Introduction of P-Glycoprotein in BBB

glycoprotein (P-gp), also known as multidrug-resistant transporter protein-1, is a member of the ATP-binding cassette (ABC) superfamily and is one of the most important efflux transporter proteins. P-gp is mainly expressed in the lumen of the brain microvascular endothelial cells (BMVECs), and it is an important drug-transporting protein on the blood-brain barrier (BBB). It can transport a variety of structurally and functionally diverse compounds from the brain to the bloodstream, thereby maintaining homeostasis in the brain. A recent report indicated that P-gp abnormality contributes to the development of many CNS disorders. The capillary endothelial cells are proliferated by P-gp following focal cerebral ischemia, breaking the BBB and leading to infiltration of inflammatory cells and the secretion of cytokines. But the precise processes or upstream molecules still need to be defined. It's essential to determine whether or not P-gp is modified or regulated in cerebral ischemia in order to help drug reach the brain.

Fig.1. Most therapeutic compounds cannot attain the brain because of the BBB and its expression of efflux transporters. (Montesinos, 2017)

P-gp Aggravates BBB Dysfunction in Ischemic Stroke

In experimental ischemic stroke, there was in vivo and in vitro upregulation of P-gp. P-gp silencing or drug inhibition can prevent ischemic stroke by strengthening and activating the BBB. In this way, P-gp overexpression contributes to BBB dysregulation and stroke progression by inhibiting tight junction proteins (TJPs) and provoking the inflammatory cascade.

By regulating leukocyte adhesion and migration on BMVEC

Q-gp is predominantly expressed on the BMVEC of the BBB. BMVEC is activated soon after stroke, releases pro-inflammatory cytokines, has an abundance of adhesion molecules, releases MMPs that destroy the extracellular matrix, ruptures the basement membrane and ruptures blood vessels. In this way, P-gp might act on BBB damage in ischemic stroke by inhibiting leukocyte adhesion and migration on BMVEC.

By disrupting TJPs

The TJPs, such as ZO-1, Claudin-5, and Occludin, control BBB integrity and permeability, which is altered and redistributed in the wake of ischemic stroke. P-gp silencing upregulated TJP levels, and P-gp overexpression suppressed TJP levels.

By inhibiting endothelial autophagy

Autophagy is an important degradative pathway that maintains cellular and energetic homeostasis and prevents cell death. Activation of autophagy prevents apoptosis in BMVEC after ischemia/reperfusion and maintains the integrity of the blood-brain barrier after ischemic stroke. P-gp silencing activates endothelial autophagy as evidenced by the increase in the LC3-II/LC3-I ratio and Beclin 1 levels, and a decrease in the P62 level. In addition, P-gp silencing inhibited the activity of the Akt/mTOR pathway, which is important for endothelial autophagy. In ischemic brain tissue, P-gp restricts cerebral delivery of glucocorticoids, leading to inhibition of glucocorticoid receptor nuclear translocation, which in turn activates Akt/mTOR signaling. In conclusion, elevated P-gp levels after ischemic stroke exacerbate BBB destruction and brain inflammatory responses by increasing Akt/mTOR activity and inhibiting autophagy activation.

P-gp Exacerbates Brain Injury Following Cerebral Ischemia by Promoting Proinflammatory Microglia Activation

Microglia are activated after cerebral ischemic injury. Once activated, microglia undergo two distinct functional activations/polarizations: classical pro-inflammatory activation and alternating anti-inflammatory activation. It has been shown that P-gp can exacerbate the pro-inflammatory phenotype microglia polarization and inflammatory response induced by ischemic stroke. Mechanistically, it may be mediated by inhibition of glucocorticoid receptor-mediated mRNA decay altering C-C motif chemokine ligand 2 (CCL2) release. P-gp may be a valuable therapeutic target for the development of new drugs for ischemic stroke.

Imaging P-gp Function at the BBB After Cerebral Ischemia

P-gp can limit the distribution of many drugs in the brain. Thus, P-gp is a major obstacle to drug delivery to the ischemic brain. Changes in P-gp function in the BBB may result in changes in response to stroke drugs.

Studying P-gp function in living subjects is a challenging task, especially when the goal is to study the role of P-gp in human disease. Positron emission tomography (PET) imaging using radiolabeled P-gp substrates opens up the possibility of directly and non-invasively measuring drug concentrations in the human brain. This makes it possible to study the effect of altered P-gp function in the BBB on the distribution of drugs to the brain. (R)-[¹¹C]Verapamil and [¹¹C]dLop have been the most frequently used P-gp substrate tracers for PET studies of

P-gp function at the BBB.

Once the PET tracer is injected, radioactivity in the region of interest is measured by PET scanner. They can then be re-interpreted to see the amount of PET tracer and the binding site interaction over time. That's the principle with which labeled P-gp substrates were used as PET tracers — low brain concentrations denote efficient P-gp function, and high brain concentrations denote less efficient P-gp function.

Fig.2. Average [¹¹C]verapamil PET images before (A) and after (B) intervention with P-gp inhibitor Cyclosporine A 25 mg/kg. (Syvänen, et al., 2013)

1. Montesinos, R. (2017). Liposomal drug delivery to the central nervous system. Liposomes, 213-242.
2. Syvänen, S., & Eriksson, J. (2013). Advances in PET imaging of P-glycoprotein function at the blood-brain barrier. ACS chemical neuroscience, 4(2), 225-237.