Restricted expression of homeobox genes distinguishes fetal from adult human smooth muscle cells.

Smooth muscle cell plasticity is considered a prerequisite for atherosclerosis and restenosis following angioplasty and bypass surgery. Identification of transcription factors that specify one smooth muscle cell phenotype over another therefore may be of major importance in understanding the molecular basis of these vascular disorders. Homeobox genes exemplify one class of transcription factors that could govern smooth muscle cell phenotypic diversity. Accordingly, we screened adult and fetal human smooth muscle cell cDNA libraries with a degenerate oligonucleotide corresponding to a highly conserved region of the homeodomain with the idea that homeobox genes, if present, would display a smooth muscle cell phenotype-dependent pattern of expression. No homeobox genes were detected in the adult human smooth muscle cell library; however, five nonparalogous homeobox genes were uncovered from the fetal library (HoxA5, HoxA11, HoxB1, HoxB7, and HoxC9). Northern blotting of adult and fetal tissues revealed low and restricted expression of all five homeobox genes. No significant differences in transcripts of HoxA5, HoxA11, and HoxB1 were detected between adult or fetal human smooth muscle cells in culture. HoxB7 and HoxC9, however, showed preferential mRNA expression in fetal human smooth muscle cells that appeared to correlate with the age of the donor. This phenotype-dependent expression of homeobox genes was also noted in rat pup versus adult smooth muscle cells. While similar differences in gene expression have been reported between subsets of smooth muscle cells from rat vessels of different-aged animals or clones of rat smooth muscle, our findings represent a demonstration of a transcription factor distinguishing two human smooth muscle cell phenotypes.

Epigallocathechin-3 Gallate Selectively Inhibits the PDGF-BB–induced Intracellular Signaling Transduction Pathway in Vascular Smooth Muscle Cells and Inhibits Transformation of sis-transfected NIH 3T3 Fibroblasts and Human Glioblastoma Cells (A172)

Enhanced activity of receptor tyrosine kinases such as the PDGF β-receptor and EGF receptor has been implicated as a contributing factor in the development of malignant and nonmalignant proliferative diseases such as cancer and atherosclerosis. Several epidemiological studies suggest that green tea may prevent the development of cancer and atherosclerosis. One of the major constituents of green tea is the polyphenol epigallocathechin-3 gallate (EGCG). In an attempt to offer a possible explanation for the anti-cancer and anti-atherosclerotic activity of EGCG, we examined the effect of EGCG on the PDGF-BB–, EGF-, angiotensin II-, and FCS-induced activation of the 44 kDa and 42 kDa mitogen-activated protein (MAP) kinase isoforms (p44mapk/p42mapk) in cultured vascular smooth muscle cells (VSMCs) from rat aorta. VSMCs were treated with EGCG (1–100 μM) for 24 h and stimulated with the above mentioned agonists for different time periods. Stimulation of the p44mapk/p42mapk was detected by the enhanced Western blotting method using phospho-specific MAP kinase antibodies that recognized the Tyr204-phosphorylated (active) isoforms. Treatment of VSMCs with 10 and 50 μM EGCG resulted in an 80% and a complete inhibition of the PDGF-BB–induced activation of MAP kinase isoforms, respectively. In striking contrast, EGCG (1–100 μM) did not influence MAP kinase activation by EGF, angiotensin II, and FCS. Similarly, the maximal effect of PDGF-BB on the c-fos and egr-1 mRNA expression as well as on intracellular free Ca2+ concentration was completely inhibited in EGCG-treated VSMCs, whereas the effect of EGF was not affected. Quantification of the immunoprecipitated tyrosine-phosphorylated PDGF-Rβ, phosphatidylinositol 3′-kinase, and phospholipase C-γ1 by the enhanced Western blotting method revealed that EGCG treatment effectively inhibits tyrosine phosphorylation of these kinases in VSMCs. Furthermore, we show that spheroid formation of human glioblastoma cells (A172) and colony formation of sis-transfected NIH 3T3 cells in semisolid agar are completely inhibited by 20–50 μM EGCG. Our findings demonstrate that EGCG is a selective inhibitor of the tyrosine phosphorylation of PDGF-Rβ and its downstream signaling pathway. The present findings may partly explain the anti-cancer and anti-atherosclerotic activity of green tea.

Platelet-derived growth factor stimulates the secretion of hyaluronic acid by proliferating human vascular smooth muscle cells.

Total glycans from the cell layer and the culture medium of human vascular smooth muscle cells (VSMC) that had been cultivated in the presence of platelet-derived growth factor (PDGF) were isolated and purified by gel filtration after Pronase and DNase digestion and alkaliborohydride treatment. Measurements of the content of neutral hexoses and uronic acids revealed that PDGF stimulates total glycan synthesis by proliferating VSMC in a linear fashion from 24 h to 72 h of incubation. In contrast, total glycan synthesis by human fibroblasts, epithelial cells, or endothelial cells was not affected by PDGF, indicating cell-type specificity. Chemical, biochemical, and enzymological characterization of the total glycans synthesized by VSMC showed that PDGF stimulates the secretion of a 340-kDa glycan molecule in a time-dependent manner from 24 h to 72 h. This molecule is highly acidic, shares a common structure with hyaluronic acid, and exhibits a potent antiproliferative activity on VSMC. These results suggest that VSMC in response to PDGF are capable of controlling their own growth and migration by the synthesis of a specific form of hyaluronic acid with antiproliferative potency, which may be involved in the regulation of the local inflammatory responses associated with atherosclerosis.

Cloning, expression, and characterization of a cDNA encoding a novel human growth factor for primitive hematopoietic progenitor cells

Multiple growth factors synergistically stimulate proliferation of primitive hematopoietic progenitor cells. A human myeloid cell line, KPB-M15, constitutively produces a novel hematopoietic cytokine, termed stem cell growth factor (SCGF), possessing species-specific proliferative activities. Here we report the molecular cloning, expression, and characterization of a cDNA encoding human SCGF using a newly developed λSHDM vector that is more efficient for differential and expression cloning. cDNA for SCGF encodes a 29-kDa polypeptide without N-linked glycosylation. SCGF transiently produced by COS-1 cells supports growth of hematopoietic progenitor cells through a short-term liquid culture of bone marrow cells and exhibits promoting activities on erythroid and granulocyte/macrophage progenitor cells in primary semisolid culture with erythropoietin and granulocyte/macrophage colony-stimulating factor, respectively. Expression of SCGF mRNA is restricted to myeloid cells and fibroblasts, suggesting that SCGF is a growth factor functioning within the hematopoietic microenvironment. SCGF could disclose some human-specific mechanisms as yet unidentified from studies on the murine hematopoietic system.

The Thrombospondin Receptor CD47 (IAP) Modulates and Associates with α2β1 Integrin in Vascular Smooth Muscle Cells

The carboxyl-terminal domain of thrombospondin-1 enhances the migration and proliferation of smooth muscle cells. Integrin-associated protein (IAP or CD47) is a receptor for the thrombospondin-1 carboxyl-terminal cell-binding domain and binds the agonist peptide 4N1K (kRFYVVMWKk) from this domain. 4N1K peptide stimulates chemotaxis of both human and rat aortic smooth muscle cells on gelatin-coated filters. The migration on gelatin is specifically blocked by monoclonal antibodies against IAP and a β1 integrin, rather than αvβ3 as found previously for 4N1K-stimulated chemotaxis of endothelial cells on gelatin. Both human and rat smooth muscle cells displayed a weak migratory response to soluble type I collagen; however, the presence of 4N1K peptide or intact thrombospondin-1 provoked a synergistic chemotactic response that was partially blocked by antibodies to α2 and β1 integrin subunits and to IAP. A combination of antiα2 and IAP monoclonal antibodies completely blocked chemotaxis. RGD peptide and antiαvβ3 mAb were without effect. 4N1K and thrombospondin-1 did not augment the chemotactic response of smooth muscle cells to fibronectin, vitronectin, or collagenase-digested type I collagen. Complex formation between α2β1 and IAP was detected by the coimmunoprecipitation of both α2 and β1 integrin subunits with IAP. These data suggest that IAP can associate with α2β1 integrin and modulate its function.

Ligand occupancy of the alpha-V-beta3 integrin is necessary for smooth muscle cells to migrate in response to insulin-like growth factor.

Smooth muscle cells (SMCs) have been shown to migrate in response to insulin-like growth factor I (IGF-I). However, the mechanism mediating this response has not been determined. The migration rates of porcine and human vascular SMCs were assessed in a monolayer wounding assay. IGF-I and IGF-II induced increases of 141% and 97%, respectively, in the number of cells that migrated in 4 days. The presence of 0.2% fetal bovine serum in the culture medium was necessary for the IGFs to stimulate migration over uncoated plastic surfaces. However, if vitronectin was used as the substratum, IGF-I stimulated migration by 162% even in the absence of serum. To determine the role of integrins in mediating this migration, SMC surface proteins were labeled with 125I and immunoprecipitated with specific anti-integrin antibodies. Integrins containing alpha-V (vitronectin receptor), alpha5 (fibronectin receptor), and alpha3 (collagen/laminin receptor) subunits were the most abundant. IGF-I treatment caused a 73% reduction in alpha5-integrin subunit protein and a 25% increase in alpha-V subunit. More importantly, ligand binding of alpha-V-beta3 was increased by 2.4-fold. We therefore examined whether the function of the alpha-V-beta3 integrin was important for IGF-I-mediated migration. The disintegrin kistrin was shown by affinity crosslinking to specifically bind with high affinity to alpha-V-beta3 and not to alpha5-beta1 or other abundant integrins. The related disintegrin echistatin specifically inhibited 125I-labeled kistrin binding to alpha-V-beta3, while a structurally distinct disintegrin, decorsin, had 1000-fold lower affinity. The addition of increasing concentrations of either kistrin or echistatin inhibited IGF-I-induced migration, whereas decorsin had a minimal effect. The potency of these disintegrins in inhibiting IGF-I-induced migration paralleled their apparent affinity for the alpha-V integrin. Furthermore, an alpha-V-beta3 blocking antibody inhibited SMC migration by 80%. In summary, vitronectin receptor activation is a necessary component of IGF-I-mediated stimulation of smooth muscle migration, and alpha-V-beta3 integrin antagonists appear to be important reagents for modulating this process.

20-Hydroxyeicosatetraenoic acid mediates calcium/calmodulin-dependent protein kinase II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells

Norepinephrine (NE) and angiotensin II (Ang II), by promoting extracellular Ca2+ influx, increase Ca2+/calmodulin-dependent kinase II (CaMKII) activity, leading to activation of mitogen-activated protein kinase (MAPK) and cytosolic phospholipase A2 (cPLA2), resulting in release of arachidonic acid (AA) for prostacyclin synthesis in rabbit vascular smooth muscle cells. However, the mechanism by which CaMKII activates MAPK is unclear. The present study was conducted to determine the contribution of AA and its metabolites as possible mediators of CaMKII-induced MAPK activation by NE, Ang II, and epidermal growth factor (EGF) in vascular smooth muscle cells. NE-, Ang II-, and EGF-stimulated MAPK and cPLA2 were reduced by inhibitors of cytochrome P450 (CYP450) and lipoxygenase but not by cyclooxygenase. NE-, Ang II-, and EGF-induced increases in Ras activity, measured by its translocation to plasma membrane, were abolished by CYP450, lipoxygenase, and farnesyltransferase inhibitors. An AA metabolite of CYP450, 20-hydroxyeicosatetraenoic acid (20-HETE), increased the activities of MAPK and cPLA2 and caused translocation of Ras. These data suggest that activation of MAPK by NE, Ang II, and EGF is mediated by a signaling mechanism involving 20-HETE, which is generated by stimulation of cPLA2 by CaMKII. Activation of Ras/MAPK by 20-HETE amplifies cPLA2 activity and releases additional AA by a positive feedback mechanism. This mechanism of Ras/MAPK activation by 20-HETE may play a central role in the regulation of other cellular signaling molecules involved in cell proliferation and growth.

Proteomic definition of normal human luminal and myoepithelial breast cells purified from reduction mammoplasties

Normal human luminal and myoepithelial breast cells separately purified from a set of 10 reduction mammoplasties by using a double antibody magnetic affinity cell sorting and Dynabead immunomagnetic technique were used in two-dimensional gel proteome studies. A total of 43,302 proteins were detected across the 20 samples, and a master image for each cell type comprising a total of 1,738 unique proteins was derived. Differential analysis identified 170 proteins that were elevated 2-fold or more between the two breast cell types, and 51 of these were annotated by tandem mass spectrometry. Muscle-specific enzyme isoforms and contractile intermediate filaments including tropomyosin and smooth muscle (SM22) alpha protein were detected in the myoepithelial cells, and a large number of cytokeratin subclasses and isoforms characteristic of luminal cells were detected in this cell type. A further 134 nondifferentially regulated proteins were also annotated from the two breast cell types, making this the most extensive study to date of the protein expression map of the normal human breast and the basis for future studies of purified breast cancer cells.

Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic–scid/scid mice

Human hematopoiesis originates in a population of stem cells with transplantable lympho-myeloid reconstituting potential, but a method for quantitating such cells has not been available. We now describe a simple assay that meets this need. It is based on the ability of sublethally irradiated immunodeficient nonobese diabetic–scid/scid (NOD/SCID) mice to be engrafted by intravenously injected human hematopoietic cells and uses limiting dilution analysis to measure the frequency of human cells that produce both CD34−CD19+ (B-lymphoid) and CD34+ (myeloid) colony-forming cell progeny in the marrow of such recipients 6 to 8 weeks post-transplant. Human cord blood (CB) contains ≈5 of these competitive repopulating units (CRU) per ml that have a similar distribution between the CD38− and CD38+ subsets of CD34+ CB cells as long-term culture-initiating cells (LTC-IC) (4:1 vs. 2:1). Incubation of purified CD34+CD38− human CB cells in serum-free medium containing flt-3 ligand, Steel factor, interleukin 3, interleukin 6, and granulocyte colony-stimulating factor for 5–8 days resulted in a 100-fold expansion of colony-forming cells, a 4-fold expansion of LTC-IC, and a 2-fold (but significant, P < 0.02) increase in CRU. The culture-derived CRU, like the original CB CRU, generated pluripotent, erythroid, granulopoietic, megakaryopoietic, and pre-B cell progeny upon transplantation into NOD/SCID mice. These findings demonstrate an equivalent phenotypic heterogeneity amongst human CB cells detectable as CRU and LTC-IC. In addition, their similarly modest response to stimulation by a combination of cytokines that extensively amplify LTC-IC from normal adult marrow underscores the importance of ontogeny-dependent changes in human hematopoietic stem cell proliferation and self-renewal.

Formation of heteromeric gap junction channels by connexins 40 and 43 in vascular smooth muscle cells

Connexin (Cx) 43 and Cx40 are coexpressed in several tissues, including cardiac atrial and ventricular myocytes and vascular smooth muscle. It has been shown that these Cxs form homomeric/homotypic channels with distinct permeability and gating properties but do not form functional homomeric/heterotypic channels. If these Cxs were to form heteromeric channels, they could display functional properties not well predicted by the homomeric forms. We assessed this possibility by using A7r5 cells, an embryonic rat aortic smooth muscle cell line that coexpresses Cxs 43 and 40. Connexons (hemichannels), which were isolated from these cells by density centrifugation and immunoprecipitated with antibody against Cx43, contained Cx40. Similarly, antibody against Cx40 coimmunoprecipitated Cx43 from the same connexon fraction but only Cx40 from Cx (monomer) fractions. These results indicate that heteromeric connexons are formed by these Cxs in the A7r5 cells. The gap junction channels formed in the A7r5 cells display many unitary conductances distinct from homomeric/homotypic Cx43 or Cx40 channels. Voltage-dependent gating parameters in the A7r5 cells are also quite variable compared with cells that express only Cx40 or Cx43. These data indicate that Cxs 43 and 40 form functional heteromeric channels with unique gating and conductance properties.

Opposing mitogenic and anti-mitogenic actions of parathyroid hormone-related protein in vascular smooth muscle cells: A critical role for nuclear targeting

Parathyroid hormone-related protein (PTHrP) is a prohormone that is posttranslationally processed to a family of mature secretory forms, each of which has its own cognate receptor(s) on the cell surface that mediate the actions of PTHrP. In addition to being secreted via the classical secretory pathway and interacting with cell surface receptors in a paracrine/autocrine fashion, PTHrP appears to be able to enter the nucleus directly following translation and influence cellular events in an “intracrine” fashion. In this report, we demonstrate that PTHrP can be targeted to the nucleus in vascular smooth muscle cells, that this nuclear targeting is associated with a striking increase in mitogenesis, that this nuclear effect on proliferation is the diametric opposite of the effects of PTHrP resulting from interaction with cell surface receptors on vascular smooth muscle cells, and that the regions of the PTHrP sequence responsible for this nuclear targeting represent a classical bipartite nuclear localization signal. This report describes the activation of the cell cycle in association with nuclear localization of PTHrP in any cell type. These findings have important implications for the normal physiology of PTHrP in the many tissues which produce it, and suggest that gene delivery of PTHrP or modified variants may be useful in the management of atherosclerotic vascular disease.

The pattern of gene expression in human CD15+ myeloid progenitor cells

We performed a genome-wide analysis of gene expression in primary human CD15+ myeloid progenitor cells. By using the serial analysis of gene expression (SAGE) technique, we obtained quantitative information for the expression of 37,519 unique SAGE-tag sequences. Of these unique tags, (i) 25% were detected at high and intermediate levels, whereas 75% were present as single copies, (ii) 53% of the tags matched known expressed sequences, 34% of which were matched to more than one known expressed sequence, and (iii) 47% of the tags had no matches and represent potentially novel genes. The correct genes were confirmed by application of the generation of longer cDNA fragments from SAGE tags for gene identification (GLGI) technique for high-copy tags with multiple matches. A set of genes known to be important in myeloid differentiation were expressed at various levels and used different spliced forms. This study provides a normal baseline for comparison of gene expression in myeloid diseases. The strategy of using SAGE and GLGI techniques in this study has broad applications to the genome-wide identification of expressed genes.

NO hyperpolarizes pulmonary artery smooth muscle cells and decreases the intracellular Ca2+ concentration by activating voltage-gated K+ channels.

NO causes pulmonary vasodilation in patients with pulmonary hypertension. In pulmonary arterial smooth muscle cells, the activity of voltage-gated K+ (Kv) channels controls resting membrane potential. In turn, membrane potential is an important regulator of the intracellular free calcium concentration ([Ca2+]i) and pulmonary vascular tone. We used patch clamp methods to determine whether the NO-induced pulmonary vasodilation is mediated by activation of Kv channels. Quantitative fluorescence microscopy was employed to test the effect of NO on the depolarization-induced rise in [Ca2+]i. Blockade of Kv channels by 4-aminopyridine (5 mM) depolarized pulmonary artery myocytes to threshold for initiation of Ca2+ action potentials, and thereby increased [Ca2+]i. NO (approximately 3 microM) and the NO-generating compound sodium nitroprusside (5-10 microM) opened Kv channels in rat pulmonary artery smooth muscle cells. The enhanced K+ currents then hyperpolarized the cells, and blocked Ca(2+)-dependent action potentials, thereby preventing the evoked increases in [Ca2+]i. Nitroprusside also increased the probability of Kv channel opening in excised, outside-out membrane patches. This raises the possibility that NO may act either directly on the channel protein or on a closely associated molecule rather than via soluble guanylate cyclase. In isolated pulmonary arteries, 4-aminopyridine significantly inhibited NO-induced relaxation. We conclude that NO promotes the opening of Kv channels in pulmonary arterial smooth muscle cells. The resulting membrane hyperpolarization, which lowers [Ca2+]i, is apparently one of the mechanisms by which NO induces pulmonary vasodilation.

DNA strand breakage, activation of poly (ADP-ribose) synthetase, and cellular energy depletion are involved in the cytotoxicity of macrophages and smooth muscle cells exposed to peroxynitrite.

The free radicals nitric oxide and superoxide anion react to form peroxynitrite (ONOO-), a highly toxic oxidant species. In vivo formation of ONOO- has been demonstrated in shock and inflammation. Herein we provide evidence that cytotoxicity in cells exposed to ONOO- is mediated by DNA strand breakage and the subsequent activation of the DNA repair enzyme poly(ADP ribose) synthetase (PARS). Exposure to ONOO- (100 microM to 1 mM) inhibited mitochondrial respiration in cultured J774 macrophages and in rat aortic smooth muscle cells. The loss of cellular respiration was rapid, peaking 1-3 h after ONOO- exposure, and reversible, with recovery after a period of 6-24 h. The inhibition of mitochondrial respiration was paralleled by a dose-dependent increase in DNA strand breakage, reaching its maximum at 20-30 min after exposure to ONOO-. We observed a dose-dependent increase in the activity of PARS in cells exposed to ONOO-. Inhibitors of PARS such as 3-aminobenzamide (1 mM) prevented the inhibition of cellular respiration in cells exposed to ONOO-. Activation of PARS by ONOO--mediated DNA strand breakage resulted in a significant decrease in intracellular energy stores, as reflected by a decline of intracellular NAD+ and ATP content. 3-Aminobenzamide prevented the loss of NAD+ and ATP in cells exposed to ONOO-. In contrast, impairment of cellular respiration by the addition of the nitric oxide donors S-nitroso-N-acetyl-DL-penicillamine or diethyltriamine nitric oxide complex, was not associated with the development of DNA strand breaks, in concentrations up to 1 mM, and was largely refractory to PARS inhibition. Our results suggest that DNA damage and activation of PARS, an energy-consuming futile repair cycle, play a central role in ONOO--mediated cellular injury.

Core binding factor beta-smooth muscle myosin heavy chain chimeric protein involved in acute myeloid leukemia forms unusual nuclear rod-like structures in transformed NIH 3T3 cells.

Patients with the M4Eo subtype of acute myeloid leukemia almost invariably are found to have an inversion of chromosome 16 in their leukemic cells, which results in a gene fusion between the transcription factor called core binding factor beta (CBFbeta) on 16q and a smooth muscle myosin heavy chain (SMMHC) gene on 16p. Subcellular localizations of the wild-type CBFbeta and the CBFbeta-SMMHC fusion protein were determined by immunofluorescence of NIH 3T3 cells that overexpress wild-type or fusion protein. Normal CBFbeta showed an unexpected perinuclear pattern consistent with primary localization in the Golgi complex. The CBFbeta-SMMHC fusion protein had a very different pattern. Nuclear staining included rod-like crystalline structures as long as 11 microm. The heterodimeric partner of CBFbeta, CBFalpha, formed part of this complex. Cytoplasmic staining included stress fibers that colocalized with actin, probably as a consequence of the myosin heavy chain component of the fusion protein. Deletion of different regions of the CBFbeta portion of the fusion protein showed that binding to CBFalpha was not required for nuclear translocation. However, deletion of parts of the SMMHC domain of the fusion protein involved in myosin-mediated filament formation resulted in proteins that did not form rod-like structures. These observations confirm previous indirect evidence that the CBFbeta-SMMHC fusion protein is capable of forming macromolecular nuclear aggregates and suggests possible models for the mechanism of leukemic transformation.