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Comparative Study
. 2010 Dec 31:11:102.
doi: 10.1186/1471-2121-11-102.

Lack of α8 integrin leads to morphological changes in renal mesangial cells, but not in vascular smooth muscle cells

Affiliations
Comparative Study

Lack of α8 integrin leads to morphological changes in renal mesangial cells, but not in vascular smooth muscle cells

Ines Marek et al. BMC Cell Biol. .

Abstract

Background: Extracellular matrix receptors of the integrin family are known to regulate cell adhesion, shape and functions. The α8 integrin chain is expressed in glomerular mesangial cells and in vascular smooth muscle cells. Mice deficient for α8 integrin have structural alterations in glomeruli but not in renal arteries. For this reason we hypothesized that mesangial cells and vascular smooth muscle cells differ in their respective capacity to compensate for the lack of α8 integrin.

Results: Wild type and α8 integrin-deficient mesangial cells varied markedly in cell morphology and expression or localization of cytoskeletal molecules. In α8 integrin-deficient mesangial cells α-smooth muscle actin and CTGF were downregulated. In contrast, there were no comparable differences between α8 integrin-deficient and wild type vascular smooth muscle cells. Expression patterns of integrins were altered in α8 integrin-deficient mesangial cells compared to wild type mesangial cells, displaying a prominent overexpression of α2 and α6 integrins, while expression patterns of the these integrins were not different between wild type and α8 integrin-deficient vascular smooth muscle cells, respectively. Cell proliferation was augmented in α8 integrin-deficient mesangial cells, but not in vascular smooth muscle cells, compared to wild type cells.

Conclusions: Our findings suggest that α8 integrin deficiency has differential effects in mesangial cells and vascular smooth muscle cells. While the phenotype of vascular smooth muscle cells lacking α8 integrin is not altered, mesangial cells lacking α8 integrin differ considerably from wild type mesangial cells which might be a consequence of compensatory changes in the expression patterns of other integrins. This could result in glomerular changes in α8 integrin-deficient mice, while the vasculature is not affected in these mice.

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Figures

Figure 1
Figure 1
Mesangial cell expression of α8 integrin in wild type (wt) and α8 integrin-deficient mesangial cells (α8-/-). A: Real-time RT-PCR analysis of α8 integrin expression in wt and α8-/- mesangial cells. B: Western blot analysis of α8 integrin protein in wt and α8-/- mesangial cells. Amido black (Abl) staining of the blot served as loading control. Results are representative for at least 3 similar experiments. Data are means ± sd. * p < 0.05 vs. wt.
Figure 2
Figure 2
Comparison of wild type (wt) and α8 integrin-deficient (α8-/-) mesangial cell morphology after hematoxylin stain (A+B), immunofluorescent double staining for f-actin in green and vinculin in red (high magnification C+D, low magnification E+F), immunofluorescent staining for f-actin alone (G+H) or immunofluorescent staining for vinculin alone (I+K). White arrows indicate focal contacts of the cells and white arrowheads indicate bundles of focal contacts in α8-/- mesangial cells.
Figure 3
Figure 3
Comparison of α-smooth muscle actin expression in wild type (wt) and α8 integrin-deficient (α8-/-) mesangial cells. mRNA expression analysis by real-time RT-PCR analysis (A), representative Western blot analysis with amido black (Abl) staining as loading control (B) and immunofluorescent detection of α-smooth muscle actin (C). Arrows indicate cell nuclei of cells negative for α-smooth muscle actin. D: Doublestaining of wild type mesangial cells for α-smooth muscle actin (left) and f-actin (middle panel). Detection of f-actin in α8-/- mesangial cells (right). Results are representative for at least 3 similar experiments. Data are means ± sd. * p < 0.05 vs. wt.
Figure 4
Figure 4
Expression of α8 integrin in vascular smooth muscle cells. Real-time RT-PCR analysis of α8 integrin expression in wild type (wt) vascular smooth muscle cells cultivated in passage 1 or 10 (A). Real-time RT-PCR analysis of α8 integrin expression in freshly isolated wt and α8-/- vascular smooth muscle cells (B). Western blot analysis of α8 integrin expression in freshly isolated wt and α8-/- vascular smooth muscle cells (C). Amido black (Abl) staining of the blot served as loading control. Results are representative for at least 3 similar experiments. Data are means ± sd. * p < 0.05 vs. freshly isolated cells or wt, respectively.
Figure 5
Figure 5
Comparison of wild type (wt) and α8 integrin-deficient (α8-/-) vascular smooth muscle cell morphology after hematoxylin stain (A + B) or immunofluorescent staining for f-actin (C +D) or immunofluorescent staining for vinculin (E + F).
Figure 6
Figure 6
Comparison of α-smooth muscle actin expression in wild type (wt) and α8 integrin-deficient (α8-/-) freshly isolated vascular smooth muscle cells. α8 integrin expression by real-time RT-PCR analysis (A), Western blot analysis with amido black (Abl) staining as loading control. (B) and immunofluorescent detection (C) (x1000). Results are representative for at least 3 similar experiments.
Figure 7
Figure 7
Connective tissue growth factor (CTGF; 36 kDa) protein expression in wild type (wt) and α8 integrin-deficient (α8-/-) mesangial cells (MCs) and vascular smooth muscle cells (VSCMs). Staining for β-actin was used as a loading control. Results are representative for 3 similar experiments.
Figure 8
Figure 8
Real-time RT-PCR analysis of integrin chain α1, α2, α3, α5, α6, αv and β1 expression profiles in wild type (wt) and α8 integrin-deficient (α8-/-) mesangial cells (A) and freshly isolated vascular smooth muscle cells (B). Results are representative for at least 3 similar experiments. Data are means ± sd. * p < 0.05 vs. wt.
Figure 9
Figure 9
Real-time RT-PCR analyses of desmin (Des), vimentin (Vim) and E-cadherin (E-cad) in wild type (wt) and α8 integrin-deficient (α8-/-) mesangial cells (MCs) and freshly isolated vascular smooth muscle cells (VSMCs). For E-cadherin expression in MCs, a positive control (liver cells) was used. Results are representative for at least 3 similar experiments. Data are means ± sd. * p < 0.05 vs. wt.
Figure 10
Figure 10
Cell proliferation of wild type (wt) and α8 integrin-deficient (α8-/-) mesangial cells (A) and vascular smooth muscle cells (B) in response to stimulation with 10% FCS. Results are representative for at least 3 similar experiments. Data are means ± sd. * p < 0.05 vs. wt.

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