Regulation of Blood-Brain Barrier Transporters by Transforming Growth Factor-ß/Activin Receptor-Like Kinase 1 Signaling: Relevance to the Brain Disposition of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors (i.e., Statins).

Our laboratory has shown that activation of transforming growth factor- ß (TGF- ß )/activin receptor-like kinase 1 (ALK1) signaling can increase protein expression and transport activity of organic anion transporting polypeptide 1a4 (Oatp1a4) at the blood-brain barrier (BBB). These results are relevant to treatment of ischemic stroke because Oatp transport substrates such as 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (i.e., statins) improve functional neurologic outcomes in patients. Advancement of our work requires determination if TGF- ß /ALK1 signaling alters Oatp1a4 functional expression differently across brain regions and if such disparities affect central nervous system (CNS) statin disposition. Therefore, we studied regulation of Oatp1a4 by the TGF- ß /ALK1 pathway, in vivo, in rat brain microvessels isolated from cerebral cortex, hippocampus, and cerebellum using the ALK1 agonist bone morphogenetic protein-9 (BMP-9) and the ALK1 inhibitor 4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline dihydrochloride 193189. We showed that Oatp1a4 protein expression and brain distribution of three currently marketed statin drugs (i.e., atorvastatin, pravastatin, and rosuvastatin) were increased in cortex relative to hippocampus and cerebellum. Additionally, BMP-9 treatment enhanced Oatp-mediated statin transport in cortical tissue but not in hippocampus or cerebellum. Although brain drug delivery is also dependent upon efflux transporters, such as P-glycoprotein and/or Breast Cancer Resistance Protein, our data showed that administration of BMP-9 did not alter the relative contribution of these transporters to CNS disposition of statins. Overall, this study provides evidence for differential regulation of Oatp1a4 by TGF- ß /ALK1 signaling across brain regions, knowledge that is critical for development of therapeutic strategies to target Oatps at the BBB for CNS drug delivery. SIGNIFICANCE STATEMENT: Organic anion transporting polypeptides (Oatps) represent transporter targets for brain drug delivery. We have shown that Oatp1a4 statin uptake is higher in cortex versus hippocampus and cerebellum. Additionally, we report that the transforming growth factor- ß /activin receptor-like kinase 1 agonist bone morphogenetic protein-9 increases Oatp1a4 functional expression, but not efflux transporters P-glycoprotein and Breast Cancer Resistance Protein, in cortical brain microvessels. Overall, this study provides critical data that will advance treatment for neurological diseases where drug development has been challenging.

Transport Properties of Statins by Organic Anion Transporting Polypeptide 1A2 and Regulation by Transforming Growth Factor-ß Signaling in Human Endothelial Cells.

Our in vivo rodent studies have shown that organic anion transporting polypeptide (Oatp) 1a4 is critical for blood-to-brain transport of statins, drugs that are effective neuroprotectants. Additionally, transforming growth factor-ß (TGF-ß) signaling via the activin receptor-like kinase 1 (ALK1) receptor regulates Oatp1a4 functional expression. The human ortholog of Oatp1a4 is OATP1A2. Therefore, the translational significance of our work requires demonstration that OATP1A2 can transport statins and is regulated by TGF-ß/ALK1 signaling. Cellular uptake and monolayer permeability of atorvastatin, pravastatin, and rosuvastatin were investigated in vitro using human umbilical vein endothelial cells (HUVECs). Regulation of OATP1A2 by the TGF-ß/ALK1 pathway was evaluated using bone morphogenetic protein 9 (BMP-9), a selective ALK1 agonist, and LDN193189, an ALK1 antagonist. We showed that statin accumulation in HUVECs requires OATP1A2-mediated uptake but is also affected by efflux transporters (i.e., P-glycoprotein, breast cancer resistance protein). Absorptive flux (i.e., apical-to-basolateral) for all statins was higher than secretory flux (i.e., basolateral-to-apical) and was decreased by an OATP inhibitor (i.e., estrone-3-sulfate). OATP1A2 protein expression, statin uptake, and cellular monolayer permeability were increased by BMP-9 treatment. This effect was attenuated in the presence of LDN193189. Apical-to-basolateral statin transport across human endothelial cellular monolayers requires functional expression of OATP1A2, which can be controlled by therapeutically targeting TGF-ß/ALK1 signaling. Taken together with our previous work, the present data show that OATP-mediated drug transport is a critical mechanism in facilitating neuroprotective drug disposition across endothelial barriers of the blood-brain barrier. SIGNIFICANCE STATEMENT: Transporter data derived from rodent models requires validation in human models. Using human umbilical vein endothelial cells, this study has shown that statin transport is mediated by OATP1A2. Additionally, we demonstrated that OATP1A2 is regulated by transforming growth factor-ß/activin receptor-like kinase 1 signaling. This work emphasizes the need to consider endothelial transporter kinetics and regulation during preclinical drug development. Furthermore, our forward-thinking approach can identify effective therapeutics for diseases for which drug development has been challenging (i.e., neurological diseases).

Sex-specific differences in organic anion transporting polypeptide 1a4 (Oatp1a4) functional expression at the blood-brain barrier in Sprague-Dawley rats.

BACKGROUND: Targeting endogenous blood-brain barrier (BBB) transporters such as organic anion transporting polypeptide 1a4 (Oatp1a4) can facilitate drug delivery for treatment of neurological diseases. Advancement of Oatp targeting for optimization of CNS drug delivery requires characterization of sex-specific differences in BBB expression and/or activity of this transporter. METHODS: In this study, we investigated sex differences in Oatp1a4 functional expression at the BBB in adult and prepubertal (i.e., 6-week-old) Sprague-Dawley rats. We also performed castration or ovariectomy surgeries to assess the role of gonadal hormones on Oatp1a4 protein expression and transport activity at the BBB. Slco1a4 (i.e., the gene encoding Oatp1a4) mRNA expression and Oatp1a4 protein expression in brain microvessels was determined using quantitative real-time PCR and western blot analysis, respectively. Oatp transport function at the BBB was determined via in situ brain perfusion using [3H]taurocholate and [3H]atorvastatin as probe substrates. Data were expressed as mean ± SD and analyzed via one-way ANOVA followed by the post hoc Bonferroni t-test. RESULTS: Our results showed increased brain microvascular Slco1a4 mRNA and Oatp1a4 protein expression as well as increased brain uptake of [3H]taurocholate and [3H]atorvastatin in female rats as compared to males. Oatp1a4 expression at the BBB was enhanced in castrated male animals but was not affected by ovariectomy in female animals. In prepubertal rats, no sex-specific differences in brain microvascular Oatp1a4 expression were observed. Brain accumulation of [3H]taurocholate in male rats was increased following castration as compared to controls. In contrast, there was no difference in [3H]taurocholate brain uptake between ovariectomized and control female rats. CONCLUSIONS: These novel data confirm sex-specific differences in BBB Oatp1a4 functional expression, findings that have profound implications for treatment of CNS diseases. Studies are ongoing to fully characterize molecular pathways that regulate sex differences in Oatp1a4 expression and activity.

Functional Expression of Organic Anion Transporting Polypeptide 1a4 Is Regulated by Transforming Growth Factor-ß/Activin Receptor-like Kinase 1 Signaling at the Blood-Brain Barrier.

Central nervous system (CNS) drug delivery can be achieved by targeting drug uptake transporters such as Oatp1a4. In fact, many drugs that can improve neurologic outcomes in CNS diseases [3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (i.e., statins)] are organic anion transporting polypeptide (OATP) transport substrates. To date, transport properties and regulatory mechanisms of Oatp1a4 at the blood-brain barrier (BBB) have not been rigorously studied. Such knowledge is critical to develop Oatp1a4 for optimization of CNS drug delivery and for improved treatment of neurological diseases. Our laboratory has demonstrated that the transforming growth factor-ß (TGF-ß)/activin receptor-like kinase 1 (ALK1) signaling agonist bone morphogenetic protein 9 (BMP-9) increases functional expression of Oatp1a4 in rat brain microvessels. Here, we expand on this work and show that BMP-9 treatment increases blood-to-brain transport and brain exposure of established OATP transport substrates (i.e., taurocholate, atorvastatin, and pravastatin). We also demonstrate that BMP-9 activates the TGF-ß/ALK1 pathway in brain microvessels as indicated by increased nuclear translocation of specific Smad proteins associated with signaling mediated by the ALK1 receptor (i.e., pSmad1/5/8). Furthermore, we report that an activated Smad protein complex comprised of phosphorylated Smad1/5/8 and Smad4 is formed following BMP-9 treatment and binds to the promoter of the Slco1a4 gene (i.e., the gene that encodes Oatp1a4). This signaling mechanism causes increased expression of Slco1a4 mRNA. Overall, this study provides evidence that Oatp1a4 transport activity at the BBB is directly regulated by TGF-ß/ALK1 signaling and indicates that this pathway can be targeted for control of CNS delivery of OATP substrate drugs.

Blood-brain barrier dysfunction in ischemic stroke: targeting tight junctions and transporters for vascular protection.

The blood-brain barrier (BBB) is a physical and biochemical barrier that precisely controls cerebral homeostasis. It also plays a central role in the regulation of blood-to-brain flux of endogenous and exogenous xenobiotics and associated metabolites. This is accomplished by molecular characteristics of brain microvessel endothelial cells such as tight junction protein complexes and functional expression of influx and efflux transporters. One of the pathophysiological features of ischemic stroke is disruption of the BBB, which significantly contributes to development of brain injury and subsequent neurological impairment. Biochemical characteristics of BBB damage include decreased expression and altered organization of tight junction constituent proteins as well as modulation of functional expression of endogenous BBB transporters. Therefore, there is a critical need for development of novel therapeutic strategies that can protect against BBB dysfunction (i.e., vascular protection) in the setting of ischemic stroke. Such strategies include targeting tight junctions to ensure that they maintain their correct structure or targeting transporters to control flux of physiological substrates for protection of endothelial homeostasis. In this review, we will describe the pathophysiological mechanisms in cerebral microvascular endothelial cells that lead to BBB dysfunction following onset of stroke. Additionally, we will utilize this state-of-the-art knowledge to provide insights on novel pharmacological strategies that can be developed to confer BBB protection in the setting of ischemic stroke.

A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels.

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting from these stimuli can greatly modulate drug delivery to the brain and, by extension, cause considerable challenges in the treatment of central nervous system (CNS) diseases. Many BBB changes that affect pharmacotherapy, involve proteins that are localized and expressed at the level of endothelial cells. Indeed, such knowledge on BBB physiology in health and disease has sparked considerable interest in the study of these membrane proteins. From a basic science research standpoint, this implies a requirement for a simple but robust and reproducible method for isolation of microvessels from brain tissue harvested from experimental animals. In order to prepare membrane samples from freshly isolated microvessels, it is essential that sample preparations be enriched in endothelial cells but limited in the presence of other cell types of the neurovascular unit (i.e., astrocytes, microglia, neurons, pericytes). An added benefit is the ability to prepare samples from individual animals in order to capture the true variability of protein expression in an experimental population. In this manuscript, details regarding a method that is utilized for isolation of rat brain microvessels and preparation of membrane samples are provided. Microvessel enrichment, from samples derived, is achieved by using four centrifugation steps where dextran is included in the sample buffer. This protocol can easily be adapted by other laboratories for their own specific applications. Samples generated from this protocol have been shown to yield robust experimental data from protein analysis experiments that can greatly aid the understanding of BBB responses to physiological, pathophysiological, and pharmacological stimuli.

Role of Transporters in Central Nervous System Drug Delivery and Blood-Brain Barrier Protection: Relevance to Treatment of Stroke.

Ischemic stroke is a leading cause of morbidity and mortality in the United States. The only approved pharmacologic treatment for ischemic stroke is thrombolysis via recombinant tissue plasminogen activator (r-tPA). A short therapeutic window and serious adverse events (ie, hemorrhage, excitotoxicity) greatly limit r-tPA therapy, which indicates an essential need to develop novel stroke treatment paradigms. Transporters expressed at the blood-brain barrier (BBB) provide a significant opportunity to advance stroke therapy via central nervous system delivery of drugs that have neuroprotective properties. Examples of such transporters include organic anion-transporting polypeptides (Oatps) and organic cation transporters (Octs). In addition, multidrug resistance proteins (Mrps) are transporter targets in brain microvascular endothelial cells that can be exploited to preserve BBB integrity in the setting of stroke. Here, we review current knowledge on stroke pharmacotherapy and demonstrate how endogenous BBB transporters can be targeted for improvement of ischemic stroke treatment.

Functional Expression of P-glycoprotein and Organic Anion Transporting Polypeptides at the Blood-Brain Barrier: Understanding Transport Mechanisms for Improved CNS Drug Delivery?

Drug delivery to the central nervous system (CNS) is greatly limited by the blood-brain barrier (BBB). Physical and biochemical properties of the BBB have rendered treatment of CNS diseases, including those with a hypoxia/reoxygenation (H/R) component, extremely difficult. Targeting endogenous BBB transporters from the ATP-binding cassette (ABC) superfamily (i.e., P-glycoprotein (P-gp)) or from the solute carrier (SLC) family (i.e., organic anion transporting polypeptides (OATPs in humans; Oatps in rodents)) has been suggested as a strategy that can improve delivery of drugs to the brain. With respect to P-gp, direct pharmacological inhibition using small molecules or selective regulation by targeting intracellular signaling pathways has been explored. These approaches have been largely unsuccessful due to toxicity issues and unpredictable pharmacokinetics. Therefore, our laboratory has proposed that optimization of CNS drug delivery, particularly for treatment of diseases with an H/R component, can be achieved by targeting Oatp isoforms at the BBB. As the major drug transporting Oatp isoform, Oatp1a4 has demonstrated blood-to-brain transport of substrate drugs with neuroprotective properties. Furthermore, our laboratory has shown that targeting Oatp1a4 regulation (i.e., TGF-ß signaling mediated via the ALK-1 and ALK-5 transmembrane receptors) represents an opportunity to control Oatp1a4 functional expression for the purpose of delivering therapeutics to the CNS. In this review, we will discuss limitations of targeting P-gp-mediated transport activity and the advantages of targeting Oatp-mediated transport. Through this discussion, we will also provide critical information on novel approaches to improve CNS drug delivery by targeting endogenous uptake transporters expressed at the BBB.

Bone morphogenetic protein-9 increases the functional expression of organic anion transporting polypeptide 1a4 at the blood-brain barrier via the activin receptor-like kinase-1 receptor.

Targeting uptake transporters such as organic anion transporting polypeptide 1a4 (Oatp1a4) at the blood-brain barrier (BBB) can facilitate central nervous system (CNS) drug delivery. Effective blood-to-brain drug transport via this strategy requires characterization of mechanisms that modulate BBB transporter expression and/or activity. Here, we show that activation of activin receptor-like kinase (ALK)-1 using bone morphogenetic protein (BMP)-9 increases Oatp1a4 protein expression in rat brain microvessels in vivo. These data indicate that targeting ALK1 signaling with BMP-9 modulates BBB Oatp1a4 expression, presenting a unique opportunity to optimize drug delivery and improve pharmacotherapy for CNS diseases.

Role of buried polar residues in helix bundle stability and lipid binding of apolipophorin III: destabilization by threonine 31.

Apolipophorin III (apoLp-III) from Locusta migratoria is a model exchangeable apolipoprotein that plays a key role in neutral lipid transport. The protein is comprised of a bundle of five amphipathic alpha-helices, with most hydrophobic residues buried in the protein interior. The low stability of apoLp-III is thought to be crucial for lipid-induced helix bundle opening, to allow protein-lipid interactions. The presence of polar residues in the hydrophobic protein interior may facilitate this role. To test this, two buried polar residues, Thr-31 and Thr-144, were changed into alanine by site-directed mutagenesis. Secondary structure analysis and GdnHCl- and temperature-induced denaturation studies indicated an increase in alpha-helical content and protein stability for T31A apoLp-III compared to wild-type apoLp-III. In contrast, T144A had a decreased alpha-helical content and protein stability, while tryptophan fluorescence indicated increased exposure of the hydrophobic interior to buffer. Two mutant proteins that had lysine residues introduced in the hydrophobic core displayed a more pronounced decrease in secondary structure and protein stability. Lipid binding studies using phospholipid vesicles showed that T31A apoLp-III was able to transform phospholipid vesicles into discoidal particles but at a 3-fold reduced rate compared to wild-type apoLp-III. In contrast, the less stable apoLp-III mutants displayed an increased ability to transform phospholipid vesicles. These results demonstrate the inverse correlation between protein stability and the ability to transform phospholipid vesicles into discoidal protein-lipid complexes and that Thr-31 is a key determinant of the relatively low protein stability, thereby promoting apoLp-III to interact with lipid surfaces.