Lingling Yang

Substance P promotes diabetic corneal epithelial wound healing through molecular mechanisms mediated via the neurokinin-1 receptor

Substance P (SP) is a neuropeptide, predominantly released from sensory nerve fibers, with a potentially protective role in diabetic corneal epithelial wound healing. However, the molecular mechanism remains unclear. We investigated the protective mechanism of SP against hyperglycemia-induced corneal epithelial wound healing defects, using type 1 diabetic mice and high glucose–treated corneal epithelial cells. Hyperglycemia induced delayed corneal epithelial wound healing, accompanied by attenuated corneal sensation, mitochondrial dysfunction, and impairments of Akt, epidermal growth factor receptor (EGFR), and Sirt1 activation, as well as decreased reactive oxygen species (ROS) scavenging capacity. However, SP application promoted epithelial wound healing, recovery of corneal sensation, improvement of mitochondrial function, and reactivation of Akt, EGFR, and Sirt1, as well as increased ROS scavenging capacity, in both diabetic mouse corneal epithelium and high glucose–treated corneal epithelial cells. The promotion of SP on diabetic corneal epithelial healing was completely abolished by a neurokinin-1 (NK-1) receptor antagonist. Moreover, the subconjunctival injection of NK-1 receptor antagonist also caused diabetic corneal pathological changes in normal mice. In conclusion, the results suggest that SP-NK-1 receptor signaling plays a critical role in the maintenance of corneal epithelium homeostasis, and that SP signaling through the NK-1 receptor contributes to the promotion of diabetic corneal epithelial wound healing by rescued activation of Akt, EGFR, and Sirt1, improvement of mitochondrial function, and increased ROS scavenging capacity.

Overexpression of SIRT1 promotes high glucose-attenuated corneal epithelial wound healing via p53 regulation of the IGFBP3/IGF-1R/AKT pathway.

PURPOSE To investigate how Sirtuin (silent mating type information regulation 2 homolog) 1 (SIRT1) promotes high glucose-attenuated corneal epithelial wound healing. METHODS The effects of high glucose on SIRT1 expression were assessed in primary human corneal epithelial cells (CECs) in treatment of 5 mM d-glucose (normal glucose [NG]) and 25 mM D-glucose (high glucose [HG]) and corneas from Ins2(Akita/+) mice by Western blotting. The osmotic pressure of the NG medium was adjusted to that of the HG medium by adding 20 mM mannitol. Pifithrin-α (PFT-α) was used to inhibit the expression of p53 and an adenovirus was used for overexpression of SIRT1 in vivo and in vitro. How overexpression of SIRT1 promotes HG-attenuated corneal epithelial wound healing via p53 regulation of the IGFBP3 (insulin-like growth factor binding protein-3)/IGF-1 (insulin-like growth factor-1)/AKT pathway was investigated in CECs and Ins2(Akita/+) mice. RESULTS HG induced the downregulation of SIRT1 and the upregulation of p53 acetylation in primary human CECs and corneas from Ins2(Akita/+) mice. The results of cell migration assay and corneal wound healing from Ins2(Akita/+) mice demonstrated that SIRT1 overexpression strongly promoted wound healing in the presence of HG levels via the downregulation of the IGFBP3 protein. The levels of total p53 expression and acetylated p53 decreased dramatically in the presence of PFT-α, whereas the IGF-1R/AKT pathway was activated in CECs. The results of cell migration assay suggested this posttranslational modification of p53 was involved in the response to cell injury under HG conditions in CECs. CONCLUSIONS The molecular mechanism by which SIRT1 promotes corneal epithelial wound healing was involved in an enhancement of the IGFBP3/IGF-1/AKT pathway through the deacetylation of p53. This study also suggests that SIRT1 has a protective role in the pathogenesis of diabetic keratopathy.

Trichostatin A inhibits transforming growth factor-β-induced reactive oxygen species accumulation and myofibroblast differentiation via enhanced NF-E2-related factor 2-antioxidant response element signaling.

Trichostatin A (TSA) has been shown to prevent fibrosis in vitro and in vivo. The present study aimed at investigating the role of reactive oxygen species (ROS) scavenging by TSA on transforming growth factor-β (TGF-β)-induced myofibroblast differentiation of corneal fibroblasts in vitro. Human immortalized corneal fibroblasts were treated with TGF-β in the presence of TSA, the NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI), the antioxidant N-acetyl-cysteine (NAC), the NF-E2–related factor 2-antioxidant response element (Nrf2-ARE) activator sulforaphane, or small interfering RNA. Myofibroblast differentiation was assessed by α-smooth muscle actin (α-SMA) expression, F-actin bundle formation, and collagen gel contraction. ROS, H2O2, intracellular glutathione (GSH) level, cellular total antioxidant capacity, and the activation of Nrf2-ARE signaling were determined with various assays. Treatment with TSA and the Nrf2-ARE activator resulted in increased inhibition of the TGF-β–induced myofibroblast differentiation as compared with treatment with DPI or NAC. Furthermore, TSA also decreased cellular ROS and H2O2 accumulation induced by TGF-β, whereas it elevated intracellular GSH level and cellular total antioxidant capacity. In addition, TSA induced Nrf2 nuclear translocation and up-regulated the expression of Nrf2-ARE downstream antioxidant genes, whereas Nrf2 knockdown by RNA interference blocked the inhibition of TSA on myofibroblast differentiation. In conclusion, this study provides the first evidence implicating that TSA inhibits TGF-β–induced ROS accumulation and myofibroblast differentiation via enhanced Nrf2-ARE signaling.

TGFβ mediated transition of corneal fibroblasts from a proinflammatory state to a profibrotic state through modulation of histone acetylation

Corneal fibroblasts exhibit different phenotypes in different phases of corneal wound healing. In the inflammatory phase, the cells assume a proinflammatory phenotype and produce large amounts of cytokines and chemokines, but in the proliferative and remodeling phases, they adapt a profibrotic state, differentiate into myofibroblasts and increase extracellular matrix protein synthesis, secretion, and deposition. In the present study, the molecular mechanisms regulating the transition of corneal fibroblasts from the proinflammatory state to the profibrotic state were investigated. Corneal fibroblasts were treated with TGFβ, a known profibrotic and anti‐inflammatory factor in wound healing, in the absence or presence of trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor. The results revealed that TGFβ induced the profibrotic transition of corneal fibroblasts, including increased extracellular matrix synthesis, morphological changes, and assembly of actin filaments. Meanwhile, proinflammatory gene expressions of corneal fibroblasts were down‐regulated with the treatment of TGFβ, as confirmed by cDNA microarray, real time PCR and ELISA. Moreover, TSA reversed the TGFβ‐mediated transition of corneal fibroblasts from the proinflammatory state to the profibrotic state, as accompanied by histone hyperacetylations. In conclusion, TGFβ suppressed the production of proinflammatory factors and enhanced the expression of matrix remodeling genes of corneal fibroblasts in the transition from the proinflammatory state to the profibrotic state, and the dual roles of TGFβ on the phenotype regulations of corneal fibroblasts were mediated by altered histone acetylation. J. Cell. Physiol. 224:135–143, 2010

Low Concentration of S100A8/9 Promotes Angiogenesis-Related Activity of Vascular Endothelial Cells: Bridges among Inflammation, Angiogenesis, and Tumorigenesis?

Previous studies showed that several members of the S100A family are involved in neovascularization and tumor development. This study checked whether low concentrations of S100A8 or S100A9 has any effect on the behaviour of vascular endothelial cells. A human umbilical vascular endothelial cell (HUVEC) line was used to measure vascular endothelial cell bioactivity related to angiogenesis, such as cell proliferation, migration, and vessel formation. In the low concentration range up to 10 μg/mL, either each alone or in combination, S100A8 and S100A9 proteins promoted proliferation of HUVEC cells in a dose-dependent manner. The presence of both proteins in culture showed additive effects over each single protein. Both proteins enhanced HUVEC cells to migrate across the transwell membrane and to form tube-like structures on the Matrigel surface. When mixed in Matrigel and injected subcutaneously in Balb/c mice, both proteins increased vessel development in the gel plugs. Microarray assay of HUVEC cells treated with 10 μg/mL S100A8 revealed that ribosome pathway, pathogenic Escherichia coli infection pathway, apoptosis, and stress response genes were modulated by S100A8 treatment. We propose that S100A8 and S100A9 proteins from either infiltrating inflammatory cells or tumor cells play an important role in the interplay among inflammation, angiogenesis, and tumorigenesis.

Nicotine alters morphology and function of retinal pigment epithelial cells in mice.

To study the effects of nicotine on retinal pigment epithelium (RPE) cells in vivo and in vitro, (Balb/c×C57Bl/6) F1 mice were given water containing 100 µg/mL nicotine for six months. Cultured fetal RPE cells were treated with nicotine or lipopolysaccharide for seventy-two hours. Expression of matrix metalloproteinase protein (MMP)2, MMP9, and VEGF was measured using Western blot. Expression of IL6 and IL8 was measured using real-time polymerase chain reaction or enzyme-linked immunosorbent assay. Electron microscopy was performed to observe the effects of nicotine on morphological changes of mice retina or cultured RPE cells, and filamentous actin in RPE cells was stained with phalloidin. Electron microscopy revealed that nicotine-treated mice showed thinner outer nuclear layers, fewer pigment granules in RPE cells, and a damaged photoreceptor–RPE interface when compared with age-control mice. When added to cultured RPE cells, nicotine induced accumulation of osmiophilic lamellated intracellular inclusions in cytoplasm, mitochondrion hypertrophy and vacuolar degeneration, and redistribution of actin in cells without affecting cell proliferation. Expression of MMP2 and MMP9 in nicotine-treated RPE cells was decreased. Nicotine-induced changes in RPE morphology and function provide insight into pathogenesis of smoking-related retinal diseases.

Substance P Inhibits Hyperosmotic Stress-Induced Apoptosis in Corneal Epithelial Cells through the Mechanism of Akt Activation and Reactive Oxygen Species Scavenging via the Neurokinin-1 Receptor.

Hyperosmolarity has been recognized as an important pathological factor in dry eye leading to ocular discomfort and damage. As one of the major neuropeptides of corneal innervation, substance P (SP) has been shown to possess anti-apoptotic effects in various cells. The aim of this study was to determine the capacity and mechanism of SP against hyperosmotic stress-induced apoptosis in cultured corneal epithelial cells. The cells were exposed to hyperosmotic stress by the addition of high glucose in the presence or absence of SP. The results showed that SP inhibited hyperosmotic stress-induced apoptosis of mouse corneal epithelial cells. Moreover, SP promoted the recovery of phosphorylated Akt level, mitochondrial membrane potential, Ca2+ contents, intracellular reactive oxygen species (ROS) and glutathione levels that impaired by hyperosmotic stress. However, the antiapoptotic capacity of SP was partially suppressed by Akt inhibitor or glutathione depleting agent, while the neurokinin-1 (NK-1) receptor antagonist impaired Akt activation and ROS scavenging that promoted by SP addition. In conclusion, SP protects corneal epithelial cells from hyperosmotic stress-induced apoptosis through the mechanism of Akt activation and ROS scavenging via the NK-1 receptor.

Comparison of the therapeutic effects of extracts from Spirulina platensis and amnion membrane on inflammation-associated corneal neovascularization.

AIM To compare the therapeutic effects of polysaccharide extract from Spirulina platensis (PSP) and extract from amnion membrane (AME) on alkali burn-induced corneal neovascularization (CorNV). METHODS PSP and AME were extracted from dry powder of Spirulina platensis and human aminion membrane respectively. Murine CorNV was induced by applying 1N sodiumhydroxide (NaOH) solution directly on the mice corneas. PSP and AME extracts were administered topically on the corneas 4 times daily for 7 days. The therapy effects of PSP and AME extracts were evaluated daily using slit-lamp. At the end of the therapy, corneas were harvested for H&E staining, masson trichrome staining, immunohistochemical study, and semi-quantification reverse transcriptive PCR (RT-PCR) was utilized for measurement of inflammation-related molecules. RESULTS Topical application of PSP extract had significant therapeutic effects on CorNV that could be shown in various assays of the corneas. Compared with AME extract, PSP extract treatment was more effective in suppressing CorNV in terms of vessel length and levels of cluster of differentiation 31 (CD31) proteins or the angiogenesis related genes like vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9). PSP also inhibited inflammation more markedly by more effectively inhibiting mononuclear and polymorphonuclear cells infiltration into the corneal stroma and reducing levels of stromal cell-derived factor-1 (SDF1), tumor necrosis factor-alpha (TNFα) and macrophage inflammatory protein-3 (MIP3a). In additon, corneas of PSP group had a more regular and compact architecture of collagen with thinner corneal thickness than in the AME group. CONCLUSION Polysaccharide extract from Spirulina platensis inhibited alkali burn-induced inflammation and CorNV more effectively than AME extract at the studied doses, thus may be used for the therapy of corneal diseases involving neovascularization and inflammation.

Differentiation of human bone marrow-derived mesenchymal stem cells into neural-like cells by co-culture with retinal pigmented epithelial cells

AIM To detect the differentiation effects of retinal cells or extracts on bone marrow-derived mesenchymal stem cells (BMSC). METHODS Human fetal BMSC were previously labelled by carboxyfluorescein succinimidyl ester (CFSE), and co-cultured with retinal pigment epithelial (RPE) cells which were pre-treated with ultraviolet irradiation at a ratio of 1:1 to induce the differentiation of BMSC for up to 14 days. In some assays, a retinal extract of bovine retinal extract (BRE) was added to detect the potential effects of retinal component on the differentiation of BMSC. In addition, Neuron-specific enolase (NSE), Nestin and Glial fibrillary acidic protein (GFAP) immunostaining were performed to determine the characteristics of BMSC. RESULTS The results indicated that by co-cultured with RPE cells, fetal BMSC were differentiated into neural-like cells expressing special neuronal markers Nestin, GFAP and NSE. And the expression of these markers was obviously increased by BRE. CONCLUSION Retina derived cells and extracts can induce the differentiation of BMSC into neural-like cells.

Role of senescent fibroblasts on alkali-induced corneal neovascularization.

Cellular senescence acts as a potent regulator of tumor suppression and fibrosis limitation; however, its contribution and crosstalk with neovascularization during normal wound healing has not been examined. Here, we explored the role of senescent fibroblasts on neovascularization with a mouse model of alkali‐induced corneal wound healing. Senescent cells accumulated in corneal stroma from day 7 to 27 after alkali burn and peaked on day 14, which was consistent with the development of corneal neovascularization (CNV). In vitro and in vivo assays confirmed that the senescent cells were derived primarily from activated corneal fibroblasts. Furthermore, senescent corneal fibroblasts exhibited enhanced synthesis and secretion of extracellular matrix‐degrading enzymes (matrix metalloproteinases 2, 3, and 14 and tissue‐ and urokinase‐type plasminogen activators) and angiogenic factors (vascular endothelial growth factor) and decreased expression of anti‐angiogenic factors (pigment epithelium‐derived factor and thrombospondins), which supported the proliferation, migration, and promotion of tube formation of vascular endothelial cells. Intrastromal injection of premature senescent fibroblasts induced CNV earlier than that of normal fibroblasts, while matrix metalloproteinase inhibitors blocked the early onset of senescent cell‐induced CNV. Therefore, senescent fibroblasts promoted the alkali‐induced CNV partially via the enhanced secretion of matrix metalloproteases. J. Cell. Physiol. 227: 1148–1156, 2012.