Critical Role of Cytosolic DNA and Its Sensing Adaptor STING in Aortic Degeneration, Dissection, and Rupture.

BACKGROUND: Sporadic aortic aneurysm and dissection (AAD), caused by progressive aortic smooth muscle cell (SMC) loss and extracellular matrix degradation, is a highly lethal condition. Identifying mechanisms that drive aortic degeneration is a crucial step in developing an effective pharmacologic treatment to prevent disease progression. Recent evidence has indicated that cytosolic DNA and abnormal activation of the cytosolic DNA sensing adaptor STING (stimulator of interferon genes) play a critical role in vascular inflammation and destruction. Here, we examined the involvement of this mechanism in aortic degeneration and sporadic AAD formation. METHODS: The presence of cytosolic DNA in aortic cells and activation of the STING pathway were examined in aortic tissues from patients with sporadic ascending thoracic AAD. The role of STING in AAD development was evaluated in Sting-deficient (Stinggt/gt) mice in a sporadic AAD model induced by challenging mice with a combination of a high-fat diet and angiotensin II. We also examined the direct effects of STING on SMC death and macrophage activation in vitro. RESULTS: In human sporadic AAD tissues, we observed the presence of cytosolic DNA in SMCs and macrophages and significant activation of the STING pathway. In the sporadic AAD model, Stinggt/gt mice showed significant reductions in challenge-induced aortic enlargement, dissection, and rupture in both the thoracic and abdominal aortic regions. Single-cell transcriptome analysis revealed that aortic challenge in wild-type mice induced the DNA damage response, the inflammatory response, dedifferentiation and cell death in SMCs, and matrix metalloproteinase expression in macrophages. These changes were attenuated in challenged Stinggt/gt mice. Mechanistically, nuclear and mitochondrial DNA damage in SMCs and the subsequent leak of DNA to the cytosol activated STING signaling, which induced cell death through apoptosis and necroptosis. In addition, DNA from damaged SMCs was engulfed by macrophages in which it activated STING and its target interferon regulatory factor 3, which directly induced matrix metalloproteinase-9 expression. We also found that pharmacologically inhibiting STING activation partially prevented AAD development. CONCLUSIONS: Our findings indicate that the presence of cytosolic DNA and subsequent activation of cytosolic DNA sensing adaptor STING signaling represent a key mechanism in aortic degeneration and that targeting STING may prevent sporadic AAD development.

Activation of Bone Marrow-Derived Cells and Resident Aortic Cells During Aortic Injury.

BACKGROUND: The process of aortic injury, repair, and remodeling during aortic aneurysm and dissection is poorly understood. We examined the activation of bone marrow (BM)-derived and resident aortic cells in response to aortic injury in a mouse model of sporadic aortic aneurysm and dissection. MATERIALS AND METHODS: Wild-type C57BL/6 mice were transplanted with green fluorescent protein (GFP)+ BM cells. For 4 wk, these mice were either unchallenged with chow diet and saline infusion or challenged with high-fat diet and angiotensin II infusion. We then examined the aortic recruitment of GFP+ BM-derived cells, growth factor production, and the differentiation potential of GFP+ BM-derived and GFP- resident aortic cells. RESULTS: Aortic challenge induced recruitment of GFP+ BM cells and activation of GFP- resident aortic cells, both of which produced growth factors. Although BM cells and resident aortic cells equally contributed to the fibroblast populations, we did not detect the differentiation of BM cells into smooth muscle cells. Interestingly, aortic macrophages were both of BM-derived (45%) and of non-BM-derived (55%) origin. We also observed a significant increase in stem cell antigen-1 (Sca-1)+ stem/progenitor cells and neural/glial antigen 2 (NG2+) cells in the aortic wall of challenged mice. Although some of the Sca-1+ cells and NG2+ cells were BM derived, most of these cells were resident aortic cells. Sca-1+ cells produced growth factors and differentiated into fibroblasts and NG2+ cells. CONCLUSIONS: BM-derived and resident aortic cells are activated in response to aortic injury and contribute to aortic inflammation, repair, and remodeling by producing growth factors and differentiating into fibroblasts and inflammatory cells.

Celf1 regulates cell cycle and is partially responsible for defective myoblast differentiation in myotonic dystrophy RNA toxicity.

Myotonic dystrophy is a neuromuscular disease of RNA toxicity. The disease gene DMPK harbors expanded CTG trinucleotide repeats on its 3'-UTR. The transcripts of this mutant DMPK led to misregulation of RNA-binding proteins including MBNL1 and Celf1. In myoblasts, CUG-expansion impaired terminal differentiation. In this study, we formally tested how the abundance of Celf1 regulates normal myocyte differentiation, and how Celf1 expression level mediates CUG-expansion RNA toxicity-triggered impairment of myocyte differentiation. As the results, overexpression of Celf1 largely recapitulated the defects of myocytes with CUG-expansion, by increasing myocyte cycling. Knockdown of endogenous Celf1 level led to precocious myotube formation, supporting a negative connection between Celf1 abundance and myocyte terminal differentiation. Finally, knockdown of Celf1 in myocyte with CUG-expansion led to partial rescue, by promoting cell cycle exit. Our results suggest that Celf1 plays a distinctive and negative role in terminal myocyte differentiation, which partially contribute to DM1 RNA toxicity. Targeting Celf1 may be a valid strategy in correcting DM1 muscle phenotypes, especially for congenital cases.

The Composition of Adipose-Derived Regenerative Cells Isolated from Lipoaspirate Using a Point of Care System Does Not Depend on the Subject's Individual Age, Sex, Body Mass Index and Ethnicity.

Uncultured, unmodified, autologous, adipose-derived regenerative cells (UA-ADRCs) are a safe and effective treatment option for various musculoskeletal pathologies. However, it is unknown whether the composition of the final cell suspension systematically varies with the subject's individual age, sex, body mass index and ethnicity. UA-ADRCs were isolated from lipoaspirate from n = 232 subjects undergoing elective lipoplasty using the Transpose RT system (InGeneron, Inc.; Houston, TX, USA). The UA-ADRCs were assessed for the number of nucleated cells, cell viability and the number of viable nucleated cells per gram of adipose tissue harvested. Cells from n = 37 subjects were further characterized using four-channel flow cytometry. The present study shows, for the first time, that key characteristics of UA-ADRCs can be independent of the subject's age, sex, BMI and ethnicity. This result has important implications for the general applicability of UA-ADRCs in regeneration of musculoskeletal tissue. Future studies must determine whether the independence of key characteristics of UA-ADRCs of the subject's individual age, sex, BMI and ethnicity only applies to the system used in the present study, or also to others of the more than 25 different experimental methods and commercially available systems used to isolate UA-ADRCs from lipoaspirate that have been described in the literature.

Multidomain peptide hydrogel adjuvants elicit strong bias towards humoral immunity.

Adjuvants play a critical role in enhancing vaccine efficacy; however, there is a need to develop new immunomodulatory compounds to address emerging pathogens and to expand the use of immunotherapies. Multidomain peptides (MDPs) are materials composed of canonical amino acids that form injectable supramolecular hydrogels under physiological salt and pH conditions. MDP hydrogels are rapidly infiltrated by immune cells in vivo and have previously been shown to influence cytokine production. Therefore, we hypothesized that these immunostimulatory characteristics would allow MDPs to function as vaccine adjuvants. Herein, we demonstrate that loading antigen into MDP hydrogels does not interfere with their rheological properties and that positively charged MDPs can act as antigen depots, as demonstrated by their ability to release ovalbumin (OVA) over a period of 7-9 days in vivo. Mice vaccinated with MDP-adjuvanted antigen generated significantly higher IgG titers than mice treated with the unadjuvanted control, suggesting that these hydrogels potentiate humoral immunity. Interestingly, MDP hydrogels did not elicit a robust cellular immune response, as indicated by the lower production of IgG2c and smaller populations of tetramer-positive CD8+ T splenocytes compared to mice vaccinated alum-adjuvanted OVA. Together, the data suggest that MDP hydrogel adjuvants strongly bias the immune response towards humoral immunity while evoking a very limited cellular immune response. As a result, MDPs may have the potential to serve as adjuvants for applications that benefit exclusively from humoral immunity.

Single-Cell Analysis of Aneurysmal Aortic Tissue in Patients with Marfan Syndrome Reveals Dysfunctional TGF-ß Signaling.

The molecular and cellular processes leading to aortic aneurysm development in Marfan syndrome (MFS) remain poorly understood. In this study, we examined the changes of aortic cell populations and gene expression in MFS by performing single-cell RNA sequencing (scRNA seq) on ascending aortic aneurysm tissues from patients with MFS (n = 3) and age-matched non-aneurysmal control tissues from cardiac donors and recipients (n = 4). The expression of key molecules was confirmed by immunostaining. We detected diverse populations of smooth muscle cells (SMCs), fibroblasts, and endothelial cells (ECs) in the aortic wall. Aortic tissues from MFS showed alterations of cell populations with increased de-differentiated proliferative SMCs compared to controls. Furthermore, there was a downregulation of MYOCD and MYH11 in SMCs, and an upregulation of COL1A1/2 in fibroblasts in MFS samples compared to controls. We also examined TGF-ß signaling, an important pathway in aortic homeostasis. We found that TGFB1 was significantly upregulated in two fibroblast clusters in MFS tissues. However, TGF-ß receptor genes (predominantly TGFBR2) and SMAD genes were downregulated in SMCs, fibroblasts, and ECs in MFS, indicating impairment in TGF-ß signaling. In conclusion, despite upregulation of TGFB1, the rest of the canonical TGF-ß pathway and mature SMCs were consistently downregulated in MFS, indicating a potential compromise of TGF-ß signaling and lack of stimulus for SMC differentiation.

Effect of Ciprofloxacin on Susceptibility to Aortic Dissection and Rupture in Mice.

Importance: Fluoroquinolones are among the most commonly prescribed antibiotics. Recent clinical studies indicated an association between fluoroquinolone use and increased risk of aortic aneurysm and dissection (AAD). This alarming association has raised concern, especially in patients with AAD with risk of rupture and in individuals at risk for developing AAD. Objective: To examine the effect of ciprofloxacin on AAD development in mice. Design, Setting, and Participants: In a mouse model of moderate, sporadic AAD, 4-week-old male and female C57BL/6J mice were challenged with a high-fat diet and low-dose angiotensin infusion (1000 ng/min/kg). Control unchallenged mice were fed a normal diet and infused with saline. After randomization, challenged and unchallenged mice received ciprofloxacin (100 mg/kg/d) or vehicle through daily gavage during angiotensin or saline infusion. Aortic aneurysm and dissection development and aortic destruction were compared between mice. The direct effects of ciprofloxacin on aortic smooth muscle cells were examined in cultured cells. Results: No notable aortic destruction was observed in unchallenged mice that received ciprofloxacin alone. Aortic challenge induced moderate aortic destruction with development of AAD in 17 of 38 mice (45%) and severe AAD in 9 (24%) but no rupture or death. However, challenged mice that received ciprofloxacin had severe aortic destruction and a significantly increased incidence of AAD (38 of 48 [79%]; P = .001; χ2 = 10.9), severe AAD (32 of 48 [67%]; P < .001; χ2 = 15.7), and rupture and premature death (7 of 48 [15%]; P = .01; χ2 = 6.0). The increased AAD incidence was observed in different aortic segments and was similar between male and female mice. Compared with aortic tissues from challenged control mice, those from challenged mice that received ciprofloxacin showed decreased expression of lysyl oxidase, an enzyme that is critical in the assembly and stabilization of elastic fibers and collagen. These aortas also showed increased matrix metalloproteinase levels and activity, elastic fiber fragmentation, and aortic cell injury. In cultured smooth muscle cells, ciprofloxacin treatment significantly reduced lysyl oxidase expression and activity, increased matrix metalloproteinase expression and activity, suppressed cell proliferation, and induced cell death. Furthermore, ciprofloxacin-a DNA topoisomerase inhibitor-caused nuclear and mitochondrial DNA damage and the release of DNA into the cytosol, subsequently inducing mitochondrial dysfunction, reactive oxygen species production, and activation of the cytosolic DNA sensor STING, which we further showed was involved in the suppression of lysyl oxidase expression and induction of matrix metalloproteinase expression. Conclusions and Relevance: Ciprofloxacin increases susceptibility to aortic dissection and rupture in a mouse model of moderate, sporadic AAD. Ciprofloxacin should be used with caution in patients with aortic dilatation, as well as in those at high risk for AAD.

AKT2 Promotes Bone Marrow Cell-Mediated Aortic Protection in Mice.

BACKGROUND: Insufficient aortic protection and repair may contribute to the development of aortic aneurysms and dissections (AAD). However, mechanisms of aortic protection and repair are poorly understood. We have shown that the multifunctional kinase AKT2 plays an important role in protecting the aortic wall. Here, we examined whether AKT2 protects against AAD by promoting bone marrow cell (BMC)-mediated aortic protection. METHODS: Irradiated wild-type mice received green fluorescent protein-expressing BMCs from wild-type mice or Akt2(-/-) mice, followed by challenge with angiotensin II (1000 ng/kg/min) infusion for 4 weeks. We compared BMC recruitment, aortic destruction, and AAD development between groups. The direct effects of wild-type and Akt2(-/-) BMCs on smooth muscle cell survival were examined in coculture experiments. RESULTS: After angiotensin II infusion, no (0 of 14) wild-type BMC recipients had AAD; in contrast, 64% (9 of 14) of Akt2(-/-) BMC recipients had AAD (p = 0.002) with severe aortic destruction. Compared with aortas from challenged wild-type BMC recipients, aortas from challenged Akt2(-/-) BMC recipients showed significantly less BMC recruitment, NG2 (neuron-glial antigen 2) progenitor activation, and FSP1 (fibroblast-specific protein 1) fibroblast activation. In addition, aortas from challenged Akt2(-/-) BMC recipients showed increased apoptosis and inflammation. In coculture experiments, wild-type but not Akt2(-/-) BMCs prevented smooth muscle cells from undergoing oxidative stress-induced apoptosis. CONCLUSIONS: After aortic challenge, BMCs are recruited to the aortic wall and provide protection by activating progenitors and fibroblasts and by promoting aortic cell survival. Our findings indicate that AKT2 is involved in these processes and that defects in this pathway may promote progressive degeneration during AAD development.