The ability of Mycobacterium tuberculosis to form serpentine cords is intrinsically related to its virulence, but specifically how M. tuberculosis cording contributes to pathogenesis remains obscure. We show that several M. tuberculosis clinical isolates form intracellular cords in primary human lymphatic endothelial cells (hLEC) in vitro and also in the lymph nodes of patients with tuberculosis. We identified via RNA-seq a transcriptional programme that activates, in infected-hLECs, cell-survival and cytosolic surveillance of pathogens pathways. Consistent with this, cytosolic access is required for intracellular M. tuberculosis cording. Mycobacteria lacking ESX-1 type VII secretion system or PDIM expression, which fail to access to the cytosol, are indeed unable to cords within hLECs. Finally, we show that M. tuberculosis cording is a size-dependent mechanism used by the pathogen to avoid its recognition by cytosolic sensors and evade either resting or IFN-γ-induced hLEC immunity. These results explain the long-standing association between M. tuberculosis cording and virulence and how virulent mycobacteria use intracellular cording as strategy to successfully adapt and persist in the lymphatic tracts.
Thomas R. Lerner, Christophe J. Queval, Rachel PJ Lai, Matthew Robert Geoffrey Russell, Antony Fearns, Daniel J. Greenwood, Lucy Collinson, Robert J. Wilkinson, Maximiliano G. Gutierrez
Ischemic retinopathies are major causes of blindness worldwide. Local hypoxia created by loss of vascular supply leads to tissue injury and aberrant neovascularization in the retina. There is a great need for therapies that enhance revascularization of hypoxic neuroretinal tissue. To test the therapeutic feasibility of human-induced pluripotent stem cell–derived endothelial cells (hiPSC-ECs) for the treatment of ischemic retinopathies, we compared the angiogenic potential of hiPSC-ECs with mature human retinal endothelial cells (HRECs) in response to hypoxia. hiPSC-ECs formed more robust and complex vascular networks in collagen gels, whereas HRECs displayed minimal sprouting. The cells were further tested in the mouse oxygen-induced retinopathy (OIR) model. Retinas with hiPSC-EC injection showed colocalization with host vessels, whereas HRECs lacked such responses. hiPSC-ECs markedly reduced vaso-obliteration and pathological neovascularization. This beneficial effect of hiPSC-ECs was explained by the stromal cell–derived factor-1a (SDF1a)/CXCR4 axis; hiPSC-ECs exhibited much higher cell-surface expression of CXCR4 than HRECs and greater chemotaxis toward SDF1a-embedded 3D collagen hydrogel. Furthermore, treatment with neutralizing antibody to CXCR4 abolished recruitment of hiPSCs in the OIR model. These findings suggest superior angiogenic potential of hiPSC-ECs under hypoxia and underscore the importance of SDF1a/CXCR4 in the reparative function of hiPSC-ECs in ischemic diseases.
Hongkwan Cho, Bria L. Macklin, Ying-Yu Lin, Lingli Zhou, Michael J. Lai, Grace Lee, Sharon Gerecht, Elia J. Duh
Background Salt-sensitive hypertension is often accompanied by insulin resistance in obese individuals, but the underlying mechanisms are obscure. Microvascular function is known to affect both salt-sensitivity of blood pressure and metabolic insulin sensitivity. We hypothesized that excessive salt intake increases blood pressure and decreases insulin-mediated glucose disposal, at least in part by impairing insulin-mediated muscle microvascular recruitment (IMMR). Methods In 20 lean and 20 abdominally obese individuals, we assessed mean arterial pressure (MAP; 24h ABPM), insulin-mediated whole body glucose disposal (M/I-value; hyperinsulinemic, euglycemic clamp technique), IMMR (contrast enhanced ultrasound), osmolyte and water balance, and excretion of mineralocorticoids, glucocorticoids, and amino and organic acids after a low and high salt diet during seven days in a randomized double-blind cross-over design. Results On a low, as compared to a high salt intake, MAP was lower, M/I-value was lower and IMMR was greater in both lean and abdominally obese individuals. In addition, Ln IMMR was inversely associated with MAP in lean participants on a low salt diet only. On a high salt diet, free water clearance decreased, and excretion of glucocorticoids and of amino acids involved in the urea cycle increased. Conclusion Our findings imply that hemodynamic and metabolic changes resulting from alterations in salt intake are not necessarily associated. Moreover, they are consistent with the concept that a high salt intake increases muscle glucose uptake as a response to high-salt-induced, glucocorticoid-drive muscle catabolism to stimulate urea production and thereby renal water conservation. Clinical Trial Registration Number: NCT02068781
Monica T.J. Schütten, Yvo H.A.M. Kusters, Alfons J.H.M. Houben, Hanneke E. C. Niessen, Jos op 't Roodt, Jean L.J. M. Scheijen, Marjo P. van de Waarenburg, Casper G. Schalkwijk, Peter W. de Leeuw, Coen D.A. Stehouwer
Vascular inflammation is present in many cardiovascular diseases, and exogenous glucocorticoids have traditionally been used as a therapy to suppress inflammation. However, recent data have shown that endogenous glucocorticoids, acting through the endothelial glucocorticoid receptor, act as negative regulators of inflammation. Here, we performed ChIP for the glucocorticoid receptor, followed by next-generation sequencing in mouse endothelial cells to investigate how the endothelial glucocorticoid receptor regulates vascular inflammation. We identified a role of the Wnt signaling pathway in this setting and show that loss of the endothelial glucocorticoid receptor results in upregulation of Wnt signaling both in vitro and in vivo using our validated mouse model. Furthermore, we demonstrate glucocorticoid receptor regulation of a key gene in the Wnt pathway, Frzb, via a glucocorticoid response element gleaned from our genomic data. These results suggest a role for endothelial Wnt signaling modulation in states of vascular inflammation.
Han Zhou, Sameet Mehta, Swayam Prakash Srivastava, Kariona Grabinska, Xinbo Zhang, Chris Wong, Ahmad Hedayat, Paola Perrotta, Carlos Fernández-Hernando, William C. Sessa, Julie E. Goodwin
CADASIL leads to premature stroke and vascular dementia. Mechanism-specific therapies for this aggressive cerebral small vessel disease are lacking. CADASIL is caused by NOTCH3 mutations that influence vascular smooth muscle cell (VSMC) function through unknown processes. We investigated molecular mechanisms underlying the vasculopathy in CADASIL focusing on ER stress and RhoA/Rho kinase (ROCK). Peripheral small arteries and VSMCs were isolated from gluteal biopsies of CADASIL patients and mesentery of TgNotch3R169C mice (CADASIL model). CADASIL vessels exhibited impaired vasorelaxation, blunted vasoconstriction and hypertrophic remodelling. Expression of NOTCH3 and ER stress target genes was amplified and ER stress response, Rho kinase activity, superoxide production and cytoskeletal-associated protein phosphorylation were increased in CADASIL, processes associated with Nox5 upregulation. Aberrant vascular responses and signalling in CADASIL were ameliorated by inhibitors of Notch3 (gamma-secretase inhibitor), Nox5 (mellitin), ER stress (4-PBA) and ROCK (fasudil). Observations in human CADASIL were recapitulated in TgNotch3R169C mice. These findings indicate that vascular dysfunction in CADASIL involves ER stress/ROCK interplay driven by Notch3-induced Nox5 activation and that NOTCH3 mutation-associated vascular pathology, typical in cerebral vessels, also manifests peripherally. We define Notch3-Nox5/ERstress/ROCK signaling as a novel putative mechanism-specific target and suggest that peripheral artery responses may be an accessible biomarker in CADASIL.
Karla B. Neves, Adam P. Harvey, Fiona Moreton, Augusto C. Montezano, Francisco J. Rios, Rhéure Alves-Lopes, Aurelie Nguyen Dinh Cat, Paul Rocchiccioli, Christian Delles, Anne Joutel, Keith Muir, Rhian M. Touyz
Lymphatic malformations (LMs) are congenital, non-neoplastic vascular malformations associated with post-zygotic activating PIK3CA mutations. The mutation spectrum within LMs is narrow, with the majority having one of three “hotspot” mutations. Despite this relative genetic homogeneity, clinical presentations differ dramatically. We used molecular inversion probes and droplet digital polymerase chain reaction to perform deep, targeted sequencing of PIK3CA in 271 affected and unaffected tissue samples from 81 individuals with isolated LMs and retrospectively collected clinical data. Pathogenic PIK3CA mutations were identified in affected LM tissue in 64 individuals (79%) with isolated LMs, with variant allele fractions (VAFs) ranging from 0.1 to 13%. Initial analyses revealed no correlation between VAF and phenotype variables. Recognizing that different mutations activate PI3K to varying degrees, we developed a metric, the genotype-adjusted VAF (GVAF), to account for differences in mutation strength, and found significantly higher GVAFs in LMs with more severe clinical characteristics including orofacial location or microcystic structure. In addition to providing insight into LM pathogenesis, we believe GVAF may have broad applicability for genotype-phenotype analyses in mosaic disorders.
Kaitlyn Zenner, Chi Vicky Cheng, Dana M. Jensen, Andrew E. Timms, Giridhar Shivaram, Randall Bly, Sheila Ganti, Kathryn B. Whitlock, William B. Dobyns, Jonathan Perkins, James T. Bennett
Nitric oxide (NO) regulates blood pressure (BP) by binding the reduced heme iron (Fe2+) in soluble guanylyl cyclase (sGC) and relaxing vascular smooth muscle cells (SMC). We previously showed that sGC heme iron reduction (Fe3+ → Fe2+) is modulated by cytochrome b5 reductase 3 (CYB5R3). However, the in vivo role of SMC CYB5R3 in BP regulation remains elusive. Here, we generated conditional smooth muscle cell-specific Cyb5r3 knockout mice (SMC CYB5R3 KO) to test if SMC CYB5R3 loss impacts systemic BP in normotension and hypertension via regulation of sGC redox state. SMC CYB5R3 KO mice exhibited a 5.84 mmHg increase in BP and impaired acetylcholine-induced vasodilation in mesenteric arteries compared to controls. To drive sGC oxidation and elevate BP, we infused mice with angiotensin-II. We found SMC CYB5R3 KO mice exhibited a 14.75 mmHg BP increase and mesenteric arteries had diminished NO-dependent vasodilation, but increased responsiveness to sGC heme-independent activator BAY 58-2667 over controls. Furthermore, acute injection of BAY 58-2667 in angiotensin-II treated SMC CYB5R3 KO mice showed greater BP reduction compared to controls. Together, these data provide the first in vivo evidence that SMC CYB5R3 is a sGC heme reductase in resistance arteries and provides resilience against systemic hypertension development.
Brittany G. Durgin, Scott A. Hahn, Heidi M. Schmidt, Megan P. Miller, Neha Hafeez, Ilka Mathar, Daniel Freitag, Peter Sandner, Adam C. Straub
Age-related macular degeneration (AMD) is the leading cause of central retinal vision loss worldwide, with an estimated 1 in 10 people over the age of 55 showing early signs of the condition. There are currently no forms of therapy available for the end stage of dry AMD, geographic atrophy (GA). Here, we show that the inner blood-retina barrier (iBRB) is highly dynamic and may play a contributory role in GA development. We have discovered that the gene CLDN5, which encodes claudin-5, a tight junction protein abundantly expressed at the iBRB, is regulated by BMAL1 and the circadian clock. Persistent suppression of claudin-5 expression in mice exposed to a cholesterol-enriched diet induced striking retinal pigment epithelium (RPE) cell atrophy, and persistent targeted suppression of claudin-5 in the macular region of nonhuman primates induced RPE cell atrophy. Moreover, fundus fluorescein angiography in human and nonhuman primate subjects showed increased retinal vascular permeability in the evening compared with the morning. These findings implicate an inner retina–derived component in the early pathophysiological changes observed in AMD, and we suggest that restoring the integrity of the iBRB may represent a novel therapeutic target for the prevention and treatment of GA secondary to dry AMD.
Natalie Hudson, Lucia Celkova, Alan Hopkins, Chris Greene, Federica Storti, Ema Ozaki, Erin Fahey, Sofia Theodoropoulou, Paul F. Kenna, Marian M. Humphries, Annie M. Curtis, Eleanor Demmons, Akeem Browne, Shervin Liddie, Matthew S. Lawrence, Christian Grimm, Mark T. Cahill, Pete Humphries, Sarah L. Doyle, Matthew Campbell
Circulating macrophages recruited to the lung contribute to pulmonary vascular remodeling in various forms of pulmonary hypertension (PH). In this study we investigated a macrophage phenotype characterized by intracellular iron accumulation and expression of antioxidant (HO-1), vasoactive (ET-1), and proinflammatory (IL-6) mediators observed in the lung tissue of deceased sickle cell disease (SCD) patients with diagnosed PH. To this end, we evaluated an established rat model of group 5 PH that is simultaneously exposed to free hemoglobin (Hb) and hypobaric hypoxia (HX). Here, we tested the hypothesis that pulmonary vascular remodeling observed in human SCD with concomitant PH could be replicated and mechanistically driven in our rat model by a similar macrophage phenotype with iron accumulation and expression of a similar mixture of antioxidant (HO-1), vasoactive (ET-1), and inflammatory (IL-6) proteins. Our data suggest phenotypic similarities between pulmonary perivascular macrophages in our rat model and human SCD with PH, indicating a potentially novel maladaptive immune response to concomitant bouts of Hb and HX exposure. Moreover, by knocking out circulating macrophages with gadolinium trichloride (GdCl3), the response to combined Hb and hypobaric HX was significantly attenuated in rats, suggesting a critical role for macrophages in the exacerbation of SCD PH.
Katherine Redinus, Jin Hyen Baek, Ayla Yalamanoglu, Hye Kyung H. Shin, Radu Moldova, Julie W. Harral, Delaney Swindle, David Pak, Scott K. Ferguson, Rachelle Nuss, Kathryn Hassell, Eva Nozik-Grayck, Andre F. Palmer, Mehdi A. Fini, Vijaya Karoor, Kurt R. Stenmark, Paul W. Buehler, David C. Irwin
Bone provides supportive microenvironments for hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) and is a frequent site of metastasis. While incidences of bone metastases increase with age, the properties of the bone marrow microenvironment that regulate dormancy and reactivation of disseminated tumor cells (DTCs) remain poorly understood. Here, we elucidate the age-associated changes in the bone secretome that trigger proliferation of HSCs, MSCs, and DTCs in the aging bone marrow microenvironment. Remarkably, a bone-specific mechanism involving expansion of pericytes and induction of quiescence-promoting secretome rendered this proliferative microenvironment resistant to radiation and chemotherapy. This bone-specific expansion of pericytes was triggered by an increase in PDGF signaling via remodeling of specialized type H blood vessels in response to therapy. The decline in bone marrow pericytes upon aging provides an explanation for loss of quiescence and expansion of cancer cells in the aged bone marrow microenvironment. Manipulation of blood flow — specifically, reduced blood flow — inhibited pericyte expansion, regulated endothelial PDGF-B expression, and rendered bone metastatic cancer cells susceptible to radiation and chemotherapy. Thus, our study provides a framework to recognize bone marrow vascular niches in age-associated increases in metastasis and to target angiocrine signals in therapeutic strategies to manage bone metastasis.
Amit Singh, Vimal Veeriah, Pengjun Xi, Rossella Labella, Junyu Chen, Sara G. Romeo, Saravana K. Ramasamy, Anjali P. Kusumbe
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