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In vivo bioluminescence imaging of
hyperglycemia exacerbating stem cells on choroidal neovascularization in mice
Xiang
Gao1, Yu Wang1, Hui-Yuan Hou1, Yang Lyu1,
Hai-Yan Wang1, Li-Bo Yao2, Jian Zhang2, Feng
Cao3, Yu-Sheng Wang1
1Department of
Ophthalmology, Xijing Hospital, Eye
Institute of Chinese PLA, Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China
2State
Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular
Biology, Fourth Military Medical University, Xi'an 710032, Shaanxi Province,
China
3Department
of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an
710032, Shaanxi Province, China
Co-first authors: Xiang
Gao and Yu Wang
Correspondence to:
Yu-Sheng Wang. Department of Ophthalmology, Xijing Hospital, Eye Institute of
Chinese PLA, Fourth Military Medical University, No.15 West Changle Road, Xi’an
710032, Shaanxi Province, China. wangys003@126.com. Feng Cao. Department of
Cardiology, Xijing Hospital, Fourth Military Medical University No.15 West
Changle Road, Xi’an 710032, Shaanxi Province, China. fengcao8828@163.com. Jian
Zhang. State Key Laboratory of Cancer Biology, Department of Biochemistry and
Molecular Biology, Fourth Military Medical University No.15 West Changle Road,
Xi’an 710032, Shaanxi Province, China. biozhangj@yahoo.com.cn
Received:
2015-10-08
Accepted: 2015-11-20
Abstract
AIM: To investigate
the influence of hyperglycemia on the severity of choroidal neovascularization
(CNV), especially the involvement of bone marrow-derived cells (BMCs) and
underlying mechanisms.
METHODS: BMCs from
firefly luciferase (Fluc)/green fluorescent protein (GFP) double transgenic
mice were transplanted into C57BL/6J wide-type mice. The recipient mice were
injected intraperitoneally with streptozotocin (STZ) daily for 5 consecutive
days to induce diabetes mellitus (DM), followed by CNV laser photocoagulation.
The BMCs recruitment in CNV exposed to hyperglycemia was firstly
examined in Fluc/GFP chimeric mice by in
vivo optical bioluminescence imaging (BLI) and in vitro Fluc assays. The CNV severity
was evaluated by H&E staining and choroidal flatmount. The expression of
vascular endothelial growth factor (VEGF) and stromal cell derived factor-1
(SDF-1) was detected by Western Blot.
RESULTS: BLI showed that
the BMCs exerted dynamic effects in CNV model in Fluc/GFP chimeric mice exposed
to hyperglycemia. The signal intensity of transplanted Fluc+GFP+
BMCs in the DM chimeric mice was significantly
higher than that in the control chimeric mice with CNV induction at days 5, 7,
14 and 21 (121861.67±9948.81 vs 144998.33±13787.13 photons/second/cm2/sr
for control and DM mice, P5d<0.05;
178791.67±30350.8 vs 240166.67±22605.3, P7d<0.05; 124176.67±16253.52 vs
196376.67±18556.79, P14d<0.05;
97951.60±10343.09 vs 119510.00±14383.76, P21d<0.05), which was consistent with in vitro
Fluc assay at day 7 [relative light units of Fluc (RLU1)], 215.00±52.05 vs
707.33±88.65, P<0.05; RLU1/
relative light units of renilla luciferase (RLU2), 0.90±0.17 vs
1.83±0.17, P<0.05]. The CNVs in
the DM mice were wider than those in the control group at days 5, 7, 14 and 21
(147.83±17.36 vs 220.33±20.17 μm,
P5d<0.05; 212.17±24.63 vs
326.83±19.49, P7d<0.05;
163.17±18.24 vs 265.17±20.55, P14d<0.05;
132.00±10.88 vs 205.33±12.98, P21d<0.05).
The average area of CNV in the DM group was larger at 7d (20688.67±3644.96 vs
32218.00±4132.69 μm2,
P<0.05). The expression of
VEGF and SDF-1 was enhanced in the DM mice.
CONCLUSION: Hyperglycemia
promots the vasculogenesis of CNV, especially the contribution of BMCs, which
might be triggered by VEGF and SDF-1 production.
KEYWORDS: hyperglycemia; choroidal
neovascularization; bone marrow-derived cells; molecular imaging; in
vivo optical bioluminescence imaging
DOI:10.18240/ijo.2016.04.07
Citation: Gao
X, Wang Y, Hou HY, Lyu Y, Wang HY, Yao LB, Zhang J, Cao F, Wang YS. In vivo bioluminescence
imaging of hyperglycemia exacerbating stem cells on choroidal
neovascularization in mice. Int J
Ophthalmol 2016;9(4):519-527
INTRODUCTION
Age-related
macular degeneration (AMD) is a common and devastating disease resulting in
irreversible visual loss[1]. The hallmark
of neovascular AMD is choroidal neovascularization (CNV), which is
characterized by naturally occurring new blood vessels in the choroid to grow
aberrantly into the subretinal space through breaking the RPE and Bruch’s
membrane, thus causing exudative or hemorrhagic retinal detachments[2]. The pathogenesis of CNV is clearly multifactorial,
involving both angiogenesis (development of new blood vessels from resident
adjacent preexisting capillaries) and postnatal vasculogenesis (the new vessel
complex derived from bone marrow-derived circulating vascular progenitors)[3-4]. Previous studies have shown that bone
marrow-derived cells (BMCs) are recruited into CNV by vascular endothelial
growth factor (VEGF), stromal cell derived factor (SDF)-1 and differentiated
into endothelial cells (ECs) and vascular smooth muscle cells (VSMCs),
incorporating into the new blood vessel wall and forming vascular tubes[5-7].
Diabetes
mellitus (DM), a condition characterized by micro- and macroangiopathy, is a
global health problem. Vascular complications in diabetes are major causes of
human morbidity and mortality, affecting multiple organs and persisting despite
tight glucose control[8].
Epidemiological studies have verified that diabetes is a risk factor for AMD[9-10]. Growing evidence has shown that diabetes
exacerbated the development of laser-induced CNV in mice but the underlying
mechanism for this enhancement remained unsolved[11]. Our
previous studies have demonstrated that hyperglycemia promoted the progression
of CNV in diabetic mice, by enhancing the expression of VEGF [induced by
oxidative stress and activation of signal transducer and activator of transcription
3 (STAT3)
signaling] and SDF-1 in RPE cells, promoting the recruitment and incorporation
of BMCs, and affecting the differentiation of BMCs in CNV[11-12]. By taking advantage of this recruitment potential,
a therapeutic strategy for CNV due to hyperglycemia has been described, using
BMCs as a delivery vehicle carrying antiangiogenic factors, thereby inhibiting
the growth of CNVs and stimulating regressive features[13]. However, it seemed contrary to the
findings in several studies revealing that diabetes led to multiple bone marrow
microenvironmental defects, such as impaired stem cell mobilization
(mobilopathy)[8,14]. Therefore, it
is necessary to explore a method real-time monitoring BMCs’ cellular kinetics
after their transplantation into diabetic mice with CNV model and ultimately to
verify our recent conclusion that hyperglycemia aggravated the severity of CNV
by recruiting more BMCs in CNV.
In
vivo
bioluminescence imaging (BLI) is now the most sensitive optical technique for
longitudinally tracking cell behavior in
vivo, based on detection of light emission (photoproteins and luciferases)
from cells or tissues[15]. This molecular
imaging modality has several advantages over traditional screening methods, not
least the ability to analyse ongoing biological processes and quantitatively
monitor pharmacodynamic changes at the cellular and molecular level in living
animals non-invasively in real time[16-18]. As the most
conventional technique for BLI, the firefly luciferin-luciferase system is
exceptionally functional in vitro and in vivo[19]. When the engineered stem cells labeled
with firefly luciferase (Fluc) are injected into the mice, BLI provides
valuable information about their dissemination and functional status using the
location and intensity of the light signal, which is released by Fluc-luciferin
reaction with limited loss or attenuation, resulting in consecutive images
obtained from the same mice, instead of point data from conventional approaches[20-21]. Our previous study suggested that the noninvasive
BLI analysis has been extensively used to provide detailed cellular and
molecular characterization of the CNV pathology and engrafted stem cells[17].
In
the present study, we firstly applied molecular reporter gene imaging
techniques in conjunction with conventional histological methods, to
dynamically verify the positive effect of hyperglycemia on the recruitment and
participation of BMCs in laser-induced CNV mice model. Further, we also
investigated the role of local VEGF and SDF-1 expression in engrafted BMCs in
CNV under high glucose.
MATERIALS AND METHODS
Animals, Diabetes Induction and Grouping
The animal
experiments were performed in accordance with the guidelines of the Association
for Research in Vision and Ophthalmology (ARVO) statement for the use of
animals in ophthalmic and vision research. The animal protocol was approved by
the Animal Care Committee of the Fourth Military Medical University. Eight
week-old female C57BL/6J mice, expressing Fluc and green fluorescent protein
(GFP) reporter genes induced by β-actin promoter (Department of Cardiology,
Xijing Hospital, Fourth Military Medical University, Xi’an, China) and age
matched female congenic wide-type (WT) mice (Experimental Animal Center, Fourth
Military Medical University, Xi’an, China) were used.
The
animals were randomly divided into five groups (n=6/group): 1) control
chimeric mice (normal); 2) diabetic chimeric mice (DM); 3) control chimeric
mice with CNV (normal+CNV); 4) diabetic chimeric mice with CNV (DM+CNV); 5)
control WT mice.
In
the normal and normal+CNV groups, mice were injected intraperitoneally with
sodium citrate buffer (0.05 mol/L, pH 4.5) only daily for five consecutive
days. In the DM and DM+CNV groups, mice were injected intraperitoneally with 60
mg/kg streptozotocin (STZ; Sigma Chemical, St. Louis, MO, USA) in sodium
citrate buffer (0.05 mol/L, pH 4.5) daily for 5 consecutive days to induce
diabetes. Seven days after the fifth injection, blood glucose levels from tail
vein were measured using a glucomonitor. Mice with glucose levels >300 mg/dL
were included in the study[22-23]. Three weeks after the fifth injection, all of mice
underwent laser-induction of CNV as described below.
Choroidal Neovascularization Model CNV was induced
by laser
photocoagulation as previously reported[24-25]. Briefly, mice were anesthetized, and their pupils
were dilated. Laser (532 nm wavelength, 75 mm spot size, 0.1s duration, and 90
mW intensity) was delivered into the eyes using a slit lamp and a cornea
contact lens. The laser burns were performed on the fundus 1.5-2 disc diameters
away from the optic nerve. Only laser spots where Bruch’s membrane was ruptured
(confirmed via the presence of a
vaporization bubble and the absence of hemorrhage) were considered effective
and included in the study.
Bone Marrow Transplantation The bone marrow
transplantation was conducted as previously described, with slight
modifications[3]. Fluc/GFP
double transgenic mice (8 weeks old, Department of Cardiology, Xijing Hospital,
Fourth Military Medical University, Xi’an, China), as donor mice, were
sacrificed by cervical dislocation, and were placed in 75% ethanol water for
5min. The femurs and tibias of donor mice were obtained with skin and muscles
away, then washed in PBS and immersed in DMEM culture medium (Gibco, Grand
Island, NY, USA) with 1% penicillin and 1% streptomycin. The BMCs were isolated
from the femurs and tibias of donor mice by slowly flushing DMEM culture medium
into the diaphyseal channel, and erythrocytes were schizolysed. The isolated
cells were centrifuged at 800 rpm for 5min and resuspended into PBS. The
chimeric mice were injected with 4×106 cells into the tail vein
within 10h after irradiation (8.0 Gy). The level of chimerism in all recipients
for subsequent use in this study was above the standard by flow cytometry. So
the engraftment of BMCs was similar between the diabetic group and control
group.
In Vivo Optical Bioluminescence Imaging The recipient
mice were anesthetized with 2% isoflurane and injected with D-luciferin (375
mg/kg body weight; Xenogen, USA) intraperitoneally. The mice were placed in a
prone position in the IVIS kinetic system (Xenogen, USA) and their mouths were
covered with black papers in order to prevent interference[26]. The mice were imaged at days 1, 3, 5,
7, 14, 21 and 28 after CNV induction. Photon emissions from predefined regions
of interest (ROIs) were normalized as photons per second per square centimeter
per steradian. The ROIs in every chimeric mice had fixed shape and area, with
two eyes covered in and avoiding signals from ears and nose. Peak signals of
ROIs were evaluated using Living Image 4.0 software (Xenogen, USA).
In Vitro Firefly Luciferase Assays The recipient
mice were sacrificed at 7d after CNV induction. The choroidal organization of
each mouse was dissected and a hand-held pestle was used to homogenize the
organizations into cell suspensions. The suspensions were lysed using 200 μL
1×passive lysis buffer (Promega, Madison, Wisc., USA) and centrifuged at 1200
rpm for 2min. Each sample was placed into luminometer tubes and consisted of 20
μL supernatant in 100 μL Fluc [relative light units of Fluc (RLU1)] activitiy
assay reagent (LAR II; Promega) and 100 μL renilla luciferase [relative light
units of renilla luciferase (RLU2)] activitiy assay reagent (Stop & Glo®;
Promega). Both Fluc and renilla luciferase activities were measured by using
luciferase photometer.
Evaluation of Choroidal
Neovascularization Severity Choroidal
flatmount was prepared at 7d after CNV induction (6 eyes in each group, 6 spots
per eye) in accordance with a previously described protocol[13]. Anesthetized mice were perfused
transcardially with 0.9% saline followed by 4% paraformaldehyde. The entire eye
globes were enucleated, followed by the anterior segment and the neural retina
removed. The remained RPE-choroid-sclera complex was flatmounted with six
radial cuts or more, permeabilized in 0.2% Triton X-100 for 24h, then
transferred into 1:1000 rhodamine-conjugated Ricinus communisagglutinin (Vector
Laboratories, Burlingame, CA, USA) for 24h, and washed in 0.01 mol
Tris-buffered saline Tween-20 (TBST) for 24h. The flatmounts were examined and
photographed by a confocal laser scanning microscopy (Fv1000, Olympus
Corporation, Tokyo, Japan) and CNV area was assessed automatically with image
pro plus 6.0 software (IPP 6.0). For measuring CNV surface area, the
agglutinin-positive red area at the laser spots in the flatmounts was measured
and expressed in μm2. Individual lesions with surface areas more
than 0.50 disc areas (DAs) were defined as having CNV[13,27-28].
Histopathological
analysis was performed as described previously[7] (3
eyes in each group, 6 spots per eye). The mice were killed at days 1, 3, 5, 7,
14, 21 and 28 after photocoagulation and eyes were enucleated, then eyecup
preparations were fixed in Bouin’s fixative (Zhongshan Biotechnology Company,
Beijing, China) at 4℃ for 24h. The fixed tissues were embedded in paraffin,
serially sectioned at 3 μm, and stained with hematoxylin and eosin (H&E).
Serial slices of each CNV were examined and digitized using a light microscope
(BX51, Olympus Corporation, Tokyo, Japan). CNV thickness was measured
vertically from the adjacent RPE layer to the top of the CNV and CNV length was
measured as horizontal maximize distance of CNV using IPP 6.0 software, which
expressed in μm.
Western Blot Assay The
RPE-choroid-sclera complex was collected and lysed, and the expression levels
of VEGF and SDF-1 were measured using standard techniques[11]. The monoclonal primary antibodies to
VEGF (1:200, Abcam Biotechnology, USA) and SDF-1 (1:200, Abcam Biotechnology,
USA) were used. The β-actin levels were used to normalize the quantity of the
proteins and all experiments were repeated at least three times.
Statistical Analysis Statistical
analyses were performed using SPSS 19.0 software. The data from several
experiments were pooled and then presented as the mean±SEM. Analysis of
variance followed by LSD-t test was used to compare two groups.
Student’s t-test was used for the rest statistical analyses. All
experimental data sets were scrutinized for normal distribution of variance,
and nonparametric tests were appropriately applied when necessary. A two-tailed
value of P<0.05 was considered significant.
RESULTS
Kinetic Observation of Hyperglycemia
Promoting the Transplanted Stem Cells’ Participation in Choroidal Neovascularization by
Bioluminescence Imaging To
confirm the hypotheses that hyperglycemia promotes the participation of stem
cells in CNV formation, we conducted the bone marrow transplantation, STZ
intraperitoneal injection and laser photocoagulation to induce the chimeric
mice, DM and CNV model, respectively. By using in vivo optical BLI, the
recruitment of Fluc+GFP+ BMCs was detected at 1d after
laser photocoagulation, with the peak signals occupied at 7d, then weakened at
subsequent time points, indicating the donor cell death. The fact that the
DM+CNV group showed a stronger signal than the Normal+CNV group indicated that
hyperglycemia promoted the transplanted BMCs to participate in CNV (Figure 1A).
Quantitative
analysis of ROIs showed that the radiance (photons per second per square
centimeter per steradian) of Fluc+GFP+ BMCs increased
from 1d to 7d; after reaching a maximum intensity at 7d, the radiance decreased
in the following days. At each time point, the radiance in the CNV groups were
stronger than that in the control groups no matter the blood glucose
concentration was normal or high, and the radiance in the DM+CNV group was
stronger than that in the Normal+CNV group at days 5, 7, 14 and 21
(121861.67±9948.81 vs 144998.33±13787.13 photons/second/cm2/sr
for control and DM mice, P5d<0.05;
178791.67±30350.8 vs 240166.67±22605.3, P7d<0.05; 124176.67±16253.52 vs
196376.67±18556.79, P14d<0.05;
97951.60±10343.09 vs 119510.00±14383.76, P21d<0.05; Figure 1B), consistent with the BLI
signals mentioned above.
Figure 1 Hyperglycemia promoted the participation of
BMCs in CNV after laser photocoagulation
A: In vivo optical BLI signals of transplanted Fluc+GFP+
BMCs in living mice among five groups at days 1, 3, 5, 7, 14, 21 and 28
after laser photocoagulation. The BLI signals were observed at 1d after laser
photocoagulation, with the peak signals occupied at 7d, then decreased at the
subsequent time points. Ⅰ: control chimeric mice (Normal); Ⅱ: diabetic chimeric
mice (DM); Ⅲ: control chimeric mice with CNV (Normal+CNV); Ⅳ: diabetic chimeric
mice with CNV (DM+CNV); Ⅴ: control WT mice. Scale bars represent BLI signals in
photons per second per square centimeter per steradian (p/s/cm2/sr).
B: Quantitative analysis of the BLI signals in ROIs. The radiance in the
Normal+CNV and DM+CNV groups were stronger than that in the Normal and DM
control groups, respectively, and the radiance in the DM+CNV group was stronger
than that in the Normal+CNV group at days 5, 7, 14 and 21 after laser induction
(P<0.05). There was no signal in ROIs in the control WT mice, and the
control WT group was not included in the analyses.
Histopathological Analysis of
Hyperglycemia Promoting the Development of Choroidal Neovascularization in Mice
Vascular
complexes that formed after the damage to Bruch’s membrane extended from the
choroid to the subretinal space. To confirm the effects of hyperglycemia on the
progression of CNV, we used the histopathological assay at days 1, 3, 5, 7, 14, 21 and 28 after CNV
induction (Figure 2A) and found that the CNVs in the cross-sectional slices
stained with H&E from the hyperglycemic mice were wider than those in the
control group at days 5, 7, 14 and 21, with the maximum at 7d (147.83±17.36 vs
220.33±20.17 μm for control and
DM mice, P5d<0.05;
212.17±24.63 vs 326.83±19.49, P7d<0.05;
163.17±18.24 vs 265.17±20.55, P14d<0.05;
132.00±10.88 vs 205.33±12.98, P21d<0.05;
Figure 2B), but the average thickness of CNVs between the two groups had no
significant difference (Figure 2C).
Figure 2 Hyperglycemia aggravated the
development of CNV after laser photocoagulation A:
Representative images of the
H&E-stained serial cross-sections of the eyecups from control (left) and
diabetic (right) mice at days 1, 3, 5, 7, 14, 21 and 28 after laser
photocoagulation (Red lines: area of CNV). B: Statistical analysis of the average length of CNV between the control and diabetic groups in A. The
CNVs in the hyperglycemic mice were wider than those in the control group at
days 5, 7, 14 and 21 after laser photocoagulation (P<0.05), with the
maximum at 7d. C: Statistical
analysis of the average thickness of CNV between the two groups in A. There was no significant difference.
In Vivo Flatmount and in Vitro Fluc
Assay of Hyperglycemia Exacerbating the Choroidal Neovascularization at 7d After
Laser Photocoagulation Based
on the fact that the largest CNV and the maximum amount of transplanted stem
cells were appeared at 7d after laser photocoagulation, we conducted the
flatmount assay in vivo and the quantitative analysis of Fluc reporter
enzyme in vitro at 7d. Compared with the control group, the average area
of CNV in the diabetic group was notably larger at 7d (20688.67±3644.96 vs
32218.00±4132.69 μm2 for
control and diabetic mice, respectively; P<0.05; Figure 3A, 3B). In
view of the BLI data only showing the differential recruitment and
participation of BMCs in CNV but unable to afford accurate quantitative
analysis, a Fluc assay was used to quantify the Fluc reporter enzyme in the
diabetic and control mice with CNV model. Three sets of data were obtained
using a luminometer for each group: RLU1, RLU2 and the
ratio of RLU1 and RLU2. RLU1 and ratio are standard for the comparisons of Fluc
reporter enzyme. Compared with the control group, RLU1 and ratio were markedly
higher at 7d in the diabetic group (215.00±52.05 vs 707.33±88.65, P<0.05;
0.90±0.17 vs 1.83±0.17, P<0.05; for control and diabetic mice,
respectively; Figure 3C, 3D).
Figure 3 Hyperglycemia exacerbated the
CNV lesion at 7d after laser photocoagulation A: Representative flatmount preparations of the eyecups from the control
(left) and diabetic (right) mice at 7d after laser photocoagulation (OD:
Optic disc). B: Statistical analysis of CNV area in A.
The average area of CNV in the diabetic mice was markedly larger than that in
the control group at 7d (P<0.05). C, D: In vitro
characterization of reporter gene expression at 7d. RLU1 and the RLU1/RLU2
ratio in the diabetic group were significantly higher than those in the control
group (P<0.05), determined using the luminometer assay.
Mechanism of Hyperglycemia Promoting the
Participation of Bone Marrow-derived Cells in Choroidal Neovascularization After
Laser Photocoagulation To
explore the underlying mechanism that hyperglycemia exacerbated the recruitment
and participation of BMCs in CNV after laser photocoagulation, we investigated
the levels of VEGF and SDF-1 production in
the diabetic and control mice with CNV model by Western Blot examination (Figure 4A) and found that the
level of VEGF protein expression significantly increased from 1d to 7d; after
reaching a maximum at 7d, it decreased subsequently, and at each time point,
the amount of VEGF protein in the diabetic group was higher than that in
control group (Figure 4B, P<0.05).
In addition, the amount of SDF-1 protein was also up-regulated in the diabetic
group, and the changing timecourse of the up-regulation was similar to the VEGF
protein expression (Fig. 4C, P<0.05).
Figure 4 Mechanism of hyperglycemia
exacerbating the participation of BMCs in CNV after laser photocoagulation A: Expression of VEGF and SDF-1 in the control (left) and diabetic (right)
mice at days 1, 3, 5, 7, 14, 21 and 28 after laser photocoagulation by Western Blot examination. B, C: Statistical
analysis of the data in A. The level of VEGF and SDF-1 protein expression in hyperglycemic mice significantly
increased in a time-dependent manner, with the peak value occupied at 7d (P<0.05).
DISCUSSION
Neovascular
AMD, the primary cause of blindness among elderly people in developed
countries, is characterized by the presence of CNV under the macula[29]. Because diabetes functions on vascular
systems, most epidemiological studies have focused on the relationship between diabetes
and AMD. Diabetes-related changes have been considered as risk factors for
developing AMD and the underlying mechanism needs to be further carried out[30].
In
previous studies of our research group, we found that BMCs (GFP-positive)
recruited to CNV expressed the mature vascular ECs marker CD31, VSMCs marker
α-SMA and the macrophage marker F4/80, indicating BMCs recruited to CNV
differentiated into these three types of cells and actually integrated into the
CNV structures, that is the vasculogenesis of CNV[6,13,24].
Furthermore, hyperglycemia significantly increased the constituent ratio of
vascular ECs and macrophages labelled GFP, up-regulated the expression of VEGF
and SDF-1 in diabetic group, suggesting that hyperglycemia promoted
recruitment, differentiation and incorporation of BMCs in CNV[12]. In addition, hyperglycemia promoted
the development of CNV by inducing oxidative stress, which in turn activated
STAT3-regulated VEGF expression in RPE cells[11]. In
this study, molecular imaging techniques, including in vivo BLI and in
vitro Fluc assay, were used to observe the in vivo behaviors and
migration of BMCs implanted into diabetic mice with CNV model for the first
time and to verify our previous conclusion that hyperglycemia exacerbated the
vasculogenesis of CNV due to more BMCs recruited. In combination with
conventional histological methods, we demonstrated that hyperglycemia enhanced
the progression of CNV and the recruitment of BMCs in CNV associated with
elevation of VEGF and SDF-1 in the eye; molecular imaging using the Fluc
reporter enzyme was a reliable method for monitoring stem cell survival in
vivo.
In
previous studies, in vivo stem cell research has been predominantly
relied on postmortem histological analysis and molecular biology examination,
which are inappropriate to reveal the time scale of the dynamic interplay
between the stem cell graft, the CNV lesion and the endogenous related
mechanisms[31-33]. In the present
study, the noninvasive molecular imaging technique using the Fluc reporter gene
and D-luciferin reporter probe provided complementary in vivo
information and showed great potential for in vivo monitoring stem cell
events in CNV lesions in a diabetic mouse model. This in vivo optical
BLI method might avoid sample biases caused by the sacrifice of multiple
animals at different time points, permit simple animal preparation, repetitive
experimental conditions and relatively medium-cost instrumentation, and be
performed under mild anesthesia, thus nearly under physiological conditions[34]. Furthermore, molecular imaging
technology is a valuable, unpolluted tool for the analysis of disease processes
at the molecular level in living intact animals, which would extend our
understanding of the basis of optimal cell administration system and dose, and
would greatly influence our views on the efficacy of future cell-based
therapies[20].
Although
cells in situ play an angiogenetic role in CNV, BMCs-mediated postnatal
vasculogenesis has been reported as the main responsible for the regulation of
CNV progression[7]. We
demonstrated that hyperglycemia exacerbated the severity of CNV in diabetic
mice by choroidal flatmount and histological sections as reported before,
detected that more BMCs were recruited to CNV lesions in diabetic chimeric mice
by in vivo BLI monitoring and in vitro Fluc assay for the first
time, and further revealed that more severe CNV and the increased number of
BMCs in CNV exposed to high glucose were associated with up-regulation
expression of VEGF and SDF-1 by RT-PCR and Western blot, consistent with our
previous studies. However, the results seemed contrary to the conclusion that
BMCs were decreased and disordered in diabetes as reported. Hyperglycemia was
related to the dysfunction of bone marrow-derived endothelial progenitor cells
(EPCs) at each step of their lifespan (bone marrow mobilization, trafficking
into the bloodstream, survival, differentiation into ECs, and homing in damaged
tissues/organs)[35-36]. Hyperglycemia alone, through the mitochondrial
overproduction of reactive oxygen species (ROS), induced changes in gene
expression and cellular behavior, would explain the impairments in
vasculogenesis, the process by which circulating EPCs contributed to new vessel
formation in diabetes[14]. EPCs were
functionally impaired in hyperglycemia through the p38 MAPK signaling pathway[37]. These conflicts might be due to the
different animal models being used. The CNV model has been used in most studies
on CNV, but still exists some defects such as its relative short disease
duration (about 4wk), which might be insufficient to produce significant
reduction and dysfunction of BMCs in diabetic mice, forcing us optimize the CNV
model in the future. In addition, BMCs consist of mesenchymal stem cells,
hematopoietic stem cells, EPCs and various other cell subtypes, all of which
need further detection using molecular imaging technologies in order to
ascertain which components of BMCs predominantly participate in CNV development
in diabetic mice[38].
In
conclusion, the present study used dynamic molecular imaging techniques for the
first time to observe the in vivo behavior of stem cells exposed to
hyperglycemia in CNV mice and demonstrated the diabetes-aggravated
vasculogenesis of CNV, suggesting the potential of BMCs as powerful delivery
vehicles carrying antiangiogenic agents targeting VEGF in CNV. The underlying
mechanisms might be the increased levels of VEGF and SDF-1 and needs further
investigation. Our findings reveal that diabetes is a risk factor for disorders
involving the vasculogenesis of CNV, and cell-based protocol may serve as a
therapeutic strategy for the treatment and prevention of these diseases.
ACKNOWLEDGEMENTS
Foundations: Supported by
the National Natural Science Foundation of China (No. 81070748; No. 81200708);
National Basic Research Program of China (973 Program).
Conflicts of Interest: Gao X, None; Wang Y, None; Hou HY, None;
Lyu Y, None; Wang HY, None; Yao LB,
None; Zhang J, None; Cao F, None; Wang YS, None.
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