Although cardiovascular disease (CVD) is the leading cause of morbimortality worldwide, promising new drug candidates are lacking. We compared the arterial high-resolution proteome of patients with advanced versus early-stage CVD to predict, from a library of small bioactive molecules, drug candidates able to reverse this disease signature. Of the approximately 4000 identified proteins, 100 proteins were upregulated and 52 were downregulated in advanced-stage CVD. Arachidonyl trifluoromethyl ketone (AACOCF3), a cytosolic phospholipase A2 (cPLA2) inhibitor was predicted as the top drug able to reverse the advanced-stage CVD signature. Vascular cPLA2 expression was increased in patients with advanced-stage CVD. Treatment with AACOCF3 significantly reduced vascular calcification in a cholecalciferol-overload mouse model and inhibited osteoinductive signaling in vivo and in vitro in human aortic smooth muscle cells. In conclusion, using a systems biology approach, we have identified a potentially new compound that prevented typical vascular calcification in CVD in vivo. Apart from the clear effect of this approach in CVD, such strategy should also be able to generate novel drug candidates in other complex diseases.
Joost P. Schanstra, Trang T.D. Luong, Manousos Makridakis, Sophie Van Linthout, Vasiliki Lygirou, Agnieszka Latosisnska, Ioana Alesutan, Beate Boehme, Nadeshda Schelski, Dirk Von Lewinski, William Mullen, Stuart Nicklin, Christian Delles, Guylène Feuillet, Colette Denis, Florian Lang, Burkert Pieske, Jean-Loup Bascands, Harald Mischak, Jean-Sebastien Saulnier-Blache, Jakob Voelkl, Antonia Vlahou, Julie Klein
Hypercholesterolemia and hypertension are two major risk factors for coronary artery diseases, which remain the major cause of mortality in the industrialized world. Current animal models of atherosclerosis do not recapitulate coronary plaque disruption, thrombosis, and myocardial infarction occurring in humans. Recently, we demonstrated that exposure of the heart to high pressure, by transverse aortic constriction (TAC), induced coronary lesions in ApoE–/– mice on chow diet. The aim of this study was to characterize the magnitude and location of coronary lesions in ApoE–/– mice after TAC and to assess the susceptibility of coronary plaque to disruption, leading to myocardial events. Here, we describe a reliable pathological condition in mice characterized by the development of coronary lesions and its progression, leading to myocardial infarction; this model better recapitulates human disease. Following TAC surgery, about 90% of ApoE–/– mice developed coronary lesions, especially in the left anterior descending artery, with 59% of the mice manifesting a different magnitude of LAD stenosis. Myocardial events, identified in 74% of the mice, were mainly due to coronary plaque thrombosis and occlusion. That TAC-induced development and progression of coronary lesions in ApoE–/– mice, leading to myocardial events, represents a potentially novel and important tool to investigate the development of coronary lesions and its sequelae in a setting that better resemble human conditions.
Alice Marino, Yi Zhang, Luisa Rubinelli, Maria Antonietta Riemma, James E. Ip, Annarita Di Lorenzo
The interplay among signaling events for endothelial cell (EC) senescence, apoptosis, and activation and how these pathological conditions promote atherosclerosis in the area exposed to disturbed flow (d-flow) in concert remain unclear. The aim of this study was to determine whether telomeric repeat-binding factor 2–interacting protein (TERF2IP), a member of the shelterin complex at the telomere, can regulate EC senescence, apoptosis, and activation simultaneously, and if so, by what molecular mechanisms. We found that d-flow induced p90RSK and TERF2IP interaction in a p90RSK kinase activity–dependent manner. An in vitro kinase assay revealed that p90RSK directly phosphorylated TERF2IP at the serine 205 (S205) residue, and d-flow increased TERF2IP S205 phosphorylation as well as EC senescence, apoptosis, and activation by activating p90RSK. TERF2IP phosphorylation was crucial for nuclear export of the TERF2IP-TRF2 complex, which led to EC activation by cytosolic TERF2IP-mediated NF-κB activation and also to senescence and apoptosis of ECs by depleting TRF2 from the nucleus. Lastly, using EC-specific TERF2IP-knockout (TERF2IP-KO) mice, we found that the depletion of TERF2IP inhibited d-flow–induced EC senescence, apoptosis, and activation, as well as atherosclerotic plaque formation. These findings demonstrate that TERF2IP is an important molecular switch that simultaneously accelerates EC senescence, apoptosis, and activation by S205 phosphorylation.
Sivareddy Kotla, Hang Thi Vu, Kyung Ae Ko, Yin Wang, Masaki Imanishi, Kyung-Sun Heo, Yuka Fujii, Tamlyn N. Thomas, Young Jin Gi, Hira Mazhar, Jesus Paez-Mayorga, Ji-Hyun Shin, Yunting Tao, Carolyn J. Giancursio, Jan L.M. Medina, Jack Taunton, Aldos J. Lusis, John P. Cooke, Keigi Fujiwara, Nhat-Tu Le, Jun-ichi Abe
Extracellular mRNAs (ex-mRNAs) potentially supersede extracellular miRNAs (ex-miRNAs) and other RNA classes as biomarkers. We performed conventional small-RNA-sequencing (sRNA-seq) and sRNA-seq with T4 polynucleotide kinase (PNK) end-treatment of total exRNA isolated from serum and platelet-poor EDTA, ACD, and heparin plasma to study the effect on ex-mRNA capture. Compared to conventional sRNA-seq PNK-treatment increased the detection of informative ex-mRNAs reads up to 50-fold. The exRNA pool was dominated by hematopoietic cells and platelets, with additional contribution from the liver. About 60% of the 15- to 42-nt reads originated from the coding sequences, in a pattern reminiscent of ribosome-profiling. Blood sample type had a considerable influence on the exRNA profile. On average approximately 350 to 1,100 distinct ex-mRNA transcripts were detected depending on plasma type. In serum, additional transcripts from neutrophils and hematopoietic cells increased this number to near 2,300. EDTA and ACD plasma showed a destabilizing effect on ex mRNA and non-coding RNA ribonucleoprotein complexes compared to other plasma types. In a proof-of-concept study, we investigated differences between the exRNA profiles of patients with acute coronary syndrome (ACS) and healthy controls. The improved tissue resolution of ex mRNAs after PNK-treatment enabled us to detect a neutrophil-signature in ACS that escaped detection by ex miRNA analysis.
Kemal M. Akat, Youngmin A. Lee, Arlene Hurley, Pavel Morozov, Klaas E.A. Max, Miguel Brown, Kimberly Bogardus, Anuoluwapo Sopeyin, Kai Hildner, Thomas G. Diacovo, Markus F. Neurath, Martin Borggrefe, Thomas Tuschl
Changes in neuronal activity alter blood flow to match energy demand with the supply of oxygen and nutrients. This functional hyperemia is maintained by interactions between neurons, vascular cells, and glia. However, how changing neuronal activity prevalent at the onset of neurodegenerative disease affects neurovascular elements is unclear. Here, in mice with photoreceptor degeneration, a model of neuron-specific dysfunction, we combined assessment of visual function, neurovascular unit structure, and the blood-retina barrier permeability. We found that the rod loss paralleled remodeling of the neurovascular unit, comprised of photoreceptors, retinal pigment epithelium, and Muller glia. When significant visual function was still present, blood flow became disrupted and blood-retina barrier began to fail, facilitating cone loss and vision decline. Thus, in contrast to the established view, vascular deficit in neuronal degeneration is not a late consequence of neuronal dysfunction, but is present early in the course of disease. These findings further establish the importance of vascular deficit and blood retina barrier function in neuron-specific loss, and highlight it as a target for early therapeutic intervention.
Elena Ivanova, Nazia M. Alam, Glen T. Prusky, Botir T. Sagdullaev
The purpose of this study was to determine important genes, functions, and networks contributing to the pathobiology of cerebral cavernous malformation (CCM) from transcriptomic analyses across 3 species and 2 disease genotypes. Sequencing of RNA from laser microdissected neurovascular units of 5 human surgically resected CCM lesions, mouse brain microvascular endothelial cells, Caenorhabditis elegans with induced Ccm gene loss, and their respective controls provided differentially expressed genes (DEGs). DEGs from mouse and C. elegans were annotated into human homologous genes. Cross-comparisons of DEGs between species and genotypes, as well as network and gene ontology (GO) enrichment analyses, were performed. Among hundreds of DEGs identified in each model, common genes and 1 GO term (GO:0051656, establishment of organelle localization) were commonly identified across the different species and genotypes. In addition, 24 GO functions were present in 4 of 5 models and were related to cell-to-cell adhesion, neutrophil-mediated immunity, ion transmembrane transporter activity, and responses to oxidative stress. We have provided a comprehensive transcriptome library of CCM disease across species and for the first time to our knowledge in Ccm1/Krit1 versus Ccm3/Pdcd10 genotypes. We have provided examples of how results can be used in hypothesis generation or mechanistic confirmatory studies.
Janne Koskimäki, Romuald Girard, Yan Li, Laleh Saadat, Hussein A. Zeineddine, Rhonda Lightle, Thomas Moore, Seán Lyne, Kenneth Avner, Robert Shenkar, Ying Cao, Changbin Shi, Sean P. Polster, Dongdong Zhang, Julián Carrión-Penagos, Sharbel Romanos, Gregory Fonseca, Miguel A. Lopez-Ramirez, Eric M. Chapman, Evelyn Popiel, Alan T. Tang, Amy Akers, Pieter Faber, Jorge Andrade, Mark Ginsberg, W. Brent Derry, Mark L. Kahn, Douglas A. Marchuk, Issam A. Awad
Kawasaki disease (KD), the leading cause of acquired cardiac disease among children, is often associated with myocarditis that may lead to long-term myocardial dysfunction and fibrosis. Although those myocardial changes develop during the acute phase, they may persist for decades and closely correlate with long-term myocardial sequelae. Using the Lactobacillus casei cell wall extract–induced (LCWE-induced) KD vasculitis murine model, we investigated long-term cardiovascular sequelae, such as myocardial dysfunction, fibrosis, and coronary microvascular lesions following adrenergic stimuli after established KD vasculitis. We found that adrenergic stimulation with isoproterenol following LCWE-induced KD vasculitis in mice was associated with increased risk of cardiac hypertrophy and myocardial fibrosis, diminished ejection fraction, and increased serum levels of brain natriuretic peptide. Myocardial fibrosis resulting from pharmacologic-induced exercise after KD development was IL-1 signaling dependent and was associated with a significant reduction in myocardial capillary CD31 expression, indicative of a rarefied myocardial capillary bed. These observations suggest that adrenergic stimulation after KD vasculitis may lead to cardiac hypertrophy and bridging fibrosis in the myocardium in the LCWE-induced KD vasculitis mouse model and that this process involves IL-1 signaling and diminished microvascular circulation in the myocardium.
Harry H. Matundan, Jon Sin, Magali Noval Rivas, Michael C. Fishbein, Thomas J. Lehman, Shuang Chen, Roberta A. Gottlieb, Timothy R. Crother, Masanori Abe, Moshe Arditi
Acid aspiration, which can result from several etiologies, including postoperative complications, leads to direct contact of concentrated hydrochloric acid (HCl) with the alveolar epithelium. As a result, rapid endothelial activation induces alveolar inflammation, leading to life-threatening pulmonary edema. Because mechanisms underlying the rapid endothelial activation are not understood, here we determined responses in real time through optical imaging of alveoli of live mouse lungs. By alveolar micropuncture, we microinfused concentrated HCl in the alveolar lumen. As expected, acid contact with the epithelium caused rapid, but transient, apical injury. However, there was no concomitant membrane injury to the endothelium. Nevertheless, H2O2-mediated epithelial-endothelial paracrine signaling induced endothelial barrier failure, as detected by microvascular dextran leakage and lung water quantification. Remarkably, endothelial mitochondria regulated the barrier failure by activating uncoupling protein 2 (UCP2), thereby inducing transient mitochondrial depolarization that led to cofilin-induced actin depolymerization. Knockdown, or endothelium-targeted deletion of UCP2 expression, blocked these responses, including pulmonary edema. To our knowledge, these findings are the first to mechanistically implicate endothelial mitochondria in acid-induced barrier deterioration and pulmonary edema. We suggest endothelial UCP2 may be a therapeutic target for acid-induced acute lung injury.
Rebecca F. Hough, Mohammad N. Islam, Galina A. Gusarova, Guangchun Jin, Shonit Das, Jahar Bhattacharya
Beta-2 microglobulin (β2M) is a molecular chaperone for the major histocompatibility class I (MHC I) complex, hemochromatosis factor protein (HFE), and the neonatal Fc receptor (FcRn), but β2M may also have less understood chaperoneindependent functions. Elevated plasma β2M has a direct role in neurocognitive decline and is a risk factor for adverse cardiovascular events. β2M mRNA is present in platelets at very high levels and β2M is part of the activated platelet releasate. In addition to their more well studied thrombotic functions, platelets are important immune regulatory cells that release inflammatory molecules and contribute to leukocyte trafficking, activation, and differentiation. We have now found that platelet-derived β2M is a mediator of monocyte pro-inflammatory differentiation through non-canonical TGF-β receptor signaling. Circulating monocytes from mice lacking β2M only in platelets (Plt-β2M–/–) had a more pro-reparative monocyte phenotype, in part dependent on increased platelet-derived TGF-β signaling in the absence of β2M. Using a mouse myocardial infarction (MI) model, Plt-β2M–/– mice had limited post-MI pro-inflammatory monocyte responses, and instead demonstrated early pro-reparative monocyte differentiation, profibrotic myofibroblast responses, and a rapid decline in heart function compared to WT mice. These data demonstrate a novel chaperone-independent, monocyte phenotype regulatory function for platelet β2M, and that platelet-derived β2M and TGF-β have opposing roles in monocyte differentiation that may be important in tissue injury responses.
Zachary T. Hilt, Daphne N. Pariser, Sara K. Ture, Amy Mohan, Pearl Quijada, Akua Asante, Scott J. Cameron, Julie A. Sterling, Alyssa R. Merkel, Andrew L. Johanson, Jermaine L. Jenkins, Eric M. Small, Kathleen E. McGrath, James Palis, Michael R. Elliott, Craig N. Morrell
The lymphatic system plays crucial roles in tissue homeostasis, lipid absorption and immune cell trafficking. While lymphatic valves ensure unidirectional lymph flows, the flow itself controls lymphatic valve formation. Here, we demonstrate that a mechanically activated ion channel Piezo1 senses oscillating shear stress (OSS), and incorporates the signal into the genetic program controlling lymphatic valve development and maintenance. Time-controlled deletion of Piezo1 using a pan-endothelial Cre driver (Cdh5(PAC)-CreERT2) or lymphatic-specific Cre driver (Prox1-CreERT2) equally inhibited lymphatic valve formation in newborn mouse. Furthermore, Piezo1 deletion in adult lymphatics caused substantial lymphatic valve degeneration. Piezo1 knockdown in cultured lymphatic endothelial cells (LECs) largely abrogated the OSS-induced upregulation of the lymphatic valve-signature genes. Conversely, ectopic Piezo1 overexpression upregulated the lymphatic valve genes in the absence of OSS. Remarkably, activation of Piezo1 using a chemical agonist Yoda1 not only accelerated lymphatic valve formation in animals, but also triggered upregulation of some lymphatic valve genes in cultured LECs without exposure to OSS. In summary, our studies together demonstrate that Piezo1 is the force sensor in the mechanotransduction pathway controlling lymphatic valve development and maintenance, and Piezo1 activation is a potential novel therapeutic strategy for congenital and surgery-associated lymphedema.
Dongwon Choi, Eunkyung Park, Eunson Jung, Boksik Cha, Somin Lee, James Yu, Paul M. Kim, Sunju Lee, Yeo Jin Hong, Chester J. Koh, Chang-Won Cho, Yifan Wu, Noo Li Jeon, Alex K. Wong, Laura Shin, S. Ram Kumar, Ivan Bermejo-Moreno, R. Sathish Srinivasan, Il-Taeg Cho, Young-Kwon Hong
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