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(Circulation. 1996;93:161-167.)
© 1996 American Heart Association, Inc.

Articles

Diabetes Exacerbates Inflammatory Responses to Ischemia-Reperfusion

Julián Panés, MD; Iwao Kurose, MD; M. Dolores Rodriguez-Vaca, MD; Donald C. Anderson, MD; Masayuki Miyasaka, PhD; Patrick Tso, PhD; D. Neil Granger, PhD

From the Department of Physiology, Louisiana State University Medical Center, Shreveport; the Discovery Research, UpJohn Laboratories, Kalamazoo, Mich (D.C.A.); and the Department of Bioregulation, Biomedical Research Center, Osaka University Medical School, Japan (M.M.).

Correspondence to D. Neil Granger, PhD, Department of Physiology, LSU Medical Center, 1501 Kings Hwy, PO Box 33932, Shreveport, LA 71130-3932. E-mail dgrang@lsumc.edu.

	Abstract

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Background Diabetes is associated with an increased incidence of ischemic organ damage. The objectives of present study were to compare the leukocyte–endothelial cell adhesive interactions and albumin leakage response of mesenteric venules to ischemia-reperfusion between control rats, rats with streptozotocin-induced diabetes, and rats with hyperglycemia induced by glucose infusion and to define the molecular determinants of the leukocyte accumulation elicited by ischemia-reperfusion in diabetic rats.

Methods and Results Under baseline conditions, lower venular shear rates and an increased number of rolling leukocytes were noted in diabetic rats, whereas the number of adherent and emigrated leukocytes did not differ from that in control rats. Spontaneous albumin leakage from mesenteric venules was markedly increased in diabetic rats but not in hyperglycemic nondiabetic rats. Ischemia-reperfusion elicited significantly larger increases in leukocyte adhesion and emigration and albumin leakage in diabetic rats. Acute elevation of glucose levels did not modify the microvascular responses to ischemia-reperfusion compared with control rats. Antibodies directed against CD11/CD18, intercellular adhesion molecule–1 (ICAM-1), or P-selectin but not L-selectin significantly decreased the number of adherent and emigrated leukocytes after ischemia-reperfusion in diabetic rats. However, none of the antibodies significantly attenuated the increased albumin leakage response to ischemia-reperfusion in diabetic rats.

Conclusions Thes results indicate that diabetes mellitus is associated with exaggerated leukocyte–endothelial cell adhesion and albumin leakage responses to ischemia-reperfusion. The enhanced leukocyte accumulation in response to ischemia-reperfusion is mediated by CD11/CD18–ICAM-1 interactions (firm adhesion) and P-selectin (rolling). The exaggerated albumin leakage response to ischemia-reperfusion in diabetics is not mediated by the recruited inflammatory cells.

Key Words: ischemia • reperfusion • leukocytes • microcirculation • risk factors

	Introduction

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Accelerated ischemic vascular disease is the principal cause of mortality in patients with diabetes mellitus.¹ Hyperglycemia, hypertension, and abnormalities in lipid and lipoprotein metabolism are risk factors that may predispose the diabetic vasculature to the deleterious consequences of ischemia and reperfusion. However, none of these cardiovascular risk factors, either alone or in combination, appear to fully account for the marked increase in mortality associated with diabetes.²

Over the past decade, a large body of evidence has accumulated that implicates neutrophils in the pathobiology of different cardiovascular diseases, including hypertension,³ atherosclerosis,⁴ stroke,⁵ and myocardial ischemia.⁶ A role for neutrophils in these cardiovascular diseases has been invoked on the basis of studies demonstrating that (1) neutrophils are activated and accumulate in affected tissues, (2) neutrophil depletion attenuates the accompanying microvascular and parenchymal cell dysfunction, and (3) the extent of tissue damage and organ dysfunction is reduced by agents that prevent the activation or accumulation of neutrophils.^{7 8 9 10} One means of preventing leukocyte sequestration that has proved to be very effective is immunoneutralization of adhesion glycoproteins expressed on the surface of leukocytes or endothelial cells.¹¹ Monoclonal antibodies (mAbs) directed against these molecular determinants of leukocyte–endothelial cell adhesion have been shown to afford protection in a variety of experimental models of ischemic disease.^{8 9 10}

A growing body of evidence also shows that the function and mechanical properties of granulocytes are altered in diabetes. Granulocytes isolated from diabetic animals and humans are less deformable^{12 13 14} and generate larger quantities of toxic oxygen radicals¹⁵ than granulocytes isolated from nondiabetics. Moreover, a larger percentage of the circulating neutrophils is activated in human diabetics compared with control populations.¹⁶ Taken together, these observations suggest that the activated and less deformable polymorphonuclear leukocytes may predispose diabetics to neutrophil-mediated tissue injury. Although it would appear likely that diabetes predisposes tissues to the deleterious consequences of ischemia and reperfusion, an injury process that is largely mediated by neutrophils in some tissues, there is no experimental evidence that directly addresses this important issue. Thus, the overall objective of this study was to determine whether diabetes renders the microvasculature more vulnerable to the deleterious effects of ischemia-reperfusion. In addition, we addressed the possibility that an enhanced accumulation of adherent leukocytes within postcapillary venules or hyperglycemia per se contributes to the microvascular dysfunction observed in diabetic tissues exposed to ischemia-reperfusion.

	Methods

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Animal Model
Male Sprague-Dawley rats weighing 200 to 225 g were obtained from Harlan. After a 5-day adaptation period, diabetes mellitus was induced by injection of streptozotocin (65 mg/kg IP; Sigma Chemical Co)¹⁷ diluted in citrate buffer at a concentration of 6.5 mg/mL. Control rats received an intraperitoneal injection of the corresponding amount of citrate buffer alone. Induction of the diabetic condition, defined as a serum glucose concentration >200 mg/dL, was confirmed by glucose measurements in serum samples obtained from the tail vein at 1 and 3 weeks (520±27 and 480±35 mg/dL, respectively) after streptozotocin administration. All studies were performed 3 weeks after treatment, preceded by a 24-hour fasting period with free access to water. The experimental protocols were approved and performed in accordance with the guidelines of the Louisiana State University Medical Center Animal Care and Use Committee, Shreveport.

To assess the effects of acutely increased glucose levels on the microvascular responses to ischemia-reperfusion, two groups of previously untreated control rats were studied. In one group, the region of the mesentery under study was superfused with bicarbonate buffer containing glucose at a concentration of 480 mL/dL, which corresponds to the mean value of plasma glucose in the diabetic group at the time of study. In a second series of experiments, acute hyperglycemia was induced by continuous infusion of 50% glucose at 200 mg·kg^-1·min^-1 IV during the first 5 minutes and 75 mg·kg^-1·min^-1 thereafter.

Surgical Techniques
The animals were anesthetized with thiobutabarbital (Inactin) 90 mg/kg IP in nondiabetic rats and 60 mg/kg IP in diabetic rats. A tracheotomy was performed on each rat to facilitate breathing throughout the experiment, and the right carotid artery and right jugular vein were cannulated. Systemic arterial pressure was measured with a Statham P23A pressure transducer and recorded with a polygraph DC driver amplifier (Grass Instrument Co). A midline abdominal incision was made to allow for exteriorization of a section of the mesentery from the small intestine. A blood sample was obtained before the beginning of the study to determine total white blood cell and neutrophil counts and plasma glucose concentration.

Intravital Microscopy
The rats were positioned on a 20x30-cm Plexiglas board in a manner that allowed a selected section of mesentery to be placed over a glass slide covering a 3.5x3.5-cm hole centered in the Plexiglas. All exposed tissue was covered with saline-soaked gauze to minimize tissue dehydration. The board was mounted onto the stage of an orthostatic microscope (Nikon Optihot). The image produced with a x40 objective (Nikon E-Plan 40) was captured on videotape (BR–S601MU videocassette recorder, JVC) with a color camera (VK-C150, Hitachi). The time and date were displayed on both taped and live images (Trinitron monitor, Sony) with a date-time generator (WJ-810, Panasonic). The mesentery was superfused at 2.5 mL/min with bicarbonate-buffered saline bubbled with a 95% N₂/5% CO₂ gas mixture to reduce the oxygen tension to the physiological intraperitoneal level (40 to 50 mm Hg). The superfusate was maintained at 37°C by pumping the solution through a heat exchanger warmed with a constant-temperature circulator (model 801, Fisher Scientific). Rectal and mesenteric temperatures were monitored with an electrothermometer. Body temperature was kept between 36.5°C and 37.5°C with an infrared heat lamp.

Single unbranched venules with diameters of 25 to 35 µm and lengths >150 µm were selected for study. Venular diameter (D_V) was measured on-line with a video caliper (Microcirculation Research Institute, Texas A&M University, College Station). The number of adherent, emigrated, and rolling leukocytes was determined off-line during playback of videotaped images. A leukocyte was considered adherent to venular endothelium if it remained stationary for 30 seconds or longer. Adherent leukocytes were quantified as the number per 100-µm length of venule. Leukocyte emigration was expressed as the number per microscopic field (2.12x10^-2 mm²). Rolling leukocytes were defined as those white blood cells that moved at a velocity less than that of erythrocytes in the same vessel. Leukocyte rolling velocity (V_LR) was determined from the time required for a leukocyte to traverse a 50-µm distance along the length of the venule and is expressed as micrometers per second. The flux of rolling leukocytes was measured as those white cells that could be seen moving within a small (10-µm) viewing area of the vessel with the same area used throughout the experiment. The number of rolling leukocytes per 100-µm venule length was calculated as the leukocyte flux divided by V_LR.

Centerline red blood cell velocity (V_RBC) was measured with an optical Doppler velocimeter (Microcirculation Research Institute, Texas A&M University) that was calibrated against a rotating glass disk coated with red cells. Venular blood flow was calculated from the product of mean red blood cell velocity (V_mean=Centerline Velocity/1.6) and microvascular cross-sectional area, with cylindrical geometry assumed. Venular wall shear rate () was calculated from the newtonian definition: =8(V_mean/D_v).

To quantify albumin leakage across mesenteric venules, 25 mg/kg IV FITC-labeled rat albumin (Sigma Chemical Co) was administered to the rats. Fluorescence intensity (excitation wavelength, 420 to 490 nm; emission wavelength, 520 nm) was detected with a CCD camera model XC-77 (Hamamatsu Photonics), a C2400-60 CCD camera control unit, and a C2400-68 intensifier head (Hamamatsu Photonics) attached to the camera. The fluorescence intensity of the venule under study (I_v), the fluorescence intensity of contiguous perivenular interstitium within 10 to 50 µm of the venular wall (I_i), and the background fluorescence before injection of FITC-albumin (I_b) were analyzed with a Macintosh Quadra computer (Apple Computer Inc) equipped with a 24STV graphics display board (Rasterops Co) with the public-domain NIH IMAGE computer-assisted digital imaging processor. An index of vascular albumin leakage (permeability index) was determined according to the formula (I_i-I_b)/(I_v-I_b). Measurements of I_v and I_i were obtained at least 30 minutes after FITC-albumin injection.

Experimental Protocols
After a stabilization period of 30 minutes, images from the mesenteric preparation were recorded on videotape for 10 minutes. Thereafter, ischemia was induced by placing a ligature of polyethylene P-50 tubing around the superior mesenteric artery and a piece of polyethylene P-280 tubing around the superior mesenteric vein. Ischemia was maintained for 10 minutes; then the P-280 tubing was removed, allowing blood to recirculate. Only rats in which ligation of the superior mesenteric vessels induced a decrease in centerline red blood cell velocity 80% were used in the study. All parameters were measured again 5 to 15 minutes and 30 to 40 minutes after reperfusion. In some experiments, animals received mAbs directed against the ß subunit (CD18) of CD11/CD18 (mAb CL26, 100 µg per rat),¹⁸ intercellular adhesion molecule–1 (ICAM-1; mAb 1A29, 2.0 mg/kg),¹⁹ L-selectin (HLR3, 1 mg/kg),²⁰ P-selectin (PB1.3, 2 mg/kg),²¹ or a nonbinding antibody (PNB1.6, 2.0 mg/kg),²² with the mAb administered immediately after the baseline measurements were obtained (10 minutes before ischemia was induced). The concentration of mAbs used in this study was based on experiments that determined the amount of mAb needed to maximally reduce leukocyte adherence and emigration in rat mesenteric venules induced by inflammatory mediators²³ or ischemia-reperfusion.¹⁰

In the experiments designed to assess the effects of acutely increased glucose levels on the microvascular response to ischemia-reperfusion, glucose was either superfused directly onto the mesentery or infused intravenously (as described above) after the baseline measurements were obtained. Thirty minutes after the onset of exposure to high glucose levels, all parameters were measured again. Thereafter, ischemia-reperfusion was induced with the same protocol as described previously.

Statistical Analysis
The data were analyzed with standard statistical analyses, ie, ANOVA with Scheffé's (post hoc) test and Student's paired or unpaired t test where appropriate. All values are reported as mean±SEM from six to eight rats. Statistical significance was set at P<.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Under baseline conditions, diabetic rats had significantly lower blood pressure than control rats (104±4 versus 119±2 mm Hg; P<.05). Diabetic and control rats had very similar total white cell (7.1±0.7 versus 7.9±0.6 cells/mm³) and neutrophil (2.4±0.5 versus 2.5±0.5 cells/mm³) counts in blood. A proliferation of small blood vessels (10 to 50 µm in diameter) was observed in the mesentery of diabetic rats. The diameters of the venules studied were similar in diabetic and control rats (34±1 versus 33±1 µm). Mesenteric venules of diabetic rats had a significantly lower centerline red blood cell velocity (2.50±0.15 versus 3.68±0.39 mm/s, P<.01) and shear rate (368±29 versus 555±52 s^-1, P<.01) than control rats. In diabetic rats, the basal leukocyte rolling velocity was significantly lower and the number of rolling leukocytes was significantly higher compared with control rats (Fig 1). The decrease in rolling velocity was determined largely by a reduction in blood flow velocity because after normalization of leukocyte rolling velocity to the corresponding red blood cell velocity (V_LR/V_RBC), no significant differences were observed between control and diabetic rats (0.016±0.002 versus 0.013±0.001). Under baseline conditions, control and diabetic rats had similar numbers of adherent and emigrated leukocytes (Fig 2), whereas diabetic rats exhibited a markedly increased albumin leakage relative to control rats (39.0±8.7 versus 9.7±2.3%, P<.01; Fig 3).

View larger version (46K): [in this window] [in a new window]   Figure 1. Bar graphs showing the characteristics of leukocyte rolling in mesenteric venules in diabetic (stippled bar) and control (nondiabetic; solid bar) rats under baseline conditions. *P<.05 vs control rats.

View larger version (47K): [in this window] [in a new window]   Figure 2. Bar graphs showing leukocyte adhesion (A) and emigration (B) in mesenteric postcapillary venules in diabetic and control rats under baseline conditions and after ischemia-reperfusion (I/R). +P<.05 vs basal; *P<.05 vs control rats.

View larger version (55K): [in this window] [in a new window]   Figure 3. Bar graph showing the influence of the diabetic state and ischemia-reperfusion (I/R) on mesenteric venular permeability to albumin. +P<.05 vs basal; *P<.05 vs control rats.

During the ischemic period, centerline red blood cell velocity (0.43±0.06 versus 0.46±0.06 mm/s, P>.05) and shear rate (60.3±9.0 versus 69.5±11.1 s^-1, P>.05) were similar in both groups of rats. After reperfusion, diabetic rats again exhibited a significantly lower centerline red blood cell velocity (1.52±0.22 versus 2.7±0.24 mm/s, P<.01) and shear rate (221±36 versus 406±27 s^-1) than control rats. In both groups of rats, the values of red blood cell velocity and shear rate were significantly lower after reperfusion compared with values obtained under baseline conditions (P<.05, paired t test).

Diabetic rats exhibited a significant reduction in leukocyte rolling velocity and significant increases in the flux of rolling leukocytes and the number of rolling leukocytes immediately after reperfusion (Fig 4). Control (nondiabetic) rats exhibited similar increases in the flux and number of rolling leukocytes, but leukocyte rolling velocity was not significantly altered (Fig 4). The alterations in leukocyte rolling induced by ischemia-reperfusion were short-lived and had returned entirely to control levels 30 minutes after reperfusion (Fig 4). Ischemia-reperfusion caused a significant increase in the number of adherent and emigrated leukocytes in both diabetic and nondiabetic rats, but leukocyte adhesion and emigration after reperfusion were significantly higher in diabetic than control rats (Fig 2). Albumin leakage increased significantly in response to ischemia-reperfusion in both groups of rats; however, diabetic rats exhibited a significantly higher albumin leakage than control rats after reperfusion (Fig 3).

View larger version (18K): [in this window] [in a new window]   Figure 4. Plots showing the effects of ischemia-reperfusion (Rep) on the leukocyte rolling phenomenon in diabetic () and control () rats. +P<.05 vs basal; *P<.05 vs controls.

In control rats, superfusion of the mesentery with bicarbonate buffer containing 480 mg/dL glucose did not produce any significant change in red blood cell velocity, shear rate, leukocyte rolling, adhesion, emigration, or microvascular permeability (data not shown). In this series of experiments, the responses of leukocyte rolling (0.28±0.06 cells/100 µm), adherence (6.66±0.88 cells/100 µm), and emigration (5.00±0.57 cells per field) to ischemia-reperfusion and the increased albumin leakage (0.24±0.05) were very similar to those observed in control preparations and significantly different from the response observed in the diabetic group.

Infusion of 50% glucose at a rate that increased plasma glucose concentration to diabetic levels (498±21 and 534±33 mg/dL IV at 30 and 80 minutes of continuous infusion, respectively) produced a rapid and sustained increase in red blood cell velocity (3.26±0.25 versus 3.72±0.28 mm/s, P<.01), an increase in shear rate (556±22 versus 634±24 seconds, P<.01), and a decrease in the number of rolling leukocytes (0.59±13 versus 0.29±.06 cells/100 µm, P<.01) without significant changes in leukocyte adhesion (2.2±0.58 versus 2.4±0.51 cells/100 µm), emigration (1.81±0.58 versus 1.99±0.68 cells per field), or albumin leakage (0.077±0.024 versus 0.076±0.019) compared with baseline values. In these experiments, the ischemia-reperfusion–induced changes in leukocyte rolling (0.31±0.10 cells/100 µm), adherence (4.60±1.20 cells/100 µm), and emigration (2.80±0.49 cells per field) and the increased albumin leakage (0.29±0.06) were very similar to responses observed in the control group and significantly different from those noted in the diabetic group.

In diabetic rats, administration of a P-selectin mAb had a marked effect on the pattern of leukocyte rolling observed 5 minutes after reperfusion. The P-selectin mAb resulted in a significant increase in leukocyte rolling velocity and reduced both the flux and number of rolling leukocytes compared with untreated diabetic rats and those receiving a nonbinding mAb (Fig 5). At 30 minutes after reperfusion, rats treated with the P-selectin mAb still exhibited a significantly higher leukocyte rolling velocity compared with baseline values, but differences among groups were not significant. P-selectin mAb treatment also was associated with a significant reduction in the number of adherent (Fig 6A) and emigrated (Fig 6B) leukocytes. Treatment with an L-selectin mAb had less effect on leukocyte rolling. The L-selectin mAb prevented the reduction in leukocyte rolling velocity and the increases in the flux and number of rolling leukocytes that usually were observed 5 minutes after reperfusion in untreated rats and rats treated with a nonbinding mAb (Fig 5). Although there was a trend for reduced ischemia-reperfusion–induced leukocyte adhesion and emigration in rats treated with the L-selectin mAb (relative to untreated rats and those receiving a nonbinding mAb), these differences were not statistically significant. Administration of mAbs directed against either CD11/CD18 or ICAM-1 had no effect on leukocyte rolling at reperfusion (data not shown) but markedly decreased leukocyte adhesion and emigration after reperfusion (Fig 6). In diabetic rats, none of the mAbs studied exerted a significant effect on albumin leakage after ischemia-reperfusion (Fig 7).

View larger version (22K): [in this window] [in a new window]   Figure 5. Plots showing the effects of immunoneutralization of different leukocyte or endothelial cell adhesion molecules on ischemia-reperfusion (I/R)–induced changes in leukocyte rolling. Rep indicates reperfusion; , untreated diabetes; , treated with L-selectin monoclonal antibodies (mAB); and , treated with P-selectin mAb. +P<.05 vs basal; *P<.05 vs controls.

View larger version (60K): [in this window] [in a new window]   Figure 6. Bar graphs showing the effects of immunoneutralization of different leukocyte or endothelial cell adhesion molecules on ischemia-reperfusion (I/R)–induced leukocyte adhesion (A) and emigration (B). ICAM-1 indicates intercellular adhesion molecule–1; Non-Bind, nonbinding; and mAb, monoclonal antibodies. *P<.05 vs controls.

View larger version (91K): [in this window] [in a new window]   Figure 7. Bar graphs showing the effects of immunoneutralization of different leukocyte or endothelial cell adhesion molecules on venular permeability to albumin after ischemia-reperfusion (I/R). Abbreviations as in Fig 6. *P<.05 vs controls.

Baseline and ischemia-reperfusion values for centerline red blood cell velocity and shear rate in postcapillary venules of diabetic rats were not altered by treatment with any of the mAbs (binding or nonbinding).

	Discussion

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The results of this study indicate that the diabetic state induces an exaggerated inflammatory response to ischemia-reperfusion manifested as a greater accumulation of adherent and emigrated leukocytes and a larger increase in albumin extravasation. The present study also shows that diabetes is associated with an increased baseline level of leukocyte rolling (Fig 1). Although we cannot exclude the possibility of altered adhesion molecule expression in diabetic rats as a basis for the observed changes in leukocyte rolling, a more likely explanation for this response is the corresponding reduction in venular blood flow noted in mesenteric venules of diabetic rats. Previous studies^{24 25} have demonstrated a fairly constant ratio of leukocyte rolling velocity to red cell velocity over a wide range of shear rates in mesenteric venules, and our finding that this ratio does not differ between control and diabetic rats suggests that changes in blood flow velocity account largely for reduced leukocyte rolling velocity in diabetic venules. The decreased red blood cell velocity and shear rates observed in diabetic rats might be related to a redistribution of blood flow through the net of proliferating small vessels that we observed in the mesentery 3 weeks after the induction of diabetes.

Despite the higher basal number of rolling leukocytes in diabetic rats relative to control rats, the former group also exhibited a more marked recruitment of rolling leukocytes in response to ischemia-reperfusion (Fig 4). In control rats, ischemia-reperfusion induced a transient increase in the number of rolling leukocytes that was determined primarily by an increased flux of rolling leukocytes without significant changes in leukocyte rolling velocity. However, the increased number of rolling leukocytes observed in diabetic rats after ischemia-reperfusion resulted from both an increased flux of rolling leukocytes and a further decrease in leukocyte rolling velocity. The latter observation suggests that the additional increase in rolling leukocytes in diabetic rats may result from a shear rate–dependent recruitment of rollers.²⁵

Our study also provides some insights into the contributions of leukocyte (L-selectin, CD11/CD18) and endothelial cell (P-selectin, ICAM-1) adhesion molecules to the ischemia-reperfusion–induced recruitment of rolling leukocytes in diabetic rats (Fig 5). We noted that mAbs directed against either P-selectin or L-selectin were effective in blunting the leukocyte rolling response elicited by ischemia-reperfusion, whereas mAbs against CD11/CD18 or ICAM-1 and a nonbinding (control) mAb had no effect. These observations suggest that the altered leukocyte rolling response elicited by ischemia-reperfusion is mediated primarily by adhesive interactions of L-selectin on leukocytes and P-selectin on endothelial cells. The involvement of P-selectin in mediating the rapid rolling response elicited by ischemia-reperfusion is consistent with the rapidity of expression of the lectinlike adhesion molecule after activation of endothelial cells in culture²⁶ and the well-characterized role of P-selectin in mediating leukocyte rolling in mesenteric venules exposed to normal^{27 28} and low²⁵ shear rates. Our inability to demonstrate a contribution of CD11/CD18 and ICAM-1 to the enhanced leukocyte rolling in diabetes is also in accordance with published reports that failed to invoke a role for these adhesion molecules in the modulation of leukocyte rolling.^{23 29}

Kurose and associates¹⁰ recently demonstrated that rat mesenteric venules exposed to ischemia-reperfusion sustain an increased number of firmly adherent and emigrating leukocytes. Our findings in control rats confirm their observations, and our results in diabetic rats extend their work to show that the leukocyte–endothelial cell adhesive interactions that are manifested as adherence and emigration in postischemic microvessels are greatly exaggerated in this animal model of human disease. The quantitatively different responses to ischemia-reperfusion between control and diabetic rats are not due to differential ischemic insults inasmuch as venular red blood cell velocity during ischemia fell to a similar level in both groups of rats. However, a lower venular shear rate or a higher number of rolling leukocytes in diabetic venules after reperfusion could account for at least some of the differences in leukocyte adherence and emigration between control and diabetic rats. A dependency of firm leukocyte adhesion on shear rates has been demonstrated in vitro³⁰ and in vivo.²⁴ It also has been shown that graded reductions in rolling leukocyte flux are accompanied by proportional reductions in chemoattractant-induced leukocyte adhesion.³¹ Taken together, these observations suggest that the lower shear rates in diabetic compared with control rats may contribute to the increased leukocyte adhesion observed after ischemia-reperfusion in diabetic rats. Nonetheless, we cannot invoke differences in shear rate as the sole explanation for the exaggerated inflammatory response to ischemia-reperfusion in diabetic rats, particularly because these differences in shear rate were already present under baseline conditions, when similar numbers of adherent leukocytes were observed in control and diabetic rats.

The present study also demonstrates that hyperglycemia per se cannot explain the altered inflammatory responses observed in diabetic rats. Elevation of blood glucose concentration in control rats to a level measured in diabetic rats induced significant increases in red blood cell velocity and venular shear rate and a decrease in the number of rolling leukocytes relative to baseline conditions. These changes contrast the microvascular alterations noted in diabetic rats. In addition, acute hyperglycemia did not enhance the inflammatory responses (leukocyte–endothelial cell adhesion and albumin leakage) usually elicited by ischemia-reperfusion.

In postcapillary venules of control (nondiabetic) rats subjected to ischemia-reperfusion^{32 33 34} and on endothelial cell monolayers exposed to anoxia or reoxygenation,³⁵ the increased leukocyte adherence is mediated by an interaction between CD11/CD18 on leukocytes and ICAM-1 on endothelial cells. A recent study from our group¹⁰ characterized the molecular determinants of ischemia-reperfusion–induced leukocyte adhesion and emigration in nondiabetic rats. That study showed that mAbs directed against CD18 and ICAM-1 but not P- or L-selectin effectively blunted ischemia-reperfusion–induced leukocyte adherence and emigration in rat mesenteric venules; these results are summarized in the Table for comparison with the current findings in diabetic rats. In the present study, we provide evidence that CD11/CD18–ICAM interactions also are important in mediating the leukocyte adherence and emigration observed in mesenteric venules of diabetic rats exposed to ischemia-reperfusion (Fig 6). However, a more substantial role for selectins as a determinant of ischemia-reperfusion–induced leukocyte adherence can be inferred from our experiments on diabetic rats. The P-selectin mAb, which had a very potent and lasting effect on leukocyte rolling after ischemia-reperfusion in diabetic rats, effectively reduced both leukocyte adherence and emigration. The L-selectin mAb, which had a less potent and short-lived effect on leukocyte rolling, was less effective in reducing leukocyte adherence and had no significant effect on leukocyte emigration. These observations suggest that a very effective blockade of leukocyte rolling is needed to completely prevent firm adhesion. An explanation for the relatively striking effect of the P-selectin mAb in reducing leukocyte adherence and emigration in diabetic rats (compared with published studies on control rats) is not readily available; however, it may reflect differences in the ability of ischemia-reperfusion to mobilize P-selectin to the endothelial cell surface between control and diabetic rats.

View this table: [in this window] [in a new window]   Table 1. Effects of Monoclonal Antibodies Directed Against the Adhesion Molecules CD18, ICAM-1, L-Selectin, and P-Selectin on Ischemia-Reperfusion–Induced Leukocyte Adhesion and Emigration and Albumin Leakage

Numerous reports demonstrate an increased microvascular permeability to plasma proteins in tissues exposed to ischemia-reperfusion and show that the ischemia-reperfusion–induced vascular protein leakage is mediated by adherent leukocytes.^{32 36 37 38} Thus, another primary objective of this study was to determine whether diabetes alters the phenomenon of ischemia-reperfusion–induced endothelial barrier dysfunction. Our results indicate that even under basal conditions, albumin leakage from mesenteric venules is significantly higher in diabetic compared with control rats (Fig 3). This observation is consistent with published evidence demonstrating an increased microvascular permeability in different organs of diabetic rats.^{39 40} However, despite this basal level of endothelial cell barrier dysfunction, diabetic rats exhibited a significantly enhanced albumin leakage in response to ischemia-reperfusion. However, unlike control rats in which mAbs that attenuate ischemia-reperfusion–induced leukocyte–endothelial cell adhesion also blunt the accompanying albumin leakage,¹⁰ postcapillary venules of diabetic rats are largely unresponsive to immunoneutralization of adhesion molecules on leukocytes or endothelial cells (see the Table). These observations indicate that the enhanced albumin extravasation observed in control and postischemic venules of diabetic rats is mediated by leukocyte-independent processes.

It has been proposed that hyperglycemia per se may contribute to the impaired endothelial cell barrier function in diabetes.⁴¹ However, the findings of the present study do not favor this hypothesis; we observed that exposure of the mesenteric microvasculature to high glucose levels did not alter albumin leakage, either under baseline conditions or after ischemia-reperfusion. Other leukocyte-independent mechanisms that may contribute to the enhanced microvascular protein transfer in diabetes include changes in redox state in endothelial cells as a result of glucose metabolism through the polyol pathway,⁴² activation of protein kinase C,⁴³ and quenching of nitric oxide by advanced glycosylation products.^{44 45}

	Acknowledgments

 
This work was supported by NIH grants HL-26441 and DK-32288. Dr Panés is a recipient of a grant from Fundación Ramón Areces.

Received April 25, 1995; revision received August 16, 1995; accepted August 20, 1995.

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