Differentiation and function of cardiac valves in the adult Drosophila heart.

Drosophila, like all insects, has an open circulatory system for the distribution of haemolymph and its components. The circulation of the haemolymph is essentially driven by the pumping activity of the linear heart. The heart is constructed as a tube into which the haemolymph is sucked and pumped forward by rhythmic contractions running from the posterior to the anterior, where it leaves the heart tube. The heart harbours cardiac valves to regulate flow directionality, with a single heart valve differentiating during larval development to separate the heart tube into two chambers. During metamorphosis, the heart is partially restructured, with the linear heart tube with one terminal wide-lumen heart chamber being converted into a linear four-chambered heart tube with three valves. As in all metazoan circulatory systems, the cardiac valves play an essential role in regulating the direction of blood flow. We provide evidence that the valves in adult flies arise via transdifferentiation, converting lumen-forming contractile cardiomyocytes into differently structured valve cells. Interestingly, adult cardiac valves exhibit a similar morphology to their larval counterparts, but act differently upon heart beating. Applying calcium imaging in living specimens to analyse activity in valve cells, we show that adult cardiac valves operate owing to muscle contraction. However, valve cell shape dynamics are altered compared with larval valves, which led us to propose our current model of the opening and closing mechanisms in the fly heart.

Drosophila collagens in specialised extracellular matrices.

The basement membrane (BM) constitutes a specialised form of the extracellular matrix (ECM) and plays important roles in many biological processes, such as cell migration, organ and tissue integrity, cell polarity, and the formation of metastases. In metazoans, a canonical BM is formed by only a few conserved structural core proteins: Laminin, Collagen IV, Nidogen and Perlecan. Depending on the tissue's function and mechanical load, additional matrix proteins interact with, or are incorporated into the BM, resulting in tissue-specific mechanical properties, such as higher stiffness or elasticity, or special resistance to mechanical stress or harmful environmental conditions. In flies, the collagen IV-like protein Pericardin forms an integral constituent of matrices around the heart and tension sensors (chordotonal organs) of the peripheral nervous system. The function and integrity of both organ systems strongly relies on the appropriate establishment of a Pericardin (Prc) matrix and the function of its adapter protein-Lonely heart (Loh). In this review, we provide an overview of the four collagens present in flies, and will discuss our recent work on the formation and function of Pericardin-containing matrices, the role of the adapter protein Lonely heart and the necessity of specialised ECM molecules in tissue architecture and function.

Tailup expression in Drosophila larval and adult cardiac valve cells.

In Drosophila larvae, the direction of blood flow within the heart tube, as well as the diastolic filling of the posterior heart chamber, is regulated by a single cardiac valve. This valve is sufficient to close the heart tube at the junction of the ventricle and the aorta and is formed by only two cells; both are integral parts of the heart tube. The valve cells regulate hemolymph flow by oscillating between a spherical and a flattened cell shape during heartbeats. At the spherical stage, the opposing valve cells close the heart lumen. The dynamic cell shape changes of valve cells are supported by a dense, criss-cross orientation of myofibrils and the presence of the valvosomal compartment, a large intracellular cavity. Both structures are essential for the valve cells' function. In a screen for factors specifically expressed in cardiac valve cells, we identified the transcription factor Tailup. Knockdown of tailup causes abnormal orientation and differentiation of cardiac muscle fibers in the larval aorta and inhibits the formation of the ventral longitudinal muscle layer located underneath the heart tube in the adult fly and affects myofibrillar orientation of valve cells. Furthermore, we have identified regulatory sequences of tup that control the expression of tailup in the larval and adult valve cells.

Formation and function of a highly specialised type of organelle in cardiac valve cells.

Within a cell, vesicles play a crucial role in the transport of membrane material and proteins to a given target membrane, and thus regulate a variety of cellular functions. Vesicular transport occurs by means of, among others, endocytosis, where cargoes are taken up by the cell and are processed further upon vesicular trafficking, i.e. transported back to the plasma membrane via recycling endosomes or the degraded by fusion of the vesicles with lysosomes. During evolution, a variety of vesicles with individual functions arose, with some of them building up highly specialised subcellular compartments. In this study, we have analysed the biosynthesis of a new vesicular compartment present in the valve cells of Drosophila melanogaster. We show that the compartment is formed by invaginations of the plasma membrane and grows via re-routing of the recycling endosomal pathway. This is achieved by inactivation of other membrane-consuming pathways and a plasma membrane-like molecular signature of the compartment in these highly specialised heart cells.

Optical flow analysis reveals that Kinesin-mediated advection impacts the orientation of microtubules in the Drosophila oocyte.

The orientation of microtubule (MT) networks is exploited by motors to deliver cargoes to specific intracellular destinations and is thus essential for cell polarity and function. Reconstituted in vitro systems have largely contributed to understanding the molecular framework regulating the behavior of MT filaments. In cells, however, MTs are exposed to various biomechanical forces that might impact on their orientation, but little is known about it. Oocytes, which display forceful cytoplasmic streaming, are excellent model systems to study the impact of motion forces on cytoskeletons in vivo. Here we implement variational optical flow analysis as a new approach to analyze the polarity of MTs in the Drosophila oocyte, a cell that displays distinct Kinesin-dependent streaming. After validating the method as robust for describing MT orientation from confocal movies, we find that increasing the speed of flows results in aberrant plus end growth direction. Furthermore, we find that in oocytes where Kinesin is unable to induce cytoplasmic streaming, the growth direction of MT plus ends is also altered. These findings lead us to propose that cytoplasmic streaming - and thus motion by advection - contributes to the correct orientation of MTs in vivo. Finally, we propose a possible mechanism for a specialized cytoplasmic actin network (the actin mesh) to act as a regulator of flow speeds to counteract the recruitment of Kinesin to MTs.

Hematopoietic ChemR23 (Chemerin Receptor 23) Fuels Atherosclerosis by Sustaining an M1 Macrophage-Phenotype and Guidance of Plasmacytoid Dendritic Cells to Murine Lesions-Brief Report.

Objective- Expression of the chemokine-like receptor ChemR23 (chemerin receptor 23) has been specifically attributed to plasmacytoid dendritic cells (pDCs) and macrophages and ChemR23 has been suggested to mediate an inflammatory immune response in these cells. Because chemokine receptors are important in perpetuating chronic inflammation, we aimed to establish the role of ChemR23-deficiency on macrophages and pDCs in atherosclerosis. Approach and Results- ChemR23-knockout/knockin mice expressing eGFP (enhanced green fluorescent protein) were generated and after crossing with apolipoprotein E-deficient ( Apoe-/- ChemR23 e/e) animals were fed a western-type diet for 4 and 12 weeks. Apoe-/- ChemR23 e/e mice displayed reduced lesion formation and reduced leukocyte adhesion to the vessel wall after 4 weeks, as well as diminished plaque growth, a decreased number of lesional macrophages with an increased proportion of M2 cells and a less inflammatory lesion composition after 12 weeks of western-type diet feeding. Hematopoietic ChemR23-deficiency similarly reduced atherosclerosis. Additional experiments revealed that ChemR23-deficiency induces an alternatively activated macrophage phenotype, an increased cholesterol efflux and a systemic reduction in pDC frequencies. Consequently, expression of the pDC marker SiglecH in atherosclerotic plaques of Apoe-/- ChemR23 e/e mice was declined. ChemR23-knockout pDCs also exhibited a reduced migratory capacity and decreased CCR (CC-type chemokine receptor)7 expression. Finally, adoptive transfer of sorted wild-type and knockout pDCs into Apoe-/- recipient mice revealed reduced accumulation of ChemR23-deficient pDCs in atherosclerotic lesions. Conclusions- Hematopoietic ChemR23-deficiency increases the proportion of alternatively activated M2 macrophages in atherosclerotic lesions and attenuates pDC homing to lymphatic organs and recruitment to atherosclerotic lesions, which synergistically restricts atherosclerotic plaque formation and progression.

Characterization of Drosophila ATPsynC mutants as a new model of mitochondrial ATP synthase disorders.

Mitochondrial disorders associated with genetic defects of the ATP synthase are among the most deleterious diseases of the neuromuscular system that primarily manifest in newborns. Nevertheless, the number of established animal models for the elucidation of the molecular mechanisms behind such pathologies is limited. In this paper, we target the Drosophila melanogaster gene encoding for the ATP synthase subunit c, ATPsynC, in order to create a fruit fly model for investigating defects in mitochondrial bioenergetics and to better understand the comprehensive pathological spectrum associated with mitochondrial ATP synthase dysfunctions. Using P-element and EMS mutagenesis, we isolated a set of mutations showing a wide range of effects, from larval lethality to complex pleiotropic phenotypes encompassing developmental delay, early adult lethality, hypoactivity, sterility, hypofertility, aberrant male courtship behavior, locomotor defects and aberrant gonadogenesis. ATPsynC mutations impair ATP synthesis and mitochondrial morphology, and represent a powerful toolkit for the screening of genetic modifiers that can lead to potential therapeutic solutions. Furthermore, the molecular characterization of ATPsynC mutations allowed us to better understand the genetics of the ATPsynC locus and to define three broad pathological consequences of mutations affecting the mitochondrial ATP synthase functionality in Drosophila: i) pre-adult lethality; ii) multi-trait pathology accompanied by early adult lethality; iii) multi-trait adult pathology. We finally predict plausible parallelisms with genetic defects of mitochondrial ATP synthase in humans.

APC/CFzr regulates cardiac and myoblast cell numbers, and plays a crucial role during myoblast fusion.

Somatic muscles are formed by the iterative fusion of myoblasts into muscle fibres. This process is driven by the recurrent recruitment of proteins to the cell membrane to induce F-actin nucleation at the fusion site. Although several proteins involved in myoblast fusion have been identified, knowledge about their subcellular regulation is rather elusive. We identified the anaphase-promoting complex (APC/C) adaptor Fizzy related (Fzr) as an essential regulator of heart and muscle development. We show that APC/CFzr regulates the fusion of myoblasts as well as the mitotic exit of pericardial cells, cardioblasts and myoblasts. Surprisingly, overproliferation is not causative for the observed fusion defects. Instead, fzr mutants exhibit smaller F-actin foci at the fusion site and display reduced membrane breakdown between adjacent myoblasts. We show that lack of APC/CFzr causes accumulation and mislocalisation of Rols and Duf, two proteins involved in the fusion process. Duf seems to serve as direct substrate of the APC/CFzr and its destruction depends on the presence of distinct degron sequences. These novel findings indicate that protein destruction and turnover constitute major events during myoblast fusion.

Chrono-pharmacological Targeting of the CCL2-CCR2 Axis Ameliorates Atherosclerosis.

Onset of cardiovascular complications as a consequence of atherosclerosis exhibits a circadian incidence with a peak in the morning hours. Although development of atherosclerosis extends for long periods of time through arterial leukocyte recruitment, we hypothesized that discrete diurnal invasion of the arterial wall could sustain atherogenic growth. Here, we show that myeloid cell recruitment to atherosclerotic lesions oscillates with a peak during the transition from the activity to the resting phase. This diurnal phenotype is regulated by rhythmic release of myeloid cell-derived CCL2, and blockade of its signaling abolished oscillatory leukocyte adhesion. In contrast, we show that myeloid cell adhesion to microvascular beds peaks during the early activity phase. Consequently, timed pharmacological CCR2 neutralization during the activity phase caused inhibition of atherosclerosis without disturbing microvascular recruitment. These findings demonstrate that chronic inflammation of large vessels feeds on rhythmic myeloid cell recruitment, and lay the foundation for chrono-pharmacology-based therapy.

Myosin 1f is specifically required for neutrophil migration in 3D environments during acute inflammation.

Neutrophil extravasation and interstitial migration are important steps during the recruitment of neutrophils to sites of inflammation. In the present study, we addressed the functional importance of the unconventional class I myosin 1f (Myo1f) for neutrophil trafficking during acute inflammation. In contrast to leukocyte rolling and adhesion, the genetic absence of Myo1f severely compromised neutrophil extravasation into the inflamed mouse cremaster tissue when compared with Myo1f+/+ mice as studied by intravital microscopy. Similar results were obtained in experimental models of acute peritonitis and acute lung injury. In contrast to 2-dimensional migration, which occurred independently of Myo1f, Myo1f was indispensable for neutrophil migration in 3-dimensional (3D) environments, that is, transmigration and migration in collagen networks as it regulated squeezing and dynamic deformation of the neutrophil nucleus during migration through physical barriers. Thus, we provide evidence for an important role of Myo1f in neutrophil trafficking during inflammation by specifically regulating neutrophil extravasation and migration in 3D environments.

Active diffusion and advection in Drosophila oocytes result from the interplay of actin and microtubules.

Transport in cells occurs via a delicate interplay of passive and active processes, including diffusion, directed transport and advection. Despite progress in super-resolution microscopy, discriminating and quantifying these processes is a challenge, requiring tracking of rapidly moving, sub-diffraction objects in a crowded, noisy environment. Here we use differential dynamic microscopy with different contrast mechanisms to provide a thorough characterization of the dynamics in the Drosophila oocyte. We study the movement of vesicles and the elusive motion of a cytoplasmic F-actin mesh, a known regulator of cytoplasmic flows. We find that cytoplasmic motility constitutes a combination of directed motion and random diffusion. While advection is mainly attributed to microtubules, we find that active diffusion is driven by the actin cytoskeleton, although it is also enhanced by the flow. We also find that an important dynamic link exists between vesicles and cytoplasmic F-actin motion, as recently suggested in mouse oocytes.

Vascular CXCR4 Limits Atherosclerosis by Maintaining Arterial Integrity: Evidence From Mouse and Human Studies.

BACKGROUND: The CXCL12/CXCR4 chemokine ligand/receptor axis controls (progenitor) cell homeostasis and trafficking. So far, an atheroprotective role of CXCL12/CXCR4 has only been implied through pharmacological intervention, in particular, because the somatic deletion of the CXCR4 gene in mice is embryonically lethal. Moreover, cell-specific effects of CXCR4 in the arterial wall and underlying mechanisms remain elusive, prompting us to investigate the relevance of CXCR4 in vascular cell types for atheroprotection. METHODS: We examined the role of vascular CXCR4 in atherosclerosis and plaque composition by inducing an endothelial cell (BmxCreERT2-driven)-specific or smooth muscle cell (SMC, SmmhcCreERT2- or TaglnCre-driven)-specific deficiency of CXCR4 in an apolipoprotein E-deficient mouse model. To identify underlying mechanisms for effects of CXCR4, we studied endothelial permeability, intravital leukocyte adhesion, involvement of the Akt/WNT/ß-catenin signaling pathway and relevant phosphatases in VE-cadherin expression and function, vascular tone in aortic rings, cholesterol efflux from macrophages, and expression of SMC phenotypic markers. Finally, we analyzed associations of common genetic variants at the CXCR4 locus with the risk for coronary heart disease, along with CXCR4 transcript expression in human atherosclerotic plaques. RESULTS: The cell-specific deletion of CXCR4 in arterial endothelial cells (n=12-15) or SMCs (n=13-24) markedly increased atherosclerotic lesion formation in hyperlipidemic mice. Endothelial barrier function was promoted by CXCL12/CXCR4, which triggered Akt/WNT/ß-catenin signaling to drive VE-cadherin expression and stabilized junctional VE-cadherin complexes through associated phosphatases. Conversely, endothelial CXCR4 deficiency caused arterial leakage and inflammatory leukocyte recruitment during atherogenesis. In arterial SMCs, CXCR4 sustained normal vascular reactivity and contractile responses, whereas CXCR4 deficiency favored a synthetic phenotype, the occurrence of macrophage-like SMCs in the lesions, and impaired cholesterol efflux. Regression analyses in humans (n=259 796) identified the C-allele at rs2322864 within the CXCR4 locus to be associated with increased risk for coronary heart disease. In line, C/C risk genotype carriers showed reduced CXCR4 expression in carotid artery plaques (n=188), which was furthermore associated with symptomatic disease. CONCLUSIONS: Our data clearly establish that vascular CXCR4 limits atherosclerosis by maintaining arterial integrity, preserving endothelial barrier function, and a normal contractile SMC phenotype. Enhancing these beneficial functions of arterial CXCR4 by selective modulators might open novel therapeutic options in atherosclerosis.

Chemokine interactome mapping enables tailored intervention in acute and chronic inflammation.

Chemokines orchestrate leukocyte trafficking and function in health and disease. Heterophilic interactions between chemokines in a given microenvironment may amplify, inhibit, or modulate their activity; however, a systematic evaluation of the chemokine interactome has not been performed. We used immunoligand blotting and surface plasmon resonance to obtain a comprehensive map of chemokine-chemokine interactions and to confirm their specificity. Structure-function analyses revealed that chemokine activity can be enhanced by CC-type heterodimers but inhibited by CXC-type heterodimers. Functional synergism was achieved through receptor heteromerization induced by CCL5-CCL17 or receptor retention at the cell surface via auxiliary proteoglycan binding of CCL5-CXCL4. In contrast, inhibitory activity relied on conformational changes (in CXCL12), affecting receptor signaling. Obligate CC-type heterodimers showed high efficacy and potency and drove acute lung injury and atherosclerosis, processes abrogated by specific CCL5-derived peptide inhibitors or knock-in of an interaction-deficient CXCL4 variant. Atheroprotective effects of CCL17 deficiency were phenocopied by a CCL5-derived peptide disrupting CCL5-CCL17 heterodimers, whereas a CCL5 α-helix peptide mimicked inhibitory effects on CXCL12-driven platelet aggregation. Thus, formation of specific chemokine heterodimers differentially dictates functional activity and can be exploited for therapeutic targeting.

Neutrophils orchestrate post-myocardial infarction healing by polarizing macrophages towards a reparative phenotype.

Aims: Acute myocardial infarction (MI) is the leading cause of mortality worldwide. Anti-inflammatory strategies to reduce neutrophil-driven acute post-MI injury have been shown to limit acute cardiac tissue damage. On the other hand, whether neutrophils are required for resolving post-MI inflammation and repair is unknown. Methods and Results: We show that neutrophil-depleted mice subjected to MI had worsened cardiac function, increased fibrosis, and progressively developed heart failure. Flow cytometry of blood, lymphoid organs and digested hearts revealed reduced numbers of Ly6Chigh monocytes in infarcts of neutrophil-depleted mice, whereas the number of macrophages increased, which was paralleled by reduced splenic Ly6Chigh monocyte mobilization but enhanced proliferation of cardiac macrophages. Macrophage subtype analysis revealed reduced cardiac expression of M1 markers, whereas M2 markers were increased in neutrophil-depleted mice. Surprisingly, we found reduced expression of phagocytosis receptor myeloid-epithelial-reproductive tyrosine kinase, a marker of reparative M2c macrophages which mediate clearance of apoptotic cells. In agreement with this finding, neutrophil-depleted mice had increased numbers of TUNEL-positive cells within infarcts. We identified neutrophil gelatinase-associated lipocalin (NGAL) in the neutrophil secretome as a key inducer of macrophages with high capacity to engulf apoptotic cells. The cardiac macrophage phenotype in neutrophil-depleted mice was restored by administration of neutrophil secretome or NGAL. Conclusion: Neutrophils are crucially involved in cardiac repair after MI by polarizing macrophages towards a reparative phenotype. Therapeutic strategies to reduce acute neutrophil-driven inflammation after MI should be carefully balanced as they might interfere with the healing response and cardiac remodelling.

Human Neutrophil Peptide 1 Limits Hypercholesterolemia-induced Atherosclerosis by Increasing Hepatic LDL Clearance.

Increases in plasma LDL-cholesterol have unequivocally been established as a causal risk factor for atherosclerosis. Hence, strategies for lowering of LDL-cholesterol may have immediate therapeutic relevance. Here we study the role of human neutrophil peptide 1 (HNP1) in a mouse model of atherosclerosis and identify its potent atheroprotective effect both upon transgenic overexpression and therapeutic delivery. The effect was found to be due to a reduction of plasma LDL-cholesterol. Mechanistically, HNP1 binds to apolipoproteins enriched in LDL. This interaction facilitates clearance of LDL particles in the liver via LDL receptor. Thus, we here identify a non-redundant mechanism by which HNP1 allows for reduction of LDL-cholesterol, a process that may be therapeutically instructed to lower cardiovascular risk.

Protective Aptitude of Annexin A1 in Arterial Neointima Formation in Atherosclerosis-Prone Mice-Brief Report.

OBJECTIVE: Restenosis as a consequence of arterial injury is aggravated by inflammatory pathways. Here, we investigate the role of the proresolving protein annexin A1 (AnxA1) in healing after wire injury. APPROACH AND RESULTS: Apoe-/- and Apoe-/-Anxa1-/- mice were subjected to wire injury while fed a high-cholesterol diet. Subsequently, localization of AnxA1 and AnxA1 plasma levels were examined. AnxA1 was found to localize within endothelial cells and macrophages in the neointima. Levels of AnxA1 in the plasma and its lesional expression negatively correlated with neointima size, and in the absence of AnxA1, neointima formation was aggravated by the accumulation and proliferation of macrophages. In contrast, reendothelialization and smooth muscle cell infiltration were not affected in Apoe-/-Anxa1-/- mice. CONCLUSIONS: AnxA1 is protective in healing after wire injury and could, therefore, be an attractive therapeutic compound to prevent from restenosis after vascular damage.

The advantageous role of annexin A1 in cardiovascular disease.

The inflammatory response protects the human body against infection and injury. However, uncontrolled and unresolved inflammation can lead to tissue damage and chronic inflammatory diseases. Therefore, active resolution of inflammation is essential to restore tissue homeostasis. This review focuses on the pro-resolving molecule annexin A1 (ANXA1) and its derived peptides. Mechanisms instructed by ANXA1 are multidisciplinary and affect leukocytes as well as endothelial cells and tissue resident cells like macrophages and mast cells. ANXA1 has an outstanding role in limiting leukocyte recruitment and different aspects of ANXA1 as modulator of the leukocyte adhesion cascade are discussed here. Additionally, this review details the therapeutic relevance of ANXA1 and its derived peptides in cardiovascular diseases since atherosclerosis stands out as a chronic inflammatory disease with impaired resolution and continuous leukocyte recruitment.

Cathepsin G Controls Arterial But Not Venular Myeloid Cell Recruitment.

BACKGROUND: Therapeutic targeting of arterial leukocyte recruitment in the context of atherosclerosis has been disappointing in clinical studies. Reasons for such failures include the lack of knowledge of arterial-specific recruitment patterns. Here we establish the importance of the cathepsin G (CatG) in the context of arterial myeloid cell recruitment. METHODS: Intravital microscopy of the carotid artery, the jugular vein, and cremasteric arterioles and venules in Apoe-/-and CatG-deficient mice (Apoe-/-Ctsg-/-) was used to study site-specific myeloid cell behavior after high-fat diet feeding or tumor necrosis factor stimulation. Atherosclerosis development was assessed in aortic root sections after 4 weeks of high-fat diet, whereas lung inflammation was assessed after inhalation of lipopolysaccharide. Endothelial deposition of CatG and CCL5 was quantified in whole-mount preparations using 2-photon and confocal microscopy. RESULTS: Our observations elucidated a crucial role for CatG during arterial leukocyte adhesion, an effect not found during venular adhesion. Consequently, CatG deficiency attenuates atherosclerosis but not acute lung inflammation. Mechanistically, CatG is immobilized on arterial endothelium where it activates leukocytes to firmly adhere engaging integrin clustering, a process of crucial importance to achieve effective adherence under high-shear flow. Therapeutic neutralization of CatG specifically abrogated arterial leukocyte adhesion without affecting myeloid cell adhesion in the microcirculation. Repetitive application of CatG-neutralizing antibodies permitted inhibition of atherogenesis in mice. CONCLUSIONS: Taken together, these findings present evidence of an arterial-specific recruitment pattern centered on CatG-instructed adhesion strengthening. The inhibition of this process could provide a novel strategy for treatment of arterial inflammation with limited side effects.