To investigate macular perfusion in healthy Chinese individuals and examine its dependence on age and sex.
Healthy adult Chinese individuals were recruited. Macular perfusion was measured by ...spectral-domain optical coherence tomography (OCT) using the split-spectrum amplitude-decorrelation angiography (SSADA) algorithm. The parafoveal flow index and vessel area density as well as the area of the foveal capillary-free zone (CFZ) were quantified.
A total of 76 eyes in 45 subjects were included (20 males and 25 females, mean age 36 ± 11 years). The mean parafoveal flow index was 0.099 ± 0.013; the mean vessel area density was 0.891 ± 0.073; and the mean CFZ area was 0.474 ± 0.172 mm2. All three parameters were significantly correlated with age (flow index: P = 0.00; vessel area density: P = 0.00; CFZ area: P = 0.02). The flow index and vessel area density decreased annually by 0.6% and 0.4%, respectively, and CFZ area increased by 1.48% annually. The CFZ area was larger in females than in males, while all three parameters seemed to change more rapidly with age in males than in females.
In healthy Chinese eyes, macular perfusion decreased with increasing age, and decreased more rapidly in males than in females. The application of OCT angiograms may provide a useful approach for monitoring macular perfusion, although caution must be exercised with regard to age- and sex-related variations.
Pathological stimuli cause mitochondrial damage and leakage of mitochondrial DNA (mtDNA) into the cytosol, as demonstrated in many cell types. The cytosolic mtDNA then drives the activation of ...noninfectious inflammation. Retinal microvascular endothelial cells (RMECs) play an important role in the inner endothelial blood-retinal barrier (BRB). RMEC dysfunction frequently occurs in posterior-segment eye diseases, causing loss of vision. In this study, we investigated the involvement of cytosolic mtDNA in noninfectious immune inflammation in RMECs under pathological stimuli.
RMECs were stimulated with 100 ng/ml lipopolysaccharide (LPS), 200 μM hydrogen peroxide (H
O
), or 25 mM D-glucose. After 24 h, immunofluorescent staining was used to detect the opening of the mitochondrial permeability transition pore (MPTP). Cytosolic mtDNA was detected with immunofluorescent staining and PCR after stimulation. mtDNA was then isolated and used to transfect RMECs in vitro, and the protein levels of cGAS were evaluated with western blotting. Real-time PCR was used to examine cGAS mRNA expression levels at different time points after mtDNA stimulation. The activation of STING was detected with immunofluorescent staining 6 h after mtDNA stimulation. Western blotting was used to determine the expression of STING and IFNβ, the phosphorylation status of TBK1, IRF3, and nuclear factor-κB (NF-κB) P65, and the nuclear translocation of IRF3 and NF-κB P65 at 0, 3, 6, 12, and 24 h. The mRNA expression of proinflammatory cytokines CCL4, CXCL10, and IFNB1, and transcription factor IRF1 were determined with real-time PCR, together with the concentrations of intercellular adhesion molecule 1 (ICAM-1) mRNA.
Pathological stimuli caused mtDNA to leak into the cytosol by opening the MPTP in RMECs after 24 h. Cytosolic mtDNA regulated the expression of cGAS and the distribution of STING in RMECs. It promoted ICAM-1, STING and IFNβ expression, TBK1, IRF3, and NF-κB phosphorylation and the nuclear translocation in RMECs at 12 and 24 h after its transfection. The mRNAs of proinflammatory cytokines CCL4, CXCL10, and IFNB1, and transcription factor IRF1 were significantly elevated at 12 and 24 h after mtDNA stimulation.
Pathological stimulation induces mtDNA escape into the cytosol of RMECs. This cytoplasmic mtDNA is recognized by the DNA sensor cGAS, increasing the expression of inflammatory cytokines through the STING-TBK1 signaling pathway. Video Abstract. (MP4 37490 kb).
To investigate the relationship between retinal perfusion and retinal thickness in the peripapillary and macular areas of healthy subjects.
Using spectral-domain optic coherence tomography and ...split-spectrum amplitude decorrelation angiography (SSADA) algorithm, retinal perfusion and retinal thicknesses in the macular and peripapillary areas were measured in healthy volunteers, and correlations among these variables were analyzed.
Overall, 64 subjects (121 eyes) including 28 males and 36 females with a mean ± SD age of 38 ± 13 years participated. Linear mixed-models showed that vessel area density was significantly correlated with the inner retinal thickness (from the inner limiting membrane to the outer border of the inner nucleus layer; P < 0.05), but not with the thickness of the full retina (P > 0.05) in the parafoveal area. The area of the foveal capillary-free zone was negatively correlated with the inner and full foveal thicknesses (all P < 0.001). In the peripapillary area, the vessel area density was positively correlated with the thickness of the retinal nerve fiber layer (P < 0.001).
In healthy subjects, retinal perfusion in small vessels was closely correlated with the thickness of the inner retinal layers in both the macular and peripapillary areas.
Attention is increasingly being given to microglia-related inflammation in neovascular diseases, such as diabetic retinopathy and age-related macular disease. Evidence shows that activated microglia ...contribute to disruption of the blood-retinal barrier, however, the mechanism is unclear. In this study, we aimed to clarify whether and how microglia affect the function of retinal microvascular endothelial cells (RMECs).
We activated microglia by Lipopolysaccharides (LPS) stimulation. After co-culturing static or activated microglia with RMECs using the Transwell system, we evaluated the function of RMECs. Vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor-BB (PDGF-BB) levels in the supernatant from the lower chamber were evaluated by ELISA. Angiogenesis, migration, and proliferation of RMECs were assessed by tube formation, wound healing, and WST-1 assays. The expression levels of tight junction proteins (ZO-1 and occludin) and endothelial markers (CD31 and CD34) were examined by Western blot analysis.
We successfully established an LPS-activated microglia model and co-culture system of static or activated microglia with RMECs. In the co-culture system, we showed that microglia, especially activated microglia stimulated VEGF-A and PDGF-BB expression, enhanced angiogenesis, migration, proliferation, and permeability, and altered the phenotype of co-cultured RMECs.
Microglia, especially activated microglia, play important roles in angiogenesis and maintenance of vascular function hemostasis in the retinal microvasculature. The mechanism needs further investigation and clarification.
The aim of the present study was to investigate the neuroprotective effects of glucocorticoid-induced leucine zipper (GILZ) in a light-induced retinal degeneration model and to explore the underlying ...mechanisms.
Intravitreal injection of recombinant GILZ-overexpressing lentivirus (OE-GILZ-rLV) and short hairpin RNA targeting GILZ recombinant lentivirus (shRNA-GILZ-rLV) was performed to up- and downregulate retinal GILZ, respectively. Three days after stable transduction, rats were exposed to continuous bright light (5000 lux) for 2 days. Retinal function was assessed by full-field electroretinography (ERG), and the retinal structure was examined for photoreceptor survival and death in rats kept under a 12-hour light:2-hour dark cycle following light exposure. The expression levels of retinal Bcl-xL, caspase-9, and caspase-3 were examined by Western blotting or real-time PCR at 1, 3, 5, and 7 days after light exposure.
Exposure to bright light downregulated retinal GILZ in parallel with the downregulation of Bcl-xL and the upregulation of active caspase-3. Overexpression of retinal GILZ attenuated the decrease of Bcl-xL and the activation of caspase-9 and caspase-3 at 1, 3, 5, and 7 days after bright light exposure, respectively. GILZ silencing aggravated the downregulation of Bcl-xL induced by bright light exposure. Bright light exposure reduced the amplitude of ERG, increased the number of apoptotic photoreceptor cells, and decreased retinal thickness; and GILZ overexpression could attenuate all these effects.
Overexpression of GILZ by OE-GILZ-rLV transduction protected the retina from light-induced cellular damage by activating antiapoptotic pathways.
: Aberrant neovascularization resulting from inappropriate angiogenic signaling is closely related to many diseases, such as cancer, cardiovascular disease, and proliferative retinopathy. Although ...some factors involved in regulating pathogenic angiogenesis have been identified, the molecular mechanisms of proliferative retinopathy remain largely unknown. In the present study, we determined the role of platelet-derived growth factor-B (PDGF-B), one of the HIF-1-responsive gene products, in cell proliferation and angiogenesis in retinal microvascular endothelial cells (RMECs) and explored its regulatory mechanism.
: Cell counting kit-8 (CCK-8), bromodeoxyuridine (BrdU) incorporation, tube formation, cell migration, and Western blot assays were used in our study.
: Our results showed that PDGF-B promoted cell proliferation and angiogenesis by increasing the activity of Src homology 2 domain-containing tyrosine phosphatase 2 (SHP-2) in RMECs, which was attenuated by the inhibition of PDGF receptor (PDGFR) or SHP-2 knockdown. Moreover, activation of c-Myc was involved in the processes of PDGF-B/SHP-2-driven cell proliferation in RMECs. The promoting effects of PDGF-B/SHP-2 on c-Myc expression were mediated by the Erk pathway.
: These results indicate that PDGF-B facilitates cell proliferation and angiogenesis, at least in part,
the SHP-2/Erk/c-Myc pathway in RMECs, implying new potential treatment candidates for retinal microangiopathy.
Lipocalin 2 (LCN2), an important mediator of a variety of cellular processes, is involved in regulating the inflammatory response, but its roles in different inflammatory diseases are controversial. ...Because the role of LCN2 in ocular inflammation has been unclear until now, we explored the function of LCN2 in lipopolysaccharide (LPS)-induced ocular inflammation in vivo and in vitro.
Endotoxin-induced uveitis (EIU) was induced in male Sprague Dawley rats by the intravitreal injection of LPS. The expression and location of LCN2 in the retina were detected with western blotting and immunohistochemistry, respectively. We determined the clinical scores for anterior inflammation, quantified the infiltrated inflammatory cells, and measured the pro-inflammatory factors to determine the anti-inflammatory effects of LCN2 in EIU eyes. Cultured primary rat Müller cells were stimulated with LPS and the expression and secretion of LCN2 were measured with real-time PCR, western blotting, and an ELISA. After Müller cells were cotreated with LPS and LCN2 or PBS, the expression and secretion of TNF-α, IL-6, and MCP-1 were examined with realtime PCR, western blotting, and ELISAs. Western blotting and immunofluorescence were used to detect the phosphorylation and cellular distribution of nuclear factor kappaB (NF-κB) subunit p65.
In EIU, the expression of LCN2 was significantly upregulated in the retina, especially in the outer nuclear layer (mainly composed of Müller cells). LPS stimulation of cultured Müller cells also markedly elevated LCN2 expression. Intravitreal injection of LCN2 significantly reduced the clinical scores, inflammatory infiltration, and protein leakage in EIU, which correlated with the reduced levels of proinflammatory factors in the aqueous humor and retina. LCN2 treatment also reduced the expression and secretion of TNF-α, IL-6, and MCP-1 in LPS-stimulated Müller cells. LCN2 inhibited the inflammatory response by inhibiting the phosphorylation and translocation of NF-κB p65.
LCN2 protects against ocular inflammation, at least in part, by negatively regulating the activation of the NF-κB signaling pathway. LCN2 may be a promising anti-inflammatory therapy for ocular diseases, such as uveitis.
Retinal neurodegeneration is induced by a variety of environmental insults and stresses, but the exact mechanisms are unclear. In the present study, we explored the involvement of cytosolic ...mitochondrial DNA (mtDNA), resulting in the cGAS-STING dependent inflammatory response and apoptosis in retinal damage in vivo.
Retinal injury was induced with white light or intravitreal injection of lipopolysaccharide (LPS). After light- or LPS-induced injury, the amount of cytosolic mtDNA in the retina was detected by PCR. The mtDNA was isolated and used to transfect retinas in vivo. WB and real-time PCR were used to evaluate the activation of cGAS-STING pathway and the levels of apoptosis-associated protein at different times after mtDNA injection. Retinal cell apoptosis rate was detected by TUNEL staining. Full-field electroretinography (ERG) was used to assess the retinal function.
Light injury and the intravitreal injection of LPS both caused the leakage of mtDNA into the cytoplasm in retinal tissue. After the transfection of mtDNA in vivo, the levels of cGAS, STING, and IFN-β mRNAs and the protein levels of STING, phosph-TBK1, phospho-IRF3, and IFN-β were upregulated. mtDNA injection also induced the activation of caspase 3 and caspase 9. BAX and BAK were increased at both the mRNA and protein levels. The release of cytochrome c from the mitochondria to the cytosol was increased after mtDNA injection. The wave amplitudes on ERG decreased and retinal cell apoptosis was detected after mtDNA injection.
Cytosolic mtDNA triggers an inflammatory response. It also promotes apoptosis and the dysfunction of the retina.
The anti-inflammatory activities of protein glucocorticoid-induced leucine zipper (GILZ) have been demonstrated
and
. Here, we examined the potential effect of a synthetic peptide derived from the ...leucine zipper motif and proline-rich region of GILZ on suppressing inflammatory responses in primary cultured rat Müller cells.
Peptides were selected from amino acids 98-134 of the GILZ protein (GILZ-p). Solid-phase peptide synthesis was used to generate the cell-penetrating peptide TAT, which was bound to the amino terminus of GILZ-p. Primary cultured retinal Müller cells were stimulated with lipopolysaccharide (LPS) alone or in combination with different concentrations of GILZ-p, and the interaction of GILZ-p with nuclear factor (NF)-κB p65 in Müller cells was investigated by western blotting, immunoprecipitation, and immunofluorescence. The expression of the Müller cell gliosis marker glial fibrillary acidic protein (GFAP), functional protein aquaporin (AQP)-4, and the inflammatory cytokines interleukin (IL)-1β, tumor necrosis factor (TNF) α, intercellular adhesion molecule (ICAM)-1, and monocyte chemoattractant protein (MCP)-1 was measured by Western Blotting. The concentration of those cytokines in culture medium was measured by using Enzyme-Linked Immunosorbent Assay.
The synthesized GILZ-p, which was water-soluble, entered cells and bound with NF-κB p65, inhibiting p65 nuclear translocation. GILZ-p inhibited the LPS-induced expression of GFAP, IL-1β, TNFα, ICAM-1, and MCP-1 in Müller cells and prevented the LPS-induced downregulation of AQP4.
These results indicate that GILZ-p interacted with NF-κB p65 and suppressed p65 nuclear translocation, thereby inhibiting inflammatory cytokine release and Müller cell gliosis.
Numerous studies have suggested that the integrity of the cone interdigitation zone (IZ) could be considered to be a marker of photoreceptor damage and its recovery. However, little is known about ...the IZ in healthy eyes. Our present study was to measure the cone IZ area by optical coherence tomography (OCT), and determine its distribution in healthy adults.
This was a cross-sectional non-interventional study. We involved a group of 158 emmetropic or low myopic (from -3D to + 0.5D) eyes in 97 healthy adult volunteers. All subjects underwent thorough ophthalmologic examinations and the posterior pole was scanned by OCT. The cone IZ area in healthy adults and its correlation with macular volume and other factors was analyzed.
The cone IZ was visible and clear in all 158 eyes, and the IZ area was successfully measured by 6 radical scans centered on the fovea. The mean IZ area was 30.22 ± 12.70 mm
, and ranged from 5.91 to 57.47 mm
. The IZ area exhibited a normal distribution (P = 0.635) with 95% confidence interval of 28.06-32.29 mm
. The IZ area was significantly correlated with the retinal and outer nuclear layer (ONL) volumes within the macula.
The cone IZ area could be measured using a commercially available OCT system. The IZ area showed high variability among healthy adults, and this might be related to the variability in the photoreceptor distribution in healthy adults.
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