Transthyretin represses neovascularization in diabetic retinopathy

Materials

This study followed the tenets of the Declaration of Helsinki and the ARVO statement for research involving human subjects and was approved by the Ethics Committee of Nanjing Medical University. hRECs and hRPECs were purchased from Shanghai Bioleaf Biotech Co., Ltd (Shanghai, China). Dulbecco’s Modified Eagle's medium (DMEM), fetal bovine serum (FBS), PBS (1X; 155.2 mM NaCl, 2.7 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.4), Lipofectamine® 2000 Transfection Reagent, TRIzol® Plus RNA Purification Kit, SuperScript® One-Step RT–PCR System, pCDNA3.1 plasmid, EcoRI, BamHI, and T4 DNA ligase were from Life Technologies (Carlsbad, CA). CoCl₂ and glucose were from Sigma. The human TTR enzyme-linked immunosorbent assay (ELISA) kit was from Abnova (Taipei City, Taiwan), and the human TTR was from Sino Biologic Inc. (Shanghai, China). Other reagents and chemicals were purchased from local companies and were of superior analytical grade.

Culture of hRECs in natural and simulated DR environments

hRECs from passages 3 to 6 were used for the following experiments. Low glucose, high glucose, and hypoxia media were prepared according to previous reports [26-28] (Table 1). For natural medium (LG), cells were cultured at 37 °C in a Series 8000 WJ cell incubator (ThermoScientific, Waltham, MA) in DMEM containing 5.5 mM glucose and 10% FBS. To simulate hyperglycemia (HG) in the DR environment, the glucose in the DMEM was increased to 25 mM; hypoxia was induced with 200 μM CoCl₂ (LG+Hypoxia and HG+Hypoxia). The cells were then cultured for 72 h, and the cell index was detected with xCELLigence RTCA MP (Roche, Basel, Swiss).

The effects of TTR on hREC proliferation

As previously reported regarding post-liver transplant familial amyloid polyneuropathy (FAP), the growth of human umbilical vein endothelial cells is inhibited by TTR, and proangiogenic genes for NV are regulated [19]. Therefore, approximately 4 μM TTR was used to induce cell proliferation (Table 1), following which, the hRECs were cultured at 37 °C for 72 h, and the cell index was detected with xCELLigence RTCA MP.

Overexpression of TTR in hRECs

Total RNA was isolated from hRPECs using a TRIzol® Plus RNA Purification Kit according to the manufacturer’s instructions. Then, poly (A) RNA was enriched and amplified using a SuperScript® One-Step RT–PCR System. To clone the cDNA, according to the mRNA sequence of human TTR (cDNA clone MGC: 23689 IMAGE: 4734010), a sense primer (5′-AAT CGA GAA TTC AGT CCA CTC ATT CTT GGC AGG-3′) containing an EcoRI site and an antisense primer (5′-ATT CGA GGA TCC CAC TGG AGG AGA AGT CCC TCA-3′) containing a BamHI site were used for the PCR. The PCR conditions were as follows: an initial denaturation at 98 °C for 30 s; followed by 30 cycles of 98 °C for 10 s, 60 °C for 30 s, and 72 °C for 20 s; and a final extension of 72 °C for 4 min.

The amplified DNA and pCDNA3.1 plasmid were digested by EcoRI and BamHI at 37 °C for 1 h, respectively. The fragments were purified and cleaned on a DNA clear column and then linked using T4 DNA ligase. The pCDNA3.1 plasmid containing the human TTR gene was submitted for sequencing and then transfected into hRECs, using Lipofectamine 2000 Transfection Reagent, and an empty plasmid was used as the control. After transfection, the cells were cultured in natural and experimental media for 72 h, and the cell index was detected with xCELLigence RTCA MP (Roche).

At 24, 48, and 72 h after transfection, the cells cultured in the natural environment were collected, lysed in radioimmune precipitation assay (RIPA) buffer containing 1 mM phenylmethanesulfonyl fluoride (PMSF), incubated in ice water for 10 min, and centrifuged at 10,000 ×g for 5 min. The supernatants were used for ELISA to detect the overexpression of TTR, according to the manufacturer’s protocol. Briefly, 50 μl of TTR standards and 50 μl of test samples were added into antibody-precoated wells in the plate; then, 100 μl of 5% bovine serum albumin was added to each well. The wells were covered, incubated for 1 h at 37 °C, and washed five times using distilled water. Next, horseradish peroxidase (HRP)-coupled antibody was added to all the wells and covered and incubated for another 1 h at 37 °C. The washing step was repeated. Tetramethylbenzidine (TMB) substrate (50 μl of a 0.1% solution) was added to each well, covered, and incubated for 15 min at 25 °C. Finally, the reactions were stopped with 50 μl of 2M H₂SO₄ added to each well. The optical delnsity (OD) at 450 nm was detected for each sample within 30 min using a microtiter plate reader.

Wound healing assay

hRECs were allowed to grow until confluency, and wounds were created in the monolayer by scraping the plate with a pipette tip. Immediately afterward, 4 μM TTR was added to all four test environments: LG, LG+Hypoxic, HG, and HG+Hypoxic. Wounded monolayers were photographed immediately after wounding and 6, 12, 24, 32, and 36 h later. The relative wound density (RWD) was calculated using IncuCyte ZOOM (ESSEN BioScience, Ann Arbor, MI) software.

Tube formation assay

A Matrigel tube formation assay was performed as previously reported [29]. The culture plates were coated with 300 μl of growth factor-reduced Matrigel per well and then allowed to polymerize for 30 min at 37 °C. hRECs cultured for 24 h in natural and abnormal environments with or without TTR were seeded on coated plates at a density of approximately 2×10⁵ cells per well and then incubated for 24 h at 37 °C. Images were obtained at 40X magnification with an inverted phase-contrast microscope (Olympus IX70, Toyko, Japan). The total tube length was measured using the NIH Image program (National Institutes of Health, Bethesda, MD).

qRT-PCR

Total RNA was isolated from hRECs cultured for 48 h with 4 μM TTR proteins in natural and DR environments using the TRIzol® Plus RNA Purification Kit. For each sample, 1 μg RNA was reverse transcribed into cDNA using the SuperScript® One-Step RT–PCR System. The mRNA levels of the following target genes including Tie2 (ID: 7010; OMIM: 600221), VEGFR1 (ID: 2321; OMIM: 165070), VEGFR2 (ID: 3791; OMIM: 191306), Angpt1 (ID: 284; OMIM: 601667), and Angpt2 (ID: 285; OMIM: 601922) were detected with quantitative real-time PCR (qRT-PCR) using Power SYBR® Green (Life Technologies) and Applied Biosystems 7500 Fast Real-time PCR, following the manufacturer’s protocol.

GAPDH was used as the housekeeping gene. Information about the primers is listed in Table 2. The RT–PCR program comprised an initial denaturing step at 95 °C for 5 min, followed by 45 cycles of 95 °C for 20 s, 60 °C for 20 s, and 70 °C for 20 s. Relative quantification was performed according to the comparative method (2^-△△CT; Applied Biosystems User Bulletin 2P/N 4303859).

Statistical analysis

SPSS 13.0 for Windows (Chicago, IL) was used for data analysis, and statistical significance was determined using the one-way ANOVA test. P and F values were calculated. A p value of less than 0.05 was considered statistically significant.

hREC proliferation in DR environment

When 80–90% confluent, hRECs-P3 were washed twice in PBS and then treated with 0.05% trypsin for 2–3 min. Trypsin was neutralized after low-glucose (5.5 mM) DMEM medium was added. The cell density in the low-glucose DMEM medium plates was 3×10³ cells/well; after overnight incubation, the attached pericytes were washed twice and incubated with 200 μl fresh DMEM, for all the following conditions: low glucose (LG), low glucose and hypoxia (LG+Hypoxia), high glucose (HG), and high glucose and hypoxia (HG+Hypoxia; Table 1). These cells were cultured for 72 h, and each plate was scanned using xCELLigence RTCA MP.

After 72 h, the proliferation of hRECs in natural medium (LG) was much higher than that of the cells in the simulated high-glucose medium (HG; #: p = 0.003, F = 22.970) and the simulated hypoxia medium (*: p = 0.01; F = 13.962). In addition, in the simulated HG and hypoxia media (HG+Hypoxia), the growth of hRECs was further repressed (**: p < 0.001, F = 146.184; Figure 1A).

TTR affects the proliferation of hRECs

The hRECs was trypsinized for 2–3 min, followed by neutralization with low-glucose DMEM. Cells were harvested, spun down, washed three times with natural medium, and plated at a density of 3×10³ cells/well. After overnight incubation, the attached pericytes were washed twice and cultured with 200 μl fresh DMEM under the following conditions: LG, LG+Hypoxia, HG, and HG+Hypoxia, with or without 4 μM additional TTR (Table 1).

After 72 h, the growth of the hRECs was significantly decreased in all high-glucose cultures, including HG and HG+Hypoxia, by the 4 μM exogenous TTR (**: p < 0.001, F = 188.278; ##: p = 0.003; F = 24.106). However, in all low-glucose cultures, including LG and LG+Hypoxia, the cell proliferation was slightly enhanced by TTR (*: p = 0.014, F = 11.877; #: p = 0.034; F=7.158). This revealed that TTR significantly inhibited the growth of hRECs in the DR environment but only under hyperglycemic conditions (Figure 1B).

Overexpression of TTR in hRECs

Total mRNA was extracted from hRPECs, and cDNA of human TTR was amplified from the total cDNA products; then, it was inserted into the pCDNA3.1 plasmid through the EcoRI and BamHI sites. After the DNA sequence had been confirmed with sequencing, the plasmid was transfected into hRECs, using the empty pCDNA3.1 plasmid that contained no foreign sequence as the control. The cells were cultured in natural and abnormal media for 72 h, and the cell index was detected with xCELLigence RTCA MP.

The overexpression of TTR was confirmed with ELISA. The 24-, 48-, and 72-h cell cultures were harvested and lysed, and the TTR in the supernatants was detected. As shown in Figure 2A, the TTR content of the overexpressed cells (TTR-OE) was almost three times or higher than that in the cells containing an empty plasmid (Control; *: p < 0.001, F = 113.347 (24 h); p < 0.001, F = 55.084 (48 h); p < 0.001, F = 78.254 (72 h)).

The growth of hRECs was compared after culturing in natural and abnormal environments. As shown in Figure 2B, after 72 h, regardless of the hypoxic conditions, the TTR-OE significantly inhibited hREC proliferation only in the HG media (HG+Control, HG+TTR-OE, HG+Hypoxia+Control, and HG+Hypoxia+TTR-OE; **: p = 0.003, F = 21.997; ##: p < 0.001, F = 105.887). In contrast, in the low-glucose media (LG+Control, LG+TTR-OE, LG+Hypoxia+Control, and LG+Hypoxia+TTR-OE), TTR-OE enhanced the growth of hRECs only slightly (*: p = 0.002, F = 27.308; #: p = 0.002, F = 22.291). This finding was consistent with the results of cells cultured with 4 μM exogenous TTR.

TTR affects the migration of hRECs

The hRECs were seeded in 96-well plates and grown to confluence, wounds were created by scraping with a pipette tip, and the plates were transferred to the IncuCyte after the medium had been replaced with fresh media (natural and simulated DR environments) with or without 4 μM TTR. The plates were incubated in the IncuCyte for 36 h, and an integrated metric denoted as the RWD was used to quantify the effects on cell migration. This metric measured the cell density in the wound area relative to the cell density outside the wound area. Multiple regions (n = 3) for each treatment performed in triplicate were recorded, and the means of each treatment were calculated.

As shown in Figure 3A, the wound-healing efficiencies of the hRECs were quite different from each other after 36 h of culture. Using the RWD to reveal the effects of TTR on the migration of the cells (Figure 3B), in the natural environment (LG and LG+TTR), TTR slightly enhanced the healing efficiency (*: p = 0.002, F = 29.378). However, in the low-glucose and hypoxia environment (LG+Hypoxia and LG+Hypoxia+TTR), although proliferation was slightly enhanced, the migration of hRECs was inhibited (#: p = 0.034, F = 7.476). In the high-glucose media, TTR significantly reduced the wound healing process, regardless of hypoxia (HG and HG+Hypoxia), (**: p < 0.001, F = 77.118 and ##: p = 0.005, F = 18.206).

TTR affects the tube formation process of hRECs

To study the effects of TTR on the NV formation in the DR environment, a Matrigel tube formation assay was performed. hRECs cultured for 24 h in natural and simulated DR environments with or without TTR were seeded on coated plates at a density of approximately 2×10⁵ cells/well and then incubated for 24 h at 37 °C. Images were obtained and scanned with the NIH Image program (National Institutes of Health, Bethesda, MD), and then the total tube length was calculated with the same software to quantify the tube formation process.

TTR affected the tube formation processes of the hRECs in natural and abnormal media (Figure 4A). In addition, as calculated by the NIH Image program (Image J; Figure 4B), in the natural medium (LG and LG+TTR), the tube length was slightly increased by more than 20% with 4 μM TTR (*: p = 0.009, F = 22.311). In the natural medium under hypoxia (LG+Hypoxia and LG+Hypoxia+TTR), the tube length was approximately 25% greater with TTR (#: p < 0.001, F = 465.592). Conversely, in the HG media without hypoxia (HG and HG+TTR), the tube length was reduced by nearly 25% with TTR (**: p < 0.001, F = 933.495). Furthermore, in the simulated DR environment with HG and hypoxia (HG+Hypoxia and HG+Hypoxia+TTR), the tube formation process of the hRECs was significantly decreased by TTR (about 55%; ##: p < 0.001, F = 364.605). This result suggested that the tube formation process was repressed by TTR only in HG environments.

qRT-PCR analysis

To evaluate the effect of TTR on the proliferation, migration, and tube formation processes of hRECs in the simulated DR environment, the mRNA levels of several key genes for NV development in DR were detected. The results showed that TTR-mediated repression of hRECs required hyperglycemic conditions, while hypoxia was not a necessary factor; therefore, the genes directly associated with hypoxia-inducible factor-1 (HIF-1) were not selected. Instead, the expression of certain Tie2 pathway genes, such as Tie2, VEGFR1, VEGFR2, Angpt1, and Angpt2, were determined in natural and simulated DR environments.

After incubation with 4 μM TTR, the levels of these key genes were regulated. As shown in Figure 5, in natural medium (LG), the VEGFR1 and VEGFR2 levels were upregulated to 1.2-fold, Tie2 to 1.8-fold, and Angpt2 to 2.7-fold. The Angpt1 levels remained unchanged. In the LG+Hypoxia medium, Tie2 was downregulated to 0.72-fold, VEGFR2 to 1.45-fold, Angpt1 to 2.98-fold, and Angpt2 to 1.88-fold. The results, however, were quite interesting when the hRECs were cultured in HG environments with TTR. In the HG medium, Tie2 was upregulated to 5.95-fold and Angpt1 to 5.30-fold, while Angpt2 was downregulated to 0.06-fold and VEGFR1 and VEGF2 to 0.86- and 0.63-fold, respectively. In the HG+Hypoxia medium, Tie2 was upregulated to 3.13-fold and Angpt1, to 4.67-fold, whereas Angpt2 was downregulated to 0.04-fold and VEGFR1 and VEGF2, to 0.09- and 0.02-fold, respectively (Figure 5). This suggested that in abnormal environments with TTR, especially in HG, the significant downregulation of VEGFR1, VEGF2, and Angpt2 and the upregulation of Tie2 and Angpt1 were directly associated with the repression phenomenon of TTR on hRECs.