Vascular cellular adhesion molecule-1 (VCAM-1) expression in mice retinal vessels is affected by both hyperglycemia and hyperlipidemia - PubMed (original) (raw)

Vascular cellular adhesion molecule-1 (VCAM-1) expression in mice retinal vessels is affected by both hyperglycemia and hyperlipidemia

Carin Gustavsson et al. PLoS One. 2010.

Abstract

Background: Inflammation has been proposed to be important in the pathogenesis of diabetic retinopathy. An early feature of inflammation is the release of cytokines leading to increased expression of endothelial activation markers such as vascular cellular adhesion molecule-1 (VCAM-1). Here we investigated the impact of diabetes and dyslipidemia on VCAM-1 expression in mouse retinal vessels, as well as the potential role of tumor necrosis factor-α (TNFα).

Methodology/principal findings: Expression of VCAM-1 was examined by confocal immunofluorescence microscopy in vessels of wild type (wt), hyperlipidemic (ApoE(-/-)) and TNFα deficient (TNFα(-/-), ApoE(-/-)/TNFα(-/-)) mice. Eight weeks of streptozotocin-induced diabetes resulted in increased VCAM-1 in wt mice, predominantly in small vessels (<10 µm). Diabetic wt mice had higher total retinal TNFα, IL-6 and IL-1β mRNA than controls; as well as higher soluble VCAM-1 (sVCAM-1) in plasma. Lack of TNFα increased higher basal VCAM-1 protein and sVCAM-1, but failed to up-regulate IL-6 and IL-1β mRNA and VCAM-1 protein in response to diabetes. Basal VCAM-1 expression was higher in ApoE(-/-) than in wt mice and both VCAM-1 mRNA and protein levels were further increased by high fat diet. These changes correlated to plasma cholesterol, LDL- and HDL-cholesterol, but not to triglycerides levels. Diabetes, despite further increasing plasma cholesterol in ApoE(-/-) mice, had no effects on VCAM-1 protein expression or on sVCAM-1. However, it increased ICAM-1 mRNA expression in retinal vessels, which correlated to plasma triglycerides.

Conclusions/significance: Hyperglycemia triggers an inflammatory response in the retina of normolipidemic mice and up-regulation of VCAM-1 in retinal vessels. Hypercholesterolemia effectively promotes VCAM-1 expression without evident stimulation of inflammation. Diabetes-induced endothelial activation in ApoE(-/-) mice seems driven by elevated plasma triglycerides but not by cholesterol. Results also suggest a complex role for TNFα in the regulation of VCAM-1 expression, being protective under basal conditions but pro-inflammatory in response to diabetes.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Effects of diabetes on VCAM-1 protein expression in retinal vessels.

(A) Confocal immunofluorescence microscopy images showing VCAM-1 staining (red) in retinal whole-mounts from control non-diabetic and diabetic wild type (wt), ApoE−/−, TNFα−/− and ApoE−/−/TNFα−/− mice. The DNA-binding dye SYTOX (green) was used for nuclear localization. Bars = 50 µm. Measurements were performed 8 weeks after the first STZ-injection, when mice were 30 weeks of age. (B) Summarized data from experiments in A showing mean fluorescence intensity of VCAM-1 in the different groups. White bars represent control and black bars diabetic mice. Two-way analysis of variance (for the effects of diabetes and genotype) revealed significant interactions between factors. Bonferroni posttests yielded *p<0.05 and ***p<0.001 for comparisons between ApoE−/− and TNFα−/−, respectively vs. wt control; ***p<0.001 for comparison between ApoE−/− and ApoE−/−/TNFα−/−; and ### p<0.001 for comparisons between control and diabetes of the same genotype.

Figure 2

Figure 2. The effect of diabetes on VCAM-1 expression is dependent on retinal vessel size.

Summarized data from confocal immunofluorescence microscopy experiments showing mean fluorescence intensity of VCAM-1 in retinal vessels divided into quartiles depending on vessel diameter (<10 µm, 10–15 µm, 16–23 µm and >23 µm). Each graph represents the expression-diameter relationship in the different genotypes. White bars are for control and grey bars for diabetes. The two first bars in each graph (in white and black, for control and diabetes respectively) show the same data as in Figure 1, but are displayed here as references. *p<0.05 and ***p<0.001 indicate differences between control and diabetes of the same size and genotype.

Figure 3

Figure 3. Changes in the expression of vascular adhesion markers in retinal vessels are difficult to resolve if measured in total retinal RNA.

(A) Expression of VCAM-1, ICAM-1, P- and E-selectin mRNA was measured by real time RT-PCR in intact retinas of control (white bars) and diabetic (black bars) wt, ApoE−/−, TNFα−/− and ApoE−/−/TNFα−/− mice. No statistically significant differences were seen between genotypes or between retinas from control and diabetic mice.Results are normalized to the expression of the housekeeping control cyclophilin B. (B) VCAM-1, ICAM-1 and E-selectin mRNA were measured in isolated retinal vessels from control (white) and diabetic (black) ApoE−/− mice. Values are normalized to the expression of cyclophilin B and GAPDH. *p<0.05 indicates difference to the control group. (C) Mean sVCAM-1 concentrations in plasma (ng/ml), measured by ELISA are depicted for control (white bars) and diabetic (black bars) wt, ApoE−/− and ApoE−/−/TNFα−/− mice. *p<0.05 vs. wt control and ***p<0.001 for differences between non-diabetic ApoE−/− and ApoE−/−/TNFα−/− mice. For A–C, measurements were performed 8 weeks after the first STZ-injection, when mice were 30 weeks of age.

Figure 4

Figure 4. Diabetes enhances the expression of inflammatory cytokines in the retina of wt mice but not of ApoE- or TNFα deficient mice.

Expression of _TNF_α, IL-6, IL-1β, IFNγ and caspase-1 mRNA was measured by real time RT-PCR in intact retinas of control (white bars) and diabetic (black bars) wt, ApoE−/−, TNFα−/− and ApoE−/−/TNFα−/− mice. Values are normalized to the expression of cyclophilin B. *p<0.05 and **p<0.01 indicates differences to control wt mice.

Figure 5

Figure 5. High fat diet (HFD) increases VCAM-1 expression in retinal vessels.

(A) Confocal immunofluorescence microscopy images showing VCAM-1 expression (red) and nuclei stained by SYTOX (green) in retinal whole-mounts from wt mice fed normal chow diet and from ApoE−/− mice fed normal chow or 4 weeks of HFD. Bars = 50 µm. Measurements were performed when mice were 17 weeks of age. (B) Summarized data from experiments as in A, showing increased mean fluorescence intensity of VCAM-1 in ApoE−/− (light gray bar) when compared to wt controls (white bar; ***p<0.001) and further increased VCAM-1 expression in response to HFD (dark gray bar; ## p<0.01). (C) Mean sVCAM-1 concentrations in plasma from the same mice used in A and B. ***p<0.001 vs. wt chow and # p<0.05 for differences between ApoE−/− groups (chow vs. HFD). (D) Expression of VCAM-1 mRNA was studied by real time RT-PCR in normolipidemic FVBN mice. Expression was enhanced by 4 or 8 weeks of HFD (plain and patterned gray bars, respectively) when compared to mice fed regular chow diet (white bars). The effects were significant after 8 weeks (*p<0.05). Values are normalized to the expression of cyclophilin B and GAPDH. (E) Mean sVCAM-1 concentrations in plasma from the same mice used in D. **p<0.01 vs. wt chow.

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