Background/Aims: Proliferative vitreoretinopathy (PVR) is a severe blinding complication of rhegmatogenous retinal detachment. Epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is thought to play a pivotal role in the pathogenesis of PVR. Fucoidan, a marine extract, reportedly has many benefits effects in a variety of tissues and organs such as anti-inflammation, anti-oxidative stress, and anti-carcinogenesis. In this study, we investigated the potential role of fucoidan on EMT in RPE cells and its effect on the development of PVR. Methods: MTS, Transwell, and collagen gel contraction assays were employed to measure the viability, migration, and contraction of RPE cells, respectively. mRNA and protein expression were evaluated via real-time quantitative PCR and western blot analysis, respectively. In vivo, a pigmented rabbit model of PVR was established to examine the anti-PVR effect of fucoidan. Results: Fucoidan reversed the transforming growth factor (TGF)-β1-induced EMT of RPE cells, including the increased expression of α-smooth muscle actin (α-SMA) and fibronectin and down-regulation of E-cadherin in human primary RPE cells. Moreover, the upregulation of phosphorylated Smad2/3 induced by TGF-β1 was suppressed by fucoidan. Fucoidan also inhibited the migration and contraction of RPE cells induced by TGF-β1. In vivo, fucoidan inhibited the progression of experimental PVR in rabbit eyes. Histological findings showed that fucoidan suppressed the formation of α-SMA-positive epiretinal membranes. Conclusion: Our findings regarding the protective effects of fucoidan on the EMT of RPE cells and experimental PVR suggest the potential clinical application of fucoidan as an anti-PVR agent.

Proliferative vitreoretinopathy (PVR), a severe blinding complication of retinal detachment surgery, is characterized by the formation of an epi/sub-retinal membrane and traction of the reattached retina [1, 2]. The retinal pigment epithelium (RPE) is thought to play a pivotal role in the pathogenesis of PVR due to its primary appearance among several cell types established in PVR fibrotic membranes [3]. Epithelial-mesenchymal transition (EMT), which enables RPE cells to lose their epithelial properties and transform into mesenchymal cells, is considered as the fundamental mechanism underlying the formation of the PVR membrane [4, 5]. Similar to EMT in carcinogenesis, the EMT of RPE cells involves the activation of the relevant cellular pathway, rearrangement of the cytoskeleton, and disassembly of the junctions between RPE cells. Transforming growth factor (TGF)- β, a classic EMT trigger, is also found in the eye of PVR patients. Therefore, blocking the EMT of RPE cells might be an efficient way to prevent PVR. However, despite the study of the EMT of RPE cells in PVR for decades, there is no currently available drug to prevent it.

Brown algae and other marine products have long been regarded as beneficial for the human body. The exact molecules underlying these beneficial effects are being discovered gradually with the development of biopharmaceutical techniques. Fucoidan, a sulfated polysaccharide extracted from brown algae and marine invertebrates, was first isolated by Kylin in 1913. Fucoidan reportedly exerts many beneficial effects in inflammation, oxidative stress, and carcinogenesis [6-8]. In ophthalmic studies, fucoidan appears to have a therapeutic effect in several blinding diseases such as age-related macular degeneration (AMD) and diabetic retinopathy. In 2013, Yang et al. demonstrated that fucoidan alleviates diabetic retinal neovascularization and damage through the inhibition of hypoxia-inducible factor-1α and vascular endothelial growth factor [9]. In 2015, Li et al. suggested that fucoidan normalizes reactive oxygen species and protects RPE cells against oxidative damage, which plays an essential role in the pathogenesis of AMD [10]. Fucoidan was also reported to block EMT by regulating the ERK1/2, Akt, p38, and Smad3 pathways and subsequently prevent renal interstitial fibrosis and ameliorate the progression of diabetic nephropathy [11]. However, it is still unclear whether fucoidan could inhibit the EMT of RPE cells, and its effect on the progression of PVR has not been investigated. In this study, we examined the effect of fucoidan on the EMT of RPE cells and the development of PVR.

Reagents and antibodies

Fucoidan from Fucus vesiculosus was purchased from Sigma-Aldrich (St. Louis, MO, USA). Human recombinant TGF-β1 was purchased from Gibco (Carlsbad, CA, USA). The following antibodies were used for western blotting and immunofluorescence analyses: anti-α-smooth muscle actin (SMA; A2547; Sigma-Aldrich), anti-fibronectin (F7387; Sigma-Aldrich), anti-E-cadherin (610182; BD Biosciences, San Jose, CA, USA), anti-β actin (ab119716; Abcam Ltd., Cambridge, MA, USA), anti-GAPDH (ab8245; Abcam Ltd.), anti-ZO-1 (14-9776-80; Invitrogen, Carlsbad, CA, USA), anti-phospho-Smad2/3 (8828; Cell Signaling Technology, Danvers, MA, USA), and anti-Smad2/3 (3102; Cell Signaling Technology). Secondary antibodies for western blotting were IRDye 800CW and IRD 680LT (Li Cor Biosciences, Lincoln, NE, USA). Most of the other reagents, such as salt and buffer components, were analytical grade and obtained from Sigma-Aldrich.

Ethics statements

The current research involving human participants was approved by the Ethics Committee of Shanghai Tenth People’s Hospital and was in compliance with the Declaration of Helsinki. Donors’ eyes were obtained from the Eye Bank of Shanghai Tenth People’s Hospital. All animal experimental designs were approved by the Animal Care and Use Committee of Shanghai Tenth People’s Hospital, Tongji University. This study was approved by the Science and Technology Commission of Shanghai Municipality (ID: SYXK 2011-0111). All methods were performed in accordance with the relevant guidelines and regulations.

Cell culture

Human primary RPE cells (obtained from the Eye Bank of Shanghai Tenth People’s Hospital) were used in this study. Human primary RPE cells were isolated from human donors according to previously published protocols [12]. Passage 2–4 cells were used in this study. Primary RPE cells were cultured at 37°C in a 5% CO2 humidified incubator with a 1: 1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium (DMEM/F12; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco).The medium was changed every 2–3 days. For further experiments, the cells were seeded and cultivated for 12 h. Then, the cells were starved with DMEM/F12 medium supplemented with 1% penicillin-streptomycin without FBS for 24 h before treatment with 10 ng/mL TGF-β1.

Cell viability assay

Primary RPE cells were seeded on 96-well plates at a density of 1.0 × 104 cells per well. RPE cells were exposed to serum-free medium containing fucoidan at different concentrations. Each experiment was performed in 6 independent wells. After 48 h, cell viability was determined using a CellTiter 96® Aqueous One Solution Cell Proliferation Assay (MTS) Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Absorbance was measured at 490 nm using a microplate spectrophotometer (Thermo, Waltham, MA, USA).

Real-time quantitative PCR

Total RNA was extracted at the indicated time points using the TRIzol reagent (Invitrogen) according to manufacturer’s protocol and quantified using a NanoDrop 2000 Spectrophotometer (Thermo Scientific, Inc., Carlsbad, CA, USA). cDNA was synthesized using a PrimerScriptTM RT Reagent Kit (Takara Clontech, Kyoto, Japan). Real-time PCR was performed in triplicate using a SuperReal PreMix Plus (SYBR Green) Kit (Takara Clontech) on a CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA). Each reaction contained 12.5 µL of 2× SYBR ® Premix Ex TaqTM (with SYBR Green I), 300 nM oligonucleotide primers synthesized by Generay Corp. (Nanjing, China), and 1 µL cDNA in a final volume of 25 µL. The thermal cycling conditions included an initial denaturation step at 95°C for 30 s and 40 cycles of 95°C for 5 s and 60°C for 30 s. mRNA expression was normalized to the level of β actin mRNA. The sequences for the real-time quantitative PCR primers were as follows: human E-cadherin sense: 5′-TCACGCTGTGTCATCCAACGG-3′ and antisense: 5′-TAGGTGTTCACATCATCGTCCGC-3′; human α-SMA sense: 5′-CAGAAGGAGATCACGGCCCTAG-3′ and antisense: 5′-CGGCTTCATCGTATTCCTGTTTG-3′; human fibronectin sense: 5′-AAGACCATACCCGCCGAATG-3′ and antisense: 5′-GGCATTTGGATTGAGTCCCG-3′; and human β actin sense: 5′-GGCTGTATTCCCCTCCATCG-3′ and antisense: 5′-CCAGTTGGTAACAATGCCATGT-3′.

Western blot analysis

The cells were lysed in RIPA buffer (Beyotime, Shanghai, China) supplemented with phenylmethylsulfonyl fluoride and PhoSTOP EASY pack phosphatase inhibitor (Roche, Mannheim, Germany) on ice for 30 min at 48 h after treatment. The lysates were clarified by centrifugation at 12000 rpm for 5 min at 4°C. Total protein concentration was quantified by a bicinchoninic acid assay kit (Thermo Scientific). Protein (40 µg) was loaded and separated on a sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred onto a nitrocellulose membrane (Bio-Rad). The membrane was blocked using 5% bovine serum albumin (BSA; Sigma Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS) for 45 min at room temperature to prevent non-specific binding. The membrane was then incubated with primary antibodies diluted in 2% BSA in PBS with 0.1% Tween-20 (PBS-T) at 4°C overnight. After rinsing 3 times with PBS-T, the membrane was incubated with IRDye® 680LT goat anti-rabbit or IRDye® 800CW goat anti-mouse secondary antibodies (Li Cor Biosciences) at room temperature for 1 h. After washing 3 times with PBS-T, the bound antibodies were detected using an Odyssey Infrared Imaging System (Li Cor Biosciences). Band intensities were analyzed with Odyssey software and normalized to β actin or GAPDH.

Immunofluorescence analysis

Immunofluorescence analysis of RPE cells: the cells were seeded and cultured in a 24-well plate inlaid with glass coverslips. After treatment, the cells were washed and fixed in cold acetone for 5 min. After washing 3 times with PBS, the cells were blocked with 2% BSA for 1 h at room temperature and incubated with the primary antibodies overnight at 4°C. After being rinsed 3 times with PBS, the coverslips were then incubated with FITC-conjugated secondary antibodies for 1 h at room temperature. After counterstaining with 4, 6-diamidino-2-phenylindole (DAPI), the stained coverslips were mounted and visualized under a confocal microscope (LSM710; Carl Zeiss, Jena, Germany).

Immunofluorescence analysis of rabbit retina: prepared slides were incubated in PBS for 10 min and then permeabilized in 0.25% Triton X-100 for 10 min. After washing 3 times in PBS, the slides were blocked with 1% BSA for 30 min and then incubated with the primary antibody anti-α-SMA at 4°C overnight. After washing for 15 min in PBS, the slides were incubated with FITC-conjugated secondary antibodies for 1 h at room temperature. After counterstaining with DAPI, the stained coverslips were mounted and visualized under a confocal microscope (LSM710; Carl Zeiss).

Transwell migration assay

After treatment, the cells were trypsinized and seeded in the upper chamber of a 24-well Transwell plate (8 mm pore size; Costar, Conning, CA, USA) in 100 µL DMEM/F12 containing 0.5% FBS at a density of 5.0 × 104 cells per chamber. The lower compartment was filled with 600 µL DMEM/F12 containing 10% FBS. After incubation at 37°C for 18 h, the cells on the upper surface of the filter were removed and the migrated cells on the lower surface of the culture inserts were fixed with ice-cold methanol and stained with 0.1% crystal violet for 30 min. The number of migrated cells in each chamber was then determined by counting 5 random fields. All experiments were performed in triplicate.

Collagen gel contraction assay

Collagen gel contraction assays were performed as described previously [13]. In brief, 24-well culture plates were coated with 1 mL of 1% BSA for 1 h at 37°C. Primary RPE cells pre-treated by TGF-β1 with or without fucoidan (100 µg/mL) were harvested and suspended in serum-free DMEM/F12. Collagen I (final concentration, 2 mg/mL; Gibco), 10× DMEM/F12, cell suspension (final density, 2.5 × 105 cells/well), sterile distilled water, and sterile 1 N NaOH (0.025-fold of the volume of collagen) were prepared and mixed on ice. The mixture (total volume, 0.5 mL) was added to the BSA-coated wells and incubated for 1 h at 37°C under 5% CO2 to promote polymerization of the gels. The gels were freed from the sides with a pipette tip and serum-free DMEM/F12 (0.5 mL) was then added to the top of the gel. Photographs were taken after 48 h to quantify the gel contraction area using ImageJ software (version 1.46r; National Institutes of Health, Bethesda, MD, USA).

Rabbit PVR model

PVR was induced in the right eye of pigmented rabbits. Twelve rabbits were divided into 2 groups. Both groups were PVR models, one served as the control and the other was treated with fucoidan. Briefly, at 7 days before the experiment, 0.2 mL aqueous humor was removed and 0.4 mL perfluoropropane was injected into the vitreous cavity at 3 mm posterior to the corneal limbus. PBS (100 µL) with or without fucoidan (2000 µg/mL) containing human primary RPE cells (passage 2–3, 2.0 × 105 cells) and 50 ng PDGF-BB (R&D Systems, Minneapolis, MN, USA) were injected into the vitreous cavity on day 1. All procedures were conducted under anesthesia. Fundus examinations were conducted initially and at day 1 (before injection), day 7, and day 14 via the combination of a microscope (EZ4; Leica, Wetzlar, Germany) and a Volk lens (SuperQuad®160; Volk, Mentor, OH, USA). Representative eyes in each group were chosen to conduct optical coherence tomography (OCT) and B-ultrasonic examinations according to the condition of the fundus. PVR was classified according to Fastenberg et al. [14]. In brief, the stage of PVR was based on the findings of retinal fundus examination as follows: stage 1, intravitreal membrane; stage 2, focal traction, localized vascular changes, hyperemia, engorgement, and blood vessel elevation; stage 3, localized detachment of the medullary ray; stage 4, extensive retinal detachment, total medullary ray detachment, and peripapillary retinal detachment; and stage 5, total retinal detachment, and retinal folds and holes. The eyes were harvested on day 14 for histologic and immunofluorescence analyses.

Statistical analysis

All experiments were performed at least 3 times. The mean and standard error of the mean (SEM) were calculated for all parameters determined in this study. Data were analyzed statistically using one-way ANOVA, two-tailed Student’s t test, and Mann-Whitney U test (*P < 0.05, **P < 0.01).

Fucoidan inhibits EMT in human primary RPE cells by inhibiting TGF-β1-induced Smad2/3 phosphorylation

To investigate whether fucoidan can inhibit EMT in RPE cells, we first testified the cell viability of primary RPE cells treated with fucoidan at the recommended concentrations [15]. At 48 h after exposure to fucoidan, no significant influence on cell viability was observed within the range of concentrations tested (Fig. 1A).

Fig. 1.

Fucoidan attenuates TGF-β1-induced EMT in human primary RPE cells. (A) Cell viability analysis of fucoidan on primary RPE cells. Primary RPE cells were treated with fucoidan at different concentrations (0, 50, 60,70, 80, 90, and 100 µg/mL) for 48 h and then cell viability was examined with the MTS assay. Cell viability is expressed as the percentage of the normal control, which was defined as 100%. Data are expressed as the mean ± SEM (n = 3). (B) Real-time quanititative PCR analysis. Primary RPE cells were treated with 10 ng/mL TGF-β1 with or without fucoidan (50 µg/mL and 100 µg/mL) for 48 h. The mRNA expression levels of E-cadherin, α-SMA, and fibronectin were detected with real-time quantitative PCR. The data are presented as the mean ± SEM. n = 3. *P< 0.05. (C) Western blot analysis of the expression of EMT-related proteins. Primary RPE cells were treated by 10 ng/mL TGF-β1 with or without fucoidan (50 µg/mL and 100 µg/mL) for 48 h. Relative protein expression (normalized to β actin) was quantified in the western blots by determing their gray scale value. The data are presented as the mean ± SEM. n = 3. *P< 0.05. Two-tailed Student’s t test was used to calculate P-values. Abbreviations: EMT, epithelial-mesenchymal transition; α-SMA, α-smooth muscle actin.

Fig. 1.

Fucoidan attenuates TGF-β1-induced EMT in human primary RPE cells. (A) Cell viability analysis of fucoidan on primary RPE cells. Primary RPE cells were treated with fucoidan at different concentrations (0, 50, 60,70, 80, 90, and 100 µg/mL) for 48 h and then cell viability was examined with the MTS assay. Cell viability is expressed as the percentage of the normal control, which was defined as 100%. Data are expressed as the mean ± SEM (n = 3). (B) Real-time quanititative PCR analysis. Primary RPE cells were treated with 10 ng/mL TGF-β1 with or without fucoidan (50 µg/mL and 100 µg/mL) for 48 h. The mRNA expression levels of E-cadherin, α-SMA, and fibronectin were detected with real-time quantitative PCR. The data are presented as the mean ± SEM. n = 3. *P< 0.05. (C) Western blot analysis of the expression of EMT-related proteins. Primary RPE cells were treated by 10 ng/mL TGF-β1 with or without fucoidan (50 µg/mL and 100 µg/mL) for 48 h. Relative protein expression (normalized to β actin) was quantified in the western blots by determing their gray scale value. The data are presented as the mean ± SEM. n = 3. *P< 0.05. Two-tailed Student’s t test was used to calculate P-values. Abbreviations: EMT, epithelial-mesenchymal transition; α-SMA, α-smooth muscle actin.

Close modal

We next examined changes in the expression of E-cadherin, α-SMA, and fibronectin in RPE cells. Treatment with TGF-β1 (10 ng/mL for 48 h) significantly reduced the expression of E-cadherin and increased the expression of α-SMA and fibronectin at both the mRNA and protein level. The TGF-β1-induced decrease of E-cadherin and increase of α-SMA and fibronectin could be reversed by fucoidan at both 50 µg/mL and 100 µg/mL (Fig. 1B and C). The relative changes of E-cadherin, α-SMA, and fibronectin were further validated by immunofluorescence analysis. Moreover, the morphologic continuity of ZO-1 was disrupted when the cells were treated with TGF-β1, and this phenomenon could also be prevented by fucoidan (Fig. 2). Our results suggest that fucoidan could reverse the TGF-β1-induced EMT of RPE cells.

Fig. 2.

Immunofluorescence analysis of EMT-related proteins in human primary RPE cells. After primary RPE cells were treated with 10 ng/mL TGF-β1 with or without fucoidan (100 µg/mL) for 48 h, ZO-1, E cadherin, α-SMA, and fibronectin were detected using the corresponding antibody. Nuclei were stained with DAPI. The slides were examined by confocal microscopy. Original magnification: 630×, oil. Scale bar: 10 µm. Abbreviations: N, normal control; EMT, epithelial-mesenchymal transition; ZO-1, zonula occludens-1; α-SMA, α-smooth muscle actin. (Top to bottom: ZO-1, E-cadherin, α-SMA, fibronectin).

Fig. 2.

Immunofluorescence analysis of EMT-related proteins in human primary RPE cells. After primary RPE cells were treated with 10 ng/mL TGF-β1 with or without fucoidan (100 µg/mL) for 48 h, ZO-1, E cadherin, α-SMA, and fibronectin were detected using the corresponding antibody. Nuclei were stained with DAPI. The slides were examined by confocal microscopy. Original magnification: 630×, oil. Scale bar: 10 µm. Abbreviations: N, normal control; EMT, epithelial-mesenchymal transition; ZO-1, zonula occludens-1; α-SMA, α-smooth muscle actin. (Top to bottom: ZO-1, E-cadherin, α-SMA, fibronectin).

Close modal

We then investigated the effect of fucoidan on the TGF-β signaling pathway. Smad2/3 and phosphorylated-Smad2/3 were extracted from RPE cells treated with TGF-β1 with or without fucoidan. Significant Smad2/3 phosphorylation was detected at 1 h after TGF-β1 treatment, while treatment with TGF-β1 and fucoidan inhibited Smad2/3 phosphorylation (Fig. 3). Taken together, these findings suggest that fucoidan could inhibit TGF-β1-induced EMT in human primary RPE cells by suppressing the phosphorylation of Smad2/3.

Fig. 3.

Fucoidan attenuates TGF-β1-induced Smad2/3 phosphorylation in primary RPE cells. Primary RPE cells were treated with 10 ng/mL TGF-β1 with or without fucoidan (50 µg/mL and 100 µg/mL) for 48 h. Phosphorylated Smad2/3 was analyzed by western blotting and quantified against relative total Smad2/3 protein. (A) Western blot analysis. (B) Relative Smad2/3 protein level. (C) Relative phosphorylated Smad2/3 protein level. The data are presented as the mean ± SEM. n = 3. *P< 0.05. Two-tailed Student’s t test was used to calculate P-values.

Fig. 3.

Fucoidan attenuates TGF-β1-induced Smad2/3 phosphorylation in primary RPE cells. Primary RPE cells were treated with 10 ng/mL TGF-β1 with or without fucoidan (50 µg/mL and 100 µg/mL) for 48 h. Phosphorylated Smad2/3 was analyzed by western blotting and quantified against relative total Smad2/3 protein. (A) Western blot analysis. (B) Relative Smad2/3 protein level. (C) Relative phosphorylated Smad2/3 protein level. The data are presented as the mean ± SEM. n = 3. *P< 0.05. Two-tailed Student’s t test was used to calculate P-values.

Close modal

Fucoidan attenuates TGF-β1-induced migration of RPE cells and collagen gel contraction

We examined TGF-β1-induced functional changes using a Transwell migration assay and collagen gel contraction assay. For Transwell migration, primary RPE cells pre-treated with TGF-β1 with or without Fucoidan (100 µg/mL) for 48 h were seeded in the Transwell migration system. Our results suggested that TGF-β1 significantly promoted RPE cell migration, while fucoidan treatment significantly inhibited cell migration (Fig. 4A). The gel contraction assay also indicated that TGF-β1 facilitated the contraction of RPE cells and that fucoidan could suppress this effect (Fig. 4B).

Fig. 4.

Fucoidan attenuates TGF-β1-induced migration and collagen gel contraction in primary RPE cells. (A) Transwell migration analysis. After treatment with 10 ng/mL TGF-β1 with or without fucoidan (100 µg/mL) for 48 h, RPE cells were harvested and seeded in the Transwell system for another 18 h. The number of migrated cells was quantified by counting 3 random vision fields under a microscope (magnification ×200). The data are presented as the mean ± SEM. n = 3. *P< 0.05. Scale bar: 100 µm. (B) Collagen gel contraction analysis. After treatment with 10 ng/mL TGF-β1 with or without fucoidan (100 µg/mL) for 48 h, primary RPE cells were harvested and seeded in a collagen gel system for another 48 h. The extent of gel contraction was quantified. The data are presented as the mean ± SEM. n = 3.*P< 0.05. Two-tailed Student’s t test was used to calculate P-values.

Fig. 4.

Fucoidan attenuates TGF-β1-induced migration and collagen gel contraction in primary RPE cells. (A) Transwell migration analysis. After treatment with 10 ng/mL TGF-β1 with or without fucoidan (100 µg/mL) for 48 h, RPE cells were harvested and seeded in the Transwell system for another 18 h. The number of migrated cells was quantified by counting 3 random vision fields under a microscope (magnification ×200). The data are presented as the mean ± SEM. n = 3. *P< 0.05. Scale bar: 100 µm. (B) Collagen gel contraction analysis. After treatment with 10 ng/mL TGF-β1 with or without fucoidan (100 µg/mL) for 48 h, primary RPE cells were harvested and seeded in a collagen gel system for another 48 h. The extent of gel contraction was quantified. The data are presented as the mean ± SEM. n = 3.*P< 0.05. Two-tailed Student’s t test was used to calculate P-values.

Close modal

Fucoidan inhibits the progression of PVR in vivo

We investigated the effect of fucoidan on the progression of PVR in vivo. Among the 12 pigmented rabbits involved in this study, 6 were assigned randomly to the PBS group and the other 6 were assigned to the fucoidan group. Fundus examinations ensured the fitness of the retina and the visibility of the normal pigmented rabbit fundus structure before the experiment (Fig. 5E). One rabbit was excluded from the PBS group on day 7 due to endophthalmitis, and another was excluded from the fucoidan group on day 14 due to cataract. The included rabbits and detailed information of the experiment are shown in Table 1 and Fig. 5.

Table 1.

Summary of the stage of PVR formation

Summary of the stage of PVR formation
Summary of the stage of PVR formation
Fig. 5.

Fucoidan inhibits the progression of experimental PVR in rabbits. Progression of PVR stages in an experimental rabbit PVR model treated with PBS or fucoidan is presented as mean ± SEM. n = 5/group. **P< 0.01. (a–e) Ocular fundus photographs of normal eyes and eyes with PVR after treatment with PBS or fucoidan (200 µg). For the PBS group, the light blue arrows represent the localized detachment of the medullary ray and extensive retinal detachment on day 7 and day 14, respectively; the green arrow represents a retinal hole. For the fucoidan group, the light blue arrows represent localized vascular changes and engorgements. Details of the PVR grading procedures for Grades 1–5 for the experimental PVR model are described in the Methods section. (f–k) OCT and B-ultrasonic report of normal eyes and eyes with PVR after treatment with PBS or fucoidan (200 µg) on day 14. The arrows represent retinal detachment. The Mann-Whitney U test was used to calculate P-values. Abbreviations: OCT, optical coherence tomography.

Fig. 5.

Fucoidan inhibits the progression of experimental PVR in rabbits. Progression of PVR stages in an experimental rabbit PVR model treated with PBS or fucoidan is presented as mean ± SEM. n = 5/group. **P< 0.01. (a–e) Ocular fundus photographs of normal eyes and eyes with PVR after treatment with PBS or fucoidan (200 µg). For the PBS group, the light blue arrows represent the localized detachment of the medullary ray and extensive retinal detachment on day 7 and day 14, respectively; the green arrow represents a retinal hole. For the fucoidan group, the light blue arrows represent localized vascular changes and engorgements. Details of the PVR grading procedures for Grades 1–5 for the experimental PVR model are described in the Methods section. (f–k) OCT and B-ultrasonic report of normal eyes and eyes with PVR after treatment with PBS or fucoidan (200 µg) on day 14. The arrows represent retinal detachment. The Mann-Whitney U test was used to calculate P-values. Abbreviations: OCT, optical coherence tomography.

Close modal

Representative fundus photos from the PBS group demonstrated focal traction and localized detachment of the medullary ray (Fig. 5a) on day 7 and extensive retinal detachment (Fig. 5b) with a retinal hole (green arrow) on day 14. OCT and B-ultrasonic examinations confirmed retinal detachment on day 14 (Fig. 5f–k). However, among the 5 eyes treated simultaneously with fucoidan (200 µg), the most advanced PVR was stage 3 in 1 eye, and the other 4 eyes were all stage 2 with slight focal contraction and moderate vessel tortuosity (Fig. 5c and d). Taken together, these findings show that PVR progression was significantly inhibited in the eyes injected with fucoidan compared with the eyes injected with PBS as a control. Histological hematoxylin and eosin (H&E) staining confirmed the presence of extensive retinal detachment and an obvious epiretinal membrane in the eyes injected with PBS, while no apparent retinal detachment was seen in the eyes injected with fucoidan, despite the presence of an unsmooth retinal surface (Fig. 6A). Immunofluorescence analysis showed an α-SMA-positive epiretinal membrane in the eyes treated with PBS, while slight α-SMA staining was seen in the eyes treated with fucoidan (Fig. 6B).

Fig. 6.

Fucoidan inhibits the formation of the epiretinal membrane. (A) Histological H&E staining of a normal eye (a), eye with PVR after treatment with PBS (b), or after treatment with 200 µg fucoidan (c). **Epithelial membrane. (B) Immuno-fluorescence of α-SMA in a normal eye (a), eye with PVR after treatment with PBS (b, arrows represent the α-SMA-positive epiretinal membrane with massive cell proliferation), or after treatment with 200 µg fucoidan (c, arrow represents the unsmooth surface of the retina with slight cell proliferation). Original magnification: 400×. Scale bar: 20 µm. Abbreviations: α-SMA, α-smooth muscle actin.

Fig. 6.

Fucoidan inhibits the formation of the epiretinal membrane. (A) Histological H&E staining of a normal eye (a), eye with PVR after treatment with PBS (b), or after treatment with 200 µg fucoidan (c). **Epithelial membrane. (B) Immuno-fluorescence of α-SMA in a normal eye (a), eye with PVR after treatment with PBS (b, arrows represent the α-SMA-positive epiretinal membrane with massive cell proliferation), or after treatment with 200 µg fucoidan (c, arrow represents the unsmooth surface of the retina with slight cell proliferation). Original magnification: 400×. Scale bar: 20 µm. Abbreviations: α-SMA, α-smooth muscle actin.

Close modal

Our study showed that fucoidan inhibits the TGF-β1-induced EMT of RPE cells through Smad2/3 signaling. We further demonstrated the suppressive effect of fucoidan on the progression of PVR in a rabbit model, suggesting that fucoidan might be a potential therapeutic agent for PVR intervention.

PVR, which is characterized by the formation of an epi/sub-retinal membrane and postoperative tractional retinal re-detachment, occurs as a complication in 5–10% of patients with rhegmatogenous retinal detachment [2]. Modern secondary surgical repair may only lead to anatomical recovery with poor functional visual acuity [16]. Despite the great efforts made by researchers, PVR remains the primary reason for the failure of retinal re-attachment surgery. The current interpretation of the pathogenesis of PVR involves the migration of cells into the vitreous cavity, where they proliferate into membranes and their subsequent contractions lead to the development of retinal wrinkles and detachment [17, 18]. The RPE, a monolayer of pigmented cells located between the nerve retina and Bruch’s membrane, is an important partner for the fit of the retina, and disturbance of the RPE may ultimately lead to retinal damage and disease. Various studies have investigated the relationship and underlying mechanism between the RPE and ocular diseases [19-21]. The RPE also participates in the pathogenesis of PVR. Among several cell types established in the PVR membrane, RPE cells are regarded to play a primary role within the disease as they represent the largest cellular component [22]. Since EMT represents the dedifferentiation of the RPE and was shown to underlie the development of PVR by Casaroli-Marano et al. in 1999 [4], EMT of RPE cells has been regarded as the trigger of PVR pathogenesis, and TGF-β-induced EMT in cultured RPE cells has been widely adopted to study the mechanism of PVR in vitro [23-26].

In our study, we tested whether inhibition of EMT could block or ameliorate the progression of PVR. We first used TGF-β to induce EMT in RPE cells in vitro. Both isoforms of TGF-β (1 and 2) have been used to induce EMT in a variety of cell types including RPE cells. However, which isoform exerts the greatest contribution to PVR pathogenesis is still under debate. On one hand, the concentration of TGF-β2 is much higher than that of TGF-β1 in PVR vitreous [27]; on the other hand, the concentration of TGF-β1 is significantly elevated during PVR development, while that of TGF-β2 is increased to a much lesser extent [28, 29]. Our team successfully established a model of RPE cell EMT by TGF-β1 in 2011 [30]. In our latest published study, we compared the effect of both isoforms in inducing human RPE cell EMT. Both TGF-β isoforms exhibited a similar effect in inducing EMT of RPE cells, such as the upregulation of the epithelial marker E-cadherin and the inhibition of the mesenchymal markers α-SMA and fibronectin [31]. Therefore, we utilized TGF-β1 to induce EMT in RPE cells in the present study.

In our experiments, TGF-β1 was used to induce EMT in human primary RPE cells. At 48 h after TGF-β1 treatment, the expression of the epithelial differentiation marker E-cadherin was significantly suppressed in RPE cells and accompanied with an increase in the expression of the mesenchymal markers α-SMA and fibronectin. Our data also suggested that the classical TGF-β signaling component Smad2/3 was phosphorylated under TGF-β1 treatment in RPE cells.

Fucoidan is a heterogeneous compound of sulfated polysaccharides with a high content of L-fucose and sulfate ester groups, and it was first isolated from marine brown algae by Kylin in 1913. Fucoidan reportedly has many beneficial effects in a variety of tissues and systems such as anti-inflammation, anti-oxidative stress, and anti-carcinogenesis [32, 33]. Fucoidan also displays protective effects against several ocular diseases. For example, fucoidan reportedly attenuates diabetic retinopathy via the inhibition of vascular endothelial growth factor [9]. Fucoidan was also demonstrated to scavenge superoxide radicals and inhibit oxidative stress and protect ARPE-19 cells against oxidative damage via normalization of the generation of reactive oxygen species, indicating a beneficial role for fucoidan in AMD [10]. Recently, several studies suggested that fucoidan could inhibit EMT in various cell types. For instance, Hsu et al. found that fucoidan could regulate EMT by modulating TGFR/Smad signaling, which leads to the inhibition of breast cancer metastasis in vitro and in vivo [34]. Yan et al. found that fucoidan regulates the miR-29b/DNMT3B/MTSS1 axis and inhibits EMT in human hepatocellular carcinoma cells [35]. Chen et al. demonstrated that fucoidan inhibits EMT in HK2 cells and attenuates renal fibrosis in both type 1 and type 2 diabetic rats [11].

Similar to the results in diabetic renal fibrosis, its inhibitory effect on RPE EMT in our study suggested that fucoidan may contribute to the maintenance of the epithelial properties of RPE cells and prevent the acquisition of a mesenchymal phenotype. The retention of E-cadherin may strengthen cell-cell adhesions among RPE cells, and the elimination of α-SMA and fibronectin could prevent the release of RPE cells. The restoration of the regular morphologic features of ZO-1 suggested that fucoidan may also reinforce tight junctions to ensure that RPE cells stay in situ. Meanwhile, the attenuated contraction ability of RPE cells indicated that apart from protecting the RPE in situ, fucoidan can weaken the damage caused by released RPE cells.

Apart from the in vitro protective effects of fucoidan, we also performed in vivo experiments. Our method for the establishment of PVR involves gas vitrectomy and the injection of human primary RPE cells into the vitreous of pigmented rabbits. This method was according to Fastenberg et al. [14] and Hida et al. [36]. We chose a cell injection method to establish a PVR model due to its better reproducibility, safety, and stability compared with other methods such as lensectomy [37] or retinal cryopexy with an ocular wound [38]. For animal species, including non-human primate models, cats, mice, and rabbits, have all been adopted in previous studies [39-41]. However, considering their suitability for the experiment, the ease of conducting the procedure and convenience for observing the development of PVR, we selected pigmented rabbits because their large eyeball is easy to manipulate, the relatively smaller size of their lens helps to avoid cataract formation, and the typical structure of the medullary wing can be used to identify definite retinal detachment. For the choice of cell type, autologous fibroblasts [42, 43], homologous fibroblasts [14, 44, 45], autologous RPE cells [46], homologous RPE cells [47], activated macrophages [48, 49], and human primary RPE cells [50] have all been used to induce PVR in pigmented rabbits. We chose human primary RPE cells to conduct the in vivo experiment so it was in accordance with the in vitro experiment. Gas vitrectomy was performed using perfluoropropane to compress the vitreous and allow the RPE cells to settle on the retinal surface rather than being trapped in the vitreous. The injection of RPE cells is more relevant to the natural pathogenesis of human PVR as it simulates the detached RPE cells in the vitreous of humans. Funduscopy, OCT, and B-ultrasonic examinations were employed to investigate the progression of PVR. On day 14, 5 rabbits in the PBS control group had developed PVR. While in the fucoidan group, 4 rabbits were stage 2 and only 1 rabbit was stage 3, due to the appearance of the localized minor detachment of the medullary ray.

Histologic H&E staining and immunofluorescence analysis were used to observe the morphologically detached retina and to evaluate cell proliferation upon the epi/sub-retinal surface. Our results showed an obvious epiretinal proliferative membrane accompanied by α-SMA-positive staining in the stage 5 eyes of the PBS control group. However, a few cells on the retinal surface with slight α-SMA staining were observed in the fucoidan group. α-SMA, one of six different actin isoforms that have been identified, is involved in cell motility, structure, and integrity. During actin stress fiber formation in EMT, the enhanced expression of α-SMA enables the target cell to acquire the abilities of migration and contraction, which may play a pivotal role in the pathogenesis of PVR. Our findings indicated that fucoidan not only inhibited the formation of the proliferative membrane but also suppressed the expression of α-SMA in the experimental PVR model.

In conclusion, these data showed that fucoidan inhibits the EMT of RPE cells and the progression of PVR. Our findings suggest the potential clinical application of fucoidan as an anti-PVR agent. However, the safety and detailed mechanism of fucoidan still need further investigation prior to its clinical administration.

This work was supported by the National Natural Foundation of China (Nos. 81271029, 81300772, 81500727, 81570852, 81670867, and 81770939) and the Shanghai Pujiang Program (No. 15PJ1408700).

Y.Z. conceived and performed the experiments, analyzed the data, and wrote the main manuscript text; H.L. and F.W. conceived the experiments and analyzed the data; D.Z., S.Y., and H.Y. carried out the experiments. M.L., H.L., J.Z., and G.X. participated in writing and revising the manuscript. All authors reviewed the manuscript and had final approval for the submitted version.

The authors declare to have no conflict of interests.

1.
Kim IK, Arroyo JG: Mechanisms in proliferative vitreoretinopathy. Ophthalmol Clin North Am 2002; 15: 81-86.
2.
Cardillo JA, Stout JT, LaBree L, Azen SP, Omphroy L, Cui JZ, Kimura H, Hinton DR, Ryan SJ: Post-traumatic proliferative vitreoretinopathy. The epidemiologic profile, onset, risk factors, and visual outcome. Ophthalmology 1997; 104: 1166-1173.
3.
Machemer R, van Horn D, Aaberg TM: Pigment epithelial proliferation in human retinal detachment with massive periretinal proliferation. Am J Ophthalmol 1978; 85: 181-191.
4.
Casaroli-Marano RP, Pagan R, Vilaro S: Epithelial-mesenchymal transition in proliferative vitreoretinopathy: intermediate filament protein expression in retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 1999; 40: 2062-2072.
5.
Tosi GM, Marigliani D, Romeo N, Toti P: Disease pathways in proliferative vitreoretinopathy: an ongoing challenge. J Cell Physiol 2014; 229: 1577-1583.
6.
Fitton JH: Therapies from fucoidan; multifunctional marine polymers. Mar Drugs 2011; 9: 1731-1760.
7.
Kwak JY: Fucoidan as a marine anticancer agent in preclinical development. Mar Drugs 2014; 12: 851-870.
8.
Senthilkumar K, Manivasagan P, Venkatesan J, Kim SK: Brown seaweed fucoidan: biological activity and apoptosis, growth signaling mechanism in cancer. Int J Biol Macromol 2013; 60: 366-374.
9.
Yang W, Yu X, Zhang Q, Lu Q, Wang J, Cui W, Zheng Y, Wang X, Luo D: Attenuation of streptozotocin-induced diabetic retinopathy with low molecular weight fucoidan via inhibition of vascular endothelial growth factor. Exp Eye Res 2013; 115: 96-105.
10.
Li X, Zhao H, Wang Q, Liang H, Jiang X: Fucoidan protects ARPE-19 cells from oxidative stress via normalization of reactive oxygen species generation through the Ca(2)(+)-dependent ERK signaling pathway. Mol Med Rep 2015; 11: 3746-3752.
11.
Chen J, Cui W, Zhang Q, Jia Y, Sun Y, Weng L, Luo D, Zhou H, Yang B: Low molecular weight fucoidan ameliorates diabetic nephropathy via inhibiting epithelial-mesenchymal transition and fibrotic processes. Am J Transl Res 2015; 7: 1553-1563.
12.
Parapuram SK, Ganti R, Hunt RC, Hunt DM: Vitreous induces components of the prostaglandin E2 pathway in human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 2003; 44: 1767-1774.
13.
Kimura K, Orita T, Fujitsu Y, Liu Y, Wakuta M, Morishige N, Suzuki K, Sonoda KH: Inhibition by female sex hormones of collagen gel contraction mediated by retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 2014; 55: 2621-2630.
14.
Fastenberg DM, Diddie KR, Dorey K, Ryan SJ: The role of cellular proliferation in an experimental model of massive periretinal proliferation. Am J Ophthalmol 1982; 93: 565-572.
15.
Dithmer M, Fuchs S, Shi Y, Schmidt H, Richert E, Roider J, Klettner A: Fucoidan reduces secretion and expression of vascular endothelial growth factor in the retinal pigment epithelium and reduces angiogenesis in vitro. PLoS One 2014; 9:e89150.
16.
Sadaka A, Giuliari GP: Proliferative vitreoretinopathy: current and emerging treatments. Clin Ophthalmol 2012; 6: 1325-1333.
17.
Lei H, Rheaume MA, Kazlauskas A: Recent developments in our understanding of how platelet-derived growth factor (PDGF) and its receptors contribute to proliferative vitreoretinopathy. Exp Eye Res 2010; 90: 376-381.
18.
Pastor JC, de la Rua ER, Martin F: Proliferative vitreoretinopathy: risk factors and pathobiology. Prog Retin Eye Res 2002; 21: 127-144.
19.
Sun YZ, Cai N, Liu NN: Celecoxib Down-Regulates the Hypoxia-Induced Expression of HIF-1alpha and VEGF Through the PI3K/AKT Pathway in Retinal Pigment Epithelial Cells. Cell Physiol Biochem 2017; 44: 1640-1650.
20.
Chen J, Wang W, Li Q: Increased Th1/Th17 Responses Contribute to Low-Grade Inflammation in Age-Related Macular Degeneration. Cell Physiol Biochem 2017; 44: 357-367.
21.
Ranjbar M, Brinkmann MP, Zapf D, Miura Y, Rudolf M, Grisanti S: Fc Receptor Inhibition Reduces Susceptibility to Oxidative Stress in Human RPE Cells Treated with Bevacizumab, but not Aflibercept. Cell Physiol Biochem 2016; 38: 737-747.
22.
Pennock S, Haddock LJ, Eliott D, Mukai S, Kazlauskas A: Is neutralizing vitreal growth factors a viable strategy to prevent proliferative vitreoretinopathy? Prog Retin Eye Res 2014; 40: 16-34.
23.
Chiba C: The retinal pigment epithelium: an important player of retinal disorders and regeneration. Exp Eye Res 2014; 123: 107-114.
24.
Grisanti S, Guidry C: Transdifferentiation of retinal pigment epithelial cells from epithelial to mesenchymal phenotype. Invest Ophthalmol Vis Sci 1995; 36: 391-405.
25.
Tamiya S, Liu L, Kaplan HJ: Epithelial-mesenchymal transition and proliferation of retinal pigment epithelial cells initiated upon loss of cell-cell contact. Invest Ophthalmol Vis Sci 2010; 51: 2755-2763.
26.
Umazume K, Barak Y, McDonald K, Liu L, Kaplan HJ, Tamiya S: Proliferative vitreoretinopathy in the Swine-a new model. Invest Ophthalmol Vis Sci 2012; 53: 4910-4916.
27.
Kita T, Hata Y, Arita R, Kawahara S, Miura M, Nakao S, Mochizuki Y, Enaida H, Goto Y, Shimokawa H, Hafezi-Moghadam A, Ishibashi T: Role of TGF-beta in proliferative vitreoretinal diseases and ROCK as a therapeutic target. Proc Natl Acad Sci U S A 2008; 105: 17504-17509.
28.
Pfeffer BA, Flanders KC, Guerin CJ, Danielpour D, Anderson DH: Transforming growth factor beta 2 is the predominant isoform in the neural retina, retinal pigment epithelium-choroid and vitreous of the monkey eye. Exp Eye Res 1994; 59: 323-333.
29.
Shah M, Foreman DM, Ferguson MW: Neutralisation of TGF-beta 1 and TGF-beta 2 or exogenous addition of TGF-beta 3 to cutaneous rat wounds reduces scarring. J Cell Sci 1995; 108 ( Pt 3): 985-1002.
30.
Li H, Wang H, Wang F, Gu Q, Xu X: Snail involves in the transforming growth factor beta1-mediated epithelial-mesenchymal transition of retinal pigment epithelial cells. PLoS One 2011; 6:e23322.
31.
Yao H, Li H, Yang S, Li M, Zhao C, Zhang J, Xu G, Wang F: Inhibitory Effect of Bone Morphogenetic Protein 4 in Retinal Pigment Epithelial-Mesenchymal Transition. Sci Rep 2016; 6: 32182.
32.
Chizhov AO, Dell A, Morris HR, Haslam SM, McDowell RA, Shashkov AS, Nifant'ev NE, Khatuntseva EA, Usov AI: A study of fucoidan from the brown seaweed Chorda filum. Carbohydr Res 1999; 320: 108-119.
33.
Bilan MI, Grachev AA, Ustuzhanina NE, Shashkov AS, Nifantiev NE, Usov AI: Structure of a fucoidan from the brown seaweed Fucus evanescens C.Ag. Carbohydr Res 2002; 337: 719-730.
34.
Hsu HY, Lin TY, Hwang PA, Tseng LM, Chen RH, Tsao SM, Hsu J: Fucoidan induces changes in the epithelial to mesenchymal transition and decreases metastasis by enhancing ubiquitin-dependent TGFbeta receptor degradation in breast cancer. Carcinogenesis 2013; 34: 874-884.
35.
Yan MD, Yao CJ, Chow JM, Chang CL, Hwang PA, Chuang SE, Whang-Peng J, Lai GM: Fucoidan Elevates MicroRNA-29b to Regulate DNMT3B-MTSS1 Axis and Inhibit EMT in Human Hepatocellular Carcinoma Cells. Mar Drugs 2015; 13: 6099-6116.
36.
Hida T, Chandler DB, Sheta SM: Classification of the stages of proliferative vitreoretinopathy in a refined experimental model in the rabbit eye. Graefes Arch Clin Exp Ophthalmol 1987; 225: 303-307.
37.
Lean JS, van der Zee WA, Ryan SJ: Experimental model of proliferative vitreoretinopathy (PVR) in the vitrectomised eye: effect of silicone oil. Br J Ophthalmol 1984; 68: 332-335.
38.
Campochiaro PA, Gaskin HC, Vinores SA: Retinal cryopexy stimulates traction retinal detachment formation in the presence of an ocular wound. Arch Ophthalmol 1987; 105: 1567-1570.
39.
Cleary PE, Ryan SJ: Method of production and natural history of experimental posterior penetrating eye injury in the rhesus monkey. Am J Ophthalmol 1979; 88: 212-220.
40.
Wilson CA, Khawly JA, Hatchell DL, Machemer R: Experimental traction retinal detachment in the cat. Graefes Arch Clin Exp Ophthalmol 1991; 229: 568-573.
41.
Canto Soler MV, Gallo JE, Dodds RA, Suburo AM: A mouse model of proliferative vitreoretinopathy induced by dispase. Exp Eye Res 2002; 75: 491-504.
42.
Sugita G, Tano Y, Machemer R, Abrams G, Claflin A, Fiorentino G: Intravitreal autotransplantation of fibroblasts. Am J Ophthalmol 1980; 89: 121-130.
43.
Algvere P, Kock E: Experimental Fibroplasia in the rabbit vitreous. Retinal detachment induced by autologous fibroblasts. Albrecht Von Graefes Arch Klin Exp Ophthalmol 1976; 199: 215-222.
44.
de Souza OF, Sakamoto T, Kimura H, Koda RP, Gabrielian K, Spee C, Ryan SJ: Inhibition of experimental proliferative vitreoretinopathy in rabbits by suramin. Ophthalmologica 1995; 209: 212-216.
45.
Wiedemann P, Sorgente N, Ryan SJ: Proliferative vitreoretinopathy: the rabbit cell injection model for screening of antiproliferative drugs. J Pharmacol Methods 1984; 12: 69-78.
46.
Radtke ND, Tano Y, Chandler D, Machemer R: Simulation of massive periretinal proliferation by autotransplantation of retinal pigment epithelial cells in rabbits. Am J Ophthalmol 1981; 91: 76-87.
47.
Fastenberg DM, Diddie KR, Sorgente N, Ryan SJ: A comparison of different cellular inocula in an experimental model of massive periretinal proliferation. Am J Ophthalmol 1982; 93: 559-564.
48.
Hui YN, Sorgente N, Ryan SJ: Posterior vitreous separation and retinal detachment induced by macrophages. Graefes Arch Clin Exp Ophthalmol 1987; 225: 279-284.
49.
Hui YN, Goodnight R, Sorgente N, Ryan SJ: Fibrovascular proliferation and retinal detachment after intravitreal injection of activated macrophages in the rabbit eye. Am J Ophthalmol 1989; 108: 176-184.
50.
Wong CA, Potter MJ, Cui JZ, Chang TS, Ma P, Maberley AL, Ross WH, White VA, Samad A, Jia W, Hornan D, Matsubara JA: Induction of proliferative vitreoretinopathy by a unique line of human retinal pigment epithelial cells. Can J Ophthalmol 2002; 37: 211-220.
Open Access License / Drug Dosage / Disclaimer
This article is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND). Usage and distribution for commercial purposes as well as any distribution of modified material requires written permission. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.