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Xabier L Aranguren,
Jonathan D McCue,
Benoit Hendrickx,
Xiao-Hong Zhu,
Fei Du, Eleanor Chen,
Beatriz Pelacho,
Ivan Peñuelas,
Gloria Abizanda,
Maialen Uriz, [......],
Nathan H Harris,
Yi Zhang,
Xiaoliang Zhang,
Molly H Nelson-Holte,
Yuehua Jiang,
An D Billiau,
Wei Chen,
Felipe Prósper,
Catherine M Verfaillie,
Aernout Luttun
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ABSTRACT: Despite progress in cardiovascular research, a cure for peripheral vascular disease has not been found. We compared the vascularization and tissue regeneration potential of murine and human undifferentiated multipotent adult progenitor cells (mMAPC-U and hMAPC-U), murine MAPC-derived vascular progenitors (mMAPC-VP), and unselected murine BM cells (mBMCs) in mice with moderate limb ischemia, reminiscent of intermittent claudication in human patients. mMAPC-U durably restored blood flow and muscle function and stimulated muscle regeneration, by direct and trophic contribution to vascular and skeletal muscle growth. This was in contrast to mBMCs and mMAPC-VP, which did not affect muscle regeneration and provided only limited and transient improvement. Moreover, mBMCs participated in a sustained inflammatory response in the lower limb, associated with progressive deterioration in muscle function. Importantly, mMAPC-U and hMAPC-U also remedied vascular and muscular deficiency in severe limb ischemia, representative of critical limb ischemia in humans. Thus, unlike BMCs or vascular-committed progenitors, undifferentiated multipotent adult progenitor cells offer the potential to durably repair ischemic damage in peripheral vascular disease patients.
Journal of Clinical Investigation 03/2008; 118(2):505-14. · 15.39 Impact Factor
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ABSTRACT: Mutations in fibrillin-1 (FBN1) result in Marfan syndrome, demonstrating a critical requirement for microfibrils in vessel structure and function. However, the identity and function of many microfibril-associated molecules essential for vascular development and function have yet to be characterized. In our morpholino-based screen for members of the secretome required for vascular development, we identified a key player in microfibril formation in zebrafish embryogenesis. Microfibril-associated glycoprotein-1 (MAGP1) is a conserved protein found in mammalian and zebrafish microfibrils. Expression of magp1 mRNA is detected in microfibril-producing cells. Analysis of a functional Magp1-mRFP fusion protein reveals localization along the midline and in the vasculature during embryogenesis. Underexpression and overexpression analyses demonstrate that specific Magp1 protein levels are critical for vascular development. Integrin function is compromised in magp1 morphant embryos, suggesting that reduced integrin-matrix interaction is the main mechanism for the vascular defects in magp1 morphants. We further show that Magp1 and fibrillin-1 interact in vivo. This study implicates MAGP1 as a key player in microfibril formation and integrity during development. The essential role for MAGP1 in vascular morphogenesis and function also supports a wide range of clinical applications, including therapeutic targets in vascular disease and cardiovascular tissue engineering.
Blood 06/2006; 107(11):4364-74. · 9.90 Impact Factor
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ABSTRACT: The members of the Syndecan family of heparan sulfate proteoglycans play diverse roles in cell adhesion and cell communication by serving as co-receptors for both cell-signaling and extracellular matrix molecules. Syndecan-2 has been implicated in the formation of specialized membrane domains and functions as a direct link between the extracellular environment and the organization of the cortical cytoplasm. Recent studies have shown that syndecan-2 is required for angiogenesis, possibly by serving as a co-receptor for vascular endothelial growth factor, and cell-to-cell signaling during development of left-right asymmetry. This unique combination of activities suggests that syndecan-2 can function as a potential drug target for the development of multi-functional, anti-cancer therapeutics.
The International Journal of Biochemistry & Cell Biology 03/2006; 38(2):152-6. · 4.63 Impact Factor
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Michael A Pickart,
Eric W Klee,
Aubrey L Nielsen,
Sridhar Sivasubbu,
Eric M Mendenhall,
Brent R Bill, Eleanor Chen,
Craig E Eckfeldt,
Michelle Knowlton,
Mara E Robu,
Jon D Larson,
Yun Deng,
Lisa A Schimmenti,
Lynda B M Ellis,
Catherine M Verfaillie,
Matthias Hammerschmidt,
Steven A Farber,
Stephen C Ekker
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ABSTRACT: Understanding the functional role(s) of the more than 20,000 proteins of the vertebrate genome is a major next step in the post-genome era. The approximately 4,000 co-translationally translocated (CTT) proteins - representing the vertebrate secretome - are important for such vertebrate-critical processes as organogenesis. However, the role(s) for most of these genes is currently unknown.
We identified 585 putative full-length zebrafish CTT proteins using cross-species genomic and EST-based comparative sequence analyses. We further investigated 150 of these genes (Figure 1) for unique function using morpholino-based analysis in zebrafish embryos. 12% of the CTT protein-deficient embryos resulted in specific developmental defects, a notably higher rate of gene function annotation than the 2%-3% estimate from random gene mutagenesis studies.
This initial collection includes novel genes required for the development of vascular, hematopoietic, pigmentation, and craniofacial tissues, as well as lipid metabolism, and organogenesis. This study provides a framework utilizing zebrafish for the systematic assignment of biological function in a vertebrate genome.
PLoS ONE 02/2006; 1:e104. · 4.09 Impact Factor
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John R Ohlfest,
Zachary L Demorest,
Yasuhiko Motooka,
Isabelita Vengco,
Seunguk Oh, Eleanor Chen,
Frank A Scappaticci,
Rachel J Saplis,
Stephen C Ekker,
Walter C Low,
Andrew B Freese,
David A Largaespada
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ABSTRACT: Glioblastoma is a fatal brain tumor that becomes highly vascularized by secreting proangiogenic factors and depends on continued angiogenesis to increase in size. Consequently, a successful antiangiogenic therapy should provide long-term inhibition of tumor-induced angiogenesis, suggesting long-term gene transfer as a therapeutic strategy. In this study a soluble vascular endothelial growth factor receptor (sFlt-1) and an angiostatin-endostatin fusion gene (statin-AE) were codelivered to human glioblastoma xenografts by nonviral gene transfer using the Sleeping Beauty (SB) transposon. In subcutaneously implanted xenografts, co-injection of both transgenes showed marked anti-tumor activity as demonstrated by reduction of tumor vessel density, inhibition or abolition of glioma growth, and increase in animal survival (P = 0.003). Using luciferase-stable engrafted intracranial gliomas, the anti-tumor effect of convection-enhanced delivery of plasmid DNA into the tumor was assessed by luciferase in vivo imaging. Sustained tumor regression of intracranial gliomas was achieved only when statin-AE and sFlt-1 transposons were coadministered with SB-transposase-encoding DNA to facilitate long-term expression. We show that SB can be used to increase animal survival significantly (P = 0.008) by combinatorial antiangiogenic gene transfer in an intracranial glioma model.
Molecular Therapy 12/2005; 12(5):778-88. · 6.87 Impact Factor
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ABSTRACT: Heparan sulfate proteoglycans are important modulators of growth factor signaling in a variety of patterning processes. Secreted growth factors that play critical roles in angiogenesis bind to heparan sulfate, and this association is affected by 6-O-sulfation of the heparan sulfate chains. Addition of 6-O-sulfate is catalyzed by a family of sulfotransferases (HS6STs), and genetic manipulation of their function permits an assessment of their contribution to vascular assembly. We report on the biochemical activity and expression patterns of two zebrafish HS6ST genes. In situ hybridization reveals dynamic and distinct expression patterns of these two genes during development. Structural analysis of heparan sulfate from wild-type and morpholino antisense 'knockdown' embryos suggests that HS6ST-1 and HS6ST-2 have similar biochemical activity. HS6ST-2, but not HS6ST-1, morphants exhibit abnormalities in the branching morphogenesis of the caudal vein during embryonic development of the zebrafish. Our finding that HS6ST-2 is required for the branching morphogenesis of the caudal vein is the first in vivo evidence for an essential role of a gene encoding a heparan sulfate modifying enzyme in vertebrate angiogenesis. Our analysis of two zebrafish HS6ST genes suggests that a wide range of biological processes may be regulated by an array of sulfation-modifying enzymes in the vertebrate genome.
Developmental Biology 09/2005; 284(2):364-76. · 4.07 Impact Factor
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ABSTRACT: We investigated the mechanisms of intermediate cell mass (ICM) expansion in zebrafish chordin (Chd) morphant embryos and examined the role of BMPs in relation to this phenotype. At 24 h post-fertilization (hpf), the expanded ICM of embryos injected with chd morpholino (MO) (ChdMO embryos) contained a monotonous population of hematopoietic progenitors. In situ hybridization showed that hematopoietic transcription factors were ubiquitously expressed in the ICM whereas vascular gene expression was confined to the periphery. BMP4 (but not BMP2b or 7) and smad5 mRNA were ectopically expressed in the ChdMO ICM. At 48 hpf, monocytic cells were evident in both the ICM and circulation of ChdMO but not WT embryos. While injection of BMP4 MO had no effect on WT hematopoiesis, co-injecting BMP4 with chd MOs significantly reduced ICM expansion. Microarray studies revealed a number of genes that were differentially expressed in ChdMO and WT embryos and their roles in hematopoiesis has yet to be determined. In conclusion, the expanded ICM in ChdMO embryos represented an expansion of embryonic hematopoiesis that was skewed towards a monocytic lineage. BMP4, but not BMP2b or 7, was involved in this process. The results provide ground for further research into the mechanisms of embryonic hematopoietic cell expansion.
Developmental Biology 02/2005; 277(1):235-54. · 4.07 Impact Factor
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ABSTRACT: The zebrafish (Danio rerio) is a recent addition to the genomic scientists' repertoire of vertebrate animal model systems. Unlike simple invertebrates such as the fly or the nematode, this teleost maintains the biological and genomic complexity found in higher vertebrates. Furthermore, the zebrafish has many advantageous technical and genomic properties that open the door to experimental approaches not practical using more classical models. The zebrafish genome can be functionally accessed using both forward and reverse genetics based approaches. A notable recent addition to the zebrafish genomics toolbox is the development of morpholino-based antisense gene inhibition for sequence-based 'knockdown' screening. This method offers the opportunity to examine the role of significant subsets of the vertebrate genome for specific gene function in vivo. The zebrafish embryo can rapidly provide critical information for drug target discovery purposes when examined with an emphasis on clinically-relevant biological processes. Finally, the advent of chemical genetics in zebrafish suggests that, in addition to the identification and understanding of drug targets and their biology, this system will be a powerful tool in the direct development of novel pharmaceuticals in the near future.
Current Pharmaceutical Biotechnology 11/2004; 5(5):409-13. · 2.81 Impact Factor
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Jon D Larson,
Shannon A Wadman, Eleanor Chen,
Lesa Kerley,
Karl J Clark,
Mark Eide,
Sarah Lippert,
Aidas Nasevicius,
Stephen C Ekker,
Perry B Hackett,
Jeffrey J Essner
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ABSTRACT: We have identified the zebrafish homologue of VE-cadherin and documented its expression in the developing vascular system. The zebrafish VE-cadherin gene is specifically expressed in the vascular endothelial cell lineage beginning with the differentiation and migration of angioblasts and persists throughout vasculogenesis, angiogenesis, and endocardium development. Staining zebrafish embryos by whole-mount in situ hybridization with the VE-cadherin probe provides a method to screen embryos for vascular defects. To illustrate this utility, we used VE-cadherin expression to demonstrate a conservation of vascular endothelial growth factor-A (VEGF-A) function. The morpholino antisense oligonucleotide knockdown of VEGF-A function in zebrafish embryos results in a loss of angiogenic blood vessels, as indicated by the lack of VE-cadherin expression in the intersegmental vasculature. This loss can be restored in embryos supplemented with either zebrafish or human VEGF-A, the latter indicating that genes crucial to angiogenesis have highly conserved functional activities in vertebrates.
Developmental Dynamics 10/2004; 231(1):204-13. · 2.54 Impact Factor
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ABSTRACT: We used a morpholino-based gene-targeting screen to identify a novel protein essential for vascular development using the zebrafish, Danio rerio. We show that syndecan-2, a cell-surface heparan sulfate proteoglycan, is essential for angiogenic sprouting during embryogenesis. The vascular function of syndecan-2 is likely conserved, as zebrafish and mouse syndecan-2 show similar expression patterns around major trunk vessels, and human syndecan-2 can restore angiogenic sprouting in syndecan-2 morphants. In contrast, forced expression of a truncated form of syndecan-2 results in embryos with defects in angiogenesis, indicating that the highly conserved cytoplasmic tail is important for the vascular function of syndecan-2. We further show that vascular endothelial growth factor (VEGF) and syndecan-2 genetically interact in vivo using both gain-of-function and loss-of-function studies in zebrafish. VEGF-mediated ectopic signaling is compromised in syndecan-2 morphants, and ectopic syndecan-2 potentiates ectopic VEGF signaling. Syndecan-2 as a novel angiogenic factor is a potential candidate for use in the development of angiogenesis-based therapies.
Blood 04/2004; 103(5):1710-9. · 9.90 Impact Factor
