DuP 532, an angiotensin II receptor antagonist: first administration and comparison with losartan.

We investigated the tolerability and angiotensin II antagonist activity of oral DuP 532 in healthy male subjects. DuP 532 (1 to 200 mg) was well tolerated, with no effect on blood pressure or heart rate. Compared with losartan (100 mg), DuP 532 (200 mg) was a weak antagonist of pressor responses to intravenous angiotensin II. Maximum inhibition of diastolic pressor response was 86% (95% confidence interval [CI], 84%, 88%) approximately 4.6 hours after losartan and 48% (95% CI, 38%, 56%) 8.7 hours after DuP 532. Twenty-four hours after dosing, inhibition by losartan and DuP 532 was similar (40% to 45%). DUP 532 is extensively bound in human plasma, with an in vitro free fraction of 0.06. Although DuP 532 and EXP3174 (losartan's active metabolite) have similar AT1-receptor potency, and plasma concentrations of DuP 532 were much greater than losartan/EXP3174, the level of antagonism was much less for DuP 532. These results indicate that multiple factors determine the in vivo potency of angiotensin II antagonists, including affinity for and distribution to the receptor as modulated by plasma binding.

Antagonistic regulation of a proline-rich transcription factor by transforming growth factor beta and tumor necrosis factor alpha.

Transforming growth factor beta (TGF-beta) and tumor necrosis factor alpha (TNF-alpha) often exhibit antagonistic actions on the regulation of various activities such as immune responses, cell growth, and gene expression. However, the molecular mechanisms involved in the mutually opposing effects of TGF-beta and TNF-alpha are unknown. Here, we report that binding sites for the transcription factor CTF/NF-I mediate antagonistic TGF-beta and TNF-alpha transcriptional regulation in NIH3T3 fibroblasts. TGF-beta induces the proline-rich transactivation domain of specific CTF/NF-I family members, such as CTF-1, whereas TNF-alpha represses both the uninduced as well as the TGF-beta-induced CTF-1 transcriptional activity. CTF-1 is thus the first transcription factor reported to be repressed by TNF-alpha. The previously identified TGF-beta-responsive domain in the proline-rich transcriptional activation sequence of CTF-1 mediates both transcriptional induction and repression by the two growth factors. Analysis of potential signal transduction intermediates does not support a role for known mediators of TNF-alpha action, such as arachidonic acid, in CTF-1 regulation. However, overexpression of oncogenic forms of the small GTPase Ras or of the Raf-1 kinase represses CTF-1 transcriptional activity, as does TNF-alpha. Furthermore, TNF-alpha is unable to repress CTF-1 activity in NIH3T3 cells overexpressing ras or raf, suggesting that TNF-alpha regulates CTF-1 by a Ras-Raf kinase-dependent pathway. Mutagenesis studies demonstrated that the CTF-1 TGF-beta-responsive domain is not the primary target of regulatory phosphorylations. Interestingly, however, the domain mediating TGF-beta and TNF-alpha antagonistic regulation overlapped precisely the previously identified histone H3 interaction domain of CTF-1. These results identify CTF-1 as a molecular target of mutually antagonistic TGF-beta and TNF-alpha regulation, and they further suggest a molecular mechanism for the opposing effects of these growth factors on gene expression.

Ultraweak photon emission in assessing bone growth factor efficiency using fibroblastic differentiation.

Photons participate in many atomic and molecular interactions and changes. Recent biophysical research has shown the induction of ultraweak photons in biological tissue. It is now established that plants, animal and human cells emit a very weak radiation which can be readily detected with an appropriate photomultiplier system. Although the emission is extremely low in mammalian cells, it can be efficiently induced by ultraviolet light. In our studies, we used the differentiation system of human skin fibroblasts from a patient with Xeroderma Pigmentosum of complementation group A in order to test the growth stimulation efficiency of various bone growth factors at concentrations as low as 5 ng/ml of cell culture medium. In additional experiments, the cells were irradiated with a moderate fluence of ultraviolet A. The different batches of growth factors showed various proliferation of skin fibroblasts in culture which could be correlated with the ultraweak photon emission. The growth factors reduced the acceleration of the fibroblast differentiation induced by mitomycin C by a factor of 10-30%. In view that fibroblasts play an essential role in skin aging and wound healing, the fibroblast differentiation system is a very useful tool in order to elucidate the efficacy of growth factors.

Effect of SR 49059, a V1a vasopressin receptor antagonist, in Raynaud's phenomenon.

OBJECTIVE: To assess whether vasopressin V1a receptor blockade reduces the abnormal vasoactive response to cold in patients suffering from Raynaud's phenomenon (RP). METHODS: SR 49059, an orally active, non-peptidic vasopressin V1a receptor antagonist, was given orally (300 mg once daily) to 20 patients with RP in a single-centre, double-blind, placebo-controlled, randomized cross-over study with two 7-day periods of treatment separated by 21 days of washout. Bilateral finger systolic blood pressure and skin temperature were assessed before and after immersion of the hand in cold water for 3 min (15 degrees C) during the screening phase and three times (before and 2 and 4 h after drug intake) on days 1 and 7 of each of the two treatment periods. Recovery of digital pressure and skin temperature was measured 0, 10, 20 and 32 min after the end of the cold immersion test. RESULTS: SR 49059 significantly attenuated the cold-induced fall in systolic pressure by 14.5% (95% confidence interval 0-29; P = 0.045) on the most affected hand on day 7 compared with placebo. Temperature recovery after the end of the cold test showed a trend to enhancement 2 and 4 h after SR 49059 on day 7 (P = 0.060 and P = 0.062 respectively). The beneficial effects on finger pressure and temperature recovery were obtained without changes in supine blood pressure or in heart rate. CONCLUSION: SR 49059 given orally once a day for 7 days to patients with RP showed favourable effects compared with placebo on finger systolic pressure and temperature recovery after cold immersion, without inducing side-effects.

Drug concentration response relationships in normal volunteers after oral administration of losartan, an angiotensin II receptor antagonist.

The aim of this study was to investigate the relationships between plasma concentrations of losartan, an orally active angiotensin II inhibitor, its active metabolite EXP3174, and angiotensin II blockade. Six healthy subjects received single oral doses of 40, 80, or 120 mg losartan and placebo at 1-week intervals in a crossover study. Angiotensin II blockade was assessed by the blood pressure response to exogenous angiotensin II before and after losartan administration. EXP3174 reached higher plasma concentrations and was eliminated more slowly than its parent compound; its levels paralleled the profile of angiotensin II blockade closer than losartan. Inhibition of the pressure response was dose dependent. The Hill-shaped relationship between response and EXP3174 concentration (or time-integrated variables) approached a plateau with 80 mg. The dose-dependent increase in plasma renin and angiotensin II exhibited a considerable individual scatter. We conclude that losartan produces a dose-dependent, effective angiotensin II blockade that is largely determined by the active metabolite EXP3174.

Regulation of the transforming growth factor beta-responsive transcription factor CTF-1 by calcineurin and calcium/calmodulin-dependent protein kinase IV.

Transforming growth factor beta (TGF-beta) is a pluripotent peptide hormone that regulates various cellular activities, including growth, differentiation, and extracellular matrix protein gene expression. We previously showed that TGF-beta induces the transcriptional activation domain (TAD) of CTF-1, the prototypic member of the CTF/NF-I family of transcription factors. This induction correlates with the proposed role of CTF/NF-I binding sites in collagen gene induction by TGF-beta. However, the mechanisms of TGF-beta signal transduction remain poorly understood. Here, we analyzed the role of free calcium signaling in the induction of CTF-1 transcriptional activity by TGF-beta. We found that TGF-beta stimulates calcium influx and mediates an increase of the cytoplasmic calcium concentration in NIH3T3 cells. TGF-beta induction of CTF-1 is inhibited in cells pretreated with thapsigargin, which depletes the endoplasmic reticulum calcium stores, thus further arguing for the potential relevance of calcium mobilization in TGF-beta action. Consistent with this possibility, expression of a constitutively active form of the calcium/calmodulin-dependent phosphatase calcineurin or of the calcium/calmodulin-dependent kinase IV (DeltaCaMKIV) specifically induces the CTF-1 TAD and the endogenous mouse CTF/NF-I proteins. Both calcineurin- and DeltaCaMKIV-mediated induction require the previously identified TGF-beta-responsive domain of CTF-1. The immunosuppressants cyclosporin A and FK506 abolish calcineurin-mediated induction of CTF-1 activity. However, TGF-beta still induces the CTF-1 TAD in cells treated with these compounds or in cells overexpressing both calcineurin and DeltaCaMKIV, suggesting that other calcium-sensitive enzymes might mediate TGF-beta action. These results identify CTF/NF-I as a novel calcium signaling pathway-responsive transcription factor and further suggest multiple molecular mechanisms for the induction of CTF/NF-I transcriptional activity by growth factors.

Nuclear localization of mouse fibroblast growth factor 2 requires N-terminal and C-terminal sequences.

In vertebrates, different isoforms of fibroblast growth factor 2 (FGF2) exist, which differ by their N-terminal extension. They show different localization and expression levels and exert distinct biological effects. Nevertheless, genetic inactivation of all FGF2 isoforms in the mouse results in only mild phenotypes. Here, we analyzed mouse FGF2, and show that, as in the human, mouse FGF2 contains CTG-initiated high molecular-weight (HMW) isoforms, which contain a nuclear localization signal, and which mediate localization of this isoform to the nucleus. Using green fluorescent protein-FGF2 fusions, we furthermore observed, that C-terminal deletions disable nuclear localization of the short low-molecular-weight (LMW) 18-kDa isoform. This loss of specific localization is accompanied by a loss in heparin binding. We therefore suggest that, first, localization of mouse FGF2 is comparable to that in other vertebrates and, second, FGF2 contains at least two sequences important for nuclear localization, a nuclear localization sequence at the N terminus which is only contained in the HMW isoform, and another sequence at the C terminus, which is only required for localization of the LMW 18-kDa isoform.

Prospective study of insulin-like growth factor-I, insulin-like growth factor-binding protein 3, genetic variants in the IGF1 and IGFBP3 genes and risk of coronary artery disease.

Although experimental studies have suggested that insulin-like growth factor I (IGF-I) and its binding protein IGFBP-3 might have a role in the aetiology of coronary artery disease (CAD), the relevance of circulating IGFs and their binding proteins in the development of CAD in human populations is unclear. We conducted a nested case-control study, with a mean follow-up of six years, within the EPIC-Norfolk cohort to assess the association between circulating levels of IGF-I and IGFBP-3 and risk of CAD in up to 1,013 cases and 2,055 controls matched for age, sex and study enrolment date. After adjustment for cardiovascular risk factors, we found no association between circulating levels of IGF-I or IGFBP-3 and risk of CAD (odds ratio: 0.98 (95% Cl 0.90-1.06) per 1 SD increase in circulating IGF-I; odds ratio: 1.02 (95% Cl 0.94-1.12) for IGFBP-3). We examined associations between tagging single nucleotide polymorphisms (tSNPs) at the IGF1 and IGFBP3 loci and circulating IGF-I and IGFBP-3 levels in up to 1,133 cases and 2,223 controls and identified three tSNPs (rs1520220, rs3730204, rs2132571) that showed independent association with either circulating IGF-I or IGFBP-3 levels. In an assessment of 31 SNPs spanning the IGF1 or IGFBP3 loci, none were associated with risk of CAD in a meta-analysis that included EPIC-Norfolk and eight additional studies comprising up to 9,319 cases and 19,964 controls. Our results indicate that IGF-I and IGFBP-3 are unlikely to be importantly involved in the aetiology of CAD in human populations.

Combinations of vascular endothelial growth factor pathway inhibitors with metronomic chemotherapy: Rational and current status.

Chemotherapy given in a metronomic manner can be administered with less adverse effects which are common with conventional schedules such as myelotoxicity and gastrointestinal toxicity and thus may be appropriate for older patients and patients with decreased performance status. Efficacy has been observed in several settings. An opportunity to improve the efficacy of metronomic schedules without significantly increasing toxicity presents with the addition of anti-angiogenic targeted treatments. These combinations rational stems from the understanding of the importance of angiogenesis in the mechanism of action of metronomic chemotherapy which may be augmented by specific targeting of the vascular endothelial growth factor (VEGF) pathway by antibodies or small tyrosine kinase inhibitors. Combinations of metronomic chemotherapy schedules with VEGF pathway targeting drugs will be discussed in this paper.

The Ret receptor tyrosine kinase pathway functionally interacts with the ERalpha pathway in breast cancer.

A limited number of receptor tyrosine kinases (e.g., ErbB and fibroblast growth factor receptor families) have been genetically linked to breast cancer development. Here, we investigated the contribution of the Ret receptor tyrosine kinase to breast tumor biology. Ret was expressed in primary breast tumors and cell lines. In estrogen receptor (ER)alpha-positive MCF7 and T47D lines, the ligand (glial-derived neurotrophic factor) activated signaling pathways and increased anchorage-independent proliferation in a Ret-dependent manner, showing that Ret signaling is functional in breast tumor cells. Ret expression was induced by estrogens and Ret signaling enhanced estrogen-driven proliferation, highlighting the functional interaction of Ret and ER pathways. Furthermore, Ret was detected in primary cancers, and there were higher Ret levels in ERalpha-positive tumors. In summary, we showed that Ret is a novel proliferative pathway interacting with ER signaling in vitro. Expression of Ret in primary breast tumors suggests that Ret might be a novel therapeutic target in breast cancer.

Cellular mechanisms underlying the regulation of dendritic development by hepatocyte growth factor.

Acquisition of a mature dendritic morphology is critical for neural information processing. In particular, hepatocyte growth factor (HGF) controls dendritic arborization during brain development. However, the cellular mechanisms underlying the effects of HGF on dendritic growth remain elusive. Here, we show that HGF increases dendritic length and branching of rat cortical neurons through activation of the mitogen-activated protein kinase (MAPK) signaling pathway. Activation of MAPK by HGF leads to the rapid and transient phosphorylation of cAMP response element-binding protein (CREB), a key step necessary for the control of dendritic development by HGF. In addition to CREB phosphorylation, regulation of dendritic growth by HGF requires the interaction between CREB and CREB-regulated transcription coactivator 1 (CRTC1), as expression of a mutated form of CREB unable to bind CRTC1 completely abolished the effects of HGF on dendritic morphology. Treatment of cortical neurons with HGF in combination with brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family that regulates dendritic development via similar mechanisms, showed additive effects on MAPK activation, CREB phosphorylation and dendritic growth. Collectively, these results support the conclusion that regulation of cortical dendritic morphology by HGF is mediated by activation of the MAPK pathway, phosphorylation of CREB and interaction of CREB with CRTC1.

Pharmacology of valsartan, an angiotensin II receptor antagonist.

Valsartan is the second orally-active, non-peptide angiotensin II receptor blocker to reach the market in Europe and the USA for the treatment of hypertension. Preclinical studies have demonstrated that this blocker is specific for the AT(1) receptor and has no affinity for the angiotensin II AT(2) receptor. Experimentally, valsartan dose-dependently inhibits the vasoconstriction induced by angiotensin II and lowers blood pressure in renin-dependent models of hypertension. Pharmacologically, oral valsartan is characterised by a low bioavailability but a rapid absorption and distribution with a half-life in keeping with once-daily administration. Thus, after oral administration, the maximal plasma concentration is reached 2 h after dosing and the elimination half-life is about 6 h. Clinically, several dose-finding and comparative studies have demonstrated that valsartan is an effective and well-tolerated antihypertensive drug in patients with mild to moderate hypertension. Valsartan has also been shown to be effective in severe hypertension. Valsartan is at least as effective as ACE inhibitors, diuretics, beta-blockers and calcium antagonists. However, none of the side-effects observed with these latter agents, including cough and lower limb oedema, has been observed with the administration of valsartan. Three large clinical trials are now underway to demonstrate whether valsartan can reduce morbidity and mortality: one in hypertensives with a high cardiovascular risk profile (VALUE), one in patients with heart failure previously treated with an angiotensin-converting enzyme inhibitor (VAL-HeFT) and one in post-myocardial infarct patients (VALIANT). These studies will further define the place of valsartan beyond the treatment of hypertension.

BAFF binds to the tumor necrosis factor receptor-like molecule B cell maturation antigen and is important for maintaining the peripheral B cell population.

The tumor necrosis factor (TNF) family member B cell activating factor (BAFF) binds B cells and enhances B cell receptor-triggered proliferation. We find that B cell maturation antigen (BCMA), a predicted member of the TNF receptor family expressed primarily in mature B cells, is a receptor for BAFF. Although BCMA was previously localized to the Golgi apparatus, BCMA was found to be expressed on the surface of transfected cells and tonsillar B cells. A soluble form of BCMA, which inhibited the binding of BAFF to a B cell line, induced a dramatic decrease in the number of peripheral B cells when administered in vivo. Moreover, culturing splenic cells in the presence of BAFF increased survival of a percentage of the B cells. These results are consistent with a role for BAFF in maintaining homeostasis of the B cell population.