Increasing donor age adversely impacts beneficial effects of bone marrow but not smooth muscle myocardial cell therapy

We evaluated the impact of donor age on the efficacy of myocardial cellular therapy for ischemic cardiomyopathy. Characteristics of smooth muscle cells (SMC), bone marrow stromal cells (MSCs), and skeletal muscle cells (SKMCs) from young, adult, and old rats were compared in vitro. Three weeks after coronary ligation, 3.5 million SMCs ( n = 11) or MSCs ( n = 9) from old syngenic rats or culture medium ( n = 6) were injected into the ischemic region. Five weeks after implantation, cardiac function was assessed by echocardiography and the Langendorff apparatus. In the in vitro study, the numbers and proliferation of MSCs from fresh bone marrow and SKMCs from fresh tissue but not SMCs were markedly diminished in old animals ( P < 0.05 both groups). SKMCs from old animals did not reach confluence. After treatment with 5-azacytidine (azacitidine), the myogenic potential of old MSCs was decreased compared with young MSCs. In the in vivo study, both SMC and MSC transplantation induced significant angiogenesis compared with media injections ( P < 0.05 both groups). Transplantation of SMCs but not MSCs prevented scar thinning ( P = 0.03) and improved ejection fraction and fractional shortening ( P < 0.05). Load-independent indices of cardiac function in a Langendorff preparation confirmed improved function in the aged SMC group ( P = 0.01) but not in the MSC group compared with the control group. In conclusion, donor age adversely impacts the efficacy of cellular therapy for myocardial regeneration and is cell-type dependent. SMCs from old donors retain their ability to improve cardiac function after implantation into ischemic myocardium.

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Grb10 Mediated Akt Activation Is Required for Induction of CML Like Myeloproliferative Disease in Mice by BCR-ABL.

Blood ◽

10.1182/blood.v110.11.1012.1012 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1012-1012

Author(s):

Corinna Albers ◽

Anna L. Illert ◽

Cornelius Miething ◽

Christian Peschel ◽

Justus Duyster

Keyword(s):

Bone Marrow ◽

Tyrosine Kinases ◽

Chromosomal Translocation ◽

Control Group ◽

Myeloproliferative Disease ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Vitro Analysis ◽

The Impact

Abstract Chronic myelogenous leukaemia (CML) results from the neoplastic transformation of hematopoietic stem cells (HSC) and is characterized by a chromosomal translocation t(9;22)(q34;q11). This aberration leads to the expression of the oncogenic tyrosine kinase BCR-ABL, which mediates signals for proliferation, transformation and anti-apoptosis via various signalling pathways. Grb10, a member of the growth factor bound proteins, is known to bind activated tyrosine kinases like BCR-ABL and might be involved in the activation of the Akt signalling pathway. Here we report the impact of Grb10 for BCR-ABL mediated transformation. We exerted a siRNA based approach in combination with a murine bone marrow transplantation model. To this end we designed a MSCV based retrovirus encoding both a Grb10 microRNA and the BCR-ABL oncogene on a single construct. This approach ensured knockdowns of more than 90% in every BCR-ABL transformed cell. Methylcellulose assays demonstrated that bone marrow coexpressing Grb10 microRNA and BCR-ABL had a 4-fold decreased colony forming ability compared to control cells. We then transduced bone marrow (BM) with retrovirus coexpressing Grb10 microRNA and p185 BCR-ABL and transplanted lethally irradiated recipient Balb/C mice. The onset and progression of leukaemia was significantly delayed in mice transplanted with Grb10 microRNA and BCR-ABL compared with the BCR-ABL transduced control microRNA group. However, we were not able to completely avoid the development of leukaemia by Grb10 knockdown. Mice transplanted with the Grb10 knockdown construct showed a delayed lymphoblastic disease, positive for B220, whereas the control group developed a rapid myeloproliferative disease, characterized by CD11b and Gr-1. In vitro analysis of BaF/3 and 32D cells showed that Grb10 knockdown in combination with BCR-ABL expression leads to a reduced phosphorylation of Akt. Taken together our data demonstrate that Grb10 is required for the development of a myeloproliferative disease by BCR-ABL in mice. Hereby, Grb10 seems to be critical for the BCR-ABL induced activation of the Akt pathway. In addition, this study describes a novel approach to express an oncogene and a microRNA using a single retroviral construct. This tool can be used to systematically screen for drugable signalling targets involved in oncogenesis.

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Protective Effects of Adiponectin against Cobalt Chloride-Induced Apoptosis of Smooth Muscle Cells via cAMP/PKA Pathway

BioMed Research International ◽

10.1155/2020/7169348 ◽

2020 ◽

Vol 2020 ◽

pp. 1-11

Author(s):

Jingjie Xiao ◽

Yingying Zhang ◽

Wei Zhang ◽

Liang Zhang ◽

Li Li ◽

...

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Cell Viability ◽

Cobalt Chloride ◽

Muscle Cells ◽

Camp Level ◽

Control Group ◽

Dependent Manner ◽

Sod Activity

Adiponectin (APN) is an adipokine secreted from adipose tissue and exhibits biological functions such as microcirculation-regulating, hearing-protective, and antiapoptotic. However, the effect of APN on the apoptosis of spiral arterial smooth muscle cells (SMCs) under hypoxic conditions in vitro is not clear. We used cobalt chloride (CoCl2) to simulate chemical hypoxia in vitro, and the SMCs were pretreated with APN and then stimulated with CoCl2. The viability of cells and apoptosis were assessed by CCK-8 and flow cytometry, respectively. Superoxide dismutase (SOD) activity, malondialdehyde (MDA) levels, cAMP level, and the activity of PKA were detected by ELISA. Protein expression and localization were studied by Western blot and immunofluorescence analysis. In the present study, we found that APN exhibits antiapoptosis effects. CoCl2 exhibited decreased cell viability, increased apoptosis and MDA levels, and decreased SOD activity in a concentration-dependent manner, compared with the control group. Moreover, CoCl2 upregulated the expression levels of Bax and cleaved caspase-3 and then downregulated Bcl-2 levels in a time-dependent manner. Compared with the CoCl2 group, the group pretreated with APN had increased cell viability, SOD activity, PKA activity, cAMP level, and PKA expression, but decreased MDA levels and apoptosis. Lastly, the protective effect of APN was blocked by cAMP inhibitor SQ22536 and PKA inhibitor H 89. These results showed that APN protected SMCs against CoCl2-induced hypoxic injury via the cAMP/PKA signaling pathway.

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Impact of Bone Marrow Mesenchymal Stem Cells That Express Vascular Endothelial Growth Factor on Cardiac Function After Myocardial Infarction

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2021.2384 ◽

2021 ◽

Vol 11 (1) ◽

pp. 44-50

Author(s):

Yongming He ◽

Ping Li ◽

Yunlong Chen ◽

Youmei Li

Keyword(s):

Myocardial Infarction ◽

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Cardiac Function ◽

Troponin T ◽

Gene Transfection ◽

Control Group ◽

Marrow Mesenchymal Stem Cells ◽

The Impact

Transplanted bone marrow mesenchymal stem cells (MSCs) can differentiate into cardiomyocytes and may have the potential to replace necrotic cardiomyocytes resulting from myocardial infarction (MI). Here we established a method for transfection of MSCs with an expression vector encoding human vascular Eedothelial Ggowth Ffctor (hVEGF). We evaluated the impact of transplantation of transfected MSCs on the recovery cardiac function and angiogenesis in a rat model of MI. Rat MSCs were separated by density gradient centrifugation; their specific surface markers were examined as was their ability to differentiate. MSCs were then transfected with pcDNA 3.1-hVEGF 165 or control-containing liposomes. Rats in the experimental MI groups received transfected MSCs, MSCs alone, or gene-transfection alone; controls included a no intervention MI group and a group that was not subjected to ischemia. Among the results, MSCs were successfully isolated and cultured. Among the intervention groups, those that received transplantation of MSCs expressing hVEGF 165 included the smallest areas of infarction and demonstrated the best recovery of cardiac function overall. Moreover, capillary density detected in this group was significantly greater than in the control group and likewise greater than in rats transplanted with MSCs alone. BrdU and Troponin-T staining revealed differential increases in the number of viable cardiomyocytes within the infarction areas; some cardiomyocytes were double-positive. Likewise, evaluation using RT-PCR revealed higher expression levels of hVEGF in rats transplanted with transfected cells compared to those treated with gene transfection alone.

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The effect of donor age on the in vitro life span of cultured human arterial smooth-muscle cells

In Vitro ◽

10.1007/bf02616126 ◽

1978 ◽

Vol 14 (11) ◽

pp. 951-955 ◽

Cited By ~ 108

Author(s):

Edwin L. Bierman

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Life Span ◽

Muscle Cells ◽

Donor Age ◽

Arterial Smooth Muscle ◽

Arterial Smooth Muscle Cells

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CGRP protects bladder smooth muscle cells stimulated by high glucose through inhibiting p38 MAPK pathway in vitro

Scientific Reports ◽

10.1038/s41598-021-87140-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jun Xue ◽

Yadong Liu ◽

Sichong Zhang ◽

Liucheng Ding ◽

Baixin Shen ◽

...

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Cell Viability ◽

P38 Mapk ◽

High Glucose ◽

Muscle Cells ◽

Control Group ◽

Asiatic Acid ◽

Bladder Smooth Muscle

AbstractThis study aimed to explore the effect of calcitonin gene-related peptide (CGRP) on bladder smooth muscle cells (BSMCs) under high glucose (HG) treatment in vitro. BSMCs from Sprague–Dawley rat bladders were cultured and passaged in vitro. The third-generation cells were cultured and divided into control group, HG group, HG + CGRP group, HG + CGRP + asiatic acid (AA, p-p38 activator) group, CGRP group, AA group, HG + CGRP + CGRP-8-37 (CGRP receptor antagonist) group and HG + LY2228820 (p38 MAPK inhibitor) group. The cell viability, apoptosis, malondialdehyde (MDA) and superoxide dismutase (SOD) levels of BSMCs were observed by the relevant detection kits. The expressions of α-SM-actin, p38 and p-p38 were detected by qRT-PCR or Western blot analysis. Compared with the control group, the cell viability, SOD and α-SM-actin levels of BSMCs were decreased and apoptotic cells, MDA and p-p38 levels were increased after HG treatment, while these changes could be partly reversed when BSMCs were treated with HG and CGRP or LY2228820 together. Moreover, AA or CGRP-8-37 could suppress the effect of CGRP on BSMCs under HG condition. Our data indicate that CGRP protects BSMCs from oxidative stress induced by HG in vitro, and inhibit the α-SM-actin expression decrease through inhibiting the intracellular p38 MAPK signaling pathway.

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212 PORCINE BONE MARROW-DERIVED MESENCHYMAL STEM CELLS DIFFERENTIATE IN VITRO INTO SMOOTH MUSCLE CELLS.

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab212 ◽

2012 ◽

Vol 24 (1) ◽

pp. 218

Author(s):

B. Mohana Kumar ◽

G. H. Maeng ◽

Y. M. Lee ◽

T. H. Kim ◽

W. J. Lee ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Muscle Cells ◽

Class Ii ◽

Alizarin Red ◽

Expression Levels

In the context of multipotent stem cells, mesenchymal stem cells (MSC) derived from bone marrow have been identified as most promising cell types for the treatment of smooth muscle related injured tissues and organs. In the present study, the ability of porcine bone marrow derived MSC to differentiate in vitro into smooth muscle cells (SMC) was examined. MSC were isolated from domestic pig bone marrow by their readily adherent property to tissue culture plastic with fibroblast-like morphology. Cells were analysed for the expression of MSC specific markers by flow cytometer and mesenchymal lineage differentiation by following previously published protocols. Differences in values were analysed by one-way ANOVA using SPSS and data are presented as mean ± SD. Flow cytometry analysis of MSC showed the positive expression of markers, such as CD29 (97.33 ± 2.08%), CD44 (97.67 ± 1.15%), CD73 (62.33 ± 2.89%), CD90 (96.67 ± 2.08%) and vimentin (59.33 ± 2.52%). In contrast, the expression levels were significantly lower for CD34 (3.33 ± 1.53%), CD45 (3.67 ± 1.53%), major histocompatibility complex class II (MHC class II, 10.33 ± 2.52%) and swine leukocyte antigen-DR (SLA-DR, 9.67 ± 2.08%). The MSC were further confirmed by their ability to differentiate in vitro along the distinct lineages of adipocytes (Oil red O), osteocytes (von Kossa and Alizarin red) and chondrocytes (Alcian blue). Induction of SMC differentiation was performed with supplementation of porcine transforming growth factor-β (TGF-β) and recombinant human bone morphogenic protein 4 (BMP4) as described earlier (Wang et al. 2010 Tissue Eng. A 1201–1213) with minor modifications. Upon induction, porcine MSC acquired myoblast-like morphology with intracellular thin filaments. Immunofluorescence staining showed the presence of early and late markers of smooth muscle differentiation, such as α-smooth muscle actin (α-SMA), calponin, smooth muscle 22 α (SM22α) and smooth muscle-myosin heavy chain (SM-MHC) and their expression levels varied from 22.65% to 56.75%. Later, the expression of selected markers was demonstrated by Western blotting analysis. Consistent with this phenotypic characterisation, reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (RT-qPCR) further showed the expression and a sequential up-regulation of transcripts for α-SMA, calponin, SM22α and SM-MHC. However, no expression of SMC-specific markers was observed in untreated MSC. In conclusion, these findings suggest the ability of porcine MSC from bone marrow to differentiate in vitro into SMC in the presence of growth factors. Further understanding of SMC differentiation with functional properties would be essential for employing porcine MSC as a useful model for cell-based tissue engineering and regeneration strategies. This work was supported by Basic Science Research Program through the National Research Foundation (NRF) funded by the Ministry of Education, Science and Technology (2010-0010528) and BioGreen 21 (20070301034040), Republic of Korea.

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Metformin Inhibits the Phenotypic Transformation of Aortic Vascular Smooth Muscle Cells Induced by Hyperglycemia Through Regulation of the Mir-21-mediated PTEN/pAKT/Egr-1 Pathway

10.21203/rs.3.rs-907826/v1 ◽

2021 ◽

Author(s):

Hui Zheng ◽

Ying Liu ◽

Xiaoyue Sun ◽

Xiaochen Li ◽

Le Liu ◽

...

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Muscle Cells ◽

Control Group ◽

Diabetic Mice ◽

Marker Protein ◽

Phenotypic Transformation

Abstract Background The phenotypic transformation of arterial vascular smooth muscle cells (VSMC) is one of the key mechanisms in the formation of atherosclerotic plaque. It is unclear whether metformin can inhibit the phenotypic transformation of VSMC. In this study, we observed the effect of metformin on the phenotypic transformation of VSMC in vivo and in vitro and its mechanism.Methods Patients who underwent simple coronary artery bypass grafting (CABG) were divided into non-diabetic(non-DM) group and diabetes (DM) group according to whether diabetes was combined. The miR-21 and VSMC contractile marker protein, smooth muscle contractile protein (αSMA) and synthetic marker protein osteopontin (OPN) were isolated from the internal mammary artery.30 male, clean, and 6 week old wild type C57/BL6J mice were randomly divided into 3 groups:the control group (NC), the diabetes mellitus group (DM) and the metformin intervention group(DM+MET).Diabetic mice model was established by high-fat diet combined with low-dose streptozotocin. Metformin was given by gavage for 8 weeks.The aortic tissue from aortic root to renal artery was retained, miR-21 was determined by real-time PCR, and SMC and OPN were determined by Western Blotting (WB). In vitro, human aortic smooth muscle cells (HA-SMC) were divided into three groups: control group, HG group and HG+ group. The content of miR-21/PTEN/pAKT/Egr-1 protein was determined by WB, the migration ability was determined by MTT and scratch method.Results 1.In vivo, the expression of miR-21 and OPN in the aortic VSMC of DM petients and diabetic mice increased and the intima media thickness increased. Metformin treatment reduced the expression of miR-21 and OPN in the aorta and decreased the thickness of intima media. 2.In vitro, glucose concentration dependently upregulates the expression of miR-21 and osteopontin in HA-SMC.Conclusion Metformin can inhibit the phenotype transformation of VSMC induced by HG, which may inhibit the migration of VSMC through miR-21/PTEN/pAkt/Egr-1.

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Cannabinoid receptor CB2 protects against balloon-induced neointima formation

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00444.2011 ◽

2012 ◽

Vol 302 (5) ◽

pp. H1064-H1074 ◽

Cited By ~ 15

Author(s):

Filippo Molica ◽

Christian M. Matter ◽

Fabienne Burger ◽

Graziano Pelli ◽

Sébastien Lenglet ◽

...

Keyword(s):

Bone Marrow ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Cannabinoid Receptor ◽

Muscle Cells ◽

Mrna Levels ◽

Neointima Formation ◽

Balloon Injury ◽

And Migration

Cannabinoid receptor CB2 activation inhibits inflammatory proliferation and migration of vascular smooth muscle cells in vitro. The potential in vivo relevance of these findings is unclear. We performed carotid balloon distension injury in hypercholesterolemic apolipoprotein E knockout (ApoE−/−) mice receiving daily intraperitoneal injection of the CB2 agonist JWH133 (5 mg/kg) or vehicle, with the first injection given 30 min before injury. Alternatively, we subjected CB2−/− and wild-type (WT) mice to balloon injury. We determined CB2 mRNA and protein expression in dilated arteries of ApoE−/− mice. Neointima formation was assessed histologically. We used bone marrow-derived murine CB2−/− and WT macrophages to study adhesion to plastic, fibronectin, or collagen, and migration was assayed by modified Boyden chamber. Aortic smooth muscle cells were isolated to determine in vitro proliferation rates. We found increased vascular CB2 expression in ApoE−/− mice in response to balloon injury. Seven to twenty-one days after dilatation, injured vessels of JWH133-treated mice had less intimal nuclei numbers as well as intimal and medial areas, associated with less staining for proliferating cells, smooth muscle cells, and macrophages. Complete endothelial repair was observed after 14 days in both JWH133- and vehicle-treated mice. CB2 deficiency resulted in increased intima formation compared with WT, whereas JWH133 did not affect intimal formation in CB2−/− mice. Apoptosis rates assessed by in situ terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining 1 h postballooning were significantly higher in the CB2 knockouts. In vitro, bone marrow-derived CB2−/− macrophages showed enhanced adherence and migration compared with WT cells and elevated mRNA levels of adhesion molecules, chemokine receptors CCR1 and 5, and chemokine CCL2. Proliferation rates were significantly increased in CB2−/− smooth muscle cells compared with WT. In conclusion, pharmacological activation or genetic deletion of CB2 receptors modulate neointima formation via protective effects in macrophages and smooth muscle cells.

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