Gabriel M. Gordon

Virally delivered Channelrhodopsin-2 Safely and Effectively Restores Visual Function in Multiple Mouse Models of Blindness

Previous work established retinal expression of channelrhodopsin-2 (ChR2), an algal cation channel gated by light, restored physiological and behavioral visual responses in otherwise blind rd1 mice. However, a viable ChR2-based human therapy must meet several key criteria: (i) ChR2 expression must be targeted, robust, and long-term, (ii) ChR2 must provide long-term and continuous therapeutic efficacy, and (iii) both viral vector delivery and ChR2 expression must be safe. Here, we demonstrate the development of a clinically relevant therapy for late stage retinal degeneration using ChR2. We achieved specific and stable expression of ChR2 in ON bipolar cells using a recombinant adeno-associated viral vector (rAAV) packaged in a tyrosine-mutated capsid. Targeted expression led to ChR2-driven electrophysiological ON responses in postsynaptic retinal ganglion cells and significant improvement in visually guided behavior for multiple models of blindness up to 10 months postinjection. Light levels to elicit visually guided behavioral responses were within the physiological range of cone photoreceptors. Finally, chronic ChR2 expression was nontoxic, with transgene biodistribution limited to the eye. No measurable immune or inflammatory response was observed following intraocular vector administration. Together, these data indicate that virally delivered ChR2 can provide a viable and efficacious clinical therapy for photoreceptor disease-related blindness.

Cytokines and signaling pathways regulating matrix metalloproteinase-9 (MMP-9) expression in corneal epithelial cells.

Matrix metalloproteinase‐9 (MMP‐9) is a well‐known regulator and effecter of many cellular processes including wound healing. In the cornea, either too much or too little MMP‐9 can be detrimental to overall wound repair. We investigated the secreted factors as well as the intracellular signaling pathways and the promoter sequences that mediate this regulation. Primary culture rabbit corneal epithelial cells were treated with various cytokines alone or in different combinations and MMP‐9 induction was assessed by gel zymography. Pharmacological inhibitors were used to determine the intracellular signaling pathways induced by the cytokines tested and deletion promoter constructs were created to determine the regions of the MMP‐9 promoter involved in the cytokine regulation, thereby assessing the exact transcription factors binding the MMP‐9 promoter. We found that two cytokine families, transforming growth factor β (TGF‐β) and interleukin 1 (IL‐1), act additively in an isoform non‐specific manner to induce MMP‐9 in this cell type. Our data suggest TGF‐β mediated MMP‐9 induction may be regulated by the NF‐κB, Smad3, and JNK pathways, whereas the IL‐1β mediated induction may be regulated by the NF‐κB and p38 pathways. Inhibition of the p38, NF‐κB, or JNK pathways significantly reduced, but did not abrogate, basal MMP‐9 levels. Inhibition of the ERK pathway did not have an effect on MMP‐9 mediated expression in either the treated or untreated co‐transfected cells. J. Cell. Physiol. 221: 402–411, 2009.

Systemic Safety of Prolonged Monthly Anti-Vascular Endothelial Growth Factor Therapy for Diabetic Macular Edema: A Systematic Review and Meta-analysis.

IMPORTANCE Anti-vascular endothelial growth factor (VEGF) therapy is commonly used to treat numerous retinal conditions and appears safe, yet controversy remains regarding systemic safety. OBJECTIVE To evaluate the systemic safety of intravitreous anti-VEGF injections in high-risk patients with diabetic macular edema (DME) and to investigate separately the subgroup of these patients with the highest level of exposure to anti-VEGF monthly treatment for 2 years. DATA SOURCES A search of MEDLINE, Cochrane Central Register of Controlled Trials, clincaltrials.gov, and ophthalmology congress abstracts January 1, 1947, to May 19, 2015. STUDY SELECTION Randomized clinical trials were selected that evaluated monthly anti-VEGF injections for DME for 2 years and reported the outcome measures of cerebrovascular accidents, myocardial infarctions, arteriothrombotic events, and mortality. DATA EXTRACTION AND SYNTHESIS Two reviewers collected data independently from each study for the meta-analysis. Data were pooled using a fixed-effects model and analyzed from November 6, 2014, to June 28, 2015. Peto odds ratios with 95% CIs were calculated. MAIN OUTCOMES AND MEASURES Primary end points included cerebrovascular accidents and all-cause mortality in the highest-dose arms. Secondary outcomes included myocardial infarctions, arteriothrombotic events, and vascular-related death. RESULTS Of 1126 articles reviewed, 598 were removed as duplicate studies and 524, for lack of monthly treatment data for 2 years, leaving 4 studies for the meta-analysis that met the search criteria: 2 trials using monthly aflibercept and 2 using monthly ranibizumab, representing 1328 patients. The primary evaluation (1078 patients) combined the monthly aflibercept and the 0.5-mg ranibizumab arms and yielded an increased risk for death compared with sham and laser treatments (odds ratio [OR], 2.98; 95% CI, 1.44-6.14; P = .003). Analysis including monthly aflibercept and 0.5-mg ranibizumab yielded an increased risk for cerebrovascular accidents (OR, 2.33; 95% CI, 1.04-5.22; P = .04) and vascular death (OR, 2.51; 95% CI, 1.08-5.82; P = .03). No definitive increased risk for myocardial infarctions and arteriothrombotic events was seen with all dose combinations. CONCLUSIONS AND RELEVANCE In this meta-analysis of anti-VEGF agents for patients with DME, assessment of the highest-level exposure group (those high-risk patients with DME who received 2 years of monthly treatment) revealed a possible increased risk for death and potentially for cerebrovascular accidents. Consideration of total exposure to anti-VEGF agents when treating those at high risk for vascular disease may be important.

Interaction of Clusterin and Matrix Metalloproteinase-9 and Its Implication for Epithelial Homeostasis and Inflammation

Uncontrolled increases of matrix metalloproteinase-9 (MMP-9) activity have been causally linked to epithelial barrier disruption and severe symptoms of inflammatory diseases such as dry eye (DE). The data presented here show that the anti-inflammatory, cytoprotective intracellular and extracellular chaperone protein clusterin (CLU) interacts with MMP-9 both inside and outside epithelial cells. CLU bound very strongly to active MMP-9, with an affinity constant K(D) of 2.63 nmol/L. Unexpectedly, CLU had a much higher affinity for pro-MMP-9 than for active MMP-9 or pro-MMP-2, requiring the N-terminal propeptide domain of pro-MMP-9. The significance of the interaction between CLU and MMP-9 was demonstrated by the observation that CLU prevents stress-induced MMP-9 aggregation and inhibits MMP-9 enzymatic activity. Furthermore, CLU inhibited MMP-9-mediated disintegration of the tight junction structure formed between human epithelial cells. Additionally, CLU inhibited enzymatic activities of MMP-2, MMP-3, and MMP-7. Treatment with proinflammatory cytokines, which are known to increase MMP-9 transcription under inflammatory conditions, reduced the expression of CLU in human epithelial cells. Similarly, in a mouse model of human DE, inflammatory stress depleted CLU in the ocular surface epithelium but allowed MMP-9 to prevail therein. The present results thus provide novel insights into previously unrecognized mechanisms by which CLU maintains fluid-epithelial interface homeostasis, thereby preventing the onset of inflammatory conditions, especially where MMP-9 is actively involved.

Comprehensive gene expression profiling and functional analysis of matrix metalloproteinases and TIMPs, and identification of ADAM-10 gene expression, in a corneal model of epithelial resurfacing

This study provides a comprehensive expression analysis for the entire matrix metalloproteinase (MMP) gene family during the process of epithelial resurfacing following corneal abrasion injury in the mouse. The mRNA levels for all known MMP genes expressed in mouse, the related enzyme ADAM‐10, and the known tissue inhibitors of metalloproteinases (TIMPs) were determined semi‐quantitatively by reverse transcriptase‐polymerase chain reaction (RT‐PCR) in the uninjured epithelium, and in the epithelial tissue resurfacing the abraded area or residing in its periphery at two time points: during the epithelial migration phase and immediately following wound closure. The mRNA levels for MMP‐1a, ‐1b, ‐9, ‐10, ‐12, and ‐13 as well as TIMP‐1 were significantly up‐regulated in the migrating corneal epithelium. After wound resurfacing, the mRNA levels for all of these MMPs were down‐regulated, although MMP‐1a, ‐1b, and ‐13 remained significantly elevated in comparison to the uninjured epithelium. The only gene found to be down‐regulated was TIMP‐3, which occurred throughout the wound‐healing process. During resurfacing, MMP‐9 was localized to the front of the migrating epithelium, MMP‐10 and ‐13 were localized throughout the migrating epithelium, and MMP‐13 could also be found in the periphery. Following epithelial closure, immunoreactive MMPs‐9 and ‐10 became undetectable, but MMP‐13 continued to be found throughout the epithelium. Functional analysis of MMP‐10 revealed no effects on epithelial migration or cell proliferation. In conclusion, distinct MMP temporal–spatial profiles define the uninjured corneal epithelium and the corneal epithelium at different stages of regeneration. An extensive review of the literature is also provided in the discussion. J. Cell. Physiol. 226: 1461–1470, 2011.

Correlation Between Epithelial Ingrowth and Basement Membrane Remodeling in Human Corneas After Laser-Assisted In Situ Keratomileusis

OBJECTIVE To further investigate the hypothesis that epithelial ingrowth in human corneas after laser-assisted in situ keratomileusis (LASIK) correlates with basement membrane remodeling, as suggested by the presence of matrix metalloproteinase 9 around epithelial cells in the lamellar scar. METHODS Immunohistochemical analysis and transmission electron microscopy were applied to human postmortem corneas with post-LASIK epithelial ingrowth. RESULTS Epithelial ingrowth into the flap margin was observed in 8 of 18 corneas (44%). Matrix metalloproteinase 9 immunolocalized around ingrown epithelium in 6 of these 8 corneas (75%). There was a positive correlation between the presence of matrix metalloproteinase 9 at the wound margin and discontinuities in the basement membrane, as determined by laminin and beta(4) integrin immunofluorescence. Transforming growth factor beta2 was present into the stroma of some corneas with epithelial ingrowth and interrupted basement membrane, suggesting some degree of epithelial-stromal interaction. Transmission electron microscopy confirmed large areas of remodeled basement membrane along ingrown epithelial cells. CONCLUSIONS The neo-basement membrane components underlying the ingrown cells in human corneas with epithelial ingrowth after LASIK appear to be partially disassembled. Epithelial-stromal interaction over time may be related to prolonged wound healing remodeling, which calls into question the stability of the flap.

A Novel Mechanism of Increased Infections in Contact Lens Wearers

PURPOSE It is well documented that contact lens wearers have much higher incidences of corneal infections compared with those of non-contact lens wearers, although the exact cause(s) of this increased susceptibility has not been identified. A distinct subset of mucins (MUCs) is present on the ocular surface, acting to protect the integrity of the corneal epithelium. This study was performed to determine whether multipurpose contact lens solutions (MPCLSs) can cause increased infections in the cornea by destroying the protective cell-bound mucin layer. METHODS An immortalized human corneal limbal epithelial cell line was treated in the presence of four commonly used MPCLSs or PBS and the expression and release of MUC-16 was assessed. Cells were also cultured with Pseudomonas aeruginosa after MPCLS treatment and internalization of bacteria was assessed by quantitative genomic PCR. Loss of MUC-16 was then correlated with infection rates. RESULTS Each of the four commonly used MPCLSs examined in this study differentially affected mucin release. The relative effect was correlated with an increase in infection of corneal epithelial cells by P. aeruginosa. CONCLUSIONS The results of this study are consistent with the hypothesis that MPCLSs cause increased infections in the cornea by destroying the protective cell-bound mucin layer.

Correlations of long-term matrix metalloproteinase localization in human corneas after successful laser-assisted in situ keratomileusis with minor complications at the flap margin.

OBJECTIVE To determine whether matrix metalloproteinases (MMPs) are present long-term in human corneas after successful laser-assisted in situ keratomileusis (LASIK). METHODS Eighteen postmortem corneas from 10 patients with postoperative intervals of 2 to 8 years after LASIK surgery and 4 normal control corneas from 2 patients were collected from US eye banks and processed for histologic analysis and immunolocalization with antibodies to MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, and MMP-14. RESULTS Matrix metalloproteinase 7 was present in the epithelium of all corneas. Other MMPs were localized to the wound margin in some post-LASIK corneas. Matrix metalloproteinase 9 was detected around epithelial cells trapped in the lamellar scar in 5 of 6 corneas with epithelial ingrowth. Various MMPs were detected in fibrotic tissue at the wound margin in 2 of 2 corneas with flap retraction. CONCLUSIONS The presence of MMPs in post-LASIK corneas correlates with an ongoing wound healing process associated with minor post-LASIK complications. Matrix metalloproteinases might contribute to instances of ongoing flap instability, and if so, judicious use of MMP inhibitors could provide benefit.

Mechanisms for PDGF, a serum cytokine, stimulating loss of corneal keratocyte crystallins.

Purpose: As corneal stromal cells (keratocytes) become activated before transition to the fibroblastic repair phenotype in response to injury (in situ) or serum (in culture), the corneal crystallins, transketolase (TKT) and aldehyde dehydrogenase (ALDH1A1), are lost. The authors previously showed that the serum cytokine platelet-derived growth factor-BB (PDGF), but not transforming growth factor beta2 (TGF-beta2), stimulates TKT loss. The goal of this study was to further define the molecular mechanisms for PDGF-stimulated loss of crystallins to elucidate the pathway for keratocyte activation. Methods: Freshly isolated rabbit corneal keratocytes were plated in serum-free medium with or without PDGF and/or specific inhibitors of the PDGF-relevant signal pathway components, PDGF receptor, PI3K/AKT, or ras-initiated MAPK proteins. Intracellular TKT protein levels were quantified by immunoblotting. Ubiquitinated TKT levels were assessed by immunoprecipitation, and TKT messenger RNA (mRNA) levels were quantified by quantitative reverse transcription–polymerase chain reaction. Results: PDGF treatment at the same time as inhibition of PDGF receptor, Akt, JNK, and ubiquitin–proteasome pathway prevented PDGF-induced TKT protein loss. In contrast, treatment with PDGF did not affect TKT mRNA levels. Conclusions: The results suggest that PDGF-stimulated TKT loss is mediated through cross talk between PI3K-independent Akt and JNK. This signaling pathway leads to the degradation of existing TKT protein but does not compromise the accumulation of TKT mRNA. Therefore, cells retain the potential to reaccumulate TKT protein that is enabled by PDGF removal. These findings suggest that targeting PDGF signaling could improve repair outcomes after surgical procedures in the cornea.

Large Silicone Droplets After Intravitreal Bevacizumab (avastin)

Purpose: Despite its off-label status, intravitreal bevacizumab is the most commonly used intraocular anti–vascular endothelial growth factor agent. Regulation of compounding pharmacies has recently increased to make compounded pharmaceuticals safer. Despite these changes, a marked increase in symptomatic, large silicone oil droplets following intravitreal bevacizumab injections was noticed. Methods: Retrospective chart review was performed. Within a single private practice, patients who were noted to have large or symptomatic silicone oil bubbles after an intravitreal injection were reviewed. Results: A recent, dramatic increase in the incidence of large or symptomatic silicone oil droplets was noted, with 23 cases noted in the past 5 months, compared with 1 in the previous decade. Patients frequently noted a circular floater consisting of a dark ring surrounding a bright central area immediately following an injection of intravitreal bevacizumab. All bevacizumab injections were from single-piece insulin syringes. B-scan ultrasonography produced a very characteristic reverberation pattern. No inflammation or visual acuity loss was noted because of the droplets; however, some patients were annoyed enough to consider vitrectomy. Conclusion: Patients should be carefully evaluated for this possibility, and the characteristic symptom of a round floater consisting of a dark ring surrounding a bright center, and the prominent reverberation pattern on B-scan ultrasonography may help increase detection. Changes in consent forms and discussion of this possibility are indicated while investigation into the cause of this increased incidence continues, especially if one is administering bevacizumab via the one-piece insulin syringes commonly used by compound pharmacies.