microvascular-macrovascular-endothelial-cell-isolation-purification

Microvascular and Macrovascular Endothelial Cell Isolation and Purification from Lung-Derived Samples

The availability of cells isolated from healthy and diseased tissues and organs represents a key element for personalized medicine approaches. Although ...

Lipoxin (LX) A4, a main stop signal of inflammation, exerts potent bioactions by activating a specific G protein-coupled receptor, termed formyl peptide receptor 2 and recently renamed ALX/FPR2. Knowledge of the regulatory mechanisms that drive ALX/FPR2 gene expression is key for the development of innovative anti-inflammatory pharmacology. Here, we examined chromatin patterns of the ALX/FPR2 gene. We report that in MDA-MB231 breast cancer cells, the ALX/FPR2 gene undergoes epigenetic silencing characterized by low acetylation at lysine 27 and trimethylation at lysine 4, associated with high methylation at lysine 27 of histone 3. This pattern, which is consistent with transcriptionally inaccessible chromatin leading to low ALX/FPR2 mRNA and protein expression, is reversed in polymorphonuclear leukocytes that express high ALX/FPR2 levels. Activation of p300 histone acetyltransferase and inhibition of DNA methyltransferase restored chromatin accessibility and significantly increased ALX/FPR2 mRNA transcription and protein levels in MDA-MB231 cells, as well as in pulmonary artery endothelial cells. In both cells types, changes in the histone acetylation/methylation status enhanced ALX/FPR2 signaling in response to LXA4. Collectively, these results uncover unappreciated epigenetic regulation of ALX/FPR2 expression that can be exploited for innovative approaches to inflammatory disorders.

Epigenetic regulation of the formyl peptide receptor 2 gene.

Endothelial cell (EC) dysfunction has been reported in cystic fibrosis (CF) patients. Thus, the availability of CF EC is paramount to uncover mechanisms of endothelial dysfunction in CF. Using collagenase digestion, we isolated cells from small fragments of pulmonary artery dissected from non-CF lobes or explanted CF lungs. These cells were a heterogeneous mixture, containing variable percentages of EC. To obtain virtually pure pulmonary artery endothelial cells (PAEC), we developed an easy, inexpensive, and reliable method, based on the differential adhesion time of pulmonary artery cells collected after collagenase digestion. With this method, we obtained up to 95% pure non-CF and CF-PAEC. Moreover, we also succeed at immortalizing both PAEC and CF-PAEC, which remained viable and with unchanged phenotype and proliferation rate over the 30th passage. These cells recapitulated cystic fibrosis transmembrane conductance regulator expression and functions of the parental cells. Thus, we isolated for the first time endothelial cells from CF patients, providing a valuable tool to define the emerging role of EC in CF lung and vascular disease.

Establishment and long-term culture of human cystic fibrosis endothelial cells.

The involvement of microRNA (miR) in cystic fibrosis (CF) pathobiology is rapidly emerging. We previously documented that miR-181b controls the expression of the ALX/FPR2 receptor, which is recognized by the endogenous proresolution ligand, lipoxin (LX)A. Here, we examined whether the miR-181b-ALX/FPR2 circuit was altered in CF. We examined human airways epithelial cells, normal (16HBE14o-), carrying the ΔF508 mutation (CFBE41o-) or corrected for this mutation (CFBE41o-/CEP-CFTR wt 6.2 kb), as well as monocyte-derived macrophages (MΦs) from CF patients. CFBE41o- cells exhibited higher miR-181b and reduced ALX/FPR2 levels compared to 16HBE14o- and CFBE41o-/CEP-CFTR wt 6.2 kb cells. An anti-mir-181b significantly enhanced ALX/FPR2 expression (+ 60%) as well as LXA-induced increase in transepithelial electric resistance (+ 25%) in CFBE41o- cells. MΦs from CF patients also displayed increased miR-181b (+ 100%) and lower ALX/FPR2 levels (- 20%) compared to healthy cells. An anti-mir-181b enhanced ALX/FPR2 expression (+ 40%) and normalized receptor-dependent LXA-induced phagocytosis of fluorescent-labeled zymosan particles as well as of Pseudomonas aeruginosa by CF-MΦs. These results provide the first evidence that miR-181b is overexpressed in CF cells, impairing some mechanisms of the ALX/FPR2-dependent pathway of inflammation resolution. Thus, targeting miR-181b may represent a strategy to enhance anti-inflammatory and anti-microbial defense mechanisms in CF.

microRNA-181b is increased in cystic fibrosis cells and impairs lipoxin A receptor-dependent mechanisms of inflammation resolution and antimicrobial defense.

Despite the high expectations associated with the recent introduction of CFTR modulators, airway inflammation still remains a relevant clinical issue in cystic fibrosis (CF). The classical anti-inflammatory drugs have shown very limited efficacy, when not being harmful, raising the question of whether alternative approaches should be undertaken. Thus, a better knowledge of the mechanisms underlying the aberrant inflammation observed in CF is pivotal to develop more efficacious pharmacology. In this respect, the observation that endogenous proresolving pathways are defective in CF and that proresolving mediators, physiologically generated during an acute inflammatory reaction, do not completely suppress inflammation, but promote resolution, tissue healing and microbial clearance, without compromising immune host defense mechanisms, opens interesting therapeutic scenarios for CF. In this mini-review, we present the current knowledge and perspectives of proresolving pharmacology in CF, focusing on the specialized proresolving lipid mediators and selected peptides.

The Resolution Approach to Cystic Fibrosis Inflammation.

The rapid repurposing of antivirals is particularly pressing during pandemics. However, rapid assays for assessing candidate drugs typically involve in vitro screens and cell lines that do not recapitulate human physiology at the tissue and organ levels. Here we show that a microfluidic bronchial-airway-on-a-chip lined by highly differentiated human bronchial-airway epithelium and pulmonary endothelium can model viral infection, strain-dependent virulence, cytokine production and the recruitment of circulating immune cells. In airway chips infected with influenza A, the co-administration of nafamostat with oseltamivir doubled the treatment-time window for oseltamivir. In chips infected with pseudotyped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), clinically relevant doses of the antimalarial drug amodiaquine inhibited infection but clinical doses of hydroxychloroquine and other antiviral drugs that inhibit the entry of pseudotyped SARS-CoV-2 in cell lines under static conditions did not. We also show that amodiaquine showed substantial prophylactic and therapeutic activities in hamsters challenged with native SARS-CoV-2. The human airway-on-a-chip may accelerate the identification of therapeutics and prophylactics with repurposing potential.

A human-airway-on-a-chip for the rapid identification of candidate antiviral therapeutics and prophylactics.

Neutrophilic inflammation is a key determinant of cystic fibrosis (CF) lung disease. Neutrophil-derived free DNA, released in the form of extracellular traps (NETs), significantly correlates with impaired lung function in patients with CF, underlying their pathogenetic role in CF lung disease. Thus, specific approaches to control NETosis of neutrophils migrated into the lungs may be clinically relevant in CF. We investigated the efficacy of phosphodiesterase (PDE) type-4 inhibitors, , on NET release by neutrophils from healthy volunteers and individuals with CF, and , on NET accumulation and lung inflammation in mice infected with . PDE4 blockade curbed endotoxin-induced NET production and preserved cellular integrity and apoptosis in neutrophils, from healthy subjects and patients with CF, challenged with endotoxin, . The pharmacological effects of PDE4 inhibitors were significantly more evident on CF neutrophils. In a mouse model of  chronic infection, aerosol treatment with roflumilast, a selective PDE4 inhibitor, gave a significant reduction in free DNA in the BALF. This was accompanied by reduced citrullination of histone H3 in neutrophils migrated into the airways. Roflumilast-treated mice showed a significant improvement in weight recovery. Our study provides the first evidence that PDE4 blockade controls NETosis  and , in CF-relevant models. Since selective PDE4 inhibitors have been recently approved for the treatment of COPD and psoriasis, our present results encourage clinical trials to test the efficacy of this class of drugs in CF.

Type-4 Phosphodiesterase (PDE4) Blockade Reduces NETosis in Cystic Fibrosis.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), which results in impaired airway mucociliary clearance, inflammation, infection, and respiratory insufficiency. The development of new therapeutics for CF are limited by the lack of reliable preclinical models that recapitulate the structural, immunological, and bioelectrical features of human CF lungs.

Modeling pulmonary cystic fibrosis in a human lung airway-on-a-chip.

Mechanical breathing motions have a fundamental function in lung development and disease, but little is known about how they contribute to host innate immunity. Here we use a human lung alveolus chip that experiences cyclic breathing-like deformations to investigate whether physical forces influence innate immune responses to viral infection. Influenza H3N2 infection of mechanically active chips induces a cascade of host responses including increased lung permeability, apoptosis, cell regeneration, cytokines production, and recruitment of circulating immune cells. Comparison with static chips reveals that breathing motions suppress viral replication by activating protective innate immune responses in epithelial and endothelial cells, which are mediated in part through activation of the mechanosensitive ion channel TRPV4 and signaling via receptor for advanced glycation end products (RAGE). RAGE inhibitors suppress cytokines induction, while TRPV4 inhibition attenuates both inflammation and viral burden, in infected chips with breathing motions. Therefore, TRPV4 and RAGE may serve as new targets for therapeutic intervention in patients infected with influenza and other potential pandemic viruses that cause life-threatening lung inflammation.

Mechanical control of innate immune responses against viral infection revealed in a human lung alveolus chip.

People with cystic fibrosis should be considered at increased risk of developing severe symptoms of COVID-19. Strikingly, a broad array of evidence shows reduced spread of SARS-CoV-2 in these subjects, suggesting a potential role for CFTR in the regulation of SARS-CoV-2 infection/replication. Here, we analyzed SARS-CoV-2 replication in wild-type and CFTR-modified human bronchial epithelial cell lines and primary cells to investigate SARS-CoV-2 infection in people with cystic fibrosis. Both immortalized and primary human bronchial epithelial cells expressing wt or F508del-CFTR along with CRISPR/Cas9 CFTR-ablated clones were infected with SARS-CoV-2 and samples were harvested before and from 24 to 72 h post-infection. CFTR function was also inhibited in wt-CFTR cells with the CFTR-specific inhibitor IOWH-032 and partially restored in F508del-CFTR cells with a combination of CFTR modulators (VX-661+VX-445). Viral load was evaluated by real-time RT-PCR in both supernatant and cell extracts, and ACE-2 expression was analyzed by both western blotting and flow cytometry. SARS-CoV-2 replication was reduced in CFTR-modified bronchial cells compared with wild-type cell lines. No major difference in ACE-2 expression was detected before infection between wild-type and CFTR-modified cells, while a higher expression in wild-type compared to CFTR-modified cells was detectable at 72 h post-infection. Furthermore, inhibition of CFTR channel function elicited significant inhibition of viral replication in cells with wt-CFTR, and correction of CFTR function in F508del-CFTR cells increased the release of SARS-CoV-2 viral particles. Our study provides evidence that CFTR expression/function is involved in the regulation of SARS-CoV-2 replication, thus providing novel insights into the role of CFTR in SARS-CoV-2 infection and the development of therapeutic strategies for COVID-19.

CFTR Modulation Reduces SARS-CoV-2 Infection in Human Bronchial Epithelial Cells.

The usefulness of live attenuated virus vaccines has been limited by suboptimal immunogenicity, safety concerns or cumbersome manufacturing processes and techniques. Here we describe the generation of a live attenuated influenza A virus vaccine using proteolysis-targeting chimeric (PROTAC) technology to degrade viral proteins via the endogenous ubiquitin-proteasome system of host cells. We engineered the genome of influenza A viruses in stable cell lines engineered for virus production to introduce a conditionally removable proteasome-targeting domain, generating fully infective PROTAC viruses that were live attenuated by the host protein degradation machinery upon infection. In mouse and ferret models, PROTAC viruses were highly attenuated and able to elicit robust and broad humoral, mucosal and cellular immunity against homologous and heterologous virus challenges. PROTAC-mediated attenuation of viruses may be broadly applicable for generating live attenuated vaccines.

Generation of a live attenuated influenza A vaccine by proteolysis targeting.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the coronavirus disease 2019 (COVID-19), has provoked more than six million deaths worldwide and continues to pose a major threat to global health. Enormous efforts have been made by researchers around the world to elucidate COVID-19 pathophysiology, design efficacious therapy and develop new vaccines to control the pandemic. To this end, experimental models are essential. While animal models and conventional cell cultures have been widely utilized during these research endeavors, they often do not adequately reflect the human responses to SARS-CoV-2 infection. Therefore, models that emulate with high fidelity the SARS-CoV-2 infection in human organs are needed for discovering new antiviral drugs and vaccines against COVID-19. Three-dimensional (3D) cell cultures, such as lung organoids and bioengineered organs-on-chips, are emerging as crucial tools for research on respiratory diseases. The lung airway, small airway and alveolus organ chips have been successfully used for studies on lung response to infection by various pathogens, including corona and influenza A viruses. In this review, we provide an overview of these new tools and their use in studies on COVID-19 pathogenesis and drug testing. We also discuss the limitations of the existing models and indicate some improvements for their use in research against COVID-19 as well as future emerging epidemics.

3D Lung Tissue Models for Studies on SARS-CoV-2 Pathophysiology and Therapeutics.

A dominance of non-iners Lactobacillus species in the vaginal microbiome is optimal and strongly associated with gynecological and obstetric health, while the presence of diverse obligate or facultative anaerobic bacteria and a paucity in Lactobacillus species, similar to communities found in bacterial vaginosis (BV), is considered non-optimal and associated with adverse health outcomes. Various therapeutic strategies are being explored to modulate the composition of the vaginal microbiome; however, there is no human model that faithfully reproduces the vaginal epithelial microenvironment for preclinical validation of potential therapeutics or testing hypotheses about vaginal epithelium-microbiome interactions.

Article	Year
Epigenetic regulation of the formyl peptide receptor 2 gene. Biochimica et biophysica acta\| PubMed ID: 27424221	2016
Establishment and long-term culture of human cystic fibrosis endothelial cells. Laboratory investigation; a journal of technical methods and pathology\| PubMed ID: 28759010	2017
microRNA-181b is increased in cystic fibrosis cells and impairs lipoxin A receptor-dependent mechanisms of inflammation resolution and antimicrobial defense. Scientific reports\| PubMed ID: 29044225	2017
The Resolution Approach to Cystic Fibrosis Inflammation. Frontiers in pharmacology\| PubMed ID: 32848748	2020
A human-airway-on-a-chip for the rapid identification of candidate antiviral therapeutics and prophylactics. Nature biomedical engineering\| PubMed ID: 33941899	2021
Type-4 Phosphodiesterase (PDE4) Blockade Reduces NETosis in Cystic Fibrosis. Frontiers in pharmacology\| PubMed ID: 34566635	2021
Modeling pulmonary cystic fibrosis in a human lung airway-on-a-chip. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society\| PubMed ID: 34799298	2022
Mechanical control of innate immune responses against viral infection revealed in a human lung alveolus chip. Nature communications\| PubMed ID: 35396513	2022
CFTR Modulation Reduces SARS-CoV-2 Infection in Human Bronchial Epithelial Cells. Cells\| PubMed ID: 35456026	2022
Generation of a live attenuated influenza A vaccine by proteolysis targeting. Nature biotechnology\| PubMed ID: 35788567	2022
3D Lung Tissue Models for Studies on SARS-CoV-2 Pathophysiology and Therapeutics. International journal of molecular sciences\| PubMed ID: 36077471	2022
Vaginal microbiome-host interactions modeled in a human vagina-on-a-chip. Microbiome\| PubMed ID: 36434666	2022

Roberto Plebani

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