The mechanisms of uremic serum-induced expression of bone matrix proteins in bovine vascular smooth muscle cells.

We have previously found that uremic human serum upregulates RUNX2 in vascular smooth muscle cells (VSMCs), and that RUNX2 is upregulated in areas of vascular calcification in vivo. To confirm the role of RUNX2, we transiently transfected a dominant-negative RUNX2 (DeltaRUNX2) construct in bovine vascular smooth muscle cells (BVSMCs). Blocking RUNX2 transcriptional activity significantly decreased uremic serum induced alkaline phosphatase (ALP) activity (268+/-34 vs 188+/-9.5 U/g protein, P<0.05) and osteocalcin expression (172+/-17 vs 125+/-9 ODU, P<0.05). To determine the mechanism by which uremic serum upregulates RUNX2, we examined cell signaling pathways. BVSMCs were incubated in the presence or absence of inhibitors and RUNX2 expression and ALP activity were determined. The results demonstrate that the cyclic AMP (cAMP)/protein kinase A (PKA), but not protein kinase C, signaling pathway is involved in uremic serum-induced RUNX2 expression and ALP activity in BVSMCs. To examine potential uremic 'toxins', we measured bone morphogenetic protein (BMP)-2 concentration and found that uremic serum contained increased BMP-2 (uremic serum=169+/-33 pg/ml, normal serum=117+/-15 pg/ml, P<0.05). The incubation of BVSMCs with noggin, an inhibitor of BMP, decreased RUNX2 expression. In addition, BMP-2 secretion progressively increased during calcification and uremic serum enhanced its secretion compared to normal serum. In conclusion, this study demonstrates that RUNX2 transcriptional activity is critical in uremic serum-induced bone matrix protein expression in BVSMCs and that the cAMP/PKA pathway is involved. BMP-2 is also increased in uremic serum and can upregulate RUNX2 and calcification in vitro in VSMCs.

A new method of mounting and directing chronically implanted microdrives.

A new method of mounting a microdrive on the skull and adjusting the trajectories of microelectrodes is described. The key to this system is a swiveling guide tube held in a small, skull-mounted base by a low-melting-point metal alloy. The microelectrode is advanced via a modified, commercially available, miniature microdrive screwed onto the guide tube. To change the trajectory of the electrode, the alloy is melted in place, and the guide tube swiveled to a different angle. The microelectrode or the trajectory of the pass may be changed in a few minutes while easily maintaining aseptic conditions. The entire assembly is small enough so that several can be simultaneously implanted on the skull of a cat. A metal crown is used to fix the head during recording sessions or to hold a fiberglass cap that protects all skull implants between sessions. Since the microdrives need not be removed between recording sessions, an electrode pass may be continued from day to day. Although guide tubes are generally employed only for subcortical structures, this arrangement also works well when recording from cortex in deeper parts of gyri.