Published online before print August 28, 2006, doi: 10.1161/CIRCULATIONAHA.106.631457
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(Circulation. 2006;114:1293-1300.)
© 2006 American Heart Association, Inc.
Vascular Medicine |
Heparin Displaces Interferon-
–Inducible Chemokines (IP-10, I-TAC, and Mig) Sequestered in the Vasculature and Inhibits the Transendothelial Migration and Arterial Recruitment of T Cells
From the Interdepartmental Program in Vascular Biology and Transplantation and the Departments of Surgery (H.R., Y.W., A.O.Y., G.T.), Pathology (T.D.M., J.S.P.), Anesthesiology (S.A.), and Dermatology (M.S.K., J.S.P.), Yale University School of Medicine, New Haven, Conn.
Correspondence to George Tellides, MD, PhD, 295 Congress Ave, BCMM 454, New Haven, CT 06510. E-mail george.tellides{at}yale.edu
Received April 3, 2006; revision received July 18, 2006; accepted July 19, 2006.
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Abstract |
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Background— Heparin, used clinically as an anticoagulant, also has antiinflammatory properties and has been described to inhibit interferon (IFN)-
responses in endothelial cells. We investigated the effects of heparin on the IFN-–inducible chemokines IP-10/CXCL10, I-TAC/CXCL11, and Mig/CXCL9, which play important roles in the vascular recruitment of IFN-–producing Th1 cells through interactions with their cognate receptor, CXCR3.
Methods and Results— Patients undergoing coronary artery bypass grafting were studied because coronary atherosclerosis is recognized as a Th1-type inflammatory disease and the subjects required systemic heparinization. Plasma levels of IP-10, I-TAC, and Mig increased immediately after heparin administration and diminished promptly after heparin antagonism with protamine. These effects were independent of detectable circulating IFN- or the IFN- inducer interleukin-12. We confirmed previous reports that heparin inhibits the IFN-–dependent production of CXCR3 chemokine ligands using atherosclerotic coronary arteries in organ culture. In addition to prolonged treatment decreasing chemokine secretion, heparin rapidly displaced membrane-associated IP-10 from cultured endothelial cells that did not express CXCR3 and reduced the IP-10–dependent transendothelial migration of T helper cells under conditions of venular shear stress. Finally, heparin administration to immunodeficient mouse hosts decreased both the recruitment and accumulation of memory T cells within allogeneic human coronary arteries.
Conclusions— Besides inhibiting IFN- responses, heparin has further immunomodulatory effects by competing for binding with IP-10, I-TAC, and Mig on endothelial cells. Disruption of CXCR3+ Th1 cell trafficking to arteriosclerotic arteries may contribute to the therapeutic efficacy of heparin in inflammatory arterial diseases, and nonanticoagulant heparin derivatives may represent a novel antiinflammatory strategy.
Key Words: chemokines • endothelium • heparin • inflammation • lymphocytes
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Introduction |
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Immune responses contribute to host defense and are capable of causing tissue injury and disease under pathological conditions. Innate immunocytes, eg, macrophages and dendritic cells, are the first line of defense against pathogens and have bidirectional interactions with lymphocytes through cell-surface molecules and secreted cytokines. T lymphocytes, cellular mediators of adaptive immune responses, are functionally classified as CD4+ helper (Th) or CD8+ cytotoxic (Tc) cells and are further subdivided on the basis of the profile of cytokines that they elaborate on activation. Th1 and Tc1 cells produce the signature type I cytokine interferon (IFN)-
.1 The differentiation of naïve CD45RA+ T cells to cytokine-polarized, memory CD45RO+ Th1 cells is regulated by the monokine interleukin (IL)-12 and further promoted by IL-18. Th1 cells preferentially express C-X-C chemokine receptor 3 (CXCR3), which binds 3 chemokines with high affinity: IFN-–inducible protein of 10 kDa (IP-10)/CXCL10, IFN-inducible T-cell 
chemoattractant (I-TAC)/CXCL11, and monokine induced by IFN- (Mig)/CXCL9.2 The CXCR3 ligands are strongly inducible by IFN- (as their names imply) in a variety of cell types, most robustly in vascular cells, and their receptor-mediated signaling has important chemotaxis, activation, and effector functions for T cells.
Clinical Perspective p 1300
Innate and adaptive immune responses play central roles in the pathogenesis of inflammatory arterial diseases such as atherosclerosis and graft arteriosclerosis.3 Analyses of clinical specimens have found T-cell infiltrates with predominantly IFN-–secreting cytokine profiles, expression of IFN-–inducing cytokines, and a strong upregulation of IFN-–inducible molecules in arteriosclerotic lesions.3–5 CXCR3-bearing T cells and the chemokines IP-10, I-TAC, and Mig are also present in arterial plaques.6 These studies in humans support the concept that chemokine-dependent migration of Th1 cells through inflamed endothelium into the arterial intima may be a critical process in the pathogenesis of arteriosclerosis.
The cellular surface and extracellular matrix of the vasculature constitute a complex structure containing proteins and carbohydrates, including conjugate proteoglycans. Proteoglycans consist of a core protein that is covalently linked to 
1 linear carbohydrate polymers of the glycosaminoglycan (GAG) polysaccharide family. Heparan sulfate, the most abundant GAG of the endothelium, is negatively charged because of its sulfated domains and has nonspecific ionic interactions with many positively charged proinflammatory molecules, including IFN-, certain interleukins, and many chemokines.7 The electrostatic interactions of acidic cell surface GAG and basic proinflammatory molecules are generally of lower affinity than the binding of ligands to their specific receptors and often serve to enhance cytokine and chemokine activity and stability.8–11 Heparin, a soluble GAG produced by mast cells and used therapeutically as an anticoagulant, is structurally similar to heparan sulfate but is more uniformly sulfated and negatively charged. Heparin competitively binds and antagonizes several proinflammatory molecules, resulting in immunosuppressive properties in experimental and clinical settings.7,12,13
We investigated the effects of heparin on the IFN- axis (the IFN- inducer IL-12, the prototypic Th1 cytokine IFN-, and the IFN-–inducible chemokines IP-10, I-TAC, and Mig) in patients undergoing coronary artery bypass graft (CABG) surgery. The rationale for the experimental design is that coronary atherosclerosis is considered a Th1-mediated disease, the subjects require systemic heparinization to undergo extracorporeal circulatory support by cardiopulmonary bypass (CPB), and the heparin activity is fully reversed on completion of CPB to avoid excessive postoperative bleeding. We further examined the effects of heparin on CXCR3 chemokine ligands in experimental systems of organ culture, cell culture, and chimeric humanized animals. We found that heparin mobilizes IP-10, I-TAC, and Mig in the circulation, competes for binding with these chemokines to endothelial cells, and inhibits the transendothelial migration and arterial recruitment of memory T cells.
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Methods |
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Plasma and Tissue Samples
Research protocols were approved by the institutional review boards, and informed consent was obtained. Blood samples from patients undergoing CABG surgery (n=29) were collected in EDTA tubes from an intra-arterial line before and after administration of porcine intestinal heparin (Elkins-Sinn, Cherry Hill, NJ) or protamine sulfate (American Pharmaceutical Partners, Schaumburg, Ill) and kept at 4°C until the plasma was separated by centrifugation at 3000 rpm for 10 minutes. The specimens were stored at –80°C until batch analysis. Atherosclerotic coronary arteries were obtained from the explanted hearts of cardiac transplant recipients or cadaver organ donors (n=7).
Reagents and Cells
Recombinant tumor necrosis factor (TNF), IL-2, IL-12, IL-18, IFN-, IP-10, I-TAC, and Mig and sandwich ELISA kits for IL-12 p70, IFN-, IP-10, I-TAC, and Mig were from R&D Systems (Minneapolis, Minn). The lower limit of detection for ELISA was 
15 pg/mL. Peripheral blood mononuclear cells (PBMCs) were prepared by density gradient centrifugation of blood from healthy donors and were depleted of monocytes by incubation on plastic for 1 hour. CD4+ Th cells were isolated by positive selection with magnetic beads (Dynal Biotech, Oslo, Norway) from which memory CD45RO+ cells were negatively selected through the use of mouse anti-human CD45RA (eBioscience, San Diego, Calif), anti-human HLA-DR (BD Pharmingen, San Diego, Calif), and anti-mouse IgG magnetic beads (Dynal Biotech). Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords and cultured in gelatin-coated flasks at 37°C in M-199 medium containing 20% FBS (Life Technologies, Grand Island, NY), 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin (Invitrogen, Carlsbad, Calif), 50 µg/mL fibroblast growth factor-1 (Collaborative Research, Bedford, Mass), and 100 µg/mL (10 U/mL) porcine intestinal heparin (Sigma-Aldrich, St Louis, Mo).
Organ Culture
Atherosclerotic coronary arteries were divided into 3-mm rings and cultured in 24-well plates at 37°C in M-199 supplemented with 20% FBS, glutamine, and antibiotics. The media was changed after 6 hours to remove any basal chemokine production in response to host stimuli, and the rings were then treated with various doses of cytokines and heparin.
CXCR3 Expression
HUVECs and CD4+/CD45RO+ T cells were labeled with an isotype-matched control antibody or mouse anti-human CXCR3 (clone 1C6/CXCR3, BD Pharmingen, or clone 49801 against CXCR3-A and CXCR3-B,14 R&D Systems), followed by FITC-conjugated goat anti-mouse F(ab')2 (Boehringer Mannheim, Indianapolis, Ind), and analyzed with FACSort (BD Biosciences, San Jose, Calif). Real-time reverse-transcription polymerase chain reaction (RT-PCR) was performed on total RNA isolated from 3x105 cells with a Multiscribe RT system protocol (Applied Biosystems, Foster City, Calif), Taqman PCR reagents (Applied Biosystems), and an iCycler (Bio-Rad Laboratories, Hercules, Calif), as previously described.15
Heparin/IP-10 Competition Assay
Confluent HUVECs were washed, resuspended in media without fibroblast growth factor-1/heparin, and then incubated with 100 ng/mL IP-10 at 4°C for 2 hours. The cells were washed, mobilized with 5 mmol/L EDTA or 0.05% trypsin (Invitrogen), and then transferred to 24-well plates at 3x105 cells/mm2. Various concentrations of heparin were added for 30 minutes at 37°C, the supernatants were removed, and the IP-10 levels were determined.
Transendothelial Migration Assay
HUVECs, grown to confluence on 35-mm fibronectin-coated cover glasses, were treated with TNF at 10 ng/mL overnight, incubated with IP-10 for 5 minutes, and then treated with heparin for 5 minutes. The cells were washed and assembled in a parallel-plate flow chamber apparatus (Glycotech, Rockville, Md) on a 37°C heating surface. CD4+ T cells were treated with 400 U/mL IL-2 and 50 µmol/L ß-mercaptoethanol for 7 days and then labeled with 0.3 µg/mL calcein-AM (Molecular Probes, Eugene, Ore) for 1 hour. Then, 1x106 CD4+ T cells in 500 µL RPMI 1640/10% FBS were loaded onto the HUVEC monolayer at 0.75 dyne/cm2 for 2 minutes, followed by media only at 1 dyne/cm2 for 15 minutes. Samples were fixed with 2% formaldehyde in PBS and placed on slides with mounting medium (Biomeda Gel/Mount, Foster City, Calif) containing DAPI (Molecular Probes). A Zeiss Axiovert 200M fluorescence microscope equipped with GFP and DAPI filters was used to detect calcein-labeled T cells and nuclei, respectively. Phase-contrast optics was used to determine whether T cells remained adherent or had transmigrated.
Artery Grafts
Human epicardial coronary arteries, 1 mm in diameter and 3 to 4 mm in length, were interposed into the infrarenal aortas of "nonleaky" (serum IgG <1 µg/mL), 8- to 12-week-old female C.B-17 SCID/beige mice (Taconic, Germantown, NY) using an end-to-end anastomotic technique as previously described.15 The grafts were analyzed by immunohistochemistry and morphometry, as previously described.15
Statistical Analysis
Statistical analyses were performed with Prism 4 (GraphPad Software, San Diego, Calif). Data are represented as mean±SEM. Groups were compared by paired t test, 1-way ANOVA, or repeated-measures ANOVA. Differences with P<0.05 were considered statistically significant.
The authors had full access to the data and take full responsibility for their integrity. All authors have read and agree to the manuscript as written.
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Systemic Heparinization Increased Chemokine Plasma Levels
To determine the effect of heparin on the IFN-
axis, we measured the plasma levels of IL-12, IFN-, IP-10, I-TAC, and Mig in patients with coronary atherosclerosis undergoing CABG surgery (Figure 1A). Samples were drawn after the induction of anesthesia, after initial heparin administration, and during CPB. The CXCR3-binding chemokines were significantly elevated early after heparinization and did not increase further with CPB. In contrast, IL-12 and IFN- remained at low or undetectable levels throughout the study period, suggesting that they were not responsible for the induction in circulating chemokines.
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Because the increase in CXCR3 ligands may have been due to the surgical procedure of median sternotomy and conduit dissection in the 1- to 2-hour period after anesthesia induction and before heparin administration, we measured the chemokine levels immediately before and 3 minutes after the initial intravenous bolus of heparin in an additional group of patients. Heparin administration was associated with an immediate increase in circulating IP-10, I-TAC, and Mig (Figure 1B, 1D, and 1F). Furthermore, this effect was promptly reversed by neutralization of heparin with an infusion of protamine on completion of CPB (Figure 1C, 1E, and 1G). We excluded a trivial explanation for these results by confirming that heparin and protamine did not interfere with the ELISA measurements of either endogenous chemokines or known concentrations of recombinant IP-10, I-TAC, and Mig added to plasma (data not shown). It should be noted that many statistical tests were conducted in our analysis that increased the likelihood of false-positive results. However, confirmation of the effects of heparin on chemokine plasma levels in an independent group of patients and verification by the use of a pharmacological antagonist diminished this possibility. Thus, these observations demonstrated that heparin rapidly and specifically increased plasma chemokine levels.
Heparin Suppressed Chemokine Secretion in Atherosclerotic Coronary Arteries
To gain mechanistic insight into the regulation of CXCR3 ligands, we investigated the effects of heparin on Th1-type immune responses in segments of atherosclerotic human coronary arteries maintained in organ culture. In the absence of exogenous cytokine stimulation, the artery rings secreted very low levels of chemokines. Preliminary experiments revealed that treatment with IL-12/IL-18 resulted in robust production of IP-10 and Mig and less abundant I-TAC secretion in an IFN-–dependent and a dose- and time-dependent fashion (data not shown). Heparin 1000 µg/mL (100 U/mL) but not 100 µg/mL (10 U/mL) inhibited IP-10 and Mig production in response to a submaximal IL-12/IL-18 stimulus (Figure 2A and 2B). Furthermore, heparin 1000 µg/mL (100 U/mL) suppressed IP-10 and Mig secretion at nonsaturating doses of IFN-, and heparin did not induce chemokine production in the absence of cytokines (Figure 2C and 2D). Low-molecular-weight heparin had effects similar to those of unfractionated heparin (data not shown). The organ culture data established that heparin did not induce the secretion or de novo production of CXCR3-binding chemokines by vascular cells or artery-infiltrating leukocytes to account for the elevated chemokine plasma levels seen in the CABG patients but that heparin at relatively high concentrations inhibited chemokine secretion in response to endogenous production or exogenous sources of IFN-.
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Heparin Displaces Chemokines Bound to Endothelial Low-Affinity Receptors
We investigated whether the rapid mobilization of chemokines by heparin in patients may have been due to displacement of cell surface–bound molecules. Endothelial cells, unlike T cells, do not express the chemokine cognate receptor CXCR3 (Figure 3A) but do express membrane GAG, which can bind chemokines with low affinity.10–12 We confirmed that HUVECs did not express alternative isoforms of CXCR3 using a second antibody that recognizes both CXCR3-A and CXCR3-B (data not shown). We further excluded low levels of CXCR3 expression that may not be detected by FACS analysis using a more sensitive technique of real-time RT-PCR. HUVECs did not express detectable levels of CXCR3 transcripts even after cytokine treatment or under flow conditions (Figure 3B).
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To determine whether heparin competes for binding with chemokines to the endothelial cell surface, EDTA-mobilized HUVECs were preincubated with IP-10 and then exposed to increasing concentrations of heparin. In agreement with a competitive binding model, heparin increased IP-10 supernatant levels compared with untreated cells (Figure 3C). In contrast, no release of IP-10 was detected from HUVECs harvested with trypsin (which detaches cells by cleaving membrane and matrix proteoglycans as opposed to Ca2+ sequestration by EDTA), further suggesting that heparin releases surface-bound, not intracellular, chemokine (data not shown). IP-10 also was not detected in the supernatant of heparin-treated PBMCs preincubated with chemokine under the same conditions as HUVECs (data not shown), suggesting that at pharmacologically relevant concentrations, heparin did not effectively compete with binding of chemokines to the high-affinity CXCR3 receptor or that PBMCs sequester insignificant amounts of chemokine. We verified that heparin did not interfere with ELISA measurements of media spiked with chemokines (data not shown). Together, these in vitro results imply that the elevation of plasma CXCR3 ligands by heparin in patients was due to displacement of chemokines sequestered within the vasculature.
Heparin Inhibited IP-10–Dependent Transendothelial Migration of T Cells
We investigated whether the competition of heparin for cell surface–bound chemokine can affect endothelial–T-cell interactions in vitro. We used a transendothelial migration assay of IL-2–cultured CD4+ Th cells that is dependent on the presence of endothelial bound IP-10 under conditions of physiological shear stress. Neither IP-10 nor heparin influenced the attachment of T cells to endothelial cells under flow (Figure 4A). However, heparin inhibited the absolute and relative number of T cells migrating through the endothelial monolayer expressing IP-10 (Figure 4B and 4C). These results confirmed that displacement of membrane-associated chemokines by heparin modulates vascular inflammatory responses.
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Heparin Inhibited Arterial Accumulation and Recruitment of Allogeneic T Cells
Finally, we tested whether the immunomodulatory effects of heparin were relevant in an in vivo model of human arterial inflammation and injury. Immunodeficient mouse hosts bearing human coronary arteries and reconstituted with allogeneic human PBMCs treated with heparin for 2 weeks had a decreased accumulation of memory CD45RO+ T cells within the vessel wall, particularly within the intima (Figure 5A through 5C). There was an associated modest decrease in intimal expansion, whereas differences in outward vascular remodeling did not reach statistical significance (Figure 5D). The artery grafts from heparin-treated recipients displayed diminished IP-10 immunoreactivity compared with those from saline-treated hosts (data not shown). Because the relatively long-term administration of heparin may have decreased chemokine production by inhibiting IFN- responses, we assessed the effects of shorter heparin treatment on T-cell recruitment rather than accumulation. SCID/beige mouse recipients of human coronary artery grafts were reconstituted with allogeneic PBMCs, 2 weeks later received a single dose of heparin, together with fluorescent dye-labeled leukocytes, and were killed after 24 hours. The number of PKH26+ graft-infiltrating cells was decreased in treated compared with control animals (Figure 5E through 5G), suggesting a rapid effect of heparin on T-cell trafficking independent of inhibiting IFN- responses.
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Discussion |
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In this study, we have made a number of related observations in patients and in the laboratory that have led to a greater understanding of the immunomodulatory effects of heparin. First, heparin at clinical doses rapidly and specifically elevates plasma levels of IP-10, I-TAC, and Mig in subjects with coronary atherosclerosis. Second, although heparin at relatively high concentrations may inhibit the production of IFN-
–inducible chemokines in atherosclerotic coronary arteries, at lower concentrations, it displaces surface-bound IP-10 from cultured endothelial cells that lack CXCR3 and are known to express low-affinity proteoglycan binding sites for chemokines. Third, heparin completely inhibits the IP-10–dependent transendothelial migration of IL-2-cultured Th cells under flow conditions. Finally, heparin diminishes both the recruitment and accumulation of allogeneic T cells in human coronary artery grafts in vivo.
The antiinflammatory effects of heparin result from its ability to compete for binding with proinflammatory molecules and inhibit their function.7 The underlying mechanism is that heparin, a soluble GAG, has a structure and biological activities that are similar to those of cell-surface GAG such as heparan sulfate. During homeostasis and inflammation, the electrostatic interactions of membrane-associated GAG to cytokines and chemokines generally enhance their functions and stability. For example, binding of IFN- to heparan sulfate proteoglycans is important for tissue localization and accumulation, protection from proteolytic inactivation, and augmentation of specific receptor activity.8,9 Additionally, immobilization and oligomerization of chemokines on endothelial surface GAG facilitate the formation of concentration gradients and leukocyte migration.10–12 The low-affinity ionic interactions depend on certain acidic and basic residues in GAG and proinflammatory molecules, respectively.8,9,11–13 Specifically, the binding sites for heparin and cognate receptor are distinct on IFN-16 but are partially overlapping on IP-10.17 In both cases, GAG and specific receptor binding are mutually exclusive for a ligand monomer. Binding of cytokine or chemokine to GAG also may cause oligomerization or conformational changes of the ligand, which influence interactions with the specific receptor.10,16 Furthermore, ligands bound to soluble GAG may not be accessible for binding to their cognate receptors because of electrostatic repulsion between the mutually acidic molecules.12 These complex interactions between membrane-bound and circulating factors determine the ability of heparin to inhibit immune responses.
Suppression of IFN- responses by heparin in cultured endothelial cells involves competitive inhibition of the high-affinity–type IFN- receptor,13 whereas chemotaxis of leukocytes under flow conditions may be blocked at the level of low-affinity endothelial GAG binding.18 In addition, heparin competes more effectively than soluble heparan sulfate for binding to cationic proinflammatory molecules.12 It is estimated that heparan sulfate is present at the cell surface at an effective concentration between 100 and 200 µg/mL.13 Interestingly, we required higher concentrations of heparin (1000 µg/mL or 100 U/mL) to inhibit IFN-–dependent chemokine production and lower levels (10 to 100 µg/mL or 1 to 10 U/mL) to displace and block IP-10 in vitro. Although we did not measure plasma concentrations of heparin that mobilized chemokines but not IFN- in our patients, circulating levels of heparin have been measured at 24 to 70 µg/mL before CPB and <1.5 µg/mL after protamine administration.19 It is possible that at concentrations used clinically, heparin may selectively modulate chemokine but not IFN- activity. We also speculate that an excessive release of free chemokines when heparin activity is too rapidly reversed with protamine may cause leukocyte aggregation in the pulmonary circulation such as described for intravenous injection of IL-8/CXCL820 and contribute to the not uncommonly observed protamine hemodynamic reaction characterized by pulmonary hypertension and systemic hypotension.21 A heparin-mediated increase in plasma levels of chemokines may result in greater binding to leukocyte cognate receptors, but vascular recruitment is unlikely if endothelial low-affinity GAG receptors required for a haptotactic gradient are masked by heparin.
The paradigm of soluble GAG mobilizing cationic proinflammatory factors sequestered in the vasculature is well established. In clinical studies >20 years ago, it was observed that heparin increases the circulating levels of platelet factor 4 (PF4)/CXCL4 in both healthy volunteers and patients with coronary atherosclerosis.22,23 Further investigations revealed that heparin displaced PF4 from cultured endothelial cells24 but not whole blood,22 similar to our findings that IP-10 was detected in the supernatant of heparin-treated HUVECs but not PBMCs. Moreover, PF4 and IP-10 share the same binding site on cell-surface GAG, and endothelial cells contain >1000-fold more binding sites for IP-10 than leukocytes.25 The cytokines midkine and pleiotrophin also were shown to be heparin-releasable proteins in the blood of normal volunteers.26 In contrast, TNF was not systemically mobilized by heparin despite binding to GAG in vitro.27 Similar to the TNF study, we may not have detected IFN- in the circulation after heparin infusion because of an absent pool of cell surface–bound IFN-, an insufficient concentration of heparin to compete for binding with high-affinity cognate receptors, or competition by excess unbound IFN- receptors in the vasculature. Finally, intravenous administration of fucoidan, another sulfated GAG, increases the plasma levels of IL-6, IL-8, monocyte chemotactic protein-1/CCL2, macrophage colony-stimulating factor, granulocyte colony-stimulating factor, soluble kit ligand, and stromal-derived factor-1/CXCL12 in mice and monkeys.28,29 We extend these previous observations by demonstrating the mobilization of IP-10, I-TAC, and Mig by heparin in patients, an observation of particular relevance to coronary atherosclerosis because the recruitment of CXCR3-bearing Th1 cells is important in the pathogenesis of the disease.30
Beneficial clinical effects of heparin have been demonstrated in certain inflammatory diseases such as asthma.31 In experimental models, the immunosuppressive properties of heparin can prolong skin allograft survival in mice32 and cardiac allograft and xenograft survival in rats33,34; however, these studies did not assess arterial pathology. We found that heparin inhibits both the recruitment and accumulation of memory T cells to allogeneic human coronary arteries using a chimeric animal model with IFN-–dependent vascular remodeling15 and CXCR3 chemokine expression within the grafts.35 In addition to our findings with regard to T lymphocyte transendothelial migration and arterial recruitment, heparin has been shown to inhibit other atherogenic processes such as monocyte adhesion and recruitment to activated or injured endothelium18,36 and the proliferation of vascular smooth muscle cells.37 In the clinical setting, heparin is a standard therapy for unstable angina,38 but it does not prevent postangioplasty restenosis,39 possibly because of dosing issues.40 It is possible that the antiinflammatory effects of heparin may contribute to its therapeutic efficacy in the treatment of unstable angina, despite certain limitations as an antithrombotic agent. A rebound release of proinflammatory factors may contribute to the relapse specifically associated with discontinuation of heparin in acute coronary syndromes.38 Currently, a number of groups have expressed a great deal of interest in developing synthetic GAG derivatives capable of competitively binding cytokines and chemokines41 and in producing modified chemokines with an alteration in the GAG binding site so that they do not oligomerize on endothelial cell surfaces.11 These complementary and novel therapeutic approaches may hold great promise in the treatment of inflammatory arterial diseases.
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Conclusions |
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We have described an immunomodulatory effect of heparin on IP-10, I-TAC, and Mig in patients, and we have investigated its mechanisms and functional relevance in vitro and in vivo. Our results support the model that endothelial GAG sequester chemokines and play an important role in the chemokine-dependent transendothelial migration and arterial recruitment of T cells. Inhibition of Th1 cell vascular trafficking may contribute to the therapeutic efficacy of heparin in inflammatory arterial diseases, and heparin analogues without anticoagulant activity may translate to a novel antiinflammatory pharmacological strategy.
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Acknowledgments |
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We thank Liping Zhao for expert technical assistance.
Sources of Funding
This work was supported by the NIH (PO1 HL70295). Dr Pober has also received funding from Yale-Boehringer-Ingelheim Pharmaceuticals, Inc., Research Alliance and from other NIH grants.
Disclosures
None.
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