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(Circulation Research. 1995;77:1129-1135.)
© 1995 American Heart Association, Inc.

Articles

_vß₃ Integrin Expression in Normal and Atherosclerotic Artery

Masaaki Hoshiga, Charles E. Alpers, Laura L. Smith, Cecilia M. Giachelli, Stephen M. Schwartz

From the Department of Pathology, University of Washington, Seattle.

Correspondence to Masaaki Hoshiga, MD, PhD, University of Washington, Vascular Biology, Box 357335, Seattle, WA 98195.E-mail hoshiga@u.washington.edu.

	Abstract

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Abstract Recent evidence suggests that _vß₃ integrin is a critical molecule in several processes involved in atherosclerosis progression and in restenosis, eg, smooth muscle cell (SMC) migration and angiogenesis. While several ligands for this integrin are known to be present in atherosclerotic plaque, little is known about the presence of _vß₃ integrin at this site. In the present study, we have examined _vß₃ expression in normal and atherosclerotic arteries. Thirty-six coronary artery segments from the recipient hearts of 24 patients undergoing heart transplantation were classified into two groups: nonatherosclerotic diffuse intimal thickening (DIT) and atherosclerotic plaques. Serial frozen sections were examined immunohistochemically with four different monoclonal antibodies directed against human _vß₃ complex or the ß₃ subunit and with cell markers for SMCs, macrophages, and endothelial cells. The endothelium along the lumen of both DIT and plaque arteries showed high expression of _vß₃. The media of both DIT and plaque arteries showed less intense but extensive expression of _vß₃. Immunoprecipitation and reverse-transcribed polymerase chain reaction (RT-PCR) analyses performed on extracts from the aortic media confirmed the presence of _vß₃ in the media. In the intima of both DIT and plaque arteries, _vß₃ expression generally colocalized with SMCs but rarely with macrophages. The microvessels in the adventitia as well as in the plaque showed prominent expression of _vß₃, in contrast to low expression in similar-sized microvessels of the skin. These results suggest that _vß₃ is present both in the normal artery and in sites of SMC accumulation and angiogenesis in atherosclerotic plaques.

Key Words: immunohistochemistry • _vß₃ integrin • atherosclerosis • angiogenesis • human

	Introduction

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Surprisingly little is known about the presence of integrins in atherosclerotic plaques. However, there is much current speculation about the role of integrins in atherosclerosis based on cell-culture studies. For example, a number of groups have implicated ₂ß₁ as a critical molecule in SMC migration^{1 2} and in contraction of collagen gels.^{3 4 5} Other groups, in studies of fibrin clots with transfected cells, have implicated ß₃ integrins in contraction and possible remodeling of fibrin clots.^{6 7} Finally, a recent clinical trial (the EPIC trial) of a ß₃ neutralizing antibody suggests that vessel-wall ß₃ might play a critical role in human restenosis lesions.⁸

All of this initial data, however, has to be viewed with caution, as little is known about expression of integrins in atherosclerotic plaques. For example, despite the potentially significant in vitro functions of ₂ß₁ in modulating SMC behavior, Glukhova et al⁹ were unable to detect this integrin complex in normal or atherosclerotic arteries. We decided to focus attention on atherosclerotic plaque expression of ß₃ integrins in view of the possibility, suggested by the EPIC trial,⁸ that antagonism of _IIbß₃ and _vß₃ ligand-receptor interactions interrupt pathophysiological processes leading to restenosis after angioplasty-associated vessel injury. We were specifically interested in the possibility that additional integrin complexes containing ß₃, beyond the _IIbß₃ integrins on platelets, were involved in the long-term beneficial effect after treatment of restenosis with 7E3, an antibody directed against ß₃ integrin. Toward this end, we present immunohistochemical localization data and evidence of mRNA expression to demonstrate patterns of the _vß₃ integrin complex expression in normal and atherosclerotic arteries.

	Materials and Methods

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Antibodies
Anti-VnR1 has been previously characterized to be specific for human integrin _vß₃¹⁰ and was kindly provided by Dr M. H. Ginsberg (Research Institute of Scripps, La Jolla, Calif). A second antibody directed against human _vß₃, LM609,¹¹ was purchased from Chemicon Inc. Two monoclonal antibodies directed against human integrin ß₃ subunit, Y2/51¹² and SZ 21,¹³ were purchased from Dako Corp and Immunotech Inc, respectively.

Tissues
Thirty-six coronary artery segments were obtained from hearts removed from 24 patients (18 men and 6 women, 31 to 63 years old) undergoing heart transplantation at the University of Washington. These segments were classified into two groups by classic histological criteria: one demonstrating DIT, characteristic of aging human arteries without clinical or morphological evidence of atherosclerosis (n=16), and the other demonstrating advanced and complicated atherosclerotic plaques (n=20). Normal human kidney was obtained fresh from uninvolved portions of kidneys surgically resected for localized renal cell carcinoma. Human brachial artery was obtained from an arm surgically amputated due to trauma. Normal skin was obtained from surgically resected abdomen. Tissues were snap frozen in OCT compound (Miles) and stored at -70°C. For IP and RT-PCR analyses, adult thoracic aorta was obtained from heart-transplant-donor specimens. The whole vessel and the dissected media were stored at -70°C. These human tissue studies received appropriate University of Washington Human Subjects Review approval.

Immunohistochemistry
Immunoperoxidase staining of tissues was performed on cryostat sections of human coronary arteries. Serial sections (5 µm thick) were placed onto poly-L-lysine–coated slides and fixed in 10% neutral-buffered formalin for 10 minutes at room temperature. Before staining, cryosections were blocked with 10% normal goat serum in PBS containing 1% BSA. Slides were incubated with primary antibody for 30 minutes at room temperature: the titer used was 1:500 for LM609 and 1:200 for anti-VnR1, Y2/51, and SZ 21. A biotinylated horse anti-mouse secondary antibody was then applied for 30 minutes, followed by an avidin-biotin-peroxidase conjugate (ABC Elite, Vector Labs) for 30 minutes at room temperature. Then 3,3'-diaminobenzidine with nickel chloride was added to yield a black reaction product, and methyl green was used as nuclear counterstain. Human normal kidney was used as a positive control tissue for _vß₃ immunohistochemistry. In addition, the following immunohistochemical controls were performed on each tissue: (1) deletion of the primary antibody, (2) replacement of the primary antibody with mouse IgG1 antibody (clone DAK-GO 1, Dako Corp) against Aspergillus niger glucose oxidase, an enzyme that is neither present nor inducible in mammalian tissues. The following antibodies were used as cell markers on tissue sections obtained from a subset of cases of DIT (n=4) and atherosclerotic plaque (n=7): anti–smooth muscle -actin (1:250 dilution; Boehringer Mannheim Corp) to identify SMCs, anti–CD-68 (1:8000; Dako Corp) to identify macrophages, and ulex europaeus agglutinin I (1:1000; Vector Labs) to identify endothelial cells. The use of these antibodies and lectins with these techniques and references to their characterization has been described previously.¹⁴

IP and Western Blotting
Cell lysates were made from frozen human kidney, intact segments of aorta, and dissected arterial media tissue samples. Tissues were homogenized in extraction buffer (PBS with 1% Triton X-100 and protease inhibitors) and allowed to incubate on ice for 20 minutes. The cell lysate was centrifuged twice at 16 000g for 10 minutes. The equivalent of 50 µg of tissue from the cell lysate was then diluted to 0.5 mL with extraction buffer, and BSA was added to 0.5 mg/mL. Lysates were precleared with 40 µL of 50% (vol/vol) protein A–sepharose CL-4B (Pharmacia) at 4°C for 30 minutes. The supernatants were immunoprecipitated with either LM609, Y2/51, or, as a negative control, mouse IgG at 4°C for 16 hours. Immune complexes were recovered by binding to protein A–Sepharose and washing five times with IP wash buffer (50 mmol/L Tris, pH 7.4, 0.5 mol/L NaCl, 2 mmol/L PMSF, 0.1% Triton X-100, and 0.1% Tween 20). After samples were separated by electrophoresis on 8% polyacrylamide-SDS gels under nonreducing condition, the proteins were transferred to polyvinylidene difluoride membrane (DuPont NEN). The membrane was blocked with 10% nonfat dry milk in buffer (10 mmol/L Tris base, pH 8, 150 mmol/L NaCl, and 0.05% Tween 20) at room temperature for 1 hour. After washing, blots were incubated for an additional hour with 1:120 Y2/51 followed by 1:5000 horseradish peroxidase–conjugated goat anti-mouse antibody (Jackson Immunolabs), and proteins were visualized by the addition of a chemiluminescence reagent to the membrane according to the manufacturer's instructions (DuPont NEN).

RT-PCR
Two micrograms of total RNA from the aortic media was treated with DNase (Pharmacia), and 1 µg was reverse transcribed. Primers for the ß₃ integrin subunit were used to amplify 4 µg of the resulting cDNA by PCR. The PCR primers were synthesized to match the sequences of human ß₃ integrin¹⁵ : ß₃ forward 5'-GTGCTGACGCTAACTGACC-3' and ß₃ reverse 5'-CATGGTAGTGGAGGCAGAGT-3'. PCR reactions were denatured at 94°C for 5 minutes and then amplified for 40 cycles at 94°C for 1 minute, 54°C for 1 minute, and 72°C for 1 minute. This procedure was followed by an extension period at 72°C for 10 minutes. The resulting 284-bp fragment was then resolved in a 2% agarose gel after staining with ethidium bromide. Southern blot analysis with an oligonucleotide that recognizes an internal sequence of the ß₃ subunit (5'-ATCACAGACTGTAGTAGCCTGCATG-3') verified the amplified product as a ß₃ integrin.

	Results

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Immunohistochemistry of Integrin _vß₃ in Human Coronary Arteries
Immunohistochemistry was performed with two different monoclonal antibodies directed against the human _vß₃ integrin complex and with two different monoclonal antibodies directed against the human ß₃ integrin subunit on frozen sections of 36 different human coronary arteries. The endothelium along the lumen of both atherosclerotic and nonatherosclerotic coronary arteries showed strong expression of _vß₃ (Fig 1), and the expression of _vß₃ by luminal endothelium was similar in all specimens. The media of both atherosclerotic and nonatherosclerotic arteries was similar in showing low but diffuse and extensive expression of _vß₃ (Fig 1b and 1c). In coronary arteries with DIT but without atherosclerosis, _vß₃ expression colocalized with SMC marker (-actin) in the intima as well as the media (Fig 2), demonstrating that SMCs that accumulate in the intima express integrin _vß₃.

View larger version (107K): [in this window] [in a new window]   Figure 1. Photomicrographs showing vß3 expression in the human coronary artery with DIT. a, Hematoxylin-eosin staining shows a mildly thickened intima that is thinner than the media (m). Serial frozen sections were immunostained with monoclonal antibodies against human vß3 (LM609) (b) and ß3 (Y2/51) (c) and with an irrelevant antibody against Aspergillus niger glucose oxidase (d). The endothelium highly expressed vß3 or ß3 and the media showed relatively low but extensive expression. Arrowheads indicate internal elastic lamina, which is the border between the intima and the media. Original magnification x400.

View larger version (43K): [in this window] [in a new window]   Figure 2. Photomicrographs showing vascular SMCs in the coronary artery that express vß3. Serial frozen sections were immunostained with monoclonal antibodies against vß3 (LM609) (a), the SMC marker -actin (b), and macrophage marker CD-68 (c). Arrowheads point to the internal elastic lamina. In this coronary artery, in which the intima is thicker than the media (m), vß3 was colocalized with SMCs, identified by -actin expression in the intima as well as the media. Only a few macrophages are identified. Original magnification x400.

Expression of _vß₃ was variable within the atherosclerotic plaques. Most often, large parts of the plaques, generally those parts with the greatest accumulations of matrix and the least degree of cellularity, had undetectable expression of _vß₃. However, in some foci, generally the most cellular components of the plaque, _vß₃ expression was prominent, and cells expressing _vß₃ were identified as SMCs by their expression of -actin on serial sections. Not all -actin expressing SMCs within the plaques expressed _vß₃. Uncommonly, _vß₃ expression appeared to colocalize with CD-68–expressing macrophages, but overall we could not detect a pattern of colocalization of _vß₃ with these leukocytes. For example, clusters of CD-68–positive cells at the shoulders of the necrotic centers were _vß₃ negative. However, there was widespread expression of _vß₃, -actin, CD-68, and ulex lectin within the regions of neovascularization in the plaque so that no confident assignment of cells expressing _vß₃ could be made in these areas.

In the adventitia, the vasa vasorum showed intense staining (Fig 3). No staining of adventitial fibroblasts was observed. In atherosclerosis, the intraplaque microvessels as well as the vasa vasorum in the adventitia showed the most prominent and uniform expression of _vß₃ observed in this study (Fig 3). To determine whether this strong expression of _vß₃ in the intraplaque microvessels and vasa vasorum was localized to endothelial cells or SMCs, we stained serial sections for endothelial cell (ulex agglutinin) and SMC (-actin) markers. The comparison between _vß₃ immunohistochemistry and cell-marker staining in microvessels gave the following results: (1) vasa vasorum in the adventitia showed strong _vß₃ expression both on endothelial cells and SMCs (Fig 4); (2) about two thirds of intraplaque microvessels lacking an identifiable SMC layer showed _vß₃ expression of the endothelial lining, whereas the rest did not stain with _vß₃ (data not shown); and (3) intraplaque microvessels containing -actin–positive SMCs showed strong _vß₃ expression on SMCs as well as endothelial cells. No similar process of neovascularization is present in the intima of nonatherosclerotic arteries with DIT.

View larger version (114K): [in this window] [in a new window]   Figure 3. Photomicrograph of vasa vasorum and intraplaque microvessels that express vß3. This atherosclerotic human coronary artery was immunostained with anti-ß3 antibody (Y2/51). Arrowheads indicate the internal elastic lamina. Vasa vasorum in the adventitia (black arrow) and the intraplaque microvessels (open arrows) show strong and uniform expression of ß3. m indicates media. Original magnification x400.

View larger version (81K): [in this window] [in a new window]   Figure 4. Photomicrographs of endothelial cells and SMCs of the vasa vasorum containing vß3. Serial frozen sections were immunostained with anti-vß3 antibody (LM609) (a), endothelial cell marker (ulex agglutinin) (b), and SMC marker (–smooth muscle actin) (c). Arrowheads indicate the luminal endothelium of vasa vasorum in the adventitia. Original magnification x400.

We obtained similar immunohistochemical results with four different antibodies. The only difference between _vß₃ and ß₃ expression was that occasional mural thrombi in the vessel wall showed ß₃ expression but not _vß₃ (data not shown). This finding was expected, since the ß₃ antibody can recognize both _vß₃ and _IIbß₃ complex proteins but _vß₃ antibody (LM609) can recognize the _vß₃ integrin complex only.¹²

_vß₃ Expression in Other Vessels
We performed immunohistochemistry on normal skin and brachial artery to determine _vß₃ expression in vessels other than the coronary artery (Fig 5). The subepidermal microvessels of the skin (Fig 5a) expressed _vß_3, but this expression as detected immunohistochemically was less than that of the endothelium and media of the vasa vasorum in the coronary artery (Fig 4a). In contrast, the brachial artery (Fig 5b) showed high expression of _vß₃ in the endothelium but lower and diffuse expression in the media, similar to that observed in coronary arteries with DIT.

View larger version (87K): [in this window] [in a new window]   Figure 5. Photomicrographs showing vß3 expression in the microvessels in normal skin and the brachial artery. Frozen sections from human normal skin and brachial artery were immunostained with anti-vß3 (LM609). In the subepidermal microvessels (a), ß3 expression was low in the endothelium (arrowheads) as well as the media. Arrows show positive staining of ß3 in the neural sheath. The weak expression of ß3 detected immunohistochemically in this endothelium as well as in the media is in contrast to the strong expression detected in the vasa vasorum in coronary arteries (Fig 4a). The brachial artery (b) expressed vß3 highly in the endothelium and to a lesser extent in the media (m), similar to the pattern seen in coronary arteries. Arrowheads indicate the internal elastic lamina. Original magnification: a x400; b x200.

IP and RT-PCR
To confirm the presence of _vß₃ integrin in arterial media, we performed IP and RT-PCR analyses by using protein and RNA extracts, respectively, from the media of human aorta. As shown in Fig 6a, IP with the complex-specific anti-_vß₃ (LM609) followed by immunoblotting with anti-ß₃ antibody clearly demonstrated the presence of this integrin complex in the media of human aorta. RT-PCR analysis demonstrated ß₃ mRNA expression in the media of human aorta (Fig 6b).

View larger version (51K): [in this window] [in a new window]   Figure 6. IP and RT-PCR analyses showing vß3 in the media of aorta. a, Cell lysates of tissue were immunoprecipitated with saturating amounts of monoclonal antibodies to vß3 (LM609; lanes 1, 3, and 5) or to ß3 (Y2/51; lanes 2, 4, and 6). Samples were analyzed by SDS-PAGE under nonreducing conditions and immunoblotted with the antibody Y2/51. Lanes 1 and 2, kidney; 3 and 4, whole aorta; 5 and 6, media. Arrows with Ig and ß3 indicate molecular weight of immunoglobulin chain and ß3 integrin subunit (95 kD). Higher-molecular-weight bands may represent antibody complexes. b, Agarose gel of ß3 integrin subunit cDNA from the media of human aortic tissue RNA. ß3 cDNA was detectable by PCR amplification as a 284-bp band. Lane 1, negative control reactions in which cDNA templates were omitted. Lane 2, plasmid control ß3 integrin. Lane 3, media from human aorta.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Integrins can mediate cell attachment and cell movement¹⁶ and appear to play roles in cell differentiation,¹⁷ stimulation of protease expression,^{18 19} wound contraction,²⁰ apoptosis,²¹ and angiogenesis.²² It is therefore surprising that so little is known about the expression of this gene family in atherosclerotic plaques. In this study, we report the distribution of integrin _vß₃ in human coronary arteries. By use of four different antibodies against _vß₃ or ß₃, we could demonstrate the following results: (1) _vß₃ was highly expressed on endothelial cells of coronary artery; (2) the SMCs comprising the media of coronary arteries showed extensive staining with _vß₃; (3) SMCs accumulating in the intima expressed _vß₃; and (4) intraplaque microvessels as well as vasa vasorum showed high _vß₃ expression by both SMC and endothelial cell components.

Several lines of evidence suggest that _vß₃ integrin contributes to SMC accumulation in the intima. (1) Human aortic SMCs migrate toward osteopontin,²³ an _vß₃ ligand, which is enriched in human atherosclerotic plaques.^{24 25 26 27} In vitro studies indicate that this migration is dependent on expression of _vß₃ on the cell surface of the SMCs.^{28 29} (2) Platelet-derived growth factor and transforming growth factor–ß, both of which have been identified in human atherosclerotic plaques^{30 31} and in the neointima in experimental models of arterial injury,^{32 33} are potent inducers of SMC migration, and both induce ß₃ expression in rabbit and bovine SMCs.^{34 35} (3) Peptides that bind selectively to the _vß₃ receptor reduce neointima formation in rabbit³⁶ and hamster³⁷ balloon-injury models. The presence of _vß₃ in the plaques provides a necessary condition for this hypothesis. Moreover, since _vß₃ can bind multiple ligands,^{17 38} eg, fibrinogen, fibronectin, vitronectin, thrombospondin, and denatured collagen type I, as well as osteopontin, all known to be present in atherosclerotic plaques,³⁹ several ligands are available to interact with this receptor. The data, however, do not allow us to determine whether accumulating SMCs upregulate _vß₃ expression or whether the integrin is bound to any specific ligand of those cited.

Recent reports have emphasized the role of _vß₃ in angiogenesis.^{22 40} These reports show that _vß₃ is upregulated in angiogenic vessels found in the granulation tissue of wounds as well as those associated with neoplasms. SMCs and endothelial cells in intraplaque microvessels (Fig 3) expressed _vß₃ at much higher levels than are seen in microvessels in the skin (Fig 5a). Intraplaque microvessels are believed to be derived from the adventitial vasa vasorum⁴¹ by invasion, migration, and proliferation of endothelial cells. In a recent study we reported that many of these vessels have very high replication rates.⁴² This high level of cell replication implies either that endothelial cells in these sites have high rates of cell turnover or that angiogenesis may be present in advanced atherosclerotic lesions. Of course, turnover and angiogenesis could occur together. Our data show prominent _vß₃ expression in SMCs as well as endothelial cells of these vessels. As a candidate ligand for _vß₃ in the intraplaque microvessel, we have recently shown colocalization of osteopontin with intraplaque microvessels.²⁵ The association of _vß₃ with vasa vasorum in the adventitia as well as the intraplaque microvessels is consistent with the hypothesis that the vasa vasorum are actively angiogenic in the region of plaques.⁴¹ We recognize this is not a necessary or exclusive feature of angiogenesis, since our study also revealed _vß₃ expression by endothelial cells of larger muscular arteries, such as brachial arteries.

The ability of ß₃ antibodies to recognize vascular SMCs is at variance with a recent analysis by Skinner et al.² They reported that no significant ß₃ subunit could be detected in fresh SMCs isolated by collagenase and elastase digestion from adult thoracic aorta. In contrast to the present study, which used immunohistochemistry of frozen tissue, they determined ß₃ expression on the surface of freshly dissociated SMCs by flow cytometry. It is therefore possible that the differences observed stem from differences in tissue preparation. Validity of the present data was confirmed with four different antibodies and an IP analysis. Moreover, RT-PCR analysis detected ß₃ mRNA in the aortic media. Because the brachial artery is known as an atherosclerosis-resistant vessel,⁴³ our observations that the brachial artery media expressed _vß₃ suggest that SMCs express this integrin even in the absence of disease. A previous report of ß₃ expression in the pulmonary artery⁴⁴ showed quite similar patterns, with predominant staining in the endothelium and low and diffuse staining in the underlying media.

In summary, we have shown that _vß₃ is a prominent feature of the normal as well as the atherosclerotic vessel wall. The integrin is expressed by endothelial cells, medial SMCs, some intimal SMCs, and microvessels in the adventitia as well as in the plaque. A variety of clinical data and in vitro studies suggest that this integrin might play a role in neointimal events, such as SMC migration and angiogenesis, that could be important for atherosclerotic lesion development.

	Selected Abbreviations and Acronyms

 

anti-VnR1 = monoclonal antibody against human vitronectin receptor-1 DIT = diffuse intimal thickening IP = immunoprecipitation RT-PCR = reverse-transcribed polymerase chain reaction SMC = smooth muscle cell

	Acknowledgments

 
This work was supported by National Institutes of Health grants HL47151, HL18645, and DK47659. This work was also supported by a grant from Hoffmann–La Roche Company. L. Smith is supported by National Institutes of Health training grant 5T32 GM07270-20. We thank Marina Ferguson for her technical advice, Randy Small for cutting frozen sections, Dr Kevin O'Brien for collecting human coronary arteries, Dr Frank Isik and Dr Charles Murry for obtaining human skin and human aorta specimens, respectively, and Dr Mark Ginsberg for providing anti-VnR1.

Received May 18, 1995; accepted August 23, 1995.

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