Basic Fibroblast Growth Factor Opens and Closes the Endothelial Blood-Brain Barrier in a Concentration-Dependent Manner.

Multiple paracrine factors are implicated in the regulation of barrier properties of human brain endothelial cells (BECs) in different physiologic and pathologic settings. We have recently demonstrated that autocrine secretion of basic fibroblast growth factor (bFGF) by BECs is necessary for the establishment of endothelial barrier (as demonstrated by high trans-endothelial electric resistance, TEER), whereas exogenous bFGF inhibits TEER in a concentration-dependent manner. In the present study we analysed the contribution of MAPK/ERK and STAT3 signalling pathways to the inhibitory effects of exogenous bFGF. Treatment with bFGF (8 ng/ml) for 3 days increased phosphorylation of ERK1/2 and STAT3. Treatment with FGF receptor 1 (FGFR1) inhibitor PD173074 (15 µM) suppressed both basal and bFGF-induced activation of ERK1/2 and STAT3. Suppression of STAT signalling with Janus kinase inhibitor JAKi (15 nM) alone or in the presence of bFGF did not change TEER in BEC monolayers. Exposure to JAKi affected neither proliferation, nor expression and distribution of tight junction (TJ) proteins claudin-5, occludin and zonula occludens-1 (ZO-1). In contrast, treatment with MEK 1/2 inhibitor U0126 (10 µM) partially neutralised inhibitory effect of bFGF thus increasing TEER, whereas U0126 alone did not affect resistance of endothelial barrier. Our findings demonstrate that MAPK/ERK signalling pathway does not affect autocrine bFGF signalling-dependent BECs barrier function but is largely responsible for the disruptive effects of the exogenous bFGF. We speculate that bFGF may (depending on concentration and possibly origin) dynamically regulate permeability of the endothelial blood-brain barrier.

Concentration-dependent duality of bFGF in regulation of barrier properties of human brain endothelial cells.

Multiple paracrine factors regulate the barrier properties of human brain capillary endothelial cells (BCECs). Understanding the precise mode of action of these factors remains a challenging task, because of the limited availability of functionally competent BCECs and the use of serum-containing medium. In the present study, we employed a defined protocol for producing BCECs from human inducible pluripotent stem cells. We found that autocrine secretion of basic fibroblast growth factor (bFGF) is necessary for the establishment a tight BCECs barrier, as revealed by measurements of transendothelial electric resistance (TEER). In contrast, addition of exogenous bFGF in concentrations higher than 4 ng/ml inhibited TEER in a concentration-dependent manner. Exogenous bFGF did not significantly affect expression and distribution of tight junction proteins claudin-5, occludin and zonula occludens (ZO)-1. Treatment with FGF receptor blocker PD173074 (15 µM) suppressed inhibitory effects of bFGF and induced nuclear translocation of protein ZO-1. Inhibition of phosphoinositide 3-Kinase (PI-3K) with LY294002 (25 µM) significantly potentiated an inhibitory effect of bFGF on TEER indicating that PI-3K signalling pathway counteracts bFGF modulation of TEER. In conclusion, we show that autocrine bFGF secretion is necessary for the proper barrier function of BCECs, whereas exogenous bFGF in higher doses suppresses barrier resistance. Our findings demonstrate a dual role for bFGF in the regulation of BCEC barrier function.

Immortalised Hippocampal Astrocytes from 3xTG-AD Mice Fail to Support BBB Integrity In Vitro: Role of Extracellular Vesicles in Glial-Endothelial Communication.

Impairments of the blood-brain barrier (BBB) and vascular dysfunction contribute to Alzheimer's disease (AD) from the earliest stages. However, the influence of AD-affected astrocytes on the BBB remain largely unexplored. In the present study, we created an in vitro BBB using human-immortalized endothelial cells in combination with immortalized astroglial cell lines from the hippocampus of 3xTG-AD and wild-type mice (3Tg-iAstro and WT-iAstro, respectively). We found that co-culturing endothelial monolayers with WT-iAstro upregulates expression of endothelial tight junction proteins (claudin-5, occludin, ZO-1) and increases the trans-endothelial electrical resistance (TEER). In contrast, co-culturing with 3Tg-iAstro does not affect expression of tight junction proteins and does not change the TEER of endothelial monolayers. The same in vitro model has been used to evaluate the effects of extracellular vesicles (EVs) derived from the WT-iAstro and 3Tg-iAstro. The EVs derived from WT-iAstro increased TEER and upregulated expression of tight junction proteins, whereas EVs from 3Tg-iAstro were ineffective. In conclusion, we show for the first time that immortalized hippocampal astrocytes from 3xTG-AD mice exhibit impaired capacity to support BBB integrity in vitro through paracrine mechanisms and may represent an important factor underlying vascular abnormalities during development of AD.

Time-Dependent Memory and Gait Improvement by Intranasally-Administered Extracellular Vesicles in Parkinson's Disease Model Rats.

We have recently demonstrated that extracellular vesicles (EVs) derived from the human teeth stem cells improve motor symptoms and normalize tyrosine hydroxylase (TH) expression in the nigrostriatal structures of Parkinson's disease (PD) model rats obtained by 6-hydroxydopamine (6-OHDA) unilateral injection into the medial forebrain bundle (MFB). The aim of this study was to clarify: (1) how long therapeutic effects persist after discontinuation of 17-day intranasal administration of EVs in 6-OHDA rats; (2) may EVs reverse cognitive (learning/memory) dysfunction in these PD model rats; (3) whether and how the behavioral improvement may be related to the expression of TH and Nissl bodies count in the nigrostriatal structures. Our results demonstrated that in 6-OHDA rats, gait was normalized even ten days after discontinuation of EVs administration. EVs successfully reversed 6-OHDA-induced impairment in spatial learning/memory performance; however, the beneficial effect was shorter (up to post-treatment day 6) than that revealed for gait improvement. The shorter effect of EVs coincided with both full normalization of TH expression and Nissl bodies count in the nigrostriatal structures, while slight but significant increase in the 6-OHDA-decreased Nissl count persisted in the substantia nigra even on the post-treatment day 20, supposedly due to the continuation of protein synthesis in the living cells. The obtained data indicate the usefulness of further studies to find the optimal administration regimen which could be translated into clinical trials on PD patients, as well as to clarify other-apart from dopaminergic-neuromodulatory pathways involved in the EVs mechanism of action.

Extracellular Vesicles from Human Teeth Stem Cells Trigger ATP Release and Promote Migration of Human Microglia through P2X4 Receptor/MFG-E8-Dependent Mechanisms.

Extracellular vesicles (EVs) effectively suppress neuroinflammation and induce neuroprotective effects in different disease models. However, the mechanisms by which EVs regulate the neuroinflammatory response of microglia remains largely unexplored. Here, we addressed this issue by testing the action of EVs derived from human exfoliated deciduous teeth stem cells (SHEDs) on immortalized human microglial cells. We found that EVs induced a rapid increase in intracellular Ca2+ and promoted significant ATP release in microglial cells after 20 min of treatment. Boyden chamber assays revealed that EVs promoted microglial migration by 20%. Pharmacological inhibition of different subtypes of purinergic receptors demonstrated that EVs activated microglial migration preferentially through the P2X4 receptor (P2X4R) pathway. Proximity ligation and co-immunoprecipitation assays revealed that EVs promote association between milk fat globule-epidermal growth factor-factor VIII (MFG-E8) and P2X4R proteins. Furthermore, pharmacological inhibition of αVß3/αVß5 integrin suppressed EV-induced cell migration and formation of lipid rafts in microglia. These results demonstrate that EVs promote microglial motility through P2X4R/MFG-E8-dependent mechanisms. Our findings provide novel insights into the molecular mechanisms through which EVs target human microglia that may be exploited for the development of new therapeutic strategies targeting disease-associated neuroinflammation.

Plasma of COVID-19 Patients Does Not Alter Electrical Resistance of Human Endothelial Blood-Brain Barrier In Vitro.

The pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 instigated the most serious global health crisis. Clinical presentation of COVID-19 frequently includes severe neurological and neuropsychiatric symptoms. However, it is presently unknown whether and to which extent pathological impairment of blood-brain barrier (BBB) contributes to the development of neuropathology during COVID-19 progression. In the present study, we used human induced pluripotent stem cells-derived brain endothelial cells (iBECs) to study the effects of blood plasma derived from COVID-19 patients on the BBB integrity in vitro. We also performed a comprehensive analysis of the cytokine and chemokine profiles in the plasma of COVID-19 patients, healthy and recovered individuals. We found significantly increased levels of interferon γ-induced protein 10 kDa, hepatocyte growth factor, and interleukin-18 in the plasma of COVID-19 patients. However, blood plasma from COVID-19 patients did not affect transendothelial electrical resistance in iBEC monolayers. Our results demonstrate that COVID-19-associated blood plasma inflammatory factors do not affect BBB paracellular pathway directly and suggest that pathological remodeling (if any) of BBB during COVID-19 may occur through indirect or yet unknown mechanisms.

Thymus Subset Alterations Accompanying Concomitant Tumor Immunity Mimics Phenotypic Patterns of Cytotoxic Drug Doxorubicin.

BACKGROUND/AIM: Concomitant immunity (CIM) is a phenomenon that elicits an antitumor response not sufficient enough to destroy the primary tumor but prevents a secondary implant from growing and spreading. This study aimed to develop a method of identification of serum tumoricidal factors released into circulation during CIM and to compare the CIM-related effect to the effect elicited by the cytotoxic drug doxorubicin. MATERIALS AND METHODS: SL2 tumor-bearing mice were studied at three time points - day 4, day 7, and day 11 following i.p. 5×105 cell implantation. Hematological effects and thymocyte immunophenotyping (CD4/CD8) data were compared to the effects induced by intravenous 10 mg/kg doxorubicin (DOX) administration to intact DBA 2 mice. The level of plasma colony stimulating factor-granulocyte macrophage (CSF-GM) was evaluated by ELISA. RESULTS: Identical thymus histopathology and an extent of double-positive CD4+CD8+ subset depletion was found in day 11 tumor-bearing mice (TBM-11) and in DOX-administered animals. TBM-11 exhibited a leukemoid reaction with an increase in monocyte and granulocyte counts. Conversely, DOX administration was followed by severe leukocytopenia at the 72-h time point. No increase in CSF-GM was observed in mice with or without a leukemoid reaction. CONCLUSION: The complexity of CIM can be examined by tracking alterations in the most fragile cortical CD8+CD4+ double positive population. Thymocyte apoptosis induced by DOX and TBM-11 might be associated with different mechanisms. TBM-11 did not exhibit severe myelotoxicity as DOX did. CIM-related serum factors can be assessed and screened via thymocyte subset analysis.

Extracellular Vesicles Suppress Basal and Lipopolysaccharide-Induced NFκB Activity in Human Periodontal Ligament Stem Cells.

Periodontitis is an infectious disease characterized by chronic inflammation and progressive destruction of periodontal tissues. Chronic inflammatory environment may affect immunomodulatory function of periodontal ligament stem cells (PDLSCs) and promote shift toward proinflammatory phenotype contributing to propagation of periodontitis. Therefore, suppression of inflammatory response in PDLSCs represents a novel therapeutic approach. Extracellular vesicles (EVs) have been shown to display anti-inflammatory and immunosuppressive actions in different tissues and could represent a potent therapeutic tools against chronic inflammation during periodontitis. In the present study, we investigated the effects of EVs on the basal and lipopolysaccharide (LPS)-induced activity of NFκB signaling pathway in PDLSCs. We also examined the impact of EVs on the osteogenic differentiation and expression of osteogenesis-related genes. EVs were purified by differential ultracentrifugation from PDLSCs grown on gelatin-coated alginate microcarriers in a bioreactor. NFκB reporter assays demonstrated that EVs permanently suppressed basal and LPS-induced activity of NFκB in PDLSCs. Combined treatment with EVs and anti-TLR4 antibody (Ab) resulted in attenuation of the inhibitory effect on the NFκB activity, suggesting a possible interference through a competition for TLR4 signaling pathway. EVs also increased phosphorylation of Akt and its downstream target GSK3ß (Ser 9) indicating that PI3K/Akt signaling pathway may act as suppressor of NFκB activity. LPS stimulated osteogenic mineralization of PDLSCs. Unexpectedly, anti-TLR4 blocking Ab per se significantly decreased osteogenic mineralization of PDLSCs. EVs did not affect osteogenic mineralization, but partially suppressed inhibitory effect of anti-TLR4 blocking Ab. Gene expression studies revealed significant effects of EVs on osteogenesis-related genes and possible interference with TLR4 signaling in PDLSCs. In conclusion, our study demonstrates that EVs suppress basal and LPS-induced activity of NFκB signaling pathway in PDLSCs and could potentially be used for targeting of chronic inflammation during periodontitis.

Extracellular vesicles can act as a potent immunomodulators of human microglial cells.

Functional impairments of microglia have been recently associated with several neurological conditions. Therefore, modulation of anti-inflammatory and phagocytic properties of microglial cells could represent a novel therapeutic approach. In the present study, we investigated the effects of extracellular vesicles (EVs) derived from stem cells from the dental pulp of human exfoliated deciduous teeth (SHEDs) on the inflammatory response and functional properties of immortalized human microglial cells. NFκB reporter assays demonstrated that EVs suppressed LPS-induced activation of NFκB signalling pathway in human microglial cells. The effect was similar to that obtained with anti-TLR4 blocking antibody. We also show that EVs differentially affected phagocytic activity of unpolarized (M0) and polarized (M1 and M2) microglial cells. EVs induced significant upregulation of phagocytic activity in M0 cells (by 39%), slight decrease in M1 cells, and moderate increase (by 21%) in M2 cells. The Seahorse XF Glycolysis Stress Test revealed that EVs induced an immediate and sustained increase of glycolytic activity in M0, M1, and M2 cells. Interestingly, EVs acted in an inverse dose-dependent manner. These findings indicate that EVs can induce glycolytic reprogramming of unpolarized and polarized human microglial cells. In conclusion, our pilot study demonstrates that EVs derived from SHEDs can act as a potent immunomodulators of human microglial cells. These findings could be potentially exploited for the development of new therapeutic strategies targeting neuroinflammatory microglia.

Intranasal Administration of Extracellular Vesicles Derived from Human Teeth Stem Cells Improves Motor Symptoms and Normalizes Tyrosine Hydroxylase Expression in the Substantia Nigra and Striatum of the 6-Hydroxydopamine-Treated Rats.

Parkinson's disease (PD) is the second most common neurodegenerative disorder affecting millions of people worldwide. At present, there is no effective cure for PD; treatments are symptomatic and do not halt progression of neurodegeneration. Extracellular vesicles (EVs) can cross the blood-brain barrier and represent promising alternative to the classical treatment strategies. In the present study, we examined therapeutic effects of intranasal administration of EVs derived from human exfoliated deciduous teeth stem cells (SHEDs) on unilateral 6-hydroxydopamine (6-OHDA) medial forebrain bundle (MFB) rat model of PD. CatWalk gait tests revealed that EVs effectively suppressed 6-OHDA-induced gait impairments. All tested gait parameters (stand, stride length, step cycle, and duty cycle) were significantly improved in EV-treated animals when compared with 6-OHDA-lesion group rats. Furthermore, EVs slowed down numbers of 6-OHDA-induced contralateral rotations in apomorphine test. Improvements in motor function correlated with normalization of tyrosine hydroxylase expression in the striatum and substantia nigra. In conclusion, we demonstrated, for the first time, the therapeutic efficacy of intranasal administration of EVs derived from SHEDs in a rat model of PD induced by 6-OHDA intra-MFB lesion. Our findings could be potentially exploited for the development of new treatment strategies against PD.