Bioorthogonal probes for imaging sterols in cells.

Cholesterol is a fundamental lipid component of eukaryotic membranes and a precursor of potent signaling molecules, such as oxysterols and steroid hormones. Cholesterol and oxysterols are also essential for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Despite their importance, the use of imaging sterols in cells is currently very limited. We introduce a robust and versatile method for sterol microscopy based on C19 alkyne cholesterol and oxysterol analogues. These sterol analogues are fully functional; they rescue growth of cholesterol auxotrophic cells and faithfully recapitulate the multiple roles that sterols play in Hedgehog signal transduction. Alkyne sterol analogues incorporate efficiently into cellular membranes and can be imaged with high resolution after copper(I)-catalyzed azide-alkyne cycloaddition reaction with fluorescent azides. We demonstrate the use of alkyne sterol probes for visualizing the subcellular distribution of cholesterol and for two-color imaging of sterols and choline phospholipids. Our imaging strategy should be broadly applicable to studying the role of sterols in normal physiology and disease.

Genome-wide scan identifies novel QTLs for cholesterol and LDL levels in F2[Dahl RxS]-intercross rats.

Hypercholesterolemia is a significant risk factor for coronary artery disease development. Genes influencing nonmonogenic hypercholesterolemia susceptibility in humans remain to be identified. Animal models are key investigative systems because major confounding variables such as diet, activity, and genetic background can be controlled. We performed a 121-marker, total genome-analysis of an F2[Dahl RxS]-intercross selected for contrasting parental strain susceptibilities for hyperlipidemia on regular rat diets at 6 months of age. Quantitative traits studied were plasma total cholesterol, triglyceride, HDL, and LDL levels adjusted for obesity. Genome-wide analysis of 200 F2-intercross male rats detects two QTLs with highly significant linkage for total cholesterol (TC) on chromosome (chr) 5-133.3 Mbp (LOD 5.8), and chr5-54.2 Mbp (LOD 4.8), and two QTLs with significant linkage for TC: on chromosome 8, chr8-60.4 Mbp (LOD 3.8), and chromosome 2, chr2-243.5 Mbp (LOD 3.4). A QTL for LDL with significant linkage is detected on chromosome 5, chr5-104 Mbp (LOD 3.7). These QTLs contribute from 7% to 12% of total trait variance, respectively, with Dahl-S allele effects resulting in increased TC and LDL levels consistent with hyperlipidemia susceptibility in the parental Dahl-S rat strain. Predicted QTL-peaks do not coincide with previous genome scans. Human homologues of two TC-QTLs span genes listed in a LocusLink profile for cholesterol. Only suggestive loci were detected for HDL and total triglyceride levels. Altogether, the data demonstrates the contribution of multiple QTLs to hypercholesterolemia making a multipathway pathogenic framework imperative. QTL-peak candidate genes delineated are syntenic between rat and human genomes, increasing clinical relevance and mandating further study.

X-linked locus associated with hypertensive renal disease susceptibility in Dahl rats.

OBJECTIVE: There is increasing evidence that genetic factors contribute to renal disease susceptibility associated with essential hypertension. To what extent these genetic factors act independently of hypertension susceptibility remains undetermined. The present study was undertaken to assess the potential chromosome X influence on target organ renal disease in the Dahl rat model of salt-sensitive hypertension. SUBJECTS AND METHODS: Dahl S, Dahl R, F1(RXS), F1(SXR) and F2(RXS) rat male populations were phenotyped for hypertensive renal disease by measuring the percent of incidence of the Grade IV Raij renal pathology score. Six chromosome X markers informative for our (RXS) intercross were analyzed in our F2 rat population (n = 105) for co-segregation with hypertensive renal disease and blood pressure characterized by radiotelemetry. RESULTS: Comparison of the incidence of renal disease (histologically determined) between F1 reciprocal intercross male progenies reveals a significant chromosome X effect on renal disease [percent incidence of Grade IV Raij renal pathology score in F1 (R female S male) male rats = 2.75 +/- 0.66, and in F1 (S female R male) male rats = 0.67 +/- 0.42; = 0.02]. QTL analysis on an F2(RXS) male population phenotyped for renal disease susceptibility (percent incidence of Grade IV Raij renal pathology score) detects significant linkage to DXRat98 (likelihood ratio statistic = 9.4, P = 0.00223) on chromosome X, corroborating X-linkage of renal disease susceptibility in Dahl rats. CONCLUSIONS: Our results demonstrate the existence of an X-linked locus associated with hypertensive renal disease susceptibility in Dahl rats. Furthermore, the chromosome X markers tested did not co-segregate with hypertension, indicating that the gene(s) on chromosome X influence renal disease susceptibility independent of blood pressure.

A mechanism for vertebrate Hedgehog signaling: recruitment to cilia and dissociation of SuFu-Gli protein complexes.

In vertebrates, Hedgehog (Hh) signaling initiated in primary cilia activates the membrane protein Smoothened (Smo) and leads to activation of Gli proteins, the transcriptional effectors of the pathway. In the absence of signaling, Gli proteins are inhibited by the cytoplasmic protein Suppressor of Fused (SuFu). It is unclear how Hh activates Gli and whether it directly regulates SuFu. We find that Hh stimulation quickly recruits endogenous SuFu-Gli complexes to cilia, suggesting a model in which Smo activates Gli by relieving inhibition by SuFu. In support of this model, we find that Hh causes rapid dissociation of the SuFu-Gli complex, thus allowing Gli to enter the nucleus and activate transcription. Activation of protein kinase A (PKA), an inhibitor of Hh signaling, blocks ciliary localization of SuFu-Gli complexes, which in turn prevents their dissociation by signaling. Our results support a simple mechanism in which Hh signals at vertebrate cilia cause dissociation of inactive SuFu-Gli complexes, a process inhibited by PKA.

Both p16(Ink4a) and the p19(Arf)-p53 pathway constrain progression of pancreatic adenocarcinoma in the mouse.

Activating KRAS mutations and p16(Ink4a) inactivation are near universal events in human pancreatic ductal adenocarcinoma (PDAC). In mouse models, Kras(G12D) initiates formation of premalignant pancreatic ductal lesions, and loss of either Ink4a/Arf (p16(Ink4a)/p19(Arf)) or p53 enables their malignant progression. As recent mouse modeling studies have suggested a less prominent role for p16(Ink4a) in constraining malignant progression, we sought to assess the pathological and genomic impact of inactivation of p16(Ink4a), p19(Arf), and/or p53 in the Kras(G12D) model. Rapidly progressive PDAC was observed in the setting of homozygous deletion of either p53 or p16(Ink4a), the latter with intact germ-line p53 and p19(Arf) sequences. Additionally, Kras(G12D) in the context of heterozygosity either for p53 plus p16(Ink4a) or for p16(Ink4a)/p19(Arf) produced PDAC with longer latency and greater propensity for distant metastases relative to mice with homozygous deletion of p53 or p16(Ink4a)/p19(Arf). Tumors from the double-heterozygous cohorts showed frequent p16(Ink4a) inactivation and loss of either p53 or p19(Arf). Different genotypes were associated with specific histopathologic characteristics, most notably a trend toward less differentiated features in the homozygous p16(Ink4a)/p19(Arf) mutant model. High-resolution genomic analysis revealed that the tumor suppressor genotype influenced the specific genomic patterns of these tumors and showed overlap in regional chromosomal alterations between murine and human PDAC. Collectively, our results establish that disruptions of p16(Ink4a) and the p19(ARF)-p53 circuit play critical and cooperative roles in PDAC progression, with specific tumor suppressor genotypes provocatively influencing the tumor biological phenotypes and genomic profiles of the resultant tumors.

Modulation of learning and memory in Dahl rats by dietary salt restriction.

The Dahl rat represents a robust animal model of salt-sensitive hypertension, with Dahl S rats being salt sensitive and Dahl R rats (the Dahl S counterparts) being salt resistant for the development of hypertension. Here we evaluate the effect of reduced dietary salt intake on learning and memory in the Dahl rat model. Salt restriction produced a significant impairment in social transmission of food preference and social recognition memory in Dahl S rats without affecting spatial learning. In contrast, social transmission of food preference and social recognition memory remained unaffected in Dahl R rats, whereas navigation performance was significantly improved. This effect on learning and memory was not generalized because sodium restriction did not influence object recognition memory in either Dahl S or Dahl R rats. The significant decrement in select cognitive functions in Dahl S rats produced by salt restriction are in sharp contrast to the well known positive effect of dietary salt restriction in alleviating high blood pressure and associated target organ complications, suggesting that caution must be exercised when weighing the benefits of salt restriction in improving cardiovascular health in salt-sensitive hypertension against the potential undesirable effects of reduced cognitive function.

The dual AngII/AVP receptor gene N119S/C163R variant exhibits sodium-induced dysfunction and cosegregates with salt-sensitive hypertension in the Dahl salt-sensitive hypertensive rat model.

BACKGROUND: Essential hypertension is a prevalent complex polygenic disease and a major risk factor for cardiovascular disease, the leading cause of death in developed countries. Because of its complex and multifactorial nature, its genetic determinants still remain largely unknown. The Dahl salt-sensitive hypertensive rat model exhibits impaired sodium handling, which is hypothesized to play a key role in the pathophysiology of polygenic hypertension. Thus, genes associated with renal regulation of salt and water balance are a priori likely candidates for a causative role in hypertension pathogenesis. The functional properties and renal-specific expression of the recently characterized AngII/AVP receptor suggest a putative modulator role in tubular sodium and fluid reabsorption. Based on these observations, we investigated the potential involvement of the AngII/AVP receptor in salt-sensitive hypertension. MATERIALS AND METHODS: We performed cosegregation analysis of the AngII/AVP receptor locus with salt-sensitive hypertension in an F2 (Dahl S X Dahl salt-resistant [R]) hybrid male cohort characterized for blood pressure by radiotelemetry after 8 weeks of high salt challenge. Further molecular analysis was done to identify putative AngII/AVP receptor molecular variants that could account for the AngII/ AVP receptor involvement in salt-sensitive hypertension pathogenesis. RESULTS: The AngII/AVP receptor was mapped to rat chromosome 1, 1.7 cM centromeric to the D1Rat188 marker by radiation hybrid mapping analysis. Quantitative trait locus (QTL) analysis detected a highly significant linkage of the AngII/AVP receptor locus with high blood pressure (LRS = 13.8, p= 0.0002). Molecular characterization of the Dahl S and Dahl R AngII/AVP receptor cDNAs revealed two amino acid substitutions in the Dahl S AngII/AVP receptor (N119S, C163R) when compared to the Dahl R AngII/AVP receptor. These mutations are associated with an increased receptor affinity for both ligands (AVP and AngII) and an enhanced G(s)-coupling by the receptor resulting in increased activation of adenylate cyclase with concomitant increase in cAMP production. CONCLUSIONS: The observed molecular dysfunction in the Dahl S AngII/AVP receptor is consistent with increased tubular sodium and fluid reabsorption observed in Dahl S rats. Interestingly, the AngII/AVPr locus is within the narrowed chromosome 1 QTL region for blood pressure detected in different rat intercross linkage analyses. Altogether, the data strongly suggest that the AngII/AVP receptor is a hypertension susceptibility gene in the Dahl S rat model, as well as raises the hypothesis that it too underlies the chromosome 1 blood pressure QTL identified in other hypertension rat models.

Differential regulation of functional gene clusters in overt coronary artery disease in a transgenic atherosclerosis-hypertensive rat model.

BACKGROUND: Human acute coronary syndrome refers to the spectrum of clinical manifestations of overt coronary artery (CAD) disease characterized by atherosclerotic plaque destabilization and resultant myocardial injury. Typically studied as distinct pathologies, emerging pathogenic paradigms implicate multiple processes beyond thrombosis and ischemic cell injury respectively, with significant pathway overlap involving inflammation, apoptosis, matrix degradation, and oxidative stress. However, all these pathways have also been implicated in still-quiescent coronary plaque progression, thus making it harder to pinpoint the turnkey events leading to overt-CAD. Analysis of transcription profiles could identify a working framework of pathogenesis distinguishing overt-CAD. MATERIALS AND METHODS: We investigated the transcription profile associated with overt-coronary artery disease (CAD), in contrast to quiescent-CAD and attenuated, quiescent-CAD using the Tg 53 transgenic atherosclerosis-hypertensive rat model, which exhibits end-stage coronary heart disease simulating human acute coronary syndromes. Using a rat-specific known-gene oligonucleotide array, twice corroborated transcription profiles from four individual Tg 53 rats exhibiting overt-CAD were analyzed and contrasted to transcription profiles of age-matched Tg 53 rats with quiescent-CAD (pooled n = 4) and attenuated, quiescent-CAD (pooled n = 4). RESULTS: Tg 53 male rats with overt-CAD exhibited distinct transcription profiles compared with both quiescent-CAD control groups. Functional gene cluster analysis detects upregulation of genes involved in inflammation (interleukin-1, interleukin-18, Fc gamma II receptor, thyrotropin releasing hormone), matrix balance (membrane type metalloproteinase, TIMP-1, lysyl oxidase), oxidized LDL entry (endothelial oxLDL receptor), which contrast deinduced gene clusters involved in angiogenesis, proliferation, metabolism, ion transport and adrenergic receptors. CONCLUSION: The data demonstrate that transcriptionally mediated events distinguish the onset of overt-CAD and identify a first list of putative "turnkey" genes. This altered molecular framework implies an altered "hardwiring" which a priori would require multifaceted, targeted intervention- currently not implemented to date. Although more studies are necessary, early concordance with current pathogenic paradigms of human coronary plaque destabilization and post-ischemic myocardial response provides translational significance to observations and hypotheses.

Attenuated hippocampus-dependent learning and memory decline in transgenic TgAPPswe Fischer-344 rats.

Alzheimer's disease (AD) is characterized by increased beta amyloid (Abeta) levels, extracellular Abeta deposits in senile plaques, neurofibrillary tangles, and neuronal loss. However, the physiological role of normal levels of Abeta and its parent protein, the amyloid precursor protein (APP) are unknown. Here we report that low-level transgenic (Tg) expression of the Swedish APP mutant gene (APPswe) in Fischer-344 rats results in attenuated age-dependent cognitive performance decline in 2 hippocampus-dependent learning and memory tasks compared with age-matched nontransgenic Fischer-344 controls. TgAPPswe rats exhibit mild increases in brain APP mRNA (56.8%), Abeta-42 (21%), and Abeta-40 (6.1%) peptide levels at 12 mo of age, with no extracellular Abeta deposits or senile plaques at 6, 12, and 18 mo of age, whereas 3- to 6-fold increases in Abeta levels are detected in plaque-positive human AD patients and transgenic mouse models. The data support the hypothesis that a threshold paradigm underlies Abeta-related pathology, below which APP expression may play a physiological role in specific hippocampus-dependent tasks, most likely related to its neurotrophic role.

Chlamydia pneumoniae accelerates coronary artery disease progression in transgenic hyperlipidemia-genetic hypertension rat model.

Chlamydia pneumoniae (Cpn) has been associated with human coronary artery disease but causal relevance as a risk factor has not been shown. Several rabbit and mouse model studies demonstrate exacerbation of aortic atherosclerosis by Cpn, however impact of Cpn on coronary artery disease (CAD) and survival outcomes has not been shown. To study this, we used specific pathogen-free, inbred, transgenic-CAD Dahl salt-sensitive (S) hypertensive (Tg53) rats and control inbred, non-transgenic Dahl S (nonTg) rats to analyze the effects of Cpn infection on macrophage foam cell formation, coronary artery disease progression, and effect on survival. Cpn infection induced acceleration of foam cell formation in hyperlipidemic Tg53 recruited peritoneal macrophages. This effect is hyperlipidemia-dependent. The transcription profile of Tg53-Cpn macrophage foam cells is different from control mock-inoculated (Tg53-spg) and heat-inactivated (Tg53-iCpn) macrophages (ANOVA P < 0.0001). Decreased survival was detected in Tg53-Cpn compared with control nonTg-Cpn and mock-infected Tg53-mouse pneumonitic rats (P = 0.009) and was associated with "culprit" coronary plaques and left atrial thrombi. These data demonstrate that in the presence of significant hyperlipidemia and hypertension, one-time Cpn infection at 5 mo of age (associated with early CAD stage) accelerates progression to overt-CAD in the Tg53 rat model. The data support the hypothesis that untreated Cpn infection is a causal risk factor for CAD progression most likely mediated by Cpn-induced accelerated macrophage foam cell formation.