Expression Of Pericyte Markers Research Articles

EDNRA-Expressing Mesenchymal Cells Are Expanded in Myeloma Interstitial Bone Marrow and Associated with Disease Progression.

Multiple myeloma (MM) induces dysfunctional bone marrow (BM) mesenchymal cells and neoangiogenesis. Pericytes and smooth muscle cells (SMCs) could detach from vessels and become cancer-associated fibroblasts. We found that the pericyte and SMC marker endothelin receptor type A (EDNRA) is overexpressed in whole MM bone biopsies; we sought to characterize its expression. EDNRA expression gradually increased with disease progression. High-risk MM patients had higher EDNRA expression than low-risk MM patients and EDNRA expression was highest in focal lesions. High EDNRA expression was associated with high expression of pericyte markers (e.g., RGS5, POSTN, and CD146) and the angiogenic marker FLT1. A single-cell analysis of unexpanded BM mesenchymal cells detected EDNRA expression in a subset of cells that coexpressed mesenchymal cell markers and had higher expression of proliferation genes. Immunohistochemistry revealed that the number of EDNRA+ cells in the interstitial BM increased as MM progressed; EDNRA+ cells were prevalent in areas near the MM focal growth. EDNRA+ cells were detached from CD34+ angiogenic cells and coexpressed RGS5 and periostin. Therefore, they likely originated from pericytes or SMCs. These findings identify a novel microenvironmental biomarker in MM and suggest that the presence of detached EDNRA+ cells indicates disrupted vasculature and increased angiogenesis.

Differential pericyte marker expression in craniofacial benign and malignant vascular tumors.

Vascular anomalies and tumors are common in the head, neck, and craniofacial areas and are associated with abnormalities in the angiomatous architecture. However, the etiology and molecular basis for the pathogenesis of most vascular lesions are still unknown. Pericytes are mural cells that surround endothelial cells. Besides angiogenesis and other physiological functions, pericytes play an important role in vascularized tissue repair and as resident mesenchymal stem/progenitor cells. Perivascular cells demonstrate a distinct immunohistochemical profile, including expression of alpha-smooth muscle actin (α-SMA), CD146, CD105, and PDGFRβ, without endothelial differentiation (absence of CD31 and CD34 immunoreactivity). These pericyte markers have been shown to be expressed in soft tissue hemangiomas. However, they have not been fully examined in intraosseous hemangiomas. In this study, we compared mesenchymal stem cell (MSC)expression of CD146 and α-SMA markers inpericytes from hemangiomas from different tissues and malignant vascular tumors. The results demonstrated an increased expression of pericyte markers in perivascular cells of benign hemangiomas, especially intraosseous hemangiomas and a significantly reduced expression of pericyte markers in malignant angiosarcomas. The evidence provides insight into the function of pericytes in vascular tumors and suggests their role in vascular tumor disease types.

MiR-24-3p regulates the differentiation of adipose-derived stem cells toward pericytes and promotes fat grafting vascularization.

Adipose-derived stem cells (ADSCs) enhance fat graft survival by promoting neovascularization. The mechanism that promotes ADSCs differentiation toward pericytes was not known. We treated ADSCs with conditional medium (CM) from endothelial cells (ECs) or human recombinant transforming growth factor β (TGF-β) to induce differentiation into pericytes. Pericytes markers, including platelet-derived growth factor receptor β (PDGFRβ), alpha-smooth muscle actin (α-SMA), and desmin, were examined. Pericytes differentiation markers, migration, and their association with ECs were examined in ADSCs transfected with miR-24-3p mimics and inhibitors. Bioinformatics target prediction platforms and luciferase assays were used to investigate whether PDGFRβ was directly targeted by miR-24-3p. In vivo, fat mixed with ADSCs transfected with miR-24-3p mimics or inhibitors was implanted subcutaneously on the lower back region of nude mice. Fat grafts were harvested and analyzed at 2, 4, 6, and 8 weeks. Results showed that endogenous TGF-β derived from CM from EC or human recombinant TGF-β promoted migration, association with ECs, and induced expression of pericyte markers (PDGFRβ, α-SMA, Desmin) in ADSCs. MiR-24-3p directly targeted PDGFRβ in ADSCs by lucifer reporter assays. Inhibition of miR-24-3p promoted pericytes differentiation, migration, and association with ECs in ADSCs. Inhibition of miR-24-3p in ADSCs promoted survival, integrity, adipocyte viability, vascularization, pericytes association with ECs, and reduced fibrosis, whereas overexpression of miR-24-3p in ADSCs yielded the opposite results. Collectively, TGF-β released by ECs induced ADSCs differentiation toward pericytes through miR-24-3p. Downregulation of miR-24-3p in ADSCs induced survival, integrity, adipocyte viability, vascularization, pericytes association with ECs, and reduced fibrosis after fat grafting.

Adult-induced genetic ablation distinguishes PDGFB roles in blood-brain barrier maintenance and development

Platelet-derived growth factor B (PDGFB) released from endothelial cells is indispensable for pericyte recruitment during angiogenesis in embryonic and postnatal organ growth. Constitutive genetic loss-of-function of PDGFB leads to pericyte hypoplasia and the formation of a sparse, dilated and venous-shifted brain microvasculature with dysfunctional blood-brain barrier (BBB) in mice, as well as the formation of microvascular calcification in both mice and humans. Endothelial PDGFB is also expressed in the adult quiescent microvasculature, but here its importance is unknown. We show that deletion of Pdgfb in endothelial cells in 2-months-old mice causes a slowly progressing pericyte loss leading, at 12–18 months of age, to ≈50% decrease in endothelial:pericyte cell ratio, ≈60% decrease in pericyte longitudinal capillary coverage and >70% decrease in pericyte marker expression. Similar to constitutive loss of Pdgfb, this correlates with increased BBB permeability. However, in contrast to the constitutive loss of Pdgfb, adult-induced loss does not lead to vessel dilation, impaired arterio-venous zonation or the formation of microvascular calcifications. We conclude that PDFGB expression in quiescent adult microvascular brain endothelium is critical for the maintenance of pericyte coverage and normal BBB function, but that microvessel dilation, rarefaction, arterio-venous skewing and calcification reflect developmental roles of PDGFB.

OMO-1 reduces progression and enhances cisplatin efficacy in a 4T1-based non-c-MET addicted intraductal mouse model for triple-negative breast cancer

c-MET is considered a driver of cancer progression, impacting tumor growth and tumor-supporting stroma. Here, we investigated the therapeutic efficacy of OMO-1, a potent and selective c-MET inhibitor, in an immunocompetent intraductal mouse model for triple-negative breast cancer (TNBC). OMO-1 reduced non-c-MET addicted 4T1 tumor progression dose dependently as monotherapeutic and provided additional disease reduction in combination with cisplatin. At the stromal level, OMO-1 significantly reduced neutrophil infiltration in 4T1 tumors, promoted immune activation, and enhanced cisplatin-mediated reduction of tumor-associated macrophages. OMO-1 treatment also reduced 4T1 tumor hypoxia and increased expression of pericyte markers, indicative for vascular maturation. Corroborating this finding, cisplatin delivery to the 4T1 primary tumor was enhanced upon OMO-1 treatment, increasing cisplatin DNA-adduct levels and tumor cell death. Although verification in additional cell lines is warranted, our findings provide initial evidence that TNBC patients may benefit from OMO-1 treatment, even in cases of non-c-MET addicted tumors.

In Vitro Conditioning Determines the Capacity of Dental Pulp Stem Cells to Function as Pericyte-Like Cells.

Dental pulp has been revealed as an accessible and a rich source of mesenchymal stem cells (MSCs) and its biological potential is currently under intense investigation. MSCs from dental pulp stem cells (DPSCs) have been indicated as a heterogeneous population oriented not only in repairing dentine but also in maintaining vascular and nervous homeostasis of the teeth. We sought to verify the phenotype of cells isolated from dental pulp of young donors and to investigate in vitro their role as pericyte-like cells. Specifically, we evaluated how culture conditions can modulate expression of pericyte markers in DPSCs and their capacity to stabilize endothelial tubes in vitro. DPSCs cultured in standard conditions expressed MSC markers and demonstrated to contain a population expressing the pericyte marker NG2. These DPSCs were associated with low sprouting capacity in extra-cellular (EC) Matrix and limited ability in retaining tubes formed by endothelial cells in a coculture angiogenesis model. When cultured in endothelial growth medium (EGM)-2, DPSCs significantly upregulated NG2, and partially alpha-smooth muscle actin. The resulting population conserved the stem marker CD73, but was negative for calponin and endothelial markers. EGM-2-conditioned DPSCs showed a higher sprouting ability in EC Matrix and efficient association with human umbilical vein endothelial cells allowing the partial retention of endothelial tubes for several days. Among growth factors contained in EGM-2 we identified basic fibroblast growth factor (bFGF) as mainly responsible for NG2 upregulation and long-term stabilization of endothelial tubes. According to the in vitro analysis, DPSCs represent an effective source of pericytes and the appropriate culture conditions could result in a population with a promising ability to stabilize vessels and promote vascular maturation.

MicroRNA‐26b‐5p Regulates Bone Marrow Mesenchymal Stem Cell Differentiation to Pericytes via Targeting Platelet‐Derived Growth Factor Receptor‐β and Interacting with Long non‐Coding RNA Maternally Expressed Gene 3 in Liver Fibrosis and Angiogenesis

Background/AimsPericytes are important regulators of vascular morphogenesis and function in multiple biological processes, including liver fibrosis, which is closely associated with angiogenesis. However, the origin and functional role of pericytes during liver fibrosis have not been deciphered yet.MethodsMice received irradiation and transplantation of EGFP‐labeled bone marrow (BM) mesenchymal stem cells (BMSCs), which obtained from EGFP transgenic mice and had been sorted and purified by immunomagnetic cell sorting with anti‐CD146 microbeads. Four weeks later, the BM was reconstructed and the chimera mice with EGFP‐labeled BMSCs were fed with methionine‐choline‐deficient and high fat (MCDHF) diet to build liver injury model. Expression of pericyte markers or fibrosis markers was characterized by qRT‐PCR, Western blot, immunofluerescent staining and microarray analysis. Biotin‐avidin pull‐down and lucifer reporter assay were performed to validate the binding of microRNA (miR)‐26b‐5p and its target platelet‐derived growth factor receptor beta (PDGFR‐β). GO, KEGG and Reactome enrichment analysis was conducted to clarify the function of differentially expressed genes.ResultsIncreased expression of pericyte markers (e.g. PDGFR‐β, neural glial antigen 2, regulator of G protein signaling 5) was detected in MCDHF mice with a positive correlation with fibrosis markers (e.g. TGF‐β1, α‐smooth muscle actin, procollagen α1(I) and (III)). Numerous PDGFR‐β+ pericytes was found in the fibrotic liver not only around mature vessels but also around nascent vessels, and a significant proportion (72.5%) of pericytes in mouse fibrotic liver derived from BMSCs. BMSCs differentiated into pericytes under TGF‐β1 treatment in vitro. Long non‐coding RNA maternally expressed gene 3 (LncMEG3), which had been focused on its effects on angiogenesis recently, positively regulated PDGFR‐β expression during BMSC differentiation to pericytes. Bioinformatics analysis from three databases (miRanda, TargetScan, and RNA22v2) suggested that miR‐26b‐5p was predicted to target PDGFR‐β. MiR‐26b‐5p negatively regulated BMSC differentiation to pericytes via directly targeting PDGFR‐β in cultured BMSCs. GO, KEGG and Reactome enrichment analysis revealed that miR‐26b‐5p overexpression affected a list of genes associated with cell differentiation, angiogenesis and extracellular matrix. LncMEG3 acted as a molecular sponge of miR‐26b‐5p thus contributed to the de‐repression of miRNA target PDGFR‐β. MiR‐26b‐5p agomir blocked BMSC differentiation to pericytes and attenuates liver fibrosis and angiogenesis in vivo.ConclusionmiR‐26b‐5p regulates BMSC differentiation to pericytes via targeting PDGFR‐β and interacting with lncMEG3, which may represent an effective therapeutic strategy for liver fibrosis.Support or Funding InformationThis work was supported by grants from the National Natural and Science Foundation of China (81430013, 81500465) and the Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges under Beijing Municipality(IDHT20150502)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Modelling physiological and pathological conditions to study pericyte biology in brain function and dysfunction

BackgroundBrain pericytes ensheathe the endothelium and contribute to formation and maintenance of the blood–brain-barrier. Additionally, pericytes are involved in several aspects of the CNS immune response including scarring, adhesion molecule expression, chemokine secretion, and phagocytosis. In vitro cultures are routinely used to investigate these functions of brain pericytes, however, these are highly plastic cells and can display differing phenotypes and functional responses depending on their culture conditions. Here we sought to investigate how two commonly used culture media, high serum containing DMEM/F12 and low serum containing Pericyte Medium (ScienCell), altered the phenotype of human brain pericytes and neuroinflammatory responses.MethodsPericytes were isolated from adult human brain biopsy tissue and cultured in DMEM/F12 (D-pericytes) or Pericyte Medium (P-pericytes). Immunocytochemistry, qRT-PCR, and EdU incorporation were used to determine how this altered their basal phenotype, including the expression of pericyte markers, proliferation, and cell morphology. To determine whether culture media altered the inflammatory response in human brain pericytes, immunocytochemistry, qRT-PCR, cytometric bead arrays, and flow cytometry were used to investigate transcription factor induction, chemokine secretion, adhesion molecule expression, migration, phagocytosis, and response to inflammatory-related growth factors.ResultsP-pericytes displayed elevated proliferation and a distinct bipolar morphology compared to D-pericytes. Additionally, P-pericytes displayed lower expression of pericyte-associated markers NG2, PDGFRβ, and fibronectin, with notably lower αSMA, CD146, P4H and desmin, and higher Col-IV expression. Nuclear NF-kB translocation in response to IL-1β stimulation was observed in both cultures, however, P-pericytes displayed elevated expression of the transcription factor C/EBPδ, and lower expression of the adhesion molecule ICAM-1. P-pericytes displayed elevated phagocytic and migratory ability. Both cultures responded similarly to stimulation by the growth factors TGFβ1 and PDGF-BB.ConclusionsDespite differences in their phenotype and magnitude of response, both P-pericytes and D-pericytes responded similarly to all examined functions, indicating that the neuroinflammatory phenotype of these cells is robust to culture conditions.

Human predecidual stromal cells have distinctive characteristics of pericytes: Cell contractility, chemotactic activity, and expression of pericyte markers and angiogenic factors

IntroductionHuman decidual stromal cells (DSCs) play a key role in maternal–fetal interactions. Precursors of DSCs (preDSCs) localize around vessels in both the endometrium and decidua. Previous studies suggested a relationship between preDSCs and pericytes because these cells share a perivascular location, alpha smooth muscle actin (α-SM actin) expression and the ability to contract under the effects of cytokines. MethodsTo further study this relationship, we established 15 human preDSC lines and 3 preDSC clones. The preDSC lines and clones were tested by flow cytometry with a panel of 29 monoclonal antibodies, 14 of which are pericyte markers. The expression of angiogenic factors was determined by RT-PCR, chemotactic activity was studied with the migration assay, and cell contractility was evaluated with the collagen cell contraction assay. Confocal microscopy was used to study decidual sections. ResultsUnder the effect of progesterone and cAMP, these lines decidualized in vitro: the cells became rounder and secreted prolactin, a marker of physiological DSC differentiation (decidualization). The antigen phenotype of these preDSC lines and clones was fully compatible with that reported for pericytes. PreDSC lines displayed pericyte characteristics: they expressed angiogenic factors and showed chemotactic and cytokine-induced contractile activity. Confocal microscopic examination of decidual sections revealed the expression of antigens detected in preDSC lines: α-SM actin colocalized with CD146, CD140b, MFG-E8, nestin, and STRO-1 (all of which are pericyte markers) in cells located around the vessels, a distinctive location of preDSCs and pericytes. DiscussionTaken together, our results show that preDSCs are pericyte-like cells.

Equine Mesenchymal Stromal Cells Retain a Pericyte-Like Phenotype.

Mesenchymal stem/stromal cells (MSCs) have been used in human and equine regenerative medicine, and interest in exploiting their potential has increased dramatically over the years. Despite significant effort to characterize equine MSCs, the actual origin of these cells and how much of their native phenotype is maintained in culture have not been determined. In this study, we investigated the relationship between MSCs, derived from adipose tissue (AT) and bone marrow (BM), and pericytes in the horse. Both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD90, and CD73) markers were detected in equine AT and colocalized around blood vessels. Importantly, as assessed by flow cytometry, both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD44, CD90, and CD105) markers were present in a majority (≥90%) of cells in cultures of AT-MSCs and BM-MSCs; however, levels of pericyte markers were variable within each of those populations. Moreover, the expression of pericyte markers was maintained for at least eight passages in both AT-MSCs and BM-MSCs. Hematopoietic (CD45) and endothelial (CD144) markers were also detected at low levels in MSCs by quantitative polymerase chain reaction (qPCR). Finally, in coculture experiments, AT-MSCs closely associated with networks produced by endothelial cells, resembling the natural perivascular location of pericytes in vivo. Our results indicate that equine MSCs originate from perivascular cells and moreover maintain a pericyte-like phenotype in culture. Therefore, we suggest that, in addition to classical MSC markers, pericyte markers such as CD146 could be used when assessing and characterizing equine MSCs.

Cytoskeletal Priming of Mesenchymal Stem Cells to a Medicinal Phenotype

Mesenchymal stem cell (MSC) therapy is a promising approach for the treatment of cardiovascular disease, demonstrating pronounced trophic, immunomodulatory, and pro-angiogenic activity. However, clinical efficacy has suffered from broad variability, presumably due to cell death upon implantation, and the heterogeneous population of autologous cells. Micropatterning single cells in the same geometry can normalize the phenotype in a population, and variations in subcellular curvature will guide focal adhesion, cytoskeletal organization, and the regulation of distinct epigenetic marks to orchestrate a medicinal secretome. Within 2 days, activated cells show elevated expression of pericyte markers and will recapitulate functional pericyte activity through enhanced association with endothelial cell tubules in co-culture. MSCs are believed to undergo a temporary switch in vivo to an activated state in response to injury; thus, we propose engineering actomyosin contractility after isolation can similarly activate MSCs, which may serve as a general approach to prime a medicinal phenotype for cell-based therapies. Patient-derived mesenchymal stem cells will secrete molecules that promote new vasculature and have demonstrated clinical efficacy as a therapy for treating myocardial infarction. While autologous cell implantation is promising, there is considerable variability in current treatments and controversy over the underlying mechanisms. In this work, we reveal that careful control of actomyosin contractility in mesenchymal stem cells can prime a pericyte state with a medicinal secretome that shows sustained pro-angiogenic activity in vitro and in vivo. Activation of mesenchymal stem cells to a medicinal phenotype may increase the efficacy and reproducibility of cell-based therapies for cardiovascular disease

Isolation and Characterization of Pericytes from Murine Skeletal Muscle

Pericytes are multipotent stem cells located in the perivascular space throughout the microvasculature. Their ability to differentiate into mesodermal cell types and modulate the local tissue environment through paracrine signaling makes pericytes a novel therapeutic target, but their prospective isolation and identification preclude their widespread use in therapeutic research. PURPOSE: To use novel flow cytometry methods to isolate and characterize pericytes from murine skeletal muscle. METHODS: Gastrocnemius and soleus complexes were dissected from CD1 mice, aged 3 months. Muscles were digested using collagenase into a single cell suspension. Cells were filtered through 70 um and 40 um mesh then resuspended in staining medium. Cells were stained at 4°C for 20 minutes with the following antibodies: CD45-BB515 (1:400), CD34-BV421 (1:100), CD56-PE (1:100) and CD146-Alexa647 (1:100). Fluorescence activated cell sorting (FACS) was used to sort CD45-CD56-CD34-CD146+ pericytes using appropriate unstained, single stained, and fluorescence minus one controls. P0 cells were plated on 0.2% gelatin coated plates in EBM-2 complete medium. At 70% confluence, P0 cells were split 1:1 on polystyrene tissue culture plates, and split 1:2 thereafter. Flow cytometry was used to determine the expression of pericyte markers (NG2 and PDGFβ), mesenchymal cell markers (CD73, CD90, and CD105), endothelial (CD31) and hematopoietic cells (CD45) markers on P5 cells. Characterization data were analyzed for the percentage of positive cells and median fluorescent intensity relative to unstained control (nMFI). RESULTS: Pericytes were positive for the pericyte marker CD146 (15.5%, nMFI 102.4), weakly positive for PDGFRβ (10.8%, nMFI 7.5), and weakly positive for NG2 (7.0%, nMFI 6.9). Cultured pericytes were positive for mesenchymal stem cell markers CD73 (52.9%, nMFI 56.9), and CD105 (35.3%, nMFI 9.5), but negative for CD90 (4.9%, nMFI 6.3). Pericytes were negative for the endothelial marker CD31 (2.1%, nMFI 4.4) and hematopoietic marker CD45 (5.4%, nMFI 7.9). CONCLUSIONS: FACS sorting for CD45-CD56-CD34-CD146+ cells yields murine pericytes that can be reliably isolated, definitively characterized, and may be suitable for in vivo and in vitro experiments to test the efficacy of pericyte cell therapy for various diseases.

Dysfunction of brain pericytes in chronic neuroinflammation

Brain pericytes are uniquely positioned within the neurovascular unit to provide support to blood brain barrier (BBB) maintenance. Neurologic conditions, such as HIV-1-associated neurocognitive disorder, are associated with BBB compromise due to chronic inflammation. Little is known about pericyte dysfunction during HIV-1 infection. We found decreased expression of pericyte markers in human brains from HIV-1-infected patients (even those on antiretroviral therapy). Using primary human brain pericytes, we assessed expression of pericyte markers (α1-integrin, α-smooth muscle actin, platelet-derived growth factor-B receptor β, CX-43) and found their downregulation after treatment with tumor necrosis factor-α (TNFα) or interleukin-1 β (IL-1β). Pericyte exposure to virus or cytokines resulted in decreased secretion of factors promoting BBB formation (angiopoietin-1, transforming growth factor-β1) and mRNA for basement membrane components. TNFα and IL-1β enhanced expression of adhesion molecules in pericytes paralleling increased monocyte adhesion to pericytes. Monocyte migration across BBB models composed of human brain endothelial cells and pericytes demonstrated a diminished rate in baseline migration compared to constructs composed only of brain endothelial cells. However, exposure to the relevant chemokine, CCL2, enhanced the magnitude of monocyte migration when compared to BBB models composed of brain endothelial cells only. These data suggest an important role of pericytes in BBB regulation in neuroinflammation.

Pericyte Antigens in Perivascular Soft Tissue Tumors.

Perivascular soft tissue tumors are relatively uncommon neoplasms of unclear line of differentiation, although most are presumed to originate from pericytes or modified perivascular cells. Among these, glomus tumor, myopericytoma, and angioleiomyoma share a spectrum of histologic findings and a perivascular growth pattern. In contrast, solitary fibrous tumor (previously termed hemangiopericytoma) was once hypothesized to have pericytic differentiation. Here, we systematically examine pericyte immunohistochemical markers among glomus tumor (including malignant glomus tumor), myopericytoma, angioleiomyoma, and solitary fibrous tumor. Immunohistochemical staining and semiquantification was performed using well-defined pericyte antigens, including αSMA, CD146, and PDGFRβ. Glomus tumor and myopericytoma demonstrate diffuse staining for all pericyte markers, including immunohistochemical reactivity for αSMA, CD146, and PDGFRβ. Malignant glomus tumors all showed some degree of pericyte marker immunoreactivity, although it was significantly reduced. Angioleiomyoma shared a similar αSMA + CD146 + PDGFRβ+ immunophenotype; however, this was predominantly seen in the areas of perivascular tumor growth. Solitary fibrous tumors showed patchy PDGFRβ immunoreactivity only. In summary, pericyte marker expression is a ubiquitous finding in glomus tumor, myopericytoma, and angioleiomyoma. Malignant glomus tumor shows a comparative reduction in pericyte marker expression, which may represent partial loss of pericytic differentiation. Pericyte markers are essentially not seen in solitary fibrous tumor. The combination of αSMA, CD146, and PDGFRβ immunohistochemical stainings may be of utility for the evaluation of pericytic differentiation in soft tissue tumors.

BMP2 Regulation of CXCL12 Cellular, Temporal, and Spatial Expression is Essential During Fracture Repair.

The cellular and humoral responses that orchestrate fracture healing are still elusive. Here we report that bone morphogenic protein 2 (BMP2)-dependent fracture healing occurs through a tight control of chemokine C-X-C motif-ligand-12 (CXCL12) cellular, spatial, and temporal expression. We found that the fracture repair process elicited an early site-specific response of CXCL12(+)-BMP2(+) endosteal cells and osteocytes that was not present in unfractured bones and gradually decreased as healing progressed. Absence of a full complement of BMP2 in mesenchyme osteoprogenitors (BMP2(cKO/+)) prevented healing and led to a dysregulated temporal and cellular upregulation of CXCL12 expression associated with a deranged angiogenic response. Healing was rescued when BMP2(cKO/+) mice were systemically treated with AMD3100, an antagonist of CXCR4 and agonist for CXCR7 both receptors for CXCL12. We further found that mesenchymal stromal cells (MSCs), capable of delivering BMP2 at the endosteal site, restored fracture healing when transplanted into BMP2(cKO/+) mice by rectifying the CXCL12 expression pattern. Our in vitro studies showed that in isolated endosteal cells, BMP2, while inducing osteoblastic differentiation, stimulated expression of pericyte markers that was coupled with a decrease in CXCL12. Furthermore, in isolated BMP2(cKO/cKO) endosteal cells, high expression levels of CXCL12 inhibited osteoblastic differentiation that was restored by AMD3100 treatment or coculture with BMP2-expressing MSCs that led to an upregulation of pericyte markers while decreasing platelet endothelial cell adhesion molecule (PECAM). Taken together, our studies show that following fracture, a CXCL12(+)-BMP2(+) perivascular cell population is recruited along the endosteum, then a timely increase of BMP2 leads to downregulation of CXCL12 that is essential to determine the fate of the CXCL12(+)-BMP2(+) to osteogenesis while departing their supportive role to angiogenesis. Our findings have far-reaching implications for understanding mechanisms regulating the selective recruitment of distinct cells into the repairing niches and the development of novel pharmacological (by targeting BMP2/CXCL12) and cellular (MSCs, endosteal cells) interventions to promote fracture healing.

In vitro study on regeneration of periodontal tissue microvasculature using human dedifferentiated fat cells.

Human dedifferentiated fat cells (HDFATs) may be a new cell type suitable for regenerative therapies. The aim of this study is to assess the potential of HDFATs for vascular regeneration of periodontal tissue. To do this, HDFATs and human gingival endothelial cells (HGECs) were cocultivated, and vascular regeneration was examined in vitro. HDFATs were isolated from subcutaneous adipose tissue, and HGECs were isolated from gingival cells using anti-cluster of differentiation 31 antibody-coated magnetic beads. HDFATs were cocultured with HGECs in microvascular endothelial cell growth medium-2 (EGM-2MV) for 7 days. Expression of endothelial cell (EC) markers, the formation of capillary-like tubes, and the expression of pericyte markers were determined. HDFATs, cultured in EGM-2MV or cocultured with HGECs, expressed EC markers. HDFATs in both conditions initiated tube formation within 5 hours of seeding and formed extensive capillary-like structures within 12 hours. These structures disintegrated within 24 hours when cells were cultured in EGM-2MV alone, whereas cocultured HDFATs maintained tubes for >24 hours. Cocultured HDFATs significantly increased expression of pericyte markers, a cell type associated with microvasculature. HDFATs possess the ability to express EC markers, and coculture with HGECs promotes differentiation into pericytes involved in the maturation and stabilization of the microvasculature.

547: Amniotic fluid stem cells accelerate muscle regeneration

Structural birth defects in muscles (such as diaphragmatic defects in congenital diaphragmatic hernia) can be repaired by the use of synthetic and biological patches, with or without the addition of (stem) cells. Amniotic fluid stem cells (AFSc) are a heterogeneous subpopulation of stem cells. Herein we investigate the expression of pericyte markers, progenitors of the skeletal muscle, their in vitro myogenic potential using a well-established cardiotoxin (CTX) induced muscle injury rodent model.

EWS‐FLI‐1 regulates the neuronal repressor gene REST, which controls Ewing sarcoma growth and vascular morphology

RE1-silencing transcription factor (REST), a neuronal repressor gene, regulates neuronal stem cell differentiation. Ewing sarcoma may originate from neural crest cells. In the current study, the authors investigated whether REST plays a role in the growth of this tumor. REST expression was determined by Western blot analysis and reverse transcription-polymerase chain reaction in 3 human Ewing sarcoma cell lines and 7 patient tumor samples. The role of REST in tumor growth and tumor vascular morphology was determined using a Ewing sarcoma xenograft model. Immunofluorescence staining, Hypoxyprobe, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays were performed to investigate the impact of REST on pericyte marker expression, hypoxia, and apoptosis in vivo. High levels of REST were expressed in all 3 human Ewing sarcoma cell lines and in 6 of the 7 patient tumor samples. Overexpression of EWS-FLI-1 in human mesenchymal stem cells and human neural progenitor cells was found to increase REST expression. Inhibition of EWS-FLI-1 using small interfering RNA decreased REST expression in human Ewing sarcoma cells. Inhibition of REST did not affect EWS-FLI-1, but significantly suppressed tumor growth in vivo, reduced the tumor vessel pericyte markers α- smooth muscle actin (SMA) and desmin, increased hypoxia and apoptosis in tumor tissues, and decreased the expression of delta-like ligand 4 (DLL4) and Hes1. Inhibition of REST suppressed tumor growth, inhibited pericyte marker expression, and increased tumor hypoxia and apoptosis. Because tumor vessel function has been linked to tumor growth and metastases, REST may be a new therapeutic target in patients with Ewing sarcoma.

Periodontal Ligament Stem Cells Possess the Characteristics of Pericytes

Periodontal ligament (PDL) contributes to maintaining homeostasis in periodontal tissues by supplying stem/progenitor cells. It has long been suggested that PDL stem cells/progenitors are located around blood vessels. Recently mesenchymal stem cells (MSCs) have been isolated and cultured from PDL in vitro, although the location of the stem cells in PDL is unclear. The purpose of this study is to test the characteristics of human PDL stem cells (PDLSCs) and examine their similarity to related vascular cell types, such as pericytes and endothelial cells. PDLSCs were obtained from healthy extracted teeth using the collagenase/dispase enzyme digestion method. MSC and pericyte characteristics of PDLSCs were examined by cell surface marker expression using flow cytometry. The expression of pericyte markers was tested using immunohistochemistry. Pericyte-like functions of PDLSCs were examined in co-culture of PDLSCs and umbilical vein endothelial cells on a gel matrix. Cultured PDLSCs were positive for both MSC markers and pericyte markers, including cluster of differentiation 146 (CD146), neural/glial antigen 2 (NG2), and CD140b. When pericyte marker expression was explored in rat periodontal tissue sections, CD146- and NG2-positive signals were observed in the perivascular area of the PDL. Further, when the cells were cultured with human umbilical cord endothelial cells under conditions for forming capillary-like structures in vitro, PDLSCs localized adjacent to endothelial cells and contributed to the stability of the capillary-like structure. PDLSCs possess pericyte-like characteristics and may localize as pericytes in the PDL. These data provide useful information for stem cell biology in periodontal research and stem cell-based periodontal therapy.

Association of tumor perivascular desmin expression with survival in patients with sunitinib-treated renal carcinoma.

11097 Background: The introduction of tyrosine kinase inhibitors has markedly improved the outcome for patients with metastatic renal carcinoma (mRCC), but lack of reliable response-predictive markers makes it difficult to individualize treatment. Pericytes surround endothelial cells of capillaries and regulate vessel function. Associations between pericyte marker expression and response to sunitinib have not been well characterized. Methods: A tissue micro array with samples from 67 mRCC patients ( 34 males, 33 females, age 42 – 84) treated at Dept. of Oncology, Karolinska University Hospital where constructed from primary tumors. All patients included had received at least one course with the tyrosine kinase receptor inhibitor sunitinib. Data of treatment, progression free survival and overall survival was collected in a clinical registry. Tissues were double-stained with the endothelial cell marker CD 34 and either of three pericyte markers desmin, PDGF beta receptor and alpha smoth muscle actin (ASMA). By semi-quantitative visual scoring, the perivascular staining of these markers was evaluated and cases dichotomized into negative/low- or positive/high-expressing groups. Results: For patients with desmin positive/high vessels ( n = 12), median PFS where 5.0 months (2.9 – 7.1, 95% CI) vs 7.0 months for patients with desmin negative/low vessels (n = 55, 3.8 – 10.2, 95% CI, Mantel Cox log rank test p<0.02). The median OS in the desmin-positive/high group was 24 months ( 7 – 41, 95% CI) vs 46 months in the negative/low group ( 34 – 58, 95 % CI, Mantel Cox log rank test p<0.014). No significant associations were seen between PFS or OS and the expression of perivascular PDGF beta receptor or ASMA. Conclusions: Metastatic RCC displays a clinically relevant variation in perivascular desmin expression which is associated with survival in sunitinib-treated patients. Future studies are warranted which further explores the prognostic and/or response-predicative capacity of this novel candidate renal cell cancer biomarker.