Fatty Acid-Binding Protein 4 Inhibition Promotes Locomotor and Autonomic Recovery in Rats following Spinal Cord Injury.

The inflammatory response associated with traumatic spinal cord injury (SCI) contributes to locomotor and sensory impairments. Pro-inflammatory (M1) macrophages/microglia (MϕMG) are the major cellular players in this response as they promote chronic inflammation resulting in injury expansion and tissue damage. Fatty acid-binding protein 4 (FABP4) promotes M1 MϕMG differentiation; however, it is unknown if FABP4 also plays a role in the etiology of SCI. The present study investigates whether FABP4's gene expression influences functional recovery following SCI. Analysis of quantitative polymerase chain reaction data shows a robust induction of FABP4 messenger RNA (mRNA; >100 fold) in rats subjected to a T9-T10 contusion injury compared with control. Western blot experiments reveal significant upregulation of FABP4 protein at the injury epicenter, and immunofluorescence analysis identifies that this upregulation occurs in CD11b+ MϕMG. Further, upregulation of FABP4 gene expression correlates with peroxisome proliferator-activated receptor γ (PPARγ) downregulation, inactivation of Iκßα, and the activation of the NF-κB pathway. Analysis of locomotor recovery using the Basso-Beattie-Bresnahan's locomotor scale and the CatWalk gait analysis system shows that injured rats treated with FABP4 inhibitor BMS309403 have significant improvements in locomotion compared with vehicle controls. Additionally, inhibitor-treated rats exhibit enhanced autonomic bladder reflex recovery. Immunofluorescence experiments also show the administration of the FABP4 inhibitor increases the number of CD163+ and liver arginase+ M2 MϕMG within the epicenter and penumbra of the injured spinal cord 28 days post-injury. These findings show that FABP4 may significantly exacerbate locomotor and sensory impairments during SCI by modulating macrophage/microglial activity.

Protective effect of docosahexaenoic acid on lipotoxicity-mediated cell death in Schwann cells: Implication of PI3K/AKT and mTORC2 pathways.

BACKGROUND AND AIM: Docosahexaenoic acid (DHA) exhibits neuroprotective properties and has been shown to preserve nerve cells following trauma and ischemic injury. Recently, we showed that DHA pretreatment improved locomotion and reduced neuropathic pain after acute spinal cord injury in adult rats. These improvements were associated with an increase in the levels of AKT in spinal cord injury neurons. In this study, we investigate the implication of PI3K/AKT and mTOR pathway in DHA-mediated protection of primary cultured Schwann cells (pSC) undergoing palmitic acid-induced lipotoxicity (PA-LTx). METHODS: Primary cultured Schwann cells were treated with PA (PA:BSA, 2:1) in the presence or absence of DHA (1-200 µM) for 24-48 hr. Cell viability was determined by crystal violet staining and nuclear morphology was examined using Hoechst staining. RESULTS: We found that pSC cultures exposed to palmitic acid (PA) overload showed chromatin condensation, a decrease in cell viability and an inhibition of AKT phosphorylation in a time-dependent manner. Next, pSC exposed to PA overload were treated with DHA. The data show that co-treatment with DHA inhibited the loss of cell viability and apoptosis caused by PA. Moreover, treatment with DHA inhibited chromatin condensation, significantly stimulated p-AKT phosphorylation under PA-LTx condition, and DHA alone increased AKT phosphorylation. Additionally, when these pSC cultures were treated with PI3K inhibitors LY294002 and, BKM120 and mTOR inhibitors Torin 1 (mTORC1/mTORC2), but not rapamycin (mTORC1), the protective effects of DHA were not observed. CONCLUSION: These findings suggest PI3K/AKT and mTORC2 kinase pathways are involved in the protective function (s) of DHA in PA-induced Schwann cell death.

Hyperglycemia magnifies Schwann cell dysfunction and cell death triggered by PA-induced lipotoxicity.

Lipid overload resulting in lipotoxicity is prominent in a number of chronic diseases and has been associated with cellular dysfunction and cell death. This study characterizes palmitic acid-induced lipotoxicity (PA-LTx) in Schwann cell cultures grown in normal and high glucose concentrations. The study shows for the first time that Schwann cell (SC) cultures exposed to elevated levels of PA exhibit a dose- and time-dependent loss in cell viability. Hoescht and Annexin V/7AAD staining confirmed cell death through apoptosis and the lipotoxic effect was more dramatic in SC cultures grown under high glucose conditions. The first indication of cellular dysfunction in treated SC cultures was a decrease in Ca(++) levels in the endoplasmic reticulum (ER, [Ca(++)](ER)) observed five minutes following the initial challenge with PA. This decrease in [Ca(++) ](ER) was followed by a significant increase in the expression of ER stress signature genes CHOP, Xbp1 and GRP78. The early ER stress response induced by PA-LTx was followed by a strong mitochondrial membrane depolarization. Flow cytometry using 2', 7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA) showed an increase in oxidative stress within three to six hours after PA treatment. Treatment of cultures undergoing PA-LTx with the calcium chelator BAPTA-AM and the anti-oxidant MCI-186 significantly reversed the lipotoxic effect by decreasing the generation of ROS and significantly increasing cell viability. We conclude that lipotoxicity in Schwann cells results in cellular dysfunction and cell death that involves a robust ER stress response, mitochondrial dysfunction and an augmented state of cellular oxidative stress (ASCOS).

Role of oxidative stress in high-glucose- and diabetes-induced increased expression of Gq/11α proteins and associated signaling in vascular smooth muscle cells.

We have recently shown that aortic vascular smooth muscle cells (VSMCs) from streptozotocin (STZ)-induced diabetic rats and A10 VSMCs exposed to high glucose exhibited increased levels of Gqα and PLCß proteins. In the present study, we investigated whether the enhanced oxidative stress in hyperglycemia/diabetes contributes to the increased expression of the Gq/11α and PLCß proteins and the associated signaling in VSMCs by using antioxidants. The levels of Gq/11α and PLCß1/2 proteins, as determined by Western blotting, were significantly increased in A10 VSMCs exposed to high glucose and in aortic VSMCs from STZ-diabetic rats compared with control cells and were restored to control levels by antioxidants: apocynin, an NADPH oxidase inhibitor, and catalase, a scavenger of hydrogen peroxide. However, (111)Mn-tetrakis(benzoic acid porphyrin) and uric acid, scavengers of peroxynitrite, did not affect the high-glucose-induced enhanced levels of Gq/11α and PLCß proteins in A10 cells. Furthermore, the levels of superoxide dismutase-1 (SOD-1) but not of SOD-2 protein were augmented by high glucose. In addition, high-glucose-induced endothelin-1-stimulated production of IP(3) was also restored toward control levels by catalase. These results suggest that hyperglycemia/diabetes-induced enhanced expression of Gq/11α and PLCß proteins and signaling may be attributable to the enhanced oxidative stress due to augmented levels of H(2)O(2) and not to peroxynitrite.

Role of vasoactive peptides in high glucose-induced increased expression of Gαq/11 proteins and associated signaling in vascular smooth muscle cells.

We have recently shown that A10 vascular smooth muscle cells (VSMCs) exposed to high glucose exhibited enhanced expression of G(alpha)q and PLCbeta proteins. Since high glucose has been reported to increase the levels of vasoactive peptides and oxidative stress, the present study was undertaken to investigate the implication of angiotensin II (Ang II), endothelin (ET)-1, and oxidative stress in the high glucose-induced enhanced expression of G(alpha)q/11 and PLCbeta proteins and associated signaling in A10 VSMCs. The levels of G(alpha)q, G(alpha)11, PLCbeta-1, and PLCbeta-2 proteins, as determined by Western blotting, were significantly higher in A10 VSMCs exposed to high glucose than in control cells. The elevated levels were restored to control values by the antioxidant diphenyleneiodonium (DPI), as well as by the antagonist of Ang II AT1 receptor losartan and the antagonists of ETA and ETB receptors BQ123 and BQ788, respectively. In addition, ET-1-stimulated production of inositol trisphosphate (IP3), which was enhanced by high glucose, was also restored toward control levels by DPI. Furthermore, the enhanced production of superoxide anion (O2-), increased NADPH oxidase activity, and enhanced expression of p22phox and p47phox proteins induced by high glucose were restored to control levels by losartan, BQ123, and BQ788. These results suggest that through increased oxidative stress, high glucose-induced enhanced levels of endogenous Ang II and ET-1 may contribute to the increased levels of G(alpha)q/11 and mediated signaling in A10 VSMCs.

Role of growth factor receptor transactivation in high glucose-induced increased levels of Gq/11alpha and signaling in vascular smooth muscle cells.

We have recently shown that high glucose increased the expression of Gq/11alpha, PLCbeta and mediated signaling in A10 vascular smooth muscle cells (VSMC). Since high glucose has been shown to increase growth factor receptor activation, we investigated the role of epidermal growth factor receptor (EGF-R) and platelet-derived growth factor receptor (PDGF-R) transactivation in high glucose-induced enhanced expression of Gq/11alpha and PLCbeta. Pre-treatment of A10 VSMC with high glucose (26 mM) for 3 days, increased the levels of Gqalpha, G11alpha, PLCbeta-1 and PLCbeta-2 proteins which were restored to control levels by AG1478, an inhibitor of EGF-R, AG1295, an inhibitor of PDGF-R and PP2, an inhibitor of c-Src but not by PP3. In addition, endothelin-1 (ET-1)-stimulated production of IP(3) that was enhanced by high glucose was also restored towards control levels by AG1478, AG1295 and PP2. High glucose also increased the phosphorylation of EGF-R and PDGF-R which was abolished by AG1478, AG1295 and PP2. Furthermore, high glucose-induced enhanced levels of Gqalpha, G11alpha and PLCbeta were also attenuated by PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) and wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K). In addition, AG1478 and AG1295, also attenuated high glucose-induced enhanced phosphorylation of ERK1/2 and AKT. Furthermore, high glucose augmented the phosphorylation of c-Src which was attenuated by antioxidant, DPI. These results suggest that oxidative stress through the activation of c-Src and resultant transactivation of growth factor receptor contributes to the high glucose-induced enhanced expression of Gq/11alpha/PLC and -mediated cell signaling through MAPK/PI3K pathway.

High glucose increases the expression of Gq/11alpha and PLC-beta proteins and associated signaling in vascular smooth muscle cells.

The levels and activity of protein kinase C and diacylglycerol were shown to be upregulated in diabetes/hyperglycemia; however, studies on the expression of upstream signaling molecules of phosphatidylinositol turnover were lacking. The present study was therefore undertaken to examine whether hyperglycemia/diabetes could also modulate the expression of Gqalpha and phospholipase C-beta (PLC-beta) proteins and associated phosphatidylinositol turnover signaling in aortic vascular smooth muscle cells (VSMCs) and A10 VSMCs exposed to high glucose. Aortic VSMCs from streptozotocin-diabetic rats exhibited an increased expression of Gqalpha and PLC-beta1 proteins (60% and 30%, respectively) compared with control cells as determined by Western blot analysis. The pretreatment of A10 VSMCs with high glucose (26 mM) for 3 days also augmented the levels of Gqalpha, G11alpha, PLC-beta1 and -beta2 proteins by about 50, 35, 30, and 30%, respectively, compared with control cells that were restored to control levels by endothelin-1 (ET-1), ET types A and B (ET(A) and ET(B)) receptors, and angiotensin II type 1 (AT1) receptor antagonists. In addition, ET-1-stimulated inositol triphosphate formation was also significantly higher in VSMCs exposed to high glucose, whereas the basal levels of inositol triphosphate were not different between the two groups. Furthermore, the treatment of A10 VSMCs with angiotensin II and ET-1 also significantly increased the levels of Gq/11alpha and PLC-beta proteins that were restored toward control levels by ET(A)/ET(B) and AT1 receptor antagonists. These results suggest that high glucose augments the expression of Gq/11alpha, PLC-beta, and mediated signaling in VSMCs, which may be attributed to AT1, ET(A), and ET(B) receptors.

Role of oxidative stress in high glucose-induced decreased expression of Gialpha proteins and adenylyl cyclase signaling in vascular smooth muscle cells.

We have recently shown that aorta from streptozotocin (STZ)-induced diabetic rats and A10 vascular smooth muscle cells (VSMCs) exposed to high glucose exhibited decreased levels of inhibitory guanine nucleotide regulatory protein (Gi)alpha proteins. In the present studies, we investigated the implication of oxidative stress in the hyperglycemia/diabetes-induced decreased expression of the Gialpha protein and adenylyl cyclase signaling in VSMCs by using antioxidants. The levels of Gialpha proteins were significantly decreased in A10 VSMCs exposed to high glucose and in aortic VSMCs from STZ-diabetic rats compared with control cells and were restored to control levels by antioxidants. In addition, (111)Mn-tetralis(benzoic acid porphyrin) and uric acid, scavengers of peroxynitrite, and NG-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase but not catalase, also restored the high glucose-induced decreased expression of Gialpha proteins to the control levels in A10 VSMCs. Furthermore, the enhanced production of superoxide anion (O2-) and increased activity of NADPH oxidase in these cells were also restored to control levels by diphenyleneiodonium, an inhibitor of NADPH oxidase. In addition, the diminished inhibition of adenylyl cyclase activity by inhibitory hormones and forskolin-stimulated adenylyl cyclase activity by low concentrations of GTPgammaS as well as the enhanced stimulation of adenylyl cyclase by stimulatory agonists in hyperglycemic cells were restored to control levels by antioxidant treatments. These results suggest that high glucose-induced decreased levels of Gialpha proteins and associated signaling in A10 VSMCs may be attributed to the enhanced oxidative stress due to augmented levels of peroxynitrite and not to H2O2.

The distribution and density of ET-1 and its receptors are different in human right and left ventricular endocardial endothelial cells.

Evidence suggests that endocardial endothelial cells (EECs) may play a role in the regulation of cardiac function by releasing ET-1. Furthermore, reports in the literature suggested that differences may exist in peptide receptor distribution between the left and right EECs. In this study, we verified if the distribution and density of ET-1 and its receptors could be different in right as compared to left ventricular EECs, and whether this difference may affect ET-1-induced increase of intracellular calcium. Using immunofluorescence and 3D confocal microscopy, our results showed that in both cell types, the ET(A) receptor is present and is homogeneously distributed throughout the two cell types. The relative density of the ET(A) receptor is similar in both right and left ventricular EECs. The ET(B) receptor is also present in right and left ventricular EECs, however, the relative density of the ET(B) receptor is higher in the nucleus as compared to the cytosol. In addition, the ET(B) receptor density was found to be higher in left EECs as compared to right EECs. In addition, our results showed that ET-1 is present in the cytosol and the nucleus of both types of cells and that the relative density of ET-1 is higher in right as compared to left ventricular EECs. Moreover, using the Fura-2 calcium measurement technique, our results showed that in left ventricular EECs, both ET(A) and ET(B) receptor activation mediated the effect of ET-1 on intracellular calcium, whereas in right ventricular EECs, this effect was solely mediated by the ET(A) receptor. In conclusion, our results showed that ET-1 and its receptors are present in both right and left ventricular EECs. However, the distribution and relative density of ET-1 and its receptors seem to be different in right EECs as compared to left EECs.

Presence of neuropeptide Y and the Y1 receptor in the plasma membrane and nuclear envelope of human endocardial endothelial cells: modulation of intracellular calcium.

The aims of the present study were to investigate the presence and distribution of NPY and the Y1 receptor in endocardial endothelial cells (EECs), to verify if EECs can release NPY, and to determine if the effect of NPY on intracellular calcium is mediated via the Y1 receptor. Immunofluorescence, 3-D confocal microscopy and radioimmunoassay techniques were used on 20-week-old human fetal EECs. Our results showed that NPY and the Y1 receptor are present in human EECs (hEECs) and that their distributions are similar, the fluorescence labelling being higher in the nucleus and more particularly at the level of the nuclear envelope when compared with the cytosol. Using radioimmunoassay, we demonstrated that EECs are a source of NPY and can secrete this peptide upon a sustained increase of intracellular calcium ([Ca]i). Using fluo-3 and 3-D confocal microscopy technique, superfusion of hEECs as well as EECs isolated from rat adult hearts with increasing concentrations of NPY induced a dose-dependent, sustained increase in free cytosolic and nuclear Ca2+ levels. This effect of NPY on EEC [Ca]i was completely reversible upon washout of NPY and was partially blocked by BIBP3226, a selective Y1 receptor antagonist. The results suggest that NPY and Y1 receptors are present in the EECs of 20-week-old human fetal heart and they share the same distribution and localization inside the cell. In addition, EECs are able to secrete NPY in response to an increase in [Ca]i, and the Y1 receptor as well as other NPY receptors seem to participate in mediating the effects of NPY on [Ca]i in these cells. Thus, NPY released by EECs may modulate excitation-secretion coupling of these cells.