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.

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.

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.

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.

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.

Hypertension and abnormal fat distribution but not insulin resistance in mice with P465L PPARgamma.

Peroxisome proliferator-activated receptor gamma (PPARgamma), the molecular target of a class of insulin sensitizers, regulates adipocyte differentiation and lipid metabolism. A dominant negative P467L mutation in the ligand-binding domain of PPARgamma in humans is associated with severe insulin resistance and hypertension. Homozygous mice with the equivalent P465L mutation die in utero. Heterozygous mice grow normally and have normal total adipose tissue weight. However, they have reduced interscapular brown adipose tissue and intra-abdominal fat mass, and increased extra-abdominal subcutaneous fat, compared with wild-type mice. They have normal plasma glucose levels and insulin sensitivity, and increased glucose tolerance. However, during high-fat feeding, their plasma insulin levels are mildly elevated in association with a significant increase in pancreatic islet mass. They are hypertensive, and expression of the angiotensinogen gene is increased in their subcutaneous adipose tissues. The effects of P465L on blood pressure, fat distribution, and insulin sensitivity are the same in both male and female mice regardless of diet and age. Thus the P465L mutation alone is sufficient to cause abnormal fat distribution and hypertension but not insulin resistance in mice. These results provide genetic evidence for a critical role for PPARgamma in blood pressure regulation that is not dependent on altered insulin sensitivity.

Cardiac hypertrophy and sudden death in mice with a genetically clamped renin transgene.

Several mouse models have already proved valuable for investigating hypertrophic responses to cardiac stress. Here, we characterize one caused by a well defined single copy transgene, RenTgMK, that genetically clamps plasma renin and thence angiotensin II at high levels. All of the transgenic males develop concentric cardiac hypertrophy with fibrosis but without dilatation. Over half die suddenly aged 6-8 months. Telemetry showed disturbances in diurnal rhythms a few days before death and, later, electrocardiographic disturbances comparable to those in humans with congestive heart failure. Expression of seven hypertrophy-related genes in this and two categorically different models (lack of atrial natriuretic peptide receptor A; overexpression of calsequestrin) were compared. Statistical analyses show that ventricular expressions of the genes coding for atrial natriuretic peptide, beta myosin heavy chain, medium chain acyl-CoA dehydrogenase, and adrenomedullin correlate equally well with the degree of hypertrophy, although their ranges of expression are, respectively, 50-, 30-, 10-, and 3-fold.

Minireview: computer simulations of blood pressure regulation by the renin-angiotensin system.

Gene targeting experiments in mice have been used by us and others to test whether quantitative changes in gene expression in the renin-angiotensin system affect blood pressure. Surprisingly, these studies showed that blood pressure does not change with mild quantitative changes in the expression of the angiotensin converting enzyme (ACE). Yet, ACE inhibitors are widely used for the treatment of hypertension. This apparent paradox motivated us to develop a simple computer simulation, which qualitatively reconciled the paradox. We have now improved the simulation by including blood pressure as an explicit variable and by adding the kallikrein-kinin system and feedback control of plasma renin via plasma angiotensin II levels. The new simulation now matches quantitative aspects of the experimental data and suggests that a decrease in bradykinin plays an important role in the increased risk of diabetic nephropathy associated with genetically determined higher levels of ACE activity. This emphasizes that the value of these types of simulation lies in the thoughts that they provoke rather than in their ability to replicate experimental data.