Improved Trimethylangelicin Analogs for Cystic Fibrosis: Design, Synthesis and Preliminary Screening.

A small library of new angelicin derivatives was designed and synthesized with the aim of bypassing the side effects of trimethylangelicin (TMA), a promising agent for the treatment of cystic fibrosis. To prevent photoreactions with DNA, hindered substituents were inserted at the 4 and/or 6 positions. Unlike the parent TMA, none of the new derivatives exhibited significant cytotoxicity or mutagenic effects. Among the synthesized compounds, the 4-phenylderivative 12 and the 6-phenylderivative 25 exerted a promising F508del CFTR rescue ability. On these compounds, preliminary in vivo pharmacokinetic (PK) studies were carried out, evidencing a favorable PK profile per se or after incorporation into lipid formulations. Therefore, the selected compounds are good candidates for future extensive investigation to evaluate and develop novel CFTR correctors based on the angelicin structure.

New TMA (4,6,4'-Trimethyl angelicin) Analogues as Anti-Inflammatory Agents in the Treatment of Cystic Fibrosis Lung Disease.

A series of new-generation TMA (4,6,4'-trimethyl angelicin) analogues was projected and synthetized in order to ameliorate anti-inflammatory activity, with reduced or absent toxicity. Since the NF-κB transcription factor (TF) plays a critical role in the expression of IL-8 (Interluekin 8), a typical marker of lung inflammation in Cystic Fibrosis (CF), the use of agents able to interfere with the NF-κB pathway represents an interesting therapeutic strategy. Through preliminary EMSA experiments, we identified several new TMA derivatives able to inhibit the NF-κB/DNA complex. The selected active molecules were then analyzed to evaluate the anti-inflammatory effect using both Pseudomonas aeruginosa (PAO1) infection and TNF-alpha stimulus on the CF IB3-1 cell line. It was demonstrated that mainly two TMA analogues, GY971a mesylate salt (6-p-minophenyl-4,4'-dimethyl-angelicin) and GY964 (4-phenyl-6,4'-dimethyl-angelicin), were able to decrease the IL-8 gene expression. At the same time, these molecules were found to have no pro-apoptotic, mutagenic and phototoxic effects, facilitating our decision to test the efficacy in vivo by using a mouse model of acute P. aeruginosa lung infection. The anti-inflammatory effect of GY971a was confirmed in vivo; this derivative was able to deeply decrease the total number of inflammatory cells, the neutrophil count and the cytokine/chemokine profile in the P. aeruginosa acute infection model, without evident toxicity. Considering all the obtained and reported in vitro and in vivo pre-clinical results, GY971a seems to have interesting anti-inflammatory effects, modulating the NF-κB pathway, as well as the starting lead compound TMA, but without side effects.

Sphingolipids and plasma membrane hydrolases in human primary bronchial cells during differentiation and their altered patterns in cystic fibrosis.

Human primary bronchial epithelial cells differentiated in vitro represent a valuable tool to study lung diseases such as cystic fibrosis (CF), an inherited disorder caused by mutations in the gene coding for the Cystic Fibrosis Transmembrane Conductance Regulator. In CF, sphingolipids, a ubiquitous class of bioactive lipids mainly associated with the outer layer of the plasma membrane, seem to play a crucial role in the establishment of the severe lung complications. Nevertheless, no information on the involvement of sphingolipids and their metabolism in the differentiation of primary bronchial epithelial cells are available so far. Here we show that ceramide and globotriaosylceramide increased during cell differentiation, whereas glucosylceramide and gangliosides content decreased. In addition, we found that apical plasma membrane of differentiated bronchial cells is characterized by a higher content of sphingolipids in comparison to the other cell membranes and that activity of sphingolipids catabolic enzymes associated with this membrane results altered with respect to the total cell activities. In particular, the apical membrane of CF cells was characterized by high levels of ceramide and glucosylceramide, known to have proinflammatory activity. On this basis, our data further support the role of sphingolipids in the onset of CF lung pathology.

Synthesis and Therapeutic Applications of Iminosugars in Cystic Fibrosis.

Iminosugars are sugar analogues endowed with a high pharmacological potential. The wide range of biological activities exhibited by these glycomimetics associated with their excellent drug profile make them attractive therapeutic candidates for several medical interventions. The ability of iminosugars to act as inhibitors or enhancers of carbohydrate-processing enzymes suggests their potential use as therapeutics for the treatment of cystic fibrosis (CF). Herein we review the most relevant advances in the field, paying attention to both the chemical synthesis of the iminosugars and their biological evaluations, resulting from in vitro and in vivo assays. Starting from the example of the marketed drug NBDNJ (N-butyl deoxynojirimycin), a variety of iminosugars have exhibited the capacity to rescue the trafficking of F508del-CFTR (deletion of F508 residue in the CF transmembrane conductance regulator), either alone or in combination with other correctors. Interesting results have also been obtained when iminosugars were considered as anti-inflammatory agents in CF lung disease. The data herein reported demonstrate that iminosugars hold considerable potential to be applied for both therapeutic purposes.

GM1 as Adjuvant of Innovative Therapies for Cystic Fibrosis Disease.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein is expressed at the apical plasma membrane (PM) of different epithelial cells. The most common mutation responsible for the onset of cystic fibrosis (CF), F508del, inhibits the biosynthesis and transport of the protein at PM, and also presents gating and stability defects of the membrane anion channel upon its rescue by the use of correctors and potentiators. This prompted a multiple drug strategy for F508delCFTR aimed simultaneously at its rescue, functional potentiation and PM stabilization. Since ganglioside GM1 is involved in the functional stabilization of transmembrane proteins, we investigated its role as an adjuvant to increase the effectiveness of CFTR modulators. According to our results, we found that GM1 resides in the same PM microenvironment as CFTR. In CF cells, the expression of the mutated channel is accompanied by a decrease in the PM GM1 content. Interestingly, by the exogenous administration of GM1, it becomes a component of the PM, reducing the destabilizing effect of the potentiator VX-770 on rescued CFTR protein expression/function and improving its stabilization. This evidence could represent a starting point for developing innovative therapeutic strategies based on the co-administration of GM1, correctors and potentiators, with the aim of improving F508del CFTR function.

PLCB3 Loss of Function Reduces Pseudomonas aeruginosa-Dependent IL-8 Release in Cystic Fibrosis.

The lungs of patients with cystic fibrosis (CF) are characterized by an exaggerated inflammation driven by secretion of IL-8 from bronchial epithelial cells and worsened by Pseudomonas aeruginosa infection. To identify novel antiinflammatory molecular targets, we previously performed a genetic study of 135 genes of the immune response, which identified the c.2534C>T (p.S845L) variant of phospholipase C-ß3 (PLCB3) as being significantly associated with mild progression of pulmonary disease. Silencing PLCB3 revealed that it potentiates the Toll-like receptor's inflammatory signaling cascade originating from CF bronchial epithelial cells. In the present study, we investigated the role of the PLCB3-S845L variant together with two synthetic mutants paradigmatic of impaired catalytic activity or lacking functional activation in CF bronchial epithelial cells. In experiments in which cells were exposed to P. aeruginosa, the supernatant of mucopurulent material from the airways of patients with CF or different agonists revealed that PLCB3-S845L has defects of 1) agonist-induced Ca2+ release from endoplasmic reticulum and rise of Ca2+ concentration, 2) activation of conventional protein kinase C isoform ß, and 3) induction of IL-8 release. These results, besides identifying S845L as a loss-of-function variant, strengthen the importance of targeting PLCB3 to mitigate the CF inflammatory response in bronchial epithelial cells without blunting the immune response.

A microRNA signature from serum exosomes of patients with glioma as complementary diagnostic biomarker.

Malignant gliomas, the most frequent primary brain tumors, are characterized by a dismal prognosis. Reliable biomarkers complementary to neuroradiology in the differential diagnosis of gliomas and monitoring for post-surgical progression are unmet needs. Altered expression of several microRNAs in tumour tissues from patients with gliomas compared to normal brain tissue have been described, thus supporting the rationale of using microRNA-based biomarkers. Although different circulating microRNAs were proposed in association with gliomas, they have not been introduced into clinical practice so far. Blood samples were collected from patients with high and low grade gliomas, both before and after surgical resection, and the expression of miR-21, miR-222 and miR-124-3p was measured in exosomes isolated from serum. The expression levels of miR-21, miR-222 and miR-124-3p in serum exosomes of patients with high grade gliomas were significantly higher than those of low grade gliomas and healthy controls and were sharply decreased in samples obtained after surgery. The analysis of miR-21, miR-222 and miR-124-3p in serum exosomes of patients affected by gliomas can provide a minimally invasive and innovative tool to help the differential diagnosis of gliomas at their onset in the brain and predict glioma grading and non glial metastases before surgery.

Differential Effects of Angelicin Analogues on NF-κB Activity and IL-8 Gene Expression in Cystic Fibrosis IB3-1 Cells.

The angelicin analogue 4,6,4'-trimethylangelicin (TMA) was recently reported as a strong inhibitor of nuclear factor-κB (NF-κB) activity and of the expression of the interleukin-8 (IL-8) gene in bronchial epithelial cells in which the inflammatory response has been challenged with P. aeruginosa, the most common bacterium found in the airways of patients affected by cystic fibrosis (CF). These findings encouraged us to analyze new synthetic analogues of TMA in order to evaluate their biological activities on human bronchial epithelial CF IB3-1 cells and to find more potent anti-NF-κB agents exhibiting only minor antiproliferative effects. Analogues able to inhibit NF-κB/DNA interaction at lower concentration than TMA were found and selected to investigate their biological activity on IB3-1 cells induced with TNF-α. In this biological system, NF-κB-mediated IL-8 gene expression was investigated. Some analogues showed similar activity to the lead compound TMA. Other analogues displayed higher activities; in particular, the most interesting compounds showing relevant anti-inflammatory effects were found to cause 56-83% reduction of IL-8 mRNA expression at low concentrations (1-10 µM), without changes in cell proliferation pattern, demonstrating their potential interest for a possible development of anti-inflammatory therapy of cystic fibrosis.

Evidence for the Involvement of Lipid Rafts and Plasma Membrane Sphingolipid Hydrolases in Pseudomonas aeruginosa Infection of Cystic Fibrosis Bronchial Epithelial Cells.

Cystic fibrosis (CF) is the most common autosomal genetic recessive disease caused by mutations of gene encoding for the cystic fibrosis transmembrane conductance regulator. Patients with CF display a wide spectrum of symptoms, the most severe being chronic lung infection and inflammation, which lead to onset of cystic fibrosis lung disease. Several studies indicate that sphingolipids play a regulatory role in airway inflammation. The inhibition and downregulation of GBA2, the enzyme catabolizing glucosylceramide to ceramide, are associated with a significant reduction of IL-8 production in CF bronchial epithelial cells. Herein, we demonstrate that GBA2 plays a role in the proinflammatory state characterizing CF cells. We also report for the first time that Pseudomonas aeruginosa infection causes a recruitment of plasma membrane-associated glycosphingolipid hydrolases into lipid rafts of CuFi-1-infected cells. This reorganization of cell membrane may be responsible for activation of a signaling cascade, culminating in aberrant inflammatory response in CF bronchial epithelial cells upon bacterial infection. Taken together, the presented data further support the role of sphingolipids and their metabolic enzymes in controlling the inflammatory response in CF.

A Peptide Nucleic Acid against MicroRNA miR-145-5p Enhances the Expression of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Calu-3 Cells.

Peptide nucleic acids (PNAs) are very useful tools for gene regulation at different levels, but in particular in the last years their use for targeting microRNA (anti-miR PNAs) has provided impressive advancements. In this respect, microRNAs related to the repression of cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is defective in cystic fibrosis, are of great importance in the development of new type of treatments. In this paper we propose the use of an anti-miR PNA for targeting miR-145, a microRNA reported to suppress CFTR expression. Octaarginine-anti-miR PNA conjugates were delivered to Calu-3 cells, exerting sequence dependent targeting of miR-145-5p. This allowed to enhance expression of the miR-145 regulated CFTR gene, analyzed at mRNA (RT-qPCR, Reverse Transcription quantitative Polymerase Chain Reaction) and CFTR protein (Western blotting) level.

Transient Receptor Potential Ankyrin 1 Channels Modulate Inflammatory Response in Respiratory Cells from Patients with Cystic Fibrosis.

Pseudomonas aeruginosa colonization, prominent inflammation with massive expression of the neutrophil chemokine IL-8, and luminal infiltrates of neutrophils are hallmarks of chronic lung disease in patients with cystic fibrosis (CF). The nociceptive transient receptor potential ankyrin (TRPA) 1 calcium channels have been recently found to be involved in nonneurogenic inflammation. Here, we investigate the role of TRPA1 in CF respiratory inflammatory models in vitro. Expression of TRPA1 was evaluated in CF lung tissue sections and cells by immunohistochemistry and immunofluorescence. Epithelial cell lines (A549, IB3-1, CuFi-1, CFBE41o-) and primary cells from patients with CF were used to: (1) check TRPA1 function modulation, by Fura-2 calcium imaging; (2) down-modulate TRPA1 function and expression, by pharmacological inhibitors (HC-030031 and A-967079) and small interfering RNA silencing; and (3) assess the effect of TRPA1 down-modulation on expression and release of cytokines upon exposure to proinflammatory challenges, by quantitative RT-PCR and 27-protein Bioplex assay. TRPA1 channels are expressed in the CF pseudostratified columnar epithelium facing the bronchial lumina exposed to bacteria, where IL-8 is coexpressed. Inhibition of TRPA1 expression results in a relevant reduction of release of several cytokines, including IL-8 and the proinflammatory cytokines IL-1ß and TNF-α, in CF primary bronchial epithelial cells exposed to P. aeruginosa and to the supernatant of mucopurulent material derived from the chronically infected airways of patients with CF. In conclusion, TRPA1 channels are involved in regulating the extent of airway inflammation driven by CF bronchial epithelial cells.

Expression of microRNA-93 and Interleukin-8 during Pseudomonas aeruginosa-mediated induction of proinflammatory responses.

In this study we analyzed the microRNA profile of cystic fibrosis (CF) bronchial epithelial IB3-1 cells infected with Pseudomonas aeruginosa by microarray and quantitative RT-PCR, demonstrating that microRNA 93 (miR-93), which is highly expressed in basal conditions, decreases during infection in parallel with increased expression of the IL-8 gene. The down-regulation of miR-93 after P. aeruginosa infection was confirmed in other bronchial cell lines derived from subjects with and without CF, namely CuFi-1 and NuLi-1 cells. Sequence analysis shows that the 3'-UTR region of IL-8 mRNA is a potential target of miR-93 and that the consensus sequence is highly conserved throughout molecular evolution. The possible involvement of miR-93 in IL-8 gene regulation was validated using three luciferase vectors, including one carrying the complete 3'-UTR region of the IL-8 mRNA and one carrying the same region with a mutated miR-93 site. Up-modulation of IL-8 after P. aeruginosa infection was counteracted in IB3-1, CuFi-1, and NuLi-1 cells by pre-miR-93 transfection. In addition, IL-8 was up-regulated in uninfected cells treated with antagomiR-93. Our results support the concept of a possible link between microRNA expression and IL-8 induction in bronchial epithelial cells infected with P. aeruginosa. Specifically, the data presented here indicate that, in addition to NF-κB-dependent up-regulation of IL-8 gene transcription, IL-8 protein expression is posttranscriptionally regulated by interactions of the IL-8 mRNA with the inhibitory miR-93.

Antibacterial and anti-inflammatory activity of a temporin B peptide analogue on an in vitro model of cystic fibrosis.

Natural peptides with antimicrobial properties are deeply investigated as tools to fight bacteria resistant to common antibiotics. Small peptides, as those belonging to the temporin family, are very attractive because their activity can easily be tuned after small modification to their primary sequence. Structure-activity studies previously reported by us allowed the identification of one peptide, analogue of temporin B, TB_KKG6A, showing, unlike temporin B, antimicrobial activity against both Gram-positive and Gram-negative bacteria. In this paper, we investigated the antimicrobial and anti-inflammatory activity of the peptide TB_KKG6A against Pseudomonas aeruginosa. Interestingly, we found that the peptide exhibits antimicrobial activity at low concentrations, being able to downregulate the pro-inflammatory chemokines and cytokines interleukin (IL)-8, IL-1ß, IL-6 and tumor necrosis factor-α produced downstream infected human bronchial epithelial cells. Experiments were carried out also with temporin B, which was found to show pro-inflammatory activity. Details on the interaction between TB_KKG6A and the P. aeruginosa LPS were obtained by circular dichroism and fluorescence studies.

Assessing the Potential of N-Butyl-l-deoxynojirimycin (l-NBDNJ) in Models of Cystic Fibrosis as a Promising Antibacterial Agent.

Over the past few years, l-iminosugars have revealed attractive pharmacological properties for managing rare diseases including Cystic Fibrosis (CF). The iminosugar N-butyl-l-deoxynojirimycin (l-NBDNJ, ent-1), prepared by a carbohydrate-based route, was herein evaluated for its anti-inflammatory and anti-infective potential in models of CF lung disease infection. A significant decrease in the bacterial load in the airways was observed in the murine model of Pseudomonas aeruginosa chronic infection in the presence of l-NBDNJ, also accompanied by a modest reduction of inflammatory cells. Mechanistic insights into the observed activity revealed that l-NBDNJ interferes with the expression of proteins regulating cytoskeleton assembly and organization of the host cell, downregulates the main virulence factors of P. aeruginosa involved in the host response, and affects pathogen adhesion to human cells. These findings along with the observation of the absence of an in vitro bacteriostatic/bactericidal action of l-NBDNJ suggest the potential use of this glycomimetic as an antivirulence agent in the management of CF lung disease.

Oxidative stress and antioxidant therapy in cystic fibrosis.

Cystic fibrosis is a lethal autosomal recessive condition caused by a defect of the transmembrane conductance regulator gene that has a key role in cell homeostasis. A dysfunctional cystic fibrosis transmembrane conductance regulator impairs the efflux of cell anions such as chloride and bicarbonate, and also that of other solutes such as reduced glutathione. This defect produces an increased viscosity of secretions together with other metabolic defects of epithelia that ultimately promote the obstruction and fibrosis of organs. Recurrent pulmonary infections and respiratory dysfunction are main clinical consequences of these pathogenetic events, followed by pancreatic and liver insufficiency, diabetes, protein-energy malnutrition, etc. This complex comorbidity is associated with the extensive injury of different biomolecular targets by reactive oxygen species, which is the biochemical hallmark of oxidative stress. These biological lesions are particularly pronounced in the lung, in which the extent of oxidative markers parallels that of inflammatory markers between chronic events and acute exacerbations along the progression of the disease. Herein, an abnormal flux of reactive oxygen species is present by the sustained activation of neutrophils and other cystic fibrosis-derived defects in the homeostatic processes of pulmonary epithelia and lining fluids. A sub-optimal antioxidant protection is believed to represent a main contributor to oxidative stress and to the poor control of immuno-inflammatory pathways in these patients. Observed defects include an impaired reduced glutathione metabolism and lowered intake and absorption of fat-soluble antioxidants (vitamin E, carotenoids, coenzyme Q-10, some polyunsaturated fatty acids, etc.) and oligoelements (such as Se, Cu and Zn) that are involved in reactive oxygen species detoxification by means of enzymatic defenses. Oral supplements and aerosolized formulations of thiols have been used in the antioxidant therapy of this inherited disease with the main aim of reducing the extent of oxidative lesions and the rate of lung deterioration. Despite positive effects on laboratory end points, poor evidence was obtained on the side of clinical outcome so far. These aspects examined in this critical review of the literature clearly suggest that further and more rigorous trials are needed together with new generations of pharmacological tools to a more effective antioxidant and anti-inflammatory therapy of cystic fibrosis patients. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.

ß-sitosterol ameliorates inflammation and Pseudomonas aeruginosa lung infection in a mouse model.

We previously demonstrated that ß-sitosterol (BSS) inhibits the expression of the chemokine IL-8 in CF bronchial epithelial cells exposed to P. aeruginosa. In the mouse model of lung chronic infection, herein shown, BSS significantly reduced leukocyte recruitment in the bronchoalveolar lavage fluid and decreased bacteria recovered in the airways. Treatment with BSS decreased the expression of key cytokines involved in immune response, mainly neutrophil chemotaxis, in the lung homogenate. This anti-inflammatory activity is accompanied by a beneficial protecting activity against infection and improvement of health status. Our data suggest that BSS has the potential to become a new drug to target the excessive neutrophil recruitment in lungs chronically infected by P. aeruginosa and encourage future investigations on mechanism of protection driven by BSS.

miRNAs in Serum Exosomes for Differential Diagnosis of Brain Metastases.

Circulating miRNAs are increasingly studied and proposed as tumor markers with the aim of investigating their role in monitoring the response to therapy as well as the natural evolution of primary or secondary brain tumors. This study aimed to evaluate the modulation of the expression of three miRNAs, miR-21, miR-222 and miR-124-3p, in the serum exosomes of patients with high-grade gliomas (HGGs) and brain metastases (BMs) to verify their usefulness in the differential diagnosis of brain masses; then, it focused on their variations following the surgical and/or radiosurgical treatment of the BMs. A total of 105 patients with BMs from primary lung or breast cancer, or melanoma underwent neurosurgery or radiosurgery treatment, and 91 patients with HGGs were enrolled, along with 30 healthy controls. A significant increase in miR-21 expression in serum exosomes was observed in both HGGs and BMs compared with healthy controls; on the other hand, miR-124-3p was significantly decreased in BMs, and it was increased in HGGs. After the surgical or radiosurgical treatment of patients with BMs, a significant reduction in miR-21 was noted with both types of treatments. This study identified a signature of exosomal miRNAs that could be useful as a noninvasive complementary analysis both in the differential diagnosis of BMs from glial tumors and in providing information on tumor evolution over time.

Modulators of sphingolipid metabolism reduce lung inflammation.

The investigation of novel targets for the treatment of cystic fibrosis (CF) lung inflammation is a major priority, considering that no effective therapy is available for this purpose. Consistent with the evidence that the sphingolipid (SL) ceramide regulates airway inflammation and infection in mice and patients with CF, SLs were identified as targets for treating pulmonary disorders, including CF. Because miglustat, an inhibitor of the synthesis of glycosphingolipids, reduces the Pseudomonas aeruginosa-dependent transcription of the IL-8 gene in bronchial cells, we examined the effects of miglustat and amitriptyline, another drug affecting ceramide metabolism, on the expression of 92 genes implicated in host immune defense. Infection with the P. aeruginosa strain PAO1 up-modulated the expression of 14 (27%) genes in IB3-1 cells and 15 (29%) genes in CF primary respiratory epithelia grown at an air-liquid interface, including chemokines (IL-8, growth-regulated Gro-α/ß/γ proteins, and granulocyte chemotactic peptide-2 [GCP-2]), proinflammatory cytokines (IL-1α/ß, IL-6, and TNF-α), and the intercellular adhesion molecule-1, nuclear factor kB1, toll like receptor 2, and human defensin B4 genes, confirming that bronchial epithelium is an important source of inflammatory mediators. Both miglustat and amitriptyline reduced the immune response, an effect that paralleled a decrease in the P. aeruginosa-induced accumulation of ceramide. Miglustat (100 mg/kg), given to C57BL/6 mice once daily for a period of 3 consecutive days before lipopolysaccharide (LPS) challenge, strongly reduced the number of neutrophils recruited in the airways and the expression of the keratinocyte-derived chemokine in lung extracts. Collectively, these results indicate that targeting the metabolism of SLs can down-modulate the recruitment of neutrophils into the lung.

Serum Exosomal microRNA-21, 222 and 124-3p as Noninvasive Predictive Biomarkers in Newly Diagnosed High-Grade Gliomas: A Prospective Study.

Background: High-grade gliomas (HGG) are malignant brain tumors associated with frequent recurrent disease. Clinical management of HGG patients is currently devoid of blood biomarkers for early diagnosis, monitoring therapeutic effects and predicting recurrence. Different circulating miRNAs, both free and associated with exosomes, are described in patients with HGG. We previously identified miR-21, miR-222 and miR-124-3p purified from serum exosomes as molecular signature to help pre-operative clinical diagnosis and grading of gliomas. The aim of the present study was to verify this signature as a tool to assess the effect of treatment and for the early identification of progression in newly diagnosed HGG patients. Material and Methods: Major inclusion criteria were newly diagnosed, histologically confirmed HGG patients, no prior chemotherapy, ECOG PS 0-2 and patients scheduled for radiochemotherapy with temozolomide as first-line treatment after surgery. RANO criteria were used for response assessment. Serum was collected at baseline and subsequently at each neuroradiological assessment. mir-21, -222 and -124-3p expression in serum exosomes was measured in all samples. Results: A total number of 57 patients were enrolled; 41 were male, 52 with glioblastoma and 5 with anaplastic astrocytoma; 18 received radical surgery. HGG patients with higher exosomal miRNA expression displayed a statistically significant lower progression-free survival and overall survival. Increased expression of miR-21, -222 and -124-3p during post-operative follow-up was associated with HGG progression. Conclusions: These data indicate that miR-21, -222 and -124-3p in serum exosomes may be useful molecular biomarkers for complementing clinical evaluation of early tumor progression during post-surgical therapy in patients with HGG.