Impairment of pericyte-endothelium crosstalk leads to blood-brain barrier dysfunction following traumatic brain injury

Highlights

• Traumatic brain injury impairs pericyte-endothelium integrity.

• TBI causes impairment in the platelet-derived growth factor B (PDGF-B)/PDGF receptor-β signaling

• Impairment of pericyte-endothelium interaction leads to brain edema.

• Impairment of pericyte-endothelium increases the permeability of BBB and its dysfunctioning following TBI.

Abstract

The blood-brain barrier (BBB) constitutes a neurovascular unit formed by microvascular endothelial cells, pericytes, and astrocytes. Brain pericytes are important regulators of BBB integrity, permeability, and blood flow. Pericyte loss has been implicated in injury; however, how the crosstalk among pericytes, endothelial cells, and astrocytes ultimately leads to BBB dysfunction in traumatic brain injury (TBI) remains elusive. In this study, we demonstrate the importance of pericyte-endothelium interaction in maintaining the BBB function. TBI causes the platelet-derived growth factor-B (PDGF-B)/PDGF receptor-β signaling impairment that results in loss of interaction with endothelium and leads to neurovascular dysfunction. Using in vivo mild (7 psi) and moderate (15 psi) fluid percussion injury (FPI) in mice, we demonstrate the expression of various pericyte markers including PDGFR-β, NG2 and CD13 that were significantly reduced with a subsequent reduction in the expression of various integrins; adherent junction protein, N-cadherin; gap junction protein, connexin-43; and tight junction proteins such as occludin, claudin-5, ZO-1, and JAM-a. Impairment of pericyte-endothelium interaction increases the BBB permeability to water that is marked by a significant increase in aquaporin4 expression in injured animals. Similarly, pericyte-endothelium integrity impairment in FPI animals greatly increases the permeability of small-molecular-weight sodium fluorescein and high-molecular-weight-tracer Evans blue across the BBB. In addition, the injury-inflicted animals show significantly higher levels of S100β and NSE in the blood samples compared with controls. In conclusion, our data provide an insight that brain trauma causes an early impairment of pericyte-endothelium integrity and results in BBB dysregulation that initiates pathological consequences associated with TBI.

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.