Thy-1, a Novel Marker for Angiogenesis Upregulated by Inflammatory Cytokines

From the Cardiovascular Biology Laboratory, Harvard School of Public Health (W.-S.L., M.K.J., B.M.A., D.Z., S.K., K.M., S.-L.L., M.-E.L., E.H.), the Department of Medicine, Harvard Medical School (N.K.H., M.-E.L., E.H.), and the Divisions of Cardiology (M.K.J., M.-E.L.) and Radiology (N.K.H.), Brigham and Women's Hospital, Boston, Mass, and the Department of Chemistry, National Cheng Kung University (S.-Y.S.), Tainan, Taiwan, ROC.

Correspondence to Mu-En Lee, MD, PhD, Cardiovascular Biology Laboratory, Harvard School of Public Health, 677 Huntington Ave, Boston, MA 02115. E-mail lee{at}cvlab.harvard.edu

	Abstract

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Abstract—We identified the cell surface glycoprotein Thy-1 on the endothelium of newly formed blood vessels in four models of angiogenesis in adult rats. Anti–Thy-1 staining showed that Thy-1 was upregulated in adventitial blood vessels after balloon injury to the carotid artery. Preabsorption with a rat Thy-1–Ig fusion construct eliminated all immunoreactivity and thus confirmed the specificity of the Thy-1 staining. Thy-1 was also expressed in the endothelium of small blood vessels formed after tumor implantation in the cornea, in periureteral vessels formed after ligation of the renal artery, and in small blood vessels of the uterus formed during pregnancy. In contrast with its expression during adult angiogenesis, Thy-1 was not expressed in the endothelium of blood vessels during embryonic angiogenesis. In vitro, the inflammatory cytokines interleukin-1ß and tumor necrosis factor- upregulated Thy-1 mRNA by 8- and 14-fold, respectively. Vascular endothelial growth factor, basic fibroblast growth factor, transforming growth factor-ß, and platelet-derived growth factor-BB had no effect on Thy-1 mRNA. Thus, Thy-1 appears to be a marker of adult but not embryonic angiogenesis. The upregulation of Thy-1 by cytokines but not growth factors indicates the importance of inflammation in the pathogenesis of adult angiogenesis.

Key Words: endothelium • interleukin-1 • tumor necrosis factor • inflammation • angiogenesis

	Introduction

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Angiogenesis is fundamental to reproduction, fetal development, and tissue repair. However, angiogenesis also plays an important role in the pathogenesis of tumor growth and ophthalmic and rheumatic diseases.¹ Although the sequence of events governing angiogenesis associated with pathological or reparative processes in adults no doubt resembles that associated with developmental processes in embryos, it probably involves additional mechanisms more distinct to adults. In pathological conditions, for example, angiogenesis is thought to be accompanied by, and may possibly require, inflammation.² Indeed, the elaboration of inflammatory cytokines has been shown to induce angiogenesis, even though the mechanisms regulating this elaboration remain poorly understood.^{3 4}

Thy-1 is a cell-surface glycoprotein that belongs to the immunoglobulin-like supergene family.⁵ Originally described as a marker for thymocyte differentiation in mice,⁶ Thy-1 was subsequently found to be expressed highly in neuronal cells.^{7 8} Variable Thy-1 expression has been observed in other cell types, including vascular endothelial cells.⁹

Thy-1 appears to be involved in cellular growth and differentiation.^{8 10} Although the natural ligand for Thy-1 has not been identified, cross-linking antibodies specific to Thy-1 have been shown to initiate signaling events in murine neuronal cells¹¹ and to increase cytoplasmic calcium in murine T cells and Thy-1–transfected B cells.^{12 13} Also, in mice bearing a targeted disruption of the Thy-1 gene, long-term potentiation in the hippocampus is impaired.¹⁴

Using antibodies specific to Thy-1, we identified it as a cell-surface marker expressed on microvascular endothelial cells during new blood vessel formation. Thy-1 was expressed during pathological and physiological angiogenesis in adult rats but not during embryonic angiogenesis. In cultured microvascular endothelial cells, the inflammatory cytokines interleukin-1ß and tumor necrosis factor- selectively upregulated Thy-1 expression. Vascular endothelial growth factor, basic fibroblast growth factor, transforming growth factor-ß, and platelet-derived growth factor-BB had no effect on Thy-1 expression.

	Materials and Methods

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Animal Models
Thy-1 expression in endothelial cells was studied in four rat models: (1) Renal artery stenosis was performed according to the method of Ilich et al.¹⁵ The main renal artery was occluded partially by the application of a silver clip. In "sham"-operated rats, the silver clip was removed. Ureters were harvested 5 days after ligation. Tissue was provided by N.K.H. (2) Implantation of a tumor in the cornea was performed according to the method of Gimbrone et al.¹⁶ Corneas were harvested 10 days after tumor implantation. Tissue was provided by Dr M. Judah Folkman (Children's Hospital, Boston, Mass). (3) Balloon angioplasty of the carotid artery was performed by the Zivic Miller Co (Zelienople, Penn). Carotid arteries were harvested from adult male Sprague-Dawley rats 14 days after they had been subjected to balloon injury. (4) Neovascularization was assessed in the early pregnant uterus. Uteruses, including placentas and embryos, were harvested after 6 to 18 days of gestation by Novagen, who provided slides of embryonic tissue.

Immunocytochemistry
All tissue was fixed with 4% paraformaldehyde and processed for paraffin embedding in an automated system (Hypercenter XP, Shandon Scientific). Immunocytochemical staining was performed as described.^{17 18} Paraffin was removed from the tissue sections, and they were incubated with 10% normal goat serum for 30 minutes at room temperature to reduce nonspecific binding. The sections were incubated with one of four primary antibodies for 1 hour at room temperature and then overnight at 4°C, after which they were rinsed twice with high-salt PBS (0.5 mol/L NaCl) and once with regular PBS for 5 minutes (each wash) and then incubated with one of four secondary antibodies for 1 hour at room temperature. The antibody combinations were (1) mouse anti–rat Thy-1 antibody (clone OX-7, Pharmingen) at 1:300 dilution and biotinylated goat anti–mouse IgG1 (Amersham) at 1:100 dilution, (2) mouse anti–rat Thy-1 antibody (clone HIS51, Pharmingen) at 1:100 dilution and biotinylated goat anti–mouse IgG2a (Amersham) at 1:100 dilution, (3) rabbit anti–human von Willebrand factor antibody (DAKO) at 1:1000 dilution and biotinylated goat anti–rabbit IgG (heavy and light chains) (Vector Laboratories) at 1:2000 dilution, and (4) rat anti–mouse CD31 (platelet endothelial cell adhesion molecule-1) antibody (Pharmingen) at 1:100 dilution and biotinylated rabbit anti–rat IgG (heavy and light chains) (Vector Laboratories) at 1:100 dilution.

The tissue sections were then rinsed twice with high-salt PBS and once with regular PBS for 5 minutes (each wash) and incubated with avidin-biotin complex (ABC reagent, Vector Laboratories) at 1:100 dilution for 1 hour at room temperature. After a wash with PBS, the sections were developed with 3,3'-diaminobenzidine or 3,3'-diaminobenzidine plus nickel sulfate in PBS-H₂O_2. A brown color showed positive staining for 3,3'-diaminobenzidine; a blue/black color showed positive staining for 3,3'-diaminobenzidine plus nickel sulfate. To control for anti–Thy-1 antibody specificity, we incubated tissue in anti–Thy-1 antibody (clone OX-7) that had been preabsorbed with 25 µg/mL Thy-1–Ig fusion protein (see below); this procedure blocked all staining. Counterstaining was performed with 1% methyl green.

Immunostaining for the presence of macrophages and proliferating cell nuclear antigen (PCNA) was performed with the antibodies ED-1 (Serotec) and Ab-1 (Calbiochem), respectively. Tissue was treated with methyl Carnoy's fixative and embedded in paraffin. Sections were cut at a thickness of 5 µm. The antibody combinations were as follows: (1) ED-1 (1:300 dilution) and biotinylated horse anti–mouse IgG (heavy and light chains) (rat adsorbed) (Vector Laboratories) (1:100 dilution) and (2) Ab-1 (1:100 dilution) and goat anti–mouse IgG2a (1:100 dilution).

Construction and Expression of Rat Thy-1–Ig Fusion Protein
A rat Thy-1–Ig expression plasmid (pThy-1–Ig) was constructed by connecting the extracellular fragment of rat Thy-1 (residues Gln1-Gly113)¹⁹ to a human IgG1 constant region containing the hinge, CH2, and CH3 regions. The rat Thy-1 and human IgG1 DNAs were obtained by polymerase chain reaction with templates from rat brain cDNA (Clontech) and human spleen cDNA (Clontech), respectively. The Thy-1 forward primer, 5'-GCG CAG AAG CTT ATT GGC ACC ATG AAC CCA GTC ATC-3', corresponded to the N-terminus of the rat Thy-1 signal sequence plus a HindIII restriction endonuclease site. The Thy-1 reverse primer, 5'-CCT CGA GAG ATC TCC ACC ACA CTT GAC CAG CTT GTC-3', corresponded to residues Asp106-Gly113 of rat Thy-1 plus a BglII restriction endonuclease site. The IgG1 constant region forward primer, 5'-A GAT CTC TCG AGT AGA CCC AAA TCT TCT GAC AAA ACT CAC ACA TCC CCA CCG TCC CCA-3', corresponded to residues Pro227-Pro243 of the human IgG1 hinge region plus a BglII restriction endonuclease site. Three mutations were also introduced in the nucleotides of the hinge region to change cysteines to serines. The IgG1 constant region reverse primer, 5'-TCT AGA CGG CGG TCG CAC TCA TTT ACC-3', corresponded to the C-terminus of the human IgG1 CH3 region plus an XbaI restriction endonuclease site. The polymerase chain reaction products of the Thy-1 and human IgG1 constant regions were digested with HindIII-BglII and BglII-XbaI, respectively. The digested fragments were ligated to the HindIII-XbaI site of pcDNA1.1/Amp (Invitrogen) to make the final expression plasmid pThy-1–Ig.

The Thy-1–Ig fusion protein was transiently expressed in COS cells as described by Linsley et al²⁰ and purified from the conditioned medium by protein-G affinity chromatography (Pharmacia). The purity of the protein was examined by SDS-PAGE, and its concentration was determined by the DC protein assay (Bio-Rad). We assayed for the presence of rat Thy-1 epitopes by Western blot analysis with a biotin-conjugated mouse anti–rat Thy-1 primary antibody (clone OX-7) and a streptavidin-conjugated alkaline phosphatase secondary antibody (Pharmingen).

Cell Culture and RNA Extraction
Human microvascular endothelial cells were obtained from Clonetics Corp and grown in microvascular endothelial cell growth medium (Clonetics, No. CC-3125). Cells were passaged every 4 to 5 days. Cells from passages 6 to 7 were used for all experiments. After the cells had grown to 80% confluence, they were placed in quiescence medium (endothelial cell basal medium-2, serum free, Clonetics, No. CC-3156) overnight. Total RNA was prepared by guanidinium isothiocyanate extraction and centrifugation through cesium chloride.²¹ Total RNA from cells was fractionated on a 1.3% formaldehyde-agarose gel and transferred to nitrocellulose paper. The filters were hybridized with a randomly primed ³²P-labeled human Thy-1 full-length cDNA. The hybridized filters were then washed in 30 mmol/L sodium chloride, 3 mmol/L sodium citrate, and 0.1% SDS solution at 55°C and autoradiographed on Kodak XAR film at –80°C. The blots were hybridized with an 18S oligonucleotide probe to correct for differences in RNA loading. Filters were scanned on a PhosphorImager running ImageQuant software (Molecular Dynamics).

	Results

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Detection of Thy-1 Immunoreactivity During Angiogenesis in Adult Rats
We evaluated expression of Thy-1 in four adult rat models of angiogenesis by immunostaining with an antibody to rat Thy-1 (clone OX-7). In the first model, balloon injury to the carotid artery,^{22 23} new blood vessels form in the tunica adventitia. As Figure 1, top, shows, no Thy-1 immunoreactivity was visible in the endothelium of an uninjured carotid artery or in the few small blood vessels of its tunica adventitia. After injury, however, robust staining was visible in many of the small blood vessels of the tunica adventitia (Figure 1, bottom).

View larger version (88K): [in this window] [in a new window]   Figure 1. Immunohistochemical analysis of Thy-1 expression 14 days after balloon injury to the rat carotid artery. Uninjured (top) and injured (bottom) contralateral carotid arteries were stained with anti–Thy-1 antibody OX-7. Final magnification x155.

We then performed two additional experiments to be certain that this immunoreactivity was specific to Thy-1. First, we immunostained carotid artery tissue with another anti–Thy-1 antibody, clone HIS51. The results obtained with HIS51 were identical to those obtained with OX-7 (data not shown). Second, we constructed the chimeric fusion protein Thy-1–Ig to obtain a water-soluble form of Thy-1 (which is a glycosyl phosphatidylinositol–anchored protein). The N-terminus of Thy-1–Ig contained the extracellular domain of rat Thy-1 (residues Gln1-Gly113), and its C-terminus contained the constant region of human IgG1 (hinge, CH2, and CH3). To prevent nonspecific disulfide linkage with the Cys111 in the rat Thy-1 protein, we mutated the three cysteine residues in the IgG hinge region to serines.

The chimeric protein was purified by protein-G affinity chromatography and characterized by SDS–polyacrylamide gel electrophoresis. The Coomassie blue–stained gel (Figure 2A, left) revealed one band at 104 kD under nonreducing conditions and one band at 52 kD under reducing conditions. Epitopes for rat Thy-1, as determined by Western blot analysis with anti–Thy-1 antibody OX-7 (Figure 2A, right), were present in both the 104-kD and the 52-kD bands. These experiments indicate that Thy-1–Ig is a disulfide-linked homodimer. The molecular mass of a monomer would approximate the sum of rat Thy-1 at 25 kD²⁴ and the human immunoglobulin Fc fragment at 25 kD.

View larger version (60K): [in this window] [in a new window]   Figure 2. A, Expression of rat Thy-1–Ig fusion protein. The extracellular fragment of rat Thy-1 was fused to the human IgG1 constant region and expressed transiently in COS cells. SDS-PAGE (left) under nonreducing and reducing conditions revealed protein bands at the expected sizes of 104 and 52 kD, respectively. Western blot analysis (right) confirmed the presence of the rat Thy-1 epitope. Species at 104 and 52 kD were recognized by the anti–Thy-1 antibody OX-7. B, Elimination of Thy-1 immunoreactivity by preabsorption with rat Thy-1–Ig fusion protein. Immunostaining for Thy-1 is visible in a small adventitial blood vessel after balloon injury to the carotid artery (top) but is blocked in an adjacent section by preabsorption with the Thy-1–Ig fusion construct (bottom). Final magnification x379.

We then used the Thy-1–Ig fusion protein to confirm the specificity of the Thy-1 immunostaining visible in Figure 1 (bottom). Preabsorption with the fusion protein eliminated all Thy-1 immunoreactivity (Figure 2B). Together, these data suggest strongly that Thy-1 is the antigen detected in small blood vessels formed after balloon injury to the carotid artery.

In the second rat model of angiogenesis, renal arteries are clipped for 5 days, and ureters are harvested for sectioning and immunohistochemistry.^{15 25} New blood vessels form in the periureteral area.^{15 25} We detected strong Thy-1 immunostaining in small blood vessels beneath the ureteral epithelium in a sample from a rat with a clipped renal artery (Figure 3, top) but not in a sample from a control rat (not shown). Immunostaining for von Willebrand factor in an adjacent section from the experimental rat (Figure 3, bottom) was also strong in some small blood vessels. Note that the small vessels expressing Thy-1 were distinct from those expressing von Willebrand factor. We also observed this differential pattern of Thy-1 expression in the other models studied (not shown).

View larger version (122K): [in this window] [in a new window]   Figure 3. Immunohistochemical analysis of Thy-1 expression after renal artery ligation. Anti–Thy-1 antibody OX-7 staining is visible in the small blood vessels beneath the ureteral epithelium (top). Anti–von Willebrand factor antibody staining is also visible in an adjacent section (bottom). Final magnification x246.

The third model, implantation of a tumor in the rat cornea, is another well-established model of pathological angiogenesis. After a glioblastoma is placed in the anterior chamber of the normally avascular cornea,¹⁶ new blood vessels form. Corneas were harvested for immunostaining 10 days after implantation. Although Thy-1 staining was absent in the control sample (Figure 4, top), strong Thy-1 immunoreactivity was present in the small blood vessels of the invading tumor in the experimental sample (Figure 4, bottom).

View larger version (109K): [in this window] [in a new window]   Figure 4. Immunohistochemical analysis of Thy-1 expression after glioblastoma placement in the rat cornea. Anti–Thy-1 antibody OX-7 staining is not visible in blood vessels in control (uninjured) tissue (top) but is visible in small blood vessels formed after tumor placement (bottom). Final magnification x246.

The fourth model of adult angiogenesis we studied was the pregnant rat uterus. Formation of new blood vessels in the uterus is critical during pregnancy.²⁶ At days 8 and 11 of pregnancy, robust immunoreactivity for Thy-1 was present in small blood vessels of the uterus (not shown).

Absence of Thy-1 Immunoreactivity During Angiogenesis in Rat Embryos
While conducting the studies described above, we found that Thy-1 was not expressed in the endothelium of small or large blood vessels in control samples. This observation suggested that Thy-1 was expressed selectively on the endothelium of blood vessels only during pathological settings in the adult or during specific physiological settings (such as pregnancy). Therefore, we also studied Thy-1 expression during physiological vasculogenesis and angiogenesis in developing rat embryos. Tissue sections were immunostained for von Willebrand factor as a positive control. At embryonic day 11, von Willebrand factor staining was readily detectable in a large vessel such as the aorta (Figure 5, top left) and in small vessels of the yolk sac (Figure 5, bottom left). In contrast, no Thy-1 immunostaining was detectable in the aorta (Figure 5, top right) or the small vessels of the yolk sac (Figure 5, bottom right). We were also unable to detect Thy-1 immunoreactivity in the endothelium of large or small blood vessels at embryonic days 10 and 12 to 18 (data not shown). We did, however, detect immunoreactivity for Thy-1 in the developing brain, as described elsewhere,^{14 27} and, as noted here, in the small blood vessels of the pregnant uterus. Thus, Thy-1 does not appear to be expressed in the vasculature of the developing rat embryo.

View larger version (91K): [in this window] [in a new window]   Figure 5. Immunohistochemical analysis of Thy-1 and von Willebrand factor expression during rat embryogenesis. Adjacent rat sections at embryonic day 11 were stained for von Willebrand factor (left) and Thy-1 (right). Robust anti–von Willebrand factor antibody staining is present in the dorsal aorta (top) and blood islands of the yolk sac (bottom). Anti–Thy-1 antibody OX-7 staining is absent in both locations. Final magnification x172.

Upregulation of Thy-1 mRNA by Cytokines but Not Growth Factors
The data presented so far suggest that Thy-1 is upregulated in settings of pathological angiogenesis or pregnancy but not during embryonic vasculogenesis or angiogenesis. Because a number of inflammatory cytokines and growth factors¹ play important roles in the angiogenic response, we studied the effect of such factors on Thy-1 expression in cultured microvascular endothelial cells. Thy-1 mRNA was expressed at barely detectable levels in control microvascular endothelial cells (Figure 6A). Treatment with the cytokine interleukin-1ß, however, increased Thy-1 mRNA expression by 8-fold at 24 hours after stimulation. Treatment with another cytokine, tumor necrosis factor-, resulted in 5- and 14-fold increases, respectively, in Thy-1 mRNA at 8 and 24 hours after stimulation. In contrast, treatment with vascular endothelial growth factor or basic fibroblast growth factor did not induce Thy-1 expression in these cells (Figure 6A). We also found that neither platelet-derived growth factor nor transforming growth factor-ß induced Thy-1 mRNA expression in microvascular endothelial cells (not shown).

View larger version (72K): [in this window] [in a new window]   Figure 6. A, Expression of Thy-1 mRNA in cultured human microvascular endothelial cells. Cells were made quiescent for 24 hours before stimulation with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) and the cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor- (TNF-). RNA was harvested 48 hours after treatment. Northern analysis was performed with 7.5 µg of total RNA per lane. After electrophoresis, RNA was transferred to nitrocellulose filters and hybridized with a 32P-labeled human Thy-1 cDNA. A single Thy-1 transcript is visible at 1.8 kb. Filters were hybridized with 18S to verify equivalent loading. B, Immunohistochemical analysis for Thy-1 (left), macrophage (middle), and proliferating cell nuclear antigen (PCNA) (right) expression after balloon injury to the rat carotid artery. Arrows in middle panel mark macrophages. Original magnification x1000.

Macrophages are an important source of inflammatory cytokines, and the presence of macrophages is associated with angiogenesis.³ To show that Thy-1 expression during angiogenesis occurred within the context of inflammation, we stained rat carotid arteries subjected to balloon injury with the rat macrophage–specific antibody ED-1. We found robust immunostaining for macrophages adjacent to newly formed blood vessels in the tunica adventitia (Figure 6B, middle, arrows). The presence of PCNA (Figure 6B, right) confirmed that cellular proliferation was occurring. These small blood vessels in the tunica adventitia also expressed Thy-1 after injury to the vessel wall (Figures 1 and 6B, left panel).

	Discussion

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The paracrine effects of the angiopoietins and platelet-derived growth factor, vascular endothelial growth factor, transforming growth factor-ß, and others appear to be critical in the steps leading to development of the vascular system.²⁸ Although these growth factor participants in embryonic angiogenesis also participate in adult angiogenesis (vascular endothelial growth factor, for example, affects tumor angiogenesis and tumor growth^{29 30} ), other factors are specific to the process in adults. Factors affecting progression of the cell cycle, remodeling of cell adhesion molecules, induction of proteolytic activity, and neutralization of inhibitors² have all been found to influence adult angiogenesis.

An inflammatory response is also thought to be a feature of the process in adults, possibly a prerequisite for it.² The modulation of cytokine production is a common homeostatic response to neoplasia, hypoxia, and infection,³¹ and the elaboration of cytokines has been implicated in the blood vessel's response to injury and the pathogenesis of atherosclerosis.³²

The models tested in the present study represent a spectrum of conditions that are accompanied by new blood vessel formation. Stimuli such as mechanical injury (carotid injury model), tumorigenesis (eye model), and ischemia (renal artery ligation model) are accompanied by the release of inflammatory cytokines such as interleukin-1ß and tumor necrosis factor-.^{31 33 34} Moreover, both interleukin-1ß and tumor necrosis factor- mRNA and protein have been reported in the pregnant uterus.^{35 36} In each model we tested, we found that Thy-1 was highly expressed in small blood vessels formed after the pathological stimuli or after pregnancy. This expression of Thy-1 during angiogenesis does not appear to be limited to the rat, as similar expression has also been observed in a mouse model of angiogenesis (M.K. Jain and E. Haber, unpublished data, 1997).

Inflammatory cytokines were probably responsible for this upregulation of Thy-1, since, in vitro, we found that tumor necrosis factor- and interleukin-1ß but not basic fibroblast growth factor, vascular endothelial growth factor, platelet-derived growth factor, or transforming growth factor-ß induced Thy-1 mRNA in microvascular endothelial cells, an observation in agreement with a recent demonstration that tumor necrosis factor- affects Thy-1 expression.³⁷ One potential source for these inflammatory cytokines is the macrophage (Figure 6B). The fact that tumor necrosis factor- has a stimulatory effect on Thy-1 is especially interesting because this cytokine is also known to promote endothelial cell chemotaxis and to induce capillary vessel formation in vivo.^{3 31} Inhibition of tumor necrosis factor- protects tissue from ischemic injury.³⁸ The absence of Thy-1 mRNA upregulation in microvascular endothelial cells after growth factor stimulation is consistent with the absence of Thy-1 expression during embryonic vasculogenesis and angiogenesis, two processes in which the role of growth factors is essential.²⁸ Also, tumor necrosis factor- does not appear to be expressed in the vasculature during embryogenesis.^{39 40}

To our knowledge, the present study is first to establish that Thy-1 is expressed on blood vessel endothelium in settings of pathological angiogenesis in adult animals and in a specific setting of physiological angiogenesis but not during embryonic angiogenesis. Our finding that Thy-1 is upregulated selectively in cultured microvascular endothelial cells in response to inflammatory cytokines but not growth factors provides insight into the pathogenesis of adult angiogenesis, and it suggests that Thy-1 may serve as a target for therapeutic intervention. An identification of mechanisms controlling the upregulation of Thy-1 by cytokines and the effect of Thy-1 on endothelial cell function may add greatly to our understanding of the contribution of inflammation in pathological angiogenesis.

	Acknowledgments

 
This study was supported in part by National Institutes of Health grant RO1 GM-53249 (to Dr M.-E. Lee). Dr Shaw was supported by National Science Council of the Republic of China grant NSC86-2113-M006-013. Dr Hollenberg was supported by National Institutes of Health grant 1P50 HL-55000-02. We thank Bonna Ith for tissue culture assistance and Thomas McVarish for editorial assistance.

	Footnotes

 
¹ Both authors contributed equally to this study.

² Deceased.

Received July 17, 1997; accepted February 4, 1998.

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