The estrogen receptor (ER) is a transcription factor whose activity is normally activated by the hormone estradiol and inhibited by antiestrogen. It has been found that certain mutational changes in the activation function-2 region in the hormone-binding domain of the human ER result in ligand activity inversion mutants, i.e. receptors that are now activated by antiestrogen and inhibited by estrogen. The ER point mutant L540Q is activated by several antiestrogens (the more pure antiestrogens ICI 164,384 and RU 54,876 or the partial antiestrogen trans-hydroxytamoxifen) but not by estradiol. The presence of the F domain and an intact activation function-i in the A/B domain are required for this activity, as is the DNA-binding ability of the receptor. This inverted ligand activity is observed with several estrogen-responsive promoters, both simple and complex; however, the activating ability of antiestrogens is observed only in some cells, highlighting the important role of cell-specific factors in ligand interpretation. The introduction of two additional amino acid changes close to 540 results in receptors that are still not activated by estradiol but are now able to distinguish between partial antiestrogens (which remain agonistic) and pure antiestrogens (which show a greatly reduced stimulatory activity). These ligand activity inversion mutants remain stable in cells in the presence of the antiestrogen ICI 164,384, as does a related ER mutant receptor that shows the normal, wild type ER ligand activity profile in which ICI 164,384 is transcriptionally inactive. Thus, the presence of adequate levels of mutant ER may be necessary but not sufficient for ICI 164,384 to elicit transcriptional activity. These findings highlight the means by which the carboxyl-terminal region in domain E functions to interpret the activity of a ligand, and they demonstrate that rather minimal changes in the ER can result in receptors with inverted response to antiestrogen and estrogen. Such point mutations, if present in estrogen target cells, would result in antiestrogens being seen as growth stimulators, rather than suppressors, with potentially detrimental consequences in terms of breast cancer treatment with antiestrogens.

Summary Insulin-like growth factor (IGF)-1 has been implicated in the development of occlusive vascular lesions. Although its role in vascular smooth muscle cell (VSMC) growth and migration are fairly well characterized, antiapoptotic signals of IGF-1 in human VSMC remain largely unknown. In this study, we examined IGF-1 signals that protect human and rat VSMC from staurosporine (STAU)-and c-myc -induced apoptosis, respectively. Treatment with STAU resulted in apoptotic DNA fragmentation, phosphatidylserine externalization and cell shrinkage, but only occasional VSMC 'blebbing'. STAU-induced death and IGF-1-mediated survival were concentration dependent, while time-lapse video microscopy showed that IGF-1 inhibited c-myc -induced apoptosis by 90%. Pretreatment with mitogen-activated protein kinase/extracellular signal regulated kinase kinase (MEK) inhibitors UO126 and PD098059, or with the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin, reversed IGF-1-mediated human VSMC survival by 25-27% and 66%, respectively. Translocation studies showed that IGF-1 activated protein kinase C (PKC)-ε , but not PKC-α or PKC-δ , even in the presence of STAU, while pharmacological PKC inhibition (Ro-318220 or Go6976) implicated PKC-ζ or a novel PKC isozyme in IGF-1-mediated survival. Transient expression of activated PKC-ε but not activated PKC-ζ decreased myc -induced apoptosis in rat VSMC. In human VSMC, antisense oligodeoxynucleotides to PKC-ε partially reversed IGF-1-induced survival. In addition, IGF-1 elicited a mild but sustained activation of extracellular signal regulated kinase (ERK)1/2 in human VSMC that was abolished after 1 h in the presence of STAU. PKC downregulation reversed both IGF-1-and PMA-induced ERK activity, but platelet-derived growth factor (PDGF)-induced activity was unchanged. These results indicate for the first time that IGF-1 can protect human VSMC via multiple signals, including PKC-ε , PI3-K and mitogenactivated protein kinase pathways.

Characteristics of hVSMC apoptosis and its inhibition by insulin-like growth factor-1 (IGF-1) remain unclear. Also unclear is whether a balance in hVSMCs exists whereby c-Jun N-terminal stress kinases (JNK) promote apoptosis while extracellular signal-regulated (ERK1/2) MAP kinases inhibit cell death. In this study, we examined the involvement of Akt/PKB and its upstream kinase, PDK1 and whether JNK activation correlated with human and rat VSMC apoptosis induced by staurosporine and by c-myc, respectively. We observed a strong, sustained JNK activation (and c-Jun phosphorylation), which correlated with VSMC apoptosis. IGF-1 (13.3 nM), during apoptosis inhibition, transiently inhibited JNK activity at 1 h in a phosphatidylinositol 3-kinase (PI3-K)- and MEK-ERK-dependent manner, as wortmannin (100 nM) or PD98059 (30 muM) partially attenuated the IGF-1 effect. PKC down-regulation had no effect on JNK inhibition by IGF-1. While IGF-1 alone produced a strong phosphorylation of Akt/PKB in hVSMCs up to 6 h, it was notably stronger and more sustained during ratmyc and hVSMCs apoptosis inhibition. Further, whereas transient expression of phosphorylated Akt protected VSMCs from apoptosis by nearly 50%, expression of dominant interfering alleles of Akt or PDK1 strongly inhibited IGF-1-mediated VSMC survival. These results demonstrate for the first time that transient inhibition of a pro-apoptotic stimulus in VSMCs may be sufficient to inhibit a programmed cell death and that sustained anti-apoptotic signals (Akt) elicited by IGF-1 are augmented during a death stimulus. Furthermore, PI3-K and ERK-MAPK pathways may cooperate to protect VSMCs from cell death.

These results show that coronary vessels are more prone to develop significant intimal hyperplasia and subsequent restenosis than internal iliac vessels. A better insight into how different arteries and arterial components behave is important in understanding and developing newer and better therapeutic measures for restenosis.

A 72-year-old man with a history of dilated cardiomyopathy, prosthetic aortic valve, biventricular intracardiac pacemaker, and permanent atrial fibrillation presented with uncontrolled ventricular response from atrial fibrillation despite pharmacological therapy. He underwent radiofrequency (RF) catheter ablation of the atrioventricular (AV) node to achieve optimal biventricular pacing.AV node ablation was performed via the right femoral vein with a 4-mm tip ablation catheter (EPT, BostonScientific) positioned at the AV node just proximal to the His bundle region. RF energy was applied at this site for 60 seconds (55°C) resulting in complete heart block with an escape rhythm of 32 bpm. The next day he was discharged uneventfully. An echocardiogram at 5 months follow-up showed a left ventricular to right atrial (LV-RA) shunt across the membranous septum immediately above the tricuspid valve (online-only Data Supplement Movies I and II and Figure 1A and 1B). This LV-RA shunt (Gerbode-type defect) had a 76-mm Hg gradient across the defect ( Figure 1C). Left ventricular ejection fraction was depressed (25% to 30%) and the right ventricle was mildly to moderately dilated. The mechanical aortic prosthetic valve functioned normally with a mean gradient of 17 mm Hg. Review of the patient's preablation echocardiographic studies did not show any evidence of LV-RA shunt. He remained stable with New York Heart Association class I to II and Ͼ85% biventricular pacing. A repeat echocardiographic study 14 months after the ablation procedure did not show any progression in the size of iatrogenic Gerbode-type defect or shunting.In a second case, a 68-year-old man with a history of severe chronic obstructive pulmonary disease and symptomatic permanent atrial fibrillation underwent RF catheter ablation of the AV node because of ineffective ventricular rate control by medical therapy. Three days after the procedure, the patient developed ventricular fibrillation and could not be resuscitated. Autopsy findings showed lesions at the RF ablation sites. Although he did not develop a frank defect, the ablation lesions extended to the left ventricular side of the membranous septum (Figure 2) at a site similar to the Gerbode-type defect in the first patient.