Agonist of growth hormone-releasing hormone enhances retinal ganglion cell protection induced by macrophages after optic nerve injury.

Optic neuropathies are leading causes of irreversible visual impairment and blindness, currently affecting more than 100 million people worldwide. Glaucoma is a group of optic neuropathies attributed to progressive degeneration of retinal ganglion cells (RGCs). We have previously demonstrated an increase in survival of RGCs by the activation of macrophages, whereas the inhibition of macrophages was involved in the alleviation on endotoxin-induced inflammation by antagonist of growth hormone-releasing hormone (GHRH). Herein, we hypothesized that GHRH receptor (GHRH-R) signaling could be involved in the survival of RGCs mediated by inflammation. We found the expression of GHRH-R in RGCs of adult rat retina. After optic nerve crush, subcutaneous application of GHRH agonist MR-409 or antagonist MIA-602 promoted the survival of RGCs. Both the GHRH agonist and antagonist increased the phosphorylation of Akt in the retina, but only agonist MR-409 promoted microglia activation in the retina. The antagonist MIA-602 reduced significantly the expression of inflammation-related genes Il1b, Il6, and Tnf Moreover, agonist MR-409 further enhanced the promotion of RGC survival by lens injury or zymosan-induced macrophage activation, whereas antagonist MIA-602 attenuated the enhancement in RGC survival. Our findings reveal the protective effect of agonistic analogs of GHRH on RGCs in rats after optic nerve injury and its additive effect to macrophage activation, indicating a therapeutic potential of GHRH agonists for the protection of RGCs against optic neuropathies especially in glaucoma.

Glucose sensing based on hydrogel grating incorporating phenylboronic acid groups.

We proposed a hydrogel grating sensor functionalized with phenylboronic acid (PBA) group for glucose concentration detection. A PBA functionalized polyacrylamide hydrogel film was first prepared via ultraviolet polymerization. Then, the diffraction grating was written on the hydrogel film via the femto-second (fs) laser point-by-point direct inscription. Binding between the PBA groups in the hydrogel and glucose molecules would lead to the swelling of hydrogel and the thus grating structure, thus modifying the diffraction properties of the grating. We experimentally characterized the swelling and transmission of the grating with different glucose concentrations. Sensitivity of the sensor was defined as variations in relative diffraction efficiency in response to glucose concentration changes, and was experimentally found to 0.61%/mM. The proposed sensor showed fast response towards the presence of glucose, and its reusability and biocompatibility were also confirmed. The use of fs-laser inscription technique does not require a pre-fabricated template, and would allow to directly modify the fabrication parameters such as scanning speed, pulse energy and frequency. Therefore, one is able to conveniently optimize the grating structure and improve the inscription efficiency. The proposed hydrogel grating could be potentially fabricated into wearable sensors, namely, contact lenses, for continuous monitoring of tear glucose level with rapid response.

Attenuated regenerative properties in human periodontal ligament-derived stem cells of older donor ages with shorter telomere length and lower SSEA4 expression.

Periodontal ligament (PDL) stem cell properties are critical in the periodontal tissue regeneration for periodontitis. Previously, we have demonstrated that cigarette smoking attenuates PDL-derived stem cell (PDLSC) regenerative properties. Here, we report the findings on the regenerative properties of human PDLSCs with different donor ages and the underlying mechanisms. Human PDLSCs from 18 independent donors were divided into different age groups (≤ 20, 20-40, and > 40 years old). The proliferation of PDLSCs with donor age of ≤ 20 years old was significantly higher than that of the 20-40- and > 40-years-old groups, whereas the migration of PDLSCs with donor age of ≤ 20 and 20-40 years old was significantly higher than that of the > 40-years-old group. Moreover, the mesodermal lineage differentiation capabilities of PDLSCs were also higher in the donor age group of ≤ 20 years old than the donor age of > 40 years old. In addition, shorter telomere length and lower expression of SSEA4 were found in PDLSCs with donor age of > 40 years old, compared with those with donor age of ≤ 20-years-old group. Besides, PDLSCs with donor age of 20-40 and > 40 years old had higher IL6 and CXCL8 gene expressions. In summary, results from this study revealed the attenuated proliferation, migration, and mesodermal lineage differentiation properties in human PDLSCs with older donor ages. Donor age of PDLSCs should be considered as the selection criteria for the periodontal tissue regeneration treatment.

Longitudinal evaluation of immediate inflammatory responses after intravitreal AAV2 injection in rats by optical coherence tomography.

Gene therapy has been proposed as a feasible strategy for RGC survival and optic nerve regeneration. Some preclinical and clinical studies revealed intraocular inflammation after intravitreal injection of adeno-associated virus (AAV) by slit-lamp or indirect ophthalmoscope. Here we evaluate the longitudinal profile of immediate inflammatory responses after AAV2 injection in rat retina and vitreous body by optical coherence tomography (OCT). Adult Fischer F344 rats were intravitreally injected once with saline, AAV2 or zymosan. Retinal thickness and cell infiltration were recorded by OCT longitudinally for 2 months and verified by histological analysis. The transduction rate of single intravitreal AAV2 injection was 21.3 ± 4.9% of whole retina, and the transduction efficiency on RGCs was 91.5 ± 2.5% in the transduced area. Significant increase in cell infiltration was observed from Day 1-3 after AAV2 injection, compared to very few infiltrating cells observed in the saline-injected group. The infiltrating cells ceased at Day 5 after intravitreal injection and remained absent at 2 months. The thicknesses of total and inner retina were increased along Day 1-3 after AAV2 injection, but reverted to normal afterwards. The surviving RGCs in the AAV2-injected groups at Day 14 showed no significant difference compared to saline-injected group. In summary, this study revealed the immediate inflammatory responses and retinal edema after intravitreal AAV2 injection in normal rats, without influencing long-term retinal thickness and RGC survival. OCT can be implemented for the time-lapse in vivo evaluation of inflammatory response after AAV-mediated gene therapy through intravitreal injection.

Caveolin-1 regulates human trabecular meshwork cell adhesion, endocytosis, and autophagy.

Impaired trabecular meshwork (TM) outflow is implicated in the pathogenesis of primary open-angle glaucoma (POAG). We previously identified the association of a caveolin-1 (CAV1) variant with POAG by genome-wide association study. Here we report a study of CAV1 knockout (KO) effect on human TM cell properties. We generated human CAV1-KO TM cells by CRISPR/Cas9 technology, and we found that the CAV1-KO TM cells less adhered to the surface coating than the wildtype TM cells by 69.34% ( P < 0.05), but showed no difference in apoptosis. Higher endocytosis ability of dextran and transferrin was also observed in the CAV1-KO TM cells (4.37 and 1.89-fold respectively, P < 0.001), compared to the wildtype TM cells. Moreover, the CAV1-KO TM cells had higher expression of extracellular matrix-degrading enzyme genes ( ADMTS13 and MMP14) as well as autophagy-related genes ( ATG7 and BECN1) and protein (LC3B-II) than the wildtype TM cells. In summary, results from this study showed that the CAV1-KO TM cells have reduced adhesion with higher extracellular matrix-degrading enzyme expression, but increased endocytosis and autophagy activities, indicating that CAV1 could be involved in the regulation of adhesion, endocytosis, and autophagy in human TM cells.

Mutations of RagA GTPase in mTORC1 Pathway Are Associated with Autosomal Dominant Cataracts.

Cataracts are a significant public health problem with no proven methods for prevention. Discovery of novel disease mechanisms to delineate new therapeutic targets is of importance in cataract prevention and therapy. Herein, we report that mutations in the RagA GTPase (RRAGA), a key regulator of the mechanistic rapamycin complex 1 (mTORC1), are associated with autosomal dominant cataracts. We performed whole exome sequencing in a family with autosomal dominant juvenile-onset cataracts, and identified a novel p.Leu60Arg mutation in RRAGA that co-segregated with the disease, after filtering against the dbSNP database, and at least 123,000 control chromosomes from public and in-house exome databases. In a follow-up direct screening of RRAGA in another 22 families and 142 unrelated patients with congenital or juvenile-onset cataracts, RRAGA was found to be mutated in two unrelated patients (p.Leu60Arg and c.-16G>A respectively). Functional studies in human lens epithelial cells revealed that the RRAGA mutations exerted deleterious effects on mTORC1 signaling, including increased relocation of RRAGA to the lysosomes, up-regulated mTORC1 phosphorylation, down-regulated autophagy, altered cell growth or compromised promoter activity. These data indicate that the RRAGA mutations, associated with autosomal dominant cataracts, play a role in the disease by acting through disruption of mTORC1 signaling.

CircSlc17a5 controlled by VLDLR/QKI pathway regulated the choroidal angiogenesis.

RATIONALE: Very-low-density lipoprotein receptor (VLDLR) involves in ocular neovascularization, a major cause of severe vision loss. However, the underlying molecular mechanisms were not completely clarified. Here, we aimed to investigate roles of circular RNAs (circRNAs) in VLDLR-associated ocular neovascularization. METHODS: Vldlr knockout (Vldlr-/-, ko), Robo4 knockout (Robo4-/-, ko) and wild-type (WT) mice were used. Mouse model of oxygen induced retinopathy (OIR) and high-throughput sequence were performed to profile the differential expression of circRNA and transcripts. RNase R treatment, Sanger PCR sequencing and quantitative polymerase chain reaction (qPCR) were used to validate candidate circRNAs and their expression patterns. Choroidal sprouting assay ex vivo and laser induction choroid neovascularization were used to determine the expression and functions of QKI/CircSlc17a5 on choroidal neovascularization. RESULTS: In macrophage and ocular tissues derived from Vldlr (Vldlr-/-,Vldlr ko) or Robo4 (Robo4-/-,Robo4 ko) deficiency as well as wild-type (WT) mice, Quaking (Qki) expression was significantly down-regulated in Vldlr deficiency compared to WT and Robo4 deficiency groups. Ectopic VLDLR expression or Reelin stimulation increased expression of QKI in bEnd.3 cells. Circular RNA sequencing uncovered that VLDLR regulated the biogenesis of certain circular RNAs, including the circSlc17a5. The number of Circular RNAs increased in mice treated with OIR. QKI mediated the biogenesis of circSlc17a5, which was an important regulator of choroidal angiogenesis. CONCLUSION: CircSlc17a5 regulated by VLDLR/QKI plays important roles in the choroidal angiogenesis.

Peritoneal macrophages attenuate retinal ganglion cell survival and neurite outgrowth.

Inflammation is a critical pathophysiological process that modulates neuronal survival in the central nervous system after disease or injury. However, the effects and mechanisms of macrophage activation on neuronal survival remain unclear. In the present study, we co-cultured adult Fischer rat retinas with primary peritoneal macrophages or zymosan-treated peritoneal macrophages for 7 days. Immunofluorescence analysis revealed that peritoneal macrophages reduced retinal ganglion cell survival and neurite outgrowth in the retinal explant compared with the control group. The addition of zymosan to peritoneal macrophages attenuated the survival and neurite outgrowth of retinal ganglion cells. Conditioned media from peritoneal macrophages also reduced retinal ganglion cell survival and neurite outgrowth. This result suggests that secretions from peritoneal macrophages mediate the inhibitory effects of these macrophages. In addition, increased inflammation- and oxidation-related gene expression may be related to the enhanced retinal ganglion cell degeneration caused by zymosan activation. In summary, this study revealed that primary rat peritoneal macrophages attenuated retinal ganglion cell survival and neurite outgrowth, and that macrophage activation further aggravated retinal ganglion cell degeneration. This study was approved by the Animal Ethics Committee of the Joint Shantou International Eye Center of Shantou University and the Chinese University of Hong Kong, Shantou, Guangdong Province, China, on March 11, 2014 (approval no. EC20140311(2)-P01).

CXCL5/CXCR2 modulates inflammation-mediated neural repair after optic nerve injury.

BACKGROUND: Previous studies reported that mild inflammation promotes retinal ganglion cell (RGC) survival and axonal regeneration after optic nerve (ON) injury with involvement of infiltrating macrophages and neutrophils. Here we aimed to evaluate the involvement and regulation of the main inflammatory chemokine pathway CXCL5/CXCR2 in the inflammation-mediated RGC survival and axonal regeneration in mice after ON injury. METHODS: The expressions and cellular locations of CXCL5 and CXCR2 were confirmed in mouse retina. Treatment effects of recombinant CXCL5 and CXCR2 antagonist SB225002 were studied in the explant culture and the ON injury model with or without lens injury. The number of RGCs, regenerating axons, and inflammatory cells were determined, and the activation of Akt andSTAT3 signaling pathways were evaluated. RESULTS: Cxcr2 and Cxcl5 expressions were increased after ON and lens injury. Addition of recombinant CXCL5 promoted RGC survival and neurite outgrowth in retinal explant culture with increase in the number of activated microglia, which was inhibited by SB225002 or clodronate liposomes. Recombinant CXCL5 also alleviated RGC death and promoted axonal regeneration in mice after ON injury, and promoted the lens injury-induced RGC protection with increase in the number of activated CD68+ cells. SB225002 inhibited lens injury-induced cell infiltration and activation, and attenuated the promotion effect on RGC survival and axonal regeneration through reduction of lens injury-induced Akt activation. CONCLUSIONS: CXCL5 promotes RGC survival and axonal regeneration after ON injury and further enhances RGC protection induced by lens injury with CD68+ cell activation, which is attenuated by CXCR2 antagonist. CXCL5/CXCR2 could be a potential therapeutic target for RGC survival promotion after ON injury.

ROBO4 deletion ameliorates PAF-mediated skin inflammation via regulating the mRNA translation efficiency of LPCAT1/LPCAT2 and the expression of PAF receptor.

The diminished level of platelet-activating factor acetylhydrolase (PAFAH) in milk causes an enhanced level of platelet activating factor (PAF) in the skin, leading to a severe hair loss phenotype during neonatal pup's lactation. The deletion of very-low-density-lipoprotein receptor (VLDLR) prevents the expression and secretion of PAFAH. Here we revealed that deletion of Roundabout 4 (ROBO4) in mice ameliorated hair loss phenotype via reducing PAF concentration in skin. As a consequence, the neonatal pups with ROBO4 deletion lactated by mother with VLDLR deletion showed normal hair phenotype during lactation. In details,ROBO4 deletion reduced the protein but not mRNA expression of two PAF synthetic enzymes LPCAT1/LPCAT2 in macrophage as well as the expression of PAF receptor in both macrophage and ocular tissue, but increased PAFAH protein in serum. On the other hand, RNA expression profile analysis in macrophages revealed that the genes involving in oxidative phosphorylation and ribosome obviously decreased their expression in response to ROBO4 deletion. Moreover, through High Performance Liquid Chromatography (HPLC) analysis, we found that ATP concentration also reduced in ROBO4 deletion macrophages. Because ribosome and energy are very important factors for the mRNA translation, we then tested whether ROBO4 deletion affects LPCAT1/LPCAT2 mRNA translation using polyribosome assay. As expected, the mRNA level of LPCAT1/LPCAT2 significantly decreased in polyribosome in ROBO4 deletion macrophage comparing to that of wild type. Additionally, mice with ROBO4 deletion suppressed LPS-induced IL-6 expression as well as the phosphorylation of p44/42 and p65, but enhanced the AKT phosphorylation. Collectively, ROBO4 deletion alleviates PAF- and LPS-mediated inflammation. And above results also indicate PAF signal might be a crosstalk point of ROBO4- and VLDLR-activated pathways.

Expression of SARS-CoV-2 receptor ACE2 and TMPRSS2 in human primary conjunctival and pterygium cell lines and in mouse cornea.

PURPOSE: To determine the expressions of SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) and type II transmembrane serine protease (TMPRSS2) genes in human and mouse ocular cells and comparison to other tissue cells. METHODS: Human conjunctiva and primary pterygium tissues were collected from pterygium patients who underwent surgery. The expression of ACE2 and TMPRSS2 genes was determined in human primary conjunctival and pterygium cells, human ocular and other tissue cell lines, mesenchymal stem cells as well as mouse ocular and other tissues by reverse transcription-polymerase chain reaction (RT-PCR) and SYBR green PCR. RESULTS: RT-PCR analysis showed consistent expression by 2 ACE2 gene primers in 2 out of 3 human conjunctival cells and pterygium cell lines. Expression by 2 TMPRSS2 gene primers could only be found in 1 out of 3 pterygium cell lines, but not in any conjunctival cells. Compared with the lung A549 cells, similar expression was noted in conjunctival and pterygium cells. In addition, mouse cornea had comparable expression of Tmprss2 gene and lower but prominent Ace2 gene expression compared with the lung tissue. CONCLUSION: Considering the necessity of both ACE2 and TMPRSS2 for SARS-CoV-2 infection, our results suggest that conjunctiva would be less likely to be infected by SARS-CoV-2, whereas pterygium possesses some possibility of SARS-CoV-2 infection. With high and consistent expression of Ace2 and Tmprss2 in cornea, cornea rather than conjunctiva has higher potential to be infected by SARS-CoV-2. Precaution is necessary to prevent possible SARS-CoV-2 infection through ocular surface in clinical practice.

Green Tea Extract Ameliorates Ischemia-Induced Retinal Ganglion Cell Degeneration in Rats.

PURPOSE: Oxidative stress induced by reduced blood circulation is a critical pathological damage to retinal ganglion cells (RGCs) in glaucoma. We previously showed that green tea extract (GTE) and its catechin constituents alleviate sodium iodate-induced retinal degeneration in rats. Here, we investigated the therapeutic effect of GTE on ischemia-induced RGC degeneration in rats. METHODS: RGC degeneration was induced by ischemic reperfusion in adult Fischer F344 rats. Green tea extract (Theaphenon E) was intragastrically administered 4 times within 48 hours after ischemia. RGC survival, pupillary light reflex, expressions of cell apoptosis, oxidative stress, and inflammation-related proteins were studied. RESULTS: Ischemic reperfusion significantly induced apoptotic RGCs, RGC loss, and larger constricted pupil area compared to the untreated normal rats. Expressions of activated caspase-3 and caspase-8, Sod2, and inflammation-related proteins as well as p38 phosphorylation were significantly upregulated in the ischemia-injured rats. Compared to the saline-fed ischemic rats, significantly higher number of surviving RGCs, less apoptotic RGCs, and smaller constricted pupil area were observed in the GTE-fed ischemic rats. GTE also reduced the increased protein expressions caused by ischemic injury but enhanced the Jak phosphorylation in the retina. Notably, green tea extract did not affect the survival of RGCs in the uninjured normal rats. CONCLUSIONS: In summary, GTE offers neuroprotection to RGCs under ischemic challenge, suggesting a potential therapeutic strategy for glaucoma and optic neuropathies.

Induction of mast cell accumulation by chymase via an enzymatic activity- and intercellular adhesion molecule-1-dependent mechanism.

BACKGROUND AND PURPOSE: Chymase is a unique, abundant secretory product of mast cells and a potent chemoattractant for eosinophils, monocytes and neutrophils, but little is known of its influence on mast cell accumulation. EXPERIMENTAL APPROACH: A mouse peritoneal inflammation model, cell migration assay and flowcytometry analysis, were used to investigate the role of chymase in recruiting mast cells. KEY RESULTS: Chymase increased, by up to 5.4-fold, mast cell numbers in mouse peritoneum. Inhibitors of chymase, heat-inactivation of the enzyme, sodium cromoglycate and terfenadine, and pretreatment of mice with anti-intercellular adhesion molecule 1, anti-L-selectin, anti-CD11a and anti-CD18 antibodies dramatically diminished the chymase-induced increase in mast cell accumulation. These findings indicate that this effect of chymase is dependent on its enzymatic activity and activation of adhesion molecules. In addition, chymase provoked a significant increase in 5-HT and eotaxin release (up to 1.8- and 2.2-fold, respectively) in mouse peritoneum. Since 5-HT, eotaxin and RANTES can induce marked mast cell accumulation, these indirect mechanisms may also contribute to chymase-induced mast cell accumulation. Moreover, chymase increased the trans-endothelium migration of mast cells in vitro indicating it also acts as a chemoattractant. CONCLUSION AND IMPLICATIONS: The finding that mast cells accumulate in response to chymase implies further that chymase is a major pro-inflammatory mediator of mast cells. This effect of chymase, a major product of mast cell granules, suggests a novel self-amplification mechanism for mast cell accumulation in allergic inflammation. Mast cell stabilizers and inhibitors of chymase may have potential as a treatment of allergic disorders.

[Increase expression of neurotrophins mRNA in peripheral blood of patients with allergic rhinitis].

OBJECTIVE: To assess the expression of NGF, BDNF, NT-3 mRNA in the peripheral blood of patients with allergic rhinitis (AR). Meanwhile, to analysis whether the expression of NGF, BDNF, NT-3 mRNA correlate with the severity of rhinitis. METHOD: This study is a group controlled trial, which takes the healthy adults as control group. The total RNA have been extracted from the peripheral blood of AR patients. The expression of NGF, BDNF and NT-3 mRNA have been tested by real-time quantitative RT-PCR. RESULT: Comparing with the healthy adults, the expression of NGF, BDNF and NT-3 mRNA as 2(-deltadeltaCt) are 2.436 8, 4.4588 and 1.781 8 respectively. The increasing expression of NT-3 correlated positively with the scores of visual analog scale. CONCLUSION: The expression of NGF, BDNF and NT-3 mRNA are as high as 2.4368, 4.4588 and 1.7818 times to healthy adults. We propose NGF, BDNF and NT-3 may contribute to the pathogenesis of AR. NT-3 could reflect the severity of rhinitis as a molecular biological index.

Expression of Sirt1 and Sirt2 in injured optic retina of calorie restricted rats.

PURPOSE: To investigate retinal ganglion cell (RGC) survival after optic nerve injury in calorie-restricted (CR) rats, and analyze the potential role of Sirtuins. METHODS: Ten-month old male Sprague-Dawley rats (n=14) were divided into calorie restricted (CR) and ad libitum (AL) groups. In the CR group (n=7), the rats were denied access to food every other day. Animals in the AL group (n=7) had had free access to food. PN-ON grafting was carried out on the right eyes of all subjects after 5 months of feeding. Three weeks postoperatively, retina samples were collected, half of which were fixed in 4% paraformaldehyde (PFA) and subjected to standard immunofluorescence staining for TUJ-1. The remaining samples were subjected to total RNA analysis and RT-PCR for Sirt1 and 2 expression. RESULTS: Comparing the amount of TUJ-1 staining between the groups, the mean density and the total number of RGCs showed no significant difference. RT-PCR results indicated that mRNA expression of Sirtuin2 in the CR group was significantly lower than that in the AL group, whereas no statistically-significant difference was observed between the two groups regarding the mRNA expression of Sirt1. CONCLUSION: Calorie restriction had no impact on the survival of injured RGCs. The down-regulated mRNA expression of Sirt2 in the CR group may indicate an improved capacity for regeneration among these animals, but more work is needed to explore this possibility.