Abstract: P262 OXIDIZED LDL INDUCED THE BONE MARROW-DERIVED SMOOTH MUSCLE-LIKE CELLS TRANSDIFFERENTIATION INTO FOAM-LIKE CELLS IN VITRO

Background: Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators in various biological processes and diseases. Here we sought to identify and functionally characterize the lncRNA MIAT as a novel regulator in atherosclerotic plaque stability. Methods and results: We profiled RNA transcript expression in patients with advanced atherosclerotic lesions from the Biobank of Karolinska Endarterectomies (BiKE). By microarray analysis, lncRNA MIAT was identified as one of the most highly up-regulated non-coding RNAs in carotid plaques compared to iliac artery controls, which was confirmed by qRT-PCR and in situ hybridization. Additional in silico analysis indicated a substantial positive correlation of MIAT with markers of inflammation, apoptosis and matrix degradation in carotid plaques. Experimental knock-down of MIAT, utilizing site-specific antisense oligonucleotides (LNA-GapmeRs) not only markedly decreased proliferation and migration rates of cultured human carotid artery smooth muscle cells (hCASMCs), but also increased their levels of apoptosis. In addition, MIAT inhibition significantly impaired oxidized LDL (oxLDL) uptake of murine peritoneal as well as human monocyte-differentiated macrophages in vitro. In contrast, induction of MIAT expression by lipoprotein-a (LPa) treatment, displayed the opposite effect. Conditioned medium from macrophage cultures after MIAT knock-down substantially decreased hCASMC proliferation, indicating a potential involvement of MIAT in macrophage-SMC interactions during advanced stages of atherosclerosis. Conclusion: The lncRNA MIAT is a novel regulator of cellular processes in atherosclerosis and plaque stability, which influences SMC proliferation and apoptosis and interacts with disease-triggering macrophages.

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Abstract 263: Role of Micro RNA-21 in Atherosclerosis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.263 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Rihab E Hamed-Berair ◽

Srinivas D Sithu ◽

Nalinie Wickramasinghe ◽

Jasmit Shah ◽

Abhinav Agawral ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Oxidized Ldl ◽

Ldl Receptor ◽

Micro Rna ◽

Limited Information ◽

Micro Rnas ◽

Late Apoptosis ◽

Apoptosis And Inflammation

Micro RNAs (miR) are short non-coding RNAs that regulate several genes under pathophysiological conditions. Accumulating evidence suggest the involvement of miR in atherogenesis. However, limited information is available about atherogenic miR and the underling mechanisms by which miR affect atherogenesis. Our data shows that 12 weeks of western diet (WD) in LDL receptor-knockout (LDLR-KO) mice upregulated 99 and downregulated 50 miR in the aorta. Among the 41 differentially expressed miR associated with macrophage inflammation and apoptosis, expression of micro RNA-21 (miR-21) was increased by 1.4-fold (P<0.05). WD also increased the expression of miR-21 by 1.5-fold in bone marrow derived macrophages (BMDM). In vitro , LDL, oxidized LDL, acetylated LDL and LPS induced miR-21 by 2-3-fold (P<0.05) and down regulated its target protein PDCD4 in BMDM. Basally, miR-21 deficient BMDM showed increased secretion of IL-6, IL-9 and CXCL-2,-3,-4, and -10 (P<0.05)); and increased early and late apoptosis (2-3-fold, P<0.05). We also observed 40% decrease in the survival of F4/80+ cells during differentiation of bone marrow derived cells isolated from miR-21-KO mice. Stimulation of miR-21-KO BMDM with LPS significantly increased the activation of NF-κB and enhanced the secretion of several pro-inflammatory cytokines including TNFα, IL-6, IL-12 and CXCL-2 (2-10 fold; P<0.05); interferon gamma+LPS polarized the macrophages to pro-inflammatory M1 phenotype (increased expression of CD11c and CD86). Staurosporin and oxidized lipids derived aldehyde 4-hydroxynonenal significantly increased both early and late apoptosis of miR-21-KO BMDM (2-4-fold, P<0.05). This was accompanied by increased cleavage of caspase -3, -7 and -9. Transplantation of bone marrow cells from miR-21-KO into LDLR-KO mice, followed by 12 weeks of WD increased the lesion formation (1.7-fold, P<0.05), apoptosis (3-fold, P<0.05) and necrosis (1.6-fold, P<0.05) in the aortic valve of the chimeric mice. Collectively, these data suggest that miR-21 prevents atherosclerosis, at least in parts, by preventing macrophage apoptosis and inflammation.

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Activation of heme oxygenase-1 by Ginkgo biloba extract differentially modulates endothelial and smooth muscle-like progenitor cells for vascular repair

Scientific Reports ◽

10.1038/s41598-019-53818-7 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Tao-Cheng Wu ◽

Jia-Shiong Chen ◽

Chao-Hung Wang ◽

Po-Hsun Huang ◽

Feng-Yen Lin ◽

...

Keyword(s):

Bone Marrow ◽

Smooth Muscle ◽

Progenitor Cells ◽

Heme Oxygenase ◽

Neointimal Hyperplasia ◽

Vascular Repair ◽

Vascular Progenitor Cells

AbstractVascular progenitors such as endothelial progenitor cells (EPCs) and smooth muscle-like progenitor cells (SMPCs) may play different roles in vascular repair. Ginkgo biloba extract (GBE) is an exogenous activator of heme oxygenase (HO)-1, which has been suggested to improve vascular repair; however, the detailed mechanisms have yet to be elucidated. This study aimed to investigate whether GBE can modulate different vascular progenitor cells by activating HO-1 for vascular repair. A bone marrow transplantation mouse model was used to evaluate the in vivo effects of GBE treatment on wire-injury induced neointimal hyperplasia, which is representative of impaired vascular repair. On day 14 of GBE treatment, the mice were subjected to wire injury of the femoral artery to identify vascular reendothelialization. Compared to the mice without treatment, neointimal hyperplasia was reduced in the mice that received GBE treatment for 28 days in a dose-dependent manner. Furthermore, GBE treatment increased bone marrow-derived EPCs, accelerated endothelial recovery, and reduced the number of SMPCs attached to vascular injury sites. The effects of GBE treatment on neointimal hyperplasia could be abolished by co-treatment with zinc protoporphyrin IX, an HO-1 inhibitor, suggesting the in vivo role of HO-1. In this in vitro study, treatment with GBE activated human early and late EPCs and suppressed SMPC migration. These effects were abolished by HO-1 siRNA and an HO-1 inhibitor. Furthermore, GBE induced the expression of HO-1 by activating PI3K/Akt/eNOS signaling in human late EPCs and via p38 pathways in SMPCs, suggesting that GBE can induce HO-1 in vitro through different molecular mechanisms in different vascular progenitor cells. Accordingly, GBE could activate early and late EPCs, suppress the migration of SMPCs, and improve in vivo vascular repair after mechanical injury by activating HO-1, suggesting the potential role of pharmacological HO-1 activators, such as GBE, for vascular protection in atherosclerotic diseases.

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Abstract 386: Sonic Hedgehog Promotes Stem Cell Differentiation to Vascular Smooth Muscle in vitro

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.386 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Maryam Alshamrani ◽

Emma Fitzpatrick ◽

Eileen M Redmond ◽

Paul A Cahill

Keyword(s):

Bone Marrow ◽

Smooth Muscle ◽

Stem Cell ◽

Cell Differentiation ◽

Cell Growth ◽

Vascular Smooth Muscle ◽

Sonic Hedgehog ◽

Stem Cell Differentiation ◽

Differentiation Marker

Background: The morphogen Sonic Hedgehog (SHh) and its signaling pathway components are significantly up-regulated within adventitial and medial segments from arteriosclerotic vessels in mice concomitant with enhanced accumulation of SMCs. This vessel remodelling is attenuated in vivo following Hh receptor, Patched 1, depletion. There is evidence supporting a role for stem cell-derived vascular smooth muscle (vSMCs) in contributing to arteriosclerotic vascular disease. In this context, SHh signaling may be an important regulator of stem cell self-renewal and differentiation to SMC in vitro. Aim: Determine the effects of SHh on bone-marrow derived mesenchymal stem cell (MSC) differentiation to SMC in vitro. Methods: Murine CD44+ bone-marrow derived MSCs and Sca1+ rat adventitial progenitor stem cells (APCs) were examined for SHh components and their capacity to differentiate to SMCs before and after treatment with sonic hedgehog (rSHh, 0.5 μg/ml) for 7 d, in the absence or presence of Hh inhibitors cyclopamine (10μM) or HPI-4 (50μM). The transition to SMC was determined be examining intermediate (calponin1, CNN1) and late (myosin heavy chain, Myh11) SMC differentiation marker expression by western blot analysis and immunocytochemistry, respectively. Results: Hh signaling components were present on MSCs and APCs. Stem cell growth was unaffected by treatment with Hh inhibitors cyclopamine or HPI-4 at concentrations that inhibited Gli signalling in vitro. Recombinant SHh increased SMC differentiation marker protein protein expression after 7 days, an effect that was inhibited following SHh inhibition with smoothened inhibitors cyclopamine and HPI-4. Conclusion: in the absence of any effect on cell growth, Sonic Hedgehog controls mesenchymal stem-like cell differentiation to SMC.

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The Hematopoietic Supportive Function of Adult Bone Marrow Mesenchymal Stem Cells Is Lost after Differentiation Induction towards Adipocytic, Osteoblastic and Vascular Smooth Muscle Lineages

Blood ◽

10.1182/blood.v112.11.4742.4742 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4742-4742

Author(s):

Tatiana Ribeiro ◽

Aurelie Picard ◽

Elfi Ducrocq ◽

Alain Langonne ◽

Philippe Rosset ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Stromal Cells ◽

Expansion Medium ◽

Hsc Niche ◽

And Migration

Abstract The bone marrow (BM) hematopoietic stem cell (HSC) niche is a specialized structure of the microenvironment, which supports survival and regulates HSC function (i.e. the HSC control of the self-renewal/differentiation balance and migration). The supportive cells involved in the HSC niche are usually named as “stromal cells” but their precise nature remains a matter of debate (in particular, to know whether these cells belong to osteoblastic or to vascular smooth muscle lineage). Mesenchymal stem cells (MSCs) that are present into the BM are characterized by a broad differentiation potential including adipocytic (A), osteoblastic (O) and vascular smooth muscle (V) pathways. Although MSCs are believed to be at the origin of stromal cells, their real function within the niche is unknown. The aim of this study was to investigate in vitro the hematopoietic function (HSC support and migration) of cultured adult BM MSCs non-differentiated and during induced differentiation along A, O and V lineages. MSCs were obtained from BM nucleated cells of patients undergoing orthopedic surgery by culture in expansion medium (alpha-MEM medium with 10% FCS and 1 ng/mL FGF-2). The MSCs were tested before (cultured in expansion medium) and during differentiation induction in appropriate medium for A, O or V lineages (from 3 to 21 days). Interestingly, non-differentiated MSCs already co-expressed O (PTH-receptor), A (leptin) and V (ASMA) markers as assessed by Western blotting. Capacity of MSCs to support hematopoiesis was evaluated by long-term cultures (for 5 wks) with BM CD34+ cells in limiting dilution (CAFC assay), and capacity to control CD34+ cell migration by using Transwells seeded with MSCs (trans-stromal migration assay). We showed that non-differentiated MSCs have the most important capacity to support hematopoiesis (5-week CAFC frequency) and that this capacity was quickly and dramatically lost from 3 days of differentiation towards A (36±2% of non-differentiated values), O (40±3%) and V (38±1%) lineages. This capacity was almost abolished after 14 days of A, O and V differentiation (<5%). In parallel, CD34+ cell migration was clearly reduced through 3-day A and O differentiated MSCs, while it was increased through 3-day V differentiated MSCs (5 fold). These results show that MSCs maintained in vitro in non-differentiated state (although already expressing some A, O and V markers), display the strongest hematopoietic supportive activity compared to MSCs induced to differentiate into adipocytic, osteoblastic, or vascular smooth muscle lineages. Therefore, the stromal cell function could be supported by a cell close to a non-differentiated MSC in endosteal or perivascular niches as well. In contrast, vascular smooth muscle differentiated MSCs at advanced stages could be devoted rather to HSC migration control than to HSC support.

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Delta-like ligand 4–Notch signaling regulates bone marrow–derived pericyte/vascular smooth muscle cell formation

Blood ◽

10.1182/blood-2010-05-284869 ◽

2011 ◽

Vol 117 (2) ◽

pp. 719-726 ◽

Cited By ~ 20

Author(s):

Keri Schadler Stewart ◽

Zhichao Zhou ◽

Patrick Zweidler-McKay ◽

Eugenie S. Kleinerman

Keyword(s):

Bone Marrow ◽

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cell ◽

Notch Signaling ◽

Muscle Cell ◽

Vascular Smooth Muscle Cell ◽

Ewing Sarcoma

Abstract Delta-like ligand 4 (DLL4) is essential for the formation of mature vasculature. However, the role of DLL4-Notch signaling in pericyte/vascular smooth muscle cell (vSMC) development is poorly understood. We sought to determine whether DLL4-Notch signaling is involved in pericyte/vSMC formation in vitro and during vasculogenesis in vivo using 2 Ewing sarcoma mouse models. Inhibition of DLL4 with the antibody YW152F inhibited pericyte/vSMC marker expression by bone marrow (BM) cells in vitro. Conversely, transfection of 10T1/2 cells with the active domains of Notch receptors led to increased expression of pericyte/vSMC markers. Furthermore, the blood vessels of Ewing sarcoma tumors from mice treated with YW152F had reduced numbers of BM-derived pericytes/vSMCs, fewer open lumens, and were less functional than the vessels in tumors of control-treated mice. Tumor growth was also inhibited. These data demonstrate a specific role for DLL4 in the formation of BM-derived pericytes/vSMCs and indicate that DLL4 may be a novel therapeutic target for the inhibition of vasculogenesis.

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TGFβ-1 and epithelial-mesenchymal interactions promote smooth muscle gene expression in bone marrow stromal cells: Possible application in therapies for urological defects

The International Journal of Artificial Organs ◽

10.1177/039139880803101105 ◽

2008 ◽

Vol 31 (11) ◽

pp. 951-959 ◽

Cited By ~ 12

Author(s):

C. Becker ◽

T. Laeufer ◽

J. Arikkat ◽

G. Jakse

Keyword(s):

Bone Marrow ◽

Smooth Muscle ◽

Growth Factor ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Smooth Muscle Actin ◽

Muscle Actin ◽

Muscle Gene Expression ◽

Muscle Gene

Purpose For regenerative and cellular therapies of the urinary tract system, autologous bladder smooth muscle cells (SMCs) have several limitations, including constricted in vitro proliferation capacity and, more importantly, inability to be used in malignant conditions. The use of in vitro (pre-)differentiated multipotential adult progenitor cells may help to overcome the shortcomings associated with primary cells. Methods By mimicking environmental conditions of the bladder wall, we investigated in vitro effects of growth factor applications and epithelial-mesenchymal interactions on smooth muscle gene expression and on the morphological appearance of adherent bone marrow stromal cells (BMSCs). Results Transcription growth factor beta-1 (TGFβ-1) upregulated the transcription of myogenic gene desmin and smooth muscle actin-γ2 in cultured BMSCs. Stimulatory effects were significantly increased by coculture with urothelial cells. Prolonged stimulation times and epigenetic modifications further enhanced transcription levels, indicating a dose-response relationship. Immunocytochemical staining of in vitro-differentiated BMSCs revealed expression of myogenic protein α-smooth muscle actin and desmin, and changes in morphological appearance from a fusiform convex shape to a laminar flattened shape with filamentous inclusions similar to the appearance of bladder SMCs. In contrast to the TGFβ-1 action, application of vascular endothelial growth factor (VEGF) did not affect the cells. Conclusions The combined application of TGFβ-1 and epithelial-mesenchymal interactions promoted in vitro outgrowth of cells with a smooth muscle-like phenotype from a selected adherent murine bone marrow-derived cell population.

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Expression of alpha-smooth muscle actin in murine bone marrow stromal cells

Blood ◽

10.1182/blood.v78.2.304.bloodjournal782304 ◽

1991 ◽

Vol 78 (2) ◽

pp. 304-309 ◽

Cited By ~ 1

Author(s):

A Peled ◽

D Zipori ◽

O Abramsky ◽

H Ovadia ◽

E Shezen

Keyword(s):

Bone Marrow ◽

Smooth Muscle ◽

Cell Lines ◽

Smooth Muscle Actin ◽

Muscle Actin ◽

Alpha Smooth Muscle Actin ◽

Murine Bone Marrow ◽

Cellular Origin ◽

Hematopoietic Activity

Human fibrotic bone marrow (BM) stroma has been shown to contain alpha- smooth muscle actin (alpha-SMA)-positive cells. These closely resemble myofibroblasts that were described in other fibrotic tissues. We studied the expression of alpha-SMA in a series of murine BM-derived stromal cell lines to investigate the cellular origin and functional significance of myofibroblast-like cells in hematopoietic tissues. Although these cell lines differed in their biologic properties, most of them expressed alpha-SMA under certain conditions. Cells expressing alpha-SMA constituted a minor population in post-confluent, growth- arrested cultures. However, the incidence of cells expressing alpha-SMA increased significantly when cultures were transferred to nonconfluent conditions. A similar increase in alpha-SMA-positive cells occurred after a strip of cells was scraped away from the confluent cell layer; the cells of the affected area acquired alpha-SMA-positive contractile phenotype. The relationship between alpha-SMA expression and hematopoietic activity was studied using a cloned cell line of BM origin (14F1.1). The ability of these endothelial-adipocyte cells to support hematopoiesis in vitro was maximal under confluent conditions, whereas their expression of alpha-SMA under such conditions was residual. Moreover, in long-term BM cultures supported by confluent 14F1.1 cells, stromal areas associated with proliferating hematopoietic precursors, known as “cobblestone areas,” were devoid of alpha-SMA- positive cells. These observations suggest that the expression of alpha- SMA is reversible and inversely related to hematopoietic activity.

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Expression of alpha-smooth muscle actin in murine bone marrow stromal cells

Blood ◽

10.1182/blood.v78.2.304.304 ◽

1991 ◽

Vol 78 (2) ◽

pp. 304-309 ◽

Cited By ~ 60

Author(s):

A Peled ◽

D Zipori ◽

O Abramsky ◽

H Ovadia ◽

E Shezen

Keyword(s):

Bone Marrow ◽

Smooth Muscle ◽

Cell Lines ◽

Smooth Muscle Actin ◽

Muscle Actin ◽

Alpha Smooth Muscle Actin ◽

Murine Bone Marrow ◽

Cellular Origin ◽

Hematopoietic Activity

Abstract Human fibrotic bone marrow (BM) stroma has been shown to contain alpha- smooth muscle actin (alpha-SMA)-positive cells. These closely resemble myofibroblasts that were described in other fibrotic tissues. We studied the expression of alpha-SMA in a series of murine BM-derived stromal cell lines to investigate the cellular origin and functional significance of myofibroblast-like cells in hematopoietic tissues. Although these cell lines differed in their biologic properties, most of them expressed alpha-SMA under certain conditions. Cells expressing alpha-SMA constituted a minor population in post-confluent, growth- arrested cultures. However, the incidence of cells expressing alpha-SMA increased significantly when cultures were transferred to nonconfluent conditions. A similar increase in alpha-SMA-positive cells occurred after a strip of cells was scraped away from the confluent cell layer; the cells of the affected area acquired alpha-SMA-positive contractile phenotype. The relationship between alpha-SMA expression and hematopoietic activity was studied using a cloned cell line of BM origin (14F1.1). The ability of these endothelial-adipocyte cells to support hematopoiesis in vitro was maximal under confluent conditions, whereas their expression of alpha-SMA under such conditions was residual. Moreover, in long-term BM cultures supported by confluent 14F1.1 cells, stromal areas associated with proliferating hematopoietic precursors, known as “cobblestone areas,” were devoid of alpha-SMA- positive cells. These observations suggest that the expression of alpha- SMA is reversible and inversely related to hematopoietic activity.

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