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
Current clinical methods for the evaluation of lymphatic vessel function, crucial for early diagnosis and evaluation of treatment-response of several pathological conditions, in particular of post-surgical lymphedema, are based on complex and mainly qualitative imaging techniques. To address this unmet medical need, we established a simple strategy for the painless and quantitative assessment of cutaneous lymphatic function. We prepared a lymphatic-specific tracer formulation, consisting of the clinically approved near-infrared fluorescent dye, indocyanine green, and the solubilizing surfactant Kolliphor HS15. The tracer is non-invasively delivered to the dermal layer of the skin using MicronJet600TM hollow microneedles, and the fluorescence signal decay at the injection site is measured over time using a custom-made, portable detection device. The decay rate of fluorescence signal in the skin was used as a direct measure of lymphatic vessel drainage function. With this new method, we could quantify impaired lymphatic clearance in transgenic mice lacking dermal lymphatics and distinguish distinct lymphatic clearance patterns in pigs in different body locations and under manual stimulus. Overall, this method has the potential for becoming a non-invasive and quantitative clinical “office-test” for lymphatic function assessment.
Anna K. Polomska, Steven T. Proulx, Davide Brambilla, Daniel Fehr, Mathias Bonmarin, Simon Brändli, Mirko Meboldt, Christian Steuer, Tsvetina Vasileva, Nils Reinke, Jean-Christophe Leroux, Michael Detmar
The angiopoietin (Ang)-Tie2 signaling pathway is essential for maintaining vascular homeostasis and its dysregulation is associated with several diseases. Interactions between Tie2 and α5β1 integrin have emerged as part of this control; however, the mechanism is incompletely understood. AXT107, a collagen IV-derived peptide, has strong anti-permeability activity and has enabled the elucidation of this previously undetermined mechanism. Previously, AXT107 was shown to inhibit VEGFR2 and other growth factor signaling via receptor tyrosine kinase association with specific integrins. AXT107 disrupts α5β1 and stimulates the relocation of Tie2 and α5 to cell junctions. In the presence of Ang2 and AXT107, junctional Tie2 is activated, downstream survival signals are upregulated, F-actin is rearranged to strengthen junctions, and, as a result, endothelial junctional permeability is reduced. These data suggest that α5β1 sequesters Tie2 in non-junctional locations in endothelial cell membranes and that AXT107-induced disruption of α5β1 promotes clustering of Tie2 at junctions and converts Ang2 into a strong agonist, similar to responses observed when Ang1 levels greatly exceed those of Ang2. The potentiation of Tie2 activation by Ang2 even extended in to mouse models in which AXT107 induced Tie2 phosphorylation in a model of hypoxia and inhibited vascular leakage in an Ang2-overexpression transgenic model and an LPS-induced inflammation model. Since Ang2 levels are very high in ischemic diseases, such as diabetic macular edema, neovascular age-related macular degeneration, uveitis, and cancer, targeting α5β1 with AXT107 provides a novel and potentially more effective approach to treat these diseases.
Adam C. Mirando, Jikui Shen, Raquel Lima e Silva, Zenny Chu, Nicholas Sass, Valeria E. Lorenc, Jordan J. Green, Peter A. Campochiaro, Aleksander S. Popel, Niranjan B. Pandey
Arterial stiffening is a consequence of aging and a cholesterol-independent risk factor for cardiovascular disease (CVD). Arterial stiffening and CVD show a sex bias, with men more susceptible than premenopausal women. How arterial stiffness and sex interact at a molecular level to confer risk of CVD is not well understood. Here, we used the sexual dimorphism in LDLR-null mice to show that the protective effect of female sex on atherosclerosis is linked to reduced aortic stiffness and reduced expression of matrix metalloproteinase-12 (MMP12) by lesional macrophages. Deletion of MMP12 in LDLR-null mice attenuated the male sex bias for both arterial stiffness and atherosclerosis, and these effects occurred despite high serum cholesterol. Mechanistically, we found that oxidized LDL stimulates secretion of MMP12 in human as well as mouse macrophages. Estrogen antagonizes this effect by downregulating MMP12 expression. Our data support cholesterol-independent causal relationships between estrogen, oxidized LDL–induced secretion of macrophage MMP12, and arterial stiffness that protect against atherosclerosis in females and emphasize that reduced MMP12 functionality can confer atheroprotection to males.
Shu-lin Liu, Anamika Bajpai, Elizabeth A. Hawthorne, Yongho Bae, Paola Castagnino, James Monslow, Ellen Puré, Kara L. Spiller, Richard K. Assoian
The contribution of intracellular hemoglobin (Hb) oxidation to RBC-derived microparticle (MP) formation is poorly defined in sickle cell disease (SCD). Here we report that sickle Hb (HbS) oxidation, coupled with changes in cytosolic antioxidative proteins, is associated with membrane alterations and MP formation in homozygous Townes–sickle cell (Townes-SS) mice. Photometric and proteomic analyses confirmed the presence of high levels of Hb oxidation intermediates (ferric/ferryl) and consequent β-globin posttranslational modifications, including the irreversible oxidation of βCys93 and the ubiquitination of βLys96 and βLys145. This is the first report to our knowledge to link the UPS (via ubiquitinated Hb and other proteins) to oxidative stress. Ferryl Hb also induced complex formation with band 3 and RBC membrane proteins. Incubation of Townes-SS MPs with human endothelial cells caused greater loss of monolayer integrity, apoptotic activation, heme oxygenase-1 induction, and concomitant bioenergetic imbalance compared with control Townes-AA MPs. MPs obtained from Townes-SS mice treated with hydroxyurea produced fewer posttranslational Hb modifications. In vitro, hydroxyurea reduced the levels of ferryl Hb and shielded its target residue, βCys93, by a process of S-nitrosylation. These mechanistic analyses suggest potential antioxidative therapeutic modalities that may interrupt MP heme-mediated pathophysiology in SCD patients.
Sirsendu Jana, Michael Brad Strader, Fantao Meng, Wayne Hicks, Tigist Kassa, Ivan Tarandovskiy, Silvia De Paoli, Jan Simak, Michael R. Heaven, John D. Belcher, Gregory M. Vercellotti, Abdu I. Alayash
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