[Show abstract][Hide abstract] ABSTRACT: Adult stem cell treatment is a potential novel therapeutic approach for acute respiratory distress syndrome. Given the extremely low rate of cell engraftment, it is believed that these cells exert their beneficial effects via paracrine mechanisms. However, the endogenous mediator(s) in the pulmonary vasculature remains unclear. Employing the mouse model with endothelial cell (EC)-restricted disruption of FoxM1 (FoxM1 CKO), here we show that endothelial expression of the reparative transcriptional factor FoxM1 is required for the protective effects of bone marrow progenitor cells (BMPC) against LPS-induced inflammatory lung injury and mortality. BMPC treatment resulted in rapid induction of FoxM1 expression in WT but not FoxM1 CKO lungs. BMPC-induced inhibition of lung vascular injury, resolution of lung inflammation, and survival, as seen in WT mice, were abrogated in FoxM1 CKO mice following LPS challenge. Mechanistically, BMPC treatment failed to induce lung EC proliferation in FoxM1 CKO mice, which was associated with impaired expression of FoxM1 target genes essential for cell cycle progression. We also observed that BMPC treatment enhanced endothelial barrier function in WT, but not in FoxM1-deficient EC monolayers. Restoration of β-catenin expression in FoxM1-deficient ECs normalized endothelial barrier enhancement in response to BMPC treatment. These data demonstrate the requisite role of endothelial FoxM1 in the mechanism of BMPC-induced vascular repair to restore vascular integrity and accelerate resolution of inflammation, thereby promoting survival following inflammatory lung injury. Stem Cells 2014.
[Show abstract][Hide abstract] ABSTRACT: Here we found that the transcription repressor DREAM bound to the promoter of the gene encoding A20 to repress expression of this deubiquitinase that suppresses inflammatory NF-κB signaling. DREAM-deficient mice displayed persistent and unchecked A20 expression in response to endotoxin. DREAM functioned by transcriptionally repressing A20 through binding to downstream regulatory elements (DREs). In contrast, binding of the transcription factor USF1 to the DRE-associated E-box domain in the gene encoding A20 activated its expression in response to inflammatory stimuli. Our studies define the critical opposing functions of DREAM and USF1 in inhibiting and inducing A20 expression, respectively, and thereby the strength of NF-κB signaling. Targeting of DREAM to induce USF1-mediated A20 expression is therefore a potential anti-inflammatory strategy for the treatment of diseases associated with unconstrained NF-κB activity, such as acute lung injury.
[Show abstract][Hide abstract] ABSTRACT: Excessive reactive oxygen/nitrogen species have been associated with the onset, progression and outcome of sepsis, both in pre-clinical and clinical studies. However, the signaling pathways regulating oxidative/nitrative stress in the pathogenesis of sepsis-induced acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are not fully understood. Employing the novel mouse model with genetic deletions of both Cav1 and ADPN (DKO mice), we have demonstrated the critical role of Cav1 and ADPN signaling cross-talk in regulating oxidative/nitrative stress and resulting inflammatory lung injury following LPS challenge. In contrast to the inhibited inflammatory lung injury in Cav1(-/-) mice, we observed severe lung inflammation and markedly increased lung vascular permeability in DKO mice in response to LPS challenge. Accordingly, the DKO mice exhibited an 80% mortality rate following a sublethal dose of LPS challenge. At basal state, loss of Cav1 and ADPN resulted in a drastic increase of oxidative stress and resultant nitrative stress in DKO lungs. Scavenging of superoxide by pretreating the DKO mice with MnTMPYP (a superoxide dismutase mimetic) restored the inflammatory responses to LPS challenge including reduced lung MPO activity and vascular permeability. Thus, oxidative/nitrative stress collectively modulated by Cav1 and ADPN signalings is a critical determinant of inflammatory lung injury in response to LPS challenge.
[Show abstract][Hide abstract] ABSTRACT: Adult stem cell-based therapy is a promising novel approach for treatment of acute lung injury. Here we investigated the therapeutic potential of freshly isolated human umbilical cord blood CD34(+) progenitor cells (fCB-CD34(+) cells) in a mouse model of acute lung injury. At 3 h post-lipopolysaccharide (LPS) challenge, fCB-CD34(+) cells were transplanted i.v. to mice while CD34(-) cells or PBS were administered as controls in separate cohorts of mice. We observed that fCB-CD34(+) cell treatment inhibited lung vascular injury evident by decreased lung vascular permeability. In contrast, CD34(-) cells had no effects on lung vascular injury. Lung inflammation determined by myeloperoxidase activity, neutrophil sequestration and expression of pro-inflammatory mediators was attenuated in fCB-CD34(+) cell-treated mice at 26 h post-LPS challenge compared to PBS or CD34(-) cell-treated controls. Importantly, lung inflammation in fCB-CD34(+) cell-treated mice was returned to normal levels as seen in basal mice at 52 h post-LPS challenge whereas PBS or CD34(-) cell-treated control mice exhibited persistent lung inflammation. Accordingly, fCB-CD34(+) cell-treated mice exhibited a marked increase of survival rate. Employing in vivo 5-bromo-2'-deoxyuridine incorporation assay, we found a drastic induction of lung endothelial proliferation in fCB-CD34(+) cell-treated mice at 52 h post-LPS compared to PBS or CD34(-) cell-treated controls, which contributed to restoration of vascular integrity and thereby inhibition of lung inflammation. Taken together, these data have demonstrated the protective effects of fCB-CD34(+) cell on acute lung injury induced by LPS challenge, suggesting fCB-CD34(+) cells are an important source of stem cells for the treatment of acute lung injury.
PLoS ONE 01/2014; 9(2):e88814. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hypoxic pulmonary vasoconstriction (HPV) is a compensatory physiological mechanism in the lung that optimizes the matching of ventilation to perfusion and thereby maximizes gas exchange. Historically, HPV has been primarily studied in isolated perfused/ventilated lungs; however, the results of these studies have varied greatly due to different experimental conditions and species. Therefore, in the present study, we utilized the mouse isolated perfused/ventilated lung model for investigation of the role of extracellular Ca(2+) and caveolin-1 and endothelial nitric oxide synthase expression on HPV. We also compared HPV using different perfusate solutions: Physiological salt solution (PSS) with albumin, Ficoll, rat blood, fetal bovine serum (FBS), or Dulbecco's Modified Eagle Medium (DMEM). After stabilization of the pulmonary arterial pressure (PAP), hypoxic (1% O2) and normoxic (21% O2) gases were applied via a ventilator in five-minute intervals to measure HPV. The addition of albumin or Ficoll with PSS did not induce persistent and strong HPV with or without a pretone agent. DMEM with the inclusion of FBS in the perfusate induced strong HPV in the first hypoxic challenge, but the HPV was neither persistent nor repetitive. PSS with rat blood only induced a small increase in HPV amplitude. Persistent and repetitive HPV occurred with PSS with 20% FBS as perfusate. HPV was significantly decreased by the removal of extracellular Ca(2+) along with addition of 1 mM EGTA to chelate residual Ca(2+) and voltage-dependent Ca(2+) channel blocker (nifedipine 1 μM). PAP was also reactive to contractile stimulation by high K(+) depolarization and U46619 (a stable analogue of thromboxane A2). In summary, optimal conditions for measuring HPV were established in the isolated perfused/ventilated mouse lung. Using this method, we further confirmed that HPV is dependent on Ca(2+) influx.
[Show abstract][Hide abstract] ABSTRACT: Protein kinase G (PKG) plays an important role in the regulation of vascular smooth cell contractility and is a critical mediator of nitric oxide signaling, which regulates cardiovascular homeostasis. PKG-I-knockout (Prkg1(-/-)) mice exhibit impaired nitric oxide/cGMP-dependent vasorelaxation and systemic hypertension. However, it remains unknown whether PKG-I deficiency induces pulmonary hypertension. In this study, we characterized the hypertensive pulmonary phenotypes in Prkg1(-/-) mice and delineated the underlying molecular basis. We observed a significant increase in right ventricular systolic pressure in Prkg1(-/-) mice in the absence of systemic hypertension and left-sided heart dysfunction. In addition, we observed marked muscularization of distal pulmonary vessels in Prkg1(-/-) mice. Microangiography revealed impaired integrity of the pulmonary vasculature in Prkg1(-/-) mice. Mechanistically, PKG-I-mediated phosphorylation of Rho A Ser188 was markedly decreased, and the resultant Rho A activation was significantly increased in Prkg1(-/-) lung tissues, which resulted in Rho kinase activation. The i.t. administration of fasudil, a Rho kinase inhibitor, reversed the hypertensive pulmonary phenotype in Prkg1(-/-) mice. Taken together, these data show that PKG-I deficiency induces pulmonary hypertension through Rho A/Rho kinase activation-mediated vasoconstriction and pulmonary vascular remodeling.
American Journal Of Pathology 06/2012; 180(6):2268-75. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Enhancing endothelial barrier integrity for the treatment of acute lung injury (ALI) is an emerging novel therapeutic strategy. Our previous studies have demonstrated the essential role of FoxM1 in mediating endothelial regeneration and barrier repair following lipopolysaccharide-induced lung injury. However, it remains unclear whether FoxM1 expression is sufficient to promote endothelial repair in experimental models of sepsis. Here, employing the FoxM1 transgenic (FoxM1 Tg) mice, we showed that transgenic expression of FoxM1 promoted rapid recovery of endothelial barrier function and survival in a clinically relevant model of sepsis induced by cecal ligation and puncture (CLP). We observed lung vascular permeability was rapidly recovered and returned to levels similar to baseline at 48 h post-CLP challenge in FoxM1 Tg mice whereas it remained markedly elevated in WT mice. Lung edema and inflammation were resolved only in FoxM1 Tg mice at 24 h post-CLP. 5-bromo-2-deoxyuridine incorporation assay revealed a drastic induction of endothelial proliferation in FoxM1 Tg lungs at 24h post-CLP, correlating with early induction of expression of FoxM1 target genes essential for cell cycle progression. Additionally, deletion of FoxM1 in endothelial cells, employing the mouse model with endothelial cell-restricted disruption of FoxM1 (FoxM1 CKO) resulted in impaired endothelial repair following CLP challenge. Together, these data suggest FoxM1 expression in endothelial cells is necessary and sufficient to mediate endothelial repair and thereby promote survival following sepsis challenge.
PLoS ONE 01/2012; 7(11):e50094. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The alveolar epithelium is composed of the flat type I cells comprising 95% of the gas-exchange surface area and cuboidal type II cells comprising the rest. Type II cells are described as facultative progenitor cells based on their ability to proliferate and trans-differentiate into type I cells. In this study, we observed that pneumonia induced by intratracheal instillation of Pseudomonas aeruginosa (PA) in mice increased the expression of the forkhead transcription factor FoxM1 in type II cells coincidentally with the induction of alveolar epithelial barrier repair. FoxM1 was preferentially expressed in the Sca-1(+) subpopulation of progenitor type II cells. In mice lacking FoxM1 specifically in type II cells, type II cells showed decreased proliferation and impaired trans-differentiation into type I cells. Lungs of these mice also displayed defective alveolar barrier repair after injury. Expression of FoxM1 in the knockout mouse lungs partially rescued the defective trans-differentiation phenotype. Thus, expression of FoxM1 in type II cells is essential for their proliferation and transition into type I cells and for restoring alveolar barrier homeostasis after PA-induced lung injury.
Journal of Experimental Medicine 06/2011; 208(7):1473-84. · 13.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Endothelial barrier function is regulated by adherens junctions (AJs) and caveolae-mediated transcellular pathways. The opening of AJs that is observed in caveolin-1(-/-) (Cav-1(-/-)) endothelium suggests that Cav-1 is necessary for AJ assembly or maintenance. Here, using endothelial cells isolated from Cav-1(-/-) mice, we show that Cav-1 deficiency induced the activation of endothelial nitric oxide synthase (eNOS) and the generation of nitric oxide (NO) and peroxynitrite. We assessed S-nitrosylation and nitration of AJ-associated proteins to identify downstream NO redox signaling targets. We found that the GTPase-activating protein (GAP) p190RhoGAP-A was selectively nitrated at Tyr1105, resulting in impaired GAP activity and RhoA activation. Inhibition of eNOS or RhoA restored AJ integrity and diminished endothelial hyperpermeability in Cav-1(-/-) mice. Thrombin, a mediator of increased endothelial permeability, also induced nitration of p120-catenin-associated p190RhoGAP-A. Thus, eNOS-dependent nitration of p190RhoGAP-A represents a crucial mechanism for AJ disassembly and resultant increased endothelial permeability.
The Journal of Cell Biology 05/2011; 193(5):841-50. · 10.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Repair of the injured vascular intima requires a series of coordinated events that mediate both endothelial regeneration and reannealing of adherens junctions (AJs) to form a restrictive endothelial barrier. The forkhead transcription factor FoxM1 is essential for endothelial proliferation after vascular injury. However, little is known about mechanisms by which FoxM1 regulates endothelial barrier reannealing. Here, using a mouse model with endothelial cell (EC)-restricted disruption of FoxM1 (FoxM1 CKO) and primary cultures of ECs with small interfering RNA (siRNA)-mediated knockdown of FoxM1, we demonstrate a novel requisite role of FoxM1 in mediating endothelial AJ barrier repair through the transcriptional control of beta-catenin. In the FoxM1 CKO lung vasculature, we observed persistent microvessel leakage characterized by impaired reannealing of endothelial AJs after endothelial injury. We also showed that FoxM1 directly regulated beta-catenin transcription and that reexpression of beta-catenin rescued the defective AJ barrier-reannealing phenotype of FoxM1-deficient ECs. Knockdown of beta-catenin mimicked the phenotype of defective barrier recovery seen in FoxM1-deficient ECs. These data demonstrate that FoxM1 is required for reannealing of endothelial AJs in order to form a restrictive endothelial barrier through transcriptional control of beta-catenin expression. Therefore, means of activating FoxM1-mediated endothelial repair represent a new therapeutic strategy for the treatment of inflammatory vascular diseases associated with persistent vascular barrier leakiness such as acute lung injury.
Journal of Experimental Medicine 08/2010; 207(8):1675-85. · 13.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mechanism underlying the protective effect of sphingosine kinase 1 (SphK1) in inflammatory injury is not clear. We demonstrated
using SphK1-null mice (SphK1−/−) the crucial role of SphK1 in suppressing lipopolysaccharide-induced neutrophil oxidant production and sequestration in lungs
and mitigating lung inflammatory injury. This effect of SphK1 was independent of the production of sphingosine 1-phosphate,
the product of SphK1 activity. The anti-inflammatory effect of SphK1 in the lipopolysaccharide model was mediated through
SphK1 interaction with JNK. SphK1 stabilization of JNK in turn inhibited JNK binding to the JNK-interacting protein 3 (JIP3)
and thus abrogated the activation of NADPH oxidase and oxidant generation and resultant NF-κB activation. Therefore, SphK1-mediated
down-regulation of JNK activity serves to dampen inflammation and tissue injury.
Journal of Biological Chemistry 05/2010; 285(21):15848-15857. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mechanism underlying the protective effect of sphingosine kinase 1 (SphK1) in inflammatory injury is not clear. We demonstrated using SphK1-null mice (SphK1(-/-)) the crucial role of SphK1 in suppressing lipopolysaccharide-induced neutrophil oxidant production and sequestration in lungs and mitigating lung inflammatory injury. This effect of SphK1 was independent of the production of sphingosine 1-phosphate, the product of SphK1 activity. The anti-inflammatory effect of SphK1 in the lipopolysaccharide model was mediated through SphK1 interaction with JNK. SphK1 stabilization of JNK in turn inhibited JNK binding to the JNK-interacting protein 3 (JIP3) and thus abrogated the activation of NADPH oxidase and oxidant generation and resultant NF-kappaB activation. Therefore, SphK1-mediated down-regulation of JNK activity serves to dampen inflammation and tissue injury.
Journal of Biological Chemistry 03/2010; 285(21):15848-57. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Caveolin-1 (Cav1), the scaffolding protein of caveolae, has been shown to play an important role in host defense and inflammation. However, the underlying molecular basis for these actions remains elusive. Here, using double mutant mice with genetic deletions of Cav1 and NOS3, we show that chronic endothelial nitric oxide synthase (eNOS) activation secondary to loss of Cav1 serves a crucial immunomodulatory function through tyrosine nitration-mediated impairment of interleukin-1 receptor associated kinase (IRAK)4, a signaling component required for nuclear factor-kappaB activation and innate immunity. We observed an eNOS-dependent decrease in the plasma concentration of pro-inflammatory cytokines and marked improvement of survival in Cav1(-/-) mice following lipopolysaccharide challenge. Activation of eNOS secondary to loss of Cav1 resulted in decreased activation of nuclear factor-kappaB in response to lipopolysaccharide challenge, and thereby protected the animals from lipopolysaccharide-induced lung injury. IRAK4 was prominently nitrated in Cav1-deficient endothelial cells, whereas eNOS deletion in Cav1-deficient endothelial cells resulted in marked decrease of IRAK4 nitration and restored the inflammatory response after lipopolysaccharide challenge. Furthermore, in vitro nitration of IRAK4 resulted in impairment of the kinase activity. Thus, eNOS activation secondary to loss of Cav1 signals dampening of the innate immune response to lipopolysaccharide through IRAK4 nitration and the resultant impairment of kinase activity, and consequently mitigates inflammatory lung injury.
American Journal Of Pathology 03/2010; 176(5):2344-51. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Since thrombin activation of endothelial cells (ECs) is well-known to increase endothelial permeability by disassembly of adherens junctions (AJs) and actinomyosin contractility mechanism involving myosin light chain (MLC) phosphorylation, we investigated the effects of bone marrow-derived progenitor cells (BMPCs) on the thrombin-induced endothelial permeability response. We observed that addition of BMPCs to endothelial monolayers at a fixed ratio prevented the thrombin-induced decrease in transendothelial electrical resistance, a measure of AJ integrity, and increased mouse pulmonary microvessel filtration coefficient, a measure of transvascular liquid permeability. The barrier protection was coupled to increased vascular endothelial cadherin expression and increased Cdc42 activity in ECs. Using small interfering RNA (siRNA) to deplete Cdc42 in ECs, we demonstrated a key role of Cdc42 in signaling the BMPC-induced endothelial barrier protection. Endothelial integrity induced by BMPCs was also secondary to inhibition of MLC phosphorylation in ECs. Thus BMPCs interacting with ECs prevent thrombin-induced endothelial hyperpermeability by a mechanism involving AJ barrier annealing, inhibition of MLC phosphorylation, and activation of Cdc42.
[Show abstract][Hide abstract] ABSTRACT: Severe pulmonary hypertension (PH) is characterized by a progressive increase in pulmonary vascular resistance and vascular remodeling leading to right heart failure and early death. Our recent studies with the use of the novel mouse model with genetic deletions of caveolin-1 (Cav1) and endothelial nitric oxide synthase (eNOS) (NOS3) have demonstrated that persistent eNOS activation in Cav1(-/-) lungs results in tyrosine nitration of protein kinase G (PKG) and impairment of its activity, which thereby induces PH. The finding of eNOS activation and PKG nitration concomitant with Cav1 deficiency was recapitulated in lungs from patients with idiopathic pulmonary arterial hypertension. These data suggest targeting PKG nitration has potential value for the treatment of PH. Here, we will review the current knowledge about Cav1-regulated eNOS activity and its fundamental role in the pathogenesis of PH.
Trends in cardiovascular medicine 10/2009; 19(7):238-42. · 4.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Little is known about the contribution of bone marrow-derived progenitor cells (BMPCs) in the regulation endothelial barrier function as defined by microvascular permeability alterations at the level of adherens junctions (AJs).
We investigated the role of BMPCs in annealing AJs and thereby in preventing lung edema formation induced by endotoxin (LPS).
We observed that BMPCs enhanced basal endothelial barrier function and prevented the increase in pulmonary microvascular permeability and edema formation in mice after LPS challenge. Coculture of BMPCs with endothelial cells induced Rac1 and Cdc42 activation and AJ assembly in endothelial cells. However, transplantation of BMPCs isolated from sphingosine kinase-1-null mice (SPHK1(-/-)), having impaired S1P production, failed to activate Rac1 and Cdc42 or protect the endothelial barrier.
These results demonstrate that BMPCs have the ability to reanneal endothelial AJs by paracrine S1P release in the inflammatory milieu and the consequent activation of Rac-1 and Cdc42 in endothelial cells.
Circulation Research 09/2009; 105(7):696-704, 8 p following 704. · 11.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pulmonary hypertension (PH) is an unremitting disease defined by a progressive increase in pulmonary vascular resistance leading to right-sided heart failure. Using mice with genetic deletions of caveolin 1 (Cav1) and eNOS (Nos3), we demonstrate here that chronic eNOS activation secondary to loss of caveolin-1 can lead to PH. Consistent with a role for eNOS in the pathogenesis of PH, the pulmonary vascular remodeling and PH phenotype of Cav1-/- mice were absent in Cav1-/-Nos3-/- mice. Further, treatment of Cav1-/- mice with either MnTMPyP (a superoxide scavenger) or l-NAME (a NOS inhibitor) reversed their pulmonary vascular pathology and PH phenotype. Activation of eNOS in Cav1-/- lungs led to the impairment of PKG activity through tyrosine nitration. Moreover, the PH phenotype in Cav1-/- lungs could be rescued by overexpression of PKG-1. The clinical relevance of the data was indicated by the observation that lung tissue from patients with idiopathic pulmonary arterial hypertension demonstrated increased eNOS activation and PKG nitration and reduced caveolin-1 expression. Together, these data show that loss of caveolin-1 leads to hyperactive eNOS and subsequent tyrosine nitration-dependent impairment of PKG activity, which results in PH. Thus, targeting of PKG nitration represents a potential novel therapeutic strategy for the treatment of PH.
The Journal of clinical investigation 07/2009; 119(7):2009-18. · 15.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nonmuscle myosin light-chain kinase (MYLK) mediates increased lung vascular endothelial permeability in lipopolysaccharide-induced lung inflammatory injury, the chief cause of the acute respiratory distress syndrome. In a lung injury model, we demonstrate here that MYLK was also essential for neutrophil transmigration, but that this function was mostly independent of myosin II regulatory light chain, the only known substrate of MYLK. Instead, MYLK in neutrophils was required for the recruitment and activation of the tyrosine kinase Pyk2, which mediated full activation of beta(2) integrins. Our results demonstrate that MYLK-mediated activation of beta(2) integrins through Pyk2 links beta(2) integrin signaling to the actin motile machinery of neutrophils.