Suppression of angiotensin II stimulated responses in aortic vascular smooth muscle cells of experimental cirrhotic rats.

Functional responses to angiotensin II(AT-II) were determined in aortic vascular smooth muscle cells (VSMCs) from experimental cirrhotic rats.Our data showed that AT-II-stimulated extracellular acidification rate (ECAR),which was measured by Cytosesor microphysiometry,was significantly reduced in the aortic VSMCs from the cirrhotic rats as compared to those from the control animals.The ability of AT-II to promote formation of inositol phosphates,the second messenger produced by the activation of Gq-coupled receptors,was also considerably suppressed in the cirrhotic VSMCs.Furthermore,the maximal p42/44 MAPK phosphorylation stimulated by AT-II was significantly reduced in the cirrhotic VSMCs in contrast to that in the normal VSMCs.Taken together,our data clearly demonstrated that the functional responses to AT-II was severely suppressed in aortic VSMCs in cirrhosis,indicating the impairment of general Gq-coupled receptor signaling and subsequent biological function in the cirrhotic VSMCs.     

Differential susceptibility of human primary aortic and coronary artery vascular cells to RNA interference

BackgroundRNAi technology is a promising tool for gene therapy of vascular disease. However, the biological heterogeneity between endothelial (EC) and vascular smooth muscle cells (SMC) and within different vascular beds make them differentially susceptible to siRNA. This is further complicated by the task of choosing the right transfection reagent that leads to consistent gene silencing across all cell types with minimal toxicity. The goal of this study was to investigate the intrinsic RNAi susceptibility of primary human aortic and coronary artery endothelial and vascular smooth muscle cells (AoEC, CoEC, AoSMC and CoSMC) using adherent cell cytometry.MethodsCells were seeded at a density of 5000 cells/well of a 96well plate. Twenty four hours later cells were transfected with either non-targeting unlabeled control siRNA (50 nM), or non-targeting red fluorescence labeled siRNA (siGLO Red, 5 or 50 nM) using no transfection reagent, HiPerFect or Lipofectamine RNAiMAX. Hoechst nuclei stain was used to label cells for counting. For data analysis an adherent cell cytometer, Celigo was used.ResultsRed fluorescence counts were normalized to the cell count. EC displayed a higher susceptibility towards siRNA delivery than SMC from the corresponding artery. CoSMC were more susceptible than AoSMC. In all cell types RNAiMAX was more potent compared to HiPerFect or no transfection reagent. However, after 24 h, RNAiMAX led to a significant cell loss in both AoEC and CoEC. None of the other transfection conditions led to a significant cell loss.ConclusionThis study confirms our prior observation that EC are more susceptible to siRNA than SMC based on intracellular siRNA delivery. RNAiMax treatment led to significant cell loss in AoEC and CoEC, but not in the SMC populations. Additionally, this study is the first to demonstrate that coronary SMC are more susceptible to siRNA than aortic SMC.     

Differential expression of guinea pig class II major histocompatibility complex antigens on vascular endothelial cells in vitro and in experimental allergic encephalomyelitis

Previous studies have shown that vascular endothelial cells do not normally express major histocompatibility complex (MHC) Class II antigens either in vivo or in vitro. In this investigation it was found that endothelial in the central nervous system (CNS) of normal guinea pigs constitutively express MHC Class II antigens recognized by the monoclonal antibodies HLA-DR, 27E7, and MSgp8. This phenotype is retained when these CNS-derived endothelial cells are propagated in tissue culture. Furthermore, examination of CNS tissue taken from animals in the acute phase of chronic relapsing experimental allergic encephalomyelitis shows that additional epitopes of the MHC Class II antigen, detected by the monoclonal antibodies CI.13.1 and 22C4, are present during the diseased state. This study not only demonstrates constitutive expression of certain MHC Class II determinants by guinea pig endothelial cells, but also shows that other Class II determinants can be differentially expressed in certain disease states.     

Vitronectin-induced haptotaxis of vascular smooth muscle cells in vitro.

Vitronectin, a multifunctional glycoprotein present in the plasma and interstitial tissues, has recently been found to be localized in atherosclerotic lesions. In this study we examined the effects of vitronectin on the migration of cultured bovine aortic smooth muscle cells using a modified Boyden chamber assay. The cells migrated to fluid-phase vitronectin in a concentration-dependent fashion. The cells also migrated to membrane filter surfaces precoated with vitronectin for a few minutes in the absence of additional vitronectin in the fluid phase, suggesting that this substance binds easily to the filters and stimulates cell migration by haptotaxis under the conditions described. These observations suggest that vitronectin deposited in the intima may be involved in the pathogenesis of atherosclerosis by recruiting smooth muscle cells from the media into the intima.     

Stellate cells of aortic intima: II. Arborization of intimal cells in culture.

The present study analyzed effects of different cAMP-elevators on cell morphology in primary culture of human intimal and medial cells from grossly normal and atherosclerotic areas. In primary culture of human aortic cells adenylate cyclase activator forskolin and other cAMP elevators induced arborization of cells, i.e. they reversibly changed the shape of cells. This resulted in the formation of thin branching processes and in the concentration of cytoplasm around the nucleus. In the culture, the shape of the arborized cells resembled that of stellate ones detected in the aortic intima in situ. The arborization of cells was accompanied by destruction of myofilaments. Due to cAMP elevators' effect, most of the arborized cells were exhibited in the cultures isolated from the elastic-hyperplastic layer of the intima. The number of arborized cells was significantly less in the cultures isolated from the musculo-elastic layer and still lesser in those isolated from media. We failed to reveal any significant difference in the number of arborized cells cultured from fatty streaks, atherosclerotic plaques and grossly normal aortic areas. Obtained results suggest that the previously revealed polymorphism of human aortic intimal cells may be accounted for by the cell shape transformations underlined by the mechanism similar to that of arborization in vitro.     

Murine mammary tumour cells in vitro. II. Recruitment of quiescent cells

The development of a pure quiescent (Q) tumour cell population can be induced in three mouse mammary tumour lines (66, 67 and 68H) by nutrient deprivation. When these Q cells were removed from nutrient-deprived cultures and replated in fresh medium at a lower cell concentration within 72 hr of entering quiescence virtually all of the Q cells could re-enter the proliferating (P) state. This recruitment was characterized by an increase in cell volume, an increase in total cellular RNA, and a resumption of cell division. The length of the Q to P transition varied among the three cell lines and the depth of the quiescent state depended on the amount of time the cells had been quiescent. Once re-entry into the P compartment was completed, cell-cycle times, as estimated by the culture doubling time, were the same as the cells that had not entered the Q state, however, after 72 hr in quiescence, not all of the 66 cells could reattach after trypsinization and of those that could reattach approximately equal to 50% were incapable of either increasing their RNA levels to that of proliferating G1 cells or entering S. Clonogenicity of the nutrient-deprived Q cells in these lines decreases exponentially from time the cells enter quiescence with approximate half-times of 32, 34, and 96 hr for the 66, 68H and 67 cells, respectively. Since clonogenicity was already declining at a time when all the Q cells could re-enter the P compartment, the ability of a Q cell to form a colony is not determined solely by its capacity to re-enter the proliferating compartment.     

Differential proliferation of rat aortic and mesenteric smooth muscle cells in culture.

Smooth muscle cells (SMC) from various arterial origins have been successfully maintained in culture. The present study evaluates the proliferative activity of aortic and mesenteric SMC in culture. Aortic and mesenteric SMC were obtained from male Wistar rats by explant and enzyme digestion techniques, respectively. Vascular SMC obtained by either method exhibited a characteristic hill-and-valley growth pattern in culture after confluence and were positively labelled with either anti-smooth muscle actin or myosin by an indirect immunofluorescent method. The rate of incorporation of thymidine into DNA and cell number counting were used as indices of proliferation in vitro. Vascular SMC from passages 4-33 were first synchronized with either Dullbecco's Modified Eagle's Medium (DME) or Ham's F-12 medium, supplemented with insulin-transferring-selenium (ITS), for 72 hours. SMC were then stimulated with 10% bovine serum for either 24 or 72 hours with the former processed for scintillation counting, the latter for cell number determination. The incorporation of tritiated thymidine into DNA following a 2 hour incubation was determined by scintillation counting after perchloric acid extraction. In terms of cell numbers, proliferative responses to bovine serum were determined by Coulter counting. Autoradiography was also carried out in some cultures to determine both thymidine and mitotic labelling indices. The rate of thymidine incorporation in aortic cells was 2-3 fold higher than in mesenteric cells. Aortic and mesenteric SMC lines exhibited similar cell cycle intervals in terms of total duration and individuals cycle parameters. However, the total thymidine index was higher in the aortic than mesenteric SMC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of tissue plasminogen activator to human aortic endothelial cells.

The experiments described in this paper were designed to examine the specific binding of tissue plasminogen activator (tPA) to cultured human aortic endothelial (HAE) cells. When 125I-labelled tPA was incubated with the cells at 4 degrees C, binding was found to plateau within 90 min after incubations were begun. Binding was saturable and the bound enzyme dissociated from the sites with a half-time of approx. 48 min. Scatchard analyses were performed using tPA molecules isolated from human melanoma and colon cells as well as from C127 and Chinese hamster ovary cells that had been transfected with the human tPA gene. These enzymes showed very similar binding characteristics in spite of the fact that they differ substantially in the types of sugars which comprise their side chains. Neither the chainedness of the molecules (one-chain or two-chain) nor the sites at which they are glycosylated (type I or type II) appear to affect their ability to interact with binding sites. The tPA molecules were found to have an average equilibrium dissociation constant of (1.15 +/- 0.10) x 10(-9) M and HAE cells appeared to have a single, homogeneous population of independent binding sites present at a concentration of (1.57 +/- 0.13) x 10(6) sites per cell. Lowering the pH of the binding buffer from 7.4 to 6.5 resulted in a reversible increase in specific binding of between 2-fold and 7-fold depending upon the particular preparation of cells. Preincubation of tPA with plasminogen activator inhibitor 1 (PAI-1) was found to have little effect on binding, suggesting that tPA interacts at sites distinct from surface-bound PAI-1. No evidence for either internalization or degradation of tPA was observed in assays run at 37 degrees C. This suggests that, like urokinase, tPA remains on cell surfaces for an extended period of time.     

Differential regulation of biglycan and decorin synthesis by connective tissue growth factor in cultured vascular endothelial cells

It is possible that connective tissue growth factor (CTGF) serves as either an independent regulator or a downstream effector of transforming growth factor-beta (TGF-beta) on the proteoglycan synthesis in vascular endothelial cells. Since TGF-beta regulates endothelial proteoglycan synthesis in a cell density-dependent manner, dense and sparse cultures of bovine aortic endothelial cells were metabolically labeled with [(35)S]sulfate or (35)S-labeled amino acids in the presence of CTGF, and the labeled proteoglycans were characterized by biochemical techniques. The results indicate that CTGF suppresses the synthesis of biglycan but newly induced that of decorin in the cells when the cell density is low; in addition, no change was observed in the hydrodynamic size and the glycosaminoglycan chain length of these two small chondroitin/dermatan sulfate proteoglycans. The regulation of endothelial proteoglycan synthesis by CTGF is completely different from that by TGF-beta, suggesting that CTGF is not a downstream effector of TGF-beta but an independent regulator in vascular endothelial cells with respect to the proteoglycan synthesis.     

Differential expression of tissue transglutaminase in human cells

Summary Tissue transglutaminase is an intracellular enzyme without established physiological function. Biochemically it can be detected in all organs, but no systematic in situ localization has been carried out so far. Here we report the immunohistochemical localization of transglutaminase in human tissues using an affinity purified, monospecific anti-human transglutaminase antibody. It is shown that the widespread organ distribution of the enzyme is the consequence of its occurrence in ubiquitous cell types such as endothelium and smooth muscle cells. Some organ-specific cell types express the enzyme constitutively (mesangial cells, renomedullary interstitial cells, thymic subcapsular epithelium, colonic pericryptal fibroblasts), while in others it seems to be induced either by external stimuli (epithelium of the female breast) or as part of their differentiation/maturation program (developing nephrons, enterocytes of the small intestine). The presence of tissue transglutaminase can be demonstrated in derivatives of all germ layers and in the trophoblast. The functional implications of these findings are presently unknown; however, based on its distribution the role of this enzyme in compartmentation and preservation of tissue integrity against stress may be suggested.Part of this work was presented at the symposium Transglutaminase and Protein Crosslinking Reactions (Noble Conference in Cellular and Molecular Biology, March 29–April 1, 1987, Miami, Fla.), and at the 1st International Symposium on Post-translational Modifications of Proteins and Ageing (Lacco Ameno d'Ischia, Naples, Italy, May 11–15, 1987)     

In vitro growth of murine T cells. II. Growth of in vitro sensitized cells cytotoxic for alloantigens.

Growth factors (GM), produced by murine lymphoid cells incubated with Concanavalin A, have been used to grow cytotoxic lymphoid cells in culture. C57BL/6 and DBA/2 lymphoid cells were sensitized against each other in primary, secondary, and tertiary in vitro cultures. These sensitized cells were grown in vitro in GM and retained their cytotoxic properties. Cells grew in culture about 10-fold every 5 to 7 days for over 2 months. Initial growth of cytotoxic cells in GM resulted in marked enhancement of specific cytotoxicity that returned to original levels after subsequent subcultures. After five 10-fold cell culture generations some nonspecific cytotoxicity directed against the responding target cell strain appeared in continuous cultures. This technique for growing large numbers of cytotoxic cells may be of value in the development of adoptive immunotherapies.