Fibrovascular Tissues Research Articles

Single Cell Isolation from Human Diabetic Fibrovascular Membranes for Single-Cell RNA Sequencing.

Single-cell transcriptomic analyses have emerged as very powerful tools to query the gene expression changes at the single-cell level in physiological and pathological conditions. The quality of the analysis is heavily dependent on tissue digestion protocols, with the goal of preserving thousands of single live cells to submit to the subsequent processing steps and analysis. Multiple digestion protocols that use different enzymes to digest the tissues have been described. Harsh digestion can damage certain cell types, but this might be required to digest especially fibrotic tissue as in our experimental condition. In this paper, we summarize a collagenase type I digestion protocol for preparing the single-cell suspension from fibrovascular tissues surgically removed from patients with proliferative diabetic retinopathy (PDR) for single-cell RNA sequencing (scRNA-Seq) analyses. We also provide a detailed description of the data analysis that we implemented in a previously published study. Key features • Single-cell suspension from fibrovascular membranes isolated from PDR patients. • Single-cell RNA sequencing analyses performed using Seurat package in RStudio. • Trajectory analyses or pseudotime analyses to study the trajectory over (pseudo)time of specific cell types. • This protocol requires Illumina HiSEQ4000 instrument and knowledge of R and RStudio language for the analyses.

Primary intra-axial Ewing sarcoma of the central nervous system: report of a rare case with literature review

BackgroundPrimary intra-axial Ewing sarcoma (EWS) is extremely rare and a highly aggressive small blue round cell tumor in the central nervous system (CNS). We reported a case of primary intra-axial CNS-EWS in a 30-year-old male and presented an extensive literature review of CNS-EWS cases.Case presentationA 30-year-old male had been diagnosed with a grade 2 astrocytoma in the left frontal lobe a year ago and had received chemotherapy and radiotherapy. He presented with a right-sided mass, which was revealed to be a 6 cm contrast-enhancing lesion with heterogeneous hemorrhagic areas in the right frontal lobe. The patient underwent emergent craniotomy for surgical excision. Histologically, the tumor was composed of high-grade, small, blue, round cells forming trabecular islands and rosettes which were separated by fibrovascular tissues. In immunohistochemical (IHC) examination, the tumor cells were positive for CD99 and FLI-1. Fluorescence in situ hybridization analysis revealed the presence of EWSR1 gene rearrangement. The histopathological, IHC, and molecular findings were consistent with a diagnosis of EWS. Further imaging did not show evidence of another primary site. The patient was followed up without further therapy and succumbed to the disease three months after the diagnosis.ConclusionEWS is very rare but an aggressive neoplasm in the CNS. Chemoradiotherapy may promote secondary cancers but it is unclear whether the development of EWS was associated with chemoradiotherapy that our case had received for astrocytoma. EWS should be considered when diagnosing a CNS tumor that shows small, blue, round cell tumor morphology. A combination of IHC and molecular tests is required for accurate diagnosis to ensure that all patients receive the most appropriate treatment to optimize outcomes.

Computer-Assisted Pterygium Screening System: A Review.

Pterygium is an eye condition that causes the fibrovascular tissues to grow towards the corneal region. At the early stage, it is not a harmful condition, except for slight discomfort for the patients. However, it will start to affect the eyesight of the patient once the tissues encroach towards the corneal region, with a more serious impact if it has grown into the pupil region. Therefore, this condition needs to be identified as early as possible to halt its growth, with the use of simple eye drops and sunglasses. One of the associated risk factors for this condition is a low educational level, which explains the reason that the majority of the patients are not aware of this condition. Hence, it is important to develop an automated pterygium screening system based on simple imaging modalities such as a mobile phone camera so that it can be assessed by many people. During the early stage of automated pterygium screening system development, conventional machine learning techniques such as support vector machines and artificial neural networks are the de facto algorithms to detect the presence of pterygium tissues. However, with the arrival of the deep learning era, coupled with the availability of large training data, deep learning networks have replaced the conventional networks in screening for the pterygium condition. The deep learning networks have been successfully implemented for three major purposes, which are to classify an image regarding whether there is the presence of pterygium tissues or not, to localize the lesion tissues through object detection methodology, and to semantically segment the lesion tissues at the pixel level. This review paper summarizes the type, severity, risk factors, and existing state-of-the-art technology in automated pterygium screening systems. A few available datasets are also discussed in this paper for both classification and segmentation tasks. In conclusion, a computer-assisted pterygium screening system will benefit many people all over the world, especially in alerting them to the possibility of having this condition so that preventive actions can be advised at an early stage.

Transfer, transportation, and accumulation of cerium-doped carbon quantum dots: Promoting growth and development in wheat

New cerium-doped carbon quantum dots (CDs:Ce) were developed in this study using hydrothermal synthesis method. The small and uniform sizes and nearly spherical lattice of CDs:Ce indicate its high stability, satisfactory water solubility, and biocompatibility. Wheat was treated with Ce, CDs, and different concentrations (0.01, 0.025, 0.05, 0.1, 0.2, and 0.4 mg/mL) of CDs:Ce. The results showed that, compared with the control group, Ce, CDs, and CDs:Ce could promote the growth and development of wheat in a certain concentration range. Wheat demonstrated the optimal morphological index (compared with the control, the root number, root length, leaf length, and plant height were increased by 45%, 57%, 28%, and 46%, respectively), maximum chlorophyll content (increased by 51%) and peroxidase activity (increased by 76%), and minimum malondialdehyde content (reduced by 68%) after treatment of 0.025 mg/mL of CDs:Ce. Hence, wheat plants can adsorb and transport CDs:Ce from roots to stems and leaves through fibrovascular tissues. The majority of CDs:Ce are concentrated in roots while some accumulate in leaves. A considerable amount of CDs:Ce gather in cell walls, fibrovascular tissues, leaf veins, and stomata. CDs:Ce can be applied to agricultural production activities as a new agricultural nanofertilizer and technology of plant in vivo imaging.

Phytochemical & Pharmacological Studies of Moltkiopsis ciliata

The stem and branches have silvery white colour with a pink tint. They bear stiff covering trichomes and leaves or leaves remaining are found at their numerous inter nodes. Dark brown scars are left by falling leaves. The branches are brittle and their outer layer separate on breaking. Microscopically، the powder shown many free conical، warty، tapering covering trichomes of various lengths and sizes; the comparatively smaller ones belong to leaves while the significantly large ones are detached from stem and branches. The powder also shows light orange-brown fragments of leaf exhibiting compact endings of palisade cells rounded in outlines; some of these fragments bear many covering trichomes. There are also many grey or grayish-brown fragments of fibro-vascular tissues of branches، some are thick and closely packed، in addition to many fragments of light brown bark cells polygonal or almost square in outlines.

ROCK1 Mediates Retinal Glial Cell Migration Promoted by Acrolein.

Objective: Acrolein is a highly reactive aldehyde that covalently binds to cellular macromolecules and subsequently modulates cellular function. Our previous study demonstrated that acrolein induces glial cell migration, a pathological hallmark of diabetic retinopathy; however, the detailed cellular mechanism remains unclear. The purpose of this study was to investigate the role of acrolein in retinal glial cell migration by focusing on rho-associated coiled-coil-containing protein kinases (ROCKs).Methods: Immunofluorescence staining for ROCK isoforms was performed using sections of fibrovascular tissue obtained from the eyes of patients with proliferative diabetic retinopathy (PDR). Rat retinal Müller glial cell line, TR-MUL5, was stimulated with acrolein and the levels of ROCK1 were evaluated using real-time PCR and western blotting. Phosphorylation of the myosin-binding subunit of myosin light chain phosphatase [myosin phosphatase target subunit 1, (MYPT1)] and myosin light chain 2 (MLC2) was assessed. The cell migration rate of TR-MUL5 cells exposed to acrolein and/or ripasudil, a non-selective ROCK inhibitor, was measured using the Oris cell migration assay.Results: ROCK isoforms, ROCK1 and ROCK2, were positively stained in the cytosol of glial cells in fibrovascular tissues. In TR-MUL5 cells, the mRNA expression level of Rock1, but not Rock2, was increased following acrolein stimulation. In line with the PCR data, western blotting showed increase in ROCK1 and cleaved ROCK1 protein in TR-MUL5 cells stimulated with acrolein. N-acetylcysteine (NAC) suppressed acrolein-associated Rock1 upregulation in TR-MUL5 cells. Acrolein augmented the phosphorylation of MYPT1 and MLC2 and increased the cell migration rate of TR-MUL5 cells, both of which were abrogated by ripasudil.Conclusions: Our study demonstrated that ROCK1 mediates the migration of retinal glial cells promoted by the unsaturated aldehyde acrolein.

Known data on applied regenerative medicine in tendon healing.

Tendons and ligaments are important structures in the musculoskeletal system. Ligaments connect various bones and provide stability in complex movements of joints in the knee. Tendon is made of dense connective tissue and transmits the force of contraction from muscle to bone. They are injured due to direct trauma in sports or roadside accidents. Tendon healing after repair is often poor due to the formation of fibro vascular scar tissues with low mechanical property. Regenerative techniques such as PRP (platelet-rich plasma), stem cells, scaffolds, gene therapy, cell sheets, and scaffolds help augment repair and regenerate tissue in this context. Therefore, it is of interest to document known data (repair process, tissue regeneration, mechanical strength, and clinical outcome) on applied regenerative medicine in tendon healing.

Involvement of Müller Glial Autoinduction of TGF-β in Diabetic Fibrovascular Proliferation Via Glial-Mesenchymal Transition.

PurposeMüller glial–mesenchymal transition (GMT) is reported as the fibrogenic mechanism promoted by TGF-β–SNAIL axis in Müller cells transdifferentiated into myofibroblasts. Here we show the multifaceted involvement of TGF-β in diabetic fibrovascular proliferation via Müller GMT and VEGF-A production.MethodsSurgically excised fibrovascular tissues from the eyes of patients with proliferative diabetic retinopathy were processed for immunofluorescence analyses of TGF-β downstream molecules. Human Müller glial cells were used to evaluate changes in gene and protein expression with real-time quantitative PCR and ELISA, respectively. Immunoblot analyses were performed to detect TGF-β signal activation.ResultsMüller glial cells in patient fibrovascular tissues were immunopositive for GMT-related molecular markers, including SNAIL and smooth muscle protein 22, together with colocalization of VEGF-A and TGF-β receptors. In vitro administration of TGF-β1/2 upregulated TGFB1 and TGFB2, both of which were suppressed by inhibitors for nuclear factor-κB, glycogen synthase kinase-3, and p38 mitogen-activated protein kinase. Of the various profibrotic cytokines, TGF-β1/2 application exclusively induced Müller glial VEGFA mRNA expression, which was decreased by pretreatment with small interfering RNA for SMAD2 and inhibitors for p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase. Supporting these findings, TGF-β1/2 stimulation to Müller cells increased the phosphorylation of these intracellular signaling molecules, all of which were also activated in Müller glial cells in patient fibrovascular tissues.ConclusionsThis study underscored the significance of Müller glial autoinduction of TGF-β as a pathogenic cue to facilitate diabetic fibrovascular proliferation via TGF-β–driven GMT and VEGF-A–driven angiogenesis.

Regulation of Spermine Oxidase through Hypoxia-Inducible Factor-1α Signaling in Retinal Glial Cells under Hypoxic Conditions.

PurposeAcrolein, a highly reactive unsaturated aldehyde, is known to facilitate glial cell migration, one of the pathological hallmarks in diabetic retinopathy. However, cellular mechanisms of acrolein generation in retinal glial cells remains elusive. In the present study, we investigated the role and regulation of spermine oxidase (SMOX), one of the enzymes related to acrolein generation, in retinal glial cells under hypoxic condition.MethodsImmunofluorescence staining for SMOX was performed using sections of fibrovascular tissues obtained from patients with proliferative diabetic retinopathy. Expression levels of polyamine oxidation enzymes including SMOX were analyzed in rat retinal Müller cell line 5 (TR-MUL5) cells under either normoxic or hypoxic conditions. The transcriptional activity of Smox in TR-MUL5 cells was evaluated using the luciferase assay. Levels of acrolein-conjugated protein, Nε-(3-formyl-3,4-dehydropiperidino) lysine adduct (FDP-Lys), and hydrogen peroxide were measured.ResultsSMOX was localized in glial cells in fibrovascular tissues. Hypoxia induced SMOX production in TR-MUL5 cells, which was suppressed by silencing of hypoxia-inducible factor-1α (Hif1a), but not Hif2a. Transcriptional activity of Smox was regulated through HIF-1 binding to hypoxia response elements 2, 3, and 4 sites in the promoter region of Smox. Generation of FDP-Lys and hydrogen peroxide increased in TR-MUL5 cells under hypoxic condition, which was abrogated by SMOX inhibitor MDL72527.ConclusionsThe current data demonstrated that hypoxia regulates production of SMOX, which plays a role in the generation of oxidative stress inducers, through HIF-1α signaling in Müller glial cells under hypoxic condition.

Glucocorticoid-transactivated TSC22D3 attenuates hypoxia- and diabetes-induced Müller glial galectin-1 expression via HIF-1α destabilization.

Galectin‐1/LGALS1, a newly recognized angiogenic factor, contributes to the pathogenesis of diabetic retinopathy (DR). Recently, we demonstrated that glucocorticoids suppressed an interleukin‐1β‐driven inflammatory pathway for galectin‐1 expression in vitro and in vivo. Here, we show glucocorticoid‐mediated inhibitory mechanism against hypoxia‐inducible factor (HIF)‐1α‐involved galectin‐1 expression in human Müller glial cells and the retina of diabetic mice. Hypoxia‐induced increases in galectin‐1/LGALS1 expression and promoter activity were attenuated by dexamethasone and triamcinolone acetonide in vitro. Glucocorticoid application to hypoxia‐stimulated cells decreased HIF‐1α protein, but not mRNA, together with its DNA‐binding activity, while transactivating TSC22 domain family member (TSC22D)3 mRNA and protein expression. Co‐immunoprecipitation revealed that glucocorticoid‐transactivated TSC22D3 interacted with HIF‐1α, leading to degradation of hypoxia‐stabilized HIF‐1α via the ubiquitin‐proteasome pathway. Silencing TSC22D3 reversed glucocorticoid‐mediated ubiquitination of HIF‐1α and subsequent down‐regulation of HIF‐1α and galectin‐1/LGALS1 levels. Glucocorticoid treatment to mice significantly alleviated diabetes‐induced retinal HIF‐1α and galectin‐1/Lgals1 levels, while increasing TSC22D3 expression. Fibrovascular tissues from patients with proliferative DR demonstrated co‐localization of galectin‐1 and HIF‐1α in glial cells partially positive for TSC22D3. These results indicate that glucocorticoid‐transactivated TSC22D3 attenuates hypoxia‐ and diabetes‐induced retinal glial galectin‐1/LGALS1 expression via HIF‐1α destabilization, highlighting therapeutic implications for DR in the era of anti‐vascular endothelial growth factor treatment.

Increased Expression of Growth Hormone–Releasing Hormone in Fibrinous Inflammation of Proliferative Diabetic Retinopathy

To investigate the involvement of growth hormone-releasing hormone (GHRH) - growth hormone (GH) signaling in pathogenesis of proliferative diabetic retinopathy (PDR). Experimental laboratory study. Vitreous humor, aqueous humor, and serum were obtained from 36 eyes of 36 patients with or without type 2 diabetes from 2017 to 2019. For histologic examination, 6 fibrovascular membranes were excised from eyes with active PDR. Three fibrovascular membranes were excised from nondiabetic patients with proliferative vitreoretinopathy (PVR) as controls. In PDR, the fibrovascular tissues consisted of a mature region containing fibrocytes, and an immature region populated by abundant polymorphonuclear leukocytes in a fibrinogen meshwork. Clusters of leukocytes were found adhering to the vascular walls. In PVR, no fibrinogen and polymorphonuclear leukocyte was observed in the fibrovascular membranes. The levels of GHRH and GH in PDR were significantly increased (P < .001), with 1.8-fold and 72.8-fold in vitreous humor, and 2-fold and 4.9-fold in aqueous humor, respectively, when compared with corresponding levels in controls. No significant difference was detected for insulin-like growth factor-1. Immunohistochemistry showed intense expression of GHRH and its receptor GHRH-R in polymorphonuclear leukocytes, vascular endothelial cells, and fibrocytes in fibrovascular membranes of PDR. GHRH staining was not detectable in infiltrating cells within the fibrovascular membrane of PVR. These findings reveal a possible involvement of GHRH/GHRH-R in fibrinous inflammation that might contribute to the formation of fibrovascular membrane in PDR through mediating activities of leukocytes, vascular endothelial cells, and fibrocytes. Targeting GHRH/GHRH-R may be considered as a potential therapeutic approach for the treatment of PDR.

Unsaturated Aldehyde Acrolein Promotes Retinal Glial Cell Migration.

To investigate the effect of the unsaturated aldehyde acrolein on retinal glial cell migration. Müller glial cell markers expression in TR-MUL5 were confirmed by RT-PCR and immunostaining. Cell viability and migration rate of TR-MUL5 cells were assessed after the stimulation with acrolein. DNA microarray analysis was performed to analyze changes in the expression levels of migration-related genes in Müller glial cells stimulated with acrolein. Real-time PCR and ELISA were performed to validate DNA microarray analysis results. Inhibitors of C-X-C motif chemokine ligand 1 (CXCL1), one of the genes highly upregulated after the exposure to acrolein, and blockers of its receptor, CXCR2, were used to investigate the role of the CXCL1-CXCR2 axis on glial cell migration. CXCL1 concentration was measured in vitreous fluid samples obtained from proliferative diabetic retinopathy (PDR) and nondiabetic control eyes. CXCL1 and CXCR2 expression in glial cells of fibrovascular tissues obtained from PDR patients was examined by immunostaining. At a high concentration, acrolein (100 μM) significantly decreased cell viability. However, in moderate, sublethal concentrations (25-50 μM), acrolein induced cell migration and substantially increased the production of CXCL1 in TR-MUL5 cells. CXCL1 concentration was significantly elevated in vitreous fluids of PDR patients, and CXCL1 and CXCR2 were present in glial cells in fibrovascular tissues of PDR patients. CXCL1 stimulation increased glial cell migration in a dose-dependent manner, which was abrogated by the neutralization of the CXCL1-CXCR2 axis. Our data demonstrate that acrolein promotes retinal Müller glial cell migration by enhancing CXCL1 production.

Glucocorticoid receptor inhibits Müller glial galectin-1 expression via DUSP1-dependent and -independent deactivation of AP-1 signalling.

Galectin‐1/LGALS1 is a hypoxia‐induced angiogenic factor associated with diabetic retinopathy (DR). Recently, we elucidated a hypoxia‐independent pathway to produce galectin‐1 in Müller glial cells stimulated by interleukin (IL)‐1β. Here we revealed glucocorticoid receptor (GR)‐mediated inhibitory mechanisms for Müller glial galectin‐1/LGALS1 expression. Activator protein (AP)‐1 site in the LGALS1 enhancer region, to which activating transcription factor2, c‐Fos and c‐Jun bind, was shown to be essential for IL‐1β‐induced galectin‐1/LGALS1 expression in Müller cells. Ligand (dexamethasone or triamcinolone acetonide)‐activated GR induced dual specificity phosphatase (DUSP)1 expression via the glucocorticoid response element and attenuated IL‐1β‐induced galectin‐1/LGALS1 expression by reducing phosphorylation of these AP‐1 subunits following AKT and extracellular signal‐regulated kinase (ERK)1/2 deactivation. Moreover, activated GR also caused DUSP1‐independent down‐regulation of IL‐1β‐induced LGALS1 expression via its binding to AP‐1. Administration of glucocorticoids to mice attenuated diabetes‐induced retinal galectin‐1/Lgals1 expression together with AKT/AP‐1 and ERK/AP‐1 pathways. Supporting these in vitro and in vivo findings, immunofluorescence analyses showed co‐localization of galectin‐1 with GR and phosphorylated AP‐1 in DUSP1‐positive glial cells in fibrovascular tissues from patients with DR. Our present data demonstrated the inhibitory effects of glucocorticoids on glial galectin‐1 expression via DUSP1‐dependent and ‐independent deactivation of AP‐1 signalling (transactivation and transrepression), highlighting therapeutic implications for DR.

Histopathologic Evaluation of Polymer Supports for Pintucci-type Keratoprostheses: An Animal Study.

PurposeTo report histopathological findings for different types of polymers proposed as support for a Pintucci-type keratoprosthesis.MethodsSix polymers, including three types of polyesters (1-3), one type of polytetrafluoroethylene (PTFE, 4), polyethylene (5), and expanded polytetrafluoroethylene (ePTFE, 6) were evaluated. Four samples of each material were placed under the orbicularis oculi muscles of 12 rabbits. After five weeks, the samples were removed and evaluated histopathologically. Fibrovascular tissue ingrowths were investigated in terms of tissue penetration depth into the materials (graded as none, mild, moderate, and intense) and fibrovascular ingrowth area at the ultimate level of tissue penetrance. ImageJ software was used to calculate fibrovascular tissue area between the material fibers, and the mean area values were compared between the materials.ResultsPolyester materials 1 and 3 demonstrated intense fibrovascular tissue penetration with a large fibrovascular ingrowth area; no overt tissue ingrowth was observed into material #6. The mean area of penetrated fibrovascular tissues was significantly different between materials ( < 0.001). Materials 2, 4, and 5 showed moderate fibrovascular tissue ingrowth and the area of presented fibrovascular tissue at the paracentral parts of material 4 was significantly smaller than that of materials 1 ( = 0.02) and 3 ( = 0.01).ConclusionTwo polyester materials that had relatively large pore sizes demonstrated a deep and large area of fibrovascular ingrowth. Given that material #3 is thicker and more consistent than material 1, the former can be used as the appropriate material for supporting the Pintucci-type keratoprosthesis.

White Adipocytes of the Uterine Cervix Tend to Appear in Women of Older Age, Postmenopause Status, and Higher Body Mass Index.

The human uterine cervix consists mainly of epithelium and stroma, including smooth muscle cells and fibrovascular tissues. Fat cells in the uterine cervix have been rarely reported, and the only previous research article has shown that intracervical adipocytes are unrelated to clinical factors. The aim of this study was to investigate the frequency of fat cells in the uterine cervix, as well as to evaluate the relationship between intracervical adipocytes and clinicopathologic factors. We retrospectively selected 405 cases in Japanese women who received cervical conization at our hospital between 2003 and 2017. Cervical conization was not performed during pregnancy or within 1 yr after childbirth. The prepared histologic specimens for pathologic diagnosis were available in all cases. Age, menopause status, body mass index, gravidity, and parity were selected clinical factors, which were obtained in 214 patients. The mean patient age was 42 yr (range, 22-80 yr). Intracervical white adipocytes were observed in 13% of patients (53/405), with no brown adipocytes detected. The existence of intracervical adipocytes was significantly correlated to older age (P<0.0001), postmenopause status (P<0.0001), and higher body mass index (P=0.0018). Intracervical adipocytes might undergo adipocytic metaplasia from cervical stromal cells in accordance with aging, postmenopause status, or weight gain. Our result also suggest that cervical malignancy involving fat cells does not necessarily imply parametrial invasion.

In vivo detecting mouse persistent hyperplastic primary vitreous by Spectralis Optical Coherence Tomography

To identify imaging characteristics of mouse persistent hyperplastic primary vitreous (PHPV) by Spectralis Optical Coherence Tomography (OCT), as well as to assess and compare the sensitivity and precision of OCT with color photography (CP) and Fundus Fluorescein Angiography (FFA) imaging in detecting mouse PHPV. Notch4−/− C57BL/6J mice (224 eyes) aged from 3 months to 7 months were examined in this study. CP, FFA and OCT imaging were utilized to examine vitreous cavity and retina of mouse eyes. Horizontal and radial OCT scan volume was centered on the optic nerve head. Hematoxylin and eosin (H&E) staining was performed to validate PHPV. For color photography and FFA imaging, retrolental irregular fibrovascular membrane-like tissues were found in 33 eyes with/without blood vessels in vitreous cavity. Among them, 31 eyes were visualized with lateral and oblique linear hyperreflective opacities in vitreous cavity using Spectralis OCT. Position of PHPV in posterior segment of eyes was also measured via OCT. Mouse PHPV was validated by H&E staining. Typical hyperreflective opacities in vitreous cavity were detected in PHPV mouse using Spectralis OCT. Spectralis OCT imaging can effectively detect mouse PHPV as color photography and FFA.

Advanced glycation endproducts link inflammatory cues to upregulation of galectin-1 in diabetic retinopathy

Diabetic retinopathy (DR) is an inflammatory and progressive vaso-occlusive disease resulting in angiogenesis. Galectin-1 is a hypoxia-induced angiogenic factor associated with cancer and proliferative DR. Here we reveal a significant upregulation of galectin-1 in eyes of DR patients along with progression of clinical stages beginning from the pre-ischemic, inflammatory stage with diabetic macular edema, but not in eyes with non-diabetic retinal vascular occlusions. As for its regulatory mechanism unrelated to hypoxia but selective to DR, in vitro galectin-1/LGALS1 expression was shown to increase after application to Müller glial cells with interleukin (IL)-1β, which was induced in monocyte-derived macrophages and microglial cells via toll-like receptor (TLR) 4 signaling stimulated by advanced glycation endproducts (AGE). In vivo inhibition of AGE generation with aminoguanidine, macrophage depletion with clodronate liposomes, and antibody-based blockade of Il-1β and Tlr4 attenuated diabetes-induced retinal Lgals1 expression in mice. Fibrovascular tissues from proliferative DR eyes were immunoreactive for AGE, TRL4 and IL-1β in macrophages, and IL-1β receptor-positive glial cells expressed galectin-1. Therefore, diabetes-induced retinal AGE accumulation was suggested to activate IL-1β-related inflammatory cues in macrophages followed by Müller cells, linking to galectin-1 upregulation in human DR with time. Our data highlight AGE-triggered inflammation as the DR-selective inducer of galectin-1.

Soluble Vascular Adhesion Protein-1 Mediates Spermine Oxidation as Semicarbazide-Sensitive Amine Oxidase: Possible Role in Proliferative Diabetic Retinopathy

ABSTRACTPurpose/Aim of the study: To explore the possible role of vascular adhesion protein-1 (VAP-1) via its enzymatic function as a semicarbazide-sensitive amine oxidase (SSAO) in the pathogenesis of proliferative diabetic retinopathy (PDR).Materials and Methods: The levels of soluble VAP-1/SSAO and the unsaturated aldehyde acrolein (ACR)-conjugated protein, Nε-(3-formyl-3, 4-dehydropiperidino) lysine adduct (FDP-Lys), were measured in vitreous fluid samples of PDR and non-diabetic patients using ELISA. Recombinant human VAP-1/SSAO (rhVAP-1/SSAO) was incubated with spermine, with or without semicarbazide or RTU-1096 (a specific inhibitor for VAP-1/SSAO). Immunofluorescence assays were performed to assess the localization of VAP-1/SSAO and FDP-Lys in fibrovascular tissues from patients with PDR. The impact of ACR on cultured retinal capillary endothelial cells was assessed using a cell viability assay and total glutathione (GSH) measurements.Results: The levels of sVAP-1/SSAO and FDP-Lys were elevated in the vitreous fluid of patients with PDR. Incubation of rhVAP-1 with spermine resulted in the generation of hydrogen peroxide and FDP-Lys and the production was inhibited by semicarbazide and RTU-1096. In fibrovascular tissues, FDP-Lys and VAP-1/SSAO were present in endothelial cells. ACR stimulation reduced GSH levels in the cultured endothelial cells in a dose-dependent manner and caused cellular toxicity.Conclusions: Our results indicate the pathological role of sVAP-1/SSAO to generate hydrogen peroxide and toxic aldehyde ACR, both of which are associated with oxidative stress, as a consequence of spermine oxidation in eyes with PDR.

Enhancement of rotator cuff tendon–bone healing with fibroblast growth factor 2 impregnated in gelatin hydrogel sheets in a rabbit model

Application of fibroblast growth factor 2 (FGF-2) may improve the healing response after rotator cuff (RC) surgical repair. This study aimed to determine whether FGF-2-impregnated gelatin hydrogel sheet (GHS) incorporation into the bony trough on the greater tuberosity facilitates healing after RC surgical repair in rabbits. We assigned 120 adult male Japanese white rabbits treated with unilateral surgery for supraspinatus tendon repair into the following groups: suture-only group (suture); suture and GHS with phosphate-buffered saline (carrier); suture and GHS with 3 µg of FGF-2 (F3); and suture and GHS with 30 µg of FGF-2 (F30). The effect of FGF-2 was assessed using histologic, biomechanical, and microcomputed tomography evaluations at 2, 6, and 12 weeks. At 12 weeks, loose fibrovascular tissues emerged at the repair site in the suture and carrier groups and dense tendon-like tissues in the F3 and F30 groups, which demonstrated significantly higher ultimate load-to-failure and stress-to-failure at 12 weeks than that in the suture and carrier groups. Microcomputed tomography imaging showed ectopic calcification formation in some specimens from each group. Appearances or frequencies were similar among groups. The histologic and biomechanical effects of FGF-2 on RC healing were obvious at ≥6 weeks postoperatively. FGF-2-impregnated GHS incorporation into the bony trough on the greater tuberosity before RC surgical repair is feasible and results in histologic and biomechanical improvements during RC healing in rabbits. No detrimental effect on ectopic calcification was observed.

The effect of intraarticular serum rich in growth factors (SRGF) on knee osteoarthritis in the rat model

Osteoarthritis (OA) is a degenerative joint disease with high incidence in world. Various treatments have been used for decreasing pain and improving physical function without clearly beneficial evidences. Nowadays, platelet-rich therapy is used as a novel approach in field of regenerative medicine. The present study evaluated the effect of intraarticular serum rich in growth factors (SRGF) on the monosodium iodoacetate induced-osteoarthritis in rat model. SRGF was injected 3 times weekly. Femorotibial joints were examined 2 weeks after last injection. Histopathologic results showed remarkable articular regeneration in the SRGF treated rats. Our findings were less surface irregularities and articular clefts, proliferation of chondrocytes and increasing synthesis of matrix proteoglycan with toluidine blue staining. Destroyed articular cartilage was repaired by fibro-hyaline cartilage in contrast to the OA rats that filled with fibrovascular tissues. The microscopic score had decreased in the treatment group. We concluded that SRGF, as a source of growth factors, have chondroinductive capacity with regard to enhancement of cartilage regeneration.