Exploring smooth muscle phenotype and function in a bioreactor model of abdominal aortic aneurysm

Abstract

BackgroundVascular smooth muscle cells (SMC) are central to arterial structure and function yet their involvement in the progression of abdominal aortic aneurysm (AAA) disease is not well studied. The progressive and silent nature of AAA in man essentially restricts research to the use of “end-stage” tissue recovered during surgical repair. This study aimed to generate an ex vivo model of AAA using protease-treated porcine carotid arteries maintained in a novel bioreactor, and to compare the structural and functional changes in SMC cultured from the recovered vessels with those from human tissue acquired at elective surgical repair.MethodsFreshly isolated porcine arteries were pretreated with collagenase and/or elastase before culturing under flow in a bioreactor for 12 days. Human end-stage aneurysmal tissue and saphenous veins from age-matched controls were collected from patients undergoing surgery. SMC were cultured and characterised (immunocytochemistry, measurement of spread cell area) and assessed functionally at the level of proliferation (cell-counting) and matrix-metalloproteinase (MMP) secretion (gelatin zymography). Cellular senescence was investigated using β-galactosidase staining and apoptosis was quantified using a fluorescence-based caspase 3 assay.ResultsCo-expression of alpha-smooth muscle actin and smooth muscle myosin heavy chain confirmed all cell populations as SMC. Porcine SMC harvested and cultivated after collagenase/elastase pretreatment displayed a prominent “rhomboid” morphology, increased spread area (32%, P < 0.01), impaired proliferation (47% reduction, P < 0.05), increased senescence (52%, P < 0.001), susceptibility to apoptosis and reduced MMP-2 secretion (60% decrease, P < 0.01) compared with SMC from vehicle, collagenase or elastase pre-treated vessels. Notably, these changes were comparable to those observed in human AAA SMC which were 2.4-fold larger than non-aneurysmal SMC (P < 0.001) and exhibited reduced proliferation (39% reduction, P < 0.001), greater apoptosis (4-fold increase, P < 0.001), and increased senescence (61%, P < 0.05).ConclusionsCombined collagenase/elastase exposure of porcine artery maintained in a bioreactor under flow conditions induced a SMC phenotype characteristic of those cultured from end-stage AAA specimens. This model has potential and versatility to examine temporal changes in SMC biology and to identify the molecular mechanisms leading to early aberrancies in SMC function. In the longer term this may inform new targets to maintain aortic SMC content and drive cells to a “reparative” phenotype at early stages of the disease.

Progressive Development of Aberrant Smooth Muscle Cell Phenotype in Abdominal Aortic Aneurysm Disease

“…Furthermore, we established a novel ex vivo bioreactor model whereby porcine carotid arteries (PCA) were briefly treated with a combination of collagenase and elastase (CCE) or control vehicle gel (VEH) and then maintained under flow for 12 days in a bioreactor. During this time, dilatation of the vessel was apparent and importantly CCE-SMC derived from the vessels retrieved at the end of the culture period were compatible with those of end-stage human AAA-SMC with respect to morphology and behaviour in culture [9]. There is clearly a demand for models that may help identify factors that precede aneurysm development in order to find targets for new therapeutics.…”

“…SMC isolated from end-stage human tissue exhibit diverse features in culture [7,8,9]. Recently, we described a distinct SMC phenotype isolated from end-stage human AAA tissue (aortic diameter ≥5 cm) characterised by a large rhomboid morphology, a slow proliferative rate, increased senescence, and reduced levels of secreted MMP-2 compared to SMC cultured from non-aneurysmal human saphenous vein (SV) [9].…”

“…Some of the phenotypic features of AAA-SMC that we have described, i.e., an aberrant morphology, increased senescence, and a poor proliferative capacity [9], correspond to features apparent in aged SMC [13]; however, our observation of reduced MMP-2 secretion is at variance with animal models of aging [14]. Aging is associated with an increased production of proinflammatory cytokines [15] and the development of cellular senescence, which has a multitude of effects on cell phenotype and behaviour [16].…”

“…They do this through switching between contractile (differentiated) and synthetic (dedifferentiated) phenotypes [5]. Whilst this plasticity is essential to adaptive remodelling, it is also implicated in the development of human AAA [6,7,8,9] and animal models of abdominal and thoracic aneurysm [6,10]. Undeniably the involvement of SMC in AAA progression is an area worthy of research; however, the key challenge facing investigators is that human tissue is accessible only at the end stage of the disease.…”

“…Recently, we described a distinct SMC phenotype isolated from end-stage human AAA tissue (aortic diameter ≥5 cm) characterised by a large rhomboid morphology, a slow proliferative rate, increased senescence, and reduced levels of secreted MMP-2 compared to SMC cultured from non-aneurysmal human saphenous vein (SV) [9]. Furthermore, we established a novel ex vivo bioreactor model whereby porcine carotid arteries (PCA) were briefly treated with a combination of collagenase and elastase (CCE) or control vehicle gel (VEH) and then maintained under flow for 12 days in a bioreactor.…”