Culture of quiescent arterial smooth muscle cells in a defined serum-free medium

An ideal medium for metabolic studies would maintain cultured vascular smooth muscle cells in a quiescent, viable state, as they are in normal arteries in vivo, and would be chemically defined so that the concentrations of hormones and nutrients could be manipulated precisely. In unsupplemented serum-free media these cultures lose protein and DNA, indicating impaired viability. Addition of maximally effective concentrations of insulin (10^?6 M) and transferrin (5 m?g/ml) prevents loss of DNA and produces near neutral protein balance. Further addition of ascorbic acid (10^?4 M) actually promotes net gain of protein with little or no increase in DNA. Ascorbate consistently increased noncollagen protein synthesis by cultured aortic smooth muscle cells. This novel action of the vitamin did not require insulin but was additive to the effect of this hormone, and was produced by isoascorbate, but not by a variety of other reducing agents. Thus, vascular smooth muscle cells can be maintained in a quiescent but noncatabolic state in simple chemically defined culture media. This finding should facilitate studies of the effects of nutrients and hormones on the metabolism of these cells under conditions that resemble those in the normal artery in vivo. Such an approach may also prove valuable for culture of other differentiated cell types that do not usually divide in the intact organism.     

Adult human arterial smooth muscle cells in primary culture Modulation from contractile to synthetic phenotype

Summary Smooth muscle cells were isolated enzymatically from adult human arteries, grown in primary culture in medium containing 10% whole blood serum, and studied by transmission electron microscopy and [³H]thymidine autoradiography. In the intact arterial wall and directly after isolation, each smooth muscle cell had a nucleus with a wide peripheral zone of condensed chromatin and a cytoplasm dominated by myofilament bundles with associated dense bodies. After 1–2 days of culture, the cells had attached to the substrate and started to spread out. At the same time, a characteristic fine-structural modification took place. It included nuclear enlargement, dispersion of the chromatin and formation of large nucleoli. Moreover, myofilament bundles disappeared and an extensive rough endoplasmic reticulum and a large Golgi complex were organized in the cytoplasm. This morphological transformation of the cells was completed in 3–4 days. It was accompanied by initiation of DNA replication and mitosis.The observations demonstrate that adult human arterial smooth muscle cells, when cultivated in vitro, pass through a phenotypic modulation of the same type as arterial smooth muscle cells from experimental animals. This modulation gives the cells morphological and functional properties resembling those of the modified smooth muscle cells found in fibroproliferative lesions of atherosclerosis. Further studies of the regulation of smooth muscle phenotype and growth may provide important clues for a better understanding of the pathogenesis of atherosclerosis.     

Replication of cytomegalovirus in human arterial smooth muscle cells.

Cytomegalovirus (CMV) strain AD-169 replicated in smooth muscle cell (SMC) cultures derived from human umbilical arteries, producing enveloped infectious virions. However, unlike the effects of CMV on fully permissive human lung fibroblasts, the effects of strain AD-169 on SMC cultures were delayed and prolonged, resulting in extended survival of a fraction of the starting population. This period of survival did not exceed the life-span of the control SMC cultures. Infectious CMV continued to be isolated from the surviving SMC cultures after extinction of the original inoculum by dilution and after treatment of the cultures with CMV neutralizing antibody. The implications of these findings for the pathogenesis of atherosclerosis are discussed.     

The characterization of human uterine smooth muscle cells in culture

Primary cultures initiated from normal human uterine endometrium after total enzymatic dissociation contained epithelioid cells and smooth muscle cells. The smooth muscle cells were subsequently isolated by differential trypsinization and grown in culture for 36 +/- 4 generations. Ultrastructural examination of log and post-confluent cultures of cells at low and high population doubling levels revealed characteristics similar to those of published reports on other smooth muscle cells studied in vivo and in vitro. Among the common features present were: (a) abundant bundles of 60--70 A myofilaments; (b) branched mitochondria; (c) stacks of cisternae of rough endoplasmic reticulum; (d) caveolae intracellulares; (e) nexuses. Other features included ovoid nuclei, a well developed Golgi apparatus and abundant free ribosomes. The subcultured cells exhibited features of dedifferentiation in the log phase of growth and at post-confluency. However, the post-confluent cells showed characteristics indicating redifferentiation back towards their in vivo morphology. Smooth muscle cells isolated from endometrial curettings may provide a useful model for biochemical and pharmacological studies of a cell type derived from a hormonal target tissue as the cells "age" in culture.     

Oxidized low-density lipoprotein is chemotactic for arterial smooth muscle cells in culture

The effects of human native and Cu2(+)-oxidized low-density lipoprotein (LDL) were tested on the migration of cultured bovine aortic smooth muscle cells (SMCs) in blind-well chambers. LDL oxidation was controlled by measuring the formation of conjugated dienes and lipid hydroperoxides, and by agarose gel electrophoresis. Oxidized LDL stimulated SMC migration, and the effect was dose-dependent up to 200 microgram/ml. The stimulation was chemotactic in nature. Native LDL was without significant activity. The results suggest that oxidized LDL may contribute to the migration of medial SMCs into the intima during atherogenesis.     

Isolation and culture of human intestinal smooth muscle cells

Intestinal smooth muscle cells were isolated from human bowel and maintained in culture through several passages. These cells were obtained by enzyme digestion of slices taken from the circular layer of the muscularis propria of human jejunum. When subcultured, they initially flattened out and then began proliferating after 3 days. After 3 weeks in culture, they began aggregating into ridges. Fluorohistochemical staining revealed numerous prominent actin stress fibers. When these cells were exposed to the C-terminal octapeptide of cholecystokinin they contracted in a dose-dependent fashion. The availability of human intestinal smooth muscle cells in culture will considerably enhance our ability to study the contractile, proliferative and connective tissue responses of the smooth muscle of the human gastrointestinal tract.     

The proteome and secretome of human arterial smooth muscle cells

Smooth muscle cells (SMCs) play a crucial role in cardiovascular disorders. A differential proteomic approach should help to elucidate SMC dysfunctions involved in these diseases. With this goal in mind, we plotted the first 2-dimensional (2-D) maps of the proteome and secretome of human arterial smooth muscle cell (ASMC). Intracellular and secreted proteins were extracted from a primary culture of SMCs obtained from patients undergoing coronary artery bypass surgery (n = 11) and separated by 2-dimensional gel electrophoresis. Silver-stained gels were analyzed using Progenesis software. A high level of between-gel reproducibility was obtained, allowing us to generate two protein patterns specific to the ASMC proteome and secretome, respectively. A total of 121 and 40 distinct intracellular and secreted polypeptide spots, corresponding to 83 and 18 different proteins, respectively, were identified by matrix-assisted laser desorption/ionization mass spectrometry. The 2-D reference maps and database resulting from this study confirm that SMCs are involved in a wide range of biological functions. They could constitute a useful tool for a wide range of investigators involved in vascular biology, allowing them to investigate SMC protein changes associated with cardiovascular disorders or environmental stimuli.     

Lipoprotein uptake by cultured human arterial smooth muscle cells.

Human arterial smooth muscle cells growing in tissue culture, in contrast to rat cells, preferentially bind and take up large, lipid-rich lipoproteins (125I-labeled low density and very low density lipoproteins) in comparison to the known difference in the propensity of these two species to develop atherosclerosis.     

Properties and subcellular localization of adenosine diphosphatase in arterial smooth muscle cells in culture

The properties and subcellular localization of adenosine diphosphatase (ADPase) activity in smooth muscle cells cultured from pig aortas have been investigated. The pH optimum of ADPase activity was 7.3 and the apparent K_m for ADP was 10.3 μM. ADPase activity was inhibited completely by EDTA and was restored by the addition of divalent cations. The enzyme activity was not inhibited by 2-glycerophosphate, a substrate for non-specific phosphatases, nor by levamisole, a specific inhibitor of alkaline phosphatase. Smooth muscle cells were homogenized and a post-nuclear supernatant was applied to a sucrose density gradient in a Beaufay automatic zonal rotor. The distribution of ADPase activity in the density gradient was similar to that of 5′-nucleotidase activity, a marker enzyme for the plasma membrane, and distinct from the distributions of the marker enzymes for the other organelles. When the cells were homogenized in the presence of digitonin, an agent which binds to cholesterol and increases the equilibrium density of the plasma membrane, the modal equilibrium densities of ADPase activity and of 5′-nucleotidase activity were increased to similar extents, thus confirming the plasma membrane localization of ADPase activity.     

Prostaglandin 12 reduces activation of human arterial smooth muscle cells in-vivo

Patients suffering from peripheral vascular disease have been “ultima ratio”-treated with PGI2 at a rate of 5 ng/kg/min for 6 hours a day and 5 consecutive days i.v. 20 of them underwent surgery thereafter as therapy was not sufficient. A histological examination and quantification of vascular tissue revealed that the number of activated smooth muscle cells was significantly lower in treated patients vascular segments than in untreated ones in all the different age groups. A comparable suppression was found in the intima and the media as well. It is thus concluded, that PGI2 inhibits smooth muscle cell proliferation most probably by inhibiting PDGF-release from the platelets and stimulation of smooth muscle cell cAMP. To achieve a more beneficial PGI2-effect at the vascular level, a prolonged PGI2-therapy looks rather promising.     

Properties and subcellular localization of elastase-like activities of arterial smooth muscle cells in culture

The interaction of cardiolipin with Ca2+ was assessed by measuring the cardiolipin-mediated extraction of 45Ca2+ from an aqueous to an organic (methylene chloride) phase. Cardiolipin binds Ca2+ with high affinity [Kd(apparent) = 0.70 +/- 0.17 microM (S.D.)]. Cation-cardiolipin interactions are selective. Interaction of cardiolipin with Ca2+ is insensitive to Na+, but is inhibited by divalent cations with Mn2+ greater than Zn2+ greater than Mg2+. In addition La3+ and Ruthenium red are particularly potent inhibitors of Ca2+ binding by cardiolipin. Cardiolipin-mediated extraction of Ca2+ into an aqueous phase is also inhibited by phosphatidylcholine. Inhibition of Ca2+-cardiolipin interaction by phosphatidylcholine (a phospholipid known to stabilize the bilayer conformation) may implicate inverted, non-bilayer lipid structures in the binding.     

大鼠细小肺动脉平滑肌细胞原代培养和鉴定方法的研究

目的：建立一种重复性好、培养周期短及传代次数多的大鼠细小肺动脉平滑肌细胞（PASMCs）培养方法。方法：在无菌条件下,分离雄性SD大鼠肺细小动脉,剥离外膜和剔除内皮细胞,经胶原酶I消化,培养PASMCs。0.4%台盼蓝染色测定细胞活力;倒置相差显微镜观察;免疫细胞化学法和免疫荧光染色法,进行平滑肌α-肌动蛋白（α-SMactin）鉴定。结果：形态学观察、免疫细胞化学法及免疫荧光染色法鉴定表明培养细胞为PASMCs;细胞存活率在96.5%以上;原代培养后4～7d即可传代,并且生长特点、细胞形态不易发生改变。结论：采用胶原酶I消化法培养PASMCs,方法简单、酶消化时间易控制、培养周期短、重复性好,培养的原代PASMCs具有数量多和生长迅速的特点。     

Biochemical markers of contraction in human myometrial smooth muscle cells in culture

Summary Phosphorylation of a light chain subunit of myosin by Ca²⁺ and calmodulin-dependent myosin light chain kinase is believed to be essential for smooth muscle contraction. The biochemical properties of the myosin phosphorylation system in human myometrial smooth muscle cells in monolayer culture were compared with those of human myometrial tissue and nonmuscle cells in culture. Native myosin was isolated from other cellular proteins of crude homogenates by polyacrylamide gel electrophoresis (in the presence of pyrophosphate) and quantified by densitometry. The myosin content of myometrial smooth muscle cells in culture and that of myometrial tissue were similar and four- to five-fold greater than that of human endometrial stromal cells or skin fibroblasts in culture. The specific activities of myosin light chain kinase in homogenates of myometrial smooth muscle cells that were maintained in culture and in myometrial tissue were similar (2.05±0.18 and 1.60±0.37 nmol phosphate incorporated per min per mg protein, respectively). On the other hand, enzyme activity in skin fibroblasts was only 5% of that in myometrial smooth muscle cells. Myosin light chain kinase activity in myometrial smooth muscle cells was dependent upon Ca²⁺ and was inhibited reversibly by the calmodulin antagonist, calmidazolium. The intracellular Ca²⁺ concentration measured by quin2 fluorescence was 0.12 μM in resting cells and increased in a concentration-dependent manner with KC1 to a maximal value of 0.47 μM. These results indicate that biochemical processes important for smooth muscle contraction are retained in human myometrial smooth muscle cells in culture. This research was supported by grants HL26043, HD11149, and GM07062 from the National Institutes of Health, Bethesda, MD.     

Puerarin induces mitochondria-dependent apoptosis in hypoxic human pulmonary arterial smooth muscle cells

Background

Pulmonary vascular medial hypertrophy in hypoxic pulmonary arterial hypertension (PAH) is caused in part by decreased apoptosis in pulmonary artery smooth muscle cells (PASMCs). Puerarin, an isoflavone purified from the Chinese medicinal herb kudzu, ameliorates chronic hypoxic PAH in animal models. Here we investigated the effects of puerarin on apoptosis of hypoxic human PASMCs (HPASMCs), and to determine the possible underlying mechanisms.

Methodology/Principal Findings

HPASMCs were cultured for 24 h in normoxia or hypoxia (5% O₂) conditions with and without puerarin. Cell number and viability were determined with a hemacytometer or a cell counting kit. Apoptosis was detected with a TUNEL test, rhodamine-123 (R-123) fluorescence, a colorimetric assay, western blots, immunohistochemical staining and RT-PCR. Hypoxia inhibited mitochondria-dependent apoptosis and promoted HPASMC growth. In contrast, after puerarin (50 µM or more) intervention, cell growth was inhibited and apoptosis was observed. Puerarin-induced apoptosis in hypoxic HPASMCs was accompanied by reduced mitochondrial membrane potential, cytochrome c release from the mitochondria, caspase-9 activation, and Bcl-2 down-regulation with concurrent Bax up-regulation.

Conclusions/Significance

Puerarin promoted apoptosis in hypoxic HPASMCs by acting on the mitochondria-dependent pathway. These results suggest a new mechanism of puerarin relevant to the management of clinical hypoxic pulmonary hypertension.     

The smooth muscle cell. III. Elastin synthesis in arterial smooth muscle cell culture

Primate arterial smooth muscle cells and skin fibroblasts were examined for their ability to synthesize elastin in culture. In the presence of the lathyrogen beta-aminopropionitrile, the smooth muscle cells incorporate [3H]lysine into a lysyl oxidase substrate that was present in the medium and associated with the cell layer. A component having a mol wt of 72,000 and an electrophoretic mobility similar to that of authentic tropoelastin was isolated from the labeled smooth muscle cells by coacervation and fractionation with organic solvents. In the absence of beta-aminopropionitrile, long-term cultures of smooth muscle cells incorporated [14C]lysine into desmosine and isodesmosine, the cross-link amino acids unique to elastin. In contrast, no desmosine formation occurred in the fibroblast cultures. These characteristics demonstrate that arterial smooth muscle cells are capable of synthesizing both soluble and cross-lined elastin in culture.     

Collagen synthesis by monkey arterial smooth muscle cells during proliferation and quiescence in culture

Monkey arterial smooth muscle cells (SMC) which are stimulated to proliferate in the presence of 5% monkey blood serum (MBS) and which remain quiescent in 5% monkey platelet-poor plasma serum (MPPPS) were examined for their ability to synthesize collagen in each of these conditions in culture. Collagen synthesis was measured by determining amounts of newly formed labeled hydroxyproline, following labelling in the presence of [³H]proline and ascorbic acid. Ascorbate requirements of SMC were examined to assure maximal hydroxylation. SMC synthesize the same amount of collagen/cell in 5% whole blood serum (MBS) during the early phase of rapid proliferation as during slow growth in later phases in culture. SMC grown in the presence of serum-lacking platelet factors synthesize 60–90% less collagen and 60–90% less non-collagen protein (per cell or per mg protein) than cells grown in MBS. Non-collagen protein synthesis was measured as incorporation of both [³H]proline and of [³H]leucine, determined as trichloroacetic acid (TCA)-precipitable material. Previous studies indicate that a factor derived from platelets is the principal mitogen present in whole blood serum for diploid cells such as SMC and fibroblasts in culture. Similarly derived factors are potent stimulators of both collagen and non-collagen protein synthesis by SMC. SMC, quiescent in medium lacking platelet derived material (MPPPS), is being used to investigate factors important in SMC proliferation since this is a significant event in atherogenesis in vivo. An increased deposition of collagen also occurs during atherogenesis. Consequently it will be useful to employ similar cultures of quiescent SMC to examine agents which affect production of this connective tissue matrix protein.     

Uterine smooth muscle cells in primary culture

Summary Smooth muscle cells (SMC) were enzymatically isolated from the myometrium of adult rat and human uteri and grown in primary culture. Cell fine structure and cytoskeletal organization were followed by transmission electron microscopy and cytochemical demonstration of actin filaments, microtubules and intermediate filaments, and initiation of DNA synthesis was investigated by thymidine autoradiography. During the first few days in culture the cells spread out on the substrate and went through a morphological transformation including loss of myofilaments followed by formation of an extensive rough endoplasmic reticulum and a large Golgi complex. Actin filaments aggregated in stress fibers spanning the entire length of the cells and microtubules and intermediate filaments formed a radiating system originating in the juxtanuclear region. In vivo, the SMC contained intermediate filaments reactive for desmin, but as early as the first day of culture expressed vimentin as well. For five days at least, all cells remained positive for both proteins, but the staining for desmin decreased while that for vimentin increased. This structural modification was accompanied by initiation of DNA synthesis, with a peak on day 3 (45–55% labeled nuclei). Subconfluent, growth-arrested primary cultures responded weakly to purified platelet-derived growth factor and serum, and in secondary cultures no response to the mitogenic stimulation was obtained. The observations indicate that uterine SMC cultivated in vitro undergo a transformation from contractile to synthetic phenotype, similar to the transformation described previously for arterial SMC under the same conditions. The proliferative potential of the uterine cells is, however, markedly lower. The findings support the notions that the transition into synthetic phenotype is a necessary but not sufficient requirement for initiation of DNA synthesis in SMC and that visceral and vascular SMC represent separate differentiation pathways.