Research paperImpairment of pericyte-endothelium crosstalk leads to blood-brain barrier dysfunction following traumatic brain injury
Introduction
Pericytes in the central nervous system (CNS) plays a critical and complex regulatory role interacting with other cell types of the neurovascular unit, especially endothelial cells and astrocytes (Sweeney et al., 2016). Within the neurovascular unit, the CNS pericytes are uniquely positioned between the neurons, astrocytes and endothelial cells (Fig. 1A). Pericytes ensheath the capillary wall, making direct contacts with endothelial cells (Armulik et al., 2005; Diaz-Flores et al., 2009). Brain pericytes interact with their neighboring cells and process signals to execute diverse functional response such as regulation of blood-brain barrier (BBB) permeability, angiogenesis, clearance of toxic metabolites, capillary hemodynamic responses, neuroinflammation, and stem cell activity that are critical for CNS functions in health and disease. Pericyte-endothelial interactions play an important role in the maintenance of BBB with critical effects on the structure and function of the basement membrane and endothelial tight junction (TJ) (Daneman and Prat, 2015). Endothelial-secreted platelet-derived growth factor B (PDGF-B) binds to the platelet-derived growth factor receptor beta (PDGFR-β) on pericytes, initiating multiple signal transduction pathways regulating proliferation, migration, and pericytes recruitment to the vascular wall (Lebrin et al., 2010). Signaling mediated by PDGFR-β promotes pericyte attachment to endothelial cells, migration, and proliferation (Lindahl et al., 1999; Tallquist et al., 2003; Tallquist and Soriano, 2003).
Pericyte loss or ablation is one of the hallmarks of BBB dysfunction and has been suggested to trigger several pathological conditions such as abnormal BBB leakage, edema, micro-aneurysm formation, ischemia and so forth (Hellstrom et al., 1999; Lindahl et al., 1997). Under physiological conditions, BBB integrity is highly dependent on the ability of pericytes, endothelial cells and astrocytes to maintain a highly restricted environment in the brain against the entry of blood-borne factors and circulating immune cells. In previous studies, we have demonstrated that the induction of oxidative stress activates TGF-β1 (Patel et al., 2017) and matrix metalloproteinases (MMPs) that lead to disruption of BBB, and induction of inflammatory signaling following TBI (Abdul-Muneer et al., 2017b; Abdul-Muneer et al., 2017d). Another study demonstrated that MMP-9 induces migration of the pericytes from the endothelium leading to pericyte loss and disruption of BBB (Takata et al., 2011). Recent studies in adult and aging brain demonstrated that pericyte is required for capillary perfusion, cerebral blood flow (CBF) and BBB integrity (Bell et al., 2010). Furthermore, in 2010, Armulik et al. and Daneman et al. independently showed that pericytes are required for BBB function during development using pericyte-deficient mouse models resulting from defective PDGF/PDGFR-β signaling (Armulik et al., 2010; Daneman et al., 2010). In an aging brain, pericyte loss and subsequent increase of vessel permeability promote neuroinflammation and neurodegeneration (Sengillo et al., 2013). In spite of the important role of brain pericytes in human health, the molecular mechanisms that regulate their development, survival, and distribution remain poorly understood.
In the present study, we propose pericytes loss following TBI is a consequence of down-regulation in the PDGF-B/PDGFR-β signaling pathway that results in the impairment of pericyte-endothelium interaction in BBB and leads to neurovascular dysfunction. Here, we show that PDGF-B/PDGFR-β signaling is critical for pericyte maintenance and is dispensable for BBB integrity. Pericyte loss following TBI results in significant reduction in the expression of pericyte markers such as PDGFR-β, NG2 (chondroitin sulfate proteoglycan 4) and CD13 (alanyl (membrane) aminopeptidase) and leads to permeability of BBB marked by a significant increase in Aquaporin4 (AQP4). Moreover, our data also provide strong evidence that mechanical disruption of vascular integrity and/or increased permeability with functional changes at the BBB occurring after trauma leads to subsequent reduction in the expression of extracellular matrix (ECM) proteins such as N-cadherin and Connexin-43 that connect endothelium and pericyte and TJ proteins such as Occludin, Claudin 5, ZO-1 and JAM-a. Hence, restoration of pericyte ablation using a pharmacological approach and keeping pericyte-endothelium integrity following TBI presents a new therapeutic possibility in maintaining BBB integrity and thus provides a better avenue for the treatment of TBI-related neurological disorders.
Section snippets
Reagents
The primary antibodies rabbit anti-PDGFR-β (Cat. No: 3169; RRID: AB_2162497), anti-NG2 (Cat. No: 4235; RRID: AB_2087604), anti-CD13 (Cat. No: 13721), anti-N-cadherin (Cat. No: 13116; RRID: AB_2687616), anti-connexin-43 (Cat. No: 3512; RRID: AB_2294590), and anti-integrin α6 (Cat. No: 3750S; RRID: AB_2249263) were purchased from Cell Signaling Technology, Danvers, MA. Antibodies anti-integrin α3β1 (Cat. No: ab217145), anti-integrin β1 (Cat. No: ab183666), anti-occludin (Cat. No: ab31721; RRID: AB_881773
TBI down regulates pericyte markers
Our first aim was to show the expression level and role of pericytes in the integrity of BBB, and how brain injury compromises this integrity. Using immunofluorescence imaging analysis for PDGFR-β, a specific marker for pericytes (Winkler et al., 2010), we show that PDGFR-β co-localizes with pericytes on brain capillaries, as illustrated in a brain cortex tissue section of injured and uninjured mice (Fig. 1B). We have not seen the expression of PDGFR-β in other cell types such as neurons,
Discussion
Pericytes within the neurovascular unit are capable of regulating many neurovascular functions, including development and maintenance of structural elements of the BBB, integrity of BBB, vascular stability and angiogenesis, and regulation of blood flow at the capillary level (Winkler et al., 2011). Pericytes are required for the formation of an integral part of BBB, the tight junctions, and transendothelial vesicle trafficking (Armulik et al., 2010; Daneman et al., 2010). Pericytes exert their
Conflict of interest
The authors state that they have no conflict of interest.
Acknowledgments
This work was supported by the Neuroscience Institute at Hackensack Meridian Health JFK Medical Center, Edison, NJ to P.M. Abdul-Muneer.
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