Temporal trends in the use of invasive cardiac procedures for non-ST segment elevation acute coronary syndromes according to initial risk stratification.

BACKGROUND: Current guidelines support an early invasive strategy in the management of high-risk non-ST elevation acute coronary syndromes (NSTE-ACS). Although studies in the 1990s suggested that highrisk patients received less aggressive treatment, there are limited data on the contemporary management patterns of NSTE-ACS in Canada. OBJECTIVE: To examine the in-hospital use of coronary angiography and revascularization in relation to risk among less selected patients with NSTE-ACS. METHODS: Data from the prospective, multicentre Global Registry of Acute Coronary Events (main GRACE and expanded GRACE2) were used. Between June 1999 and September 2007, 7131 patients from across Canada with a final diagnosis of NSTE-ACS were included the study. The study population was stratified into low-, intermediate- and high-risk groups, based on their calculated GRACE risk score (a validated predictor of in-hospital mortality) and according to time of enrollment. RESULTS: While rates of in-hospital death and reinfarction were significantly (P<0.001) greater in higher-risk patients, the in-hospital use of cardiac catheterization in low- (64.7%), intermediate- (60.3%) and highrisk (42.3%) patients showed an inverse relationship (P<0.001). This trend persisted despite the increase in the overall rates of cardiac catheterization over time (47.9% in 1999 to 2003 versus 51.6% in 2004 to 2005 versus 63.8% in 2006 to 2007; P<0.001). After adjusting for confounders, intermediate-risk (adjusted OR 0.80 [95% CI 0.70 to 0.92], P=0.002) and high-risk (adjusted OR 0.38 [95% CI 0.29 to 0.48], P<0.001) patients remained less likely to undergo in-hospital cardiac catheterization. CONCLUSION: Despite the temporal increase in the use of invasive cardiac procedures, they remain paradoxically targeted toward low-risk patients with NSTE-ACS in contemporary practice. This treatment-risk paradox needs to be further addressed to maximize the benefits of invasive therapies in Canada.

Cross-talk between the p42/p44 MAP kinase and Smad pathways in transforming growth factor beta 1-induced furin gene transactivation.

Furin, a predominant convertase of the cellular constitutive secretory pathway, is known to be involved in the maturation of a number of growth/differentiation factors, but the mechanisms governing its expression remain elusive. We have previously demonstrated that transforming growth factor (TGF) beta 1, through the activation of Smad transducers, regulates its own converting enzyme, furin, creating a unique activation/regulation loop of potential importance in a variety of cell fate and functions. Here we studied the involvement of the p42/p44 MAPK pathway in such regulation. Using HepG2 cells transfected with fur P1 LUC (luciferase) promoter construct, we observed that forced expression of a dominant negative mutant form of the small G protein p21(ras) (RasN17) inhibited TGF beta 1-induced fur gene transcription, suggesting the involvement of the p42/p44 MAPK cascade. In addition, TGF beta induced sustained activation/phosphorylation of endogenous p42/p44 MAPK. Further-more, the role of MAPK cascade in fur gene transcription was highlighted by the use of the MEK1/2 inhibitors, PD98059 or U0126, or co-expression of a p44 antisense construct that repressed the induction of fur promoter transactivation. Conversely, overexpression of a constitutively active form of MEK1 increased unstimulated, TGF beta 1-stimulated, and Smad2-stimulated promoter P1 transactivation, and the universal Smad inhibitor, Smad7, inhibited this effect. Activation of Smad2 by MEK1 or TGF beta 1 resulted in an enhanced nuclear localization of Smad2, which was inhibited upon blocking MEK1 activity. Our findings clearly show that the activation of the p42/p44 MAPK pathway is involved in fur gene expression and led us to propose a co-operative model whereby TGF beta 1-induced receptor activation stimulates not only a Smad pathway but also a parallel p42/p44 MAPK pathway that targets Smad2 for an increased nuclear translocation and enhanced fur gene transactivation. Such an uncovered mechanism may be a key determinant for the regulation of furin in embryogenesis and growth-related physiopathological conditions.

Involvement of Smads in TGFbeta1-induced furin (fur) transcription.

Furin is recognized as being one of the main convertases of the cellular constitutive secretion pathway but the mechanisms regulating its expression are still unknown. We have previously demonstrated that TGFbeta1 up-regulates its own converting enzyme, furin, creating a novel activation/regulation cycle of potential importance in a variety of physiological and pathophysiological conditions. The fur (fes upstream region) gene is regulated via three alternative promoters; P1, P1A, and P1B. To gain insight into the molecular mechanism(s) underlying this up-regulation, we performed transient cell transfections with P1, P1A, and P1B promoter luciferase constructs. Transfection experiments in HepG2 cells revealed that fur P1 promoter is the strongest and the most sensitive to TGFbeta1 stimulation (5 ng/ml) (3.2-fold vs. 2.4-fold for P1A and 2.1-fold for P1B). Cotransfection with either a dominant negative mutant form of Smad2 [Smad2(3SA)] or a known Smad inhibitor [Smad7] inhibit constitutive and TGFbeta1-induced luciferase activity indicating the participation of endogenous Smads. Increased levels of TGFbeta1-induced transcriptional activation of the P1 promoter by overexpression of Smad2 and/or Smad4 is greatly reduced in the presence of Smad2(3SA) and completely inhibited by Smad7, suggesting the participation of endogenous Smad2/Smad4 complexes. Furthermore, the fork-head activin signal transducer (FAST-1), known to interact with Smad2/Smad4 complexes, is a potent stimulator of TGFbeta1-induced transactivation of the fur P1 promoter. Five prime-deletion analysis of this promoter identified the proximal region (between positions -8734 and -7925), as the nucleotide stretch that carries most of the transcriptional activation of fur P1 promoter by Smad2. Overall, the present data demonstrate that Smad2 and Smad4 possibly in complex with FAST-1 or other DNA binding partners participate in the constitutive and inducible transactivation of the fur P1 promoter. This represents the first detailed study of the transcriptional regulation of the fur gene.

Evidence that furin is an authentic transforming growth factor-beta1-converting enzyme.

Transforming growth factor (TGF)-beta1 plays an essential role in cell growth and differentiation. It is also considered as a gatekeeper of immune homeostasis with gene disruption leading to autoimmune and inflammatory diseases. TGF-beta1 is produced as an inactive precursor polypeptide that can be efficiently secreted but correct proteolytic cleavage is an essential step for its activation. Assessment of the cleavage site has revealed a unique R-H-R-R sequence reminiscent of proprotein convertase (PC) recognition motifs and has previously demonstrated that this PC-like cleavage site is correctly cleaved by furin, a member of the PC family. Here we report that among PC members, furin more closely satisfies the requirements needed to fulfill the role of a genuine TGF-beta1 convertase. Even though six members of the PC family have the ability to cleave TGF-beta1, ectopic expression of alpha(1)-antitrypsin Portland (alpha(1)-AT-PDX), a potent furin inhibitor, blocked 80% of TGF-beta1 processing mediated by endogenous enzymes as demonstrated in an in vitro digestion assay. Genetic complementation of a furin-deficient LoVo cell line with the wild-type gene restores the production of mature and bioactivable TGF-beta1. Moreover, both furin and TGF-beta are coordinately expressed and regulated in vitro and in vivo in the hematopoietic and immune system, an important tissue target. These results demonstrate for the first time that furin is an authentic and adaptive TGF-beta1-converting enzyme whereas other members of the PC family might substitute or supplement furin activity. Our study advances our comprehension of the complexity of the TGF-beta system and should facilitate the development of therapeutically useful TGF-beta inhibitors.

Proprotein cleavage of E-cadherin by furin in baculovirus over-expression system: potential role of other convertases in mammalian cells.

Sequence analysis of the adhesion molecule E-cadherin had revealed a multibasic motif [4PArg-Gln-Lys-Arg1P], reminiscent of the minimal cleavage signal for furin, the prototype of the proprotein convertase family, and/or other members sharing similar sequence specificity. Mutation of this site was sufficient to abolish processing of E-cadherin in fibroblasts reinforcing the possibility that proprotein convertases are involved in the maturation of this adhesion molecule. Here we demonstrate that even though furin can efficiently and specifically cleave proE-cadherin in a baculovirus-based co-expression system, the furin-deficient LoVo cells were found to process endogenous E-cadherin as efficiently as normal cell lines. This suggests, for the first time, that E-cadherin is not only a substrate for furin but for other mammalian convertases sharing similar sequence specificity.

Enhanced TGFbeta1 maturation in high five cells coinfected with recombinant baculovirus encoding the convertase furin/pace: improved technology for the production of recombinant proproteins in insect cells.

One important limitation of the widely used insect baculovirus overexpression system is its inefficiency to properly process heterologous proteins which are initially biosynthesized as larger inactive precursor proteins. One example is transforming growth factor beta 1 (TGFbeta1), a 25-kDa homodimeric protein with pleiotropic functions. As many growth factors, the inactive TGFbeta1 precursor molecule needs to be proteolytically cleaved C-terminal to a basic sequence to yield the mature and active homodimer. In insect cells, a large proportion of overexpressed TGFbeta1 was found in an inactive precursor form suggesting that the levels of endogenous convertases are limiting for the production of mature and bioactive TGFbeta1 in this system. We have demonstrated that furin, a member of a novel family of mammalian prohormone convertases (PCs) can efficiently process TGFbeta1 precursor resulting in the production of the mature and active growth factor. Taking advantage of this observation, we have developed an improved overproduction system for TGFbeta1 by coexpressing prohTGFbeta1 and human furin convertase in High Five cells. Using this system, the production of mature active TGFbeta1 increased in a dose-dependent fashion reaching up to 7. 8-fold the amount obtained with the growth factor only. Thus, eliminating the rate-limiting step in recombinant TGFbeta1 production maximizes its processing efficiency and the yield of the mature active growth factor. Such simple and efficient technology could be useful for large scale production of other proproteins which undergo similar maturation processes and share furin recognition sequences at the junction between the proregion and the mature polypeptide.

Antiphospholipid antibody syndrome associated with large aortic valve vegetation and stroke.

An unusual case of an aortic valve mass in a patient with a stroke and primary antiphospholipid antibody syndrome is discussed. The association of cardiac abnormalities and antiphospholipid syndrome is reviewed.

Some effects of chitosan on liver function in the rat.

Chitosan, a natural product derived from chitin, possesses hypocholesterolemic properties similar to those of cholestyramine, but there has been no report concerning its effects on the equilibrium between dietary cholesterol and de novo cholesterol synthesis in the liver. In this work, we studied the effects of chitosan on plasma and liver cholesterol levels, liver weight, and the key regulatory enzyme of cholesterogenesis 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase in rats fed a sterol diet containing 1% cholesterol and 0.2% cholic acid. The animals given the sterol diet showed increases in plasma and liver cholesterol, which were lowered by 54% in plasma and 64% in liver by 5% chitosan, while cholestyramine completely blocked such increases. HMG-CoA reductase activity was considerably increased in the sterol-cholestyramine group, but was greatly decreased in both sterol and sterol-chitosan groups. There was no change in liver weight or appearance after treatment with chitosan, but cholestyramine-treated animals manifested secondary effects from the treatment, including smaller yellowish livers. High mol wt chitosans [> 750 kilodaltons (kDa)] were found to be less effective as hypocholesterolemia than a 70-kDa preparation. Also, when the 70-kDa chitosan was used at 2.5%, 5%, and 7.5% of the total diet, its effectiveness was greatest at the higher concentrations; indeed, incorporation of 7.5% chitosan in the sterol diet for 3 weeks completely prevented any decrease in plasma high density lipoprotein cholesterol or increase in the plasma cholesterol level and liver weight. This formula greatly reduced the increase in liver cholesterol content due to the sterol diet, with values of 8.8 +/- 1.3 for the sterol-chitosan diet vs. 18.2 +/- 0.8 mg/g tissue for the sterol diet. The increased intake of sterols considerably lowered both HMG-CoA reductase activity (33-fold) and HMG-CoA reductase mRNA levels (3-fold) in rat liver, but in the sterol-chitosan group, HMG-CoA reductase activity was 7.7 times more elevated than in the sterol group, although it was still lower than the control value, whereas HMG-CoA reductase mRNA levels were normal. The results obtained did not differ significantly when rats were studied for 1, 3, or 6 weeks. These results taken collectively indicate that the 7.5% chitosan formula maintained adequate cholesterol homeostasis in rats, despite a greatly increased intake of cholesterol.

The expression of mRNA for glucocorticoid receptor gene and functional glucocorticoid receptors detected in PA-III rat prostate adenocarcinoma cells.

The metastatic capacity of PA-III rat prostate adenocarcinoma cells has been well documented, although little is known about the biological and biochemical characteristics of PA-III cells. This study characterizes PA-III cells with regard to the presence or absence of glucocorticoid and androgen receptors. Cytosols of PA-III cells possessed [3H]-dexamethasone binding sites with association constant (Ka) 0.46 +/- 0.17 x 10(9) M-1 and number 341 +/- 175 fmols/mg protein. Displacement of [3H]-dexamethasone binding from PA-III cytosols achieved by increasing doses of unlabelled dexamethasone, corticosterone, cortisol, progesterone, deoxycorticosterone and aldosterone documented glucocorticoid binding specificity. Northern and dot blot analyses detected the expression of mRNA for glucocorticoid receptor using a 750 bp cDNA probe of the glucocorticoid receptor gene. Twenty-four hours incubation of PA-III cells with increasing amounts of dexamethasone resulted in a remarkable inhibition of the growth of PA-III cells. Binding studies with [3H]-R1881 as well as dot blot and Northern blot analyses using a 500 bp cDNA probe of androgen receptor gene could not detect the presence of androgen receptors in PA-III cells. The present study documented functional glucocorticoid receptors in the androgen-independent PA-III rat prostate adenocarcinoma cells. These results suggest that glucocorticoids may regulate important aspects of androgen-independent prostate cancer cells growth and functions.

The protein kinase C content is increased in the nuclear fraction of rat adrenal zona glomerulosa following long-term ACTH administration.

It is well known that the adrenal zona glomerulosa is transformed to zona fasciculata-reticularis in rats exposed chronically to ACTH. This model was used to study the intracellular distribution of protein kinase C, which is known to be involved in differentiation processes. Under basal conditions, in zona glomerulosa, 70, 23, and 7% of the protein kinase C was located in the cytosol, membrane and nuclear fractions, respectively. At 30 min after ACTH administration to rats, the protein kinase C content remained unchanged in the nuclear fraction, whereas that of the cytosolic fraction was decreased to 43% while in the membranes it was increased to 48%. After 2 days of ACTH treatment, we observed a significant increase, up to 25%, of protein kinase C in the nuclear fraction, a decrease to 47% in the cytosol, whereas the membrane fraction content had returned to its basal value. The intracellular distribution of inner zones was 17% in nuclear fraction, 47% in cytosol and 36% in membranes. ACTH treatments did not change these proportions. The total protein kinase C content of ACTH-treated groups was not different than that of their respective controls, in zona glomerulosa and in inner zones respectively. The cytosolic protein kinase C formed complexes with detergent-treated nuclei; this association was saturable, and could be measured by the ability of the kinase to bind [3H]PDBu ([20(n)-3H]phorbol-12,13-dibutyrate). The number of nuclear 'acceptor sites' thus measured was calculated to be 5245 fmol/mg DNA in the zona glomerulosa; this did not change significantly following a 3-day administration of ACTH. Protein kinase C prepared from the adrenal inner zones also bound zona glomerulosa detergent-treated nuclei but occupied fewer sites than the protein kinase C from the zona glomerulosa. In conclusion, the effects of chronic ACTH treatment on rat adrenal zona glomerulosa could be mediated by an increased level of protein kinase C in the nuclear fraction and possibly through its binding to specific 'acceptor sites'.

Characterization and partial purification of the 8-9S androgen receptor from benign prostatic hyperplasia tissues.

The present study reports a 3,800-fold purification of the 8-9S androgen-receptor complex from benign prostate hyperplasia (BPH) tissues using differential chromatography. In addition, the BPH androgen receptor complexes have been characterized using sucrose density gradient (SDG) ultracentrifugation, gel permeation, and anion exchange high performance liquid chromatography (HPLC). Results indicate that a) under nontransforming conditions, BPH cytosols contained both 8-9S (40-78%) and 4S (22-60%) androgen-receptor forms, b) apparent molecular weights of these androgen-receptor apparent molecular weights of these androgen-receptor complexes, as analyzed by gel permeation HPLC, were estimated to correspond at 270 kDa, and 90 kDa respectively, c) 8-9S androgen-receptor complexes were retained on an anion exchange HPLC column and could be eluted at 0.22 M KCl at a linear gradient, whereas 4S complexes were not retained on anion exchange columns under identical experimental conditions, d) 10X dilution of BPH cytosols containing only the 4S (0.6 M KCl) form and subsequent chromatography on anion exchange HPLC system was indicative of fragmentation (these fragments were retained on anion exchange columns and could be eluted by 0.33 M KCl on a linear gradient HPLC), and e) increased temperature (22 C) was permissive of proteolytic fragmentation (fragments were estimated to correspond at 30, 15, and 5 kDa). The results are discussed in relationship with the composition of the nontransformed androgen-receptor molecules.

Characterization of androgen receptor by high performance liquid chromatography and sucrose density gradient ultracentrifugation in normal and malignant human prostatic tissues.

In the present study we have characterized androgen-receptor complexes of normal and malignant human prostate cytosols using sucrose density gradient centrifugation, gel permeation and anion exchange high performance liquid chromatography (HPLC). Our results indicated that: 1) malignant tissue cytosols differed from normal by the presence of a 4-5S androgen receptor form which accounted for 30% of total specific-binding of malignant tissue cytosols, 2) 8-9S androgen-receptor complexes in normal and malignant prostate cytosols were estimated as 270kDa by gel permeation HPLC, 3) 8-9S complexes were retained and could be eluted by 0.22M KCl on a linear gradient anion exchange HPLC, 4) 4-5S androgen-receptor complexes were estimated as 90kDa by gel permeation HPLC and were not retained on anion exchange HPLC in our experimental conditions, and 5) either 10X dilution of the 4-5S complexes and subsequent anion exchange HPLC, or anion exchange chromatography of 8-9S complexes at 22 degrees C were causing fragmentation of the androgen receptor molecule from normal and malignant tissues. These fragments had enhanced affinity for anion exchange columns. These results are discussed in relation to the composition of the nontransformed androgen receptor macromolecule.

Sucrose density gradient and exchange high performance liquid chromatography characteristics of rat prostate androgen receptor forms.

In this study, we characterized rat prostate androgen receptor using sucrose density gradient ultracentrifugation, gel permeation and anion exchange high performance liquid chromatography (HPLC). Our results indicated that 1) androgen receptor complexes in rat prostate cytosols containing no KCl were of 8-9S form corresponding to 270kDa, and in cytosols containing 0.6M KCl were of 5-6S form corresponding to 90kDa; 2) 8-9S androgen receptor complexes were retained and could be eluted by 0.22M KCl in a linear gradient anion exchange HPLC; 3) Dilution (10X) of the 8-9S and of the 5-6S receptor complexes and subsequent chromatography on anion exchange columns was indicative of fragmentation. Fragments were of 3-4S form and were eluted by 0.33M KCl on anion exchange HPLC. Temperature (22 degrees C) was permissive of proteolysis generating a 30kDa androgen receptor fragment. These results are discussed in the context of the composition of non-transformed rat prostate androgen receptor.

Application of octadecylsilyl-silica in purification studies related to the nontransformed rat ventral prostate androgen receptor.

We investigated the application of octadecylsilyl (ODS)-silica in studies related to characterization and purification of the nontransformed rat ventral prostate androgen receptor. The results indicated that ODS-silica successfully separates the free [3H]R1881 from the labeled [3H]R1881 transformed (4-5S) and nontransformed (8-9S) rat ventral prostate androgen receptors. Partial purification of the 8-9S receptor form was performed by the fractionation of rat cytosol using cartridges of the ODS-silica and fast-flow-rate phosphocellulose chromatography. Further purification was accomplished by differential chromatography (DEAE-cellulose and slow-flow-rate phosphocellulose chromatography). This partially purified 8-9S receptor, when analyzed on a gel permeation high-performance liquid chromatography column, resulted in a three-peak pattern of UV absorbance. One of these peaks corresponded to a 59-kD non[3H]R1881-binding protein and the remaining two corresponded to 270-kD and 190-kD[3H]R1881-binding proteins. These results demonstrate the usefulness of ODS-silica in androgen receptor studies. The association of a 59-kD nonsteroid binding protein with the nontransformed rat ventral prostate androgen receptor is discussed.