β4 integrin is not essential for localization of hemidesmosome proteins plectin and CD151 in cerebral vessels

Abstract

Objective: In the central nervous system (CNS), β4 integrin is predominantly expressed by endothelial cells lining arterioles. As β4 integrin plays an essential role in epithelial tissues, organizing structural proteins into specialized adhesive structures called hemidesmosomes (HD), the aim of this study was to determine whether it plays a similar role in CNS endothelium.

Methods: Dual-immunofluorescence was used to examine the relationship between β4 integrin expression and co-expression of the HD proteins plectin and CD151 in frozen sections of mouse brain, both under normoxic (control) conditions and following chronic mild hypoxia. The requirement of β4 integrin for the localization of HD proteins was examined in transgenic mice lacking β4 integrin expression specifically in endothelial cells (β4-EC-KO mice).

Results: Immunofluorescence revealed that in the normal adult CNS, plectin and CD151 strongly co-localized with β4 integrin in arterioles. However, in the chronic mild hypoxia model, in which extensive cerebrovascular remodeling is observed, plectin and CD151 were strongly upregulated on all cerebral vessels, but surprisingly, in capillaries, this occurred in a β4 integrin-independent manner. Unexpectedly, absence of endothelial β4 integrin (in β4-EC-KO mice) had no impact on the expression level or distribution pattern of plectin and CD151 within stable or remodeling cerebral vessels.

Conclusions: These results demonstrate that the HD proteins plectin and CD151 are closely associated with β4 integrin on arterioles in normal brain, and are strongly upregulated on remodeling blood vessels. However, unlike its described role in the epidermis, β4 integrin is not essential for localization or regulation of expression of plectin and CD151 in cerebral vessels.

Keywords: CD151; central nervous system; hemidesmosome; integrin; plectin; vessel.

Figure 1

Colocalization of β4 integrin with plectin and CD151 on cerebral blood vessels. Dual-immunofluorescence was performed on frozen sections of the frontal lobe from adult mice using antibodies specific for β4 integrin (AlexaFluor-488, green), plectin (Cy3, red), CD151 (Cy3, red), or the endothelial marker CD31 (AlexaFluor-488, green). Scale bar = 100 μm. Note that plectin expression strongly colocalized with β4 integrin and that CD151 was also expressed at high levels by the same medium-sized cerebral blood vessels (diameter range 10–25 μm)

Figure 2

Increased expression of plectin and CD151 on remodeling blood vessels in the hypoxic central nervous system. Frozen sections of frontal lobe taken from adult mice exposed to normoxia or 4, 7, or 14 days mild hypoxia (8% O₂) were stained with antibodies specific for plectin (Cy3, red) and β4 integrin (AlexaFluor-488, green) (a), or antibodies specific for CD151 (Cy3, red) and β4 integrin (AlexaFluor-488, green) (b). Scale bar = 100 μm. Note that in the normoxic brain, plectin and CD151 strongly colocalize with β4 integrin. However, after 4 and 7 days of mild hypoxia, while all β4 integrin-positive vessels still expressed plectin and CD151, a large number of small caliber (<8 μm diameter) β4 integrin-negative vessels now expressed plectin and CD151. Quantification of the influence of mild hypoxia on vascular plectin (c) and CD151 (d) expression in the frontal lobe and medulla oblongata. Experiments were performed with three different animals, and the results were expressed as the mean ± standard error of the mean of the number of vessels positive for each antigen per field of view. Note that hypoxia promoted a transient increase in vascular plectin and CD151 expression in both areas of the brain, which peaked after 7 days hypoxia, *P < 0.01

Figure 3

Absence of endothelial β4 integrin had no impact on the expression or distribution pattern of plectin within cerebral vessels. Frozen sections of frontal lobe taken from wild-type (a) or β4-EC-KO mice (b) that had been exposed to normoxia or 4, 7, or 14 days mild hypoxia (8% O2) were stained with antibodies specific for plectin (AlexaFluor-488, green) or α-SMA (Cy3, red). Scale bar = 100 μm. Note that plectin and α-SMA strongly colocalized in the normoxic brain and that after 4 and 7 days of mild hypoxia, many α-SMA-negative small vessels strongly upregulated plectin expression. However, surprisingly, the expression pattern of plectin in the brain of β4-EC-KO mice (b) was no different from wild-type controls (a). (c) Comparison of vascular plectin upregulation in wild-type and β4-EC-KO mice exposed to mild hypoxia. Expression was evaluated in two areas of the brain (frontal lobe and medulla oblongata) with three different animals per strain, and the results were expressed as the mean ± standard error of the mean of the number of plectin-positive vessels per field of view. Note that hypoxia promoted a transient increase in plectin expression in both strains of mice, with no detectable differences between the two strains

Figure 4

Absence of endothelial β4 integrin had no impact on the expression or distribution pattern of CD151 within cerebral vessels. Frozen sections of frontal lobe taken from wild-type (a) or β4-EC-KO mice (b) that had been exposed to normoxia or 4, 7, or 14 days mild hypoxia (8% O2) were stained with antibodies specific for CD151 (AlexaFluor-488, green) or α-SMA (Cy3, red). Scale bar = 100 μm. Note that CD151 and α-SMA strongly colocalized in the normoxic brain and that after 4 and 7 days of mild hypoxia, many α-SMA-negative small vessels strongly upregulated CD151 expression. However, surprisingly, the expression pattern of CD151 in the brain of β4-EC-KO mice (b) was no different from wild-type controls (a). (c) Comparison of vascular CD151 upregulation in wild-type and β4-EC-KO mice exposed to mild hypoxia. Expression was evaluated in two areas of the brain (frontal lobe and medulla oblongata) using three different animals per strain, and the results were expressed as the mean ± standard error of the mean of the number of CD151-positive vessels per field of view. Note that hypoxia promoted a transient increase in vascular CD151 expression in both strains of mice, with no detectable differences between the two strains