Smooth Muscle Progenitor Cells Research Articles

Isolation of bone marrow stromal cell-derived smooth muscle cells by a human SM22alpha promoter: in vitro differentiation of putative smooth muscle progenitor cells of bone marrow.

Bone marrow stromal cells (BMSCs) have many characteristics of mesenchymal stem cells that can differentiate into smooth muscle cells (SMCs). However, there have been few studies closely following the cell development of smooth muscle lineage among BMSCs. To investigate the possible existence of a cell population committed to the SMC lineage among bone marrow adhesion cells, we tried to detect and follow the in vitro differentiation of such a cell type by using a promoter-sorting method with a human SM22alpha promoter (-480 bp)/green fluorescent protein (GFP) construct. The construct was transfected to adhesion cells that appeared 5 days after the seeding of mononuclear cells from bone marrow. GFP was first detectable 5 days after the transfection in a cell population [Ad(G) cells], which expressed PDGF-beta but neither mature (calponin) nor immature (SMemb) SMC-specific proteins at that time. However, the cells were eventually grown into individual clones that expressed SMC-specific proteins (alpha-smooth muscle actin, calponin, and SM-1), suggesting that Ad(G) cells have partly at least progenitor properties. Because early studies have reported that PDGF-beta signaling plays pivotal roles in the differentiation of mesenchymal smooth muscle progenitor cells, Ad(G) cells might be putative mesenchymal smooth muscle progenitors expressing PDGF-beta. We demonstrated the presence of a cell population fated to become SMCs and followed their differentiation into SMCs among BMSCs.

Bone Marrow-derived Vascular Cells in Response to Injury.

Intimal hyperplasia is a key lesion for various vascular disorders such as atherosclerosis, postangioplasty restenosis and transplant arteriopathy. It has widely been accepted that intimal smooth muscle cells (SMC) originate from the medial layer in the same artery. However, recent studies suggest that bone marrow can also provide circulating progenitors for vascular SMC. Bone marrow-derived SMC participate in neointimal formation in animal models of allotransplantation, severe mechanical injury and hyperlipidemia-induced atherosclerosis. In human, transplantation arteriopathy also seems to involve circulating SMC, but their role in atherosclerosis and restenosis remains to be elucidated. Mobilization, differentiation and proliferation steps of SMC progenitors will provide promising targets for novel therapeutic approaches against proliferative vascular diseases.

Sonic hedgehog regulates proliferation and differentiation of mesenchymal cells in the mouse metanephric kidney.

Signaling by the ureteric bud epithelium is essential for survival, proliferation and differentiation of the metanephric mesenchyme during kidney development. Most studies that have addressed ureteric signaling have focused on the proximal, branching, ureteric epithelium. We demonstrate that sonic hedgehog is expressed in the ureteric epithelium of the distal, non-branching medullary collecting ducts and continues into the epithelium of the ureter -- the urinary outflow tract that connects the kidney with the bladder. Upregulation of patched 1, the sonic hedgehog receptor and a downstream target gene of the signaling pathway in the mesenchyme surrounding the distal collecting ducts and the ureter suggests that sonic hedgehog acts as a paracrine signal. In vivo and in vitro analyses demonstrate that sonic hedgehog promotes mesenchymal cell proliferation, regulates the timing of differentiation of smooth muscle progenitor cells, and sets the pattern of mesenchymal differentiation through its dose-dependent inhibition of smooth muscle formation. In addition, we also show that bone morphogenetic protein 4 is a downstream target gene of sonic hedgehog signaling in kidney stroma and ureteral mesenchyme, but does not mediate the effects of sonic hedgehog in the control of mesenchymal proliferation.

Smooth Muscle Progenitor Cells of Bone Marrow Origin Contribute to the Development of Neointimal Thickenings in Rat Aortic Allografts and Injured Rat Carotid Arteries

Smooth Muscle Progenitor Cells of Bone Marrow Origin Contribute to the Development of Neointimal Thickenings in Rat Aortic Allografts and Injured Rat Carotid Arteries

Smooth-muscle progenitor cells of bone marrow origin contribute to the development of neointimal thickenings in rat aortic allografts and injured rat carotid arteries.

This study indicates that circulating progenitors of bone marrow origin give rise to cells with smooth muscle-like properties during formation of neointimal thickenings in the arterial wall after allotransplantation and after balloon injury. A segment of abdominal aorta was transplanted from female F344 to male LEW rats, and the grafts were analyzed for male cells by using the gene as a marker. Immunostaining demonstrated that CD45-positive leukocytes made up 35-45% of the neointimal cells during the 8-week period examined. Concurrently, up to 70% of the neointimal cells were of host origin, as shown by real-time polymerase chain reaction for the gene (Y chromosome). This suggests that the neointima contained host cells also of noninflammatory character. Accordingly, many cells positive for smooth-muscle alpha-actin were detected in this layer. To explore the possible bone marrow origin of allograft cells, female LEW rats were irradiated and substituted with bone marrow from male LEW rats. Subsequently, the animals received an aortic transplant from female F344 rats or were exposed to a balloon injury of the carotid artery. Immunostaining and real-time polymerase chain reaction confirmed the above findings, but the fractions of leukocytes and -positive cells were lower in the carotids than in the allografts. Combined primed in situ labeling and immunostaining verified that not only inflammatory but also smooth muscle-like cells of male origin appeared in the vessel wall in both situations. These observations suggest that the smooth-muscle cells that participate in the development of neointimal lesions during vascular disease may, in part, originate from circulating progenitors.

Smooth muscle progenitor cells in human blood.

Recent animal data suggest that vascular smooth muscle cells within the neointima of the vessel wall may originate from bone marrow, providing indirect evidence for circulating smooth muscle progenitor cells (SPCs). Evidence for circulating SPCs in human subjects does not exist, and the mechanism whereby such putative SPCs may home to sites of plaque formation is presently not understood but is likely to involve expression of specific surface adhesion molecules, such as integrins. In this study, we aimed to culture smooth muscle outgrowth cells (SOCs) from SPCs in human peripheral blood and characterize surface integrin expression on these cells. Human mononuclear cells isolated from buffy coat were seeded on collagen type 1 matrix and outgrowth cells selected in endothelial growth medium (EGM-2) or EGM-2 and platelet-derived growth factor BB. Selection in platelet-derived growth factor BB-enriched medium caused rapid outgrowth and expansion of SOC to >40 population doublings in a 4-month period. These SOCs were positive for smooth muscle cell-specific alpha actin (alphaSMA), myosin heavy chain, and calponin on immunofluorescence and Western blotting and were also positive for CD34, Flt1, and Flk1 receptor but negative for Tie-2 receptor expression, suggesting a potential bone marrow angioblastic origin. In contrast, endothelial outgrowth cells (EOCs) grown in EGM-2 alone and the initial MNC population were negative for these smooth muscle-specific markers. Integrin alpha5beta1 expression by FACS and Western blotting was significantly increased in SOCs compared with EOCs, and this was confirmed by 8-fold greater adhesion of SOC to fibronectin (P<0.001), an effect that could be decreased using an alpha5beta1 antibody. Finally, SOC showed a significantly greater in vitro proliferative potential compared with EOCs of similar passage (P<0.001). This study demonstrates for the first time outgrowth of smooth muscle cells with a specific growth, adhesion, and integrin profile from putative SPC in human blood. These data have implications for our understanding of adult vascular smooth muscle cell differentiation, proliferation, and homing.

Expression of TGFbeta3 RNA during chick embryogenesis: a possible important role in cardiovascular development.

We examined the temporal and spatial expression pattern of transforming growth factor (TGF)-beta3 RNA during chick embryogenesis from stage 6 to stage 33 (Hamburger and Hamilton scale) by using in situ hybridization. During cardiogenesis, TGFbeta3 mRNA was first expressed in the premyocardium at stage 8 and thereafter it was localized in endocardial cushion tissue and the ventricular myocardium until the end of embryogenesis. During the formation of the major arteries, mRNA for TGFbeta3 was found in smooth muscle progenitor cells, but not in endothelium. In addition, TGFbeta3 mRNA was detectable in various mesoderm-derived tissues, such as the notochord, myotome, pleura, peritoneum, mesenchymal cells in the limb, and developing bone. These results suggest that TGFbeta3 plays an important role in the development of the cardiovascular system and of other mesodermal derivatives during chicken embryogenesis.

MONOCLONAL ANTIBODY CONFIRMATION OF A PRIMARY LEIOMYOMA OF THE TESTIS

Primary mesenchymal tumors of the testis are rare, with fewer than 30 cases reported in the world literature. Although the data are scant, primary mesenchymal testis tumors appear to exhibit the same clinical characteristics as their counterparts in other tissues and organs.' Leiomyoma has been reported throughout the genitourinary tract, yet it is extremely rare in the testis, with only 6 cases reported. Previous authors have based their diagnosis on nonspecific histological stains or gross histological analy~is.~,~ We report diagnostic confirmation of a testis leiomyoma using novel monoclonal antibodies. CASE RE PORT An 18-year-old man presented with a 3-year history of a painless right testicular mass. He denied any constitutional symptoms, weight loss, lower urinary tract symptoms or gynecomastia. Physical examination was remarkable for a 1 cm. indurated mass located over the upper pole of the right testis. Serum lactic dehydrogenase, a-fetoprotein and p-human chorionic gonadotropin were within normal limits. The chest x-ray was normal. Scrota1 ultrasound revealed a 1 cm. hypoechoic mass in the superomedial aspect of the right testis (fig. 1). Inguinal exploration with wedge resection of the mass was performed. A frozen section of the mass was interpreted as a spindle cell neoplasm of uncertain malignant potential and radical orchiectomy was completed. Followup was uneventful 1 year after surgery. DISCUSSION Previously, the diagnosis of a primary testis leiomyoma was based on nonspecific histological stain^,^,^ which leads to uncertainty with regard to neoplasm identity, especially in view of its rarity. In our case actin specific monoclonal antibodies were used to detect smooth muscle specific actin in the neoplastic cells (fig. 2). Previous study has proved that the monoclonal antibodies used in our case (clone 14A and HHF 35) reliably mark the product of the a and y isoactin genes, thus detecting neoplastic cells of smooth muscle origin.4 The morphological appearance of the tumor did not reveal mitotic figures, hemorrhage or necrosis, thus supporting the diagnosis of a clinically benign leiomyoma. The patient is diseasefree at 1 year postoperatively. Myoid cells, or potentially their progenitors, may give rise to these particular tumors in the testis. Myoid cells can be found in the tunica albuginea, tunica propria of the seminiferous tubules and smooth muscle of blood vessel walls. Testis leiomyomas most likely originate from the progenitors of smooth muscle cells and not the actual smooth muscle cells themselves. These progenitor cells remain to be identified.

Immunohistochemical evidence for a mesothelial contribution to the ventral wall of the avian aorta.

We report morphological and immunohistochemical evidence for a translocation of cells from the coelomic mesothelium to the aortic wall between the developmental stages HH16 and HH22 of the quail embryos. The coelomic mesothelial cells closest to the aorta showed, at these stages, increased mitotic activity, reduced intercellular adhesion, loss of tight junctions, and long basal cytoplasmic processes. Coinciding with these morphological traits, cytokeratin immunoreactivity was found in the mesothelium, in cells of the aortic wall and throughout the ventral periaortic mesenchyme (but not in the lateral and dorsal aortic regions). Vimentin immunoreactivity colocalized with cytokeratin in the mesothelial cells adjacent to the aorta. In the ventral aortic wall, cytokeratin colocalized with smooth muscle alpha-actin and with the 1E12 antigen (a smooth muscle-specific alpha-actinin isoform). We think that the morphological and immunolocalization data observed are compatible with an epithelial-mesenchymal transition by which mesothelial-derived cells contribute to the splanchnic mesoderm and aortic wall. The precise coincidence between the mesothelial contribution and the emergence of the aortic smooth muscle cells progenitors, as well as the immunolocalization data, suggest a potential relationship of the mesothelial-derived cells with this cell lineage. This may explain the observed ventrodorsal asymmetry in the distribution of smooth muscle cells progenitors in the aortic wall.

Cloning of capsulin, a basic helix-loop-helix factor expressed in progenitor cells of the pericardium and the coronary arteries

The basic helix-loop-helix (bHLH) class of transcription factors have been linked to a variety of cellular differentiation processes, including myogenesis, neurogenesis and hematopoiesis. Here we report the cloning of a new member of this family of factors, capsulin. Capsulin was shown to be expressed as early as 9.5 days of mouse development, with expression in mesodermal cells that are progenitors of the epicardium and the coronary arteries. At later stages of development, expression is seen in mesenchymal cells that are closely associated with the epithelium of the developing lung, gut and kidney. In the proepicardial organ, and in the organs where it is expressed in later development, Capsulin is expressed in cells that give will give rise to smooth muscle. Given the likely expression of Capsulin in smooth muscle cell progenitors, and significant sequence similarity through the bHLH domain, capsulin may be a functional ortholog of a Drosophila gene that is expressed in cells that give rise to the longitudinal visceral muscle. Capsulin alone or in combination with other bHLH proteins, was shown to function as a transcription factor by its ability to transactivate both a synthetic and a native promoter, each of which contains multiple E-boxes. These studies extend the growing family of bHLH factors that are expressed in the early mesoderm, and suggest that capsulin may have a functional role in development of the coronary vasculature and organs containing epithelial lined tubular structures.

Not all myofibroblasts are alike: revisiting the role of PDGF-A and PDGF-B using PDGF-targeted mice.

Analyses of platelet-derived growth factor (PDGF) and PDGF-receptor knockout mice show that the development of specific subsets of smooth muscle cells (SMCs) depend on PDGF. Pericytes and mesangial cells failed to develop in PDGF-B deficient embryos, whereas alveolar SMCs failed to develop in PDGF-A deficient embryos. The ontogeny of pericytes and alveolar SMCs suggests a common theme in the formation of PDGF-dependent SMCs, in which PDGF-receptor positive SMC progenitors spread from proximal to distal sites along PDGF-expressing epithelial, or endothelial, tubes.

Alveogenesis failure in PDGF-A-deficient mice is coupled to lack of distal spreading of alveolar smooth muscle cell progenitors during lung development.

PDGF-A(-/-) mice lack lung alveolar smooth muscle cells (SMC), exhibit reduced deposition of elastin fibres in the lung parenchyma, and develop lung emphysema due to complete failure of alveogenesis. We have mapped the expression of PDGF-A, PDGF receptor-alpha, tropoelastin, smooth muscle alpha-actin and desmin in developing lungs from wild type and PDGF-A(-/-) mice of pre- and postnatal ages in order to get insight into the mechanisms of PDGF-A-induced alveolar SMC formation and elastin deposition. PDGF-A was expressed by developing lung epithelium. Clusters of PDGF-Ralpha-positive (PDGF-Ralpha+) mesenchymal cells occurred at the distal epithelial branches until embryonic day (E) 15.5. Between E16.5 and E17.5, PDGF-Ralpha+ cells multiplied and spread to acquire positions as solitary cells in the terminal sac walls, where they remained until the onset of alveogenesis. In PDGF-A(-/-) lungs PDGF-Ralpha+ cells failed to multiply and spread and instead remained in prospective bronchiolar walls. Three phases of tropoelastin expression were seen in the developing lung, each phase characterized by a distinct pattern of expression. The third phase, tropoelastin expression by developing alveolar SMC in conjunction with alveogenesis, was specifically and completely absent in PDGF-A(-/-) lungs. We propose that lung PDGF-Ralpha+ cells are progenitors of the tropoelastin-positive alveolar SMC. We also propose that postnatal alveogenesis failure in PDGF-A(-/-) mice is due to a prenatal block in the distal spreading of PDGF-Ralpha+ cells along the tubular lung epithelium during the canalicular stage of lung development.

Expression of collagens and decorin during aortic arch artery development: Implications for matrix pattern formation

The elastic matrix of the large arteries shows a high level of spatial order. However, the mechanisms by which such order is established and maintained are largely unknown. The embryonic development of the avian heart and great vessels provides an appropriate model to investigate these mechanisms. In control embryos, an elastic matrix with a high level of spatial order develops in the nascent great vessels. But after the normal vascular smooth muscle (VSM) progenitor cells in the great vessels are experimentally replaced by other VSM progenitor cells, the elastic extracellular matrix is congenitally disordered. The present study used this model to test the hypothesis that the proteoglycan decorin was involved in the establishment and maintenance of the normal three-dimensional spatial order of the vascular elastic matrix. The temporospatial expression of decorin was analysed during development of normal vessels and in experimental vessels with surrogate VSM. The results showed the following: (1) the expression of decorin was related in time and space to the establishment of large helical collagen type III fibers that are characteristic of the normal elastic extracellular matrix; (2) in the experimental extracellular matrix there were few helical fibers of collagen type III, but those that were present remained positive for decorin; and (3) in both control and experimental vessels, decorin associated with neither fibers of collagen type I nor fibers of collagen type III in any conformation other than the large helical fibers. These data indicate a previously unrecognized relationship between decorin and the spatial order of the physiologically significant helical fibers of collagen type III.