Activation of PKC modulates blood-brain barrier endothelial cell permeability changes induced by hypoxia and posthypoxic reoxygenation

Abstract

-The blood-brain barrier (BBB) is a metabolic and physiological barrier important for maintaining brain homeostasis. The aim of this study was to determine the role of PKC activation in BBB paracellular permeability changes induced by hypoxia and posthypoxic reoxygenation using in vitro and in vivo BBB models. In rat brain microvessel endothelial cells (RMECs) exposed to hypoxia (1% O 2-99% N2; 24 h), a significant increase in total PKC activity was observed, and this was reduced by posthypoxic reoxygenation (95% room air-5% CO 2) for 2 h. The expression of PKC-␤II, PKC-␥, PKC-, PKC-, and PKC-also increased following hypoxia (1% O 2-99% N2; 24 h), and these protein levels remained elevated following posthypoxic reoxygenation (95% room air-5% CO 2; 2 h). Increases in the expression of PKC-⑀ and PKC-were also observed following posthypoxic reoxygenation (95% room air-5% CO 2; 2 h). Moreover, inhibition of PKC with chelerythrine chloride (10 M) attenuated the hypoxiainduced increases in [ 14 C]sucrose permeability. Similar to what was observed in RMECs, total PKC activity was also stimulated in cerebral microvessels isolated from rats exposed to hypoxia (6% O 2-94% N2; 1 h) and posthypoxic reoxygenation (room air; 10 min). In contrast, hypoxia (6% O2-94% N2; 1 h) and posthypoxic reoxygenation (room air; 10 min) significantly increased the expression levels of only PKC-␥ and PKC-in the in vivo hypoxia model. These data demonstrate that hypoxia-induced BBB paracellular permeability changes occur via a PKC-dependent mechanism, possibly by differentially regulating the protein expression of the 11 PKC isozymes. protein kinase C; paracellular; neurovascular unit; rat THE BLOOD-BRAIN BARRIER (BBB) is a metabolic and physiological barrier important for maintaining cerebral homeostasis. Brain microvessels that form the BBB are lined with specialized endothelial cells surrounded by pericytes, astroglial processes, and the extracellular matrix. Compared with the peripheral microvasculature, cerebral microvessels are highly specialized because they lack vesicular transport and fenestrations while having a high level of metabolic activity. This lack of fenestrations is due to the presence of tight junctions (TJ) and adherens junctions, which restrict paracellular movement of molecules across the BBB (29,31,40).Stroke is a leading cause of death and disability in the United States (3). It has been demonstrated that the BBB is compromised during stroke (29). The effects of stroke on the cerebral vasculature significantly contribute to the brain damage caused by stroke. It has been determined that the lack of oxygen (hypoxia, H) followed by reperfusion (posthypoxic reoxygenation, H/R) during stroke contributes to both neuronal and vascular damage. Both H and H/R cause increases in cerebrovascular permeability with concomitant increases in vasogenic cerebral edema (1,36,47).Previous studies (36, 47) have demonstrated that H and H/R cause changes in paracellular permeability to [ 14 C]sucrose in cerebral vascular endothelial cells....

Protein Kinase C Activation Modulates Reversible Increase in Cortical Blood–Brain Barrier Permeability and Tight Junction Protein Expression during Hypoxia and Posthypoxic Reoxygenation

“…We have shown increased expression and activation of nPKC-y and aPKC-z after Hx. Our biochemical results are in agreement with an earlier in vitro and in vivo study (Fleegal et al, 2005), which showed that Hx and HR increased total PKC activity through altered expression of specific PKC isozymes. In vivo, Hx induced a large increase in activity of cPKC-g, -bII, and nPKC-e, whereas nPKC-y and aPKC-z showed modest increases, although nPKC-y showed much greater levels of expression during HR.…”

“…There was also increased membrane expression of the 47.9-kDa occludin band, whereas the 51.5-kDa band showed little change. Chelerythrine chloride has been shown to attenuate Hx-induced paracellular permeability changes rat brain microvessel endothelial cell cultures (Fleegal et al, 2005). We believe this is the first in vivo demonstration that PKC isozyme inhibition attenuates Hx-induced increased vascular permeability and supports the earlier in vitro work.…”

Interendothelial Claudin-5 Expression Depends on Cerebral Endothelial Cell–Matrix Adhesion by β1-Integrins

“…After 48 hours hypoxia, an increase in the permeability of brain capillary endothelial cell monolayers was observed that was associated with rearrangements of F-actin filaments and decreased ATP levels (Plateel et al, 1995). Changes in blood-brain barrier permeability are associated with protein phosphorylations that are regulated by protein kinase C isotypes, tyrosine phosphatase, or kinase activity (Staddon et al, 1995;Tsukamoto and Nigam, 1999;Fleegal et al, 2005). It is likely that cytoskeletal reorganization has an important role in the relation between b 1 -integrin adhesion and tight junction integrity.…”

Angiotensin II Modulates BBB Permeability via Activation of the AT1 Receptor in Brain Endothelial Cells

“…In BBB endothelial cells treated with Ang II (100 nmol/L) for 0 to 6 h, there was a significant increase in PKC activity seen as early as 1 h, which remained elevated at both 2 and 6 h ( Figure 5). As studies have shown that PKC can regulate paracellular permeability and several studies have suggested that PKC regulates the TJs (Clarke et al, 2000;Fleegal et al, 2005;Wosik et al, 2007), we also looked at whether Ang II altered the expression of the TJ proteins occludin and claudin 5. There was no change in the expression of these two proteins or the cytoskeletal protein, actin ( Figure 6; Table 1).…”