Lysyl oxidase like-1 deficiency in optic nerve head astrocytes elicits reactive astrocytosis and alters functional effects of astrocyte derived exosomes.

Glaucoma is a multifactorial progressive ocular pathology that manifests clinically with damage to the optic nerve (ON) and the retina, ultimately leading to blindness. The optic nerve head (ONH) shows the earliest signs of glaucoma pathology, and therefore, is an attractive target for drug discovery. The goal of this study was to elucidate the effects of reactive astrocytosis on the elastin metabolism pathway in primary rat optic nerve head astrocytes (ONHA), the primary glial cell type in the unmyelinated ONH. Following exposure to static equibiaxial mechanical strain, we observed prototypic molecular and biochemical signatures of reactive astrocytosis that were associated with a decrease in lysyl oxidase like 1 (Loxl1) expression and a concomitant decrease in elastin (Eln) gene expression. We subsequently investigated the role of Loxl1 in reactive astrocytosis by generating primary rat ONHA cultures with ∼50% decreased Loxl1 expression. Our results suggest that reduced Loxl1 expression is sufficient to elicit molecular signatures of elastinopathy in ONHA. Astrocyte derived exosomes (ADE) significantly increased the length of primary neurites of primary neurons in vitro. In contrast, ADE from Loxl1-deficient ONHA were deficient of trophic effects on neurite outgrowth in vitro, positing that Loxl1 dysfunction and the ensuing impaired elastin synthesis during reactive astrocytosis in the ONH may contribute to impaired neuron-glia signaling in glaucoma. Our data support a role of dysregulated Loxl1 function in eliciting reactive astrocytosis in glaucoma subtypes associated with increased IOP, even in the absence of genetic polymorphisms in LOXL1 typically associated with exfoliation glaucoma. This suggests the need for a paradigm shift toward considering lysyl oxidase activity and elastin metabolism and signaling as contributors to an altered secretome of the ONH that may lead to the progression of glaucomatous changes. Future research is needed to investigate cargo of exosomes in the context of reactive astrocytosis and identify the pathways leading to the observed transcriptome changes during reactive astrocytosis.

Isoprenylation of Monomeric GTPases in Human Trabecular Meshwork Cells.

Small monomeric GTPases, including those belonging to the Rho family, regulate a diverse array of intracellular signaling pathways which affect vesicle transport/trafficking, endocytosis, cell cycle progression, cell contractility, and formation of stress fibers or focal adhesions. Functional activation of newly synthesized small monomeric GTPases is facilitated by a multi-step posttranslational process involving transferase-catalyzed addition of farnesyl or geranylgeranyl isoprenoids to conserved cysteine residues within a unique carboxy terminal -CaaX motif. Here, using well-established and widely available contemporary methodologies, detailed protocols by which to semi-quantitatively evaluate the functional consequence of posttranslational isoprenylation in human trabecular meshwork cells are described. We propose the novel concept that posttranslational isoprenylation itself is a key regulator of mammalian Rho GTPase protein expression and turnover.

Testosterone treatment restores vestibular function by enhancing neuronal survival in an experimental closed-head repetitive mild traumatic brain injury model.

Repetitive mild traumatic brain injury (rmTBI) results in a myriad of symptoms, including vestibular impairment. The mechanisms underlying vestibular dysfunction in rmTBI patients remain poorly understood. Concomitantly, acute hypogonadism occurs following TBI and can persist chronically in many patients. Using a repetitive mild closed-head animal model of TBI, the role of testosterone on vestibular function was tested. Male Long Evans Hooded rats were randomly divided into sham or rmTBI groups. Significant vestibular deficits were observed both acutely and chronically in the rmTBI groups. Systemic testosterone was administered after the development of chronic vestibular dysfunction. rmTBI animals given testosterone showed improved vestibular function that was sustained for 175 days post-rmTBI. Significant vestibular neuronal cell loss was, however, observed in the rmTBI animals compared to Sham animals at 175 days post-rmTBI and testosterone treatment significantly improved vestibular neuronal survival. Taken together, these data demonstrate a critical restorative role of testosterone in vestibular function following rmTBI. This study has important clinical implications because it identifies testosterone treatment as a viable therapeutic strategy for the long-term recovery of vestibular function following TBI.

TGF-ß2 Promotes Oxidative Stress in Human Trabecular Meshwork Cells by Selectively Enhancing NADPH Oxidase 4 Expression.

Purpose: The multifunctional profibrotic cytokine TGF-ß2 is implicated in the pathophysiology of primary open angle glaucoma (POAG). While the underlying cause of POAG remains unclear, TGF-ß2 dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment is considered an early pathologic consequence associated with impaired aqueous humor (AH) outflow and elevated IOP. Mitochondrial-targeted antioxidants have been recently shown by our group to markedly attenuate TGF-ß2 profibrotic responses, strongly implicating oxidative stress as a key facilitator of TGF-ß2 signaling in human TM cells. In this study, we determined the mechanism by which oxidative stress facilitates TGF-ß2 profibrotic responses in cultured primary human TM cells. Methods: Semiconfluent cultures of primary or transformed human TM cells were conditioned overnight in serum-free media and subsequently challenged without or with TGF-ß2 (5 ng/mL). Relative changes in the mRNA content of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) isoforms, connective tissue growth factor (CTGF), collagen 1α1 and 4α1 isoforms or relative changes in the protein content of Nox4, phospho- and total-Smad2 and -Smad3, collagens I and IV were determined in the absence or presence of GKT137831, a Nox1-Nox4 dual enzyme inhibitor, and quantified by real-time qPCR or by immunoblot, respectively. Relative in situ changes in collagens I and IV and in alpha smooth muscle actin (αSMA) were semiquantified by immunocytochemistry, whereas relative changes in filamentous actin stress fiber formation was semiquantified by phalloidin staining. Results: Quiescent primary human TM cells cultured in the presence of TGF-ß2 exhibited a marked selective increase in endogenous Nox4 mRNA and Nox4 protein expression. Actinomycin D prevented TGF-ß2 mediated increases in Nox4 mRNA expression. TM cells reverse transfected with siRNA against Smad3 prevented TGF-ß2 mediated increases in Nox4 mRNA expression. Pre-incubating TM cells with GKT137831 attenuated TGF-ß2 mediated increases in intracellular reactive oxygen species (ROS), in COL1A1, COL4A1, and CTGF mRNA expression, in Smad3 protein phosphorylation, in collagens I, collagens IV, and αSMA protein expression, and in filamentous actin stress fiber formation. Conclusions: TGF-ß2 promotes oxidative stress in primary human TM cells by selectively increasing expression of NADPH oxidase 4. Dysregulation of redox equilibrium by induction of NADPH oxidase 4 expression appears to be a key early event involved in the pathologic profibrotic responses elicited by TGF-ß2 canonical signaling, including ECM remodeling, filamentous actin stress fiber formation, and αSMA expression. Selective inhibition of Nox4 expression/activation, in combination with mitochondrial-targeted antioxidants, represents a novel strategy by which to slow the progression of TGF-ß2 elicited profibrotic responses within the TM.

Targeting the blood-nerve barrier for the management of immune-mediated peripheral neuropathies.

Healthy peripheral nerves encounter, with increased frequency, numerous chemical, biological, and biomechanical forces. Over time and with increasing age, these forces collectively contribute to the pathophysiology of a spectrum of traumatic, metabolic, and/or immune-mediated peripheral nerve disorders. The blood-nerve barrier (BNB) serves as a critical first-line defense against chemical and biologic insults while biomechanical forces are continuously buffered by a dense array of longitudinally orientated epineural collagen fibers exhibiting high-tensile strength. As emphasized throughout this Experimental Neurology Special Issue, the BNB is best characterized as a functionally dynamic multicellular vascular unit comprised of not only highly specialized endoneurial endothelial cells, but also associated perineurial cells, pericytes, Schwann cells, basement membrane, and invested axons. The composition of the BNB, while anatomically distinct, is not functionally dissimilar to that of the well characterized neurovascular unit of the central nervous system. While the BNB lacks a glial limitans and an astrocytic endfoot layer, the primary function of both vascular units is to establish, maintain, and protect an optimal endoneurial (PNS) or interstitial (CNS) fluid microenvironment that is vital for proper neuronal function. Altered endoneurial homeostasis as a secondary consequence of BNB dysregulation is considered an early pathological event in the course of a variety of traumatic, immune-mediated, or metabolically acquired peripheral neuropathies. In this review, emerging experimental advancements targeting the endoneurial microvasculature for the therapeutic management of immune-mediated inflammatory peripheral neuropathies, including the AIDP variant of Guillain-Barré syndrome, are discussed.

Differential Activation of Glioprotective Intracellular Signaling Pathways in Primary Optic Nerve Head Astrocytes after Treatment with Different Classes of Antioxidants.

Optic nerve head astrocytes are the specialized glia cells that provide structural and trophic support to the optic nerve head. In response to cellular injury, optic nerve head astrocytes undergo reactive astrocytosis, the process of cellular activation associated with cytoskeletal remodeling, increases in the rate of proliferation and motility, and the generation of Reactive Oxygen Species. Antioxidant intervention has previously been proposed as a therapeutic approach for glaucomatous optic neuropathy, however, little is known regarding the response of optic nerve head astrocytes to antioxidants under physiological versus pathological conditions. The goal of this study was to determine the effects of three different antioxidants, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (Mn-TM-2-PyP), resveratrol and xanthohumol in primary optic nerve head astrocytes. Effects on the expression of the master regulator nuclear factor erythroid 2-related factor 2 (Nrf2), the antioxidant enzyme, manganese-dependent superoxide dismutase 2 (SOD2), and the pro-oxidant enzyme, nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), were determined by quantitative immunoblotting. Furthermore, efficacy in preventing chemically and reactive astrocytosis-induced increases in cellular oxidative stress was quantified using cell viability assays. The results were compared to the effects of the prototypic antioxidant, Trolox. Antioxidants elicited highly differential changes in the expression levels of Nrf2, SOD2, and NOX4. Notably, Mn-TM-2-PyP increased SOD2 expression eight-fold, while resveratrol increased Nrf2 expression three-fold. In contrast, xanthohumol exerted no statistically significant changes in expression levels. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake and lactate dehydrogenase (LDH) release assays were performed to assess cell viability after chemically and reactive astrocytosis-induced oxidative stress. Mn-TM-2-PyP exerted the most potent glioprotection by fully preventing the loss of cell viability, whereas resveratrol and xanthohumol partially restored cell viability. Our data provide the first evidence for a well-developed antioxidant defense system in optic nerve head astrocytes, which can be pharmacologically targeted by different classes of antioxidants.

Mitochondrial-Targeted Antioxidants Attenuate TGF-ß2 Signaling in Human Trabecular Meshwork Cells.

Purpose: POAG is a progressive optic neuropathy that is currently the leading cause of irreversible blindness worldwide. While the underlying cause of POAG remains unclear, TGF-ß2-dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment is considered an early pathologic consequence associated with impaired aqueous humor (AH) outflow and elevated IOP. Early studies have also demonstrated markedly elevated levels of oxidative stress markers in AH from POAG patients along with altered expression of antioxidant defenses. Here, using cultured primary or transformed human TM cells, we investigated the role oxidative stress plays at regulating TGF-ß2-mediated remodeling of the ECM. Methods: Primary or transformed (GTM3) human TM cells conditioned in serum-free media were incubated in the absence or presence of TGF-ß2 and relative changes in intracellular reactive oxygen species (ROS) were measured using oxidation-sensitive fluorogenic dyes CellROX green or 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA). TGF-ß2-mediated changes in the content of connective tissue growth factor (CTGF) and collagen types 1α1 (COL1A1) and 4α1 (COL4A1) mRNA or collagens I and IV isoform proteins were determined in the absence or presence of mitochondrial-targeted antioxidants (XJB-5-131 or MitoQ) and quantified by quantitative PCR or by immunoblot and immunocytochemistry. Smad-dependent canonic signaling was determined by immunoblot, whereas Smad-dependent transcriptional activity was quantified using a Smad2/3-responsive SBE-luciferase reporter assay. Results: Primary or transformed human TM cells cultured in the presence of TGF-ß2 (5 ng/mL; 2 hours) exhibited marked increases in CellROX or fluorescein fluorescence. Consistent with previous reports, challenging cultured human TM cells with TGF-ß2 elicited measurable increases in regulated Smad2/3 signaling as well as increases in CTGF, COL1A1, and COL4A1 mRNA and collagen protein content. Pretreating human TM cells with mitochondrial-targeted antioxidants XJB-5-131 (10 µM) or MitoQ (10 nM) attenuated TGF-ß2-mediated changes in Smad-dependent transcriptional activity. Conclusions: The multifunctional profibrotic cytokine TGF-ß2 elicits a marked increase in oxidative stress in human TM cells. Mitochondrial-targeted antioxidants attenuate TGF-ß2-mediated changes in Smad-dependent transcriptional activity, including marked reductions in CTGF and collagen isoform gene and protein expression. These findings suggest that mitochondrial-targeted antioxidants, when delivered directly to the TM, exhibit potential as a novel strategy by which to slow the progression of TGF-ß2-mediated remodeling of the ECM within the TM.

Randomized Controlled Trial of Physical Exercise in Diabetic Veterans With Length-Dependent Distal Symmetric Polyneuropathy.

Rationale: Physical exercise is an essential adjunct to the management of patients with type 2 diabetes mellitus. Therapeutic interventions that improve blood flow to peripheral nerves, such as exercise, may slow the progression of neuropathy in the diabetic patient. Aims: This randomized clinical trial was conducted to determine whether a structured program of aerobic, isokinetic strength, or the combination of aerobic-isokinetic strength exercise intervention alters peripheral nerve function in glycemic-controlled diabetic patients with advanced length-dependent distal symmetric polyneuropathy. Methods: Forty-five patients with type 2 diabetes mellitus exhibiting tight glycemic control (HbA1c intergroup range 7.2-8.0%) were randomized by block design across four experimental groups: sedentary controls (n = 12), aerobic exercise (n = 11), isokinetic strength (n = 11), or the combination of aerobic-isokinetic strength training (n = 11). Patients randomized to training groups exercised 3× per week for 12 weeks, whereas patients randomized to the sedentary control group received standard of care. To minimize attention and educational bias, all patients attended a 12-session health promotion educational series. At baseline, immediately following intervention, and again at 12-week post-intervention, detailed nerve conduction studies were conducted as a primary outcome measure. At these same intervals, all patients completed as secondary measures quantitative sensory testing, symptom-limited treadmill stress tests, and a Short-Form 36-Veterans Questionnaire (SF-36V). Results: Of the 45 patients randomized into this study, 37 (82%) had absent sural nerve responses, 19 (42%) had absent median sensory nerve responses, and 17 (38%) had absent ulnar sensory nerve responses. By comparison, responses from tibial nerves were absent in only three (7%) subjects while responses from peroneal nerves were absent in five (11%) subjects. Eleven (92%) of 12 patients that had volunteered to be biopsied exhibited abnormal levels of epidermal nerve fiber densities. Exercise, regardless of type, did not alter sensory or motor nerve electrodiagnostic findings among those patients exhibiting measurable responses (ANOVA). There was, however, a modest (p = 0.01) beneficial effect of exercise on sensory nerve function (Fisher's Exact Test). Importantly, the beneficial effect of exercise on sensory nerve function was enhanced (p = 0.03) during the post-intervention interval. In addition, three of six patients that had undergone exercise intervention exhibited a marked 1.9 ± 0.3-fold improvement in epidermal nerve fiber density. By comparison, none of three sedentary patients whom agreed to be biopsied a second time showed improvement in epidermal nerve fiber density. Compared to baseline values within groups, and compared with sedentary values across groups, neither aerobic, isokinetic strength, or the combination of aerobic-isokinetic strength exercise intervention altered peak oxygen uptake. Patients that underwent aerobic or the combined aerobic-isokinetic strength exercise intervention, however, demonstrated an increase in treadmill test duration that was sustained over the 12-week post-intervention period. Conclusion: A 12-week course of physical exercise, regardless of type, does not alter sensory or motor nerve electrodiagnostic findings. In a subset of patients, a short-term structured program of aerobic exercise may selectively improve sensory nerve fiber function. Large-scale exercise lifestyle intervention trials are warranted to further evaluate the impact of aerobic exercise on sensory nerve fiber function in diabetic neuropathic patients. Clinical Trial Registration: www.ClinicalTrials.gov, identifier NCT00955201.

Consensus recommendations for trabecular meshwork cell isolation, characterization and culture.

Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings.

Isoprenylation of Monomeric GTPases in Human Trabecular Meshwork Cells.

Small monomeric GTPases, including those belonging to the Rho family, regulate a diverse array of intracellular signaling pathways which affect vesicle transport/trafficking, endocytosis, cell cycle progression, cell contractility, and formation of stress fibers or focal adhesions. Functional activation of newly synthesized small monomeric GTPases is facilitated by a multistep post-translational process involving transferase-catalyzed addition of farnesyl or geranylgeranyl isoprenoids to conserved cysteine residues within a unique carboxy terminal CaaX motif. Here, using well-established and widely available contemporary methodologies, detailed protocols by which to semi-quantitatively evaluate the functional consequence of post-translational isoprenylation in human trabecular meshwork cells are described. We introduce the concept that isoprenylation alone is itself a key regulator of mammalian Rho GTPase expression and turnover.

Attenuation of experimental autoimmune neuritis with locally administered lovastatin-encapsulating poly(lactic-co-glycolic) acid nanoparticles.

Acute inflammatory demyelinating polyneuropathy (AIDP) is an aggressive antibody- and T-cell-mediated variant of Guillain-Barré Syndrome (GBS), a prominent and debilitating autoimmune disorder of the peripheral nervous system. Despite advancements in clinical management, treatment of patients with AIDP/GBS and its chronic variant CIDP remains palliative and relies on the use of non-specific immunemodulating therapies. Our laboratory has previously reported that therapeutic administration of statins safely attenuates the clinical severity of experimental autoimmune neuritis (EAN), a well-characterized animal model of AIDP/GBS, by restricting the migration of autoreactive leukocytes across peripheral nerve microvascular endoneurial endothelial cells that form the blood-nerve barrier. Despite these advancements, the clinical application of systemically administered statins for the management of inflammatory disorders remains controversial as a result of disappointingly inconclusive phase trials. Here, poly(lactic-co-glycolic) acid (PLGA) nanoparticles were evaluated as an alternative strategy by which to locally administer statins for the management of EAN. When tested in vitro, lovastatin-encapsulating PLGA nanoparticles elicited a marked increase in RhoB mRNA content in peripheral nerve microvascular endoneurial endothelial cells, similar to cells treated with activated unencapsulated lovastatin. Unilateral peri-neural administration of lovastatin-encapsulating PLGA nanoparticles, but not empty nanoparticles, to naïve Lewis rats similarly enhanced RhoB mRNA content in adjacent nerve and muscle tissue. When administered in this manner, serum levels of lovastatin were below the level of detection. Bilateral peri-neural administration of lovastatin-encapsulating PLGA nanoparticles to EAN-induced Lewis rats significantly attenuated EAN clinical severity while protecting against EAN-induced peripheral nerve morphological and functional deficits. This study provides the first proof-of-concept approach for the application of a nanoparticle-based local drug delivery platform for the management of inflammatory demyelinating diseases, including AIDP/GBS.

Th17 Cell Response in SOD1G93A Mice following Motor Nerve Injury.

An increased risk of ALS has been reported for veterans, varsity athletes, and professional football players. The mechanism underlying the increased risk in these populations has not been identified; however, it has been proposed that motor nerve injury may trigger immune responses which, in turn, can accelerate the progression of ALS. Accumulating evidence indicates that abnormal immune reactions and inflammation are involved in the pathogenesis of ALS, but the specific immune cells involved have not been clearly defined. To understand how nerve injury and immune responses may contribute to ALS development, we investigated responses of CD4(+) T cell after facial motor nerve axotomy (FNA) at a presymptomatic stage in a transgenic mouse model of ALS (B6SJL SOD1(G93A)). SOD1(G93A) mice, compared with WT mice, displayed an increase in the basal activation state of CD4(+) T cells and higher frequency of Th17 cells, which were further enhanced by FNA. In conclusion, SOD1(G93A) mice exhibit abnormal CD4(+) T cell activation with increased levels of Th17 cells prior to the onset of neurological symptoms. Motor nerve injury exacerbates Th17 cell responses and may contribute to the development of ALS, especially in those who carry genetic susceptibility to this disease.

Rho GTPase signaling promotes constitutive expression and release of TGF-ß2 by human trabecular meshwork cells.

Elevated intraocular pressure (IOP) is causally implicated in the pathophysiology of primary open-angle glaucoma (POAG). The molecular mechanisms responsible for elevated IOP remain elusive, but may involve aberrant expression and signaling of transforming growth factor (TGF)-ß2 within the trabecular meshwork (TM). Consistent with previously published studies, we show here that exogenous addition of TGF-ß2 to cultured porcine anterior segments significantly attenuates outflow facility in a time-dependent manner. By comparison, perfusing segments with a TGFßRI/ALK-5 antagonist (SB-431542) unexpectedly elicited a significant and sustained increase in outflow facility, implicating a role for TM-localized constitutive expression and release of TGF-ß2. Consistent with this thesis, cultured primary or transformed (GTM3) quiescent human TM cells were found to constitutively express and secrete measurable amounts of biologically-active TGF-ß2. Disrupting monomeric GTPase post-translational prenylation and activation with lovastatin or GGTI-298 markedly reduced constitutive TGF-ß2 expression and release. Specifically, inhibiting the Rho subfamily of GTPases with C3 exoenzyme similarly reduced constitutive expression and secretion of TGF-ß2. These findings suggest that Rho GTPase signaling, in part, regulates constitutive expression and release of biologically-active TGF-ß2 from human TM cells. Localized constitutive expression and release of TGF-ß2 by TM cells may promote or exacerbate elevation of IOP in POAG.

Forced Exercise Preconditioning Attenuates Experimental Autoimmune Neuritis by Altering Th1 Lymphocyte Composition and Egress.

A short-term exposure to moderately intense physical exercise affords a novel measure of protection against autoimmune-mediated peripheral nerve injury. Here, we investigated the mechanism by which forced exercise attenuates the development and progression of experimental autoimmune neuritis (EAN), an established animal model of Guillain-Barré syndrome. Adult male Lewis rats remained sedentary (control) or were preconditioned with forced exercise (1.2 km/day × 3 weeks) prior to P2-antigen induction of EAN. Sedentary rats developed a monophasic course of EAN beginning on postimmunization day 12.3 ± 0.2 and reaching peak severity on day 17.0 ± 0.3 (N = 12). By comparison, forced-exercise preconditioned rats exhibited a similar monophasic course but with significant (p < .05) reduction of disease severity. Analysis of popliteal lymph nodes revealed a protective effect of exercise preconditioning on leukocyte composition and egress. Compared with sedentary controls, forced exercise preconditioning promoted a sustained twofold retention of P2-antigen responsive leukocytes. The percentage distribution of pro-inflammatory (Th1) lymphocytes retained in the nodes from sedentary EAN rats (5.1 ± 0.9%) was significantly greater than that present in nodes from forced-exercise preconditioned EAN rats (2.9 ± 0.6%) or from adjuvant controls (2.0 ± 0.3%). In contrast, the percentage of anti-inflammatory (Th2) lymphocytes (7-10%) and that of cytotoxic T lymphocytes (∼20%) remained unaltered by forced exercise preconditioning. These data do not support an exercise-inducible shift in Th1:Th2 cell bias. Rather, preconditioning with forced exercise elicits a sustained attenuation of EAN severity, in part, by altering the composition and egress of autoreactive proinflammatory (Th1) lymphocytes from draining lymph nodes.

Novel role of Cdc42 and RalA GTPases in TNF-α mediated secretion of CCL2.

Transendothelial migration of autoreactive leukocytes into peripheral nerves is an early pathological hallmark of acute inflammatory demyelinating polyneuropathy (AIDP), a North American and European variant of Guillain-Barré Syndrome. Whereas the clinical management of AIDP is currently limited to non-selective immune modulating therapies, recent experimental studies support selective targeting of leukocyte trafficking as a promising alternative therapeutic strategy. Here, using a combination of targeted siRNA knockdown and pharmacological inhibitors, we report a novel role of both Cdc42 and RalA GTPases in facilitating TNF-α mediated CCL2 trafficking and release from immortalized rat peripheral nerve microvascular endoneurial endothelial cells. These findings raise interest in Cdc42 and RalA GTPases as potential therapeutic targets for the management of autoimmune inflammatory peripheral nerve disease.

Cdc42 GTPases facilitate TNF-α-mediated secretion of CCL2 from peripheral nerve microvascular endoneurial endothelial cells.

Trafficking of autoreactive leukocytes across the blood-nerve barrier and into peripheral nerves is an early pathological hallmark of Guillain-Barré syndrome (GBS). Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, promotes transendothelial migration by upregulating endothelial expression of inflammatory mediators, including CCL2, a chemokine implicated in GBS. We sought to determine the mechanism by which TNF-α induces expression and secretion of CCL2 from peripheral nerve microvascular endoneurial endothelial cells (PNMECs). Expression of CCL2 mRNA and protein in quiescent PNMEC cultures was minimal. In contrast, cultures treated with TNF-α exhibited increased CCL2 mRNA and protein content, as well as protein secretion. Simvastatin significantly attenuated TNF-α-induced CCL2 secretion without affecting CCL2 mRNA or protein expression. Co-incubation with geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate, prevented the effect of simvastatin. By comparison, inhibiting protein isoprenylation with GGTI-298, but not FTI-277, mimicked the effect of simvastatin and significantly attenuated transendothelial migration in vitro. Inhibition of the monomeric GTPase Cdc42, but not Rac1 or RhoA-C, attenuated TNF-α-mediated CCL2 secretion. TNF-α-mediated trafficking of autoreactive leukocytes into peripheral nerves during GBS may proceed by a mechanism that involves Cdc42-facilitated secretion of CCL2.

Neuroprotective effect of resveratrol prophylaxis on experimental retinal ischemic injury.

The purpose of the present study was to investigate whether systemically administered resveratrol can protect against acute retinal ischemic reperfusion injury. Two groups of adult male Sprague Dawley rats (n = 6 per group) were used for this study. Resveratrol (30 mg/kg) or an equal volume of vehicle (30% Solutol HS 15 in 0.9% saline) was administered daily for 5 days via intraperitoneal injection. On the third day of treatment, retinal ischemic injury was induced by elevation of intraocular pressure for 45 min. Prior to resveratrol administration and one-week following ischemic insult, retinal function was measured by scotopic electroretinography (ERG). Retinas were harvested and morphologically analyzed one week after ischemic insult. ERG a- and b-wave amplitudes were significantly reduced following ischemic reperfusion injury. Resveratrol treatment attenuated ischemic-induced loss of retinal function. In control vehicle-treated rats, ischemic reperfusion injury elicited marked thinning of inner retinal layers. Resveratrol prophylactic treatment reduced ischemia-mediated thinning of the whole retina and in particular the inner retinal layers. Therefore, resveratrol may have therapeutic value for the management of retinal ischemic disorders.

Tumour necrosis factor α enhances CCL2 and ICAM-1 expression in peripheral nerve microvascular endoneurial endothelial cells.

Recruitment and trafficking of autoreactive leucocytes across the BNB (blood-nerve barrier) is an early pathological insult in GBS (Guillain-Barré syndrome), an aggressive autoimmune disorder of the PNS (peripheral nervous system). Whereas the aetiology and pathogenesis of GBS remain unclear, pro-inflammatory cytokines, including TNFα (tumour necrosis factor α), are reported to be elevated early in the course of GBS and may initiate nerve injury by activating the BNB. Previously, we reported that disrupting leucocyte trafficking in vivo therapeutically attenuates the course of an established animal model of GBS. Here, PNMECs (peripheral nerve microvascular endothelial cells) that form the BNB were harvested from rat sciatic nerves, immortalized by SV40 (simian virus 40) large T antigen transduction and subsequently challenged with TNFα. Relative changes in CCL2 (chemokine ligand 2) and ICAM-1 (intercellular adhesion molecule 1) expression were determined. We report that TNFα elicits marked dose- and time-dependent increases in CCL2 and ICAM-1 mRNA and protein content and promotes secretion of functional CCL2 from immortalized and primary PNMEC cultures. TNFα-mediated secretion of CCL2 promotes, in vitro, the transendothelial migration of CCR2-expressing THP-1 monocytes. Increased CCL2 and ICAM-1 expression in response to TNFα may facilitate recruitment and trafficking of autoreactive leucocytes across the BNB in autoimmune disorders, including GBS.

Transforming growth factor-ß2 induces synthesis and secretion of endothelin-1 in human trabecular meshwork cells.

PURPOSE: Analysis of aqueous humor from patients with primary open-angle glaucoma (POAG) revealed marked increases in the content of endothelin-1 (ET-1) and transforming growth factor-beta (TGF-ß). We determined the consequences of TGF-ß signaling on ET-1 expression and secretion by human trabecular meshwork (TM) cells. METHODS: Primary or transformed (NTM5 and GTM3) human TM cells conditioned in serum-free media were incubated in the absence or presence of TGF-ß1 or -ß2. Relative changes in preproendothelin (ppET)-1 mRNA content and secreted ET-1 peptide were quantified by real-time PCR and ELISA, respectively. In some experiments, TGF-ß or ET-1 receptor antagonists, or Rho G-protein inhibitors, were evaluated for effects on TGF-ß signaling. Filamentous actin organization was visualized by phalloidin. RESULTS: Primary or transformed human TM cells cultured in the presence of TGF-ß1 or -ß2 exhibit a marked (>8-fold) increase in ppET-1 mRNA content compared to vehicle controls. Coincubation with SB-505124, an inhibitor of TGFßRI/ALK-5 signaling, prevented TGF-ß-mediated ppET-1 mRNA expression. In contrast, coincubation with ET(A) (BQ-123) or ET(B) (BQ-788) receptor antagonists had no effect on TGF-ß-mediated ppET-1 mRNA expression. TGF-ß1 and -ß2 each elicited a robust (>7-fold) secretion of ET-1 while enhancing stress fiber organization. Inhibition of Rho signaling attenuated TGF-ß-mediated increases in ppET-1 mRNA content, ET-1 secretion, and stress fiber organization. CONCLUSIONS: TGF-ß, signaling through the TGFßRI/ALK-5 receptor, elicits marked increases in ET-1 mRNA content and ET-1 secretion from cultured primary or transformed human TM cells. Elevated levels of TGF-ß2 present in AH of POAG patients may elevate intraocular pressure, in part, by eliciting aberrant Rho G-protein dependent cell contraction, and increasing ET-1 synthesis and secretion, in human TM cells.

Forced-exercise attenuates experimental autoimmune neuritis.

Physical inactivity in combination with a sedentary lifestyle is strongly associated with an increased risk of development of inflammatory-mediated diseases, including autoimmune disorders. Recent studies suggest that anti-inflammatory effects of physical exercise may be of therapeutic value in some affected individuals. In this study, we determined the effects of forced-exercise (treadmill running) on the development and progression of experimental autoimmune neuritis (EAN), an established animal model of Guillain-Barré syndrome. Adult male Lewis rats were subjected to sedentary (control) or forced-exercise (1.2 km per day, 5 days a week) for three weeks prior to induction of EAN. P2 (53-78)-immunized sedentary control rats developed a monophasic course of EAN beginning on post-injection day 12.33 ± 0.59 (n = 18) and reaching peak severity on day 15.83 ± 0.35 (n = 18). At near peak of disease, ankle- and sciatic notch-evoked compound muscle action potential (CMAP) amplitudes in sedentary control rats were reduced (~50%) while motor nerve conduction velocity (MNCV) was slowed (~30%) compared with pre-induction evoked responses. In marked contrast, rats undergoing forced-exercise exhibited a significantly less severe clinical course of EAN beginning on post-injection day 12.63 ± 0.53 (n = 16) and reaching peaking severity on day 14.69 ± 0.73 (n = 16). At near peak of disease, ankle- and sciatic-notch-evoked CMAP amplitudes in forced-exercised rats were preserved while EAN-associated slowing of MNCV was modestly attenuated by exercise. Three weeks of forced-exercise reduced by 46% total plasma corticosterone content while elevating the levels of corticosteroid binding globulin. We conclude from this study that forced-exercise administered prior to and during development of EAN affords a novel measure of protection against autoimmune-associated deficits in peripheral nerve evoked responses independent of steroid-induced immune suppression.