Ephrin-B2 PB-mononuclear cells reduce early post-stroke deficit in diabetic mice but not long-term memory impairment.

BACKGROUND AND PURPOSE: Post-stroke cognitive impairment (PSCI) has become a major public health issue, as a leading cause of dementia. The inflammation that develops soon after cerebral artery occlusion and may persist for weeks or months after stroke is a key component of PSCI. Our aim was to take advantage of the immunomodulatory properties of peripheral blood mononuclear cells (PB-MNC) stimulated with ephrin-B2/fc (PB-MNC+) for preventing PSCI. METHODS: Cortical infarct was induced by thermocoagulation of the middle cerebral artery in male diabetic mice (streptozotocin IP). PB-MNC were isolated from diabetic human donors, washed with recombinant ephrin-B2/Fc and injected into the mice intravenously on the following day. Infarct volume, sensorimotor deficit, cell death and immune cell densities were assessed on day 3. Six weeks later, cognitive assessment was performed using the Barnes maze. RESULTS: PB-MNC+ transplanted in post-stroke diabetic mice reduced the neurological deficit, infarct volume and apoptosis at D3, without modification of microglial cells, astrocytes and T-lymphocytes densities in the brain. Barnes maze assessment of memory showed that the learning, retention and reversal phases were not significantly modified by cell therapy. CONCLUSIONS: Intravenous PB-MNC+ administration the day after stroke induction in diabetic mice improved sensorimotor deficit and reduced infarct volume at the short term, but was unable to prevent long-term memory loss. To what extent diabetes impacts on cell therapy efficacy will have to be specifically investigated in the future. Including vascular risk factors systematically in preclinical studies of cell therapy will provide a comprehensive understanding of the mechanisms potentially limiting cell efficacy and also to identify good and bad responders, particularly in the long term.

COX-2 is required for the modulation of spinal nociceptive information related to ephrinB/EphB signalling.

BACKGROUND: EphB receptors and their ephrinB ligands are implicated in modulating spinal nociceptive information processing. Here, we investigated whether cyclooxygenase-2 (COX-2), acts as a downstream effector, participates in the modulation of spinal nociceptive information related to ephrinB/EphB signalling. METHODS: Thermal hyperalgesia and mechanical allodynia were measured by using radiant heat and von Frey filaments test, respectively. Real-time PCR (RT-PCR) was used to detect the expression of spinal COX-2 mRNA. Spinal COX-2 and extracellular signal-regulated kinase (ERK) protein were determined by Western blot analysis. RESULTS: Intrathecal injection of ephrinB2-Fc caused thermal hyperalgesia and mechanical allodynia, which were accompanied by increased expression of spinal COX-2 mRNA and protein. Inhibition of spinal COX-2 prevented and reversed pain behaviours induced by the intrathecal injection of ephrinB2-Fc. Blockade of EphB receptors by intrathecal injection of EphB2-Fc reduced complete Freund's adjuvant (CFA)-induced inflammatory pain behaviours, which were accompanied by decreased expression of spinal COX-2 mRNA and protein. Furthermore, treatment with U0126, a mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitor, suppressed spinal ERK activation and COX-2 mRNA and protein expression induced by intrathecal injection of ephrinB1-Fc. CONCLUSIONS: These results confirmed the important involvement of COX-2 in the modulation of spinal nociceptive information related to ephrinBs-EphBs signalling.

PKA is required for the modulation of spinal nociceptive information related to ephrinB-EphB signaling in mice.

EphB receptors and their ephrinB ligands are implicated in modulating of spinal nociceptive information processing. Here, we investigated whether protein kinase A (PKA), acts as a downstream effector, participates in the modulation spinal nociceptive information related to ephrinB-EphB signaling. Intrathecal injection of ephrinB2-Fc caused thermal hyperalgesia and mechanical allodynia, which were accompanied by increased expression of spinal PKA catalytic subunit (PKAca) and phosphorylated cAMP-response element-binding protein (p-CREB). Pre-treatment with H89, a PKA inhibitor, prevented the activation of CREB by ephrinB2-Fc. Inhibition of spinal PKA signaling prevented and reversed pain behaviors induced by the intrathecal injection of ephrinB2-Fc. Furthermore, blockade of the EphB receptors by intrathecal injection of EphB2-Fc reduced formalin-induced inflammatory, chronic constrictive injury (CCI)-induced neuropathic, and tibia bone cavity tumor cell implantation (TCI)-induced bone cancer pain behaviors, which were accompanied by decreased expression of spinal PKAca and p-CREB. Overall, these results confirmed the important involvement of PKA in the modulation of spinal nociceptive information related to ephrinBs-EphBs signaling. This finding may have important implications for exploring the roles and mechanisms of ephrinB-EphB signaling in physiologic and pathologic pain.

EphrinB-EphB receptor signaling contributes to bone cancer pain via Toll-like receptor and proinflammatory cytokines in rat spinal cord.

Treating bone cancer pain poses a major clinical challenge, and the mechanisms underlying bone cancer pain remain elusive. EphrinB-EphB receptor signaling may contribute to bone cancer pain through N-methyl-d-aspartate receptor neuronal mechanisms. Here, we report that ephrinB-EphB signaling may also act through a Toll-like receptor 4 (TLR4)-glial cell mechanism in the spinal cord. Bone cancer pain was induced by tibia bone cavity tumor cell implantation (TCI) in rats. TCI increased the expression of TLR4 and the EphB1 receptor, the activation of astrocytes and microglial cells, and increased levels of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α). The increased expression of TLR4 and EphB1 were colocalized with each other in astrocytes and microglial cells. Spinal knockdown of TLR4 suppressed TCI-induced behavioral signs of bone cancer pain. The TCI-induced activation of astrocytes and microglial cells, as well as the increased levels of IL-1ß and TNF-α, were inhibited by intrathecal administration of TLR4-targeting siRNA2 and the EphB receptor antagonist EphB2-Fc, respectively. The administration of EphB2-Fc suppressed the TCI-induced increase of TLR4 expression but siRNA2 failed to affect TCI-induced EphB1 expression. Intrathecal administration of an exogenous EphB1 receptor activator, ephrinB2-Fc, increased the expression of TLR4 and the levels of IL-1ß and TNF-α, activated astrocytes and microglial cells, and induced thermal hypersensitivity. These ephrinB2-Fc-induced alterations were suppressed by spinal knockdown of TLR4. This study suggests that TLR4 may be a potential target for preventing or reversing bone cancer pain and other similar painful processes mediated by ephrinB-EphB receptor signaling.

Therapeutic interference with EphrinB2 signalling inhibits oxygen-induced angioproliferative retinopathy.

PURPOSE: To investigate whether EphrinB2 (EfnB2) or EphB4 influence retinal angiogenesis under physiological or pathological conditions. METHODS: Using the mouse model of oxygen-induced proliferative retinopathy (OIR), the expression of EfnB2, EphB4, vascular endothelial growth factor (VEGF), VEGFR1 and VEGFR2 was quantified by quantitative polymerase chain reaction (qPCR) and localized in EfnB2- and EphB4-lacZ mice. Angioproliferative retinopathy was manipulated by intravitreal injection of dimeric EfnB2 and monomeric or dimeric EphB4. RESULTS: Dimeric EphB4 (EphB4-Fc) and EfnB2 (EfnB2-Fc) enhanced hypoxia-induced angioproliferative retinopathy but not physiological angiogenesis. Monomeric EphB4 (sEphB4) reduced angiogenesis. The messenger RNA (mRNA) level of EfnB2 increased significantly in the hyperoxic phase (P7-P12), while EphB4, VEGF, VEGFR1 and VEGFR2 showed a significant - up to fivefold - increased expression at P14, the start of morphologically visible vasoproliferation caused by relative hypoxia. CONCLUSION: The ephrin/Eph system is involved in angioproliferative retinopathy. Stimulation of EphB4 and EfnB2 signalling using EfnB2-Fc and EphB4-Fc, respectively, enhanced hypoxia-induced angiogenesis. In contrast, sEphB4 inhibited hypoxia-induced angiogenesis. Therefore, angiogenesis is enhanced by signalling through both EphB4 (forward) and EfnB2 (reverse). The distinction in the expression kinetics of EphB4 and EfnB2 indicates that they govern two different signalling pathways and are regulated in diverse ways. sEphB4 might be a useful drug for antiangiogenic therapy.

Ocular safety profile and intraocular pharmacokinetics of an antagonist of EphB4/EphrinB2 signalling.

AIMS: To characterise the ocular safety profile of sEphB4 and its pharmacokinetics in rabbit eyes. METHODS: 15 rabbits with single intravitreal injection of sEphB4 in the right eye (1000 µg, 465 µg, 160 µg or 80 µg) and phosphate-buffered saline in the left eye were studied at different time points by monitoring inflammatory changes, intraocular pressure, electroretinogram and histological changes. The dose of 80 µg/eye was injected intravitreally into 21 rabbits, and the fellow eyes were used as controls for sEphB4 ocular pharmacokinetics. sEphB4 concentrations were measured in the vitreous, retina, choroids and plasma using ELISA at the designated time points. RESULTS: The study showed that there was no evidence of intraocular toxicity at any time point with any dose tested. No statistically significant differences were seen in the intraocular pressure, scotopic and photopic ERGs, and histopathology between the control and sEphB4 injected eyes. A pharmacokinetic study demonstrated a vitreous half-life of 4.1 days and 6.3 days in the retina. The mean residence time of the drug was 10.45 days in the retina and 7.95 days in the choroid. CONCLUSION: It seems that sEphB4 at the concentrations studied did not appear to be toxic to rabbit eyes and may be a longer-acting treatment option to the current therapies for ocular abnormal neovascularisation.

Engineered fibrin matrices for functional display of cell membrane-bound growth factor-like activities: study of angiogenic signaling by ephrin-B2.

With the rapid increase in approaches to pro- or anti-angiogenic therapy, new and effective methodologies for administration of cell-bound growth factors will be required. We sought to develop the natural hydrogel matrix fibrin as platform for extensive interactions and continuous signaling by the vascular morphogen ephrin-B2 that normally resides in the plasma membrane and requires multivalent presentation for ligation and activation of Eph receptors on apposing endothelial cell surfaces. Using fibrin and protein engineering technology to induce multivalent ligand presentation, a recombinant mutant ephrin-B2 receptor binding domain was covalently coupled to fibrin networks at variably high densities. The ability of fibrin-bound ephrin-B2 to act as ligand for endothelial cells was preserved, as demonstrated by a concomitant, dose-dependent increase of endothelial cell binding to engineered ephrin-B2-fibrin substrates in vitro. The therapeutic relevance of ephrin-B2-fibrin implant matrices was demonstrated by a local angiogenic response in the chick embryo chorioallontoic membrane evoked by the local and prolonged presentation of matrix-bound ephrin-B2 to tissue adjacing the implant. This new knowledge on biomimetic fibrin vehicles for precise local delivery of membrane-bound growth factor signals may help to elucidate specific biological growth factor function, and serve as starting point for development of new treatment strategies.