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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5029fjev1 20/12/2159    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Roy, H. Articles by Ylä-Herttuala, S. Search for Related Content PubMed PubMed Citation Articles by Roy, H. Articles by 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

Himadri Roy^*, Shalini Bhardwaj^*, Mohan Babu^*, Ilze Kokina^*, Sanna Uotila^*, Tiia Ahtialansaari^*, Teemu Laitinen, Juhana Hakumaki, Markku Laakso, Karl-Heinz Herzig^*, and Seppo Ylä-Herttuala^*,,,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

SPECIFIC AIMS

The objectives of this study were to 1) to develop a diabetic hyperlipidemic animal model that would allow studies of the combined effects of these risk factors on atherogenesis and 2) investigate the effects of these risk factors on the expression of vascular endothelial growth factor (VEGF)-A, VEGF-D, VEGFR-1, VEGFR-2, and angiogenesis in atherosclerotic lesions.

PRINCIPAL FINDINGS

1. Accelerated atherogenesis in diabetic rabbits
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. A significant increase (P<0.05) in intramyocellular lipids (IMCL), as determined by ¹H-NMR spectroscopy, was observed in the diabetic rabbits. Atherosclerosis was significantly increased in the 2 month (P<0.01) and 6 month (P<0.001) diabetic groups (see online manuscript) along with a significant (P<0.001) increase in the immunostained area positive for macrophages in the diabetic rabbits at 6 months (Fig. 1 A).

View larger version (32K): [in this window] [in a new window]   Figure 1. Immunohistochemical analysis of atherosclerotic lesions in diabetic rabbits. A) RAM-11 immunostaining for macrophages was significantly increased in the 6 month subgroup. B) HHF-35 immunostaining for smooth muscle cells in diabetic and control rabbits was similar. C) Areas of RAGE and D) NF-B p65 immunostainings were significantly increased in diabetic rabbits. E) VEGF-A staining was significantly increased in diabetic rabbits at both time points whereas F) VEGF-D staining was increased only at the 6 month time point. G) A significant increase in VEGFR-2 and (H) VEGFR-1 immunostaining was present at the 6 month time point in diabetic rabbits. I) The number of capillaries in the atherosclerotic lesions of the diabetic rabbits tended to be increased compared with controls, but the difference was not statistically significant. *P < 0.05; **P < 0.01; ***P < 0.001.

2. Increased RAGE and NF-B p65 expression in atherosclerotic lesions of diabetic rabbits
There was a significant increase in RAGE staining in both the 2 and the 6 month diabetic rabbits. RAGE localized predominantly in the endothelial cells, subendothelial region, macrophages, and foam cells (Fig. 1C ). Immunohistochemical staining for NF-B p65 was significantly higher in both the 2 and the 6 month diabetic rabbits (Fig. 1D ). NF-B p65 staining was most prominent in macrophages, but positive immunostaining was also observed in some endothelial cells and smooth muscle cells.

3. Angiogenic growth factors and angiogenesis in atherosclerotic plaques
Real-time quantitative RT-polymerase chain reaction (RT-PCR) from aortas showed that VEGF-A₁₆₅ mRNA expression was significantly increased (P<0.05) in the diabetic rabbits (see online manuscript). VEGF-A₁₂₁ mRNA expression levels were comparable in the aortas of diabetic and control animals. VEGF-A immunostaining was significantly increased in both the 2 and the 6 month diabetic subgroups (Fig. 1E ). VEGF-A immunostaining was localized mostly in lesion macrophages and smooth muscle cells.

VEGF-D immunostaining tended to be increased in the 2 month diabetic group, but the difference with the controls was not significant. In the 6 month diabetic group, however, a significant increase was found (Fig. 1F ). VEGF-D was detected in macrophages, smooth muscle cells, and endothelial cells.

VEGFR-1 immunostaining was seen in endothelial cells, macrophages, and some smooth muscle cells
VEGFR-2 immunostaining was detected in smooth muscle cells and in some endothelial cells. VEGFR-1 and VEGFR-2 stainings in the atherosclerotic plaques were significantly increased in the 6 month diabetic rabbits (Fig. 1G, H ). VEGFR-2 mRNA levels were also significantly increased in the aortas of the diabetic rabbits (see online manuscript).

Positive cluster of differentiation-31 (CD-31) immunostaining was localized in the endothelium. Some medium-sized blood vessels were also seen in the aortic wall in the 6 month diabetic rabbits. The number of vessels in the atherosclerotic lesions of the diabetic rabbits tended to be higher than the controls, but the difference did not reach statistical significance (Fig 1I ).

CONCLUSIONS AND SIGNIFICANCE

Alloxan-induced diabetes in WHHL rabbits significantly increased serum cholesterol, triglyceride, and FFA levels. There was also a significant increase in IMCL content and atherosclerosis in these animals. 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 atherogenesis and plaque angiogenesis. This study demonstrated a significant increase in the presence of VEGF-A, VEGF-D, VEGF receptor-1, and VEGF receptor-2 and a trend toward increased vascularization in the atherosclerotic lesions of the diabetic WHHL rabbits. The results also show a significant increase in VEGF-A₁₆₅ but not VEGF-A₁₂₁ mRNA expression in the diabetic rabbits. This suggests that diabetes has a selective effect on the expression of different VEGF-A isoforms. An increased presence of NF-B p65 and RAGE was also found in diabetic atherosclerotic lesions. This indicates that diabetes increases proinflammatory responses in the atherosclerotic lesions. Induction of VEGF-A and VEGF-D through a pathway involving RAGE and NF-B/activating protein-1 could explain the increased expression of VEGFs in the atherosclerotic lesions of the diabetic rabbits. In conclusion, we generated a model of diabetic macrovascular disease. Our results suggest that diabetes accelerates atherogenesis, up-regulates VEGF-A, VEGF-D, VEGF receptor-1 expression and VEGF receptor-2 expression and increases NF-B, RAGE, and proinflammatory responses in atherosclerotic leisons.

View larger version (12K): [in this window] [in a new window]   Figure 2. Accelerated atherosclerosis in WHHL rabbit model of diabetes and dyslipidemia. It has been hypothesized that in diabetes there is an induction of VEGF-A and VEGF-D expression through a pathway involving RAGE and NF-B that enhances inflammation and plaque vascularization. Macrophages also contribute to accelerated atherosclerosis in diabetic rabbits.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5029fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5029fjev1 20/12/2159    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Roy, H. Articles by Ylä-Herttuala, S. Search for Related Content PubMed PubMed Citation Articles by Roy, H. Articles by Ylä-Herttuala, S.