Vascular biology
Abstract
Red blood cells (RBCs) induce endothelial dysfunction in type 2 diabetes (T2D), but the mechanism by which RBCs communicate with the vessel is unknown. This study tested the hypothesis that extracellular vesicles (EVs) secreted by RBCs act as mediators of endothelial dysfunction in T2D. Despite a lower production of EVs derived from RBCs of T2D patients (T2D RBC-EVs), their uptake by endothelial cells was greater than that of EVs derived from RBCs of healthy individuals (H RBC-EVs). T2D RBC-EVs impaired endothelium-dependent relaxation and this effect was attenuated following inhibition of arginase in EVs. Inhibition of vascular arginase or oxidative stress also attenuated endothelial dysfunction induced by T2D RBC-EVs. Arginase-1 was detected in RBC-derived EVs, and arginase-1 and oxidative stress were increased in endothelial cells following co-incubation with T2D RBC-EVs. T2D RBC-EVs also increased arginase-1 protein in endothelial cells following mRNA silencing and in the endothelium of aortas from endothelial cell arginase 1 knockout mice. It is concluded that T2D-RBCs induce endothelial dysfunction through increased uptake of EVs that transfer arginase-1 from RBCs to the endothelium to induce oxidative stress and endothelial dysfunction. These results shed important light on the mechanism underlying endothelial injury mediated by RBCs in T2D.
Authors
Aida Collado, Rawan Humoud, Eftychia Kontidou, Maria Eldh, Jasmin Swaich, Allan Zhao, Jiangning Yang, Tong Jiao, Elena Domingo, Emelie Carlestål, Ali Mahdi, John Tengbom, Ákos Végvári, Qiaolin Deng, Michael Alvarsson, Susanne Gabrielsson, Per Eriksson, Zhichao Zhou, John Pernow
Abstract
Aortic aneurysms are potentially fatal focal enlargements of the aortic lumen; the disease burden disease is increasing as the human population ages. Pathological oxidative stress is implicated in development of aortic aneurysms. We pursued a chemogenetic approach to create an animal model of aortic aneurysm formation using a transgenic mouse line DAAO-TGTie2 that expresses yeast D-amino acid oxidase (DAAO) under control of the endothelial Tie2 promoter. In DAAO-TGTie2 mice, DAAO generates the reactive oxygen species hydrogen peroxide (H2O2) in endothelial cells only when provided with D-amino acids. When DAAO-TGTie2 mice are chronically fed D-alanine, the animals become hypertensive and develop abdominal but not thoracic aortic aneurysms. Generation of H2O2 in the endothelium leads to oxidative stress throughout the vascular wall. Proteomic analyses indicate that the oxidant-modulated protein kinase JNK1 is dephosphorylated by the phophoprotein phosphatase DUSP3 in abdominal but not thoracic aorta, causing activation of KLF4-dependent transcriptional pathways that trigger phenotypic switching and aneurysm formation. Pharmacological DUSP3 inhibition completely blocks aneurysm formation caused by chemogenetic oxidative stress. These studies establish that regional differences in oxidant-modulated signaling pathways lead to differential disease progression in discrete vascular beds, and identify DUSP3 as a potential pharmacological target for the treatment of aortic aneurysms.
Authors
Apabrita Ayan Das, Markus Waldeck-Weiermair, Shambhu Yadav, Fotios Spyropoulos, Arvind Pandey, Tanoy Dutta, Taylor A. Covington, Thomas Michel
Abstract
Osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) has been recognized as the principal mechanism underlying vascular calcification (VC). Runt-related transcription factor 2 (RUNX2) in VSMCs plays a pivotal role because it constitutes an essential osteogenic transcription factor for bone formation. As a key DNA demethylation enzyme, ten-eleven translocation 2 (TET2) is crucial in maintaining the VSMC phenotype. However, whether TET2 involves in VC progression remains elusive. Here we identified a substantial downregulation of TET2 in calcified human and mouse arteries, as well as human primary VSMCs. In vitro gain- and loss-of function experiments demonstrated TET2 regulated VC. Subsequently, in vivo knockdown of TET2 significantly exacerbated VC in both vitamin D3 and adenine-diet-induced chronic kidney disease (CKD) mice models. Mechanistically, TET2 binds to and suppresses the activity of the P2 promoter within the RUNX2 gene, whereas an enzymatic loss-of-function mutation of TET2 has a comparable effect. Furthermore, TET2 forms a complex with histone deacetylases 1/2 (HDAC1/2 ) to deacetylate H3K27ac on the P2 promoter, thereby inhibiting its transcription. Moreover, SNIP1 is indispensable for TET2 to interact with HDAC1/2 to exert inhibitory effect on VC, and knockdown of SNIP1 accelerated VC in mice. Collectively, our findings imply that TET2 might serve as a potential therapeutic target for VC.
Authors
Dayu He, Jianshuai Ma, Ziting Zhou, Yanli Qi, Yaxin Lian, Feng Wang, Huiyong Yin, Huanji Zhang, Tingting Zhang, Hui Huang
Abstract
Elevated Angiopoietin-2 is associated with diverse inflammatory conditions including sepsis, a leading global cause of mortality. During inflammation, Angiopoietin-2 antagonizes the endothelium-enriched receptor Tie2 to destabilize the vasculature. In other contexts, Angiopoietin-2 stimulates Tie2. The basis for context-dependent antagonism remains incompletely understood. Here we show that inflammation-induced proteolytic cleavage of Angiopoietin-2 converts this ligand from Tie2 agonist to antagonist. Conditioned media from stimulated macrophages induced endothelial Angiopoietin-2 secretion. Unexpectedly, this was associated with reduction of the 75 kDa full-length protein and appearance of new 25 and 50 kDa C-terminal fragments. Peptide sequencing proposed cathepsin K as a candidate protease. Cathepsin K was necessary and sufficient to cleave Angiopoietin-2. Recombinant 25 and 50 kDa Angiopoietin-2 fragments (cANGPT225, cANGPT250) bound and antagonized Tie2. Cathepsin K inhibition with the Phase-3 small molecule inhibitor odanacatib improved survival in distinct murine sepsis models. Full-length Angiopoietin-2 enhanced survival in endotoxemic mice administered odanacatib and, conversely, increased mortality in the drug’s absence. Odanacatib’s benefit was reversed by heterologous cANGPT225. Septic humans accumulated circulating Angiopoietin-2 fragments, which were associated with adverse outcomes. These results identify cathepsin K as a candidate marker of sepsis and a proteolytic mechanism for the conversion of Angiopoietin-2 from Tie2 agonist to antagonist with therapeutic implications for inflammatory conditions associated with Angiopoietin-2 induction.
Authors
Takashi Suzuki, Erik Loyde, Sara Chen, Valerie Etzrodt, Temitayo O. Idowu, Amanda J. Clark, Marie Christelle Saade, Brenda Mendoza Flores, Shulin Lu, Gabriel Birrane, Vamsidhara Vemireddy, Benjamin Seeliger, Sascha David, Samir M. Parikh
An endothelial SOX18-mevalonate pathway axis enables repurposing of statins for infantile hemangioma
Abstract
Infantile hemangioma (IH) is the most common tumor in children and a paradigm for pathological vasculogenesis, angiogenesis, and regression. Propranolol, the mainstay treatment, inhibits IH vessel formation via a β-adrenergic receptor independent off-target effect of its R(+) enantiomer on the endothelial SRY box transcription factor 18 (SOX18). Transcriptomic profiling of patient-derived hemangioma stem cells (HemSC) uncovered the mevalonate pathway (MVP) as a target of R(+) propranolol. Loss and gain of function of SOX18 confirmed it is both necessary and sufficient for R(+) propranolol suppression of the MVP, including regulation of sterol regulatory element binding protein 2 (SREBP2) and the rate-limiting enzyme HMG-CoA reductase (HMGCR). AThe biological relevance of the endothelial SOX18-MVP axis in IH patient tissue was demonstrated by nuclear co-localization of SOX18 and SREBP2. Functional validation in a preclinical IH xenograft model revealed that statins – competitive inhibitors of HMGCR – efficiently suppress IH vessel formation. We propose an novel endothelial SOX18-MVP-axis as a central regulator of IH pathogenesis and suggest statin repurposing to treat IH. The pleiotropic effects of R(+) propranolol and statins along the SOX18-MVP axis to disable an endothelial-specific program may have therapeutic implications for other vascular disease entities involving pathological vasculogenesis and angiogenesis.
Authors
Annegret Holm, Matthew S. Graus, Jill Wylie-Sears, Jerry Wei Heng Tan, Maya Alvarez-Harmon, Luke Borgelt, Sana Nasim, Long Chung, Ashish Jain, Mingwei Sun, Liang Sun, Pascal Brouillard, Ramrada Lekwuttikarn, Yanfei Qi, Joyce Teng, Miikka Vikkula, Harry Kozakewich, John B. Mulliken, Mathias Francois, Joyce Bischoff
Abstract
Polymorphisms in Nos3 increases risk for glaucoma, the leading cause of irreversible blindness worldwide. A key modifiable risk factor for glaucoma is intraocular pressure (IOP), which is regulated by nitric oxide (NO), a product of nitric oxide synthase-3 (Nos3) in Schlemm’s canal of the conventional outflow pathway. We studied the effects of a conditional, endothelial-specific postnatal deletion of Nos3 (Endo-SclCre-ERT;Nos3flox/flox) on tissues of the outflow pathway. We observed that Cre-ERT expression spontaneously and gradually increased with time in vascular endothelia including Schlemm’s canal, beginning at P10, with complete Nos3 deletion occurring around P90. Unlike the reduced outflow resistance in global Nos3 knockout mice, outflow resistance and IOP in Endo-SclCre-ERT;Nos3flox/flox mice were normal. Coinciding with Nos3 deletion, we observed recruitment of macrophages to, and induction of both ELAM-1 and NOS2 expression by endothelia in the distal portion of the outflow pathway, which increased vessel diameter. These adjustments reduced outflow resistance to maintain IOP in these Endo-SclCre-ERT;Nos3flox/flox mice. Selective inhibition of iNOS by 1400W resulted in narrowing of distal vessels and IOP elevation. Together, results emphasize the pliability of the outflow system, the importance of NO signaling in IOP control and implicates an important role for macrophages in IOP homeostasis.
Authors
Ruth A. Kelly, Megan S. Kuhn, Ester Reina-Torres, Revathi Balasubramanian, Kristin M. Perkumas, Guorong Li, Takamune Takahashi, Simon W.M. John, Michael H. Elliott, Darryl R. Overby, W. Daniel Stamer
Abstract
The osteogenic environment promotes vascular calcium phosphate deposition and aggregation of unfolded and misfolded proteins, resulting in endoplasmic reticulum (ER) stress in chronic renal disease (CKD). Controlling ER stress through genetic intervention is a promising approach for treating vascular calcification. In this study, we demonstrated a positive correlation between ER stress-induced tribble 3 (TRIB3) expression and progression of vascular calcification in human and rodent CKD. Increased TRIB3 expression promoted vascular smooth muscle cell (VSMC) calcification by interacting with the C2 domain of the E3 ubiquitin-protein ligase Smurf1, facilitating its K48-related self-ubiquitination at Lys381 and Lys383 and subsequent dissociation from the plasma membrane and nuclei. This degeneration of Smurf1 accelerated the stabilization of the osteogenic transcription factors RUNX Family Transcription Factor 2 (Runx2) and SMAD Family Member 1 (Smad1). C/EBP homologous protein and activating transcription factor 4 are upstream transcription factors of TRIB3 in an osteogenic environment. Genetic knockout of TRIB3 or rescue of Smurf1 ameliorated VSMC and vascular calcification by stabilizing Smurf1 and enhancing the degradation of Runx2 and Smad1. Our findings shed light on the vital role of TRIB3 as a scaffold in ER stress and vascular calcification and offer a potential therapeutic option for chronic renal disease.
Authors
Yihui Li, Chang Ma, Yanan Sheng, Shanying Huang, Huaibing Sun, Yun Ti, Zhihao Wang, Feng Wang, Fangfang Chen, Chen Li, Haipeng Guo, Mengxiong Tang, Fangqiang Song, Hao Wang, Ming Zhong
Abstract
Translational control shapes the proteome and is particularly important in regulating gene expression under stress. A key source of endothelial stress is treatment with tyrosine kinase inhibitors (TKIs), which lowers cancer mortality but increases cardiovascular mortality. Using a human induced pluripotent stem cell–derived endothelial cell (hiPSC-EC) model of sunitinib-induced vascular dysfunction combined with ribosome profiling, we assessed the role of translational control in hiPSC-ECs in response to stress. We identified staphylococcal nuclease and tudor domain–containing protein 1 (SND1) as a sunitinib-dependent translationally repressed gene. SND1 translational repression was mediated by the mTORC1/4E-BP1 pathway. SND1 inhibition led to endothelial dysfunction, whereas SND1 OE protected against sunitinib-induced endothelial dysfunction. Mechanistically, SND1 transcriptionally regulated UBE2N, an E2-conjugating enzyme that mediates K63-linked ubiquitination. UBE2N along with the E3 ligases RNF8 and RNF168 regulated the DNA damage repair response pathway to mitigate the deleterious effects of sunitinib. In silico analysis of FDA-approved drugs led to the identification of an ACE inhibitor, ramipril, that protected against sunitinib-induced vascular dysfunction in vitro and in vivo, all while preserving the efficacy of cancer therapy. Our study established a central role for translational control of SND1 in sunitinib-induced endothelial dysfunction that could potentially be therapeutically targeted to reduce sunitinib-induced vascular toxicity.
Authors
Zhenbo Han, Gege Yan, Jordan Jousma, Sarath Babu Nukala, Mehdi Amiri, Stephen Kiniry, Negar Tabatabaei, Youjeong Kwon, Sen Zhang, Jalees Rehman, Sandra Pinho, Sang-Bing Ong, Pavel V. Baranov, Soroush Tahmasebi, Sang-Ging Ong
Abstract
Hypoxia is a major cause of pulmonary hypertension (PH) worldwide, and it is likely that interstitial pulmonary macrophages contribute to this vascular pathology. We observed in hypoxia-exposed mice an increase in resident interstitial macrophages, which expanded through proliferation and expressed the monocyte recruitment ligand CCL2. We also observed an increase in CCR2+ macrophages through recruitment, which express the protein thrombospondin-1 that functionally activates TGF-beta to cause vascular disease. Blockade of monocyte recruitment with either CCL2 neutralizing antibody treatment or CCR2 deficiency in the bone marrow compartment suppressed hypoxic PH. These data were supported by analysis of plasma samples from humans who travelled from low (225m) to high (3500m) elevation, revealing an increase in thrombospondin-1 and TGF-beta expression following ascent, which was blocked by dexamethasone prophylaxis. In the hypoxic mouse model, dexamethasone prophylaxis recapitulated these findings by mechanistically suppressing CCL2 expression and CCR2+ monocyte recruitment. These data suggest a pathologic cross-talk between two discrete interstitial macrophage populations, which can be therapeutically targeted.
Authors
Rahul Kumar, Kevin Nolan, Biruk Kassa, Neha Chanana, Tsering Palmo, Kavita Sharma, Kanika Singh, Claudia Mickael, Dara Fonseca Balladares, Julia Nilsson, Amit Prabhakar, Aastha Mishra, Michael H. Lee, Linda Sanders, Sushil Kumar, Ari B. Molofsky, Kurt R. Stenmark, Dean Sheppard, Rubin M. Tuder, Mohit D. Gupta, Tashi Thinlas, Qadar Pasha, Brian B. Graham
Abstract
The pathogenesis of thoracic aortic aneurysm (TAA) in Marfan syndrome (MFS) is generally attributed to vascular smooth muscle cell (VSMC) pathologies. However, the role of immune cell–mediated inflammation remains elusive. Single-cell RNA sequencing identified a subset of CX3CR1+ macrophages mainly located in the intima in the aortic roots and ascending aortas of Fbn1C1041G/+ mice, further validated in MFS patients. Specific elimination of CX3CR1+ cells by diphtheria toxin in Cx3cr1-CreERT2iDTRF/+Fbn1C1041G/+ mice efficiently ameliorated TAA progression. Administering the monoclonal antibodies to respectively neutralize TNF-α and IGF1 produced by CX3CR1+ cells from MFS patients greatly suppressed the cocultured MFS patient–specific induced pluripotent stem cell–derived VSMC inflammation. BM transplantation and parabiosis revealed that CX3CR1+ macrophages are mainly originated from BM-derived monocytes. Targeting TNF-α and IGF1 in CX3CR1+ macrophages via shRNA lentivirus transduction in BM cells efficiently suppressed TAA development in BM-transplanted Fbn1C1041G/+ mice. Application of the CCR2 antagonist RS504393 to inhibit monocyte infiltration markedly reduced the accumulation of CX3CR1+ macrophages and subsequently alleviated TAA progression in Fbn1C1041G/+ mice. In summary, CX3CR1+ macrophages mainly located in aortic intima mediate TAA formation by paracrinally causing VSMC inflammation, and targeting them offers a potential antiinflammatory therapeutic strategy for MFS-related TAA.
Authors
Jiaqi Huang, Hao Liu, Zhujiang Liu, Zhenting Wang, Hanshi Xu, Zhuofan Li, Shan Huang, Xueyuan Yang, Yicong Shen, Fang Yu, Yulin Li, Junming Zhu, Wei Li, Li Wang, Wei Kong, Yi Fu
Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)