Posts Tagged ‘nitric oxide’

Nitric Oxide Synthase Inhibitors (NOS-I)

Author: Larry H Bernstein, MD, FCAP

Curator: Stephen J. Williams, PhD


Co-Curator: Aviva Lev-Ari, PhD, RN


This recent article sheds a new light on nitric oxide and the activity of NOS in reactive oxygen species generation and the effect of NOS inhibitors in bacteria.

Structural and Biological Studies on Bacterial Nitric Oxide Synthase Inhibitors

Jeffrey K. Holdena, Huiying Lia, Qing Jingb, Soosung Kangb, Jerry Richoa, Richard B. Silvermanb,1, and Thomas L. Poulosb,1
Author contributions: J.K.H. designed research; J.K.H. and J.R. performed research; Q.J. and S.K. contributed new reagents/analytic tools; J.K.H., H.L., R.B.S., and T.L.P. analyzed data; and J.K.H., R.B.S., and T.L.P. wrote the paper.

PNAS Oct 21, 2013;       http://dx.doi.org/10.1073/pnas.1314080110
This article is a PNAS Direct Submission
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank
Edited by Douglas C. Rees, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, and approved September 23, 2013 (received for review July 29, 2013)
Keywords:  crystallography, antibiotics, nitric oxide, NOS inhibitors, Bacillus subtilis, gram positive bacteria


Nitric oxide (NO) produced by bacterial nitric oxide synthase has recently been shown to

Using Bacillus subtilis as a model system, we identified

  • two NOS inhibitors that work in conjunction with an antibiotic to kill B. subtilis.

Moreover, comparison of inhibitor-bound crystal structures between the bacterial NOS and mammalian NOS revealed an unprecedented

  • mode of binding to the bacterial NOS that can be further exploited for future structure-based drug design.

Overall, this work is an important advance in developing inhibitors against gram-positive pathogens.


Nitric oxide (NO) produced by bacterial NOS functions as

  • a cytoprotective agent against oxidative stress in Staphylococcus aureusBacillus anthracis, and Bacillus subtilis.

The screening of several NOS-selective inhibitors uncovered two inhibitors with potential antimicrobial properties. These two compounds

  • impede the growth of B. subtilis under oxidative stress, and
  • crystal structures show that each compound exhibits a unique binding mode.

Both compounds serve as excellent leads for the future development of antimicrobials against bacterial NOS-containing bacteria.

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Reporter/Curator: Stephen J. Williams, Ph.D.

Picture of a human melanoma cell line growing in tissue culture

Cultured human melanocytes .

Nitric oxide (NO), a gas with many biological functions in healthy cells, has also been implicated in the development of pathologies such as cancer.  Nitric oxide may also play a role in chemotherapeutic reisitance. For example it had been known (in the 1996 Melanoma study by Joshi et al. curated below) that nitric oxide synthase activity (the enzyme system which produces NO) was significantly elevated in cultured melanoma cell lines versus normal melanocytes.   Although it is known that many protein and enzymes systems could be directly covalently-modified by nitric oxide, either by S-nitrosylation or NO-NAD+ modifications (one of my earlier postings described one such protein modified by nitric oxide, GAPDH, and the effect these NO-modifications of GAPDH has on the etiology of various pathologies.), the molecular mechanisms by which these modifications affect cellular processes, lead to disease etiology, the proteins which are affected, and mechanisms related to chemotherapeutic sensitivity need to be further characterized. A new study from MIT reveals how NO-induced modifications may reduce cisplatin sensitivity in melanoma cells.  This study focuses on how decreasing nitric oxide levels in melanoma cells increases their cisplatin sensitivity.  The study also describes a possible mechanism for this effect: NO-induced modifications of the proapoptotic enzyme caspace-3 and prolyl-hdroxylase-2 (responsible for targeting prosurvival HIF-1α for proteosomal degradation).  Also, for a description of other cancer-related targets of nitric oxide please see the posting by Dr. Saxena at Crucial role of Nitric Oxide in Cancer on this site.

To read more background on nitric oxide and its role in disease etiology please see our e-book Perspectives on Nitric Oxide in Disease Mechanisms (Biomed e-Books) available on Amazon at:


      It is important, however, to note that most of these relationships between NO-induced protein modification and its relationship to disease mechanisms are causal, meaning that, in general, one notices a nitric-induced modification of a protein/enzyme with concomitant alteration of protein/enzyme function occurring in a disease/phenotype.  However, unlike reversible modifications, which have a cadre of pharmacologic inhibitors, nitric oxide induced modifications are covalent and nonenzymatic, therefore hindering easy cause/effect relationships.

With that said, the following was adapted from the MIT site at http://web.mit.edu/newsoffice/2013/how-melanoma-evades-chemotherapy-0407.html.



The findings from Dr. Luiz Godoy’s PNAS paper ENDOGENOUSLY PRODUCED NITRIC OXIDE MITIGATES SENSITIVITY OF MELANOMA CELLS TO CISPLATIN,  were presented at the 2013 annual meeting of the American Association for Cancer Research. The prognosis is generally worse for patients whose tumors have high levels of NO, said Luiz Godoy, an MIT research associate and lead author of the study.

Godoy and his colleagues have unraveled the mechanism behind melanoma’s resistance to cisplatin, a commonly used chemotherapy drug, and, in ongoing studies, have found that cisplatin treatment also increases NO levels in breast and colon cancers.

“This could be a mechanism that is widely shared in different cancers, and if you use the drugs that are already used to treat cancer, along with other drugs that could scavenge or decrease the production of NO, you may have a synergistic effect,” said Godoy, who works in the lab of Gerald Wogan, an MIT professor emeritus of biological engineering and senior author of the study.

NO has many roles within living cells. At low concentrations, it helps regulate processes such as cell death and muscle contraction. NO, which is a free radical, is also important for immune-system function. Immune cells, such as macrophages, produce large amounts of NO during infection, helping to kill invading microbes by damaging their DNA or other cell components.

“It’s really a molecule that has a dual effect,” Godoy said. “At low concentrations it can act as a signaling molecule, while high concentrations will be toxic.”

Knocking out NO

In the new study, the researchers treated melanoma cells grown in the lab with drugs that capture NO before it can act. They then treated the cells with cisplatin and tracked cell-death rates. The NO-depleted cells became much more sensitive to the drug, confirming earlier findings.

The MIT team then went a step further, investigating how NO confers its survival benefits. It was already known that NO can alter protein function through a process known as S-nitrosation, which involves attaching NO to the target protein. S-nitrosation can affect many proteins, but in this study the researchers focused on two that are strongly linked with cell death and survival, known as caspase-3 and PHD2.

The role of caspase-3 is to stimulate cell suicide, under the appropriate conditions, but adding NO to the protein deactivates it. This prevents the cell from dying even when treated with cisplatin, a drug that produces massive DNA damage.

PHD2 is also involved in cell death; its role is to help break down another protein called HIF-1 alpha, which is a pro-survival protein. When NO inactivates PHD2, HIF-1 alpha stays intact and keeps the cell alive.

“Now we have a mechanistic link between nitric oxide and the increased aggressiveness of melanoma,” said Douglas Thomas, an assistant professor of medicinal chemistry and pharmacognosy at the University of Illinois at Chicago, who was not part of the research team. “It certainly would be worth exploring whether this mechanism is also present in different tumor types as well.”

The MIT researchers also found in some cancer cells, NO levels were five times higher than normal following cisplatin treatment. Godoy is now investigating how cisplatin stimulates that NO boost, and is also looking for other proteins that NO may be targeting.

Source: http://web.mit.edu/newsoffice/2013/how-melanoma-evades-chemotherapy-0407.html

Melanoma Res. 1996 Apr;6(2):121-6.

Nitric oxide synthase activity is up-regulated in melanoma cell lines: a potential mechanism for metastases formation.

Joshi M, Strandhoy J, White WL.


Department of Dermatology, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27157, USA.


Nitric oxide (NO) may be an important mediator of tumour angiogenesis and metastasis formation. Tumour cell derived NO may be important in the regulation of angiogenesis and vasodilatation of the blood vessels surrounding a tumour. The aims of the present study were, firstly, to determine whether malignant melanoma cells and normal melanocytes had nitric oxide synthase (NOS) activity (measured by the conversion of L-arginine to L-citrulline) and, secondly, to determine whether there was a difference in NOS activity between malignant and normal cell types. This paper assays NOS activity directly in lysates from normal human melanocyte and malignant melanoma cell lines. The enzyme activity was not inducible with bacterial lipopolysaccharide and could be heat denatured. The activity of NOS was demonstrated to be both NADPH- and calcium-dependent and it was inhibitable in a dose-dependent manner by the NOS inhibitor Nw-nitro-L-arginine methyl ester. We conclude that melanoma and melanocyte cells express a constitutive form of NOS. Finally, nitric oxide synthase activity in melanoma cell lines was found to be significantly greater than in normal melanocytes. These findings suggest that NO synthesis is elevated in malignant melanoma. An elevated NO concentration in melanoma is expected to promote metastases by maintaining a vasodilator tone in the blood vessels in and around the melanoma.

Proc Natl Acad Sci U S A. 2012 Dec 11;109(50):20373-8. doi: 10.1073/pnas.1218938109. Epub 2012 Nov 26.

Endogenously produced nitric oxide mitigates sensitivity of melanoma cells to cisplatin.

Godoy LC, Anderson CT, Chowdhury R, Trudel LJ, Wogan GN.


Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.


Melanoma patients experience inferior survival after biochemotherapy when their tumors contain numerous cells expressing the inducible isoform of NO synthase (iNOS) and elevated levels of nitrotyrosine, a product derived from NO. Although several lines of evidence suggest that NO promotes tumor growth and increases resistance to chemotherapy, it is unclear how it shapes these outcomes. Here we demonstrate that modulation of NO-mediated S-nitrosation of cellular proteins is strongly associated with the pattern of response to the anticancer agent cisplatin in human melanoma cells in vitro. Cells were shown to express iNOS constitutively, and to generate sustained nanomolar levels of NO intracellularly. Inhibition of NO synthesis or scavenging of NO enhanced cisplatin-induced apoptotic cell death. Additionally, pharmacologic agents disrupting S-nitrosation markedly increased cisplatin toxicity, whereas treatments favoring stabilization of S-nitrosothiols (SNOs) decreased its cytotoxic potency. Activity of the proapoptotic enzyme caspase-3 was higher in cells treated with a combination of cisplatin and chemicals that decreased NO/SNOs, whereas lower activity resulted from cisplatin combined with stabilization of SNOs. Constitutive protein S-nitrosation in cells was detected by analysis with biotin switch and reduction/chemiluminescence techniques. Moreover, intracellular NO concentration increased significantly in cells that survived cisplatin treatment, resulting in augmented S-nitrosation of caspase-3 and prolyl-hydroxylase-2, the enzyme responsible for targeting the prosurvival transcription factor hypoxia-inducible factor-1α for proteasomal degradation. Because activities of these enzymes are inhibited by S-nitrosation, our data thus indicate that modulation of intrinsic intracellular NO levels substantially affects cisplatin toxicity in melanoma cells. The underlying mechanisms may thus represent potential targets for adjuvant strategies to improve the efficacy of chemotherapy.

Other posts on this site regarding Nitric Oxide and Cancer include:

Crucial role of Nitric Oxide in Cancer

Nitric Oxide Covalent Modifications: A Putative Therapeutic Target?

Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function

Nitric Oxide Signalling Pathways

In focus: Melanoma therapeutics

Combined anti-CTLA4 and anti-PD1 immunotherapy shows promising results against advanced melanoma

Whole exome somatic mutations analysis of malignant melanoma contributes to the development of personalized cancer therapy for this disease

In focus: Melanoma therapeutics

In focus: Melanoma Genetics

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Cardiovascular Original Research: Cases in Methodology Design for Content Curation and Co-Curation

Author: Aviva Lev-Ari, PhD, RN

For a general article on Science and Curation, go to

Science and Curation: the New Practice of Web 2.0

Since 4/2012, Leaders in Pharmaceutical Business Intelligence, is developing an innovative methodology for the facilitation of Global access to Biomedical knowledge rather than the access to sheer search results on Scientific subject matters in the Life Sciences and Medicine. For the methodology to attain this complex goal it is to be dealing with popularization of ORIGINAL Scientific Research via Content Curation of Scientific Research Results by Experts, Authors, Writers using the critical  thinking process of expert interpretation of the original research results. We demonstrate in this article two approaches to the process of reaching that goal successfully.

Editorial Team Members and Five Series of e-Bookd in BioMed

Series A: e-Books on Cardiovascular Diseases

Content Consultant: Justin D Pearlman, MD, PhD, FACC

Volume One: Perspectives on Nitric Oxide

Sr. Editor: Larry Bernstein

Editor: Aviral Vatsa

Content Consultant: Stephen J Williams

available on Kindle Store @ Amazon.com


Volume Two: Cardiovascular Original Research: Cases in Methodology Design for Content Co-Curation

Curators: Justin D Pearlman, Larry H Bernstein, Aviva Lev-Ari

  • Causes
  • Risks and Biomarkers
  • Therapeutic Implications

Volume Three: Etiologies of CVD: Epigenetics, Genetics & Genomics

Curators: Larry H Bernstein and Aviva Lev-Ari

  • Causes
  • Risks and Biomarkers
  • Therapeutic Implications

Chapter 1: Genomics and Medicine by Marcus Feldman

Volume Four: Therapeutic Promise: CVD, Regenerative & Translational Medicine

Curators: Larry H Bernstein and Aviva Lev-Ari

  • Causes
  • Risks and Biomarkers
  • Therapeutic Implications

Volume Five: Pharmaco-Therapies for CVD

Curators: Vivek Lal, Larry H Bernstein and Aviva Lev-Ari

  • Causes
  • Risks and Biomarkers
  • Therapeutic Implications

Volume Six: Interventional Cardiology and Cardiac Surgery

Curators: Justin D Pearlman, Larry H Bernstein, Aviva Lev-Ari

  • Causes
  • Risks and Biomarkers
  • Therapeutic Implications

Volume Seven: CVD Imaging for Disease Diagnosis and Guidance of Treatment

Curators: Justin D Pearlman and Aviva Lev-Ari

  • Causes
  • Risks and Biomarkers
  • Therapeutic Implications

Series B: e-Books on Genomics & Medicine

Content Consultant: Larry H Bernstein, MD, FCAP

Volume 1: Genomics and Individualized Medicine

Sr. Editor: Stephen J Williams

Editors: Larry H Bernstein and Aviva Lev-Ari

Volume 2: Methodological Breakthroughs in NGS

Editor: Marcus Feldman

Volume 3: Institutional Leadership in Genomics

Editors: Marcus Feldman and Aviva Lev-Ari 

Series C: e-Books on Cancer & Oncology

Content Consultant: Larry H Bernstein, MD, FCAP

Volume 1: Cancer and Genomics

Sr. Editor: Stephen J Williams

Editors: Ritu Saxena, Tilda Barliya

Volume 2: Immunotherapy in Oncology

Sr. Editor: Stephen J Williams

Editors: Tilda Barliya and Demet Sag

Volume 3: Nanotechnology and Drug Delivery

Editor and Author: Tilda Barliya

Series D: e-Books on BioMedicine

Volume 1: Metabolomics

Sr. Editors: Larry H Bernstein and

Editor: Ritu Saxena 

Volume 2: Infectious Diseases

Editor: TBA

Volume 3: Immunology and Therapeutics

Editor: TBA

Series E: Titles in the Strategic Plan for 2014 – 2015

Volume 1: The Patient’s Voice: Personal Experience with Invasive Medical Procedures

Editor: TBA 

Volume 2: Interviews with Scientific Leaders

Editor: TBA

Volume 3: Infectious Milestones in Physiology – Discoveries in Medicine

Editor: TBA

[affiliate] Dr. Pnina G. Abir-Am, Belmont, MA – Independent AUTHOR, History of Molecular Biology

Dr. Aviva Lev-Ari, Boston, MA – Editor-in-Chief, BioMed Series, Editor – Genomics Volume One

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This article has two parts:

Part I: The Curator as a Scientific Content Critique for the Architecture of Knowledge, its meaning and its societal implications.

Part II: Cases in Co-Curation and Scientific Content Critique

In Part I, one curator edifies the e-Reader via his/hers OWN creative mental processes of knowledge synthesis following the creative mental process of analytical critique. The outcome is a new FORM of writing Science and of writing about Science, as well as, a new FORM of framework been created for the organization of the interrelations exposed in the analytical phase of a dialectically generated original synthesis, the process of which is manifold: the structure of the knowledge presented, culling in the midst of inclusion/exclusion dialectics and finally the Curator’s own original synthetic statements of the new Art, a new conceptual perspective on Science.

  • For our VISION, See


  • For periodic updates to the List of Cases developed by this Author/Curator, see


  • For a complete contribution to the Open Access Online Scientific Journal by the Author/Curator, see

http://pharmaceuticalintelligence.com — Search by Author/Curator’s Last Name, 567 articles on 7/30/2013

  • For the BioMed e-Books Series in Production, see


  • FIRST book of their BioMedical E-Book Series, Perspectives on Nitric Oxide in Disease Mechanisms, now available on Amazon.com Kindle Store


  • For CV of our entire Team of Experts, Authors, Writers, see


In part Part II: Cases in Co-Curation and Scientific Content Critique, are presented. A similar process to the one in Part I, is been applied. However, the Co-Curation, brings on stage several players. The Actors in the Scientific Writers Theater,  all own scientific knowledge and master the process of creation of a new Synthesis for most writing engagements. Since the Co-curators are educated in different disciplines, they are skillfully providing interpretations for others’ and their own new conception of ideas. Thus, they are developing new views of the original scientific results presented in peer reviewed journals, just the leading ones in every field. The Co-Curators, their creation is a new layer of comprehension for the processes at hand.

Example #1:

Action Potential, a well define concept in Physiology. For us,  Action Potential was a conceptual creation for the process of Co-Curation. Dr. Lev-Ari, requesting Dr. Bernstein to elaborate creatively, on the function of actin in cytoskeleton mobility, he did,  THEN a new conceptual creation process emerged and had YIELDED the following article:

Identification of Biomarkers that are Related to the Actin Cytoskeleton

Curator: Larry H Bernstein, MD, FCAP


Example #2:

The e-Reader reads first

High Serum Calcium Linked to Developing Diabetes: IRAS Study

 Sep 24, 2013


The e-Reader reads second the curation of that Source Interview

Diabetes-risk Forecasts: Serum Calcium in Upper-Normal Range (>2.5 mmol/L) as a New Biomarker


The e-Reader will compare which of the two is more beneficial for the e-Reader.

We believe that the curation of the Source Interview has remarkable value added analysis that the Reader can benefit from.

The unique process as described for Part I and for Part II, above, will be demonstrated, below,  in concrete cases, as we applied the methodology of curation by one or by several Experts, Authors, Writers in the field of Cardiovascular Diseases.

The Process: We culled the scene for Cardiovascular Original Research in +24 Journals, we pre-select domains of research to cover: The Etiology of the Disease, the Risks of dysfunction at cellular, tissue, organelle, organ, anatomy, physiology, pathophysiology and diagnostics for all of the above. We interpret the Disease Management Options in a comprehensive fashion, exposing the e-Reader to an integrative approach for the treatment of Cardiovascular Disease.

Below,  the e-Reader finds selective cases exemplifying the methodology described, making

the one and only on the Internet and in e-Book Stores, to date.


Part I       

The Curator as a Scientific Content Critique for the Architecture of Knowledge

Lev-Ari, A. 8/6/2013 Stent Design and Thrombosis:  Bifurcation Intervention, Drug Eluting Stents (DES) and Biodegrable Stents


Lev-Ari, A. 8/1/2013 Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD


Lev-Ari, A. 7/19/2013 3D Cardiovascular Theater – Hybrid Cath Lab/OR Suite, Hybrid Surgery, Complications Post PCI and Repeat Sternotomy


Lev-Ari, A. 7/14/2013 Vascular Surgery: International, Multispecialty Position Statement on Carotid Stenting, 2013 and Contributions of a Vascular Surgeon at Peak Career – Richard Paul Cambria, MD


Lev-Ari, A. 7/9/2013 Heart Transplant (HT) Indication for Heart Failure (HF): Procedure Outcomes and Research on HF, HT @ Two Nation’s Leading HF & HT Centers


Lev-Ari, A. 7/8/2013 Becoming a Cardiothoracic Surgeon: An Emerging Profile in the Surgery Theater and through Scientific Publications


Lev-Ari, A. 7/1/22013 Endovascular Lower-extremity Revascularization Effectiveness: Vascular Surgeons (VSs), Interventional Cardiologists (ICs) and Interventional Radiologists (IRs)


Lev-Ari, A. 6/10/2013 No Early Symptoms – An Aortic Aneurysm Before It Ruptures – Is There A Way To Know If I Have it?


Lev-Ari, A. 6/9/2013 Congenital Heart Disease (CHD) at Birth and into Adulthood: The Role of Spontaneous Mutations


Lev-Ari, A. 6/3/2013 Clinical Indications for Use of Inhaled Nitric Oxide (iNO) in the Adult Patient Market: Clinical Outcomes after Use, Therapy Demand and Cost of Care


Lev-Ari, A. 6/2/2013 Inhaled Nitric Oxide in Adults: Clinical Trials and Meta Analysis Studies – Recent Findings


Lev-Ari, A. 5/17/2013 Synthetic Biology: On Advanced Genome Interpretation for Gene Variants and Pathways: What is the Genetic Base of Atherosclerosis and Loss of Arterial Elasticity with Aging


Lev-Ari, A. 4/28/2013 Genetics of Conduction Disease: Atrioventricular (AV) Conduction Disease (block): Gene Mutations – Transcription, Excitability, and Energy Homeostasis


Lev-Ari, A. 2/28/2013 The Heart: Vasculature Protection – A Concept-based Pharmacological Therapy including THYMOSIN


Part II         

Cases in Co-Curation and Scientific Content Critique

Pearlman, JD, and A.  Lev-Ari, 9/30/2013

State of Cardiology on Wall Stress, Ventricular Workload and Myocardial Contractile Reserve: Aspects of Translational Medicine(TM)


Lal, V, Pearlman JD, and A. Lev-Ari, 9/23/2013

Do Novel Anticoagulants Affect the PT/INR? The Cases of  XARELTO (rivaroxaban) or PRADAXA (dabigatran)


Bernstein LH, SJ Williams and A. Lev-Ari, 8/26/2013

Part II: Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility


Bernstein LH, SJ Williams and A. Lev-Ari,  9/2/2013

Part III: Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease


Bernstein LH, Pearlman JD and A. Lev-Ari, 9/8/2013

Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets


Bernstein LH, Pearlman JD and A. Lev-Ari, 8/26/2013

Part V: Heart, Vascular Smooth Muscle, Excitation-Contraction Coupling (E-CC), Cytoskeleton, Cellular Dynamics and Ca2 Signaling


Pearlman, JD, Bernstein, HL and A. Lev-Ari 8/28/2013

Part VII: Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure – Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses


Pearlman, JD, Bernstein, LH and A. Lev-Ari, 9/12/2013

Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism


Pearlman, JD, Bernstein, LH and A. Lev-Ari, 9/16/2013

Part IX: Calcium-Channel Blockers, Calcium Release-related Contractile Dysfunction (Ryanopathy) and Calcium as Neurotransmitter Sensor


Bernstein, LH and A. Lev-Ari, 9/10/2013

Part X: Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of vesicles with cell membranes during Neurotransmission


Pearlman JD and A. Lev-Ari 8/25/2013

Coronary Circulation Combined Assessment: Optical Coherence Tomography (OCT), Near-Infrared Spectroscopy (NIRS) and Intravascular Ultrasound (IVUS) – Detection of Lipid-Rich Plaque and Prevention of Acute Coronary Syndrome (ACS)


Pearlman, JD, Bernstein, LH and A. Lev-Ari 8/5/2013

Alternative Designs for the Human Artificial Heart: The Patients in Heart Failure – Outcomes of Transplant (donor)/Implantation (artificial) and Monitoring Technologies for the Transplant/Implant Patient in the Community. To be submitted to Heart Failure Society of America (HFSA)


Pearlman, JD and A. Lev-Ari 7/23/2013

Cardiovascular Complications: Death from Reoperative Sternotomy after prior CABG, MVR, AVR, or Radiation; Complications of PCI; Sepsis from Cardiovascular Interventions


Pearlman, JD and A. Lev-Ari 7/22/2013

Cardiac Resynchronization Therapy (CRT) to Arrhythmias: Pacemaker/Implantable Cardioverter Defibrillator (ICD) Insertion


Pearlman, JD and A. Lev-Ari 7/17/2013

Emerging Clinical Applications for Cardiac CT: Plaque Characterization, SPECT Functionality, Angiogram’s and Non-Invasive FFR


Pearlman, JD and A. Lev-Ari 7/4/2013

Fractional Flow Reserve (FFR) & Instantaneous wave-free ratio (iFR): An Evaluation of Catheterization Lab Tools for Ischemic Assessment


Pearlman, JD and A. Lev-Ari 5/24/2013

Imaging Biomarker for Arterial Stiffness: Pathways in Pharmacotherapy for Hypertension and Hypercholesterolemia Management


Pearlman, JD and A. Lev-Ari 5/22/2013

Acute and Chronic Myocardial Infarction: Quantification of Myocardial Perfusion Viability – FDG-PET/MRI vs. MRI or PET alone


Pearlman JD, LH Bernstein and A. Lev-Ari 5/15/2013

Diagnosis of Cardiovascular Disease, Treatment and Prevention: Current & Predicted Cost of Care and the Promise of Individualized Medicine Using Clinical Decision Support Systems


Pearlman, JD and A. Lev-Ari 5/11/2013

Hypertension and Vascular Compliance: 2013 Thought Frontier – An Arterial Elasticity Focus


Pearlman, JD and A. Lev-Ari 5/7/2013

On Devices and On Algorithms: Arrhythmia after Cardiac Surgery Prediction and ECG Prediction of Paroxysmal Atrial Fibrillation Onset


Pearlman, JD and A. Lev-Ari 5/4/2013

Clinical Decision Support Systems for Management Decision Making of Cardiovascular Diseases


Lev-Ari, A. and LH Bernstein 3/7/2013

Genomics & Genetics of Cardiovascular Disease Diagnoses: A Literature Survey of AHA’s Circulation Cardiovascular Genetics, 3/2010 – 3/2013


Find out more:

« Curation is the new research, »… et le nouveau média, Benoit Raphael, 2011http://benoitraphael.com/2011/01/17/curation-is-the-new-search/

La curation : la révolution du webjournalisme?, non-fiction.fr http://www.nonfiction.fr/article-4158-la_curation__la_revolution_du_webjournalisme_.htm

La curation : les 10 raisons de s’y intéresser, Pierre Tran http://pro.01net.com/editorial/529947/la-curation-les-10-raisons-de-sy-interesser/

Curation : quelle valeur pour les entreprises, les médias, et sa « marque personnelle »?, Marie-Laure Vie http://marilor.posterous.com/curation-et-marketing-de-linformation

Cracking Open the Scientific Process, Thomas Lin, New York Timeshttp://www.nytimes.com/2012/01/17/science/open-science-challenges-journal-tradition-with-web-collaboration.html?_r=4&pagewanted=1

La « massification » du web transforme les relations sociales, Valérie Varandat, INRIAhttp://www.inria.fr/actualite/actualites-inria/internet-du-futur

Internet a révolutionné le métier de chercheur, AgoraVoxhttp://www.agoravox.fr/actualites/technologies/article/internet-a-revolutionne-le-metier-103514

Gérer ses références numériques, Université de Genèvehttp://www.unige.ch/medecine/udrem/Unit/actualites/biblioManager.html

Notre liste Scoop-it : Scientific Social Network, MyScienceWork

SOURCE on Curation and Science

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Curator: Aviva Lev-Ari, PhD, RN

Clinical Indications for Use of Inhaled Nitric Oxide (iNO) in the Adult Patient Market: Clinical Outcomes after Use of iNO in the Institutional Market,  Therapy Demand and Cost of Care vs. Existing Supply Solutions

Inhaled NO

Word Cloud Created by Noam Steiner Tomer 8/10/2020

Introduction  to Inhaled Nitric Oxide Therapy in Adults

Part 1:             Clinical Indications for Use of Inhaled Nitric Oxide (iNO) in the Adult Patient Market

Part 2:            Clinical Outcomes after Use of iNO in the Institutional Market

Part 3:            Therapy Demand and Cost of Care vs. Existing Supply Solutions

Part 4:            Product Development Concepts for New Medical Devices to Deliver Inhaled Nitric Oxide

Introduction  to Inhaled Nitric Oxide Therapy in Adults: Evidence-based Medicine 

This Introduction section of the article is based on research results and literature survey in:

Mark J.D. Griffiths, M.R.C.P., Ph.D., and Timothy W. Evans, M.D., Ph.D.

Inhaled Nitric Oxide Therapy in Adults, n engl j med 353;25 http://www.nejm.org December 22, 2005


  • On the basis of the evidence, inhaled nitric oxide is not an effective therapeutic intervention in patients with acute lung injury or ARDS, and its routine use to achieve this end is inappropriate. However, inhaled nitric oxide may be useful as a short-term adjunct to cardiorespiratory support in patients with acute hypoxemia, life-threatening pulmonary hypertension, or both.
  • Inhaled nitric oxide is a selective pulmonary vasodilator that improves ventilation–perfusion matching at low doses in patients with acute respiratory failure, potentially improving oxygenation and lowering pulmonary vascular resistance.
  • Large clinical trials have indicated that physiologic benefits are short-lived in adults with acute lung injury or ARDS, and no associated improvement in mortality rates has been demonstrated. However, clinical trials involving patients with acute lung injury or ARDS have been statistically underpowered to show a decrease in mortality rates and have not considered recent insights into the effect of continuous inhalation on the dose– response relationship of this agent. In patients with acute respiratory failure, the potential toxicity or protective effects of inhaled nitric oxide, particularly any effects on cell survival and inflammation, are poorly understood.
  • Ideal Treatment Goals for Inhaled Nitric Oxide
  1. Improved oxygenation
  2. Decreased pulmonary vascular resistance
  3. Decreased pulmonary edema
  4. Reduction or prevention of inflammation – rebound phenomena may be avoided by withdrawing inhaled nitric oxide gradually. Despite these concerns, in large clinical studies of patients with ARDS, the abrupt discontinuation of inhaled nitric oxide has not caused a deterioration in oxygenation
  5. Cytoprotection
  6. Protection against infection
  • Administration of Inhaled Nitric Oxide to Adults: Routes and Safety Monitoring

Nitric oxide is most commonly administered to patients receiving mechanical ventilation, although it may also be given through a face mask or nasal cannulae. Limiting the mixing of nitric oxide and high concentrations of inspired oxygen reduces the risk of adverse effects resulting from the formation of nitrogen dioxide. This is minimized further by introducing the mixture of nitric oxide and nitrogen into the inspiratory limb of the ventilator tubing as near to the patient as possible and synchronizing injection of the mixture with inspiration

  • Electrochemical analyzers can be used to monitor the concentrations of nitric oxide and nitrogen dioxide in the inspired gas mixture to an accuracy of 1 ppm.
  • More sensitive Chemiluminescence monitors can detect nitric oxide and its oxidative derivatives in parts per billion.
  • Dose-Response for Respiratory Failure in the Adult Patient – a response is defined as a 20 percent increase in oxygenation. For example, a 10 percentage point improvement in hemoglobin saturation in a patient with hypoxemia who is breathing 100 percent oxygen may be clinically very important.
  • Dose-Response for Pulmonary Hypertension in the Adult Patient – a 30 percent decrease in pulmonary vascular resistance during the inhalation of nitric oxide (10 ppm for 10 minutes) has been used to identify an association with vascular responsiveness to agents that can be helpful in the long term. A positive response to nitric oxide was associated with a favorable response to calcium-channel blockers in a small cohort of patients with primary pulmonary hypertension
  • Time-dependent variation in the dose–response relationship of inhaled nitric oxide in patients with severe ARDS – Observations imply that the optimal dose of inhaled nitric oxide must be determined by titration against the therapeutic target in each patient at least every two days, and probably more frequently.
  • Other Inhaled Vasodilators – Alternatives and Adjuncts to Inhaled Nitric Oxide
  1. Aerosolized sodium nitrite caused potent, selective, nitric oxide–dependent pulmonary vasodilatation through its reaction with deoxyhemoglobin at a low pH, suggesting that nitrite may be a cheap and stable alternative to inhaled nitric oxide
  2. Epoprostenol, the most extensively studied alternative to inhaled nitric oxide, is also an endothelium- derived vasodilator with antithrombotic effects. Inhaled epoprostenol has an effect on hemodynamics and oxygenation similar to that of nitric oxide in patients with ARDS, sepsis, or severe heart failure. Nebulized epoprostenol has been studied less frequently than inhaled nitric oxide, but at therapeutic doses (10 to 50 ng per kilogram per minute), the rates of predicted side effects, such as systemic hypotension and bleeding after surgery, have not been clinically important.
  3. Iloprost, a long-acting prostacyclin analogue (half-life, 20 to 30 minutes), improves the exercise tolerance of patients with severe pulmonary hypertension when administered by intermittent rather than by continuous nebulization. Inhaled prostaglandin E1 (6 to 15 ng per kilogram of body weight per minute) has effects similar to those of inhaled nitric oxide (2 to 10 ppm) in patients with ARDS
  • Agonists to Nitric Oxide – Adjunctive Therapies That Increase the Effectiveness of Inhaled Nitric Oxide

1. Orally administered sildenafil, an inhibitor of phosphodiesterase type 5, is a selective pulmonary vasodilator, partially because phosphodiesterase type 5 is highly expressed in the lung. Sildenafil has augmented pulmonary vasodilatation induced by inhaled nitric oxide,  although a second inhibitor of phosphodiesterase type 5, zaprinast, predictably worsened oxygenation through the attenuation of hypoxic pulmonary vasoconstriction in an ovine model of acute lung injury.  Such agents may therefore be most useful when pulmonary hypertension rather than respiratory failure is the chief concern.

2. Almitrine, an agonist at peripheral arterial chemoreceptors, is a selective pulmonary vasoconstrictor that specifically enhances hypoxic pulmonary vasoconstriction. The addition of almitrine to low-dose inhaled nitric oxide improves oxygenation in patients with ARDS, but concern about the effects of long-term infusion has hampered the wider investigation of this combination. In patients with acute respiratory failure, the effect of nitric oxide depends on the degree of recruitment of injured lung units by — for example — positive end-expiratory pressure, prone positioning, or ventilatory maneuvers designed to inflate collapsed lung, which may explain how the response to nitric oxide varies over short periods. Partial liquid ventilation with perfluorocarbons facilitates the delivery of dissolved gases to alveoli by enhancing recruitment of the injured lung units. Inhaled nitric oxide has enhanced the effects of partial liquid ventilation on gas exchange in animal models, demonstrating the potential benefit of combining therapeutic strategies in patients with ARDS.

For 2005 – 2013 List of References on Inhaled Nitric Oxide Therapy in Adults, see the list of article that has cited  at the bottom of the following seminal paper:



Part 1:

Clinical Indications for Use of Inhaled Nitric Oxide (iNO) in the Adult Patient Market:


George, Isaac, Xydas, Steve, Topkara, Veli K., Ferdinando, Corrina, Barnwell, Eileen C., Gableman, Larissa, Sladen, Robert N., Naka, Yoshifumi, Oz, Mehmet C.
Clinical Indication for Use and Outcomes After Inhaled Nitric Oxide Therapy
Ann Thorac Surg 2006 82: 2161-2169

Abbreviations and Acronyms

ARDS  adult respiratory distress syndrome

iNO  inhaled nitric oxide

OHT  orthotopic heart transplantation

OLT  orthotopic lung transplantation

PAP  pulmonary artery pressure

PVR  pulmonary vascular resistance

ROC  receiver operating curve

RV  right ventricular

VAD  ventricular assist device

Institutional Guidelines for Inhaled Nitric Oxide Administration – Table 1 in the Study

1. Heart transplantation with evidence of pulmonary hypertension

2. Complicated coronary surgery with evidence of right ventricular failure based on at least one of the following


  • Mean pulmonary artery pressure 25 mm Hg
  • Echocardiographic evidence of moderate to severe right
  • ventricular dysfunction; severe right atrial or ventricular enlargement
  • Cardiac index 2.2 L · min1 · m2

3. Precapillary pulmonary hypertension diagnosis

4. Congenital cardiac disease

5. Acute chest syndrome in sickle cell disease

6. The starting dose for all above indications was 10 to 20 ppm, with an initial trial for 60 minutes before up-titration.

Indication for inhaled nitric oxide (iNO) use – Surgical Patient

1.  orthotopic heart transplantation [OHT] with pulmonary hypertension;

2. precapillary pulmonary hypertension;

3. coronary surgery with right ventricular failure;

4. congenital cardiac disease;

  • OLT – orthotopic lung transplantation- patients received iNO for treatment of pulmonary hypertension, 
  • OHT – orthotopic heart transplant  –  right ventricular failure was the most common indication for patients undergoing cardiac surgery and ventricular assist device (VAD) implantation.

Indication for inhaled nitric oxide (iNO) use – Medical Patients in ICU

5. hypoxemia                                                                                                                                                                                                    

  • Other surgical and medical patients received iNO predominantly for hypoxemia use.

A trend toward a lower average duration of iNO use was seen:

  • after OHT (n 67) and OLT (n 45)


  • cardiac surgery (n 105),
  • VAD (n 66),
  • other surgery (n 34), and
  • medical patients (n 59; p 0.09).

Primary Surgical Procedure –  Table 4. in the Study – All Patients in the Study

Abbreviations and Acronyms

BiVAD biventricular assist device;

CABG coronary artery bypass grafting;

LVAD left ventricular assist device;

MVR mitral valve replacement or repair;

OHT orthotopic heart transplantation;

OLT orthotopic lung transplanatation;

RVAD right ventricular assist device;

Txp transplant;

VAD ventricular assist device.

AVR aortic valve replacement;

OHT = 67 OLT = 45 Cardiac Surgery = 105  VAD = 66  Other Surgery = 34  Medical (No Surgery) = 59

N (%)

OHT – Heart Txp – 67 (100)

OLT – Lung Txp – 45 (100)

Cardiac Surgery = 105

  • AVR, 10 (9.5) 59 (100)
  • AVR/MVR, 10 (9.5)
  • CABG, 23 (21.9)
  • CABG/Valve, 23 (21.9)
  • MVR, 22 (20.9)
  • Other cardiac, 11 (10.5)
  • Other valve, 3 (2.9)

VAD = 66

  • LVAD, 54 (81.8)
  • BiVAD, 12 (18.2)
  • RVAD, 0

Other surgery = 34

  • Other surgery 21 (61.8)
  • Thoracic surgery, 8 (23.5)
  • Other Txp. 5 (14.7)

Medical =59 in ICU

  • No Surgery, 59 (100)


Part 2:

Clinical Outcomes after Use of iNO in the Institutional Market

Use of iNO for pulmonary hypertension in patients undergoing

  • OHT and orthotopic lung transplantation was associated with a significantly lower overall mortality rate compared with its use after cardiac surgery or for hypoxemia in medical patients.
  • Inhaled nitric oxide does not appear to be cost effective when treating hypoxemia in medical patients with high-risk scores and irreversible disease.

In conclusion,

  • the present study reports comprehensive long-term survival data from a critically ill adult population receiving iNO therapy.
  • Inhaled nitric oxide treatment is a valuable pharmacologic adjunct in OHT and OLT for short-term hemodynamic improvements, and long-term data from the present study suggest a translation into long-term survival benefits.
  • Mortality outcomes after iNO are directly related to the clinical indication for use, and prolonged therapy for patients with irreversible systemic disease processes, such as hypoxemia or respiratory failure in medical patients, is not warranted.
  • Poor outcomes and high cost for medical patients with respiratory failure and hypoxemia in this study require further investigation to determine the appropriate duration of iNO use based on clinical response and appropriate endpoints of treatment.
  • A prospective clinical study controlling for severity of illness and addressing clinical efficacy in both surgical and medical populations is needed to definitively answer these questions, and may help reduce the burden of intensive care expenses.


Inhaled nitric oxide therapy has been shown to lead to reductions in PAP and PVR and improvement in oxygenation in several populations, including neonates and adult patients with ARDS and RV dysfunction, and after OHT or OLT [3, 6, 9, 10, 14]. These effects may improve short-term outcomes, but a study of long-term outcomes, costs, and clinical use of iNO use in other populations has not been conducted to date. This study is the first to describe outcomes and cost of iNO therapy in an unselected population of critically ill adult patients in a tertiary care center. These study results demonstrate that (1) outcomes after iNO vary substantially based on clinical indication of use, (2) iNO may benefit transplant patients more than other patients, and (3) iNO does not appear to alter the natural history or long-term clinical course of hypoxemic respiratory failure. This study also identifies the medical patient population with respiratory failure as one with substantial morbidity whose high mortality after iNO precludes prolonged therapy.

In the present study, OHT and OLT patients had a 1-year survival rate four times greater than medical patients not undergoing surgery, as well as higher survival rates compared with patients undergoing other types of surgery. The large differences in mortality after iNO therapy may be attributed to differences in the underlying etiology of the cardiac or respiratory failure (pulmonary hypertension versus hypoxemia) and the reversibility of pulmonary hypertension versus respira- tory failure.

In OHT, acutely elevated PAP, which accounts for 19% of early deaths after heart transplantation [24], may be secondary to both increases in flow (increased backward transmission of elevated left ventricular pressure) and increases in resistance in the pulmonary bed. With iNO use, PVR and PAP are reduced [25], decreasing RV afterload, ameliorating the wean from cardiopulmonary bypass, and preventing RV failure without affecting systemic vascular resistance. By providing temporary support, iNO therapy after transplant allows for the stabilization of hemodynamics until PVR returns to normal levels, which is attained in 80% of patients 1 year after OHT [26], reinforcing its reversible nature after cardiac transplantation. Short-term use of iNO after OHT has been demonstrated to improve RV function, PVR, and mean PAP after 12 to 76 hours of iNO use in 16 OHT patients, although there were no statistically significant differences in survival [9]. In 23 OLT patients, iNO therapy has been shown to reduce reimplantation edema, increase PaO2/FIO2, decrease the need for mechanical ventilation, and reduce the 2-month mortality rate [10].

The observed improvement in pulmonary hypertension also predicts significant outcome benefits, as OHT patients with reversible preoperative PVR have a much lower mortality than do those with a fixed elevated PVR [27, 28]. Survival at 4 years after iNO therapy was 68% in the transplant cohort in the present study, comparing favorably to reported 5-year survival rates of 71% for OHT [29] and 63% for OLT [30]. This study confirms prior studies that have shown acute benefits with iNO therapy after transplantation and shows that long-term survival in OHT and OLT after iNO therapy is comparable to that of patients not requiring iNO. In addition, although mortality in the VAD group was not appreciably different than that in the cardiac surgery group, a likely benefit of iNO in these patients was the avoidance of right ventricular assist device placement, as evidenced by the low rate of left ventricular assist device patients requiring a right ventricular assist device (5 of 66, 7.6%).

Furthermore, iNO therapy has not been shown to lead to long-term benefits in the treatment of severe respiratory failure, which was present in 80% of the medical cohort in this study, or hypoxemia, which was the primary indication in 85% of the medical patients. No benefit beyond 1 day of therapy was seen in indices of lung function in a randomized controlled clinical trial of 30 medical patients with severe respiratory failure and ARDS, yielding a 30-day mortality rate of 60% in iNOtreated patients and 53% in nontreated patients (p _ 0.71) [31]. More importantly, nonresponders had a 30-day mortality rate of 80%, whereas responders had a 50% mortality rate. The lack of short-term mortality benefit was confirmed by Michael and colleagues [32] in a randomized controlled trial of iNO in ARDS patients that showed transient improvements after 1 hour but no sustained improvements after 72 hours in PaO2, FIO2, and PaO2/FIO2. These two studies highlight important findings that iNO initially improves indices of lung function but does not produce lasting effects on oxygenation.

The inability to produce sustained effects on hypoxia and respiratory failure may explain the striking 1-year survival of only 17.3% and 4-year survival of 0% in our medical cohort, rates higher than the 90-day mortality rates of 40% to 50% that have been previously reported [33, 34]. Medical patients with severe cardiac or respiratory failure requiring iNO therapy represent a critically ill, challenging population with numerous comorbidities.

Judicious use of iNO is warranted for such patients if the immediate mortality risk is estimated to be high. The risk-scoring model reported here allows stratification of patients based on clinical history and provides prognostic information on mortality outcomes. The model predicted a mortality of 76.5% versus 37.2% (p _ 0.001) for a risk score greater than 1, with a sensitivity of 60%, specificity of 79%, and area under ROC of 0.731.

For cases in which the benefit is likely to be limited with a risk score greater than 1 (namely, respiratory failure in any non-OHT patient), efforts should be made to determine whether a patient responds to iNO therapy before prolonged administration is undertaken. As expected, hours of iNO use were highest in the medical group at 133 hours, and lowest after OHT and OLT at 71 and 57 hours, respectively. However, longer average duration of use did not produce higher iNO costs using the 2000 to 2003 charging practice, as many patients in all subgroups reached the maximal monthly charge after the first 4 days of therapy. This cap on iNO charges served to equalize costs in surgical and nonsurgical groups, and healthcare providers may continue iNO use in nonresponders as salvage therapy, given that it may not increase iNO-associated charges. However, the cost difference was more pronounced for OLT patients compared with medical and VAD patients using the current hourly charging practice, which was intended to reduce the overall cost of iNO therapy through more precise hourly billing. These findings confirm that prolonged iNO use is associated with higher cost and provides a financial rationale for limiting therapy for patients without expected survival benefit.

The study limitations include those inherent to an observational study. The lack of a randomized design and a control cohort not receiving iNO therapy precludes any definitive conclusions regarding the long-term clinical efficacy or cost effectiveness of iNO use, as long-term hemodynamics were unable to be measured and costeffectiveness measurements were not calculated. The transient but clinically important appearance of RV dysfunction in the operating room may only be apparent on hemodynamic analysis rather than on echocardiography, and RV dysfunction may be underreported using our echocardiographic definition. The poor survival rates observed in the medical cohort may be attributed to late initiation of iNO therapy in this group; it cannot, therefore, be excluded that earlier iNO administration may have led to higher survival rates. Finally, the absence of indirect hospital costs is a major limiting factor in the description of iNO costs, which may be significant.

Ann Thorac Surg 2006;82:2161-2169


Part 3:

Therapy Demand and Cost of Care vs. Existing Supply Solutions

Acquisition Cost of Inhaled Nitric Oxide Therapy

Charges for each iNO therapy encounter were calculated based on the charging practice of INO Therapeutics (AGA Healthcare, Clinton, New Jersey) between 2000 and 2003, and recalculated using the current 2005 charging practice. For the years 2000 to 2003, the charge to hospitals was $3,000 per 24 hours of therapy, up to a maximum charge of $12,000 per month, independent of  total hourly usage. Using the current 2005 charging practice, the charge for iNO was changed to an hourly rate of $125, with a maximum charge of $12,000 per month, independent of hourly usage. Indirect costs associated with iNO administration, including those for respiratory personnel, intensive care unit care, and daily monitoring were not included in this analysis.

Estimated Cost of iNO Therapy

The cost for iNO therapy is summarized in Table 6 using the 2000 to 2003 charging practice and current 2005 charging practice, demonstrating a higher cost of therapy in VAD and medical patients. Under the current 2005 pricing, a significantly lower proportion of OHT and OLT patients reached the maximal charge versus medical patients (23% versus 51%, 0.001).

Acquisition Cost of Inhaled Nitric Oxide Therapy – Table 6 in the Study

2000–2003 Charge Scale ($) || Current Charge Scale ($)

OHT 9,121 + or – 4,226  ||  7,010 + or – 5,072

OLT 8,040 + or – 3,659a  ||  5,710 + or – 4,132b

Cardiac surgery 9,179  + or – 5,319   ||  7,349 + or – 6,543

VAD 10,726 + or – 4,121   ||  8,722 + or – 4,966

Other surgery 9,324 + or – 4,110 ||  7,056 + or – 4,826

Medical 10,075 + or – 5,215  || 8,867 + or – 7,233

a p 0.05 versus VAD. b p 0.05 versus VAD, medical.

OHT orthotopic heart transplantation; OLT orthotopic lung transplantation; VAD ventricular assist device.

Ann Thorac Surg 2006;82:2161-2169

Present Market Demand for inhaled Nitric Oxide Gas

Clinical Policy Bulletin: Nitric Oxide, Inhalational (INO) Number: 0518

Aetna Policy

  • Aetna considers inhaled nitric oxide (INO) therapy medically necessary as a component of the treatment of hypoxic respiratory

failure in term and near-term (born at 34 or more weeks of gestation) neonates when both of the following criteria are met:

Neonates do not have congenital diaphragmatic hernia; and  When conventional therapies such as administration of high concentrations of oxygen, hyperventilation, high-frequency ventilation, the induction of alkalosis, neuromuscular blockade, and sedation have failed or are expected to fail.

Note: Use of INO therapy for more than 4 days is subject to medical necessity review.

  • Aetna considers the diagnostic use of INO medically necessary as a method of assessing pulmonary vaso-reactivity in persons

with pulmonary hypertension.

  • Aetna considers INO therapy experimental and investigational for all other indications because of insufficient evidence in the

peer-reviewed literature, including any of the following:

  • Acute bronchiolitis; or
  • Acute hypoxemic respiratory failure in children (other than those who meet the medical necessity criteria above) and in adults; or
  • Adult respiratory distress syndrome or acute lung injury; or
  • Post-operative management of pulmonary hypertension in infants and children with congenital heart disease; or
  • Premature neonates (less than 34 weeks of gestation); or
  • Prevention of ischemia-reperfusion injury/acute rejection following lung transplantation; or
  • Treatment of persons with congenital diaphragmatic hernia; or
  • Treatment of vaso-occlusive crises or acute chest syndrome in persons with sickle cell disease (sickle cell vasculopathy).

Part 4:

Product Development Concepts

A. Institutional Applications – Adult Patient Market

Dr. Pearlman’s Flywheel Concept, presents a solution in this Space, with potential NEW product design for POC for the Institutional Market and the HomeCare Market

Protected: Flywheel iNO, Three Novel Adult Patient Inhaled Nitric Oxide Product Concepts by Justin D. Pearlman MD ME PhD FACC

INDICATIONS for Flywheel

a.                  Hypoxic respiratory failure (HRF)

Aa.1      Neonatal market – Solution in Existence, [NOT COVERED BY LPBI]

Aa.2     Adult market hypoxic respiratory failure (HRF) associated with pulmonary hypertension or from other etiologies

b.                  Pulmonary Arterial Hypertension (PAH)

Ab.1    Neonatal market [NOT COVERED BY LPBI]

Ab.2   Adult market

c.                  Diagnostic Use of inhaled Nitric Oxide

Ac.1 Pulmonary Vasoreactivity Testing in the Cardiac Catheterization Laboratory

Ac2 Treatment of Perioperative Pulmonary Hypertension With Inhaled NO for  Congenital Heart Disease

Ac3 Cardiac Transplantation

Ac4 Insertion of Left Ventricular Assist Device

Ac5 Inhaled NO to Treat Ischemia-Reperfusion Injury

Ac6 Inhaled NO and Acute Respiratory Distress Syndrome

Ac7 Lung Transplantation

Ac8 Sickle Cell Disease

Ac9 Airway chronic inflammation: Nebulized epoprostenol, Iloprost, a long acting prostacyclin analogue, inhaled prostaglandin E1, Adjuctive therapy with inhaled Nitric Oxide

B. Home Care Applications –

Applications for the HomeCare Segment, as the POC is the Home – Types of Products:

For the Institutional Market:

A1. PiNO
A2. SiNO

For the HomeCare Market

Bx. HiNO –   Dr. Pearlman’s solution

B1. HiNO –    LPBI’s PORTABLE inspiratory pulsing device with option to turn off pulsing feature

B2. HiNO –   LPBI’s Home Care Facial Inhaling Device

a.                 COPD

b                  Unstable Angina

Present Market Supply for inhaled Nitric Oxide gas

The market supply of inhaled Nitric Oxide gas experience the structure of a Monopoly. No competition, one product type very expensive in use by Institutions, i.e., Hospitals, only AND Pediatric population, primarily

The Massachusetts General Hospital owns patents covering the use of nitric oxide inhalation, which it has licensed to INO Therapeutics, a division of AGA Linde, and Dr Zapol receives a portion of the royalties.

Dr Roberts is a member of the Scientific Advisory Board of INOTherapeutics, a company that sells inhaled nitric oxide gas. Dr Roberts is not compensated for this activity by the company.


Clinical Trials – Newborns, full-term and nearly full-term infants

Hypoxic Respiratory Failure (HRF)

Clinical trials have shown that INOMAX is effective and well tolerated in the treatment of HRF associated with pulmonary hypertension.3 Its safety has been demonstrated in clinical trials and through post-marketing experience.

Neonatal Inhaled Nitric Oxide Study Group (NINOS). Inhaled nitric oxide in full-term and nearly full-term infants with hypoxic respiratory failure. N Engl J Med. 1997;336:597-604. Detailed description.

Clark RH, Kuesser RJ, Walker MW, et al. Clinical Inhaled Nitric Oxide Research Group (CINRGI). Low-dose nitric oxide therapy for persistent pulmonary hypertension of the newborn. N Engl J Med. 2000;342:469-474. Detailed description.

Davidson D, Barefield ES, Kattwinkel J, et al. Inhaled nitric oxide for the early treatment of persistent pulmonary hypertension of the term newborn: a randomized, double-masked, placebo-controlled, dose-response, multicenter study. Pediatrics. 1998;101:325-334.

Wessel DL, Adatia I, Van Marter LJ, Thompson JE, Kane JW, Stark AR, Kourebanas S. Improved oxygenation in a randomized trial of inhaled nitric oxide for persistent pulmonary hypertension of the newborn. J Pediatr. 1997;100:E7. [PubMed]

Neonatal Inhaled Nitric Oxide Group. Inhaled nitric oxide in full term and nearly full term infants with hypoxic respiratory failure. N Engl J Med. 1997;336:597–604. [PubMed]

Roberts JD, Fineman JR, Morin FC, Shaul PW, Rimer S, Schreiber MD, et al. Inhaled nitric oxide and persistent pulmonary hypertension of the newborn. The Inhaled Nitric Oxide Group. N Engl J Med. 1997;336:605–610. [PubMed]

Wessel DL, Adatia I, Giglia TM, Thompson JE, Kulik TJ. Use of inhaled nitric oxide and acetylcholine in the evaluation of pulmonary hypertension and endothelial function after cardiopulmonary bypass. Circulation. 1993;88:2128–2138. [PubMed]

Petros AJ, Turner SC, Nunn AJ. Cost implications of using inhaled nitric oxide compared with epoprostenol for pulmonary hypertension. J Pharm Technol. 1995;11:163–166. [PubMed]


Industry LEADER for the Neonatal Market : INOMAX®


Nitric oxide delivery systems designed for critical care

With the INOMAX® delivery systems, you can be confident that you have continual innovative devices.

Dedication to developing next-generation technologies.

Continuous innovation supports evolving information and technology needs

Compatible with 60 ventilation systems, including HFOV and noninvasive modalities

Allow for operator-determined concentrations of nitric oxide (NO) in the breathing unit

Provide for a concentration that is constant throughout the respiratory cycle

Monitor for NO, oxygen (FiO2), and nitrogen dioxide (NO2)

Prevent generation of excessive inhaled NO2

INOMAX® demostrates safety and efficacy in the treatment of hypoxic respiratory failure (HRF)

Clinical trials have shown that INOMAX is effective and well tolerated in the treatment of HRF associated with pulmonary hypertension.3 Its safety has been demonstrated in clinical trials and through post-marketing experience.

INOMAX has a well-established safety profile

More than 530,000 patients treated worldwide*2

Meet all FDA-required specifications

In the US in 2013 – Inhaled Nitric Oxide is NOT a FDA approved Drug  Therapy for the Adult Patient

CLINICAL TRIALS on the Use of Inhaled Nitric Oxide by Adult Patients, include:

Inhaled Nitric Oxide for Acute Respiratory Distress Syndrome and Acute Lung Injury in Adults and Children: A Systematic Review with Meta-Analysis and Trial Sequential Analysis

  1. Arash Afshari, MD*,
  2. Jesper Brok, MD, PhD§,
  3. Ann M. Møller, MD, MSDC and
  4. Jørn Wetterslev, MD, PhD§

Published online before print March 3, 2011, doi:10.1213/​ANE.0b013e31820bd185A & A June 2011 vol. 112 no. 6 1411-1421


CONCLUSION: iNO cannot be recommended for patients with acute hypoxemic respiratory failure. iNO results in a transient improvement in oxygenation but does not reduce mortality and may be harmful.

Michael JR, Barton RG, Saffle JR, Mone M, Markewitz BA. Inhaled nitric oxide versus conventional therapy: effect on oxygenation in ARDS Am J Resp Crit Care Med 1998;157:1361-1362. [Free Full Text]

Abstract  A randomized, controlled clinical trial was performed with patients with acute respiratory distress syndrome (ARDS) to compare the effect of conventional therapy or inhaled nitric oxide (iNO) on oxygenation. Patients were randomized to either conventional therapy or conventional therapy plus iNO for 72 h. We tested the following hypotheses: (1) that iNO would improve oxygenation during the 72 h after randomization, as compared with conventional therapy; and (2) that iNO would increase the likelihood that patients would improve to the extent that the FI(O2) could be decreased by > or = 0.15 within 72 h after randomization. There were two major findings. First, That iNO as compared with conventional therapy increased Pa(O2)/FI(O2) at 1 h, 12 h, and possibly 24 h. Beyond 24 h, the two groups had an equivalent improvement in Pa(O2)/FI(O2). Second, that patients treated with iNO therapy were no more likely to improve so that they could be managed with a persistent decrease in FI(O2) > or = 0.15 during the 72 h following randomization (11 of 20 patients with iNO versus 9 of 20 patients with conventional therapy, p = 0.55). In patients with severe ARDS, our results indicate that iNO does not lead to a sustained improvement in oxygenation as compared with conventional therapy.

Dellinger RPZimmerman JLTaylor RWStraube RCHauser DLCriner GJDavis K JrHyers TMPapadakos PEffects of inhaled nitric oxide in patients with acute respiratory distress syndrome: results of a randomized phase II trial. Inhaled Nitric Oxide in ARDS Study Group.

Conclusions: From this placebo-controlled study, inhaled NO appears to be well tolerated in the population of ARDS patients studied. With mechanical ventilation held constant, inhaled NO is associated with a significant improvement in oxygenation compared with placebo over the first 4 hrs of treatment. An improvement in oxygenation index was observed over the first 4 days. Larger phase III studies are needed to ascertain if these acute physiologic improvements can lead to altered clinical outcome.

Conclusions: Inhaled nitric oxide at a dose of 5 ppm in patients with acute lung injury not due to sepsis and without evidence of nonpulmonary organ system dysfunction results in short-term oxygenation improvements but has no substantial impact on the duration of ventilatory support or mortality.

Lundin S, Mang H, Smithies M, Stenqvist O, Frostell C. Inhalation of nitric oxide in acute lung injury: results of a European multicentre study. Intensive Care Med 1999;25:911-9.

Conclusions: Improvement of oxygenation by INO did not increase the frequency of reversal of ALI. Use of inhaled NO in early ALI did not alter mortality although it did reduce the frequency of severe respiratory failure in patients developing severe hypoxaemia.

Inhaled Nitric Oxide as Drug Therapy continue to be a very HOT research subject as 2004 article was cited by the following studies, 2004-2013:

The Pharmacological Treatment of Pulmonary Arterial Hypertension 
Pharmacol. Rev.. 2012;64:583-620,

AbstractFull TextPDF

Transpulmonary Flux of S-Nitrosothiols and Pulmonary Vasodilation during Nitric Oxide Inhalation: Role of Transport 
Am. J. Respir. Cell Mol. Bio.. 2012;47:37-43,

AbstractFull TextPDF

Stimulation of soluble guanylate cyclase reduces experimental dermal fibrosis 
Ann Rheum Dis. 2012;71:1019-1026,

AbstractFull TextPDF

Inhaled Nitric Oxide for Elevated Cavopulmonary Pressure and Hypoxemia After Cavopulmonary Operations 
World Journal for Pediatric and Congenital Heart Surgery. 2012;3:26-31,

AbstractFull TextPDF

Inhaled Nitric Oxide Improves Outcomes After Successful Cardiopulmonary Resuscitation in Mice 
Circulation. 2011;124:1645-1653,

AbstractFull TextPDF

Nitrite Potently Inhibits Hypoxic and Inflammatory Pulmonary Arterial Hypertension and Smooth Muscle Proliferation via Xanthine Oxidoreductase-Dependent Nitric Oxide Generation 
Circulation. 2010;121:98-109,

AbstractFull TextPDF

Soluble guanylate cyclase stimulation: an emerging option in pulmonary hypertension therapy 
Eur Respir Rev. 2009;18:35-41,

AbstractFull TextPDF

Intravenous Magnesium Sulphate vs. Inhaled Nitric Oxide for Moderate, Persistent Pulmonary Hypertension of the Newborn. A Multicentre, Retrospective Study 
J Trop Pediatr. 2008;54:196-199,

AbstractFull TextPDF

RETRACTED: Treating pulmonary hypertension post cardiopulmonary bypass in pigs: milrinone vs. sildenafil analog 
Perfusion. 2008;23:117-125,


Inhaled Agonists of Soluble Guanylate Cyclase Induce Selective Pulmonary Vasodilation 
Am. J. Respir. Crit. Care Med.. 2007;176:1138-1145,

AbstractFull TextPDF

Nitric Oxide in the Pulmonary Vasculature 
Arterioscler. Thromb. Vasc. Bio.. 2007;27:1877-1885,

AbstractFull TextPDF

Soluble Guanylate Cyclase-{alpha}1 Deficiency Selectively Inhibits the Pulmonary Vasodilator Response to Nitric Oxide and Increases the Pulmonary Vascular Remodeling Response to Chronic Hypoxia 
Circulation. 2007;116:936-943,

AbstractFull TextPDF

Nitric Oxide and Peroxynitrite in Health and Disease 
Physiol. Rev.. 2007;87:315-424,

AbstractFull TextPDF

Sleeping Beauty-mediated eNOS gene therapy attenuates monocrotaline-induced pulmonary hypertension in rats 
FASEB J.. 2006;20:2594-2596,

AbstractFull TextPDF

Inhaled nitric oxide decreases infarction size and improves left ventricular function in a murine model of myocardial ischemia-reperfusion injury 
Am. J. Physiol. Heart Circ. Physiol.. 2006;291:H379-H384,

AbstractFull TextPDF

Inhaled nitric oxide does not reduce systemic vascular resistance in mice 
Am. J. Physiol. Heart Circ. Physiol.. 2006;290:H1826-H1829,

AbstractFull TextPDF

Inhibition of phosphodiesterase 1 augments the pulmonary vasodilator response to inhaled nitric oxide in awake lambs with acute pulmonary hypertension 
Am. J. Physiol. Lung Cell. Mol. Physiol.. 2006;290:L723-L729,

AbstractFull TextPDF

Treatment with phosphodiesterase inhibitors type III and V: milrinone and sildenafil is an effective combination during thromboxane-induced acute pulmonary hypertension 
Br J Anaesth. 2006;96:317-322,

AbstractFull TextPDF

Extrapulmonary effects of inhaled nitric oxide: role of reversible s-nitrosylation of erythrocytic hemoglobin. 
Proc Am Thorac Soc. 2006;3:153-160,

AbstractFull TextPDF

Soluble Guanylate Cyclase Activator Reverses Acute Pulmonary Hypertension and Augments the Pulmonary Vasodilator Response to Inhaled Nitric Oxide in Awake Lambs 
Circulation. 2004;110:2253-2259,

AbstractFull TextPDF

REFERENCES for the Introduction, Part 1,2,3,4



Ann Thorac Surg 2006;82:2161-2169
© 2006 The Society of Thoracic Surgeons

Clinical Indication for Use and Outcomes After Inhaled Nitric Oxide Therapy

Isaac George, MDa,*, Steve Xydas, MDa, Veli K. Topkara, MDa, Corrina Ferdinando, MDa, Eileen C. Barnwell, MS, RRTb,Larissa Gablemana, Robert N. Sladen, MDc, Yoshifumi Naka, MD, PhDa, Mehmet C. Oz, MDa

a Department of Surgery, Division of Cardiothoracic Surgery, Columbia University College of Physicians and Surgeons, New York, New York
b Department of Respiratory Therapy, Columbia-Presbyterian Medical Center, New York, New York
c Department of Anesthesia and Critical Care, Columbia-Presbyterian Medical Center, New York, New York 

References in this article

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  6. Rossaint R, Falke KJ, Lopez F, Slama K, Pison U, Zapol WM. Inhaled nitric oxide for the adult respiratory distress syndrome N Engl J Med 2004;328:399-405.
  7. Gladwin MT, Schecter AN, Shelhamer JH, Pannell LK, Conway DA. Inhaled NO augments NO transport on sickle cell hemoglobin without affecting oxygen affinity J Clin Invest 1999;104:847-848.[Medline]
  8. Stobierska-Dzierzek B, Awad H, Michler RE. The evolving management of acute right-sided heart failure in cardiac transplant recipients J Am Coll Card 2001;38:923-931.[Medline]
  9. Ardehali A, Hughes K, Sadeghi A, Esmailian F, Marelli D, Moriguchi J. Inhaled NO for pulmonary hypertension after heart transplantation Transplantation 2001;72:638-641.[Medline]
  10. Thabut G, Brugiere O, Leseche G, Stern JB, Fradj K. Preventive effect of inhaled NO and pentoxyfylline on ischemia-reperfusion injury after lung transplantation Transplantation 2001;71:1295-1300.[Medline]
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  12. Semigran MJ, Cockrill BA, Kacmarek R, et al. Hemodynamic effects of inhaled nitric oxide in heart failure J Am Coll Cardiol 1994;24:982-988.[Medline]
  13. Girard C, Lehot J, Pannetier J, Filley S, Ffrench P, Estenove S. Inhaled nitric oxide after mitral valve replacement in patients with chronic pulmonary artery hypertension Anesthesiology 1992;77:880-883.[Medline]
  14. Bhorade S, Christenson J, O’Connor M, Lavoie A, Pohman A, Hall JB. Response to inhaled nitric oxide in patients with acute right heart syndrome Am J Respir Crit Care Med 1999;159:571-579.[Abstract/Free Full Text]
  15. Radermacher P, Santak B, Wust HJ, Tarnon J, Falke KJ. Prostacyclin and right ventricular function in patients with pulmonary hypertension associated with ARDS Intens Care Med 1990;16:227-232.[Medline]
  16. Neonatal Inhaled Nitric Oxide Study Group Inhaled nitric oxide in full-term and nearly full-term infants with hypoxic respiratory failure N Engl J Med 1997;336:597-604.[Medline]
  17. Roberts JD, Fineman JR, Morin FC, et al. Inhaled nitric oxide and persistent pulmonary hypertension in the newborn N Engl J Med 1997;336:605-610.[Medline]
  18. Lonnquist PA. Efficacy and economy of inhaled nitric oxide in neonates accepted for extra-corporeal membrane oxygenation Acta Physiol Scand 1999;167:175-179.[Medline]
  19. Baigorri F, Joseph D, Artigas A, Blanch L. Inhaled NO does not improve cardiac or pulmonary function in patients with an exacerbation of chronic obstructive pulmonary disease Crit Care Med 1999;27:2153-2158.[Medline]
  20. Kaisers U, Busch T, Deja M, Donaubauer B, Falke K. Selective pulmonary vasodilatation in acute respiratory distress syndrome Crit Care Med 2003;31(Suppl):337-342.[Medline]
  21. Abman AH, Griebel JL, Parker DK, et al. Acute effects of inhaled nitric oxide in children with severe hypoxemic respiratory failure J Pediatr 1994;124:881-888.[Medline]
  22. Dellinger RP, Zimmerman JL, Taylor RW, et al. Effects of inhaled nitric oxide in patients with acute respiratory distress syndrome Crit Care Med 1998;26:15-23.[Medline]
  23. Jacobs PD, Finer NN, Robertson CMT, Etches P, Hall E, Saunders LD. A cost-effectiveness analysis of the application of nitric oxide versus oxygen gas for near-term newborns with respiratory failure: results from a Canadian randomized clinical trial Crit Care Med 2000;28:872-878.[Medline]
  24. Hosenpud JD, Bennett LE, Keck BM, Boucek MM, Novick RJ. The Registry of the International Society for Heart and Lung Transplantation: seventeeth official report–2000 J Heart Lung Transplant 2000;19:909-931.[Medline]
  25. Doyle AR, Dhir AK, Moors AH, Latimer RD. Treatment of perioperative low cardiac output syndrome Ann Thorac Surg 1995;59(Suppl 2):3-11.
  26. Bhatia SJ, Kirshenbaum JM, Shemin RJ, et al. Time course of resolution of pulmonary hypertension and right ventricular remodeling after orthotopic cardiac transplantation Circulation 1987;76:819-826.[Abstract/Free Full Text]
  27. Chen JM, Levin HR, Micheler RE, et al. Reevaluating the significance of pulmonary hypertension before cardiac transplantation: determination of optimal thresholds and quantification of the effect of reversibility on perioperative mortality J Thorac Cardiovasc Surg 1997;114:627-634.[Abstract/Free Full Text]
  28. Tenderich G, Koerner MM, Stuettgen B, et al. Does preexisting elevated pulmonary vascular resistance (transpulmonary gradient >15 mmHg or >5 Wood) predict early and long-term results after othotopic heart transplantation? Transplant Proc 1998;30:1130-1131.[Medline]
  29. Bennett LE, Keck BM, Hertz MI, Trulock EP, Taylor DO. Worldwide thoracic organ transplantation: a report from the UNO/ISHLT international registry for thoracic organ transplantation Clin Transplant 2001;15:25-40.
  30. Harringer W, Wiebe K, Struber M, et al. Lung transplantation—10 year experience Eur J CardioThorac Surg 1999;16:546-554.[Abstract/Free Full Text]
  31. Troncy E, Collet JP, Shapiro S, et al. Should we treat acute respiratory distress syndrome with inhaled nitric oxide? Lancet 1997;350:111-118.[Medline]
  32. Michael JR, Barton RG, Saffle JR, Mone M, Markewitz BA. Inhaled nitric oxide versus conventional therapy: effect on oxygenation in ARDS Am J Resp Crit Care Med 1998;157:1361-1362.[Free Full Text]
  33. Luhr OR, Antonsen K, Karlsson M, et al. Incidence and mortality after acute respiratory failure and acute respiratory distress syndrome in Sweden, Denmark, and Iceland Am J Resp Crit Care Med 1999;159:1849-1861.[Abstract/Free Full Text]
  34. Krafft P, Fridrich P, Pernerstorfer T, et al. The acute respiratory distress syndrome: definitions, severity, and clinical outcome Intens Care Med 1996;22:519-529.[Medline]

This article has been cited by other articles:

M. M. Hoeper and J. Granton
Intensive Care Unit Management of Patients with Severe Pulmonary Hypertension and Right Heart Failure
, November 15, 2011; 184(10): 1114 – 1124.
[Abstract] [Full Text] [PDF]

A. N. Tavare and T. Tsakok
Does prophylactic inhaled nitric oxide reduce morbidity and mortality after lung transplantation?
Interact CardioVasc Thorac Surg, November 1, 2011; 13(5): 516 – 520.
[Abstract] [Full Text] [PDF]

A. Hoskote, C. Carter, P. Rees, M. Elliott, M. Burch, and K. Brown
Acute right ventricular failure after pediatric cardiac transplant: Predictors and long-term outcome in current era of transplantation medicine
J. Thorac. Cardiovasc. Surg., January 1, 2010; 139(1): 146 – 153.
[Abstract] [Full Text] [PDF]


RESOURCES on this Open Access Online Scientific Journal

1. electronic Book on Nitric Oxide by Nitric Oxide Team @ Leaders in Pharmaceutical Business Intelligence (LPBI), Amazon-Kindle, 2013

Perspectives on Nitric Oxide in Disease Mechanisms

 The Nitric Oxide Discovery, Function, and Targeted Therapy  Opportunities

From Discovery to Innovation

     From Innovation to Therapeutic Targets

From Therapeutic Targets to Clinical Applications

Aviral Vatsa, PhD, Editor

Larry H Bernstein, MD, Editor

2. The rationale and use of inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure Larry H. Bernstein 8/20/2012

3. Inhaled Nitric Oxide in Adults: Clinical Trials and Meta Analysis Studies – Recent Findings Aviva Lev-Ari, PhD, RN, 6/2/2013

Read Full Post »

Reporter: Aviva Lev-Ari, PhD, RN

Prostacyclin and Nitric Oxide: Adventures in vascular biology –  a tale of two mediators

The e-Readers are encouraged to review two additional Sources on this topic on this Open Access Online Scientific Journal

Perspectives on Nitric Oxide in Disease Mechanisms


Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium

S Moncada*

The Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BT, UK
* (Email: s.moncada@ucl.ac.uk)

Prof. Moncada:

I would like to thank the Royal Society for inviting me to deliver the Croonian Lecture. In so doing, the Society is adding my name to a list of very distinguished scientists who, since 1738, have preceded me in this task. This is, indeed, a great honour.

For most of my research career my main interest has been the understanding of the normal functioning of the blood vessel wall and the way this is affected in pathology. During this time, our knowledge of these subjects has grown to such an extent that many people now believe that the conquering of vascular disease is a real possibility in the foreseeable future.

My lecture concerns the discovery of two substances, prostacyclin and nitric oxide. I would like to describe the moments of insight and some of the critical experiments that contributed significantly to the uncovering of their roles in vascular biology. The process was often adventurous, hence the title of this lecture. It is the excitement of the adventure that I would like to convey in the text that follows.

Keywords: prostacyclin, aspirin, nitric oxide, oxidative stress, free radicals, cardiovascular pathology
Full article 
Philos Trans R Soc Lond B Biol Sci. 2006 May 29; 361(1469): 735–759.
Published online 2006 February 8. doi:  10.1098/rstb.2005.1775
PMCID: PMC1609404


Although the research fields of prostacyclin/thromboxane and NO are now mature, they have developed mostly as parallel research activities with few points of contact between them. Thus, our understanding of how both might operate in relation to each other in physiology and pathophysiology remains to be developed. Table 2 shows some of the similarities between prostacyclin and NO. Both mediators, from very different biochemical pathways, play a variety of roles in the modulation and protection of the vascular wall. The release of both mediators is dependent on constitutive enzymes, the activity of which seems to be regulated locally, predominantly by the shear stress caused by the blood passing over the endothelial surface (Grabowski et al. 1985Frangos et al. 1985; for review see Boo & Jo 2003). However, while the constitutive eNOS—localized only in the vascular endothelium—is the enzyme that responds to shear stress, the generation of prostacyclin is dependent on the activity of two enzymes, COX-1 and COX-2, in relation to which several questions remain unanswered. These include whether COX-2 is a constitutive as well as an inducible enzyme, and whether COX-1 or COX-2, or both, respond to shear stress by increases in their mRNA, their activity, or both (Topper et al. 1996Okahara et al. 1998;McCormick et al. 2000Garcia-Cardena et al. 2001). Prostacyclin, unlike NO, is constitutively generated throughout the vessel wall (Moncada et al. 1977c) and at this stage we also do not know whether the ratio between COX-1 and COX-2 changes in the different layers. In addition, the similarities and differences between regulation of NO and prostacyclin by shear stress are only now being investigated (Osanai et al. 2000McAllister et al. 2000Walshe et al. 2005).

Table 2

Table 2

Comparison of the properties of nitric oxide and prostacyclin.

A clear synergism between NO and prostacyclin has been demonstrated in regard to inhibition of platelet aggregation; however, only one of them (NO) plays a role in inhibiting platelet adhesion. The significance of this difference remains to be understood. Many years ago a physiological role for platelets in repairing the vessel wall was investigated (for discussion see Higgs et al. 1978). This subject has not been re-evaluated in the light of all this new knowledge about the roles of NO and prostacyclin in platelet/vessel wall interactions. Both mediators also regulate vascular smooth muscle proliferation and white cell vessel wall interactions through similar mechanisms which include, at least in part, the activation of adenylate cyclase and the soluble guanylate cyclase. The interactions between NO and prostacyclin in the control of these functions are not fully understood.

Both mediators are further increased by inflammatory stimuli; however, while in the case of prostacyclin the same COX-2 which responds to shear stress responds to such stimuli by a further increase in its expression, NO is generated during inflammation by a specific ‘inducible’ NO synthase which is not normally present physiologically in the vessel wall. The induction of both is inhibited by anti-inflammatory glucocorticoids (Axelrod 1983Knowles et al. 1990). It is remarkable that both compounds possess antioxidant properties (Wink et al. 1995Egan et al. 2004) but are themselves affected by oxidative stress, which inhibits the synthesis of prostacyclin and decreases the bioavailability of NO. This mechanism might be relevant to the ‘malfunctioning’ of the constitutive generation of both mediators and therefore to the genesis of endothelial dysfunction. This, however, is an early phenomenon. In advanced disease the situation is far more complex, akin to chronic inflammation in other parts of the body and, as such, probably varies significantly in the different stages of the disease. A simple hypothesis would suggest that any amount of prostacyclin which is bioavailable, although pro-inflammatory, will provide anti-thrombotic protection, while in the case of NO the balance will vary between bioavailable NO which is protective and cytotoxic peroxynitrite formed from the interaction of NO with O2. Currently, however, the results are not clear and on the crucial question of the role of both mediators in the progression of atherosclerosis, the information in relation to prostacyclin is contradictory (Burleigh et al. 2002Olesen et al. 2002Rott et al. 2003). The evidence in relation to NO, on the other hand, seems to suggest that, while constitutive NO generated by eNOS is protective (e.g. Kawashima & Yokoyama 2004), NO generated by the inducible enzyme favours the development of atherosclerosis (Chyu et al. 1999). Studies of genetically manipulated animals are providing some important clues. For example, knockout of the prostacyclin receptor (IP) leads to mice with normal blood pressure but an increased tendency to thrombosis when the endothelium is damaged (Murata et al. 1997) These animals also exhibit an increased platelet activation and proliferative response to injury that can be prevented by deletion or antagonism of the TXA2 receptor (Cheng et al. 2002). Furthermore, deletion of the IP receptor in animals prone to spontaneous atherosclerosis accelerates the development of the disease (Egan et al. 2004;Kobayashi et al. 2004). On the other hand, knocking out the thromboxane receptor or the thromboxane synthase gives rise to a mild bleeding tendency and a resistance to platelet aggregation and sudden death induced by arachidonic acid infusion (Thomas et al. 1998Yu et al. 2004). Deletion of the thromboxane receptor also seems to retard atherogenesis in murine models of atherosclerosis (Cayatte et al. 2000;Egan et al. 2005).

Although the lack of either mediator has been shown to increase the risk of thrombosis and atherosclerosis, especially in animals with additional risk factors such as ApoE deficiencies (Kuhlencordtet al. 2001Belton et al. 2003), there seems to be a certain specialization in their actions, so that NO has a more significant role in the regulation of blood pressure and blood flow, while prostacyclin has a clearer role in regulating platelet/vessel wall interactions. For example, inhibition of NO generation has an immediate and dramatic effect on blood flow and blood pressure and the eNOS−/− animal exhibits a clear hypertensive phenotype. On the other hand, inhibition of prostacyclin synthesis by the coxibs leads to a slow effect on blood pressure and apparently to a more thrombotic situation (Muscara et al. 2000;FitzGerald 2003). Similarly, COX-1−/− and COX-2−/− animals show no change in blood pressure (Norwood et al. 2000Cheung et al. 2002) and manipulation of COX or IP results in a prothrombotic phenotype.

Protection against decreases in the generation of constitutive NO and prostacyclin in the vasculature may prevent the development of vascular disease. In relation to NO, the most often tried interventions relate to the use of antioxidants (see Carr & Frei 2000) and the manipulation of eNOS expression by genetic means (Von der Leyen & Dzau 2001). Each of these interventions has shown promise in both animal experiments and in humans. An unexpected and highly interesting development relates to the effects of statins which, in the last few years, have been shown to increase the production of endothelial NO in endothelial cell cultures and in animals (for review see Laufs 2003). Many mechanisms have been claimed for this action. However, of interest in the context of our discussion is the fact that statins have been claimed to reduce oxidative stress by increasing the synthesis of BH4 (Hattori et al. 2002), increasing the coupling of the eNOS (Brouet et al. 2001) or reducing the activation of NADPH oxidase (Wagner et al. 2000). Reduction of oxidative stress is likely to preserve the generation of prostacyclin, and to our knowledge there is at least one report suggesting that statins also increase prostacyclin in endothelial cell cultures of human coronary arteries (Mueck et al. 2001). Studies on the transfection of COX-1 or COX-2 into endothelial and other cells, on the other hand, are at an early stage and clear results are not conclusive (Murakami et al. 1999Shyue et al. 2001). The full consequences of overexpression of both NO and prostacyclin in the vasculature remain to be investigated.

Also relevant to this discussion are studies of the role that NO and prostacyclin play in the protection of the cardiovascular system provided by oestrogens, and therefore in the difference between genders in susceptibility to cardiovascular disease. Oestrogens increase the expression and the activity of eNOS (Weiner et al. 1994Yang et al. 2000) and the activity of the COX-2 enzyme (Akarasereenont et al. 2000;Egan et al. 2004). They could therefore reduce oxidative stress by simply increasing both mediators. Alternatively, it has been claimed that oestrogens increase the efficiency of the NO synthase, thus reducing free radical formation (Barbacanne et al. 1999).

In summary, the concept of the balance between prostacyclin and TXA2 has to be expanded to include NO. Furthermore, although not discussed in this review, the way in which these compounds interact with many other systems known to be involved in vessel wall physiology and pathophysiology requires further investigation. Both prostacyclin and NO synergize in the protection of the vessel wall. TXA2, however, lies on the negative side of this balance being responsible for, among other things, platelet aggregation and vasoconstriction. The investigation into the interplay between these three molecules is just beginning. This is a sobering thought when one is contemplating probably close to 100 000 papers and over 30 years of research! However, it is clear that the discoveries of prostacyclin and NO have transformed our comprehension of vascular physiology and opened avenues for further understanding of pathophysiological processes. This knowledge has already benefited clinical medicine and no doubt will continue providing clues that will guide future therapy and prevention of vascular disease. I have had the good fortune to be intimately involved with both discoveries. More importantly, many of the colleagues that I have interacted with in the process of doing this work have become life-long personal friends. To those with whom I have managed to combine scientific excitement with friendship I owe a double debt of gratitude.

Philos Trans R Soc Lond B Biol Sci. 2006 May 29; 361(1469): 735–759.
Published online 2006 February 8. doi:  10.1098/rstb.2005.1775

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Perspectives on Nitric Oxide in Disease Mechanisms


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Nitric Oxide and it’s impact on Cardiothoracic Surgery

Author, curator: Tilda Barliya PhD


In the past few weeks we’ve had extensive in-depth series about nitric oxide (NO) and it’s role in renal function and donors in renal disorders, coagulation, endothelium and hemostasis. This inspired this new post regarding the impact of NO on cardiothoratic surgery.  You can read and follow up on these posts here: https://pharmaceuticalintelligence.com/category/nitric-oxide-in-health-and-disease/

Atherosclerosis in the form of peripheral arterial disease (PAD) affects approximately eight million Americans, which includes 12 to 20% of individuals over the age of 65.  Approximately 20% of patients with PAD have typical symptoms of lower extremity claudication, rest pain, ulceration, or gangrene, and one-third have atypical exertional symptoms. Persons with PAD have impaired function and quality of life even if they do not report symptoms and experience a decline in lower extremity function over time. Cardiovascular disease is the major cause of death in patients with intermittent claudication; the annual rate of cardiovascular events (myocardial infarction, stroke, or death from cardiovascular causes) is 5 to 7%.  Thus, PAD represents a significant source of morbidity and mortality. (1) (http://www.medscape.com/viewarticle/569812).

Several options exist for treating atherosclerotic lesions, including:

  • percutaneous transluminal angioplasty with and without stenting,
  • endarterectomy
  • bypass grafting

Unfortunately, patency rates for each of these procedures continue to be suboptimal secondary to the development of neointimal hyperplasia. A universal feature of all vascular surgical procedures is the removal of or damage to the endothelial cell monolayer that occurs whether the procedure performed is endovascular or open. This endothelial damage leads to a decreased or absent production of nitric oxide (NO) at the site of injury.


he relationship between NO and the cardiovascular system has proven to be a landmark discovery, and the scientists credited for its discovery were awarded the Nobel Prize in Medicine in 1998. Since its discovery, NO has proven to be one of the most important molecules in vascular homeostasis. In fact, the term endothelial dysfunction has now become synonymous with the reduced biologic activity of NO.

NO produced by endothelial cells has been shown to have many beneficial effects on the vasculature.

As described above,

  • NO stimulates vascular smooth muscle cells (VSMC) relaxation, which leads to vessel vasodilatation.  
  • NO has opposite beneficial affects on endothelial cells compared with VSMCs.
  • Whereas NO stimulates endothelial cell proliferation and prevents endothelial cell apoptosis,  it inhibits VSMC growth and migration  and stimulates VSMC apoptosis.  
  • NO also has many thromboresistant properties, such as inhibition of platelet aggregation, adhesion, and activation;  inhibition of leukocyte adhesion and migration;  and inhibition of matrix formation

 As stated before, the endothelial cell monolayer is often removed or damaged during the time of vascular procedures, which leads to a local decrease in the production of NO. It is now understood that this loss of local NO synthesis by endothelial cells at the site of vascular injury is one of the inciting events that allows platelet aggregation, inflammatory cell infiltration, and VSMC proliferation and migration to occur in excess, which, taken together, leads to neointimal hyperplasia.

Reendothelialization of the injured artery can restore proper function to the artery and potentially halt the restenotic process. Many studies have attempted to improve the patency of bypass grafts and stents by coating them with endothelial cells in the hope that this would restore the thromboresistant nature of native blood vessels.

Unfortunately, although it has been possible to coat these devices with endothelial cells, these cells do not behave like normal endothelial cells and their NO production is often diminished or absent. Because the vasoprotective properties of endothelial cells are largely carried out by NO alone, investigators are engaged in research to improve the bioavailability of NO at the site of vascular injury in an attempt to reduce the risk of thrombosis and restenosis after successful revascularization. The overall goal of using a NO-based approach is to reproduce the same thromboresistive moiety observed with normal NO production.

Why of delivering NO to the injured site:

  • Systemic delivery
  • Local delivery

Systemic Delivery

One simple mechanism by which to deliver NO to the body is via inhalational therapy. Inhaled NO has been used clinically in the past to selectively reduce pulmonary vascular resistance in patients with pulmonary hypertension, as well as a potential therapy for patients with acute respiratory distress syndrome. Because the gas is delivered only to the pulmonary system and has a very short half-life, it was thought that there would be no systemic effects of the drug. Subsequently, studies in the mid- to late 1990s suggested that inhaled NO had beneficial antiplatelet and antileukocyte properties without adverse systemic side effects (2,3)

To test if inhaled NO had any beneficial systemic properties specifically on the vasculature, Lee and colleagues evaluated the effect of inhaled NO on neointimal hyperplasia in rats undergoing carotid balloon injury, Unfortunately, the treatment was required for the full 2 weeks to see any difference between the treatment and the control group, thereby limiting its clinical utility.

Despite some of the early animal studies, investigations with healthy human volunteers failed to reproduce these findings.I t was speculated that despite the obvious effects of inhaled NO on the pulmonary vasculature, systemic bioavailability could not be reliably achieved because of the immediate binding and depletion of NO by hemoglobin as soon as it entered the systemic circulation.

Hamon and colleagues tested the ability of orally supplementing l-arginine (2.25%), the precursor to NO, in the drinking water of rabbits to reduce the formation of neointimal hyperplasia after injuring the iliac arteries with a balloon.  This amount of l-arginine is approximately sixfold higher than normal daily intake. When the arteries were studied 4 weeks after injury, the l-arginine-fed group exhibited less neointimal hyperplasia and greater acetylcholine-induced relaxation compared with the control animals. The authors speculated that the improved outcomes were due to increased bioavailability of NO secondary to the l-arginine-supplemented diets. To test the ability of this supplemented diet to reduce neointimal hyperplasia in a vein bypass graft model, Davies and colleagues fed rabbits l-arginine (2.25%) 7 days prior to and 28 days after common carotid vein bypass grafts. A 51% decrease in the formation of neointimal hyperplasia was demonstrated in the l-arginine-fed groups, and their vein grafts exhibited preserved NO-mediated relaxation.

Despite some of the positive findings in animals, similar studies in humans have failed to show any benefit with l-arginine supplementation. Shiraki and colleagues studied the effects of short-term high-dose l-arginine on restenosis after PTCA.  Thirty-four patients undergoing cardiac catheterization and PTCA for angina pectoris received 500 mg of l-arginine administered through the cardiac catheter immediately prior to PTCA and 30 g per day of l-arginine administered via the peripheral vein for 5 days after PTCA. No significant statistical differences in restenosis were observed between the two groups (34% vs 44%). The authors speculated that the lack of effect was secondary to the fact that although the levels of l-arginine in the plasma increased significantly, NO and cyclic guanosine monophosphate (cGMP) did not. (4)

Table 1.  Comparison of Different Nitric Oxide Donor Drugs Currently Used for Clinical or Research Purposes
Drug Mechanism of NO Release Unique Properties
Diazeniumdiolates Spontaneous when in contact with physiologic fluidsNO release follows first-order kinetics Stable as solidsVarious reliable half-lives depending on the structure of the nucleophile it is attached to
Nitrosamines can form as by-products
S-Nitrosothiols Copper ion-mediated decomposition Stable as a solid
Direct reaction with ascorbate Must be protected from light
Homeolytic cleavage by light Present in circulating blood
Potential for unlimited NO release
Sydnonimines Requires enzymatic cleavage by liver esterases to form active metabolite Stable as a solidMust be protected from light
Requires molecular oxygen as an electron acceptor Requires alkaline pHReleases superoxide as a by-product, which may have negative effects
l-Arginine Substrate for NOS genes Stable as a solid
Ease of administration
Dependent on presence of NOS for NO production
Sodium nitroprusside Requires a one-electron reduction to release NO Stable as a solid
Must be protected from light
Light can induce NO release Must be given intravenously
Releases cyanide as a by-product
Organic nitrates Either by enzymatic cleavage or nonenzymatic bioactivation with sulfhydryl or thiol groups Stable as a solid
Must be protected from light
Ease of administration
Development of tolerance limits efficacy
NO-releasing aspirin Require enzymatic cleavage to break the covalent bond between the aspirin and the NO moiety Stable as a solid
Ease of administration
Inherent benefits of aspirin also
Does not affect systemic blood pressure

Despite the ease of administration, the reliability of drug delivery, and the relative safety of these NO-donating drugs, there are limitations associated with systemic administration. One such limitation is that NO is rapidly inactivated by hemoglobin in the circulating blood, resulting in limited bioavailability. Furthermore, in attempts to increase the amount of drug delivered to obtain the desired clinical effect, unwanted systemic circulatory effects (eg, vasodilation) and unwanted hemostatic effects (eg, bleeding) often preclude administration of biologically effective doses of NO.

Because NO produces systemic side effects, lower doses of NO have been used in many of the human studies. One of the reasons for the differences observed between the animal studies and the human studies was the 10- to 50-fold lower doses of drugs used in the human studies compared with the animal studies. Thus, local delivery of NO may achieve improved results.

Local Delivery

The local delivery of drugs allows for the administration of the maximally effective dose of a drug without the unwanted systemic side effects. Because the target vessels are easily accessible during most vascular procedures, a local pharmacologic approach to administer a drug during the intervention can be easily performed.

Suzuki and colleagues performed a prospective, randomized, single-center clinical trial. (7)

The study population consisted of patients with symptomatic ischemic heart disease who were undergoing coronary artery stent placement. After stent deployment, l-arginine (600 mg/6 mL) or saline (6 mL) was locally delivered via a catheter over 15 minutes. The patients were followed with serial angiography and intravascular ultrasonography to assess for neointimal thickness for up to 6 months. The authors found that in the l-arginine-treated groups, there was slightly less neointimal volume, but this was not statistically significant.

Because it was not known if the addition of l-arginine actually translated to increased NO production, several studies have focused on the addition of NO donors directly to the site of injury.However, Critics of some of the highlighted animal studies point out that the evaluation of neointimal hyperplasia was performed radiographically, which could be subjectively biased. Furthermore, infusing the drug through a catheter for an extended period of time during the procedure to achieve an effect is not clinically feasible. Because of this, other studies have aimed to develop a clinically applicable approach to deliver NO locally to the site of injury.

  • Hydrogels
  • Vascular grafts
  • Gene therapy

represents another method by which to locally increase the level of NO at the site of vascular injury, tested in different multiple creative animal models. Thought, most of this studies shown great preliminary results, only the gene therapy moved forward into randomized clinical trial in humans using gene therapy to reduce neointimal hyperplasia.

In December 2000, the Recombinant DNA Advisory Committee at the National Institutes of Health voted unanimously to proceed with the first phase of clinical evaluation of iNOS lipoplex-mediated gene transfer, called REGENT-1: Restenosis Gene Therapy Trial. (8). The primary objective of this multicenter, prospective, single-blind, dose escalation study was to obtain safety and tolerability information of iNOS-lipoplex gene therapy for reducing restenosis following coronary angioplasty. As of 2002, 27 patients had been enrolled overseas and the process had been determined to be safe. To date, no results have been published as it appears that this trial lost its funding and closed. On April 5, 2002, a notification was issued that the trial had been closed without enrolling any individuals in the United States.

Unfortunately, despite the promising findings shown with NOS therapy, the field of gene therapy has been mottled by two widely known complications. One case occurred as the result of administering a large viral load that led to the death of a patient. In addition, in France, there were at least two cases of malignancy following retroviral gene therapy.  (9)


Atherosclerosis in the form of coronary artery disease and peripheral vascular disease continues to be a major source of morbidity and mortality. Unfortunately, the procedures and materials that are currently used to alleviate these disease states are temporary at best because of the inevitable injury to the native endothelium and the subsequent impairment of NO release. Since the discovery of NO and its role in vascular biology, a main focus in vascular research has been to create novel mechanisms to use NO to combat neointimal hyperplasia. To date, numerous animal studies have restored NO production to the vasculature and have shown that this inhibits neointimal hyperplasia, improves patency rates, and is safe to the animal. Clinical studies using these novel NO-releasing compounds in humans are on the horizon.


1. Daniel A. Popowich, Vinit Varu, Melina R. Kibbe. Nitric Oxide: What a Vascular Surgeon Needs to Know. Vascular. 2007;15(6):324-335. (http://www.medscape.com/viewarticle/569812).

2.  Gries A, Bode C, Peter K, et al. Inhaled nitric oxide inhibits human platelet aggregation, P-selectin expression, and fibrinogen binding in vitro and in vivo Circulation 1998;97:1481-7.

3.  Lee JS, Adrie C, Jacob HJ, et al. Chronic inhalation of nitric oxide inhibits neointimal formation after balloon-induced arterial injury Circ Res 1996;78:337-42.

4.  Shiraki T, Takamura T, Kajiyama A, et al. Effect of short-term administration of high dose l-arginine on restenosis after percutaneous transluminal coronary angioplasty J Cardiol 2004;44:13-20.

5. David A. Fullerton, MD, Robert C. McIntyre, Jr, MD. Inhaled Nitric Oxide: Therapeutic Applications in Cardiothoracic Surgery. Ann Thorac Surg 1996;61:1856-1864. http://ats.ctsnetjournals.org/cgi/content/abstract/61/6/1856

6. Owen I.Miller,Swee Fong Tang, Anthony Keech,Nicholas B.Pigott, Elaine Beller and David S. Celemajer.  Inhaled nitric oxide and prevention of pulmonary hypertension after congenital heart surgery: a randomised double-blind study. The Lancet,2000:356; 9240 Pages 1464 – 1469,  http://www.thelancet.com/journals/lancet/article/PIIS0140-6736(00)02869-5/abstract

7. Suzuki T, Hayase M, Hibi K, et al. Effect of local delivery of l-arginine on in-stent restenosis in humans Am J Cardiol 2002;89:363-7.

8. von der Leyen HE, Chew N. Nitric oxide synthase gene transfer and treatment of restenosis: from bench to bedside Eur J Clin Pharmacol 2006;62:83-89

9.  Barbato JE, Tzeng E. iNOS gene transfer for graft disease Trends Cardiovasc Med 2004;14:267-72.

10. E. Matevossian, A. Novotny, C. Knebel, T. Brill, M. Werner, I. Sinicina, M. Kriner, M. Stangl, S. Thorban, and N. Hüser. The Effect of Selective Inhibition of Inducible Nitric Oxide Synthase on Cytochrome P450 After Liver Transplantation in a Rat Model. Transplantation Proceedings 2008, 40, 983–985.

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Telling NO to Cardiac Risk

DDAH Says NO to ADMA(1); The DDAH/ADMA/NOS Pathway(2)

Author-Writer-Reporter:  Stephen J. Williams, PhD

Endothelium-derived nitric oxide (NO) has been shown to be vasoprotective.  Nitric oxide enhances endothelial cell survival, inhibits excessive proliferation of vascular smooth muscle cells, regulates vascular smooth muscle tone, and prevents platelets from sticking to the endothelial wall.  Together with evidence from preclinical and human studies, it is clear that impairment of the NOS pathway increases risk of cardiovascular disease (3-5).

This post contains two articles on the physiological regulation of nitric oxide (NO) by an endogenous NO synthase inhibitor asymmetrical dimethylarginine (ADMA) and ADMA metabolism by the enzyme DDAH(1,2).  Previous posts on nitric oxide, referenced at the bottom of the page, provides excellent background and further insight for this posting. In summary plasma ADMA levels are elevated in patients with cardiovascular disease and several large studies have shown that plasma ADMA is an independent biomarker for cardiovascular-related morbidity and mortality(6-8).



Figure 1 A. Cardiac risks of ADMA B. Effects of ADMA (Photo credit: Wikipedia)

ADMA Production and Metabolism

Nuclear proteins such as histones can be methylated on arginine residues by protein-arginine methyltransferases, enzymes which use S-adenosylmethionine as methyl groups.  This methylation event is thought to regulate protein function, much in the way of protein acetylation and phosphorylation (9).  And much like phosphorylation, these modifications are reversible through methylesterases.   The proteolysis of these arginine-methyl modifications lead to the liberation of free guanidine-methylated arginine residues such as L-NMMA, asymmetric dimethylarginine (ADMA) and symmetrical methylarginine (SDMA).

The first two, L-NMMA and ADMA, have been shown to inhibit the activity of the endothelial NOS.  This protein turnover is substantial: for instance the authors note that each day 40% of constitutive protein in adult liver is newly synthesized protein. And in several diseases, such as muscular dystrophy, ischemic heart disease, and diabetes, it has been known since the 1970’s that protein catabolism rates are very high, with corresponding increased urinary excretion of ADMA(10-13).  Methylarginines are excreted in the urine by cationic transport.  However, the majority of ADMA and L-NMMA are degraded within the cell by dimethylaminohydrolase (DDAH), first cloned and purified in rat(14).

endogenous NO inhibitors from pubchem

Figure 2.  Endogenous inhibitors of NO synthase.  Chemical structures generated from PubChem.


DDAH specifically hydrolyzes ADMA and L-NMMA to yield citruline and demethylamine and usually shows co-localization with NOS. Pharmacologic inhibition of DDAH activity causes accumulation of ADMA and can reverse the NO-mediated bradykinin-induced relaxation of human saphenous vein.

Two isoforms have been found in human:

  • DDAH1 (found in brain and kidney and associated with nNOS) and
  • DDAH2 (highly expressed in heart, placenta, and kidney and associated with eNOS).

DDAH2 can be upregulated by all-trans retinoic acid (atRA can increase NO production).  Increased reactive oxygen species and possibly homocysteine, a risk factor for cardiovascular disease, can decrease DDAH activity(15,16).

  • The importance of DDAH activity can also be seen in transgenic mice which overexpress DDAH, exhibiting increased NO production, increased insulin sensitivity, and reduced vascular resistance  (17).  Likewise,
  • Transgenic mice, null for the DDAH1, showed increase in blood pressure, decreased NO production, and significant increase in tissue and plasma ADMA and L-NMMA.


Figure 3.  The DDAH/ADMA/NOS cycle. Figure adapted from Cooke and Ghebremarian (1).

As mentioned in the article by Cooke and Ghebremariam, the authors state: the weight of the evidence indicates that DDAH is a worthy therapeutic target. Agents that increase DDAH expression are known, and 1 of these, a farnesoid X receptor agonist, is in clinical trials


An alternate approach is to

  • develop an allosteric activator of the enzyme.  Although
  • development of an allosteric activator is not a typical pharmaceutical approach, recent studies indicate that this may be achievable aim(18).


1.            Cooke, J. P., and Ghebremariam, Y. T. : DDAH says NO to ADMA.(2011) Arteriosclerosis, thrombosis, and vascular biology 31, 1462-1464

2.            Tran, C. T., Leiper, J. M., and Vallance, P. : The DDAH/ADMA/NOS pathway.(2003) Atherosclerosis. Supplements 4, 33-40

3.            Niebauer, J., Maxwell, A. J., Lin, P. S., Wang, D., Tsao, P. S., and Cooke, J. P.: NOS inhibition accelerates atherogenesis: reversal by exercise. (2003) American journal of physiology. Heart and circulatory physiology 285, H535-540

4.            Miyazaki, H., Matsuoka, H., Cooke, J. P., Usui, M., Ueda, S., Okuda, S., and Imaizumi, T. : Endogenous nitric oxide synthase inhibitor: a novel marker of atherosclerosis.(1999) Circulation 99, 1141-1146

5.            Wilson, A. M., Shin, D. S., Weatherby, C., Harada, R. K., Ng, M. K., Nair, N., Kielstein, J., and Cooke, J. P. (2010): Asymmetric dimethylarginine correlates with measures of disease severity, major adverse cardiovascular events and all-cause mortality in patients with peripheral arterial disease. Vasc Med 15, 267-274

6.            Kielstein, J. T., Impraim, B., Simmel, S., Bode-Boger, S. M., Tsikas, D., Frolich, J. C., Hoeper, M. M., Haller, H., and Fliser, D. : Cardiovascular effects of systemic nitric oxide synthase inhibition with asymmetrical dimethylarginine in humans.(2004) Circulation 109, 172-177

7.            Kielstein, J. T., Donnerstag, F., Gasper, S., Menne, J., Kielstein, A., Martens-Lobenhoffer, J., Scalera, F., Cooke, J. P., Fliser, D., and Bode-Boger, S. M. : ADMA increases arterial stiffness and decreases cerebral blood flow in humans.(2006) Stroke; a journal of cerebral circulation 37, 2024-2029

8.            Mittermayer, F., Krzyzanowska, K., Exner, M., Mlekusch, W., Amighi, J., Sabeti, S., Minar, E., Muller, M., Wolzt, M., and Schillinger, M. : Asymmetric dimethylarginine predicts major adverse cardiovascular events in patients with advanced peripheral artery disease.(2006) Arteriosclerosis, thrombosis, and vascular biology 26, 2536-2540

9.            Kakimoto, Y., and Akazawa, S.: Isolation and identification of N-G,N-G- and N-G,N’-G-dimethyl-arginine, N-epsilon-mono-, di-, and trimethyllysine, and glucosylgalactosyl- and galactosyl-delta-hydroxylysine from human urine. (1970) The Journal of biological chemistry 245, 5751-5758

10.          Inoue, R., Miyake, M., Kanazawa, A., Sato, M., and Kakimoto, Y.: Decrease of 3-methylhistidine and increase of NG,NG-dimethylarginine in the urine of patients with muscular dystrophy. (1979) Metabolism: clinical and experimental 28, 801-804

11.          Millward, D. J.: Protein turnover in skeletal muscle. II. The effect of starvation and a protein-free diet on the synthesis and catabolism of skeletal muscle proteins in comparison to liver. (1970) Clinical science 39, 591-603

12.          Goldberg, A. L., and St John, A. C.: Intracellular protein degradation in mammalian and bacterial cells: Part 2. (1976) Annual review of biochemistry 45, 747-803

13.          Dice, J. F., and Walker, C. D.: Protein degradation in metabolic and nutritional disorders. (1979) Ciba Foundation symposium, 331-350

14.          Ogawa, T., Kimoto, M., and Sasaoka, K.: Purification and properties of a new enzyme, NG,NG-dimethylarginine dimethylaminohydrolase, from rat kidney. (1989) The Journal of biological chemistry 264, 10205-10209

15.          Ito, A., Tsao, P. S., Adimoolam, S., Kimoto, M., Ogawa, T., and Cooke, J. P.: Novel mechanism for endothelial dysfunction: dysregulation of dimethylarginine dimethylaminohydrolase. (1999) Circulation 99, 3092-3095

16.          Stuhlinger, M. C., Tsao, P. S., Her, J. H., Kimoto, M., Balint, R. F., and Cooke, J. P. : Homocysteine impairs the nitric oxide synthase pathway: role of asymmetric dimethylarginine.(2001) Circulation 104, 2569-2575

17.          Sydow, K., Mondon, C. E., Schrader, J., Konishi, H., and Cooke, J. P.: Dimethylarginine dimethylaminohydrolase overexpression enhances insulin sensitivity. (2008) Arteriosclerosis, thrombosis, and vascular biology 28, 692-697

18.          Zorn, J. A., and Wells, J. A.: Turning enzymes ON with small molecules. (2010) Nature chemical biology 6, 179-188

Other research papers on Nitric Oxide and Cardiac Risk  were published on this Scientific Web site as follows:

The Nitric Oxide and Renal is presented in FOUR parts:

Part I: The Amazing Structure and Adaptive Functioning of the Kidneys: Nitric Oxide

Part II: Nitric Oxide and iNOS have Key Roles in Kidney Diseases

Part III: The Molecular Biology of Renal Disorders: Nitric Oxide

Part IV: New Insights on Nitric Oxide donors

Cardiac Arrhythmias: A Risk for Extreme Performance Athletes

What is the role of plasma viscosity in hemostasis and vascular disease risk?

Cardiovascular Risk Inflammatory Marker: Risk Assessment for Coronary Heart Disease and Ischemic Stroke – Atherosclerosis.

Endothelial Dysfunction, Diminished Availability of cEPCs, Increasing CVD Risk for Macrovascular Disease – Therapeutic Potential of cEPCs

Biochemistry of the Coagulation Cascade and Platelet Aggregation – Part I

Nitric Oxide Function in Coagulation

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