The phenotypic and functional properties of mouse yolk-sac-derived embryonic macrophages.

Macrophages are well characterized as immune cells. However, in recent years, a multitude of non-immune functions have emerged many of which play essential roles in a variety of developmental processes (Wynn et al., 2013; DeFalco et al., 2014). In adult animals, macrophages are derived from circulating monocytes originating in the bone marrow, but much of the tissue-resident population arise from erythro-myeloid progenitors (EMPs) in the extra-embryonic yolk sac, appearing around the same time as primitive erythroblasts (Schulz et al., 2012; Kierdorf et al., 2013; McGrath et al., 2015; Gomez Perdiguero et al., 2015; Mass et al., 2016). Of particular interest to our group, macrophages have been shown to act as pro-angiogenic regulators during development (Wynn et al., 2013; DeFalco et al., 2014; Hsu et al., 2015), but there is still much to learn about these early cells. The goal of the present study was to isolate and expand progenitors of yolk-sac-derived Embryonic Macrophages (EMs) in vitro to generate a new platform for mechanistic studies of EM differentiation. To accomplish this goal, we isolated pure (>98%) EGFP+ populations by flow cytometry from embryonic day 9.5 (E9.5) Csf1r-EGFP+/tg mice, then evaluated the angiogenic potential of EMs relative to Bone Marrow-Derived Macrophages (BMDMs). We found that EMs expressed more pro-angiogenic and less pro-inflammatory macrophage markers than BMDMs. EMs also promoted more endothelial cell (EC) cord formation in vitro, as compared to BMDMs in a manner that required direct cell-to-cell contact. Importantly, EMs preferentially matured into microglia when co-cultured with mouse Neural Stem/Progenitor Cells (NSPCs). In conclusion, we have established a protocol to isolate and propagate EMs in vitro, have further defined specialized properties of yolk-sac-derived macrophages, and have identified EM-EC and EM-NSPC interactions as key inducers of EC tube formation and microglial cell maturation, respectively.

An immortalized human blood-nerve barrier endothelial cell line for in vitro permeability studies.

Solute and macromolecular transport studies may elucidate nutritional requirements and drug effects in healthy and diseased peripheral nerves. Endoneurial endothelial cells are specialized microvascular cells that form the restrictive blood-nerve barrier (BNB). Primary human endoneurial endothelial cells (pHEndECs) are difficult to isolate, limiting their widespread availability for biomedical research. We developed a simian virus-40 large T-antigen (SV40-LTA) immortalized human BNB cell line via stable transfection of low passage pHEndECs and observed continuous growth in culture for >45 population doublings. As observed with pHEndECs, the immortalized BNB endothelial cells were Ulex Europaeus agglutinin-1-positive and endocytosed low density lipoprotein, but lost von Willebrand factor expression. Glucose transporter-1, P-glycoprotein (P-gp), γ-glutamyl transpeptidase (γ-GT), large neutral amino acid transporter-1 (LAT-1), creatine transporter (CRT), and monocarboxylate transporter-1 (MCT-1) mRNA expression were retained at all passages with loss of alkaline phosphatase (AP) expression after passages 16-20. Compared with an SV40-LTA immortalized human blood-brain barrier endothelial cell line, there was increased γ-GT protein expression, equivalent expression of organic anion transporting polypeptide-C (OATP-C), organic anion transporter 3 (OAT-3), MCT-1, and LAT-1, and reduced expression of AP, CRT, and P-gp by the BNB cell line at passage 20. Further studies demonstrated lower transendothelial electrical resistance (~181 vs. 191 Ω cm(2)), equivalent permeability to fluoresceinated sodium (4.84 vs. 4.39 %), and lower permeability to fluoresceinated high molecular weight (70 kDa) dextran (0.39 vs. 0.52 %) by the BNB cell line. This cell line retained essential molecular and biophysical properties suitable for in vitro peripheral nerve permeability studies.

VEGF-A165 potently induces human blood-nerve barrier endothelial cell proliferation, angiogenesis, and wound healing in vitro.

Several mitogens such as vascular endothelial growth factor (VEGF) have been implicated in mammalian vascular proliferation and repair. However, the molecular mediators of human blood-nerve barrier (BNB) development and specialization are unknown. Primary human endoneurial endothelial cells (pHEndECs) were expanded in vitro and specific mitogen receptors detected by western blot. pHEndECs were cultured with basal medium containing different mitogen concentrations with or without heparin. Non-radioactive cell proliferation, Matrigel(™)-induced angiogenesis and sterile micropipette injury wound healing assays were performed. Proliferation rates, number and total length of induced microvessels, and rate of endothelial cell monolayer wound healing were determined and compared to basal conditions. VEGF-A165 in the presence of heparin, was the most potent inducer of pHEndEC proliferation, angiogenesis, and wound healing in vitro. 1.31 nM VEGF-A165 induced ~110 % increase in cell proliferation relative to basal conditions (∼51 % without heparin). 2.62 pM VEGF-A165 induced a three-fold increase in mean number of microvessels and 3.9-fold increase in total capillary length/field relative to basal conditions. In addition, 0.26 nM VEGF-A165 induced ∼1.3-fold increased average rate of endothelial wound healing 4-18 h after endothelial monolayer injury, mediated by increased cell migration. VEGF-A165 was the only mitogen capable of complete wound closure, occurring within 30 h following injury via increased cell proliferation. This study demonstrates that VEGF-A165, in the presence of heparin, is a potent inducer of pHEndEC proliferation, angiogenesis, and wound healing in vitro. VEGF-A165 may be an important mitogen necessary for human BNB development and recovery in response to peripheral nerve injury.

α(M)ß(2)-integrin-intercellular adhesion molecule-1 interactions drive the flow-dependent trafficking of Guillain-Barré syndrome patient derived mononuclear leukocytes at the blood-nerve barrier in vitro.

The mechanisms of hematogenous leukocyte trafficking at the human blood-nerve barrier (BNB) are largely unknown. Intercellular adhesion molecule-1 (ICAM-1) has been implicated in the pathogenesis of Guillain-Barré syndrome (GBS). We developed a cytokine-activated human in vitro BNB model using primary endoneurial endothelial cells. Endothelial treatment with 10 U/ml tissue necrosis factor-α and 20 U/ml interferon-γ resulted in de novo expression of pro-inflammatory chemokines CCL2, CXCL9, CXCL11, and CCL20, with increased expression of CXCL2-3, CXCL8, and CXCL10 relative to basal levels. Cytokine treatment induced/enhanced ICAM-1, E- and P-selectin, vascular cell adhesion molecule-1 and the alternatively spliced pro-adhesive fibronectin variant, fibronectin connecting segment-1 expression in a time-dependent manner, without alterations in junctional adhesion molecule-A expression. Lymphocytes and monocytes from untreated GBS patients express ICAM-1 counterligands, α(M)- and α(L)-integrin, with differential regulation of α(M) -integrin expression compared to healthy controls. Under flow conditions that mimic capillary hemodynamics in vivo, there was a >3-fold increase in total GBS patient and healthy control mononuclear leukocyte adhesion/migration at the BNB following cytokine treatment relative to the untreated state. Function neutralizing monoclonal antibodies against human α(M)-integrin (CD11b) and ICAM-1 reduced untreated GBS patient mononuclear leukocyte trafficking at the BNB by 59% and 64.2%, respectively. Monoclonal antibodies against α(L)-integrin (CD11a) and human intravenous immunoglobulin reduced total leukocyte adhesion/migration by 22.8% and 17.6%, respectively. This study demonstrates differential regulation of α(M)-integrin on circulating mononuclear cells in GBS, as well as an important role for α(M)-integrin-ICAM-1 interactions in pathogenic GBS patient leukocyte trafficking at the human BNB in vitro.

GDNF restores human blood-nerve barrier function via RET tyrosine kinase-mediated cytoskeletal reorganization.

Endoneurial microvessels and the perineurium are responsible for maintaining homeostasis in peripheral nerves. Endoneurial endothelial cells form the blood-nerve barrier (BNB). The molecular pathways responsible for endoneurial microvascular barrier formation in humans are not fully understood. We tested the effect of different mitogens on the transendothelial electrical resistance (TEER) of confluent primary human endoneurial endothelial cell (pHEndEC) cultures following serum withdrawal (mimicking diffuse endothelial injury) in vitro. We show that glial-derived neurotrophic factor (GDNF, 1 ng/mL) sufficiently induced a maximal 114.2% recovery in TEER over basal conditions 48 h after serum withdrawal. Solute permeability to high molecular weight dextran was reduced by 52.4% following GDNF treatment. GDNF-mediated increase in TEER was dependent on RET tyrosine-kinase signaling pathways and mildly enhanced by cyclic adenosine monophosphate in combination with maximal concentrations of multiple redundant mitogens. There was no significant increase in adherens or tight junction proteins ß-catenin, VE-Cadherin, zona occludens-1 and occludin following GDNF treatment. GDNF induced a small increase in total claudin-5 protein expression without significant increase in messenger RNA or modulation in tyrosine phosphorylation following serum withdrawal. Indirect immunocytochemistry revealed membrane relocation of longitudinal F-actin cytoskeletal filaments in pHEndECs following GDNF treatment, resulting in more continuous intercellular contacts that formed adherens and tight junctions. Together, these results demonstrate a sufficient role for GDNF in human BNB recovery following serum withdrawal in vitro, facilitated primarily by endothelial cell cytoskeletal reorganization. These observations provide insights into the regulation of human BNB function during recovery from peripheral nerve injury.

Behavioral, electrophysiological, and histopathological characterization of a severe murine chronic demyelinating polyneuritis model.

The objective of this study was to define the behavioral, electrophysiological, and morphological characteristics of spontaneous autoimmune peripheral polyneuropathy (SAPP) in female B7-2 deficient non-obese diabetic (NOD) mice. A cohort of 77 female B7-2 deficient and 31 wild-type control NOD mice were studied from 18 to 40 weeks of age. At pre-defined time points, the dorsal caudal tail and sciatic motor nerve conduction studies (MNCS) were performed. Sciatic nerves were harvested for morphological evaluation. SAPP mice showed slowly progressive severe weakness in hind and forelimbs without significant recovery after 30 weeks of age. MNCS showed progressive reduction in mean compound motor action potential amplitudes and conduction velocities, and increase in mean total waveform duration from 24 to 27 weeks of age, peaking between 32 and 35 weeks of age. Toluidine blue-stained, semi-thin plastic-embedded sections demonstrated focal demyelination associated with mononuclear cell infiltration early in the disease course, with progressively diffuse demyelination and axonal loss associated with more intense mononuclear infiltration at peak severity. Immunohistochemistry confirmed macrophage-predominant inflammation. This study verifies SAPP as a progressive, unremitting chronic inflammatory demyelinating polyneuropathy with axonal loss.

Dorsal caudal tail and sciatic motor nerve conduction studies in adult mice: technical aspects and normative data.

Mice provide an important tool to investigate human neuromuscular disorders. The variability of electrophysiological techniques limits direct comparison between studies. The purpose of this study was to establish normative motor nerve conduction data in adult mice. The dorsal caudal tail nerve and sciatic nerve motor conduction studies were performed bilaterally on restrained anesthetized adult mice. The means and standard deviations for each electrophysiological parameter were determined in normal mice. Data were compared with inflammatory demyelinating polyneuropathy mice to determine whether these parameters discriminate between normal and abnormal peripheral nerves. Normal adult mice had a distal latency of 0.89 (+/-0.17) ms and 0.75 (+/-0.09) ms, distal compound motor unit action potential amplitude of 13.2 (+/-5.9) mV and 28.1 (+/-8.3) mV, and conduction velocity of 74.6 (+/-9.0) m/s and 76.5 (+/-8.3) m/s, respectively. These data were validated by the finding of statistically significant differences in several electrophysiological parameters that compared normal and polyneuropathy-affected mice. A standardized method for motor nerve conduction studies and associated normative data in mice should facilitate comparisons of disease severity and response to treatment between studies that use similar models. This would assist in the process of translational therapeutic drug design in neuromuscular disorders.

Isolation and transcriptional characterization of mouse perivascular astrocytes.

In the post-natal mammalian brain perivascular astrocytes (PAs) ensheath blood vessels to regulate their unique permeability properties known as the blood-brain barrier (BBB). Very little is known about PA-expressed genes and signaling pathways that mediate contact and communication with endothelial cells (ECs) to regulate BBB physiology. This is due, in part, to lack of suitable models to distinguish PAs from other astrocyte sub-populations in the brain. To decipher the unique biology of PAs, we used in vivo gene knock-in technology to fluorescently label these cells in the adult mouse brain followed by fractionation and quantitative single cell RNA sequencing. In addition, PAs and non-PAs were also distinguished with transgenic fluorescent reporters followed by gene expression comparisons using bulk RNA sequencing. These efforts have identified several genes and pathways in PAs with potential roles in contact and communication with brain ECs. These genes encode various extracellular matrix (ECM) proteins and adhesion receptors, secreted growth factors, and intracellular signaling enzymes. Collectively, our experimental data reveal a set of genes that are expressed in PAs with putative roles in BBB physiology.

Antioxidant Carbon Nanoparticles Inhibit Fibroblast-Like Synoviocyte Invasiveness and Reduce Disease Severity in a Rat Model of Rheumatoid Arthritis.

Reactive oxygen species have been involved in the pathogenesis of rheumatoid arthritis (RA). Our goal was to determine the effects of selectively scavenging superoxide (O2•-) and hydroxyl radicals with antioxidant nanoparticles, called poly(ethylene glycol)-functionalized hydrophilic carbon clusters (PEG-HCCs), on the pathogenic functions of fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and on the progression of an animal model of RA. We used human FLS from patients with RA to determine PEG-HCC internalization and effects on FLS cytotoxicity, invasiveness, proliferation, and production of proteases. We used the pristane-induced arthritis (PIA) rat model of RA to assess the benefits of PEG-HCCs on reducing disease severity. PEG-HCCs were internalized by RA-FLS, reduced their intracellular O2•-, and reduced multiple measures of their pathogenicity in vitro, including proliferation and invasion. In PIA, PEG-HCCs caused a 65% reduction in disease severity, as measured by a standardized scoring system of paw inflammation and caused a significant reduction in bone and tissue damage, and circulating rheumatoid factor. PEG-HCCs did not induce lymphopenia during PIA. Our study demonstrated a role for O2•- and hydroxyl radicals in the pathogenesis of a rat model of RA and showed efficacy of PEG-HCCs in treating a rat model of RA.

Hypothesized kinetic models for describing the growth of globular and encrusting demosponges.

The marine sponges Dysidea avara and Chondrosia reniformis (globular forms) were cultured in the laboratory on a diet of viable Phaeodactylum tricornutum cells and dissolved nutrients (algae and fish powders). Our growth data were combined with literature data for Pseudosuberites andrewsi (a globular sponge) and for the encrusting sponges Oscarella lobularis, Hemimycale columella, and Crambe crambe. The suitability of three growth models-linear, exponential, and radial accretive-for describing the growth of globular and encrusting sponges was assessed. Radial accretive growth was determined to be the best model to describe growth of both encrusting and globular sponges. Average growth rates of 0.051+/-0.016 and 0.019+/-0.003 mm/day (calculated as the increase of the radius of the sponge per day) were obtained experimentally for D. avara and C. reniformis, respectively.

Improved Angiogenesis in Response to Localized Delivery of Macrophage-Recruiting Molecules.

Successful engineering of complex organs requires improved methods to promote rapid and stable vascularization of artificial tissue scaffolds. Toward this goal, tissue engineering strategies utilize the release of pro-angiogenic growth factors, alone or in combination, from biomaterials to induce angiogenesis. In this study we have used intravital microscopy to define key, dynamic cellular changes induced by the release of pro-angiogenic factors from polyethylene glycol diacrylate hydrogels transplanted in vivo. Our data show robust macrophage recruitment when the potent and synergistic angiogenic factors, PDGFBB and FGF2 were used as compared with VEGF alone and intravital imaging suggested roles for macrophages in endothelial tip cell migration and anastomosis, as well as pericyte-like behavior. Further data from in vivo experiments show that delivery of CSF1 with VEGF can dramatically improve the poor angiogenic response seen with VEGF alone. These studies show that incorporating macrophage-recruiting factors into the design of pro-angiogenic biomaterial scaffolds is a key strategy likely to be necessary for stable vascularization and survival of implanted artificial tissues.

CCR2 gene deletion and pharmacologic blockade ameliorate a severe murine experimental autoimmune neuritis model of Guillain-Barré syndrome.

The molecular determinants and signaling pathways responsible for hematogenous leukocyte trafficking during peripheral neuroinflammation are incompletely elucidated. Chemokine ligand/receptor pair CCL2/CCR2 has been pathogenically implicated in the acute inflammatory demyelinating polyradiculoneuropathy variant of Guillain-Barré syndrome (GBS). We evaluated the role of CCR2 in peripheral neuroinflammation utilizing a severe murine experimental autoimmune neuritis (sm-EAN) model. Sm-EAN was induced in 8-12 week old female SJL CCR2 knockout (CCR2KO), heterozygote (CCR2HT) and wild type (CCR2WT) mice, and daily neuromuscular severity scores and weights recorded. In vitro and in vivo splenocyte proliferation and cytokine expression assays, and sciatic nerve Toll-like receptor (TLR) 2, TLR4 and CCL2 expression assays were performed to evaluate systemic and local innate immune activation at disease onset. Motor nerve electrophysiology and sciatic nerve histology were also performed to characterize the inflammatory neuropathy at expected peak severity. To further determine the functional relevance of CCR2 in sm-EAN, 20 mg/kg CCR2 antagonist, RS 102895 was administered daily for 5 days to a cohort of CCR2WT mice following sm-EAN disease onset, with efficacy compared to 400 mg/kg human intravenous immunoglobulin (IVIg). CCR2KO mice were relatively resistant to sm-EAN compared to CCR2WT and CCR2HT mice, associated with attenuated peripheral nerve demyelinating neuritis. Partial CCR2 gene deletion did not confer any protection against sm-EAN. CCR2KO mice demonstrated similar splenocyte activation or proliferation profiles, as well as TLR2, TLR4 and CCL2 expression to CCR2WT or CCR2HT mice, implying a direct role for CCR2 in sm-EAN pathogenesis. CCR2 signaling blockade resulted in rapid, near complete recovery from sm-EAN following disease onset. RS 102895 was significantly more efficacious than IVIg. CCR2 mediates pathogenic hematogenous monocyte trafficking into peripheral nerves, with consequential demyelination in sm-EAN. CCR2 is amenable to pharmacologic blockade, making it a plausible drug target for GBS.

Development and characterization of a novel human in vitro blood-nerve barrier model using primary endoneurial endothelial cells.

There are phenotypic and functional differences between vascular endothelium from different tissues and between microvascular and macrovascular endothelial cells (ECs) from the same tissue. Relatively little is known about the human blood-nerve barrier (BNB). We report the development of an in vitro BNB model using primary human endoneurial ECs freshly isolated and purified from decedent sciatic nerves via endoneurial stripping, connective tissue enzymatic digestion, and density centrifugation. Primary human endoneurial ECs are spindle shaped and contact inhibited. They rapidly differentiate to form capillary-like networks and microvessels, bind Ulex Europaeus Agglutinin 1 lectin, express von Willebrand factor, and endocytose acetylated low-density lipoprotein. They also express specific transport and cellular adhesion molecules and tight junction proteins, consistent with cells that form a highly restrictive endothelial barrier similar to the blood-brain barrier. When cultured on collagen-coated transwell inserts, the primary human endoneurial ECs develop an in vitro BNB with high transendothelial electrical resistances (160 Omega x cm(2); maximal 12 days after seeding) and low solute permeability coefficient to fluoresceinated high-molecular weight (70 kDa) dextran (2.75 x 10(-3) cm/minute). This in vitro BNB model retains essential known or expected characteristics of the human BNB and has many potential applications for studies of solute, macromolecule, microbial, virus, and leukocyte interactions with this highly specialized endothelial barrier.

Clinical, electrophysiological and pathologic correlations in a severe murine experimental autoimmune neuritis model of Guillain-Barré syndrome.

Severe murine experimental autoimmune neuritis (sm-EAN) in SJL/J mice is a recently described, but incompletely characterized mouse model of Guillain-Barré syndrome (GBS). Electrophysiological and pathologic characterization during the disease course is a necessary prerequisite to designing mechanistic studies that may be relevant to GBS pathogenesis. Sm-EAN is a monophasic disorder with electrophysiological evidence for a diffuse demyelinating polyneuropathy with axonal loss at peak severity. Regression analyses demonstrated strong correlations between neuromuscular severity scores and electrophysiological parameters during the disease course. Progressive multi-focal or diffuse demyelination with axonal loss was observed pathologically in sciatic nerves in association with mononuclear cell infiltrates (F4/80+ macrophages>CD3+ T-lymphocytes>CD19+ B-lymphocytes), peaking at maximal severity. Regression analyses demonstrated strong correlations between severity scores and inflammatory cell counts. The correlative data imply that mononuclear infiltration, as well as demyelination and axonal loss are directly related to the observed neuromuscular weakness in sm-EAN. The high induction rates, as well as pathologic similarities with AIDP make sm-EAN a robust model to study the pathogenesis of human peripheral nerve inflammation using objective outcome measures.