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 ...

“G. d’Annunzio” University of Chieti-Pescara

University “G. d’Annunzio” of Chieti-Pescara

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 epithelial mesenchymal transition (EMT) program is defined as a cellular transition from an epithelial to a mesenchymal state. This process occurs to provide the cell with new phenotypic assets and new skills to perform complex processes. EMT is regulated at multilayer levels, including transcriptional control of gene expression, regulation of RNA splicing, and translational/post-translational control. Although transcriptional regulation by EMT-inducing transcription factors (EMT-TFs), including Zeb, Snail and Slug members, is generally considered the master step in this process, emerging data indicate that all these regulatory networks may have a role in the control of EMT. There is a sort of parallelism between the biological and still unrevealed EMT complexity and the cosmological hypothesis that sustains the universe may exist as a multiverse. The presence of different EMT transition states together with the occurrence of multiple layers of regulation support the idea that EMT is just one on many out there. Is the activation of a single layer of regulation sufficient to initiate the whole EMT program? Can we postulate the activation of different EMT "dimensions"? If we think about these layers as multiple separate "universes", various scenarios can be revealed.

The multiverse nature of epithelial to mesenchymal transition.

With the advent of novel molecular platforms for high-throughput/next-generation sequencing, the communities of commensal and pathogenic microorganisms that inhabit the human body have been defined in depth. In the last decade, the role of microbiota-host interactions in driving human cancer plasticity and malignant progression has been well documented. Germ-free preclinical models provided an invaluable tool to demonstrate that the human microbiota can confer susceptibility to various types of cancer and can also modulate the host response to therapeutic treatments. Of interest, besides the detrimental effects of dysbiosis on cancer etiopathogenesis, specific microorganisms have been shown to exert protective activities against cancer growth. This has strong clinical implications, as restoration of the physiologic microbiota is being rapidly implemented as a novel anticancer therapeutic strategy. Here, we reviewed past and recent literature depicting the role of microbiota-host interactions in modulating key molecular mechanisms that drive human cancer plasticity and lead to malignant progression. We analyzed microbiota-host interactions occurring in the gut as well as in other anatomic sites, such as oral and nasal cavities, lungs, breast, esophagus, stomach, reproductive tract, and skin. We revealed a common ground of biological alterations and pathways modulated by a dysbiotic microbiota and potentially involved in the control of cancer progression. The molecular mechanisms most frequently affected by the pathogenic microorganisms to induce malignant progression involve epithelial-mesenchymal transition- (EMT-) dependent barrier alterations and tumor-promoting inflammation. This evidence may pave the way to better stratify high-risk cancer patients based on unique microenvironmental/microbial signatures and to develop novel, personalized, biological therapies.

The Cancer Microbiota: EMT and Inflammation as Shared Molecular Mechanisms Associated with Plasticity and Progression.

Molecular changes associated with the initiation of the epithelial to mesenchymal transition (EMT) program involve alterations of large proteome-based networks. The role of protein products mapping to non-coding genomic regions is still unexplored.

A Hidden Human Proteome Signature Characterizes the Epithelial Mesenchymal Transition Program.

Extracellular vesicles (EVs) actively participate in inter-cellular crosstalk and have progressively emerged as key players of organized communities of cells within multicellular organisms in health and disease. For these reasons, EVs are attracting the attention of many investigators across different biomedical fields. In this scenario, the possibility to study specific placental-derived EVs in the maternal peripheral blood may open novel perspectives in the development of new early biomarkers for major obstetric pathological conditions. Here we reviewed the involvement of EVs in feto-maternal crosstalk mechanisms, both in physiological and pathological conditions (preeclampsia, fetal growth restriction, preterm labor, gestational diabetes mellitus), also underlining the usefulness of EV characterization in maternal-fetal medicine.

Extracellular Vesicles in Feto-Maternal Crosstalk and Pregnancy Disorders.

We recently reported that activation of Trop-2 through its cleavage at R87-T88 by ADAM10 underlies Trop-2-driven progression of colon cancer. However, the mechanism of action and pathological impact of Trop-2 in metastatic diffusion remain unexplored. Through searches for molecular determinants of cancer metastasis, we identified TROP2 as unique in its up-regulation across independent colon cancer metastasis models. Overexpression of wild-type Trop-2 in KM12SM human colon cancer cells increased liver metastasis rates in vivo in immunosuppressed mice. Metastatic growth was further enhanced by a tail-less, activated ΔcytoTrop-2 mutant, indicating the Trop-2 tail as a pivotal inhibitory signaling element. In primary tumors and metastases, transcriptome analysis showed no down-regulation of CDH1 by transcription factors for epithelial-to-mesenchymal transition, thus suggesting that the pro-metastatic activity of Trop-2 is through alternative mechanisms. Trop-2 can tightly interact with ADAM10. Here, Trop-2 bound E-cadherin and stimulated ADAM10-mediated proteolytic cleavage of E-cadherin intracellular domain. This induced detachment of E-cadherin from β-actin, and loss of cell-cell adhesion, acquisition of invasive capability, and membrane-driven activation of β-catenin signaling, which were further enhanced by the ΔcytoTrop-2 mutant. This Trop-2/E-cadherin/β-catenin program led to anti-apoptotic signaling, increased cell migration, and enhanced cancer-cell survival. In patients with colon cancer, activation of this Trop-2-centered program led to significantly reduced relapse-free and overall survival, indicating a major impact on progression to metastatic disease. Recently, the anti-Trop-2 mAb Sacituzumab govitecan-hziy was shown to be active against metastatic breast cancer. Our findings define the key relevance of Trop-2 as a target in metastatic colon cancer.

Trop-2 induces ADAM10-mediated cleavage of E-cadherin and drives EMT-less metastasis in colon cancer.

COVID-19 pandemic has hit people's health, economy, and society worldwide. Great confidence in returning to normality has been placed in the vaccination campaign. The knowledge of individual immune profiles and the time required to achieve immunological protection is crucial to choose the best vaccination strategy. We compared anti-S1 antibody levels produced over time by BNT162b2 and AZD1222 vaccines and evaluated the induction of antigen-specific T-cells. A total of 2569 anti-SARS-CoV-2 IgG determination on dried blood spot samples were carried out, firstly in a cohort of 1181 individuals at random time-points, and subsequently, in an independent cohort of 88 vaccinated subjects, up to the seventeenth week from the first dose administration. Spike-specific T-cells were analysed in seronegative subjects between the two doses. AZD1222 induced lower anti-S1 IgG levels as compared to BNT162b2. Moreover, 40% of AZD1222 vaccinated subjects and 3% of BNT162b2 individuals resulted in seronegative during all the time-points, between the two doses. All these subjects developed antigen-specific T cells, already after the first dose. These results suggest that this test represents an excellent tool for a wide sero-surveillance. Both vaccines induce a favourable immune profile guaranteeing efficacy against severe adverse effects of SARS-CoV-2 infection, already after the first dose administration.

Picture of the Favourable Immune Profile Induced by Anti-SARS-CoV-2 Vaccination.

The efficacy of SARS-CoV-2 mRNA-based vaccines in preventing COVID-19 disease has been extensively demonstrated; however, it is of uttermost importance to acquire knowledge on the persistence of immune-protection both in terms of levels of neutralizing antibodies and specialized memory cells. This can provide important scientific basis for decisions on the need of additional vaccine doses and on when these should be administered thus resulting in an improvement in vaccination schedules. Here, we briefly report the changes in antibody levels and cellular immunity following BNT162b2 administration. We show an important fall in anti S1-Spike antibodies in BNT162b2 vaccinated subjects overtime, paralleled by a contextual consolidation of specific spike (S) T-cells, mainly of the CD8+ compartment. Contrariwise, CD4+ S-specific response shows a considerable interindividual variability. These data suggest that the well-known antibody drop in vaccinated subjects is replaced by memory cell consolidation that can protect from severe adverse effects of SARS-CoV-2 infection.

BNT162b2 mRNA Vaccination Leads to Long-Term Protection from COVID-19 Disease.

, a Gram-negative neutrophilic pathogen, is the cause of chronic gastritis, peptic ulcers, and gastric cancer in humans. Current therapeutic regimens suffer from an emerging bacterial resistance rate and poor patience compliance. To improve the discovery of compounds targeting bacterial alternative enzymes or essential pathways such as carbonic anhydrases (CAs), we assessed the anti- activity of thymol and carvacrol in terms of CA inhibition, isoform selectivity, growth impairment, biofilm production, and release of associated outer membrane vesicles-eDNA. The microbiological results were correlated by the evaluation in vitro of  CA inhibition, in silico analysis of the structural requirements to display such isoform selectivity, and the assessment of their limited toxicity against three probiotic species with respect to amoxicillin. Carvacrol and thymol could thus be considered as new lead compounds as alternative  CA inhibitors or to be used in association with current drugs for the management of  infection and limiting the spread of antibiotic resistance.

Pasquale Simeone

Center on Advanced Studies and Technology (CAST)

“G. d’Annunzio” University of Chieti-Pescara

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

Pasquale Simeone

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Center on Advanced Studies and Technology (CAST)

“G. d’Annunzio” University of Chieti-Pescara

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

Center on Advanced Studies and Technology (CAST)