Glycolysis regulated transglutaminase 2 activation in cardiopulmonary fibrogenic remodeling.

The pathophysiology of pulmonary hypertension (PH) and heart failure (HF) includes fibrogenic remodeling associated with the loss of pulmonary arterial (PA) and cardiac compliance. We and others have previously identified transglutaminase 2 (TG2) as a participant in adverse fibrogenic remodeling. However, little is known about the biologic mechanisms that regulate TG2 function. We examined physiological mouse models of experimental PH, HF, and type 1 diabetes that are associated with altered glucose metabolism/glycolysis and report here that TG2 expression and activity are elevated in pulmonary and cardiac tissues under all these conditions. We additionally used PA adventitial fibroblasts to test the hypothesis that TG2 is an intermediary between enhanced tissue glycolysis and fibrogenesis. Our in vitro results show that glycolytic enzymes and TG2 are upregulated in fibroblasts exposed to high glucose, which stimulates cellular glycolysis as measured by Seahorse analysis. We examined the relationship of TG2 to a terminal glycolytic enzyme, pyruvate kinase M2 (PKM2), and found that PKM2 regulates glucose-induced TG2 expression and activity as well as fibrogenesis. Our studies further show that TG2 inhibition blocks glucose-induced fibrogenesis and cell proliferation. Our findings support a novel role for glycolysis-mediated TG2 induction and tissue fibrosis associated with experimental PH, HF, and hyperglycemia.

Targeted disruption of glycogen synthase kinase-3ß in cardiomyocytes attenuates cardiac parasympathetic dysfunction in type 1 diabetic Akita mice.

Type 1 diabetic Akita mice develop severe cardiac parasympathetic dysfunction that we have previously demonstrated is due at least in part to an abnormality in the response of the end organ to parasympathetic stimulation. Specifically, we had shown that hypoinsulinemia in the diabetic heart results in attenuation of the G-protein coupled inward rectifying K channel (GIRK) which mediates the negative chronotropic response to parasympathetic stimulation due at least in part to decreased expression of the GIRK1 and GIRK4 subunits of the channel. We further demonstrated that the expression of GIRK1 and GIRK4 is under the control of the Sterol Regulatory element Binding Protein (SREBP-1), which is also decreased in response to hypoinsulinemia. Finally, given that hyperactivity of Glycogen Synthase Kinase (GSK)3ß, had been demonstrated in the diabetic heart, we demonstrated that treatment of Akita mice with Li+, an inhibitor of GSK3ß, increased parasympathetic responsiveness and SREBP-1 levels consistent with the conclusion that GSK3ß might regulate IKACh via an effect on SREBP-1. However, inhibitor studies were complicated by lack of specificity for GSK3ß. Here we generated an Akita mouse with cardiac specific inducible knockout of GSK3ß. Using this mouse, we demonstrate that attenuation of GSK3ß expression is associated with an increase in parasympathetic responsiveness measured as an increase in the heart rate response to atropine from 17.3 ± 3.5% (n = 8) prior to 41.2 ± 5.4% (n = 8, P = 0.017), an increase in the duration of carbamylcholine mediated bradycardia from 8.43 ± 1.60 min (n = 7) to 12.71 ± 2.26 min (n = 7, P = 0.028) and an increase in HRV as measured by an increase in the high frequency fraction from 40.78 ± 3.86% to 65.04 ± 5.64 (n = 10, P = 0.005). Furthermore, patch clamp measurements demonstrated a 3-fold increase in acetylcholine stimulated peak IKACh in atrial myocytes from GSK3ß deficiency mice compared with control. Finally, western blot analysis of atrial extracts from knockout mice demonstrated increased levels of SREBP-1, GIRK1 and GIRK4 compared with control. Taken together with our prior observations, these data establish a role of increased GSK3ß activity in the pathogenesis of parasympathetic dysfunction in type 1 diabetes via the regulation of IKACh and GIRK1/4 expression.

QRS/T-wave and calcium alternans in a type I diabetic mouse model for spontaneous postmyocardial infarction ventricular tachycardia: A mechanism for the antiarrhythmic effect of statins.

BACKGROUND: The incidence of sudden arrhythmic death is markedly increased in diabetics. OBJECTIVE: The purpose of this study was to develop a mouse model for postmyocardial infarction (post-MI) ventricular tachycardia (VT) in the diabetic heart and determine the mechanism of an antiarrhythmic effect of statins. METHODS: ECG transmitters were implanted in wild-type (WT), placebo, and pravastatin-treated type I diabetic Akita mice. MIs were induced by coronary ligation, and Ca2+ transients were studied by optical mapping, and Ca2+ transients and sparks in left ventricular myocytes (VM) by the Ionoptix system and confocal microscopy. RESULTS: Burst pacing of Akita mouse hearts resulted in rate-related QRS/T-wave alternans, which was attenuated in pravastatin-treated mice. Post-MI Akita mice developed QRS/T-wave alternans and VT at 2820 ± 879 beats per mouse, which decreased to 343 ± 115 in pravastatin-treated mice (n = 13, P <.05). Optical mapping demonstrated pacing-induced VT originating in the peri-infarction zone and Ca2+ alternans, both attenuated in hearts of statin-treated mice. Akita VM displayed Ca2+ alternans, and triggered activity as well as increased Ca2+ transient decay time (Tau), Ca2+ sparks, and cytosolic Ca2+ and decreased SR Ca2+ stores all of which were in part reversed in cells from statin treated mice. Homogenates of Akita ventricles demonstrated decreased SERCA2a/PLB ratio and increased ratio of protein phosphatase (PP-1) to the PP-1 inhibitor PPI-1 which were reversed in homogenates of pravastatin-treated Akita mice. CONCLUSION: Pravastatin decreased the incidence of post-MI VT and Ca2+ alternans in Akita mouse hearts in part by revering abnormalities of Ca2+ handling via the PP-1/PPI-1 pathway.

Angiotensin II-induced TLR4 mediated abdominal aortic aneurysm in apolipoprotein E knockout mice is dependent on STAT3.

Abdominal Aortic Aneurysm (AAA) is a major cause of mortality and morbidity in men over 65 years of age. Male apolipoprotein E knockout (ApoE(-/-)) mice infused with angiotensin II (AngII) develop AAA. Although AngII stimulates both JAK/STAT and Toll-like receptor 4 (TLR4) signaling pathways, their involvement in AngII mediated AAA formation is unclear. Here we used the small molecule STAT3 inhibitor, S3I-201, the TLR4 inhibitor Eritoran and ApoE(-/-)TLR4(-/-) mice to evaluate the interaction between STAT3 and TLR4 signaling in AngII-induced AAA formation. ApoE(-/-) mice infused for 28 days with AngII developed AAAs and increased STAT3 activation and TLR4 expression. Moreover, AngII increased macrophage infiltration and the ratio of M1 (pro-inflammatory)/M2 (healing) macrophages in aneurysmal tissue as early as 7-10 days after AngII infusion. STAT3 inhibition with S3I-201 decreased the incidence and severity of AngII-induced AAA formation and decreased MMP activity and the ratio of M1/M2 macrophages. Furthermore, AngII-mediated AAA formation, MMP secretion, STAT3 phosphorylation and the ratio of M1/M2 macrophages were markedly decreased in ApoE(-/-)TLR4(-/-) mice, and in Eritoran-treated ApoE(-/-) mice. TLR4 and pSTAT3 levels were also increased in human aneurysmal tissue. These data support a role of pSTAT3 in TLR4 dependent AAA formation and possible therapeutic roles for TLR4 and/or STAT3 inhibition in AAA.

Glycogen synthase kinase-3ß inhibition ameliorates cardiac parasympathetic dysfunction in type 1 diabetic Akita mice.

Decreased heart rate variability (HRV) is a major risk factor for sudden death and cardiovascular disease. We previously demonstrated that parasympathetic dysfunction in the heart of the Akita type 1 diabetic mouse was due to a decrease in the level of the sterol response element-binding protein (SREBP-1). Here we demonstrate that hyperactivity of glycogen synthase kinase-3ß (GSK3ß) in the atrium of the Akita mouse results in decreased SREBP-1, attenuation of parasympathetic modulation of heart rate, measured as a decrease in the high-frequency (HF) fraction of HRV in the presence of propranolol, and a decrease in expression of the G-protein coupled inward rectifying K(+) (GIRK4) subunit of the acetylcholine (ACh)-activated inward-rectifying K(+) channel (IKACh), the ion channel that mediates the heart rate response to parasympathetic stimulation. Treatment of atrial myocytes with the GSK3ß inhibitor Kenpaullone increased levels of SREBP-1 and expression of GIRK4 and IKACh, whereas a dominant-active GSK3ß mutant decreased SREBP-1 and GIRK4 expression. In Akita mice treated with GSK3ß inhibitors Li(+) and/or CHIR-99021, Li(+) increased IKACh, and Li(+) and CHIR-99021 both partially reversed the decrease in HF fraction while increasing GIRK4 and SREBP-1 expression. These data support the conclusion that increased GSK3ß activity in the type 1 diabetic heart plays a critical role in parasympathetic dysfunction through an effect on SREBP-1, supporting GSK3ß as a new therapeutic target for diabetic autonomic neuropathy.

Increased inducibility of ventricular tachycardia and decreased heart rate variability in a mouse model for type 1 diabetes: effect of pravastatin.

Although a reduction in the high-frequency (HF) component of heart rate variability (HRV) is a major complication of diabetes and a risk factor for sudden death, its relationship to ventricular tachycardia (VT) is unknown. We developed a mouse model for the study of VT and its relationship to changes in HRV in the Akita type 1 diabetic mouse. Programmed ventricular stimulation of anesthetized mice demonstrated that Akita mice were more inducible for VT compared with wild-type mice: 78.6% versus 28.6% (P = 0.007). Optical mapping of perfused hearts demonstrated multifocal breakthroughs that occasionally gave rise to short-lived rotors consistent with focal initiation and maintenance of VT. Treatment of Akita mice with pravastatin, which had been previously shown clinically to decrease ventricular ectopy and to increase HRV, decreased the inducibility of VT: 36.8% compared with 75.0% with placebo treatment (P = 0.022). The HF fraction of HRV was reduced in Akita mice (48.6 ± 5.2% vs. 70.9 ± 4.8% in wild-type mice, P = 0.005) and was increased compared with placebo treatment in pravastatin-treated mice. Pretreatment of Akita mice with the muscarinic agonist carbamylcholine or the ß-adrenergic receptor blocker propranolol decreased the inducibility of VT (P = 0.001). In conclusion, the increased inducibility of focally initiated VT and reduced HF fraction in Akita mice were partially reversed by both pravastatin treatment and pharmacologic reversal of parasympathetic dysfunction. In this new animal model for the study of the pathogenesis of VT in type 1 diabetes, pravastatin may play a role in the prevention of VT by attenuating parasympathetic dysfunction.

Phenylephrine as a simulated intravascular epidural test dose in pediatrics: a pilot study.

BACKGROUND: A test dose is used to detect intravascular injection during neuraxial block in pediatrics. Accidental intravascular epidural local anesthetic injection might be unrecognized in anesthetized children leading to potential life-threatening complications. In children, sevoflurane anesthesia blunts the hemodynamic response when intravascular cathecolamines are administered. No studies have explored the hemodynamics and the criteria for a positive test dose result following phenylephrine in sevoflurane anesthetized children. METHODS: Healthy children undergoing minor procedures were randomly assigned to receive intravenous placebo, or 5 µg∙kg(-1) phenylephrine (n = 11/group) during sevoflurane anesthesia. Hemodynamic response was assessed using electrocardiography, pulse oxymetry and non-invasive blood pressure monitoring for 5 min following drug administration in anesthetized patients. RESULTS: All patients receiving phenylephrine showed a decreased heart rate (HR) but not all of them met the positive criterion for test dose response. Overall, at 1 min, patients receiving phenylephrine showed a 25% decrease in HR from the baseline while an increase in blood pressure was noticed in 54% of patients receiving phenylephrine. DISCUSSION: Phenylephrine might be a future indicator of positive intravascular test dose. Further investigation is needed to find out the phenylephrine dose that elicits a reliable hemodynamic response and whether phenylephrine needs to be dose age-adjusted in order to appreciate relevant hemodynamic changes in children receiving neuraxial blocks undergoing general anesthesia.

Role of toll-like receptor 4 in intimal foam cell accumulation in apolipoprotein E-deficient mice.

OBJECTIVE: Atherosclerosis encompasses a conspicuously maladaptive inflammatory response that might involve innate immunity. Here, we compared the role of Toll-like receptor 4 (TLR4) with that of TLR2 in intimal foam cell accumulation and inflammation in apolipoprotein E (ApoE) knockout (KO) mice in vivo and determined potential mechanisms of upstream activation and downstream action. METHODS AND RESULTS: We measured lipid accumulation and gene expression in the lesion-prone lesser curvature of the aortic arch. TLR4 deficiency reduced intimal lipid by ≈75% in ApoE KO mice, despite unaltered total serum cholesterol and triglyceride levels, whereas TLR2 deficiency reduced it by ≈45%. TLR4 deficiency prevented the increased interleukin-1α (IL-1α) and monocyte chemoattractant protein-1 mRNA levels seen within lesional tissue, and it also lowered serum IL-1α levels. Smooth muscle cells (SMC) were present within the intima of the lesser curvature of the aortic arch at this early lesion stage, and they enveloped and permeated nascent lesions, which consisted of focal clusters of foam cells. Cholesterol enrichment of SMC in vitro stimulated acyl-coenzyme A:cholesterol acyltransferase-1 mRNA expression, cytoplasmic cholesterol ester accumulation, and monocyte chemoattractant protein-1 mRNA and protein expression in a TLR4-dependent manner. CONCLUSIONS: TLR4 contributes to early-stage intimal foam cell accumulation at lesion-prone aortic sites in ApoE KO mice, as does TLR2 to a lesser extent. Intimal SMC surround and penetrate early lesions, where TLR4 signaling within them may influence lesion progression.

Differential effects of statins (pravastatin or simvastatin) on ventricular ectopic complexes: Galpha(i2), a possible molecular marker for ventricular irritability.

Retrospective studies suggest that statins might exert an antiarrhythmic effect on the heart. The mechanism of this effect is unclear. Parasympathetic stimulation of the heart has been shown to protect against ventricular arrhythmias. The goal of this study was to determine the effect of statins on ventricular arrhythmias and its correlation with changes in parasympathetic responsiveness and Galpha(i2) expression. Patients were randomized to pravastatin and simvastatin in a double-blind crossover design. Ventricular arrhythmias were determined by analysis of 24-hour Holter recordings. Spectral RR interval analysis of Holter studies determined peak high-frequency power fraction, which reflects parasympathetic modulation of heart rate. Expression of Galpha(i2), a molecular component of the parasympathetic response pathway, was determined by Western blots of patients' lymphocytes. Pravastatin treatment decreased the incidence of ventricular premature complexes by 22.5 + or - 3.4% (n = 20, p <0.05), couplets, and runs of 3 to 6 beats of nonsustained ventricular tachycardia from 9.8 + or - 2.67 to 3.9 + or - 1.25 events/patient/24 hours (n = 12, p <0.05). Pravastatin increased peak high-frequency fraction by 29.8 + or - 4.3% (n = 33, p <0.001), while Galpha(i2) expression increased by 51.3 + or - 22.5% (n = 21, p <0.05). Effects of simvastatin on ventricular premature complexes and nonsustained ventricular tachycardia were not significant. Relative changes in couplets and nonsustained ventricular tachycardia in pravastatin-treated patients correlated negatively with changes in Galpha(i2) and high-frequency fraction (rho = -0.588 and rho = -0.763, respectively, n = 12, p <0.05). In conclusion, these data suggest that pravastatin might decrease cardiac irritability via an increase in parasympathetic responsiveness and that changes in Galpha(i2) expression might serve as a molecular marker for this effect, which might play a role in the molecular mechanism of the antiarrhythmic effect of statins.

Simvastatin inhibits angiotensin II-induced abdominal aortic aneurysm formation in apolipoprotein E-knockout mice: possible role of ERK.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a life-threatening disease affecting almost 10% of the population over age 65. Generation of AAAs by infusion of angiotensin (Ang) II in apolipoprotein E-knockout (ApoE(-/-)) mice is an animal model which supports an imbalance of the renin-angiotensin system in the pathogenesis of AAA. The effect of statins on AngII-mediated AAA formation and the associated neovascularization is not known. Here we determined the effect of simvastatin and the ERK inhibitor, CI1040, on AngII-stimulated AAA formation. METHODS AND RESULTS: ApoE(-/-) mice infused for 28 days with AngII using osmotic minipumps were treated with placebo, 10 mg/kg/d simvastatin, or 100 mg/kg/d CI1040. 95% of AngII-treated mice developed AAA with neovascularization of the lesion, increased ERK phosphorylation, MCP-1 secretion, and MMP activity. These effects were markedly reversed by simvastatin and in part by CI1040. Furthermore, simvastatin and the ERK inhibitor U0126 reversed AngII-stimulated angiogenesis and MMP secretion by human umbilical vein endothelial cells. CONCLUSIONS: These data support the conclusion that simvastatin interferes with AAA formation induced by AngII in ApoE(-/-) mice at least in part via ERK inhibition.

Role of SREBP-1 in the development of parasympathetic dysfunction in the hearts of type 1 diabetic Akita mice.

RATIONALE: Diabetic autonomic neuropathy (DAN), a major complication of diabetes mellitus, is characterized, in part, by impaired cardiac parasympathetic responsiveness. Parasympathetic stimulation of the heart involves activation of an acetylcholine-gated K+ current, I(KAch), via a (GIRK1)2/(GIRK4)2 K+ channel. Sterol regulatory element binding protein-1 (SREBP-1) is a lipid-sensitive transcription factor. OBJECTIVE: We describe a unique SREBP-1-dependent mechanism for insulin regulation of cardiac parasympathetic response in a mouse model for DAN. METHODS AND RESULTS: Using implantable EKG transmitters, we demonstrated that compared with wild-type, Ins2(Akita) type I diabetic mice demonstrated a decrease in the negative chronotropic response to carbamylcholine characterized by a 2.4-fold decrease in the duration of bradycardia, a 52+/-8% decrease in atrial expression of GIRK1 (P<0.01), and a 31.3+/-2.1% decrease in SREBP-1 (P<0.05). Whole-cell patch-clamp studies of atrial myocytes from Akita mice exhibited a markedly decreased carbamylcholine stimulation of I(KAch) with a peak value of -181+/-31 pA/pF compared with -451+/-62 pA/pF (P<0.01) in cells from wild-type mice. Western blot analysis of extracts of Akita mice demonstrated that insulin treatment increased the expression of GIRK1, SREBP-1, and I(KAch) activity in atrial myocytes from these mice to levels in wild-type mice. Insulin treatment of cultured atrial myocytes stimulated GIRK1 expression 2.68+/-0.12-fold (P<0.01), which was reversed by overexpression of dominant negative SREBP-1. Finally, adenoviral expression of SREBP-1 in Akita atrial myocytes reversed the impaired I(KAch) to levels in cells from wild-type mice. CONCLUSIONS: These results support a unique molecular mechanism for insulin regulation of GIRK1 expression and parasympathetic response via SREBP-1, which might play a role in the pathogenesis of DAN in response to insulin deficiency in the diabetic heart.

Parasympathetic response in chick myocytes and mouse heart is controlled by SREBP.

Parasympathetic stimulation of the heart, which provides protection from arrhythmias and sudden death, involves activation of the G protein-coupled inward rectifying K+ channel GIRK1/4 and results in an acetylcholine-sensitive K+ current, I KACh. We describe a unique relationship between lipid homeostasis, the lipid-sensitive transcription factor SREBP-1, regulation of the cardiac parasympathetic response, and the development of ventricular arrhythmia. In embryonic chick atrial myocytes, lipid lowering by culture in lipoprotein-depleted serum increased SREBP-1 levels, GIRK1 expression, and I KACh activation. Regulation of the GIRK1 promoter by SREBP-1 and lipid lowering was dependent on interaction with 2 tandem sterol response elements and an upstream E-box motif. Expression of dominant negative SREBP-1 (DN-SREBP-1) reversed the effect of lipid lowering on I KACh and GIRK1. In SREBP-1 knockout mice, both the response of the heart to parasympathetic stimulation and the expression of GIRK1 were reduced compared with WT. I KACh, attenuated in atrial myocytes from SREBP-1 knockout mice, was stimulated by SREBP-1 expression. Following myocardial infarction, SREBP-1 knockout mice were twice as likely as WT mice to develop ventricular tachycardia in response to programmed ventricular stimulation. These results demonstrate a relationship between lipid metabolism and parasympathetic response that may play a role in arrhythmogenesis.

Simvastatin potentiates tumor necrosis factor alpha-mediated apoptosis of human vascular endothelial cells via the inhibition of the geranylgeranylation of RhoA.

HMG-CoA reductase inhibitors (statins) are widely used in the treatment and prevention of atherosclerosis. Here we demonstrate that the HMG-CoA reductase inhibitor simvastatin potentiates TNFalpha-mediated apoptosis and TNFalpha signaling in human umbilical vein endothelial cells (HUVECs). While 2.5 microM simvastatin or 40 ng/ml TNFalpha alone had only a small effect on apoptosis in HUVECs, co-incubation with simvastatin and TNFalpha markedly increased apoptosis in a time- and dose-dependent manner as measured by FACS analysis of propidium iodide-stained cells. Geranylgeraniol, which serves as a substrate for the geranylgeranylation of small GTP binding proteins such as RhoA, which is required for the function and membrane localization of Rho, reversed the effect of simvastatin on apoptosis. GGTI, an inhibitor of protein geranylgeranylation, mimicked the effect of simvastatin on apoptosis and interfered with the membrane localization of RhoA. Furthermore, simvastatin increased the expression of the TNFalpha type I receptor (TNFalphaRI) with a dose dependence and a dependence on geranylgeranylation similar to that demonstrated for the potentiation of TNFalpha-mediated apoptosis. Adenoviral expression of a dominant-negative RhoA mimicked the effect of simvastatin on the expression of TNFalphaRI, while adenoviral expression of a dominant-activating RhoA mutant reversed the effect of simvastatin on the expression of TNFalphaRI. Simvastatin also potentiated TNFalpha signaling as determined by increased TNFalpha-mediated E-selectin expression. These data support the conclusion that TNFalpha signaling is under the negative control of RhoA and that statins potentiate TNFalpha signaling at least in part via interference with RhoA inhibition of TNFalpha type I receptor expression.

Transforming growth factor beta regulates the expression of the M2 muscarinic receptor in atrial myocytes via an effect on RhoA and p190RhoGAP.

Transforming growth factor beta (TGFbeta) signaling is involved in the development and regulation of multiple organ systems and cellular signaling pathways. We recently demonstrated that TGFbeta regulates the response of atrial myocytes to parasympathetic stimulation. Here, TGFbeta(1) is shown to inhibit expression of the M(2) muscarinic receptor (M(2)), which plays a critical role in the parasympathetic response of the heart. This effect is mimicked by overexpression of a dominant negative mutant of RhoA and by the RhoA kinase inhibitor Y27632, whereas adenoviral expression of a dominant activating-RhoA reverses TGFbeta inhibition of M(2) expression. TGFbeta(1) also mediates a decrease in GTP-bound RhoA and a reciprocal increase in the expression of the RhoA GTPase-activating protein, p190RhoGAP, whereas total RhoA is unchanged. Inhibition of M(2) promoter activity by TGFbeta(1) is mimicked by overexpression of p190RhoGAP, whereas a dominant negative mutant of p190RhoGAP reverses this effect of TGFbeta(1). In contrast to atrial myocytes, in mink lung epithelial cells, in which TGFbeta signaling through activation of RhoA has been previously identified, TGFbeta(1) stimulated an increase in GTP-bound RhoA in association with a reciprocal decrease in the expression of p190RhoGAP. Both effects demonstrated a similar dose dependence on TGFbeta(1). Thus TGFbeta regulation of M(2) muscarinic receptor expression is dependent on RhoA, and TGFbeta regulation of p190RhoGAP expression may be a cell type-specific mechanism for TGFbeta signaling through RhoA.

Human umbilical vein endothelial cells and human dermal microvascular endothelial cells offer new insights into the relationship between lipid metabolism and angiogenesis.

Human umbilical vein endothelial cells (HUVECs) have played a major role as a model system for the study of the regulation of endothelial cell function and the role of the endothelium in the response of the blood vessel wall to stretch, shear forces, and the development of atherosclerotic plaques and angiogenesis. Here, we use HUVECs and human microvascular endothelial cells to study the role of the HMG-CoA reductase inhibitor, simvastatin, and the small GTP-binding protein Rho in the regulation of angiogenesis. Simvastatin inhibited angiogenesis in response to FGF-2 in the corneal pocket assay of the mouse and in vascular endothelial growth factor (VEGF)-stimulated angiogenesis in the chick chorioallontoic membrane. Furthermore, simvastatin inhibited VEGF-stimulated tube formation by human dermal microvascular endothelial cells and the formation of honeycomb-like structures by HUVECs. The effect was dose-dependent and was not secondary to apoptosis. Geranylgeranyl-pyrophosphate (GGPP), a product of the cholesterol metabolic pathway that serves as a substrate for the posttranslational lipidation of RhoA, was required for membrane localization, but not farnesylpyrophosphate (FPP), the substrate for the lipidation of Ras. Furthermore, GGTI, a specific inhibitor of GGPP, mimicked the effect of simvastatin of tube formation and the formation of honeycombs whereas FTI, a specific inhibitor of the farnesylation of Ras, had no effect. Adenoviral expression of a DN-RhoA mutant mimicked the effect of simvastatin on tube formation and the formation of honeycombs, whereas a dominant activating mutant of RhoA reversed the effect of simvastatin on tube formation. Finally, simvastatin interfered with the membrane localization of RhoA with a dose-dependence similar to that for the inhibition of tube formation. Simvastatin also inhibited the VEGFstimulated phosphorylation of the VEGF receptor KDR, and the tyrosine kinase FAK, which plays a role in cell migration. These data demonstrate that simvastatin interfered with angiogenesis via the inhibition of RhoA. Data supporting a role for angiogenesis in the development and growth of atherosclerotic plaques suggest that this antiangiogenic effect of Statins might prevent the progression of atherosclerosis via the inhibition of plaque angiogenesis.

Lipid lowering by pravastatin increases parasympathetic modulation of heart rate: Galpha(i2), a possible molecular marker for parasympathetic responsiveness.

BACKGROUND: We have previously demonstrated in an in vitro model for lipid lowering that lipoprotein depletion resulted in a marked increase in the negative chronotropic response to the acetylcholine analogue carbamylcholine. In this study we used heart rate variability analysis to determine the effect of lipid lowering by statins on the response of the heart to parasympathetic stimulation. In parallel, we examined whether changes in parasympathetic responsiveness correlated with changes in the expression of Galpha(i2), a molecular component of the parasympathetic signaling pathway in the heart. METHODS AND RESULTS: Patients were randomized in a crossover study of pravastatin and simvastatin. R-R interval analysis of Holter monitor studies demonstrated that in patients treated initially with pravastatin, the peak high-frequency power fraction during sleep, which reflects parasympathetic modulation of heart rate, increased by 24.0+/-5.02% (SEM, n=13, P<0.001) compared with the untreated control value. Simvastatin had no significant effect. Western blot analysis of lymphocytes from patients treated with pravastatin demonstrated a 90.1+/-27.3% (n=10, P=0.009) increase in Galpha(i2) expression, whereas simvastatin had no effect. Relative changes in Galpha(i2) correlated significantly with the changes in the fraction of high-frequency power (rho=0.574, P=0.016). CONCLUSIONS: Taken together with our in vitro data, these data are the first to suggest that cholesterol lowering by pravastatin might increase the response of the heart to parasympathetic stimulation and that changes in Galpha(i2) expression might serve as a molecular marker for this effect.

Transforming growth factor beta (TGFbeta ) signaling via differential activation of activin receptor-like kinases 2 and 5 during cardiac development. Role in regulating parasympathetic responsiveness.

Little is known regarding factors that induce parasympathetic responsiveness during cardiac development. We demonstrated previously that in atrial cells cultured from chicks 14 days in ovo, transforming growth factor beta (TGFbeta) decreased parasympathetic inhibition of beat rate by the muscarinic agonist, carbamylcholine, by 5-fold and decreased expression of Galpha(i2). Here in atrial cells 5 days in ovo, TGFbeta increased carbamylcholine inhibition of beat rate 2.5-fold and increased expression of Galpha(i2). TGFbeta also stimulated Galpha(i2) mRNA expression and promoter activity at day 5 while inhibiting them at day 14 in ovo. Over the same time course expression of type I TGFbeta receptors, chick activin receptor-like kinase 2 and 5 increased with a 2.3-fold higher increase in activin receptor-like kinase 2. Constitutively active activin receptor-like kinase 2 inhibited Galpha(i2) promoter activity, whereas constitutively active activin receptor-like kinase 5 stimulated Galpha(i2) promoter activity independent of embryonic age. In 5-day atrial cells, TGFbeta stimulated the p3TP-lux reporter, which is downstream of activin receptor-like kinase 5 and had no effect on the activity of the pVent reporter, which is downstream of activin receptor-like kinase 2. In 14-day cells, TGFbeta stimulated both pVent and p3TP-lux. Thus TGFbeta exerts opposing effects on parasympathetic response and Galpha(i2) expression by activating different type I TGFbeta receptors at distinct stages during cardiac development.

TGFbeta regulates the expression of G alpha(i2) via an effect on the localization of ras.

The negative chronotropic response of the heart to parasympathetic stimulation is mediated via the interaction of M(2) muscarinic receptors, Galpha(i2) and the G-protein coupled inward rectifying K(+) channel, GIRK1. Here TGFbeta(1) is shown to decrease the expression of Galpha(i2) in cultured chick atrial cells in parallel with attenuation of the negative chronotropic response to parasympathetic stimulation. The response to the acetylcholine analogue, carbamylcholine, decreased from a 95+/-2% (+/-SEM, n=8) inhibition of beat rate in control cells to 18+/-2% (+/-SEM,n =8) in TGFbeta(1) treated cells. Data support the conclusion that TGFbeta regulation of Galpha(i2) expression was mediated via an effect on Ras. TGFbeta(1) inhibited Galpha(i2) promoter activity by 56+/-6% (+/-SEM, n=4) compared to control. A dominant activating Ras mutant reversed the effect of TGFbeta on Galpha(i2) expression and stimulated Galpha(i2) promoter activity 1.7 fold above control. A dominant negative Ras mutant mimicked the effect of TGFbeta(1) on Galpha(i2) promoter activity. TGFbeta had no effect on the ratio of GDP/GTP bound Ras, but markedly decreased the level of membrane associated Ras and increased the level of cytoplasmic Ras compared to control. Furthermore, farnesol, a precursor to farnesylpyrophosphate, the substrate for the farnesylation of Ras, not only reversed TGFbeta(1) inhibition of Ras localization to the membrane, but also reversed TGFbeta(1) inhibition of Galpha(i2)promoter activity. FTI-277, a specific inhibitor of the farnesylation of Ras, mimicked the effect of TGFbeta(1) on Ras localization and Galpha(i2) promoter activity. These data suggest a novel relationship between TGFbeta signaling, regulation of Ras function and the autonomic response of the heart.

Role of sterol regulatory element binding proteins in the regulation of Galpha(i2) expression in cultured atrial cells.

We have previously demonstrated that growth of embryonic chick atrial cells in medium supplemented with lipoprotein-depleted serum (LPDS) resulted in a coordinate increase in the expression of genes involved in the parasympathetic response of the heart (the M2 muscarinic receptor; the alpha-subunit of the heterotrimeric G protein, Galpha(i2); and the inward rectifying K+ channel protein, GIRK1) and a marked increase in the negative chronotropic response of atrial cells to muscarinic stimulation. In the present study, we demonstrate that regulation of Galpha(i2) promoter activity by LPDS is mediated by the binding of a sterol regulatory element binding protein (SREBP) to a sterol regulatory element (SRE) in the Galpha(i2) promoter. Deletion and point mutation of this putative SRE interfered with the regulation of the Galpha(i2) promoter by SREBP and LPDS. Furthermore gel shift assays demonstrated that point mutations in the putative Galpha(i2) SRE markedly inhibited the binding of purified SREBP to oligonucleotides containing the Galpha(i2) SRE sequence. The expression of a dominant-negative SREBP mutant interfered with LPDS stimulation of Galpha(i2) promoter activity. Finally, we demonstrate that SREBP-1 is markedly more potent than SREBP-2 for the stimulation of Galpha(i2) promoter activity, suggesting that SREBP1 may play a role in the regulation of Galpha(i2) expression. These are the first data to demonstrate SREBP regulation of a protein not involved in lipid homeostasis and suggest a new relationship between lipid metabolism and the parasympathetic response of the heart.

3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors interfere with angiogenesis by inhibiting the geranylgeranylation of RhoA.

Angiogenesis is implicated in the pathogenesis of cancer, rheumatoid arthritis, and atherosclerosis and in the treatment of coronary artery and peripheral vascular disease. Here, cholesterol-lowering agents, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are shown to interfere with angiogenesis. In vivo, the HMG-CoA reductase inhibitor simvastatin dose-dependently inhibited capillary growth in both vascular endothelial growth factor-stimulated chick chorioallantoic membranes and basic fibroblast growth factor-stimulated mouse corneas. In vitro, the development of tubelike structures by human microvascular endothelial cells cultured on 3D collagen gels was inhibited at simvastatin concentrations similar to those found in the serum of patients on therapeutic doses of this agent. HMG-CoA reductase inhibitors interfered with angiogenesis via inhibition of the geranylgeranylation and membrane localization of RhoA. Simvastatin inhibited membrane localization of RhoA with a concentration dependence similar to that for the inhibition of tube formation, whereas geranylgeranyl pyrophosphate, the substrate for the geranylgeranylation of Rho, reversed the effect of simvastatin on tube formation and on the membrane localization of RhoA. Furthermore, tube formation was inhibited by GGTI, a specific inhibitor of the geranylgeranylation of Rho; by C3 exotoxin, which inactivates Rho; and by the adenoviral expression of a dominant-negative RhoA mutant. The expression of a dominant-activating RhoA mutant reversed the effect of simvastatin on tube formation. Finally, HMG-CoA reductase inhibitors inhibited signaling by vascular endothelial growth factor, Akt, and focal adhesion kinase, three RhoA-dependent pathways known to be involved in angiogenesis. This study demonstrates a new relationship between lipid metabolism and angiogenesis and an antiangiogenic effect of HMG-CoA reductase inhibitors with possible important therapeutic implications.