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B, and RAGE in atherosclerotic lesions of diabetic Watanabe heritable hyperlipidemic rabbits
,
,1
* Department of Biotechnology and Molecular Medicine, A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, Finland;
Department of Medicine, University of Kuopio, Finland;
Gene Therapy Unit, Kuopio, University Hospital, Finland; and
Department of Biomedical NMR, A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, Finland
1Correspondence: Department of Molecular Medicine, A.I. Virtanen Institute, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. E-mail: seppo.ylaherttuala{at}uku.fi
ABSTRACT
Plaque angiogenesis may be associated with the development of unstable and vulnerable plaques. Vascular endothelial growth factors (VEGFs) are potent angiogenic factors that can affect plaque neovascularization. Our objective was to determine the effect of diabetes on atherosclerosis and on the expression of angiogenesis-related genes in atherosclerotic lesions. Alloxan was used to induce diabetes in male Watanabe heritable hyperlipidemic (WHHL) rabbits that were sacrificed 2 and 6 months after the induction of diabetes. Nondiabetic WHHL rabbits served as controls. Blood glucose (Glc), serum-free fatty acids (FFA), and serum triglyceride levels were significantly higher in diabetic rabbits. Accelerated atherogenesis was observed in the diabetic WHHL rabbits together with increased intramyocellular lipids (IMCL), as determined by 1H-NMR spectroscopy. Atherosclerotic lesions in the diabetic rabbits had an increased content of macrophages and showed significant increases in immunostainings for vascular endothelial growth factor (VEGF)-A, VEGF-D, VEGF receptor-1, VEGF receptor-2, RAGE, and NF-
B. VEGF-A165 and VEGFR-2 mRNA levels were significantly increased in aortas of the diabetic rabbits, where a trend toward increased plaque vascularization was also observed. These results suggest that diabetes accelerates atherogenesis, up-regulates VEGF-A, VEGF-D, and VEGF receptor-2 expression, and increases NF-B, RAGE, and inflammatory responses in atherosclerotic lesions in WHHL rabbits.—Roy, H., Bhardwaj, S., Babu, M., Kokina, I., Uotila, S., Ahtialansaari, T., Laitinen, T., Hakumaki, J., Laakso, M., Herzig, K-H., Ylä-Herttuala, S. VEGF-A, VEGF-D, VEGF receptor-1, VEGF receptor-2, NF-B, and RAGE in atherosclerotic lesions of diabetic Watanabe heritable hyperlipidemic rabbits.
Key Words: diabetes • dyslipidemia • atherosclerosis • vascular endothelial growth factor • plaque angiogenesis
DIABETES MELLITUS IS a major risk factor for the development of atherosclerotic vascular disease (1)
. Metabolic abnormalities that characterize diabetes, particularly hyperglycemia, insulin resistance, increased levels of free fatty acids (FFA), and dyslipidemia (2
, 3)
, provoke cellular and molecular mechanisms that can alter the function and structure of blood vessels and have discrete effects on the initiation and progression of atherosclerotic lesions. Depending on the site of lesions, fatal events such as a myocardial infarction or a cerebral stroke can occur. Plaque angiogenesis may be associated with increased atherogenesis and unstable vulnerable plaques (4
, 5)
. Vascular endothelial growth factors (VEGFs) are potent angiogenic factors that can affect plaque neovascularization. It is therefore important to determine the effects of diabetes on VEGF expression and plaque angiogenesis in atherosclerotic lesions.
In 1980, Watanabe et al. reported the breeding of Watanabe heritable hyperlipidemic (WHHL) rabbits (6)
, which have markedly elevated levels of serum lipids. WHHL rabbits have a defective LDL receptor (LDLr) due to a spontaneous 4 amino acid deletion in the cysteine-rich ligand binding domain of the receptor (7)
in exon-4 of the LDLr gene. WHHL rabbits are also characterized by hyperinsulinemia, both in the fasting state and during an intravenous (i.v.) Glc tolerance test, but they have a relatively normal Glc tolerance (8)
. Even though WHHL rabbits have been used extensively in hyperlipidemia and atherosclerosis research, to the best of our knowledge no data are available from well-characterized diabetes models in WHHL rabbits. Here we report induction of diabetes in WHHL rabbits using alloxan injection. This model was then used to characterize factors associated with accelerated atherosclerosis and plaque neovascularization.
MATERIALS AND METHODS
Diabetes was induced in male WHHL rabbits (n=15, between 6 and 10 months of age) by a single i.v. injection of alloxan (Sigma Aldrich, St. Louis, MO, USA), 100 mg/kg dissolved in 10 ml of 0.9% NaCl. Blood Glc was monitored before alloxan injection, then every 2 h for 8 h after the alloxan injection. Blood Glc levels were measured every morning using a standard Glucometer (ELITA XL- Bayer). To prevent life-threatening hyperglycemia, diabetic rabbits were treated with injections of Ultratard Insulin (1–2 U/kg, Novo Nordisk) whenever the morning blood Glc level was >18 mmol/l. The majority of the rabbits (n=13) developed severe hyperglycemia within 24–48 h of alloxan injection. Rabbits that died (n=3) of diabetic ketoacidosis and those that remained euglycemic after alloxan injection (n=2) or became euglycemic 3 months after the injection (n=1) were excluded from the study. All deaths occurred in the first week after induction of diabetes. Diabetic WHHL rabbits (n=9) and nondiabetic WHHL controls (n=9) were used for this study. Diabetic and control animals were divided into two subgroups. Animals from the diabetic (n=6) and the control subgroups (n=5) were sacrificed 2 months after the induction of diabetes. A second group of diabetic rabbits (n=3) and controls (n=4) was sacrificed 6 months after the induction of diabetes. All rabbits received standard chow and water ad libitum. Experiments were approved by the Experimental Animal Committee, University of Kuopio, Finland.
Oral Glc tolerance test (OGTT)
Rabbits were fasted overnight and a fasting blood Glc level was measured. Thereafter, rabbits were given orally 1.2 g of Glc/kg body wt. Blood Glc levels were measured at 15, 30, 60, 90, and 120 min using a glucometer (ELITA XL- Bayer).
Serum lipids
Arterial blood was collected at baseline and before sacrificing the animals. Total serum cholesterol and triglyceride levels were measured by standard enzymatic procedures using Ecoline® reagent kits (E. Merck, Darmstadt, Germany). FFA were measured by an enzymatic colorimetric method (Wako NEFA C test kit; Wako Chemicals GmbH, Neuss, Germany)
1H-NMR spectroscopy
Two months after the induction of diabetes, nuclear magnetic resonance (1H-NMR) spectrum was obtained from the biceps femoris muscles of the diabetic rabbits (n=6) and nondiabetic controls (n=5). MRS was performed with a horizontal bore 4.7 T magnet equipped with actively shielded field gradients interfaced to a Varian UNITYINOVA console. A quadrature surface coil was used both as a transmitter and a receiver. Gradient echo images were acquired to place the voxel to the muscle. For metabolite analysis, LASER method incorporating water suppression was used (9)
(repetition time TR=6s, echo time TE=34ms and number of scans NS=128). Non-water-suppressed spectra were obtained similarly (NS=8) to provide a reference. For measurements, the major axis of the muscle was aligned parallel to the magnetic field in order to prevent differences in orientation-dependent effects between animals. Spectral analysis was performed in the time domain using jMRUI software (http://carbon.uab.es/mrui). Metabolite concentrations were calculated based on the water content. Since relaxation times of the signals from lipids and creatine are intra- and interindividually constant and the applied echo times were short; results were not corrected for relaxation effects (10)
.
Cell culture, RNA isolation, and cDNA synthesis
Rabbit aortic smooth muscle cells (RAASMCs) were cultured in 100 mm plates in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS (Life Technologies, Inc., Grand Island, NY, USA) and 100 U/ml streptomycin/ampicillin at 37°C in a humidified atmosphere of 5% CO2 until 60% to 70% confluent. Total RNA was extracted from RAASMC using RNeasy kit (Qiagen, Valencia, CA, USA). Genomic DNA was digested by thorough treatment with DNase I (Qiagen). Reverse transcription was done using random hexamers with "You prime First strand cDNA synthesis kit"® (Amersham Biosciences, Freiburg, Germany).
Quantitative RT-polymerase chain reaction (RT-PCR)
Aortic samples from 2 month diabetic and control groups were snap frozen in liquid nitrogen for RNA extraction. Total RNA was extracted from the segments using Trizol reagent (Gibco BRL). RT-PCR to detect VEGFR-1, VEGFR-2, VEGF-A121, and VEGF-A165 expression in aortic samples was performed. SYBR® Green (Applied Biosystems, Foster City, CA, USA) real-time polymerase chain reaction (PCR) using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems) was used to quantify mRNA expression levels of VEGFR-2 and endogenous VEGF-A121 and VEGF-A165. A common VEGF-A forward primer and specific reverse primers to amplify VEGF-A121 and VEGF-A165 isoforms were used (Table 1
). 18S was selected as a reference gene. Reactions contained 100 ng of cDNA, 15 µl of SYBR Green master mix (Applied Biosystems), and 7.5 pmol of primers. All samples were done as duplicates under the following conditions: 30 s at 50°C and 10 min at 95°C, followed by 42 cycles of 15 s at 95°C and 1 min at 60°C. Each assay included a standard curve of three serial dilutions of rabbit aortic cDNA and no template controls. Ratios of target gene and 18S expression (relative gene expression numbers) were calculated. Results were calculated as instructed in ABI PRISM 7700 Sequence Detection System User Bulletin #2.
|
Histology
After sacrificing the animals, the lower thoracic aorta and upper abdominal aorta were dissected and immersion fixed in 4% paraformaldehyde/15% sucrose (pH 7.4) overnight, processed, and embedded in paraffin for further histological evaluation (11)
. To determine the effect of diabetes on atherosclerosis, hematoxylin-eosin and immunohistochemical stainings were performed on 6 µm-thin paraffin sections from aortas. Immunohistochemistry on paraffin sections was done with the avidin-biotin-HRP method and 3,3'-diaminobenzidine was used as a substrate (Vector Laboratories, Burlingame CA, USA). Fluorescein avidin D and 4',6'-diam idino-2-phenylidole (Vector Laboratories) were used in fluorescent stainings. The following antibodies were used for the immunostainings: macrophages, monoclonal antibody (mAb) RAM-11, (DAKO, Carpinteria, CA, USA; dilution 1:100); smooth muscle cells, mAb HHF-35 (DAKO, dilution 1:100); receptor for advanced glycation end products, mAb RAGE (Chemicon International, dilution 1:500); NF-B, mAb NF-B-C terminus of p65 (Pharmingen, San Diego, CA, USA; dilution 1:50); endothelium, mAb CD31, (DAKO, dilution 1:50); mAb VEGF-A (Santa Cruz Biotechnology, Santa Cruz, CA, USA; dilution 1:200); mAb VEGF-D (R&D Systems, Minneapolis, MN, USA; dilution 1:100), VEGFR-1 poyclonal antibody (Ab) (Santa Cruz Biotechnology, 1:50), and mAb VEGFR-2 (Santa Cruz Biotechnology, 1:500). Incubations with class and species-matched unrelated antibodies and incubations where primary antibodies were omitted were used as controls (11
, 12)
.
Morphometry and image analysis
Morphometry and image analysis were done using AnalySIS (Soft Imaging Systems, Munster, Germany) software with Olympus AX70 microscope (Olympus Optical, Japan). The specimens were analyzed by an expert operator (S.B.) in a blinded manner. Total area of atherosclerotic plaques was measured from five standardized sections (stained with hematoxylin-eosin) of each animal using the imaging software and the mean value of the affected area is reported (13)
. The total number of cluster of differentiation (CD)-31-positive capillaries in the intima was counted and data were expressed as a number of capillaries/mm2. Semiquantitative immunohistochemical analysis was performed by the image processing software that was programmed to select immunostained or immunofluorescent areas based on preset thresholds (14)
. The positively stained areas were then calculated as percentages of the total intimal cross-sectional area (12)
.
Statistical analysis
Data were tested for homogeneity of variance by the Levene’s test. Logarithmic transformations were performed to correct unequal variance in the data. Two-tailed unpaired t test was used for the comparison of two variables. Response to diabetes and duration was determined by 2-way ANOVA, and Benferroni’s post hoc test was used to compare subgroups. P 
0.05 was considered statistically significant. Data are expressed as mean ± SD. Statistical calculations were performed using GraphPad Prism version 4.00 for Windows, (GraphPad Software, San Diego California, USA).
RESULTS
Induction of diabetes
Mean blood Glc in the diabetic WHHL rabbits was significantly higher (P<0.001) in the 2 month and 6 month diabetic subgroups compared with nondiabetic controls (Fig. 1
A). OGTT showed high fasting blood Glc levels (17.96±5.14 mmol/l) in the diabetic groups, with high levels of blood Glc (24.98±5.16 mmol/l) persisting 2 h after the initial Glc load. In the control group the fasting blood Glc levels were in the normal range (4.86±0.30 mmol/l), and the blood Glc level returned back to normal (5.2±0.20 mmol/l) 2 h after the initial Glc load (Fig. 1B
).
|
Serum lipids
Serum cholesterol levels in the diabetic rabbits tended to be higher at the 2 and 6 month time points, but the differences did not reach statistical significance (Fig. 1C
). Serum triglyceride levels were significantly increased (P<0.001) in the 2 month and 6 month diabetic subgroups (Fig. 1D
).
Intramyocellular lipids and serum-free fatty acids
Increased delivery of FFA to muscles contributes to insulin resistance (15)
. Two months after the induction of diabetes, a significant increase (P<0.05) was found in serum FFA levels of the diabetic rabbits (Fig 2
B). This leads to the accumulation of triglycerides in skeletal muscle. Proton magnetic resonance spectroscopy (1H-NMR) was used to assess intramyocellular lipids (16)
(IMCL). We observed a significant increase (P<0.05) in the IMCL levels in the diabetic rabbits (Fig. 2A
).
|
Enhanced atherogenesis in diabetic rabbits
Atherosclerosis was significantly increased in the 2 month (P<0.01) and 6 month (P<0.001) diabetic subgroups (Fig 2C
). Image analysis showed a significant (P<0.001) increase in the immunostained area positive for macrophages in the diabetic rabbits at 6 months (Fig. 3
A). Macrophages and foam cells were present in the atherosclerotic lesions of both the diabetic and control rabbits. Macrophage infiltration was most prominent in the subendothelial region (Fig. 4
A–D) and in the shoulder regions of the plaques. Large numbers of smooth muscle cells were seen in the atherosclerotic lesions in both groups (Fig. 3B
, Fig. 4E-H
).
|
|
Increased RAGE expression in diabetic plaques
A strong RAGE immunoreactivity was seen in atherosclerotic plaques of the diabetic animals. There was a significant increase in RAGE staining in both the 2 and the 6 month diabetic subgroups. RAGE localized predominantly in the endothelial cells, subendothelial region, macrophages, and foam cells (Fig. 3C
, Fig. 4I-L
).
Increased presence of NF-B in diabetic atherosclerotic lesions
Immunohistochemical staining for NF-B p65 was significantly higher in both the 2 and 6 month diabetic rabbits (Fig. 3D
). NF-B staining was most prominent in macrophages, but positive immunostaining was also observed in some endothelial cells and smooth muscle cells (Fig. 4M-P
).
Angiogenic growth factors and angiogenesis
Real-time quantitative RT-PCR from aortas showed that VEGF-A165 mRNA expression was significantly increased in the diabetic rabbits. The VEGF-A121 mRNA expression levels were comparable in the aortas of the diabetic and the control animals (Fig. 5
A).
|
VEGF-A immunostaining was significantly increased both in the 2 month and 6 month diabetic subgroups. VEGF-A immunostaining was observed in some lesionmacrophages and smooth muscle cells. VEGF-A staining was also observed in the subendothelial region (Fig. 3E
, Fig. 6
A–D).
|
VEGF-D immunostaining tended to be increased in the 2 month diabetic subgroup, but the difference with the controls was not significant. On the other hand, a significant increase was found in the 6 month diabetic subgroup (Fig. 3F
). VEGF-D was detected in macrophages, smooth muscle cells, and endothelial cells. Positive VEGF-D staining was also seen in the adjacent extracellular area (Fig. 6E
–H).
RT-PCR and immunohistochemistry confirmed VEGFR-1 and VEGFR-2 expression in aortic samples. VEGFR-1 immunostainings were detected in the endothelial cells, macrophages, and SMCs (Fig. 6I-L
). VEGFR-1 immunostainings were significantly increased in the atherosclerotic lesions of the 6 month diabetic rabbits (Fig. 3H
). VEGFR-2 mRNA levels were significantly increased in aortic samples of diabetic rabbits (Fig. 5B
). VEGFR-2 immunostaining was detected in SMCs and also in some endothelial cells (Fig. 6M-P
). VEGFR-2 staining in the atherosclerotic plaques of the 2 month diabetic subgroup was not significantly different, but in the 6 month diabetic rabbits a significant increase was found (Fig. 3G
). Expression of VEGFR-1 and VEGFR-2 in SMCs was confirmed by RT-PCR of the cultured RAASMC (Fig. 5F
).
Positive CD-31 immunostaining was localized in the endothelium (Fig. 6Q-T
). Some medium-sized blood vessels were also seen in the aortic wall in the 6 month diabetic subgroup (Fig. 6U
). Positive VEGF-D and VEGF-A stainings were observed in the walls of these vessels (data not shown). The number of vessels in the atherosclerotic lesions of the diabetic rabbits tended to be higher, but the difference with the controls was not significant (Fig. 3I
). No evidence of plaque rupture or thrombosis was seen in the lesions.
DISCUSSION
In diabetes, excess FFAs, insulin resistance, and hyperglycemia generate adverse metabolic events in endothelial cells that result in endothelial dysfunction, augmented vasoconstriction, inflammation, and thrombosis (17
18)
. Elevated triglyceride and total cholesterol levels are also known to increase the risk of cardiovascular diseases in diabetes (19)
. An animal model for diabetic macrovascular disease should therefore have increased FFA, insulin resistance, hyperglycemia, and dyslipidemia.
Insulin resistance is one factor contributing to the development of diabetic atherosclerotic disease (2
, 17)
. Increased flux of FFAs into the skeletal muscle or a decrease in intracellular metabolism of FFAs leads to insulin resistance in both type-1 and type-2 diabetes (20
, 21)
. WHHL rabbits have a moderate insulin resistance (8)
, and our studies showed that IMCL levels in control WHHL rabbits are significantly higher than in New Zealand white rabbits (unpublished data). In the present study the IMCL levels were significantly increased in diabetic WHHL rabbits as compared with the nondiabetic controls, indicating an accumulation of triglycerides in skeletal muscle and an increase in insulin resistance after induction of diabetes.
Increased FFA delivery to liver also results in increased production of triglyceride-rich VLDL from the liver, which contributes to the development of hypertriglyceridemia in diabetes (22)
. In the present study, serum triglyceride levels were significantly increased in the diabetic rabbits. Hypertriglyceridemia is an important feature of diabetic dyslipidemia (23)
. Thus, induction of diabetes in WHHL rabbits produced an animal model that had increased FFA, insulin resistance, hyperglycemia, and dyslipidemia.
We used this animal model to evaluate the effects of diabetes on atherosclerosis and plaque angiogenesis. Diabetes in WHHL rabbits accelerated atherogenesis. Macrophages were more prevalent together with an increase in NF-B immunostaining in the atherosclerotic lesions of the diabetic rabbits. These findings suggest that diabetes augments inflammatory reactions in atherosclerotic lesions.
RAGE is a multiligand member of the immunoglobulin superfamily of the cell surface molecules. It has been hypothesized that the ligand-RAGE axis amplifies vascular stress and accelerates atherosclerosis (24)
. Thus, RAGE, and NF-B overexpression in atherosclerotic lesions is associated with enhanced inflammatory reactions in the vessel wall (25)
.
It has been reported that VEGF-A enhances progression of atherosclerotic plaques (26)
. VEGF-A and VEGF-D, a new member of the VEGF family, are expressed in medial smooth muscle cells and in macrophages of human atherosclerotic lesions (27)
, but the effect of diabetes on the expression of VEGFs in atherosclerotic arteries is not well documented. The results of this study support the findings that have demonstrated the expression of VEGF receptors in vascular smooth muscle cells (28)
. SMC proliferation and migration in response to VEGFs seen in various vascular pathologies presumably are at least to some extent modulated through these receptors (28
, 29)
. VEGF-A165 and VEGFR-2 expression was up-regulated in the aortas of diabetic rabbits. VEGF-A, VEGF-D, VEGFR-1, and VEGFR-2 immunostainings were also increased in the atherosclerotic plaques of the diabetic rabbits. The presence of VEGFs in macrophages and in the macrophage-rich subendothelial region of the atherosclerotic plaques in the diabetic rabbits was observed. This indicates that macrophages, RAGE, and NF-B may modulate the presence of VEGFs in atherosclerotic lesions. Induction of VEGF-A and VEGF-D through a pathway involving RAGE and NF-B/activating protein-1 (30)
could explain the increased expression of VEGFs in the atherosclerotic lesions of the diabetic rabbits.
This study shows that diabetes increases inflammatory responses and expression of VEGF-A, VEGF-D, VEGF receptor-1, and VEGF receptor-2 in atherosclerotic lesions. To the best of our knowledge this is the first study reporting a diabetic model in WHHL rabbits and the presence of small vessels in their atherosclerotic plaques. Diabetic WHHL rabbits will be a useful model to study the pathogenesis of diabetic macroangiopathy.
ACKNOWLEDGMENTS
This study was supported by grants from Finnish Foundation for Cardiovascular Research, Finnish Academy and European Union (QLK3 computed tomography-2002–01955 and LSHG computed tomography-2004–503573). We thank Mr. Tommi Heikura and Mr. Janne Kokkonen for excellent technical assistance and Ms. Marja Poikolainen for help in preparation of the manuscript.
Received for publication December 8, 2005. Accepted for publication May 8, 2006.
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