H. Roger Lijnen

Function of the Plasminogen/Plasmin and Matrix Metalloproteinase Systems After Vascular Injury in Mice With Targeted Inactivation of Fibrinolytic System Genes

The matrix metalloproteinase (MMP) system, which may be activated via the plasminogen (Plg)/plasmin system, is claimed to play a role in matrix degradation and smooth muscle cell migration. To test the role of both systems, expression of fibrinolytic and gelatinolytic activity was quantified after vascular injury in mice with targeted inactivation of tissue-type Plg activator (tPA-/-), urokinase-type Plg activator (uPA-/-), or Plg (Plg-/-). Neointima formation 1 week after vascular injury was impaired in uPA-/- and Plg-/- mice compared with wild-type (WT) mice or tPA-/- mice (reduction of neointimal area to 30% and 10% of WT, respectively). Cell accumulation at the borders of the injury was significantly (P<0.01) impaired compared with that in WT mice. One week after injury of the femoral artery, tPA-mediated fibrinolytic activity in arterial sections or extracts of WT, uPA-/-, or Plg-/- mice was not altered, whereas uPA activity levels in tPA-/- and Plg-/- mice were 2- to 3-fold higher than in uninjured controls. Total levels (latent plus active) of MMP-2 (gelatinase A) were increased by 2- to 4-fold, whereas the contribution of active MMP-2 represented 38% to 63% of the total in the different genotypes. MMP-9 (gelatinase B) was not detectable in the majority of control arteries, whereas total MMP-9 levels after injury were dramatically increased (up to 50-fold above the detection limit). Active MMP-9 represented 20% to 46% of total MMP-9 in WT, tPA-/-, and uPA-/- mice but was not consistently detectable in Plg-/- mice. Similar results were obtained in carotid arteries. Thus, the unaltered ratios of active and latent MMP-2 suggest that proMMP-2 activation may occur in the absence of tPA, uPA, or Plg, whereas no active MMP-9 was detected in the absence of Plg. The data of this study confirm a role for uPA and Plg but not for tPA in smooth muscle cell migration and neointima formation after vascular injury and indicate that impairment of these phenomena may occur despite the observed increases in MMP-2 or MMP-9 levels after vascular injury.

1 Mechanisms of physiological fibrinolysis

The fibrinolytic system comprises an inactive proenzyme, plasminogen, that is converted by plasminogen activators to the active enzyme, plasmin, which degrades fibrin. Two immunologically distinct plasminogen activators (PA) have been identified: tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). t-PA mediated plasminogen activation is mainly involved in the dissolution of fibrin in the circulation, whereas u-PA mediated plasminogen activation mainly plays a role in pericellular proteolysis. Plasminogen activation is regulated by specific molecular interactions between its main components, such as binding of plasminogen and t-PA to fibrin, or to specific cellular receptors resulting in enhanced plasminogen activation, inhibition of t-PA and u-PA by plasminogen activator inhibitors (PAI) and inhibition of plasmin by alpha 2-antiplasmin. Controlled synthesis and release of PAs and PAIs primarily from endothelial cells also contributes to the regulation of physiological fibrinolysis. The lysine binding sites situated in the kringle structures of plasminogen play a crucial role in the regulation of fibrinolysis by modulating its binding to fibrin and to cell surfaces, and by controlling the inhibition rate of plasmin by alpha 2-antiplasmin.

Pressure-Induced Matrix Metalloproteinase-9 Contributes to Early Hypertensive Remodeling

Background—High blood pressure causes a change in vascular wall structure involving altered extracellular matrix composition, but how this process occurs is not fully understood. Methods and Results—Using mouse carotid arteries maintained in organ culture for 3 days, we detected increased gelatin zymographic activity of matrix metalloproteinase (MMP)-2 (168±13%, P <0.05) in vessels kept at low intraluminal pressure (10 mm Hg) compared with vessels at 80 mm Hg (100%), whereas in vessels maintained at high pressure (150 mm Hg), both MMP-2 and MMP-9 activity was induced (182±32%, P <0.05, and 194±21%, P <0.01, respectively). MMPs were detected in endothelial and smooth muscle cells by immunohistochemistry and in situ gelatin zymography. In vessels at 150 mm Hg, MMP activation was associated with a shift in the pressure-diameter curve toward greater distensibility (P <0.01) compared with vessels at 80 mm Hg. However, distensibility was not altered in vessels at 10 mm Hg, in which only activated MMP-2 was detected. The role of MMPs in high pressure–induced vessel distensibility was confirmed by use of the MMP inhibitor FN-439, which prevented the shift in the pressure-diameter relationship. Furthermore, in carotid arteries from MMP-9–deficient mice, the pressure-dependent increase in MMP-2 and in situ gelatinolytic activity were maintained, but the upward shift in the pressure-diameter curve was abolished. Conclusions—MMP-9 seems to play a key role in the early stages of hypertensive vascular remodeling.

Matrix Metalloproteinase-9 Deficiency Impairs Host Defense against Abdominal Sepsis

Matrix metalloproteinase (MMP)-9 is involved in extracellular matrix degradation and leukocyte migration. To determine the role of MMP-9 in the innate immune response to peritonitis, MMP-9 gene-deficient (MMP-9−/−) and normal wild-type mice were i.p. infected with Escherichia coli. MMP-9 mRNA and pro-MMP-9 protein levels increased rapidly upon induction of peritonitis. Although MMP-9−/− neutrophils showed a normal phagocytosis of E. coli in vitro, MMP-9−/− mice displayed a reduced resistance against E. coli peritonitis, as indicated by an enhanced bacterial outgrowth in the peritoneal cavity and increased dissemination of the infection. Furthermore, the cytokine response to LPS was not influenced by MMP-9 deficiency. However, during E. coli peritonitis, MMP-9−/− mice showed much higher peritoneal chemokine and cytokine levels compared with wild-type mice. Despite the increased local chemokine concentrations, MMP-9−/− mice displayed a diminished recruitment of leukocytes to the site of infection, indicating that cellular migration was impaired. Moreover, MMP-9−/− mice developed more severe distant organ damage during infection. These data suggest that MMP-9 is an essential component of an effective host response to E. coli peritonitis.

Nutritionally Induced Obesity Is Attenuated in Transgenic Mice Overexpressing Plasminogen Activator Inhibitor-1

Objective—The objective of this study was to investigate the role of plasminogen activator inhibitor-1 (PAI-1) in adipose tissue development in vivo. Methods and Results—Transgenic (Tg) mice overexpressing murine PAI-1 under control of the adipocyte promoter aP2 and wild-type (WT) controls were kept on standard food (SFD) or on high-fat diet (HFD) for 15 weeks. The body weight and the weight of the isolated subcutaneous and gonadal fat deposits of the Tg mice kept on the HFD were significantly lower than those of the WT mice. The number of adipocytes in the adipose tissue was similar for Tg and WT mice on the HFD, but adipocyte hypotrophy and a significantly lower ratio of stroma cells/adipocytes were observed in the Tg mice. A significant negative correlation (P <0.01) was observed between expression of preadipocyte factor-1, which blocks adipocyte differentiation, and adipose tissue weight. Fasting insulin and total cholesterol levels on the HFD were lower in Tg than in WT mice. Conclusions—High circulating PAI-1 levels attenuate nutritionally induced obesity. This may be related to modifications in adipose tissue cellularity affecting weight and plasma metabolic parameters.

Metalloproteinases in development and progression of vascular disease.

Remodeling of the vascular wall plays a role in many physiological processes, but also in the pathogenesis of major cardiovascular diseases such as restenosis and atherosclerosis. Remodeling requires proteolytic activity to degrade components of the extracellular matrix; this can be generated by the matrix metalloproteinase(MMP) system alone or in concert with the fibrinolytic (plasminogen/plasmin) system. Several lines of evidence suggest that the MMP system plays a role in vascular smooth muscle cell migration and neointima formation after vascular injury. In atherosclerotic lesions, active MMPs may contribute to plaque destabilisation by degrading extracellular matrix components, but may also promote aneurysm formation by proteolytic degradation of the elastic lamina. The MMP system may therefore represent a potential therapeutic target for treatment of restenosis or atherosclerosis.

Induction of Vascular SMC Proliferation by Urokinase Indicates a Novel Mechanism of Action in Vasoproliferative Disorders

The urokinase-type plasminogen activator (UPA) and its receptor are expressed in the vasculature and are involved in cell migration and remodeling of the extracellular matrix in the neointima. Vessels with atherosclerosis or neointimal hyperplasia, when compared with normal vessels, contain high UPA activity as well as increased levels of UPA receptor. In this study, we have identified the stimulation of vascular smooth muscle cell proliferation as a novel activity for UPA in the vessel wall. High-molecular-weight-UPA (12-200 nmol/L range) stimulated DNA synthesis and cell proliferation, which was half that induced by fetal calf serum or by platelet-derived growth factor-BB. UPA did not induce growth of endothelial cells, and tissue-type plasminogen activator showed no activity on either cell type. Induction of proliferation required the complete UPA molecule but was independent of the proteolytic activity of UPA, whereas neither the amino-terminal fragment nor the catalytic domain by itself was mitogenic. UPA also stimulated c-fos/c-myc mRNA expression and mitogen-activated protein kinase activity in smooth muscle cells. Blocking monoclonal antibodies against the UPA receptor and the enzymatic removal of receptors were ineffective in inhibiting the mitogenic effect of UPA, suggesting a UPA receptor-independent mechanism. Thus, we provide evidence for a novel function of UPA on vascular smooth muscle cell proliferation that, together with its previously documented involvement in regulating pericellular proteolysis-related events and cell migration, provides additional evidence for a role in the pathogenesis of atherosclerosis/restenosis.

Impaired Adipose Tissue Development in Mice With Inactivation of Placental Growth Factor Function

Placental growth factor (PlGF)-deficient (PlGF−/−) and wild-type mice were kept on a standard-fat or high-fat diet for 15 weeks. With the standard-fat diet, the body weights of PlGF−/− and wild-type mice were comparable, whereas the combined weight of subcutaneous and gonadal adipose tissues was lower in PlGF−/− mice (P = 0.02). With the high-fat diet, PlGF−/− mice had a lower body weight (P < 0.05) and less total subcutaneous plus gonadal adipose tissue (P < 0.0001). Blood vessel size was lower in gonadal adipose tissue of PlGF−/− mice with both the standard-fat and high-fat diet (P < 0.05). Blood vessel density, normalized to adipocyte number, was significantly lower in subcutaneous adipose tissue of PlGF−/− mice fed the high-fat diet (P < 0.01). De novo adipose tissue development in nude mice injected with 3T3-F442A preadipocytes was reduced (P < 0.005) by administration of a PlGF-neutralizing antibody. Bone marrow transplantation from wild-type or PlGF−/− mice to wild-type or PlGF−/− recipient mice revealed significantly lower blood vessel density in PlGF−/− recipient mice without an effect on adipose tissue growth. Thus, in murine models of diet-induced obesity, inactivation of PlGF impairs adipose tissue development, at least in part as a result of reduced angiogenesis.

Tissue-type plasminogen activator (t-PA) induces stromelysin-1 (MMP-3) in endothelial cells through activation of lipoprotein receptor–related protein

Tissue-type plasminogen activator (t-PA) is approved for treatment of ischemic stroke patients, but it increases the risk of intracranial bleeding (ICB). Previously, we have shown in a mouse stroke model that stromelysin-1 (matrix metalloproteinase-3 [MMP-3]) induced in endothelial cells was critical for ICB induced by t-PA. In the present study, using bEnd.3 cells, a mouse brain-derived endothelial cell line, we showed that MMP-3 was induced by both ischemic stress and t-PA treatment. This induction by t-PA was prevented by inhibition either of low-density lipoprotein receptor-related protein (LRP) or of nuclear factor-kappaB activation. LRP was up-regulated by ischemic stress, both in bEnd.3 cells in vitro and in endothelial cells at the ischemic damage area in the mouse stroke model. Furthermore, inhibition of LRP suppressed both MMP-3 induction in endothelial cells and the increase in ICB by t-PA treatment after stroke. These findings indicate that t-PA deteriorates ICB via MMP-3 induction in endothelial cells, which is regulated through the LRP/nuclear factor-kappaB pathway.

Mouse Carotid Artery Ligation Induces Platelet-Leukocyte–Dependent Luminal Fibrin, Required for Neointima Development

Abstract — The relationship between platelet and leukocyte activation, coagulation, and neointima development was investigated in noninjured murine blood vessels subjected to blood stasis. The left common carotid artery of C57BL/6J mice was ligated proximal to the bifurcation. Tissue-factor expression in luminal leukocytes progressively increased over 2 weeks. On day 3 after ligation, in addition to infiltrated granulocytes, platelet microthrombi and platelet-covered leukocytes as well as tissue-factor–positive fibrin deposits lined the endothelium. Maximal neointima formation in carotid artery cross sections of control mice equaled 28±3.7% (n=11) and 42±5.1% (n=8) of the internal elastic lamina cross-sectional area 1 and 2 weeks after ligation. In FVIII−/− mice, stenosis was significantly lower 1 (11±3.6%, n=8) and 2 (21±4.7%, n=7) weeks after ligation (both P <0.01 versus background-matched controls). In u-PA−/− mice, luminal stenosis was significantly higher 1 (38±7.0%, n=7) and 2 (77±5.6%, n=6) weeks after ligation (P <0.05 and P <0.01, respectively, versus matched controls). In &agr;2-AP−/− mice, stenosis was lower at 1 week (14±2.6%, n=7, P <0.01) but not at 2 weeks. Responses in tissue-type plasminogen activator or plasminogen activator inhibitor-1 gene–deficient mice equaled that in controls. Reducing plasma fibrinogen levels in controls with ancrod or inducing partial thrombocytopenia with busulfan resulted in significantly less neointima, but inflammation was inhibited only in busulfan-treated mice. We conclude that stasis induces platelet activation, leading to microthrombosis and platelet-leukocyte conjugate formation, triggering inflammation and tissue-factor accumulation on the carotid artery endothelium. Delayed coagulation then results in formation of a fibrin matrix, which is used by smooth muscle cells to migrate into the lumen.