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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)

Summary

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.

Ref:

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. http://211.144.68.84:9998/91keshi/Public/File/29/40-4/pdf/1-s2.0-S0041134508004181-main.pdf