News and Publications

Novel Mechanisms of Cell Motility and Cardiovascular Disease

* Georg-August University of Göttingen, Göttingen, Germany

Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor (serpin) that regulates proteases that remove pathologic fibrin deposits from the vasculature. PAI-1 differs from most other members of the family of proteins encoded by the serpin genes because it is a trace protein in blood, it has a short half-life, and its synthesis is highly regulated by cytokines, growth factors, and hormones. Moreover, it is the product of an immediate-early gene, and it binds to the adhesive glycoprotein vitronectin.

Leptin is the 167 amino acid product of the ob gene. It is synthesized by adipocytes and acts on hypothalamic receptors to reduce food intake and to increase energy expenditure. Juvenile-onset obesity develops in mice that lack functional leptin (ob/ob mice) or its receptor (db/db mice). Results of research with cultured cells and genetically altered mice indicate that PAI-1, vitronectin, and leptin influence cell adhesion and migration in a variety of physiologic and pathologic settings and may regulate atherothrombotic cardiovascular disease in obesity.

The Somatomedin B Domain of Vitronectin

PAI-1 and the urokinase receptor (uPAR; CD87) bind to vitronectin, and these interactions not only influence the attachment, detachment, and migration of cells but also regulate local extracellular proteolysis. Vitronectin itself is organized into a number of distinct domains, including the somatomedin B (SMB) domain at the amino terminus, the adjacent connector region containing the arginine-glycine-aspartic acid site, and 2 hemopexin-like domains. We localized the high-affinity binding sites for both PAI-1 and uPAR to 2 distinct but overlapping regions in the SMB domain.

Using a variety of methods, we identified the 4 disulfide bonds in recombinant SMB (rSMB). Two pairs of disulfide bonds at the amino-terminal part of active rSMB were identified as Cys⁵-Cys⁹ and Cys¹⁹-Cys²¹. Selective reduction or S-alkylation of these 2 disulfide linkages caused the complete loss of PAI-1 binding activity. The other 2 pairs of disulfide bonds in the carboxyl-terminal part of rSMB were identified as Cys²⁵-Cys³¹ and Cys³²-Cys³⁹. These results suggest an unusual linear uncrossed pattern for the disulfide bond topology of rSMB, which is distinct from the crossed pattern present in most small proteins rich in disulfide bonds.

In separate studies, we showed that PAI-1 inhibits uPAR- and integrin-mediated cell attachment by binding to SMB. The bound PAI-1 directly blocks the uPAR site and sterically interferes with binding to the adjacent arginine-glycine-aspartic acid site. Although PAI-1 can detach cells bound to vitronectin through uPAR (e.g., U937 cells) by displacing uPAR from SMB, this vitronectin-dependent mechanism does not account for the ability of PAI-1 to detach cells bound predominantly via integrins (e.g., HT-1080 cells). For cells bound to integrins, the antiadhesive effects of PAI-1 are vitronectin independent and only require interaction of PAI-1 with urokinase bound to uPAR-integrin complexes. Binding of PAI-1 to urokinase-uPAR-integrin complexes leads to the specific inactivation and internalization of integrins bound to the extracellular matrix, thus leading to rapid cell detachment. This pathway represents a general mechanism, because PAI-1 can also detach a number of other cells from a variety of matrices, including fibronectin, type I collagen, and laminin.

Thus, PAI-1 can be added to the list of known antiadhesive molecules (e.g., thrombospondin), and its antiadhesive behavior may explain why high PAI-1 levels are associated with a poor prognosis for survival in a number of metastatic cancers in humans. PAI-1 also induces migration of smooth muscle cells and thus may contribute to the formation of the neointima common in atherosclerosis and other vascular disorders.

Cardiovascular Disease, Obesity, and Leptin

Although obese mice have high circulating levels of the prothrombotic molecule PAI-1, the expected prothrombotic state does not develop in these animals. We found that the thrombi formed when the carotid arteries of obese mice (ob/ob and db/db mice) are injured are unstable and often embolize. This instability seems to be due to the absence of leptin, because intraperitoneal administration of leptin rapidly corrected these defects in ob/ob mice but not in db/db mice, which lack the receptor for leptin.

Platelets express the receptor for leptin, and leptin also potentiated the aggregation of platelets from ob/ob mice but not from db/db mice. Leptin also potentiated the aggregation of human platelets, but only in only 50% of the samples. Further studies indicated that levels of leptin receptors in platelets from "responder" donors were consistently higher than the levels in platelets from "nonresponder" donors.

The underlying cause of death in human obesity is atherosclerosis, a chronic wound-healing process that occurs in response to endothelial injury. We found that leptin also directly promotes vascular remodeling and pathologic changes in the vessel wall in mice. For example, wild-type mice placed on an atherogenic high-fat diet had elevated (10-fold) levels of leptin and significantly enhanced neointimal thickening after injury to the carotid artery. The thickened areas histologically resembled the plaques that develop in humans with atherosclerosis, and the cells in the areas expressed leptin receptor mRNA and protein.

Unexpectedly, the atherogenic diet had no effect on injured vessels in leptin-deficient ob/ob mice despite aggravating obesity, diabetes, and hyperlipidemia in these animals. Daily administration of leptin to ob/ob mice restored the pathologic phenotype, dramatically increasing the size of the plaquelike vascular lesions. Exogenous leptin also enhanced development of plaque-like areas in injured vessels in wild-type mice, but it had no effect on vessels from leptin receptor-deficient db/db mice. Taken together, these results suggest the existence of a direct, leptin receptor-mediated link between the hyperleptinemia in obesity and the increased risk for atherosclerosis.

Publications

Czekay, R.-P., Aertgeerts, K., Curriden, S.A., Loskutoff, D.J. Plasminogen activator inhibitor-1 detaches cells from extracellular matrices by inactivating integrins. J. Cell Biol. 160:781, 2003.

Deng, G.G., Martin-McNulty, B., Sukovich, D.A., Freay, A., Halks-Miller, M., Thinnes, T., Loskutoff, D.J., Carmeliet, P., Dole, W.P., Wang, Y.-X. Urokinase-type plasminogen activator plays a critical role in angiotensin II-induced abdominal aortic aneurysm. Circ. Res. 92:510, 2003.

Kamikubo, Y., Okumura, Y., Loskutoff, D.J. Identification of the disulfide bonds in the recombinant somatomedin B domain of human vitronectin. J. Biol. Chem. 277:27109, 2002.

Konstantinides, S., Schäfer, K., Loskutoff, D.J. Do PAI-1 and vitronectin promote or inhibit neointima formation? The exact role of the fibrinolytic system in vascular remodeling remains uncertain. Arterioscler. Thromb. Vasc. Biol. 22:1943, 2002.

Sartipy, P., Loskutoff, D.J. Monocyte chemoattractant protein 1 in murine obesity and insulin resistance. Proc. Natl. Acad. Sci. U. S. A., in press.

Schäfer, K., Konstantinides, S., Riedel, C., Thinnes, T., Müller, K., Dellas, C., Hasenfuss, G., Loskutoff, D.J. Different mechanisms of increased luminal stenosis after arterial injury in mice deficient for urokinase- or tissue-type plasminogen activator. Circulation 106:1847, 2002.

Takeshita, K., Yamamoto, K., Ito, M., Kondo, T., Matsushita, T., Hirai, M., Kojima, T., Nishimura, M., Nabeshima, Y., Loskutoff, D.J., Saito, H., Murohara, T. Increased expression of plasminogen activator inhibitor-1 with fibrin deposition in a murine model of aging, "klotho" mouse. Semin. Thromb. Hemost. 28:545, 2002.

Yamamoto, K., Shimokawa, T., Yi, H., Isobe, K., Kojima, T., Loskutoff, D.J., Saito, H. Aging accelerates endotoxin-induced thrombosis: increased responses of plasminogen activator inhibitor-1 and lipopolysaccharide signaling with aging. Am. J. Pathol. 161:1805, 2002.

Yamamoto, K., Shimokawa, T., Yi, H., Isobe, K., Kojima, T., Loskutoff, D.J., Saito, H. Aging and obesity augment the stress-induced expression of tissue factor gene in the mouse. Blood 100:4011, 2002.