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PubMed2003

Failure of thrombus to resolve in urokinase-type plasminogen activator gene-knockout mice: rescue by normal bone marrow-derived cells.

BACKGROUND: Monocytes may have an important role in the resolution of venous thrombosis. Increased expression of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) is associated with an ingress of monocytes into the thrombus. This study was designed to evaluate the importance of these activators in thrombus resolution. METHODS AND RESULTS: Inferior caval vein thrombosis was induced in cohorts of adult wild-type, uPA gene-knockout (uPA-/-), and tPA gene-knockout (tPA-/-) mice in a flow model. Thrombi were harvested from wild-type and uPA-/- mice (n=60 per group) between 1 and 60 days. Thrombi were also obtained from groups of wild-type and tPA-/- mice (n=24 per group) between 1 and 28 days. Thrombus size and macrophage content were measured by computer-assisted image analysis. Thrombus resolution was significantly impaired in the uPA-/- mice compared with wild-type controls (P<0.0001) but was unaffected in tPA-/- mice. Monocyte content in wild-type mice was highest at 14 days after thrombus induction and was approximately 4 times greater than in uPA-/- mice (P=0.0043). Thrombus size in uPA-/- mice transplanted with wild-type marrow (0.29+/-0.06 mm2) was significantly smaller than in uPA-/- mice given uPA-/- bone marrow (3.9+/-1.1 mm2) (P=0.0022). Donor bone marrow-derived cells expressing LacZ were present in the thrombus after transplantation. CONCLUSIONS: The resolution of experimental venous thrombus is dependent on uPA but is unaffected by the absence of tPA. Absence of uPA is also associated with delayed monocyte recruitment into the thrombus. Transplanting wild-type bone marrow restores thrombus resolution in uPA-/- animals, suggesting an important role for bone marrow-derived cells in this process.

Animals↗

PubMed2003

Decreased atherosclerotic lesion formation in CX3CR1/apolipoprotein E double knockout mice.

BACKGROUND: Fractalkine (CX3CL1), a CX3C chemokine, is expressed in the vessel wall and mediates the firm adhesion and chemotaxis of leukocytes expressing its receptor, CX3CR1. A polymorphism in the CX3CR1 gene is associated with low CX3CR1 expression and reduced risk of acute coronary disease in humans. METHODS AND RESULTS: We generated CX3CR1-deficient mice (CX3CR1(-/-)) by targeted gene disruption and crossed them with the proatherogenic apolipoprotein E-deficient mice (apoE(-/-)). Here we show that the extent of lipid-stained lesions in the thoracic aorta was reduced by 59% in CX3CR1/apoE double knockout mice compared with their CX3CR1(+/+)/apoE(-/-) littermates. The development of atherosclerosis in the aortic sinus was also markedly altered in the double knockout mice, with 50% reduction in macrophage accumulation. Although lesions of CX3CR1(-/-) mice were smaller in size, they retained a substantial accumulation of smooth muscle cells and collagen, features consistent with a stable plaque phenotype. Finally, CX3CR1(+/-)/apoE(-/-) mice showed the same reduction in atherosclerosis as the CX3CR1(-/-)/apoE(-/-) mice. CONCLUSIONS: The CX3CR1-CX3CL1 pathway seems to play a direct and critical role in monocyte recruitment and atherosclerotic lesion development in a mouse model of human atherosclerosis.

Alleles↗

PubMed2003

Cyclooxygenase isoforms and platelet vessel wall interactions in the apolipoprotein E knockout mouse model of atherosclerosis.

BACKGROUND: Cyclooxygenase (COX) activity is induced in human atherosclerosis, and the products formed may modify the disease directly or through an effect on platelets. We examined the role of COX-1 and -2 on platelet vessel wall interactions and development of atherosclerosis in a murine model. METHODS AND RESULTS: Apolipoprotein E-deficient (apoE-/-) mice fed a 1% cholesterol diet were treated with a selective COX-1 inhibitor (SC-560), a selective COX-2 inhibitor (SC-236), or vehicle. Urinary prostacyclin and thromboxane metabolites (2,3-dinor-6-keto-PGF1alpha and 2,3-dinor-TXB2) were increased in the apoE-/- knockout mouse. There was also induction of both COX isoforms in the vascular lesions formed, which stained for CD41, a platelet-specific marker, and for CD40L. Selective inhibition of COX-2 had no effect on lesion formation and, despite selective reduction in prostacyclin generation, had no effect on platelet activity, as measured by thromboxane formation or platelet deposition. Selective inhibition of COX-1 reduced 2,3-dinor-TXB2 generation and lesion formation. However, platelet deposition on the vessel wall persisted, with well-defined monolayers seen. There was also persistent expression of the macrophage marker CD68 and increased expression of the cell death protein Bax. In contrast to lesion development, the selective COX-1 inhibitor had no effect on the regression of evolving lesions. CONCLUSIONS: COX-1 plays an important role in the early stages of lesion development in the apoE-/- knockout model of atherosclerosis, preventing gross lesion formation in the face of continued vascular injury and inflammation. Despite the inhibition of prostacyclin, COX-2 inhibition had no effect on lesion development or platelet-vessel wall interactions.

Animals↗

PubMed2000

Inducible nitric oxide synthase mediates delayed myocardial protection induced by activation of adenosine A(1) receptors: evidence from gene-knockout mice.

BACKGROUND: The mechanism of delayed preconditioning induced by activation of adenosine A(1) receptors (A(1)ARs) is not fully understood. We determined the role of inducible nitric oxide synthase (iNOS) in mediating adenosine-induced late cardioprotection using pharmacological inhibitors and iNOS gene-knockout mice. METHODS AND RESULTS: Adult male mice were treated with saline or an A(1)AR agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA). Twenty-four hours later, the hearts were perfused in Langendorff mode and subjected to 30 minutes of global ischemia followed by 30 minutes of reperfusion. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX; 0.1 mg/kg IP) and S-methylisothiourea (SMT; 3 mg/kg IP) were used to block A(1)ARs and iNOS, respectively. Infarct size (IS) was measured by triphenyltetrazolium chloride staining, and iNOS expression was measured by Western blots. Myocardial IS was reduced from 24.0+/-3. 2% in the saline group to 12.2+/-2.5% in CCPA-treated mice (P<0.05). The infarct-reducing effect of CCPA was abrogated by DPCPX (29.3+/-3. 4%) and SMT (32.3+/-2.6%) and was absent in mice with targeted ablation of iNOS (23.9+/-1.6%). CCPA produced improvement in postischemic end-diastolic pressure, developed pressure, and rate-pressure product, which was also blocked by DPCPX and SMT. Increased iNOS protein expression observed in CCPA-treated hearts was diminished by DPCPX. CONCLUSIONS: Selective activation of A(1)ARs produces delayed cardioprotection against ischemia/reperfusion injury in the mouse. Increased iNOS expression concomitant with the lack of protective effect of A(1)AR activation in iNOS gene-knockout mice suggests a direct cause-and-effect relationship of iNOS in adenosine-induced late cardioprotection.

Adenosine↗

PubMed2001

Double-outlet right ventricle and overriding tricuspid valve reflect disturbances of looping, myocardialization, endocardial cushion differentiation, and apoptosis in TGF-beta(2)-knockout mice.

BACKGROUND: Transforming growth factor-beta(2) (TGF-beta(2)) is a member of a family of growth factors with the potential to modify multiple processes. Mice deficient in the TGF-beta(2) gene die around birth and show a variety of defects of different organs, including the heart. METHODS AND RESULTS: We studied the hearts of TGF-beta(2)-null mouse embryos from 11.5 to 18.5 days of gestation to analyze the types of defects and determine which processes of cardiac morphogenesis are affected by the absence of TGF-beta(2). Analysis of serial sections revealed malformations of the outflow tract (typically a double-outlet right ventricle) in 87.5%. There was 1 case of common arterial trunk. Abnormal thickening of the semilunar valves was seen in 4.2%. Associated malformations of the atrioventricular (AV) canal were found in 62.5% and were composed of perimembranous inlet ventricular septal defects (37.5%), AV valve thickening (33.3%), overriding tricuspid valve (25.0%), and complete AV septal defects (4.2%). Anomalies of the aorta and its branches were seen in 33.3%. Immunohistochemical staining showed failure of myocardialization of the mesenchyme of the atrial septum and the ventricular outflow tract as well as deficient valve differentiation. Morphometry documented this to be associated with absence of the normal decrease of total endocardial cushion volume in the older stages. Apoptosis in TGF-beta(2)-knockout mice was increased, although regional distribution was normal. CONCLUSIONS: TGF-beta(2)-knockout mice exhibited characteristic cardiovascular anomalies comparable to malformations seen in the human population.

Animals↗

PubMed2001

Genetic deficiency of inducible nitric oxide synthase reduces atherosclerosis and lowers plasma lipid peroxides in apolipoprotein E-knockout mice.

BACKGROUND: Inducible nitric oxide synthase (iNOS) is expressed by leukocytes and smooth muscle cells in atherosclerotic lesions. To test whether NO produced by iNOS deficiency affects atherosclerosis, we studied apoE/iNOS-double knockout (dKO) and apoE-knockout (KO) control animals fed a "Western-type" diet. METHODS AND RESULTS: After 16 weeks of Western-type diet, the aortic lesion area in apoE/iNOS-dKO males and females was significantly reduced, by 22% and 21%, respectively, compared with apoE-KO males and females. This effect was more pronounced after 24 weeks of Western-type diet, after which lesion formation in male and female dKO mice was reduced by 38% and 40%, respectively. Plasma levels of lipoperoxides in apoE/iNOS-dKO mice (2.0+/-0.23 micromol/L) were significantly lower than in apoE-KO control animals (3.2+/-0.44 micromol/L; P=0.02). To test whether substrate deficiency plays a role in the proatherogenic actions of iNOS, we administered L-arginine to apoE-KO animals for 16 and 24 weeks. L-Arginine treatment did not affect lesion formation in apoE-KO animals fed a Western-type diet. CONCLUSIONS: Genetic deficiency of iNOS decreases diet-induced atherosclerosis and lowers plasma levels of lipoperoxides, a marker for oxidative stress, in apoE-KO animals. Reduction in iNOS-mediated oxidative stress could partly explain protection from lesion formation in dKO animals. L-Arginine supplementation did not change lesion area in apoE-KO mice, indicating that substrate deficiency is not a likely cause for iNOS-mediated injury in this model of atherosclerosis.

Animals↗

PubMed2001

Key role of the P2Y(1) receptor in tissue factor-induced thrombin-dependent acute thromboembolism: studies in P2Y(1)-knockout mice and mice treated with a P2Y(1) antagonist.

BACKGROUND: ADP plays a key role in hemostasis, acting through 2 platelet receptors: the P2Y(1) receptor and an unidentified P2 receptor, called P2cyc, coupled to adenylyl cyclase inhibition, which is the target of the antiplatelet drug clopidogrel. We showed that the P2Y(1) receptor is an essential cofactor in thrombotic states induced by intravenous infusion of collagen and epinephrine. The aim of the present study was to assess the role of this receptor in thrombin-dependent tissue factor-induced thromboembolism. METHODS AND RESULTS: Human thromboplastin was injected intravenously into wild-type or P2Y(1)-deficient mice, and the effects on platelet count and mortality were determined and plasma thrombin-antithrombin III (TAT) complexes were quantified. P2Y(1)-deficient mice were resistant to the thromboembolism induced by injection of thromboplastin. Whereas the platelet count decreased sharply in wild-type mice, there was no significant drop in platelets in P2Y(1)-knockout mice. The platelet consumption in wild-type mice was probably due to thrombin generation, because it was abolished by hirudin. Thromboplastin also led to a rise in TAT complexes in plasma, again reflecting thrombin formation. This effect, however, was less important in P2Y(1)-knockout mice than in wild-type mice, indicating that less thrombin was generated in the absence of P2Y(1). Similar results were obtained after intravenous administration of N:(6)-methyl-2'-deoxyadenosine-3':5'-bisphosphate, a selective antagonist of the P2Y(1) receptor, to wild-type mice. CONCLUSIONS: Our results demonstrate a role of the P2Y(1) receptor in thrombotic states involving thrombin generation and provide further evidence for the potential relevance of this receptor as a target for antithrombotic drugs.

Acute Disease↗

PubMed1997

Tamoxifen decreases cholesterol sevenfold and abolishes lipid lesion development in apolipoprotein E knockout mice.

BACKGROUND: Apolipoprotein E (apo E) knockout mice develop severe vascular lipid lesions resembling human atherosclerotic plaques, irrespective of the fat content of their diet. METHODS AND RESULTS: Oral tamoxifen (TMX) at a dose of 1.9 mg.kg body wt-1.d-1 abolished lipid lesion development, assayed by oil red O staining, whether the mice were fed a normal diet or a diet with high fat content. The TMX-treated mice showed a sevenfold decrease in total cholesterol. However, the proportion of plasma cholesterol present in VLDL remained unchanged, whereas the proportion in LDL decreased by 37%, and that in HDL increased by 64%. Consistent with the shift from LDL to HDL cholesterol, there was a 62% decrease in total triglycerides. The concentrations of active and acid-activatable latent plus active TGF-beta in the aorta were substantially elevated by TMX (87% and 24% increase, respectively). CONCLUSIONS: Although the mechanism of cardiovascular protection by TMX in apo E knockout mice is unknown, the inhibition of lipid lesion formation may be attributable to the changes in lipoprotein profile and the elevated levels of TGF-beta, both of which are thought to be protective against atherosclerosis in humans and animal models.

Animals↗

PubMed2003

Cardiac hypertrophy and microvascular deficit in kinin B2 receptor knockout mice.

Experimental and clinical evidence suggests kinin involvement in adaptive myocardial growth. Kinins are growth-inhibitory to cardiomyocytes. Knockout of kinin B2 receptor (B2R) signaling causes dilated and failing cardiomyopathy in 129/J mice, and a 9-bp deletion polymorphism of human B2R is associated with reduced receptor expression and exaggerated left ventricular growth response to physical stress. We reasoned that genetic background and aging may significantly influence the impact of B2R mutation on cardiac phenotype. The theory was challenged in C57BL/6 mice, a strain that naturally differs from the 129/J strain, carrying 1 instead of 2 renin genes. C57BL/6 B2R knockouts (B2R-KO) showed higher blood pressure and heart rate levels (P<0.05) compared with wild-type controls (WT) at all ages examined. At 12 months, left ventricular contractility and diastolic function were mildly altered (P<0.05) and histological and morphological analyses revealed ventricular hypertrophy and cardiomyocyte enlargement in B2R-KO (P<0.01). Reparative fibrosis was enhanced by 208% and capillary density reduced by 38% (P<0.01). Functional and structural alterations induced by B2R deletion in C57BL/6 mice were less severe than those reported previously in the 129/J strain. We conclude that interaction of B2R signaling with other genetic determinants influences aging-related changes in myocardial structure and function. These findings may help us understand the role of kinins in the development of cardiac failure.

Animals↗

PubMed1993

Double knockouts. Production of mutant cell lines in cardiovascular research.

Double knockouts by homologous recombination is a method for producing cell lines with an inactivating mutation in any desired gene. The biochemical analysis of genetically altered cell lines has been important in determining the function of specific proteins. Until recently, mutant cell lines have been produced by random mutagenesis and then selection for a particular phenotypic change. Recent technological advances in gene targeting by homologous recombination now enable the production of mutants in any desired gene. Diploid cells contain two copies or alleles of each gene encoded on an autosome (nonsex) chromosome. In most cases, both alleles must be inactivated to produce a phenotypic change in a mutant cell line, hence the term "double knockout." We and others have described the production of mutationally altered cell lines by inactivating both alleles by the production of two targeting vectors, two separate homologous recombination events, and selection. A simpler procedure, involving considerably less effort and time, has been used to inactivate several alpha-subunits of G proteins and other genes. This method facilitates the inactivation of more than one gene in a single cell line.

Alleles↗

PubMed1999

Enhanced blood pressure variability in eNOS knockout mice.

It has been shown previously that endogenous nitric oxide can buffer arterial blood pressure variability in dogs and rats. In these former studies, all isoforms of the nitric oxide synthase were blocked pharmacologically and an increased blood pressure variability was observed. Thus the question as to which isoform of the nitric oxide synthase is responsible for the blood pressure buffering effect of endogenous nitric oxide remains unraveled. In the present study, we therefore compared blood pressure variability in knockout mice that lack specifically the gene for endothelial nitric oxide synthase with their respective wild-type controls. One day after carotid artery cannulation, blood pressure was recorded in these conscious mice. During resting conditions, blood pressure variability was markedly enhanced in knockout mice compared with wild-type mice (10.5+/-1.5 mm Hg2 vs 6.0+/-0.8 mm Hg2, P<0.05). Power spectral analysis revealed that this increase in blood pressure variability is manifested at low frequencies that range from 0.05 to 0.40 s-1 (Hz) (5.1+/-1.0 mm Hg2 vs 2.5+/-0.5 mm Hg2, P<0.05). On the basis of these results, we conclude that the blood pressure buffering effect of endogenous nitric oxide is mediated by the endothelial isoform of the nitric oxide synthase. In addition, endothelial nitric oxide is most effective in buffering blood pressure oscillations at frequencies that range from 0.05 to 0.40 s-1 (Hz) in conscious mice.

Animals↗

PubMed2005

Excitation-contraction coupling in Na+-Ca2+ exchanger knockout mice: reduced transsarcolemmal Ca2+ flux.

Cardiac-specific Na+-Ca2+ exchanger (NCX) knockout (KO) mice surprisingly survive into adulthood without compensatory changes in protein expression levels. To determine how cardiac function is maintained in the absence of NCX, we investigated membrane currents, intracellular Ca2+, and action potentials (APs) in whole cell patch-clamped myocytes from wild-type (WT) and NCX knockout mice. There was no difference in resting Ca2+ or sarcoplasmic reticular Ca2+ load between KO and WT. During prolonged caffeine exposure, the decrease of the Ca2+ transient was drastically slowed in KO versus WT myocytes, indicating that no alternative Ca2+-extrusion mechanism is upregulated to compensate for the absence of NCX. Peak L-type Ca2+ current (ICa) was reduced by 62% in KO myocytes compared with WT. Nevertheless, the corresponding Ca2+ transients were similar, implying an increase in the gain of excitation-contraction coupling in KO cells. APs recorded from KO cells repolarized more rapidly than in WT. In WT myocytes, applying a KO AP waveform voltage clamp reduced Ca2+ influx via ICa by 59% compared with WT AP waveform clamps. Again, the corresponding Ca2+ transients remained similar. Our findings indicate that NCX KO myocytes limit Ca2+ influx to &20% of that in WT by reducing ICa and by abbreviating the AP. Contractility is maintained by an increase in the gain of excitation-contraction coupling resulting from both a more rapid repolarization of the AP and an as yet unidentified AP-independent mechanism.

Action Potentials↗

PubMed1997

beta-Arrestin1 knockout mice appear normal but demonstrate altered cardiac responses to beta-adrenergic stimulation.

beta-Arrestin1 knockout mice were studied to define the physiological role of beta-arrestin1 in the regulation of G protein-coupled receptors. beta-Arrestin1 is thought to be involved in the desensitization of many G protein-associated cell surface receptors, particularly beta-adrenergic receptors. Homozygous knockout mice are overtly normal. Resting cardiovascular parameters modulated by beta-adrenergic receptors such as heart rate, blood pressure, and left ventricular ejection fraction are not changed. However, homozygous mutants are more sensitive to beta-receptor agonist-stimulated increases in ejection fraction, consistent with a role of beta-arrestin1 in beta-adrenergic receptor desensitization. We conclude that beta-arrestin1 is important for in vivo G protein-coupled receptor desensitization and that this aspect of desensitization represents a mechanism for fine-tuning responses. However, beta-arrestin1 does not appear to be required for development or for other essential biological functions.

Animals↗

PubMed2001

Decreased flow-dependent dilation in carotid arteries of tissue kallikrein-knockout mice.

- Flow-dependent dilation is a fundamental mechanism by which large arteries ensure appropriate blood supply to tissues. We investigated whether or not the vascular kallikrein-kinin system, especially tissue kallikrein (TK), contributes to flow-dependent dilation by comparing wild-type and TK-knockout mice in which the presence or absence of TK expression was verified. We examined in vitro changes in the outer diameter of perfused carotid arteries from TK(+/+) and TK(-/-) mice. In both groups, exogenous bradykinin caused a similar dilation that was abolished by the B(2) receptor antagonist HOE-140, as well as by the NO synthase inhibitor N:(omega)-nitro-L-arginine methyl ester. However, purified kininogen dilated only TK(+/+) arteries, demonstrating the essential role of TK in the vascular formation of kinins. In TK(+/+) arteries, increasing intraluminal flow caused a larger endothelium-dependent dilation than that seen in TK(-/-). In both strains the flow response was mediated by NO and by endothelium-derived hyperpolarizing factor, whereas in TK(-/-) vasoconstrictor prostanoids participated as well. HOE-140 impaired flow-dependent dilation in TK(+/+) arteries while showing no effect in TK(-/-). This compound reduced the flow response in TK(+/+) arteries to a level similar to that in TK(-/-). After NO synthase inhibition, HOE-140 no longer affected the response of TK(+/+). Impaired flow-dependent dilation was also observed in arteries from knockout mice lacking bradykinin B(2) receptors as compared with wild-type animals. This study demonstrates the active contribution of the vascular kallikrein-kinin system to one-third of the flow-dependent dilation response via activation of B(2) receptors coupled to endothelial NO release.

Acetylcholine↗

PubMed2003

Aggravated brain damage after hypoxic ischemia in immature adenosine A2A knockout mice.

BACKGROUND AND PURPOSE: Cerebral hypoxic ischemia (HI) is an important cause of brain injury in the newborn infant. Adenosine is believed to protect against HI brain damage. However, the roles of the different adenosine receptors are unclear, particularly in young animals. We examined the role of adenosine A2A receptors (A2AR) using 7-day-old A2A knockout (A2AR(-/-)) mice in a model of HI. METHODS: HI was induced in 7-day-old CD1 mice by exposure to 8% oxygen for 30 minutes after occlusion of the left common carotid artery. The resulting unilateral focal lesion was evaluated with the use of histopathological scoring and measurements of residual brain areas at 5 days, 3 weeks, and 3 months after HI. Behavioral evaluation of brain injury by locomotor activity, rotarod, and beam-walking test was made 3 weeks and 3 months after HI. Cortical cerebral blood flow, assessed by laser-Doppler flowmetry, and rectal temperature were measured during HI. RESULTS: Reduction in cortical cerebral blood flow during HI and rectal temperature did not differ between wild-type (A2AR(+/+)) and knockout mice. In the A2AR(-/-) animals, brain injury was aggravated compared with wild-type mice. The A2AR(-/-) mice subjected to HI displayed increased forward locomotion and impaired rotarod performance in adulthood compared with A2AR(+/+) mice subjected to HI, whereas beam-walking performance was similarly defective in both groups. CONCLUSIONS: These results suggest that, in contrast to the situation in adult animals, A2AR play an important protective role in neonatal HI brain injury.

Animals↗

PubMed2001

Accelerated atherosclerosis, aortic aneurysm formation, and ischemic heart disease in apolipoprotein E/endothelial nitric oxide synthase double-knockout mice.

BACKGROUND: To test whether deficiency in endothelial nitric oxide synthase (eNOS) affects atherosclerosis development, we compared lesion formation in apolipoprotein E (apoE)/eNOS-double knockout (DKO) and apoE-knockout (KO) control animals. METHODS AND RESULTS: After 16 weeks of "Western-type" diet, apoE/eNOS-DKO males and females showed significant increases in lesion area of 93.6% and 59.2% compared with apoE-KO mice. All apoE/eNOS-DKO animals studied developed peripheral coronary arteriosclerosis, associated with perivascular and myocardial fibrosis, whereas none of the apoE-KO mice did. Transthoracic echocardiography showed a significantly increased left ventricular wall thickness and decreased fractional shortening in DKO animals. Mean arterial pressure was increased in DKO mice and was comparable in degree to eNOS-KO animals. Male DKO animals developed atherosclerotic abdominal aneurysms and aortic dissection. CONCLUSIONS: eNOS deficiency increases atherosclerosis in Western-type diet-fed apoE-KO animals and introduces coronary disease and an array of cardiovascular complications, including spontaneous aortic aneurysm and dissection. This phenotype constitutes the first murine model to demonstrate distal coronary arteriosclerosis associated with evidence of myocardial ischemia, infarction, and heart failure. Hypertrophy and reduced left ventricular function cannot be explained by increased blood pressure alone, because eNOS-KO animals do not develop these complications.

Animals↗

PubMed2001

Effects of estrogen on the vascular injury response in estrogen receptor alpha, beta (double) knockout mice.

The two known estrogen receptors, ERalpha and ERbeta, mediate the effects of estrogen in all target tissues, including blood vessels. We have shown previously that estrogen inhibits vascular injury response to the same extent in female wild-type (WT), ERalpha knockout (ERalphaKO(CH)), and ERbeta knockout (ERbetaKO(CH)) mice. We generated mice harboring disruptions of both ERalpha and ERbeta genes (ERalpha,betaKO(CH)) by breeding and studied the effect of 17beta-estradiol (E2) on vascular injury responses in ovariectomized female ERalpha,betaKO(CH) mice and WT littermates. E2 inhibited increases in vascular medial area following injury in the WT mice but not in the ERalpha,betaKO(CH) mice, demonstrating for the first time that the two known estrogen receptors are necessary and sufficient to mediate estrogen inhibition of a component of the vascular injury response. Surprisingly, as in WT littermates, E2 still significantly increased uterine weight and inhibited vascular smooth muscle cell (VSMC) proliferation following injury in the ERalpha,betaKO(CH) mice. These data support that the role of estrogen receptors differs for specific components of the vascular injury response in the ERalpha,betaKO(CH) mice. The results leave unresolved whether E2 inhibition of VSMC proliferation in ERalpha,betaKO(CH) mice is caused by a receptor-independent mechanism, an unidentified receptor responsive to estrogen, or residual activity of the ERalpha splice variant reported previously in the parental ERalphaKO(CH) mice. These possibilities may be resolved by studies of mice in which ERalpha has been fully disrupted (ERalphaKO(St)), which are in progress.

Animals↗

PubMed2001

Macrophage-specific expression of human lipoprotein lipase accelerates atherosclerosis in transgenic apolipoprotein e knockout mice but not in C57BL/6 mice.

Transgenic mice with macrophage-specific expression of human (hu) lipoprotein lipase (LPL) were generated to determine the contribution of macrophage LPL to atherogenesis. Macrophage specificity was accomplished with the scavenger receptor A promoter. Complete characterization demonstrated that macrophages from these mice expressed huLPL mRNA and secreted enzymatically active huLPL protein. Expression of huLPL was macrophage specific, because total RNA isolated from heart, thymus, lung, liver, muscle, and adipose tissues was devoid of huLPL mRNA. Macrophage-specific expression of huLPL did not exacerbate lesions in aortas of C57BL/6 mice even after 32 weeks on an atherosclerotic diet. However, when expressed in apolipoprotein E knockout background, the extent of occlusion in the aortic sinus region of male huLPL+ mice increased 51% (n=9 to 11, P<0.002) compared with huLPL- mice after they had been fed a Western diet for 8 weeks. The proatherogenic effect of macrophage LPL was confirmed in serial sections of the aorta obtained after mice had been fed a Western diet for 3 weeks. By immunohistochemical analysis, huLPL protein was detected in the lesions of huLPL+ mice but not in huLPL- mice. Our results establish that macrophage LPL accelerates atherosclerosis in male apolipoprotein E knockout mice.

Animals↗