Recent progress in the genetics of spontaneously hypertensive rats.

The spontaneously hypertensive rat (SHR) is the most widely used animal model of essential hypertension and accompanying metabolic disturbances. Recent advances in sequencing of genomes of BN-Lx and SHR progenitors of the BXH/HXB recombinant inbred (RI) strains as well as accumulation of multiple data sets of intermediary phenotypes in the RI strains, including mRNA and microRNA abundance, quantitative metabolomics, proteomics, methylomics or histone modifications, will make it possible to systematically search for genetic variants involved in regulation of gene expression and in the etiology of complex pathophysiological traits. New advances in manipulation of the rat genome, including efficient transgenesis and gene targeting, will enable in vivo functional analyses of selected candidate genes to identify QTL at the molecular level or to provide insight into mechanisms whereby targeted genes affect pathophysiological traits in the SHR.

Long-term pioglitazone treatment augments insulin sensitivity and PKC-epsilon and PKC-theta activation in skeletal muscles in sucrose fed rats.

It has been suggested that thiazolidinediones (TZDs) ameliorate insulin resistance in muscle tissue by suppressing muscle lipid storage and the activity of novel protein kinase C (nPKC) isoforms. To test this hypothesis, we analyzed long-term metabolic effects of pioglitazone and the activation of nPKC-epsilon and -theta isoforms in an animal model of the metabolic syndrome, the spontaneously hypertensive rat (a congenic SHR strain with wild type Cd36 gene) fed a diet with 60 % sucrose from the age of 4 to 8 months. Compared to untreated controls, pioglitazone treatment was associated with significantly increased basal (809+/-36 vs 527+/-47 nmol glucose/g/2h, P<0.005) and insulin-stimulated glycogenesis (1321+/-62 vs 749+/-60 nmol glucose/g/2h, P<0.0001) in isolated gastrocnemius muscles despite increased concentrations of muscle triglycerides (3.83+/-0.33 vs 2.25+/-0.12 micromol/g, P<0.005). Pioglitazone-treated rats exhibited significantly increased membrane/total (cytosolic plus membrane) ratio of both PKC-epsilon and PKC-theta isoforms compared to untreated controls. These results suggest that amelioration of insulin resistance after long-term pioglitazone treatment is associated with increased activation of PKC-epsilon and -theta isoforms in spite of increased lipid concentration in skeletal muscles.

Expression of class III beta-tubulin in colorectal carcinomas: an immunohistochemical study using TU-20 & TuJ-1 antibody.

BACKGROUND & OBJECTIVE: Expression of class III beta-tubulin represents newly discovered marker of resistance to taxol-based chemotherapy in a wide spectra of carcinomas. However, very little is known about its expression in colorectal carcinomas. This study was done to determine class III beta-tubulin expression in a large series of colonic carcinomas, covering tumours with different degree of differentiation in order to evaluate its prospective significance in resistance to taxol-based chemotherapeutics and to compare the immunostaining profile of two widely used monoclonal antibodies, TU-20 and TuJ-1 METHODS: Sixty patients with colorectal carcinoma were enrolled; all of them were treated surgically by the resection. Twenty tumours were histologically assessed as G1, 20 as G2 and 20 as G3. Routine immunohistochemical procedure using TU-20 and TuJ-1 mouse monoclonal antibodies was applied to all 60 specimen and slides were evaluated using an optical microscope. RESULTS: Expression of class III beta-tubulin was detected in 14 tumours (23.3%), while remaining tumours were negative. Relatively higher frequency of class III beta-tubulin expression was observed in G3 tumours (10 cases) in comparison with G1 (3 cases) and G2 (1 case), respectively. Seven tumours displayed positive immunostaining with both tested antibodies TU-20 and TuJ-1. Six tumours showed expression of class III beta- tubulin in more than 1 per cent of neoplastic cell population. In remaining 8 tumours only individual scattered neoplastic cells exhibited class III beta-tubulin expression either with TU-20, or with TuJ-1 antibody. INTERPRETATION & CONCLUSION: Higher frequency of immunoreactivity was observed in poorly differentiated tumours. However, more than 90 per cent of neoplastic cell population did not express class III beta-tubulin in almost all tumours. These negative cells of colonic cancer could represent the potential target for taxane-based chemotherapy in the future. Our results indicate that TU-20 and TuJ-1 antibodies exhibit very similar immunoreactivity in neoplastic tissue.

Transgenic expression of CD36 in the spontaneously hypertensive rat is associated with amelioration of metabolic disturbances but has no effect on hypertension.

Spontaneously hypertensive rats (SHR/NIH strain) harbor a deletion variant in the Cd36 fatty acid transporter and display defective fatty acid metabolism, insulin resistance and hypertension. Transgenic rescue of Cd36 in SHR ameliorates insulin resistance and improves dyslipidemia. However, the role of Cd36 in blood pressure regulation remains controversial due to inconsistent blood pressure effects that were observed with transgenic expression of Cd36 on the SHR background. In the current studies, we developed two new SHR transgenic lines, which express wild type Cd36 under the control of the universal Ef-1 alpha promoter, and examined the effects of transgenic expression of wild type Cd36 on selected metabolic and cardiovascular phenotypes. Transgenic expression of Cd36 in the new lines was associated with significantly decreased serum fatty acids, amelioration of insulin resistance and glucose intolerance but failed to induce any consistent changes in blood pressure as measured by radiotelemetry. The current findings confirm the genetic association of defective Cd36 with disordered insulin action and fatty acid metabolism in the SHR/NIH strain and suggest that Cd36 is linked to other gene(s) on rat chromosome 4 that regulate blood pressure.

Genetic isolation of quantitative trait loci for blood pressure development and renal mass on chromosome 5 in the spontaneously hypertensive rat.

Total genome scans of genetically segregating populations derived from spontaneously hypertensive rats (SHR) and other rat models of essential hypertension suggested a presence of quantitative trait loci (QTL) regulating blood pressure on multiple chromosomes, including chromosome 5. The objective of the current study was to test directly a hypothesis that chromosome 5 of the SHR carries a blood pressure regulatory QTL. A new congenic strain was derived by replacing a segment of chromosome 5 in the SHR/Ola between the D5Wox20 and D5Rat63 markers with the corresponding chromosome segment from the normotensive Brown Norway (BN/Crl) rat. Arterial pressures were directly monitored in conscious, unrestrained rats by radiotelemetry. The transfer of a segment of chromosome 5 from the BN strain onto the SHR genetic background was associated with a significant decrease of systolic blood pressure, that was accompanied by amelioration of renal hypertrophy. The heart rates were not significantly different in the SHR compared to SHR chromosome 5 congenic strain. The findings of the current study demonstrate that gene(s) with major effects on blood pressure and renal mass exist in the differential segment of chromosome 5 trapped within the new SHR.BN congenic strain.

Burns of the perineum and anus.

One of the critical sites of the thermal injuries is the region of the perineum. Burns of the perineum are relatively frequent, while affections of the anus and sphincters are rare. At the Prague Burns Centre, University Hospital Královské Vinohrady, two patients were treated with this kind of injury. The site of the burns proved in both instances decisive for treatment and for subsequent prognosis. The surgical procedures (necrectomy and autotransplantation) had to be prepared with regard to the case-histories: in the 11-year-old boy by colostomy and in the adult man suffering from muscular dystrophy by a synthetic low-residue diet.

Genetic isolation of a blood pressure quantitative trait locus on chromosome 2 in the spontaneously hypertensive rat.

OBJECTIVES: Total genome scans of genetically segregating populations derived from the spontaneously hypertensive rat (SHR) and other rat models of hypertension have suggested the presence of quantitative trait loci (QTL) regulating blood pressure and cardiac mass on multiple chromosomes, including chromosome 2. The objective of the current study was to directly test for the presence of a blood pressure QTL on rat chromosome 2. DESIGN: A new congenic strain was derived by replacing a segment of chromosome 2 in the SHR between D2Rat171 and D2Arb24 with the corresponding chromosome segment from the normotensive Brown Norway rat. Arterial pressures were directly monitored in conscious rats by radiotelemetry. RESULTS: We found that the SHR congenic strain (SHR-2) carrying a segment of chromosome 2 from the Brown Norway rat had significantly lower systolic and diastolic blood pressures than the SHR progenitor strain. The attenuation of hypertension in the SHR-2 congenic strain versus the SHR progenitor strain was accompanied by significant amelioration of cardiac hypertrophy. CONCLUSIONS: These findings demonstrate that gene(s) with major effects on blood pressure exist in the differential segment of chromosome 2 trapped within the new SHR.BN congenic strain.

Transgenic rescue of defective Cd36 ameliorates insulin resistance in spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) display several features of the human insulin-resistance syndromes. Cd36 deficiency is genetically linked to insulin resistance in SHR. We show that transgenic expression of Cd36 in SHR ameliorates insulin resistance and lowers serum fatty acids. Our results provide direct evidence that Cd36 deficiency can promote defective insulin action and disordered fatty-acid metabolism in spontaneous hypertension.

SHR.BN-congenic strains for genetic analysis of multifactorially determined traits.

The laboratory rat is an important laboratory animal with multiple well-defined inbred strains, including some of the most widely used animal models of human diseases. Recent advances in the development of rat genetic resources will enable the exploitation of the full potential of rat models of human diseases and, in addition, the rat can provide useful information for comparative genomics of humans and mice. In the current review, we describe the development of congenics strains by introgression of differential chromosome segments from the Brown Norway (BN) rat to the genetic background of the spontaneously hypertensive rat (SHR). These SHR.BN-congenic strains and recombinant sublines derived from them were developed as a model system for genetic analysis of multifactorially determined pathophysiological and morphological conditions.

Genetic analysis of cardiovascular risk factor clustering in spontaneous hypertension.

The SHR is the most widely studied animal model of hypertension. In this strain, as in many humans with essential hypertension, increased blood pressure has been reported to cluster with other risk factors for cardiovascular disease, including insulin resistance and dyslipidemia. However, the genetic mechanisms that mediate this clustering of risk factors for cardiovascular disease or the hypertension "metabolic syndrome" remain poorly understood. In the current studies, we have demonstrated (1) that a gene or genes responsible for a whole spectrum of cardiovascular risk factors mapped to a limited segment of the centromeric region of rat chromosome 4, (2) that a spontaneous deletion in the gene for Cd36 that encodes a fatty acid transporter and is located directly at the peak of QTL linkages on chromosome 4 has been indirectly linked to the transmission of insulin resistance, defective fatty acid metabolism, and increased blood pressure, and (3) based on complementation analysis in two transgenic lines expressing wild-type Cd36 on the genetic background of the SHR strain harboring the deletion variant of Cd36, we have established that defective Cd36 can be a determinant of disordered fatty acid metabolism, glucose intolerance, and insulin resistance in spontaneous hypertension.

Mapping of quantitative trait loci for seminal vesicle mass and litter size to rat chromosome 8.

The spontaneously hypertensive rat (SHR) and the Brown Norway (BN) rat differ significantly in litter size (7.6 versus 4.5 pups). In the HXB and BXH sets of recombinant inbred (RI) strains derived from SHR and BN rats, heritability of litter size and of selected male reproductive parameters such as sperm production, sperm count, sperm morphology and motility, and the mass of the testis, epididymides, and seminal vesicles were estimated and a search was undertaken for quantitative trait loci (QTL) associated with these phenotypes. The mass of seminal vesicles was significantly associated with a QTL near the D8Cebr204S21 marker on chromosome 8 (LOD score = 4.1, P = 0.00001); this QTL was responsible for 46% of the genetic variability of the trait. The same gene marker on chromosome 8 also showed a suggestive association with the litter size. Litter size was significantly correlated with the mass of seminal vesicles (r = 0.58, P = 0.003). These findings indicate that the variability in litter size among RI strains may be due in part to differences in the mass of seminal vesicles and it is possible that both mass of seminal vesicles and litter size are determined by a pleiotropic effect of the same QTL on rat chromosome 8.

HXB/Ipcv and BXH/Cub recombinant inbred strains of the rat: strain distribution patterns of 632 alleles.

The HXB/Ipcv and BXH/Cub sets of recombinant inbred (RI) strains were derived from the spontaneously hypertensive rats (SHR/OlaIpcv) and normotensive Brown Norway (BN-Lx/Cub) rats. The RI strains were produced as a model system for genetic and correlation analysis of spontaneous hypertension and other risk factors of cardiovascular disease such as insulin resistance and dyslipidemia. The RI strains were phenotyped in multiple hemodynamic and metabolic traits. In the current study, we describe strain distribution patterns of 632 genetic markers.

Hematocrit and hemoglobin values are negatively correlated with insulin resistance in spontaneous hypertension.

It has been recently reported that increased hematocrit and hemoglobin values often accompany insulin resistance and compensatory hyperinsulinemia in humans. In the current study, we analyzed the relationship between hematocrit/hemoglobin on the one hand and insulin resistance, dyslipidemia, and hypertension on the other hand in HXB/BXH recombinant inbred (RI) strains derived from the spontaneously hypertensive rat (SHR) and the Brown Norway (BN) rat. The SHR progenitor strain had a significantly increased hematocrit values and it was also hypertensive and insulin-resistant when compared with the BN progenitor. The distribution of hematocrit and hemoglobin values among RI strains was continuous, suggesting a polygenic mode of inheritance. Analysis of RI strains revealed that hemoglobin was negatively correlated with insulin and insulin/glucose ratio, and that hematocrit was negatively correlated with insulin-stimulated glucose uptake in isolated adipocytes. There was no relationship between hematological parameters and blood pressure or lipid phenotypes in RI strains. The findings of the current study suggest that hematocrit and hemoglobin values might be added to the clustering variables related to the insulin resistance syndrome in the SHR strain.

Quantitative trait loci for compensatory renal hypertrophy in the mouse.

Reduction in numbers of nephrons or decrease in kidney function due to a variety of diseases results in compensatory renal hypertrophy (CRH). Recently, it has been proposed that CRH may be a prerequisite for progression of renal injury; genetic dissection of CRH may be therefore helpful in understanding the process whereby people with partial renal insufficiency progress to end-stage renal disease. Since genetic analysis of CRH in humans is quite limited, we searched for genetic determinants of CRH after unilateral nephrectomy using a total genome scan of the mouse BXD recombinant inbred strains. We demonstrated that CRH is a highly heritable trait and we identified a quantitative trait locus on mouse chromosome 11 near the D11Mit14 marker that exerts a major effect on CRH (lod score = 3.4) and is responsible for approximately 52% of genetic variation in CRH. This marker maps near Ace, Gh, and Ngfr positional candidate genes.

Genetic dissection of testicular weight in the mouse with the BXD recombinant inbred strains.

Testicular weights were studied in the mouse BXD recombinant inbred (RI) strains. These strains were derived from DBA/2J and C57BL/6J progenitors that differ significantly in their testicular weights (0.224 g +/- 0.015 vs. 0.161 g +/- 0.03, P < 0.0001). The heritability of testicular weights was calculated to be 0.53, and the minimum number of responsible effective factors was estimated to be 5.7. The total genome scanning of the BXD RI strains with over 1000 markers revealed a quantitative trait locus (QTL) on mouse Chromosome (Chr) 13 near the D13Mit3 marker (LOD score 6.9). This QTL region was designated Twq1 and associated with over 75% of genetic variability.

A genetic and correlation analysis of liver cholesterol concentration in rat recombinant inbred strains fed a high cholesterol diet.

Liver cholesterol concentration in rats fed a high cholesterol diet, is under genetic control which is supported by significant differences observed among inbred strains. For instance, the Brown Norway (BN-Lx/Cub) rat developed a twofold higher liver cholesterol concentration than the spontaneously hypertensive rat (SHR/Ola). In the current study, we used 30 recombinant inbred (RI) strains, derived from BN-Lx and SHR progenitors, to locate quantitative trait loci (QTL) that are responsible for differences in liver cholesterol concentrations between the BN-Lx and SHR strains. The heritability of liver cholesterol was estimated to be 0.55 and a significant association was detected between concentration of liver cholesterol and the D10Cebrp1016s2 marker on chromosome 10 (lod score = 3.3); this putative QTL was responsible for nearly 64% of additive genetic variability and thus represents a major genetic determinant of liver cholesterol concentration. Liver cholesterol concentrations significantly correlated with intermediate density lipoprotein (IDL) cholesterol levels.

Genetic isolation of a chromosome 1 region affecting blood pressure in the spontaneously hypertensive rat.

Recent linkage studies in the spontaneously hypertensive rat (SHR) suggest that a blood pressure regulatory gene or genes may be located on rat chromosome 1q. To investigate this possibility, we replaced a region of chromosome 1 in the SHR (defined by the markers D1Mit3 and Igf2) with the corresponding chromosome segment from the normotensive Brown-Norway (BN) strain. In male SHR congenic rats carrying the transferred BN chromosome segment, 24-hour average systolic and diastolic blood pressures were significantly lower than in male progenitor SHR. Polymerase chain reaction genotyping using 60 polymorphic microsatellite markers dispersed throughout the genome confirmed the congenic status of the new strain designated SHR.BN-D1Mit3/Igf2. These findings provide direct evidence that a blood pressure regulatory gene exists on the differential segment of chromosome 1 that is sufficient to decrease blood pressure in the SHR. The SHR.BN-D1Mit3/Igf2 congenic strain represents an important new model for fine mapping and characterization of genes on chromosome 1 involved in the pathogenesis of spontaneous hypertension.

Quantitative trait loci influencing cholesterol and phospholipid phenotypes map to chromosomes that contain genes regulating blood pressure in the spontaneously hypertensive rat.

The frequent coincidence of hypertension and dyslipidemia suggests that related genetic factors might underlie these common risk factors for cardiovascular disease. To investigate whether quantitative trait loci (QTLs) regulating lipid levels map to chromosomes known to contain genes regulating blood pressure, we used a genome scanning approach to map QTLs influencing cholesterol and phospholipid phenotypes in a large set of recombinant inbred strains and in congenic strains derived from the spontaneously hypertensive rat and normotensive Brown-Norway (BN.Lx) rat fed normal and high cholesterol diets. QTLs regulating lipid phenotypes were mapped by scanning the genome with 534 genetic markers. A significant relationship (P < 0.00006) was found between basal HDL2 cholesterol levels and the D19Mit2 marker on chromosome 19. Analysis of congenic strains of spontaneously hypertensive rat indicated that QTLs regulating postdietary lipid phenotypes exist also on chromosomes 8 and 20. Previous studies in the recombinant inbred and congenic strains have demonstrated the presence of blood pressure regulatory genes in corresponding segments of chromosomes 8, 19, and 20. These findings provide support for the hypothesis that blood pressure and certain lipid subfractions can be modulated by linked genes or perhaps even the same genes.