Sepsis, the systemic inflammatory response to infection, is a very common disease, and although major progress has been made in its treatment, it remains one of the leading causes of death in critically ill patients. Current guidelines for resuscitation of septic patients aim at normalizing macrocirculatory perfusion indices such as mean arterial and central venous pressure, urinary output, and mixed/central venous oxygen saturation. However, it is now widely accepted that microcirculatory alterations play an important role in the pathogenesis of sepsis and particularly its most feared complication, the multiple organ failure syndrome (1).
The role of nitric oxide (NO) in the pathogenesis of sepsis is controversial (2). Nonselective NO synthase (NOS) inhibition improves hemodynamic parameters but increases mortality (3), and administration of nitroglycerin as NO donor might have a similar deleterious effect (4). Injecting low-dose Escherichia coli LPS to healthy volunteers is an accepted in vivo model of sepsis in humans (5). LPS injection has been shown to reduce NO-dependent vasodilation of resistance vessels in human skeletal muscle (6). Several animal studies have shown differential effects of endotoxemia on larger versus smaller blood vessels in various vascular beds (2). Whether the systemic inflammatory response induced by experimental endotoxemia affects NO-dependent vasodilation in the human microcirculation is currently not known.
One of the main physiological regulators of NO production is asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NOS (7). The circulating levels of ADMA are mainly regulated through its metabolizing enzyme, dimethylarginine-dimethylaminohydrolase (DDAH) (8). Leiper et al. (9) recently showed that ADMA accumulation can be a direct cause of endothelial dysfunction, a condition characterized by a decreased bioavailability of NO. In patients with septic shock, serum ADMA levels are increased (10), and this might at least partly be explained by the inhibitory effect of LPS on DDAH activity (11). Furthermore, ADMA levels are directly associated with disease severity (10) and intensive care unit (ICU) outcome (12). It has been hypothesized that accumulation of ADMA plays a causal role in the development of multiple organ failure by blocking basal endothelial NO production with reduced organ perfusion as consequence (13).
Microvascular endothelial function can easily be assessed in the human skin by the noninvasive technique of laser Doppler (14, 15). Local heating of the skin induces vasodilation mediated by two independent mechanisms: the initial peak in skin blood flow (SkBF) relies predominantly on local sensory nerves, whereas the sustained secondary plateau is mediated by endothelial NO production (16, 17). Alterations of these parameters have recently been shown to be of prognostic value for cardiovascular mortality in end-stage renal disease (18).
The aim of the present study was to test the hypothesis that a single injection of LPS causes a decrease in microvascular NO-dependent vasodilation explained by a modification of the ADMA-DDAH pathway.
We included seven obese subjects (four women and three men) with a body mass index of greater than 30 kg/m2 (mean, 34.7 ± 4.2 kg/m2) aged 19 to 40 years (mean, 33.6 ± 7.1 years). They all participated in another study on the physiological response to E. coli endotoxin in obese subjects. All subjects underwent clinical examination and were asked to complete a personal health and medical history questionnaire, which served as screening tool. They were excluded from the study if they had diabetes mellitus or a family history of diabetes mellitus, hypertension, cardiovascular disease, or hypertriglyceridemia. Furthermore, they all had a normal glucose tolerance (2-h postload plasma glucose <7.8 mmol/L) and normal 12-lead electrocardiogram. Two participants were smokers, and two used medication (levothyroxine or a selective serotonin reuptake inhibitor). Subjects were asked to avoid nonsteroidal anti-inflammatory drugs for 5 days before study onset until its termination. After an overnight fast, experiments were conducted in a quiet investigation room of the ICU with complete resuscitation facilities. Ambient temperature was systematically measured and ranged from 23.5°C to 26°C (mean, 25°C).
The study was approved by the ethics committee of the University of Lausanne and conformed with the principles outlined in the Declaration of Helsinki. All participants gave written informed consent.
Generation of acute systemic inflammation
A bolus of E. coli endotoxin (LPS, 2 ng/kg body weight; USP, Rockville, Md) was injected intravenously to induce acute inflammation. Administration of LPS is an established model to study cardiovascular and metabolic responses to acute systemic inflammation (5, 19) and causes endothelial dysfunction that is most marked 4 h after LPS bolus administration (19). Flulike symptoms such as fever, headache, nausea, muscle pain, and rarely vomiting or diarrhea are mild and disappear spontaneously after 6 to 8 h. Injection of normal saline solution served as placebo.
Measurement of SkBF
We used laser Doppler imager (Moor Instruments; Axminster, UK), as described previously (20). This device allows the measurement of dermal microvascular blood flow in a region of interest with no skin contact.
Briefly, a computer-controlled moving mirror directs a beam of laser light (633 nm) onto a square skin region, the size of which may be adjusted by the operator. Photons of the laser light scattered by moving red blood cells undergo a minor frequency shift proportional to average erythrocyte velocity. The computer then analyzes the backscattered Doppler-shifted light and calculates the microvascular blood flow in each of up to 256 × 256 adjacent spots (pixels). Finally, total SkBF, expressed in an arbitrary perfusion unit (PU) according to the principle of laser Doppler flowmetry, is calculated by averaging the pixel values in a chosen region of interest within the scanned area.
Assessment of skin microvascular endothelial function
Local thermal hyperemia of the skin was used to investigate flow-related endothelial function (14). Local heating of the skin leads to a temperature-dependent sustained increase in SkBF with maximal vasodilation achieved by skin temperatures above 42°C (14). Local thermal hyperemia induced by heating to submaximal temperatures is characterized by a biphasic rise in SkBF with an initial peak mediated by both local neurogenic reflexes and locally released substances, followed by a brief nadir and then a secondary dilation to a sustained plateau that occurs approximately 30 min after the onset of heating (14). This plateau has been shown to be primarily mediated by endothelial generation of NO because pharmacological blocking of endothelial but not neuronal NOS largely attenuates this plateau response (16, 17). Two stainless-steel, temperature-controlled, ring-shaped chambers, with inner diameter, outer diameter, and thickness of 8, 25, and 8 mm, respectively, were affixed to the skin with double-sided tape, filled to the rim with deionized water and overlaid with a transparent glass coverslip (20). The skin underneath the coverslip and deionized water was thus accessible to laser Doppler imaging. The device was programmed to repetitively scan the area comprising the chamber every 60 s, each scan being accomplished in 50 s. Each chamber was connected to an analog temperature controller with adjustable set point. Temperature was set at 34°C until stable SkBF was obtained. After 2 min of baseline SkBF measurement, temperature was raised to 39°C in one and 41°C in the second chamber in 60 s and maintained at this level for 33 min.
The increase in SkBF above baseline level after the release of a short arterial occlusion is referred to as post-occlusive skin reactive hyperemia (PORH) (14). The exact mechanism of this reaction is not known, but at least four different factors are thought to be involved (14): metabolic vasodilators, endothelial vasodilators, the myogenic response, and skin nerves. Because of this multiplicity, PORH can be considered to represent a global microvascular response to an acute period of ischemia (14). In particular, and unlike thermal hyperemia, PORH is not dependent on NO (21). We assessed the PORH response after an arterial occlusion achieved by a pressure cuff placed on the arm and inflated at a suprasystolic pressure (200 mmHg) for 3 min. This procedure was repeated three times, with a recovery period of 7 min between each occlusion.
Blood samples were collected into heparinized tubes and centrifuged (400g, 10 min, 4°C) to separate plasma, which was stored at −20°C or −80°C until analysis. Plasma TNF-α was determined by photometric enzyme-linked immunosorbent assay in streptavidin-coated microtiter plates (Roche, Mannheim, Germany); IL-6 by enzyme immunoassay using an enzyme-linked polyclonal antibody specific for IL-6 (Quantikine; R&D Systems, Wiesbaden, Germany).
Asymmetric dimethylarginine determination in plasma was performed by electrospray tandem mass spectrometry, and l-arginine levels in plasma were measured with high-pressure liquid chromatography as previously described (15).
The investigations took place on two different study days (sessions) at least 2 weeks apart in a randomized, double-blind, crossover design. Subjects arrived at 8:00 am in our research facility and were randomly allocated to receive either LPS or placebo on the first session, and the other treatment on the second. Except for drug injection, the protocol was identical on the two study days.
Subjects were examined in the supine position, with the right arm supported by a vacuum cushion. A venous cannula was inserted into an antecubital vein of the left arm. On the first session, a site on the proximal anterior face of the right forearm was chosen for the local thermal hyperemia measurements and another site for PORH assessment. The exact location of these sites was marked on a transparent acetate film, together with the anatomic outline of the forearm, to use exactly the same sites for all SkBF measurements performed (20).
One hour before LPS/placebo injection, the preinjection thermal hyperemia response was recorded. Then at t0, LPS or placebo was administered as described above. After an additional 240 min (4 h after injection), a second thermal hyperemia response was measured.
Blood samples were taken 1 h before and then every hour until 5 h after the injection of LPS/placebo for the determination of inflammatory markers (TNF-α, IL-6), whereas l-arginine and ADMA levels were determined just before and 4 h after LPS/placebo injection.
The following vital functions were recorded: heart rate, respiratory rate, rectal temperature (Servomed, Hellige, Freiburg, Germany), cardiac output by the method of thoracic bioimpedence (NCCOM3 cardiodynamic monitor; BoMed, Irvine, Calif), and noninvasive arterial blood pressure (Dinamap, Critikon, Tampa, Fla). Skin temperature was also systematically measured using a cutaneous probe (G.Métraux, Crissier, Switzerland).
Data are presented as means ± SD. The local thermal hyperemia response was quantified as baseline (average SkBF of the first two measurements before local heating) and plateau SkBF (average SkBF of the last 5 min). To summarize the PORH response, the peak postischemic increase in SkBF was expressed as the maximal SkBF minus baseline SkBF, and the area under the curve above baseline was calculated.
Considering the relatively small number of studied volunteers, data were analyzed with the Friedman test, a nonparametric equivalent to repeated-measures analysis of variance. The null hypothesis of no difference between the four experimental times (preinjection and 4 h after injection in the course of the placebo and the LPS sessions) was tested with the T2 statistic (22). When rejected, further pairwise comparisons were carried out as described (22). The α level of all tests was set at 0.05.
LPS injection caused the expected mild and transient flulike symptoms. There was no significant change in blood pressure after injection of LPS or placebo (Table 1). However, heart rate (+43%, P < 0.001), cardiac output (+16%, P < 0.01), and rectal temperature (+1.4°C, P < 0.001) significantly increased after LPS injection. Skin temperature significantly increased from preinjection to 4 h after injection, on both sessions by a similar amount.
After LPS administration, TNF-α levels markedly increased with a peak after 2 h (P < 0.01), whereas IL-6 levels peaked between 2 and 3 h (P < 0.01). Both TNF-α and IL-6 levels were still significantly increased at 4 h after injection (Table 2). The plasma concentrations of ADMA was significantly lower after LPS injection (P < 0.01 for both, Table 2) but not after placebo administration. The plasma concentration of l-arginine was lower after LPS administration, but this decrease did not reach the level of significance (P = 0.41). Of note, the relative magnitude of these changes was rather small, amounting to approximately −10% for ADMA and −15% for l-arginine. The concentration ratio of these two did not change in any statistically detectable fashion.
Microvascular vasodilatory responses
Cutaneous thermal hyperemia responses induced by local heating to 39°C and to 41°C are shown in Figure 1 and summarized in Table 2. There was no significant difference in baseline SkBF between the various sessions at which local thermal hyperemia was performed. The NO-dependent plateau response 4 h after LPS injection was significantly decreased in all subjects with both heating protocols (P < 0.01 and P < 0.05 for heating temperatures of, respectively, 39°C and 41°C; Fig. 2), whereas the placebo administration had no influence on the plateau responses.
The different parameters derived from PORH were not significantly modified by either LPS or placebo injection as illustrated in Figure 3 and listed in Table 2.
There was no correlation between the changes observed in the plateau response at 39°C or 41°C and the corresponding changes in plasma ADMA, l-arginine, or the l-arginine/ADMA ratio. However, there was a significant negative correlation between the changes in these vascular responses to local heating and the corresponding changes in plasma TNF-α levels (39°C, r2 = 0.74, P = 0.02; 41°C, r2 = 0.81, P = 0.01), whereas there was no such correlation for IL-6 levels.
Microcirculatory dysfunction has attracted much interest in recent years as one of the most important components in the pathophysiology of sepsis (1). In the present study, we show for the first time in humans that acute systemic inflammation induced by experimental endotoxemia causes a reduction of NO-dependent vasodilation in the cutaneous microcirculation (Fig. 1), whereas global reactivity of these vessels remains preserved (Fig. 3). This is an important finding because regionally decreased NO bioavailability in the microcirculation despite a state of total body NO excess might be responsible for the heterogeneous tissue perfusion that characterizes sepsis in both experimental and clinical settings (1).
There is growing evidence that acute systemic inflammation is associated with a transient increase in the risk of cardiovascular events (23), and endothelial dysfunction might be the explaining mechanism of this association (24). Several studies have documented in humans that acute infection (25) or administration of proinflammatory factors (6) may induce transient endothelial dysfunction in conduit (25) or resistance arteries (6). In a very recent study, Draisma et al. (6) showed that experimental endotoxemia in healthy volunteers also results in endothelial and microvascular dysfunction in the thenar muscular and sublingual microvascular beds. The new information added by our study is that LPS administration causes specifically a decrease in the endothelial NO-dependent microcirculatory vasodilation [the plateau response of thermal hyperemia (17); Fig. 1], whereas the general reactivity of the microcirculation tested in the form of PORH (14) (Fig. 3) remains preserved. Furthermore, we found a significant negative correlation between TNF-α but not IL-6 levels and the NO-dependent vasodilation. This is in line with the previous observations of a reduced vasodilatory response to bradykinin in human forearm veins and arteries after instillation of TNF-α or IL-1β, but not of IL-6 (26).
In 1999, Ince and Sinaasappel (27) introduced the concept of microvascular shunting and microcirculatory weak units, the latter being the first to become dysoxic in stress situations such as sepsis. In an experimental study with isolated perfused rat hearts, these authors showed that nonspecific inhibition of NOS led to the appearance of microcirculatory weak units only in endotoxemic but not control rat hearts, leading to the suggestion that endotoxemia promotes myocardial ischemia in vulnerable areas of the heart after inhibition of the NO pathway (28). This leads to the hypothesis that microvascular NO deficiency, as supported in humans for the first time in the present study, might be responsible for the heterogeneity of tissue perfusion that is characteristic for experimental and human sepsis (1). In accordance with this hypothesis, nitroglycerin infusion in patients with septic shock improved sublingual microvascular blood flow (29), although this finding was not replicated (4).
Several nonmutually exclusive mechanisms can be responsible for the attenuation of NO-dependent microvascular vasodilation observed in our study: first, the microvascular production of NO could be reduced. Second, the responsiveness of vascular smooth muscle cells to the action of NO might be blunted, and third, NO could be inactivated or destroyed by oxidative stress (6). In the present study, we addressed the first mechanism.
A reduced microvascular production of NO can be related to either an abnormally low abundance of endothelial NOS (eNOS), a decreased availability of its substrate, or the effects of inhibitors. In rodents, acute endotoxemia is well known to downregulate the expression of eNOS in blood vessels (2), but whether the same occurs in humans remains unsettled, unresolved by our data, and worth of further investigations. l-Arginine is a substrate of NOS, and sepsis is considered an arginine-deficient state (30). In another study of healthy human volunteers (19), acute endotoxemia was associated with a fall in the plasma concentration of l-arginine, and a similar, although statistically nonsignificant, trend exists in our data (Table 2). One possible mechanism for this effect of LPS might be increased catabolism through the arginase pathway (30). We must note that the changes in plasma l-arginine observed in either report were modest, suggesting that reduced substrate availability is not a cause of low NO production in these conditions. There are essentially two types of endogenous NOS inhibitors, namely, NO itself and methylated derivatives of l-arginine such as ADMA. The inhibition of eNOS by NO overproduced because of the induction of iNOS has been reported in rodents in acute rodent endotoxemia (31), but whether NO overproduction may follow exposure to a bolus of LPS in humans is controversial (32, 33). With these considerations in mind, we chose to test whether the microvascular endothelial dysfunction caused by LPS in the present study could be related to an accumulation of ADMA.
There is a continuous low-level production of ADMA from the proteolysis of methylated arginine residues on various proteins (7), and this production can be increased in situations of accelerated protein breakdown such as sepsis (13). However, the major determinant of ADMA concentrations is the activity of DDAH, the enzyme responsible for degrading this metabolite (7). Leiper and coworkers (9) recently showed that in DDAH-deficient mice, ADMA may reach levels sufficient to inhibit NOS function, and this, in turn, leads to endothelial dysfunction. In septic shock patients, serum ADMA levels have been found to be increased (10), and high ADMA levels were independently associated with disease severity (10) and ICU outcome (12). In the healthy subjects of the present study, however, acute exposure to LPS was followed after 4 h by a small, statistically significant decrease of ADMA plasma levels (Table 2). This result is essentially in agreement with data obtained by Mittermayer et al. (19) in conditions similar to ours. In that study, as in the present one, a bolus of LPS administered to healthy humans caused after a few hours a modest fall in the circulating concentrations of l-arginine and ADMA. That such changes reached statistical significance for the first but not the second parameter in the study by Mittermayer et al., in exact opposition to the pattern of P values displayed in Table 2, most likely reflects the vagaries of experiments conducted with a small number of subjects (Mittermayer et al.: n = 8, present study: n = 7). The apparent discrepancy between the high ADMA levels measured in septic shock patients and the low levels in experimental endotoxemia might be due to a certain time dependency. It is indeed conceivable that, in early sepsis, DDAH activity is induced with increased degradation of ADMA to boost the host defense response by increased NO production, whereas in advanced sepsis, DDAH inhibition causes accumulation of ADMA as a negative feedback on NO overproduction by the upregulated iNOS. In accordance with this hypothesis, Zoccali et al. (34) observed normal ADMA levels measured at the clinical outset of acute inflammation in 17 patients with acute bacterial infection, whereas the resolution of the inflammatory response was characterized by an increase in plasma concentration of ADMA. Furthermore, O'Dwyer et al. (10) also reported higher ADMA levels on day 7 than during the first 24 h after ICU admission in patients with severe sepsis.
To summarize, ADMA accumulation did not seem to underlie the microvascular endothelial dysfunction induced by LPS in the present study. Consistent with this conclusion, there was no correlation between the changes observed in the NO-dependent vasodilation and the corresponding changes in plasma ADMA, l-arginine, or the l-arginine/ADMA ratio.
Intraorgan and interorgan perfusion heterogeneity is a classic feature in sepsis. The spatial resolution of laser Doppler imaging is insufficient to make a precise statement on the heterogeneity of SkBF. Furthermore, laser Doppler specifically measures red blood cell flow, which depends not only on vessel diameter but also on perfusion pressure, vessel length, and blood viscosity. Therefore, the reduced plateau SkBF during the local heating protocol observed 4 h after LPS administration (Figs. 1 and 2) might be due to a change in of one of the latter parameters. Arguing strongly against this possibility is the fact that LPS did not modify other responses of SkBF, i.e., the initial peak of local thermal (Fig. 1) hyperemia and PORH (Fig. 3). Also, baseline SkBF did not significantly change during the different measurement sessions, despite a significant increase in cardiac output measured 4 h after LPS injection (Table 1), suggesting that hemodynamic conditions remained stable in the cutaneous microcirculation despite important systemic hemodynamic modifications (15).
Another limitation is that all subjects were participating in another study on the physiological response of obese subjects to E. coli LPS, hence the lack of a control group of lean individuals. Animal experiments have shown that obesity may enhance the microvascular impact of sepsis (35), but no corresponding data are available in humans. Whether LPS infusion would blunt NO-dependent skin vasodilation to a lesser degree in the lean than in the obese remains to be determined and is certainly worth another study.
Although experimental endotoxemia is presently the best human model for sepsis, one has to keep in mind that it still has important drawbacks. Acute intravenous administration of LPS to healthy volunteers mainly permits to study the early highly dynamic inflammatory reaction, which differs substantially from the more prolonged and more severe inflammatory response in septic shock patients (5). Therefore, the extrapolation from experimental findings to the clinical setting has to be done with caution.
In summary, we show for the first time that experimental endotoxemia causes a specific decrease in endothelial NO-dependent vasodilation in the human cutaneous microcirculation, whereas global microvascular reactivity remains preserved. Furthermore, the ADMA-DDAH pathway seems not to be responsible for the reduced NO-dependent vasodilation in this setting. Deficient NO-dependent vasodilation in the microcirculation might be one of the reasons for the heterogeneity of intratissue and intertissue perfusion typically observed in sepsis and could represent a target for future therapeutic strategies.
The authors thank Marie-Christine Cayeux for her excellent technical assistance.
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