William R. Baumbach

REGULATION OF GROWTH HORMONE RECEPTOR AND BINDING PROTEIN EXPRESSION IN DOMESTIC SPECIES

Abstract Growth hormone receptor (GHR) expression has been analyzed at the RNA level. In the rat, relative expression of the RNA species encoding the GHR and the GH-binding protein (GHBP) appears to be sensitive to endocrine status. Full-length GHR cDNA clones from ovine, porcine, and chicken were used as probes to investigate the existence of unique RNAs for GHBPs in these species. In the sheep and pig, only a single, ~4.5-kb RNA is apparent. Although quite high levels of GH binding activity are found in pig serum, a variety of methods failed to isolate a separate GHBP message, suggesting that porcine GHBP is produced via a mechanism different from that which is known for rat. One class of chicken GHR cDNA, resulting from alternative use of a splice acceptor 17 bases upstream of the Intron 6/Exon 7 junction, is also presented.

Generation of growth hormone binding protein by avian growth plate chondrocytes is dependent on cell differentiation

Growth hormone receptor (GH-R) gene expression was evaluated in avian growth-plates in situ and in cultured chondrocytes. In the epiphyseal growth-plate, chondrocytes at different stages of differentiation located at the proliferative and upper hypertrophic zones express the GH-R gene. In culture, addition of ascorbic acid facilitated chondrocyte differentiation as evaluated by decrease in collagen type II gene expression and increase in alkaline phosphatase activity and osteopontin gene expression. Both the ascorbic acid-treated and untreated chondrocytes expressed the gene coding for the chicken growth hormone receptor (cGH-R), but only the undifferentiated cells were capable of binding the hormone. This reduction in GH-binding resulted in alteration in GH-dependent regulation of the GH-R gene expression: only the undifferentiated chondrocytes responded to chicken GH (cGH) by down-regulation of the cGH-R gene expression. Chondrocyte differentiation induced by either ascorbic acid or retinoic acid was associated with the appearance of two growth hormone binding-proteins (GHBPs) in the culture medium with estimated MWs of 32 and 70 kDa, respectively. These GHBPs differ in their MW from the major GHBP found in chicken plasma. Chondrocyte GHBPs specifically bind [125I]cGH, which can be displaced by an excess of unlabeled cGH. The differentiation-dependent increase in the 70 kDa GHBP was observed also using specific chicken GHBP antiserum. Our data suggest that the reduction of the differentiated chondrocytes response to GH is due to differentiation-dependent loss of the extracellular domain of the GH-R, resulting in a lack of functional receptors on the cell surface and generation of GHBP.

In vivo delivery and long-term tissue retention of nano-encapsulated sirolimus using a novel porous balloon angioplasty system.

AIMS Among antirestenotic compounds, sirolimus displays a superior safety profile compared to paclitaxel, but its pharmacokinetic properties make it a challenging therapeutic candidate for single-time delivery. Herein we evaluate the feasibility of delivery, long-term retention and vascular effects of sirolimus nanoparticles delivered through a novel porous angioplasty balloon in normal porcine arteries and in a swine model of in-stent restenosis (ISR). METHODS AND RESULTS Sirolimus nanoparticle formulation was delivered via porous balloon angioplasty to 753 coronary artery segments for pharmacokinetic studies and 26 segments for biological effect of sirolimus delivery in different clinical scenarios (de novo [n=8], ISR [n=6] and following stent implantation [n=12]). Sirolimus coronary artery concentrations were above the target therapeutic level of 1 ng/mg after 26 days, and were >100-fold higher in coronary artery treatment sites than in distal myocardium and remote tissues at all time points. At 28 days, reduction in percent stenosis in formulation-treated sites compared to balloon angioplasty treatment was noted in all three clinical scenarios, with the largest effect seen in the de novo study. CONCLUSIONS Local coronary delivery of sirolimus nanoparticles in the porcine model using a novel porous balloon delivery system achieved therapeutic long-term intra-arterial drug levels without significant systemic residual exposure.