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Am J Physiol Heart Circ Physiol 288: H1257-H1264, 2005. First published November 11, 2004; doi:10.1152/ajpheart.00856.2004
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Fibrinogen and fragment D-induced vascular constriction

David Lominadze, Nina Tsakadze, Utpal Sen, Jeff C. Falcone, and Stanley E. D'Souza

Department of Physiology and Biophysics, Health Sciences Center, University of Louisville, Kentucky

Submitted 20 August 2004 ; accepted in final form 5 November 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Elevated fibrinogen (Fg) concentration in blood is a high risk factor for many cardiovascular diseases. We hypothesize that Fg and its early degradation product, fragment D, may result in arterial constriction by binding endothelial intercellular adhesion molecule-1 (ICAM-1). The vasoconstriction induced by Fg and fragment D was studied in third- and second-order arterioles (3As and 2As, respectively) of Sprague-Dawley rat cremaster muscle in vivo, in aortic and femoral artery rings, and in the segments of first-order arterioles (1As) isolated from rat cremaster muscle. Intravascular infusion of Fg induced significant constriction of 3As and 2As (by 33.4 ± 3.4 and 23.7 ± 4.3%, respectively) in vivo and was abolished in the presence of the specific endothelin type A receptor blocker BQ-610. Fg and fragment D produced significant constriction of both aortic and femoral artery rings. Isolated 1As constricted in response to Fg (0.3 µM) and fragment D (3 µM) by 31 ± 1.4 and 12 ± 1.5%, respectively. Fluorescently labeled Fg and fragment D bound to the vascular wall, whereas albumin bound to a significantly lesser degree. The binding of Fg and fragment D to the arteriolar wall and constriction of aortic and femoral artery rings as well as isolated 1As were abolished in the presence of anti-Fg and anti-ICAM-1 antibodies. These results indicate that binding of Fg and fragment D to the vascular wall through ICAM-1 may contribute to the increased vascular tone and resistance that compromise circulation.

arteries; endothelin; endothelium; intercellular adhesion molecule-1

ELEVATED PLASMA FIBRINOGEN (Fg) concentration typically occurs during hypertension (21, 23), diabetes (25), and stroke (9). Increased blood Fg concentration results in an increase in blood viscosity (20, 23) and, therefore, an increase in blood flow shear stress (7). These in turn contribute to increased peripheral vascular resistance and lead to a reduction of blood flow in muscle, which exacerbates complications during hypertension (40). In addition, higher blood viscosity-induced increases in blood flow shear stress can activate endothelial cells (8, 38) and platelets (36). Endothelial cell activation results in expression of adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1) (39). Fg is a ligand for ICAM-1 (18, 41), and as such, Fg binding to shear stress-activated platelets through platelet GPIIb/IIIa (34) may serve as a bridging mechanism for platelet adhesion to the endothelium (5). Therefore, elevated blood Fg content may lead to a higher predisposition for platelet thrombogenesis by triggering both platelet and endothelial activation mechanisms.

Fg deposition onto the microvascular wall of mice after ischemia-reperfusion has been demonstrated elsewhere (26). This Fg deposition was partially reduced in an ICAM-1-mutant mouse, which suggests that Fg deposition at the vascular endothelium mainly occurs through endothelial ICAM-1. Hicks et al. (16) showed that Fg has a vasodilatory effect on the human saphenous vein. The authors suggested that this vasorelaxation could be a result of Fg binding to endothelial ICAM-1 and was induced by the expression of vasoactive mediators other than nitric oxide and prostacyclin.

Various reports show the vasoactive effects of isolated peptides derived from plasmin digestion of fibrin and Fg in several vascular beds, including the lung (17), heart (27, 33), femoral artery (37), and mesenteric arteries (3). However, the mechanism of these effects still remains unclear. An attempt to study vasoconstriction of rat pulmonary artery rings induced by early digestion products of Fg failed to show any effect of these Fg fragments including fragment D (6). The effects of vascular constriction by Fg degradation products on the arterial rings were tested in the presence of 4 x 10–8 M phenylephrine (PE; Ref. 6), which induces ~30–40% of maximal contraction. Therefore, the presence of PE in the referred study (6) could have had a masking effect on fragment D-induced vasoconstriction.

The hypotheses tested in this study were whether Fg and its early digestion product fragment D bind to arterial endothelial ICAM-1 and result in vasoconstriction. We show here for the first time that Fg-induced vasoconstriction may be mediated by endothelin, and that Fg and its fragment D-induced vasoconstrictions are due to binding of these proteins to vascular ICAM-1. The results of the present study suggest that this endothelial cell adhesion molecule has a significant role in Fg- and fragment D-induced signaling that results in arterial constriction.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Approval for these experiments was obtained from the University of Louisville Animal Care and Use Committee.

Reagents and antibodies. Human plasma Fg (FIB 3L, plasminogen, fibronectin, and von Willebrand factor depleted) was purchased from Enzyme Research Laboratories (Lafayette, IN). The purity of the protein was confirmed by Coomassie-stained SDS-PAGE gel (10%) analysis under reducing conditions. Fragment D was obtained by plasmin digestion of Fg and was purified by ion-exchange chromatography using a 0–200-mM NaCl gradient as described earlier (12, 35). The molecular mass of fragment D was 100 kDa according to an estimate obtained by 5–15% gradient SDS-PAGE analysis. Fragment D was dialyzed against phosphate-buffered saline (PBS; pH 7.4).

A function-blocking monoclonal antibody (clone 94017) directed against ICAM-1 was prepared from the serum of rabbits injected with Escherichia coli-expressed ICAM-1 as described earlier (12). An isotype-matched nonblocking antibody R98 directed against the cytoplasmic sequence of ICAM-1 was also produced according to the method described previously (41). The anti-Fg antibody was obtained from Dako (Carpinteria, CA). Bovine serum albumin (BSA; minimum 99% purity) and mouse IgG were purchased from Sigma (St. Louis, MO). The secondary rabbit anti-hamster IgG antibody was obtained from Open Biosystems (Huntsville, AL). The Fg and BSA solutions were prepared fresh for each experiment by dissolution in PBS. The specific endothelin A-type (ETA) receptor blocker BQ-610 was purchased from Peninsula Laboratories (San Carlos, CA).

Protein labeling with fluorescence. Fg, fragment D, and BSA were labeled with Alexa 488 fluorescent dye using a protein labeling kit (Molecular Probes; Eugene, OR). The fluorescein-conjugated proteins were dialyzed against PBS if necessary. Concentrations of the proteins in all samples were measured using a Pierce BCA protein assay kit (Pierce; Rockford, IL).

Cremaster preparation for infusion of Fg or fragment D. Male Sprague-Dawley rats (body wt, 150–170 g) were anesthetized with pentobarbital sodium (50 mg/kg ip), and a tracheal cannula was inserted to maintain a patent airway. A carotid artery cannula was used to continuously monitor mean arterial blood pressure (MABP) and heart rate with a transducer and a Micro-Med blood pressure analyzer (Louisville, KY). The right cremaster muscle was prepared for microvascular observations as described earlier (22).

To achieve adequate perfusion of the cremaster muscle microvessels with Fg- or fragment D-containing solutions, we used a procedure developed in our department (2, 43). Briefly, a cannula (polyethylene-10) was inserted into the right femoral artery. The distal end of the cannula was connected to an infusion pump (Harvard Apparatus; Holliston, MA). The proximal end of the cannula was placed a few millimeters distal to the pedicle artery of the cremaster muscle. The arterial branches between the proximal end of the cannula and the pedicle artery were ligated during the initial surgical preparation to ensure that the infusate fully flowed into the cremaster muscle during the infusion.

After surgical preparation, the rats were positioned on the modified stage of a Nikon MM-11 microscope (Nikon; Tokyo, Japan) and allowed to equilibrate for 1 h. The images of a selected second- or third-order arteriole (2A and 3A, respectively) were video recorded for 1 h using a closed-circuit video system. The luminal diameters of the vessels were measured offline every 5 min using a video caliper device. The results for maximum vascular constriction are presented.

Fg- and fragment D-induced vasoconstriction in vivo. After equilibration, 100 µl of PBS were administered as a bolus injection through the femoral artery cannula. The bolus injection also served as a test to ensure that cannulation was proper and the cremaster muscle microvessels were perfused with the PBS. After 10 min of the PBS injection, a bolus injection of 100 µl of either Fg (10 mg·ml–1·100 g body wt–1), fragment D (300 µg·ml–1·100 g body wt–1), or BSA (10 mg·ml–1·100 g body wt–1) were performed. After the bolus injection, the infusion of the respective protein into the cremaster muscle circulation continued at a rate of 0.1 ml/h (43). This infused volume was calculated to be one-eighth of the normal blood flow to the cremaster muscle (28). As an additional control, to determine the effects of time and the vehicle (PBS), vasoreactivity to only PBS infusion was assessed.

To determine the possible role of ETA receptors, after 10 min of Fg infusion and observation of the arterial constriction, the specific ETA receptor blocker BQ-610 (1 µM) was added to the bath with the cremaster muscle. Arterial diameters were recorded, and the bath was changed in 5 min. This procedure was repeated two more times to ensure the reproducibility of the results.

Isolated thoracic aorta and femoral artery. The preparation of the aortic and femoral artery rings was performed according to a method described earlier (42). Briefly, male Sprague-Dawley rats (body wt, 250–300 g; Harlan; Indianapolis, IN) were anesthetized with pentobarbital sodium (50 mg/kg ip). The descending thoracic aortas and femoral arteries were excised, cleaned of connective tissues, and cut into 4-mm-ring segments. The rings were mounted between triangular stainless steel hooks and suspended under 2 g (for aortas) or 1 g (for femoral arteries) of passive tension in organ baths that contained physiological salt solution (PSS, pH 7.4) maintained at 37°C and aerated with a 95% O2-5% CO2 mixture. Isometric tension was recorded with Grass FT03 force-displacement transducers coupled to a Grass polygraph (model 7H). After the equilibration (1 h), the endothelial integrity of the vessels was assessed by recording acetylcholine (10 µM)-induced, endothelium-dependent relaxation of the vessel rings that were precontracted with PE (1 µM) (42). Vascular diameter changes were recorded continuously throughout the experiments (50–70 min). The results for maximum vascular constrictions are expressed as a percentage of the maximum response to 10–6 M PE. Because we found in our previous study (23) that the normal concentration of Fg in the whole blood of rats is ~5 µM, most of the experiments on aortic and femoral artery rings were performed using this concentration of Fg. An effect of Fg was studied in four series of experiments as follows: 1) 5, 10, and 15 µM of Fg alone; 2) 5 µM Fg in the presence of anti-Fg antibody (1:100 dilution); 3) 5 µM Fg in the presence 1–2 µl/ml of anti-ICAM-1 antibody (2.8 mg/ml); and 4) 5 µM Fg in the presence 1–2 µl/ml of nonblocking anti-ICAM-1 antibody (3.2 mg/ml). The role of fragment D (3 µM) was studied in two series of experiments: with fragment D alone, and in the presence of anti-Fg antibody (1:100 dilution). To test whether the osmotic stress had an effect on vascular tone of aortic or femoral artery rings, in some experiments the vascular responses to BSA were studied. The final concentration of 440 µM BSA was chosen based on the known content of albumin in rat plasma (4). The foaming of proteins due to the bubbling of the tissue bath was avoided by decreasing the rate of the aeration of the baths. At the end of the experiments, after vessels were washed and allowed to equilibrate for 15 min, the viability of an intact vascular endothelium was tested by constriction of the vessels with PE and relaxation with acetylcholine.

Isolated cremaster muscle first-order arterioles. First-order arterioles (1As) were isolated from rat cremaster muscles and cannulated according to the method described previously (10, 11). Briefly, a 2- to 3-mm segment of 1A void of branches was isolated, excised, and then cannulated with siliconized glass micropipettes. When necessary, the vessels were denuded of endothelium by sliding the glass micropipettes through the vessels several times. The preparation was then transferred to an inverted microscope (Nikon Diaphot 200) and pressurized to its approximate in vivo pressure of 90 cmH2O (29). The vessel was equilibrated for 1 h. The viability of the isolated arteriolar preparation was determined by the development of spontaneous (intrinsic) tone and reactivity to acetylcholine (10–7 M).

Before each experiment, autofluorescence of the vessel was recorded over a standard range of camera gains. One of the fluorescently labeled protein (Fg, fragment D, or BSA) solutions was then infused into the vessel at a pressure gradient of 45 cmH2O by moving the reservoirs connected to the micropipettes in equal yet opposite directions as described previously (11). After the presence of the protein inside the vessel was detected, flow was stopped, and the vessel was pressurized to its normal pressure of 90 cmH2O. During the 30-min incubation period, a modified (red) bright-field image of the vessel was continuously recorded on a videocassette recorder for 1 min with 5-min intervals for the later diameter measurements offline. After incubation, the vessel luminal contents were washed out with PBS perfusion for 20 min. The digital images of the fluorescently labeled protein bound to the vessel wall were recorded along the vessel length at one of the gain settings used during the vessel autofluorescence recordings. The images were taken from the vessel's bottom (close to the objective) wall and away from the cannulas. The washing was continued for another 20 min. After the last wash, the vessel was pressurized, and digital images of the bound protein were recorded again along the vessel length. Pixel intensities were corrected to a consistent camera gain. A greater fluorescence intensity of the bound protein after the first wash compared with that after the second wash confirmed the detachment of nonspecifically bound protein.

In the experiments with function-blocking antibodies or IgG, the vessels were perfused with an antibody (or with IgG) first and then pressurized and incubated for 30 min. The vessel's content was then substituted with the experimental protein (Fg, fragment D, or BSA) dissolved in PBS that contained the appropriate antibody (or IgG) at the same concentration used before and was incubated for another 30 min.

The effects of Fg were studied in four series of experiments where the vessels were treated with 0.3 µM Fg, as follows: 1) alone, or in the presence of 2) anti-Fg antibody (1:100 dilution), 3) anti-ICAM-1 (100 µg/ml), and 4) IgG (100 µg/ml). To determine whether fragment D binds to the vascular wall, the protocol mentioned above was repeated with fragment D infused into the segment of isolated 1As. The effects of fragment D on vascular tone were studied in three series of experiments where the vessels were incubated with 3 µM fragment D alone, in the presence of anti-Fg antibody (1:100 dilution), and in the presence of anti-ICAM-1 antibody (100 µg/ml). In a separate group of experiments, the vessels were incubated with 440 µM BSA. In yet another separate series of experiments, arteriolar responses to various doses of PE were recorded.

Video microscopy and digital image processing. Observations were made using a Nikon fluorite oil x40 objective (numerical aperture, 1.30–0.80). A multi-image module (beam-splitting device) was connected to a Hamamatsu charge-couple device (CCD) camera (red-field imaging) and a Hamamatsu intensified CCD camera system (fluorescence imaging). A video caliper device (Microcirculation Research Institute; Texas A&M University) was used for arteriolar diameter measurements in 5-min intervals. Values for maximum vascular constrictions are presented. The fluorescence images were acquired on a computer using image-acquisition software (Universal Imaging). Binding of fluorescently labeled Fg, fragment D, BSA, or ICAM-1 to the vascular wall was determined by subtracting the autofluorescence intensity of the vessel (which contained any background data as well) from the total fluorescence intensity of the vessel segment in the middle of the vessel length recorded after the second wash of the vessel with PBS. If the results were obtained at different gain settings, the results were corrected to a consistent camera gain level over the linear range of the detector by using the slope from the system's intensity vs. gain curve. The results are presented as fluorescence intensity units (FIU).

Cultured rat endothelial cells and ICAM-1 expression. Endothelial cells from rat coronary, skeletal, and cremaster muscle microcirculations were a gift from Cynthia Meininger (Texas A&M University). The cells were cultured and grown according to the method described previously until they formed a complete monolayer (24). The cell lysates were collected, and expression of ICAM-1 was tested by Western blot analysis (35).

Data analysis. All data are expressed as means ± SE. Comparisons between the groups were determined by ANOVA. For comparison between the groups before and after treatment, one-way repeated measures ANOVA was used. Effects were considered significant if P < 0.05, and a pairwise comparison of means between the treatment groups was done using the Tukey test. Differences in means were considered significant if P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The effects of Fg or fragment D on vascular constriction were studied in rat cremaster muscle arterioles in vivo. An elevated Fg level in 3As resulted in a significant decrease (by 33.4 ± 3.4%) of the vessel diameter compared with its baseline value (Fig. 1A). This vasoconstriction was noticeable during the first 5 min of Fg infusion and was sustained for 50–60 min. Infusion of vehicle (PBS) did not result in any changes of the vessel diameters (Fig. 1A). Similarly, the infusion of BSA did not result in vasoconstriction (data not presented). The infusion of fragment D into the rat cremaster microcirculation resulted in significant constriction of both 2As (by 27.5 ± 7.7%) and 3As (by 31.6 ± 5.6%; Fig. 1B). These arteriolar constrictions were noticeable (as in response to Fg infusion) during the first 5 min of fragment D infusion and were sustained for ~50–60 min. In a separate series of experiments, we tested the role of ETA receptors in Fg-induced constriction of 2As and 3As. In the presence of the specific ETA receptor blocker BQ-610, Fg-induced arteriolar constrictions were abolished (Fig. 2).



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  		Fig. 1. Fibrinogen (Fg)- and fragment D-induced arteriolar constriction in rat cremaster muscle. A: constriction of third-order arterioles (3As) as a result of Fg (10 mg·ml–1·100 g body wt–1) infusion. B: constriction of second- and third-order arterioles (2As and 3As, respectively) as a result of fragment D (300 µg·ml–1·100 g body wt–1) infusion. *P < 0.05 vs. baseline diameter values.

 


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  		Fig. 2. Effects of blocking of endothelin type A (ETA) receptors on Fg-induced arteriolar constriction in rat cremaster muscle. Diameter changes of arterioles during infusion of Fg (10 mg·ml–1·100 g body wt–1) into the cremaster muscle microcirculation in the absence and presence of ETA receptor blocker BQ-610 (1 µM) are shown. A: 2A diameter changes. B: 3A diameter changes. *P < 0.05 vs. diameter values in the presence of BQ-610.

 
There were no significant differences in MABP before (107 ± 2 mmHg) and after (118 ± 4 mmHg) infusion of Fg. Similarly, MABP (106 ± 3 mmHg) did not change significantly (109 ± 3 mmHg) after infusion of fragment D. No changes in MABP were found in response to PBS and BSA infusion (data not presented). The heart rate values did not change significantly after infusion of either Fg, fragment D, PBS, or BSA (data not presented).

To determine the vasoactive effect of Fg on larger arteries, aortic and femoral artery ring isometric tensions were recorded in response to 5, 10, and 15 µM Fg concentrations in the tissue baths. Fg induced strong constriction of both aortic and femoral artery rings. Arterial responses to 5 µM Fg are shown on Fig. 3. Femoral artery rings constricted more than aortic rings in response to Fg treatment (Fig. 3). Higher concentrations of Fg (10 and 15 µM) induced constriction of aortic rings by 47.4 ± 6.7 (n = 5) and 55.8 ± 4.9% (n = 5), respectively. Constriction values for aortic rings in response to 15, 10, or 5 µM Fg were not significantly different from one another. Treatment of the femoral artery rings with Fg at high concentrations (10 and 15 µM) resulted in constriction of these vessel rings by 126.4 ± 20.5 (n = 5) and 136.3 ± 20.4% (n = 5), respectively. Constriction values for femoral artery rings in response to various concentrations of Fg were not significantly different from one another.



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  		Fig. 3. Fg (5 µM)-induced constriction of aortic (n = 5) and femoral artery (n = 15) rings in the absence or presence of anti-Fg and function-blocking and nonblocking anti-intercellular adhesion molecule-1 (ICAM-1) antibodies (n = 3). *P < 0.05 vs. Fg alone. Clearly defined {alpha}-, {beta}-, and {gamma}-chains of Fg shown by the Coomassie-stained SDS-PAGE analysis (reducing conditions) confirms the purity of the Fg (inset).

 
Fg-induced constriction of both aortic and femoral artery rings reached a maximum value in these vessels in ~5–10 min and was sustained for ~30–40 min. The 5 µM Fg-induced vasoconstriction of both aortic and femoral artery rings was significantly less (30 ± 5 and 20 ± 6% of Fg responses, respectively) in the presence of anti-Fg antibody (Fig. 3). Treatment of the vessels with BSA (440 µM) alone did not result in any changes in vascular diameters (data not presented).

Because one of the possible receptors for Fg expressed by the endothelial cells may be ICAM-1 (18), we tested whether Fg-induced constriction of aortic and femoral artery rings was mediated by Fg binding to this endothelial cell molecule. The vasoconstriction of aortic and femoral artery rings in response to Fg was significantly decreased in the presence of anti-ICAM-1 antibody (Fig. 3). The presence of nonblocking antibody against ICAM-1 did not change Fg-induced vasoconstriction of these vessels (Fig. 3).

Fragment D induced a significant constriction of both aortic and femoral artery rings (Fig. 4). Vessels reached minimal diameter values in ~3–5 min. Constriction was sustained for ~15–25 min. The presence of anti-Fg antibody drastically decreased the vasoconstriction of rings from both vessels (Fig. 4).



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  		Fig. 4. Fragment D (3 µM)-induced constriction of aortic and femoral artery rings. *P < 0.05 vs. fragment D alone. Purity of fragment D and Fg (nonreducing conditions; inset, left) and fragment D with no visible extra bands (reducing conditions; inset, right) are confirmed by the Coomassie-stained SDS-PAGE analysis.

 
The extent of ICAM-1 expression in isolated 1As treated with fluorescently labeled anti-ICAM-1 antibody is presented in Fig. 5A. The increased fluorescence intensity on the intravascular wall compared with the vascular autofluorescence suggests that ICAM-1 is expressed on the endothelial cell surface of the arterioles used in the present study (Fig. 5B). In another group of experiments, 1As were incubated with fluorescently labeled BSA that resulted in significantly lower protein binding to the vessel wall (Fig. 5, A and B) and no alteration in vascular diameter values (data not presented). In addition, probing of stimulated or nonstimulated (control) cultured rat endothelial cells from various tissues with anti-ICAM-1 antibodies confirmed that the antibodies against ICAM-1 used in the present study recognize the ICAM-1 expressed on the surface of cultured rat endothelial cells (Fig. 5C).



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  		Fig. 5. Expression of ICAM-1 in isolated 1As. A: examples of images recorded before (autofluorescence) and after the treatments of 1As with BSA (29 mg/ml) and anti-ICAM-1 (100 µg/ml). B: binding of fluorescently labeled BSA and anti-ICAM-1. *P < 0.05 vs. BSA. C: expression of endothelial ICAM-1 on cultured rat endothelial cells shown by Western blot analysis.

 
In the series of experiments on isolated 1As designed to study Fg binding to vascular endothelium, the optimal concentration of Fg that results in binding of this protein to endothelium alone was determined by evaluating the fluorescence pattern of the Fg bound to the vessel wall. Our data show that at a concentration of 0.3 µM, Fg positively binds to vascular endothelial cells at the perinuclear bulge. This was documented by a punctuated, more random distribution of the fluorescence that follows the spiral arrangement of the endothelial cells inside the vessel wall (Fig. 6A). At a higher concentration (6 µM), in addition to binding to endothelial cells, Fg also bound to vascular smooth muscle cells (VSMCs). This fact was documented by distribution of the fluorescently labeled Fg in a more distinct, circular-shape pattern that is typical for the structure of the VSMC lining (Fig. 6A). At a concentration of 1.2 µM, Fg may also have been bound to both endothelial cells and VSMCs (Fig. 6A). Treatment of the isolated 1As with Fg resulted in significant vascular constriction regardless of the Fg dose used (Fig. 6B). Therefore, to focus on Fg-to-endothelial cell binding, additional experiments were performed using Fg at a concentration of 0.3 µM. Treatment of the vessels with fluorescent dye alone did not result in any visible binding of the dye to the vessel wall and did not affect the vascular diameter values (data not presented).



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  		Fig. 6. Binding of various concentrations of Fg and resultant constriction of isolated 1As. A: Fg binds to vascular endothelium at a concentration of 0.3 µM. At higher concentrations, Fg binding of the protein to the vascular smooth muscle cells is distinctly noticeable. B: Fg-induced arteriolar constriction. *P < 0.05 vs. baseline diameter values.

 
Treatment of the isolated 1As with fluorescently labeled Fg resulted in sustained binding of Fg to the vascular wall, which was documented by significantly increased fluorescence intensity (Fig. 7, A and B). This was accompanied by a significant decrease in the arteriolar diameter (Fig. 7C). The maximum constriction occurred during the first 5–10 min of Fg treatment. This Fg-induced constriction was absent in endothelium-denuded arterioles (data not presented). Both Fg binding and Fg-induced vasoconstriction were significantly decreased in the presence of anti-Fg or anti-ICAM-1 antibodies (Fig. 7, B and C). Treatment of the vessels with only Fg-PBS solution (without anti-ICAM-1 antibody) after the anti-ICAM-1 antibody treatments restored Fg binding to the vessel wall. This was documented by the fluorescence intensity (69 ± 8.3 FIU), which was similar to the fluorescence level after the vessels were treated with Fg alone (Fig. 7B, solid bar). Fg binding to vessel walls in the presence of IgG (86 ± 32 FIU) was not different from the binding of Fg alone (74 ± 11 FIU).



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  		Fig. 7. Binding of Fg (0.3 µM) and resultant constriction of isolated 1As. A: examples of arteriolar wall images recorded after treatment with Fg alone and with Fg in the presence of anti-Fg or anti-ICAM-1 antibodies. B: Fg (0.3 µM) binding to vascular wall and inhibition of this binding with anti-Fg and anti-ICAM-1 antibodies. *P < 0.05 vs. Fg alone. C: Fg-induced vasoconstriction and its inhibition with anti-Fg or anti-ICAM-1 antibodies. *P < 0.05 vs. baseline diameter values.

 
Similar to the experiments with Fg, fragment D bound to arterial wall (Fig. 8, A and B) led to significant constriction of the 1As (Fig. 8C). Again, both fragment D binding to vascular endothelium and fragment D-induced vasoconstriction were significantly decreased in the presence of anti-Fg or anti-ICAM-1 antibodies (Fig. 8, B and C).



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  		Fig. 8. Binding and vasoactive effects of fragment D (3 µM). A: examples of images recorded after treatment with fluorescently labeled fragment D alone and with fragment D in the presence of anti-Fg or anti-ICAM-1 antibodies. B: binding of fragment D (3 µM) to the vascular wall and inhibition of this binding with anti-Fg or anti-ICAM-1 antibodies. *P < 0.05 vs. fragment D alone. C: fragment D-induced vasoconstriction and its inhibition with anti-Fg or anti-ICAM-1 antibodies. *P < 0.05 vs. baseline diameter values.

 
The degree of arteriolar constriction in response to Fg or fragment D treatments was compared with arteriolar constrictions in response to various doses of PE. Using the PE dose-response curve as a reference, our results show that Fg-induced vasoconstriction was similar to vasoconstriction induced by ~5 x 10–7 M PE. Vasoconstriction induced by fragment D was similar to arteriolar constriction induced by ~5 x 10–8 M PE.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study shows that increased content of Fg and its early degradation product fragment D, which consists of all three ({alpha}-, {beta}-, and {gamma}-) chains that are the components of intact Fg, results in constriction of arterioles of rat cremaster muscle (see Fig. 1). Fg-induced arteriolar constriction was abolished in the presence of a specific anti-ETA receptor antagonist (see Fig. 2). These results point to the newly found properties of Fg and suggest that Fg-induced arteriolar constriction is mediated through release of the potent vasoconstrictor endothelin.

The arteries of both systemic and microvascular circulations were constricted by Fg and fragment D. These include an aorta, a femoral artery, 1As, 2As, and 3As, which are considered to be resistance vessels that contribute to peripheral vascular resistance. Treatment of the vessels with albumin or with a vehicle (PBS) did not result in any changes in vascular diameters. Arteriolar diameters did not change in response to anti-Fg antibody treatment alone. However, the presence of an anti-Fg antibody almost completely abolished both Fg- and fragment D-induced vasoconstriction in all isolated vessels. These results suggest that constriction of arteries and arterioles seen in the present study are induced by both Fg and fragment D.

Higher doses of Fg or fragment D did not significantly change the vasoconstriction of aortic and femoral artery rings. This may suggest that the number of Fg-binding vascular receptors that mediate vasoconstriction is not abundant in these vessels and may be saturated even at lower doses of Fg or fragment D. Greater vasoconstriction of femoral artery rings compared with aortic rings in response to both Fg and fragment D may be a result of the structural and functional differences between these vessels.

Because it was reported that amino acid sequence 117-133 on the {gamma}-chain of Fg and fragment D is a region involved in binding to ICAM-1 (1), the specificity of Fg and fragment D interaction with vascular ICAM-1 was tested using a function-blocking antibody against ICAM-1. Indeed, Fg and fragment D binding to vascular ICAM-1 and resulting vasoconstriction were significantly abrogated by anti-ICAM-1 antibody treatment of various arteries such as aorta, femoral artery (see Figs. 3 and 4), and 1As (see Figs. 7 and 8). These data suggest that Fg and fragment D binding to arterial wall and the resultant vasoconstriction are mediated through vascular ICAM-1. However, there may be other receptor(s) of the integrin family such as endothelial {alpha}v{beta}3 and {alpha}5{beta}1 integrins (31, 32) that are also involved in Fg and fragment D binding to arterial wall.

In the present study, higher concentrations of Fg resulted in noticeable binding of the protein to VSMCs (in addition to binding to endothelial cells), which was detected by deposition of the fluorescently labeled Fg on the vessel wall with a typical dense circular-like pattern of the VSMC lining (10, 30). This binding may be a result of the gap formation in the endothelial cell layer (13), penetration of Fg through the gaps, and binding of ICAM-1 on VSMCs (19). The constriction of the arterioles occurred regardless of Fg concentration during the first 5 min after Fg treatment. These results suggest that Fg triggered a fast-acting vasoconstriction signaling mechanism that may involve release of endothelin.

It was reported (13, 14) that fragment D induced an increase in endothelial cell permeability as a result of prolonged (at least 2–4 h) exposure of the cells to the protein. In the present study, we did not find either visible or functional impairment of the endothelium as a result of Fg or fragment D treatments at the low concentrations (0.3 and 3 µM, respectively) used for isolated 1As. In addition, it is noteworthy to mention that the vessels (1As) were treated with Fg or with fragment D for no longer than 30 min. Therefore, constriction of isolated 1As seen in the present study may be a result of Fg and fragment D binding to endothelial ICAM-1 in the arterioles. In addition, endothelium-denuded arterioles did not constrict in response to Fg treatment, which suggests the involvement of endothelial (39) and not VSMC ICAM-1 (19) in vasoconstriction. However, the possible interaction of Fg and fragment D, which both contain the RGD sequence, with endothelial cell {alpha}5{beta}1 integrin should not be ruled out.

One of the possible mechanisms of arterial constriction observed in the present study may involve ICAM-1-induced activation of vascular ERK (19). It was reported (15) that ERK is a signaling mechanism in nitric oxide synthase inhibition-induced arterial constriction. Our results coincide with data showing that binding of Fg to endothelial ICAM-1 does not result in synthesis of such dilatory mediators as nitric oxide and prostacyclin (16).

In summary, we have shown that Fg and fragment D induce vasoconstriction in systemic and small resistance arteries both in vivo and in vitro. The mechanism of this vasoconstriction involves binding of Fg and fragment D to endothelial ICAM-1, which evoke the signaling pathways that may lead to synthesis of vascular mediators such as endothelin and result in arteriolar constriction. This study provides additional evidence for the role of plasma adhesion protein Fg and its degradation product fragment D in the alteration of arterial tone through an endothelial ICAM-1-signaling mechanism. During various cardiovascular diseases and cerebrovascular disorders such as hypertension, diabetes, and stroke, elevated plasma Fg content increases the probability of binding of this protein to the arteriolar wall, and resultant vasoconstriction may exacerbate the complications of the diseases.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work is partially supported by the American Heart Association (AHA) National Affiliate Grant 0235317N (to D. Lominadze), by an AHA Ohio Valley Affiliate Grant-In-Aid (to J. C. Falcone) and Postdoctoral Fellowship (to N. Tsakadze), and by National Institutes of Health Grant HL-43721 (to S. E. D'Souza).


   ACKNOWLEDGMENTS
 
The authors are grateful to Dr. Cynthia Meininger (Texas A&M University) for the generous gift of rat endothelial cells.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Lominadze, Dept. of Physiology and Biophysics, Health Sciences Center, A-1115, Univ. of Louisville, Louisville, KY 40292 (E-mail: dglomi01{at}louisville.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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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