Enzymatic vitreolysis using reengineered Vibrio mimicus- derived collagenase

Abstract

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.

Figure 1

Cloning of VMA and iHA-VMA. A: Schematic representation of the constructs, Vibrio mimicus collagenase that remains active (VMA) and inhibitor of hyaluronic acid-VMA (iHA-VMA). B: Agarose gel showing the PCR amplicons (about 1.5 kb) corresponding to iHA-VMA and VMA, respectively, and 100 bp ladder. C: Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) profile of the purified proteins, VMA and iHA-VMA.

Figure 2

Activity profile of VMA and iHA-VMA. A: Gelatin zymography showing gelatinolytic activity corresponding to Vibrio mimicus collagenase that remains active (VMA) and inhibitor of hyaluronic acid-VMA (iHA-VMA). B: Fluorescamine-based activity assay was performed by incubating 250 μg of gelatin in the absence (control) or presence of VMA (1 µg), iHA-VMA (1 µg), or collagenase type IV (50 ng) in incubation buffer (pH 7.0) at 37 °C overnight, followed by a change in pH to 6.0 using citrate buffer, and the addition of fluorescamine. Fluorescence was recorded; the relative fluorescence intensity (RFI) is directly related to the gelatin degradation. Three independent experiments were performed, and data were plotted with mean and standard deviation (SD). C: Collagen type 1 degradation (associated with decreased intensity of β, α1, and α2 chains of collagen) was assessed in the absence or presence of 100 nU/μl VMA and iHA-VMA, respectively, on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE; p<0.05, n.s., not significant).

Figure 3

Assessment of binding affinity of iHA-VMA toward HA with flow cytometry. Binding of fluorescein isothiocyanate (FITC)-labeled hyaluronan acid (HA) to the ARPE-19 cell surface receptor (taken to be 100%) was monitored with flow cytometry. The addition of inhibitor of HA (iHA) in fusion with Vibrio mimicus collagenase that remains active (VMA) resulted in decreased fluorescence intensity. There was no change in signal intensity in the presence of VMA alone or irrelevant proteins (controls), such as bovine serum albumin (BSA) and CK or the cysteine knot domain of CTGF. The histogram corresponds to data from three independent flow cytometry experiments (p=0.0069, n.s., not significant).

Figure 4

Ex vivo OCT and SEM in goat eyeballs upon VMA treatment. A: Schematic representation of the experimental setup. B, C: Scanning electron microscopy (SEM) micrographs showing dense collagen fibrils on the retinal surface of the control eye. D, E: SEM micrographs corresponding to eyes treated with Vibrio mimicus collagenase that remains active (VMA), showing disorganization of the collagen matrix (D) or clearing of collagen fibers (E). F, G: Representative optical coherence tomography (OCT) images of control eyes injected with saline, showing no posterior vitreous detachment (PVD). H, I: Representative OCT images upon treatment with 750 μU (H) and 900 μU of VMA (I), respectively, showing detachment of the posterior hyaloid membrane of the vitreous from the inner limiting membrane (ILM) of the retina (shown by arrows). Scale bar: 10 µm.

Figure 5

Optical coherence tomography of rabbit eyes. Representative images of three pairs of rabbit eyes treated with saline (A, C, E), Vibrio mimicus collagenase that remains active (VMA; F), or inhibitor of hyaluronic acid-VMA (iHA-VMA; B, D). Floating posterior hyaloid membrane is seen floating just above the retina (B) or toward the anterior side, still attached to the optic disc (D, F), indicated by arrows.

Figure 6

Rheological studies of rabbit vitreous after euthanasia. Vitreous from four animals was removed, and storage modulus (G’) was monitored with rheology. Data from four pairs of eyes are represented here; two pairs belonged to the inhibitor of hyaluronic acid-Vibrio mimicus collagenase (VMC) that remains active (iHA-VMA) group (animals 1 and 2), and two pairs belonged to the VMA group (animals 3 and 4), with contralateral eyes serving as controls. Circle: control vitreous; square: treated vitreous.

Figure 7

Scanning electron microscopy of rabbit eyes treated with iHA-VMA and VMA. A, C: Control (contralateral) eyes showing dense vitreous cortical fibers, suggesting no induction of posterior vitreous detachment (PVD). B, D: Inner retinal surface of representative animals injected with inhibitor of hyaluronic acid-Vibrio mimicus collagenase that remains active (iHA-VMA) or VMA, showing the retinal surface devoid of any collagen fibers, suggestive of induction of complete PVD. Scale bar = 1 µm; 10,000X magnification.

Figure 8

Light micrographs of retinal cross-section (H&E staining). Hematoxylin and eosin (H&E) staining was performed for rabbits with an intravitreal injection of inhibitor of hyaluronic acid-Vibrio mimicus collagenase that remains active (iHA-VMA) or VMA, post-euthanasia. The retinal morphology of the eyes treated with iHA-VMA (A, B) was similar to that of the control eyes. Eyes injected with VMA (C, D) show intracellular edema, a significant increase in layer thickness, merging of the ONL and the INL (marked with asterisks in D), or rarification in the retinal layers and inflammatory cells above the retina (marked with arrows in C), possibly due to vascular leakage. The corresponding contralateral, control eyes (C, D) show no sign of toxicity. In each panel, the control and treated eyes belong to the same rabbit. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; PL, photoreceptor layer. Scale bar = 50 µm.