Aims The artery contains numerous cell types which contribute to multiple vascular diseases. However, the heterogeneity and cellular responses of these vascular cells during abdominal aortic aneurysm (AAA) progression have not been well characterized. Methods and results Single-cell RNA sequencing was performed on the infrarenal abdominal aortas (IAAs) from C57BL/6J mice at Days 7 and 14 post-sham or peri-adventitial elastase-induced AAA. Unbiased clustering analysis of the transcriptional profiles from >4500 aortic cells identified 17 clusters representing nine-cell lineages, encompassing vascular smooth muscle cells (VSMCs), fibroblasts, endothelial cells, immune cells (macrophages, T cells, B cells, and dendritic cells), and two types of rare cells, including neural cells and erythrocyte cells. Seurat clustering analysis identified four smooth muscle cell (SMC) subpopulations and five monocyte/macrophage subpopulations, with distinct transcriptional profiles. During AAA progression, three major SMC subpopulations were proportionally decreased, whereas the small subpopulation was increased, accompanied with down-regulation of SMC contractile markers and up-regulation of pro-inflammatory genes. Another AAA-associated cellular response is immune cell expansion, particularly monocytes/macrophages. Elastase exposure induced significant expansion and activation of aortic resident macrophages, blood-derived monocytes and inflammatory macrophages. We also identified increased blood-derived reparative macrophages expressing anti-inflammatory cytokines suggesting that resolution of inflammation and vascular repair also persist during AAA progression. Conclusion Our data identify AAA disease-relevant transcriptional signatures of vascular cells in the IAA. Furthermore, we characterize the heterogeneity and cellular responses of VSMCs and monocytes/macrophages during AAA progression, which provide insights into their function and the regulation of AAA onset and progression.

Abstract-Insulin-like growth factor binding protein (IGFBP)-5 is a conserved protein synthesized and secreted by vascular smooth muscle cells (VSMCs). IGFBP-5 binds to extracellular IGFs and modulates IGF actions in regulating VSMC proliferation, migration, and survival. IGFBP-5 also stimulates VSMC migration through an IGF-independent mechanism, but the molecular basis underlying this ligand-independent action is unknown. In this study, we show that endogenous IGFBP-5 or transiently expressed IGFBP-5-EGFP, but not IGFBP-4-EGFP, is localized in the nuclei of VSMCs. Using a series of IGFBP-4/5 chimeras and IGFBP-5 points mutants, we demonstrated that the IGFBP-5 C-domain is necessary and sufficient for its nuclear localization, and residues K206, K208, K217, and K218 are particularly critical. Intriguingly, inhibition of protein secretion abolishes IGFBP-5 nuclear localization, suggesting the nuclear IGFBP-5 is derived from the secreted protein. When added exogenously, 125 I-or Cy3-labeled IGFBP-5 is capable of cellular entry and nuclear translocation. To identify potential transcriptional factor(s) that interact with IGFBP-5, a human aorta cDNA library was screened by a yeast two-hybrid screening strategy. Although this screen identified many extracellular and cytosolic proteins that are known to interact with IGFBP-5, no known transcription factors were found. Further motif analysis revealed that the IGFBP-5 N-domain contains a putative transactivation domain. When fused to GAL-4 DNA dinging domain and tested, the IGFBP-5 N-domain has strong transactivation activity. Mutation of the IGF binding domain or treatment of cells with IGF-I has little effect on transactivation activity. These results suggest that IGFBP-5 is localized in VSMC nucleus and possesses transcription-regulatory activity that is IGF independent. The full text of this article is available online at http://circres.ahajournals.org.

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Background: Abdominal aortic aneurysm (AAA) is a severe aortic disease with a high mortality rate in the event of rupture. Pharmacological therapy is needed to inhibit AAA expansion and prevent aneurysm rupture. Transcription factor EB (TFEB), a master regulator of autophagy and lysosome biogenesis, is critical to maintain cell homeostasis. In this study, we aim to investigate the role of vascular smooth muscle cell (VSMC) TFEB in the development of AAA and establish TFEB as a novel target to treat AAA. Methods: The expression of TFEB was measured in human and mouse aortic aneurysm samples. We used loss/gain-of-function approaches to understand the role of TFEB in VSMC survival and explored the underlying mechanisms through transcriptome and functional studies. Using VSMC-selective Tfeb knockout mice and different mouse AAA models, we determined the role of VSMC TFEB and a TFEB activator in AAA in vivo. Results: We found that TFEB is downregulated in both human and mouse aortic aneurysm lesions. TFEB potently inhibits apoptosis in VSMCs, and transcriptome analysis revealed that TFEB regulates apoptotic signaling pathways, especially apoptosis inhibitor B-cell lymphoma 2. B-cell lymphoma 2 is significantly upregulated by TFEB and is required for TFEB to inhibit VSMC apoptosis. We consistently observed that TFEB deficiency increases VSMC apoptosis and promotes AAA formation in different mouse AAA models. Furthermore, we demonstrated that 2-hydroxypropyl-β-cyclodextrin, a clinical agent used to enhance the solubility of drugs, activates TFEB and inhibits AAA formation and progression in mice. Last, we found that 2-hydroxypropyl-β-cyclodextrin inhibits AAA in a VSMC TFEB–dependent manner in mouse models. Conclusions: Our study demonstrated that TFEB protects against VSMC apoptosis and AAA. TFEB activation by 2-hydroxypropyl-β-cyclodextrin may be a promising therapeutic strategy for the prevention and treatment of AAA.

Insulin-like growth factor-binding protein (IGFBP)-5 is a secreted protein that binds to IGFs and modulates IGF actions. IGFBP-5 is also found in the nuclei of cultured cells and has transactivation activity.Here we report the nuclear localization of endogenous IGFBP-5 in mouse embryonic skeletal cells. Chromatin immunoprecipitation experiments indicated that IGFBP-5 interacts with the nuclear histone-DNA complex. Using a series of deletion mutants, the transactivation domain of IGFBP-5 was mapped to its N-terminal region. Intriguingly, the transactivation activity of IGFBP-5 is masked by negative regulatory elements located in the L-and C-domains. Among the other IGFBPs, the N-domains of IGFBP-2 and -3 also had strong transactivation activity, whereas those of IGFBP-1 and -6 had no activity. The IGFBP-4 N-domain had modest activity. Sequence analysis revealed several amino acids in the IGFBP-5 N-domain that are not present in IGFBP-1. The activities of mutants in which these residues were changed to the corresponding IGFBP-1 sequence were determined. Mutations that changed acidic residues to neutral residues (e.g. E8A, D11S, E12A, E30S/P31A, E43L, and E52A) or a polar to a basic residue (e.g. Q56R) significantly reduced transactivation activity. The E8A/D11S/E12A triple mutant and E52A/Q56R double mutants showed further reduced activity. The combinatory mutants had essentially no transactivation activity. Taken together, our results indicate that there are several conserved residues in the IGFBP-5 N-terminal region that are critical for transactivation and that IGFBP-2 and -3 also have strong transactivation activity in their N-domains.The insulin-like growth factor (IGF) 2 system, consisting of two ligands (IGF-I and -II), two receptors (the IGF-I receptor and IGF-II receptor), and six high affinity IGF-binding proteins (IGFBPs), converges on a conserved signaling pathway that plays fundamental roles in vertebrate development and physiology and is implicated in several human diseases (1). The bioavailability and bioactivity of IGFs are regulated by their interactions with various members of the IGFBP family. IGFBPs all have a highly cysteine-rich N-terminal (N)-domain, a cysteine-rich C-terminal (C)-domain, and a middle linker (L)-domain with no cysteine residues except in IGFBP-4. The N-and C-domains are highly conserved within the IGFBP family, whereas the L-domain varies both within the family and across species.Besides binding to IGF and modulating its actions, IGFBP-5 has been reported to regulate cell proliferation (2, 3), migration (4 -6), and apoptosis/survival (7-11) independent of IGF. Although the ligand-dependent actions of IGFBP-5 are attributed to its interactions with the IGF ligand and other proteins (1), the mechanistic basis of the ligand-independent actions of IGFBP-5 is not yet understood.Andress et al. (12) used IGFBP-5 affinity chromatography to purify a 420-kDa membrane protein from human osteoblast cells and proposed that it was an IGFBP-5 receptor. The same study reported that IGFBP-5 ...

Insulin-like growth factor binding protein (IGFBP)-5 is a secreted protein that binds to IGF and modulates IGF actions. IGFBP-5 is also found in the nucleus of mammalian cells and has transactivation activity. The structural basis of this transactivation activity and its role in mediating IGF-independent actions are not clear. Here we report that there are 2 igfbp-5 genes in zebrafish and other teleost fish. In zebrafish, igfbp-5a and -5b are expressed in spatially restricted, mostly nonoverlapping domains during early development. The IGF binding site is conserved in both zebrafish IGFBP-5s, and they are both secreted and capable of IGF binding. Both proteins contain a consensus bipartite nuclear localization signal and were found in the nucleus when introduced into cultured cells. Although zebrafish IGFBP-5b possesses transactivation activity, zebrafish IGFBP-5a lacks this activity. Mutational analysis demonstrated that 2 unique amino acids in positions 22 and 56 of IGFBP-5a are responsible for its lack of transactivation activity. These findings suggest that the duplicated zebrafish IGFBP-5s have evolved divergent regulatory mechanisms and distinct biological properties by partitioning of ancestral structural domains and provide new evidence for a conserved role of the IGF binding, nuclear localization, and transactivation domain of this multifunctional IGFBP.

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Rationale:We reviewed 76 published cases of Doege–Potter syndrome, and non-islet cell tumor hypoglycemia (NICTH) secondary to a solitary fibrous tumor (SFT) between 1989 and 2016, to study disease pathogenesis, diagnosis, and treatment of this rare paraneoplastic disease. Further, we report 1 new case of a patient presenting with Doege–Potter syndrome.Patients concerns:The tumors originated from the pleural cavity, lung, pelvis, liver, retroperitoneum, kidney, mediastinal, the sella, uterus, bladder, intestine, mandibular, and the thigh. The most common location was the pleural cavity (left 12 cases and right 28 cases). Moreover, 28/71 (39.4%) were benign and 43/71 (60.6%) were malignant. SFTs with NICTH were more likely to be malignant and present at a higher rate than previously published (5%–10.4%). The malignancy rate of extrathoracic SFTs was higher than that of thoracic SFTs, 20 (66.7%) as compared with 23 (56.1%). Age of onset varied from 24 to 85 years (mean 59 years), with 47 males and 28 females, and gender unavailable for 1 case. When comparing clinical characteristics of patients with benign as compared malignant tumors, no significant differences in the age of onset, gender, or size of tumor were seen. Among 15/19 cases, the insulin-like growth factor II (IGF-II)/IGF-I ration was >10.0. Complete tumor resection remained the only definitive treatment.Outcomes and lessens:Glucocorticoids dose-dependently reduce the frequency and severity of hypoglycemic episodes. Low doses of prednisone were ineffective at relieving hypoglycemia. The effect of neoadjuvant treatment, consisting of chemoradiation, and consecutive selective embolization of vessels feeding the tumor were not identified.