Human Coronary Vessels - Europe PMC

American Journal of Pathology, Vol. 150, No. 3, March 1997 Copyright s American Society for Investigatite Patbology

Immunohistochemical Localization of Defensin in Human Coronary Vessels

Elliot S. Barnathan,* P. N. Raghunath,*t John E. Tomaszewski,t Tomas Ganz,* Douglas B. Cines*,t and Abd Al-Roof Higazit From the Departments of Medicine* and Pathology and

Laborator'y Medicine,t

University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, and the Division of Pulmonary and Critical Care,* University of California at Los Angeles, Los Angeles, California

Neutrophil defensins comprise a family of cationic peptides that possess potent antimicrobial activity. Defensins are normaly sequestered in cytoplasmic granules with their primary site of action in phagolysosomes, although some peptide is released into the circulation during the course of infection or infZammation. In view of the fact that neutrophils adhere to the endothelium and that defensins have been reported to bind to human endothelial ceUs in vitro, we used immunohistochemistry to study the distribution of these peptides in normal and in atherosclerotic human coronary arteries. Defensin was found primarily in the intima of normal and atherosclerotic vessels, most prominently in association with intimal smooth muscle ceUs. Both large- and small-vessel endothelium stained focallyfor defensin. Defensin was alsofound in the media near the external elastic lamina and in some periadventitial vessels. The same distribution was seen in vessels that had been perfusion fixed immediately upon procurement, excluding diffusion of defensin from PMNs ex vivoa These data indicate that neutrophil defensin is present in the waUs of human coronary arteries. The deposition of defensin in vessels may contribute to the pathophysiological consequences of inflammation in addition to their role in host defense. (AmJ Pathol 1997, 150:1009-1020)

Polymorphonuclear leukocytes (PMNs) play a critical role in host defense against microbial organisms. One mechanism by which PMNs contain microbes is by exposing them to antibiotic peptides.) Several

distinct classes of such proteins have been identified, among which are the human defensins. Human defensins comprise a family of closely related cationic peptides, each of which is 29 to 34 amino acids in length and contains three intramolecular disulfide bonds.14 Three of the defensins, human neutrophil peptides (HNPs) 1 to 3, constitute approximately 5% of the total protein in PMNs.5 Defensins are normally sequestered in neutrophil granules but are discharged into phagolysosomes upon ingestion of microbes. Within these compartments, cationic defensins are able to form hydrophobic pores, which disrupt ion fluxes and eventuate in lysis of the organisms with minimal damage to host tissue.46 Defensins are also released into the circulation when PMNs are activated5 as evidenced by the fact that the concentration of HNP-1 to -3 in plasma, which is normally less than 15 nmol/L, approaches 50 ,tmol/L in some patients with sepsis or bacterial meningitis.7 As a consequence, defensins may contribute to the pathophysiological consequences of inflammation in addition to their role in host defense. For example, we previously reported that leukocyte defensins inhibit fibrinolysis8 and that defensins bind specifically to human endothelial cells in vitro and alter the binding of plasminogen and tissue-type plasminogen activator (t-PA) to this cell type.9 One explanation for these effects may lie in the fact that defensins share certain similarities in their amino acid sequence with the lysine-binding sites found in the kringles of plasminogen and, like plasminogen, they can be purified through their affinity for lysineSepharose.6 If similar events occur in vivo, defensins released from activated PMNs could promote the development of thrombotic microvascular occlusion typical of delayed hypersensitivity reactions, some Supported in part by National Institutes of Health grants HL47839 (E. S. Barnathan), HL40387, HL50970, HL49517, and HL54749 (D. B. Cines) and American Heart Association grant 960105000 (A. A. Higazi). Accepted for publication November 13, 1996. Address reprint requests to Dr. Elliot S. Barnathan, University of Pennsylvania School of Medicine, 524 Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104-6060.

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forms of vasculitis, disseminated intravascular coagulation, or thrombosis pursuant to the rupture of atherosclerotic plaque. The present studies were designed to pursue the possibility that leukocyte defensins indeed bind in vivo to endothelial cells or other components of the vessel wall. To date, few studies have carefully addressed the issue of whether leukocyte defensins are found in normal or in pathological human tissue and no study has specifically addressed the localization of defensin in the vasculature. Therefore, the major aim of the present study was to examine the localization of defensin in normal coronary vessels as well as in atherosclerotic coronary vessels in which fibrinolytic activity may be altered.10

Materials and Methods Antibodies Rabbit polyclonal and murine monoclonal anti-defensin antibodies were prepared against highly purified HNP-1, as described.6'11 Both antibodies also recognize HNP-2 and HNP-3 but not HNP-4, -5 and -6, and both stain PMNs intensely. The results of preliminary studies indicated that staining of PMNs as well as specific vascular staining was eliminated when the monoclonal antibody (8 ,ug/ml) was preincubated with excess HNP-1 overnight followed by high-speed centrifugation. All subsequent studies were performed using a fivefold lower concentration of antibody (1.6 ,tg/ml), which continued to produce intense staining of PMNs although no staining was observed using an isotype (IgG1) control at this concentration. Sections of vessels were also stained with polyclonal anti-defensin antiserum (1:3,000 to 1:100,000). In these experiments, rabbit serum at the same concentrations or the equivalent concentration of rabbit IgG were used as controls. Antibodies to the following antigens were used to define specific cell types: von Willebrand factor (polyclonal goat anti-human at 1:3750 dilution) and CD31 (JC/70A, monoclonal at 13 jig/ml) for endothelial cells, a-smooth muscle actin (IA4, monoclonal at 0.12 ,tg/ ml) for vascular smooth muscle cells, CD68 (KP1, monoclonal at 0.36 ,ug/ml) for macrophages, and CD3 (A452, polyclonal rabbit anti-human CD3 at 1:50 dilution) for T lymphocytes. All antibodies other than those against defensin were obtained from Dako Corp. (Carpinteria, CA).

Tissue Samples Vascular samples were obtained from human hearts removed at the time of transplantation. Additional samples of coronary arteries were obtained from normal donor heart tissue not used for transplantation. A total of 25 coronary arteries from 18 patients (15 male and 3 female; 8 with severe coronary artery disease, 1 with alcoholic cardiomyopathy, 1 with familial cardiomyopathy, and 8 with idiopathic dilated cardiomyopathy) were evaluated. Replicate 2to 3-mm sections were fixed by immersing each sample in either 1) 100% ethanol overnight at 21°C, 2) 10% neutral buffered formalin overnight at 210C, or 3) 4% paraformaldehyde overnight at 4°C followed by 15% sucrose for 4 hours. No significant differences in antigen preservation were noted among the three fixation regimens. Therefore, most studies were carried out using tissue fixed in 100% ethanol. In one set of experiments, the mid-portion of a coronary artery was removed and the distal portion of the vessel was perfusion fixed immediately with a modified Karnovsky's fixative containing 1% glutaraldehyde and 4% paraformaldehyde. A second 2- to 3-mm section of the artery was then removed distal to the catheter site and immersed in Karnovsky's fixative overnight as above. All samples were then embedded in paraffin, and serial 5-,um sections were prepared for immunohistochemistry.

Immunohistochemistry For immunolabeling, a modification of the streptavidin-biotin peroxidase method was performed using capillary action technology and the MicroProbe System (Fisher Scientific, Pittsburgh, PA). Normal rabbit IgG and isotype-matched nonimmune mouse IgG1 were used as irrelevant control antibodies. Briefly, sections were deparaffinized at 55°C for 30 minutes, placed in xylene, and hydrated in 100% and then 95% ethanol, and endogenous peroxidase activity was quenched with a 2.2% (v/v) H202/methanol solution added for 10 minutes. The slides were then washed with 1X automation buffer (Biomeda Corp,, Foster City, CA) with 10% normal horse or goat serum added for 20 minutes at room temperature to block nonspecific binding. Primary and secondary antibodies were diluted in 1X automation buffer containing 10% normal horse or goat serum. The slides were then incubated with primary antibody for 60 minutes at 370C, washed in 1X automation buffer for 10 minutes, incubated with biotinylated anti-mouse or anti-rabbit antisera (Vector Laboratories, Burlingame, CA) at a 1:200 dilution for 30 minutes at 370C

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Figure 1. Localization of defiensin in histologically normal and atherosclerotic coronary art(eies: Effect of intimal thickening. Samples of' epicardial coronary arteries were taken from a donor heart uwith histologically normal-appearinig vessels (A), from a patienit with idiopathic dilated cardiomyopathy and tnormal coronary arteries by angiographv but with early atherosclerotic changes in the intima (B), andfromn a patient u'ith extensive atherosclerotic coronary artery disease (C). The prmaty antibody (detected as a brown color) in A to C was a monoclonial defensin antibody (1.6 ,g/ml) whereas in D, a serial section fronm the same artery shown in C uas incubated with an equal concentration of normal murine IgG,. Original magnification, x200 (A) and x 100 (B to D);

bar, 100pum.

and washed again with 1X automation buffer. The slides were then incubated for 30 minutes at 370C with the streptavidin-biotin system (Dako) at a 1:50 final dilution and developed by adding 0.05% (v/v) 3,3'-diaminobenzidine solution (Sigma Chemical Co., St. Louis, MO) and 0.03% (v/v) H202 for 5 minutes. After a final wash, the slides were counterstained with aqueous hematoxylin and dehydrated in ethanol and xylene, and the coverslip was secured with Permount. In other studies, the extent of staining in coronary arteries with minimal or no disease (four patients with idiopathic dilated cardiomyopathy but normal coronary angiograms and two normal coronary arteries from heart donor tissue) were compared with five coronary arteries from patients with severe ischemic cardiomyopathy and extensive coronary atherosclerosis using a single antibody (monoclonal, 1.6 ,ug/ml) without reference to clinical history. All samples were reviewed by two experienced investigators for the extent and intensity of staining. The scoring system used for extent of staining represents the percentage of cells with any staining in that compartment: 0, no cells positive; 1, 50%. Unpaired, two-tailed Student's t-test was used to compare atherosclerotic

and normal arteries.

Results The main objective of this study was to determine whether leukocyte defensins are found in the human vasculature. To address this issue, sections of normal-appearing human coronary arteries and vessels involved with atherosclerosis were studied using polyclonal and monoclonal anti-defensin antibodies previously shown to recognize HNP-1 to -3, but not HNP-4 to -6.6,1 1 Defensin was identified in the intima of normal-appearing coronary arteries (Figure 1A) as well as in arteries from patients with minimal intimal thickening (Figure 1 B). A rim of intense staining was also seen near the external elastic lamina. Similar amounts of defensin were found in normal-appearing vessels and those involved with atherosclerosis. However, the distribution of defensin was more heterogeneous in arteries with more extensive intimal

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hyperplasia (Figure 1C). The greatest intensity and extent of staining was in the intima. Medial staining was, in general, less intense and variable in more diseased arteries, although some staining was present in all vessels. Variable staining was observed in the adventitia. No staining was seen with an isotype control IgG (Figure 1D). In the most severely affected vessels (Figure 2, A and B), the heterogeneous pattern of defensin localization within the intima and media was even more evident compared with its distribution in histologically normal vessels (Figure 2C). This appearance was most evident in areas of the vessel wall expanded by acellular plaque rather than in areas of intense intimal cell proliferation. Defensin antigen was found co-localized with luminal endothelial cells at times in a patchy distribution (Figure 2B) and was also found co-localized with intimal and medial smooth muscle cells and in some but not all microvessels feeding the adventitia. Little staining was present in macrophage-rich areas (Figure 2, D and E); few, if any macrophages were present in the normal vessel (Figure 2F). Intense defensin staining (Figure 2A) was noted in some areas devoid of smooth muscle cells and macrophages. In addition, staining for defensin was not co-localized with T lymphocytes, as demonstrated by staining serial sections for defensin (Figure 2B) and CD3 (Figure 2N). No staining was seen using control IgG1 at the same concentrations (eg, Figure 2, M and 0). The distribution of defensin within the intima was studied in more detail in two epicardial coronary arteries with minimal histological abnormalities (Figure 3). Staining for defensin was most intense in and around intimal smooth muscle cells (identified in serial section by a-smooth muscle actin staining; Figure 3, E and F). Luminal endothelial cells, identified in serial sections by CD31 (Figure 3, G and H) also stained for defensin, albeit with somewhat less intensity (Figure 3A, monoclonal antibody; Figure 3B, polyclonal antibody). Staining for defensin was again noted in some small periadventitial vessels and occasionally in areas noted to contain CD68-positive cells (Figure 3C). Defensin staining was also seen in occasional cells within the myocardium that had the morphologic appearance of tissue macrophages (not shown).

To study defensin localization within the periadventitia in greater detail, serial sections of an intramyocardial coronary artery stained with anti-defensin antibodies and with histogenetic markers were analyzed at higher magnification (Figure 4). Smooth muscle cells were identified by staining for a-smooth muscle actin (Figure 4A). Even at lowpower magnification, the focal localization of defensin near the external elastic lamina was evident using monoclonal (Figure 4B) or polyclonal (Figure 4C) antibody. Analysis of sections under higher power magnification of the area within the box shown in Figure 4B indicates that defensin protein was confined to a PMN within a capillary lumen and to the surrounding capillary endothelium identified by CD31 expression (Figure 4D). Localization of defensin in endothelial cells was especially evident on a longitudinal section of a small vessel (Figure 4G, horizontal arrow). No CD68-positive cells were detected in this area (Figure 4E). Defensin was detected (Figure 4G) in association with cells around the capillary (vertical arrow) that stained positive for a-smooth muscle actin (Figure 4F). We then explored whether endothelial cells were found to contain defensin only as a result of its release from neutrophils in close proximity. To do this, serial sections were stained with very low concentrations of antibody (polyclonal serum 1:25,000 dilution) to maximize specificity and limit the halo effect associated with the intense staining of PMNs (Figure 5). In some vessels, staining of the endothelium was apparent only at sites where direct contact with PMNs was evident (Figure 5A). Multiple examples of similar focal concentrations of defensin antigen in endothelial cells were also found in large coronary arteries (Figure 5B). In a few such instances, defensin antigen could be attributed to direct diffusion from neighboring PMNs (Figure 51), whereas in others, the entire vessel stained for defensin and no PMNs were identified (Figure 5, C and H). At lower power, the heterogeneous distribution of defensin among periadventitial capillary endothelial cells (Figure 5L) compared with the uniform staining for CD31 (Figure 5M) could be readily appreciated. These results led us to address the possibility that the endothelial cells stain for defensin as a result of the protein diffusing out of PMNs ex vivo

Figure 2. Localization ofdefensin in atherosclerotic and niornmal coronary arteres. Serial sectionls qf an atherosclerotic coronary artery at low pou'er (left column; X 20; bar, 1 mm) and highpower(middle column; X 400; bar, 100 p.m) and a histologically normal coronary artery at lowpower(right column; x 40; bar, 1 mm) are shown. The arrow in A denotes the area depicted at higherpower in B. A to C: Incubated with a monoclonal defensinl anttibody ( 1.6 ,ug/ml). D to L and N: Localization of histogenetic markers. Sections incnibated wvith normal IgG, as the control ( 1.6 p.g/mnl) are shoun in M and 0. D to F: Localization of macrophages (CD68). G to l: Identification of smooth inuscle cells (a smooth mu.scle actin). J to L: Endotheliuim (CD31). N: Localization of T lymphocytes (CD3).

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Figure 3. Localizationolc)de/f',nsin in the fibroiiitinam. Serial sectiolis *f coronlary arteries

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fionm diff/erentpatients nith ischenmic cardioni -opathY antd niiild infinial thickening u'ereXchosen ./or study'. Originlal mag?nlficationi, 200

(left column) amid x 400 (right column; bars, 50 pini. 7he vessel shown in A w'as stained with a

rionoclonal dle/eInsin anltibody ( 1.6 p.ginml) whereas thci shown in B was stained with a polvclonial antisernum ( 1.25,000 dilntion). NVote al staiciied leuIkocyte near oneportion of thbe endothelinniii bitt that the imitiniia at considerable distance firon this cell is also stainied. Scerial sectionis staiiiied nith the cell-specific markers nioted in the lecgend to Figu.re 2 were used to identmJj'. cell types in the section. C and D: Macrophages. E and F: Sniooth muscle cells. G and H: Endothelial cells. The sectionis shown in and J were incubated with mouse IgGl ( 1.6 jig/ml) and rabbit IgG (3.6 jig/ml), respectively, as primary antibody controls.

before adequate fixation of the tissue sections. Some support for this possibility was noted in the occasional sample where the size of the halo detected around PMNs in the myocardium was broader in the central portions of the section (ie, further from the surface of the tissue block fixed by immersion) than near the surface. To address this concern, 2- to 3-mm samples from the mid-left anterior descending coronary artery obtained at the time of heart transplantation were immersion

fixed in 100% ethanol, as per our standard protocol. Immediately afterward, the vessel was cannulated and the distal vessel perfused with Karnovsky's fixative to induce rapid fixation from the luminal side outward. Sections of the vessel were obtained distal to the site of perfusion and additionally immersion fixed overnight (Figure 6). As before, using a monoclonal antibody in the ethanol-fixed section (left side), defensin was again co-localized with endothelial and smooth muscle

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Figure 4. Localization of defensin in periadventitial vessels. 7Te section shown was taken from an intramyocardial coronary artery that had been fixed in 100% neutral bufferedformalin. A: Low-power view (original magnification, X 100) stainedfor smooth muscle cell actin (bar, 500 gum). B: Serial section stained with monoclonal defensin antibody ( 1. 6 jig/ml). The box denotes the area studied at higher power with histogenetic markers. C: Polyclonal defensin antiserum (1:10, 000 dilution). D to are immediately adjacent 5-,im serial sections (original magnification, x 1000; bar, 50 jim) stained with antibodies to the following antigens: D, CD31 to identify endothelial cells; E, CD68 to identify macrophages (none found); F, a-smooth muscle actin to identify smooth muscle cells; and G to 1, defensin using both a monoclonal antibody (G) and a polyclonal antibody (H; 1:10,000, same slide as C). shows the adjacent section stained with the same polyclonal defensin antibody at a 1:25,000 dilution, emphasizing the reduction in halo size with dilution ofpolyclonal antiserum compared with that seen with the monoclonal antibody (G). Black arrows denote the position of two small vessels traversing the section. A leukocyte is seen within the lumen of the vessel in the top of the field in D.

cells. The identical distribution was seen in the distal sections of the same coronary artery that had been subject to immediate perfusion fixation. The focal nature of defensin localization in the endothelium is evident using both fixation techniques (Figure 6, F and H) and with monoclonal (Figure 6, A, B, and F) or polyclonal (Figure 6H)

antibody.

Discussion The main finding from this study is that defensin antigen (HNP-1 to -3) is present in the endothelium and smooth muscle cells of the human coronary vasculature. The specificity of this observation was confirmed by 1) the use of both monoclonal and polyclonal antibodies that showed the same pattern

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Figure 5. Localization of defensin in arteries, venules, and capillaries. Images are taken from small vessels uwithin the periadventitia of an epicardial coronary artery. A, B, C, G, H, 1, and L: Sections were stained u'ith a polyclonal anti-defensin antibody (1:25,000 diluition). D, E, F, and M: An-ti-CD31 was used to identify the endothelitum. J and K: Incuibated with normal rabbit IgG (.3.6 l.g/ml) as a control. All bars, 50 Am. Original magnification, x 1000 (A, C, D, F, H, 1, and K), x 400 (B, D, G, and J), and X200 (L and M). A: Two leukocytes in a ventule stainedfor defensimi. Otie leukoc'te appears to be adherentt to the v'essel wall, anid the adjoining enidothelial cell stains tbr dlefensinn. B: Def'tnsin localized to a nacrov'ascular endothelial cell in an epicardial coronarv artery. C: Dcfensin within enidothelial cells lining a ivenuile. G to 1: Defensini within capillary enidothelial cells. In onie case, shown in 1, an ad;oininig leutkocyte was detectecd. Defensin was Ibunnd in a some hoat not all periadientitial capillaries (L) o'herea%scall capillaries stained unifonrnyvfor CD31 (M).

of defensin localization in vascular tissue, 2) the use of very dilute concentrations of primary antibody to limit nonspecific binding, 3) elimination of specific binding when the monoclonal antibody was preadsorbed with highly purified defensin, and 4) utiliza-

tion of four different fixation regimens with similar results. Defensin was found to a variable extent in capillaries, arterioles, venules, arteries, and veins in both normal donor heart tissue as well as in vessels from

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Figure 6. Effe?ct ofperfjisioni fixationi oni the localizatiot of defensin in ntormnal-appearinig himan

coronary artery. A 2- to 3-mm-thick .sec-

of coronary artery uwas removed from the mid-left anterior descenditng artery of a 36year-old man ivith atn idiopathic dilatecd cartion

dionmvon athv antd hi.stolovicaltv flurxse,s normal ,LU{t.co,oJ,otutust,;vvrs nary arteries. The sample u'as immersion fixed overnight in 100% ethanol at 21C. A, C, E, and G: Serial sections from this block fixed in ethanol at an original magnification of X 400. After removing the first tissne block, a catheter u'Cas immediatel/ inserted intto the samc artery and perfuision fixed distally with inodified Karnovsky's fixative. A 2- to 3- nm section takent after perfusion from a site distal to the catheter u'as additiotnally in niersiot fixed overnight. B, D, F, and H arefrom this block. A and B show the localization of defentsin (monoclonal antibody at 1.6 gg/lnl). Smooth muiscle cells (C and D,) endothelial cells (E), and macrophages (G) ivere identified as described previously. B shous the localizatiotn of defrnsin in the sanie artery at a more distal location after perficsion fixationi. Note the presence of dcft'nsin anztigen in endothelial cells and intimal smnooth muiscle cells. D shous the localizationi of sniiooth mutscle cells in ani adjacenit perfnsion fixed section. F demonstrates decfenisin antigen in one of seieral capillaries in the same section. H is a parallel sectioni stained with a polyclonlal dcfensin antibodJy ( 1: 10, 000 diluitiot), uhich denionst ratces leukocyte and endothelial cell staiiiitig in a vessel perfuisioni fixed with inodified Karnovsky s fixative. Origitnal niagniffication, X 1000 (B, D, F, antd H); bar, 50 gtn). -""t&Jt1

patients with end-stage heart failure undergoing cardiac transplantation. Staining for defensin in epicardial coronary arteries was most intense in the intima, especially in association with the intimal smooth muscle cells, and in the media near the external elastic lamina. Defensin was also found in endothelial cells, generally in a focal manner. The same predominantly intimal distribution was seen in vessels with early fibrointimal hyperplasia. Although we found no significant correlation between the overall amount of defensin antigen and the extent of atherosclerosis in the limited number of vessels studied, the distribution of defensin was more heterogeneous

in vessel walls with more advanced atherosclerotic changes, including vessels with extensive matrix deposition and those that contained complex, acellular plaque. There is little precedent for the finding of defensin in human vessels. Indeed, expression of defensin protein or mRNA in vivo in either normal or pathological samples of human tissue has received relatively little study. Defensins have been isolated from the neutrophils of many species, including man.1 In rats, leukocyte defensin mRNA (rat NP1-4) appears to be restricted to myeloid precursors in the bone marrow, whereas mRNAs for other defensins have

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been found in murine and human Paneth cells13-15 and in bovine tongue and trachea in response to inflammation.16-18 More recently, a novel f3-defensin has been identified in human plasma, but its cellular source has not been identified19 and a defensinrelated peptide, NP-3A (corticostatin 1), has been identified in adrenal and pituitary tissue.20 Immunohistochemical studies have also suggested that leukocyte defensin is found mainly in myeloid cells in humans21 and alveolar macrophages in rabbits.12 However, there is evidence that defensin can diffuse across the blood-brain barrier and penetrate the neuropil, suggesting a mechanism by which defensin may accumulate in tissue. Similarly, defensin has been detected on the surface of adult filariae and in surrounding tissue in patients with onchocerciasis.22 These latter findings suggest that defensin, which is known to be released from activated neutrophils,7 can bind to other cell types. It is likely that defensin accumulates in human coronary arteries through a similar mechanism. The proposed existence of binding sites for defensin in human vascular tissue is in accord with our observation that defensin binds specifically to cultured endothelial cells9 and vascular smooth muscle cells (unpublished observations) in vitro. Such binding of defensin to cells is not random or nonspecific, as defensin was often seen focally in association with endothelium and vascular smooth muscle but not with other cellular components of the vessel wall such as fibroblasts or T lymphocytes. Nor is it likely that staining of the tissue sections occurred as an artifact of ex vivo release from neutrophils, as defensin was seen at the same locations, including deep in the media, in vessels that had been perfusion fixed immediately after their removal. Rather, the data suggest that defensin released as a result of neutrophil senescence or activation in the circulation permeates and is retained in the vessel wall. Alternatively, neutrophils may be activated within the vessel wall. A final possibility, although less likely, is that defensin or a related molecule is synthesized locally. Additional studies with in situ hybridization or in situ polymerase chain reaction will be required to address the latter possibility. The implications of defensin localization in the vasculature are uncertain. As recent data suggest that certain microbes may play a pathogenetic role in atherosclerosis,23 it is conceivable that defensin may function to limit microbial invasion of vascular tissue or maintain the latency of organisms that infect vascular tissue. However, the requirement for high defensin concentrations, low salt concentrations, and absence of interfering plasma proteins make an antimicrobial role less likely.2 Rather, release of de-

fensins into tissue as a consequence of neutrophil activation may mediate some of the pathophysiological consequences of inflammation. For example, defensins have been reported to be chemotactic for monocytes24 and T lymphocytes,25 stimulate the proliferation of fibroblasts,26 act as an agonist for neutrophils27 and mast cells,2" and alter intercellular permeability.29 It is easy to conceive that additional effects of defensins on the growth and behavior of endothelial cells and smooth muscle cells exist that remain to be identified. Another consequence of defensin residing within vascular tissue may relate to its effect on the binding of plasminogen to endothelial cells and the capacity of defensin to inhibit plasminogen activation by tPA.8'9 Defensins localized in or around the endothelium may also inhibit fibrinolysis in vivo, predisposing to thrombus accumulation particularly after plaque rupture. Furthermore, defensin may also modulate other plasmin-mediated effects such as matrix turnover and intimal proliferation. For example, it has been suggested that plasmin inhibits smooth muscle cell proliferation and/or migration by activating transforming growth factor-3,30'31 in which case accumulation of defensin within the intima may represent a previously unrecognized mechanism by which growth of these cells is regulated. In contrast, however, it has recently been reported that mice deficient in plasminogen activator inhibitor type 1 (PAI-1) develop more exuberant intimal thickening after mechanical vascular injury than do normal mice, whereas animals that lack urokinase-type plasminogen activator (u-PA) but not t-PA are relatively protected,32 suggesting a specific role for u-PA-mediated plasminogen activation after injury to facilitate smooth muscle migration and intimal thickening. Furthermore, profibrinolytic proteins, including urokinase and its receptor as well as t-PA are more abundant in the intima relative to the media in vessels more severely affected with atherosclerosis.10 Finally, three recently published studies33-35 demonstrate that components of the plasminogen activator system such as u-PA or its receptor, u-PAR, or t-PA can augment smooth muscle cell migration and/or adhesion, whereas PAI-1 can limit this process, in part via direct interactions with integrins. Thus, the ability of defensin to limit fibrinolysis could have opposing effects on the vessel wall depending on the type of injury or disease. Irrespective of its net effect, the results of this study suggest that the role of defensin in the pathophysiology of vessel wall disorders is deserving of more study.

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Acknowledgments We acknowledge Dr. Ehud Lavi for his helpful discussions, Robert Caron for technical assistance, and the entire heart transplantation team for helping in the expeditious procurement of tissue.

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References 1. Kagan B, Ganz T, Lehrer RI: Defensins: a family of antimicrobial and cytotoxic peptides. Toxicology 1994, 87:131-149 2. Harwig S, Ganz T, Lehrer RI: Neutrophil defensins: purification, characterization, and antimicrobial testing. Methods Enzymol 1994, 236:160-172 3. Levy 0: Antibiotic proteins of polymorphonuclear leukocytes. Eur J Haematol 1996, 56:263-277 4. White S, Wimley W, Selsted M: Structure, function, and membrane integration of defensins. Curr Opin Struct Biol 1995, 5:521-527 5. Ganz T: Extracellular release of antimicrobial defensins by human polymorphonuclear leukocytes. Infect Immun 1987, 55:568-571 6. Ganz T, Selsted M, Szklarek D, Harwig S, Daher K, Bainton D, Lehrer R: Defensins: natural peptide antibiotics of human neutrophils. J Clin Invest 1985, 75: 1427-1435 7. Panyutich A, Panyutich E, Krapivin V, Baturevich E, Ganz T: Plasma defensin concentrations are elevated in patients with sepsis or bacterial meningitis. J Lab Clin Med 1993, 122:202-207 8. Higazi A, Barghouti I, Abu-Much R: Identification of an inhibitor of tissue-type plasminogen activator-mediated fibrinolysis in human neutrophils. J Biol Chem 1995, 270:9472-9477 9. Higazi A, Ganz T, Kariko K, Cines D: Defensin modulates tPA and plasminogen binding to fibrin and endothelial cells. J Biol Chem 1996, 88:17650-17655 10. Raghunath P, Tomaszewski J, Brady S, Caron R, Okada S, Barnathan E: Plasminogen activator system in human coronary atherosclerosis. Arterioscler Thromb Vasc Biol 1995, 15:1432-1443 11. Panyutich A, Voitenok N, Lehrer R, Ganz T: An enzyme immunoassay for human defensin. J Immunol Methods 1991, 141:149-155 12. Yount N, Wang M, Yuan J, Oulette A, Selsted M: Rat neutrophil defensins: precursor structures and expression during neutrophilic myelopoiesis. J Immunol 1995, 155:4476-4484 13. Oulette A, Miller S, Henschen A, Selsted M: Purification and primary structure of murine cryptin-1, a Paneth cell defensin. FEBS Lett 1992, 304:146-148 14. Jones D, Bevins C: Defensin-6 mRNA in human Paneth cells: implications for antimicrobial peptides in host defense of the human bowel. FEBS Lett 1993, 315:187-192 15. Mallow E, Harris A, Salzmann N, Russell J, DeBaradinis R, Ruchelli E, Bevins C: Human enteric defensins: gene

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