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Repression
of retinal microvascular endothelial cells by transthyretin under simulated
diabetic retinopathy conditions
Jun Shao, Yong Yao
Department of Ophthalmology, Wuxi People’s Hospital Affiliated to
Nanjing Medical University, Wuxi 214023, Jiangsu Province, China
Correspondence to: Yong Yao. Department of Ophthalmology, Wuxi People’s Hospital Affiliated to Nanjing
Medical University, Qingyang Road 299, Wuxi 214023, Jiangsu Province, China. Pard1@126.com
Received: 2015-08-19 Accepted: 2016-04-07
Abstract
AIM: To investigate biological effects of
transthyretin (TTR) on the development of neovascularization under simulated
diabetic retinopathy (DR) condition associated with high glucose and hypoxia.
METHODS: Human retinal microvascular endothelial
cells (hRECs) were cultured in normal and simulated DR environments with high
glucose and hypoxia. The normal serum glucose concentration is approximately
5.5 mmol/L; thus, hyperglycemia was simulated with 25 mmol/L glucose, while hypoxia
was induced using 200 µmol/L CoCl2. The influence of TTR on hRECs
and human retinal pigment epithelial cells (hRPECs) was determined by
incubating the cells with 4 μmol/L TTR in normal and abnormal media. A
co-culture system was then employed to evaluate the effects of hRPECs on hRECs.
RESULTS: Decreased hRECs and hRPECs were observed
under abnormal conditions, including high-glucose and hypoxic media. In
addition, hRECs were significantly inhibited by 4 μmol/L exogenous TTR during
hyperglycemic culture. During co-culture, hRPECs inhibited hRECs in both the
normal and abnormal environments.
CONCLUSION:
hREC growth is inhibited
by exogenous TTR under simulated DR environments with high-glucose and hypoxic,
particularly in the medium containing 25 mmol/L glucose. hRPECs, which
manufacture TTR in the eye, also represses hRECs in the same environment. TTR
is predicted to inhibit the proliferation of hRECs and neovascularization.
KEYWORDS: transthyretin; diabetic
retinal; human retinal microvascular endothelial cells; human retinal pigment epithelial cells;
hyperglycemia; hypoxia
DOI:10.18240/ijo.2016.06.03
Citation: Shao J, Yao Y. Repression of retinal
microvascular endothelial cells by transthyretin under simulated diabetic
retinopathy conditions. Int
J Ophthalmol 2016;9(6):809-815
INTRODUCTION
Diabetes is a chronic disease characterized
by hyperglycemia and diabetic retinopathy (DR). DR is considered to be the most
frequent microvascular complication of diabetes and is a common cause of
moderate and severe vision loss[1-2]. In
diabetes, retinal abnormalities clinically characterized by microaneurysms,
hemorrhages, lipid exudates, macular edema, capillary occlusion, cotton-wool
spots, and neovascularization (NV) can be identified as DR[3-4].
The development of DR is directly associated with the duration of diabetes and
severity of hyperglycemia. As previously reported, ischemic retinal hypoxia
playsavital role in the molecular pathogenesis of retinal NV, and the
expression of a subunit of hypoxia-inducible factor (HIF)-1, HIF-1-α, can be
significantly increased[5-6]. In a
mouse model of ischemic retinopathy, excess vascular endothelial growth factor
(VEGF) has been related to an increased HIF-1-α level[7].
Other HIF-1-regulated genes and products, including placental growth factor,
platelet-derived growth factor-B, and stromal-derived growth factor (SDF)-1,
have also been reported. Placental growth factor is a member of the VEGF gene
family and binds with VEGF receptor-1 to recruit bone marrow-derived cells.
These factors could stimulate the development of retinal NV[8].
In transgenic mice, retina-specific expression of platelet-derived growth
factor-B has been shown to lead to severe NV and retinal detachment[9-10]. CXCR4 is the receptor for SDF-1
and reduces retinal NV in ischemic retinas; however, CXCR4 is inhibited when
SDF-1 is overexpressed, thus promoting retinal NV [11].
Transthyretin (TTR) is a 55 kDa
homotetramer that is found in serum and cerebrospinal fluid. In the eye, TTR is
synthesized and secreted by human retinal pigment epithelial cells (hRPECs)[12]. It has also been identified as a
fluid carrier for thyroxine and retinol [13-14].
According to previous reports, TTR mutants can down-regulate pro-angiogenic
genes in human umbilical vein endothelial cells, thereby inducing apoptosis and
inhibiting migration[15].
Individuals with type 1 diabetes show low serum TTR levels, and the protein is
found mainly as a monomer; whereas in type 2 diabetes, TTR is detected at
normal concentrations[16].
Significantly increased TTR serum levels have been detected in patients with
severe myopia compared with healthy controls[17];
higher proportions of misfolded TTR with abnormal secondary structures were
also found in the vitreous of said patients, causing TTR to lose their natural
bio-functions[18-19].
However, the effects of TTR on human retinal microvascular endothelial cells
(hRECs) and on the development of NV in DR remain unclear.
In this study, hRECs were cultured with
exogenous TTR or with expressing cells (hRPECs) to evaluate the effects of TTR
on the growth of hRECs under normal and simulated DR conditions.
MATERIALS
AND METHODS
Materials This study applied the principles
of the Declaration of Helsinki for the use of human subjects and was approved
by the Ethics Committee of Nanjing Medical University.
hRECs and hRPECs were purchased from
Shanghai Bioleaf Biotech Co., Ltd. (China). Dulbecco’s modified Eagle’s medium
(DMEM), fetal bovine serum, and phosphate-buffered saline were obtained from
Life Technologies (USA). CoCl2 and glucose were purchased from
Sigma. Human TTR and a human TTR ELISA kit were supplied by Sino Biological
Inc. (China) and Abnova (USA), respectively. Other reagents and chemicals were
obtained from local companies and were of analytical grade or better.
Culture of Human Retinal Microvascular
Endothelial Cells and Human Retinal Pigment Epithelial Cells Under Normal and
Simulated Diabetic Retinopathy Conditions
hRECs
and hRPECs from passage 3 were used in the following experiments. The cells
were cultured under normal and simulated DR conditions as described previously [20-22]. Media were prepared as
shown in Table 1. Under normal culture conditions, cells were grown at 37℃
using a Series 8000 WJ cell incubator (Thermo Scientific, USA) in DMEM
containing 5.5 mmol/L glucose and 10% fetal bovine serum. A total of 25 mmol/L
glucose was used in DMEM to simulate DR[20], and hypoxia was
induced using 200 μmol/L CoCl2 [21-22]. Cells were cultured
for 72h, and the cell index was detected using the xCELLigence real-time cell
analysis (RTCA) MP system (Roche). The xCELLigence RTCA system is an
established electronic cell sensor array. This system uses microelectronic
biosensor technology that is verified for real-time, label-free, dynamic, and
non-offensive monitoring of cellular features. The relative cell index was
automatically calculated and read based on the number, morphology, adhesion,
and size of cells.
Table 1 The medium for hRECs and hRPECs
|
Medium |
DMEM |
FBS (%) |
Glucose (mmol/L) |
CoCl2 (μmol/L) |
|
LG |
+ |
10 |
5.5 |
- |
|
LG+hypoxia |
+ |
10 |
5.5 |
200 |
|
HG |
+ |
10 |
25 |
- |
|
HG+hypoxia |
+ |
10 |
25 |
200 |
LG: Low-glucose; LG+hypoxia: Low-glucose with
hypoxia; HG: high-glucose; HG+hypoxia: High-glucose with hypoxia; DMEM: Dulbecco’s
modified Eagle’s medium; FBS: Fetal bovine serum.
Effects of Transthyretin on the Growth of
Human Retinal Microvascular Endothelial Cells and Human Retinal Pigment
Epithelial Cells TTR mutants can inhibit
the growth of human umbilical vein endothelial cells in liver disease by
regulating pro-angiogenic genes. hRECs and hRPECs from passage 6 were cultured
under normal and abnormal conditions, and approximately 4 μmol/L TTR was
thenadded to the mediain accordance with a previously described protocol [15]. Media were prepared as
shown in Table 2. The cells were grown for 72h, and the cell index was detected
usingthe xCELLigence RTCA MP system.
Co-culture of Human Retinal Microvascular
Endothelial Cells and Human Retinal Pigment Epithelial Cells hRECs and hRPECs from passage 9
were used in the Transwell co-culture system. TTR has been shown to be
synthesized and secreted by hRPECs in the eye[12]. The expression of TTR
in hRPECs was detected using a human TTR ELISA kit (Abnova). hRPECs were
cultured in normal low-glucose (LG) medium for 24, 48 and 72h. After this,
hRPECs were collected, lysed in radio immunoprecipitation assay lysis buffer
containing 1 mmol/L) phenylmethylsulfonyl fluoride (PMSF), incubated in
ice-water for 10min, and centrifuged at 10000 g for 5min. Supernatants were
applied to the ELISA in accordance with the manufacturer’s instructions.
A transwell co-culture was subsequently
performed, with hRPECs used as the source of TTR. About 50 μL of hRECs
(approximately 8000 cells/50 μL) in DMEM was added to an E-plate. About 50 μL
of hRPECs (approximately 2000 cells/50 μL) in DMEM was also added to the
inserts in the CCD receiver plate. After overnight growth at 37℃, the inserts
were placed into the E-plates. The cells were then co-cultured in high-glucose
(HG) and HG plus hypoxia (HG+hypoxia) media (Table 2) for 48h. The cell index
was determined using the xCELLigence RTCA MP system.
Table
2 The medium containing TTR for hRECs and hRPECs
|
Medium |
DMEM |
FBS (%) |
Glucose (mmol/L) |
CoCl2 (μmol/L) |
TTR (μmol/L) |
|
LG |
+ |
10 |
5.5 |
- |
- |
|
LG+TTR |
+ |
10 |
5.5 |
- |
4 |
|
LG+hypoxia |
+ |
10 |
5.5 |
200 |
- |
|
LG+hypoxia+TTR |
+ |
10 |
5.5 |
200 |
4 |
|
HG |
+ |
10 |
25 |
- |
- |
|
HG+TTR |
+ |
10 |
25 |
- |
4 |
|
HG+hypoxia |
+ |
10 |
25 |
200 |
- |
|
HG+hypoxia+TTR |
+ |
10 |
25 |
200 |
4 |
LG: Low-glucose; LG+TTR: Low-glucose with TTR; LG+hypoxia: Low-glucose with hypoxia; LG+hypoxia+TTR:
Low-glucose with hypoxia and TTR; HG: High-glucose; HG+TTR: High-glucose with TTR;
HG+hypoxia: High-glucose with hypoxia; HG+hypoxia+TTR: High-glucose with hypoxia and TTR; DMEM: Dulbecco’s modified Eagle’s medium; FBS: Fetal bovine serum; TTR: Transthyretin.
Statistical Analysis Origin 9.1 software (OriginLab, USA) was
used in data processing, and statistical significance was determined using the t-test and analysis of variance. P<0.05 was considered statistically
significant.
RESULTS
Growth of Human Retinal Microvascular Endothelial
Cells and Human Retinal Pigment Epithelial Cells hRECs-P3 and hRPECs-P3 were grown
to approximately 80%-90% confluence. These cells were then washed twice with
phosphate-buffered salineand treated with 0.05% trypsin for 2-3min. Trypsin was
neutralized after adding LG DMEM. A total of 3000 cells per well were cultured
on plates in LG medium after an overnight incubation. The attached pericytes
were washed twice and incubated with 200 μL fresh DMEM, including LG, LG with
hypoxia (LG+hypoxia), HG, and HG+hypoxia media (Table 1). The cells were
cultured for 72h, and the plates were scanned using the xCELLigence RTCA MP
system for 5min each. The relative cell index was automatically calculated and
read based on the number, morphology, adhesion, and size of cells.
As shown in Figure 1 and Table 3, the growth of hRECs was much higher in LG
than in LG+hypoxia or HG media (P<0.05). The growth of hRECs was further
decreased in HG+hypoxia medium (P<0.05).
Figure 1 The growth of hRECs under natural and
abnormal conditions hRECs were grown under natural and
simulated DR conditions. LG: Low-glucose;
LG+hypoxia: Low-glucose with hypoxia; HG:
High-glucose; HG+hypoxia: High-glucose with hypoxia. aLG vs
LG+hypoxia, cLG vs HG, eHG
vs HG+hypoxia. High glucose level (25
mmol/L) and hypoxia induced with 200 μmol/L CoCl2 could significantly
repressed the growth of hRECs (aP＜0.05, cP＜0.05, eP＜0.05).
Table 3 The index of hRECs under natural
and abnormal conditions
|
Parameters |
4h |
8h |
16h |
24h |
36h |
48h |
60h |
72h |
|
LG |
1.835±0.061 |
2.222±0.107 |
3.284±0.181 |
4.834±0.272 |
8.206±0.739 |
11.406±1.341 |
13.057±1.581 |
14.299±1.804 |
|
LG+hypoxia |
1.506±0.059 |
1.658±0.129 |
2.261±0.194 |
3.074±0.206 |
4.610±0.361 |
7.040±0.635 |
9.135±0.867 |
9.341±0.748 |
|
HG |
1.480±0.071 |
1.816±0.835 |
2.829±0.136 |
3.977±0.181 |
5.269±0.155 |
6.220±0.172 |
7.183±0.273 |
8.281±0.349 |
|
HG+hypoxia |
1.305±0.065 |
1.546±0.780 |
2.021±0.058 |
2.622±0.366 |
3.377±0.075 |
4.246±0.135 |
4.866±0.171 |
5.096±0.193 |
|
aP |
0.008 |
0.018 |
0.007 |
0.003 |
0.000 |
0.000 |
0.001 |
0.000 |
|
cP |
0.008 |
0.037 |
0.147 |
0.079 |
0.000 |
0.000 |
0.000 |
0.000 |
|
eP |
0.162 |
0.059 |
0.003 |
0.005 |
0.000 |
0.017 |
0.020 |
0.006 |
aLG vs
LG+hypoxia; cLG vs HG; eHG
vs HG+hypoxia.
In the hRPEC culture
(Figure 2 and Table 4), the growth of hRPECs was also higher in LG than in LG+hypoxia or HG media
during almost the whole process (P<0.05). However, the effect of the
HG+hypoxia medium was not significant (P>0.05).
Figure 2 The growth of hRPECs under natural and
abnormal conditions hRPECs were cultured under natural and
simulated DR conditions. LG: Low-glucose; LG+hypoxia: Low-glucose with
hypoxia; HG: High-glucose; HG+hypoxia:
High-glucose with hypoxia. aLG vs
LG+hypoxia, cLG vs HG, dHG vs HG+hypoxia. The growth of hRECs in LG
was higher than that in HG or LG+hypoxia medium during almost the whole process
(a,cP<0.05). But the effect of hypoxia in HG medium was not
significant (dP＞0.05).
Table 4 The index of hRPECs under natural and abnormal
conditions
|
Parameters |
4h |
8h |
16h |
24h |
36h |
48h |
60h |
72h |
|
LG |
1.086±0.011 |
1.332±0.008 |
2.013±0.400 |
3.451±0.132 |
6.875±0.353 |
11.312±0.772 |
14.381±0.938 |
15.065±1.118 |
|
LG+hypoxia |
1.304±0.009 |
1.250±0.020 |
1.734±0.494 |
2.306±0.113 |
3.770±0.391 |
6.080±0.807 |
7.985±1.018 |
8.872±0.903 |
|
HG |
1.099±0.048 |
1.178±0.069 |
1.427±0.116 |
2.167±0.248 |
3.693±0.520 |
5.998±1.131 |
9.497±1.683 |
12.946±1.916 |
|
HG+hypoxia |
1.380±0.062 |
1.417±0.061 |
1.440±0.491 |
1.857±0.076 |
2.581±0.190 |
3.552±0.294 |
4.776±0.420 |
5.911±0.431 |
|
aP |
0.000 |
0.035 |
0.005 |
0.000 |
0.000 |
0.002 |
0.002 |
0.004 |
|
cP |
0.752 |
0.204 |
0.013 |
0.011 |
0.004 |
0.007 |
0.071 |
0.771 |
|
dP |
0.000 |
0.048 |
1.000 |
0.669 |
0.244 |
0.190 |
0.107 |
0.059 |
aLG vs LG+hypoxia; cLG vs
HG; dHG vs HG+hypoxia.
Effects of Transthyretin on Cell
Growth hRECs-P6 and hRPECs-P6 cells were
trypsinized for 2-3min, and LG DMEM was added to neutralize trypsin. The cells
were harvested, spun down, and washed several times with the medium, and
3000/well cells were cultured on plates. After an overnight incubation, the attached
pericytes were washed twice and cultured with 200 μL of LG, LG+hypoxia, HG, and
HG+hypoxia media. Human TTR (4 μmol/L) was also added to the media (Table 2).
The relative cell index was read using the xCELLigence RTCA MP system for 5min
each.
In all HG cultures (Figure 3B, Table 5),
including those under the CoCl2-induced hypoxic conditions, TTR
significantly decreased the growth of hRECs (P<0.05). By contrast, in all LG cultures (Figure 3A, Table 6),
including those in LG and LG+hypoxia media, TTR slightly enhanced the growth of
hRECs (P<0.05).
Figure 3 The effect of TTR on the growth of hRECs hRECs were cultured under natural and simulated DR
conditions with 4 μmol/L TTR. LG: Low-glucose;
LG+TTR: Low-glucose with TTR; LG+hypoxia: Low-glucose with hypoxia; LG+hypoxia+TTR: Low-glucose with hypoxia
and TTR; HG: High-glucose; HG+TTR: High-glucose
with TTR; HG+hypoxia: High-glucose with hypoxia; HG+hypoxia+TTR: High-glucose with hypoxia
and TTR. aLG vs LG+TTR, cLG+hypoxia vs LG+hypoxia+TTR, eHG vs HG+TTR, gHG+hypoxia vs HG+hypoxia+TTR. A: In low glucose media, TTR slightly enhanced the growth of hRECs (a,cP＜0.05); B: Only in high
glucose media, could TTR inhibit the growth of hRECs (e,gP＜0.05).
Table 5 The index of hPECs under high glucose
conditions with TTR
|
4h |
8h |
16h |
24h |
36h |
48h |
72h |
|
|
HG |
1.047±0.022 |
1.134±0.023 |
1.454±0.034 |
1.874±0.035 |
2.576±0.048 |
3.217±0.090 |
3.513±0.145 |
|
HG+TTR |
1.119±0.008 |
1.255±0.042 |
1.546±0.112 |
1.795±0.152 |
1.950±0.134 |
2.058±0.129 |
1.974±0.097 |
|
HG+hypoxia |
1.107±0.021 |
1.143±0.034 |
1.362±0.083 |
1.477±0.129 |
1.595±0.197 |
1.635±0.263 |
1.391±0.275 |
|
HG+hypoxia+TTR |
1.181±0.025 |
0.948±0.019 |
0.836±0.022 |
0.832±0.023 |
0.781±0.039 |
0.744±0.043 |
0.488±0.056 |
|
eP |
0.039 |
0.008 |
0.478 |
0.731 |
0.056 |
0.007 |
0.002 |
|
gP |
0.030 |
0.030 |
0.000 |
0.009 |
0.015 |
0.031 |
0.001 |
eHG vs HG+TTR; gHG+hypoxia vs HG+hypoxia+TTR.
Table 6 The index of hPECs under low glucose
conditions with TTR
|
Parameters |
4h |
8h |
16h |
24h |
36h |
48h |
72h |
|
LG |
1.129±0.025 |
1.382±0.088 |
2.202±0.164 |
3.347±0.275 |
4.785±0.459 |
6.055±0.387 |
6.975±0.249 |
|
LG+TTR |
1.160±0.015 |
1.589±0.050 |
3.137±0.220 |
5.133±0.497 |
7.261±0.532 |
8.300±0.362 |
8.020±0.324 |
|
LG+hypoxia |
1.189±0.018 |
1.511±0.041 |
2.343±0.105 |
3.198±0.121 |
4.445±0.281 |
5.572±0.364 |
7.167±0.373 |
|
LG+hypoxia+TTR |
1.107±0.018 |
1.233±0.020 |
2.007±0.570 |
3.236±0.192 |
5.586±0.230 |
7.141±0.209 |
8.317±0.393 |
|
aP |
0.260 |
0.000 |
0.000 |
0.000 |
0.000 |
0.000 |
0.000 |
|
cP |
0.014 |
0.009 |
0.010 |
0.871 |
0.001 |
0.001 |
0.002 |
aLG vs LG+TTR; cLG+hypoxia vs LG+hypoxia+TTR.
TTR slightly promoted hRPEC cell growth
under normal and simulated DR conditions (Figure 4), but the differences were
not always significant (Tables 7, 8).
Figure 4 The effect of TTR on the growth of hRPECs hRPECs were cultured under natural and simulated DR
conditions with 4 μmol/L TTR. LG: Low-glucose;
LG+TTR: Low-glucose with TTR; LG+hypoxia: Low-glucose with hypoxia; LG+hypoxia+TTR: Low-glucose with hypoxia
and TTR; HG: High-glucose; HG+TTR: High-glucose
with TTR; HG+hypoxia: High-glucose with hypoxia; HG+hypoxia+TTR: High-glucose with hypoxia
and TTR. aLG vs LG+TTR, bLG+hypoxia vs LG+hypoxia+TTR, cHG vs HG+TTR, dHG+hypoxia vs HG+hypoxia+TTR. TTR slightly enhanced
the growth of hRPECs under all culture conditions, but the differences were not
always significant (a, b, c, d).
Table 7 The index of hRPECs under low glucose
conditions with TTR
|
Parameters |
4h |
8h |
16h |
24h |
36h |
48h |
72h |
|
LG |
1.094±0.020 |
1.171±0.039 |
1.347±0.081 |
1.539±0.123 |
1.871±0.262 |
2.331±0.481 |
3.990±1.317 |
|
LG+TTR |
1.178±0.018 |
1.390±0.046 |
1.687±0.100 |
1.968±0.140 |
2.353±0.180 |
2.858±0.244 |
4.751±0.596 |
|
LG+hypoxia |
1.022±0.011 |
1.056±0.012 |
1.182±0.020 |
1.344±0.023 |
1.651±0.097 |
2.103±0.251 |
2.674±0.439 |
|
LG+hypoxia+TTR |
1.115±0.013 |
1.253±0.034 |
1.555±0.080 |
1.873±0.106 |
2.224±0.167 |
2.512±0.244 |
2.944±0.365 |
|
aP |
0.009 |
0.001 |
0.011 |
0.043 |
0.21 |
0.38 |
0.621 |
|
bP |
0.039 |
0.049 |
0.049 |
0.053 |
0.068 |
0.272 |
0.598 |
aLG vs LG+TTR; bLG+hypoxia vs LG+hypoxia+TTR.
Table 8 The
index of hRPECs under high glucose conditions with TTR
|
Parameters |
4h |
8h |
16h |
24h |
36h |
48h |
72h |
|
HG |
1.045±0.016 |
1.084±0.015 |
1.173±0.018 |
1.290±0.013 |
1.513±0.054 |
1.822±0.120 |
2.831±0.294 |
|
HG+TTR |
1.216±0.017 |
1.407±0.030 |
1.702±0.055 |
1.949±.0.063 |
2.199±0.058 |
2.420±0.066 |
3.182±0.085 |
|
HG+hypoxia |
1.031±0.017 |
1.073±0.021 |
1.162±0.032 |
1.264±0.062 |
1.456±0.111 |
1.595±0.156 |
1.407±0.231 |
|
HG+hypoxia+TTR |
1.139±0.019 |
1.229±0.049 |
1.376±0.100 |
1.503±0.147 |
1.642±0.193 |
1.697±0.220 |
1.517±0.190 |
|
cP |
0.002 |
0.002 |
0.004 |
0.007 |
0.016 |
0.055 |
0.342 |
|
dP |
0.001 |
0.005 |
0.021 |
0.038 |
0.096 |
0.297 |
0.445 |
cHG vs HG+TTR; dHG+hypoxia vs HG+hypoxia+TTR.
Co-culture of Human Retinal Microvascular Endothelial
Cells and Human Retinal Pigment Epithelial Cells In the eye, TTR is expressed by the
retinal pigment epithelium. In this study, hRPECs were therefore employed as
the TTR producer in the Transwell co-culture system. TTR expression in
hRPECs-P9 was detected by ELISA. As shown in Figure 5, TTR expression in the
cells reached its peak at around 48h.
Figure 5 The expression of TTR in hRPECs The TTR contents in hRECs were detected by ELISA assay
after the cells were cultured in natural medium for 24, 48 and 72h,
respectively. It revealed that the expression of TTR reached the peak at
approximately 48h and was then remained quite stable.
hRECs-P9 and hRPECs-P9 were washed and trypsinized
when 80%-90% confluency was reached. Trypsin was then neutralized using the LG
medium. Subsequently, 8000 hRECs and 2000 hRPECs in the LG medium were added to
the E-plate and inserts of the CCD receiver plate, respectively. After an
overnight incubation at 37℃, the inserts were placed into the E-plates, and the
two cell types were co-cultured in normal, HG, LG+hypoxia, and HG+hypoxia media
(Table 1). The relative cell index was read using the xCELLigence RTCA MP
system.
In co-culture, hRECs cultured alone were
used asthe control. As shown in Figure 6, Tables 9, 10, hRECs growth was
significantly inhibited by the co-cultured hRPECs under normal and simulated DR
conditions (P<0.05).
Figure 6 The effect of hRPECs on hRECs in co-culture
system In the trans-well co-culture system, hRECs was
cultured with hRPECs under both natural and abnormal DR conditions. hREC-LG:
hRECs in low glucose; hREC&hRPEC-LG: hRECs co-cultured with hRPECs in low
glucose; hREC-LG+hypoxia: hRECs in low glucose and hypoxia; hREC&hRPEC-LG+hypoxia:
hRECs co-cultured with hRPECs in low glucose and hypoxia; hREC-HG: hRECs in
high glucose; hREC&hRPEC-HG: hRECs co-cultured with hRPECs in high glucose;
hREC-HG+hypoxia: hRECs in high glucose and hypoxia; hREC&hRPEC-HG+hypoxia:
hRECs co-cultured with hRPECs in high glucose and hypoxia. ahREC-LG vs hREC&hRPEC-LG, chREC-LG+hypoxia
vs hREC&hRPEC-LG+hypoxia, eHREC-HG
vs hREC&hRPEC-HG, ghREC-LG+hypoxia
vs hREC&hRPEC-HG+hypoxia. hRPECs
significantly repressed the growth of hRECs in low glucose (A) and high glucose
(B) media (a,c,e,gP＜0.05).
Table 9 The index of hPECs under low glucose
conditions with hRPECs
|
Parameters |
4h |
8h |
16h |
24h |
36h |
48h |
|
HREC-LG |
1.932±0.082 |
2.513±0.112 |
3.444±0.126 |
4.002±0.169 |
4.623±0.347 |
5.085±0.446 |
|
HREC&HRPEC-LG |
1.409±0.052 |
1.852±0.085 |
2.483±0.222 |
2.946±0.239 |
3.126±0.184 |
3.228±0.186 |
|
HREC-LG+Hypoxia |
1.764±0.052 |
2.230±0.059 |
3.385±0.017 |
3.961±0.136 |
3.977±0.286 |
4.055±0.306 |
|
HREC&HRPEC-LG+hypoxia |
1.270±0.016 |
1.304±0.016 |
1.589±0.047 |
1.867±0.090 |
1.856±0.207 |
1.952±0.216 |
|
aP |
0.000 |
0.019 |
0.038 |
0.049 |
0.026 |
0.045 |
|
cP |
0.000 |
0.034 |
0.000 |
0.006 |
0.028 |
0.038 |
ahREC-LG
vs hREC&hRPEC-LG; chREC-LG+hypoxia
vs hREC&hRPEC-LG+hypoxia.
Table 10 The index of hPECs under high glucose
conditions with hRPECs
|
Parameters |
4h |
8h |
16h |
24h |
36h |
48h |
|
HREC-HG |
1.775±0.123 |
2.206±0.094 |
2.604±0.256 |
2.910±0.219 |
3.247±0.375 |
3.614±0.398 |
|
HREC&HRPEC-HG |
1.341±0.049 |
1.655±0.074 |
1.710±0.107 |
1.877±0.160 |
2.013±0.230 |
2.069±0.262 |
|
HREC-HG+hypoxia |
1.669±0.040 |
2.010±0.061 |
2.471±0.148 |
2.463±0.220 |
2.306±0.312 |
2.141±0.262 |
|
HREC&HRPEC-HG+hypoxia |
1.181±0.085 |
1.186±0.092 |
1.198±0.090 |
1.265±0.110 |
1.247±0.109 |
1.268±0.114 |
|
eP |
0.000 |
0.047 |
0.045 |
0.049 |
0.048 |
0.047 |
|
gP |
0.000 |
0.007 |
0.001 |
0.048 |
0.042 |
0.046 |
eHREC-HG
vs hREC&hRPEC-HG; ghREC-LG+hypoxia
vs hREC&hRPEC-HG+hypoxia.
DISCUSSION
As previously reported, retinal NV is
closely associated with high levels of glucose in DR development, as well as
with ischemic retinal hypoxia. HIF-1-α is induced by hypoxia and plays a vital
role in the development of NV[5-6] by
regulating other significant factors for vascular endothelial growth,
angiogenesis, and apoptosis[7-11]. NV in
DR has been studied for decades; however, TTR has seldom been described in this
field. TTR has recently been reported to affect the growth, migration, and
apoptosis of human umbilical vein endothelial cells by regulating the
expression of protein factors in NV; however, this study was only performed
under normal conditions[15].
The effect of TTR on the development of NV
in DR environments remains unclear. In the current study, DR environments were
simulated, and exogenous TTR and hRPECs (the producer of TTR in the eye) were
employed to affect hREC growth. In HG and HG+hypoxia media, hREC growth was
inhibited by 4 μmol/L exogenous TTR. Incontrast, hREC growth was slightly
increased by TTR in LG and LG+hypoxia media. These resultssuggest that in the
process of DR, TTR affects the development of NV only under hyperglycemic
conditions, and hypoxia does not induce the bio-function of TTR. In addition,
in co-culture, hRPECs inhibited the growth of hRECs in both HG and LG media.
TTR was secreted by hRPECs in the eye, and ELISA showed that the expression was
stable for 726h; thus, hREC repression under LG conditions could be attributed
to some unknown mechanisms. Therefore, quantitative reverse transcription
polymerase chain reaction (qRT-PCR) should be employed in further
investigations to determine the levels of key genes in DR NV and reveal the
as-yet unknown mechanisms behind this condition.
In several clinical investigations into
severe myopia, individuals with diabetes and myopia have been found to be less
likely than those without myopia to have DR [23-24].
Higher concentrations of TTR were detected in the serum and vitreous of
individuals with severe myopia, and the presence of some abnormal TTRs with
misfolded structures was confirmed. Lower TTR concentrations havealso been
detected in the vitreous of patients with DR[17-19].
These findings agree with the experimental results ofthe current study,
potentially revealing that diabetic patients with myopia are less likely suffer
from DR.
The results of the present study suggest
that TTR might repress hREC growth and that HG, but not hypoxia, is an
important factor. Further investigations are necessary to determine whether TTR
can affect migration and tube-forming processes in DR. qRT-PCR should be
employed in further studies to detect the levels of key genesin important
pathways for the development of NV in DR.
ACKNOWLEDGEMENTS
Foundation: Supported by the
National Natural Science Foundation of China (No. 81400415).
Conflicts of Interest: Shao J, None; Yao Y, None.
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