Crosstalk between cannabinoid receptor 2 and lysophosphatidic acid receptor 5.

Cannabinoid receptor 2 (CB2) and lysophosphatidic acid receptor 5 (LPA5) are both classified as G-protein coupled receptors (GPCRs) activated by bioactive lipids and are highly expressed in colon cancer cells. However, crosstalk between two receptors and its potential effects on cancer cell physiology have not been fully elucidated. In the present study, the results of bioluminescence resonance energy transfer analysis showed that, among the LPA receptors, CB2 strongly and specifically interacted with LPA5. Both receptors were co-localized in the plasma membrane in the absence of agonists, and the receptors were co-internalized upon activation of either receptor alone or both receptors together. We further investigated the effects of expression of both receptors on cell proliferation and migration, and the molecular mechanisms underlying these effects in HCT116 colon cancer cells. Co-expression of receptors significantly increased cell proliferation and migration by increasing Akt phosphorylation and tumor progression-related gene expression, whereas no such effect was seen upon expression of either receptor alone. These results suggest the possibility of physical and functional crosstalk between CB2 and LPA5.

Interactions between lysophosphatidylinositol receptor GPR55 and sphingosine-1-phosphate receptor S1P5 in live cells.

Lysophosphatidylinositol (LPI) and sphingosine-1-phosphate (S1P) are bioactive lipids implicated in various cellular events including proliferation, migration, and cancer progression. LPI and S1P act as ligands for G-protein coupled GPR55 and S1P receptors, respectively, and activate specific signaling pathways. Both receptors are highly expressed in various cancer tissues and associated with tumor progression. However, physical and functional crosstalk between the two receptors has not been elucidated to date. Bioluminescence resonance energy transfer (BRET) experiments in the current study showed that S1P5 strongly and specifically interacts with GPR55. We observed co-internalization of both receptors upon agonist stimulation. Notably, activation of one receptor induced co-internalization of the partner receptor. Next, we examined functional crosstalk of the two receptors. Interestingly, while activation of the individual receptors augmented cell proliferation, ERK phosphorylation and cancer-associated gene expression in HCT116 cells, co-activation of both receptors inhibited these stimulatory effects. Our collective findings indicate that GPR55 and S1P5 form a heterodimer and their co-activation attenuates the stimulatory activity of each receptor on colon cancer progression.

Modulation of G-protein-coupled receptor 55-mediated signaling by regulator of G-protein signaling 2.

Activation of seven-transmembrane G-protein coupled receptor (GPCR) mediates extracellular signals into intracellular responses. G-protein coupled receptor 55 (GPR55) is one of GPCRs and activated by endogenous cannabinoids. A family of regulators of G-protein signaling (RGS) stimulates GTP hydrolysis of alpha subunit of G-protein (Gα) and inhibits GPCR/Gα-mediated signaling. RGS2 is member of R4 RGS family and mainly attenuates GPCR/Gαq signaling. Although RGS2 is known to modulate some GPCR signaling, the specific effects of RGS2 on GPR55-mediated signaling are not fully understood at present. Previously, we reported some RGS proteins interact with protease-activated receptors, one of GPCRs, and modulate their functions. Here, we investigated whether GPR55 interacts with RGS2, employing bioluminescence resonance energy transfer and co-immunoprecipitation analyses. Interestingly, GPR55 interacted with RGS2 alone and also formed a ternary complex with RGS2 and either Gαq or Gα12. In the presence of GPR55 alone and together with Gαq or Gα12, RGS2 translocated from the cytoplasm to plasma membrane while RGS1 remained in the cytoplasm. GPR55 activation significantly induced ERK phosphorylation and intracellular calcium mobilization, which were markedly inhibited by RGS2 in HCT116 colon cancer cell line. Furthermore, GPR55-mediated cell proliferation and migration of HCT116 cells, was significantly attenuated by RGS2. Our collective findings highlight a novel physiological function of RGS2, supporting its utility as a therapeutic target to control GPR55-induced pathophysiology.

Regulators of G-protein signaling, RGS2 and RGS4, inhibit protease-activated receptor 4-mediated signaling by forming a complex with the receptor and Gα in live cells.

BACKGROUND: Protease-activated receptor 4 (PAR4) is a seven transmembrane G-protein coupled receptor (GPCR) activated by endogenous proteases, such as thrombin. PAR4 is involved in various pathophysiologies including cancer, inflammation, pain, and thrombosis. Although regulators of G-protein signaling (RGS) are known to modulate GPCR/Gα-mediated pathways, their specific effects on PAR4 are not fully understood at present. We previously reported that RGS proteins attenuate PAR1- and PAR2-mediated signaling through interactions with these receptors in conjunction with distinct Gα subunits. METHODS: We employed a bioluminescence resonance energy transfer technique and confocal microscopy to examine potential interactions among PAR4, RGS, and Gα subunits. The inhibitory effects of RGS proteins on PAR4-mediated downstream signaling and cancer progression were additionally investigated by using several assays including ERK phosphorylation, calcium mobilization, RhoA activity, cancer cell proliferation, and related gene expression. RESULTS: In live cells, RGS2 interacts with PAR4 in the presence of Gαq while RGS4 binding to PAR4 occurs in the presence of Gαq and Gα12/13. Co-expression of PAR4 and Gαq induced a shift in the subcellular localization of RGS2 and RGS4 from the cytoplasm to plasma membrane. Combined PAR4 and Gα12/13 expression additionally promoted translocation of RGS4 from the cytoplasm to the membrane. Both RGS2 and RGS4 abolished PAR4-activated ERK phosphorylation, calcium mobilization and RhoA activity, as well as PAR4-mediated colon cancer cell proliferation and related gene expression. CONCLUSIONS: RGS2 and RGS4 forms ternary complex with PAR4 in Gα-dependent manner and inhibits its downstream signaling. Our findings support a novel physiological function of RGS2 and RGS4 as inhibitors of PAR4-mediated signaling through selective PAR4/RGS/Gα coupling. Video Abstract.

Development of the main olfactory system and main olfactory epithelium-dependent male mating behavior are altered in Go-deficient mice.

In mammals, initial detection of olfactory stimuli is mediated by sensory neurons in the main olfactory epithelium (MOE) and the vomeronasal organ (VNO). The heterotrimeric GTP-binding protein Go is widely expressed in the MOE and VNO of mice. Early studies indicated that Go expression in VNO sensory neurons is critical for directing social and sexual behaviors in female mice [Oboti L, et al. (2014) BMC Biol 12:31]. However, the physiological functions of Go in the MOE have remained poorly defined. Here, we examined the role of Go in the MOE using mice lacking the α subunit of Go Development of the olfactory bulb (OB) was perturbed in mutant mice as a result of reduced neurogenesis and increased cell death. The balance between cell types of OB interneurons was altered in mutant mice, with an increase in the number of tyrosine hydroxylase-positive interneurons at the expense of calbindin-positive interneurons. Sexual behavior toward female mice and preference for female urine odors by olfactory sensory neurons in the MOE were abolished in mutant male mice. Our data suggest that Go signaling is essential for the structural and functional integrity of the MOE and for specification of OB interneurons, which in turn are required for the transmission of pheromone signals and the initiation of mating behavior with the opposite sex.

Necdin modulates osteogenic cell differentiation by regulating Dlx5 and MAGE-D1.

Osteoblasts originate from mesenchymal stem cells that also differentiate into adipocytes, myoblasts, chondrocytes and fibroblasts. Osteogenic differentiation involves diverse regulatory proteins, including transcription and growth factors. Neurally differentiated embryonal carcinoma-derived protein (Necdin) has been identified as a key regulator of cell differentiation in various tissues, including neuronal, adipose, and muscular tissues; although its role in bone tissue remains to be established. Here, we investigated the potential involvement of Necdin in osteogenic differentiation. Our experiments revealed high expression of Necdin during osteoblast differentiation. Moreover, both transient and stable expression of Necdin induced osteoblast-specific markers in an osteogenic cell line through formation of a complex with melanoma-associated antigen D1 (MAGE-D1) and distal-less Homeobox 5 (Dlx5) and Runx2 promoter activation. Necdin expression was further associated with suppression of both cell proliferation and death in osteoblasts. Our results suggest that Necdin plays roles in cellular differentiation, proliferation and death in bone tissue.

The alpha subunit of Go modulates cell proliferation and differentiation through interactions with Necdin.

BACKGROUND: Heterotrimeric GTP-binding proteins (G-proteins) play an important role in mediating signal transduction generated by neurotransmitters or hormones. Go, a member of the Gi/Go subfamily, is the most abundant G-protein found in the brain. Recently, the alpha subunit of Go (Gαo) was characterized as an inducer of neuronal differentiation. However, its underlying molecular mechanisms have remained unclear to date, since the downstream effectors of Gαo are ambiguous. RESULTS: A neurally differentiated embryonal carcinoma-derived protein (Necdin) was isolated as an interacting partner for Gαo from a mouse brain cDNA library using yeast two-hybrid screening. Interactions between the proteins were confirmed with several affinity binding assays, both in vitro and in vivo. Necdin interacted directly and preferentially with activated Gαo, compared to wild-type protein. Interestingly, Gαo did not interact with Gαi, despite high sequence homology between the two proteins. We subsequently analyzed whether Gαo modulates the cellular activities of Necdin. Notably, expression of Gαo significantly augmented Necdin-mediated cellular responses, such as proliferation and differentiation. Moreover, activation of type 1 cannabinoid receptor (CB1R), a Gi/oα-coupled receptor, augmented cell growth suppression, which was mediated by Gαo and Necdin in U87MG cells containing CB1R, Gαo, and Necdin as normal components. CONCLUSIONS: These results collectively suggest that Necdin is a candidate downstream effector for Gαo. Our findings provide novel insights into the cellular roles of Gαo and its coupled receptor.

Cannabinoid receptor activation inhibits cell cycle progression by modulating 14-3-3ß.

Cannabinoids display various pharmacological activities, including tumor regression, anti-inflammatory and neuroprotective effects. To investigate the molecular mechanisms underlying the pharmacological effects of cannabinoids, we used a yeast two-hybrid system to screen a mouse brain cDNA library for proteins interacting with type 1 cannabinoid receptor (CB1R). Using the intracellular loop 3 of CB1R as bait, we identified 14-3-3ß as an interacting partner of CB1R and confirmed their interaction using affinity-binding assays. 14-3-3ß has been reported to induce a cell cycle delay at the G2/M phase. We tested the effects of cannabinoids on cell cycle progression in HeLa cells synchronized using a double-thymidine block-and-release protocol and found an increase in the population of G2/M phase cells. We further found that CB1R activation augmented the interaction of 14-3-3ß with Wee1 and Cdc25B, and promoted phosphorylation of Cdc2 at Tyr-15. These results suggest that cannabinoids induce cell cycle delay at the G2/M phase by activating 14-3-3ß.

Structure-Activity Relationship and Functional Evaluation of Cannabinoid Type-1 Receptor.

The type-1 cannabinoid receptor (CB1R) is a potential therapeutic target in several pathological conditions, including neuropsychological disorders and neurodegenerative diseases. Owing to their structural diversity, it is not easy to derive general structure-activity relationships (SARs) for CB1R ligands. In this study, CB1R ligands were classified into six structural families, and the corresponding SAR was determined for their affinities for CB1R. In addition, we determined their functional activities for the activation of extracellular signal-regulated kinases (ERKs). Among derivatives of indol-3-yl-methanone, the highest ligand affinity was observed when a pentyl and a naphthalenyl group were attached to the N1 position of the indole ring and the carbon site of the methanone moiety, respectively. In the case of adamantane indazole-3-carboxamide derivatives, the presence of fluorine in the pentyl group, the substituent at the N1 position of the indazole ring, strongly increased the affinity for CB1R. For (naphthalen-1-yl) methanone derivatives, the presence of 4-alkoxynaphthalene in the methanone moiety was more beneficial for the affinity to CB1R than that of a heterocyclic ring. The functional activities of the tested compounds, evaluated through ERK assay, were correlated with their affinity for CB1R, suggesting their agonistic nature. In conclusion, this study provides valuable insight for designing novel ligands for CB1R, which can be used to control psychiatric disorders and drug abuse.

BRET analysis reveals interaction between the lysophosphatidic acid receptor LPA2 and the lysophosphatidylinositol receptor GPR55 in live cells.

Lysophosphatidic acid (LPA) and lysophosphatidylinositol bind to the G protein-coupled receptors (GPCRs) LPA and GPR55, respectively. LPA2 , a type 2 LPA receptor, and GPR55 are highly expressed in colon cancer and involved in cancer progression. However, crosstalk between the two receptors and potential effects on cellular physiology are not fully understood. Here, using BRET analysis, we found that LPA2 and GPR55 interact in live cells. In the presence of both receptors, LPA2 and/or GPR55 activation facilitated co-internalization, and activation of GPR55, uncoupled with Gαi , induced reduction of intracellular cAMP. Notably, co-activation of receptors synergistically triggered further decline in the cAMP level, promoted cell proliferation, and increased the expression of cancer progression-related genes, suggesting that physical and functional crosstalk between LPA2 and GRR55 is involved in cancer progression.

The regulators of G protein signaling RGS16 and RGS18 inhibit protease-activated receptor 2/Gi/o signaling through distinct interactions with Gα in live cells.

Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor (GPCR) activated by endogenous proteases, in particular, trypsin. Although regulators of G protein signaling (RGS) are known to inhibit GPCR/Gα-mediated signaling, their specific effects on PAR2 are poorly understood at present. Here, we use a bioluminescence resonance energy transfer technique to investigate whether RGS16 and RGS18 bind PAR2 in live cells to regulate PAR2/Gαi/o -mediated signaling. Notably, we find that RGS16 binds to PAR2 in the presence of Gαi while RGS18 does not interact with PAR2, regardless of the presence of Gα. Both RGS16 and RGS18 inhibit PAR2/Gαi/o -mediated signaling. To our knowledge, the current study is the first to highlight the effects of RGS proteins on PAR2-mediated signaling.

Identification of warm temperature acclimation-associated 65-kDa protein-2 in Kumgang fat minnow Rhynchocypris kumgangensis.

Warm temperature acclimation-associated 65-kDa protein (Wap65) is known to respond to elevated water temperatures and the corresponding gene from several fish species has been cloned. Expression of Wap65 gene is induced by various physiological stresses, such as increase in water temperature, immune response and heavy metal exposure. Two isolated Wap65 genes, Wap65-1 and Wap65-2, display distinct tissue distribution and physiological functions despite high sequence homology. In a previous study, we identified the Wap65-1 gene (kmWap65-1) from Kumgang fat minnow, Rhynchocypris kumgangensis, a small freshwater fish endemic to Korea. The kmWap65-1 gene showed sequence homology with teleost Wap65-1 and mammalian hemopexin, and was highly expressed in response to increased water temperature and bacterial lipopolysaccharide (LPS) exposure. Here, we isolated kmWap65-2 from liver tissue of Kumgang fat minnow and compared the expression profiles of both kmWap65 genes following exposure to various physiological stresses, including thermal changes, bacterial challenge, and environmental toxins. Notably, while kmWap65-1 expression was significantly increased in response to high water temperature, LPS, cadmium, and iron, kmWap65-2 displayed no alterations in expression at high water temperature. However, kmWap65-2 expression was upregulated slightly in response to LPS and highly in presence of copper, bisphenol A, and estradiol. Based on the collective findings, we propose that kmWap65-1 and kmWap65-2 are multifunctional proteins with distinct functions that could serve as useful biomarkers for assessing physiological stress and associated responses in Kumgang fat minnow.

Regulator of G protein signaling 8 inhibits protease-activated receptor 1/Gi/o signaling by forming a distinct G protein-dependent complex in live cells.

Activation of seven-transmembrane-domain-possessing G protein-coupled receptors (GPCRs) by extracellular stimuli elicits intracellular responses. One class of GPCRs-protease-activated receptors (PARs)-is activated by endogenous proteases, such as thrombin and trypsin. Members of the regulator of G protein signaling (RGS) family stimulate GTP hydrolysis of G protein alpha (Gα) subunits, thereby inhibiting GPCR/Gα-mediated signaling. We previously reported that RGS2 and RGS4 inhibit PAR1/Gα-mediated signaling by interacting with PAR1 in a Gα-dependent manner. Here, employing the bioluminescence resonance energy transfer (BRET) technique, we identified RGS8 as a novel PAR1-interacting protein. Very little BRET activity was observed between PAR1-Venus (PAR1-Ven) and RGS8-Luciferase (RGS8-Luc) in the absence of Gα. However, in the presence of Gαo, BRET activity was specifically and significantly increased. This interaction was confirmed by biochemical and immunofluorescence assays. Notably, RGS8 inhibited PAR1/Gαi/o-mediated adenylyl cyclase and ERK activation, and prevented Gαo-induced neurite outgrowth and activation of Necdin protein, a downstream target of Gαo. Our findings suggest a novel function of RGS8 and reveal cellular mechanisms by which RGS8 mediates PAR1 inhibition.

Identification of a warm-temperature acclimation-associated 65-kDa protein encoded by a temperature- and infection-responsive gene in the Kumgang fat minnow Rhynchocypris kumgangensis.

Water temperature is one of the most important factors in fish physiology; thus, it is important to identify genes that respond to changes in water temperature. In this study, we identified a warm- temperature acclimation-associated 65-kDa protein (Wap65) in the Kumgang fat minnow Rhynchocypris kumgangensis, a small, cold-freshwater fish species endemic to Korea. Kumgang fat minnow Wap65-1 (kmWap65-1) was cloned using polymerase chain reaction (PCR)-based strategies, and was found to be highly homologous with teleost Wap65-1 and mammalian hemopexin, a heme-binding protein that transfers plasma heme into hepatocytes. kmWap65-1 mRNA was expressed mainly in the liver and its expression levels were significantly increased by both short- and long-term exposure to high temperature, which was evaluated by real-time quantitative PCR. Furthermore, the expression levels of kmWap65-1 were highly elevated by exposure to bacterial lipopolysaccharide. These results indicate that kmWap65-1 expression is associated with environmental stresses such as increases in water temperature and bacterial infection. J. Exp. Zool. 325A:65-74, 2016. © 2015 Wiley Periodicals, Inc.

cAMP induces neuronal differentiation of mesenchymal stem cells via activation of extracellular signal-regulated kinase/MAPK.

Mesenchymal stem cells are able to trans-differentiate into nonmesodermal lineage cells. Here, we identified downstream signaling molecules required for acquisition of neuron-like traits by mesenchymal stem cells following the elevation of intracellular cAMP levels. We found that forskolin induced neuron-like morphology and expression of neuron-specific enolase and neurofilament-200 in mesenchymal stem cells. Forskolin sequentially activated protein kinase A and B-regulation of alpha-fetoprotein (Raf), which led to phosphorylation of extracellular signal-regulated kinase. Importantly, blockade of extracellular signal-regulated kinase phosphorylation with a mitogen-activated protein kinase (MAPK) kinase inhibitor abrogated the forskolin-induced morphological changes and induction of neuronal proteins. These results indicate that extracellular signal-regulated kinase/MAPK mediates both cAMP-induced early cytoskeletal rearrangement and the later induction of neuronal markers in mesenchymal stem cells.

Primary cell culture method for the honeybee Apis mellifera.

Honeybees are among the most important pollinators in nature, and honeybee-associated products are useful in various areas, including the food industry. However, honeybees may be infected by various types of pathogens. The study of honeybee-associated diseases would greatly benefit from a successful cell culture system, but although some honeybee cell culture techniques have been introduced, these methods have not yet been fully established. Here, we describe a primary cell culture method for the honeybee, Apis mellifera. We isolated, sterilized, and seeded egg cells into non-coated cell culture dishes to generate cell aggregates. After approximately 10 d, aggregates were dissociated and seeded to cell culture dishes. Cell passages were continuously performed, with sub-culturing every 3-4 d. The cells expressed non-adherent phenotypes. Their growth increased with the passage number when they were cultured in growth medium based on L-15 insect medium but not Schneider's insect medium. Finally, polymerase chain reaction confirmed that the cells originated from A. mellifera. Our results suggest that the culturing methods described herein are appropriate for isolating primary cells from honeybee eggs. These methods could thus facilitate the study of honeybee-associated pathogenesis, development, and toxicology.

Modulation of the N-type calcium channel gene expression by the alpha subunit of Go.

Go, a heterotrimeric G-protein, is enriched in brain and neuronal growth cones. Although several reports suggest that Go may be involved in modulation of neuronal differentiation, the precise role of Go is not clear. To investigate the function of Go in neuronal differentiation, we determined the effect of Goalpha, the alpha subunit of Go, on the expression of Ca(v)2.2, the pore-forming unit of N-type calcium channels, at the transcription level. Treatment with cyclic AMP (cAMP), which triggers neurite outgrowth in neuroblastoma F11 cells, increased the mRNA level and the promoter activity of the Ca(v)2.2 gene. Overexpression of Goalpha inhibited neurite extension in F11 cells and simultaneously repressed the stimulatory effect of cAMP on the Ca(v)2.2 gene expression to the basal level. Targeted mutation of the Goalpha gene also increased the level of Ca(v)2.2 in the brain. These results suggest that Go may regulate neuronal differentiation through modulation of gene expression of target genes such as N-type calcium channels.

Inhibition of BETA2/NeuroD by Id2.

Id (Inhibitor of Differentiation) proteins belong to a family of transcriptional modulators that are characterized by a helix loop helix (HLH) region but lack the basic amino acid domain. Id proteins are known to interact with basic helix-loop-helix (bHLH) transcription factors and function as their negative regulators. The negative role of Id proteins has been well demonstrated in muscle development and some in neuronal cells. In this study, we investigated the effect of Id on the function of BETA2/NeuroD, a bHLH transcription factor responsible for neuron and endocrine cell specific gene expression. cDNAs of several Id isoforms were isolated by yeast two-hybrid system using the bHLH domain of E47, a ubiquitous bHLH partner as a bait. Id proteins expressed in COS M6 cells, were found in both cytosolic and nuclear fractions. Electrophoretic mobility shift assay showed that coexpression of Id2 proteins inhibited BETA2/ NeuroD binding to its target sequence, E-box. Id2 inhibited E-box mediated gene expression in a dose dependent manner in BETA2/NeuroD expressing HIT cells. Id coexpressed with BETA2/NeuroD in HeLa cells, inhibited the stimulatory activity of BETA2/NeuroD. These results suggest that Id proteins may negatively regulate tissue specific gene expression induced by BETA2/NeuroD in neuroendocrine cells and the inhibitory role of Id proteins during differentiation may be conserved in various tissues.

Overexpression of neurogenin1 induces neurite outgrowth in F11 neuroblastoma cells.

Neurogenin1 (Ngn1) is a basic helix-loop-helix (bHLH) transcription factor expressed in neuronal precursors in the developing nervous system. The function of Ngn1 in neurogenesis has been shown in various aspects. In this study, we investigated the neurogenic potential of Ngn1 using neuroblastoma cell line, F11, which could be induced to differentiate into neurons in the presence of cAMP. To investigate the expression of Ngn1, expression vectors for the full-length and the C- terminal deletion mutant of Ngn1 were constructed and their transactivation potential was verified using reporter gene containing the E-box sequence. Overexpression of the full-length Ngn1 induced neurite outgrowth in F11 cells in the absence of cAMP. A C-terminal deletion mutant, Ngn1(1-197), inhibited neurite outgrowth induced by cAMP in F11 cells. These results indicate that the Ngn1 plays an important role in differentiation of neuroblastoma cells and the C terminus of Ngn1 is essential for the efficient differentiation.

Isolation of muscle derived stem cells from rat and its smooth muscle differentiation [corrected].

We investigated whether stem cells (MDSC) from primary cultures of rat skeletal muscle can differentiate into the smooth muscle lineage in response to vascular endothelial growth factor (VEGF) and coculture with bladder smooth muscle cells. The MDSC were isolated from gastrocnemius muscle biopsies of normal 3-6 week-old Sprague-Dawley rats and purified by the preplate technique. Cells that took approximately 6 days to adhere to the collagen-coated flasks were termed late preplate (LP) cells, and were used in all the experiments. The early plate (EP) cells (pp1-pp4) contained some myogenic cells but were mostly fibroblasts (< 15% desmin+ cells) whereas the LP cells (pp5-pp6) were highly purified muscle-derived cells (pp6) (> 90% desmin+ cells). The muscle-derived stem cells (LP cells) were CD34+ or Sca-1+, CD45- and desmin+ by immunohistochemical staining. After two days of co-culture with bladder smooth muscle cells, about 25% of the muscle-derived stem cells were positive for alpha-smooth muscle actin (alpha-SMA)+. RT-PCR for alpha-SMA was positive in the VEGF stimulated MDSC, but negative in the absence of VEGF. In conclusion, rat muscle-derived stem cells exhibited stem cell properties (CD34+ or Sca-1+), and were not of hematogeous (CD45-) but of myogenic origin (desmin+). RT-PCR of alpha-SMA was positive in the VEGF stimulated muscle-derived stem cells.