Analysis of plasminogen activation by the plasmin-staphylokinase complex in plasma of alpha2-antiplasmin-deficient mice.

Staphylokinase (SAK) expresses plasminogen activator (PA) activity by forming a complex with plasmin; this PA activity is inhibited by alpha2-antiplasmin (alpha2-AP) in plasma. However, SAK's activity is protected against inhibition by alpha2-AP in the presence of fibrin because the plasmin-SAK complex binds to fibrin. In the present study, the interaction between SAK and murine plasminogen was investigated in the plasma of alpha2-AP-deficient (alpha2-AP-/-) mice or plasminogen-deficient (Plg-/-) mice. Although the human plasmin-SAK complex was formed in equimolar mixtures of plasmin and SAK, the murine plasmin-SAK complex was not formed. Human plasminogen was activated by the human plasmin-SAK complex, although equimolar mixtures of murine plasmin and SAK did not activate murine plasminogen. These findings suggest that SAK does not react with murine plasmin. However, the murine plasminogen was activated by the human plasmin-SAK complex, although this activation was approximately 100-fold weaker than human plasminogen. Human and wild-type mouse plasminogens were not activated by the human plasmin-SAK complex in their plasma. In alpha2-AP-/- mouse plasma, murine plasminogen was activated by the human plasmin-SAK complex. Human or murine plasminogen, which had been added to Plg-/- mouse plasma, was not activated by the human plasmin-SAK complex. However, plasma clot lysis by the human plasmin-SAK complex was observed in both human and murine plasma. These findings indicate that: (1) murine plasmin does not react with SAK, (2) human plasmin-SAK complex activates murine plasminogen, (3) this activation is inhibited by murine alpha2-AP, but (4) this activation is not inhibited by murine alpha2-AP in the presence of fibrin.     

The regulation of liver regeneration by the plasmin/alpha 2-antiplasmin system

BACKGROUND/AIMS: The regeneration after liver injury is regulated by the release and activation of several growth factors. The role of the plasmin/alpha(2)-antiplasmin (alpha(2)-AP) system in liver regeneration was investigated. METHODS: CCl(4) was injected intraperitoneally into the mice deficient (-/-) in fibrinolytic factors: alpha(2)-AP-/-, plasminogen (Plg) -/-, and Plg-/-.alpha(2)-AP-/-, and wild-type (WT) mice. The liver tissue was examined for its microscopic appearance, fibrinolytic activity, and fibronectin levels. RESULTS: In the gene deficient and WT mice, the livers exhibited the same extent of necrosis 2 days after the CCl(4) injection. The livers of the WT mice normalized after 7 days, and the alpha(2)-AP-/- mice normalized after 5 days. In contrast, the livers of the Plg-/- and Plg-/-.alpha(2)-AP-/- mice remained in the damaged state until 14 days after the liver injury. The injection of anti-alpha(2)-AP antibody in the WT mice improved the regeneration after the liver injury, and the injection of tranexamic acid in the alpha(2)-AP-/- mice reduced. CONCLUSIONS: These results suggest that the plasmin/alpha(2)-AP system played an important role in hepatic repair via clearance from the injury area.     

Lack of alpha 2-antiplasmin promotes re-endothelialization via over-release of VEGF after vascular injury in mice

We here report that the arterial blood flow after endothelial injury in mice deficient in alpha 2-antiplasmin (alpha 2-AP-/- mice) was well maintained compared with that of wild-type mice. Moreover, the development of neointima 4 weeks after injury in alpha 2-AP-/- mice was significantly decreased. Histologic observations showed a prompt recovery of endothelial cells with a much higher proliferating index in repaired endothelium in alpha 2-AP-/- mice. The amount of secreted vascular endothelial growth factor (VEGF) by explanted vascular smooth muscle cells (SMCs) from alpha 2-AP-/- mice was significantly increased. In separate experiments using a human endothelial cell (EC) line, we could demonstrate that plasminogen binds to ECs and that this binding can be prevented by alpha 2-AP. Finally, an injection of either an anti-VEGF receptor-1 antibody or alpha 2-AP reduced the prompt endothelial healing. alpha 2-AP is the main inactivator of plasmin, which cleaves extracellular matrix-bound VEGF to release a diffusible proteolytic fragment. Lack of alpha 2-AP, therefore, could lead to a local over-release of VEGF by the continuously active plasmin in the injured area, which could result in a prompt re-endothelialization after vascular injury. Our results provide new insight into the role of alpha 2-AP and VEGF in the pathogenesis of re-endothelialization following vascular injury.     

Vascular release of plasminogen activator inhibitor-1 impairs fibrinolysis during acute arterial thrombosis in mice

The role of plasminogen activator inhibitor-1 (PAI-1) in the plasma, blood platelets, and vessel wall during acute arterial thrombus formation was investigated in gene-deficient mice. Photochemically induced thrombosis in the carotid artery was analyzed via transillumination. In comparison to thrombosis in C57BL/6J wild-type (wt) mice (113 +/- 19 x 10(6) arbitrary light units [AU] n = 15, mean +/- SEM), thrombosis in PAI-1(-/-) mice (40 +/- 10 x 10(6) AU, n = 13) was inhibited (P <.01), indicating that PAI-1 controls fibrinolysis during thrombus formation. Systemic administration of murine PAI-1 into PAI-1(-/-) mice led to a full recovery of thrombotic response. Occurrence of fibrinolytic activity was confirmed in alpha(2)-antiplasmin (alpha(2)-AP)-deficient mice. The sizes of thrombi developing in wt mice, in alpha(2)-AP(+/-) and alpha(2)-AP(-/-) mice were 102 +/- 35, 65 +/- 8.1, and 13 +/- 6.1 x 10(6) AU, respectively (n = 6 each) (P <.05), compatible with functional plasmin inhibition by alpha(2)-AP. In contrast, thrombi in wt mice, t-PA(-/-) and u-PA(-/-) mice were comparable, substantiating efficient inhibition of fibrinolysis by the combined PAI-1/alpha(2)-AP action. Platelet depletion and reconstitution confirmed a normal thrombotic response in wt mice, reconstituted with PAI-1(-/-) platelets, but weak thrombosis in PAI-1(-/-) mice reconstituted with wt platelets. Accordingly, murine (wt) PAI-1 levels in platelet lysates and releasates were 0.43 +/- 0.09 ng/10(9) platelets and plasma concentrations equaled 0.73 +/- 0.13 ng/mL. After photochemical injury, plasma PAI-1 rose to 2.9 +/- 0.7 ng/mL (n = 9, P <.01). The plasma rise was prevented by ligating the carotid artery. Hence, during acute thrombosis, fibrinolysis is efficiently prevented by plasma alpha(2)-AP, but also by vascular PAI-1, locally released into the circulation after endothelial injury.     

Effective lysis of model thrombi by a t-PA mutant (A473S) that is resistant to alpha2-antiplasmin

This study used two mutants of tissue-type plasminogen activator (t-PA) with resistance to inhibitors of fibrinolysis to define the contribution of plasminogen activator inhibitor (PAI)-1 and alpha2-antiplasmin (alpha2-AP) to the control of fibrin lysis. Wild-type t-PA was compared with KHRR296-299AAAA, which is resistant to PAI-1, and with A473S, which is resistant to alpha2-AP. We examined these forms of t-PA in model systems that are physiologically relevant. Neutralization of alpha2-AP was essential for lysis of plasma clots, irrespective of their platelet content, by either wild-type t-PA or KHRR296-299AAAA. In marked contrast, A473S lysed plasma clots without neutralization of alpha2-AP. Model thrombi, with structures similar to in vivo thrombi, were lysed slowly by wild-type t-PA; the rate and extent of lysis were enhanced by the addition of antibodies to alpha2-AP or PAI-1. A473S was more effective than wild-type t-PA without the addition of antibodies by virtue of its resistance to alpha2-AP. This resistance was remarkable, in that no complex formed between A473S t-PA and alpha2-AP, even after extended incubation, when 50% of wild-type t-PA could be converted to complex. Comparison of A473S and KHRR296-299AAAA mutants showed their similar effectiveness in lysis of platelet-rich model thrombi. Thus, PAI-1 and alpha2-AP contribute approximately equally to the inhibition of thrombus lysis. This study underlines the functional significance of alpha2-AP as a direct inhibitor of t-PA and further explains the basis of the accepted role of alpha2-AP as a regulator of fibrin persistence and thrombus resistance to lysis.     

Restoration of thrombolytic potential in plasminogen-deficient mice by bolus administration of plasminogen

Homozygous plasminogen-deficient (Plg-/-) mice had a significantly reduced thrombolytic capacity toward intravenously injected 125I-fibrin labeled plasma clots prepared from Plg-/- murine plasma (9% +/- 3% lysis after 8 hours; (mean +/- SEM, n = 6), as compared with 82% +/- 8% in wild-type mice; P < .0001). Bolus injection of 1 mg purified murine plasminogen in 10- to 17-week-old Plg-/- mice increased the plasminogen antigen and activity levels at 8 hours to normal levels (130 +/- 5 micrograms/mL). Plasminogen administration was associated with significant restoration of thrombolytic potential (64% +/- 7% spontaneous clot lysis; P < .0001 versus lysis without plasminogen injection). Bolus injection of 1 mg plasminogen in homozygous tissue- type plasminogen activator-deficient (t-PA-/-) mice doubled the plasminogen antigen and activity levels after 8 hours and increased 125I-fibrin clot lysis at 8 hours from 13% +/- 3% to 34% +/- 5% (P = .008). Fibrinogen, t-PA antigen and alpha 2-antiplasmin activity levels after 8 hours were not significantly different in the groups with or without plasminogen injection. Injection of plasminogen induced a variable increase (on average 7- to 10-fold) of PAI-1, but no correlation with the extent of spontaneous clot lysis was observed. Histopathologic examination at the end of the experiments revealed that fibrin deposition in the liver of Plg-/- mice was slightly reduced 8 hours after bolus plasminogen injection (P = .007) and markedly reduced after 24 hours (P < .0001). Plasminogen antigen levels in liver extracts were comparable with those found in wild-type mice at 8 hours (130 +/- 20 versus 110 +/- 15 ng/mg protein) and decreased to 25 +/- 3.2 ng/mg protein at 24 hours. Thus, restoration of normal plasminogen levels in Plg-/- mice normalized the thrombolytic potential toward experimentally induced pulmonary emboli, and resulted in removal of endogenous fibrin deposits within 24 hours.     

Essential role of AT1A receptor in the development of 2K1C hypertension

The aims of this study were to delineate the relative contribution of angiotensin II (ANG II) subtype 1A (AT1A) and 1B (AT1B) receptors to the development of two-kidney, one-clip (2K1C) Goldblatt hypertension in mice, to examine if increased nitric oxide synthase (NOS) activity counteracts the vasoconstrictor influences of ANG II in 2K1C hypertensive mice, and to determine the role of ANG II type 2 (AT2) receptors in 2K1C hypertension in mice. AT(1A) ANG II receptor knockout (AT1A-/-) and wild-type (AT1A+/+) mice underwent clipping of the right renal artery. Systolic blood pressure (SBP) was significantly lower in AT1A-/- compared with AT1A+/+ mice, and neither clip placement nor AT2 receptor blockade with PD 123319 (PD) altered SBP in AT1A-/- mice. A significant and sustained rise in SBP from 119+/-5 to 163+/-6 mm Hg was observed in the 2K1C AT1A+/+ mice from day 10 to day 26. Chronic PD infusion did not alter the course of hypertension in 2K1C/AT1A+/+. Acute PD infusion did not alter mean arterial pressure (MAP) in AT1A+/+, PD/AT1A+/+, 2K1C/AT1A+/+, PD/2K1C/AT1A+/+, AT1A-/-, PD/AT1A-/-, and PD/2K1C/AT1A-/- mice compared with basal levels. In contrast, acute PD infusion caused significant increases in MAP in 2K1C/AT1A-/- mice. The subsequent acute NOS inhibition caused greater increases in MAP in 2K1C/AT1A+/+ and PD/2K1C/AT1A+/+ mice than in AT1A+/+ and PD/AT1A+/+ mice. These results support the essential role of AT1A receptors in mediating 2K1C hypertension and support the hypothesis that augmented NO production serves as a counteracting system in this model of hypertension.     

The tumor spectrum in FHIT-deficient mice

Mice carrying one inactivated Fhit allele (Fhit +/- mice) are highly susceptible to tumor induction by N-nitrosomethylbenzylamine, with 100% of Fhit +/- mice exhibiting tumors of the forestomach/squamocolumnar junction vs. 25% of Fhit +/+ controls. In the current study a single N-nitrosomethylbenzylamine dose was administered to Fhit +/+, +/-, and -/- mice to compare carcinogen susceptibility in +/- and -/- Fhit-deficient mice. At 29 weeks after treatment, 7.7% of wild-type mice had tumors. Of the Fhit -/- mice 89.5% exhibited tumors (average 3.3 tumors/mouse) of the forestomach and squamocolumnar junction; half of the -/- mice had medium (2 mm diameter) to large (>2 mm) tumors. Of the Fhit +/- mice 78% exhibited tumors (average 2.4 tumors/mouse) and 22% showed medium to large tumors. Untreated Fhit-deficient mice have been observed for up to 2 years for spontaneous tumors. Fhit +/- mice (average age 21 mo) exhibit an average of 0.94 tumors of different types; Fhit -/- mice (average age 16 mo) also showed an array of tumors (average 0.76 tumor/mouse). The similar spontaneous and induced tumor spectra observed in mice with one or both Fhit alleles inactivated suggests that Fhit may be a one-hit tumor suppressor gene in some tissues.     

Alpha(2)-antiplasmin gene deficiency in mice does not affect neointima formation after vascular injury

The hypothesis that alpha(2)-antiplasmin (alpha(2)-AP), the main physiological plasmin inhibitor, plays a role in neointima formation was tested with use of a vascular injury model in wild-type (alpha(2)-AP(+/+)) and alpha(2)-AP-deficient (alpha(2)-AP(-/-)) mice. The neointimal and medial areas were similar 1 to 3 weeks after electric injury of the femoral artery in alpha(2)-AP(+/+) and alpha(2)-AP(-/-) mice, resulting in comparable intima/media ratios (eg, 0.43+/-0.12 and 0.42+/-0.11 2 weeks after injury). Nuclear cell counts in cross-sectional areas of the intima of the injured region were also comparable in arteries from alpha(2)-AP(+/+) and alpha(2)-AP(-/-) mice (78+/-19 and 69+/-8). Fibrin deposition was not significantly different in arteries of both genotypes 1 day after injury, and no mural thrombosis was detected 1 week after injury. Fibrinolytic activity in femoral arterial sections, as monitored by fibrin zymography, was higher in alpha(2)-AP(-/-) mice 1 week after injury (P<0.001) but was comparable in both genotypes 2 and 3 weeks after injury. Staining for elastin did not reveal significant degradation of the internal elastica lamina in either genotype. Immunocytochemical analysis revealed a comparable distribution pattern of alpha-actin-positive smooth muscle cells in both genotypes. These findings indicate that the endogenous fibrinolytic system of alpha(2)-AP(+/+) mice is capable of preventing fibrin deposition after vascular injury and suggest that alpha(2)-AP does not play a major role in smooth muscle cell migration and neointima formation in vivo.     

Vitronectin inhibits the thrombotic response to arterial injury in mice

Vitronectin (VN) binds to plasminogen activator inhibitor-1 (PAI-1) and integrins and may play an important role in the vascular response to injury by regulating fibrinolysis and cell migration. However, the role of VN in the earliest response to vascular injury, thrombosis, is not well characterized. The purpose of this study was to test the hypothesis that variation in vitronectin expression alters the thrombotic response to arterial injury in mice. Ferric chloride (FeCl3) injury was used to induce platelet-rich thrombi in mouse carotid arteries. Wild-type (VN +/+, n = 14) and VN-deficient (VN -/-, n = 15) mice, matched for age and gender, were studied. Time to occlusion after FeCl3 injury was determined by application of a Doppler flowprobe to the carotid artery. Occlusion times of VN -/- mice were significantly shorter than those of VN +/+ mice (6.0 +/- 1.2 minutes v 17.8 +/- 2.3 minutes, respectively, P < .001). Histologic analysis of injured arterial segments showed that thrombi from VN +/+ and VN -/- mice consisted of dense platelet aggregates. In vitro studies of murine VN +/+ and VN -/- platelets showed no significant differences in ADP-induced aggregation, but a trend towards increased thrombin-induced aggregation in VN -/- platelets. Purified, denatured VN inhibited thrombin-induced platelet aggregation, whereas native VN did not. Thrombin times of plasma from VN -/- mice (20.5 +/- 2.1 seconds, n = 4) were significantly shorter than those of VN +/+ mice (34.2 +/- 6.7 seconds, n = 4, P < .01), and the addition of purified VN to VN -/- plasma prolonged the thrombin time into the normal range, suggesting that VN inhibits thrombin-fibrinogen interactions. PAI-1-deficient mice (n = 6) did not demonstrate significantly enhanced arterial thrombosis compared with wild-type mice (n = 6), excluding a potential indirect antithrombin function of VN mediated by interactions with PAI-1 as an explanation for the accelerated thrombosis observed in VN -/- mice. These results suggest that vitronectin plays a previously unappreciated antithrombotic role at sites of arterial injury and that this activity may be mediated, at least in part, by inhibiting platelet-platelet interactions and/or thrombin procoagulant activity.     

Lack of alpha2-antiplasmin promotes pulmonary heart failure via overrelease of VEGF after acute myocardial infarction

Identification of a novel therapy for prevention of sudden death by ischemic cardiac infarction is an area of intensive investigation. We here report that the mortality due to an experimental acute myocardial infarction (AMI) was markedly increased in mice deficient in alpha2-antiplasmin (alpha2-AP(-/-) mice) but not in mice deficient in other components acting in fibrinolysis (tissue-type PA, urokinase type PA, or plasminogen activator inhibitor-1) even if the infarct area in alpha2-AP(-/-) mice was not different from those in the other mice. Echocardiography showed in alpha2-AP(-/-) mice after AMI an overload of the right ventricle and that pulmonary permeability was increased. According to the experiments using explanted myocytes and vascular smooth muscle cells, it was found that the amount of secreted vascular endothelial cell growth factor (VEGF) in alpha2-AP(-/-) mice was markedly increased compared with that in wild-type mice. Finally, an injection of an anti-VEGF antibody decreased the mortality after AMI in alpha2-AP(-/-) mice. Plasmin cleaves extracellular matrix-bound VEGF to release a diffusible proteolytic fragment and is inactivated mainly by alpha2-AP. Therefore, lack of alpha2-AP could markedly result in overrelease of VEGF by the continuous activation of plasmin because of AMI and could result in an acute cor pulmonale. Our results provide new aspects on the role of alpha2-AP and VEGF in the pathogenesis of cardiac events.     

Effects of loss of classical estrogen response element signaling on bone in male mice

The role of estrogen signaling in the male skeleton via estrogen receptor (ER)-alpha is now well established. ERalpha can elicit responses through either classical estrogen response elements (ERE) pathways or nonclassical, non-ERE pathways. In the present study, we examined the effects of either the attenuation or loss of classical ERalpha signaling on the murine male skeleton. To accomplish this, we crossed male mice heterozygous for a knock-in mutation [nonclassical ERalpha knock-in (NERKI)], which abolishes the ERE-mediated pathway with female heterozygous ERalpha knockout mice (ERalpha+/-) and studied the F1 generation ERalpha+/+, ERalpha+/-, ERalpha+/NERKI, and ERalpha-/NERKI male progeny longitudinally using bone density and histomorphometry. The only ERalpha allele present in ERalpha-/NERKI mice is incapable of classical ERE-mediated signaling, whereas the heterozygous ERalpha+/NERKI mice have both one intact ERalpha and one NERKI allele. As compared with ERalpha+/+ littermates (n=10/genotype), male ERalpha+/NERKI and ERalpha-/NERKI mice displayed axial and appendicular skeletal osteopenia at 6, 12, 20, and 25 wk of age, as demonstrated by significant reductions in total bone mineral density (BMD) at representative sites (areal BMD by dual-energy x-ray absorptiometry at the lumbar vertebrae and femur and volumetric BMD by peripheral quantitative computed tomography at the tibia; P<0.05-0.001 vs. ERalpha+/+). The observed osteopenia in these mice was evident in both trabecular and cortical bone compartments. However, these decreases were more severe in mice lacking classical ERalpha signaling (ERalpha-/NERKI mice), compared with mice in which one wild-type ERalpha allele was present (ERalpha+/NERKI mice). Collectively, these data demonstrate that classical ERalpha signaling is crucial for the development of the murine male skeleton.     

Depletion of circulating alpha(2)-antiplasmin by intravenous plasmin or immunoneutralization reduces focal cerebral ischemic injury in the absence of arterial recanalization

In the absence of arterial recanalization, thrombolytic agents induce a dose-related extension of focal cerebral ischemic injury (FII) in experimental animals. However, FII is smaller in mice lacking alpha(2)-antiplasmin (alpha(2)-AP), the physiologic inhibitor of plasmin, suggesting its depletion might reduce FII in the absence of reperfusion. Therefore, the effect of human plasmin (Pli), human miniplasmin (mPli), and an Fab fragment neutralizing murine alpha(2)-AP (Fab-4H9) on FII after middle cerebral artery (MCA) ligation was studied in mice and in hamsters. In BALB/c mice, the median FII after 24 hours was 28 microL (range, 20-34) (n = 10) with saline and 23 microL (range, 17-26) (n = 9) with a single bolus of 0.07 mg Pli, given after MCA ligation (P =.010), which reduced alpha(2)-AP to 44% and fibrinogen from 0.75 to 0.44 g/L. FII was 20 microL (range, 13-26) (n = 6, P =.025) with 0.2 mg mPli and was 24 microL (range, 20-27) (n = 6, P =.020) with 1.7 mg Fab-4H9. Neuronal atrophy and reduction of laminin immunoreactivity were comparably observed in the infarct area after saline and Pli. In hamsters, a single bolus injection of 1 mg Pli, after MCA ligation, depleted alpha(2)-AP and fibrinogen and reduced FII at 24 hours from 20 microL (range, 9.9-38) (n = 6) to 7.0 microL (range, 0.44-31) (n = 7, P =.032). Thus, reduction of circulating alpha(2)-AP, with a single bolus of plasmin or of a neutralizing antibody fragment, significantly reduced FII after MCA ligation in mouse and hamster models, suggesting that, provided these observations can be extrapolated to human beings, transient depletion of circulating alpha(2)-AP might reduce ischemic stroke in the absence of reperfusion.     

Mouse carotid artery ligation induces platelet-leukocyte-dependent luminal fibrin, required for neointima development

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 alpha(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.     

Analysis of the role of the SA gene in blood pressure regulation by gene targeting

The SA gene is expressed in the proximal tubule of the kidney and may be involved in blood pressure (BP) regulation. However, direct evidence for this is lacking. We constructed and analyzed an SA-null mouse in which exons 2 and 3 of the SA gene (including the start codon) had been deleted by homologous recombination. Basal BP and BP changes in response to increased salt and to treatment with losartan were compared between mice homozygous for the targeted SA allele (SA-/- mice) and littermates carrying the wild-type allele (SA+/+ mice). Molecular and biochemical analysis confirmed the lack of SA gene product in SA-/- mice. SA-/- mice grew normally, were fertile, and had no overt phenotype. With both indirect and direct techniques, basal BP was similar in SA-/- and SA+/+ mice. A high salt diet for 4 weeks caused a significant increase in BP in SA-/- and SA+/+, mice but there was no difference between the 2 strains. Losartan caused a significant decrease in BP, but again the response was similar between SA-/- and SA+/+ mice, as were their kidney renin mRNA levels. SA is not involved in the regulation of either basal or salt related BP, and the lack of differential effect in SA-/- mice is not a consequence of compensatory activation of the renin-angiotensin system.     

肝硬化高同型半胱氨酸血症与MTHFR基因C667T多态性的关系

目的研究肝硬化血浆同型半胱氨酸(HCY)水平及其与N5,N10-亚甲基四氢叶酸还原酶(MTHFR)基因多态性的关系.方法采用柱前衍生化-HPLC方法检测112例健康对照者、87例肝硬化患者血浆同型半胱氨酸的水平,用多聚酶链反应-限制性内切酶片断长度多态性技术(PCR-RFLP)检测其MTHFR基因C667T多态性.结果健康对照组平均血浆HCY浓度为(8.34±3.59)μmol/L,肝硬化组平均血浆HCY浓度为(21.71±4.85)μmol/L.与健康对照组相比,肝硬化组血浆HCY水平显著升高,差异有统计学意义(P＜0.01).PCR-RFLP检测结果发现MTHFR基因型有3种,即纯合子突变TT(+/+)型,杂合子突变TC(+/)型,正常CC(-/-)型.肝硬化组中+/+型、+/型和/-型频率分别为29.9%、52.9%、17.2%;健康对照组分别为19.6%、33.9%、46.4%,两组差异有统计学意义.肝硬化组MTHFR基因突变无论是纯合子还是杂合子突变基因型,其血浆HCY水平均明显高于正常基因型.结论高同型半胱氨酸血症可能是肝硬化的一个危险因素,血浆HCY水平可作为肝硬化的一个辅助诊断指标,MTHFR基因C667T多态性可能是肝硬化高同型半胱氨酸血症的易感基因之一.     

Role of the alpha1D-adrenergic receptor in the development of salt-induced hypertension

In an attempt to elucidate whether there is a specific alpha1-adrenergic receptor (alpha1-AR) subtype involved in the genesis or maintenance of hypertension, the alpha1D-AR subtype was evaluated in a model of salt-induced hypertension. The alpha1D-AR-deficient (alpha1D-/-) and control (alpha1D+/+) mice (n=8 to 14 in each group) were submitted to subtotal nephrectomy and given 1% saline as drinking water for 35 days. Blood pressure (BP) was monitored by tail-cuff readings and confirmed at the end point by direct intraarterial BP recording. The alpha1D-/- mice had a significantly (P=0.0004) attenuated increase in BP response in this protocol (baseline 94.6+/-2.8 versus end point 107.4+/-4.5 mm Hg) compared with that of their wild-type counterparts (alpha1D+/+), from a baseline 97.4+/-2.9 to an end point 139.4+/-4.5 mm Hg. Seven of 15 alpha1D+/+ mice died with edema, probably owing to renal failure, whereas 14 of 15 alpha1D-/- mice were maintained for 35 days. Body weight, renal remnant weight, and residual renal function were similar in the 2 groups, whereas the values of plasma catecholamines (epinephrine, norepinephrine, and dopamine) were higher in alpha1D+/+ than in the alpha1D-/- mice. These data suggest that alpha1D-AR plays an important role in developing a high BP in response to dietary salt-loading, and that agents having selective alpha1D-AR antagonism could have significant therapeutic potential in the treatment of hypertension.     

Hepatic low-density lipoprotein receptor-related protein deficiency in mice increases atherosclerosis independent of plasma cholesterol

The low-density lipoprotein (LDL) receptor-related protein (LRP) has a well-established role in the hepatic removal of atherogenic apolipoprotein E (APOE)-rich remnant lipoproteins from plasma. In addition, LRP recognizes multiple distinct pro- and antiatherogenic ligands in vitro. Here, we investigated the role of hepatic LRP in atherogenesis independent of its role in removal of APOE-rich remnant lipoproteins. Mice that allow inducible inactivation of hepatic LRP were combined with LDL receptor and APOE double-deficient mice (MX1Cre(+)LRP(flox/flox)LDLR(-/-)APOE(-/-)). On an LDLR(-/-)APOE(-/-) background, hepatic LRP deficiency resulted in decreased plasma cholesterol and triglycerides (cholesterol: 17.1 +/- 5.2 vs 23.4 +/- 6.3 mM, P =.025; triglycerides: 1.1 +/- 0.5 vs 2.2 +/- 0.8 mM, P =.002, for MX1Cre(+)LRP(flox/flox)-LDLR(-/-)APOE(-/-) and control LRP(flox/flox)-LDLR(-/-)APOE(-/-) mice, respectively). Lower plasma cholesterol in MX1Cre(+)LRP(flox/flox)-LDLR(-/-)APOE(-/-) mice coincided with increased plasma lipoprotein lipase (71.2 +/- 7.5 vs 19.1 +/- 2.4 ng/ml, P =.002), coagulation factor VIII (4.4 +/- 1.1 vs 1.9 +/- 0.5 U/mL, P =.001), von Willebrand factor (2.8 +/- 0.6 vs 1.4 +/- 0.3 U/mL, P =.001), and tissue-type plasminogen activator (1.7 +/- 0.7 vs 0.9 +/- 0.5 ng/ml, P =.008) compared with controls. Strikingly, MX1Cre(+)LRP(flox/flox)LDLR(-/-)APOE(-/-) mice showed a 2-fold higher atherosclerotic lesion area compared with controls (408.5 +/- 115.1 vs 219.1 +/- 86.0 10(3)microm(2), P =.003). Our data indicate that hepatic LRP plays a clear protective role in atherogenesis independent of plasma cholesterol, possibly due to maintaining low levels of its proatherogenic ligands.     

Increased atherosclerosis in mice lacking apolipoprotein A-I attributable to both impaired reverse cholesterol transport and increased inflammation

To test the hypothesis that apolipoprotein A-I (apoA-I) functions specifically to inhibit atherosclerosis independent of the level of high-density lipoprotein cholesterol (HDL-C) by promoting both reverse cholesterol transport and HDL antiinflammatory function in vivo, we established a murine atherosclerosis model of apoA-I deficiency in which the level of HDL-C is well maintained. ApoA-I-/- mice were crossed with atherosclerosis susceptible low-density lipoprotein receptor-/-/apobec-/- (LA) mice to generate LA mice with apoA-I+/+, apoA-I+/-, and apoA-I-/- genotypes. There were no major differences in the amounts of non-HDL-C and HDL-C in the plasma between different apoA-I genotypes. A significant inverse relationship was observed, however, between apoA-I gene dose and atherosclerosis in both female and male mice. Compared with LA-apoA-I+/+ mice, serum from LA-apoA-I-/- mice had a significantly reduced capacity to function as an acceptor of ABCA1- and SR-BI-mediated cellular cholesterol efflux, and also had markedly reduced lecithin cholesterol acyltransferase activity. In addition, LA-apoA-I-/- mice had significantly reduced macrophage-derived cholesterol esterification and reverse cholesterol transport in vivo. There was significantly reduced plasma paraoxonase (PON-1) activity, impaired HDL vascular antiinflammatory function, and increased basal levels of monocyte chemotactic protein-1 in the plasma of LA-apoA-I-/- mice compared with LA-apoA-I+/+ mice. In LA-apoA-I-/- mice, there was also greater induction of some, but not all, inflammatory cytokines and chemokines in response to intraperitoneal injection of lipopolysaccharide than in LA-apoA-I+/+ mice. We conclude that apoA-I inhibits atherosclerosis by promoting both macrophage reverse cholesterol transport and HDL antiinflammatory function, and that these anti-atherogenic functions of apoA-I are largely independent of the plasma level of HDL-C in this mouse model.     

Prevention of lymphocytic thyroiditis in iodide-treated non-obese diabetic mice lacking interferon regulatory factor-1

OBJECTIVE: Interferon regulatory factor-1 (IRF-1) is a critical regulator of interferon-gamma(IFNgamma)-mediated immune responses. To determine whether IRF-1 is involved in the pathogenesis of thyroiditis in animal models, we evaluated the incidence of iodide-induced lymphocytic thyroiditis (LT) in non-obese diabetic (NOD) mice lacking IRF-1 as well as IRF-1 +/+ and +/- mice. DESIGN: IRF-1 +/+, +/- and -/- NOD mice at 6 weeks of age were fed water (group 1) or iodide water (group 2) for 8 weeks. METHODS: Thyroids were examined histopathologically and intrathyroidal lymphocytic infiltration was arbitrarily graded. Serum thyroxine (T(4)) and anti-mouse thyroglobulin antibody (anti-mTgAb) levels were measured. Spleen cell population was analyzed by flow cytometry, and IFNgamma and interleukin-10 produced by splenocytes were measured by enzyme-linked immunosorbent assay. RESULTS: In group 1, only 4.3% of NOD mice developed LT. In contrast, 67.6% of mice in group 2 developed the disease. Iodide treatment induced LT in more than 80% of IRF-1 +/+ and +/- mice. However, no IRF-1 -/- mice in group 2 developed LT. There was no difference in both serum anti-mTgAb and T(4) levels among the three IRF-1 genotypes of NOD mice. Numbers of splenic CD8(+) T cells and IFNgamma production by Concanavalin A-stimulated splenocytes were markedly decreased in IRF-1-deficient NOD mice. CONCLUSIONS: IRF-1 is involved in the development of iodide-induced LT in NOD mice.