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1.  Hepatoma Derived Growth Factor represses SET and MYND domain containing 1 gene expression through interaction with C-terminal binding protein 
Journal of molecular biology  2009;386(4):938-950.
Hepatoma Derived Growth Factor (HDGF) is a nuclear protein with both mitogenic and angiogenic activity, it is highly expressed in the developing heart and vasculature. To date the mechanisms of HDGF’s function are unknown. Oligonucleotide microarray analysis was used to gain insights into HDGF function. Adenoviral expression of HDGF significantly (≥ 2 fold) downregulated a large group (66) of genes, and increased expression of a relatively small number of genes (9). Two groups of target genes which are involved in cardiovascular development and transcriptional regulation were validated by real time PCR, including the skeletal/cardiac muscle specific SET and MYND domain containing 1 (SMYD1) gene. This suggested that HDGF could function as a transcriptional repressor. In a one-hybrid system, GBD-HDGF significantly repressed reporter gene activity in a dose dependent manner. This demonstrated that HDGF has transcriptional repressive activity. Moreover, in G-7 myoblast cells, overexpression of a GFP-HDGF fusion specifically downregulated SMYD1 mRNA expression and the activity of the human SMYD1 promoter. HDGF repressed SMYD1 gene transcription through interaction with a transcriptional corepressor C-terminal binding protein (CtBP). Overexpressing of CtBP potentiated the trans-repressive activity of HDGF; on the other hand, knocking down CtBP attenuated the trans-repressive effect of HDGF. HDGF binds CtBP through a non-canonical binding motif (PKDLF) within the PWWP domain, as substitutional mutation of DL to AS abolished HDGF and CtBP interaction and diminished the trans-repressive effect of HDGF without affecting DNA binding. Finally, fluorescent microscopy studies showed that HDGF induced the nuclear accumulation of CtBP suggesting that HDGF forms a transcriptional complex with CtBP. Taken together, our data demonstrate that HDGF functions as a transcriptional repressor of the SMYD1 gene, through interaction with the transcriptional corepressor CtBP. Because of moderate conservation of the CtBP binding motif in HDGF family members, trans-repressive activity mediated by CtBP may be a common function among HDGF proteins.
PMCID: PMC2752746  PMID: 19162039
HDGF; SMYD1; CtBP; transcription; repressor
2.  Glial Fibrillary Acidic Protein as a Biomarker for Periventricular White Matter Injury 
Periventricular white matter injury (PWMI), a precursor of cerebral palsy, is traditionally not diagnosed until 6 weeks of life by head ultrasound. We sought to determine if early neonatal glial fibrillary acidic protein (GFAP) levels could identify PWMI in low birth weight (< 2500 grams) infants.
Each case with PWMI on head ultrasound at 6 weeks from 4/09-4/11 was matched by gestational age and mode of delivery to 2 subsequent neonates with a normal head ultrasound. GFAP was measured in cord blood at birth, at neonatal intensive care unit (NICU) admission, and daily on days 1–4 of life.
During this 2 year period, 21 cases with PWMI with gestational age 27.4±3.3 weeks were compared to 42 controls. The incidence of cesarean delivery was 61.9% in both groups. GFAP was not significantly different in cord blood or at NICU admission, but was significantly elevated on day 1 (median, 5%–95%; 0, 0–0.98 ng/mL cases; 0,0–0.06 ng/mL controls, P=0.03), day 2 (0, 0–1.21 ng/mL; 0, 0–0.05 ng/mL; P=0.02), day 3 (0.05, 0–0.33 ng/mL; 0, 0–0.04 ng/mL, P=0.004) and day 4 (0.02, 0–1.03 ng/mL; 0, 0–0.05 ng/mL, P<0.001). The odds of developing PWMI significantly increased with increasing levels of GFAP from day 1 to day 4 of life adjusting for preeclampsia, antenatal steroid administration and neonatal chronic lung disease.
The ability to predict PWMI with a blood test for GFAP shortly after birth opens the possibility for rapid identification of infants for early intervention and provides a benchmark for qualifying new therapies to improve neurodevelopmental outcomes.
PMCID: PMC3708978  PMID: 23467054
Periventricular white matter injury; Glial fibrillary acidic protein; Cerebral palsy
3.  Glial Fibrillary Acidic Protein as a Biomarker for Neonatal Hypoxic-Ischemic Encephalopathy Treated with Whole Body Cooling 
GFAP is specific to astrocytes in the central nervous system. We hypothesized that serum GFAP would be increased in neonates with hypoxic-ischemic encephalopathy (HIE) treated with whole body cooling.
Study Design
We measured GFAP at birth and daily for up to 7 days for neonates in the intensive care unit. We compared neonates with HIE treated with whole body cooling to gestational age matched controls without neurologic injury and neonates with HIE by brain abnormalities on MRI.
Neonates with HIE had increased GFAP levels compared to controls. Neonates with HIE and abnormal brain imaging had elevated GFAP levels compared to neonates with HIE and normal imaging.
Serum GFAP levels during the first week of life were increased in neonates with HIE and were predictive of brain injury on MRI. Biomarkers like GFAP could help triage neonates with HIE to treatment, measure treatment efficacy and provide prognostic information.
PMCID: PMC4011017  PMID: 21784396
fetal acidosis; glial fibrillary acidic protein; hypoxic-ischemic encephalopathy; neonatal seizures; whole body cooling
4.  Frontiers in Pulmonary Hypertension in Infants and Children With Bronchopulmonary Dysplasia 
Pediatric pulmonology  2012;47(11):1042-1053.
Pulmonary hypertension (PH) is an increasingly recognized complication of premature birth and bronchopulmonary dysplasia (BPD), and is associated with increased morbidity and mortality. Extreme phenotypic variability exists among preterm infants of similar gestational ages, making it difficult to predict which infants are at increased risk for developing PH. Intrauterine growth retardation or drug exposures, postnatal therapy with prolonged positive pressure ventilation, cardiovascular shunts, poor postnatal lung and somatic growth, and genetic or epigenetic factors may all contribute to the development of PH in preterm infants with BPD. In addition to the variability of severity of PH, there is also qualitative variability seen in PH, such as the variable responses to vasoactive medications. To reduce the morbidity and mortality associated with PH, a multi-pronged approach is needed. First, improved screening for and increased recognition of PH may allow for earlier treatment and better clinical outcomes. Second, identification of both prenatal and postnatal risk factors for the development of PH may allow targeting of therapy and resources for those at highest risk. Third, understanding the pathophysiology of the preterm pulmonary vascular bed may help improve outcomes through recognizing pathways that are dysregulated in PH, identifying novel biomarkers, and testing novel treatments. Finally, the recognition of conditions and exposures that may exacerbate or lead to recurrent PH is needed to help with developing treatment guidelines and preventative strategies that can be used to reduce the burden of disease.
PMCID: PMC3963167  PMID: 22777709
pulmonary hypertension; bronchopulmonary dysplasia; prematurity; chronic lung disease
5.  Hepatoma-derived growth factor stimulates smooth muscle cell growth and is expressed in vascular development 
Journal of Clinical Investigation  2000;105(5):567-575.
Hepatoma-derived growth factor (HDGF) is the first member identified of a new family of secreted heparin-binding growth factors highly expressed in the fetal aorta. The biologic role of HDGF in vascular growth is unknown. Here, we demonstrate that HDGF mRNA is expressed in smooth muscle cells (SMCs), most prominently in proliferating SMCs, 8–24 hours after serum stimulation. Exogenous HDGF and endogenous overexpression of HDGF stimulated a significant increase in SMC number and DNA synthesis. Rat aortic SMCs transfected with a hemagglutinin-epitope–tagged rat HDGF cDNA contain HA-HDGF in their nuclei during S-phase. We also detected native HDGF in nuclei of cultured SMCs, of SMCs and endothelial cells from 19-day fetal (but not in the adult) rat aorta, of SMCs proximal to abdominal aortic constriction in adult rats, and of SMCs in the neointima formed after endothelial denudation of the rat common carotid artery. Moreover, HDGF colocalizes with the proliferating cell nuclear antigen (PCNA) in SMCs in human atherosclerotic carotid arteries, suggesting that HDGF helps regulate SMC growth during development and in response to vascular injury.
PMCID: PMC289171  PMID: 10712428
6.  Plasma proteomics of differential outcome to long-term therapy in children with idiopathic pulmonary arterial hypertension 
Proteomics. Clinical applications  2012;6(5-6):257-267.
The prognosis for children with IPAH unresponsive to therapy is poor. We investigated the plasma proteome for a molecular basis of good versus poor outcome to long-term vasodilator therapy.
Experimental design
Plasma was collected at baseline or shortly after therapy initiation and following chronic vasodilator therapy, then divided into those with good outcome (n = 8), and those with a poor outcome (n = 7). To identify proteins unique to either outcome, we used differential gel electrophoresis and mass spectrometry. Results were confirmed by commercial enzyme-linked immunosorbent assay.
Before and after therapy, SAA-4 was 4-fold lower in those with good outcome compared to those with poor outcome, while serum paraoxonase/arylesterase-1 was increased 2-fold in those with good outcome versus poor outcome. After therapy, haptoglobin and hemopexin were 1.45- and 1.8-fold lower, respectively, in those with a good versus poor outcome. Among those with a good outcome, SAP was 1.3-fold lower prior to therapy.
Conclusions and clinical relevance
SAP and SAA-4 regulate circulating mononuclear phagocytes. As such, they may contribute to the differential response to chronic vasodilator therapy in the context of inflammation in IPAH.
PMCID: PMC3569520  PMID: 22653875
Inflammation; Pulmonary hypertension; Vasodilators
7.  Plasma Glial Fibrillary Acidic Protein Levels in a Child with Sickle Cell Disease and Stroke 
Acta Haematologica  2010;125(3):103-106.
A 12-year-old boy with HbSS sickle cell disease (SCD) was admitted with an acute febrile illness and developed overt stroke 3 days later. Plasma glial fibrillary acidic protein levels were elevated, as compared to pediatric controls, 32 h prior to the clinical diagnosis of stroke, peaked immediately prior to the exchange transfusion, and remained elevated 1 year later despite chronic transfusion therapy. Stroke in SCD can occur in the setting of acute illness, and a biomarker that could predict the onset and triage ill children to therapeutic intervention more quickly would be useful.
PMCID: PMC3202928  PMID: 21099215
Chronic transfusion; Glial fibrillary acidic protein; Sickle cell; Stroke
8.  Assessment of Pulmonary Hypertension in the Pediatric Catheterization Laboratory 
To assess protocols, demographics, and hemodynamics in pediatric patients undergoing catheterization for pulmonary hypertension (PH).
Pediatric specific data is limited on PH.
Review of the Mid-Atlantic Group of Interventional Cardiology (MAGIC) collaboration PH registry dataset.
Between November 2003 and October 2008, seven institutions submitted data from 177 initial catheterizations in pediatric patients with suspected PH. Pulmonary arterial hypertension associated with congenital heart disease (APAH-CHD) (n = 61, 34%) was more common than idiopathic PAH (IPAH) (n = 36, 20%). IPAH patients were older with higher mean pulmonary arterial pressures (mPAP) (P < 0.01). Oxygen lowered mPAP in patients with IPAH (P < 0.01) and associated PAH not related to congenital heart disease (APAH-non CHD) (P < 0.01). A synergistic effect was seen with inhaled Nitric Oxide (iNO) (P < 0.01). Overall 9/30 (29%) patients with IPAH and 8/48 (16%) patients with APAH-non CHD were reactive to vasodilator testing. Oxygen lowered pulmonary vascular resistance index (PVRI) in patients with APAH-CHD (P < 0.01). There was no additive effect with iNO but a subset of patients required iNO to lower PVRI below 5 WU·m2. General anesthesia (GA) lowered systemic arterial pressure (P < 0.01) with no difference between GA and procedural sedation on mPAP or PVRI. Adverse events were rare (n = 7) with no procedural deaths.
Pediatric patients with PH demonstrate a higher incidence of APAH-CHD and neonatal specific disorders compared to adults. Pediatric PH patients may demonstrate baseline mPAP < 40 mm Hg but > 50% systemic illustrating the difficulty in applying adult criteria to children with PH. Catheterization in children with PH is relatively safe.
PMCID: PMC3116922  PMID: 20549685
pulmonary hypertension; pediatric; cardiac catheterization; hemodynamics; vasoreactivity
9.  Mitotic phosphorylation activates hepatoma-derived growth factor as a mitogen 
BMC Cell Biology  2011;12:15.
Hepatoma-derived growth factor (HDGF) is a nuclear protein that is a mitogen for a wide variety of cells. Mass spectrometry based methods have identified HDGF as a phosphoprotein without validation or a functional consequence of this post-translational modification.
We found that HDGF in primary mouse aortic vascular smooth muscle cells (VSMC) was phosphorylated. Wild type HDGF was phosphorylated in asynchronous cells and substitution of S103, S165 and S202 to alanine each demonstrated a decrease in HDGF phosphorylation. A phospho-S103 HDGF specific antibody was developed and demonstrated mitosis-specific phosphorylation. HDGF-S103A was not mitogenic and FACS analysis demonstrated a G2/M arrest in HDGF-S103A expressing cells, whereas cells expressing HDGF-S103D showed cell cycle progression. Nocodazole arrest increased S103 phosphorylation from 1.6% to 29% (P = 0.037).
Thus, HDGF is a phosphoprotein and phosphorylation of S103 is mitosis related and required for its function as a mitogen. We speculate that cell cycle regulated phosphorylation of HDGF may play an important role in vascular cell proliferation.
PMCID: PMC3094319  PMID: 21489262
10.  Milk Fat Globule Protein-Epidermal Growth Factor-8: a Pivotal Relay Element within the Angiotensin II and Monocyte Chemoattractant Protein-1 Signaling Cascade Mediating Vascular Smooth Muscle Cells Invasion 
Circulation research  2009;104(12):1337-1346.
Advancing age induces aortic wall thickening that results from the concerted effects of numerous signaling proteins, many of which have yet to be identified. To search for novel proteins associated with aortic wall thickening, we have performed a comprehensive quantitative proteomic study to analyze aortic proteins from young (8 mo) and old (30 mo) rats and identified 50 proteins that significantly change in abundance with aging. One novel protein, the milk fat globule protein epidermal growth factor 8 (MFG-E8), increases 2.3-fold in abundance in old aorta. Transcription and translation analysis demonstrated that aortic MFG-E8 mRNA and protein levels increase with aging in several mammalian species including humans. Dual immunolabeling shows that MFG-E8 colocalizes with both angiotensin II (Ang II) and monocyte chemoattractant protein-1 (MCP-1) within vascular smooth muscle cells (VSMCs) of the thickened aged aortic wall. Exposure of early passage VMSCs from young aorta to Ang II markedly increases MFG-E8 and enhances invasive capacity to levels observed in VSMCs from old rats. Treatment of VSMCs with MFG-E8 increases MCP-1 expression and VSMCs invasion that are inhibited by the MCP-1 receptor blocker, vCCI. Silencing MFG-E8 RNA substantially reduces MFG-E8 expression and VSMCs invasion capacity. The data indicate that arterial MFG-E8 significantly increases with aging and is a pivotal relay element within the Ang II/MCP-1/VSMC invasion signaling cascade. Thus, targeting of MFG-E8 within this signaling axis pathway is a potential novel therapy for the prevention and treatment of the age-associated vascular diseases such as atherosclerosis.
PMCID: PMC2764993  PMID: 19443842
MFG-E8; Angiotensin II; Monocyte chemoattractant protein-1; Vascular smooth muscle cells; Aging
Experimental cell research  2008;314(17):3107-3117.
Bone marrow-derived mesenchymal stromal cells (MSCs) localize to solid tumors. Defining the signaling mechanisms that regulate this process is important to understanding the role of MSCs in tumor growth. Using a combination of chromatography and electrospray tandem mass spectrometry we have identified novel soluble signaling molecules that induce MSC chemotaxis present in conditioned medium of the breast carcinoma cell line MDA-MB231. Previous work has employed survey strategies using ELISA assay to identify known chemokines that promote MSC chemotaxis. While these studies provide valuable insights into the intercellular signals that impact MSC behavior, many less well-described, but potentially important soluble signaling molecules could be overlooked using these methods. Through the less directed method of column chromatography we have identified novel candidate MSC chemotactic peptides. Two proteins, cyclophilin B and hepatoma-derived growth factor were then further characterized and shown to promote MSC chemotaxis.
PMCID: PMC2606107  PMID: 18722367
Mesenchymal Stromal Cell; Tumor microenvironment; chemotaxis
12.  Hepatoma derived growth factor binds DNA through the N-terminal PWWP domain 
BMC Molecular Biology  2007;8:101.
Hepatoma Derived Growth Factor (HDGF) is a nuclear protein with nuclear targeting required for mitogenic activity. Recently we demonstrated that HDGF is a transcriptional repressor, but whether HDGF binds DNA, the specificity of DNA binding and what protein domain is required are still unknown. In this study, we aimed to identify if HDGF is a DNA binding protein, map the functional DNA binding domain and DNA binding element for HDGF.
Using chromatin immunoprecipitation (ChIP) of human DNA, we isolated 10 DNA sequences sharing a conserved ~200 bp element. Homology analysis identified the binding sequences as a motif within the promoter of the SMYD1 gene, a HDGF target gene. Electrophoretic Mobility Shift Assays (EMSA) confirmed the binding of HDGF to this conserved sequence. As a result, an 80 bp conserved sequence located in the SMYD1 promoter bound GST-HDGF tightly. The binding core sequence for HDGF was narrowed down to 37 bp using a deletion mapping strategy from both the 5' and 3' ends. Moreover, ChIP and DNase I footprinting analysis revealed that HDGF binds this 80 bp DNA fragment specifically. Functionally overexpression of HDGF represses a reporter gene which is controlled by an SV-40 promoter containing the 80 bp DNA element. Using serial truncations of GST-HDGF, we mapped the DNA binding domain of HDGF to the N-terminal PWWP domain.
HDGF is a DNA binding protein, binds DNA specifically, and prefers a minimum of 37 bp long DNA fragment. The N-terminal PWWP domain of HDGF is required for DNA binding. HDGF exerts its transcription repressive effect through binding to a conserved DNA element in the promoter of target genes.
PMCID: PMC2176068  PMID: 17974029

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