Nina Wijnands

Introduction

Sepsis and inflammatory conditions accompanied by endotoxemia are an important health problem that is associated with high rates of morbidity and mortality [1]. The development of multiple organ failure (MOF) [1-6] is a major determinant to the detrimental outcome of these syndromes as a result of an auto-destructive or dysfunctional immune response [7]. Derangement of the microcirculatory flow is one of the other critical pathogenic events during sepsis and appears to be an important direct underlying cause for the development of MOF [8-10]. Preservation of the microcirculation, therefore, appears to be essential during these conditions. Especially preservation of the microcirculation in the intestinal mucosa seems to be a key factor to prevent organ failure during sepsis. The largest part of the intestinal blood flow (70-80%) passes through the mucosal layer, the metabolically most active component of the gut, since it is, in addition to nutrient absorption from the lumen, responsible for the barrier function as part of the host immune response [11].

The intestinal mucosa is also the site for absorption of amino acids [12]. Glutamine is the precursor amino acid of arginine, via conversion to citrulline [13,14]. Arginine has exhibited special attention over the past decades, as arginine is the sole precursor of nitric oxide (NO) and essential for vasodilatation. Unfortunately, enhanced arginine consumption by arginase and NOS2 and impaired arginine de novo synthesis from citrulline (possibly due to a decreased ASS activity), combined with a decreased supply of arginine, result in impaired arginine availability during inflammatory conditions [15-17]. As a result, the arginine deficiency during sepsis and endotoxemia [15,17-19] further contributes to the microcirculatory disturbances in sepsis. Basically, increasing the arginine availability with L-arginine supplementation would be a good therapeutic strategy, but this approach led to an inconclusive outcome, with some studies demonstrating beneficial results, while others showed an increased mortality or no effect on mortality [20-25]. The quest for the best intervention to enhance the arginine-NO metabolism and microcirculatory flow during sepsis and endotoxemia remained. In vitro studies have shed new light on the potential role of citrulline supplementation as arginine precursor, based on the compartmentalization of arginine as present within e.g. endothelial cells [26-29]. These studies indicated that endothelial NOS (NOS3) possibly exists in a complex with ASS and, therefore, benefits from citrulline supplementation to produce NO in case of arginine deficiency. Based on these in vitro studies and the previous work on arginine supplementation, citrulline may be able to restore intracellular arginine concentrations and the microcirculation during endotoxemia. However, in depth knowledge, at the enzyme level, of the pathophysiological changes of the arginine-NO metabolism during endotoxemia is essential. Therefore, the overall aim of this thesis was to unravel the role of the different enzymes (arginase, argininosuccinate synthetase and NOS2 and NOS3) and their interrelationship during disturbances in the arginine metabolism as present during endotoxemia. Thus far most experimental models study arginine-NO metabolism in an acute model in combination with drugs that modulate enzyme activity. However, patients with sepsis usually do no present during the acute phase of sepsis, but have developed this clinical state in the course of a few days instead of hours. Furthermore, drugs that modulate these enzymes are all arginine homologs and may, therefore, affect not only more than one enzyme, but also the activity of arginine transporters. We, therefore, developed a prolonged experimental endotoxemia model that better mimicks the human situation. We then investigated the potential effects of L-citrulline supplementation on arginine-NO metabolism and microcirculatory flow, and the role of the respective enzymes in NO production in this model. This resulted in the formulation of several aims for the studies reported in this thesis:

1. To develop an experimental model, which represents the arginine-deficient state during a prolonged inflammatory response.
2. To determine the best diagnostic tools to investigate the microcirculatory flow during modulations of arginine-NO metabolism.
3. To unravel the role of enhanced arginine consumption by increased arginase activity on NO production and microcirculatory flow.
4. To improve our knowledge on the complex interrelationship in the inflammatory response between arginase and NOS2 on the regulation of the arginine availability in our new model.
5. To investigate the consequences of an impaired arginine-NO metabolism on NO production and microcirculatory flow.
6. To investigate whether L-citrulline or L-arginine supplementation is the best therapeutic strategy in endotoxemia to improve the arginine-NO metabolism in relation to the microcirculation.
7. To provide insight into the role of endothelial NO production on microcirculatory flow during endotoxemia.
8. To investigate the influence of an impaired arginine de novo synthesis, as a result of ASS deficiency, on arginine concentrations and microcirculatory flow during endotoxemia.
9. To improve our knowledge on the key enzymatic mechanisms which control NO production during endotoxemia.
10. To provide evidence of the potential role of L-citrulline as substrate for enhanced arginine de novo synthesis during conditions with clinical impairment of the intestinal perfusion.

Model development

The first aim of this thesis was to develop an experimental model, which represents the arginine-deficient state during prolonged and severe inflammation as observed in humans with persistent sepsis. In the past, acute experimental animal models were used to study this complex interplay, especially murine endotoxemia models [30-34]. Unfortunately and in contrast to the arginine-NO metabolic derangements reported in human sepsis, a deficit of arginine in the plasma pool was not found in these acute models [30-34]. In part 1 of this thesis, in chapter 4, we describe the development of our prolonged murine endotoxemia model that mimics the characteristic arginine depletion in human inflammatory conditions (first aim). In this model, mice received a continuous infusion of LPS during an 18 hours period to determine the time-point at which the characteristic arginine deficiency developed. As in previous experimental studies and during the acute phases of human sepsis, an initial increase in arginine plasma concentrations is present as a result of an enhanced protein breakdown and increased generation of arginine from glutamine and citrulline as a response to the increased consumption [35]. In line, in our prolonged endotoxemia model, the initial increase in arginine plasma concentrations occurred around 8 hours of endotoxemia (LPS infusion). After 12 hours of infusion, a significant decrease in arginine plasma concentrations developed compared to basal concentrations and most importantly also compared to the initial increase in arginine at 8 hours of endotoxemia.

In chapter 4 we also determined the best diagnostic tools to investigate the microcirculatory flow and NO production under basal and endotoxemic conditions during modulations in arginine-NO metabolism (second aim). The approach was based on the technique described by Massberg et al. [36] to evaluate mucosal jejunal microcirculatory flow. A jejunal segment was opened to microscopically visualize the inner surface of the intestine and to reveal the microcirculatory flow in the mucosa with a sidestream dark-field (SDF) imager [37,38]. The SDF-imager is applied on tissue surfaces such as mucosa and uses 530nm light that is absorbed by haemoglobin in red blood cells to measure perfused vessels [39]. The microcirculation is thought to be the motor of sepsis [40], so that measurements of the microcirculation are probably a good indicator of the perfusion of other vascular beds [41]. We specifically selected the gut as the circulation of the intestines is the first end organ during inflammatory conditions to dysfunction and the last organ to recover [42]. Practically, it is also easily accessible, which minimizes confounding surgical trauma.

To determine NO production in tissue of control and endotoxemic animals, we used NO spin trapping [43]. NO spin trapping is a sufficiently accurate method that allows assessment of the influence of endotoxemia on NO production in our model, as we could demonstrate an impaired NO production in the jejunal villi during endotoxemia (described in chapter 6-9). Moreover, the carotid arteries of our experimental mice were used to measure NO production in endothelial cells with 2-photon laser microscopy and a NO-sensitive copper-based fluorescent probe (Cu2FL2E). In agreement with the spin trapping measurements, assays with the 2-photon laser microscope showed that endothelial NO production was significantly reduced during endotoxemia compared to control. In conclusion, the results of chapter 4 suggest that this newly developed model of prolonged endotoxemia and the above mentioned NO-detection techniques are suitable to investigate therapeutic strategies to enhance NO production and to unravel the role of the key enzymes in this pathway (described in chapter 5-9).

Role of inflammation

The enhanced arginine consumption during inflammatory conditions is a major contributor to the development of arginine deficiency during sepsis or endotoxemia [44]. Both arginase-1 and NOS2 enzymes are upregulated during inflammation and use the same substrate, contributing to the reduction in arginine availability [44-46]. Furthermore, the increased consumption of arginine by these enzymes may result in a lower availability arginine for NOS3, and ultimately results in microcirculatory dysfunction.

Since the expression of arginase is upregulated under conditions such as sepsis and endotoxemia, improving intracellular arginine availability may be effective (third aim). To address this question, we used a model in which arginase was injected into the circulation. This approach separates the effects of low circulating arginine concentration from that after upregulation of arginase and NOS2 expression in tissues. In chapter 5 we show that intravenous arginase injection caused an acute decrease in plasma and tissue arginine concentrations, resulting in an acutely occurring impairment of microvascular perfusion. L-citrulline, but not L-arginine supplementation was capable of enhancing both plasma and tissue arginine concentrations, which led to an increased NO production and improved microcirculatory flow. Thus, during an acute condition with enhanced catabolism of arginine by arginase, citrulline is capable of restoring the cellular arginine concentrations and improves the end organ microcirculation.

Reducing the effects of arginase during conditions with increased arginase activity such as sepsis and endotoxemia may therefore be a starting point for treatment. This hypothesis was tested in an arginase-1-deficient mouse model was used (fourth aim; chapter 6). These Arg1 fl/fl / Tie2-Cre tg/- mice did not express arginase-1 in endothelial and hematopoietic cells. Deficiency of arginase-1 resulted in higher plasma arginine concentrations, and enhanced NO production in endothelial cells and jejunal tissue during endotoxemia. However, this increase in NO production had no effect on microcirculatory flow, indicating that it did not occur in the endothelium. Supplementation of L-citrulline in these animals also did not enhance the NO production or microcirculatory flow. In agreement, we found that the enhancement of NO production was due to enhanced NOS2 activity and was accompanied by an increased inflammatory response. In vitro data from cultured bone-marrow-derived macrophages of arginase-1-deficient animals confirmed this higher inflammatory response to endotoxin than seen in control fl/fl / Tie2-Cre tg/- littermates. The effects of treatment of Arg1 fl/fl / Tie2-Cre tg/- mice with the specific NOS2 inhibitor 1400W further implicated NOS2 in the enhanced capacity to produce NO. In conclusion, arginase-1 deficiency facilitates a NOS2-mediated pro-inflammatory activity at the expense of NOS3-mediated endothelial relaxation. Based on the complex regulation between NOS2 and arginase, inhibition of arginase should be avoided during inflammatory conditions as this may lead to detrimental results.

Role of the endothelium

The fifth aim of this thesis was to investigate the consequences of an impaired arginine-NO metabolism on microcirculatory function. As briefly described above, arginine deficiency during endotoxemia resulted in an impaired NO production in jejunal tissue (Chapter 7). This impaired NO production was related to a decreased microcirculation in these animals. In patients with severe infection or systemic inflammatory response syndrome (SIRS), microcirculatory function is also impaired and NO production is disturbed [47-50]. Efforts to improve organ perfusion are therefore an important therapeutic target. Based on the impaired arginine availability during sepsis and decreased citrulline concentrations, and the tight coupling between NOS3 and ASS in endothelial cells [27], supplementation of L-citrulline may be a promising option to restore these reduced concentrations (sixth aim). In chapter 7, we supplemented our chronic endotoxemia model with L-citrulline, L-arginine or the isocaloric placebo L-alanine. L-citrulline supplementation increased plasma and tissue concentrations of arginine and citrulline, restored the intracellular NO production in the intestine, and improved intestinal microvascular perfusion compared to L-arginine supplementation. Furthermore, the beneficial effects of L-citrulline supplementation were accompanied by an increased phosphorylation of Ser 1177 in NOS3 (implying activation) and a decrease in NOS2 content. These effects were not present in the L-arginine supplemented animals. Therefore, these data suggest that citrulline supplementation is a key factor to increase intracellular arginine availability for NOS3-mediated NO production and thereby improve organ perfusion during endotoxemia.

Because of the beneficial effects of L-citrulline supplementation on NO production during endotoxemia require conversion of citrulline into arginine, we investigated the role of ASS on NO production and on microcirculatory flow during endotoxemia (seventh aim; chapter 8). Therefore, an endothelium- (and macrophage-) specific ASS-deficient mouse was developed (ASS fl/fl / Tie2-Cre tg/- mice). While ASS-deficient mice exhibited a higher plasma arginine concentration than controls, this was not associated with an enhanced NO production in endothelial cells or jejunal tissue during endotoxemia. Furthermore, an impaired jejunal microcirculation was observed during endotoxemia in these animals. These results show that de novo arginine synthesis in endothelial cells is necessary for the beneficial effect of L-citrulline supplementation during endotoxemia (eighth aim). This concept was confirmed by the demonstration that Nos3-deficient mice exhibited similar microcirculatory disturbances as ASS-deficient mice under endotoxemic conditions. In macrophages intracellular arginine resynthesis for NOS activity only becomes essential when extracellular arginine is deprived [51], such as in prolonged endotoxemia [52,53] or sepsis [18,30,52,54]. In our study, cultured bone marrow-derived macrophages of ASS-deficient animals developed a lower inflammatory response to endotoxin (reduced cytokine and nitrite production) than those of control littermates. Thus, reduced ASS activity in ASS fl/fl / Tie2-Cre tg/- mice resulted in endothelial and macrophage dysfunction.

Collectively, we can conclude that during endotoxemia not only substrate availability, but also enzyme functionality has to be preserved for NO synthesis and microcirculatory flow. Earlier studies had suggested that in the absence of NOS3, NOS1 is capable of maintaining the NO production in the microcirculation. This hypothesis was based on the observation that compensatory upregulation of NOS1 activity was responsible for normal angiogenesis [55] and an adequate NO production in Nos3-deficient [55-59]. These findings were the basis to clarify the role of the NOS enzymes [56-60] on NO production during endotoxemia (ninth aim). In Nos3 -/- and Nos2 -/- / Nos3 -/- double knockout mice, the negative effects of endotoxemia were absent. Moreover, the beneficial effects of citrulline supplementation during LPS infusion on the microcirculation were not present in these mice, although, as expected, citrulline significantly enhanced plasma arginine concentration in all mouse strains. These data show that only NOS3 produces NO that will increase the circulation in the jejunal mucosa. In agreement, clinical studies found that polymorphisms in the Nos3 gene [61] are associated with several different pathophysiological responses to shock, impaired organ function [62] and an increased risk for cardiovascular disease [63]. The extent of the functional NOS3 inhibition in patients with prolonged sepsis and multiple organ failure is not known at present.

Figure 1. The complex interplay of arginine-NO metabolism during endotoxemia. During endotoxemia arginine deficiency occurs as a result of an impaired conversion of glutamine, a decreased arginine uptake in the intestinal villi and an enhanced consumption by inflammatory cells. This arginine deficiency results in a diminished availability for NOS3 in the endothelial cells, which results in NOS3 uncoupling and an impaired microcirculation (left in illustration). L-citrulline supplementation during endotoxemia restores the intracellular and extracellular arginine concentrations, and leads to enhanced arginine availability for NOS3 and restores microcirculatory flow (right in illustration).

Plasma amino acid concentrations (A), and microcirculatory (B) and macrocirculatory effects (C) during L-citrulline infusion in a septic ICU patient. Arginine Citrulline Ornithine Perfused vessels Systolic Diastolic MAP.
Figure 2. Plasma arginine, citrulline and ornithine concentrations (μmol/L) (A), and microcirculatory (B) and macrocirculatory effects (C) during L-citrulline infusion in a septic ICU patient.

Transitional aspects

Based on the beneficial effects of L-citrulline supplementation in experimental studies, the effects of L-citrulline supplementation was tested in humans (tenth aim). Prior to a study in critically ill patients, the effect of L-citrulline on arginine availability and the intestinal microcirculation was studied in healthy controls. Enteral supplementation is feasible in humans as oral citrulline supplementation exhibits less gastrointestinal discomfort than arginine [64,65]. We used an exercise-induced inflammatory model to determine the potential role of L-citrulline as substrate for enhanced de novo arginine synthesis during impaired intestinal perfusion in healthy subjects. This model is characterized by strenuous exercise-induced splanchnic hypoperfusion associated with small intestinal injury and loss of gut barrier function [66].

In chapter 10, we show that oral L-citrulline supplementation significantly enhanced both plasma citrulline and arginine levels compared to placebo and enhanced arginine availability. Previous human [64,67-69] and experimental studies with L-citrulline supplementation [70] support this finding. In addition, L-citrulline intake reduces small intestinal cellular injury. In line with these data, L-citrulline was recently shown to reduce intestinal injury after experimentally induced small bowel obstruction [71] and gastric mucosal injury as a consequence of ischemia-reperfusion [72].

In accordance, our study revealed that oral supplementation of L-citrulline prior to a condition with splanchnic hypoperfusion improves sublingual microcirculation, which strongly suggests improved intestinal microcirculation [41]. The next step is to determine whether L-citrulline improves microcirculatory function in septic ICU patients without detrimental systemic hemodynamic effects. In the pilot phase of our study, preliminary data of a patient receiving a continuous enteral supplementation with L-citrulline during 8 hours, suggest improvement of microcirculatory measurements without systemic hemodynamic side-effects (Figure 2A-C).

Overall conclusion

In this thesis we demonstrated that arginine deficiency during endotoxemia and inflammatory conditions is detrimental for the microcirculatory flow and endothelial NO production. We developed a prolonged endotoxemia model which mimicked the deficiency of arginine as present during human sepsis. This arginine deficiency resulted in an impaired NO production and microcirculatory flow. L-citrulline supplementation restored NO production and microcirculatory flow during endotoxemia, whereas supplementation with L-arginine did not. Furthermore, we showed in this model that the balance between arginine consumption (arginase-1 and NOS2) and production (ASS) has to be maintained to ensure arginine availability during inflammatory conditions. Competition for arginine between the arginase/NOS2 and the ASS/NOS3 enzymes modulates the effects of therapeutic interventions that aim to improve the microcirculatory response during endotoxemia. Addressing the reduction of ASS and/or NOS3 function during sepsis may be the key to the ability to restore this arginine deficiency and microcirculatory function with L-citrulline. In this respect it is promising that L-citrulline supplementation preserves splanchnic perfusion and reduces intestinal injury in athletes. Preliminary data during sepsis also support the experimental findings in this thesis.