It has been suggested that pancreatic acinar cells can serve as progenitors for pancreatic islets, a concept with substantial implications for therapeutic efforts to increase insulin-producing β cell mass in patients with diabetes. We report what we believe to be the first in vivo lineage tracing approach to determine the plasticity potential of pancreatic acinar cells. We developed an acinar cell-specific inducible Cre recombinase transgenic mouse, which, when mated with a reporter strain and pulsed with tamoxifen, resulted in permanent and specific labeling of acinar cells and their progeny. During various time periods of observation and using several models to provoke injury, we failed to observe any chase of the labeled cells into the endocrine compartment, indicating that acinar cells do not normally transdifferentiate into islet β cells in vivo in adult mice. In contrast, we observed a substantial role for replication of preexisting acinar cells in the regeneration of new acinar cells after partial pancreatectomy. These results indicate that mature acinar cells harbor a facultative acinar but not endocrine progenitor capacity.

Loss of fluid shear stress (ischemia) to the lung endothelium causes endothelial plasma membrane depolarization via ATP-sensitive K(+) (K(ATP)) channel closure, initiating a signaling cascade that leads to NADPH oxidase (NOX2) activation and ROS production. Since wortmannin treatment significantly reduces ROS production with ischemia, we investigated the role of phosphoinositide 3-kinase (PI3K) in shear-associated signaling. Pulmonary microvascular endothelial cells in perfused lungs subjected to abrupt stop of flow showed membrane depolarization and ROS generation. Stop of flow in flow-adapted mouse pulmonary microvascular endothelial cells in vitro resulted in the activation of PI3K and Akt as well as ROS generation. ROS generation in the lungs in situ was almost abolished by the PI3K inhibitor wortmannin and the PKC inhibitor H7. The combination of the two (wortmannin and H7) did not have a greater effect. Activation of NOX2 was greatly diminished by wortmannin, knockout of Akt1, or dominant negative PI3K, whereas membrane depolarization was unaffected. Ischemia-induced Akt activation (phosphorylation) was not observed with K(ATP) channel-null cells, which showed minimal changes in membrane potential with ischemia. Activation of Akt was similar to wild-type cells in NOX2-null cells, which do not generate ROS with ischemia. Cromakalim, a K(ATP) channel agonist, prevented both membrane depolarization and Akt phosphorylation with ischemia. Thus, Akt1 phosphorylation follows cell membrane depolarization and precedes the activation of NOX2. These results indicate that PI3K/Akt and PKC serve as mediators between endothelial cell membrane depolarization and NOX2 assembly.

Rationale: Endothelial thrombomodulin (TM) regulates thrombosis and inflammation. Diverse forms of pulmonary and vascular injury are accompanied by down-regulation of TM, which aggravates tissue injury. We postulated that anchoring TM to the endothelial surface would restore its protective functions. Objectives: To design an effective and safe strategy to treat pulmonary thrombotic and inflammatory injury. Methods: We synthesized a fusion protein, designated scFv/TM, by linking the extracellular domain of mouse TM to a single-chain variable fragment of an antibody to platelet endothelial cell adhesion molecule-1 (PECAM-1). The targeting and protective functions of scFv/TM were tested in mouse models of lung ischemia-reperfusion and acute lung injury (ALI) caused by intratracheal endotoxin and hyperoxia, both of which caused approximately 50% reduction in the endogenous expression of TM. Measurements and Main Results: Biochemical assays showed that scFv/ TM accelerated protein C activation by thrombin and bound mouse PECAM-1 and cytokine high mobility group-B1. After intravenous injection, scFv/TM preferentially accumulated in the mouse pulmonary vasculature. In a lung model of ischemia-reperfusion injury, scFv/TM attenuated elevation of early growth response-1, inhibited pulmonary deposition of fibrin and leukocyte infiltration, and preserved blood oxygenation more effectively than soluble TM. In an ALI model, scFv/TM, but not soluble TM, suppressed activation of nuclear factor-kB, inflammation and edema in the lung and reduced mortality without causing hemorrhage. Conclusions: Targeting TM to the endothelium using an scFv anchor enhances its antithrombotic and antiinflammatory effectiveness in models of ALI.

Abrupt cessation of flow representing the acute loss of shear stress (simulated ischemia) to flow-adapted pulmonary microvascular endothelial cells (PMVEC) leads to reactive oxygen species (ROS) generation that signals for EC proliferation. We evaluated the role of caveolin-1 on this cellular response with mouse PMVEC that were preconditioned for 72 h to laminar flow at 5 dyn/cm(2) followed by stop of flow ("ischemia"). Preconditioning resulted in a 2.7-fold increase in cellular expression of K(ATP) (K(IR) 6.2) channels but no change in expression level of caveolin-1, gp91(phox), or MAP kinases. The initial response to ischemia in wild type cells was cell membrane depolarization that was abolished by gene targeting of K(IR) 6.2. The subsequent response was increased ROS production associated with activation of NADPH oxidase (NOX2) and then phosphorylation of MAP kinases (Erk, JNK). After 24 h of ischemia in wild type cells, the cell proliferation index increased 2.5 fold and the % of cells in S+G(2)/M phases increased 6-fold. This signaling cascade (cell membrane depolarization, ROS production, MAP kinase activation and cell proliferation) was abrogated in caveolin-1 null PMVEC or by treatment of wild type cells with filipin. These studies indicate that caveolin-1 functions as a shear sensor in flow-adapted EC resulting in ROS-mediated cell signaling and endothelial cell proliferation following the abrupt reduction in flow.

AB, Chatterjee S. PECAM-1 and caveolae form the mechanosensing complex necessary for NOX2 activation and angiogenic signaling with stopped flow in pulmonary endothelium. Am J Physiol Lung Cell Mol Physiol 305: L805-L818, 2013. First published September 27, 2013 doi:10.1152/ajplung.00123.2013.-We showed that stop of flow triggers a mechanosignaling cascade that leads to the generation of reactive oxygen species (ROS); however, a mechanosensor coupled to the cytoskeleton that could potentially transduce flow stimulus has not been identified. We showed a role for KATP channel, caveolae (caveolin-1), and NADPH oxidase 2 (NOX2) in ROS production with stop of flow. Based on reports of a mechanosensory complex that includes platelet endothelial cell adhesion molecule-1 (PECAM-1) and initiates signaling with mechanical force, we hypothesized that PECAM-1 could serve as a mechanosensor in sensing disruption of flow. Using lungs in situ, we observed that ROS production with stop of flow was significantly reduced in PECAM-1 Ϫ/Ϫ lungs compared with lungs from wild-type (WT) mice. Lack of PECAM-1 did not affect NOX2 activation machinery or the caveolin-1 expression or caveolae number in the pulmonary endothelium. Stop of flow in vitro triggered an increase in angiogenic potential of WT pulmonary microvascular endothelial cells (PMVEC) but not of PECAM-1 Ϫ/Ϫ PMVEC. Obstruction of flow in lungs in vivo showed that the neutrophil infiltration as observed in WT mice was significantly lowered in PECAM-1 Ϫ/Ϫ mice. With stop of flow, WT lungs showed higher expression of the angiogenic marker VEGF compared with untreated (sham) and PECAM-1 Ϫ/Ϫ lungs. Thus PECAM-1 (and caveolae) are parts of the mechanosensing machinery that generates superoxide with loss of shear; the resultant ROS potentially drives neutrophil influx and acts as an angiogenic signal. mechanotransduction; stop of flow; pulmonary endothelium; PECAM; K atp (Kir6.2) channel; NOX2; angiogenic potential CELLS SENSE THE PHYSICAL STIMULUS in their environment and translate these physical forces into biochemical signals (20, 37). Sensing and responding to a physical force require specialized structures and machinery that can engage in signal transduction (12,23,42).In the vascular system, with a highly distributed network of blood vessels, mechanical forces arising from blood flow initiate signaling that helps maintain vascular structure and function. Indeed, shear associated with blood flow is sensed by the endothelium and the resultant signaling regulates normal vascular physiology (such as embryonic morphogenesis and organization of the vascular tree) while irregular or abnormal shear can lead to vascular dysfunction and disease (19,27). Thus the mechanosignaling that accompanies various shear profiles and patterns, regular or aberrant, governs susceptibility to atherosclerosis, by inducing athero-protective or atheroprone phenotypes in endothelial cells (10,22). It thus becomes important to understand the link among the mechanical force, the shear sensing machinery and...

Aims: We reported earlier that ischemia results in the generation of reactive oxygen species (ROS) via the closure of a K ATP channel which causes membrane depolarization and NADPH oxidase 2 (NOX2) activation. This study was undertaken to understand the role of ischemia-mediated ROS in signaling. Results: Angiogenic potential of pulmonary microvascular endothelial cells (PMVEC) was studied in vitro and in the hind limb in vivo. Flow adapted PMVEC injected into a Matrigel matrix showed significantly higher tube formation than cells grown under static conditions or cells from mice with knockout of K ATP channels or the NOX2. Blocking of hypoxia inducible factor-1 alpha (HIF-1a) accumulation completely abrogated the tube formation in wild-type (WT) PMVEC. With ischemia in vivo (femoral artery ligation), revascularization was high in WT mice and was significantly decreased in mice with knockout of K ATP channel and in mice orally fed with a K ATP channel agonist. In transgenic mice with endothelial-specific NOX2 expression, the revascularization observed was intermediate between that of WT and knockout of K ATP channel or NOX2. Increased HIF-1a activation and vascular endothelial growth factor (VEGF) expression was observed in ischemic tissue of WT mice but not in K ATP channel and NOX2 null mice. Revascularization could be partially rescued in K ATP channel null mice by delivering VEGF into the hind limb. Innovation: This is the first report of a mechanosensitive ion channel (K ATP channel) initiating endothelial signaling that drives revascularization. Conclusion: The K ATP channel responds to the stop of flow and activates signals for revascularization to restore the impeded blood flow. Antioxid. Redox Signal. 20,[872][873][874][875][876][877][878][879][880][881][882][883][884][885][886]

A recombinant prodrug, single-chain urokinase-type plasminogen activator (scuPA) fused to an anti-PECAM-1 antibody single-chain variable fragment (anti-PECAM scFv/scuPA) targets endothelium and augments thrombolysis in the pulmonary vasculature. 1 To avoid premature activation and inactivation and to limit systemic toxicity, we replaced the native plasmin activation site in scFv/lowmolecular-weight (lmw)-scuPA with a thrombin activation site, generating anti-PECAM scFv/uPA-T that (1) is latent and activated by thrombin instead of plasmin; (2) binds to PECAM-1; (3) does not consume plasma fibrinogen; (4) accumulates in mouse lungs after intravenous injection; and (5) resists PA inhibitor PAI-1 until activated by thrombin. In mouse models of pulmonary thrombosis caused by thromboplastin and ischemiareperfusion (I/R), scFv/uPA-T provided more potent thromboprophylaxis and greater lung protection than plasminsensitive scFv/uPA. Endothelium-targeted thromboprophylaxis triggered by a prothrombotic enzyme illustrates a novel approach to time-and site-specific regulation of proteolytic reactions that can be modulated for therapeutic benefit. IntroductionPlasminogen activators (PAs) used in the treatment of thrombosis convert the zymogen plasminogen into plasmin, which lyses fibrin and restores perfusion. [2][3][4] Unfortunately, the utility of PAs is limited by inadequate delivery (rapid clearance, inactivation, and ineffective penetration of occlusive clots) and side effects, including hemorrhage and extravascular toxicity. [5][6][7] Further, postevent thrombolytic therapy is marred by inevitable delays (time needed for diagnosis, transportation, injection, and lysis) causing ischemia-reperfusion (I/R) injury that perpetuates thrombosis and worsens outcome. 8,9 Attempts to improve the effectiveness of PAs by increasing clot affinity have further diminished permeation into occluding thrombi due to retention on the clot surface 5 and have yet to provide decisively better clinical outcomes. [10][11][12][13][14] Thrombi often recur due to underlying procoagulation states, vascular damage, disturbance in blood flow, inflammation, or/and patients' immobility. 15 Rethromboses often occur within hours to days after acute myocardial infarction, transient ischemic attack, ischemic stroke, and pulmonary embolism. The effectiveness of secondary prevention using existing anticoagulants and antiplatelet agents is limited and dosing is constrained by the risk of bleeding. 16 In theory, prophylactic delivery of a PA into nascent thrombi, which are more susceptible to dissolution than mature occlusive clots, would expedite thrombolysis and minimize ischemiareperfusion injury in patients at high risk of imminent thrombosis. Unfortunately, PAs are not suitable for thromboprophylaxis due to unfavorable pharmacokinetics and toxicity.Conceivably, the failure to exploit PAs for thromboprophylaxis could be overcome by optimization of drug and targeting strategies. Single-chain urokinase-type plasminogen activator (scuPA) seems suitable for t...

Rab38 is a rat Hermansky-Pudlak syndrome gene that plays an important role in surfactant homeostasis in alveolar type II (ATII) pneumocytes. We examined Rab38 function in regulating lamellar body (LB) morphology in ATII cells. Quantitative electron microscopy revealed that LBs in ATII cells were ∼77% larger in Rab38-null fawn-hooded hypertension (FHH) than control Sprague-Dawley (SD) rats. Rab38 protein expression was restricted in lung epithelial cells but was not found in primary endothelial cells. In SD ATII cells, Rab38 protein level gradually declined during 5 days in culture. Importantly, endogenous Rab38 was present in LB fractions purified from SD rat lungs, and transiently expressed enhanced green fluorescent protein (EGFP)-tagged Rab38 labeled only the limiting membranes of a subpopulation (∼30%) of LBs in cultured ATII cells. This selective targeting was abolished by point mutations to EGFP-Rab38 and was not shared by Rab7 and Rab4b, which also function in the ATII cells. Using confocal microscopy, we established a method for quantitative evaluation of the enlarged LB phenotype temporally preserved in cultured FHH ATII cells. A direct causal relationship was established when the enlarged LB phenotype was reserved and then rescued by transiently reexpressed EGFP-Rab38 in cultured FHH ATII cells. This rescuing effect was associated with dynamic EGFP-Rab38 targeting to and on LB limiting membranes. We conclude that Rab38 plays an indispensible role in maintaining LB morphology and surfactant homeostasis in ATII pneumocytes.