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PURPOSE: To evaluate a three-compartmental semi-physiological model for analysis of uptake clearance and efflux from brain tissue of the hydrophilic markers sucrose and mannitol, compared to non-compartmental techniques presuming unidirectional uptake. METHODS: Stable isotope-labeled [(13)C]sucrose and [(13)C]mannitol (10 mg/kg each) were injected as IV bolus into the tail vein of awake young adult mice. Blood and brain samples were taken after different time intervals up to 8 h. Plasma and brain concentrations were quantified by UPLC-MS/MS. Brain uptake clearance (K(in)) was analyzed using either the single-time point analysis, the multiple time point graphical method, or by fitting the parameters of a three-compartmental model that allows for symmetrical exchange across the blood-brain barrier and an additional brain efflux clearance. RESULTS: The three-compartment model was able to describe the experimental data well, yielding estimates for K(in) of sucrose and mannitol of 0.068 ± 0.005 and 0.146 ± 0.020 μl.min(−1).g(−1), respectively, which were significantly different (p < 0.01). The separate brain efflux clearance had values of 0.693 ± 0.106 (sucrose) and 0.881 ± 0.20 (mannitol) μl.min(−1).g(−1), which were not statistically different. K(in) values obtained by single time point and multiple time point analyses were dependent on the terminal sampling time and showed declining values for later time points. CONCLUSIONS: Using the three-compartment model allows determination of K(in) for small molecule hydrophilic markers with low blood-brain barrier permeability. It also provides, for the first time, an estimate of brain efflux after systemic administration of a marker, which likely represents bulk flow clearance from brain tissue.

The dopaminergic system is involved in the regulation of immune responses in various homeostatic and disease conditions. For conditions such as Parkinson’s disease and multiple sclerosis (MS), pharmacological modulation of dopamine (DA) system activity is thought to have therapeutic relevance, providing the basis for using dopaminergic agents as a treatment of relevant states. In particular, it was proposed that restoration of DA levels may inhibit neuroinflammation. We have recently reported a new class of dopamine transporter (DAT) inhibitors with high selectivity to the DAT over other G-protein coupled receptors tested. Here, we continue their evaluation as monoamine transporter inhibitors. Furthermore, we show that the urea-like DAT inhibitor (compound 5) has statistically significant anti-inflammatory effects and attenuates motor deficits and pain behaviors in the experimental autoimmune encephalomyelitis model mimicking clinical signs of MS. To the best of our knowledge, this is the first study reporting the beneficial effects of DAT inhibitor-based treatment in animals with induced autoimmune encephalomyelitis, and the observed results provide additional support to the model of DA-related neuroinflammation.

Lymphangiogenesis is an essential physiological process but also a determining factor in vascular-related pathological conditions. Angiopoietin-2 (Ang2) plays an important role in lymphatic vascular development and function and its upregulation has been reported in several vascular-related diseases, including cancer. Given the established role of the small GTPase RhoA on cytoskeleton-dependent endothelial functions, we investigated the relationship between RhoA and Ang2-induced cellular activities. This study shows that Ang2-driven human dermal lymphatic endothelial cell migration depends on RhoA. We demonstrate that Ang2-induced migration is independent of the Tie receptors, but dependent on β1 integrin-mediated RhoA activation with knockdown, pharmacological approaches, and protein sequencing experiments. Although the key proteins downstream of RhoA, Rho kinase (ROCK) and myosin light chain, were activated, blockade of ROCK did not abrogate the Ang2-driven migratory effect. However, formins, an alternative target of RhoA, were identified as key players, and especially FHOD1. The Ang2-RhoA relationship was explored in vivo, where lymphatic endothelial RhoA deficiency blocked Ang2-induced lymphangiogenesis, highlighting RhoA as an important target for anti-lymphangiogenic treatments.

The neuronal ceroid lipofuscinoses (NCLs) commonly referred to as Batten disease are a family of rare lysosomal storage disorders (LSDs). The most common form of NCL occurs in children harboring a mutation in the CLN3 gene. This form is lethal with no existing cure or treatment beyond symptomatic relief. The pathophysiology of CLN3 disease is complex and poorly understood, with the current in vivo and in vitro models failing to identify pharmacological targets for therapeutic intervention. This study reports the characterization of the first CLN3 patient-specific induced pluripotent stem cell (iPSC)-derived model of the blood-brain barrier (BBB) and adds to the few available iPSC-derived neuron models of the disease. Upon differentiation, hallmarks of CLN3 disease were displayed including lipofuscin and subunit C of mitochondrial ATP synthase accumulation, mitochondrial dysfunction and aberrant lysosomal pH. Small molecules were identified that cleared subunit C accumulation by the mTOR-independent modulation of autophagy, conferred protective effects through induction of Bcl-2 and rescued mitochondrial dysfunction.

Pleiotrophin (PTN) has a moderate stimulatory effect on endothelial cell migration through α

Angiogenesis is a well-coordinated physiological process that leads to new blood vessel formation. Physiologically, angiogenesis is more prominent during development and wound healing and its dysregulation drives or is related to several diseases, including cancer. The endothelial cells are the main regulators of the angiogenic process, and thus the angiogenic outcome is assessed based on the effect on endothelial cell functions. Several in vitro and in vivo techniques have been developed to assess the effect of various factors on angiogenesis. Compared to the in vivo techniques, the in vitro techniques are considered less physiologically relevant. This has been partially overcome by the development of 3-dimensional (3D) in vitro models, one of which is the spheroid assay or 3D sprouting assay that exploits the effect of the extracellular matrix to endothelial cell functions. This chapter focuses on the description of the spheroid assay and mentions the variations and potential applications this assay can have.

Simple Summary Anti-angiogenic therapies targeting the vascular endothelial growth factor (VEGF) signaling are established in the arsenal of cancer treatments. Despite the expectations, their benefits are temporary in cancer patients, partly due to the compensatory function of other angiogenic growth factors. This review focuses on the role of basic fibroblast growth factor (bFGF), one of the highly implicated players in the emergence of resistance to anti-angiogenic approaches. Here, we summarize data from various tumor types where bFGF is upregulated after anti-angiogenic treatment, the molecular mechanisms involved, and we highlight the current status and future perspectives of multi-target anti-angiogenic drugs for cancer. Abstract Anti-angiogenic approaches targeting the vascular endothelial growth factor (VEGF) signaling pathway have been a significant research focus during the past decades and are well established in clinical practice. Despite the expectations, their benefit is ephemeral in several diseases, including specific cancers. One of the most prominent side effects of the current, VEGF-based, anti-angiogenic treatments remains the development of resistance, mostly due to the upregulation and compensatory mechanisms of other growth factors, with the basic fibroblast growth factor (bFGF) being at the top of the list. Over the past decade, several anti-angiogenic approaches targeting simultaneously different growth factors and their signaling pathways have been developed and some have reached the clinical practice. In the present review, we summarize the knowledge regarding resistance mechanisms upon anti-angiogenic treatment, mainly focusing on bFGF. We discuss its role in acquired resistance upon prolonged anti-angiogenic treatment in different tumor settings, outline the reported resistance mechanisms leading to bFGF upregulation, and summarize the efforts and outcome of combined anti-angiogenic approaches to date.

Lung cancer (LC) is the leading cause of cancer-related deaths. Downregulation of CDK1, 4 and 6, key regulators of cell cycle progression, correlates with decreased LC cell proliferation. Enforced expression of miRNAs (miRs) is a promising approach to regulate genes. Here, we study the combinatorial treatment of miR-143 and miR-506 to target the CDK1, 4/6 genes, respectively. We analyzed the differential expression of CDK genes by qPCR, and western blot, and evaluated changes in the cell cycle distribution upon combinatorial treatment. We used an antibody microarray analysis to evaluate protein expression, focusing on the cell cycle pathway, and performed RNA-sequencing for pathway analysis. The combinatorial miR treatment significantly downregulated CDK1, 4 and 6 expression, and induced a shift of the cell cycle populations, indicating a G1 and G2 cell cycle block. The two miRs induces strong cytotoxic activity, with potential synergism, and a significant Caspase 3/7 activation. We identified a strong inhibition of tube formation in the presence or absence VEGF in an in vitro angiogenesis model. Together with the pathways analysis of the RNA-sequencing data, our findings establish the combinatorial miR transfection as a viable strategy for lung cancer treatment that merits further investigation.