Long-term results of topical 0.02% tacrolimus ointment for refractory ocular surface inflammation in pediatric patients.

BACKGROUND: No studies have been reported on the efficacy and safety of long-term (≥12 months) use of topical tacrolimus for refractory ocular surface inflammation in pediatric patients. METHODS: Medical records of pediatric patients who were prescribed topical 0.02% tacrolimus ointment for refractory ocular surface inflammation between January of 2010 and March of 2018 were reviewed retrospectively. Changes in ocular surface signs during slit-lamp examination, clinical symptoms and concurrent steroid use were graded with a scoring system. The presence of side effects was also assessed. The changes in disease severity and patient symptoms were compared between baseline and after the treatment. RESULTS: Among 72 patients (55% males, mean age 10.8 ± 3.9 years, range 3 to 17 years), 25 patients (48% males, mean age 11.4 ± 3.9 years) fully recovered, resulting in discontinuance of the ointment treatment before 12 months. Six patients experienced intolerable burning sensation, which required treatment cessation. Cessation days of those who quit were 1,5,14,20,26, and 35 days. Seven patients were lost during follow-up. Thirty-four patients (56% males, mean age 11.2 ± 4.2 years, range 3 to 17 years) were treated with tacrolimus ointment for over 12 months (average 23.1 ± 19.1 months, range 12 to 98 months). During the follow-up period, all patients showed improved clinical signs and symptoms, and no adverse reaction was noted. CONCLUSIONS: Long-term maintenance of topical tacrolimus 0.02% ointment is safe and effective in improving refractory ocular surface inflammation in pediatric patients.

Changes in ocular surface and Meibomian gland after penetrating Keratoplasty.

BACKGROUND: To acquire desirable outcomes of penetrating keratoplasty (PKP), various factors affecting graft survival, visual function, and subjective symptom should be considered. As ocular surface and meibomian gland function are associated with these factors, this study aims to investigate changes of ocular surface and meibomian gland parameters after PKP. METHODS: This retrospective case series study included 24 eyes of 24 patients who underwent penetrating keratoplasty. Examinations on lipid layer thickness (LLT), meiboscore, tear meniscus area (TMA), tear breakup time (TBUT), corneal fluorescein staining (CFS), Schirmer I test (SIT), Ocular Surface Disease Index (OSDI), and meibomian gland functions were performed before and at 1 week, 1 month, 6 months, and 12 months after surgery. RESULTS: Compared to baseline (2.9 ± 0.6 s), TBUTs were longer at 1 week (4.4 ± 0.5 s, P = 0.027) and 6 months (4.4 ± 0.5, P = 0.048) after surgery. CFS values improved from baseline (6.5 ± 1.1) to 6 months (3.5 ± 0.6, P = 0.023) and 12 months (3.3 ± 0.7, P = 0.001) after surgery. Meibum quality value worsened at 1 week and 12 months after surgery and meibomian gland expressibility value worsened at 1 week and 6 months after surgery compared to baseline. OSDI scores improved at 6 and 12 months after surgery. Meiboscore showed no change throughout the follow up period. The patients with high preoperative meiboscore had worse meibomian gland expressibility at 6 and 12 months and meibum quality at 6 months postoperatively compared to their baseline and to those of patients with low preoperative meiboscore. CONCLUSIONS: After penetrating keratoplasty, ocular surface parameters including corneal staining, TBUT, and OSDI significantly improved whereas meibomian gland parameters showed deteriorations, which was marked in patients with high preoperative meiboscore. Thus, perioperative management of MGD is recommended for patients who undergo penetrating keratoplasty, especially in patients with advanced MGD.

3-month surgical outcomes of Implantable Collamer Lens implantation for myopic regression after laser vision correction surgeries: a retrospective case series.

BACKGROUND: To investigate the surgical outcomes of implantable collamer lens (ICL) implantation in eyes with residual myopia after primary laser vision correction (LVC) surgeries. METHODS: This study included patients who underwent ICL implantation and had a history of LVC surgery, including photorefractive keratectomy (PRK) or laser-assisted in situ keratomileusis (LASIK). Visual acuity and refractive error were assessed pre and 3-months postoperatively and the efficacy and safety indices calculated accordingly. RESULTS: A total of 30 eyes of 17 patients were included in this study. At 3 months, the mean logMAR uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), and spherical equivalent were - 0.03 ± 0.11 (include logMAR), - 0.04 ± 0.09 (include logMAR), and - 0.06 ± 0.33 diopters (D), respectively. The 3-month Snellen UDVA was better than 20/20 for 83% of eyes, and 97% of eyes showed an unchanged or improved CDVA after surgery. The mean efficacy and safety indices were 1.11 ± 0.22 and 1.13 ± 0.20, respectively. Further, 93 and 100% of eyes were within ±0.5 and ± 1.0 D of the attempted spherical equivalent refraction, respectively. CONCLUSIONS: ICL implantation in eyes with myopic regression after previous LVC surgery showed safe, effective, and predictable outcomes. TRIAL REGISTRATION: retrospectively registered.

Lysosomal dysfunction of corneal fibroblasts underlies the pathogenesis of Granular Corneal Dystrophy Type 2 and can be rescued by TFEB.

Granular corneal dystrophy type 2 (GCD2) is the most common form of transforming growth factor ß-induced (TGFBI) gene-linked corneal dystrophy and is pathologically characterized by the corneal deposition of mutant-TGFBIp. The defective autophagic degradation of pathogenic mutant-TGFBIp has been shown in GCD2; however, its exact mechanisms are unknown. To address this, we investigated lysosomal functions using corneal fibroblasts. Levels of cathepsins K and L (CTSK and CTSL) were significantly decreased in GCD2 cells, but of cathepsins B and D (CTSB and CTSD) did not change. The maturation of the pro-enzymes to their active forms (CTSB, CTSK and CTSL) was inhibited in GCD2 cells. CTSL enzymes directly degraded both LC3 (autophagosomes marker) and mutant-TGFBIp. Exogenous CTSL expression dramatically reduced mutant-TGFBIp in GCD2 cells, but not TGFBIp in WT cells. An increased lysosomal pH and clustered lysosomal perinuclear position were found in GCD2 cells. Transcription factor EB (TFEB) levels were significantly reduced in GCD2 cells, compared to WT. Notably, exogenous TFEB expression improved mutant-TGFBIp clearance and lysosomal abnormalities in GCD2 cells. Taken together, lysosomal dysfunction in the corneal fibroblasts underlies the pathogenesis of GCD2, and TFEB has a therapeutic potential in the treatment of GCD2.

Uptake of cell debris and enhanced expression of inflammatory factors in response to dead cells in corneal fibroblast cells.

Keratocytes synthesize stromal proteins and participate in wound healing through successive differentiation into corneal fibroblasts and myofibroblasts. Cultured keratocytes or corneal fibroblasts are also known as non-professional phagocytes and innate immune cells. However, whether the corneal fibroblasts phagocytize their dead cells and whether the associated innate immunity is enhanced remains unknown. We initially characterized immortalized corneal fibroblast cells with the expression of specific genes. The corneal fibroblasts strongly expressed extracellular matrix molecules (FN and COL1A1) and low or medium levels of macrophage markers (CD14, CD68, and CD36), inflammatory cytokines (IL1A, IL1B, and IL6), and chemokines (IL8 and CCL2), but not CD11b, suggesting that corneal fibroblasts are macrophage-like fibroblasts. We confirmed the phagocytic activity of the corneal fibroblasts with fluorescent dye labeled-dead E. coli and S. aureus bacteria using confocal microscopy and flow cytometry. To test corneal fibroblast phagocytosis of apoptotic and necrotic cells we co-cultured corneal fibroblasts with fluorescent dye labeled-apoptotic and -necrotic cells and analyzed their uptake using fluorescence and confocal microscopy. We observed that corneal fibroblasts can engulf digested or processed cellular debris and entire dead cells. Co-cultured dying and dead cells strongly enhanced the expression of cytokine (IL1A, IL1B, and IL6), chemokine (CCL2, CCL5, CCL20, IL8, and CXCL10), and MMP (MMP1, MMP3, and MMP9) genes through the NF-κB signaling pathway. Our findings suggest that dying and dead cells stimulate corneal fibroblasts to further induce inflammatory factors and that corneal fibroblasts contribute to the clearing of cell debris as non-professional phagocytes.

APP processing and metabolism in corneal fibroblasts and epithelium as a potential biomarker for Alzheimer's disease.

Alzheimer's disease (AD) primarily affects the brain and is the most common form of dementia worldwide. Despite more than a century of research, there are still no early biomarkers for AD. It has been reported that AD affects the eye, which is more accessible for imaging than the brain; however, links with the cornea have not been evaluated. To investigate whether the cornea could be used to identify possible diagnostic indicators of AD, we analyzed the proteolytic processing and isoforms of amyloid precursor protein (APP) and evaluated the expression of AD-related genes and proteins in corneal fibroblasts from wild-type (WT) corneas and corneas from patients with granular corneal dystrophy type 2 (GCD2), which is related to amyloid formation in the cornea. Reverse transcription polymerase chain reaction (RT-PCR) analysis was used to assess the expression of AD-related genes, i.e., APP, ADAM10, BACE1, BACE2, PSEN1, NCSTN, IDE, and NEP. RT-PCR and DNA sequencing analysis demonstrated that isoforms of APP770 and APP751, but not APP695, were expressed in corneal fibroblasts. Moreover, the mRNA ratio of APP770/APP751 isoforms was approximately 4:1. Western blot analysis also demonstrated the expression of a disintegrin and metalloprotease domain-containing protein 10 (ADAM10), beta-site APP-cleaving enzyme 1 (BACE1), nicastrin, insulin degradation enzyme, and neprilysin in corneal fibroblasts. Among these targets, the levels of immature ADAM10 and BACE1 protein were significantly increased in GCD2 cells. The expression levels of APP, ADAM10, BACE1, and transforming growth factor-beta-induced protein (TGFBIp) were also detected by western blot in human corneal epithelium. We also investigated the effects of inhibition of the autophagy-lysosomal and ubiquitin-proteasomal proteolytic systems (UPS) on APP processing and metabolism. These pathway inhibitors accumulated APP, α-carboxy-terminal fragments (CTFs), ß-CTFs, and the C-terminal APP intracellular domain (AICD) in corneal fibroblasts. Analysis of microRNAs (miRNAs) revealed that miR-9 and miR-181a negatively coregulated BACE1 and TGFBIp, which was directly associated with the pathogenesis of AD and GCD2, respectively. Immunohistochemical analysis indicated that APP and BACE1 were distributed in corneal stroma cells, epithelial cells, and the retinal layer in mice. Collectively, we propose that the cornea, which is the transparent outermost layer of the eye and thus offers easy accessibility, could be used as a potential biomarker for AD diagnosis and progression.

Comparison of objective accommodation in phakic and pseudophakic eyes between age groups.

PURPOSE: To compare objective accommodation of phakic and pseudophakic eyes between two different age groups. METHODS: Eighty-three eyes (83 participants aged ≥ 40 years) with a visual acuity of 20/25 or better, and refractive error < spherical - 1.0 diopters (D) and cylindrical 1.0 D, were included. Forty-four patients had undergone phacoemulsification and monofocal intraocular lens implantation and were examined 6 months post-surgery. Participants were divided into groups 1 (pseudophakic, age < 60 years), 2 (pseudophakic, ≥ 60 years), 3 (phakic, < 60 years), and 4 (phakic, ≥ 60 years). Objective accommodation and pupil diameter to 2.0- and 3.0-D stimuli were measured with a binocular open-field autorefractor. RESULTS: The mean objective accommodation was 0.29 ± 0.47 D, 0.01 ± 0.21 D, 1.00 ± 0.88 D, and 0.01 ± 0.13 to a 2.0-D stimulus, and 0.26 ± 0.51 D, - 0.06 ± 0.21 D, 1.42 ± 1.21 D, and - 0.06 ± 0.21 to a 3.0-D stimulus in groups 1, 2, 3, and 4, respectively. For both stimuli, the values in group 1 exceeded those in groups 2 and 4, and were smaller than those in group 3, while the values in group 3 exceeded those in groups 2 and 4. The mean pupillary diameter was - 0.5 ± 0.8 mm, - 0.3 ± 0.8 mm, - 0.6 ± 0.5 mm, and - 0.6 ± 0.9 mm to a 2.0-D stimulus, and - 0.6 ± 0.8 mm, - 0.6 ± 0.8 mm, - 0.9 ± 0.5 mm, and - 1.0 ± 1.1 mm to a 3.0-D stimulus in groups 1, 2, 3, and 4, respectively. There was significant correlation between objective accommodation and changes of pupil size for both stimuli. CONCLUSION: Age seems to play a role in objective accommodation among relatively young pseudophakic patients.

Analysis of pre-operative factors affecting range of optimal vaulting after implantation of 12.6-mm V4c implantable collamer lens in myopic eyes.

BACKGROUND: To evaluate clinical factors affecting postoperative vaulting in eyes that had achieved optimal vaulting within the range of 250-750 µm following implantation of 12.6-mm V4c implantable collamer lenses (ICL). METHODS: A total of 236 eyes of 236 patients that had achieved optimal vaulting following implantation of a 12.6-mm V4c ICL were retrospectively analyzed. Associations between postoperative vaulting and age, preoperative anterior chamber depth (ACD), preoperative axial length (AL), preoperative white-to-white diameter, preoperative pupil size, preoperative sulcus-to-sulcus diameter, and preoperative manifest refraction spherical equivalent were investigated using simple regression, stepwise multiple regression, and multinomial logistic regression analyses. RESULTS: Mean central vaulting at the 6-month follow-up was 519.0 ± 112.8 µm. Variables relevant to postoperative vaulting were, in order of influence, preoperative ACD (ß = 0.305, p <  0.001), preoperative pupil size (ß = 0.218, p <  0.001), and preoperative AL (ß = 0.171, p = 0.006). Low preoperative pupil size was associated with low optimal vaulting (250 to 450 µm), relative to that observed in the mid optimal vaulting group (451 to 550 µm) (odds ratio = 0.532, P = 0.021). Increasing preoperative ACD was associated with high optimal vaulting (551 and 750 µm), relative to that observed the mid optimal vaulting group (odds ratio = 6.340, P = 0.034). CONCLUSIONS: Myopic eyes with greater preoperative ACD, larger pupil size, and longer AL are predisposed to higher postoperative vaulting following 12.6-mm V4c ICL implantation. Therefore, the extremes of these parameters should be considered when choosing V4c ICL size.

Comparison between Wavefront-optimized and corneal Wavefront-guided Transepithelial photorefractive keratectomy in moderate to high astigmatism.

BACKGROUND: To compare the clinical outcomes of wavefront-optimized (WFO) transepithelial photorefractive keratectomy (trans-PRK) and corneal wavefront-guided (CWFG) trans-PRK for myopic eyes with moderate to high astigmatism. METHODS: One hundred ninety-six eyes (196 patients) with moderate to high astigmatism (≥ 1.75 D) treated with WFO or CWFG trans-PRK (101 and 95 eyes, respectively) were retrospectively registered. Safety, efficacy, predictability, vector analysis, and corneal aberrations were compared between groups preoperatively and at 6 months postoperatively. RESULTS: At postoperative 6 months, the mean logMAR uncorrected distance visual acuity was similar in the WFO (- 0.07 ± 0.08) and CWFG (- 0.07 ± 0.07) groups. Safety, efficacy, and predictability of refractive and visual outcomes were also similar. The correction indices were 1.02 ± 0.14 and 1.03 ± 0.13 in the WFO and CWFG groups, respectively, with no significant difference. The absolute values of the angle of error were significantly higher in the WFO group (2.28 ± 2.44 vs. 1.40 ± 1.40; P = 0.002). Corneal total root mean square higher-order aberrations and corneal spherical aberrations increased postoperatively in both groups; however, the change was smaller in the CWFG group. Corneal coma showed a significant increase postoperatively only in the WFO group. CONCLUSIONS: WFO and CWFG trans-PRK are safe and effective for correcting moderate to high astigmatism. However, CWFG trans-PRK provides a more predictable astigmatism correction axis and fewer induced corneal aberrations.

Melatonin reduces endoplasmic reticulum stress and corneal dystrophy-associated TGFBIp through activation of endoplasmic reticulum-associated protein degradation.

Endoplasmic reticulum (ER) stress is emerging as a factor for the pathogenesis of granular corneal dystrophy type 2 (GCD2). This study was designed to investigate the molecular mechanisms underlying the protective effects of melatonin on ER stress in GCD2. Our results showed that GCD2 corneal fibroblasts were more susceptible to ER stress-induced death than were wild-type cells. Melatonin significantly inhibited GCD2 corneal cell death, caspase-3 activation, and poly (ADP-ribose) polymerase 1 cleavage caused by the ER stress inducer, tunicamycin. Under ER stress, melatonin significantly suppressed the induction of immunoglobulin heavy-chain-binding protein (BiP) and activation of inositol-requiring enzyme 1α (IRE1α), and their downstream target, alternative splicing of X-box binding protein 1(XBP1). Notably, the reduction in BiP and IRE1α by melatonin was suppressed by the ubiquitin-proteasome inhibitor, MG132, but not by the autophagy inhibitor, bafilomycin A1, indicating involvement of the ER-associated protein degradation (ERAD) system. Melatonin treatment reduced the levels of transforming growth factor-ß-induced protein (TGFBIp) significantly, and this reduction was suppressed by MG132. We also found reduced mRNA expression of the ERAD system components HRD1 and SEL1L, and a reduced level of SEL1L protein in GCD2 cells. Interestingly, melatonin treatments enhanced SEL1L levels and suppressed the inhibition of SEL1L N-glycosylation caused by tunicamycin. In conclusion, this study provides new insights into the mechanisms by which melatonin confers its protective actions during ER stress. The results also indicate that melatonin might have potential as a therapeutic agent for ER stress-related diseases including GCD2.

Portable OCT-assisted surgical treatment of intracorneal pre-Descemet epithelial cyst: a case report.

BACKGROUND: Intracorneal epithelial cysts are a rare clinical condition that can occur anywhere in the corneal tissue; however, they appear most commonly in the stroma. They are sometimes challenging to treat because of their location, depth, and visual outcomes. Herein, we report a pre-Descemet epithelial cyst that was successfully treated surgically, with guidance from Fourier-domain optical coherence tomography (FD-OCT). CASE PRESENTATION: This interventional case report presents a patient with gradually decreasing vision caused by a pre-Descemet epithelial cyst. A 4-year-old girl with no history of trauma or ocular surgery showed a deep-seated intracorneal cyst in her left eye (8 o'clock corneoscleral area, dissecting into the pre-Descemet cornea). The cyst was threatening the visual axis. An epithelial cyst was diagnosed after drainage on the basis of the cyst contents. We irrigated inside the cyst using 10% trichloroacetic acid (TCA), distilled water, and 1% 5-fluorouracil (5-FU) solutions for chemical cyto-destruction of the lining epithelial cells of the cystic wall. We used a portable FD-OCT during operation to guide this procedure, without perforating the Descemet's membrane and endothelial layer. Recurrence could be prevented after removal of the cystic tissue located in the sclera area outside of the limbus. No recurrence was noted during the 4-year follow-up. CONCLUSION: When treating centrally deep-seated intracorneal epithelial cysts, clinicians must consider recurrence, endothelial damage, and visual outcome. Herein we report the case of a deep-seated, intracorneoscleral epithelial cyst that was completely resolved with chemical cyto-destruction and removal of the intrascleral cystic tissue under the guidance with FD-OCT; thus, endothelial damage could be minimized.

Visual rehabilitation in moderate keratoconus: combined corneal wavefront-guided transepithelial photorefractive keratectomy and high-fluence accelerated corneal collagen cross-linking after intracorneal ring segment implantation.

BACKGROUND: To investigate the effects of combined corneal wavefront-guided transepithelial photorefractive keratectomy (tPRK) and accelerated corneal collagen cross-linking (CXL) after intracorneal ring segment (ICRS) implantation in patients with moderate keratoconus. METHODS: Medical records of 23 eyes of 23 patients undergoing combined tPRK and CXL after ICRS implantation were retrospectively analyzed. Uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), manifest refraction spherical equivalent (MRSE), corneal indices based on Scheimpflug tomography, higher-order aberrations (HOAs), and corneal biomechanical properties were evaluated before and after ICRS implantation, and at 1, 3, and 6 months after combined tPRK and CXL. RESULTS: There were significant improvements in final logMAR UDVA and logMAR CDVA, and reductions in sphere, MRSE, and all corneal indices from baseline. Significant improvements in logMAR UDVA and reductions in sphere, MRSE, maximal keratometry, keratometry at the apex, mean keratometry, and keratoconus index were noted after ICRS implantation. After tPRK and CXL, significant improvements in logMAR UDVA and logMAR CDVA, and reductions in cylinder and all corneal indices were observed. There were significant improvements in final root mean square HOAs and coma aberrations from baseline, but no changes from baseline after ICRS implantation. Significant reductions in final radius and deformation amplitude from baseline were noted. CONCLUSIONS: Combined tPRK and accelerated CXL after ICRS implantation in moderate keratoconus appears to be a safe and effective treatment, providing an improvement in visual acuity, corneal indices, and HOAs. TRIAL REGISTRATION: retrospectively registered (identification no. NCT03355430 ). Date registered: 28/11/2017.

LASIK surgery of granular corneal dystrophy type 2 patients leads to accumulation and differential proteolytic processing of transforming growth factor beta-induced protein (TGFBIp).

More than 60 mutations in transforming growth factor beta-induced protein (TGFBIp) have been reported in humans causing a variety of phenotypic protein aggregates in the cornea, commonly termed corneal dystrophies. One mutation, generating an arginine to histidine amino acid substitution at position 124 in mature TGFBIp leads to granular corneal dystrophy type 2 (GCD2). Homozygous GCD2 cases develop massive protein accumulation early in life whereas heterozygous GCD2 cases become affected much later and generally with a much less severe outcome. However, if heterozygous GCD2 patients undergo laser-assisted in situ keratomileusis (LASIK) surgery protein accumulation is accelerated and they develop massive protein accumulations a few years after surgery. Here, we present the protein profile of aggregate-containing corneal tissue from GCD2 patients with a history of LASIK surgery using LC-MS/MS. Label-free quantification of corneal extracellular matrix proteins showed accumulation of TGFBIp. This was supported by 2DE and immunoblotting against TGFBIp that revealed the accumulation of full-length TGFBIp. In addition, a high molecular weight TGFBIp complex was more apparent in GCD2 patients after LASIK surgery, which may be important for the disease progression. Lastly, 2DE also revealed differential processing between GCD2 patients with a history of LASIK surgery when compared to healthy individuals.

TGF-ß regulates TGFBIp expression in corneal fibroblasts via miR-21, miR-181a, and Smad signaling.

Transforming growth factor-ß (TGF-ß)-induced gene (TGFBI) protein (TGFBIp) is associated with granular corneal dystrophy type 2 (GCD2). TGFBIp levels can affect GCD2 phenotypes, but the underlying molecular mechanisms have not been fully elucidated. We investigated the involvement of microRNA (miRNA) and TGF-ß in the regulation of TGFBIp expression in corneal fibroblasts. Ectopic expression of miR-9, miR-21, and miR-181a significantly decreased TGFBIp levels. Conversely, expression of miR-21 and miR-181a was induced by TGF-ß1. Expression of miR-21 was 10-fold higher than that of miR-9 and miR-181a in corneal fibroblasts. Additionally, TGF-ß1 expression was significantly higher than that of TGF-ß2 and TGF-ß3 in corneal fibroblasts, whereas expression of all three TGF-ß forms was not significantly different between wild-type (WT) and GCD2 homozygotes (HO) corneal fibroblasts. Taken together, these data indicate that TGFBIp expression is positively regulated by TGF-ß, whereas TGF-ß-induced miR-21 and miR-181a negatively regulate TGFBIp expression. In conclusion, TGFBIp levels in corneal fibroblasts are controlled via the coordinated activity of miR-21 and miR-181a and by Smad signaling. Pharmacologic modulation of these miRNAs and TGF-ß signaling could have therapeutic potential for TGFBI-associated corneal dystrophy, including GCD2.

4-Phenylbutyric acid reduces mutant-TGFBIp levels and ER stress through activation of ERAD pathway in corneal fibroblasts of granular corneal dystrophy type 2.

Granular corneal dystrophy type 2 (GCD2) is caused by a point mutation (R124H) in the transforming growth factor ß-induced (TGFBI) gene. In GCD2 corneal fibroblasts, secretion of the accumulated mutant TGFBI-encoded protein (TGFBIp) is delayed via the endoplasmic reticulum (ER)/Golgi-dependent secretory pathway. However, ER stress as the pathogenic mechanism underlying GCD2 has not been fully characterized. The aim of this study was to confirm whether ER stress is linked to GCD2 pathogenesis and whether the chemical chaperone, 4-phenylbutyric acid (4-PBA), could be exploited as a therapy for GCD2. We found that the ER chaperone binding immunoglobulin protein (BiP) and the protein disulfide isomerase (PDI) were elevated in GCD2. Western bolt analysis also showed a significant increase in both the protein levels and the phosphorylation of the key ER stress kinases, inositol-requiring enzyme 1α (IRE1α) and double stranded RNA activated protein kinase (PKR)-like ER kinase, as well as in levels of their downstream targets, X box-binding protein 1 (XBP1) and activating transcription factor 4, respectively, in GCD2 corneal fibroblasts. GCD2 cells were found to be more susceptible to ER stress-induced cell death than were wild-type corneal fibroblasts. Treatment with 4-PBA considerably reduced the levels of BiP, IRE1α, and XBP1 in GCD2 cells; notably, 4-PBA treatment significantly reduced the levels of TGFBIp without change in TGFBI mRNA levels. In addition, TGFBIp levels were significantly reduced under ER stress and this reduction was considerably suppressed by the ubiquitin proteasome inhibitor MG132, indicating TGFBIp degradation via the ER-associated degradation pathway. Treatment with 4-PBA not only protected against the GCD2 cell death induced by ER stress but also significantly suppressed the MG132-mediated increase in TGFBIp levels under ER stress. Together, these results suggest that ER stress might comprise an important factor in GCD2 pathophysiology and that the effects of 4-PBA treatment might have important implications for the development of GCD2 therapeutics.

TGFBIp regulates differentiation of EPC (CD133(+) C-kit(+) Lin(-) cells) to EC through activation of the Notch signaling pathway.

Endothelial progenitor cells (EPCs) in the circulatory system have been suggested to maintain vascular homeostasis and contribute to adult vascular regeneration and repair. These processes require that EPCs recognize the extracellular matrix (ECM), migrate, differentiate, and undergo tube morphogenesis. The ECM plays a critical role by providing biochemical and biophysical cues that regulate cellular behavior. Here, we tested the importance of transforming growth factor-beta-induced protein (TGFBIp) in regulation of the differentiation and angiogenic potential of human cord blood-derived EPCs (CD133(+) C-kit(+) Lin(-) cells). EPCs displayed increased endothelial differentiation when plated on TGFBIp compared to fibronectin. EPCs also exhibited increased adhesion and migration upon TGFBIp stimulation. Moreover, TGFBIp induced phosphorylation of the intracellular signaling molecules SRC, FAK, AKT, JNK, and ERK in EPCs. Using integrin-neutralizing antibodies, we showed that the effects of TGFBIp on EPCs are mediated by integrins α4 and α5. Furthermore, TGFBIp increased the adhesion, migration, and tube formation of CD34(+) mouse bone marrow stem cells in vitro. Gene expression analysis of EPCs plated on TGFBIp revealed that EPCs stimulated by TGFBIp exhibit increased expression of Notch ligands, such as delta-like 1 (DLL1) and Jagged1 (JAG1), through nuclear factor-kappa B signaling activation. Collectively, our findings demonstrate, for the first time, that locally generated TGFBIp at either wounds or tumor sites may contribute to differentiation and angiogenic function of EPCs by augmenting the recruitment of EPCs and regulating the expression of endothelial genes DLL1 and JAG1.

Heterochromatin Protein 1 Alpha (HP1α: CBX5) is a Key Regulator in Differentiation of Endothelial Progenitor Cells to Endothelial Cells.

As the ability to control the differentiation of endothelial stem/progenitor cells (EPCs) into vascular endothelial cell lineages could be useful for promoting neovascularization, it is important to obtain a deeper understanding of the epigenetic mechanisms that regulate EPC differentiation and neovascularization. Heterochromatin protein 1α (HP1α) is known to be involved in the epigenetic regulation of gene silencing. However, recent reports demonstrate that HP1α can also activate gene expression during cell differentiation. In this study, microarray analysis revealed that HP1α expression was induced during EPC differentiation and is associated with the expression of outgrowing endothelial cell (OEC)-specific protein markers. To explore the role of HP1α in the differentiation of EPCs to OECs, its expression was knocked-down or over-expressed in differentiating EPCs. Overexpression of HP1α promoted the differentiation and angiogenic activity of EPCs in vitro and in vivo, whereas knockdown of HP1α led to a defect in OEC migration, tube formation, and angiogenic sprouting activity. Gene expression profiling showed increased expression of angiogenic genes, including NOTCH1, cadherin-5, and angiopoietin-like-2, and decreased expression of progenitor cell marker genes, including CD133, CXCR4, and C-KIT, in HP1α-overexpressing EPCs. Also, increased HP1α at an early stage of EPC differentiation may regulate angiogenic gene transcription by interacting with chromatin that modifies epigenetic factors such as the methyl-CpG binding domain, Polycomb group ring finger 2, and DNA methyltransferases. Our findings demonstrate, for the first time, that HP1α plays an important role in the differentiation and angiogenic function of EPCs by regulating endothelial gene expression. Stem Cells 2015;33:1512-1522.

Autophagy in granular corneal dystrophy type 2.

Autophagy is a lysosomal degradative process that is essential for cellular homeostasis and metabolic stress adaptation. Defective autophagy is involved in the pathogenesis of many diseases including granular corneal dystrophy type 2 (GCD2). GCD2 is an autosomal dominant disorder caused by substitution of histidine for arginine at codon 124 (R124H) in the transforming growth factor ß-induced gene (TGFBI) on chromosome 5q31. Transforming growth factor ß-induced protein (TGFBIp) is degraded by autophagy, but mutant-TGFBIp accumulates in autophagosomes and/or lysosomes, despite significant activation of basal autophagy, in GCD2 corneal fibroblasts. Furthermore, inhibition of autophagy induces cell death of GCD2 corneal fibroblasts through active caspase-3. As there is currently no pharmacological treatment for GCD2, development of novel therapies is required. A potential strategy for preventing cytoplasmic accumulation of mutant-TGFBIp in GCD2 corneal fibroblasts is to enhance mutant-TGFBIp degradation. This could be achieved by activation of the autophagic pathway. Here, we will consider the role and the potential therapeutic benefits of autophagy in GCD2, with focus on TGFBIp degradation, in light of the recently established role of autophagy in protein degradation.

Inhibition of recurrence of epithelial ingrowth with an amniotic membrane pressure patch to a laser in situ keratomileusis flap with a central stellate laceration: a case report.

BACKGROUND: Surgical lifting and scraping is a well-known treatment for epithelial ingrowth, but treatment for epithelial ingrowth on the centrally perforated laser in situ keratomileusis (LASIK) flap has not been well studied. CASE PRESENTATION: We present a patient who had epithelial ingrowth to the backside of the flap through a central LASIK flap laceration with a stellate shape. The patient had undergone uncomplicated bilateral LASIK surgery 3 years before the trauma. Because the epithelial ingrowth was suspected during the first visit 2 weeks after trauma, and definite epithelial ingrowth was noted during the additional 2 week observation period, the ingrown epithelial tissue was removed mechanically with a number 15 blade after lifting of the flap 4 weeks after the trauma. An amniotic membrane overlay was applied over the cornea and was sutured tightly to the episclera to firmly press down the flap to the remaining posterior stroma, to prevent growth of the epithelium again to the backside of the flap. At the last follow-up visit, 5 months after surgery, the patient's visual acuity remained stabilized with no sign of recurrent epithelial ingrowth. CONCLUSION: These results showed that an amniotic membrane patch can be a useful adjuvant in the treatment of epithelial ingrowth, even on the central stellate laceration of the LASIK flap over the visual axis.