Biology subjects

Caporali, A.

Publications and source records attributed to Caporali, A..

6 recordsLinked to original sources

bioRxiv2026

Development and characterisation of an optimised in vitro differentiation protocol for deriving hepatocyte-like cells from mouse embryonic stem cells

IntroductionReliable generation of hepatocyte-like cells (HLCs) from pluripotent stem cells remains limited by heterogeneity and incomplete maturation of the cells. Derivation of induced pluripotent- and embryonic stem cells into hepatocytes typically relies on complex, and costly reagent-intensive protocols, with inconsistent reporting of differentiation efficiencies and functional maturation criteria. Variability in protocol designs highlights the need for optimisation, particularly in mouse embryonic stem cells (mESCs) systems that can be more comparable with mouse models for underpinning translational and toxicological studies. Here, we developed and evaluated two cytokine-based strategies: an advanced hepatic-inducing cocktail (A-HIC) and a simplified hepatic-inducing cocktail (HIC), both designed to reduce complexity while increasing functional maturation. MethodsHepatic differentiation and maturation were assessed by morphology, immunofluorescence, flow cytometry, and qRT-PCR. Functional competence was evaluated via urea production, glutathione synthesis, indocyanine green handling, cytochrome P450 inducibility, and impedance-based cell layer integrity monitoring. ResultsMorphological, molecular and phenotypic analyses confirmed that both protocols supported hepatic lineage progression, generating heterogeneous populations of hepatoblast-like and more mature HLCs. Gene expression confirmed the loss of pluripotency, transient endoderm induction, and subsequent hepatic specification. Functionally, cells exhibited glycogen storage, inducible urea production, glutathione depletion, and active ICG uptake and clearance, with stable monolayer formation by day 21. A-HIC-derived HLCs demonstrated enhanced maturation, with higher ASGR1 expression and stronger Cyp1a1 induction. DiscussionThese findings suggest that both protocols generate functional HLCs; however, A-HIC yields a higher proportion of functionally mature cells with reduced variability. This approach enables a simple, cost-effective, and time-efficient generation of HLCs, supported by improved functional characterisation with potential applicability to more complex pluripotent systems, including human iPSC-based models for disease modelling and toxicology.

cell biology↗

bioRxiv2022

MC1R drives cutaneous repair by promoting angiogenesis and lymphangiogenesis

Cutaneous healing results in scarring with significant functional and psychological sequelae, while chronic non-healing wounds represent repair failure often with devastating consequences, including amputation and death. Due to a lack of effective therapies, novel interventions addressing scarring and chronic wounds are urgently needed. Here, we demonstrate that harnessing melanocortin 1 receptor with a selective agonist (MC1R-Ag) confers multifaceted benefits to wound repair. MC1R-Ag accelerates wound closure and re-epithelialization while improving wound bed perfusion and lymphatic drainage by promoting angiogenesis and lymphangiogenesis. Concomitant reductions in oxidative stress, inflammation and scarring were also observed. To evaluate the therapeutic potential of targeting MC1R in pathological healing, we established a novel murine model that recapitulates the hallmarks of human non-healing wounds. This model combines advanced age and locally elevated oxidative stress. Remarkably, topical application of MC1R-Ag restored repair, whereas disrupting MC1R signalling exacerbated the chronic wound phenotype. Our study highlights MC1R agonism as a promising therapeutic approach for scarring and non-healing wound pathologies, and our chronic wound model as a valuable tool for elucidating ulcer development mechanisms.

pathology↗

bioRxiv2022

EZH2 interacts with MEG3 to directly regulate integrin signaling and endothelial cell function

Enhancer of Zeste Homologue 2 (EZH2) modulates gene transcription during endothelial cell (EC) dysfunction, via interaction with non-coding RNAs (ncRNAs). Thus, EZH2 can act as a rheostat in deposition of histone H3K27 trimethylation (H3K27me3) to repress many genes. We profiled EZH2-RNA interactions using formaldehyde/UV assisted cross-linking ligation and sequencing of hybrids (FLASH-seq) in primary human ECs. Transcriptome-wide EZH2-associated ncRNAs and RNA-RNA interactome were obtained. This approach revealed EZH2 directly binding maternally expressed gene (MEG3) and MEG3:MEG3 hybrid structures. By chromatin immunoprecipitation with sequencing (ChIP-seq) following depletion of MEG3, we discovered that MEG3 targets and controls recruitment of EZH2/H3K27me3 onto a regulatory region of integrin subunit alpha 4 (ITGA4). MEG3 knockdown or pharmacological inhibition of EZH2 de-repressed ITGA4, whilst improving endothelial cell function in vitro, and increasing ITGA4 expression in vivo. Our study demonstrates new role for MEG3, as instrumental in epigenetic regulation of EC function by EZH2, through targeting of integrin-dependent signalling.

molecular biology↗

bioRxiv2020

Contractile reprogramming of cardiac pericytes by MEK inhibition promotes arteriologenesis of the ischemic heart

BackgroundThe development of collateral arteries after a myocardial infarction (MI) was intensively studied, while the mechanism by which pericytes (PCs) contribute to arteriologenesis remains unexplored. This study aimed to 1) investigate if cardiac PCs gain functional features of contractile vascular smooth muscle cells (VSMCs) in vitro, and 2) determine if this potential can be evoked pharmacologically to encourage heart arteriologenesis in vivo. MethodsPCs were immunosorted as CD31neg/CD34pos cells from human and mouse hearts. Contractile reprogramming was induced by either depletion of growth factors or addition of PD0325901, a clinically available MEK inhibitor. Next generation RNA-Sequencing was performed in naive and differentiated human PCs to assess the whole-transcriptome profile. Three in vivo studies were conducted in C57BL6/J mice to determine: 1) the ability of human PCs to promote arteriole formation when implanted subcutaneously within PD0325901-containing Matrigel plugs, 2) the effect of orally administered PD0325901 on the arteriole density of normoperfused hearts, and 3) the possibility of promoting capillary formation and muscularization of the infarcted heart through the same pharmacological approach. ResultsRemoval of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) from the culture medium induced the differentiation of PCs into contractile VSMC-like cells. Because both growth factors induce the extracellular signal-regulated kinase 1/2 (ERK1/2) signalling, we attempted to induce PC differentiation in vitro and in vivo using PD0325901. RNA-sequencing revealed that differentiated PCs were enriched in transcripts associated with smooth muscle contraction and biological function. PD0325901-treated PCs rapidly acquired antigenic and functional features of contractile VSMCs in vitro. Moreover, human PCs formed new arterioles when implanted subcutaneously within PD0325901-containing Matrigel plugs in mice. Oral administration of PD0325901 for two weeks increased the density and expression of contractile proteins in small-calibre arterioles of the murine heart, thereby increasing myocardial perfusion. Similarly, PD0325901 induced reparative arteriologenesis and capillarization, reduced the scar, and improved left ventricular performance in a murine model of MI. ConclusionWe propose a novel method to promote the heart vascularization through the pharmacological modulation of resident mural cells. This novel approach could have an immediate impact on the treatment of coronary artery disease. Clinical perspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIHuman myocardial pericytes have intrinsic vascular plasticity that can be pharmacologically evoked using PD0325901, a clinically available MEK inhibitor. C_LIO_LIIn mice, the pharmacological inhibition of ERK1/2 signalling, by the oral administration of PD0325901 for 2 weeks, encouraged the heart arteriologenesis through pericyte differentiation. C_LIO_LIIn a preclinical mouse model of myocardial infarction, the oral administration of PD0325901 for 2 weeks induced reparative arteriologenesis and capillarization, reduced the scar, and improved left ventricular performance. C_LI What are the clinical implications?O_LIThis novel drug-based therapeutic approach is readily available to all patients. C_LIO_LITherefore, it could have an immediate clinical impact for the treatment of coronary artery disease and other heart conditions associated with deficient coronary vascularization. C_LI

cell biology↗

bioRxiv2019

Eosinophil deficiency promotes aberrant repair and adverse remodelling following acute myocardial infarction

BackgroundEosinophil count predicts outcome following myocardial infarction (MI) and eosinophils regulate tissue repair and regeneration in extra-cardiac settings.\n\nObjectivesTo investigate the role of eosinophils in regulating inflammation, repair and remodelling following MI.\n\nMethodsBlood eosinophil count was assessed in 732 patients undergoing primary percutaneous coronary intervention for ST-segment elevation MI (STEMI). Experimental MI was induced in wild-type (WT) and eosinophil-depleted mice ({Delta}dblGATA or anti-Siglec F antibody treated). Cardiac function was characterised by high-resolution ultrasound and immune cell infiltration by flow cytometry of infarct digests.\n\nResultsBlood eosinophil count declined in the hours following STEMI in patients and following MI induction in mice. Eosinophils were subsequently identified in the myocardium of patients and mice. Genetic eosinophil depletion in mice increased LV dilatation (end-systolic area: 29.0{+/-}2.2cm2 v 21.6{+/-}1.6cm2; p=0.02) and reduced ejection fraction (22.0{+/-}3.6% v 34.3{+/-}4.0% in p=0.04,) in {Delta}dblGATA v WT following MI (n=8-9/group), an outcome reproduced by pharmacological depletion. {Delta}dblGATA mice had increased scar size with disrupted collagen deposition and altered expression of the collagen cross-linking genes plod2 and lox. CD206+pro-repair macrophages were less prevalent in the infarct zone of {Delta}dblGATA mice (27.0{+/-}2.3% v WT: 39.2.4{+/-}3.5%; p=0.01, n=9-11/group) but were restored by replenishment with bone marrow-derived eosinophils. Anti-inflammatory cytokine concentrations were reduced in {Delta}dblGATA mice and IL-4 complex administration 24h after MI rescued adverse remodelling.\n\nConclusionsEosinophils are recruited to the heart following MI and are required for effective repair and to prevent adverse remodelling. IL-4 therapy has potential to limit detrimental outcomes when eosinophil availability is low.\n\nHIGHLIGHTSA drop in eosinophil blood count is associated with recruitment of eosinophils to the heart during repair following clinical and experimental myocardial infarction.\n\nGenetic & pharmacological eosinophil depletion leads to increased adverse remodelling in experimental MI.\n\nEosinophils are required for aquisition of an anti-inflammatory macrophage phenotype and a shift to resolution of inflammation during infarct repair.\n\nInterleukin-4 therapy is able to rescue the adverse remodelling phenotype in conditions of eosinophil deficiency.\n\nCondensed abstractIn ST-segment elevation myocardial infarction (STEMI) of both patients and mice, there was a decline in blood eosinophil count, with activated eosinophils recruited to the infarct zone. Eosinophil deficiency resulted in attenuated anti-inflammatory pro-repair macrophage polarization, enhanced myocardial inflammation, increased scar size and deterioration of myocardial structure and function. Adverse cardiac remodelling in the setting of eosinophil deficiency was prevented by IL-4 therapy.

immunology↗

bioRxiv2019

Trichoplein controls endothelial cell function by regulating autophagy

Autophagy is an essential cellular quality control process that emerged critical for vascular homeostasis. Here we describe, the role for Trichoplein (TCHP) protein in linking autophagy with endothelial cells (ECs) function. The depletion of TCHP in ECs impairs migration and sprouting. TCHP directly binds PCM1, to regulate degradation of GABARAP, thus leading to a defective autophagy. Mechanistically, TCHP is indispensable for autophagosome maturation and its depletion resulted in the accumulation of SQSTM1/p62 (p62) and unfolded protein aggregates in ECs. The latter process is coupled to TCHP-mediated NF-kB activation. Of note, low levels of TCHP and high p62 levels were detected in primary ECs from patients with coronary artery disease. In addition, Tchp knock-out mice showed accumulation of p62 in the heart and cardiac vessels and reduced cardiac vascularization. Here, we reveal an autophagy-mediated mechanism for TCHP down-regulation, which poses a plausible target for regulation of endothelial function.

molecular biology↗