PGE 2 -induced ICAM-1 expression is regulated by NF-κB activation through Akt signaling pathway signaling pathway

Transcription factors such as NF-κB and AP-1 are known to bind to κB and TRE consensus sequences separately to regulate ICAM-1 gene expression (Chen et al, 2001). To assess the effects of PGE2 on transcriptional activities of NF-κB and AP-1, NF-κB and AP-1-specific reporter assays were adopted. As shown in Fig 21, PGE₂ increased the transcriptional activity of NF-κB whereas it did not elicit any significant effect in AP-1 reporter assay

It has been known that NF-κB activation is mediated by variety of extracellular signals, which can activate IκB kinase (IKK). IKK phosphorylates the IκBα protein, which results in ubiquitination and dissociation of IκBα from NF-κB. The dissociated NF-κB (p65 subunit) is then translocated into the nucleus where it binds to specific RE sequences of DNA (Perkins, 2007). In this study, PGE2 induced the phosphorylation of IKKβ and IκBα and stimulated the translocation of p65 to the nucleus (Fig 22), supporting the activation of NF-κB by PGE₂. dbcAMP and Epac activator (8-Cpt-cAMP) also showed similar effects (Fig 22). dbcAMP and 8-Cpt-cAMP increased NF-κB activity in a reporter assay, which was significantly suppressed by Akti, an Akt inhibitor (Fig 23).

To further investigate the role of NF-κB activation in PGE₂-induced ICAM-1 expression, it was examined the effect of NF-κB inhibitors on ICAM-1 expression. Bay-11-7082, an IκBα phosphorylation inhibitor, and MG 132, an IκBα degradation inhibitor, significantly inhibited PGE2-induced ICAM-1 expression (Fig 24). These results suggest that cAMP/Epac-mediated signaling proceeds through PI3K, Akt and NF-κB and finally leads to

the expression of ICAM-1 in bEnd.3 brain endothelial cells exposed to PGE2. A functional relevance of this molecular signaling was demonstrated in PGE₂-induced promotion of leukocyte adhesion (Fig 25). In primary cultured mouse brain endothelial cells, PGE2 -induced ICAM-1 expression was significantly attenuated by the treatment of ONO-AE2-227 (an EP4 antagonist), brefeldin A (an Epac inhibitor), Akti (an Akt inhibitor) or MG132 (an IkBα degradation inhibitor), confirming the results obtained from bEnd.3 brain endothelial cell (Fig 26).

Fig. 21. Effects of PGE2 on NF-κB and AP-1 transcriptional activity. bEnd.3 cells were co-transfected with AP-1 binding site or NF-κB binding site(2x)-luciferase reporter plasmid and the expression plasmid of β-galactosidase. After 24 h, cells were treated with PGE2 (1 ng/mL) or LPS (1 μg/mL) for another 12 h, Luciferase activities were measured as described in Material and Methods and relative luciferase activity (RLA) was normalized with β-galactosidase activity. * P < 0.01 compared with control (CTL).

Fig. 22. Effects of PGE2 and Epac activator on IKK/IκB phosphorylation and p65 translocation. bEnd.3 cells were exposed to PGE2 (1 ng/mL), dbcAMP (cAMP analogue, 10 μM) and 8-Cpt-cAMP (Epac specific activator, 100 μM) to assess IKK and IκB phosphorylation for 15 min. The levels of IKK and IκB phosphorylation were determined by western blotting as described in Material and Methods. p65 translocation was determined by confocal microscopy (p65 : green, Alexa Fluor^○R 488; nucleus : red, Hoechst 33258)

Fig. 23. Effect of Akt inhibition on PGE₂ and Epac-induced NF-κB activation. bEnd.3 cells were transfected with NF-κB binding site (2x)-luciferase reporter plasmid. After 24 h, cells were treated Akt inhibitor VIII (Akti, 100 nM) for 30 min and further incubated with 1 ng/mL PGE2, 10 μM dbcAMP, and 100 μM 8-Cpt-cAMP for another 12 h. Data are expressed as the mean ± SEM of three independent experiments. (*P < 0.01 vs vehicle controls (CTL), #P < 0.01 vs bEnd.3 stimulated with PGE2, dbcAMP and 8-Cpt-cAMP).

Fig. 24. Effect of NF-κB inhibitors on PGE₂-induced ICAM-1 expression. bEnd.3 cells were incubated with PGE2 (1 ng/mL) in the presence of NF-κB inhibitors such as MG132 (a proteasome inhibitor, 10 μM) and Bay11-7082 (IκB phosphorylation inhibitor, 5 μM) for 4 h or 24 h to assess the levels of mRNA and protein respectively. The levels of ICAM-1 mRNA and protein were determined by RT-PCR and immunoblot at 4 h and 24 h, respectively. Data are representative of three independent experiments. *P < 0.01 vs vehicle controls (CTL), #P

< 0.01 vs bEnd.3 stimulated with PGE_2.

Fig. 25. Involvements of EP4 receptor and Epac/Akt/NF-κB signaling pathways in PGE₂-induced leukocyte adhesion. bEnd.3 cells were treated with ONO-AE2-227 (AE-227, an EP4 antagonist, 10 μM), brefeldin A (BFA, an Epac inhibitor, 10 μM), Akt inhibitor VIII (Akti, 100 nM) or MG132 (a proteasome inhibitor, 10 μM) in the presence of PGE₂ (1 ng/mL) for 24 h and further incubated with CellTracker^TM Orange prelabeled U937 cells (4x10⁵ cells/mL) at 37 °C for 1 h. Adhered monocyte cells were imaged under Axiovert 200 inverted microscope. Data are presented as mean ± S.E. of three independent measurements.

* P < 0.05 compared with control (CTL), # P < 0.05 compared with PGE₂ alone.

Fig. 26. Involvements of EP4 receptor and Epac/Akt/NF-κB signaling pathways in PGE₂-induced ICAM-1 expression in primary cultured mouse brain endothelial cells.

Cells were treated with ONO-AE2-227 (an EP4 antagonist, AE-227, 10 μM), brefeldin A (an Epac inhibitor, BFA, 10 μM), Akti (100 nM) or MG132 (10 μM) in the presence of PGE2 (1 ng/mL) for 24 h and protein levels of ICAM-1 were determined by immunoblot. Data are presented as mean ± S.E. of three independent measurements. * P < 0.05 compared with control (CTL), # P < 0.05 compared with PGE₂ alone.

V. DISCUSSION

Prostaglandin E2(PGE2) is known to be linked to alterations in BBB integrity during inflammation in the CNS (Mark et al, 2001) and its effect on inflammation vary depending on their receptor expression on target cells (McCoy et al, 2002; Kabashima et al, 2002; 2003).

In the CNS, the crosslinking of ICAM-1 with its counterpart integrin αLβ2 not only allows a leukocyte to adhere to the endothelium but also leads to a weakening of the BBB and facilitates the transendothelial leukocyte migration. ICAM-1 is potentially a critical point in the disassembling of the barrier (Simka, 2009). Our previous data showed that COX inhibitors, indomethacin and NS-398 inhibited cadmium-induced ICAM-1 expression, and PGE2 induced ICAM-1 expression without cadmium in bEnd.3 cells (Seok et al, 2006).

However, the effect of PGE₂ on ICAM-1 expression in connection with EP receptor-induced intracellular signaling pathway in mouse cerebrovascular endothelial cells remain largely unexplored, and it is unknown which subtype (s) of EP receptors is expressed in bEnd.3 and involved in ICAM-1 expression. In this study, it was observed that PGE2 markedly increase of ICAM-1 expression and monocyte adhesion to endothelial cells (Fig 4, 5) and both bEnd.3 cells and primary brain endothelial cells highly expressed EP1, EP2, and EP4 receptor (Fig 6). Its suggested antiinflammatory effect of PGE₂ on ICAM-1 expression is mediated via EP2/EP4 receptors in human gingival fibroblasts and blood mononuclear cells (Noguchi et al, 1999; 2001; Takahashi et al, 2002).

It has been known that two different signaling pathway lead to various cellular effects of PGE₂ such as Ca²⁺ dependent signaling pathway via EP1 receptor and cAMP-mediated

signaling pathways by adenylyl cyclase via EP2/4 (Akaogi et al, 2004). EP1 receptor stimulates release of intracellular calcium and activate protein kinase C (PKC) and MAPK (Watabe et al, 1993; Yun et al, 2009) and EP2/4 receptor-mediated intracellular cAMP level caused activation of PKA and Epac signaling pathways (Cheng et al, 2008). Although PGE₂ increase intracellular Ca²⁺ and cAMP levels, and sulprostone, a selective EP1/3 agonist also increase ICAM-1 expression, PGE₂-induced ICAM-1 expression was blocked only in presence of EP4 antagonist among EP antagonists. This result was further supported by knock-down of EP4 with siRNA. siRNA data suggested EP1 might be involved in PGE₂ -induced ICAM-1 expression though data obtained with specific antagonist denied the role of EP1. Ongoing study showed that intracellular calcium levels were increased upon PGE₂ treatment, which was expected to be mediated by EP1. However, inhibitors for other downstream signaling molecules of EP1 such as PKC and MAPKs did not affect PGE₂ -induced ICAM-1 expression. At present i don’t have clear explanation for these discrepancies. But this study clearly indicate that EP4, at least, has a role in PGE₂-induced ICAM-1 expression. Therefore, in this study, it was included only EP4 mediation of PGE2

effect and reserved the possible role of EP1 until it is clarified. cAMP is known to play a regulatory role in leukocyte adhesion to endothelium and transendothelial migration during inflammation. However, the effect of cAMP on cell adhesion remains controversial (Zeidler et al., 2000; Lorenowicz et al., 2006). This discrepancy has been attributed to the complexity of cAMP-driven signaling. Actions of cAMP are mediated by a variety of cAMP effector proteins such as PKA, Epac, PDZ-GEF and cyclic nucleotide-gated channels. Among these, PKA and Epac are two major targets of cAMP, which have been implicated in the regulation

of leukocyte transendothelial migration and endothelial barrier function (Lorenowicz et al., 2007). Recently it was reported that cAMP/Epac pathway might be involved in acidosis-induced endothelial cell adhesion and ICAM-1 expression (Chen et al., 2011). It was also shown that Epac specific activators completely reversed thromboxane receptor antagonist-induced reduction of ICAM-1 expression in human coronary artery endothelial cells (Sand et al., 2010). In contrast, the prime cAMP effector PKA was reported to have both negative and positive role in the regulation of ICAM-1 expression depending on the types of cAMP-inducing stimuli (Bernot et al., 2005; Yoshimoto et al., 2005). In this study, cAMP analog (dbcAMP) and adenylyl cyclase activator (forskolin) significantly increased ICAM-1 expression (Fig 13). These data indicate that PGE₂ upregulates ICAM-1 expression via cAMP-dependent signaling pathways. In addition, it was demonstrated that PGE2-induced ICAM-1 expression was mimicked by 8-Cpt-cAMP, an Epac specific activator, and suppressed by knock-down of Epac1 (Fig 17, 18). However, these results were not reproduced by the manipulation of PKA activity, indicating the implication of Epac rather than PKA in the PGE2-induced ICAM-1 expression in cerebrovascular endothelial cells.

Often, both cAMP targets are associated with the same biological process, in which they fulfill either opposite or synergistic effects (Gloerich and Bos, 2010).

Epac proteins are reported to exert diverse biological functions by signaling to a wide range of effectors such as ERKs, PKB/Akt, NF-κB and GSK-3β. These signaling molecules have been implicated in the inflammatory responses including ICAM-1 expression in vascular endothelial cells (Radisavljevic et al., 2000; Minhajuddin et al., 2009; Fan et al, 2010). However, it was observed that specific inhibitors of MAPKs did not affect PGE₂

-induced ICAM-1 expression in brain endothelial cells (Fig 11). In contrast, PGE2 effect was significantly attenuated by specific inhibitors of PI3K and Akt (Fig 20). These results were supported by data obtained from a dominant-negative and constitutively active mutant of Akt.

Akt phosphorylation was stimulated by PGE₂, of which effect was mimicked by dbcAMP and Epac activator. Taken together, our data indicate that Epac-mediated signaling proceeds through PI3K and Akt in PGE₂-stimulated brain endothelial cells. Roles of PI3K/Akt pathway in endothelial ICAM-1 expression under various pathological conditions have been documented (Radisavljevic et al., 2000; Hur et al., 2007; Minhajuddin et al., 2009; Dagia et al., 2010). However, until this time, there is no report describing the involvement of PI3K/Akt pathway in PGE₂-induced ICAM-1 expression. To my knowledge, this study is the first to demonstrate a regulatory link between activation of Epac/PI3K/Akt and upregulation of ICAM-1 in brain endothelial cells exposed to PGE₂.

NF-κB activation has been implicated in pro-inflammatory stimuli-triggered endothelial ICAM-1 expression (Balyasnikova et al., 2000; Rahman et al., 2004; Minhajuddin et al., 2009). In the present study, it was found that PGE2 induced sequential events for NF-κB activation, i.e. phosphorylation of IκBβ kinase and IκBα, translocation of p65 to nucleus and increase in NF-κB reporter gene activity (Fig 21, 22). In addition, suppression of NF-κB by Bay-11-7082 and MG-132 attenuated PGE₂-induced ICAM-1 expression (Fig 24). These results suggest that NF-κB activation is critically involved in ICAM-1 expression in cerebrovascular endothelial cells stimulated with PGE₂. Moreover, Akt inhibitor diminished NF-κB dependent reporter gene activity induced by PGE2, dbcAMP and Epac activator, indicating roles of cAMP, Epac and Akt in PGE₂-induced NF-κB activation. These findings

are consistent with previous reports that PI3K/Akt modulates NF-κB activation and ICAM-1 expression in endothelial cells (Rahman et al., 2002; Minhajuddin et al., 2009). cAMP is known to exert differential effects on NF-κB activity in a cell type- and stimulus-specific manner. Although most studies report the inhibition of NF-κB activity by cAMP-inducing stimuli, other papers have shown that cAMP stimulates NF-κB activity or does not interfere with NF-κB activation (Gerlo et al., 2011). Overwhelming reports have shown that prototypical cAMP effector PKA is implicated in either inhibitory or stimulatory modulation of NF-κB activity by cAMP effects. However, recent papers showed that the positive effect on NF-κB was not mediated by PKA, but was dependent on Epac activation in murine macrophages (Moon et al., 2007; 2011). In this study, it was also observed that Epac mediated NF-κB activation in cerebrovascular endothelial cells. These results suggest that Epac-mediated Akt/NF-κB pathway could be helpful for interpretation on various cAMP-mediated physiological responses. Indeed, it was postulated that differential effects of cAMP on NF-κB might be the result of preferential activation of PKA or Epac (Gerlo et al., 2011).

Likewise, findings in this study could provide an insight into explaining contradictory results in PGE₂ effect on ICAM-1 expression in a variety of cells.

At present, it is unclear what caused differences in PGE2 effect and participating signaling cascade, but these may include the injury context including the type, duration and strength of stimulus as well as cell types involved, expressing a specific repertoire of PGE2- or cAMP-responsive effectors (Lorenowicz et al., 2007; Andreasson, 2010). Thus, comparative study on these factors would contribute to better understanding of roles of PGE2 in brain inflammation. In summary, data obtained in the current study indicate that PGE₂ upregulates

endothelial ICAM-1 expression through ligation of EP4 receptor and sequential activation of Epac-Rap1, PI3K/Akt and NF-κB, and thereby induces leukocyte adhesion to brain endothelial cells. These findings imply the role of PGE2 and cAMP-controlled signaling pathway in a variety of inflammatory CNS injury, though additional in vivo studies are needed to address the functional significance of results.

Fig. 27. The putative signaling pathway for PGE2-induced ICAM-1 expression in mouse cerebrovascular endothelial cells. Ligation of EP4 activates the cAMP/Epac cascade to induce ICAM-1 expression via PI3K/Akt/NF-κB signaling axis. These signaling pathways are identified with various pharmacological or genetic approach. Cell were treated with sulprostone (EP1/3 agonist), Butaprost (EP2 agonist), ONO-AE-248 (EP3 agonist), 1-OH-PGE1 (EP3/4 agonist, PGE1), ONO-8713(EP1 antagonist), ONO-AE3-240(EP3 antagonist), ONO-AE2-227(EP4 antagonist), dbcAMP (cAMP analogue), forskolin (adenylyl cyclase activator), N⁶-Benz-cAMP (PKA specific activator), catalytically active form of PKA (PKA-Cα), 8-Cpt-cAMP (Epac specific activator), brefeldin A (BFA, an Epac inhibitor), H89

(PKA inhibitor), PKAi(PKA inhibitor), LY294002 (PI3K inhibitor), Akt inhibitor VIII (Akti), catalytically active Akt (Akt-CA), dominant-negative Akt (Akt-DN), chelerythrine (novel PKC inhibitor), GO 6976 (conventional PKC inhibitor), PD98059 (ERK inhibitor), SB202190 (p38 inhibitor), SP600125 (JNK inhibitor), MG132 (a proteasome inhibitor), Bay11-7082 (IκB phosphorylation inhibitor).

VI. REFERENCES

1. Andreasson K: Emerging roles of PGE2 receptors in models of neurological disease.

Prostaglandins Other Lipid Mediat 91: 104-112, 2010

2. Ahmad M, Ahmad AS, Zhuang H, Maruyama S, Dore S: Stimulation of prostaglandin E2-EP3 receptors exacerbates stroke and excitotoxic injury. J Neuroimmunol 184: 172-179, 2007

3. Aid S, Bosetti F: Targeting cyclooxygenases-1 and -2 in neuroinflammation:

Therapeutic implications. Biochimie 93: 46-51, 2011

4. Akaogi J, Yamada H, Kuroda Y, Nacionales DC, Reeves WH, Satho M: Prostaglandin E₂ receptors EP2 and EP4 are up-regulated in peritoneal macrophages and joints of pristane-treated mice and modulate TNF-α and IL-6 production. J Leukoc Biol 76: 227-236, 2004

5. Aktan S, Aykut C, Oktay S, Yegen B, Keles E, Aykac I, Ercan S: The alterations of leukotriene C4 and prostaglandin E2 levels following different ischemic periods in rat brain tissue. Prostaglandins Leukot Essent Fatty Acids 42: 67-71, 1991

6. Andersson PB, Perry VH, Gordon S: The acute inflammatory response to lipopolysaccharide in CNS parenchyma differs from that in other body tissues.

Neuroscience 48: 169–186, 1992

7. Arumugam TV, Woodruff TM, Lathia JD, Selvaraj PK, Mattson MP, Taylor SM:

Neuroprotection in stroke by complement inhibition and immunoglobulin therapy.

Neuroscience 158: 1074-1089, 2009

8. Balyasnikova IV, Pelligrino DA, Greenwood J, Adamson P, Dragon S, Raza H, Galea E: Cyclic adenosine monophosphate regulates the expression of the intercellular adhesion molecule and the inducible nitric oxide synthase in brain endothelial cells. J Cereb Blood Flow Metab 20: 688-699, 2000

9. Battacharya M, Peri K, Ribeiro-Da-Silva A, Almazan G, Shichi H, Hou X, Varma DR, Chemtob S: Localization of functional prostaglandin E2 receptors EP3 and EP4 in the nuclear envelope. J Biol Chem 274: 15719-15724, 1999

10. Baviera AM, Zanon NM, Navegantes LC, Kettelhut IC: Involvement of cAMP/Epac/PI3K-dependent pathway in the antiproteolytic effect of epinephrine on rat skeletal muscle. Mol Cell Endocrinol 315: 104-112, 2010

11. Bernot D, Peiretti F, Canault M, Juhan-Vague I, Nalbone G: Upreguation of TNF-α-induced ICAM-1 surface expression by adenylate cyclase-dependent pathway in human endothelial cells. J Cell Physiol 202: 434-441, 2005

12. Bilak M, Wu L, Wang Q, Haughey N, Conant K, St Hillaire C, Andreasson K: PGE2 receptors rescue motor neurons in a model of amyotrophic lateral sclerosis. Ann Neurol 56: 240-248, 2004

13. Bishop-Bailey D, Burke-Gaffney A, Hellewell PG, Pepper JR, Mitchell JA: Cyclo-oxygenase-2 regulates inducible ICAM-1 and VCAM-1 expression in human vascular smooth muscle cells. Biochem Biophys Res Commun 249: 44-47, 1998

14. Bos JL: Epac: a new cAMP target and new avenues in cAMP research. Nat Rev Mol Cell Biol 4: 733-738, 2003

15. Bos JL: Epac proteins : multi-purpose cAMP targets. Trends Biochem Sci 31: 680-686, 2006

16. Borland G, Smith BO, Yarwood SJ: Epac proteins transduce diverse cellular actions of cAMP. British Journal of Pharmacology 158: 70-86, 2009

17. Bowes MP, Zivin JA, Rothlein R: Monoclonal antibody to the ICAM-1 adhesion site reduces neurological damage in a rabbit cerebral embolism stroke model. Exp Neurol 119: 215-219, 1993

18. Burkey TH, Regan JW: Activation of mitogen-activated protein kinase by the human prostaglandin EP3A receptor. Bichem Biophys Res Commun 211: 152-158, 1995

19. Burks SR, Wright CL, McCarthy MM: Exploration of prostanoid receptor subtype regulating estradiol and prostaglandin E2 induction of spinophilin in developing preoptic area neurons. Neuroscience 146: 1117-1127, 2007

20. Carlos TM, Harlan JM: Leukocyte-endothelial adhesion molecules. Blood 84: 2068-2101, 1994

21. Cheng X, Ji Z, Tsalkova T, Mei F: Epac and PKA: a tale of two intracellular cAMP receptors. Acta Biochim Biophys Sin 40: 651–662, 2008

22. Cimino PJ, Keene D, Breyer RM, Montine KS, Montine TJ: Therapeutic targets in prostaglandin E2 signaling for neurologic disease. Curr Med Chem 15: 1863-1869, 2008

23. Chen NG, Han X: Dual function of troglitazone in ICAM-1 gene expression in human vascular endothelium. Biochem Biophys Res Commun 282: 717-722, 2001

24. Dagia NM, Aqarwal G, Kamath DV, Chetrapal-Kunwar A, Gupte RD, Jadhav MG, Dadarkar SS, Trivedi J, Kulkarni-Almeida AA, Kharas F, Fonseca LC, Kumar S, Bhonde MR: A preferential p110alpha/gamma PI3K inhibitor attenuates experimental inflammation by suppressing the production of proinflammatory mediators in a NF-kappaB-dependent manner. Am J Physiol Cell Physiol 298: C929-941, 2010

25. De Clercka LS, Bridts CH, Mertens AM, Moens MM, Stevens WJ: Use of fluorescent dyes in the determination of adherence of human leukocytes to endothelial cells and the effect of fluorechromes on cellular function. J Immunol Methods 172: 115-124, 1994

26. de Rooij J, Zwartkruis FJ, Verheijen MH, Cool RH, Nijman SM, Wittinghofer A, Bos JL: Epac is a Rap1 guanine-nucleotide exchage factor directly activated by cyclic AMP.

Nature 396: 474-477, 1998

27. de Vries HE, Blom-Roosemalen MC, van Oosten M, de Boer AG, van Berkel TJ, Breimer DD, Kuiper J. The influence of cytokines on the integrity of the blood-brain barrier in vitro. J Neuroimmunol 64: 37-43, 1996

28. Dietrich JB: The adhesion molecule ICAM-1 and its regulation in relation with the blood-brain barrier. J Neuroimmunol 128(1): 58-68, 2002

29. Dormond O, Bezzi M, Mariotti A, Ruegg C: Prostaglandin E₂ promotes integrin alpha Vbeta 3-dependent endothelial cell adhesion, rac-activation, and spreading through cAMP/PKA-dependent signaling. J Biol Chem 277: 45838-45846, 2002

30. Fan Y, Liu C, Qin X, Wang Y, Han Y, Zhou Y: The role of ERK1/2 signaling pathway in Nef protein upregulation of the expression of the intercellular adhesion molecule 1 in endothelial cells. Angiology 61: 669-678, 2010

31. Feinstein DL, Galea E, Roberts S, Berquist H, Wang H, Reis DJ: Induction of nitric oxide synthase in rat C6 glioma cells. J Neurochem 62: 315-321, 1994

32. Gerlo S, Kooijman R, Beck IM, Kolmus K, Spooren A, Haegeman G: Cyclic AMP: a selective modulator of NF-kB action. Cell Mol Life Sci 68: 3823-3841, 2011

33. Gloerich M, Bos JL: Epac: defining a new mechanism of cAMP action. Annu Rev Pharmacol Toxicol 50: 355-375, 2010

34. Greenwood J, Wang Y, Calder VL: Lymphocyte adhesion and transendothelial migration in the central nervous system: the role of LFA-1, ICAM-1, VLA-4 and VCAM-1. off. Immunology 86(3): 408-415, 1995

35. Griot C, Vandevelde M, Richard A, Peterhans E, Stocker R: Selective degeneration of oligodendrocytes mediated by oxygen radical species. Free Radical Res Commun 11:

181-193, 1990

36. Hewett SJ, Uliasz TF, Vidwans AS, Hewett JA: Cyclooxygenase-2 contributes to N-methyl-D-aspartate-mediated neuronal cell death in primary cortical cell culture. J Pharmacol Exp Ther 293: 417-425, 2000

37. Hickey WF: Leukocyte traffic in the central nervous system: The particifants and their roles. Semin Immunol 11: 125-137, 1999

38. Hickey WF: Basic principles of immunological surveillance of the normal central nervous system. Glia 36: 118-124, 2001

39. Hur J, Yoon CH, Lee CS, Kim TY, Oh IY, Park KW, Kim JH, Lee HS, Kang HJ, Chae IH, Oh BH, Park YB, Kim HS: Akt is a key modulator of endothelial progenitor cell trafficking in ischemic muscle. Stem Cells 25: 1769-1778, 2007

40. Hurley SD, Olschowka JA, O’Banion MK: Cyclooxygenase inhibition as a strategy to ameliorate brain injury. J Neurotrauma 19: 1-15, 2002

41. Iadecola C, Niwa K, Nogawa S, Zhao X, Nagayama M, Araki E, Morham S, Ross ME:

41. Iadecola C, Niwa K, Nogawa S, Zhao X, Nagayama M, Araki E, Morham S, Ross ME: