Dimethylarginine Dimethylaminohydrolase Research Articles

Hypertensive effects of transforming growth factor-β1 in vascular smooth muscles cells from spontaneously hypertensive rats are mediated by sulfatase 2.

Extracellular sulfatases (sulfatase 1 and sulfatase 2) mediate up- or down-regulatory effects of cytokines on angiotensin II (Ang II)-induced expression of hypertensive mediators in hypertensive cells. The overproduction of transforming growth factor-β1 (TGF-β1) is associated with chronic hypertension. In this study, we examined the role of extracellular sulfatases on TGF-β1-induced effects associated with the expression of mediators related to hypertension in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR). First, TGF-β1 increased the expression of 12-lipoxygenase (12-LO) and endothelin-1 (ET-1), inhibited dimethylarginine dimethylaminohydrolase-1 (DDAH-1) expression and showed additive effects on Ang II-induced 12-LO and ET-1 expression as well as Ang II-induced inhibition of DDAH-1 expression in SHR VSMCs. However, it had no effect on the expression of 12-LO, ET-1, and DDAH-1 in VSMCs from normotensive Wistar Kyoto rats. Downregulation of sulfatase 2 (Sulf2) inhibited all of these hypertensive effects caused by TGF-β1, while sulfatase 1 (Sulf1) had no effect on these events in SHR VSMCs. All these hypertensive effects of TGF-β1 were dependent on the Ang II subtype 1 receptor (AT1 R) pathway, and not on Ang II subtype 2 receptor (AT2 R). In addition, downregulation of Sulf2 inhibited the expression of TGF-β1-induced AT1 R and the additive effect of TGF-β1 on Ang II-induced AT1 R expression. Additionally, downregulation of Sulf2, but not Sulf1, abrogated TGF-β1-induced inhibition of AMP-activated protein kinase (AMPK) activation and the additive effect of TGF-β1 on Ang II-induced inhibition of AMPK activation via the AT1 R pathway. Moreover, TGF-β1-induced VSMCs proliferation and the additive effect of TGF-β1 on Ang II-induced VSMCs proliferation were abrogated in Sulf2 siRNA-transfected SHR VSMCs, while these effects were maintained in Sulf1 siRNA-transfected SHR VSMCs. The hypertensive effects of TGF-β1 through the AT1 R pathway were mainly dependent on Sulf2 activity in SHR VSMCs. Taken together, these results suggest that Sulf2, but not Sulf1, plays a major role in mediating the increased effects of TGF-β1 in hypertensive VSMCs.

DDAH1 SNP rs480414 that protects against the development of pulmonary hypertension in bronchopulmonary dysplasia results in lower nitric oxide production in neonatal cord blood-derived lymphoblastoid cell lines.

Bronchopulmonary dysplasia (BPD) is chronic lung disease of prematurity and pulmonary hypertension (PH) is a major contributor to morbidity and mortality in BPD patients. Nitric oxide (NO) is a vasodilator and apoptotic mediator made by NO synthase (NOS). NOS is inhibited by asymmetric dimethylarginine (ADMA), and dimethylarginine dimethylaminohydrolase (DDAH) hydrolyzes ADMA. Previously, in a BPD patient cohort, we identified single nucleotide polymorphism (SNP) DDAH1 rs480414 (G > A) that was protective against developing PH. This study aims to determine functional consequences of the DDAH1 SNP in lymphoblastoid cell lines (LCLs) derived from neonatal cord blood. We tested the hypothesis that DDAH1 SNP (AA) results in DDAH1 gain of function, leading to greater NO-mediated apoptosis compared to DDAH1 wild-type (GG) in LCLs. LCLs were analyzed by Western blot (DDAH1, cleaved and total caspase-3 and -8, and β-actin), and RT-PCR (DDAH1, iNOS). Cell media assayed for nitrites with chemiluminescence NO analyzer, and conversion of ADMA to L-citrulline was measured by spectrophotometry. LCLs with DDAH1 SNP had similar levels of DDAH1 protein and mRNA expression, as well as DDAH activity, compared to DDAH1 WT LCLs. There were also no changes in cleaved caspase-3 and -8 protein levels. LCLs with DDAH1 SNP had similar iNOS mRNA expression. Nitrite levels in media were lower for DDAH1 SNP LCLs compared to DDAH1 WT LCLs (p < 0.05). Contrary to our hypothesis, we found that NO production was lower in DDAH1 SNP LCLs, indicative of a loss of function phenotype.

A Recombinant Dimethylarginine Dimethylaminohydrolase-1-Based Biotherapeutics to Pharmacologically Lower Asymmetric Dimethyl Arginine, thus Improving Postischemic Cardiac Function and Cardiomyocyte Mitochondrial Activity.

High serum levels of asymmetric dimethyl arginine (ADMA) are associated with cardiovascular disease and mortality. Pharmacological agents to specifically lower ADMA and their potential impact on cardiovascular complications are not known. In this study, we aimed to investigate the effect of specific lowering of ADMA on myocardial response to ischemia-reperfusion injury (I/R) and direct effects on cardiomyocyte function. Effects of recombinant dimethylarginine dimethylaminohydrolase (rDDAH)-1 on I/R injury were determined using isolated mouse heart preparation. Respiration capacity and mitochondrial reactive oxygen species (ROS) generation were determined on mouse cardiomyocytes. Our results show that lowering ADMA by rDDAH-1 treatment resulted in improved recovery of cardiac function and reduction in myocardial infarct size in mouse heart response to I/R injury (control 22.24 ±4.60% versus rDDAH-1 15.90 ±4.23%, P < 0.01). In mouse cardiomyocytes, rDDAH-1 treatment improved ADMA-induced dysregulation of respiration capacity and decreased mitochondrial ROS. Furthermore, in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes with impaired contractility under hypoxia and high ADMA, rDDAH-1 treatment improved recovery and beating frequency (P < 0.05). rDDAH-1 treatment selectively modified I/R-induced myocardial cytokine expression, resulting in reduction in proinflammatory cytokine IL-17A (P < 0.001) and increased expression of anti-inflammatory cytokines IL-10 and IL-13 (P < 0.01). Further in vitro studies showed that IL-17A was the predominant and common cytokine modulated by ADMA-DDAH pathway in heart, cardiomyocytes, and endothelial cells. These studies show that lowering ADMA by pharmacological treatment with rDDAH-1 reduced I/R injury, improved cardiac function, and ameliorated cardiomyocyte bioenergetics and beating activity. These effects may be attributable to ADMA lowering in cardiomyocytes and preservation of cardiomyocyte mitochondrial function. SIGNIFICANCE STATEMENT: The pathological role of asymmetric dimethyl arginine (ADMA) has been demonstrated by its association with cardiovascular disease and mortality. Currently, pharmacological drugs to specifically lower ADMA are not available. The present study provides the first evidence that lowering of ADMA by recombinant recombinant dimethylarginine dimethylaminohydrolase (rDDAH)-1 improved postischemic cardiac function and cardiomyocyte bioenergetics and beating activity. Our studies suggest that lowering of ADMA by pharmacologic treatment offers opportunity to develop new therapies for the treatment of cardiovascular and renal disease.

Mechanical force promotes dimethylarginine dimethylaminohydrolase 1-mediated hydrolysis of the metabolite asymmetric dimethylarginine to enhance bone formation

Mechanical force is critical for the development and remodeling of bone. Here we report that mechanical force regulates the production of the metabolite asymmetric dimethylarginine (ADMA) via regulating the hydrolytic enzyme dimethylarginine dimethylaminohydrolase 1 (Ddah1) expression in osteoblasts. The presence of -394 4 N del/ins polymorphism of Ddah1 and higher serum ADMA concentration are negatively associated with bone mineral density. Global or osteoblast-specific deletion of Ddah1 leads to increased ADMA level but reduced bone formation. Further molecular study unveils that mechanical stimulation enhances TAZ/SMAD4-induced Ddah1 transcription. Deletion of Ddah1 in osteoblast-lineage cells fails to respond to mechanical stimulus-associated bone formation. Taken together, the study reveals mechanical force is capable of down-regulating ADMA to enhance bone formation.

Beyond eNOS: Genetic influence in NO pathway affecting drug response.

Nitric Oxide (NO) has important biological functions, and its production may be influenced by genetic polymorphisms. Since NO mediates the drug response, the same genetic polymorphism that alter NO levels may also impact drug therapy. The vast majority of studies in the literature that assess the genetic influence on NO-related drug response focus on NOS3 (which encodes endothelial nitric oxide synthase), however several other proteins are interconnected in the same pathway and may also impact NO availability and drug response. The aim of this study was to review the literature regarding genetic polymorphisms that influence NO in response to pharmacological agents located in genes other than NOS3. Articles were obtained from Pubmed and consisted of 17 manuscripts that assessed polymorphisms of the following targets: Arginases 1 and 2 (ARG1 and ARG2), dimethylarginine dimethylaminohydrolases 1 and 2 (DDAH1 and DDAH2), and vascular endothelial growth factor (VEGF). Here we analyze the main results of these articles, which show promising evidences that may suggest that the NO-driven pharmacological response is affected by more than the eNOS gene. The search for genetic markers may result in better understanding of the variability of drug response and turn pharmacotherapy involving NO safer and more effective.

RETRACTED ARTICLE: Interference of KLF9 relieved the development of gestational diabetes mellitus by upregulating DDAH2

Statement of Retraction Weixia Chen, Huiqin Wang, Jin Liu, and Kaixia Li- 2022, Interference of KLF9 relieved the development of gestational diabetes mellitus by upregulating DDAH2, Bioengineered 13(1), doi: 10.1080/21655979.2021.2005929 Since publication, significant concerns have been raised about the integrity of the data and reported results in the article. When approached for an explanation, the authors did not provide their original data or any necessary supporting information. As verifying the validity of published work is core to the integrity of the scholarly record, we are therefore retracting the article. All authors listed in this publication have been informed. We have been informed in our decision-making by our editorial policies and the COPE guidelines. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.

Association of Genes of the NO Pathway with Altitude Disease and Hypoxic Pulmonary Hypertension.

Chronic intermittent hypoxia leads to high-altitude pulmonary hypertension, which is associated with high asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthesis. Therefore, we aimed to understand the relation of single nucleotide polymorphisms in this pathway to high-altitude pulmonary hypertension (HAPH). We genotyped 69 healthy male Chileans subjected to chronic intermittent hypoxia. Acclimatization to altitude was determined using the Lake Louise Score and the presence of acute mountain sickness. Echocardiography was performed after six months in 24 individuals to estimate pulmonary arterial pressure. The minor allele of dimethylarginine dimethylaminohydrolase (DDAH)1 rs233112 was associated with high-baseline plasma ADMA concentration, while individuals homozygous for the major allele of DDAH2 rs805304 had a significantly greater increase in ADMA during chronic intermittent hypoxia. The major allele of alanine glyoxylate aminotransferase-2 (AGXT2) rs37369 was associated with a greater reduction of plasma symmetric dimethylarginine (SDMA). Several genes were associated with high-altitude pulmonary hypertension, and the nitric oxide synthase (NOS)3 and DDAH2 genes were related to acute mountain sickness. In conclusion, DDAH1 determines baseline plasma ADMA, while DDAH2 modulates ADMA increase in hypoxia. AGXT2 may be up-regulated in hypoxia. Genomic variation in the dimethylarginine pathway affects the development of HAPH and altitude acclimatization.

Pathways of Microcirculatory Endothelial Dysfunction in Obstructive Sleep Apnea: A Comprehensive Ex Vivo Evaluation in Human Tissue.

The mechanism and markers of cardiovascular disease (CVD) in obstructive sleep apnea (OSA) remain unknown. The microcirculation is the site of early changes in OSA patients who are free of CVD risk. Patients with newly diagnosed moderate to severe OSA (n = 7) were studied before and 12 weeks after intensive treatment with continuous positive airway pressure (CPAP), along with weight and age matched controls (n = 7). Microcirculatory vessels were isolated from gluteal biopsies and changes in critical functional genes were measured. The following genes changed after 12 weeks of intensive CPAP therapy in the microcirculatory vessels: angiotensin receptor type 1 (AGTR-1) (11.6 (3.4) to 6 (0.8); P = 0.019); NADPH oxidase (NOX4) (0.85 (0.02) to 0.79 (0.11); P = 0.016); and dimethylarginine dimethylaminohydrolase (DDAH 1) (1 (0.31) to 0.55 (0.1); P = 0.028). Despite decreased nitric oxide (NO) availability as measured indirectly through brachial artery flow-mediated dilation, endothelial NO synthase (NOS3) did not change with CPAP. Other disease markers of OSA that changed with treatment in the microcirculation were endothelin, hypoxia inducible factor 1a, nuclear factor kappa B, interleukin-8, and interleukin-6. In this ex vivo evaluation of the microcirculation of patients with OSA and no CVD risk, several pathways of CVD were activated supporting that OSA independently induces microcirculatory endothelial dysfunction and serving as disease-specific markers for future pharmacological targeting of OSA-related CVD risk. The findings support the role of renin-angiotensin activation and endothelial oxidative stress in the decreased microcirculatory NO availability in OSA.

Nitric oxide and regulators of its synthesis in chronic obstructive pulmonary disease

ABSTRACT&#x0D; The clinical picture and long-term prognosis of chronic obstructive pulmonary disease (COPD) largely depend on comorbid conditions, thereby prompting a relevant search for predictive and preventive methods in the pathogenesis of the disease. Cardiovascular diseases are highly prevalent among patients with COPD. Cardiovascular risks in patients with COPD are associated with changes in the activity of vasoactive mediators, with nitric oxide (NO) being the most important. The important role of nitric oxide in the body prompts it being studied as a biomarker of many diseases; however, its short half-life and rapid clearance prevent its direct assessment in the blood. In the body, nitric oxide is formed from L-arginine with the help of enzymes of NO-synthase group. NO oxide synthesis depends on the concentration of L-arginine, arginase and asymmetric dimethylarginine (inhibitory effect on NO-synthase). The presented literature review highlights modern views on the importance of nitric oxide and regulators of its synthesis in the pathogenesis of chronic obstructive pulmonary disease. It also indicates their role in the formation of comorbid conditions, and highlights processes of NO formation in the body.&#x0D; CONCLUSION: The components of the nitric oxide system (nitric oxide metabolites, L-arginine, arginase, dimethylarginine dimethylaminohydrolase, asymmetric and symmetric dimethylarginine) can be considered as potential biomarkers of COPD, especially in conditions of cardiovascular comorbidity. Further studies on the nitric oxide system are recommended for assessing the prognosis of the course of diseases and the effectiveness of the current therapy.

Dissecting VEGF-induced acute versus chronic vascular hyperpermeability: Essential roles of dimethylarginine dimethylaminohydrolase-1

SummaryVascular endothelial cell growth factor (VEGF) is a key regulator of vascular permeability. Herein we aim to understand how acute and chronic exposures of VEGF induce different levels of vascular permeability. We demonstrate that chronic VEGF exposure leads to decreased phosphorylation of VEGFR2 and c-Src as well as steady increases of nitric oxide (NO) as compared to that of acute exposure. Utilizing heat-inducible VEGF transgenic zebrafish (Danio rerio) and establishing an algorithm incorporating segmentation techniques for quantification, we monitored acute and chronic VEGF-induced vascular hyperpermeability in real time. Importantly, dimethylarginine dimethylaminohydrolase-1 (DDAH1), an enzyme essential for NO generation, was shown to play essential roles in both acute and chronic vascular permeability in cultured human cells, zebrafish model, and Miles assay. Taken together, our data reveal acute and chronic VEGF exposures induce divergent signaling pathways and identify DDAH1 as a critical player and potentially a therapeutic target of vascular hyperpermeability-mediated pathogenesis.

Overexpression of dimethylarginine dimethylaminohydrolase 1 protects from angiotensin II-induced cardiac hypertrophy and vascular remodeling.

Cardiovascular complications are the leading cause of death, and elevated levels of asymmetric dimethyarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, are implicated in their pathophysiology. We investigated the role of dimethylarginine dimethylaminohydrolase 1 (DDAH1), an enzyme hydrolyzing ADMA, in prevention of cardiovascular remodeling during hypertension. We hypothesized that the animals overexpressing DDAH1 will be protected from angiotensin II (ANG II)-induced end organ damage. Angiotensin II (ANG II) was infused in two doses: 0.75 and 1.5 mg/kg/day in DDAH1 transgenic mice (DDAH1 TG) and wild-type (WT) littermates for 2 or 4 wk. Echocardiography was performed in the first and fourth weeks of the infusion, systolic blood pressure (SBP) was measured weekly, and cardiac hypertrophy and vascular remodeling was assessed by histology. Increase in SBP after 1 wk of ANG II infusion was not different between the groups, whereas TG mice had lower SBP at later time points. TG mice were protected from cardiovascular remodeling after 2 wk of ANG II infusion in the high dose and after 4 wk in the moderate dose. TG mice had higher left ventricular lumen-to-wall ratio, lower cardiomyocyte cross-sectional area, and less interstitial fibrosis compared with WT controls. In aorta, TG mice had less adventitial fibrosis, lower medial thickness with preserved elastin content, lower counts of inflammatory cells, lower levels of active matrix metalloproteinase-2, and showed better endothelium-dependent relaxation. We demonstrated that overexpression of DDAH1 protects from ANG II-induced cardiovascular remodeling and progression of hypertension by preserving endothelial function and limiting inflammation.NEW & NOTEWORTHY We showed that overexpression of dimethylarginine dimethylaminohydrolase 1 (DDAH1) protects from angiotensin II-induced cardiovascular damage, progression of hypertension, and adverse vascular remodeling in vivo. This protective effect is associated with decreased levels of asymmetric dimethylarginine, preservation of endothelial function, inhibition of cardiovascular inflammation, and lower activity of matrix metalloproteinase-2. Our findings are highly clinically relevant, because they suggest that upregulation of DDAH1 might be a promising therapeutic approach against angiotensin II-induced end organ damage.

Ramipril Increases Adma Concentration in Acute Myocardial Infarction in Rats Induced by Isoproterenol

In experimental animals, the induction of isoproterenol which is a synthetic of catecholamine, can cause acute myocardial infarction where the pathophysiology and morphology are the same as myocardial infarction in humans. Isoproterenol induction will increase oxidative stress, which will damage the enzyme dimethylarginine dimethylaminohydrolase (DDAH), thus causing asymmetric dimethylarginine (ADMA) levels to increase in circulation. Increased levels of ADMA will inhibit the activity of the enzyme nitric oxide synthase, which results in decreased nitric oxide resulting in endothelial damage. This study aims to determine the effect of Ramipril on asymmetric dimethylarginine (ADMA) levels in rats (Rattus norvegicus) Wistar strain with acute myocardial infarction.&#x0D; Eighteen male Wistar rats (150-250 g) were randomly allocated into three groups: negative control group, positive control, and treatment group. The treatment group was pretreated with Ramipril at dose 3 mg/kg BW orally for seven days. Acute myocardial infarction was induced in positive control groups and treatment groups by subcutaneous injection of isoproterenol (85 mg/kg BW) for two consecutive days. Twenty-four hours after the last administration, rats from all groups were anesthetized and sacrificed for blood sample collection to evaluate the level of Asymmetric Dimethylarginine with Enzym-linked Immunosorbent Assay (ELISA) method. The result showed that ADMA levels were increased in the treatment group after pretreated with Ramipril. This study concluded that pretreatment with Ramipril increased ADMA concentration in acute myocardial infarction rats induced by isoproterenol.

Protective effects of perindopril against indomethacin-induced gastric mucosal damage through modulation of DDAH-1/ADMA and ACE-2/ANG-(1-7) signaling pathways

Indomethacin is a widely used nonsteroidal anti-inflammatory drug; however, its clinical utility is accompanied by serious adverse reactions including peptic ulcers. The current study aims to investigate the protective potential of perindopril against indomethacin-induced gastric injury in rats. Perindopril (4 mg/kg) was administered orally for 7 days and indomethacin (60 mg/kg, single oral dose) was administered on the 7th day, 1 h after perindopril administration. Pantoprazole was used as a standard agent. Ulcer index (UI), preventive index ratio (PI), histopathological examination, oxidative stress, and inflammatory biomarkers were investigated. Perindopril significantly decreased UI while increased PI and counteracted histopathological aberrations induced by indomethacin. It alleviated indomethacin-induced oxidative stress by lowering NO while increasing GSH content and superoxide dismutase activity. Perindopril significantly downregulated TNF-α and asymmetric dimethylarginine (ADMA), while significantly upregulated COX-2, PGE-2, dimethylarginine dimethylaminohydrolase-1 (DDAH-1), ANG-(1-7), and ACE-2 expression. Together, these findings suggest the gastroprotective effects of perindopril through modulation of DDAH-1/ADMA and ACE-2/ANG-(1-7) signaling. HIGHLIGHTS Perindopril attenuated gastric histopathological damage. It increased GSH content and SOD activity while decreased NO content. It modulated gastric ADMA and DDAH-1 activity. It reduced TNF-α, while increased COX-2 and PGE-2 expression. It upregulated ACE-2 activity and ANG-(1-7) protein expression.

Transgenic overexpression of dimethylarginine dimethylaminohydrolase 1 protects from angiotensin II - induced cardiac hypertrophy and vascular remodeling

Background and Aims: Dimethylarginine dimethylaminohydrolase 1 (DDAH1) hydrolyzes endogenous inhibitor of nitric oxide synthases asymmetric dimethylarginine (ADMA). DDAH1 overexpression lowers ADMA levels and protects from renal interstitial fibrosis and vascular oxidative stress in angiotensin-II-induced hypertension. The current study was designed to test the hypothesis that DDAH1 overexpression protects from angiotensin II-induced cardiac hypertrophy and vascular remodeling.

Dimethylarginine dimethylaminohydrolase 1 as a novel regulator of oligodendrocyte differentiation in the central nervous system remyelination.

Remyelination is a regenerative process that restores the lost neurological function and partially depends on oligodendrocyte differentiation. Differentiation of oligodendrocytes spontaneously occurs after demyelination, depending on the cell intrinsic mechanisms. By combining a loss-of-function genomic screen with a web-resource-based candidate gene identification approach, we identified that dimethylarginine dimethylaminohydrolase 1 (DDAH1) is a novel regulator of oligodendrocyte differentiation. Silencing DDAH1 in oligodendrocytes prevented the expression of myelin basic protein in mouse oligodendrocyte culture with the change in expression of genes annotated with oligodendrocyte development. DDAH1 inhibition attenuated spontaneous remyelination in a cuprizone-induced demyelinated mouse model. Conversely, increased DDAH1 expression enhanced remyelination capacity in experimental autoimmune encephalomyelitis. These results provide a novel therapeutic option for demyelinating diseases by modulating DDAH1 activity.

DDAH-1, via regulation of ADMA levels, protects against ischemia-induced blood-brain barrier leakage

Dimethylarginine dimethylamino hydrolase-1 (DDAH-1) is an important regulator of nitric oxide (NO) metabolism that has been implicated in the pathogenesis of cardiovascular diseases. Nevertheless, its role in cerebral ischemia still needs to be elucidated. Herein, we examined the expression of DDAH-1 in the brain of rat by double-label immunofluorescence staining. DDAH-1 knock-out (DDAH-1−/−) and wild-type rats underwent middle cerebral artery occlusion/reperfusion (MCAO/R). After 24 h, neurological scores, TTC staining and TUNEL assay were used to evaluate neurological damages. 3 and 7-days infarct outcomes were also shown. Blood-brain-barrier (BBB) permeability was examined via Evans blue extravasation and tight junction (TJ) proteins expression and mRNA levels by western blot and RT-qPCR. The levels of plasma asymmetric dimethylarginine (ADMA), NO and ADMA in brain tissue were also assessed. In addition, supplementation of L-arginine to DDAH-1−/− rats was used to explore its role in regulating NO. DDAH-1 was abundantly distributed in cerebral cortex and basal nuclei, and mainly expressed in neurons and endothelial cells. DDAH-1−/− rats showed aggravated neurological damage and BBB disruption, including decrease of TJ proteins expression but indistinguishable mRNA levels after MCAO/R. DDAH-1 depletion and neurological damages were accompanied with increased ADMA levels and decreased NO concentrations. The supplementation with L-arginine partly restored the neurological damages and BBB disruption. To sum up, DDAH-1 revealed to have a protective role in ischemia stroke (IS) and IS-induced leakage of BBB via decreasing ADMA level and possibly via preventing TJ proteins degradation.Dimethylarginine dimethylamino hydrolase-1 (DDAH-1), as the critical enzyme responsible for asymmetric dimethylarginine (ADMA) degradation, serves as a protective factor for ischemic stroke. DDAH-1 protects ischemia-induced disruption of blood-brain-barrier via regulating ADMA level and preventing tight junction proteins degradation. The supplementation of L-arginine helps restore the function of DDAH-1.

Neurochemical changes underlying cognitive impairment in olfactory bulbectomized rats and the impact of the mGlu5-positive allosteric modulator CDPPB

The olfactory bulbectomized (OBX) rat model is a well-established model of depression in which antidepressant drugs reverse deficits in the passive avoidance test 14 days after administration. Recently, the olfactory bulbectomized rat model has been proposed to be a model of Alzheimer’s disease (AD), and the available data indicate similarities between the changes that typically occur in AD and those observed in OBX animals. In the present study, the occurrence of neurochemical impairments related to AD were investigated 8 months after OB ablation. The expression of the nitric oxide synthases eNOS and nNOS, receptor for advanced glycation endproducts (RAGEs) and dimethylarginine dimethylaminohydrolase (DDAH1) in the prefrontal cortices (PFCs), hippocampi and striata of olfactory bulbectomized and sham-operated rats was evaluated. Subsequently, the impact of the administration of a positive allosteric modulator of the mGlu5 receptor, CDPPB (14 days, 2.5 or 5 mg/kg), on OBX-related changes was assessed. OB ablation induced typical deficits in passive avoidance. Significant aberrations in the expression of both isoforms of NOS were observed in the hippocampus and striatum, and the expression of DDAH1 was increased in the PFCs of OBX animals. CDPPB at a dose of 5 mg/kg ameliorated cognitive impairment in the passive avoidance test and partially reversed the changes in eNOS and nNOS expression induced by the lesion. The results of this study confirm that some of the neurochemical changes observed in OBX animals may resemble those associated with AD pathology and that activation of the mGlu5 receptor may partially counteract these pathological alterations.

Divergent Dimethylarginine Dimethylaminohydrolase Isoenzyme Expression in the Central Nervous System

The endogenous methylated derivative of ʟ-arginine, Nω,Nω′-dimethyl-ʟ-arginine (asymmetric dimethylarginine, ADMA), an independent risk factor in many diseases, inhibits the activity of nitric oxide synthases and, consequently, modulates the availability of nitric oxide. While most studies on the biological role of ADMA have focused on endothelial and inducible nitric oxide synthases modulation and its contribution to cardiovascular, metabolic, and renal diseases, a role in regulating neuronal nitric oxide synthases and pathologies of the central nervous system is less understood. The two isoforms of dimethylarginine dimethylaminohydrolase (DDAH), DDAH1 and DDAH2, are thought to be the main enzymes responsible for ADMA catabolism. A current impediment is limited knowledge on specific tissue and cellular distribution of DDAH enzymes within the brain. In this study, we provide a detailed characterization of the regional and cellular distribution of DDAH1 and DDAH2 proteins in the adult murine and human brain. Immunohistochemical analysis showed a wide distribution of DDAH1, mapping to multiple cell types, while DDAH2 was detected in a limited number of brain regions and exclusively in neurons. Our results provide key information for the investigation of the pathophysiological roles of the ADMA/DDAH system in neuropsychiatric diseases and pave the way for the development of novel selective therapeutic approaches.

Hyperoxia suppresses DDAH1 expression and promotes proliferation in human pulmonary microvascular endothelial cells

Objective Pulmonary hypertension (PH) is associated with mortality in patients with bronchopulmonary dysplasia (BPD), and BPD-PH is characterized by pulmonary vascular remodeling. Patients often receive chronic supplemental oxygen and nitric oxide (NO)/sildenifil therapy to dilate the pulmonary vasculature. In vitro studies of human pulmonary microvascular endothelial cells (hPMVEC) have shown that dimethylarginine dimethylaminohydrolase-1 (DDAH1) promotes NO-mediated apoptosis. In this study, we hypothesized that DDAH1 is regulated by hyperoxia, and that hyperoxia regulates hPMVEC proliferation. Methods hPMVEC were grown at 37 ̊C in a 60mm plate to ~70% confluence. hPMVEC were then exposed to 85% O2 or 21% O2 for 6, 12, or 24 hours, and RNA or protein harvested for RT-PCR or Western blot, respectively. In a separate experiment, hPMVEC were seeded at 1x105 cells in 30mm plates, exposed to 85% O2 or 21% O2 for 24 or 72 hours and viable cell number counted by trypan blue exclusion. Newborn wild-type DDAH1 mice were exposed to 85% O2 or 21% O2 for 14 days, followed by an additional 14 days of 21% O2, then protein was isolated from lung for DDAH1 protein by Western blot. Groups were compared by t-test, p<0.05 was significant. Results hPMVEC exposed to 24 hours of hyperoxia had lower DDAH1 RNA (N=5, p<0.05) and protein (N=6, p<0.05) compared to normoxic control cells, with no differences at 6 or 12 hours. The number of viable hPMVEC (x105) was greater (1.83 ± 0.07 vs. 1.38 ± 0.17; N=6, p<0.05) after 24 hours, and the number of viable hPMVEC (x105) was lower (1.26 ± 0.12 vs. 2.19 ± 0.22; N=6, p<0.01) after 72 hours of hyperoxia compared to normoxia control. DDAH1 protein expression in mouse lung homogenates was significantly lower after 85% O2 compared to 21% O2 mice (N=3, p<0.05). Conclusions These data support the concept that in hPMVEC, hyperoxia negatively regulates DDAH1. We observed that hyperoxia results in a proliferative phenotype that coincided with lower DDAH1 levels. In BPD-PH, where patients routinely receive life-saving hyperoxia treatment, the pathophysiology may include pulmonary endothelial-DDAH1 depletion, therefore, we speculate that therapies that up-regulate pulmonary endothelial-DDAH1 activity may protect endogenous NO production to prevent and/or treat PH. Future studies will determine the role of DDAH1 in hyperoxia-mediated hPMVEC proliferation, with a particular focus on the effects of hyperoxia-mediated DDAH1 suppression on NO-mediated apoptosis in hPMVEC.

Abnormalities of the PRMT1-ADMA-DDAH1 metabolism axis and probucol treatment in diabetic patients and diabetic rats.

Symmetrical dimethylarginine (ADMA) endogenously inhibits nitric oxide synthase (NOS) and strongly indicates oxidant stress, whose formation primarily derived from type 1 protein arginine N-methyltransferase (PRMT1) and whose metabolism was governed by type 1 dimethylarginine dimethylaminohydrolase (DDAH1). This study aimed to evaluate participation of the PRMT1-ADMA-DDAH1 metabolism axis in the kidneys of type 2 diabetes model rats and human subjects, and the effect of probucol on this axis and renal function. A total of 30 rats were randomly assigned to a normal group (NC, n=10), diabetic group (DM, n=10), and a diabetics under probucol treatment group (PM, n=10). Throughout 8 weeks of probucol treatment, plasma NOS, the malondialdehyde (MDA), superoxide dismutase (SOD), nitric oxide (NO), and catalase (CAT) activity were evaluated by chemical colorimetric approach. ADMA concentration was evaluated with an enzyme-linked immunosorbent assay (ELISA) and analysis of expression of PRMT1 and DDAH1 in kidneys with reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC) and western blotting were performed. The expression of DDAH1 in the kidney, and the plasma NOS, NO, SOD, and CAT activities in diabetic group were lower, while MDA and the expression of PRMT1 and ADMA were higher in contrast to the control group. In diabetics rats receiving probucol, the expressions of DDAH1 and ADMA were downregulated, whereas that of PRMT1 was upregulated. Probucol inhibited the indexes of oxidative stress and improved the kidney function in both diabetic rats and humans. We found that the expression of the PRMT1-ADMA-DDAH1 axis was altered in the kidneys of diabetic rats. Moreover, results indicated that probucol therapy regulates expression at both ends of this axis, which may preserve renal function by reducing oxidant stress. Therefore, probucol may partially restore expression of the PRMT1-ADMA-DDAH1 axis in diabetic kidneys, immigrate oxidant stress, and enhance renal function.