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ABSTRACT: The metalloproteinase ADAMTS4/aggrecanase-1 is highly expressed in cartilage and has been implicated in human arthritis. Although abundantly expressed in many types of cancer, its role in cancer remains unknown. In this work, we demonstrate for the first time that full-length ADAMTS4 or its catalytically more active N-terminal 53 kDa autocatalytic fragment both promote B16 melanoma growth and angiogenesis in mice. In contrast, overexpression of its catalytically-inactive E362A mutant or truncated fragments containing only the C-terminal ancillary domains suppresses melanoma growth and angiogenesis under similar conditions. Structure-function mapping revealed that the single TSR domain is essential and sufficient for the anti-tumorigenic activity displayed by the catalytically-inactive ADAMTS4 isoforms. Suppression of tumor growth and angiogenesis in mice is accompanied by a significant increase in tumor cell apoptosis while tumor cell proliferation is not affected. Importantly, we identified and demonstrated the presence of novel proteolytic fragments of ADAMTS4 containing essentially only the C-terminal ancillary domains in cultured cells, and also in human cancer tissues, co-existing with full-length and catalytically-active N-terminal fragments. The contrasting functions towards tumor growth in mice by the wild-type proteinase and its catalytically-inactive mutant correlate with their contrasting influences on angiogenesis signaling pathway molecules in B16 melanoma in mice. Our results suggest a complex role for ADAMTS4 in cancer with the functional balance of pro-tumorigenic and anti-tumorigenic isoforms likely to act as an important parameter in determining the net influence of this metalloproteinase on tumor growth in vivo. © 2013 Wiley Periodicals, Inc.
International Journal of Cancer 01/2013; · 5.44 Impact Factor
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ABSTRACT: ADAMTS5 is a member of the A Disintegrin-like And Metalloproteinase with ThromboSpondin motifs (ADAMTS) family of secreted metalloproteinases with multiple proteoglycan substrates. Although well characterized for its role in cartilage degradation and arthritis, how it influences cancer remains unclear. We have previously shown that the first thrombospondin type 1 repeat (TSR1, the central TSR) but not TSR2 (the C-terminal TSR) of ADAMTS5 is anti-angiogenic in vitro. Coupled with previous reports that ADAMTS5 expression is altered in several human cancers, we hypothesized that this proteoglycanase may play an important role in cancer and angiogenesis. Here, we demonstrated that overexpression of full-length ADAMTS5 suppressed B16 melanoma growth in mice. The reduced tumor growth is correlated with diminished tumor angiogenesis, together with reduced tumor cell proliferation and increased tumor cell apoptosis. Catalytically active ADAMTS5 proteolytic fragment also suppressed angiogenesis in vitro. The catalytic activity of ADAMTS5 is dispensable for its anti-tumorigenic function, as the full-length active site mutant E411A presented similar tumor suppression activity. Domain mapping and mechanistic studies revealed that ADAMTS5 inhibits B16 tumorigenesis through its TSR1 by suppressing tumor angiogenesis, likely by down-regulating pro-angiogenic factors such as vascular endothelial growth factor (VEGF), placenta growth factor (PlGF), and platelet-derived endothelial growth factor (PD-ECGF) in the tumor milieu. This is the first report that ADAMTS5 is an anti-angiogenic and anti-tumorigenic protein independent of its proteoglycanase activity.
American Journal Of Pathology 07/2012; 181(3):1056-68. · 4.89 Impact Factor
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ABSTRACT: Cancer is a complex organ whose behavior is not only influenced by genetic and epigenetic changes in cancer cells but also by stromal cells, local extracellular matrix and specific tissue architecture. Intercellular communications within the cancer microenvironment are critical to coordinate the assembly of multiple cell types for an amalgamated form and function of a cancer. Exosomes are small membrane vesicles with an endosome origin that are released by cells into the extracellular environment. They carry a cargo of proteins, lipids, and nucleic acids and transfer their cargo to recipient cells and altering the recipient cells' biochemical composition, signaling pathways, and gene regulation. Exosomes can thus serve as extracellular messengers mediating cell-cell communication. Both cancer cells and stromal cells release exosomes not only into the cancer microenvironment but also into the circulation. In this review, we summarize the research done so far on cancer-derived exosomes and assess their roles as extracellular messengers facilitating cancer progression and metastasis.
Cancer Microenvironment 05/2012; 5(3):323-32.
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ABSTRACT: Augmenter of Liver Regeneration (ALR) is a sulfhydryl oxidase carrying out fundamental functions facilitating protein disulfide bond formation. In mammals, it also functions as a hepatotrophic growth factor that specifically stimulates hepatocyte proliferation and promotes liver regeneration after liver damage or partial hepatectomy. Whether ALR also plays a role during vertebrate hepatogenesis is unknown. In this work, we investigated the function of alr in liver organogenesis in zebrafish model. We showed that alr is expressed in liver throughout hepatogenesis. Knockdown of alr through morpholino antisense oligonucleotide (MO) leads to suppression of liver outgrowth while overexpression of alr promotes liver growth. The small-liver phenotype in alr morphants results from a reduction of hepatocyte proliferation without affecting apoptosis. When expressed in cultured cells, zebrafish Alr exists as dimer and is localized in mitochondria as well as cytosol but not in nucleus or secreted outside of the cell. Similar to mammalian ALR, zebrafish Alr is a flavin-linked sulfhydryl oxidase and mutation of the conserved cysteine in the CxxC motif abolishes its enzymatic activity. Interestingly, overexpression of either wild type Alr or enzyme-inactive Alr(C131S) mutant promoted liver growth and rescued the liver growth defect of alr morphants. Nevertheless, alr(C131S) is less efficacious in both functions. Meantime, high doses of alr MOs lead to widespread developmental defects and early embryonic death in an alr sequence-dependent manner. These results suggest that alr promotes zebrafish liver outgrowth using mechanisms that are dependent as well as independent of its sulfhydryl oxidase activity. This is the first demonstration of a developmental role of alr in vertebrate. It exemplifies that a low-level sulfhydryl oxidase activity of Alr is essential for embryonic development and cellular survival. The dose-dependent and partial suppression of alr expression through MO-mediated knockdown allows the identification of its late developmental role in vertebrate liver organogenesis.
PLoS ONE 01/2012; 7(1):e30835. · 4.09 Impact Factor
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ABSTRACT: Chimera formation is a standard test for pluripotency of stem cells in vivo. Interspecific chimera formation between distantly related organisms offers also an attractive approach for propagating endangered species. Parameters influencing interspecies chimera formation have remained poorly elucidated. Here, we report interordinal chimera formation between medaka and zebrafish, which separated ∼320 million years ago and exhibit a more than 2-fold difference in developmental speed. We show that, on transplantation into zebrafish blastulae, both noncultivated blastomeres and long-term cultivated embryonic stem (ES) cells of medaka adopted the zebrafish developmental program and differentiated into physiologically functional cell types including pigment cells, blood cells, and cardiomyocytes. We also show that medaka ES cells express differentiation gene markers during chimeric embryogenesis. Therefore, the evolutionary distance and different embryogenesis speeds do not produce donor-host incompatibility to compromise chimera formation between medaka and zebrafish, and molecular markers are valuable for analyzing lineage commitment and cell differentiation in interspecific chimeric embryos.
Stem cells and development 12/2011; 21(12):2333-41. · 4.15 Impact Factor
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Haobin Zhao,
Mingyou Li,
Yovita Ida Purwanti,
Rong Liu,
Tiansheng Chen,
Zhendong Li,
Ni Hong,
Guijun Guan,
Ao Yin,
Ling Xiao, Ruowen Ge,
Jianxing Song,
Yunhan Hong
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ABSTRACT: Germ cells express a unique subset of genes called germ genes mostly encoding RNA-binding proteins such as Dazl, Dnd and Vasa. How germ gene expression is controlled remains illusive, because in no organism has a transcription factor been identified that regulate expression of these genes. Microphthalmia-associated transcription factor (Mitf) has been reported to show expression in male mouse germ cells of the adult testis. Here we report in the fish medaka (Oryzias latipes) that Mitf is a transcription activator of germ gene expression. Mitf is a master regulator of melanocyte development, which activates melanogenic genes through binding to the E-box containing consensus CANNTG. The E-box was found to be present in 23-26 copies in the promoters of medaka germ genes dazl, dnd and vasa. Importantly, forced Mitf expression enhanced the transcriptional activity of the three gene promoters by up to more than 10 fold and remarkably increased the level of endogenous dazl, dnd and vasa transcripts in cell culture. Transfection of Mitf expression vectors was sufficient to induce directed differentiation of medaka embryonic stem cells into melanocytes. Fluorescence in situ hybridization revealed the expression of both medaka mitf genes in adult germ cells of male and female gonads. Mitf is well-known as the melanocyte master regulator. Our results offer first evidence that Mitf may act as a transcriptional activator of germ gene expression in medaka.
Biochimie 11/2011; 94(3):759-67. · 3.02 Impact Factor
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ABSTRACT: Mammalian embryos at the blastocyst stage have three major lineages, which in culture can give rise to embryonic stem (ES) cells from the inner cell mass or epiblast, trophoblast stem cells from the trophectoderm, and primitive endoderm stem cells. None of these stem cells is totipotent, because they show gene expression profiles characteristic of their sources and usually contribute only to the lineages of their origins in chimeric embryos. It is unknown whether embryos prior to the blastocyst stage can be cultivated towards totipotent stem cell cultures. Medaka is an excellent model for stem cell research. This laboratory fish has generated diploid and even haploid ES cells from the midblastula embryo with ~2000 cells. Here we report in medaka that dispersed cells from earlier embryos can survive, proliferate and attach in culture. We show that even 32-cells embryos can be dissociated into individual cells capable of producing continuously growing ES-like cultures. Our data point to the possibility to derive stable cell culture from cleavage embryos in this organism.
International journal of biological sciences 01/2011; 7(4):418-25. · 2.70 Impact Factor
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11th International Conference on Control, Automation, Robotics and Vision, ICARCV 2010, Singapore, 7-10 December 2010, Proceedings; 01/2010
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11th International Conference on Control, Automation, Robotics and Vision, ICARCV 2010, Singapore, 7-10 December 2010, Proceedings; 01/2010
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ABSTRACT: Constantly exposed to various forms of mechanical forces inherent in their physical environment (such as gravity, stress induced by fluid flow or cell–cell interactions, etc), cellular organisms sense such forces and convert them into biochemical signals through the processes of mechanosensing and mechanotransduction that eventually lead to biological changes. The effect of external forces on the internal structures and activities in a cellular organism may manifest in changes its physical properties, such as impedance. Studying variation in the impedance of a cellular organism induced by the application of an external mechanical force represents a meaningful endeavor (from a biosystems perspective) in exploring the complex mechanosensing and mechanotransduction mechanisms that govern the behavior of a cellular organism under the influence of external mechanical stimuli. In this paper we describe the development of an explicit force-feedback control system for exerting an indentation force on a cellular organism while simultaneously measuring its impedance. To demonstrate the effectiveness of this force-control system, we have conducted experiments using zebrafish embryos as a test model of a cellular organism. We report experimental results demonstrating that the application of a properly controlled external force leads to a significant change in the impedance of a zebrafish embryo. These results offer support for a plausible explanation that activities of pore canals in the chorion are responsible for the observed change in impedance.
Journal of Micromechanics and Microengineering 12/2009; 20(2):025003. · 2.11 Impact Factor
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ABSTRACT: Hepatocellular carcinoma (HCC) is one of the deadliest cancers with few treatment options. It is a hypervascular tumor in which angiogenesis plays a critical role in its progression. Tumor capillary endothelial cells (TECs) in HCC are known to originate from liver sinusoid endothelial cells, which then go through a capillarization process to become morphologically as well as functionally different TECs. In this work, we investigated proteins differentially expressed between freshly isolated TECs and sinusoid endothelial cells from well-formed rat HCC using 2-D DIGE coupled with MALDI-TOF/TOF MS. Thirty-eight unique proteins were identified to be differentially expressed more than twofold between the two endothelial cell types. Amongst the differentially expressed proteins, two novel endothelial markers, EH domain-containing protein 3 and galectin-3, were confirmed by Western blot and immunohistochemistry in both rat and human HCC samples. We showed that EH domain-containing protein 3 is significantly down-regulated in TECs, but galectin-3 is up-regulated. We propose possible roles of these two proteins in tumor vessel development in HCC.
Proteomics 11/2009; 10(2):224-34. · 4.43 Impact Factor
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Wei Xiang,
Zhiyuan Ke,
Yong Zhang,
Grace Ho-Yuet Cheng,
Ishak Darryl Irwan,
K N Sulochana,
Padma Potturi,
Zhengyuan Wang,
He Yang,
Jingyu Wang,
Lang Zhuo,
R Manjunatha Kini, Ruowen Ge
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ABSTRACT: Anti-angiogenesis represents a promising therapeutic strategy for the treatment of various malignancies. Isthmin (ISM) is a gene highly expressed in the isthmus of the midbrain-hindbrain organizer in Xenopus with no known functions. It encodes a secreted 60 kD protein containing a thrombospondin type 1 repeat domain in the central region and an adhesion-associated domain in MUC4 and other proteins (AMOP) domain at the C-terminal. In this work, we demonstrate that ISM is a novel angiogenesis inhibitor. Recombinant mouse ISM inhibited endothelial cell (EC) capillary network formation on Matrigel through its C-terminal AMOP domain. It also suppressed vascular endothelial growth factor (VEGF)-basic fibroblast growth factor (bFGF) induced in vivo angiogenesis in mouse. It mitigated VEGF-stimulated EC proliferation without affecting EC migration. Furthermore, ISM induced EC apoptosis in the presence of VEGF through a caspase-dependent pathway. ISM binds to αvβ(5) integrin on EC surface and supports EC adhesion. Overexpression of ISM significantly suppressed mouse B16 melanoma tumour growth through inhibition of tumour angiogenesis without affecting tumour cell proliferation. Knockdown of isthmin in zebrafish embryos using morpholino antisense oligonucleotides led to disorganized intersegmental vessels in the trunk. Our results demonstrate that ISM is a novel endogenous angiogenesis inhibitor with functions likely in physiological as well as pathological angiogenesis.
Journal of Cellular and Molecular Medicine 10/2009; 15(2):359-74. · 4.13 Impact Factor
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ABSTRACT: Trocarin D is a prothrombin activator from the Tropidechis carinatus venom. It is a functional and structural homologue to mammalian blood coagulation factor Xa. Trocarin D is hypothesised to have evolved from its factor X counterpart (TrFX) through gene duplication and recruitment. The genes of trocarin D and TrFX have significant sequence identities, except for insertions/deletions in their intron 1 and promoter regions. In trocarin D intron 1 region, there are three insertions and two deletions. In trocarin D promoter region, there is a novel 264 bp insertion which has potential cis-elements. This insertion is termed as Venom Recruitment/Switch Element (VERSE) and is hypothesised to account for switching the low-level constitutive expression of factor X in the liver to the high-level inducible expression of trocarin D in the venom gland. To understand the role of VERSE in the trocarin D expression, its cis-elements were characterised by luciferase assays in mammalian cell lines as well as snake venom gland cells. The ability of VERSE to drive luciferase expression is comparable to that of the trocarin D promoter. The predicted cis-elements are important in promoting expression as their mutagenesis resulted in lower luciferase expression. VERSE minimal core promoter and three novel cis-elements (two up-regulatory and one suppressor elements) were identified using deletion/site-directed mutagenesis studies. VERSE is primarily responsible for the increase of trocarin D expression. The insertions/deletions within trocarin D intron 1 need to be characterised for their role in tissue-specific and inducible expression of trocarin D.
Thrombosis and Haemostasis 10/2009; 102(3):469-78. · 5.04 Impact Factor
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ABSTRACT: Micro-injection of zebrafish embryo is widely applied in biology for the analysis of early developmental processes. The success of a micro-injection to a large extent depends on the mechanical interaction between the micro-pipette and the membrane of the zebrafish embryo. In this paper, we present the development of (i) a maximum stress model of the deformed membrane with respect to the depth of indentation, (ii) a family-of-conics elongation model to determine the length of the deformed membrane for the estimation of the maximum strain at a given indentation depth, and (iii) an experimental system to generate the required data for these two models. The significance of these results is that the estimated maximum stress provides a performance target for the penetration process, while the estimated corresponding maximum strain serves as an indicator of the extent of deformation sustained by the embryo prior to penetration. Implications of these modeling and experimental results are discussed in the context of optimizing the process of micro-injection.
Journal of biomechanics 03/2009; 42(5):620-5. · 2.66 Impact Factor
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ABSTRACT: Angiogenesis is critical for tumour growth and metastasis where factors that regulate this process are potential targets for development of anti-cancer drugs. In this study, we show that the first TSR domain of the extracellular matrix protease ADAMTS5, unlike the second TSR, has anti-angiogenic activities where it inhibits endothelial cell tube formation on Matrigel, reduces cell proliferation and attachment, while promoting cell apoptosis and migration, all in a dose-dependent manner. Furthermore, it influences the architecture of endothelial cells by disrupting actin stress fibres and reducing focal adhesions, likely via suppressing RhoA activation. TSR1 of ADAMTS5 is therefore a novel anti-angiogenic peptide and could serve as a prototype for future development into anti-cancer drugs.
Biochemical and Biophysical Research Communications 07/2008; 371(2):215-9. · 2.48 Impact Factor
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ABSTRACT: Histone deacetylases (HDACs) are key transcription regulators that function by deacetylating histones/transcription factors and modifying chromatin structure. In this work, we showed that chemical inhibition of HDACs by valproic acid (VPA) led to impaired liver development in zebrafish mainly by inhibiting specification, budding, and differentiation. Formation of exocrine pancreas but not endocrine pancreas was also inhibited. The liver defects induced by VPA correlate with suppressed total HDAC enzymatic activity, but are independent of angiogenesis inhibition. Gene knockdown by morpholino demonstrated that hdac3 is specifically required for liver formation while hdac1 is more globally required for multiple development processes in zebrafish including liver/exocrine pancreas formation. Furthermore, overexpression of hdac3 but not hdac1 partially rescued VPA induced small liver. One mechanism by which hdac3 regulates zebrafish liver growth is through inhibiting growth differentiation factor 11 (gdf11), a unique target of hdac3 and a member of the transforming growth factor beta family. Simultaneous overexpression or morpholino knockdown showed that hdac3 and gdf11 function antagonistically in zebrafish liver development. These results revealed a novel and specific role of hdac3 in liver development and the distinct functions between hdac1 and hdac3 in zebrafish embryonic development.
Developmental Biology 06/2008; 317(1):336-53. · 4.07 Impact Factor
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ABSTRACT: Excessive angiogenesis plays critical roles in many human diseases including cancer. We have previously shown that human decorin derived 26 amino acids peptide Leucine Rich Repeat 5 (LRR5) inhibits multiple aspects of angiogenesis including vascular endothelial growth factor (VEGF) stimulated migration of endothelial cells (ECs). In this study, we have characterized the molecular mechanism of LRR5 which reveals that its anti-migratory effect on ECs is mediated by inhibiting VEGF-stimulated endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO) release. LRR5 carried out this function through signaling pathways that involves PI3 kinase and Akt, but not ERK. This anti-NO release effect is mediated by the C-terminal 13 amino acids of LRR5, correlating with the anti-migratory function of this region.
The International Journal of Biochemistry & Cell Biology 03/2008; 40(10):2120-8. · 4.63 Impact Factor
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Lei Ye,
Husnain Kh Haider,
RuSan Tan,
WeeChi Toh,
Peter K Law,
WeeBeng Tan,
LiPing Su,
Wei Zhang, RuoWen Ge,
Yong Zhang,
YeanTeng Lim,
Eugene K W Sim
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ABSTRACT: We investigated the feasibility and efficacy of polyethylenimine (PEI) based human vascular endothelial growth factor-165 (hVEGF165) gene transfer into human skeletal myoblasts (HSM) for cell based delivery to the infarcted myocardium.
Based on optimized transfection procedure using enhanced green fluorescent protein (pEGFP), HSM were transfected with plasmid-hVEGF165 (phVEGF165) carried by PEI (PEI-phVEGF165) nanoparticles. The transfected HSM were characterized for transfection and expression of hVEGF165 in vitro and transplanted into rat heart model of acute myocardial infarction (AMI): group-1=DMEM injection, group-2= HSM transplantation, group-3= PEI-phVEGF165-transfected HSM (PEI-phVEGF165 myoblast) transplantation. A total of 48 rats received cyclosporine injection from 3 days before and until 4 weeks after cell transplantation. Echocardiography was performed to assess the heart function. Animals were sacrificed for molecular and histological studies on the heart tissue at 4 weeks after treatment. Based on optimized transfection conditions, transfected HSM expressed hVEGF165 for 18 days with >90% cell viability in vitro. Apoptotic index was reduced in group-2 and group-3 as compared with group-1. Blood vessel density (x400) by immunostaining for PECAM-1 in group-3 was significantly higher (P=0.043 for both) as compared with group-1 and group-2 at 4 weeks. Regional blood flow (ml/min/g) in the left ventricular anterior wall was higher in group-3 (P=0.043 for both) as compared with group-1 and group-2. Improved ejection fraction was achieved in group-3 (58.44+/-4.92%) as compared with group-1 (P=0.004).
PEI nanoparticle mediated hVEGF165 gene transfer into HSM is feasible and safe. It may serve as a novel and efficient alternative for angiomyogenesis in cardiac repair.
Circulation 10/2007; 116(11 Suppl):I113-20. · 14.74 Impact Factor
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ABSTRACT: The microinjection is a common technique in genetic engineering for transferring genetic material into a cell. It is normally performed on a micromanipulation system that usually consists of an inverted microscope, a micromanipulator, a micropipette and an injector. Manual microinjection is a conventional and widespread practice. However, this approach suffers from low success rate, poor efficiency, and high probability of contamination. We have developed a micromanipulation system for automatic batch microinjection by automating the key process of penetration in a microinjection. The microinjection system automatically identifies and penetrates each embryo/cell with a constant and fast speed, and in doing so, eliminates the possibility of contamination caused by manual operations. The effectiveness of this force-controlled system has been experimentally demonstrated in the tasks of automatic identification and penetration zebrafish embryos, which are widely used as a model for studying vertebrate development and genetics.
Robotics and Automation, 2007 IEEE International Conference on; 05/2007
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ABSTRACT: We compare the effectiveness of direct adenoviral angiopoietin-1 (Ad-Ang-1) injection with transplantation of skeletal myoblasts (SkMs) over-expressing angiopoietin-1 (Ang-1) for angiogenic response and improvement of heart function in an experimental porcine model of myocardial infarction (MI).
Ad-Ang-1 was used for intramyocardial injection or transduction of SkMs. Three weeks after coronary artery ligation in 32 female pigs, animals were grouped to receive multiple intramyocardial injections of DMEM without cells (group-1; n=7), or containing 3 x 10(8)Lac-z labelled SkMs transduced with Ad-Null vector carrying no gene (group-2; n=7), or 1 x 10(10) PFU Ad-Ang-1 (group-3; n=9), or 3 x 10(8)Lac-z labelled SkMs transduced with Ad-Ang-1 (group-4; n=9). The animals were immunosuppressed for 6-weeks. After euthanasia, their heart tissue was processed for histological studies.
Extensive survival of Lac-z positive SkMs was observed in and around the infarct 6 and 12-weeks after transplantation. Fluorescent immunostaining for vWF-VIII at 6-weeks revealed increased blood vessel density (x100) in group-4 (p<0.05) as compared with other groups. Regional blood flow (ml/g/min) in the peri-infarct area was improved in group-4 (2.7; p<0.05) as compared with group-1 (1.2+/-0.1), group-2 (1.1+/-0.4) and group-3 (1.7+/-0.1) at 6-weeks. Similarly, ejection fraction was significantly higher in group-4 (49.2+/-5.9%, p=0.03) as compared with group-1 (36.8+/-3%) at 6 weeks.
SkMs mediated Ang-1 delivery is associated with improved angiogenic response, regional myocardial perfusion and heart function as compared with direct Ad-Ang-1 administration.
European Journal of Heart Failure 05/2007; 9(5):458-65. · 4.90 Impact Factor
