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.

Antiproliferative effect of estrogen in vascular smooth muscle cells is mediated by Kruppel-like factor-4 and manganese superoxide dismutase.

The mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD) and the zinc finger transcription factor Kruppel-like factor-4 (KLF4) are involved in the regulation of redox homeostasis, apoptosis and cell proliferation. We have shown that estrogen exerts antioxidative actions via induction of MnSOD in cultured rat aortic vascular smooth muscle cells (VSMC). The purpose of the present study was to investigate whether estrogen inhibits VSMC proliferation via alteration of KLF4 and MnSOD expression. In cultured rat aortic VSMC, estrogen binding to estrogen receptor-alpha led to rapid increase in KLF4 expression and reduction of cell proliferation by 50%. Protein separation revealed that KLF4 was shifted to the nucleus when VSMC were treated with estrogen. Estrogen-mediated induction of KLF4 and the antiproliferative effect involved activation of PI-3 kinase, Akt phosphorylation and induction of NO synthase activity. Experiments in freshly isolated denuded aortic segments revealed an increase in KLF4 abundance after estrogen treatment and demonstrated that eNOS is expressed in the media at low levels. Transfection experiments showed that estrogen-induced overexpression of MnSOD required KLF4 and that both KLF4 and MnSOD were indispensable for the observed antiproliferative effect of estrogen in VSMC. To confirm these data in vivo, we investigated neointima formation after carotid artery injury in wild-type (WT) and MnSOD+/- mice. Estrogen deficiency led to enhanced neointima formation and higher numbers of Ki67-positive proliferating cells in the neointima of ovariectomized WT and MnSOD+/- mice. Moreover, MnSOD+/- mice showed more extensive neointima formation and Ki67 immunostaining. Interestingly, estrogen replacement prevented neointima formation in WT mice but failed to completely inhibit neointima formation in MnSOD+/- mice. Cultured VSMC derived from MnSOD+/- mice showed enhanced proliferation as compared to WT VSMC, and estrogen treatment failed to inhibit proliferation in MnSOD+/- VSMC. In conclusion, these data demonstrate the importance of MnSOD and KLF4 for proliferation control in VSMC. Our results provide novel insights into how proliferation of VSMC is regulated by estrogen and may help to identify novel targets for the treatment of vascular diseases such as restenosis.

Inhibition of Rac1 GTPase Decreases Vascular Oxidative Stress, Improves Endothelial Function, and Attenuates Atherosclerosis Development in Mice.

Aims: Oxidative stress and inflammation contribute to atherogenesis. Rac1 GTPase regulates pro-oxidant NADPH oxidase activity, reactive oxygen species (ROS) formation, actin cytoskeleton organization and monocyte adhesion. We investigated the vascular effects of pharmacological inhibition of Rac1 GTPase in mice. Methods and Results: We treated wild-type and apolipoprotein E-deficient (ApoE-/-) mice with Clostridium sordellii lethal toxin (LT), a Rac1 inhibitor, and assessed vascular oxidative stress, expression and activity of involved proteins, endothelial function, macrophage infiltration, and atherosclerosis development. LT-treated wild-type mice displayed decreased vascular NADPH oxidase activity and ROS production. Therapeutic LT doses had no impact on behavior, food intake, body weight, heart rate, blood pressure, vascular and myocardial function, differential blood count, and vascular permeability. ApoE-/- mice were fed a cholesterol-rich diet and were treated with LT or vehicle. LT treatment led to decreased aortic Rac1 GTPase activity, NADPH oxidase activity and ROS production, but had no impact on expression and membrane translocation of NADPH oxidase subunits and RhoA GTPase activity. LT-treated mice showed improved aortic endothelium-dependent vasodilation, attenuated atherosclerotic lesion formation and reduced macrophage infiltration of atherosclerotic plaques. Concomitant treatment of cholesterol-fed ApoE-/- mice with LT, the specific synthetic Rac1 inhibitor NSC 23766 or simvastatin comparably reduced aortic Rac1 activity, NADPH oxidase activity, oxidative stress, endothelial dysfunction, atherosclerosis development, and macrophage infiltration. Conclusions: These findings identify an important role of the small GTPase Rac1 in atherogenesis and provide a potential target for anti-atherosclerotic therapy.

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.

Identification of a novel redox-sensitive gene, Id3, which mediates angiotensin II-induced cell growth.

BACKGROUND: Reactive oxygen species, such as superoxide (O(2)(-)), are involved in the abnormal growth of various cell types. Angiotensin II (Ang II) is one of the most potent inducers of oxidative stress in the vasculature. The molecular events involved in Ang II-induced proliferation of vascular smooth muscle cells (VSMCs) are only partially understood. METHODS AND RESULTS: Ang II as well as xanthine/xanthine oxidase (X/XO) led to enhanced DNA synthesis and proliferation of VSMCs. The effect of Ang II was abolished by diphenylene iodonium. Consequently, VSMCs were incubated with X/XO, and modulation of gene expression was monitored by differential display, leading to the identification of a novel redox-sensitive gene, the dominant-negative helix-loop-helix protein Id3, which was upregulated within 30 minutes by X/XO and Ang II. Superoxide dismutase but not catalase inhibited this effect. Overexpression of antisense Id3 via transfection in VSMCs completely abolished Ang II- and X/XO-induced cell proliferation. Ang II, X/XO, and overexpression of sense Id3 downregulated protein expression of p21(WAF1/Cip1), p27(Kip1), and p53. Overexpression of antisense Id3 abrogated the effect of Ang II on the expression of p21(WAF1/Cip1), p27(Kip1), and p53. Ang II and overexpression of sense Id3 caused hyperphosphorylation of the retinoblastoma protein. Ang II-induced phosphorylation of the retinoblastoma protein was decreased by overexpression of antisense Id3. CONCLUSIONS: Ang II induces proliferation of VSMCs via production of superoxide, which enhances the expression of Id3. Id3 governs the downstream mitogenic processing via depression of p21(WAF1/Cip1), p27(Kip1), and p53. These findings reveal a novel redox-sensitive pathway involved in growth control.

Thrombin inhibition by dabigatran attenuates atherosclerosis in ApoE deficient mice.

INTRODUCTION: Atherosclerosis is a chronic inflammatory disease characterized by endothelial cell damage, infiltration, proliferation and accumulation of macrophages, lymphocytes and transformed vascular smooth muscle cells within the vascular wall and procoagulation processes involving activation of plasmatic coagulation events and platelets. Numerous studies suggested a close interaction between thrombin action and atherogenesis, but possibly underlying mechanisms are multiple and specific treatment options were missing until now. MATERIAL AND METHODS: Atherosclerosis prone 12 weeks old ApoE(-/-) mice were fed a cholesterol rich diet for 4 weeks and were concomitantly treated orally with placebo or the thrombin inhibitor dabigatran (1.2 g/kg/day). RESULTS: The thrombin time (HEMOCLOT(®)) was significant extended in dabigatran treated animals. Vascular oxidative stress was significantly reduced during thrombin inhibition, as assessed by L012 chemiluminescence in aortic segments (212 ±84 vs. 69 ±21 RLU/s/mg dry weight, p = 0.048). Organ chamber experiments of isolated aortic rings showed that dabigatran treatment significantly improved endothelium-derived vasorelaxation (p < 0.001). Dabigatran treated mice developed less atherosclerotic lesions (6.2 ±0.2% vs. 9 ±1.1%, p = 0.037) and showed less infiltration of atherosclerotic lesions with macrophages (2.59 ±0.3% vs. 5.14 ±0.7%, p = 0.0046), as determined by systematic histological and immunohistological analyses of the aortic root. Blood pressure, body weight and food intake were not altered by the treatment. CONCLUSIONS: The thrombin inhibitor dabigatran reduces vascular oxidative stress and inflammation, improves endothelial function and decreases atherosclerosis in mice.

Acute changes of mitral valve geometry during interventional edge-to-edge repair with the MitraClip system are associated with midterm outcomes in patients with functional valve disease: preliminary results from a prospective single-center study.

BACKGROUND: Transcatheter mitral valve repair (TMVR) is a treatment option in patients with symptomatic functional or degenerative mitral regurgitation (DMR) at high surgical risk. The acute effect of MitraClip procedure on mitral valve (MV) annular geometry and its relation to functional outcomes is unclear. We sought to assess immediate effect of TMVR on MV annular geometry with 3-dimensional (3D) transesophageal echocardiography and the association of MV diameter reduction with functional response after 6 months. METHODS AND RESULTS: Consecutive patients (n = 111; age, 78.3 ± 8.1 years) at high surgical risk (logistic EuroSCORE [European System for Cardiac Operative Risk Evaluation], 29.8±21.5%) underwent TMVR. The procedure was completed successfully in 107 (96%) patients with 3D reconstruction of MV annular geometry immediately before and after clip implantation. Only patients with functional mitral regurgitation (n = 71) experienced an acute reduction of anterior-posterior MV diameters (4.0 ± 0.6 and 3.6 ± 0.6 cm; P < 0.0001), MV annulus areas (2D annulus area, 13.9 ± 3.8 and 12.8 ± 3.4 cm(2); P < 0.0001 and 3D annulus area, 14.4 ± 3.9 and 12.9 ± 3.4 cm(2); P < 0.0001), and MV annular geometry (MV sphericity index, 0.9 ± 0.1 and 0.8 ± 0.1; P < 0.0001); the lateral medial MV diameters remained unchanged (4.3 ± 0.7 and 4.4 ± 0.6 cm; P = 0.13). In subjects with DMR, all MV annular geometry-defining values were not significantly altered after TMVR (n = 36; P > 0.05). Acute anterior-posterior diameter reduction was associated with clinical response to TMVR after 6 months of follow-up (cutoff value, ≥ 6.4%; area under the curve, 0.81; P = 0.002; sensitivity, 81.6%; specificity, 81.8%), which was confirmed by additional regression analysis (P = 0.007). CONCLUSIONS: Three-dimensional transesophageal echocardiography enables assessment of acute changes of MV geometry in patients undergoing the MitraClip procedure. Only patients with functional mitral regurgitation experienced significant reduction of MV annular dimensions, which was associated with clinical response to TMVR.

Darbepoetin improves endothelial function and increases circulating endothelial progenitor cell number in patients with coronary artery disease.

BACKGROUND: Atherosclerosis is a progressive disease characterised in part by an imbalance of endothelial decline and endothelial repair. Erythropoietin has been connected to vasculoprotective effects such as enhanced nitric oxide production in endothelial cells and mobilisation of endothelial progenitor cells (EPC). OBJECTIVE: To determine the effect of erythropoietin on endothelial function and EPC mobilisation in humans with atherosclerosis. DESIGN: A prospective single-blind monocentric study of 20 patients randomly assigned to the test drug or placebo treatment over 4 weeks. METHODS: 20 Patients with stable coronary artery disease receiving optimal medical treatment with either weekly subcutaneous injections of saline (placebo) or the recombinant erythropoietin darbepoetin (60 µg/injection) over 3 consecutive weeks. At the initial and final visits, flow mediated dilatation (FMD) was determined by ultrasound. The number of EPC was determined as the number of CD34/CD133 positive mononuclear cells in peripheral blood. RESULTS: Treatment with darbepoetin resulted in a significantly improved FMD in each patient, whereas no difference was seen in placebo-treated patients. The FMD of darbepoetin-treated patients increased by 7.5±1.64%. Additionally, an increase in peripheral blood EPC of 50±24% was seen. CONCLUSION: Darbepoetin given in addition to optimal medical treatment resulted in a significantly improved endothelial function in patients with coronary artery disease, indicating a promising new atheroprotective treatment option.

Induction of p53 by GKLF is essential for inhibition of proliferation of vascular smooth muscle cells.

Proliferation of vascular smooth muscle cells (VSMC) plays an important role in the pathogenesis of atherosclerosis and restenosis. Recent studies have demonstrated that the transcription factor gut-enriched Krüppel-like factor (GKLF, KLF4) is involved in redox-sensitive growth arrest of VSMC. We investigated the role of GKLF in VSMC proliferation and differentiation and the potentially important interaction with the tumor suppressor gene p53. Cultured rat aortic VSMC were transfected with GKLF sense and antisense constructs by electroporation. GKLF enhanced the mRNA expression of the differentiation marker SM22-alpha, but had no effect on the expression of alpha-smooth muscle actin (real-time RT-PCR, Western blot). Overexpression of GKLF significantly reduced VSMC proliferation (cell count, BrdU FACS analysis). Because p53 is essential for proliferation processes, the effect of GKLF on p53 gene expression was investigated. GKLF overexpression led to an enhanced p53 promoter activity and increased p53 mRNA and protein expression (luciferase reporter assay, real-time PCR, Western blot). Consistently, GKLF overexpression induced an enhanced expression of the p53 target genes p21(WAF1/Cip1) and Mdm2. Co-transfection experiments revealed that the growth arrest induced by GKLF sense transfection was completely abolished by co-transfection of p53 antisense constructs, whereas the reduced proliferation exerted by p53 sense transfection was not inhibited by GKLF antisense transfection, suggesting that p53 induction is essential for the interference of GKLF with VSMC proliferation. Finally, stimulation of VSMC with hydroxyl radicals increased expression of GKLF and p53 and reduced cell proliferation. The transcription factor GKLF induces inhibition of proliferation of VSMC which is mechanistically linked to a GKLF-induced enhancement of the expression of the tumor suppressor gene p53.