AT1-receptor-deficiency induced atheroprotection in diabetic mice is partially mediated via PPARγ.

OBJECTIVE: Peroxisome-proliferator-activated-receptor-γ (PPARγ) acts as a transcriptional regulator of multiple genes involved in glucose and lipid metabolism. In vitro studies showed that activated PPARγ suppresses AT1R-gene expression and vice versa. However, it has not yet been determined in vivo, whether AT1R-PPARγ-interactions play a relevant role in the pathogenesis of diabetic complications and specifically in accelerated atherosclerosis. METHODS AND RESULTS: ApoE-/- and ApoE-/-/AT1R-/--mice were rendered diabetic by intraperitoneal injections of streptozotocin. Diabetic and non-diabetic ApoE-/--mice were further randomized to receive the AT1R antagonist telmisartan, the selective PPARγ antagonist GW9662, telmisartan and GW9662 or vehicle for 18 weeks. Diabetic and non-diabetic ApoE-/-/AT1R-/--mice were randomized to receive either GW9662 or vehicle. GW9662 treatment in diabetic ApoE-/- and diabetic ApoE-/-/AT1-/--mice resulted in the highest elevation of fasting blood glucose levels, whereas telmisartan treatment and AT1 deficiency in ApoE-/--mice showed the lowest fasting blood glucose levels. Diabetic ApoE-/--mice displayed severe impairment of endothelial function, enhanced oxidative stress and increased atherosclerotic lesion formation. ApoE-/-/AT1R-/- and telmisartan-treated ApoE-/--mice showed a significantly better endothelial function, decreased oxidative stress and reduced atherosclerotic lesion formation. Treatment of diabetic ApoE-/- and ApoE-/-/AT1R-/--mice with the selective PPARγ antagonist GW9662 omitted the atheroprotective effects of AT1R deficiency or AT1 antagonism. CONCLUSION: Genetic disruption or pharmacological inhibition of the AT1R attenuates atherosclerosis and improves endothelial function in diabetic ApoE-/--mice via the PPARγ pathway.

Stimulation of the AT2 receptor reduced atherogenesis in ApoE(-/-)/AT1A(-/-) double knock out mice.

AT1 receptor blockers (ARB) and in part ACE inhibitors (ACI) potentially exert beneficial effects on atherogenesis independent of AT1 receptor inhibition. These pleiotropic effects might be related to angiotensin II mediated activation of the AT2 receptor. To analyze this hypothesis we investigated the development of atherosclerosis and the role of ACIs and ARBs in apolipoprotein E-deficient (ApoE(-/-)) mice and in ApoE/AT1A receptor double knockout mice (ApoE(-/-)/AT1A(-/-)). ApoE(-/-) mice and ApoE(-/-)/AT1A(-/-) mice were fed cholesterol-rich diet for 7 weeks. Vascular oxidative stress, endothelial dysfunction, and atherosclerotic lesion formation were evident in ApoE(-/-) mice, but were markedly reduced in ApoE(-/-)/AT1A(-/-) mice. Concomitant treatment of ApoE(-/-)/AT1A(-/-) mice with either telmisartan or ramipril had no additional effect on blood pressure, vascular oxidative stress, AT2 receptor expression, and endothelial function. Remarkably, atherosclerotic lesion formation was increased in ramipril treated ApoE(-/-)/AT1A(-/-) mice compared to untreated ApoE(-/-)/AT1A(-/-) mice whereas pharmacological AT1 receptor inhibition with telmisartan had no additional effect on atherogenesis. Moreover, chronic AT2 receptor inhibition with PD123,319 significantly increased plaque development in ApoE(-/-)/AT1A(-/-) mice. In additional experiments, direct AT2 receptor stimulation reduced atherogenesis in ApoE(-/-)/AT1A(-/-) mice. Taken together, our data demonstrate a relevant antiatherosclerotic role of the AT2 receptor in atherosclerotic mice and provide novel insight in RAS-physiology.

Inhibition of leukotriene C4 action reduces oxidative stress and apoptosis in cardiomyocytes and impedes remodeling after myocardial injury.

UNLABELLED: Tissue damage leads to release of pro-inflammatory mediators. Among these, leukotriene C(4) (LTC(4)) is a powerful, intracellularly induced mediator of inflammation, which requires inside-out transport of LTC(4). We investigated whether release of LTC(4)via the multidrug resistance related protein 1 (MRP1) induces apoptosis in cardiomyocytes in vitro and in vivo. METHODS AND RESULTS: Incubation of cultured embryonic cardiomyocytes (eCM) with recombined LTC(4) caused enhanced rates of reactive oxygen species (ROS) release measured via L012-luminescence method and apoptosis. Pharmacologic LTC(4) receptor blockade antagonized this effect in vitro. To evaluate the relevance of MRP1 mediated LTC(4) release after myocardial injury in vivo, MRP1(-/-) mice and FVB wildtype mice (WT) received cryoinjury of the left ventricle. Fourteen days after injury, left-ventricular ejection fraction (EF), end-diastolic volume (EDV), and akinetic myocardial mass (AMM) were quantified via echocardiography. MRP1(-/-) mice demonstrated increased EF (MRP1(-/-): 39 ± 3%, WT: 29 ± 4%) and reduced AMM (MRP1(-/-): 13 ± 2% WT: 16 ± 4%), indicating reduced post-infarction remodeling. Mechanistically, LTC(4) serum concentrations and levels of cellular apoptosis were increased in myocardial cryosections of FVB WT mice as compared to MRP1(-/-) mice. To identify key targets for pharmacological inhibition of LTC(4) actions, WT mice were treated with the specific Cys-LT1-receptor blocker Montelukast or the MRP1-Inhibitor MK571. Treatment of WT mice resulted in significant increase of EF (WT(Montelukast): 40 ± 5%, WT(MK571): 39 ± 3%, WT(vehicle): 33 ± 3% and decrease of AMM (WT(Montelukast): 12 ± 1%, WT(MK571): 10 ± 3%, WT(vehicle): 15 ± 5%) compared to untreated WT mice. CONCLUSION: Inhibition of leukotriene C(4) reduces levels of oxidative stress and apoptosis and demonstrates beneficial effects on myocardial remodeling after left ventricular injury.

Role of the multidrug resistance protein-1 (MRP1) for endothelial progenitor cell function and survival.

The multidrug resistance related protein-1 (MRP1) is a member of the ATP binding cassette (ABC) of cell surface transport proteins expressed in multiple cell lines and tissues including endothelial cells and haematopoietic stem cells. MRP1 blockade has been shown to prevent endothelial cell apoptosis and improve endothelial function. Besides mature endothelial cells vascular homing of endothelial progenitor cells (EPC) contributes to endothelial regeneration after vascular damage. Thus, we hypothesized that MRP1 influences number and function of EPCs and mechanisms of vascular repair. To test this, we investigated the effects of MRP1 inhibition in vitro and in vivo. MRP1 is abundantly expressed in cultured human early outgrowth EPCs. Pharmacological inhibition of MRP1 by MK571 increased intracellular glutathione levels and reduced intracellular reactive oxygen species levels. This stabilization of the intracellular redox homeostasis via inhibition of MRP1 prevented angiotensin II-induced apoptosis and increased the number of early outgrowth EPCs and colony forming units in vitro. To extend the observed cytoprotective effect of MRP1 blockade in EPCs to an in vivo situation, MRP1(-/-) knockout mice were investigated. MRP1(-/-) knockout mice showed significantly increased numbers of EPCs circulating in the peripheral blood and residing in the bone marrow. Consistently, colony forming unit formation was enhanced and rate of apoptosis reduced in early outgrowth EPCs derived from MRP1(-/-) knockout mice. In addition, MRP1(-/-) knockout mice showed improved reendothelialization after carotid artery injury, and transfusion of MNCs derived from MRP1(-/-) knockout mice into wild-type mice accelerated reendothelialization compared to transfusion of wild-type cells. These findings indicate that the enhanced function and survival of EPCs in MRP1(-/-) knockout mice resulted in improved reendothelialization. In conclusion, MRP1 negatively influences EPC function and survival via perturbation of the intracellular redox homeostasis which finally leads to increased cellular apoptosis. These results reveal novel mechanistic insights and may identify MRP1 as therapeutic target to improve reendothelialization after vascular damage.

Interaction of Inhibitor of DNA binding 3 (Id3) with Gut-enriched Krüppel-like factor (GKLF) and p53 regulates proliferation of vascular smooth muscle cells.

Enhanced proliferation of vascular smooth muscle cells (VSMCs) is one of the key features of the pathogenesis of atherosclerosis. The helix-loop-helix protein Inhibitor of DNA binding 3 (Id3) contributes to regulation of VSMC proliferation in a redox-sensitive manner. We investigated the role of Id3 and its interaction with other redox-sensitive genes, the transcription factor Gut-enriched Krüppel-like factor (GKLF, KLF4) and the tumor suppressor gene p53 in the regulation of VSMC proliferation. Cultured rat aortic VSMCs were transfected with Id3 sense and antisense constructs. Overexpression of Id3 significantly enhanced VSMC proliferation. Id3 antisense transfection inhibited VSMC proliferation induced by the physiological stimuli insulin and platelet-derived growth factor (PDGF). Because p53 is essential for the regulation of proliferation processes, the effect of Id3 on p53 expression was investigated. Id3 overexpression led to decreased p53 protein expression. Co-transfection of p53 sense constructs inhibited the enhanced VSMC mitogenicity induced by Id3 sense transfection. GKLF overexpression, which causes growth arrest in VSMCs, reduced Id3 promoter activity and led to decreased Id3 expression. Id3-induced VSMC proliferation was abolished by GKLF sense co-transfection. Finally, strong Id3 expression was found in the neointima of human coronary artery atherosclerotic plaques but not in healthy coronary arteries. These findings reveal a relevant interaction of GKLF, Id3, and p53 for VSMC proliferation which might constitute a general mechanism of growth control in vascular cells.

Angiotensin II triggers release of leukotriene C4 in vascular smooth muscle cells via the multidrug resistance-related protein 1.

The multidrug resistance-related protein-1 (MRP1) is important for the management of oxidative stress in vascular cells in vivo. Substrates of MRP1 are, among others, glutathione and the leukotriene C(4) (LTC(4)), an eicosanoid and mediator of inflammation. Angiotensin (Ang) II infusion results in MRP1(-/-) mice compared to wild-type mice in improved endothelial function and reduced reactive oxygen species (ROS) formation. However, the interaction between Ang II, LTC(4) and MRP1 is not completely understood and has never been investigated in vitro. Ang II induced in vascular smooth muscle cells (VSMC) the release of LTC(4) and the generation of ROS. Pharmacologic inhibition of MRP1 via MK 571 significantly reduced Ang II-induced ROS release (L012-luminescence) in VSMC. The release of ROS after Ang II stimulation is inhibited, to a comparable degree, by blockade of the Cys-LT1 receptor with montelukast. Incubation of VSMC with recombined LTC(4) and Ang II caused enhanced rates of proliferation in VSMC. This effect can be rescued by either MRP1 or Cys-LT1 receptor inhibition. Accordingly, stimulation of VSMC with LTC(4) reduces intracellular levels of glutathione, but does not affect apoptosis. LTC(4) stimulation results in a significant activation of MRP1, but does not alter MRP1 expression. These findings indicate a connection between Ang II, MRP1 and LTC(4). Both, MRP1 and LTC(4), are potentially promising targets for atheroprotective therapy.

Multidrug resistance protein-1 affects oxidative stress, endothelial dysfunction, and atherogenesis via leukotriene C4 export.

BACKGROUND: We recently showed that the multidrug resistance related protein-1 (MRP1) is important for the management of oxidative stress in vascular cells. However, the underlying mechanism and the in vivo relevance of these findings remain elusive. We hypothesize that inside-outside transport of leukotriene C(4) (LTC(4)) via MRP1 is a substantial proatherogenic mechanism in the vasculature. To test this hypothesis, we investigated the effects of MRP1 inhibition and LTC(4) receptor blockade (Cys-LT1 receptor) in vitro and in vivo. METHODS AND RESULTS: MRP1 is expressed abundantly in vascular smooth muscle cells (VSMCs). Pharmacological inhibition of MRP1 via MK571 reduces angiotensin II-induced reactive oxygen species release by 59% (L012 fluorescence) in VSMCs. The release of reactive oxygen species after angiotensin II stimulation also is inhibited by blockade of the Cys-LT1 receptor with montelukast. Incubation of VSMCs with recombined LTC(4) causes enhanced rates of reactive oxygen species and proliferation in wild-type and MRP1(-/-) VSMCs. Accordingly, the LTC(4) release in the cell culture supernatant of MRP1(-/-) VSMCs is significantly decreased compared with wild-type cells. To extend our observations to the in vivo situation, atherosclerosis-prone apolipoprotein E-deficient mice on a high-cholesterol diet were treated with placebo, the MRP1 inhibitor MK571, or the Cys-LT1 receptor inhibitor montelukast for 6 weeks. Treatment with MK571 or montelukast reduced vascular reactive oxygen species production, significantly improved endothelial function, and ameliorated atherosclerotic plaque generation by 52% and 61%, respectively. CONCLUSIONS: These findings indicate that MRP1 and LTC(4) exert proatherosclerotic effects and that both MRP1 and LTC(4) are potentially promising targets for atheroprotective therapy.

The heterogenous nuclear riboprotein S1-1 regulates AT1 receptor gene expression via transcriptional and posttranscriptional mechanisms.

The AT1 receptor plays an essential role in the pathogenesis of atherosclerosis. AT1 receptor expression is predominately mediated via mRNA destabilization by mRNA binding proteins. We identified via MALDI-analysis the heterogenous nuclear riboprotein S1-1 as an important regulator of AT1 receptor mRNA stability. The S1-1 protein possesses multiple nucleolar and cellular functions in vascular smooth muscle cells (VSMC). Overexpression of S1-1 sense resulted in VSMC in significant stabilization of AT1 receptor mRNA. However, this stabilization of the AT1 receptor mRNA is accompanied by a significantly reduced AT1 receptor mRNA transcription as shown via nuclear run-on assay resulting finally in reduced AT1 receptor mRNA levels. Additionally, S1-1 overexpression leads to increased apoptosis in VSMC and decreases VSMC proliferation.

Differential phosphorylation of calreticulin affects AT1 receptor mRNA stability in VSMC.

The AT1 receptor plays a pivotal role for the pathogenesis of hypertension and atherosclerosis. AT1 receptor expression is regulated posttranscriptionally via destabilization of the AT1 receptor mRNA by mRNA binding proteins. Recently, we identified calreticulin as a novel binding protein within the 3'untranslated region of the AT1 receptor mRNA. Calreticulin phosphorylation is essential for binding of the AT1 receptor mRNA. In crosslink experiments, we identified src kinase as the key enzyme for calreticulin phosphorylation. Overexpression of src sense DNA resulted in vascular smooth muscle cells (VSMC) in destabilization, overexpression of src antisense resulted in stabilisation of the AT1 receptor mRNA. Furthermore, phosphorylation/dephosphorylation sites of calreticulin and their impact on the AT1 receptor mRNA stability were investigated. VSMC were stimulated with AngII before tyrosine phosphorylation as well as serine phosphorylation of calreticulin were analysed via immunoprecipitation. Stimulation of VSMC with AngII resulted in enhanced tyrosine and reduced serine phosphorylation. Both effects are essential for AT1 mRNA stability as assessed by use of pharmacological inhibitors of serine dephosphorylation (cantharidin/ocadaic acid) or tyrosine phosphorylation (tyrphostin/orthovanadat). These findings imply an important role of serine dephosphorylation and tyrosine phosphorylation on calreticulin mediated AT1 receptor mRNA stability.

Role of the multidrug resistance protein-1 in hypertension and vascular dysfunction caused by angiotensin II.

OBJECTIVE: Human endothelial cells use the multidrug resistance protein-1 (MRP1) to export glutathione disulfide (GSSG). This can promotes thiol loss during states of increased glutathione oxidation. We investigated how MRP1 modulates blood pressure and vascular function during angiotensin II-induced hypertension. METHODS AND RESULTS: Angiotensin II-induced hypertension altered vascular glutathione flux by increasing GSSG export and decreasing vascular levels of glutathione in wild-type (FVB) but not in MRP1-/- mice. Aortic endothelium-dependent vasodilatation was reduced in FVB after angiotensin II infusion, but unchanged in MRP1-/- mice. Aortic superoxide (O2*-) production and expression of several NADPH oxidase subunits were increased by angiotensin II in FVB. These effects were markedly blunted in MRP1-/- vessels. The increase in O2*- production in FVB vessels caused by angiotensin II was largely inhibited by L-NAME, suggesting eNOS uncoupling. Accordingly, aortic tetrahydrobiopterin and levels of NO were decreased by angiotensin II in FVB but were unchanged in MRP1-/-. Finally, the hypertension caused by angiotensin II was markedly blunted in MRP1-/- mice (137+/-4 versus 158+/-6 mm Hg). CONCLUSION: MRP1 plays a crucial role in the genesis of multiple vascular abnormalities that accompany hypertension and its presence is essential for the hypertensive response to angiotensin II.

The role of the multidrug resistance protein-1 in modulation of endothelial cell oxidative stress.

Glutathione (GSH) is the major source of intracellular sulfhydryl groups. Oxidized GSH (GSSG) can be recycled to GSH by the GSH reductase or exported from the cell. The mechanism by which GSSG is exported and the consequence of its export from endothelial cells has not been defined previously. We found that human endothelial cells express the multidrug resistance protein-1 (MRP1) and use this as their major exporter of GSSG. Oscillatory shear stress, which is known to stimulate endothelial cell production of reactive oxygen species, decreased intracellular GSH. In contrast, laminar shear significantly increased intracellular GSH. Oscillatory shear also caused a robust export of GSSG that was prevented by the MRP1 inhibitor MK571 and by MRP1 small interfering RNA. MRP1 inhibition prevented the decline in intracellular GSH, preserved the intracellular GSH Nernst potential, and reduced apoptosis caused by oscillatory shear. In aortas of hypertensive mice, endothelial disulfide export was doubled, and this was prevented by MK571 and was not observed in aortas of hypertensive MRP1-/- mice. Further, the altered endothelium-dependent vasodilatation caused by hypertension was ameliorated in MRP1-/- mice. GSSG export by MRP1 leads to a perturbation of endothelial redox state and ultimately endothelial cell apoptosis. Endothelial MRP1 may provide a novel therapeutic target for prevention of vascular disease.

ATVB in focus: redox mechanisms in blood vessels.

Reactive oxygen species have been implicated in the pathogenesis of virtually every stage of vascular lesion formation, hypertension, and other vascular diseases. We are currently gaining insight into important sources of reactive oxygen species in the vessel wall, including the NADPH oxidases, xanthine oxidase, uncoupled nitric oxide synthase, and mitochondrial sources. Although various reactive oxygen species have pathological roles, some serve as important signaling molecules that modulate vascular tone, growth, and remodeling. In the next several months, a series of articles in Arteriosclerosis, Thrombosis, and Vascular Biology attempt to further elucidate how reactive oxygen species are produced by vascular cells and the roles of these in vascular homeostasis. This series promises to provide a valuable update on a wide variety of issues related to the biochemistry, molecular biology, and physiology of these important and fascinating molecules. Reactive oxygen species have been implicated in the pathogenesis of virtually every stage of vascular lesion formation, hypertension, and other vascular diseases. Upcoming series of articles in Arteriosclerosis, Thrombosis, and Vascular Biology help elucidate how reactive oxygen species are produced by vascular cells and their role in vascular homeostasis.