Intrauterine growth retardation in endothelial nitric oxide synthase-deficient mice is established from early stages of pregnancy

The current study aimed to determine effects of deficiencies in nitric oxide synthase (NOS) 3 on embryo and fetal development by in vivo, noninvasive, real-time ultrasonographic assessment of phenotypic changes in Nos3-knockout pregnant mice and their wild-type counterparts. From Day 4.5 of pregnancy onwards, embryonic vesicle diameters, crown-rump lengths, and trunk diameters were obtained by serial scanning of seven adult pregnant female mice, strain B6.129P2-Nos3(tm1Unc)/J, N9 generation backcrossing with C57BL/6J mice, homozygous for the disruption of the endothelial NOS gene (group Nos3(-/-)), and 12 pregnant, wild-type C57BL/6J mice (group Nos3(+/+)). All the measurements increased in both genotypes throughout gestation. However, embryo length and width were significantly larger in Nos3(+/+) than in Nos3(-/-) mice from Day 8.5, and both longitudinal and transverse diameters of the entire gestational sacs were larger in Nos3(+/+) mice from Day 10.5. Assessment of the relative growth of embryos/fetuses and gestational annexes showed different patterns among Nos3(-/-) and Nos3(+/+) mice. Throughout pregnancy, the distance between the external limit of the gestational sac and the embryo in Nos3(+/+) mice diminished in longitudinal sections, or remained unaffected in transverse sections. In Nos3(-/-) mice, there were significant increases (P < 0.005) in the differences between embryo and gestational vesicle measurements in both longitudinal and transversal curves from Days 5.5 to 14.5, but from Day 14.5 of pregnancy onward, the changes were not significant. The results demonstrate that the processes of fetal growth retardation in the Nos3(-/-) mice are established from early pregnancy stages.     

Effect of the anti-oxidant tempol on fetal growth in a mouse model of fetal growth restriction

Fetal growth restriction (FGR) greatly increases the risk of perinatal morbidity and mortality and is associated with increased uterine artery resistance and levels of oxidative stress. There are currently no available treatments for this condition. The hypothesis that the antioxidant 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (Tempol) would improve uterine artery function and rescue fetal growth was tested in a mouse model of FGR, using the endothelial nitric oxide synthase knockout mouse (Nos3(-/-)). Pregnant Nos3(-/-) and control C57BL/6J mice were treated with the superoxide dismutase-mimetic Tempol (1 mmol/L) or vehicle from Gestational Day 12.5 to 18.5. Tempol treatment significantly increased pup weight (P < 0.05) and crown-rump length (P < 0.01) in C57BL/6J and Nos3(-/-) mice. Uterine artery resistance was increased in Nos3(-/-) mice (P < 0.05); Tempol significantly increased end diastolic velocity in Nos3(-/-) mice (P < 0.05). Superoxide production in uterine arteries did not differ between C57BL/6J and Nos3(-/-) mice but was significantly increased in placentas from Nos3(-/-) mice (P < 0.05). This was not reduced by Tempol treatment. Placental System A activity was reduced in Nos3(-/-) mice (P < 0.01); this was not improved by treatment with Tempol. Treatment of Nos3(-/-) mice with Tempol, however, was associated with reduced vascular density in the placental bed (P < 0.05). This study demonstrated that treatment with the antioxidant Tempol is able to improve fetal growth in a mouse model of FGR. This was associated with an increase in uterine artery blood flow velocity but not an improvement in uterine artery function or placental System A activity.     

The effect of embryo and maternal genotypes on prolificacy, intrauterine growth retardation and postnatal development of Nos3-knockout mice

Mice deficient for endothelial nitric oxide synthase (NOS3(-/-)) may represent a good model for studying embryo loss and intrauterine growth retardation caused by vascular deficiencies. We determined the effects of embryo genotype (homozygous vs. heterozygous descendants with paternal or maternal source of the non-functional NOS3 allele) and maternal environment (NOS3(-/-) vs. wild-type NOS3(+/+) females) on the appearance of estrus, fertility and prolificacy rates and live weight in the first week of life as well as phenotypic characteristics of offspring during the postnatal period. The results indicated that pregnancy outcomes and postnatal development of NOS3(-/-) mice seem to be related to deficiencies in fetal programming mainly determined by maternal genotype.     

Nos2 inactivation promotes the development of medulloblastoma in Ptch1(+/-) mice by deregulation of Gap43-dependent granule cell precursor migration

Medulloblastoma is the most common malignant brain tumor in children. A subset of medulloblastoma originates from granule cell precursors (GCPs) of the developing cerebellum and demonstrates aberrant hedgehog signaling, typically due to inactivating mutations in the receptor PTCH1, a pathomechanism recapitulated in Ptch1(+/-) mice. As nitric oxide may regulate GCP proliferation and differentiation, we crossed Ptch1(+/-) mice with mice lacking inducible nitric oxide synthase (Nos2) to investigate a possible influence on tumorigenesis. We observed a two-fold higher medulloblastoma rate in Ptch1(+/-) Nos2(-/-) mice compared to Ptch1(+/-) Nos2(+/+) mice. To identify the molecular mechanisms underlying this finding, we performed gene expression profiling of medulloblastomas from both genotypes, as well as normal cerebellar tissue samples of different developmental stages and genotypes. Downregulation of hedgehog target genes was observed in postnatal cerebellum from Ptch1(+/+) Nos2(-/-) mice but not from Ptch1(+/-) Nos2(-/-) mice. The most consistent effect of Nos2 deficiency was downregulation of growth-associated protein 43 (Gap43). Functional studies in neuronal progenitor cells demonstrated nitric oxide dependence of Gap43 expression and impaired migration upon Gap43 knock-down. Both effects were confirmed in situ by immunofluorescence analyses on tissue sections of the developing cerebellum. Finally, the number of proliferating GCPs at the cerebellar periphery was decreased in Ptch1(+/+) Nos2(-/-) mice but increased in Ptch1(+/-) Nos2(-/) (-) mice relative to Ptch1(+/-) Nos2(+/+) mice. Taken together, these results indicate that Nos2 deficiency promotes medulloblastoma development in Ptch1(+/-) mice through retention of proliferating GCPs in the external granular layer due to reduced Gap43 expression. This study illustrates a new role of nitric oxide signaling in cerebellar development and demonstrates that the localization of pre-neoplastic cells during morphogenesis is crucial for their malignant progression.     

Effect of inspired oxygen on periodic breathing in methy-CpG-binding protein 2 (Mecp2) deficient mice.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the X-linked gene methyl-CpG-binding protein 2 (Mecp2) that encodes a DNA binding protein involved in gene silencing. Periodic breathing (Cheyne-Stokes respiration) is commonly seen in RTT. Freely moving mice were studied with continuous recording of pleural pressure by telemetry. Episodes of periodic breathing in heterozygous Mecp2 deficient (Mecp2(+/-)) female mice (9.4 +/- 2.2 h(-1)) exceeded those in wild-type (Mecp2(+/+)) animals (2.5 +/- 0.4 h(-1)) (P = 0.010). Exposing Mecp2(+/-) animals to 40% oxygen increased the amount of periodic breathing from 118 +/- 25 s/30 min in air to 242 +/- 57 s/30 min (P = 0.001), and 12% oxygen tended to decrease it (67 +/- 29 s/30 min, P = 0.14). Relative hyperoxia and hypoxia did not affect the incidence of periodic breathing in Mecp2(+/+) animals. The ventilation/apnea ratio (V/A) was less at all levels of oxygen in heterozygous Mecp2(+/-) females compare with wild type (P = 0.003 to P < 0.001), indicating that their loop gain is larger. V/A in Mecp2(+/-) fell from 2.42 +/- 0.18 in normoxia to 1.82 +/- 0.17 in hyperoxia (P = 0.05) indicating an increase in loop gain with increased oxygen. Hyperoxia did not affect V/A in Mecp2(+/+) mice (3.73 +/- 0.28 vs. 3.5 +/- 0.28). These results show that periodic breathing in this mouse model of RTT is not dependent on enhanced peripheral chemoreceptor oxygen sensitivity. Rather, the breathing instability is of central origin.     

Placental growth retardation due to loss of imprinting of Phlda2

The maternally expressed/paternally silenced genes Phlda2 (a.k.a. Ipl/Tssc3), Slc22a1l, Cdkn1c, Kcnq1, and Ascl2 are clustered in an imprinted domain on mouse chromosome 7. Paternal deletion of a cis-acting differentially methylated DNA element, Kvdmr1, causes coordinate loss of imprinting and over-expression of all of these genes and the resulting conceptuses show intrauterine growth restriction (IUGR). To test the specific contribution of Phlda2 to IUGR in the Kvdmr1-knockout, we crossed Kvdmr1(+/-) males with Phlda2(+/-) females. Conceptuses with the (Phlda2(+/+); Kvdmr1(+/-)) genotype showed fetal and placental growth retardation. Restoration of Phlda2 dosage to normal, as occurred in the conceptuses with the (Phlda2(-/+); Kvdmr1(+/-)) genotype, had a marginally positive effect on fetal weights and no effect on post-natal weights, but significantly rescued the placental weights. As we previously reported, loss of Phlda2 expression in the wild-type background (Phlda2(-/+); Kvdmr1(+/+) genotype) caused placentomegaly. Thus Phlda2 acts as a true rheostat for placental growth, with overgrowth after gene deletion and growth retardation after loss of imprinting. Consistent with this conclusion, we observed significant placental stunting in BAC-transgenic mice that over-expressed Phlda2 and one flanking gene, Slc22a1l, but did not over-express Cdkn1c.     

Fetal origins of adult vascular dysfunction in mice lacking endothelial nitric oxide synthase

Epidemiological studies have shown increased incidence of hypertension and coronary artery disease in growth-restricted fetuses during their adult life. A novel animal model was used to test the hypothesis regarding the role of an abnormal uterine environment in fetal programming of adult vascular dysfunction. Mice lacking a functional endothelial nitric oxide synthase (NOS3-/-KO, where KO is knockout) and wild-type (WT) mice (NOS3+/+WT) were crossbred to produce homozygous NOS3-/-KO, maternally derived heterozygous (NOS3+/-mat, mother with NOS3 deficiency), paternally derived heterozygous (NOS3+/-pat, normal mother), and NOS3+/+WT litters. Number of fetuses per litter was smaller in NOS3-/-KO and NOS3+/-mat compared with NOS3+/-pat and NOS3+/+WT mice. Adult female mice from these litters (7-8 wk old) were killed, and ring preparations of carotid and mesenteric arteries were mounted in a wire myograph to evaluate the passive and reactive vascular characteristics. Slope of the length-tension plot (a measure of vascular compliance) was increased, and optimal diameter (as calculated by Laplace equation) was decreased in NOS3-/-KO and NOS3+/-mat compared with NOS3+/-pat and NOS3+/+WT mice. Acetylcholine caused vasorelaxation in NOS3+/-pat and NOS3+/+WT and contraction in NOS3-/-KO and NOS3+/-mat mice. Responses to phenylephrine and Ca2+ were increased in NOS3-/-KO and NOS3+/-mat compared with NOS3+/-pat and NOS3+/+WT mice. Relaxation to isoproterenol was decreased in NOS3-/-KO and NOS3+/-mat vs. NOS3+/-pat and NOS3+/+WT mice. Abnormalities in the passive and reactive in vitro vascular properties seen in NOS+/-mat that developed in a NOS3-deficient maternal/uterine environment compared with the genetically identical NOS3+/-pat mice that developed in a normal environment are the first direct evidence in support of a role for uterine environment in determining vascular function in later life.     

Role of leukocytes in uterine hypoperfusion and fetal growth retardation induced by ischemia-reperfusion

We investigated leukocyte involvement in uterine hypoperfusion and intrauterine fetal growth retardation (IUGR) induced by ischemia-reperfusion (I/R) in Sprague-Dawley rats. On day 17 of gestation, leukocyte accumulation in the uterus and placenta subjected to 30 min of ischemia, followed by reperfusion, was assessed by measuring myeloperoxidase (MPO) activity. Uterine MPO activity was significantly higher after 1 h of reperfusion than it was before ischemia (P < 0.05), without any increase in placental MPO activity. Immunohistochemical staining showed leukocyte accumulation in the uterus subjected to I/R. The effects of treatment with monoclonal antibodies against CD11a (WT1) and CD18 (WT3) at a dose of 0.8 mg/kg on uterine blood flow and IUGR were investigated. Laser-Doppler flowmetry demonstrated that uterine hypoperfusion at 2 h after ischemia (blood flow, -51.7 +/- 1.2%; P < 0.01) was inhibited by WT1 and WT3 treatment. I/R-induced IUGR at full term (P < 0.05 vs. nonischemic horn) was prevented by WT1 and WT3 treatment on day 17. These results indicate that leukocyte accumulation may play an important role in the pathogenesis of uterine hypoperfusion and IUGR induced by I/R in pregnant rats.     

Angiogenesis is not impaired in connective tissue growth factor (CTGF) knock-out mice.

Connective tissue growth factor (CTGF) is a member of the CCN family of growth factors. CTGF is important in scarring, wound healing, and fibrosis. It has also been implicated to play a role in angiogenesis, in addition to vascular endothelial growth factor (VEGF). In the eye, angiogenesis and subsequent fibrosis are the main causes of blindness in conditions such as diabetic retinopathy. We have applied three different models of angiogenesis to homozygous CTGF(-/-) and heterozygous CTGF(+/-) mice to establish involvement of CTGF in neovascularization. CTGF(-/-) mice die around birth. Therefore, embryonic CTGF(-/-), CTGF(+/-), and CTGF(+/+) bone explants were used to study in vitro angiogenesis, and neonatal and mature CTGF(+/-) and CTGF(+/+) mice were used in models of oxygen-induced retinopathy and laser-induced choroidal neovascularization. Angiogenesis in vitro was independent of the CTGF genotype in both the presence and the absence of VEGF. Oxygen-induced vascular pathology in the retina, as determined semi-quantitatively, and laser-induced choroidal neovascularization, as determined quantitatively, were also not affected by the CTGF genotype. Our data show that downregulation of CTGF levels does not affect neovascularization, indicating distinct roles of VEGF and CTGF in angiogenesis and fibrosis in eye conditions.     

Endothelial-derived nitric oxide and angiotensinogen: blood pressure and metabolism during mouse pregnancy

The regulation of blood pressure during pregnancy involves several biological pathways. Candidate genes implicated in hypertensive diseases during pregnancy include those of the renin-angiotensin system and nitric oxide synthase (NOS). We evaluated blood pressure and metabolic characteristics during pregnancy in mutant mice. These included mice with a null mutation in the endothelial NOS (eNOS) gene (Nos3(-/-)), four copies of the angiotensinogen gene (Agt(2/2)), and mutations in both genes [four copies of Agt and heterozygous deficient for eNOS (Agt(2/2)Nos3(+/-)), four copies of Agt and homozygous deficient for eNOS (Agt(2/2)Nos3(-/-))]. Blood pressure measurements of nulliparous females from mutant strains were compared with two common laboratory strains C57Bl6/J and SV129 throughout their first pregnancy. Serum and urine analysis for the evaluation of renal and liver physiology were measured in the prepregnant state and during the third trimester of pregnancy. Throughout pregnancy blood pressures in all mutant strains were higher compared with controls. Agt(2/2)Nos3(-/-) showed the highest blood pressures and C57Bl6/J the lowest. Control mice, but not mutant mice, showed a second trimester decline in blood pressure. No immediate differences were noted regarding behavioral characteristics, renal or liver function parameters. Mice deficient for eNOS, mice with overexpression of Agt, and mice with mutations in both genes demonstrated higher blood pressure throughout pregnancy. There was no evidence of renal dysfunction, liver dysfunction, or hemolysis among any of the strains studied. We conclude that Nos3 and Agt are important genes in the regulation of blood pressure during pregnancy.     

Activation of volume regulated chloride channels protects myocardium from ischemia/reperfusion damage in second-window ischemic preconditioning

Activation of volume regulated chloride channels (VRCCs) has been shown to be cardioprotective in ischemic preconditioning (IPC) of isolated hearts but the underlying molecular mechanisms remain unclear. Recent independent studies support that ClC-3, a ClC voltage-gated chloride channel, may function as a key component of the VRCCs. Thus, ClC-3 knockout (Clcn3(-/-)) mice and their age-matched heterozygous (Clcn3(+/-)) and wild-type (Clcn3(+/+)) littermates were used to test whether activation of VRCCs contributes to cardioprotection in early and/or second-window IPC. Targeted disruption of ClC-3 gene caused a decrease in the body weight but no changes in heart/body weight ratio. Telemetry ECG and echocardiography revealed no differences in ECG and cardiac function under resting conditions among all groups. Under treadmill stress (10 m/min for 10 min), the Clcn3(-/-) mice had significant slower heart rate (648±12 bpm) than Clcn3(+/+) littermates (737±19 bpm, n=6, P<0.05). Ex vivo IPC in the isolated working-heart preparations protected cardiac function during reperfusion and significantly decreased apoptosis and infarct size in all groups. In vivo early IPC significantly reduced infarct size in all groups including Clcn3(-/-) mice (22.7±3.7% vs control 40.1±4.3%, n=22, P=0.004). Second-window IPC significantly reduced apoptosis and infarction in Clcn3(+/+) (22.9±3.2% vs 45.7±5.4%, n=22, P<0.001) and Clcn3(+/-) mice (27.5±4.1% vs 42.2±5.7%, n=15, P<0.05) but not in Clcn3(-/-) littermates (39.8±4.9% vs 41.5±8.2%, n=13, P>0.05). Impaired cell volume regulation of the Clcn3(-/-) myocytes may contribute to the failure of cardioprotection by second-window IPC. These results strongly support that activation of VRCCs may play an important cardioprotective role in second-window IPC.     

Inhibition of stromal CXCR4 impairs development of lung metastases

Compelling evidence has emerged in recent years indicating that stromal cells play a critical role in disease progression. CXCR4 is a G-protein-coupled receptor with a major role in lymphocyte homing. Its ligand, CXCL12, is a highly efficient chemotactic factor for T cells, monocytes, pre-B cells, dendritic cells and myeloid bone marrow-derived cells (BMDCs). In addition, the CXCR4-CXCL12 axis plays a central role in tumor growth and metastasis. To evaluate the effect of genetic CXCR4 reduction on metastasis development, murine melanoma B16 cells were injected into the tail vein of C57BL/6 CXCR4(+/+) and CXCR4(+/-) mice in the presence of the CXCR4 inhibitor, Plerixafor (previously named AMD3100). Although lung metastases developed in wild-type CXCR4(+/+) and heterozygote CXCR4(+/-) mice, nodules were significantly smaller in the latter. CXCR4 pharmacological inhibition by Plerixafor further reduced lung metastases in CXCR4(+/-) mice, preserving the pulmonary architecture (4.18?±?1.38?mm(2) vs. 1.11?±?0.60?mm(2), p?=?0.038). A reduction in LY6G-positive myeloid/granulocytic cells and in p38 MAPK activation was detected in lungs from CXCR4(+/-) mice compared to CXCR4(+/+) mice [LY6G-positive myeloid CXCR4(+/-) vs. CXCR4(+/+) (p?=?0.0004); CXCR4(+/+) vs. CXCR4(+/+) Plerixafor-treated (p?=?0.0031)] suggesting that CXCR4 reduction on myeloid-derived cells reduced their recruitment to the lung, consequently impairing lung metastases. Our findings argue in favor of a specific role of CXCR4 expressed in stromal cells that condition the pro-tumor microenvironment. In this scenario, CXCR4 antagonists will target neoplastic cells as well as the pro-tumor stromal microenvironment.     

Decreased pulmonary radiation resistance of manganese superoxide dismutase (MnSOD)-deficient mice is corrected by human manganese superoxide dismutase-Plasmid/Liposome (SOD2-PL) intratracheal gene therapy

The pulmonary ionizing radiation sensitivity of C57BL/6 Sod2(+/-) mice heterozygous for MnSOD deficiency was compared to that Sod2(+/+) control littermates. Embryo fibroblast cell lines from Sod2(-/-) (neonatal lethal) or Sod2(+/-) mice produced less biochemically active MnSOD and demonstrated a significantly greater in vitro radiosensitivity. No G(2)/M-phase cell cycle arrest after 5 Gy was observed in Sod2(-/-) cells compared to the Sod2(+/-) or Sod2(+/+) lines. Subclonal Sod2(-/-) or Sod2(+/-) embryo fibroblast lines expressing the human SOD2 transgene showed increased biochemical activity of MnSOD and radioresistance. Sod2(+/-) mice receiving 18 Gy whole-lung irradiation died sooner and had an increased percentage of lung with organizing alveolitis between 100 and 160 days compared to Sod2(+/+) wild-type littermates. Both Sod2(+/-) and Sod2(+/+) littermates injected intratracheally with human manganese superoxide dismutase-plasmid/liposome (SOD2-PL) complex 24 h prior to whole-lung irradiation showed decreased DNA strand breaks and improved survival with decreased organizing alveolitis. Thus underexpression of MnSOD in the lungs of heterozygous Sod2(+/-) knockout mice is associated with increased pulmonary radiation sensitivity and parallels increased radiation sensitivity of embryo fibroblast cell lines in vitro. The restoration of cellular radioresistance in vitro and in lungs in vivo by SOD2-PL transgene expression supports a potential role for SOD2-PL gene therapy in organ-specific radioprotection.     

Angiotensin II regulates growth of the developing papillas ex vivo

We tested the hypothesis that lack of angiotensin (ANG) II production in angiotensinogen (AGT)-deficient mice or pharmacologic antagonism of ANG II AT(1) receptor (AT(1)R) impairs growth of the developing papillas ex vivo, thus contributing to the hypoplastic renal medulla phenotype observed in AGT- or AT(1)R-null mice. Papillas were dissected from Hoxb7(GFP+) or AGT(+/+), (+/-), (-/-) mouse metanephroi on postnatal day P3 and grown in three-dimentional collagen matrix gels in the presence of media (control), ANG II (10(-5) M), or the specific AT(1)R antagonist candesartan (10(-6) M) for 24 h. Percent reduction in papillary length was attenuated in AGT(+/+) and in AGT(+/-) compared with AGT(-/-) (-18.4 ± 1.3 vs. -32.2 ± 1.6%, P < 0.05, -22.8 ± 1.3 vs. -32.2 ± 1.6%, P < 0.05, respectively). ANG II blunted the decrease in papilla length observed in respective media-treated controls in Hoxb7(GFP+) (-1.5 ± 0.3 vs. -10.0 ± 1.4%, P < 0.05) or AGT(+/+), (+/-), and (-/-) papillas (-12.8 ± 0.7 vs. -18.4 ± 1.3%, P < 0.05, -16.8 ± 1.1 vs. -23 ± 1.2%, P < 0.05; -26.2 ± 1.6 vs. -32.2 ± 1.6%, P < 0.05, respectively). In contrast, percent decrease in the length of Hoxb7(GFP+) papillas in the presence of the AT(1)R antagonist candesartan was higher compared with control (-24.3 ± 2.1 vs. -10.5 ± 1.8%, P < 0.05). The number of proliferating phospho-histone H3 (pH3)-positive collecting duct cells was lower, whereas the number of caspase 3-positive cells undergoing apoptosis was higher in candesartan- vs. media-treated papillas (pH3: 12 ± 1.4 vs. 21 ± 2.1, P < 0.01; caspase 3: 3.8 ± 0.5 vs. 1.7 ± 0.2, P < 0.01). Using quantitative RT-PCR, we demonstrate that AT(1)R signaling regulates the expression of genes implicated in morphogenesis of the renal medulla. We conclude that AT(1)R prevents shrinkage of the developing papillas observed ex vivo via control of Wnt7b, FGF7, β-catenin, calcineurin B1, and α3 integrin gene expression, collecting duct cell proliferation, and survival.     

HindIII polymorphism of lipoprotein lipase gene and risk of myocardial infarction

The purpose of this study was to determine whether HindIII restriction polymorphism found in intron 8 of lipoprotein lipase gene is associated with the onset of myocardial infarction (MI) in Russians and Tartars living in Bashkortostan. HindIII polymorphism was investigated by the polymerase chain reaction in myocardial infarction survivors (males aged under 55 years (98 Russians and 68 Tartars) and in controls (53 Russians and 80 Tartars). The distribution of genotypes and allele frequencies in the controls were as follows: the frequencies of genotypes HindIII(-/-), HindIII(+/-), and HindIII(+/+) in Russians (3.77, 49.06, and 47.17%, respectively) did not differ from those in Tartars (7.50, 51.24, and 41.25%, respectively), while the frequency of HindIII(-) allele was 28.30% in Russians and 33.13% in Tartars. Among Tartars, the HindIII(+/+) genotype was more common in myocardial infarction survivors than in controls (OR 2.03). In the Russians this genotype was not associated with the risk of MI. The frequencies of HindIII(+/+) genotype and allele HindIII(+) were significantly higher (OR 8.96 and 6.71, respectively) and frequencies of HindIII(+/-) genotype and allele HindIII(-) were lower (OR 0.13 and 0.15) in Russian patients with repeated MI. These findings indicate that HindIII polymorphism may be a genetic risk factor for MI before 55 years of age in the Tartars and for repeated MI in Russians. This association prompts genotyping of HindIII polymorphism for predicting MI recurrence in Russian survivors after MI.     

Calcium-activated potassium channels and nitric oxide coregulate estrogen-induced vasodilation

Nitric oxide synthase (NOS) contributes to estradiol-17beta (E(2)beta)-induced uterine vasodilation, but additional mechanisms are involved, and the cellular pathways remain unclear. We determined if 1) uterine artery myocytes express potassium channels, 2) E(2)beta activates these channels, and 3) channel blockade plus NOS inhibition alters E(2)beta-induced uterine vasodilation. Studies of cell-attached patches identified a 107 +/- 7 pS calcium-dependent potassium channel (BK(Ca)) in uterine artery myocytes that rapidly increased single-channel open probability 70-fold (P < 0.05) after exposure to 100 nM E(2)beta through an apparent cGMP-dependent mechanism. In ovariectomized nonpregnant ewes (n = 11) with uterine artery flow probes and catheters, local BK(Ca) blockade with tetraethylammonium (TEA; 0.05-0.6 mM) dose dependently inhibited E(2)beta-induced uterine vasodilation (n = 37, R = 0.77, P < 0.0001), with maximum inhibition averaging 67 +/- 11%. Mean arterial pressure (MAP) and E(2)beta-induced increases (P 相似文献   

Salt modulates vascular response through adenosine A2A receptor in eNOS-null mice: role of CYP450 epoxygenase and soluble epoxide hydrolase

High salt (HS) intake can change the arterial tone in mice, and the nitric oxide (NO) acts as a mediator to some of the receptors mediated vascular response. The main aim of this study was to explore the mechanism behind adenosine-induced vascular response in HS-fed eNOS(+/+) and eNOS(-/-) mice The modulation of vascular response by HS was examined using aortas from mice (eNOS(+/+) and eNOS(-/-)) fed 4% (HS) or 0.45% (NS) NaCl-diet through acetylcholine (ACh), NECA (adenosine-analog), CGS 21680 (A(2A) AR-agonist), MS-PPOH (CYP epoxygenase-blocker; 10(-5) M), AUDA (sEH-blocker; 10(-5) M), and DDMS (CYP4A-blocker; 10(-5) M). ACh-response was greater in HS-eNOS(+/+) (+59.3 ± 6.3%) versus NS-eNOS(+/+) (+33.3 ± 8.0%; P < 0.05). However, there was no response in both HS-eNOS(-/-) and NS-eNOS(-/-). NECA-response was greater in HS-eNOS(-/-) (+37.4 ± 3.2%) versus NS-eNOS(-/-) (+7.4.0 ± 3.8%; P < 0.05). CGS 21680-response was also greater in HS-eNOS(-/-) (+45.4 ± 5.2%) versus NS-eNOS(-/-)(+5.1 ± 5.0%; P < 0.05). In HS-eNOS(-/-), the CGS 21680-response was reduced by MS-PPOH (+7.3 ± 3.2%; P < 0.05). In NS-eNOS(-/-), the CGS 21680-response was increased by AUDA (+38.2 ± 3.3%; P < 0.05) and DDMS (+30.1 ± 4.1%; P < 0.05). Compared to NS, HS increased CYP2J2 in eNOS(+/+) (35%; P < 0.05) and eNOS(-/-) (61%; P < 0.05), but decreased sEH in eNOS(+/+) (74%; P < 0.05) and eNOS(-/-) (40%; P < 0.05). Similarly, CYP4A decreased in HS-eNOS(+/+) (35%; P < 0.05) and HS-eNOS(-/-) (34%; P < 0.05). These data suggest that NS causes reduced-vasodilation in both eNOS(+/+) and eNOS(-/-) via sEH and CYP4A. However, HS triggers possible A(2A)AR-induced relaxation through CYP epoxygenase in both eNOS(+/+) and eNOS(-/-).     

Elastin-insufficient mice show normal cardiovascular remodeling in 2K1C hypertension despite higher baseline pressure and unique cardiovascular architecture

Mice heterozygous for the elastin gene (ELN(+/-)) show unique cardiovascular properties, including increased blood pressure and smaller, thinner arteries with an increased number of lamellar units. Some of these properties are also observed in humans with supravalvular aortic stenosis, a disease caused by functional heterozygosity of the elastin gene. The arterial geometry in ELN(+/-) mice is contrary to the increased thickness that would be expected in an animal demonstrating hypertensive remodeling. To determine whether this is due to a decreased capability for cardiovascular remodeling or to a novel adaptation of the ELN(+/-) cardiovascular system, we increased blood pressure in adult ELN(+/+) and ELN(+/-) mice using the two-kidney, one-clip Goldblatt model of hypertension. Successfully clipped mice have a systolic pressure increase of at least 15 mmHg over sham-operated animals. ELN(+/+) and ELN(+/-)-clipped mice show significant increases over sham-operated mice in cardiac weight, arterial thickness, and arterial cross-sectional area with no changes in lamellar number. There are no significant differences in most mechanical properties with clipping in either genotype. These results indicate that ELN(+/+) and ELN(+/-) hearts and arteries remodel similarly in response to adult induced hypertension. Therefore, the cardiovascular properties of ELN(+/-) mice are likely due to developmental remodeling in response to altered hemodynamics and reduced elastin levels.     

Differential ANG II generation in plasma and tissue of mice with decreased expression of the ACE gene

We utilized mice with homozygous disruption of angiotensin-converting enzyme (ACE) (-/-), mice with heterozygous deletion of ACE (+/-), and wild-type mice (+/+) to test the hypothesis that genetic variation in ACE modulates tissue and plasma angiotensin (ANG) II concentrations. With the use of ANG I as substrate, kidney, heart, and lung ACE activity was reduced 80% in -/- mice compared with +/+ mice. However, ANG II concentrations and ANG II-to-ANG I ratios in the kidney, heart, and lung did not differ among genotypes. In contrast, plasma ANG II concentrations in -/- mice were <2 fmol/ml, whereas plasma ANG I concentrations were extremely high (765 fmol/ml). Chymase activity was increased 14-fold in the kidney (P < 0.05) and 1.5-fold in the heart (P < 0.05) of -/- versus +/+ mice but did not differ among genotypes in the lung. ANG II formation from enzymes other than ACE and chymase contributed <2% of total ANG II formation in all genotypes. These data suggest that ACE is essential to ANG II formation in the vascular space, whereas chymase may provide an important mechanism in maintaining steady-state ANG II levels in tissue.     

Attenuated renal vascular responses to acute angiotensin II infusion in smooth muscle-specific Na+/Ca2+ exchanger knockout mice

Recent studies in smooth muscle-specific Na(+)/Ca(2+) exchanger-1 knockout (NCX1(sm-/-)) mice reveal reduced arterial pressure and impaired myogenic responses compared with heterozygous littermates. In this study, we determined renal function in male anesthetized NCX1(sm-/-) mice and NCX1 heterozygous (NCX1(+/-)) littermates before and during acute ANG II infusions. Systolic blood pressure in awake mice was lower in NCX1(sm-/-) mice compared with NCX1(+/-) mice (119 ± 4 vs. 131 ± 3 mmHg, P < 0.05). Acute ANG II infusions (5 ng·min(-1)·g(-1) body wt) increased mean arterial pressure in anesthetized NCX1(+/-) (109 ± 2 to 134 ± 3 mmHg, P < 0.001, n = 8) and NCX1(sm-/-) (101 ± 8 to 129 ± 8 mmHg, P < 0.01, n = 6) mice to a similar extent (Δ25 ± 1 vs. Δ28 ± 4 mmHg, P > 0.05). In response to ANG II infusions, PAH clearance (C(PAH)) decreased from 1.39 ± 0.27 to 0.98 ± 0.22 ml·min(-1)·g(-1) (P < 0.05) and glomerular filtration rate (GFR) was reduced from 0.50 ± 0.09 to 0.32 ± 0.06 ml·min(-1)·g(-1) (P < 0.05) in NCX1(+/-) mice. In contrast, the NCX1(sm-/-) did not exhibit significant reductions in either C(PAH) (1.16 ± 0.30 to 1.22 ± 0.34 ml·min(-1)·g(-1), P > 0.05) or GFR (0.48 ± 0.08 to 0.41 ± 0.05 ml·min(-1)·g(-1), P > 0.05) during acute ANG II infusions. Using flometry to measure renal blood flow continuously, NCX1(sm-/-) mice had significantly attenuated responses to ANG II infusions (-34.2 ± 3.9%, P < 0.05) compared with those in NCX1(+/-) mice (-48 ± 2%) or in wild-type mice (-69 ± 7%). These data indicate that renal vascular responses to ANG II are attenuated in NCX1(sm-/-) mice compared with NCX1(+/-) mice and that NCX1 contributes to the renal vasoconstriction response to acute ANG II infusions.