Wenru Su

Role of Mesenchymal Stem Cells on Cornea Wound Healing Induced by Acute Alkali Burn

The aim of this study was to investigate the effects of subconjunctivally administered mesenchymal stem cells (MSCs) on corneal wound healing in the acute stage of an alkali burn. A corneal alkali burn model was generated by placing a piece of 3-mm diameter filter paper soaked in NaOH on the right eye of 48 Sprague-Dawley female rats. 24 rats were administered a subconjunctival injection of a suspension of 2×106 MSCs in 0.1 ml phosphate-buffered saline (PBS) on day 0 and day 3 after the corneal alkali burn. The other 24 rats were administered a subconjunctival injection of an equal amount of PBS as a control. Deficiencies of the corneal epithelium and the area of corneal neovascularization (CNV) were evaluated on days 3 and 7 after the corneal alkali burn. Infiltrated CD68+ cells were detected by immunofluorescence staining. The mRNA expression levels of macrophage inflammatory protein-1 alpha (MIP-1α), tumor necrosis factor-alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1) and vascular endothelial growth factor (VEGF) were analyzed using real-time polymerase chain reaction (real-time PCR). In addition, VEGF protein levels were analyzed using an enzyme-linked immunosorbent assay (ELISA). MSCs significantly enhanced the recovery of the corneal epithelium and decreased the CNV area compared with the control group. On day 7, the quantity of infiltrated CD68+ cells was significantly lower in the MSC group and the mRNA levels of MIP-1α, TNF-α, and VEGF and the protein levels of VEGF were also down-regulated. However, the expression of MCP-1 was not different between the two groups. Our results suggest that subconjunctival injection of MSCs significantly accelerates corneal wound healing, attenuates inflammation and reduces CNV in alkaline-burned corneas; these effects were found to be related to a reduction of infiltrated CD68+ cells and the down-regulation of MIP-1α, TNF-α and VEGF.

Doxycycline Enhances the Inhibitory Effects of Bevacizumab on Corneal Neovascularization and Prevents Its Side Effects

PURPOSE To investigate the combination therapeutic effects of topical doxycycline temperature-sensitive hydrogel (DTSH) and bevacizumab on corneal neovascularization (CNV) and corneal wound healing (CWH) and to explore the underlying mechanisms of doxycycline on CNV and CWH. METHODS Rats were treated with a saline solution, topical DTSH (0.1%), topical bevacizumab (2.5 mg/0.1 mL), or a DTSH and bevacizumab combination. For the bFGF-induced CNV model (n = 15/group), the length and area of CNV were measured on day 7. In the alkali burn model (n = 33/group), the length and area of CNV were determined on days 3, 7, 14, and 21 after alkali burn. The activity of matrix metalloproteinase (MMP)-2 and MMP-9 was determined by a fluorogenic peptide substrate. Western blot, real-time PCR, and ELISA were used to analyze the expression of induced nitric oxide synthase (iNOS), VEGF, VEGFRS, MMP-2, MMP-9, and IL-1β. RESULTS Combination therapy more effectively inhibited CNV than therapy with topical bevacizumab or DTSH alone. DTSH combined with bevacizumab significantly accelerated delayed CWH caused by topical bevacizumab in the alkali burn model (P = 0.018). Combination therapy showed better inhibitory effects on MMP expression and phosphorylated VEGFR1 and VEGFR2. With DTSH treatment, doxycycline inhibited the activity and expression of MMPs, the expression of VEGF and of phosphorylated VEGFR1 and VEGFR2, and the production of iNOS and IL-1β in local cornea. CONCLUSIONS Doxycycline enhances the inhibitory effects of bevacizumab on CNV and prevents its side effects on CWH, possibly by inhibiting the expression and activity of MMPs, the expression of VEGF and of phosphorylated VEGFR1 and VEGFR2, and the production of iNOS and IL-1β.

Rapamycin Is Neuroprotective in a Rat Chronic Hypertensive Glaucoma Model

Glaucoma is a leading cause of irreversible blindness. Injury of retinal ganglion cells (RGCs) accounts for visual impairment of glaucoma. Here, we report rapamycin protects RGCs from death in experimental glaucoma model and the underlying mechanisms. Our results showed that treatment with rapamycin dramatically promote RGCs survival in a rat chronic ocular hypertension model. This protective action appears to be attributable to inhibition of neurotoxic mediators release and/or direct suppression of RGC apoptosis. In support of this mechanism, in vitro, rapamycin significantly inhibits the production of NO, TNF-α in BV2 microglials by modulating NF-κB signaling. In experimental animals, treatment with rapamycin also dramatically inhibited the activation of microglials. In primary RGCs, rapamycin was capable of direct suppression the apoptosis of primary RGCs induced by glutamate. Mechanistically, rapamycin-mediated suppression of RGCs apoptosis is by sparing phosphorylation of Akt at a site critical for maintenance of its survival-promoting activity in cell and animal model. These results demonstrate that rapamycin is neuroprotective in experimental glaucoma, possibly via decreasing neurotoxic releasing and suppressing directly apoptosis of RGCs.

Celastrol nanoparticles inhibit corneal neovascularization induced by suturing in rats

Purpose Celastrol, a traditional Chinese medicine, is widely used in anti-inflammation and anti-angiogenesis research. However, the poor water solubility of celastrol restricts its further application. This paper aims to study the effect of celastrol nanoparticles (CNPs) on corneal neovascularization (CNV) and determine the possible mechanism. Methods To improve the hydrophilicity of celastrol, celastrol-loaded poly(ethylene glycol)-block-poly(ɛ-caprolactone) nanopolymeric micelles were developed. The characterization of CNPs was measured by dynamic light scattering and transmission electron microscopy analysis. Celastrol loading content and release were assessed by ultraviolet-visible analysis and high performance liquid chromatography, respectively. In vitro, human umbilical vein endothelial cell proliferation and capillary-like tube formation were assayed. In vivo, suture-induced CNV was chosen to evaluate the effect of CNPs on CNV in rats. Immunohistochemistry for CD68 assessed the macrophage infiltration of the cornea on day 6 after surgery. Real-time quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay were used to evaluate the messenger ribonucleic acid and protein levels, respectively, of vascular endothelial growth factor, matrix metalloproteinase 9, and monocyte chemoattractant protein 1 in the cornea. Results The mean diameter of CNPs with spherical shape was 48 nm. The celastrol loading content was 7.36%. The release behavior of CNPs in buffered solution (pH 7.4) showed a typical two-phase release profile. CNPs inhibited the proliferation of human umbilical vein endothelial cells in a dose-independent manner and suppressed the capillary structure formation. After treatment with CNPs, the length and area of CNV reduced from 1.16 ± 0.18 mm to 0.49 ± 0.12 mm and from 7.71 ± 0.94 mm2 to 2.29 ± 0.61 mm2, respectively. Macrophage infiltration decreased significantly in the CNP-treated corneas. CNPs reduced the expression of vascular endothelial growth factor, matrix metalloproteinase 9, and monocyte chemoattractant protein 1 in the cornea on day 6 after suturing. Conclusion CNPs significantly inhibited suture-induced CNV by suppressing macrophage infiltration and the expression of vascular endothelial growth factor and matrix metalloproteinase 9 in the rat cornea.

Activated macrophages induce neovascularization through upregulation of MMP-9 and VEGF in rat corneas.

Purpose: To explore the mechanisms of activated macrophages (A-M&phgr;) involved in corneal angiogenesis. Methods: Activated macrophages were elicited by mineral oil lumbar injection and implanted into corneal micropockets in rats for the treatment group, A-M&phgr;, and phosphate-buffered saline group as control. Corneal changes were observed with a slit lamp microscope, and histopathological features were evaluated by immunofluorescence. Reverse transcription–polymerase chain reaction was used to detect the relative expression of angiogenesis-associated factors and inflammatory mediators in the activated macrophages and corneal tissue after implantation. Results: Immunofluorescence showed that peritoneal cells expressed antigens of cluster of differentiation 68 (CD68, ED1), matrix metalloproteinases-9 (MMP-9), and vascular endothelial growth factor (VEGF). Activated macrophages significantly induced corneal neovascularization (CNV), which peaked on day 5, whereas the control group and normal corneas showed less CNV. The activated macrophages and corneal tissue after implantation expressed the angiogenesis-related factors, such as cyclooxygenase-2, platelet-derived growth factor, transforming growth factor beta, interleukin-1 alpha, MMP-9, and VEGF in messenger RNA (mRNA). However, mRNA expression of MMP-9 and VEGF differed significantly only in the cornea between the A-M&phgr; group and phosphate-buffered saline group 5 days after the implantation. MMP-9 and VEGF expression of mRNA and protein was higher in the A-M&phgr; group than that in the control group and normal corneas. Conclusions: Activated macrophages induce obvious CNV and related mechanisms, which may be correlated with MMP-9 and VEGF autocrine in activated macrophages and upregulation of MMP-9 and VEGF in corneal tissue.

The effect of doxycycline temperature-sensitive hydrogel on inhibiting the corneal neovascularization induced by BFGF in rats

BackgroundTo study the effect of doxycycline temperature-sensitive hydrogel (DTSH) on inhibiting the corneal neovascularization (NV) induced by the basic fibroblast growth factor (bFGF).MethodsCorneal NV was induced by slow-release polymer pellets containing bFGF, using a rat corneal pocket model. After being implanted with bFGF pellets, the female Sprague–Dawley rats were randomly divided into seven groups (12 rats/group). The grouped rats were given topically normal saline solution and neutralized DTSH at a concentration of 0%, 0.01%, 0.05%, 0.1%, 0.5%, and 1% respectively, and treated for 6 consecutive days. After 6 days of treatment, the cornea was perfused with India ink. The length and area of the corneal vessel were measured and analyzed by Image Pro-Plus 5.1.ResultsCompared to the control group given saline solution, the study groups given DTSH at a concentration of 0.05%, 0.1%, 0.5%, and 1% showed significant reduction in the vessel length (respectively, 58%, 60%, 52%, and 37%) and the vessel area (respectively, 61%, 62%, 49%, and 39%) (p < 0.001). However, no such significant reduction was observed in the study group given 0.01% DTSH (p = 0.133 and 0.166 for vessel length and area respectively). Study groups given 0.05% and 0.1% DTSH showed better effects than groups given 0.01% and 1% DTSH with regard to reducing the vessel length and the vessel area (p < 0.05).ConclusionThe study results showed that topical DTSH effectively inhibited corneal NV at the ideal concentration of 0.05% and 0.1%. Therefore, topical DTSH could be considered as an alternative treatment for the clinical management of corneal NV.

Doxycycline inhibits inflammation-induced lymphangiogenesis in mouse cornea by multiple mechanisms.

Lymphangiogenesis is significantly involved in the pathogenesis of diseases, including graft rejection, cancer metastasis and various inflammatory conditions. The inhibition of lymphangiogenesis has become a new therapeutic target for the treatment of these diseases. Here, we explored the anti-lymphangiogenic effects of doxycycline in inflammation-induced lymphangiogenesis (ILA) in the cornea and the underlying mechanisms. In the present study, mice with ILA of the cornea were treated with topical doxycycline (0.1%) or vehicle control. Lymphangiogenesis was quantified using corneal immunostaining of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1). Human dermal lymphatic endothelial cells (HDLECs) and a murine macrophage cell line (RAW264.7) were used to further explore the underlying mechanisms of doxycycline-mediated anti-lymphangiogenesis in vitro. Our results showed that doxycycline treatment dramatically inhibited ILA in the mouse cornea (p<0.001), with a significant decrease in vascular endothelial growth factor (VEGF)-C/VEGF receptor 3 signalling, macrophage infiltration and inflammatory cytokine expression. Doxycycline also significantly inhibited VEGF-C-induced HDLEC proliferation in vitro by modulating the PI3K/Akt/endothelial nitric oxide (NO) synthase (eNOS) pathway and significantly suppressed interleukin-1β (IL-1β), TNF-α and VEGF-C production in the RAW264.7 cell line by modulating the PI3K/Akt/nuclear factor-kappaB (NF-κB) pathway. Additionally, doxycycline treatment dramatically reduced the phosphorylation of NF-κBp65, Akt and eNOS in ILA and significantly inhibited matrix metalloproteinases (MMPs) activity in vitro and in ILA. In conclusion, doxycycline inhibited ILA, possibly through suppression of VEGF-C signalling, macrophage function and MMPs activity. This observation suggests that doxycycline is a potential therapeutic agent for lymphangiogenesis-related diseases.

Doxycycline Attenuates Endotoxin-Induced Uveitis by Prostaglandin E2-EP4 Signaling.

PURPOSE We explored the anti-inflammatory effects of doxycycline in experimental uveitis and the underlying mechanisms. METHODS Rats with endotoxin-induced uveitis (EIU) received doxycycline (1.5 mg/kg) or the control vehicle via intraperitoneal injection. Clinical scores were graded under a slit lamp. Rat peritoneal macrophages were used in vitro to further explore the anti-inflammatory mechanisms of doxycycline. The levels of nitric oxide (NO), TNF-α, IL-1β, prostaglandin E2 (PGE2), cyclooxygenase (COX)-2, I kappa B-α (IκB-α), inducible nitric oxide synthase (iNOS), Akt, caspase-3, and nuclear factor-kappa B (NF-κB) were analyzed. RESULTS Treatment with doxycycline dramatically reduced the clinical scores of EIU (P < 0.001), with significant decreases in inflammatory cell infiltration, protein concentrations, and the production of NO, TNF-α, and IL-1β in the aqueous humor (AqH). In vitro, doxycycline significantly inhibited the production of NO, IL-1β, and TNF-α in peritoneal macrophages by modulating the PI3K/Akt/IκB-α/NF-κB pathway. Importantly, we found that doxycycline significantly enhanced COX2 expression and PGE2 production both in vivo and in vitro. More importantly, blockade of the EP4 receptor of PGE2 significantly reversed the doxycycline-mediated inhibition of macrophages and the PI3K/Akt pathway in vitro. Furthermore, simultaneous injection of an EP4 antagonist and doxycycline significantly blocked the doxycycline-mediated attenuation of EIU. CONCLUSIONS Doxycycline can ameliorate EIU, and PGE2-EP4 signaling is essential for the anti-inflammatory effects of doxycycline in vitro and in vivo.

Pharmacological inhibition of caspase-8 suppresses inflammation-induced lymphangiogenesis and allograft rejection in the cornea

of Corynebacterium species might more reflect the type 2 milieu than CRS disease. In a previous study increased abundance of Corynebacterium species at the time of endoscopic sinus surgery was predictive of better surgical outcomes. This indicates a potential protective role for Corynebacterium species in maintaining the health of the sinus mucosa. It is noteworthy that the genus Corynebacterium in the nasal cavity is believed to be important for maintaining a sustained and stable microbial pattern in healthy infants. In our study atopic dermatitis was associated with a higher RA of Streptococcus species. Colonization of the skin with a microbiome enriched for Streptococcus species is seen in atopic dermatitis–prone skin and associated with disease severity. This higher RA of Streptococcus species in patients with atopic CRS along with further decreased RA of Corynebacterium species in patients with allergic rhinitis suggests that the imbalance between these 2 bacterial genera might be an important factor in defying a certain atopic endotype in patients with CRS. PICRUSt analyses inferred that the resident bacterial community modifies its functional patterns in patients with CRS, and bacteria with the ability to invade the epithelium and increase production of LPS have overcome the microbial community in patients with CRS. The lack of increase of abundance of any single known LPS-producing bacterium suggests that there might be different cohorts of diverse LPS producers that are increased in individual patients with CRS. This suggests that future efforts should target discovery of the bacterial groups with functional capacities that enable them to overcome the community in disease or in a certain phenotype of disease, which could provide us with clues of how bacteria are contributing or even initiating a disease process. The results above showing a link between specific genera and CRS-related factors suggest that the nasal microbiome could be used as a tool to characterize and identify endotypes of patients with CRS. Mahboobeh Mahdavinia, MD, PhD Phillip A. Engen, BSc Phillip S. LoSavio, MD Ankur Naqib, BS Rafsa J. Khan, MD Mary C. Tobin, MD Arpita Mehta, MD Raj Kota, MD Nailliw Z. Preite, MSc Christopher D. Codispoti, MD, PhD Bobby A. Tajudeen, MD Robert P. Schleimer, PhD Stefan J. Green, PhD Ali Keshavarzian, MD Pete S. Batra, MD, FACS From the Department of Internal Medicine, Allergy/Immunology Division, the Department of Internal Medicine, Division of Gastroenterology, Hepatology and Nutrition, and the Department of Otorhinolaryngology–Head and Neck Surgery and Rush Sinus Program, Rush University Medical Center, Chicago, Ill; DNA Services Facility, Research Resources Center, and the Department of Biological Sciences, University of Illinois at Chicago, Chicago, Ill; and the Division of Allergy and Immunology, Internal Medicine Department, Northwestern University Feinberg School of Medicine, Chicago, Ill. E-mail: Mahboobeh_mahdavinia@rush.edu. This study was conducted by a research grant from Medtronic and internal departmental funding by Rush University (principal investigators: Mahboobeh Mahdavinia, MD, PhD, and Pete S. Batra, MD, FACS). M.M. is supported by a Brinson Foundation award. R.P.S. is supported in part by the Ernest S. Bazley Foundation, U19 AI106683 from the National Institute of Allergy and Infectious Diseases, and R37 HL068546 from the National Heart, Lung, and Blood Institute. Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest.

Hemin Promotes Corneal Allograft Survival Through the Suppression of Macrophage Recruitment and Activation

Purpose To explore the roles of hemin in preventing corneal allograft rejection (CGR) and the underlying mechanisms. Methods Hemin (30 mg/kg) was intraperitoneally injected into rats with a corneal allograft on alternate days, from the day of transplantation until euthanasia. The clinical signs of the corneal allografts were evaluated and recorded according to a previously published system. Corneal edema, macrophage infiltration, and phenotype, and the expression of chemokines, cytokines, and heme oxygenase (HO)-1 were detected by histology, real-time PCR, and Western blot. The rat macrophage cell line NR8383 was used to explore the mechanisms of action of hemin in vitro. Results Treatment with hemin significantly prolonged corneal allograft survival, with decreased corneal edema and fewer macrophages. Moreover, hemin treatment alleviated inflammation in the corneal grafts, as characterized by downregulated mRNA levels of proinflammatory mediators. In addition, hemin administration reduced the proportion of proinflammatory M1 macrophages and increased the proportion of anti-inflammatory M2 macrophages in the corneal grafts. Hemin treatment induced HO-1 expression in vivo and in vitro, whereas co-administration of zinc protoporphyrin IX (ZnPP), an HO-1 inhibitor, blocked the beneficial effects of hemin in preventing CGR. Conclusions Our results are the first to demonstrate that hemin, a Food and Drug Administration-approved drug, promotes corneal allograft survival. These findings indicate that hemin might be a potential alternative treatment for CGR.