Aortic root aneurysm formation is a cardinal feature of Marfan syndrome (MFS) and likely TGF-β driven via Smad (canonical) and ERK (non-canonical) signalling. The current study assesses human MFS vascular smooth muscle cell (SMC) phenotype, focusing on individual contributions by Smad and ERK, with Notch3 signalling identified as a novel compensatory mechanism against TGF-β-driven pathology. Although significant ERK activation and mixed contractile gene expression patterns were observed by traditional analysis, this did not directly correlate with the anatomic site of the aneurysm. Smooth muscle cell phenotypic changes were TGF-β-dependent and opposed by ERK in vitro, implicating the canonical Smad pathway. Bulk SMC RNA sequencing after ERK inhibition showed that ERK modulates cell proliferation, apoptosis, inflammation, and Notch signalling via Notch3 in MFS. Reversing Notch3 overexpression with siRNA demonstrated that Notch3 promotes several protective remodelling pathways, including increased SMC proliferation, decreased apoptosis and reduced matrix metalloproteinase activity, in vitro. In conclusion, in human MFS aortic SMCs: (a) ERK activation is enhanced but not specific to the site of aneurysm formation; (b) ERK opposes TGF-β-dependent negative effects on SMC phenotype; (c) multiple distinct SMC subtypes contribute to a 'mixed' contractile-synthetic phenotype in MFS aortic aneurysm; and (d) ERK drives Notch3 overexpression, a potential pathway for tissue remodelling in response to aneurysm formation. K E Y W O R D S aortic aneurysm, Marfan syndrome, smooth muscle cell phenotype 1 | INTRODUC TI ON Marfan syndrome (MFS) is an inheritable connective tissue disorder resulting from fibrillin-1 (Fbn1) mutations with an incidence of 1 in 5000 individuals. 1,2 Aortic root aneurysm formation and ensuing dissection accounts for early mortality if not surgically repaired prophylactically. 3 Transforming growth factor-beta (TGF-β) signalling is central to MFS aortic aneurysm pathogenesis. 4 Despite enhanced S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section. How to cite this article: Pedroza AJ, Koyano T, Trojan J, et al. Divergent effects of canonical and non-canonical TGF-β signalling on mixed contractile-synthetic smooth muscle cell phenotype in human Marfan syndrome aortic root aneurysms.

Background Statins reduce aneurysm growth in mouse models of Marfan syndrome, although the mechanism is unknown. In addition to reducing cholesterol, statins block farnesylation and geranylgeranylation, which participate in membrane‐bound G‐protein signaling, including Ras. We dissected the prenylation pathway to define the effect of statins on aneurysm reduction. Methods and Results Fbn1 C1039G/+ mice were treated with (1) pravastatin (HMG‐CoA [3‐hydroxy‐3‐methylglutaryl coenzyme A] reductase inhibitor), (2) manumycin A ( MA ; FPT inhibitor), (3) perillyl alcohol ( GGPT 1 and ‐2 inhibitor), or (4) vehicle control from age 4 to 8 weeks and euthanized at 12 weeks. Histological characterization was performed. Protein analysis was completed on aortic specimens to measure ERK (extracellular signal‐regulated kinase) signaling. In vitro Fbn1 C1039G/+ aortic smooth muscle cells were utilized to measure Ras‐dependent ERK signaling and MMP (matrix metalloproteinase) activity. Pravastatin and MA significantly reduced aneurysm growth compared with vehicle control (n=8 per group). In contrast, PA did not significantly decrease aneurysm size. Histology illustrated reduced elastin breakdown in MA ‐treated mice compared with vehicle control (n=5 per group). Although elevated in control Marfan mice, both phosphorylated c‐Raf and phosphorylated ERK 1/2 were significantly reduced in MA ‐treated mice (4–5 per group). In vitro smooth muscle cell studies confirmed phosphorylated cR af and phosphorylated ERK 1/2 signaling was elevated in Fbn1 C1039G/+ smooth muscle cells (n=5 per group). Fbn1 C1039G/+ smooth muscle cell Ras‐dependent ERK signaling and MMP activity were reduced following MA treatment (n=5 per group). Corroborating in vitro findings, MMP activity was also decreased in pravastatin‐treated mice. Conclusions Aneurysm reduction in Fbn1 C1039G/+ mice following pravastatin and MA treatment was associated with a decrease in Ras‐dependent ERK signaling. MMP activity can be reduced by diminishing Ras signaling.

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Central Message Saphenous vein graft aneurysms with mechanical complications should be repaired via a non-sternotomy open surgical technique.

Background Mindstep is an app that aims to improve dementia screening by assessing cognition and risk factors. It considers important clinical risk factors, including prodromal symptoms, mental health disorders, and differential diagnoses of dementia. The 9-item Patient Health Questionnaire for depression (PHQ-9) and the 7-item Generalized Anxiety Disorder Scale (GAD-7) are widely validated and commonly used scales used in screening for depression and anxiety disorders, respectively. Shortened versions of both (PHQ-2/GAD-2) have been produced. Objective We sought to develop a method that maintained the brevity of these shorter questionnaires while maintaining the better precision of the original questionnaires. Methods Single questions were designed to encompass symptoms covered in the original questionnaires. Answers to these questions were combined with PHQ-2/GAD-2, and anonymized risk factors were collected by Mindset4Dementia from 2235 users. Machine learning models were trained to use these single questions in combination with data already collected by the app: age, response to a joke, and reporting of functional impairment to predict binary and continuous outcomes as measured using PHQ-9/GAD-7. Our model was developed with a training data set by using 10-fold cross-validation and a holdout testing data set and compared to results from using the shorter questionnaires (PHQ-2/GAD-2) alone to benchmark performance. Results We were able to achieve superior performance in predicting PHQ-9/GAD-7 screening cutoffs compared to PHQ-2 (difference in area under the curve 0.04, 95% CI 0.00-0.08, P=.02) but not GAD-2 (difference in area under the curve 0.00, 95% CI –0.02 to 0.03, P=.42). Regression models were able to accurately predict total questionnaire scores in PHQ-9 (R2=0.655, mean absolute error=2.267) and GAD-7 (R2=0.837, mean absolute error=1.780). Conclusions We app-adapted PHQ-4 by adding brief summary questions about factors normally covered in the longer questionnaires. We additionally trained machine learning models that used the wide range of additional information already collected in Mindstep to make a short app-based screening tool for affective disorders, which appears to have superior or equivalent performance to well-established methods.

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BACKGROUND Mindset4Dementia is an app that aims to improve dementia screening by assessing cognition and risk factors. It considers important clinical risk factors, including prodromal symptoms, mental health disorders, and differential diagnoses of dementia. The PHQ-9 and GAD-7 are widely validated, and commonly used scales used in screening for depression and anxiety disorders respectively. Shortened versions of both (PHQ-2/GAD-2) have been produced. OBJECTIVE We sought to develop a method that maintained the brevity of these shorter questionnaires while maintaining the better precision of the original questionnaires METHODS Single questions were designed to encompass symptoms covered in the original questionnaires. Answers to these questions were combined with the PHQ-2/GAD-2 and anonymized risk factors collected by Mindset4Dementia. Machine learning models were trained to use these single questions in combination with data already collected by the app - age, response to a joke and reporting of functional impairment to predict binary and continuous outcomes as measured by the PHQ-9/GAD-7. Our model was developed with a training dataset using ten-fold cross-validation and a hold-out testing datasets and compared to results from using the shorter questionnaires (PHQ-2/GAD-2) alone to benchmark performance. RESULTS We were able to achieve superior performance in predicting PHQ-9/GAD-7 screening cut-offs than the PHQ-2 (difference In AUC 0.04, 95% CI 0.00 – 0.08, P = 0.02) but not to GAD-2 (difference in AUC 0.00, 95% CI -0.02 – 0.03, P = 0.42). In regression models we were able to accurately predict total questionnaire scores; PHQ-9 (R2 = 0.655, MAE = 2.267), GAD-7 (R2 = 0.837, MAE = 1.780). CONCLUSIONS We have developed a short screening tool for affective disorders with superior or equivalent performance to well established methods.

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