Knockdown of HSPA13 Inhibits TGFβ1-Induced Epithelial-Mesenchymal Transition of RPE by Suppressing the PI3K/Akt Signaling Pathway
Cell Culture Human embryonic stem cells (hESCs) were differentiated into RPE cells using our previously described protocol. The hESC-RPE cells were cultured onto 1% Matrigel-coated plates at a density of 1.5 × 10 6 per well in 6 well plates in RPE medium, which consisted of Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (DMEM/F-12; Gibco, USA), 10% knockout serum replacement (KSR; Gibco, USA), 1% MEM nonessential amino acids (MEM NEAA; Gibco, USA), 2 mM L-glutamine (Gibco, USA), 1% penicillin-streptomycin (Gibco, USA), and 0.1 mM b-mercaptoethanol (Gibco, USA). The medium was changed every 2 to 3 days. Cells were stained with 0.4% trypan blue dye, and the cell diameter and the number of viable cells were determined with a Countstar cell counter instrument (ALIT Life Science, China). To induce EMT, hESC-RPE cells were seeded in 1% Matrigel-coated 12-well plates at a density of 5 × 10 5 cells/well. Following 24 hours of cell plating, the cells underwent 24-hour KSR deprivation. Subsequently, the cells were subjected to incubation with a concentration of 10 ng/mL of recombinant human TGFβ1 for 48 hours. Antibodies and Reagents Antibodies against E-cadherin (E-CAD; CST, Cat# 14472, USA), FN1 (Proteintech, Cat# 66042, China), HSPA13 (Proteintech, Cat# 12667, China), α-SMA (Abcam, Cat# ab7817, UK), Cytokeratin 8 (CK8; Abcam, Cat# ab9023, UK), PI3K p85 (CST, Cat# 4292, USA), p-PI3K p85/p55 (Beyotime, Cat# AF5905, China), AKT (CST, Cat# 4691, USA), p-AKT (CST, Cat# 13038, USA), and ZO-1 (Thermo, Cat# 617300, USA) were used for Western blot or immunostaining. The BAPTA/AM was acquired from Selleck Inc. (Houston, TX, USA) and administered to hESC-RPE cells at a dose of 10 µM. Recombinant human TGFβ1 was purchased from Sino Biological Inc. (Beijing, China). Immunohistochemistry The ERMs from patients with PVR were obtained from the Department of Ophthalmology of Tongji Hospital. The ERMs were stained with 0.1% toluidine blue and subsequently fixed in 4% paraformaldehyde (PFA, Sangon Biotech, China). After dehydration treatment with a sucrose gradient, these tissues were then embedded in optimal cutting temperature compound (OCT; compound 4583; Tissue-Tek). These samples were sectioned into 10 µm thickness, and permeabilized with 0.3% Triton X-100 for 15 minutes. After washing three times with phosphate-buffered saline (PBS), these sections were blocked in 3% bovine serum albumin (BSA, Sangon Biotech, China) for 45 minutes at room temperature. Next, these sections were incubated with rabbit anti-HSPA13 antibody (diluted 1:200 in the block solution) and mouse anti-CK8 antibody (diluted 1:100 in the block solution) or anti-α-SMA antibody (diluted 1:100 in the block solution) at 4°C overnight. Following a 1-hour incubation with the corresponding secondary antibody mixture, the sections were subsequently counterstained with 4',6-diamidino-2-phenylindole (DAPI) for 2 minutes to visualize the nuclear morphology. DAPI is a fluorescent stain that binds to DNA in the nucleus. After washing three times with PBS, the coverslips were mounted on glass slides. These sections were observed using a Nikon A1R fluorescence microscope. For immunohistochemical examination of the eyeballs, rats were euthanized 4 weeks post-intravitreal injection. The eyeballs were then enucleated immediately and fixed in 4% PFA. The subsequent procedures are identical to those used for the immunohistochemistry of ERMs. Immunocytochemistry Cells were cultured on coverslips placed in 24-well plates. After the necessary treatments, cells were rinsed once with PBS, fixed in 4% PFA for 10 minutes, and permeabilized in 0.1% Triton X-100 for 10 to 15 minutes. The coverslips were washed 3 times with PBS, blocked in PBS containing 3% BSA for 30 minutes at room temperature, and stained with primary antibodies at 4°C overnight. Next, the coverslips were washed and incubated with secondary antibodies. Nuclei were confirmed by staining with DAPI for 2 minutes. After washing three times with PBS, the coverslips were mounted on glass slides and photographed using a Nikon A1R microscope. RNA Extraction and Quantitative Real-Time PCR Total RNA was extracted from cells using TRIzol reagent (Takara, Japan) according to the manufacturer's instructions. The RNA concentration was determined with a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA), and cDNA was synthesized using a reverse transcription kit (Takara, Japan). The quantitative PCR (qPCR) was performed using the SYBR Green Master Mix (Tiangen, China) on a Bio-Rad Chromo4 thermal cycler instrument (Bio-Rad, USA). The gene expression levels were standardized by comparing them to the expression of the housekeeping gene GAPDH and calculated using the 2 -ΔΔCT method. The untreated control group, specifically the RPE cells or shScramble-RPE cells, serves as the relative reference. The specific primer sequences for HSPA13, and RPE/EMT-related genes are listed in . Western Blot Analysis Total proteins from cells were extracted in ice-cold RIPA buffer (Beyotime, China) supplemented with protease inhibitor cocktails (TargetMol, USA). The samples were then centrifuged at 13,200 g at 4°C for 20 minutes. The supernatant was collected and the protein concentration was determined by a BCA protein assay kit (Pierce, USA). Then, 10 to 20 µg of the protein lysate was separated by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). After blocking with 5% BSA, the membranes were incubated with primary antibodies in 5% BSA overnight at 4°C, followed by incubation with the corresponding secondary antibodies for 1 hour at room temperature, and an enhanced chemiluminescence system (Pierce, USA) was used for band detection. Plasmid Construction and Lentivirus Infection The short interfering oligonucleotides designed to target HSPA13 with the sequence GATCCCCGGCTGACGTCTTCCACGTC were synthesized by Sangon Biotech, China, and then subsequently inserted into the pLVXpuro vector. A scrambled sequence was used as a negative control. The lentivirus-shHSPA13 and lentivirus-shScramble were produced within 293T cells. Subsequently, hESC-RPE cells were infected with shScramble or shHSPA13 lentivirus. After 36 hours of infection, the cells underwent screening with 1 ng/mL puromycin for 1 week to generate a stable cell line. Live-Cell Ca 2+ Imaging The hESC-RPE cells were cultured in 48-well plates and incubated with TGFβ1 alone or in combination with BAPTA/AM for 48 hours. Then, the cells were loaded with the fluorescence Ca 2+ probe Fluo-8/AM (2 µM, for 45 minutes loading in KSR-free culture medium; Beyotime, China). The Ca 2+ imaging in hESC-RPE cells was performed within 60 minutes of dye loading using a Nikon A1R fluorescence microscope. RNA-Sequencing Gene expression profiles were analyzed by high throughput RNA sequencing (BGI, Shenzhen, Guangdong, China), and further analysis was performed using the related software Dr. Tom (BGI, Shenzhen, Guangdong, China). The mRNAs with a fold change >2 and a P < 0.05 were defined as differentially expressed genes (DEGs) with statistical significance. The GSEA software was used to perform gene set enrichment analysis (GSEA). The RNA sequencing data were deposited in the NCBI's Gene Expression Omnibus database (GSE264212). Scratch Wound Healing Assay The scratch wound healing assay was performed as described in our previous work. Briefly, the hESC-RPE monolayers were scratched with a sterile pipette tip, gently washed twice with sterile PBS to remove the resulting debris, and incubated in KSR-free medium for 24 hours. The cells and wound were imaged at 0 and 24 hours, and the area of wound closure was calculated with ImageJ. Establishment of the PVR Rat Model Six-week-old Sprague-Dawley (S-D) rats were obtained from SLACCAS (Shanghai, China) and housed in the Tongji University Animal Center. All animals used in this study were maintained in specific pathogen-free conditions in micro isolator cages and were treated according to the guidelines provided in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Guides for the Care and Use of Animals (National Research Council and Tongji University). The protocol was approved by the Committee on the Ethics of Animal Experiments of Tongji University (Permit Number: TJAA09620204). A total of 40 S-D rats were randomly assigned to 4 groups: (1) the PBS group, (2) the hESC-RPE + platelets rich plasm (PRP) group, (3) the shScramble-hESC-RPE + PRP group, and (4) the shHSPA13-hESC-RPE + PRP group. Each group consisted of 10 rats. The PVR rat model was established as previously reported. Briefly, the rats were anesthetized and their pupils were dilated before vitreous injection. To induce PVR, a mixture of hESC-RPE cells (4 µl, 1 × 10 5 cells per µl) and 4 µl PRP were injected into the vitreous of S-D rats. Hematoxylin and Eosin Staining The eyeballs were promptly enucleated and fixed in 4% PFA. Following gradient dehydration, the eyes were embedded in paraffin and sectioned into continuous slices of 5 µm thickness. After deparaffinization and rehydration, the sections were stained using a Hematoxylin and Eosin Staining Kit (Beyotime, China). The stained eyeball slides were mounted with neutral balsam and images were captured with a light microscope. Statistical Analysis Each experiment was repeated at least three times. Data are presented as the mean ± SEM (standard error of the mean) of at least three independent experiments. The Statistical Package GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA, USA) was used to analyze the data. Statistical analysis involved a Student's t -test, and 1-way and 2-way ANOVA with Tukey's multiple comparison test as appropriate. A P value of less than 0.05 was considered to be statistically significant.   Human embryonic stem cells (hESCs) were differentiated into RPE cells using our previously described protocol. The hESC-RPE cells were cultured onto 1% Matrigel-coated plates at a density of 1.5 × 10 6 per well in 6 well plates in RPE medium, which consisted of Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (DMEM/F-12; Gibco, USA), 10% knockout serum replacement (KSR; Gibco, USA), 1% MEM nonessential amino acids (MEM NEAA; Gibco, USA), 2 mM L-glutamine (Gibco, USA), 1% penicillin-streptomycin (Gibco, USA), and 0.1 mM b-mercaptoethanol (Gibco, USA). The medium was changed every 2 to 3 days. Cells were stained with 0.4% trypan blue dye, and the cell diameter and the number of viable cells were determined with a Countstar cell counter instrument (ALIT Life Science, China). To induce EMT, hESC-RPE cells were seeded in 1% Matrigel-coated 12-well plates at a density of 5 × 10 5 cells/well. Following 24 hours of cell plating, the cells underwent 24-hour KSR deprivation. Subsequently, the cells were subjected to incubation with a concentration of 10 ng/mL of recombinant human TGFβ1 for 48 hours.   Antibodies against E-cadherin (E-CAD; CST, Cat# 14472, USA), FN1 (Proteintech, Cat# 66042, China), HSPA13 (Proteintech, Cat# 12667, China), α-SMA (Abcam, Cat# ab7817, UK), Cytokeratin 8 (CK8; Abcam, Cat# ab9023, UK), PI3K p85 (CST, Cat# 4292, USA), p-PI3K p85/p55 (Beyotime, Cat# AF5905, China), AKT (CST, Cat# 4691, USA), p-AKT (CST, Cat# 13038, USA), and ZO-1 (Thermo, Cat# 617300, USA) were used for Western blot or immunostaining. The BAPTA/AM was acquired from Selleck Inc. (Houston, TX, USA) and administered to hESC-RPE cells at a dose of 10 µM. Recombinant human TGFβ1 was purchased from Sino Biological Inc. (Beijing, China).   The ERMs from patients with PVR were obtained from the Department of Ophthalmology of Tongji Hospital. The ERMs were stained with 0.1% toluidine blue and subsequently fixed in 4% paraformaldehyde (PFA, Sangon Biotech, China). After dehydration treatment with a sucrose gradient, these tissues were then embedded in optimal cutting temperature compound (OCT; compound 4583; Tissue-Tek). These samples were sectioned into 10 µm thickness, and permeabilized with 0.3% Triton X-100 for 15 minutes. After washing three times with phosphate-buffered saline (PBS), these sections were blocked in 3% bovine serum albumin (BSA, Sangon Biotech, China) for 45 minutes at room temperature. Next, these sections were incubated with rabbit anti-HSPA13 antibody (diluted 1:200 in the block solution) and mouse anti-CK8 antibody (diluted 1:100 in the block solution) or anti-α-SMA antibody (diluted 1:100 in the block solution) at 4°C overnight. Following a 1-hour incubation with the corresponding secondary antibody mixture, the sections were subsequently counterstained with 4',6-diamidino-2-phenylindole (DAPI) for 2 minutes to visualize the nuclear morphology. DAPI is a fluorescent stain that binds to DNA in the nucleus. After washing three times with PBS, the coverslips were mounted on glass slides. These sections were observed using a Nikon A1R fluorescence microscope. For immunohistochemical examination of the eyeballs, rats were euthanized 4 weeks post-intravitreal injection. The eyeballs were then enucleated immediately and fixed in 4% PFA. The subsequent procedures are identical to those used for the immunohistochemistry of ERMs.   Cells were cultured on coverslips placed in 24-well plates. After the necessary treatments, cells were rinsed once with PBS, fixed in 4% PFA for 10 minutes, and permeabilized in 0.1% Triton X-100 for 10 to 15 minutes. The coverslips were washed 3 times with PBS, blocked in PBS containing 3% BSA for 30 minutes at room temperature, and stained with primary antibodies at 4°C overnight. Next, the coverslips were washed and incubated with secondary antibodies. Nuclei were confirmed by staining with DAPI for 2 minutes. After washing three times with PBS, the coverslips were mounted on glass slides and photographed using a Nikon A1R microscope.   Total RNA was extracted from cells using TRIzol reagent (Takara, Japan) according to the manufacturer's instructions. The RNA concentration was determined with a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA), and cDNA was synthesized using a reverse transcription kit (Takara, Japan). The quantitative PCR (qPCR) was performed using the SYBR Green Master Mix (Tiangen, China) on a Bio-Rad Chromo4 thermal cycler instrument (Bio-Rad, USA). The gene expression levels were standardized by comparing them to the expression of the housekeeping gene GAPDH and calculated using the 2 -ΔΔCT method. The untreated control group, specifically the RPE cells or shScramble-RPE cells, serves as the relative reference. The specific primer sequences for HSPA13, and RPE/EMT-related genes are listed in .   Total proteins from cells were extracted in ice-cold RIPA buffer (Beyotime, China) supplemented with protease inhibitor cocktails (TargetMol, USA). The samples were then centrifuged at 13,200 g at 4°C for 20 minutes. The supernatant was collected and the protein concentration was determined by a BCA protein assay kit (Pierce, USA). Then, 10 to 20 µg of the protein lysate was separated by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). After blocking with 5% BSA, the membranes were incubated with primary antibodies in 5% BSA overnight at 4°C, followed by incubation with the corresponding secondary antibodies for 1 hour at room temperature, and an enhanced chemiluminescence system (Pierce, USA) was used for band detection.   The short interfering oligonucleotides designed to target HSPA13 with the sequence GATCCCCGGCTGACGTCTTCCACGTC were synthesized by Sangon Biotech, China, and then subsequently inserted into the pLVXpuro vector. A scrambled sequence was used as a negative control. The lentivirus-shHSPA13 and lentivirus-shScramble were produced within 293T cells. Subsequently, hESC-RPE cells were infected with shScramble or shHSPA13 lentivirus. After 36 hours of infection, the cells underwent screening with 1 ng/mL puromycin for 1 week to generate a stable cell line.   2+ Imaging The hESC-RPE cells were cultured in 48-well plates and incubated with TGFβ1 alone or in combination with BAPTA/AM for 48 hours. Then, the cells were loaded with the fluorescence Ca 2+ probe Fluo-8/AM (2 µM, for 45 minutes loading in KSR-free culture medium; Beyotime, China). The Ca 2+ imaging in hESC-RPE cells was performed within 60 minutes of dye loading using a Nikon A1R fluorescence microscope.   Gene expression profiles were analyzed by high throughput RNA sequencing (BGI, Shenzhen, Guangdong, China), and further analysis was performed using the related software Dr. Tom (BGI, Shenzhen, Guangdong, China). The mRNAs with a fold change >2 and a P < 0.05 were defined as differentially expressed genes (DEGs) with statistical significance. The GSEA software was used to perform gene set enrichment analysis (GSEA). The RNA sequencing data were deposited in the NCBI's Gene Expression Omnibus database (GSE264212).   The scratch wound healing assay was performed as described in our previous work. Briefly, the hESC-RPE monolayers were scratched with a sterile pipette tip, gently washed twice with sterile PBS to remove the resulting debris, and incubated in KSR-free medium for 24 hours. The cells and wound were imaged at 0 and 24 hours, and the area of wound closure was calculated with ImageJ.   Six-week-old Sprague-Dawley (S-D) rats were obtained from SLACCAS (Shanghai, China) and housed in the Tongji University Animal Center. All animals used in this study were maintained in specific pathogen-free conditions in micro isolator cages and were treated according to the guidelines provided in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Guides for the Care and Use of Animals (National Research Council and Tongji University). The protocol was approved by the Committee on the Ethics of Animal Experiments of Tongji University (Permit Number: TJAA09620204). A total of 40 S-D rats were randomly assigned to 4 groups: (1) the PBS group, (2) the hESC-RPE + platelets rich plasm (PRP) group, (3) the shScramble-hESC-RPE + PRP group, and (4) the shHSPA13-hESC-RPE + PRP group. Each group consisted of 10 rats. The PVR rat model was established as previously reported. Briefly, the rats were anesthetized and their pupils were dilated before vitreous injection. To induce PVR, a mixture of hESC-RPE cells (4 µl, 1 × 10 5 cells per µl) and 4 µl PRP were injected into the vitreous of S-D rats.   The eyeballs were promptly enucleated and fixed in 4% PFA. Following gradient dehydration, the eyes were embedded in paraffin and sectioned into continuous slices of 5 µm thickness. After deparaffinization and rehydration, the sections were stained using a Hematoxylin and Eosin Staining Kit (Beyotime, China). The stained eyeball slides were mounted with neutral balsam and images were captured with a light microscope.   Each experiment was repeated at least three times. Data are presented as the mean ± SEM (standard error of the mean) of at least three independent experiments. The Statistical Package GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA, USA) was used to analyze the data. Statistical analysis involved a Student's t -test, and 1-way and 2-way ANOVA with Tukey's multiple comparison test as appropriate. A P value of less than 0.05 was considered to be statistically significant.   HSPA13 is Expressed in ERMs From Patients With Clinical PVR To investigate the potential involvement of HSPA13 in the pathogenesis of PVR, we detected the expression of HSPA13 in ERMs collected from patients with PVR by immunostaining. As shown in , the HSPA13 showed strong immunoreactivity within the ERM. Double-staining further revealed that HSPA13 co-localized with both the epithelial marker CK8 and the EMT marker α-SMA. As RPE cells are the only kind of epithelial cells seen in the ERM, this finding suggests that HSPA13-positive cells are derived from RPE cells undergoing EMT. However, there is a variability in staining patterns among the cases, potentially attributable to a range of factors, including variations in ERMs size, tissue section orientation, and EMT degree. Actually, the cell count in the Cases 1 and 2 is significantly lower than that in the Cases 3 and 4. Despite this variability, all the results consistently reveal the co-localization of HSPA13 with the epithelial marker CK8 and the EMT marker α-SMA. TGFβ1 Enhanced the Expression of HSPA13 Depending on Intracellular Ca 2+ The expression pattern of HSPA13 in PVR membranes indicates that HSPA13 might play a role in PVR pathogenesis. As the EMT of RPE cells plays a crucial role in the pathogenesis of PVR, we next investigated whether HSPA13 contributes to the EMT process in RPE cells. To stimulate the EMT of RPE cells in vitro, we used TGFβ1-induced EMT model. In this model, we treated hESC-RPE cells with 10 ng/mL TGFβ1 for 48 hours. The selection of this duration and concentration was determined by time course and dose curve studies . As expected, TGFβ1 treatment successfully induced a morphological transformation of hESC-RPE cells from polygonal to elongated spindle-shaped cells ( A). Consistent with the change in morphology, the expression of the epithelial and specific RPE markers E-CAD, RPE65, and BEST was downregulated ( B), and the expression of the mesenchymal markers FN1 ( C, E, F) and PAI-1 ( C) were upregulated. Meanwhile, we observed an increase in both the mRNA ( D) and protein ( E, G) levels of HSPA13 in RPE cells treated with TGFβ1. A similar result was also observed in ARPE-19 cells and primary mouse RPE (mRPE) cells treated with TGFβ1 . Additionally, double-immunofluorescence labeling revealed an elevated expression of both HSPA13 and α-SMA in the TGFβ1-treated RPE cells ( H), further confirming the upregulation of HSPA13 in cells undergoing EMT. On the contrary, high expression of HSPA13 was accompanied by low and cytoplasmic expression of E-cadherin, suggesting that cells with high HSPA13 expression experience a loss of cell contact . A previous study showed that treatment with TGFβ1 in megakaryocytes resulted in an increased level of intracellular Ca 2+ . In line with this result, we found that TGFβ1 treatment also increased intracellular Ca 2+ in hESC-RPE cells ( I, J). Because HSPA13 is a Ca 2+ stress-induced member of the HSP70 superfamily, we then investigated whether the increased HSPA13 expression induced by TGFβ1 is dependent on intracellular Ca 2+ . To this end, we used BAPTA/AM to chelate intracellular Ca 2+ and then examined the impact of TGFβ1 on the expression of HSPA13. As shown in K–M, chelating calcium prevented TGFβ1-induced HSPA13 upregulation in hESC-RPE cells. A similar result was also observed in ARPE-19 cells . To further confirm the role of Ca 2+ in the upregulation of EMT and HSPA13, we conducted immunofluorescence co-staining to detect the expression of α-SMA and HSPA13 under chelated calcium conditions. Our findings revealed that RPE cells treated with BAPTA and TGFβ1 exhibited reduced expression of HSPA13 and α-SMA compared to cells treated with TGFβ1 alone . Taken together, these data demonstrate that TGFβ1 can induce HSPA13 expression in hESC-RPE cells, and such induction effect is dependent on intracellular Ca 2+ . Knockdown of HSPA13 Attenuated TGFβ1-Induced EMT in hESC-RPE Cells To elucidate the function of HSPA13 in the EMT of RPE cells, we established a stable HSPA13 knockdown hESC-RPE cell line by infecting cells with lenti-shHSPA13 virus. Lenti-shScramble was used as a negative control. The mesenchymal morphology induced by TGFβ1 was suppressed in shHSPA13-RPE cells ( A). The shHSPA13 markedly reduced the level of HSPA13 in hESC-RPE cells and weakened the expression of EMT marker genes triggered by TGFβ1, compared to the shScramble group . Moreover, as anticipated, the immunofluorescence analysis targeting the specific tight junction protein ZO1 and the EMT marker α-SMA showed a marked reduction or even loss of tight junctions between cells treated with TGFβ1, concurrent with elevated α-SMA expression ( G). Importantly, suppression of HSPA13 restored the peripheral localization of ZO1 and attenuated the upregulation of α-SMA, which aligns with the morphological improvements depicted in A. These findings reinforce the notion that downregulation of HSPA13 can counteract TGFβ1-induced EMT in hESC-RPE cells. We also observed similar results in ARPE-19 cells . Notably, HSPA13 downregulation on its own (shHSPA13 without TGFβ1) could increase the expression of RPE-related genes ( B, D, F), consistent with the improved epithelial morphology observed in shHSPA13-RPE cells ( A). Given that hESC-RPE cells spontaneously undergo EMT during in vitro culture, this finding suggests that downregulating HSPA13 helps maintain an RPE identity and subsequently hinders the occurrence of EMT. To further investigate the effect of HSPA13 on TGFβ1-induced EMT of RPE cells, we performed bulk RNA sequencing (RNA-seq). The volcano plots were used to show the DEGs between different compared groups ( A–C). The Gene Ontology (GO) analysis of the DEGs between the shHSPA13 + TGFβ1 group and the shScramble + TGFβ1 group revealed that these changed genes were mainly involved in extracellular matrix, cell adhesion, and visual perception ( D, E). Gene set enrichment analysis (GSEA) revealed that gene signatures associated with EMT were significantly downregulated in the shHSPA13 + TGFβ1 group relative to the shScramble + TGFβ1 group ( F). The top EMT-related DEGs were presented in a clustering heatmap ( G). RPE migration has been implicated in the development of the PVR. A wound-healing assay was conducted to investigate the effect of HSPA13 on RPE cell migration. As shown in A and B, shScramble-RPE cells treated with TGFβ1 exhibited greater motility compared to untreated cells. However, shHSPA13-RPE cells treated with TGFβ1 showed delayed migration when compared with TGFβ1-treated shScramble-RPE cells. Once more, the knockdown of HSPA13 resulted in the suppression of TGFβ1-induced migration of ARPE-19 cells, as demonstrated by wound healing assay and Transwell migration assay . Further, RNA-seq results revealed that HSPA13 knockdown attenuated the expression of migration-related genes ( C, D). shHSPA13 Inhibits PI3K/Akt Pathway in TGFβ1-Induced RPE Cells To investigate the mechanism by which HSPA13 knockdown inhibits the EMT of RPE cells, we further analyzed our RNA-seq data. GO enrichment analysis revealed that upregulated DEGs in the shHSPA13 + TGFβ1 group compared with the shScramble + TGFβ1 group were mainly involved in visual perception, and downregulated DEGs were focused on cell adhesion and extracellular matrix organization ( A, B). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that the downregulated DEGs were predominantly associated with the PI3K/Akt signaling pathway ( C). The PI3K/Akt-related DEGs were shown in the heatmap ( D). Western blots were subsequently performed to verify the regulatory mechanisms of HSPA13 on the PI3K/Akt signaling pathway. The results showed that HSPA13 knockdown markedly downregulated the levels of phosphorylated PI3K (p-PI3K) and phosphorylated Akt (p-Akt), whereas there was no change in the expression of total PI3K and Akt ( E–G). To determine the specificity of HSPA13 toward the PI3K/Akt pathway, we further analyzed 2 additional critical signaling mechanisms: the Smads signaling, the primary signaling induced by TGFβ, and the Wnt/β-catenin signaling, another crucial regulator of EMT. The results revealed that the downregulation of HSPA13 did not significantly influence either of these two signaling pathways . This finding implies that HSPA13 exhibits a distinct specificity in regulating EMT processes, particularly for its lack of impact on the Smads and Wnt signaling pathways. Knockdown of HSPA13 Inhibits EMT In Vivo To investigate the effect of HSPA13 on EMT in vivo, we used a rat model of PVR established by intravitreal injection of hESC-RPE cells and PRP. Following 4 weeks of intravitreal injection, the hESC RPE + PRP group displayed a pronounced disruption in the retinal structure, particularly in the inner and outer retinal layers ( A). Importantly, a remarkable co-expression of α-SMA and HSPA13 was evident within the epiretinal region of this group ( A). This finding aligns with our earlier observations of HSPA13 and α-SMA co-localization within ERMs from patients with PVR , thereby further substantiating the expression of HSPA13 in the ERMs. To further validate the inhibitory effect of shHSPA13 on EMT, we separately injected shScramble hESC RPE + PRP and shHSPA13-hESC RPE + PRP into the vitreous cavity. Hematoxylin and eosin (H&E) staining revealed significant disparities between the two groups: the shScramble group exhibited dense ERMs (denoted by asterisks) and convoluted retinal tissue, whereas the shHSPA13 group presented with only mild retinal folds ( B). Immunofluorescence staining further illuminated that the injected shScramble-hESC-RPE cells (GFP-labeled) developed distinct ERMs with strong α-SMA expression ( C). In contrast, the shHSPA13-hESC RPE + PRP group displayed a reduced number of RPE cells in the epiretinal region, accompanied by absent or low levels of α-SMA expression. These data suggest that HSPA13 knockdown effectively inhibits EMT in vivo.   To investigate the potential involvement of HSPA13 in the pathogenesis of PVR, we detected the expression of HSPA13 in ERMs collected from patients with PVR by immunostaining. As shown in , the HSPA13 showed strong immunoreactivity within the ERM. Double-staining further revealed that HSPA13 co-localized with both the epithelial marker CK8 and the EMT marker α-SMA. As RPE cells are the only kind of epithelial cells seen in the ERM, this finding suggests that HSPA13-positive cells are derived from RPE cells undergoing EMT. However, there is a variability in staining patterns among the cases, potentially attributable to a range of factors, including variations in ERMs size, tissue section orientation, and EMT degree. Actually, the cell count in the Cases 1 and 2 is significantly lower than that in the Cases 3 and 4. Despite this variability, all the results consistently reveal the co-localization of HSPA13 with the epithelial marker CK8 and the EMT marker α-SMA.   2+ The expression pattern of HSPA13 in PVR membranes indicates that HSPA13 might play a role in PVR pathogenesis. As the EMT of RPE cells plays a crucial role in the pathogenesis of PVR, we next investigated whether HSPA13 contributes to the EMT process in RPE cells. To stimulate the EMT of RPE cells in vitro, we used TGFβ1-induced EMT model. In this model, we treated hESC-RPE cells with 10 ng/mL TGFβ1 for 48 hours. The selection of this duration and concentration was determined by time course and dose curve studies . As expected, TGFβ1 treatment successfully induced a morphological transformation of hESC-RPE cells from polygonal to elongated spindle-shaped cells ( A). Consistent with the change in morphology, the expression of the epithelial and specific RPE markers E-CAD, RPE65, and BEST was downregulated ( B), and the expression of the mesenchymal markers FN1 ( C, E, F) and PAI-1 ( C) were upregulated. Meanwhile, we observed an increase in both the mRNA ( D) and protein ( E, G) levels of HSPA13 in RPE cells treated with TGFβ1. A similar result was also observed in ARPE-19 cells and primary mouse RPE (mRPE) cells treated with TGFβ1 . Additionally, double-immunofluorescence labeling revealed an elevated expression of both HSPA13 and α-SMA in the TGFβ1-treated RPE cells ( H), further confirming the upregulation of HSPA13 in cells undergoing EMT. On the contrary, high expression of HSPA13 was accompanied by low and cytoplasmic expression of E-cadherin, suggesting that cells with high HSPA13 expression experience a loss of cell contact . A previous study showed that treatment with TGFβ1 in megakaryocytes resulted in an increased level of intracellular Ca 2+ . In line with this result, we found that TGFβ1 treatment also increased intracellular Ca 2+ in hESC-RPE cells ( I, J). Because HSPA13 is a Ca 2+ stress-induced member of the HSP70 superfamily, we then investigated whether the increased HSPA13 expression induced by TGFβ1 is dependent on intracellular Ca 2+ . To this end, we used BAPTA/AM to chelate intracellular Ca 2+ and then examined the impact of TGFβ1 on the expression of HSPA13. As shown in K–M, chelating calcium prevented TGFβ1-induced HSPA13 upregulation in hESC-RPE cells. A similar result was also observed in ARPE-19 cells . To further confirm the role of Ca 2+ in the upregulation of EMT and HSPA13, we conducted immunofluorescence co-staining to detect the expression of α-SMA and HSPA13 under chelated calcium conditions. Our findings revealed that RPE cells treated with BAPTA and TGFβ1 exhibited reduced expression of HSPA13 and α-SMA compared to cells treated with TGFβ1 alone . Taken together, these data demonstrate that TGFβ1 can induce HSPA13 expression in hESC-RPE cells, and such induction effect is dependent on intracellular Ca 2+ .   To elucidate the function of HSPA13 in the EMT of RPE cells, we established a stable HSPA13 knockdown hESC-RPE cell line by infecting cells with lenti-shHSPA13 virus. Lenti-shScramble was used as a negative control. The mesenchymal morphology induced by TGFβ1 was suppressed in shHSPA13-RPE cells ( A). The shHSPA13 markedly reduced the level of HSPA13 in hESC-RPE cells and weakened the expression of EMT marker genes triggered by TGFβ1, compared to the shScramble group . Moreover, as anticipated, the immunofluorescence analysis targeting the specific tight junction protein ZO1 and the EMT marker α-SMA showed a marked reduction or even loss of tight junctions between cells treated with TGFβ1, concurrent with elevated α-SMA expression ( G). Importantly, suppression of HSPA13 restored the peripheral localization of ZO1 and attenuated the upregulation of α-SMA, which aligns with the morphological improvements depicted in A. These findings reinforce the notion that downregulation of HSPA13 can counteract TGFβ1-induced EMT in hESC-RPE cells. We also observed similar results in ARPE-19 cells . Notably, HSPA13 downregulation on its own (shHSPA13 without TGFβ1) could increase the expression of RPE-related genes ( B, D, F), consistent with the improved epithelial morphology observed in shHSPA13-RPE cells ( A). Given that hESC-RPE cells spontaneously undergo EMT during in vitro culture, this finding suggests that downregulating HSPA13 helps maintain an RPE identity and subsequently hinders the occurrence of EMT. To further investigate the effect of HSPA13 on TGFβ1-induced EMT of RPE cells, we performed bulk RNA sequencing (RNA-seq). The volcano plots were used to show the DEGs between different compared groups ( A–C). The Gene Ontology (GO) analysis of the DEGs between the shHSPA13 + TGFβ1 group and the shScramble + TGFβ1 group revealed that these changed genes were mainly involved in extracellular matrix, cell adhesion, and visual perception ( D, E). Gene set enrichment analysis (GSEA) revealed that gene signatures associated with EMT were significantly downregulated in the shHSPA13 + TGFβ1 group relative to the shScramble + TGFβ1 group ( F). The top EMT-related DEGs were presented in a clustering heatmap ( G). RPE migration has been implicated in the development of the PVR. A wound-healing assay was conducted to investigate the effect of HSPA13 on RPE cell migration. As shown in A and B, shScramble-RPE cells treated with TGFβ1 exhibited greater motility compared to untreated cells. However, shHSPA13-RPE cells treated with TGFβ1 showed delayed migration when compared with TGFβ1-treated shScramble-RPE cells. Once more, the knockdown of HSPA13 resulted in the suppression of TGFβ1-induced migration of ARPE-19 cells, as demonstrated by wound healing assay and Transwell migration assay . Further, RNA-seq results revealed that HSPA13 knockdown attenuated the expression of migration-related genes ( C, D).   To investigate the mechanism by which HSPA13 knockdown inhibits the EMT of RPE cells, we further analyzed our RNA-seq data. GO enrichment analysis revealed that upregulated DEGs in the shHSPA13 + TGFβ1 group compared with the shScramble + TGFβ1 group were mainly involved in visual perception, and downregulated DEGs were focused on cell adhesion and extracellular matrix organization ( A, B). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that the downregulated DEGs were predominantly associated with the PI3K/Akt signaling pathway ( C). The PI3K/Akt-related DEGs were shown in the heatmap ( D). Western blots were subsequently performed to verify the regulatory mechanisms of HSPA13 on the PI3K/Akt signaling pathway. The results showed that HSPA13 knockdown markedly downregulated the levels of phosphorylated PI3K (p-PI3K) and phosphorylated Akt (p-Akt), whereas there was no change in the expression of total PI3K and Akt ( E–G). To determine the specificity of HSPA13 toward the PI3K/Akt pathway, we further analyzed 2 additional critical signaling mechanisms: the Smads signaling, the primary signaling induced by TGFβ, and the Wnt/β-catenin signaling, another crucial regulator of EMT. The results revealed that the downregulation of HSPA13 did not significantly influence either of these two signaling pathways . This finding implies that HSPA13 exhibits a distinct specificity in regulating EMT processes, particularly for its lack of impact on the Smads and Wnt signaling pathways.   To investigate the effect of HSPA13 on EMT in vivo, we used a rat model of PVR established by intravitreal injection of hESC-RPE cells and PRP. Following 4 weeks of intravitreal injection, the hESC RPE + PRP group displayed a pronounced disruption in the retinal structure, particularly in the inner and outer retinal layers ( A). Importantly, a remarkable co-expression of α-SMA and HSPA13 was evident within the epiretinal region of this group ( A). This finding aligns with our earlier observations of HSPA13 and α-SMA co-localization within ERMs from patients with PVR , thereby further substantiating the expression of HSPA13 in the ERMs. To further validate the inhibitory effect of shHSPA13 on EMT, we separately injected shScramble hESC RPE + PRP and shHSPA13-hESC RPE + PRP into the vitreous cavity. Hematoxylin and eosin (H&E) staining revealed significant disparities between the two groups: the shScramble group exhibited dense ERMs (denoted by asterisks) and convoluted retinal tissue, whereas the shHSPA13 group presented with only mild retinal folds ( B). Immunofluorescence staining further illuminated that the injected shScramble-hESC-RPE cells (GFP-labeled) developed distinct ERMs with strong α-SMA expression ( C). In contrast, the shHSPA13-hESC RPE + PRP group displayed a reduced number of RPE cells in the epiretinal region, accompanied by absent or low levels of α-SMA expression. These data suggest that HSPA13 knockdown effectively inhibits EMT in vivo.   RPE cells have been reported to undergo EMT in various retinal degenerative disorders, such as PVR and age-related macular degeneration (AMD). However, there is still no effective pharmacological treatment for these diseases. In this study, for the first time, we demonstrated that HSPA13 was involved in TGFβ1-induced EMT of RPE cells and implicated in the pathogenesis of PVR. We found that TGFβ1 can induce HSPA13 expression in various RPE cells, including hESC-RPE cells, ARPE-19 cells, and mRPE cells. Intriguingly, this induction effect was dependent on intracellular Ca 2+ . More importantly, knockdown of HSPA13 suppressed the EMT of RPE cells in vitro and in vivo. Mechanistically, HSPA13 knockdown inhibited activation of the PI3K/Akt signaling pathway induced by TGFβ1. These findings suggest that HSPA13 may serve as a new target for PVR treatment. It is well known that Ca 2+ influx into cells via plasma membrane protein channels is tightly regulated to maintain cellular homeostasis. Previous studies have shown that the epithelial cell marker E-cadherin and the mesenchymal cell marker vimentin are regulated by calcium. , Furthermore, emerging evidence from tumor studies indicated a strong correlation between EMT inducers and intracellular Ca 2+ levels. , TGFβ-driven EMT of MCF7 breast cancer cells was found to be correlated with an elevated influx of Ca 2+ into the cells. The transient receptor potential melastatin-like 7 (TRPM7) triggers the process of EMT in breast cancer by transiently increasing levels of Ca 2+ . Blocking this channel reduces the effects of hypoxia and epidermal growth factor (EGF) on EMT. Consistent with these studies conducted on tumor cells, , our results revealed an increased Ca 2+ level in TGFβ1-driven EMT in hESC-RPE cells (see I, J) and ARPE-19 cells (see ), suggesting that the TGFβ-Calcium-EMT axis might play an important role in the development of PVR. HSPA13, a unique member of the HSP70 superfamily, is induced by Ca 2+ stress rather than heat shock. To date, there is relatively little research on HSPA13. Several reports have shown that HSPA13 is highly expressed in tumors and involved in tumor proliferation, migration, and invasion. , Given that cell migration is a characteristic of EMT, the tumor migration promoted by HSPA13 implies its potential role in the progression of EMT. Here, we demonstrated, for the first time, that HSPA13 was expressed in the ERMs of patients with PVR (see ). Besides, the expression of HSPA13 was significantly elevated in TGFβ1-induced EMT of RPE cells (see , ). More importantly, HSPA13 knockdown inhibited EMT of RPE cells in vitro (see , ) and in vivo (see ). RPE cell migration is an important step in the development of PVR. We also found that knockdown of HSPA13 effectively inhibited TGFβ1-induced RPE cell migration (see , ). In support of these findings, our RNA-seq data revealed that genes related to EMT and cell migration were suppressed by HSPA13 knockdown (see , ). Collectively, these findings indicate that HSPA13 plays an important role in the TGFβ1-induced EMT process in RPE cells and consequently, in the development of PVR. The KEGG enrichment analysis demonstrated a significantly correlation with the PI3K/Akt pathway (see C), a critical component in EMT transformation, metastasis, and survival of tumor cells. This finding implies that the PI3K/Akt pathway participate in the HSPA13-regulated EMT process in RPE cells. Further elucidation through Western blot experiments indicated that the downregulation of HSPA13 specifically suppresses TGFβ-induced PI3K activation (see ), without affecting Smad signaling, the canonical pathway triggered by TGFβ, or Wnt/β-catenin signaling, a crucial regulator of EMT (see ). Our study primarily concentrated on the suppressive impact of HSPA13 knockdown on TGFβ1-induced EMT. Notably, downregulation of HSPA13 alone, in the absence of TGFβ1 enhances the expression of RPE-specific markers (E-cadherin, RPE65, and BEST; see ). This observation suggests that reduced HSPA13 levels contribute to the preservation of RPE characteristics, thereby hindering spontaneous EMT in vitro. Critically, we found no significant modulation of Smad2/3 signaling upon HSPA13 downregulation, indicating that the inhibition of TGFβ1-induced EMT through HSPA13 knockdown is independent of the canonical TGFβ pathway. This implies a general inhibitory effect of HSPA13 downregulation on EMT, irrespective of the specific triggering stimulus. Additionally, it is remarkable that hESC-derived RPE cells exhibit the ability to traverse the retina (see C). Intriguingly, these RPE cells that infiltrate the retina appear not to undergo EMT, evidenced by their lack of α-SMA expression. Notably, an increased proportion of shHSPA13-RPE cells was observed infiltrating the retina, suggesting a potential mechanism by which shHSPA13 inhibits EMT in RPE cells. This intriguing observation warrants further exploration. Collectively, our findings provide novel evidence that HSPA13 is involved in the EMT of RPE cells and ERMs formation in PVR. Mechanistically, stimulation with TGFβ1 treatment resulted in increased intracellular Ca 2+ levels, leading to upregulation of HSPA13 expression, which in turn activated the PI3K/Akt pathway, facilitating EMT. Targeting HSPA13-related pathways involved in regulating EMT in RPE cells may offer a promising therapeutic strategy for patients with PVR.   Supplement 1   