Down But Not Out
http://www.100md.com
动脉硬化血栓血管生物学 2005年第7期
From the Division of Cardiology (G.A.S.) and Carolina Cardiovascular Biology Center (G.A.S., A.P., R.Z., J.H.), University of North Carolina, Chapel Hill, NC.
Correspondence to George A. Stouffer, MD, Division of Cardiology, University of North Carolina, Chapel Hill, NC 27599-7075. E-mail rstouff@med.unc.edu
Integrins are heterodimeric transmembrane glycoproteins involved in cell-cell and cell-extracellular matrix interactions. Two highly homologous ?3-containing integrins have been identified: v?3 (also known as the vitronectin receptor) and IIb?3 (also known as platelet glycoprotein [GP] IIb/IIIa). v?3 can be expressed by a variety of cell types including endothelial cells, smooth muscle cells (SMCs), leukocytes, platelets, osteoclasts, and mesangial cells, whereas IIb?3 is found only on platelets and megakaryocytes. v?3 consists of a 125-kDa subunit and a 105-kDa ? subunit and, in addition to vitronectin, binds other extracellular proteins including osteopontin, fibronectin, fibrinogen, thrombospondin, proteolysed collagen, DEL1 (developmentally regulated endothelial locus-1), plasminogen activator inhibitor (PAI)-1, prothrombin, DANCE (developmental arteries and neural crest epidermal growth factor [EGF]-like), and von Willebrand factor (vWF). Similar to IIb?3 integrins on platelets, v?3 integrins exist in active and inactive conformations, with variable affinity for ligands, depending on the cell type and modulation by signaling events.1
See page 1376
In the normal artery, vascular expression of v?3 integrins is generally limited to the luminal endothelial monolayer.2,3 After vascular injury, v?3 expression increases markedly, a finding that has been consistent across numerous animal models including baboons, rats, rabbits, mice, and pigs.4 v?3 expression is observed rapidly in the media after injury and is found in the neointima within 7 days, with peak levels at 14 to 28 days.2–7 v?3 integrin expression colocalizes with -actin in the injured arteries, suggesting that SMCs within the vessel wall express v?3 integrins after vascular injury.2,7 Recently, Sadeghi et al, using an 111In-labeled radiotracer, found that expression of activated v?3 increased after carotid injury in Apo E –/– mice; the increase was maximal 1 week after injury with levels remaining elevated at 3 and 4 weeks.8
In this issue of Arteriosclerosis, Thrombosis and Vascular Biology, Honda et al9 characterize the expression of v?3 after balloon injury of rat carotid arteries and perform a detailed kinetic study of the effects of a novel v?3 antagonist, BS-1417, on neointima formation and luminal narrowing. They evaluated effects of BS-1417, a phenyl-piperazine RGD peptidemimetic compound,10 on neointimal growth in both short-term (2 weeks after injury) experiments at lower doses (0.1, 0.3, and 1.0 mg/rat/d), and long-term (4, 7, and 12 weeks after injury) experiments using a higher dose (7.2 mg/rat/d). Neointima formation commenced within 5 days after balloon injury of rat carotid arteries and increased until 8 weeks after which it subsided until 12 weeks. BS-1417 elicited a dose dependent decrease in neointima formation. The highest dose used resulted in a 59% reduction in neointima formation and 88% reduction in luminal narrowing measured two weeks after injury. These results add to the already extensive data that v?3 antagonists reduce neointima formation following vascular injury, a finding that has been consistent across various species (eg, rat, rabbit, mouse, baboon, and hamster), different vascular injury models, and diverse v?3 antagonists (including antibodies, cyclic peptides, peptidomimetics, and small molecule inhibitors).
The present study provides some mechanistic insight into the effects of v?3 antagonism after vascular injury. SMC proliferation was detectable at 3 days after injury and continued for 4 weeks. BS-1417 infusion markedly reduced the presence of proliferating SMC (PCNA-positive) in the neointima of treated rats compared with untreated injured control rats. In contrast to an inhibitory effect on proliferation, v?3 antagonism had minimal effect on gene expression. Balloon injury resulted in increased gene expression of v integrins, ?3 integrins, and fibronectin and decreased expression of collagen type 1 and matrix metalloproteinase (MMP)-2 within 6 hours. Increases in MMP-9 and PDGF-? receptor were observed at delayed time points. BS-1417 had no discernible effect on gene expression except for prolonging the increase in MMP-9. The current study did not examine migration, but previous studies found that antagonists with v?3 activity reduced SMC migration after balloon injury of rat carotid arteries.11,12
An important feature of the study of Honda et al9 is the demonstration of a "catch-up" phenomenon after withdrawal of BS-1417 therapy. Treatment with BS-1417 for 2 weeks led to a reduction in neointima formation if the vessels were examined at 2 weeks. However, if the rats were treated for 2 weeks but not euthanized for 4 weeks, an interesting phenomenon was apparent. There was a significant increase in neointima resulting in increased intima to media ratio. This growth was not accompanied by decreased lumen area or increased luminal narrowing, findings suggestive of positive remodeling. Similar results were seen when rats were treated with BS-1417 for 4 weeks but not euthanized for 7 weeks. Prolonged (12 weeks) high-dose BS-1417 treatment resulted in significant reduction in "catch-up" growth in neointima.
Lastly, the present study also examined the effects of v?3 antagonism on endothelial cell function. This is an important question because endothelium regeneration of injured arteries is mediated in part by migration of endothelial cells, and v?3 has been implicated in endothelial cell migration and apoptosis.13 Honda et al9 found that there was a decrease in eNOS expression after balloon injury which recovered to normal levels at 8 weeks. Similarly, acetylecholine-induced release of cGMP was impaired for 5 weeks after injury but increased 4- to 6-fold from the 5th to 12th week. Endothelial cell regeneration by microscopy was apparent at 4 weeks, but the cells were morphologically abnormal. Normal morphology was apparent 12 weeks after injury. Long-term (4 and 7 weeks) high dose BS-1417 treatment increased cGMP production in injured rats in response to acetylcholine, but had no effect on endothelial cell morphology or eNOS gene expression. These data, taken together with a prior study showing that v?3 antagonism had no effect on reendothelialization after rabbit iliac angioplasty,14 are reassuring, although far from conclusive, that v?3 antagonism does not inhibit endothelial cell regeneration after vascular injury.
In summary, the present study demonstrates that BS-1417 inhibited neointimal formation and enhanced positive remodeling after balloon injury of rat carotid arteries. These results further confirm multiple prior studies showing that v?3 antagonists reduce vascular response to injury. The mechanisms by which vascular healing is modified appear to be pleotrophic as v?3 antagonists have been shown, in various models, to reduce SMC proliferation and migration, transforming growth factor-? production, MMP production, and macrophage recruitment and to increase apoptosis. v?3 antagonists have also been shown to have diverse effects on remodeling and, at least ex vivo, to inhibit adhesion of activated platelets, platelet microparticles, and leukocytes to endothelial cells. The consistency of the findings in animal models of the effects of v?3 antagonism on vascular healing suggest that v?3 integrins may represent an important future therapeutic target to limit neointimal formation after revascularization.
References
Takagi J, Petre BM, Walz T, Springer TA. Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell. 2002; 110: 599–511.
Stouffer GA, Hu Z, Sajid M, Li H, Jin G, Nakada MT, Hanson SR, Runge MS. ?3 integrins are upregulated following vascular injury and mediate proliferation of cultured smooth muscle cells. Circulation. 1998; 97: 907–915.
Corjay MH, Diamond SM, Schlingmann KL, Gibbs SK, Stoltenborg JK, Racanelli AL. v?3, v?5, and osteopontin are coordinately upregulated at early time points in a rabbit model of neointima formation. J Cell Bioch. 1999; 75: 492–504.
Sajid M, Stouffer GA. The role of v?3 integrins in vascular healing. Thromb Haemost. 2002; 87: 187–193.
Chico TJA, Chamberlain J, Gunn J, Arnold N, Bullens SL, Gadek TR, Francis SE, Bunting S, Horton M, Shepherd L, Lipari MT, Quan C, Knolle J, Stilz HU, Peyman A, Crossman DC. Effect of selective or combined inhibition of integrins IIb?3 and v?3 on thrombosis and neointima after oversized porcine coronary angioplasty. Circulation. 2001; 103: 1135–1141.
Bishop GG, McPherson JA, Sanders JM, Hesselbacher SE, Feldman MJ, McNamara CA, Gimple LW, Powers ER, Mousa SA, Sarembock IJ. Selective v?3-receptor blockade reduces macrophage infiltration and restenosis after balloon angioplasty in the atherosclerotic rabbit. Circulation. 2001; 103: 1906–1911.
Srivatsa SS, Fitzpatrick LA, Tsao PW, Reilly TM, Holmes DR Jr, Schwartz RS, Mousa SA. Selective v?3 integrin blockade potently limits neointimal hyperplasia and lumen stenosis following deep coronary arterial stent injury: evidence for the functional importance of integrin v?3 and osteopontin expression during neointima formation. Cardiovasc Res. 1997; 36: 408–428.
Sadeghi MM, Krassilnikova S, Zhang J, Gharaei AA, Fassaei HR, Esmailzadeh L, Kooshkabadi A, Edwards S, Yalamanchili P, Harris TD, Sinusas AJ, Zaret BL, Bender JR. Detection of injury-induced vascular remodeling by targeting activated v?3 integrin in vivo. Circulation. 2004; 110: 84–90.
Honda Y, Kitano T, Fukuya F, Sato Y, Iwama S, Morie T, Notake M. A novel v?3 integrin antagonist suppresses neointima formation for more than 4 weeks after balloon injury in rats. Arterioscler Thromb Vasc Biol. 2005; 25: 1376–1382.
Iwama S, Kitano T, Fukuya F, Honda Y, Sato Y, Notake M, Morie T. Discovery of a potent and selective v?3 integrin antagonist with strong inhibitory activity against neointima formation in rat balloon injury model. Bioorg Med Chem Lett. 2004; 14: 2567–2570.
Bendeck MP, Nakada MT. The ?3 integrin antagonist m7E3 reduces matrix metalloproteinase activity and smooth muscle cell migration. J Vasc Res. 2001; 38: 590–599.
Slepian MJ, Massia SP, Dehdashti B, Fritz A, Whitesell L. ?3-integrins rather than beta1-integrins dominate integrin-matrix interactions involved in postinjury smooth muscle cell migration. Circulation. 1998; 97: 1818–1827.
Stupack DG, Cheresh DA. Integrins and angiogenesis. Curr Top Dev Biol. 2004; 64: 207–238.
van der Zee R, Murohara T, Passeri J, Kearney M, Cheresh DA, Isner JM. Reduced intimal thickening following v?3 blockade is associated with smooth muscle cell apoptosis. Cell Adhes Commun. 1998; 6: 371–379.(G.A. Stouffer; A. Pathak;)
Correspondence to George A. Stouffer, MD, Division of Cardiology, University of North Carolina, Chapel Hill, NC 27599-7075. E-mail rstouff@med.unc.edu
Integrins are heterodimeric transmembrane glycoproteins involved in cell-cell and cell-extracellular matrix interactions. Two highly homologous ?3-containing integrins have been identified: v?3 (also known as the vitronectin receptor) and IIb?3 (also known as platelet glycoprotein [GP] IIb/IIIa). v?3 can be expressed by a variety of cell types including endothelial cells, smooth muscle cells (SMCs), leukocytes, platelets, osteoclasts, and mesangial cells, whereas IIb?3 is found only on platelets and megakaryocytes. v?3 consists of a 125-kDa subunit and a 105-kDa ? subunit and, in addition to vitronectin, binds other extracellular proteins including osteopontin, fibronectin, fibrinogen, thrombospondin, proteolysed collagen, DEL1 (developmentally regulated endothelial locus-1), plasminogen activator inhibitor (PAI)-1, prothrombin, DANCE (developmental arteries and neural crest epidermal growth factor [EGF]-like), and von Willebrand factor (vWF). Similar to IIb?3 integrins on platelets, v?3 integrins exist in active and inactive conformations, with variable affinity for ligands, depending on the cell type and modulation by signaling events.1
See page 1376
In the normal artery, vascular expression of v?3 integrins is generally limited to the luminal endothelial monolayer.2,3 After vascular injury, v?3 expression increases markedly, a finding that has been consistent across numerous animal models including baboons, rats, rabbits, mice, and pigs.4 v?3 expression is observed rapidly in the media after injury and is found in the neointima within 7 days, with peak levels at 14 to 28 days.2–7 v?3 integrin expression colocalizes with -actin in the injured arteries, suggesting that SMCs within the vessel wall express v?3 integrins after vascular injury.2,7 Recently, Sadeghi et al, using an 111In-labeled radiotracer, found that expression of activated v?3 increased after carotid injury in Apo E –/– mice; the increase was maximal 1 week after injury with levels remaining elevated at 3 and 4 weeks.8
In this issue of Arteriosclerosis, Thrombosis and Vascular Biology, Honda et al9 characterize the expression of v?3 after balloon injury of rat carotid arteries and perform a detailed kinetic study of the effects of a novel v?3 antagonist, BS-1417, on neointima formation and luminal narrowing. They evaluated effects of BS-1417, a phenyl-piperazine RGD peptidemimetic compound,10 on neointimal growth in both short-term (2 weeks after injury) experiments at lower doses (0.1, 0.3, and 1.0 mg/rat/d), and long-term (4, 7, and 12 weeks after injury) experiments using a higher dose (7.2 mg/rat/d). Neointima formation commenced within 5 days after balloon injury of rat carotid arteries and increased until 8 weeks after which it subsided until 12 weeks. BS-1417 elicited a dose dependent decrease in neointima formation. The highest dose used resulted in a 59% reduction in neointima formation and 88% reduction in luminal narrowing measured two weeks after injury. These results add to the already extensive data that v?3 antagonists reduce neointima formation following vascular injury, a finding that has been consistent across various species (eg, rat, rabbit, mouse, baboon, and hamster), different vascular injury models, and diverse v?3 antagonists (including antibodies, cyclic peptides, peptidomimetics, and small molecule inhibitors).
The present study provides some mechanistic insight into the effects of v?3 antagonism after vascular injury. SMC proliferation was detectable at 3 days after injury and continued for 4 weeks. BS-1417 infusion markedly reduced the presence of proliferating SMC (PCNA-positive) in the neointima of treated rats compared with untreated injured control rats. In contrast to an inhibitory effect on proliferation, v?3 antagonism had minimal effect on gene expression. Balloon injury resulted in increased gene expression of v integrins, ?3 integrins, and fibronectin and decreased expression of collagen type 1 and matrix metalloproteinase (MMP)-2 within 6 hours. Increases in MMP-9 and PDGF-? receptor were observed at delayed time points. BS-1417 had no discernible effect on gene expression except for prolonging the increase in MMP-9. The current study did not examine migration, but previous studies found that antagonists with v?3 activity reduced SMC migration after balloon injury of rat carotid arteries.11,12
An important feature of the study of Honda et al9 is the demonstration of a "catch-up" phenomenon after withdrawal of BS-1417 therapy. Treatment with BS-1417 for 2 weeks led to a reduction in neointima formation if the vessels were examined at 2 weeks. However, if the rats were treated for 2 weeks but not euthanized for 4 weeks, an interesting phenomenon was apparent. There was a significant increase in neointima resulting in increased intima to media ratio. This growth was not accompanied by decreased lumen area or increased luminal narrowing, findings suggestive of positive remodeling. Similar results were seen when rats were treated with BS-1417 for 4 weeks but not euthanized for 7 weeks. Prolonged (12 weeks) high-dose BS-1417 treatment resulted in significant reduction in "catch-up" growth in neointima.
Lastly, the present study also examined the effects of v?3 antagonism on endothelial cell function. This is an important question because endothelium regeneration of injured arteries is mediated in part by migration of endothelial cells, and v?3 has been implicated in endothelial cell migration and apoptosis.13 Honda et al9 found that there was a decrease in eNOS expression after balloon injury which recovered to normal levels at 8 weeks. Similarly, acetylecholine-induced release of cGMP was impaired for 5 weeks after injury but increased 4- to 6-fold from the 5th to 12th week. Endothelial cell regeneration by microscopy was apparent at 4 weeks, but the cells were morphologically abnormal. Normal morphology was apparent 12 weeks after injury. Long-term (4 and 7 weeks) high dose BS-1417 treatment increased cGMP production in injured rats in response to acetylcholine, but had no effect on endothelial cell morphology or eNOS gene expression. These data, taken together with a prior study showing that v?3 antagonism had no effect on reendothelialization after rabbit iliac angioplasty,14 are reassuring, although far from conclusive, that v?3 antagonism does not inhibit endothelial cell regeneration after vascular injury.
In summary, the present study demonstrates that BS-1417 inhibited neointimal formation and enhanced positive remodeling after balloon injury of rat carotid arteries. These results further confirm multiple prior studies showing that v?3 antagonists reduce vascular response to injury. The mechanisms by which vascular healing is modified appear to be pleotrophic as v?3 antagonists have been shown, in various models, to reduce SMC proliferation and migration, transforming growth factor-? production, MMP production, and macrophage recruitment and to increase apoptosis. v?3 antagonists have also been shown to have diverse effects on remodeling and, at least ex vivo, to inhibit adhesion of activated platelets, platelet microparticles, and leukocytes to endothelial cells. The consistency of the findings in animal models of the effects of v?3 antagonism on vascular healing suggest that v?3 integrins may represent an important future therapeutic target to limit neointimal formation after revascularization.
References
Takagi J, Petre BM, Walz T, Springer TA. Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell. 2002; 110: 599–511.
Stouffer GA, Hu Z, Sajid M, Li H, Jin G, Nakada MT, Hanson SR, Runge MS. ?3 integrins are upregulated following vascular injury and mediate proliferation of cultured smooth muscle cells. Circulation. 1998; 97: 907–915.
Corjay MH, Diamond SM, Schlingmann KL, Gibbs SK, Stoltenborg JK, Racanelli AL. v?3, v?5, and osteopontin are coordinately upregulated at early time points in a rabbit model of neointima formation. J Cell Bioch. 1999; 75: 492–504.
Sajid M, Stouffer GA. The role of v?3 integrins in vascular healing. Thromb Haemost. 2002; 87: 187–193.
Chico TJA, Chamberlain J, Gunn J, Arnold N, Bullens SL, Gadek TR, Francis SE, Bunting S, Horton M, Shepherd L, Lipari MT, Quan C, Knolle J, Stilz HU, Peyman A, Crossman DC. Effect of selective or combined inhibition of integrins IIb?3 and v?3 on thrombosis and neointima after oversized porcine coronary angioplasty. Circulation. 2001; 103: 1135–1141.
Bishop GG, McPherson JA, Sanders JM, Hesselbacher SE, Feldman MJ, McNamara CA, Gimple LW, Powers ER, Mousa SA, Sarembock IJ. Selective v?3-receptor blockade reduces macrophage infiltration and restenosis after balloon angioplasty in the atherosclerotic rabbit. Circulation. 2001; 103: 1906–1911.
Srivatsa SS, Fitzpatrick LA, Tsao PW, Reilly TM, Holmes DR Jr, Schwartz RS, Mousa SA. Selective v?3 integrin blockade potently limits neointimal hyperplasia and lumen stenosis following deep coronary arterial stent injury: evidence for the functional importance of integrin v?3 and osteopontin expression during neointima formation. Cardiovasc Res. 1997; 36: 408–428.
Sadeghi MM, Krassilnikova S, Zhang J, Gharaei AA, Fassaei HR, Esmailzadeh L, Kooshkabadi A, Edwards S, Yalamanchili P, Harris TD, Sinusas AJ, Zaret BL, Bender JR. Detection of injury-induced vascular remodeling by targeting activated v?3 integrin in vivo. Circulation. 2004; 110: 84–90.
Honda Y, Kitano T, Fukuya F, Sato Y, Iwama S, Morie T, Notake M. A novel v?3 integrin antagonist suppresses neointima formation for more than 4 weeks after balloon injury in rats. Arterioscler Thromb Vasc Biol. 2005; 25: 1376–1382.
Iwama S, Kitano T, Fukuya F, Honda Y, Sato Y, Notake M, Morie T. Discovery of a potent and selective v?3 integrin antagonist with strong inhibitory activity against neointima formation in rat balloon injury model. Bioorg Med Chem Lett. 2004; 14: 2567–2570.
Bendeck MP, Nakada MT. The ?3 integrin antagonist m7E3 reduces matrix metalloproteinase activity and smooth muscle cell migration. J Vasc Res. 2001; 38: 590–599.
Slepian MJ, Massia SP, Dehdashti B, Fritz A, Whitesell L. ?3-integrins rather than beta1-integrins dominate integrin-matrix interactions involved in postinjury smooth muscle cell migration. Circulation. 1998; 97: 1818–1827.
Stupack DG, Cheresh DA. Integrins and angiogenesis. Curr Top Dev Biol. 2004; 64: 207–238.
van der Zee R, Murohara T, Passeri J, Kearney M, Cheresh DA, Isner JM. Reduced intimal thickening following v?3 blockade is associated with smooth muscle cell apoptosis. Cell Adhes Commun. 1998; 6: 371–379.(G.A. Stouffer; A. Pathak;)