The induction of endochondral bone formation by transforming growth factor-beta(3): experimental studies in the non-human primate Papio ursinus.

Transforming growth factor-beta(3) (TGF-beta(3)), a multi-functional growth modulator of embryonic development, tissue repair and morphogenesis, immunoregulation, fibrosis, angiogenesis and carcinogenesis, is the third mammalian isoform of the TGF-beta subfamily of proteins. The pleiotropism of the signalling proteins of the TGF-beta superfamily, including the TGF-beta proteins per se, are highlighted by the apparent redundancy of soluble molecular signals initiating de novo endochondral bone induction in the primate only. In the heterotopic bioassay for bone induction in the subcutaneous site of rodents, the TGF-beta(3) isoform does not initiate endochondral bone formation. Strikingly and in marked contrast to the rodent bioassay, recombinant human (h)TGF-beta(3), when implanted in the rectus abdominis muscle of adult non-human primates Papio ursinus at doses of 5, 25 and 125 mug per 100 mg of insoluble collagenous matrix as carrier, induces rapid endochondral bone formation resulting in large corticalized ossicles by day 30 and 90. In the same animals, the delivery of identical or higher doses of theTGF-beta(3) protein results in minimal repair of calvarial defects on day 30 with limited bone regeneration across the pericranial aspect of the defects on day 90. Partial restoration of the bone induction cascade by the hTGF-beta(3) protein is obtained by mixing the hTGF-beta(3) device with minced fragments of autogenous rectus abdominis muscle thus adding responding stem cells for further bone induction by the hTGF-beta(3) protein. The observed limited bone induction in hTGF-beta(3)/treated and untreated calvarial defects in Papio ursinus and therefore by extension to Homo sapiens, is due to the influence of Smad-6 and Smad-7 down-stream antagonists of the TGF-beta signalling pathway. RT-PCR, Western and Northern blot analyses of tissue specimens generated by the TGF-beta(3) isoform demonstrate robust expression of Smad-6 and Smad-7 in orthotopic calvarial sites with limited expression in heterotopic rectus abdominis sites. Smad-6 and -7 overexpression in hTGF-beta(3)/treated and untreated calvarial defects may be due to the vascular endothelial tissue of the arachnoids expressing signalling proteins modulating the expression of the inhibitory Smads in pre-osteoblastic and osteoblastic calvarial cell lines controlling the induction of bone in the primate calvarium.     

α2-Adrenoceptor Subtypes Identified by [3H]RX821002 Binding in the Human Brain: The Agonist Guanoxabenz Does Not Discriminate Different Forms of the Predominant α2A Subtype

Abstract: Competition [³H]RX821002 ([³H]2-methoxyidazoxan) binding experiments with α₂-adrenoceptor subtype-specific antagonists—BRL 44408 (α_2A selective), ARC 239 (α_2B selective), and others—were performed to delineate through rigorous computer modeling receptor subtypes in the postmortem human brain. In the hippocampus, hypothalamus, cerebellum, and brainstem the whole population of α₂-adrenoceptors appears to belong to the α_2A subtype (100%; B_max = 34–90 fmol/mg of protein). In the frontal cortex, the predominant receptor was the α_2A subtype (87%; B_max = 53 fmol/mg of protein), although a small population of the α_2B/C subtype (13%; B_max = 8 fmol/mg of protein) was also detected. In the caudate nucleus, a mixed population of α_2A (64%; B_max = 9 fmol/mg of protein) and α_2B/C (36%; B_max = 5 fmol/mg of protein) subtypes was detected. In the cortex and caudate and in the presence of ARC 239 (to mask the α_2B/C-adrenoceptors), competition experiments with the agonist guanoxabenz clearly modeled the high- and low-affinity states of the α_2A subtype. In the presence of ARC 239 and the GTP analogue guanylyl-5′-imidodiphosphate together with NaCl and EDTA (to eliminate the high-affinity α_2A-adrenoceptor) guanoxabenz only recognized the low-affinity α_2A-adrenoceptor. The results indicate that in the human brain the predominant α₂-adrenoceptor is of the α_2A subtype and that this functionally relevant receptor subtype is not heterogeneous in nature.