Exogenous Delivery Of Nitric Oxide Research Articles

ROS-responsive & scavenging NO nanomedicine for vascular diseases treatment by inhibiting endoplasmic reticulum stress and improving NO bioavailability

Vascular diseases seriously threaten human life and health. Exogenous delivery of nitric oxide (NO) represents an effective approach for maintaining vascular homeostasis during pathological events. However, the overproduction of reactive oxygen species (ROS) at vascular injury sites would react with NO to produce damaging peroxynitrite (ONOO−) species and limit the therapeutic effect of NO. Hence, we design a ROS-responsive NO nanomedicine (t-PBA&NO NP) with ROS scavenging ability to solve the dilemma of NO-based therapy. t-PBA&NO NP targets NO and anti-oxidant ethyl caffeate (ECA) to the injury sites via collagen IV homing peptide. The ROS-triggered ROS depletion and ECA release potently alleviate local oxidative stress via ROS scavenging, endoplasmic reticulum and mitochondrial regulation. It subsequently maximizes vascular modulation effects of NO, without production of harmful compounds, reactive nitrogen species (RNS). Therefore, it significantly increases competitiveness of human umbilical vein endothelial cells (HUVECs) over human aortic smooth muscle cells (HASMCs) both in vitro and in vivo. The strategy proved effective in inducing faster re-endothelialization, inhibiting neointimal formation and restoring vascular homeostasis. The synergy between ROS depletion and NO therapy served as a new inspiration for the treatment of cardiovascular diseases and other ROS-associated illnesses.

Applications of nitric oxide-releasing nanomaterials in dermatology: Skin infections and wound healing

Nitric oxide (NO) is produced in most cells in the skin and is an important regulator of essential cutaneous functions, including responses to UV irradiation, microbial defense, wound healing, melanogenesis and epidermal permeability barrier homeostasis. Harnessing the physiological activities of NO for therapeutic use is difficult because the molecule is highly reactive and unstable. A variety of exogenous NO delivery platforms have been developed and evaluated; however, they have limited clinical applications in dermatology due to instability and poor cutaneous penetration. NO-releasing nanomaterials overcome these limitations, providing targeted tissue delivery, and sustained and controlled NO release. This review provides a comprehensive and up-to-date evaluation of the use of NO-releasing nanomaterials in dermatology for the treatment of skin and soft tissue infections and wound healing.

An Injectable Dual-Function Hydrogel Protects Against Myocardial Ischemia/Reperfusion Injury by Modulating ROS/NO Disequilibrium.

Acute myocardial infarction (MI) is the leading cause of death worldwide. Exogenous delivery of nitric oxide (NO) to the infarcted myocardium has proven to be an effective strategy for treating MI due to the multiple physiological functions of NO. However, reperfusion of blood flow to the ischemic tissues is accompanied by the overproduction of toxic reactive oxygen species (ROS), which can further exacerbate tissue damage and compromise the therapeutic efficacy. Here, an injectable hydrogel is synthesized from the chitosan modified by boronate‐protected diazeniumdiolate (CS‐B‐NO) that can release NO in response to ROS stimulation and thereby modulate ROS/NO disequilibrium after ischemia/reperfusion (I/R) injury. Furthermore, administration of CS‐B‐NO efficiently attenuated cardiac damage and adverse cardiac remodeling, promoted repair of the heart, and ameliorated cardiac function, unlike a hydrogel that only released NO, in a mouse model of myocardial I/R injury. Mechanistically, regulation of the ROS/NO balance activated the antioxidant defense system and protected against oxidative stress induced by I/R injury via adaptive regulation of the Nrf2‐Keap1 pathway. Inflammation is then reduced by inhibition of the activation of NF‐κB signaling. Collectively, these results show that this dual‐function hydrogel may be a promising candidate for the protection of tissues and organs after I/R injury.

Deleterious effect of bone marrow-resident macrophages on hematopoietic stem cells in response to total body irradiation

AbstractBone marrow (BM) resident macrophages interact with a population of long-term hematopoietic stem cells (LT-HSCs) but their role on LT-HSC properties after stress is not well defined. Here, we show that a 2 Gy-total body irradiation (TBI)-mediated death of LT-HSCs is associated with increased percentages of LT-HSCs with reactive oxygen species (ROS) and of BM resident macrophages producing nitric oxide (NO), resulting in an increased percentage of LT-HSCs with endogenous cytotoxic peroxynitrites. Pharmacological or genetic depletion of BM resident macrophages impairs the radio-induced increases in the percentage of both ROS+ LT-HSCs and peroxynitrite+ LT-HSCs and results in a complete recovery of a functional pool of LT-HSCs. Finally, we show that after a 2 Gy-TBI, a specific decrease of NO production by BM resident macrophages improves the LT-HSC recovery, whereas an exogenous NO delivery decreases the LT-HSC compartment. Altogether, these results show that BM resident macrophages are involved in the response of LT-HSCs to a 2 Gy-TBI and suggest that regulation of NO production can be used to modulate some deleterious effects of a TBI on LT-HSCs.

Incorporation of a Nitric Oxide Donating Motif into Novel PC-PLC Inhibitors Provides Enhanced Anti-Proliferative Activity.

Inhibition of phosphatidylcholine-specific phospholipase C (PC-PLC) has previously been shown to be a potential target for novel cancer therapeutics. One downstream consequence of PC-PLC activity is the activation of NF-κB, a nuclear transcription factor responsible for transcribing genes related to oncogenic traits, such as proliferation, angiogenesis, metastasis, and cancer cell survival. Another biological pathway linked to NF-κB is the exogenous delivery of nitric oxide (NO), which decreases NF-κB activity through an apparent negative-feedback loop. In this study, we designed and synthesised 13 novel NO-releasing derivatives of our previously reported class of PC-PLC inhibitors, 2-morpholinobenzoic acids. These molecules contained a secondary benzylamine group, which was readily nitrosylated and subsequently confirmed to release NO in vitro using a DAF-FM fluorescence-based assay. It was then discovered that these NO-releasing derivatives possessed significantly improved anti-proliferative activity in both MDA-MB-231 and HCT116 cancer cell lines compared to their non-nitrosylated parent compounds. These results confirmed that the inclusion of an exogenous NO-releasing functional group onto a known PC-PLC inhibitor enhances anti-proliferative activity and that this relationship can be exploited in order to further improve the anti-proliferative activity of current/future PC-PLC inhibitors.

Controlled Nitric Oxide Release Using Poly(lactic-co-glycolic acid) Nanoparticles for Anti-Inflammatory Effects.

Nitric oxide (NO) plays a key role in several physiological functions such as inflammatory responses and immune regulation. However, despite its beneficial functions, the short half-life and diffusion radius limit NO availability in biomedical applications. Hence, controlled release is important to achieve the desired therapeutic effects with exogenous NO delivery. In this study, we fabricated a poly(lactic-co-glycolic acid) (PLGA)-based NO delivery system to release NO in a sustained manner under physiological conditions. To prevent an initial burst release, branched polyethylenimine diazeniumdiolate (BPEI/NONOate), a pH-responsive NO donor, was encapsulated into the hydrophilic core of PLGA nanoparticles. Furthermore, low concentrations of NO released at a consistent level via a stabilization effect obtained as amine groups of BPEI/NONOate interacted with the nearby NONOate. Using the controlled-release profiles, we successfully regulated the inflammatory response in lipopolysaccharide-stimulated peripheral blood mononuclear cells. This work demonstrates the potential of a NO delivery carrier in the regulation of inflammation.

Control of systemic inflammation through early nitric oxide supplementation with nitric oxide releasing nanoparticles

Amelioration of immune overactivity during sepsis is key to restoring hemodynamics, microvascular blood flow, and tissue oxygenation, and in preventing multi-organ dysfunction syndrome. The systemic inflammatory response syndrome that results from sepsis ultimately leads to degradation of the endothelial glycocalyx and subsequently increased vascular leakage. Current fluid resuscitation techniques only transiently improve outcomes in sepsis, and can cause edema. Nitric oxide (NO) treatment for sepsis has shown promise in the past, but implementation is difficult due to the challenges associated with delivery and the transient nature of NO. To address this, we tested the anti-inflammatory efficacy of sustained delivery of exogenous NO using i.v. infused NO releasing nanoparticles (NO-np). The impact of NO-np on microhemodynamics and immune response in a lipopolysaccharide (LPS) induced endotoxemia mouse model was evaluated. NO-np treatment significantly attenuated the pro-inflammatory response by promoting M2 macrophage repolarization, which reduced the presence of pro-inflammatory cytokines in the serum and slowed vascular extravasation. Combined, this resulted in significantly improved microvascular blood flow and 72-h survival of animals treated with NO-np. The results from this study suggest that sustained supplementation of endogenous NO ameliorates and may prevent the morbidities of acute systemic inflammatory conditions. Given that endothelial dysfunction is a common denominator in many acute inflammatory conditions, it is likely that NO enhancement strategies may be useful for the treatment of sepsis and other acute inflammatory insults that trigger severe systemic pro-inflammatory responses and often result in a cytokine storm, as seen in COVID-19.

Characterization of rat tail lymphatic contractility and biomechanics: incorporating nitric oxide-mediated vasoregulation.

The lymphatic system transports lymph from the interstitial space back to the great veins via a series of orchestrated contractions of chains of lymphangions. Biomechanical models of lymph transport, validated with ex vivo or in vivo experimental results, have proved useful in revealing novel insight into lymphatic pumping; however, a need remains to characterize the contributions of vasoregulatory compounds in these modelling tools. Nitric oxide (NO) is a key mediator of lymphatic pumping. We quantified the active contractile and passive biaxial biomechanical response of rat tail collecting lymphatics and changes in the contractile response to the exogenous NO administration and integrated these findings into a biomechanical model. The passive mechanical response was characterized with a three-fibre family model. Nonlinear regression and non-parametric bootstrapping were used to identify best-fit material parameters to passive cylindrical biaxial mechanical data, assessing uniqueness and parameter confidence intervals; this model yielded a good fit (R2 = 0.90). Exogenous delivery of NO via sodium nitroprusside (SNP) elicited a dose-dependent suppression of contractions; the amplitude of contractions decreased by 30% and the contraction frequency decreased by 70%. Contractile function was characterized with a modified Rachev-Hayashi model, introducing a parameter that is related to SNP concentration; the model provided a good fit (R2 = 0.89) to changes in contractile responses to varying concentrations of SNP. These results demonstrated the significant role of NO in lymphatic pumping and provide a predictive biomechanical model to integrate the combined effect of mechanical loading and NO on lymphatic contractility and mechanical response.

A three-dimensional assemblage of gingiva-derived mesenchymal stem cells and NO-releasing microspheres for improved differentiation

Stem cell therapy is an attractive approach to bone tissue regeneration. Nitric oxide (NO) has been reported to facilitate osteogenic differentiation of stem cells. To enhance osteogenic differentiation of gingiva-derived mesenchymal stem cells (GMSCs), we designed a method for in situ delivery of exogenous NO to these cells. A NO donor, polyethylenimine/NONOate, was incorporated into poly(lactic-co-glycolic acid) microspheres to deliver NO to the cells for an extended period of time under in vitro culture conditions. A hybrid aggregate of GMSCs and NO-releasing microspheres was prepared by the hanging drop technique. Confocal microscopy revealed homogeneous arrangement of the stem cells and microspheres in heterospheroids. Western blot analysis and live–dead imaging showed no significant change in cell viability. Importantly, the in situ delivery of NO within the heterospheroids enhanced osteogenic differentiation indicated by a 1.2-fold increase in alkaline phosphatase activity and an approximately 10% increase in alizarin red staining. In addition, a low dose of NO promoted proliferation of the GMSCs in this 3D system. Thus, delivery of the NO-releasing microsphers to induce differentiation of stem cells within this three dimensional system may be one of possible strategies to direct differentiation of a stem cell-based therapeutic agent toward a specific lineage.

Nitric Oxide Is Required for L-Type Ca(2+) Channel-Dependent Long-Term Potentiation in the Hippocampus.

Nitric oxide (NO) has long been implicated in the generation of long-term potentiation (LTP) and other types of synaptic plasticity, a role for which the intimate coupling between NMDA receptors (NMDARs) and the neuronal isoform of NO synthase (nNOS) is likely to be instrumental in many instances. While several types of synaptic plasticity depend on NMDARs, others do not, an example of which is LTP triggered by opening of L-type voltage-gated Ca2+ channels (L-VGCCs) in postsynaptic neurons. In CA3-CA1 synapses in the hippocampus, NMDAR-dependent LTP (LTPNMDAR) appears to be primarily expressed postsynaptically whereas L-VGCC-dependent LTP (LTPL−VGCC), which often coexists with LTPNMDAR, appears mainly to reflect enhanced presynaptic transmitter release. Since NO is an excellent candidate as a retrograde messenger mediating post-to-presynaptic signaling, we sought to determine if NO functions in LTPL−VGCC in mouse CA3-CA1 synapses. When elicited by a burst type of stimulation with NMDARs and the associated NO release blocked, LTPL−VGCC was curtailed by inhibition of NO synthase or of the NO-receptor guanylyl cyclase to the same extent as occurred with inhibition of L-VGCCs. Unlike LTPNMDAR at these synapses, LTPL−VGCC was unaffected in mice lacking endothelial NO synthase, implying that the major source of the NO is neuronal. Transient delivery of exogenous NO paired with tetanic synaptic stimulation under conditions of NMDAR blockade resulted in a long-lasting potentiation that was sensitive to inhibition of NO-receptor guanylyl cyclase but was unaffected by inhibition of L-VGCCs. The results indicate that NO, acting through its second messenger cGMP, plays an unexpectedly important role in L-VGCC-dependent, NMDAR-independent LTP, possibly as a retrograde messenger generated in response to opening of postsynaptic L-VGCCs and/or as a signal acting postsynaptically, perhaps to facilitate changes in gene expression.

Ruthenium nitrosyl grafted carbon dots as a fluorescence-trackable nanoplatform for visible light-controlled nitric oxide release and targeted intracellular delivery

Nitric oxide (NO) plays a key role in various physiological and pathological processes. It is of great significance in developing a platform that enables exogenous delivery of NO spatiotemporally to a targeted site for realizing NO-mediated therapy. We report herein a stable, multifunctional NO-delivery nanoplatform that is capable of target directing, fluorescence tracking, and light-controlled NO delivery. A ruthenium nitrosyl [Ru(TPYCOOH)(o-phenylenediamine)(NO)](PF6)3 and a target-directing molecule of folic acid (FA) were covalently grafted onto the surface of a carrier of carbon dots (CDs), forming a {Ru-NO@FA@CDs} nanoplatform. This nanoplatform is fluorescence self-trackable in a cellular environment and can recognize specific cancer cells via FA–folate receptor binding; it was selectively taken up by cancer cells to enter the cytosol, in which localized NO was produced on demand by adept control of visible light illumination. This offered the potential for treating diseases derived from NO deficiency as well as NO-mediated cancer therapy.

Delivering nitric oxide with nanoparticles

While best known for its important signalling functions in human physiology, nitric oxide is also of considerable therapeutic interest. As such, nanoparticle-based systems which enable the sustained exogenous delivery of nitric oxide have been the subject of considerable investigation in recent years. Herein we review the various nanoparticle systems that have been used to date for nitric oxide delivery, and explore the array of potential therapeutic applications that have been reported. Specifically, we discuss the modification of sol–gel based silica particles, functionalised metal/metal oxide nanoparticles, polymer-coated metal nanoparticles, dendrimers, micelles and star polymers to impart nitric oxide release capability. We also consider the various areas in which therapeutic applications are envisaged: wound healing, antimicrobial applications, cardiovascular treatments, sexual medicine and cancer treatment. Finally, we discuss possible future directions for this versatile and potentially important technology.

Nitric oxide nanoparticles for wound healing: future directions to overcome challenges

Chronic, nonhealing wounds inflict significant patient morbidity and mortality and impose considerable economic burdens to health care systems. This has prompted investigations of the potential wound healing therapies, and exogenous delivery of nitric oxide (NO) is one avenue that has been explored. NO is crucial for normal wound repair: it mediates many vital processes that take place after cutaneous injury and helps to ward off invading pathogens due to its inherent antimicrobial and cytostatic properties. Because of its dual wound repair and antimicrobial features, exogenous NO delivery for the acceleration of wound healing is an attractive therapeutic strategy. Several exogenous NO delivery methods have been studied, but limitations preclude their widespread clinical use. Nanotechnology can be exploited for therapeutic delivery of a variety of active ingredients including NO. A hybrid hydrogel/glass composite NO-releasing nanoparticles platform has demonstrated wound healing efficacy both in vitro and...

Use of nitric oxide nanoparticulate platform for the treatment of skin and soft tissue infections

The incidence of skin and soft tissue infections (SSTI) due to multi‐drug resistant pathogens is increasing. The concomitant increase in antibiotic use along with the ease with which organisms develop mechanisms of resistance have together become a medical crisis, underscoring the importance of developing innovative and effective antimicrobial strategies. Nitric oxide (NO) is an endogenously produced molecule with many physiologic functions, including broad spectrum antimicrobial activity and immunomodulatory properties. The risk of resistance to NO is minimized because NO has multiple mechanisms of antimicrobial action. NO's clinical utility has been limited largely because it is highly reactive and lacks appropriate vehicles for storage and delivery. To harness NO's antimicrobial potential, a variety exogenous NO delivery platforms have been developed and evaluated, yet limitations preclude their use in the clinical setting. Nanotechnology represents a paradigm through which these limitations can be overcome, allowing for the encapsulation, controlled release, and focused delivery of NO for the treatment of SSTI. WIREs Nanomed Nanobiotechnol 2013. doi: 10.1002/wnan.1230This article is categorized under: 1Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease2Nanotechnology Approaches to Biology > Nanoscale Systems in Biology

Clay based materials for storage and therapeutic release of nitric oxide.

Nitric oxide (NO) is one of the smallest endogenous molecules with particularly interesting aspects roles in biological systems, despite its toxicological potential. Solid carriers have potential biomedical interest in the delivery of exogenous NO for anti-bacterial, anti-thrombic and wound healing applications. In this work, we prepared nanoporous materials from clay based solids, of montmorillonite type, with the main goal of studying its potential in the field of storage and release of nitric oxide for therapeutic applications. Materials were characterized by X-ray diffraction, nitrogen adsorption at -196 °C, TG-DSC and chemical analysis. The kinetic data for the nitric oxide storage and release, in selected materials, was obtained in gas and liquid phases. The released amounts of NO in the liquid phase were within the biological range and, for some materials, a slow release kinetics was obtained with, inclusively, an almost direct relation between the released fraction and time. Toxicological assays with HeLa cells indicated that the materials have low cytotoxicity.

Effects of Slow, Sustained, and Rate‐Tunable Nitric Oxide Donors on Human Aortic Smooth Muscle Cells Proliferation

Smooth muscle cell (SMC) proliferation has been accepted as a common event in the pathophysiology of vascular diseases, including atherogenesis and intimal hyperplasia. Delivery of the nitric oxide synthase (NOS) substrate l-arginine, pharmacological nitric oxide (NO) donors, NO gas or overexpression of NOS proteins can inhibit SMC proliferation and reduce the injury responses within the blood vessel wall. Although commercial development of NO donors that attempt to provide exogenous delivery of NO has accelerated over the last few years, none of the currently available products can provide controlled, sustained, time-tunable release of NO. Nitrosamine-based NO donors, prepared in our laboratory, present a unique and innovative alternative for possible treatments for long-term NO deficiency-related diseases, including atherosclerosis, asthma, erectile dysfunction, cancer, and neurodegenerative diseases. A family of secondary amines prepared via nucleophilic aromatic displacement reactions could be readily N-nitrosated to produce NO donors. NO release takes place in three distinct phases. During the initial phase, the release rate is extremely fast. In the second phase, the release is slower and the rate remains essentially the same during the final stage. These compounds inhibited up to 35% human aortic smooth muscle cell proliferation in a concentration-dependent manner.

Invited Commentary

Pulmonary hypertension, regardless of the cause, represents a significant challenge to cardiothoracic surgeons with regard to preoperative risk assessment, perioperative management, and postoperative outcomes. Despite multiple pharmacologic strategies, including prostanoids, endothelin-receptor antagonists, and phosphodiesterase inhibitors, pulmonary hypertension remains a source of significant morbidity and mortality. One of the most recognized regulators of pulmonary vascular vasomotor tone is nitric oxide (NO). Otherwise known as endothelial-derived relaxation factor, NO is biosynthesized from L-arginine in the vasculature by two forms of nitric oxide synthase: constitutive nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS). Upregulation of endogenous NO or exogenous delivery of NO has been well studied in a variety of cardiovascular models as well as humans. Indeed adenoviral gene transfer of eNOS has been previously reported to ameliorate monocrotaline and hypoxia-induced pulmonary hypertension. Zhang and colleagues [1Zhang F. Wu S. Lu X. Wang M. Liu M. Gene transfer of endothelial nitric oxide synthase attenuates flow-induced pulmonary hypertension in rabbits.Ann Thorac Surg. 2008; 85: 581-586Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar] report a therapeutically-aimed study on the effects of tracheal-delivered adenoviral eNOS gene transfer in a rabbit model of pulmonary overcirculation. They establish a background associated with congenital heart disease by performing pre-tricuspid shunts (carotid-jugular bypass). They demonstrate successful delivery of the gene and achieve a functional response by significantly decreasing pulmonary vascular resistance. The model seems challenging as only 18 of 40 animals survived or developed significant pulmonary hypertension. Although their biochemical data is firm, some of the kinetics associated with gene transfer remains nebulous. Quite frankly, the effects of only 10 minutes of adenoviral exposure are impressive. Notably missing are the data related to transfection efficiency and time course of activity. They surveyed tissues after 4 days for gene expression, but hemodynamic testing was done at 3 months. Adenoviral gene expression is usually lost by 3 to 4 weeks after transduction. It remains unknown if pulmonary hypertension returns when transfected eNOS gene expression ceases. Furthermore, the effects of endogenous NO compared with the transgene are unable to be deciphered, and thus, the effect of exogenous eNOS on their physiologic outcomes comes into question. Although they claim that eNOS was mostly expressed in the endothelial cells of resistance size vessels, it would have been nice to see immunohistochemical evidence of uptake of eNOS in other locations in the tracheobronchial tree or cell-type (ie, vascular smooth muscle). This is especially important as many other reports have demonstrated tracheal delivery of transgenes to be located in airway epithelial cells. Finally, others have shown that this model of flow-induced pulmonary hypertension causes an increased expression of native eNOS. Why would delivery of even more eNOS help? Because there is no true control group in their study, they are unable to comment on this paradox. They speculate that although eNOS is elevated, its biologic activity is decreased because the endothelial function is impaired. As such, it would have been nice to see some assessment of endothelial function in their model. Despite some of the specific limitations just outlined, their results offer additional evidence particularly related to NO for adopting translational motivated therapies for cardiopulmonary disease. The pulmonary vasculature is easily accessible and offers a clinically relevant avenue for using such innovative technology. Indeed, inhaled NO has proven effective for short-term treatment of adult and newborn acute pulmonary hypertension. Although L-arginine has gained popularity for treating patients with chronic pulmonary hypertension, no randomized study has documented its long-term effects, particularly in light of some of its pro-proliferative metabolites. As such, direct manipulation of the nitric oxide and nitric oxide synthase axis with locally delivered eNOS could offer an effective strategy for this difficult patient population. Gene Transfer of Endothelial Nitric Oxide Synthase Attenuates Flow-Induced Pulmonary Hypertension in RabbitsThe Annals of Thoracic SurgeryVol. 85Issue 2PreviewNitric oxide, a potent vasodilator with an important role in the regulation of pulmonary vascular tone, is synthesized by a family of nitric oxide synthases. To determine whether endothelial nitric oxide synthase (eNOS) gene transfer may prevent pulmonary hypertension, the effects of transfer of the eNOS gene to the lung were studied in rabbits with pulmonary hypertension induced by high pulmonary blood flow. Full-Text PDF

Dysfunction of nitric oxide synthases as a cause and therapeutic target in delayed cerebral vasospasm after SAH.

Nitric oxide (NO), also known as endothelium-derived relaxing factor, is produced by endothelial nitric oxide synthase (eNOS) in the intima and by neuronal nitric oxide synthase (nNOS) in the adventitia of cerebral vessels. It dilates the arteries in response to shear stress, metabolic demands, pterygopalatine ganglion stimulation, and chemoregulation. Subarachnoid haemorrhage (SAH) interrupts this regulation of cerebral blood flow. Hemoglobin, gradually released from erythrocytes in the subarachnoid space destroys nNOS-containing neurons in the conductive arteries. This deprives the arteries of NO, leading to the initiation of delayed vasospasm. But such vessel narrowing increases shear stress, which stimulates eNOS. This mechanism normally would lead to increased production of NO and dilation of arteries. However, a transient eNOS dysfunction evoked by an increase of the endogenous competitive nitric oxide synthase (NOS) inhibitor, asymmetric dimethyl-arginine (ADMA), prevents this vasodilation. eNOS dysfunction has been recently shown to be evoked by increased levels of ADMA in CSF in response to the presence of bilirubin-oxidized fragments (BOXes). A direct cause of the increased ADMA CSF level is most likely decreased ADMA elimination due to the disappearance of ADMA-hydrolyzing enzyme (DDAH II) immunoreactivity in the arteries in spasm. This eNOS dysfunction sustains vasospasm. CSF ADMA levels are closely associated with the degree and time-course of vasospasm; when CSF ADMA levels decrease, vasospasm resolves. Thus, the exogenous delivery of NO, inhibiting the L-arginine-methylating enzyme (IPRMT3) or stimulating DDAH II, may provide new therapeutic modalities to prevent and treat vasospasm. This paper will present results of preclinical studies supporting the NO-based hypothesis of delayed cerebral vasospasm development and its prevention by increased NO availability.

Analysis of Nitric Oxide (NO) in Cerebral Vasospasm After Aneursymal Bleeding

Nitric oxide (NO) is produced by the endothelial NOS (eNOS) in the intima and by the neuronal NOS (nNOS) in the adventitia of cerebral vessels. By activating soluble guanylyl cyclase, NO increases the production of 3'-5'cGMP, which relaxes smooth muscle cells and dilates the arteries in response to shear stress, metabolic demands and changes of pCO(2) (chemoregulation). 3'-5'cGMP is then metabolized by phosphodiesterases (PDEs). Aneurysmal subarachnoid hemorrhage (SAH) interrupts this regulation of cerebral blood flow (CBF). Oxyhemoglobin, gradually released from the subarachnoid clot enveloping the conductive arteries, scavenges NO and destroys nNOS-containing neurons. This deprives the arteries of NO, leading to vasoconstriction which initiates delayed vasospasm. This arterial narrowing increases shear stress and stimulates eNOS, which under normal conditions would lead to increased production of NO and dilation of arteries. However, this does not occur because of transient eNOS dysfunction evoked by increased levels of an endogenous NOS inhibitor, asymmetric dimethylarginine (ADMA). Increased ADMA levels result from decreased elimination due to inhibition of the ADMA-hydrolyzing enzyme (DDAH 2) in arteries in spasm by hemoglobin metabolites, bilirubin-oxidized fragments (BOXes). This eNOS dysfunction sustains vasospasm until ADMA levels decrease and NO release from endothelial cells increases. This NO-based pathophysiological mechanism of vasospasm suggests that exogenous delivery of NO, modification of PDE activity, inhibition of the L-arginine-methylating enzyme (I PRMT 3) or stimulation of DDAH 2 may provide new therapies to prevent and treat vasospasm. This paper summarizes experimental and early clinical data that are consistent with the involvement of NO in delayed cerebral vasospasm after SAH and which suggests new therapeutic possibilities.

Dysfunction of nitric oxide synthases as a cause and therapeutic target in delayed cerebral vasospasm after SAH

Nitric oxide (NO), also known as endothelium-derived relaxing factor, is produced by endothelial nitric oxide synthase (eNOS) in the intima and by neuronal nitric oxide synthase (nNOS) in the adventitia of cerebral vessels. It dilates the arteries in response to shear stress, metabolic demands, pterygopalatine ganglion stimulation and chemoregulation. Subarachnoid hemorrhage (SAH) interrupts this regulation of cerebral blood flow. Hemoglobin, gradually released from erythrocytes in the subarachnoid space, destroys nNOS-containing neurons in the conductive arteries. This deprives the arteries of NO, leading to initiation of delayed vasospasm. But such vessel narrowing increases shear stress, which stimulates eNOS. This mechanism normally would lead to increased production of NO and dilation of arteries. However, a transient eNOS dysfunction evoked by an increase in the endogenous competitive NOS inhibitor, asymmetric dimethylarginine (ADMA), prevents this vasodilation. eNOS dysfunction has been recently shown to be evoked by increased levels of ADMA in cerebrospinal fluid (CSF) in response to the presence of bilirubin-oxidized fragments (BOXes). A direct cause of the increased ADMA CSF level is most likely decreased ADMA elimination owing to disappearance of ADMA-hydrolyzing enzyme [dimethylarginine dimethylaminohydrolase II (DDAH II)] immunoreactivity in the arteries in spasm. This eNOS dysfunction sustains vasospasm. CSF ADMA levels are closely associated with the degree and time course of vasospasm; when CSF ADMA levels decrease, vasospasm resolves. Thus, exogenous delivery of NO, inhibiting the L-arginine-methylating enzyme or stimulating DDAH II, may provide new therapeutic modalities to prevent and treat vasospasm. This paper will present results of pre-clinical studies supporting the NO-based hypothesis of delayed cerebral vasospasm development and its prevention by increased NO availability.