Phenotypic heterogeneity influences the behavior of rat aortic smooth muscle cells in collagen lattice

Phenotypic modulation of vascular smooth muscle cells (SMCs) in atherosclerosis and restenosis involves responses to the surrounding microenvironment. SMCs obtained by enzymatic digestion from tunica media of newborn, young adult (YA) and old rats and from the thickened intima (TI) and underlying media of young adult rat aortas 15 days after ballooning were entrapped in floating populated collagen lattice (PCL). TI-SMCs elongated but were poor at PCL contraction and remodeling and expressed less alpha2 integrin compared to other SMCs that appeared more dendritic. During early phases of PCL contraction, SMCs showed a marked decrease in the expression of alpha-smooth muscle actin and myosin. SMCs other than TI-SMCs required 7 days to re-express alpha-smooth muscle actin and myosin. Only TI-SMCs in PCL were able to divide in 48 h, with a greater proportion in S and G2-M cell cycle phases compared to other SMCs. Anti-alpha2 integrin antibody markedly inhibited contraction but not proliferation in YA-SMC-PLCs; anti-alpha1 and anti-alpha2 integrin antibodies induced a similar slight inhibition in TI-SMC-PCLs. Finally, TI-SMCs rapidly migrated from PCL on plastic reacquiring their epithelioid phenotype. Heterogeneity in proliferation and cytoskeleton as well the capacity to remodel the extracellular matrix are maintained, when SMCs are suspended in PCLs.     

Clonally expanded human airway smooth muscle cells exhibit morphological and functional heterogeneity

Background

Mesenchyme-derived airway cell populations including airway smooth muscle (ASM) cells, fibroblasts and myofibroblasts play key roles in the pathogenesis of airway inflammation and remodeling. Phenotypic and functional characterisation of these cell populations are confounded by their heterogeneity in vitro. It is unclear which mechanisms underlie the creation of these different sub-populations.The study objectives were to investigate whether ASM cells are capable of clonal expansion and if so (i) what proportion possess this capability and (ii) do clonal populations exhibit variation in terms of morphology, phenotype, proliferation rates and pro-relaxant or pro-contractile signaling pathways.

Methods

Early passage human ASM cells were subjected to single-cell cloning and their doubling time was recorded. Immunocytochemistry was performed to assess localization and levels of markers previously reported to be specifically associated with smooth muscle or fibroblasts. Finally functional assays were used to reveal differences between clonal populations specifically assessing mitogen-induced proliferation and pro-relaxant and pro-contractile signaling pathways.

Results

Our studies provide evidence that a high proportion (58%) of single cells present within early passage human ASM cell cultures have the potential to create expanded cell populations. Despite being clonally-originated, morphological heterogeneity was still evident within these clonal populations as assessed by the range in expression of markers associated with smooth muscle cells. Functional diversity was observed between clonal populations with 10 μM isoproterenol-induced cyclic AMP responses ranging from 1.4 - 5.4 fold cf basal and bradykinin-induced inositol phosphate from 1.8 - 5.2 fold cf basal.

Conclusion

In summary we show for the first time that primary human ASM cells are capable of clonal expansion and that the resulting clonal populations themselves exhibit phenotypic plasticity.     

Vascular smooth muscle cells direct extracellular dysregulation in aortic stiffening of hypertensive rats

Aortic stiffening is an independent risk factor that underlies cardiovascular morbidity in the elderly. We have previously shown that intrinsic mechanical properties of vascular smooth muscle cells (VSMCs) play a key role in aortic stiffening in both aging and hypertension. Here, we test the hypothesis that VSMCs also contribute to aortic stiffening through their extracellular effects. Aortic stiffening was confirmed in spontaneously hypertensive rats (SHRs) vs. Wistar‐Kyoto (WKY) rats in vivo by echocardiography and ex vivo by isometric force measurements in isolated de‐endothelized aortic vessel segments. Vascular smooth muscle cells were isolated from thoracic aorta and embedded in a collagen I matrix in an in vitro 3D model to form reconstituted vessels. Reconstituted vessel segments made with SHR VSMCs were significantly stiffer than vessels made with WKY VSMCs. SHR VSMCs in the reconstituted vessels exhibited different morphologies and diminished adaptability to stretch compared to WKY VSMCs, implying dual effects on both static and dynamic stiffness. SHR VSMCs increased the synthesis of collagen and induced collagen fibril disorganization in reconstituted vessels. Mechanistically, compared to WKY VSMCs, SHR VSMCs exhibited an increase in the levels of active integrin β1‐ and bone morphogenetic protein 1 (BMP1)‐mediated proteolytic cleavage of lysyl oxidase (LOX). These VSMC‐induced alterations in the SHR were attenuated by an inhibitor of serum response factor (SRF)/myocardin. Therefore, SHR VSMCs exhibit extracellular dysregulation through modulating integrin β1 and BMP1/LOX via SRF/myocardin signaling in aortic stiffening.     

Pigeon aortic smooth muscle cells lack a functional low density lipoprotein receptor pathway

The low density lipoprotein (LDL) receptor pathway was studied in aortic smooth muscle cells from atherosclerosis-susceptible White Carneau pigeons and compared with rhesus monkey cells whose LDL receptor pathway has been previously characterized. Pigeon LDL was bound with high affinity in a saturable manner to both pigeon and monkey aortic smooth muscle cells. The kinetics of binding were different, however. LDL binding to pigeon cells exhibited positive cooperativity at low LDL concentrations and at least two classes of binding sites. The same pigeon LDL bound to monkey cells in a manner consistent with a single class of binding sites. Thus, these differences were a property of the pigeon cells and not the result of differences in the LDL. On the average, pigeon cells bound less than 50% the amount of LDL as monkey cells. Despite the surface binding to pigeon cells, little of the LDL was internalized, whereas pigeon LDL was actively internalized by monkey cells. Consistent with this observation, chloroquine and leupeptin had no effect on accumulation of LDL or on LDL degradation by pigeon cells, and incubation of pigeon cells with LDL produced no increase in cellular cholesteryl ester content. Binding of LDL to pigeon cells also differed from that of monkey cells by being unaffected by pretreatment with the proteolytic enzyme pronase, and by not requiring calcium. Binding was not specific for LDL since acetyl-LDL, and to a lesser degree HDL, were able to compete for LDL binding. Incubation with lipoprotein-deficient serum decreased LDL binding in pigeon cells while 25-OH cholesterol caused an increase in binding; both effects are opposite of that seen with the same LDL in mammalian cells. Preincubation with LDL or cholesterol dissolved in ethanol were without effect on LDL binding in pigeon cells, even though they produced significant increases in cellular free cholesterol content. In spite of the failure to internalize LDL, there was considerable degradation of LDL. This apparently occurred on the cell surface rather than by internalization and degradation within the lysosomes as occurs in mammalian cells. The functional significance of LDL binding to pigeon smooth muscle cells is unclear. The characteristics of binding resemble that of a nonspecific lipoprotein receptor referred to by others as the "lipoprotein receptor" or the "EDTA-insensitive receptor." It is apparent, however, that White Carneau pigeon aortic smooth muscle cells lack a functional LDL receptor pathway and in this way resemble cells from human beings with homozygous familial hypercholesterolemia or from Watanabe rabbits.     

Screening and functional prediction of differentially expressed circRNAs in proliferative human aortic smooth muscle cells

Vascular smooth muscle cell (VSMC) proliferation is the pathological base of vascular remodelling diseases. Circular RNAs (circRNAs) are important regulators involved in various biological processes. However, the function of circRNAs in VSMC proliferation regulation remains largely unknown. This study was conducted to identify the key differentially expressed circRNAs (DEcircRNAs) and predict their functions in human aortic smooth muscle cell (HASMC) proliferation. To achieve this, DEcircRNAs between proliferative and quiescent HASMCs were detected using a microarray, followed by quantitative real‐time RT‐PCR validation. A DEcircRNA‐miRNA‐DEmRNA network was constructed, and functional annotation was performed using Gene Ontology (GO) and KEGG pathway analysis. The function of hsa_circ_0002579 in HASMC proliferation was analysed by Western blot. The functional annotation of the DEcircRNA‐miRNA‐DEmRNA network indicated that the four DEcircRNAs might play roles in the TGF‐β receptor signalling pathway, Ras signalling pathway, AMPK signalling pathway and Wnt signalling pathway. Twenty‐seven DEcircRNAs with coding potential were screened. Hsa_circ_0002579 might be a pro‐proliferation factor of HASMC. Overall, our study identified the key DEcircRNAs between proliferative and quiescent HASMCs, which might provide new important clues for exploring the functions of circRNAs in vascular remodelling diseases.     

Proteomic dataset of mouse aortic smooth muscle cells

In an accompanying study (in this issue, DOI 10.1002/pmic.200402044), we have characterised the proteome of Sca-1(+) progenitor cells, which may function as precursors of vascular smooth muscle cells (SMCs). In the present study, we have analysed and mapped protein expression in aortic SMCs of mice, using 2-DE, MALDI-TOF MS and MS/MS. The 2-D system comprised a non-linear immobilised pH 3-10 gradient in the first dimension (separating proteins with pI values of pH 3-10), and 12%T SDS-PAGE in the second dimension (separating proteins in the range 15,000-150,000 Da). Of the 2400 spots visualised, a subset of 267 protein spots was analysed, with 235 protein spots being identified corresponding to 154 unique proteins. The data presented here are the first map of aortic SMCs and the most extensive analysis of SMC proteins published so far. This valuable tool should provide a basis for comparative studies of protein expression in vascular smooth muscle of transgenic mice and is available on our website hhtp://www.vascular-proteomics.com.     

Vasopressin phosphorylates HSP27 in aortic smooth muscle cells

Administration of arginine vasopressin (AVP) time-dependently induced the phosphorylation of heat shock protein 27 (HSP27) at Ser-15 and Ser-85 in smooth muscle of aorta in vivo. The AVP-induced phosphorylation of HSP27 at Ser-15 and Ser-85 was inhibited by a V1a receptor antagonist but not by a V2 receptor antagonist. In cultured aortic smooth muscle A10 cells, AVP markedly stimulated the phosphorylation of HSP27 at Ser-15 and Ser-85. The AVP-induced phosphorylation of HSP27 was attenuated by SB203580 and PD169316, inhibitors of p38 mitogen-activated protein (MAP) kinase, but not by PD98059, a MEK inhibitor. These results strongly suggest that AVP phosphorylates HSP27 via p38 MAP kinase in aortic smooth muscle cells.     

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.     

Metabolism of cytoplasmic triacylglycerol in cultured aortic smooth muscle cells

A turnover of cytoplasmic triacylglycerol was studied in cultured rat, rabbit, and bovine aortic smooth muscle cells. Cytoplasmic triacylglycerol was labeled with [3H]glycerol in the presence of oleic acid in the medium and its loss from the cell was studied in the presence of carrier glycerol. Multiple additions of Isuprel or dibutyryl cyclic AMP during the chase period did not enhance the loss of labeled triacylglycerol. The rate of hydrolysis of cellular triacylglycerol was unchanged in the absence or in the presence of 100 microM chloroquine. Modulation of cellular cholesterol content by addition of low density lipoprotein or high density apolipoprotein--sphingomyelin liposomes did not affect the residence time of the cellular triacylglycerol. We conclude that cytoplasmic triacylglycerol in cultured aortic smooth muscle cells is metabolized by an extralysosomal enzyme which is neither catecholamine responsive nor affected by modulation of cellular cholesterol.     

Cholesterol metabolism in pigeon aortic smooth muscle cells lacking a functional low density lipoprotein receptor pathway

Cholesterol metabolism was examined in aortic smooth muscle cells from atherosclerosis-susceptible White Carneau pigeons that have been shown to lack a functional LDL receptor pathway. In cells incubated in the presence of whole serum or low density lipoprotein (LDL) the rate of cholesterol synthesis from [1-14C]acetate or of HMG-CoA reductase activity was 20-100 times greater than for mammalian cells incubated under the same conditions. Unexpectedly, cholesterol synthesis decreased by nearly 50% after preincubation for 24 hr with lipoprotein-deficient serum (LPDS). This occurred without a change in cellular cholesterol content. Neither the high rate of cholesterol synthesis nor the effect of LPDS could be accounted for by differences in cell turnover or state of growth. Cholesterol added in ethanol was ineffective in altering cellular cholesterol synthesis or esterification even though a near doubling in cellular free cholesterol content occurred. Cholesterol synthesis and esterification were, however, able to be regulated with 25-OH cholesterol and mevalonolactone, as indicated by their ability to suppress cholesterol synthesis and to stimulate cholesterol esterification. In spite of the high rate of endogenous cholesterol synthesis, cellular cholesterol content was maintained at a constant level by the efficient efflux of the newly synthesized cholesterol from the cell. Unlike mammalian cells that require a cholesterol acceptor in the medium for efflux to occur, cholesterol efflux from pigeon cells occurred in the absence of a cholesterol acceptor. This suggests either that pigeon cells utilize a different mechanism for cholesterol efflux or that they produce their own cholesterol acceptor. As a result of a lack of a functional LDL receptor pathway, pigeon smooth muscle cells do not maintain cholesterol homeostasis through the controlled uptake of exogenous LDL cholesterol, as do mammalian cells. Rather, pigeon smooth muscle cells would appear to regulate cholesterol concentrations at the level of either cholesterol synthesis or efflux.     

Cultured aortic smooth muscle cells from newborn and adult rats show distinct cytoskeletal features

It is well known that arterial smooth muscle cells (SMC) of adult rats, cultured in a medium containing fetal calf serum (FCS), replicate actively and lose the expression of differentiation markers, such as desmin, smooth muscle (SM) myosin and alpha-SM actin. We report here that compared to freshly isolated cells, primary cultures of SMC from newborn animals show no change in the number of alpha-SM actin containing cells and a less important decrease in the number of desmin and SM myosin containing cells than that seen in primary cultures of SMC from adult animals; moreover, contrary to what is seen in SMC cultured from adult animals, they show an increase of alpha-SM actin mRNA level, alpha-SM actin synthesis and expression per cell. These features are partially maintained at the 5th passage, when the cytoskeletal equipment of adult SMC has further evolved toward dedifferentiation. Cloned newborn rat SMC continue to express alpha-SM actin, desmin and SM myosin at the 5th passage. Thus, newborn SMC maintain, at least in part, the potential to express differentiated features in culture. Heparin has been proposed to control proliferation and differentiation of arterial SMC. When cultured in the presence of heparin, newborn SMC show an increase of alpha-SM actin synthesis and content but no modification of the proportion of alpha-SM actin total (measured by Northern blots) and functional (measured by in vitro translation in a reticulocyte lysate) mRNAs compared to control cells cultured for the same time in FCS containing medium. This suggests that heparin action is exerted at a translational or post-translational level. Cultured newborn rat aortic SMC furnish an in vitro model for the study of several aspects of SMC differentiation and possibly of mechanisms leading to the establishment and prevention of atheromatous plaques.     

Functional properties of smooth muscle cells in ascending aortic aneurysm

Thoracic aortic aneurysm (TAA) develops as a result of complex sequential events that dynamically alter the structure and composition of the aortic vascular extracellular matrix (ECM). The main cellular elements that alter the composition of aortic wall are smooth muscle cells (SMCs). The purpose of the present work was to study alterations of smooth muscle cell functions derived from the patients with TAA and from healthy donors. Since it is believed that TAA associates with bicuspid aortic valve (BAV) and with tricuspid aortic valve (TAV) differed in their pathogenesis, we have compared SMCs and tissue samples from BAV and TAV patients and healthy donors. The comparison was done by several parameters: SMC growth, migration and apoptotic dynamics, metalloproteinase MMP2 and MMP9 activity (zymography), and elastin, collagen, and fibrillin content (Western blot) in both tissue samples and cultured SMCs. Proliferation of BAV and TAV SMCs was decreased and migration ability in scratch tests was increased in TAV-derived SMCs compared to donor cells. BAV-cells migration ability was not changed compared to donor SMCs. Elastin content was decreased in TAA SMCs, whereas the content of fibrillin and collagen was not altered. At the same time, the elastin and collagen protein level was significantly higher in tissue samples of TAA patients than in donorderived samples. SMC proliferation and migration is differently affected in TAV and BAV-associated TAA that supports the idea on different nature of these two TAA groups. Our data also show that SMC functional properties are altered in TAA patients and these alterations could play a significant role in the disease pathogenesis.     

Extracellular calcium sensing in rat aortic vascular smooth muscle cells

Extracellular calcium (Ca(2+)(o)) can act as a first messenger in many cell types through a G protein-coupled receptor, calcium-sensing receptor (CaR). It is still debated whether the CaR is expressed in vascular smooth muscle cells (VSMCs). Here, we report the expression of CaR mRNA and protein in rat aortic VSMCs and show that Ca(2+)(o) stimulates proliferation of the cells. The effects of Ca(2+)(o) were attenuated by pre-treatment with MAPK kinase 1 (MEK1) inhibitor, as well as an allosteric modulator, NPS 2390. Furthermore, stimulation of the VSMCs with Ca(2+)(o)-induced phosphorylation of ERK1/2, but surprisingly did not cause inositol phosphate accumulation. We were not able to conclusively state that the CaR mediates Ca(2+)(o)-induced cell proliferation. Rather, an additional calcium-sensing mechanism may exist. Our findings may be of importance with regard to atherosclerosis, an inflammatory disease characterized by abnormal proliferation of VSMCs and high local levels of calcium.