Cell biology
Abstract
Previous reports have identified a circulating pool of CD45+ collagen I+ CXCR4+ (CD45+Col I+CXCR4+) cells, termed fibrocytes, that traffic to areas of fibrosis. No studies have demonstrated that these cells actually contribute to fibrosis, however. Pulmonary fibrosis was originally thought to be mediated solely by resident lung fibroblasts. Here we show that a population of human CD45+Col I+CXCR4+ circulating fibrocytes migrates in response to CXCL12 and traffics to the lungs in a murine model of bleomycin-induced pulmonary fibrosis. Next, we demonstrated that murine CD45+Col I+CXCR4+ fibrocytes also traffic to the lungs in response to a bleomycin challenge. Maximal intrapulmonary recruitment of CD45+Col I+CXCR4+ fibrocytes directly correlated with increased collagen deposition in the lungs. Treatment of bleomycin-exposed animals with specific neutralizing anti-CXCL12 Ab’s inhibited intrapulmonary recruitment of CD45+Col I+CXCR4+ circulating fibrocytes and attenuated lung fibrosis. Thus, our results demonstrate, we believe for the first time, that circulating fibrocytes contribute to the pathogenesis of pulmonary fibrosis.
Authors
Roderick J. Phillips, Marie D. Burdick, Kurt Hong, Marin A. Lutz, Lynne A. Murray, Ying Ying Xue, John A. Belperio, Michael P. Keane, Robert M. Strieter
Abstract
Environmental stresses converge on the mitochondria that can trigger or inhibit cell death. Excitable, postmitotic cells, in response to sublethal noxious stress, engage mechanisms that afford protection from subsequent insults. We show that reoxygenation after prolonged hypoxia reduces the reactive oxygen species (ROS) threshold for the mitochondrial permeability transition (MPT) in cardiomyocytes and that cell survival is steeply negatively correlated with the fraction of depolarized mitochondria. Cell protection that exhibits a memory (preconditioning) results from triggered mitochondrial swelling that causes enhanced substrate oxidation and ROS production, leading to redox activation of PKC, which inhibits glycogen synthase kinase-3β (GSK-3β). Alternatively, receptor tyrosine kinase or certain G protein–coupled receptor activation elicits cell protection (without mitochondrial swelling or durable memory) by inhibiting GSK-3β, via protein kinase B/Akt and mTOR/p70s6k pathways, PKC pathways, or protein kinase A pathways. The convergence of these pathways via inhibition of GSK-3β on the end effector, the permeability transition pore complex, to limit MPT induction is the general mechanism of cardiomyocyte protection.
Authors
Magdalena Juhaszova, Dmitry B. Zorov, Suhn-Hee Kim, Salvatore Pepe, Qin Fu, Kenneth W. Fishbein, Bruce D. Ziman, Su Wang, Kirsti Ytrehus, Christopher L. Antos, Eric N. Olson, Steven J. Sollott
Abstract
Isoprenylcysteine carboxyl methyltransferase (Icmt) methylates the carboxyl-terminal isoprenylcysteine of CAAX proteins (e.g., Ras and Rho proteins). In the case of the Ras proteins, carboxyl methylation is important for targeting of the proteins to the plasma membrane. We hypothesized that a knockout of Icmt would reduce the ability of cells to be transformed by K-Ras. Fibroblasts harboring a floxed Icmt allele and expressing activated K-Ras (K-Ras-Icmtflx/flx) were treated with Cre-adenovirus, producing K-Ras-IcmtΔ/Δ fibroblasts. Inactivation of Icmt inhibited cell growth and K-Ras–induced oncogenic transformation, both in soft agar assays and in a nude mice model. The inactivation of Icmt did not affect growth factor–stimulated phosphorylation of Erk1/2 or Akt1. However, levels of RhoA were greatly reduced as a consequence of accelerated protein turnover. In addition, there was a large Ras/Erk1/2-dependent increase in p21Cip1, which was probably a consequence of the reduced levels of RhoA. Deletion of p21Cip1 restored the ability of K-Ras-IcmtΔ/Δ fibroblasts to grow in soft agar. The effect of inactivating Icmt was not limited to the inhibition of K-Ras–induced transformation: inactivation of Icmt blocked transformation by an oncogenic form of B-Raf (V599E). These studies identify Icmt as a potential target for reducing the growth of K-Ras– and B-Raf–induced malignancies.
Authors
Martin O. Bergo, Bryant J. Gavino, Christine Hong, Anne P. Beigneux, Martin McMahon, Patrick J. Casey, Stephen G. Young
Abstract
Mutations in the lamin A/C gene (LMNA) cause a variety of human diseases including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy, and Hutchinson-Gilford progeria syndrome. The tissue-specific effects of lamin mutations are unclear, in part because the function of lamin A/C is incompletely defined, but the many muscle-specific phenotypes suggest that defective lamin A/C could increase cellular mechanical sensitivity. To investigate the role of lamin A/C in mechanotransduction, we subjected lamin A/C–deficient mouse embryo fibroblasts to mechanical strain and measured nuclear mechanical properties and strain-induced signaling. We found that Lmna–/– cells have increased nuclear deformation, defective mechanotransduction, and impaired viability under mechanical strain. NF-κB–regulated transcription in response to mechanical or cytokine stimulation was attenuated in Lmna–/– cells despite increased transcription factor binding. Lamin A/C deficiency is thus associated with both defective nuclear mechanics and impaired mechanically activated gene transcription. These findings suggest that the tissue-specific effects of lamin A/C mutations observed in the laminopathies may arise from varying degrees of impaired nuclear mechanics and transcriptional activation.
Authors
Jan Lammerding, P. Christian Schulze, Tomosaburo Takahashi, Serguei Kozlov, Teresa Sullivan, Roger D. Kamm, Colin L. Stewart, Richard T. Lee
Abstract
The origin of fibroblasts in pulmonary fibrosis is assumed to be intrapulmonary, but their extrapulmonary origin and especially derivation from bone marrow (BM) progenitor cells has not been ruled out. To examine this possibility directly, adult mice were durably engrafted with BM isolated from transgenic mice expressing enhanced GFP. Induction of pulmonary fibrosis in such chimera mice by endotracheal bleomycin (BLM) injection caused large numbers of GFP+ cells to appear in active fibrotic lesions, while only a few GFP+ cells could be identified in control lungs. Flow-cytometric analysis of lung cells confirmed the BLM-induced increase in GFP+ cells in chimera mice and revealed a significant increase in GFP+ cells that also express type I collagen. GFP+ lung fibroblasts isolated from chimera mice expressed collagen and telomerase reverse transcriptase but not α-smooth muscle actin. Treatment of isolated GFP+ fibroblasts with TGF-β failed to induce myofibroblast differentiation. Cultured lung fibroblasts expressed the chemokine receptors CXCR4 and CCR7 and responded chemotactically to their cognate ligands, stromal cell–derived factor-1α and secondary lymphoid chemokine, respectively. Thus the collagen-producing lung fibroblasts in pulmonary fibrosis can also be derived from BM progenitor cells.
Authors
Naozumi Hashimoto, Hong Jin, Tianju Liu, Stephen W. Chensue, Sem H. Phan
Abstract
Although capillary barrier deterioration underlies major inflammatory lung pathology, barrier-enhancing strategies are not available. To consider hyperosmolar therapy as a possible strategy, we gave 15-minute infusions of hyperosmolar sucrose in lung venular capillaries imaged in real time. Surprisingly, this treatment enhanced the capillary barrier, as indicated by quantification of the capillary hydraulic conductivity. The barrier enhancement was sufficient to block the injurious effects of thrombin, TNF-α, and H2O2 in single capillaries, and of intratracheal acid instillation in the whole lung. Capillary immunofluorescence indicated that the hyperosmolar infusion markedly augmented actin filament formation and E-cadherin expression at the endothelial cell periphery. The actin-depolymerizing agent latrunculin B abrogated the hyperosmolar barrier enhancement as well as the actin filament formation, suggesting a role for actin in the barrier response. Furthermore, hyperosmolar infusion blocked TNF-α–induced P-selectin expression in an actin-dependent manner. Our results provide the first evidence to our knowledge that in lung capillaries, hyperosmolarity remodels the endothelial barrier and the actin cytoskeleton to enhance barrier properties and block proinflammatory secretory processes. Hyperosmolar therapy may be beneficial in lung inflammatory disease.
Authors
Zeenat Safdar, Ping Wang, Hideo Ichimura, Andrew C. Issekutz, Sadiqa Quadri, Jahar Bhattacharya
Abstract
15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) has been identified as an endogenous ligand for PPARγ, inducing adipogenesis in vitro. Additional roles for this molecule in the propagation and resolution of inflammation, ligation of NF-κB, and mediation of apoptosis have been proposed. However, quantitative, physiochemical evidence for the formation of 15d-PGJ2 in vivo is lacking. We report that 15d-PGJ2 is detectable using liquid chromatography–mass spectrometry–mass spectrometry at low picomolar concentrations in the medium of 3T3-L1 preadipocytes. However, despite induction of COX-2, production of PGs, including 15d-PGJ2, does not increase during adipocyte differentiation, a process unaltered by COX inhibition. 15d-PGJ2 is detectable as a minor product of COX-2 in human urine. However, its biosynthesis is unaltered during or after COX activation in vivo by LPS. Furthermore, the biosynthesis of 15d-PGJ2 is not augmented in the joint fluid of patients with arthritis, nor is its urinary excretion increased in patients with diabetes or obesity. 15d-PGJ2 is not the endogenous mediator of PPARγ-dependent adipocyte activation and is unaltered in clinical settings in which PPARγ activation has been implicated.
Authors
L. Chastine Bell-Parikh, Tomomi Ide, John A. Lawson, Peter McNamara, Muredach Reilly, Garret A. FitzGerald
Abstract
Primary pulmonary hypertension is a fatal disease causing progressive right heart failure within 3 years after diagnosis. We describe a new concept for treatment of the disease using vasoactive intestinal peptide, a neuropeptide primarily functioning as a neurotransmitter that acts as a potent systemic and pulmonary vasodilator. Our rationale is based on the finding of a deficiency of the peptide in serum and lung tissue of patients with primary pulmonary hypertension, as evidenced by radioimmunoassay and immunohistochemistry. The relevance of this finding is underlined by an upregulation of corresponding receptor sites as shown by Northern blot analysis, Western blot analysis, and immunological techniques. Consequently, the substitution with the hormone results in substantial improvement of hemodynamic and prognostic parameters of the disease without side effects. It decreased the mean pulmonary artery pressure in our eight study patients, increased cardiac output, and mixed venous oxygen saturation. Our data provide enough proof for further investigation of vasoactive intestinal peptide and its role in primary pulmonary hypertension.
Authors
Ventzislav Petkov, Wilhelm Mosgoeller, Rolf Ziesche, Markus Raderer, Leopold Stiebellehner, Karin Vonbank, Georg-Christian Funk, Gerhard Hamilton, Clemens Novotny, Bernhard Burian, Lutz-Henning Block
Abstract
In response to Ab-complement–mediated injury, podocytes can undergo lysis, apoptosis, or, when exposed to sublytic (<5% lysis) amounts of C5b-9, become activated. Following the insertion of sublytic quantities of C5b-9, there is an increase in signaling pathways and growth factor synthesis and release of proteases, oxidants, and other molecules. Despite an increase in DNA synthesis, however, sublytic C5b-9 is associated with a delay in G2/M phase progression in podocytes. Here we induced sublytic C5b-9 injury in vitro by exposing cultured rat podocytes or differentiated postmitotic mouse podocytes to Ab and a complement source; we also studied the passive Heymann nephritis model of experimental membranous nephropathy in rats. A major finding was that sublytic C5b-9–induced injury caused an increase in DNA damage in podocytes both in vitro and in vivo. This was associated with an increase in protein levels for p53, the CDK inhibitor p21, growth-arrest DNA damage-45 (GADD45), and the checkpoint kinases-1 and -2. Sublytic C5b-9 increased extracellular signal-regulated kinase-1 and -2 (ERK-1 and -2), and inhibiting ERK-1 and -2 reduced the increase in p21 and GADD45 and augmented the DNA damage response to sublytic C5b-9–induced injury. These results show that sublytic C5b-9 induces DNA damage in vitro and in vivo and may explain why podocyte proliferation is limited following immune-mediated injury.
Authors
Jeffrey W. Pippin, Raghu Durvasula, Arndt Petermann, Keiju Hiromura, William G. Couser, Stuart J. Shankland
Abstract
Cell migration and phagocytosis are both important for controlling Mycobacterium tuberculosis infection and are critically dependent on the reorganization of the cytoskeleton. Since CD44 is an adhesion molecule involved in inflammatory responses and is connected to the actin cytoskeleton, we investigated the role of CD44 in both these processes. Macrophage (Mφ) recruitment into M. tuberculosis–infected lungs and delayed-type hypersensitivity sites was impaired in CD44-deficient (CD44–/–) mice. In addition, the number of T lymphocytes and the concentration of the protective key cytokine IFN-γ were reduced in the lungs of infected CD44–/– mice. The production of IFN-γ by splenocytes of CD44–/– mice was profoundly increased upon antigen-specific stimulation. Flow cytometry analysis revealed that soluble CD44 can directly bind to virulent M. tuberculosis. Mycobacteria also interacted with Mφ-associated CD44, as reflected by reduced binding and internalization of bacilli by CD44–/– Mφs. This suggests that CD44 is a receptor on Mφs for binding of M. tuberculosis. CD44–/– mice displayed a decreased survival and an enhanced mycobacterial outgrowth in lungs and liver during pulmonary tuberculosis. In summary, we have identified CD44 as a new Mφ binding site for M. tuberculosis that mediates mycobacterial phagocytosis, Mφ recruitment, and protective immunity against pulmonary tuberculosis.
Authors
Jaklien C. Leemans, Sandrine Florquin, Mirjam Heikens, Steven T. Pals, Ronald van der Neut, Tom van der Poll
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