Abstract-Arterial intimal thickening after endothelial injury induced in rodents has proven to be a relatively unreliable model of restenosis for testing clinically useful compounds. The same has been found for cultured rat or rabbit vascular smooth muscle cells (SMCs). To test alternative possibilities, we have studied several differentiation features of porcine coronary artery SMCs, cultured up to the 5th passage after enzymatic digestion of the media. The effects of heparin, transforming growth factor (TGF)-␤ 1 or TGF-␤ 2 , and all-trans-retinoic acid (tRA) on proliferation, migration, and differentiation of these cells also were examined. Porcine arterial SMCs in culture not only express high levels of ␣-smooth muscle (SM) actin but, contrary to rodent SMCs, also maintain an appreciable expression of SM myosin heavy chain isoforms 1 and 2, desmin, and smoothelin, a recently described late differentiation marker of vascular SMCs. We demonstrate for the first time that smoothelin is colocalized with ␣-SM actin in these cells. Finally, we show that in the porcine model, heparin is more potent than TGF-␤ 1 or TGF-␤ 2 and tRA in terms of inhibition of proliferation and migration and of increasing the expression of differentiation markers. This model should be a useful complement to in vivo studies of SMC differentiation and of pathological situations such as restenosis and atheromatosis. (Circ Res. 1999;85:99-107.)Key Words: atheromatosis Ⅲ restenosis Ⅲ actin Ⅲ smoothelin Ⅲ myosin T he arterial intimal thickenings (IT) induced in the rat or rabbit after endothelial lesion are presently the moststudied models for atheroma formation and/or restenosis and have been useful for defining several biological features of smooth muscle cells (SMCs). However, these models have many important limitations (for review, see Reference 1), which could explain the clinical failure of substances that proved to be efficient inhibitors of IT formation in these animal models.The biological features of SMCs in culture also have been systematically studied using cells derived from rat or rabbit arteries, 2-7 but these, too, have shown limitations similar to those observed in in vivo experiments. Among the models in large animals, the pig coronary artery IT has been used more and more. 8,9 Pigs may develop spontaneously coronary atheromatosis with age, and the induction of typical plaques is easily achieved by a cholesterol-rich diet. 10,11 Furthermore, angioplasty and other interventions can be performed in porcine coronary arteries with the same instruments as in humans. Although pig aortic and coronary SMCs have already been studied in vitro, no systematic description of their differentiation features during culture has been published.The present study describes the characterization of differentiation features and several biological properties of cultured porcine left anterior descending (LAD) coronary artery SMCs. We show that, contrary to rat or rabbit arterial SMCs, 2-7 porcine SMCs maintain in culture a high level of differentiati...

High-resolution magnetic resonance imaging (MRI) has evolved into one of the major non-invasive tools to study the healthy and diseased mouse heart. This study presents a Cartesian CINE MRI protocol based on a fast low-angle shot sequence with a navigator echo to generate cardiac triggering and respiratory gating signals retrospectively, making the use of ECG leads and respiratory motion sensors obsolete. MRI of the in vivo mouse heart using this sequence resulted in CINE images with no detectable cardiac and respiratory motion artefacts. The retrospective method allows for steady-state imaging of the mouse heart, which is essential for quantitative contrast-enhanced MRI studies. A comparison was made between prospective and retrospective methods in terms of the signal-to-noise ratio and the contrast-to-noise ratio between blood and myocardial wall, as well as global cardiac functional indices: end-diastolic volume, end-systolic volume, stroke volume and ejection fraction. The retrospective method resulted in almost constant left-ventricle wall signal intensity throughout the cardiac cycle, at the expense of a decrease in the signal-to-noise ratio and the contrast-to-noise ratio between blood and myocardial wall as compared with the prospective method. Prospective and retrospective sequences yielded comparable global cardiac functional indices. The largest mean relative difference found was 8% for the end-systolic volume.

Background-To characterize the cells responsible for neointima formation after porcine coronary artery wall injury, we studied the expression of smooth muscle cell (SMC) differentiation markers in 2 models: (1) self-expanding stent implantation resulting in no or little interruption of internal elastic lamina and (2) percutaneous transluminal coronary angioplasty (PTCA) resulting in complete medial rupture and exposure of adventitia to blood components. Methods and Results-The expression of ␣-smooth muscle (SM) actin, SM myosin heavy chain isoforms 1 and 2, desmin, and smoothelin was investigated by means of immunohistochemistry and Western blots in tissues of the arterial wall collected at different time points and in cell populations cultured from these tissues. The expression of smoothelin, a marker of late SMC differentiation, was used to discriminate between SMCs and myofibroblasts. Both stent-and PTCA-induced neointimal tissues and their cultured cell populations expressed all 4 markers. The adventitial tissue underlying PTCA-induced lesions temporarily expressed ␣-SM actin, desmin, and SM myosin heavy chain isoforms, but not smoothelin. When placed in culture, adventitial cells expressed only ␣-SM actin. Conclusions-Our results suggest that SMCs are the main components of coronary artery neointima after both self-expanding stent implantation and PTCA. The adventitial reaction observed after PTCA evolves with a chronology independent of that of neointima formation and probably corresponds to a myofibroblastic reaction.

Rationale: Vascular calcification, the formation of calcium phosphate crystals in the vessel wall, is mediated by vascular smooth muscle cells (VSMCs). However, the underlying molecular mechanisms remain elusive precluding mechanism-based therapies. Objective: Phenotypic switching denotes a loss of contractile proteins and an increase in migration and proliferation, whereby VSMCs are termed synthetic. We examined how VSMC phenotypic switching influences vascular calcification and the possible role of the uniquely calcium-dependent ROS-forming NADPH oxidase 5 (Nox5). Methods and Results: In vitro cultures of synthetic VSMCs showed decreased expression of contractile markers CNN1, αSMA and SM22α and an increase in synthetic marker S100A4 compared to contractile VSMCs. This was associated with increased calcification of synthetic cells in response to high extracellular Ca 2+ . Phenotypic switching was accompanied by increased levels of reactive oxygen species (ROS) and Ca 2+ -dependent Nox5 in synthetic VSMCs. Nox5 itself regulated VSMC phenotype as siRNA knock-down of Nox5 increased contractile marker expression and decreased calcification, while overexpression of Nox5 decreased contractile marker expression. ROS production in synthetic VSMCs was cytosolic Ca 2+ -dependent, in line with it being mediated by Nox5. Treatment of VSMCs with Ca 2+ loaded extracellular vesicles (EVs) lead to an increase in cytosolic Ca 2+ . Inhibiting EV endocytosis with dynasore blocked the increase in cytosolic Ca 2+ and VSMC calcification. Increased ROS production resulted in increased EV release and decreased phagocytosis by VSMCs. Conclusions: We show here that contractile VSMCs are resistant to calcification and identify Nox5 as a key regulator of VSMC phenotypic switching. Additionally, we describe a new mechanism of Ca 2+ uptake via EVs and show that Ca 2+ induces ROS production in VSMCs via Nox5. ROS production is required for release of EVs, which promote calcification. Identifying molecular pathways that control Nox5 and VSMC-derived EVs provides potential targets to modulate vascular remodelling and calcification in the context of mineral imbalance.