A simplified murine intimal hyperplasia model founded on a focal carotid stenosis.

Murine models offer a powerful tool for unraveling the mechanisms of intimal hyperplasia and vascular remodeling, although their technical complexity increases experimental variability and limits widespread application. We describe a simple and clinically relevant mouse model of arterial intimal hyperplasia and remodeling. Focal left carotid artery (LCA) stenosis was created by placing 9-0 nylon suture around the artery using an external 35-gauge mandrel needle (middle or distal location), which was then removed. The effect of adjunctive diet-induced obesity was defined. Flowmetry, wall strain analyses, biomicroscopy, and histology were completed. LCA blood flow sharply decreased by ∼85%, followed by a responsive right carotid artery increase of ∼71%. Circumferential strain decreased by ∼2.1% proximal to the stenosis in both dietary groups. At 28 days, morphologic adaptations included proximal LCA intimal hyperplasia, which was exacerbated by diet-induced obesity. The proximal and distal LCA underwent outward and negative inward remodeling, respectively, in the mid-focal stenosis (remodeling indexes, 1.10 and 0.53). A simple, defined common carotid focal stenosis yields reproducible murine intimal hyperplasia and substantial differentials in arterial wall adaptations. This model offers a tool for investigating mechanisms of hemodynamically driven intimal hyperplasia and arterial wall remodeling.

Acute reductions in mechanical wall strain precede the formation of intimal hyperplasia in a murine model of arterial occlusive disease.

OBJECTIVE: Intimal hyperplasia (IH) continues to plague the durability of vascular interventions. Employing a validated murine model, ultrasound biomicroscopy, and speckle-tracking algorithms, we tested the hypothesis that reduced cyclic arterial wall strain results in accentuated arterial wall IH. METHODS: A 9-0 suture was tied around the left mouse (n = 10) common carotid artery and a 35-gauge (outer diameter = 0.14 mm) blunt mandrel. We previously showed that mandrel removal results in a ∼78% reduction in diameter and ∼85% reduction in flow, with subsequent delayed induction of IH by day 28. Preoperative, postoperative day-4 (before measurable IH), and postoperative day-27 circumferential wall strains were measured in locations 1, 2, and 3 mm proximal to the stenosis and in the same locations on the contralateral (nonstenosed) carotid. At postoperative day 28, arteries were perfusion fixed and arterial wall morphology was assessed microscopically in the same regions. RESULTS: Strains were the same in all locations preoperatively. Wall strain was decreased in all regions proximal to the stenosis by day 4 (0.26 ± 0.01 to 0.11 ± 0.02; P < .001), while strains remained unchanged for the contralateral artery (P = .45). No statistical regional differences in mean strain or IH were noted at any time point for the experimental or contralateral artery. Based on the median, regions were divided into those with low strain (≤0.1) and high strain (>0.1). Average preoperative strains in both groups were the same (0.27 ± 0.09 and 0.27 ± 0.08). All segments in the low-strain group (n = 13) demonstrated significant IH formation by day 28, while only 31% of the high strain group demonstrated any detectable IH at day 28. (Mean low-strain intimal thickness = 32 ± 20 µm, high strain = 8.0 ± 16 µm; P < .01). Changes in cross-sectional area at diastole drove the reduction in strain in the low-strain group, increasing significantly from preoperatively to day 4 (P = .04), while lumen cross-section at systole remained unchanged (P = .46). Cross-sectional area at diastole and systole in the high-strain group remained unchanged from preoperatively to day 4 (P = .67). CONCLUSIONS: Early reduction in arterial wall strain is associated with subsequent development of hemodynamically induced IH.

Development and characterization of a 3D multicell microtissue culture model of airway smooth muscle.

Airway smooth muscle (ASM) cellular and molecular biology is typically studied with single-cell cultures grown on flat 2D substrates. However, cells in vivo exist as part of complex 3D structures, and it is well established in other cell types that altering substrate geometry exerts potent effects on phenotype and function. These factors may be especially relevant to asthma, a disease characterized by structural remodeling of the airway wall, and highlights a need for more physiologically relevant models of ASM function. We utilized a tissue engineering platform known as microfabricated tissue gauges to develop a 3D culture model of ASM featuring arrays of ∼0.4 mm long, ∼350 cell "microtissues" capable of simultaneous contractile force measurement and cell-level microscopy. ASM-only microtissues generated baseline tension, exhibited strong cellular organization, and developed actin stress fibers, but lost structural integrity and dissociated from the cantilevers within 3 days. Addition of 3T3-fibroblasts dramatically improved survival times without affecting tension development or morphology. ASM-3T3 microtissues contracted similarly to ex vivo ASM, exhibiting reproducible responses to a range of contractile and relaxant agents. Compared with 2D cultures, microtissues demonstrated identical responses to acetylcholine and KCl, but not histamine, forskolin, or cytochalasin D, suggesting that contractility is regulated by substrate geometry. Microtissues represent a novel model for studying ASM, incorporating a physiological 3D structure, realistic mechanical environment, coculture of multiple cells types, and comparable contractile properties to existing models. This new model allows for rapid screening of biochemical and mechanical factors to provide insight into ASM dysfunction in asthma.

Murine ultrasound imaging for circumferential strain analyses in the angiotensin II abdominal aortic aneurysm model.

OBJECTIVE: The underlying causes of abdominal aortic aneurysms (AAAs) remain obscure, although research tools such as the angiotensin II (Ang II) apolipoprotein E-deficient (apoE(-/-)) mouse model have aided investigations. Longitudinal imaging and determination of biomechanical forces in this small-scale model have been difficult. We hypothesized that high-frequency ultrasound biomicroscopy combined with speckle-tracking analytical strategies can be used to define the role of circumferential mechanical strain in AAA formation in the Ang II/apoE(-/-) mouse model of AAAs. We simultaneously examined dietary perturbations that might impact the biomechanical properties of the aortic wall, hypothesizing that the generalized inflammatory phenotype associated with diet-induced obesity would be associated with accelerated loss of circumferential strain and aneurysmal aortic degeneration. METHODS: Receiving either a 60 kcal% fat Western diet or standard 10 kcal% fat normal chow, Ang II-treated apoE(-/-) mice (n = 34) underwent sequential aortic duplex ultrasound scan imaging (Vevo 2100 System; VisualSonics, Toronto, Ontario, Canada) of their entire aorta. Circumferential strains were calculated using speckle-tracking algorithms and a custom MatLab analysis. RESULTS: Decreased strains in all aortic locations after just 3 days of Ang II treatment were observed, and this effect progressed during the 4-week observation period. Anatomic segments along the aorta impacted wall strain (baseline highest in ascending aorta; P < .05), whereas diet did not. At 2 and 4 weeks, there was the largest progressive decrease in strain in the paravisceral/supraceliac aorta (P < .05), which was the segment most likely to be involved in aneurysm formation in this model. CONCLUSIONS: In the Ang II/apoE(-/-) aneurysm model, the aorta significantly stiffens (with decreased strain) shortly after Ang II infusion, and this progressively continues through the next 4 weeks. High-fat feeding did not have an impact on wall strain. Delineation of biomechanical factors and AAA morphology via duplex scan and speckle-tracking algorithms in mouse models should accelerate insights into human AAAs.

Optical Method to Quantify Mechanical Contraction and Calcium Transients of Human Pluripotent Stem Cell-Derived Cardiomyocytes.

Differentiation of human pluripotent stem cells into cardiomyocytes (hPS-CMs) holds promise for myocardial regeneration therapies, drug discovery, and models of cardiac disease. Potential cardiotoxicities may affect hPS-CM mechanical contraction independent of calcium signaling. Herein, a method using an image capture system is described to measure hPS-CM contractility and intracellular calcium concurrently, with high spatial and temporal resolution. The image capture system rapidly alternates between brightfield and epifluorescent illumination of contracting cells. Mechanical contraction is quantified by a speckle tracking algorithm applied to brightfield image pairs, whereas calcium transients are measured by a fluorescent calcium reporter. This technique captured changes in contractile strain, calcium transients, and beat frequency of hPS-CMs over 21 days in culture, as well as acute responses to isoproterenol and Cytochalasin D. The technique described above can be applied without the need to alter the culture platform, allowing for determination of hPS-CM behavior over weeks in culture for drug discovery and myocardial regeneration applications.

Delivering stem cells to the healthy heart on biological sutures: effects on regional mechanical function.

Current cardiac cell therapies cannot effectively target and retain cells in a specific area of the heart. Cell-seeded biological sutures were previously developed to overcome this limitation, demonstrating targeted delivery with > 60% cell retention. In this study, both cell-seeded and non-seeded fibrin-based biological sutures were implanted into normal functioning rat hearts to determine the effects on mechanical function and fibrotic response. Human mesenchymal stem cells (hMSCs) were used based on previous work and established cardioprotective effects. Non-seeded or hMSC-seeded sutures were implanted into healthy athymic rat hearts. Before cell seeding, hMSCs were passively loaded with quantum dot nanoparticles. One week after implantation, regional stroke work index and systolic area of contraction (SAC) were evaluated on the epicardial surface above the suture. Cell delivery and retention were confirmed by quantum dot tracking, and the fibrotic tissue area was evaluated. Non-seeded biological sutures decreased SAC near the suture from 0.20 ± 0.01 measured in sham hearts to 0.08 ± 0.02, whereas hMSC-seeded biological sutures dampened the decrease in SAC (0.15 ± 0.02). Non-seeded sutures also displayed a small amount of fibrosis around the sutures (1.0 ± 0.1 mm2 ). Sutures seeded with hMSCs displayed a significant reduction in fibrosis (0.5 ± 0.1 mm2 , p < 0.001), with quantum dot-labelled hMSCs found along the suture track. These results show that the addition of hMSCs attenuates the fibrotic response observed with non-seeded sutures, leading to improved regional mechanics of the implantation region. Copyright © 2014 John Wiley & Sons, Ltd.

Functional Effects of Delivering Human Mesenchymal Stem Cell-Seeded Biological Sutures to an Infarcted Heart.

Stem cell therapy has the potential to improve cardiac function after myocardial infarction (MI); however, existing methods to deliver cells to the myocardium, including intramyocardial injection, suffer from low engraftment rates. In this study, we used a rat model of acute MI to assess the effects of human mesenchymal stem cell (hMSC)-seeded fibrin biological sutures on cardiac function at 1 week after implant. Biological sutures were seeded with quantum dot (Qdot)-loaded hMSCs for 24 h before implantation. At 1 week postinfarct, the heart was imaged to assess mechanical function in the infarct region. Regional parameters assessed were regional stroke work (RSW) and systolic area of contraction (SAC) and global parameters derived from the pressure waveform. MI (n = 6) significantly decreased RSW (0.026 ± 0.011) and SAC (0.022 ± 0.015) when compared with sham operation (RSW: 0.141 ± 0.009; SAC: 0.166 ± 0.005, n = 6) (p < 0.05). The delivery of unseeded biological sutures to the infarcted hearts did not change regional mechanical function compared with the infarcted hearts (RSW: 0.032 ± 0.004, SAC: 0.037 ± 0.008, n = 6). The delivery of hMSC-seeded sutures exerted a trend toward increase of regional mechanical function compared with the infarcted heart (RSW: 0.057 ± 0.011; SAC: 0.051 ± 0.014, n = 6). Global function showed no significant differences between any group (p > 0.05); however, there was a trend toward improved function with the addition of either unseeded or seeded biological suture. Histology demonstrated that Qdot-loaded hMSCs remained present in the infarcted myocardium after 1 week. Analysis of serial sections of Masson's trichrome staining revealed that the greatest infarct size was in the infarct group (7.0% ± 2.2%), where unseeded (3.8% ± 0.6%) and hMSC-seeded (3.7% ± 0.8%) suture groups maintained similar infarct sizes. Furthermore, the remaining suture area was significantly decreased in the unseeded group compared with that in the hMSC-seeded group (p < 0.05). This study demonstrated that hMSC-seeded biological sutures are a method to deliver cells to the infarcted myocardium and have treatment potential.