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.

Type 2 diabetes (T2DM) promotes premature atherosclerosis and inferior prognosis after arterial reconstruction. Vascular smooth muscle cells (SMC) respond to patho/physiological stimuli, switching between quiescent contractile and activated synthetic phenotypes under the control of microRNAs (miRs) that regulate multiple genes critical to SMC plasticity. The importance of miRs to SMC function specifically in T2DM is unknown. This study was performed to evaluate phenotype and function in SMC cultured from non-diabetic and T2DM patients, to explore any aberrancies and investigate underlying mechanisms. Saphenous vein SMC cultured from T2DM patients (T2DM-SMC) exhibited increased spread cell area, disorganised cytoskeleton and impaired proliferation relative to cells from non-diabetic patients (ND-SMC), accompanied by a persistent, selective up-regulation of miR-143 and miR-145. Transfection of premiR-143/145 into ND-SMC induced morphological and functional characteristics similar to native T2DM-SMC; modulating miR-143/145 targets Kruppel-like factor 4, alpha smooth muscle actin and myosin VI. Conversely, transfection of antimiR-143/145 into T2DM-SMC conferred characteristics of the ND phenotype. Exposure of ND-SMC to transforming growth factor beta (TGFβ) induced a diabetes-like phenotype; elevated miR-143/145, increased cell area and reduced proliferation. Furthermore, these effects were dependent on miR-143/145. In conclusion, aberrant expression of miR-143/145 induces a distinct saphenous vein SMC phenotype that may contribute to vascular complications in patients with T2DM, and is potentially amenable to therapeutic manipulation.

Lipoprotein(a) (Lp(a)) is an independent risk factor for the development of cardiovascular disease (CVD). Indeed, individuals with plasma concentrations >20 mg/dL carry a 2-fold increased risk of developing CVD, accounting for ~25% of the population. Circulating levels of Lp(a) are remarkably resistant to common lipid lowering therapies, and there are currently no robust treatments available for reduction of Lp(a) apart from plasma apheresis, which is costly and labour intensive. The Lp(a) molecule is composed of two parts, an LDL/apoB-100 core and a unique glycoprotein, apolipoprotein(a) (apo(a)), both of which can interact with components of the coagulation cascade, inflammatory pathways, and cells of the blood vessel wall (smooth muscle cells (SMC) and endothelial cells (EC)). Therefore, it is of key importance to determine the molecular pathways by which Lp(a) exerts its influence on the vascular system in order to design therapeutics to target its cellular effects. This paper will summarise the role of Lp(a) in modulating cell behaviour in all aspects of the vascular system including platelets, monocytes, SMC, and EC.

Madi HA, Riches K, Warburton P, O'Regan DJ, Turner NA, Porter KE. Inherent differences in morphology, proliferation, and migration in saphenous vein smooth muscle cells cultured from nondiabetic and Type 2 diabetic patients. Am J Physiol Cell Physiol 297: C1307-C1317, 2009. First published September 9, 2009 doi:10.1152/ajpcell.00608.2008.-Individuals with Type 2 diabetes mellitus (T2DM) are at increased risk of saphenous vein (SV) graft stenosis following coronary artery bypass. Graft stenosis is caused by intimal hyperplasia, a pathology characterized by smooth muscle cell (SMC) proliferation and migration. We hypothesized that SV-SMC from T2DM patients were intrinsically more proliferative and migratory than those from nondiabetic individuals. SV-SMC were cultured from nondiabetic and T2DM patients. Cell morphology (light microscopy, immunocytochemistry), S100A4 expression (real-time RT-PCR, immunoblotting), proliferation (cell counting), migration (Boyden chamber assay), and cell signaling (immunoblotting with phosphorylation state-specific antibodies) were studied. SV-SMC from T2DM patients were morphologically distinct from nondiabetic patients and exhibited a predominantly rhomboid phenotype, accompanied by disrupted F-actin cytoskeleton, disorganized ␣-smooth muscle actin network, and increased focal adhesion formation. However, no differences were observed in expression of the calcium-binding protein S100A4, a marker of rhomboid SMC phenotype, between the two cell populations. T2DM cells were less proliferative in response to fetal calf serum than nondiabetic cells, but both populations had similar proliferative responses to insulin plus PDGF. Under high glucose concentration conditions in the presence of insulin, migration of diabetic SV-SMC was greater than nondiabetic cells. Glucose concentration did not affect SV-SMC proliferation. No differences in insulin or PDGF-induced phosphorylation of ERK-1/2 or components of the Akt pathway (Akt-Ser473, Akt-Thr308, and GSK-3␤) were apparent between the two populations. In conclusion, SV-SMC from T2DM patients differ from nondiabetic SV-SMC in that they exhibit a rhomboid phenotype and are more migratory, but less proliferative, in response to serum. diabetes mellitus; vein graft stenosis; metabolic memory DIABETES MELLITUS IS A SERIOUS and escalating health problem worldwide (3) that currently affects more than 2

AimThe aim of the study was to determine the potential for KV1 potassium channel blockers as inhibitors of human neoinitimal hyperplasia.Methods and resultsBlood vessels were obtained from patients or mice and studied in culture. Reverse transcriptase–polymerase chain reaction and immunocytochemistry were used to detect gene expression. Whole-cell patch-clamp, intracellular calcium measurement, cell migration assays, and organ culture were used to assess channel function. KV1.3 was unique among the KV1 channels in showing preserved and up-regulated expression when the vascular smooth muscle cells switched to the proliferating phenotype. There was strong expression in neointimal formations. Voltage-dependent potassium current in proliferating cells was sensitive to three different blockers of KV1.3 channels. Calcium entry was also inhibited. All three blockers reduced vascular smooth muscle cell migration and the effects were non-additive. One of the blockers (margatoxin) was highly potent, suppressing cell migration with an IC50 of 85 pM. Two of the blockers were tested in organ-cultured human vein samples and both inhibited neointimal hyperplasia.ConclusionKV1.3 potassium channels are functional in proliferating mouse and human vascular smooth muscle cells and have positive effects on cell migration. Blockers of the channels may be useful as inhibitors of neointimal hyperplasia and other unwanted vascular remodelling events.