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Drug delivery systems such as liposomes are widely used to stabilize and increase the plasma half-life of therapeutics. In this article, we have investigated two strategies to increase the half-life of deoxyribonuclease I, an FDA-approved enzyme used for the treatment of cystic fibrosis, and a potential candidate for the reduction of uncontrolled inflammation induced by neutrophil extracellular traps. We demonstrate that our optimized preparation procedure resulted in nanoparticles with improved plasma half-life and total exposure relative to native protein, while maintaining enzymatic activity.

The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directs infection of the lungs and other tissues following its binding to the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2'. The "priming" of the surface S protein at S1/S2 (P

International audience; The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directs infection of the lungs and other tissues following its binding to the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2′. The “priming” of the surface S protein at S1/S2 (PRRAR685↓) [the underlined basic amino acids refer to critical residues needed for the furin recognition] by furin has been shown to be important for SARS-CoV-2 infectivity in cells and small-animal models. In this study, for the first time we unambiguously identified by proteomics the fusion activation site S2′ as KPSKR815↓ (the underlined basic amino acids refer to critical residues needed for the furin recognition) and demonstrated that this cleavage was strongly enhanced by ACE2 engagement with the S protein. Novel pharmacological furin inhibitors (BOS inhibitors) effectively blocked endogenous S protein processing at both sites in HeLa cells, and SARS-CoV-2 infection of lung-derived Calu-3 cells was completely prevented by combined inhibitors of furin (BOS) and type II transmembrane serine protease 2 (TMPRSS2) (camostat). Quantitative analyses of cell-to-cell fusion and S protein processing revealed that ACE2 shedding by TMPRSS2 was required for TMPRSS2-mediated enhancement of fusion in the absence of S1/S2 priming. We further demonstrated that the collectrin dimerization domain of ACE2 was essential for the effect of TMPRSS2 on cell-to-cell fusion. Overall, our results indicate that furin and TMPRSS2 act synergistically in viral entry and infectivity, supporting the combination of furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2.IMPORTANCE SARS-CoV-2, the etiological agent of COVID-19, has so far resulted in >6.1 million deaths worldwide. The spike protein (S) of the virus directs infection of the lungs and other tissues by binding the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2′. Cleavage at S1/S2 induces a conformational change favoring the S protein recognition by ACE2. The S2′ cleavage is critical for triggering membrane fusion and virus entry into host cells. Our study highlights the complex dynamics of interaction between the S protein, ACE2, and the host proteases furin and TMPRSS2 during SARS-CoV-2 entry and suggests that the combination of a nontoxic furin inhibitor with a TMPRSS2 inhibitor significantly reduces viral entry in lung cells, as evidenced by an average synergistic ∼95% reduction of viral infection. This represents a powerful novel antiviral approach to reduce viral spread in individuals infected by SARS-CoV-2 or future related coronaviruses.

Objective SSc is an autoimmune connective tissue disorder characterized by inflammation and fibrosis. Although constitutive activation of fibroblasts is proposed to be responsible for the fibrotic and inflammatory features of the disease, the underlying mechanism remains elusive, and effective therapeutic targets are still lacking. The aim of this study was to evaluate the role of oxidative stress-induced senescence and its contribution to the pro-fibrotic and pro-inflammatory phenotypes of fibroblasts from SSc patients. Methods Dermal fibroblasts were isolated from SSc (n = 13) and healthy (n = 10) donors. Fibroblasts’ intracellular and mitochondrial reactive oxygen species (ROS) were determined by flow cytometry. Mitochondrial function was measured by Seahorse XF24 analyser. Fibrotic and inflammatory gene expressions were assessed by qPCR and key pro-inflammatory components of the fibroblasts’ secretome (IL-6 and IL-8) were quantified by ELISA. Results Compared with healthy fibroblasts, SSc fibroblasts displayed higher levels of both intracellular and mitochondrial ROS. Oxidative stress in SSc fibroblasts induced the expression of fibrotic genes and activated the TGF-β-activated kinase 1 (TAK1)–IκB kinase β (IKKβ)–IFN regulatory factor 5 (IRF5) inflammatory signalling cascade. These cellular responses paralleled the presence of a DNA damage response, a senescence-associated secretory phenotype and a fibrotic response. Treatment of SSc fibroblasts with ROS scavengers reduced their pro-inflammatory secretome production and fibrotic gene expression. Conclusions Oxidative stress-induced cellular senescence in SSc fibroblasts underlies their pro-inflammatory and pro-fibrotic phenotypes. Targeting redox imbalance of SSc fibroblasts enhances their in vitro functions and could be of relevance for SSc therapy.

Aims Diabetes is a conventional risk factor for atherosclerotic cardiovascular disease and myocardial infarction (MI) is the most common cause of death among these patients. Mesenchymal stromal cells (MSCs) in patients with type 2 diabetes mellitus (T2DM) and atherosclerosis have impaired ability to suppress activated T-cells (i.e. reduced immunopotency). This is mediated by an inflammatory shift in MSC-secreted soluble factors (i.e. pro-inflammatory secretome) and can contribute to the reduced therapeutic effects of autologous T2DM and atherosclerosis-MSC post-MI. The signalling pathways driving the altered secretome of atherosclerosis- and T2DM-MSC are unknown. Specifically, the effect of IκB kinase β (IKKβ) modulation, a key regulator of inflammatory responses, on the immunopotency of MSCs from T2DM patients with advanced atherosclerosis has not been studied. Methods and results MSCs were isolated from adipose tissue obtained from patients with (i) atherosclerosis and T2DM (atherosclerosis+T2DM MSCs, n = 17) and (ii) atherosclerosis without T2DM (atherosclerosis MSCs, n = 17). MSCs from atherosclerosis+T2DM individuals displayed an inflammatory senescent phenotype and constitutively expressed active forms of effectors of the canonical IKKβ nuclear factor-κB transcription factors inflammatory pathway. Importantly, this constitutive pro-inflammatory IKKβ signature resulted in an altered secretome and impaired in vitro immunopotency and in vivo healing capacity in an acute MI model. Notably, treatment with a selective IKKβ inhibitor or IKKβ knockdown (KD) (clustered regularly interspaced short palindromic repeats/Cas9-mediated IKKβ KD) in atherosclerosis+T2DM MSCs reduced the production of pro-inflammatory secretome, increased survival, and rescued their immunopotency both in vitro and in vivo. Conclusions Constitutively active IKKβ reduces the immunopotency of atherosclerosis+T2DM MSC by changing their secretome composition. Modulation of IKKβ in atherosclerosis+T2DM MSCs enhances their myocardial repair ability.

Unacylated ghrelin (UAG), the most abundant form of ghrelin in circulation, has been shown to exert cardioprotective effect in experimental cardiopathies. The present study aimed to investigate the cardioprotective effect of a linear bioactive fragment of UAG against myocardial ischemia-induced injury and dysfunction in C57BL/6 wild type mice and the mechanisms involved. Treatments were administered at doses of 100 (UAG), 1,000 and 3,000 (UAG6−13) nmol/kg at 12 h interval during 14 days prior to 30 min left coronary artery ligation and reperfusion for a period of 6 or 48 h. The infarct area was decreased in a dose-dependent manner at 48 h of reperfusion, with a reduction of 54% at the highest dose of UAG6−13 tested. Myocardial hemodynamics were improved as demonstrated by an increase in cardiac output, maximum first derivative of left ventricular pressure, and preload recruitable stroke work, a load-independent contractility index. Six hours after reperfusion, circulating levels of IL-6 and TNF-α pro-inflammatory cytokines were reduced, and the effect was maintained at 48 h for TNF-α. 5′ AMP-activated protein kinase (AMPK) was activated, while acetyl-CoA carboxylase (ACC) activity was inhibited, along with a decrease in apoptotic protein levels. In isolated hearts, the effect of UAG6−13 was unaffected by the presence of D-Lys3-GHRP-6, a ghrelin receptor (GHSR1a) antagonist, suggesting that the peptide acted through a GHSR1a-independent pathway. The results support the therapeutic application of UAG bioactive peptide fragments against myocardial ischemia/reperfusion injury.

Unacylated ghrelin (UAG), the most abundant form of ghrelin in circulation, has been shown to exert cardioprotective effect in experimental cardiopathies. The present study aimed to investigate the cardioprotective effect of a linear bioactive fragment of UAG against myocardial ischemia-induced injury and dysfunction in C57BL/6 wild type mice and the mechanisms involved. Treatments were administered at doses of 100 (UAG), 1,000 and 3,000 (UAG6−13) nmol/kg at 12 h interval during 14 days prior to 30 min left coronary artery ligation and reperfusion for a period of 6 or 48 h. The infarct area was decreased in a dose-dependent manner at 48 h of reperfusion, with a reduction of 54% at the highest dose of UAG6−13 tested. Myocardial hemodynamics were improved as demonstrated by an increase in cardiac output, maximum first derivative of left ventricular pressure, and preload recruitable stroke work, a load-independent contractility index. Six hours after reperfusion, circulating levels of IL-6 and TNF-α pro-inflammatory cytokines were reduced, and the effect was maintained at 48 h for TNF-α. 5′ AMP-activated protein kinase (AMPK) was activated, while acetyl-CoA carboxylase (ACC) activity was inhibited, along with a decrease in apoptotic protein levels. In isolated hearts, the effect of UAG6−13 was unaffected by the presence of D-Lys3-GHRP-6, a ghrelin receptor (GHSR1a) antagonist, suggesting that the peptide acted through a GHSR1a-independent pathway. The results support the therapeutic application of UAG bioactive peptide fragments against myocardial ischemia/reperfusion injury.

CD36 is a multiligand receptor involved in lipid metabolism. We investigated the mechanisms underlying the cardioprotective effect of CP-3(iv), an azapeptide belonging to a new class of selective CD36 ligands. The role of CP-3(iv) in mediating cardioprotection was investigated because CD36 signaling leads to activation of peroxisome proliferator-activated receptor-γ, a transcriptional regulator of adiponectin. CP-3(iv) pretreatment reduced infarct size by 54% and preserved hemodynamics in C57BL/6 mice subjected to 30 min coronary ligation and reperfusion but had no effect in CD36-deficient mice. The effects of CP-3(iv) were associated with an increase in circulating adiponectin levels, epididymal fat adiponectin gene expression, and adiponectin transcriptional regulators ( Pparg, Cebpb, Sirt1) after 6 h of reperfusion. Reduced myocardial oxidative stress and apoptosis were observed along with an increase in expression of myocardial adiponectin target proteins, including cyclooxygenase-2, phospho-AMPK, and phospho-Akt. Moreover, CP-3(iv) increased myocardial performance in isolated hearts, whereas blockade of adiponectin with an anti-adiponectin antibody abrogated it. CP-3(iv) exerts cardioprotection against myocardial ischemia and reperfusion (MI/R) injury and dysfunction, at least in part, by increasing circulating and myocardial adiponectin levels. Hence, both paracrine and endocrine effects of adiponectin may contribute to reduced reactive oxygen species generation and apoptosis after MI/R, in a CD36-dependent manner.-Huynh, D. N., Bessi, V. L., Menard, L., Piquereau, J., Proulx, C., Febbraio, M., Lubell, W. D., Carpentier, A. C., Burelle, Y., Ong, H., Marleau, S. Adiponectin has a pivotal role in the cardioprotective effect of CP-3(iv), a selective CD36 azapeptide ligand, after transient coronary artery occlusion in mice.

Insufficient oxygen delivery to organs leads to tissue dysfunction and cell death. Reperfusion, although vital to organ survival, initiates an inflammatory response that may both aggravate local tissue injury and elicit remote organ damage. Polymorphonuclear neutrophil (PMN) trafficking to remote organs following ischaemia/reperfusion (I/R) is associated with the release of lipid mediators, including leucotriene (LT) B4 , cysteinyl-LTs (CysLTs) and platelet-activating factor (PAF). Yet, their potentially cooperative role in regulating I/R-mediated inflammation has not been thoroughly assessed. The present study aimed to determine the cooperative role of lipid mediators in regulating PMN migration, tissue oedema and injury using selective receptor antagonists in selected models of I/R and dermal inflammation. Our results show that rabbits, pre-treated orally with BIIL 284 and/or WEB 2086 and MK-0571, were protected from remote tissue injury following I/R or dermal inflammation in an additive or synergistic manner when the animals were pre-treated with two drugs concomitantly. The functional selectivity of the antagonists towards their respective agonists was assessed in vitro, showing that neither BIIL 284 nor WEB 2086 prevented the inflammatory response to IL-8, C5a and zymosan-activated plasma stimulation. However, these agonists elicited LTB4 biosynthesis in isolated rabbit PMNs. Similarly, a cardioprotective effect of PAF and LTB4 receptor antagonists was shown following myocardial I/R in mice. Taken together, these results underscore the intricate involvement of LTB4 and PAF in each other's responses and provide further evidence that targeting both LTs and PAF receptors provides a much stronger anti-inflammatory effect, regulating PMN migration and oedema formation.

Aims The CD36 receptor plays an important role in facilitating fatty acid transport to the heart. The present study aimed to assess whether EP 80317, a selective synthetic peptide ligand of CD36, is cardioprotective in a murine model of myocardial ischaemia and reperfusion (MI/R) injury. Methods and results Mice were pretreated with daily subcutaneous injections of EP 80317 for 14 days before being subjected to a 30 min ligation of the left anterior descending coronary artery. The treatment reduced the infarct area and improved myocardial haemodynamics and function, as shown by an increase in cardiac output, ejection fraction and stroke work, and a reduced total peripheral resistance. In contrast, administration of EP 51389, a tripeptide analogue devoid of binding affinity to CD36, did not protect against myocardial injury. Six hours after myocardial reperfusion, EP 80317-treated mice showed reduced myocardial fatty acid uptake, as assessed by micro-positron emission tomography, in agreement with reduced levels of circulating non-esterified fatty acids. Studies using [14C]-palmitate infusion revealed reduced lipolysis, although no significant change in insulin or catecholamine plasma levels were observed. Increased expression levels of adipogenic and anti-lipolytic genes further supported an effect of EP 80317 in preventing fatty acid mobilization from adipose tissue. No effect of the treatment was observed in CD36−/− mice. Conclusion Our results show that pretreatment with EP 80317 protected the heart against damage and dysfunction elicited by MI/R, along with a transient reduction in peripheral lipolysis. Our findings support CD36 as a novel target for the treatment of ischaemic cardiopathy.

Atherosclerosis is the main underlying cause of ischemic heart disease and related acute cardiovascular complications, including myocardial infarction and stroke. In view of the failure of statins to demonstrate a beneficial effect in all patients, exhaustive research efforts have unfold into different research avenues, in close relation to the increase in basic knowledge regarding lipoprotein metabolism, macrophage function and inflammatory conditions associated with atherosclerosis. This review focuses specifically on potential therapeutic peptides targeting dyslipidemia, macrophage scavenger receptors, cholesterol metabolism and anti-inflammatory cytokines as novel therapeutic avenues in atherosclerosis.

Atherosclerosis is the main underlying cause of ischemic heart disease and related acute cardiovascular complications, including myocardial infarction and stroke. In view of the failure of statins to demonstrate a beneficial effect in all patients, exhaustive research efforts have unfold into different research avenues, in close relation to the increase in basic knowledge regarding lipoprotein metabolism, macrophage function and inflammatory conditions associated with atherosclerosis. This review focuses specifically on potential therapeutic peptides targeting dyslipidemia, macrophage scavenger receptors, cholesterol metabolism and anti-inflammatory cytokines as novel therapeutic avenues in atherosclerosis.