Evidence for soluble and nuclear site I binding of estrogens in human aorta

The purpose of this study was to establish the estrogen receptor (ER) expression and content in human aorta fragments removed at the time of by-pass surgery. To this end, we adopted a radioligand binding assay to evaluate either soluble (S) or nuclear (N) ER using dextran-coated charcoal (DCC) and filtration methods, respectively. To better define the intratissular distribution and content of ER, we also measured the presence of a 27 kDa heat shock protein (HSP27), a well established ER-associated protein, using D5 monoclonal antibody. Finally, we analysed the different molecular isoforms of both S and N ER using size exclusion-high performance liquid chromatography (SE-HPLC). High affinity (type I) sites of estrogen binding were detected in 17 out of 19 samples in either S or N fraction, although only 9 out of 19 cases displayed site 1 ER in both cell compartments. ER levels in aortic tissues, detected by radioligand method, compare well with those we have found in other hormone-sensitive human cancer tissues and cells. SE-HPLC analysis revealed two main receptor isoforms in the soluble fraction, having 65 kDa and 18 kDa molecular mass, while a minor component of 29 kDa was also found; the nuclear fraction displayed again two major components of 38 and 23 kDa. Using the HSP27 immunohistochemistry we observed a major staining occurring in smooth muscle cells (SMC), with an increasing intensity towards the lumen. All samples, including the ER negative ones, exhibited some degree of histochemical staining. Using an arbitrary cut-off value, 7 out of 12 samples displayed a highly positive staining, 6 of which showed nuclear ER. Furthermore, SE-HPLC separation indicated the presence of a 64.9 kDa component in the soluble fraction, according to the well known relative molecular mass of ER. Following HSP27 immunohistochemistry, the overall staining intensity in aortic SMC approaches that seen in endometrial and breast epithelia, whilst the muscle ER content is generally lower. Although our data are compatible with a direct role of estrogens in arterial function, the extent of the link with arterial disease remains to be established.