The developmental origin of vascular smooth muscle cells (VSMCs) has been increasingly recognized as a major determinant for regional susceptibility or resistance to vascular diseases. As a human material-based complement to animal models and human primary cultures, patient induced pluripotent stem cell iPSC-derived VSMCs have been leveraged to conduct basic research and develop therapeutic applications in vascular diseases. However, iPSC-VSMCs (induced pluripotent stem cell VSMCs) derived by most existing induction protocols are heterogeneous in developmental origins. In this review, we summarize signaling networks that govern in vivo cell fate decisions and in vitro derivation of distinct VSMC progenitors, as well as key regulators that terminally specify lineage-specific VSMCs. We then highlight the significance of leveraging patient-derived iPSC-VSMCs for vascular disease modeling, drug discovery, and vascular tissue engineering and discuss several obstacles that need to be circumvented to fully unleash the potential of induced pluripotent stem cells for precision vascular medicine.

Objective

Current guidelines for prophylactic replacement of the thoracic aorta, primarily based on size alone, may not be adequate in identifying patients at risk for either progression of disease or aortic catastrophe. We undertook the current study to determine whether the mechanical properties of the aorta might be able to predict aneurysmal dilatation of the aorta using a clinical database and benchtop mechanical testing of human aortic tissue.

Methods

Using over 400 samples from 31 patients, mechanical properties were studied in (a) normal aorta and then (b) between normal and diseased aorta using linear mixed-effects models. A machine learning technique was used to predict aortic growth rate over time using mechanical properties and baseline clinical characteristics.

Results

Healthy aortic tissue under in vivo loading conditions, after accounting for aortic segment location, had lower longitudinal elastic modulus compared with circumferential elastic modulus: -166.8 kPa (95% confidence interval [CI]: -210.8 to -122.7, P < .001). Fracture toughness was also lower in the longitudinal vs circumferential direction: -201.2 J/m³ (95% CI: -272.9 to -129.5, P < .001). Finally, relative strain was lower in the longitudinal direction compared with the circumferential direction: -0.01 (95% CI: -0.02 to -0.004, P = .002). Patients with diseased aorta, after accounting for segment location and sample direction, had decreased toughness compared with normal aorta, -431.7 J/m³ (95% CI: -628.6 to -234.8, P < .001), and increased relative strain, 0.09 (95% CI: 0.04 to 0.14, P = .003).

Conclusions

Increasing relative strain was identified as a novel independent predictor of aneurysmal degeneration. Noninvasive measurement of relative strain may aid in the identification and monitoring of patients at risk for aneurysmal degeneration. (JVS-Vascular Science 2021;2:1-12.).

Clinical relevance

Aortic aneurysm surveillance and prophylactic surgical recommendations are based on computed tomographic angiogram aortic dimensions and growth rate measurements. However, aortic catastrophes may occur at small sizes, confounding current risk stratification models. Herein, we report that increasing aortic relative strain, that is, greater distensibility, is associated with growth over time, thus potentially identifying patients at risk for dissection/rupture.

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.

Aortic root aneurysm formation and subsequent dissection and/or rupture remain the leading cause of death in patients with Marfan syndrome. Our laboratory has reported that miR-29b participates in aortic root/ascending aorta extracellular matrix remodeling during early aneurysm formation in Fbn1C1039G/+ Marfan mice. Herein, we sought to determine whether miR-29b suppression can reduce aneurysm formation long-term. Fbn1C1039G/+ Marfan mice were treated with retro-orbital LNA-anti-miR-29b inhibitor or scrambled-control-miR before aneurysms develop either (1) a single dose prenatally (pregnant Fbn1C1039G/+ mice at 14.5 days post-coitum) (n = 8-10, each group) or (2) postnatally every other week, from 2 to 22 weeks of age, and sacrificed at 24 weeks (n = 8-10, each group). To determine if miR-29b blockade was beneficial even after aneurysms develop, a third group of animals were treated every other week, starting at 8 weeks of age, until sacrificed (n = 4-6, each group). miR-29b inhibition resulted in aneurysm reduction, increased elastogenesis, decreased matrix metalloproteinase activity and decreased elastin breakdown. Prenatal LNA-anti-miR-29b inhibitor treatment decreased aneurysm formation up to age 32 weeks, whereas postnatal treatment was effective up to 16 weeks. miR-29b blockade did not slow aortic growth once aneurysms already developed. Systemic miR-29b inhibition significantly reduces aneurysm development long-term in a Marfan mouse model. Drug administration during aortic wall embryologic development appears fundamental. miR-29b suppression could be a potential therapeutic target for reducing aneurysm formation in Marfan syndrome patients.

Marfan syndrome (MFS) is a connective tissue disorder that results in aortic root aneurysm formation. Reactive oxygen species (ROS) seem to play a role in aortic wall remodelling in MFS, although the mechanism remains unknown. MFS Fbn1^C1039G/+ mouse root/ascending (AS) and descending (DES) aortic samples were examined using DHE staining, lucigenin-enhanced chemiluminescence (LGCL), Verhoeff's elastin-Van Gieson staining (elastin breakdown) and in situ zymography for protease activity. Fbn1^C1039G/+ AS- or DES-derived smooth muscle cells (SMC) were treated with anti-TGF-β antibody, angiotensin II (AngII), anti-TGF-β antibody + AngII, or isotype control. ROS were detected during early aneurysm formation in the Fbn1^C1039G/+ AS aorta, but absent in normal-sized DES aorta. Fbn1^C1039G/+ mice treated with the unspecific NADPH oxidase inhibitor, apocynin reduced AS aneurysm formation, with attenuated elastin fragmentation. In situ zymography revealed apocynin treatment decreased protease activity. In vitro SMC studies showed Fbn1^C1039G/+ -derived AS SMC had increased NADPH activity compared to DES-derived SMC. AS SMC NADPH activity increased with AngII treatment and appeared TGF-β dependent. In conclusion, ROS play a role in MFS aneurysm development and correspond anatomically with aneurysmal aortic segments. ROS inhibition via apocynin treatment attenuates MFS aneurysm progression. AngII enhances ROS production in MFS AS SMCs and is likely TGF-β dependent.

OBJECTIVES: Establishing the reproducibility of expert-derived measurements on CTA exams of aortic dissection is clinically important and paramount for ground-truth determination for machine learning. METHODS: Four independent observers retrospectively evaluated CTA exams of 72 patients with uncomplicated Stanford type B aortic dissection and assessed the reproducibility of a recently proposed combination of four morphologic risk predictors (maximum aortic diameter, false lumen circumferential angle, false lumen outflow, and intercostal arteries). For the first inter-observer variability assessment, 47 CTA scans from one aortic center were evaluated by expert-observer 1 in an unconstrained clinical assessment without a standardized workflow and compared to a composite of three expert-observers (observers 2-4) using a standardized workflow. A second inter-observer variability assessment on 30 out of the 47 CTA scans compared observers 3 and 4 with a constrained, standardized workflow. A third inter-observer variability assessment was done after specialized training and tested between observers 3 and 4 in an external population of 25 CTA scans. Inter-observer agreement was assessed with intraclass correlation coefficients (ICCs) and Bland-Altman plots. RESULTS: Pre-training ICCs of the four morphologic features ranged from 0.04 (-0.05 to 0.13) to 0.68 (0.49-0.81) between observer 1 and observers 2-4 and from 0.50 (0.32-0.69) to 0.89 (0.78-0.95) between observers 3 and 4. ICCs improved after training ranging from 0.69 (0.52-0.87) to 0.97 (0.94-0.99), and Bland-Altman analysis showed decreased bias and limits of agreement. CONCLUSIONS: Manual morphologic feature measurements on CTA images can be optimized resulting in improved inter-observer reliability. This is essential for robust ground-truth determination for machine learning models. KEY POINTS: • Clinical fashion manual measurements of aortic CTA imaging features showed poor inter-observer reproducibility. • A standardized workflow with standardized training resulted in substantial improvements with excellent inter-observer reproducibility. • Robust ground truth labels obtained manually with excellent inter-observer reproducibility are key to develop reliable machine learning models.