In critical illness, severe microcirculatory disturbances causing tissue hypoxia and transmigration of activated leukocytes into the tissue compromise the vascular endothelium (1, 2). Many studies investigating critical illness and sepsis were able to show that perfusion at the microvascular level becomes very heterogenous within organ systems despite successful restoration of the macrohemodynamics. This maldistribution of blood flow contributes to abnormal tissue oxygen delivery and extraction, leading to tissue injury and, eventually, organ dysfunction (3-5). Endothelial cells play a critical role in integrating local stimulatory signals or acting as signal transducers of local shear stress (6, 7). Under these conditions, only few interventions with clinical effectiveness have been proposed to date, which may preserve the vascular endothelium in its functional integrity. One promising option may be the application of activated protein C in sepsis, which has been shown to reduce the inflammatory alteration in microcirculatory function (8, 9). However, this has been proven only for selected severe septic patients with a high risk of death, whereas the anticoagulant nature of this substance is associated with an increased risk of bleeding (10).
Erythropoietin (EPO) has gained widespread preclinical acceptance as a tissue-protective cytokine in critically endangered organs. Physiologically, EPO is secreted from the kidney in response to hypobaric hypoxia and primarily acts on burst- and colony-forming unit erythroid progenitor cells, causing the maturation to erythrocytes by inhibiting their apoptotic cell death.
The question arises, whether EPO can improve microvascular perfusion and oxygenation in hypoxic tissue with impaired bioavailability of NO. An emerging number of studies have demonstrated protective nonhematopoietic effects of exogenously administered EPO on the vascular endothelium. These effects have primarily been achieved by enhancing angiogenesis and preventing apoptosis (11-13).
A study in rats showed a tissue-protective effect of EPO in I/R of the heart and hemorrhagic shock (14). After mechanical vascular injury, EPO has been demonstrated to exert an NO-dependent repair on injured vascular endothelium in vivo (15, 16). A predominant role of endothelial NO synthase (eNOS) in EPO-mediated endothelial protection has been suggested in vitro, demonstrating a greater sensitivity to produce vasoregulating NO under hypoxic conditions (17). The importance of such fine tuning of eNOS is underlined by recent findings, demonstrating that an increased hematocrit after EPO treatment does not increase the risk for thrombus formation as long as endothelial NO production serves as compensatory mechanism (18).
Little is known about the anti-inflammatory effect of EPO. In an experimental model of colitis, EPO treatment attenuated intercellular adhesion molecule 1-mediated neutrophil infiltration (19). In addition, it has been demonstrated that EPO reduces polymorphonuclear cell migration in critically ill animals in a nonseptic zymosan-induced shock model (20). Most recently, EPO has been shown to attenuate leukocyte recruitment in LPS-induced acute liver failure (21). To date, there is no study exploring the in vivo effect of EPO on the microcirculation in hypoxic tissues. The present study investigates the efficacy of EPO to increase oxygenation in hypoxic, collateralized ischemic tissue and to elucidate the involvement of NO.
MATERIALS AND METHODS
Animals and drugs
Twenty-one male Syrian golden hamsters (Harlan, Germany), weighing 75 to 105 g and 8 to 12 weeks old, were included in this study. The animals received humane care according to the guidelines of the University Hospital of Zurich. The study protocol was approved by the Federal Veterinary Office of Zurich. The animals were randomly assigned and equally distributed to the control group (n = 7) and two treatment groups receiving epoetin alfa (Recormon) 5,000 U/kg of body weight (BW), with (n = 7) or without (n = 7) blocking NO synthase by l-NAME (Nω-nitro-l-arginine methyl ester hydrochloride; Sigma-Aldrich, Buchs, Switzerland). l-NAME was dissolved in saline 0.9% and infused intravenously at a concentration of 30 mg/kg BW (11 mmol/L). The volume for i.v. infusion of l-NAME solution in saline was approximately 10% of the total blood volume, which was estimated to be ∼7% of the BW. The other groups received the same amount of saline 0.9%.
Flap preparation and animal monitoring
A hamster skin flap model was used as previously described in detail (22). Anesthesia was induced by i.p. injection of 100 mg/kg BW pentobarbital sodium (Nembutal; Abbott Laboratories, Chicago, Ill). The left carotid artery and external jugular vein were cannulated for administration of anesthesia, infusion of the substances, blood sampling for laboratory analysis, and continuous monitoring of mean arterial blood pressure (MAP) and heart rate. An island flap measuring 4×3 cm was dissected from the shaved and epilated back skin of the animal. The flap consisted of skin and a thin layer of panniculus carnosus muscle and was perfused by one vascular axis that bifurcated into two equal-size branches within the flap, each of them supplying a separate vascular territory. One of the branches was transected after being secured with microsurgical ligatures, thus rendering the corresponding vascular territory ischemic. This tissue was perfused by a collateral vasculature connecting the two vascular networks.
A heating pad was used to keep the animal's skin temperature constant at ∼32°C, which was verified with a microthermometer placed on the abdominal skin. Blood samples were taken from the carotid artery catheter and collected in heparin-washed microtubes (Clinitubes D957G-70; Radiometer, Copenhagen, Denmark) for immediate measurements of total hemoglobin concentration, hematocrit, pH, and systemic arterial oxygen tension (PO2) and arterial carbon dioxide tension (PCO2) (ABL 715; Radiometer).
Partial tissue oxygen tension and microhemodynamics
Tissue oxygenation was monitored with Clarktype microprobes (7 mm2, Revoxode CC1; GMS, Kiel, Germany). The probes were inserted into the subcutaneous tissue in the center of each vascular territory under visual control and microscopic magnification (22).
Microhemodynamic measurements were performed using an epi-illumination intravital microscope (Leica DM/LM; Leica Microsystems, Wetzlar, Germany) attached to a blue (450 - 490/>520 nm), a green (530 - 560/>580 nm), and an ultraviolet (330 - 390/>430 nm excitation/emission wavelength) filter system. Microscopic images were captured by a CCD television camera (charge-coupled device camera; Kappa Messtechnik, Gleichen, Germany), displayed on a television screen (Trinitron PVM-20N5E; Sony, Pencoed, UK), and recorded on video (50 Hz, Panasonic AG-7350-SVHS; Panasonic, Tokyo, Japan) for subsequent off-line analysis. The preparation was observed visually with a water-immersion objective ×20 with a numerical aperture of 0.50, which resulted in total optical magnification of ×800 on the video monitor. Animals received a tail vein injection of 0.05 mL fluorescein isothiocyanate-dextran (50 mg/mL saline; Sigma) for vascular contrast enhancement and 0.05 mL rhodamine 6G (0.1 mg/mL saline; Sigma) for leukocyte staining in vivo. The microvessels were classified according to physiological and anatomical features into conduit and end arterioles, capillaries, and small collecting venules (23).
Before vessel ligation, both parts of the flap were scanned for random selection of distinct observation areas, which included four to six conduit and end arterioles, six to nine nutritive capillary fields, and four to six draining postcapillary venules. The total number of vessels studied in each animal group is given in Table 2 as sample size. Video printouts were made during videography and initially marked to indicate the exact localization for repetitive measurements of vessel diameter and red blood cell velocity.
The recorded video sequences were analyzed using a computer-assisted image analysis system (CapImage; Zeintl Software, Heidelberg, Germany). Functional capillary density (FCD) was defined as the length of red blood cell-perfused capillaries per observation area (cm/cm2). Vessel diameters were measured in micrometers perpendicularly to the vessel path, and centerline red blood cell velocity was analyzed using the line-shift method. Volumetric blood flow was calculated from diameter (d) and red blood cell velocity (v) by Q = π (d/2)2 × v/1.6 [picoliter/second (pl/s)], where 1.6 represents the Baker-Wayland factor to correct for the parabolic velocity profile in microvessels with diameters of more than 20 μm (24). The number of permanent adherent leukocytes defined as cells that remained stationary for at least 30 s to the microvascular endothelium was evaluated at ×800 magnification as number of cells per square millimeter of venular endothelial cell surface (calculated from diameter and length of the vessel segment studied, assuming cylindrical geometry). Rolling leukocytes were defined as cells moving with a velocity of less than two fifths of the centerline velocity and are given as number of cells per minute passing a reference point within the microvessel (25). Transmigrated leukocytes were defined as those cells in the perivenular tissue within a distance of 50 μm above or below the 100-μm venular segment under study (26).
Immunohistochemistry and TUNEL analysis
Tissue samples were obtained from the middle of each vascular territory at the end of the experiment. They were fixed in 4% paraformaldehyde, washed in phosphate-buffered saline, stored in 70% ethanol, and finally embedded in paraffin blocks. For analysis of endothelial expression of eNOS, 4-µm sections were incubated with a primary rabbit anti-eNOS antibody (1:30, KAP-NO002; Stressgen, Ann Arbor, Mich). As secondary antibody, the antirabbit EnVision was used (EnVision+ System Labelled Polymer-HRP Anti-Rabbit K4003; DakoCytomation, Zug, Switzerland), and as chromogen, AEC (Aminoethyl Carbazole Substrate Kit; Zymed Laboratories, San Francisco, Calif) was applied.
The intensity of the staining reactions in endothelial cells was evaluated by a Zeiss Axioplan 2 imaging system (Carl Zeiss, Oberkochen, Germany), using a semiquantitative score (graded as 0 = no, 1 = weak, 2 = moderate, and 3 = strong staining).
Apoptotic cell death was assessed within the ischemic tissues with the transferase-mediated dUTP nick end labeling (TUNEL) assay (in situ cell death detection kit, tetramethylrhodamine red; Roche Diagnostics, Rotkreuz, Switzerland) according to the supplier's instructions. The labeled DNA fragments were visualized by incubating the sections with tetramethylrhodamine used as a fluorescence marker, and the sections were examined by the Zeiss Axioplan 2 imaging system (Carl Zeiss). Data are given as the averages of fluorescent cells counted in five randomly selected visual fields (0.5 × 0.5 mm). Sebaceous glands and hair follicles were identified and excluded from the cell counts because of their consistently high apoptosis rate.
The animals were kept under light anesthesia with a continuous infusion of 50 mg/mL pentobarbital given at a rate of approximately 0.5 mg · min−1 · kg−1 BW throughout the experiment. The depth of anesthesia was regulated by tolerating a noxious reflex due to pinching of the hind paw, but no nonaversive reflexes (palpebral, corneal, and jaw reflexes) (22).
The timing of the interventions is illustrated in Figure 1. Baseline measurements were taken after a postoperative stabilization period of 30 min had elapsed. Thereafter, animals received either an infusion of 30 mg/kg BW l-NAME or saline. After another 30 min, the treatment groups received 5,000 IU/kg BW Recormon intraperitoneally. After another 60 min, ligation of one bifurcated artery was performed. Microhemodynamic measurements were repeated after 1 and 5 h of collateral flap ischemia. Exclusion criteria were abnormalities of the vascular anatomy, insufficient optical clarity, MAP of less than 60 mmHg, and systemic arterial pH, PO2, and PCO2 levels that are outside the reference ranges at baseline (7.25 - 7.35, 35 - 50 mmHg, and 45 - 65 mmHg, respectively) (22). The animals were killed with an overdose of pentobarbital at the end of the experiment after having harvested the tissue samples.
All values are expressed as means ± SD. For comparison between individual time points, ANOVA for repeated measures was performed, which was followed by the appropriate post hoc test, including the correction of the α error according to Bonferroni probabilities. Comparison between the groups was done by ANOVA for comparison of multiple groups, followed by Student-Newman-Keuls test for appropriate post hoc analysis (SigmaStat; Jandel, San Rafael, Calif). P < 0.05 was taken to indicate statistically significant differences.
A total of four animals, that is, two control animals (one showing suboptimal optical clarity, the other abnormal vascular anatomy), one of the EPO-treated groups (showing abnormal vascular anatomy), and one of the EPO/l-NAME groups (showing severe hypotension), did not fulfill the inclusion criteria and were excluded from the study. The systemic data are presented in Table 1. Infusion of l-NAME caused an immediate hypertension when compared with the animals of the other groups (P < 0.01). Hypertension diminished after 5 h of ischemia. MAP remained virtually unchanged over time in the other groups. The control animals showed a slight but significant increase of arterial PO2 compared with baseline (P < 0.05), whereas arterial PCO2 did not significantly decrease. Accordingly, pH was found slightly although not significantly elevated after 5-h ischemia when compared with baseline. Hemoglobin concentration and hematocrit remained stable in all groups over time. At baseline, the microhemodynamic data did not differ between the three groups studied (Table 2).
In the anatomical part of the flap, we found a distinct reduction of FCD, minimally increasing diameters, and a slight reduction of microvascular blood flow at 5 h after induction of ischemia (Fig. 2). In the ischemic part of the flap, however, we observed a reduction of 48% of FCD in control animals after 5 h of ischemia (P<0.05 vs. baseline) (Fig. 2). Erythropoietin pretreatment was capable of partly preventing this impairment of nutritive perfusion by improving FCD by 21% (P < 0.05 vs. baseline and other groups; Fig. 2). Of interest, the blockade of the NO synthase by l-NAME completely abolished the protective effect of EPO pretreatment. This was indicated by FCD values comparable to those measured in control animals (Fig. 2). Capillary diameters substantially dilated by 43% and revealed a marked reduction of microvascular blood flow by 65% of baseline after 5 h of ischemia (both, P < 0.05 vs. baseline). This time-related deterioration of capillary hemodynamics was significantly attenuated by EPO pretreatment, as shown by capillaries that dilated only by 24% and a microvascular blood flow that was decreased by only 29% (both, P < 0.05 vs. baseline and vs. control). The infusion of l-NAME completely abolished the beneficial effects of EPO on the capillary microhemodynamics (Fig. 2).
Diameters of arterioles and venules remained virtually unchanged in all groups in both parts of the flaps throughout the experiment (Table 2). Five hours of ischemia reduced conduit arteriolar flow to 28% and venular flow to 12% of baseline (Fig. 3). Erythropoietin pretreatment was capable of substantially maintaining arteriolar flow at 61% and venular flow at 48% of baseline (Fig. 3). The administration of l-NAME completely abolished the beneficial effects of EPO on the arteriolar and venular blood flow (Fig. 3).
Partial tissue oxygen tension (tPO2) was significantly reduced in the ischemic tissue compared with the anatomically perfused part (P < 0.01; Fig. 4). Erythropoietin pretreatment improved PTO2 from ∼9 to 15 mmHg in the ischemic tissue (P < 0.01 vs. control). Of interest, PTO2 was not improved after EPO pretreatment when animals received l-NAME instead of saline (Fig. 4).
Analysis of leukocyte-endothelial cell interactions at 5 h after ischemia demonstrated a ∼30-fold increase of the number of transmigrated leukocytes compared with baseline (P < 0.01), whereas both leukocyte rolling and firm adherence were not substantially increased at this time point, indicating at 5 h of ischemia the late phase of the inflammatory response (Fig. 5). Erythropoietin pretreatment substantially attenuated the inflammatory response, which was characterized by a shift toward an increased rolling and firmly adherent fraction of leukocytes (P < 0.01 vs. control), but a ∼4-fold decreased number of transmigrating leukocytes (P < 0.01 vs. control) (Fig. 5). Notably, infusion of l-NAME completely blunted the anti-inflammatory action of EPO (Fig. 5).
Apoptotic cell counts were analyzed in the ischemic part of the flap 5 h after induction of ischemia. In the control group, a massive accumulation of TUNEL-positive nuclei was observed in the ischemic part of the flap (Fig. 6). Erythropoietin was effective to significantly reduce the apoptotic cell counts by ∼50% (P < 0.01 vs. control). Of interest, l-NAME almost completely abolished this protective antiapoptotic effect of EPO (P < 0.01 vs. and vs. EPO) (Fig. 6).
The density of endothelial eNOS expression in the anatomically perfused and ischemic tissues was analyzed from immunohistochemically stained tissue sections by a semiquantitative score. Expression of eNOS could be detected in the endoluminal aspects of endothelial cells in the anatomically perfused tissues, representing physiological conditions. A slightly increased expression of eNOS was observed in the ischemic tissues of saline-treated control animals. In contrast, after EPO pretreatment, the whole cytoplasm showed substantial eNOS expression (P < 0.05 vs. control and vs. ischemic), which was not affected by the infusion of l-NAME (Fig. 7).
The principal findings of this study are that tissue hypoxia, microcirculatory damage, apoptosis, and inflammation as a consequence of leukocyte transmigration from postcapillary venules into the interstitial space are significantly reduced in ischemic, collateralized flap tissue by pretreatment with a single dose of 5,000 U/kg EPO. Furthermore, we demonstrate that these beneficial effects are most likely related to enhanced eNOS expression with restorative effects on endothelial function through an upregulation of NO production.
Hypoxemia is common in critically ill patients. Applying orthogonal polarization spectral imaging techniques, various investigators have observed microcirculatory alterations in these patients, especially under septicemic conditions, potentially leading to organ failure (27). These alterations include a decrease in oxygenating capillary density and an increased proportion of nonperfused or intermittently perfused capillaries (28). In addition, the combination with arteriolar-venular shunting eventually leads to a reduction of the number of perfused capillaries, resulting in ischemia and hypoxia. Although a mitochondrial malfunction has been proposed in the past, it is nowadays generally accepted that the main reason for tissue hypoxia in the critically ill patient is an impaired microvascular perfusion (29). Moreover, additional phenomena occurring in critically ill patients, such as inflammation, coagulation activation, edema formation, and rheologic dysfunction may further contribute to the alteration of the microvasculature (30). In line with this, our model displayed a significant reduction of nutritive perfusion, a markedly diminished arteriolar and venular blood flow, and a compromised PTO2 within the collateralized flap part. Thus, it represents ideal in vivo conditions to mimic critically ischemic tissue. Collateralized ischemia ultimately terminates in inflammation, entailing the generation of chemokines and the upregulation of endothelial cell adhesion molecules eventually leading to leukocyte-endothelial adherence and migration through the vessel wall (31). These inflammatory conditions significantly impair the nutritive microcirculation, and subsequent microvascular patency has been shown to be markedly improved by neutrophil depletion in the setting of an ischemic insult (32). There seem to be several assumed mechanisms for the occlusion of capillaries early in sepsis: endothelial cell swelling, fibrin clots as a consequence of intravascular coagulation, and maybe even stiff and sticking leukocytes and erythrocytes (30). Next to this, a reduced erythrocyte deformability has been detected in a rat sepsis model at 37°C very early in sepsis, making this another potential cause of capillary perfusion failure (33). Finally, disseminated intravascular coagulation certainly may play a role in microvascular plugging because treatment with activated protein C, which targets coagulation and inflammation pathways, has been shown to be effective (34, 35). In addition, vascular permeability and transcapillary fluid filtration and therefore interstitial tissue pressure are increased, causing capillary compression and obstruction (36).
One of the major findings of the presented study is that EPO pretreatment significantly reduced all these detrimental consequences of severe collateralized ischemia by inhibition of leukocyte transmigration into the perivascular tissue and shifting leukocyte distribution within the postcapillary venules toward the rolling and sticking fraction. The reason for this seems to be a stabilizing effect on the endothelium, resulting in an improved generation of protective NO. The island flap model applied in this study involves a severe ischemic damage to the musculocutaneous tissue with the consequence of massive accumulation and transmigration of activated leukocytes within less than an hour (37). It is known from the literature that the entire process from rolling to transmigration may take less than half a minute in vivo, making this a reasonable explanation for the observed rapid extravasation of leukocytes (38).
It has become apparent over the past 5 years that EPO has beneficial effects that reach well beyond the stimulation of red blood cell production in anemic patients (39). Next to neuroprotective properties, it has been found to protect the vascular endothelium against ischemic injury (11). Recent studies were able to show that both EPO and the EPO receptor are expressed in a number of tissues including vascular endothelium and smooth muscle, which emphasizes that modulation of endothelial cell function might be a potential effect of EPO (40). The fact that leukocyte transmigration and subsequent microvascular perfusion failure could be repressed suggests potent NO-related anti-inflammatory properties of EPO. NO interferes with vascular smooth muscle cells, and reduces platelet and endothelial activation as well as neutrophil aggregation and adherence to postcapillary venules (41). Due to this NO-related vasoregulation, increased arteriolar and venular blood flow probably also contributed to the improved tissue oxygenation in our study. Kubes et al. (42) were able to show in cat mesenteric venules that NO donors prevent neutrophil adhesion, but not P selectin-dependent rolling. Thus, the increase in rolling leukocytes does not contradict the protective effects of NO.
The preferential mechanism by which EPO maintains and increases erythropoiesis is the prevention of apoptosis (43). In in vivo models of diseases of the central nervous system, EPO exerted protective effects by antiapoptotic properties and a decrease of proinflammatory cytokines such as TNF-α, IL-6, and monocyte chemoattractant protein 1 (44). In line with this, we found a significant reduction of the number of ischemia-associated apoptotic cells in the musculocutaneous tissue, an effect which was completely blunted by blocking NO synthase with l-NAME. Indeed, it has been presumed before that NO may act antiapoptotic, because the physiological continuous low production and release of NO by eNOS were able to prevent apoptosis (45). Several studies suggested that NO inhibits apoptosis through its interaction with the caspase cysteine proteases by specific inhibition of caspase-3 activity after S-nitrosation (46). Thus, next to antiadhesive and anti-inflammatory properties, the antiapoptotic effect seen in this study may as well be a consequence of restored endothelial NO production by EPO.
Endothelial response to produce NO after EPO stimulation in vitro has been reported to be more pronounced under hypoxemic conditions (17). As immunohistochemistry revealed a significant upregulation of eNOS after EPO pretreatment in our study, this most likely accounts for the vasculoprotective properties. This is strongly underlined by the fact that inhibition of NO production by l-NAME completely blunted these positive effects on microvascular perfusion, tPO2, leukocyte transmigration, and apoptotic cell death.
Because the restoration of endothelial integrity by treatment with EPO prevented only the transmigration, but not rolling and sticking of leukocytes, it is also thinkable that other distinct vascular mechanisms may be responsible for this fact. Initial adhesive interaction between leukocytes and activated endothelium is mediated by both endothelial P and E selectins and leukocyte L selectin, whereas firm adherence involves the interaction of the leukocyte integrin CD11/18 and the endothelial counter-receptor intercellular adhesion molecule 1 (47). Blocking platelet-endothelial cell adhesion molecule with antibodies abrogated the ability of neutrophils to migrate from a vessel to the site of inflammation as shown in past studies, although they were able to roll (48). Thus, a direct inhibitory effect of EPO on platelet-endothelial cell adhesion molecule expression cannot be ruled out as a reason for our observations and should be addressed in future studies.
Erythropoietin significantly attenuated ischemia-induced leukocytic inflammation, capillary perfusion failure, tissue hypoxia, and apoptotic cell death, which is most probably due to an EPO-mediated eNOS induction. As a consequence of these new protective properties, the application of EPO in critically ill patients might be a promising strategy to restore endothelial function and to ameliorate ischemia- and hypoxia-related complications in critically ill patients.
The authors thank the group of Prof. Dr. Med. Vet. Brigitte von Rechenberg (Institute of Veterinary Pathology, University of Zurich) for assistance in the immunohistochemical analysis.
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