Search

One goal of the NIH SEPA (Science Education Partnership Awards) program is to create partnerships between biomedical researchers and teachers and schools. The MSOE Center for BioMolecular Modeling (CBM) has leveraged the support of a series of SEPA awards to create a multifaceted outreach program that connects biomolecular researchers and their stories of active research with high school teachers and the foundational concepts of chemistry and biology they teach in their classrooms. The enabling technology that supports this outreach program is 3D printing. The CBM has pioneered the use of 3D printing to create physical models of proteins that allow researchers to tell the story of their research project while at the same time engaging teachers and their students as active participants in this process of science.

The rat has been widely used as a disease model in a laboratory setting, resulting in an abundance of genetic and phenotype data from a wide variety of studies. These data can be found at the Rat Genome Database (RGD, http://rgd.mcw.edu/ ), which provides a platform for researchers interested in linking genomic variations to phenotypes. Quantitative trait loci (QTLs) form one of the earliest and core datasets, allowing researchers to identify loci harboring genes associated with disease. These QTLs are not only important for those using the rat to identify genes and regions associated with disease, but also for cross-organism analyses of syntenic regions on the mouse and the human genomes to identify potential regions for study in these organisms. Currently, RGD has data on >1,900 rat QTLs that include details about the methods and animals used to determine the respective QTL along with the genomic positions and markers that define the region. RGD also curates human QTLs (>1,900) and houses >4,000 mouse QTLs (imported from Mouse Genome Informatics). Multiple ontologies are used to standardize traits, phenotypes, diseases, and experimental methods to facilitate queries, analyses, and cross-organism comparisons. QTLs are visualized in tools such as GBrowse and GViewer, with additional tools for analysis of gene sets within QTL regions. The QTL data at RGD provide valuable information for the study of mapped phenotypes and identification of candidate genes for disease associations.

The Rat Genome Database (RGD) was started >10 years ago to provide a core genomic resource for rat researchers. Currently, RGD combines genetic, genomic, pathway, phenotype and strain information with a focus on disease. RGD users are provided with access to structured and curated data from the molecular level through the organismal level. Those users access RGD from all over the world. End users are not only rat researchers but also researchers working with mouse and human data. Translational research is supported by RGD’s comparative genetics/genomics data in disease portals, in GBrowse, in VCMap and on gene report pages. The impact of RGD also goes beyond the traditional biomedical researcher, as the influence of RGD reaches bioinformaticians, tool developers and curators. Import of RGD data into other publicly available databases expands the influence of RGD to a larger set of end users than those who avail themselves of the RGD website. The value of RGD continues to grow as more types of data and more tools are added, while reaching more types of end users.

Elevated dietary salt intake has previously been demonstrated to have dramatic effects on microvascular structure and function. The purpose of this study was to determine whether a high-salt diet modulates physiological angiogenesis in skeletal muscle. Male Sprague-Dawley rats were placed on a control diet (0.4% NaCl by weight) or a high-salt diet (4.0% NaCl) before implantation of a chronic electrical stimulator. After seven consecutive days of unilateral hindlimb muscle stimulation, animals on control diets demonstrated a significant increase in microvessel density in the tibialis anterior muscle of the stimulated hindlimb relative to the contralateral control leg. High salt-fed rats demonstrated a complete inhibition of this angiogenic response, as well as a significant reduction in plasma ANG II levels compared with those of control animals. To investigate the role of ANG II suppression on the inhibitory effect of high-salt diets, a group of rats that were fed high salt were chronically infused with ANG II at a low dose. Maintenance of ANG II levels restored stimulated angiogenesis to control levels in animals fed a high-salt diet. Western blot analysis indicated that inhibition of angiogenesis in high salt-fed rats was not due to changes in VEGF or VEGF receptor type 1 protein expression in response to stimulation; however, the degree to which VEGF receptor 2 protein increased with stimulation was significantly lower in high salt-fed animals. This study demonstrates an inhibitory effect of high salt intake on stimulated angiogenesis and suggests a critical role for ANG II suppression in mediating this antiangiogenic effect.

Angiogenesis within an ischemic region of the brain may increase tissue viability and act to limit the extent of an infarct. The ANG II pathway can both stimulate and inhibit angiogenesis depending on the tissue and the activated receptors. Previous work showed that 2-wk losartan administration (ANG II type 1 receptor blockade) initiates a significant cerebral angiogenic response. We hypothesized that administration of losartan in the drinking water of rats for 2 wk before initiation of focal ischemia would decrease the extent of the resulting infarct. Adult male Sprague-Dawley rats were given losartan (50 mg/day) in drinking water for 2 wk before initiation of cerebral focal ischemia produced by cauterization of cortical surface vessels. Controls received normal drinking water. In control animals, three main vessels feeding the whisker barrel cortex were cauterized, resulting in cessation of blood flow. The same protocol was followed for losartan-treated animals but did not result in cessation of blood flow in the whisker barrel cortex. Another group of losartan-treated animals received between 8 and 14 cauterizations of surface vessels feeding the whisker barrel cortex, and cessation of blood flow was verified. Rats were killed 72 h after surgery. Morphological examination revealed angiogenesis, maintained vascular delivery, and significantly decreased infarct size in losartan-treated animals compared with controls. These results demonstrate that pretreatment with losartan reduces infarct size after cerebral focal ischemia and support the hypothesis that cerebral angiogenesis may be one of the mechanisms responsible.

We recently described a chymostatin-sensitive elastase-2 as the major angiotensin (ANG) II-forming enzyme in the perfusate of the rat mesenteric arterial bed (MAB) with the same cDNA sequence as rat pancreatic elastase-2. The role of this enzyme in generating ANG II was examined in the rat isolated and perfused MAB. The vasoconstrictor effect elicited by ANG I and the renin substrate tetradecapeptide was only partially inhibited by captopril but abolished by the combination of captopril and chymostatin or N-acetyl-Ala-Ala-Pro-Leu-chloromethylketone (Ac-AAPL-CK; inhibitor originally developed for human elastase-2). The effect induced by [Pro11,d-Ala12]-ANG I, an ANG I-converting enzyme (ACE)-resistant biologically inactive precursor of ANG II, was blocked by chymostatin or Ac-AAPL-CK. It was also demonstrated that cultured rat mesenteric endothelial cells synthesize elastase-2 and that mRNA for this enzyme can be detected in different rat tissues such as the pancreas, MAB, lung, heart, kidney, liver, and spleen. In conclusion, the demonstration of a functional alternative pathway to ACE for ANG II generation in the rat MAB and the fact that cultured MAB endothelial cells are capable of producing and secreting elastase-2 represent strong evidence of a physiological role for this enzyme in the rat vasculature.

The Rat Genome Database (RGD) is the premier resource for genetic, genomic and phenotype data for the laboratory rat, Rattus norvegicus. In addition to organizing biological data from rats, the RGD team focuses on manual curation of gene–disease associations for rat, human and mouse. In this work, we have analyzed disease-associated strains, quantitative trait loci (QTL) and genes from rats. These disease objects form the basis for seven disease portals. Among disease portals, the cardiovascular disease and obesity/metabolic syndrome portals have the highest number of rat strains and QTL. These two portals share 398 rat QTL, and these shared QTL are highly concentrated on rat chromosomes 1 and 2. For disease-associated genes, we performed gene ontology (GO) enrichment analysis across portals using RatMine enrichment widgets. Fifteen GO terms, five from each GO aspect, were selected to profile enrichment patterns of each portal. Of the selected biological process (BP) terms, ‘regulation of programmed cell death’ was the top enriched term across all disease portals except in the obesity/metabolic syndrome portal where ‘lipid metabolic process’ was the most enriched term. ‘Cytosol’ and ‘nucleus’ were common cellular component (CC) annotations for disease genes, but only the cancer portal genes were highly enriched with ‘nucleus’ annotations. Similar enrichment patterns were observed in a parallel analysis using the DAVID functional annotation tool. The relationship between the preselected 15 GO terms and disease terms was examined reciprocally by retrieving rat genes annotated with these preselected terms. The individual GO term–annotated gene list showed enrichment in physiologically related diseases. For example, the ‘regulation of blood pressure’ genes were enriched with cardiovascular disease annotations, and the ‘lipid metabolic process’ genes with obesity annotations. Furthermore, we were able to enhance enrichment of neurological diseases by combining ‘G-protein coupled receptor binding’ annotated genes with ‘protein kinase binding’ annotated genes. Database URL: http://rgd.mcw.edu

Abstract We performed studies to further elucidate the mechanisms of angiotensin II (Ang II)–induced angiogenesis of the microvasculature. Rats were placed on a high salt diet (4% NaCl), and Ang II was infused at a subpressor rate (5 ng/kg per minute) for 3 days. Blood pressure was measured daily for 2 control and 3 infusion days. Microvessel density in the cremaster muscle was measured at the end of the infusion. Vessel density in rats that received subpressor Ang II infusion increased by 12.6% compared with rats that received vehicle infusion. When the angiotensin type 2 (AT 2 ) receptor antagonist PD 123319 was coinfused with Ang II, blood pressure was elevated and vessel density increased above that observed with Ang II infusion alone (23% increase). When the AT 1 receptor antagonist losartan was coinfused with Ang II, blood pressure was lower than control and vessel density was reduced compared with the Ang II group but was still greater than control (7.8% increase). In this study, Ang II stimulated angiogenesis in the rat cremaster muscle; this effect was enhanced by AT 2 antagonism and inhibited by AT 1 antagonism. Ang II infusion at a subpressor dose resulted in a pressor response with AT 2 antagonism and a depressor response with AT 1 antagonism. This suggests that in the microvasculature, the AT 1 receptor mediates angiogenesis and vasoconstriction, and the AT 2 receptor mediates an inhibition of angiogenesis and vasodilation.

Alterations in nuclear calcium levels in response to angiotensin II (ANG II) may play an important role in the trophic actions of ANG II. This study utilized confocal microscopy and nuclear staining to test the hypothesis that both nuclear and cytoplasmic calcium levels are altered in response to ANG II stimulation of freshly dissociated aortic smooth muscle cells. Cells were loaded with the calcium indicator fluo 3 acetoxymethyl ester, and the calcium response to ANG II stimulation was analyzed over time with a laser-scanning confocal microscope. Additionally, the ratiometric calcium indicator fura 2 acetoxymethyl ester and conventional fluorescence microscopy were used to verify these observations. Results show that basal nuclear calcium exceeds cytoplasmic calcium in these cells. Stimulation by ANG II induces a sustained rise (separate from the rapid transient) in both nuclear and cytoplasmic calcium in excess of 20 min in duration. This rise was blockable by either the AT1 receptor antagonist DuP-753 or by the AT2 antagonist PD-123319. Thus ANG II stimulates a sustained rise in nuclear calcium by a mechanism that necessitates activation of both AT1 and AT2 receptors.

At model organism databases data is curated for numerous biological categories using various ontologies and vocabularies. The Rat Genome Database (RGD) uses four different ontologies to standardize annotation information for genes and their associations with disease, phenotypes, and pathways. For manual gene curation this is all done in a single user interface of a web-based annotation tool developed at RGD. The same interface can be used for the curation of QTLs and strains. The development of the tool has been achieved through a collaboration of curators and software developers. Features have been tailored to the needs of the curators to allow optimum efficiency of the data entry portion of the curation process. Annotations using the Gene Ontology, Mammalian Phenotype Ontology, Pathway Ontology, and Disease Ontology can be done simultaneously in the same user interface. The search function of the tool has multiple options, giving the curators the opportunity to specify the search according to the immediate need. For ontology terms, gene, QTL, and strain objects the tool searches RGD data tables. For references the tool searches both internal and PubMed reference IDs. If an abstract reference is not already found at RGD, the tool automatically downloads the reference and assigns an RGD ID to it. Data objects, ontology terms, and references can be stored in multiples, providing various options for composing annotations. An editing step allows the composed annotation line to be altered before entering it in the database. The tool displays all current annotations for any gene that is selected. A second editing function is available by hyperlink for any pre-existing annotation displayed in the tool. The curation software has more than doubled literature curation production since replacing spreadsheet curation.

Angiogenesis, under normal conditions, is a tightly regulated balance between pro- and antiangiogenic factors. The goal of this study was to investigate the mechanisms involved in the control of the skeletal muscle angiogenic response induced by electrical stimulation during the suppression of plasma renin activity (PRA) with a high-salt diet. Rats fed 0.4% or 4% salt diets were exposed to electrical stimulation for 7 days. The tibialis anterior (TA) muscles from stimulated and unstimulated hindlimbs were removed and prepared for gene expression analysis, CD31-terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) double-staining assay, and Bcl-2 and Bax protein expression by Western blot. Rats fed a low-salt diet showed a dramatic angiogenesis response in the stimulated limb compared with the unstimulated limb. This angiogenesis response was significantly attenuated when rats were placed on a high-salt diet. Microarray analysis showed that in the stimulated limb of rats fed a low-salt diet many genes related to angiogenesis were upregulated. In contrast, in rats fed a high-salt diet most of the genes upregulated in the stimulated limb function in apoptosis and cell cycle arrest. Endothelial cell apoptosis, as analyzed by CD31-TUNEL staining, increased by fourfold in the stimulated limb compared with the unstimulated limb. There was also a 48% decrease in the Bcl-2-to-Bax ratio in stimulated compared with unstimulated limbs of rats fed a high-salt diet, confirming severe apoptosis. This study suggests that the increase in endothelial cell apoptosis in TA muscle might contribute to the attenuation of angiogenesis response observed in rats fed a high-salt diet.

Angiotensin II is known to stimulate angiogenesis in the peripheral circulation through activation of the angiotensin II type 1 (AT1) receptor. This study investigated the effect of angiotensin receptor blockade on cerebral cortical microvessel density. Rats (6–7 wk old, n = 5–17) were instrumented with femoral arterial and venous indwelling catheters for arterial blood pressure measurement and drug administration. Rats were treated for 3 or 14 days with the AT1 receptor blocker losartan (50 mg/day in drinking water) or vehicle. Brains were sectioned and immunostained for CD31, and microvessel density was measured. Treatment with losartan for 3 or 14 days resulted in a slight decrease in mean arterial blood pressure (3 days, 92 ± 1 mmHg; and 14 days, 99 ± 2 mmHg) compared with vehicle (109 ± 3 and 125 ± 4 mmHg, respectively). A furosemide + captopril 14-day treatment group was added to control for the blood pressure change (96 ± 3 mmHg). Microvessel density increased in groups treated with losartan for 14 days (429 ± 13 vessels/mm2) compared with vehicle (383 ± 11 vessels/mm2) but did not change with furosemide + captopril (364 ± 7 vessels/mm2). Thus AT1 receptor blockade for 14 days resulted in increased cerebral microvessel density in a blood pressure-independent manner.

Background and Purpose— Female, compared with male, animals are protected from cerebral ischemic injury. Physiological concentrations of 17β-estradiol (E2) reduce damage in experimental stroke. E2 augments angiogenesis in reproductive organs and noncerebral vascular beds. We hypothesized that E2 protects brain in stroke through modulation of angiogenesis. We quantified molecular markers of angiogenesis and capillary density before and after unilateral middle cerebral artery occlusion (MCAO). Methods— Female animals were ovariectomized, treated with 25 μg E2 or placebo implants, and subjected to 2-hour MCAO and 22 hours of reperfusion. Brain angiopoietin-1 (Ang-1), Ang-2, Tie-1, Tie-2, vascular endothelial growth factor (VEGF), VEGF R1, and VEGF R2 mRNA levels were determined by RNAse protection assays, and CD31-positive vessels were counted. Results— E2, but not ischemia, upregulated cerebral Ang-1 mRNA by 49%. Capillary density was higher in the brains of E2-treated animals. In estrogen receptor-α knockout (ERKO) mice, E2-mediated induction of Ang-1 mRNA was absent relative to wild-type littermates. Conclusions— These results suggest that E2 increases Ang-1 and enhances capillary density in brain under basal conditions, priming the MCA territory for survival after experimental focal ischemia.

Cerebral microvascular endothelial cells (CMVEC) form tubes when cocultured with astrocytes (AS). Therefore, it appears that AS may be important in mediating angiogenesis in the brain. We hypothesized that AS modulate CMVEC tube formation by releasing a soluble factor. Thymidine incorporation in cultured CMVEC increased 305% when incubated with 50% conditioned AS medium for 24 h [control: 52,755 ± 4,838 counts per minute (cpm) per well, conditioned 161,082 ± 12,099 cpm/well, n = 8]. Because our laboratory has previously shown that AS can produce epoxyeicosatrienoic acids (EETs), which are known mitogens, we investigated whether release of EETs by AS is responsible for tube formation in the CMVEC-AS coculture. AS were seeded on Lab-Tek slides, CMVEC were seeded on the AS the next day, and cultures were allowed to progress for another 5 days with and without cytochrome P-450 epoxygenase blockade by 17-octadecynoic acid (17-ODYA). Tube formation in cocultures receiving 17-ODYA was significantly inhibited compared with control (93.8%). These data suggest that tube formation requires the release of EETs by AS.

Only functional studies have suggested the presence of the ANG II type 2 (AT2) receptor in the microcirculation. To determine the distribution of this receptor in the rat skeletal muscle microcirculation, a polyclonal rabbit anti-rat antiserum was developed and used for immunohistochemistry and Western blot analysis. The antiserum was prepared against a highly specific and antigenic AT2-receptor synthetic peptide and was validated by competition and sensitivity assays. Western blot analysis demonstrated a prominent, single band at ∼40 kDa in cremaster and soleus muscle. Immunohistochemical analysis revealed a wide distribution of AT2 receptors throughout the skeletal muscle microcirculation in large and small microvessels. Microanatomic studies displayed an endothelial localization of the AT2 receptor, whereas dual labeling with smooth muscle α-actin also showed colocalization of the AT2 receptor with vascular smooth muscle cells. Other cells associated with the microvessels also stained positive for AT2 receptors. Briefly, this study confirms previous functional data and localizes the AT2 receptor to the microcirculation. These studies demonstrate that the AT2 receptor is present on a variety of vascular cell types and that it is situated in a fashion that would allow it to directly oppose ANG II type 1 receptor actions.

Background The use of ontologies to standardize biological data and facilitate comparisons among datasets has steadily grown as the complexity and amount of available data have increased. Despite the numerous ontologies available, one area currently lacking a robust ontology is the description of vertebrate traits. A trait is defined as any measurable or observable characteristic pertaining to an organism or any of its substructures. While there are several ontologies to describe entities and processes in phenotypes, diseases, and clinical measurements, one has not been developed for vertebrate traits; the Vertebrate Trait Ontology (VT) was created to fill this void. Description Significant inconsistencies in trait nomenclature exist in the literature, and additional difficulties arise when trait data are compared across species. The VT is a unified trait vocabulary created to aid in the transfer of data within and between species and to facilitate investigation of the genetic basis of traits. Trait information provides a valuable link between the measurements that are used to assess the trait, the phenotypes related to the traits, and the diseases associated with one or more phenotypes. Because multiple clinical and morphological measurements are often used to assess a single trait, and a single measurement can be used to assess multiple physiological processes, providing investigators with standardized annotations for trait data will allow them to investigate connections among these data types. Conclusions The annotation of genomic data with ontology terms provides unique opportunities for data mining and analysis. Links between data in disparate databases can be identified and explored, a strategy that is particularly useful for cross-species comparisons or in situations involving inconsistent terminology. The VT provides a common basis for the description of traits in multiple vertebrate species. It is being used in the Rat Genome Database and Animal QTL Database for annotation of QTL data for rat, cattle, chicken, swine, sheep, and rainbow trout, and in the Mouse Phenome Database to annotate strain characterization data. In these databases, data are also cross-referenced to applicable terms from other ontologies, providing additional avenues for data mining and analysis. The ontology is available at http://bioportal.bioontology.org/ontologies/50138.

The Rat Genome Database (RGD) ( http://rgd.mcw.edu/ ) is the premier site for comprehensive data on the different strains of the laboratory rat (Rattus norvegicus). The strain data are collected from various publications, direct submissions from individual researchers, and rat providers worldwide. Rat strain, substrain designation and nomenclature follow the Guidelines for Nomenclature of Mouse and Rat Strains, instituted by the International Committee on Standardized Genetic Nomenclature for Mice. While symbols and names aid in identifying strains correctly, the flat nature of this information prohibits easy search and retrieval, as well as other data mining functions. In order to improve these functionalities, particularly in ontology-based tools, the Rat Strain Ontology (RS) was developed. The Rat Strain Ontology (RS) reflects the breeding history, parental background, and genetic manipulation of rat strains. This controlled vocabulary organizes strains by type: inbred, outbred, chromosome altered, congenic, mutant and so on. In addition, under the chromosome altered category, strains are organized by chromosome, and further by type of manipulations, such as mutant or congenic. This allows users to easily retrieve strains of interest with modifications in specific genomic regions. The ontology was developed using the Open Biological and Biomedical Ontology (OBO) file format, and is organized on the Directed Acyclic Graph (DAG) structure. Rat Strain Ontology IDs are included as part of the strain report (RS: ######). As rat researchers are often unaware of the number of substrains or altered strains within a breeding line, this vocabulary now provides an easy way to retrieve all substrains and accompanying information. Its usefulness is particularly evident in tools such as the PhenoMiner at RGD, where users can now easily retrieve phenotype measurement data for related strains, strains with similar backgrounds or those with similar introgressed regions. This controlled vocabulary also allows better retrieval and filtering for QTLs and in genomic tools such as the GViewer. The Rat Strain Ontology has been incorporated into the RGD Ontology Browser ( http://rgd.mcw.edu/rgdweb/ontology/view.html?acc_id=RS:0000457#s ) and is available through the National Center for Biomedical Ontology ( http://bioportal.bioontology.org/ontologies/1150 ) or the RGD ftp site ( ftp://rgd.mcw.edu/pub/ontology/rat_strain/ ).

Leaders in the fields of Temporomandibular Disorders (TMD) and its accompanying overlapping pain conditions presented their latest findings at the Seventh Scientific Meeting of The TMJ Association, September 7–9, 2014, in Bethesda, MD. The meeting was co-sponsored by The TMJ Association and the National Institutes of Health. Topics of the scientific sessions included epidemiology and diagnostic criteria, basic mechanisms of chronic pain including the genetic and epigenetic basis of chronic pain, and the development of novel drugs for treatment of these conditions. Discussions were directed toward formulating a set of recommendations to advance research in this field.