Kawasaki disease (KD) is the leading cause of non-congenital heart disease in children. Studies in mice and humans propound the NLRP3-IL-1β pathway as the principal driver of KD pathophysiology. Endoplasmic reticulum (ER) stress can activate the NLRP3 inflammasome, but the potential implication of ER stress in KD pathophysiology has not been investigated. We used human patient data and the Lactobacillus casei cell wall extract (LCWE) murine model of KD vasculitis to characterize the impact of ER stress on the development of cardiovascular lesions. KD patient transcriptomics and single-cell RNA sequencing of the abdominal aorta from LCWE-injected mice revealed changes in the expression of ER stress genes. Alleviating ER stress genetically, by conditional deletion of Inositol Requiring Enzyme-1 (IRE1) in myeloid cells, or pharmacologically, by inhibition of IRE1 endoribonuclease (RNase) activity, led to significant reduction of LCWE-induced cardiovascular lesion formation as well as reduced caspase-1 activity and IL-1β secretion. These results demonstrate the causal relationship of ER stress to KD pathogenesis, and highlight IRE1 RNase activity as a potential new therapeutic target.
Stefanie Marek-Iannucci, Asli D. Yildirim, Syed M. Hamid, Asli B. Ozdemir, Angela C. Gomez, Begüm Kocatürk, Rebecca A. Porritt, Michael C. Fishbein, Takao Iwawaki, Magali Noval Rivas, Ebru Erbay, Moshe Arditi
Cilia, microtubule-based organelles that project from the apical luminal surface of endothelial cells (ECs), are widely regarded as a low flow-sensors. Previous reports suggest that upon high shear stress, cilia on the EC surface are lost, and more recent evidence suggests that deciliation - the physical removal of cilia from the cell surface - is a predominant mechanism for cilia loss in mammalian cells. Thus, we hypothesized that EC deciliation facilitated by changes in shear stress will manifest in increased abundance of cilia-related proteins in circulation. To test this hypothesis, we performed shear stress experiments that mimicked flow conditions from low to high shear stress in human primary cells and a zebrafish model system. In the primary cells, we showed that upon shear stress induction, indeed, ciliary fragments were observed in the effluent in vitro and effluents contained ciliary proteins normally expressed in both endothelial and epithelial cells. In zebrafish, upon shear stress induction, fewer ciliary-expressing ECs were observed. To test the translational relevance of these findings, we investigated our hypothesis using patient blood samples from sickle cell disease and found that plasma levels of ciliary proteins were elevated compared to healthy controls. Further, sickled red blood cells demonstrated high levels of ciliary protein (Arl13b) on their surface post-adhesion to brain ECs. Brain ECs post interaction with sickle RBCs show high reactive oxygen species (ROS) levels. Attenuating ROS levels in brain ECs decreases cilia protein levels on RBCs and rescues ciliary protein levels in brain ECs. Collectively, these data suggest that cilia and ciliary proteins in circulation are detectable under various altered flow conditions, which could serve as a surrogate biomarker of the damaged endothelium.
Ankan Gupta, Karthikeyan Thirugnanam, Madhan Thamilarasan, Ashraf M. Mohieldin, Hadeel T. Zedan, Shubhangi Prabhudesai, Meghan R. Griffin, Andrew D. Spearman, Amy Pan, Sean P. Palecek, Huseyin C. Yalcin, Surya M. Nauli, Kevin R. Rarick, Rahima Zennadi, Ramani Ramchandran
Aortic dissection and rupture are triggered by decreased vascular wall strength and/or increased mechanical loads. We investigated the role of mTOR signaling in aortopathy using a well-described model of angiotensin II–induced dissection, aneurysm, or rupture of the suprarenal abdominal aorta in Apoe-deficient mice. Although not widely appreciated, nonlethal hemorrhagic lesions present as pseudoaneurysms without significant dissection in this model. Angiotensin II–induced aortic tears result in free rupture, contained rupture with subadventitial hematoma (forming pseudoaneurysms), dilatation, or healing, while the media invariably thickens regardless of mural tears. Medial thickening results from smooth muscle cell hypertrophy and extracellular matrix accumulation, including matricellular proteins. Angiotensin II activates mTOR signaling in vascular wall cells, and inhibition of mTOR signaling by rapamycin prevents aortic rupture but promotes dissection. Decreased aortic rupture correlates with decreased inflammation and metalloproteinase expression, whereas extensive dissection correlates with induction of matricellular proteins that modulate adhesion of vascular cells. Thus, mTOR activation in vascular wall cells determines whether aortic tears progress to dissection or rupture. Previous mechanistic studies of aortic aneurysm and dissection by angiotensin II in Apoe-deficient mice should be reinterpreted as clinically relevant to pseudoaneurysms, and mTOR inhibition for aortic disease should be explored with caution.
Changshun He, Bo Jiang, Mo Wang, Pengwei Ren, Sae-Il Murtada, Alexander W. Caulk, Guangxin Li, Lingfeng Qin, Roland Assi, Constantinos J. Lovoulos, Martin A. Schwartz, Jay D. Humphrey, George Tellides
BACKGROUND. Accumulation of advanced glycation endproducts (AGEs) may contribute to the pathophysiology of type 2 diabetes and its vascular complications. AGEs are widely present in food, but whether restricting AGE intake improves risk factors for type 2 diabetes and vascular dysfunction is controversial. RESEARCH DESIGN AND METHODS. Abdominally obese but otherwise healthy individuals were randomly assigned to a specifically designed 4-week diet low or high in AGEs in a double blind parallel-design. Insulin sensitivity, secretion, and clearance were assessed by a combined hyperinsulinemic-euglycemic and hyperglycemic clamp. Micro- and macrovascular function, inflammation, and lipid profile were assessed by state-of-art in vivo measurements and biomarkers. Specific urinary and plasma AGEs Nε-(carboxymethyl)lysine (CML), Nε-(1-carboxyethyl)lysine (CEL), and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1) were assessed by mass spectrometry. RESULTS. In 73 individuals (22 males, mean ± SD age and BMI 52 y ± 14, 30.6 kg/m2 ± 4.0), intake of CML, CEL, and MG-H1 differed 2.7, 5.3, and 3.7-fold between the low and high AGE diets, which led to corresponding changes of these AGEs in urine and plasma. Despite this, there was no difference in insulin sensitivity, secretion, or clearance, micro- and macrovascular function, overall inflammation, or lipid profile between the low and high dietary AGE groups (all p for treatment effects > 0.05). CONCLUSIONS. This comprehensive RCT demonstrates very limited biological consequences of a 4-week diet low or high in AGEs in abdominally obese individuals. TRIAL REGISTRATION. clinicaltrials.gov: NCT03866343, trialregister.nl: NTR7594. FUNDING. Diabetesfonds and ZonMw.
Armand M A Linkens, Alfons J. Houben, Petra M Niessen, Nicole Wijckmans, Erica de Goei, Mathias D.G. Van den Eynde, Jean L. J. M. Scheijen, Marjo Waarenburg, Andrea Mari, Tos T.J.M. Berendschot, Lukas Streese, Henner Hanssen, Martien C.J.M. van Dongen, Christel van Gool, Coen D.A. Stehouwer, Simone JPM Eussen, Casper Schalkwijk.
Capillary malformation-arteriovenous malformation (CM-AVM) is a blood vascular anomaly caused by inherited loss of function mutations in RASA1 or EPHB4 genes that encode p120 Ras GTPase-activating protein (p120 RasGAP/RASA1) and Ephrin receptor B4 (EPHB4) respectively. However, whether RASA1 and EPHB4 function in the same molecular signaling pathway to regulate the blood vasculature is uncertain. Here, we show that induced endothelial cell (EC)-specific disruption of Ephb4 in mice results in accumulation of collagen IV in the EC endoplasmic reticulum leading to EC apoptotic death and defective developmental, neonatal and pathological angiogenesis, as reported previously in induced EC-specific RASA1-deficient mice. Moreover, defects in angiogenic responses in EPHB4-deficient mice can be rescued by drugs that inhibit signaling through the Ras pathway and drugs that promote collagen IV export from the ER. However, EPHB4 mutant mice that express a form of EPHB4 that is unable to physically engage RASA1 but retains protein tyrosine kinase activity show normal angiogenic responses. These findings provide strong evidence that RASA1 and EPHB4 function in the same signaling pathway to protect against the development of CM-AVM independent of physical interaction and have important implications with regards possible means of treatment of this disease.
Di Chen, Elizabeth D. Hughes, Thomas L. Saunders, Jiangping Wu, Magda N. Hernández Vásquez, Taija Makinen, Philip D. King
Systemic hypoxia is characterized by peripheral vasodilation and pulmonary vasoconstriction. However, the system-wide mechanism for signaling hypoxia remains unknown. Accumulating evidence suggests that hemoglobin in RBCs may serve as an O2 sensor and O2-responsive NO signal transducer to regulate systemic and pulmonary vascular tone, but this remains unexamined at the integrated system level. One residue invariant in mammalian hemoglobins (Hb), β-globin Cys93 (βCys93), carries NO as vasorelaxant S-nitrosothiol (SNO) to autoregulate blood flow during oxygen delivery. βCys93Ala mutant mice thus exhibit systemic hypoxia despite transporting oxygen normally. Here we show that βCys93Ala mutant mice have reduced S-nitrosohemoglobin (SNO-Hb) at baseline and upon targeted SNO repletion, and that hypoxic vasodilation by RBCs is impaired in vitro and in vivo, recapitulating hypoxic pathophysiology. Notably, βCys93Ala mutant mice show marked impairment of hypoxic peripheral vasodilation and develop signs of pulmonary hypertension with age. Mutant mice also die prematurely with cor pulmonale (pulmonary hypertension with right ventricular dysfunction) when living under low oxygen. Altogether, we identify a major role for RBC-SNO in clinically-relevant vasodilatory responses attributed previously to endothelial NO. We conclude that SNO-Hb transduces the integrated, system-wide response to hypoxia in the mammalian respiratory cycle, expanding a core physiological principle.
Rongli Zhang, Alfred Hausladen, Zhaoxia Qian, Xudong Liao, Richard T. Premont, Jonathan S. Stamler
The anatomical routes for the clearance of cerebrospinal fluid (CSF) remain incompletely understood. However, recent evidence has given strong support for routes leading to lymphatic vessels. A current debate centers upon the routes through which CSF can access lymphatics, with evidence emerging for either direct routes to meningeal lymphatics or along cranial nerves to reach lymphatics outside the skull. Here, a method was established to infuse contrast agent into the ventricles using indwelling cannulae during imaging of mice at 2 and 12 months of age by magnetic resonance imaging. As expected, a significant decline in overall CSF turnover was found with aging. Quantifications demonstrated that the bulk of the contrast agent flowed from the ventricles to the subarachnoid space in the basal cisterns. Comparatively little contrast agent signal was found at the dorsal aspect of the skull. The imaging dynamics from the two cohorts revealed that the contrast agent cleared from the cranium through the cribriform plate to the nasopharyngeal lymphatics. On decalcified sections, we confirmed that fluorescentlylabeled ovalbumin drains through the cribriform plate and can be found within lymphatics surrounding the nasopharynx. In conclusion, routes leading to nasopharyngeal lymphatics appear to be a major efflux pathway for cranial CSF.
Yann Decker, Jonas Krämer, Li Xin, Andreas Müller, Anja Scheller, Klaus Fassbender, Steven T. Proulx
Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with limited treatment options. Despite endothelial cells (ECs) comprising 30% of the lung cellular composition, the role of EC dysfunction in pulmonary fibrosis (PF) remains unclear. We hypothesize that sterol regulatory element-binding protein 2 (SREBP2) plays a critical role in the pathogenesis of PF via EC phenotypic modifications. Transcriptome data demonstrate that SREBP2 overexpression in ECs led to the induction of the TGF, Wnt, and cytoskeleton remodeling gene ontology pathways and the increased expression of mesenchymal genes, such as snail family transcriptional repressor 1 (snai1), α-smooth muscle actin, vimentin, and neural cadherin. Furthermore, SREBP2 directly bound to the promoter regions and transactivated these mesenchymal genes. This transcriptomic change was associated with an epigenetic and phenotypic switch in ECs, leading to increased proliferation, stress fiber formation, and ECM deposition. Mice with endothelial-specific transgenic overexpression of SREBP2 (EC-SREBP2[N]-Tg mice) that were administered bleomycin to induce PF demonstrated exacerbated vascular remodeling and increased mesenchymal transition in the lung. SREBP2 was also found to be markedly increased in lung specimens from patients with IPF. These results suggest that SREBP2, induced by lung injury, can exacerbate PF in rodent models and in human patients with IPF.
Marcy Martin, Jiao Zhang, Yifei Miao, Ming He, Jian Kang, Hsi-Yuan Huang, Chih-Hung Chou, Tse-Shun Huang, Hsiao-Chin Hong, Shu-Han Su, Simon S. Wong, Rebecca L. Harper, Lingli Wang, Rakesh Bhattacharjee, Hsien-Da Huang, Zhen Bouman Chen, Atul Malhotra, Marlene Rabinovitch, James S. Hagood, John Y-J. Shyy
Inhibitors of the renin-angiotensin system (RAS) are widely used to treat hypertension. Using mice harboring fluorescent cell lineage tracers, single-cell RNA-seq, and long-term inhibition of RAS in both mice and humans, we found that deletion of renin or inhibition of the RAS leads to concentric thickening of the intrarenal arteries and arterioles. This severe disease is caused by the multiclonal expansion and transformation of renin cells from a classical endocrine phenotype to a matrix-secretory phenotype: the cells surround the vessel walls and induce the accumulation of adjacent smooth muscle cells and extracellular matrix, resulting in blood flow obstruction, focal ischemia, and fibrosis. Ablation of the renin cells via conditional deletion of β1integrin prevents arteriolar hypertrophy, indicating that renin cells are responsible for vascular disease. Given these findings, prospective morphological studies in humans are necessary to determine the extent of renal-vascular damage caused by the widespread use of inhibitors of RAS.
Hirofumi Watanabe, Alexandre G. Martini, Evan A. Brown, Xiuyin Liang, Silvia Medrano, Shin Goto, Ichiei Narita, Lois J. Arend, Maria Luisa S. Sequeira-Lopez, R. Ariel Gomez
Patients with diabetes with coronary microvascular disease (CMD) exhibit higher cardiac mortality than patients without CMD. However, the molecular mechanism by which diabetes promotes CMD is poorly understood. RNA-binding protein human antigen R (HuR) is a key regulator of mRNA stability and translation; therefore, we investigated the role of HuR in the development of CMD in mice with type 2 diabetes. Diabetic mice exhibited decreases in coronary flow velocity reserve (CFVR; a determinant of coronary microvascular function) and capillary density in the left ventricle. HuR levels in cardiac endothelial cells (CECs) were significantly lower in diabetic mice and patients with diabetes than the controls. Endothelial-specific HuR-KO mice also displayed significant reductions in CFVR and capillary density. By examining mRNA levels of 92 genes associated with endothelial function, we found that HuR, Cx40, and Nox4 levels were decreased in CECs from diabetic and HuR-KO mice compared with control mice. Cx40 expression and HuR binding to Cx40 mRNA were downregulated in CECs from diabetic mice. Cx40-KO mice exhibited decreased CFVR and capillary density, whereas endothelium-specific Cx40 overexpression increased capillary density and improved CFVR in diabetic mice. These data suggest that decreased HuR contributes to the development of CMD in diabetes through downregulation of gap junction protein Cx40 in CECs.
Rui Si, Jody Tori O. Cabrera, Atsumi Tsuji-Hosokawa, Rui Guo, Makiko Watanabe, Lei Gao, Yun Sok Lee, Jae-Su Moon, Brian T. Scott, Jian Wang, Anthony W. Ashton, Jaladanki N. Rao, Jian-Ying Wang, Jason X.-J. Yuan, Ayako Makino
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