Receptor and Molecular Mechanism of AGGF1 Signaling in Endothelial Cell Functions and Angiogenesis.

Objective: Angiogenic factor AGGF1 (angiogenic factor with G-patch and FHA [Forkhead-associated] domain 1) promotes angiogenesis as potently as VEGFA (vascular endothelial growth factor A) and regulates endothelial cell (EC) proliferation, migration, specification of multipotent hemangioblasts and venous ECs, hematopoiesis, and vascular development and causes vascular disease Klippel-Trenaunay syndrome when mutated. However, the receptor for AGGF1 and the underlying molecular mechanisms remain to be defined. Approach and Results: Using functional blocking studies with neutralizing antibodies, we identified [alpha]5[beta]1 as the receptor for AGGF1 on ECs. AGGF1 interacts with [alpha]5[beta]1 and activates FAK (focal adhesion kinase), Src (proto-oncogene tyrosine-protein kinase), and AKT (protein kinase B). Functional analysis of 12 serial N-terminal deletions and 13 C-terminal deletions by every 50 amino acids mapped the angiogenic domain of AGGF1 to a domain between amino acids 604-613 (FQRDDAPAS). The angiogenic domain is required for EC adhesion and migration, capillary tube formation, and AKT activation. The deletion of the angiogenic domain eliminated the effects of AGGF1 on therapeutic angiogenesis and increased blood flow in a mouse model for peripheral artery disease. A 40-mer or 15-mer peptide containing the angiogenic domain blocks AGGF1 function, however, a 15-mer peptide containing a single amino acid mutation from -RDD- to -RGD- (a classical RGD integrin-binding motif) failed to block AGGF1 function. Conclusions: We have identified integrin [alpha]5[beta]1 as an EC receptor for AGGF1 and a novel AGGF1-mediated signaling pathway of [alpha]5[beta]1-FAK-Src-AKT for angiogenesis. Our results identify an FQRDDAPAS angiogenic domain of AGGF1 crucial for its interaction with [alpha]5[beta]1 and signaling.

Ultrasensitive and selective recognition of peptide hormone using close-packed arrays of hPTHR-conjugated polymer nanoparticles.

Recognition of diverse hormones in the human body is a highly significant challenge because numerous diseases can be affected by hormonal imbalances. However, the methodologies reported to date for detecting hormones have exhibited limited performance. Therefore, development of innovative methods is still a major concern in hormone-sensing applications. In this study, we report an immobilization-based approach to facilitate formation of close-packed arrays of carboxylated polypyrrole nanoparticles (CPPyNPs) and their integration with human parathyroid hormone receptor (hPTHR), which is a B-class family of G-protein-coupled receptors (GPCRs). Our devices enabled use of an electrically controllable liquid-ion-gated field-effect transistor by using the surrounding phosphate-buffered saline solution (pH 7.4) as electrolyte solution. Field-induced signals from the peptide hormone sensors were observed and provided highly sensitive and selective recognition of target molecules at unprecedentedly low concentrations (ca. 48 fM). This hormone sensor also showed long-term stability and excellent selectivity in fetal bovine serum. Importantly, the hormone receptor attached on the surface of CPPyNPs enabled GPCR functional studies; synergistic effects corresponding to increased hPTH peptide length were monitored. These results demonstrate that close-packed CPPyNP arrays are a promising approach for high-performance biosensing devices.

Multidimensional conducting polymer nanotubes for ultrasensitive chemical nerve agent sensing.

Tailoring the morphology of materials in the nanometer regime is vital to realizing enhanced device performance. Here, we demonstrate flexible nerve agent sensors, based on hydroxylated poly(3,4-ethylenedioxythiophene) (PEDOT) nanotubes (HPNTs) with surface substructures such as nanonodules (NNs) and nanorods (NRs). The surface substructures can be grown on a nanofiber surface by controlling critical synthetic conditions during vapor deposition polymerization (VDP) on the polymer nanotemplate, leading to the formation of multidimensional conducting polymer nanostructures. Hydroxyl groups are found to interact with the nerve agents. Representatively, the sensing response of dimethyl methylphosphonate (DMMP) as a simulant for sarin is highly sensitive and reversible from the aligned nanotubes. The minimum detection limit is as low as 10 ppt. Additionally, the sensor had excellent mechanical bendability and durability.

Fabrication of ultrafine metal-oxide-decorated carbon nanofibers for DMMP sensor application.

Ultrafine metal-oxide-decorated hybrid carbon nanofibers (CNFs) were fabricated by a single-nozzle co-electrospinning process using a phase-separated mixed polymer composite solution and heat treatment. To decorate metal oxides on the CNF surface, core (PAN) and shell (PVP) structured nanofibers (NFs) were fabricated as starting materials. The core-shell NF structure was prepared by single-nozzle co-electrospinning because of the incompatibility of the two polymers. Ultrafine hybrid CNFs were then formed by decomposing the PVP phase, converting the metal precursors to metal oxide nanonodules, and transforming the PAN to CNFs of ca. 40 nm diameter during heat treatment. The decoration morphology of the metal oxide nanonodules could be controlled by precursor concentration in the PVP solution. These ultrafine hybrid CNFs were applied to a dimethyl methylphosphonate (DMMP) chemical sensor at room temperature with excellent sensitivity. The minimum detectable level (MDL) of hybrid CNFs was as low as 0.1 ppb, which is 10-100 times higher than for a chemical sensor based on carbon nanotubes. This is because the metal oxide nanonodules of hybrid CNFs increase the surface area and affinity to DMMP vapor. Our new synthetic methodology promises to be an effective approach to fabricating hybrid CNF/inorganic nanostructures for future sensing technologies.

Novel roles of GATA1 in regulation of angiogenic factor AGGF1 and endothelial cell function.

AGGF1 is an angiogenic factor, and its deregulation is associated with a vascular malformation consistent with Klippel-Trenaunay syndrome (KTS). This study defines the molecular mechanism for transcriptional regulation of AGGF1 expression. Transcription of AGGF1 starts at two nearby sites, -367 and -364 bp upstream of the translation start site. Analyses of 5'- and 3'-serial promoter deletions defined the core promoter/regulatory elements, including two repressor sites (from -1971 to -3990 and from -7521 to -8391, respectively) and two activator sites (a GATA1 consensus binding site from -295 to -300 and a second activator site from -129 to -159). Both the GATA1 site and the second activator site are essential for AGGF1 expression. A similar expression profile was found for GATA1 and AGGF1 in cells (including various endothelial cells) and tissues. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays demonstrated that GATA1 was able to bind to the AGGF1 DNA in vitro and in vivo. Overexpression of GATA1 increased expression of AGGF1. We identified one rare polymorphism -294C>T in a sporadic KTS patient, which is located in the GATA1 site, disrupts binding of GATA1 to DNA, and abolishes the GATA1 stimulatory effect on transcription of AGGF1. Knockdown of GATA1 expression by siRNA reduced expression of AGGF1, and resulted in endothelial cell apoptosis and inhibition of endothelial capillary vessel formation and cell migration, which was rescued by purified recombinant human AGGF1 protein. These results demonstrate that GATA1 regulates expression of AGGF1 and reveal a novel role for GATA1 in endothelial cell biology and angiogenesis.

Proteomics with two-dimensional gel electrophoresis and mass spectrometry analysis in cardiovascular research.

Proteomics is a large-scale, comprehensive study of the proteins of a cell or organism. It is a unique means of characterizing proteins that are expressed in a cell or tissue at any given time-point and of identifying any modifications that they may undergo. Thus, it is a powerful technology that can detect and identify the changes of the structure and function of proteins in response to intra- and extracellular environmental signals or disease states. As proteomics can establish a link for genes and proteins with a disease, it will play an important role in defining the molecular determinants of a disease and in identifying targets for drug discoveries and diagnostics. We have carried out the first proteomics study for coronary artery disease (CAD) and found that the expression of the ferritin light chain was significantly increased in CAD tissues. In this chapter, we use the CAD study as an example to demonstrate the procedures involved in proteomics analysis. The proteome is visualized by two-dimensional gel electrophoresis, a powerful and widely used method for proteomics, and the proteins of interest are then identified by mass spectrometry. This technique should be useful in characterizing cardiovascular diseases and in defining signaling pathways for cardiovascular development and physiology.

Calcium-sensitive potassium channelopathy in human epilepsy and paroxysmal movement disorder.

The large conductance calcium-sensitive potassium (BK) channel is widely expressed in many organs and tissues, but its in vivo physiological functions have not been fully defined. Here we report a genetic locus associated with a human syndrome of coexistent generalized epilepsy and paroxysmal dyskinesia on chromosome 10q22 and show that a mutation of the alpha subunit of the BK channel causes this syndrome. The mutant BK channel had a markedly greater macroscopic current. Single-channel recordings showed an increase in open-channel probability due to a three- to fivefold increase in Ca(2+) sensitivity. We propose that enhancement of BK channels in vivo leads to increased excitability by inducing rapid repolarization of action potentials, resulting in generalized epilepsy and paroxysmal dyskinesia by allowing neurons to fire at a faster rate. These results identify a gene that is mutated in generalized epilepsy and paroxysmal dyskinesia and have implications for the pathogenesis of human epilepsy, the neurophysiology of paroxysmal movement disorders and the role of BK channels in neurological disease.

Ferritin in atherosclerosis.

Iron, an essential element for many important cellular functions in all living organisms, can catalyze the formation of potentially toxic free radicals. Excessive iron is sequestered by ferritin in a nontoxic and readily available form in a cell. Ferritin is composed of 24 subunits of different proportions of two functionally distinct subunits: ferritin H and L. The former is involved in ferroxidase activity necessary for iron uptake and oxidation of ferrous iron, while the latter is involved in nucleation of the iron core. The expression of ferritin is under delicate control and is regulated at both the transcriptional and posttranscriptional levels by iron, cytokines and oxidative stress. Elevated ferritin levels are associated with an increased risk of atherosclerotic coronary artery disease (CAD), the leading cause of death and illness in developed countries. Serum ferritin levels are a good indicator of iron stores in the body. In fact, epidemiological studies have suggested that elevated serum ferritin levels are associated with an increased risk of CAD and myocardial infarction (MI), though inconsistent results were obtained in some other studies. Moreover, recent proteomics and molecular biology studies have shown that ferritin levels in arteries are increased in diseased tissues, which further supports the link of ferritin to CAD/MI. Future studies will determine whether increased ferritin levels can serve as a distinct biomarker for the incidence of CAD/MI and distinguish whether increased ferritin levels are a cause of CAD or a consequence of the disease process.

Identification of an angiogenic factor that when mutated causes susceptibility to Klippel-Trenaunay syndrome.

Angiogenic factors are critical to the initiation of angiogenesis and maintenance of the vascular network. Here we use human genetics as an approach to identify an angiogenic factor, VG5Q, and further define two genetic defects of VG5Q in patients with the vascular disease Klippel-Trenaunay syndrome (KTS). One mutation is chromosomal translocation t(5;11), which increases VG5Q transcription. The second is mutation E133K identified in five KTS patients, but not in 200 matched controls. VG5Q protein acts as a potent angiogenic factor in promoting angiogenesis, and suppression of VG5Q expression inhibits vessel formation. E133K is a functional mutation that substantially enhances the angiogenic effect of VG5Q. VG5Q shows strong expression in blood vessels and is secreted as vessel formation is initiated. VG5Q can bind to endothelial cells and promote cell proliferation, suggesting that it may act in an autocrine fashion. We also demonstrate a direct interaction of VG5Q with another secreted angiogenic factor, TWEAK (also known as TNFSF12). These results define VG5Q as an angiogenic factor, establish VG5Q as a susceptibility gene for KTS, and show that increased angiogenesis is a molecular pathogenic mechanism of KTS.

Proteomic approach to coronary atherosclerosis shows ferritin light chain as a significant marker: evidence consistent with iron hypothesis in atherosclerosis.

Coronary artery disease (CAD) is the leading cause of mortality and morbidity in developed nations. We hypothesized that CAD is associated with distinct patterns of protein expression in the coronary arteries, and we have begun to employ proteomics to identify differentially expressed proteins in diseased coronary arteries. Two-dimensional (2-D) gel electrophoresis of proteins and subsequent mass spectrometric analysis identified the ferritin light chain as differentially expressed between 10 coronary arteries from patients with CAD and 7 coronary arteries from normal individuals. Western blot analysis indicated significantly increased expression of the ferritin light chain in the diseased coronary arteries (1.41 vs. 0.75; P = 0.01). Quantitative real-time PCR analysis showed that expression of ferritin light chain mRNA was decreased in diseased tissues (0.70 vs. 1.17; P = 0.013), suggesting that increased expression of ferritin light chain in CAD coronary arteries may be related to increased protein stability or upregulation of expression at the posttranscriptional level in the diseased tissues. Ferritin light chain protein mediates storage of iron in cells. We speculate that increased expression of the ferritin light chain may contribute to pathogenesis of CAD by modulating oxidation of lipids within the vessel wall through the generation of reactive oxygen species. Our results provide in situ proteomic evidence consistent with the "iron hypothesis," which proposes an association between excessive iron storage and a high risk of CAD. However, it is also possible that the increased ferritin expression in diseased coronary arteries is a consequence, rather than a cause, of CAD.