Luca Cucullo

In Vitro Blood–Brain Barrier Models: Current and Perspective Technologies

Even in the 21st century, studies aimed at characterizing the pathological paradigms associated with the development and progression of central nervous system diseases are primarily performed in laboratory animals. However, limited translational significance, high cost, and labor to develop the appropriate model (e.g., transgenic or inbred strains) have favored parallel in vitro approaches. In vitro models are of particular interest for cerebrovascular studies of the blood-brain barrier (BBB), which plays a critical role in maintaining the brain homeostasis and neuronal functions. Because the BBB dynamically responds to many events associated with rheological and systemic impairments (e.g., hypoperfusion), including the exposure of potentially harmful xenobiotics, the development of more sophisticated artificial systems capable of replicating the vascular properties of the brain microcapillaries are becoming a major focus in basic, translational, and pharmaceutical research. In vitro BBB models are valuable and easy to use supporting tools that can precede and complement animal and human studies. In this article, we provide a detailed review and analysis of currently available in vitro BBB models ranging from static culture systems to the most advanced flow-based and three-dimensional coculture apparatus. We also discuss recent and perspective developments in this ever expanding research field.

Development of a humanized in vitro blood-brain barrier model to screen for brain penetration of antiepileptic drugs.

Summary:  Purpose: A biotechnologic breakthrough for the study of drug permeability across the blood–brain barrier (BBB) would be the use of a reproducible in vitro model that recapitulates the functional, structural, and pathologic properties of the BBB in situ. We developed a humanized dynamic in vitro BBB model (DIV‐BBB) based on cocultures of human microvascular endothelial cells (HBMECs) from “normal” and drug‐resistant epileptic brain tissue with human brain astrocytes (HAs) from epilepsy patients or controls.

Oxidative and pro-inflammatory impact of regular and denicotinized cigarettes on blood brain barrier endothelial cells: is smoking reduced or nicotine-free products really safe?

BackgroundBoth active and passive tobacco smoke (TS) potentially impair the vascular endothelial function in a causative and dose-dependent manner, largely related to the content of reactive oxygen species (ROS), nicotine, and pro-inflammatory activity. Together these factors can compromise the restrictive properties of the blood–brain barrier (BBB) and trigger the pathogenesis/progression of several neurological disorders including silent cerebral infarction, stroke, multiple sclerosis and Alzheimer’s disease. Based on these premises, we analyzed and assessed the toxic impact of smoke extract from a range of tobacco products (with varying levels of nicotine) on brain microvascular endothelial cell line (hCMEC/D3), a well characterized human BBB model.ResultsInitial profiling of TS showed a significant release of reactive oxygen (ROS) and reactive nitrogen species (RNS) in full flavor, nicotine-free (NF, “reduced-exposure” brand) and ultralow nicotine products. This release correlated with increased oxidative cell damage. In parallel, membrane expression of endothelial tight junction proteins ZO-1 and occludin were significantly down-regulated suggesting the impairment of barrier function. Expression of VE-cadherin and claudin-5 were also increased by the ultralow or nicotine free tobacco smoke extract. TS extract from these cigarettes also induced an inflammatory response in BBB ECs as demonstrated by increased IL-6 and MMP-2 levels and up-regulation of vascular adhesion molecules, such as VCAM-1 and PECAM-1.ConclusionsIn summary, our results indicate that NF and ultralow nicotine cigarettes are potentially more harmful to the BBB endothelium than regular tobacco products. In addition, this study demonstrates that the TS-induced toxicity at BBB ECs is strongly correlated to the TAR and NO levels in the cigarettes rather than the nicotine content.

Impact of altered glycaemia on blood-brain barrier endothelium: an in vitro study using the hCMEC/D3 cell line

BackgroundCerebrovascular complications involving endothelial dysfunction at the blood-brain barrier (BBB) are central to the pathogenesis of diabetes-related CNS disorders. However, clinical and experimental studies have reported contrasting evidence in relation to the effects of hyperglycemia on BBB permeability and function. Similarly the effect of hypoglycemia on BBB integrity is not well understood. Therefore, we assessed the differential impact of hypo and hyperglycemic conditions on BBB integrity and endothelial function in vitro using hCMEC/D3, a well characterized human brain microvascular endothelial cell line.MethodsParallel monolayers of hCMEC/D3 were exposed to normal, hypo- or hyperglycemic media, containing 5.5, 2.2 or 35 mM D-glucose, respectively. Following 3-24h exposure, the expression and distribution of BBB tight junction (ZO-1 and claudin-5) adherence junction (VE-cadherin) proteins, and glucose transporters as well as inflammatory (VCAM-1) and oxidative stress (Nrf-2) markers were analyzed by immunofluorescence and western blotting. Endothelial release of growth factors and pro-inflammatory cytokines were determined by ELISA. Further, the impact of altered glycemia on BBB permeability was assessed in hCMEC/D3 – astrocyte co-cultures on Transwell supports using fluorescent dextrans (4–70 kDa).ResultsCompared to controls, exposure to hypoglycemia (3 and 24h) down-regulated the expression of claudin-5 and disrupted the ZO-1 localization at cell-cell contacts, while hyperglycemia marginally reduced claudin-5 expression without affecting ZO-1 distribution. Permeability to dextrans (4-10 kDa) and VEGF release at 24h were significantly increased by hypo- and hyperglycemia, although 70 kDa dextran permeability was increased only under hypoglycemic conditions. The expression of SGLT-1 was up-regulated at 24h hypoglycemic exposure while only a modest increase of GLUT-1 expression was observed. In addition, the expression of Nrf-2 and release of interleukin-6 and PDGF-BB, were down-regulated by hypoglycemia (but not hyperglycemia), while both conditions induced a marginal and transient increase in VCAM-1 expression from 3 to 24h, including a significant increase in VE-cadherin expression at 3 h following hyperglycemia.ConclusionsIn summary, our findings demonstrate a potential impairment of BBB integrity and function by hypo or hyperglycemia, through altered expression/distribution of TJ proteins and nutrient transporters. In addition, hypoglycemic exposure severely affects the expression of oxidative and inflammatory stress markers of BBB endothelium.

A decade of e-cigarettes: Limited research & unresolved safety concerns.

It is well known that tobacco consumption is a leading cause of preventable deaths worldwide and has been linked to major diseases ranging from cancer to chronic obstructive pulmonary disease, atherosclerosis, stroke and a host of neurological/neurodegenerative disorders. In the past decade a number of alternative vaping products have hit the market, rapidly gaining consumers especially among the younger population. Electronic nicotine delivery systems or e-cigarettes have become the sought-after product due to the belief that they are much safer than traditional cigarettes. However, inadequate research and lack of regulatory guidelines for both the manufacturing process and the content of the vaping solution of the e-cigarette has become a major concern. Highly debated and unresolved questions such as whether e-cigarettes may help smokers quit and whether e-cigarettes will promote the use of nicotine among non-smokers add to the confusion of the safety of e-cigarettes. In this review article, we summarize the current understanding (and lack thereof) of the potential health impacts of e-cigarettes. We will also highlight the most recent studies (in vivo/in vitro) which seem to conflict with the broad safety claims put forward by the manufacturers. Finally, we provide potential solutions to overcome the research gap of the short and long-term health impact of e-cigarettes.

In Vitro Assessment of Tobacco Smoke Toxicity at the BBB: Do Antioxidant Supplements Have a Protective Role?

BackgroundTobacco smoke (TS) contains highly reactive oxygen species (such as hydrogen peroxide, peroxynitrite, etc), which cause oxidative damage in vascular tissue and may exacerbate inflammatory events leading to the blood-brain barrier damage (BBBD) which accompanies the development of a variety of neurological disorders. Smokers often have elevated leukocyte counts (primarily neutrophils and monocytes), and significant decreases in plasma alpha-tocopherol (vitamin E) and ascorbic acid (vitamin C) levels due to increased anti-oxidative mobilization in response to oxidative stress evoked by TS. For this purpose, using static culture systems and a well-established dynamic in vitro BBB model (DIV-BBB) we tested the hypothesis that antioxidant vitamin supplementation (E and/or C) can protect the BBB during exposure to whole soluble TS.ResultsTS exacerbates inflammatory events and leads to endothelial overexpression of vascular adhesion molecules (VCAM-1, P-selectin and E-selectin), release of pro-inflammatory cytokines (TNF-α and IL-6) and nitric oxide (NO), release and activation of matrix metalloproteinases (MMP-2 and MMP-9), monocytic maturation into macrophages, and adhesion to the vascular endothelium. Furthermore, TS altered the normal glucose metabolic behaviour of in vitro BBB capillaries and caused a period of transient anaerobic respiration to meet the cellular bioenergetic demand. Pre-treatment with antioxidant vitamins (C and/or E) effectively reduced the pro-inflammatory activity associated with TS, protecting the viability and functions of the BBB.ConclusionOur results have shown that loss of endothelial viability as well as BBB function and integrity caused by TS exposure can be prevented or at least reduced by normal physiologic concentrations of antioxidant vitamins in vitro.

Altered Nrf2 Signaling Mediates Hypoglycemia-Induced Blood–Brain Barrier Endothelial Dysfunction In Vitro

Hypoglycemia impairs blood-brain barrier (BBB) endothelial function; a major hallmark in the pathogenesis of various CNS disorders. Previously, we have demonstrated that prolonged hypoglycemic exposure down-regulated BBB endothelial NF-E2 related factor-2 (Nrf2) expression; a redox-sensitive transcriptional factor that regulates endothelial function. Here, we sought to determine the functional role of Nrf2 in preserving BBB integrity and molecular mechanisms underlying hypoglycemia-induced Nrf2 down-regulation in vitro using human cerebral microvascular endothelial cell line (hCMEC/D3). Cell monolayers were exposed to normal or hypoglycemic (5.5 or 2.2mM D-glucose) media for 3-24h. Pharmacological or gene manipulation (by silencing RNA) approaches were used to investigate specific molecular pathways implicated in hypoglycemia-induced Nrf2 degradation. BBB integrity was assessed by paracellular permeability to labeled dextrans of increasing molecular sizes (4-70kDa). Silencing Nrf2 expression in hCMEC/D3 cells abrogated the expression of claudin-5 and VE-cadherin, while ZO-1 was up-regulated. These effects were paralleled by a decrease in electrical resistance of hCMEC/D3 monolayers and potential increase in permeability to all labeled dextrans. Hypoglycemic exposure (3-24h) led to progressive and sustained down-regulation of Nrf2 (without affecting mRNA) and its target, NQO-1, with a concomitant increase in the cytosolic pool of E3 ubiquitin ligase, Siah2 (but not Keap1). Pretreatment with protease inhibitor MG132, or selective knock-down of Siah2 (but not Keap1) significantly attenuated hypoglycemia-induced Nrf2 destabilization. While hypoglycemic exposure triggered a significant increase in BBB permeability to dextrans, silencing Siah2 gene abrogated the effects of hypoglycemia and restored BBB integrity. In summary, our data indicate a potential role for Nrf2 signaling in regulating tight junction integrity and maintaining BBB function. Nrf2 suppression by increased Siah2-driven proteasomal degradation mediates hypoglycemia-evoked endothelial dysfunction and loss of BBB integrity. Overall, this study suggests that sustained activation of endothelial Nrf2 signaling could have therapeutic potential to prevent hypoglycemia-induced cerebrovascular dysfunction.

Drugs of abuse and blood-brain barrier endothelial dysfunction: A focus on the role of oxidative stress

Psychostimulants and nicotine are the most widely abused drugs with a detrimental impact on public health globally. While the long-term neurobehavioral deficits and synaptic perturbations are well documented with chronic use of methamphetamine, cocaine, and nicotine, emerging human and experimental studies also suggest an increasing incidence of neurovascular complications associated with drug abuse. Short- or long-term administration of psychostimulants or nicotine is known to disrupt blood-brain barrier (BBB) integrity/function, thus leading to an increased risk of brain edema and neuroinflammation. Various pathophysiological mechanisms have been proposed to underlie drug abuse-induced BBB dysfunction suggesting a central and unifying role for oxidative stress in BBB endothelium and perivascular cells. This review discusses drug-specific effects of methamphetamine, cocaine, and tobacco smoking on brain microvascular crisis and provides critical assessment of oxidative stress-dependent molecular pathways focal to the global compromise of BBB. Additionally, given the increased risk of human immunodeficiency virus (HIV) encephalitis in drug abusers, we have summarized the synergistic pathological impact of psychostimulants and HIV infection on BBB integrity with an emphasis on unifying role of endothelial oxidative stress. This mechanistic framework would guide further investigations on specific molecular pathways to accelerate therapeutic approaches for the prevention of neurovascular deficits by drugs of abuse.

HMGB1 and thrombin mediate the blood-brain barrier dysfunction acting as biomarkers of neuroinflammation and progression to neurodegeneration in Alzheimer’s disease

BackgroundThe blood-brain barrier (BBB) dysfunction represents an early feature of Alzheimer’s disease (AD) that precedes the hallmarks of amyloid beta (amyloid β) plaque deposition and neuronal neurofibrillary tangle (NFT) formation. A damaged BBB correlates directly with neuroinflammation involving microglial activation and reactive astrogliosis, which is associated with increased expression and/or release of high-mobility group box protein 1 (HMGB1) and thrombin. However, the link between the presence of these molecules, BBB damage, and progression to neurodegeneration in AD is still elusive. Therefore, we aimed to profile and validate non-invasive clinical biomarkers of BBB dysfunction and neuroinflammation to assess the progression to neurodegeneration in mild cognitive impairment (MCI) and AD patients.MethodsWe determined the serum levels of various proinflammatory damage-associated molecules in aged control subjects and patients with MCI or AD using validated ELISA kits. We then assessed the specific and direct effects of such molecules on BBB integrity in vitro using human primary brain microvascular endothelial cells or a cell line.ResultsWe observed a significant increase in serum HMGB1 and soluble receptor for advanced glycation end products (sRAGE) that correlated well with amyloid beta levels in AD patients (vs. control subjects). Interestingly, serum HMGB1 levels were significantly elevated in MCI patients compared to controls or AD patients. In addition, as a marker of BBB damage, soluble thrombomodulin (sTM) antigen, and activity were significantly (and distinctly) increased in MCI and AD patients. Direct in vitro BBB integrity assessment further revealed a significant and concentration-dependent increase in paracellular permeability to dextrans by HMGB1 or α-thrombin, possibly through disruption of zona occludins-1 bands. Pre-treatment with anti-HMGB1 monoclonal antibody blocked HMGB1 effects and leaving BBB integrity intact.ConclusionsOur current studies indicate that thrombin and HMGB1 are causal proximate proinflammatory mediators of BBB dysfunction, while sTM levels may indicate BBB endothelial damage; HMGB1 and sRAGE might serve as clinical biomarkers for progression and/or therapeutic efficacy along the AD spectrum.

In Vitro Cerebrovascular Modeling in the 21st Century: Current and Prospective Technologies

The blood-brain barrier (BBB) maintains the brain homeostasis and dynamically responds to events associated with systemic and/or rheological impairments (e.g., inflammation, ischemia) including the exposure to harmful xenobiotics. Thus, understanding the BBB physiology is crucial for the resolution of major central nervous system CNS) disorders challenging both health care providers and the pharmaceutical industry. These challenges include drug delivery to the brain, neurological disorders, toxicological studies, and biodefense. Studies aimed at advancing our understanding of CNS diseases and promoting the development of more effective therapeutics are primarily performed in laboratory animals. However, there are major hindering factors inherent to in vivo studies such as cost, limited throughput and translational significance to humans. These factors promoted the development of alternative in vitro strategies for studying the physiology and pathophysiology of the BBB in relation to brain disorders as well as screening tools to aid in the development of novel CNS drugs. Herein, we provide a detailed review including pros and cons of current and prospective technologies for modelling the BBB in vitro including ex situ, cell based and computational (in silico) models. A special section is dedicated to microfluidic systems including micro-BBB, BBB-on-a-chip, Neurovascular Unit-on-a-Chip and Synthetic Microvasculature Blood-brain Barrier.