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.

The primary macrophage chemokine, CCL2, is not necessary after a peripheral nerve injury for macrophage recruitment and activation or for conditioning lesion enhanced peripheral regeneration.

BACKGROUND: Peripheral nerve injuries stimulate the regenerative capacity of injured neurons through a neuroimmune phenomenon termed the conditioning lesion (CL) response. This response depends on macrophage accumulation in affected dorsal root ganglia (DRGs) and peripheral nerves. The macrophage chemokine CCL2 is upregulated after injury and is allegedly required for stimulating macrophage recruitment and pro-regenerative signaling through its receptor, CCR2. In these tissues, CCL2 is putatively produced by neurons in the DRG and Schwann cells in the distal nerve. METHODS: Ccl2fl/fl mice were crossed with Advillin-Cre, P0-Cre, or both to create conditional Ccl2 knockouts (CKOs) in sensory neurons, Schwann cells, or both to hypothetically remove CCL2 and macrophages from DRGs, nerves or both. CCL2 was localized using Ccl2-RFPfl/fl mice. CCL2-CCR2 signaling was further examined using global Ccl2 KOs and Ccr2gfp knock-in/knock-outs. Unilateral sciatic nerve transection was used as the injury model, and at various timepoints, chemokine expression, macrophage accumulation and function, and in vivo regeneration were examined using qPCR, immunohistochemistry, and luxol fast blue staining. RESULTS: Surprisingly, in all CKOs, DRG Ccl2 gene expression was decreased, while nerve Ccl2 was not. CCL2-RFP reporter mice revealed CCL2 expression in several cell types beyond the expected neurons and Schwann cells. Furthermore, macrophage accumulation, myelin clearance, and in vivo regeneration were unaffected in all CKOs, suggesting CCL2 may not be necessary for the CL response. Indeed, Ccl2 global knockout mice showed normal macrophage accumulation, myelin clearance, and in vivo regeneration, indicating these responses do not require CCL2. CCR2 ligands, Ccl7 and Ccl12, were upregulated after nerve injury and perhaps could compensate for the absence of Ccl2. Finally, Ccr2gfp knock-in/knock-out animals were used to differentiate resident and recruited macrophages in the injured tissues. Ccr2gfp/gfp KOs showed a 50% decrease in macrophages in the distal nerve compared to controls with a relative increase in resident macrophages. In the DRG there was a small but insignificant decrease in macrophages. CONCLUSIONS: CCL2 is not necessary for macrophage accumulation, myelin clearance, and axon regeneration in the peripheral nervous system. Without CCL2, other CCR2 chemokines, resident macrophage proliferation, and CCR2-independent monocyte recruitment can compensate and allow for normal macrophage accumulation.

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.

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.

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.

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.

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.

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.

Proteomic interrogation of the meninges reveals the molecular identities of structural components and regional distinctions along the CNS axis.

The meninges surround the brain and spinal cord, affording physical protection while also serving as a niche of neuroimmune activity. Though possessing stromal qualities, its complex cellular and extracellular makeup has yet to be elaborated, and it remains unclear whether the meninges vary along the neuroaxis. Hence, studies were carried-out to elucidate the protein composition and structural organization of brain and spinal cord meninges in normal, adult Biozzi ABH mice. First, shotgun, bottom-up proteomics was carried-out. Prominent proteins at both brain and spinal levels included Type II collagen and Type II keratins, representing extracellular matrix (ECM) and cytoskeletal categories, respectively. While the vast majority of total proteins detected was shared between both meningeal locales, more were uniquely detected in brain than in spine. This pattern was also seen when total proteins were subdivided by cellular compartment, except in the case of the ECM category where brain and spinal meninges each had near equal number of unique proteins, and Type V and type III collagen registered exclusively in the spine. Quantitative analysis revealed differential expression of several collagens and cytoskeletal proteins between brain and spinal meninges. High-resolution immunofluorescence and immunogold-scanning electronmicroscopy on sections from whole brain and spinal cord - still encased within bone -identified major proteins detected by proteomics, and highlighted their association with cellular and extracellular elements of variously shaped arachnoid trabeculae. Western blotting aligned with the proteomic and immunohistological analyses, reinforcing differential appearance of proteins in brain vs spinal meninges. Results could reflect regional distinctions in meninges that govern protective and/or neuroimmune functions.

Host extracellular vesicles confer cytosolic access to systemic LPS licensing non-canonical inflammasome sensing and pyroptosis.

Intracellular surveillance for systemic microbial components during homeostasis and infections governs host physiology and immunity. However, a long-standing question is how circulating microbial ligands become accessible to intracellular receptors. Here we show a role for host-derived extracellular vesicles (EVs) in this process; human and murine plasma-derived and cell culture-derived EVs have an intrinsic capacity to bind bacterial lipopolysaccharide (LPS). Remarkably, circulating host EVs capture blood-borne LPS in vivo, and the LPS-laden EVs confer cytosolic access for LPS, triggering non-canonical inflammasome activation of gasdermin D and pyroptosis. Mechanistically, the interaction between the lipid bilayer of EVs and the lipid A of LPS underlies EV capture of LPS, and the intracellular transfer of LPS by EVs is mediated by CD14. Overall, this study demonstrates that EVs capture and escort systemic LPS to the cytosol licensing inflammasome responses, uncovering EVs as a previously unrecognized link between systemic microbial ligands and intracellular surveillance.

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.

Mitofusin-2 regulates leukocyte adhesion and ß2 integrin activation.

Neutrophils are critical for inflammation and innate immunity, and their adhesion to vascular endothelium is a crucial step in neutrophil recruitment. Mitofusin-2 (MFN2) is required for neutrophil adhesion, but molecular details are unclear. Here, we demonstrated that ß2 -integrin-mediated slow-rolling and arrest, but not PSGL-1-mediated cell rolling, are defective in MFN2-deficient neutrophil-like HL60 cells. This adhesion defect is associated with reduced expression of fMLP (N-formylmethionyl-leucyl-phenylalanine) receptor FPR1 as well as the inhibited ß2 integrin activation, as assessed by conformation-specific monoclonal antibodies. MFN2 deficiency also leads to decreased actin polymerization, which is important for ß2 integrin activation. Mn2+ -induced cell spreading is also inhibited after MFN2 knockdown. MFN2 deficiency limited the maturation of ß2 integrin activation during the neutrophil-directed differentiation of HL60 cells, which is indicated by CD35 and CD87 markers. MFN2 knockdown in ß2-integrin activation-matured cells (CD87high population) also inhibits integrin activation, indicating that MFN2 directly affects ß2 integrin activation. Our study illustrates the function of MFN2 in leukocyte adhesion and may provide new insights into the development and treatment of MFN2 deficiency-related diseases.

Endothelial cell heterogeneity of blood-brain barrier gene expression along the cerebral microvasculature.

The blood-brain barrier (BBB) refers to the network of microvessels that selectively restricts the passage of substances between the circulation and the central nervous system (CNS). This microvascular network is comprised of arterioles, capillaries and venules, yet the respective contribution of each of these to the BBB awaits clarification. In this regard, it has been postulated that brain microvascular endothelial cells (BMEC) from these different tributaries might exhibit considerable heterogeneity in form and function, with such diversity underlying unique roles in physiological and pathophysiological processes. Means to begin exploring such endothelial differences in situ, free from caveats associated with cell isolation and culturing procedures, are crucial to comprehending the nature and treatment of CNS diseases with vascular involvement. Here, the recently validated approach of immuno-laser capture microdissection (immuno-LCM) coupled to quantitative real-time PCR (qRT-PCR) was used to analyze gene expression patterns of BMEC retrieved in situ from either capillaries or venules. From profiling 87 genes known to play a role in BBB function and/or be enriched in isolated brain microvessels, results imply that most BBB properties reside in both segments, but that capillaries preferentially express some genes related to solute transport, while venules tend toward higher expression of an assortment of genes involved in inflammatory-related tasks. Fuller appreciation of such heterogeneity will be critical for efficient therapeutic targeting of the endothelium and the management of CNS disease.

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.

Transcellular transport of CCL2 across brain microvascular endothelial cells.

The means by which the chemokine CCL2 produced in the brain parenchyma can recruit leukocytes lying behind the highly impervious endothelium of the blood-brain barrier (BBB) has remained a paradox. As other chemokines have been evidenced to stimulate their own synthesis and release by peripheral microvascular endothelial cells, and/or undergo transcytosis in the abluminal-to-luminal direction, we determined whether CCL2 experiences similar fates across brain microvascular endothelial cells (BMEC). Using cultured BMEC as a paradigm of the BBB, it was observed that exogenous unlabeled CCL2 actually depressed the release of endogenous CCL2, and further caused diminished CCL2 mRNA levels in these cells. On the other hand, exogenous (125)I-labeled CCL2 exhibited transport across BMEC in a manner that was sensitive to temperature, competition by excess unlabeled CCL2 but not unlabeled CCL3, knockdown of caveolin-1/caveolae, and elimination of the cognate CCL2 receptor CCR2. These results implied a facet of CCL2 transport by a transcellular mechanism partly involving binding of CCL2 to CCR2, and subsequent transfer to caveolae vesicles for transcytosis. This notion was supported by double-label immuno-electronmicroscopy, which revealed co-localization of caveolin-1 with exogenous CCL2, during this chemokine's transit across BMEC. Collectively, these findings provide a rationale by which CCL2, deposited on the abluminal side of the brain microvasculature during inflammatory episodes, can be relayed across the BBB to foster leukocyte recruitment.

Validation of immuno-laser capture microdissection coupled with quantitative RT-PCR to probe blood-brain barrier gene expression in situ.

Laser capture microdissection (LCM) holds great potential for analyzing gene expression profiles in situ. Most recently, this laboratory employed a novel immunostain-based LCM protocol (immuno-LCM) to selectively retrieve brain microvascular endothelial cells (BMEC) from intimately associated perivascular cells. However, before this protocol can be confidently coupled to downstream analytical platforms, it must be demonstrated that any variability associated with it is minimal, so as not to obscure data interpretation. As various factors could contribute to variability, this study focused on determining whether technical inconsistency and/or biological diversity of sample populations, played such a role. Specifically, two separate immuno-LCM-derived BMEC samples derived from adjacent tissue sections of a single mouse (to detect only technical variability), and from analogous tissue sections of three different mice (to detect technical and biological variability) were compared for their relative expression of 16 genes, using quantitative-RT-PCR (qRT-PCR). Both significant linear and rank-order correlations were observed between different sections from the same animal, underscoring lack of technical variability in this LCM application. Furthermore, a three-dimensional scatter plot of gene expression profiles from the three animals was linear, and ANOVA showed absence of statistically significant differences between any of the animals, confirming lack of biological variability. These findings argue that immuno-LCM coupled to qRT-PCR affords a reproducible means to assay gene expression in situ.

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.

Laser-Capture Microdissection and RNA Extraction from Perfusion-Fixed Cartilage and Bone Tissue from Mice Implanted with Human iPSC-Derived MSCs in a Calvarial Defect Model.

Laser-capture microdissection (LCM) coupled to downstream RNA analysis poses unique difficulties for the evaluation of mineralized tissues. A rapid protocol was thus developed to enable sufficient integrity of bone and cartilage tissue for reliable sectioning, while minimizing RNA loss associated with prolonged decalcification and purification steps. Specifically, the protocol involves pump-assisted, cardiac perfusion-fixation with paraformaldehyde, and moderate digestion of LCM-acquired tissue with proteinase K followed by DNase treatment and separation of RNA using magnetic beads. Reverse transcription and cDNA synthesis are performed immediately after RNA purification, without need for further protein removal.

Isolation and culture of microvascular endothelial cells from murine spinal cord.

The isolation and culture of spinal cord microvascular endothelial cells (SCMEC), which form the blood-spinal cord barrier (BSCB), is described. Though morphologically similar to brain microvascular endothelial cells (BMEC) that form the blood-brain barrier (BBB), SCMEC express reduced amounts of several prominent BBB proteins, including tight junction-associated proteins ZO-1 and occludin, adherens junction-associated proteins beta-catenin and VE-cadherin, and the efflux transporter P-glycoprotein. These distinguishing features may reflect more widespread differences between the BBB and BSCB that impact physiological and pathophysiological processes.