Rong Wang, Ph.D. - US grants

This program has a long term objective to provide a superior low- noble metal content alloy system having metal/ceramics compatibility, and biocompatibility at a lower cost than the present high-noble alloys. The combination of the performance and cost factors of this alloy system will allow it to replace noble metal and base metal alloys for dental restoration. The aim of the Phase I project is to explore the design and fabrication of Duplex alloys and crowns using rapid solidification and casting technology. Consequently, materials will be characterized and tested for demonstration of metal/ porcelain compatibility, corrosion resistance, and fit accuracy. In Phase II of the program, both alloy design and process parameters will be optimized. Initial evaluation of the material performance will include regional dental practitioners and a clinical evaluation program at the dental clinic of the University of Washington. Market assessment will be conducted in several regions of the U.S. aiming for commercial development. The improved performance Duplex alloys would be synthesized using current well-known dental materials, suggesting there could be rapid acceptance of these Duplex alloys by the dentistry community. These materials offer the opportunity to substitute for base metal alloys, which have toxicity problems, with a material having the desirable characteristics of high-noble alloys, but at reduced cost.

This program has a long term objective to provide superior composite dental alloys having metal/ceramics compatibility, and biocompatibility at a lower cost than the present high-noble alloys. The combination of the performance and cost factors of these alloys will allow them to replace noble metal and base metal alloys for dental restoration. The aim of Phase I research was to explore the design and prove the fabrication concept of duplex alloys using rapid solidification and casting technology. In Phase II, both alloy design and processing parameters will be optimized. Performance evaluations would be conducted for demonstration of metal/porcelain compatibility, and corrosion resistance. Optionally, a task is offered providing duplex alloy design principles to form duplex Ti implant alloys. With the duplex alloys, a strong and ductile metal/ceramic interface could be formed to enhance the durability and strength of Ti implants.

technology /technique development; biomedical resource; biomedical equipment development;

technology /technique development; proteins; nucleic acids; biomedical resource; biomedical equipment development; biological products;

technology /technique development; proteins; biomedical resource; biological products;

technology /technique development; proteins; biomedical resource; biomedical equipment development;

Facile, effective means for determining the carboxyl-terminal amino acid sequence of proteins have long been sought. One promising approach utilizes mass spectrometric analysis of peptide fragments produced by enzymatic degradation of the polypeptide of interest with carboxypeptidases, wherein differences in the measured masses of the degradation products define the carboxyl-terminal sequence. However, the utility of this approach has been limited by large discrepancies in the rates of digestion of different terminal amino acid residues, leading to difficulties in producing uninterrupted sequence-defining peptide fragments. In order to ensure a mixture of uninterrupted sequence-defining peptide fragments (i.e., a continuous "peptide ladder"), it is necessary to either diminish differential rates of terminal amino acid removal or to render a fraction of each peptide product resistant to further degradation. Towards this objective, we have explored a new strategy in which hydrolysis and aminolysis are set up as competing reactions catalyzed by the same exopeptidase. The hydrolysis reaction removes the terminal amino acid residue, while the aminolysis (reverse proteolysis) reaction is designed to add a terminating group to the newly formed termini. We use an amino acid-amide (lysinamide) as a terminating reagent because it competes effectively as a nucleophile with water for the acyl-enzyme intermediate and because the resulting peptide amide is relatively resistant to hydrolysis. Our results demonstrate that kinetic effects resulting from the addition of a large molar excess of lysinamide can considerably improve the control of carboxypeptidase digestion for carboxyl-terminal sequencing by mass spectrometric readout of the resulting peptide ladders. Large discrepancies in enzyme digestion rates tend to be evened out because both hydrolysis and aminolysis are catalyzed by the enzyme. Although further optimization is desirable, the present strategy has the potential to provide an easy and reliable method for obtaining limited carboxyl-terminal sequences of peptides and proteins. To assist in the c-terminal sequencing of proteins by the method outlined above, we have also begun to develop a practical means for isolating the C-terminal peptide from a lys-C digest of a protein (see following subproject).

Affinity MS is being used extensively in a detailed series of studies concerning proteolytic degradation of P-amyloid protein AD). In particular, the new approach is being used to help: Identify proteolytic fragments of AD in cultured cells. Measure degradation rates for AD 1 -40 and A P 1 -42. Characterize proteolytic enzymes involved in AD degradation. Examine pH dependency of proteolytic degradation of AD. Correlate proteolytic AD degradation and AD peptide composition in Alzheimer's disease patient's brain.

Evidence for the significant advances made in the Rockefeller Mass Spectrometry Resource in Technological Research and Development can be assessed from the 14 presentations given by members of The Resource at the 45 ASMS Conference in Palms Springs, CA. May 1997. ( One page Summaries of each presentation follow below).

Protein ladder sequencing is a technique developed in our laboratory to sequence polypeptides including those with modified amino acids. One of the important potential applications of this technique is the study of protein phosphorylation. To understand the specificities of different protein kinases and to further explore the potentials of ladder sequencing, a model peptide, which contains multiple serine-phosphorylation sites, was synthesized chemically. This peptide is being used to study the specificities of several different protein kinases and the stochiometries of phosphorylation. In addition, variously phosphorylated (site and number) model peptides (same sequence as above peptide) will be synthesized to examine whether our mass spectrometric technique can be used to obtain detailed information concerning the stochiometry of phosphorylation. A paper describing these results is in preparation.

The amino acid selenomethionine has been used in x-ray crystallography as a label to determine the phase in place of heavy metal derivatives. However, in some cases, the selenomethioninecontaining protein crystals give poor diffraction patterns. One hypothesis for this phenomenon is that selenomethionine is oxidized more easily than methionine. To examine this hypothesis, we have used matrix-assisted laser desorption mass spectrometry to determine the oxidized products generated by hydrogen peroxide oxidation. To better characterize the sensitivity to oxidation for selenomethionine residue in peptide, we employed reverse-phase HPLC to quantitatively analyze the oxidized products and on-line HPLC/ESI mass spectrometry to identify the products. By this means, we have characterized the oxidation properties of selenomethionine residue in peptide. A paper describing this work is in preparation.

We have constructed a cell line for expressing human APP and PSI proteins using mouse neuroplastorna cells (N2a cell) and studied the effect of PSI mutations on AP production by analyzing AP peptide composition in cell culture media conditioned by cells transfected with human APP and PSI cDNAs. We have also studied the effect of PSI mutations on AP degradation by analyzing proteolytic degradation products of synthetic AP peptide (API-40 and API-42) in cultured cells transfectes with wildtype and mutant human PSI cDNAs. Finally, we are analyzing AP peptides in human cerebrospinal. fluid (CSF) and brain tissue specimens of AD patient with PSI mutations to determine whether we can determine abnon-nal pattern and levels in patients with disease.

Recent progress in elucidating the pathogenesis of Alzheimer's disease (AD) has centered on the apparent role of the 40-42 residue amyloid beta- peptide (Abeta) as a unifying pathological feature of the genetically diverse forms of this complex disorder. For example, DNA mutations have been identified in several distinct genes (four of which have been discovered to date) associating with familial AD (FAD). In addition, others have shown that the proteolytic processing of beta-amyloid precursor protein (betaAPP) is altered by many of these mutations in a way that results in increased production of Abeta peptides, particularly of the highly hydrophobic (and thus amyloidogenic) 42-residue form of Abeta (Abeta/42). Elevated production of Abeta/42 by FAD-linked genetic mutations indicates that the proteolytic cleavage of betaAPP by gamma- secretase may be a key element in the formation of amyloidogenic form of Abeta. A full understanding of the molecular basis of the substrate specificity of gamma-secretase requires knowledge of how FAD-associated genetic mutations affect the processing of betaAPP in AD. However, none of these enzymes responsible for the proteolytic cleavage of betaAPP have yet been identified. The objective of this study is to investigate the molecular mechanisms of gamma-secretase in the processing of betaAPP, with three specific aims. (1) Characterizing the substrate specificity of gamma-secretase cleavage of betaAPP. (2) Investigating the effects of the association of the transmembrane domain of betaAPP with membrane on gamma- secretase cleavage. (3) Investigating the effects of the localization of betaAPP to different cellular compartment on the function of gamma- secretase. Site directed mutagenesis will be used to vary either the properties of amino acid residues around the gamma-secretase cleavage site or to vary the length of the transmembrane domain of betaAPP. The exact identities of secreted Abeta-related peptides will be determined be measuring their molecular basses using immunoprecipitation and mass spectrometry. These studies will reveal fine detains concerning the cleavage specificities of gamma-secretase towards betaAPP, leading to better strategies to identify the as yet undiscovered enzyme(s).

Facile, effective means for determining the caThoxyl-terminal amino acid sequence of proteins have long been sought. One promising approach utilizes mass spectrometric analysis of peptide fi-agments produced by enzymatic degradation of the polypeptide of interest with carboxypeptidases, wherein differences in the measured masses of the degradation products define the carboxyl-terminal sequence. However, the utility of this approach has been limited by large discrepancies in the rates of digestion of different terminal amino acid residues, leading to difficulties in producing uninterrupted sequence-defining peptide firagments. In order to ensure a mixture of uninterrupted sequence-defining peptide ftagrnents (i.e., a continuous "peptide ladder), it is necessary to either diminish differential rates of terminal amino acid removal or to render a fraction of each peptide product resistant to finther degradation. Towards this ob ective, we have explored a new strategy in which hydrolysis and aminolysis are set up as competing reactions catalyzed by the same exopeptidase. The hydrolysis reaction removes the terminal amino acid residue, while the aminolysis (reverse proteolysis) reaction is designed to add a terminating group to the newly fonned termini. We use an amino acid-amide (lysinamide) as a terminating reagent because it competes effectively as a nucleophile with water for the acyl-enzyme intermediate and because the resulting peptide amide is relatively resistant to hydrolysis. Our results demonstrate that kinetic effects resulting from the addition of a large molar excess of lysinamide can considerably improve the control of carboxypeptidase digestion for carboxyl-terminal sequencing by mass spectrometric readout of the resulting peptide ladders. Large discrepancies in enzyme digestion rates tend to be evened out because both hydrolysis and arninolysis are catalyzed by the enzyme. Although fin-dier optimization is desirable, the present strategy has the potential to provide an easy and reliable method for obtaining limited carboxyl-terminal sequences of peptides and proteins. To assist in the c-terminal sequencing of proteins by the method outlined above, we have also begun to develop a practical means for isolating the C-terminal peptide from a lys-C digest of a protein (see following subproject).

We have initiated a study using immuno-affmity technique combined with matrix-assisted laser desorption mass spectrometry to characterize P-Amyloid related peptides. In this study, monoclonal anti-p-amyloid peptide antibody was used as a "fishhook" to specifically and sensitively capture the target peptide-amyloid related peptides in samples. Amyloid related peptides was first immunoprecipitated by monoclonal Anti-amyloid antibodies. The antibody-antigen complex was precipitated by protein A/Gagarose. After washing away non-specific bonded peptides, the complex was analyzed by matrix-assisted laser desorption mass spectrometry. The sensitivity and quantitative aspects of this method has been carefully evaluated. Cell culture equipment was set up in our laboratory to facilitate cell biological studies and studies of the processing of P-Amyloid in vitro. We have used our method to investigate the processing of P-Amyloid protein in a series of cultured cell lines (e.g.,we have investigated the effects of the S 182 gene mutation on chromosome- 14 on the production and degradation of amyloid P protein) and in the near future plan to extend the investigation to an analysis of amyloid peptides in CSF of patients with Alzheimer's disease. A paper describing these results has been published in the J. Biol. Chem. 272, 43234326, 1997. Currently we are: Analyze the effect of detergents on immunoprecipitation/mass spectrometry analysis of proteins. Compare the effeciency of different detergents on extracting intracellular AP peptides. Optimize the washing condition for removing non-specific boned protein and detergents used in immunoprecipitation.

The Rad51 protein, a homolog of bacterial RecA, functions in DNA double-strand break repair and genetic recombination. Whereas Rad51 catalyzes ATP-dependent pairing and strand exchange between homologous DNA molecules, regulation of this function is unknown. The c-Abl tyrosine kinase is activated by ionizing radiation and certain other DNA-damaging agents. Here we demonstrate that c-Abl interacts constitutively with Rad51. We show that c-Abl phosphorylates Rad51 on Tyr-54 in vitro. The results also show that treatment of cells with ionizing radiation induces c-Abl-dependent phosphorylation of Rad51. Phosphorylation of Rad51 by c-Abl inhibits the binding of Rad51 to DNA and the function of Rad51 in ATP-dependent DNA strand exchange reactions. These findings represent the first demonstration that Rad51 is regulated by phosphorylation and support a functional role for c-Abl in regulating Rad51-dependent recombination in the response to DNA damage. Zhi-Min Yuan, Yinyin Huang, Takatoshi Ishiko, Shuji Nakada, Taiju Utsugisawa, Surender Kharbanda, Rong Wang, Patrick Sung, Akira Shinohara, Ralph Weichselbaum, and Donald Kufe (1998), J. Biol. Chem. 273, 3799-3802.

We have constructed a new electrospray apparatus that is capable of stable spray for flow rates between 50ml/min - 5(l/min. We are using this spray apparatus to couple capillary HPLC to our Finnigan LCQ ion trap mass spectrometer and inusion electrospray to our Finnigan TSQ700 triple quadrupole apparatus. The use of this source has improved sensitivity by a factor of ~8 and has made electrospray MS considerably more robust.

Amyloid (-protein (A() is the major protein constituent found in the senile plaques of Alzheimer's disease (AD) and is produced by proteolytic processing of a large type-I transmembrane protein, (-amyloid precursor protein ((APP). Biochemical studies have demonstrated that genetic mutations in presenilin genes (PS1 and PS2) give rise elevated A(1-42. We have recently reported that A(1-43 can be detected from cultured cell media conditioned by mouse neuroblastoma (N2a) cells stably transfected with human APP695 cDNA and PS1 cDNA carrying exon-9 deletion mutation in addition to other carboxy-terminal heterogeneous A( peptides using immunoprecipitation/mass spectrometry assay. We have further investigated the generation of this A(1-43 species. The results from these studies have revealed several molecular characteristics of this A(1-43 species. Its generation is not correlated to the total A( level and neither the ratio of A(1-42 to A(1-40. It has only being detected from cell culture media conditioned by cells expressing PS1 with exon-9 deletion mutation but not other PS1 mutations including M146L and A246E point mutations. The effect of the exon-9 deletion of PS1 on production of this A(1-43 peptide is same for both wild type APP and mutant APP with Swedish mutation. Intracellular A( peptide profile and subcellular A( peptide distribution were analyzed by subcellular fractionation and cont.... immunoprecipitation/mass spectrometry, which reveals detailed information on the effect of PS1 mutations on intracellular A( peptide production. These ex vivo experimental data will be reported in this presentation and should help us to understand how PS1 mutations function on APP processing and production of A(.

In the course of investigating biotransformation of Alzheimer's amyloid (-protein (A(), a hydrophobic protein, we have developed a micro-scale sample preparation procedure for analyzing soluble A( directly from biological fluid. In this micro-scale sample preparation, the immunoprecipitation technique was utilized to enhance the specificity and sensitivity for A( detection. This method was further developed for analyzing intracellular soluble and aggregate forms of A(s directly from cell lysates in the presence of detergents. Three type of detergents (nonionic, zwitterionic and anionic) were tested for the efficiency of solubilizing hydrophobic A(s and effect on mass spectrometric signal-to-noise ratio (S/N) of A( peaks. Our data showed that both Triton X-100 and CHAPS can efficiently lyse cells and solubilize A(s without harming the S/N of spectra. The nonionic detergent, N-octylglucoside (NOG), did not result useful spectra although NOG has been reported have no harmfu l effect on MALDI-MS assay for proteins. Our experimental results indicate that this micro-scale sample preparation method is a reliable method and should be a general and useful method for studying intracellular and hydrophobic proteins by MALDI-MS directly from complex biological preparations.

(-Secretase activity is the final cleavage event that releases the A( or P3, respectively, from the corresponding (- or (-secretase cleaved carboxyl terminal fragments of the APP. No protease responsible for this highly unusual, purportedly intramembranous, cleavage has been isolated. We examined the substrate specificity of (-secretase by mutating various residues within or adjacent to the transmembrane domain of the APP and then analyzing A( production from cells transfected with these mutant APPs by ELISA and mass spectrometry. A( production was also analyzed from a subset of TMD APP mutants that showed dramatic shifts in (-secretase cleavage in the presence or absence of pepstatin, an inhibitor of (-secretase activity. These studies demonstrate that (-secretase's cleavage specificity is primarily determined by location of the (-secretase cleavage site of APP with respect to the membrane, and that (-secretase activity is due to the action of multiple proteases exhibiting both a pepstatin-sensitive activity and a pepstatin-insensitive activity. Given that (-secretase is a major therapeutic target in Alzheimer's disease these studies provide important information with respect to the mechanism of A( production that will direct efforts to isolate the (-secretases and develop effective therapeutic inhibitors of pathologically relevant (-secretase activities.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been successfully used in measuring intact protein molecular masses, identifying post-translational modifications of proteins, elucidating structure domains of proteins, and identifying unknown proteins. In most of these applications, mass (exactly should be mass/charge) is the only information being utilized. The peak intensity as the second part information provided by mass spectrometry is hardly used because the complexity of matrix-assisted laser desorption ionization processes. From a practical point view, we investigated the effects of matrix, solvent on peak intensity as well as sample distribution on sample probe of proteins and peptides with different size and different hydrophobicity. Based on these investigations, we formalized an experimental protocol on how to select solvent, select internal standard, and collect spectra for quantitative analysis of prote in and pep tide using MALDI-TOF-MS. The plot of relative peak intensity vs. protein concentration showed linear range of three to four orders of magnitude for quantitation. Our data indicated that the coefficient variation of quantitation by MALDI can be limited to less than 20%. The quantitative analysis of protein by MALDI-TOF-MS has its own advantages comparing with other mass spectrometric method, which all of the ions of protein and peptide are detected in one spectrum and the concentration of these protein and peptide can be measured at once.

The amino acid selenomethionine has been used in x-ray crystallography as a label to determine the phase in place of heavy metal derivatives. However, in some cases, the selenomethionine-containing protein crystals give poor diffraction patterns. One hypothesis for this phenomenon is that selenomethionine is oxidized more easily than methionine. To examine this hypothesis, we have used matrix-assisted laser desorption mass spectrometry to determine the oxidized products generated by hydrogen peroxide oxidation. To better characterize the sensitivity to oxidation for selenomethionine residue in peptide, we employed reverse-phase HPLC to quantitatively analyze the oxidized products and on-line HPLC/ESI mass spectrometry to identify the products. By this means, we have characterized the oxidation properties of selenomethionine residue in peptide. A paper describing this work is in preparation.

This research project, supported by the Division of Integrative Biology and Neuroscience, will be carried out by Dr. Rong Wang, Dr. Nickolas Menhart and their students at the Illinois Institute of Technology. The target of the research is to develop a novel approach that allows a real-time study of the dynamic behavior of individual ligand-receptor pairs (typical size of several nanometers) in the living cell environment. The essence of this approach is to guide the tip of the atomic force microscope (AFM) to desired receptor proteins regardless of the roughness and complexity on the cell membrane surface. Besides imaging of biomolecules at the submolecular level under physiological conditions, dynamic and kinetic processes of the biorecognition events can be clarified on the nanoscale.
The research will involve novel experiments aimed at fundamental studies of single ligand-receptor interaction in the natural environment. This will provide the molecular basis for biological activities and molecular communications within cells. One of the promising applications is to elucidate a vaccine or drug target at the particular cell-surface protein in a diseased cell or an activator target in a growth cell. This revolutionary approach shows strong promise to elevate the development of the fundamental understanding of molecular functions in bioscience to an entirely new level, and will stimulate progress in the study of biological and biologically inspired systems in which nanostructures play an important role.

serine threonine protein kinase; biomedical resource; macromolecule; clinical research; mass spectrometry;

mass spectrometry

DESCRIPTION (provided by applicant): The formation of arterial and venous (AV) branches must be exquisitely coordinated to generate proper AV circuitry essential for vascular function. The mechanism of AV coordination particularly that between paired, parallel arteries and veins is poorly understood. Our long-term objective is to elucidate the genetic program of mammalian AV circuitry. In the previous funding period, we reported that luminal sizes of the developing dorsal aorta (DA) and cardinal vein (CV) are synchronized. Notch signaling controls arterial specification and the allocation of both arterial and venous endothelial cells (ECs) into their respective vessels, thereby balancing the sizes of the developing DA and CV. We have also obtained preliminary data in mice suggesting that DA and CV formation is not initiated by pre-determined arterial and venous ECs, as previously thought. Instead, our work suggests a new step-wise model of mammalian parallel AV pair morphogenesis: the primitive unspecified artery assembles prior to the vein;followed by a phase of mixed AV identities in both vessels;finally the mixed ECs are segregated into uniformly-specified vessels with coordinated sizes. The specific aims of this grant are designed to test this new paradigm and to define the cellular mechanisms mediated by AV signaling in the morphogenesis of parallel AV pairs in mice. Our strategy is to take a cross- disciplinary approach including cutting-edge mouse genetics, cell lineage fate mapping, and imaging technologies. We recently built a custom 2-photon excited fluorescence microscope that is capable of imaging vasculature 1000 5m deep in living mouse tissue, achieving unprecedented resolution of previously inaccessible vascular structures. Aim 1 Examine Vascular Endothelial Growth Factor (VEGF)-mediated cell differentiation as a mechanism underlying heterogeneous arterial- and venous- fated ECs in the primordial DA (pDA) and CV (pCV). Aim 2 Examine cell segregation as a mechanism to sort venous-fated ECs in the pDA to the pCV. Aim 3 Determine the role of Notch signaling in coordinating the development of parallel artery and vein pairs. Aim 4 Determine the requirement of endothelial Notch1 and Coup-TFII in AV specification of adult parallel artery and vein pairs. Successful completion of this study will conceptually advance our knowledge of the morphogenesis and maintenance of parallel AV pairs, providing evidence regarding the origins of arteries and veins. Basic knowledge of the molecular mechanism of AV specification will inspire novel approaches to study blood vessel regeneration and vein graft engineering in disease settings. The combination of 2-photon high-resolution imaging with cutting-edge cell lineage tracing in living mouse embryos will be a major technological innovation for the field of mammalian vascular development at large. PUBLIC HEALTH RELEVANCE: Our study aims to reveal the role of the Notch signaling pathway in vascular differentiation and maintenance and illuminate potential molecular mechanisms underlying these processes. In the future, this basic understanding of Notch in pre- and post-natal mammalian vascular function will guide investigations into its function in vessel regeneration under pathological conditions, such as heart attack, stroke, and other ischemic diseases. Ultimately, our understanding of the Notch pathway may lead to the identification of novel drug targets and therapeutic interventions for cardiovascular diseases.

DESCRIPTION (provided by applicant): Brain arteriovenous malformations (BAVMs) can cause stroke and epilepsy and have no effective treatment. BAVMs are abnormal arteriovenous (AV) shunts that are not believed to regress spontaneously, but rather are prone to dangerous rupture. The cellular and molecular basis of BAVM pathogenesis remains enigmatic. Our long-term objectives are to elucidate the mechanisms of BAVM pathogenesis and to identify novel therapeutic targets to ameliorate this disease. Our general strategy is to take a cross-disciplinary approach fusing cutting-edge mouse genetics and imaging technologies to determine the function of critical molecular pathways that normally regulate AV differentiation, such as Notch signaling, in the pathogenesis of BAVM. We have reported a faithful transgenic mouse model of BAVMs, in which expression of constitutively-active Notch4 (Notch4*) specifically in endothelium elicits hallmarks of BAVMs in immature mice. Furthermore, the areas within the developing brain which grow most rapidly, likely the most angiogenic, were most susceptible to Notch4* effects, suggesting that angiogenesis underlies BAVM formation. Repression of Notch4* expression in severely affected mice resulted in a reversal of neurologic symptoms and recovery from the illness, suggesting that BAVM-like lesions can regress in animals when the molecular cause is removed. We have also reported that Notch activity is increased in the endothelium of human BAVMs, suggesting that Notch signaling may act as a molecular mediator in the human disease. Here we hypothesize that Notch4* during angiogenesis inhibits a capillary number increase, thus promoting the enlargement of capillary diameter, which initiates and sustains AV shunts that catalyze BAVM formation. Our specific aims are designed to elucidate the mechanisms of Notch4*-mediated onset, progression, and regression of BAVM-like lesions in mice. We will combine our mouse model of BAVM with advanced 2-photon imaging to obtain 4D vascular morphology at cellular resolution and blood velocity data in living brains. Our custom-built 2-photon microscope, optimal for cerebral vascular imaging, makes this innovative study possible. Aim1 Examine the angiogenic mechanism by which Notch4* elicits BAVM-like lesions in mice. Aim2 Examine lateral induction as a potential mechanism by which Notch4* propagates Notch signaling in cerebral endothelium. Aim3 Determine the cellular mechanism underlying the regression of AV shunting upon Notch4* repression. Successful completion of this study will conceptually advance our understanding of the cellular and molecular mechanisms of BAVM pathogenesis and help establish new paradigms in the knowledge and treatment of BAVMs. Our establishment of 2-photon high resolution imaging to study BAVM development in living animals will be a major technological innovation for BAVM research at large. PUBLIC HEALTH RELEVANCE: Brain arteriovenous malformations (BAVMs) are abnormal connections between arteries and veins that can cause stroke and epilepsy. There is currently no effective treatment for BAVMs, which are conventionally believed to not regress, although recent evidence suggests regression is possible. This proposal is designed to determine the molecular pathways underlying BAVM formation and regression, with the hope of identifying novel therapeutic targets to treat this disease.

DESCRIPTION (provided by applicant): The formation of arterial and venous (AV) branches must be exquisitely coordinated to generate proper AV circuitry essential for vascular function. The mechanism of AV coordination particularly that between paired, parallel arteries and veins is poorly understood. Our long-term objective is to elucidate the genetic program of mammalian AV circuitry. In the previous funding period, we reported that luminal sizes of the developing dorsal aorta (DA) and cardinal vein (CV) are synchronized. Notch signaling controls arterial specification and the allocation of both arterial and venous endothelial cells (ECs) into their respective vessels, thereby balancing the sizes of the developing DA and CV. We have also obtained preliminary data in mice suggesting that DA and CV formation is not initiated by pre-determined arterial and venous ECs, as previously thought. Instead, our work suggests a new step-wise model of mammalian parallel AV pair morphogenesis: the primitive unspecified artery assembles prior to the vein; followed by a phase of mixed AV identities in both vessels; finally the mixed ECs are segregated into uniformly-specified vessels with coordinated sizes. The specific aims of this grant are designed to test this new paradigm and to define the cellular mechanisms mediated by AV signaling in the morphogenesis of parallel AV pairs in mice. Our strategy is to take a cross- disciplinary approach including cutting-edge mouse genetics, cell lineage fate mapping, and imaging technologies. We recently built a custom 2-photon excited fluorescence microscope that is capable of imaging vasculature 1000 5m deep in living mouse tissue, achieving unprecedented resolution of previously inaccessible vascular structures. Aim 1 Examine Vascular Endothelial Growth Factor (VEGF)-mediated cell differentiation as a mechanism underlying heterogeneous arterial- and venous- fated ECs in the primordial DA (pDA) and CV (pCV). Aim 2 Examine cell segregation as a mechanism to sort venous-fated ECs in the pDA to the pCV. Aim 3 Determine the role of Notch signaling in coordinating the development of parallel artery and vein pairs. Aim 4 Determine the requirement of endothelial Notch1 and Coup-TFII in AV specification of adult parallel artery and vein pairs. Successful completion of this study will conceptually advance our knowledge of the morphogenesis and maintenance of parallel AV pairs, providing evidence regarding the origins of arteries and veins. Basic knowledge of the molecular mechanism of AV specification will inspire novel approaches to study blood vessel regeneration and vein graft engineering in disease settings. The combination of 2-photon high-resolution imaging with cutting-edge cell lineage tracing in living mouse embryos will be a major technological innovation for the field of mammalian vascular development at large. PUBLIC HEALTH RELEVANCE: Our study aims to reveal the role of the Notch signaling pathway in vascular differentiation and maintenance and illuminate potential molecular mechanisms underlying these processes. In the future, this basic understanding of Notch in pre- and post-natal mammalian vascular function will guide investigations into its function in vessel regeneration under pathological conditions, such as heart attack, stroke, and other ischemic diseases. Ultimately, our understanding of the Notch pathway may lead to the identification of novel drug targets and therapeutic interventions for cardiovascular diseases.

DESCRIPTION (provided by applicant): Brain arteriovenous (AV) malformations (BAVMs) can cause life-threatening strokes and have limited treatment options. The goal for this project is to elucidate the cellular and molecular mechanisms underlying BAVM pathogenesis to identify novel candidates as therapeutic targets to ameliorate this disease. AVMs are characterized by abnormal AV shunts that displace intervening capillaries. We propose a cross-disciplinary approach, fusing cutting-edge mouse genetics and imaging technologies, to test our hypothesis that Notch mutations can reprogram AV identity and alter AV differential nitric oxide (NO) signaling, and thus endothelial dysfunction, to elicit BAVMs. Notch receptors and ligands are expressed in arteries but not veins. Notch signaling promotes arterial at the expense of venous differentiation by enhancing arterial and suppressing venous molecular markers. We have reported that endothelial expression of a constitutively active Notch4 mutation (Notch4*) elicits BAVMs in mice. Notch4* reprograms veins to gain arterial and lose venous molecular identity, and correcting the causal Notch4* leads to normalization of established BAVMs. We have built a custom two-photon microscope, optimal for structural and hemodynamic imaging of cerebral vasculature in live mice. We can thus obtain 5D data (3D plus blood velocity over time) through a cranial window to reveal the process of BAVM formation in mice. Built on our strong background and preliminary data, we propose: Aim 1 - Determine the effect of endothelial Notch4* on venous endothelial dysfunction and BAVM formation. We will test our hypothesis that Notch4* upregulates NO levels in the veins, alters venous endothelial response to blood flow, and thus permits AVM formation. We will examine the effect of Notch4* on venous NO signaling, endothelial response to blood flow, and flow mediated BAVM formation; Aim 2 - Determine the effect of endothelial Notch deficiency on arterial reprogramming, arterial endothelial dysfunction, and BAVM formation. We will test our hypothesis that loss of endothelial Notch gene function reprograms arteries to lose arterial and gain venous molecular identity and reduces arterial NO signaling, leading to arterial dysfunction and thus AVMs. We will analyze mice with endothelial deletion of Notch for BAVM pathology, arterial NO signaling, and endothelial response to blood flow stimuli; Aim 3 - Ascertain the interaction between Notch and HHT in mouse dorsal aorta and cardinal vein development. We will test our hypothesis that Notch functions downstream of Hereditary Hemorrhagic Telangectasia (HHT) to mediate HHT function in AV specification. We will compare the Notch and HHT mutant phenotypes using two-photon imaging and 3D rendering and perform genetic rescue. The findings from this study will conceptually advance our understanding of the cellular and molecular mechanisms of AVM pathogenesis, reveal novel functions for Notch in regulating the unique physiology of arteries and veins, and uncover interactions between the Notch and HHT pathways. The success of this work will inspire new areas of investigation in the fields of AVMs, Notch signaling, and vascular pathophysiology.

DESCRIPTION (provided by applicant): Acute myeloid leukemia (AML) is characterized by arrest of differentiation in the myeloid lineage and an over proliferation of blast cells. It is th most common type of leukemia in the United States and is primarily a disease of the elderly. Compared with their younger counterparts, elderly AML patients (age e 65 years) have worse outcome, with a median survival far less than one year. The poorer survival in the elderly has been attributed to less effective therapy, more comorbidities, and other patient characteristics. Although intensive chemotherapy is a standard treatment for younger AML patients, whether intensive or low-intensive chemotherapy will benefit elderly AML patients is not established. To date, most studies evaluating the effects of chemotherapy in elderly AML patients have been limited to small patient series from one or few clinical institutions. Medicare expenditure for elderly AML patients has steadily increased over recent years. The cost for elderly AML patients who received chemotherapy was almost three times higher than those who did not. However, there is no existing study comparing the cost-effectiveness of intensive chemotherapy versus low- intensive chemotherapy for the treatment of elderly AML patients. In the proposed study, we will assemble a cohort of approximately 5,000 elderly AML patients who were diagnosed in the Surveillance, Epidemiology and End Results program area during 2005-2009 and follow the medical care they received through the end of 2010. We will evaluate the comparative effectiveness of intensive and low-intensive chemotherapy for the treatment of elderly AML patients in two aspects, i.e., the clinical effectiveness and the cost-effectiveness. The clinical effectiveness of intensive and low-intensive treatment will be measured by 8-week and 1-year survival, and the cost-effectiveness will be measured by the length of survival, quality adjusted survival, and incremental cost-effectiveness ratio. Given the aging of the population and the continuous rise in Medicare expenditure, findings from the proposed study will not only provide valuable information for physicians and patients to choose treatment options, but also have significant health policy implications.

Project Summary/Abstract: POP has become a significant medical burden. In the United States, almost one in four women suffer from one or more symptoms of POP. In this disorder, pelvic organs protrude through the vaginal inlet due to pelvic floor dysfunction. While pelvic floor muscle plays an important role in visceral support, deficiencies in the strength of endopelvic fascia have shown to cause prolapse even without muscle damage. Collagen is the most abundant constituent of endopelvic fascia. With elastin and smooth muscle cells (SMCs), it forms a suspensory system to support the pelvic organs. Evidence suggests that individuals with prolapse have abnormal quantity and function of fibroblast cells, which maintain and remodel the collagen matrix. Consistent with reports in literature, we found reduced collagen content, increased collagen types I (COLI) to III (COLIII) ratio and increased MMP expression in vaginal wall connective tissues of POP patients leading to a loose and fragile fiber network, reduced collagen-smooth muscle integration and reduced load-bearing mechanical capacity. We hypothesize that by transforming the extracted cells from POP patients in vitro and reviving their matrix protein productivity, the application of these transformed cells in vivo can restore the biomechanics of pelvic floor connective tissues. The objective of the proposal is to attest the functionality of the transformed fibroblast cells in decellularized matrices of patient tissues, and to evaluate the feasibility of transplanting the transformed fibroblasts to restore the biological function of vaginal wall connective tissues in the POP mouse model. The proposed research has a great translational potential as the use of autologous fibroblast cells and the choice of local cell administration are simple, safe and direct, making the procedure affordable for most patients.

PROJECT SUMMARY/ABSTRACT Arteriovenous (AV) malformations (AVMs) are vascular anomalies that shunt blood from an artery directly to a vein, causing organ dysfunction. AVM pathogenesis is poorly understood, limiting the rational design of molecular interventions. Our long-term goal is to develop better therapeutic treatments for AVMs, as current treatment options are limited and risky. Our strategy is to focus on brain AVMs (BAVMs), as they are the most dangerous AVMs, and findings in BAVM are applicable to AVMs elsewhere in the body. Most BAVMs are sporadic, but hereditary BAVMs, such as those seen in hereditary hemorrhagic telangiectasia (HHT), offer an excellent opportunity to study the molecular mechanism underlying disease processes. HHT is an autosomal dominant genetic disorder characterized by multifocal AVMs throughout the body, including the brain. Mutations in activin receptor-like kinase (ALK1) are responsible for Type 2 HHT (HHT2), which represents 25- 57% of all HHT cases. Alk1 is a type I TGF? receptor for BMP ligands, and the mechanism through which Alk1 leads to AVMs is poorly understood. Building on our strong preliminary data, we propose to establish a novel HHT2-BAVM mouse model, with which to identify molecular regulators crucial for AVM pathogenesis, using both a targeted approach and unbiased genome-wide expression profiling. To this end, we propose to establish a much-needed robust preclinical animal model that faithfully models certain aspects of disease presentations in HHT2 patients. Existing mouse models of HHT are limited in recapitulating clinical manifestations. Using a cutting-edge strategy, we have developed a useful mouse model of HHT2-BAVM by deleting both Alk1 alleles specifically in brain endothelial cells, and have obtained strong preliminary data that this deletion results in robust BAVM, intracranial hemorrhages, and neurological consequences, without detectable defects elsewhere in the body. We will first fully characterize this model using innovative, high- resolution two-photon imaging through a cranial window to access the vasculature in live brains, achieving a 5D perspective (3D vascular structure plus blood velocity over time). W candidate molecular regulators that promote BAVM formation including AV programming, endothelial barrier, inflammation, endothelial-to-mesenchymal transition, and superoxide production in mice with Alk1 deletion in the brain endothelium. Finally, we will perform cutting-edge genomic expression profiling to elucidate Alk1 target genes, and then use bioinformatics tools to categorize identified genes based on their functional characteristics. Our proposed Aims comprise a combination of technical and conceptual innovations that will advance the knowledge of the molecular mechanisms underlying AVM formation and HHT pathogenesis. Our work will establish a robust preclinical model for these diseases, uncover new molecular mechanisms underlying the disease etiology, and impact future clinical practice for patients with HHT and BAVM. e will also investigate