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News and Publications
Novel Mechanisms of Cell Motility and Cardiovascular Disease
D. Loskutoff, R.P. Czekay, B. Degryse, Y. Kamikubo, J. Neels,
S. Konstantinides, Y. Okumura, F. Samad, P. Sartipy, K. Schäfer
Plasminogen activator inhibitor-1 (PAI-1) is a serine protease
inhibitor (serpin) that regulates tissue-type plasminogen activator
and urokinase plasminogen activator (uPA), proteases that remove
pathologic fibrin deposits from the vasculature. PAI-1 differs from
most other members of the serpin gene family 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 in 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 suggest that PAI-1, vitronectin, and leptin may influence cell
adhesion and migration in a variety of physiologic and pathologic
settings and may regulate atherothrombotic cardiovascular disease
in obesity.
CELL MOTILITY
Binding of uPA to its cell-surface receptor (uPAR) promotes cell
adhesion to vitronectin and to integrins. PAI-1 disrupts these interactions
and detaches the cells by binding to uPA-uPAR complexes at the cell
surface. Interestingly, the detached cells cannot reattach unless
they are first treated with manganese chloride to activate integrins.
Thus, the addition of PAI-1 appears to deactivate aV integrins. Immunoprecipitation and subcellular
fractionation experiments revealed that treatment with PAI-1 triggers
internalization of complexes consisting of uPA, uPAR, and aV integrin by the
low-density lipoprotein receptor-related protein. PAI-1 also can
detach cells from type 1 collagen and fibronectin by a similar mechanism.
In all instances, the degree of cell detachment depends on the number
of uPA-uPAR-integrin complexes that are engaged by PAI-1. PAI-1
also induces smooth muscle cell migration by binding to the low-density
lipoprotein receptor-related protein and activating the Jak/Stat
pathway. Because PAI-1 is present at sites of vascular injury, it
may promote migration of smooth muscle cells in vivo, leading to
the formation of the neointima common in atherosclerosis and other
vascular disorders.
CARDIOVASCULAR DISEASE AND OBESITY
Although obese mice have high circulating levels of 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) are injured are unstable and often embolize.
Intraperitoneal administration of leptin corrects these defects
in the ob/ob mice but not in the db/db mice. Platelets express the
leptin receptor, and leptin potentiates the aggregation of platelets
from ob/ob mice but not from db/db mice. These results reveal a
novel receptor-dependent effect of leptin on platelet function and
hemostasis and may provide new insights into the molecular basis
of cardiovascular complications in obese individuals.
Leptin also potentiated the aggregation of human platelets, but
it did so in platelets from only 50% of the donors. Thus, 2 distinct
groups of donors exist: responders and nonresponders. Statistical
analysis indicated that the variability in leptin responsiveness
was not due to differences in sex, age, plasma levels of leptin,
or plasma levels of insulin. Moreover, washing platelets from nonresponders
and resuspending the cells in plasma from responders, and vice versa,
did not alter the responsiveness of the platelets to leptin. Western
blotting revealed the presence of consistently higher levels of
the leptin receptor on platelets from the responders. Thus, the
variability in platelet sensitivity to leptin may reflect differences
in the expression of the long form of the leptin receptor.
The hyperinsulinemia that often accompanies obesity and insulin
resistance may contribute to altered gene expression in the target
tissues of insulin. We found that the gene for monocyte chemoattractant
protein 1 (MCP-1) is an insulin-sensitive gene. In fact, insulin
continues to induce expression of MCP-1 in vitro in both normal
and insulin-resistant 3T3-L1 adipocytes and in vivo in insulin-resistant
obese mice (ob/ob). Thus, the gene for MCP-1 resembles other previously
described genes that remain sensitive to insulin in insulin-resistant
mice and adipocytes.
We found that compared with their lean controls, obese mice had
overexpression of MCP-1 and that white adipose tissue was a major
source of MCP-1 in vivo. Incubation of differentiated adipocytes
with low concentrations of MCP-1 in vitro decreased both insulin-stimulated
glucose uptake and the expression of lipoprotein lipase. Thus, MCP-1
may directly contribute to the pathologic changes associated with
hyperinsulinemia and obesity, including type II diabetes.
The microcirculation of adipose tissue is unique within the vascular
system because the tissue can grow throughout adult life. We are
using an in vivo model of fat pad development to study angiogenesis
in expanding adipose tissue. Briefly, 3T3-F442A preadipocytes were
implanted subcutaneously into athymic Balb/c nude mice. Within 24
hours, the cells were encapsulated by a fascia that formed a flat
layer of fibrous tissue surrounding the implant. Although the central
core of the implant rapidly became necrotic, the outer layer survived
and by 48 hours after implantation began to differentiate into mature
adipocytes. By 21 days after implantation, this layer of cells had
developed into a highly vascularized fat pad that was indistinguishable
from normal adipose tissue.
The results of histologic studies suggested that the differentiation
of preadipocytes into mature adipocytes preceded angiogenesis; a
new microvasculature was evident by 5 days after cell implantation.
Reverse transcriptase-polymerase chain reaction revealed the temporal
expression of adipocyte and endothelial markers by 3-5 days after
injection of the cells. PAI-1 levels also rapidly but transiently
increased and were 500-fold higher in 1-day-old fat pads than in
control adipose tissue. Results of immunohistochemistry and in situ
hybridization studies localized PAI-1 to the layer of preadipocytes
that survived the initial ischemia. PAI-1 has been implicated in
the regulation of angiogenesis, raising the possibility that the
dramatic induction of PAI-1 gene expression may be necessary for
some early event in the angiogenic process.
PUBLICATIONS
Hollestelle, M.J., Thinnes, T., Crain, K., Stiko, A., Kruijt,
J.K., van Berkel, T.J.C., Loskutoff, D.J., van Mourik, J.A.
Tissue distribution of factor VIII gene expression in vivo: a closer
look. Thromb. Haemost. 86:855, 2001.
Konstantinides, S., Schäfer, K., Koschnick, K., Loskutoff,
D.J. Leptin-dependent platelet aggregation and arterial thrombosis
suggests a mechanism for atherothrombotic disease in obesity. J.
Clin. Invest. 108:1533, 2001.
Konstantinides, S., Schäfer, K., Loskutoff, D.J. The
prothrombotic effects of leptin: possible implications for the risk
of cardiovascular disease in obesity. Ann. N. Y. Acad. Sci. 947:134,
2001.
Loskutoff, D.J. Alterations in hemostatic gene expression
in obesity. In: Vascular Protection: Molecular Mechanisms,
Novel Therapeutic Principles, and Clinical Application, Vol. 9.
Rubanyi, G.M., Dzau, V.J., Cooke, J.P. (Eds.). Taylor & Francis,
New York, 2002, p. 347.
Okumura, Y., Kamikubo, Y., Curriden, S.A., Wang, J., Kiwada,
T., Futaki, S., Kitagawa, K., Loskutoff, D.J. Kinetic analysis
of the interaction between vitronectin and the urokinase receptor.
J. Biol. Chem. 277:9395, 2002.
Royle, G., Deng, G., Seiffert, D., Loskutoff, D.J. A method
for defining binding sites involved in protein-protein interactions:
analysis of the binding of plasminogen activator inhibitor 1 to
the somatomedin domain of vitronectin. Anal. Biochem. 296:245, 2001.
Samad, F., Pandey, M., Loskutoff, D.J. Regulation of tissue
factor gene expression in obesity. Blood 98:3353, 2001.
Yamamoto, K., Takeshita, K., Shimokawa, T., Yi, H., Isobe,
K., Loskutoff, D.J., Saito, H. Plasminogen activator inhibitor-1
is a major stress-regulated gene: implications for stress-induced
thrombosis in aged individuals. Proc. Natl. Acad. Sci. U. S. A.
99:890, 2002.
Zhang, L., Seiffert, D., Fowler, B.J., Jenkins, G.R., Thinnes,
T.C., Loskutoff, D.J., Parmer, R.J., Miles, L.A. Plasminogen
has a broad extrahepatic distribution. Thromb. Haemost. 87:493,
2002.
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