Engineering of Ocriplasmin Variants by Bioinformatics Methods for the Reduction of Proteolytic and Autolytic Activities.

Background: Ocriplasmin has been developed for the induction of posterior vitreous detachment in patients with vitreomacular adhesion. At physiological pH, ocriplasmin is susceptible to autolytic and proteolytic degradation, limiting its activity duration. These undesirable properties of ocriplasmin can be reduced by site-directed mutagenesis, so that its enzymatic activities can be augmented. This study aimed to design ocriplasmin variants with improved biological/physicochemical characteristics via bioinformatics tools. Methods: This study was performed in Tabriz University of Medical Sciences, Tabriz, Iran, 2019. Through site-directed mutagenesis, three ocriplasmin variants were designed. Structural analysis was performed on the wild-type variant and the mutant variants using the Protein Interactions Calculator (PIC) server. The interactions between the S-2403 substrate and the ocriplasmin variants were studied by molecular docking simulations, and binding capability was evaluated by the calculation of free binding energy. The conformational features of protein-substrate complex systems for all the variants were evaluated using molecular dynamic simulations at 100 nanoseconds. Results: The structural analysis of ocriplasmin revealed that the substitution of threonine for alanine 59 significantly reduced proteolytic activity, while the substitution of glutamic acid for lysine 156 influenced autolytic function. The molecular docking simulation results indicated the appropriate binding of the substrate to the ocriplasmin variants with high-to-low affinities. The binding affinity of the wild-type variant for the substrate was higher than that between the mutant variants and the substrate. Simulation analyses, consisting of the root-mean-square deviation, the root-mean-square fluctuation, and the center-of-mass average distance showed a higher affinity of the substrate for the wild type than for the mutant variants. Conclusion: The mutational analysis of ocriplasmin revealed that A59T and K156E mutagenesis could be used for the development of a new variant with higher therapeutic efficacy.

Enzymatic vitreolysis using reengineered Vibrio mimicus-derived collagenase.

Purpose: Collagen is a key player contributing to vitreoelasticity and vitreoretinal adhesions. Molecular reorganization causes spontaneous weakening of these adhesions with age, resulting in the separation of the posterior hyaloid membrane (PHM) from the retina in what is called complete posterior vitreous detachment (PVD). Incomplete separation of the posterior hyaloid or tight adherence or both can lead to retinal detachment, vitreomacular traction syndrome, or epiretinal membrane formation, which requires surgical intervention. Pharmacological vitrectomy has the potential of avoiding surgical vitrectomy; it is also useful as an adjunct during retinal surgery to induce PVD. Previously studied enzymatic reagents, such as collagenase derived from Clostridium histolyticum, are nonspecific and potentially toxic. We studied a novel collagenase from Vibrio mimicus (VMC) which remains active (VMA), even after deletion of 51 C-terminal amino acids. To limit the activity of VMA to the vitreous cavity, a fusion construct (inhibitor of hyaluronic acid-VMA [iHA-VMA]) was made in which a 12-mer peptide (iHA, which binds to HA) was fused to the N-terminus of VMA. The construct was evaluated in the context of PVD. Methods: VMA and iHA-VMA were expressed in Escherichia coli, purified, and characterized with gelatin zymography, collagen degradation assay, fluorescamine-based assay, and cell-based assays. Two sets of experiments were performed in New Zealand albino rabbits. Group A (n = 10) received iHA-VMA, while group B (n = 5) received the equivalent dose of VMA. In both groups, saline was injected as a control in the contralateral eyes. Animals were monitored with indirect ophthalmoscopy, optical coherence tomography (OCT), and B-scan ultrasonography. Retinal toxicity was assessed with hematoxylin and eosin (H&E) staining of retinal tissue. Results: The activity of iHA-VMA and VMA was comparable and 65-fold lower than that of C. histolyticum collagenase Type IV. In the iHA-VMA group, all the rabbits (n = 10) developed PVD, with complete PVD seen in six animals. No statistically significant histomorphological changes were seen. In the VMA group, four of the five rabbits developed complete PVD; however, retinal morphological changes were seen in two animals. Conclusions: iHA-VMA displays targeted action confined to the vitreous and shows potential for safe pharmacologic vitreolysis.

[Retinal detachment after intravitreal ocriplasmin injection]. / Ablatio retinae nach intravitrealer Ocriplasmin-Injektion.

After an uneventful intravitreal injection (IVI) of Ocriplasmin in a patient with reduced visual acuity due to vitreomacular traction (VMT) and a small macular hole, retinal detachment occurred within a few days after the operation. Although retinal detachment is known as a risk factor of IVI this case is noteworthy: an excessive reaction occurred in the region of the vitreous body, which resulted in the development of severe traction on the retina leading to a posterior vitreous body detachment, retinal holes and complete retinal detachment. This possible complication should be discussed in the preoperative patient informed consent and the reason for this excessive reaction should be the subject of further investigations.

[Vitreomacular Traction Following Anti-VEGF Therapy - Two Cases]. / Vitreomakuläres Traktionssyndrom im Rahmen der Anti-VEGF-Therapie ­ 2 Kasuistiken.

Vitreomacular traction syndrome (VMTS) is defined as an incomplete or anomalous posterior vitreous detachment resulting in tractional forces at the macular region. In the context of anti-VEGF therapy, the formation of vitreoretinal traction has mainly been reported as a potential complication of VEGF inhibition in ischemic proliferative retinal disease, such as proliferative diabetic retinopathy. In this report, we present two patients who developed VMTS during anti-VEGF therapy for exudative age-related macular degeneration and diabetic macular edema. VMTS following anti-VEGF therapy of exudative macular diseases is rare. The exact pathomechanism remains unclear. However, there is a hypothesis that in eyes with adherent vitreous cortex, the induction of fibrosis as a result of the VEGF inhibition may lead to vitreomacular traction.

VITREOMACULAR TRACTION AFTER DEXAMETHASONE INTRAVITREAL IMPLANT (OZURDEX) INJECTION: THE EFFECT OF ANOMALOUS POSTERIOR VITREOUS DETACHMENT.

PURPOSE: To describe an unusual macular complication after dexamethasone intravitreal implant (Ozurdex) injection. METHODS: Case history and macular optical coherence tomography findings are described. The report describes a patient with proliferative diabetic retinopathy and vitreoschisis who received Ozurdex treatment for diabetic macular edema. RESULTS: After Ozurdex injection, visual acuity worsened because of newly formed vitreomacular traction that was demonstrated on optical coherence tomography imaging. A mechanism is proposed by which the medication might have contributed to this outcome. CONCLUSION: In this patient with vitreoschisis, a thickened posterior hyaloid membrane and traction developed after Ozurdex injection. The authors recommend careful evaluation of the macula before injection, particularly in diabetic patients with vitreoschisis.

[Research update of ocriplasmin for symptomatic vitreomacular adhesion].

Ocriplasmin is a recombinant truncated form of human serine protease plasmin with proteolytic activity to induce vitreous liquefaction and weaken vitreomacular adhesion, thereby facilitating posterior vitreous detachment and acting as a potential alternative to replace the more traumatic vitrectomy which can result in incomplete vitreoretinal separation. Intravitreal ocriplasmin for symptomatic vitreomacular adhesion has been systematically evaluated in clinical trials. Ocriplasmin at the recommended dose of a single 125 µg intravitreal injection has recently been approved for the treatment of symptomatic VMA in the USA and EU. This review is aiming to highlight the research update of ocriplasmin.

Review and perspectives on pharmacological vitreolysis.

The vitreous is involved in multiple diseases when an incomplete posterior vitreous detachment (PVD) occurs. An incomplete PVD can lead to several pathological conditions. Such visually threatening conditions are traditionally of exclusive surgical interest. In contrast, pharmacological vitreolysis is the effort to reduce or eliminate the pathogenetic role of the vitreous solely by means of drug delivery. Here we aim to review and summarize the evidence available to date about this challenging new approach.

Oxidative responses induced by pharmacologic vitreolysis and/or long-term hyperoxia treatment in rat lenses.

PURPOSE: The aim of the study was to investigate the protective effects of intact vitreous gel on the lens after pharmacologic vitreolysis and hyperoxia exposure in rats in vivo. METHODS: Eyes of Sprague-Dawley rats were induced to posterior vitreous detachment (PVD) by pharmacologic vitreolysis, and the rats with and without PVD were treated with hyperoxia 3 h per day for 5 months. Lens transparency was monitored by a slit-lamp biomicroscope. A series of biochemical measurements were made in extracts of the lens cortex and nucleus. Ascorbate levels were measured in the aqueous and vitreous humors. RESULTS: No significant differences in lens transparency or morphology were observed in all groups, and no significant biochemical changes were observed in the cortex or nucleus of lenses of the PVD group. In the lens nucleus, the values of water-soluble protein concentration in PVD + hyperoxia group were lower than that of the PVD group. The levels of water-soluble proteins, glutathione (GSH) and ascorbate decreased in the hyperoxia group with an intact vitreous body. Vitreolysis enhanced the effect of hyperoxia, decreasing soluble protein, GSH and ascorbate below the levels seen in eyes with vitreolysis alone. The levels of antioxidants and soluble proteins were lower in the lens nucleus, and the effects of vitreolysis plus hyperoxia were more significant in the nucleus. Hyperoxia and hyperoxia plus vitreolysis reduced catalase activity and increased oxidized GSH to a greater extent in the lens cortex, although these treatments increased protein-GSH mixed disulfides in both regions. Long-term hyperoxia also lowered ascorbate levels in the vitreous and aqueous humors, an effect that was enhanced by vitreolysis. CONCLUSIONS: Exposure to excess molecular oxygen produces significant oxidative damage to the lens, especially the lens nucleus. These effects were enhanced by pharmacologic vitreolysis, indicating that intact vitreous gel protects the lens from oxidative damage.

Efficient vitreolysis by combining plasmin and sulfur hexafluoride injection in a preclinical study in rabbit eyes.

PURPOSE: To investigate the efficacy of plasmin and sulfur hexafluoride (SF(6)) on the vitreoretinal junction, as well as the long-term safety in the eye and effect on the recipient's general health after application in the eye. METHODS: The study design included four groups of rabbits with three animals in each group. Group 1 received an intravitreal injection (IVI) of plasmin and SF(6) in the right eye; group 2 received an IVI of plasmin in the right eye; group 3 received an IVI of SF(6) in the right eye; and group 4 received an IVI of balanced salt solution in the right eye, which served as a normal control. Long-term safety (up to approximately three months) after plasmin and/or SF(6) injection was evaluated morphologically by clinical examination, histology, and immunohistochemistry, and functionally by electroretinograms (ERGs). General health evaluations after intravitreal injection included the assessment of weight gain, food intake, body temperature, and complete blood count analysis. RESULTS: Plasmin plus SF(6) injection resulted in complete posterior vitreous detachment (PVD), whereas plasmin or SF(6) injection alone resulted in only partial PVD. Balanced salt solution did not induce PVD. Eighty days after intravitreal injection, there were no major differences among the eyes of the three groups of animals compared with the normal control animals upon clinical evaluation, or regarding retinal morphology and ERGs. The lenses examined remained clear for up to 80 days following the intravitreal injection of plasmin plus SF(6), except one eye in the plasmin-treated group. ERGs decreased transiently one week after intravitreal injection in groups 1 through 3, but animals recovered fully to normal status afterward. General health was not affected after the injection of plasmin plus SF(6). CONCLUSIONS: Efficient vitreoretinal separation could be achieved, and an acceptable long-term safety profile was noted after plasmin plus SF(6) injection in the eye. No major ocular toxicity or systemic toxicity was found after the injection of plasmin plus SF(6). These results provide good support for the future clinical use of plasmin plus SF(6) for treatment of a variety of vitreoretinopathies.

Intravitreal tissue plasminogen activator to treat refractory diabetic macular edema by induction of posterior vitreous detachment.

PURPOSE: To evaluate the effects of intravitreal injection of recombinant tissue plasminogen activator (TPA) for the treatment of refractory diabetic macular edema. METHODS: A total of 27 patients with refractory diabetic macular edema with no evidence of posterior vitreous detachment were randomly assigned into follow-up (F/U) or TPA treatment groups. To control for the effects of intravitreal injection, an additional 14 patients with diabetic macular edema who were candidates for first-time intravitreal bevacizumab injection were enrolled as the IVB group. For the TPA and IVB groups, 25 µg of TPA or 1.25 mg of bevacizumab, respectively, were intravitreally injected. Fundoscopy, optical coherence tomography, and B-scan ultrasonography were performed at 1 week, 1 month, and 3 months after initiation of the study. RESULTS: The incidence of posterior vitreous detachment in fundoscopy over the follow-up period was 69.2% in the TPA group, which was significantly higher than that of the F/U and IVB groups (P = 0.001). Best-corrected visual acuity and changes in macular thickness did not significantly differ between the TPA and F/U groups over the 3-month period. CONCLUSION: Intravitreal TPA injection induces posterior vitreous detachment in patients with diabetic macular edema refractory to standard treatment but has no effect on macular thickness or best-corrected visual acuity within 3 months.

Predictability of vitreous detachment following intravitreal plasmin injection in diabetic macular edema associated with vitreomacular traction.

PURPOSE: To assess preoperative factors associated with postoperative posterior vitreous detachment (PVD) following intravitreal autologous plasmin injection in diabetic macular edema associated with vitreomacular traction. METHODS: Twenty-five eyes with diabetic macular edema associated with vitreomacular traction as documented with optical coherence tomography were included. Approximately 0.2 IU/0.2 ml of autologous plasmin was injected intravitreally. Condition of the posterior vitreous face (degree of detachment, thickness, reflectivity, and diameter of attached vitreous base) was evaluated preoperatively and postoperatively up to 3 months. RESULTS: PVD was achieved in ten eyes (41.7%). There was a significant difference (P = 0.03) in mean posterior vitreous face thickness between the eyes with PVD and the eyes with failed PVD. There was a significant correlation between PVD and both posterior vitreous face thickness (P < 0.03%) and degree of posterior vitreous face reflectivity (P = 0.002). CONCLUSION: In diabetic eyes with vitreomacular traction, the prediction of PVD after plasmin injection is governed by the condition of posterior vitreous face; mainly posterior vitreous face thickness and reflectivity. Eyes with thinner, less reflective posterior vitreous face are more likely to develop PVD.

Retinal tears after posterior vitreous detachment and vitreous hemorrhage in patients on systemic anticoagulants.

UNLABELLED: AIMS OR PURPOSE: To determine the rate of retinal tears (RTs) after posterior vitreous detachment (PVD) and vitreous hemorrhage (VH) in patients on systemic anticoagulants. METHODS: In all, 260 eyes of 260 patients with an acute PVD and VH were followed for evidence of an RT or detachment. Patients were divided into those taking systemic anticoagulants and those not taking anticoagulants. RESULTS: A total of 137 patients (53%) were taking anticoagulants, 123 (47%) were not. Overall, 72% of patients not taking any anticoagulant had evidence of an RT, whereas 46% of patients taking an anticoagulant had an RT (P-value 0.0002). Also, 37% of patients not taking an anticoagulant had a retinal detachment (RD), whereas 23% of patients taking any anticoagulant had an RD (P-value 0.01). CONCLUSIONS: In patients with an acute PVD and VH using anticoagulants, RTs and RDs were common. Anticoagulation status may be an important contributing factor in predicting the incidence of an RT or detachment.

Plasmin treatment accelerates vascular endothelial growth factor clearance from rabbit eyes.

PURPOSE: To investigate the clearance of vascular endothelial growth factor (VEGF) after the induction of posterior vitreous detachment by plasmin and/or SF(6). METHODS: The study design included four groups of rabbits: group 1 received an intravitreal injection of plasmin and SF(6) in the right eye, group 2 received an intravitreal injection of plasmin in the right eye, group 3 received an intravitreal injection of SF(6) in the right eye, and group 4 received an intravitreal injection of balanced salt solution in the right eye. Intravitreal injection of human VEGF (50 µL, 10 ng/µL) was performed in study eyes and control eyes 1 month after plasmin and/or SF(6) injection. Serum and vitreous samples were collected on days 1, 3, and 7 after VEGF injection to determine the serum and vitreous concentrations of VEGF. RESULTS: One day after VEGF injection, residual human VEGF concentration in the vitreous cavity was significantly lower in the plasmin- and SF(6)-treated eyes (group 1) and the plasmin-treated eyes (group 2) when compared with the control eyes (group 4) (P = 0.047 and 0.027, respectively). Three days after VEGF injection, the residual VEGF concentration in the vitreous cavity was still significantly lower in the plasmin- and SF(6)-treated eyes (group 1) when compared with the control eyes (group 4) (P = 0.025). CONCLUSIONS: Eyes treated with plasmin exhibit a more rapid clearance of exogenous VEGF than control eyes. This finding suggests a novel treatment for retinopathies associated with vitreous traction and VEGF elevation.

Novel vitreous modulators for pharmacologic vitreolysis in the treatment of diabetic retinopathy.

Vitreous constitutes about 80% of the volume of the human eye. It is an extended extracellular matrix that is composed of collagen, hyaluronan, and other extracellular matrix molecules, but mostly water. In both health as well as disease, especially diabetic retinopathy (DR), special attention should be drawn to the posterior vitreous cortex and its relation to the retinal surface. The important role of vitreous in the pathogenesis of proliferative DR has already been demonstrated by several experimental and clinical studies. Thus, vitreo-retinal separation by pharmacologic vitreolysis and/or removal by surgical means are appropriate approaches to interrupt the pathogenic contribution of vitreous and prevent progression of diabetic retinopathy to more advanced stages. This review describes various aspects of the molecular morphology and structural anatomy of vitreous and the vitreo-retinal interface, as well as the role of vitreous in the pathophysiology of DR. Lastly, this treatise provides a critical analysis of novel vitreous modulators for pharmacologic vitreolysis in the treatment of DR. Microplasmin is currently the most promising approach to treat vitreoretinal traction by pharmacologic vitreolysis.

Pharmacologic vitreolysis in diabetic retinopathy.

Diabetic retinopathy remains a major cause of worldwide preventable blindness. The vitreo-retinal interface plays a critical role in the pathogenesis of diabetic retinopathy. The term pharmacologic vitreolysis refers to the use of enzymes to liquefy the vitreous gel, and to induce posterior vitreous detachment (PVD). Intravitreal ovine hyaluronidase injection was effective in clearing vitreous hemorrhage. Several human case series demonstrated that intravitreal injection of autologous plasmin enzyme was a safe and effective adjunct to vitreous surgery for the treatment of diabetic macular edema and proliferative diabetic retinopathy. Recently, it was shown that intravitreal injection of plasmin enzyme without the performance of vitrectomy induced complete PVD and reduced macular thickening due to refractory diabetic macular edema.

Characterization of a stabilized form of microplasmin for the induction of posterior vitreous detachment.

PURPOSE: To investigate the stability and safety of a diluted acidified form of microplasmin and its ability to induce a posterior vitreous detachment (PVD) following intravitreal injection in post-mortem porcine eyes. METHODS: Microplasmin diluted in normal saline (NS) and balanced salt solution (BSS+) was assayed for residual activity by hydrolysis of the chromogenic substrate Glu-Phe-Lys-pNA. Residual activity on vitreous was determined by injecting aliquots of microplasmin reconstituted in balanced salt solution (BSS+) or normal saline (NS) kept at room temperature (RT) for up to 1 hr, then injected in aliquots of porcine vitreous and incubated for 2 hr at 37°C. The breakdown products were submitted to SDS Page electrophoresis and compared to determine the level of enzymatic activity. Pig eyes were incubated with graded concentrations of microplasmin 0.625, 1.25, or 2.50 mg/mL reconstituted in BBS+ or NS. Morphologic alterations and the ability to induce a PVD was assessed by light and electron microscopy. RESULTS: Microplasmin's enzymatic activity in an in vitro assay in BSS+ was 70% of its baseline value after 30 min, and about 50% after 60 min at RT. The corresponding effect on degradation of vitreous gel was 60 and 40% baseline at 30 and 60 min. There was no loss of activity in the microplasmin diluted in normal saline over this time period. Dilution of acidified microplasmin in normal saline did not lead to structural changes within the retina. A dose dependent PVD was observed in eyes treated with microplasmin diluted in NS. CONCLUSIONS: Acidified (stabilized) microplasmin has the same intraocular activity profile as microplasmin at a neutral pH. Better retention of activity at room temperature makes it a better candidate for use in clinical practice.

Microplasmin degrades fibronectin and laminin at vitreoretinal interface and outer retina during enzymatic vitrectomy.

PURPOSE: To determine whether intravitreal administration of microplasmin (microPlm) will degrade fibronectin (FN) and laminin (LN) in rat retina during microPlm-induced posterior vitreous detachment (PVD). METHODS: Increasing doses of microPlm, from 0.01 U to 0.03 U, were injected into the left eyes of 60 Sprague-Dawley rats to induce PVD. The right eyes were injected with the same volume of balanced salt solution (BSS). Histochemistry, scanning electron microscopy (SEM), and phase contrast microscopy were performed after 1 day and 7 days, to assess the remnant vitreous cortex. The FN and LN level located at the vitreoretinal interface and the outer retina were detected by immunohistochemistry. RESULTS: microPlm induced complete PVD in a dose-dependent fashion, without internal limiting membrane (ILM) damage (P = 0.0001, r = -0.479). The FN and LN in the photoreceptor cell layer (PCL) were completely degraded in all microPlm-treated eyes. In eyes with complete PVD, the FN, but not the LN, was completely removed from the ILM by microPlm treatment. CONCLUSION: Intravitreal injection of microplasmin degraded FN and LN at the vitreoretinal junction as well as at the outer retina.

[Pharmacologic vitreolysis in diabetic rats].

OBJECTIVE: To evaluate the efficacy and safety of plasmin or hyaluronidase in the inducing of posterior vitreous detachment in diabetic rats. METHODS: Forty SD rats were induced diabetes by Streptozotocin (STZ). Four weeks later, these rats were randomized into 4 groups: rats in group A received 5 U hyaluronidase intravitreal injection in right eyes; rats in group B received 0.5 U plasmin intravitreal injection in right eyes; rats in group C received 0.5 U plasmin +5 U hyaluronidase intravitreal injection in right eyes; rats in group D received BSS (balance salt solution) 2 microl intravitreal injection in right eyes. Clinical examination were performed at 1, 3, 7 days after injection. After 1 week, scanning electron microscope (SEM) was performed to judge whether the PVD was induced. ERG and histology were examined to evaluate the toxicity after the intravitreal injection of these two drugs. RESULTS: No PVD was found in SEM disclosed group A and group D. Forty percent eyes were induced complete PVD in groups B, and one hundred percent eyes were induced complete PVD in group C. ERG and histology showed no toxicity changes in any group. CONCLUSION: Intravitreal injection of 0.5 U plasmin + 5 U hyaluronidase can induce complete PVD without obvious toxicity in diabetic rats. Solo usage of plasmin or hyaluronidase can not induce complete PVD.