Lucy Liaw - Academia.edu (original) (raw)

Papers by Lucy Liaw

Notch signaling regulates smooth muscle cell phenotype and is critical for vascular development. ... more Notch signaling regulates smooth muscle cell phenotype and is critical for vascular development. One Notch target is smooth muscle -actin (SMA), a differentiated smooth muscle cell marker. The Notch intracellular domain (NotchICD) forms a complex with CBF-1 (C-promoter-binding factor-1) and directly induces SMA expression. Using primary human smooth muscle cells, we show that expression of the constitutive active ICD of

Matrix Biology, 2004

Osteopontin (OPN) is a secreted, arginine-glycine-aspartic acid (RGD)-containing phosphoprotein p... more Osteopontin (OPN) is a secreted, arginine-glycine-aspartic acid (RGD)-containing phosphoprotein proteolytically modified by members of the matrix metalloproteinase (MMP) family. We previously defined the MMP-3 and MMP-7 cleavage sites in OPN and found increased adhesive and migratory activity of a pool of MMP-cleaved fragments compared to full-length OPN. In the present study, we performed mutational analysis of recombinant full-length OPN and

Obesity (Silver Spring, Md.), Jan 7, 2015

This study investigated the effects of loss of Cthrc1 on adipogenesis, body composition, metaboli... more This study investigated the effects of loss of Cthrc1 on adipogenesis, body composition, metabolism, physical activity, and muscle physiology. Complete metabolic and activity monitoring as well as grip strength measurements and muscle myography was performed in Cthrc1 null and wildtype mice. Compared to wildtypes, Cthrc1 null mice had similar body weights but significantly reduced energy expenditure, decreased lean mass, and increased fat mass, especially visceral fat. In vitro studies demonstrated that Cthrc1 inhibited adipocyte differentiation as well as PPAR and CREB reporter activity, while preadipocytes isolated from Cthrc1 null mice exhibited enhanced adipogenic differentiation. Voluntary physical activity in Cthrc1 null mice as assessed by wheel running was reduced to approximately half the distance covered by wildtypes. Reduced grip strength was observed in Cthrc1 null mice at the age of 15 weeks or older with reduced performance and mass of hyphenate muscle. In the brain, C...

PloS one, 2015

Endoglin is a type III TGFβ auxiliary receptor that is upregulated in endothelial cells during an... more Endoglin is a type III TGFβ auxiliary receptor that is upregulated in endothelial cells during angiogenesis and, when mutated in humans, results in the vascular disease hereditary hemorrhagic telangiectasia (HHT). Though endoglin has been implicated in cell adhesion, the underlying molecular mechanisms are still poorly understood. Here we show endoglin expression in endothelial cells regulates subcellular localization of zyxin in focal adhesions in response to BMP9. RNA knockdown of endoglin resulted in mislocalization of zyxin and altered formation of focal adhesions. The mechanotransduction role of focal adhesions and their ability to transmit regulatory signals through binding of the extracellular matrix are altered by endoglin deficiency. BMP/TGFβ transcription factors, SMADs, and zyxin have recently been implicated in a newly emerging signaling cascade, the Hippo pathway. The Hippo transcription coactivator, YAP1 (yes-associated protein 1), has been suggested to play a crucial ...

The open medical imaging journal, Jan 31, 2013

The quantitative analysis of blood vessel volumes from magnetic resonance angiograms (MRA) or μCT... more The quantitative analysis of blood vessel volumes from magnetic resonance angiograms (MRA) or μCT images is difficult and time-consuming. This fact, when combined with a study that involves multiple scans of multiple subjects, can represent a significant portion of research time. In order to enhance analysis options and to provide an automated and fast analysis method, we developed a software plugin for the ImageJ and Fiji image processing frameworks that enables the quick and reproducible volume quantification of blood vessel segments. The novel plugin named Volume Calculator (VolCal), accepts any binary (thresholded) image and produces a three-dimensional schematic representation of the vasculature that can be directly manipulated by the investigator. Using MRAs of the mouse hindlimb ischemia model, we demonstrate quick and reproducible blood vessel volume calculations with 95 - 98% accuracy. In clinical settings this software may enhance image interpretation and the speed of data...

Infection and immunity, 1999

Expression of the cytokine osteopontin (OPN) is elevated in granulomas caused by Mycobacterium tu... more Expression of the cytokine osteopontin (OPN) is elevated in granulomas caused by Mycobacterium tuberculosis. We tested the hypothesis that OPN contributes to host protection in a mouse model of mycobacterial infection. When infected with Mycobacterium bovis BCG, mice lacking a functional OPN gene had more severe infections characterized by heavier bacterial loads and a delayed clearance of the bacteria. The OPN-null mice had greater granuloma burdens consistent with the elevated bacterial load. The ability of osteopontin to facilitate the clearance of mycobacteria was most pronounced early after infection and appeared to be independent of known mediators of resistance to infection by mycobacteria: antigen-specific T-cell immunity, gamma interferon production, and nitric oxide production. BCG grew more rapidly in macrophages derived from OPN-null mice than in those from wild-type mice, demonstrating that the null phenotype was due to an intrinsic macrophage defect. These results indi...

Cancer research, Jan 15, 1998

Secreted phosphoprotein 1 (spp1), the gene encoding osteopontin (OPN), is expressed in many human... more Secreted phosphoprotein 1 (spp1), the gene encoding osteopontin (OPN), is expressed in many human carcinomas, although its in vivo functions remain unclear. To delineate the role of OPN during tumor progression, we have subjected OPN null mutant mice to repeated applications of a mutagen/carcinogen to induce cutaneous squamous cell carcinoma. OPN null animals exhibited accelerated tumor growth and progression and had a greater number of metastases per animal compared with wild-type animals. However, metastases in the OPN null animals were significantly smaller than in controls. When injected into nude mice, the growth of OPN null tumor lines and the same lines engineered to reexpress spp1 recapitulated the growth differences observed in the progression study. These differences in tumor growth inversely correlated with the degree of macrophage infiltration. Slower-growing, OPN-producing tumors contained significantly more macrophages, although a higher proportion were mannose recepto...

Frontiers in Genetics, 2014

The human genome encodes for over 1800 microRNAs (miRNAs), which are short non-coding RNA molecul... more The human genome encodes for over 1800 microRNAs (miRNAs), which are short non-coding RNA molecules that function to regulate gene expression post-transcriptionally. Due to the potential for one miRNA to target multiple gene transcripts, miRNAs are recognized as a major mechanism to regulate gene expression and mRNA translation. Computational prediction of miRNA targets is a critical initial step in identifying miRNA:mRNA target interactions for experimental validation. The available tools for miRNA target prediction encompass a range of different computational approaches, from the modeling of physical interactions to the incorporation of machine learning. This review provides an overview of the major computational approaches to miRNA target prediction. Our discussion highlights three tools for their ease of use, reliance on relatively updated versions of miRBase, and range of capabilities, and these are DIANA-microT-CDS, miRanda-mirSVR, and TargetScan. In comparison across all miRNA target prediction tools, four main aspects of the miRNA:mRNA target interaction emerge as common features on which most target prediction is based: seed match, conservation, free energy, and site accessibility. This review explains these features and identifies how they are incorporated into currently available target prediction tools. MiRNA target prediction is a dynamic field with increasing attention on development of new analysis tools. This review attempts to provide a comprehensive assessment of these tools in a manner that is accessible across disciplines. Understanding the basis of these prediction methodologies will aid in user selection of the appropriate tools and interpretation of the tool output.

Trends in Cardiovascular Medicine, 1995

Molecular biology has greatly enhanced our ability to identify molecules expressed as a part of t... more Molecular biology has greatly enhanced our ability to identify molecules expressed as a part of the vascular response to injury. Among these, osteopontin is of particular interest. Osteopontin is a multifunctional adhesive glycoprotein implicated in the pathogenesis of a growing number of cardiovascular lesions, including restenosis, atherosclerosis, aortic valve calcification, and repair of myocardial wounds. In this review, we focus on structural, functional, and regulatory properties of osteopontin, which might be particularly relevant to cardiovascular lesion development.

Transgenic Research, 2006

Mouse strains expressing the site-specific Cre recombinase facilitate conditional ablation or act... more Mouse strains expressing the site-specific Cre recombinase facilitate conditional ablation or activation of genomic sequences when one or several exons of a gene of interest are flanked by loxP sites. Recently, several strains targeting Cre expression to adipocytes have been developed using promoter sequences from the aP2 (Fatty Acid Binding Protein 4, FABP4) gene for adipose tissue-specific gene expression studies. aP2/FABP4 is predominantly expressed in adipose tissue, and while this promoter provides adipocyte-restricted expression postnatally, its expression throughout embryonic development had not been previously characterized. In this report, we demonstrate that the aP2-Cre transgene is expressed and consistently localized within the embryo from mid-gestation stage 9.5 dpc. By 15.5 dpc, beta-gal activity was detected primarily in the brown adipose tissue, trigeminal ganglia, dorsal root ganglia, cartilage primordia and vertebrae. Immunofluorescence staining for Cre recombinase and FABP4 protein showed a corresponding staining pattern similar to that of beta-gal, confirming that Cre recombinase was produced in the transgenic line at late stages of development, and overlapped with endogenous aP2/FABP4 production. Further, fat-specific oil red O staining of tissue sections validated the presence of lipids in the stained tissues indicating that adipocytes and/or adipocyte-like cells were indeed present in these tissues. This is the first report to our knowledge to describe and confirm aP2/FABP4 promoter expression in this transgenic line during development in the mouse embryo and indicates that aP2/FABP4 expression occurs not only in mature adipocytes, but has a wider embryonic expression pattern than previously appreciated.

Transgenic Research, 2006

Cre recombinase has become a ubiquitous tool in transgenic strategies for regulation of transgene... more Cre recombinase has become a ubiquitous tool in transgenic strategies for regulation of transgene expression in a tissue-specific manner. We report analysis of two SM22alphaCre lines and their ability to mediate genomic recombination in five independent Cre-responsive transgenic lines. One of the SM22alphaCre lines developed was a tet-on system based on the reverse tetracycline transactivator. Our goal was to use this strategy to inhibit the Notch signaling pathway specifically in smooth muscle cells. Our responder transgenes contained a constitutively expressed marker gene (chloramphenicol acetyltransferase, CAT), flanked by loxP sites in direct orientation, upstream of Notch-related transgenes. We developed two dominant negative Notch transgenic responder lines activated by Cre-mediated DNA recombination. The first is the extracellular domain of human Jagged1, and the second is the extracellular domain of the human Notch2 receptor. Despite high expression of the marker gene in all responder lines, we found that Cre-mediated genomic recombination between these five lines was highly variable, ranging from 46 to 93% of individuals using an SM22alphaCre activating strain, or 8-58% of individuals using an inducible SM22alphartTACre. In all cases examined, detection of recombination by PCR correlated with expression of the transgene as determined by Western blot analysis. Our studies reflect the variability in recombination success based on the responder strain, presumably due to inaccessibility of the locus of integration of the responder allele.

Neurobiology of Disease, 2007

In the pathogenesis of Parkinson's disease (PD), oxidative and nitrosative stress, apoptosis, mit... more In the pathogenesis of Parkinson's disease (PD), oxidative and nitrosative stress, apoptosis, mitochondrial dysfunction, and excitotoxicity are involved, i.e., processes in which osteopontin (OPN) may also play a role. We have studied in PD patients serum and cerebrospinal fluid (CSF) concentrations of OPN, its immunohistochemical presence in substantia nigra (SN) and tested in OPN-null mice the impact of this protein on MPTP-induced neurodegeneration. PD was accompanied by increased OPN levels in the body fluids. Higher serum levels were associated with more severe motor symptoms. CSF levels were positively associated with concomitant dementia and negatively associated with dopaminergic treatment. In human SN, OPN was expressed in neurons, in their Lewy bodies and in microglia. Loss of tyrosine-hydroxylase-positive cells in the SN and of dopaminergic fibers in the striatum was reduced 3 weeks after MPTP intoxication in OPN-null mice. These data suggest that OPN is involved in PD-associated neurodegeneration.

Molecular Biology of the Cell, 2008

Angiogenesis is controlled by several regulatory mechanisms, including the Notch and fibroblast g... more Angiogenesis is controlled by several regulatory mechanisms, including the Notch and fibroblast growth factor (FGF) signaling pathways. FGF1, a prototype member of FGF family, lacks a signal peptide and is released through an endoplasmic reticulum-Golgi-independent mechanism. A soluble extracellular domain of the Notch ligand Jagged1 (sJ1) inhibits Notch signaling and induces FGF1 release. Thrombin, a key protease of the blood coagulation cascade and a potent inducer of angiogenesis, stimulates rapid FGF1 release through a mechanism dependent on the major thrombin receptor protease-activated receptor (PAR) 1. This study demonstrates that thrombin cleaves Jagged1 in its extracellular

Kidney International, 1999

Kidney International, 2002

Journal of Vascular Research, 2008

Our study characterizes Delta-like 1 (Dll1) in the adult mouse, particularly in normal versus inj... more Our study characterizes Delta-like 1 (Dll1) in the adult mouse, particularly in normal versus injured vasculature, with the aid of the transgenic Dll1(LacZ) line. Normal mouse adult tissues or those from the Dll1(LacZ) reporter line were analyzed for Dll1 expression and promoter activity. Vascular tissue was analyzed before and after carotid artery ligation. In wild-type mice, Dll1 transcript expression was widespread. Similarly, the Dll1(LacZ) reporter had beta-galactosidase activity detectable in the cerebellum, cerebrum, spinal cord, liver, lung and cornea, although the normal adult vasculature had no reporter expression. Following arterial ligation, there was acute induction of Dll1(LacZ) reporter expression, both in the ligated left carotid artery, and the uninjured right contralateral artery. Expression returned to low/undetectable levels 4-10 days after arterial ligation. The expression of Dll1 in the adult mouse is more widespread than previously realized, although not in resting large arteries in the adult mouse. Following arterial injury, Dll1 promoter activity is induced selectively in the endothelial cells of both the injured artery and the contralateral uninjured artery. Our results show that while overall expression in the adult mouse is widespread, Dll1 may be selectively expressed in the endothelium of injured vasculature, similar to the endothelial-restricted expression of Dll4.

Journal of Vascular Research, 2011

Background: Transforming growth factor-␤ (TGF-␤ ) plays an important role in vascular homeostasis... more Background: Transforming growth factor-␤ (TGF-␤ ) plays an important role in vascular homeostasis through effects on vascular smooth muscle cells (SMC). Fine-tuning of TGF-␤ signaling occurs at the level of ALK receptors or Smads, and is regulated with cell type specificity. Methods: Our goal was to understand TGF-␤ signaling in regulating SMC differentiation marker expression in human SMC. Activation of Smads was characterized, and loss-and gain-of-function reagents used to define ALK pathways. In addition, Smad-independent mechanisms were determined. Results: TGF-␤ type I receptors, ALK1 and ALK5, are expressed in human SMC, and TGF-␤ 1 phosphorylates Smad1/5/8 and Smad2/3 in a timeand dosage-dependent pattern. ALK5 activity, not bone morphogenetic protein type I receptors, is required for Smad phosphorylation. Endoglin, a TGF-␤ type III receptor, is a TGF-␤ 1 target in SMC, yet endoglin does not modify TGF-␤ 1 responsiveness. ALK5, not ALK1, is required for TGF-␤ 1-induction of SMC differentiation markers, and ALK5 signals through an ALK5/Smad3-and MAP kinase-dependent pathway. Conclusion: The definition of the specific signaling downstream of TGF-␤ regulating SMC differentiation markers will contribute to a better understanding of vascular disorders involving changes in SMC phenotype.

Journal of the American Society of Nephrology, 2003

Calcium nephrolithiasis is the most common form of renal stone disease, with calcium oxalate (CaO... more Calcium nephrolithiasis is the most common form of renal stone disease, with calcium oxalate (CaOx) being the predominant constituent of renal stones. Current in vitro evidence implicates osteopontin (OPN) as one of several macromolecular inhibitors of urinary crystallization with potentially important actions at several stages of CaOx crystal formation and retention. To determine the importance of OPN in vivo, hyperoxaluria was induced in mice targeted for the deletion of the OPN gene together with wild-type control mice. Both groups were given 1% ethylene glycol, an oxalate precursor, in their drinking water for up to 4 wk. At 4 wk, OPN-deficient mice demonstrated significant intratubular deposits of CaOx crystals, whereas wild-type mice were completely unaffected. Retained crystals in tissue sections were positively identified as CaOx monohydrate by both polarized optical microscopy and x-ray powder diffraction analysis. Furthermore, hyperoxaluria in the OPN wild-type mice was associated with a significant 2to 4-fold upregulation of renal OPN expression by immunocytochemistry, lending further support to a renoprotective role for OPN. These data indicate that OPN plays a critical renoprotective role in vivo as an inhibitor of CaOx crystal formation and retention in renal tubules.

Journal of Investigative Dermatology, 2008

Journal of Experimental Medicine, 2001

Osteopontin (OPN) is a chemotactic protein that attracts immune cells, to inflammatory sites. The... more Osteopontin (OPN) is a chemotactic protein that attracts immune cells, to inflammatory sites. The sensitization phase of allergic cutaneous contact hypersensitivity (CHS) requires the migration of Langerhans cells/dendritic cells (LCs/DCs) from skin to draining lymph nodes. Characterizing OPN function for LC/DC migration we found upregulated OPN expression in hapten sensitized skin and draining lymph nodes. OPN induces chemotactic LC/DC migration, initiates their emigration from the epidermis, and attracts LCs/DCs to draining lymph nodes by interacting with CD44 and ␣ v integrin. Furthermore, OPN-deficient mice have a significantly reduced CHS response that correlates with an impaired ability of OPN-deficient mice to attract LCs/DCs to draining lymph nodes. In conclusion, OPN is an important factor in the initiation of CHS by guiding LCs/DCs from skin into lymphatic organs.

Notch signaling regulates smooth muscle cell phenotype and is critical for vascular development. ... more Notch signaling regulates smooth muscle cell phenotype and is critical for vascular development. One Notch target is smooth muscle -actin (SMA), a differentiated smooth muscle cell marker. The Notch intracellular domain (NotchICD) forms a complex with CBF-1 (C-promoter-binding factor-1) and directly induces SMA expression. Using primary human smooth muscle cells, we show that expression of the constitutive active ICD of

Matrix Biology, 2004

Osteopontin (OPN) is a secreted, arginine-glycine-aspartic acid (RGD)-containing phosphoprotein p... more Osteopontin (OPN) is a secreted, arginine-glycine-aspartic acid (RGD)-containing phosphoprotein proteolytically modified by members of the matrix metalloproteinase (MMP) family. We previously defined the MMP-3 and MMP-7 cleavage sites in OPN and found increased adhesive and migratory activity of a pool of MMP-cleaved fragments compared to full-length OPN. In the present study, we performed mutational analysis of recombinant full-length OPN and

Obesity (Silver Spring, Md.), Jan 7, 2015

This study investigated the effects of loss of Cthrc1 on adipogenesis, body composition, metaboli... more This study investigated the effects of loss of Cthrc1 on adipogenesis, body composition, metabolism, physical activity, and muscle physiology. Complete metabolic and activity monitoring as well as grip strength measurements and muscle myography was performed in Cthrc1 null and wildtype mice. Compared to wildtypes, Cthrc1 null mice had similar body weights but significantly reduced energy expenditure, decreased lean mass, and increased fat mass, especially visceral fat. In vitro studies demonstrated that Cthrc1 inhibited adipocyte differentiation as well as PPAR and CREB reporter activity, while preadipocytes isolated from Cthrc1 null mice exhibited enhanced adipogenic differentiation. Voluntary physical activity in Cthrc1 null mice as assessed by wheel running was reduced to approximately half the distance covered by wildtypes. Reduced grip strength was observed in Cthrc1 null mice at the age of 15 weeks or older with reduced performance and mass of hyphenate muscle. In the brain, C...

PloS one, 2015

Endoglin is a type III TGFβ auxiliary receptor that is upregulated in endothelial cells during an... more Endoglin is a type III TGFβ auxiliary receptor that is upregulated in endothelial cells during angiogenesis and, when mutated in humans, results in the vascular disease hereditary hemorrhagic telangiectasia (HHT). Though endoglin has been implicated in cell adhesion, the underlying molecular mechanisms are still poorly understood. Here we show endoglin expression in endothelial cells regulates subcellular localization of zyxin in focal adhesions in response to BMP9. RNA knockdown of endoglin resulted in mislocalization of zyxin and altered formation of focal adhesions. The mechanotransduction role of focal adhesions and their ability to transmit regulatory signals through binding of the extracellular matrix are altered by endoglin deficiency. BMP/TGFβ transcription factors, SMADs, and zyxin have recently been implicated in a newly emerging signaling cascade, the Hippo pathway. The Hippo transcription coactivator, YAP1 (yes-associated protein 1), has been suggested to play a crucial ...

The open medical imaging journal, Jan 31, 2013

The quantitative analysis of blood vessel volumes from magnetic resonance angiograms (MRA) or μCT... more The quantitative analysis of blood vessel volumes from magnetic resonance angiograms (MRA) or μCT images is difficult and time-consuming. This fact, when combined with a study that involves multiple scans of multiple subjects, can represent a significant portion of research time. In order to enhance analysis options and to provide an automated and fast analysis method, we developed a software plugin for the ImageJ and Fiji image processing frameworks that enables the quick and reproducible volume quantification of blood vessel segments. The novel plugin named Volume Calculator (VolCal), accepts any binary (thresholded) image and produces a three-dimensional schematic representation of the vasculature that can be directly manipulated by the investigator. Using MRAs of the mouse hindlimb ischemia model, we demonstrate quick and reproducible blood vessel volume calculations with 95 - 98% accuracy. In clinical settings this software may enhance image interpretation and the speed of data...

Infection and immunity, 1999

Expression of the cytokine osteopontin (OPN) is elevated in granulomas caused by Mycobacterium tu... more Expression of the cytokine osteopontin (OPN) is elevated in granulomas caused by Mycobacterium tuberculosis. We tested the hypothesis that OPN contributes to host protection in a mouse model of mycobacterial infection. When infected with Mycobacterium bovis BCG, mice lacking a functional OPN gene had more severe infections characterized by heavier bacterial loads and a delayed clearance of the bacteria. The OPN-null mice had greater granuloma burdens consistent with the elevated bacterial load. The ability of osteopontin to facilitate the clearance of mycobacteria was most pronounced early after infection and appeared to be independent of known mediators of resistance to infection by mycobacteria: antigen-specific T-cell immunity, gamma interferon production, and nitric oxide production. BCG grew more rapidly in macrophages derived from OPN-null mice than in those from wild-type mice, demonstrating that the null phenotype was due to an intrinsic macrophage defect. These results indi...

Cancer research, Jan 15, 1998

Secreted phosphoprotein 1 (spp1), the gene encoding osteopontin (OPN), is expressed in many human... more Secreted phosphoprotein 1 (spp1), the gene encoding osteopontin (OPN), is expressed in many human carcinomas, although its in vivo functions remain unclear. To delineate the role of OPN during tumor progression, we have subjected OPN null mutant mice to repeated applications of a mutagen/carcinogen to induce cutaneous squamous cell carcinoma. OPN null animals exhibited accelerated tumor growth and progression and had a greater number of metastases per animal compared with wild-type animals. However, metastases in the OPN null animals were significantly smaller than in controls. When injected into nude mice, the growth of OPN null tumor lines and the same lines engineered to reexpress spp1 recapitulated the growth differences observed in the progression study. These differences in tumor growth inversely correlated with the degree of macrophage infiltration. Slower-growing, OPN-producing tumors contained significantly more macrophages, although a higher proportion were mannose recepto...

Frontiers in Genetics, 2014

The human genome encodes for over 1800 microRNAs (miRNAs), which are short non-coding RNA molecul... more The human genome encodes for over 1800 microRNAs (miRNAs), which are short non-coding RNA molecules that function to regulate gene expression post-transcriptionally. Due to the potential for one miRNA to target multiple gene transcripts, miRNAs are recognized as a major mechanism to regulate gene expression and mRNA translation. Computational prediction of miRNA targets is a critical initial step in identifying miRNA:mRNA target interactions for experimental validation. The available tools for miRNA target prediction encompass a range of different computational approaches, from the modeling of physical interactions to the incorporation of machine learning. This review provides an overview of the major computational approaches to miRNA target prediction. Our discussion highlights three tools for their ease of use, reliance on relatively updated versions of miRBase, and range of capabilities, and these are DIANA-microT-CDS, miRanda-mirSVR, and TargetScan. In comparison across all miRNA target prediction tools, four main aspects of the miRNA:mRNA target interaction emerge as common features on which most target prediction is based: seed match, conservation, free energy, and site accessibility. This review explains these features and identifies how they are incorporated into currently available target prediction tools. MiRNA target prediction is a dynamic field with increasing attention on development of new analysis tools. This review attempts to provide a comprehensive assessment of these tools in a manner that is accessible across disciplines. Understanding the basis of these prediction methodologies will aid in user selection of the appropriate tools and interpretation of the tool output.

Trends in Cardiovascular Medicine, 1995

Molecular biology has greatly enhanced our ability to identify molecules expressed as a part of t... more Molecular biology has greatly enhanced our ability to identify molecules expressed as a part of the vascular response to injury. Among these, osteopontin is of particular interest. Osteopontin is a multifunctional adhesive glycoprotein implicated in the pathogenesis of a growing number of cardiovascular lesions, including restenosis, atherosclerosis, aortic valve calcification, and repair of myocardial wounds. In this review, we focus on structural, functional, and regulatory properties of osteopontin, which might be particularly relevant to cardiovascular lesion development.

Transgenic Research, 2006

Mouse strains expressing the site-specific Cre recombinase facilitate conditional ablation or act... more Mouse strains expressing the site-specific Cre recombinase facilitate conditional ablation or activation of genomic sequences when one or several exons of a gene of interest are flanked by loxP sites. Recently, several strains targeting Cre expression to adipocytes have been developed using promoter sequences from the aP2 (Fatty Acid Binding Protein 4, FABP4) gene for adipose tissue-specific gene expression studies. aP2/FABP4 is predominantly expressed in adipose tissue, and while this promoter provides adipocyte-restricted expression postnatally, its expression throughout embryonic development had not been previously characterized. In this report, we demonstrate that the aP2-Cre transgene is expressed and consistently localized within the embryo from mid-gestation stage 9.5 dpc. By 15.5 dpc, beta-gal activity was detected primarily in the brown adipose tissue, trigeminal ganglia, dorsal root ganglia, cartilage primordia and vertebrae. Immunofluorescence staining for Cre recombinase and FABP4 protein showed a corresponding staining pattern similar to that of beta-gal, confirming that Cre recombinase was produced in the transgenic line at late stages of development, and overlapped with endogenous aP2/FABP4 production. Further, fat-specific oil red O staining of tissue sections validated the presence of lipids in the stained tissues indicating that adipocytes and/or adipocyte-like cells were indeed present in these tissues. This is the first report to our knowledge to describe and confirm aP2/FABP4 promoter expression in this transgenic line during development in the mouse embryo and indicates that aP2/FABP4 expression occurs not only in mature adipocytes, but has a wider embryonic expression pattern than previously appreciated.

Transgenic Research, 2006

Cre recombinase has become a ubiquitous tool in transgenic strategies for regulation of transgene... more Cre recombinase has become a ubiquitous tool in transgenic strategies for regulation of transgene expression in a tissue-specific manner. We report analysis of two SM22alphaCre lines and their ability to mediate genomic recombination in five independent Cre-responsive transgenic lines. One of the SM22alphaCre lines developed was a tet-on system based on the reverse tetracycline transactivator. Our goal was to use this strategy to inhibit the Notch signaling pathway specifically in smooth muscle cells. Our responder transgenes contained a constitutively expressed marker gene (chloramphenicol acetyltransferase, CAT), flanked by loxP sites in direct orientation, upstream of Notch-related transgenes. We developed two dominant negative Notch transgenic responder lines activated by Cre-mediated DNA recombination. The first is the extracellular domain of human Jagged1, and the second is the extracellular domain of the human Notch2 receptor. Despite high expression of the marker gene in all responder lines, we found that Cre-mediated genomic recombination between these five lines was highly variable, ranging from 46 to 93% of individuals using an SM22alphaCre activating strain, or 8-58% of individuals using an inducible SM22alphartTACre. In all cases examined, detection of recombination by PCR correlated with expression of the transgene as determined by Western blot analysis. Our studies reflect the variability in recombination success based on the responder strain, presumably due to inaccessibility of the locus of integration of the responder allele.

Neurobiology of Disease, 2007

In the pathogenesis of Parkinson's disease (PD), oxidative and nitrosative stress, apoptosis, mit... more In the pathogenesis of Parkinson's disease (PD), oxidative and nitrosative stress, apoptosis, mitochondrial dysfunction, and excitotoxicity are involved, i.e., processes in which osteopontin (OPN) may also play a role. We have studied in PD patients serum and cerebrospinal fluid (CSF) concentrations of OPN, its immunohistochemical presence in substantia nigra (SN) and tested in OPN-null mice the impact of this protein on MPTP-induced neurodegeneration. PD was accompanied by increased OPN levels in the body fluids. Higher serum levels were associated with more severe motor symptoms. CSF levels were positively associated with concomitant dementia and negatively associated with dopaminergic treatment. In human SN, OPN was expressed in neurons, in their Lewy bodies and in microglia. Loss of tyrosine-hydroxylase-positive cells in the SN and of dopaminergic fibers in the striatum was reduced 3 weeks after MPTP intoxication in OPN-null mice. These data suggest that OPN is involved in PD-associated neurodegeneration.

Molecular Biology of the Cell, 2008

Angiogenesis is controlled by several regulatory mechanisms, including the Notch and fibroblast g... more Angiogenesis is controlled by several regulatory mechanisms, including the Notch and fibroblast growth factor (FGF) signaling pathways. FGF1, a prototype member of FGF family, lacks a signal peptide and is released through an endoplasmic reticulum-Golgi-independent mechanism. A soluble extracellular domain of the Notch ligand Jagged1 (sJ1) inhibits Notch signaling and induces FGF1 release. Thrombin, a key protease of the blood coagulation cascade and a potent inducer of angiogenesis, stimulates rapid FGF1 release through a mechanism dependent on the major thrombin receptor protease-activated receptor (PAR) 1. This study demonstrates that thrombin cleaves Jagged1 in its extracellular

Kidney International, 1999

Kidney International, 2002

Journal of Vascular Research, 2008

Our study characterizes Delta-like 1 (Dll1) in the adult mouse, particularly in normal versus inj... more Our study characterizes Delta-like 1 (Dll1) in the adult mouse, particularly in normal versus injured vasculature, with the aid of the transgenic Dll1(LacZ) line. Normal mouse adult tissues or those from the Dll1(LacZ) reporter line were analyzed for Dll1 expression and promoter activity. Vascular tissue was analyzed before and after carotid artery ligation. In wild-type mice, Dll1 transcript expression was widespread. Similarly, the Dll1(LacZ) reporter had beta-galactosidase activity detectable in the cerebellum, cerebrum, spinal cord, liver, lung and cornea, although the normal adult vasculature had no reporter expression. Following arterial ligation, there was acute induction of Dll1(LacZ) reporter expression, both in the ligated left carotid artery, and the uninjured right contralateral artery. Expression returned to low/undetectable levels 4-10 days after arterial ligation. The expression of Dll1 in the adult mouse is more widespread than previously realized, although not in resting large arteries in the adult mouse. Following arterial injury, Dll1 promoter activity is induced selectively in the endothelial cells of both the injured artery and the contralateral uninjured artery. Our results show that while overall expression in the adult mouse is widespread, Dll1 may be selectively expressed in the endothelium of injured vasculature, similar to the endothelial-restricted expression of Dll4.

Journal of Vascular Research, 2011

Background: Transforming growth factor-␤ (TGF-␤ ) plays an important role in vascular homeostasis... more Background: Transforming growth factor-␤ (TGF-␤ ) plays an important role in vascular homeostasis through effects on vascular smooth muscle cells (SMC). Fine-tuning of TGF-␤ signaling occurs at the level of ALK receptors or Smads, and is regulated with cell type specificity. Methods: Our goal was to understand TGF-␤ signaling in regulating SMC differentiation marker expression in human SMC. Activation of Smads was characterized, and loss-and gain-of-function reagents used to define ALK pathways. In addition, Smad-independent mechanisms were determined. Results: TGF-␤ type I receptors, ALK1 and ALK5, are expressed in human SMC, and TGF-␤ 1 phosphorylates Smad1/5/8 and Smad2/3 in a timeand dosage-dependent pattern. ALK5 activity, not bone morphogenetic protein type I receptors, is required for Smad phosphorylation. Endoglin, a TGF-␤ type III receptor, is a TGF-␤ 1 target in SMC, yet endoglin does not modify TGF-␤ 1 responsiveness. ALK5, not ALK1, is required for TGF-␤ 1-induction of SMC differentiation markers, and ALK5 signals through an ALK5/Smad3-and MAP kinase-dependent pathway. Conclusion: The definition of the specific signaling downstream of TGF-␤ regulating SMC differentiation markers will contribute to a better understanding of vascular disorders involving changes in SMC phenotype.

Journal of the American Society of Nephrology, 2003

Calcium nephrolithiasis is the most common form of renal stone disease, with calcium oxalate (CaO... more Calcium nephrolithiasis is the most common form of renal stone disease, with calcium oxalate (CaOx) being the predominant constituent of renal stones. Current in vitro evidence implicates osteopontin (OPN) as one of several macromolecular inhibitors of urinary crystallization with potentially important actions at several stages of CaOx crystal formation and retention. To determine the importance of OPN in vivo, hyperoxaluria was induced in mice targeted for the deletion of the OPN gene together with wild-type control mice. Both groups were given 1% ethylene glycol, an oxalate precursor, in their drinking water for up to 4 wk. At 4 wk, OPN-deficient mice demonstrated significant intratubular deposits of CaOx crystals, whereas wild-type mice were completely unaffected. Retained crystals in tissue sections were positively identified as CaOx monohydrate by both polarized optical microscopy and x-ray powder diffraction analysis. Furthermore, hyperoxaluria in the OPN wild-type mice was associated with a significant 2to 4-fold upregulation of renal OPN expression by immunocytochemistry, lending further support to a renoprotective role for OPN. These data indicate that OPN plays a critical renoprotective role in vivo as an inhibitor of CaOx crystal formation and retention in renal tubules.

Journal of Investigative Dermatology, 2008

Journal of Experimental Medicine, 2001

Osteopontin (OPN) is a chemotactic protein that attracts immune cells, to inflammatory sites. The... more Osteopontin (OPN) is a chemotactic protein that attracts immune cells, to inflammatory sites. The sensitization phase of allergic cutaneous contact hypersensitivity (CHS) requires the migration of Langerhans cells/dendritic cells (LCs/DCs) from skin to draining lymph nodes. Characterizing OPN function for LC/DC migration we found upregulated OPN expression in hapten sensitized skin and draining lymph nodes. OPN induces chemotactic LC/DC migration, initiates their emigration from the epidermis, and attracts LCs/DCs to draining lymph nodes by interacting with CD44 and ␣ v integrin. Furthermore, OPN-deficient mice have a significantly reduced CHS response that correlates with an impaired ability of OPN-deficient mice to attract LCs/DCs to draining lymph nodes. In conclusion, OPN is an important factor in the initiation of CHS by guiding LCs/DCs from skin into lymphatic organs.