Precision Culture Scaling to Establish High-Throughput Vasculogenesis Models.

Hydrogel-based 3D cell cultures can recapitulate (patho)physiological phenomena ex vivo. However, due to their complex multifactorial regulation, adapting these tissue and disease models for high-throughput screening workflows remains challenging. In this study, a new precision culture scaling (PCS-X) methodology combines statistical techniques (design of experiment and multiple linear regression) with automated, parallelized experiments and analyses to customize hydrogel-based vasculogenesis cultures using human umbilical vein endothelial cells and retinal microvascular endothelial cells. Variations of cell density, growth factor supplementation, and media composition are systematically explored to induce vasculogenesis in endothelial mono- and cocultures with mesenchymal stromal cells or retinal microvascular pericytes in 384-well plate formats. The developed cultures are shown to respond to vasculogenesis inhibitors in a compound- and dose-dependent manner, demonstrating the scope and power of PCS-X in creating parallelized tissue and disease models for drug discovery and individualized therapies.

Nogo-A is secreted in extracellular vesicles, occurs in blood and can influence vascular permeability.

Nogo-A is a transmembrane protein with multiple functions in the central nervous system (CNS), including restriction of neurite growth and synaptic plasticity. Thus far, Nogo-A has been predominantly considered a cell contact-dependent ligand signaling via cell surface receptors. Here, we show that Nogo-A can be secreted by cultured cells of neuronal and glial origin in association with extracellular vesicles (EVs). Neuron- and oligodendrocyte-derived Nogo-A containing EVs inhibited fibroblast spreading, and this effect was partially reversed by Nogo-A receptor S1PR2 blockage. EVs purified from HEK cells only inhibited fibroblast spreading upon Nogo-A over-expression. Nogo-A-containing EVs were found in vivo in the blood of healthy mice and rats, as well as in human plasma. Blood Nogo-A concentrations were elevated after acute stroke lesions in mice and rats. Nogo-A active peptides decreased barrier integrity in an in vitro blood-brain barrier model. Stroked mice showed increased dye permeability in peripheral organs when tested 2 weeks after injury. In the Miles assay, an in vivo test to assess leakage of the skin vasculature, a Nogo-A active peptide increased dye permeability. These findings suggest that blood borne, possibly EV-associated Nogo-A could exert long-range regulatory actions on vascular permeability.

Selenium mediates exercise-induced adult neurogenesis and reverses learning deficits induced by hippocampal injury and aging.

Although the neurogenesis-enhancing effects of exercise have been extensively studied, the molecular mechanisms underlying this response remain unclear. Here, we propose that this is mediated by the exercise-induced systemic release of the antioxidant selenium transport protein, selenoprotein P (SEPP1). Using knockout mouse models, we confirmed that SEPP1 and its receptor low-density lipoprotein receptor-related protein 8 (LRP8) are required for the exercise-induced increase in adult hippocampal neurogenesis. In vivo selenium infusion increased hippocampal neural precursor cell (NPC) proliferation and adult neurogenesis. Mimicking the effect of exercise through dietary selenium supplementation restored neurogenesis and reversed the cognitive decline associated with aging and hippocampal injury, suggesting potential therapeutic relevance. These results provide a molecular mechanism linking exercise-induced changes in the systemic environment to the activation of quiescent hippocampal NPCs and their subsequent recruitment into the neurogenic trajectory.

Characterization of the Blood Brain Barrier Disruption in the Photothrombotic Stroke Model.

Blood brain barrier (BBB) damage is an important pathophysiological feature of ischemic stroke which significantly contributes to development of severe brain injury and therefore is an interesting target for therapeutic intervention. A popular permanent occlusion model to study long term recovery following stroke is the photothrombotic model, which so far has not been anatomically characterized for BBB leakage beyond the acute phase. Here, we observed enhanced BBB permeability over a time course of 3 weeks in peri-infarct and core regions of the ischemic cortex. Slight increases in BBB permeability could also be seen in the contralesional cortex, hippocampus and the cerebellum at different time points, regions where lesion-induced degeneration of pathways is prominent. Severe damage of tight and adherens junctions and loss of pericytes was observed within the peri-infarct region. Overall, the photothrombotic stroke model reproduces a variety of features observed in human stroke and thus, represents a suitable model to study BBB damage and therapeutic approaches interfering with this process.

Author Correction: Mast cells increase adult neural precursor proliferation and differentiation but this potential is not realized in vivo under physiological conditions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A Practical Guide to the Automated Analysis of Vascular Growth, Maturation and Injury in the Brain.

The distinct organization of the brain's vasculature ensures the adequate delivery of oxygen and nutrients during development and adulthood. Acute and chronic pathological changes of the vascular system have been implicated in many neurological disorders including stroke and dementia. Here, we describe a fast, automated method that allows the highly reproducible, quantitative assessment of distinct vascular parameters and their changes based on the open source software Fiji (ImageJ). In particular, we developed a practical guide to reliably measure aspects of growth, repair and maturation of the brain's vasculature during development and neurovascular disease in mice and humans. The script can be used to assess the effects of different external factors including pharmacological treatments or disease states. Moreover, the procedure is expandable to blood vessels of other organs and vascular in vitro models.

Anti-Nogo-A antibodies prevent vascular leakage and act as pro-angiogenic factors following stroke.

Angiogenesis is a key restorative process following stroke but has also been linked to increased vascular permeability and blood brain barrier (BBB) disruption. Previous pre-clinical approaches primarily focused on the administration of vascular endothelial growth factor (VEGF) to promote vascular repair after stroke. Although shown to improve angiogenesis and functional recovery from stroke, VEGF increased the risk of blood brain barrier disruption and bleedings to such an extent that its clinical use is contraindicated. As an alternative strategy, antibodies against the neurite growth inhibitory factor Nogo-A have recently been shown to enhance vascular regeneration in the ischemic central nervous system (CNS); however, their effect on vascular permeability is unknown. Here, we demonstrate that antibody-mediated Nogo-A neutralization following stroke has strong pro-angiogenic effects but does not increase vascular permeability as opposed to VEGF. Moreover, VEGF-induced vascular permeability was partially prevented when VEGF was co-administered with anti-Nogo-A antibodies. This study may provide a novel therapeutic strategy for vascular repair and maturation in the ischemic brain.

Refueling the Ischemic CNS: Guidance Molecules for Vascular Repair.

Stroke patients have only limited therapeutic options and often remain with considerable disabilities. To promote neurological recovery, angiogenesis in the ischemic peri-infarct region has been recognized as an encouraging therapeutic target. Despite advances in mechanistic understanding of vascular growth and repair, effective and safe angiogenic treatments are currently missing. Besides the most intensively studied angiogenic growth factors, recent research has indicated that the process of vascular sprouting and migration also requires the participation of guidance molecules, many of which were initially identified as regulators of axonal growth. Here, we review the inhibitory and growth-promoting effects of guidance molecules on the vascular system and discuss their potential as novel angiogenic targets for neurovascular diseases.

Nogo-A targeted therapy promotes vascular repair and functional recovery following stroke.

Stroke is a major cause of serious disability due to the brain's limited capacity to regenerate damaged tissue and neuronal circuits. After ischemic injury, a multiphasic degenerative and inflammatory response is coupled with severely restricted vascular and neuronal repair, resulting in permanent functional deficits. Although clinical evidence indicates that revascularization of the ischemic brain regions is crucial for functional recovery, no therapeutics that promote angiogenesis after cerebral stroke are currently available. Besides vascular growth factors, guidance molecules have been identified to regulate aspects of angiogenesis in the central nervous system (CNS) and may provide targets for therapeutic angiogenesis. In this study, we demonstrate that genetic deletion of the neurite outgrowth inhibitor Nogo-A or one of its corresponding receptors, S1PR2, improves vascular sprouting and repair and reduces neurological deficits after cerebral ischemia in mice. These findings were reproduced in a therapeutic approach using intrathecal anti-Nogo-A antibodies; such a therapy is currently in clinical testing for spinal cord injury. These results provide a basis for a therapeutic blockage of inhibitory guidance molecules to improve vascular and neural repair after ischemic CNS injuries.

A Revised View on Growth and Remodeling in the Retinal Vasculature.

The mouse retina provides an excellent model for studying angiogenesis. Recent advancements in high-throughput microscopy and image analysis provide great tools to visualize and describe the complexity of the retinal vascular architecture in a detailed and comprehensive way. Most developmental studies have focused on only a few parameters mostly in the inner-most layers that do not describe the entirety of the three-dimensional vascular network. Here, we analyzed the entire three-dimensional retinal vascular architecture and its growth and remodeling starting from the age of postnatal day 3 to 4 months in mice. We show plexus specific characteristics of the vasculature in terms of vascular tissue fraction, branching and length of the blood vessels, and distance and distribution between single capillaries. Such detailed knowledge is of particular interest, as it has become apparent that disease-specific mechanisms and treatments affect the retinal vasculature often in a plexus specific way.

Inflammation after Stroke: A Local Rather Than Systemic Response?

After injury, activation and recruitment of inflammatory and immune cells has been thought to occur throughout the whole body. A recent study shows that after brain injury in mice, immune cells are primarily recruited from nearby skull bone marrow and invade the brain through microscopic vascular channels. Manipulation of this process may provide new therapeutic options.

T Lymphocytes Contribute to the Control of Baseline Neural Precursor Cell Proliferation but Not the Exercise-Induced Up-Regulation of Adult Hippocampal Neurogenesis.

Cross-talk between the peripheral immune system and the central nervous system is important for physiological brain health. T cells are required to maintain normal baseline levels of neural precursor proliferation in the hippocampus of adult mice. We show here that neither T cells, B cells, natural killer cells nor natural killer T cells are required for the increase in hippocampal precursor proliferation that occurs in response to physical exercise. In addition, we demonstrate that a subpopulation of T cells, regulatory T cells, is not involved in maintaining baseline levels of neural precursor proliferation. Even when applied at supraphysiological numbers, populations of both naive and stimulated lymphocytes had no effect on hippocampal precursor proliferation in vitro. In addition, physical activity had no effect on peripheral immune cells in terms of distribution in the bone marrow, lymph nodes or spleen, activation state or chemokine receptor (CXCR4 and CCR9) expression. Together these results suggest that lymphocytes are not involved in translating the peripheral effects of exercise to the neurogenic niche in the hippocampus and further support the idea that the exercise-induced regulation of adult neurogenesis is mechanistically distinct from its baseline control.

Mast cells increase adult neural precursor proliferation and differentiation but this potential is not realized in vivo under physiological conditions.

There is growing evidence that both peripheral and resident immune cells play an important part in regulating adult neural stem cell proliferation and neurogenesis, although the contribution of the various immune cell types is still unclear. Mast cells, a population of immune cells known for their role in the allergic response, have been implicated in the regulation of adult hippocampal neurogenesis. Mast cell-deficient c-kitW-sh/W-sh mice have previously been shown to exhibit significantly decreased adult hippocampal neurogenesis and associated learning and memory deficits. However, given that numerous other cell types also express high levels of c-kit, the utility of these mice as a reliable model of mast cell-specific depletion is questionable. We show here, using a different model of mast cell deficiency (Mcpt5CreR26DTA/DTA), that precursor proliferation and adult neurogenesis are not influenced by mast cells in vivo. Interestingly, when applied at supraphysiological doses, mast cells can activate latent hippocampal precursor cells and increase subventricular zone precursor proliferation in vitro, an effect that can be blocked with specific histamine-receptor antagonists. Thus, we conclude that while both mast cells and their major chemical mediator histamine have the potential to affect neural precursor proliferation and neurogenesis, this is unlikely to occur under physiological conditions.