sirolimus and Corneal-Neovascularization

Alkali burn-induced corneal injury often causes inflammation and neovascularization and leads to compromised vision. We previously reported that rapamycin ameliorated corneal injury after alkali burns by methylation modification. In this study, we aimed to investigate the rapamycin-medicated mechanism against corneal inflammation and neovascularization. Our data showed that alkali burn could induce a range of different inflammatory response, including a stark upregulation of pro-inflammatory factor expression and an increase in the infiltration of myeloperoxidase- and F4/80-positive cells from the corneal limbus to the central stroma. Rapamycin effectively downregulated the mRNA expression levels of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), toll-like receptor 4 (TLR4), nucleotide binding oligomerization domain-like receptors (NLR) family pyrin domain-containing 3 (NLRP3), and Caspase-1, and suppressed the infiltration of neutrophils and macrophages. Inflammation-related angiogenesis mediated by matrix metalloproteinase-2 (MMP-2) and rapamycin restrained this process by inhibiting the TNF-α upregulation in burned corneas of mice. Rapamycin also restrained corneal alkali burn-induced inflammation by regulating HIF-1α/VEGF-mediated angiogenesis and the serum cytokines TNF-α, IL-6, Interferon-gamma (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The findings of this study indicated rapamycin may reduce inflammation-associated infiltration of inflammatory cells, shape the expression of cytokines, and balance the regulation of MMP-2 and HIF-1α-mediated inflammation and angiogenesis by suppressing mTOR activation in corneal wound healing induced by an alkali injury. It offered novel insights relevant for a potent drug for treating corneal alkali burn.

Topics: Alkalies; Animals; Burns, Chemical; Cornea; Corneal Injuries; Corneal Neovascularization; Cytokines; Disease Models, Animal; Eye Burns; Inflammation; Matrix Metalloproteinase 2; Mice; Neovascularization, Pathologic; Sirolimus; Tumor Necrosis Factor-alpha

To investigate the role of macrophage autophagy in the process of corneal neovascularization (CNV).. In vivo, mice CNV was induced by alkali injury and compared with rapamycin-treated alkaline burn mice. Western blot was used to determine the autophagic status of the macrophages. We quantified the levels of macrophage polarization markers (CD86, INOS, CD163, CD206) by RT-qPCR and measured inflammatory factors through ELISA (IL-6 and TNF-α) in the early phase after injury. In vitro, the human umbilical vein endothelial cells (HUVECs) were co-cultured with macrophage-conditioned medium (MCM) induced by the THP-1 cell line to simulate the neovascular microenvironment. The vascularization capacity of HUVECs was examined using the CCK-8 assay kit, tube formation assay, and scratch wound-healing assay.. In vivo, the mRNA expression of Beclin-1 and ATG5 was increased, together with the upregulation of M1 macrophage markers (CD86 and INOS) in corneas after early alkali injury. The area of CNV is effectively relieved in the rapamycin-treated mice. In vitro, upregulation of autophagy level by pretreatment with 3-methyladenine (3-MA) could increase the mRNA expression of the M1 markers. Macrophage-conditioned medium with impaired autophagy contains more IL-6 and TNF-α compared to the M1 macrophage-conditioned medium, promoting HUVEC proliferation, migration, and tube formation capacity. Enhancing the autophagy level with rapamycin (RAPA) could reverse this phenomenon.. Impaired autophagy promoted macrophage polarization toward M1 type and increased the expression of IL-6 and TNF-α, which led to severe CNV. Using the autophagy activator (RAPA) could effectively alleviate CNV by promoting autophagy.

Topics: Alkalies; Animals; Autophagy; Corneal Neovascularization; Culture Media, Conditioned; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-6; Macrophages; Mice; RNA, Messenger; Sirolimus; Tumor Necrosis Factor-alpha

Topics: Administration, Ophthalmic; Animals; Burns, Chemical; Cell Proliferation; Cells, Cultured; Cornea; Corneal Injuries; Corneal Neovascularization; Corneal Opacity; Disease Models, Animal; Drug Carriers; Drug Compounding; Eye Burns; Fibroblasts; Fibrosis; Humans; Lipids; Male; Mice, Inbred BALB C; Nanomedicine; Nanoparticles; Sirolimus; Sodium Hydroxide; Wound Healing

To investigate the protective effect of rapamycin against alkali burn-induced corneal damage in mice.. BALB/c mice were treated with 0.1 N NaOH to the cornea for 30 seconds. Corneal neovascularization and opacity were clinically evaluated at 1, 2, and 4 weeks after chemical burn injury. Rapamycin was delivered topically to right eyes (1 mg/mL) and injected intraperitoneally (0.2 mg/kg) once a day. Concentrations of interleukin-6 (IL-6) and transforming growth factor-beta1 (TGF-β1) in the cornea were measured by enzyme-linked immunosorbent assay (ELISA). In vitro-cultured human corneal stromal cells were treated with 0 to 500 nM rapamycin for 3 days and then assessed by immunofluorescence staining of vimentin and alpha-smooth muscle actin (α-SMA). Western blotting assays for α-SMA, phosphorylated extracellular signal-regulated kinase (ρ-ERK 1/2), and total ERK 1/2 were also performed.. Corneal neovascularization and corneal opacity scores measured 4 weeks after the chemical burn corneal injury were lower in the rapamycin group than in the control group. Two weeks after the chemical burn injury, a significant elevation in the corneal IL-6 levels of the positive control group was observed, compared to the levels in the negative control group or the rapamycin group (P < 0.05). Corneal TGF-β1 levels were lower in the rapamycin-treated group than in the control group at 4 weeks after chemical burn injury (P < 0.05). Moreover, rapamycin inhibited TGF-β1-induced α-SMA expression and augmented ERK 1/2 phosphorylation.. Rapamycin treatment reduced corneal opacity and corneal neovascularization in BALB/c mice. Rapamycin protected the cornea from chemical damage via reduction of IL-6 and TGF-β1 expression. Rapamycin reduced α-SMA expression through the ERK 1/2 pathway.

Topics: Animals; Blotting, Western; Burns, Chemical; Cells, Cultured; Corneal Neovascularization; Corneal Opacity; Corneal Stroma; Enzyme-Linked Immunosorbent Assay; Eye Burns; Immunosuppressive Agents; Injections, Intraperitoneal; Interleukin-6; Male; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Signal Transduction; Sirolimus

To explore the inhibiting effect of rapamycin (RAPA) on corneal neovascularization (CNV) of rats and the functional mechanism.. A design group was adopted. 102 Wistar rats were divided into four groups at random, including rapamycin liposome treated group (24 rats), the rapamycin solved in bean oil treated group (24 rats), blank liposome treated group (24 rats), blank treated group (24 rats) and normal control group (6 rats). All right eyes of 96 rats were induced by alkali cauterization. Rapamycin liposome were prepared by thin film hydration and the major factors were studied by the method of orthogonal design. After alkali burn, cauterized rats were observed by slitlamp biomicroscope every day. On the 1st, 4th, 7th, 14th days after operation, the expression of HIF-1alpha and VEGF were examined by immunohistochemical method and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The analysis of variance and q test groups for analysis were adopted to analyze the results.. (1) The bodies of RAPA liposome were intact kinds of spheres, the average diameter was 145.2 nm, and the envelopment rate was 90.02%. (2) After the burn of 14 d, CNV area of B, C, D and E group were (28.289 +/- 0.703), (28.005 +/- 0.801), (20.002 +/- 1.005) and (22.300 +/- 0.853) mm(2) (F = 159.62, P < 0.05). The CNV of both the rapamycin liposome treated group and the rapamycin solved in bean oil group grew slowly and smaller than that of blank liposome treated group and blank treated group (q = 47.80, 46.20, 34.60, 32.90;P = 0.00). While the rapamycin liposome treated group changed more obviously than the rapamycin solved in bean oil group (q = 13.20, P = 0.00). After alkali burn, the expression of HIF-1alpha and VEGF increased dramatically, meanwhile the expression of HIF-1alpha and VEGF were significantly decreased by RAPA.. Liposome body is an excellent medicine carrier for the RAPA. RAPA can obviously suppress the growth of CNV. The possibly mechanism is weakening VEGF expression by inhibiting the transcription factor HIF-1alpha.

Topics: Animals; Corneal Neovascularization; Drug Carriers; Hypoxia-Inducible Factor 1, alpha Subunit; Liposomes; Ophthalmic Solutions; Rats; Rats, Wistar; Sirolimus; Vascular Endothelial Growth Factor A

To evaluate the immunosuppressive and antiangiogenesis effects of rapamycin drug delivery system (RAPA DDS) in high risk rabbit model of penetrating keratoplasty (PK).. (1) RAPA DDS preparation: 50 mg of PGLC and 50 mg of RAPA were mixed as a RAPA drug delivery system. (2) High risk rabbit model: Corneal vascularization was induced in 45 New Zealand white rabbits (45 eyes) by passing 5 - 0 silk sutures in corneal stroma in each quadrant. (3) 40 rabbits with corneal neovascularization beyond three quadrants were received a unilateral 7 mm diameter central PK. The 40 were divided into four treatment groups: Group A, control group and received no therapy; Group B, 1 mg PGLC carrier was implanted in the anterior chamber; Group C, 1% RAPA eye drops was applied four times daily; Group D, 0.5 mg RAPA DDS was implanted in the anterior chamber. (4) Postoperative examination: The cornea allografts (opacity, edema and neovascularization) were examined by the slit-lamp biomicroscopy for ninety days. Rejection index (RI) and neovascularization index (NI) of these animal models were recorded. RAPA concentration in the aqueous humor was detected on 2, 4, 8 and 12 weeks in group C and D after surgery; the expressions of IL-2R, MCP-1, Fas/FasL in samples were detected with in situ hybridization; TNF-alpha and VEGF were detected with immuno-histochemical technique three weeks after the operation in all groups. Histochemical method was carried out on the procured specimens of cornea, retina, liver and kidney at ninety days.. (1) Allografts rejection: Mean survival times in 4 trial groups were (16.5 +/- 2.5), (16.0 +/- 2.6), (47.1 +/- 13.2), (87.6 +/- 5.8) d respectively (P = 0.000). (2) Corneal neovascularization: Mean NI was 2.4 +/- 0.7, 2.1 +/- 0.5, 0.6 +/- 0.5, 0.3 +/- 0.5 (P = 0.000) 2 weeks after the operation, and the NI value was 3.8 +/- 0.5, 3.8 +/- 0.4, 0.8 +/- 0.7, 0.4 +/- 0.8 (P = 0.000) 12 weeks after the operation in groups A, B, C and D respectively. (3) RAPA concentration in aqueous humor: Mean RAPA concentration in aqueous humor was 10.7, 12.0, 9.2, 7.0 ng/ml in group D in the 2, 4, 8 and 12 weeks after the operation respectively. RAPA can not be detected in group C. (4) Cytokine expression: IL-2R, MCP-1, TNF-alpha and VEGF were overexpression in group A and B, and undetectable in group C and D. Fas and FasL were negative in all groups. (5) No inflammatory cell infiltration was found in retina, liver and kidney tissue ninety days after the surgery.. Sustained RAPA DDS and eyedrops can prolong allograft survival and inhibit cornea neovascularization in rabbit model. However, RAPA DDS is better than eyedrops.

Topics: Animals; Corneal Neovascularization; Drug Delivery Systems; Female; Graft Rejection; Graft Survival; Immunosuppressive Agents; Keratoplasty, Penetrating; Male; Rabbits; Sirolimus

The purpose of this study was to determine whether rapamycin could inhibit corneal angiogenesis induced by basic fibroblast growth factor (bFGF). Using human dermal microvascular endothelial cells (HDMECs), we examined the effect of rapamycin on cell proliferation and migration, and the expression of vascular endothelial growth factor (VEGF). The rabbit's eye was implanted intrastromally into the superior cornea with pellet containing bFGF for the control group and pellet containing bFGF and rapamycin for the rapamycin group. Biomicrographically, corneal angiogenesis was evaluated for 10 days after pellet implantation. The neovascularized cornea also was examined histologically. bFGF induced corneal neovascularization was significantly reduced by treatment with rapamycin. Using in vitro model, rapamycin strongly inhibited bFGF induced proliferation, migration, and VEGF secretion of HDMECs. We could observe that the bFGF induced corneal angiogenesis was inhibited by rapamycin in a micropocket rabbit model. The score of neovascularization was significantly decreased in the rapamycin group than in the control group at 10 days after pellet implantation. Histologically, the cornea of rapamycin group also showed much less new vessels than that of control group. Collectively, rapamycin appears to inhibit bFGF induced angiogenesis in a rabbit corneal micropocket assay and may have therapeutic potential as an antiangiogenic agent.

Topics: Angiogenesis Inhibitors; Animals; Cell Movement; Cell Proliferation; Cells, Cultured; Corneal Neovascularization; Dose-Response Relationship, Drug; Drug Implants; Endothelial Cells; Female; Fibroblast Growth Factor 2; Humans; Rabbits; Sirolimus; Vascular Endothelial Growth Factor A

To evaluate the immunosuppressive and antiangiogenic activities of an intraocular rapamycin (RAPA) drug delivery system (DDS) in a rabbit model of high-risk penetrating keratoplasty.. Forty New Zealand White rabbits with corneal neovascularization underwent allograft cornea transplantation and were randomly divided into four groups: a control group, a glycolide-co-lactide-co-caprolactone copolymer (PGLC)-implanted group, a RAPA eye drop group, and a RAPA-PGLC DDS-implanted group. Graft survival, corneal neovascularization, and RAPA concentration in the aqueous humor were monitored for 90 days. Corneal grafts were also examined by in situ hybridization and immunohistochemistry for proinflammatory gene expression.. In the control and PGLC groups, graft rejection occurred within 3 weeks of keratoplasty. In the RAPA eye drop and RAPA-PGLC groups, corneal rejection was significantly delayed, and neovascularization was markedly inhibited. Median graft survival times were 36 and >90 days in the eye drop and RAPA-PGLC groups, respectively. Mean RAPA concentrations in the aqueous humor were 10.7 ng/mL, 12.0 ng/mL, 9.2 ng/mL, and 7.0 ng/mL in the RAPA-PGLC group 2, 4, 8, and 12 weeks after surgery, respectively. By contrast, RAPA was undetectable in the aqueous humor in the eye drop group. High levels of IL-2R, MCP-1, TNF-alpha, and VEGF were detected in the corneal grafts of the control and PGLC groups but not in those of the RAPA-treated groups.. RAPA-PGLC DDS and RAPA eye drops can significantly prolong the survival of allografts at high risk and inhibit corneal neovascularization. However, RAPA-PGLC DDS is far more effective than RAPA eye drops in preventing corneal graft rejection.

Topics: Animals; Chemokine CCL2; Corneal Neovascularization; Drug Delivery Systems; Graft Rejection; Graft Survival; Immunosuppressive Agents; In Situ Hybridization; Keratoplasty, Penetrating; Polyesters; Rabbits; Receptors, Interleukin-2; Risk Factors; Sirolimus; Transplantation, Homologous; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

To examine the effect of rapamycin on the proliferation and the migration of human umbilical vein endothelial cells (HUVECs) and on the corneal neovascularization in the corneal alkaline burn murine model.. HUVEC proliferation, migration, and apoptosis were examined after treatment with rapamycin. The effect of rapamycin on the mRNA expression of FK506 binding protein (FKBP)-12 and mammalian target of rapamycin (mTOR) was also evaluated in vitro. Corneal neovascularization was induced in vivo by an alkaline burn of the cornea with 1 N NaOH on BALB/c mice. Rapamycin was given intraperitoneally at 2 mg/kg body weight once a day for 12 days after the corneal alkaline burn. Growth factors and cytokines related with neovascularization and inflammation were evaluated in the corneal tissue and the peripheral blood by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The corneal neovascularization was evaluated by a slit lamp biomicroscopy.. Rapamycin at the concentration of 1000 ng/mL for >48 hours' exposure significantly inhibited the growth of HUVECs. The double chamber assay showed that rapamycin dramatically inhibited the migration of HUVECs at concentrations of 10 and 100 ng/mL and that these concentrations did not affect endothelial cell growth. When TUNEL assays were performed, the number of apoptotic cells increased 1.9-, 2.1-, and 2.6-fold compared with the control at 10, 100, and 1000 ng/mL, respectively, of rapamycin at 48 hours of exposure. RT-PCR showed that the expression of mTOR was suppressed in the HUVECs after rapamycin treatment; however, FKBP-12 expression was not affected. Among the angiogenic factors, gene expression of substance P and hypoxia inducible factor (HIF)-1 alpha was inhibited by rapamycin earlier (1-3 days), with vascular endothelial growth factor (VEGFR)-1 gene expression being suppressed for the first 7 days in the corneal tissue. The protein level of substance P and vascular endothelial growth factor (VEGF) was significantly decreased--more in mice treated with rapamycin than the control mice--as shown by ELISA assay of peripheral blood. Furthermore, rapamycin significantly inhibited corneal neovascularization in the alkaline-burned cornea.. Rapamycin strongly inhibited HUVEC migration at doses that did not cause cytotoxicity and apoptosis in this in vitro model. Rapamycin also suppressed corneal neovascularization, possibly by inhibiting proinflammatory cytokines, as shown by the in vivo study. Therefore, rapamycin may be useful as an angiogenic regulator in the treatment of corneal diseases that manifest with neovascularization.

Topics: Animals; Apoptosis; Carrier Proteins; Cell Movement; Cell Proliferation; Corneal Neovascularization; Cytokines; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Growth Substances; Immunosuppressive Agents; In Situ Nick-End Labeling; Injections, Intraperitoneal; Male; Mice; Mice, Inbred BALB C; Phosphotransferases (Alcohol Group Acceptor); Protein Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sirolimus; Sodium Hydroxide; TOR Serine-Threonine Kinases; Umbilical Veins

To investigate the effects of rapamycin on prevention of corneal allograft rejection in murine corneal transplantation.. The outbred strain SD and Wistar rats were used as donors and recipients, respectively. Sixty-eight Wistar rats were divided into four groups: Group A, autograft control; Group B, allograft control (the control groups were given placebo only); Group C and D, allograft groups, were treated with orally RAPA (3 mg.kg(-1).d(-1)) and CsA (10 mg.kg(-1).d(-1)), respectively. The drugs were delivered for 12 days beginning at the day of transplantation. Each animal was examined by operating microscopy. The grafts were evaluated clinically by means of Holland's scoring system and graft survival was assessed by Kaplan-Meier analysis. The neovascular indexes of rejection were compared among different groups. Histological examination on ocular tissues was performed on day 14 to confirm the clinical diagnosis of rejection.. The average transplant survival time in the allogenic control (Group B) was (11.0 +/- 1.5) d. Treatment with RAPA (Group C) led to a statistically significant prolongation of transplant survival to (36.1 +/- 14.9) d (P < 0.05). Treatment with RAPA 3 mg.kg(-1).d(-1) prolonged transplant survival as compared with treatment with CsA (Group D), but the difference was not statistically significant (P > 0.05). Corneal neovascularization was induced after the surgery. In RAPA group, corneal neovascularization was markedly reduced as compared with allograft control (Group B) (P < 0.05) and CsA group (Group D) (P < 0.05). Fewer inflammatory cells were found in the corneal stroma of the RAPA group.. These results show that oral immunosuppression with RAPA can prevent corneal graft rejection and corneal neovascularization.

Topics: Animals; Corneal Neovascularization; Corneal Transplantation; Female; Graft Rejection; Graft Survival; Immunosuppressive Agents; Random Allocation; Rats; Rats, Sprague-Dawley; Rats, Wistar; Sirolimus

To investigate the immunosuppressive effect of rapamycin in prolonging allograft survival in the rat model of orthotopic allogeneic penetrating keratoplasty.. Thirty inbred Lewis rats received corneal allografts from Brown Norway donors. Animals were divided into two rapamycin treatment groups and one allogeneic control group.. By the second week after surgery, all of the control animals had experienced allograft failure due to allograft rejection. However, allografts in seven of 10 animals in the low-dose treatment group and allografts in seven of nine animals in the high-dose treatment group remained clear. In addition, corneal neovascularization was markedly reduced in the treated animals.. The systemic administration of rapamycin prolongs corneal allograft survival and significantly inhibits the neovascular component of rejection in the rat model of orthotopic allogeneic penetrating keratoplasty.

Topics: Animals; Cornea; Corneal Neovascularization; Graft Rejection; Graft Survival; Injections, Intramuscular; Keratoplasty, Penetrating; Polyenes; Rats; Rats, Inbred BN; Rats, Inbred Lew; Sirolimus; Transplantation, Homologous