Basic Science Aspects
Heparin Use in a Rat Hemorrhagic Shock Model Induces Biologic Activity in Mesenteric Lymph Separate from Shock
Qin, Yong; Prescott, Lauriston M.; Deitch, Edwin A.; Kaiser, Vicki L.
Department of Surgery, UMDNJ-New Jersey Medical School, Newark, New Jersey
Received 27 Aug 2010; first review completed 16 Sep 2010; accepted in final form 14 Oct 2010
Address reprint requests to Vicki L. Kaiser, Department of Surgery, UMDNJ-New Jersey Medical School, 185 S Orange Ave, MSB G576, Newark, NJ 07103. E-mail: firstname.lastname@example.org.
This study was supported by grants from the National Institutes of Health (R01-GM059841 and 1P50GM069790).
The authors have no conflict of interest to disclose.
Experimental data have shown that mesenteric lymph from rats subjected to trauma-hemorrhagic shock (THS) but not trauma-sham shock induces neutrophil activation, cytotoxicity, decreased red blood cell (RBC) deformability, and bone marrow colony growth suppression. These data have led to the hypothesis that gut factors produced from THS enter the systemic circulation via the mesenteric lymphatics and contribute to the progression of multiple organ failure after THS. Ongoing studies designed to identify bioactive lymph agents implicated factors associated with the heparin use in the THS procedure. We investigated if heparin itself was responsible for reported toxicity to human umbilical vein endothelial cells (HUVECs). Human umbilical vein endothelial cell toxicity was not induced by lymph when alternate anticoagulants (citrate and EDTA) were used in THS. Human umbilical vein endothelial cell toxicity was induced by lymph after heparin but not saline or citrate injection into trauma-sham shock and naive animals and was dose dependent. Activities of both heparin-releasable lipases (lipoprotein and hepatic) were detected in the plasma and lymph from THS and naive animals receiving heparin but not citrate or saline. Lymph-induced HUVEC toxicity correlated with lymph lipase activities. Finally, incubation of HUVECs with purified lipoprotein lipase added to naive lymph-induced toxicity in vitro. These data show that heparin, not THS, is responsible for the reported lymph-mediated HUVEC toxicity through its release of lipases into the lymph. These findings can provide alternative explanations for several of the THS effects reported in the literature using heparin models, thus necessitating a review of previous work in this field.
Multiple organ failure (MOF) is the cause of 50% to 80% of deaths in the surgical intensive care units (1). Because the development of MOF is poorly understood, current treatment is mainly supportive (2). It is therefore critical to identify key events and factors that lead to the progression of MOF to identify new and important points of intervention.
Glenn and Lefer (3) first proposed that deleterious factors created in the hypoperfused splanchnic region due to trauma with hemorrhagic shock (THS) were transported to the systemic circulation via the lymphatics. These authors theorized that proteolytic enzymes from the ischemic pancreas created a myocardial depressant factor localized in thoracic lymph after THS based on the finding that pancreatic enzymes were detected in lymph (4). This concept has been expanded in recent years in the Deitch et al. (5) and Moore laboratories (Hassoun et al. 6) after clinical data emerged correlating gut permeability with extent of the injury in patients (7-10). It is currently hypothesized that THS end-organ damage results from factors derived from the ischemic gut that enter the mesenteric lymphatics and are introduced to the general circulation via the subclavian vein. This theory is supported by in vivo data showing that lymph duct ligation (LDL) or diversion abrogates THS-induced lung injury (11, 12), neutrophil activation (PMNs, 13, 14), increased rigidity of RBCs (15), endothelial cell dysfunction (16-18), and bone marrow colony suppression (19). Furthermore, incubation of THS but not sham-shocked (TSS) lymph with PMNs, RBCs, bone marrow, and endothelial cells in vitro could reproduce in vivo effects (12, 13, 15, 19-21). More recently, it was shown that lung injury could be recreated in animals after injection of THS but not TSS lymph (22).
Isolation studies designed to identify the bioactive components in THS lymph are ongoing, and both lipid and protein components have been implicated for neutrophil activation and cytotoxicity (8, 23, 24). Our current isolation methods have implicated lipoproteins in the induction of post-THS lymph-mediated human umbilical vein endothelial cell (HUVEC) toxicity (unpublished). This finding necessitated the investigation into the role of heparin used in the THS but not TSS procedure because it is well established that heparin promotes lipoprotein metabolism through its release of lipoprotein lipase (LPL) and hepatic lipase (HL) into the circulation (25, 26). Furthermore, LPL activity has been linked to endothelial cell damage in atherosclerosis (27) and cytotoxicity to macrophages and endothelial cells in vitro when active within hypertriglyceremic serum and triglyceride-rich lipoproteins (28, 29). We therefore tested the hypothesis that HUVEC toxicity induced by mesenteric lymph is dependent on heparin and results from heparin's release of lipases and not to THS itself.
MATERIALS AND METHODS
Pathogen-free, male Sprague-Dawley rats (Charles River, Troy, NY) were maintained under 12-h light-dark cycles in barrier-sustained conditions and allowed access to water and chow ad libitum (Teklad 22/5 rodent diet W-8640; Harlan Teklad, Madison, Wis). For experiments using fasted animals, food was withdrawn 18 h before surgery. Procedures were approved by the New Jersey Medical School Animal Care Committee, and animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals.
Male rats (average weight, 380 ± 40 g) were anesthetized with pentobarbital (50 mg/kg, i.p.) and secured to an operating board (Fisher, Pittsburgh, Pa), which was placed on a heating pad to maintain the animal's temperature. Lymph duct cannulation and collection were as previously described (30) with minor modifications: (a) the silastic tubing (internal diameter, 0.51 mm; Dow Corning, Midland, Mich) used to cannulate the mesenteric lymph duct was flushed with saline rather than heparin, was decreased in length from 50 cm to 8 cm, and was secured in place using 6-0 silk suture rather than glue; (b) lymph was collected by placing the end of the lymph catheter into a sterile 1-mL syringe (BD Biosciences, Franklin Lakes, NJ) that was secured to an ice pack (Polar Tech Industries, Inc, Genoa, Ill); (c) lymph volumes (average range, 0.2-0.45 mL) were determined using the syringe calibrations, then the lymph was transferred to a sterile 1.5-mL Eppendorf tube (VWR, Westchester, Pa), and an antiprotease cocktail (Sigma, St Louis, Mo) was added (1:100 vol/vol) to the sample, which was then mixed by inversion; and (d) cells were removed from lymph by centrifugation in a tabletop microfuge (8,000g, 2 min, 4°C), and a 12-μL aliquot was dispensed into a 0.5-mL Eppendorf tube and placed on ice for cell viability testing. In most cases, cell viability was performed on the same day as collection.
Naive lymph used for in vitro experiments was collected for 4 h then processed as above, stored at 4°C, and used within 72 h. In experiments examining the effects of LDL, the isolated lymph duct was ligated with 6-0 silk suture before abdominal closure.
In experiments using the standard fixed-pressure shock model (17), after the laparotomy to cannulate the lymphatic duct (trauma), the external jugular vein was isolated and cannulated with silastic tubing, and the femoral artery was cannulated with polyethylene tubing (PE-50; internal diameter, 0.58 mm; length, 20 cm; Becton Dickinson, Sparks, Md). Both cannulas were filled (primed) with heparinized saline (100 U/mL). The femoral catheter for THS animals was attached to a blood pressure recorder (BPA Blood Pressure Analyzer; Micro-Med, Louisville, Ky) used to continuously monitor the animals' blood pressure during the shock period. The animals were subjected to THS or TSS after a 30-min stabilization period.
Hemorrhagic shock was induced by the withdrawal of blood from the external jugular vein to a MAP of 30 mmHg into a 10-mL syringe containing heparin (heparin amounts are provided in the figure legends for specific experiments). Blood was withdrawn or reinfused during this period as needed to maintain this blood pressure for 90 min. At the end of the shock period, the animals were resuscitated by reinfusion of all shed blood using a syringe pump (1 mL/min; KD Scientific, Holliston, Mass). The sham-shock animals were subjected to the same surgical procedures, but no blood was withdrawn or reinfused, nor was pressure monitored. Throughout the procedure, the animal's temperature was maintained with the use of heating pads.
In THS procedures with alternate anticoagulants or lactated Ringer's (RL) solution as a resuscitation fluid, all lines were primed with either normal saline, sodium citrate, or EDTA diluted in saline (1:10 dilution from BD Vacutainers; BD Biosciences), and sodium citrate or EDTA replaced heparin in the syringe for blood withdrawal. For clarity, additional details are provided in the figure legends for these specific experiments.
In THS procedures without blood pressure monitoring, the femoral catheter was omitted. The jugular catheter was primed with saline. Blood was withdrawn into separate heparinized syringes in volumes and at times analogous to the standard fixed-pressure model as follows: 5 mL (1 min), 0.5 mL (10, 15, 20, 25, and 30 min), and 0.3 mL (35, 40, 50, 60, and 70 min). A total volume of 9 mL was withdrawn based on an average withdrawal of 9.9 mL calculated for 30 rats. Animals were resuscitated with RL solution as described above. No anticoagulant was given to the animals at any point.
In experiments in which the effects of heparin alone were investigated, the femoral catheter was omitted. Heparin, saline, or citrate was injected into the external jugular vein using a 1-mL syringe. The final calculated volume of heparin per weight (250, 125, 60, or 30 U/kg) was increased by 50 μL to account for the void volume of the syringe tip and catheter. For clarity, additional details are provided in the figure legends for these specific experiments.
Plasma used in lipase measurements was obtained from blood withdrawn (100 μL) at 30-min intervals from the external jugular vein into a 1-mL syringe with no anticoagulant. The plasma sample collected during THS was taken from the syringe used for blood withdrawal immediately before reinfusion of the shed blood. After each collection, samples were immediately subjected to centrifugation (8,000g, 2 min, 4°C), transferred to a sterile Eppendorf tube, then stored at −80°C.
For in vitro incubation assays, naive nonheparinized and post-THS plasmas were obtained by exsanguination from the ascending aorta into a 10-mL syringe containing 100 U of heparin. Cells were removed by centrifugation (8,000g, 10 min, 4°C), and plasma was aliquoted and stored at −40° or −80°C. Heparinized rat plasma was collected 30 min after a bolus injection of heparin (250 U/kg) in the same manner as other plasmas.
Superior mesenteric arterial occlusion
In experiments investigating the source of lymph lipases, the superior mesenteric artery was occluded (superior mesenteric arterial occlusion [SMAO]) as previously described (31) except occlusion was for 60 min. The lymph duct and external jugular vein were cannulated as described above. Heparin (250 U/kg) was injected into the external jugular vein using a 1-mL syringe immediately after occlusion. Lymph was collected for 30 min before occlusion, 60 min during occlusion-postheparin injection, and for six 30-min intervals after reperfusion of blood. Lymph was processed as described above.
Reagents and cell lines
Human umbilical vein endothelial cells, endothelial growth media, endothelial basal media, and Hanks balanced salt solution were from Lonza Walkersville (Walkersville, Md). The mitochondrial cell viability assay (MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), antiprotease cocktail, gum arabic, isopropanol, heptane, unlabeled triolein (glyceryl trioleate), NaCl, sodium dodecyl sulfate (SDS), bovine serum albumin (BSA) (fraction V), orlistat, and sodium oleate were purchased from Sigma. The LPL monoclonal antibody (5D2) was purchased from Roar Biomedical (New York, NY). Mouse isogenic control IgG was purchased from Santa Cruz Biotechnology (Santa Cruz, Calif). Heparin (1,000 U/mL) was purchased from Cardinal Health (Dublin, Ohio). Labeled tri[9,10(n)-3H]oleate and scintillation fluid (Optiphase HiSafe 3) were from PerkinElmer (Boston, Mass). Tris base was from Invitrogen (Carlsbad, Calif).
Endothelial cell viability assay
The MTT cell viability assay was performed according to the manufacturer's instructions. Owing to their availability, HUVECs were used in viability assays since previous work has shown that THS lymph affects HUVECs to the same degree as rat pulmonary microvascular endothelial cells (32). Briefly, HUVECs (passage three or four) were grown to 100% confluency in 75 cm2 tissue culture flasks (Corning, Corning, NY) then trypsinized and resuspended in endothelial growth media at 2.5 × 105 cells/mL. Cells were seeded (100 μL/well) onto 96-well tissue culture plate (Corning), and the plate was placed in a humidified incubator overnight at 37°C/5%CO2. Lymph was tested at 3% or 5% vol/vol. Cells incubated with 3% to 5% fetal calf serum or 10% dimethyl sulfoxide served as negative or positive controls for cell death, respectively. After incubation (3 or 18 h) at 37°C/5%CO2, the lymph/media were removed and replaced with 90 μL endothelial basal media and 10 μL of a 5 mg/mL MTT solution. The formazan crystals were solubilized after additional incubation at 37°C/5%CO2. The absorbance570 (SpectraMax 384 Plus; Molecular Dynamics, Sunnyvale, Calif) was determined, and viability was calculated as a percentage of the negative control. For clarity, specific lymph volumes and incubation times are detailed in figure legends.
Measurements of LPL and HL were made using a gum arabic emulsion as described (33), with adjustments made for a smaller sonication probe. Briefly, unlabeled (6.25 mg) and labeled tri[9,10(n)-3H] oleate (8 × 106 disintegrations per minute [dpm]) was dispensed into the bottom of a disposable 16 × 100-mm borosilicate tube (Corning) and evaporated under a stream of nitrogen. Gum arabic and Tris-Cl (pH 8.5) were added to final concentrations of 2.35% and 0.3 M, respectively, in a final volume of 1.0625 mL. The tube was placed in a beaker packed with ice and sonicated using a 3-mm-diameter probe (Vibra Cell; Sonics and Materials, Danbury, Conn) using a 50% pulsed mode at medium setting until no oil droplets were visible on the surface (2 × 10-min pulses). Emulsions were placed on ice and used within 3 h.
In assays for the measure of HL, LPL was inactivated using high salt (1.3 M NaCl) in the assay buffer. Samples (30-μL naive or preheparin plasma or lymph, 3- or 10-μL postheparin plasma or lymph, respectively) were diluted in 10 mM Tris-Cl (pH7.7) to a final volume of 50 μL. For the measure of LPL, HL was inactivated by diluting the sample (3 μL) 1:1 with 10 mM Tris-Cl (pH 7.7) followed by a 1:1 dilution with 0.721% SDS and incubated at 28°C for 1 h followed by dilution as above. No HL activity was detected in control experiments with heparinized plasma using this method. Higher volumes of sample could not be used in the LPL assays because of interference by SDS. Duplicate sample measures were made in all assays.
Lipase assay solutions were made by mixing the emulsion with NaCl (5 M in 0.2 M Tris-Cl, pH 8.8), BSA (20% in water, pH 8.4), and heat-inactivated rat serum (56°C for 30 min) to final concentrations of 2 mg/mL triolein, 8% BSA, 0.15 M NaCl (LPL)/1.3 M NaCl (HL), 9% heat-inactivated rat serum (LPL), 0.02% (wt/vol) heparin (LPL), and water to volume. Assay solution (150 μL) was dispensed into borosilicate tubes and incubated for 5 min in a shaking water bath (50 revolutions per minute, 30°C). The assay was initiated by the addition of sample (50 μL) prepared as above. Each reaction tube was individually timed. Reactions were stopped, and fatty acids (FAs) were extracted according to Dole (34) as described (33). Radioactive counts were determined as described (35), and activity was calculated according to Briquet-Laugier et al. (36) and expressed as nanomoles of FAs released in 1 min per 1 mL (nmol FA/min per mL).
Isolation of bovine LPL
Bovine LPL was isolated from milk as described (37). Bovine milk was a generous gift from Rob Kibbe at Fosterfields Farm, Morristown, NJ.
Free calcium was measured in lymph using the Quantichrom Calcium Assay kit (BioAssay Systems, Hayward, Calif) according to instructions. Interference due to the opacity of lymph was controlled for in 2 ways: (a) the kit buffer contains reagents to limit interference with lipids/chylomicrons (CMs), and (b) the OD612 of corresponding time points in THS-heparin (THS-hep) and THS-citrate (THS-cit) samples was compared in a water background. Because there was no significant difference in this value, any interference from opacity was equal between like samples. Because our measurements were relative, and not absolute, any errors using a buffer-based standard were negated.
The pH was measured in each lymph sample immediately after each lymph collection period after centrifugation and antiprotease addition using a Symphony pH meter (VWR) with an Orion micro pH electrode (Thermo Fisher Scientific, Waltham, Mass).
Lipoprotein profile analysis
Mesenteric lymph (2 μL) from individual time points was applied to Titan gels (Helena Labs, Beaumont, Tex) and electrophoresed and stained according to manufacturer's instructions. Dried gels were photographed, and densitometry was performed using an AlphaImager IS-3400 (AlphaInnotech, San Leandro, Calif). A box of equal size was drawn in each gel lane corresponding to the area of the fast migrating band observed after heparin injection. Densities were standardized to the preinjection lane, and data are expressed as a ratio of the integrated density value for each band from each time point relative to the preinjection standard. Background values were determined by the software.
Creation of cytotoxic lymph in vitro
Lipase activities were measured in all plasmas, and LPL activity was normalized to either 40 or 115 nmol FA/min per mL in each reaction tube (final volume, 20 μL). Plasma was mixed with 1× phosphate-buffered saline (pH 7.6) to a final volume of 13 μL in 0.5-mL Eppendorf tubes and placed on ice. Working with each tube separately, 7 μL of naive lymph was added to the plasma, mixed, then dispensed in duplicate aliquots (8.6 μL) to endothelial cells to achieve a final lymph concentration of 3% vol/vol. No more than 15 min passed between the first and last sample.
For lipase inhibition studies, the LPL monoclonal antibody (5D2, 0.08-0.16 mg/mL final concentration) or orlistat (252-μM final concentration) was incubated with plasma, lymph, or purified LPL on ice. After 1 h, phosphate-buffered saline (pH7.7) was added to a volume of 13 μL followed by addition of lymph and tested as described above. Mouse IgG and solvent (10% dimethyl sulfoxide/90% EtOH) served as negative controls for the antibody and orlistat, respectively.
For CM depletion experiments, naive lymph (two tubes of 0.25 mL each) was subjected to centrifugation (10,000g, 10 min, 21°C). The subnatant (125 μL) from each tube was removed and combined in a fresh tube (CM depleted, CMD). The remaining CM-enriched (CME) lymph (125 μL) from both tubes was combined. This was repeated two more times to obtain equal volumes (250 μL) of CMD and CME. An additional sample was remixed after each centrifugation and was used as the whole lymph control.
For oleic acid experiments, naive lymph (18 μL) was incubated with 2 μL of oleic acid stock (in water) to yield final concentrations ranging from 0.328 μM to 32.8 mM. Samples were analyzed on Titan gels as described above.
In studies investigating if heparin alone induced HUVEC toxicity, heparin (1, 2, and 4 U/mL, final concentration) was incubated with HUVECs for 3 h. In studies investigating if heparin added to naive lymph could induce HUVEC toxicity, the same concentrations of heparin were incubated with naive lymph and incubated at 37°C to mimic in vivo effects. Aliquots were removed at timed intervals (0, 30, 60, 90, and 120 min) and incubated with HUVECs (3% vol/vol) for 3 h. The high concentration of 4 U/mL was based on the assumption that a 400-g rat receiving 250 U/kg of heparin (100 U total) would have a maximum of 4 U of heparin/mL in 24 mL [blood volume (in milliliters) = 0.06 * body weight (in grams) + 0.77] (38) of circulating blood.
Analysis of data were performed using Sigma Stat 8.0 (SPSS, and IBM company, Somers, NY) using a Student paired t test or repeated-measures ANOVA at an overall significance level of P = 0.05. The Tukey method was used for pairwise multiple comparisons within a group using at an overall significance level of P = 0.05. The correlation between lipase activities and endothelial cell viability was determined using Sigma-Plot 3.0 (SPSS) Spearman rank-order correlation test at an overall significance level of P = 0.05.
Endothelial cell toxicity is the direct result of heparin injection and not of THS
To discern the role of heparin in mesenteric lymph-mediated HUVEC toxicity in a THS model, blood was withdrawn into a syringe containing either heparin (THS-hep, approximately 100 U) or sodium citrate (THS-cit, 1.5 mL of 0.105 M sodium citrate) as an anticoagulant in amounts sufficient to inhibit clot formation in 10 mL of whole blood. Control TSS animals received an equal volume and concentration of the respective anticoagulant at the end of the TSS period, but no blood was withdrawn. Jugular and femoral cannulas were both primed with sodium citrate in both the THS and TSS groups. Lymph collected from each group was tested for induction of HUVEC toxicity. Toxicity to HUVECs was induced by lymph only from animals in which heparin was used as an anticoagulant in blood withdrawal (Fig. 1A). Although the anticoagulant properties of citrate are due to its ability to chelate calcium, no overall significance was found in calcium levels between THS rats receiving citrate and those receiving heparin (data not shown).
To test if the lack of post-THS lymph-induced HUVEC toxicity was specific to citrate, EDTA (21.6 mg in 200 μL of saline) was used as an alternate anticoagulant. No HUVEC toxicity was induced by post-THS lymph when EDTA was used for both blood withdrawal and priming of jugular and femoral cannulas; however, lymph-induced HUVEC toxicity was detected if heparin replaced EDTA only in priming the femoral cannula used for blood pressure monitoring (Fig. 1B). Although differences in the viabilities did not reach significance between the two EDTA groups, HUVEC toxicity was detected at one or more time points in three of the four animals in the heparin group as reflected by the high standard deviations.
Post-THS lymph-induced HUVEC toxicity has also been reported after resuscitation with RL solution, rather than shed blood (39-41). We therefore investigated heparin's influence in this model. Toxicity to HUVECs was induced by lymph only when heparin was used for blood withdrawal and priming of all cannulas. In this model, heparin is introduced not only via the femoral catheter but also by the partial reinfusion of shed blood as needed to maintain target blood pressure over the 90-min period (0.2-2 mL) before resuscitation with RL solution. When citrate was substituted for the heparin in the syringe and cannulas, no HUVEC toxicity was induced by lymph (Fig. 1C).
We next conducted a set of experiments to test whether the lack of post-THS lymph-induced HUVEC toxicity was due to a protective effect conferred by citrate or EDTA. To accomplish this, the jugular cannula was primed with saline, and THS was induced using a methodology analogous to a fixed-volume model in which a percentage of total blood volume is withdrawn, thus eliminating the need for the femoral catheter for pressure monitoring and the jugular cannula. Blood was withdrawn from the animals in fixed volumes at specified times as determined using our fixed pressure model, thus maintaining the same shock time frame. Animals were first resuscitated with their shed, heparinized blood to ensure that the modification did not alter results. Lymph from these animals induced HUVEC toxicity (Fig. 1D). The next group of animals was resuscitated with shed blood that contained both citrate and heparin using the rationale that if citrate was protective, then the lymph should not induce HUVEC toxicity. Toxicity to HUVECs equivalent to THS-hep was induced by lymph collected from these THS-animals (Fig. 1D). Another set of animals was resuscitated with RL solution, thus omitting all anticoagulants. Lymph collected from these animals were incubated at a higher concentration (10%) and longer period (18 h) to compensate for the dilution of lymph due to the high volume of RL solution used for resuscitation. The viabilities of HUVECs were slightly reduced by the lymph collected from these animals at 30 to 90 min after THS (Fig. 1E) but were not reduced to the levels when heparin was used. To further test that this lack of HUVEC toxicity was not simply due to the dilution of lymph by RL solution, heparin was added to the RL solution used for resuscitation. In contrast to the RL-solution group, lymph from the RL-hep group significantly induced HUVEC toxicity after only a 3-h incubation with cells (Fig. 1E) at the same concentration as RL solution. Because of the charge differences between heparin (negative) and citrate (positive), the pH was measured in the lymph from THS animals receiving either citrate or heparin. Although the pH of the THS-cit lymph was slightly higher at all time points and reached significance at 150 min after THS (Fig. 2), there was no correlation between pH and cell viability after incubation with the lymph (correlation coefficient = −0.231).
Intravenous injection of heparin into naive rats produces cytotoxic lymph
To separate the contribution of heparin from THS in the production of lymph that induced HUVEC toxicity, heparin alone was injected into naive animals. The amount of heparin (250 U/kg) was based on the THS model using 100 U for a 400-g rat. Toxicity to HUVECs was induced by mesenteric lymph collected after heparin injection and was attributed to the supernatant and not the cellular component, consistent with original reports of THS lymph (20) (Fig. 3). Lymph from animals injected with saline or citrate as a vehicle and anticoagulant control, respectively, did not induce HUVEC toxicity (Fig. 3).
HUVEC toxicity is lipase dependent
Because it is well established that heparin injection releases both HL and LPL into the general blood circulation (25, 26), we hypothesized that lipase activity would be present in the plasma and lymph of naive and THS animals receiving heparin but not citrate in the surgical procedure. Consistent with this hypothesis, both HL and LPL activities were detected in both the plasma and mesenteric lymph after, but not before, heparin injection (naive-hep) or THS blood reperfusion (THS-hep) (Fig. 4, A-D). Maximum activity in the plasma is reached by 30 min and sharply decreases for the following 60 min (Fig. 4, A and B). The maximum activities measured in lymph lag the maximum plasma activities by 30 to 60 min (compare Fig. 4C with Fig. 4D) and were sustained until 120 min after heparin injection. This period of maximal lipase activity in lymph directly correlates with a decrease in cell viability (Fig. 4E, Table 1). From 90 to 180 min, lipase activities were equally distributed between the two fluids. Lipase activities in lymph after saline or citrate injection into naive animals or after blood reperfusion in THS-cit animals never exceeded the activity measured in the preinjection sample (data not shown).
Because mesenteric lymph is reintroduced to general circulation and mesenteric LDL has been shown to abrogate THS end-organ injury (11, 12), we hypothesized that LDL should reduce the amount of lipase activity measured within the plasma. Only the LPL, and not HL, was affected (Fig. 4F).
Fasted animals were next used to substantiate a direct in vivo role for LPL in heparin-induced lymph-mediated HUVEC toxicity because fasting has been shown to decrease the amount of lipase in animals (42-45). The lymph from the fasted animals was less toxic to cells when compared with fed animals in both naive-hep and THS-hep groups (Table 2).
Because lipases are released into circulation because of their high affinity for heparin, we theorized that lower doses of heparin should release less lipase and therefore result in less HUVEC toxicity. Consistent with this theory, lymph-mediated HUVEC toxicity was less with lower doses of heparin in naive-hep and to a greater degree in THS-hep (Table 2). No average cell viability below 89% was measured after incubation of endothelial cells with increasing doses of heparin alone (1, 2, and 4 U/mL) or with naive lymph that had been preincubated (0, 30, 60, 90, 120 min) with the same doses of heparin (Fig. 5).
Lipases are derived from the plasma
Because lymph is a filtrate of plasma and the lymph lipase profiles mirrored those of the plasma, we hypothesized that the lipases were derived from the plasma. To test this, we reasoned that the temporary interruption of the blood supply to the area drained by the mesenteric lymphatics would delay or diminish the appearance of lipase activity and, in turn, lymph-induced HUVEC toxicity. The superior mesenteric artery was therefore occluded (SMAO) immediately before heparin injection and remained so for 60 min before the return of blood flow. Only lymph collected immediately after the return of blood flow in the animals receiving heparin reduced cell viability (Fig. 6A). The detection of this reduced activity was delayed 30 min (90 min after heparin injection) compared with a nonoccluded naive-hep animal (60 min after heparin injection; Fig. 4A and Table 2). Lipase activity was also detected only in the animals receiving heparin (Fig. 6B).
Bioactive lymph can be created in vitro
To begin establishing a direct role for lipase activity in the production of postheparin lymph-mediated HUVEC toxicity and because our data indicate that postheparin mesenteric lymph lipases enter from the plasma, we hypothesized that lymph-induced HUVEC toxicity could be recreated in vitro by combining lymph with postheparin plasma. Lymph collected from naive rats (no i.v. injections) was combined with plasma collected from naive-hep rats 30 min after receiving a bolus i.v. injection of 250 U/kg heparin or with plasma from THS-hep rats collected 1 and 2 h after resuscitation with shed blood. The amount of plasma used in each experiment was normalized to an LPL activity of either 40 or 115 nmol FA/min per mL. These values corresponded to the lowest (THS-hep) and median (naive-hep) LPL activity, respectively, that yielded toxicity at 60 to 90 min after heparin injection. To reflect the lymph at 60 to 120 min after heparin injection, samples were mixed and immediately tested for the induction of HUVEC toxicity. Lymph incubated with both naive-hep and THS-hep plasma at 115 nmol FA/min per mL induced HUVEC toxicity within 3 h (Fig. 7A). Cell viability was reduced up to 40% with LPL normalized to 40 nmol FA/min per mL after 3 h and up to 90% after 18 h (data not shown). The induced HUVEC toxicity was abrogated by preincubating the naive-hep or THS-hep plasma with either the general lipase inhibitor, orlistat, or the monoclonal antibody 5D2 specific for LPL (Fig. 7A). Depleting the lymph of CMs (Fig. 7B), the substrate for LPL, before incubation, also reduced the amount of HUVEC toxicity (Fig. 7A). Naive lymph mixed with plasma collected from nonheparinized rats (n = 3 different plasmas) did not induce HUVEC toxicity nor did naive plasma, CMD lymph, or CME lymph alone (data not shown).
We next purified bovine milk LPL to test if LPL alone mixed with naive rat lymph could induce HUVEC toxicity. The presence of LPL was verified by Western blot and by limited protein sequencing (data not shown). The purified enzyme had lipase activity that could be neutralized with orlistat and the 5D2 antibody in the lipase assay (data not shown). Toxicity to HUVECs was induced when the purified LPL was incubated with naive rat lymph using the same procedure and activity as described for heparinized plasma (Fig. 8A). We next tested if LPL was responsible for the induced HUVEC toxicity within our cell viability assays by preincubating lymph shown to induce HUVEC toxicity with both the 5D2 antibody and the general lipase inhibitor, orlistat. Both inhibitors were equally protective (Fig. 8B). Neither inhibitor was protective for three lymph samples from a single naive-hep rat (not included in the figure). It was noted that these lymph samples had very high lipase activities (both LPL and HL >150 nmol FA/min per mL).
Mesenteric lymph lipoprotein profiles are altered after heparin injection
It has been reported that electrophoretic migration of plasma lipoproteins are altered in postheparin plasma, which the authors attributed to the action of lipase on plasma lipoprotein substrates (46). Because our data indicate that lipases are plasma derived, and processing of lymph lipoproteins produces cytotoxic factors, we reasoned that similar postheparin lipoprotein profiles should be observed in lymph. We therefore compared the electrophoretic migration patterns in the lymph collected from naive-hep, naive-sal, THS-hep, and THS-cit animals on agarose gels. Postheparin lymph consistently showed the appearance of fast migrating bands in a pattern identical to that previously reported for postheparin plasma (Fig. 9, arrow). These bands did not appear in preheparin samples or in any time point after saline or citrate injection. Furthermore, these bands appeared in both naive-hep and THS-hep lymph in the time points corresponding to maximal HUVEC toxicity (right lower panels, 60-120 min, compare with Table 2). In addition, the lipoprotein profile of lymph after in vitro incubation with heparinized plasma was analogous to those collected from naive-hep and THS-hep animals (data not shown).
It has been shown that the simple association of free fatty acids with lipoproteins can alter their electrophoretic migration (47). Because lipase activity creates free fatty acids, we investigated if this mechanism could account for the new bands detected in postheparin lymph by mixing oleic acid (0.328 μM to 32.8 mM) with naive lymph as previously reported (47) followed by electrophoretic analysis. No bands were observed at any concentration when oleic acid alone was applied to the gels, and no alterations in the banding patterns were observed in naive lymph incubated with oleic acid (data not shown).
The study of factors created during THS is paramount to understanding events leading to MOF. To this end, animal models have been developed to study the impact of THS on inflammation and end-organ injury. Within each model, the means of resuscitation may vary and include RL solution (39-41), hypertonic saline (40, 41), shed blood (11-21), or a combination of these (39). Despite these differences, these surgical procedures have a single commonality: heparin. Heparin is highly effective in preventing coagulation of shed blood and preventing clot formation in the vessel cannulas used for blood withdrawal or pressure monitoring. In controlling for this drug in our current model of THS, we found that injection of heparin into control animals could produce a factor in lymph that induced HUVEC toxicity. We therefore investigated the respective roles of THS and heparin in the creation of this lymph-induced HUVEC toxicity.
Two key findings show that lymph-induced HUVEC toxicity is the direct result of heparin injection and not of THS. First, post-THS lymph did not induce HUVEC toxicity when heparin was omitted from the surgical procedure. This result was independent of the anticoagulant (citrate, EDTA, none) and the resuscitation fluid (shed blood or RL solution) because all combinations of the two yielded the same results. This lack of cytotoxicity could not be attributed to the calcium-chelating abilities of citrate or the anionic charge of heparin because calcium levels between THS-cit and THS-hep lymph were not significantly different, and there was no correlation between pH and cell viability. The use of citrate did not confer protection because lymph collected from THS animals receiving both citrate and heparin with shed blood could induce HUVEC toxicity to the same extent as heparin alone. Second, heparin injection alone, but not citrate or saline, into TSS and naive animals produced lymph-induced HUVEC toxicity. Because citrate and saline were given under identical conditions, this induced HUVEC toxicity was not due to injection itself or to injection rate (Fig. 3). Together, these data clearly show that heparin, not THS, is the precipitating factor in lymph-induced HUVEC toxicity.
Four key findings show that heparin-released lipases are responsible for the lymph-induced HUVEC toxicity in both naive-hep and THS-hep animals. First, heparin-induced LPL and HL activities were detected in plasma and lymph in both heparin groups. Second, the lipase activity had similar time-course profiles. Third, the lipase activity directly correlated with lymph-induced HUVEC toxicity and fasting, a well-established method of lowering LPL levels in vivo (42-45), significantly reduced the ability of lymph to induce HUVEC toxicity in both heparin groups. Lastly, HUVEC toxicity could be both created in vitro by incubating cells with purified bovine LPL with lymph from naive animals and inhibited after preincubating toxic lymph with both a general lipase inhibitor and an LPL-specific neutralizing antibody. The clear heparin-lipase link to lymph-induced in both naive-hep and THS-hep groups is fully consistent with the notion that THS-hep does not contribute to the formation of novel cytotoxic factors.
Our data are consistent with a previous report of LPL and HL activity in dog skeletal and peripheral lymph (48), where plasma levels peaked 30 min after heparin injection and 60 to 90 min in lymph. Contrary to their study, our absolute plasma and lymph lipase activities were lower, and our lymph-plasma lipase ratios were higher. These differences could be species specific because our plasma lipase activities are fully consistent with those reported in rat (49).
In this prior study, it was also suggested that heparin exiting the circulation into the interstitial space displaces LPL attached to tissues, thus freeing the lipases to enter the lymph (48). Within the THS model, it is proposed that lymph factors responsible for the post-THS lymph-induced endothelial cell damage are derived from the ischemic gut (21). Our data, however, lead us to hypothesize that the lymph lipases are derived directly from the circulation. First, the similarity of plasma-lymph lipase profiles coupled with the 30-min delay of lipase appearance in the lymph compared with plasma (Fig. 4) supports this concept. Second, incubation of postheparin plasma with naive lymph induced HUVEC toxicity. Third, the temporary interruption of blood (SMAO) flow before heparin injection delayed the appearance of both lymph-induced HUVEC toxicity and lipase activity. The lack of detectable HUVEC toxicity in the lymph collected from SMAO animals in the absence of heparin indicates that the injured gut is not a source of the lipases. Furthermore, if lipases were exiting the gut into the lymph because of ischemia, lymph-induced HUVEC toxicity would be expected to increase after occlusion, which is inconsistent with our observed decrease. Lymph lipases were also not due to blood contamination during the surgical or collection procedure because no RBCs were detected in the pellet after centrifugation of the lymph from either naive-hep or THS-hep animals. Collectively, the data support the hypothesis that postheparin mesenteric lymph lipases in THS and naive animals are plasma derived.
Because the half-life of a circulating CM is 5 min (50), it could be argued that the detected toxicity is an artifact of collection. Although we have not rigorously tested this concept, it has not been our experience that lymph samples remaining on ice for the duration of the entire collection period become more toxic than those frozen immediately after collection. We do recognize that pooling the lymph samples most likely skews the true physiologic concentrations at any given time. However, this is not simply an in vitro phenomenon because LDL prevents organ injury in the THS-hep model (11, 12). In addition, lymph collected after heparin injection in shorter time intervals (15 min) was still able to induce HUVEC toxicity. Pooling lymph is unavoidable to do several difference measurements on a single sample because of low lymph volumes. Still, we have attempted to avoid any possible processing during collection by reducing the lymph cannula length to 8 cm, collecting lymph directly into a syringe affixed to an ice pack, and processing and freezing (−80°C) lymph at each time point immediately after each is collected.
There are two important points that have come from these data. First, the sole use of heparin in the femoral catheter used for monitoring blood pressure is sufficient to induce HUVEC toxicity in the mesenteric lymph as shown in the experiments using EDTA as an anticoagulant (Fig. 1B). That this is a significant source of heparin is underscored in some large animal models using flow-through transducers to negate the need for heparin (51, 52). Second, heparin can be used as a tool not only to visualize the reperfusion process after THS by comparing lipase activities within blood at different areas but also to investigate organ damage that could be contributed by lipoprotein metabolism during THS. Indeed, heparin (200 U/kg per h) is used to amplify the effects of lipids in TLR4 signaling in a mouse model (53).
The deleterious effects of heparin-induced lipases shown in our current study can, in part, provide an explanation for several experimental findings attributed to THS when heparinized shed blood is returned in the shock model. For example, a series of experiments showed that heparinization of rats before THS was protective, which the authors attributed to improved microcirculation during THS due to the anticoagulation effects of heparin (54, 55). In their study, shock was induced by blood withdrawal immediately after a high dose (1 U/g) of heparin and then resuscitated with RL solution, not shed blood. Therefore, the protective effects of preheparinization could be due to the removal of heparin-released lipases from the shocked animal, thus reducing lipase contributions to end-organ damage. The protective effects of hypertonic saline (1.3 M NaCl) (40, 41) can be attributed to the irreversible inhibition of LPL, which is sensitive to salt concentrations as low as 0.5 M NaCl (26, 33). In fact, concentrations of 1 M are used routinely to inhibit LPL activity in assays of HL measurements in vitro. The protective effects of high-density lipoproteins in THS end organ-injury (56) could be due to the pivotal role of high-density lipoprotein particles in CM remnant clearance (57), which are implicated in endothelial damage in atherosclerosis [reviewed in Botham ()]. Estrogen has been shown to decrease LPL in adipose tissue (59), and injected estradiol has been shown to preferentially bind to and inhibit HL activity (60), which could therefore account, in part, for protective effects seen in female rats subjected to THS (61, 62). Furthermore, heparin-induced lymph-mediated effects may also explain the reported decrease in post-THS RBC deformability (15) because the same post-THS lymph fractions that we reported to induce endothelial cell toxicity (23) also affect RBC deformability (unpublished). Finally, myeloperoxidase, a marker used for neutrophil activation in post-THS animals (12, 17, 40), is not limited to the neutrophil but is also bound to the vascular endothelium and released by heparin into circulation (63). These examples do not negate the fact that THS clearly has a systemic impact because gut injury, inflammation, sex differences, and organ injury are seen in models not using heparin (52, 64, 65) and slightly reduce endothelial cell viability in our RL solution resuscitation (Fig. 1E) model in the absence of any anticoagulants. However, viewed within the context of our study, heparin-related factors can provide alternate explanations for the results obtained from specific end assays used to evaluate damage due to THS when heparin is used.
Our data clearly show that the use of heparin in a rat shock model causes biologic activity in mesenteric lymph that is independent of THS by demonstrating that the biologic activity in mesenteric lymph collected from naive animals receiving an i.v. injection of heparin is indistinguishable from the biologic activity measured in lymph collected from postshock animals using a heparin model. Our data are fully consistent with a model in which heparin injection into both naive and THS animals releases LPL and HL into circulation, and the lipases are filtered into the mesenteric lymph. Here, action of LPL on the nascent lipoproteins induces factor(s) that are toxic to HUVECs. Furthermore, the presence of the HL, a liver-specific enzyme, within postheparin lymph indicates that the gut may not be the sole source for mesenteric lymph components and suggests that factors generated by the liver during THS may also be potential cytotoxic candidates contributing to MOF via the lymphatics. Finally, these data show that the use of heparin in animal models causes confounding effects as it is clear that lipoprotein metabolism is instrumental in the observed biologic effects in these models.
The authors thank Dr. Patricia Fitzgerald-Bocarsly for support throughout this project. they also thank Drs. George Dikdan, Robert Donnelly, and Jeffrey Wilusz for critical reading of the manuscript; Dr. Qi Lu for technical assistance; and Chloe Sibona for help in manuscript preparation.
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