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.

Immunomodulation in progeny from thymectomized primiparous mice exposed to benzo(a)pyrene during mid-pregnancy.

Previous studies have shown that Benzo(a)pyrene (B(a)P3) given to non-thymectomized (NTX) female mice alters expression of T cell subsets and suppresses cell mediated immunity (CMI) and humoral immunity (HI) in the progeny. Thus, maternal exposure to B(a)P may influence changes in progeny immune status. To understand how maternal cellular and humoral factors influence embryonic development of progeny immunity, adult female mice were thymectomized (TX) at 6 weeks, mated and injected with 150 microg B(a)P)/g body weight at 12 days of pregnancy. After B(a)P exposure, the following studies were performed: (A) Maternal reproductive capacity and survival rate of progeny; (B) Detection of T cells in progeny thymus; (C) Functional characteristics of progeny thymus or spleen. Maternal thymectomy and B(a)P exposure reduced average litter size by 40%. Serological sensitivity of thymus cells with anti-Thyl + complement occurred at a higher dilution of mAb in progeny from TX mothers exposed to B(a)P, suggesting that B(a)P-thymectomy led to increased sensitivity of developing thymocytes to mAb plus complement. Progeny from TX mothers exposed to B(a)P showed enhanced thymic CMI, but suppressed splenic CMI and HI. Thus, thymectomy prevents CMI immunosuppression by B(a)P, while HI is still suppressed. These results indicate that the maternal thymus is necessary for incurring the effect of B(a)P on progeny CMI.