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Am J Physiol Heart Circ Physiol 293: H1038-H1045, 2007. First published May 4, 2007; doi:10.1152/ajpheart.01054.2006
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Small-molecule cyclic {alpha}Vbeta3 antagonists inhibit sickle red cell adhesion to vascular endothelium and vasoocclusion

Eileen M. Finnegan, Gilda A. Barabino, Xiao-du Liu, Hee-Yoon Chang, Alfred Jonczyk, and Dhananjay K. Kaul

Department of Chemical Engineering, Northeastern University, Boston, Massachusetts; Division of Hematology, Albert Einstein College of Medicine, Bronx, New York; and Merck, Darmstadt, Germany

Submitted 26 September 2006 ; accepted in final form 30 April 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Abnormal adhesion of sickle red blood cells (SS RBCs) to vascular endothelium may play an important role in vasoocclusion in sickle cell disease. Accruing evidence shows that endothelial {alpha}Vbeta3-integrin has an important role in SS RBC adhesion because of its ability to bind several adhesive proteins implicated in this interaction. In the present studies, we tested therapeutic efficacy of small-molecule cyclic pentapeptides for their ability to block {alpha}Vbeta3-mediated SS RBC adhesion by using two well-established assay systems, i.e., cultured human umbilical vein endothelial cells (HUVEC) and artificially perfused mesocecum vasculature of the rat under flow conditions. We tested the efficacy of two RGD-containing cyclic pentapeptides, i.e., cRGDFV (EMD 66203) and cRGDF-ACHA ({alpha}-amino cyclohexyl carboxylic acid) (EMD 270179), based on their known ability to bind {alpha}Vbeta3. An inactive peptide, EMD 135981 (cRbeta-ADFV) was used as control. Cyclization and the introduction of D-Phe (F) results in a marked increase in the ability of cyclic peptides to selectively bind {alpha}Vbeta3 receptors. In the mesocecum vasculature, both EMD 66203 and EMD 270179 ameliorated platelet-activating factor-induced enhanced SS RBC adhesion, postcapillary blockage, and significantly improved hemodynamic behavior. Infusion of a fluorescent derivative of EMD 66203 resulted in colocalization of the antagonist with vascular endothelium. Also, pretreatment of HUVEC with either {alpha}Vbeta3 antagonist resulted in a significant decrease in SS RBC adhesion. Because of their metabolic stability, the use of these cyclic {alpha}Vbeta3 antagonists may constitute a novel therapeutic strategy to block SS RBC adhesion and associated vasoocclusion under flow conditions.

sickle cell disease; {alpha}Vbeta3-integrin; peripheral resistance unit; sickle erythrocytes

ABNORMAL ADHESION OF SICKLE red blood cells (SS RBCs) to vascular endothelium has been implicated as a contributing factor in vasoocclusion in sickle cell disease (SCD) (13, 14, 19). Multiple-receptor-ligand interactions have been defined in SS RBC adhesion to vascular endothelium involving adhesion molecules expressed on both SS RBCs and endothelium. For example, CD36 and {alpha}4beta1-integrin expressed on stress sickle reticulocytes can mediate adhesion of these cells by interacting, respectively, with thrombospondin (TSP) and vascular cell adhesion molecule-1, the latter expressed on cytokine-stimulated endothelial cells (4, 33, 34). Also, binding of sulfated glycolipids exposed on SS RBCs to von Willebrand factor (vWF) and {alpha}Vbeta3-integrin expressed on endothelium can contribute to sickle cell adhesion (3, 16, 23). In addition, a new category of adhesion molecules on SS RBCs has been described that require activation via signal transduction; these include basal cell adhesion molecule-1/Lutheran, intercellular adhesion molecule (ICAM)-4/Landsteiner-Weiner (LW) (37), and integrinassociated protein (IAP) (5, 17). Other described mechanisms include a role for P-selectin (26) and increased adhesion of SS RBCs that express exposed phosphatidylserine (31).

Lately, considerable attention has focused on the role of the endothelial integrin {alpha}Vbeta3 in mediating SS RBC adhesion to endothelium. The LW blood group glycoprotein, otherwise known as ICAM-4/LW, is expressed on sickle red cells and interacts directly with endothelial {alpha}Vbeta3 (15, 25) and has been shown to be modulated by a PKA-dependent erythroid signaling pathway (37). In addition, endothelial {alpha}Vbeta3 binds to TSP and vWF (7, 8, 10), leading to the possible formation of tripartite complexes (RBC receptor-adhesive protein-endothelial receptor), which may contribute to SS RBC adhesion. Potential complexes include CD36-TSP-{alpha}Vbeta3, IAP-TSP-{alpha}Vbeta3, sulfated glycolipids-TSP-{alpha}Vbeta3, and sulfated glycolipids-vWF-{alpha}Vbeta3.

Our laboratory has previously demonstrated that the blockade of {alpha}Vbeta3-integrin by monoclonal antibodies (MAb) 7E3 and LM609 ameliorates platelet-activating factor (PAF)-induced SS RBC adhesion and associated vasoocclusion in an ex vivo vasculature under flow conditions (23). LM609 specifically targets {alpha}Vbeta3-integrin, whereas 7E3 interacts with {alpha}Vbeta3-integrin, as well as with the closely related integrin {alpha}IIbbeta3 (GP IIb/IIIa). However, MAb 10E5, directed against {alpha}IIbbeta3 alone, failed to inhibit SS RBC adhesion, revealing the specific involvement of {alpha}Vbeta3-integrin in this interaction. Our laboratory's recent studies have demonstrated that synthetic peptides based on {alpha}V-binding domains of ICAM-4 ameliorate SS RBC adhesion and related vasoocclusion in PAF-stimulated ex vivo vasculature, and that the interaction of ICAM-4 peptides with endothelium is blocked by a function blocking antibody to {alpha}Vbeta3-integrin (22). These studies show that {alpha}Vbeta3 plays a prominent role in SS RBC adhesion and adhesion-induced vasoocclusion in SCD.

Cyclic peptides containing the integrin recognition motif Arg-Gly-Asp (RGD) have been shown to inhibit the adhesion of a variety of cells to endothelium (6, 27, 36). In a previous study, large-molecule cyclic decapeptides containing RGD sequence were shown to have an inhibitory effect on SS RBC adhesion to cultured endothelial cells (24), but those examined were not absolutely specific for {alpha}Vbeta3. Moreover, in vivo metabolic stability and adhesion-inhibiting efficacy of these large-molecule RGD peptides under microvascular flow was not ascertained. Therefore, in the present studies, we have explored the therapeutic potential of small-molecule antagonists (cyclic pentapeptides) in blocking {alpha}Vbeta3-mediated SS RBC adhesion and vasoocclusion using cultured human endothelial cells and an ex vivo microcirculatory bed.

Aumailley et al. (1) reported that conformationally constrained, cyclic RGD peptides containing a D-Phe (F), but no disulfide bonds, were inhibitors of {alpha}Vbeta3-ligand interactions. One such pentapeptide, cyclic Arg-Gly-Asp-D-Phe-Val (cRGDFV, EMD 66203), shown to selectively target {alpha}Vbeta3-integrin (29), reduced joint damage in a rabbit model of rheumatoid arthritis, which is associated with intense angiogenesis where {alpha}Vbeta3-integrin plays a major role (32). Importantly, this compound was shown to be metabolically stable as a fluorescent form was detected in vivo 24 h after administration. Moreover, the ability of cRGDFV to inhibit cell adhesion to vitronectin is increased by ~100-fold compared with a linear variant (1). These and other studies reveal that the enhanced potency and specificity of its inhibition of {alpha}Vbeta3 result from the introduction of D-Phe as well as cyclization of the peptide (11, 12, 30). Such observations make this new generation of {alpha}Vbeta3 antagonists attractive candidates for anti-adhesive therapy in SCD.

Because {alpha}Vbeta3 is a receptor for several adhesive proteins, inhibiting SS RBC adhesion by blocking this endothelial integrin may constitute an important therapeutic strategy. In the present study, we tested the efficacy of two small-molecule cyclic pentapeptides, cRGDFV (EMD 66203) and cRGDF-ACHA (ACHA = {alpha}-amino cyclohexyl carboxylic acid) (EMD 270179), to inhibit adhesion of SS RBCs to endothelium using two well-established complementary dynamic assay systems. In one assay, SS RBCs were infused into a parallel plate flow chamber lined with human umbilical vein endothelial cells (HUVECs). In the second assay, we performed hemodynamic and microcirculatory observations in the ex vivo mesocecum vasculature of the rat treated with PAF. In the ex vivo vasculature, PAF causes endothelial oxidant generation and endothelial activation (21), both of which characterize SCD. PAF is elevated twofold in sickle patients (28) and enhances SS RBC adhesion in the ex vivo microcirculatory preparation (21–23). In both assay systems, small-molecule {alpha}Vbeta3 antagonists resulted in significant inhibition of SS RBC adhesion. Furthermore, in the ex vivo microvasculature, both EMD 66203 and EMD 270179 decreased not only adhesion, but also vasoocclusion, resulting in improved hemodynamics.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cyclic Peptides

Conformationally constrained cyclic pentapeptides, cyclic Arg-Gly-Asp-D-Phe-Val (EMD 66203), cyclic Arg-Gly-Asp-D-Phe-ACHA (EMD 270179), cyclic Arg-beta-Ala-D-Phe-Val (EMD 135981), cyclic Arg-Gly-Asp-D-Phe-Lys (fluorescein carboxylic acid) (EMD 80838), and cyclic Arg-beta-Ala-Asp-D-Phe-Lys (fluorescein carboxylic acid) (EMD 486904) were a kind gift of Merck (Darmstadt, Germany). The peptides were dissolved in normal (0.9%) saline at a concentration of 2 mg/ml and stored at –20°C until use.

Preparation of Cells

Heparinized blood was obtained with informed consent from normal (AA) adults (n = 3) and from sickle cell anemia patients (n = 12) who were not in crisis and had not received blood transfusion in the preceding 4 mo. The blood samples were drawn under a protocol approved by the Institutional Review Board (Albert Einstein College of Medicine, Bronx, NY).

For the adhesion assay involving HUVEC, after removal of the buffy coat, blood was washed three times in Hanks' balanced salt solution (without Ca2+ and Mg2+) and resuspended in M199/1% bovine serum albumin (BSA) at a hematocrit (Hct) of 1%. For microcirculatory studies involving ex vivo mesocecum preparation of the rat, blood was resuspended in autologous plasma following removal of the buffy coat, and Hct was adjusted to 30%.

Adhesion Studies with Human Endothelial Cells Under Flow Conditions

HUVECs were purchased from Clonetics (Cambrex Bio Science, Walkersville, MD). Cultures were serially passaged with Clonetics endothelial growth medium (EGM)-2 culture medium in Corning tissue culture flasks (Cambridge, MA). Experiments were performed using second- to third-passage HUVECs that were grown to confluence on fibronectin-coated, single-well LabTek slides (LabTek, Naperville, IL).

Endothelial monolayers were incubated for 30 min with EGM-2 culture medium containing a given cyclic peptide (100 µg/ml). The HUVEC-coated slide was attached to a parallel plate flow chamber to assess the adhesion of RBCs under defined fluid dynamic conditions, as previously described (3). Briefly, RBC suspensions (Hct 1%) were drawn into the chamber using a syringe pump (model 55–143, Harvard Apparatus, South Natick, MA) for 10 min at a controlled flow rate to give a venular wall shear stress of 1 dyn/cm2. Experiments were visualized with an inverted phase-contrast microscope (DIAPHOT-TMD, Nikon) equipped with a charge-coupled device (CCD) video camera (model 72, Dage-MTI) and recorded in real time on a 0.5-in. video cassette recorder (model BV-1000, Mitsubishi, Cypress, CA). The number of adherent erythrocytes remaining after a 10-min rinse period were counted in a minimum of 24 fields and reported as the number of adherent RBCs per millimeter squared.

HUVEC Expression of {alpha}Vbeta3-Integrin by Flow Cytometry

Naive HUVEC cultures were harvested in the second to third passage by nonenzymatic cell dissociation using PBS supplemented with 5 mM EDTA and 1% BSA. Individual HUVEC suspensions containing a minimum of 0.5 x 105 cells were incubated for 30 min with one of the following mouse anti-human MAb antibodies, diluted 1:200 in PBS with 1% BSA: LM609 (anti-{alpha}Vbeta3, kindly provided by Dr. David Cheresh) or MOPC-21 (mouse IgG1,{kappa}, BD Biosciences) as a negative isotype control. After washing, cell suspensions were incubated 30 min with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (BD Biosciences) diluted 1:50 in PBS with 1% BSA. Labeled cells were fixed in 2% formaldehyde and then analyzed using a FACScan flow cytometer.

Hemodynamic and Microcirculatory Evaluation in Ex Vivo Mesocecum Preparation

Preparation and perfusion of rat mesocecum vasculature. Perfusion studies were performed in the isolated, acutely denervated, and artificially perfused rat mesocecum vasculature (n = 32), according to the method of Baez et al. (2), as modified by Kaul et al. (18) for the infusion of erythrocytes. Details of the procedure have been described elsewhere (18, 23). All experimental protocols were approved by the Animal Care and Use Committee of the Albert Einstein College of Medicine. Briefly, arterial perfusion pressure was maintained at 60 mmHg, and venous outflow pressure at 3.8 mmHg. During perfusion with Ringer-albumin solution containing 2% bovine albumin, a 0.2-ml bolus of a given red cell sample (Hct 30%) was infused via the arterial injection port over ~5 s. Peripheral resistance units (PRU) were determined as described (9) and expressed in millimeters of mercury per milliliter per minute per gram. PRU = {Delta}P/Q, where {Delta}P is the arteriovenous pressure difference and Q is the rate of venous outflow (ml/min) per gram of tissue weight. Pressure flow recovery time (Tpf) is defined as the time (seconds) required for the arterial pressure and the venous outflow to return to their baseline levels after the passage of infused RBCs.

Intravital microscopic observations and adhesion quantification. Intravital microscopic observations and simultaneous video recording of the microcirculatory events were carried out using a Nikon microscope (model E400; Nikon, Melville, NY) equipped with Dage-MTI-CCD television camera (model CCD-300T-RC; Dage-MTI, Michigan City, IN) and a Sony U-matic video recorder (model VO5800; Sony, Teaneck, NJ). The number of adherent SS cells per 100 µm2 was calculated from the counts of individual adherent cells and the surface area (µm2) of the inner wall of the vessel segment, as described previously (20). The number of venules where counts were made varied from 8 to 26 venules in individual preparations (3–4 preparations in each group). Because of topological variations, no two venular networks are alike in branching pattern, vessel bending, and vessel length. Therefore, adhesion data for each experimental group were pooled for statistical comparisons.

Protocol of ex vivo perfusion experiments with cyclic {alpha}Vbeta3 antagonists. PAF was used to enhance SS RBC adhesion in the ex vivo mesocecum preparations, as described in our laboratory's previous studies (23, 24). Rat mesocecum was isolated and perfused with 40 ml of Ringer-albumin containing PAF (200 pg/ml) for 10 min. After a 5-min incubation period, the preparation was perfused as above, and a bolus of SS RBCs was infused.

In experiments designed to evaluate the effects of cyclic {alpha}Vbeta3 antagonist peptides (EMD 66203 and EMD 270179), the preparation was first infused with a given peptide (200 µg/ml in 4 ml Ringer-albumin). After 30-min incubation at room temperature, the preparation was perfused with PAF solution (200 pg/ml in 40 ml) over a 10-min period followed by a repeat infusion with a given peptide. Control experiments were done using the control cyclic peptide EMD 135981, in which the glycine residue in the RGD sequence is replaced by beta-alanine. Identical results were obtained when the preparation was first treated with PAF followed by infusion with a given peptide, suggesting that post-PAF treatment with {alpha}Vbeta3 antagonists was sufficient to inhibit PAF-induced SS RBC adhesion and vasoocclusion. Following the incubation with peptide, a bolus of SS RBCs was infused into the preparation during perfusion with Ringer-albumin solution.

Immunofluorescence

In two preparations each, EMD 80838 [cyclic Arg-Gly-Asp-D-Phe-Lys (fluorescein carboxylic acid)], a fluorescent derivative of EMD 66203, or EMD 486904 [cyclic Arg-beta-Ala-Asp-D-Phe-Lys (fluorescein carboxylic acid)], a fluorescent derivative of the control peptide EMD 135981, was infused (100 µg/ml) in combination with PAF treatment using the above protocol to ascertain if the infused antagonist (EMD 66203) was targeted to endothelium. After a brief perfusion with Ringer-albumin (5 ml), the mesocecum tissue was frozen in tissue-freezing medium (Miles Laboratories, Elkhart, IN) at –80°C.

Cryostat sections (6 µm) of fresh-frozen mesocecum tissue were postfixed in acetone. The sections were stained for vWF, a marker for blood vessels. The sections were treated with primary rabbit anti-human vWF polyclonal antibody (Dakopatts, Glostrup, Denmark), followed by secondary antibody, i.e., tetrarhodamine isothiocyanate (TRITC)-conjugated affinity-isolated swine anti-rabbit IgG (Dakopatts), as described (22). Digital fluorescent images of the same vessels were processed to determine vessel localization of peptides and vWF using MetaMorph image processing program (Universal Imaging, Dowingtown, PA).

Statistical Analysis

One-way ANOVA with Duncan's multiple-range comparisons was applied to analyze hemodynamic data as indicated in Table 1 and RESULTS. Regression line analysis of the number of adherent RBCs/100 µm2 (Y) vs. the venular diameter (X) was performed using the equation Y = aX–b for the best fit. Logarithmic transformation of both variables allowed comparison of intercepts and slopes of the regression lines between experimental groups, using multiple linear regression analysis (21). For groups compared, homogeneity of variance for Y values in relation to X was confirmed using Bartlett's test. The various statistical tests or tests for hypotheses were performed using a type I error and were two-tailed. The statistical analysis was performed using STATGRAPHICS Plus 5.0 for Windows (Manugistics, Rockville, MD).


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  		Table 1. The effect of PAF and {alpha}Vbeta3 antagonists on hemodynamic behavior of SS RBCs in the ex vivo mesocecum preparation

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The anti-adhesive potential of cyclic {alpha}Vbeta3 antagonists (EMD 66203 and EMD 270179) was investigated using two complementary dynamic assays: 1) a parallel-plate flow chamber lined with HUVEC to evaluate the efficacy of the antagonists; and 2) ex vivo mesocecum vasculature to ascertain hemodynamic and microcirculatory effects of inhibition of SS RBC adhesion to microvascular endothelium.

Studies with HUVEC Under Flow Conditions

Expression of {alpha}Vbeta3-integrin on HUVEC. Fluorescence-activated cell sorting analysis of cultured human endothelial cells was performed to ascertain expression of {alpha}Vbeta3-integrin. As shown in Fig. 1, 94% of untreated HUVECs expressed {alpha}Vbeta3 as determined by the range of fluorescent intensity (M1). Nevertheless, all of the cells analyzed for {alpha}Vbeta3 expression essentially followed a Gaussian distribution.


Figure 1
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  		Fig. 1. Fluorescence-activated cell sorting analysis of cultured human endothelial cells. Ninety-four percent of the human umbilical vein endothelial cells (HUVECs) were positive for {alpha}Vbeta3-integrin, as determined by staining with a fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (LM609) directed against {alpha}Vbeta3. M1 is the range of fluorescent intensity for {alpha}Vbeta3-positive cells.

 
Inhibition of SS RBC adhesion to HUVEC by cyclic {alpha}Vbeta3 antagonists. Since HUVEC expressed {alpha}Vbeta3, we investigated the ability of two {alpha}Vbeta3 antagonists (EMD 66203 and EMD 270179) to inhibit adhesion of SS RBCs to HUVEC under flow conditions. Blood samples from three SS patients were tested at least in duplicate runs, except in experiments involving EMD 270179, wherein RBCs from two of these patients were used in duplicate runs. As shown in Fig. 2, treatment of HUVEC with inactive control peptide EMD 135981 (3 patients, 7 experiments) did not affect SS RBC adhesion compared with the untreated control HUVEC (3 patients, 6 experiments). In contrast, both active peptides EMD 66203 (3 patients, 6 experiments) and EMD 270179 (2 patients, 4 experiments) caused marked inhibition of SS RBC adhesion (i.e., 55 and 62%, respectively) to HUVEC compared with the control peptide group (adherent SS RBCs/mm2: EMD 66203 = 33.8 ± 12.0, P < 0.018; EMD 27179 = 29.4 ± 7.8, P < 0.015). Both active peptides were able to inhibit adhesion of SS RBCs by ~70% (each P < 0.01) compared with untreated control HUVEC.


Figure 2
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  		Fig. 2. The effect of {alpha}Vbeta3 antagonists (EMD 66203 and EMD 270179) on sickle red blood cell (SS RBC) adhesion to HUVEC. Human endothelial monolayers were incubated with either peptide (100 µg/ml) for 30 min before the perfusion of the endothelialized flow chamber with SS RBC suspension (hematocrit 1%) at 1 dyn/cm2. SS RBC adhesion from the same three SS patients was tested at least in duplicate runs, except in experiments using EMD 270179, wherein RBCs from two of these patients were used in duplicate runs. The HUVEC treated with control peptide (3 patients, 7 experiments) showed no significant difference in adhesion of SS RBC/mm2 compared with the baseline adhesion of these cells to untreated control HUVEC (n = 6) (P > 0.51). In contrast, both EMD 66203 (3 patients, 6 experiments) and EMD 270179 (2 patients, 4 experiments) caused marked inhibition of adhesion (i.e., 55 and 62%, respectively) of SS RBCs to HUVEC compared with the control peptide group (P < 0.018 and P < 0.015, respectively).

 
Ex Vivo Microcirculatory Studies

The effect of {alpha}Vbeta3 antagonists on the hemodynamic behavior of SS RBCs. Table 1 depicts hemodynamic behavior of SS RBCs infused into the ex vivo vasculature with or without PAF pretreatment and following treatment with a given peptide (control peptide or an {alpha}Vbeta3 antagonist) in PAF-perfused preparation. In the untreated preparations (infusion groups 1 and 2), infusion of SS RBCs resulted in 50% greater PRU than for normal (AA) RBCs (P < 0.05) (Table 1). In group 3, infusion of SS RBCs into PAF-treated preparations resulted in 63% greater PRU (P < 0.05) compared with the values for SS RBCs in untreated preparations (group 2) (Table 1). The observed increase in PRU in PAF-treated preparations compared with preparations perfused with Ringer-albumin alone is in accordance with our laboratory's previous studies (21, 23).

To determine the effect of {alpha}Vbeta3 antagonists (EMD 66203 and EMD 270179), the ex vivo preparations were incubated with a given peptide combined with PAF treatment (see MATERIALS AND METHODS). In the presence of PAF and the inactive control peptide EMD 135981, SS RBCs caused ~70% increase in PRU (group 4: 5 patients, 7 experiments), which was not different from that observed after the infusion of SS RBCs into preparations treated with PAF alone (group 3). In contrast, when the preparations were treated with PAF and {alpha}Vbeta3 antagonist EMD 66203 (group 5), SS RBCs from the same patients resulted in almost 40% lower PRU compared with the control peptide group (P < 0.05, Table 1). Infusion of SS RBCs into preparations treated with PAF and EMD 270179 (group 6) resulted in an almost 50% decrease in PRU (P < 0.05) compared with the control peptide group (Table 1). In the presence of either active peptide (EMD 66203 or EMD 270179), the resulting PRU was not significantly different from the values obtained in untreated preparations (group 2), suggesting equal efficacy of each antagonist.

The Tpf in the above experiments were in agreement with the PRU changes. Accordingly, in untreated preparations, infusion of SS RBCs was followed by an ~70% increase in Tpf compared with that for AA RBCs (Table 1, groups 1 and 2; P < 0.05). The Tpf values following infusion of SS RBCs either into PAF-treated preparations (group 3) or into the preparations treated with PAF and the control peptide EMD 135981 (group 4) showed similar increases compared with the values for SS RBCs in untreated preparations (group 2, 82–97%; P < 0.05). Treatment with {alpha}Vbeta3 antagonists (EMD 66203 and EMD 270179) (groups 5 and 6) resulted in significant decreases in Tpf compared with the values obtained for the control peptide group (each P < 0.05, Table 1) or the untreated preparations (group 2).

The effect of cyclic {alpha}Vbeta3 antagonists on SS RBC adhesion in microcirculation. Direct microscopic observations and video analysis showed that treatment with PAF or PAF and control peptide EMD 135981 caused a prominent increase in SS RBC adhesion to endothelium in mesocecum venules compared with SS RBC adhesion in untreated preparations. Figure 3, A–C, shows that infusion of SS RBCs into PAF and control peptide-treated preparations invariably resulted in adhesion of these cells to venular endothelium. Maximal adhesion was observed in small-diameter postcapillary venules (Fig. 3, B and C) and often resulted in postcapillary blockage.


Figure 3
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  		Fig. 3. Videomicrographs showing inhibitory effect of EMD 66203 on platelet-activating factor (PAF)-induced SS RBC adhesion in the mesocecum microvasculature. A–C: ex vivo preparation treated with PAF and control peptide EMD 135981. A: clear vessel lumen during perfusion with Ringer-albumin (a, arteriole; v, venule). B: a bolus infusion of SS RBCs is accompanied by adhesion of these cells in the venules. The small-diameter venule (small arrow) is almost completely blocked with adherent cells. The large arrow indicates the direction of the flow. C: following the passage of bolus, a large number of SS RBCs are seen adhering to the vessel wall and result in almost complete blockage of the small-diameter venule (small arrow). D–F: ex vivo preparation treated with PAF and EMD 66203. D: venules during artificial perfusion with Ringer-albumin. E: rapid flow of SS RBCs after a bolus infusion. F: little or no adhesion of SS RBC is seen after the passage of the bolus. Bar in B = 20 µm.

 
Adhesion of SS RBCs to endothelium in preparations treated with PAF was inversely correlated with the venular diameter (r = –0.77, P < 0.0001, using the equation Y = aX–b) (Fig. 4A). The observed increase in PRU in PAF-treated preparations (Table 1, groups 3 and 4) probably reflects the effect of postcapillary blockage secondary to enhanced adhesion of SS RBCs. In marked contrast, PAF treatment combined with infusion of active peptides EMD 270179 or EMD 66203 largely inhibited SS RBC adhesion in venules of all diameters (Fig. 4, B and C). In both of these cases, adhesion showed a weak or no correlation with venular diameter (EMD 270179: r = 0.29, P < 0.01; EMD 660203: r = 0.14, P > 0.14) (Fig. 4, B and C). It is important to note that no postcapillary blockage was evident in preparations treated with either EMD 270179 or EMD 66203. In contrast, the SS RBC adhesion in preparations treated with control peptide EMD 135981 was similar to that observed in PAF-treated preparations (r = –0.77, P < 0.0001) (Fig. 4D).


Figure 4
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  		Fig. 4. Regression plots for the number of adherent SS RBC/100 µm2 relative to venular diameter in the ex vivo mesocecum using the multiplicative equation of the form Y = aX–b. A: in preparations treated with PAF alone, SS RBC adhesion showed a strong correlation with the venular diameter. B: {alpha}Vbeta3 antagonist EMD 270179 markedly inhibited SS RBC adhesion in venules of all diameters in PAF-treated preparations. C: in PAF-treated preparations, EMD 66203 had a greater inhibitory effect on the adhesion in small-diameter venules, the site of frequent blockage. D: treatment with PAF and the inactive peptide EMD 135981 resulted in similar adhesion as observed with PAF alone. E: regression analysis using logarithmic transformations of the data confirmed marked inhibition of SS RBC adhesion by EMD 66203 compared with preparations treated with PAF alone. Because log of zero is undefined, the values of Y (number of adherent cells) were coded by the addition of 1. Note the significantly lower Y-intercept (P < 0.001) and reduced slope of the regression lines for the peptide EMD 66203. F: a similar comparison of regression lines revealed no significant differences between SS RBC adhesion in preparations treated with PAF alone or with PAF and the control peptide EMD 135981.

 
Regression analysis using logarithmic transformation of the data confirmed that both of the {alpha}Vbeta3 antagonists inhibited SS RBC adhesion with equal effectiveness. In each case, the marked inhibition of SS RBC adhesion compared with preparations treated with PAF alone was evidenced by significantly lower Y-intercepts (P < 0.001) and reduced slopes of the regression lines (P < 0.001), as shown in Fig. 4E for peptide EMD 66203. On the other hand, the adhesion of SS RBCs in preparations treated with control peptide EMD 135981 was essentially similar to that in preparations treated with PAF alone (Fig. 4F).

Colocalization of {alpha}Vbeta3 Antagonist With Vascular Endothelial Lining

EMD 80838, a fluorescent derivative of the {alpha}Vbeta3 antagonist EMD 66203, was used to verify that the infused {alpha}Vbeta3 antagonist was targeted to vascular endothelium (see MATERIALS AND METHODS). Cryostat sections were treated with an anti-vWF antibody and a TRITC-conjugated secondary antibody for the identification of vascular endothelium. Figure 5, A–C, shows colocalization of the fluorescent {alpha}Vbeta3 antagonist with vascular endothelium in a mesocecum vessel. In contrast, preparations treated with EMD 486904, a fluorescent derivative of the control peptide EMD 135981, showed no evidence of colocalization of EMD 486904 with vWF in blood vessels (Fig. 5, D–F). These observations confirm a specific interaction of the {alpha}Vbeta3 antagonist with vascular endothelium.


Figure 5
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  		Fig. 5. A–C: colocalization of EMD 80838 [cRGD-FK (fluorescein carboxylic acid)], a fluorescent derivative of EMD 66203, with vascular endothelial lining in a mesocecum vessel. A: the presence of the fluorescent peptide is shown in green. B: in the middle, the blood vessel was identified by a polyclonal primary antibody to von Willebrand factor (vWF) and a secondary tetrarhodamine isothiocyanate-conjugated antibody (red). C: on the right, the image signals were merged to confirm colocalization of {alpha}Vbeta3 antagonist (arrows) with the endothelial lining. D–F: EMD 486904 [cRbetaADFK (fluorescein carboxylic acid)], a fluorescent derivative of the control peptide EMD 135981, failed to colocalize with vascular endothelial lining of mesocecum vessel. D: infusion of EMD 486904 resulted in weak or no fluorescent staining. E: blood vessel was identified by a polyclonal antibody to vWF as in B (red). F: merged image signals showed no colocalization of the control peptide with vWF.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present studies show that cyclic RGD pentapeptide antagonists of {alpha}Vbeta3-integrin, EMD 66203 and EMD 270179, markedly inhibit PAF-induced SS RBC adhesion in the ex vivo mesocecum vasculature. We show that treatment with {alpha}Vbeta3 antagonists significantly decreases peripheral resistance and Tpf in the ex vivo vasculature and abolishes adhesion-induced blockage of postcapillary venules in PAF-treated preparations. In contrast, treatment with a control peptide (EMD 135981) had no effect. The inhibitory effect of these antagonists on SS RBC adhesion was reaffirmed using cultured HUVEC in parallel plate cell adhesion assays. Thus the blockade of {alpha}Vbeta3 may prevent binding of this integrin to its putative ligands (ICAM-4, TSP, and vWF) and thereby inhibit SS RBC-endothelial interaction and related vessel blockage.

PAF was chosen in our ex vivo studies because it invariably results in enhanced SS RBC adhesion in the mesocecum preparation (21, 23). We tested PAF at a dose approximating that reported in sickle cell patients (28). Moreover, PAF induces endothelial oxidant generation in this preparation (21) and upregulates endothelial adhesion molecules such as vWF and {alpha}Vbeta3 (22, 23). Infusion of SS RBCs into ex vivo preparations treated with PAF alone or PAF and the control peptide resulted in adhesion of these cells in an inverse correlation with venular diameter and resulted in a frequent blockage of small-diameter venules (the sites of maximal adhesion), in agreement with our laboratory's previous studies (20, 23). In contrast, AA RBCs do not adhere in PAF-treated or untreated ex vivo preparations as reported by our laboratory (23), suggesting that only SS RBCs express the adhesion molecules required for interactions with endothelium.

Treatment of the ex vivo preparation with either EMD 66203 or EMD 270179 resulted in a dramatic inhibition of SS RBC adhesion in venules of all diameters, and no postcapillary blockage was evident. Immunofluorescence analysis of the ex vivo preparation infused with a fluorescent derivative of EMD 66203 (EMD 80838) clearly demonstrated that the peptide targeted the vessel wall, while a fluoresceinated control peptide failed to colocalize with the endothelium. Taken together, the ability of these antagonists to inhibit PAF-induced SS RBC adhesion in this ex vivo model and to HUVEC provides insight into potential contribution of {alpha}Vbeta3-integrin to vascular obstruction in SCD. Thus the inhibition of {alpha}Vbeta3-integrin by small-molecule cyclic peptides containing Arg-Gly-Asp is a powerful approach to block the pathological interaction between SS RBC and vascular endothelium and prevent adhesion-induced vessel blockage.

Small-molecule cyclic peptides, such as EMD 66203, bind {alpha}Vbeta3-integrin with high specificity (29). In addition to the Arg-Gly-Asp (RGD) sequence, both EMD 66203 and EMD 270179 have a D-Phe residue. They differ only in the fifth amino acid: in EMD 66203, the fifth residue is Val, while in EMD 270179, Val is replaced by ACHA. Similar efficacy in blocking SS RBC adhesion and preserving hemodynamic behavior signifies that Val is not critical to attachment to {alpha}Vbeta3. The control peptide, EMD 135981, differs from EMD 66203 by the substitution of Gly with beta-Ala within the RGD sequence. That EMD 135981 was ineffective in preventing SS RBC adhesion both in vitro and ex vivo reveals that the blockade of {alpha}Vbeta3 via RGD is the likely mechanism in the effective inhibition of SS RBC adhesion by these antagonists. Small size, cyclization, and the introduction of D-Phe may contribute to the marked increase in the ability of these antagonists to selectively bind {alpha}Vbeta3-integrin (29, 32).

Although we find SS RBC adhesion to apparently intact vascular endothelium in PAF-treated preparations, it is possible some of the adhesion occurs to the exposed subendothelial matrix and could involve the contribution of laminin as reported before (16, 35). We also cannot rule out the contribution of thrombin (26), or substances released from platelets, such as TSP, vWF, and fibrinogen. Nevertheless, the significant reduction in SS RBC adhesion and related vessel blockage caused by {alpha}Vbeta3 antagonists suggests that {alpha}Vbeta3 blockade may mitigate the potential contribution of specific adhesive molecules (e.g., TSP, vWF, and ICAM-4) implicated in SS RBC adhesion via their known interaction with {alpha}Vbeta3 (7, 8, 10, 37).

In conclusion, we have shown that {alpha}Vbeta3 antagonists (i.e., EMD 66203 and EMD 270179) cause a significant inhibition of SS RBC adhesion in ex vivo mesocecum vasculature and to human endothelial monolayers under venular shear flow conditions. In the mesocecum, the antagonists not only inhibited adhesion, but also abolished related vasoocclusion and maintained flow patency, as evidenced by smaller increases in PRU. Thus the use of metabolically stable, small-molecule cyclic {alpha}Vbeta3 antagonists may constitute a novel therapeutic strategy to ameliorate SS RBC adhesion and related vascular obstruction.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants U54 38655 (D. K. Kaul, G. A. Barabino), HL070047 (D. K. Kaul), U54 HL070819 (G. A. Barabino), and HL071631 (G. A. Barabino) and a Grant-in-Aid from American Heart Association-Heritage Affiliate (D. K. Kaul).


   ACKNOWLEDGMENTS
 
The authors thank Michael Cammer (Analytical Image Facility, Albert Einstein College of Medicine) for help in photomicrographs of fluorescent images.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. K. Kaul, Dept. of Medicine, Rm. U-917, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 (e-mail: kaul{at}aecom.yu.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
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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