Pulsatile shear and Gja5 modulate arterial identity and remodeling events during flow-driven arteriogenesis

In the developing chicken embryo yolk sac vasculature, the expression of arterial identity genes requires arterial hemodynamic conditions. We hypothesize that arterial flow must provide a unique signal that is relevant for supporting arterial identity gene expression and is absent in veins. We analyzed factors related to flow, pressure and oxygenation in the chicken embryo vitelline vasculature in vivo. The best discrimination between arteries and veins was obtained by calculating the maximal pulsatile increase in shear rate relative to the time-averaged shear rate in the same vessel: the relative pulse slope index (RPSI). RPSI was significantly higher in arteries than veins. Arterial endothelial cells exposed to pulsatile shear in vitro augmented arterial marker expression as compared with exposure to constant shear. The expression of Gja5 correlated with arterial flow patterns: the redistribution of arterial flow provoked by vitelline artery ligation resulted in flow-driven collateral arterial network formation and was associated with increased expression of Gja5. In situ hybridization in normal and ligation embryos confirmed that Gja5 expression is confined to arteries and regulated by flow. In mice, Gja5 (connexin 40) was also expressed in arteries. In the adult, increased flow drives arteriogenesis and the formation of collateral arterial networks in peripheral occlusive diseases. Genetic ablation of Gja5 function in mice resulted in reduced arteriogenesis in two occlusion models. We conclude that pulsatile shear patterns may be central for supporting arterial identity, and that arterial Gja5 expression plays a functional role in flow-driven arteriogenesis.

Porcine circovirus type 2 DNA influences cytoskeleton rearrangements in plasmacytoid and monocyte-derived dendritic cells

Functional disruption of dendritic cells (DC) is an important strategy for viral pathogens to evade host defences. In this context, porcine circovirus type 2 (PCV2), a single-stranded DNA virus, impairs plasmacytoid DC (pDC) and conventional DC activation by certain viruses or Toll-like receptor (TLR) ligands. This inhibitory capacity is associated with the viral DNA, but the impairment does not affect all signalling cascades; TLR7 ligation by small chemical molecules will still induce interleukin-6 (IL-6) and tumour necrosis factor-α secretion, but not interferon-α or IL-12. In this study, the molecular mechanisms by which silencing occurs were investigated. PP2, a potent inhibitor of the Lyn and Hck kinases, produced a similar profile to the PCV2 DNA interference with cytokine secretion by pDC, efficiently inhibiting cell activation induced through TLR9, but not TLR7, ligation. Confocal microscopy and cytometry analysis strongly suggested that PCV2 DNA impairs actin polymerization and endocytosis in pDC and monocyte-derived DC, respectively. Altogether, this study delineates for the first time particular molecular mechanisms involved in PCV2 interference with DC danger recognition, which may be responsible for the virus-induced immunosuppression observed in infected pigs.

Hemizygous deletion of COL3A1, COL5A2, and MSTN causes a complex phenotype with aortic dissection: a lesson for and from true haploinsufficiency

Aortic dilatation/dissection (AD) can occur spontaneously or in association with genetic syndromes, such as Marfan syndrome (MFS; caused by FBN1 mutations), MFS type 2 and Loeys-Dietz syndrome (associated with TGFBR1/TGFBR2 mutations), and Ehlers-Danlos syndrome (EDS) vascular type (caused by COL3A1 mutations). Although mutations in FBN1 and TGFBR1/TGFBR2 account for the majority of AD cases referred to us for molecular genetic testing, we have obtained negative results for these genes in a large cohort of AD patients, suggesting the involvement of additional genes or acquired factors. In this study we assessed the effect of COL3A1 deletions/duplications in this cohort. Multiplex ligation-dependent probe amplification (MLPA) analysis of 100 unrelated patients identified one hemizygous deletion of the entire COL3A1 gene. Subsequent microarray analyses and sequencing of breakpoints revealed the deletion size of 3,408,306 bp at 2q32.1q32.3. This deletion affects not only COL3A1 but also 21 other known genes (GULP1, DIRC1, COL5A2, WDR75, SLC40A1, ASNSD1, ANKAR, OSGEPL1, ORMDL1, LOC100129592, PMS1, MSTN, C2orf88, HIBCH, INPP1, MFSD6, TMEM194B, NAB1, GLS, STAT1, and STAT4), mutations in three of which (COL5A2, SLC40A1, and MSTN) have also been associated with an autosomal dominant disorder (EDS classical type, hemochromatosis type 4, and muscle hypertrophy). Physical and laboratory examinations revealed that true haploinsufficiency of COL3A1, COL5A2, and MSTN, but not that of SLC40A1, leads to a clinical phenotype. Our data not only emphasize the impact/role of COL3A1 in AD patients but also extend the molecular etiology of several disorders by providing hitherto unreported evidence for true haploinsufficiency of the underlying gene.

Long-term evaluation of myoblast seeded patches implanted on infarcted rat hearts

Cell transplantation presents great potential for treatment of patients with severe heart failure. However, its clinical application was revealed to be more challenging than initially expected in experimental studies. Further investigations need to be undertaken to define the optimal treatment conditions. We previously reported on the epicardial implantation of a bio-engineered construct of skeletal myoblast-seeded polyurethane and its preventive effect on progression toward heart failure. In the present study, we present a long-term evaluation of this functional outcome. Left anterior descending coronary ligation was performed in female Lewis rats. Two weeks later, animals were treated with either epicardial implantation of biograft, acellular scaffold, sham operation, or direct intramyocardial skeletal myoblast injection. Functional assessments were performed with serial echocardiographies every 3 months and end point left ventricle pressure was assessed. Hearts were then harvested for histological examinations. Myocardial infarction induced a slow and progressive reduction in fractional shortening after 3 months. Progression toward heart failure was significantly prevented for up to 6 months after injection of myoblasts and for up to 9 months following biograft implantation. Nevertheless, this effect vanished after 12 months, with immunohistological examinations revealing an absence of the transplanted myoblasts within the scaffold. We demonstrated that tissue therapy is superior to cell therapy for stabilization of heart function. However, beneficial effects are transient.

Inflammation-associated autophagy-related programmed necrotic death of human neutrophils characterized by organelle fusion events

The most common form of neutrophil death, under both physiological and inflammatory conditions, is apoptosis. In this study, we report a novel form of programmed necrotic cell death, associated with cytoplasmic organelle fusion events, that occurs in neutrophils exposed to GM-CSF and other inflammatory cytokines upon ligation of CD44. Strikingly, this type of neutrophil death requires PI3K activation, a signaling event usually involved in cellular survival pathways. In the death pathway reported in this study, PI3K is required for the generation of reactive oxygen species, which somehow trigger the generation of large cytoplasmic vacuoles, generated by the fusion of CD44-containing endosomes with autophagosomes and secondary, but not primary, granules. Neutrophils demonstrating vacuolization undergo rapid cell death that depends on receptor-interacting protein 1 kinase activity and papain family protease(s), but not caspases, that are most likely activated and released, respectively, during or as a consequence of organelle fusion. Vacuolized neutrophils are present in infectious and autoimmune diseases under in vivo conditions. Moreover, isolated neutrophils from such patients are highly sensitive toward CD44-mediated PI3K activation, reactive oxygen species production, and cell death, suggesting that the newly described autophagy-related form of programmed neutrophil necrosis plays an important role in inflammatory responses.

Bone morphogenetic protein receptor 2 in patients with idiopathic portal hypertension

In idiopathic portal hypertension (IPH) typical vascular lesions are present in the branches of the portal vein or in the perisinusoidal area of the liver. Similar histological alterations have been reported in the pulmonary vasculature of patients with idiopathic pulmonary artery hypertension (IPAH). As IPAH is associated with mutations of the bone morphogenetic protein receptor 2 (BMPR2) gene, the aim of this study was to investigate whether this association might also be found in patients with IPH. Twenty-three samples belonging to 21 unrelated caucasian patients with IPH followed in the hepatic haemodynamic laboratory of the Hospital Clinic in Barcelona were included in the study. All patients were studied for the entire open reading frame and splice site of the BMPR2 gene by direct sequencing and multiple ligation probe amplification (MLPA) in order to detect large deletions/duplications. None of the 23 patients had pulmonary artery hypertension. Four patients presented one single nucleotide polymorphism (SNP) in intron 5, four patients had a SNP in exon 12 and a SNP in exon 1 was found in two cases. Two patients had both intron 5 and exon 12 polymorphisms. All SNPs were previously described. Except for these three SNPs, neither mutations nor rearrangements have been identified in the BMPR2 gene in this population. We did not detect mutations or rearrangements in the coding region of the BMPR2 gene in our patients with IPH. These findings suggest that, in contrast to IPAH, mutations in BMPR2 are not involved in the pathogenesis of IPH.

Modulating CD27 signaling to treat cancer

CD27 signaling can either improve T-cell function or lead to T-cell dysfunction, depending on the duration and conditions of receptor ligation. Recent studies have shown that modulating the CD70-CD27 interaction is an attractive strategy to treat solid tumors and also to directly target leukemia stem cells.

Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages

There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the "Beijing" sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive.

Consensus Document of the International Union of Angiology (IUA)-2013. Current concept on the management of arterio-venous management

Arterio-venous malformations (AVMs) are congenital vascular malformations (CVMs) that result from birth defects involving the vessels of both arterial and venous origins, resulting in direct communications between the different size vessels or a meshwork of primitive reticular networks of dysplastic minute vessels which have failed to mature to become 'capillary' vessels termed "nidus". These lesions are defined by shunting of high velocity, low resistance flow from the arterial vasculature into the venous system in a variety of fistulous conditions. A systematic classification system developed by various groups of experts (Hamburg classification, ISSVA classification, Schobinger classification, angiographic classification of AVMs,) has resulted in a better understanding of the biology and natural history of these lesions and improved management of CVMs and AVMs. The Hamburg classification, based on the embryological differentiation between extratruncular and truncular type of lesions, allows the determination of the potential of progression and recurrence of these lesions. The majority of all AVMs are extra-truncular lesions with persistent proliferative potential, whereas truncular AVM lesions are exceedingly rare. Regardless of the type, AV shunting may ultimately result in significant anatomical, pathophysiological and hemodynamic consequences. Therefore, despite their relative rarity (10-20% of all CVMs), AVMs remain the most challenging and potentially limb or life-threatening form of vascular anomalies. The initial diagnosis and assessment may be facilitated by non- to minimally invasive investigations such as duplex ultrasound, magnetic resonance imaging (MRI), MR angiography (MRA), computerized tomography (CT) and CT angiography (CTA). Arteriography remains the diagnostic gold standard, and is required for planning subsequent treatment. A multidisciplinary team approach should be utilized to integrate surgical and non-surgical interventions for optimum care. Currently available treatments are associated with significant risk of complications and morbidity. However, an early aggressive approach to elimiate the nidus (if present) may be undertaken if the benefits exceed the risks. Trans-arterial coil embolization or ligation of feeding arteries where the nidus is left intact, are incorrect approaches and may result in proliferation of the lesion. Furthermore, such procedures would prevent future endovascular access to the lesions via the arterial route. Surgically inaccessible, infiltrating, extra-truncular AVMs can be treated with endovascular therapy as an independent modality. Among various embolo-sclerotherapy agents, ethanol sclerotherapy produces the best long term outcomes with minimum recurrence. However, this procedure requires extensive training and sufficient experience to minimize complications and associated morbidity. For the surgically accessible lesions, surgical resection may be the treatment of choice with a chance of optimal control. Preoperative sclerotherapy or embolization may supplement the subsequent surgical excision by reducing the morbidity (e.g. operative bleeding) and defining the lesion borders. Such a combined approach may provide an excellent potential for a curative result. Conclusion. AVMs are high flow congenital vascular malformations that may occur in any part of the body. The clinical presentation depends on the extent and size of the lesion and can range from an asymptomatic birthmark to congestive heart failure. Detailed investigations including duplex ultrasound, MRI/MRA and CT/CTA are required to develop an appropriate treatment plan. Appropriate management is best achieved via a multi-disciplinary approach and interventions should be undertaken by appropriately trained physicians.

Caspase-3 mediates in part hippocampal apoptosis in sepsis

The brain is one of the first organs affected during sepsis development resulting in apoptosis for a short-term and cognitive impairment for a long-term. Despite its importance, the mechanisms of brain dysfunction during sepsis are not fully elucidated. Thus, we here, in an animal model of sepsis, evaluated apoptosis in the dentate gyrus cell layer of the hippocampus to document the involvement of caspase-3 in the pathogenesis of neuronal apoptosis. Wistar rats sham-operated or submitted to the cecal ligation and perforation (CLP) procedure were killed at 12, 24, 48 h, and 10 days after surgery for the determination of caspase-3 and apoptosis rate. In a separate cohort of animals, a caspase-3-specific inhibitor was administered and animals were killed at 12 h after sepsis. An increase in the number of apoptotic cells 12, 24, and 48 h by histopathological evaluations and an increase of caspase-3 apoptotic cells 12 and 24 h after sepsis induction were observed. The caspase-3 inhibitor decreases the number of apoptotic cells by histopathological evaluations but not by immunohistochemistry evaluations. Caspase-3 is involved in part in apoptosis in the dentate gyrus cell layer of the hippocampus in septic rats submitted by CLP.

17β-estradiol protects 7-day old rats from acute brain injury and reduces the number of apoptotic cells

To test a possible neuroprotective activity of 17β-estradiol in the neonatal rat brain exposed to hypoxic-ischemia (controlled hypoxia after unilateral carotid artery ligation).

[Oesophageal and fundic variceal bleeding]

Oesophageal and fundic varices belong to the most frequent complications of cirrhosis and portal hypertension. Due to their significant morbidity and mortality, bleedings from oesophageal or fundic varices represent a challenge for the emergency medical team as well as for the gastroenterologist. The patient with a variceal bleeding should be accurately monitored and his/her hemodynamic parameters should be maintained stable with the administration of plasma expanders and blood units when indicated. An antibiotic prophylaxis in this setting--norfloxacin or ceftriaxon--has been demonstrated to significantly reduce morbidity and mortality. Additionally, the early administration of vasoactive compounds, such as terlipressin, somatostatin or octreotide, is associated with beneficial effects in reducing the bleeding. An upper gastrointestinal endoscopy should be generally performed within the first twelve hours from the beginning of the bleeding in order to obtain an accurate diagnosis and to provide an adequate treatment. Endoscopic procedures to control the bleeding include the rubber band ligation, the treatment of the varix with a sclerosing agent or the injection of tissue glue into the varix. In case of recurrent bleeding, beyond the above methods, different techniques, such as the transjugular porto-caval shunt, surgical shunt procedures, as well as embolisation of splanchnic blood vessels, represent additional therapeutic options. However, they are associated with very high mortality rates and their indication has to be discussed case by case by an interdisciplinary team of experts. Future therapies include the optimisation and the improvement of the current medical and endoscopic armamentarium, as well as the application of treatments to novel targets, such as the coagulation cascade.

Low-dose oral rapamycin treatment reduces fibrogenesis, improves liver function, and prolongs survival in rats with established liver cirrhosis

BACKGROUND/AIMS: Mammalian target of rapamycin (mTOR) signalling is central in the activation of hepatic stellate cells (HSCs), the key source of extracellular matrix (ECM) in fibrotic liver. We tested the therapeutic potential of the mTOR inhibitor rapamycin in advanced cirrhosis. METHODS: Cirrhosis was induced by bile duct-ligation (BDL) or thioacetamide injections (TAA). Rats received oral rapamycin (0.5 mg/kg/day) for either 14 or 28 days. Untreated BDL and TAA-rats served as controls. Liver function was quantified by aminopyrine breath test. ECM and ECM-producing cells were quantified by morphometry. MMP-2 activity was measured by zymography. mRNA expression of procollagen-alpha1, transforming growth factor-beta1 (TGF-beta1) and beta2 was quantified by RT-PCR. RESULTS: Fourteen days of rapamycin improved liver function. Accumulation of ECM was decreased together with numbers of activated HSCs and MMP-2 activity in both animal models. TGF-beta1 mRNA was downregulated in TAA, TGF-beta2 mRNA was downregulated in BDL. 28 days of rapamycin treatment entailed a survival advantage of long-term treated BDL-rats. CONCLUSIONS: Low-dose rapamycin treatment is effectively antifibrotic and attenuates disease progression in advanced fibrosis. Our results warrant the clinical evaluation of rapamycin as an antifibrotic drug.

Noninvasive Doppler-derived myocardial performance index in rats with myocardial infarction: validation and correlation by conductance catheter

The rodent model of myocardial infarction (MI) is extensively used in heart failure studies. However, long-term follow-up of echocardiographic left ventricular (LV) function parameters such as the myocardial performance index (MPI) and its ratio with the fractional shortening (LVFS/MPI) has not been validated in conjunction with invasive indexes, such as those derived from the conductance catheter (CC). Sprague-Dawley rats with left anterior descending coronary artery ligation (MI group, n = 9) were compared with a sham-operated control group (n = 10) without MI. Transthoracic echocardiography (TTE) was performed every 2 wk over an 8-wk period, after which classic TTE parameters, especially MPI and LVFS/MPI, were compared with invasive indexes obtained by using a CC. Serial TTE data showed significant alterations in the majority of the noninvasive functional and structural parameters (classic and novel) studied in the presence of MI. Both MPI and LVFS/MPI significantly (P < 0.05 for all reported values) correlated with body weight (r = -0.58 and 0.76 for MPI and LVFS/MPI, respectively), preload recruitable stroke work (r = -0.61 and 0.63), LV end-diastolic pressure (LVEDP) (r = 0.82 and -0.80), end-diastolic volume (r = 0.61 and -0.58), and end-systolic volume (r = 0.46 and -0.48). Forward stepwise linear regression analysis revealed that, of all variables tested, LVEDP was the only independent determinant of MPI (r = 0.84) and LVFS/MPI (r = 0.83). We conclude that MPI and LVFS/MPI correlate strongly and better than the classic noninvasive TTE parameters with established, invasively assessed indexes of contractility, preload, and volumetry. These findings support the use of these two new noninvasive indexes for long-term analysis of the post-MI LV remodeling.

Autophagic-like cell death in neutrophils induced by autoantibodies

Human neutrophils undergo autophagic-like cell death following Sialic acid binding immunoglobulin-like lectin-9 (Siglec-9) ligation and concurrent stimulation with certain, but not all, neutrophil survival cytokines. Caspase inhibition by these cytokines is required, but is not sufficient, to trigger this particular form of cell death. Additional mechanisms may involve reactive oxygen species (ROS), and blocking of ROS or prevention of ROS production prevents autophagic-like neutrophil death. Interestingly, human intravenous immunoglobulin (IVIg) preparations contain natural anti-Siglec-9 autoantibodies, which are able to ligate Siglec-9 on neutrophils and induce autophagic-like cell death in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and some other survival cytokines. Here, we discuss the pathophysiological and therapeutic implications of these recent findings.