Atrial natriuretic peptide attenuates agonist-induced pulmonary edema in mice with targeted disruption of the gene for natriuretic peptide receptor-A.

Atrial natriuretic peptide (ANP) inhibits agonist-induced pulmonary edema formation, but the signaling pathway responsible is not well defined. To investigate the role of the particulate guanylate cyclase-linked receptor, natriuretic peptide receptor-A (NPR-A), we measured acute lung injury responses in intact mice and pulmonary microvascular endothelial cells (PMVEC) with normal and disrupted expression of NPR-A. NPR-A wild-type (NPR-A+/+), heterozygous (NPR-A+/-), and knockout (NPR-A-/-) mice were anesthetized and treated with thrombin receptor agonist peptide (TRAP) or lipopolysaccharide (LPS). Lung injury was assessed by lung wet-to-dry (W/D) weight and by protein and cell concentration of bronchoalveolar lavage (BAL) fluid. No difference in pulmonary edema formation was seen between NPR-A genotypes under baseline conditions. TRAP and LPS increased lung W/D weight and BAL fluid cell counts more in NPR-A-/- mice than in NPR-A+/- or NPR-A+/+ mice, but no genotype-related differences were seen in TRAP-induced increases in bloodless lung W/D weight or LPS-induced increases in BAL protein concentration. Pretreatment with ANP infusion completely blocked TRAP-induced increases in lung W/D weight and blunted LPS-induced increases in BAL cell counts and protein concentration in both NPR-A-/- and NPR-A+/+ mice. Thrombin decreased transmembrane electrical resistance in monolayers of PMVECs in vitro, and this effect was attenuated by ANP in PMVECs isolated from both genotypes. Administration of the NPR-C-specific ligand, cANF, also blocked TRAP-induced increases in lung W/D weight and LPS-induced increases in BAL cell count and protein concentration in NPR-A+/+ and NPR-A-/- mice. We conclude that ANP is capable of attenuating agonist-induced lung edema in the absence of NPR-A. The protective effect of ANP on agonist-induced lung injury and pulmonary barrier function may be mediated by NPR-C.

Genetic disruption of protein kinase Cδ reduces endotoxin-induced lung injury.

The pathogenesis of acute lung injury and acute respiratory distress syndrome is characterized by sequestration of leukocytes in lung tissue, disruption of capillary integrity, and pulmonary edema. PKCδ plays a critical role in RhoA-mediated endothelial barrier function and inflammatory responses. We used mice with genetic deletion of PKCδ (PKCδ(-/-)) to assess the role of PKCδ in susceptibility to LPS-induced lung injury and pulmonary edema. Under baseline conditions or in settings of increased capillary hydrostatic pressures, no differences were noted in the filtration coefficients (k(f)) or wet-to-dry weight ratios between PKCδ(+/+) and PKCδ(-/-) mice. However, at 24 h after exposure to LPS, the k(f) values were significantly higher in lungs isolated from PKCδ(+/+) than PKCδ(-/-) mice. In addition, bronchoalveolar lavage fluid obtained from LPS-exposed PKCδ(+/+) mice displayed increased protein and cell content compared with LPS-exposed PKCδ(-/-) mice, but similar changes in inflammatory cytokines were measured. Histology indicated elevated LPS-induced cellularity and inflammation within PKCδ(+/+) mouse lung parenchyma relative to PKCδ(-/-) mouse lungs. Transient overexpression of catalytically inactive PKCδ cDNA in the endothelium significantly attenuated LPS-induced endothelial barrier dysfunction in vitro and increased k(f) lung values in PKCδ(+/+) mice. However, transient overexpression of wild-type PKCδ cDNA in PKCδ(-/-) mouse lung vasculature did not alter the protective effects of PKCδ deficiency against LPS-induced acute lung injury. We conclude that PKCδ plays a role in the pathological progression of endotoxin-induced lung injury, likely mediated through modulation of inflammatory signaling and pulmonary vascular barrier function.

Interplay between FAK, PKCδ, and p190RhoGAP in the regulation of endothelial barrier function.

Disruption of either intercellular or extracellular junctions involved in maintaining endothelial barrier function can result in increased endothelial permeability. Increased endothelial permeability, in turn, allows for the unregulated movement of fluid and solutes out of the vasculature and into the surrounding connective tissue, contributing to a number of disease states, including stroke and pulmonary edema (Ermert et al., 1995; Lee and Slutsky, 2010; van Hinsbergh, 1997; Waller et al., 1996; Warboys et al., 2010). Thus, a better understanding of the molecular mechanisms by which endothelial cell junction integrity is controlled is necessary for development of therapies aimed at treating such conditions. In this review, we will discuss the functions of three signaling molecules known to be involved in regulation of endothelial permeability: focal adhesion kinase (FAK), protein kinase C delta (PKCδ), and p190RhoGAP (p190). We will discuss the independent functions of each protein, as well as the interplay that exists between them and the effects of such interactions on endothelial function.

Heterogeneity in apoptotic responses of microvascular endothelial cells to oxidative stress.

Oxidative stress contributes to disease and can alter endothelial cell (EC) function. EC from different vascular beds are heterogeneous in structure and function, thus we assessed the apoptotic responses of EC from lung and heart to oxidative stress. Since protein kinase Cδ (PKCδ) is activated by oxidative stress and is an important modulator of apoptosis, experiments assessed the level of apoptosis in fixed lung and heart sections of PKCδ wild-type (PKCδ(+/+)) and null (PKCδ(-/-)) mice housed under normoxia (21% O(2)) or hyperoxia (~95% O(2)). We noted a significantly greater number of TUNEL-positive cells in lungs of hyperoxic PKCδ(+/+) mice, compared to matched hearts or normoxic organs. We found that 33% of apoptotic cells identified in hyperoxic lungs of PKCδ(+/+) mice were EC, compared to 7% EC in hyperoxic hearts. We further noted that EC apoptosis was significantly reduced in lungs of PKCδ(-/-) hyperoxic mice, compared to lungs of PKCδ(+/+) hyperoxic mice. In vitro, both hyperoxia and H(2)O(2) promoted apoptosis in EC isolated from microvasculature of lung (LMVEC), but not from the heart (HMVEC). H(2)O(2) treatment significantly increased p38 activity in LMVEC, but not in HMVEC. Inhibition of p38 attenuated H(2)O(2)-induced LMVEC apoptosis. Baseline expression of total PKCδ protein, as well as the caspase-mediated, catalytically active PKCδ cleavage fragment, was higher in LMVEC, compared to HMVEC. PKCδ inhibition significantly attenuated H(2)O(2)-induced LMVEC p38 activation. Conversely, overexpression of wild-type PKCδ or the catalytically active PKCδ cleavage product greatly increased H(2)O(2)-induced HMVEC caspase and p38 activation. We propose that enhanced susceptibility of lung EC to oxidant-induced apoptosis is due to increased PKCδ→p38 signaling, and we describe a PKCδ-centric pathway which dictates the differential response of EC from distinct vascular beds to oxidative stress.

Protection against LPS-induced pulmonary edema through the attenuation of protein tyrosine phosphatase-1B oxidation.

One hallmark of acute lung injury is the disruption of the pulmonary endothelial barrier. Such disruption correlates with increased endothelial permeability, partly through the disruption of cell-cell contacts. Protein tyrosine phosphatases (PTPs) are known to affect the stability of both cell-extracellular matrix adhesions and intercellular adherens junctions (AJs). However, evidence for the role of select PTPs in regulating endothelial permeability is limited. Our investigations noted that the inhibition of PTP1B in cultured pulmonary endothelial cells (ECs), as well as in the vasculature of intact murine lungs via the transient overexpression of a catalytically inactive PTP1B, decreased the baseline resistance of cultured EC monolayers and increased the formation of edema in murine lungs, respectively. In addition, we observed that the overexpression of wild-type PTP1B enhanced basal barrier function in vitro. Immunohistochemical analyses of pulmonary ECs and the coimmunoprecipitation of murine lung homogenates demonstrated the association of PTP1B with the AJ proteins ß-catenin, p120-catenin, and VE-cadherin both in vitro and ex vivo. Using LPS in a model of sepsis-induced acute lung injury, we showed that reactive oxygen species were generated in response to LPS, which correlated with enhanced PTP1B oxidation, inhibited phosphatase activity, and attenuation of the interactions between PTP1B and ß-catenin, as well as enhanced ß-catenin tyrosine phosphorylation. Finally, the overexpression of a cytosolic PTP1B fragment, shown to be resistant to nicotinamide adenine dinucleotide phosphate-reduced oxidase-4 (Nox4)-mediated oxidation, significantly attenuated LPS-induced endothelial barrier dysfunction and the formation of lung edema, and preserved the associations of PTP1B with AJ protein components, independent of PTP1B phosphatase activity. We conclude that PTP1B plays an important role in maintaining the pulmonary endothelial barrier, and PTP1B oxidation appears to contribute to sepsis-induced pulmonary vascular dysfunction, possibly through the disruption of AJs.

Cigarette smoke causes lung vascular barrier dysfunction via oxidative stress-mediated inhibition of RhoA and focal adhesion kinase.

Cigarette smoke (CS) is a major cause of chronic lung and cardiovascular diseases. Recent studies indicate that tobacco use is also a risk factor for acute lung injury (ALI) associated with blunt trauma. Increased endothelial cell (EC) permeability is a hallmark of ALI. CS increases EC permeability in vitro and in vivo; however, the underlying mechanism is not well understood. In this study, we found that only 6 h of exposure to CS impaired endothelial barrier function in vivo, an effect associated with increased oxidative stress in the lungs and attenuated by the antioxidant N-acetylcysteine (NAC). CS also exacerbated lipopolysaccharide (LPS)-induced increase in vascular permeability in vivo. Similar additive effects were also seen in cultured lung EC exposed to cigarette smoke extract (CSE) and LPS. We further demonstrated that CSE caused disruption of focal adhesion complexes (FAC), F-actin fibers, and adherens junctions (AJ) and decreased activities of RhoA and focal adhesion kinase (FAK) in cultured lung EC. CSE-induced inhibition of RhoA and FAK, endothelial barrier dysfunction, and disassembly of FAC, F-actin, and AJ were prevented by NAC. In addition, the deleterious effects of CSE on FAC, F-actin fibers, and AJ were blunted by overexpression of constitutively active RhoA and of FAK. Our data indicate that CS causes endothelial barrier dysfunction via oxidative stress-mediated inhibition of RhoA and FAK.

De-differentiation of mouse interfollicular keratinocytes by the embryonic transcription factor Oct-4.

The embryonic transcription factor Oct-4 is often referred to as the master regulator of the undifferentiated state. Although its role in maintaining embryonic stem (ES) cell pluripotency is well established, its ability to directly reprogram committed somatic cells is not well defined. Using transient transfection, we tested its ability to revert mouse interfollicular epidermal basal keratinocytes to a more ES cell-like state. We found that the Oct-4-transfected keratinocytes expressed the Oct-4 target genes, Sox-2, Nanog, undifferentiated transcription factor 1 (Utf1), and Rex-1. We also noted an increase in developmental potential caused by Oct-4, with the transfected cells able to differentiate into neuronal cells when exposed to neuroectodermal differentiation medium. Control-transfected keratinocytes were unable to respond to the medium, and remained as keratinocytes. These findings suggest that Oct-4 may be the master regulator of the pluripotent state and demonstrate that differentiated somatic cells can be reverted into more developmentally potent cells through the use of a single factor. The latter finding has great implications for therapeutic cell-replacement applications using cells from easily accessible adult tissues, such as the skin.

Epidermal stem cells have the potential to assist in healing damaged tissues.

Homeostasis of continuously renewing tissues, such as the epidermis, is maintained by somatic undifferentiated, self-renewing stem cells, which are thought to persist throughout life. Through a series of labeling experiments, we previously showed that stem cells from mouse skin did not divide often, but they did divide at a steady rate in vivo. Using our recently redefined sorting method, we isolated epidermal stem and transit amplifying (TA) cells from mouse skin. When injected into a developing blastocyst or into damaged tissues, the stem cells, but not the TA cells, could participate in the formation of new tissues. We hypothesize that all tissues contain reserved undifferentiated stem cells that are primed to react if needed. These reserve stem cells could restore the tissue in which they reside or they could be called upon to help restore another tissue that was severely damage.

Epidermal stem cells: interactions in developmental environments.

Homeostasis of continuously renewing adult tissues, such as the epidermis of the skin, is maintained by epidermal stem cells (EpiSC), which are a small population of undifferentiated, self-renewing basal keratinocyte cells that produce daughter transit amplifying (TA) cells to make up the majority of the proliferative basal cell population in the epidermis. We have isolated EpiSC from neonatal and adult skin, and shown that these cells can regenerate an epidermis that lasts long term in vitro and in vivo, and that permanently expresses a recombinant gene in the regenerated tissue (Bickenbach and Dunnwald, 2000; Dunnwald et al., 2001). When we injected murine EpiSC into the developing blastocyst environment of the mouse, we found that both neonatal and adult EpiSC retained some ability to participate in the formation of tissues from all three germ layers (Liang and Bickenbach, 2002; Bickenbach and Chinnathambi, 2004; Liang et al., 2004). Although it appears evident that EpiSC act as pluripotent stem cells, how this reprogramming takes place is not understood. EpiSC might directly transdifferentiate into other cell types or they might first dedifferentiate into a more primitive cell type, and then proceed to develop along a cell lineage pathway. To begin to unravel this, we co-cultured EpiSC with embryonic stem (ES) cells, and found that EpiSC could alter their cell lineage protein expression to that of a more primitive cell type. We also placed EpiSC in a wounded environment and found that EpiSC interacted with the mesenchymal cells repopulating the wound bed. Our findings indicate that the population of cells that we isolate as EpiSC has a pluripotent capability. This has led us to postulate a paradigm shift for somatic stem cells. We propose that tissues maintain a sequestered population of uncommitted stem cells that retain a regenerative response which is enhanced when the cells are exposed to developmental or stress influences.