Multipotent Vascular Stem Cells Research Articles

Native extracellular matrix orientation determines multipotent vascular stem cell proliferation in response to cyclic uniaxial tensile strain and simulated stent indentation

Cardiovascular disease is the leading cause of death worldwide, with multipotent vascular stem cells (MVSC) implicated in contributing to diseased vessels. MVSC are mechanosensitive cells which align perpendicular to cyclic uniaxial tensile strain. Within the blood vessel wall, collagen fibers constrain cells so that they are forced to align circumferentially, in the primary direction of tensile strain. In these experiments, MVSC were seeded onto the medial layer of decellularized porcine carotid arteries, then exposed to 10%, 1 Hz cyclic tensile strain for 10 days with the collagen fiber direction either parallel or perpendicular to the direction of strain. Cells aligned with the direction of the collagen fibers regardless of the orientation to strain. Cells aligned with the direction of strain showed an increased number of proliferative Ki67 positive cells, while those strained perpendicular to the direction of cell alignment showed no change in cell proliferation. A bioreactor system was designed to simulate the indentation of a single, wire stent strut. After 10 days of cyclic loading to 10% strain, MVSC showed regions of densely packed, highly proliferative cells. Therefore, MVSC may play a significant role in in-stent restenosis, and this proliferative response could potentially be controlled by controlling MVSC orientation relative to applied strain.

Resident multipotent vascular stem cells exhibit amplitude dependent strain avoidance similar to that of vascular smooth muscle cells

Atherosclerosis is one of the leading causes of mortality worldwide, and presents as a narrowing or occlusion of the arterial lumen. Interventions to re-open the arterial lumen can result in re-occlusion through intimal hyperplasia. Historically only de-differentiated vascular smooth muscle cells were thought to contribute to intimal hyperplasia. However recent significant evidence suggests that resident medial multipotent vascular stem cells (MVSC) may also play a role. We therefore investigated the strain response of MVSC since these resident cells are also subjected to strain within their native environment. Accordingly, we applied uniaxial 1 Hz cyclic uniaxial tensile strain at three amplitudes around a mean strain of 5%, (4–6%, 2–8% and 0–10%) to either rat MVSC or rat VSMC before their strain response was evaluated. While both cell types strain avoid, the strain avoidant response was greater for MVSC after 24 h, while VSMC strain avoid to a greater degree after 72 h. Additionally, both cell types increase strain avoidance as strain amplitude is increased. Moreover, MVSC and VSMC both demonstrate a strain-induced decrease in cell number, an effect more pronounced for MVSC. These experiments demonstrate for the first time the mechano-sensitivity of MVSC that may influence intimal thickening, and emphasizes the importance of strain amplitude in controlling the response of vascular cells in tissue engineering applications.

Multipotent vascular stem cells contribute to neurovascular regeneration of peripheral nerve

BackgroundNeurovascular unit restoration is crucial for nerve regeneration, especially in critical gaps of injured peripheral nerve. Multipotent vascular stem cells (MVSCs) harvested from an adult blood vessel are involved in vascular remodeling; however, the therapeutic benefit for nerve regeneration is not clear.MethodsMVSCs were isolated from rats expressing green fluorescence protein (GFP), expanded, mixed with Matrigel matrix, and loaded into the nerve conduits. A nerve autograft or a nerve conduit (with acellular matrigel or MVSCs in matrigel) was used to bridge a transected sciatic nerve (10-mm critical gap) in rats. The functional motor recovery and cell fate in the regenerated nerve were investigated to understand the therapeutic benefit.ResultsMVSCs expressed markers such as Sox 17 and Sox10 and could differentiate into neural cells in vitro. One month following MVSC transplantation, the compound muscle action potential (CMAP) significantly increased as compared to the acellular group. MVSCs facilitated the recruitment of Schwann cell to regenerated axons. The transplanted cells, traced by GFP, differentiated into perineurial cells around the bundles of regenerated myelinated axons. In addition, MVSCs enhanced tight junction formation as a part of the blood-nerve barrier (BNB). Furthermore, MVSCs differentiated into perivascular cells and enhanced microvessel formation within regenerated neurovascular bundles.ConclusionsIn rats with peripheral nerve injuries, the transplantation of MVSCs into the nerve conduits improved the recovery of neuromuscular function; MVSCs differentiated into perineural cells and perivascular cells and enhanced the formation of tight junctions in perineural BNB. This study demonstrates the in vivo therapeutic benefit of adult MVSCs for peripheral nerve regeneration and provides insight into the role of MVSCs in BNB regeneration.

Abstract 478: Resident Multipotent Vascular Stem Cells Isolated From Susceptible and Non-susceptible Arteriosclerotic Regions of the Mouse Aorta Are Sca1/s100β/nestin + and Respond Similarly to the Same Myogenic Inductive Stimulus

Vascular remodeling leading to arterial obstruction is a hallmark of arteriosclerosis and in-stent restenosis and is due in part to the accumulation of vascular smooth muscle (SMC)-like cells within the vessel wall. The source of these vascular cells has been controversial with many studies providing compelling evidence for a putative role for stem cell-derived myogenic progeny. It is known that neuroectoderm-derived vascular regions (ascending aorta, aortic arch, carotid artery) are more susceptible to arteriosclerotic lesion formation in comparison with mesoderm derived regions (descending and abdominal aorta, femoral artery). Our aim was to isolate and characterize stem cells from arteriosclerotic-susceptible and non-susceptible regions and determine their differential responsiveness to discrete myogenic inductive stimuli. A population of myosin heavy chain (Myh11 - ) negative, Sca1/S100β/Nestin + multipotent vascular stem cells (MVSCs) was first shown to accumulate within the intima of murine carotid arteries following injury using Sca1 and S100β e-GFP transgenic mice. Resident MVSCs were isolated from mouse aorta arch (susceptible) and descending aorta (non-susceptible) by enzymatic dissociation and initial seeding on non-adherent plates for 48 h before suspended cells were re-seeded on adherent plates and grown in B27 supplemented maintenance media. Cells were characterized by fluorescent immunocytochemistry using stem (Sca1/S100β/Nestin) and vascular SMC cell markers (CNN1 and Myh11). MVSCs from both aortic arch and descending aorta were positive for stem cell markers but negative for Myh11 and Cnn1. Treatment of both cell populations with TGF-β1 or the Notch ligand, Jagged-1 for 7 days promoted myogenic differentiation by increasing the number of cells expressing SMC markers concomitant with increased Myh11 and CNN1 mRNA levels, respectively. There was no difference in the responsiveness of stem cells from either arteriosclerotic-prone and non-prone regions. We conclude that the aortic arch and descending aortic region both house a neuroectoderm-derived Sca1/S100β/Nestin + stem cell population that responds similarly to myogenic inductive stimulation.

Abstract 480: Injury-Activated Vascular Cells Share a Common Photonic Fingerprint with Stem Cell-Derived Myogenic Progeny Following Interrogation Using a Lab-on-a-Disc (Load) Platform

The accumulation of vascular smooth muscle (SMC)-like cells within the intima contributes significantly to intimal medial thickening (IMT) and vascular remodeling typical of arteriosclerotic disease. Light has emerged as a powerful tool to interrogate cells label-free and facilitates discriminant observations both in vitro and in vivo . The auto-fluorescence (AF) profile of individual cells isolated from arteriosclerotic vessels, captured on V-cup array and interrogated across five wavelengths using a novel Lab-on-a-Disc platform, was significantly increased at the 565 ± 20nm wavelength concomitant with a reduction in Myh11 expression, when compared to differentiated vascular smooth muscle (SMC) cells from control vessels. In vitro, TGF-β1 promoted myogenic differentiation of murine bone-marrow derived Sca1 + /CD44 + mesenchymal stem cells (MSC) and murine Sca1 + C3H 10T1/2 cells concomitant with enrichment of the specific SMC epigenetic histone mark, H3K4me2 at the Myh11 promoter, Myh11 promoter transactivation and increased SMC differentiation marker mRNA and protein expression. Myogenic differentiation resulted in a significant increase in the AF intensity across 565 ± 20nm wavelength, an effect not observed for TGF-β1 treated RAMOS human B lymphocytes but mimicked by Notch activation of resident Sca1 + multipotent vascular stem cells (MVSCs) with Jagged1 and inhibited following elastin and collagen III depletion, respectively. Moreover, the temporal increase in the AF intensity at 565 ± 20nm wavelength during myogenic differentiation was similar to the AF profile of dissociated cells from arteriosclerotic vessels at this same wavelength. These data suggest that an AF photonic fingerprint of stem cell-derived myogenic progeny in vitro mimics that of vascular cells ex vivo following injury.

Abstract 648: Resident S100β + /Sca1 + Multipotent Vascular Stem Cells Undergo Myogenic and Vasculogenic Differentiation In Vitro

Arteriosclerosis is an important age-dependent disease that encompasses atherosclerosis, in-stent restenosis, autologous bypass grafting and transplant arteriosclerosis. Vascular smooth muscle (vSMC)-like accumulation is a key event leading to intimal-medial thickening (IMT), vessel remodelling and an important marker of subclinical disease. Vascular stem cell progeny in addition to de-differentiated SMC and SMC derived from endothelial-mesenchymal transition (EndoMT) are all reported to contribute to IMT as they become activated/dysfunctional, differentiate down vascular and myeloid lineages and subsequently dictate, in-part, vessel remodelling. In this study we examined the multipotent potential of a specialised population of rat adult resident multipotent vascular stem cells (rMVSC) located within the vessel wall and their capability to differentiate down both myogenic (muscle) and vasculogenic (endothelial) lineages when given the appropriate stimulus. Using Sca1-eGFP transgenic mice, in vivo , there was a significant increase in the number of Sca1 + cells within the intima of the left carotid artery (LCA) following partial carotid artery ligation-induced injury after 3 days that co-localised with endothelial nitic oxide synthase (eNOS) and CD31 + positive cells, when compared to the sham-operated control vessels and the contralateral right carotid artery (RCA). The number of Sca1-eGFP + cells significantly increased over time within the adventitial, medial and neointimal layers following ligation-induced injury after 7 and 14 days, respectively. In vitro, S100β/Sca1 + rat MVSCs cultured in vasculogenic inductive media for 7 days underwent differentiation to an endothelial cell phenotype characterised by the appearance of a cobblestone morphology and increased eNOS expression. In contrast, MVSC exposed to media supplemented with TGF-β1 for 7 days underwent myogenic differentiation to SMC. These data suggest that resident S100β/Sca1 + MVSCs are capable of both myogenic and vasculogenic differentiation depending on the inductive stimulus and may contribute in part to intimal medial thickening (IMT) and endothelial regeneration following injury in vivo.

Atherosclerosis is a vascular stem cell disease caused by insulin

The present article proposes the hypothesis that when multipotent vascular stem cells are exposed to excessive insulin in a rhythmic pattern of sharply rising and falling concentrations, their differentiation is misdirected toward adipogenic and osteogenic cell lineages. This results in plaque-like accumulation of adipocytes with fat and cholesterol deposition from adipocyte debris, and osteogenic (progenitor) cells with a calcified matrix in advanced lesions. The ingrowth of capillaries and infiltration with macrophages, which upon uptake of lipids turn into foam cells, are unspecific pro-resolving reactions. Epidemiological, histopathological, pharmacological, and experimental evidence in favour of this hypothesis is summarised.

Oxidation Prevents HMGB1 Inhibition on PDGF-Induced Differentiation of Multipotent Vascular Stem Cells to Smooth Muscle Cells: A Possible Mechanism Linking Oxidative Stress to Atherosclerosis.

Atherosclerosis is considered as a multifactorial disease in terms of the pathogenic mechanisms. Oxidative stress has been implicated in atherogenesis, and the putative mechanisms of its action include oxidative modification of redox-sensitive signaling factors. High mobility group box 1 (HMGB1) is a key inflammatory mediator in atherosclerosis, but if oxidized it loses its activity. Thus, whether and how it participates in oxidative stress-induced atherosclerosis are not clear. The current study found that exogenous HMGB1 dose-dependently inhibited the proliferation of multipotent vascular stem cells and their differentiation to smooth muscle cells induced by platelet-derived growth factor. But oxidative modification impaired the activity of HMGB1 to produce the effect. The stem cells were regarded as the source of smooth muscle cells in vascular remodeling and neointimal hyperplasia. Therefore, the findings suggested that HMGB1 participated in oxidative stress-induced atherosclerosis presumably by targeting multipotent vascular stem cells.

Cell Based Therapeutic Approach in Vascular Surgery: Application and Review.

Multipotent stem cells - such as mesenchymal stem/stromal cells and stem cells derived from different sources like vascular wall are intensely studied to try to rapidly translate their discovered features from bench to bedside. Vascular wall resident stem cells recruitment, differentiation, survival, proliferation, growth factor production, and signaling pathways transduced were analyzed. We studied biological properties of vascular resident stem cells and explored the relationship from several factors as Matrix Metalloproteinases (MMPs) and regulations of biological, translational and clinical features of these cells. In this review we described a translational and clinical approach to Adult Vascular Wall Resident Multipotent Vascular Stem Cells (VW-SCs) and reported their involvement in alternative clinical approach as cells based therapy in vascular disease like arterial aneurysms or peripheral arterial obstructive disease.

Abstract 143: Enrichment of Dimethylation of Lysine 4 on Histone 3 and Resident Vascular Stem Cell Transition to Vascular Smooth Muscle Cells

Data support a role for stem cell-derived vascular smooth muscle cells (SMC) in arteriosclerosis. Epigenetics play a critical role in SMC differentiation where histone proteins associated with the myosin heavy chain (Myh11) promoter are post-translationally modified by dimethylation of lysine 4 on histone 3 (H3K4me2). Studies report that ‘de-differentiated’ SMC do not exhibit loss of H3K4me2 at the Myh11 promoter even when mRNA levels decrease thereby allowing this modification to be used to track differentiated SMC. Our aim was to determine the level of H3K4me2 and H3K27me3 methylation of differentiated SMCs and compare to resident vascular stem cells and stem cell-derived SMC. Murine adventitial Sca1 + stem cells (APCs), rat medial Sox10 + multipotent vascular stem cells (MVSCs) and CD44 + bone-marrow derived mesenchymal stem cells (MSCs) were all examined for methylation of lysine 4 (H3K4me2) and lysine 27 (H3K27me3) on histone 3 associated with the Myh11 promoter, before and after SMC differentiation and compared to fresh aortic differentiated SMC and sub-cultured de-differentiated SMC in vitro by Chromatin Immunoprecipitation (ChIP) assay. Cells were also examined for Myh11 expression, stemness (telomerase activity) and multipotentcy. Differentiated rat and murine SMC were enriched for H3K4Me2 at the Myh11 promoter, compared to H3K27me3. Phenotypically de-differentiated sub-cutured rat and murine SMC were enriched for H3K4Me2 when compared to H3K27me3, but to a much lesser extent when compared to differentiated aortic SMC. In contrast, resident APCs and MVSCs, and bone-marrow derived MSCs, were all enriched for H3K27me3, concomitant with significant telomerase activity and multipotent differentiation capacity. The levels of enrichment of H3K27me3 dropped significantly after SMC differentiation with TGF-βeta1 concomitant with a significant enrichment of H3K4me2 to levels that mimicked the level of enrichment in de-differentiated SMC when compared to aortic differentiated SMC. De-differentiated SMC exhibit reduced enrichment of H3K4me2 at the Myh11 promoter region when compared to differentiated aortic SMCs, but mimic the level of enrichment of H3K4me2 observed following resident vascular stem cell differentiation to SMCs.

Brain pericytes serve as microglia-generating multipotent vascular stem cells following ischemic stroke.

BackgroundMicroglia are the resident macrophage population of the central nervous system (CNS) and play essential roles, particularly in inflammation-mediated pathological conditions such as ischemic stroke. Increasing evidence shows that the population of vascular cells located around the blood vessels, rather than circulating cells, harbor stem cells and that these resident vascular stem cells (VSCs) are the likely source of some microglia. However, the precise traits and origins of these cells under pathological CNS conditions remain unclear.MethodsIn this study, we used a mouse model of cerebral infarction to investigate whether reactive pericytes (PCs) acquire microglia-producing VSC activity following ischemia.ResultsWe demonstrated the localization of ionized calcium-binding adaptor molecule 1 (Iba1)-expressing microglia to perivascular regions within ischemic areas. These cells expressed platelet-derived growth factor receptor-β (PDGFRβ), a hallmark of vascular PCs. PDGFRβ+ PCs isolated from ischemic, but not non-ischemic, areas expressed stem/undifferentiated cell markers and subsequently differentiated into various cell types, including microglia-like cells with phagocytic capacity.ConclusionsThe study results suggest that vascular PCs acquire multipotent VSC activity under pathological conditions and may thus be a novel source of microglia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0523-9) contains supplementary material, which is available to authorized users.

Abstract 42: Differentiation of Human Multipotent Vascular Stem Cells Contributes to Vascular Diseases

Vascular disease, such as neointimal hyperplasia and atherosclerosis, involves in migration and proliferation of vascular cells in blood vessel wall. It is generally accepted that the de-differentiation of vascular smooth muscle cells (SMCs) contributes to vascular diseases. However, previous studies suggest that resident multipotent vascular stem cells (MVSCs) are also involved in neointima formation. In this study, we isolated MVSCs from the medial layer of human carotid artery and thoracic aorta by explant cultures, characterized these cells, and identified the origin of these vascular cells populated in lesion areas. Human MVSCs could be isolated from healthy and diseased blood vessels, were cloneable, and were positive for stem cell markers SOX10 and SOX17 but not SMC markers α-actin and calponin-1. MVSCs were able to differentiate into neural cells, SMCs and other mesenchymal lineages in vitro. In addition, we examined the location of MVSCs in diseased vascular wall by immunohistochemistry. The majority of cells within tunica media were calponin-positive cells, whereas a small population of cells was proliferative and double positive for SOX10 and Ki67 in the border between tunica intima and media. The number of SOX10+ cells in atherosclerotic lesions was more than that in healthy blood vessels, and some of these cells were double positive for SOX10 and chondrogenic markers. Furthermore, we performed in situ PCR and proximity ligation assays to stain for the epigenetic markers of SMC lineage. The results suggested that SOX10+ cells in cell cultures and tissue sections were not derived from de-differentiated SMCs. In conclusion, these findings support that MVSCs contributes to human vascular diseases.

Inhibitory role of reactive oxygen species in the differentiation of multipotent vascular stem cells into vascular smooth muscle cells in rats: a novel aspect of traditional culture of rat aortic smooth muscle cells.

Proliferative or synthetic vascular smooth muscle cells (VSMCs) are widely accepted to be mainly derived from the dedifferentiation or phenotypic modulation of mature contractile VSMCs, i.e., a phenotype switch from a normally quiescent and contractile type into a proliferative or synthetic form. However, this theory has been challenged by recent evidence that synthetic VSMCs predominantly originate instead from media-derived multipotent vascular stem cells (MVSCs). To test these hypotheses further, we re-examine whether the conventional rat aortic SMC (RASMC) culture involves the VSMC differentiation of MVSCs or the dedifferentiation of mature VSMCs and the potential mechanism for controlling the synthetic phenotype of RASMCs. We enzymatically isolated RASMCs and cultured the cells in both a regular growth medium (RGM) and a stem cell growth medium (SCGM). Regardless of culture conditions, only a small portion of freshly isolated RASMCs attaches, survives and grows slowly during the first 7days of primary culture, while expressing both SMC- and MVSC-specific markers. RGM-cultured cells undergo a process of synthetic SMC differentiation, whereas SCGM-cultured cells can be differentiated into not only synthetic SMCs but also other somatic cells. Notably, compared with the RGM-cultured differentiated RASMCs, the SCGM-cultured undifferentiated cells exhibit the phenotype of MVSCs and generate greater amounts of reactive oxygen species (ROS) that act as a negative regulator of differentiation into synthetic VSMCs. Knockdown of phospholipase A2, group 7 (Pla2g7) suppresses ROS formation in the MVSCs while enhancing SMC differentiation of MVSCs. These results suggest that cultured synthetic VSMCs can be derived from the SMC differentiation of MVSCs with ROS as a negative regulator.

Do Vascular Pericytes Contribute to Neurovasculogenesis in the Central Nervous System as Multipotent Vascular Stem Cells?

Increasing evidence suggests that multipotent stem cells are harbored within a vascular niche inside various organs. Although a precise phenotype of resident vascular stem cells (VSCs) that can function as multipotent stem cells remains unclear, accumulating evidence shows that multipotent VSCs are likely vascular pericytes (PCs) that localize within blood vessels. These PCs are multipotent, possessing the ability to differentiate into various cell types, including vascular lineage cells. In addition, brain PCs are unique: They are derived from neural crest and can differentiate into neural lineage cells. Because PCs in the central nervous system (CNS) can contribute to both neurogenesis and vasculogenesis, they may mediate the reparative process of neurovascular units that are constructed by neural and vascular cells. Here, we describe the activity of PCs when viewed as multipotent VSCs, primarily regarding their neurogenic and vasculogenic potential in the CNS. We also discuss similarities between PCs and other candidates for multipotent VSCs, including perivascular mesenchymal stem cells, neural crest-derived stem cells, adventitial progenitor cells, and adipose-derived stem cells.

Adventitial vessel growth and progenitor cells activation in an ex vivo culture system mimicking human saphenous vein wall strain after coronary artery bypass grafting.

Saphenous vein graft disease is a timely problem in coronary artery bypass grafting. Indeed, after exposure of the vein to arterial blood flow, a progressive modification in the wall begins, due to proliferation of smooth muscle cells in the intima. As a consequence, the graft progressively occludes and this leads to recurrent ischemia. In the present study we employed a novel ex vivo culture system to assess the biological effects of arterial-like pressure on the human saphenous vein structure and physiology, and to compare the results to those achieved in the presence of a constant low pressure and flow mimicking the physiologic vein perfusion. While under both conditions we found an activation of Matrix Metallo-Proteases 2/9 and of microRNAs-21/146a/221, a specific effect of the arterial-like pressure was observed. This consisted in a marked geometrical remodeling, in the suppression of Tissue Inhibitor of Metallo-Protease-1, in the enhanced expression of TGF-β1 and BMP-2 mRNAs and, finally, in the upregulation of microRNAs-138/200b/200c. In addition, the veins exposed to arterial-like pressure showed an increase in the density of the adventitial vasa vasorum and of cells co-expressing NG2, CD44 and SM22α markers in the adventitia. Cells with nuclear expression of Sox-10, a transcription factor characterizing multipotent vascular stem cells, were finally found in adventitial vessels. Our findings suggest, for the first time, a role of arterial-like wall strain in the activation of pro-pathologic pathways resulting in adventitial vessels growth, activation of vasa vasorum cells, and upregulation of specific gene products associated to vascular remodeling and inflammation.

Hedgehog and Resident Vascular Stem Cell Fate.

The Hedgehog pathway is a pivotal morphogenic driver during embryonic development and a key regulator of adult stem cell self-renewal. The discovery of resident multipotent vascular stem cells and adventitial progenitors within the vessel wall has transformed our understanding of the origin of medial and neointimal vascular smooth muscle cells (SMCs) during vessel repair in response to injury, lesion formation, and overall disease progression. This review highlights the importance of components of the Hh and Notch signalling pathways within the medial and adventitial regions of adult vessels, their recapitulation following vascular injury and disease progression, and their putative role in the maintenance and differentiation of resident vascular stem cells to vascular lineages from discrete niches within the vessel wall.

Adult vascular wall resident multipotent vascular stem cells, matrix metalloproteinases, and arterial aneurysms.

Evidences have shown the presence of multipotent stem cells (SCs) at sites of arterial aneurysms: they can differentiate into smooth muscle cells (SMCs) and are activated after residing in a quiescent state in the vascular wall. Recent studies have implicated the role of matrix metalloproteinases in the pathogenesis of arterial aneurysms: in fact the increased synthesis of MMPs by arterial SMCs is thought to be a pivotal mechanism in aneurysm formation. The factors and signaling pathways involved in regulating wall resident SC recruitment, survival, proliferation, growth factor production, and differentiation may be also related to selective expression of different MMPs. This review explores the relationship between adult vascular wall resident multipotent vascular SCs, MMPs, and arterial aneurysms.

Adult vascular smooth muscle cells in culture express neural stem cell markers typical of resident multipotent vascular stem cells

Differentiation of resident multipotent vascular stem cells (MVSCs) or de-differentiation of vascular smooth muscle cells (vSMCs) might be responsible for the SMC phenotype that plays a major role in vascular diseases such as arteriosclerosis and restenosis. We examined vSMCs from three different species (rat, murine and bovine) to establish whether they exhibit neural stem cell characteristics typical of MVSCs. We determined their SMC differentiation, neural stem cell marker expression and multipotency following induction in vitro by using immunocytochemistry, confocal microscopy, fluorescence-activated cell sorting analysis and quantitative real-time polymerase chain reaction. MVSCs isolated from rat aortic explants, enzymatically dispersed rat SMCs and rat bone-marrow-derived mesenchymal stem cells served as controls. Murine carotid artery lysates and primary rat aortic vSMCs were both myosin-heavy-chain-positive but weakly expressed the neural crest stem cell marker, Sox10. Each vSMC line examined expressed SMC differentiation markers (smooth muscle α-actin, myosin heavy chain and calponin), neural crest stem cell markers (Sox10(+), Sox17(+)) and a glia marker (S100β(+)). Serum deprivation significantly increased calponin and myosin heavy chain expression and decreased stem cell marker expression, when compared with serum-rich conditions. vSMCs did not differentiate to adipocytes or osteoblasts following adipogenic or osteogenic inductive stimulation, respectively, or respond to transforming growth factor-β1 or Notch following γ-secretase inhibition. Thus, vascular SMCs in culture express neural stem cell markers typical of MVSCs, concomitant with SMC differentiation markers, but do not retain their multipotency. The ultimate origin of these cells might have important implications for their use in investigations of vascular proliferative disease in vitro.

Engineering micropatterned surfaces to modulate the function of vascular stem cells

Multipotent vascular stem cells have been implicated in vascular disease and in tissue remodeling post therapeutic intervention. Hyper-proliferation and calcified extracellular matrix deposition of VSC cause blood vessel narrowing and plaque hardening thereby increasing the risk of myocardial infarct. In this study, to optimize the surface design of vascular implants, we determined whether micropatterned polymer surfaces can modulate VSC differentiation and calcified matrix deposition. Undifferentiated rat VSC were cultured on microgrooved surfaces of varied groove widths, and on micropost surfaces. 10μm microgrooved surfaces elongated VSC and decreased cell proliferation. However, microgrooved surfaces did not attenuate calcified extracellular matrix deposition by VSC cultured in osteogenic media conditions. In contrast, VSC cultured on micropost surfaces assumed a dendritic morphology, were significantly less proliferative, and deposited minimal calcified extracellular matrix. These results have significant implications for optimizing the design of cardiovascular implant surfaces.

Embryonic rat vascular smooth muscle cells revisited - a model for neonatal, neointimal SMC or differentiated vascular stem cells?

BackgroundThe A10 and A7r5 cell lines derived from the thoracic aorta of embryonic rat are widely used as models of non-differentiated, neonatal and neointimal vascular smooth muscle cells in culture. The recent discovery of resident multipotent vascular stem cells within the vessel wall has necessitated the identity and origin of these vascular cells be revisited. In this context, we examined A10 and A7r5 cell lines to establish the similarities and differences between these cell lines and multipotent vascular stem cells isolated from adult rat aortas by determining their differentiation state, stem cell marker expression and their multipotency potential in vitro.MethodsVascular smooth muscle cell differentiation markers (alpha-actin, myosin heavy chain, calponin) and stem cell marker expression (Sox10, Sox17 and S100β) were assessed using immunocytochemistry, confocal microscopy, FACS analysis and real-time quantitative PCR.ResultsBoth A10 and A7r5 expressed vascular smooth muscle differentiation, markers, smooth muscle alpha - actin, smooth muscle myosin heavy chain and calponin. In parallel analysis, multipotent vascular stem cells isolated from rat aortic explants were immunocytochemically myosin heavy chain negative but positive for the neural stem cell markers Sox10+, a neural crest marker, Sox17+ the endoderm marker, and the glia marker, S100β+. This multipotent vascular stem cell marker profile was detected in both embryonic vascular cell lines in addition to the adventitial progenitor stem cell marker, stem cell antigen-1, Sca1+. Serum deprivation resulted in a significant increase in stem cell and smooth muscle cell differentiation marker expression, when compared to serum treated cells. Both cell types exhibited weak multipotency following adipocyte inductive stimulation. Moreover, Notch signaling blockade following γ-secretase inhibition with DAPT enhanced the expression of both vascular smooth muscle and stem cell markers.ConclusionsWe conclude that A10 and A7r5 cells share similar neural stem cell markers to both multipotent vascular stem cells and adventitial progenitors that are indicative of neointimal stem-derived smooth muscle cells. This may have important implications for their use in examining vascular contractile and proliferative phenotypes in vitro.