C3H/HeJ Research Articles

Genes Within the MHC Region Have a Dramatic Influence on Radiation-Enhanced Atherosclerosis in Mice

C3H/HeJ (C3H) mice develop much smaller atherosclerotic lesions than C57BL/6 (B6) mice when deficient in apolipoprotein E (apoE⁻(/)⁻) or fed an atherogenic diet. The 2 strains differ in H2 haplotypes, with B6 having H2(b) and C3H having H2(k). C3.SW-H2(b)/SnJ (C3.SW) is a congenic strain of C3H/HeJ in which H2(k) is replaced with H2(b). We performed bone marrow transplantation and found that atherosclerosis-resistant C3.SW.apoE⁻(/)⁻ mice reconstituted with bone marrow from either C3.SW.apoE⁻(/)⁻ or B6.apoE⁻(/)⁻ mice after lethal irradiation had significantly larger atherosclerotic lesions than B6.apoE⁻(/)⁻ mice receiving identical treatments and much larger lesions than C3H.apoE⁻(/)⁻ mice reconstituted with syngeneic bone marrow. For syngeneic transplantation, C3.SW.apoE⁻(/)⁻ mice exhibited a 21-fold increase in lesion size over C3H.apoE⁻(/)⁻ mice (152 800±21 937 versus 7060±2290 μm²/section) and a near 4-fold increase over B6.apoE⁻(/)⁻ mice (40 529±4675 μm²/section). C3.SW.apoE⁻(/)⁻ mice reconstituted with syngeneic marrow exhibited enhanced lesion formation relative to those reconstituted with B6 marrow (152 800±21 937 versus 107 000±9374 μm²/section; P=0.067). Sublethal irradiation led to a 6-fold increase of lesion size in C3.SW.apoE⁻(/)⁻ mice (9795±2804 versus 1550±607 μm²/section; P=0.008). Wild-type C3.SW mice reconstituted with apoE(+/+) or apoE⁻(/)⁻ bone marrow had significantly larger atherosclerotic lesions than C3H mice receiving identical treatments on an atherogenic diet. These results indicate that gene(s) within the H2 region have a dramatic impact on radiation-enhanced atherosclerosis, and their effect is conveyed partially through bone marrow-derived cells.

Collagen fibril morphology and mechanical properties of the Achilles tendon in two inbred mouse strains

The relationship between collagen fibril morphology and the functional behavior of tendon tissue has been investigated in numerous experimental studies. Several of these studies suggest that larger fibril radius is a primary determinant of higher tendon stiffness and strength; others have shown that factors apart from fibril radius (such as fibril-fibril interactions) may be critical to improved tendon strength. In the present study, we investigate these factors in two inbred mouse strains that are widely used in skeletal structure-function research: C57BL/6J (B6) and C3H/HeJ (C3H). The aim was to establish a quantitative baseline that will allow one to assess how regulation of tendon extracellular matrix architecture affects tensile mechanical properties. We specifically focused on collagen fibril structure and glycosaminoglycan (GAG) content--the two primary constituents of tendon by dry weight--and their potential functional interactions. For this purpose, Achilles tendons from both groups were tested to failure in tension. Tendon collagen morphology was analyzed from transmission electron microscopy images of tendon sections perpendicular to the longitudinal axis. Our results showed that the two inbred strains are macroscopically similar, but C3H mice have a higher elastic modulus (P < 0.05). Structurally, C3H mice showed a larger collagen fibril radius compared to B6 mice (96 +/- 7 nm and 80 +/- 10 nm respectively). Tendons from C3H mice also showed smaller specific fibril surface (0.015 +/- 0.001 nm nm(-2) vs. 0.017 +/- 0.003 nm nm(-2) in the B6 tendons, P < 0.05), and accordingly a lower concentration of GAGs (0.60 +/- 0.07 microg mg(-1) vs. 0.83 +/- 0.11 microg mg(-1), P < 0.05). As in other studies of tendon structure and function, larger collagen fibril radius appears to be associated with stiffer tendon, but this functional difference could also be attributed to reduced potential surface area exchange between fibrils and the surrounding proteoglycan-rich matrix, in which the hydrophilic GAG side chains may promote inter-fibril sliding. This study provides an architectural and functional baseline for a comparative murine model that can be used to investigate the genetic regulation of tendon biomechanics.

Mechanical loading of mouse caudal vertebrae increases trabecular and cortical bone mass-dependence on dose and genotype

Most in vivo studies addressing the skeletal responses of mice to mechanical loading have targeted cortical bone. To investigate trabecular bone responses also we have developed a caudal vertebral axial compression device (CVAD) that transmits mechanical loads to compress the fifth caudal vertebra via stainless steel pins inserted into the forth and sixth caudal vertebral bodies. Here, we used the CVAD in C57BL/6 (B6) and C3H/Hej (C3H) female mice (15weeks of age) to investigate whether the effect of regular bouts of mechanical stimulation on bone modeling and bone mass was dependent on dose and genotype. A combined micro-computed tomographic and dynamic histomorphometric analysis was carried out at the end of a 4-week loading regimen (3,000 cycles, 10 Hz, 3 xweek) for load amplitudes of 0N, 2N, 4N and 8N. Significant increases in trabecular bone mass of 9 and 21% for loads of 4N and 8N, respectively, were observed in B6 mice. A significant increase of 10% in trabecular bone mass occurred for a load of 8N in the C3H strain. For other loads, no significant increases were detected. Both mouse strains exhibited substantial increases in trabecular bone formation rates for all loads, B6: 111% (2N), 86% (4N), 164% (8N), C3H: 41% (2N), 38% (4N), 141% (8N). Significant decreases in osteoclast number of 146 and 93% for a load of 8N were detected in B6 and C3H mice, respectively. These findings demonstrate that the effect of loading on the structural and functional parameters of bone is dose and genotype dependent. The caudal vertebral loading model established here is proposed for further studies addressing the molecular processes involved in the skeletal responses to mechanical stimuli.

Quantitative trait loci for tibial bone strength in C57BL/6J and C3H/HeJ inbred strains of mice

Three-point bending technology has been widely used in the measurement of bone strength. Quantitative trait loci (QTLs) for bone strength have been identified using mouse femurs. In this study, we investigate the use of mouse tibiae in identification of QTLs that regulate bone strength. Mouse tibiae were from a F(2) population derived from C57BL/6J (B6) and C3H/HeJ (C3H). Three-point bending was measured using ISO 4049, with the support width adjustable to accommodate specimen sizes outside the scope of ISO 4049. The strain rate is selectable from 0.05 to 500 mm per min. All stress strain diagrams are recorded and retrieved in digital electronic form. Genome scan was performed in The Jackson Laboratory (TJL). QTL mapping was conducted using Map Manager QTX software. Data show that (i) both elastic modulus (stiffness) and maximum loading (strength) value appear as normal distributions, suggesting that multiple genetic factors control the bone strength; (ii) 11 QTLs, accounting for 90% of variation for strength, have been detected. More than half QTLs of three-point bending are located on the same locations of bone density earlier identified from mouse femurs; (iii) a major QTL of femoral and vertebral bone mineral density (BMD) was not detected for bone strength of tibiae; (iv) the QTL on chromosome 4 has extremely high LOD score of 31.8 and represents 60% of the variation of bone strength; and (v) four QTLs of stiffness (chromosomes 2, 11, 15 and 19) have been identified.

DYNAMICS OF MICRONUCLEI IN MOUSE SKIN FIBROBLASTS AFTER GAMMA IRRADIATION

Micronuclei (MN) were assessed in dermal fibroblasts from C3H HeJ and C57 Bl6 mice (6-10 mo of age) irradiated in vivo as a potential method of biodosimetry. Radiation-induced MN [per 1,000 binucleated (BN) cells], assessed in fibroblasts obtained 1 d post-irradiation, increased in a dose-dependent manner in the range of 1 - 10 Gy per single dose. Analysis at 1 wk post irradiation showed some attenuation of MN response in C3H HeJ male mice, suggesting partial recovery of DNA damage. This was not observed in C57 Bl6 mice. Monomicronucleated cells predominated in unirradiated fibroblasts, whereas in irradiated fibroblasts multimicronucleated cells predominated at dose levels above about 5 Gy (more than 1,000 MN per 1,000 BN cells). Modeling of the data indicate that assaying total MN in dermal fibroblasts from samples of irradiated skin taken up to 1 wk after irradiation can provide biodosimetric information (with an accuracy of 1-2 Gy) for doses up to at least 6 Gy with lesser accuracy at higher doses. Percentage of multimicronucleated cells may be useful as a substitute of total MN at the higher doses, but total percentage of micronucleated cells is of limited value as a substitute.

Genetic analysis of albuminuria in aging mice and concordance with loci for human diabetic nephropathy found in a genome-wide association scan

Aging in the kidney can cause albuminuria, and discovering molecular mechanisms responsible for this might offer a new perspective on the etiology of this abnormality. Haplotype association mapping in the mouse is a novel approach which uses the haplotypes of the relatively closely related mouse inbred strains and the phenotypic variation among these strains to find associations between haplotypes and phenotype. The albumin-to-creatinine ratios, a measure of urinary albumin excretion, were determined in 30 inbred mouse strains at 12, 18, and 24 months of age. Mapping was performed for males and females separately at all three time points using a high density set of 63,222 single-nucleotide polymorphisms to determine genetic loci involved in albuminuria. One significant and eight suggestive loci were found, some of which map to previously identified loci for traits associated with kidney damage in the mouse, but with a much higher resolution thus narrowing their chromosomal location. These nine loci were then compared with genome-wide association scans for diabetic nephropathy (DN) in human type I diabetes. Our study found that two of the nine mouse loci for age-related albuminuria were significantly associated with DN and consistent across male and female strata. This suggests common underlying genes predispose to kidney disease in mice and humans.

Indications of IL1β, IL18 and VEGF cascade in maintenance of homeostasis following transient pulmonary inflammation due to carbon nanoparticle (CNP) challenge in mice

Aims: Carbonaceous nanoparticles possess an emerging source of human exposure. The pulmonary inflammation caused by deposited nanoparticles is a key event for their adverse health effects. Epidemiological studies suggest that individuals with favourable lung physiology are at lower risk for particulate matter associated respiratory diseases. We selected a mouse strain C3H/HeJ (C3) with robust lung physiology and exposed it to moderately toxic carbon nanoparticles (CNP) to study the elicited pulmonary inflammation and its resolution. Methods: 5µg, 20µg and 50µg CNP were intratracheally (i.t.) instilled in C3 mice. Inflammation was assessed using histology, broncheoalveolar lavage (BAL) analysis and by a panel of 62 protein markers to analyze the molecular response. Results: 24h after instillation of CNP, C3 mice exhibited a typical dose response, with the lowest dose (5µg) representing the 'no effect level' as reflected by polymorphonuclear leucocyte (PMN) influx, and BAL/lung concentrations of pro-inflammatory cytokines and proteins. Accordingly time course assessment of the inflammatory response was performed after 3 and 7days with 20µg CNP instilled animals. Compared to 24h, BAL PMN counts were significantly decreased at day 3 and by day 7 complete resolution of PMN influx was observed. We have identified protein markers related to acute response and also for time course response in lung and BAL. After complete resolution of PMN influx on day7, protein concentrations of FGA, CRP, APCS, CCL5, CCL9, FGF2, CCL11, vWF, XCL1, CXCL5, THPO, VEGFA, IL1B, IL18, CSF1, EDN1, CCL3, CCL4, F3 and CD40L were significantly elevated in the lung and BAL. These factors are involved in a closely regulated molecular cascade with IL1β/IL18at the upstream and FGF2/EDN1/VEGF at the downstream. BAL-protein concentration and histological analysis did not reveal any evidence of tissue injury in 20µg CNP instilled animals. Conclusion: Considering the role of VEGF, FGF2 and EDN1 in lung development and morphogenesis together with the lack of any evident tissue damage we suggest a protective/homeostatic machinery is activated to counter the CNP challenge as a precautionary measure.

Lipidomic Analysis of Dynamic Eicosanoid Responses during the Induction and Resolution of Lyme Arthritis

Eicosanoids and other bioactive lipid mediators are indispensable regulators of biological processes, as demonstrated by the numerous inflammatory diseases resulting from their dysregulation, including cancer, hyperalgesia, atherosclerosis, and arthritis. Despite their importance, a robust strategy comparable with gene or protein array technology for comprehensively analyzing the eicosanoid metabolome has not been forthcoming. We have developed liquid chromatography-tandem mass spectrometry methodology that quantitatively and comprehensively analyzes the eicosanoid metabolome and utilized this approach to characterize eicosanoid production during experimental Lyme arthritis in mice infected with the bacterium Borrelia burgdorferi. Eicosanoids were extracted throughout infection from the joints of Lyme arthritis-resistant and -susceptible mice and subjected to lipidomic profiling. We identified temporal and quantitative differences between these mouse strains in the production of eicosanoids, which correlated with differences in arthritis development. The eicosanoid biosynthetic enzyme cyclooxygenase (COX)-2 has been implicated in the regulation of Lyme arthritis pathology, and subsequent lipidomic profiling of B. burgdorferi-infected COX-2(-/-) mice identified reductions not only in COX-2 products but, surprisingly, also significant off-target reductions in 5-lipoxygenase metabolites. Our results demonstrate the utility of a comprehensive lipidomic approach for identifying potential contributors to disease pathology and may facilitate the development of more precisely targeted treatment strategies.

Quantitative Trait Locus Analysis of Neointimal Formation in an Intercross Between C57BL/6 and C3H/HeJ Apolipoprotein E–Deficient Mice

Inbred mouse strains C57BL/6J (B6) and C3H/HeJ (C3H) exhibit marked differences in neointimal formation after arterial injury when deficient in apolipoprotein E (apoE(-/-)) and fed a Western diet. Quantitative trait locus (QTL) analysis was performed on an intercross between B6.apoE(-/-) and C3H.apoE(-/-) mice to determine genetic factors contributing to the phenotype. Female B6.apoE(-/-) mice were crossed with male C3H.apoE(-/-) mice to generate F(1)s, which were intercrossed to generate 204 male F(2) progeny. At 10 weeks of age, F(2)s underwent endothelium denudation injury to the left common carotid artery. Mice were fed a Western diet for 1 week before and 4 weeks after injury and analyzed for neointimal lesion size, plasma lipid and MCP-1 levels. One significant QTL, named Nih1 (61cM, LOD score: 5.02), on chromosome 12 and a suggestive locus on chromosome 13 (35cM, LOD: 2.67) were identified to influence lesion size. One significant QTL on distal chromosome 1 accounted for major variations in plasma non-HDL cholesterol and triglyceride levels. Four suggestive QTLs on chromosomes 1, 2, and 3 were detected for circulating MCP-1 levels. No correlations were observed between neointimal lesion size and plasma lipid levels or between lesion size and plasma MCP-1 levels. Neointimal formation is controlled by genetic factors independent of those affecting plasma lipid levels and circulating MCP-1 levels in the B6 and C3H mouse model.

Modulation of murine bone marrow‐derived CFU‐F and CFU‐OB by in vivo bisphosphonate and fluoride treatments

Bisphosphonates (BPN) have actions on a variety of cell types including: osteoclasts, osteoblasts, osteocytes, and endothelial cells. The objectives of this report are to review the current state of understanding of the effects of BPNs on orthodontic tooth movement and to provide evidence on BPN's in vivo effects on bone marrow-derived osteoprogenitor cells. Mice from the C3H/HeJ (C3H), C57BL/6J (B6), FVB/NJ (FVB), and BALB/cByJ (BALB) strains were treated for 3 weeks with 0, 3, 30, or 150 mcg/kg/week alendronate (ALN) administered subcutaneous alone or in combination with 50 ppm fluoride (F). Bone marrow cells were harvested and subjected to in vitro colony-forming unit fibroblast (CFU-F) and colony-forming unit osteoblasts (CFU-OB) assays. Baseline differences in CFU-F, CFU-OB/ALP+, and CFU-OB/total were observed among the four strains. Strain-specific responses to ALN and F treatments were observed for CFU-F, CFU-OB/ALP+, and CFU-OB/total. F treatment alone resulted in decreases in CFU-F (p = 0.013), CFU-OB/ALP+ (p = 0.005), and CFU-OB/total (p = 0.003) in the C3H strain. CFU-F (p = 0.036) were decreased by F in the B6 strain. No significant (NS) effects of F were observed for FVB and BALB. ALN treatment resulted in a significant decrease in CFU-F (p = 0.0014) and CFU-OB/total (p = 0.028) in C3H only. ALN treatment had NS effect on CFU-OB/ALP+ in all four strains. Genetic factors appear to play a role in ALN's effects on CFU-F and CFU-OB/total but not on CFU-OB/ALP+.

Variable Effects of Caloric Restriction on Metabolic and Breathing Regulation Between Two Inbred Mouse Strains

The World Health Organization reports that ~800 million people worldwide are undernourished, corresponding to a daily mortality rate of 25,000 people. While altered metabolic and breathing characteristics occur with severe caloric restriction (CR), the mechanisms are unclear. We hypothesize that a robust genetic difference between C3H/HeJ (C3) and C57BL/6 (B6) mice lead to variation in metabolic and breathing regulation with CR. C3 and B6 mice (n = 8 per strain) were fed a diet equal to 33% of the daily food intake for 21 days. Using plethysmography, breathing characteristics were measured weekly on room air, and following acute hypercapnic (5% CO2) and hypoxic (10% O2) challenges. While the decline in body weight through 21 days of CR was similar in C3 vs. B6 strains (~40%), reductions in body temperature (10.0±1.8 vs. 2.8±1.0°C) and O2 consumption (1.36±0.32 vs. ‐0.04±0.16 ml/hr/g) were significantly (P &lt; 0.01) lower in C3 relative to B6 mice. Minute ventilation (VE) on room air remained unchanged with CR in both strains, but hypercapnic and hypoxic VE were depressed incrementally though 14 days. By day 21 of CR, hypercapnic and hypoxic VE recovered in B6 but not in C3 mice. In conclusion, genetic factors determine variation in metabolic and breathing regulation during CR, allowing B6 mice to maintain indices of homeostasis closer to control values than C3 mice.

Microarray analysis of gene expression in mouse aorta reveals role of the calcium signaling pathway in control of atherosclerosis susceptibility

Inbred mouse strains C57BL/6J (B6) and C3H/HeJ (C3H) exhibit a marked difference in atherosclerotic lesion formation when deficient in apolipoprotein E (apoE(-/-)), and the arterial wall has been identified as a source of the difference in atherosclerosis susceptibility. In the present study, differences in gene expression in aortic walls of the two strains were analyzed by microarrays. Total RNA was extracted from the aorta of 6-wk-old female B6 and C3H apoE(-/-) mice fed a chow or Western diet. There were 1,514 genes in chow fed mice and 590 genes in Western fed mice that were found to be differentially expressed between the two strains. Pathway analysis of differentially expressed genes suggested a role for the calcium signaling pathway in regulating atherosclerosis susceptibility. Oxidized LDL (oxLDL) induced a dose-dependent rise in cytosolic calcium levels in B6 endothelial cells. oxLDL-induced monocyte chemoattractant protein-1 production was inhibited by pretreatment with calcium chelator EGTA or intracellular calcium trapping compound BAPTA, indicating that calcium ions mediate the effect of oxLDL on monocyte chemoattractant protein-1 induction. The present findings demonstrate involvement of the calcium signaling pathway in the inflammatory process of atherogenesis.

Pathway focused protein profiling indicates differential function for IL-1B, -18 and VEGF during initiation and resolution of lung inflammation evoked by carbon nanoparticle exposure in mice

BackgroundCarbonaceous nanoparticles possess an emerging source of human exposure due to the massive release of combustion products and the ongoing revolution in nanotechnology. Pulmonary inflammation caused by deposited nanoparticles is central for their adverse health effects. Epidemiological studies suggest that individuals with favourable lung physiology are at lower risk for particulate matter associated respiratory diseases probably due to efficient control of inflammation and repair process. Therefore we selected a mouse strain C3H/HeJ (C3) with robust lung physiology and exposed it to moderately toxic carbon nanoparticles (CNP) to study the elicited pulmonary inflammation and its resolution.Methods5 μg, 20 μg and 50 μg CNP were intratracheally (i.t.) instilled in C3 mice to identify the optimal dose for subsequent time course studies. Pulmonary inflammation was assessed using histology, bronchoalveolar lavage (BAL) analysis and by a panel of 62 protein markers.Results1 day after instillation of CNP, C3 mice exhibited a typical dose response, with the lowest dose (5 μg) representing the 'no effect level' as reflected by polymorphonuclear leucocyte (PMN), and BAL/lung concentrations of pro-inflammatory proteins. Histological analysis and BAL-protein concentration did not reveal any evidence of tissue injury in 20 μg CNP instilled animals. Accordingly time course assessment of the inflammatory response was performed after 3 and 7 days with this dose (20 μg). Compared to day 1, BAL PMN counts were significantly decreased at day 3 and completely returned to normal by day 7. We have identified protein markers related to the acute response and also to the time dependent response in lung and BAL. After complete resolution of PMN influx on day 7, we detected elevated concentrations of 20 markers that included IL1B, IL18, FGF2, EDN1, and VEGF in lung and/or BAL. Biological pathway analysis revealed these factors to be involved in a closely regulated molecular cascade with IL1B/IL18 as upstream and FGF2/EDN1/VEGF as downstream molecules.ConclusionConsidering the role of VEGF, FGF2 and EDN1 in lung development and morphogenesis together with the lack of any evident tissue damage we suggest a protective/homeostatic machinery to be associated in lungs of stable organisms to counter the CNP challenge as a precautionary measure.

Fourier Transform Infrared Imaging Microspectroscopy and Tissue-Level Mechanical Testing Reveal Intraspecies Variation in Mouse Bone Mineral and Matrix Composition

Fracture susceptibility is heritable and dependent upon bone morphology and quality. However, studies of bone quality are typically overshadowed by emphasis on bone geometry and bone mineral density. Given that differences in mineral and matrix composition exist in a variety of species, we hypothesized that genetic variation in bone quality and tissue-level mechanical properties would also exist within species. Sixteen-week-old female A/J, C57BL/6J (B6), and C3H/HeJ (C3H) inbred mouse femora were analyzed using Fourier transform infrared imaging and tissue-level mechanical testing for variation in mineral composition, mineral maturity, collagen cross-link ratio, and tissue-level mechanical properties. A/J femora had an increased mineral-to-matrix ratio compared to B6. The C3H mineral-to-matrix ratio was intermediate of A/J and B6. C3H femora had reduced acid phosphate and carbonate levels and an increased collagen cross-link ratio compared to A/J and B6. Modulus values paralleled mineral-to-matrix values, with A/J femora being the most stiff, B6 being the least stiff, and C3H having intermediate stiffness. In addition, work-to-failure varied among the strains, with the highly mineralized and brittle A/J femora performing the least amount of work-to-failure. Inbred mice are therefore able to differentially modulate the composition of their bone mineral and the maturity of their bone matrix in conjunction with tissue-level mechanical properties. These results suggest that specific combinations of bone quality and morphological traits are genetically regulated such that mechanically functional bones can be constructed in different ways.

Prenatal activation of 5‐HT2A receptor induces expression of 5‐HT1B receptor in phrenic motoneurons and alters the organization of their premotor network in newborn mice

In newborn mice of the control [C3H/HeJ (C3H)] and monoamine oxidase A-deficient (Tg8) strains, in which levels of endogenous serotonin (5-HT) were drastically increased, we investigated how 5-HT system dysregulation affected the maturation of phrenic motoneurons (PhMns), which innervate the diaphragm. First, using immunocytochemistry and confocal microscopy, we observed a 5-HT(2A) receptor (5-HT(2A)-R) expression in PhMns of both C3H and Tg8 neonates at the somatic and dendritic levels, whereas 5-HT(1B) receptor (5-HT(1B)-R) expression was observed only in Tg8 PhMns at the somatic level. We investigated the interactions between 5-HT(2A)-R and 5-HT(1B)-R during maturation by treating pregnant C3H mice with a 5-HT(2A)-R agonist (2,5-dimethoxy-4-iodoamphetamine hydrochloride). This pharmacological overactivation of 5-HT(2A)-R induced a somatic expression of 5-HT(1B)-R in PhMns of their progeny. Conversely, treatment of pregnant Tg8 mice with a 5-HT(2A)-R antagonist (ketanserin) decreased the 5-HT(1B)-R density in PhMns of their progeny. Second, using retrograde transneuronal tracing with rabies virus injected into the diaphragm of Tg8 and C3H neonates, we studied the organization of the premotor network driving PhMns. The interneuronal network monosynaptically connected to PhMns was much more extensive in Tg8 than in C3H neonates. However, treatment of pregnant C3H mice with 2,5-dimethoxy-4-iodoamphetamine hydrochloride switched the premotoneuronal network of their progeny from a C3H- to a Tg8-like pattern. These results show that a prenatal 5-HT excess affects, via the overactivation of 5-HT(2A)-R, the expression of 5-HT(1B)-R in PhMns and the organization of their premotor network.

PPARG by Dietary Fat Interaction Influences Bone Mass in Mice and Humans

Adult BMD, an important risk factor for fracture, is the result of genetic and environmental interactions. A quantitative trait locus (QTL) for the phenotype of volumetric BMD (vBMD), named Bmd8, was found on mid-distal chromosome (Chr) 6 in mice. This region is homologous to human Chr 3p25. The B6.C3H-6T (6T) congenic mouse was previously created to study this QTL. Using block haplotyping of the 6T congenic region, expression analysis in the mouse, and examination of nonsynonymous SNPs, peroxisome proliferator activated receptor gamma (Pparg) was determined to be the most likely candidate gene for the Bmd8 QTL of the 630 genes located in the congenic region. Furthermore, in the C3H/HeJ (C3H) strain, which is the donor strain for the 6T congenic, several polymorphisms were found in the Pparg gene. On challenge with a high-fat diet, we found that the 6T mouse has a lower areal BMD (aBMD) and volume fraction of trabecular bone (BV/TV%) of the distal femur compared with B6 mice. Interactions between SNPs in the PPARG gene and dietary fat for the phenotype of BMD were examined in the Framingham Offspring Cohort. This analysis showed that there was a similar interaction of the PPARG gene and diet (fat intake) on aBMD in both men and women. These findings suggest that dietary fat has a significant influence on BMD that is dependent on the alleles present for the PPARG gene.

A Dominant X-Linked QTL Regulating Pubertal Timing in Mice Found by Whole Genome Scanning and Modified Interval-Specific Congenic Strain Analysis

BackgroundPubertal timing in mammals is triggered by reactivation of the hypothalamic-pituitary-gonadal (HPG) axis and modulated by both genetic and environmental factors. Strain-dependent differences in vaginal opening among inbred mouse strains suggest that genetic background contribute significantly to the puberty timing, although the exact mechanism remains unknown.Methodology/Principal FindingsWe performed a genome-wide scanning for linkage in reciprocal crosses between two strains, C3H/HeJ (C3H) and C57BL6/J (B6), which differed significantly in the pubertal timing. Vaginal opening (VO) was used to characterize pubertal timing in female mice, and the age at VO of all female mice (two parental strains, F1 and F2 progeny) was recorded. A genome-wide search was performed in 260 phenotypically extreme F2 mice out of 464 female progeny of the F1 intercrosses to identify quantitative trait loci (QTLs) controlling this trait. A QTL significantly associated was mapped to the DXMit166 marker (15.5 cM, LOD = 3.86, p<0.01) in the reciprocal cross population (C3HB6F2). This QTL contributed 2.1 days to the timing of VO, which accounted for 32.31% of the difference between the original strains. Further study showed that the QTL was B6-dominant and explained 10.5% of variation to this trait with a power of 99.4% at an alpha level of 0.05.The location of the significant ChrX QTL found by genome scanning was then fine-mapped to a region of ∼2.5 cM between marker DXMit68 and rs29053133 by generating and phenotyping a panel of 10 modified interval-specific congenic strains (mISCSs).Conclusions/SignificanceSuch findings in our study lay a foundation for positional cloning of genes regulating the timing of puberty, and also reveal the fact that chromosome X (the sex chromosome) does carry gene(s) which take part in the regulative pathway of the pubertal timing in mice.

Genetic Variation in Mouse Femoral Tissue-Level Mineral Content Underlies Differences in Whole Bone Mechanical Properties

A/J mice, as compared to C57BL/6J (B6) mice, have a significantly greater total femoral mineral (ash) content which correlates with an increased femoral stiffness (resistance to deformation), but also with an increased brittleness (catastrophic failure). To determine if this whole bone variation in mineral content is indicative of significant mineral and/or matrix variation at the tissue level, femora from 16-week-old female A/J and B6 mice were isolated, embedded in PMMA, sectioned and mounted on barium fluoride infrared windows for FTIRI analyses. In addition, preliminary studies of femora from C3H/HeJ (C3H) mice were conducted, since they have an ash content intermediate to A/J and B6. Mean values for mineral-to-matrix ratio were significantly different for A/J (8.4 ± 0.8) and B6 (7.5 ± 0.4), as were values for collagen cross-link maturity (1.8 ± 0.05 and 3.2 ± 0.1, respectively). C3H mice appeared to have a mineral-to-matrix ratio intermediate of A/J and B6, and cross-link maturity greater than both A/J and B6. B6 femora had similar carbonate-to-amide ratios, carbonate-to-mineral ratios and acid phosphate levels. Thus, whole bone differences in mineral content are concurrent with tissue-level variation in mineral content and collagen maturity in inbred mice. The greater stiffness and brittleness of A/J femora are likely due to differential biological control (osteoblast activity) of the amount of mineral.

Quantitative trait locus analysis of circulating adhesion molecules in hyperlipidemic apolipoprotein E-deficient mice.

Circulating soluble adhesion molecules have been suggested as useful markers to predict several clinical conditions such as atherosclerosis, type 2 diabetes, obesity, and hypertension. To determine genetic factors influencing plasma levels of soluble vascular cell adhesion molecule-1 (VCAM-1) and P-selectin, quantitative trait locus (QTL) analysis was performed on an intercross between C57BL/6J (B6) and C3H/HeJ (C3H) mouse strains deficient in apolipoprotein E-deficient (apoE-/-). Female F2 mice were fed a western diet for 12 weeks. One significant QTL, named sVcam1 (71 cM, LOD 3.9), on chromosome 9 and three suggestive QTLs on chromosomes 5, 13 and 15 were identified to affect soluble VCAM-1 levels. Soluble P-selectin levels were controlled by one significant QTL, named sSelp1 (8.5 cM, LOD 3.4), on chromosome 16 and two suggestive QTLs on chromosomes 10 and 13. Both adhesion molecules showed significant or an apparent trend of correlations with body weight, total cholesterol, and LDL/VLDL cholesterol levels in the F2 population. These results indicate that plasma VCAM-1 and P-selectin levels are complex traits regulated by multiple genes, and this regulation is conferred, at least partially, by acting on body weight and lipid metabolism in hyperlipidemic apoE-/- mice.

Genetic Variation in the Patterns of Skeletal Progenitor Cell Differentiation and Progression During Endochondral Bone Formation Affects the Rate of Fracture Healing

These studies examined how genetic differences that regulate architectural and bone material properties would be expressed during fracture healing and determine whether any of these features would affect rates of healing as defined by regain of strength. Controlled fractures were generated in three inbred strains of mice: A/J, C57Bl/6J (B6), and C3H/HeJ (C3H). Both the A/J and B6 strains showed faster healing than the C3H strain based on regains in strength and stiffness. Strain-specific architectural features such as moment of inertia, cross-sectional area, and cortical thickness were all recapitulated during the development of the callus tissues. None of these traits were directly relatable to rates of fracture healing. However, rates of healing were related to variations in the temporal patterns of chondrogenic and osteogenic lineage development. The B6 strain expressed the highest percentage of cartilage gene products and had the longest period of chondrocyte maturation and hypertrophy. The slowest healing strain (C3H) had the shortest period of chondrogenic development and earliest initiation of osteogenic development. Although the A/J strain showed an almost identical pattern of chondrogenic development as the C3H strain, A/J initiated osteogenic development several days later than C3H during fracture healing. Long bone growth plates at 28 days after birth showed similar strain-specific variation in cartilage tissue development as seen in fracture healing. Thus, the B6 strain had the largest growth plate heights, cell numbers per column, and the largest cell size, whereas the C3H columns were the shortest, had the smallest number of cells per column, and showed the smallest cell sizes. These results show that (1) different strains of mice express variations of skeletal stem cell lineage differentiation and (2) that these variations affect the rate of fracture healing.