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ComiXology Thousands of Digital Comics. East Dane Designer Men's Fashion. Shopbop Designer Fashion Brands. Volume acquisition z-stack and time t-series of ascending venules were rendered in 3-D and analyzed for the number of attached leukocytes. Using tracking software, the distinction between adhesion and rolling was discerned by evaluating the 4-dimensional movement of leukocytes inside the vessels. Figure 5 top panels shows the lack of adhesion in untreated animals, with streaking asterisk labeled leukocytes in the lumen.
As expected, the LPS insult revealed significant immune-endothelial interaction Figure 5 , middle panels , with red-stained leukocytes attached to the endothelium.
Introduction of O with LPS reduced the number of adherent cells, thus confirming the previous results in surface vessels. Of note, similar results were achieved with JWH data not shown. Figure 5C, provides a quantitative assessment of the findings from multiphoton microscopy. A Representative images of 3-D reconstructions of z-stacks taken by multicolor two-photon intravital microscopy using animals with the indicted experimental conditions. The intraparenchymal microvasculature of the brain cortex down to microns below the cortex surface was outlined by i.
Leukocytes were fluorescently labeled by i. Leukocyte adhesion events were identified in ascending postcapillary venules ranging from microns in diameter. In untreated control animals only moving leukocytes were apparent streaking cells; asterisks. Corner insert shows max projection composites providing a top view of the z-stack. B The results of particle counting, providing a measure of the number of attached leukocytes per vessel surface area assuming cylindrical dimensions and using microns of vessel length.
Adhesion molecules play an essential role in the engagement of immune cells to the endothelium. Immunofluorescence histology was performed on frozen brain tissue sections 15 microns thick from animals treated as indicated. Sections from animals with LPS-associated encephalitis displayed a high level of immunopositive staining for ICAM-1 in the vasculature arrows compared to the marginally positive staining in the control animals middle and top panels respectively.
BMVEC were treated for 4 hrs and the treatments removed prior to the addition of monocytes. Next, we performed experiments in primary human BMVEC in order to 1 determine if the observations in vivo could be confirmed in human brain endothelial cells, 2 isolate the effects of the CB2R agonist on endothelial cells from leukocytes and 3 determine whether the same effects on adhesion molecule expression are also observed.
Purified primary human monocytes were placed on the BMVEC to initiate adhesion only after all treatments were removed and the medium was changed. Unlike the in-vivo experiments, the change in medium prior to the monocyte introduction allows for the evaluation of the effects of the CB2R agonist on BMVEC only. As shown in Figure 6C , the results from the adhesion assays indicate that human brain endothelial cells respond similarly to CB2R agonists in preventing immune cell adhesion.
In addition, the effects with either CB2R agonist are dose dependent for the reduction in adhesion. O diminished monocyte adhesion from 2. Thus CB2R activation during inflammatory insult can regulate the expression profile of adhesion molecules leading to the inhibition in immune-endothelial interaction. Loss of BBB integrity results in the leakiness of blood components from the vessel lumen into the brain parenchyma. LPS exposure induces an acute opening of the BBB which is also potentiated by the presence of inflammatory mediators and activated immune cells.
To assess permeability, animals were i. In contrast, 4 hrs after LPS injection, animals had considerable vascular permeability, which was greatly attenuated by the presence of O Figure 7A. To arrive at a more precise quantitative comparison of permeability, animals were injected with the small molecular tracer sodium fluorescein Na-F, Da and the brain tissue content of Na-F was measured Figure 7B.
Taken together, the results indicate that CB2R agonist provides a barrier protective effect, thus limiting the degree of permeability that is induced during inflammation. A Images from intravital microscopy showing no dextran permeability out of the vessel at baseline, while LPS significantly induced dextran leakage into peri-vascular spaces.
The dextran leakage was attenuated by the treatment with O B BBB permeability was evaluated by administration of the tracer, sodium fluorescein Na-F in saline via i. Following perfusion, the content of Na-F in homogenized brain tissue was measured. LPS significantly increased Na-F leakage into the brain parenchyma compared to untreated mice, which was attenuated by the treatment with O The results are expressed in fold change from calculating the relative fluorescence RFU in mg of brain tissue, and dividing the treated by untreated.
The resistance was measured at Hz in 30 min intervals for the duration of the time shown. Treatments were initiated arrow after stable resistance was reached.
The data are presented as percent change from baseline, which is the resistance measured post-treatment divided by the resistance acquired before treatment introduction. CB2R agonists induced a further tightening of the barrier, evident by a rise in the degree of resistance above the basal level B and C. Our next set of experiments was designed to understand whether CB2R activation could increase barrier structural integrity under physiologic conditions.
Although various signaling mechanisms can influence BBB tightness, the culminating effect is on TJ located between brain endothelial cells assuring tightness of the BBB.
Longer exposure to JWH resulted in further increases in occludin 3. In summary, these results suggest CB2R protective effects on BBB function are mediated in part by the increased presence of TJ proteins in the membrane fraction of brain endothelium. Previous studies have shown that activation of the CB2 receptor CB2R by pharmacological ligands promotes anti-inflammatory outcomes.
Although suppression of inflammatory responses resulting from CB2R activation has been observed in various cell types, little is known about how it affects the brain endothelium or the BBB. Here we provide compelling evidence that CB2R ligands, including a novel resorcinol-based agonist, not only decrease immune-endothelial interactions, but also render direct BBB protection during neuroinflammation.
Earlier studies indicated that CB2R expression was restricted to peripheral tissues, particularly in immune cells Cabral and Griffin-Thomas, However, it is now known that CB2R can also be found in cells of the CNS, but predominantly as a consequence of neuroinflammation reviewed in Benito et al. Thus far, this augmentation in CB2R expression has been reported to be largely localized to microglial cells and perivascular macrophages.
In this study, our findings point to the induction of CB2R in yet another cell type, the brain endothelial cell. Under normal physiological conditions the vessels of the brain express very low levels of CB2R Schley et al. Our observations confirm this expression however; analysis of brain tissue sections from cases of HIV-1 encephalitis revealed a strong upregulated immuno-positive staining for CB2R on the vasculature.
To our knowledge this is the first demonstration of increased CB2R levels in brain endothelium during neuroinflammation. Another study also showed an increase in CB2R in vessels of the brain, but only in proliferating microvessels of glioblastomas Schley et al. Given the complexity of the inflammatory response within the neurovascular unit, we sought to determine whether isolated human BMVEC would also upregulate CB2R by pro-inflammatory factors.
Indeed, all tested inflammatory insults enhanced CB2R in the cultured cells with varying degrees of expression. Whether the inducibility of CB2R triggered by inflammation is part of the pathogenesis process or part of the mechanism to resolve the injury remains unclear. Evidence for the later suggests that endothelial production of endocannabinoids after inflammatory insult may act to regulate endothelial activation Golech et al.
In any case, the presence of upregulated CB2Roffers the opportunity to target the receptor with synthetic and highly selective CB2R agonists in order to dampen neuroinflammation. Using LPS induced encephalitis and intravital microscopy, we showed that leukocyte adhesion in cerebral vessels was significantly reduced after JWH or O administration.
Interestingly, a recent study using cannabidiol, a CB1R antagonist and a CB2R inverse agonist, in LPS-associated encephalitis was shown to decrease leukocyte adhesion and vasodilatation Ruiz-Valdepenas et al. Given that the brain endothelium also expresses the CB1R Golech et al. The changes in adhesion with CB2R agonists were noted both in pial vessels and deep cortical ascending post capillary venules, the sites of most leukocyte-endothelial interaction during neuroinflammation Owens et al.
Evaluation of CB2R agonists in surface and deep cerebral vessels was important because surface pial vessels do not have an integrated neurovascular unit and thus anti-inflammatory responses differ between the two sites Persidsky et al. However, these investigations did not address whether the effects of CB2R apply only to the immune cell, the endothelial cell or both; given that both cell types express CB2R. Consistent with previous reports Yamashita et al.
Thus, the reduction in leukocyte adhesion seen in the intravital analysis was in part due to the lack of firm attachment between endothelial adhesion molecules and immune cell integrins.
Interestingly, it is possible that intercellular adhesion is abrogated by CB2R signaling even prior to adhesion i. Therefore further analysis need be performed to identify whether regulation of selectins may also be a target of CB2R activaiton. Therefore signaling events triggered by CB2R agonists that prevent the increase in surface expression of adhesion molecules may apply universally to endothelial cells of brain and non-brain origin.
Undoubtedly, activated immune cells are affected by CB2R agonists in many ways, including cytokine production and adhesion-migration ability Pacher and Steffens, For instance, adoptive transfer of immune cells exposed to the CB2R agonist, JWH, prevented the attachment and transendothelial migration of leukocytes in inflamed retinas Xu et al. In an attempt to uncouple the effects of CB2R agonists on both immune and endothelial cell, we continued our investigations in-vitro with primary human BMVEC.
Therefore BMVEC were activated with cytokines in the presence of CB2R agonist, then the agonist was removed and adhesion with human monocytes evaluated. Our experiments revealed a dose dependent decrease in monocyte adhesion to cytokine activated endothelium when CB2R agonists were present. The results presented clarify that these effects are not only one-sided i. BBB permeability is increased in neuroinflammation and is one of the contributing factors associated with neurodegeneration.
Distinct from the vascular beds in the periphery, the vasculature of the brain features a high concentration of intercellular tight junctions that form the BBB Abbott, We observed that CB2R agonists did maintain barrier integrity as visualized by the absence of fluorescence labeled high molecular weight dextrans 70 kDa entering the brain parenchyma Figure 7. Since BBB permeability evaluations with large molecular tracers may fail to detect permeability to smaller molecules, we also used a low molecular weight tracer.
Although significantly reduced, global assessment of BBB permeability with sodium-flourescein Da showed that permeability still remained at half a fold higher than the basal level. Perhaps this is due to the low number of leukocytes that still remained adherent. The measurements showed the expected sustained decrease in TEER after LPS; conversely, O was able to prevent the full drop in resistance and allowed the barrier to rebound back to normal levels more rapidly Figure 8.
Surprisingly, the protection in barrier integrity was achieved beyond the anti-inflammatory effects of CB2R, since the TEER showed a further enhancement of barrier tightness with just the CB2R agonist present. These results are reminiscent of other barrier enhancing agents such as steroids, cAMP or growth factors Liu et al.
We also found that the barrier enhancing effects could be explained in part by the augmentation in the TJ proteins, occludin and claudin-5, in membrane fractions of CB2R agonist treated BMVEC. Together these analyses provide compelling evidence that CB2R agonists, independent of their anti-inflammatory effects, promote and enhance BBB integrity.
At present it is unclear how CB2R signaling cascades may act on both the TJ complex and inflammatory pathways. Whether concurrently or sequentially, it is also possible that CB2R activation inhibits pathways that alter normal TJ protein expression or stability. One of the pathways previously describes to be critical for tight junction protein stability, is the Rho GTPase pathway Persidsky et al.
Specifically, RhoA activity negatively impacts the stability of the TJ complex. This a reasonable possibility in light of a recent report by Kurihara and colleagues describing that in T-Cells the activity of RhoA decreases in response to CB2R stimulation Kurihara et al. Alternatively CB2R signaling may attenuate oxidative stress signals that are known for example via MMPs to affect the status of tight junction proteins Lischper et al.
Undoubtedly future investigations will help shed light into how CB2R signaling events in the BMVEC contribute to anti-inflammation and barrier protection.
In summary, CB2R receptors may provide a therapeutic target, not only for their effects on attenuating immune cell activation, but also in protecting the BBB during neuroinflammation.
Are you Anu Mahadevan? Pozzuoli Napoli , Italy. Sign In Join Now. Register this Author 31 Publications. Combined inhibition of monoacylglycerol lipase and cyclooxygenases synergistically reduces neuropathic pain in mice. Br J Pharmacol Apr 20; 7: Epub Jan Targeting multiple cannabinoid anti-tumour pathways with a resorcinol derivative leads to inhibition of advanced stages of breast cancer.
Br J Pharmacol Oct 5; Epub Sep 5. A novel fluorophosphonate inhibitor of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol with potential anti-obesity effects. Br J Pharmacol Jun; 4: The monoacylglycerol lipase inhibitor JZL suppresses inflammatory pain in the mouse carrageenan model.
Life Sci Mar 28;92 Epub Jun Structural analogs of pyrazole and sulfonamide cannabinoids: Eur J Pharmacol Nov 6; Epub Sep 6. Unique effects of compounds active at both cannabinoid and serotonin receptors during stroke. Transl Stroke Res Sep 26;3 3: Epub Jul Br J Pharmacol Apr; 8: Activation of cannabinoid receptor 2 attenuates leukocyte-endothelial cell interactions and blood-brain barrier dysfunction under inflammatory conditions.
J Neurosci Mar;32 The CB2 cannabinoid receptor-selective agonist O reduces pain and inflammation without apparent cannabinoid behavioral effects. Neuropharmacology Feb-Mar;60
T Bisogno, MG Cascio, B Saha, A Mahadevan, P Urbani, A Minassi, Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids , View the profiles of people named Anu Mahadevan. Join Facebook to connect with Anu Mahadevan and others you may know. Facebook gives people the. Anu Mahadevan is on Facebook. Join Facebook to connect with Anu Mahadevan and others you may know. Facebook gives people the power to share and.