Mechanism of action of collagenase on the permeability of the blood-brain barrier

Some proteolytic enzymes are able to increase reversibly the permeability of the blood-brain barrier (BBB) to different tracers such as trypan blue. Intraventricularly injected collagenase is the most potent of the enzymes tested. It was assumed that collagenase acts on basement membrane collagen, the partial hydrolysis of which increases BBB permeability, and that the recovery of normal permeability requires resynthesis of the degraded substrate. In this paper, it is shown that injection of collagenase in lateral brain ventricles of rats increases the level of hydroxyproline (hypro) in the CSF, suggesting that collagen is indeed degraded by the enzyme. We also demonstrate that treatment with inhibitors of protein synthesis—puromycin and cycloheximide—delays considerably the recovery of normal BBB permeability, which occurs 140 h after collagenase treatment instead of 70–72 h without inhibitors. This fact indicates that protein synthesis is necessary for the recovery of normal BBB permeability. The demonstration of release of hypro in the cerebrospinal fluid (CSF) after collagenase action, and of the necessity of protein synthesis for the recovery of normal permeability, supports the above-mentioned hypothesis, according to which basement membrane collagen plays a role in the regulation of the permeability of the BBB.     

Histochemical localization of capillary enzyme activities in brain smears.

Since the regulation of the vascular permeability in the CNS is dependent partly on the enzymes associated with the wall of brain capillaries, the histochemical demonstration of these enzymes may furnish further data on the function of the blood brain barrier (BBB), a new methodological approach, using brain smears was developed for the histochemical demonstration of several enzymes participating in the function of the BBB. The method presented renders possible also the subsequent demonstration of monoamines and the activity of different enzymes in the same tissue preparation. The usefulness of this simple technique in the study of brain capillary functions is discussed.     

Role of VEGF in an experimental model of cortical micronecrosis

Vascular endothelial growth factor (VEGF) is a major mediator in angiogenesis and vascular permeability. In central nervous system (CNS) it plays a pivotal role as: 1. inductor of endothelial cell proliferation, migration and inhibition of apoptosis, and 2. mediator of vascular permeability and subsequently of brain edema. This ubiquitous epiphenomenon is a major complication in several CNS pathologies, including head trauma and stroke.After brain injury the expression of VEGF is increased contributing to disruption of the blood brain barrier (BBB). VEGF increase the permeability of BBB via the synthesis/release of nitric oxide and subsequent activation of soluble guanylate cyclase. The immunohistochemistry shows an increase of stained astrocytes and endothelial cells around cortical micronecrosis. VEGF immunopositivity distribution shows some correspondence with the blood brain barrier breakdown following a cortical micronecrosis.     

Different vascular permeability between the sensory and secretory circumventricular organs of adult mouse brain

The blood-brain barrier (BBB) prevents free access of circulating molecules to the brain and maintains a specialized brain environment to protect the brain from blood-derived bioactive and toxic molecules; however, the circumventricular organs (CVOs) have fenestrated vasculature. The fenestrated vasculature in the sensory CVOs, including the organum vasculosum of lamina terminalis (OVLT), subfornical organ (SFO) and area postrema (AP), allows neurons and astrocytes to sense a variety of plasma molecules and convey their information into other brain regions and the vasculature in the secretory CVOs, including median eminence (ME) and neurohypophysis (NH), permits neuronal terminals to secrete many peptides into the blood stream. The present study showed that vascular permeability of low-molecular-mass tracers such as fluorescein isothiocyanate (FITC) and Evans Blue was higher in the secretory CVOs and kidney as compared with that in the sensory CVOs. On the other hand, vascular permeability of high-molecular-mass tracers such as FITC-labeled bovine serum albumin and Dextran 70,000 was lower in the CVOs as compared with that in the kidney. Prominent vascular permeability of low- and high-molecular-mass tracers was also observed in the arcuate nucleus. These data demonstrate that vascular permeability for low-molecular-mass molecules is higher in the secretory CVOs as compared with that in the sensory CVOs, possibly for large secretion of peptides to the blood stream. Moreover, vascular permeability for high-molecular-mass tracers in the CVOs is smaller than that of the kidney, indicating that the CVOs are not totally without a BBB.     

Connexin Channels at the Glio-Vascular Interface: Gatekeepers of the Brain

Neuronal survival, electrical signaling and synaptic activity require a well-balanced micro-environment in the central nervous system. This is achieved by the blood–brain barrier (BBB), an endothelial barrier situated in the brain capillaries, that controls near-to-all passage in and out of the brain. The endothelial barrier function is highly dependent on signaling interactions with surrounding glial, neuronal and vascular cells, together forming the neuro-glio-vascular unit. Within this functional unit, connexin (Cx) channels are of utmost importance for intercellular communication between the different cellular compartments. Connexins are best known as the building blocks of gap junction (GJ) channels that enable direct cell–cell transfer of metabolic, biochemical and electric signals. In addition, beyond their role in direct intercellular communication, Cxs also form unapposed, non-junctional hemichannels in the plasma membrane that allow the passage of several paracrine messengers, complementing direct GJ communication. Within the NGVU, Cxs are expressed in vascular endothelial cells, including those that form the BBB, and are eminent in astrocytes, especially at their endfoot processes that wrap around cerebral vessels. However, despite the density of Cx channels at this so-called gliovascular interface, it remains unclear as to how Cx-based signaling between astrocytes and BBB endothelial cells may converge control over BBB permeability in health and disease. In this review we describe available evidence that supports a role for astroglial as well as endothelial Cxs in the regulation of BBB permeability during development as well as in disease states.     

Effects of hypoxia-reoxygenation on rat blood-brain barrier permeability and tight junctional protein expression

Cerebral microvessel endothelial cells that form the blood-brain barrier (BBB) have tight junctions (TJs) that are critical for maintaining brain homeostasis. The effects of initial reoxygenation after a hypoxic insult (H/R) on functional and molecular properties of the BBB and TJs remain unclear. In situ brain perfusion and Western blot analyses were performed to assess in vivo BBB integrity on reoxygenation after a hypoxic insult of 6% O2 for 1 h. Model conditions [blood pressure, blood gas chemistries, cerebral blood flow (CBF), and brain ATP concentration] were also assessed to ensure consistent levels and criteria for insult. In situ brain perfusion revealed that initial reoxygenation (10 min) significantly increased the uptake of [14C]sucrose into brain parenchyma. Capillary depletion and CBF analyses indicated the perturbations were due to increased paracellular permeability rather than vascular volume changes. Hypoxia with reoxygenation (10 min) produced an increase in BBB permeability with associated alterations in tight junctional protein expression. These results suggest that H/R leads to reorganization of TJs and increased paracellular diffusion at the BBB, which is not a result of increased CBF, vascular volume change, or endothelial uptake of marker. Additionally, the tight junctional protein occludin had a shift in bands that correlated with functional changes (i.e., increased permeability) without significant change in expression of claudin-3, zonula occludens-1, or actin. H/R-induced changes in the BBB may result in edema and/or associated pathological outcomes.     

Differential effects of HIF-1 inhibition by YC-1 on the overall outcome and blood-brain barrier damage in a rat model of ischemic stroke

Hypoxia-inducible factor 1 (HIF-1) is a master regulator of cellular adaptation to hypoxia and has been suggested as a potent therapeutic target in cerebral ischemia. Here we show in an ischemic stroke model of rats that inhibiting HIF-1 and its downstream genes by 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) significantly increases mortality and enlarges infarct volume evaluated by MRI and histological staining. Interestingly, the HIF-1 inhibition remarkably ameliorates ischemia-induced blood-brain barrier (BBB) disruption determined by Evans blue leakage although it does not affect brain edema. The result demonstrates that HIF-1 inhibition has differential effects on ischemic outcomes and BBB permeability. It indicates that HIF-1 may have different functions in different brain cells. Further analyses show that ischemia upregulates HIF-1 and its downstream genes erythropoietin (EPO), vascular endothelial growth factor (VEGF), and glucose transporter (Glut) in neurons and brain endothelial cells and that YC-1 inhibits their expression. We postulate that HIF-1-induced VEGF increases BBB permeability while certain other proteins coded by HIF-1's downstream genes such as epo and glut provide neuroprotection in an ischemic brain. The results indicate that YC-1 lacks the potential as a cerebral ischemic treatment although it confers certain protection to the cerebral vascular system.     

Effects of radiofrequency radiation exposure on blood-brain barrier permeability in male and female rats

During the last several decades, numerous studies have been performed aiming at the question of whether or not exposure to radiofrequency radiation (RFR) influences the permeability of the blood-brain barrier (BBB). The objective of this study was to investigate the effect of RFR on the permeability of BBB in male and female Wistar albino rats. Right brain, left brain, cerebellum, and total brain were analyzed separately in the study. Rats were exposed to 0.9 and 1.8 GHz continuous-wave (CW) RFR for 20 min (at SARs of 4.26 mW/kg and 1.46 mW/kg, respectively) while under anesthesia. Control rats were sham-exposed. Disruption of BBB integrity was detected spectrophotometrically using the Evans-blue dye, which has been used as a BBB tracer and is known to be bound to serum albumin. Right brain, left brain, cerebellum, and total brain were evaluated for BBB permeability. In female rats, no albumin extravasation was found in in the brain after RFR exposure. A significant increase in albumin was found in the brains of the RF-exposed male rats when compared to sham-exposed male brains. These results suggest that exposure to 0.9 and 1.8 GHz CW RFR at levels below the international limits can affect the vascular permeability in the brain of male rats. The possible risk of RFR exposure in humans is a major concern for the society. Thus, this topic should be investigated more thoroughly in the future.     

Effects of radiofrequency radiation exposure on blood-brain barrier permeability in male and female rats

During the last several decades, numerous studies have been performed aiming at the question of whether or not exposure to radiofrequency radiation (RFR) influences the permeability of the blood-brain barrier (BBB). The objective of this study was to investigate the effect of RFR on the permeability of BBB in male and female Wistar albino rats. Right brain, left brain, cerebellum, and total brain were analyzed separately in the study. Rats were exposed to 0.9 and 1.8?GHz continuous-wave (CW) RFR for 20?min (at SARs of 4.26?mW/kg and 1.46?mW/kg, respectively) while under anesthesia. Control rats were sham-exposed. Disruption of BBB integrity was detected spectrophotometrically using the Evans-blue dye, which has been used as a BBB tracer and is known to be bound to serum albumin. Right brain, left brain, cerebellum, and total brain were evaluated for BBB permeability. In female rats, no albumin extravasation was found in in the brain after RFR exposure. A significant increase in albumin was found in the brains of the RF-exposed male rats when compared to sham-exposed male brains. These results suggest that exposure to 0.9 and 1.8?GHz CW RFR at levels below the international limits can affect the vascular permeability in the brain of male rats. The possible risk of RFR exposure in humans is a major concern for the society. Thus, this topic should be investigated more thoroughly in the future.     

SSeCKS regulates angiogenesis and tight junction formation in blood-brain barrier

The blood-brain barrier (BBB) is essential for maintaining brain homeostasis and low permeability. BBB maintenance is important in the central nervous system (CNS) because disruption of the BBB may contribute to many brain disorders, including Alzheimer disease and ischemic stroke. The molecular mechanisms of BBB development remain ill-defined, however. Here we report that src-suppressed C-kinase substrate (SSeCKS) decreases the expression of vascular endothelial growth factor (VEGF) through AP-1 reduction and stimulates expression of angiopoietin-1 (Ang-1), an antipermeability factor in astrocytes. Conditioned media from SSeCKS-overexpressing astrocytes (SSeCKS-CM) blocked angiogenesis in vivo and in vitro. Moreover, SSeCKS-CM increased tight junction proteins in endothelial cells, consequently decreasing [3H]sucrose permeability. Furthermore, immunoreactivity to SSeCKS gradually increased during the BBB maturation period, and SSeCKS-expressing astrocytes closely interacted with zonula occludens (ZO)-1-expressing blood vessels in vivo. Collectively, our results suggest that SSeCKS regulates BBB differentiation by modulating both brain angiogenesis and tight junction formation.     

Induction of blood brain barrier tight junction protein alterations by CD8 T cells

Disruption of the blood brain barrier (BBB) is a hallmark feature of immune-mediated neurological disorders as diverse as viral hemorrhagic fevers, cerebral malaria and acute hemorrhagic leukoencephalitis. Although current models hypothesize that immune cells promote vascular permeability in human disease, the role CD8 T cells play in BBB breakdown remains poorly defined. Our laboratory has developed a novel murine model of CD8 T cell mediated central nervous system (CNS) vascular permeability using a variation of the Theiler's virus model of multiple sclerosis. In previous studies, we observed that MHC class II(-/-) (CD4 T cell deficient), IFN-gammaR(-/-), TNF-alpha(-/-), TNFR1(-/-), TNFR2(-/-), and TNFR1/TNFR2 double knockout mice as well as those with inhibition of IL-1 and LTbeta activity were susceptible to CNS vascular permeability. Therefore, the objective of this study was to determine the extent immune effector proteins utilized by CD8 T cells, perforin and FasL, contributed to CNS vascular permeability. Using techniques such as fluorescent activated cell sorting (FACS), T1 gadolinium-enhanced magnetic resonance imaging (MRI), FITC-albumin leakage assays, microvessel isolation, western blotting and immunofluorescent microscopy, we show that in vivo stimulation of CNS infiltrating antigen-specific CD8 T cells initiates astrocyte activation, alteration of BBB tight junction proteins and increased CNS vascular permeability in a non-apoptotic manner. Using the aforementioned techniques, we found that despite having similar expansion of CD8 T cells in the brain as wildtype and Fas Ligand deficient animals, perforin deficient mice were resistant to tight junction alterations and CNS vascular permeability. To our knowledge, this study is the first to demonstrate that CNS infiltrating antigen-specific CD8 T cells have the capacity to initiate BBB tight junction disruption through a non-apoptotic perforin dependent mechanism and our model is one of few that are useful for studies in this field. These novel findings are highly relevant to the development of therapies designed to control immune mediated CNS vascular permeability.     

Phase changes in the permeability of the mouse blood-brain barrier after uneven gamma-irradiation

Two phases of changes in blood-brain-barrier permeability for sour fuchsin have been observed in the early period after head or body irradiation at 2.58 C/kg: an increase (up to 2 h) and a decrease (from 2 to 6 h). The correlation between the decrease in BBB permeability and the frequency of clinical manifestations of radiation disease has been established. BBB permeability progressively increased later after head irradiation (24-120 h) which was indicative of its absolute dysfunction. The changes observed may reflect redistribution of fluid and electrolytes in the vascular bed and tissues leading to the onset of brain tissue edema.     

The blood-brain barrier: an alternative hypothesis

Brain blood vessels, unlike most vessels elsewhere in the body, exhibit a blood-brain barrier (BBB) to certain substances, e.g. trypan blue. Under some circumstances this barrier is no longer effective and the permeability of the vessels increases. Although capillarization is much less in the brain than in many other organs, e.g. heart muscle, total cerebral blood flow per minute is enormous. Consequently, to accommodate a large blood volume with a limited capillary bed, the velocity of blood through brain vessels must be extremely fast. The hypothesis presented in this paper is that this rapid flow results in a low or negative pressure on the endothelium, and plasma and trypan blue are prevented from passing through the wall. The tight junctions of cerebral endothelial cells may be able to withstand only a limited amount of pressure on their luminal surface. If the velocity of blood in brain capillaries decreases, pressure on the endothelium should increase, and brain vessels, like blood vessels elsewhere in the body, become permeable to vital dyes. Other conditions also increase capillary permeability, e.g. acute arterial hypertension or venous congestion. Although brain vessels can adapt to a moderate, gradual change in systemic pressure, when a significant rise in cerebral arterial pressure is abrupt, the compensatory changes in the postcapillary venous bed may be inadequate and consequently intracapillary pressure and vascular permeability are increased. Venous congestion increases intracapillary pressure by restricting capillary outflow as well as by reducing velocity through capillary beds. Under such conditions increased capillary permeability may be indicated by cerebral edema, and even, on occasion, by petechial hemorrhages. In short, if the flow is fast and unimpeded the BBB will be effective; if the velocity decreases, or intracapillary pressure increases for whatever reason, the permeability of the brain endothelium will be abnormally increased.     

P-Glycoprotein Mediated Efflux Limits the Transport of the Novel Anti-Parkinson's Disease Candidate Drug FLZ across the Physiological and PD Pathological In Vitro BBB Models

FLZ, a novel anti-Parkinson''s disease (PD) candidate drug, has shown poor blood-brain barrier (BBB) penetration based on the pharmacokinetic study using rat brain. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two important transporters obstructing substrates entry into the CNS as well as in relation to PD neuropathology. However, it is unclear whether P-gp and BCRP are involved in low BBB permeability of FLZ and what the differences of FLZ brain penetration are between normal and Parkinson''s conditions. For this purpose, in vitro BBB models mimicking physiological and PD pathological-related BBB properties were constructed by C6 astroglial cells co-cultured with primary normal or PD rat cerebral microvessel endothelial cells (rCMECs) and in vitro permeability experiments of FLZ were carried out. High transepithelial electrical resistance (TEER) and low permeability for sodium fluorescein (NaF) confirmed the BBB functionality of the two models. Significantly greater expressions of P-gp and BCRP were detected in PD rCMECs associated with the lower in vitro BBB permeability of FLZ in pathological BBB model compared with physiological model. In transport studies only P-gp blocker effectively inhibited the efflux of FLZ, which was consistent with the in vivo permeability data. This result was also confirmed by ATPase assays, suggesting FLZ is a substrate for P-gp but not BCRP. The present study first established in vitro BBB models reproducing PD-related changes of BBB functions in vivo and demonstrated that poor brain penetration of FLZ and low BBB permeability were due to the P-gp transport.     

VEGF increases permeability of the blood-brain barrier via a nitric oxide synthase/cGMP-dependent pathway

It appears thatthe expression of vascular endothelial growth factor (VEGF) isincreased during brain injury and thus may contribute to disruption ofthe blood-brain barrier (BBB) during cerebrovascular trauma. The firstgoal of this study was to determine the effect of VEGF on permeabilityof the BBB in vivo. The second goal was to determine possible cellularmechanisms by which VEGF increases permeability of the BBB. We examinedthe pial microcirculation in rats using intravital fluorescencemicroscopy. Permeability of the BBB [clearance of FITC-labeleddextran of molecular mass 10,000 Da (FITC-dextran-10K)] anddiameter of pial arterioles were measured in absence and presence ofVEGF (0.01 and 0.1 nM). During superfusion with vehicle (saline),clearance of FITC-dextran-10K from pial vessels was minimal anddiameter of pial arterioles remained constant. Topical application ofVEGF (0.01 nM) did not alter permeability of the BBB toFITC-dextran-10K or arteriolar diameter. However, superfusion with VEGF(0.1 nM) produced a marked increase in clearance of FITC-dextran-10Kand a modest dilatation of pial arterioles. To determine a potentialrole for nitric oxide and stimulation of soluble guanylate cyclase inVEGF-induced increases in permeability of the BBB and arteriolardilatation, we examined the effects ofN^G-monomethyl-L-arginine(L-NMMA; 10 µM) and1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1.0 µM), respectively.L-NMMA and ODQ inhibitedVEGF-induced increases in permeability of the BBB and arteriolardilatation. The findings of the present study suggest that VEGF, whichappears to be increased in brain tissue during cerebrovascular trauma, increases the permeability of the BBB via the synthesis/release ofnitric oxide and subsequent activation of soluble guanylate cyclase.     

Pericytes Contribute to the Disruption of the Cerebral Endothelial Barrier via Increasing VEGF Expression: Implications for Stroke

Disruption of the blood-brain barrier (BBB) integrity occurring during the early onset of stroke is not only a consequence of, but also contributes to the further progression of stroke. Although it has been well documented that brain microvascular endothelial cells and astrocytes play a critical role in the maintenance of BBB integrity, pericytes, sandwiched between endothelial cells and astrocytes, remain poorly studied in the pathogenesis of stroke. Our findings demonstrated that treatment of human brain microvascular pericytes with sodium cyanide (NaCN) and glucose deprivation resulted in increased expression of vascular endothelial growth factor (VEGF) via the activation of tyrosine kinase Src, with downstream activation of mitogen activated protein kinase and PI3K/Akt pathways and subsequent translocation of NF-κB into the nucleus. Conditioned medium from NaCN-treated pericytes led to increased permeability of endothelial cells, and this effect was significantly inhibited by VEGF-neutralizing antibody. The in vivo relevance of these findings was further corroborated in the stroke model of mice wherein the mice, demonstrated disruption of the BBB integrity and concomitant increase in the expression of VEGF in the brain tissue as well as in the isolated microvessel. These findings thus suggest the role of pericyte-derived VEGF in modulating increased permeability of BBB during stroke. Understanding the regulation of VEGF expression could open new avenues for the development of potential therapeutic targets for stroke and other neurological disease.     

Increase of tumor necrosis factor‐α in the blood induces early activation of matrix metalloproteinase‐9 in the brain

Increases of cytokine in the blood play important roles in the pathogenesis of influenza‐associated encephalopathy. TNF‐α was administered intravenously to wild‐type mice, after which blood, CSF and brain tissue were obtained, and changes in BBB permeability, the amounts of MMP‐9 and TIMP‐1, and the localization of activated MMP were assessed. There was a significant increase in BBB permeability after 6 and 12 hr. MMP‐9 was increased after 3 hr in the brain and cerebrospinal fluid, which was earlier than in the serum. TIMP‐1 protein in the brain increased significantly after MMP‐9 had increased. Activation of MMP‐9 was observed in neurons in the cerebral cortex and hippocampus, and in vascular endothelial cells. These findings suggest that an increase in blood TNF‐α promotes activation of MMP‐9 in the brain, and may also induce an increase in permeability of the BBB. Early activation of MMP‐9 in the brain may contribute to an early onset of neurological disorders and brain edema prior to multiple organ failure in those inflammatory diseases associated with highly increased concentrations of TNF‐α in the blood, such as sepsis, burns, trauma and influenza‐associated encephalopathy.     

In vivo monitoring blood‐brain barrier permeability using spectral imaging through optical clearing skull window

The blood‐brain barrier (BBB) plays a key role in the health of the central nervous system. Opening the BBB is very important for drug delivery to brain tissues to enhance the therapeutic effect on brain diseases. It is necessary to in vivo monitor the BBB permeability for assessing drug release with high resolution; however, an effective method is lacking. In this work, we developed a new method that combined spectral imaging with an optical clearing skull window to in vivo dynamically monitor BBB opening caused by 5‐aminolevulinic acid (5‐ALA)‐mediated photodynamic therapy (PDT), in which the Evans blue dye (EBd) acted as an indicator of the BBB permeability. Using this method, we effectively monitored the cerebrovascular EBd leakage process. Moreover, the analysis of changes in the vascular and extravascular EBd concentrations demonstrated that the PDT‐induced BBB opening exhibited spatiotemporal differences in the cortex. This spectral imaging method based on the optical clearing skull window provides a low‐cost and simply operated tool for in vivo monitoring BBB opening process. This has a high potential for the visualization of drug delivery to the central nervous system. Thus, it is of tremendous significance in brain disease therapy. Monitoring the changes in PDT‐induced BBB permeability by evaluating the EBd concentration using an optical clearing skull window. (A) Entire brains and coronal sections following treatment of PDT with/without an optical clearing skull window after injection of EBd. (B) Typical EBd distribution maps before and after laser irradiation captured by the spectral imaging method. (Colorbar represents the EBd concentration).      

HIV-1 Tat-mediated effects on focal adhesion assembly and permeability in brain microvascular endothelial cells

The blood-brain barrier (BBB) is a network formed mainly by brain microvascular endothelial cells (BMECs). The integrity of the BBB is critical for brain function. Breakdown of the BBB is commonly seen in AIDS patients with HIV-1-associated dementia despite the lack of productive HIV infection of the brain endothelium. The processes by which HIV causes these pathological conditions are not well understood. In this study we characterized the molecular mechanisms by which Tat mediates its pathogenic effects in vitro on primary human BMECs (HBMECs). Tat treatment of HBMECs stimulated cytoskeletal organization and increased focal adhesion sites compared with control cells or cells treated with heat-inactivated Tat. Pretreatment with Tat Abs or with the specific inhibitor SU-1498, which interferes with vascular endothelial growth factor receptor type 2 (Flk-1/KDR) phosphorylation, blocked the ability of Tat to stimulate focal adhesion assembly and the migration of HBMECs. Focal adhesion kinase (FAK) was tyrosine-phosphorylated by Tat and was found to be an important component of focal adhesion sites. Inhibition of FAK by the dominant interfering mutant form, FAK-related nonkinase, significantly blocked HBMEC migration and disrupted focal adhesions upon Tat activation. Furthermore, HIV-Tat induced permeability changes in HBMECs in a time-dependent manner. Tat also impaired BBB permeability, as observed in HIV-1 Tat transgenic mice. These studies define a mechanism for HIV-1 Tat in focal adhesion complex assembly in HBMECs via activation of FAK, leading to cytoskeletal reorganization and permeability changes.