Expression of angiogenesis-related factors in human corneas after cultivated oral mucosal epithelial transplantation.

PURPOSE: We analyzed the expression of angiogenesis-related factors in corneal tissues that had undergone previously autologous cultivated oral mucosal epithelial transplantation (COMET). METHODS: Six eyes from four chemically- and two thermally-injured patients with limbal stem cell deficiency who received COMET to promote wound healing were studied retrospectively. Immunoconfocal microscopy was performed on corneal specimens from the patients after COMET, as well on normal corneas, conjunctiva, and oral mucosa for keratin 8, fibroblast growth factor-2 (FGF-2), VEGF, collagen XVIII (endostatin), pigment epithelium-derived factor (PEDF), soluble fms-like tyrosine kinase-1 (sFlt-1), tissue inhibitor of metalloproteinase-3 (TIMP-3), thrombospondin-1 (TSP-1), and interleukin-1 receptor antagonist (IL-1ra). RESULTS: FGF-2, VEGF, endostatin, PEDF, and IL-1ra were detected in all the samples, with signals for FGF-2, VEGF, and IL-1ra localized to the full-thickness epithelial layer, as signals for endostatin limited to the basement membrane. Expression of PEDF varied in tissues, with a preferential expression in the suprabasal epithelial layer. FGF-2 and IL-1ra were abundantly expressed in the basal epithelial layer in specimens with increased stratification. Signals for sFlt-1, TIMP-3, and TSP-1 were detected in normal corneal epithelium, and in a specimen containing corneal epithelium, but were negative in all other specimens. CONCLUSIONS: Expression of FGF-2, VEGF, PEDF, endostatin, and IL-1ra was similar in normal corneas, conjunctiva, oral mucosa, and corneas after COMET. Expression of sFlt-1, TIMP-3, and TSP-1 was limited to normal corneas and negative for other tissues. A lack of the aforementioned antiangiogenic factors may contribute to the peripheral corneal neovascularization seen after COMET.

Up-regulation of heat shock protein 70-1 (Hsp70-1) in human limbo-corneal epithelial cells cultivated on amniotic membrane: A proteomic study.

Transplantation of cultivated human limbo-corneal epithelial (HLE) cells has been recognized as an effective stem cell (SC) therapy for treating corneal epithelial SC deficiency caused by burn or other diseases. With this technique, cryo-preserved human intact amniotic membrane (IAM) has been successfully used as a cell culture substrate and carrier, and is reported to preferentially preserve HLE stem/progenitor cells in vitro. However, little is known about what factors released by HLE cells are involved in the progenitor cell-preserving mechanism. Using proteomic method, we identified 13 proteins over-expressed by HLE cells cultured on IAM, which included heat shock protein 70-1 (Hsp70-1), Hsp-27, glutathione (GSH) S-transferase, annexin A2, galectin-7, and protein S100-A9. Increased Hsp70-1 expression was confirmed by Western blot and real-time PCR. The role of Hsp70-1 in promoting HLE cell survival was demonstrated by increased apoptosis index and increased cleaved poly ADP-ribose polymerase (CPARP) formation in hsp70-1-silenced, but not normal HLE cells after exposure to sublethal UVB irradiation or hydrogen peroxide. To understand the regulatory mechanism of Hsp70-1 expression in HLE cells, the role of transcription factor deltaNp63 (a well-recognized HLE stem cell; SC marker) was studied. We found that over-expression of deltaNp63α by plasmid vector induced a corresponding increase in Hsp70-1 protein production. Likewise, Hsp70-1 expression decreased in HLE cells after addition of deltaNp63α SiRNA. Immunoconfocal microscopy also revealed a paralleled expression of both proteins in corneal specimens. Thus, deltaNp63α-associated Hsp70-1 over-expression may promote HLE progenitor cell survival on IAM, possibly through the cytoprotective and anti-apoptotic effect of Hsp70-1.

Enteric-coated mycophenolate sodium as a corticosteroid-sparing agent for the treatment of autoimmune scleritis.

PURPOSE: To evaluate enteric-coated mycophenolate sodium (EC-MPS) as a corticosteroid-sparing agent in the treatment of autoimmune scleritis. METHODS: A retrospective, interventional, noncomparative review of EC-MPS use in patients with autoimmune scleritis. RESULTS: Seven eyes of 5 patients (all female; median age: 47 years, range: 20-55 years) with inflammatory scleral disease were treated with EC-MPS. The mean follow-up duration was 16.4 months (range, 12-20 months). EC-MPS was started at 360 mg twice daily. The mean time to treatment success was 1.6 months (range, 1-3 months). The mean prednisolone dosage at the onset of EC-MPS was 24 mg daily (range, 15-30 mg), and this was reduced to 6.5 mg daily (range, 0-10 mg) as inflammation control was achieved. No severe adverse events except for 1 patient with transient knee pain were reported; the incidence of adverse events after using EC-MPS was 1/6.83 person-years. There was no recurrence of scleral inflammation during the follow-up period. CONCLUSIONS: EC-MPS can be used as a corticosteroid-sparing agent to safely suppress inflammatory autoimmune scleritis.

Corneal arcus and cardiovascular risk factors in middle-aged subjects in Taiwan.

BACKGROUND: To delineate the association between corneal arcus and potential cardiovascular risk factors in middle-aged population. METHODS: This cross-sectional study randomly enrolled 119 subjects with corneal arcus and 119 subjects without arcus, aged between 30 and 60 years, from community-based East Asian population. Corneal arcus identification was completed by a single ophthalmologist using slit-lamp biomicroscopy examination. Cardiovascular risk factor parameters were measured by standardized techniques. RESULTS: Subjects with higher levels of total-cholesterol, low-density lipoprotein cholesterol (LDL-C), non-high-density lipoprotein cholesterol (non-HDL-C), and total/HDL cholesterol ratio had increased risk of having corneal arcus, whereas subjects with higher systolic blood pressure (SBP) and diastolic blood pressure had decreased risk. Using stepwise logistic regression analysis, we found that male gender was a strong independent risk factor for arcus formation [odds ratio (OR): 2.87; 95% confidence interval (CI): 1.59-5.17; P < 0.001]. Higher non-HDL-C level also had significant but mildly increased risk (OR: 1.02; 95% CI: 1.01-1.02; P 0.008) of having arcus, whereas higher SBP had significant but mildly decreased risk (OR: 0.98; 95% CI: 0.96-0.99; P < 0.001). Besides, subjects who had circumferential arcus had significantly higher risk of having LDL-C >or=160 mg/dL than those who had only partial arcus (OR: 5.79 versus OR: 4.60; P < 0.001). CONCLUSIONS: In addition to serum LDL-C, male gender and serum non-HDL-C level are significantly correlated to corneal arcus. Conversely, SBP is negatively correlated to corneal arcus. Presence of corneal arcus in middle-aged men may be an indicator for dyslipidemia, and we speculate that the relationship between arcus and coronary heart disease may be dependent of dyslipidemia.

Matrix revolution: molecular mechanism for inflammatory corneal neovascularization and restoration of corneal avascularity by epithelial stem cell transplantation.

Corneal neovascularization (CNV) associated with severe limbal stem cell (LSC) deficiency remains a challenging ocular surface disease in that corneal inflammation may persist and progress, and the condition will not improve without LSC transplantation. A prominent feature after successful LSC transplantation is the suppression of corneal inflammation and CNV, which is generally attributed to the endogenous anti-angiogenic/anti-inflammatory factors secreted by corneal epithelial cells. In addition, corneal epithelial basement membrane (EBM) plays a unique role in the regulation of angiogenesis; several potent anti-angiogenic factors are derived from the matrix component of EBM, such as endostatin (from collagen XVIII) and restin (from collagen XV). Also, angio-inhibitory thrombospondin and tissue inhibitor of metalloproteinase-3 are deposited in EBM. Moreover, the heparan sulphate proteoglycan in EBM can bind and sequester VEGF and FGF-2 from activation. Recently, cultivated corneal epithelial transplantation (CCET) and cultivated oral mucosal epithelial transplantation (COMET) have emerged as promising techniques for the treatment of LSC deficiency. When human limbo-corneal epithelial (HLE) cells are cultivated on cryopreserved amniotic membrane, production of endostatin, restin, and IL-1ra is enhanced. This highlights the significance of delicate epithelial-matrix interactions in the generation of anti-angiogenic/anti-inflammatory factors by HLE cells, and this may, in part, explain the rapid restoration of corneal avascularity following CCET. In addition, whether epithelial stem cells can persist after transplantation is the key for CCET and COMET. Emerging evidence of long-term survival of cultivated epithelial cells after transplantation suggest that epithelial stem cells can be isolated and cultivated in vitro, and can re-establish the epithelial phenotype in vivo. Taken together, the merits of enhanced anti-angiogenic activity and the preservation of corneal epithelial stem cells encourage further application of this tissue engineering technique for ocular surface reconstruction.

Persistence of transplanted oral mucosal epithelial cells in human cornea.

PURPOSE: To determine the expression of differentiation and progenitor cell markers in corneal tissues that previously underwent autologous cultivated oral mucosal epithelial transplantation (COMET). METHODS: Four eyes from three alkaline-injured patients and one thermally injured patient underwent COMET to promote re-epithelialization or corneal reconstruction. Between 10 and 22 months (mean, 14.2 +/- 5.5 months [SD]) after COMET, the corneal tissues were obtained after penetrating keratoplasty (n = 1) or autologous limbal transplantation (n = 3). Immunoconfocal microscopy for keratin (K)3, -12, -4, -13, and -8; connexin (Cx)43; MUC5AC; laminin-5; pan-p63; ABCG2; and p75 was performed in those specimens as well as in the oral mucosa and cultivated oral mucosal epithelial cells (OMECs). RESULTS: All four specimens were unanimously positive for K3, -4, and -13 but negative for K8 and MUC5AC, suggesting that the keratinocytes were oral mucosa-derived. However, peripheral K12 staining was positive only in patient 2, suggesting a mixed oral and corneal epithelium in that case. Cx43 staining in the basal epithelium was negative in patients 1, 2, and 3, but was positive in patient 4. Small, compact keratinocytes in the basal epithelium preferentially expressed pan-p63, ABCG2, and p75. Although the staining of pan-p63 and ABCG2 tended to be more than one layer, signal for p75 was consistently localized only to the basal layer. CONCLUSIONS: The study demonstrated the persistence of transplanted OMECs in human corneas. In addition, small, compact cells in the basal epithelium preferentially expressed the keratinocyte stem/progenitor cell markers, which may be indicative of the engraftment of the progenitor cells after transplantation.

Overview of laser refractive surgery.

Since approval of the use of the excimer laser in 1995 to reshape the cornea, significant developments in the correction of refractive errors such as myopia, hyperopia, and astigmatism have been achieved. Combined with other advanced ophthalmological instruments (e.g. anterior segment imaging systems, the femtosecond laser, wavefront-guided customized ablation) and the knowledge accumulated concerning the basic science of refractive errors (e.g. biomechanics and wound healing of the cornea, higher-order aberrations), laser refractive surgery has promisingly outshone other conventional techniques (e.g. radial keratotomy [RK], automated lamellar keratectomy [ALK]) in terms of both safety and efficacy. Photorefractive keratectomy (PRK) produces stable and predictable results with a safe profile. Similarly, laser in situ keratomileusis (LASIK) is also safe and efficacious with the additional advantages of rapid visual recovery and minimal postoperative pain. The choice between the two methods is made only after thoughtful discussion between the surgeon and the patient. Despite these advances, certain limitations and complications do exist. There are also specific and controversial circumstances for which studies should be conducted to make further breakthroughs and avoid annoying complications. In this review, the basic knowledge, surgical issues, and clinical outcomes, of laser refractive surgery, as well as complex cases, will be presented.