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Maternal viral infection and immune response are known to increase the risk of altered development of the foetal brain. Given the ongoing global pandemic of coronavirus disease 2019 (COVID-19), investigating the impact of SARS-CoV-2 on foetal brain health is of critical importance. Here, we report the presence of SARS-CoV-2 in first and second trimester foetal brain tissue in association with cortical haemorrhages. SARS-CoV-2 spike protein was sparsely detected within progenitors and neurons of the cortex itself, but was abundant in the choroid plexus of haemorrhagic samples. SARS-CoV-2 was also sparsely detected in placenta, amnion and umbilical cord tissues. Cortical haemorrhages were linked to a reduction in blood vessel integrity and an increase in immune cell infiltration into the foetal brain. Our findings indicate that SARS-CoV-2 infection may affect the foetal brain during early gestation and highlight the need for further study of its impact on subsequent neurological development.

Several papers have been published about the risk of recurrence after an attack of diverticulitis treated conservatively. However, very few papers have been devoted to the risk of postoperative recurrence of diverticulitis (PRD) after prophylactic sigmoidectomy (PS). The aim of this work was to report the rate of PRD after PS and to assess possible risk factors for recurrence after surgery.All consecutive patients who underwent elective laparoscopic PS for diverticulitis between 2005 and 2019 were retrospectively included. PRD was assessed.Three hundred and sixty four patients (199 men, mean age 54 ± 13 years) were included. Among these, 26 (7%) presented with 1.7 ± 1 (range 1-4) episodes of recurrence of diverticulitis after a mean delay of 44 ± 39 months (1 month-11 years) after surgery. Patients who presented with postoperative recurrence of diverticulitis were younger (46 ± 11 vs. 55 ± 13 years, p = 0.002) and more frequently had uncomplicated diverticulitis [15/26 (58%) vs. 97/338 (29%), p = 0.002] and more than two previous episodes before PS [17/26 (65%) vs. 132/338 (39%), p = 0.009] than patients without PRD. After multivariate analysis, two independent risk factors for PRD were identified: patients with more than two episodes before PS (OR = 3.3, 95% CI = 1.2-9, p = 0.005) and age 50 years (OR = 4.5, 95% CI = 2-11, p = 0.001). If both factors were present, recurrence reached 18% (9/51).Postoperative recurrence of diverticulitis is rare (7%) after PS for diverticulitis. Some patients (i.e. those with more than two episodes before PS and/or age50 years) could be exposed to a higher risk of recurrence (up to 18%), making prophylactic surgery questionable in these patients.

Aims The adult mammalian heart is a post-mitotic organ. Even in response to necrotic injuries, where regeneration would be essential to reinstate cardiac structure and function, only a minor percentage of cardiomyocytes undergo cytokinesis. The gene programme that promotes cell division within this population of cardiomyocytes is not fully understood. In this study, we aimed to determine the gene expression profile of proliferating adult cardiomyocytes in the mammalian heart after myocardial ischaemia, to identify factors to can promote cardiac regeneration. Methods and results Here, we demonstrate increased 5-ethynyl-2’deoxyuridine incorporation in cardiomyocytes 3 days post-myocardial infarction in mice. By applying multi-colour lineage tracing, we show that this is paralleled by clonal expansion of cardiomyocytes in the borderzone of the infarcted tissue. Bioinformatic analysis of single-cell RNA sequencing data from cardiomyocytes at 3 days post ischaemic injury revealed a distinct transcriptional profile in cardiomyocytes expressing cell cycle markers. Combinatorial overexpression of the enriched genes within this population in neonatal rat cardiomyocytes and mice at postnatal day 12 (P12) unveiled key genes that promoted increased cardiomyocyte proliferation. Therapeutic delivery of these gene cocktails into the myocardial wall after ischaemic injury demonstrated that a combination of thymosin beta 4 (TMSB4) and prothymosin alpha (PTMA) provide a permissive environment for cardiomyocyte proliferation and thereby attenuated cardiac dysfunction. Conclusion This study reveals the transcriptional profile of proliferating cardiomyocytes in the ischaemic heart and shows that overexpression of the two identified factors, TMSB4 and PTMA, can promote cardiac regeneration. This work indicates that in addition to activating cardiomyocyte proliferation, a supportive environment is a key for regeneration to occur.

Iridoviridae, such as the lymphocystis disease virus-1 (LCDV-1) and other viruses, encode viral insulin–like peptides (VILPs) which are capable of triggering insulin receptors (IRs) and insulin-like growth factor receptors. The homology of VILPs includes highly conserved disulfide bridges. However, the binding affinities to IRs were reported to be 200- to 500-fold less effective compared to the endogenous ligands. We therefore speculated that these peptides also have noninsulin functions. Here, we report that the LCDV-1 VILP can function as a potent and highly specific inhibitor of ferroptosis. Induction of cell death by the ferroptosis inducers erastin, RSL3, FIN56, and FINO2 and nonferroptotic necrosis produced by the thioredoxin-reductase inhibitor ferroptocide were potently prevented by LCDV-1, while human insulin had no effect. Fas-induced apoptosis, necroptosis, mitotane-induced cell death and growth hormone–releasing hormone antagonist–induced necrosis were unaffected, suggesting the specificity to ferroptosis inhibition by the LCDV-1 VILP. Mechanistically, we identified the viral C-peptide to be required for inhibition of lipid peroxidation and ferroptosis inhibition, while the human C-peptide exhibited no antiferroptotic properties. In addition, the deletion of the viral C-peptide abolishes radical trapping activity in cell-free systems. We conclude that iridoviridae, through the expression of insulin-like viral peptides, are capable of preventing ferroptosis. In analogy to the viral mitochondrial inhibitor of apoptosis and the viral inhibitor of RIP activation (vIRA) that prevents necroptosis, we rename the LCDV-1 VILP a viral peptide inhibitor of ferroptosis-1. Finally, our findings indicate that ferroptosis may function as a viral defense mechanism in lower organisms.

Aims Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disorder that is characterized by progressive loss of myocardium that is replaced by fibro-fatty cells, arrhythmias, and sudden cardiac death. While myocardial degeneration and fibro-fatty replacement occur in specific locations, the underlying molecular changes remain poorly characterized. Here, we aim to delineate local changes in gene expression to identify new genes and pathways that are relevant for specific remodelling processes occurring during ACM. Methods and results Using Tomo-Seq, genome-wide transcriptional profiling with high spatial resolution, we created transmural epicardial-to-endocardial gene expression atlases of explanted ACM hearts to gain molecular insights into disease-driving processes. This enabled us to link gene expression profiles to the different regional remodelling responses and allowed us to identify genes that are potentially relevant for disease progression. In doing so, we identified distinct gene expression profiles marking regions of cardiomyocyte degeneration and fibro-fatty remodelling and revealed Zinc finger and BTB domain-containing protein 11 (ZBTB11) to be specifically enriched at sites of active fibro-fatty replacement of myocardium. Immunohistochemistry indicated ZBTB11 to be induced in cardiomyocytes flanking fibro-fatty areas, which could be confirmed in multiple cardiomyopathy patients. Forced overexpression of ZBTB11 induced autophagy and cell death-related gene programmes in human cardiomyocytes, leading to increased apoptosis. Conclusion Our study shows the power of Tomo-Seq to unveil new molecular mechanisms in human cardiomyopathy and uncovers ZBTB11 as a novel driver of cardiomyocyte loss.

Hypoglycemia is a major complication associated with insulin therapy in people with diabetes that could cause life-threatening conditions if untreated. Glucagon, a counter-acting hormone, is thus administered for rescue of severe hypoglycemia. However, due to the instability of glucagon, only limited medications are available for emergency use, which are unsuitable for patients with hypoglycemia unawareness or with the inability to self-administer, especially during sleep (namely, nocturnal hypoglycemia). To prevent unattended and extended hypoglycemia, we designed a "smart" composite microneedle (cMN) patch capable of stabilizing glucagon, sensing hypoglycemia, and delivering glucagon automatically on demand. In this design, native glucagon was encapsulated in glucose-responsive microgels containing a glucagon-stabilizing component rationally selected by molecular dynamics (MD) simulation. A cMN patch was then prepared by incorporating the glucagon microgels with poly(methyl vinyl ether

Objective Despite growing evidence about myocardial injury in hospitalized COronaVIrus Disease 2019 (COVID-19) patients, the mechanism behind this injury is only poorly understood and little is known about its association with SARS-CoV-2-mediated myocarditis. Furthermore, definite evidence of the presence and role of SARS-CoV-2 in cardiomyocytes in the clinical scenario is still lacking. Methods We histologically characterized myocardial tissue of 40 patients deceased with severe SARS-CoV-2 infection during the first wave of the pandemic. Clinical data were also recorded and analyzed. In case of findings supportive of myocardial inflammation, histological analysis was complemented by RT-PCR and immunohistochemistry for SARS-CoV-2 viral antigens and in situ RNA hybridization for the detection of viral genomes. Results Both chronic and acute myocardial damage was invariably present, correlating with the age and comorbidities of our population. Myocarditis of overt entity was found in one case (2.5%). SARS-CoV-2 genome was not found in the cardiomyocytes of the patient with myocarditis, while it was focally and negligibly present in cardiomyocytes of patients with known viral persistence in the lungs and no signs of myocardial inflammation. The presence of myocardial injury was not associated with myocardial inflammatory infiltrates. Conclusions In this autopsy cohort of COVID-19 patients, myocarditis is rarely found and not associated with SARS-CoV-2 presence in cardiomyocytes. Chronic and acute forms of myocardial damage are constantly found and correlate with the severity of COVID-19 disease and pre-existing comorbidities. Graphic abstract

Therapies for patients with myocardial infarction and heart failure are urgently needed, in light of the breadth of these conditions and lack of curative treatments. To systematically identify previously unidentified cardioactive biologicals in an unbiased manner in vivo, we developed cardiac FunSel, a method for the systematic, functional selection of effective factors using a library of 1198 barcoded adeno-associated virus (AAV) vectors encoding for the mouse secretome. By pooled vector injection into the heart, this library was screened to functionally select for factors that confer cardioprotection against myocardial infarction. After two rounds of iterative selection in mice, cardiac FunSel identified three proteins [chordin-like 1 (Chrdl1), family with sequence similarity 3 member C (Fam3c), and Fam3b] that preserve cardiomyocyte viability, sustain cardiac function, and prevent pathological remodeling. In particular, Chrdl1 exerted its protective activity by binding and inhibiting extracellular bone morphogenetic protein 4 (BMP4), which resulted in protection against cardiomyocyte death and induction of autophagy in cardiomyocytes after myocardial infarction. Chrdl1 also inhibited fibrosis and maladaptive cardiac remodeling by binding transforming growth factor–β (TGF-β) and preventing cardiac fibroblast differentiation into myofibroblasts. Production of secreted and circulating Chrdl1, Fam3c, and Fam3b from the liver also protected the heart from myocardial infarction, thus supporting the use of the three proteins as recombinant factors. Together, these findings disclose a powerful method for the in vivo, unbiased selection of tissue-protective factors and describe potential cardiac therapeutics.

PDF file - 91K, Supplementary Table 1. List of genes analyzed for expression by real-time, quantitative RT-PCR. All the amplifications were performed on a 7000 ABI Prism Instrument (Applied Biosystems), using pre-developed assays ("Mm" code), custom assays (for CCL5 and CXCR4) (Applied Biosystems) or SYBR Green (for Gr-1 and HPRT). The housekeeping genes GAPDH and HPRT were used to normalize the results.

PDF file - 1560K, Quantification of the flow cytometry profiles for the characterization of the four bone marrow cell populations sorted according to the expression of Nrp1 and Gr1. The results (shown as bar charts in the upper part of the figure and as a table in the bottom part) refer to four independent experiments; shown are the mean � s.d. The markers analyzed are the same as in the representative experiment shown in Figure 1D.

PDF file - 3704K, The vessel normalization effect of NEMs is independent of tumor size. B16.F10 tumors were inoculated in mice, which were then injected at days 11 and 13 after tumor inoculation with PBS, Gr1+ cells and NEMs. Mice were sacrificed when tumors reached 1700 mm3 , irrespective of the day. A. Immunofluorescence analysis of the vasculature - representative images. Frozen sections were stained with antibodies against CD31 (green) and α-SMA (red).Nuclei are counterstained with DAPI. Scale bar: 100 μm. B. Quantitative evaluation of the α-SMA/CD31 ratio in vessels of tumors injected with PBS, Gr1+ cells or NEMs. Data are mean�s.e.m. **=P

PDF file - 1471K, Levels of expression of plexins in CD11b+ Gr1+ Nrp1+ cells and in NEMs. Real-time PCR quantifications were normalized to the levels of GAPDH mRNA. Data are mean �s.e.m. **=P

PDF file - 1214K, Levels of expression of HIF-1 target genes in tumors inoculated with bone marrow Gr1+ cells or NEMs. Quantification of the mRNA levels of the indicated factors (VEGF-A, Angiopoietin-2 and Interleukin-6) was obtained by real-time PCR. The results are shown after normalization for the GAPDH gene. Data are mean �s.e.m. **=P

PDF file - 3108K, Flow cytometry analysis of NEMs purified from the bone marrow. Flow cytometry analysis of bone marrow CD11b+/Gr1-/Nrp1+ cells (NEMs) using the indicated antibodies conjugated with phycoerythrin (PE). Percentages of positive cells, referred to the total number of NEMs, are reported. The plots reported in the first row are also shown in Figure 1D, fourth column.

PDF file - 3482K, Proliferative activity of 4T1 tumors in vivo. A. Immunofluorescence staining of frozen tumor sections from mice injected with PBS, Gr1+ cells or NEMs using an antibody against PCNA (red); nuclei are counterstained in blue with DAPI. Analysis was performed after the animal sacrifice at day 18 after tumor inoculation. Scale bar: 100 �m. B. Quantitative analysis showing the percentage of PCNA+ nuclei in the tumor sections. Data are mean�s.e.m.; n=5 per group.

Myeloid cells can restrain antitumor immunity by metabolic pathways, such as the degradation of l -arginine, whose concentrations are regulated by the arginase 1 (ARG1) enzyme. Results from preclinical studies indicate the important role of arginine metabolism in pancreatic ductal adenocarcinoma (PDAC) progression, suggesting a potential for clinical application; however, divergent evolution in ARG1 expression and function in rodents and humans has restricted clinical translation. To overcome this dichotomy, here, we show that neutrophil extracellular traps (NETs), released by spontaneously activated neutrophils isolated from patients with PDAC, create a microdomain where cathepsin S (CTSS) cleaves human (h)ARG1 into different molecular forms endowed with enhanced enzymatic activity at physiological pH. NET-associated hARG1 suppresses T lymphocytes whose proliferation is restored by either adding a hARG1-specific monoclonal antibody (mAb) or preventing CTSS-mediated cleavage, whereas small-molecule inhibitors are not effective. We show that ARG1 blockade, combined with immune checkpoint inhibitors, can restore CD8 + T cell function in ex vivo PDAC tumors. Furthermore, anti-hARG1 mAbs increase the frequency of adoptively transferred tumor-specific CD8 + T cells in tumor and enhance the effectiveness of immune checkpoint therapy in humanized mice. Thus, this study shows that extracellular ARG1, released by activated myeloid cells, localizes in NETs, where it interacts with CTSS that in turn cleaves ARG1, producing major molecular forms endowed with different enzymatic activity at physiological pH. Once exocytosed, ARG1 activity can be targeted by mAbs, which bear potential for clinical application for the treatment of PDAC and require further exploration.

Thrombosis of the lung microvasculature is a characteristic of COVID-19 disease, which is observed in large excess compared to other forms of acute respiratory distress syndrome and thus suggests a trigger for thrombosis that is endogenous to the lung. Our recent work has shown that the SARS-CoV-2 Spike protein activates the cellular TMEM16F chloride channel and scramblase. Through a screening on >3,000 FDA/EMA approved drugs, we identified Niclosamide and Clofazimine as the most effective molecules at inhibiting Spike-induced TMEM16 activation. As TMEM16F plays an important role in stimulating the procoagulant activity of platelets, we investigated whether Spike directly affects platelet activation and pro-thrombotic function and tested the effect of Niclosamide and Clofazimine on these processes. Here we show that Spike, present either on the virion envelope or on the cell plasma membrane, promotes platelet activation, adhesion and spreading. Spike was active as a sole agonist or, even more effectively, by enhancing the function of known platelet activators. In particular, Spike-induced a marked procoagulant phenotype in platelets, by enhancing Ca2+ flux, phosphatidylserine externalization on the platelet outer cell membrane, and thrombin generation. Eventually, this increased thrombin-induced clot formation and retraction. Both Niclosamide and Clofazimine blocked this Spike-induced procoagulant response. These findings provide a pathogenic mechanism to explain lung thrombosis-associated with severe COVID-19 infection. We propose that Spike, present in SARS-CoV-2 virions or exposed on the surface of infected cells in the lungs, enhances the effects of inflammation and leads to local platelet stimulation and subsequent activation of the coagulation cascade. As platelet TMEM16F is central in this process, these findings reinforce the rationale of repurposing Niclosamide for COVID-19 therapy.

Heart failure with preserved ejection fraction (HFpEF) remains an elusive entity, due to its heterogeneous clinical profile and an arbitrarily defined nosology. Several pathophysiological mechanisms recognized as central for the development of HFpEF appear to be in common with the process of physiological aging of the heart. Both conditions are characterized by progressive impairment in cardiac function, accompanied by left ventricular hypertrophy, diastolic dysfunction, sarcomeric, and metabolic abnormalities. The neurological paradigm of dementia-intended as a progressive, multifactorial organ damage with decline of functional reserve, eventually leading to irreversible dysfunction-is well suited to represent HFpEF. In such perspective, certain phenotypes of HFpEF may be viewed as a maladaptive response to environmental modifiers, causing premature and pathological aging of the heart. We here propose that the 'HFpEF syndrome' may reflect the interplay of adverse structural remodelling and erosion of functional reserve, mirroring the processes leading to dementia in the brain. The resulting conceptual framework may help advance our understanding of HFpEF and unravel potential therapeutical targets.

Following insults or injury, microglia cells are activated contributing to the cytotoxic response or by promoting an immune-mediated damage resolution. Microglia cells express HCA2R, a hydroxy carboxylic acid (HCA) re-ceptor, which has been shown to mediate neuroprotective and anti-inflammatory effects.In this study we found that HCAR2 expression levels were increased in cultured rat microglia cells after Lipopolysaccharide (LPS) exposure. In a similar fashion, the treatment with MK 1903, a potent full agonist of HCAR2, increased the receptor protein levels. Moreover, HCAR2 stimulation prevented i) cells viability ii) morphological activation iii) pro/anti-inflammatory mediators production in LPS-treated cells. Likewise, HCAR2 stimulation reduced the proinflammatory mediators mRNA expression induced by neuronal chemokine frac-talkine (FKN), a neuronal derived chemokine activating its unique receptor, chemokine receptor 1 (CX3CR1) on microglia surface. Interestingly, electrophysiological recordings in vivo revealed that MK1903 was able to prevent the increase of the nociceptive neurons (NS) firing activity mediated by the spinal FKN application in healthy rats. Collectively, our data demonstrate that HCAR2 is functionally expressed in microglia, by showing its capa-bility to shift microglia toward an anti-inflammatory phenotype. Moreover, we indicated the contribute of HCAR2 in the FKN signaling and suggested a possible HCAR2/CX3CR1 functional interaction. This study paves the way for further investigations aimed at understanding the role HCAR2 as potential target in neuroinflammation-based CNS disorders.This article is part of the Special Issue on "The receptor-receptor interaction as a new target for therapy".

Background: Diverse genetic backgrounds often lead to phenotypic heterogeneity in cardiomyopathies (CMPs). Previous genotype-phenotype studies have primarily focused on the analysis of a single phenotype, and the diagnostic and prognostic features of the CMP genotype across different phenotypic expressions remain poorly understood. Objectives: We sought to define differences in outcome prediction when stratifying patients based on phenotype at presentation compared with genotype in a large cohort of patients with CMPs and positive genetic testing. Methods: Dilated cardiomyopathy (DCM), arrhythmogenic right ventricular cardiomyopathy, left-dominant arrhythmogenic cardiomyopathy, and biventricular arrhythmogenic cardiomyopathy were examined in this study. A total of 281 patients (80% DCM) with pathogenic or likely pathogenic variants were included. The primary and secondary outcomes were: 1) all-cause mortality (D)/heart transplant (HT); 2) sudden cardiac death/major ventricular arrhythmias (SCD/MVA); and 3) heart failure-related death (DHF)/HT/left ventricular assist device implantation (LVAD). Results: Survival analysis revealed that SCD/MVA events occurred more frequently in patients without a DCM phenotype and in carriers of DSP, PKP2, LMNA, and FLNC variants. However, after adjustment for age and sex, genotype-based classification, but not phenotype-based classification, was predictive of SCD/MVA. LMNA showed the worst trends in terms of D/HT and DHF/HT/LVAD. Conclusions: Genotypes were associated with significant phenotypic heterogeneity in genetic cardiomyopathies. Nevertheless, in our study, genotypic-based classification showed higher precision in predicting the outcome of patients with CMP than phenotype-based classification. These findings add to our current understanding of inherited CMPs and contribute to the risk stratification of patients with positive genetic testing.

Aims Acute myocarditis (AM) is a heterogeneous condition with variable estimates of survival. Contemporary criteria for the diagnosis of clinically suspected AM enable non-invasive assessment, resulting in greater sensitivity and more representative cohorts. We aimed to describe the demographic characteristics and long-term outcomes of patients with AM diagnosed using non-invasive criteria. Methods and Results A total of 199 patients with cardiac magnetic resonance (CMR)-confirmed AM were included. The majority (n=130, 65%) were male and the average age was 39±16 years. Half of patients were White (n=99, 52%) with the remainder from Black and Minority Ethnic (BAME) groups. The most common clinical presentation was with chest pain (n=156, 78%), with smaller numbers presenting with breathlessness (n=25, 13%) and arrhythmias (n=18, 9%). Patients admitted with breathlessness were sicker and more often required inotropes, steroids, and renal replacement therapy (p Conclusions AM is highly heterogeneous with an overall favourable prognosis. Three quarters of patients with AM present with chest pain and this is associated with a benign prognosis. AM presenting with life-threatening arrhythmias is associated with higher risk of adverse events.

Background Gene therapy based on viral vectors is a promising new approach and offers great potential for the study and the treatment of cardiac diseases. Nucleic acid-based transfection of human heart tissue could be considered a valid and translational alternative to the use of transgenic animal models in cardiovascular research. Purpose Here we explore the use of Living Myocardial Slices (LMS) as a platform for nucleic acid-based therapies. Rat LMS and Adeno-Associated viruses (AAV) were used to optimise and analyse gene transfer efficiency, viability, tissue functionality, and cell tropism in cardiac tissue. Human cardiac samples from failing (dilated cardiomyopathy) hearts were also used to validate the human model. In addition, we transfected and tested for the first time the regenerative effect of AAV-miR-199a in both healthy and pathologically overloaded LMS. Methods LMS (300 μm thick) were prepared and glued to PTFE-coated holders, mounted on custom stretchers, and cultured at physiological sarcomere length for 72-hours under electrical stimulation. Two recombinant AAV serotypes (AAV-6 and AAV-9) at different multiplicity of infection (MOI) expressing green fluorescent protein (GFP) or the miR-199 transgene were added to the surface of the LMS. Results AAV6 proved to be the most suitable serotype and 20,000 MOI the most efficient dose, as these conditions were proven not to affect LMS contractility and to have the highest transduction and penetrability efficiency. This serotype exhibited preferential cell tropism to cardiomyocytes and stromal cells (40% of transduction in both) in rat LMS, with lower efficiency in endothelial cells, (20% of transduction). In contrast, AAV6 in human failing LMS showed higher transduction efficiency in endothelial cells and myofibroblasts. Treatment with miR-199a significantly increased the active force of pathologically overstretched LMS, downregulated the expression of its target genes, and affected the proliferative capacity of LMS. Conclusion LMS can be used as a model for nucleic acid-based therapies and their transfection protocol can be adapted to samples obtained from failing human hearts. With great translational relevance, this model would accelerate preclinical studies of novel nucleic acid-based therapies for heart failure. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation

Aims Cardiac ischaemia does not elicit an efficient angiogenic response. Indeed, lack of surgical revascularization upon myocardial infarction results in cardiomyocyte death, scarring, and loss of contractile function. Clinical trials aimed at inducing therapeutic revascularization through the delivery of pro-angiogenic molecules after cardiac ischaemia have invariably failed, suggesting that endothelial cells in the heart cannot mount an efficient angiogenic response. To understand why the heart is a poorly angiogenic environment, here we compare the angiogenic response of the cardiac and skeletal muscle using a lineage tracing approach to genetically label sprouting endothelial cells. Methods and results We observed that overexpression of the vascular endothelial growth factor in the skeletal muscle potently stimulated angiogenesis, resulting in the formation of a massive number of new capillaries and arterioles. In contrast, response to the same dose of the same factor in the heart was blunted and consisted in a modest increase in the number of new arterioles. By using Apelin-CreER mice to genetically label sprouting endothelial cells we observed that different pro-angiogenic stimuli activated Apelin expression in both muscle types to a similar extent, however, only in the skeletal muscle, these cells were able to sprout, form elongated vascular tubes activating Notch signalling, and became incorporated into arteries. In the heart, Apelin-positive cells transiently persisted and failed to give rise to new vessels. When we implanted cancer cells in different organs, the abortive angiogenic response in the heart resulted in a reduced expansion of the tumour mass. Conclusion Our genetic lineage tracing indicates that cardiac endothelial cells activate Apelin expression in response to pro-angiogenic stimuli but, different from those of the skeletal muscle, fail to proliferate and form mature and structured vessels. The poor angiogenic potential of the heart is associated with reduced tumour angiogenesis and growth of cancer cells.

The disorganized and inefficient tumor vasculature is a major obstacle to the delivery and efficacy of antineoplastic treatments. Antiangiogenic agents can normalize the tumor vessels, improving vessel function and boosting the distribution and activity of chemotherapy. The type III repeats (T3R) domain of thrombospondin-1 contains different potential antiangiogenic sequences. We therefore hypothesized that it might affect the tumor vasculature. Ectopic expression of the T3R domain by the tumor cells or by the host, or administration of recombinant T3R, delayed the in vivo growth of experimental tumors. Tumors presented marked reorganization of the vasculature, with abundant but smaller vessels, associated with substantially less necrosis. Mechanistically, the use of truncated forms of the domain, containing different active sequences, pointed to the FGF2/FGFR/ERK axis as a target for T3R activity. Along with reduced necrosis, the expression of T3R promoted tumor distribution of chemotherapy (paclitaxel), with a higher drug concentration and more homogeneous distribution, as assessed by HPLC and MALDI imaging mass spectrometry. T3R-expressing tumors were more responsive to paclitaxel and cisplatin. This study shows that together with its known role as a canonical inhibitor of angiogenesis, thrombospondin-1 can also remodel tumor blood vessels, affecting the morphological and functional properties of the tumor vasculature. The ability of T3R to reduce tumor growth and improve the response to chemotherapy opens new perspectives for therapeutic strategies based on T3R to be used in combination therapies.

AimAcute myocarditis (AM) is a heterogeneous condition with variable estimates of survival. Contemporary criteria for the diagnosis of clinically suspected AM enable non-invasive assessment, resulting in greater sensitivity and more representative cohorts. We aimed to describe the demographic characteristics and long-term outcomes of patients with AM diagnosed using non-invasive criteria.Methods and resultsA total of 199 patients with cardiac magnetic resonance (CMR)-confirmed AM were included. The majority (n = 130, 65%) were male, and the average age was 39 ± 16 years. Half of the patients were White (n = 99, 52%), with the remainder from Black and Minority Ethnic (BAME) groups. The most common clinical presentation was chest pain (n = 156, 78%), with smaller numbers presenting with breathlessness (n = 25, 13%) and arrhythmias (n = 18, 9%). Patients admitted with breathlessness were sicker and more often required inotropes, steroids, and renal replacement therapy (p < 0.001, p < 0.001, and p = 0.01, respectively). Over a median follow-up of 53 (IQR 34–76) months, 11 patients (6%) experienced an adverse outcome, defined as a composite of all-cause mortality, resuscitated cardiac arrest, and appropriate implantable cardioverter defibrillator (ICD) therapy. Patients in the arrhythmia group had a worse prognosis, with a nearly sevenfold risk of adverse events [hazard ratio (HR) 6.97; 95% confidence interval (CI) 1.87–26.00, p = 0.004]. Sex and ethnicity were not significantly associated with the outcome.ConclusionAM is highly heterogeneous with an overall favourable prognosis. Three-quarters of patients with AM present with chest pain, which is associated with a benign prognosis. AM presenting with life-threatening arrhythmias is associated with a higher risk of adverse events.

To what extent cardiac renewal occurs in adulthood and can be stimulated by therapeutic interventions is still a matter of investigation. In particular, over the last few years, significant effort has been made into awakening the endogenous regenerative potential of the adult mammalian heart after injury using different strategies. However, these studies are significantly hindered by the lack of tools to properly assess cardiomyocyte renewal over time. We have developed a genetic strategy, which we named CycleTrack, allowing a cumulative and accurate estimate of cardiomyocyte divisions in vivo. This method is based on the expression, in cardiomyocytes, of the Cre recombinase under the control of a 312-bp fragment of the Cyclin B2 promoter, which is specifically sensitive to cell proliferation. Replication-activated Cre acts on floxed GFP that is either present in the genome of transgenic mice or is exogenously delivered using Adeno-Associated Viral (AAV) vectors. As a result, cardiomyocytes traversing G2/M become irreversibly labeled. Using CycleTrack, we could monitor cardiomyocyte turnover in several physiological and pathological conditions. These included the measurement of the rate of proliferation of cardiomyocytes after apical resection in neonatal mice, the pro-regenerative effect of AAV9-mediated delivery of miRNA-199a and miRNA-590 after myocardial infarction in adult mice and the effect of pregnancy on myocardial hyperplasia. We also delivered the CycleTrack vectors into pig hearts to visualize cardiomyocyte turnover after myocardial infarction. Considering the large availability of Cre reporter mouse lines and the efficacy of AAVs to transfer genes into cardiomyocytes, we propose CycleTrack as a robust and straightforward tool to trace mitotic events for cardiac regeneration studies in small and large animal models.

Despite the development of new pharmacological and device-based therapies, no approaches currently exist to compensate for the irreversible loss of cardiomyocytes occurring after myocardial infarction. In a search for novel treatments that induce cardiomyocyte proliferation, we performed a high-content, fluorescence-microscopy-based, high-throughput screening in neonatal rat cardiomyocytes, using a library of FDA/EMA-approved drug library compounds. The screening, which was based on EdU incorporation as an S-phase marker, identified harmine as the most powerful drug enhancing proliferation in combination with miR-199a-3p and miR-590-3p, the most effective microRNAs that the laboratory had previously identified. Harmine treatment of CD1 mice showed an improved cardiac outcome upon harmine treatment compared to controls. Harmine is a potent inhibitor of the Dyrk1 family of protein kinases, with high specificity for the Dyrk1a isoform. Dyrk1a downregulation experiments in neonatal rat ventricular cardiomyocytes showed increased proliferation, consistent with a negative role of this kinase. The simultaneous knockdown of YAP1 blunted the effect of Dyrk1a downregulation. Of note, the YAP total protein levels were unchanged upon either Dyrk1a silencing or overexpression, but there was a marked modification of YAP subcellular localisation, being YAP1 nuclear upon knockdown and mostly cytoplasmic upon overexpression. Dyrk1a downregulation and harmine treatment activated YAP-controlled gene expression. In the course of these experiments, we also noticed a reciprocal regulation of YAP onto DYRK1a, as YAP downregulation markedly decreased Dyrk1a protein levels. This finding was reinforced by the observations that, in cardiomyocytes, the YAP-TEAD inhibitor verteporfin also decreased Dyrk1a protein levels. Of note, this downregulation was not rescued by the proteasome inhibitor MG132, thus indicating that the level of regulation is most likely transcriptional. These results support the role of Dyrk1a as an anti-proliferative factor in cardiomyocytes and highlight a novel feedback connection between Dyrk1a and YAP.

SARS-CoV-2 enters cells via its spike glycoprotein which must be cleaved sequentially at the S1/S2, then the S2’ cleavage sites (CS) to mediate membrane fusion. SARS-CoV-2 has a unique polybasic insertion at the S1/S2 CS, which we demonstrate can be cleaved by furin. Using lentiviral pseudotypes and a cell-culture adapted SARS-CoV-2 virus with a S1/S2 deletion, we show that the polybasic insertion is selected for in lung cells and primary human airway epithelial cultures but selected against in Vero E6, a cell line used for passaging SARS-CoV-2. We find this selective advantage depends on expression of the cell surface protease, TMPRSS2, that allows virus entry independent of endosomes thus avoiding antiviral IFITM proteins. SARS-CoV-2 virus lacking the S1/S2 furin CS was shed to lower titres from infected ferrets and was not transmitted to cohoused sentinel animals. Thus, the polybasic CS is a key determinant for efficient SARS-CoV-2 transmission.

CRISPR activation and interference (CRISPRa/i) technology offers the unprecedented possibility of achieving regulated gene expression both in vitro and in vivo. The DNA pairing specificity of a nuclease dead Cas9 (dCas9) is exploited to precisely target a transcriptional activator or repressor in proximity to a gene promoter. This permits both the study of phenotypes arising from gene modulation for investigative purposes, and the development of potential therapeutics. As with virtually all other organ systems, the cardiovascular system can deeply benefit from a broader utilisation of CRISPRa/i. However, application of this technology is still in its infancy. Significant areas for improvement include the identification of novel and more effective transcriptional regulators that can be docked to dCas9, and the development of more efficient methods for their delivery and expression in vivo.

Aims After a myocardial infarction, the adult human heart lacks sufficient regenerative capacity to restore lost tissue, leading to heart failure progression. Finding novel ways to reprogram adult cardiomyocytes into a regenerative state is a major therapeutic goal. The epicardium, the outermost layer of the heart, contributes cardiovascular cell types to the forming heart and is a source of trophic signals to promote heart muscle growth during embryonic development. The epicardium is also essential for heart regeneration in zebrafish and neonatal mice and can be reactivated after injury in adult hearts to improve outcome. A recently identified mechanism of cell–cell communication and signalling is that mediated by extracellular vesicles (EVs). Here, we aimed to investigate epicardial signalling via EV release in response to cardiac injury and as a means to optimize cardiac repair and regeneration. Methods and results We isolated epicardial EVs from mouse and human sources and targeted the cardiomyocyte population. Epicardial EVs enhanced proliferation in H9C2 cells and in primary neonatal murine cardiomyocytes in vitro and promoted cell cycle re-entry when injected into the injured area of infarcted neonatal hearts. These EVs also enhanced regeneration in cryoinjured engineered human myocardium (EHM) as a novel model of human myocardial injury. Deep RNA-sequencing of epicardial EV cargo revealed conserved microRNAs (miRs) between human and mouse epicardial-derived exosomes, and the effects on cell cycle re-entry were recapitulated by administration of cargo miR-30a, miR-100, miR-27a, and miR-30e to human stem cell-derived cardiomyocytes and cryoinjured EHM constructs. Conclusion Here, we describe the first characterization of epicardial EV secretion, which can signal to promote proliferation of cardiomyocytes in infarcted mouse hearts and in a human model of myocardial injury, resulting in enhanced contractile function. Analysis of exosome cargo in mouse and human identified conserved pro-regenerative miRs, which in combination recapitulated the therapeutic effects of promoting cardiomyocyte proliferation., Graphical Abstract

Antipsychotic use is associated with an increased risk of type 2 diabetes. Recent work suggests antipsychotics can induce insulin resistance immediately and independently of weight gain, and that this may occur via the central nervous system (CNS). We have previously shown that the highly effective and widely prescribed antipsychotic, olanzapine inhibits CNS insulin-mediated suppression of hepatic glucose production, but the mechanisms remain unknown. The ATP-sensitive potassium (KATP) channel is a key metabolic sensor downstream of hypothalamic insulin signalling, involved in the maintenance of glucose homeostasis. Thus, the possibility arises that olanzapine inhibits central KATP channel activation to disrupt glucose metabolism. We replicate that intracerebroventricular (ICV) administration of the KATP channel activator, diazoxide, suppresses hepatic glucose production and additionally demonstrate stimulation of peripheral glucose utilization. We report that olanzapine inhibits the effects of central KATP channel activation resulting in perturbation of whole body insulin sensitivity, specifically via inhibition of glucose utilization, while leaving central KATP channel-mediated suppression of glucose production intact. Perturbation of KATP channel action in the CNS could represent a novel mechanism of antipsychotic-induced diabetes.

Adeno-associated virus (AAV) is a small, non-enveloped virus used as vector in gene therapy, mainly produced in human cells and in baculovirus systems. Intense studies on these platforms led to the production of vectors with titers between 103 and 105 viral genomes (vg) per cells. In spite of this, vector yields need to be improved to satisfy the high product demands of clinical trials and future commercialization. Our studies and those of other groups have explored the possibility to exploit the yeast Saccharomyces cerevisiae to produce rAAV. We previously demonstrated that yeast supports AAV genome replication and capsid assembly. The purpose of this study was to evaluate the quality of the encapsidated AAV DNA. Here, we report the construction of a yeast strain expressing Rep68/40 from an integrated copy of the Rep gene under the control of the yeast constitutive ADH promoter and Capsid proteins from the Cap gene under the control of an inducible GAL promoter. Our results indicate that a portion of AAV particles generated by this system contains encapsidated AAV DNA. However, the majority of encapsidated DNA consists of fragmented regions of the transgene cassette, with ITRs being the most represented sequences. Altogether, these data indicate that, in yeast, encapsidation occurs with low efficiency and that rAAVs resemble pseudo-vectors that are present in clinical-grade rAAV preparations.

Management of recurrent rectovaginal fistula (rRVF) remains challenging despite the good results of graciloplasty reported in the literature. However, little is known about how to avoid a permanent stoma if graciloplasty fails. The aim of our study was to report the management of rRVF after failure of graciloplasty.A retrospective study was performed on consecutive patients with rRVF after failure of graciloplasty treated at our institution in January 2005-December 2021.There were 19 patients, with a median age at graciloplasty of 39 years (range 25-64 years). Etiologies of RVF were Crohn's disease (CD) (n = 10), postoperative (n = 5), post-obstetrical (n = 3), and unknown (n = 1). After failure of graciloplasty, 45 new procedures were performed, all of them with a covering stoma: trans-anal repairs (n = 31), delayed colo-anal anastomosis (DCAA) (n = 4), biological mesh interposition (n = 3), second graciloplasty (n = 3), stoma only (n = 2) and redo ileal pouch-anal anastomosis (IPAA) (n = 2). One patient was not re-operated on and instead treated medically for CD. After a mean follow-up of 63 ± 49 months, success (i.e., absence of stoma or RVF) was obtained in 11 patients (58%): 4/4 DCAA (100%), 5/31 after local repair (16%), 1 after stoma creation alone (50%) and 1 after redo IPAA (50%). Second graciloplasty and biologic mesh interposition all failed. All 8 patients with failed intervention had CD.In cases of rRVF after failed graciloplasty, reoperation is possible, although the chance of success is relatively low. The best results were obtained with DCAA. CD is a predictor of poor outcome.

C-reactive protein (CRP) is a common biomarker of inflammation which has largely been used to predict the risk of postoperative septic complications after colorectal surgery. However, no data exist concerning its potential benefit after ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC). The aim of this study was to evaluate a CRP-driven monitoring discharge strategy after laparoscopic IPAA for UC.Since 2012, 158 patients undergoing a laparoscopic IPAA for UC have been included: 66 patients (CRP group) operated since 2016 had a CRP-driven monitoring discharge on postoperative day 5 (POD 5) and were discharged on POD 6 if CRP 100 mg/L; these patients were matched (according to age, gender, body mass index, IPAA in two or three steps) to 92 patients operated between 2012 and 2016 without any CRP monitoring (control group).Median length of hospital stay was shorter in the CRP than the control group (7 vs. 9 days; P 0.001) and discharge on POD 6 occurred more frequently in the CRP group (47% vs. 7%, P 0.001). No difference was observed between the two groups concerning overall morbidity (P = 0.980), surgical site infection (P = 0.554), Clavien-Dindo ≥ IIIa morbidity (P = 0.523), unplanned rehospitalization (P = 0.734) and 30-day reoperation (P = 0.240).CRP-driven monitoring discharge strategy after laparoscopic IPAA for UC is associated with a significant reduction in length of hospital stay, without increasing morbidity, reoperation or rehospitalization rates.

Background: Obesity and type 2 diabetes (T2D) are associated with chronic low-grade inflammation. Elevated free fatty acids (FFAs) , as seen in obesity and T2D, can act as damage associated molecular patterns (DAMPs) activating the innate immune system. Recent studies have identified nucleotide binding oligomerization domain 1 (NOD1) , an intracellular pattern recognition receptor, can be activated by saturated fatty acids (sFA) . NOD1 activates inflammatory kinase RIPK2, leading to increased cytokine production by the innate immune system. Our study aimed to determine whether wild type mice treated with GSK583, a RIPK2 inhibitor, are protected against FFA-induced glucose intolerance. Material and Methods: C57Bl/6J mice were surgically cannulated in their jugular veins and were infused with ethylpalmitate (EtP) for 48h, which results in increased plasma palmitate levels and mimics the elevated FFA levels seen in vivo. Furthermore, mice were also infused with EtP and GSK583 for 48h. After infusion, the mice were subjected to a hyperglycemic clamp and the glucose infusion rate was measured to assess glucose tolerance that under hyperglycemic clamp conditions, is mainly determined by the beta cell ability to compensate for FFA-induced insulin resistance. Results: Preliminary data revealed that mice infused with EtP and GSK583 had a higher glucose infusion rate (GIR; mg/kgmin) (83+20, mean+SEM) than mice infused with EtP alone (57+4) . The EtP and GSK583 was comparable to the GIR of control (EtP vehicle infused) mice (84+7) . Conclusions: Our data indicate that pharmacological inhibition of NOD1 signaling protects against FFA-induced glucose intolerance in mice. Targeting NOD1 may be an effective strategy for treating beta cell dysfunction in T2D. Disclosure N.Tsakiridis: None. A.Ivovic: None. S.Rahman: None. Y.Tan: None. A.Giacca: None.

Percutaneous transluminal coronary angioplasty is an effective treatment for stenotic lesions, but restenosis remains as a common adverse effect and contributes to post-angioplasty failure. Patients with type 2 diabetes have higher risk of post-angioplasty restenosis due to insulin resistance. In vitro studies showed that insulin has a vasculoprotective effect on endothelial cells (ECs) which is likely mediated by phosphoinositide 3-kinase (PI3K) /Akt pathway, while its mitogenic effects are mainly manifested on vascular smooth muscle cells (VSMCs) and likely mediated via mitogen-activated protein kinase (MAPK) pathway. Currently, there is not much information about pathway-specificity of insulin’s vascular effects in in vivo models. This study aims to determine the Akt2- and MEK1 (MAPK kinase activator) -mediated effects on neointimal growth in a murine model of restenosis. Akt2/MEK1 floxed mice were crossed with tamoxifen-inducible Cre recombinase under the control of vascular endothelial cadherin (Cdh5) or smooth muscle myosin heavy chain (SMMHC) promoters. Mice were implanted subcutaneously with either insulin pellets (0.U/day) or blank pellets three days prior to femoral artery wire injury surgery. Femoral arteries were collected 28 days after surgery for histology analysis. We found that both neointimal area (NA) and intima-to-media ratio (I/M) were lowered in Akt2 floxed Cre- and and wild type Cre+ control mice following insulin treatment, however, insulin failed to have the same effect in both EC and SMC Akt2 deficient mice. Preliminary data suggest that the effect of insulin on decreasing NA and I/M may be greater in EC MEK1 deficient mice but not in SMC MEK1 deficient mice than controls. These data suggest that both EC and VSMC Akt2 contribute to the beneficial effects of insulin, while EC-specific MEK1 knockdown may accentuate the beneficial effects of insulin. Disclosure Z.Liu: None. M.Gonzalez: None. S.Ou: None. L.Qiu: None. A.Giacca: None.

Angioplasty and stenting are widely used in the treatment of atherosclerosis. However, these procedures may fail due to post-angioplasty restenosis, which remains a problem for patients with diabetes. Currently, the vascular role of insulin is unclear. Its role on endothelial cells (EC) is vasculoprotective, whereas its effects on vascular smooth muscle cells (SMC) are mainly mitogenic according to in vitro data. We have previously shown a suppressive effect of insulin treatment on neointimal growth in rodent models of restenosis in insulin sensitive conditions that was abolished in insulin resistant conditions. The objective of this study was to determine the EC-and SMC-specific effects of insulin treatment on neointimal growth in a murine model of restenosis in insulin sensitive and insulin resistant conditions. Mice were generated by crossing insulin receptor (IR) floxed mice with tamoxifen inducible Cre recombinant mice under the control of Cdh5 (vascular endothelial cadherin) or SMMHC (smooth muscle heavy chain) promoters. Mice were fed with a low-fat (LFD) or a high-fat-high-sucrose (HFSD) diet and implanted with insulin pellet (0.U/day) or vehicle (control) prior to femoral artery wire injury. We found that compared to IR-floxed controls, tamoxifen-induced IRf/f-Cdh5-Cre+ or IRf/f-SMMHC-Cre+ mice showed a 94% and 80% decrease in IR expression in ECs or SMCs, respectively. In LFD-fed insulin sensitive conditions, insulin decreased neointimal area (NA) and intima-to-media ratio (I/M) in controls (Cdh5 and-SMMHC-floxed and wildtype-Cre+ mice) but not in endothelial and SMC IR deficient mice. In HFSD-fed insulin resistant conditions, insulin had no effect in either controls or endothelial and SMC IR deficient mice. These data demonstrate that insulin action in both ECs and SMCs is required for the anti-restenotic effect of insulin in insulin sensitive conditions, which is abolished but not reversed in insulin resistance even when endothelial IR is deficient. Disclosure J.Z. Wang: None. C.X. Zhang: None. A. Giacca: None.

Restenosis following percutaneous transluminal angioplasty and stenting is increased in subjects with insulin resistance and diabetes. Our preclinical models of restenosis show that insulin treatment is vasculoprotective in insulin sensitive conditions but insulin's effect is diminished or lost in Western diet induced insulin resistant conditions. Accordingly, early insulin treatment has not proven to provide cardiovascular disease (CVD) protection in subjects with dysglycemia, while the effect of intensive insulin treatment of type 2 diabetes (T2D) is debated. Glucagon-like peptide-1 receptor agonists (GLP-1RA) are a new class of antidiabetic drugs that reduce the CVD risk of T2D. Several animal studies have shown that GLP-1RA decrease neointimal growth in models of restenosis; however, the mechanisms of the CVD actions of these drugs remain elusive. GLP-1RA are insulin secretagogues, therefore their effect may be partly mediated by the insulin receptor (IR) . The objective of this study was to determine if insulin receptor (IR) deficiency in endothelial cells (EC) attenuates the effect of GLP-1RA to decrease neointimal growth after arterial injury. Endothelial deficient IR mice were generated by crossing IR floxed mice with mice expressing tamoxifen-inducible Cre recombinase under the control of Tie2 promoter. Mice were fed a low-fat (LFD) or a high-fat-high-sucrose (HFSD) Western diet and implanted with an osmotic pump delivering liraglutide, a GLP-1RA prototype (1mol/kg/day) , or vehicle prior to femoral artery wire injury. In LFD- as well as in HFSD-fed IR-floxed controls, liraglutide decreased neointimal area (NA) and intima-to-media ratio (I/M) . In LFD-fed endothelial IR deficient mice liraglutide had no effect. These data suggest that IR in ECs is important for the anti-restenotic effect of GLP1-RA, which is preserved in Western diet induced insulin resistant conditions unlike the effect of insulin. *First and second authors contributed equally Disclosure M.Gonzalez: None. Z.Liu: None. A.Giacca: None.