High Resolution Imaging of Vascular Function in Zebrafish

Rationale: The role of the endothelium in the pathogenesis of cardiovascular disease is an emerging field of study, necessitating the development of appropriate model systems and methodologies to investigate the multifaceted nature of endothelial dysfunction including disturbed barrier function and impaired vascular reactivity. Objective: We aimed to develop and test an optimized high-speed imaging platform to obtain quantitative real-time measures of blood flow, vessel diameter and endothelial barrier function in order to assess vascular function in live vertebrate models. Methods and Results: We used a combination of cutting-edge optical imaging techniques, including high-speed, camera-based imaging (up to 1000 frames/second), and 3D confocal methods to collect real time metrics of vascular performance and assess the dynamic response to the thromboxane A2 (TXA2) analogue, U-46619 (1 μM), in transgenic zebrafish larvae. Data obtained in 3 and 5 day post-fertilization larvae show that these methods are capable of imaging blood flow in a large (1 mm) segment of the vessel of interest over many cardiac cycles, with sufficient speed and sensitivity such that the trajectories of individual erythrocytes can be resolved in real time. Further, we are able to map changes in the three dimensional sizes of vessels and assess barrier function by visualizing the continuity of the endothelial layer combined with measurements of extravasation of fluorescent microspheres. Conclusions: We propose that this system-based microscopic approach can be used to combine measures of physiologic function with molecular behavior in zebrafish models of human vascular disease. © 2012 Watkins et al

Chlamydia Hijacks ARF GTPases To Coordinate Microtubule Posttranslational Modifications and Golgi Complex Positioning.

The intracellular bacterium Chlamydia trachomatis develops in a parasitic compartment called the inclusion. Posttranslationally modified microtubules encase the inclusion, controlling the positioning of Golgi complex fragments around the inclusion. The molecular mechanisms by which Chlamydia coopts the host cytoskeleton and the Golgi complex to sustain its infectious compartment are unknown. Here, using a genetically modified Chlamydia strain, we discovered that both posttranslationally modified microtubules and Golgi complex positioning around the inclusion are controlled by the chlamydial inclusion protein CT813/CTL0184/InaC and host ARF GTPases. CT813 recruits ARF1 and ARF4 to the inclusion membrane, where they induce posttranslationally modified microtubules. Similarly, both ARF isoforms are required for the repositioning of Golgi complex fragments around the inclusion. We demonstrate that CT813 directly recruits ARF GTPases on the inclusion membrane and plays a pivotal role in their activation. Together, these results reveal that Chlamydia uses CT813 to hijack ARF GTPases to couple posttranslationally modified microtubules and Golgi complex repositioning at the inclusion.IMPORTANCEChlamydia trachomatis is an important cause of morbidity and a significant economic burden in the world. However, how Chlamydia develops its intracellular compartment, the so-called inclusion, is poorly understood. Using genetically engineered Chlamydia mutants, we discovered that the effector protein CT813 recruits and activates host ADP-ribosylation factor 1 (ARF1) and ARF4 to regulate microtubules. In this context, CT813 acts as a molecular platform that induces the posttranslational modification of microtubules around the inclusion. These cages are then used to reposition the Golgi complex during infection and promote the development of the inclusion. This study provides the first evidence that ARF1 and ARF4 play critical roles in controlling posttranslationally modified microtubules around the inclusion and that Chlamydia trachomatis hijacks this novel function of ARF to reposition the Golgi complex

Fatty acid nitroalkenes ameliorate glucose intolerance and pulmonary hypertension in high-fat diet-induced obesity

Aims Obesity is a risk factor for diabetes and cardiovascular diseases, with the incidence of these disorders becoming epidemic. Pathogenic responses to obesity have been ascribed to adipose tissue (AT) dysfunction that promotes bioactive mediator secretion from visceral AT and the initiation of pro-inflammatory events that induce oxidative stress and tissue dysfunction. Current understanding supports that suppressing pro-inflammatory and oxidative events promotes improved metabolic and cardiovascular function. In this regard, electrophilic nitro-fatty acids display pleiotropic anti-inflammatory signalling actions. Methods and results It was hypothesized that high-fat diet (HFD)-induced inflammatory and metabolic responses, manifested by loss of glucose tolerance and vascular dysfunction, would be attenuated by systemic administration of nitrooctadecenoic acid (OA-NO2). Male C57BL/6j mice subjected to a HFD for 20 weeks displayed increased adiposity, fasting glucose, and insulin levels, which led to glucose intolerance and pulmonary hypertension, characterized by increased right ventricular (RV) end-systolic pressure (RVESP) and pulmonary vascular resistance (PVR). This was associated with increased lung xanthine oxidoreductase (XO) activity, macrophage infiltration, and enhanced expression of pro-inflammatory cytokines. Left ventricular (LV) end-diastolic pressure remained unaltered, indicating that the HFD produces pulmonary vascular remodelling, rather than LV dysfunction and pulmonary venous hypertension. Administration of OA-NO2 for the final 6.5 weeks of HFD improved glucose tolerance and significantly attenuated HFD-induced RVESP, PVR, RV hypertrophy, lung XO activity, oxidative stress, and pro-inflammatory pulmonary cytokine levels. Conclusions These observations support that the pleiotropic signalling actions of electrophilic fatty acids represent a therapeutic strategy for limiting the complex pathogenic responses instigated by obesity.Fil: Kelley, Eric E.. University of Pittsburgh; Estados UnidosFil: Baust, Jeff. University of Pittsburgh; Estados UnidosFil: Bonacci, Gustavo Roberto. University of Pittsburgh; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Golin Bisello, Franca. University of Pittsburgh; Estados UnidosFil: Devlin, Jason E.. University of Pittsburgh; Estados UnidosFil: Croix, Claudette M. St.. University of Pittsburgh; Estados UnidosFil: Watkins, Simon C.. University of Pittsburgh; Estados UnidosFil: Gor, Sonia. University of Pittsburgh; Estados UnidosFil: Cantu Medellin, Nadiezhda. University of Pittsburgh; Estados UnidosFil: Weidert, Eric R.. University of Pittsburgh; Estados UnidosFil: Frisbee,Jefferson C.. University of Virginia; Estados UnidosFil: Gladwin, Mark T.. University of Pittsburgh; Estados UnidosFil: Champion, Hunter C.. University of Pittsburgh; Estados UnidosFil: Freeman, Bruce A.. University of Pittsburgh; Estados UnidosFil: Khoo, Nicholas K.H.. University of Pittsburgh; Estados Unido

Oxidation products of 5-methyl cytosine are decreased in senescent cells and tissues of progeroid mice

5-Hydroxymethylcytosine and 5-formylcytosine are stable DNA base modifications generated from 5-methylcytosine by the ten-eleven translocation protein family that function as epigenetic markers. 5-Hydroxymethyluracil may also be generated from thymine by ten-eleven translocation enzymes. Here, we asked if these epigenetic changes accumulate in senescent cells, since they are thought to be inversely correlated with proliferation. Testing this in ERCC1-XPF-deficient cells and mice also enabled discovery if these DNA base changes are repaired by nucleotide excision repair. Epigenetic marks were measured in proliferating, quiescent and senescent wild-type (WT) and Ercc1−/− primary mouse embryonic fibroblasts. The pattern of epigenetic marks depended more on the proliferation status of the cells than their DNA repair capacity. The cytosine modifications were all decreased in senescent cells compared to quiescent or proliferating cells, whereas 5-(hydroxymethyl)-2′-deoxyuridine was increased. In vivo, both 5-(hydroxymethyl)-2′-deoxyuridine and 5-(hydroxymethyl)-2′-deoxycytidine were significantly increased in liver tissues of aged WT mice compared to young adult WT mice. Livers of Ercc1-deficient mice with premature senescence and aging had reduced level of 5-(hydroxymethyl)- 2′-deoxycytidine and 5-formyl-2′-deoxycytidine compared to aged-matched WT controls. Taken together, we demonstrate for the first time, that 5-(hydroxymethyl)-2′-deoxycytidine is significantly reduced in senescent cells and tissue, potentially yielding a novel marker of senescence

Interleukin-22 inhibits respiratory syncytial virus production by blocking virus-mediated subversion of cellular autophagy

Respiratory syncytial virus (RSV) infection can cause severe bronchiolitis in infants requiring hospitalization, whereas the elderly and immunocompromised are prone to RSV-induced pneumonia. RSV primarily infects lung epithelial cells. Given that no vaccine against RSV is currently available, we tested the ability of the epithelial-barrier protective cytokine interleukin-22 (IL-22) to control RSV production. When used in a therapeutic modality, IL-22 efficiently blunted RSV production from infected human airway and alveolar epithelial cells and IL-22 administration drastically reduced virus titer in the lungs of infected newborn mice. RSV infection resulted in increased expression of LC3B, a key component of the cellular autophagic machinery, and knockdown of LC3B ablated virus production. RSV subverted LC3B with evidence of co-localization and caused a significant reduction in autophagic flux, both reversed by IL-22 treatment. Our findings inform a previously unrecognized anti-viral effect of IL-22 that can be harnessed to prevent RSV-induced severe respiratory disease

Dysregulation of DAF-16/FOXO3A-mediated stress responses accelerates T oxidative DNA damage induced aging

DNA damage is presumed to be one type of stochastic macromolecular damage that contributes to aging, yet little is known about the precise mechanism by which DNA damage drives aging. Here, we attempt to address this gap in knowledge using DNA repair-deficient C. elegans and mice. ERCC1-XPF is a nuclear endonuclease required for genomic stability and loss of ERCC1 in humans and mice accelerates the incidence of age-related pathologies. Like mice, ercc-1 worms are UV sensitive, shorter lived, display premature functional decline and they accumulate spontaneous oxidative DNA lesions (cyclopurines) more rapidly than wild-type worms. We found that ercc-1 worms displayed early activation of DAF-16 relative to wild-type worms, which conferred resistance to multiple stressors and was important for maximal longevity of the mutant worms. However, DAF- 16 activity was not maintained over the lifespan of ercc-1 animals and this decline in DAF-16 activation cor- responded with a loss of stress resistance, a rise in oxidant levels and increased morbidity, all of which were cep- 1/ p53 dependent. A similar early activation of FOXO3A (the mammalian homolog of DAF-16), with increased resistance to oxidative stress, followed by a decline in FOXO3A activity and an increase in oxidant abundance was observed in Ercc1-/- primary mouse embryonic fibroblasts. Likewise, in vivo, ERCC1-deficient mice had transient activation of FOXO3A in early adulthood as did middle-aged wild-type mice, followed by a late life decline. The healthspan and mean lifespan of ERCC1 deficient mice was rescued by inactivation of p53. These data indicate that activation of DAF-16/FOXO3A is a highly conserved response to genotoxic stress that is important for suppressing consequent oxidative stress. Correspondingly, dysregulation of DAF-16/FOXO3A appears to underpin shortened healthspan and lifespan, rather than the increased DNA damage burden itself

Lower respiratory tract myeloid cells harbor SARS-CoV-2 and display an inflammatory phenotype

SARS-CoV-2 pneumonia may induce an aberrant immune response with brisk recruitment of myeloid cells into the airspaces. Although the clinical implications are unclear, others have suggested that infiltrating myeloid cells may contribute to morbidity and mortality during SARS-CoV-2 infection.1–3 However, few reports have characterized myeloid cells from the lower respiratory tract, which appears to be the primary site of viral-induced pathology, during severe SARS-CoV-2 pneumonia

Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation

NADPH oxidase 4 (NOX4) regulates endothelial inflammation by producing hydrogen peroxide (H2O2) and to a lesser extent O2•-. The ratio of NOX4-derived H2O2 and O2•- can be altered by coenzyme Q (CoQ) mimics. Therefore, we hypothesize that cytochrome b5 reductase 3 (CYB5R3), a CoQ reductase abundant in vascular endothelial cells, regulates inflammatory activation. To examine endothelial CYB5R3 in vivo, we created tamoxifen-inducible endothelium-specific Cyb5r3 knockout mice (R3 KO). Radiotelemetry measurements of systolic blood pressure showed systemic hypotension in lipopolysaccharides (LPS) challenged mice, which was exacerbated in R3 KO mice. Meanwhile, LPS treatment caused greater endothelial dysfunction in R3 KO mice, evaluated by acetylcholine-induced vasodilation in the isolated aorta, accompanied by elevated mRNA expression of vascular adhesion molecule 1 (Vcam-1). Similarly, in cultured human aortic endothelial cells (HAEC), LPS and tumor necrosis factor α (TNF-α) induced VCAM-1 protein expression was enhanced by Cyb5r3 siRNA, which was ablated by silencing the Nox4 gene simultaneously. Moreover, super-resolution confocal microscopy indicated mitochondrial co-localization of CYB5R3 and NOX4 in HAECs. APEX2-based electron microscopy and proximity biotinylation also demonstrated CYB5R3's localization on the mitochondrial outer membrane and its interaction with NOX4, which was further confirmed by the proximity ligation assay. Notably, Cyb5r3 knockdown HAECs showed less total H2O2 but more mitochondrial O2•-. Using inactive or non-membrane bound active CYB5R3, we found that CYB5R3 activity and membrane translocation are needed for optimal generation of H2O2 by NOX4. Lastly, cells lacking the CoQ synthesizing enzyme COQ6 showed decreased NOX4-derived H2O2, indicating a requirement for endogenous CoQ in NOX4 activity. In conclusion, CYB5R3 mitigates endothelial inflammatory activation by assisting in NOX4-dependent H2O2 generation via CoQ.This work was supported by National Institutes of Health (NIH) R01 awards [R01 HL 133864 (A.C.S), R01 HL 128304 (A.C.S), R01 HL 149825 (A.C.S), R01 HL 153532 (A.C.S), R01 GM 125944 (F.J.S.), R01 DK 112854 (F.J.S.), 1S10OD021540-01 (Center for Biologic Imaging, University of Pittsburgh), 1S10RR019003-01 (Simon Watkins (S.W.)), 1S10RR025488-01 (S.W.), 1S10RR016236-01 (S.W)]. American Heart Association (AHA) [Established Investigator Award 19EIA34770095 (A.C.S.)], Post-doctoral Fellowship 19POST34410028 (S.Y.)]. American Society of Hematology (ASH) Minority Hematology Graduate Award (A.M.D-O.). Junta de Andalucía grant BIO-177 (P.N.), the FEDER Funding Program from the European Union and Spanish Ministry of Science, Innovation and Universities grant RED2018-102576-T (P.N.)