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(The FASEB Journal. 2000;14:2357-2364.)
© 2000 FASEB

C-peptide inhibits leukocyte–endothelium interaction in the microcirculation during acute endothelial dysfunction

ROSARIO SCALIA1, KATHLEEN M. COYLE, BRIAN J. LEVINE, GREGORY BOOTH and ALLAN M. LEFER

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

1Correspondence: Department of Physiology, Jefferson Medical College, Thomas Jefferson University, 1020 Locust St., Philadelphia, PA 19107-6799, USA. E-mail: Rosario. Scalia{at}mail.tju.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
C-peptide is a cleavage product that comes from processing proinsulin to insulin that induces nitric oxide (NO) -mediated vasodilation. NO modulates leukocyte–endothelium interaction. We hypothesized that C-peptide might inhibit leukocyte–endothelium interaction via increased release of endothelial NO. Using intravital microscopy of the rat mesentery, we measured leukocyte–endothelium interactions after administration of C-peptide to the rat. Superfusion of the rat mesentery with either thrombin or L-NAME consistently and significantly increased the number of rolling, adhering, and transmigrated leukocytes. C-peptide significantly attenuated either thrombin- or L-NAME-induced leukocyte–endothelium interactions in rat mesenteric venules. A control scrambled sequence of C-peptide characterized by the same amino acid composition in a randomized sequence failed to inhibit leukocyte–endothelium interactions. These effects of C-peptide were associated with decreased surface expression of the cell adhesion molecules P-selectin and ICAM-1 on the microvascular endothelium. Endothelial nitric oxide synthase (eNOS) mRNA levels were increased in rats injected with C-peptide. This enhanced eNOS expression was associated with a marked increase in basal NO release from the aorta of C-peptide-treated rats. We conclude that C-peptide is a potent inhibitor of leukocyte–endothelium interaction and that this effect is specifically related to inhibition of endothelial cell adhesion molecules via maintenance of NO release from the vascular endothelium.—Scalia, R., Coyle, K. M., Levine, B. J., Booth, G., Lefer, A. M. C-peptide inhibits leukocyte–endothelium interaction in the microcirculation during acute endothelial dysfunction.


Key Words: intravital microscopy • inflammation • rat • diabetes • nitric oxide synthase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
C-PEPTIDE IS A cleavage product that comes from processing proinsulin to insulin that has recently been shown to possess biological activity in laboratory animals (1 , 2) and in humans (3 4 5) . Systemic administration of C-peptide for 5 wk has been shown to ameliorate vascular and neural dysfunction in diabetic rats (1) . Low rates of C-peptide infusion in type I diabetic patients decrease glomerular filtration and increases effective renal plasma flow (6) . Moreover, administration of C-peptide during exercise significantly increases forearm blood flow, capillary diffusion capacity, and glucose uptake (5) . More recently, Jensen et al. (2) , have reported that C-peptide evokes arteriolar dilation in skeletal muscle via a nitric oxide (NO) -mediated mechanism that appears to be enhanced by interaction with insulin.

A widely accepted feature of physiological concentrations of NO is its protective role in several models of inflammation, including ischemia-reperfusion (7 , 8) , hypercholesterolemia (9 , 10) , and diabetic microangiopathy (11) . Several investigators (12 13 14) have shown that impaired release of NO from inflamed vascular beds results in increased leukocyte–endothelium interaction via up-regulation of endothelial cell adhesion molecules (15) . Under these conditions, numerous leukocytes adhere to the vascular endothelium (16) and many transmigrate across the endothelium, thus potentiating endothelial dysfunction and tissue injury (17 18 19) . In addition, systemic administration of NO donors during acute (8 , 20 , 21) and chronic experimental models (10) of inflammation preserves endothelial function and attenuates proinflammatory interactions between circulating leukocytes and the vascular endothelium.

Therefore, the purpose of the present study was to examine the effect of C-peptide on leukocyte–endothelium interaction in vivo. Using intravital microscopy of rat mesenteric venules, we illustrated that systemic administration of C-peptide inhibits leukocyte–endothelium interaction induced by either thrombin or L-NAME. This protective effect of C-peptide was associated with inhibition of endothelial cell adhesion molecule expression in the microvascular endothelium. Finally, this immunomodulatory action of C-peptide was associated with increased expression of endothelial nitric oxide synthase (eNOS) mRNA, as well as augmented release of nitric oxide from the vascular endothelium. Therefore, our data suggest a novel role for C-peptide in attenuating inflammatory disorders of the vasculature. These data may also help to explain the mechanism of the beneficial effects of C-peptide in diabetic vascular dysfunction reported by other investigators.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND 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.

Human C-peptide
Biosynthetic human C-peptide was purchased from Sigma (St. Louis, Mo.) and an additional supply donated by Eli Lilly (Indianapolis, Ind.). Both batches behaved identically in our system. C-peptide was dissolved in 0.5 M acetic acid, with further dilutions being made with 0.9% saline as previously reported (2) . Each animal received a single administration of C-peptide at a dose of 7 or 70 nmol/kg as an intravenous (i.v.) bolus. Previous observations had shown that injection of 130 nmol/kg of human C-peptide to rats increases physiological plasma levels of C-peptide by 10-fold, peaking at 3 h postinjection (1) . Therefore, the higher dose of C-peptide used in the present study was likely to result in approximately a fivefold increase in plasma level of C-peptide. This dose of C-peptide (i.e., 70 nmol/kg) was selected on the basis of its activity in correcting vascular permeability and neuronal dysfunction in diabetic rats (1) and in improving cutaneous microvascular blood flow in humans (4) . In control experiments, a scrambled peptide (Eli Lilly) was used in which the amino acid composition was identical to that of the human C-peptide, but the sequence was randomized. This peptide was inactive and had the following sequence: L-G-G-G-P-Q-V-G-S-L-L-A-Q-V-E-Q-E-A-E-D-G-L-E-G-S-L-A-G-Q-P-L (left, N-term to right, C-term).

Intravital microscopy recordings
Male Sprague-Dawley rats, weighing 250–275 g, were anesthetized with sodium pentobarbital (65 mg/kg) injected intraperitoneally. A tracheotomy was performed to maintain a patent airway throughout the experiment. A PE-50 polyethylene catheter was inserted in the left carotid artery and the right jugular vein for monitoring of mean arterial blood pressure and infusion of anesthetic. The abdominal cavity was opened via a midline laparotomy, and a loop of ileal mesentery was exteriorized for observation of the mesenteric microcirculation via intravital microscopy. Rats were randomly divided into one of five groups: 1) rats given C-peptide (70 nmol/kg/iv bolus) and superfused with Krebs-Henseleit (K-H) buffer, 2) rats given 0.9% saline or scrambled peptide (70 nmol/kg/iv bolus) and superfused with 50 µM L-NAME, 3) rats given 0.9% saline or scrambled peptide (70 nmol/kg/iv bolus) and superfused with 0.5 U/ml thrombin, 4) rats given 7 or 70 nmol/kg C-peptide and superfused with 50 µM L-NAME, 5) rats given 7 or 70 nmol/kg C-peptide and superfused with 0.5 U/ml thrombin. Intravital microscopy experiments were performed 30 min after administration of C-peptide and leukocyte–endothelium interactions were studied for a 2 h period. The jugular vein was used as the route for C-peptide administration. In control experiments, an equal volume of saline containing less then 0.001% acetic acid (vehicle) was given to the rats; leukocyte–endothelium interactions were monitored over 120 min after K-H superfusion as well as 0.5 U/ml thrombin or 50 µM L-NAME. The concentration of L-NAME used does not result in full inhibition of NOS.

Intravital microscopy of rat mesenteric tissue was performed according to a previously described method (22) . Briefly, the ileum and mesentery of anesthetized animals were placed in a temperature controlled Plexiglas chamber. A modified K-H solution alone or K-H solution containing either 50 µM L-NAME or 0.5 U/ml thrombin was used to superfuse the rat mesentery and the mouse intestine. A Microphot microscope, (Nikon Corp., Tokyo, Japan) was used to visualize the mesenteric venules in the rat. The image was projected by a high-resolution color video camera (DC-330, DAGE-MTI, Inc. Michigan City, Ind.) onto a color Sony high-resolution 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 Inc, Nampa, Idaho). Red blood cell velocity was determined on-line using an optical Doppler velocimeter (23) . Red blood cell velocity (V) and venular diameter (D) were used to calculate venular wall shear rate (g) using the formula g = 8 (Vmean/D) (Vmean=Vrbc/1.6), where V = velocity and D = diameter (24) . Leukocytes were considered to be rolling if they were moving at a velocity significantly slower than that of red blood cells. A leukocyte was judged to be adherent if it remained stationary for > 30 s. Adherence is expressed as the number of leukocytes adhering to the endothelium/100 µm of vessel length. The number of extravasated leukocytes in the rat mesenteric tissue was counted in the tissue area adjacent to the postcapillary venules and normalized with respect to perivascular area (20x100 µm). Control experiments performed in rat given 0.9% NaCl and superfused with K-H buffer alone exhibit a steady, low number of rolling, adherent, and transmigrated leukocytes in the mesenteric microcirculation over the 2 h observation time used (data not shown). This result is not different from that observed in C-peptide-treated rats superfused with KH buffer alone.

Immunohistochemistry
Immunohistochemical localization of P-selectin and ICAM-1 was determined after intravital microscopy was completed. Both the superior mesenteric artery and superior mesenteric vein were rapidly cannulated for perfusion fixation of the small bowel as described previously (22) . Briefly, the ileum was first washed free of blood by perfusion with K-H buffer and then perfused with iced 4% paraformaldehyde in phosphate-buffered 0.9% NaCl for 5 min. A 3 to 4 cm segment of ileum was isolated from the perfused intestine and fixed in 4% paraformaldehyde for 90 min at 4°C. The ileum was then cut into smaller rings, and the tissue dehydrated using graded acetone washes at 4°C. Tissue sections were embedded in plastic (Immunobed: Polysciences Inc., Warrington, Pa.), and 4 µm-thick sections were cut and transferred to Vectabond coated slides (Vector Laboratories, Burlingame, Calif.).

Immunohistochemical localization of P-selectin and ICAM-1 was investigated using the avidin-biotin immunoperoxidase technique (Vectastain ABC Reagent; Vector Laboratories) according to a described method (25) . Tissue sections were treated with 0.25% trypsin (Sigma) to improve reagent penetration. Blocking serum (horse) was applied to the tissue for 30 min to reduce nonspecific binding and the tissue sections were incubated for 24 h with specific primary antibodies. P-selectin was detected with the monoclonal antibody PB1.3 (Cytel Corp. San Diego, Calif.) at a dilution of 1/100, while ICAM-1 was immunolocalized using the monoclonal antibody 1A29 (Genzyme, Cambridge, Mass.) at a dilution of 1/500. PB1.3 is a monoclonal antibody that only recognizes P-selectin expressed on the endothelial cell surface and does not bind to intracellular P-selectin (26) . 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 adhesion molecules was determined by microscopic observation of the brown peroxidase reaction product on the microvascular endothelium of the tissue sections. Positive staining was defined as a vessel displaying brown reaction product on greater than 50% of the circumference of its endothelium. Fifty ileal venules per tissue section were examined in each of 20 sections and the percentage of positive staining vessels was tallied.

Effect of C-peptide on eNOS activity and NO release
Quantification of eNOS mRNA expression by ribonuclease protection assays
Immediately after intravital microscopy was performed, rat lungs were removed for total RNA extraction. Lungs were used since they are a good source of microvascular endothelial cells due to their high density in the microvascular bed. Total RNA was extracted from the lung tissue using the acid guanidium-phenol-chloroform extraction method described by Chomczynski and Sacchi (27) . Subsequently, 0.4 ml of 2 M sodium acetate, pH 4.0, 4 ml of phenol (water saturated), and 0.8 ml of chloroform-isoamyl alcohol mixture (49:1) were added to homogenate. RNA was precipitated with equal volumes of 2-propanol. RNA pellet was washed with 75% ethanol and dissolved in nuclease-free water. First-strand cDNA synthesis was performed at 42°C for 20 min using 2 mg total RNA from rat lung in a 20 ml reaction mixture containing 50 mM Tris/HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 20 mM dNTP (i.e., an equal mixture of dATP, dGTP, dCTP and dTTP), 1 mM oligo(dT), and 200 U superscript reverse transcriptase (RT; Life Technologies, Inc./BRL, Gaithersburg, Md.), as described previously (28) . The eNOS fragment was amplified using forward primer (5'-GACTGGCATTGCACCCTTCCGG-3'), corresponding to 3049 to 3070, and reverse primer (5'-GTTGCCAGAATTCTCTGCACGG-3'), corresponding to 3402 to 3381 of the mouse eNOS gene (GenBankTM accession number U53142). This polymerase chain reaction (PCR) product was cloned using PCR 2.1-TOPO Cloning Kit (Invitrogen Corp., Carlsbad, Calif.). The fragment of eNOS cDNA was verified by sequencing the insert in the plasmid (GenBankTM accession number AF093837). For ribonuclease protection assays, a XbaI-SstII fragment containing 251 bp of rat eNOS cDNA was cloned in the pBluescript II SK+ vector (Stratagene, La Jolla, Calif.). The plasmid was digested with HindIII and used as a template for in vitro transcription of a 367-base radiolabeled antisense probe containing a 251 base-protected fragment using T3 RNA-polymerase (Boehringer Mannheim, Indianapolis, Ind.) in the presence of {alpha}[32P-UTP] (Amersham Corp., Arlington Heights, Ill.). Expression of mRNA was quantified using storage phosphor technology (Amersham Corp.). Intensity of each eNOS band mRNA was normalized for ß-actin mRNA. Mouse ß-actin plasmid for synthesis of antisense RNA probe was obtained from Ambion (Austin, Tex.).

Quantification of NO released from isolated aortic segments
Freshly isolated rat aortic rings were used as the source of primary endothelial cells. Thoracic aortas were rapidly isolated from control rats given 0.9% saline and C-peptide-injected rats at either 1 or 3 h postinjection. Aortas were immersed into warm oxygenated K-H solution and cleansed of adherent fat and connective tissue. Rings of 6–7 mm length were subsequently cut and opened from randomly selected areas of the aorta and finally fixed by small pins, with the endothelial surface up, in 24-well culture dishes containing 1 ml K-H solution. After equilibration at 37°C, NO released into the K-H solution was measured according to the method of Guo et al. (29) , using an internally shielded polarographic NO electrode connected to a NO meter (Iso-NO, World Precision Instruments, Inc., Sarasota, Fla.). Calibration of the NO electrode was performed daily before each experimental protocol.

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, adherence, and transmigration and arterial blood pressure and shear rates were analyzed by ANOVA, incorporating repeated measurements. Probabilities of 0.05 or less were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Intravital microscopy
We examined mesenteric venules ranging from 36 to 41 µm in diameter (mean±SE), and there was no difference in venular diameter among any of the groups studied. The mean venular shear rates were also very similar in all groups, ranging from 605 ± 44 to 631 ± 52 s-1. Similarly, there was no significant difference in initial mean arterial blood pressures among any of the groups of rats after all surgical procedures were completed. Mean arterial blood pressures ranged between 138 ± 10 and 145 ± 6 mmHg over the 2 h observation period. In addition, no significant systemic effect was recorded after the exposure of the rat mesentery to either 0.5 U/ml thrombin or 50 µM L-NAME. These findings clearly indicate that the adhesive interactions observed between circulating leukocytes and the microvascular endothelium were not due to changes in hemodynamics brought about by the superfusion with thrombin or L-NAME or by systemic administration of either 7 or 70 nmol/kg C-peptide.

Thrombin (0.5 U/ml) markedly increased both leukocyte rolling (Fig. 1 ) and adherence (Fig. 2 ) 60–120 min after superfusion. However, both leukocyte rolling and adherence were virtually abolished in thrombin-stimulated rats pretreated with 70 nmol/kg C-peptide (Fig. 1 and 2) . Similarly, after thrombin stimulation leukocyte transmigration significantly increased from 2 ± 1.2 to 8.5 ± 0.8 cells/100 x 20 µm perivascular space (P<0.01), and this response was also attenuated by C-peptide treatment (3.6±0.2 cells/100x20 µm perivascular space; P<0.05 vs. thrombin alone). These effects of C-peptide were also dose related, as shown in Figs. 1 and 2 . Thus, administration of a lower concentration of C-peptide (i.e., 7 nmol/kg) failed to inhibit thrombin-stimulated leukocyte rolling (Fig. 1) and only partially attenuated leukocyte adherence (Fig. 2) . To exclude potential nonspecific actions of C-peptide on leukocyte–endothelium interaction in the rat mesenteric microcirculation, we also studied leukocyte rolling, adherence, and transmigration in rats given 70 nmol/kg of a control scrambled sequence of C-peptide. The scrambled sequence of C-peptide was unable to attenuate thrombin-induced leukocyte rolling, adherence, and transmigration in mesenteric venules exposed to 0.5 U/ml thrombin (Figs. 1 and 2) . Thus, in rats given the scrambled sequence of C-peptide, thrombin superfusion increased leukocyte rolling from 12 ± 4 to 60 ± 8 cells/min, leukocyte adherence from 3 ± 1 to 12 ± 3 cells/100 µm, and leukocyte transmigration from 1.5 ± 0.5 to 9 ± 2.5 cells/100 x 20 µm perivascular space (P>0.05 from thrombin-superfused rats given 0.9% NaCl). Therefore, the physiological properties observed for the naturally occurring sequence of C-peptide were not due to nonspecific interaction of its peptidic structure with biological systems.



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  		Figure 1. Leukocyte rolling in the rat mesenteric microvasculature after superfusion of the mesentery with 0.5 U/ml thrombin. Bar heights show number of rolling leukocytes per minute. All values are means ± SE for number of rolling cells observed at 0, 30, 60, 90, and 120 min for each group. Numbers in parentheses indicate numbers of rats studied. C-peptide was administered to the rats at a dose of 7 or 70 nmol/kg, as an i.v. bolus.



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  		Figure 2. Leukocyte adherence in the rat mesenteric microvasculature after superfusion of the mesentery with 0.5 U/ml thrombin. Bar heights show number of adhering leukocytes per 100 µm of postcapillary venular endothelium. All values are means ± SE for number of adherent cells observed at 0, 30, 60, 90, and 120 min for each group. Numbers in parentheses indicate numbers of rats studied. C-peptide was administered to the rats at a dose of 7 or 70 nmol/kg, as an i.v. bolus.

In parallel experimental studies, we investigated the effects of C-peptide treatment on leukocyte rolling, adherence, and transmigration induced by superfusion of the rat mesentery with 50 µM L-NAME, a concentration partially inhibiting NO biosynthesis. Compared to K-H-superfused mesenteries, superfusion of the mesenteric tissue with 50 µM L-NAME for 120 min increased leukocyte rolling fourfold (from 13±5 to 54±9 cells/min; P<0.01), leukocyte adherence fivefold (from 2.5±0.8 13±3 cells/100 µm P<0.01), and leukocyte transmigration fivefold (1.5±0.4 to 8.5±2 cells/area; P<0.01). Systemic administration of C-peptide also significantly inhibited the L-NAME-induced increase in leukocyte–endothelium interactions to a degree comparable to its effects on thrombin-stimulated mesenteries. In particular, leukocyte rolling, adherence, and transmigration induced by L-NAME superfusion were attenuated to 10 ± 2 cell/min (P<0.01), 3 ± 1 cells/100 µm (P<0.01), and 4 ± 1 cells/area (P<0.05), respectively. Therefore, systemic administration of C-peptide to the rat is able to inhibit leukocyte–endothelium interaction after exposure of the mesenteric microvasculature to either inflammatory stimuli or NOS inhibition.

Immunohistochemical localization of cell adhesion molecules
Surface expression of two major adhesion molecules, P-selectin and ICAM-1, was investigated on the microvascular mesenteric endothelium of control rats, thrombin-superfused rats given 0.9% saline, and thrombin-superfused rats treated with 70 nmol/kg C-peptide.

The percentage of venules staining positively for P-selectin in ileal sections from control rats superfused only with K-H buffer was consistently low (Fig. 3 , upper panel). However, superfusion with 0.5 U/ml thrombin for 120 min resulted in an increased expression of P-selectin as quantified by the percentage of venules staining positively for P-selectin (P<0.001). This represents a statistically significant increase in the surface expression of P-selectin under these experimental conditions. This increased P-selectin expression on the venular endothelium was significantly attenuated by the i.v. infusion of 70 nmol/kg C-peptide (Fig. 3 , upper panel). The degree of endothelial ICAM-1 expression was also investigated (Fig. 3 , lower panel). There was minimal expression of ICAM-1 in the microvascular endothelium of control rats superfused with K-H. However, after thrombin superfusion, the number of positive-staining venules increased significantly to 52 ± 3 in the untreated rat group (P<0.01 vs. control). In contrast, thrombin-superfused rats given C-peptide exhibited a significantly lower expression of ICAM-1 on the microvascular endothelium. Thus, systemic administration of C-peptide significantly attenuates increased venular surface expression of P-selectin and ICAM-1 during acute inflammation of the microcirculation. These data are consistent with functional changes in leukocyte rolling and adherence shown in Figs. 1 and 2 .



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  		Figure 3. Immunohistochemical summary of P-selectin expression (upper panel) and ICAM-1 expression (lower panel) on rat ileal venules. Percentage of venules staining positive for P-selectin and ICAM-1 in all experimental groups of rats. Bar heights represent mean values; brackets indicate ± SE. Numbers at base of bars indicate the numbers of rats studied in each group. Twenty sections were studied in each rat.

Effect of C-peptide administration on NO synthesis/release
C-peptide increases NOS mRNA expression
Levels of mRNA expression codifying for eNOS were assessed in control rats given saline or rats injected with 70 nmol/kg C-peptide, using a ribonuclease protection assay. As shown in Fig. 4 , upper panel, the intensity of each eNOS mRNA band was normalized to that of ß-actin. After systemic administration of C-peptide to the rat, eNOS transcripts were significantly increased in the lung. In particular, eNOS transcripts increased 43 ± 10% (P<0.01 vs. control rat tissue), as quantified by densitometric analysis (Fig. 4 , lower panel). Thus, systemic administration of the human sequence of C-peptide induces de novo mRNA expression codifying for eNOS in the rat.



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  		Figure 4. Effect of C-peptide on eNOS mRNA levels. Upper panel: Representative polyacrylamide gels used in typical ribonuclease protection assay comparing eNOS mRNA expression in lungs of control and C-peptide-injected rats. Gels were run at the end of the 150 min intravital microscopy observation period. Compared to lane 1 containing eNOS mRNA levels from control rats, there is a significant increase in eNOS mRNA in lung isolated from C-peptide-injected rats (lane 2, P<0.01). Lower panel: Densitometric analyses of eNOS mRNA in rat lung. Bar heights represent mean values; brackets indicate ± SE for 4 experiments.

C-peptide increases basal release of NO from isolated rat aortic segments
We detected a small basal level of NO release in the range of 12 ± 1 nmol/g of tissue in aortic rings isolated from control rats given saline (Fig. 5 ). One and 3 h after rats were given the bolus dose of C-peptide, the basal release of NO measured in aortic rings increased from 12 ± 1 to 24 ± 3 nmol/g tissue at 1 h and 35 ± 4 nmol/g tissue at 3 h, respectively (P<0.01). In contrast, aortic rings isolated from rats injected with control scrambled C-peptide (70 nmol/kg) did not show significant increased release of NO both at 1 and 3 h [basal release of NO was 10±2 nmol/g (n=12) and 11 ± 3 nmol/g (n=12) of tissue, respectively; P>0.05 vs. aortic rings from saline-injected rats]. Moreover, the addition of a full NOS inhibitory concentration of L-NAME (i.e., 100 µM) totally inhibited NO release in aortic rings obtained from both control rats and C-peptide-treated rats (Fig. 4 , lower panel). Therefore, systemic administration of C-peptide to the rats increases endothelium-derived nitric oxide release in the rat aorta. C-peptide-induced NO release is likely mediated via potentiation of NOS activity on the vascular endothelium as confirmed by increased mRNA NOS levels.



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  		Figure 5. Effect of C-peptide on NO release. Basal release of nitric oxide expressed as nanomoles per gram of tissue. NO release was measured in isolated rat aortic rings obtained from control rats given vehicle and rats injected with 70 nmol/kg C-peptide. Bar heights are means; brackets are ± SE. L-NAME (100 µM), inhibited basal release of NO in all experimental groups of rats.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The major finding in this study is that i.v. administration of a single bolus of C-peptide attenuates inflammation induced leukocyte–endothelium interaction in the microcirculation. The mechanism of this inhibitory effect of C-peptide on leukocyte–endothelium interaction was mediated by inhibition of vascular cell adhesion molecule expression associated with increased release of endothelium-derived nitric oxide. These conclusions are based on the following observations: 1) C-peptide inhibits leukocyte–endothelium interaction in vivo in the splanchnic microvasculature of rats; 2) C-peptide markedly suppresses up-regulation of endothelial cell adhesion molecules in this vascular bed during inflammation; 3) eNOS mRNA is increased in lung tissue obtained from C-peptide-treated rats; and 4) a threefold increase in the basal release of NO from the aortic endothelium occurs in rats injected with C-peptide.

C-peptide, which is released from the pancreatic ß-cells into the circulation in equimolar amounts to insulin, fulfills an important function in the assembly of the two-chain insulin structure, but has otherwise been considered biologically inactive. However, recent experimental and clinical studies have demonstrated that systemic administration of C-peptide to diabetic animals as well as to patients with insulin-dependent diabetes mellitus elicits several important regulatory effects. Thus, injection of human C-peptide prevents vascular and neuronal dysfunction in diabetic rats (1) . In humans, short-term substitution of C-peptide (1–3 h) decreases glomerular hyperfiltration (6) , augments whole body and skeletal muscle glucose utilization (5) , improves autonomic nerve function (30) , and promotes redistribution of microvascular skin blood flow (4) . More recently, Rigler et al. (31) have shown that C-peptide binds to specific G-protein-coupled receptors on human endothelial cell membrane, thus providing a specific molecular basis for its biological effects.

However, the mechanisms underlying the cytoprotective effects of C-peptide during inflammatory events of the microcirculation remain unclear (3) . Our results are the first to show a novel, important mechanism of the protective effect of C-peptide during acute inflammatory states in the splanchnic microcirculation. Recent investigation has indicated that stimulation of eNOS may contribute to the observed physiological effects of C-peptide (2) . It is well established that NO is a physiologically important modulator of leukocyte adhesion to the vascular endothelium (13 , 14) . A number of previous studies have clearly demonstrated antiinflammatory properties of NO both in vivo and in vitro (8 , 14 , 32 , 33) . Administration of authentic NO (21 , 34) and NO donors (8) have proved to be highly beneficial in a variety of inflammatory disorders including ischemia-reperfusion and hypercholesterolemia (10 , 35) . Furthermore, we have recently shown that NO-releasing agents inhibit leukocyte–endothelium interactions induced by activation of the microvascular endothelium with either L-NAME or thrombin (36) . These two stimuli are of particular interest because they affect either the NO pathway or the homeostasis of the blood-endothelial cell interface. In addition, Davenpeck et al. (37) have shown that that nitric oxide inhibits the expression of cell adhesion molecules on the vascular endothelium. More recently, this effect was shown to be due in part by inhibiting NF-{kappa}B (38) . Increased expression of adhesion molecules, such as P-selectin and ICAM-1, on the endothelial cell surface exerts a crucial proinflammatory role by promoting leukocyte–endothelium interactions (39 40) and subsequent leukocyte extravasation into inflamed tissue (41) . Therefore, inhibition of adhesion molecule expression is likely to be an essential mechanism of the antileukocytic actions of NO.

In this study we provide strong evidence that systemic administration of C-peptide increases release of NO from the vascular endothelium in the face of enhanced leukocyte–endothelium interaction induced by thrombin or L-NAME. This result is consistent with recent findings showing that C-peptide dilates skeletal muscle arterioles from normal rats via a nitric oxide mediated mechanism that is synergized by insulin (2) . Although we did not administer exogenous insulin together with C-peptide, it is likely that physiological levels of endogenous insulin in the rat blood exerted a synergistic effect with C-peptide in our experimental model of inflammation.

In the present study, we found that C-peptide increases eNOS mRNA levels in endothelial cells, which was associated with an increased release of NO from the aortic endothelium. The mechanism by which C-peptide up-regulates eNOS activity needs to be further investigated. However, one possibility is that C-peptide inhibits degradation of eNOS mRNA. Other proteins such as glycosylated products of albumin were recently found able to enhance eNOS mRNA degradation (42) . In addition, C-peptide induces release of endothelial NO in the low nanomolar range, thus not affecting systemic blood pressure. This result agrees with previous work showing that with authentic NO gas, much higher concentrations of 170–350 nmol/l are required in order to induce arterial relaxation using authentic NO gas (43) .

Due to this NO-potentiating property of C-peptide, it is likely that the C-peptide-induced NO release is responsible for down-regulating leukocyte–endothelium interaction. The precise mechanism of the immunomodulatory function of C-peptide it is related to expression of cell adhesion molecules in vivo. We have demonstrated that C-peptide inhibits leukocyte–endothelium interactions by attenuating P-selectin and ICAM-1 expression on the vascular endothelium. However, other adhesion molecules, such as VCAM-1 on the vascular endothelium and CD11/CD18 on circulating leukocytes, may also be modulated by C-peptide in chronic models of inflammation (e.g., diabetes). Our experiments were conducted using nondiabetic rats. Previous studies have indicated that the vascular effects of C-peptide are most marked in type I diabetic animals (1) and insulin-dependent diabetic patients (3) . Our investigations provide a preliminary understanding of the antiinflammatory action of C-peptide. Further experimentation in diabetic animal models will be necessary to elucidate the significance of the role of C-peptide as well as its potential therapeutic effect in diabetic microvascular diseases.

In summary, we have demonstrated that a single i.v. dose of human C-peptide markedly attenuates leukocyte–endothelium interaction in a well-established experimental model of inflammation. In particular, C-peptide inhibited leukocyte rolling, adhesion, and transmigration in rat mesenteric venules, attenuated cell surface expression of vascular adhesion molecules in the microcirculation, and increased release of nitric oxide from the aortic endothelium. To our knowledge, this is the first in vivo evidence showing that potentiation of NO release via systemic administration of C-peptide results in attenuation of leukocyte recruitment in inflamed vascular areas. Since inhibition of leukocyte–endothelium interaction has been found beneficial in several cardiovascular disorders, this effect may be important in understanding the physiological role and therapeutic significance of C-peptide in chronic disorders of the microcirculation such those observed in diabetes mellitus.


   ACKNOWLEDGMENTS
 
This work was supported in part by research grant no. 1–2000-68 from the Juvenile Diabetes Foundation International to R.S. We thank Dr. Alex Michenko and Ms. Irina Opentanova for their expert technical assistance in the molecular biological measurements used in this study. K.M.C. and B.J.L. are NIH Summer Medical Student Research Trainees (HL-07845); G.B. is a predoctoral trainee at NIH (HL-07599).

Received for publication March 23, 2000. Revision received May 5, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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