Inhibition of activin A signalling in a mouse model of pre-eclampsia.

INTRODUCTION: Pre-eclampsia remains a major cause of maternal and fetal morbidity and mortality. Despite intensive research over the last 50 years, significant therapeutic advances have yet to be realised. We recently reported on the role of activin A in the pathophysiology of pre-eclampsia, whereby a pre-eclampsia-like disease state was induced in pregnant mice through activin A infusion. Using the same animal model, the effects of inhibiting activin A signalling on this pre-eclampsia-like disease state have now been assessed with low molecular weight compounds structurally related to activin-receptor-like kinase (ALK) inhibitors. METHODS: 23 synthetic compounds were screened for ability to reduce activin A-induced free radical production in HUVECs. Further, following administration of activin A (50 µg) via a subcutaneous mini-osmotic pump from day 10 of pregnancy, the most active inhibitor, MKP-1-140A, (1 mg/kg) was also concomitantly administered via subcutaneous injections. RESULTS: Significant reductions in activin A-induced systolic blood pressure and urine albumin:creatinine ratio were observed with inhibitor-treated animals. However, these findings were accompanied by sustained elevation of liver enzymes and albumin extravasation in the brains of pregnant mice that received MKP-1-140A. Furthermore, inhibition of activin A signalling with MKP-1-140A failed to rescue fetal growth restriction, and treatment with MKP-1-140A alone resulted in craniofacial and karyotypic abnormalities. DISCUSSION: These data indicate that whilst inhibition of activin A signalling by the low molecular weight ALK kinase inhibitor, MKP-1-140A, reduced some of the physiological manifestations of pre-eclampsia, the potential for serious maternal and fetal side effects may preclude it from clinical applications.

The CEC behaviour of several synthetic peptides related to the activin betaA-betaD subunits.

The resolution of several structurally related synthetic peptides, derived from the loop 3 region of the activin betaA-betaD subunits, has been studied using capillary electrochromatography (CEC) with Hypersil n-octadecylsilica as the sorbent. The results confirm that the CEC migration of these peptides can be varied in a charge-state-specific manner as the properties of the background electrolyte, such as pH, salt concentration and content of organic modifier, or temperature are systematically changed. Acidic peptides followed similar trends in retention behaviour, which was distinctly different to that shown by more basic peptides. The CEC separation of these peptides with the Hypersil n-octadecyl-silica involved distinguishable contributions from both electrophoretic mobility and chromatographic retention. Temperature effects were reflected as variations in both the electro-osmotic flow and the electrophoretic mobility of the peptides. When the separation forces acting on the peptides were synergistic with the electro-osmotic flow, as, for example, with the positively charged peptides at a particular pH and buffer electrolyte composition, their retention coefficient, kappacec, decreased with increasing capillary temperature, whereas when the separation forces worked in opposite directions, as for example with negatively charged peptides, their kappacec values increased slightly with increasing temperature. Moreover, when the content of organic modifier, acetonitrile, was sufficiently high, e.g. > 40% (v/v) and nonpolar interactions with the Hypersil n-octadecyl-silica sorbent were suppressed, mixtures of both the basic and acidic synthetic peptides could be baseline resolved under isocratic conditions by exploiting the mutual processes of electrophoretic mobility and electrostatic interaction. A linear relationship between the ln kappacec values and the volume fractions, psi, of the organic modifier over a limited range of psi-values, was established for the negatively charged peptides under these isocratic conditions. These findings thus provide useful guidelines in a more general context for the resolution and analysis of structurally related synthetic peptides using CEC methods.

Application of CEC procedures for the analysis of synthetic peptides: characterization of linear immunogenic peptides that mimic a HIV-1 gp120 epitope.

In this study, we describe the application of a new analytical procedure based on capillary electrochromatographic(CEC) techniques for the characterization of different basic and acidic peptides using isocratic eluent conditions containing acetonitrile and ammonium acetate buffers of different molarities between pH 3.8 and 5.2. In particular,10 immunogenic peptide analogs with isoelectric points ranging from 3.7 to 10.1 were investigated; nine of these peptides, 1-9, were truncated analogs of the parent peptide, 10, which is a peptidomimetic related to a HIV-1 gp120 epitope. Several of these peptides have the propensity to form alpha-helical secondary structures in solution. Electrochromatographic separations of these peptides were achieved with packed fused silica capillaries(25 cm packed length, 100 microm i.d.) containing 3 microm n-octadecylsilica particles. The influence of temperature on the CEC elution behavior of these peptides, as well as the impact of changes in the eluent composition, e.g. pH, buffer concentration and acetonitrile content, were examined. The results confirm that improvements in the resolution and analysis of synthetic peptides by CEC procedures result from the increase inelectroosmotic flow (EOF) as the temperature is increased. These findings emphasize the dominant influence of the temperature-dependent viscosity parameter, eta, on the EOF and thus on peptide resolution in CEC. Moreover, these investigations have shown that eluent properties can be specifically chosen to favor either electrophoretic mobility or chromatographic retention, with the overall CEC selectivity peptides of different sequence or composition reflecting the summated contributions from both separation mechanisms. Over the pH range 4.0-5.0, and using eluents with ionic strengths ranging from 6.2 to 15 mM ammonium acetate but containing a fixed volume fraction, psi, of acetonitrile above psi = 0.40, the CEC retention behavior of peptides 1-10 correlated with a linear relationship linking the retention coefficient, kappta(cec), and the differential frictional size-to-mass ratio parameter, Xi(fric), of these peptides. However, using eluents with a low acetonitrile content and low pH values, linear correlations were also observed between the incremental retention coefficient, Delta(Kappa)cec, and the product term [-0.66(Delta(Sigma[Xn]) log(Mi/Mj)], which links the difference in intrinsic hydrophobicities and molecular masses of two peptides, Pi and Pj. This study thus demonstrates the power of CEC procedures in the analysis of synthetic bioactive peptides and provides a general experimental framework to evaluate,using CEC procedures, the influence of the key molecular attributes of peptides on their structure-retention dependencies.Finally, these studies provide additional, practical insights into the use of CEC procedures for the analysis, resolution and biophysical characterization of closely related peptide analogs derived from solid-state peptide synthesis under conditions of different eluent composition or temperature.