Reduction of Stabilin-2 Contributes to a Protection Against Atherosclerosis.

We have previously identified a novel atherosclerosis quantitative trait locus (QTL), Arch atherosclerosis 5 (Aath5), on mouse chromosome 10 by three-way QTL analyses between Apoe -/- mice on a DBA/2J, 129S6 and C57BL/6J background. The DBA/2J haplotype at the Aath5 locus was associated with smaller plaque size. One of the candidate genes underlying Aath5 was Stabilin-2 (Stab2), which encodes a clearance receptor for hyaluronan (HA) predominantly expressed in liver sinusoidal endothelial cells (LSECs). However, the role of Stab2 in atherosclerosis is unknown. A congenic line of Apoe -/- mice carrying Aath5 covering the Stab2 DBA allele on a background of 129S6 confirmed the small reductions of atherosclerotic plaque development. To further determine whether Stab2 is an underlying gene for Aath5, we generated Stab2 -/- Apoe -/- mice on a C57BL/6J background. When fed with a Western diet for 8 weeks, Stab2 -/- Apoe -/- males developed approximately 30% smaller plaques than Stab2 +/+ Apoe -/- mice. HA was accumulated in circulation but not in major organs in the Stab2 deficient mice. STAB2-binding molecules that are involved in atherosclerosis, including acLDL, apoptotic cells, heparin and vWF were not likely the direct cause of the protection in the Stab2 -/- Apoe -/- males. These data indicate that reduction of Stab2 is protective against atherosclerotic plaque development, and that Stab2 is a contributing gene underlying Aath5, although its effect is small. To test whether non-synonymous amino acid changes unique to DBA/2J affect the function of STAB2 protein, we made HEK293 cell lines expressing STAB2129 or STAB2DBA proteins, as well as STAB2129 proteins carrying each of five DBA-unique replacements that have been predicted to be deleterious. These mutant cells were capable of internalizing 125I -HA and DiI-acLDL similarly to the control cells. These results indicate that the amino acid changes unique to DBA/2J are not affecting the function of STAB2 protein, and support our previous observation that the reduced transcription of Stab2 in the liver sinusoid as a consequence of the insertion of a viral-derived sequence, intracisternal A particle, is the primary contributor to the athero-protection conferred by the DBA/2J allele.

Cyanocobalamin prevents cardiomyopathy in type 1 diabetes by modulating oxidative stress and DNMT-SOCS1/3-IGF-1 signaling.

Patients with long-standing diabetes have a high risk for cardiac complications that is exacerbated by increased reactive oxygen species (ROS) production. We found that feeding cyanocobalamin (B12), a scavenger of superoxide, not only prevented but reversed signs of cardiomyopathy in type 1 diabetic Elmo1H/H Ins2Akita/+ mice. ROS reductions in plasma and hearts were comparable to those in mice treated with other antioxidants, N-acetyl-L-cysteine or tempol, but B12 produced better cardioprotective effects. Diabetes markedly decreased plasma insulin-like growth factor (IGF)-1 levels, while B12, but not N-acetyl-L-cysteine nor tempol, restored them. B12 activated hepatic IGF-1 production via normalization of S-adenosylmethionine levels, DNA methyltransferase (DNMT)-1/3a/3b mRNA, and DNA methylation of promoters for suppressor of cytokine signaling (SOCS)-1/3. Reductions of cardiac IGF-1 mRNA and phosphorylated IGF-1 receptors were also restored. Thus, B12 is a promising option for preventing diabetic cardiomyopathy via ROS reduction and IGF-1 retrieval through DNMT-SOCS1/3 signaling.

Beneficial effects of nicotinamide on hypertensive mice with impaired endothelial nitric oxide function.

Nicotinamide (Nam, amide form of niacin acid or nicotinate), a precursor for nicotinamide adenine dinucleotide (NAD+), is important for normal physiological function of organisms. Nam also suppresses mobilization of Ca2+ from sarcoplasmic reticulum into cytoplasm through inhibiting ADP-ribose cyclase. Previously, we have demonstrated that a pharmacological dose of Nam normalizes maternal blood pressure in mouse models of preeclampsia, a pregnancy related hypertensive disorder. We hypothesized that Nam could decrease blood pressure in hypertensive conditions unrelated to pregnancy. Nam at a dose of 500 mg/kg/day was given to wild type (WT) mice treated with L-NAME, endothelial nitric oxide synthase (eNOS)-null and renin transgenic (Renin-Tg) mice via drinking water. Blood pressure was measured by tail-cuff at different stages of treatment. The function and structure of kidneys of WT mice with L-NAME were determined at the end of the study. The gene expression of markers of inflammation and fibrosis in the kidneys of WT mice with L-NAME was also measured. Nam effectively prevented increase in blood pressure in L-NAME treated mice and decreased elevated blood pressure in eNOS-null mice. However, it did not alter high blood pressure in Renin-Tg mice. Nam prevented increase in urinary albumin excretion and collagen deposit in kidneys of WT mice treated with L-NAME. In addition, Nam significantly decreased the mRNA levels of the markers of inflammation and fibrosis in the kidneys of WT mice treated with L-NAME. Nam may execute beneficial effects on hypertensive conditions associated with eNOS dysfunction via suppressing inflammation. Because Nam is generally regarded as safe in humans, it merits further evaluation for the tailored treatment for the subgroup of hypertensive cases associated with impaired eNOS system.

Oral high dose vitamin B12 decreases renal superoxide and post-ischemia/reperfusion injury in mice.

Renal ischemia/reperfusion injury (IRI) is a leading cause of acute kidney injury (AKI), a potentially fatal syndrome characterized by a rapid decline in kidney function. Excess production of superoxide contributes to the injury. We hypothesized that oral administration of a high dose of vitamin B12 (B12 - cyanocobalamin), which possesses a superoxide scavenging function, would protect kidneys against IRI and provide a safe means of treatment. Following unilateral renal IR surgery, C57BL/6J wild type (WT) mice were administered B12 via drinking water at a dose of 50 mg/L. After 5 days of the treatment, plasma B12 levels increased by 1.2-1.5x, and kidney B12 levels increased by 7-8x. IRI mice treated with B12 showed near normal renal function and morphology. Further, IRI-induced changes in RNA and protein markers of inflammation, fibrosis, apoptosis, and DNA damage response (DDR) were significantly attenuated by at least 50% compared to those in untreated mice. Moreover, the presence of B12 at 0.3 µM in the culture medium of mouse proximal tubular cells subjected to 3 hr of hypoxia followed by 1 hr of reperfusion in vitro showed similar protective effects, including increased cell viability and decreased reactive oxygen species (ROS) level. We conclude that a high dose of B12 protects against perfusion injury both in vivo and in vitro without observable adverse effects in mice and suggest that B12 merits evaluation as a treatment for I/R-mediated AKI in humans.

Engulfment and cell motility protein 1 potentiates diabetic cardiomyopathy via Rac-dependent and Rac-independent ROS production.

Engulfment and cell motility protein 1 (ELMO1) is part of a guanine nucleotide exchange factor for Ras-related C3 botulinum toxin substrate (Rac), and ELMO1 polymorphisms were identified to be associated with diabetic nephropathy in genome-wide association studies. We generated a set of Akita Ins2C96Y diabetic mice having 5 graded cardiac mRNA levels of ELMO1 from 30% to 200% of normal and found that severe dilated cardiomyopathy develops in ELMO1-hypermorphic mice independent of renal function at age 16 weeks, whereas ELMO1-hypomorphic mice were completely protected. As ELMO1 expression increased, reactive oxygen species indicators, dissociation of the intercalated disc, mitochondrial fragmentation/dysfunction, cleaved caspase-3 levels, and actin polymerization increased in hearts from Akita mice. Cardiomyocyte-specific overexpression in otherwise ELMO1-hypomorphic Akita mice was sufficient to promote cardiomyopathy. Cardiac Rac1 activity was positively correlated with the ELMO1 levels, and oral administration of a pan-Rac inhibitor, EHT1864, partially mitigated cardiomyopathy of the ELMO1 hypermorphs. Disrupting Nox4, a Rac-independent NADPH oxidase, also partially mitigated it. In contrast, a pan-NADPH oxidase inhibitor, VAS3947, markedly prevented cardiomyopathy. Our data demonstrate that in diabetes mellitus ELMO1 is the "rate-limiting" factor of reactive oxygen species production via both Rac-dependent and Rac-independent NADPH oxidases, which in turn trigger cellular signaling cascades toward cardiomyopathy.

Nicotinamide benefits both mothers and pups in two contrasting mouse models of preeclampsia.

Preeclampsia (PE) complicates ∼5% of human pregnancies and is one of the leading causes of pregnancy-related maternal deaths. The only definitive treatment, induced delivery, invariably results in prematurity, and in severe early-onset cases may lead to fetal death. Many currently available antihypertensive drugs are teratogenic and therefore precluded from use. Nonteratogenic antihypertensives help control maternal blood pressure in PE, but results in preventing preterm delivery and correcting fetal growth restriction (FGR) that also occurs in PE have been disappointing. Here we show that dietary nicotinamide, a nonteratogenic amide of vitamin B3, improves the maternal condition, prolongs pregnancies, and prevents FGR in two contrasting mouse models of PE. The first is caused by endotheliosis due to excess levels in the mothers of a soluble form of the receptor for vascular endothelial growth factor (VEGF), which binds to and inactivates VEGF. The second is caused by genetic absence of Ankiryn-repeat-and-SOCS-box-containing-protein 4, a factor that contributes to the differentiation of trophoblast stem cells into the giant trophoblast cells necessary for embryo implantation in mice; its absence leads to impaired placental development. In both models, fetal production of ATP is impaired and FGR is observed. We show here that nicotinamide decreases blood pressure and endotheliosis in the mothers, probably by inhibiting ADP ribosyl cyclase (ADPRC), and prevents FGR, probably by normalizing fetal ATP synthesis via the nucleotide salvage pathway. Because nicotinamide benefits both dams and pups, it merits evaluation for preventing or treating PE in humans.

Endothelin-1 critically influences cardiac function via superoxide-MMP9 cascade.

We have generated low-expressing and high-expressing endothelin-1 genes (L and H) and have bred mice with four levels of expression: L/L, ∼20%; L/+, ∼65%; +/+ (wild type), 100%; and H/+, ∼350%. The hypomorphic L allele can be spatiotemporally switched to the hypermorphic H allele by Cre-loxP recombination. Young adult L/L and L/+ mice have dilated cardiomyopathy, hypertension, and increased plasma volumes, together with increased ventricular superoxide levels, increased matrix metalloproteinase 9 (Mmp9) expression, and reduced ventricular stiffness. H/+ mice have decreased plasma volumes and significantly heavy stiff hearts. Global or cardiomyocyte-specific switching expression from L to H normalized the abnormalities already present in young adult L/L mice. An epithelial sodium channel antagonist normalized plasma volume and blood pressure, but only partially corrected the cardiomyopathy. A superoxide dismutase mimetic made superoxide levels subnormal, reduced Mmp9 overexpression, and substantially improved cardiac function. Genetic absence of Mmp9 also improved cardiac function, but increased superoxide remained. We conclude that endothelin-1 is critical for maintaining normal contractile function, for controlling superoxide and Mmp9 levels, and for ensuring that the myocardium has sufficient collagen to prevent overstretching. Even a modest (∼35%) decrease in endothelin-1 gene (Edn1) expression is sufficient to cause cardiac dysfunction.

Primary aldosteronism and impaired natriuresis in mice underexpressing TGFß1.

To uncover the potential cardiovascular effects of human polymorphisms influencing transforming growth factor ß1 (TGFß1) expression, we generated mice with Tgfb1 mRNA expression graded in five steps from 10% to 300% normal. Adrenal expression of the genes for mineralocorticoid-producing enzymes ranged from 50% normal in the hypermorphs at age 12 wk to 400% normal in the hypomorphs accompanied with proportionate changes in plasma aldosterone levels, whereas plasma volumes ranged from 50% to 150% normal accompanied by marked compensatory changes in plasma angiotensin II and renin levels. The aldosterone/renin ratio ranged from 0.3 times normal in the 300% hypermorphs to six times in the 10% hypomorphs, which have elevated blood pressure. Urinary output of water and electrolytes are markedly decreased in the 10% hypomorphs without significant change in the glomerular filtration rate. Renal activities for the Na(+), K(+)-ATPase, and epithelial sodium channel are markedly increased in the 10% hypomorphs. The hypertension in the 10% hypomorphs is corrected by spironolactone or amiloride at doses that do not change blood pressure in wild-type mice. Thus, changes in Tgfb1 expression cause marked progressive changes in multiple systems that regulate blood pressure and fluid homeostasis, with the major effects being mediated by changes in adrenocortical function.

eNOS deficiency acts through endothelin to aggravate sFlt-1-induced pre-eclampsia-like phenotype.

Excess soluble fms-like tyrosine kinase 1 (sFlt-1) of vascular endothelial growth factor receptor 1 secreted from the placenta causes pre-eclampsia-like features by antagonizing vascular endothelial growth factor signaling, which can lead to reduced endothelial nitric oxide synthase (eNOS) activity; the effect of this concomitant decrease in eNOS activity is unknown. We tested whether the decrease in nitric oxide occurring in female mice lacking eNOS aggravates the pre-eclampsia-like phenotype induced by increased sFlt-1. Untreated eNOS-deficient female mice had higher BP than wild-type mice. Adenovirus-mediated overexpression of sFlt-1 increased systolic BP by approximately 27 mmHg and led to severe loss of fenestration of glomerular capillary endothelial cells in both eNOS-deficient and wild-type mice. However, only the eNOS-deficient sFlt-1 mice exhibited severe foot process effacement. Compared with wild-type sFlt-1 mice, eNOS-deficient sFlt-1 mice also showed markedly higher urinary albumin excretion (467±74 versus 174±23 µg/d), lower creatinine clearance (126±29 versus 452±63 µl/min), and more severe endotheliosis. Expression of preproendothelin-1 (ET-1) and its ET(A) receptor in the kidney was higher in eNOS-deficient sFlt-1 mice than in wild-type sFlt-1 mice. Furthermore, the selective ET(A) receptor antagonist ambrisentan attenuated the increases in BP and urinary albumin excretion and ameliorated endotheliosis in both wild-type and eNOS-deficient sFlt-1 mice. Ambrisentan improved creatinine clearance and podocyte effacement in eNOS-deficient sFlt-1 mice. In conclusion, reduced maternal eNOS/nitric oxide exacerbates the sFlt1-related pre-eclampsia-like phenotype through activation of the endothelin system.

Relationship between hemodynamics and atherosclerosis in aortic arches of apolipoprotein E-null mice on 129S6/SvEvTac and C57BL/6J genetic backgrounds.

OBJECTIVE: We investigated the relationships between hemodynamics and differential plaque development at the aortic arch of apolipoprotein E (apoE)-null mice on 129S6/SvEvTac (129) and C57BL/6J (B6) genetic backgrounds. METHODS: Mean flow velocities at the ascending and descending aorta (mVAA and mVDA) were measured by Doppler ultrasound in wild type and apoE-null male mice at 3 and 9 months of age. Following dissection of the aortic arches, anatomical parameters and plaque areas were evaluated. RESULTS: Arch plaques were five times bigger in 129-apoE than in B6-apoE mice at 3 months, and twice as large at 9 months. The geometric differences, namely larger vessel diameter in the B6 strain and broader inner curvature of the aortic arch in the 129 strain, were exaggerated in 9-month-old apoE-null mice. Cardiac output and heart rate under anesthesia were significantly higher in the B6 strain than in the 129 strain. The values of mVAA were similar in the two strains, while mVDA was lower in the 129 strain. However, there was a 129-apoE-specific reduction of flow velocities with age, and both mVAA and mVDA were significantly lower in 129-apoE than in B6-apoE mice at 9 months. The mean relative wall shear stress (rWSS) over the aortic arch in 129-apoE and B6-apoE mice were not different, but animals with lower mean rWSS had larger arch plaques within each strain. CONCLUSIONS: The plaque formation in the arch of apoE-null mice is accompanied by strain-dependent changes in both arch geometry and hemodynamics. While arch plaque sizes negatively correlate with mean rWSS, additional factors are necessary to account for the strain differences in arch plaque development.

Differences in aortic arch geometry, hemodynamics, and plaque patterns between C57BL/6 and 129/SvEv mice.

Atherosclerotic plaques are distributed differently in the aortic arches of C57BL/6 (B6) and 129/SvEv (129) apolipoprotein E (apoE)-deficient mice. It is now recognized that hemodynamic wall shear stress (WSS) plays an important role in the localization of atherosclerotic development. Since the blood flow field in the vessel is modulated by the vascular geometry, we quantitatively examined the difference in the aortic arch geometry and hemodynamic WSS between the two corresponding wild-type mouse strains. The three-dimensional (3D) geometry of 14 murine aortic arches, seven from each strain, was characterized using casts and stereo microscopic imaging. Based on the geometry of each cast, an average 3D geometry of the aortic arch for each mouse strain was obtained, and computational fluid dynamic calculations were performed in the two average aortic arches. Many geometric features, including aortic arch shape, vessel diameter, and branch locations, were significantly different at p<0.05 between the two mouse strains. Lower shear stress was found at the inner curvature of the aortic arch in the 129 strain, corresponding to greater involvement in the corresponding apoE-deficient mice relative to the B6 strain. These results support the notion that heritable features of arterial geometry can contribute to individual differences in local susceptibility to arterial disease.

Discordant on/off switching of gene expression in myocytes during cardiac hypertrophy in vivo.

To determine whether the expression of cardiac genes changes in a graded manner or by on/off switching when cardiac myocytes change genetic programs in living animals, we have studied two indicator genes that change their expression oppositely in mouse binucleate ventricular cardiomyocytes during development and in response to cardiac hypertrophy. One is a single-copy transgene controlled by an alpha-myosin heavy chain (aMHC) promoter and coding for CFP. The other is the endogenous beta-myosin heavy chain (bMHC) gene modified to code for a YFP-bMHC fusion protein. Using high-resolution confocal microscopy, we determined the expression of the two indicator genes in individual cardiomyocytes perinatally and after inducing cardiac hypertrophy by transverse aortic constriction. Our results provide strong evidence that the cardiac genes respond by switching their expression in an on/off rather than graded manner, and that responding genes within a single cell and within the two nuclei of cardiomyocytes do not necessarily switch concordantly.

Salt-sensitive blood pressure in mice with increased expression of aldosterone synthase.

To study the effects of modestly increased expression of aldosterone synthase (AS), we generated mice (AS(hi/hi)) by replacing the 3' untranslated region of AS mRNA with that from a stable mRNA. AS(hi/hi) mice on a normal-salt diet had 1.5 times the wild-type AS mRNA in adrenals, although their blood pressure and plasma aldosterone did not differ from wild-type mice. Changes in dietary salt did not affect the blood pressure of wild-type mice, but AS(hi/hi) mice had approximately 10-mm Hg higher blood pressure on a high-salt diet than on a low-salt diet and than wild-type mice on either diet. The AS(hi/hi) mice on a high-salt diet also had higher plasma aldosterone, lower plasma potassium, and greater renal expression of the alpha subunit of epithelial sodium channel compared with wild-type mice. The AS(hi/hi) mice on a high-salt diet also had more water intake and urine volume and less urine osmolality than wild-type mice. On a low-salt diet, AS(hi/hi) mice maintained normal blood pressure with less activation of the renin-angiotensin-aldosterone system than wild-type mice. The AS(hi/hi) mice also had less water intake and urine volume and higher urine osmolality than wild-type mice. On a medium high-salt diet, AS(hi/hi) mice were more susceptible than wild-type mice to infusion of angiotensin II, having a higher blood pressure, greater cardiac hypertrophy, and increased oxidative stress. Thus, a modest increase in AS expression makes blood pressure more sensitive to salt, suggesting that genetically increased AS expression in humans may contribute to hypertension and cardiovascular complications in societies with high-salt diets.

In vivo response to vascular injury in the absence of factor IX: examination in factor IX knockout mice.

INTRODUCTION: Recently, in vitro models of coagulation have called into question the traditional conception of Factor IX as an intrinsic pathway protein, essential to propagation of coagulation but not central to the initiation of hemostatic plug, which has been thought instead to involve TF/FVIIa interactions with factor X and platelets. We hypothesized that the activation of factor IX, and its role in a factor IXa/FVIIa "tenase" complex leading to thrombin generation, plays a more important role than that of TF/FVIIa complex activation of factor X in the early hemostatic response to vascular injury. In vivo modeling is possible because of the generation of factor IX(-/-) mice. MATERIALS AND METHODS: We used two models of arterial vascular injury, histological examination following mechanical carotid artery disruption and intravital microscopy of a mesenteric arteriole subsequent to ferric chloride arteriolar injury to examine mice having complete deficiency of factor IX (FIX(-/-)). RESULTS: Both injury models demonstrate that platelet rich thrombi /hemostatic plug in FIX(-/-) mice is dramatically reduced as compared to wild type mice under conditions of high shear; in fact, no platelet thrombi (>20 mum) were observed in the intravital experiments. Interestingly, the platelet defect is more striking than that described in mice lacking fibrinogen and/or von Willebrand factor. CONCLUSIONS: The results suggest TF/FVIIa-->FX pathway is insufficient for effective platelet aggregation in the presence of high flow, requiring factor IX at the convergence of both intrinsic and extrinsic pathways. Following platelet adhesion, factor IX is required for normal platelet aggregation in vivo, as well as thrombin generation and propagation of occlusive thrombus at the site of vascular injury.

Adrenomedullin gene expression differences in mice do not affect blood pressure but modulate hypertension-induced pathology in males.

Adrenomedullin (AM) is a potent vasodilator peptide in plasma at picomolar levels. Polymorphisms in the human AM gene have been associated with genetic predisposition to diabetic nephropathy and proteinuria with essential hypertension, and numerous studies have demonstrated that endogenous AM plays a role in protecting the heart and kidneys from fibrosis resulting from cardiovascular disease. Elevated plasma levels of AM are associated with pregnancy and sepsis and with cardiovascular stress and hypertension. However, there are no reports of the effects of genetic differences in the expression of the endogenous AM gene and of gender on blood pressure in these circumstances or on the pathological changes accompanying hypertension. To address these questions, we have generated mice having genetically controlled levels of AM mRNA ranging from approximately 50% to approximately 140% of wild-type levels. These modest changes in AM gene expression have no effect on basal blood pressure. Although pregnancy and sepsis increase plasma AM levels, genetically reducing AM production does not affect the transient hypotension that occurs during normal pregnancy or that is induced by treatment with lipopolysaccharide. Nor does the reduction of AM affect chronic hypertension caused by a renin transgene. However, 50% normal expression of AM enhances cardiac hypertrophy and renal damage in male, but not female, mice with a renin transgene. These observations suggest that the effect of gender on the role of AM in counteracting cardiovascular damage in humans merits careful evaluation.

Anatomical differences and atherosclerosis in apolipoprotein E-deficient mice with 129/SvEv and C57BL/6 genetic backgrounds.

There are well-known genetic background effects on atherosclerosis susceptibility in mice. To study the basis of these effects, we have generated the apolipoprotein E-null mutation in mouse embryonic stem cells of 129/SvEv origin, maintained it in the inbred strain (129-apoE), and compared these mice with those previously made in strain 129/Ola and backcrossed to a C57BL/6 genetic background (B6-apoE). Plasma cholesterol and triglyceride levels in the apoE-129 mice are twice the levels in apoE-B6, and both VLDL/chylomicron remnants and HDL particles are increased. Regression analysis of plaque size relative to the age of mice suggests that the initiation of atherosclerotic plaque development at the aortic root is slower in 129-apoE mice (intercept at 3.9 months in females and 4.1 months in males) than in B6-apoE mice (1.3 months in females and 2.8 months in males). In contrast, 129-apoE mice develop extensive plaques in the aortic arches earlier than B6-apoE mice. Distinct differences in the geometry of the aortic arch between the two strains suggest that anatomical differences may contribute to the effects of genetic background on atherosclerosis. The 129-apoE/B6-apoE pair thus provides a tool to study factors governing the relation between arterial geometry and the location of plaque development.

Characterizing 3-D geometry of mouse aortic arch using light stereo-microscopic imaging.

The vascular geometry may play an important role in the development of atherosclerosis by modulating the local hemodynamics and mechanical stresses of the vessel wall. The mouse is now the most popular animal model to study cardiovascular disease. Here, we present a method to characterize the 3-D geometry of mouse aortic arches by casting and light stereo-microscopic imaging. After calibration of the stereo-microscopic imaging system, the 3-D axis of an aortic cast is reconstructed using two stereo images. Based on the analysis of this 3-D curve, a geometry-based definition of the arch segment is given, and some geometric features, including curvature, torsion, and symmetry, have been derived. Casts from a C57BL6 and a SVEV mouse have been processed. This method will be used to detect the geometric difference of the aortic arch among different mouse strains.

An improved technique for tail-cuff blood pressure measurements with dark-tailed mice.

Study of the genetics of hypertension has been facilitated greatly by the use of mice with modified genes that affect blood pressure. A current successful method for measuring blood pressure in mice relies on detection of light passing through the tail to determine the pressure in a tail-cuff necessary to stop pulsed flow. Success in obtaining reliable blood pressure measurements in light-tailed strains of mice (e.g., C57BL/6J) has been excellent. However, in our and others' experience, mice having highly pigmented tails (e.g., 129S6/SvEvTac) have yielded less consistent measurements. We report here that simple modifications to the channel containing the pulse detection sensor can greatly improve the pulse detection of dark-tailed mice. The first modification--lining the sensor channel with four layers of clear plastic wrap--increased the frequency of successful blood pressure measurements of 129S6/SvEvTac mice twofold and reduced variability by one-third. The second modification--lining the sides of the channel with reflective foil--also improved the success rate with dark-tailed mice. Mean blood pressures were unaffected by these modifications, which enhance detection of the pulse wave and likely will be helpful in diverse applications in which blood pressure is measured in rodent strains with pigmented tails.

Increased susceptibility to heart failure in response to volume overload in mice lacking natriuretic peptide receptor-A gene.

OBJECT: Contribution of the natriuretic peptide system to the development of heart failure (HF) in vivo was examined using mice lacking or having decreased natriuretic peptide receptor-A (NPRA), a guanylyl cyclase-linked receptor. METHODS: Volume-overloaded HF was produced by aortocaval fistula in mice with wild-type (+/+), heterozygous (+/-), and homozygous null mutants (-/-) of the NPRA gene. Severity of HF was assessed 4 weeks after operation on the basis of organ weight, hemodynamics, echocardiographic indices, urinary variables, neurohumoral factors, and myocardial gene expression. RESULTS: There were no significant differences in lung weight, kidney weight, left ventricular end-diastolic pressure (LVEDP), left ventricular systolic function, or urinary variables among the three sham-operated groups; however, sham-operated (-/-) mice had higher blood pressure and individual cardiac chamber weights than did (+/+) mice. In contrast, (-/-) mice with aortocaval fistula had higher LVEDP, left and right ventricular weights, lung weight, and left ventricular dimension, as well as lower fractional shortening and urinary sodium and cyclic guanosine monophosphate (cGMP) excretion than did (+/+) mice with aortocaval fistula. In addition, ventricular mRNA expression of natriuretic peptides and beta-myosin heavy chain increased markedly only in (-/-) mice. Plasma atrial natriuretic peptide, renin, and aldosterone, but not cGMP, showed greater responses to aortocaval fistula in (-/-) mice than in (+/+) mice. Both sham-operated and aortocaval fistula NPRA (+/-) mice almost consistently showed a phenotype intermediate between those of NPRA (-/-) and NPRA (+/+) mice. CONCLUSION: These results provide genetic evidence that NPRA signaling protects against HF induced by volume overload in mice.