Abstract
Aims
The pathogenesis of endocarditis is not well understood resulting in unsuccessful attempts at prevention. Clinical observations suggest that Staphylococcus aureus infects either damaged ...or inflamed heart valves. Using a newly developed endocarditis mouse model, we therefore studied the initial adhesion of S. aureus in both risk states.
Methods and results
Using 3D confocal microscopy, we examined the adhesion of fluorescent S. aureus to murine aortic valves. To mimic different risk states we either damaged the valves with a surgically placed catheter or simulated valve inflammation by local endothelium activation. We used von Willebrand factor (VWF) gene-deficient mice, induced platelet and fibrinogen depletion and used several S. aureus mutant strains to investigate the contribution of both host and bacterial factors in early bacterial adhesion. Both cardiac valve damage and inflammation predisposed to endocarditis, but by distinct mechanisms. Following valve damage, S. aureus adhered directly to VWF and fibrin, deposited on the damaged valve. This was mediated by Sortase A-dependent adhesins such as VWF-binding protein and Clumping factor A. Platelets did not contribute. In contrast, upon cardiac valve inflammation, widespread endothelial activation led to endothelial cell-bound VWF release. This recruited large amounts of platelets, capturing S. aureus to the valve surface. Here, neither fibrinogen, nor Sortase A were essential.
Conclusion
Cardiac valve damage and inflammation predispose to S. aureus endocarditis via distinct mechanisms. These findings may have important implications for the development of new preventive strategies, as some interventions might be effective in one risk state, but not in the other.
Implementation of in vivo high-resolution micro-computed tomography (µCT), a powerful tool for longitudinal analysis of murine lung disease models, is hampered by the lack of data on cumulative ...low-dose radiation effects on the investigated disease models. We aimed to measure radiation doses and effects of repeated µCT scans, to establish cumulative radiation levels and scan protocols without relevant toxicity. Lung metastasis, inflammation and fibrosis models and healthy mice were weekly scanned over one-month with µCT using high-resolution respiratory-gated 4D and expiration-weighted 3D protocols, comparing 5-times weekly scanned animals with controls. Radiation dose was measured by ionization chamber, optical fiberradioluminescence probe and thermoluminescent detectors in a mouse phantom. Dose effects were evaluated by in vivo µCT and bioluminescence imaging read-outs, gold standard endpoint evaluation and blood cell counts. Weekly exposure to 4D µCT, dose of 540-699 mGy/scan, did not alter lung metastatic load nor affected healthy mice. We found a disease-independent decrease in circulating blood platelets and lymphocytes after repeated 4D µCT. This effect was eliminated by optimizing a 3D protocol, reducing dose to 180-233 mGy/scan while maintaining equally high-quality images. We established µCT safety limits and protocols for weekly repeated whole-body acquisitions with proven safety for the overall health status, lung, disease process and host responses under investigation, including the radiosensitive blood cell compartment.
Vascular development is an orchestrated process of vessel formation from pre-existing vessels via sprouting and intussusceptive angiogenesis as well as vascular remodeling to generate the mature ...vasculature. Bone morphogenetic protein (BMP) signaling via intracellular SMAD1 and SMAD5 effectors regulates sprouting angiogenesis in the early mouse embryo, but its role in other processes of vascular development and in other vascular beds remains incompletely understood. Here, we investigate the function of SMAD1/5 during early postnatal retinal vascular development using inducible, endothelium-specific deletion of
and
. We observe the formation of arterial-venous malformations in areas with high blood flow, and fewer and less functional tip cells at the angiogenic front. The vascular plexus region is remarkably hyperdense and this is associated with reduced vessel regression and aberrant vascular loop formation. Taken together, our results highlight important functions of SMAD1/5 during vessel formation and remodeling in the early postnatal retina.
Background Arteriovenous fistulae (AVFs) are the gold standard for vascular access in those requiring hemodialysis but may put an extra hemodynamic stress on the cardiovascular system. The complex ...interactions between the heart, kidney, and AVFs remain incompletely understood. Methods and Results We characterized a novel rat model of five‐sixths partial nephrectomy (NX) and AVFs. NX induced increases in urea, creatinine, and hippuric acid. The addition of an AVF (AVF+NX) further increased urea and a number of uremic toxins such as trimethylamine N‐oxide and led to increases in cardiac index, left and right ventricular volumes, and right ventricular mass. Plasma levels of uremic toxins correlated well with ventricular morphology and function. Heart transcriptomes identified altered expression of 8 genes following NX and 894 genes following AVF+NX, whereas 290 and 1431 genes were altered in the kidney transcriptomes, respectively. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis revealed gene expression changes related to cell division and immune activation in both organs, suppression of ribosomes and transcriptional activity in the heart, and altered renin‐angiotensin signaling as well as chronodisruption in the kidney. All except the latter were worsened in AVF+NX compared with NX. Conclusions Inflammation and organ dysfunction in chronic kidney disease are exacerbated following AVF creation. Furthermore, our study provides important information for the discovery of novel biomarkers and therapeutic targets in the management of cardiorenal syndrome.
New platforms are needed for the design of novel prophylactic vaccines and advanced immune therapies. Live-attenuated yellow fever vaccine YF17D serves as a vector for several licensed vaccines and ...platform for novel candidates. On the basis of YF17D, we developed an exceptionally potent COVID-19 vaccine candidate called YF-S0. However, use of such live RNA viruses raises safety concerns, such as adverse events linked to original YF17D (yellow fever vaccine-associated neurotropic disease YEL-AND and yellow fever vaccine-associated viscerotropic disease YEL-AVD). In this study, we investigated the biodistribution and shedding of YF-S0 in hamsters. Likewise, we introduced hamsters deficient in signal transducer and activator of transcription 2 (STAT2) signaling as a new preclinical model of YEL-AND/AVD. Compared with YF17D, YF-S0 showed improved safety with limited dissemination to brain and visceral tissues, absent or low viremia, and no shedding of infectious virus. Considering that yellow fever virus is transmitted by Aedes mosquitoes, any inadvertent exposure to the live recombinant vector via mosquito bites is to be excluded. The transmission risk of YF-S0 was hence compared with readily transmitting YF-Asibi strain and non-transmitting YF17D vaccine, with no evidence for productive infection of mosquitoes. The overall favorable safety profile of YF-S0 is expected to translate to other vaccines based on the same YF17D platform.
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Little is known about the biodistribution and environmental safety of vaccines using live yellow fever YF17D virus as a vector. In hamsters, detection of such replicating COVID-19 vaccine was limited and transient, with no evidence for shedding or mosquito-borne transmission. Comparable safety features may apply to YF17D-vectored vaccines in general.
Recovered COVID-19 patients often display cardiac dysfunction, even after a mild infection. Most current histological results come from patients that are hospitalized and therefore represent more ...severe outcomes than most COVID-19 patients face. To overcome this limitation, we investigated the cardiac effects of SARS-CoV-2 infection in a hamster model. SARS-CoV-2 infected hamsters developed diastolic dysfunction after recovering from COVID-19. Histologically, increased cardiomyocyte size was present at the peak of viral load and remained at all time points investigated. As this increase is too rapid for hypertrophic remodeling, we found instead that the heart was oedemic. Moreover, cardiomyocyte swelling is associated with the presence of ischemia. Fibrin-rich microthrombi and pericyte loss were observed at the peak of viral load, resulting in increased HIF1α in cardiomyocytes. Surprisingly, SARS-CoV-2 infection inhibited the translocation of HIF1α to the nucleus both in hamster hearts, in cultured cardiomyocytes, as well as in an epithelial cell line. We propose that the observed diastolic dysfunction is the consequence of cardiac oedema, downstream of microvascular cardiac ischemia. Additionally, our data suggest that inhibition of HIF1α translocation could contribute to an exaggerated response upon SARS-CoV-2 infection.
Obesity has become a global health-threat for every age group. It is well known that young mice (10-12 weeks of age) fed a western-type diet (WD) become obese and develop higher cholesterol levels ...and liver steatosis whereas insulin sensitivity is reduced. Less is known, however, about the effect of a WD on advanced-age mice. Therefore, 10 week-old (young) and 22 month-old (advanced-age), male C57BL/6JRj mice were kept on either a WD or a control diet (SFD) for 15 weeks. In contrast to young mice, advanced-age mice on WD did not show a higher body weight or adipose tissue (AT)-masses, suggesting a protection against diet-induced obesity. Furthermore, plasma adiponectin and leptin levels were not affected upon WD-feeding. A WD, however, did induce more hepatic lipid accumulation as well as increased hepatic expression of the macrophage marker F4/80, in advanced-age mice. There were no significant differences in mRNA levels of uncoupling protein-1 or F4/80 in brown AT (BAT) or of several intestinal integrity markers in colon suggesting that the protection against obesity is not due to excessive BAT or to impaired intestinal absorption of fat. Thus, advanced-age mice, in contrast to their younger counterparts, appeared to be protected against diet-induced obesity.
Atherosclerosis is characterized by an early inflammatory response involving proinflammatory mediators such as platelet-activating factor (PAF)-like phospholipids, which are inactivated by ...PAF-acetylhydrolase (PAF-AH). The effect of adenovirus-mediated expression of PAF-AH on injury-induced neointima formation and spontaneous atherosclerosis was studied in apolipoprotein E-deficient mice.
Intravenous administration of an adenovirus (5 x 10(8) plaque-forming units) directing liver-specific expression of human PAF-AH resulted in a 3.5-fold increase of plasma PAF-AH activity at day 7 (P<0.001); this was associated with a 2.4- and 2.3-fold decrease in malondialdehyde-modified LDL autoantibodies and the lysophosphatidylcholine/phosphatidylcholine ratio, respectively (P<0.001 for both). Non-HDL and HDL cholesterol levels in PAF-AH-treated mice were similar to those of control virus-treated mice. Seven days after virus injection, endothelial denudation of the common left carotid artery was induced with a guidewire. Neointima formation was assessed 18 days later. PAF-AH gene transfer reduced oxidized lipoproteins by 82% (P<0.001), macrophages by 69% (P=0.006), and smooth muscle cells by 84% (P=0.002) in the arterial wall. This resulted in a 77% reduction (P<0.001) of neointimal area. Six weeks after adenovirus-mediated gene transfer, spontaneous atherosclerotic lesions in the aortic root were analyzed. PAF-AH gene transfer reduced atherosclerotic lesions by 42% (P=0.02) in male mice, whereas a nonsignificant 14% reduction was observed in female mice. Basal and PAF-AH activity after gene transfer were higher in male mice than in female mice (P=0.01 and P=0.04, respectively).
Gene transfer of PAF-AH inhibited injury-induced neointima formation and spontaneous atherosclerosis in apolipoprotein E-deficient mice. Our data indicate that PAF-AH, by reducing oxidized lipoprotein accumulation, is a potent protective enzyme against atherosclerosis.
Abstract only Background: Neutrophil extracellular traps (NETs) and von Willebrand factor (VWF) have been proposed as key drivers of immunothrombosis and thromboinflammation in Staphylococcus aureus ...sepsis. Peptidylarginine deiminase 4 (PAD4) contributes to NET formation and citrullinates coagulation proteins. NETs interact with VWF and platelets, thus providing a scaffold for the formation of microthrombi that cause microvascular perfusion deficits. Aims: To study the relationship between PAD4, NETs, and VWF in murine S. aureus sepsis. Methods: Murine sepsis was induced by injecting methicillin-resistant S. aureus (MRSA, USA300 strain) intravenously in C57BL/6J, MRP8Cre + PAD4 flfl (NET-defective) or PAD4 fl/fl littermates. In separate experiments, granulocyte colony stimulating factor (G-CSF) was used to induce neutrophilia prior to administration of USA300. Mice were followed for up to 8 days for clinical scoring and reaching humane endpoints as a surrogate readout for survival. Cell counts and plasma levels of citrullinated histone H3-DNA (H3Cit-DNA) complexes, VWF antigen, and PAD4 were measured at days 1, 4, and 8. Results: WT mice injected with USA300 developed thrombocytopenia and had higher plasma levels of NETs, VWF, and PAD4 as compared to vehicle (saline)-injected mice. MRP8Cre + PAD4 flfl mice had significantly reduced H3Cit-DNA and lower levels of VWF antigen 1d after USA300 injection as compared to littermate controls, with comparable VWF levels in the later disease phase. There was no difference in levels of cell-free PAD4 in plasma indicating a non-neutrophil source of PAD4, but the substantial reduction of plasma NET levels supporting that neutrophil PAD4 was essential for NET release. Neutrophilic mice (prior to sepsis induction) had lower sepsis disease scores and significantly improved survival rates as compared to mice receiving saline control. This protection was absent in the NET-defective mice, who surprisingly retained higher VWF levels. Conclusion: Individually eliminating NETs or VWF had little impact in MRSA sepsis in mice, and circulating PAD4 likely does not originate from neutrophils. Inducing neutrophilia protected mice against S. aureus sepsis, an effect which was abrogated in the absence of NET formation.
Abstract only Background: Interleukin 6 (IL6) is a pleiotropic cytokine mostly known as a proinflammatory cytokine. There is a need for a reliable in vivo model to study IL6 mediated chronic ...inflammation. Hypothesis: The aim of our study was to create a mouse model mimicking the chronic inflammatory state as observed in cancer and inflammatory diseases. We opted for a recombinant adeno-associated viral vector (rAAV) to mimic the acute initiation of chronic inflammation later in life. Methods: We opted for a rAAV2/8 subtype, human EF1a short (EFS) promoter and firefly luciferase led by an internal ribosome entry site (IL6-IRES-fLuc) DNA cassette to create a stable low-grade expression of IL6 in liver and serum, with minimal expression in other organs, particularly the heart. rAAV2/8_EFS-IL6-IRES-fLuc and the control vector rAAV2/8_EFS-MCS-IRES-fLuc were generated and different dosages were tested. Results: All mice treated with high dose AAV2/8-IL6 vector (8.8* 10 10 genome copies (GC)/animal) showed severe weight loss and hepatosplenomegaly, starting between week 4-6 post injection. Histological examination of the liver showed features of chronic hepatitis. Infiltrating cells were found in both the liver and the heart. In the spleen, the histological structure of red and white pulp was disturbed and more giant cells could be detected. Cardiac function remained normal at 6 weeks post injection. Assuming that the previous results were due to an overexpression of IL6 due to too high amounts of rAAV2/8_EFS-IL6-IRES-fLuc, we subsequently challenged mice with a rAAV2/8_EFS-IL6-IRES-fLuc dilution series to test which vector dose could be used for reliable long-term low grade IL6 overexpression. After extensive testing, the ideal dose for a 12 weeks IL6 overexpression model was between 2.2 and 2.93* 10 10 GC. Using this dose range, we saw stable overexpression of IL6 without weight loss. Hepatosplenomegaly, disturbed anatomy of the spleen and infiltrating cells in the liver were still present yet less pronounced. Conclusion: We successfully generated and characterized a mouse model of IL6 overexpression to mimic chronic IL6 mediated inflammation. To the best of our knowledge this is the first model employing IL6 overexpression using a rAAV viral vector.
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