A Promising Combination: PACAP and PARP Inhibitor Have Therapeutic Potential in Models of Diabetic and Hypertensive Retinopathies.

Diabetes and hypertension are complex pathologies with increasing prevalence nowadays. Their interconnected pathways are frequently manifested in retinopathies. Severe retinal consequences and their tight connections as well as their possible treatments are particularly important to retinal research. In the present, work we induced diabetes with streptozotocin in spontaneously hypertensive rats and treated them either with PACAP or olaparib and alternatively with both agents. Morphological and immunohistochemical analyses were carried out to describe cell-specific changes during pathologies and after different treatments. Diabetes and hypertension caused massive structural and cellular changes especially when they were elicited together. Hypertension was crucial in the formation of ONL and OPL damage while diabetes caused significant differences in retinal thickness, OPL thickness and in the cell number of the GCL. In diabetes, double neuroprotective treatment ameliorated changes of calbindin-positive cells, rod bipolar cells and dopaminergic amacrine cells. Double treatment was curative in hypertensive diabetic rat retinas, especially in the case of rod bipolar and parvalbumin-positive cells compared to untreated or single-treated retinas. Our results highlighted the promising therapeutic benefits of olaparib and PACAP in these severe metabolic retinal disorders.

Role of complement 3 in renin generation during the differentiation of mesenchymal stem cells to smooth muscle cells.

We showed that increased expression of complement 3 (C3) induces dedifferentiation of mesenchymal cells and epithelial mesenchymal transition, which activate the local renin-angiotensin system (RAS) that contributes to cardiovascular and renal remodeling in spontaneously hypertensive rats (SHRs). In the present study, to investigate contributions of C3 to the development of the pathogenesis of hypertension, we evaluated the formation of renin-producing cells and roles of C3 in renin generation during differentiation of primary bone marrow-mesenchymal stem cells (MSCs) from C57BL/6 mice, Wistar-Kyoto (WKY) rats, and SHRs to smooth muscle cells (SMCs) with transforming growth factor-ß1. The expression of renin transiently increased with increases in transcription factor liver X receptor α (LXRα), and expression of C3 and Krüppel-like factor 5 (KLF5) increased during differentiation of MSCs from C57BL/6 mice, WKY rats, and SHRs to SMCs. Exogenous C3a stimulated renin and LXRα expression accompanied by nuclear translocation of LXRα. C3a receptor antagonist SB290157 suppressed renin and LXRα expression, with inhibition of nuclear translocation of LXRα during the differentiation of mouse MSCs to SMCs. The expression of C3 and KLF5 was significantly higher in the differentiated cells from SHRs compared with the cells from WKY rats during differentiation. Renin-producing cells were formed during differentiation of MSCs to SMCs, and renin generation was observed in undifferentiated SMCs, in which transient expression of renin in the differentiated cells with lower differentiation stage was stronger from SHRs than that from WKY rats. Expression and nuclear localization of LXRα in the differentiated cells from SHRs were stronger than that from WKY rats. C3 was important in forming and maintaining this undifferentiated state of SMCs from MSCs to generate renin with increases in transcription factor LXRα and KLF5. Increases in C3 expression maintain the undifferentiated state of SMCs from MSCs to generate renin that activates RAS and contributes to the pathogenesis of hypertension in SHRs.

Design and evaluation of four novel tripeptides as potent angiotensin converting enzyme (ACE) inhibitors with anti-hypertension activity.

The current study investigated the angiotensin-converting enzyme (ACE) inhibitory activity of 4 synthetic tripeptides. All the peptides showed enzyme inhibitory activity, especially two promising ones, TTP (Thea-Thea-Pro) and gAgAP (GABA-GABA-Pro), with IC50 values of 0.92 and 3.4 µmol/L, respectively. Enzyme inhibition kinetics determined by Lineweaver-Burk plots revealed that TTP and gAgAP were competitive inhibitors with Ki values of 0.87 and 3.12 µmol/L, respectively. Molecular docking experiments confirmed that the higher inhibitory potency of TTP and gAgAP might be attributed to the formation of several critical hydrogen bonds with the active site residues in ACE. We further demonstrated that TTP and gAgAP initiated a rapid and significant decrease in systolic blood pressure (SBP) in spontaneously hypertensive rats (SHRs). TTP treatment lowered SBP to the same extent as captopril, although the duration of anti-hypertensive effect was shorter in TTP group than that observed in captopril group. Moreover, the transcription levels of angiotensin II receptor type 1 (agtr1) and miR-132/-212 were downregulated in SHRs after administration of TTP and gAgAP. In particular, TTP treatment caused a comparable reduction of agtr1 levels compared to captopril treatment, while miR-132/212 expression was significantly decreased. These results showed that compound TTP might be served as a potential antihypertensive candidate.

The immunomodulatory mechanism of brain injury induced by hyperhomocysteinemia in spontaneously hypertensive rats.

BACKGROUND: Elevated plasma homocysteine (Hcy) concentration is considered as the diagnostic criteria of Hyperhomocysteinemia (HHcy), which is associated with the inflammatory response and blood-brain barrier disruption. Previous studies have proposed that HHcy with hypertension was associated with the brain injury by enhancing the cerebrovascular permeability, however, the immune mechanism remains obscure. The purpose of the study is to explore the immunomodulatory mechanism of brain injury in spontaneously hypertensive rats (SHRs) induced by HHcy. MATERIALS AND METHODS: Sixty SHRs were randomly assigned to three groups: SHR-C (control group), SHR-M (methionine group) and SHR-T (treatment group). Physical examination of body weight, systolic blood pressure (SBP) and plasma Hcy content was measured every 4 weeks. Besides, T-helper cell 17 and regulatory T cells (Treg)-related inflammatory cytokines (interleukin [IL]-6, IL-17, IL-10, and transforming growth factor beta [TGF-ß]) and genes (RORγt and FoxP3) were detected by enzyme-linked immunosorbent assay, quantitative polymerase chain reaction , Western blot, and immunohistochemistry. RESULTS: High methionine diet could cause weight loss, SBP rising, and plasma Hcy content significantly elevated. IL-16 and IL-17A levels in peripheral blood and in brain tissue both lifted, while IL-10 and TGF-ß levels dropped; RORγt expression raised in brain, nevertheless, FoxP3 levels were the opposite. After the intervention with vitamin B6, B12, and folic acid in SHR-T group, these trends would be eased or completely changed. Furthermore, brain tissue slices showed that IL-17-positive cells tended to decrease, and IL-10-positive cells increased in SHR-T group, which was reversed in SHR-M group. CONCLUSIONS: HHcy may promote inflammation that can lead to brain lesions and down-regulate immune response to protect the brain.

Genetically determined folate deficiency is associated with abnormal hepatic folate profiles in the spontaneously hypertensive rat.

Increased levels of plasma cysteine are associated with obesity and metabolic disturbances. Our recent genetic analyses in spontaneously hypertensive rats (SHR) revealed a mutated Folr1 (folate receptor 1) as the quantitative trait gene associated with diminished renal Folr1 expression, lower plasma folate levels, hypercysteinemia, hyperhomocysteinemia and metabolic disturbances. To further analyse the effects of the Folr1 gene expression on folate metabolism, we used mass spectrometry to quantify folate profiles in the plasma and liver of an SHR-1 congenic strain, with wild type Folr1 allele on the SHR genetic background, and compared them with the SHR strain. In the plasma, concentration of 5-methyltetrahydrofolate (5mTHF) was significantly higher in SHR-1 congenic rats compared to SHR (60+/-6 vs. 42+/-2 nmol/l, P<0.01) and 5mTHF monoglutamate was the predominant form in both strains (>99 % of total folate). In the liver, SHR-1 congenic rats showed a significantly increased level of 5mTHF and decreased concentrations of dihydrofolate (DHF), tetrahydrofolate (THF) and formyl-THF when compared to the SHR strain. We also analysed the extent of folate glutamylation in the liver. Compared with the SHR strain, congenic wild-type Folr1 rats had significantly higher levels of 5mTHF monoglutamate. On the other hand, 5mTHF penta- and hexaglutamates were significantly higher in SHR when compared to SHR-1 rats. This inverse relationship of rat hepatic folate polyglutamate chain length and folate sufficiency was also true for other folate species. These results strongly indicate that the whole body homeostasis of folates is substantially impaired in SHR rats compared to the SHR-1 congenic strain and might be contributing to the associated metabolic disturbances observed in our previous studies.

Anti-Renal Fibrotic Effect of Exercise Training in Hypertension.

The purpose of this study was to evaluate the effects of exercise training on renal fibrosis in hypertensive rats. Masson's trichrome staining and Western blotting were performed on the excised renal cortex from sixteen male spontaneously hypertensive rats (SHR), which were randomly divided into either a sedentary hypertensive group (SHR) or exercise hypertensive group (SHR-EX, running on an exercise treadmill for 60 min/day, 5 sessions/week, for 12 weeks), and from eight male Wistar-Kyoto rats which served as a sedentary normotensive group (WKY). The systolic blood pressure (SBP) and renal fibrosis in hypertensive rats improved after exercise training. The inflammatory-related protein levels of interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2), as well as the fibrotic-related protein levels of transforming growth factor-beta (TGF-ß), phospho-Smad2/3 (p-Smad2/3), connective tissue growth factor (CTGF), matrix metalloproteinase-9 (MMP-9), and matrix metalloproteinase-2 (MMP-2) were decreased in the SHR-EX group when compared with the SHR group. Exercise training suppressed the hypertension-induced renal cortical inflammatory and fibrotic pathways in hypertensive rat models. These findings might indicate a new therapeutic effect for exercise training to prevent renal fibrosis in hypertensive nephropathy.

Connexin 43 in splenic lymphocytes is involved in the regulation of CD4+CD25+ T lymphocyte proliferation and cytokine production in hypertensive inflammation.

Chronic inflammation promotes the development of hypertension and is associated with increased T cell infiltration and cytokine production in impaired organs. Gap junction protein connexin 43 (Cx43), is ubiquitously expressed in immune cells and plays an important role in T cell proliferation and activation, and cytokine production. However, the correlation between Cx43 in T cells and the hypertensive inflammatory response remains unknown. Thus, in this study, we wished to examine this correlation. First, our results revealed that hypertension caused significant thickening of the vascular wall, inflammatory cell infiltration into part of the renal interstitium and glomerular atrophy, and it increased the tubular damage scores in the kidneys of spontaneously hypertensive rats (SHRs). Moreover, the SHRs exhibited stenosis in the central artery wall ofthe spleen with increased serum levels of interleukin (IL)-2 and IL-6 compared with normotensive Wistar-Kyoto (WKY) rats. The spleens of the SHRs exhibited a significantly decreased percentage of CD4+CD25+ (Treg) T cells. However, the percentages of CD3+, CD4+ and CD8+ T cell and the levels of CD4+Cx43 and CD8+Cx43 did not differ significantly between the SHRs and WKY rats. In cultured lymphocytes from the SHRs and WKY rats, low percentages of Treg cells and reduced cytokine (IL-2 and IL-6) mRNA expression levels were observed in the lymphocytes obtained from the SHRs and WKY rats treated with the connexin blocker, Gap27, or concanavalin A (ConA) plus Gap27. The effects of ConA and Gap27 differed between the SHRs and WKY rats. On the whole, our findings demonstrate that the splenic Treg cell-mediated suppression in SHRs may be involved in hypertensive inflammatory responses. Cx43 in the gap junctional channel may regulate lymphocyte activation and inflammatory cytokine production.

Gallic Acid Reduces Blood Pressure and Attenuates Oxidative Stress and Cardiac Hypertrophy in Spontaneously Hypertensive Rats.

Gallic acid (GA) has been reported to have beneficial effects on cancer, vascular calcification, and diabetes-induced myocardial dysfunction. We hypothesized that GA controls hypertension via oxidative stress response regulation in an animal model for essential hypertension. Spontaneously hypertensive rats (SHRs) were administered GA for 16 weeks. GA treatment lowered elevated systolic blood pressure in SHRs through the inhibition of vascular contractility and components of the renin-angiotensin II system. In addition, GA administration reduced aortic wall thickness and body weight in SHRs. In SHRs, GA attenuated left ventricular hypertrophy and reduced the expression of cardiac-specific transcription factors. NADPH oxidase 2 (Nox2) and GATA4 mRNA expression was induced in SHR hearts and angiotensin II-treated H9c2 cells; this expression was downregulated by GA treatment. Nox2 promoter activity was increased by the synergistic action of GATA4 and Nkx2-5. GA seems to regulate oxidative stress by inhibiting the DNA binding activity of GATA4 in the rat Nox2 promoter. GA reduced the GATA4-induced Nox activity in SHRs and angiotensin II-treated H9c2 cells. GA administration reduced the elevation of malondialdehyde levels in heart tissue obtained from SHRs. These findings suggest that GA is a potential therapeutic agent for treating cardiac hypertrophy and oxidative stress in SHRs.

SHRSP/Izm and WKY/NCrlCrlj rats having a missense mutation in Abcg5 deposited plant sterols in the body, but did not change their biliary secretion and lymphatic absorption-comparison with Jcl:Wistar and WKY/Izm rats.

We had previously found plant sterols deposited in the bodies of stroke-prone spontaneously hypertensive rats (SHRSP)/Sea and Wistar Kyoto (WKY)/NCrlCrlj rats that had a missense mutation in the Abcg5 cDNA sequence that coded for ATP-binding cassette transporter (ABC) G5. We used SHRSP/Izm, WKY/NCrlCrlj, and WKY/Izm rats in the present study to determine the mechanisms for plant sterol deposition in the body. Jcl:Wistar rats were used as a control strain. A diet containing 0.5% plant sterols fed to the rats resulted in plant sterol deposition in the body of SHRSP/Izm, but not in WKY/Izm or Jcl:Wistar rats. Only a single non-synonymous nucleotide change, G1747T, resulting in a conservative cysteine substitution for glycine at amino acid 583 (Gly583Cys) in Abcg5 cDNA was identified in the SHRSP/Izm and WKY/NCrlCrlj rats. However, this mutation was not found in the WKY/Izm or Jcl:Wistar rats. No significant difference in the biliary secretion or lymphatic absorption of plant sterols was apparent between the rat strains with or without the missense mutation in Abcg5 cDNA. Our observations suggest that plant sterol deposition in rat strains with the missense mutation in Abcg5 cDNA can occur, despite there being no significant change in the biliary secretion or lymphatic absorption of plant sterols.

Reduced ischemic brain injury by partial rejuvenation of bone marrow cells in aged rats.

Circulating bone marrow-derived immature cells, including endothelial progenitor cells, have been implicated in homeostasis of the microvasculature. Decreased levels of circulating endothelial progenitor cells, associated with aging and/or cardiovascular risk factors, correlate with poor clinical outcomes in a range of cardiovascular diseases. Herein, we transplanted bone marrow cells from young stroke-prone spontaneously hypertensive rats (SHR-SP) into aged SHR-SP, the latter not exposed to radiation or chemotherapy. Analysis of recipient peripheral blood 28 days after transplantation revealed that 5% of circulating blood cells were of donor origin. Cerebral infarction was induced on day 30 posttransplantation. Animals transplanted with bone marrow from young SHR-SP displayed an increase in density of the microvasculature in the periinfarction zone, reduced ischemic brain damage and improved neurologic function. In vitro analysis revealed enhanced activation of endothelial nitric oxide synthase and reduced activation p38 microtubule-associated protein (MAP) kinase, the latter associated with endothelial apoptosis, in cultures exposed to bone marrow-derived mononuclear cells from young animals versus cells from aged counterparts. Our findings indicate that partial rejuvenation of bone marrow from aged rats with cells from young animals enhances the response to ischemic injury, potentially at the level of endothelial/vascular activation, providing insight into a novel approach ameliorate chronic vascular diseases.

Recombinant adeno-associated virus-mediated human kallikrein gene therapy protects against hypertensive target organ injuries through inhibiting cell apoptosis.

AIM: Overexpression of human tissue kallikrein (HK), mediated by recombinant adeno-associated virus (rAAV), decreased blood pressure in spontaneous hypertensive rats (SHRs) and reduced injury to the heart, aorta and kidney. In this study, we used both an in vivo animal model and in vitro cell culture system to investigate whether rAAV-mediated HK gene therapy protects against organ damage by inhibiting cell apoptosis. METHODS: rAAV encoding HK (rAAV-HK) or LacZ (rAAV-lacZ) were delivered as a control to spontaneously hypertensive rats (SHRs) and cultured human embryonic kidney (HEK) 293 cells. RESULTS: Treatment with rAAV-HK decreased cell apoptosis in the target organs of SHRs and also inhibited lipopolysaccharide (LPS)-induced HEK 293 apoptosis. The rAAV-HK delivery system also increased the levels of apoptosis-inhibiting proteins bcl-2 and bcl-x(L), and decreased the level of Bax and the activity of caspase 3, two promoters of apoptosis. In addition to its role in the inhibition of apoptosis, rAAV-HK also activated the cell survival and proliferation signaling pathways ERK1/2 and PI3K/AKT. CONCLUSION: rAAV-mediated HK gene delivery has multiple therapeutic possibilities for treating hypertension, not only by decreasing blood pressure, but also by directly inhibiting end-organ damage.

Delivery of sry1, but not sry2, to the kidney increases blood pressure and sns indices in normotensive wky rats.

BACKGROUND: Our laboratory has shown that a locus on the SHR Y chromosome increases blood pressure (BP) in the SHR rat and in WKY rats with the SHR Y chromosome (SHR/y rat). A candidate for this Y chromosome hypertension locus is Sry, a gene that encodes a transcription factor responsible for testes determination. The SHR Y chromosome has six divergent Sry loci. The following study examined if exogenous Sry1 or Sry2 delivered to the kidney would elevate renal tyrosine hydroxylase, renal catecholamines, plasma catecholamines and telemetered BP over a 28 day period. We delivered 50 mug of either the expression construct Sry1/pcDNA 3.1, Sry2/pcDNA 3.1, or control vector into the medulla of the left kidney of normotensive WKY rats by electroporation. Weekly air stress was performed to determine BP responsiveness. Separate groups of animals were tested for renal function and plasma hormone patterns and pharmacological intervention using alpha adrenergic receptor blockade. Pre-surgery baseline and weekly blood samples were taken from Sry1 electroporated and control vector males for plasma renin, aldosterone, and corticosterone. BP was measured by telemetry and tyrosine hydroxylase and catecholamines by HPLC with electrochemical detection. RESULTS: In the animals receiving the Sry1 plasmid there were significant increases after 21 days in resting plasma norepinephrine (NE, 27%) and renal tyrosine hydroxylase content (41%, p < .05) compared to controls. BP was higher in animals electroporated with Sry1 (143 mmHg, p < .05) compared to controls (125 mmHg) between 2-4 weeks. Also the pressor response to air stress was significantly elevated in males electroporated with Sry1 (41 mmHg) compared to controls (28 mmHg, p < .001). Sry2 did not elevate BP or SNS indices and further tests were not done. The hormone profiles for plasma renin, aldosterone, and corticosterone between electroporated Sry1 and control vector males showed no significant differences over the 28 day period. Alpha adrenergic receptor blockade prevented the air stress pressor response in both strains. Urinary dopamine significantly increased after 7 days post Sry electroporation. CONCLUSION: These results are consistent with a role for Sry1 in increasing BP by directly or indirectly activating renal sympathetic nervous system activity.

The spontaneously hypertensive rat: an experimental model of sulfur dioxide-induced airways disease.

Chronic obstructive pulmonary disease (COPD) is characterized by airway obstruction, inflammation, and mucus hypersecretion, features that are common in bronchitis, emphysema, and often asthma. However, current rodent models do not reflect this human disease. Because genetically predisposed spontaneously hypertensive (SH) rats display phenotypes such as systemic inflammation, hypercoagulation, oxidative stress, and suppressed immune function that are also apparent in COPD patients, we hypothesized that SH rat may offer a better model of experimental bronchitis. We, therefore, exposed SH and commonly used Sprague Dawley (SD) rats (male, 13- to 15-weeks old) to 0, 250, or 350 ppm sulfur dioxide (SO(2)), 5 h/day for 4 consecutive days to induce airway injury. SO(2) caused dose-dependent changes in breathing parameters in both strains with SH rats being slightly more affected than SD rats. Increases in bronchoalveolar lavage fluid (BALF) total cells and neutrophilic inflammation were dose dependent and significantly greater in SH than in SD rats. The recovery was incomplete at 4 days following SO(2) exposure in SH rats. Pulmonary protein leakage was modest in either strain, but lactate dehydrogenase and N-acetyl glucosaminidase activity were increased in BALF of SH rats. Airway pathology and morphometric evaluation of mucin demonstrated significantly greater impact of SO(2) in SH than in SD rats. Baseline differences in lung gene expression pattern suggested marked immune dysregulation, oxidative stress, impairment of cell signaling, and fatty acid metabolism in SH rats. SO(2) effects on these genes were more pronounced in SH than in SD rats. Thus, SO(2) exposure in SH rats may yield a relevant experimental model of bronchitis.

Single nucleotide polymorphisms associated with rat expressed sequences.

Single nucleotide polymorphisms (SNPs) are the most common source of genetic variation in populations and are thus most likely to account for the majority of phenotypic and behavioral differences between individuals or strains. Although the rat is extensively studied for the latter, data on naturally occurring polymorphisms are mostly lacking. We have used publicly available sequences consisting of whole-genome shotgun (WGS), expressed sequence tag (EST), and mRNA data as a source for the in silico identification of SNPs in gene-coding regions and have identified a large collection of 33,305 high-quality candidate SNPs. Experimental verification of 471 candidate SNPs using a limited set of rat isolates revealed a confirmation rate of approximately 50%. Although the majority of SNPs were identified between Sprague-Dawley (EST data) and Brown Norway (WGS data) strains, we found that 66% of the verified variations are common among different rat strains. All SNPs were extensively annotated, including chromosomal and genetic map information, and nonsynonymous SNPs were analyzed by SIFT and PolyPhen prediction programs for their potential deleterious effect on protein function. Interestingly, we retrieved three SNPs from the database that result in the introduction of a premature stop codon and that could be confirmed experimentally. Two of these "in silico-identified knockouts" reside in interesting QTL regions. Data are publicly available via a Web interface (http://cascad.niob.knaw.nl), allowing simple and advanced search queries.

Dietary canola and soybean oil fed to SHRSP rat dams differently affect the growth and survival of their male pups.

Canola oil (Can), as well as some other oils, shortens the survival of SHRSP rats compared with soybean oil (Soy). Although detrimental factors other than phytosterols have not been identified, they are likely to be hydrophobic and transmissible to pups. To test this possibility, female SHRSP rats (F0) were fed a diet supplemented with Can or Soy and mated at 11 wk of age. The growth of suckling pups (F1) from the Can-fed dams was significantly retarded compared with that of pups from the Soy-fed dams. Half of the male pups (F1) were weaned to the same diet as their dams (Can-->Can and Soy-->Soy groups) and the rest were weaned to the other diet (Can-->Soy and Soy-->Can groups). The survival rate of the male pups (F1) was significantly lower in the Can-->Can group than in the Soy-->Can group, and in the Can-->Soy group than in the Soy-->Soy group, indicating that the oils fed to dams differently affected the growth and survival of pups. There were fewer pups per dam in the Can-fed dams (F0) than in the Soy-fed dams, and in the dams (F1) of the Can-->Can and Soy-->Can groups than in those of the Can-->Soy and Soy-->Soy groups. Although Can is nutritionally detrimental to SHRSP rats compared with Soy, no direct evidence has been obtained thus far relating these observations to human nutrition.

Prenatal exposure to nicotine modifies kidney weight and blood pressure in genetically susceptible rats: a case of gene-environment interaction.

BACKGROUND: Epidemiologic studies suggest that in utero exposure to maternal smoking is associated with elevated blood pressure (BP) later in life. Our aims were: (1) to examine effects of intrauterine exposure to nicotine on BP and hypertension target-organ size in rats; and (2) to investigate whether such effects depend on genetic background, by studying two genetically distinct strains of rats: the spontaneously hypertensive rat (SHR) and the normotensive Brown Norway (BN) rat. METHODS: Nicotine or saline was administered to dams via subcutaneous osmotic minipumps throughout gestation. In nine-week-old male offspring, we measured BP and heart rate, assessed the weight of kidneys and heart, and determined fasting levels of glucose, insulin, triglycerides (TG), and cholesterol. We also measured gene expression of the insulin-like growth factor (IGF) system in the liver and kidneys. RESULTS: SHR and BN offspring differed in their response to intrauterine exposure to nicotine. SHR exposed to nicotine (vs. saline) exhibited higher BP (P < 0.02) and serum cholesterol levels (P = 0.01), and lower kidney weight (P < 0.0001). In contrast, BN rats did not demonstrate differences between the nicotine and saline groups in these variables, but the nicotine-exposed BN rats showed a significant up-regulation in the gene expression of IGF-1 in the liver (P < 0.0001) and IGF receptor in the kidney (P = 0.006). CONCLUSION: These results suggest that intrauterine exposure to nicotine alters the cardiovascular system depending on the genetic background and, as such, supports the notion that the intrauterine environment interacts with genes in determining an individual's health later in life.

Up-regulation of cyr61 in vascular smooth muscle cells of spontaneously hypertensive rats.

In the present study, we applied a fluorescent differential display method to mRNAs from aortae of spontaneously hypertensive rats (SHRs), stroke-prone spontaneously hypertensive rats (SPSHRs), and their parental strain, Wistar Kyoto rats (WKYRs), to identify the genes involved in the development of hypertension. Through this screen we came across a gene that is consistently up-regulated in hypertensive rats. Nucleotide sequence determination of the corresponding cDNA revealed that the gene is the rat orthologue of cyr61. Northern blot analysis showed that cyr61 expression increases in SHR and SPSHR before the onset of hypertension and is sustained thereafter at higher levels than in age-matched WKYRs. In situ hybridization analysis demonstrated that cyr61 is expressed strongly in smooth muscle cells (SMCs) in media of SHR and SPSHR but not WKYR aorta. Fluorescent in situ hybridization mapped the cyr61 gene to rat chromosome 1p12-13, which is located in close proximity to a recently defined quantitative trait locus including NHE3 Na(+)/H(+) exchanger. Overexpression of the cyr61 gene in stably transfected rat SMC line A7r5 caused rather inhibitory effects on the proliferation and DNA and protein synthesis. Our results thus demonstrate for the first time that cyr61 can also act as a growth inhibitor in SMC of genetically hypertensive rats. This may reveal a new route for investigation of the pathogenesis of hypertension.

A missense mutation in the Abcg5 gene causes phytosterolemia in SHR, stroke-prone SHR, and WKY rats.

Sitosterolemia is an autosomal recessive disorder caused by mutations in the ABCG5 or ABCG8 half-transporter genes. These mutations disrupt the mechanism that distinguishes between absorbed sterols and is most prominently characterized by hyperabsorption and impaired biliary elimination of dietary plant sterols. Sitosterolemia patients retain 15-20% of dietary plant sterols, whereas normal individuals absorb less than 1-5%. Normotensive Wistar Kyoto inbred (WKY inbred), spontaneously hypertensive rat (SHR), and stroke-prone spontaneously hypertensive rat (SHRSP) strains also display increased absorption and decreased elimination of dietary plant sterols. To determine if the genes responsible for sitosterolemia in humans are also responsible for phytosterolemia in rats, we sequenced the Abcg5 and Abcg8 genes in WKY inbred, SHR, and SHRSP rat strains. All three strains possessed a homozygous guanine-to-thymine transversion in exon 12 of the Abcg5 gene that results in the substitution of a conserved glycine residue for a cysteine amino acid in the extracellular loop between the fifth and sixth membrane-spanning domains of the ATP binding cassette half-transporter, sterolin-1. The identification of this naturally occurring mutation confirms that these rat strains are important animal models of sitosterolemia in which to study the mechanisms of sterol trafficking.

Molecular basis of the Cd36 chromosomal deletion underlying SHR defects in insulin action and fatty acid metabolism.

The human insulin resistance syndromes---type 2 diabetes, obesity, combined hyperlipidemia, and essential hypertension---are genetically complex disorders whose molecular basis is largely unknown. The spontaneously hypertensive rate (SHR) is a model of these human syndromes. In the SHR/NCrlBR strain, a chromosomal deletion event that occurred at the Cd36 locus during the evolution of this SHR strain has been proposed as a cause of defective insulin action and fatty acid metabolism. In this study, three copies of the Cd36 gene, one transcribed copy and two pseudogenes, were identified in normal rat strains, but only a single gene in SHR/NCrlBR. Analysis of SHR genomic sequence localized the chromosomal deletion event between intron 4 of the normally transcribed copy of the gene and intron 4 of the second pseudogene. The deletion led to the creation of a single chimeric Cd36 gene in SHR/NCrlBR. The boundaries of the recombination/deletion junction identified within intron 4 were surrounded by long interspersed nuclear elements (LINEs) and DNA topoisomerase I recognition sequences. An 8-bp deletion at the intron 14/exon 15 boundary of the second pseudogene abolishes the putative splice acceptor site and is the cause of an aberrant 3' UTR previously observed in SHR/NCrlBR. We conclude that in SHR/NCrlBR, the complex trait of insulin resistance and defective fatty acid metabolism is caused by Cd36 deficiency, resulting from a chromosomal deletion caused by unequal recombination. This demonstrates that chromosomal deletions caused by unequal recombination can be a cause of quantitative or complex mammalian phenotypes.

Genetics of Cd36 and the hypertension metabolic syndrome.

Although genetic mapping of quantitative trait loci (QTL) for complex traits related to hypertension is relatively straightforward, the identification of QTL at the molecular level has proven far more difficult. By combining techniques of gene mapping and gene expression profiling with studies in congenic and transgenic strains, a specific molecular defect in the Cd36 fatty acid transporter has been identified that contributes to the pathogenesis of 2 complex traits in the spontaneously hypertensive rat (SHR), namely, insulin resistance and disordered fatty acid metabolism. After mapping QTL linked to insulin resistance and dyslipidemia to the telomeric region of SHR chromosome 4, gene expression studies were used to identify candidate genes within the target chromosome segment that were differentially expressed in white adipose tissue between SHR congenic strains. This led to the identification of a major mutation in the SHR gene encoding Cd36, a fatty acid transporter involved in the transmembrane transport of long-chain fatty acids. The role for mutant Cd36 in the pathogenesis of insulin resistance and dyslipidemia was confirmed by rescuing the abnormal metabolic phenotypes through transgenic expression of wild-type Cd36 on the SHR background. These findings show that a primary defect in fatty acid transport can promote disordered carbohydrate metabolism in the SHR and show the power of advanced genome technologies for identifying QTL at the molecular level.