Cytogenetic analysis reveals clonal proliferation of smooth muscle cells in atherosclerotic plaques

Summary Cytogenetic analysis of primary cell cultures from human atherosclerotic fibrous plaques revealed clonal chromosome abnormalities in 13 of the 18 cases studied. Loss of the Y chromosome and del(13)(q14) were present as single clonal abnormalities in eight cases; in five cases separate clones were found involving loss of the Y and a XXY karyotype, trisomy 10 and 18, loss of the Y and trisomy 7. A variety of single numerical and structural abnormalities were present in all but two of the 18 cases. Immunocytochemical studies were performed on cells from the same cultures used for cytogenetic analysis using monoclonal antibodies to human leucocyte common antigen, to human vimentin and to muscle actin. The immunoreactivity was positive for actin in 70–80% of the cells; 100% of the cells were positive for vimentin and all cells were ALC negative. These results indicated that the chromosomal abnormalities are present in the smooth muscle cells of the plaque. The hypothesis is proposed that the proliferation leading to the atherosclerotic lesion may primarily represent a hyperplastic response to mechanical and biological injuries and that this reactive proliferation is, in turn, associated with a tendency to chromosome instability.     

Rapid effects of aldosterone on clonal human vascular smooth muscle cells

It has been increasingly appreciated that aldosterone elicits acute vascular effects through nongenomic signaling pathways. Our previous studies demonstrated that aldosterone attenuated phenylephrine-mediated constriction in intact vessels [via phosphatidylinositol 3-kinase-dependent nitric oxide synthase activation] but enhanced vasoconstrictor responses in endothelium-denuded arteries. To determine the mechanism of this vasoconstrictor response, we assessed the effect of aldosterone on myosin light-chain phosphorylation and contraction in clonal adult human vascular smooth muscle cells. Acute aldosterone exposure mediated dose-dependent myosin light-chain phosphorylation, inhibited by spironolactone and phosphatidylinositol 3-kinase inhibition. These rapid effects of aldosterone were mimicked by estradiol and hydrocortisone and were also inhibitable by both spironolactone and eplerenone. In parallel to its effects on myosin light-chain phosphorylation, aldosterone mediated dose-dependent contraction responses that were inhibited by spironolactone. Comparable contractile responses were seen with both 17-estradiol and hydrocortisone. In total, these data are consistent with a mechanism of acute aldosterone-mediated contraction common to both glucocorticoids and estrogen. Steroid-mediated vasoconstriction may represent an important pathobiological mechanism of vascular disease, especially in the setting of preexisting endothelial dysfunction. steroid hormones; contraction; nongenomic     

Expression of smooth muscle myosin in relation to growth kinetics of cultured aortic smooth muscle cells

The goal of this study is to quantify smooth muscle myosin (SMM) expression at the level of the individual cell and to ascertain whether SMM expression in cultured aortic smooth muscle cells is related to definite growth phases, and whether the initial seeding density affects growth or SMM staining. Rabbit aortic smooth muscle cells (SMCs) were harvested by enzyme digestion of aortic tissue and plated at low (100 cells cm-2), medium (1000 cells cm-2), and high (10,000 cells cm-2) densities. Independent of seeding density, the lag phase lasted 2 to 3 days and, at all three densities, the growth rate during the logarithmic growth phase was almost the same. However, the time, the number of population doubling needed to reach the plateau phase and the cell number in the plateau were influenced by the initial seeding density. Immunofluorescence staining with anti-smooth muscle myosin (ASMM) revealed intensive staining of striated and filamentous patterns in all cells during the lag and early logarithmic growth phases. During the late logarithmic growth phase, two subpopulations of cells appeared, one showing a positive and the other no reaction with SMM antiserum. The lowest relative number of cells which showed positive reactions with SMM antiserum was observed toward the end of the logarithmic growth phase. During the plateau phase, the SMM-positive subpopulation increased, amounting to about 60% of the total number of cells, independent of the seeding density. In terms of absolute numbers, the number of SMM-positive cells increased over the course of 21 days by factors of 13, 72, and 342 for high, medium, and low seeded cultures, respectively. We conclude that a SMC subpopulation can divide without loss of SMM and that some, but not all, cells which lose their SMM may possibly regain it in the postconfluent state.     

Isolation and clonal analysis of satellite cells from chicken pectoralis muscle

Satellite cells, liberated from the breast muscle of young adult chickens by sequential treatment with collagenase and trypsin, were fractionated by Percoll density centrifugation to remove myofibril fragments and cell debris which otherwise heavily contaminate the preparation. This procedure allowed direct measurements of cell yields (1.5-4 X 10(5) cells/g tissue), plating efficiencies (27-40%) and identification of single cells in culture. In mass cultures, satellite cells gave rise to myotubes on the fifth day, and the progeny of these cells were sequentially passaged several times without losing myogenic traits. In clonal studies, over 90% of the satellite cells gave rise to large clones of which more than 99% were myogenic as demonstrated by the appearance of myotubes. The results obtained with satellite cells differ from observations made using embryonic muscle cell preparation from chicks. In the embryonic system massive formation of myotubes was observed following the third day of culture; sequential subculturing led to overgrowth of fibroblast-like cells following the first passage; and cells gave rise to both small myogenic clones (up to 16 terminally differentiated cells per clone) and non-myogenic clones in addition to large myogenic clones. We conclude that the isolated satellite cells represent a homogeneous cell population and reside in a stem cell compartment.     

Lipoproteins increase growth of mitogen-stimulated arterial smooth muscle cells

The relationship between lipoproteins and growth of aortic smooth muscle cells has been a matter of controversy. We therefore reexamined this issue using serum-free defined media methodology. By themselves, LDL or HDL (50-500 micrograms/ml) from normolipemic human or bovine plasma produced little or no growth of homologous aortic smooth muscle cells incubated in serum-free medium that was supplemented with insulin and transferrin to maintain cell viability. In fact, LDL prepared in the absence of an antioxidant (BHT) was toxic to these cells. However, in the presence of maximally effective concentrations of platelet-derived growth factor (PDGF), LDL or HDL consistently increased the growth of homologous smooth muscle cells (up to twofold increased in DNA accumulation in 48 hr). Lipoproteins also augmented the growth response of arterial smooth muscle cells to fibroblast growth factor or epidermal growth factor. The mechanism of this effect was investigated further with HDL, because, in contrast to LDL, HDL apoproteins are water-soluble. Neither HDL delipidated by solvent extraction (apoHDL), purified bovine apoA-I, nor cholesterol added in the form of phospholipid vesicles appreciably increased PDGF-induced growth of bovine smooth muscle cells. However, HDL-like particles reconstituted by sonication of apoHDL with cholesterol and phospholipids did increase the growth of cultures of bovine smooth muscle cells treated with PDGF. Uptake of tritiated thymidine by cultures incubated with partially purified PDGF alone (10 micrograms/ml) was 5,693 +/- 235 dpm/24 hr compared to 10,381 +/- 645 dpm/24 hr (p less than 0.01) in the presence of both PDGF and reconstituted HDL-like particles (250 micrograms protein/ml). Thus both the lipid and protein components of HDL may be necessary for optimal potentiation of growth of mitogen-stimulated cells. These results indicate that lipoproteins from normolipemic sera are not bona fide growth factors but can potentiate the growth of mitogen-stimulated cells, perhaps by supplying exogenous cholesterol required for membrane biogenesis. This finding might be important in arterial injury when the release of PDGF and exposure to plasma lipoproteins could act in concert to stimulate the proliferation of smooth muscle cells.     

Platelet-derived growth factor is a chemoattractant for vascular smooth muscle cells

In previous experiments (Grotendorst et al, 1981), we showed that platelet-derived growth factor promotes the migration of smooth muscle cells in vitro. Using a "checkerboard" analysis, we now establish that platelet-derived growth factor (PDGF) acts as a true chemoattractant for cultured aortic smooth muscle cells. Other growth factors such as epidermal growth factor, fibroblast growth factor, and insulin are not chemoattractants. The chemotactic response occurs before the initiation of DNA synthesis and is not affected by inhibition of DNA synthesis. Chemotaxis occurs at levels of PDGF lower than required for mitogenesis. RNA and protein synthesis are required for the chemotactic response. As found previously in bacteria and leucocytes, we find that methylation reactions are required for the chemotactic response. The possibility is discussed that PDGF acts in vivo at sites of vascular injury to attract smooth muscle cells from the medial layer to the luminal surface, and is involved in the early stages of the formation of atherosclerotic plaques.     

Improved mean estimation and its application to diagonal discriminant analysis

MOTIVATION: High-dimensional data such as microarrays have created new challenges to traditional statistical methods. One such example is on class prediction with high-dimension, low-sample size data. Due to the small sample size, the sample mean estimates are usually unreliable. As a consequence, the performance of the class prediction methods using the sample mean may also be unsatisfactory. To obtain more accurate estimation of parameters some statistical methods, such as regularizations through shrinkage, are often desired. RESULTS: In this article, we investigate the family of shrinkage estimators for the mean value under the quadratic loss function. The optimal shrinkage parameter is proposed under the scenario when the sample size is fixed and the dimension is large. We then construct a shrinkage-based diagonal discriminant rule by replacing the sample mean by the proposed shrinkage mean. Finally, we demonstrate via simulation studies and real data analysis that the proposed shrinkage-based rule outperforms its original competitor in a wide range of settings.     

Impedance analysis of renal vascular smooth muscle cells

Impedance of renal vascular smooth muscle cells (VSMCs) cultured on microelectrodes was measured by electric cell-substrate impedance sensing. Changes in measured impedance as a function of frequency were compared with the calculated values obtained from an extended cell-electrode model to estimate the junctional resistance, distance between the ventral cell surface and the substratum, and apical and basolateral membrane capacitances of renal VSMCs. This cell-electrode model was derived to accommodate the slender and rectangular shape of VSMCs. The calculated changes in impedance (Z_cal) based on the model agreed well with the experimental measurement (Z_exp), and the percentage error defined as |(Z_cal – Z_exp)/Z_exp| was 1.0%. To test the sensitivity of the new model for capturing changes in cell-cell and cell-substrate interactions induced by changes in cellular environment, we then applied this model to analyze timpedance changes induced by an integrin binding peptide in renal VSMCs. Our result demonstrates that integrin binding peptide decreases junctional resistance between cells, increases the distance between the basolateral cell surface and substratum, and increases the apical membrane capacitance, whereas the basolateral membrane capacitance stays relatively stable. This model provides a generic approach for impedance analysis of cell layers composed of slender, rectangular cells. electric cell-substrate impedance sensing; cell attachment; cell adhesion; extracellular matrix; integrin     

Analysis of smooth muscle myosin phosphorylation with native pyrophosphate gels and its application to studies on myosin phosphorylation in contracting smooth muscle

Gizzard smooth muscle myosin, the 20,000 Mr light chain (L20) of which had been phosphorylated in vitro with a calmodulin-myosin light chain kinase system, was separated into 5 isolated bands in a pyrophosphate polyacrylamide gel. Their mobilities were in the following order: myosin with 2 unphosphorylated L20 (GM) less than myosin with 1 unphosphorylated and 1 mono-phosphorylated L20 (GMP1) less than myosin with 2 mono-phosphorylated L20 (GMP2) less than myosin with 1 mono-phosphorylated and 1 di-phosphorylated L20 (GMP3) less than myosin with 2 di-phosphorylated L20 (GMP4). We used this pyrophosphate polyacrylamide gel electrophoresis to analyze the phosphorylated state of taenia coli smooth muscle during K+-induced contraction. During the initial 2 min contraction, phosphorylated forms corresponding to GMP1 and GMP2 were detected in addition to the unphosphorylated form.     

Improved media for normal human muscle satellite cells: Serum-free clonal growth and enhanced growth with low serum

Summary We have developed a serum-free medium for clonal growth of normal human muscle satellite cells (HMSC). It consists of an optimized nutrient medium MCDB 120, plus a serum-free supplement, designated SF, that contains epidermal growth factor (EGF), insulin, dexamethasone, bovine serum albumin, and fetuin. Fibroblast growth factor was needed with dialyzed fetal bovine serum (dFBS) as the only other supplement, but in media containing SF, it was only slightly beneficial, and was omitted from the final medium without significant loss. Clonal growth of HMSC in MCDB 120 plus SF is as good as with 15% serum and 0.5% chicken embryo or bovine pituitary extract. However, growth is further improved by use of a doubly-supplemented (DS) medium containing both SF and 5% dFBS. Clonal growth of HMSC in the DS medium far exceeds that in previous media with any amount of serum, and monolayer growth is at least equal to that in conventional media with higher levels of serum. Cells grown in these media exhibit little differentiation, even when grown to high densities. However, they retain the capacity for extensive fusion and synthesis of increased creatine kinase when transferred to a serum-free differentiation-promoting medium, such as Dulbecco's modified Eagle's medium plus insulin. All experiments were done with clonal cultures of HMSC to insure that observed growth responses were always those of muscle cells. This research was supported by a grant from the Muscular, Dystrophy Association. Editor's statement This article describes the optimization of both the basal nutrient medium and growth factor requirements for human muscle cells in vitro. This system is critical for studies of normal muscle cell and molecular biology, as well as for understanding diseases of muscle such as Duchenne, Muscular Dystrophy.     

A method for automated pathogenic content estimation with application to rheumatoid arthritis

Background

Sequencing technologies applied to mammals’ microbiomes have revolutionized our understanding of health and disease. Hence, to assess diseases’ progression as well as therapies longterm effects, the impact of maladies and drugs on the gut-intestinal (GI) microbiome has to be evaluated. Typical metagenomic analyses are run to associate to a condition (disease, therapy, diet) a pool of bacteria, whose eubiotic/dysbiotic potential is assessed either by α-diversity, a measure of the varieties populating the microbiome, or by Firmicutes to Bacteroides ratio, associated to systemic inflammation, and finally by manual and direct inspection of bacteria’s biological functions, when known. These approaches lead to results sometimes difficult to interpret in terms of the evolution towards a specific microbial composition, harmed by large areas of unknown.

Results

We propose to additionally evaluate a microbiome based on its global composition, by automatic annotation of pathogenic genera and statistical assessment of the net varied frequency of harmless versus harmful organisms. This application is intuitive, quantitative and computationally efficient and designed to cope with the currently incomplete species’ functional knowledge. Our results, applied to human GI-microbiome data exemplify how this layer of information provides additional insights into treatments’ impact on the GI microbiome, allowing to characterize a more physiologic effects of Prednisone versus Methotrexate, two treatments for rheumatoid arthritis (RA) a complex autoimmune systemic disease.

Conclusions

Our quantitative analysis integrates with previous approaches offering an additional systemic level of interpretation here applied, for its potential to translate into clinically relevant information, to the therapies for RA.

     

Vascular smooth muscle cells exhibit increased growth in response to elevated glucose.

Diabetes mellitus is associated with an increased risk of cardiovascular disease. In order to elucidate the association between hyperglycemia and vascular complications, the growth patterns of vascular smooth muscle cells were studied under high glucose conditions. We examined the effect of culturing porcine aortic smooth muscle cells (PVSMC) in high glucose (25 mM, HG) on total cell protein, cell volume, DNA synthesis and cell number. We observed that cells cultured in HG had higher total cell protein content which was associated with increased cell volume as compared to the cells cultured under normoglycemic conditions (5.5 mM glucose, NG). PVSMC cultured in HG also had 1.4 fold increased growth rate and a greater fetal calf serum-induced DNA synthesis rate compared to cells cultured in NG. These observations suggest for the first time that elevated glucose could lead to both hypertrophic and hyperplastic effects in PVSMC. We also examined protein kinase C (PKC) activities as well as the cellular levels of the 12-lipoxygenase product, 12-hydroxyeicosatetraenoic acid (12-HETE) in NG and HG as possible mechanisms for the enhanced growth effects in HG. The results show that PVSMC cultured in HG have increased PKC activity as well as increased levels of 12-HETE. Therefore hyperglycemia may be linked to accelerated vascular disease by increasing smooth muscle cell growth and proliferation.     

Transforming growth factor-beta1 signaling contributes to development of smooth muscle cells from embryonic stem cells

Knockout of transforming growth factor (TGF)-1 or components of its signaling pathway leads to embryonic death in mice due to impaired yolk sac vascular development before significant smooth muscle cell (SMC) maturation occurs. Thus the role of TGF-1 in SMC development remains unclear. Embryonic stem cell (ESC)-derived embryoid bodies (EBs) recapitulate many of the events of early embryonic development and represent a more physiological context in which to study SMC development than most other in vitro systems. The present studies showed induction of the SMC-selective genes smooth muscle -actin (SMA), SM22, myocardin, smoothelin-B, and smooth muscle myosin heavy chain (SMMHC) within a mouse ESC-EB model system. Significantly, SM2, the SMMHC isoform associated with fully differentiated SMCs, was expressed. Importantly, the results showed that aggregates of SMMHC-expressing cells exhibited visible contractile activity, suggesting that all regulatory pathways essential for development of contractile SMCs were functional in this in vitro model system. Inhibition of endogenous TGF- with an adenovirus expressing a soluble truncated TGF- type II receptor attenuated the increase in SMC-selective gene expression in the ESC-EBs, as did an antibody specific for TGF-1. Of interest, the results of small interfering (si)RNA experiments provided evidence for differential TGF--Smad signaling for an early vs. late SMC marker gene in that SMA promoter activity was dependent on both Smad2 and Smad3 whereas SMMHC activity was Smad2 dependent. These results are the first to provide direct evidence that TGF-1 signaling through Smad2 and Smad3 plays an important role in the development of SMCs from totipotential ESCs. embryoid body; Smad     

Inhibition of capillary endothelial cell growth by pericytes and smooth muscle cells

Morphological studies of developing capillaries and observations of alterations in capillaries associated with pathologic neovascularization indicate that pericytes may act as suppressors of endothelial cell (EC) growth. We have developed systems that enable us to investigate this possibility in vitro. Two models were used: a co-culture system that allowed direct contact between pericytes and ECs and a co-culture system that prevented physical contact but allowed diffusion of soluble factors. For these studies, co-cultures were established between bovine capillary ECs and the following growth-arrested cells (hereafter referred to as modulating cells): pericytes, smooth muscle cells (SMCs), fibroblasts, epithelial cells, and 3T3 cells. The modulating cell type was growth arrested by treatment with mitomycin C before co-culture with ECs. In experiments where cells were co-cultured directly, the effect of co-culture on EC growth was determined by comparing the mean number of cells in the co-cultures to the mean for each cell type (EC and modulating cell) cultured separately. Since pericytes and other modulating cells were growth arrested, any cell number change in co-cultures was due to EC growth. In the co-cultures, pericytes inhibited all EC proliferation throughout the 14-d time course; similar levels of EC inhibition were observed in SMC-EC co-cultures. Co-culture of ECs with fibroblasts, epithelial cells, and 3T3 cells significantly stimulated EC growth over the same time course (30-192% as compared to EC cultured alone). To determine if cell contact was required for inhibition, cells were co-cultured using Millicell chambers (Millipore Corp., Bedford, MA), which separated the cell types by 1-2 mm but allowed the exchange of diffusible materials. There was no inhibition of EC proliferation by pericytes or SMCs in this co-culture system. The influence of the cell ratios on observed inhibition was assessed by co-culturing the cells at EC/pericyte ratios of 1:1, 2:1, 5:1, 10:1, and 20:1. Comparable levels of EC inhibition were observed at ratios from 1:1 to 10:1. When the cells were co-cultured at a ratio of 20 ECs to 1 pericyte, inhibition of EC growth at 3 d was similar to that observed at other ratios. However, at higher ratios, the inhibition diminished so that by the end of the time course the co-cultured ECs were growing at the same rate as the controls. These results suggest that pericytes and SMCs can modulate EC growth by a mechanism that requires contact or proximity. We postulate that similar interactions may operate to modulate vascular growth in vivo.     

Insulin-like growth factor-I promotes proliferation and migration of cavernous smooth muscle cells

To better understand the physiology of cavernous smooth muscle cells (CSMC), particularly their regulation by IGF-I, we isolated CSMC from rats of various ages and grew them as cell cultures. CSMC from very young (1 week of age) and very old (28 months of age) rats secreted the least amounts of IGF-I, and those from 16-week-old rats the most. IGF-I stimulated growth of CSMC at an optimal concentration of 12.5 ng/ml. At this concentration, CSMC from 11-week-old rats showed the highest growth rate and CSMC from 28-month-old rats showed the lowest. The optimal IGF-I concentration for migration of CSMC was 10 ng/ml. At this concentration, CSMC from 4-week-old rats showed the highest migratory rate and CSMC from 28-month-old rats showed the lowest. IGF-I also stimulated VEGF secretion from CSMC at an optimal concentration of 10 ng/ml. At this concentration, CSMC from 16-week-old rats secreted VEGF the most and CSMC from 28-month-old rats secreted the least. The expression levels of IGF-IR paralleled the IGF-I-regulated growth rates of these cells. Expression of IGF-IR was identified in the cavernous smooth muscle and the urethra epithelium of the penis.     

Transforming growth factor-beta-induced growth inhibition and cellular hypertrophy in cultured vascular smooth muscle cells

We have explored the hypothesis that hypertrophy of vascular smooth muscle cells may be regulated, in part, by growth inhibitory factors that alter the pattern of the growth response to serum mitogens by characterizing the effects of the potent growth inhibitor, transforming growth factor-beta (TGF-beta), on both hyperplastic and hypertrophic growth of cultured rat aortic smooth muscle cells. TGF-beta inhibited serum-induced proliferation of rat aortic smooth muscle cells (ED50 = 2 pM); this is consistent with previously reported observations in bovine aortic smooth muscle cells (Assoian et al. 1982. J. Biol. Chem. 258:7155-7160). Growth inhibition was due in part to a greater than twofold increase in the cell cycle transit time in cells that continued to proliferate in the presence of TGF-beta. TGF-beta concurrently induced cellular hypertrophy as assessed by flow cytometric analysis of cellular protein content (47% increase) and forward angle light scatter (32-50% increase), an index of cell size. In addition to being time and concentration dependent, this hypertrophy was reversible. Simultaneous flow cytometric evaluation of forward angle light scatter and cellular DNA content demonstrated that TGF-beta-induced hypertrophy was not dependent on withdrawal of cells from the cell cycle nor was it dependent on growth arrest of cells at a particular point in the cell cycle in that both cycling cells in the G2 phase of the cell cycle and those in G1 were hypertrophied with respect to the corresponding cells in vehicle-treated controls. Chronic treatment with TGF-beta (100 pM, 9 d) was associated with accumulation of cells in the G2 phase of the cell cycle in the virtual absence of cells in S phase, whereas subsequent removal of TGF-beta from these cultures was associated with the appearance of a significant fraction of cycling cells with greater than 4c DNA content, consistent with development of tetraploidy. Results of these studies support a role for TGF-beta in the control of smooth muscle cell growth and suggest that at least one mechanism whereby hypertrophy and hyperploidy may occur in this, as well as other cell types, is by alterations in the response to serum mitogens by potent growth inhibitors such as TGF-beta.