Manasi Nandi

The therapeutic potential of targeting endogenous inhibitors of nitric oxide synthesis

Asymmetric dimethylarginine (ADMA) — a naturally occurring amino acid that is a product of protein breakdown — is released into the cytoplasm following the post-translational methylation of arginine residues within proteins and the subsequent proteolysis of these arginine-methylated proteins. ADMA inhibits all three isoforms of nitric oxide synthase and therefore has the potential to produce diverse biological effects, particularly in the cardiovascular system. In addition to its renal clearance, endogenously produced ADMA is metabolized to L-citrulline and dimethylamine by the dimethylarginine dimethylaminohydrolase (DDAH) enzymes. Pharmacological modification of DDAH has therefore been proposed as a mechanism for manipulating endogenous ADMA concentrations and regulating the production of nitric oxide in situations where alterations in nitric oxide signalling have been shown to contribute to pathophysiology. This Review describes the biology of ADMA and the potential therapeutic utility of manipulating DDAH activity.

Pulmonary Hypertension in a GTP-Cyclohydrolase 1–Deficient Mouse

Background—GTP-cyclohydrolase 1 (GTP-CH1) catalyzes the first step for the de novo production of tetrahydrobiopterin (BH4), a cofactor for nitric oxide synthase (NOS). The hyperphenylalaninemic mutant mouse (hph-1) displays a 90% reduction in GTP-CH1 activity. Reduced BH4 decreases NOS activity and may lead to endothelial dysfunction, and there is increasing evidence that a dysfunction of the NOS pathway may be implicated in pulmonary hypertension. The aim of the study was to investigate whether reduced BH4 in the hph-1 mouse results in a pulmonary hypertensive phenotype. Methods and Results—Morphological characterization of the heart, lung, and kidney and measurements of systemic and right ventricular blood pressures were performed in both hph-1 and wild-type mice. BH4 and NOx levels were also measured. Hph-1 mice had significantly lower NOx and BH4 levels, consistent with previous findings. Both morphological and in vivo data were indicative of a pulmonary but not systemic hypertensive phenotype. We observed increased right ventricle–left ventricle plus septum ratios attributable only to an increase in right ventricular mass, increased smooth muscle medial area in pulmonary resistance vessels, and significantly higher right ventricular pressures in vivo. There were no significant differences between left ventricular masses and systemic pressures, and there was no observed evidence of systemic hypertension in kidney sections between wild-type and hph-1. Conclusions—This study demonstrates that mice deficient in GTP-CH1/BH4 display a pulmonary hypertensive but not systemic hypertensive phenotype.

A Role for TRPV1 in Influencing the Onset of Cardiovascular Disease in Obesity

Obesity induced by Western diets is associated with type 2 diabetes mellitus and cardiovascular diseases, although underlying mechanisms are unclear. We investigated a murine model of diet-induced obesity to determine the effect of transient potential receptor vanilloid 1 (TRPV1) deletion on hypertension and metabolic syndrome. Wild-type and TRPV1 knockout mice were fed normal or high-fat diet from 3 to 15 weeks. High-fat diet-fed mice from both genotypes became obese, with similar increases in body and adipose tissue weights. High-fat diet-fed TRPV1 knockout mice showed significantly improved handling of glucose compared with high-fat diet-fed wild-type mice. Hypertension, vascular hypertrophy, and altered nociception were observed in high-fat diet-fed wild-type but not high-fat diet-fed TRPV1 knockout mice. Wild-type, but not high-fat diet-fed TRPV1 knockout, mice demonstrated remodeling in terms of aortic vascular hypertrophy and increased heart and kidney weight, although resistance vessel responses were similar in each. Moreover, the wild-type mice had significantly increased plasma levels of leptin, interleukin 10 and interleukin 1&bgr;, whereas samples from TRPV1 knockout mice did not show significant increases. Our results do not support the concept that TRPV1 plays a major role in influencing weight gain. However, we identified a role of TRPV1 in the deleterious effects observed with high-fat feeding in terms of inducing hypertension, impairing thermal nociception sensitivity, and reducing glucose tolerance. The observation of raised levels of adipokines in wild-type but not TRPV1 knockout mice is in keeping with TRPV1 involvement in stimulating the proinflammatory network that is central to obesity-induced hypertension and sensory neuronal dysfunction.

Spontaneous contractions of myometrium from humans, non-human primate and rodents are sensitive to selective oxytocin receptor antagonism in vitro

Objectives To determine whether: 1. oxytocin receptor antagonists influence spontaneous contractions of myometrium from humans, non‐human primates and rodents (in vitro), and 2. vasopressin V1a receptor antagonism is important for inhibition of spontaneous contractions in human myometrium.

Genetic and Pharmacological Inhibition of Dimethylarginine Dimethylaminohydrolase 1 Is Protective in Endotoxic Shock

Objective—The overproduction of vascular NO contributes toward the circulatory collapse observed in patients with septic shock. Dimethylarginine dimethylaminohydrolase (DDAH), which has 2 isoforms, metabolizes asymmetrically methylated arginines (asymmetric mono- or di-methylarginine), endogenously produced NO synthase inhibitors. We wished to investigate whether reducing DDAH1 activity, using genetic and pharmacological approaches, is protective during lipopolysaccharide-induced endotoxic shock. Methods and Results—Experiments were conducted in DDAH1 heterozygous knockout mice (DDAH1+/−) or naive rats treated with a synthetic pharmacological DDAH inhibitor (L-257). We demonstrate for the first time that L-257 is DDAH1 selective using recombinant human DDAH proteins. DDAH1 mRNA was expressed in aortic but not macrophage cDNA, and consistent with this expression profile, L-257 selectively inhibited NO production from lipopolysaccharide-treated aorta but not macrophages, in culture. Conscious and anesthetized cardiovascular hemodynamics were monitored using implanted radiotelemetry devices or invasive catheters, respectively. Lipopolysaccharide was administered intravenously to model endotoxemia, and all animals presented with circulatory shock. DDAH1+/− mice or L-257–treated rats displayed attenuation in the rate of developed hypotension compared with wild-type littermates or vehicle control animals, respectively. Conclusion—Pharmacological and genetic reduction of DDAH1 activity is protective against the vascular changes observed during endotoxic shock.

Adenoviral-mediated overexpression of DDAH improves vascular tone regulation

Dimethylarginine dimethylaminohydrolase (DDAH) degrades asymmetric dimethylarginine (ADMA), an endogenously produced nitric oxide (NO) synthase inhibitor. In mammals, two isoforms of DDAH, DDAH1 and DDAH2, are expressed in the cardiovascular system, suggesting that ADMA concentrations are actively regulated in blood vessels, raising the possibility that cardiovascular metabolism of ADMA constitutes a novel mechanism for the regulation of NO production. The purpose of this study was to determine the role of DDAH-catalyzed asymmetric methylarginine metabolism in the regulation of vascular function. We developed adenoviral vectors for the expression of human DDAH1 and 2. Overexpression of DDAH1 or 2 in human umbilical vein endothelial cells (HUVEC) increases DDAH activity, reduces ADMA concentrations and increases NO production. Similarly, overexpression of DDAH1 or 2 in DDAH1+/ — mice carotid vessels increases NO production and attenuates the response to phenylephrine (PE), enhances acetylcholine (ACh) relaxation and attenuates the effect of exogenously applied ADMA. Finally, overexpression of either DDAH1 or 2 completely reversed the vascular dysfunction seen in DDAH1+/— mice. These data indicate that basal concentrations of ADMA in blood vessels are sufficient to regulate NO production, that increases in the level of either DDAH1 or 2, improves vascular function and that overexpression of either DDAH1 or 2 is sufficient to compensate for life-long exposure to elevated ADMA. Thus, therapeutic manipulation of DDAH expression or activity may represent a novel approach to improve vascular dysfunction in various cardiovascular diseases.

A role for Dimethylarginine Dimethylaminohydrolase 1 (DDAH1) in mammalian development

Nitric oxide has been linked to a number of embryonic processes, yet the role of nitric oxide signalling in development remains largely unknown. Dimethylarginine Dimethylaminohydrolase 1 and 2 (DDAH1/2) catalyse the breakdown of asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide production, and may also have nitric oxide-independent functions. We have generated transgenic mice targeting the DDAH1 and DDAH2 loci. Here we report that homozygous DDAH1 null embryos are generated at low frequency, and do not progress through embryonic development. During normal development DDAH1 RNA is expressed in the left ventricle, cardiac outflow tract and developing vasculature. In contrast, DDAH2 homozygous null mice are viable and fertile, with a normal lifespan. DDAH2 expression is seen in the developing left ventricle and cardiac outflow tract, and additionally in the peripheral nervous system. Both DDAH1 and 2 are expressed in the developing limb buds in patterns overlapping areas with high nitric oxide synthase activity. These expression patterns implicate DDAH1 and DDAH2 in embryonic development, possibly through specific effects on nitric oxide pathways.

Refinement of animal models of sepsis and septic shock.

ABSTRACT This report aims to facilitate the implementation of the Three Rs (replacement, reduction, and refinement) in the use of animal models or procedures involving sepsis and septic shock, an area where there is the potential of high levels of suffering for animals. The emphasis is on refinement because this has the greatest potential for immediate implementation. Specific welfare issues are identified and discussed, and practical measures are proposed to reduce animal use and suffering as well as reducing experimental variability and increasing translatability. The report is based on discussions and submissions from a nonregulatory expert working group consisting of veterinarians, animal technologists, and scientists with expert knowledge relevant to the field.

Developmental Regulation of GTP-CH1 in the Porcine Lung and Its Relationship to Pulmonary Vascular Relaxation

Nitric oxide (NO) plays an important role in lowering pulmonary vascular resistance after birth. However, in persistent pulmonary hypertension of the newborn (PPHN) NO-mediated dilation is dysfunctional. GTP-cyclohydrolase 1 (GTP-CH1) is the rate-limiting enzyme for the biosynthesis of 6R-l-erythro-5,6,7,8-tetrahydrobiopterin (BH4) an essential cofactor for nitric oxide synthase (NOS) activity. Suboptimal levels of BH4 may result in NOS uncoupling and the subsequent generation of harmful superoxide anions. We therefore investigated the functional effects of supplementing BH4 and/or a superoxide dismutase mimetic (MnTMPyP) in porcine intrapulmonary arteries from normal animals and from a porcine model of PPHN. We investigated whether any functional effects of BH4 could be explained by changes in GTP-CH1 expression. Supplementation of BH4 significantly improved endothelium-dependent relaxations in arteries from 3- and 14-d-old healthy animals, whereas no effect was seen in vessels from younger animals and adults. GTP-CH1 protein expression was lowest at 3 and 14 d, suggesting a rate limitation of BH4 at this time. BH4 supplementation alone did not improve the relaxant response to acetylcholine in arteries obtained in a porcine model of PPHN. Furthermore, GTP-CH1 protein expression was normal for age. However, co-treatment with both BH4 and MnTMPyP restored endothelial function. GTP-CH1 is developmentally regulated in the pulmonary vasculature of neonates and this results in a functionally significant limitation of BH4. Although GTP-CH-1/BH4 levels alone do not explain the profound endothelial dysfunction seen in PPHN, increasing NO bioavailability by supplementing BH4 and quenching superoxide may prove to be therapeutically beneficial.

Quantification of microcirculatory blood flow: a sensitive and clinically relevant prognostic marker in murine models of sepsis

Sepsis and sepsis-associated multiorgan failure represent the major cause of mortality in intensive care units worldwide. Cardiovascular dysfunction, a key component of sepsis pathogenesis, has received much research interest, although research translatability remains severely limited. There is a critical need for more comprehensive preclinical sepsis models, with more clinically relevant end points, such as microvascular perfusion. The purpose of this study was to compare microcirculatory blood flow measurements, using a novel application of laser speckle contrast imaging technology, with more traditional hemodynamic end points, as part of a multiparameter monitoring system in preclinical models of sepsis. Our aim, in measuring mesenteric blood flow, was to increase the prognostic sensitivity of preclinical studies. In two commonly used sepsis models (cecal ligation and puncture, and lipopolysaccharide), we demonstrate that blood pressure and cardiac output are compromised postsepsis, but subsequently stabilize over the 24-h recording period. In contrast, mesenteric blood flow continuously declines in a time-dependent manner and in parallel with the development of metabolic acidosis and organ dysfunction. Importantly, these microcirculatory perturbations are reversed by fluid resuscitation, a mainstay intervention associated with improved outcome in patients. These data suggest that global hemodynamics are maintained at the expense of the microcirculation and are, therefore, not sufficiently predictive of outcome. We demonstrate that microcirculatory blood flow is a more sensitive biomarker of sepsis syndrome progression and believe that incorporation of this biomarker into preclinical models will facilitate sophisticated proof-of-concept studies for novel sepsis interventions, providing more robust data on which to base future clinical trials.