BACKGROUND
the blood-brain barrier is a dynamic interface between the blood and the central nervous system , that controls the influx and efflux of biological substances needed for the brain metabolic processes, as well as for neuronal function.
therefore the functional and structural integrity of the bbb is vital to maintain the homeostasis of the brain microenvironment.
at the cellular level, the bbb consists of microvascular endothelial cells  lining the brain microvessels together with the closely associated astrocytic end-feet processes  <cit> .
the microcapillary endothelium is characterized by the presence of tight junctions, lack of fenestrations, and minimal pinocytotic vesicles.
in particular, tight junctions between the cerebral endothelial cells form a diffusion barrier, which selectively excludes most blood-borne substances from entering the brain, protecting it from systemic influences mediated by substances of all size or polar molecules such as water soluble compounds .
transport for nutrients  from the peripheral circulation into brain parenchyma requires translocation through the capillary endothelium by specialized carrier-mediated transport systems.
membrane localization of these enzymes is indicative of the polarity of the endothelial functions in the control of the blood-brain interface  <cit> .
the bbb endothelial cytoplasm is richly endowed with enzymes, including monoamine oxidase, acid and alkaline phosphatases, p <dig> enzymes  <cit>  and is also characterized by very high density of mitochondria denoting high metabolic activity  <cit> .
furthermore, the cellular membrane hosts a variety of adhesion molecules and integrins that allow for the interaction with the host immune system when activated by pro-inflammatory stimuli  <cit> .
this plethora of highly specialized functions is indicative of a significant level of differentiation that sets apart the bbb endothelium from that of other vascular beds.
while the physiological environment is certainly responsible for the differentiation of these endothelial cells into a bbb phenotype, the mechanisms involved are not fully understood.
the surrounding cellular elements  by means of trophic stimuli  are crucially important for the ec differentiation however, there is an underestimated and poorly understood mechanical stimuli that also plays a major role in this process, such as the exposure to shear stress .
ss is a tangential force generated by flow across the apical surface of vascular endothelium.
in this study we show that ss affects endothelial cell by modulating the induction/suppression of genes, which impact the development of bbb properties and functions.
RESULTS
shear stress promoted bbb tightness
gene array analysis from ec samples grown under static and dynamic conditions in presence of abluminal astrocytes showed  that exposure to capillary-like ss levels   <cit>  increases the rna levels of a variety of tight and adherens junction components such zonula occludens- <dig> , claudin  <dig> and  <dig>  cadherins, catenin α <dig> and β <dig>  and actin α <dig>  <cit> .
actin filaments in conjunction with catenin molecules provide the structural cellular connection with the transmembrane proteins forming the inter-endothelial tight and adherens junctional complexes  <cit> .
the functional significance of this response to ss is quite evident if one looks at the structural integrity and tightness of the corresponding vascular endothelial beds.
as shown in figure 1b ec co-cultured with ah under the influence of ss formed a significantly more stringent barrier  than parallel co-cultures grown under static condition .
barrier selectivity is significantly affected by the tightness of the vascular bed formed.
as shown in figures 1c and 1d ec grown under static conditions did not form a viable barrier capable of differentiating the passage of substances in relation to their real  permeability distributions.
results from gene array analysis were supported by comparative protein quantification of tight and adherens junction proteins in 2d gels of endothelial protein extracts from membrane sub-cellular fractions of ec harvested from the co-culture systems.
figure 1e shows the expression ratio  of key tj components as well as that of adherens junction protein identified on the 2d gels.
exposure to a physiological capillary level of ss significantly increased the expression level of tj components occludin  and claudin  <dig> .
the expression level of the adherens junctions cadherin- <dig> , - <dig> , - <dig>  were also unregulated.
data were expressed as fold changes .
shear stress induces the endothelial expression of drug transporters and metabolic properties that allow the bbb to shield the cns from potentially harmful substances
pivotal properties of the bbb are the shielding from potentially harmful substances and the selective permeability to ions and other nutrients/substances.
exposure to physiological levels of ss increased the rna level of multidrug resistance transporter  commonly expressed at the bbb in vivo.
these included abcb <dig> , abcc <dig> , abcc <dig> , and abcc <dig>   <cit> .
recent studies have also shown that in addition to multidrug resistance proteins, bbb endothelial cells express a variety of cytochrome  p <dig> enzymes which may synergistically contribute to regulate the passage of substances into the brain  <cit> .
we now report that exposure to ss increased the endothelial rna levels of several cyp <dig> enzymes .
these included members of the cyp <dig>  cyp <dig>  and cyp <dig> families , which are mainly involved with steroid and drug metabolism  <cit> ; cyp4b <dig> is a monooxygenase engaged in the metabolic transformation of fatty acids and the synthesis of cholesterol; cyp11b <dig> is a steroid 11β-hydroxylase involved in the biosynthesis of steroids.
additional cyp rna levels that were significantly upregulated are that of cyp19a <dig>  and that of cyp27a <dig>  the latter is a mitochondrial p- <dig> enzyme with broad substrate specificity for c <dig> sterols .
exposure to flow promotes the endothelial expression of ion channels and specialized transport systems
our results  showed that exposure to flow increased the rna levels of different classes of ion channels.
these included voltage-gated potassium channels , voltage-dependent calcium channels  inwardly rectifying potassium channel  delayed-rectifier potassium channels  and other voltage-dependent anion channel .
among the many roles played by the bbb that of supplying essential nutrients to the cns and regulating ionic trafficking and fluid movements between the blood and the brain are considered among the most crucial functions.
the bbb has a very low permeability to ions and polar molecules thus, the presence of specific transporters is essential to regulate the trafficking of these substances across the vascular endothelium.
our results  showed that the rna level of a number of crucial endothelial transporters was significantly altered by the exposure to physiological capillary levels of ss.
rna level of members of the "facilitate glucose transporters family" such as glut- <dig> , glut- <dig> , glut- <dig>  and glut- <dig>  were significantly increased.
that of acetyl-coa transporter  was also significantly upregulated .
similar trend was observed for the rna level of organic anions and cations transporters  as well as that of cationic aminoacids , folate , sulfate , mitocondrial carriers .
several ions transporters  were also upregulated .
by contrast, the rna level of sodium/hydrogen exchanger , the copper transporter , and that of a mitochondrial phosphate carrier  were decreased.
all the results were statistically significant .
shear stress facilitates endothelial-leukocyte cross-talk to respond to pro-inflammatory stimuli
the recruitment of leukocytes from the blood stream and their subsequent interaction  with the vascular endothelium are critical stages of the immune response during inflammation.
the detail mechanisms controlling leukocyte-endothelial interaction at the bbb as well as the molecular mediators that are involved in the process are not fully understood.
it is clear however, that the vascular endothelium plays a crucial role in this process through the presentation of various adhesion ligands at sites of inflammation.
our study  has shown that exposure to ss increased the rna levels of several key adhesion molecules including icam <dig>  vcam <dig> and pecam <dig>  which play an important role on the adhesion and migration of t lymphocytes  <cit> .
e and p-selectin rna was instead downregulated.
some data suggest that they do not have a critical role in leukocyte recruitment across the bbb  <cit>  but their function is not fully understood.
integrins play a role in cell signaling and therefore they are involved with the interactions between ecs and the surrounding environment.
exposure to ss increases the rna level of several integrins .
for example the alpha chains  <dig>   <dig>   <dig>  and the beta chains  <dig>   <dig>  and  <dig>  interact with the extracellular matrix   <cit> .
shear stress modulate the bioenergetic behaviour on the bbb endothelial cells
our results  have shown that the ecs rna level of the key metabolic switch controller  pyruvate dehydrogenase is significantly increased by the exposure to flow.
by contrast that of lactate dehydrogenase  is decreased.
in addition to that, the rna level of many dehydrogenases and other key enzyme controlling the krebb cycle  including the acetyl-coa transporter  were also increased.
the effect of flow on the glucose metabolism was confirmed by measurements of glucose consumption and lactate production over a period of  <dig> weeks from the establishment of the co-culture systems .
our experimental data showed that endothelial cells grown under dynamic conditions develop a metabolic behavior that makes significant use of aerobic respiration.
this is demonstrated by the comparative measurements of lactate produced over glucose consumed  showing that at least 50% of the glucose is channeled into the citric acid cycle and metabolically converted in co <dig> and h2o with a significant release of bioenergetic equivalents .
this was not observed in parallel co-cultures where ecs were not exposed to flow.
in this case we measured a lactate produced over glucose consumed ratio ≈  <dig>  in this case each molecule of glucose underwent anaerobic metabolic transformation into two molecules of lactate.
these data further strengthen previous funding  <cit>  suggesting that the exposure to intraluminal flow plays a key role in determining the intracellular bioenergetic pathway .
this hypothesis is also supported by the fact that the corresponding expression levels of the key enzymes controlling the aerobic metabolic pattern were also upregulated .
by contrast, the expression of lactate dehydrogenase,  was downregulated.
laminar shear stress inhibits endothelial cell proliferation
inhibition of cell proliferation is a common prodromic event to cell differentiation.
our results showed that ss affects the transcription of genes involved in the cell cycle regulation.
btg proteins are in this specific case novel regulators of transcription engaged in the control of the cell cycle.
although the biological array of functions of btg proteins is not fully understood, it is known that btg <dig> act as a growth arrest gene responsible for the maintenance of the quiescent state, while btg <dig> acts as a negative regulator of the cell cycle.
therefore both btg <dig> and  <dig> act as negative regulators of cell proliferation .
our results have shown that ss significantly increases the rna level of both b-cell translocation gene  <dig>  and gene  <dig>  .
btg <dig> and  <dig> also interact with protein arginine n-methyltransferase  <dig>   <cit> , which activity is critical for growth factor-induced cell differentiation  <cit> .
by contrast, the rna level of key regulators g1/s checkpoint phase which favor cell division such as cdk4/6-cyclin d, cyclin d <dig>  the transcription factor e2f <dig> as well as that of histone deacetylase  <dig>   <cit>  was significantly decreased.
gene array data were also supported by parallel analysis and comparative quantification of the corresponding proteins .
our data have shown that ss significantly increased the expression of prmt <dig>  as well as that of cyclin-dependent kinase inhibitor  .
by contrast, retinoblastoma binding protein and e2f transcription factor  <dig> were significantly downregulated .
these results were also indirectly corroborated by measurement of the sub-cellular  and total protein content .
our results showed that the exposure to flow significantly increased the expression of cytoskeletal proteins  while reducing that of the other sub-cellular fractions.
the total amount of protein content/cell was increased by flow and the sub-cellular protein content showed that the largest component of the total cellular protein pool  was made by cytoskeletal proteins  with cytosolic, nuclear and membrane protein pools significantly reduced .
discussion
bbb endothelial cells in vivo are continuously exposed to laminar shear stress to which they respond by structural  and functional remodeling showing significant evidences of differentiation  <cit> .
one of the main functions of the bbb is shielding of the brain from unwanted and potentially harmful substances.
tight junction protein complexes provide a mechanical means to seal the paracellular pathways between adjacent endothelial cells  <cit> .
our study shows that exposure to flow increases the rna levels of genes encoding for a variety of tight junctional proteins  including the intracellular scaffold proteins zonula occludens- <dig>  and  <dig>  which link the junctional molecules claudin and occludin to intracellular actin and the cytoskeleton.
claudin  <dig>   <dig> and actin gene espression were also upregulated by the exposure to laminar shear stress thus strongly suggesting that acting as a key regulator of tj expression.
in addition to tj protein upregulation we also found a significant increase the rna and protein levels of several cadherins.
these are a class of type- <dig> transmembrane proteins which play important roles in cell adhesion, through the formation of adherens junctions.
our data showed the up-regulation of n-, p- and ve-cadherin.
we also observed an increase in the rna and protein levels of e-cadherin.
however, even though the upregulation of e-cadherin  was statistically significant; its overall rna and total protein expression levels were very modest in comparison to that of the other adherens junction components.
the result is the formation of tight barrier with high trans-endothelial electrical resistance that can efficiently discriminate the passage of substances according to their permeability coefficient.
in the absence of intraluminal flow despite the presence of abluminal astrocytes the resulting barrier is much less stringent and does not provide selective permeability .
this demonstrates that glial stimuli are important but not sufficient to establish a functional bbb and that exposure to flow provides the missing modulatory element to enable bbb properties.
bbb shielding function however, is also achieved at other levels: 1) by mechanisms of drugs extrusion.
this is carried out by atp-binding cassette transporters  which are transmembrane proteins that utilize the energy of adenosine triphosphate  hydrolysis to translocate a broad range of substrates across the cellular membranes  <cit> ; 2) by enzymatic metabolism of substances that enter the cell.
this function performed is by cytochrome p <dig> enzymes similar to those present in the hepatocytes.
these enzymes can use a variety of molecules  as substrates in enzymatic reactions  <cit> .
our study shows that exposure to flow increases gene expression of several efflux transporters including the multidrug resistance  <dig>  gene , and members of the multidrug resistance associated protein family mrp <dig> , mrp <dig> , and mrp <dig>  .
p-glycoprotein  the products of the mdr <dig> gene is an effluxer with specificity for cationic or electrically neutral substrates as well as a broad spectrum of amphiphilic substrates  <cit>  while mrps are multispecific organic anion efflux transporters.
togheter these efflux transporters can drastically reduce brain penetration of a broad range of endogenous and exogenous substances which do not follow a paracellular pathway to cross the vascular endothelium.
the "third" barrier is instead metabolic in nature.
the bbb endothelium is also provided with cytochrome p <dig> enzymes which catalyze the oxidation of organic substrates such as lipids and steroidal hormones but also exogenous substances including drugs  <cit> .
recent studies  <cit>  have shown that bbb endothelial p <dig> enzymes may be co-responsible for the onset of drug resistance to pharmacological treatments.
for example, carbamazepine  is an anticonvulsant currently used in the pharmacological treatment of epileptic patients.
cbz is a p <dig> cyp3a <dig> substrate.
overexpression of cyp3a <dig> has been shown to correlate with that of mdr <dig> at the blood-brain barrier interface of patients with drug refractory epilepsy.
our study has shown that exposure to flow significantly increases gene expression levels of most members of the cytochrome p <dig> families cyp <dig> , cyp <dig>  and cyp <dig>  taken togheter the increased gene expression of multidrug resistance proteins and p <dig> enzymes provides a complex mechanism to regulate the cns drug bioavailability, or to shield the cns.
the rna level of cyp11b <dig>  and cyp19a <dig> were also upregulated by flow.
these enzymes are involved in the biosynthesis of steroids and estrogen, which modulate many biological activities including bbb permeability and leukocytes adhesion  <cit> .
cyp27a <dig> rna level was also significantly upregulated by flow.
these p <dig> enzymes are responsible for the degradation of cholesterol and play an in important role in the maintenance of cholesterol pool in the cns  <cit> .
this is in agreement with the fact that enhancement of endothelial permeability and lipid deposition preferentially occurs at turbulent, low shear stress regions such as in the arteries  <cit> .
the shielding provided by the bbb may on the other end limit the paracellular diffusion of polar substances including ions and essential nutrients  necessary for the maintenance of neuronal activity.
therefore the bbb endothelium must be equipped with specific transporters to supply the cns with these substances without compromising its barrier functions and its ability to maintain the homeostasis of the brain environment.
our data have shown that the exposure to ss positively modulates the translational expression of ca2+ and k+ ion channels.
calcium and potassium homeostasis play an important role in the control of neuronal excitability but are also involved in macrophage transmigration across the vascular endothelium at the bbb  <cit> .
furthermore, ca2+ is involved in the modulation of bbb integrity and endothelial morphology  <cit> .
in addition to ion channels the gene expression level of other specialized solute carriers was also found to be modulated by flow.
these include members of the glucose transporters family glut- <dig>  glut- <dig>  glut- <dig> and glut- <dig>  <cit> ; solute carriers specific for cationic aminoacids, organic anions and cations, folate, sulfate as well as the na-k-cl symporter and the potassium-chloride cotransporter.
all together these specialized functions represent the hallmark of a drastic differentiation process that set the brain microcapillary endothelium aside from those forming other vascular beds.
transporters allow nutrients and other biologically important substances to transit from the blood into the brain across the bbb.
to efficiently meet the bioenergetic demand of the many active transport systems that are critical for sustaining neuronal function the bbb ecs make a substantial use of aerobic respiration or citric acid cycle.
this is an oxigen-dependent 8-step enzimatic process that allows the full conversion of  <dig> molecules of acetyl-coa  into carbon dioxide  and water .
the theoretical energetic yield of the process from the complete oxidation of one glucose molecule to co <dig> and h2o is of  <dig> nadh,  <dig> fadh <dig>  and  <dig> atp .
the aerobic pathway also provides reductant equivalents  to counteract the harmful effect of reactive oxidative species generated intracellularly or originating by exposure to xenobiotic substances.
our results show that exposure to flow plays a critical role in the modulation of the bbb-ec bioenergitic metabolism favoring the expression of genes encoding for the key enzymes operating the conversion of acetyl-coa into co <dig> and h2o during the citric acid cycle.
furthermore, shear stress increases the rna levels of pyruvate dehydrogenase .
the aerobic metabolic induction operated by flow on the bbb endothelium was further confirmed by lactate production and glucose consumption measurements demonstrating that at least 50% of the glucose was processed by aerobic respiration.
an important implication of this "metabolic modulation by flow" is that bbb endothelial cells can adapt their metabolic process according to the availability of oxygen.
this is dependent on blood flow therefore, it is logic to postulate that whether blood flow is interrupted  or the oxygen content in the blood is reduced , the vascular endothelium can switch to an anaerobic respiration to meet its energetic demand.
controls on cell proliferation and differentiation are interrelated even though it is still not clear to what extent.
our data  show that exposure to flow increases the rna levels of btg proteins  which are negative regulators of the cell cycle  <cit> .
btg <dig> or btg <dig> bound to prmt  <dig>  is required to induce growth inhibition  <cit> .
furthermore, the rna level of cyclin d, cdk <dig>  and  <dig>  which control the re-entry of resting g <dig> cells into the g <dig> phase of cell cycle were downregulated.
cells that exit the cell cycle to enter and stay at resting  phase is characteristic of terminally differentiated cells.
quantitative analysis of the protein content  relative to nucleus, cytosol, cytoskeleton, and membrane sub-cellular fractions have shown a drastic reduction of the nuclear protein content in ec exposed to flow.
this is in agreement with the lack of dna synthesis in resting cells and the fact that in vivo the endothelial dna synthesis preferentially occurs at branch orifices characterized by low and turbulent flow  <cit> .
furthermore, we observed a substantial increase in cytoskeletal protein content.
this can be related to a morphological adaptation of the bbb endothelium to flow and the necessity to provide structural support for tj protein complexes  <cit> .
taken togheter our results suggest that the exposure to a capillary level laminar shear stress on one hand favors ec differentiation into a bbb phenotype and on the other hand inhibits the cell cycle.
however, whether the two processes are simultaneously regulated is still unclear.
CONCLUSIONS
shear stress plays a key role in modulating endothelial structure and function.
our study shows that ss-dependent effects on ec are modulated by induction/suppression of genes regulating multiple aspects of endothelial physiology; from the formation of inter-endothelial tight junction to the expression of specific carrier-mediated transporters and drug resistance mechanisms.
studies by other have also shown that ss induces the production of vasoactive substances  <cit> .
turbulent, but not laminar shear stress stimulates ec turnover  <cit> .
accordingly, in vivo dna synthesis of ec preferentially occurs at branch orifices with low flow rates  <cit>  which is paralleled by the enhancement of endothelial permeability  <cit> .
genomic and proteomic analyses are currently being used to study the bbb and how it relates to the pathogenesis of major neurological diseases  <cit> .
in this respect this study provides the bases for new hypotheses and future studies to unveil novel unexploited clinical targets that can facilitate the development of innovative therapeutic strategies to reduce the burden of bbb-related cns diseases.

 the blood-brain barrier is a dynamic interface between the blood and the central nervous system, that controls the influx and efflux of biological substances needed for the brain metabolic processes, as well as for neuronal function. therefore the functional and structural integrity of the bbb is vital to maintain the homeostasis of the brain microenvironment. at the cellular level, the bbb consists of microvascular endothelial cells lining the brain microvessels together with the closely associated astrocytic end-feet processes. the microcapillary endothelium is characterized by the presence of tight junctions, lack of fenestrations, and minimal pinocytotic vesicles. in particular, tight junctions between the cerebral endothelial cells form a diffusion barrier, which selectively excludes most blood-borne substances from entering the brain, protecting it from systemic influences mediated by substances of all size or polar molecules such as water soluble compounds. transport for nutrients from the peripheral circulation into brain parenchyma requires translocation through the capillary endothelium by specialized carrier-mediated transport systems. membrane localization of these enzymes is indicative of the polarity of the endothelial functions in the control of the blood-brain interface. the bbb endothelial cytoplasm is richly endowed with enzymes, including monoamine oxidase, acid and alkaline phosphatases, p enzymes and is also characterized by very high density of mitochondria denoting high metabolic activity. furthermore, the cellular membrane hosts a variety of adhesion molecules and integrins that allow for the interaction with the host immune system when activated by pro-inflammatory stimuli. this plethora of highly specialized functions is indicative of a significant level of differentiation that sets apart the bbb endothelium from that of other vascular beds. while the physiological environment is certainly responsible for the differentiation of these endothelial cells into a bbb phenotype, the mechanisms involved are not fully understood. the surrounding cellular elements by means of trophic stimuli are crucially important for the ec differentiation however, there is an underestimated and poorly understood mechanical stimuli that also plays a major role in this process, such as the exposure to shear stress. ss is a tangential force generated by flow across the apical surface of vascular endothelium. in this study we show that ss affects endothelial cell by modulating the induction/suppression of genes, which impact the development of bbb properties and functions.  shear stress promoted bbb tightness gene array analysis from ec samples grown under static and dynamic conditions in presence of abluminal astrocytes showed that exposure to capillary-like ss levels increases the rna levels of a variety of tight and adherens junction components such zonula occludens-, claudin and cadherins, catenin α and β and actin α. actin filaments in conjunction with catenin molecules provide the structural cellular connection with the transmembrane proteins forming the inter-endothelial tight and adherens junctional complexes. the functional significance of this response to ss is quite evident if one looks at the structural integrity and tightness of the corresponding vascular endothelial beds. as shown in figure 1b ec co-cultured with ah under the influence of ss formed a significantly more stringent barrier than parallel co-cultures grown under static condition. barrier selectivity is significantly affected by the tightness of the vascular bed formed. as shown in figures 1c and 1d ec grown under static conditions did not form a viable barrier capable of differentiating the passage of substances in relation to their real permeability distributions. results from gene array analysis were supported by comparative protein quantification of tight and adherens junction proteins in 2d gels of endothelial protein extracts from membrane sub-cellular fractions of ec harvested from the co-culture systems. figure 1e shows the expression ratio of key tj components as well as that of adherens junction protein identified on the 2d gels. exposure to a physiological capillary level of ss significantly increased the expression level of tj components occludin and claudin. the expression level of the adherens junctions cadherin-, -, - were also unregulated. data were expressed as fold changes. shear stress induces the endothelial expression of drug transporters and metabolic properties that allow the bbb to shield the cns from potentially harmful substances pivotal properties of the bbb are the shielding from potentially harmful substances and the selective permeability to ions and other nutrients/substances. exposure to physiological levels of ss increased the rna level of multidrug resistance transporter commonly expressed at the bbb in vivo. these included abcb, abcc, abcc, and abcc. recent studies have also shown that in addition to multidrug resistance proteins, bbb endothelial cells express a variety of cytochrome p enzymes which may synergistically contribute to regulate the passage of substances into the brain. we now report that exposure to ss increased the endothelial rna levels of several cyp enzymes. these included members of the cyp cyp and cyp families, which are mainly involved with steroid and drug metabolism; cyp4b is a monooxygenase engaged in the metabolic transformation of fatty acids and the synthesis of cholesterol; cyp11b is a steroid 11β-hydroxylase involved in the biosynthesis of steroids. additional cyp rna levels that were significantly upregulated are that of cyp19a and that of cyp27a the latter is a mitochondrial p- enzyme with broad substrate specificity for c sterols. exposure to flow promotes the endothelial expression of ion channels and specialized transport systems our results showed that exposure to flow increased the rna levels of different classes of ion channels. these included voltage-gated potassium channels, voltage-dependent calcium channels inwardly rectifying potassium channel delayed-rectifier potassium channels and other voltage-dependent anion channel. among the many roles played by the bbb that of supplying essential nutrients to the cns and regulating ionic trafficking and fluid movements between the blood and the brain are considered among the most crucial functions. the bbb has a very low permeability to ions and polar molecules thus, the presence of specific transporters is essential to regulate the trafficking of these substances across the vascular endothelium. our results showed that the rna level of a number of crucial endothelial transporters was significantly altered by the exposure to physiological capillary levels of ss. rna level of members of the "facilitate glucose transporters family" such as glut-, glut-, glut- and glut- were significantly increased. that of acetyl-coa transporter was also significantly upregulated. similar trend was observed for the rna level of organic anions and cations transporters as well as that of cationic aminoacids, folate, sulfate, mitocondrial carriers. several ions transporters were also upregulated. by contrast, the rna level of sodium/hydrogen exchanger, the copper transporter, and that of a mitochondrial phosphate carrier were decreased. all the results were statistically significant. shear stress facilitates endothelial-leukocyte cross-talk to respond to pro-inflammatory stimuli the recruitment of leukocytes from the blood stream and their subsequent interaction with the vascular endothelium are critical stages of the immune response during inflammation. the detail mechanisms controlling leukocyte-endothelial interaction at the bbb as well as the molecular mediators that are involved in the process are not fully understood. it is clear however, that the vascular endothelium plays a crucial role in this process through the presentation of various adhesion ligands at sites of inflammation. our study has shown that exposure to ss increased the rna levels of several key adhesion molecules including icam vcam and pecam which play an important role on the adhesion and migration of t lymphocytes. e and p-selectin rna was instead downregulated. some data suggest that they do not have a critical role in leukocyte recruitment across the bbb but their function is not fully understood. integrins play a role in cell signaling and therefore they are involved with the interactions between ecs and the surrounding environment. exposure to ss increases the rna level of several integrins. for example the alpha chains and the beta chains and interact with the extracellular matrix. shear stress modulate the bioenergetic behaviour on the bbb endothelial cells our results have shown that the ecs rna level of the key metabolic switch controller pyruvate dehydrogenase is significantly increased by the exposure to flow. by contrast that of lactate dehydrogenase is decreased. in addition to that, the rna level of many dehydrogenases and other key enzyme controlling the krebb cycle including the acetyl-coa transporter were also increased. the effect of flow on the glucose metabolism was confirmed by measurements of glucose consumption and lactate production over a period of weeks from the establishment of the co-culture systems. our experimental data showed that endothelial cells grown under dynamic conditions develop a metabolic behavior that makes significant use of aerobic respiration. this is demonstrated by the comparative measurements of lactate produced over glucose consumed showing that at least 50% of the glucose is channeled into the citric acid cycle and metabolically converted in co and h2o with a significant release of bioenergetic equivalents. this was not observed in parallel co-cultures where ecs were not exposed to flow. in this case we measured a lactate produced over glucose consumed ratio ≈ in this case each molecule of glucose underwent anaerobic metabolic transformation into two molecules of lactate. these data further strengthen previous funding suggesting that the exposure to intraluminal flow plays a key role in determining the intracellular bioenergetic pathway. this hypothesis is also supported by the fact that the corresponding expression levels of the key enzymes controlling the aerobic metabolic pattern were also upregulated. by contrast, the expression of lactate dehydrogenase, was downregulated. laminar shear stress inhibits endothelial cell proliferation inhibition of cell proliferation is a common prodromic event to cell differentiation. our results showed that ss affects the transcription of genes involved in the cell cycle regulation. btg proteins are in this specific case novel regulators of transcription engaged in the control of the cell cycle. although the biological array of functions of btg proteins is not fully understood, it is known that btg act as a growth arrest gene responsible for the maintenance of the quiescent state, while btg acts as a negative regulator of the cell cycle. therefore both btg and act as negative regulators of cell proliferation. our results have shown that ss significantly increases the rna level of both b-cell translocation gene and gene. btg and also interact with protein arginine n-methyltransferase, which activity is critical for growth factor-induced cell differentiation. by contrast, the rna level of key regulators g1/s checkpoint phase which favor cell division such as cdk4/6-cyclin d, cyclin d the transcription factor e2f as well as that of histone deacetylase was significantly decreased. gene array data were also supported by parallel analysis and comparative quantification of the corresponding proteins. our data have shown that ss significantly increased the expression of prmt as well as that of cyclin-dependent kinase inhibitor. by contrast, retinoblastoma binding protein and e2f transcription factor were significantly downregulated. these results were also indirectly corroborated by measurement of the sub-cellular and total protein content. our results showed that the exposure to flow significantly increased the expression of cytoskeletal proteins while reducing that of the other sub-cellular fractions. the total amount of protein content/cell was increased by flow and the sub-cellular protein content showed that the largest component of the total cellular protein pool was made by cytoskeletal proteins with cytosolic, nuclear and membrane protein pools significantly reduced. discussion bbb endothelial cells in vivo are continuously exposed to laminar shear stress to which they respond by structural and functional remodeling showing significant evidences of differentiation. one of the main functions of the bbb is shielding of the brain from unwanted and potentially harmful substances. tight junction protein complexes provide a mechanical means to seal the paracellular pathways between adjacent endothelial cells. our study shows that exposure to flow increases the rna levels of genes encoding for a variety of tight junctional proteins including the intracellular scaffold proteins zonula occludens- and which link the junctional molecules claudin and occludin to intracellular actin and the cytoskeleton. claudin and actin gene espression were also upregulated by the exposure to laminar shear stress thus strongly suggesting that acting as a key regulator of tj expression. in addition to tj protein upregulation we also found a significant increase the rna and protein levels of several cadherins. these are a class of type- transmembrane proteins which play important roles in cell adhesion, through the formation of adherens junctions. our data showed the up-regulation of n-, p- and ve-cadherin. we also observed an increase in the rna and protein levels of e-cadherin. however, even though the upregulation of e-cadherin was statistically significant; its overall rna and total protein expression levels were very modest in comparison to that of the other adherens junction components. the result is the formation of tight barrier with high trans-endothelial electrical resistance that can efficiently discriminate the passage of substances according to their permeability coefficient. in the absence of intraluminal flow despite the presence of abluminal astrocytes the resulting barrier is much less stringent and does not provide selective permeability. this demonstrates that glial stimuli are important but not sufficient to establish a functional bbb and that exposure to flow provides the missing modulatory element to enable bbb properties. bbb shielding function however, is also achieved at other levels: 1) by mechanisms of drugs extrusion. this is carried out by atp-binding cassette transporters which are transmembrane proteins that utilize the energy of adenosine triphosphate hydrolysis to translocate a broad range of substrates across the cellular membranes; 2) by enzymatic metabolism of substances that enter the cell. this function performed is by cytochrome p enzymes similar to those present in the hepatocytes. these enzymes can use a variety of molecules as substrates in enzymatic reactions. our study shows that exposure to flow increases gene expression of several efflux transporters including the multidrug resistance gene, and members of the multidrug resistance associated protein family mrp, mrp, and mrp. p-glycoprotein the products of the mdr gene is an effluxer with specificity for cationic or electrically neutral substrates as well as a broad spectrum of amphiphilic substrates while mrps are multispecific organic anion efflux transporters. togheter these efflux transporters can drastically reduce brain penetration of a broad range of endogenous and exogenous substances which do not follow a paracellular pathway to cross the vascular endothelium. the "third" barrier is instead metabolic in nature. the bbb endothelium is also provided with cytochrome p enzymes which catalyze the oxidation of organic substrates such as lipids and steroidal hormones but also exogenous substances including drugs. recent studies have shown that bbb endothelial p enzymes may be co-responsible for the onset of drug resistance to pharmacological treatments. for example, carbamazepine is an anticonvulsant currently used in the pharmacological treatment of epileptic patients. cbz is a p cyp3a substrate. overexpression of cyp3a has been shown to correlate with that of mdr at the blood-brain barrier interface of patients with drug refractory epilepsy. our study has shown that exposure to flow significantly increases gene expression levels of most members of the cytochrome p families cyp, cyp and cyp taken togheter the increased gene expression of multidrug resistance proteins and p enzymes provides a complex mechanism to regulate the cns drug bioavailability, or to shield the cns. the rna level of cyp11b and cyp19a were also upregulated by flow. these enzymes are involved in the biosynthesis of steroids and estrogen, which modulate many biological activities including bbb permeability and leukocytes adhesion. cyp27a rna level was also significantly upregulated by flow. these p enzymes are responsible for the degradation of cholesterol and play an in important role in the maintenance of cholesterol pool in the cns. this is in agreement with the fact that enhancement of endothelial permeability and lipid deposition preferentially occurs at turbulent, low shear stress regions such as in the arteries. the shielding provided by the bbb may on the other end limit the paracellular diffusion of polar substances including ions and essential nutrients necessary for the maintenance of neuronal activity. therefore the bbb endothelium must be equipped with specific transporters to supply the cns with these substances without compromising its barrier functions and its ability to maintain the homeostasis of the brain environment. our data have shown that the exposure to ss positively modulates the translational expression of ca2+ and k+ ion channels. calcium and potassium homeostasis play an important role in the control of neuronal excitability but are also involved in macrophage transmigration across the vascular endothelium at the bbb. furthermore, ca2+ is involved in the modulation of bbb integrity and endothelial morphology. in addition to ion channels the gene expression level of other specialized solute carriers was also found to be modulated by flow. these include members of the glucose transporters family glut- glut- glut- and glut-; solute carriers specific for cationic aminoacids, organic anions and cations, folate, sulfate as well as the na-k-cl symporter and the potassium-chloride cotransporter. all together these specialized functions represent the hallmark of a drastic differentiation process that set the brain microcapillary endothelium aside from those forming other vascular beds. transporters allow nutrients and other biologically important substances to transit from the blood into the brain across the bbb. to efficiently meet the bioenergetic demand of the many active transport systems that are critical for sustaining neuronal function the bbb ecs make a substantial use of aerobic respiration or citric acid cycle. this is an oxigen-dependent 8-step enzimatic process that allows the full conversion of molecules of acetyl-coa into carbon dioxide and water. the theoretical energetic yield of the process from the complete oxidation of one glucose molecule to co and h2o is of nadh, fadh and atp. the aerobic pathway also provides reductant equivalents to counteract the harmful effect of reactive oxidative species generated intracellularly or originating by exposure to xenobiotic substances. our results show that exposure to flow plays a critical role in the modulation of the bbb-ec bioenergitic metabolism favoring the expression of genes encoding for the key enzymes operating the conversion of acetyl-coa into co and h2o during the citric acid cycle. furthermore, shear stress increases the rna levels of pyruvate dehydrogenase. the aerobic metabolic induction operated by flow on the bbb endothelium was further confirmed by lactate production and glucose consumption measurements demonstrating that at least 50% of the glucose was processed by aerobic respiration. an important implication of this "metabolic modulation by flow" is that bbb endothelial cells can adapt their metabolic process according to the availability of oxygen. this is dependent on blood flow therefore, it is logic to postulate that whether blood flow is interrupted or the oxygen content in the blood is reduced, the vascular endothelium can switch to an anaerobic respiration to meet its energetic demand. controls on cell proliferation and differentiation are interrelated even though it is still not clear to what extent. our data show that exposure to flow increases the rna levels of btg proteins which are negative regulators of the cell cycle. btg or btg bound to prmt is required to induce growth inhibition. furthermore, the rna level of cyclin d, cdk and which control the re-entry of resting g cells into the g phase of cell cycle were downregulated. cells that exit the cell cycle to enter and stay at resting phase is characteristic of terminally differentiated cells. quantitative analysis of the protein content relative to nucleus, cytosol, cytoskeleton, and membrane sub-cellular fractions have shown a drastic reduction of the nuclear protein content in ec exposed to flow. this is in agreement with the lack of dna synthesis in resting cells and the fact that in vivo the endothelial dna synthesis preferentially occurs at branch orifices characterized by low and turbulent flow. furthermore, we observed a substantial increase in cytoskeletal protein content. this can be related to a morphological adaptation of the bbb endothelium to flow and the necessity to provide structural support for tj protein complexes. taken togheter our results suggest that the exposure to a capillary level laminar shear stress on one hand favors ec differentiation into a bbb phenotype and on the other hand inhibits the cell cycle. however, whether the two processes are simultaneously regulated is still unclear.  shear stress plays a key role in modulating endothelial structure and function. our study shows that ss-dependent effects on ec are modulated by induction/suppression of genes regulating multiple aspects of endothelial physiology; from the formation of inter-endothelial tight junction to the expression of specific carrier-mediated transporters and drug resistance mechanisms. studies by other have also shown that ss induces the production of vasoactive substances. turbulent, but not laminar shear stress stimulates ec turnover. accordingly, in vivo dna synthesis of ec preferentially occurs at branch orifices with low flow rates which is paralleled by the enhancement of endothelial permeability. genomic and proteomic analyses are currently being used to study the bbb and how it relates to the pathogenesis of major neurological diseases. in this respect this study provides the bases for new hypotheses and future studies to unveil novel unexploited clinical targets that can facilitate the development of innovative therapeutic strategies to reduce the burden of bbb-related cns diseases. 
rna levels of modulatory enzymes of the glycolytic pathway were downregulated by ss while those involved in the krebs cycle were upregulated. our data showed that ss increased the rna levels of integrins and vascular adhesion molecules. bbb ecs are responsive to inflammatory stimuli. one of the most important and often neglected physiological stimuli contributing to the differentiation of vascular endothelial cells into a blood-brain barrier phenotype is shear stress. cerebral blood flowshear stresscell cyclealternativein vitroinflammation ss also increased the rna level of multidrug resistance transporters, ion channels, and several p enzymes. the rna level of a number of specialized carrier-mediated transport systems was also upregulated. genomic and proteomic analyses are currently used to examine bbb function in healthy and diseased brain and characterize this dynamic interface. ss affected multiple aspect of the endothelial physiology spanning from tight junctions formation to cell division as well as the expression of multidrug resistance transporters. trans-endothelial electrical resistance and permeability measurements to know substances have shown that ss promoted the formation of a tight and highly selective bbb. in this study we showed that ss plays a key role in promoting the differentiation of vascular endothelial cells into a truly bbb phenotype. ss also inhibited endothelial cell cycle via regulation of btg family proteins encoding genes. furthermore, the expression pattern of key protein regulators of the cell cycle and parallel gene array data supported a cell proliferation inhibitory role for ss. this was paralleled by significant increase in the cytoskeletal protein content while that of membrane, cytosol, and nuclear sub-cellular fractions decreased. measurements of glucose consumption versus lactate production showed that ss negatively modulated the glycolytic bioenergetic pathways of glucose metabolism in favor of the more efficient aerobic respiration.
 one of the most important and often neglected physiological stimuli contributing to the differentiation of vascular endothelial cells into a blood-brain barrier phenotype is shear stress. with the use of a well established humanized dynamic in vitro bbb model and cdna microarrays, we have profiled the effect of ss in the induction/suppression of ecs genes and related functions.  specifically, we found a significant upregulation of tight and adherens junctions proteins and genes. trans-endothelial electrical resistance and permeability measurements to know substances have shown that ss promoted the formation of a tight and highly selective bbb. ss also increased the rna level of multidrug resistance transporters, ion channels, and several p enzymes. the rna level of a number of specialized carrier-mediated transport systems was also upregulated. rna levels of modulatory enzymes of the glycolytic pathway were downregulated by ss while those involved in the krebs cycle were upregulated. measurements of glucose consumption versus lactate production showed that ss negatively modulated the glycolytic bioenergetic pathways of glucose metabolism in favor of the more efficient aerobic respiration. bbb ecs are responsive to inflammatory stimuli. our data showed that ss increased the rna levels of integrins and vascular adhesion molecules. ss also inhibited endothelial cell cycle via regulation of btg family proteins encoding genes. this was paralleled by significant increase in the cytoskeletal protein content while that of membrane, cytosol, and nuclear sub-cellular fractions decreased. furthermore, analysis of 2d gel electrophoresis of ec proteins extracted from membrane sub-cellular endothelial fractions showed that ss increased the expression levels of tight junction proteins. in addition, regulatory enzymes of the krebb's cycle were also upregulated. furthermore, the expression pattern of key protein regulators of the cell cycle and parallel gene array data supported a cell proliferation inhibitory role for ss.  genomic and proteomic analyses are currently used to examine bbb function in healthy and diseased brain and characterize this dynamic interface. in this study we showed that ss plays a key role in promoting the differentiation of vascular endothelial cells into a truly bbb phenotype. ss affected multiple aspect of the endothelial physiology spanning from tight junctions formation to cell division as well as the expression of multidrug resistance transporters. bbb dysfunction has been observed in many neurological diseases, but the causes are generally unknown. our study provides essential insights to understand the role played by ss in the bbb formation and maintenance. cerebral blood flowshear stresscell cyclealternativein vitroinflammation 