Nadine S. Weich

Activated human hepatic stellate cells express the renin-angiotensin system and synthesize angiotensin II.

BACKGROUND & AIMS The renin-angiotensin system plays an important role in hepatic fibrogenesis. In other organs, myofibroblasts accumulated in damaged tissues generate angiotensin II, which promotes inflammation and extracellular matrix synthesis. It is unknown whether myofibroblastic hepatic stellate cells, the main hepatic fibrogenic cell type, express the renin-angiotensin system and synthesize angiotensin II. The aim of this study was to investigate whether quiescent and activated human hepatic stellate cells contain the components of the renin-angiotensin system and synthesize angiotensin II. METHODS Hepatic stellate cells were freshly isolated from normal human livers (quiescent hepatic stellate cells) and from human cirrhotic livers (in vivo activated hepatic stellate cells). Culture-activated hepatic stellate cells were used after a second passage of quiescent hepatic stellate cells. Angiotensinogen, renin, and angiotensin-converting enzyme were assessed by quantitative polymerase chain reaction. Angiotensin II production was assessed by enzyme-linked immunosorbent assay and immunohistochemistry. RESULTS Quiescent hepatic stellate cells barely express the renin-angiotensin system components--angiotensinogen, renin, and angiotensin-converting enzyme--and do not secrete angiotensin II. In contrast, both in vivo activated hepatic stellate cells and culture-activated hepatic stellate cells highly express active renin and angiotensin-converting enzyme and secrete angiotensin II to the culture media. Mature angiotensin II protein is also detected in the cytoplasm of in vivo activated and culture-activated hepatic stellate cells. Growth factors (platelet-derived growth factor and epidermal growth factor) and vasoconstrictor substances (endothelin-1 and thrombin) stimulate angiotensin II synthesis, whereas transforming growth factor-beta and proinflammatory cytokines have no effect. Vasodilator substances markedly attenuate the effect of endothelin-1. CONCLUSIONS After activation, human hepatic stellate cells express the components of the renin-angiotensin system and synthesize angiotensin II. These results suggest that locally generated angiotensin II could participate in tissue remodeling in the human liver.

Interleukin-6 is required in vivo for the regulation of stem cells and committed progenitors of the hematopoietic system

The development of blood cells from hematopoietic stem cells is controlled by multiple cytokines. These growth factors influence survival, cell cycle status, differentiation into lineage-committed progenitors, final maturation into blood cells, and perhaps self-renewal of stem cells. The specific contribution of IL-6 to these processes in vivo was evaluated in mice with a targeted disruption of the IL-6 gene. Decreases in the absolute numbers of CFU-Sd12 and preCFU-S, as well as in the functionality of LTRSC in these mutant mice, suggests a role for IL-6 in the survival, self-renewal, or both of hematopoietic stem cells and early progenitors. In addition, as a result of the IL-6 deficiency, the control between proliferation and differentiation of the progenitor cells of the granulocytic-monocytic, megakaryocytic, and erythroid lineages into mature blood cells is altered, leading to abnormal levels of committed progenitors of these lineages and to a slow recovery from hematopoietic ablation.

Mycobacterium tuberculosis prcBA genes encode a gated proteasome with broad oligopeptide specificity

Genes predicted to be associated with the putative proteasome of Mycobacterium tuberculosis (Mtb) play a critical role in defence of the bacillus against nitrosative stress. However, proteasomes are uncommon in eubacteria and it remains to be established whether Mtbs prcBA genes in fact encode a proteasome. We found that coexpression of recombinant PrcB and PrcA in Escherichia coli over a prolonged period at 37°C allowed formation of an α7β7β7α7, 750 kDa cylindrical stack of four rings in which all 14 β‐subunits were proteolytically processed to expose the active site threonine. In contrast to another Actinomycete, Rhodococcus erythropolis, Mtbs β‐chain propeptide was not required for particle assembly. Peptidolytic activity of the 750 kDa particle towards a hydrophobic oligopeptide was nearly two orders of magnitude less than that of the Rhodococcus 20S proteasome, and unlike eukaryotic and archaeal proteasomes, activity of the Mtb 750 kDa particle could not be stimulated by SDS, Mg2+ or Ca2+. Electron microscopy revealed what appeared to be obstructed α‐rings in the Mtb 750 kDa particle. Deletion of the N‐terminal octapeptide from Mtbs α‐chain led to disappearance of the apparent obstruction and a marked increase of peptidolytic activity. Unlike proteasomes isolated from other Actinomycetes, the open‐gate Mtb mutant 750 kDa particle cleaved oligopeptides not only after hydrophobic residues but also after basic, acidic and small, neutral amino acids. Thus, Mtb encodes a broadly active, gated proteasome that may work in concert with an endogenous activator.

Suppressive effects of TNF-alpha, TGF-beta1, and chemokines on megakaryocytic colony formation in CD34+ cells derived from umbilical cord blood compared with mobilized peripheral blood and bone marrow.

CD34+ cells from human umbilical cord blood (CB) were isolated and investigated for megakaryocytic (MK) colony formation in response to recombinant human (rh) stimulatory and suppressive cytokines and compared with their counterparts in normal BM and G-CSF-mobilized peripheral blood (mPBL). First, we observed that IL-11 by itself at any dosage had no stimulator activity on MK colony formation derived from CD34+ cells in CB, mPBL, and BM. IL-3, steel factor (SLF), or thrombopoietin (Tpo) alone stimulated numbers of colony-forming unit-megakaryocyte (CFU-MK) in a dose-dependent fashion. Maximum growth of MK progenitor cells was noted in the presence of a combination of cytokines: IL-11, IL-3, SLF, and Tpo. The frequency of CFU-MK in CB and mPBL was significantly greater than that in BM, and the size of colonies in CB and mPBL was significantly greater than that in BM, and the size of colonies was larger as well. In addition, an increased number of big mixed colonies containing MK were observed in CB and mPBL. In the presence of IL-11, IL-3, SLF, and Tpo, CFU-MK derived from CB, mPBL, and BM was suppressed by tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1 (TGF-beta1). CFU-MK derived from normal BM was inhibited by some chemokines evaluated, whereas CFU-MK derived from CB was suppressed only by platelet factor-4 (PF-4), IFN-inducible protein-10 (IP-10), Exodus-1, Exodus-2, and Exodus-3, but to a lesser degree. In CB, unlike granulocyte-macrophage (CFU-GM), erythroid (BFU-E), high-proliferative potential (HPP-CFC), or multipotential (CFU-GEMM) progenitors, at least a subpopulation of MK progenitors are in S-phase. Therefore, CB MK progenitors respond to the suppressive effects of some members of the chemokine family. Similar results were noted for burst-forming unit-MK (BFU-MK). Our results indicate that CB and mPBL are rich sources of MK progenitors and that MK progenitors in CB are responsive to the suppressive effects of TNF-alpha and TGF-beta1 and some members of the chemokine family.

Sodium valproate directly inhibits thrombopoietin induced megakaryocytopoiesis from human bone marrow CD34+ cells in vitro

Abstract Sodium valproate is a commonly used anticonvulsant in the management of complex seizure disorders. The primary reported hematologic side effect is thrombocytopenia. To investigate whether the effects observed in vivo are due to direct actions of sodium valproate on progenitor cell differentiation, we studied the in vitro formation of megakaryocytes in liquid cultures containing the drug. We then compared the ability of sodium valproate to suppress megakaryocytopoiesis with that of a second agent previously described to inhibit this process, TGFβ. Human mPBCD34 + cells were purified from apheresis samples by immunomagnetic separation techniques. These progenitor cells were cultured in serum-free media with 20 ng/ml SF, 20 ng/ml Tpo and increasing concentrations of the agents for 10 days. Differentiation was assessed by monitoring the expression of CD34 and CD41 on the cell surface using fluorescence activated cell sorting analysis. Addition of 50 μg/ml sodium valproate did not appear to be toxic to the mPBCD34 + cells as it did not inhibit cell growth. On day 7, the number of cells expressing CD34 in these cultures equaled that detected in cultures not containing the drug. However, in the presence of sodium valproate approximately 23% fewer cells expressed CD41. A 2-fold increase in the number of CD34 + CD41 + cells was observed in these cultures. Ten days of exposure to the drug produced a 3-fold increase in CD34 + cells over that detected in cultures without the drug. The majority of these CD34 + cells (72–75%) were single positives that did not express CD41. Increasing the concentration of sodium valproate to 100 μg/ml was toxic and by day 10 resulted in approximately 66% fewer cells than in control cultures. Thirty percent of these cells were CD34 + (23% CD34 + , 7% CD34 + CD41 + ) as compared to 6–7% CD34 + cells in cultures grown in the absence of the drug. TGFβ was added to mPBCD34 + cell cultures at concentrations of 5, 50 and 500 pg/ml. Toxicity was observed at 50 pg/ml on day 10 with the majority of the surviving cells expressing CD41 (approximately 90%) and few cells expressing CD34 (3–4%). These results suggest that the observed thrombocytopenia in patients receiving sodium valproate is likely to be due at least in part to direct suppression of megakaryocyte development from progenitor cells and that the mechanism by which it inhibits megakaryocytopoiesis differs from that of TGFβ.