Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiomyopathy. The molecular mechanisms determining HCM phenotypes are incompletely understood. Myocardial biopsies were obtained from a group of patients with obstructive HCM (n = 23) selected for surgical myectomy and from 9 unused donor hearts (controls). A subset of tissue-abundant myectomy samples from HCM (n = 10) and controls (n = 6) was submitted to laser-capture microdissection to isolate cardiomyocytes. We investigated the relationship among clinical phenotype, cardiac myosin proteins (MyHC6, MyHC7, and MyHC7b) measured by optimized label-free mass spectrometry, the relative genes (MYH7, MYH7B and MYLC2), and the MyomiR network (myosin-encoded microRNA (miRs) and long-noncoding RNAs (Mhrt)) measured using RNA sequencing and RT-qPCR. MyHC6 was lower in HCM vs. controls, whilst MyHC7, MyHC7b, and MyLC2 were comparable. MYH7, MYH7B, and MYLC2 were higher in HCM whilst MYH6, miR-208a, miR-208b, miR-499 were comparable in HCM and controls. These results are compatible with defective transcription by active genes in HCM. Mhrt and two miR-499-target genes, SOX6 and PTBP3, were upregulated in HCM. The presence of HCM-associated mutations correlated with PTBP3 in myectomies and with SOX6 in cardiomyocytes. Additionally, iPSC-derived cardiomyocytes, transiently transfected with either miR-208a or miR-499, demonstrated a time-dependent relationship between MyomiRs and myosin genes. The transfection end-stage pattern was at least in part similar to findings in HCM myectomies. These data support uncoupling between myosin protein/genes and a modulatory role for the myosin/MyomiR network in the HCM myocardium, possibly contributing to phenotypic diversity and providing putative therapeutic targets.

Hypertrophic cardiomyopathy (HCM) is the most common genetic disease of the myocardium associated to mutations in sarcomeric genes, but the link between genotype and phenotype remains poorly understood. Magnetic resonance spectroscopy studies have demonstrated impaired cardiac energetics in patients with HCM, and altered mitochondria were described in biopsies, but little is known about possible perturbations of mitochondrial function and adenosine triphosphate (ATP) production/consumption. The aim of this study was to investigate possible abnormalities in mitochondrial enzymes generating/scavenging reactive oxygen species, and changes in the Ca²⁺-activated ATPases in myocardial tissue from patients with obstructive HCM undergoing surgical myectomy compared to unused donor hearts (CTRL). Methods and Results: Both the amount and activity of mitochondrial Complex I (nicotinamide adenine dinucleotide -reduced form, NADH, dehydrogenase) were upregulated in HCM vs. CTRL, whilst the activity of Complex V (ATP synthase) was not reduced and ATP levels were significantly higher in HCM vs. CTRL. Antioxidant Mn-activated superoxide dismutase (SOD2) and (m)-aconitase activities were increased in HCM vs. CTRL. The Cu/Zn-activated superoxide dismutase (SOD1) amount and mtDNA copy number were unaltered in HCM. Total Ca²⁺-activated ATPase activity and absolute amount were not different HCM vs. CTRL, but the ratio between ATPase sarcoplasmic/endoplasmic reticulum Ca²⁺ transporting type 2 (ATP2A2) and type 1 (ATP2A1), ATP2A2/ATP2A1, was increased in HCM in favor of the slow isoform (ATP2A2). Conclusion: HCM is characterized by mitochondrial Complex I hyperactivity and preserved Ca²⁺-activated ATPase activity with a partial switch towards slow ATP2A2. This data may give insight into the abnormal cellular energetics observed in HCM cardiomyopathy but other studies would need to be performed to confirm the observations described here.

Atherothrombosis exposes vascular components to blood. Currently, new antithrombotic therapies are emerging. Herein we investigated thrombogenesis of human arteries with/without atherosclerosis, and the interaction of coagulation and vascular components, we and explored the anti-thrombogenic efficacy of blockade of the P2X purinoceptor 7 (P2X7). A confocal blood flow videomicroscopy system was performed on cryosections of internal mammary artery (IMA) or carotid plaque (CPL) determining/localizing platelets and fibrin. Blood from healthy donors elicited thrombi over arterial layers. Confocal microscopy associated thrombus with tissue presence of collagen type I, laminin, fibrin(ogen) and tissue factor (TF). The addition of antibodies blocking TF (aTF) or factor XI (aFXI) to blood significantly reduced fibrin deposition, variable platelet aggregation and aTF + aFXI almost abolished thrombus formation, showing synergy between coagulation pathways. A scarce effect of aTF over sub-endothelial regions, more abundant in tissue TF and bundles of laminin and collagen type I than deep intima, may suggest tissue thrombogenicity as molecular structure-related. Consistently with TF-related vascular function and expression of P2X7, the sections from CPL but not IMA tissue cultures pre-treated with the P2X7 antagonist A740003 demonstrated poor thrombogenesis in flow experiments. These data hint to local targeting studies on P2X7 modulation for atherothrombosis prevention/therapy.

Abdominal aortic aneurysm (AAA) is a multifactorial, mainly silent disease leading to aortic dilatation through alteration of all the artery layers, extracellular matrix (ECM) hyperdegradation and inflammation, prevalent in men older than 60 years.¹ AAA ruptures, accounting for about 200 000 deaths/year worldwide, are associated to the aortic diameter. Surgical repair remains the elective treatment for large AAA.² Following discrepant results from clinical trials on AAA pharmacotherapy,³ non-surgical management of AAA and identification of a medical treatment slowing AAA growth are unmet medical needs. The MMP9 upregulation is a key event during AAA growth² and modulation of ECM degradation an emerging target (e.g. MAAAGI trial, NCT0422405). The CXCR1/CXCR2 antagonism by non-competitive allosteric inhibitor ladarixin (Dompé Farmaceutici S.p.A., Milano, Italy) reduced MMP9 and leucocyte infiltration in a murine AAA model.⁴ Nevertheless, the clinical relevance of ladarixin (approved for the treatment of type I diabetes mellitus, EMEA-002642-PIP01-19-M03) in cardiovascular diseases and of CXCR2 as therapeutic target in human AAA remain both unaddressed. CXCR2 interacts with multiple cytokines including CXCL1 and its homologue CXCL8 (synonym: IL-8), which play chemotactic and angiogenic roles, e.g. immune cell recruitment at site of inflammation.^5,6 Circulating IL-8 is related to the diameter of small AAA, and human AAA hyper-express IL-8.⁴ MMP9 release from in vitro–activated neutrophils is induced by IL-8 and CXCL1 and reduced by pharmacological blockade of CXCR2.⁵ In turn, MMP9 proteolytically activates IL-8 but inactivates CXCL1.⁵

An unusual case of a patient with ovarian carcinoma carrying the p53 point mutation in both metastases (omentum and lymph node), but not in the primary tumor, is described. The presence of a p53 single mutation (G:A) at the second base of codon 248 was examined by polymerase chain reaction-amplification refractory mutation system (PCR-ARMS) analysis. This case was examined also by fluorescent in situ hybrization (FISH) analysis and flow cytometry (FCM) to obtain further information at the single cell level and to detect heterogeneity within a population of cells. FCM analysis evidenced the same multiple aneuploid cell subpopulations in primary and in metastatic samples showing the presence of a cellular heterogeneity. FISH analysis showed a disomic condition for the 17 chromosome in the primary and in one metastasis, while in the other metastasis a monosomic together with a disomic subpopulation was revealed. Our results confirm the independent clonal evolution of the metastasis. The late mutation event observed only in metastatic specimens suggests the hypothesis that in the primary tumor the wild-type gene either does not perform its control role for unknown genetic structural events or the p53 gene in this case does not play a critical role in carcinogenesis.

Bicompartmental in vitro cultures are performed using culture inserts in multi‐well plates. The main challenge of these systems is sample retrieval, which potentially impacts the experimental outcomes. To overcome this challenge, we developed a novel cartridge‐based device called true‐tissue‐on‐platform (TTOP). Our device offers the same handling than multi‐well inserts with a key difference: the cartridge can be easily retrieved and reused without compromising sample morphology. The functionality of TTOP is demonstrated by building gut and vascular barrier in‐vitro models. Barrier tightness is assessed by measuring trans epithelial/endothelial electrical resistance (TEER) and permeability by Lucifer Yellow assay (LY). Geometry‐related effects on TEER are evaluated using a finite element model, comparing TTOP to conventional inserts, which showed an improvement in data robustness and accuracy. Moreover, the device enabled the detection of inflammation‐related changes by comparing untreated versus Tumor‐Necrosis‐Factor‐α‐treated cell barriers, revealing significant differences both with TEER and LY assays. Preliminary data on coupled gut/vascular barriers in the two compartments supported the feasibility of mimicking the gut–vascular barrier in vitro. This new cartridge‐based concept may provide a simple and modular device, accessible to any biological laboratory, enabling controlled handling of the biological samples during and after the experiment.