Appearance of claudin-5+ leukocyte subtypes in the blood and CNS during progression of EAE.

BACKGROUND: Tight junctions (TJs) are membrane specializations characteristic of barrier-forming membranes, which function to seal the aqueous pathway between endothelial cells or epithelial cells and, thereby, obstruct intercellular solute and cellular movement. However, previous work from our laboratory found that claudin-5 (CLN-5), a TJ protein prominent at the blood-brain barrier (BBB), was also detected, ectopically, on leukocytes (CLN-5+) in the blood and central nervous system (CNS) of mice with experimental autoimmune encephalomyelitis (EAE), a neuroinflammatory, demyelinating disease that is a model for multiple sclerosis. CLN-5 was further shown to be transferred from endothelial cells to circulating leukocytes during disease, prompting consideration this action is coupled to leukocyte transendothelial migration (TEM) into the CNS by fostering transient interactions between corresponding leukocyte and endothelial junctional proteins at the BBB. METHODS: To begin clarifying the significance of CLN-5+ leukocytes, flow cytometry was used to determine their appearance in the blood and CNS during EAE. RESULTS: Flow cytometric analysis revealed CLN-5+ populations among CD4 and CD8 T cells, B cells, monocytes and neutrophils, and these appeared with varying kinetics and to different extents in both blood and CNS. CLN-5 levels on circulating T cells further correlated highly with activation state. And, the percentage of CLN-5+ cells among each of the subtypes analyzed was considerably higher in CNS tissue than in blood, consistent with the interpretation that CLN-5+ leukocytes gain preferred access to the CNS. CONCLUSION: Several leukocyte subtypes variably acquire CLN-5 in blood before they enter the CNS, an event that may represent a novel mechanism to guide leukocytes to sites for paracellular diapedesis across the BBB.

Biodegradable nanofiber-based piezoelectric transducer.

Piezoelectric materials, a type of "smart" material that generates electricity while deforming and vice versa, have been used extensively for many important implantable medical devices such as sensors, transducers, and actuators. However, commonly utilized piezoelectric materials are either toxic or nondegradable. Thus, implanted devices employing these materials raise a significant concern in terms of safety issues and often require an invasive removal surgery, which can damage directly interfaced tissues/organs. Here, we present a strategy for materials processing, device assembly, and electronic integration to 1) create biodegradable and biocompatible piezoelectric PLLA [poly(l-lactic acid)] nanofibers with a highly controllable, efficient, and stable piezoelectric performance, and 2) demonstrate device applications of this nanomaterial, including a highly sensitive biodegradable pressure sensor for monitoring vital physiological pressures and a biodegradable ultrasonic transducer for blood-brain barrier opening that can be used to facilitate the delivery of drugs into the brain. These significant applications, which have not been achieved so far by conventional piezoelectric materials and bulk piezoelectric PLLA, demonstrate the PLLA nanofibers as a powerful material platform that offers a profound impact on various medical fields including drug delivery, tissue engineering, and implanted medical devices.

Human ES-derived MSCs correct TNF-α-mediated alterations in a blood-brain barrier model.

BACKGROUND: Immune cell trafficking into the CNS is considered to contribute to pathogenesis in MS and its animal model, EAE. Disruption of the blood-brain barrier (BBB) is a hallmark of these pathologies and a potential target of therapeutics. Human embryonic stem cell-derived mesenchymal stem/stromal cells (hES-MSCs) have shown superior therapeutic efficacy, compared to bone marrow-derived MSCs, in reducing clinical symptoms and neuropathology of EAE. However, it has not yet been reported whether hES-MSCs inhibit and/or repair the BBB damage associated with neuroinflammation that accompanies EAE. METHODS: BMECs were cultured on Transwell inserts as a BBB model for all the experiments. Disruption of BBB models was induced by TNF-α, a pro-inflammatory cytokine that is a hallmark of acute and chronic neuroinflammation. RESULTS: Results indicated that hES-MSCs reversed the TNF-α-induced changes in tight junction proteins, permeability, transendothelial electrical resistance, and expression of adhesion molecules, especially when these cells were placed in direct contact with BMEC. CONCLUSIONS: hES-MSCs and/or products derived from them could potentially serve as novel therapeutics to repair BBB disturbances in MS.

Systemic TLR2 tolerance enhances central nervous system remyelination.

BACKGROUND: Multiple sclerosis (MS) is a central nervous system (CNS) autoimmune disease characterized by both inflammatory demyelination and impaired remyelination. Studies indicate that Toll-like receptor 2 (TLR2) signaling contributes to both the inflammatory component and the defective remyelination in MS. While most MS therapeutics target adaptive immunity, we recently reported that reducing TLR2 signaling in innate immune cells by inducing TLR2 tolerance attenuates adoptively transferred experimental autoimmune encephalomyelitis. Given that previous reports suggest TLR2 signaling also inhibits myelin repair, the objective of this study was to assess how reducing TLR2 signaling through TLR2 tolerance induction affects CNS myelin repair. METHODS: Chow containing 0.2% cuprizone was fed to male and female wild-type (WT) C57BL/6 mice or TLR2-deficient (TLR2-/-) mice for 5 weeks to induce demyelination. During a 2-week remyelination period following discontinuation of cuprizone, WT mice received either low dose TLR2 ligands to induce systemic TLR2 tolerance or vehicle control (VC). Remyelination was evaluated via electron microscopy and immunohistochemical analysis of microglia and oligodendrocytes in the corpus callosum. Statistical tests included 2-way ANOVA and Mann-Whitney U analyses. RESULTS: Inducing TLR2 tolerance in WT mice during remyelination significantly enhanced myelin recovery, restoring unmyelinated axon frequency and myelin thickness to baseline levels compared to VC-treated mice. Mechanistically, enhanced remyelination in TLR2 tolerized mice was associated with a shift in corpus callosum microglia from a pro-inflammatory iNOS+ phenotype to a non-inflammatory/pro-repair Arg1+ phenotype. This result was confirmed in vitro by inducing TLR2 tolerance in WT microglia cultures. TLR2-/- mice, without TLR2 tolerance induction, also significantly enhanced myelin recovery compared to WT mice, adding confirmation that reduced TLR2 signaling is associated with enhanced remyelination. DISCUSSION: Our results suggest that reducing TLR2 signaling in vivo by inducing TLR2 tolerance significantly enhances myelin repair. Furthermore, the enhanced remyelination resulting from TLR2 tolerance induction is associated with a shift in corpus callosum microglia from a pro-inflammatory iNOS+ phenotype to a non-inflammatory/pro-repair Arg1+ phenotype. While deletion of TLR2 would be an impractical approach in vivo, reducing innate immune signaling through TLR2 tolerance induction may represent a novel, two-pronged approach for treating both inflammatory and myelin repair components of MS.

Extracellular vesicles: mediators and biomarkers of pathology along CNS barriers.

Extracellular vesicles (EVs) are heterogeneous, nano-sized vesicles that are shed into the blood and other body fluids, which disperse a variety of bioactive molecules (e.g., protein, mRNA, miRNA, DNA and lipids) to cellular targets over long and short distances. EVs are thought to be produced by nearly every cell type, however this review will focus specifically on EVs that originate from cells at the interface of CNS barriers. Highlighted topics include, EV biogenesis, the production of EVs in response to neuroinflammation, role in intercellular communication and their utility as a therapeutic platform. In this review, novel concepts regarding the use of EVs as biomarkers for BBB status and as facilitators for immune neuroinvasion are also discussed. Future directions and prospective are covered along with important unanswered questions in the field of CNS endothelial EV biology.

Spatiotemporal resolution of spinal meningeal and parenchymal inflammation during experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) induced by active immunization of C57BL/6 mice with peptide from myelin oligodendrocyte protein (MOG35-55), is a neuroinflammatory, demyelinating disease widely recognized as an animal model of multiple sclerosis (MS). Typically, EAE presents with an ascending course of paralysis, and inflammation that is predominantly localized to the spinal cord. Recent studies have further indicated that inflammation - in both MS and EAE - might initiate within the meninges and propagate from there to the underlying parenchyma. However, the patterns of inflammation within the respective meningeal and parenchymal compartments along the length of the spinal cord, and the progression with which these patterns develop during EAE, have yet to be detailed. Such analysis could hold key to identifying factors critical for spreading, as well as constraining, inflammation along the neuraxis. To address this issue, high-resolution 3-dimensional (3D) confocal microscopy was performed to visualize, in detail, the sequence of leukocyte infiltration at distinct regions of the spinal cord. High quality virtual slide scanning for imaging the entire spinal cord using epifluorescence was further conducted to highlight the directionality and relative degree of inflammation. Meningeal inflammation was found to precede parenchymal inflammation at all levels of the spinal cord, but did not develop equally or simultaneously throughout the subarachnoid space (SAS) of the meninges. Instead, meningeal inflammation was initially most obvious in the caudal SAS, from which it progressed to the immediate underlying parenchyma, paralleling the first signs of clinical disease in the tail and hind limbs. Meningeal inflammation could then be seen to extend in the caudal-to-rostral direction, followed by a similar, but delayed, trajectory of parenchymal inflammation. To additionally determine whether the course of ascending paralysis and leukocyte infiltration during EAE is reflected in differences in inflammatory gene expression by meningeal and parenchymal microvessels along the spinal cord, laser capture microdissection (LCM) coupled with gene expression profiling was performed. Expression profiles varied between these respective vessel populations at both the cervical and caudal levels of the spinal cord during disease progression, and within each vessel population at different levels of the cord at a given time during disease. These results reinforce a significant role for the meninges in the development and propagation of central nervous system inflammation associated with MS and EAE.

Appearance of claudin-5+ leukocytes in the central nervous system during neuroinflammation: a novel role for endothelial-derived extracellular vesicles.

BACKGROUND: The mechanism of leukocyte transendothelial migration (TEM) across the highly restrictive blood-brain barrier (BBB) remains enigmatic, with paracellular TEM thought to require leukocytes to somehow navigate the obstructive endothelial tight junctions (TJs). Transient interactions between TJ proteins on the respective leukocyte and endothelial surfaces have been proposed as one mechanism for TEM. Given the expanding role of extracellular vesicles (EVs) in intercellular communication, we investigated whether EVs derived from brain microvascular endothelial cells (BMEC) of the BBB may play a role in transferring a major TJ protein, claudin-5 (CLN-5), to leukocytes as a possible basis for such a mechanism during neuroinflammation. METHODS: High-resolution 3D confocal imaging was used to highlight CLN-5 immunoreactivity in the central nervous system (CNS) and on leukocytes of mice with the neuroinflammatory condition experimental autoimmune encephalomyelitis (EAE). Both Western blotting of circulating leukocytes from wild-type mice and fluorescence imaging of leukocyte-associated eGFP-CLN-5 in the blood and CNS of endothelial-targeted, Tie-2-eGFP-CLN-5 transgenic mice were used to confirm the presence of CLN-5 protein on these cells. EVs were isolated from TNF-α-stimulated BMEC cultures and blood plasma of Tie-2-eGFP-CLN-5 mice with EAE and evaluated for CLN-5 protein by Western blotting and fluorescence-activated cell sorting (FACS), respectively. Confocal imaging and FACS were used to detect binding of endothelial-derived EVs from these two sources to leukocytes in vitro. Serial electron microscopy (serial EM) and 3D contour-based surface reconstruction were employed to view EV-like structures at the leukocyte:BBB interface in situ in inflamed CNS microvessels. RESULTS: A subpopulation of leukocytes immunoreactive for CLN-5 on their surface was seen to infiltrate the CNS of mice with EAE and reside in close apposition to inflamed vessels. Confocal imaging of immunostained samples and Western blotting established the presence of CLN-5+ leukocytes in blood as well, implying these cells are present prior to TEM. Moreover, imaging of inflamed CNS vessels and the associated perivascular cell infiltrates from Tie-2-eGFP-CLN-5 mice with EAE revealed leukocytes bearing the eGFP label, further supporting the hypothesis CLN-5 is transferred from endothelial cells to circulating leukocytes in vivo. Western blotting of BMEC-derived EVs, corresponding in size to both exosomes and microvesicles, and FACS analysis of plasma-derived EVs from Tie-2-eGFP-CLN-5 mice with EAE validated expression of CLN-5 by EVs of endothelial origin. Confocal imaging and FACS further revealed both PKH-67-labeled EVs from cultured BMECs and eGFP-CLN-5+ EVs from plasma of Tie-2-eGFP-CLN-5 mice with EAE can bind to leukocytes. Lastly, serial EM and 3D contour-based surface reconstruction revealed a close association of EV-like structures between the marginating leukocytes and BMECs in situ during EAE. CONCLUSIONS: During neuroinflammation, CLN-5+ leukocytes appear in the CNS, and both CLN-5+ leukocytes and CLN-5+ EVs are detected in the blood. As endothelial cells transfer CLN-5+ to leukocytes in vivo, and EVs released from BMEC bind to leukocytes in vitro, EVs may serve as the vehicles to transfer CLN-5 protein at sites of leukocyte:endothelial contact along the BBB. This action may be a prelude to facilitate TEM through the formation of temporary TJ protein bridges between these two cell types.

Alterations in tight junction protein and IgG permeability accompany leukocyte extravasation across the choroid plexus during neuroinflammation.

The choroid plexus (CP) is considered to be a point of leukocyte entry into the CNS during normal immune surveillance and in neuroinflammatory diseases. The structural and functional alterations within the CP that support this migration are not understood. We used quantitative, high-resolution, 3-dimensional (3-D) fluorescence imaging to analyze CP alterations associated with inflammatory responses in C57/Bl6 mice after the induction of experimental autoimmune encephalomyelitis by immunization with myelin oligodendrocyte glycoprotein (MOG) and complete Freund adjuvant/pertussis toxin (MOG-CFA/PTX) or adjuvants alone (CFA-PTX). The MOG-CFA/PTX and CFA/PTX produced similar effects, although those caused by the former were consistently more marked. Both treatments resulted in the accumulation of serum immunoglobulin G and leukocytes in the CP stroma, consistent with elevated stromal capillary permeability. They also provoked distortions and diminished immunostaining patterns of the tight junction adaptor protein ZO-1 in the choroidal epithelium but no obvious change in the patterns of the tight junction associated protein claudin-2. Only MOG-CFA/PTX triggered visible extravasation of immunoglobulin G and leukocytes across the choroidal epithelium. Our results suggest that CFA/PTX primes the CP for neuroinflammation by inducing structural changes that are exacerbated when there is an immune response to MOG and reinforce the CP as a gateway for leukocytes to enter the CNS by accessing the CSF and leptomeninges.

Human ESC-derived MSCs outperform bone marrow MSCs in the treatment of an EAE model of multiple sclerosis.

Current therapies for multiple sclerosis (MS) are largely palliative, not curative. Mesenchymal stem cells (MSCs) harbor regenerative and immunosuppressive functions, indicating a potential therapy for MS, yet the variability and low potency of MSCs from adult sources hinder their therapeutic potential. MSCs derived from human embryonic stem cells (hES-MSCs) may be better suited for clinical treatment of MS because of their unlimited and stable supply. Here, we show that hES-MSCs significantly reduce clinical symptoms and prevent neuronal demyelination in a mouse experimental autoimmune encephalitis (EAE) model of MS, and that the EAE disease-modifying effect of hES-MSCs is significantly greater than that of human bone-marrow-derived MSCs (BM-MSCs). Our evidence also suggests that increased IL-6 expression by BM-MSCs contributes to the reduced anti-EAE therapeutic activity of these cells. A distinct ability to extravasate and migrate into inflamed CNS tissues may also be associated with the robust therapeutic effects of hES-MSCs on EAE.

P-glycoprotein regulates trafficking of CD8(+) T cells to the brain parenchyma.

The trafficking of cytotoxic CD8(+) T lymphocytes across the lining of the cerebral vasculature is key to the onset of the chronic neuro-inflammatory disorder multiple sclerosis. However, the mechanisms controlling their final transmigration across the brain endothelium remain unknown. Here, we describe that CD8(+) T lymphocyte trafficking into the brain is dependent on the activity of the brain endothelial adenosine triphosphate-binding cassette transporter P-glycoprotein. Silencing P-glycoprotein activity selectively reduced the trafficking of CD8(+) T cells across the brain endothelium in vitro as well as in vivo. In response to formation of the T cell-endothelial synapse, P-glycoprotein was found to regulate secretion of endothelial (C-C motif) ligand 2 (CCL2), a chemokine that mediates CD8(+) T cell migration in vitro. Notably, CCL2 levels were significantly enhanced in microvessels isolated from human multiple sclerosis lesions in comparison with non-neurological controls. Endothelial cell-specific elimination of CCL2 in mice subjected to experimental autoimmune encephalomyelitis also significantly diminished the accumulation of CD8(+) T cells compared to wild-type animals. Collectively, these results highlight a novel (patho)physiological role for P-glycoprotein in CD8(+) T cell trafficking into the central nervous system during neuro-inflammation and illustrate CCL2 secretion as a potential link in this mechanism.

Cell-selective knockout and 3D confocal image analysis reveals separate roles for astrocyte-and endothelial-derived CCL2 in neuroinflammation.

BACKGROUND: Expression of chemokine CCL2 in the normal central nervous system (CNS) is nearly undetectable, but is significantly upregulated and drives neuroinflammation during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis which is considered a contributing factor in the human disease. As astrocytes and brain microvascular endothelial cells (BMEC) forming the blood-brain barrier (BBB) are sources of CCL2 in EAE and other neuroinflammatory conditions, it is unclear if one or both CCL2 pools are critical to disease and by what mechanism(s). METHODS: Mice with selective CCL2 gene knockout (KO) in astrocytes (Astro KO) or endothelial cells (Endo KO) were used to evaluate the respective contributions of these sources to neuroinflammation, i.e., clinical disease progression, BBB damage, and parenchymal leukocyte invasion in a myelin oligodendrocyte glycoprotein peptide (MOG35-55)-induced EAE model. High-resolution 3-dimensional (3D) immunofluorescence confocal microscopy and colloidal gold immuno-electron microscopy were employed to confirm sites of CCL2 expression, and 3D immunofluorescence confocal microscopy utilized to assess inflammatory responses along the CNS microvasculature. RESULTS: Cell-selective loss of CCL2 immunoreactivity was demonstrated in the respective KO mice. Compared to wild-type (WT) mice, Astro KO mice showed reduced EAE severity but similar onset, while Endo KO mice displayed near normal severity but significantly delayed onset. Neither of the KO mice showed deficits in T cell proliferation, or IL-17 and IFN-γ production, following MOG35-55 exposure in vitro, or altered MOG-major histocompatibility complex class II tetramer binding. 3D confocal imaging further revealed distinct actions of the two CCL2 pools in the CNS. Astro KOs lacked the CNS leukocyte penetration and disrupted immunostaining of CLN-5 at the BBB seen during early EAE in WT mice, while Endo KOs uniquely displayed leukocytes stalled in the microvascular lumen. CONCLUSIONS: These results point to astrocyte and endothelial pools of CCL2 each regulating different stages of neuroinflammation in EAE, and carry implications for drug delivery in neuroinflammatory disease.

Novel 3D analysis of Claudin-5 reveals significant endothelial heterogeneity among CNS microvessels.

Tight junctions (TJs) feature critically in maintaining the integrity of the blood-brain barrier (BBB), and undergo significant disruption during neuroinflammatory diseases. Accordingly, the expression and distribution of CLN-5, a prominent TJ protein in central nervous system (CNS) microvessels and BBB determinant, has been shown to parallel physiological and pathophysiological changes in microvascular function. However, efforts to quantify CLN-5 within the CNS microvasculature in situ, by using conventional two-dimensional immunohistochemical analysis of thin sections, are encumbered by the tortuosity of capillaries and distorted diameters of inflamed venules. Herein, we describe a novel contour-based 3D image visualization and quantification method, employing high-resolution confocal z-stacks from thick immunofluorescently-stained thoraco-lumbar spinal cord cryosections, to analyze CLN-5 along the junctional regions of different-sized CNS microvascular segments. Analysis was performed on spinal cords of both healthy mice, and mice experiencing experimental autoimmune encephalomyelitis (EAE), an animal model of the neuroinflammatory disease multiple sclerosis. Results indicated that, under normal conditions, the density of CLN-5 staining (CLN-5 intensity/ endothelial surface area) was greatest in the capillaries and smaller venules, and least in the larger venules. This heterogeneity in junctional CLN-5 staining was exacerbated during EAE, as spinal venules revealed a significant loss of junctional CLN-5 staining that was associated with focal leukocyte extravasation, while adjacent capillaries exhibited neither CLN-5 loss nor infiltrating leukocytes. However, despite only venules displaying these behaviors, both capillaries and venules evidenced leakage of IgG during disease, further underscoring the heterogeneity of the inflammatory response in CNS microvessels. This method should be readily adaptable to analyzing other junctional proteins of the CNS and peripheral microvasculature, and serve to highlight their role(s) in health and disease.

Active induction of experimental autoimmune encephalomyelitis by MOG35-55 peptide immunization is associated with differential responses in separate compartments of the choroid plexus.

BACKGROUND: There is increasing awareness that, aside from producing cerebrospinal fluid, the choroid plexus (CP) might be a key regulator of immune activity in the central nervous system (CNS) during neuroinflammation. Specifically, the CP has recently been posited to control entry of sentinel T cells into the uninflamed CNS during the early stages of neuroinflammatory diseases, like multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). As the CP is compartmentalized into a stromal core containing fenestrated capillaries devoid of typical blood-brain barrier properties, surrounded by a tight junction-expressing choroidal epithelium, each of these compartments might mount unique responses that instigate the neuroinflammatory process. METHODS: To discern responses of the respective CP stromal capillary and choroidal epithelial tissues during evolving neuroinflammation, we investigated morphology and in situ expression of 93 immune-related genes during early stages of EAE induced by immunization with myelin oligodendrocyte glycoprotein peptide (MOG35-55). Specifically, 3-D immunofluorescent imaging was employed to gauge morphological changes, and laser capture microdissection was coupled to an Immune Panel TaqMan Low Density Array to detail alterations in gene expression patterns at these separate CP sites on days 9 and 15 post-immunization (p.i.). To resolve CP effects due to autoimmunity against MOG peptide, from those due to complete Freund's adjuvant (CFA) and pertussis toxin (PTX) included in the immunization, analysis was performed on MOG-CFA/PTX-treated, CFA/PTX-treated, and naïve cohorts. RESULTS: The CP became swollen and displayed significant molecular changes in response to MOG-CFA/PTX immunization. Both stromal capillary and choroidal epithelial tissues mounted vigorous, yet different, changes in expression of numerous genes over the time course analyzed - including those encoding adhesion molecules, cytokines, chemokines, statins, interleukins, T cell activation markers, costimulatory molecules, cyclooxygenase, pro-inflammatory transcription factors and pro-apoptotic markers. Moreover, CFA/PTX-treatment, alone, resulted in extensive, though less robust, alterations in both CP compartments. CONCLUSIONS: MOG-CFA/PTX immunization significantly affects CP morphology and stimulates distinct expression patterns of immune-related genes in CP stromal capillary and epithelial tissues during evolving EAE. CFA/PTX treatment, alone, causes widespread gene alterations that could prime the CP to unlock the CNS to T cell infiltration during neuroinflammatory disease.

The CCL2 synthesis inhibitor bindarit targets cells of the neurovascular unit, and suppresses experimental autoimmune encephalomyelitis.

BACKGROUND: Production of the chemokine CCL2 by cells of the neurovascular unit (NVU) drives critical aspects of neuroinflammation. Suppression of CCL2 therefore holds promise in treating neuroinflammatory disease. Accordingly, we sought to determine if the compound bindarit, which inhibits CCL2 synthesis, could repress the three NVU sources of CCL2 most commonly reported in neuroinflammation--astrocytes, microglia and brain microvascular endothelial cells (BMEC)--as well as modify the clinical course of neuroinflammatory disease. METHODS: The effect of bindarit on CCL2 expression by cultured murine astrocytes, microglia and BMEC was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Bindarit action on mouse brain and spinal cord in vivo was similarly investigated by qRT-PCR following LPS injection in mice. And to further gauge the potential remedial effects of bindarit on neuroinflammatory disease, its impact on the clinical course of experimental autoimmune encephalomyelitis (EAE) in mice was also explored. RESULTS: Bindarit repressed CCL2 expression by all three cultured cells, and antagonized upregulated expression of CCL2 in both brain and spinal cord in vivo following LPS administration. Bindarit also significantly modified the course and severity of clinical EAE, diminished the incidence and onset of disease, and evidenced signs of disease reversal. CONCLUSION: Bindarit was effective in suppressing CCL2 expression by cultured NVU cells as well as brain and spinal cord tissue in vivo. It further modulated the course of clinical EAE in both preventative and therapeutic ways. Collectively, these results suggest that bindarit might prove an effective treatment for neuroinflammatory disease.

Coadministration of adenoviral vascular endothelial growth factor and angiopoietin-1 enhances vascularization and reduces ventricular remodeling in the infarcted myocardium of type 1 diabetic rats.

OBJECTIVE: Hyperglycemia impairs angiogenesis in response to ischemia, leading to ventricular remodeling. Although the effects of overexpressing angiogenic growth factors have been studied in inducing angiogenesis, the formation of functional vessels remains a challenge. The present study evaluates the reversal of diabetes-mediated impairment of angiogenesis in the infarcted diabetic rat myocardium by proangiogenic gene therapy. RESEARCH DESIGN AND METHODS: Ad*VEGF and Ad*Ang1 were intramyocardially administered in combination immediately after myocardial infarction to nondiabetic and diabetic rats. Ad*LacZ was similarly administered to the respective control groups. The hearts were excised for molecular and immunohistochemical analysis at predetermined time points. The myocardial function was measured by echocardiography 30 days after the intervention. RESULTS: We observed reduced fibrosis and increased capillary/arteriolar density along with reduced ventricular remodeling, as assessed by echocardiography in the treated diabetic animals compared with the nontreated diabetic controls. We also observed increased phosphorylated mitogen-activated protein kinase-activated protein kinase-2, 2 days after the treatment and increased expression of vascular endothelial growth factor (VEGF), Flk-1, angiopoietin-1 (Ang-1), Tie-2, and survivin, 4 days after treatment in the diabetic animals. Gel shift analysis revealed that the combination gene therapy stimulated the DNA binding activity of nuclear factor-kappaB in the diabetic animals. CONCLUSIONS: Our preclinical data demonstrate the efficacy of coadministration of adenoviral VEGF and Ang-1 in increasing angiogenesis and reducing ventricular remodeling in the infarcted diabetic myocardium. These unique results call for the initiation of a clinical trial to assess the efficacy of this therapeutic strategy in the treatment of diabetes-related human heart failure.

Mesenchymal stem cell: present challenges and prospective cellular cardiomyoplasty approaches for myocardial regeneration.

Myocardial ischemia and cardiac dysfunction have been known to follow ischemic heart diseases (IHDs). Despite a plethora of conventional treatment options, their efficacies are associated with skepticism. Cell therapies harbor a promising potential for vascular and cardiac repair, which is corroborated by adequate preclinical evidence. The underlying objectives behind cardiac regenerative therapies subsume enhancing angiomyogenesis in the ischemic myocardium, ameliorating cellular apoptosis, regenerating the damaged myocardium, repopulating the lost resident myocardial cells (smooth muscle, cardiomyocyte, and endothelial cells), and finally, decreasing fibrosis with a consequent reduction in ventricular remodeling. Although-cell based cardiomyoplasty approaches have an immense potential, their clinical utilization is limited owing to the increased need for better candidates for cellular cardiomyoplasty, better routes of delivery, appropriate dose for efficient engraftment, and better preconditioning or genetic-modification strategies for the progenitor and stem cells. Mesenchymal stem cells (MSCs) have emerged as powerful candidates in mediating myocardial repair owing to their unique properties of multipotency, transdifferentiation, intercellular connection with the resident cardiomyocytes via connexin 43 (Cx43)-positive gap junctions in the myocardium, and most important, immunomodulation. In this review, we present an in-depth discussion on the complexities associated with stem and progenitor cell therapies, the potential of preclinical approaches involving MSCs for myocardial repair, and an account of the past milestones and ongoing MSC-based trials in humans.

Akt/FOXO3a/SIRT1-mediated cardioprotection by n-tyrosol against ischemic stress in rat in vivo model of myocardial infarction: switching gears toward survival and longevity.

Moderate consumption of wine has been associated with decreased risk of cardiovascular events. Recently we have shown that white wine is equally as cardioprotective as red wine. However, unlike resveratrol (polyphenol in red wine), the white wine component, n-tyrosol [2-(4-hydroxyphenyl)ethanol] has not been explored for its cardioprotective effect and mechanism of action. Therefore, the present study was designed to evaluate the effect of tyrosol treatment (5 mg/kg/day for 30 days) on myocardial ischemic stress in a rat in vivo model of Myocardial Infarction (MI) and to identify key molecular targets involved in this mechanism. MI was induced by Left Anterior Descending (LAD) coronary artery ligation. Reduced infarct size (32.42 vs 48.03%) and cardiomyocyte apoptosis (171 vs 256 counts/100 HPF) were observed along with improvement in the myocardial functional parameters such as LVIDs (5.89 vs 6.58 mm), ejection fraction (51.91 vs 45.09%), and fractional shortening (28.46 vs 23.52%) as assessed by echocardiography in the tyrosol-treated animals when compared to the nontreated controls. We have also observed significant increase in the phosphorylation of Akt (1.4-fold), eNOS (3-fold) and FOXO3a (2.6-fold). In addition, tyrosol induced the expression of longevity protein SIRT1 (3.2-fold) in the MI group as compared to the non-treated MI control. Therefore tyrosol's SIRT1, Akt and eNOS activating power adds another dimension to the white wine research, because it adds a great link to the French paradox. In conclusion these findings suggest that tyrosol induces myocardial protection against ischemia related stress by inducing survival and longevity proteins that may be considered as anti-aging therapy for the heart. However, human intervention studies would be necessary before establishing any recommendations about dietary habits for tyrosol intake or administration of dietary supplements containing tyrosol.