proteins for which the expression profiles are altered with maturation and aging. Protein profiles were compared in the newborn, juvenile, middle-age, and aged retinas using 2-dimensional gel electrophoresis. Matrix-assisted desorption ionization-time-of-flight mass spectrometry revealed common proteins in rats and marmosets that exhibited changes in concentration throughout life. Western blot, quantitative reverse-transcriptase polymerase chain reaction, and immunohistochemistry analyses of selected proteins and their mRNA were used to determine whether the changes identified by proteomics were verifiable at the cellular and molecular levels. We found 4 proteins common to both species (Parkinson disease [autosomal recessive, early onset] 7/DJ-1, stathmin, peroxiredoxin, and ß-synuclein) whose concentrations were regulated with age. These findings were confirmed by Western blot, immunohistochemistry, and quantitative reverse-transcriptase polymerase chain reaction analyses. The proteins were localized in certain layers and subsets of cells within the retinas of both species. The expression of these proteins in the adult human retina was confirmed with immunohistochemistry. The present study is the first to provide evidence that the retina is physiologically characterized by specific lifelong changes in its proteome. These changes are independent of whether the retina bears a macula, occur in key functional pathways during the processing of visual signals, and might be involved in the development of age-related pathologic entities.

Dissecting the inter-substrate navigation of migrating glioblastoma cells with the stripe assay reveals a causative role of ROCK.

A hallmark of gliomas is the growth and migration of cells over long distances within the brain and proliferation within selected niches, indicating that the migrating cells navigate between complex substrates. We demonstrate in the present study a differential preference for migration that depends on Rho-associated coil kinase (ROCK) signaling, using the alternating Bonhoeffer stripe assay. Membrane fractions from nonmyelinated and myelinated brain areas from female rats, purified myelin also from female rats, and commercial extracellular matrix were used as substrates, with each substrate being tested against the others. The human tumor cell lines exhibited a clear preference for extracellular matrix over all other substrates and for myelinated over nonmyelinated tissue. ROCK signaling was different when cells were cultured on either substrate. The ROCK inhibitor Y27632 significantly attenuated and neutralized the preference for extracellular matrix and myelin, indicating that ROCK controls the substrate selectivity. The findings of this study pave the way for navigation-targeted therapeutics.