Department of Immunopathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
Department of Otolaryngology and Head & Neck surgery, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
Mol Vis. 2021 Apr 1;27:125-141. eCollection 2021.
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 are nonspecific and potentially toxic. We studied a novel collagenase from (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.
VMA and iHA-VMA were expressed in 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.
The activity of iHA-VMA and VMA was comparable and 65-fold lower than that of 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.
iHA-VMA displays targeted action confined to the vitreous and shows potential for safe pharmacologic vitreolysis.
胶原蛋白是玻璃体弹性和玻璃体视网膜粘连的主要成分。随着年龄的增长，分子重组导致这些粘连自发减弱，从而导致后玻璃体膜（PHM）与视网膜分离，这被称为完全玻璃体后脱离（PVD）。后玻璃体不完全分离或紧密粘连或两者兼而有之，可导致视网膜脱离、玻璃体黄斑牵引综合征或视网膜内膜形成，需要手术干预。药物玻璃体切除术有可能避免手术玻璃体切除术; 它在视网膜手术中也可用作辅助手段，以诱导 PVD。以前研究过的酶类试剂，如来源于 的胶原酶，是非特异性的，且具有潜在毒性。我们研究了一种新型胶原酶，来源于 （VMC），即使在删除 51 个 C 末端氨基酸后，它仍然保持活性（VMA）。为了将 VMA 的活性限制在玻璃体腔内，构建了一个融合构建体（透明质酸-VMA 的抑制剂 [iHA-VMA]），其中将 12 个氨基酸的肽（iHA，与 HA 结合）融合到 VMA 的 N 末端。该构建体在 PVD 背景下进行了评估。
VMA 和 iHA-VMA 在 中表达并纯化，并通过明胶酶谱、胶原蛋白降解测定、荧光胺测定和基于细胞的测定进行表征。在新西兰白化兔中进行了两组实验。A 组（n = 10）接受 iHA-VMA，B 组（n = 5）接受等量的 VMA。在两组中，对侧眼注射生理盐水作为对照。通过间接检眼镜、光学相干断层扫描（OCT）和 B 型超声检查对动物进行监测。通过视网膜组织的苏木精和伊红（H&E）染色评估视网膜毒性。
iHA-VMA 和 VMA 的活性相当，比 型胶原酶 IV 低 65 倍。在 iHA-VMA 组中，所有兔子（n = 10）均发生 PVD，6 只动物完全发生 PVD。未见明显的组织形态学变化。在 VMA 组中，5 只兔子中有 4 只发生完全 PVD;然而，在两只动物中观察到视网膜形态变化。
iHA-VMA 显示出靶向作用局限于玻璃体，并显示出安全的药物玻璃体溶解的潜力。
