We studied the impact of natural killer T (NKT) cell activation by alpha-galactocysylceramide (?-GalCer, ?-GC) on cancer cell repopulation during chemotherapy in murine mesothelioma. The number of NKT cells was found to be increased during the development of murine mesothelioma. NKT cells specifically recognize ?-GC through CD1d resulting in their activation and expansion. Tumor-bearing mice were treated with chemotherapy once weekly, and ?-GC was followed after each cycle of chemotherapy. Anti-tumor effect was evaluated on wild-type (WT) and CD1d knockout (CD1dKO) mice. Cancer cell proliferation and apoptosis were evaluated by Ki67 and TUNEL immunohistochemistry. CD4(+) and CD8(+) T cell proportion and activation in tumor, spleen, draining lymph node and peripheral blood were determined by flow cytometry, and gene expression of activated T cell-related cytokines was quantified by reverse transcription PCR. NKT cells were identified by CD1d-?-GC-tetramer staining. In WT mice, tumor growth delay was achieved by cisplatin (Cis), and this effect was improved in combination with ?-GC, but ?-GC alone had little effect. Cancer cell proliferation during chemotherapy was significantly inhibited by ?-GC, while cancer cell death was significantly upregulated. ?-GC following chemotherapy resulted in NKT cell expansion and an increase of interferon-? production in the draining lymph node, blood and spleen. Gene expression of immune-associated cytokines was upregulated. Strikingly, the percentage of inducible T cell co-stimulator(+)CD4 T cells, Th17/Tc17 cells increased in splenocytes. In CD1d KO mice, however, Cis alone was less effective and Cis + ?-GC provided no additional benefit over Cis alone. ?-GC alone had minimal effect in both mice. NKT activation between cycles of chemotherapy could improve the outcome of mesothelioma treatment.
Although multiple, complex molecular studies have been done for understanding the development and progression of pulmonary hypertension (PAH), little is known about the metabolic heterogeneity of PAH. Using a combination of high-throughput liquid-and-gas-chromatography-based mass spectrometry, we found bile acid metabolites, which are normally product derivatives of the liver and gallbladder, were highly increased in the PAH lung. Microarray showed that the gene encoding cytochrome P450 7B1 (CYP7B1), an isozyme for bile acid synthesis, was highly expressed in the PAH lung compared with the control. CYP7B1 protein was found to be primarily localized on pulmonary vascular endothelial cells suggesting de novo bile acid synthesis may be involved in the development of PAH. Here, by profiling the metabolomic heterogeneity of the PAH lung, we reveal a newly discovered pathogenesis mechanism of PAH.
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. Using 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 wild type (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 ?-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.
Excessive reactive oxygen/nitrogen species have been associated with the onset, progression, and outcome of sepsis, both in preclinical and clinical studies. However, the signaling pathways regulating oxidative/nitrative stress in the pathogenesis of sepsis-induced acute lung injury and acute respiratory distress syndrome are not fully understood. Employing the novel mouse model with genetic deletions of both caveolin-1 (Cav1) and adiponectin (ADPN) [double knockout (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 myeloperoxidase 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.
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.
Although multiple gene and protein expression have been extensively profiled in human pulmonary arterial hypertension (PAH), the mechanism for the development and progression of pulmonary hypertension remains elusive. Analysis of the global metabolomic heterogeneity within the pulmonary vascular system leads to a better understanding of disease progression. Using a combination of high-throughput liquid-and-gas-chromatography-based mass spectrometry, we showed unbiased metabolomic profiles of disrupted glycolysis, increased TCA cycle, and fatty acid metabolites with altered oxidation pathways in the human PAH lung. The results suggest that PAH has specific metabolic pathways contributing to increased ATP synthesis for the vascular remodeling process in severe pulmonary hypertension. These identified metabolites may serve as potential biomarkers for the diagnosis of PAH. By profiling metabolomic alterations of the PAH lung, we reveal new pathogenic mechanisms of PAH, opening an avenue of exploration for therapeutics that target metabolic pathway alterations in the progression of PAH.
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.
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.
The goal of this study was to determine the role of integrin-mediated adhesion of bone-marrow-derived progenitor cells (BMPCs) as a requirement for the endothelial barrier protection in a lung injury model. C57BL mice were used as the source for BMPCs, which were characterized as CD34(+) and fetal liver kinase-1 (Flk1)(+) and also an expression of a repertoire of integrins. We used a mouse model of bacterial lipopolysaccharide (LPS)-induced lung vascular injury and edema formation to test the effects of BMPC integrin expression in preventing endothelial barrier injury. Adhesion of BMPCs to purified extracellular matrix proteins induced focal adhesion kinase (Fak) phosphorylation and formation of branching point structures in a alpha(4) and alpha(5) integrin-dependent manner. BMPCs expressing red fluorescent protein (RFP) were administered via the retro-orbital venous route in mice treated intraperitonially with LPS (7.5 mg/kg body weight). We observed increased retention of RFP-labeled Flk1(+) and CD34(+) BMPCs for up to 8 weeks in mice injured with LPS. BMPC transplantation increased survival by 50% (at 72-96 hours after LPS) and reduced lung vascular injury and extravascular water content induced by LPS. However, blocking with anti-alpha(4) or anti-alpha(5) integrin antibody or shRNA-mediated silencing of alpha(4) or alpha(5) integrins in donor BMPCs failed to prevent the vascular injury or edema formation and mortality. Thus, alpha(4) and alpha(5) integrin-dependent adhesion of BMPCs in lung tissue plays a critical role in preventing lung vascular injury and increasing survival in a mouse model of LPS-induced acute lung injury.
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).
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.
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.
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