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1.
Article in English | MEDLINE | ID: mdl-23308073

ABSTRACT

Numerous animal and clinical investigations have pointed to a potential role of the renin-angiotensin system (RAS) in the development of insulin resistance and diabetes in conditions of expanded fat mass. However, the mechanisms underlying this association remain unclear. We used a transgenic mouse model overexpressing renin in the liver (RenTgMK) to examine the effects of chronic activation of RAS on adiposity and insulin sensitivity. Hepatic overexpression of renin resulted in constitutively elevated plasma angiotensin II (four- to six-fold increase vs. wild-type, WT). Surprisingly, RenTgMK mice developed glucose intolerance despite low levels of adiposity and insulinemia. The transgenics also had lower plasma triglyceride levels. Glucose intolerance in transgenic mice fed a low-fat diet was comparable to that observed in high-fat fed WT mice. These studies demonstrate that overexpression of renin and associated hyperangiotensinemia impair glucose tolerance in a diet-dependent manner and further support a consistent role of RAS in the pathogenesis of diabetes and insulin resistance, independent of changes in fat mass.

2.
Methods Enzymol ; 499: 77-104, 2011.
Article in English | MEDLINE | ID: mdl-21683250

ABSTRACT

Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) and therefore plays an important role in the plasminogen/plasmin system. PAI-1 is involved in a variety of cardiovascular diseases (mainly through inhibition of t-PA) as well as in cell migration and tumor development (mainly through inhibition of u-PA and interaction with vitronectin). PAI-1 is a unique member of the serpin superfamily, exhibiting particular unique conformational and functional properties. Since its involvement in various biological and pathophysiological processes PAI-1 has been the subject of many in vivo studies in mouse models. We briefly discuss structural and physiological differences between human and mouse PAI-1 that should be taken into account prior to extrapolation of data obtained in mouse models to the human situation. The current review provides an overview of the various models, with a focus on cardiovascular disease and cancer, using wild-type mice or genetically modified mice, either deficient in PAI-1 or overexpressing different variants of PAI-1.


Subject(s)
Plasminogen Activator Inhibitor 1/metabolism , Animals , Atherosclerosis/genetics , Atherosclerosis/metabolism , Cardiovascular Diseases/genetics , Cardiovascular Diseases/metabolism , Fibrosis/genetics , Fibrosis/metabolism , Humans , Mice , Mice, Transgenic , Neoplasms/genetics , Neoplasms/metabolism , Plasminogen Activator Inhibitor 1/genetics , Thrombosis/genetics , Thrombosis/metabolism
3.
Circulation ; 122(12): 1200-9, 2010 Sep 21.
Article in English | MEDLINE | ID: mdl-20823384

ABSTRACT

BACKGROUND: Elevated levels of plasminogen activator inhibitor-1 (PAI-1), a potent inhibitor of urokinase plasminogen activator and tissue plasminogen activator, are implicated in the pathogenesis of tissue fibrosis. Paradoxically, lack of PAI-1 in the heart is associated with the development of cardiac fibrosis in aged mice. However, the molecular basis of cardiac fibrosis in aged PAI-1-deficient mice is unknown. Here, we investigated the molecular and cellular bases of myocardial fibrosis. METHODS AND RESULTS: Histological evaluation of myocardial tissues derived from aged PAI-1-deficient mice revealed myocardial fibrosis resulting from excessive accumulation of collagen. Immunohistochemical characterization revealed that the levels of matrix metalloproteinase-2, matrix metalloproteinase-9, and transforming growth factor-ß1/2 and the number of Mac3-positive and fibroblast specific protein-1-positive cells were significantly elevated in aged PAI-1-deficient myocardial tissues compared with controls. Zymographic analysis revealed that matrix metalloproteinase-2 enzymatic activity was elevated in PAI-1-deficient mouse cardiac endothelial cells. Real-time quantitative polymerase chain reaction analyses of RNA from myocardial tissues revealed the upregulation of profibrotic markers in aged PAI-1-deficient mice. The numbers of phosphorylated Smad2-, phosphorylated Smad3-, and phosphorylated ERK1/2 MAPK-, but not pAkt/PKB-, positive cells were significantly increased in PAI-1-deficient myocardial tissues. Western blot and immunocytochemical analysis revealed that PAI-1-deficient mouse cardiac endothelial cells were more susceptible to endothelial-to-mesenchymal transition in response to transforming growth factor-ß2. CONCLUSIONS: These results indicate that spontaneous activation of both Smad and non-Smad transforming growth factor-ß signaling may contribute to profibrotic responses in aged PAI-1-deficient mice hearts and establish a possible link between endothelial-to-mesenchymal transition and cardiac fibrosis in PAI-1-deficient mice.


Subject(s)
Aging/pathology , Endothelium, Vascular/pathology , Heart Diseases/genetics , Heart Diseases/pathology , Mesoderm/pathology , Serpins/genetics , Transforming Growth Factor beta/metabolism , Animals , Cell Differentiation/physiology , Collagen/metabolism , Disease Models, Animal , Fibrosis/genetics , Fibrosis/metabolism , Fibrosis/pathology , Heart Diseases/metabolism , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Myocardium/metabolism , Myocardium/pathology , S100 Calcium-Binding Protein A4 , S100 Proteins/metabolism , Serpin E2 , Serpins/deficiency , Serpins/metabolism , Signal Transduction/physiology
4.
Obesity (Silver Spring) ; 18(3): 489-98, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19644452

ABSTRACT

Obesity is an increasingly important public health issue reaching epidemic proportions. Visceral obesity has been defined as an important element of the metabolic syndrome and expansion of the visceral fat mass has been shown to contribute to the development of insulin resistance and cardiovascular disease. To identify novel contributors to cardiovascular and metabolic abnormalities in obesity, we analyzed the adipose proteome and identified soluble epoxide hydrolase (sEH) in the epididymal fat pad from C57BL/6J mice that received either a regular diet or a "western diet." sEH was synthesized in adipocytes and expression levels increased upon differentiation of 3T3-L1 preadipocytes. Although normalized sEH mRNA and protein levels did not differ in the fat pads from mice receiving a regular or a "western diet," total adipose sEH activity was higher in the obese mice, even after normalization for body weight. Furthermore, peroxisome proliferator-activated receptor gamma (PPARgamma) agonists increased the expression of sEH in mature 3T3-L1 adipocytes in vitro and in adipose tissue in vivo. Considering the established role for sEH in inflammation, cardiovascular diseases, and lipid metabolism, and the suggested involvement of sEH in the development of type 2 diabetes, our study has identified adipose sEH as a potential novel therapeutic target that might affect the development of metabolic and cardiovascular abnormalities in obesity.


Subject(s)
Adipocytes/enzymology , Adipose Tissue/enzymology , Diet , Epoxide Hydrolases/metabolism , Obesity/enzymology , 3T3 Cells , Animals , Epididymis , Male , Mice , Mice, Inbred C57BL , PPAR gamma/agonists , Proteome
5.
Am J Physiol Endocrinol Metab ; 297(2): E375-83, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19458064

ABSTRACT

Soluble epoxide hydrolase (Ephx2, sEH) is a bifunctional enzyme with COOH-terminal hydrolase and NH(2)-terminal phosphatase activities. sEH converts epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs), and the phosphatase activity is suggested to be involved in cholesterol metabolism. EETs participate in a wide range of biological functions, including regulation of vascular tone, renal tubular transport, cardiac contractility, and inflammation. Inhibition of sEH is a potential approach for enhancing the biological activity of EETs. Therefore, disruption of sEH activity is becoming an attractive therapeutic target for both cardiovascular and inflammatory diseases. To define the physiological role of sEH, we characterized a knockout mouse colony lacking expression of the Ephx2 gene. Lack of sEH enzyme is characterized by elevation of EET to DHET ratios in both the linoleate and arachidonate series in plasma and tissues of both female and male mice. In male mice, this lack of expression was also associated with decreased plasma testosterone levels, sperm count, and testicular size. However, this genotype was still able to sire litters. Plasma cholesterol levels also declined in this genotype. Behavior tests such as anxiety-like behavior and hedonic response were also examined in Ephx2-null and WT mice, as all can be related to hormonal changes. Null mice showed a level of anxiety with a decreased hedonic response. In conclusion, this study provides a broad biochemical, physiological, and behavioral characterization of the Ephx2-null mouse colony and suggests a mechanism by which sEH and its substrates may regulate circulating levels of testosterone through cholesterol biosynthesis and metabolism.


Subject(s)
Epoxide Hydrolases/genetics , Testosterone/blood , Animals , Anxiety/genetics , Anxiety/physiopathology , Behavior, Animal/physiology , Cholesterol/blood , Cholesterol/metabolism , Eicosanoids/metabolism , Epoxy Compounds/metabolism , Female , Fertility/genetics , Genotype , Hydrolysis , Lipid Metabolism/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Solubility
6.
Curr Drug Targets ; 8(9): 962-70, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17896947

ABSTRACT

At present, thrombolytic agents represent the only direct way of augmenting fibrinolytic activity in humans. While these agents are proven to be efficacious in the treatment of acute thrombotic events, they are not a viable option for long-term administration. There are numerous drugs available that indirectly to increase fibrinolytic activity by reducing plasma levels of plasminogen activator inhibitor-1 (PAI-1), including ACE inhibitors, insulin-sensitizing agents, and hormone replacement therapy in women. At present, efforts are underway to develop and test synthetic, selective PAI-1 antagonists. The potential applications of PAI-1 antagonists include thrombotic disorders (arterial and venous), amyloidosis, obesity, polycystic ovarian syndrome, and perhaps even type 2 diabetes mellitus. The availability of specific PAI-1 antagonists promises to expand the limits of understanding the role the fibrinolytic system plays in human disease and break through the current confines of therapeutic options that can effectively restore and augment the activity of the fibrinolytic system.


Subject(s)
Plasminogen Activator Inhibitor 1/metabolism , Angiotensin-Converting Enzyme Inhibitors/therapeutic use , Animals , Arteriosclerosis/drug therapy , Diabetes Mellitus, Type 2/blood , Female , Fibrosis/drug therapy , Humans , Obesity/blood , Obesity/prevention & control , Plasminogen Activator Inhibitor 1/blood , Polycystic Ovary Syndrome/metabolism , Serine Proteinase Inhibitors/blood , Serine Proteinase Inhibitors/metabolism , Thrombosis/drug therapy
7.
Am J Physiol Endocrinol Metab ; 293(3): E713-25, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17578885

ABSTRACT

Obesity is commonly associated with development of insulin resistance and systemic evidence of inflammation. Macrophages contribute to inflammatory amplification in obesity and may contribute directly to insulin resistance and the development of nonalcoholic fatty liver disease through the production of inflammatory cytokines, including tumor necrosis factor (TNF)-alpha. To test this hypothesis, we transplanted male wild-type (WT) and TNF-alpha deficient (KO) mice with either TNF-alpha-sufficient (TNF-alpha(+/+)) or TNF-alpha-deficient (TNF-alpha(-/-)) bone marrow. After consuming a high-fat diet for 26 wk, metabolic and morphometric characteristics of the animals were analyzed. While there were no differences in terms of relative weight gain, body composition analysis yielded a lower relative adipose and higher relative lean mass in mice lacking TNF-alpha, which was partially explained by reduced epididymal fat pad and liver weight. TNF-alpha(-/-) -->KO mice exhibited enhanced insulin sensitivity compared with that observed in TNF-alpha(+/+)-->KO mice; remarkably, no protection against insulin resistance was provided by transplanting TNF-alpha(-/-) bone marrow in WT mice compared with TNF-alpha(+/+)-->WT. The preserved insulin sensitivity seen in TNF-alpha(-/-)-->KO mice provided protection against the development of hepatic steatosis. Taken together, these data indicate that macrophage-derived TNF-alpha contributes to the pattern and extent of fat accumulation and insulin resistance in diet-induced obesity; however, this contribution is negligible in the presence of host-derived TNF-alpha.


Subject(s)
Dietary Fats , Fatty Liver/metabolism , Insulin Resistance , Macrophages/metabolism , Obesity/metabolism , Tumor Necrosis Factor-alpha/metabolism , Animals , Mice , Mice, Knockout , Obesity/chemically induced
8.
J Biol Chem ; 281(43): 32796-805, 2006 Oct 27.
Article in English | MEDLINE | ID: mdl-16931518

ABSTRACT

Plasma levels of plasminogen activator inhibitor-1 (PAI-1) are elevated in obesity and correlate with body mass index. The increase in PAI-1 associated with obesity likely contributes to increased cardiovascular risk and may predict the development of type 2 diabetes mellitus. Although adipocytes are capable of synthesizing PAI-1, the bulk of evidence indicates that cells residing in the stromal fraction of visceral fat are the primary source of PAI-1. We hypothesized that bone marrow-derived PAI-1, e.g. derived from macrophages located in visceral fat, contributes to the development of diet-induced obesity. To test this hypothesis, male C57BL/6 wild-type mice and C57BL/6 PAI-1 deficient mice were transplanted with either PAI-1(-/-), PAI-1(+/-), or PAI-1(+/+) bone marrow. The transplanted animals were subsequently fed a high fat diet for 24 weeks. Our findings show that only the complete absence of PAI-1 protects from the development of diet-induced obesity, whereas the absence of bone marrow-derived PAI-1 protects against expansion of the visceral fat mass. Remarkably, there is a link between the PAI-1 levels, the degree of inflammation in adipose tissue, and the development of obesity. Based on these findings we suggest that bone marrow-derived PAI-1 has an effect on the development of obesity through its effect on inflammation.


Subject(s)
Bone Marrow/chemistry , Obesity/blood , Obesity/metabolism , Plasminogen Activator Inhibitor 1/physiology , Adiponectin/blood , Adipose Tissue/metabolism , Adipose Tissue/pathology , Animals , Blood Glucose/analysis , Body Composition , Bone Marrow Cells/cytology , Bone Marrow Transplantation , Dietary Fats/administration & dosage , Energy Metabolism , Fasting , Glucose Tolerance Test , Insulin/blood , Leptin/blood , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Motor Activity , Organ Size , Oxygen Consumption , Plasminogen Activator Inhibitor 1/deficiency , RNA, Messenger/analysis , Resistin/blood , Time Factors
9.
Curr Opin Pharmacol ; 5(2): 149-54, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15780823

ABSTRACT

A classical perspective of cardiovascular risk does not adequately account for all of the cardiovascular events associated with obesity and diabetes. The combination of hypertriglyceridemia, glucose intolerance and inflammation is linked with increased production of the primary inhibitor of endogenous thrombolysis, plasminogen activator inhibitor-1 (PAI-1). Recent data suggest that PAI-1 contributes directly to the complications of obesity, including type 2 diabetes, coronary arterial thrombi, and may even influence the accumulation of visceral fat. Therefore, direct inhibition of PAI-1 might not only provide a new therapeutic strategy for reducing cardiovascular risk, but may also have beneficial effects on obesity and insulin resistance.


Subject(s)
Cardiovascular Diseases/metabolism , Diabetes Mellitus, Type 2/metabolism , Diabetic Angiopathies/metabolism , Obesity/metabolism , Plasminogen Activator Inhibitor 1/biosynthesis , Animals , Cardiovascular Diseases/etiology , Cardiovascular Diseases/therapy , Diabetes Mellitus, Type 2/etiology , Diabetes Mellitus, Type 2/therapy , Diabetic Angiopathies/etiology , Diabetic Angiopathies/therapy , Humans , Obesity/etiology , Obesity/therapy , Plasminogen Activator Inhibitor 1/genetics
10.
Thromb Haemost ; 92(5): 898-924, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15543316

ABSTRACT

The importance of obtaining insight in the structure/function relationship in the serpin plasminogen activator inhibitor type-1 can be understood from the major role PAI-1 plays in different (patho)physiological processes, mainly because of its involvement in the plasminogen/plasmin system. Moreover, during the past years, studies indicated a contribution of PAI-1 to the development of cardiovascular disease in common syndromes such as atherosclerosis, diabetes and hypertension. Furthermore, PAI-1 also inhibits u-PA, attributing a role in phenomena such as cell migration and tissue remodelling. Considering the role of PAI-1 in such various pathogenic path-ways, detailed insight into the structure/function relationship in PAI-1 might provide a means of interfering with a given pathological situation without disturbing other physiological processes. Therefore, since the discovery of PAI-1 and the cloning of its cDNA 20 years ago, over 600 PAI-1 variants have been constructed, elucidating the most important structural features of PAI-1. This review gives an overview of the contribution of the different PAI-1 variants to the understanding of the structure/function relationship in PAI-1, based on the different functional features of PAI-1.


Subject(s)
Mutation , Plasminogen Activator Inhibitor 1/chemistry , Plasminogen Activator Inhibitor 1/physiology , Animals , Genetic Variation , Humans , Plasminogen Activator Inhibitor 1/genetics , Protein Binding/genetics , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/genetics , Serine Proteinase Inhibitors/physiology , Structure-Activity Relationship , Substrate Specificity/genetics
11.
Biochem Biophys Res Commun ; 321(3): 746-51, 2004 Aug 27.
Article in English | MEDLINE | ID: mdl-15358169

ABSTRACT

PAI-1, the physiological inhibitor of tissue-type and urokinase-type plasminogen activator, is a unique member of the serpins as it exists in three distinct conformations: an active inhibitory conformation, a non-inhibitory substrate conformation, and a non-reactive latent conformation. Proline substitution of single residues in the P16-P20 region (situated at the proximal hinge of the reactive site loop) of wild-type PAI-1 (wtPAI-1) and a stabilized PAI-1-variant (PAI-1-stab; N150H, K154T, Q301P, Q319L, and M354I, t(1/2)=150), respectively, resulted in two series of PAI-1-variants with different properties. In wtPAI-1 only substitution at P18 resulted in a pronounced u-PA specificity and substrate behaviour towards t-PA. In contrast, in PAI-1-stab substitution at either P18, P19 or P20 resulted in a u-PA specificity and a significantly increased substrate behaviour towards t-PA and u-PA. Importantly, analysis of the kinetics of inhibition did not reveal any differences in the second-order rate constants of inhibition (k approximately 10(7)M(-1)s(-1)). The pronounced differences observed for identical mutations in wtPAI-1 vs PAI-1-stab demonstrate that not merely the sequence of the reactive loop but also intramolecular interactions between the hF/s3A-loop and the main part of the molecule govern the functional and conformational behaviour of PAI-1.


Subject(s)
Plasminogen Activator Inhibitor 1/chemistry , Plasminogen Activator Inhibitor 1/metabolism , Protein Structure, Tertiary , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/metabolism , Models, Molecular , Mutagenesis, Site-Directed , Plasminogen Activator Inhibitor 1/genetics , Serine Proteinase Inhibitors/genetics , Substrate Specificity , Tissue Plasminogen Activator/chemistry , Tissue Plasminogen Activator/metabolism , Urokinase-Type Plasminogen Activator/chemistry , Urokinase-Type Plasminogen Activator/metabolism
12.
J Biol Chem ; 279(19): 20447-50, 2004 May 07.
Article in English | MEDLINE | ID: mdl-14988411

ABSTRACT

As plasminogen activator inhibitor-1 (PAI-1), the physiological inhibitor of tissue-type plasminogen activator, is considered to be an important risk factor in several (patho)physiological conditions, many research activities focus on attempts to inhibit this serpin. The approach illustrated in the current study focuses on elucidating important interaction sites allowing the inhibition of PAI-1. Since monoclonal antibodies are in most cases not ideal for therapeutic use, the question of whether smaller molecules exert comparable effects is a hot issue. To answer this question, Cys residues were introduced in PAI-1 at positions previously identified as determining the epitope of a PAI-1-inhibiting antibody, MA-8H9D4, resulting in PAI-1-R300C, PAI-1-Q303C, and PAI-1-D305C. Subsequently, low molecular mass sulfhydryl-specific reagents (i.e. BODIPY 530/550 IA (molecular mass 626 Da) and BODIPY FL C(1)-IA (molecular mass 417 Da)) were allowed to react covalently with the cysteine. The functional distribution (inhibitory versus substrate) toward tissue-type plasminogen activator was determined for the labeled and the unlabeled samples. Labeling at position 300 leads to a 1.7- and 2.2-fold increase in SI value for BODIPY 530/550 IA and BODIPY FL C(1)-IA, respectively. Labeling at position 303 results in a 3.3- and 1.9-fold increase of the SI value for the large and the small label, respectively. At position 305, the SI values are 3.1-fold increased for both labels. The effect (on SI and on serpin activity) of the manipulations at these positions is in good agreement with the effect exerted by MA-8H9D4. In conclusion, our study provides proof of concept for the proposed approach in evaluating whether targeting a functional epitope with a small synthetic compound may be a feasible strategy in rational drug design.


Subject(s)
Drug Design , Plasminogen Activator Inhibitor 1/chemistry , Antibodies, Monoclonal/chemistry , Boron Compounds/pharmacology , Coloring Agents/pharmacology , Cysteine/chemistry , Epitopes/chemistry , Humans , Kinetics , Models, Chemical , Models, Molecular , Mutagenesis, Site-Directed , Plasminogen Activator Inhibitor 1/metabolism , Protein Structure, Secondary , Risk Factors
13.
J Biol Chem ; 278(26): 23899-905, 2003 Jun 27.
Article in English | MEDLINE | ID: mdl-12686544

ABSTRACT

The serpin plasminogen activator inhibitor-1 (PAI-1) plays an important role in the regulation of the fibrinolytic activity in blood. In plasma, PAI-1 circulates mainly in the active conformation. However, PAI-1 spontaneously converts to a latent conformation. This conversion comprises drastic conformational changes in both the distal and the proximal hinge region of the reactive center loop. To study the functional and conformational rearrangements associated solely with the mobility of the proximal hinge, disulfide bonds were introduced to immobilize the distal hinge region. These mutants exhibited specific activities comparable with that of PAI-1-wt. However, the engineered disulfide bond had a major effect on the conformational and associated functional transitions. Strikingly, in contrast to PAI-1-wt, inactivation of these mutants yielded a virtually complete conversion to a substrate-like conformation. Comparison of the digestion pattern (with trypsin and elastase) of the mutants and PAI-1-wt revealed that the inactivated mutants have a conformation differing from that of latent and active PAI-1-wt. Unique trypsin-susceptible cleavage sites arose upon inactivation of these mutants. The localization of these exposed residues provides evidence that a displacement of alphahF has occurred, indicating that the proximal hinge is partly inserted between s3A and s5A. In conclusion, immobilization of the distal hinge region in PAI-1 allowed the identification of an "intermediate" conformation characterized by a partial insertion of the proximal hinge region. We hypothesize that locking PAI-1 in this transition state between active and latent conformations is associated with a displacement of alphahF, subsequently resulting in substrate behavior.


Subject(s)
Plasminogen Activator Inhibitor 1/chemistry , Disulfides , Enzyme Inhibitors/chemistry , Humans , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Peptide Mapping , Protein Conformation , Protein Denaturation , Temperature , Tissue Plasminogen Activator/antagonists & inhibitors , Urokinase-Type Plasminogen Activator/antagonists & inhibitors
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