Purpose: To compare the capsulotomy rim morphology of anterior lens capsules obtained by 5 femtosecond laser-assisted cataract surgery (FLACS) platforms: Catalys, FEMTO LDV Z8, LensAR, LenSx, and Victus. Setting: University of Basel, Basel, Switzerland. Design: Experimental study. Methods: Anterior lens capsules from 6 interventions per laser platform were collected (N = 30). 4 of each were assessed under light microscopy (LM; n = 20), and 2 were assessed under scanning electron microscopy (n = 10). A qualitative evaluation of all samples was conducted, as well as a semiquantitative analysis of the extent of irregularities present in LM samples. The Dunn multiple comparisons test was conducted to determine which lasers produced significantly smoother capsulotomies. Results: Irregularities along the lens capsule rim were observed in all samples. Distinct irregularities were observed for Catalys (unconnected laser pulses bordering the capsulotomy), FEMTO LDV Z8 (numerous additional rows of laser pulses), and LensAR (at least one section with multiple rows of laser pulses). Victus demonstrated a significantly higher degree of smoothness of capsulotomy (median = 306 degrees) compared with Catalys (median = 171 degrees) ( P = .02). The medians for other laser platforms fell between these values. Conclusions: Different laser platforms produce morphologically and quantitatively distinct irregularities, which may influence capsulotomy strength differently. FEMTO LDV Z8, a low-energy laser, did not produce a smoother capsulotomy than the high-energy lasers. Our results suggest that, with current technologies, the precision of the laser is more important than the pulse energy in determining the regularity of the capsulotomy surface.

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.

Pluripotent stem cells are promising source of cells for tissue engineering, regenerative medicine and drug discovery applications. The process of stem cell differentiation is regulated by multi-parametric cues from the surrounding microenvironment, one of the critical one being cell interaction with extracellular matrix (ECM). The ECM is a complex tissue-specific structure which is an important physiological regulator of stem cell function and fate. Recapitulating this native ECM microenvironment niche is best facilitated by decellularized tissue/organ derived ECM, which can faithfully reproduce the physiological environment with high fidelity toin vivocondition and promote tissue-specific cellular development and maturation. Recognizing the need for organ specific ECM in a 3D culture environment in driving phenotypic differentiation and maturation of hPSCs, we fabricated an ECM array platform using native-mimicry ECM from decellularized organs (namely pancreas, liver and heart), which allows cell-ECM interactions in both 2D and 3D configuration. The ECM array was integrated with rapid quantitative imaging for a systematic investigation of matrix protein profiles and sensitive measurement of cell-ECM interaction during hPSC differentiation. We tested our platform by elucidating the role of the three different organ-specific ECM in supporting induced pancreatic differentiation of hPSCs. While the focus of this report is on pancreatic differentiation, the developed platform is versatile to be applied to characterize any lineage specific differentiation.

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.

Abstract The onset of metastasis occurs when cancer cells invade and breach the basement membrane (BM) that provides mechanical support to epithelial tissues. Yet, it remains unclear what triggers cancer cells to breach the BM, and how ‘triggered’ cells in fact breach the BM. We have established an in vitro assay using native BM interface for culturing epithelial cells. Using atomic force microscopy (AFM) with other high-resolution microscopies and TER (trans-epithelial resistance), we have correlated the mechano-cellular attributes of the BM/epithelium interface to its biochemical and structural properties. We demonstrated that the internal limiting membrane (ILM) isolated from human retinas acts as a native substrate for culturing epithelial cells in terms of BM composition, architecture and stiffness. These are required to act jointly in order to achieve apico-basal polarity, tissue barrier formation and stiffness properties of the epithelial layer similarly to secretory epithelia in vivo. The native BM serves several advantageous over reconstituted Matrigel, an ECM extracts that originates from mouse tumor ascites. Besides variations in thickness and biochemical composition, we find that Matrigel is mechanically 100-fold more compliant (i.e., softer) than native BMs. When tumorigenic variants of cells are used, we demonstrated that cancer cell invasion is associated with a decrease in cellular stiffness correlated to changes in cell and BM morphology. In addition, we showed that activation of ß1 integrin by the stiffness and architecture of the native alpha - 5 laminin chain has a key role, not only as previously thought for maintenance of cell polarity but also for the establishment of a physiological mechanophenotype. On the other hand it is well know that during cancer progression in vivo, cancer cells can perforate the BM using proteolysis. Whether stromal cells also play a role and what kind of role in this process is unclear. Therefore, we asked if carcinoma-associated fibroblasts (CAFs) isolated from cancer patients promote cancer cell invasion through a BM. In the presence of CAFs, moderately invasive cancer cells invade in a matrix metalloproteinase-independent manner. Using live imaging and atomic force microscopy, we showed that CAFs actively pull, stretch and soften the BM, forming gaps through which cancer cells can migrate. By exerting contractile forces, CAFs alter the organization and the physical properties of the BM, making it permissive for cancer cell invasion. Finally, we propose that, in addition to proteolysis, mechanical interactions between CAFs and BM represent an alternative mechanism of BM breaching. Given their mechano-biological relevance, native BMs allow us to further understand how mechanosignaling occurs between the epithelia and the surrounding stromal layers at the BM interface during cancer invasion and progression. Note: This abstract was not presented at the meeting. Citation Format: Marija Plodinec, Philipp Oertle, Daphne Assgeirson, Willi Halfter, Serenella Eppenberger Castori, Ellen C. Obermann, Alexandre Glentis, Roderick Y. LIM. Mechanobiology of epithelia on native basement membrane and relevance for cancer invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2962. doi:10.1158/1538-7445.AM2017-2962

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.

Tenascin-C (TN-C) is an extracellular matrix glycoprotein linked to inflammatory processes in pathological conditions including Alzheimer disease (AD). We examined the distribution of TN-C immunoreactivity (ir) in relation to amyloid-β (Aβ) plaques and vascular Aβ deposits in autopsy brain tissues from 14 patients with clinical and neuropathological AD and 10 aged-matched controls with no cognitive impairment; 5 of the controls had Aβ plaques and 5 did not. TN-C ir was abundant in cortical white matter and subpial cerebral gray matter in all cases, whereas TN-C ir was weak in blood vessels. In all cases with Aβ plaques but not in plaque-free controls, TN-C ir was detected as large (>100 µm in diameter) diffuse extracellular deposits in cortical grey matter. TN-C plaques completely overlapped and surrounded cored Aβ plaques labeled with X-34, a fluorescent derivative of Congo red, and they were associated with reactive astrocytes astrocytes, microglia and phosphorylated tau-containing dystrophic neurites. Diffuse Aβ plaques lacking amyloid cores, reactive glia or dystrophic neurites showed no TN-C ir. In cases with cerebral amyloid angiopathy, TN-C ir in vessel walls did not spread into the surrounding neuropil. These results suggest a role for TN-C in Aβ plaque pathogenesis and its potential as a biomarker and therapy target.

PURPOSECataract 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.METHODSLens 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.RESULTSContinuous 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.CONCLUSIONSFinite-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.

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 BM s have led to new and unexpected insights. Proteomic analysis combined with ultrastructural studies showed that many BM s 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 BM s are more than two‐fold thicker than previously estimated, providing greater freedom for modelling the large protein polymers within BM s. In addition, data gathered using BM s extracted from mutant mice showed that laminin has a crucial role in BM stability. Finally, recent evidence demonstrate that BM s 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 BM s are ancient structures with tissue‐organizing functions and were essential in the evolution of metazoan species.

Basement membranes (BMs) are extracellular matrix sheets comprising the laminins, type-IV collagens, nidogens, and the heparan sulfate proteoglycans, perlecan, collagen XVIII, and agrin. In intact BMs, BM proteins are physiologically insoluble and partially resistant to proteolytic digestion, making BMs a challenge to study. Here three types of BMs from adult human eyes, the inner limiting membrane (ILM), the retinal vascular BMs, and the lens capsule, were isolated for analysis by 1D-SDS-PAGE and LC-MS/MS. Peptide and protein identifications were done using MaxQuant. 1129 proteins were identified with a 1% false discovery rate. Data showed that BMs are composed of multiple laminins, collagen IVs, nidogens, and proteoglycans. The dominant laminin family member in all BMs was laminin α5β2γ1. The dominant collagen IV trimer in lens capsule (LC) and blood vessel (BV) BMs had a chain composition of α1(IV)2, α2 (IV), whereas the dominant collagen IV in the ILM had the α3(IV), α4(IV), α5(IV) chain composition. The data also showed that the ratio of laminin and collagen IVs varied among different BM types: the ratio of collagen IV to the other BM proteins is highest in LC, followed by BV and lowest for the ILM. The data have been deposited to the ProteomeXchange with identifier PXD001025.

The basement membranes (BMs) of the nervous system include (a) the pial BM that surrounds the entire CNS, (b) the BMs that outline the vascular system of the CNS and PNS and (c) the BMs that are associated with Schwann cells. We previously found that isolated BMs are bi-functionally organized, whereby the two surfaces have different compositional, biomechanical and cell adhesion properties. To find out whether the bi-functional nature of BMs has an instructive function in organizing the tissue architecture of the developing nervous system, segments of human BMs were inserted into (a) the parasomitic mesoderm of chick embryos, intersecting with the pathways of axons and neural crest cells, or (b) into the midline of the embryonic chick spinal cord. The implanted BMs integrated into the embryonic tissues within 24h and were impenetrable to growing axons and migrating neural crests cells. Host axons and neural crest cells contacted the epithelial side but avoided the stromal side of the implanted BM. When the BMs were inserted into the spinal cord, neurons, glia cells and axons assembled at the epithelial side of the implanted BMs, while a connective tissue layer formed at the stromal side, resembling the tissue architecture of the spinal cord at the pial surface. Since the spinal cord is a-vascular at the time of BM implantation, we propose that the bi-functional nature of BMs has the function of segregating epithelial and connective cells into two adjacent compartments and participates in establishing the tissue architecture at the pial surface of the CNS.

Mutations in glycosyltransferases, such as protein O-mannose N-acetylglucosaminyltransferase 1 (POMGnT1), causes disruptions of basement membranes (BMs) that results in neuronal ectopias and muscular dystrophy. While the mutations diminish dystroglycan-mediated cell–ECM interactions, the cause and mechanism of BM disruptions remain unclear. In this study, we established an in vitro model to measure BM assembly on the surface of neural stem cells. Compared to control cells, the rate of BM assembly on POMGnT1 knockout neural stem cells was significantly reduced. Further, immunofluorescence staining and quantitative proteomic analysis of the inner limiting membrane (ILM), a BM of the retina, revealed that laminin-111 and nidogen-1 were reduced in POMGnT1 knockout mice. Finally, atomic force microscopy showed that the ILM from POMGnT1 knockout mice was thinner with an altered surface topography. The results combined demonstrate that reduced levels of key BM components cause physical changes that weaken the BM in POMGnT1 knockout mice. These changes are caused by a reduced rate of BM assembly during the developmental expansion of the neural tissue.

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.

Accumulation of globotriaosylceramide (Gb3) and other neutral glycosphingolipids with galactosyl residues is the hallmark of Fabry disease, a lysosomal storage disorder caused by deficiency of the enzyme alpha-galactosidase A (α-gal A). These lipids are incorporated into the plasma membrane and intracellular membranes, with a preference for lipid rafts. Disruption of raft mediated cell processes is implicated in the pathogenesis of several human diseases, but little is known about the effects of the accumulation of glycosphingolipids on raft dynamics in the context of Fabry disease. Using siRNA technology, we have generated a polarized renal epithelial cell model of Fabry disease in Madin–Darby canine kidney cells. These cells present increased levels of Gb3 and enlarged lysosomes, and progressively accumulate zebra bodies. The polarized delivery of both raft-associated and raft-independent proteins was unaffected by α-gal A knockdown, suggesting that accumulation of Gb3 does not disrupt biosynthetic trafficking pathways. To assess the effect of α-gal A silencing on lipid raft dynamics, we employed number and brightness (N&B) analysis to measure the oligomeric status and mobility of the model glycosylphosphatidylinositol (GPI)-anchored protein GFP-GPI. We observed a significant increase in the oligomeric size of antibody-induced clusters of GFP-GPI at the plasma membrane of α-gal A silenced cells compared with control cells. Our results suggest that the interaction of GFP-GPI with lipid rafts may be altered in the presence of accumulated Gb3. The implications of our results with respect to the pathogenesis of Fabry disease are discussed.

Basement membranes (BMs) evolved together with the first metazoan species approximately 500 million years ago. Main functions of BMs are stabilizing epithelial cell layers and connecting different types of tissues to functional, multicellular organisms. Mutations of BM proteins from worms to humans are either embryonic lethal or result in severe diseases, including muscular dystrophy, blindness, deafness, kidney defects, cardio-vascular abnormalities or retinal and cortical malformations. In vivo-derived BMs are difficult to come by; they are very thin and sticky and, therefore, difficult to handle and probe. In addition, BMs are difficult to solubilize complicating their biochemical analysis. For these reasons, most of our knowledge of BM biology is based on studies of the BM-like extracellular matrix (ECM) of mouse yolk sac tumors or from studies of the lens capsule, an unusually thick BM. Recently, isolation procedures for a variety of BMs have been described, and new techniques have been developed to directly analyze the protein compositions, the biomechanical properties and the biological functions of BMs. New findings show that native BMs consist of approximately 20 proteins. BMs are four times thicker than previously recorded, and proteoglycans are mainly responsible to determine the thickness of BMs by binding large quantities of water to the matrix. The mechanical stiffness of BMs is similar to that of articular cartilage. In mice with mutation of BM proteins, the stiffness of BMs is often reduced. As a consequence, these BMs rupture due to mechanical instability explaining many of the pathological phenotypes. Finally, the morphology and protein composition of human BMs changes with age, thus BMs are dynamic in their structure, composition and biomechanical properties.

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.

Approximately 285 million people worldwide suffer from diabetes, with insulin supplementation as the most common treatment measure. Regenerative medicine approaches such as a bioengineered pancreas has been proposed as potential therapeutic alternatives. A bioengineered pancreas will benefit from the development of a bioscaffold that supports and enhances cellular function and tissue development. Perfusion-decellularized organs are a likely candidate for use in such scaffolds since they mimic compositional, architectural and biomechanical nature of a native organ. In this study, we investigate perfusion-decellularization of whole pancreas and the feasibility to recellularize the whole pancreas scaffold with pancreatic cell types. Our result demonstrates that perfusion-decellularization of whole pancreas effectively removes cellular and nuclear material while retaining intricate three-dimensional microarchitecture with perfusable vasculature and ductal network and crucial extracellular matrix (ECM) components. To mimic pancreatic cell composition, we recellularized the whole pancreas scaffold with acinar and beta cell lines and cultured up to 5 days. Our result shows successful cellular engraftment within the decellularized pancreas, and the resulting graft gave rise to strong up-regulation of insulin gene expression. These findings support biological utility of whole pancreas ECM as a biomaterials scaffold for supporting and enhancing pancreatic cell functionality and represent a step toward bioengineered pancreas using regenerative medicine approaches.

Kidney function requires the appropriate distribution of membrane proteins between the apical and basolateral surfaces along the kidney tubule. Further, the absolute amount of a protein at the cell surface versus intracellular compartments must be attuned to specific physiological needs. Endolyn (CD164) is a transmembrane protein that is expressed at the brush border and in apical endosomes of the proximal convoluted tubule and in lysosomes of more distal segments of the kidney. Endolyn has been shown to regulate CXCR4 signaling in hematopoietic precursor cells and myoblasts; however, little is known about endolyn function in the adult or developing kidney. Here we identify endolyn as a gene important for zebrafish pronephric kidney function. Zebrafish endolyn lacks the N-terminal mucin-like domain of the mammalian protein, but is otherwise highly conserved. Using in situ hybridization we show that endolyn is expressed early during development in zebrafish brain, eye, gut and pronephric kidney. Embryos injected with a translation-inhibiting morpholino oligonucleotide targeted against endolyn developed pericardial edema, hydrocephaly and body curvature. The pronephric kidney appeared normal morphologically, but clearance of fluorescent dextran injected into the common cardinal vein was delayed, consistent with a defect in the regulation of water balance in morphant embryos. Heterologous expression of rat endolyn rescued the morphant phenotypes. Interestingly, rescue experiments using mutant rat endolyn constructs revealed that both apical sorting and endocytic/lysosomal targeting motifs are required for normal pronephric kidney function. This suggests that both polarized targeting and postendocytic trafficking of endolyn are essential for the protein's proper function in mammalian kidney.