Mapping baroreceptor function to genome: a mathematical modeling approach.

To gain information about the genetic basis of a complex disease such as hypertension, blood pressure averages are often obtained and used as phenotypes in genetic mapping studies. In contrast, direct measurements of physiological regulatory mechanisms are not often obtained, due in large part to the time and expense required. As a result, little information about the genetic basis of physiological controlling mechanisms is available. Such information is important for disease diagnosis and treatment. In this article, we use a mathematical model of blood pressure to derive phenotypes related to the baroreceptor reflex, a short-term controller of blood pressure. The phenotypes are then used in a quantitative trait loci (QTL) mapping study to identify a potential genetic basis of this controller.

Automated construction of high-density comparative maps between rat, human, and mouse.

Animal models have been used primarily as surrogates for humans, having similar disease-based phenotypes. Genomic organization also tends to be conserved between species, leading to the generation of comparative genome maps. The emergence of radiation hybrid (RH) maps, coupled with the large numbers of available Expressed Sequence Tags (ESTs), has revolutionized the way comparative maps can be built. We used publicly available rat, mouse, and human data to identify genes and ESTs with interspecies sequence identity (homology), identified their UniGene relationships, and incorporated their RH map positions to build integrated comparative maps with >2100 homologous UniGenes mapped in more than one species (approximately 6% of all mammalian genes). The generation of these maps is iterative and labor intensive; therefore, we developed a series of computer tools (not described here) based on our algorithm that identifies anchors between species and produces printable and on-line clickable comparative maps that link to a wide variety of useful tools and databases. The maps were constructed using sequence-based comparisons, thus creating "hooks" for further sequence-based annotation of human, mouse, and rat sequences. Currently, this map enables investigators to link the physiology of the rat with the genetics of the mouse and the clinical significance of the human.

A genomic-systems biology map for cardiovascular function.

With the draft sequence of the human genome available, there is a need to better define gene function in the context of systems biology. We studied 239 cardiovascular and renal phenotypes in 113 male rats derived from an F2 intercross and mapped 81 of these traits onto the genome. Aggregates of traits were identified on chromosomes 1, 2, 7, and 18. Systems biology was assessed by examining patterns of correlations ("physiological profiles") that can be used for gene hunting, mechanism-based physiological studies, and, with comparative genomics, translating these data to the human genome.

High-throughput scanning of the rat genome using interspersed repetitive sequence-PCR markers.

We report the establishment of a hybridization-based marker system for the rat genome based on the PCR amplification of interspersed repetitive sequences (IRS). Overall, 351 IRS markers were mapped within the rat genome. The IRS marker panel consists of 210 nonpolymorphic and 141 polymorphic markers that were screened for presence/absence polymorphism patterns in 38 different rat strains and substrains that are commonly used in biomedical research. The IRS marker panel was demonstrated to be useful for rapid genome screening in experimental rat crosses and high-throughput characterization of large-insert genomic library clones. Information on corresponding YAC clones is made available for this IRS marker set distributed over the whole rat genome. The two existing rat radiation hybrid maps were integrated by placing the IRS markers in both maps. The genetic and physical mapping data presented provide substantial information for ongoing positional cloning projects in the rat.

Microvascular flow and tissue PO(2) in skeletal muscle of chronic reduced renal mass hypertensive rats.

This study determined whether arteriolar blood flow, capillary red blood cell (RBC) velocity, capillary hematocrit (Hct(cap)), and tissue PO(2) are altered in cremaster muscles of rats with chronic reduced renal mass hypertension (RRM-HT) relative to normotensive rats on high- or low-salt (NT-HS vs. NT-LS) diet. The blood flow in first- through third-order arterioles was not different between NT and HT rats, either at rest or during maximal relaxation of the vessels with 10(-4) M adenosine. Capillary RBC velocity was similar between the groups at rest but was elevated in RRM-HT and NT-HS rats during adenosine superfusion. Hct(cap) was reduced at rest in RRM-HT and NT-HS rats compared with NT-LS and was reduced in RRM-HT rats during adenosine-induced dilation. Tissue PO(2) was reduced in RRM-HT and NT-HS rats compared with NT-LS rats during control conditions and was lower in RRM-HT than in NT-LS rats during adenosine-induced dilation. These results indicate that both RRM-HT and chronic exposure of normotensive rats to a high-salt diet lead to reduced tissue oxygenation, despite the maintenance of normal arteriolar blood flow.

Genetically defined risk of salt sensitivity in an intercross of Brown Norway and Dahl S rats.

A genetic segregation analysis was performed to identify genes that cosegregate with arterial blood pressure traits reflective of salt sensitivity. A population of 113 F2 male rats was derived from an intercross of inbred SS/JrHsd/Mcw (Dahl salt-sensitive) and BN/SsN/Mcw (Brown Norway) rats. Rats were maintained on an 8% salt diet from the age of 9 to 13 wk, and arterial pressure was measured for 3 h daily during the 4th wk of high salt intake in unanesthetized rats using implanted arterial catheters. At the end of the 3rd day of high-salt pressure recordings, the arterial pressure response to salt depletion was determined 1.5 days following treatment with Lasix and a low-sodium (0. 4%) diet. A genome-wide scan using 265 polymorphic simple sequence length polymorphism (SSLP) markers found that seven arterial pressure phenotypes determined at different times and circumstances, and representing two distinct indexes of salt sensitivity, mapped to the same region of rat chromosome 18. The trait of salt sensitivity was strongly influenced by the presence of SS alleles in this region of chromosome 18, and those rats which were homozygote SS/SS exhibited a significantly greater reduction of mean arterial pressure following sodium depletion (29 +/- 2 mmHg) than homozygote BN/BN (17 +/- 3 mmHg) or heterozygotic (22 +/- 2 mmHg) rats. This region of rat chromosome 18 corresponds to the long arm of human chromosome 5 and a region of human chromosome 18 that has been linked to hypertension in humans. Given the unlikely chance of these different blood pressure traits mapping to the same region, we believe these data provide evidence that this region of rat chromosome 18 plays an important role in salt-induced hypertension.

Quantification of the contribution of type 1 and type 2 angiotensin II receptors to the net tissue specific effect of angiotensin II.

Numerous studies have demonstrated changes in receptor number, protein concentration, or mRNA levels and have proposed that these subcellular changes produce physiologic effects. To date, no adequate mathematical analysis has been available to provide a framework for interpretation of such data. In the present study we have combined measurements of angiotensin receptor protein levels with the development of a mathematical model that includes two receptors with opposing actions for a single ligand. This model was used to quantify the net, physiologic response of each receptor population to ANG II stimulation and the effect of altering the expression of receptor populations by a physiologic stimulus. Altered sodium intake was used as the physiologic stimulus and quantification of Western blot analysis and revealed that high sodium diet significantly suppressed AT1 receptor protein in the adrenal gland and aorta and augmented AT2 receptor protein in the aorta. A high sodium diet did not significantly alter AT2 receptor protein in the adrenal gland. Modeling the measured sodium-induced changes in receptor concentration demonstrated that small, subcellular changes in receptor concentration can have a large impact on the net physiologic effect. This model for dual receptor-single ligand interactions should be amenable for other systems.

A high-density integrated genetic linkage and radiation hybrid map of the laboratory rat.

The laboratory rat (Rattus norvegicus) is a key animal model for biomedical research. However, the genetic infrastructure required for connecting phenotype and genotype in the rat is currently incomplete. Here, we report the construction and integration of two genomic maps: a dense genetic linkage map of the rat and the first radiation hybrid (RH) map of the rat. The genetic map was constructed in two F2 intercrosses (SHRSP x BN and FHH x ACI), containing a total of 4736 simple sequence length polymorphism (SSLP) markers. Allele sizes for 4328 of the genetic markers were characterized in 48 of the most commonly used inbred strains. The RH map is a lod >/= 3 framework map, including 983 SSLPs, thereby allowing integration with markers on various genetic maps and with markers mapped on the RH panel. Together, the maps provide an integrated reference to >3000 genes and ESTs and >8500 genetic markers (5211 of our SSLPs and >3500 SSLPs developed by other groups). [Bihoreau et al. (1997); James and Tanigami, RHdb (http:www.ebi.ac.uk/RHdb/index.html); Wilder (http://www.nih.gov/niams/scientific/ratgbase); Serikawa et al. (1992); RATMAP server (http://ratmap.gen.gu.se)] RH maps (v. 2.0) have been posted on our web sites at http://goliath.ifrc.mcw.edu/LGR/index.html or http://curatools.curagen.com/ratmap. Both web sites provide an RH mapping server where investigators can localize their own RH vectors relative to this map. The raw data have been deposited in the RHdb database. Taken together, these maps provide the basic tools for rat genomics. The RH map provides the means to rapidly localize genetic markers, genes, and ESTs within the rat genome. These maps provide the basic tools for rat genomics. They will facilitate studies of multifactorial disease and functional genomics, allow construction of physical maps, and provide a scaffold for both directed and large-scale sequencing efforts and comparative genomics in this important experimental organism.

Evaluating maximum likelihood estimation methods to determine the Hurst coeficient.

A maximum likelihood estimation method implemented in S-PLUS (S-MLE) to estimate the Hurst coefficient (H) is evaluated. The Hurst coefficient, with 0.5 < H <1, characterizes long memory time series by quantifying the rate of decay of the autocorrelation function. S-MLE was developed to estimate H for fractionally differenced (fd) processes. However, in practice it is difficult to distinguish between fd processes and fractional Gaussian noise (fGn) processes. Thus, the method is evaluated for estimating H for both fd and fGn processes. S-MLE gave biased results of H for fGn processes of any length and for fd processes of lengths less than 2(10). A modified method is proposed to correct for this bias. It gives reliable estimates of H for both fd and fGn processes of length greater than or equal to 2(11).

The Microcirculation Physiome Project.

Presented is a discussion of steps towards the creation of a database of the microcirculation encompassing anatomical and functional experimental data, and conceptual and computational models. The discussion includes issues of database utility, organization, data deposition, and linkage to other databases. The database will span levels from gene to tissue and will serve both research and educational purposes.

Mathematical analysis of type-I and type-IIb muscle fiber force generation in renal hypertension.

Previous results from our laboratory have shown that isometric tension development is significantly lower in reduced renal mass (RRM) hypertensive rats when compared to sham-operated controls. The current study was designed to mathematically analyze isometric tetanic contraction profiles and determine the relative contribution of fast-glycolytic (FG) and slow-oxidative (SO) muscle fibers produced by the isolated gastrocnemius-plantaris-soleus muscle group of RRM and sham rats. Because renal hypertension has been shown to be associated with a reduction in microvascular density, we hypothesized that renal hypertension leads to a decrease in SO muscle fiber contribution to force generation. The mathematical model determined the force contribution of two independent muscle fiber components, SO and FG, to the contraction and relaxation phase of isometric tetanic contractions. Each phase was modeled as having an exponentially rising contraction phase during the stimulus period and an exponentially decaying relaxation phase when the stimulus was removed. Each fiber type's tension was also scaled by an envelope function describing the fatigue over the contraction bout. The model, which included 10 parameters, was fit to experimental data by using a nonlinear optimization method and described certain limited characteristics of both fiber types. Results from this model suggest that renal hypertension affects skeletal muscle force generation primarily by decreasing the SO muscle fiber contribution to the total developed tension, decreasing performance and increasing muscle fatigue in RRM rats.

Effect of microvascular rarefaction on tissue oxygen delivery in hypertension.

A mathematical model of oxygen transport in tissue was used to analyze the effects of microvessel rarefaction and nonhomogeneous oxygen consumption on tissue oxygen distribution. The model was based on the diffusion equation with a nonhomogeneous consumption term. Solutions were computed for several configurations of vessel and oxygen sink distributions on a finite domain using the finite element method. A microcirculatory unit consisting of a tissue slice of 100-microns depth and 40-microns width was chosen. Symmetry boundary conditions were applied so that the entire tissue consisted of a series of such microvascular units placed side by side. The boundary condition at the surface of the unit was chosen to simulate a tissue suffusion experiment in which the suffusion oxygen was varied from 0 to 37 mmHg. Results of the model, which were compared with direct measurements with oxygen microelectrodes, indicate that vascular oxygen delivery strongly dominates the tissue oxygen field for suffusion PO2 values of less than 20 mmHg, whereas above this level tissue oxygen is dominated by the suffusion PO2. Reduction of vessel density within the tissue was found to have the largest effect on tissue oxygen levels at low suffusion oxygen. Finally, under some configurations of oxygen sources (vessels) and sinks (mitochondria), extremely low PO2 levels may exist within the area of high consumption, which could limit the metabolic activity of the tissue.

Microvascular rarefaction and tissue vascular resistance in hypertension.

The purpose of this study was to quantitatively estimate the relative contribution of arteriolar rarefaction (disappearance of microvessels) and arteriolar constriction to the increases in total peripheral resistance and changes in the patterns of flow distribution observed in hypertension. A mathematical model of the hamster cheek pouch intraluminal microcirculation was constructed based on data from the literature and observations from our own laboratory. Separate rarefaction and constriction of third-order (3A) and fourth-order (4A) arterioles were performed on the model, and the results were quantified based on the changes of the computed vascular resistance. The degree of increase in resistance depended both on the number and the order of vessels rarefied or constricted and also on the position of those vessels in the network. The maximum increases in resistance obtained in the model runs were 21% for rarefaction and 75% for constriction. Rarefaction, but not constriction, produced large increases in the degree of heterogeneity of blood flow in the various vessel orders. These results demonstrate that vessel rarefaction significantly influences tissue blood flow resistance to a degree comparable with vessel constriction; however, unlike constriction, microvascular rarefaction markedly altered blood flow distribution in our model of the hamster cheek pouch vascular bed. These findings conform with the hypothesis that a significant component of the increase in total peripheral resistance in hypertension may be due to vessel rarefaction.