Adherens junction proteins are expressed in collagen corneal equivalents produced in vitro with human cells

Purpose To test whether adherens junction proteins are present in the epithelium and the endothelium of corneal equivalents. Methods Corneal cell types were harvested from human eyes and grown separately. Stromal equivalents were constructed by seeding fibroblasts into a collagen gel on which epithelial and endothelial cells were added on each side. Alternatively, bovine endothelial cells were used. At maturity, sections of stromal equivalents were processed for Masson's trichrome or indirect immunofluorescence using antibodies against pan-, N-, or E-cadherins or a- or ß-catenins. Alternatively, stromal equivalents were dissected, to separate the proteins from the epithelium, endothelium, and stroma with sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Western blots of the transferred proteins exposed to these primary antibodies were detected with chemiluminescence. Native corneas were processed similarly. Results Three or four layers of epithelial cells reminiscent of the native cornea (basal cuboidal and superficial flatter cells) lay over a stromal construct containing fibroblastic cells under which an endothelium is present. Western blots and indirect immunofluorescence revealed that, similarly to the native cornea, the epithelium reacted positively to antibodies against catenins (a and ß) and E-cadherin. The endothelium of corneal constructs, whether of human or bovine origin, reacted mildly to catenins and N-cadherin. Conclusions This collagen-based corneal equivalent simulated the native cornea. Cells from the epithelial and endothelial layers expressed adherens junction proteins, indicating the presence of cell–cell contacts and the existence of polarized morphology of these layers over corneal equivalents

Is Desmin Propensity to Aggregate Part of its Protective Function?

Desmin is the major protein component of the intermediate filaments (IFs) cytoskeleton in muscle cells, including cardiac. The accumulation of cleaved and misfolded desmin is a cellular hallmark of heart failure (HF). These desmin alterations are reversed by therapy, suggesting a causal role for the IFs in the development of HF. Though IFs are known to play a role in the protection from stress, a mechanistic model of how that occurs is currently lacking. On the other hand, the heart is uniquely suited to study the function of the IFs, due to its inherent, cyclic contraction. That is, HF can be used as a model to address how IFs afford protection from mechanical, and possibly redox, stress. In this review we provide a brief summary of the current views on the function of the IFs, focusing on desmin. We also propose a new model according to which the propensity of desmin to aggregate may have been selected during evolution as a way to dissipate excessive mechanical and possibly redox stress. According to this model, though desmin misfolding may afford protection from acute injury, the sustained or excessive accumulation of desmin aggregates could impair proteostasis and contribute to disease

Nebivolol Desensitizes Myofilaments of a Hypertrophic Cardiomyopathy Mouse Model

Background: Hypertrophic cardiomyopathy (HCM) patients often present with diastolic dysfunction and a normal to supranormal systolic function. To counteract this hypercontractility, guideline therapies advocate treatment with beta-adrenoceptor and Ca2+ channel blockers. One well established pathomechanism for the hypercontractile phenotype frequently observed in HCM patients and several HCM mouse models is an increased myofilament Ca2+ sensitivity. Nebivolol, a commonly used beta-adrenoceptor antagonist, has been reported to lower maximal force development and myofilament Ca2+ sensitivity in rabbit and human heart tissues. The aim of this study was to evaluate the effect of nebivolol in cardiac muscle strips of an established HCM Mybpc3 mouse model. Furthermore, we investigated actions of nebivolol and epigallocatechin-gallate, which has been shown to desensitize myofilaments for Ca2+ in mouse and human HCM models, in cardiac strips of HCM patients with a mutation in the most frequently mutated HCM gene MYBPC3.Methods and Results: Nebivolol effects were tested on contractile parameters and force-Ca2+ relationship of skinned ventricular muscle strips isolated from Mybpc3-targeted knock-in (KI), wild-type (WT) mice and cardiac strips of three HCM patients with MYBPC3 mutations. At baseline, KI strips showed no difference in maximal force development compared to WT mouse heart strips. Neither 1 nor 10 μM nebivolol had an effect on maximal force development in both genotypes. 10 μM nebivolol induced myofilament Ca2+ desensitization in WT strips and to a greater extent in KI strips. Neither 1 nor 10 μM nebivolol had an effect on Ca2+ sensitivity in cardiac muscle strips of three HCM patients with MYBPC3 mutations, whereas epigallocatechin-gallate induced a right shift in the force-Ca2+ curve.Conclusion: Nebivolol induced a myofilament Ca2+ desensitization in both WT and KI strips, which was more pronounced in KI muscle strips. In human cardiac muscle strips of three HCM patients nebivolol had no effect on myofilament Ca2+ sensitivity

Influence of Sp1/Sp3 expression on corneal epithelial cells proliferation and differentiation properties in reconstructed tissues

PURPOSE : Primary cultured epithelial cells are widely used for the production of tissue-engineered substitutes and are gaining popularity as a model for gene expression studies. However, as such cells are passaged in culture, they often lose their ability to proliferate by progressing toward terminal cell differentiation, a process likely to be determined by altered expression of transcription factors that have functions critical for cell adhesion and differentiation. This study was designed to determine whether the variable life span of primary cultured human corneal epithelial cells (HCECs) might be the consequence of varying expression levels of the well-known transcription factors Sp1 and Sp3 (Sp1/Sp3). METHODS : HCECs were obtained from donor eyes and cultured on irradiated Swiss-3T3. Sp1/Sp3 expression was monitored by Western blot and electrophoretic mobility shift assay (EMSA). The Sp1/Sp3 regulatory influence was evaluated by transfection of HCECs with a recombinant plasmid bearing the Sp1/Sp3-dependent poly(ADP-ribose) polymerase (rPARP) promoter fused to the CAT reporter gene. HCECs that expressed various levels of Sp1/Sp3 were also used for the production of corneal substitutes. RESULTS : Expression of Sp1/Sp3 was dramatically inconsistent between HCECs isolated from the eyes of different donors. Both factors were highly expressed during one passage and then totally disappeared as cells terminally differentiated. Proper stratification of HCECs on reconstructed tissue substitutes could be obtained only with cells that also had a delayed peak of Sp1/Sp3 expression when cultured in vitro. CONCLUSIONS : Expression of Sp1/Sp3 may represent a good predictor for selecting HCECs that are most likely to proliferate, stratify, and differentiate properly when used for the production of reconstructed corneal substitutes

Transcriptional regulation of the human α6 integrin gene by the transcription factor NFI during corneal wound healing

Purpose. Wound healing of the corneal epithelium is highly influenced by regulation of integrin gene expression. A recent study demonstrated that laminin (LM), a major constituent of the extracellular matrix (ECM), reduces expression of the human α6 integrin subunit gene by altering the properties of the transcription factor (TF) Sp1. In this work, a target site was identified for the TF nuclear factor I (NFI) on the human α6 gene, and its regulatory influence was characterized in corneal epithelial cells. Methods. Plasmids bearing the α6 promoter fused to the CAT gene were transfected into human (HCECs) and rabbit (RCECs) corneal epithelial cells grown on LM. The DNA-binding site for NFI in the α6 promoter was identified by DNase I footprinting. Expression and DNA binding of NFI was monitored by Western blot, RT-PCR, and electrophoretic mobility shift assays (EMSAs), and its function was investigated through RNAi and NFI overexpression assays. Results. All NFI isoforms were found to be expressed in HCECs and RCECs. Transfection analyses revealed that NFI is a repressor of α6 expression in both types of cells. LM increases expression of NFI, whereas inhibition of each NFI isoform increases promoter activity suggesting that NFI is a key repressor of α6 transcription. In addition, the negative influence of NFI appears to be potentiated by the degradation of Sp1 when cells are grown on LM. Conclusions. Repression of α6 expression therefore contributes to the final steps of corneal wound healing by both reducing proliferation and allowing attachment of the epithelium to the basal membrane

The ubiquitin-proteasome system and nonsense-mediated mRNA decay in hypertrophic cardiomyopathy

Cardiomyopathies represent an important cause of cardiovascular morbidity and mortality due to heart failure, arrhythmias, and sudden death. Most forms of hypertrophic cardiomyopathy (HCM) are familial with an autosomal-dominant mode of inheritance. Over the last 20 years, the genetic basis of the disease has been largely unravelled. HCM is considered as a sarcomeropathy involving mutations in sarcomeric proteins, most often β-myosin heavy chain and cardiac myosin-binding protein C. ‘Missense’ mutations, more common in the former, are associated with dysfunctional proteins stably integrated into the sarcomere. ‘Nonsense’ and frameshift mutations, more common in the latter, are associated with low mRNA and protein levels derived from the diseased allele, leading to haploinsufficiency of the remaining healthy allele. The two quality control systems responsible for the removal of the affected mRNAs and proteins are the nonsense-mediated mRNA decay (NMD) and the ubiquitin-proteasome system (UPS), respectively. This review discusses clinical and genetic aspects of HCM and the role of NMD and UPS in the regulation of mutant proteins, evidence for impairment of UPS as a pathogenic factor, as well as potential therapies for HCM

The ubiquitin–proteasome system in cardiac dysfunction

Abstract Since proteins play crucial roles in all biological processes, the finely tuned equilibrium between their synthesis and degradation regulates cellular homeostasis. Controlling the quality of proteome informational content is essential for cell survival and function. After initial synthesis, membrane and secretory proteins are modified, folded, and assembled in the endoplasmic reticulum, whereas other proteins are synthesized and processed in the cytosol. Cells have different protein quality control systems, the molecular chaperones, which help protein folding and stabilization, and the ubiquitin–proteasome system (UPS) and lysosomes, which degrade proteins. It has generally been assumed that UPS and lysosomes are regulated independently and serve distinct functions. The UPS degrades both cytosolic, nuclear proteins, and myofibrillar proteins, whereas the lysosomes degrade most membrane and extracellular proteins by endocytosis as well as cytosolic proteins and organelles via autophagy. Over the last two decades, the UPS has been increasingly recognized as a major system in several biological processes including cell proliferation, adaptation to stress and cell death. More recently, activation or impairment of the UPS has been reported in cardiac disease and recent evidence indicate that autophagy is a key mechanism to maintain cardiac structure and function. This review mainly focuses on the UPS and its various components in healthy and diseased heart, but also summarizes recent data suggesting parallel activation of the UPS and autophagy in cardiac disease

Autologous transplantation of rabbit limbal epithelia cultured on fibrin gels for ocular surface reconstruction

Purpose: Regeneration of the corneal epithelium could be severely impaired in patients suffering from limbal stem cell deficiency. The purpose of this study was to evaluate the restoration of the corneal epithelium by grafting onto denuded corneas autologous limbal cells cultured on fibrin gels. The rabbit model was chosen to allow the microscopic evaluation over time after grafting. Methods: Rabbit limbal epithelial cells (RLECs) were isolated and cultured from small limbal biopsies (3 mm2 ). The epithelium was separated from stroma after dispase digestion and put in culture on lethally irradiated fibroblasts used as a feeder layer. At the first passage, RLECs were cultured on a fibrin gel matrix. At confluence, the cultured epithelia were grafted in vivo on denuded autologous rabbit corneas. At different postoperative times, grafted and control (without graft or grafted with fibrin gels only) rabbit corneas were compared in vivo with a slit lamp microscope, and in situ by histological and immunohistological microscopy of harvested biopsies. Results: A small limbal biopsy was sufficient to generate enough RLECs to prepare several grafts and to perform cell analysis. Only two weeks were required to produce a cultured epithelium suitable for autologous transplantation. One month after grafting, a normal corneal phenotype was observed on the ocular surface of grafted rabbits in contrast to the control rabbits (ungrafted or grafted with fibrin gel only) where histological signs of conjunctivalization were found. The absence of goblet cells and negative staining for keratin 4 confirmed that the cultured cells persisted and that the epithelium regenerated after grafting was not from conjunctival origin. Conclusions: Our results demonstrate that an autologous epithelium cultured on a physiologically biodegradable matrix can be prepared from a small biopsy and grafted on denuded cornea. The autologous graft allows epithelial regeneration from cultured cells and promotes corneal healing of unilateral total stem cell deficiency

Impact of cell source on human cornea reconstructed by tissue engineering

Purpose: To investigate the effect of the tissue origin of stromal fibroblasts and epithelial cells on reconstructed corneas in vitro. Methods: Four types of constructs were produced by the self-assembly approach using the following combinations of human cells: corneal fibroblasts/corneal epithelial cells, corneal fibroblasts/skin epithelial cells, skin fibroblasts/corneal epithelial cells, skin fibroblasts/skin epithelial cells. Fibroblasts were cultured with ascorbic acid to produce stromal sheets on which epithelial cells were cultured. After 2 weeks at the air-liquid interface, the reconstructed tissues were photographed, absorption spectra were measured, and tissues were fixed for histologic analysis. Cytokine expression in corneal- or skin-fibroblast-conditioned media was determined with the use of protein array membranes. The effect of culturing reconstructed tissues with conditioned media, or media supplemented with a cytokine secreted mainly by corneal fibroblasts, was determined. Results: The tissue source from which epithelial and mesenchymal cells were isolated had a great impact on the macroscopic and histologic features (epithelium thickness and differentiation) and the functional properties (transparency) of the reconstructed tissues. The reconstructed cornea had ultraviolet-absorption characteristics resembling those of native human cornea. The regulation of epithelial differentiation and thickness was mesenchyme-dependent and mediated by diffusible factors. IL-6, which is secreted in greater amounts by corneal fibroblasts than skin fibroblasts, decreased the expression of the differentiation marker DLK in the reconstructed epidermis. Conclusions: The tissue origin of fibroblasts and epithelial cells plays a significant role in the properties of the reconstructed tissues. These human models are promising tools for gaining a thorough understanding of epithelial-stromal interactions and regulation of epithelia homeostasis

Reconstruction of a human cornea by the self-assembly approach of tissue engineering using the three native cell types

Purpose: The purpose of this study was to produce and characterize human tissue-engineered corneas reconstructed using all three corneal cell types (epithelial, stromal, and endothelial cells) by the self-assembly approach. Methods: Fibroblasts cultured in medium containing serum and ascorbic acid secreted their own extracellular matrix and formed sheets that were superposed to reconstruct a stromal tissue. Endothelial and epithelial cells were seeded on each side of the reconstructed stroma. After culturing at the air-liquid interface, the engineered corneas were fixed for histology and transmission electron microscopy (TEM). Immunofluorescence labeling of epithelial keratins, basement membrane components, Na+/K+-ATPase α1, and collagen type I was also performed. Results: Epithelial and endothelial cells adhered to the reconstructed stroma. After 10 days at the air-liquid interface, the corneal epithelial cells stratified (4 to 5 cell layers) and differentiated into well defined basal and wing cells that also expressed Na+/K+-ATPase α1 protein, keratin 3/12, and basic keratins. Basal epithelial cells from the reconstructed epithelium formed many hemidesmosomes and secreted a well defined basement membrane rich in laminin V and collagen VII. Endothelial cells formed a monolayer of tightly-packed cells and also expressed the function related protein Na+/K +-ATPase α1. Conclusions: This study demonstrates the feasibility of producing a complete tissue-engineered human cornea, similar to native corneas, using untransformed fibroblasts, epithelial and endothelial cells, without the need for exogenous biomaterial