A Potential Role for MAGI-1 in the Bi-Directional Relationship Between Major Depressive Disorder and Cardiovascular Disease.

PURPOSE OF REVIEW: Major Depressive Disorder (MDD) is characterized by persistent symptoms such as fatigue, loss of interest in activities, feelings of sadness and worthlessness. MDD often coexist with cardiovascular disease (CVD), yet the precise link between these conditions remains unclear. This review explores factors underlying the development of MDD and CVD, including genetic, epigenetic, platelet activation, inflammation, hypothalamic-pituitary-adrenal (HPA) axis activation, endothelial cell (EC) dysfunction, and blood-brain barrier (BBB) disruption. RECENT FINDINGS: Single nucleotide polymorphisms (SNPs) in the membrane-associated guanylate kinase WW and PDZ domain-containing protein 1 (MAGI-1) are associated with neuroticism and psychiatric disorders including MDD. SNPs in MAGI-1 are also linked to chronic inflammatory disorders such as spontaneous glomerulosclerosis, celiac disease, ulcerative colitis, and Crohn's disease. Increased MAGI-1 expression has been observed in colonic epithelial samples from Crohn's disease and ulcerative colitis patients. MAGI-1 also plays a role in regulating EC activation and atherogenesis in mice and is essential for Influenza A virus (IAV) infection, endoplasmic reticulum stress-induced EC apoptosis, and thrombin-induced EC permeability. Despite being understudied in human disease; evidence suggests that MAGI-1 may play a role in linking CVD and MDD. Therefore, further investigation of MAG-1 could be warranted to elucidate its potential involvement in these conditions.

TNIK regulation of interferon signaling and endothelial cell response to virus infection.

Background: Traf2 and Nck-interacting kinase (TNIK) is known for its regulatory role in various processes within cancer cells. However, its role within endothelial cells (ECs) has remained relatively unexplored. Methods: Leveraging RNA-seq data and Ingenuity Pathway Analysis (IPA), we probed the potential impact of TNIK depletion on ECs. Results: Examination of RNA-seq data uncovered more than 450 Differentially Expressed Genes (DEGs) in TNIK-depleted ECs, displaying a fold change exceeding 2 with a false discovery rate (FDR) below 0.05. IPA analysis unveiled that TNIK depletion leads to the inhibition of the interferon (IFN) pathway [-log (p-value) >11], downregulation of IFN-related genes, and inhibition of Hypercytokinemia/Hyperchemokinemia [-log (p-value) >8]. The validation process encompassed qRT-PCR to evaluate mRNA expression of crucial IFN-related genes, immunoblotting to gauge STAT1 and STAT2 protein levels, and ELISA for the quantification of IFN and cytokine secretion in siTNIK-depleted ECs. These assessments consistently revealed substantial reductions upon TNIK depletion. When transducing HUVECs with replication incompetent E1-E4 deleted adenovirus expressing green fluorescent protein (Ad-GFP), it was demonstrated that TNIK depletion did not affect the uptake of Ad-GFP. Nonetheless, TNIK depletion induced cytopathic effects (CPE) in ECs transduced with wild-type human adenovirus serotype 5 (Ad-WT). Summary: Our findings suggest that TNIK plays a crucial role in regulating the EC response to virus infections through modulation of the IFN pathway.

Characterization of neuromas in peripheral nerves and their effects on heterotopic bone formation.

The formation of neuromas involves expansion of the cellular components of peripheral nerves. The onset of these disorganized tumors involves activation of sensory nerves and neuroinflammation. Particularly problematic in neuroma is arborization of axons leading to extreme, neuropathic pain. The most common sites for neuroma are the ends of transected nerves following injury; however, this rodent model does not reliably result in neuroma formation. In this study, we established a rodent model of neuroma in which the sciatic nerve was loosely ligated with two chromic gut sutures. This model formed neuromas reliably (∼95%), presumably through activation of the neural inflammatory cascade. Resulting neuromas had a disorganized structure and a significant number of replicating cells. Quantification of changes in perineurial and Schwann cells showed a significant increase in these populations. Immunohistochemical analysis showed the presence of ß-tubulin 3 in the rapidly expanding nerve and a decrease in neurofilament heavy chain compared to the normal nerve, suggesting the axons forming a disorganized structure. Measurement of the permeability of the blood-nerve barrier shows that it opened almost immediately and remained open as long as 10 days. Studies using an antagonist of the ß3-adrenergic receptor (L-748,337) or cromolyn showed a significant reduction in tumor size and cell expansion as determined by flow cytometry, with an improvement in the animal's gait detected using a Catwalk system. Previous studies in our laboratory have shown that heterotopic ossification is also a result of the activation of neuroinflammation. Since heterotopic ossification and neuroma often occur together in amputees, they were induced in the same limbs of the study animals. More heterotopic bone was formed in animals with neuromas as compared to those without. These data collectively suggest that perturbation of early neuroinflammation with compounds such as L-748,337 and cromolyn may reduce formation of neuromas.

Cell-Based Gene Therapy System for Delivering BMPs.

The use of an adenoviral vector to transduce cells allows for certain secreted proteins or growth factors to be generated in vivo in eukaryotic cells with accurate posttranslational processing. The use of transduced cells eliminates viral toxicity, allows for targeted expression of the secreted factor at a specific site, and ensures that the therapy will be turned off when the cells are cleared by the organism. Here we describe the delivery system which utilizes cells transduced with a non-replicating adenovirus containing bone morphogenetic protein 2 (BMP-2) in the E1 region of the cassette. With this method of delivery, small amounts of the protein can incite de novo bone formation.

Trauma-Induced Heterotopic Ossification Regulates the Blood-Nerve Barrier.

De novo bone formation can occur in soft tissues as a result of traumatic injury. This process, known as heterotopic ossification (HO), has recently been linked to the peripheral nervous system. Studies suggest that HO may resemble neural crest-derived bone formation and is activated through the release of key bone matrix proteins leading to opening of the blood-nerve barrier (BNB). One of the first steps in this process is the activation of a neuro-inflammatory cascade, which results in migration of chondro-osseous progenitors, and other cells from both the endoneurial and perineurial regions of the peripheral nerves. The perineurial cells undergo brown adipogenesis, to form essential support cells, which regulate expression and activation of matrix metallopeptidase 9 (MMP9) an essential regulatory protein involved in opening the BNB. However, recent studies suggest that, in mice, a key bone matrix protein, bone morphogenetic protein 2 (BMP2) is able to immediately cross the BNB to activate signaling in specific cells within the endoneurial compartment. BMP signaling correlates with bone formation and appears critical for the induction of HO. Surprisingly, several other bone matrix proteins have also been reported to regulate the BNB, leading us to question whether these matrix proteins are important in regulating the BNB. However, this temporary regulation of the BNB does not appear to result in degeneration of the peripheral nerve, but rather may represent one of the first steps in innervation of the newly forming bone.

Is heterotopic ossification getting nervous?: The role of the peripheral nervous system in heterotopic ossification.

Heterotopic ossification (HO), or de novo bone formation in soft tissue, is often observed following traumatic injury. Recent studies suggest that peripheral nerves may play a key functional role in this process. The results supporting a neurological basis for HO are examined in this article. Evidence supports the fact that BMPs released from bone matrix possess the capacity to induce HO. However, the process cannot be recapitulated using recombinant proteins without extremely high doses suggesting other components are required for this process. Study of injuries that increase risk for HO, i.e. amputation, hip replacement, elbow fracture, burn, and CNS injury suggests that a likely candidate is traumatic injury of adjacent peripheral nerves. Recent studies suggest neuroinflammation may play a key functional role, by its ability to open the blood-nerve barrier (BNB). Barrier opening is characterized by a change in permeability and is experimentally assessed by the ability of Evans blue dye to enter the endoneurium of peripheral nerves. A combination of BMP and barrier opening is required to activate bone progenitors in the endoneurial compartment. This process is referred to as "neurogenic HO".

Development of a Cell-Based Gene Therapy Approach to Selectively Turn Off Bone Formation.

Cell and gene therapy approaches are safer when they possess a system that enables the therapy to be rapidly halted. Human mesenchymal stem cells were transduced with an adenoviral vector containing the cDNA for bone morphogenetic protein 2 (AdBMP2) to induce bone formation. To make this method safer, a system to quickly kill these virally transduced cells was designed and evaluated. Cells were encapsulated inside poly(ethylene glycol) diacrylate (PEG-Da) hydrogels that are able to shield the cells from immunological destruction. The system involves an inducible caspase-9 (iCasp9) activated using a specific chemical inducer of dimerization (CID). Delivering AdBMP2-transduced human mesenchymal stem cells encapsulated in PEG-Da hydrogel promoted ectopic ossification in vivo, and the iCasp9 system allowed direct control of the timing of apoptosis of the injected cells. The iCasp9-CID system enhances the safety of delivering AdBMP2-transduced cells, making it a more compelling therapeutic for bone repair and spine fusion. J. Cell. Biochem. 118: 3627-3634, 2017. © 2017 Wiley Periodicals, Inc.

Progenitors in Peripheral Nerves Launch Heterotopic Ossification.

Studies presented here, using a murine model of bone morphogenetic protein type 2 (BMP2)-induced heterotopic ossification (HO) show that the protein initiates HO by signaling through progenitors in the endoneurium of peripheral nerves. In the mouse, these cells were identified in the endoneurium one day after BMP2 induction using antibody against phosphoSMAD (PS) 1, 5, and 8. Studies conducted in a tracking mouse that contains a tamoxifen-regulated Wnt1-Cre recombinase crossed with a td Tomato red (TR) reporter (Wnt1CreErt :Ai9Tm) confirmed their neural origin. In this model both BMP2 induction and tamoxifen are absolutely required to induce TR. SP7+ (osterix+ )TR+ cells were found in the endoneurium on day 1 and associated with bone on day 7. Quantification of TR+ and TR- cells isolated by fluorescence-activated cell sorting showed that all SP7+ cells were found in the TR+ population, whereas only about 80% of the TR+ cells expressed SP7. Pre-chondrocytes (Sox 9+ ) and transient brown fat (tBAT, UCP1+ ) also coexpressed TR, suggesting that the progenitor in nerves is multi-potential. The endoneurium of human nerves near the site of HO contained many PS+ cells, and SP7+ cells were found in nerves and on bone in tissue from patients with HO. Control tissues and nerves did not contain these PS+ and SP7+ cells. Some osteoblasts on bone from patients with HO were positive for PS, suggesting the continued presence of BMP during bone formation. The data suggests that the progenitors for HO are derived from the endoneurium in both the mouse model of HO and in humans with HO. Stem Cells Translational Medicine 2017;6:1109-1119.

Encapsulation of Adenovirus BMP2-Transduced Cells with PEGDA Hydrogels Allows Bone Formation in the Presence of Immune Response.

Gene therapy approaches have been difficult to implement due to pre-existing immunity against the virus used for delivery. To circumvent this problem, a cell-based approach was developed that avoided the use of free virus within the animal. However, even cells transduced in vitro with E1- to E3-deleted adenovirus encoding bone morphogenetic protein 2 (AdBMP2) resulted in the production of virus-neutralizing antibodies in mice. Furthermore, when mice received an intramuscular injection of nonencoding adenovirus (AdEmpty)-transduced cells, AdBMP2-transduced cells were unable to launch bone formation when an intramuscular injection of these BMP2-producing cells was delivered 1 week later. This phenomenon was not observed in NOD/SCID mice, and could be overcome in C57BL/6 mice by encapsulating the adenovirus-transduced cells in a nondegradable hydrogel poly(ethylene glycol) diacrylate (PEGDA). Data collectively suggest that PEGDA hydrogel encapsulation of AdBMP2-transduced cells prevents pre-existing immunity from suppressing BMP2-induced bone formation.

Location-dependent heterotopic ossification in the rat model: The role of activated matrix metalloproteinase 9.

Extremity amputation or traumatic injury can often lead to the formation of heterotopic ossification (HO). Studies to induce HO in rat muscle using cell-based gene therapy show that this process appears to be location dependent. In the present study, HO was induced in mice and rats through injection of immunologically matched cells transduced with either a replication-defective adenovirus possessing bone morphogenetic protein 2 (BMP2) or an empty adenovirus vector (control). Injection in rat near the skeletal bone resulted in HO, whereas cells injected into the same muscle group but distal from the bone did not result in bone formation. When cells were injected in the same limb at both locations at the same time, HO was formed at both sites. Characterization of the bone formation in rats versus mice demonstrated that different sources of osteogenic progenitors were involved, which may account for the location dependent bone formation observed in the rat. Further experimentation has shown that a potential reason for this difference may be the inability of rat to activate matrix metalloproteinase 9 (MMP9), an essential protease in mice necessary for recruitment of progenitors. Inhibition of active MMP9 in mice led to a significant decrease in HO. The studies reported here provide insight into the mechanisms and pathways leading to bone formation in different animals and species. It appears that not all animal models are appropriate for testing HO therapies, and our studies also challenge the conventional wisdom that larger animal models are better for testing treatments affecting bone. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1894-1904, 2016.

Anaplerotic Accumulation of Tricarboxylic Acid Cycle Intermediates as Well as Changes in Other Key Metabolites During Heterotopic Ossification.

Heterotopic ossification (HO) is the de novo formation of bone that occurs in soft tissue, through recruitment, expansion, and differentiation of multiple cells types including transient brown adipocytes, osteoblasts, chondrocytes, mast cells, and platelets to name a few. Much evidence is accumulating that suggests changes in metabolism may be required to accomplish this bone formation. Recent work using a mouse model of heterotopic bone formation reliant on delivery of adenovirus-transduced cells expressing low levels of BMP2 showed the immediate expansion of a unique brown adipocyte-like cell. These cells are undergoing robust uncoupled oxidative phosphorylation to a level such that oxygen in the microenvironment is dramatically lowered creating areas of hypoxia. It is unclear how these oxygen changes ultimately affect metabolism and bone formation. To identify the processes and changes occurring over the course of bone formation, HO was established in the mice, and tissues isolated at early and late times were subjected to a global metabolomic screen. Results show that there are significant changes in both glucose levels, as well as TCA cycle intermediates. Additionally, metabolites necessary for oxidation of stored lipids were also found to be significantly elevated. The complete results of this screen are presented here, and provide a unique picture of the metabolic changes occurring during heterotopic bone formation.

Osteoblasts Have a Neural Origin in Heterotopic Ossification.

BACKGROUND: Heterotopic ossification (HO) is the process of bone formation at a nonskeletal site. Recently, we showed that the earliest steps occur in sensory nerves. We now extend these studies by identifying unique osteogenic progenitors within the endoneurial compartment of sensory nerves. QUESTIONS/PURPOSES: We asked: (1) What is the nature of the osteoprogenitor in the endoneurium of peripheral nerves? (2) How do osteoprogenitors travel from the nerve to the site of new bone formation? METHODS: HO was induced by intramuscular injection of Ad5BMP-2-transduced cells in mice. Osteoprogenitors were identified through immunohistochemistry and then quantified and further characterized by fluorescence-activated cell sorting and immunocytochemistry. The kinetics of the appearance of markers of extravasation was determined by quantitative reverse transcription-polymerase chain reaction. In each experiment mice were injected with bone morphogenetic protein-2 (BMP-2)-producing cells (experimental) or with cells transduced with empty vector or, in some cases, a group receiving no injection (control). RESULTS: Induction of HO leads to the expression, within 24 hours, of osteoblast-specific transcription factors in cells in the endoneurium followed by their coordinate disappearance from the nerve at 48 hours. They reappear in blood also at 48 hours after induction. During vessel entrance they begin to express the tight junction molecule, claudin 5. The cells expressing both the osteoblast-specific transcription factor, osterix, as well as claudin 5, then disappear from circulation at approximately 3 to 4 days by extravasation into the site of new bone formation. These endoneurial osteoprogenitors express neural markers PDGFRα, musashi-1, and the low-affinity nerve growth factor receptor p75(NTR) as well as the endothelial marker Tie-2. In a key experiment, cells that were obtained from mice that were injected with cells transduced with an empty vector, at 2 days after injection, contained 0.83% (SD, 0.07; 95% confidence interval [CI], 0.59-1.05) cells expressing claudin 5. However, cells that were obtained from mice 2 days after injection of BMP-2-producing cells contained 4.5% cells expressing claudin 5 (SD, 0.72%; 95% CI, 2.01-6.94; p < 0.0015). Further analysis revealed that all of the cells expressing claudin 5 were found to be positive for osteoblast-specific markers, whereas cells not expressing claudin 5 were negative for these same markers. CONCLUSIONS: The findings suggest that the endoneurial progenitors are the major osteogenic precursors that are used for HO. They exit the nerve through the endoneurial vessels, flow through vessels to the site of new bone formation, and then extravasate out of the vessels into this site. CLINICAL RELEVANCE: The biogenesis of osteoblasts in HO is very different than expected and shows that HO is, at least in part, a neurological disorder. This could result in a major shift in orthopaedic methodologies to prevent or treat this disease. The fact that nerves are intimately involved in the process may also provide clues that will lead to an explanation of the clinical fact that HO often occurs as a result of traumatic brain injury.

On developing a process for conducting extractable-leachable assessment of components used for storage of biopharmaceuticals.

Extractables and leachables are product-related impurities that result from product contact with components such as gaskets, stoppers, storage bags, cartridges, and prefilled syringes that are used for processing, storage, and/or delivery of biopharmaceuticals. These impurities are a concern for patients due to potential effects on product quality and safety. It is possible that such an impurity could directly impact the patient or indirectly impact the patient by interacting with the protein therapeutics and forming protein adducts. Adducts and leachables may or may not be detected as product-related impurities in routine stability indicating assays depending on the rigor of the analytical program. The need for the development of a thorough and holistic extractable and leachable program based on risk assessment, review of existing literature, and consolidation of industry best practices is discussed. Standardizing component use within an organization enables streamlining of the extractable-leachable program. Our strategy for an extractable-leachable program is divided into different stages, each stage detailing the activities and the department within the organization that is responsible for execution of these activities. The roles and responsibilities of the key stakeholders are identified. The integration of analytical activities with health-based risk-assessment information into the design of an extractable-leachable program is highlighted.

A study in glycation of a therapeutic recombinant humanized monoclonal antibody: where it is, how it got there, and how it affects charge-based behavior.

The glycated form of a basic recombinant humanized monoclonal antibody (rhuMAb) was separated and quantitated by boronate affinity chromatography using optimized shielding reagents. Characterization on the isolated glycated material by peptide mapping analysis, using liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS) sequencing techniques, identified eight reactive lysine primary amine sites. The glycation reaction extent was similar among the various reactive sites, ranging from approximately 1 to 12%, and a single histidine residue separated the most and least reactive sites. Boronate chromatography run in a linear gradient mode separated monoglycated rhuMAb from higher order glycated species and indicated that the majority ( approximately 90%) of glycated rhuMAb is monoglycated. Low-level glycation on a heavy chain lysine located within a complementarity-determining region (CDR) did not significantly affect binding activity in potency measurements. The glycated forms also behaved as slightly more acidic than the nonglycated antibody in charge-based separation techniques, observable by capillary isoelectric focusing (cIEF) and ion exchange chromatography (IEC). The boronate column has significantly increased retention of aggregated rhuMAb material under separation conditions optimized for the monomer form. Recombinant protein glycation initially occurred during production in mammalian cell culture, where feed sugar and protein concentrations contribute to the total overall glycation on this antibody product.

Differential in vivo effects of rituximab on two B-cell subsets in cynomolgus monkeys.

Cynomolgus monkeys (Macaca fascicularis) are widely used animal models in biomedical research and have been used to study new therapeutics aimed at B-cell depletion. We have recently identified two different B-cell subsets in cynomolgus monkey, with the CD20lowCD40highCD21+ subset being phenotypically closer to human B cells and having a similar responsivness to anti-CD20 mAb, rituximab, in in vitro depletion assays. Here, we show that similar to in vitro findings CD20highCD40lowCD21- and CD20lowCD40highCD21+ cynomolgus monkey B cells differ significantly in their in vivo susceptibility to rituximab, as the low dose of 0.05 mg/kg of rituximab resulted in more than 70% depletion of the former B-cell subset and virtually no depletion of the latter B-cell subset. Our data suggest that for the B-cell-targeting anti-CD20 therapeutics, depletion of CD20lowCD40highCD21+ subset rather than depletion of all cynomolgus monkey B cells is more relevant to dose-efficacy projections for humans. In addition, we show that differential cell surface expression of CD80/CD86 costimulatory molecules on the two different cynomolgus monkey B-cell subsets is similar to that identified in rhesus monkeys, suggesting that our in vivo study may be relevant to other monkey models.

Effect of anti-CD20 monoclonal antibody, Rituxan, on cynomolgus monkey and human B cells in a whole blood matrix.

BACKGROUND: Cynomolgus monkeys are widely used animal models in biomedical research. The differences between cynomolgus monkey and human B cells are not completely understood. However, these differences are of crucial importance for interpretation of data from studies on new therapeutic agents aimed at B-cell depletion, such as anti-CD20 monoclonal antibodies. METHODS: Multicolor fluorescence-activated cell sorting analysis of peripheral blood B cells was performed on samples treated ex vivo with the anti-CD20 therapeutic monoclonal antibody, Rituxan, in a whole blood matrix. RESULTS: In contrast to humans, cynomolgus monkeys had two distinct B-cell subsets, CD20highCD40lowCD21- and CD20lowCD40highCD21+. These B-cell subsets had a 2.5-fold difference in the EC50 for Rituxan binding and differed significantly in their in vitro susceptibility to Rituxan depletion. Human B cells were similar to the CD20lowCD40highCD21+ cynomolgus monkey B cells with regard to their EC50 for Rituxan and response to Rituxan in a whole blood matrix assay. CD21 was upregulated, whereas CD40 was downregulated at incubation with Rituxan in the CD20lowCD40highCD21+ monkey and human B cells in a concentration-dependent manner. CONCLUSIONS: These findings have direct implications for in vivo studies of therapeutic agents that target B cells in cynomolgus monkeys and for extrapolation of the results to humans. In addition, our data are consistent with the model in which CD20, CD21, and CD40 exist in a supramolecular complex that is affected by anti-CD20 monoclonal antibodies.

Letters.

SPINAL INJURY AND PRESSURE SORE PREVENTION TREATMENT FOR OVERGRANULATION.