Advances in Clinical and Experimental Medicine

Title abbreviation: Adv Clin Exp Med
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Advances in Clinical and Experimental Medicine

2024, vol. 33, nr 6, June, p. 641–651

doi: 10.17219/acem/170999

Publication type: original article

Language: English

License: Creative Commons Attribution 3.0 Unported (CC BY 3.0)

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Zhang Y, Zhang Y, He S, Wang W. Lysyl oxidase-mediated elastin upregulation promotes the proliferation and migration of human retinal endothelial cells. Adv Clin Exp Med. 2024;33(6):641–651. doi:10.17219/acem/170999

Lysyl oxidase-mediated elastin upregulation promotes the proliferation and migration of human retinal endothelial cells

Yu Zhang1,A,D, Yurong Zhang1,B,C, Siyu He1,C,E, Weixing Wang1,E,F

1 Department of Ophthalmology, Nantong Haimen People’s Hospital, China

Graphical abstract


Graphical abstracts

Abstract

Background. Proliferative diabetic retinopathy (PDR) is a major cause of irreversible blindness in the working age population. The dysfunction of retinal vascular endothelial cells (RVECs) is the primary cause of PDR. Extracellular matrix (ECM) accumulation promotes intracellular signaling required for RVEC proliferation, migration, survival, and tube morphogenesis.

Objectives. This study aimed to investigate the role of lysyl oxidase (LOX) in the cellular function of RVECs and PDR pathogenesis and to identify the underlying mechanisms.

Materials and methods. Protein expression was determined with western blot. The interaction between LOX and elastin (ELN) was detected using a co-immunoprecipitation (Co-IP) assay, and the Cell Counting Kit-8 (CCK-8) assay evaluated cell viability. A colony formation assay was employed to assess the proliferation of human RVECs (hRVECs), and a transwell assay to determine their migration ability. Streptozotocin was used to establish PDR in mice in vivo. A histological analysis was conducted using hematoxylin and eosin (H&E) staining.

Results. The results showed that LOX was overexpressed in PDR patients. The LOX knockdown suppressed ECM formation and hRVEC proliferation and migration. Additionally, LOX upregulated ELN expression. However, overexpressed ELN promoted hRVEC proliferation and migration. In vivo experiments showed that curcumin-mediated LOX deficiency restored retinal tissue structure.

Conclusions. The LOX-knockdown suppressed ECM formation and hRVEC proliferation and migration by inactivating ELN. Therefore, LOX/ELN signaling may be a potential PDR biomarker.

Key words: extracellular matrix, proliferative diabetic retinopathy, LOX, ELN

Background

Diabetic retinopathy (DR) is a common complication inducing ophthalmic diseases.1 It can be divided into non-proliferative DR (NPDR) and proliferative DR (PDR) based on the neovascularization status.2 The dysfunction of retinal vascular endothelial cells (RVECs) is the primary cause of PDR.3 Recent evidence reveals that metabolic imbalance induced by high glucose levels promotes RVEC degradation and PDR pathogenesis.4, 5, 6 In recent years, great advances have been made in PDR treatment, such as anti-vascular endothelial growth factor (anti-VEGF) strategies.7, 8, 9 However, the mechanisms of the disease are still unclear. Therefore, unveiling the underlying molecular mechanisms may help uncover the Achilles’ heel of PDR.

Lysyl oxidase (LOX) is a copper-dependent amine oxidase that catalyzes lysine-derived cross-links in collagen and elastin (ELN), resulting in extracellular matrix (ECM) reprogramming.10 The LOX plays an important role in vascular diseases. For instance, LOX deficiency is closely associated with cardiovascular dysfunction.11 However, aberrantly high levels of LOX promote angiogenesis and tumor metastasis.12

In ophthalmic diseases, LOX is frequently downregulated in keratoconus patients.13 However, LOX is overexpressed in PDR patients and the retinas of diabetic mice.14 Interestingly, increasing evidence reports that LOX inhibition may be a promising strategy for PDR treatment. For instance, LOX knockdown protects against the development of vascular lesions characteristic of DR by suppressing retinal vascular permeability.15 Meanwhile, β-aminopropionitrile-mediated LOX deficiency inhibits the angiogenesis and migration of human umbilical vein endothelial cells.16 However, the underlying mechanisms are still unclear.

The ECM is rich in ELN glycoprotein,17 and abnormal ELN expression is closely associated with vascular endothelial cell (VEC) functions. For instance, idiopathic portal hypertension-induced overexpression of ELN in VECs induces the pathogenesis of hepatoportal sclerosis.18 In eye disorders, ELN-mediated choroidal endothelial cell migration contributes to age-related macular degeneration.19 Also, ELN-derived peptides (EDPs) promote the migration and tubulogenesis of human corneal endothelial cells.20 However, the role of ELN in PDR has not been elucidated.

Objectives

This study aimed to investigate the role of LOX in PDR and uncover its underlying mechanisms. Gene levels and expression were determined using enzyme-linked immunosorbent assay (ELISA) and western blot. The interaction between LOX and ELN was investigated using a co-immunoprecipitation (Co-IP) assay. We hypothesized that LOX-mediated ELN upregulation promoted the development of PDR.

Materials and methods

Ethical approval

The Ethical Committee of Nantong Haimen People’s Hospital, China, approved the study (approval No. 2021[07]). All patients provided informed consent.

Cell culture

Primary human RVECs (hRVECs) were purchased from American Type Culture Collection (ATCC; Manassas, USA) and cultured in Dulbecco’s modified Eagles’s medium (DMEM) (Gibco, Waltham, USA) containing 10% fetal bovine serum (FBS) at 37°C in 5% carbon dioxide (CO2). Cells were exposed to high glucose (HG; 30 mmol/L of d-glucose) or normal glucose (NG; 5.55 mmol/L of d-glucose) conditions.

Cell transfection

The negative control (NC) and short hairpin LOX (shLOX) (GenePharma, Shanghai, China) were transfected using a Lipofectamine™ 2000 kit (Thermo Fisher Scientific, Waltham, USA), according to the manufacturer’s protocol. After 48 h, cells were used in the experiments outlined below. The sequences of sh ribonucleic acids (shRNAs) and their NCs are listed in Supplementary Table 1.

Real-time quantitative polymerase chain reaction

Total RNA was extracted from cells or tissues using RNA isolation reagents and then quantified using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific). Afterward, complementary deoxyribonucleic acid (cDNA) was synthesized using a PrimeScript® RT Reagent Kit (Takara, Shiga, Japan), according to the manufacturer’s protocols. Polymerase chain reaction (PCR) was performed using a PrimeScript real-time (RT)-PCR kit (Takara) on an ABI 7500 Real-Time PCR system (Thermo Fisher Scientific). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the loading control. Relative messenger RNA (mRNA) levels were determined with the 2−ΔΔCq method.21 The primers used are listed in Supplementary Table 2.

Immunofluorescence (IF)

Cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. Afterward, cells were blocked with 5% FBS and incubated with primary antibodies against LOX (ab174316), collagen I alpha-1 (Col1A1) (ab138492) and Col4A1 (ab308360) (all from Abcam, Cambridge, USA). Then, the cells were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) and visualized with the use of an immunofluorescence microscope (Axio Imager 5; Carl Zeiss, Oberkochen, Germany).

Western blot

Cells were collected and lysed with radioimmunoprecipitation assay (RIPA) lysis buffer. Then, the protein was collected and concentrated using a bicinchoninic acid assay (BCA) kit (Abcam, Cambridge, UK) according to the manufacturer’s protocols. After isolation using 12% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the protein was transferred onto a polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific). The membranes were blocked with 5% skimmed milk and then incubated with primary antibodies against LOX (ab221936; 1:1,000), ELN (ab307150; 1:1,000) or GAPDH (ab9485; 1:2,500), and goat-anti-rabbit secondary antibodies (ab6721; 1:10,000) (all from Abcam). Finally, the bands were captured using an enhanced chemiluminescence (ECL) kit (Abcam).

Co-immunoprecipitation

The Co-IP was performed using the Pierce™ Classic Magnetic Co-IP Kit (Thermo Fisher Scientific) according to the manufacturer’s protocols.22 Briefly, the cells were lysed with IP lysis buffer and homogenized. Afterward, they were centrifuged at 12,000 × g, and the supernatants were collected. The protein was incubated with antibodies against immunoglobulin G (IgG) (ab174316) or LOX (ab174316) (both from Abcam) and immobilized on protein A/G beads. The beads were then washed and microcentrifuged. Finally, the bands were analyzed using western blot.

Bioinformatics analysis

The genes interacting with LOX were predicted using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (https://cn.string-db.org).

Colony formation assay

Following transfection, the cells were transferred into a 96-well plate and cultured for 2 weeks. After being washed with phosphate-buffered saline (PBS), the cells were fixed in 95% ethanol and stained with 0.1% crystal violet. Finally, the colonies were counted under a microscope (Primovert; Carl Zeiss AG).

Transwell assay

The migration assays used transwell chambers without a Matrigel mix coating (BD Biosciences, Franklin Lakes, USA). Homogeneous serum-free single-cell suspensions (1 × 105 cells/well) were added to the upper chambers, and the medium containing 10% FBS was added into the lower chambers and incubated for 24 h. The migratory and invasive cells were fixed, stained and counted.

Propidium iodide staining

After transfection, the cells were plated onto a 24-well plate and treated with a propidium iodide (PI) solution (2 µg/mL). Finally, PI-positive cells were captured using a fluorescence microscope (Leica DFC9000 sCMOS; Leica Camera AG, Wetzlar, Germany).

Flow cytometry

After transfection, the cells were collected and cultured at 37°C. Then, following trypsinization, the cells were cultured with Annexin-V-FITC/PI for 15 min at 37°C. Finally, the apoptotic cells were determined using a FACSCalibur flow cytometer (BD Biosciences) with FlowJo software 7.6 (FlowJo, LLC, Ashland, USA).

Gene set enrichment database analysis

The LOX expression was analyzed using the GSE60436 online microarray database, and the differentially expressed genes were used for Gene Ontology (GO; https://geneontology.org/) and Kyoto Encyclopedia of Genes and Genome (KEGG) analysis (https://www.kegg.jp/kegg/mapper/).

In vivo assay

Male C57BL/6J mice (n = 18, 6–8 weeks, 18–22 g) were purchased from the Experimental Animal Center of Nanjing Medical University (Nanjing, China) and used to establish a previously described diabetic model.23 Mice were housed in pathogen-free cages under a 12-hour light/dark cycle with free access to food and water. The animals were randomly divided into 3 groups: sham group (n = 6), streptozocin (STZ) group (n = 6) and STZ + curcumin group (n = 6). Mice in the sham group were intraperitoneally injected with 10% dimethyl sulfoxide (DMSO), while those in the STZ or STZ+GA groups were injected intraperitoneally with STZ (60 mg/kg) once a day for 5 consecutive days. After 4 weeks, mice were injected intraperitoneally with 100 mg/kg/day curcumin for 4 weeks. Afterward, the animals were euthanized by sodium pentobarbital injection, and retinal samples were collected and stained with hematoxylin and eosin (H&E) for histological analysis.

Statistical analyses

Data were analyzed using GraphPad Prism 9.0 (GraphPad Software, San Diego, USA). Each experiment was performed in triplicate. The Mann–Whitney U test was employed to compare differences between 2 groups, while the Kruskal–Wallis test followed by Dunn’s post hoc test were used to analyze the differences between multiple groups (Supplementary Table 3). Statistical significance was set at p < 0.05.

Results

Lysyl oxidase was upregulated in diabetic retinopathy

The GSE60436 database was applied to analyze the differentially expressed genes in DR patients. As shown in Figure 1A, 32 genes were significantly upregulated and 39 were downregulated in DR patients. Moreover, GO and KEGG analysis showed that the upregulated genes were enriched in amino acid metabolism (Figure 1B). To further analyze the role of these genes in DR, we divided DR patients into 2 subtype groups: NPDR and PDR. We found that, among the 35 upregulated genes, the expression of GrpE protein homolog 2 (GRPEL2), phosphatidylinositol binding clathrin assembly protein interacting mitotic regulator (PIMREG), kinesin family member 20A (KIF20A), and LOX increased significantly in PDR patients compared to NPDR patients (Figure 1C). Among the upregulated genes in PDR, high levels of LOX promote the pathogenesis of PDR.14, 24 Therefore, we selected LOX for further study. Cells were exposed to HG conditions to further confirm the roles of LOX in PDR. The results showed that LOX mRNA expression increased markedly in the HG group (Figure 1D), consistent with the Co-immunofluorescence assay results (Figure 1E).

Lysyl oxidase was required for extracellular matrix production

The LOX, a crucial ECM remodeler, is a potential therapeutic target for fibrosis and cardiovascular disease. Therefore, we determined the roles of LOX in the ECM and found that the fluorescence intensity of Col1A1 was significantly increased after HG exposure (Figure 2A). However, LOX knockdown markedly inhibited the increase in HG-induced Col1A1 expression, and markedly reduced Col4A1 expression (Figure 2B). These findings suggested that LOX promoted ECM formation.

Lysyl oxidase promoted human retinal vascular endothelial cell proliferation and migration

Figure 3A shows the transfection efficiency of shLOX. The sh2 had remarkable effects and was used in the following experiment. The LOX knockdown markedly suppressed the increased cell viability induced by HG (Figure 3B). Moreover, the LOX knockdown significantly inhibited hRVEC colony formation (Figure 3C). Transwell assays showed that HG-induced hRVEC migration was dampened by LOX knockdown (Figure 3D). Whether it could induce hRVEC death required further verification, with cellular functions determined using PI and flow cytometry. The results showed that HG and/or LOX knockdown had no significant impact on hRVEC death (Figure 3E and Supplementary Fig. 1), suggesting that LOX is required for promoting hRVEC morbidity.

Lysyl oxidase upregulated elastin

The STRING database was used to analyze the genes interacting with LOX (Figure 4A). We found that LOX interacted with fibronectin 1 (FN1), matrix metalloproteinase 2 (MMP2) and ELN. The ELN encodes proteins enriched in hydrophobic amino acids such as glycine and proline. Therefore, we speculated that LOX may participate in amino acid metabolism via ELN upregulation. The Co-IP assay confirmed the interaction between LOX and ELN (Figure 4B). Moreover, LOX knockdown decreased ELN protein expression, which was reversed by ELN overexpression (Figure 4C).

Overexpressed elastin promoted human retinal endothelial cell proliferation and migration

Rescue assays were performed to verify the role of ELN in PDR. The experiments were conducted using cells exposed to HG conditions. The ELN mRNA expression was significantly increased by ELN overexpression plasmids (Figure 5A), suggesting that the cells were successfully transfected. Overexpressed ELN markedly increased hRVEC viability (Figure 5B). Moreover, overexpressed ELN significantly increased hRVEC colony formation (Figure 5C). Additionally, ELN overexpression remarkably increased hRVEC migration ability (Figure 5D).

Curcumin alleviated proliferative diabetic retinopathy in vivo

The GeneCards online database (https://www.genecards.
org) was used to choose drugs approved for LOX. Among the approved drugs, acetylsalicylic acid (aspirin) increases cardiovascular vitality, and bleomycin and cisplatin are used for cancer therapy, with outstanding side effects, such as vascular malformation. Therefore, we chose curcumin to induce LOX deficiency in vivo. As shown in Figure 6A, the layers of retinal tissues were loose and irregular in the STZ group. Moreover, the number of ganglion cells was reduced, and the ganglion cell layer displayed obvious vacuolar degeneration. Additionally, LOX, Col1A1, Col4A1, and ELN expression significantly decreased after curcumin injection (Figure 6B–E).

Discussion

In this study, LOX was overexpressed in PDR patients. The LOX deficiency markedly suppressed ECM formation and hRVEC proliferation and migration, and alleviated PDR in vivo. Furthermore, LOX upregulated ELN, thereby promoting hRVEC proliferation and migration.

The LOX is often abnormally expressed in ophthalmic diseases.12, 13, 14 For instance, HG-induced upregulation of LOX promotes monolayer permeability and compromises barrier functional integrity.25 Moreover, the overexpression of LOX is associated with PDR and rhegmatogenous retinal detachment.14 Therefore, LOX may promote the pathogenesis of ophthalmic diseases. Increasing evidence demonstrates that LOX inhibition using specific shRNAs or LOX inhibitors may be a promising PDR treatment strategy.14, 15 In this study, the expression of LOX was increased in PDR patients, suggesting that LOX may play a positive role in PDR development. However, LOX knockdown markedly suppressed ECM formation and hRVEC proliferation and migration, suggesting that LOX deficiency mediated hRVEC morbidity inhibition and may be a promising strategy for PDR therapy.

The pathogenesis of DR is influenced by ECM capillary basement membrane alterations,26 which may induce changes in the characteristics of the endothelium.27 The ECM accumulation promotes intracellular signaling required for cell proliferation, migration, survival, and tube morphogenesis.28, 29 As an ECM constituent, ELN is a key gene modulating endothelial cell functions via stiff substrate and stenotic phenotype regulation.30 However, ELN mutation or abnormal expression contributes to polypoidal choroidal vasculopathy.31 In this study, LOX-mediated ELN upregulation promoted ECM synthesis and enhanced hRVEC proliferation and migration. Therefore, LOX/ELN signaling may be a therapeutic target for PDR.

Curcumin is a natural compound widely used in the treatment of retinal diseases.32 For instance, curcumin suppresses the release of pro-inflammatory cytokines and alleviates DR.33 Moreover, it inhibits the development of PDR by inactivating vascular endothelial growth factor (VEGF).34 These findings suggest that curcumin, with anti-inflammatory, anti-oxidant and anti-angiogenic effects, has beneficial effects in DR treatment. Moreover, curcumin suppresses the accumulation of pro-inflammatory cytokines and alleviates retinal inflammatory injury, contributing to decreased endothelial cell permeability and pathological angiogenesis, which is frequently found in the late stages of DR and PDR.35, 36 Furthermore, curcumin represses ECM-receptor interactions in the diabetic retina.37 In this study, curcumin treatment improved the retinal structure and suppressed endothelial cell permeability, suggesting that curcumin-mediated LOX deficiency may be a promising strategy for PDR treatment.

Limitations

Due to the limitations of the experimental conditions, future studies should assess the role of LOX in PDR patients. As a collagen cross-linking enzyme, LOX regulates cellular functions by inducing metabolic reprogramming, such as glycolysis and amino acid metabolism; whether LOX-mediated collagen formation and cell proliferation and migration by inducing metabolic reprogramming requires further study.

Conclusions

In conclusion, the overexpression of LOX was associated with PDR development. The LOX deficiency suppressed ECM formation and hRVEC proliferation and migration by inactivating ELN (see the graphical abstract). Therefore, LOX/ELN may be a novel therapeutic target for PDR.

Supplementary data

The supplementary materials are available at https://doi.org/10.5281/zenodo.8202183. The package includes the following files:

Supplementary Fig. 1. The apoptosis rates of hRVECs.

Supplementary Table 1. The sequences of shRNAs used in transfection.

Supplementary Table 2. The sequences of the primers used in PCR.

Supplementary Table 3. Statistical significance of the differences presented in figures.

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Consent for publication

Not applicable.

Figures


Fig. 1. Lysyl oxidase (LOX) is overexpressed in proliferative diabetic retinopathy (PDR). A. Differentially expressed genes in diabetic retinopathy (DR) patients were analyzed using GSE60436; B. Differentially expressed genes in PDR compared to non-proliferative PDR (NPDR) patients; C. Kyoto Encyclopedia of Genes and Genome (KEGG) and Gene Ontology (GO) analysis of the differentially expressed genes; D. LOX messenger ribonucleic acid (mRNA) expression in human retinal vascular endothelial cells (hRVECs) was detected using real-time quantitative polymerase chain reaction (qPCR); E. LOX expression in hRVECs was detected using immunofluorescence (IF). Results were statistically analyzed using the Kruskal–Wallis test followed by Dunn’s post hoc test. Data were expressed as data points. The horizontal lines represent the median value
NG – normal glucose; HG – high glucose; **p < 0.05 compared to short hairpin negative control (shNC).
Fig. 2. Lysyl oxidase (LOX) is required for extracellular matrix (ECM) production. A. Collagen 1 alpha-1 (Col1A1) expression in human retinal vascular endothelial cells (hRVECs) was detected with immunofluorescence (IF); B. Col4A1 expression in hRVECs was detected using IF. Results were statistically analyzed using the Kruskal–Wallis test followed by Dunn’s post hoc test. Data were expressed as data points. The horizontal lines represent the median value
NG – normal glucose; HG – high glucose; **p < 0.01 compared to short hairpin negative control (shNC); *p < 0.05 compared to HG+shNC.
Fig. 3. Lysyl oxidase (LOX) promotes human retinal vascular endothelial cell (hRVEC) proliferation and migration. A. LOX messenger ribonucleic acid (mRNA) expression in hRVECs was detected using real-time quantitative polymerase chain reaction (qPCR); B. Cell viability was detected using the Cell Counting Kit-8 (CCK-8) assay; C. Cell proliferation determined with colony formation assay (CFA; ×40 magnification); D. Cell migration detected using transwell assay (×100 magnification); E. Cell death detected with propidium iodide (PI) staining (×100 magnification). Results were statistically analyzed using the Kruskal–Wallis test followed by Dunn’s post hoc test. Data were expressed as data points. The horizontal lines represent the median value
NG – normal glucose; HG – high glucose; **p < 0.01 compared to short hairpin negative control (shNC); *p < 0.05 compared to HG+shNC.
Fig. 4. Lysyl oxidase (LOX) upregulates elastin (ELN). A. The genes interacting with LOX were predicted using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database; B. The interaction between ELN and LOX was verified with co-immunoprecipitation (Co-IP) assay; C. ELN protein expression was determined with western blot (n = 3)
IgG – immunoglobulin G; GAPDH – glyceraldehyde-3-phosphate dehydrogenase; shNC – short hairpin negative control.
Fig. 5. Overexpressed elastin (ELN) promoted human retinal vascular endothelial cell (hRVEC) proliferation and migration. A. ELN messenger ribonucleic acid (mRNA) expression in hRVECs was detected using real-time quantitative polymerase chain reaction (qPCR); B. Cell viability was detected using Cell Counting Kit-8 (CCK-8) assay; C. Cell proliferation determined with colony formation assay (CFA; ×40 magnification); D. Cell migration detected using transwell assay (×100 magnification) (n = 3). Results were statistically analyzed using the Kruskal–Wallis test followed by Dunn’s post hoc test or a Mann–Whitney U test. Data were expressed as data points. The horizontal lines represent the median value
**p < 0.01 compared to short hairpin negative control (shNC) or vector; *p < 0.05 compared to sh2+vector.
Fig. 6. Curcumin alleviated proliferative diabetic retinopathy (PDR) in vivo. A. Histological analysis was performed using hematoxylin and eosin (H&E) staining; B. Lysyl oxidase (LOX) expression in mice determined with real-time quantitative polymerase chain reaction (qPCR); C. Col1A1 expression in mice determined with qPCR; D. Col4 expression in mice determined with qPCR; E. ELN expression in mice determined with qPCR. Results were statistically analyzed using the Kruskal–Wallis test followed by Dunn’s post hoc test. Data were expressed as data points. The horizontal lines represent the median value
**p < 0.01 compared to sham; *p < 0.05 compared to streptozocin (STZ).

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