The present protocol describes the extraction of lumican from the amniotic membrane (AM) and their storing conditions as AM extract (AME) at -20 °C, 4 °C, and room temperature (RT) for 6, 12, 20, and 32 days to quantify its proteins and lumican concentration.
Lumican is a small leucine-rich proteoglycan in the human amniotic membrane (AM) that promotes corneal epithelialization and the organization of collagen fibers, maintaining corneal transparency. In the present work, a method for protein extraction from AM to obtain lumican is proposed. Additionally, the stability of lumican in the AM extract (AME) stored at different temperatures and time periods is evaluated. 100 mg of AM were thawed and mechanical de-epithelialized. The de-epithelialized AM was frozen and crushed until a fine powder was obtained, which was solubilized with 2.5 mL of saline buffer with protease inhibitors and centrifuged for protein extraction. The supernatant was collected and stored at -20 °C, 4 °C, and room temperature (RT) for 6, 12, 20, and 32 days. Afterward, lumican was quantified in each AME. This technique allows an accessible and acquirable protocol for lumican extraction from AM. Lumican concentration was affected by storage time and temperature conditions. Lumican in the AME of 12 days stored at -20 °C and 4 °C was significantly higher than other AME. This lumican extraction could be useful for developing treatments and pharmaceutical solutions. Further studies are needed to determine the uses of AME lumican in re-epithelialization and wound healing process.
One of the most used treatments for corneal affections is amniotic membrane transplantation; however, in recent years, new proposals have emerged for using various components of amniotic tissue as alternative and adjuvant treatments. Among the most studied components of AM are those obtained from the AM extract (AME)1,2,3,4,5,6,7. AM contains multiple soluble factors such as antiangiogenic proteins, interleukins (IL), tissue inhibitors of metalloproteinases (TIMPs), anti-inflammatory proteins mediated by TSG-6 that inhibit neutrophil extracellular traps, growth factors: epidermal growth factor (EGF), transforming growth factor (TGF) (alpha and beta), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and lumican, which maintains corneal transparency by regulating collagen fibrillogenesis1,2,3,4,5,6,7,8,9.
Lumican is a small leucine-rich proteoglycan (SLRP), one of the main extracellular components of interstitial collagenase in the corneal stroma matrix, responsible for organizing collagen fibers and maintaining corneal transparency4,10,11. Proteoglycans are molecules in the extracellular matrix (ECM), which are the main ones in carrying out cell signaling and maintaining intracellular homeostasis12. ECM proteins have been reported to drive the cellular processes of proliferation, differentiation, and migration during wound healing11.
Evidence indicates the possible participation of lumican in the process of corneal re-epithelialization. Saika et al., in a study, showed that after a corneal injury, lumican could be detected in corneal keratocytes between the first 8 h and up to 3 days after injury. Presenting the highest concentration of lumican on the second and third day, this proteoglycan is subsequently undetectable on the seventh day13. These data suggest the participation of lumican in the activation of the corneal re-epithelialization process. On the other hand, in another study, it was reported that the absence of lumican delays re-epithelialization; interestingly, adding lumican could accelerate the re-epithelialization process4,11,13. Likewise, a recent study has reported that lumican can modulate the inflammatory functions of corneal limbus fibroblasts14, which suggests a role for lumican as a modulator of the inflammatory, antifibrotic and re-epithelializing response. Similarly, lumican can modulate the corneal response by interacting with signaling molecules such as Fas-FasL. Also, the absence of lumican in a knockout Lum-/- mouse model demonstrated that the lack of lumican signaling prevents adequate corneal repair15.
Primarily, this method aims to demonstrate a feasible and approachable way to extract lumican from AM. With this advantageous method of lumican extraction, it is possible to obtain similar concentrations of proteins, decreasing the processing time and making it more convenient for investigators compared to the previous studies16. Furthermore, this AME lumican could be used as an adjuvant for corneal repair and re-epithelialization processes.
All the experimental procedures were approved by the Institutional Review Board (Project No. CEI-2020/06/04). The AM was obtained from the Instituto de Oftalmologia Conde de Valenciana amnion bank (from deidentified human subjects), which is prepared as described by Chávez-García et al.17.
1. Preparation of the amniotic membrane extract
Figure 1: Process of AME preparation and lumican concentration measurement. 100 mg of AM were incubated with dispase II at 37 °C for 30 min and mechanically de-epithelialized. The de-epithelialized AM was washed and immersed in liquid nitrogen for 40 min, and then crushed until a fine powder was obtained, which was solubilized with 2.5 mL of saline buffer with protease inhibitors and centrifuged. The supernatant was collected and stored at -20 °C, 4 °C and RT for 6, 12, 20, and 32 days until total protein and lumican quantitation. Please click here to view a larger version of this figure.
2. AME protein quantification
NOTE: The quantification of total protein in the AME must be carried out immediately after obtention. Quantify proteins using Lowry protein assay and follow the manufacturer's instructions (see Table of Materials). It is recommended that all standards and samples be assayed in triplicate.
3. Quantification of Lumican in AME
NOTE: The concentration of lumican must be measured in the AME stored at different storage conditions and time periods. Quantify lumican using sandwich ELISA and follow the manufacturer's instructions. It is recommended that all standards and samples be assayed in duplicate.
Results are reported as the mean value ± standard deviation (SD). Student's t-tests and analysis of variance (ANOVA) were performed. P-values < 0.05 was considered statistically significant. Statistical analysis was performed using statistics software (see Table of Materials).
The total protein quantity in the AME was affected by time and storage conditions. The basal protein concentration was similar among all AME; the range of total protein was 2.7 ± 0.3 µg/mL without a significant difference between the samples evaluated. However, when the samples were stored for 12, 20, and 32 days, variability in protein concentration with respect to basal concentration was observed. Interestingly, protein concentration increased in the AME at 4 °C and -20 °C with respect to the RT at all times of storage.
Similarly, when protein concentration was compared between storage times, it changed after 12, 20, and 32 days. A significant difference (p < 0.05) was found in the AME of 32 and 20 days at 4 °C and -20 °C in comparison to the RT condition (Figure 2), suggesting that temperature is important for protein conservation in the different AME obtained.
Figure 2: Total protein concentration in AME affected by time and storage temperature. The concentration of protein extraction on AME was quantified before and after temperature and time storage conditions. Storage time evaluated was 6 days (black triangles), 12 days (pink triangles), 20 days (purple square), and 32 days (brown circles) in comparison with three different temperature conditions (RT °C, 4 °C, and -20 °C). Basal protein concentration was similar among all AME. There was a significant difference in protein concentration in the AME of 20 and 32 days with respect to basal protein concentration at different temperature conditions. In each condition n = 3. Data is expressed as median of µg/mL of protein ± SE *p < 0.05 (S1 32 days vs. S1 20, 12, and 6 days at 4 °C); (S1 32 days vs. S1 20, 12, and 6 days at -20 °C). Please click here to view a larger version of this figure.
Lumican concentration was affected by storage time and temperature conditions. Fewer concentration of lumican was found in the AME stored for 6, 20, and 32 days, compared with 12 days of storage. Significantly, the AME of 12 days had a higher concentration of lumican than 20 and 32 days of storage (p < 0.05).
When lumican concentration in the AME was compared between storage temperatures, a higher concentration of lumican was found if stored at -20 °C and 4 °C for 12 days (Figure 3). Interestingly, even a higher (p < 0.05) concentration of lumican was found in the 12 days AME if stored at -20 °C in comparison with 4 °C.
This suggests that the concentration of lumican is affected by temperature conditions and storage time, suggesting that the appropriate storage time and temperature to achieve the highest concentration of lumican is 12 days at -20 °C.
Figure 3: Total lumican concentration in AME affected by time and storage temperature. Lumican concentration was affected by storage time and temperature conditions. The concentration of lumican in AME was quantified before and after temperature and time storage conditions. Storage time evaluated was 6 days (black triangles), 12 days (pink triangles), 20 days (purple square), and 32 days (brown circles) in comparison with three different temperature conditions (RT °C, 4 °C, and -20 °C). Lumican in the AME of 12 days was significantly higher in comparison with the AME of 32, 20, and 6 days, at temperature conditions of -20 °C and 4 °C. *p < 0.05 (S1 12 days vs. S1 32, 20, and 6 days at 4 °C); ***p < 0.001 (S1 12 days vs. S1 32, 20 and 6 days at -20°C). The highest concentration of lumican in AME was at 12 days stored at -20 °C. ++p < 0.01 (S1 12 days 4 °C vs. S1 12 days -20 °C). There was no significant difference between lumican in the AME of 32 and 20 days compared with storage temperature conditions (n.s). In each condition (n = 3), data are expressed as the median of ng/mL of protein. Data were normalized with respect to mg of tissue ± SE. (n.s.) not statistical significance. Please click here to view a larger version of this figure.
In this study, the presence of lumican was analyzed in the AME and its direct correlation with its stability under different storage conditions. Interestingly, when the total protein concentration in AME was quantified, protein concentration increased after storage. Evidence suggests three mechanisms that could change protein concentration in frozen storage: cold denaturation, the frozen concentration of solutes, and ice-induced partial unfolding of protein structure19. The freezing process could affect protein concentration on storage samples due to the crystallization of the liquid phase in the sample. The results suggest that this could have occurred as a process of freezing the concentration, affected by the storage time in the freezer. A higher concentration of proteins was observed at longer times of storage (32 and 20 days) and at the coldest temperatures (4 °C and -20 °C). However, the period with the highest concentration of protein did not have the highest concentration of lumican; this suggests that lumican could be affected by frozen temperatures and time conditions.
According to the results, lumican concentration was higher and more stable at 12 days of storage at 4 °C and -20 °C. Nonetheless, a lesser concentration of lumican was found at 6 days compared to 12 days. Some reports suggest that protein concentration could change after frozen storage conditions20. The results could be caused by a thermodynamic mechanism named ice-induced partial unfolding of protein structure, which happens during the freezing of samples. The interaction of proteins with water in the aqueous solutions reduces their interactions with other molecules. The crystallization process of water under frozen conditions allows proteins and some functional regions to interact with other molecules19. By the aforementioned, after 12 days of freezing lumican in an aqueous solution, it could be able to interact with the antibodies present in the ELISA quantification kit to result in a higher concentration. On the other hand, probably on the sixth day, the lumican could have been secluded in the aqueous solution.
In advancing innovations, the use of AM is the state-of-the-art treatment for corneal re-epithelialization regardless of etiology. As aforementioned, the benefits of AM are vast21,22,23,24,25,26. Many authors have demonstrated the benefits of lumican in AME, making it an affordable alternative specifically for developing countries1,2,3,4,5,6,7,8,9. Currently, there are many benefits of lumican in AME; its use as a treatment favors corneal re-epithelialization and improves the prognosis of corneal ulcers4,5,6,7,8,9,10,11,12,13,14,15. AM transplantation (AMT) has become a treatment with great benefits for improving various corneal disorders24,25. However, there are some chronic affections of the corneal tissue, such as persistent epithelial defects (PED) and limbal stem cell deficiencies (LESCD), that require constant treatment and maintenance of the presence of biological factors that help corneal repair21,24. Currently, there are no adjuvant treatments that allow long-term maintenance of the factors released by AMT on the corneal surface. However, a constant replacement of the AMT could not be recommended for patient safety26. For this reason, it is necessary to develop alternatives that allow the functions of AMT to assist and helps maintain the presence of factors released by AM in the corneal tissue such as lumican for a longer time, intending to favor the treatment of persistent problems of the cornea27.
Lumican is one of the factors present in AM with anti-inflammatory and antifibrotic functions, which has been reported to have functions in the corneal repair process6,7,8,9,10,11,12,13,14,15. That is why lumican suggests being a good candidate to aid in treating corneal affections; however, additional research is required to determine the efficacy of lumican in AME to achieve corneal re-epithelialization.
Lumican is a proteoglycan that has been shown to regulate the secretion of extracellular matrix compounds as collagen; also, it is involved in fibroblast activation and modulation of inflammatory cells and the angiogenesis process, having an important role in wound healing. According to the results, lumican can be extracted from AM tissue. Therapeutic applications of lumican are numerous; the use of lumican in the AME allows an attainable therapeutic option for ocular disorders4,13. The primary advantage of using AME is that it provides an easy application as a topical treatment for the ocular surface, given its aqueous composition. Likewise, other proteins with anti-inflammatory and immunoregulatory characteristics can be found within the components extracted from the AM tissue, which could present a greater benefit in treating de-epithelializing problems in the eye. For example, other anti-inflammatory factors such as TSG-6 present in the AM and cellular components have previously been reported to have immunoregulatory properties8. Hereby, a combined therapy of lumican and other extracellular matrix compounds and immunomodulatory molecules present in the AME could be useful in the re-epithelialization and wound healing process.
This method aims to demonstrate a straightforward technique for protein extraction, useful for the obtention of bountiful proteins and factors. One of the critical considerations for this method is the use of protease inhibitors, as it is fundamental for successful protein extraction as the AM is a tissue with loads of enzymatic compounds to prevent protein degradation27. Evidence has reported that using protein inhibitors together with an aqueous solution increases the extraction of other factors, such as HGF, in AM tissue26. The obtention of a fine powder after freezing and grounding the AM is necessary for the optimal obtention of AME, as this process is suitable for tissue and cellular disruption of structures needed for the extraction of cytosolic and other nuclear compounds28,29.
A few limitations of this extraction method are that the AM quantity used could not allow a high-scale extraction. This protocol allows protein extraction from a limited amount of tissue; since there is no evidence that this method allows obtaining a greater amount of protein from a larger tissue area. Troubleshooting to be considered in comparison with other extraction methods is to use a suitable concentration of protease inhibitor and incubation time, as the excess enzymes could affect proteins30 and reduce the technique's efficacy.
Part of this technique was modified from that reported by Mahbod et al.16, which describes that repeating the centrifugation and extraction process increases protein extraction. On contrary to Mahbod, the results did not report protein concentration after three cycles of centrifugation. When the total protein concentration was determined, it decreased by 80% in a second extraction and up to 97% in a third extraction. Performing only one extraction reduces the processing time and does not decrease the amount of total protein. With the preceding, the extraction method reported here requires only one centrifugation step with favorable results.
This technique could be used to extract other factors and proteins present in the AM and further, to obtain factors from other sources such as animals or vegetables. It could also have an application for obtaining proteins to carry out basic research or even the development of formulations and treatments.
These results suggest that lumican can be extracted from AM and stored for 12 days as AME under both -20 °C and 4 °C temperature conditions. It is important to consider its half-life to achieve the therapeutic effects of lumican as AME. Further studies are needed to determine the role of AME lumican in corneal epithelial cells and to determine the ideal dose of lumican for corneal re-epithelialization.
In conclusion, the results suggest obtaining factors such as lumican in the AME is possible. Likewise, temperature and storage time conditions influence the concentration of lumican present in the AME.
The authors have nothing to disclose.
The authors have no competing financial interests.
1 N H2SO4 stop solution | R&D Systems | DY994 | |
100 μL micropipette | Eppendorf | ||
1000 μL micropipette | Eppendorf | ||
15 mm Petri dish | Symlaboratorios | ||
18 G Needle (1.2 mm x 40 mm) | BD Becton Dickinson | 305211 | |
2 mL microcentrifuge tube | Eppendorf | Z606340 | |
20 mL plastic syringe | BD Becton Dickinson | 302562 | |
20 μL micropipette | Eppendorf | ||
20-200 μL micropipette | Eppendorf | ||
5 mL microcentrifuge tube | Eppendorf | 30119401 | |
96-well microplate | SARSTEDT | 821581 | |
Aluminum foil | N/A | N/A | |
Amniotic membrane | Instituto de Oftalmologia Conde de Valenciana Amnion Bank | 100 mg | |
Balanced salt solution | Bausch + Lomb | BSS-403802 | |
Beaker | N/A | N/A | |
BioRender | BioRender | figures design | |
Compact Rocker | BioRad | 970822DD | Mod. 5202SD-BIO |
complete, EDTA-free, Protease inhibitor cocktail tablets |
Roche | 11 873 580 001 | Protease Inhibitor |
Daiggner vortex Genie 2 | A.Daigger & Co. , INC | 22220A | |
Dispase II | Gibco | 17105-041 | |
ELISA plate spectrometer | Thermo Labsystems | 35401106 | Multiscan |
Freezer | |||
GraphPad Prism | GraphPad Software, Inc | version 9 | statistical analysis and graphic program |
Human lumican DuoSet ELISA kit | R&D Systems | DY2846-05 | includes human Lumican capture antibody |
Incubator | Forma Scientific | 3326 S/N 36481-7002 | |
Inverted light Microscope | Olympus | 6A13921 | to confirm de-epithelialization Mod.CK2 |
Laminar flow hood | Forma Scientific | 14753-567 | Mod.1184 |
Liquid nitrogen | N/A | N/A | |
Mortar | N/A | N/A | |
Multi-channel pipettor | Eppendorf | ||
Nitrogen Tank | Thermo Scientific | Mod. Biocan 20 | |
Paper towels | N/A | N/A | |
Phosphate-buffered saline | R&D Systems | DY006 | |
Pierce Modified Lowry Protein Assay Kit | Thermo Scientific | 23240 | |
Plate sealers | R&D Systems | DY992 | |
Reagent diluent | R&D Systems | DY995 | 1% BSA in PBS, pH 7.2-7.4, 0.2 μm filtered |
Refrigerated centrifuge | centurion scientific Ltd | 15877 | Mod. K2015R |
Rubber policeman cell scraper | NEST | 710001 | for mechanical de-epithelialization |
Scalpel knife | Braun | BB521 | No. 10 or 21 |
Streptavidin-HRP 40-fold concentrated | R&D Systems | part 893975 | |
Substrate tetramethylbenzidine (TMB) solution | R&D Systems | DY999 | |
Toothed tweezers | Invent Germany | 6b | inox |
Ultrapure water | PISA | ||
Wash buffer | R&D Systems | WA126 | 0.05% Tween 20 in PBS, pH 7.2-7.4 |