Ruolo dei fibroblasti cardiaci nella cardiotossicit\ue0 da doxorubicina

Le terapie antitumorali sono note cause di complicazioni cardiache irreversibili nei pazienti trattati. Numerosi sono gli studi che hanno definito i meccanismi con cui i trattamenti chemioterapici portano a danni cardiaci. In modo intrinseco, le antracicline influenzano l\u2019integrit\ue0 dei cardiomiociti, provocando in combinazione stress ossidativo, disfunzione mitocondriale, danni nella struttura del DNA ed alterazioni nel metabolismo energetico. Negli ultimi anni sono state numerose le intuizioni circa i meccanismi alla base della patogenesi dei cardiomiociti, indotta dalla Doxorubicina (DOX): un antibiotico antraciclinico, ad azione antineoplastica, che possiede un ampio spettro antitumorale. Ben poco, invece, \ue8 stato scoperto sull\u2019effetto di questi farmaci sui fibroblasti cardiaci: l\u2019attivazione di queste cellule e la fibrosi miocardica, che ne deriva, sono gli aspetti che caratterizzano la cardiomiopatia da DOX. L\u2019insieme di questi eventi, tuttavia, sono considerati secondari alla lesione dei cardiomiociti, e si ritiene che abbiano un esordio tardivo nel corso della cardiotossicit\ue0, e quindi un impatto biologico minore. La componente cardiomiocitica, in un cuore adulto, rappresenta circa il 25-35% della popolazione cellulare; la restante parte \ue8 occupata da cellule di natura non cardiomiocitica (endoteliali, leucociti, cellule muscolari lisce, periciti e fibroblasti): un sistema equilibrato e sinergico, che garantisce la stabilit\ue0 organica, solo se ogni componente \ue8 in grado di mantenere la sua omeostasi. Per tale ragione abbiamo ipotizzato che la DOX potesse esercitare effetti diretti e biologicamente rilevanti sui fibroblasti. Per verificare la nostra ipotesi, abbiamo, in un primo momento, studiato il comportamento dei fibroblasti cardiaci primari (HCF, Human Cardiac Fibroblast), isolati dalle appendici atriali destre, di pazienti sottoposti a chirurgia cardiaca elettiva, con circolazione extracorporea. Successivamente ci siamo avvalsi di colture primarie di cardiomiociti e fibroblasti cardiaci, ottenuti dai cuori di topi neonatali, per comprendere se i fibroblasti trattati con la DOX fossero in grado di influenzare le cellule contigue del miocardio. Per i nostri esperimenti, HCF, ottenuti per digestione enzimatica, sono stati trattati con DOX 0,3 \ub5M, una concentrazione subapoptotica; \ue8 stata studiato, in queste condizioni, lo stato di senescenza cellulare (SA \u3b2-Gal ed immunofluorescenza per p16INK4), lo stato di proliferazione (saggio citofluorimetrico per CFSE), l\u2019apoptosi (immunofluorescenza per caspasi 3 clivata), l\u2019attivazione fibroblastica (immunofluorescenza per \u3b1-SMA e colorazione con Picro-Sirius red) ed il coinvolgimento di p53 (western blot). Esperimenti di tipo funzionale sono stati condotti per meglio comprendere il coinvolgimento dei pathway di p53 e TGF-\u3b2 nella disfunzione cardiaca da DOX: le cellule sono state, per tanto, trattate con pifitrina-\u3b1 (PFT, inibitore di p53) e GW788388 (GW, inibitore del recettore di TGF-\u3b2). La DOX, nella concentrazione testata, induce la senescenza nei HCF trattati, i quali appaiono morfologicamente pi\uf9 estesi ed appiattiti, con aumentata espressione di SA \u3b2-Gal e p16INK4a, rispetto alle cellule di controllo, guidata da un aumento nell\u2019espressione di p53, e con un\u2019attivit\ue0 proliferativa contenuta. La DOX induce anche la deposizione di collagene e \u3b1-SMA nei miofibroblasti. I pretrattamenti con PFT e GW si sono rivelati essenziali per prevenire lo switch fenotipico dei HCF in miofibroblasti. Questi effetti sono altres\uec evidenti nel caso dell\u2019effetto autocrino di HCF, trattati con il mezzo di coltura condizionato di altri HCF (precedentemente trattati con DOX 0,3 \ub5M per 3 ore e mantenuti in coltura per 2 ore in condizioni serum free, per ottenere il mezzo di coltura condizionato). Per meglio comprendere la presenza di un cross-talk tra le cellule che compongono il cuore, cardiomiociti (nMCM, neonatal Mouse Cardiomyocyte) e fibroblasti (nMCF, neonatal Mouse Cardiac Fibroblast) murini neonatali sono stati ottenuti per digestione enzimatica, da cuori di topi di 1-2 giorni (C57BL/6), e cresciuti nel loro specifico terreno di crescita. nMCF sono stati trattati con DOX 0,5 \ub5M per tre ore e mantenuti in coltura per le due ore successive in terreno serum free; il mezzo condizionato (CM, Conditioned Medium), contenente il secretoma dei miofibroblasti \ue8 stato utilizzato per trattare i nMCM e studiarne l\u2019effetto paracrino. L\u2019analisi del CM di nMCF ci ha permesso di escludere la presenza di DOX o suoi metaboliti, ma il secretoma dei nMCF \ue8 sufficiente ad indurre la senescenza nei nMCM, che mostrano un\u2019incrementata attivit\ue0 SA \u3b2-Gal, senza intaccare l\u2019integrit\ue0 mitocondriale. I fibroblasti, diversamente da quanto noto fino ad oggi, sono profondamente danneggiati dalla DOX, ed influenzano in maniera paracrina i cardiomiociti contigui, provocando lesioni, per mezzo del loro secretoma, e non per effetto dei metaboliti della DOX. Ulteriori studi saranno necessari per caratterizzare i fattori secreti dai fibroblasti, implicati nella patogenesi della cardiotossicit\ue0.Cancer therapies are known cause of irreversible cardiac complications in treated patients. Many studies defined the mechanisms of heart damage in chemotherapy treatments. Intrinsically, anthracyclines affect the integrity of cardiomyocytes, causing in combination oxidative stress, mitochondrial dysfunction, damage to DNA structure and alterations in energy metabolism. In recent years, numerous insights underlined the mechanisms of cardiomyocytes pathogenesis, induced by Doxorubicin (DOX): an anthracyclinic antibiotic, with antineoplastic action, with large antitumor spectrum. However, very little has been discovered about the effect of these drugs on cardiac fibroblasts: the activation of these cells and the derived myocardial fibrosis are the aspects that characterize DOX cardiomyopathy. All of these events, however, are considered secondary to cardiomyocyte injury, with a late onset during cardiotoxicity, and therefore a minor biological impact. The cardiomyocytic component, in an adult heart, represents only 25-35% of the cell population; the other portion is occupied by non-cardiomyocytic cells (endothelial cells, leukocytes, smooth muscle cells, pericytes and fibroblasts): a balanced and synergic system, which guarantees organic stability, only if each component is able to maintain its homeostasis. For this reason, we hypothesized that DOX could exert a direct and biologically relevant effects on fibroblasts. To test our hypothesis, we first studied the behavior of primary cardiac fibroblasts (HCFs, Human Cardiac Fibroblasts), isolated from the right atrial appendages, of patients undergoing elective cardiac surgery, with extra-corporeal circulation. We then made use of primary cultures of cardiomyocytes and cardiac fibroblasts, obtained from the hearts of neonatal mice, to understand if the fibroblasts treated with DOX were able to influence the contiguous cells in the myocardium. For our experiments, HCFs, obtained by enzymatic digestion, were treated with DOX 0.3 \ub5M, a subapoptotic concentration; under these conditions, the state of cellular senescence (SA \u3b2-Gal and immunofluorescence for p16INK4), the state of proliferation (cytofluorimetric assay for CFSE), apoptosis (immunofluorescence for cleaved caspase 3), fibroblastic activation (immunofluorescence for \u3b1-SMA and staining with Picro-Sirius red) and the involvement of p53 (western blot) were studied. Functional experiments have been conducted to better understand the involvement of the p53 and TGF-\u3b2 pathways in cardiac DOX dysfunction: the cells were therefore treated with pifitrin-\u3b1 (PFT, p53 inhibitor) and GW788388 (GW , TGF-\u3b2 receptor inhibitor). The DOX, with tested concentration, induces senescence in the treated HCFs, which appear morphologically more extended and flattened, with increased expression of SA \u3b2-Gal and p16INK4a, compared to the control cells, guided by an increase in the expression of p53, and with a contained proliferative activity. DOX also induces the deposition of collagen and \u3b1-SMA in myofibroblasts. Pretreatments with PFT and GW have proven to be essential to prevent the phenotypic switch of HCFs in myofibroblasts. These effects are also evident in the case of the autocrine effect of HCFs, treated with the conditioned culture medium of other HCFs (previously treated with DOX 0.3 \ub5M for 3 hours and maintained in culture for 2 hours in serum free conditions, to obtain the conditioned culture medium). To better understand the presence of a cross-talk between the cells that compose the heart, murine neonatal cardiomyocytes (nMCMs, neonatal Mouse Cardiomyocytes) and fibroblasts (nMCFs, neonatal Mouse Cardiac Fibroblasts) were obtained by enzymatic digestion, from hearts of mice of 1-2 days (C57BL / 6), and grown in their specific growth medium. nMCFs were treated with DOX 0.5 \ub5M for three hours and kept in culture for the next two hours in serum free medium; the conditioned medium (CM, Conditioned Medium), containing the secretome of myofibroblasts has been used to treat the nMCMs and study its paracrine effect. The analysis of the nMCFs CM allowed us to exclude the presence of DOX or its metabolites, but the secretome of the nMCFs is sufficient to induce senescence in the nMCMs, which show an increased SA \u3b2-Gal activity, without affecting the integrity mitochondrial. The fibroblasts, unlike what is known to date, are deeply damaged by DOX, and and influence contiguous cardiomyocytes in paracrine manner, causing lesions, by means of their secretome, and not by effect of the metabolites of DOX. Further studies will be necessary to characterize the factors secreted by fibroblasts, implicated in the pathogenesis of cardiotoxicity

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

From Exosome Glycobiology to Exosome Glycotechnology, the Role of Natural Occurring Polysaccharides

Exosomes (EXOs) are nano-sized informative shuttles acting as endogenous mediators of cell-to-cell communication. Their innate ability to target specific cells and deliver functional cargo is recently claimed as a promising theranostic strategy. The glycan profile, actively involved in the EXO biogenesis, release, sorting and function, is highly cell type-specific and frequently altered in pathological conditions. Therefore, the modulation of EXO glyco-composition has recently been considered an attractive tool in the design of novel therapeutics. In addition to the available approaches involving conventional glyco-engineering, soft technology is becoming more and more attractive for better exploiting EXO glycan tasks and optimizing EXO delivery platforms. This review, first, explores the main functions of EXO glycans and associates the potential implications of the reported new findings across the nanomedicine applications. The state-of-the-art of the last decade concerning the role of natural polysaccharides—as targeting molecules and in 3D soft structure manufacture matrices—is then analysed and highlighted, as an advancing EXO biofunction toolkit. The promising results, integrating the biopolymers area to the EXO-based bio-nanofabrication and bio-nanotechnology field, lay the foundation for further investigation and offer a new perspective in drug delivery and personalized medicine progress

5-fluorouracil causes endothelial cell senescence: Potential protective role of glucagon-like peptide 1

Background and Purpose: 5-fluorouracil (5FU) and its prodrug, capecitabine, can damage endothelial cells, whilst endothelial integrity is preserved by glucagon-like peptide 1 (GLP-1). Here, we studied the effect of 5FU on endothelial senescence and whether GLP-1 antagonizes it. Experimental Approach: EA.hy926 cells were exposed to 5FU or sera from patients taking capecitabine, with or without pre-incubation with GLP-1. Senescence was identified by expression of senescence-associated \u3b2-galactosidase and p16INK4a and reduced cell proliferation. Soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular adhesion molecule-1 (sICAM-1) and CD146 (marker of endothelial injury) were measured by ELISA before and at completion of capecitabine chemotherapy. RT-PCR, western blotting, functional experiments with signalling inhibitors and ERK1/2 silencing were performed to characterize 5FU-induced phenotype and elucidate the pathways underlying 5FU and GLP-1 activity. Key Results: Both 5FU and sera from capecitabine-treated patients stimulated endothelial cell senescence. 5FU-elicited senescence occurred via activation of p38 and JNK, and was associated with decreased eNOS and SIRT-1 levels. Furthermore, 5FU up-regulated VCAM1 and TYMP (encodes enzyme activating capecitabine and 5FU), and sVCAM-1 and CD146 concentrations were higher after than before capecitabine chemotherapy. A non-significant trend for higher ICAM1 levels was also observed. GLP-1 counteracted 5FU-initiated senescence and reduced eNOS and SIRT-1 expression, this protection being mediated by GLP-1 receptor, ERK1/2 and, possibly, PKA and PI3K. Conclusions and Implications: 5FU causes endothelial cell senescence and dysfunction, which may contribute to its cardiovascular side effects. 5FU-triggered senescence was prevented by GLP-1, raising the possibility of using GLP-1 analogues and degradation inhibitors to treat 5FU and capecitabine vascular toxicity

The Human Fetal and Adult Stem Cell Secretome Can Exert Cardioprotective Paracrine Effects against Cardiotoxicity and Oxidative Stress from Cancer Treatment.

Cardiovascular side effects are major shortcomings of cancer treatments causing cardiotoxicity and late-onset cardiomyopathy. While doxorubicin (Dox) has been reported as an effective chemotherapy agent, unspecific impairment in cardiomyocyte mitochondria activity has been documented. We demonstrated that the human fetal amniotic fluid-stem cell (hAFS) secretome, namely the secreted paracrine factors within the hAFS-conditioned medium (hAFS-CM), exerts pro-survival effects on Dox-exposed cardiomyocytes. Here, we provide a detailed comparison of the cardioprotective potential of hAFS-CM over the secretome of mesenchymal stromal cells from adipose tissue (hMSC-CM). hAFS and hMSC were preconditioned under hypoxia to enrich their secretome. The cardioprotective effects of hAFS/hMSC-CM were evaluated on murine neonatal ventricular cardiomyocytes (mNVCM) and on their fibroblast counterpart (mNVFib), and their long-term paracrine effects were investigated in a mouse model of Dox-induced cardiomyopathy. Both secretomes significantly contributed to preserving mitochondrial metabolism within Dox-injured cardiac cells. hAFS-CM and hMSC-CM inhibited body weight loss, improved myocardial function, reduced lipid peroxidation and counteracted the impairment of mitochondrial complex I activity, oxygen consumption, and ATP synthesis induced by Dox. The hAFS and hMSC secretomes can be exploited for inhibiting cardiotoxic detrimental side effects of Dox during cancer therapy, thus ensuring cardioprotection via combinatorial paracrine therapy in association with standard oncological treatments