The human Descemet's membrane and lens capsule: Protein composition and biomechanical properties.

The Descemet's membrane (DM) and the lens capsule (LC) are two ocular basement membranes (BMs) that are essential in maintaining stability and structure of the cornea and lens. In this study, we investigated the proteomes and biomechanical properties of these two materials to uncover common and unique properties. We also screened for possible protein changes during diabetes. LC-MS/MS was used to determine the proteomes of both BMs. Biomechanical measurements were conducted by atomic force microscopy (AFM) in force spectroscopy mode, and complemented with immunofluorescence microscopy. Proteome analysis showed that all six existing collagen IV chains represent 70% of all LC-protein, and are thus the dominant components of the LC. The DM on the other hand is predominantly composed of a single protein, TGF-induced protein, which accounted for around 50% of all DM-protein. Four collagen IV-family members in DM accounted for only 10% of the DM protein. Unlike the retinal vascular BMs, the LC and DM do not undergo significant changes in their protein compositions during diabetes. Nanomechanical measurements showed that the endothelial/epithelial sides of both BMs are stiffer than their respective stromal/anterior-chamber sides, and both endothelial and stromal sides of the DM were stiffer than the epithelial and anterior-chamber sides of the LC. Long-term diabetes did not change the stiffness of the DM and LC. In summary, our analyses show that the protein composition and biomechanical properties of the DM and LC are different, i.e., the LC is softer than DM despite a significantly higher concentration of collagen IV family members. This finding is unexpected, as collagen IV members are presumed to be responsible for BM stiffness. Diabetes had no significant effect on the protein composition and the biomechanical properties of both the DM and LC.

The biophysical and compositional properties of human basement membranes.

Basement membranes are among the most widespread, non-cellular functional materials in metazoan organisms. Despite this ubiquity, the links between their compositional and biophysical properties are often difficult to establish due to their thin and delicate nature. In this article, we examine these features on a molecular level by combining results from proteomics, elastic, and nanomechanical analyses across a selection of human basement membranes. Comparing results between these different membranes connects certain compositional attributes to distinct nanomechanical signatures and further demonstrates to what extent water defines these properties. In all, these data underline BMs as stiff yet highly elastic connective tissue layers and highlight how the interplay between composition, mechanics and hydration yields such exceptionally adaptable materials.

Diabetes-induced morphological, biomechanical, and compositional changes in ocular basement membranes.

The current study investigates the structural and compositional changes of ocular basement membranes (BMs) during long-term diabetes. By comparing retinal vascular BMs and the inner limiting membrane (ILM) from diabetic and non-diabetic human eyes by light and transmission electron microscopy (TEM), a massive, diabetes-related increase in the thickness of these BMs was detected. The increase in ILM thickness was confirmed by atomic force microscopy (AFM) on native ILM flat-mount preparations. AFM also detected a diabetes-induced increase in ILM stiffness. The changes in BM morphology and biophysical properties were accompanied by partial changes in the biochemical composition as shown by immunocytochemistry and western blots: agrin, fibronectin and tenascin underwent relative increases in concentration in diabetic BMs as compared to non-diabetic BMs. Fibronectin and tenascin were particularly high in the BMs of outlining microvascular aneurisms. The present data showed that retinal vascular BMs and the ILM undergo morphological, biomechanical and compositional changes during long-term diabetes. The increase in BM thickness not only resulted from an up-regulation of the standard BM proteins, but also from the expression of diabetes-specific extracellular matrix proteins that are not normally found in retinal BMs.

Correction: Diabetes-related changes in the protein composition and the biomechanical properties of human retinal vascular basement membranes.

[This corrects the article DOI: 10.1371/journal.pone.0189857.].

Diabetes-related changes in the protein composition and the biomechanical properties of human retinal vascular basement membranes.

Basement membranes (BMs) are specialized sheets of extracellular matrix that outline epithelial cell layers, muscle fibers, blood vessels, and peripheral nerves. A well-documented histological hallmark of progressing diabetes is a major increase in vascular BM thickness. In order to investigate whether this structural change is accompanied by a change in the protein composition, we compared the proteomes of retinal vascular BMs from diabetic and non-diabetic donors by using LC-MS/MS. Data analysis showed that seventeen extracellular matrix (ECM)-associated proteins were more abundant in diabetic than non-diabetic vascular BMs. Four ECM proteins were more abundant in non-diabetic than in diabetic BMs. Most of the over-expressed proteins implicate a complement-mediated chronic inflammatory process in the diabetic retinal vasculature. We also found an up-regulation of norrin, a protein that is known to promote vascular proliferation, possibly contributing to the vascular remodeling during diabetes. Many of the over-expressed proteins were localized to microvascular aneurisms. Further, the overall stoichiometry of proteins was changed, such that the relative abundance of collagens in BMs from diabetic patients was higher than normal. Biomechanical measurements of vascular BM flat mounts using AFM showed that their outer surface was softer than normal.

Superior Rim Stability of the Lens Capsule Following Manual Over Femtosecond Laser Capsulotomy.

PURPOSE: Cataract surgery requires the removal of a circular segment of the anterior lens capsule (LC) by manual or femtosecond laser (FL) capsulotomy. Tears in the remaining anterior LC may compromise surgical outcome. We investigated whether biophysical differences in the rim properties of the LC remaining in the patient after manual or FL capsulotomy (FLC) lead to different risks with regard to anterior tear formation. METHODS: Lens capsule samples obtained by either continuous curvilinear capsulorhexis (CCC) or FLC were investigated by light microscopy, laser scanning confocal microscopy, and scanning electron microscopy; atomic force microscopy (AFM) was used to test the biomechanical properties of the LC. The mechanical stability of the LC following either of the two capsulotomy techniques was simulated by using finite-element modeling. RESULTS: Continuous curvilinear capsulorhexis produced wedge-shaped, uniform rims, while FLC resulted in nearly perpendicular, frayed rims with numerous notches. The LC is composed of two sublayers: a stiff epithelial layer that is abundant with laminin and a softer anterior chamber layer that is predominantly made from collagen IV. Computer models show that stress is uniformly distributed over the entire rim after CCC, while focal high stress concentrations are observed in the frayed profiles of LC after FLC, making the latter procedure more prone to anterior tear formation. CONCLUSIONS: Finite-element modeling based on three-dimensional AFM maps indicated that CCC leads to a capsulotomy rim with higher stress resistance, leading to a lower propensity for anterior radial tears than FLC.

New concepts in basement membrane biology.

Basement membranes (BMs) are thin sheets of extracellular matrix that outline epithelia, muscle fibers, blood vessels and peripheral nerves. The current view of BM structure and functions is based mainly on transmission electron microscopy imaging, in vitro protein binding assays, and phenotype analysis of human patients, mutant mice and invertebrata. Recently, MS-based protein analysis, biomechanical testing and cell adhesion assays with in vivo derived BMs have led to new and unexpected insights. Proteomic analysis combined with ultrastructural studies showed that many BMs undergo compositional and structural changes with advancing age. Atomic force microscopy measurements in combination with phenotype analysis have revealed an altered mechanical stiffness that correlates with specific BM pathologies in mutant mice and human patients. Atomic force microscopy-based height measurements strongly suggest that BMs are more than two-fold thicker than previously estimated, providing greater freedom for modelling the large protein polymers within BMs. In addition, data gathered using BMs extracted from mutant mice showed that laminin has a crucial role in BM stability. Finally, recent evidence demonstrate that BMs are bi-functionally organized, leading to the proposition that BM-sidedness contributes to the alternating epithelial and stromal tissue arrangements that are found in all metazoan species. We propose that BMs are ancient structures with tissue-organizing functions and were essential in the evolution of metazoan species.

Clinical risk factors for poor anatomic response to ranibizumab in neovascular age-related macular degeneration.

PURPOSE: To identify OCT-based anatomical features and clinical characteristics for poor central retinal thickness (CRT) response to ranibizumab in neovascular age-related macular degeneration (AMD). PATIENTS AND METHODS: Investigating our electronic patient records (Eyeswide), patients with neovascular AMD treated with intravitreal injections of 0.5mg/0.05ml ranibizumab were identified and their notes reviewed. Data collected included gender, age, initial best-corrected visual acuity (BCVA), prior photodynamic therapy, lesion type (classic versus occult), type of macular edema (intraretinal fluid, subretinal fluid, pigment epithelium detachment) and the total number of previous ranibizumab injections. RESULTS: A total of 210 eyes of 182 patients with neovascular AMD were identified. Mean follow-up time was 1.34 years (SD ± 0.77). Central retinal thickness reduction in women was significantly inferior to that in men (p=0.05). Patients with cystoid type macular edema had significantly greater reduction in CRT compared to patients with subretinal fluid (p<0.001) or pigment epithelium detachment (p<0.001). The percentage drop of CRT was no longer statistically significant after the sixth injection. Age, initial BCVA, prior photodynamic therapy and lesion type had no statistically effect on CRT response. CONCLUSION: Risk factors for poor central retinal thickness response to ranibizumab include female gender and patients with predominant subretinal fluid or pigment epithelium detachment. Furthermore, the anatomical response decreased after the sixth injection of ranibizumab.

The bi-functional organization of human basement membranes.

The current basement membrane (BM) model proposes a single-layered extracellular matrix (ECM) sheet that is predominantly composed of laminins, collagen IVs and proteoglycans. The present data show that BM proteins and their domains are asymmetrically organized providing human BMs with side-specific properties: A) isolated human BMs roll up in a side-specific pattern, with the epithelial side facing outward and the stromal side inward. The rolling is independent of the curvature of the tissue from which the BMs were isolated. B) The epithelial side of BMs is twice as stiff as the stromal side, and C) epithelial cells adhere to the epithelial side of BMs only. Side-selective cell adhesion was also confirmed for BMs from mice and from chick embryos. We propose that the bi-functional organization of BMs is an inherent property of BMs and helps build the basic tissue architecture of metazoans with alternating epithelial and connective tissue layers.

Proliferative diabetic retinopathy: predictive and preventive measures at hypoxia induced retinal changes.

Retinal vasculature changes in diabetic patients are most common cause of blindness among eye diseases. Numerous studies have explored the role of the agiogenic factors in the progression of diabetic retinopathy (DR). The balance between angiogenic and antiangiogenic factors has a determining role in the DR progression. Current treatment modalities include laser photocoagulation, intravitreal drug application, and pars plana vitrectomy (ppv). These maneuvers are employed with occurrence of advanced retinal changes. New diagnostic approaches can provide better information for the initial retinal changes thereby requiring a new DR classification and treatment guidelines. The results that are expected from Diabetic Retinopathy Clinical Research Network (DRCR) are at the level where prediction and prevention can not be made. Innovative molecular-imaging technology, can pave the way for application of novel clinical approaches. Identification of pathology-specific biomarkers and their application to diagnosis and treatment, support the individualized treatment algorithms.