Control of soluble fms-like tyrosine-1 (sFlt-1) production response to placental ischemia/hypoxia: role of tumor necrosis factor-α.

Although abnormal soluble fms-like tyrosine kinase-1 (sFlt-1) production is thought to be an important factor in the pathogenesis of preeclampsia (PE), the mechanisms that regulate the production of sFlt-1 during PE are unclear. While our laboratory has shown tumor necrosis factor-α (TNF-α) and sFlt-1 to be elevated in pregnant rats in response to placental ischemia, the importance of TNF-α in the regulation of sFlt-1 production is unknown. Therefore, the purpose of this study was to determine the role of TNF-α in mediating the increase in sFlt-1 in response to placental ischemia or hypoxia. Reductions in uterine perfusion pressure in pregnant rats significantly increased plasma levels of sFlt-1 and tended to increase TNF-α, an effect markedly attenuated by pretreatment with a TNF-α inhibitor etanercept (0.4 mg/kg). To further assess chronic interactions between TNF-α and sFlt-1, we examined a chronic effect of TNF-α infusion (50 ng/day) into normal pregnant rats to increase plasma sFlt-1 levels, as well as the effects of acute hypoxia on placental sFlt-1 production in the absence and presence of TNF-α blockade. Placental explants exposed to hypoxic conditions had enhanced TNF-α levels versus normoxic conditions, as well as increased sFlt-1 production. Pretreatment of placental explants with etanercept (15 µM) significantly reduced TNF-α levels in response to hypoxia but did not attenuate sFlt-1 production. These data suggest that while TNF-α may not play an important role in stimulating sFlt-1 production in response to acute hypoxia, a more chronic hypoxia, or placental ischemia may be an important stimulus for enhanced sFlt-l production.

Role of endothelin in mediating soluble fms-like tyrosine kinase 1-induced hypertension in pregnant rats.

Although soluble fms-like tyrosine kinase 1 (sFlt-1), an antagonist of vascular endothelial growth factor and placental growth factor, has been implicated in the pathogenesis of hypertension during preeclampsia, the mechanisms whereby enhanced sFlt-1 production leads to hypertension remain unclear. Both sFlt-1 and endothelin 1 productions are elevated in women with preeclampsia and in placental ischemic animal models of preeclampsia; however, the importance of endothelin 1 and sFlt-1 interactions in the control of blood pressure during pregnancy is unknown. The purpose of this study was to determine the role of endothelin 1 in mediating sFlt-1-induced hypertension in pregnant rats. To achieve this goal, sFlt-1 (3.7 microg/kg per day for 6 days) was infused into normal pregnant rats and pregnant rats treated with a selective endothelin type A receptor antagonist, ABT 627 (5 mg/kg per day for 6 days). Plasma concentration of sFlt-1 increased from 735+/-34 pg/mL in normal pregnant rats to 2498+/-645 pg/mL (P<0.05) with infusion of sFlt-1. Arterial pressure increased from 100+/-1 mm Hg in normal pregnant rats to 122+/-3 mm Hg (P<0.05) in sFlt-1-infused rats. Chronic increases in plasma sFlt-1 in normal pregnant rats increased preproendothelin mRNA expression in the renal cortices by approximately 3-fold. In addition, chronic endothelin type A receptor blockade completely abolished the blood pressure response to sFlt-1 in pregnant rats (104+/-3 versus 100+/-1 mm Hg; P<0.05), whereas the endothelin A receptor antagonist had no effect on arterial pressure in NP rats (105+/-2 versus 100+/-1 mm Hg). In conclusion, this study demonstrates that endothelin 1, via endothelin type A receptor activation, plays an important role in mediating the hypertension in response to excess sFlt-1 during pregnancy.

Hypertension produced by reductions in uterine perfusion in the pregnant rat: role of interleukin 6.

The purpose of this study was to determine the role of interleukin (IL) 6 in mediating the increase in arterial pressure (AP) in response to chronic reductions in uterine perfusion pressure (RUPP) in pregnant rats. AP was higher in RUPP rats (138+/-1 mm Hg) than in normal pregnant (NP) rats (104+/-1 mm Hg). Serum IL-6 levels in the RUPP rats were 104.5+/-28.6 pg/mL as compared with 36.6+/-7.4 pg/mL in NP rats. To determine the long-term effects of a 2- to 3-fold elevation in plasma IL-6 on renal function and AP in pregnant rats, we infused IL-6 for 5 days (2.5 ng/day) in NP rats starting at day 14 of gestation. Five days later, serum IL-6 levels were 55.5+/-6.5 pg/mL in the control NP rats and 157.0+/-36.1 pg/mL in the IL-6-treated NP rats. AP was higher in the IL-6-treated NP rats (115+/-3 mm Hg) as compared with NP controls (101+/-1 mm Hg) at day 19 of gestation. Renal plasma flow and GFR were lower in the IL-6-treated NP rats than in the NP group. IL-6 increased plasma renin activity but did not affect endothelin in IL-6-treated NP rats. In contrast to the NP rats, IL-6 had no effect on AP or renal hemodynamics in virgin rats. In summary, these data indicate that plasma IL-6 is elevated in response to chronic reductions in uterine perfusion in pregnant rats and that a comparable elevation in plasma IL-6 increases AP and reduces renal function in pregnant rats.

Reduced uterine perfusion pressure (RUPP) model for studying cardiovascular-renal dysfunction in response to placental ischemia.

Despite being one of the leading causes of maternal death and a major contributor of maternal and perinatal morbidity, the mechanisms responsible for the pathogenesis of preeclampsia are unknown. The initiating event in preeclampsia has been postulated to be reduced uteroplacental perfusion. Placental ischemia/hypoxia is thought to lead to widespread activation/dysfunction of the maternal vascular endothelium, vasoconstriction and hypertension. Experimental induction of chronic uteroplacental ischemia appears to be the most promising animal model to study potential mechanisms of preeclampsia since reductions in uteroplacental blood flow in a variety of animal models lead to a hypertensive state that closely resembles preeclampsia in women. This chapter details the methods we use in our laboratory to produce the reduced uterine perfusion pressure (RUPP) model in the pregnant rat.

Enhanced endothelin synthesis by endothelial cells exposed to sera from pregnant rats with decreased uterine perfusion.

The initiating event in preeclampsia is thought be to reduced uteroplacental perfusion. Although we have reported previously that chronic reductions in uterine perfusion pressure (RUPP) in pregnant rats results in hypertension and enhanced endothelin production, the factors linking placental ischemia and endothelial cell activation remain unclear. The purpose of this study was to determine the role of angiotensin II type-1 (AT1) receptor activation on endothelin production induced by serum from pregnant rats exposed to reductions in uterine perfusion. To achieve this goal, human umbilical vein endothelial cells were exposed to sera collected from RUPP rats or normal pregnant rats. Arterial pressure was significantly higher in RUPP rats (135+/-2 mm Hg) than in pregnant rats (106+/-1 mm Hg). Six hours after exposure to RUPP serum (n=17), cell media endothelin concentration was 18.4+/-2.7 pg/mL as compared with 9.22+/-1.3 pg/mL from cells exposed to serum from normal pregnant rats (n=9). Eighteen hours after exposure to RUPP serum (n=7), endothelin concentration was 30.5+/-3.8 pg/mL as compared with 12.8+/-5.3 pg/mL from cells exposed to normal pregnant rat serum (n=6). In contrast, serum from RUPP rats did not increase endothelin production in human umbilical vein endothelial cells pretreated with an AT1 receptor antagonist, losartan (15 micromol/L). Eighteen hours after exposure to RUPP serum and losartan (n=14), endothelin concentration was 21.3+/-2.2 pg/mL as compared with 16.4+/-3.3 pg/mL from cells exposed to normal pregnant rat serum and losartan (n=10). These data indicate that serum from pregnant rats exposed to reductions in uterine perfusion enhances endothelin production by endothelial cells via by AT1 receptor activation.

Hypertension produced by reductions in uterine perfusion in the pregnant rat: role of tumor necrosis factor-alpha.

Inflammatory cytokines such as tumor necrosis factor (TNF)-alpha are elevated in preeclamptic women and are thought to be an important link between placental ischemia and endothelial dysfunction. The purpose of this study was to determine the role of TNF in mediating hypertension in response to chronic reductions in uterine perfusion (RUPPs) in pregnant rats. Arterial pressure was significantly higher in RUPP rats (138+/-1 mm Hg) than in pregnant rats (107+/-1 mm Hg). Serum TNF-alpha levels in the RUPP rats were 17+/-4 pg/mL compared with 8+/-1 pg/mL in normal pregnant rats. To determine the long-term effects of a 2- to 3-fold elevation in plasma TNF-alpha on renal and systemic hemodynamics in pregnant rats, we infused TNF-alpha for 5 days at a rate of 50 ng/d during days 14 to 19 of gestation in pregnant rats. Serum levels were 7+/-2 pg/mL in the control pregnant rats and 14+/-2 pg/mL in the TNF-alpha-treated pregnant rats. Mean arterial pressure was higher in the TNF-alpha-treated pregnant rats (123+/-3 mm Hg) compared with pregnant controls (96+/-3 mm Hg) at day 19 of gestation. TNF-alpha increased renal vascular resistance in pregnant rats by 182%. Renal plasma flow was 5.4+/-1.2 mL/min in the TNF-alpha-treated group and 9.2+/-1.6 mL/min in the control group. Glomerular filtration rate was 1.7+/-0.4 mL/min in the TNF-alpha-treated group and 2.6+/-0.4 mL/min in the control group. In summary, these data suggest that TNF-alpha may play an important role in mediating the increased arterial pressure in response to chronic RUPPs in pregnant rats.

Role of endothelin in mediating tumor necrosis factor-induced hypertension in pregnant rats.

Hypertension during preeclampsia is associated with an increase in plasma levels of tumor necrosis factor (TNF)-alpha, a cytokine known to contribute to endothelial dysfunction. Recently, our laboratory reported that a 2-fold increase in plasma TNF-alpha produces hypertension in pregnant rats. Endothelin is also elevated in preeclampsia and endothelin synthesis is enhanced by TNF-alpha. The purpose of this study was to determine the role of endothlelin in mediating TNF-alpha-induced hypertension in pregnant rats. To achieve this goal, TNF-alpha (50 ng/d for 5 days) was infused into control pregnant rats and pregnant rats treated with an endothelin receptor A antagonist, ABT 627 (5 mg/kg per day for 5 days). At day 19 of gestation, arterial pressure was measured and aorta, kidneys, and placentas were harvested. Infusion of TNF-alpha into pregnant rats increased plasma concentration of TNF-alpha (13.5+/-0.8 to 28.0+/-3.7 pg/mL) and arterial pressure (101+/-2 to 122+/-1 mm Hg). The increase in arterial pressure was associated with an increase in preproendothelin mRNA expression in placenta, aorta, and kidneys measured by real-time polymerase chain reaction (PCR). Pretreatment with the endothelin receptor A antagonist completely abolished the blood pressure response to TNF-alpha in pregnant rats (105+/-1 versus 97+/-2 mm Hg). In sharp contrast, the ETA receptor antagonist had no effect on arterial pressure in normal pregnant rats (97+/-2 versus 101+/-2 mm Hg). Moreover, chronic infusion of TNF-alpha had no significant effect on arterial pressure or renal preproendothelin levels in virgin rats. These results suggest an important role for endothelin in mediating TNF-alpha-induced hypertension in pregnant rats.

Participation of fad and mbt genes in synthesis of mycobactin in Mycobacterium smegmatis.

Colonies of Mycobacterium smegmatis LR222 on iron-limiting (0.1 micro M Fe) minimal medium agar fluoresce under UV light due to the accumulation in the cells of the deferri form of the siderophore mycobactin. Two mutants with little or no fluorescence, designated LUN8 and LUN9, were isolated by screening colonies of transposon (Tn611)-mutagenized M. smegmatis. Ferrimycobactin prepared from iron-restricted cells of the wild type had an R(f) of 0.62 on high-performance thin-layer chromatography (HPTLC) and a characteristic visible absorption spectrum with a peak near 450 nm. Similar extracts from LUN8 cells contained a small amount of ferrimycobactin with an R(f) of 0.58 on HPTLC and an absorption spectrum with the peak shifted to a wavelength lower than that of the wild-type ferrimycobactin. Nuclear magnetic resonance spectroscopy studies suggested that the LUN8 mycobactin may have an altered fatty acid side chain. Mutant strain LUN9 produced no detectable mycobactin. Neither mutant strain produced measurable amounts of excreted mycobactin, although both excreted exochelin (the mycobacterial peptido-hydroxamate siderophore), and both mutants were more sensitive than the wild-type strain to growth inhibition by the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid). The transposon insertion sites were identified, and sequence analyses of the cloned flanking chromosome regions showed that the mutated gene in LUN9 was an orthologue of the Mycobacterium tuberculosis mycobactin biosynthetic gene mbtE. The mutated gene in LUN8 had homology with M. tuberculosis fadD33 (Rv1345), a gene that may encode an acyl-coenzyme A synthase and which previously was not known to participate in synthesis of mycobactin.