Abstract
BackgroundIncreased matrix metalloproteinase (MMP)-9 in the plasma and brain is associated with blood-brain barrier (BBB) disruption through proteolytic activity in neuroinflammatory diseases. MMP-9 is present in the brain microvasculature and its vicinity, where brain microvascular endothelial cells (BMECs), pericytes and astrocytes constitute the BBB. Little is known about the cellular source and role of MMP-9 at the BBB. Here, we examined the ability of pericytes to release MMP-9 and migrate in response to inflammatory mediators in comparison with BMECs and astrocytes, using primary cultures isolated from rat brains.MethodsThe culture supernatants were collected from primary cultures of rat brain endothelial cells, pericytes, or astrocytes. MMP-9 activities and levels in the supernatants were measured by gelatin zymography and western blot, respectively. The involvement of signaling molecules including mitogen-activated protein kinases (MAPKs) and phosphoinositide-3-kinase (PI3K)/Akt in the mediation of tumor necrosis factor (TNF)-α-induced MMP-9 release was examined using specific inhibitors. The functional activity of MMP-9 was evaluated by a cell migration assay.ResultsZymographic and western blot analyses demonstrated that TNF-α stimulated pericytes to release MMP-9, and this release was much higher than from BMECs or astrocytes. Other inflammatory mediators [interleukin (IL)-1β, interferon-γ, IL-6 and lipopolysaccharide] failed to induce MMP-9 release from pericytes. TNF-α-induced MMP-9 release from pericytes was found to be mediated by MAPKs and PI3K. Scratch wound healing assay showed that in contrast to BMECs and astrocytes the extent of pericyte migration was significantly increased by TNF-α. This pericyte migration was inhibited by anti-MMP-9 antibody.ConclusionThese findings suggest that pericytes are most sensitive to TNF-α in terms of MMP-9 release, and are the major source of MMP-9 at the BBB. This pericyte-derived MMP-9 initiated cellular migration of pericytes, which might be involved in pericyte loss in the damaged BBB.
Highlights
Increased matrix metalloproteinase (MMP)-9 in the plasma and brain is associated with blood-brain barrier (BBB) disruption through proteolytic activity in neuroinflammatory diseases
To test whether MMP-9 production and subsequent migration of pericytes contribute to BBB disruption associated with neuroinflammation, we examined the ability of pericytes to release MMP-9 and migrate in response to tumor necrosis factor (TNF)-a, and compared it with that of brain microvascular endothelial cells (BMECs) and astrocytes
TNF-a induces MMP-9 release from brain pericytes Gelatin zymographic analysis revealed a band at the position approximately under the standard pro-MMP-9 band, indicating that the supernatant of the pericytes had MMP9 activity (Figure 1A, arrowed band)
Summary
Increased matrix metalloproteinase (MMP)-9 in the plasma and brain is associated with blood-brain barrier (BBB) disruption through proteolytic activity in neuroinflammatory diseases. Brain pericytes are located adjacent to capillaries and share a common basement membrane with brain microvascular endothelial cells (BMECs). This allows pericytes to communicate directly with BMECs through gap junctions and peg-and-socket contacts to stabilize microvessels and progressive pericyte loss with age have shown that BBB integrity is determined by the extent of pericyte coverage of cerebral microvessels [9]. It has been reported that brain pericytes extend toward the parenchyma, and the basal lamina becomes thin in the early stage of brain hypoxia [14] and traumatic injury [15] These morphological alterations were interpreted as the initial step of pericyte migration [16]. Pericytes appear to exhibit high proteolytic activities
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