Recombinant human IL-1 beta and TNF-alpha stimulate production of IL-1 alpha and IL-1 beta by vascular smooth muscle cells and IL-1 alpha by vascular endothelial cells

Vascular endothelial cells (EC) produced IL-1 alpha but not IL-1 beta into extracellular fluids. Vascular smooth muscle cells (SMC), on the other hand, produced both IL-1 alpha and IL-1 beta, and IL-1 beta produced was much higher than IL-1 alpha. The addition of recombinant human IL-1 beta or recombinant human TNF-alpha significantly enhanced IL-1 alpha production in EC, and IL-1 alpha and IL-1 beta production in SMC. IL-1 beta release was not observed even when EC were stimulated with TNF-alpha. These results suggest that the species of released form of IL-1 are different in different cell types and that cytokines enhance IL-1 alpha and IL-1 beta production in SMC and IL-1 alpha production in EC.     

Assembly, activation, and signaling by kinin-forming proteins on human vascular smooth muscle cells

Cardiovascular disease is the number one cause of death in the United States. Vascular smooth muscle cells (VSMC) are an important constituent of the vessel wall that can bring about pathological changes leading to vascular disease. Depending on the environment, the function of VSMC can deviate profoundly from its normal contractile role. Despite advances in research, the underlying mechanisms that activate VSMC toward vascular disease are poorly understood. For the first time, we have observed that factor XII and high-molecular-weight kininogen, constituents of the blood plasma, can bind to VSMC in a Zn2+-dependent manner. In the presence of prekallikrein, this assembly of factor XII and high-molecular-weight kininogen on VSMC leads to the activation of prekallikrein to kallikrein with a rapid formation of bradykinin. The amount of bradykinin in the culture medium then decreases, presumably because of the presence of a kininase activity. p44/42 mitogen-activated protein kinase is rapidly phosphorylated in response to in situ-generated or in vitro-added bradykinin and is inhibited by bradykinin antagonist HOE-140. Binding of factor XII to VSMC also results in a concentration-dependent phosphorylation of p44/42 mitogen-activated protein kinase. This early mitogenic signal, which is also implicated in atherogenesis, may change the metabolic and proliferative activity of VSMC, which are key steps in the progression of atherosclerosis.     

Stem cell-derived Sca-1+ progenitors differentiate into smooth muscle cells, which is mediated by collagen IV-integrin alpha1/beta1/alphav and PDGF receptor pathways

Embryonic stem (ES) cells can differentiate into smooth muscle cells (SMCs) that can be used for tissue engineering and repair of damaged organs. However, little is known about the molecular mechanisms of differentiation in these cells. In the present study, we found collagen IV can promote ES cells to differentiate into stem cell antigen-1-positive (Sca-1⁺) progenitor cells and SMCs. Pretreatment of ES cells with antibodies against collagen IV significantly inhibited SMC marker expression. To further elucidate the effect of collagen IV on the induction and maintenance of SMC differentiation, Sca-1⁺ progenitor cells were isolated with magnetic beads, placed in collagen-IV-coated flasks, and cultured in differentiation medium with or without platelet-derived growth factor (PDGF)-BB for 6–90 days. Both immunostaining and fluorescence-activated cell sorter analyses revealed that the majority of these cells were positive for SMC-specific markers. Pretreatment of Sca-1⁺ progenitors with antibodies against integrin ₁, _v, and ₁, but not ₃, inhibited focal adhesion kinase (FAK) and paxillin phosphorylation and resulted in a marked inhibition of SMC differentiation. Various tyrosine kinase inhibitors, and specific siRNA for phosphatidylinositol 3-kinase (PI 3-kinase) and PDGF receptor- significantly inhibited SMC marker expression. Taken together, we demonstrate for the first time that collagen IV plays a crucial role in the early stage of SMC differentiation and that integrin (₁, ₁, and _v)-FAK-PI 3-kinase-mitogen-activated protein kinase and PDGF receptor- signaling pathways are involved in SMC differentiation. progenitor cells; extracellular matrix; growth factor receptors; platelet-derived growth factor     

Functional analysis of the murine T-cell receptor beta enhancer and characteristics of its DNA-binding proteins.

The minimal T-cell receptor (TCR) beta-chain (TCR beta) enhancer has been identified by transfection into lymphoid cells. The minimal enhancer was active in T cells and in some B-lineage cells. When a larger fragment containing the minimal enhancer was used, its activity was apparent only in T cells. Studies with phytohemagglutinin and 4 beta-phorbol-12,13-dibutyrate revealed that the enhancer activity was increased by these agents. By a combination of DNase I footprinting, gel mobility shift assay, and methylation interference analysis, seven different motifs were identified within the minimal enhancer. Furthermore, competition experiments showed that some of these elements bound identical or similar factors that are known to bind to the TCR V beta promoter decamer or to the immunoglobulin enhancer kappa E2 or muEBP-E motif. These shared motifs may be important in the differential gene activity among the different lymphoid subsets.     

Presynapsis and synapsis of DNA promoted by the STP alpha and single-stranded DNA-binding proteins from Saccharomyces cerevisiae

We previously purified an activity from meiotic cell extracts of Saccharomyces cerevisiae that promotes the transfer of a strand from a duplex linear DNA molecule to complementary circular single-stranded DNA, naming it Strand Transfer Protein alpha (STP alpha) (Sugino, A., Nitiss, J., and Resnick, M. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3683-3687). This activity requires no nucleotide cofactor but is stimulated more than 10-fold by the addition of yeast single-stranded DNA-binding proteins (ySSBs). In this paper, we describe the aggregation and strand transfer of double-stranded and single-stranded DNA promoted by STP alpha and ySSB. There is a good correlation between the aggregation induced by various DNA-binding proteins (ySSBs, DBPs and histone proteins) and the stimulation of STP alpha-mediated DNA strand transfer. This implies that the stimulation by ySSBs and other binding proteins is probably due to the condensation of single-stranded and double-stranded DNA substrates into coaggregates. Within these coaggregates there is a higher probability of pairing between homologous double-stranded and single-stranded DNA, favoring the initiation of strand transfer. The aggregation reaction is rapid and precedes any reactions related to DNA strand transfer. We propose that condensation into coaggregates is a presynaptic step in DNA strand transfer promoted by STP alpha and that pairing between homologous double- and single-stranded DNA (synapsis) occurs in these coaggregates. Synapsis promoted by STP alpha and ySSBs also occurs between covalently closed double-stranded DNA and single-stranded linear DNA as well as linear double-stranded and linear single-stranded DNAs in the absence of any nucleotide cofactors.     

Expression of CCAAT/enhancer binding proteins alpha and beta in the porcine ovary and regulation in primary cultures of granulosa cells

CCAAT/enhancer binding proteins alpha and beta (CEBPA/ CEBPB) were evaluated in the porcine ovary during the estrous cycle. CEBPB mRNA was present in antral follicles and was significantly increased in healthy corpora lutea (CL), whereas CEBPA mRNA was constitutively expressed in these structures. Both isoforms of CEBPA (42 and 30 kDa) exhibited greater expression in preovulatory follicles, and the 42-kDa isoform increased in CL, whereas the 30-kDa isoform decreased. All major isoforms of CEBPB (38, 34, and 20 kDa) were expressed, with the 34- and 20-kDa isoforms being more abundant in preovulatory follicles and further increased in CL. The effects of FSH and cAMP analogue on the distribution of CEBP isoforms were evaluated in primary cultures of porcine granulosa cells. FSH and 8-Br-cAMP had little stimulatory effect on isoform distribution, but cAMP treatment for 24 h tended to decrease the 30-kDa form of CEBPA and the 34-kDa form of CEBPB. The 34-kDa form of CEBPB was decreased by the protein kinase A inhibitor H89 at 4 h (with FSH treatment), and by both protein kinase A and phosphatidylinositol 3-kinase inhibitors at 24 h of treatment. In transfected granulosa cells, FSH and cAMP analogue stimulated a CEBP consensus sequence-reporter construct that was blocked by H89. These data implicate protein kinase A as the major regulator of CEBPB isoform distribution and CEBP-mediated transactivation in granulosa cells. The differential expression of specific CEBPA/B isoforms observed in maturing follicles and CL may contribute to changes in follicular cell differentiation and increasing steroidogenic capacity.     

6-Ketoprostaglandin F1 alpha, prostaglandins E2, F2 alpha and thromboxane B2 production by endothelial cells, smooth muscle cells and fibroblasts cultured from piglet aorta

After [3H]arachidonic acid labeling, cyclooxygenase products were qualitatively analysed in the media of each cultured vascular cell type by reverse-phase high-performance liquid chromatography (rp-HPLC). The prostaglandin E2, prostaglandin F2 alpha, 6-ketoprostaglandin F1 alpha and thromboxane B2 detected in the rp-HPLC radioactive profile were then quantified by radioimmunoassay (RIA) in separate sets of experiments. In preconfluent endothelial cells prostaglandin F2 alpha and 6-ketoprostaglandin F1 alpha were detected in equal amounts (49%), whereas after confluence 6-ketoprostaglandin F1 alpha represented 57% of total secretion (P less than 0.05). Smooth muscle cells secreted mainly prostaglandin F2 alpha (48%) and fibroblasts prostaglandin E2 (44%). Using the bioassay method, antiaggregatory activity was detected only in endothelial cells, though a small percentage of immunoreactive 6-ketoprostaglandin F1 alpha was encountered in smooth muscle cells and fibroblasts (13 and 10%, respectively). Radioimmunological analysis after rp-HPLC separation of the medium of endothelial cells showed that the anti-6-ketoprostaglandin F1 alpha antibody recognized, among other substances, an unidentified compound. Its retention time was similar to that of prostaglandin F2 alpha. This unidentified compound was not detected in the media from smooth muscle cells and fibroblasts.     

alpha 1-Proteinase inhibitor, alpha 1-antichymotrypsin, and alpha 2-macroglobulin are the antiapoptotic factors of vascular smooth muscle cells

Serum depletion induces cell death. Whereas serum contains growth factors and adhesion molecules that are important for survival, serum is also likely to have antiapoptotic factor(s). We show here that the plasma proteinase inhibitors alpha1-proteinase inhibitor, alpha1-antichymotrypsin, and alpha2-macroglobulin function as critical antiapoptotic factors for human vascular smooth muscle cells. Cell survival was assured when serum-free medium was supplemented with any one or all of the above serine proteinase inhibitors. In contrast, the cells were sensitive to apoptosis when cultured in medium containing serum from which the proteinase inhibitors were removed. The antiapoptotic effect conferred by the proteinase inhibitors was proportional to proteinase inhibitory activity. Without proteinase inhibitors, the extracellular matrix was degraded, and cells could not attach to the matrix. Cell survival was dependent on the intact extracellular matrix. In the presence of the caspase inhibitor z-VAD, the cells detached but did not die. The activity of caspases was elevated without proteinase inhibitors; in contrast, caspases were not activated when medium was supplemented with one of the proteinase inhibitors. In conclusion, the plasma proteinase inhibitors prevent degradation of extracellular matrix by proteinases derived from cells. Presumably an intact cell-matrix interaction inhibits caspase activation and supports cell survival.