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.

GRAF3 serves as a blood volume-sensitive rheostat to control smooth muscle contractility and blood pressure.

Vascular resistance is a major determinant of BP and is controlled, in large part, by RhoA-dependent smooth muscle cell (SMC) contraction within small peripheral arterioles and previous studies from our lab indicate that GRAF3 is a critical regulator of RhoA in vascular SMC. The elevated contractile responses we observed in GRAF3 deficient vessels coupled with the hypertensive phenotype provided a mechanistic link for the hypertensive locus recently identified within the GRAF3 gene. On the basis of our previous findings that the RhoA signaling axis also controls SMC contractile gene expression and that GRAF3 expression was itself controlled by this pathway, we postulated that GRAF3 serves as an important counter-regulator of SMC phenotype. Indeed, our new findings presented herein indicate that GRAF3 expression acts as a pressure-sensitive rheostat to control vessel tone by both reducing calcium sensitivity and restraining expression of the SMC-specific contractile proteins that support this function. Collectively, these studies highlight the potential therapeutic value of GRAF3 in the control of human hypertension.

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.

How It All Began: A Personal History of Gel Electrophoresis.

Arne Tiselius' moving boundary electrophoresis method was still in general use in 1951 when this personal history begins, although zonal electrophoresis with a variety of supporting media (e.g., filter paper or starch grains) was beginning to replace it. This chapter is an account of 10 years of experiments carried out by the author during which molecular sieving gel electrophoresis was developed and common genetic variants of two proteins, haptoglobin and transferrin, were discovered in normal individuals. Most of the figures are images of pages from the author's laboratory notebooks, which are still available, so that some of the excitement of the time and the humorous moments are perhaps apparent. Alkaline gels, acidic gels with and without denaturants, vertical gels, two-dimensional gels and gels with differences in starch concentration are presented. The subtle details that can be discerned in these various gels played an indispensable role in determining the nature of the change in the haptoglobin gene (Hp) that leads to the polymeric series characteristic of Hp 2/Hp 2 homozygotes. Where possible the names of scientific friends who made this saga of gel electrophoresis so memorable and enjoyable are gratefully included.

Characterizing the role of atrial natriuretic peptide signaling in the development of embryonic ventricular conduction system.

Patients born with congenital heart defects frequently encounter arrhythmias due to defects in the ventricular conduction system (VCS) development. Although recent studies identified transcriptional networks essential for the heart development, there is scant information on the mechanisms regulating VCS development. Based on the association of atrial natriuretic peptide (ANP) expression with VCS forming regions, it was reasoned that ANP could play a critical role in differentiation of cardiac progenitor cells (CPCs) and cardiomyocytes (CMs) toward a VCS cell lineage. The present study showed that treatment of embryonic ventricular cells with ANP or cell permeable 8-Br-cGMP can induce gene expression of important VCS markers such as hyperpolarization-activated cyclic nucleotide-gated channel-4 (HCN4) and connexin 40 (Cx40). Inhibition of protein kinase G (PKG) via Rp-8-pCPT-cGMPS further confirmed the role of ANP/NPRA/cGMP/PKG pathway in the regulation of HCN4 and Cx40 gene expression. Additional experiments indicated that ANP may regulate VCS marker gene expression by modulating levels of miRNAs that are known to control the stability of transcripts encoding HCN4 and Cx40. Genetic ablation of NPRA revealed significant decreases in VCS marker gene expression and defects in Purkinje fiber arborisation. These results provide mechanistic insights into the role of ANP/NPRA signaling in VCS formation.

Causative Effects of Genetically Determined High Maternal/Fetal Endothelin-1 on Preeclampsia-Like Conditions in Mice.

Endothelin-1 (ET-1) is implicated in the pathophysiology of preeclampsia. An association between an EDN1 gene polymorphism with high ET-1 and preeclampsia was reported in humans, but their cause and effect relationships have not been defined. We examined the pregnancy effects in mice with a modified Edn1 allele that increases mRNA stability and thus ET-1 production. Heterozygous Edn1H/+ females showed no obvious abnormalities before pregnancy, but when mated with wild-type (WT) males developed a full spectrum of preeclampsia-like phenotypes, including increased systolic blood pressure, proteinuria, glomerular endotheliosis, and intrauterine fetal growth restriction. At 7.5 days post-coitus, the embryos from Edn1H/+ dams, regardless of their Edn1 genotype, lagged 12 hours in development compared with embryos from WT dams, had disoriented ectoplacental cones, and retained high E-cadherin expression. In contrast, WT females mated with Edn1H/+ males, which also carried half of the fetuses with Edn1H/+ genotype, showed a mild systolic blood pressure increase only. These WT dams had 2× higher plasma soluble fms-like tyrosine kinase-1 than WT dams mated with WT males. In human first trimester trophoblast cells, pharmacological doses of ET-1 increased the cellular sFlt1 transcripts and protein secretion via both type A and B ET-1 receptors. Our data demonstrate that high maternal ET-1 production causes preeclampsia-like phenotypes during pregnancy, affecting both initial stage of trophoblast differentiation/invasion and maternal peripheral vasculature during late gestation. High fetal ET-1 production, however, could cause increased soluble fms-like tyrosine kinase-1 in the maternal circulation and contribute to blood pressure elevation.

A tetraethylene glycol coat gives gold nanoparticles long in vivo half-lives with minimal increase in size.

In this study, we describe the experiments determining whether coating gold nanoparticles with tetraethylene glycol (TEG) provides pharmacologically relevant advantages, such as increased serum half-life and resistance to protein adsorption. Monodisperse TEG-coated, NaBH4-reduced gold nanoparticles with a hydrodynamic size comparable to albumin were synthesized by reducing gold chloride with NaBH4 under alkaline conditions in the presence of TEG-SH. The particles were characterized by gel electrophoresis, column chromatography, and transmission electron microscopy. The nanoparticles were subsequently injected intravenously into mice, and their half-lives and final destinations were determined via photometric analysis, light microscopy (LM), and transmission electron microscopy. The TEG particles had a long half-life (~400 minutes) that was not influenced by splenectomy. After 500 minutes of injection, TEG particles were found in kidney proximal tubule cell vesicles and in spleen red and white pulp. The particles induced apoptosis in the spleen red pulp but not in white pulp or the kidney. Some of the TEG particles appeared to have undergone ligand exchange reactions that increased their charge. The TEG particles were shown to be resistant to nonspecific protein adsorption, as judged by gel electrophoresis and column chromatography. These results demonstrate that naturally monodisperse, small-sized gold nanoparticles coated with TEG have long in vivo plasma half-lives, are minimally toxic, and are resistant to protein adsorption. This suggests that a TEG coating should be considered as an alternative to a polyethylene glycol coating, which is polydisperse and of much larger size.

Permeation of macromolecules into the renal glomerular basement membrane and capture by the tubules.

How the kidney prevents urinary excretion of plasma proteins continues to be debated. Here, using unfixed whole-mount mouse kidneys, we show that fluorescent-tagged proteins and neutral dextrans permeate into the glomerular basement membrane (GBM), in general agreement with Ogston's 1958 equation describing how permeation into gels is related to molecular size. Electron-microscopic analyses of kidneys fixed seconds to hours after injecting gold-tagged albumin, negatively charged gold nanoparticles, and stable oligoclusters of gold nanoparticles show that permeation into the lamina densa of the GBM is size-sensitive. Nanoparticles comparable in size with IgG dimers do not permeate into it. IgG monomer-sized particles permeate to some extent. Albumin-sized particles permeate extensively into the lamina densa. Particles traversing the lamina densa tend to accumulate upstream of the podocyte glycocalyx that spans the slit, but none are observed upstream of the slit diaphragm. At low concentrations, ovalbumin-sized nanoparticles reach the primary filtrate, are captured by proximal tubule cells, and are endocytosed. At higher concentrations, tubular capture is saturated, and they reach the urine. In mouse models of Pierson's or Alport's proteinuric syndromes resulting from defects in GBM structural proteins (laminin ß2 or collagen α3 IV), the GBM is irregularly swollen, the lamina densa is absent, and permeation is increased. Our observations indicate that size-dependent permeation into the lamina densa of the GBM and the podocyte glycocalyx, together with saturable tubular capture, determines which macromolecules reach the urine without the need to invoke direct size selection by the slit diaphragm.

Vitamin B3 modulates mitochondrial vulnerability and prevents glaucoma in aged mice.

Glaucomas are neurodegenerative diseases that cause vision loss, especially in the elderly. The mechanisms initiating glaucoma and driving neuronal vulnerability during normal aging are unknown. Studying glaucoma-prone mice, we show that mitochondrial abnormalities are an early driver of neuronal dysfunction, occurring before detectable degeneration. Retinal levels of nicotinamide adenine dinucleotide (NAD+, a key molecule in energy and redox metabolism) decrease with age and render aging neurons vulnerable to disease-related insults. Oral administration of the NAD+ precursor nicotinamide (vitamin B3), and/or gene therapy (driving expression of Nmnat1, a key NAD+-producing enzyme), was protective both prophylactically and as an intervention. At the highest dose tested, 93% of eyes did not develop glaucoma. This supports therapeutic use of vitamin B3 in glaucoma and potentially other age-related neurodegenerations.

Prolactin alters blood pressure by modulating the activity of endothelial nitric oxide synthase.

Increased levels of a cleaved form of prolactin (molecular weight 16 kDa) have been associated with preeclampsia. To study the effects of prolactin on blood pressure (BP), we generated male mice with a single-copy transgene (Tg; inserted into the hypoxanthine-guanine phosphoribosyltransferase locus) that enables inducible hepatic production of prolactin and its cleavage product. The Tg is driven by the indole-3-carbinol (I3C)-inducible rat cytochrome P450 1A1 promoter. When the Tg mice were fed normal chow (NC), plasma prolactin concentrations were comparable to those in female WT mice in the last third of pregnancy, and BP was lower than in WT mice (∼95 mm Hg vs. ∼105 mm Hg). When the Tg mice were fed chow containing IC3, plasma prolactin concentrations increased threefold, BP increased to ∼130 mm Hg, and cardiac function became markedly impaired. IC3 chow did not affect the WT mice. Urinary excretion of nitrite/nitrate and the amount of Ser1177-phosphorylated endothelial nitric oxide (NO) synthase (eNOS) were significantly greater in the Tg mice fed NC than in WT mice, as they are during pregnancy. However, when I3C was fed, these indicators of NO production became significantly less in the Tg mice than in WT mice. The effects of increased plasma prolactin were abolished by a genetic absence of eNOS. Thus, a threefold increase in plasma prolactin is sufficient to increase BP significantly and to markedly impair cardiac function, with effects mediated by NO produced by eNOS. We suggest that pregnant women with abnormally high prolactin levels may need special attention.

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.

High Elmo1 expression aggravates and low Elmo1 expression prevents diabetic nephropathy.

Human genome-wide association studies have demonstrated that polymorphisms in the engulfment and cell motility protein 1 gene (ELMO1) are strongly associated with susceptibility to diabetic nephropathy. However, proof of causation is lacking. To test whether modest changes in its expression alter the severity of the renal phenotype in diabetic mice, we have generated mice that are type 1 diabetic because they have the Ins2(Akita) gene, and also have genetically graded expression of Elmo1 in all tissues ranging in five steps from ∼30% to ∼200% normal. We here show that the Elmo1 hypermorphs have albuminuria, glomerulosclerosis, and changes in the ultrastructure of the glomerular basement membrane that increase in severity in parallel with the expression of Elmo 1. Progressive changes in renal mRNA expression of transforming growth factor ß1 (TGFß1), endothelin-1, and NAD(P)H oxidase 4 also occur in parallel with Elmo1, as do the plasma levels of cystatin C, lipid peroxides, and TGFß1, and erythrocyte levels of reduced glutathione. In contrast, Akita type 1 diabetic mice with below-normal Elmo1 expression have reduced expression of these various factors and less severe diabetic complications. Remarkably, the reduced Elmo1 expression in the 30% hypomorphs almost abolishes the pathological features of diabetic nephropathy, although it does not affect the hyperglycemia caused by the Akita mutation. Thus, ELMO1 plays an important role in the development of type 1 diabetic nephropathy, and its inhibition could be a promising option for slowing or preventing progression of the condition to end-stage renal disease.

A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions.

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like oligomeric clusters of these yellow nanoparticles of narrow size distribution are synthesized under ambient conditions via two methods. The delay-time method controls the number of subunits in the oligoclusters by varying the time between the addition of HAuCl4 to alkaline solution and the subsequent addition of reducing agent, NaSCN. The yellow oligoclusters produced range in size from ~3 to ~25 nm. This size range can be further extended by an add-on method utilizing hydroxylated gold chloride (Na(+)[Au(OH4-x)Clx](-)) to auto-catalytically increase the number of subunits in the as-synthesized oligocluster nanoparticles, providing a total range of 3 nm to 70 nm. The crude oligocluster preparations display narrow size distributions and do not require further fractionation for most purposes. The oligoclusters formed can be concentrated >300 fold without aggregation and the crude reaction mixtures remain stable for weeks without further processing. Because these oligomeric clusters can be concentrated before derivatization they allow expensive derivatizing agents to be used economically. In addition, we present two models by which predictions of particle size can be made with great accuracy.

Transforming growth factor-ß1 and diabetic nephropathy.

Transforming growth factor-ß1 (TGF-ß1) is established to be involved in the pathogenesis of diabetic nephropathy. The diabetic milieu enhances oxidative stress and induces the expression of TGF-ß1. TGF-ß1 promotes cell hypertrophy and extracellular matrix accumulation in the mesangium, which decreases glomerular filtration rate and leads to chronic renal failure. Recently, TGF-ß1 has been demonstrated to regulate urinary albumin excretion by both increasing glomerular permeability and decreasing reabsorption in the proximal tubules. TGF-ß1 also increases urinary excretion of water, electrolytes and glucose by suppressing tubular reabsorption in both normal and diabetic conditions. Although TGF-ß1 exerts hypertrophic and fibrogenic effects in diabetic nephropathy, whether suppression of the function of TGF-ß1 can be an option to prevent or treat the complication is still controversial. This is partly because adrenal production of mineralocorticoids could be augmented by the suppression of TGF-ß1. However, differentiating the molecular mechanisms for glomerulosclerosis from those for the suppression of the effects of mineralocorticoids by TGF-ß1 may assist in developing novel therapeutic strategies for diabetic nephropathy. In this review, we discuss recent findings on the role of TGF-ß1 in diabetic nephropathy.

Low TGFß1 expression prevents and high expression exacerbates diabetic nephropathy in mice.

Nephropathy develops in many but not all patients with long-standing type 1 diabetes. Substantial efforts to identify genotypic differences explaining this differential susceptibility have been made, with limited success. Here, we show that the expression of the transforming growth factor ß1 gene (Tgfb1) affects the development of diabetic nephropathy in mice. To do this we genetically varied Tgfb1 expression in five steps, 10%, 60%, 100%, 150%, and 300% of normal, in mice with type 1 diabetes caused by the Akita mutation in the insulin gene (Ins2(Akita)). Although plasma glucose levels were not affected by Tgfb1 genotype, many features of diabetic nephropathy (mesangial expansion, elevated plasma creatinine and urea, decreased creatinine clearance and albuminuria) were progressively ameliorated as Tgfb1 expression decreased and were progressively exacerbated when expression was increased. The diabetic 10% hypomorphs had comparable creatinine clearance and albumin excretion to wild-type mice and no harmful changes in renal morphology. The diabetic 300% hypermorphs had ∼1/3 the creatinine clearance of wild-type mice, >20× their albumin excretion, ∼3× thicker glomerular basement membranes and severe podocyte effacement, matching human diabetic nephropathy. Switching Tgfb1 expression from low to high in the tubules of the hypomorphs increased their albumin excretion more than 10-fold but creatinine clearance remained high. Switching Tgfb1 expression from low to high in the podocytes markedly decreased creatinine clearance, but minimally increased albumin excretion. Decreasing expression of Tgfb1 could be a promising option for preventing loss of renal function in diabetes.

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.

A conversation with Oliver Smithies.

Professor Oliver Smithies is the Weatherspoon Eminent Distinguished Professor of Pathology and Laboratory Medicine at the University of North Carolina, Chapel Hill. Along with Mario Capecchi and Martin Evans, Oliver was awarded the Nobel Prize in Medicine in Physiology or Medicine in 2007 for his contributions to the development of gene targeting using homologous recombination in embryonic stem cells. This technique has had an immense impact on biomedical research over the past two decades. Professor Smithies has had a long and distinguished career as a researcher and mentor. Here, he provides an entertaining and enlightening discussion of his life in science.

Transforming growth factor beta1 and aldosterone.

PURPOSE OF REVIEW: It is well established that blocking the renin-angiotensin-aldosterone system (RAAS) is effective for the treatment of cardiovascular and renal complications in hypertension and diabetes mellitus. Although the induction of transforming growth factor beta1 (TGFbeta1) by components of the RAAS mediates the hypertrophic and fibrogenic changes in cardiovascular-renal complications, it is still controversial as to whether TGFbeta1 can be a target to prevent such complications. Here, we review recent findings on the role of TGFbeta1 in fluid homeostasis, focusing on the relationship with aldosterone. RECENT FINDINGS: TGFbeta1 suppresses the adrenal production of aldosterone and renal tubular sodium reabsorption. We have generated mice with TGFbeta1 mRNA expression graded in five steps, from 10 to 300% of normal, and found that blood pressure and plasma volume are negatively regulated by TGFbeta1. Notably, the 10% hypomorph exhibits primary aldosteronism and sodium and water retention due to markedly impaired urinary excretion of water and electrolytes. SUMMARY: These results identify TGFbeta signalling as an important counterregulatory system against aldosterone. Understanding the molecular mechanisms for the suppressive effects of TGFbeta1 on adrenocortical and renal function may further our understanding of primary aldosteronism, as well as assist in the development of novel therapeutic strategies for hypertension.