Androgen receptor independent cardiovascular action of the antiandrogen flutamide

We have previously shown that flutamide (specific antagonist of the androgen receptor) has antihypertensive effects. In the present study we examined the mechanisms of flutamide action in the vasculature. The vascular effects of flutamide were assayed in aortae isolated from male or female Sprague-Dawley rats and from rats or mice lacking a functional androgen receptor ( tfm, testicular feminization mutation). The effect of flutamide on coronary flow was tested in isolated hearts. In addition, male hypertensive rats with tfm mutation were treated with flutamide, and blood pressure was monitored. Flutamide induced a relaxation of rat aortae from all the strains used (maximum relaxation at 10 microM: 51.3+/-5.2% of phenylephrine contraction) and increased the coronary flow. The aortic relaxation to flutamide was abolished by endothelium removal, or by inhibition of nitric oxide synthase, guanylyl cyclase, and tyrosine kinase but not by calmodulin inhibition. Flutamide treatment attenuated the development of hypertension in mouse renin transgenic rats with the tfm mutation. Flutamide produces direct vasodilation by inducing release of NO from the endothelium and causes subsequent activation of soluble guanylyl cyclase in an active androgen receptor independent manner. This response may contribute to the observed antihypertensive actions of flutamide.     

Evaluation of Murex CMV DNA Hybrid Capture Assay for Detection and Quantitation of Cytomegalovirus Infection in Patients following Allogeneic Stem Cell Transplantation

Murex hybrid capture DNA assay (HCS) is a solution hybridization antibody capture assay for detection and quantitation of cytomegalovirus (CMV) DNA in leukocytes. To determine whether CMV HCS is sensitive enough to initiate and monitor antiviral therapy after allogeneic stem cell transplantation (SCT), 51 consecutive SCT recipients were prospectively screened for the appearance of CMV infection by HCS, PCR, and culture assays from blood samples. Preemptive antiviral therapy was initiated after the second positive PCR result in all patients, as previously reported, and HCS was not considered for clinical decision making. A total of 417 samples were analyzed. Of these, 21 samples were found to be positive by PCR and HCS, 88 samples were PCR positive but HCS negative, and 308 were negative by both assays. Concordance of results between PCR and HCS and between HCS and blood culture was observed in 78.9 and 95.9% of the samples assayed, respectively. PCR was found to be more sensitive than HCS, and HCS was more sensitive than the blood culture assay (P < 0.0001). Four patients with symptomatic CMV infection were PCR positive prior to the onset of CMV-related symptoms, whereas HCS detected CMV DNA in three patients prior to and one at onset of CMV disease. The numbers of genomes per milliliter of blood were higher in patients with symptomatic CMV infection than in those with asymptomatic CMV infection (P = 0.06). None of the HCS-negative patients developed CMV disease. Thus, all patients with CMV disease were correctly identified by HCS; however, the lower sensitivity limit of the HCS assay may still be insufficient to allow diagnosis of CMV infection early enough to prevent CMV disease in patients following allogeneic SCT.     

1141610241Altered expression of HuR, an mRNA stabilizing protein, mediates aberrant Placenta Growth Factor (PlGF) expression in preeclampsia

Background:  Control of mRNA stability is an essential regulatory process in eukaryotic gene expression. HuR, a 3'UTR mRNA binding protein, can protect AU-rich mRNA from degradation in response to stresses. PlGF, an angiogenic growth factor, contains two consensus AU-rich sites suggesting that under normal conditions HuR may protect PlGF mRNA from degradation. Trophoblast expression of PlGF is significantly decreased in preeclampsia and by hypoxia in vitro . We hypothesize that decreased levels of cytoplasmic HuR may contribute to decreased PlGF expression in hypoxic and preeclamptic trophoblast.
Methods:  Western blots were used to determine relative effects of in vitro hypoxia on HuR protein expression and subcellular localization in trophoblast. Immunohistochemistry was used to compare HuR expression patterns in trophoblast of preeclamptic and normal placentae.
Results:  Cytoplasmic expression of HuR was decreased 1.4 fold in the cytoplasm and 1.2 fold in the nucleus of JEG3 cells. A shift in HuR was more apparent in primary trophoblast with a greater than 2-fold decrease in the cytoplasm and a 1.4 fold decrease in the nucleus following 24 hr of hypoxia. Immunohistochemical analyses detected HuR expression in near term trophoblast in situ . However, this technical approach did not detect a significant change in HuR expression between normal and preeclamptic trophoblast.
Conclusions:  HuR expression is decreased in hypoxic trophoblast, at least in vitro , which may provide a causal link to decreased PlGF mRNA expression. Down regulation of trophoblast PlGF expression is thought to contribute to the pathophysiology associated with preeclampsia including the relative lack of perfusion of the placenta and systemic renal effects.     

Functional characterization of the human atrial essential myosin light chain (hALC-1) in a transgenic rat model

Most patients with hypertrophic cardiomyopathy and congenital heart diseases express the atrial essential myosin light chains (ALC-1) in their ventricles, partially replacing the ventricular essential light chains (VLC-1). This VLC-1/ALC-1 isoform shift is correlated with an increase in cross-bridge cycling kinetics as measured using skinned fibers from the hypertrophied ventricles of human hearts.To study the functional importance of hALC-1 in the intact perfused heart, we generated a transgenic rat model (TGR) overexpressing hALC-1 in the heart. Twelve-week-old TGR rats expressed 17±4 g hALC-1 per mg of whole SDS-soluble protein. Their perfused heart contractility parameters were evaluated using the Langendorff preparation. Expression of hALC-1 was accompanied by statistically significant improvements (P<0.001) in the contractile parameters of the hearts of the TGR compared to the age matched control (WKY) animals, represented by increases from 20.8±2.3 to 45.1±3.6 mmHg/g heart weight in the developed left ventricular pressure, 1,035.7±89.8 to 2,181±135.4 mmHg/s in the contraction rate, and 713±60.2 to 1,364±137.4 mmHg/s in the relaxation rate in the WKY and the TGR groups respectively. Characterizing the functional effects of hALC-1 at the whole organ level represents a step towards gene therapy of heart failure.     

Serosal mesothelium retains vasculogenic potential.

Mesothelia comprise the epithelial covering of coelomic organs and line the cavities in which they are housed. Mesothelia contribute to the vasculature of the heart and the intestinal tract by developmental processes of epithelial-mesenchymal transition (EMT), migration, and differentiation into endothelial cells, vascular smooth muscle cells, and pericytes. Here, we establish a novel in vitro system to analyze the differentiative potential of mesothelia. Using explants from serosal mesothelium (the mesothelial covering of the gut), we demonstrate that much of the developmental program observed in embryonic mesothelia is retained in the adult structure. Namely, processes of epithelial spreading, EMT, and differentiation into smooth muscle cells from these cells are observed. Interestingly, we were unable to stimulate endothelial cell differentiation using serum or various signaling factors. Taken together, these data reveal that differentiated serosal cells retain vasculogenic potential and provide a generalizable model for future studies on the developmental and differentiative capacity of the mesothelial cell type.