Review: Mechanisms of TIMP-3 accumulation and pathogenesis in Sorsby fundus dystrophy.

Sorsby fundus dystrophy (SFD) is a rare, inherited form of macular degeneration caused by mutations in the gene encoding tissue inhibitor of metalloproteinases 3 (TIMP-3). There are 21 mutations currently associated with SFD, with some variants (e.g., Ser179Cys, Tyr191Cys, and Ser204Cys) having been studied much more than others. We review what is currently known about the identified SFD variants in terms of their dimerization, metalloproteinase inhibition, and impact on angiogenesis, with a focus on disparities between reports and areas requiring further study. We also explore the potential molecular mechanisms leading to the accumulation of extracellular TIMP-3 in SFD and consider how accumulated TIMP-3 causes macular damage. Recent reports have identified extraocular pathologies in a small number of SFD patients. We discuss these intriguing findings and consider the apparent discrepancy between the widespread expression of TIMP-3 and the primarily retinal manifestations of SFD. The potential benefits of novel experimental approaches (e.g., metabolomics and stem cell models) in terms of investigating SFD pathology are presented. The review thus highlights gaps in our current molecular understanding of SFD and suggests ways to support the development of novel therapies.

Suramin analogues protect cartilage against osteoarthritic breakdown by increasing levels of tissue inhibitor of metalloproteinases 3 (TIMP-3) in the tissue.

Osteoarthritis is a chronic degenerative joint disease affecting millions of people worldwide, with no disease-modifying drugs currently available to treat the disease. Tissue inhibitor of metalloproteinases 3 (TIMP-3) is a potential therapeutic target in osteoarthritis because of its ability to inhibit the catabolic metalloproteinases that drive joint damage by degrading the cartilage extracellular matrix. We previously found that suramin inhibits cartilage degradation through its ability to block endocytosis and intracellular degradation of TIMP-3 by low-density lipoprotein receptor-related protein 1 (LRP1), and analysis of commercially available suramin analogues indicated the importance of the 1,3,5-trisulfonic acid substitutions on the terminal naphthalene rings for this activity. Here we describe synthesis and structure-activity relationship analysis of additional suramin analogues using ex vivo models of TIMP-3 trafficking and cartilage degradation. This showed that 1,3,6-trisulfonic acid substitution of the terminal naphthalene rings was also effective, and that the protective activity of suramin analogues depended on the presence of a rigid phenyl-containing central region, with para/para substitution of these phenyl rings being most favourable. Truncated analogues lost protective activity. The physicochemical characteristics of suramin and its analogues indicate that approaches such as intra-articular injection would be required to develop them for therapeutic use.

Effects of chromosome 17 on features of the metabolic syndrome in the Lyon hypertensive rat.

The metabolic syndrome (involving obesity, hypertension, dyslipidemia, insulin resistance, and a proinflammatory/prethrombotic state) is a major risk factor for cardiovascular disease. Its incidence continues to rise, in part because of the epidemic increase in obesity. The Lyon hypertensive (LH) rat is a model for hypertension and several other features of the metabolic syndrome, having high body weight, plasma cholesterol, and triglycerides, increased insulin-to-glucose ratio, and salt-sensitive hypertension. Previous genetic studies in LH/Mav rats and a normotensive control (LN/Mav) identified quantitative trait loci (QTLs) on rat chromosome (RNO)17 for multiple features of the metabolic syndrome. To further evaluate the role of RNO17 in the LH rat, we generated a consomic strain (LH-17(BN)) by substituting LH RNO17 with that of the sequenced Brown Norway (BN/NHsdMcwi) rat. Male LH and BN rats and LH-17(BN) rats were characterized for blood pressure and metabolic and morphological parameters. Similar to the protective effect of LN alleles, the LH-17(BN) rat also showed decreased body weight, triglycerides, and blood pressure; however, there was no significant difference in cholesterol or insulin-to-glucose ratio. Therefore, the substitution of the LH chromosome 17 is sufficient to recapitulate some, but not all, of the traits previously mapped to this chromosome. This could be due to the lack of a susceptible LH genome background or due to the introgression of chromosome 17 from another strain. Regardless, this study provides a single-chromosome genetic model for further dissection of blood pressure and morphological and metabolic traits on this chromosome.

Rat survival to anthrax lethal toxin is likely controlled by a single gene.

We examined whether survival of different rat strains administered anthrax lethal toxin is genetically determined. A reproducible test population of first filial generation hybrid rats was bred based on the susceptibility of progenitors to anthrax lethal toxin and to maximize genetic diversity across the strains. These rats were then tested with varying doses of anthrax lethal toxin. We found that all 'sensitive' strains died within 2 h following systemic administration of 240 mug/kg lethal toxin, while one strain survived following a five times higher dose (1.4 mg/kg). The ability of lethal toxin to lyse macrophage cultures derived from the bone marrow of these strains corresponded with in vivo results. We conclude that a rat test population can detect strain differences in response to anthrax lethal toxin. Survival is influenced by the host genome background and is likely due to a single gene with a recessive mode of inheritance.

Synergistic QTL interactions between Rf-1 and Rf-3 increase renal damage susceptibility in double congenic rats.

The FHH (fawn-hooded hypertensive) rat is a model of hypertension-associated chronic kidney damage. Five interacting quantitative trait loci (QTLs), named Rf-1-Rf-5, determine the high renal susceptibility. The aim of the present study was to investigate a possible interaction between Rf-1 and Rf-3. Differences in renal susceptibility between ACI (August x Copenhagen Irish) controls, Rf-1A and Rf-3 single congenics, and Rf-1A+3 double congenic rats were assessed using four different treatments: two-kidney control (2K), 2K plus N(omega)-nitro-L-arginine methyl ester (L-NAME)-induced hypertension (2K+L-NAME), unilateral nephrectomy (UNX), and UNX plus L-NAME-induced hypertension (UNX+L-NAME). Proteinuria (UPV) and systolic blood pressure (SBP) were assessed after 6, 12, and 18 weeks, while the incidence of glomerulosclerosis (%FGS) was determined at the end of the experiment. In a separate experiment, renal autoregulation was assessed in 13-15-week old 2K rats of all four strains. Compared to ACI rats, small increases in renal susceptibility were found in Rf-1A and Rf-3 single congenics following 2K+L-NAME, UNX, and UNX+L-NAME treatments. However, in the Rf-1A+3 double congenics, a major increase in renal susceptibility was found with all four treatments. Both Rf-1A and Rf-1A+3 congenic rats had an impaired renal autoregulation. In contrast, the Rf-3 had a normal autoregulation, similar to that of the ACI rat. These findings indicate that Rf-1 and Rf-3 alone slightly increase the susceptibility to the development of renal damage. However, a synergistic interaction between these two QTLs markedly enhances renal susceptibility. In contrast to the Rf-1 region, the Rf-3 region does not carry genes influencing renal autoregulation.

Genetic determinants on rat chromosome 6 modulate variation in the hypercapnic ventilatory response using consomic strains.

To understand the genetic basis of pathways involved in the control of breathing, a large scale, high-throughput study using chromosomal substitution strains of rats is underway. Eight new consomic rat stains (SS-2(BN), SS-4(BN), SS-6(BN), SS-7(BN), SS-8(BN), SS-11(BN), SS-12(BN), SS-14(BN), SS-Y(BN)), containing one homozygous BN/NHsdMcwi (BN) chromosome on a background of SS/JrHsdMcwi (SS), were created by PhysGen (http://pga.mcw.edu) Program for Genomic Applications. Male and female rats were studied using standard plethysmography under control conditions and during acute hypoxia (inspired oxygen fraction = 0.12) and hypercapnia (inspired CO(2) fraction = 0.07). The rats were also studied during treadmill exercise. Both male and female BN rats had a significantly lower ventilatory response during 7% CO(2) compared with SS rats of the same gender. SS-6(BN) female rats had a significantly reduced ventilatory response, similar to BN rats due primarily to a reduced tidal volume. Male SS-6(BN) rats had a significantly reduced tidal volume response to hypercapnia but a slightly increased frequency response during hypercapnia. Gene(s) on the Y chromosome may play a role in this increased frequency response in the male rats because the SS-Y(BN) hypercapnic ventilatory response involves a significantly increased frequency response. Several chromosomal substitutions slightly altered the ventilatory responses to hypoxia and exercise. However, genes on chromosomes 6 and Y of those studied are of primary importance in aspects of ventilatory control currently studied.

Consomic rat model systems for physiological genomics.

A consomic rat strain is one in which an entire chromosome is introgressed into the isogenic background of another inbred strain using marker-assisted selection. The development and physiological screening of two inbred consomic rat panels on two genetic backgrounds (44 strains) is well underway. Consomic strains enable one to assign traits and quantitative trait loci (QTL) to chromosomes by surveying the panel of strains with substituted chromosomes. They enable the rapid development of congenic strains over a narrow region and enable one to perform F2 linkage studies to positionally locate QTL on a single chromosome with a fixed genetic background. These rodent model systems overcome many of the problems encountered with segregating crosses where even if linkage is found, each individual in the cross is genetically unique and the combination of genes cannot be reproduced or studied in detail. For physiologists, consomics enable studies to be performed in a replicative or longitudinal manner to elucidate in greater detail the sequential expression of genes responsible for the observed phenotypes of these animals. They often provide the best available inbred control strains for physiological comparisons with the parental strains and they enable one to assess the impact of a causal gene region in a genome by allowing comparisons of the effect of replacement of a specific chromosome on a disease susceptible or a resistant genomic background. Consomic rat strains are proving to be a unique scientific resource that can greatly extend our understanding of genes and their role in the regulation of complex function and disease.

Gender-specific genetic determinants of blood pressure and organ weight: pharmacogenetic approach.

A total genome scan and pharmacogenetic study were designed to search for genetic determinants of blood pressure (BP) as well as heart and kidney weights. Genome scanning was carried out in 266 F(2) intercrosses from Prague hypertensive hypertriglyceridemic rats for phenotypes of organ weights, baseline BP, BP after blockade of the renin-angiotensin system (RAS) by losartan, of the sympathetic nervous system (SNS) by pentolinium, and of the nitric oxide (NO) synthase by N(G)-nitro-L-arginine methyl ester. Pharmacogenetic analysis showed that, in males, BP was controlled by two loci on chromosomes 1 and 5 (Chr1, Chr5) through the SNS, and these loci showed a positive contribution for relative kidney weight (KW/BW). On the other hand, baseline BP in females was controlled by two loci on Chr3 and Chr7. The effect of these loci was not mediated by the RAS, SNS or NO system. These loci did not show any effect for KW/BW. Negatively-linked loci for KW/BW and relative heart weight (HW/BW) were identified on Chr2 in both genders. Another negatively-linked locus for KW/BW, located on Chr8 in males, affected BP through the SNS. This locus on Chr8 overlapped with a previously-reported modifier locus for polycystic kidney disease (PKD). In conclusion, this pharmacogenetic study determined two loci for BP and relative organ mass implicating sympathetic overactivity. Concordance of the identified locus for KW/BW and BP through the SNS on Chr8 with the PKD locus revealed the importance of this region for renal complications in various diseases.

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.

The newly inbred cohen diabetic rat: a nonobese normolipidemic genetic model of diet-induced type 2 diabetes expressing sex differences.

The newly inbred Cohen diabetic rat is an exceptional experimental model of diet-induced type 2 diabetes mellitus that is the result of secondary inbreeding nearly 30 years after it originally had been established. Animals from the original colony were selectively inbred by stringent criteria for 10 additional generations, bringing overall inbreeding to >50 generations. The metabolic phenotypes of the resulting contrasting strains, designated as the Cohen diabetic-sensitive (CDs) and -resistant (CDr) rats, were characterized. The phenotype of the CDs strain that was fed a regular diet consisted of fasting normoglycemia, normal glucose tolerance to intraperitoneal glucose loading, normal fasting insulin levels, and a normal insulin response to glucose loading. In contrast, CDs rats that were fed a custom-prepared high-sucrose low-copper diabetogenic diet became overtly diabetic: fasting glucose levels were normal or elevated, and the blood glucose insulin response to glucose loading was markedly abnormal. CDr rats that were fed a regular or diabetogenic diet did not develop diabetes and maintained normal glucose tolerance and insulin secretion. A striking sex difference was observed in CDs rats that were fed a diabetogenic diet: males had a lower growth rate and a more severe glucose intolerance pattern than females. Gonadectomy shortly after weaning did not prevent the development of the diabetic phenotype in its early phase in either sex but markedly attenuated its expression in males at a later phase, abolishing the sex differences. Alternate-day feeding, as opposed to daily feeding, also attenuated the metabolic phenotype in males. The development of the diabetic phenotype in CDs rats that were fed a diabetogenic diet was not accompanied by obesity or hyperlipidemia. The genetic profile of the strains was established using 550 microsatellite markers evenly distributed throughout the rat genome. The rate of homozygosity within strain was > or = 96%. The rate of polymorphism between the contrasting strains was 43%. We conclude that the metabolic phenotypes of the rebred colony of CDs and CDr rats and their genetic makeup render the Cohen diabetic rat a useful experimental model that is highly suitable for studying the interaction between nutritional-metabolic environmental factors and genetic susceptibility (sensitivity and resistance) for the development of type 2 diabetes. The model is also distinctively useful for investigating the effect of sex on the expression of the diabetic phenotype.

The genetic basis of plasma variation in adiponectin, a global endophenotype for obesity and the metabolic syndrome.

Here we present the first genetic analysis of adiponectin levels, a newly identified adipocyte-derived protein. Recent work has suggested that adiponectin may play a role in mediating the effects of body weight as a risk factor for coronary artery disease. For this analysis we assayed serum levels of adiponectin in 1100 adults of predominantly northern European ancestry distributed across 170 families. Quantitative genetic analysis of adiponectin levels detected an additive genetic heritability of 46%. The maximum LOD score detected in a genome wide scan for adiponectin levels was 4.06 (P = 7.7 x 10(-6)), 35 cM from pter on chromosome 5. The second largest LOD score (LOD = 3.2; P = 6.2 x 10(-5)) was detected on chromosome 14, 29 cM from pter. The detection of a significant linkage with a quantitative trait locus on chromosome 5 provides strong evidence for a replication of a previously reported quantitative trait locus for obesity-related phenotypes. In addition, several secondary signals offer potential evidence of replications for additional previously reported obesity-related quantitative trait loci on chromosomes 2 and 10. Not only do these results identify quantitative trait loci with significant effects on a newly described, and potentially very important, adipocyte-derived protein, they also reveal the emergence of a consistent pattern of linkage results for obesity-related traits across a number of human populations.

Identification of two susceptibility loci for vascular fragility in the Brown Norway rat.

A trait of vascular fragility, characterized by the formation of abrupt defects within the elastic laminae of the abdominal aorta, has been identified in Brown Norway (BN) rats. These lesions are greatly exacerbated in F(1) rats from a BN x New Zealand genetically hypertensive (GH) intercross, implying that the genetic background provided by the GH rat influences lesion severity. The F(2) progeny of the BN x GH intercross were used to identify susceptibility loci for the lesions as well as exacerbating loci. Two major quantitative trait loci (QTLs) for number of internal elastic lamina lesions were identified on rat chromosomes 5 and 10, with the maximum "log of the odds ratio" (LOD) scores at D5Rat119 (LOD 5.0) and at D10Mit2 (LOD 4.5), respectively, together contributing 33.5% to the genetic variance. Further analysis revealed that the chromosome 10 locus exhibits a dominant mode of inheritance, with BN alleles being associated with increased lesion number (P < 0.0002) compared with GH homozygotes. This locus was in epistasis to a modifier locus on rat chromosome 2 at D2Mit14 (LOD score 2.12). A second major locus was identified on chromosome 5, exhibiting a semidominant mode of inheritance, again with the BN allele being significantly associated with increased lesion number (P < 0.0001). Furthermore, a locus influencing lesion severity was identified on chromosome 3 wherein GH alleles associated with increased severity. This is the first study to identify susceptibility loci for vascular elastic tissue fragility.

Radiation hybrid mapping of 11 alpha and beta nicotinic acetylcholine receptor genes in Rattus norvegicus.

Acetylcholine is the main neurotransmitter of the vestibular efferents and a wide variety of muscarinic and nicotinic acetylcholine receptors are expressed in the vestibular periphery. To date, 11 nicotinic subunits (alpha and beta) have been reported in mammals. Previously, our group [Brain Res. 778 (1997) 409] reported that these nicotinic acetylcholine receptor alpha and beta subunits were differentially expressed in the vestibular periphery of the rat. To begin an understanding of the molecular genetics of these vestibular efferents, this study examined the chromosomal locations of these nicotinic acetylcholine receptor genes in the rat (Rattus norvegicus). Using radiation hybrid mapping and a rat radiation hybrid map server (www.rgd.mcw.edu/RHMAP SERVER/), we determined the chromosomal position for each of these genes. The alpha2-7, alpha9, alpha10, and beta2-4 nicotinic subunits mapped to the following chromosomes: alpha2, chr. 15; alpha3, chr. 8; alpha4, chr. 3; alpha5, chr. 8; alpha6, chr. 16; alpha7, chr. 1; alpha9, chr. 14; alpha10, chr. 7; beta2, chr. 2; beta3, chr. 16; and beta4, chr. 8. With the location for each of these nicotinic subunits known, it is now possible to develop consomic and/or congenic strains of rats that can be used to study the functional genomics of each of these subunits.

Brown Norway chromosome 13 confers protection from high salt to consomic Dahl S rat.

Consomic rats (SS.BN13), in which chromosome 13 from normotensive inbred Brown Norway rats from a colony maintained at the Medical College of Wisconsin (BN/Mcw) was introgressed into the background of Dahl salt-sensitive (SS/Mcw) rats, also maintained in a colony at the Medical College of Wisconsin, were bred. The present studies determined the mean arterial pressure (MAP) responses to salt and renal and peripheral vascular responses to norepinephrine and angiotensin II; 24-hour protein excretion and histological analyses were used to assess renal pathology in rats that received a high salt (4% NaCl) diet for 4 weeks. MAP of rats measured daily during the fourth week averaged 170+/-3.3 mm Hg in SS/Mcw rats, 119+/-2.1 mm Hg in SS.BN13 rats, and 103+/-1.3 mm Hg in BN/Mcw rats. After salt depletion, MAP fell an average of 27+/-4.5 mm Hg in SS/Mcw rats, 9+/-2.6 mm Hg in SS.BN13 rats, and 11+/-3.0 mm Hg in BN/Mcw rats. Protein excretion of SS/Mcw rats on a high salt diet averaged 189+/-30 mg/24 h, 63+/-18 mg/24 h in SS.BN13 rats, and 40+/-6.4 mg/24 h in BN/Mcw rats. Compared with SS.BN13 and BN/Mcw rats, SS/Mcw rats exhibited significantly greater increases of renal vascular resistance in response to intravenous norepinephrine and angiotensin II. Severe medullary interstitial fibrosis and tubular necrosis after a high salt diet were found consistently in SS/Mcw rat kidneys but were largely absent in the SS.BN13 and BN/Mcw rat kidneys. A similar degree of glomerular sclerosis was found in both SS/Mcw and SS.BN13 rats. In rats fed a 0.4% salt diet, the glomerular filtration rate of SS/Mcw rats was significantly less than that of BN/Mcw and SS.BN13 rats. These results reveal a powerful gene, or set of genes, within chromosome 13 of BN/Mcw rats that confers protection from the detrimental effects of high salt to the SS/Mcw rats.

Genetic rat models of hypertension: relationship to human hypertension.

Experimental models of human disease are frequently used to investigate the pathophysiology of disease as well as the mechanisms of action of therapeutics. However, as long as models have been used there have been debates about the utility of experimental models and their applicability for human disease on the phenotypic and genomic level. The recent advances in molecular genetics and genomics have provided powerful tools to study the genetics of multifactorial diseases, such as hypertension. However, studies of such diseases in humans remain challenging in part due to lack of statistical power and genetic heterogeneity within patient populations. For hypertension, various rat models have been developed and used for the identification of susceptibility loci for genetic hypertension. With the advent of "comparative genomics," the application of genetic studies to both human and animal model systems allows for a new paradigm, where comparative genomics can be used to bridge between model utility and clinical relevance. This review discusses recent approaches in genetics to facilitate gene discovery for polygenic disorders with specific focus on how comparative mapping can be used to select target regions in the human genome for large-scale association studies and linkage disequilibrium testing in clinical populations.

The use of designer rats in the genetic dissection of hypertension.

The rat is a well-established model for hypertension research, in both physiologic and pharmacologic study. Quantitative trait loci (QTL) for blood pressure and related phenotypes have been described on every rat chromosome; therefore, more simplified models must be generated to identify and study the function of the gene(s) located by QTL analysis. Designer rat strains, such as congenic and consomic strains, which share phenotypic and genotypic characteristics with humans but with a greatly simplified genetic background, would yield a powerful platform for functional studies, especially when combined with microarray technologies. Development of these designer rats would result in better-defined disease models that can be used in physiologic and applied pharmacologic studies to better treat human essential hypertension.