Elevated ambient glucose induces acute inflammatory events in the microvasculature: effects of insulin

Gregory Booth, Timothy J. Stalker, Allan M. Lefer, and Rosario Scalia

Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We employed intravital microscopy of the rat mesenteric microvasculature to study the effects of local hyperglycemia on leukocyte-endothelial cell interactions. Intraperitoneal injection of 6, 12.5, and 25 mmol/l D-glucose to the rat significantly and time-dependently increased leukocyte rolling and leukocyte adherence in, and leukocyte transmigration through mesenteric venules compared with control rats injected with Krebs-Henseleit (K-H) solution alone or given 25 mmol/l L-glucose intraperitoneally. The response elicited by D-glucose was associated with significant attenuation of endothelial nitric oxide (NO) release, as demonstrated by direct measurement of NO release in inferior vena caval segments isolated from rats exposed to 25 mmol/l D-glucose for 4 h (P < 0.01 vs. vena caval segments from control rats). Local application of 0.05 U/min insulin for 90 min significantly attenuated glucose-induced leukocyte rolling, adherence, and migration (P < 0.01 from 25 mmol/l D-glucose alone). Immunohistochemical localization of P-selectin expressed on endothelial surface was significantly increased 4 h after exposure of the mesenteric tissue to high ambient glucose (P < 0.01 vs. ileal venules from rats injected with K-H solution alone or 25 mmol/l L-glucose). Insulin markedly inhibited endothelial cell surface expression of P-selectin in ileal venules exposed to elevated ambient glucose in vivo (P < 0.01 vs. control rats injected with 25 mmol/l L-glucose). These data demonstrate that acute increases in ambient glucose comparable to those seen in diabetic patients are able to initiate an inflammatory response within the microcirculation. This inflammatory response to glucose is associated with upregulation of the endothelial cell adhesion molecule P-selectin and can be blocked by local application of insulin.

rat mesentery; diabetes; P-selectin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE VASCULAR ENDOTHELIUM is a structurally simple but functionally complex cell layer whose integrity is essential for normal vascular function (23). Several acute and chronic pathological conditions are associated with impaired endothelial cell function (i.e., endothelial dysfunction) (15, 42). Among these conditions, diabetes mellitus is of particular relevance because of its high prevalence of microvascular disease (18, 40). In this regard, several studies have described specific functional changes in diabetic microangiopathy, such as increased vascular permeability (1), sequestration of circulating white blood cells in the microcirculation (26, 29), and, ultimately, morphological alterations resulting in thickening of the basement membrane of microvessels (8, 12).

The mechanism(s) of the relationship between diabetes mellitus and microangiopathy remains only partially understood. Hyperglycemia appears to be an important contributing factor to the diabetic microangiopathy (13). In this regard, it now is well appreciated that exposure of the vascular endothelium to elevated ambient glucose causes endothelial dysfunction characterized by reduced release of nitric oxide (NO) (5, 21). Reduced release of endothelial NO represents the primary early hallmark of endothelial dysfunction (23). Endothelial dysfunction plays a key role during inflammation because of the modulatory action of NO on leukocyte-endothelium interaction (20). In this setting, NO suppresses expression of P-selectin (9) and other endothelial cell adhesion molecules such as ICAM-1 and VCAM-1 (10), thus attenuating interaction between the vascular endothelium and circulating blood cells (e.g., particularly leukocytes).

In light of these findings, the goal of the present study was to investigate 1) concentration-response and time course relationships of regional hyperglycemia on leukocyte rolling, leukocyte adherence, and leukocyte transmigration, 2) effects of hyperglycemia on endothelial cell surface expression of P-selectin, 3) effects of hyperglycemia on release of basal NO on the vascular endothelium, and 4) effects of insulin on glucose-induced leukocyte-endothelium interaction in the mesenteric microcirculation.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study was performed in accordance with the National Institutes of Health guidelines for the use of experimental animals, and all animal protocols were approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University.

Experimental Protocol

We used male Sprague-Dawley rats weighing 250-270 g. Leukocyte-endothelium interactions were studied in rat mesenteric venules 12 h after exposure of the mesentery to 6, 12.5, and 25 mmol/l D-glucose. Rats were randomly divided into one of the following experimental groups: 1) control rats intraperitoneally injected with 0.9% NaCl, 2) control rats injected with 25 mmol/l L-glucose intraperitoneally, 3) rats injected with 6 mmol/l D-glucose, 4) rats injected with 12.5 mmol/l D-glucose, and 5) rats injected with 25 mmol/l D-glucose. The effects of 0.05 U/min insulin on leukocyte-endothelium interaction were also tested in rat mesenteries exposed to 25 mmol/l D-glucose for a 12-h period.

In an additional set of experiments, the time course of glucose-induced leukocyte-endothelium interactions were investigated at 2, 4, and 12 h after a single intraperitoneal administration of 25 mmol/l D-glucose.

D-Glucose (Sigma Chemicals, St. Louis, MO) was freshly prepared daily in sterile 0.9% NaCl. A total volume of 2.5 ml was injected into each rat intraperitoneally. L-Glucose (Sigma) was used at a concentration of 25 mmol/l as control to exclude nonspecific osmolarity effects of D-glucose. Porcine insulin was obtained from Sigma and was locally superfused during intravital microscopy experiments on top of the exposed mesentery at a concentration of 0.05 U/min for a 90-min period of time. This concentration of insulin was used because it does not change blood glucose levels under these experimental conditions (data not shown), and it has been proved to promote wound healing in diabetic patients (16). This dose is physiologically relevant, because it corresponds to one within the clinically used range of insulin doses administered to human diabetics.

Intravital Microscopy

After anesthesia with pentobarbital sodium (60 mg/kg ip), rats were prepared for intravital microscopy according to a previously described technique (37). A tracheotomy was performed to maintain a patent airway throughout the experiment. A polyethylene catheter was inserted in the left carotid artery to monitor mean arterial blood pressure. The abdominal cavity was opened via a midline laparotomy, and a loop of ileal mesentery was exteriorized through the midline incision and placed in a temperature-controlled fluid-filled Plexiglas chamber for observation of the mesentery. The ileum and mesentery were superfused throughout the experiment with a modified Krebs-Henseleit (K-H) solution (containing in mmol/l: 118 NaCl, 4.74 KCl, 2.45 CaCl2, 1.19 KH2PO4, 1.19 MgSO4, 12.5 NaHCO3) warmed to 37°C and bubbled with 95% N2-5% CO2. A Microphot microscope and a ×40 water immersion lens (Nikon, Tokyo, Japan) were used to visualize the mesenteric microcirculation and the mesenteric tissue. The image was projected by a high-resolution color video camera (DC-330, DAGE-MTI) onto a Sony high-resolution color video monitor (Multiscan 200-sf), and the image was recorded with a videocassette recorder. All images were then analyzed using computerized imaging software (Phase 3 Image System, Media Cybernetics) on a Pentium-based IBM-compatible computer (Micron Millenia Mxe, Micron Electronics). Red blood cell velocity was determined on-line using an optical Doppler velocimeter (6) obtained from the Microcirculation Research Institute (College Station, TX). After a 20- to 30-min stabilization period, a 30- to 50-µm-diameter postcapillary venule was chosen for observation. Video recordings were made at 30, 60, 90, and 120 min for quantification of leukocyte rolling, adherence, and transmigration. Leukocyte rolling is expressed as the number of cells moving past a designated point per minute (i.e., leukocyte flux); adherence is expressed as the number of leukocytes adhering to the endothelium per 100 µm of vessel length; transmigrated leukocytes were determined in an area covering a distance of 10 µm in either direction from the vessel wall. The number of extravasated leukocytes was counted and normalized with respect to the immediate perivascular area surrounding the venule (10 µm from the endothelium × 100 µm long along the venule). Red blood cell velocity (Vrbc) and venular diameter (D) were used to calculate venular wall shear rate (g) employing the formula g = 8 × (Vmean/D); (Vmean = Vrbc/1.6), where V = velocity, and D = diameter (14).

Immunohistochemistry

Immunohistochemical localization of P-selectin was determined after intravital microscopy was completed, as previously described (37). Both the superior mesenteric artery and superior mesenteric vein were then rapidly cannulated for perfusion fixation of the small bowel. The ileum was first washed free of blood by perfusion with K-H buffer and then fixed in situ with iced 4% paraformaldehyde. A 3- to 4-cm-long segment of ileum was isolated, cut into rings, and dehydrated using graded acetone washes at 4°C. Tissue sections were embedded in plastic (Immunobed, Polysciences, Warrington, PA), and 4-µm-thick sections were cut and transferred to Vectabond-coated slides (Vector Laboratories, Burlingame, CA).

Immunohistochemical localization of P-selectin was accomplished using the avidin-biotin-immunoperoxidase technique (Vectastain ABC Reagent, Vector Laboratories) (46). Tissue sections were incubated with the primary antibody directed against P-selectin (PB1.3) at a dilution of 1:100 for 24 h. The tissue was then incubated with the biotinylated secondary antibody, and the peroxidase staining was carried out using 3,3'-diaminobenzidine. Control preparations consisted of omission of the primary antibody or omission of the secondary antibody. Expression of P-selectin was determined by microscopic observation of the brown peroxidase reaction product on the venular endothelium of the tissue sections. Positive staining was defined as a venule displaying brown reaction product on >50% of the circumference of its endothelium. Nine sections were studied from each rat, 50 venules per tissue section were examined, and the percentage of positive staining venules was tallied.

Quantification of NO released from isolated vena caval segments. We used freshly isolated inferior vena cava (IVC) segments as the source of primary endothelial cells. IVCs were rapidly isolated from glucose-injected rats at 4 h postinjection. Isolated vein segments were immersed into warm, oxygenated K-H solution, where they were cleaned of adherent fat and connective tissue. Rings of 4-5 mm in length were subsequently cut and opened from randomly selected areas of the vena cava and fixed by small pins with the endothelial surface up in 24-well culture dishes. In some experiments, vena caval segments from control rats and 25 mM D-glucose-injected rats were incubated with 0.04 U/ml insulin for 30 min. NO released into K-H solution warmed at 37°C was measured according to the method of Guo et al. (16) by use of an internally shielded polarographic NO electrode connected to a NO meter (Iso-NO Mark II, World Precision Instruments, Sarasota, FL). Calibration of the NO electrode was performed daily before each experimental protocol (17).

Determination of glucose concentrations in the blood and in the peritoneal fluid. Glucose concentrations in blood samples and in the peritoneal fluid samples were analyzed using an Accu-check Advantage blood glucose monitor (model no. 768, Roche Diagnostic, Boehringer Mannheim, Indianapolis, IN). Samples were analyzed by applying a drop of blood or peritoneal fluid to a control strip inserted into the monitor. Quality control checks were made periodically using a test solution supplied by the manufacturer. Peritoneal fluid was collected immediately before intravital microscopy experiments by means of a sterile capillary pipette.

Determination of plasma insulin concentrations. Circulating blood concentrations of insulin were determined in control rats and rats injected with 25 mM L-glucose at 4 h postinjection. Blood samples were withdrawn from pentobarbital-anesthetized rats via tail vein puncture. Blood samples were anticoagulated with heparin and centrifuged at 3,000 g for 10 min to collect the plasma fraction. Insulin plasma concentrations were determined by an ELISA kit obtained from Calbiochem (Chicago, IL).

Data Analysis

All data are presented as means ± SE. Data were compared by analysis of variance (ANOVA) using post hoc analysis with Fisher's corrected t-test. All data on leukocyte rolling and adherence and arterial blood pressure and shear rates were analyzed by ANOVA incorporating repeated measurements. Probabilities of <= 0.05 were considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Intravital Microscopy

Effects of increasing concentrations of D-glucose on leukocyte-endothelium interactions. Single intraperitoneal injection of 6, 12.5, or 25 mmol/l glucose to rats did not result in any overt systemic hemodynamic effect, as confirmed by the absence of changes in mean arterial blood pressure in all experimental groups of rats. Mean arterial blood pressure values ranged from 137 ± 12 to 142 ± 17 mmHg [not significant (NS)]. Moreover, we examined mesenteric venules ranging from 26 ± 2 to 38 ± 3 µm in diameter (mean ± SE), and there was no difference in venular diameter among any of the groups studied. Shear rates in mesenteric venules were within the physiological range, with values ranging from 496 ± 58 to 739 ± 84 s-1. This clearly indicates that a single exposure of the rat mesentery to 6, 12.5, and 25 mmol/l D-glucose does not change systemic hemodynamic or rheological factors over the 12-h observation time used in this study.

After single intraperitoneal injection of 25 mmol/l D-glucose, glucose levels in the peritoneal fluid increased to 160 ± 12 and 150 ± 8 mg/dl at 2 and 4 h, respectively. Subsequently, peritoneal fluid glucose levels declined 12 h postinjection (Fig. 1, left). Despite this intraperitoneal hyperglycemia, glucose levels in the blood of rats injected with 25 mmol/l D-glucose remained within the normal physiological range over the entire 12-h observation time with the highest value of 119 ± 4 mg/dl being recorded 4 h postinjection (Fig. 1, right). Because no significant changes in systemic blood glucose levels occurred, increased release of endogenous insulin was not a factor in this model of local hyperglycemia. In this regard, plasma insulin concentrations were found to be not significantly elevated 4 h after glucose loading (2.2 ± 0.4 vs. 1.8 ± 0.3 ng/ml insulin, in six 0.9% NaCl-injected rats vs. six 25 mM D-glucose-injected rats; NS).


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 1.   Levels of glucose in peritoneal fluid (left) and in blood (right) 2, 4, and 12 h after single intraperitoneal (ip) injection with 25 mmol/l D-glucose. NS, not significant. All values are means ± SE; n = 6 rats in each group.

A 12-h exposure of the rat mesentery to D-glucose at concentrations as low as 12.5 mmol/l induced a concentration-dependent increase in leukocyte rolling in postcapillary venules of the rat mesenteric microvasculature (Fig. 2, top). This phenomenon exhibited a more clear-cut pattern when 25 mmol/l D-glucose was injected in the rat peritoneal cavity, thus suggesting that the effect of D-glucose on leukocyte-endothelial cell interaction in vivo is concentration dependent.


View larger version (46K):
[in this window]
[in a new window]
 
Fig. 2.   Concentration response of glucose-induced leukocyte-endothelium interaction. Leukocyte rolling (top) and leukocyte adherence (bottom) were observed in rat mesenteric venules from control rats given a single ip dose of 25 mmol/l L-glucose, and rats given single ip doses of 6, 12.5, and 25 mmol/l D-glucose. Observations were made 12 h after ip injections. All values are means ± SE; nos. in parentheses indicate nos. of rats in each group. D-glucose significantly increased leukocyte rolling in a concentration-dependent manner.

Similarly, a concentration response to D-glucose was observed in the case of leukocyte adherence (Fig. 2, bottom). The number of leukocytes adhering to the venular endothelium was increased fivefold (P < 0.01) and sevenfold (P < 0.01) after intraperitoneal injection of 12.5 and 25 mmol/l D-glucose, respectively. Moreover, the number of transmigrated leukocytes was maximally increased by 25 mmol/l D-glucose from 1.8 ± 0.4 to 6 ± 0.5 cells/100 × 10 µm area (Fig. 3; P < 0.01), with most of the white cells being found in the perivascular area. Thus the biological signal resulting from a 12-h exposure to D-glucose is not a transient one.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3.   Leukocyte transmigration in the rat mesentery after ip administration of single dose of 25 mmol/l D-glucose. Extravasated leukocytes were counted 12 h after injection of D-glucose. All values are means ± SE. Nos. at bottom of bars are nos. of rats studied in each group. D-glucose significantly increased leukocyte extravasation in the rat mesentery.

None of these effects of D-glucose could be attributed to changes in ambient osmolarity, as demonstrated by the fact that intraperitoneal administration of 25 mmol/l L-glucose did not increase leukocyte rolling, adherence, and transmigration in rat mesenteric venules (Figs. 2 and 3).

In an additional set of experiments, we investigated the time course of the microvascular inflammatory response evoked by D-glucose in the rat mesenteric microcirculation. Analysis of leukocyte-endothelium interaction 2, 4, and 12 h after injection with 25 mmol/l D-glucose revealed a distinct time course for upregulation of leukocyte-endothelium interaction (Fig. 4). The resulting inflammatory response was characterized by a lag time of 2 h, with a significant increase in leukocyte rolling and leukocyte adherence beginning at 4 h postinjection (Fig. 4, top and bottom). Thus increases in ambient glucose lasting as long as 4 h initiate a microvascular inflammatory response that is concentration dependent, with concentrations as low as 12.5 mmol/l causing a significant yet submaximal effect.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 4.   Time course of leukocyte-endothelium interaction upregulation in response to a single ip administration of 25 mmol/l D-glucose. Leukocyte rolling (top) and leukocyte adherence (bottom) were observed in rat mesenteric venules at 2, 4, and 12 h after ip injection of 25 mmol/l of either L-glucose or D-glucose. All values are means ± SE. Nos. at bottom of bars are nos. of rats studied in each group.

We also studied the response to a single intraperitoneal injection of 25 mM D-glucose. In rat mesenteric venules, glucose-induced leukocyte-endothelium interactions started to decline 24 h postinjection and were completely recovered at 48 h postinjection. Thus only 22 ± 7 leukocytes/min were rolling and 3.5 ± 0.4 leukocytes/100 µm adhered in rat mesenteric venules 48 h postinjection. These values are not statistically different from values observed in control rats injected with saline.

Reversal of glucose-induced leukocyte-endothelium interaction by insulin. Because of the well known physiological role of insulin in regulating systemic and local glucose metabolism, we sought to investigate the effect of insulin on glucose-induced leukocyte-endothelium interactions in vivo. Local application of 0.05 U/min insulin onto the mesentery for 90 min markedly attenuated leukocyte rolling and leukocyte adherence induced by a 12-h exposure of the rat mesentery to intraperitoneal application of 25 mmol/l D-glucose (Fig. 5, top and bottom). In particular, leukocyte rolling and leukocyte adherence were attenuated 55% (P < 0.01) and 45% (P < 0.05), respectively. In addition, insulin inhibited D-glucose-induced transmigration of leukocytes across mesenteric venules from 6.7 ± 1.5 to 2.4 ± 0.7 cells/100 × 10-µm area (P < 0.01; n = 6). These values are significantly lower than those observed with glucose alone, indicating that insulin significantly and effectively prevented glucose-induced rolling and adherence.


View larger version (44K):
[in this window]
[in a new window]
 
Fig. 5.   Effects of insulin on leukocyte-endothelium interaction induced by D-glucose. Insulin was superfused on the exposed mesentery at a concentration of 0.05 U/min for 90 min. The effect of insulin on leukocyte rolling (top) and leukocyte adherence (bottom) was studied in rat mesenteries exposed to a single ip administration of 25 mmol/l D-glucose. Observations were made 12 h after glucose injection. All values are means ± SE. Nos. in parentheses are nos. of rats in each group. Insulin markedly attenuated glucose-induced leukocyte-endothelium interaction.

Immunohistochemistry

Expression of the endothelial cell adhesion molecule P-selectin is illustrated in Fig. 6. Localization of P-selectin was accomplished using a modified avidin-biotin-immunoperoxidase technique. The percentage of venules staining positively for P-selectin in ileal sections from control rats receiving only K-H buffer or 25 mmol/l L-glucose was consistently low (i.e., ~15%; Fig. 6). However, exposure to 25 mmol/l D-glucose resulted in a time-dependent, increased expression of P-selectin as quantified by the percentage of venules staining positive for P-selectin from two- to threefold above control (P < 0.05 and P < 0.01 at 4 and 12 h, respectively). This represents a significant increase in the surface expression of P-selectin under these conditions. This increase in expression of P-selectin on ileal venules was significantly attenuated by application of 0.05 U/min of insulin for 90 min (Fig. 6). Thus glucose-induced P-selectin expression on the endothelial cell surface of the rat mesenteric microvasculature can be suppressed by exogenous insulin. These data are consistent with our functional data on leukocyte rolling, adherence, and transmigration.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 6.   Immunohistochemistry of rat ileal venules. Percentage of venules staining positive for P-selectin in all experimental groups of rats. Bar heights represent mean values; brackets indicate ±SE. Three rats were studied in each group, and >= 9 sections were studied from each rat. A single exposure of the rat mesentery to 25 mM D-glucose significantly increased endothelial cell surface of P-selectin, which was attenuated by local application of insulin.

Effect of Glucose on NO Release from Isolated Rat Vena Caval Segments

We detected a basal level of NO release averaging 12 ± 1.2 nmol/mg of tissue in inferior vena caval segments isolated from control rats injected with saline (Fig. 7). In contrast, release of NO from the inferior vena caval endothelium of rats given a single intraperitoneal injection of 25 mM D-glucose was reduced by 45% (P < 0.01; Fig. 7). In addition, incubation of vena caval segments with 0.04 U/ml for 30 min resulted in a 2.5-fold increase in NO release (P < 0.001 vs. control vena caval tissue from glucose injected animals; Fig. 7). Therefore, exposure of the peritoneal cavity to 25 mM D-glucose induced a marked degree of endothelial dysfunction in the vascular endothelium of the inferior vena cava characterized by a reduction in NO release, and this endothelial dysfunction is attenuated by insulin.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 7.   Basal release of nitric oxide (NO) expressed as nanomoles per milligram of tissue. NO release was measured in segments of inferior vena cava isolated from control rats and rats injected ip with a single dose of 25 mM D-glucose. Release of NO was measured in Krebs-Henseleit (K-H) solution or K-H solution containing 0.04 U/ml insulin. NO measurements were taken at 4 h after ip injection of D-glucose. Bar heights are means; brackets are ± SE. Four to seven rats were studied in each group, and >= 2 segments of inferior vena cava were studied from each rat.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates that acute exposure of the vascular endothelium to elevated ambient glucose upregulates leukocyte-endothelium interaction in the rat mesenteric microcirculation in vivo, via a P-selectin-dependent mechanism, and depresses release of NO in the inferior vena cava. In addition, we provide evidence that local application of insulin attenuates leukocyte-endothelial cell interactions induced by concentrations of glucose comparable to those found in the blood of diabetic patients.

Hyperglycemia is considered to be a major contributor to diabetic angiopathy (7). In vitro and in vivo studies have demonstrated that acute exposure of the vascular endothelium to elevated glucose levels results in a reduced release of NO (21, 47) and increased expression of endothelial cell adhesion molecules (30, 35). Circulating levels of cell adhesion molecules have been found to be significantly elevated also in the plasma of diabetic patients (27). In addition, several experimental animal models of diabetes have clearly demonstrated that exposure of the vascular wall to increased blood glucose levels rapidly results in profound alterations of the homeostasis of the entire vessel wall. In this regard, increased generation of oxygen-derived free radicals has been reported throughout the carotid artery of diabetic rabbits (25). Similarly, daily intraperitoneal injections of glucose to the rats for 4-5 wk induce functional and morphological changes that are similar to those observed in diabetic animals (4). Despite the numerous reports of endothelial dysfunction in diabetes, the time course as well as the concentration response of glucose-induced endothelial dysfunction in the pathophysiology of the diabetic microangiopathy remain unclear.

Reduced release of NO is a common pathophysiological event of acute (43) and chronic (19) inflammatory conditions of the vasculature. Because of the anti-inflammatory role of physiological concentrations of NO, it is likely that loss of endothelium-derived NO during hyperglycemia initiates an inflammatory response via increased synthesis and cell surface expression of adhesion molecules (24). In this regard, NO has been shown to downregulate surface expression of endothelial cell adhesion molecules via inhibition of NF-kappa B activity (10). Moreover, because NO quenches oxygen radicals (36), reduced synthesis or release of NO leads to the existence of enhanced superoxide anions with greater ability to upregulate endothelial cell surface expression of cell adhesion molecules such as P-selectin (2).

Several investigators have demonstrated that acute hyperglycemia depresses arteriolar NO formation in the intestinal (5) and cerebral (28) microvasculature of rats as well as in skeletal muscle arterioles (21). Acute hyperglycemia impairs endothelium-dependent vasodilation also in healthy humans in vivo (47), with endothelial dysfunction occurring 6 h after exposure of the brachial artery to 300 mg/dl of D-glucose.

During diabetes, impaired endothelial function has been associated with either reduced NO bioavailability or decreased sensitivity of the vascular cells (e.g., smooth muscle layer) to NO (11). Thus several mechanisms of endothelial dysfunction have been reported, including impaired signal transduction or substrate availability, impaired release of NO, increased degradation of NO, enhanced release of endothelium-derived constricting factors, and decreased sensitivity of vascular smooth muscle cells to NO (11). In this regard, this is the first study to demonstrate that exposure of the vascular endothelium to elevated ambient glucose for as short a duration as 4 h results in a significant and acute loss of NO, as confirmed by direct measurement of NO release in inferior vena cava of glucose-injected rats.

This key finding correlates with our observation of increased leukocyte-endothelium interaction in the rat mesenteric microvasculature beginning 4 h after exposure of the microcirculation to elevated concentrations of glucose as low as 225 mg/dl. Therefore, a direct correlation can be established between loss of endothelial NO and increased leukocyte-endothelium interactions in the diabetic microcirculation.

Under physiological conditions, NO exerts distinct anti-leukocytic effects by preserving endothelial cell integrity (24) and preventing both cell surface expression (32) and de novo synthesis of adhesion molecules (2, 10). In the absence of this immunomodulatory effect of NO, circulating leukocytes become tethered to the vascular endothelium, where they become activated by a process involving cell-to-cell signaling. In vitro studies have clearly shown that elevated ambient glucose increases the adhesiveness of isolated white cells to cultured endothelial cells (30, 34). This phenomenon is associated with upregulation of ICAM-1 (3) and PECAM-1 (34) glycoproteins. In addition, high levels of circulating adhesion molecules were found in the blood of healthy volunteers after intravenous infusion of glucose, as well in type 2 diabetic patients (27).

Among the adhesion molecules, P-selectin plays a strategic role in the inflammatory process as it regulates leukocyte rolling, the first step of leukocyte-endothelium interactions. Leukocyte rolling is a prerequisite for firm adherence, because integrin-mediated adherence is relatively ineffective at physiological shear rates (22). In this regard, several investigators have demonstrated that inhibition of the rolling phase of leukocytes plays a key role in moderating the inflammatory response (9, 44).

Contact of circulating leukocytes with the vascular endothelium promotes a cascade of events that leads to leukocyte activation. Once activated, leukocytes are able to release oxygen-derived free radicals, proteolytic enzymes, and cytokines (45). Superoxide radicals released from leukocytes have been shown to inactivate NO (36), induce vasoconstriction (31), and disrupt cellular membranes through lipid peroxidation (45). All these processes lead to further leukocyte activation and aggravate vascular endothelial dysfunction. In particular, leukocyte stasis, as well as infiltration of circulating leukocytes in the perivascular area, can have detrimental effects in diabetes, as leukocytes contribute to vasoconstriction and endothelial cell injury in diabetic microangiopathy (26).

In our experimental model of local hyperglycemia, local application of insulin was able to significantly attenuate leukocyte-endothelium interactions induced by glucose. In this regard, it has been demonstrated that insulin stimulates the release of NO in endothelial cells (41) via two different signaling pathways that involve both phosphatidylinositol 3-kinase and protein kinase B (48). Therefore, it is conceivable that insulin inhibited leukocyte rolling and leukocyte adherence via increased release of NO from the vascular endothelium of the rat mesenteric microvasculature. However, the nature of this immunomodulatory action of insulin during hyperglycemia and of how it relates to the NO biosynthetic pathway needs further investigation.

Taken together, these data clearly demonstrate that upregulation of leukocyte-endothelium interactions associated with loss of endothelial NO release occurs very early after exposure of the vascular endothelium to elevated glucose levels. These data also help to further explain previous observations showing a heightened inflammatory response in the mesenteric microcirculation of streptozotocin-induced diabetic rats (33) and nondiabetic rats given a continuous intravenous infusion of glucose (38). More recently, Schaffler et al. (39) showed that the inflammatory response induced by elevated blood glucose levels in the rat mesentery in vivo can be partially blocked via inhibition of both protein kinase C (PKC) and p38 mitogen-activated protein (MAP) kinase. However, the mechanism by which inhibition of PKC and p38 MAP kinase pathways suppresses leukocyte-endothelium interactions remains unclear. One possibility is that the PKC activity in endothelial cells is physiologically regulated by basal release of NO. In this regard, we have previously demonstrated that activation of PKC by atherogenic phospholipids rapidly induces P-selectin expression in both platelets and endothelial cells, and that NO-generating agents are able to inhibit PKC-induced upregulation of P-selectin (32). The present study demonstrates that upregulation of P-selectin in the rat mesenteric microvasculature, as well as reduced release of NO from the rat aortic endothelium, occurs in response to elevated ambient glucose. Therefore, these data may help explain a key mechanism by which glucose-induced activation of protein kinase pathways in endothelial cells initiates inflammatory events within the microcirculation.

In conclusion, this is the first in vivo study to demonstrate that an acute increase in ambient glucose causes a rapid inflammatory response in the microcirculation. This rapid response appears to be triggered via upregulation of P-selectin on the endothelial cell surface, probably due to reduced endothelium-derived NO. This phenomenon may represent an important early mechanism of the diabetic microangiopathy.


    ACKNOWLEDGEMENTS

This study was supported by Research Grant 1-2000-68 from the Juvenile Diabetes Foundation International and Research Grant 0050816U from the Pennsylvania-Delaware Affiliate Research Committee of the American Heart Association. G. Booth and T. J. Stalker are supported by National Institutes of Health Training Grant HL-07599.


    FOOTNOTES

Address for reprint requests and other correspondence: R. Scalia, Dept. of Physiology, Jefferson Medical College, Thomas Jefferson University, 1020 Locust St., Philadelphia, PA 19107-6799 (E-Mail: Rosario.scalia{at}mail.tju.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12 September 2000; accepted in final form 5 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Antonetti, DA, Lieth E, Barber AJ, and Gardner TW. Molecular mechanisms of vascular permeability in diabetic retinopathy. Semin Ophthalmol 14: 240-248, 1999[Medline].

2.   Armstead, VE, Minchenko AG, Schuhl RA, Hayward R, Nossuli TO, and Lefer AM. Regulation of P-selectin expression in human endothelial cells by nitric oxide. Am J Physiol Heart Circ Physiol 273: H740-H746, 1997[Abstract/Free Full Text].

3.   Baumgartner-Parzer, SM, Wagner L, Pettermann M, Gessl A, and Waldhausl W. Modulation by high glucose of adhesion molecule expression in cultured endothelial cells. Diabetologia 38: 1367-1370, 1995[ISI][Medline].

4.   Bohlen, HG, and Hankins KD. Early arteriolar and capillary changes in streptozotocin-induced diabetic rats and intraperitoneal hyperglycaemic rats. Diabetologia 22: 344-348, 1982[ISI][Medline].

5.   Bohlen, HG, and Lash JM. Topical hyperglycemia rapidly suppresses EDRF-mediated vasodilation of normal rat arterioles. Am J Physiol Heart Circ Physiol 265: H219-H225, 1993[Abstract/Free Full Text].

6.   Borders, JL, and Granger HJ. An optical doppler intravital velocimeter. Microvasc Res 27: 117-127, 1984[ISI][Medline].

7.   Chittenden, SJ, and Shami SK. Microangiopathy in diabetes mellitus. I. Causes, prevention and treatment. Diabetes Res 17: 105-114, 1991[ISI][Medline].

8.   Cooper, ME, Rumble J, Komers R, Du HC, Jandeleit K, and Chou ST. Diabetes-associated mesenteric vascular hypertrophy is attenuated by angiotensin-converting enzyme inhibition. Diabetes 43: 1221-1228, 1994[Abstract].

9.   Davenpeck, KL, Gauthier TW, and Lefer AM. Inhibition of endothelial-derived nitric oxide promotes P-selectin expression and actions in the rat microcirculation. Gastroenterology 107: 1050-1058, 1994[ISI][Medline].

10.   De Caterina, R, Libby P, Peng HB, Thannickal VJ, Rajavashisth TB, Gimbrone MA, Jr, Shin WS, and Liao JK. Nitric oxide decreases cytokine-induced endothelial activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines. J Clin Invest 96: 60-68, 1995[ISI][Medline].

11.   De Vriese, AS, Verbeuren TJ, Van De Voorde J, Lameire NH, and Vanhoutte PM. Endothelial dysfunction in diabetes. Br J Pharmacol 130: 963-974, 2000[Abstract/Free Full Text].

12.   Evans, T, Deng DX, Chen S, and Chakrabarti S. Endothelin receptor blockade prevents augmented extracellular matrix component mRNA expression and capillary basement membrane thickening in the retina of diabetic and galactose-fed rats. Diabetes 49: 662-666, 2000[Abstract].

13.   Friedman, EA. Advanced glycosylated end products and hyperglycemia in the pathogenesis of diabetic complications. Diabetes Care 22, Suppl2: B65-B71, 1999[ISI][Medline].

14.   Granger, DN, Benoit JN, Suzuki M, and Grisham MB. Leukocyte adherence to venular endothelium during ischemia-reperfusion. Am J Physiol Gastrointest Liver Physiol 257: G683-G688, 1989[Abstract/Free Full Text].

15.   Granger, DN, and Kubes P. The microcirculation and inflammation: modulation of leukocyte-endothelial cell adhesion. J Leukoc Biol 55: 662-675, 1994[Abstract].

16.   Greenway, SE, Filler LE, and Greenway FL. Topical insulin in wound healing: a randomised, double-blind, placebo-controlled trial. J Wound Care 8: 526-528, 1999[Medline].

17.   Guo, JP, Murohara T, Buerke M, Scalia R, and Lefer AM. Direct measurement of nitric oxide release from vascular endothelial cells. J Appl Physiol 81: 774-779, 1996[Abstract/Free Full Text].

18.   Haffner, SM, Lehto S, Ronnemaa T, Pyorala K, and Laakso M. Mortality from coronary heart disease in subjects with type 2 diabetes and in nondiabetic subjects with and without prior myocardial infarction. N Engl J Med 339: 229-234, 1998[Abstract/Free Full Text].

19.   Harrison, DG. Endothelial dysfunction in atherosclerosis. Basic Res Cardiol 89: 87-102, 1994[ISI][Medline].

20.   Kubes, P, Suzuki M, and Granger DN. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci USA 88: 4651-4655, 1991[Abstract].

21.   Lash, JM, Nase GP, and Bohlen HG. Acute hyperglycemia depresses arteriolar NO formation in skeletal muscle. Am J Physiol Heart Circ Physiol 277: H1513-H1520, 1999[Abstract/Free Full Text].

22.   Lawrence, MB, and Springer TA. Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. Cell 65: 859-873, 1991[ISI][Medline].

23.   Lefer, AM, and Lefer DJ. Pharmacology of the endothelium in ischemia-reperfusion and circulatory shock. Annu Rev Pharmacol Toxicol 33: 71-90, 1993[ISI][Medline].

24.   Lefer, AM, and Lefer DJ. Nitric oxide. II. Nitric oxide protects in intestinal inflammation. Am J Physiol Gastrointest Liver Physiol 276: G572-G575, 1999[Abstract/Free Full Text].

25.   Lund, DD, Faraci FM, Miller FJ, Jr, and Heistad DD. Gene transfer of endothelial nitric oxide synthase improves relaxation of carotid arteries from diabetic rabbits. Circulation 101: 1027-1033, 2000[Abstract/Free Full Text].

26.   Lutty, GA, Cao J, and McLeod DS. Relationship of polymorphonuclear leukocytes to capillary dropout in the human diabetic choroid. Am J Pathol 151: 707-714, 1997[Abstract].

27.   Marfella, R, Esposito K, Giunta R, Coppola G, De Angelis L, Farzati B, Paolisso G, and Giugliano D. Circulating adhesion molecules in humans: role of hyperglycemia and hyperinsulinemia. Circulation 101: 2247-2251, 2000[Abstract/Free Full Text].

28.   Mayhan, WG, Simmons LK, and Sharpe GM. Mechanism of impaired responses of cerebral arterioles during diabetes mellitus. Am J Physiol Heart Circ Physiol 260: H319-H326, 1991[Abstract/Free Full Text].

29.   Miyamoto, K, Hiroshiba N, Tsujikawa A, and Ogura Y. In vivo demonstration of increased leukocyte entrapment in retinal microcirculation of diabetic rats. Invest Ophthalmol Vis Sci 39: 2190-2194, 1998[Abstract].

30.   Morigi, M, Angioletti S, Imberti B, Donadelli R, Micheletti G, Figliuzzi M, Remuzzi A, Zoja C, and Remuzzi G. Leukocyte-endothelial interaction is augmented by high glucose concentrations and hyperglycemia in a NF-kappa B-dependent fashion. J Clin Invest 101: 1905-1915, 1998[Abstract/Free Full Text].

31.   Murohara, T, Buerke M, and Lefer AM. Polymorphonuclear leukocyte-induced vasocontraction and endothelial dysfunction. Role of selectins. Arterioscler Thromb 14: 1509-1519, 1994[Abstract].

32.   Murohara, T, Scalia R, and Lefer AM. Lysophosphatidylcholine promotes P-selectin expression in platelets and endothelial cells. Possible involvement of protein kinase C activation and its inhibition by nitric oxide donors. Circ Res 78: 780-789, 1996[Abstract/Free Full Text].

33.   Panes, J, Kurose I, Rodriguez-Vaca D, Anderson DC, Miyasaka M, Tso P, and Granger DN. Diabetes exacerbates inflammatory responses to ischemia-reperfusion. Circulation 93: 161-167, 1996[Abstract/Free Full Text].

34.   Rattan, V, Shen Y, Sultana C, Kumar D, and Kalra VK. Glucose-induced transmigration of monocytes is linked to phosphorylation of PECAM-1 in cultured endothelial cells. Am J Physiol Endocrinol Metab 271: E711-E717, 1996[Abstract/Free Full Text].

35.   Ribau, JC, Hadcock SJ, Teoh K, DeReske M, and Richardson M. Endothelial adhesion molecule expression is enhanced in the aorta and internal mammary artery of diabetic patients. J Surg Res 85: 225-233, 1999[ISI][Medline].

36.   Rubanyi, GM, and Vanhoutte PM. Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor. Am J Physiol Heart Circ Physiol 250: H822-H827, 1986[Abstract/Free Full Text].

37.   Scalia, R, Gefen J, Petasis NA, Serhan CN, and Lefer AM. Lipoxin A4 stable analogs inhibit leukocyte rolling and adherence in the rat mesenteric microvasculature: role of P-selectin. Proc Natl Acad Sci USA 94: 9967-9972, 1997[Abstract/Free Full Text].

38.   Schaffler, A, Arndt H, Scholmerich J, and Palitzsch K. Acute hyperglycaemia causes severe disturbances of mesenteric microcirculation in an in vivo rat model. Eur J Clin Invest 28: 886-893, 1998[ISI][Medline].

39.   Schaffler, A, Arndt H, Scholmerich J, and Palitzsch K. Amelioration of hyperglycemic and hyperosmotic induced vascular dysfunction by in vivo inhibition of protein kinase C and p38 MAP kinase pathway in the rat mesenteric microcirculation. Eur J Clin Invest 30: 586-593, 2000[ISI][Medline].

40.   Stehouwer, CD, Lambert J, Donker AJ, and van Hinsbergh VW. Endothelial dysfunction and pathogenesis of diabetic angiopathy. Cardiovasc Res 34: 55-68, 1997[ISI][Medline].

41.   Steinberg, HO, Brechtel G, Johnson A, Fineberg N, and Baron AD. Insulin-mediated skeletal muscle vasodilation is nitric oxide dependent. A novel action of insulin to increase nitric oxide release. J Clin Invest 94: 1172-1179, 1994[ISI][Medline].

42.   Tooke, JE. Microvasculature in diabetes. Cardiovasc Res 32: 764-771, 1996[ISI][Medline].

43.   Tsao, PS, Aoki N, Lefer DJ, Johnson G, III, and Lefer AM. Time course of endothelial dysfunction and myocardial injury during myocardial ischemia and reperfusion in the cat. Circulation 82: 1402-1412, 1990[Abstract].

44.   Von Andrian, UH, Hasslen SR, Nelson RD, Erlandsen SL, and Butcher EC. A central role for microvillous receptor presentation in leukocyte adhesion under flow. Cell 82: 989-999, 1995[ISI][Medline].

45.   Weiss, SJ. Tissue destruction by neutrophils. N Engl J Med 320: 365-376, 1989[ISI][Medline].

46.   Weyrich, AS, Buerke M, Albertine KH, and Lefer AM. Time course of coronary vascular endothelial adhesion molecule expression during reperfusion of the ischemic feline myocardium. J Leukoc Biol 57: 45-55, 1995[Abstract].

47.   Williams, SB, Goldfine AB, Timimi FK, Ting HH, Roddy MA, Simonson DC, and Creager MA. Acute hyperglycemia attenuates endothelium-dependent vasodilation in humans in vivo. Circulation 97: 1695-1701, 1998[Abstract/Free Full Text].

48.   Zeng, G, Nystrom FH, Ravichandran LV, Cong LN, Kirby M, Mostowski H, and Quon MJ. Roles for insulin receptor, PI3-kinase, and Akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells. Circulation 101: 1539-1545, 2000[Abstract/Free Full Text].


Am J Physiol Endocrinol Metab 280(6):E848-E856
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society