Contraction and relaxation of smooth muscle are regulated by myosin light-chain kinase and myosin phosphatase through phosphorylation and dephosphorylation of myosin light chains. Cyclic guanosine monophosphate (cGMP)-dependent protein kinase Ialpha (cGKIalpha) mediates physiologic relaxation of vascular smooth muscle in response to nitric oxide and cGMP. It is shown here that cGKIalpha is targeted to the smooth muscle cell contractile apparatus by a leucine zipper interaction with the myosin-binding subunit (MBS) of myosin phosphatase. Uncoupling of the cGKIalpha-MBS interaction prevents cGMP-dependent dephosphorylation of myosin light chain, demonstrating that this interaction is essential to the regulation of vascular smooth muscle cell tone.

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.

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.

In this study, we investigate a role for p8 in autophagy in vitro and in vivo, by using p8 −/− mice. In both settings, silencing of p8 is associated with basal up-regulation of autophagy and apoptosis. In vivo, the hearts of p8 knockout mice develop features that provoke a decreased left ventricular functionality.

Key Words: diabetic autonomic neuropathy Ⅲ SREBP Ⅲ insulin deficiency Ⅲ GIRK channel D iabetes mellitus is associated with severe debilitating complications that include a diabetic autonomic neuropathy (DAN) characterized by impairment of vascular reflexes, occasional hypotension, and decreased sympathetic and parasympathetic responsiveness of the heart. 1 Approximately 50% of patients with diabetes for 10 years or more demonstrate an impaired response of the heart to parasympathetic stimulation. 2 The presence of DAN is a significant risk factor, as demonstrated by a 5-fold higher 5-year mortality compared with diabetics without DAN. 3 Parasympathetic regulation of the heart has both a neuronal component involving vagal stimulation of parasympathetic ganglia in the atria and atrioventricular node, followed by release of acetylcholine, and a molecular component involving an intrinsic cardiac signaling pathway that mediates the parasympathetic response to acetylcholine. The latter involves the binding of acetylcholine to M 2 muscarinic receptors on the surface of cardiomyocytes and dissociation of the heterotrimeric G protein G i2 into G␣ i2 and G␤␥ subunits. G␤␥ binds to and activates the G protein-coupled inward rectifying K ϩ channel (GIRK1) 2 /(GIRK4) 2 , the channel that is responsible for I KAch (acetylcholine-gated K ϩ current), resulting in a decrease in the rate of diastolic depolarization and a decrease in heart rate (negative chronotropic effect). 4,5

Anchoring proteins to cell surface membranes by glycosylphosphatidylinositols (GPIs) is important. We have isolated a component of the putative transamidase machinery, hGaa1p (human GPI anchor attachment protein). hGAA1 cDNA is approximately 2 kb in length and codes 621 amino acids. The amino acid sequence of hGaa1p is 25% identical and 57% homologous to that of yeast Gaa1p. Moreover, Kite-Dolittle hydrophobicity plots of both proteins show marked similarity. hGAA1 gene is expressed ubiquitously and mRNA levels are higher in the undifferentiated state. Overexpression of antisense hGAA1 in human K562 cells significantly reduced the production of a reporter GPI-anchored protein.z 1998 Federation of European Biochemical Societies.

Extradural post-traumatic posterior fossa hematoma is a rare condition estimated to complicate about 0.13% of all craniocerebral injuries, and represents 5.8% of the entire group of extradural hematomas in our records. Nine cases of posterior fossa extradural hematomas are presented. The clinical picture was dominated by headache, vomiting, and gait ataxia. An occipital fracture was seen in 77.7% of the patients. In all cases, the diagnosis was made by computed tomography. The postoperative evolution was good.

Left ventricular remodeling, including the deposition of excess extracellular matrix, is key to the pathogenesis of heart failure. The stress-inducible transcriptional regulator p8 is increased in failing human hearts and is required both for agonist-stimulated cardiomyocyte hypertrophy and for cardiac fibroblasts matrix metalloprotease-9 (MMP9) induction. In the heart, upregulation of autophagy is an adaptive response to stress and plays a causative role in cardiomyopathies. We have recently shown that p8 ablation in cardiac cells upregulates autophagy and that, in vivo, loss of p8 results in a decrease of cardiac function. Here we investigated the effects of p8 genetic deletion in mediating adverse myocardial remodeling. Unstressed p8-/- mouse hearts manifested complex alterations in the expression of fibrosis markers. In addition, these mice displayed elevated autophagy and apoptosis compared with p8+/+ mice. Transverse aortic constriction (TAC) induced left ventricular p8 expression in p8+/+ mice. Pressure overload caused left ventricular remodeling in both genotypes, however, p8-/- mice showed less cardiac fibrosis induction. Consistent with this, although MMP9 induction was attenuated in the p8-/- mice, induction of MMP2 and MMP3 were strikingly upregulated while TIMP2 was downregulated. Left ventricular autophagy increased after TAC and was significantly higher in the p8-/- mice. Thus p8-deletion results in reduced collagen fibrosis after TAC, but in turn, is associated with a detrimental higher increase in autophagy. These findings suggest a role for p8 in regulating in vivo key signaling pathways involved in the pathogenesis of heart failure.