Vascular biology
Abstract
Pulmonary arterial hypertension (PAH) is characterized by progressive increase of pulmonary vascular resistance and remodeling that result in right heart failure. Recessive mutations of EIF2AK4 gene (encoding GCN2, General control nonderepressibe 2 kinase) are linked to heritable pulmonary veno-occlusive disease (PVOD) in patients but rarely (approximately one percent) in PAH patients. The role of GCN2 kinase activation in the pathogenesis of PAH remains unclear. Here we show that GCN2 was hyperphosphorylated and activated in pulmonary vascular endothelial cells (ECs) of hypoxic mice, monocrotaline-treated rats, and PAH patients. Unexpectedly, loss of GCN2 kinase activity in Eif2ak4–/– mice with genetic disruption of the kinase domain induced neither PVOD nor PH but inhibited hypoxia-induced PH. RNA sequencing analysis suggested Endothelin-1 (Edn1) as a downstream target of GCN2. GCN2 mediated hypoxia-induced Edn1 expression in human lung ECs via HIF-2α. Restored Edn1 expression in ECs of Eif2ak4–/– mice partially reversed the reduced phenotype of hypoxia-induced PH. Furthermore, GCN2 kinase inhibitor A-92 treatment attenuated PAH in monocrotaline-treated rats. These studies demonstrate that GCN2 kinase activation mediates pulmonary vascular remodeling and PAH at least partially through Edn1. Thus, targeting GCN2 kinase activation is a promising therapeutic strategy for treatment of PAH in patients without EIF2AK4 loss of function mutations.
Authors
Maggie M. Zhu, Jingbo Dai, Zhiyu Dai, Yi Peng, You-Yang Zhao
Abstract
Pulmonary veno-occlusive disease (PVOD) is a rare but severe form of pulmonary hypertension characterized by the obstruction of pulmonary arteries and veins, causing increased pulmonary artery pressure and leading to right ventricular (RV) heart failure. PVOD is often resistant to conventional pulmonary arterial hypertension (PAH) treatments and has a poor prognosis, with a median survival time of 2 to 3 years after diagnosis. We previously showed that the administration of a chemotherapy agent mitomycin C (MMC) in rats mediates PVOD through the activation of the eukaryotic initiation factor 2 (eIF2) kinase protein kinase R (PKR) and the integrated stress response (ISR), resulting in the impairment of vascular endothelial junctional structure and barrier function. Here, we demonstrate that aged rats over one year exhibit more severe vascular remodeling and RV hypertrophy than young adult rats following MMC treatment. This is attributed to an age-associated elevation of basal ISR activity and depletion of protein phosphatase 1, leading to prolonged eIF2 phosphorylation and sustained ISR activation. Pharmacological blockade of PKR or ISR mitigates PVOD phenotypes in both age groups, suggesting that targeting the PKR-ISR axis could be a potential therapeutic strategy for PVOD.
Authors
Amit Prabhakar, Meetu Wadhwa, Rahul Kumar, Prajakta Ghatpande, Aneta Gandjeva, Rubin M. Tuder, Brian B. Graham, Giorgio Lagna, Akiko Hata
Abstract
Emerging studies suggest that various parental exposures affect offspring cardiovascular health, yet the specific mechanisms, particularly the influence of paternal cardiovascular disease (CVD) risk factors on offspring cardiovascular health, remain elusive. The present study explores how paternal hypercholesterolemia affects offspring atherosclerosis development using the LDL receptor-deficient (LDLR–/–) mouse model. We found that paternal high-cholesterol diet feeding led to significantly increased atherosclerosis in F1 female, but not male, LDLR–/– offspring. Transcriptomic analysis highlighted that paternal hypercholesterolemia stimulated proatherogenic genes, including Ccn1 and Ccn2, in the intima of female offspring. Sperm small noncoding RNAs (sncRNAs), particularly transfer RNA–derived (tRNA-derived) small RNAs (tsRNAs) and rRNA-derived small RNAs (rsRNAs), contribute to the intergenerational transmission of paternally acquired metabolic phenotypes. Using a newly developed PANDORA-Seq method, we identified that high-cholesterol feeding elicited changes in sperm tsRNA/rsRNA profiles that were undetectable by traditional RNA-Seq, and these altered sperm sncRNAs were potentially key factors mediating paternal hypercholesterolemia-elicited atherogenesis in offspring. Interestingly, high-cholesterol feeding altered sncRNA biogenesis–related gene expression in the epididymis but not testis of LDLR–/– sires; this may have led to the modified sperm sncRNA landscape. Our results underscore the sex-specific intergenerational effect of paternal hypercholesterolemia on offspring cardiovascular health and contribute to the understanding of chronic disease etiology originating from parental exposures.
Authors
Rebecca Hernandez, Xiuchun Li, Junchao Shi, Tejasvi R. Dave, Tong Zhou, Qi Chen, Changcheng Zhou
Abstract
Lung endothelium plays a pivotal role in the orchestration of inflammatory responses to acute pulmonary insults. Mammalian sterile 20-like kinase 1 (Mst1) is a serine/threonine kinase that has been shown to play an important role in the regulation of apoptosis, stress responses, and organ growth. This study investigated the role of Mst1 in lung endothelial activation and acute lung injury (ALI). We found that Mst1 was significantly activated in inflamed lung endothelial cells (ECs) and mouse lung tissues. Overexpression of Mst1 promoted nuclear factor κ-B (NF-κB) activation through promoting JNK and p38 activation in lung ECs. Inhibition of Mst1 by either its dominant negative form (DN-Mst1) or its pharmacological inhibitor markedly attenuated cytokine-induced expression of cytokines, chemokines, and adhesion molecules in lung ECs. Importantly, in a mouse model of lipopolysaccharide-induced (LPS-induced) ALI, both deletion of Mst1 in lung endothelium and treatment of WT mice with a pharmacological Mst1 inhibitor significantly protected mice from LPS-induced ALI. Together, our findings identified Mst1 kinase as a key regulator in controlling lung EC activation and suggest that therapeutic strategies aimed at inhibiting Mst1 activation might be effective in the prevention and treatment of inflammatory lung diseases.
Authors
Zhi-Fu Guo, Nopprarat Tongmuang, Chao Li, Chen Zhang, Louis Hu, Daniel Capreri, Mei-Xing Zuo, Ross Summer, Jianxin Sun
Abstract
Abdominal aortic aneurysm (AAA) is one of the most life-threatening cardiovascular diseases; however, effective drug treatments are still lacking. The formation of neutrophil extracellular traps (NETs) has been shown to be a crucial trigger of AAA, and identifying upstream regulatory targets is thus key to discovering therapeutic agents for AAA. We revealed that phosphoinositide-3-kinase γ (PI3Kγ) acted as an upstream regulatory molecule and that PI3Kγ inhibition reduced NET formation and aortic wall inflammation, thereby markedly ameliorating AAA. However, the mechanism of NET formation regulated by PI3Kγ remains unclear. In this study, we showed that PI3Kγ deficiency inactivated the noncanonical pyroptosis pathway, which suppressed downstream NET formation. In addition, PI3Kγ regulation of noncanonical pyroptosis was dependent on cyclic AMP/protein kinase A signaling. These results clarify the molecular mechanism and crosstalk between PI3Kγ and NETosis in the development of AAA, potentially facilitating the discovery of therapeutic options for AAA.
Authors
Yacheng Xiong, Shuai Liu, Yu Liu, Jiani Zhao, Jinjian Sun, Yongqing Li, Baihong Pan, Wei Wang
Abstract
To determine whether hyperlipidemia and chronic kidney disease (CKD) have a synergy in accelerating vascular inflammation via trained immunity (TI), we performed aortic pathological analysis and RNA-Seq of high-fat diet–fed (HFD-fed) 5/6 nephrectomy CKD (HFD+CKD) mice. We made the following findings: (a) HFD+CKD increased aortic cytosolic LPS levels, caspase-11 (CASP11) activation, and 998 gene expressions of TI pathways in the aorta (first-tier TI mechanism); (b) CASP11–/– decreased aortic neointima hyperplasia, aortic recruitment of macrophages, and casp11–gasdermin D–mediated cytokine secretion; (c) CASP11–/– decreased N-terminal gasdermin D (N-GSDMD) membrane expression on aortic endothelial cells and aortic IL-1B levels; (d) LPS transfection into human aortic endothelial cells resulted in CASP4 (human)/CASP11 (mouse) activation and increased N-GSDMD membrane expression; and (e) IL-1B served as the second-tier mechanism underlying HFD+CKD-promoted TI. Taken together, hyperlipidemia and CKD accelerated vascular inflammation by promoting 2-tier trained immunity.
Authors
Yu Sun, Yifan Lu, Lu Liu, Fatma Saaoud, Ying Shao, Keman Xu, Charles Drummer IV, Ramon Cueto, Huimin Shan, Xiaohua Jiang, Huaqing Zhao, Hong Wang, Xiaofeng Yang
Abstract
Some endothelial cells in the tumor vasculature expressed a system L amino acid transporter LAT1. To elucidate the role of LAT1 in tumor related endothelial cells, tumor cells were injected into endothelial specific LAT1 conditional knockout mice (Slc7a5flox/flox; Cdh5-Cre-ERT2) and found that the shape of the tumor vasculature was normalized and that the size and numbers of lung metastasis was reduced. TNFα-induced expression of VCAM1 and E-selectin at the surface of HUVEC, both of which are responsible for enhanced monocyte attachment and pre-metastatic niche formation, was reduced in the presence of LAT1 inhibitor, nanvuranlat. Deprivation of tryptophan, an LAT1 substrate, mimicked LAT1 inhibition, which led to activation of MEK1/2-ERK1/2 pathway and subsequent cystathionine γ lyase (CTH) induction. Increased production of hydrogen sulfide (H2S) by CTH was at least partially responsible for tumor vascular normalization, leading to decreased leakiness and enhanced delivery of chemotherapeutic agents to the tumor.
Authors
Jun-ichi Suehiro, Toru Kimura, Toshiyuki Fukutomi, Hisamichi Naito, Yasuharu Kanki, Youichiro Wada, Yoshiaki Kubota, Nobuyuki Takakura, Hiroyuki Sakurai
Abstract
Cantú syndrome is a multisystem disorder caused by gain-of-function (GOF) mutations in KCNJ8 and ABCC9, the genes encoding the pore-forming inward rectifier Kir6.1 and regulatory sulfonylurea receptor SUR2B subunits, respectively, of vascular ATP-sensitive K+ channels (KATP). In this study, we investigated changes in the vascular endothelium in mice in which Cantú syndrome -associated Kcnj8 or Abcc9 mutations were knocked-in to the endogenous loci. We found that endothelium-dependent dilation was impaired in small mesenteric arteries from Cantú mice. Loss of endothelium-dependent vasodilation led to increased vasoconstriction in response to intraluminal pressure or treatment with the adrenergic receptor agonist phenylephrine. We also found that either KATP GOF or acute activation of KATP channels with pinacidil increased the amplitude and frequency of wave-like Ca2+ events generated in the endothelium in response to the vasodilator agonist carbachol. Increased cytosolic Ca2+ signaling activity in arterial endothelial cells from Cantú mice was associated with elevated mitochondrial [Ca2+] and enhanced reactive oxygen species (ROS) and peroxynitrite levels. Scavenging intracellular or mitochondrial ROS restored endothelium-dependent vasodilation in the arteries of mice with KATP GOF mutations. We conclude that mitochondrial Ca2+ overload and ROS generation, which subsequently leads to nitric oxide consumption and peroxynitrite formation, cause endothelial dysfunction in mice with Cantú syndrome.
Authors
Elsayed Metwally, Alfredo Sanchez Solano, Boris Lavanderos, Evan Yamasaki, Pratish Thakore, Conor McClenaghan, Natalia Rios, Rafael Radi, Yumei Feng Earley, Colin G. Nichols, Scott Earley
Abstract
The expression of the gap junction molecule connexin-45 (Cx45; GJC1) in lymphatic endothelium and its functional relevance were not previously known. We found that Cx45 was expressed widely in the endothelium of murine lymphatics, in both valve and non-valve regions. Cell-specific deletion of Cx45, driven by a constitutive Cre line (Lyve1-Cre) or an inducible Cre line (Prox1-CreERT2), compromised the function of lymphatic valves, as assessed by physiological tests (back leak and closure) of isolated, single-valve vessel segments. The defects were comparable to those previously reported for loss of Cx43 and, like Cx43, deletion of Cx45 resulted in shortening and/or increased asymmetry of lymphatic valve leaflets, providing an explanation for the compromised valve function. In contrast to Cx43, LEC-specific deletion of Cx45 did not alter the number of valves in mesenteric or dermal lymphatic networks, or the expression patterns of the canonical valve-associated proteins PROX1, ITGA9 or CLAUDIN5. Constitutive deletion of Cx45 from LECs resulted in increased backflow of injected tracer in popliteal networks in vivo and compromised the integrity of the LEC permeability barrier in a subset of collecting vessels. These findings provide evidence for an unexpected role of Cx45 in the development and maintenance of lymphatic valves.
Authors
Michael J. Davis, Jorge A. Castorena-Gonzalez, Min Li, Scott D. Zawieja, Alexander M. Simon, Xin Geng, R. Sathish Srinivasan
Abstract
In rheumatoid arthritis, inflammatory mediators extravasate from blood into joints via gaps between endothelial cells (ECs), but the contribution of ECs is not known. Sphingosine 1-phosphate receptor 1 (S1PR1), widely expressed on ECs, maintains the vascular barrier. Here, we assessed the contribution of vascular integrity and EC S1PR1 signaling to joint damage in mice exposed to serum-induced arthritis (SIA). EC-specific deletion of S1PR1 or pharmacological blockade of S1PR1 promoted vascular leak and amplified SIA, whereas overexpression of EC S1PR1 or treatment with an S1PR1 agonist delayed SIA. Blockade of EC S1PR1 induced membrane metalloproteinase-dependent cleavage of vascular endothelial cadherin (VE-cadherin), a principal adhesion molecule that maintains EC junctional integrity. We identified a disintegrin and a metalloproteinase domain 10 (ADAM10) as the principal VE-cadherin “sheddase.” Mice expressing a stabilized VE-cadherin construct had decreased extravascular VE-cadherin and vascular leakage in response to S1PR1 blockade, and they were protected from SIA. Importantly, patients with active rheumatoid arthritis had decreased circulating S1P and microvascular expression of S1PR1, suggesting a dysregulated S1P/S1PR1 axis favoring vascular permeability and vulnerability. We present a model in which EC S1PR1 signaling maintains homeostatic vascular barrier function by limiting VE-cadherin shedding mediated by ADAM10 and suggest this signaling axis as a therapeutic target in inflammatory arthritis.
Authors
Nathalie Burg, Ryan Malpass, Linda Alex, Miles Tran, Eric Englebrecht, Andrew Kuo, Tania Pannelini, Margaret Minett, Kalana Athukorala, Tilla Worgall, Heather J. Faust, Susan Goodman, Bella Mehta, Michael Brenner, Dietmar Vestweber, Kevin Wei, Carl Blobel, Timothy Hla, Jane E. Salmon
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