Midkine (MK) is expressed in a precise temporal-spatial pattern during lung morphogenesis; however, its role in pulmonary homeostasis is unknown. Increased MK staining and mRNA expression were observed in the lungs of hypoxia-susceptible CAST/eiJ mice during hypoxia. MK expression was induced by hypoxia in cell lines in vitro. Because the transcription factor hypoxiainducible factor-1α (HIF-1α) modulates cellular responses to hypoxia, we tested whether increased expression of MK in the lung was mediated by HIF-1α. HIF-1α enhanced the transcription of MK, acting on HIF-1α regulatory elements located in the MK gene promoter. Site-directed mutagenesis of the 3′ HIF response element in the MK promoter blocked the stimulatory effects of HIF-1α. To directly assess the role of MK on lung morphogenesis, transgenic mice were generated in which MK was expressed in the respiratory epithelial cells of the developing lung. MK increased muscularization of small pulmonary arteries, increasing α-smooth muscle actin and caldesmon staining and the expression of myocardin. MK directly enhanced the expression of myocardin and the smooth muscle-specific genes α-smooth muscle actin, calponin, and SM-22 in vascular smooth muscle precursor cells. Expression of MK in the respiratory epithelium is regulated by hypoxia and HIF-1α. These data provide a model wherein the respiratory epithelium responds to hypoxia via HIF-1α-dependent regulation of MK, enhancing myocardin expression to influence pulmonary vascular gene expression.

While heparin has been used almost exclusively as a blood anticoagulant, important literature demonstrates that it also has broad anti-inflammatory activity. Herein, using low anti-coagulant 2- O,3- O-desulfated heparin (ODSH), we demonstrate that most of the anti-inflammatory pharmacology of heparin is unrelated to anticoagulant activity. ODSH has low affinity for anti-thrombin III, low anti-Xa, and anti-IIa anticoagulant activities and does not activate Hageman factor (factor XII). Unlike heparin, ODSH does not interact with heparin-platelet factor-4 antibodies present in patients with heparin-induced thrombocytopenia and even suppresses platelet activation in the presence of activating concentrations of heparin. Like heparin, ODSH inhibits complement activation, binding to the leukocyte adhesion molecule P-selectin, and the leukocyte cationic granular proteins azurocidin, human leukocyte elastase, and cathepsin G. In addition, ODSH and heparin disrupt Mac-1 (CD11b/CD18)-mediated leukocyte adhesion to the receptor for advanced glycation end products (RAGE) and inhibit ligation of RAGE by its many proinflammatory ligands, including the advanced glycation end-product carboxymethyl lysine-bovine serum albumin, the nuclear protein high mobility group box protein-1 (HMGB-1), and S100 calgranulins. In mice, ODSH is more effective than heparin in reducing selectin-mediated lung metastasis from melanoma and inhibits RAGE-mediated airway inflammation from intratracheal HMGB-1. In humans, 50% inhibitory concentrations of ODSH for these anti-inflammatory activities can be achieved in the blood without anticoagulation. These results demonstrate that the anticoagulant activity of heparin is distinct from its anti-inflammatory actions and indicate that 2- O and 3- O sulfate groups can be removed to reduce anticoagulant activity of heparin without impairing its anti-inflammatory pharmacology.

The receptor for advanced glycation end-products (RAGE) is a member of the immunoglobin superfamily of multiligand receptors. Following ligand binding, mechanisms associated with host defense, tissue remodeling, and inflammation are activated. RAGE is highly expressed in pulmonary epithelium transitioning from alveolar type (AT) II to ATI cells and is upregulated in the presence of ligand; however, the regulation and function of RAGE during development are less clear. Herein, immunohistochemistry demonstrated a temporal-spatial pattern of RAGE expression in pulmonary epithelial cells from embryonic day 17.5 to postnatal day 10. Cotransfection experiments revealed that the mouse RAGE promoter was activated by early growth response gene 1 (Egr-1) and inhibited by thyroid transcription factor-1 (TTF-1) via interaction with specific regulatory elements. A rat ATI cell line (R3/1) with endogenous RAGE expression also differentially regulated RAGE when transfected with TTF-1 or Egr-1. Because Egr-1 is markedly induced in pulmonary epithelial cells exposed to cigarette smoke extract (CSE; Reynolds PR, Hoidal JR. Am J Respir Cell Mol Biol 35: 314–319, 2006.), we sought to investigate RAGE induction by CSE. Employing RT-PCR and Western blotting, RAGE and common ligands (amphoterin and S100A12) were upregulated in epithelial (R3/1 and A549) and macrophage (RAW) cell lines following exposure to CSE. Immunostaining for RAGE in cells similarly exposed and in lungs from mice exposed to cigarette smoke for 6 mo revealed elevated RAGE expression in pulmonary epithelium. After the addition of glyoxylated BSA, an advanced glycation end-product that binds RAGE, real-time RT-PCR detected a 200-fold increase in Egr-1. These results indicate that Egr-1 regulates RAGE expression during development and the likelihood of positive feedback involving Egr-1 and RAGE in cigarette smoke-related disease.

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide and is a progressive and irreversible disorder. Cigarette smoking is associated with 80–90% of COPD cases; however, the genes involved in COPD-associated emphysema and chronic inflammation are poorly understood. It was recently demonstrated that early growth response gene 1 (Egr-1) is significantly upregulated in the lungs of smokers with COPD (Ning W and coworkers, Proc Natl Acad Sci 2004;101:14895–14900). We hypothesized that Egr-1 is activated in pulmonary epithelial cells during exposure to cigarette smoke extract (CSE). Using immunohistochemistry, we demonstrated that pulmonary adenocarcinoma cells (A-549) and primary epithelial cells lacking basal Egr-1 markedly induce Egr-1 expression after CSE exposure. To evaluate Egr-1–specific effects, we used antisense (αS) oligodeoxynucleotides (ODN) to knock down Egr-1 expression. Incorporation of Egr-1 αS ODN significantly decreased CSE-induced Egr-1 mRNA and protein, while sense ODN had no effect. Via Egr-1–mediated mechanisms, IL-1β and TNF-α were significantly upregulated in pulmonary epithelial cells exposed to CSE or transfected with Egr-1. To investigate the relationship between Egr-1 induction by smoking and susceptibility to emphysema, we determined Egr-1 expression in strains of mice with different susceptibilities for the development of smoking-induced emphysema. Egr-1 was markedly increased in the lungs of emphysema-susceptible AKR/J mice chronically exposed to cigarette smoke, but only minimally increased in resistant NZWLac/J mice. In conclusion, Egr-1 is induced by cigarette smoke and functions in proinflammatory mechanisms that likely contribute to the development of COPD in the lungs of smokers.

Bone morphogenetic proteins (BMPs) are ubiquitous regulators of cellular growth and differentiation. A variety of processes modulate BMP activity, including negative regulation by several distinct binding proteins. One such BMP antagonist chordin has a role in axis determination and neural induction in the early embryo. In this study, a role for chordin during endochondral ossification has been investigated. During limb development, Chordin expression was detected only at the distal ends of the skeletal elements. In cultured embryonic sternal chondrocytes, Chordin expression was related inversely to the stages of maturation. Further, treating cultured chondrocytes with chordin interfered with maturation induced by treatment with BMP-2. These results suggest that chordin may negatively regulate chondrocyte maturation and limb growth in vivo. To address this hypothesis, chordin protein was expressed ectopically in Hamburger-Hamilton (HH) stage 25-27 embryonic chick limbs. The phenotypic changes and alteration of gene expression in treated limbs revealed that overexpression of chordin protein delayed chondrocyte maturation in developing skeletal elements. In summary, these findings strongly support a role for chordin as a negative regulator of endochondral ossification.

The combination of patient-specific cells with scaffolds obtained from natural sources may result in improved regeneration of human tissues. Decellularization of the native tissue is the first step in this technology. Effective decellularization uses agents that lyse cells and remove all cellular materials, leaving intact collagenous extracellular matrices (ECMs). Removing cellular remnants prevents an immune response while preserving the underlying structure. In this study, the impact of five decellularization agents (0.1 N NaOH, 1% peracetic acid, 3% Triton X-100, 1% sodium dodecyl sulfate (SDS), and 0.05% trypsin/EDTA) on renal tissue was examined using slices of porcine kidneys. The NaOH solution induced the most efficient cell removal, and resulted in the highest amount of cell viability and proliferation after recellularization, although it also produced the most significant damage to collagenous fiber networks, glycosaminoglycans (GAGs) and fibroblast growth factor (FGF). The SDS solution led to less severe damage to the ECM structure but it resulted in lower metabolic activity and less proliferation. Peracetic acid and Triton X-100 resulted in minimum disruption of ECMs and the most preserved GAGs and FGF. However, these last two agents were not as efficient in removing cellular materials as NaOH and SDS, especially peracetic acid, which left more than 80% of cellular material within the ECM. As a proof of principle, after completing the comparison studies using slices of renal ECM, the NaOH process was used to decellularize a whole kidney, with good results. The overall results demonstrate the significant effect of cell lysing agents and the importance of developing an optimized protocol to avoid extensive damage to the ECM while retaining the ability to support cell growth.