Vascular smooth muscle cells vsmcs are the cellular components of the normal. Phenotypic modulation of primary vascular smooth muscle. Double the smooth muscle cell growth medium volume when the culture is 60% confluent or for weekend feedings. Technical information for working with human aortic smooth muscle cells including thawing. This study investigated the potential role of sirt7 in regulating the proliferation and migration of human vascular smooth muscle cells havsmcs and its possible molecular mechanism. Human vascular smooth muscle cells vsmcs in culture are an important tool in understanding how vsmcs function and contribute to vessel wall contraction as well as disease. Calcification of vascular smooth muscle cells and imaging. Smooth musclealpha actin inhibits vascular smooth muscle. Generating the wg59 cell line from primary vsmc culture to generate a human ascending aorta vascular smooth muscle cell line with an extended life span, primary smooth muscle cells isolated from the biopsy material were transfected with a. Vascular smooth muscle cells vsmcs play a crucial role in both the pathogenesis of aneurysms and dissections of the ascending thoracic aorta taad in humans and in the associated adaptive. Smooth muscle cell culture thermo fisher scientific us. Cells and tissues were fixed and stained using a standard protocol.
Vascular smooth muscle cells vsmc play a critical role in 3 aortic aneurysm hallmark processes. Keywords atherosclerosis, coculture, macrophage, smooth muscle cell, inflammation 1surgical services, va palo alto health. Pkm2dependent glycolysis promotes the proliferation and. Vascular smooth muscle cell isolation and culture from mouse aorta. Human aortic smooth muscle cells culture sigmaaldrich. Ray jl, leach r, herbert jm, benson m 2001 isolation of vascular smooth muscle cells from a single murine aorta. Selective expression of tspan2 in vascular smooth muscle.
Human vascular smooth muscle cell culture springerlink. In this chapter, we describe methodologies that enable the investigator to culture large numbers of proliferative vsmcs. The excessive proliferation and migration of abnormal vascular smooth muscle cells vsmcs are the major causes of atherogenesis 1. Vascular smooth muscle cells vsmcs are the cellular components of the normal blood vessel wall that provides structural integrity and regulates the diameter by contracting and relaxing. Human cell culture protocols, methods in molecular biology, vol. In vitro cultivation of vascular smooth muscle cells.
Herein, we describe a new reproducible method for isolating vsmcs, endothelial cells, and fibroblasts of high viability from fetal rat da using immunomagnetic cell. Despite available treatments, cvd prevalence continues, suggesting an urgent need to identify the pathogenic molecular mechanisms and develop effective therapeutic approaches. Isolation and culture of fibroblasts, vascular smooth muscle, and endothelial cells from the fetal rat ductus arteriosus skip to main content thank you for visiting. However, in contrast to the response of medial smooth muscle cells, neointimal smooth muscle cells proved to be remarkably resistant to the induction of cell death by distention injury. Isolation and culture of vascular smooth muscle cells. The pulsatile nature of blood flow exposes vascular smooth muscle cells vsmcs in the vessel wall to mechanical stress, in the form of circumferential and longitudinal stretch. Acute type a aortic dissection taad is a lifethreatening vascular disease. Proliferation of vascular smooth muscle cells vsmcs is a central axiom of most models of atherosclerosis, promoting atherogenesis as a response to injury 1 or inflammation. If there are 20% cells left in the flask, repeat steps 29. How can i quantify smooth muscle contraction in vitro. Initially in embryonic vasculature development, endothelial precursor cells form a common progenitor vessel which then gives rise to the first artery dorsal aorta and vein cardinal vein by selective sprouting and subsequent arterialvenous cell segregation 4 see also chapter 1. The goal of this method is to isolate vascular smooth muscle cells vsmcs from the murine coronary circulation for use in cell culture and standard cell culture assays.
Isolation and culture of smooth muscle cells from human. Building blood vessels with vascular progenitor cells. The right choice of endothelial cells for your vascular. Smooth muscle tissue is found in the tunica media layer of. Development of an optimized protocol for primary culture of smooth muscle cells from rat thoracic aortas. Primary culture of smooth muscle cells smcs is an established method in the study of vascular physiology and pathophysiology 1 2 3. In this chapter, we will discuss in detail the explant method for isolation of.
Determinants of vascular smooth muscle cell apoptosis. Induced osteogenic differentiation of human smooth muscle cells as a model of vascular calcification. Monocyte chemotactic protein1 regulates proliferation and contractility of human bladder smooth muscle cells under hydrostatic pressure. Isolation of murine coronary vascular smooth muscle cells. To what extent sma regulates migration and proliferation of smcs is unclear and putative signaling pathways involved remain. We examined the effect of hypoxia on proliferation and osteopontin opn expression in cultured rat aortic vascular smooth muscle vsm cells. Currently, tissue explants and enzymatic digestion methods are frequently applied to produce smooth muscle cells. These discrepancies are likely due to several factors related to vsmcs, such as the vascular bed from where cells were isolated, age of the animal, cell population number, cell phenotype in culture and whether cells are stimulated or not.
The vascular smooth muscle cell in arterial pathology. Vascular injury induces cellular responses which lead to clinical events, including atherosclerosis, hypertension and restenosis. Generation and characterization of vascular smooth muscle. Accelerated cerebral vascular injury in diabetes is associated with vascular smooth muscle cell dysfunction.
In this study, pulmonary artery smooth muscle cells hpasmcs isolated from human pulmonary arteries of healthy donors were used innoprot, spain. Vsmcs, found in the tunica media, are terminally undifferentiated cells, in that they alter their phenotype based on the surrounding microenvironment. In addition, we determined whether hypoxiainduced increases in opn and cell proliferation are altered under hyperglycemic conditions. The vascular smooth muscle cell vsmc is the effector cell for the regulation of vessel tone. Specification and diversification of pericytes and smooth. The method described in this chapter employs enzymatic dissociation to isolate vascular smooth muscle cells for primary cell cultures. Smooth muscle alphaactin sma is a marker for the contractile, nonproliferative phenotype of adult smooth muscle cells smcs. Cells were cultured in hpasmc basal medium supplemented with endothelial cell growth supplement. Change smooth muscle cell growth medium every other day until the cells reach 60% confluency. One common feature of vascular lesions is the migration of vascular smooth muscle cells vsmcs. Diagram 1 is an illustration of some of the basic steps used to establish a primary cell culture14. Growth of smooth muscle in culture and formation of elastic fibers. Antioxidant activity mediates pirfenidone antifibrotic.
A wide variety of primary smooth muscle cells from different tissues, species, and vascular bed smooth muscle are offered. Culture of vascular smooth muscle cells from small. Vascular smooth muscle cells vsmcs, in addition to endothelial cells ecs and fibroblasts, are one of the three main cell types that compose the blood vessel wall 1, 2. Isolation and culture of fibroblasts, vascular smooth muscle, and. Origins of vascular smooth muscle cells during embryonic development. Pdf human vascular smooth muscle cell culture researchgate. The mouse is critical to basic cardiovascular research, largely because techniques for genetic manipulation are more fully developed in the mouse than in any othermammalian species. Design and utilization of macrophage and vascular smooth. Quiescent cultures of vsm cells were exposed to hypoxia 3% o2 or normoxia 18% o2 in a serumfree medium. The pulsatile nature of blood flow exposes vascular smooth muscle cells vsmcs in the vessel wall to cyclic mechanical stretch cms, which evokes vsmc proliferation, cell death, phenotypic. Selective expression of tspan2 in vascular smooth muscle is independently regulated by tgfb1smad and myocardinserum response factor jinjing zhao,1 wen wu,1 wei zhang, yao wei lu, emiley tou, jiemei ye, ping gao, david jourdheuil, harold a. Chemokines protect vascular smooth muscle cells from cell. Deletion of bmal1 in smooth muscle cells protects mice.
Vascular smooth muscle cell marker tissue and cell culture. These vascular progenitors share the ability to generate some of the vascular lineages, including endothelial cells, smooth muscle cells, and pericytes. Using the explant technique, we were able to obtain only 7501500 cells per murine aorta after two weeks in culture. Cyclic stretch evokes vsmc proliferation, apoptosis, phenotypic switching. Balloon distention injury induced medial smooth muscle cell apoptosis within hours after vascular injury. Vascular smooth muscle cell isolation and culture from. Serum deprivation of vascular smooth muscle cells enhances collagen gene expression received for publication, march 16, 1988 mark s. Development of an optimized protocol for primary culture of smooth. We describe a protocol for isolation and culture of rodent smooth muscle cells derived from the aorta andor mesenteric artery.
Development of an optimized protocol for primary culture. There is no proper cell culture system allowing differentiation of vsmcs so that it is difficult to delineate the molecular mechanism responsible for proliferative vasculopathy. Pdf human vascular smooth muscle cells vsmcs in culture are an. The vascular smooth muscle cell plays a significant role in many important cardiovascular disorders, and smooth muscle biology is therefore important to cardiovascular research. Vascular smooth muscle cell dysfunction is a major factor contributing to the devel. Upon arterial injury, expression of sma and other structural proteins decreases and smcs acquire a promigratory and proliferative phenotype. Hi, i have been culturing a mixed culture of enteric neurons and smooth muscle cells for an experiment. Isolation of vascular smooth muscle cells from a single. Jci uhrf1 epigenetically orchestrates smooth muscle cell. The right choice of endothelial cells for your vascular research. Taad tissues were obtained from acute taad patients who. Distinct roles of e2f proteins in vascular smooth muscle cell proliferation and intimal hyperplasia.
The first involves explanting vascular tissue and then allowing time for smooth muscle cells to migrate out from the explanted tissue fragments 4. Loss of contractility and acquisition of an epithelial phenotype of vascular smooth muscle cells vsmcs are key events in proliferative vascular pathologies such as atherosclerosis and postangioplastic restenosis. Smcs play important roles in cardiovascular development and aortic aneurysm formation. All smooth muscle cells are high purity, low passage. One involves direct isolation of cells from enzymati cally digested vessels enzymatic isolation method, and the other involves.
Rat aortic smooth muscle cells raosmc provide an excellent model system to study all aspects of cardiovascular function and disease, especially those related to mechanisms of hyperplasia and hypertrophy of intimal smooth muscle cells leading to vascular occlusion in. The potential therapeutic uses for vascular progenitor cells are broad and relate to stroke, ischemic. Vascular smooth muscle cells smc in the ascending thoracic aorta arise from neural crest cells, whereas smcs in the descending aorta are derived from the presomitic mesoderm. Recent studies have shown that enhanced glycolysis is involved in plateletderived growth factorinduced vsmc proliferation and migration 2,3. Vascular smooth muscle cells line the vessel walls of large and small arteries, arterioles and veins. The monolayer culture was passaged by standard trypsin dispersion and resuspended in culture medium. Change to fresh smooth muscle cell growth medium after 24 hours or overnight to remove all traces of dmso.
Induced osteogenic differentiation of human smooth muscle. Vascular progenitor cells have been identified from perivascular cell fractions and peripheral blood and bone marrow mononuclear fractions. This protocol describes the detailed process for explanting ascending and descending smcs from mouse aortic tissue. Calcification of vascular smooth muscle cells and imaging of aortic calcification and inflammation. Renal vascular tone and hence hemodynamics are primarily controlled by the immediate action of a variety of vasoactive hormones 1.
Vsmcs are mostly found in the media layer of normal arteries. The aim of this work was to investigate whether the smcs of acute taad could be isolated and cultured for further research. I used antialpha smooth muscle actin antibody do any one know other than this. Vascular smooth muscle cells vsmcs are the cellular components of the normal blood vessel wall that provides structural integrity and regulates the diameter by contracting and relaxing dynamically in response to vasoactive stimuli. Vascular smooth muscle refers to the particular type of smooth muscle found within, and composing the majority of the wall of blood vessels. Each cell type has an important role in vascular homeostasis.
Vascular smooth muscle is innervated primarily by the sympathetic nervous system through adrenergic receptors adrenoceptors. Variability in vascular smooth muscle cell stretchinduced. Blood vessel walls are comprised primarily of endothelial cells ec and vascular smooth muscle cells vsmc. Coculture of endothelial cells and smooth muscle cells. It is well established that arterioles as well as small arteries participate in the regulation of hemodynamics in various nonrenal vascular beds 2, 3. Vascular smooth muscle cells in the pathogenesis of vascular calcification. Primary human aortic smooth muscle cells hasmcprimary human coronary artery smooth muscle cells hcasmcprimary human pulmonary artery smooth muscle cells hpasmc 500,000 cellsvial, cryopreserved at the end of 3 culture 70% viability. We developed this technique to assess the molecular mechanisms of vascular remodeling in diabetes. Smooth muscle cells play a critical role in cardiac and vascular diseases, including stroke and atherosclerosis. Human umbilical vein cells huvecs are easy to access, so they have been the major source of.
Subodh verma, variability in vascular smooth muscle cell stretchinduced responses in 2d culture, vascular cell, 2015, 7. Hi, am looking for very specific marker for vascular smooth muscle cell marker. Isolation and culture of vascular smooth muscle cells manual of. The journal of biological chemistry 0 19% by the american society for biochemistry and molecular biology, inc. The vascular smooth muscle cell growth kit contains components that when added to vascular cell basal medium atcc pcs30 create a complete cell culture environment for smooth muscle cells isolated from large vessels. Smooth muscle cells smcs are the main composition of aortic media and dysfunction of smcs may lead to acute taad. In this study, human vascular smooth muscle cells havsmcs were induced by oxidized lowdensity lipoprotein oxldl to establish atherosclerosis as cell model.
The final concentration for each component in complete vascular smooth muscle cell growth medium is as follows. Primary cell culture growth requirements primary cells, except for those derived from peripheral blood, are anchoragedependent, adherent. Vascular smooth muscle cells vsmcs are the only cell type present in the blood vessel wall of mammalian arteries. Jinfu lu, jiaojun wei, xiaohong li, micropatterned coculture of vascular endothelial and smooth muscle cells on layered electrospun fibrous mats toward blood vessel. Magnobovatol inhibits smooth muscle cell migration by.
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