A new perspective on NO pathway in sepsis and ADMA lowering as a potential therapeutic approach.

The nitric oxide pathway plays a critical role in vascular homeostasis. Increased levels of systemic nitric oxide (NO) are observed in preclinical models of sepsis and endotoxemia. This has led to the postulation that vasodilation by inducible nitric oxide synthase (iNOS) generated NO may be a mechanism of hypotension in sepsis. However, contrary to the expected pharmacological action of a nitric oxide synthase (NOS) inhibitor, clinical studies with L-NAME produced adverse cardiac and pulmonary events, and higher mortality in sepsis patients. Thus, the potential adverse effects of NO in human sepsis and shock have not been fully established. In recent years, the emerging new understanding of the NO pathway has shown that an endogenously produced inhibitor of NOS, asymmetric dimethylarginine (ADMA), a host response to infection, may play an important role in the pathophysiology of sepsis as well as organ damage during ischemia-reperfusion. ADMA induces microvascular dysfunction, proinflammatory and prothrombotic state in endothelium, release of inflammatory cytokines, oxidative stress and mitochondrial dysfunction. High levels of ADMA exist in sepsis patients, which may produce adverse effects like those observed with L-NAME. Several studies have demonstrated the association of plasma ADMA levels with mortality in sepsis patients. Preclinical studies in sepsis and ischemia-reperfusion animal models have shown that lowering of ADMA reduced organ damage and improved survival. The clinical finding with L-NAME and the preclinical research on ADMA "bed to bench" suggest that ADMA lowering could be a potential therapeutic approach to attenuate progressive organ damage and mortality in sepsis. Testing of this approach is now feasible by using the pharmacological molecules that specifically lower ADMA.

A Therapeutic Extracorporeal Device for Specific Removal of Pathologic Asymmetric Dimethylarginine from the Blood.

INTRODUCTION: Blood levels of uremic toxin, asymmetric dimethylarginine (ADMA), are strongly associated with mortality in sepsis, renal failure, and cardiovascular and renal disease patients. METHODS: An extracorporeal approach to reduce pathological ADMA was developed. The dimethylarginine dimethylaminohydrolase (DDAH) was immobilized on agarose beads to prepare a cartridge. The efficacy of cartridge for ADMA lowering in blood was investigated. RESULTS: The DDAH beads and cartridge reduced ADMA from solution or plasma. The magnitude of ADMA removal was dependent on the quantity of DDAH linked to the beads and the flow rate. When tested in association with plasmapheresis, the DDAH-cartridge was highly effective in ADMA removal from the blood and improved the arginine/ADMA ratio in a pig model. CONCLUSION: A new, safe, and effective extracorporeal approach to lower ADMA was developed which may have application in improving outcomes in patients with vascular complications and risk of mortality associated with high ADMA.

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.

Specific Lowering of Asymmetric Dimethylarginine by Pharmacological Dimethylarginine Dimethylaminohydrolase Improves Endothelial Function, Reduces Blood Pressure and Ischemia-Reperfusion Injury.

Multiple clinical and preclinical studies have demonstrated that plasma levels of asymmetric dimethylarginine (ADMA) are strongly associated with hypertension, diabetes, and cardiovascular and renal disease. Genetic studies in rodents have provided evidence that ADMA metabolizing dimethylarginine dimethylaminohydrolase (DDAH)-1 plays a role in hypertension and cardiovascular disease. However, it remains to be established whether ADMA is a bystander, biomarker, or sufficient contributor to the pathogenesis of hypertension and cardiovascular and renal disease. The goal of the present investigation was to develop a pharmacological molecule to specifically lower ADMA and determine the physiologic consequences of ADMA lowering in animal models. Further, we sought to determine whether ADMA lowering will produce therapeutic benefits in vascular disease in which high ADMA levels are produced. A novel long-acting recombinant DDAH (M-DDAH) was produced by post-translational modification, which effectively lowered ADMA in vitro and in vivo. Treatment with M-DDAH improved endothelial function as measured by increase in cGMP and in vitro angiogenesis. In a rat model of hypertension, M-DDAH significantly reduced blood pressure (vehicle: 187 ± 19 mm Hg vs. M-DDAH: 157 ± 23 mm Hg; P < 0.05). Similarly, in a rat model of ischemia-reperfusion injury, M-DDAH significantly improved renal function as measured by reduction in serum creatinine (vehicle: 3.14 ± 0.74 mg/dl vs. M-DDAH: 1.1 ± 0.75 mg/dl; P < 0.01), inflammation, and injured tubules (vehicle: 73.1 ± 11.1% vs. M-DDAH: 22.1 ± 18.4%; P < 0.001). These pharmacological studies have provided direct evidence for a pathologic role of ADMA and the therapeutic benefits of ADMA lowering in preclinical models of endothelial dysfunction, hypertension, and ischemia-reperfusion injury. SIGNIFICANCE STATEMENT: High levels of ADMA occur in patients with cardiovascular and renal disease. A novel modified dimethylarginine dimethylaminohydrolase by PEGylation effectively lowers ADMA, improves endothelial function, reduces blood pressure and protects from ischemia-reperfusion renal injury.

Cerivastatin Nanoliposome as a Potential Disease Modifying Approach for the Treatment of Pulmonary Arterial Hypertension.

In this study we investigated nanoliposome as an approach to tailoring the pharmacology of cerivastatin as a disease-modifying drug for pulmonary arterial hypertension (PAH). Cerivastatin encapsulated liposomes with an average diameter of 98 ± 27 nm were generated by a thin film and freeze-thaw process. The nanoliposomes demonstrated sustained drug-release kinetics in vitro and inhibited proliferation of pulmonary artery (PA) smooth muscle cells with significantly less cellular cytotoxicity as compared with free cerivastatin. When delivered by inhalation to a rat model of monocrotaline-induced PAH, cerivastatin significantly reduced PA pressure from 55.13 ± 9.82 to 35.56 ± 6.59 mm Hg (P < 0.001) and diminished PA wall thickening. Echocardiography showed that cerivastatin significantly reduced right ventricle thickening (monocrotaline: 0.34 ± 0.02 cm vs. cerivastatin: 0.26 ± 0.02 cm; P < 0.001) and increased PA acceleration time (monocrotaline: 13.98 ± 1.14 milliseconds vs. cerivastatin: 21.07 ± 2.80 milliseconds; P < 0.001). Nanoliposomal cerivastatin was equally effective or slightly better than cerivastatin in reducing PA pressure (monocrotaline: 67.06 ± 13.64 mm Hg; cerivastatin: 46.31 ± 7.64 mm Hg vs. liposomal cerivastatin: 37.32 ± 9.50 mm Hg) and improving parameters of right ventricular function as measured by increasing PA acceleration time (monocrotaline: 24.68 ± 3.92 milliseconds; cerivastatin: 32.59 ± 6.10 milliseconds vs. liposomal cerivastatin: 34.96 ± 7.51 milliseconds). More importantly, the rate and magnitude of toxic cerivastatin metabolite lactone generation from the intratracheally administered nanoliposomes was significantly lower as compared with intravenously administered free cerivastatin. These studies show that nanoliposome encapsulation improved in vitro and in vivo pharmacologic and safety profile of cerivastatin and may represent a safer approach as a disease-modifying therapy for PAH.

Use of T.M.J. Disc as a Soft Tissue Interpositional Graft Material for Functional Rehabilitation of Ankylosed T.M. Joint.

INTRODUCTION: Many surgical techniques have been described for the treatment of TMJ ankylosis, but no strategy has been uniformly agreed upon underscoring the difficulty of the problem. Despite new guidelines and updated methods, treating patients with TMJ Ankylosis remains a challenge as the incidence of recurrence after treatment is soaring. This study exemplifies our experience in using an unsullied method to treat TMJ Ankylosis to restore the structure of TMJ in conjunction with convalescing secondary maxillofacial deformity. MATERIALS AND METHODS: A total of 56 cases of unilateral bony TMJ ankylosis were included in the study, and postoperative results of T.M.J disc as a soft tissue interposition graft was evaluated. The operative protocol comprised of (1) resection of ankylotic mass, (2) intraoral ipsilateral coronoidectomy or contralateral coronoidectomy when needed, (4) interpositioning disc as soft tissue graft, (5) interposing and fixing sternoclavicular or costocondral graft with lag screws and (6) early mobilization, aggressive physiotherapy. RESULTS: The study assessed patients with regular follow-up checks for a period of 3 years. The average preoperative mouth opening was found to be 5.46 mm (range 2-10 mm). Mean post-operative mouth opening was 33.05 mm (range 24-43 mm), while 3 years post operative mouth opening (mean) was 39.75 mm. No cases of reankylosis were reported during this period suggesting it as a viable and satisfactory approach. CONCLUSION: The use of TMJ disc as a soft tissue interpositional graft material is an effectual method for functional rehabilitation of ankylosis cases and serves as an effective means of preventing recurrence.

Nobori stent shows less vascular inflammation and early recovery of endothelial function compared with Cypher stent.

OBJECTIVES: The current study sought to examine inflammation at the stented segments of Nobori (Terumo Corporation, Tokyo, Japan) and Cypher (Cordis, Miami, Florida) drug-eluting stents (DES), as well as free radical production and endothelial function of the adjacent nonstented segments in a pig coronary model. BACKGROUND: Nobori is a novel DES, incorporating a biolimus A9-eluting biodegradable polymer coated only on the abluminal surface of the stent. These unique features may favorably affect inflammation and endothelial function, as compared to the currently marketed DES. Presently, pre-clinical data on direct comparison of the various generations of DES are not available. METHODS: A total of 18 DES were implanted in pig coronary arteries and subsequently explanted at 1 month. Stented segments were assessed by angiography and histology. Ex vivo vasomotor function and superoxide production in segments proximal and distal to the stent were determined. The vasoconstriction, endothelial-dependent relaxation, and endothelial-independent relaxation of proximal and distal nonstented segments were measured. RESULTS: Histological evaluation revealed lower inflammatory response with Nobori than with Cypher DES. There is trend for lower angiographic percentage diameter stenosis in Nobori versus Cypher groups (p = 0.054). There was increased endothelium-dependent relaxation, decreased endothelin-1-mediated contraction, and less superoxide production in the vessel segments proximal and distal to Nobori versus Cypher stents. CONCLUSIONS: Our data show significantly lower inflammatory response in the stented segments, and rapid recovery of endothelial function of peristent segments in the Nobori group compared with Cypher DES group at 1 month in porcine coronary artery model.

Plasma protein thiols, ceruloplasmin, C-reactive protein and red blood cell acetylcholinesterase in patients undergoing intrauterine insemination.

OBJECTIVE: To estimate acetylcholinesterase (AChE), protein thiols (PT), ceruloplasmin (CP) and C-reactive proteins (CRPs) to assess any change in their levels following intrauterine insemination (IUI). MATERIALS AND METHODS: Forty-two patients aged 31 +/- 4.65 years (mean +/- SD) with primary infertility selected for IUI. All of them had induced ovulation with clomiphene citrate 50 mg from day 2 to day 6. After taking the consent, 2 ml of blood was withdrawn before and after 24 h of IUI for biochemical estimations. RESULTS: We observed a significant decrease in plasma CP, PT and RBC AChE (P < 0.001) following IUI compared with the respective pre-procedure levels. Highly sensitive CRP showed a marginal increase after IUI. CONCLUSION: Fluctuations in levels of the above parameters point to their role in the female reproductive system and in the outcome of the IUI.

A novel splicing variant of proprotein convertase subtilisin/kexin type 9.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is the most recently identified member of the proprotein convertase family. Genetic and cell biology studies have suggested a critical role of PCSK9 in regulating low-density lipoprotein receptor (LDLR) protein levels and thus modulating plasma LDL cholesterol. Recent data on the molecular basis for PCSK9 action support the model in which PCSK9 is self-cleaved, secreted, and tightly bound to the EGF-A repeat of LDLR extracellular domain. PCSK9 binding to LDLR is essential for the ensuing receptor-mediated endocytosis and is speculated to lock LDLR in a specific conformation that favors degradation in lysosomal compartment instead of recycling back to plasma membrane. We report here a novel human PCSK9 splicing variant, which we named PCSK9sv. PCSK9sv had an in-frame deletion of the eighth exon of 58 amino acids and was expressed in multiple tissues, including liver, small intestine, prostate, uterus, brain, and adipose tissue. Unlike wild-type PCSK9, which is secreted, PCSK9sv expressed in human embryonic kidney HEK293 cells failed to process the prosegment intracellularly and thus was not secreted into the medium. Examination of potential functions revealed that PCSK9sv did not change the LDLR protein levels. Two mutations that have been reported in humans with the associated changes in plasma LDL cholesterol were within exon 8, and thus the expression and function of the two mutants were studied. Both N425S and A443T mutants were processed normally, secreted, and reduced LDLR levels. However, the physiological function of this novel splicing variant of PCSK9 has yet to be determined.

High-resolution quantitative computed tomography demonstrating selective enhancement of medium-size collaterals by placental growth factor-1 in the mouse ischemic hindlimb.

BACKGROUND: The process of arteriogenesis after occlusion of a major artery is poorly understood. We have used high-resolution microcomputed tomography (mu-CT) imaging to define the arteriogenic response in the mouse model of hindlimb ischemia and to examine the effect of placental growth factor-1 (PlGF-1) on this process. METHODS AND RESULTS: After common femoral artery ligation, mu-CT imaging demonstrated formation of collateral vessels originating near the ligation site in the upper limb and connecting to the ischemic calf muscle region. Three-dimensional mu-CT and quantitative image analysis revealed changes in the number of segments and the segmental volume of vessels, ranging from 8 to 160 microm in diameter. The medium-size vessels (48 to 160 microm) comprising 85% of the vascular volume were the major contributor (188%) to the change in vascular volume in response to ischemia. Intramuscular injections of Ad-PlGF-1 significantly increased Sca1+ cells in the circulation, alpha-actin-stained vessels, and perfusion of the ischemic hindlimb. These effects were predominantly associated with an increase in vascular volume contributed by the medium-size (96 to 144 microm) vessels as determined by mu-CT. CONCLUSIONS: High-resolution mu-CT delineated the formation of medium-size collaterals representing a major vascular change that contributed to the restoration of vascular volume after ischemia. This effect is selectively potentiated by PlGF-1. Such selective enhancement of arteriogenesis by therapeutically administered PlGF-1 demonstrates a desirable biological activity for promoting the growth of functionally relevant vasculature.

Delayed arteriogenesis in hypercholesterolemic mice.

BACKGROUND: Hypercholesterolemia has been reported to inhibit ischemia-induced angiogenesis. To address its effects on arteriogenesis, we investigated arterial growth in hypercholesterolemic low-density lipoprotein receptor(-/-)/ApoB-48(-/-) (HCE) mice. METHODS AND RESULTS: The extent and the time course of arteriogenesis after femoral artery ligation was evaluated in HCE and strain-matched control mice. Distal limb perfusion was measured by laser Doppler imaging, whereas MRI was used to visualize arterial flow and micro-computed tomography to assess vascular growth. After femoral artery ligation, serial laser Doppler imaging demonstrated significantly delayed restoration of perfusion in untreated HCE compared with control mice (day 3, 0.09 versus 0.19, P<0.05). Treatment with Ad-PR39 in control mice led to a significant restoration of arterial blood flow and tissue perfusion at day 3, whereas in HCE mice, hindlimb perfusion began increasing only by day 7. Micro-CT analysis confirmed increased growth of smaller arterioles (16 to 63 microm in diameter) in the Ad-PR39-treated control compared with HCE mice. The delay in arteriogenesis in HCE mice correlated with delayed tissue appearance of F4/80+ cells. Analysis of gene expression after Ad-PR39 treatment demonstrated that HCE mice had significantly reduced expression of FGF receptor 1, hypoxia-inducible factor-1alpha, vascular cell adhesion molecule-1, macrophage scavenger receptor-1, and cyclophilin A compared with controls 3 days after arterial ligation that equalized by day 7, mimicking relative changes in arteriogenesis and tissue perfusion. CONCLUSIONS: Hypercholesterolemia results in delayed native arteriogenesis because of reduced early monocyte/macrophage influx and delayed and impaired arterial growth response to growth factor therapy.

Design and synthesis of a potent and selective triazolone-based peroxisome proliferator-activated receptor alpha agonist.

A new series of hPPARalpha agonists containing a 2,4-dihydro-3H-1,2,4-triazol-3-one (triazolone) core is described leading to the discovery of 5 (LY518674), a highly potent and selective PPARalpha agonist.