Microcirculatory Function during Endotoxemia—A Functional Citrulline-Arginine-NO Pathway and NOS3 Complex Is Essential to Maintain the Microcirculation

Competition for the amino acid arginine by endothelial nitric-oxide synthase (NOS3) and (pro-)inflammatory NO-synthase (NOS2) during endotoxemia appears essential in the derangement of the microcirculatory flow. This study investigated the role of NOS2 and NOS3 combined with/without citrulline supplementation on the NO-production and microcirculation during endotoxemia. Wildtype (C57BL6/N background; control; n = 36), Nos2-deficient, (n = 40), Nos3-deficient (n = 39) and Nos2/Nos3-deficient mice (n = 42) received a continuous intravenous LPS infusion alone (200 μg total, 18 h) or combined with L-citrulline (37.5 mg, last 6 h). The intestinal microcirculatory flow was measured by side-stream dark field (SDF)-imaging. The jejunal intracellular NO production was quantified by in vivo NO-spin trapping combined with electron spin-resonance (ESR) spectrometry. Amino-acid concentrations were measured by high-performance liquid chromatography (HPLC). LPS infusion decreased plasma arginine concentration in control and Nos3−/− compared to Nos2−/− mice. Jejunal NO production and the microcirculation were significantly decreased in control and Nos2−/− mice after LPS infusion. No beneficial effects of L-citrulline supplementation on microcirculatory flow were found in Nos3−/− or Nos2−/−/Nos3−/− mice. This study confirms that L-citrulline supplementation enhances de novo arginine synthesis and NO production in mice during endotoxemia with a functional NOS3-enzyme (control and Nos2−/− mice), as this beneficial effect was absent in Nos3−/− or Nos2−/−/Nos3−/− mice.


Introduction
Derangement of the microcirculatory flow is one of the critical pathogenic events in sepsis and appears to be an important directly underling cause for the development of multiple organ failure and mortality [1][2][3][4]. These alterations in the microcirculation, if not reversed within the first 24 h after the onset of sepsis (prolonged sepsis), are hypothesized to be the single independent factor in predicting patient outcome [4]. Endothelial damage plays an important role in the development of microcirculatory collapse during prolonged sepsis due to leakage of plasma, and impeded vasodilatation due to a compromised nitric oxide (NO) production [1,2,[5][6][7][8][9]. The conversion of arginine to citrulline and NO is the sole source of NO and is mediated by one of the three NO-synthase enzymes (NOS1, NOS2 and NOS3) [10][11][12][13]. Several mechanisms are hypothesized to be responsible for the decrease in endothelial NO production during prolonged endotoxin-induced inflammation. The impaired NO production in the microcirculation is thought to be mediated by dysfunction of NOS3 [14], which is mediated at least partly by arginine deficiency [9,15,16]. This arginine deficiency, in turn, results from an enhanced utilization of arginine upon the pro-inflammatory upregulation of NOS2 and arginase [15,17,18], and from a decreased import of arginine and/or an impaired resynthesis of arginine from citrulline [15,[19][20][21][22]. The reduced citrulline bioavailability and production may further deteriorate the already impaired substrate availability in the arginine-NO pathway [15,19,21,22]. Furthermore, excessive NOS2-mediated NO production during endotoxemia in non-homogeneously distributed inflammatory cells results in a maldistribution of NO and a "hyperdynamic" microcirculation by shunting [23].
Supplementation of L-citrulline during endotoxemia restored intracellular arginine availability and improved NO production and microcirculatory function [9]. Based on the intracellular co-localization of NOS3 and ASS [24], It is generally hypothesized, but not yet proven that this beneficial effect of L-citrulline is mediated by endothelial NOS3 [25]. We therefore investigated the effect of L-citrulline supplementation in Nos2 −/− , Nos3 −/− , and Nos2 −/− /Nos3 −/− mice on intracellular arginine availability, jejunal NO production and microcirculatory flow in a prolonged endotoxemia model.

Plasma Amino Acid Concentrations in Control and Nos-Deficient Mice under Basal and Endotoxemic Conditions
Under basal conditions Nos2 −/− and Nos2 −/− /Nos3 −/− mice exhibited significantly lower plasma concentrations of arginine than control and Nos3 −/− mice ( Figure 1A). Upon LPS infusion, only the plasma arginine concentrations of control and Nos3 −/− mice decreased significantly ( Figure 1A). Plasma citrulline concentrations were similar in all mouse lines under both basal and endotoxemic conditions, except for a higher concentration under basal conditions in Nos3 −/− mice ( Figure 1B). Plasma ornithine concentrations were not different between Nos-deficient mice and control mice under basal conditions ( Figure 1C). LPS administration increased plasma ornithine concentrations in all mouse lines.

L-Citrulline Enhances the Intestinal NO Production in Control and Nos2-Deficient Mice
Under basal conditions, intestinal intracellular NO concentrations were lower in Nosdeficient than in control mice, but reached significance in Nos2 −/− /Nos3 −/− mice only ( Figure 3). The intestinal NO production in Nos2 −/− /Nos3 −/− mice was~twofold lower than that in control mice (p < 0.05; Figure 3). LPS infusion resulted in a significant decrease in the jejunal NO production of control and Nos2 −/− mice ( Figure 3, n = 5), whereas it increased jejunal NO production~1.5fold in Nos3 −/− mice (p < 0.05, n = 5; Figure 3). Interestingly, the NO production in Nos2 −/− /Nos3 −/− mice was not altered and remained significantly lower than in control mice. L-citrulline supplementation during endotoxemia enhanced jejunal NO production in control and Nos2 −/− mice ( Figure 3, n = 5, both p < 0.05) and was~3-fold higher in control than in Nos2 −/− mice. The increased NO production due to endotoxemia in Nos3 −/− mice was not further increased by L-citrulline supplementation (p = 0.9; Figure 3).

Detrimental Effects of Nos3 Deficiency on Microcirculatory Flow during Endotoxemia
Under basal conditions the total number of perfused vessels and the number of perfused vessels per villus were lower in the Nos3 −/− and Nos2 −/− /Nos3 −/− mice than in control or Nos2 −/− mice ( Figure 4A,B). Endotoxemia reduced the total number of perfused vessels and the number of perfused vessels per villus in control and Nos2 −/− mice, but did not alter these parameters in Nos3 −/− or Nos2 −/− /Nos3 −/− mice ( Figure 4A,B). Under endotoxemic conditions, the total number of perfused vessels and vessels per villus were similar in all mouse lines. L-Citrulline supplementation during endotoxemia increased both parameters in control and Nos2 −/− mice ( Figure 4A,B, p < 0.05 for both), but not in Figure 4A,B).

Discussion
The results presented in this study demonstrate that Nos3 deficiency does not decrease jejunal tissue NO production, but does result in an impaired microcirculation as judged from the decreased number of perfused vessels under basal and endotoxemic conditions. Our findings further reveal that, in the absence of Nos3, endotoxemia and supplementation of L-citrulline does not affect the microcirculation, demonstrating that the positive effects of L-citrulline supplementation depend on the presence of a functional NOS3 enzyme. The effects of endotoxemia and L-citrulline supplementation were similar in control and Nos2 −/− mice, which shows that NOS2 does not contribute to the vasodilatory effect of NO in jejunal villi.
The microcirculation is a complex system, in which endothelial NO production plays a key role in regulating blood flow [1,2,4,5,26]. The role of NOS3 in endothelial NO production and microcirculatory homeostasis is well established under physiological conditions, but it has remained unclear thus far whether NOS3-dependent NO production is the exclusive source of NO-dependent microcirculatory flow. Endotoxemic conditions increase Nos2 expression and decrease that of Nos3 [27], which is accompanied by the development of an impaired or shunted microvascular perfusion [2,4,5,23,27,28]. Historically, deletion or inhibition of NOS2 was hypothesized to be the key to prevent or treat the detrimental effects of endotoxemia on microvascular perfusion [23,[29][30][31]. Normally, endotoxemia results in an increased expression of Nos2 mRNA and protein, which leads to an increased NO-production [32], but this overexpression of Nos2 does not enhance hypotension [32,33]. However, due to non-homogeneous expression of Nos2 in tissues, shunting of the microvascular flow during endotoxemia occurs [23]. A better microvascular responsiveness and a lower mortality during experimental sepsis induced by cecal ligation and puncture were reported in Nos2-deficient animals compared to control animals [34]. Our findings are at variance with these data, as the Nos2-deficient mice in our study demonstrated an impaired NO-production and a decreased number of perfused vessels during prolonged endotoxemia, similar to control mice. These results indicate that NOS2 does not play a significant role in the intestinal NO-production and the regulation of the microcirculation during prolonged endotoxemia. Furthermore, downregulation of Nos3 expression during endotoxemia [9] may further contribute to the impaired NO production during endotox-emia in these Nos2-deficient animals. In addition, administration of LPS, which induced an increased NOS2-derived NO-production, did not result in an altered microcirculation in Nos3-deficient mice. Therefore, our data imply a key function for NOS3 in the maintenance of the intestinal microcirculation under basal and endotoxemic conditions.
The influence of NO on the vascular tone has led to several experimental studies investigating the influence of genetic ablation of Nos genes [8,31,35]. In the absence of NOS3, a 50% decrease in basal NO concentrations was observed in brain tissue of Nos3-deficient compared to wild-type mice [36]. Despite the lower NO-production in the absence of NOS3, angiogenesis was normal [35]. Compensation by NOS1 was shown to be responsible for the maintenance of an adequate NO production [36][37][38][39][40]. In this respect it is of interest that the observed lower basal NO production in the current study in Nos3 −/− and Nos2 −/− /Nos3 −/− compared to control mice was accompanied by a decrease in the number of perfused vessels, which indicates that intestinal NOS1 does not compensate for the absence of NOS3, and that NOS3 is essential to maintain the intestinal perfusion. In order to receive a sufficient amount of NOS3-derived NO production, a continuous intracellular arginine availability is key.
This intracellular availability of arginine depends on arginine regeneration from Lcitrulline or protein degradation. Regeneration of intracellular arginine from citrulline via argininosuccinate synthetase is essential to maintain NOS3-dependent NO production in endothelial cells [25,41]. As many as three independent intracellular arginine pools have been postulated [24], but more recent literature suggests that, at least in case of enhanced arginine consumption by arginase in endothelial cells, the subcellular location of arginine is less important in maintaining NOS3-derived NO production [42]. We previously observed enhanced arginase activity during endotoxemia [9,43], as determined from the increased ornithine plasma and tissue concentrations in control mice. Furthermore, we showed that, in the absence of arginase-1 in endothelial cells or macrophages, only NOS2-derived NO production significantly increased during endotoxemia, whereas NOS3-derived NO production did not benefit from the arginase-1 deficiency [43]. This discrepancy between our results and those of Elms et al. [42] may result from the enhanced NOS2 activity in arginase-1 deficient animals.
We previously demonstrated that L-citrulline supplementation during endotoxemia resulted in enhanced arginine concentrations both in plasma and tissue [9], while arginine supplementation in this model did not increase tissue arginine concentration. In addition, in Nos2 −/− mice, L-citrulline supplementation exhibited similar effects as in control mice: increased plasma arginine concentrations, an increased intracellular NO production and an enhanced microcirculatory flow. These results suggest that the beneficial effects of L-citrulline supplementation on NO production and microcirculatory flow are mediated by NOS3. The lack of effects of citrulline supplementation in the Nos2 −/− /Nos3 −/− double knockout mice further implies that NOS1 is not able to compensate for the lack of NOS3. Therefore, our results further underline the subcellular localization of citrulline and NOS3 to maintain a NOS3-specific arginine pool as a substrate for NOS3-derived NO-production in the microcirculation.
To determine the possible potential clinical administration of citrulline and further in vivo implications, we previously conducted a randomized double-blinded control, placebo-controlled crossover study in healthy athletes in which oral citrulline was administrated prior to 1 h of strenuous exercise at 70% of the individual pre-assessed maximal workload capacity. The enhanced arginine availability in the L-citrulline supplemented group resulted in a preserved splanchnic perfusion and reduced intestinal injury during exercise [44]. In line with this previous study, the current study results suggest that L-citrulline is a potential clinical application for patients during endotoxemic or septic conditions accompanied by an impaired microcirculation and arginine deficient state. In conclusion, our study demonstrates that L-citrulline supplementation results in an enhanced de novo arginine synthesis and NO production in control and Nos2 −/− mice during endotoxemia. However, these beneficial effects of L-citrulline supplementation on the microcirculation depend entirely on a functional Nos3 gene.

Animals
To investigate the role of NOS2 and NOS3 on the microcirculatory flow and the NO production during endotoxemia, male C57Bl6 (control; n = 36), Nos2 −/− [45] (n = 40), Nos3 −/− [39] (n = 39) and Nos2 −/− /Nos3 −/− mice (n = 42) were obtained from the Department for Molecular Biomedical Research, University Ghent, Belgium. The Nos2 −/− [45] and Nos3 −/− mice [39] were initially obtained from Jackson laboratories. Nos2 −/− /Nos3 −/− double knockout mice were produced at the Department of Biomedical Molecular Biology. All mice were transported to the Centralized Animal Facilities at Maastricht University and allowed a three weeks adaptation period. The mice were two to three months old, weighed 23-28 g and were individually housed at room temperature on a 12 h light-dark cycle, fed standard lab chow (Hope Farms, Woerden, The Netherlands) and water ad libitum until the experimental phase of the study. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Maastricht University Medical Center (Permit Number: 2010-172).

Experimental Design
The experimental protocol and surgical procedures were described in detail elsewhere [9]. In brief, a jugular vein catheter was implanted 4 days prior to the experimental phase of the study. At day four, mice were randomly allocated to receive lipopolysaccharide (LPS; E. coli O55:B5, Sigma Aldrich, St. Louis, MO, USA, 0.4 µg × g bodyweight −1 × h −1 ; n = 109) or sterile 0.9% saline (n = 48). In total 13 mice died during the endotoxemia infusion (4 Nos2 −/− , 3 Nos3 −/− and 6 Nos2 −/− /Nos3 −/− mice). During the final 6 h L-citrulline (6.25 mg/h; n = 48; "LPS-Cit" group) or the placebo amino acid L-alanine (12.5 mg/h; n = 48; "LPS" group) was administered simultaneously with the LPS infusate. The control group was treated with 0.9% saline and L-alanine (n = 48; "control" group). In total, mice received 1.5 mL fluid during the 18 h infusion protocol (83 µL/h). At the end of the 18 h infusion period, mice were anesthetized as described [9]. In vivo tissue NO production (n = 60) and jejunal microcirculation (n = 84) were measured as described in brief below. Arterial blood was sampled via a cardiac puncture. Organs were then harvested, snap frozen in liquid nitrogen and stored in −80 • C until further analysis.

Plasma and Tissue Amino-Acid Concentration Measurements
After deproteinization, plasma and jejunal amino-acid concentrations were determined by high-performance liquid chromatography (HPLC) [9].

In Vivo Tissue NO Production
The in vivo NO production in jejunal tissue was determined in mice (n = 60) injected with spin trap agents and quantified as mono-nitrosyl iron complexes (MNIC) with Fe 2+dithiocarbamate complexes with electron spin resonance (ESR) spectroscopy [9]. NO concentrations were calculated from the height of the three-line NO amplitude using Bruker WINEPR software as previously described in more detail [9].

Jejunal Microcirculation Measurements with SDF Imaging
The mucosal microcirculation in the jejunal villi was microscopically visualized with the side-stream dark-field (SDF) imager (Microscan, Amsterdam, the Netherlands) [46,47], as described [9]. In brief, circulation, quantified as the total number of perfused vessels per view and the total number of perfused vessels per villus, was determined using Automated Vascular Analysis software 3.0 (Microscan, Amsterdam, the Netherlands), adjusted according to de Backer et al. [28,48,49]. The average microvascular flow index (MFI), a parameter for the predominant type of flow in the villi in the four quadrants of an image, was scored semiquantitatively (0 = absent, 1 = intermittent, with at least 50% of the time no flow, 2 = sludging, 3 = normal or 4 = hyperdynamic flow) [28]. All imaging experiments were done by an experienced investigator and images were analyzed by two independent blinded experienced researchers.

Statistical Analysis
Statistical analysis of the data was performed using GraphPad Prism 6 (GraphPad, San Diego, CA, USA). One-way analysis of variance (ANOVA) was performed with posthoc Bonferroni correction between groups to determine significant differences. Data are represented as mean and standard error of the mean (SEM). p-values smaller than 0.05 were considered as statistically significant.