Self-assembling nanofiber hydrogels to attenuate epithelial mesenchymal transition in lens
epithelial cells
da Cruz Barros, Raquel Sofia
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Publication date:
2018
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da Cruz Barros, R. S. (2018). Self-assembling nanofiber hydrogels to attenuate epithelial mesenchymal
transition in lens epithelial cells. University of Groningen.
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on bottom condition and α -SMA on top condition where Tukey tests were performed. Values were significantly different between groups when p<0.05 which are represented on tables a1 and a2 for ALK2; b1 and b2 for ALK5; c1 and c2 for collagen type III; d1 and d2 for collagen type VI; e1 and e2 for β1; f1 and f2 for
TGF-β2; g1 and g2 for α-SMA on top and bottom condition respectively.
CHAPTER 4
EMT reduction in an ex vivo lens
model by using bioactive
Submitted to Journal of Cataract and Refractive Surgery: Barros RC, Gelens E, de Jong MR, Kuijer R, van Kooten TG. EMT reduction in an ex vivo lens model by using bioactive nanofiber-based hydrogels (2018)
HIGHLIGHTS
Bioactive nanofiber-based hydrogels can reduce EMT in ex-vivo lens epithelial cells An ex vivo model to study the effects of hydrogels on EMT was developed by using
the anterior lens capsule bag
Adhesive peptide sequences incorporated in the core of the hydrogel improved LEC response
The delay of EMT is archived in the presence of a mixture that resembles the basement membrane
This bioactive hydrogel can also be used as a coating
New approaches using commercial IOL coated with this hydrogel could be explored
113 Bioactive nanofiber-based hydrogels can reduce EMT in ex-vivo lens epithelial cells An ex vivo model to study the effects of hydrogels on EMT was developed by using
the anterior lens capsule bag
Adhesive peptide sequences incorporated in the core of the hydrogel improved LEC response
The delay of EMT is archived in the presence of a mixture that resembles the basement membrane
This bioactive hydrogel can also be used as a coating
4
Submitted to Journal of Cataract and Refractive Surgery: Barros RC, Gelens E, de Jong MR, Kuijer R, van Kooten TG. EMT reduction in an ex vivo lens model by using bioactive nanofiber-based hydrogels (2018)
HIGHLIGHTS
Bioactive nanofiber-based hydrogels can reduce EMT in ex-vivo lens epithelial cells An ex vivo model to study the effects of hydrogels on EMT was developed by using
the anterior lens capsule bag
Adhesive peptide sequences incorporated in the core of the hydrogel improved LEC response
The delay of EMT is archived in the presence of a mixture that resembles the basement membrane
This bioactive hydrogel can also be used as a coating
New approaches using commercial IOL coated with this hydrogel could be explored
113 Bioactive nanofiber-based hydrogels can reduce EMT in ex-vivo lens epithelial cells An ex vivo model to study the effects of hydrogels on EMT was developed by using
the anterior lens capsule bag
Adhesive peptide sequences incorporated in the core of the hydrogel improved LEC response
The delay of EMT is archived in the presence of a mixture that resembles the basement membrane
This bioactive hydrogel can also be used as a coating
ABSTRACT
After a cataract surgery, lens epithelial cells undergo an epithelial-mesenchymal transition (EMT) that creates a posterior capsule opacification (PCO). The EMT in the eye lens is confined to the interior of the capsule bag, where the lens epithelial cells have a primordial role. During EMT these cells are detached from the basement membrane and become fiber-like cells. The use of injectable bioactive low molecular weight gelators to prevent EMT can be study using an ex-vivo lens model. In this study, a lens capsule model to analyze the effects of lens epithelial cells exposure to bioactive hydrogels towards EMT was investigated. Hydrogels incorporating adhesive peptide motifs present in fibronectin, laminin and collagens were in contact with porcine capsule bags and effects on EMT were determined. The laminin motifs created a large cellular apoptosis. Despite the large cell regeneration provided by the mixture of fibronectin and collagen motifs, EMT was enhanced. The hydrogel that most resembled the basement membrane have shown a large delay in EMT. We create an ex vivo model that allowed the interaction of lens epithelial cells with hydrogels in a similar postoperative environment. Besides the prevention on EMT by one of the bioactive hydrogels, the percentage of peptides should be further investigated toward a total inhibition of EMT.
KEYWORDS
Capsule eye lens, epithelial-mesenchymal transition, bioactive nanofibers, hydrogels, posterior capsule opacification
INTRODUCTION
Posterior capsule opacification (PCO) is a well know pathological condition that may affect vision and consequently daily life in patients who received an intraocular lens (IOL). The opacification of the implanted lens is due to a fibrotic process confined to the interior of the capsule bag. Due to the presence of an epithelial cell monolayer on the inside of the anterior side, lens capsular bag models can be an interesting tool to study the effect of biomaterials on the evolution of fibrosis [1]. It is also well known that epithelial-mesenchymal transition (EMT) is the process behind the formation of fibrotic tissue in the eye lens [2]. This process is characterized by the loss of the epithelial identity to mesenchymal like cells – myofibroblasts. As a consequence, expression of integrins, cadherins and other proteins changes. The process starts, after cataract surgery, with the presence of inflammatory molecules (e.g. TGF-βs) which interferes with the signal transduction by integrins [3]. Fibronectin, collagens and laminin are examples of available ligands for these integrins. Changes in E-cadherins to N-cadherins are also a signal for cell (de)differentiation towards EMT. Associated new proteins being expressed are collagen
type I, α-SMA and vimentin [4]. In parallel the cells lose the connection with the basement
membrane (BM) and gain the ability to migrate along the capsule bag. They go to the posterior side and can create a fibrotic tissue, hence the term PCO [5]. During this process the epithelial cells have become myofibroblast-like cells.
Several surgical techniques and IOLs with different materials and design shapes have been used to avoid PCO, although without a 100 percent success rate. Considering the quest for lens replacement materials that restore accommodation and acknowledging that this approach requires a normal functioning capsular bag, it is evident that the avoidance of the capsular opacification is the prime concern [6] Replacement of the lens material can be achieved by injecting hydrogels, such as low molecular weight gelators (LMWG). These are self-assembling hydrogels with a high capacity to link water molecules, containing both hydrophobic and hydrophilic molecular sites that can be functionalized. Hydrogels in general are considered to be suitable as drug carrier or cell depot [7].
Collagen type IV and laminin are the major constituents of the basement membrane. During EMT the epithelial cells lose their typical polarity and get detached from the basement membrane. Simultaneously, their cytoskeleton elongates and creates much larger cells than the original epithelial cells. Both phenomena are associated with changes in the interaction of integrins with the ECM [8]. Fibronectin is barely detected in the native basement membrane but is upregulated during EMT. The RGD (arginine, glycine and L-aspartic acid) sequence is a well know cell adhesion motif in fibronectin. The IKVAV (Ile-Lys-Val-Ala-Val) sequence derived from laminin α1 chain is also know to benefit cell
adherence, together with YIGSR (Tyr-Ile-Gly-Ser-Arg) derived from the laminin β1 chain.
Asp-Gly-Glu-Ala (DGEA) derived from collagen type I is recognized by integrins binding collagens (α2β1) and can be used as antagonist for collagen type I receptors [9]. Therefore
DGEA could avoid the development of EMT by blocking collagen type I receptors. These integrins are associated with the development of tumor progression and metastasis [10, 11]. In turn, collagen type IV is described as an apoptosis protector for epithelial lens [12].
4
ABSTRACT
After a cataract surgery, lens epithelial cells undergo an epithelial-mesenchymal transition (EMT) that creates a posterior capsule opacification (PCO). The EMT in the eye lens is confined to the interior of the capsule bag, where the lens epithelial cells have a primordial role. During EMT these cells are detached from the basement membrane and become fiber-like cells. The use of injectable bioactive low molecular weight gelators to prevent EMT can be study using an ex-vivo lens model. In this study, a lens capsule model to analyze the effects of lens epithelial cells exposure to bioactive hydrogels towards EMT was investigated. Hydrogels incorporating adhesive peptide motifs present in fibronectin, laminin and collagens were in contact with porcine capsule bags and effects on EMT were determined. The laminin motifs created a large cellular apoptosis. Despite the large cell regeneration provided by the mixture of fibronectin and collagen motifs, EMT was enhanced. The hydrogel that most resembled the basement membrane have shown a large delay in EMT. We create an ex vivo model that allowed the interaction of lens epithelial cells with hydrogels in a similar postoperative environment. Besides the prevention on EMT by one of the bioactive hydrogels, the percentage of peptides should be further investigated toward a total inhibition of EMT.
KEYWORDS
Capsule eye lens, epithelial-mesenchymal transition, bioactive nanofibers, hydrogels, posterior capsule opacification
INTRODUCTION
Posterior capsule opacification (PCO) is a well know pathological condition that may affect vision and consequently daily life in patients who received an intraocular lens (IOL). The opacification of the implanted lens is due to a fibrotic process confined to the interior of the capsule bag. Due to the presence of an epithelial cell monolayer on the inside of the anterior side, lens capsular bag models can be an interesting tool to study the effect of biomaterials on the evolution of fibrosis [1]. It is also well known that epithelial-mesenchymal transition (EMT) is the process behind the formation of fibrotic tissue in the eye lens [2]. This process is characterized by the loss of the epithelial identity to mesenchymal like cells – myofibroblasts. As a consequence, expression of integrins, cadherins and other proteins changes. The process starts, after cataract surgery, with the presence of inflammatory molecules (e.g. TGF-βs) which interferes with the signal transduction by integrins [3]. Fibronectin, collagens and laminin are examples of available ligands for these integrins. Changes in E-cadherins to N-cadherins are also a signal for cell (de)differentiation towards EMT. Associated new proteins being expressed are collagen
type I, α-SMA and vimentin [4]. In parallel the cells lose the connection with the basement
membrane (BM) and gain the ability to migrate along the capsule bag. They go to the posterior side and can create a fibrotic tissue, hence the term PCO [5]. During this process the epithelial cells have become myofibroblast-like cells.
Several surgical techniques and IOLs with different materials and design shapes have been used to avoid PCO, although without a 100 percent success rate. Considering the quest for lens replacement materials that restore accommodation and acknowledging that this approach requires a normal functioning capsular bag, it is evident that the avoidance of the capsular opacification is the prime concern [6] Replacement of the lens material can be achieved by injecting hydrogels, such as low molecular weight gelators (LMWG). These are self-assembling hydrogels with a high capacity to link water molecules, containing both hydrophobic and hydrophilic molecular sites that can be functionalized. Hydrogels in general are considered to be suitable as drug carrier or cell depot [7].
Collagen type IV and laminin are the major constituents of the basement membrane. During EMT the epithelial cells lose their typical polarity and get detached from the basement membrane. Simultaneously, their cytoskeleton elongates and creates much larger cells than the original epithelial cells. Both phenomena are associated with changes in the interaction of integrins with the ECM [8]. Fibronectin is barely detected in the native basement membrane but is upregulated during EMT. The RGD (arginine, glycine and L-aspartic acid) sequence is a well know cell adhesion motif in fibronectin. The IKVAV (Ile-Lys-Val-Ala-Val) sequence derived from laminin α1 chain is also know to benefit cell
adherence, together with YIGSR (Tyr-Ile-Gly-Ser-Arg) derived from the laminin β1 chain.
Asp-Gly-Glu-Ala (DGEA) derived from collagen type I is recognized by integrins binding collagens (α2β1) and can be used as antagonist for collagen type I receptors [9]. Therefore
DGEA could avoid the development of EMT by blocking collagen type I receptors. These integrins are associated with the development of tumor progression and metastasis [10, 11]. In turn, collagen type IV is described as an apoptosis protector for epithelial lens [12].
The aim of this study is to develop a porcine lens capsular bag model in which hydrogel formulations can be tested for their ability to interfere with the EMT process. In addition to the experimental LMWG hydrogels equipped with cell adhesion peptides, we included Matrigel as a control. This gel is derived from Engelbroth-Holm-Swarm (EHS) mouse sarcoma cells and it is rich in basement membrane constituents including laminin, collagens and proteoglycans [13]. Its matrix composition is batch dependent due to its origin [14].
MATERIALS AND METHODS
Materials
Culture medium: Minimum Essential Media containing Earle’s salts (EMEM), fetal bovine serum (FBS), GlutaMax, penicillin, streptomycin and sodium pyruvate were all purchased from Life Technologies, Inc. (Bleiswijk, The Netherlands)). Acid chloride (HCl) and sodium chloride (NaCl) are from J.T. Baker, analytical grade. Matrigel ® matrix basement membrane with reduced growth factor content was obtained from Corning BD Biosciences. Mouse-α-human α-smooth muscle actin antibody, DAPI (4',6-diamidino-2-phenylindole) and phalloidin-TRITC (tetramethylrhodamine isothiocyanate), were from Sigma-Aldrich (Zwijndrecht, The Netherlands). The secondary antibody, FITC (fluorescein isothiocyanate)-labelled goat-α-mouse IgG was from Jackson Immunoresearch Europe (Suffolk, UK). Live/Dead viability/ cytotoxicity kit for mammalian cells was from Molecular Probes. All the surgical instruments were from Carl Teufel GMBH&Co (Germany).
Methods
Samples collection
Porcine pig eyes (Sus domesticus) were collected from the local slaughterhouse. All the eyes were from pigs not older than 6 month old. The lens was removed from the eye and only part of the capsule bag was used in the experiments. Each hydrogel was analyzed with 4 to 6 different capsule bags and each experiment was repeated 3 times.
Lens extraction
Eyes were cleaned from muscle and other tissues. The eyes were then submerged in 70% ethanol for 10 seconds. Further preparations were performed using a stereo microscope. The cornea of the eye was cut using curve tip scissors and fine-tipped tweezers without touching the lens of the eye. To avoid pressure before removal of the lens, the ciliary body around the lens was carefully cut. Using round tip tweezers the lens was slowly pushed out of the vitreous humor and at the same time the jelly vitreous humor was being cut away from the capsular bag. The full lens and remain vitreous humour was placed in a sterile petri dish and it was turned around (posterior part of the capsule bag facing up). A hole in the posterior capsule bag was made by using a fine tip scalpel and tweezer, with an approximate diameter of 2/3 of the lens diameter. This capsule bag was
then put in a 48 wells-plate with sterile medium previously warmed up at 37°C, again with the anterior side facing down to the bottom of the well.
Preparation of low molecular weight gelators and matrigel
The self-assemble low molecular weight gelators (LMWG) were produced as mentioned in Bommel et al. The functionalized maleimide building blocks (characterized by 1H-NMR and HPLC-MS) for the LMWG-peptides were prepared by reaction of N-succinimidyl 3-maleimidopropionate (TCI, +95%) with cysteine-containing peptides (Think Peptides, +95% purity). These products were dissolved to a 0.15M solutions by adding 0.18M HCl and filtered using a 0.45 µM Whatman filter. 5% w/t hyaluronic acid solution was prepared and filtered before used.
Assemble of peptides and their combinations
LMWG was linkage to the appropriated percentage of peptides as mention on table 1 (NanoFiber Matrices B.V., Groningen, The Netherlands). A sterilized 5% wt solution of hyaluronic acid was mixture with the medium. Each combination of LMWG – peptides was then added to the previous mixture in the proportions of 1:1:12.3 (LMWG-peptide: hyaluronic acid: medium). Matrigel was diluted in medium in 1:1 proportion.
200 µL of each hydrogel was quickly added to the wells with the remaining capsule bag. The gelification took process at RT for 30 minutes followed by the addition of 250 µL of medium in each well.
4
The aim of this study is to develop a porcine lens capsular bag model in whichhydrogel formulations can be tested for their ability to interfere with the EMT process. In addition to the experimental LMWG hydrogels equipped with cell adhesion peptides, we included Matrigel as a control. This gel is derived from Engelbroth-Holm-Swarm (EHS) mouse sarcoma cells and it is rich in basement membrane constituents including laminin, collagens and proteoglycans [13]. Its matrix composition is batch dependent due to its origin [14].
MATERIALS AND METHODS
Materials
Culture medium: Minimum Essential Media containing Earle’s salts (EMEM), fetal bovine serum (FBS), GlutaMax, penicillin, streptomycin and sodium pyruvate were all purchased from Life Technologies, Inc. (Bleiswijk, The Netherlands)). Acid chloride (HCl) and sodium chloride (NaCl) are from J.T. Baker, analytical grade. Matrigel ® matrix basement membrane with reduced growth factor content was obtained from Corning BD Biosciences. Mouse-α-human α-smooth muscle actin antibody, DAPI (4',6-diamidino-2-phenylindole) and phalloidin-TRITC (tetramethylrhodamine isothiocyanate), were from Sigma-Aldrich (Zwijndrecht, The Netherlands). The secondary antibody, FITC (fluorescein isothiocyanate)-labelled goat-α-mouse IgG was from Jackson Immunoresearch Europe (Suffolk, UK). Live/Dead viability/ cytotoxicity kit for mammalian cells was from Molecular Probes. All the surgical instruments were from Carl Teufel GMBH&Co (Germany).
Methods
Samples collection
Porcine pig eyes (Sus domesticus) were collected from the local slaughterhouse. All the eyes were from pigs not older than 6 month old. The lens was removed from the eye and only part of the capsule bag was used in the experiments. Each hydrogel was analyzed with 4 to 6 different capsule bags and each experiment was repeated 3 times.
Lens extraction
Eyes were cleaned from muscle and other tissues. The eyes were then submerged in 70% ethanol for 10 seconds. Further preparations were performed using a stereo microscope. The cornea of the eye was cut using curve tip scissors and fine-tipped tweezers without touching the lens of the eye. To avoid pressure before removal of the lens, the ciliary body around the lens was carefully cut. Using round tip tweezers the lens was slowly pushed out of the vitreous humor and at the same time the jelly vitreous humor was being cut away from the capsular bag. The full lens and remain vitreous humour was placed in a sterile petri dish and it was turned around (posterior part of the capsule bag facing up). A hole in the posterior capsule bag was made by using a fine tip scalpel and tweezer, with an approximate diameter of 2/3 of the lens diameter. This capsule bag was
then put in a 48 wells-plate with sterile medium previously warmed up at 37°C, again with the anterior side facing down to the bottom of the well.
Preparation of low molecular weight gelators and matrigel
The self-assemble low molecular weight gelators (LMWG) were produced as mentioned in Bommel et al. The functionalized maleimide building blocks (characterized by 1H-NMR and HPLC-MS) for the LMWG-peptides were prepared by reaction of N-succinimidyl 3-maleimidopropionate (TCI, +95%) with cysteine-containing peptides (Think Peptides, +95% purity). These products were dissolved to a 0.15M solutions by adding 0.18M HCl and filtered using a 0.45 µM Whatman filter. 5% w/t hyaluronic acid solution was prepared and filtered before used.
Assemble of peptides and their combinations
LMWG was linkage to the appropriated percentage of peptides as mention on table 1 (NanoFiber Matrices B.V., Groningen, The Netherlands). A sterilized 5% wt solution of hyaluronic acid was mixture with the medium. Each combination of LMWG – peptides was then added to the previous mixture in the proportions of 1:1:12.3 (LMWG-peptide: hyaluronic acid: medium). Matrigel was diluted in medium in 1:1 proportion.
200 µL of each hydrogel was quickly added to the wells with the remaining capsule bag. The gelification took process at RT for 30 minutes followed by the addition of 250 µL of medium in each well.
Table 1: Peptides composition and percentage for each low molecular weight gelator (LMWG).
Lens model preparation
The lens capsule without the posterior capsule part was kept in medium at 37°C under 5% CO2 during 16 hours. The hole in the posterior capsule bag allowed the diffusion of
medium in between the lens interior and the remaining capsular bag. This technique provided the removal of the lens material while preserving the lens epithelial cells on the anterior capsule. After this period the anterior capsule bag was placed in a new 48 wells-plate and 200 µL of the different hydrogels were applied above it. During this procedure it was paid attention that the monolayer of lens epithelial cells was in contact with the hydrogel and not with the plate. After the gelification of the hydrogels, 250µL of medium was carefully added into the wells. As a control an anterior capsule bag only cultured in medium was used.
Culture procedures
Anterior capsule bags alone or in contact with hydrogels were cultured in EMEM supplemented with 17% FBS, 1% Pen/Strep, 1% GlutaMAX and 1% of sodium pyruvate in a cell culture incubator at 37°C and 5%CO2. Medium was changed every day. The cultures were performed until 5 days of incubation with a time point at day 2. Days started to be count since the addition of the hydrogelators.
Immunohistochemistry assay
All samples were fixated with 3.7% paraformaldehyde during 30 minutes and washed in PBS. 0.5% triton X-100 was added and washed away after 10 minutes. After, samples were incubated with blocking solution of 5% BSA in PBS during 30 minutes, washed and incubated with the primary antibody α-SMA mouse-anti-human diluted at 1:100 in 1%BSA (1% PBSA) in PBS for 1 hour. Samples were washed 3 times in 1% PBSA and the secondary antibodies were applied for 2 hours. The nuclei were stained by DAPI (2 μg/mL f.c.), the actin filaments were stained by TRITC-labelled phalloidin (2 μg/mL) and the α-SMA were stained by FITC- goat-anti-mouse (2 μg/mL). All the incubations were done at RT. Stainings were applied above the hydrogelators and they were removed as much as possible without interfere with the capsule bag for visualization. Samples were analyzed by confocal laser microscopy (LEICA TCS SP2) with a UV laser, Argon laser and Helium laser. Images were acquiring through a 40x immersion objective lens (NA 0.80) with a resolution of 1024x1024 pixels.
Images analysis
The number and size of the nuclei were calculated using the blue channel from the confocal images. In Image J, the threshold of each image was evaluated followed by the analysis of particles. After this process a summary table with the area in pixels for each particle were copied to an excel sheet and the (too) low values for the area were excluded. Values smaller than 100 pixels were always as noise or fragments of dead cells. The remaining values were then copied to Sigma Plot where a histogram was made. From the histogram the averages of the single cells were chose as the highest bar value. This value should also be similar with the most frequent value of the excel sheet. The primary area values were filtered again for equal or higher values to the average. This average area is denominated the average size of the nuclei and the number of nuclei per image was calculated by dividing each area value by the average area. These values were then rounded to integer and finally summed up.
For the quantification of α-SMA images with the channels green and blue were used in a combined fashion. The number of positive nuclei for α-SMA was counting by Image J. This value was divided by the total number of nuclei per image and multiplied per 100.
Since α-SMA was not present in all images, the number of positive images for α-SMA (α) was divided by the number of total images per sample (β) and multiplied per 100. And to analyze the quantity of α-SMA only in the positive images (α), the number of positive nuclei for α-SMA (α1) was divided by the number of total nuclei per image (γ) and multiplied with 100.
# 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑜𝑜𝑜𝑜𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑝𝑝𝑝𝑝𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑓𝑓𝑓𝑓 𝑖𝑖𝑖𝑖𝑎𝑎𝑎𝑎𝑝𝑝𝑝𝑝ℎ𝑖𝑖𝑖𝑖 − 𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 (𝛼𝛼𝛼𝛼) 𝑝𝑝𝑝𝑝𝑜𝑜𝑜𝑜𝑝𝑝𝑝𝑝𝑖𝑖𝑖𝑖𝑎𝑎𝑎𝑎 # 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 (𝛽𝛽𝛽𝛽)
4
Table 1: Peptides composition and percentage for each low molecular weight gelator (LMWG).
Lens model preparation
The lens capsule without the posterior capsule part was kept in medium at 37°C under 5% CO2 during 16 hours. The hole in the posterior capsule bag allowed the diffusion of
medium in between the lens interior and the remaining capsular bag. This technique provided the removal of the lens material while preserving the lens epithelial cells on the anterior capsule. After this period the anterior capsule bag was placed in a new 48 wells-plate and 200 µL of the different hydrogels were applied above it. During this procedure it was paid attention that the monolayer of lens epithelial cells was in contact with the hydrogel and not with the plate. After the gelification of the hydrogels, 250µL of medium was carefully added into the wells. As a control an anterior capsule bag only cultured in medium was used.
Culture procedures
Anterior capsule bags alone or in contact with hydrogels were cultured in EMEM supplemented with 17% FBS, 1% Pen/Strep, 1% GlutaMAX and 1% of sodium pyruvate in a cell culture incubator at 37°C and 5%CO2. Medium was changed every day. The cultures were performed until 5 days of incubation with a time point at day 2. Days started to be count since the addition of the hydrogelators.
Immunohistochemistry assay
All samples were fixated with 3.7% paraformaldehyde during 30 minutes and washed in PBS. 0.5% triton X-100 was added and washed away after 10 minutes. After, samples were incubated with blocking solution of 5% BSA in PBS during 30 minutes, washed and incubated with the primary antibody α-SMA mouse-anti-human diluted at 1:100 in 1%BSA (1% PBSA) in PBS for 1 hour. Samples were washed 3 times in 1% PBSA and the secondary antibodies were applied for 2 hours. The nuclei were stained by DAPI (2 μg/mL f.c.), the actin filaments were stained by TRITC-labelled phalloidin (2 μg/mL) and the α-SMA were stained by FITC- goat-anti-mouse (2 μg/mL). All the incubations were done at RT. Stainings were applied above the hydrogelators and they were removed as much as possible without interfere with the capsule bag for visualization. Samples were analyzed by confocal laser microscopy (LEICA TCS SP2) with a UV laser, Argon laser and Helium laser. Images were acquiring through a 40x immersion objective lens (NA 0.80) with a resolution of 1024x1024 pixels.
Images analysis
The number and size of the nuclei were calculated using the blue channel from the confocal images. In Image J, the threshold of each image was evaluated followed by the analysis of particles. After this process a summary table with the area in pixels for each particle were copied to an excel sheet and the (too) low values for the area were excluded. Values smaller than 100 pixels were always as noise or fragments of dead cells. The remaining values were then copied to Sigma Plot where a histogram was made. From the histogram the averages of the single cells were chose as the highest bar value. This value should also be similar with the most frequent value of the excel sheet. The primary area values were filtered again for equal or higher values to the average. This average area is denominated the average size of the nuclei and the number of nuclei per image was calculated by dividing each area value by the average area. These values were then rounded to integer and finally summed up.
For the quantification of α-SMA images with the channels green and blue were used in a combined fashion. The number of positive nuclei for α-SMA was counting by Image J. This value was divided by the total number of nuclei per image and multiplied per 100.
Since α-SMA was not present in all images, the number of positive images for α-SMA (α) was divided by the number of total images per sample (β) and multiplied per 100. And to analyze the quantity of α-SMA only in the positive images (α), the number of positive nuclei for α-SMA (α1) was divided by the number of total nuclei per image (γ) and multiplied with 100.
# 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑜𝑜𝑜𝑜𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑝𝑝𝑝𝑝𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑓𝑓𝑓𝑓 𝑖𝑖𝑖𝑖𝑎𝑎𝑎𝑎𝑝𝑝𝑝𝑝ℎ𝑖𝑖𝑖𝑖 − 𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 (𝛼𝛼𝛼𝛼) 𝑝𝑝𝑝𝑝𝑜𝑜𝑜𝑜𝑝𝑝𝑝𝑝𝑖𝑖𝑖𝑖𝑎𝑎𝑎𝑎 # 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 (𝛽𝛽𝛽𝛽)
# 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑜𝑜𝑜𝑜𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑝𝑝𝑝𝑝𝑛𝑛𝑛𝑛𝑝𝑝𝑝𝑝 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑓𝑓𝑓𝑓 𝑎𝑎𝑎𝑎𝑛𝑛𝑛𝑛𝑝𝑝𝑝𝑝ℎ𝑎𝑎𝑎𝑎 − 𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 (𝛼𝛼𝛼𝛼1) 𝑝𝑝𝑝𝑝𝑜𝑜𝑜𝑜𝑝𝑝𝑝𝑝𝑎𝑎𝑎𝑎𝑛𝑛𝑛𝑛 # 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑝𝑝𝑝𝑝𝑛𝑛𝑛𝑛𝑝𝑝𝑝𝑝 (𝛾𝛾𝛾𝛾)
Live-dead assay
200 µL of calcein AM and ethidium homodimer-1 mixture diluted in medium was added to the wells. Calcein AM for the detection of live cells was used in the concentration of 2µM and ethidium homodimer-1 for dead cells was used in 4 µM. The samples were incubated at 37°C for 45 minutes and then washed in PBS. Directly after that the visualization was performed under confocal microscope, green staining (live cells) at 495 nm and 515 nm and red staining (dead cells) at 495 nm and 635 nm, excitation and emission range respectively. To avoid false dead cells only 2 samples were stained per each 30 minutes.
Statistical analyses
Statistical analyses were performed by applying One-Way ANOVA on the differences in the number and size of the nucleus. A comparison between days using t-test was performed for the quantification of α-SMA-FITC staining. Values were considered significantly different between each other when p<0.05. All the statistical analysis was performed using SigmaPlot 11.0.
RESULTS
Immunohistochemistry
Analyses of number and size of nuclei, morphology of the cytoskeleton and expression of α-SMA were determined in cultured capsule-associated LEC cells in contact with hydrogelators and Matrigel. Fresh capsules and capsules without contact with hydrogels were used as controls.
When comparing fresh capsules with capsules cultured during 2 and 5 days, it was observed by confocal imaging that there were clear differences in cytoskeleton organization as well as the numbers of nuclei (Fig.1). The cultured capsule-associated LEC cells decreased in number and showed less actin fibers. However, their cytoskeleton appeared well organized without signs of morphologic alterations. After contact of the capsule with the hydrogelators significant changes in number and size of the nuclei was found, and likewise in the quantity and organization of the cytoskeleton. On day 2 the number of nuclei was not significantly decreased but their size already changed dramatically, either becoming very small with residual actin filaments (IKVAV+YIGSR, RGD+DGEA and Matrigel) or increasing in size and with a reorganization of the actin (LMWG and BM) (Fig. 1A). On day 5, all the hydrogels showed a significantly decrease in the quantity of cells when compared with fresh capsules but not compared with the
cultured capsule only. Although the number and size of nuclei at this stage did not appear to be significantly different between hydrogels the overall morphology changed drastically from day 2 to day 5 (Fig.1B). The layer of LEC in contact with LMWG created clusters of small cells, totally changing their typical phenotype. On the other side, cells on IKVAV+YIGSR and RGD+DGEA appeared with a regeneration of the cytoskeleton and with a significant increase in nucleus size. On BM cells showed a stable cytoskeleton from day 2 to day 5 without changes in size and number of nuclei. With Matrigel the size of LEC was significant smaller than on LMWG after 2 days of culture, with only a few actin fibers. On day 5 these fibers increase in quantity and changed in morphology (Fig.1B).
On day 2, IKVAV+YIGSR and RGD+DGEA hydrogels yielded the smallest nuclei but with significantly higher numbers when exposed to RGD+DGEA (Fig.1A). On day 5, cells in contact with IKVAV+YIGSR significantly increased in size and number but on RGD+DGEA there was a significant increase in size, yet a significant decrease in number (Fig.1C). While on day 2 the cytoskeleton of cells in contact with both hydrogels had almost disappeared, on day 5 it manifested itself in large quantity and with a different organization mostly with RGD+DGEA (Fig.1B).
4
# 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑜𝑜𝑜𝑜𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑝𝑝𝑝𝑝𝑛𝑛𝑛𝑛𝑝𝑝𝑝𝑝 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑓𝑓𝑓𝑓 𝑎𝑎𝑎𝑎𝑛𝑛𝑛𝑛𝑝𝑝𝑝𝑝ℎ𝑎𝑎𝑎𝑎 − 𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 (𝛼𝛼𝛼𝛼1)𝑝𝑝𝑝𝑝𝑜𝑜𝑜𝑜𝑝𝑝𝑝𝑝𝑎𝑎𝑎𝑎𝑛𝑛𝑛𝑛 # 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑝𝑝𝑝𝑝𝑛𝑛𝑛𝑛𝑝𝑝𝑝𝑝 (𝛾𝛾𝛾𝛾)
Live-dead assay
200 µL of calcein AM and ethidium homodimer-1 mixture diluted in medium was added to the wells. Calcein AM for the detection of live cells was used in the concentration of 2µM and ethidium homodimer-1 for dead cells was used in 4 µM. The samples were incubated at 37°C for 45 minutes and then washed in PBS. Directly after that the visualization was performed under confocal microscope, green staining (live cells) at 495 nm and 515 nm and red staining (dead cells) at 495 nm and 635 nm, excitation and emission range respectively. To avoid false dead cells only 2 samples were stained per each 30 minutes.
Statistical analyses
Statistical analyses were performed by applying One-Way ANOVA on the differences in the number and size of the nucleus. A comparison between days using t-test was performed for the quantification of α-SMA-FITC staining. Values were considered significantly different between each other when p<0.05. All the statistical analysis was performed using SigmaPlot 11.0.
RESULTS
Immunohistochemistry
Analyses of number and size of nuclei, morphology of the cytoskeleton and expression of α-SMA were determined in cultured capsule-associated LEC cells in contact with hydrogelators and Matrigel. Fresh capsules and capsules without contact with hydrogels were used as controls.
When comparing fresh capsules with capsules cultured during 2 and 5 days, it was observed by confocal imaging that there were clear differences in cytoskeleton organization as well as the numbers of nuclei (Fig.1). The cultured capsule-associated LEC cells decreased in number and showed less actin fibers. However, their cytoskeleton appeared well organized without signs of morphologic alterations. After contact of the capsule with the hydrogelators significant changes in number and size of the nuclei was found, and likewise in the quantity and organization of the cytoskeleton. On day 2 the number of nuclei was not significantly decreased but their size already changed dramatically, either becoming very small with residual actin filaments (IKVAV+YIGSR, RGD+DGEA and Matrigel) or increasing in size and with a reorganization of the actin (LMWG and BM) (Fig. 1A). On day 5, all the hydrogels showed a significantly decrease in the quantity of cells when compared with fresh capsules but not compared with the
cultured capsule only. Although the number and size of nuclei at this stage did not appear to be significantly different between hydrogels the overall morphology changed drastically from day 2 to day 5 (Fig.1B). The layer of LEC in contact with LMWG created clusters of small cells, totally changing their typical phenotype. On the other side, cells on IKVAV+YIGSR and RGD+DGEA appeared with a regeneration of the cytoskeleton and with a significant increase in nucleus size. On BM cells showed a stable cytoskeleton from day 2 to day 5 without changes in size and number of nuclei. With Matrigel the size of LEC was significant smaller than on LMWG after 2 days of culture, with only a few actin fibers. On day 5 these fibers increase in quantity and changed in morphology (Fig.1B).
On day 2, IKVAV+YIGSR and RGD+DGEA hydrogels yielded the smallest nuclei but with significantly higher numbers when exposed to RGD+DGEA (Fig.1A). On day 5, cells in contact with IKVAV+YIGSR significantly increased in size and number but on RGD+DGEA there was a significant increase in size, yet a significant decrease in number (Fig.1C). While on day 2 the cytoskeleton of cells in contact with both hydrogels had almost disappeared, on day 5 it manifested itself in large quantity and with a different organization mostly with RGD+DGEA (Fig.1B).
Figure 1: A) Images of confocal microscopy from the nucleus (in blue: DAPI) and actin cytoskeleton (in red:
TRITC-phalloidin) on fresh capsules and on day 2 and day 5 of capsules without materials and capsules in contact with LMWG, LMWG+ IKVAV YIGSR, LMWG+ RDG DGEA, LMWG+ BM and Matrigel. B) Images of confocal microscopy from the nucleus (in blue: DAPI) on the capsules referred in C). Resolution 1024x1024 pixels corresponding to 375 x 375 µm. C) Graphic representing the number of nucleus on fresh capsules and on day 2 and day 5 of capsules without materials and capsules in contact with LMWG, LMWG+ IKVAV YIGSR, LMWG+ RDG DGEA, LMWG+ BM and Matrigel. Analysis performed by One-way ANOVA. Significant difference for p<0.05. Error bars depict standard deviations for three different experiments. Each experiment consisted in 4 to 6 capsules from different porcine eyes. * significant difference between fresh capsule and others at day 2; ** significant difference between RGD DGEA and others at day 2; # significant difference between fresh capsule and others at day 5. Θ significant difference between day 2 and day 5. D) Graphic representing the size of nucleus on fresh capsules and on day 2 and day 5 of capsules without materials and capsules in contact with LMWG, LMWG+ IKVAV YIGSR, LMWG+ RDG DGEA, LMWG+ BM and Matrigel. Error bars depict standard deviations for three different experiments. Size of nucleus. * significant difference between LMWG and others at day 2. ** significant difference between capsule and others at day 2. # significant difference between RGD DGEA and others at day 5. Θ significant difference between day 2 and day 5.
As expected fresh capsules did not show any sign of α-SMA. After incubation cells generally displayed α-SMA expression, with the exception of cells in contact with RGD+DGEA at day 2 (Fig.2A). Capsules cultured in medium during 2 days showed low quantity of α-SMA with a large increase at day 5. Cells in contact with LMWG showed large quantities of α-SMA since day 2 of culture but the protein rarely appeared as a fiber. The α-SMA in cells exposed to IKVAV+YIGSR containing hydrogels slightly decreased from day 2 to 5 with the protein predominantly expressed near the nucleus. Cells in contact with RGD+DGEA created a mature α-SMA only on day 5 of culture. This protein was concentrated near the nucleus and started to elongate as fibers. With BM cells showed a slight increase of α-SMA production from day 2 to 5, with the protein appearing as a diffuse stain near the nucleus. Matrigel gave the largest decrease of α-SMA production (Fig.2A, B).
Figure 2: A) Confocal images representing nucleus (in blue: DAPI), actin cytoskeleton (in red:
TRITC-phalloidin) and α-SMA filaments (in green: FITC) for fresh capsules, capsules without materials and capsules in contact with LMWG, LMWG IKVAV+YIGSR, LMWG RDG+DGEA, LMWG BM and matrigel at day 2 and 5. Resolution 1024x1024 pixels. B) Quantification of total α-SMA – FITC (green) staining on capsules without materials and capsules in contact with LMWG, LMWG IKVAV+YIGSR, LMWG RDG+DGEA, LMWG BM and matrigel at day 2 and 5. Normalization was done by # positive cells per # total cells. Statistical analysis of comparison between day 2 and 5 was performed by using t-test. Significant difference for p<0.05. Error bars depict standard deviations for three different experiments. Each treatment included 4 to 6 capsules from different porcine eyes.
4
Figure 1: A) Images of confocal microscopy from the nucleus (in blue: DAPI) and actin cytoskeleton (in red:
TRITC-phalloidin) on fresh capsules and on day 2 and day 5 of capsules without materials and capsules in contact with LMWG, LMWG+ IKVAV YIGSR, LMWG+ RDG DGEA, LMWG+ BM and Matrigel. B) Images of confocal microscopy from the nucleus (in blue: DAPI) on the capsules referred in C). Resolution 1024x1024 pixels corresponding to 375 x 375 µm. C) Graphic representing the number of nucleus on fresh capsules and on day 2 and day 5 of capsules without materials and capsules in contact with LMWG, LMWG+ IKVAV YIGSR, LMWG+ RDG DGEA, LMWG+ BM and Matrigel. Analysis performed by One-way ANOVA. Significant difference for p<0.05. Error bars depict standard deviations for three different experiments. Each experiment consisted in 4 to 6 capsules from different porcine eyes. * significant difference between fresh capsule and others at day 2; ** significant difference between RGD DGEA and others at day 2; # significant difference between fresh capsule and others at day 5. Θ significant difference between day 2 and day 5. D) Graphic representing the size of nucleus on fresh capsules and on day 2 and day 5 of capsules without materials and capsules in contact with LMWG, LMWG+ IKVAV YIGSR, LMWG+ RDG DGEA, LMWG+ BM and Matrigel. Error bars depict standard deviations for three different experiments. Size of nucleus. * significant difference between LMWG and others at day 2. ** significant difference between capsule and others at day 2. # significant difference between RGD DGEA and others at day 5. Θ significant difference between day 2 and day 5.
As expected fresh capsules did not show any sign of α-SMA. After incubation cells generally displayed α-SMA expression, with the exception of cells in contact with RGD+DGEA at day 2 (Fig.2A). Capsules cultured in medium during 2 days showed low quantity of α-SMA with a large increase at day 5. Cells in contact with LMWG showed large quantities of α-SMA since day 2 of culture but the protein rarely appeared as a fiber. The α-SMA in cells exposed to IKVAV+YIGSR containing hydrogels slightly decreased from day 2 to 5 with the protein predominantly expressed near the nucleus. Cells in contact with RGD+DGEA created a mature α-SMA only on day 5 of culture. This protein was concentrated near the nucleus and started to elongate as fibers. With BM cells showed a slight increase of α-SMA production from day 2 to 5, with the protein appearing as a diffuse stain near the nucleus. Matrigel gave the largest decrease of α-SMA production (Fig.2A, B).
Figure 2: A) Confocal images representing nucleus (in blue: DAPI), actin cytoskeleton (in red:
TRITC-phalloidin) and α-SMA filaments (in green: FITC) for fresh capsules, capsules without materials and capsules in contact with LMWG, LMWG IKVAV+YIGSR, LMWG RDG+DGEA, LMWG BM and matrigel at day 2 and 5. Resolution 1024x1024 pixels. B) Quantification of total α-SMA – FITC (green) staining on capsules without materials and capsules in contact with LMWG, LMWG IKVAV+YIGSR, LMWG RDG+DGEA, LMWG BM and matrigel at day 2 and 5. Normalization was done by # positive cells per # total cells. Statistical analysis of comparison between day 2 and 5 was performed by using t-test. Significant difference for p<0.05. Error bars depict standard deviations for three different experiments. Each treatment included 4 to 6 capsules from different porcine eyes.
α-SMA images analysis
The use of pig eye lens capsular bags was associated with a relatively large variance between different samples. To account for this variation in quantifying α-SMA, we looked into each image with its nuclei (Fig.3). We analyzed the percentage of images with presence of α-SMA and for these positive images we analyzed how many nuclei were positive for α-SMA, this means detection of staining around the nuclei or in fibers. It was found that the percentage of images with capsule-associated, α-SMA-positive LEC slightly decreased from day 2 to day 5, but that in the positive images the quantity of α-SMA increased from 7% to 15%, which corroborates with the general quantification of α-SMA shown in figure 2B. For cells in contact with LMWG there was a slight increase in the number of positive images on day 5 but the quantity of positive nuclei did not change, as also indicated on Fig. 2B. With IKVAV+YIGSR the number of positive images decreased at day 5 as well as the number of positive nuclei, from 11% to 4%. The combination RGD+DGEA did not show any positive signals of α-SMA on day 2 but on day 5 more than half (54%) of the images contained α-SMA positive cells. The BM had the largest number of positive images on day 2 (78%) and this number was decreased at day 5 (56%) however, most of the nuclei were negative for α-SMA, 85% and 61% at day 2 and 5 respectively. The percentage of positive images for LEC in contact with Matrigel was similar at day 2 and 5 but the positive nuclei decreased from 38% to 17%, as is also visible in Fig. 2B.
Figure 3: Details on the quantification of α-SMA. First circular graphics represent the percentage of confocal
images where it was no detected α-SMA staining -in red- and percentage of images with presence of α-SMA staining - in dark blue. Second circular graphics represent details of the images with α-SMA. The dark blue represents the quantity of positive nuclei for SMA and the light blue the percentage of negative nuclei, no α-SMA detected.
4
α-SMA images analysis
The use of pig eye lens capsular bags was associated with a relatively large variance between different samples. To account for this variation in quantifying α-SMA, we looked into each image with its nuclei (Fig.3). We analyzed the percentage of images with presence of α-SMA and for these positive images we analyzed how many nuclei were positive for α-SMA, this means detection of staining around the nuclei or in fibers. It was found that the percentage of images with capsule-associated, α-SMA-positive LEC slightly decreased from day 2 to day 5, but that in the positive images the quantity of α-SMA increased from 7% to 15%, which corroborates with the general quantification of α-SMA shown in figure 2B. For cells in contact with LMWG there was a slight increase in the number of positive images on day 5 but the quantity of positive nuclei did not change, as also indicated on Fig. 2B. With IKVAV+YIGSR the number of positive images decreased at day 5 as well as the number of positive nuclei, from 11% to 4%. The combination RGD+DGEA did not show any positive signals of α-SMA on day 2 but on day 5 more than half (54%) of the images contained α-SMA positive cells. The BM had the largest number of positive images on day 2 (78%) and this number was decreased at day 5 (56%) however, most of the nuclei were negative for α-SMA, 85% and 61% at day 2 and 5 respectively. The percentage of positive images for LEC in contact with Matrigel was similar at day 2 and 5 but the positive nuclei decreased from 38% to 17%, as is also visible in Fig. 2B.
Figure 3: Details on the quantification of α-SMA. First circular graphics represent the percentage of confocal
images where it was no detected α-SMA staining -in red- and percentage of images with presence of α-SMA staining - in dark blue. Second circular graphics represent details of the images with α-SMA. The dark blue represents the quantity of positive nuclei for SMA and the light blue the percentage of negative nuclei, no α-SMA detected.
Live dead analysis
The live dead assay was performed as a qualitative measurement to identify the survival of LEC. It was shown in figure 1B that cells changed their cytoskeleton, number and size of their nucleus and it was also shown that the contact with the different hydrogels can result in cell death. On the fresh capsule and on capsules cultured in medium for 2 days all cells were alive. However, LEC in contact with any hydrogel partly died, but to a lesser extent with RGD+DGEA (Fig.4). After 5 days of culture, the increase of dead cells was even more significant. LMWG was the hydrogel with the largest quantity of dead cells followed by IKVAV+YIGSR. RGD+DGEA and BM were the hydrogels that performed best in the survival of the cells.
Figure 4: Confocal images for live-dead staining performed on fresh porcine lens capsules, cultured only in
medium or in contact with LMWG, LMWG+ IKVAV YIGSR, LMWG+ RDG DGEA, LMWG+ BM and matrigel at day 2 and 5. Green-fluorescent calcein-AM represented live LEC cells and red-fluorescent ethidium homodimer-1 represented dead cells. Resolution 1024x1024 pixels.
DISCUSSION
One of the characteristics of epithelial cells is their ability to (de)differentiate through the process of epithelial-mesenchymal transition (EMT). EMT is directly associated with PCO, a fibrotic process in the eye lens. In this study we developed a lens capsular bag model for establishing the effects of LEC exposure to bioactive hydrogels with respect to EMT development. Bioactivity was generated by the incorporation of adhesive peptide motifs present in fibronectin, collagens and laminin.
LEC in the natural state appeared in a single layer of small and circular/hexagonal cells. The mere fact of culturing them can already create modification in the cytoskeletal
organization [15]. With this ex vivo model, LEC were in a semi-natural stage while allowing them to interact with the hydrogels (LMWGs in our case), therewith resembling the post-operative situation in case these hydrogels were injected in the capsular bag. It was reported by Wormstone et al. [15] that LEC collected from the capsular bag can not only proliferate but also maintain their activity in a serum-free medium. We have chosen to culture the cells in serum-containing medium to create conditions towards EMT, so that the effect of LMWGs in attenuating EMT can be evaluated. The goal of using LMWG linked with different combinations of peptides was to keep LECs alive and at the same time avoid their transition to myofibroblasts. We analyzed cell transition through several parameters such as elongation of the cytoskeleton, increase of the nucleus size and the decrease in cell number. The elongation of the cytoskeleton and the augmentation of the nucleus size are associated with the dedifferentiation from the epithelial phenotype to fiber-like cells. It is likely that the increase in cell size is associated with a decrease in cell numbers per unit area.
The morphologic alterations come together with the production of fibrotic proteins such as α-SMA [16-18]. The mRNA production of this protein was visible within 24h in explants of bovine lens epithelial cells without the use of any treatment. But mRNA for fibronectin and type I collagen was not detected [19]. In our study there was no evidence of α-SMA in cells in the fresh capsular bags but this protein was present after 5 days of culture. Two of the bioactive hydrogels (LMWG RGD+DGEA and BM) gave rise to an increase of α-SMA, which could reflect the surviving behavior of the cells as demonstrated by the live dead assay, as the largest cell survival was observed in these hydrogel exposures. Although the presence of α-SMA could be related with cell survival, this can compromise the cell function. Nagamoto et al. reported that in the early stage, α-SMA is expressed near the nucleus and in a mature stage of EMT it starts to appear as a fiber [17]. Fibers of α-SMA were visible in small quantities in only one of our hydrogelators, LMWG RGD+DGEA, possibly due to the RGD motif that has been shown to promote cell migration [20].
Our data demonstrate that the use of adhesive peptide sequences incorporated in the LMWG gave an improved LEC response when compared with the LWMG alone. The combination of fibronectin and collagen type I motifs (RDG+DGEA) clearly showed a regeneration of LEC associated with cell differentiation. Olivero et al. [20] studied the effects of type IV collagen, laminin and fibronectin in promoting the adhesion of rabbit LEC and concluded that type IV collagen and fibronectin improved the adhesion and migration of LEC, respectively. This can explain the cell regeneration when in contact with LMWG-RGD+DGEA. LEC had access to both active domains of fibronectin (migration) and type IV collagen (adhesion). Due to the large initial decrease of actin filaments on this hydrogelator, the presence of cells on day 5 of culture was expected to be low. But this mixture had stimulated cultured capsule-associated LEC cells into cell survival and EMT. The combination of laminin motifs (IKVAV+YIGSR) followed a similar process although these peptides seemed to slow down the process of cell differentiation and increased cell death. The effects of type I, IV collagen, fibronectin and laminin in porcine LEC in vitro were also studied by Jong-Hesse et al. [21]. They showed that cell proliferation is enhanced by fibronectin and type I and IV collagens but not by laminin. The α-SMA
4
Live dead analysis
The live dead assay was performed as a qualitative measurement to identify the survival of LEC. It was shown in figure 1B that cells changed their cytoskeleton, number and size of their nucleus and it was also shown that the contact with the different hydrogels can result in cell death. On the fresh capsule and on capsules cultured in medium for 2 days all cells were alive. However, LEC in contact with any hydrogel partly died, but to a lesser extent with RGD+DGEA (Fig.4). After 5 days of culture, the increase of dead cells was even more significant. LMWG was the hydrogel with the largest quantity of dead cells followed by IKVAV+YIGSR. RGD+DGEA and BM were the hydrogels that performed best in the survival of the cells.
Figure 4: Confocal images for live-dead staining performed on fresh porcine lens capsules, cultured only in
medium or in contact with LMWG, LMWG+ IKVAV YIGSR, LMWG+ RDG DGEA, LMWG+ BM and matrigel at day 2 and 5. Green-fluorescent calcein-AM represented live LEC cells and red-fluorescent ethidium homodimer-1 represented dead cells. Resolution 1024x1024 pixels.
DISCUSSION
One of the characteristics of epithelial cells is their ability to (de)differentiate through the process of epithelial-mesenchymal transition (EMT). EMT is directly associated with PCO, a fibrotic process in the eye lens. In this study we developed a lens capsular bag model for establishing the effects of LEC exposure to bioactive hydrogels with respect to EMT development. Bioactivity was generated by the incorporation of adhesive peptide motifs present in fibronectin, collagens and laminin.
LEC in the natural state appeared in a single layer of small and circular/hexagonal cells. The mere fact of culturing them can already create modification in the cytoskeletal
organization [15]. With this ex vivo model, LEC were in a semi-natural stage while allowing them to interact with the hydrogels (LMWGs in our case), therewith resembling the post-operative situation in case these hydrogels were injected in the capsular bag. It was reported by Wormstone et al. [15] that LEC collected from the capsular bag can not only proliferate but also maintain their activity in a serum-free medium. We have chosen to culture the cells in serum-containing medium to create conditions towards EMT, so that the effect of LMWGs in attenuating EMT can be evaluated. The goal of using LMWG linked with different combinations of peptides was to keep LECs alive and at the same time avoid their transition to myofibroblasts. We analyzed cell transition through several parameters such as elongation of the cytoskeleton, increase of the nucleus size and the decrease in cell number. The elongation of the cytoskeleton and the augmentation of the nucleus size are associated with the dedifferentiation from the epithelial phenotype to fiber-like cells. It is likely that the increase in cell size is associated with a decrease in cell numbers per unit area.
The morphologic alterations come together with the production of fibrotic proteins such as α-SMA [16-18]. The mRNA production of this protein was visible within 24h in explants of bovine lens epithelial cells without the use of any treatment. But mRNA for fibronectin and type I collagen was not detected [19]. In our study there was no evidence of α-SMA in cells in the fresh capsular bags but this protein was present after 5 days of culture. Two of the bioactive hydrogels (LMWG RGD+DGEA and BM) gave rise to an increase of α-SMA, which could reflect the surviving behavior of the cells as demonstrated by the live dead assay, as the largest cell survival was observed in these hydrogel exposures. Although the presence of α-SMA could be related with cell survival, this can compromise the cell function. Nagamoto et al. reported that in the early stage, α-SMA is expressed near the nucleus and in a mature stage of EMT it starts to appear as a fiber [17]. Fibers of α-SMA were visible in small quantities in only one of our hydrogelators, LMWG RGD+DGEA, possibly due to the RGD motif that has been shown to promote cell migration [20].
Our data demonstrate that the use of adhesive peptide sequences incorporated in the LMWG gave an improved LEC response when compared with the LWMG alone. The combination of fibronectin and collagen type I motifs (RDG+DGEA) clearly showed a regeneration of LEC associated with cell differentiation. Olivero et al. [20] studied the effects of type IV collagen, laminin and fibronectin in promoting the adhesion of rabbit LEC and concluded that type IV collagen and fibronectin improved the adhesion and migration of LEC, respectively. This can explain the cell regeneration when in contact with LMWG-RGD+DGEA. LEC had access to both active domains of fibronectin (migration) and type IV collagen (adhesion). Due to the large initial decrease of actin filaments on this hydrogelator, the presence of cells on day 5 of culture was expected to be low. But this mixture had stimulated cultured capsule-associated LEC cells into cell survival and EMT. The combination of laminin motifs (IKVAV+YIGSR) followed a similar process although these peptides seemed to slow down the process of cell differentiation and increased cell death. The effects of type I, IV collagen, fibronectin and laminin in porcine LEC in vitro were also studied by Jong-Hesse et al. [21]. They showed that cell proliferation is enhanced by fibronectin and type I and IV collagens but not by laminin. The α-SMA
production increased on fibronectin and type I collagen but there was no significant increase on laminin and type IV collagen, the two basal lamina proteins. This is corroborated by the larger cell differentiation on LMWG RGD+DGEA and the minor survival of LEC on LMWG IKVAV+YIGSR and also the lower cell differentiation compared with LMWG RGD+DGEA in our study. The mixture resembling the basement membrane (BM) possesses a combination of fibronectin, collagen and laminin motifs, the latter in larger quantities which can explain the good cell attachment and proliferation since day 2. This combination also showed a low quantity of α-SMA and absence of α-SMA fibers. Besides having the best LEC survival, LMWG BM slowed down cell transition.
It has been proposed that the stress induced by the cataract surgery is the major cause for LEC transition into myofibroblasts [2, 22]. Fibronectin is commonly found in plasma and it is an important protein in lens development during embryogenesis; but its presence in adult lenses is still under debate. Fibronectin has been identified throughout the posterior and equatorial capsule but not in the anterior capsule [23]. Its source is unknown but some studies of bovine and rabbit cultured lenses already excluded the hypothesis of fibronectin being produced by epithelial cells [19]. Nevertheless this protein is described as a promoter of LEC attachment and migration in humans [20, 24] and rabbits [20] as well as, having an important role in lens wound healing and PCO [19]. The α5β1 integrin is the receptor for the fibronectin RGD sequence and it is associated with
adhesion and proliferation of LEC [25, 26]. It was also found that α5β1 is present in cataract
lenses and during the differentiation of LEC to myofibroblasts. In in vitro cultures TGF-β2
activates α5β1 and fibronectin in the motile cells and seems to help their elongation
towards the myofibroblasts morphology [27]. Both fibronectin and TGF- βs have not been reported to be present before cataract extraction but TGF- βs receptors are activated after an eye lens injury and soluble fibronectin is available in the aqueous humour as a consequence of breaching the blood-aqueous barrier. The integrin α6β1, a laminin
receptor, is expressed during lens development and after a cataract surgery, it seems to be primarily associated with the healing process [28]. All these findings explain the large cell regeneration and proliferation with the fibronectin combination (RGD DGEA), but not with the laminin combination (IKVAV YIGSR).
Other effects of epithelial (de)differentiation into EMT is based on loss of the contact with the basement membrane. This clearly happens during fibrosis in the eye lens when LEC, that only exist in the anterior capsule bag, start to migrate to the posterior capsule bag as myofibroblasts. We expect that the interaction of LEC with a mix of peptides resembling the basement membrane, as in the BM combination, could reassure epithelial behavior of LEC and avoid EMT. Despite that LMWG BM did not accomplish the total avoidance of EMT, cells that interact with this hydrogel slowed down the transition into fiber-like cells and did not enhance α-SMA production as in the other combinations.
In our study we still did not reach a perfect peptide composition to totally avoid EMT but we found a favorable hydrogel (LMWG BM) to delay EMT. This hydrogel showed superior cell behavior when compared with the standard in vitro gel Matrigel, with hyaluronic acid [29], a hydrogel used in surgery, or any in vitro IOL [30]. Huang et al.
functionalized hydrophilic acrylic IOLs with RGD and found enhanced cell proliferation that was, however, different from our results as the quantity of α-SMA and cell differentiation was much higher [31]. The LMWG BM already showed the lowest cell differentiation without compromising cell survival and proliferation. It is recommended to also investigate different percentages and ratios of peptides or new combinations. Along the use of this hydrogel as a 3D environment, this gel can also be used as a coating of for example commercial intraocular lenses.
CONCLUSION
The capsular bag model developed in this study turned out to yield different LEC responses when exposed to different bioactive hydrogels. Our data showed that gels can modulate EMT reduction in a model that emulates post cataract surgery. Therefore, we conclude that the LMWG BM hydrogel may delay EMT.
ACKNOWLEDGEMENTS
This project was funded by the European Fund for Regional Development of the Ministry of Economic Affairs of the Netherlands, Samenwerkingsverband Noord-Nederland (grant number 249- Bioactive interface layers for medical implants) and by NanoNextNL, a micro and nanotechnology consortium of the Government of the Netherlands and 130 partners.
4
production increased on fibronectin and type I collagen but there was no significantincrease on laminin and type IV collagen, the two basal lamina proteins. This is corroborated by the larger cell differentiation on LMWG RGD+DGEA and the minor survival of LEC on LMWG IKVAV+YIGSR and also the lower cell differentiation compared with LMWG RGD+DGEA in our study. The mixture resembling the basement membrane (BM) possesses a combination of fibronectin, collagen and laminin motifs, the latter in larger quantities which can explain the good cell attachment and proliferation since day 2. This combination also showed a low quantity of α-SMA and absence of α-SMA fibers. Besides having the best LEC survival, LMWG BM slowed down cell transition.
It has been proposed that the stress induced by the cataract surgery is the major cause for LEC transition into myofibroblasts [2, 22]. Fibronectin is commonly found in plasma and it is an important protein in lens development during embryogenesis; but its presence in adult lenses is still under debate. Fibronectin has been identified throughout the posterior and equatorial capsule but not in the anterior capsule [23]. Its source is unknown but some studies of bovine and rabbit cultured lenses already excluded the hypothesis of fibronectin being produced by epithelial cells [19]. Nevertheless this protein is described as a promoter of LEC attachment and migration in humans [20, 24] and rabbits [20] as well as, having an important role in lens wound healing and PCO [19]. The α5β1 integrin is the receptor for the fibronectin RGD sequence and it is associated with
adhesion and proliferation of LEC [25, 26]. It was also found that α5β1 is present in cataract
lenses and during the differentiation of LEC to myofibroblasts. In in vitro cultures TGF-β2
activates α5β1 and fibronectin in the motile cells and seems to help their elongation
towards the myofibroblasts morphology [27]. Both fibronectin and TGF- βs have not been reported to be present before cataract extraction but TGF- βs receptors are activated after an eye lens injury and soluble fibronectin is available in the aqueous humour as a consequence of breaching the blood-aqueous barrier. The integrin α6β1, a laminin
receptor, is expressed during lens development and after a cataract surgery, it seems to be primarily associated with the healing process [28]. All these findings explain the large cell regeneration and proliferation with the fibronectin combination (RGD DGEA), but not with the laminin combination (IKVAV YIGSR).
Other effects of epithelial (de)differentiation into EMT is based on loss of the contact with the basement membrane. This clearly happens during fibrosis in the eye lens when LEC, that only exist in the anterior capsule bag, start to migrate to the posterior capsule bag as myofibroblasts. We expect that the interaction of LEC with a mix of peptides resembling the basement membrane, as in the BM combination, could reassure epithelial behavior of LEC and avoid EMT. Despite that LMWG BM did not accomplish the total avoidance of EMT, cells that interact with this hydrogel slowed down the transition into fiber-like cells and did not enhance α-SMA production as in the other combinations.
In our study we still did not reach a perfect peptide composition to totally avoid EMT but we found a favorable hydrogel (LMWG BM) to delay EMT. This hydrogel showed superior cell behavior when compared with the standard in vitro gel Matrigel, with hyaluronic acid [29], a hydrogel used in surgery, or any in vitro IOL [30]. Huang et al.
functionalized hydrophilic acrylic IOLs with RGD and found enhanced cell proliferation that was, however, different from our results as the quantity of α-SMA and cell differentiation was much higher [31]. The LMWG BM already showed the lowest cell differentiation without compromising cell survival and proliferation. It is recommended to also investigate different percentages and ratios of peptides or new combinations. Along the use of this hydrogel as a 3D environment, this gel can also be used as a coating of for example commercial intraocular lenses.
CONCLUSION
The capsular bag model developed in this study turned out to yield different LEC responses when exposed to different bioactive hydrogels. Our data showed that gels can modulate EMT reduction in a model that emulates post cataract surgery. Therefore, we conclude that the LMWG BM hydrogel may delay EMT.
ACKNOWLEDGEMENTS
This project was funded by the European Fund for Regional Development of the Ministry of Economic Affairs of the Netherlands, Samenwerkingsverband Noord-Nederland (grant number 249- Bioactive interface layers for medical implants) and by NanoNextNL, a micro and nanotechnology consortium of the Government of the Netherlands and 130 partners.
