Kerstin Strehlow

Estrogen Increases Bone Marrow–Derived Endothelial Progenitor Cell Production and Diminishes Neointima Formation

Background Estrogens improve endothelial function and accelerate reendothelialization after vascular injury via largely unknown mechanisms. Bone marrow‐derived endothelial progenitor cells (EPCs) are thought to positively influence endothelialization, vascular repair, and angiogenesis. Methods and Results In mice subjected to sham operation, ovariectomy, or ovariectomy and estrogen replacement treatment, estrogen deficiency significantly decreased EPCs circulating in the peripheral blood and residing in the bone marrow, as well as EPCs that were in vitro expanded from spleen‐derived mononuclear cells. These effects were completely prevented by estrogen replacement. Human women with increased estrogen plasma concentrations also displayed profoundly increased levels of circulating EPCs. Estrogens increase EPC numbers through a decreased apoptosis rate, which is mediated via a caspase‐8‐dependent pathway. Estrogen deficiency increased neointima formation after carotid artery injury in mice, but this effect was diminished by estrogen replacement therapy. In mice transplanted with green fluorescent protein‐positive bone marrow, reendothelialization of injured vessel segments by bone marrow‐derived cells was decreased during estrogen deficiency and increased in response to estrogen treatment. Conclusions Estrogens increase numbers of EPCs by antiapoptotic effects leading to accelerated vascular repair and decreased neointima formation. (Circulation. 2003;107:3059‐3065.)

Estrogen Modulates AT1 Receptor Gene Expression In Vitro and In Vivo

BACKGROUND The AT1 receptor has been implicated in the pathogenesis of hypertension and atherosclerosis. Estrogen deficiency is also associated with cardiovascular diseases. Therefore, we examined the AT1 receptor gene expression in ovariectomized rats with and without estrogen replacement therapy and the influence of estrogen on AT1 receptor expression in cultured vascular smooth muscle cells. METHODS AND RESULTS Rat aortic tissue was examined 5 weeks after ovariectomy. In one group, estrogen (1.7 mg estradiol) was administered during the 5-week period. Functional experiments assessed angiotensin II-induced contraction of aortic rings. AT1 receptor mRNA levels were measured by quantitative polymerase chain reaction and Northern blotting. AT1 receptor density was assessed by radioligand binding assays. These techniques were also applied in cultured vascular smooth muscle cells. The efficacy of angiotensin II on vasoconstriction was significantly increased in aortas from ovariectomized rats. As assessed by radioligand binding assays, AT1 receptor density was increased to 160% without changes in receptor affinity during estrogen deficiency. AT1 receptor mRNA levels were consistently increased to 187% in ovariectomized rats compared with sham-operated animals. Estrogen substitution therapy in ovariectomized rats reversed this AT1 receptor overexpression. To explore the underlying mechanisms, the direct influence of estradiol on AT1 receptor expression was investigated in VSMCs. Estradiol (1 micromol/L) led to a time-dependent downregulation of AT1 receptor mRNA, with a maximum of 33.3% at 12 hours. There was a correlative decrease in AT1 receptor density. CONCLUSIONS This novel observation of estrogen-induced downregulation of AT1 receptor expression could explain the association of estrogen deficiency with hypertension and atherosclerosis, because activation of the AT1 receptor plays a key role in the regulation of blood pressure, fluid homeostasis, and vascular cell growth.

Modulation of Antioxidant Enzyme Expression and Function by Estrogen

&NA; Oxidative stress plays a pivotal role in the pathogenesis of atherosclerosis and can be effectively influenced by radical scavenging enzyme activity and expression. The vasoprotective effects of estrogens may be related to antioxidative properties. Therefore, effects of 17&bgr;‐estradiol on production of reactive oxygen species and radical scavenging enzymes were investigated. 17&bgr;‐estradiol diminished angiotensin II‐induced free radical production in vascular smooth muscle cells (DCF fluorescence laser microscopy). 17&bgr;‐estradiol time‐ and concentration‐dependently upregulated manganese (MnSOD) and extracellular superoxide dismutase (ecSOD) expression (Northern and Western blotting) and enzyme activity (photometric assay). Nuclear run‐on assays demonstrated that 17&bgr;‐estradiol increases MnSOD and ecSOD transcription rate. Half‐life of MnSOD mRNA was not influenced, whereas ecSOD mRNA was stabilized by estrogen. Copper‐zinc SOD, glutathione‐peroxidase, and catalase were not affected by estrogen. Estrogen deficiency in ovariectomized mice induced a downregulation of ecSOD and MnSOD expression, which was associated with increased production of vascular free radicals and prevented by estrogen replacement or treatment with PEG‐SOD. In humans, increased estrogen levels led to enhanced ecSOD and MnSOD expression in circulating monocytes. Estrogen acts antioxidative at least to some extent via stimulation of MnSOD and ecSOD expression and activity, which may contribute to its vasoprotective effects. (Circ Res. 2003;93:170‐177.)

Interleukin-6 Induces Oxidative Stress and Endothelial Dysfunction by Overexpression of the Angiotensin II Type 1 Receptor

Abstract— Angiotensin II type 1 (AT1) receptor activation as well as proinflammatory cytokines such as interleukin-6 (IL-6) are involved in the development and progression of atherosclerosis. The detailed underlying mechanisms including interactions between inflammatory agonists and the renin-angiotensin system are poorly understood. Stimulation of cultured rat aortic vascular smooth muscle cells (VSMCs) with IL-6 led to upregulation of AT1 receptor mRNA and protein expression, as assessed by Northern and Western blot experiments. Nuclear run-on and transcription blockade experiments showed that IL-6 increases AT1 receptor mRNA de novo synthesis but not mRNA stability. Preincubation of VSMCs with IL-6 resulted in an enhanced angiotensin II–induced production of reactive oxygen species, as assessed by DCF fluorescence laser microscopy. Treatment of C57BL/6J mice with IL-6 for 18 days increased vascular AT1 receptor expression (real-time RT-PCR) and angiotensin II–induced vasoconstriction, enhanced vascular superoxide production (L-012 chemiluminescence, DHE fluorescence), and impaired endothelium-dependent vasodilatation. These effects were completely omitted in AT1 receptor knockout mice (AT1A−/− mice). Upregulation of vascular AT1 receptor expression in vitro and in vivo is decisively involved in IL-6–induced propagation of oxidative stress and endothelial dysfunction. This interaction of the proinflammatory cytokine IL-6 with the renin-angiotensin system may represent an important pathogenetic mechanism in the atherosclerotic process.

Differential Effects of Estrogen and Progesterone on AT1 Receptor Gene Expression in Vascular Smooth Muscle Cells

BackgroundThe beneficial vasoprotective effects of a postmenopausal estrogen replacement therapy may be prevented by a concomitant administration of progestins. To investigate the differential effects of estrogens and progesterone, we examined their influence on AT1 receptor gene expression in vascular smooth muscle cells (VSMCs). Methods and Results17&bgr;-Estradiol caused downregulation of AT1 receptor mRNA expression to 46±14%, whereas progesterone led to a significant upregulation to 201±29%, as assessed by Northern analysis. Western blots revealed that estrogen induced a downregulation and progesterone an upregulation of the AT1 receptor protein. Estrogen-induced decrease of AT1 receptor expression was mediated through activation of estrogen receptors. Nuclear run-on assays revealed that 17&bgr;-estradiol did not alter AT1 receptor mRNA transcription rate, whereas progesterone caused an enhanced AT1 receptor mRNA transcription rate. 17&bgr;-Estradiol decreased the AT1 receptor mRNA half-life from 5 to 2 hours, whereas progesterone induced a stabilization of AT1 receptor mRNA to a half-life of 10 hours. Preincubation of VSMCs with PD98059, SB203580, herbimycin, wortmannin, or N&ohgr;-nitro-l-arginine suggested that 17&bgr;-estradiol caused AT1 receptor downregulation through nitric oxide–dependent pathways. Progesterone caused AT1 receptor overexpression via PI3-kinase activation. Angiotensin II–induced release of reactive oxygen species was inhibited by estrogens. Progesterone itself enhanced the production of reactive oxygen species. ConclusionsBecause AT1 receptor regulation plays a pivotal role in the pathogenesis of hypertension and atherosclerosis, the differential effects of estrogen and progesterone on the expression of this gene may in part explain the potentially counteracting effects of these reproductive hormones on the incidence of postmenopausal cardiovascular diseases.

Salt Induces Vascular AT1 Receptor Overexpression In Vitro and In Vivo

The molecular events governing salt-sensitive hypertension are currently unknown. Because the renin-angiotensin system plays a central role in blood pressure regulation, as well as electrolyte balance, it may be closely involved in the phenomenon of salt sensitivity. Therefore, we examined the effect of a high salt diet (8%) on aortic angiotensin type 1 (AT1) receptor expression in Sprague-Dawley rats by means of radioligand binding assays and quantitative polymerase chain reaction. High salt intake caused an increase of AT1 receptor mRNA and AT1 receptor density to approximately 160% compared with control levels. Northern analysis revealed that incubation of vascular smooth muscle cells (VSMCs) with an increased concentration of sodium chloride (by 10 mmol/L) caused a time-dependent elevation of AT1 receptor mRNA levels, with a maximum of 241+/-28% after 24 hours. There was a similar increase in AT1 receptor density in VSMCs in response to sodium chloride, as assessed by radioligand binding assays. The salt-induced AT1 receptor upregulation led to an enhanced functional response of VSMCs on stimulation with angiotensin II, since the angiotensin II-elicited intracellular calcium response was significantly increased in cells preincubated for 24 hours with the high salt concentration. Thus, sodium chloride may directly induce AT1 receptor upregulation in vitro as well as in vivo; this suggests a potential mechanism participating in salt-induced hypertension because the AT1 receptor activation is tightly coupled to blood pressure regulation.

Redox-sensitive vascular smooth muscle cell proliferation is mediated by GKLF and Id3 in vitro and in vivo

Reactive oxygen species such as super‐oxide and hydroxyl radicals have been implicated in the pathogenic growth of various cell types. The molecular mechanisms involved in redox‐sensitive cell growth control are poorly understood. Stimulation of cultured vascular smooth muscle cells (VSMC) with xanthin/ xanthin oxidase (X/XO) increases proliferation, whereas stimulation with hydrogen peroxide and Fe3+NTA (H‐Fe) causes growth arrest of VSMC. Differential Display led to the identification of two novel, differentially regulated redox‐sensitive genes. The dominant negative helix‐loop‐helix protein Id3 is induced by X/XO and down‐regulated by H‐Fe. The transcription factor gut‐enriched Kruppel‐like factor (GKLF) is induced by H‐Fe but not by X/XO. Induction of GKLF and inhibition of Id3 via transfection experiments leads to growth arrest, whereas overexpression of Id3 and inhibition of GKLF cause cell growth. Id3 down‐regulation is induced via binding of GKLF to the Id3 promotor and concomitantly reduced Id3 gene transcription rate. GKLF induction by H‐Fe is mediated through hydroxyl radicals, p38MAP kinase‐, calcium‐, and protein synthesis‐dependent pathways. Id3 is induced by X/XO via superoxide, calcium, p38, and p42/44 MAP kinase. GKLF induces and Id3 depresses expression of p21WAF1/Cip1, p27KIP1, p53. Induction of Id3 is accomplished by angiotensin II via superoxide release. A vascular injury mouse model revealed that Id3 is overexpressed in proliferating vascular tissue in vivo. These findings reveal novel mechanisms of redoxcontrolled cellular proliferation involving GKLF and Id3 that may have general implications for our understanding of vascular and nonvascular growth control.—Nickenig, G., Baudler, S., Müller, C., Werner, N., Welzel, H., Strehlow, K., Böhm, M. redox‐sensitive vascular smooth muscle cell proliferation is mediated by GKLF and Id3 in vitro and in vivo. FASEB J. 16, 1077–1086 (2002)

Angiotensin AT1 receptor over-expression in hypercholesterolaemia

Angiotensin II mediates most of the biological effects of the renin-angiotensin system (RAS), such as vasoconstriction and cell proliferation, via stimulation of the angiotensin II type 1 (AT1) receptor. The AT1 receptor plays a central role in the pathogenesis of atherosclerosis and hypertension. In parallel, hypercholesterolaemia is a major risk factor for the development and progression of cardiovascular diseases. The underlying molecular events, however, are understood only partially. An important mechanism may be the interaction between hypercholesterolaemia and AT1 receptor expression in vascular tissue. Low-density lipoprotein (LDL) cholesterol leads to a profound increase in AT1 receptor expression in cultured vascular smooth muscle cells as well as in hypercholesterolaemic rabbits. This up-regulation is associated with an enhanced functional response upon stimulation with angiotensin II. Over-expression of the vascular AT1 receptor can also be observed in hypercholesterolaemic men and is prevented by treatment with 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors. These findings may explain why hypercholesterolaemia is frequently associated with hypertension and why blockade of the RAS attenuates the progression of atherosclerosis.

Negative feedback regulation of reactive oxygen species on AT1 receptor gene expression

Free radicals as well as the AT1 receptor are involved in the pathogenesis of cardiovascular disease. Both the intracellular mechanisms of AT1 receptor regulation and the effect of free radicals on AT1 receptor expression are currently unknown. This study investigates the role of free radicals in the modulation of AT1 receptor expression and in the angiotensin II‐induced AT1 receptor regulation. AT1 receptor mRNA was assessed by Northern blotting and AT1 receptor density by radioligand binding assays, respectively, in vascular smooth muscle cells (VSMC). Free radical release was measured by confocal laser scanning microscopy. AT1 receptor mRNA transcription rate was determined by nuclear run‐on assays and AT1 receptor mRNA half‐life was measured under transcriptional blockade. Angiotensin II caused a time‐dependent decrease of AT1 receptor mRNA expression in rat VSMC in culture (30±6% at 4 h with 100 nM angiotensin II). This was followed by a consistent decrease in AT1 receptor density. Angiotensin II caused release of reactive oxygen species in VSMC which was abolished by preincubation with 100 μM diphenylene iodonium (DPI). DPI inhibited partially the down‐regulating effect of angiotensin II on the AT1 receptor. Incubation of VSMC with either hydrogen peroxide or xanthine/xanthine oxidase caused a dose‐dependent decrease in AT1 receptor mRNA expression which was not mediated by a decreased rate of transcription but rather through destabilization of AT1 receptor mRNA. Experiments which included preincubation of VSMC with various intracellular inhibitors suggested that free radicals caused AT1 receptor downregulation through activation of p38‐MAP kinase and intracellular release of calcium. However, angiotensin II‐induced AT1 receptor expression was not inhibited by blockade of p38‐MAP kinase activation or intracellular calcium release. Free radicals may at least in part mediate angiotensin II‐induced AT1 receptor regulation through direct post‐transcriptional effects on AT1 receptor mRNA expression which involves intracellular release of calcium and activation of p38‐MAP kinase. These findings may help to clarify the intracellular mechanisms involved in AT1 receptor regulation and reveal a novel biological feature for reactive oxygen species.

AT1 receptor regulation in salt-sensitive hypertension

The molecular events governing salt-sensitive hypertension are currently unknown. Because the renin-ANG system plays a central role in blood pressure regulation and electrolyte balance, it may be closely involved in the phenomenon of salt sensitivity. Therefore, we examined the effect of a high-salt diet (8%) and a low-salt diet (0.4%) on ANG II-caused vascular constriction and ANG II type 1 (AT(1)) receptor expression in aorta, brain, and kidney of Dahl S (salt-sensitive) and Dahl R (salt-resistant) rats by means of radioligand binding assays and quantitative PCR. NaCl diet at 8% led to a significant increase of blood pressure in Dahl S but not in Dahl R rats. High-sodium intake caused a profound decrease of ANG II-induced aortic vasoconstriction in both Dahl R and Dahl S rats. The underlying mechanism was a downregulation of aortic AT(1) receptor density and AT(1) receptor mRNA. AT(1) receptor mRNA was downregulated to 57.8% in Dahl R and 59.0% in Dahl S rats by an 8% NaCl diet compared with a 0.4% NaCl diet (P < 0.05). There was a similar decrease in aortic AT(1) receptor density. Additionally, AT(1) receptor mRNA was also downregulated in the kidney but upregulated the brain of Dahl R and S rats on a high-salt diet. Thus high NaCl intake causes organ-specific AT(1) receptor regulation in Dahl R and in Dahl S rats despite the differential blood pressure regulation in these animal models in response to a high-salt diet. These findings suggest that the regulation of vascular AT(1) receptors is influenced by numerous factors such as the renin-ANG system and obviously by various other events that are currently only partly understood.The molecular events governing salt-sensitive hypertension are currently unknown. Because the renin-ANG system plays a central role in blood pressure regulation and electrolyte balance, it may be closely involved in the phenomenon of salt sensitivity. Therefore, we examined the effect of a high-salt diet (8%) and a low-salt diet (0.4%) on ANG II-caused vascular constriction and ANG II type 1 (AT1) receptor expression in aorta, brain, and kidney of Dahl S (salt-sensitive) and Dahl R (salt-resistant) rats by means of radioligand binding assays and quantitative PCR. NaCl diet at 8% led to a significant increase of blood pressure in Dahl S but not in Dahl R rats. High-sodium intake caused a profound decrease of ANG II-induced aortic vasoconstriction in both Dahl R and Dahl S rats. The underlying mechanism was a downregulation of aortic AT1receptor density and AT1 receptor mRNA. AT1 receptor mRNA was downregulated to 57.8% in Dahl R and 59.0% in Dahl S rats by an 8% NaCl diet compared with a 0.4% NaCl diet ( P < 0.05). There was a similar decrease in aortic AT1 receptor density. Additionally, AT1receptor mRNA was also downregulated in the kidney but upregulated the brain of Dahl R and S rats on a high-salt diet. Thus high NaCl intake causes organ-specific AT1 receptor regulation in Dahl R and in Dahl S rats despite the differential blood pressure regulation in these animal models in response to a high-salt diet. These findings suggest that the regulation of vascular AT1 receptors is influenced by numerous factors such as the renin-ANG system and obviously by various other events that are currently only partly understood.