Sylvia J. P. Bogaards

Bisoprolol Delays Progression Towards Right Heart Failure in Experimental Pulmonary Hypertension

Background— In pulmonary arterial hypertension (PH), sympathetic adrenergic activity is highly elevated. Sympathetic overactivity is a compensatory mechanism at first, but might be detrimental for cardiac function in the long run. We therefore investigated whether chronic low-dose treatment with bisoprolol (a cardioselective &bgr;-blocker) has beneficial effects on cardiac function in experimental PH. Methods and Results— PH was induced in rats by a single injection of monocrotaline (60 mg/kg). Pressure telemetry in PH rats revealed that 10 mg/kg bisoprolol was the lowest dose that blunted heart rate response during daily activity. Ten days after monocrotaline injection, echocardiography was performed and PH rats were randomized for bisoprolol treatment (oral gavage) or vehicle (n=7/group). At end of study (body mass loss >5%), echocardiography was repeated, with additional pressure-volume measurements and histomolecular analyses. Compared with control, right ventricular (RV) systolic pressure and arterial elastance (measure of vascular resistance) more than tripled in PH. Bisoprolol delayed time to right heart failure (P<0.05). RV afterload was unaffected, however, bisoprolol treatment increased RV contractility and filling (both P<0.01), and partially restored right ventriculo-arterial coupling and cardiac output (both P<0.05). Bisoprolol restored RV &bgr;-adrenergic receptor signaling. Histology revealed significantly less RV fibrosis and myocardial inflammation in bisoprolol treated PH rats. Conclusions— In experimental PH, treatment with bisoprolol delays progression toward right heart failure, and partially preserves RV systolic and diastolic function. These promising results suggest a therapeutic role for &bgr;-blockers in PH that warrants further clinical investigation.

The Synthetic N-Terminal Peptide of Human Lactoferrin, hLF(1-11), Is Highly Effective against Experimental Infection Caused by Multidrug-Resistant Acinetobacter baumannii

ABSTRACT The lactoferrin-derived peptide hLF(1-11), but not its control peptide, was highly effective against five multidrug-resistant Acinetobacter baumannii strains in vitro (3 to 4 log reduction) and against four of these strains in an experimental infection in mice (2 to 3 log reduction). Therefore, this peptide is a promising candidate as a novel agent against infections with multidrug-resistant A. baumannii.

Antimicrobial Peptide hLF1-11 Directs Granulocyte-Macrophage Colony-Stimulating Factor-Driven Monocyte Differentiation toward Macrophages with Enhanced Recognition and Clearance of Pathogens

ABSTRACT The human lactoferrin-derived peptide hLF1-11 displays antimicrobial activities in vitro and is effective against infections with antibiotic-resistant bacteria and fluconazole-resistant Candida albicans in animals. However, the mechanisms underlying these activities remain largely unclear. Since hLF1-11 is ineffective in vitro at physiological salt concentrations, we suggested modulation of the immune system as an additional mechanism of action of the peptide. We investigated whether hLF1-11 affects human monocyte-macrophage differentiation and determined the antimicrobial activities of the resulting macrophages. Monocytes were cultured for 7 days with GM-CSF in the presence of hLF1-11, control peptide, or saline for various intervals. At day 6, the cells were stimulated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or heat-killed C. albicans for 24 h. Thereafter, the levels of cytokines in the culture supernatants, the expression of pathogen recognition receptors, and the antimicrobial activities of these macrophages were determined. The results showed that a short exposure of monocytes to hLF1-11 during GM-CSF-driven differentiation is sufficient to direct differentiation of monocytes toward a macrophage subset characterized by both pro- and anti-inflammatory cytokine production and increased responsiveness to microbial structures. Moreover, these macrophages are highly effective against C. albicans and Staphylococcus aureus. In conclusion, hLF1-11 directs GM-CSF-driven differentiation of monocytes toward macrophages with enhanced effector functions.

Synthetic peptides derived from human antimicrobial peptide ubiquicidin accumulate at sites of infections and eradicate (multi-drug resistant) Staphylococcus aureus in mice

The presence and antimicrobial activity of antimicrobial peptides (AMPs) has been widely recognized as an evolutionary preserved part of the innate immune system. Based on evidence in animal models and humans, AMPs are now positioned as novel anti-infective agents. The current study aimed to evaluate the potential antimicrobial activity of ubiquicidin and small synthetic fragments thereof towards methicillin resistant Staphylococcus aureus (MRSA), as a high priority target for novel antibiotics. In vitro killing of MRSA by synthetic peptides derived from the alpha-helix or beta-sheet domains of the human cationic peptide ubiquicidin (UBI 1-59), allowed selection of AMPs for possible treatment of MRSA infections. The strongest antibacterial activity was observed for the entire peptide UBI 1-59 and for synthetic fragments comprising amino acids 31-38. The availability, chemical synthesis opportunities, and size of these small peptides, combined with their strong antimicrobial activity towards MRSA make these compounds promising candidates for antimicrobial therapy and detection of infections in man.

The Human Lactoferrin-Derived Peptide hLF1-11 Exerts Immunomodulatory Effects by Specific Inhibition of Myeloperoxidase Activity

Because of their ability to eliminate pathogens and to modulate various host immune responses, antimicrobial peptides are considered as candidate agents to fight infections by (antibiotic-resistant) pathogens. We recently reported that hLF1-11 (GRRRRSVQWCA), an antimicrobial peptide derived from the N terminus of human lactoferrin, displays diverse modulatory activities on monocytes, thereby enhancing their actions in innate immune responses. The aim of this study was to identify the cellular target of hLF1-11 that mediates these effects. Results revealed that hLF1-11 binds and subsequently penetrates human monocytes, after which it inhibits the enzymatic activities of myeloperoxidase (MPO). Moreover, a chemical inhibitor of MPO (aminobenzoic acid hydrazide) mimicked the effects of hLF1-11 on the inflammatory response by monocytes and on monocyte–macrophage differentiation. Computer-assisted molecular modeling predicted that hLF1-11 can bind to the edge of and within the crevice of the active site of MPO. Experiments with a set of hLF1-11 peptides with amino acid substitutions identified the stretch of arginines and the cysteine at position 10 as pivotal in these immunomodulatory properties of hLF1-11. We conclude that hLF1-11 may exert its modulatory effects on human monocytes by specific inhibition of MPO activity.

The Antimicrobial Peptide hLF1–11 Drives Monocyte-Dendritic Cell Differentiation toward Dendritic Cells That Promote Antifungal Responses and Enhance Th17 Polarization

The hLF1–11 peptide comprising the first 11 N-terminal residues of human lactoferrin exerts antimicrobial activity in vivo, enhances the inflammatory response of monocytes and directs monocyte-macrophage differentiation toward cells with enhanced antimicrobial properties. In this study, we investigated the effects of hLF1–11 on human monocyte-dendritic cell (DC) differentiation and subsequent T cell activation. Results revealed that – compared to control (peptide-incubated) DCs – hLF1–11-differentiated DCs displayed enhanced expression of HLA class II antigens and dectin-1, and increased phagocytosis of Candida albicans. In addition, hLF1–11-differentiated DCs produced enhanced amounts of reactive oxygen species, IL-6 and IL-10, but not IL-12p40 and TNF-α, upon stimulation with C. albicans. Moreover, 6-day-cultured hLF1–11-differentiated DCs and control (peptide-incubated) DCs that had been stimulated with a Th17-inducing mix of antigens (including C. albicans) for 24 h were cocultured with autologous CD4+ T cells for 72 h and then the levels of IL-10, IL-17 and IFN-γ production and the percentage of cytokine-producing T cells were assessed. The results revealed that the hLF1–11-differentiated DCs induced an enhanced IL-17, but reduced IFN-γ, production by T cells as compared to control (peptide-incubated) DCs. Collectively, the hLF1–11 peptide drives monocyte-DC differentiation toward DCs that promote antifungal responses and enhance Th17 polarization.

Impaired microcirculatory perfusion in a rat model of cardiopulmonary bypass: the role of hemodilution

Although hemodilution is attributed as the main cause of microcirculatory impairment during cardiopulmonary bypass (CPB), this relationship has never been investigated. We investigated the distinct effects of hemodilution with or without CPB on microvascular perfusion and subsequent renal tissue injury in a rat model. Male Wistar rats (375-425 g) were anesthetized, prepared for cremaster muscle intravital microscopy, and subjected to CPB (n = 9), hemodilution alone (n = 9), or a sham procedure (n = 6). Microcirculatory recordings were performed at multiple time points and analyzed for perfusion characteristics. Kidney and lung tissue were investigated for mRNA expression for genes regulating inflammation and endothelial adhesion molecule expression. Renal injury was assessed with immunohistochemistry. Hematocrit levels dropped to 0.24 ± 0.03 l/l and 0.22 ± 0.02 l/l after onset of hemodilution with or without CPB. Microcirculatory perfusion remained unaltered in sham rats. Hemodilution alone induced a 13% decrease in perfused capillaries, after which recovery was observed. Onset of CPB reduced the perfused capillaries by 40% (9.2 ± 0.9 to 5.5 ± 1.5 perfused capillaries per microscope field; P < 0.001), and this reduction persisted throughout the experiment. Endothelial and inflammatory activation and renal histological injury were increased after CPB compared with hemodilution or sham procedure. Hemodilution leads to minor and transient disturbances in microcirculatory perfusion, which cannot fully explain impaired microcirculation following cardiopulmonary bypass. CPB led to increased renal injury and endothelial adhesion molecule expression in the kidney and lung compared with hemodilution. Our findings suggest that microcirculatory impairment during CPB may play a role in the development of kidney injury.

ULTRASOUND AND MICROBUBBLE-INDUCED LOCAL DELIVERY OF MICRORNA-BASED THERAPEUTICS

MicroRNAs are involved in many pathologic processes and are a promising target for therapeutic intervention. However, successful, localized delivery of microRNA-based therapeutics is lacking. In this study, cationic ultrasound-responsive microbubbles (MBs) were used to deliver microRNA blockers and mimics in vitro and in vivo. Cationic MBs successfully delivered microRNA blockers to human endothelial cells on ultrasound (US) exposure in vitro. This in vitro US protocol did not successfully deliver microRNA mimics to skeletal muscle of mice, whereas an US protocol that is routinely used for contrast imaging did. Additionally, we used cationic MBs and US to locally deliver antimiR and antagomiR molecules with US causing inertial cavitation. Delivery of antimiR to the extracellular compartments of the muscle was only slightly increased, whereas delivery of antagomiR to the capillaries, myocytes and extracellular space was significantly increased. AntagomiR seems to be a more suitable microRNA blocker than antimiR for use in combination with MBs and US for local delivery.

Positive End-expiratory Pressure Ventilation Induces Longitudinal Atrophy in Diaphragm Fibers

Rationale: Diaphragm weakness in critically ill patients prolongs ventilator dependency and duration of hospital stay and increases mortality and healthcare costs. The mechanisms underlying diaphragm weakness include cross‐sectional fiber atrophy and contractile protein dysfunction, but whether additional mechanisms are at play is unknown. Objectives: To test the hypothesis that mechanical ventilation with positive end‐expiratory pressure (PEEP) induces longitudinal atrophy by displacing the diaphragm in the caudal direction and reducing the length of fibers. Methods: We studied structure and function of diaphragm fibers of mechanically ventilated critically ill patients and mechanically ventilated rats with normal and increased titin compliance. Measurements and Main Results: PEEP causes a caudal movement of the diaphragm, both in critically ill patients and in rats, and this caudal movement reduces fiber length. Diaphragm fibers of 18‐hour mechanically ventilated rats (PEEP of 2.5 cm H2O) adapt to the reduced length by absorbing serially linked sarcomeres, the smallest contractile units in muscle (i.e., longitudinal atrophy). Increasing the compliance of titin molecules reduces longitudinal atrophy. Conclusions: Mechanical ventilation with PEEP results in longitudinal atrophy of diaphragm fibers, a response that is modulated by the elasticity of the giant sarcomeric protein titin. We postulate that longitudinal atrophy, in concert with the aforementioned cross‐sectional atrophy, hampers spontaneous breathing trials in critically ill patients: during these efforts, end‐expiratory lung volume is reduced, and the shortened diaphragm fibers are stretched to excessive sarcomere lengths. At these lengths, muscle fibers generate less force, and diaphragm weakness ensues.

Dysfunctional sarcomere contractility contributes to muscle weakness in ACTA1-related nemaline myopathy (NEM3): ACTA1-Related Myopathy

Nemaline myopathy (NM) is one of the most common congenital nondystrophic myopathies and is characterized by muscle weakness, often from birth. Mutations in ACTA1 are a frequent cause of NM (ie, NEM3). ACTA1 encodes alpha‐actin 1, the main constituent of the sarcomeric thin filament. The mechanisms by which mutations in ACTA1 contribute to muscle weakness in NEM3 are incompletely understood. We hypothesized that sarcomeric dysfunction contributes to muscle weakness in NEM3 patients.