Immunolocalization of matrix metalloproteinases in rabbit carotid arteries after balloon denudation

 Extracellular matrix-degrading enzymes may play a key role in vascular remodeling after arterial wall injury. We investigated the immunolocalization of matrix metalloproteinases (MMPs) in rabbit carotid arteries after balloon denudation. Positive immunostaining for MMP-1, -2, -3, and -9 appeared through the neointima 1 week after balloon denudation. The localization of immunopositive smooth muscle cells (SMCs) for MMP-1, -3, and -9, particularly for MMP-9, was almost similar to that of replicative SMCs and became confined to the luminal surface layer of the neointima at later time periods. However, MMP-2-positive SMCs appeared also in the basal layer of the neointima at 2 weeks, increased at 4 weeks, and then totally occupied the neointima at 6 weeks. The MMP-2-positive SMCs in the basal layer of the neointima at 4 and 6 weeks were negative for proliferation-associated antigens and were surrounded by extracellular matrix proteins. Our results suggest that all MMPs act in coordination to promote replication and migration of SMCs in the earlier phases of neointimal formation and that MMP-2 independently contributes to the later stages by facilitating the migration but not replication of SMCs from the media to the intima. Accepted: 25 June 1997     

Identification of the phenotypic modulation of rabbit arterial smooth muscle cells in primary culture by flow cytometry.

In atherosclerotic lesions, smooth muscle cells (SMC) change from a contractile to a synthetic phenotype. The in vivo and in vitro phenotypic transformations of SMC have been confirmed by transmission electron microscopy (TEM), but the relationship between this change and the cell cycle is still unknown. We demonstrated the structural modulation of rabbit arterial SMC in primary culture by TEM and immunocytochemistry and simultaneously studied changes in two-dimensional histograms of the relative DNA and RNA contents by flow cytometry. During the first day of primary culture, the cells exhibited the contractile phenotype and were composed of a population in the G0 phase characterized by low contents of DNA and RNA. On the second day of culture, some of the cells (18.2%) had started but not completed the transition into the synthetic phenotype and a cell population in the G1A phase with an RNA content above the G0 level appeared in almost the same proportion. This cell population could be categorized as an "intermediate" type. Moreover, after 3 days when about three-quarters of the cells had undergone structural transition, the same proportion of cells had entered into the cycling phase, while some cells still remained in the G0 and G1A phases. Thus, cell cycle analysis by flow cytometry corresponded well with the observations obtained by TEM and immunocytochemistry. These results show that flow cytometry can rapidly and relatively conveniently monitor the process of phenotypic modulation in SMC and is a useful method for the analysis of such transitions.     

O2 Level Controls Hematopoietic Circulating Progenitor Cells Differentiation into Endothelial or Smooth Muscle Cells

Background

Recent studies showed that progenitor cells could differentiate into mature vascular cells. The main physiological factors implicated in cell differentiation are specific growth factors. We hypothesized that simply by varying the oxygen content, progenitor cells can be differentiated either in mature endothelial cells (ECs) or contractile smooth muscle cells (SMCs) while keeping exactly the same culture medium.

Methodology/Principal Findings

Mononuclear cells were isolated by density gradient were cultivated under hypoxic (5% O₂) or normoxic (21% O₂) environment. Differentiated cells characterization was performed by confocal microscopy examination and flow cytometry analyses. The phenotype stability over a longer time period was also performed. The morphological examination of the confluent obtained cells after several weeks (between 2 and 4 weeks) showed two distinct morphologies: cobblestone shape in normoxia and a spindle like shape in hypoxia. The cell characterization showed that cobblestone cells were positive to ECs markers while spindle like shape cells were positive to contractile SMCs markers. Moreover, after several further amplification (until 3^rd passage) in hypoxic or normoxic conditions of the previously differentiated SMC, immunofluorescence studies showed that more than 80% cells continued to express SMCs markers whatever the cell environmental culture conditions with a higher contractile markers expression compared to control (aorta SMCs) signature of phenotype stability.

Conclusion/Significance

We demonstrate in this paper that in vitro culture of peripheral blood mononuclear cells with specific angiogenic growth factors under hypoxic conditions leads to SMCs differentiation into a contractile phenotype, signature of their physiological state. Moreover after amplification, the differentiated SMC did not reverse and keep their contractile phenotype after the 3^rd passage performed under hypoxic and normoxic conditions. These aspects are of the highest importance for tissue engineering strategies. These results highlight also the determinant role of the tissue environment in the differentiation process of vascular progenitor cells.     

Localization and effects of hepatocyte growth factor on smooth muscle cells during neointimal formation after balloon denudation

The migration and proliferation of smooth muscle cells (SMCs) may play a key role in tissue remodeling after arterial wall injury. We investigated the localization and effects of hepatocyte growth factor (HGF) in rabbit carotid arteries after balloon denudation. Immunoreactivity for HGF and the c-Met receptor was clearly observed in neointimal SMCs. The immunoreactivity was not restricted to proliferating cells but was seen even in non-dividing cells in the basal layer of the neointima 4 and 6 weeks after balloon denudation. The distribution of platelet-derived growth factor (PDGF)-positive cells paralleled that of proliferating SMCs. The SMCs in the basal layer of the neointima at 4 and 6 weeks were positive for matrix metalloproteinase (MMP)-2 and membrane type 1-MMP which can activate the proform of MMP-2. HGF significantly stimulated the migration but not proliferation of cultured SMCs. Our results suggest that HGF and PDGF act in coordination to promote the proliferation and migration of SMCs in the earlier phases of neointimal formation and that HGF as well as MMP-2 contribute to the later stages by facilitating the migration but not replication of SMCs. Accepted: 19 March 1999     

Changes in three-dimensional architecture of microfilaments in cultured vascular smooth muscle cells during phenotypic modulation

To investigate changes in the three-dimensional microfilament architecture of vascular smooth muscle cells (SMC) during the process of phenotypic modulation, rabbit aortic SMCs cultured under different conditions and at different time points were either labelled with fluorescein-conjugated probes to cytoskeletal and contractile proteins for observation by confocal laser scanning microscopy, or extracted with Triton X-100 for scanning electron microscopy. Densely seeded SMCs in primary culture, which maintain a contractile phenotype, display prominent linear myofilament bundles (stress fibres) that are present throughout the cytoplasm with alpha-actin filaments predominant in the central part and beta-actin filaments in the periphery of the cell. Intermediate filaments form a meshed network interconnecting the stress fibres and linking directly to the nucleus. Moderately and sparsely seeded SMCs, which modulate toward the synthetic phenotype during the first 5 days of culture, undergo a gradual redistribution of intermediate filaments from the perinuclear region toward the peripheral cytoplasm and a partial disassembly of stress fibres in the central part of the upper cortex of the cytoplasm, with an obvious decrease in alpha-actin and myosin staining. These changes are reversed in moderately seeded SMCs by day 8 of culture when they have reached confluence. The results reveal two changes in microfilament architecture in SMCs as they undergo a change in phenotype: the redistribution of intermediate filaments probably due to an increase in synthetic organelles in the perinuclear area, and the partial disassembly of stress fibres which may reflect a degradation of contractile components.     

Complement 3 is involved in changing the phenotype of human glomerular mesangial cells

Complement activation contributes to tissue injury in various forms of glomerulopathy and is characterized by deposition of complement components, which accelerates the progression of chronic renal damage. We recently reported that complement 3 (C3), a critical component of the complement system, is associated with the synthetic phenotype of vascular smooth muscle cells. It is possible that C3 stimulates mesangial cells to assume the synthetic phenotype to, in turn, induce glomerular injury and sclerosis. We investigated the role of C3 in the growth and phenotype of mesangial cells. Cultured human mesangial cells (HMCs) expressed C3 mRNA and protein, and levels were increased in response to IFN-gamma and TNF-alpha. HMCs also expressed C3a receptor mRNA and protein. Exogenous C3a stimulated DNA synthesis in HMCs in a dose-dependent manner. C3a decreased expression h-caldesmon mRNA, a marker of the contractile phenotype, and increased the expression of osteopontin, matrix Gla, and collagen type1 alpha1 (collagen IV) mRNAs, which are markers of the synthetic phenotype. C3a decreased expression of alpha-smooth muscle actin in HMCs. Small interfering RNA (siRNA) targeting C3 reduced the DNA synthesis and proliferation of HMCs, increased expression of h-caldesmon mRNA, and decreased expression of osteopontin, matrix Gla, and collagen IV mRNAs in HMCs. These results indicate that C3 causes HMCs to convert to the synthetic phenotype and stimulates growth of mesangial cells, suggesting that C3 may play an important role in phenotypic regulation of mesangial cells in renal diseases.     

Plasma fibronectin promotes modulation of arterial smooth-muscle cells from contractile to synthetic phenotype

Isolated arterial smooth-muscle cells (SMCs) cultured in medium containing whole blood serum or plasma-derived serum undergo modulation from a contractile to a synthetic phenotype. This process includes the loss of myofilaments and cessation of the ability to contract. Instead, an extensive rough endoplasmic reticulum and a large Golgi complex are formed and, if properly stimulated, the cells start to proliferate actively and to produce extracellular-matrix components. In vivo, a similar change in the differentiated properties of SMCs appears to be an early key event in atherogenesis. The purpose of the present investigation was to try to identify plasma components that promote the modulation of the smooth-muscle phenotype. SMCs were enzymatically isolated from rat aorta and cultured in a defined, serum-free medium. The phenotypic state of the cells was determined by transmission electron microscopy, and their growth status was followed by 3H-thymidine autoradiography and cell counting. Under these conditions, Cohn fractions I (fibrinogen) and V (albumin) were found to partially support cell attachment and transition from the contractile to the synthetic phenotype, whereas fractions II-III and IV (globulins) were inactive in this respect. Analysis on adsorptive columns of gelatin Sepharose 4B indicated that Cohn fraction I, but not fraction V, contained fibronectin, an adhesive protein that is present in plasma and binds to fibrinogen. When seeded on a substrate of plasma fibronectin, the cells attached with high efficiency and modulated into the synthetic phenotype at a rate similar to that observed in serum-containing medium. In the absence of exogenous mitogens, the structural transformation of the cells was not accompanied by a proliferative response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differentiation of Human Induced-Pluripotent Stem Cells into Smooth-Muscle Cells: Two Novel Protocols

Conventional protocols for differentiating human induced-pluripotent stem cells (hiPSCs) into smooth-muscle cells (SMCs) can be inefficient and generally fail to yield cells with a specific SMC phenotype (i.e., contractile or synthetic SMCs). Here, we present two novel hiPSC-SMC differentiation protocols that yield SMCs with predominantly contractile or synthetic phenotypes. Flow cytometry analyses of smooth-muscle actin (SMA) expression indicated that ~45% of the cells obtained with each protocol assumed an SMC phenotype, and that the populations could be purified to ~95% via metabolic selection. Assessments of cellular mRNA and/or protein levels indicated that SMA, myosin heavy chain II, collagen 1, calponin, transgelin, connexin 43, and vimentin expression in the SMCs obtained via the Contractile SMC protocol and in SMCs differentiated via a traditional protocol were similar, while SMCs produced via the Sythetic SMC protocol expressed less calponin, more collagen 1, and more connexin 43. Differences were also observed in functional assessments of the two SMC populations: the two-dimensional surface area of Contractile SMCs declined more extensively (to 12% versus 44% of original size) in response to carbachol treatment, while quantification of cell migration and proliferation were greater in Synthetic SMCs. Collectively, these data demonstrate that our novel differentiation protocols can efficiently generate SMCs from hiPSCs.     

The regulatory expression of procollagen COOH-terminal proteinase enhancer in the proliferation of vascular smooth muscle cells

Intimal hyperplasia following arterial endothelial denudation results in large part from the proliferation of vascular smooth muscle cells (SMCs) and matrix accumulation. Procollagen COOH-terminal proteinase enhancer (PCPE) binds procollagen COOH-propeptides and potentiates procollagen COOH-proteinase activity to cleave COOH-propeptides of procollagens I-III. Here we report the enhanced expression of PCPE in cultured SMCs and in intimal thickening induced by arterial injury. The levels of PCPE mRNA in parallel with the level of p21(Cip1) mRNA, as a negative regulator of cellular proliferation, increased under serum deprivation or reduced cellular proliferation in cultured SMCs. In contrast, rapidly proliferating cells show the decreased levels of PCPE mRNA. In vivo, the marked induction of PCPE in injured rat arteries occurred at 14 days after endothelial denudation. The induced expression levels of PCPE as well as p21(Cip1) were maintained until 42 days, although cyclin E expression declined. Furthermore, transforming growth factor beta1 (TGF-beta1), an important regulator of cellular proliferation in atheroma, increased the levels of the PCPE mRNA in cultured SMCs. Thus, the regulatory expression of PCPE dependent on cellular proliferation, and particularly contact inhibition, may play a key role in the proliferation of SMCs and matrix production during the process of atheroma formation.     

Synchronization of Cultured Vascular Smooth Muscle Cells Following Reversal of Quiescence Induced by Treatment with the AntioxidantN-Acetylcysteine

Smooth muscle cell (SMC) proliferation plays an important role in the pathogenesis of vascular diseases such as atherosclerosis and postangioplasty restenosis. Recently we demonstrated the thiol antioxidantN-acetylcysteine (NAC) inhibits constitutive NF-κB/Rel activity and growth of vascular SMCs. Here we show that treatment of human and bovine aortic SMC with the thiol antioxidant NAC causes cells to exit the cell cycle and remain quiescent as determined by a greatly reduced incorporation of [³H]thymidine and G₀/G₁DNA content. Removal of NAC from the culture medium stimulates SMCs to synchronously reenter the cell cycle as judged by induction of cyclin D1 and B-mybgene expression during mid and late G₁phase, respectively, and induction of histone gene expression and [³H]thymidine incorporation during S phase. The time course of cyclin D1, B-myb,and histone gene expression after NAC removal was similar to that of serum-deprived cells induced to resume cell cycle progression by the addition of fetal bovine serum to the culture medium. Taken together, these results indicate that NAC treatment causes SMCs to enter a reversible G₀quiescent, growth-arrested state. Thus, NAC provides an important new method for synchronizing SMCs in culture.     

Phenotype modulation in primary cultures of arterial smooth-muscle cells. Dual effect of prostaglandin E1

The effects of prostaglandin E1 (PGE1) on the phenotypic state of enzymatically isolated arterial smooth-muscle cells in primary culture were studied by transmission electron microscopy, thymidine autoradiography, and cell counting. Early in culture (day 0-2), PGE1 stimulated conversion of the cells from contractile (less euchromatic nucleus and cytoplasm dominated by myofilament bundles) to synthetic state (more euchromatic nucleus and cytoplasm dominated by cisternae of rough endoplasmic reticulum and a large Golgi complex). The rate of entrance of the cells into DNA synthesis and mitosis was also increased at this time. Later on (day 3-6), when the majority of the cells had entered synthetic state, PGE1 inhibited DNA synthesis and cellular proliferation. These observations indicate that the effect of prostaglandins on arterial smooth muscle is dual in nature and dependent on the state of differentiation of the cells.     

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 7 days 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.     

Endothelial Cells Can Regulate Smooth Muscle Cells in Contractile Phenotype through the miR-206/ARF6&NCX1/Exosome Axis

Active interactions between endothelial cells and smooth muscle cells (SMCs) are critical to maintaining the SMC phenotype. Exosomes play an important role in intercellular communication. However, little is known about the mechanisms that regulate endothelial cells and SMCs crosstalk. We aimed to determine the mechanisms underlying the regulation of the SMC phenotype by human umbilical vein endothelial cells (HUVECs) through exosomes. We found that HUVECs overexpressing miR-206 upregulated contractile marker (α-SMA, Smoothelin and Calponin) mRNA expression in SMCs. We also found that the expression of miR-206 by HUVECs reduced exosome production by regulating ADP-Ribosylation Factor 6 (ARF6) and sodium/calcium exchanger 1 (NCX1). Using real-time PCR and western blot analysis, we showed that HUVEC-derived exosomes decreased the expression of contractile phenotype marker genes (α-SMA, Smoothelin and Calponin) in SMCs. Furthermore, a reduction of the miR-26a-containing exosomes secreted from HUVECs affects the SMC phenotype. We propose a novel mechanism in which miR-206 expression in HUVECs maintains the contractile phenotype of SMCs by suppressing exosome secretion from HUVECs, particularly miR-26a in exosomes, through targeting ARF6 and NCX1.