Marnie A. Ryan

Altered cellular dynamics and endosteal location of aged early hematopoietic progenitor cells revealed by time-lapse intravital imaging in long bones

Aged hematopoietic stem cells (HSCs) are impaired in supporting hematopoiesis. The molecular and cellular mechanisms of stem cell aging are not well defined. HSCs interact with nonhematopoietic stroma cells in the bone marrow forming the niche. Interactions of hematopoietic cells with the stroma/microenvironment inside bone cavities are central to hematopoiesis as they regulate cell proliferation, self-renewal, and differentiation. We recently hypothesized that one underlying cause of altered hematopoiesis in aging might be due to altered interactions of aged stem cells with the microenvironment/niche. We developed time-lapse 2-photon microscopy and novel image analysis algorithms to quantify the dynamics of young and aged hematopoietic cells inside the marrow of long bones of mice in vivo. We report in this study that aged early hematopoietic progenitor cells (eHPCs) present with increased cell protrusion movement in vivo and localize more distantly to the endosteum compared with young eHPCs. This correlated with reduced adhesion to stroma cells as well as reduced cell polarity upon adhesion of aged eHPCs. These data support a role of altered eHPC dynamics and altered cell polarity, and thus altered niche biology in mechanisms of mammalian aging.

Increased expression of heat shock protein-70 protects A549 cells against hyperoxia

Acute and chronic lung injury secondary to hyperoxia remains an important complication in critically ill patients, and, consequently, there is interest in developing strategies to protect the lung against hyperoxia. Heat shock proteins (HSPs) confer protection against a broad array of cytotoxic agents. In this study, we tested the hypothesis that increased expression of the 70-kDa HSP (HSP70) would protect cultured human respiratory epithelium against hyperoxia. Recombinant A549 cells were generated in which human HSP70 was increased by stable transfection with a plasmid containing human HSP70 cDNA under control of the cytomegalovirus promoter (A549-HSP70 cells). A549-HSP70 cells exposed to hyperoxia had greater acute survival rates and clonogenic capacity compared with wild-type A549 cells and with control cells stably transfected with the empty expression plasmid. Hyperoxia-mediated lipid peroxidation and ATP depletion were also attenuated in A549-HSP70 cells exposed to hyperoxia. Increased expression of HSP70 did not detectably alter mRNA levels of the intracellular antioxidants manganese superoxide dismutase, catalase, and glutathione peroxidase. Collectively, these data demonstrate a specific in vitro protective role for HSP70 against hyperoxia and suggest that potential mechanisms of protection involve attenuation of hyperoxia-mediated lipid peroxidation and ATP depletion.

Pharmacological targeting of the thrombomodulin–activated protein C pathway mitigates radiation toxicity

Tissue damage induced by ionizing radiation in the hematopoietic and gastrointestinal systems is the major cause of lethality in radiological emergency scenarios and underlies some deleterious side effects in patients undergoing radiation therapy. The identification of target-specific interventions that confer radiomitigating activity is an unmet challenge. Here we identify the thrombomodulin (Thbd)–activated protein C (aPC) pathway as a new mechanism for the mitigation of total body irradiation (TBI)-induced mortality. Although the effects of the endogenous Thbd-aPC pathway were largely confined to the local microenvironment of Thbd-expressing cells, systemic administration of soluble Thbd or aPC could reproduce and augment the radioprotective effect of the endogenous Thbd-aPC pathway. Therapeutic administration of recombinant, soluble Thbd or aPC to lethally irradiated wild-type mice resulted in an accelerated recovery of hematopoietic progenitor activity in bone marrow and a mitigation of lethal TBI. Starting infusion of aPC as late as 24 h after exposure to radiation was sufficient to mitigate radiation-induced mortality in these mice. These findings suggest that pharmacologic augmentation of the activity of the Thbd-aPC pathway by recombinant Thbd or aPC might offer a rational approach to the mitigation of tissue injury and lethality caused by ionizing radiation.

Heat shock inhibits phosphorylation of I-κBα

Previous studies demonstrated that induction of the heat shock response is associated with inhibition of the proinflammatory transcription factor NF-kappaB by a mechanism involving inhibition of I-kappaBalpha degradation. To provide further insight regarding the interactions of these fundamental cellular responses, the present experiments were designed to elucidate the mechanism(s) by which heat shock inhibits degradation of I-kappaBalpha. In an in vitro model of inflammatory cell signaling, treatment of RAW 264.7 murine macrophages with LPS (100 ng/mL) caused rapid degradation of I-kappaBalpha. Heat shock, 1 h before treatment with LPS, completely inhibited LPS-mediated degradation of I-kappaBalpha. Immunoprecipitation studies demonstrated that heat shock inhibited LPS-mediated ubiquitination of I-kappaBalpha. Western-blot analyses using a phosphorylated I-kappaBalpha-specific antibody demonstrated that heat shock inhibited LPS-mediated phosphorylation of I-kappaBalpha. In contrast, heat shock induced phosphorylation of c-jun. In murine fibroblasts having genetic ablation of the heat shock factor-1 gene, heat shock inhibited tumor necrosis factor-alpha mediated degradation of I-kappaBalpha. We conclude that the mechanism by which heat shock inhibits LPS-mediated degradation of I-kappaBalpha involves specific inhibition of I-kappaBalpha phosphorylation and subsequent I-kappaBalpha ubiquitination. In addition, this mechanism does not involve activation of heat shock factor-1 or the heat shock proteins regulated by heat shock factor-1.

Heat shock activates the I-κBα promoter and increases I-κBα mRNA expression

Recent data indicate that the heat shock response inhibits nuclear translocation of the proinflammatory transcription factor NF-κB. Under basal conditions NF-κB is retained in the cytoplasm by an inhibitory protein called I-κB which exists as two major isoforms: I-κBα and I-κBβ. Induction of the heat shock response in BEAS-2B cells, a human cell line representative of bronchial epithelium, increased expression of I-κBα mRNA in a time-dependent manner. Coincubation with actinomycin-D inhibited heat shock-mediated expression of I-KBα mRNA. Transient transfection assays with a plasmid containing the reporter gene firefly luciferase, under the control of the human I-κBα promoter, demonstrated that heat shock activated the I-κBα promoter. Heat shock-mediated induction of I-κBα was associated with inhibition of NF-κB activation. We conclude that heat shock increases I-κBα mRNA expression in BEAS-2B cells by activating the I-κBα promoter, and propose that heat shock-mediated up-regulation of I-κBα is a potential mechanism by which the heat shock response inhibits proinflammatory responses in lung cells.

Antimicrobial Activity of Native and Synthetic Surfactant Protein B Peptides

Surfactant protein B (SP-B) is secreted into the airspaces with surfactant phospholipids where it reduces surface tension and prevents alveolar collapse at end expiration. SP-B is a member of the saposin-like family of proteins, several of which have antimicrobial properties. SP-B lyses negatively charged liposomes and was previously reported to inhibit the growth of Escherichia coli in vitro; however, a separate study indicated that elevated levels of SP-B in the airspaces of transgenic mice did not confer resistance to infection. The goal of this study was to assess the antimicrobial properties of native SP-B and synthetic peptides derived from the native peptide. Native SP-B aggregated and killed clinical isolates of Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and group B streptococcus by increasing membrane permeability; however, SP-B also lysed RBC, indicating that the membranolytic activity was not selective for bacteria. Both the antimicrobial and hemolytic activities of native SP-B were inhibited by surfactant phospholipids, suggesting that endogenous SP-B may not play a significant role in alveolar host defense. Synthetic peptides derived from native SP-B were effective at killing both Gram-positive and Gram-negative bacteria at low peptide concentrations (0.15–5.0 μM). The SP-B derivatives selectively lysed bacterial membranes and were more resistant to inhibition by phospholipids; furthermore, helix 1 (residues 7–22) retained significant antimicrobial activity in the presence of native surfactant. These results suggest that the role of endogenous SP-B in host defense may be limited; however, synthetic peptides derived from SP-B may be useful in the treatment of bacterial pneumonias.

HEAT SHOCK PROTEIN INDUCTION PROTECTS HUMAN RESPIRATORY EPITHELIUM AGAINST NITRIC OXIDE-MEDIATED CYTOTOXICITY

Induction of heat shock proteins (HSPs) confers protection against a variety of cytotoxic agents. We hypothesized that induction of HSPs would protect cultured human respiratory epithelium against nitric oxide (NO)-mediated injury. Incubation of a human bronchial epithelial cell line (BEAS-2B cells) at 43°C for 1.5 h induced expression of several HSPs. Prior induction of HSPs was associated with protection against the NO-donors S-nitroso-N-acetyl penicillamine and 3-morpholinsydnonimine. Protection was evident as improved short term survival and improved ability of cells to recover and proliferate after exposure to NO. Prior induction of HSPs also attenuated NO-mediated decreases in cellular ATP levels, but did not decrease nitrotyrosine formation. Specific overexpression of HSP-70 by plasmid-directed gene transfer protected murine respiratory epithelial cells against S-nitroso-N-acetyl penicillamine. We conclude that in cultured human respiratory epithelium induction of HSPs confers protection against NO-mediated cytotoxicity, possibly by preservation of cellular energetics. We also suggest that HSP-70 may play a specific role in protection.

Induction of the stress response with prostaglandin A1 increases I-κBα gene expression

I‐κBα is an intracellular protein that functions as a primary inhibitor of the proinflammatory transcription factor NF‐κB. Induction of the stress response with heat shock was previously demonstrated to induce I‐κBα gene expression. Because the stress response can also be induced by nonthermal stimuli, we determined whether induction of the stress response with prostaglandin A1 (PGA1) would induce I‐κBα gene expression. Treatment of human bronchial epithelium (BEAS‐2B cells) with PGA1 induced nuclear translocation of heat shock factor 1, thus confirming that PGA1 induces the stress response in BEAS‐2B cells. Induction of the stress response with PGA1 increased I‐κBα mRNA expression in a time‐dependent manner and increased I‐κBα peptide expression. Transient transfection assays involving a human I‐κBα promoter‐luciferase reporter construct demonstrated that induction of the stress response with PGA1 activated the I‐κBα promoter. Induction of the stress response with PGA1 and concomitant induction of I‐κBα were associated with inhibition of TNF‐α‐mediated secretion of interleukin 8 and with inhibition of TNF‐α‐mediated nuclear translocation and activation of NF‐κB. These data demonstrate that induction of the stress response, by a nonthermal stimulus, increases I‐κBα gene expression by a mechanism involving activation of the I‐κBα promoter. Coupled with previous data demonstrating heat shock‐mediated induction of I‐κBα gene expression, these data suggest that I‐κBα may be considered to be one of the stress proteins. The functional consequences of stress response‐mediated I‐κBα gene expression may involve attenuation of cellular proinflammatory responses.—Thomas, S. C., Ryan, M. A., Shanley, T. P., Wong, H. R. Induction of the stress response with prostaglandin A1 increases I‐ĸBα gene expression. FASEB J. 12, 1371–1378 (1998)

Stem cell specific mechanisms ensure genomic fidelity within HSCs and upon aging of HSCs

Whether aged hematopoietic stem and progenitor cells (HSPCs) have impaired DNA damage repair is controversial. Using a combination of DNA mutation indicator assays, we observe a 2- to 3-fold increase in the number of DNA mutations in the hematopoietic system upon aging. Young and aged hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) do not show an increase in mutation upon irradiation-induced DNA damage repair, and young and aged HSPCs respond very similarly to DNA damage with respect to cell-cycle checkpoint activation and apoptosis. Both young and aged HSPCs show impaired activation of the DNA-damage-induced G1-S checkpoint. Induction of chronic DNA double-strand breaks by zinc-finger nucleases suggests that HSPCs undergo apoptosis rather than faulty repair. These data reveal a protective mechanism in both the young and aged hematopoietic system against accumulation of mutations in response to DNA damage.