Human aortic vascular smooth muscle cells digest extracellular matrix by elaboration of plasminogen activators: Implications for atherogenesis

Migration and proliferation of vascular smooth muscle cells (SMCs) are hallmarks of atherogenesis and restenosis after angioplasty. Digestion of surrounding extracellular matrix (ECM) may be a critical link. To determine whether invasion of ECM by human aortic SMCs (HASMCs) depends on proteolytic digestion mediated by the cells themselves, we characterized ECM digestion in terms of solubilization of³H-proline-labeled ECM, produced by the use of rat aortic SMCs, by HASMCs under various conditions. Pasmin alone (10 g/ml) digested 80% of ECM in 2 hours. HASMCs in 10% fetal bovine serum cultured on ECM that was not exposed to plasmin digested 48% of the ECM in 7 days. When HASMCs were cultured on plasmin-pretreated ECM, only 14% of the residual ECM was digested. Conditioned media or cells cultured on porous membrane 1 mm removed from the ECM had no effect. Baseline secretion of tissue-type plasminogen activator (t-PA) into the media by HASMCs averaged 3.9 ng/10⁵ cells/24 hr and baseline secretion of type-1 plasminogen activator inhibitor (PAI-1) averaged 1300 ng/10⁵ cells/24 hr. Thrombin (5 U/ml) increased t-PA antigen production by 184% without altering PAI-1 activity and increased ECM degradation by 43% in 7 days. Transforming growth factor- (TGF-) decreased t-PA antigen production, increased PAI-1 activity, and decreased ECM degradation. These results suggest that (1) HASMCs can digest naturally produced ECM; (2) plasminogen-dependent mechanisms requiring cell contact are important in the initiation of this phenomenon; and (3) thrombin in the vicinity of clots may modulate the fibrinolytic and proteolytic properties of SMC through t-PA after vascular injury.     

Flow cytometric measurement of DNA S-phase in human bone marrow cells: correcting for peripheral blood contamination

Abstract: The relationship between bone marrow (BM) cells with S-phase DNA content and the amount of peripheral blood contamination estimated as percentage lymphocytes+monocytes (L+MO) present in BM samples has been investigated in a total of 136 BM aspirates and biopsy expellates from 35 hematologically healthy individuals. A significant negative correlation was demonstrated between total, erythroid and myeloid BM cells in S-phase and the percentage of L+MO in the aspirates (r = 0.84, 0.57 and 0.49, respectively; p<0.0001). Based on the equation of the slope of the regression line, a correction formula adjusting the measured value of BM cells in S-phase to varying amounts of L+MO percentage has been worked out for the total and erythroid BM cells. In contrast, highly proliferating myelomonocytic cells and CD34⁺ cells did not show any significant correlation between cells in S-phase and percentage L+MO, indicating that peripheral blood contamination of BM aspirates is not a problem regarding kinetic investigations of these cells. In conclusion, the described flow cytometric method of analysing BM aspirates estimates the degree of peripheral blood contamination, as well as make possible a correct estimation of the DNA synthesis of several BM populations. The method is especially applicable when frequent BM sampling is required.