Biomedicine & Pharmacotherapy 167 (2023) 115549
Contents lists available at ScienceDirect 
Biomedicine & Pharmacotherapy 
journal homepage: www.elsevier.com/locate/biopha 
Review 
Therapeutic benefits of nitric oxide in lung transplantation 
George J. Dugbartey a,b,* 
a Department of Pharmacology and Toxicology, School of Pharmacy, College of Health Sciences, University of Ghana, Legon, Accra, Ghana 
b Accra College of Medicine, Magnolia St, JVX5+FX9, East Legon, Accra, Ghana   
A R T I C L E  I N F O   A B S T R A C T   
Keywords: Lung transplantation is an evolutionary procedure from its experimental origin in the twentieth century and is 
Lung transplantation now recognized as an established and routine life-saving intervention for a variety of end-stage pulmonary 
Primary graft dysfunction (PGD) diseases refractory to medical management. Despite the success and continuous refinement in lung trans-
Lung allograft rejection, nitric oxide (NO) plantation techniques, the widespread application of this important life-saving intervention is severely hampered 
NO donors 
Donation-after-cardiac-death (DCD) by poor allograft quality offered from donors-after-brain-death. This has necessitated the use of lung allografts 
from donors-after-cardiac-death (DCD) as an additional source to expand the pool of donor lungs. Remarkably, 
the lung exhibits unique properties that may make it ideally suitable for DCD lung transplantation. However, 
primary graft dysfunction (PGD), allograft rejection and other post-transplant complications arising from un-
avoidable ischemia-reperfusion injury (IRI) of transplanted lungs, increase morbidity and mortality of lung 
transplant recipients annually. In the light of this, nitric oxide (NO), a selective pulmonary vasodilator, has been 
identified as a suitable agent that attenuates lung IRI and prevents PGD when administered directly to lung 
donors prior to donor lung procurement, or to recipients during and after transplantation, or administered 
indirectly by supplementing lung preservation solutions. This review presents a historical account of clinical lung 
transplantation and discusses the lung as an ideal organ for DCD. Next, the author highlights IRI and its clinical 
effects in lung transplantation. Finally, the author discusses preservation solutions suitable for lung trans-
plantation, and the protective effects and mechanisms of NO in experimental and clinical lung transplantation.   
1. Introduction century and after decades of a hiatus because of failed clinical attempts, 
lung transplantation has evolved into a well-established and routine life- 
Lung transplantation is currently the mainstay of therapy for patients saving intervention for patients with a variety of terminal lung diseases 
with different types of end-stage respiratory diseases worldwide. It refractory to medical management [1–5]. The first human lung trans-
provides these patients with a better quality of life and survival benefits. plantation was performed by James Hardy and his team in 1963 in a 
This form of surgical treatment involves the transfer of donor lungs 58-year-old man who was diagnosed with a squamous cell carcinoma of 
procured from circulation-intact, brain-dead individuals and from those the left main bronchus with retro-obstructive pneumonitis [6]. How-
with circulatory arrest into transplant recipients. Advances in both basic ever, the procedure was not successful enough to be accepted as a lung 
science and clinical research aspects of this field have resulted in success replacement therapy for terminal lung diseases, as the lung transplant 
in clinical lung transplantation in terms of patient outcomes due to recipient unfortunately died on post-operative day 18 due to renal 
continuous refinement in donor selection criteria, and improvements in dysfunction. Interestingly, autopsy revealed no graft rejection [7]. 
donor lung preservation techniques, perioperative and post-transplant About 3 weeks later, the second lung transplantation in human was 
management and better treatment of post-transplant complications performed by Magovern and Yates in a 44-year-old man but the patient 
including immunosuppressive therapy [1–5]. died on post-operative day 8 [8]. Following these failed attempts, a total 
of 23 lung transplants were performed by 20 lung transplant surgeons 
1.1. Historical account of clinical lung transplantation globally, which also resulted in no success, as the survial periods were 
consistently less than 1 month [9]. The breakthrough finally came in 
From its experimental origin at the beginning of the twentieth 1971 when Derom et al. [10] in Belgium performed the first successful 
* Corresponding author at: Department of Pharmacology and Toxicology, School of Pharmacy, College of Health Sciences, University of Ghana, Legon, Accra, 
Ghana. 
E-mail addresses: gjdugbartey@ug.edu.gh, profduu@yahoo.com.  
https://doi.org/10.1016/j.biopha.2023.115549 
Received 6 August 2023; Received in revised form 6 September 2023; Accepted 18 September 2023   
Available online 19 September 2023
0753-3322/© 2023 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
G.J. Dugbartey                                                                                                                                                                                    B  i o  m   e d  i c i n  e   &   P  h  a  r m  a  c  o t h  e  r a  p y  167 (2023) 115549
lung transplantation in a 23-year-old man, who worked as a sandblaster after death, and the inflated lung will remain viable for at least 2 hours 
and was diagnosed with end-stage silicosis due to heavy exposure to after death without perfusion since its oxygen delivery to tissues is in-
silicon dust for 2 years. After transplantation, the patient survived for 10 dependent of perfusion, and respiration of lung parenchymal cells oc-
months although he spent most of his post-operative life in intensive curs through diffusion from air spaces [22,23]. Using a rat model of 
care unit [10]. In 1995, a group led by Robert Love also performed DCD, non-ventilated and nitrogen-ventilated lungs retrieved at 2, 4, 8 
another successful lung transplantation [11], and 6 years later, Steen and 12 hours after cardiac death showed progression of ischemic injury 
and his surgical team also performed another successful lung trans- and ultrastructural damage, characterized by nuclear chromatin 
plantation in a 54-year-old woman with chronic obstructive pulmonary clumping, mitochondrial degeneration, intracellular edema, and loss of 
disease [12]. In this remarkable lung transplantation, the transplanted cellular membrane integrity [24,25]. However, postmortem at 2, 4, 8 
lung exhibited excellent function only 5 min after reperfusion and and 12 hours after cardiac death showed preservation of lung ultra-
ventilation, and during the first 5 months of follow-up [12]. This success structural integrity and delay in cell death in oxygen-ventilated lungs 
continued in lung transplants performed in the 21st century, with after circulatory arrest [24,25], with maintenance in the levels of 
improved survival rate [156]. It is important to note that all the human adenosine triphosphate (ATP) and total adenine nucleotide (TAN) 
lung transplantations performed thus far were single-lung transplants compared to heart and liver, which exhibited progressively reduced ATP 
including the first successful heart-lung transplantation performed by and TAN levels after cardiac death [26–29]. A similar observation was 
Reitz and his team in 1981 in a 45-year-old woman with primary pul- made in a canine model of DCD in which dogs were mechanically 
monary hypertension [13]. Patterson and Cooper were the first to ventilated with room air for various periods after cardiac death until 6 
perform a successful double-lung transplantation in 1986 in a 42-year-- hours before retrieval of the lungs [30]. Thus, compared to other solid 
old woman, who was diagnosed with emphysema secondary to alpha-1 organs, these experimental findings suggest a unique nature of the lung, 
antitrypsin deficiency-associated lung disease. In this patient, the lungs which allows it to be ideally suitable for procurement at substantial time 
were transplanted en bloc [14]. Although the surgeons observed tracheal intervals following cardiac death, and therefore, support the application 
dehiscence as one of the post-transplant complications in their trans- of postmortem mechanical ventilation with oxygen or room air for DCD 
plant patient, their surgical technique laid the foundation for the lung transplantation. The findings do not only suggest a prospect in 
development of the modern technique of sequential double-lung trans- controlled DCD (i.e. donors whose deaths are expected in a hospital) but 
plantation and anastomosis performed at the level of the mainstem also in several scenarios of uncontrolled DCD (i.e. donors that experi-
bronchus. In summary, lung transplantation has evolved into the ence cardiac arrest unexpectedly and usually outside a hospital). 
mainstay of therapy, with short- and long-term outcomes for many pa-
tients with different types of end-stage pulmonary disease. 3. Ischemia-reperfusion injury in lung transplantation 
2. The lung as an ideal organ for donation-after-cardiac-death In lung transplantation, ischemia (temporary cessation of blood 
flow) begins during the surgical procedure of lung procurement from the 
Despite the success and continuous refinement in lung trans- donor. This causes an imbalance between metabolic supply and demand, 
plantation techniques, the widespread application of this life-saving leading to cell death and tissue injury. This type of ischemia, referred to 
intervention is severely hampered by poor allograft quality offered as warm ischemia, is followed by cold ischemia when the lung allograft 
from donors-after-brain-death (DBD; heart-beating neurologically is intravascularly flushed with and stored in cold preservation solution 
deceased donors). Although DBD was introduced and accepted since at 4 ◦C to decrease its metabolic activity and energy requirement prior to 
1968 and has provided most of the organs used in transplantation today, transplantation. While hypothermic preservation is beneficial, it induces 
it negatively impacts donor lung quality, as it leads to hemodynamic, a series of pathological events in the donor lung, such as ischemia- 
metabolic, and neuroendocrine abnormalities, and ruptured alveolar induced oxidative stress from over-production of reactive oxygen spe-
capillary endothelium, culminating in neurogenic pulmonary edema, cies (ROS; a destructive mediator of cell and tissue injury) [31], 
along with acute inflammatory lung injury and acute respiratory distress Na+/K+-ATPase pump inhibition [32], Na+ and Cl- influx, along with 
syndrome [15,16]. Also, the lungs in DBD may be subjected to airway flow of water into intracellular space, endothelial cell membrane de-
aspiration, respiratory tract infection, atelectasis, and pulmonary polarization (due to absence of mechanotransduction from lack of blood 
contusion, which may contribute to graft injury prior to procurement flow) [33,34], intracellular calcium overload [31,35,36], release of 
[15,16], and may further be amplified by ischemia and reperfusion pro-inflammatory and pro-apoptotic factors [37,38], all of which 
during graft procurement, preservation and implantation. This has mediate cell death and tissue injury (Fig. 1). Prolonged ischemia results 
increased the scarcity of suitable lung donors, which has resulted in an in “no-reflow phenomenon”, which is characterized by continuous 
annual increase in the number of patients on the transplant waiting list, obstruction to blood flow (due to significant microvascular damages) 
with longer waiting times and an annual increase in morbidity and and subsequent ischemia despite reperfusion [39]. 
mortality while on the waiting list. It has also resulted in strict selection Following the ischemic phase is reperfusion phase during which 
criteria for lung transplant recipients [17–19]. This problem suggests warm oxygenated blood is restored to the ischemic lung allograft after 
that many more lives could be extended or improved with sufficient implantation. Interestingly, reperfusion is associated with more cell 
supply of donor lungs. In addition to the donor lung shortage crisis, early death, as it exacerbates ischemic-related responses. During reperfusion, 
graft dysfunction (ischemia-reperfusion injury) and late graft dysfunc- there is further production of ROS, extravasation of leukocytes such as 
tion (bronchiolitis obliterans syndrome) also complicate the long-term macrophages and neutrophils (forming neutrophil extracellular traps) 
success of DBD lung transplantation [20,21]. [40,41], activation of the complement system, upregulated expression 
Avoiding the stresses of DBD and resistance to ischemia suggests of adhesion molecules (e.g. selectins and integrins), increased release of 
donors-after-cardiac-death (DCD; previously known as non-heart- pro-inflammatory mediators (e.g. cytokines and damage-associated 
beating donors) as an additional source to expand the pool of donor molecular patterns) and pro-apoptotic factors [41–43], as well as acti-
lungs, and could serve as a valuable option especially in countries such vation of toll-like receptors, and formation of endothelial gap from 
as Japan, where the concept of DBD is not widely accepted. Unlike other increased endothelial permeability [44]. All these pathological changes 
solid organs, the lung may be ideally suitable for DCD due to its toler- worsen lung tissue injury during and after reperfusion, and contribute to 
ance for warm ischemia at substantial time intervals because of its low pulmonary dysfunction after lung transplantation (Fig. 1). Collectively, 
metabolic requirement, in addition to being normally well-perfused and this inevitable paradoxical phenomenon of blood cessation and resto-
its alveoli filled with oxygen. It has been demonstrated that lung ration is referred to as ischemia-reperfusion injury (IRI), and negatively 
epithelial cells can be cultured from specimens obtained several hours impacts lung allograft quality and increases post-transplant 
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G.J. Dugbartey                                                                                                                                                                                    B  i o  m   e d  i c i n  e   &   P  h  a  r m  a  c  o t h  e  r a  p y  167 (2023) 115549
Fig. 1. Schematic view of pulmonary ischemia-reperfusion injury. NF-кB; nuclear factor-kappaB, ROS; reactive oxygen species, ELAM-1; endothelial leucocyte 
adhesion molecule-1, ICAM-1; intercellular adhesion molecule-1, VCAM-1; vascular cell adhesion molecule-1, DAMPs; damage-associated molecular patterns, TLRs; 
toll-like receptors, i[Ca2+]; intracellular calcium ion concentration, Na+/K+-ATPase; sodium/potassium-adenosine triphosphatase, BID; BH3 interacting domain 
death agonist; BAD; Bcl-2-associated death promoter; BAX; Bcl-2-associated X protein. 
complications. transplant recipients. Compared to patients without PGD, the mortality 
rate of lung transplant recipients with PGD is 7-folds higher, which 
3.1. Clinical effect of ischemia-reperfusion injury in lung transplantation represents 42% of mortality in the month after transplantation [49]. It is 
worth mentioning that IRI in clinical lung transplantation causes robust 
According to available data at the registry of the International So- inflammation, alveolar damage (decreased alveolar compliance), pul-
ciety of Heart and Lung Transplantation, the current 5-year survival rate monary edema (due to increased endothelial permeability) and 
of lung transplant recipients is approximately 55%, which suggests that increased pulmonary vascular resistance within 72 hours after trans-
lung transplantation is a satisfactory and viable treatment for end-stage plantation, which culminate in PGD [49–53]. PGD represents a major 
respiratory diseases [45]. However, despite the incremental advances in risk factor for the development of chronic lung dysfunction such as 
lung transplantation, including refinement of surgical techniques and bronchiolitis obliterans, which has been identified as the major cause of 
improvement in peri-operative care, lung transplantation is still asso- mortality among transplant recipients after 1 year of lung trans-
ciated with various problems such as primary graft dysfunction (PGD), plantation [54,55]. In addition, IRI also contributes to acute lung allo-
allograft rejection, infection, surgical complications, malignancy and graft rejection, leading to long-term graft dysfunction [54,56,57]. 
chronic lung allograft dysfunction [46]. Among these problems, PGD is Considering this major clinical consequence of IRI in lung trans-
the most significant cause of short- and long-term morbidity and mor- plantation, there is the need for lung transplant surgeons and their staff 
tality in lung transplant recipients. It is also the cause of prolonged to redefine the selection criteria for assessment of donor lungs (i.e. focus 
mechanical ventilation and longer hospital stays beyond 72 hours in the on donor lungs that can tolerate several hours of ischemia without losing 
post-transplant period [47,48]. PGD occurs in about 10% of lung their function after reperfusion). In addition, assessment of effective 
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G.J. Dugbartey                                                                                                                                                                                    B  i o  m   e d  i c i n  e   &   P  h  a  r m  a  c  o t h  e  r a  p y  167 (2023) 115549
lung preservation technique and improved management of transplanted reperfusion, mitigates IRI, and reduces post-transplant complications. 
lungs after reperfusion will help reduce the severity of lung IRI, prevent Modified extracellular-type preservation solution has been proven to 
PGD and improve both short- and long-term outcomes following be better than intracellular-type in maintaining lung allograft function 
transplantation. [71,72]. This conclusion became increasingly evident after results from 
several experimental studies in canine, porcine and primate models of 
4. Preservation solution in lung transplantation single- and double-lung transplantation demonstrated that addition of 
1% glucose to LPD solution provided a suitable substrate for aerobic 
As PGD due to IRI remains a major cause of morbidity and mortality metabolism in inflated lungs and maintained ATP and phosphocreatine 
after lung transplantation, one of the practical approaches to mitigate levels, and thereby allowing a prolonged cold ischemic (preservation) 
IRI, improve pulmonary graft performance and reduce the increasing time for 12–24 h, with preserved lung allograft integrity [73–77]. In 
incidence of PGD is to optimize existing lung preservation techniques or addition to serving as an energy source during cold ischemia, saccha-
develop highly effective and reliable alternative lung preservation so- rides such as glucose (monosaccharide), trehalose (disaccharide) and 
lutions to minimize lung injury during the period of ischemia. Due to its raffinose (trisaccharide) in preservation solutions also act as an imper-
technical simplicity, most transplant centers have adopted cold single meant to prevent cellular edema. In a rat model of left-lung trans-
pulmonary artery flush with modified Euro-Collins solution as the plantation, for example, supplementation of LPD preservation solution 
standard preservation technique in lung transplantation, with a varying with 30 mmol/L raffinose (superior to other saccharides for this pur-
cold ischemic (preservation) time from 4 to 12 hours [58]. Euro-Collins pose) during 24 hours of lung allograft preservation at 4ºC resulted in 
solution, which was originally developed for renal graft preservation in significantly higher oxygenation, lower peak airway pressures at 2 hours 
the 1960s and later introduced 3 decades ago for lung allograft preser- after allograft reperfusion and a lower wet-to-dry weight ratio (an in-
vation, is in the same category of intracellular-type preservation solution dicator of pulmonary edema), culminating in marked improvement in 
(containing high K+ and low Na+) with University of Wisconsin solution lung allograft function compared to control allografts without raffinose 
(UW; historically for liver graft preservation), whose high potassium supplementation [78]. Using the same model, the same group of re-
levels produce pulmonary vasoconstriction [59]. In the quest for a more searchers conducted a follow-up study a year later in which they 
reliable preservation solution for lung allografts, extracellular-type observed minimal interstitial edematous expansion, fewer damaged 
preservation solution (containing low K+ and high Na+) was devel- type II pneumocytes, and minimal capillary injury in raffinose-LPD lungs 
oped. This includes low-potassium dextran (LPD) and Celsior solution compared to control lung allografts that exhibited significant weight 
(for cardiac graft preservation) [60]. Unlike intracellular-type preser- gain, more dead cells, more damaged type II pneumocytes, cellular ne-
vation solution, the low potassium content in extracellular-type pres- crosis, collapsed alveolar capillaries, and interstitial and alveolar edema, 
ervation solution supports the integrity of endothelial cells and reduces with influx of interstitial macrophages [79]. These laboratory results 
oxidative stress and pulmonary vasoconstriction [61–63]. show that modification of LPD solution with saccharides during hypo-
Although both intracellular- and extracellular-type solutions are thermic lung allograft preservation exhibits a stronger cytoprotective 
used to preserve lung allografts, LPD is the only one specifically devel- effect, and could be applied clinically in extending lung allograft pres-
oped for lung allograft preservation, and its modification with glucose ervation. Nonetheless, continuous refinement is still needed with other 
(LPD-glucose; also known as Perfadex) has been widely used at many components such as antioxidants to further improve allograft quality 
transplant centers due to its superiority over the other solutions for after preservation and limit IRI. 
prolonged lung allograft preservation [64]. In a canine model of 
single-lung transplantation to determine the individual contributions of 5. Nitric oxide in lung transplantation 
dextran and low potassium concentration during prolonged (12-hour) 
preservation of lung allografts, Keshavjee et al. [65] observed that Despite the significant strides that have been made within the realm 
addition of dextran 40 produced excellent immediate pulmonary func- of lung transplantation including optimal preservation of lung allografts, 
tion (gas exchange, pulmonary hemodynamics and mechanics), which IRI is still a major problem in lung transplantation. This has necessitated 
continued on post-transplant day 3 upon follow-up, while its absence in continuous search for further improvement of the transplantation pro-
the low-potassium preservation solution resulted in marked deteriora- tocol. In the light of this, there are studies investigating the effect of 
tion in pulmonary function. In the same study, high-potassium dextran selective pulmonary vasodilators such as nitric oxide (NO), administered 
solution produced very poor pulmonary function, characterized by directly in its gaseous form to lung donors prior to donor lung pro-
rupture of alveolar septa and severe alveolar edema and hemorrhage, curement, or to recipients, or administered indirectly by supplementing 
which resulted in death of some of the animals in this group at 6 hours lung preservation solutions with NO donor compounds. This approach 
after transplantation [65]. These observations indicate that both dextran may provide significantly superior graft protection, considering that 
40 and low potassium concentration contribute significantly to preser- endogenous pulmonary NO production is decreased during ischemia and 
ving lung allograft viability and function after prolonged preservation. reperfusion. Thus, NO pathway might be a therapeutic target, whose 
This report was later confirmed in a swine model of lung transplantation activation might be beneficial in attenuating IRI after lung trans-
in which lung allografts were preserved for 8 hours. In addition to plantation. It is important to note that NO is a member of a family of 
preserving pulmonary microcirculation, LPD solution also prevented small endogenously produced gaseous signaling molecules that include 
no-reflow phenomenon and pulmonary edema during reperfusion [66]. carbon monoxide and hydrogen sulfide, which are also showing promise 
At the cellular level, LPD solution significantly suppressed human in experimental models of organ transplantation [157-160]. NO is pro-
neutrophil chemotaxis [67], exhibited less cytotoxic effect on type II duced in the endothelium of blood vessels by a family of enzymes called 
pneumocytes of rats and humans [68,69], preserved the activity of nitric oxide synthase (NOS), in a reaction in which L-arginine is used as a 
Na+/K+-ATPase in type II pneumocytes of rats [70], and maintained substrate to produce L-citrulline [80]. The NO produced by NOS, acts on 
intact endothelial-epithelial barrier during prolonged hypothermic several target proteins and enzymes to exert its physiological function 
preservation compared to intracellular-type preservation solutions [66]. such as vasodilation, through activation of soluble guanylate cyclase, an 
As type II pneumocytes are synthesizing cells of alveolar surfactant, enzyme that converts guanosine triphosphate (GTP) to cyclic guanosine 
which lowers surface tension at the air-water interface in the alveoli, monophosphate (cGMP) to mediate many of the biological effects of NO 
thereby preventing alveolar collapse after exhalation, these in vitro [81]. NOS exists in three isoenzymes, namely neuronal NOS (nNOS or 
findings suggest that lung allograft preservation in LPD solution pro- NOSI), inducible NOS (iNOS or NOSII) and endothelial NOS (eNOS or 
duces higher levels of metabolic activity in recovering epithelial cells, NOSIII), all of which are expressed in various cell types despite their 
better surfactant function at the end of cold ischemia and after names. eNOS and nNOS are constitutively expressed and mediate many 
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G.J. Dugbartey                                                                                                                                                                                    B  i o  m   e d  i c i n  e   &   P  h  a  r m  a  c  o t h  e  r a  p y  167 (2023) 115549
of the beneficial actions of NO while iNOS is a pathological isoform that after donor lung procurement protects against lung IRI and improves 
contributes to pathophysiology of inflammatory conditions [80,81]. lung allograft function after transplantation partly by activating 
Interestingly, results from preclinical and clinical studies have shown NO/cGMP signaling pathway. Interestingly, while NO administration 
marked reduction in endogenous NO level following ischemia and had no effect on inflammation-associated transcription factor nuclear 
reperfusion of lung grafts, which contributed to lung IRI after trans- factor-kappaB (NF-κB) and mitogen-activated protein kinase (MAPK) in 
plantation [82–85]. This finding suggests that augmenting endogenous the lung allografts [86], other studies reported that NO inhibited the 
NO level by increasing the activity of NOS in the lung graft could be a activation of these two principal mediators of inflammation in rat 
novel approach to minimizing lung IRI and preventing its complications vascular smooth muscle cells and mesangial cells via activation of the 
after lung transplantation. To this end, exogenous NO gas has been NO/cGMP signaling pathway [87,88]. This may suggest that the effect of 
directly administered by inhalation or indirectly via NO donor com- NO administration on NF-κB and MAPK is dependent on the cell type. 
pounds such as nitroglycerine, nitroprusside, nicorandil, FK409, SIN-1, This promising experimental finding from the rat model of DCD 
isosorbide mononitrate and isoamyl nitrate [161]. In addition, the single-lung allotransplantation supports results from previous studies of 
lung donor could also be transfected with an adenovirus containing a similar rat model in which donor lungs were retrieved immediately 
eNOS before lung graft procurement. after cardiac arrest or 2 or 3 hours postmortem [89,90]. In these studies, 
the lung allografts were flushed with cold Celsior solution and stored in 
the same solution in an inflated state for 2 hours at 4 ◦C after ventilated 
5.1. Nitric oxide gas in lung transplantation with 100% oxygen supplemented with 30 ppm and 40 ppm of gaseous 
NO either during the period of warm ischemia, during reperfusion or 
Mounting experimental and clinical evidence shows that adminis- both. While control lung allografts without NO supplementation suf-
tration of gaseous nitric oxide (by inhalation) to lung donors prior to fered severe IRI, which was characterized by high wet-to-dry weight 
donor lung procurement and/or to recipients during and after reperfu- ratio, pulmonary vascular resistance and filtration coefficient, signifi-
sion reduces IRI and improves lung graft function after transplantation. cantly lower values of these measurable variables, with improved lung 
function and reduced neutrophil sequestration as well as increased lung 
5.1.1. Nitric oxide gas in rat models of lung transplantation cGMP were observed in NO-treated allografts [89,90]. These observa-
In a rat model of DCD single-lung allotransplantation, at 1 h after tions imply that in addition to activating the NO/cGMP signaling 
cardiac arrest, ventilation of lung allografts with 40 ppm of NO gas in pathway, administration of NO either during warm ischemia, reperfu-
60% oxygen for another 1 h followed by a 1-hour storage in an inflated sion or during both periods also suppresses inflammation and oxidative 
state in Perfadex (LPD-glucose) solution at 4 ◦C and ex vivo perfusion at stress-mediated damage in lung allografts as seen by decreased levels of 
37 ◦C with alveolar gas (5% CO2, 20% O2, 75% N2) supplemented with iNOS and TNF-α and reduced activity of myeloperoxidase (MPO; an 
40 ppm of gaseous NO. This resulted in marked reduction in wet-to-dry important inflammatory enzyme that triggers inflammation and oxida-
weight ratio, pulmonary vascular resistance, improved oxygenation and tive stress) [90] (Fig. 2). Contrary to the positive results from all these 
significantly increased cGMP level in the lung allograft, with improved studies, one study found that 20 ppm of inhaled gaseous NO together 
eNOS level and reduced levels of iNOS and TNF-α compared to control with room air in a closed chamber immediately after lung trans-
allografts without NO administration [86] (Fig. 2). This finding suggests plantation in rats, had no positive impact on IRI and lung allograft 
that administration of gaseous NO to donors before cardiac arrest and rejection [91]. This isolated contradictory observation might be due to 
technical differences, as other researchers used the same concentration 
of gaseous NO and reported beneficial effects. 
5.1.2. Nitric oxide gas in porcine models of lung transplantation 
Using a porcine model of DCD single-lung allotransplantation as a 
large animal model that is directly transferrable to clinical setting, 
20 ppm of gaseous NO was added at 2 h after hypoxic cardiac arrest and 
immediately before perfusion assessment followed by storage in Perfa-
dex solution at 4 ◦C in an inflated state prior to transplantation. The 
authors observed significant improvement in pulmonary venous 
oxygenation, airway pressure and pulmonary vascular resistance along 
with a marked reduction in neutrophil sequestration in NO-treated lung 
allografts after transplantation relative to control allografts without NO 
treatment [92]. Other studies involving DCD single-lung transplantation 
in minipigs also showed that inhalation of gaseous NO at 20 ppm before 
and after 2 h of in situ warm ischemia followed by a 2-hour preservation 
period in modified Euro-Collins solution at 4 ◦C, and inhalation by re-
cipients after allograft reperfusion for 2 h strongly reduced mean pul-
monary artery pressure, vascular resistance, lung tissue MPO activity, 
bronchoalveolar lavage fluid protein content and neutrophils, and 
increased arterial oxygen tension and pulmonary dynamic compliance, 
while preserving lung architecture [93,94]. In a porcine model of DCD 
Fig. 2. Mechanism of lung allograft protection by NO. Endogenous NO pro- single-lung autotransplantation in which left lung grafts were preserved 
duction or exogenous administration of NO donors mediates lung allograft in LPD solution in an inflated state for 24 h at 6–8 ◦C and transplanted 
protection by activating antioxidant and vasodilatory pathways while inhibit- into the same donors followed by right pneumonectomy, a 24-hour 
ing vasoconstrictive and pro-inflammatory pathways. cGMP; cyclic guanosine observation period following transplantation showed that inhalation 
monophosphate, GTP; guanosine triphosphate, GC; guanylate cyclase, KATP; 
potassium-sensitive adenosine triphosphate channel, ET-1; endothelin-1, of gaseous NO in sequential concentration of 5 ppm, 20 ppm and 
MAPK; mitogen-activated protein kinase, NF-кB; nuclear factor-kappaB, NO; 80 ppm after autotransplantation attenuated endothelial dysfunction by 
nitric oxide, sGC; ROS; reactive oxygen species, MPO; myeloperoxidase, eNOS; decreasing pulmonary vascular resistance and producing pulmonary 
endothelium nitric oxide synthase, nNOS; neuronal nitric oxide synthase, iNOS; vasodilation in proportion to the endothelial dysfunction [95]. To 
inducible nitric oxide synthase, and TNF-α; tumor necrosis factor-alpha. highlight the involvement of neutrophils in lung IRI after lung 
5
G.J. Dugbartey                                                                                                                                                                                    B  i o  m   e d  i c i n  e   &   P  h  a  r m  a  c  o t h  e  r a  p y  167 (2023) 115549
transplantation, Gómez and colleagues observed in another porcine gaseous NO on early allograft function, continuous inhalation of 40 ppm 
model of DCD single-lung allotransplantation that administration of of NO gas throughout 6 hours of reperfusion resulted in significantly 
20 ppm of gaseous NO after cardiac arrest and 30 min before donor lung higher oxygen tension, lower pulmonary artery pressure and pulmonary 
retrieval followed by reperfusion, resulted in significant improvement in vascular resistance along with reduced wet-to-dry ratio and MPO ac-
allograft function, which was evidenced by higher dynamic and static tivity, and thus culminating in increased recipient survival rate 
compliance and gas exchange, with markedly reduced production of compared to control group that received nitrogen gas in the same 
interleukin 8 (a potent neutrophil-specific chemotactic manner as NO gas [101]. This report further attests the vasodilatory 
pro-inflammatory cytokine) when compared to control group without effect of gaseous NO in the pulmonary vasculature as well as its inhib-
NO administration [96]. This result emphasizes the role of neutrophils in itory effect on neutrophil activation and thereby attenuating IRI after 
allograft IRI after lung transplantation, and also draws attention to the lung transplantation. 
anti-inflammatory property of NO within the pulmonary vessel wall, 
which contributes to its therapeutic benefit by inhibiting neutrophil 5.1.4. Nitric oxide gas in human lung transplantation 
activation, aggregation and migration [97] (Fig. 2). This was further Following the promising results from the preclinical studies dis-
demonstrated by Bacha et al. [98] in a porcine model of DCD single-lung cussed above, several human clinical trials were conducted in which 
allotransplantation in which 30 ppm of gaseous NO was administered to inhalation of gaseous NO was shown to be indeed safe and beneficial in 
deceased donor and recipient pigs after ventilation with oxygen. In this the treatment of complications arising from lung transplantation. For 
investigation, lung allografts were flushed with cold Wallworks solution example, in a prospective non-randomized clinical trial involving 14 
(a type of LPD solution) and the inflated allografts were preserved in the patients undergoing lung transplantation due to end-stage lung disease 
same solution at 4 ◦C for 2 h after 3 h of postmortem in situ warm and pulmonary hypertension (indicator of PGD) with mean pulmonary 
ischemia, followed by a 9-hour observation period during reperfusion. artery pressure higher than 30 mmHg, inhalation of 20 ppm of gaseous 
The authors observed marked inhibition of neutrophil adhesion to pul- NO resulted in significantly less incidence of acute allograft rejection in 
monary artery endothelial cells in NO-treated group before and after the first month after transplantation, with shorter hospital stay 
reperfusion, which positively correlated with reduced neutrophil compared to 22 historical control subjects with matching age, diagnosis 
sequestration in lung allografts, along with improved preservation of and disease severity, who underwent lung transplantation 2 years before 
lung architecture relative to control group which did not receive NO this study [102]. This impressive clinical outcome corroborates the 
treatment [98]. As reported by other groups, significantly reduced result of a previous prospective clinical dose-response study to assess the 
pulmonary vascular resistance as well as improved oxygenation and impact of low-dose NO gas on cardiopulmonary parameters in early lung 
prolonged survival were also observed in NO-treated group compared to allograft dysfunction after transplantation. In this clinical trial, inhala-
control group [98], which indeed underscores the important detrimental tion of gaseous NO in sequential concentrations of 1 ppm, 4 ppm and 
contribution of neutrophils in the incidence of lung IRI after lung 8 ppm by 8 patients, who had undergone single- or double-lung trans-
transplantation. plantation, markedly lowered their elevated mean pulmonary artery 
pressure and improved their arterial oxygen tension/fractional inspired 
5.1.3. Nitric oxide gas in canine models of lung transplantation oxygen ratio (PaO2/FiO2; indicator of PGD) in a dose-dependent manner 
The beneficial effect of gaseous NO in experimental lung trans- [103]. This suggests that low-dose gaseous NO could provide thera-
plantation has also been studied in dogs. In a canine model of single-lung peutic benefit in complications such as impaired gas exchange and 
DCD allotransplantation to investigate the impact of inhaled NO gas at pulmonary hypertension resulting from PGD after lung transplantation. 
the time of donor lung retrieval on graft function, lung allografts were In another clinical study, prolonged treatment of lung transplant re-
flushed with modified Euro-Collins solution and preserved in the same cipients (who developed pulmonary hypertension) with low-dose 
solution at 1 ◦C for 21 hours after gaseous NO was administered in gaseous NO (10 ppm) over 40–69 hours decreased pulmonary artery 
sequential concentration of 20 ppm, 40 ppm, 60 ppm and 80 ppm for pressure, pulmonary vascular resistance and intrapulmonary shunt 
10 minutes per sequence prior to cardiac arrest [99]. Hemodynamics fraction (the main cause of hypoxemia) as well as mean arterial pressure 
and arterial blood gas assessment during 6 hours of reperfusion at 37 ◦C and systemic vascular resistance without any side effects [104]. In 
showed significant decrease in pulmonary vascular resistance and addition, low-dose gaseous NO produced a stained improvement in 
wet-to-dry weight ratio, and marked increase in mean arterial oxygen oxygenation. Interestingly, high dose (80 ppm) produced the same 
tension in dogs that received NO-treated lung allografts (regardless of salutary effect without affecting systemic hemodynamics [104,105]. 
NO concentration) in comparison with control group without NO These observations indicate that NO therapy could prevent PGD 
administration. Also, ROS production and MPO activity were signifi- following lung transplantation. 
cantly reduced in NO-treated allografts compared to control lungs [99], In a retrospective clinical study in which 15 lung transplant re-
indicating that administration of gaseous NO at the time of donor lung cipients inhaled 20–60 ppm of gaseous NO for 15–217 hours, the au-
retrieval improves allograft function, at least in part, by suppressing free thors reported that NO therapy attenuated PGD by improving PaO2/FiO2 
radical production and neutrophil infiltration (Fig. 2). Following this ratio within 1 hour of therapy, and reducing pulmonary artery pressure, 
empirical finding, another group also confirmed the salutary effect of with sustained improvement during prolonged treatment compared to 
NO inhalation in a similar canine model of single-lung DCD allo- 17 lung transplant recipients without NO therapy [106]. Remarkably, no 
transplantation. In their study, Takashima et al. [99] reported that systemic circulatory effects were recorded in NO-treated patients. 
administration of 40 ppm of gaseous NO for the initial 1 hour during Neither were any complications associated with NO therapy. Also, 
reperfusion after 3 hours of warm ischemia, and continuous NO NO-treated patients had a significantly shorter post-operative mechan-
administration during 6 hours of reperfusion greatly reduced pulmonary ical ventilation time, with reduced mortality in comparison with their 
vascular resistance and MPO activity, improved oxygenation and pro- counterparts without NO administration [106]. In another clinical 
longed recipient survival compared to control group without NO investigation to evaluate retrospectively the protective effect of coad-
administration. Interestingly, no significant difference in these ministration of gaseous NO (10 ppm) and 400 mg pentoxifylline (an 
measurable parameters were observed between the groups that received activator of endogenous NO production) after lung transplantation, 
1 hour and 6 hours of NO during reperfusion [99,100]. This observation coadministration of these two agents to 23 lung transplant recipients 
shows that not only does NO inhalation reduce lung IRI and prolong lung prophylactically during reperfusion resulted in a marked decrease in the 
allograft function and recipient survival, but also points out that this incidence of PGD by reducing reimplantation edema, improving 
benefit occurs during the early hours of reperfusion. In a canine model of PaO2/FiO2 ratio, shortening post-operative mechanical ventilation time, 
living-donor double-lung allotransplantation to assess the effect of and reducing 2-month mortality rate when compared to two historical 
6
G.J. Dugbartey                                                                                                                                                                                    B  i o  m   e d  i c i n  e   &   P  h  a  r m  a  c  o t h  e  r a  p y  167 (2023) 115549
control groups (23 and 95 patients) [107]. This clinical report shows pharmacologically active compounds spontaneously release NO or are 
that prophylactic treatment with gaseous NO (and NO donor) is bene- metabolized to NO independent of its endogenous sources. Thus, they 
ficial in the early post-operative course in lung transplant recipients. are exogenous sources of NO. Interestingly, NO donors exhibit different 
Likewise, inhalation of 40 ppm of NO gas followed by a gradual dose pharmacological properties that determine the type and degree of their 
reduction before and after single- and double-lung transplantation effects on biological systems [113]. In addition, their experimental 
resulted in markedly reduced mean pulmonary artery pressure, with utilization has shed more light on the protective molecular mechanisms 
improvement in arterial oxygenation and overall respiratory function, of NO in lung transplantation. 
and no significant effect on systemic circulation. Also, duration of me-
chanical ventilation and mortality rate were significantly reduced [108]. 5.2.1. Nitric oxide donors in rat models of lung transplantation 
To assess the effect NO inhalation on the endogenous NOS system, Using an in situ isolated perfused rat model to investigate the effect of 
Cardella and associates [109] performed lung biopsy on lung transplant nitroglycerin on IRI, rat lungs were procured at varying intervals 
recipients who inhaled 20 ppm of gaseous NO or placebo (10 minutes following cardiac death and reperfused at intervals with Earle’s 
after start of reperfusion) in a randomized phase II clinical trial. In this balanced salt solution (a physiological solution). In this study, the au-
study, the authors collected lung tissues after warm and cold ischemia, thors reported an increase in capillary filtration coefficient (Kfc) at all 
1 hour and 2 hours after the start of reperfusion, and reported that post-mortem ischemic times and decreases in the levels of cGMP and 
protein expression of constitutive NOS (eNOS and nNOS) increased adenine nucleotides. However, addition of 0.1 mg/mL of nitroglycerin 
significantly after 2 hour of reperfusion in NO-treated lung allografts but to the solution caused a substantial decrease in Kfc and increases in 
decreased in placebo group, while iNOS protein expression did not cGMP and adenine nucleotide levels compared to reperfused lungs 
change significantly in both groups [109] (Fig. 2). Although the ran- without nitroglycerin supplementation [114]. This suggests that addi-
domized phase II clinical trial did not report on the effect of NO inha- tion of nitroglycerin to preservation solutions may prevent capillary leak 
lation in the lung transplant recipients, the upregulated expression of after reperfusion and thus, may improve DCD lung transplantation 
constitutive NOS proteins in the NO-treated lung allografts points to a outcomes. The observed increase in cGMP level following nitroglycerin 
possible interaction between gaseous NO molecules and the lung tissues. supplementation supports the established evidence that nitroglycerin is 
Despite these exciting clinical outcomes with gaseous NO, there are a an organic nitrate that releases NO and intermediates intracellularly to 
few conflicting reports. In a prospective clinical trial to investigate the directly stimulate cGMP production from GTP [81,115] (Fig. 2). In 
role of gaseous NO in preventing IRI in lung transplant recipients, pro- another rat model to specifically assess the effect of nitroglycerin during 
phylactic administration of gaseous 20 ppm of NO to 28 lung transplant early post-ischemic period, donor lungs inflated with room air, were 
recipients during reperfusion followed by withdrawal for 15 minutes at preserved in Perfadex solution at 10 ºC and supplemented with 
6 and 12 hours after reperfusion caused marked increase in pulmonary 0.1 mg/mL of nitroglycerin followed by reventilation and reperfusion 
artery pressure and decrease in oxygenation index in 5 out of the 28 for 50 minutes. This resulted in significantly reduced flush-perfusion 
recipients who developed IRI, while the remaining 23 recipients had no time, higher oxygenation capacity and reduced intrapulmonary 
IRI and no adverse events in the early post-operative course [110]. edema, with markedly improved pulmonary vascular resistance and 
Meade et al. [110] also reported that there was no effect of gaseous NO peak inspiratory pressures relative to control lungs without nitroglyc-
on the outcome after lung transplantation. In their randomized, erin treatment [116]. The result indicates that stimulation of the NO 
double-blinded, placebo-controlled clinical trial involving 84 lung pathway by NO donors may enhance early functional outcome of lung 
transplant recipients, inhalation of 20 ppm of gaseous NO by 42 lung allografts in clinical lung transplantation. Similar improvement in pul-
transplant recipients at 10 minutes after reperfusion produced no sig- monary function was obtained in an ex vivo rat lung perfusion model in 
nificant difference in hemodynamics, severity of IRI, immediate which the donor lungs were flushed with and stored in 
oxygenation, duration of mechanical ventilation and hospital stay, and nitroglycerin-supplemented extracellular-type Kyoto solution at 4 ºC for 
30-day mortality compared to 42 placebo-treated group [111]. 15 hours and then reperfused for 60 minutes [117]. Compared to con-
Following these findings, Botha and colleagues [112] also observed a trol lungs, addition of nitroglycerin (0.44 mM) to the preservation so-
similar result in which 20 bilateral sequential lung transplant recipients lution also lowered shunt fractions substantially throughout 
were administered either 20 ppm gaseous NO or a standard anesthetic reperfusion, reduced lung wet-to-dry weight ratio, maintained cGMP 
gas mixture (control group) during the first 30 minutes of reperfusion. level in lung tissue, which decreased during preservation and reperfu-
Between both groups, there was no statistically significant difference in sion, and attenuated oxidative stressed-induced DNA damage evidenced 
the effect of gaseous NO and the standard anesthetic gas mixture on the by decreased expression of 8-hydroxy-2’-deoxyguanosine (8-OHdG) in 
development of Grade II to III PGD, PaO2/FiO2 ratio, pulmonary alveolar epithelium, pulmonary endothelium and bronchial epithelium 
neutrophil sequestration and the concentrations and levels of to levels comparable to fresh lungs, and thus improved post-reperfusion 
interleukin-8, nitrotyrosine (an oxidative stress marker) and MPO ac- pulmonary function [117]. The antioxidant property of nitroglycerin has 
tivity in epithelial lining fluid and bronchoalveolar lavage fluid during been reported in in vivo and in vitro models as well as in clinical studies of 
transplantation [112]. In the face of these refuting clinical findings, the myocardial ischemia and reperfusion in which it scavenged ROS and 
therapeutic benefits of inhaled NO after clinical lung transplantation decreased toxic metabolites [118–120], and therefore may have partly 
cannot be entirely ruled out, as NO is superior to other vasodilators, and contributed to the attenuation of IRI in the ex vivo rat lung perfusion 
its selectivity nature makes it suitable to target the pulmonary vascu- model. The study also confirmed previous rat models of lung trans-
lature without significant adverse effects on systemic circulation. plantation, showing that addition of nitroglycerin to different preser-
However, additional prospective randomized studies would be neces- vation solutions attenuates lung IRI, improves pulmonary function and 
sary to further demonstrate and standardize gaseous NO therapy against prolongs recipient survival after transplantation [121–124]. 
IRI and its associated complications after clinical lung transplantation. Minanoto and colleagues [125] also noted in a rat model of isogeneic 
single-lung transplantation that early administration of 0.1 mg/mL of 
5.2. Nitric oxide donors in lung transplantation nitroglycerin during flushing and 6 hours of preservation in modified 
Euro-Collins solution at 4 ºC followed by ex vivo flushing with normal 
In addition to gaseous NO, several NO donor compounds such as saline, caused a decrease in vascular tone as well as pulmonary artery 
nitroglycerine, nitroprusside, nicorandil, FK409, SIN-1, isosorbide pressure and pulmonary vascular resistance in lung isografts, and 
mononitrate and isoamyl nitrate, have also been investigated and found improved gas exchange and recipient survival after lung transplantation. 
to exhibit similar therapeutic benefits as gaseous NO in various animal Interestingly, administration of nitroglycerin to grafts immediately 
models of lung transplantation. Following their administration, these before transplantation, did not produce the post-transplantation benefits 
7
G.J. Dugbartey                                                                                                                                                                                    B  i o  m   e d  i c i n  e   &   P  h  a  r m  a  c  o t h  e  r a  p y  167 (2023) 115549
of early nitroglycerin treatment. Surprisingly, the result of late nitro- onset of ischemia or 10 min prior to reperfusion, reduced precapillary 
glycerin treatment was similar to control group, which did not receive elevation of vascular resistance, Kfc and preserved normal pulmonary 
nitroglycerin administration [125], suggesting that early treatments of hemodynamics and microvascular integrity. Interestingly, nitroprusside 
donor lungs during procurement and preservation creates sufficient administration at the onset of reperfusion was less effective [136]. Esme 
priming and protective internal environment against IRI after trans- et al. [137] also reported in a rabbit model of in situ normothermic 
plantation. Mechanistically, the vascular response of the isografts to ischemic lung that administration of nitroglycerin during flush perfusion 
early treatment with nitroglycerin was reported to be via downregulated and reperfusion markedly improved arterial oxygenation, decreased 
expression of endothelin-1 (ET-1; a potent vasoconstrictor) mRNA and neutrophil level in bronchoalveolar lavage fluid, with lower tissue his-
protein [125] (Fig. 2). This was confirmed by a later study of normo- topathological lesion scores and higher nitrate level when compared to 
thermic ex vivo perfusion of transplantation-declined human lungs ob- control lungs without nitroglycerin treatment. Interestingly, adminis-
tained from DCD. In this ex vivo study, the authors reported significantly tration of nitroglycerin during flush perfusion period alone did not 
higher levels of ET-1 and Big ET-1 (the precursor of ET-1) in the per- produce these beneficial effects [137], demonstrating that nitroprusside 
fusates at 1 and 4 hours of ex vivo perfusion in comparison with control administration during in situ flush perfusion and reperfusion is more 
lungs from bilateral transplantation with good early outcomes [126]. protective against lung IRI than other treatment modalities. L-arginine 
This result may imply that perfusate ET-1 and Big ET-1 could serve as (20 mg/kg; NO precursor) and pentoxifylline (50 mg; enhancer of 
biomarkers of lung allograft function during ex vivo lung perfusion and endogenous NO production [138] added to lactated Ringer solution just 
following clinical lung transplantation. Besides its role in promoting before reperfusion after 4–48 h preservation of lungs at 10 ºC, preserved 
vasoconstriction, it is worth noting that ET-1 also increases vascular endothelium functional integrity and reduced IRI in a rabbit model of 
permeability, stimulates neutrophil accumulation, and promotes coag- lung transplantation [139]. Taken together, administration of nitric 
ulation, which have been reported in animals and humans with pul- oxide donors protects against IRI and preserves allograft function after 
monary hypertension and also observed in lung allografts after lung transplantation. 
transplantation [127–133]. NO is known to prevent adherence of neu-
trophils and platelets to vascular endothelium, and thereby decreasing 5.2.3. Nitric oxide donors in canine models of lung transplantation 
leukocytes extravasation into lung parenchyma. This action inhibits In canine model of single-lung allotransplantation, addition of 
cytokine production and thereby prevents vascular permeability. 10 mg/L of nitroprusside during flush perfusion in one experimental 
Considering that both ET-1 and NO are in reciprocal balance to regulate group, followed by storage of the inflated lung allografts in modified 
vascular tone, including pulmonary vasomotor tone, the observation Euro-Collins solution at 1 ºC for 21 hours, and then bolus injection 
that nitroglycerine suppresses ET-1 mRNA and protein expression sug- (0.2 mg/kg) in recipient animals prior to reperfusion as well as contin-
gests that the vasodilatory effect of NO may be via inhibition of ET-1 uous infusion at a rate of 0.1 mg/kg/hr during a 6-hour reperfusion 
production (Fig. 2). On the whole, supplementation of preservation so- period in another experimental group showed amelioration of IRI and 
lutions attenuates lung IRI, improves lung allograft quality and function, preserved lung allograft function in both nitroprusside-treated groups 
reduces post-transplant complications, and prolongs recipient survival [140,141]. This was characterized by markedly improved respiratory 
after transplantation. gas exchange and pulmonary hemodynamics, lower wet-to-dry weight 
ratio and reduced neutrophil accumulation compared to control group 
5.2.2. Nitric oxide donors in rabbit models of lung transplantation without nitroprusside supplementation. Interestingly, no significant 
In an isolated, ventilated, whole-blood-perfusion rabbit lung model, difference was observed between both nitroprusside-treated groups 
lungs were retrieved en bloc, flushed and preserved in an inflated state in [140,141], indicating that both treatment modalities are effective in 
Euro-Collins solution for 18 hours at 4 ºC followed by reperfusion at a limiting IRI after lung allotransplantation. In another study by the same 
physiologic flow rate of 60 mL/min for 30 minutes with nitroprusside authors, replacing nitroprusside with pentoxifylline using the same 
infusion at 0.2, 1.0 and 5.0 µg/kg/min through the pulmonary artery experimental protocol produced the same salutary effects, with signifi-
during reperfusion. Compared to control lungs without nitroprusside cantly decreased neutrophil adhesion to endothelium as well as reduced 
supplementation, nitroprusside infusion markedly decreased pulmonary protein levels and neutrophil concentration in bronchoalveolar lavage 
artery pressure and pulmonary vascular resistance in a dose-dependent fluid [142]. Akin to this finding, Yamashita and associates [143] also 
manner, with additional substantial improvements in wet-to-dry lung reported in another canine model of single-lung allotransplantation that 
weight ratio, arteriovenous oxygen gradient and dynamic airway nicorandil enhances allograft preservation, improves gas exchange and 
compliance without significant systemic hypotension [134]. This shows prevents allograft dysfunction after transplantation. In their investiga-
that direct intravascular infusion with NO donors during reperfusion tion, they used the same protocol and treatment modalities in which 
ameliorates IRI immediately after lung transplantation. Although the supplementation of flush solution with 24 mg/L of nicorandil, and 
authors did not measure endogenous NO level in their model, nitro- intravenous administration (0.5 mg/kg) to recipient dogs at the onset of 
prusside is a short-acting, non-selective, and direct NO donor that re- reperfusion followed by continuous infusion (0.74 mg/kg/hr) during 
leases NO into the vascular smooth muscle cell without requiring 6 hours of reperfusion and assessment period. Considering that nicor-
enzymatic conversion, leading to dilation of all blood vessels. Thus, andil is an opener of adenosine triphosphate-sensitive potassium (KATP) 
nitroprusside increases endogenous NO level and thus activates channel in addition to being a generator of endogenous NO, intravenous 
NO/cGMP signaling pathway that leads to vasodilation (Fig. 2). In administration of glibenclamide (3.0 mg/kg; a specific KATP channel 
another rabbit model of IRI in buffer-perfused rabbit lungs in which blocker) 15 minutes prior to donor lung flush and before nicorandil 
warm ischemic time lasted for 150 min and anoxic ventilation and a administration, as well as to recipients (1.0 mg/kg) 15 minutes before 
positive intravascular pressure were maintained throughout the nicorandil bolus injection and also infused during reperfusion at a rate of 
ischemic period and followed by 30 min of reperfusion, aerosol delivery 0.3 mg/kg/hr, abrogated the therapeutic effects of nicorandil [143]. 
of 0.126 μmol of nitroprusside over 5 min into alveoli at 5 min after the This observation provides another mechanism of protection, which 
onset of ischemia significantly lowered pulmonary artery pressure shows that NO reduces lung allograft IRI partly by activating and 
elevation and capillary leakage response, and preserved physiological opening KATP channels (Fig. 2). KATP channels regulate pulmonary 
gas exchange conditions after reperfusion, leading to less lung edema vascular tone, and their inhibition has been recently found to induce and 
formation in comparison with control lungs without nitroprusside increase hypoxic pulmonary vasoconstriction in murine endotoxemic 
treatment [135]. A similar isolated buffer-perfused rabbit lung model lungs [144], implying that activation of KATP channels favors pulmonary 
with longer ischemic time of 180–210 min, with the same dose and route vasodilation. Besides activating KATP channels in lung allografts, nicor-
of administration of nitroprusside but delivered at either 5 min after the andil may have also activated NO-sGC-cGMP signaling pathway, as was 
8
G.J. Dugbartey                                                                                                                                                                                    B  i o  m   e d  i c i n  e   &   P  h  a  r m  a  c  o t h  e  r a  p y  167 (2023) 115549
reported to protect against IRI in isolated rat lungs [145]. At the sub- a substantial decline in systemic vascular resistance suggests that the 
cellular level, NO was found to directly activate mitochondrial KATP infusion rate was too high and may have mediated prolonged systemic 
channels and contributed to cardioprotection [146]. Although this has vasodilation. Interestingly, lower infusion rates of 1.0 and 
not been investigated in lung transplantation, it is possible that nicor- 3.0 µg/kg/min did not have significant effect on pulmonary hemody-
andil and endogenous NO may have activated mitochondrial KATP namics. In summary, infusion of NO donors during reperfusion improves 
channels in the lung allografts, and thereby contributing to attenuating allograft function in porcine models of lung allotransplantation. 
IRI after lung transplantation. 
The therapeutic impact of NO donors in canine lung transplantation 5.3. Role of inducible nitric oxide synthase in lung transplantation 
was further studied using FK409, an organic NO donor which sponta-
neously releases NO. In a canine model of orthotopic single-lung allo- As mentioned in Section 5.0, inducible nitric oxide synthase (iNOS) 
transplantation, 5 µg/kg/min of FK409 was intravenously infusion is a pathological isoform of NOS, and its expression has been shown to be 
30 minutes before ischemia until the onset of ischemia in donor dogs, upregulated in lung IRI and contributes to acute graft rejection following 
followed by preservation of lung allografts in Euro-Collins solutions at 4 lung transplantation. Therefore, inhibition of iNOS expression is 
ºC for 8 hours, and then FK409 administration from 15 minutes before emerging as an attractive therapeutic approach for prevention of IRI and 
reperfusion until 45 minutes after reperfusion. Compared to control acute allograft rejection after lung transplantation. In a study to inves-
lung allografts which did not receive FK409 treatment, FK409-treated tigate the role of iNOS in acute lung allograft rejection, intraperitoneal 
allografts functioned adequately after reperfusion as revealed by pul- administration of 200 mg/kg of aminoguanidine (a selective iNOS in-
monary perfusion and ventilation scintigraphy [147]. This suggests hibitor) every 6 hours for 6–12 days in recipient rats after lung allo-
good pulmonary hemodynamics and blood-gas exchange, leading to transplantation significantly reduced acute allograft rejection as 
prolonged post-transplant survival. Histopathologically, FK409 mark- observed histologically and radiographically compared to saline-treated 
edly reduced alveolar damage, which was increased with severe inter- control group [151,152]. iNOS inhibition also resulted in reduced NO 
stitial, alveolar and alveolar-septal edema in control lung allografts production and prolonged recipient survival without inducing immu-
[147]. Also, serum NO level in FK409-treated group significantly nological tolerance when compared to control group [151,152]. It is 
increased at the end of ischemia and reperfusion, which corresponded important to note that iNOS is an important immunomodulation mole-
with markedly decreased serum ET-1 level in comparison with control cule in allograft rejection, and overproduction of NO can induce cyto-
group [147]. This observation further highlights the vasodilatory toxicity via its reaction with superoxide to produce peroxynitrite, a toxic 
property of NO and its beneficial effect after lung transplantation. reactive nitrogen species [153]. Therefore, the observation that iNOS 
Collectively, NO donors improve lung allograft function and prolong inhibition reduced NO production and suppressed early lung allograft 
recipient survival in canine models of lung allotransplantation via rejection suggests that NO produced by iNOS during early lung allograft 
mechanisms including activation and opening of KATP channels and in- rejection may serve as a sensitive biomarker that indicates the functional 
hibition of ET-1 activity (Fig. 2). status of the lung allograft while mediating early graft rejection. In a 
similar rat model of acute lung allograft rejection, upregulated expres-
5.2.4. Nitric oxide donors in porcine models of lung transplantation sion of iNOS mRNA and protein in transplanted lung along with 
Besides rat, rabbit and canine models, the effect of nitric oxide do- increased influx of inflammatory cells and NO production (as seen in 
nors in lung transplantation has also been studied experimentally in increased levels of serum nitrate and nitrite) was observed [154,155]. 
pigs. Using a porcine model of DCD single-lung transplantation, lung However, iNOS inhibition with 250 mg/kg aminoguanidine (adminis-
allografts were ventilated with 100% oxygen and flushed with Perfadex tered subcutaneously) every 12 hours beginning immediately after 
solution 90 minutes after cardiac arrest and then preserved in an infla- transplantation until post-operative day 5 (day of sacrifice) resulted in 
ted state in Ringer’s solution at 4 ºC for 18 hours. Continuous infusion of significant downregulation of lung allograft iNOS mRNA and protein 
nitroglycerin at an increased stepwise rate of 0.3, 0.4 and 2.4 mg/kg/ expression, reduction in NO production and improvement in histological 
min in the reperfusion circuit and at a rate of 2.0 µg/kg/min in recipient rejection scores, and thus preventing allograft rejection [154,155]. In 
pigs starting 5 minutes prior to reperfusion preserved pulmonary gas addition, iNOS inhibition also preserved allograft function, which was 
exchange, which was significantly impaired in control lungs that were impaired in the control group. These results further support experi-
flushed and retrieved immediately after cardiac arrest [148]. However, mental evidence showing that iNOS-derived NO could be used as an 
neutrophil count and protein concentration in bronchoalveolar lavage excellent diagnostic indicator of early graft rejection, and that iNOS 
fluid as well as histological changes were unchanged compared to could be an important therapeutic target for the prevention of acute 
control group [148]. This result demonstrates that administration of allograft rejection after lung allotransplantation. 
nitric oxide donors in the early phase and during reperfusion improves 
DCD lung allograft function following prolonged preservation. Along the 6. Conclusion 
same train of evidence, Clark and colleagues [149] also showed in 
another porcine model of DCD single-lung allotransplantation that after Lung transplantation has become a routine clinical practice and the 
flushing and preservation of lung allografts in modified Euro-Collins only therapeutic option for patients suffering from a variety of end-stage 
solution for 18 hours, intravenous infusion of 0.02 mg/kg/h of SIN-1 pulmonary diseases. However, despite significant success achieved in 
(NO donor which spontaneously releases NO) during reperfusion this field, including improvement in surgical techniques and modifica-
significantly lowered pulmonary vascular resistance, improved tion of preservation solutions, PGD and other post-transplant compli-
oxygenation and attenuated neutrophil sequestration relative to control cations arising from IRI continue to increase morbidity and mortality 
allografts without SIN-1 infusion. In the same study, pentoxifylline rates after transplantation, and thereby limiting the success of these 
infusion at 2 mg/kg/h also produced the same result as SIN-1, with important life-saving undertakings. Among the factors that contribute to 
superior oxygenation and a further decrease in ROS production [149]. IRI after lung transplantation is the significant decrease in endogenous 
Similarly, administration of nitroprusside attenuated IRI and improved NO production, which suggests that NO pathway can be a potential 
porcine allograft function. In their study of single-lung allo- therapeutic target, whose activation might be beneficial in attenuating 
transplantation in pigs, Kukkonen et al. [150] observed that continuous IRI after lung transplantation. Bearing this in mind, there has been 
infusion of nitroprusside at a rate of 9.0 µg/kg/min markedly lowered several preclinical studies and clinical evidence showing that direct 
pulmonary vascular resistance, however, with a 44% decline in systemic administration of gaseous NO to lung donors prior to donor lung pro-
vascular resistance compared to control group that received equal curement, or to recipients, or administered indirectly by supplementing 
amount of vehicle [150]. The observation that nitroprusside caused such preservation solutions with NO donor compounds attenuates IRI, 
9
G.J. Dugbartey                                                                                                                                                                                    B  i o  m   e d  i c i n  e   &   P  h  a  r m  a  c  o t h  e  r a  p y  167 (2023) 115549
improves graft quality and function and prolongs recipient survival [19] J.R. Maurer, A.E. Frost, M. Estenne, T. Higenbottam, A.R. Glanville, International 
without causing any unfavorable systemic hemodynamic changes. guidelines for the selection of lung transplant candidates. The International 
Society for Heart and Lung Transplantation, the American Thoracic Society, the 
Therefore, these empirical findings suggest that NO should be routinely American Society of Transplant Physicians, the European Respiratory Society, 
used in clinical lung transplantation. Additionally, high expression Heart Lung 27 (4) (1998) 223–229. 
levels of iNOS along with its increased NO production, has been [20] Van Raemdonck, M. Strüber, F. Venuta, D. Vlasselaers, W. Wisser, M.E. Erasmus, 
Strategies in the prevention and the treatment of ischaemia-reperfusion injury 
consistently associated with increased incidence of IRI, PGD, post- after lung transplantation, Book.: Surg. Non-Neoplast. Disord. Chest: A Clin. 
transplant morbidity and mortality in experimental and clinical lung Update (2004). DOI: 10.1183/1025448x.00029005. 
transplantation. Therefore, iNOS-derived NO may be considered a po- [21] G.M. Verleden, Bronchiolitis obliterans syndrome after lung transplantation: 
tential and reliable diagnostic biomarker for allograft rejection after Medical treatment, Monaldi Arch. Chest Dis. 55 (2000) 140–145. [22] T.M. Egan, C.J. Lambert Jr, R. Reddick, K.S. Ulicny Jr, B.A. Keagy, B.R. Wilcox, 
lung transplantation. A strategy to increase the donor pool: use of cadaver lungs for transplantation, 
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[23] T.M. Egan, Non-heart-beating donors in thoracic transplantation, J. Heart Lung 
Funding Transpl. 23 (1) (2004) 3–10. 
[24] A.M. D’Armini, C.S. Roberts, P.K. Griffith, J.J. Lemasters, T.M. Egan, When does 
This work received no funding. the lung die? I. Histochemical evidence of pulmonary viability after "death, 
J. Heart Lung Transpl. 13 (5) (1994) 741–747. 
[25] F. Alessandrini, A.M. D’Armini, C.S. Roberts, R.L. Reddick, T.M. Egan, When does 
CRediT authorship contribution statement the lung die? II. Ultrastructural evidence of pulmonary viability after "death, 
J. Heart Lung Transpl. 13 (5) (1994) 748–757. 
[26] A.M. D’Armini, E.J. Tom, C.S. Roberts, D.C. Henke, J.J. Lemasters, T.M. Egan, 
Conceptualization: George J. Dugbartey; Literature search: George When does the lung die? Time course of high energy phosphate depletion and 
J. Dugbartey; Manuscript writing: George J. Dugbartey; Manuscript relationship to lung viability after "death’’, J. Surg. Res. 59 (4) (1995) 468–474. 
review and editing: George J. Dugbartey; Approval: George J. [27] P.R. De Leyn, T.E. Lerut, H.H. Schreinemakers, Van Raemdonck DE, K. Mubagwa, 
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G.J. Dugbartey                                                                                                                                                                                    B  i o  m   e d  i c i n  e   &   P  h  a  r m  a  c  o t h  e  r a  p y  167 (2023) 115549
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