Cristina Alvira, MD

Abstract

RATIONALE: Premature infants exposed to oxygen are at risk for bronchopulmonary dysplasia (BPD), which is characterised by lung growth arrest. Inflammation is important, but the mechanisms remain elusive. Here, we investigated inflammatory pathways and therapeutic targets in severe clinical and experimental BPD.METHODS AND RESULTS: First, transcriptomic analysis with in-silico cellular deconvolution identified a lung-intrinsic M1-like-driven cytokine pattern in newborn mice after hyperoxia. These findings were confirmed by gene expression of macrophage-regulating chemokines (Ccl2, Ccl7, Cxcl5) and markers (Il6, Il17A, Mmp12). Second, hyperoxia-activated IL-6/STAT3 signaling was measured in vivo and related to loss of alveolar epithelial type II cells (ATII) as well as increased mesenchymal marker. Il6 null mice exhibited preserved ATII survival, reduced myofibroblasts and improved elastic fiber assembly, thus enabling lung growth and protecting lung function. Pharmacological inhibition of global IL-6 signaling and IL-6 trans-signaling promoted alveolarisation and ATII survival after hyperoxia. Third, hyperoxia triggered M1-like polarisation, possibly via Klf4; hyperoxia-conditioned medium of macrophages and IL-6 impaired ATII proliferation. Finally, clinical data demonstrate elevated macrophage-related plasma cytokines as potential biomarkers that identify infants receiving oxygen at increased risk of developing BPD. Moreover, macrophage-derived IL6 and active STAT3 were related to loss of epithelial cells in BPD lungs.CONCLUSION: We present a novel IL-6-mediated mechanism by which hyperoxia activates macrophages in immature lungs, impairs ATII homeostasis, and disrupts elastic fiber formation, thereby inhibiting lung growth. The data provide evidence that IL-6 trans-signaling could offer an innovative pharmacological target to enable lung growth in severe neonatal chronic lung disease.

View details for DOI 10.1183/13993003.02248-2020

View details for PubMedID 34446466

View details for Web of Science ID 000675441000191

View details for DOI 10.1164/rccm.202110-2316ED

View details for PubMedID 34797738

View details for Web of Science ID 000525432600027

View details for Web of Science ID 000525432600132

View details for DOI 10.1164/rccm.202004-0933ED

View details for PubMedID 32338996

Abstract

A wealth of evidence supports the broad therapeutic potential of NF-kappaB and EZH2 inhibitors as adjuvants for breast cancer treatment. We contribute to this knowledge by elucidating, for the first time, unique regulatory crosstalk between EZH2, NF-kappaB and the NF-kappaB interacting long non-coding RNA (NKILA). We define a novel signaling loop encompassing canonical and non-canonical actions of EZH2 on the regulation of NF-kappaB/NKILA homeostasis, with relevance to breast cancer treatment. We applied a respective silencing approach in non-transformed breast epithelial cells, triple negative MDA-MB-231 cells and hormone responsive MCF-7 cells, and measured changes in EZH2/NF-kappaB/NKILA levels to confirm their interdependence. We demonstrate cell line-specific fluctuations in these factors that functionally contribute to epithelial-to-mesenchymal transition (EMT) remodelling and cell fate response. EZH2 inhibition attenuates MDA-MB-231 cell motility and CDK4-mediated MCF-7 cell cycle regulation, while inducing global H3K27 methylation and an EMT phenotype in non-transformed cells. Notably, these events are mediated by a cell-context dependent gain or loss of NKILA and NF-kappaB. Depletion of NF-kappaB in non-transformed cells enhances their sensitivity to growth factor signaling and suggests a role for the host microenvironment milieu in regulating EZH2/NF-kappaB/NKILA homeostasis. Taken together, this knowledge critically informs the delivery and assessment of EZH2 inhibitors in breast cancer.

View details for DOI 10.1111/jcmm.14500

View details for PubMedID 31282094

View details for Web of Science ID 000459610400138

Abstract

Intrauterine growth restriction (IUGR) in premature newborns increases the risk for bronchopulmonary dysplasia, a chronic lung disease characterized by disrupted pulmonary angiogenesis and alveolarization. We previously showed that experimental IUGR impairs angiogenesis; however, mechanisms that impair pulmonary artery endothelial cell (PAEC) function are uncertain. The NF-B pathway promotes vascular growth in the developing mouse lung, and we hypothesized that IUGR disrupts NF-B-regulated proangiogenic targets in fetal PAEC. PAECs were isolated from the lungs of control fetal sheep and sheep with experimental IUGR from an established model of chronic placental insufficiency. Microarray analysis identified suppression of NF-B signaling and significant alterations in extracellular matrix (ECM) pathways in IUGR PAEC, including decreases in collagen 41 and laminin 4, components of the basement membrane and putative NF-B targets. In comparison with controls, immunostaining of active NF-B complexes, NF-B-DNA binding, baseline expression of NF-B subunits p65 and p50, and LPS-mediated inducible activation of NF-B signaling were decreased in IUGR PAEC. Although pharmacological NF-B inhibition did not affect angiogenic function in IUGR PAEC, angiogenic function of control PAEC was reduced to a similar degree as that observed in IUGR PAEC. These data identify reductions in endothelial NF-B signaling as central to the disrupted angiogenesis observed in IUGR, likely by impairing both intrinsic PAEC angiogenic function and NF-B-mediated regulation of ECM components necessary for vascular development. These data further suggest that strategies that preserve endothelial NF-B activation may be useful in lung diseases marked by disrupted angiogenesis such as IUGR.

View details for PubMedID 29722560

Abstract

Pulmonary angiogenesis is essential for alveolarization, the final stage of lung development that markedly increases gas exchange surface area. We recently demonstrated that activation of the nuclear factor kappa-B (NFB) pathway promotes pulmonary angiogenesis during alveolarization. However, the mechanisms activating NFB in the pulmonary endothelium, and its downstream targets are not known. In this study, we sought to delineate the specific roles for the NFB activating kinases, IKK and IKK, in promoting developmental pulmonary angiogenesis. Microarray analysis of primary pulmonary endothelial cells (PECs) after silencing IKK or IKK demonstrated that the 2 kinases regulate unique panels of genes, with few shared targets. Although silencing IKK induced mild impairments in angiogenic function, silencing IKK induced more severe angiogenic defects and decreased vascular cell adhesion molecule expression, an IKK regulated target essential for both PEC adhesion and migration. Taken together, these data show that IKK and IKK regulate unique genes in PEC, resulting in differential effects on angiogenesis upon inhibition, and identify IKK as the predominant regulator of pulmonary angiogenesis during alveolarization. These data suggest that therapeutic strategies to specifically enhance IKK activity in the pulmonary endothelium may hold promise to enhance lung growth in diseases marked by altered alveolarization.

View details for DOI 10.1111/jcmm.13741

View details for PubMedID 29993183

View details for PubMedCentralID PMC6111877

View details for DOI 10.1152/ajplung.00475.2017

View details for Web of Science ID 000443901300009

Abstract

Pulmonary artery smooth muscle cells (PASMCs) express endothelin (ET-1), which modulates the pulmonary vascular response to hypoxia. Although cross-talk between hypoxia-inducible factor-1alpha (HIF-1alpha), an O2-sensitive transcription factor, and ET-1 is established, the cell-specific relationship between HIF-1alpha and ET-1 expression remains incompletely understood. We tested the hypotheses that in PASMCs 1) HIF-1alpha expression constrains ET-1 expression, and 2) a specific microRNA (miRNA) links HIF-1alpha and ET-1 expression. In human (h)PASMCs, depletion of HIF-1alpha with siRNA increased ET-1 expression at both the mRNA and protein levels ( P < 0.01). In HIF-1alpha-/- murine PASMCs, ET-1 gene and protein expression was increased ( P < 0.0001) compared with HIF-1alpha+/+ cells. miRNA profiles were screened in hPASMCs transfected with siRNA-HIF-1alpha, and RNA hybridization was performed on the Agilent (Santa Clara, CA) human miRNA microarray. With HIF-1alpha depletion, miRNA-543 increased 2.4-fold ( P < 0.01). In hPASMCs, miRNA-543 overexpression increased ET-1 gene ( P < 0.01) and protein ( P < 0.01) expression, decreased TWIST gene expression ( P < 0.05), and increased ET-1 gene and protein expression, compared with nontargeting controls ( P < 0.01). Moreover, we evaluated low passage hPASMCs from control and patients with idiopathic pulmonary arterial hypertension (IPAH). Compared with controls, protein expression of HIF-1alpha and Twist-related protein-1 (TWIST1) was decreased ( P < 0.05), and miRNA-543 and ET-1 expression increased ( P < 0.001) in hPASMCs from patients with IPAH. Thus, in PASMCs, loss of HIF-1alpha increases miRNA-543, which decreases Twist expression, leading to an increase in PASMC ET-1 expression. This previously undescribed link between HIF-1alpha and ET-1 via miRNA-543 mediated Twist suppression represents another layer of molecular regulation that might determine pulmonary vascular tone.

View details for PubMedID 29745253

View details for DOI 10.1152/ajplung.00060.2018

View details for Web of Science ID 000442116600002

View details for PubMedID 30055815

View details for DOI 10.1152/ajplung.00363.2017

View details for Web of Science ID 000440952000007

Abstract

Accessory subunits associated with the calcium-sensitive potassium channel (BKCa), a major determinant of vascular tone, confer functional and anatomical diversity. The beta1 subunit increases Ca2+-, and voltage-sensitivity of the BKCa channel and is expressed exclusively in smooth muscle cells (SMC). Evidence supporting the physiologic significance of the beta1 subunit includes the observations that murine models with deletion of the beta1 subunit are hypertensive and that humans with a gain-of-function beta1 mutation are at decreased risk of diastolic hypertension, However, whether the beta1 subunit of the BKCa channel contributes to the low tone that characterizes the normal pulmonary circulation or modulates the pulmonary vascular response to hypoxia remains unknown. To determine the role of the BKCa channel beta1 subunit in the regulation of pulmonary vascular tone and the response to acute and chronic hypoxia, mice with deletion of the Kcnmb1 gene that encodes for the beta1 subunit ( Kcnmb1+/+) were placed in chronic hypoxia (10% O2) for 21-24 days. In normoxia, right ventricular systolic pressure (RVSP) did not differ between Kcnmb1+/+ (controls) and Kcnmb1-/- mice. After exposure to either acute or chronic hypoxia, RVSP was higher in Kcnmb1+/+ mice compared to Kcnmb1+/+ mice, without increased vascular remodeling. beta1 subunit expression was predominantly confined to pulmonary artery smooth muscle cells (PASMC) from vessels <150m. Peripheral PASMC contracted collagen gels irrespective of beta1 expression. Focal adhesion expression and Rho kinase activity were greater in Kcnmb1-/- compared to Kcnmb1+/+PASMC. Compromised PASMC beta1 function may contribute to the heightened microvascular vasoconstriction that characterizes pulmonary hypertension.

View details for PubMedID 29644895

Abstract

Compromised pulmonary endothelial cell (PEC) barrier function characterizes acute respiratory distress syndrome (ARDS), a cause of substantial morbidity and mortality. Survival from ARDS is greater in children compared to adults. Whether developmental differences intrinsic to PEC barrier function contribute to this survival advantage remains unknown. To test the hypothesis that PEC barrier function is more well preserved in neonatal compared to adult lungs in response to inflammation, we induced lung injury in neonatal and adult mice with systemic lipopolysaccharide (LPS). We assessed PEC barrier function in vivo and in vitro, evaluated changes in the expression of focal adhesion kinase (FAK1) and phosphorylation in response to LPS, and determined the effect of FAK silencing and over-expression on PEC barrier function. We found that LPS induced a greater increase in lung permeability and PEC barrier disruption in the adult, despite similar degrees of inflammation and apoptosis. Although baseline expression was similar, LPS increased FAK1 expression in neonatal PEC but increased FAK1 phosphorylation and decreased FAK1 expression in adult PEC. Pharmacologic inhibition of FAK1 accentuated LPS-induced barrier disruption most in adult PEC. Finally, in response to LPS, FAK silencing markedly impaired neonatal PEC barrier function, while FAK over-expression preserved adult PEC barrier function. Thus, developmental differences in FAK expression during inflammatory injury serve to preserve neonatal pulmonary endothelial barrier function as compared to adult, and suggest that intrinsic differences in the immature versus pulmonary endothelium, especially relative to FAK1 phosphorylation, may contribute to the improved outcomes of children with ARDS.

View details for PubMedID 29597831

View details for Web of Science ID 000429928200405

Abstract

Pulmonary angiogenesis is essential for alveolarization, the final stage of lung development that markedly increases gas exchange surface area. We recently demonstrated that activation of the nuclear factor kappa-B (NFB) pathway promotes pulmonary angiogenesis during alveolarization. However, the mechanisms activating NFB in the pulmonary endothelium, and its downstream targets are not known. In this study, we sought to delineate the specific roles for the NFB activating kinases, IKK and IKK, in promoting developmental pulmonary angiogenesis. Microarray analysis of primary pulmonary endothelial cells (PECs) after silencing IKK or IKK demonstrated that the 2 kinases regulate unique panels of genes, with few shared targets. Although silencing IKK induced mild impairments in angiogenic function, silencing IKK induced more severe angiogenic defects and decreased vascular cell adhesion molecule expression, an IKK regulated target essential for both PEC adhesion and migration. Taken together, these data show that IKK and IKK regulate unique genes in PEC, resulting in differential effects on angiogenesis upon inhibition, and identify IKK as the predominant regulator of pulmonary angiogenesis during alveolarization. These data suggest that therapeutic strategies to specifically enhance IKK activity in the pulmonary endothelium may hold promise to enhance lung growth in diseases marked by altered alveolarization.

View details for DOI 10.1111/jcmm.13741

View details for PubMedCentralID PMC6111877

View details for PubMedID 29144252

View details for PubMedCentralID PMC5726414

Abstract

Aortic root aneurysm formation and subsequent dissection and/or rupture remain the leading cause of death in patients with Marfan syndrome. Our laboratory has reported that miR-29b participates in aortic root/ascending aorta extracellular matrix remodeling during early aneurysm formation in Fbn1(C1039G/+) Marfan mice. Herein, we sought to determine whether miR-29b suppression can reduce aneurysm formation long-term. Fbn1(C1039G/+) Marfan mice were treated with retro-orbital LNA-anti-miR-29b inhibitor or scrambled-control-miR before aneurysms develop either (1) a single dose prenatally (pregnant Fbn1(C1039G/+) mice at 14.5days post-coitum) (n=8-10, each group) or (2) postnatally every other week, from 2 to 22weeks of age, and sacrificed at 24weeks (n=8-10, each group). To determine if miR-29b blockade was beneficial even after aneurysms develop, a third group of animals were treated every other week, starting at 8weeks of age, until sacrificed (n=4-6, each group). miR-29b inhibition resulted in aneurysm reduction, increased elastogenesis, decreased matrix metalloproteinase activity and decreased elastin breakdown. Prenatal LNA-anti-miR-29b inhibitor treatment decreased aneurysm formation up to age 32weeks, whereas postnatal treatment was effective up to 16weeks. miR-29b blockade did not slow aortic growth once aneurysms already developed. Systemic miR-29b inhibition significantly reduces aneurysm development long-term in a Marfan mouse model. Drug administration during aortic wall embryologic development appears fundamental. miR-29b suppression could be a potential therapeutic target for reducing aneurysm formation in Marfan syndrome patients.

View details for DOI 10.14814/phy2.13257

View details for PubMedID 28455451

View details for DOI 10.14814/phy2.13257

View details for Web of Science ID 000400380800012

View details for Web of Science ID 000405986503017

View details for Web of Science ID 000405461403418

Abstract

Bronchopulmonary dysplasia (BPD), characterized by impaired alveolarization and vascularization in association with lung inflammation and apoptosis, often occurs after mechanical ventilation with oxygen-rich gas (MV-O2). As heightened expression of the proinflammatory cytokine TNF- has been described in infants with BPD, we hypothesized that absence of TNF- would reduce pulmonary inflammation, and attenuate structural changes in newborn mice undergoing MV-O2 Neonatal TNF- null (TNF-(-/-)) and wild type (TNF-(+/+)) mice received MV-O2 for 8 h; controls spontaneously breathed 40% O2 Histologic, mRNA, and protein analysis in vivo were complemented by in vitro studies subjecting primary pulmonary myofibroblasts to mechanical stretch. Finally, TNF- level in tracheal aspirates from preterm infants were determined by ELISA. Although MV-O2 induced larger and fewer alveoli in both, TNF-(-/-) and TNF-(+/+) mice, it caused enhanced lung apoptosis (TUNEL, caspase-3/-6/-8), infiltration of macrophages and neutrophils, and proinflammatory mediator expression (IL-1, CXCL-1, MCP-1) in TNF-(-/-) mice. These differences were associated with increased pulmonary transforming growth factor- (TGF-) signaling, decreased TGF- inhibitor SMAD-7 expression, and reduced pulmonary NF-B activity in ventilated TNF-(-/-) mice. Preterm infants who went on to develop BPD showed significantly lower TNF- levels at birth. Our results suggest a critical balance between TNF- and TGF- signaling in the developing lung, and underscore the critical importance of these key pathways in the pathogenesis of BPD. Future treatment strategies need to weigh the potential benefits of inhibiting pathologic cytokine expression against the potential of altering key developmental pathways.

View details for DOI 10.1152/ajplung.00367.2015

View details for PubMedID 27016588

Abstract

Bronchopulmonary dysplasia (BPD), characterized by impaired alveolarization and vascularization in association with lung inflammation and apoptosis, often occurs after mechanical ventilation with oxygen-rich gas (MV-O2). As heightened expression of the proinflammatory cytokine TNF- has been described in infants with BPD, we hypothesized that absence of TNF- would reduce pulmonary inflammation, and attenuate structural changes in newborn mice undergoing MV-O2 Neonatal TNF- null (TNF-(-/-)) and wild type (TNF-(+/+)) mice received MV-O2 for 8 h; controls spontaneously breathed 40% O2 Histologic, mRNA, and protein analysis in vivo were complemented by in vitro studies subjecting primary pulmonary myofibroblasts to mechanical stretch. Finally, TNF- level in tracheal aspirates from preterm infants were determined by ELISA. Although MV-O2 induced larger and fewer alveoli in both, TNF-(-/-) and TNF-(+/+) mice, it caused enhanced lung apoptosis (TUNEL, caspase-3/-6/-8), infiltration of macrophages and neutrophils, and proinflammatory mediator expression (IL-1, CXCL-1, MCP-1) in TNF-(-/-) mice. These differences were associated with increased pulmonary transforming growth factor- (TGF-) signaling, decreased TGF- inhibitor SMAD-7 expression, and reduced pulmonary NF-B activity in ventilated TNF-(-/-) mice. Preterm infants who went on to develop BPD showed significantly lower TNF- levels at birth. Our results suggest a critical balance between TNF- and TGF- signaling in the developing lung, and underscore the critical importance of these key pathways in the pathogenesis of BPD. Future treatment strategies need to weigh the potential benefits of inhibiting pathologic cytokine expression against the potential of altering key developmental pathways.

View details for DOI 10.1152/ajplung.00367.2015

View details for Web of Science ID 000376464500003

View details for PubMedCentralID PMC4896101

View details for Web of Science ID 000406444705552

View details for Web of Science ID 000406444001556

View details for Web of Science ID 000406444006555

Abstract

Tie2 promoter-mediated loss of peroxisome proliferator-activated receptor gamma (PPAR) in mice leads to osteopetrosis and pulmonary arterial hypertension. Vascular disease is associated with loss of PPAR in pulmonary microvascular endothelial cells (PMVEC), we evaluated the role of PPAR in PMVEC functions, such as angiogenesis and migration. The role of PPAR in angiogenesis was evaluated in Tie2CrePPAR(flox/flox) and wild type (WT) mice, and in mouse and human PMVECs. RNA-sequencing and bioinformatic approaches were utilized to reveal angiogenesis-associated targets for PPAR. Tie2CrePPAR(flox/flox) mice showed an impaired angiogenic capacity. Analysis of endothelial progenitor-like cells using bone marrow transplantation combined with evaluation of isolated PMVECs revealed that loss of PPAR attenuates the migration and angiogenic capacity of mature PMVECs. PPAR-deficient human PMVECs showed a similar migration defect in culture. Bioinformatic and experimental analyses revealed E2F1 as a novel target of PPAR in the regulation of PMVEC migration. Disruption of the PPAR-E2F1 axis was associated with a dysregulated Wnt pathway related to the GSK3 interaction protein. In conclusion, PPAR plays an important role in sustaining angiogenic potential in mature PMVECs through E2F1-mediated gene regulation.

View details for DOI 10.1242/jcs.169011

View details for PubMedID 26743080

Abstract

Tie2 promoter-mediated loss of peroxisome proliferator-activated receptor gamma (PPAR) in mice leads to osteopetrosis and pulmonary arterial hypertension. Vascular disease is associated with loss of PPAR in pulmonary microvascular endothelial cells (PMVEC), we evaluated the role of PPAR in PMVEC functions, such as angiogenesis and migration. The role of PPAR in angiogenesis was evaluated in Tie2CrePPAR(flox/flox) and wild type (WT) mice, and in mouse and human PMVECs. RNA-sequencing and bioinformatic approaches were utilized to reveal angiogenesis-associated targets for PPAR. Tie2CrePPAR(flox/flox) mice showed an impaired angiogenic capacity. Analysis of endothelial progenitor-like cells using bone marrow transplantation combined with evaluation of isolated PMVECs revealed that loss of PPAR attenuates the migration and angiogenic capacity of mature PMVECs. PPAR-deficient human PMVECs showed a similar migration defect in culture. Bioinformatic and experimental analyses revealed E2F1 as a novel target of PPAR in the regulation of PMVEC migration. Disruption of the PPAR-E2F1 axis was associated with a dysregulated Wnt pathway related to the GSK3 interaction protein. In conclusion, PPAR plays an important role in sustaining angiogenic potential in mature PMVECs through E2F1-mediated gene regulation.

View details for DOI 10.1242/jcs.169011

View details for Web of Science ID 000370240900006

Abstract

In contrast to many other organs, a significant portion of lung development occurs after birth during alveolarization, thus rendering the lung highly susceptible to injuries that may disrupt this developmental process. Premature birth heightens this susceptibility, with many premature infants developing the chronic lung disease, bronchopulmonary dysplasia (BPD), a disease characterized by arrested alveolarization. Over the past decade, tremendous progress has been made in the elucidation of mechanisms that promote postnatal lung development, including extensive data suggesting that impaired pulmonary angiogenesis contributes to the pathogenesis of BPD. Moreover, in addition to impaired vascular growth, patients with BPD also frequently demonstrate alterations in pulmonary vascular remodeling and tone, increasing the risk for persistent hypoxemia and the development of pulmonary hypertension. In this review, an overview of normal lung development will be presented, and the pathologic features of arrested development observed in BPD will be described, with a specific emphasis on the pulmonary vascular abnormalities. Key pathways that promote normal pulmonary vascular development will be reviewed, and the experimental and clinical evidence demonstrating alterations of these essential pathways in BPD summarized.

View details for DOI 10.3389/fmed.2016.00021

View details for PubMedID 27243014

Abstract

A significant portion of lung development is completed postnatally during alveolarization, rendering the immature lung vulnerable to inflammatory stimuli that can disrupt lung structure and function. Although the NF-B pathway has well-recognized pro-inflammatory functions, novel anti-inflammatory and developmental roles for NF-B have recently been described. Thus, to determine how NF-B modulates alveolarization during inflammation, we exposed postnatal day 6 mice to vehicle (PBS), systemic lipopolysaccharide (LPS), or the combination of LPS and the global NF-B pathway inhibitor BAY 11-7082 (LPS + BAY). LPS impaired alveolarization, decreased lung cell proliferation, and reduced epithelial growth factor expression. BAY exaggerated these detrimental effects of LPS, further suppressing proliferation and disrupting pulmonary angiogenesis, an essential component of alveolarization. The more severe pathology induced by LPS + BAY was associated with marked increases in lung and plasma levels of macrophage inflammatory protein-2 (MIP-2). Experiments using primary neonatal pulmonary endothelial cells (PEC) demonstrated that MIP-2 directly impaired neonatal PEC migration in vitro; and neutralization of MIP-2 in vivo preserved lung cell proliferation and pulmonary angiogenesis and prevented the more severe alveolar disruption induced by the combined treatment of LPS + BAY. Taken together, these studies demonstrate a key anti-inflammatory function of the NF-B pathway in the early alveolar lung that functions to mitigate the detrimental effects of inflammation on pulmonary angiogenesis and alveolarization. Furthermore, these data suggest that neutralization of MIP-2 may represent a novel therapeutic target that could be beneficial in preserving lung growth in premature infants exposed to inflammatory stress.

View details for DOI 10.1152/ajplung.00029.2015

View details for PubMedID 26163511

View details for PubMedCentralID PMC4572419

View details for Web of Science ID 000361470506141

View details for Web of Science ID 000361470506125

Abstract

Endothelin-1 (ET-1) increases pulmonary vascular tone through direct effects on pulmonary artery smooth muscle cells (PASMC) via membrane-bound ET-1 receptors. Circulating ET-1 contributes to vascular remodeling by promoting SMC proliferation and migration and inhibiting SMC apoptosis. Although endothelial cells (EC) are the primary source of ET-1, whether ET-1 produced by SMC modulates pulmonary vascular tone is unknown. Using transgenic mice created by crossbreeding SM22-Cre mice with ET-1(flox/flox) mice to selectively delete ET-1 in SMC, we tested the hypothesis that PASMC ET-1 gene expression modulates the pulmonary vascular response to hypoxia. ET-1 gene deletion and selective activity of SM22 promoter-driven Cre recombinase were confirmed. Functional assays were performed under normoxic (21% O2) or hypoxic (5% O2) conditions using murine PASMC obtained from ET-1(+/+) and ET-1(-/-) mic and in human PASMC (hPASMC) after silencing of ET-1 using siRNA. Under baseline conditions, there was no difference in right ventricular systolic pressure (RVSP) between SM22-ET-1(-/-) and SM22-ET-1(+/+) (control) littermates. After exposure to hypoxia (10% O2, 21-24 days), RVSP was and vascular remodeling were less in SM22-ET-1(-/-) mice compared with control littermates (P < 0.01). Loss of ET-1 decreased PASMC proliferation and migration and increased apoptosis under normoxic and hypoxic conditions. Exposure to selective ET-1 receptor antagonists had no effect on either the hypoxia-induced hPASMC proliferative or migratory response. SMC-specific ET-1 deletion attenuates hypoxia-induced increases in pulmonary vascular tone and structural remodeling. The observation that loss of ET-1 inhibited SMC proliferation, survival, and migration represents evidence that ET-1 derived from SMC plays a previously undescribed role in modulating the response of the pulmonary circulation to hypoxia. Thus PASMC ET-1 may modulate vascular tone independently of ET-1 produced by EC.

View details for DOI 10.1152/ajplung.00253.2014

View details for PubMedID 25399435

View details for DOI 10.1152/ajplung.00253.2014

View details for Web of Science ID 000349305400006

View details for PubMedID 25399435

Abstract

Rupture and dissection of aortic root aneurysms remain the leading causes of death in patients with the Marfan syndrome, a hereditary connective tissue disorder that affects 1 in 5000 individuals worldwide. In the present study, we use a Marfan mouse model (Fbn1(C1039G/+)) to investigate the biological importance of apoptosis during aneurysm development in Marfan syndrome.Using in vivo single-photon emission computed tomographic-imaging and ex vivo autoradiography for Tc99m-annexin, we discovered increased apoptosis in the Fbn1(C1039G/+) ascending aorta during early aneurysm development peaking at 4 weeks. Immunofluorescence colocalization studies identified smooth muscle cells (SMCs) as the apoptotic cell population. As biological proof of concept that early aortic wall apoptosis plays a role in aneurysm development in Marfan syndrome, Fbn1(C1039G/+) mice were treated daily from 2 to 6 weeks with either (1) a pan-caspase inhibitor, Q-VD-OPh (20 mg/kg), or (2) vehicle control intraperitoneally. Q-VD-OPh treatment led to a significant reduction in aneurysm size and decreased extracellular matrix degradation in the aortic wall compared with control mice. In vitro studies using Fbn1(C1039G/+) ascending SMCs showed that apoptotic SMCs have increased elastolytic potential compared with viable cells, mostly because of caspase activity. Moreover, in vitro (1) cell membrane isolation, (2) immunofluorescence staining, and (3) scanning electron microscopy studies illustrate that caspases are expressed on the exterior cell surface of apoptotic SMCs.Caspase inhibition attenuates aneurysm development in an Fbn1(C1039G/+) Marfan mouse model. Mechanistically, during apoptosis, caspases are expressed on the cell surface of SMCs and likely contribute to elastin degradation and aneurysm development in Marfan syndrome.

View details for DOI 10.1161/ATVBAHA.114.304364

View details for PubMedID 25359856

Abstract

Advances in medical therapy have increased survival of extremely premature infants and changed the pathology of bronchopulmonary dysplasia (BPD) from one of acute lung injury to a disease of disrupted lung development. With this evolution, new questions emerge regarding the molecular mechanisms that control postnatal lung development, the effect of early disruptions of postnatal lung development on long-term lung function, and the existence of endogenous mechanisms that permit lung regeneration after injury.Recent data demonstrate that a significant component of alveolarization, the final stage of lung development, occurs postnatally. Further, clinical and experimental studies demonstrate that premature birth disrupts alveolarization, decreasing the gas exchange surface area of the lung and causing BPD. BPD is associated with significant short-term morbidity, and new longitudinal, clinical data demonstrate that survivors of BPD have long-standing deficits in lung function and may be at risk for the development of additional lung disease as adults. Unfortunately, current care is mainly supportive with few effective therapies that prevent or treat established BPD. These studies underscore the need to further elucidate the mechanisms that direct postnatal lung growth and develop innovative strategies to stimulate lung regeneration.Despite significant improvements in the care and survival of extremely premature infants, BPD remains a major clinical problem. Although efforts should remain focused on the prevention of preterm labor and BPD, novel research aimed at promoting postnatal alveolarization offers a unique opportunity to develop effective strategies to treat established BPD.

View details for DOI 10.1097/MOP.0000000000000095

View details for PubMedID 24739494

Abstract

In contrast to other organs, the lung completes a significant portion of its development after term birth. During this stage of alveolarization, division of the alveolar ducts into alveolar sacs by secondary septation, and expansion of the pulmonary vasculature by means of angiogenesis markedly increase the gas exchange surface area of the lung. However, postnatal completion of growth renders the lung highly susceptible to environmental insults such as inflammation that disrupt this developmental program. This is particularly evident in the setting of preterm birth, where impairment of alveolarization causes bronchopulmonary dysplasia, a chronic lung disease associated with significant morbidity. The nuclear factor -B (NFB) family of transcription factors are ubiquitously expressed, and function to regulate diverse cellular processes including proliferation, survival, and immunity. Extensive evidence suggests that activation of NFB is important in the regulation of inflammation and in the control of angiogenesis. Therefore, NFB-mediated downstream effects likely influence the lung response to injury and may also mediate normal alveolar development. This review summarizes the main biologic functions of NFB, and highlights the regulatory mechanisms that allow for diversity and specificity in downstream gene activation. This is followed by a description of the pro and anti-inflammatory functions of NFB in the lung, and of NFB-mediated angiogenic effects. Finally, this review summarizes the clinical and experimental data that support a role for NFB in mediating postnatal angiogenesis and alveolarization, and discusses the challenges that remain in developing therapies that can selectively block the detrimental functions of NFB yet preserve the beneficial effects. Birth Defects Research (Part A) 100:202-216, 2014. 2014 Wiley Periodicals, Inc.

View details for DOI 10.1002/bdra.23233

View details for PubMedID 24639404

Abstract

Neonatal chronic lung disease, also known as bronchopulmonary dysplasia (BPD), is the most common complication of premature birth, affecting up to 30% of very low birth weight infants. Improved medical care has allowed for the survival of the most premature infants and has significantly changed the pathology of BPD from a disease marked by severe lung injury to the "new" form characterized by alveolar hypoplasia and impaired vascular development. However, increased patient survival has led to a paucity of pathologic specimens available from infants with BPD. This, combined with the lack of a system to model alveolarization in vitro, has resulted in a great need for animal models that mimic key features of the disease. To this end, a number of animal models have been created by exposing the immature lung to injuries induced by hyperoxia, mechanical stretch, and inflammation and most recently by the genetic modification of mice. These animal studies have 1) allowed insight into the mechanisms that determine alveolar growth, 2) delineated factors central to the pathogenesis of neonatal chronic lung disease, and 3) informed the development of new therapies. In this review, we summarize the key findings and limitations of the most common animal models of BPD and discuss how knowledge obtained from these studies has informed clinical care. Future studies should aim to provide a more complete understanding of the pathways that preserve and repair alveolar growth during injury, which might be translated into novel strategies to treat lung diseases in infants and adults.

View details for DOI 10.1165/rcmb.2013-0014TR

View details for Web of Science ID 000331795600001

View details for PubMedID 24024524

Abstract

Haemophagocytic lymphohistiocytosis (HLH) is a rapidly fatal disease caused by dysregulated histiocytes leading to an excessive inflammatory reaction. While genetic forms of HLH exist, the most common form is acquired, frequently associated with infection. Here we report the first case of HLH associated with a coccidiomycosis infection. This patient is a 13-year-old previously healthy boy who presented with a flu-like illness, which rapidly progressed to refractory shock, severe ARDS, multiorgan failure and death despite maximal medical therapy, including broad-spectrum antibiotics to treat well-established causes of acquired HLH. Autopsy findings revealed the diagnosis of HLH in the setting of pulmonary coccidiomycosis. Antifungal therapy should be considered in cases of acquired HLH when the underlying aetiology is not clear.

View details for DOI 10.1136/bcr-2014-205681

View details for PubMedID 25139924

View details for Web of Science ID 000332162907356

View details for Web of Science ID 000332162904488

Abstract

Hypoxia-inducible factor-1 (HIF-1), an oxygen (O2)-sensitive transcription factor, mediates transcriptional responses to low-O2 tension states. Although acute hypoxia causes pulmonary vasoconstriction and chronic hypoxia can cause vascular remodeling and pulmonary hypertension, conflicting data exist on the role of HIF-1 in modulating pulmonary vascular tone.To investigate the role of smooth muscle cell (SMC)-specific HIF-1 in regulating pulmonary vascular tone.Mice with an SMC-specific deletion of HIF-1 (SM22-HIF-1(-/-)) were created to test the hypothesis that pulmonary artery SMC (PASMC) HIF-1 modulates pulmonary vascular tone and the response to hypoxia. SM22-HIF-1(-/-) mice exhibited significantly higher right ventricular systolic pressure compared with wild-type littermates under normoxia and with exposure to either acute or chronic hypoxia in the absence of histological evidence of accentuated vascular remodeling. Moreover, myosin light chain phosphorylation, a determinant of SMC tone, was higher in PASMCs isolated from SM22-HIF-1(-/-) mice compared with wild-type PASMCs, during both normoxia and after acute hypoxia. Further, overexpression of HIF-1 decreased myosin light chain phosphorylation in HIF-1-null SMCs.In both normoxia and hypoxia, PASMC HIF-1 maintains low pulmonary vascular tone by decreasing myosin light chain phosphorylation. Compromised PASMC HIF-1 expression may contribute to the heightened vasoconstriction that characterizes pulmonary hypertension.

View details for DOI 10.1161/CIRCRESAHA.112.300646

View details for PubMedID 23513056

View details for DOI 10.1161/CIRCRESAHA.112.300646

View details for PubMedID 23513056

Abstract

Increased pulmonary artery endothelial cell (PAEC) endothelium-dependent nitric oxide synthase (eNOS) activity mediates perinatal pulmonary vasodilation. Compromised eNOS activity is central to the pathogenesis of persistent pulmonary hypertension of the newborn (PPHN). Voltage-derived anion channel (VDAC)-1 was recently demonstrated to bind eNOS in the systemic circulation. We hypothesized that VDAC isoforms modulate eNOS activity in the pulmonary circulation, and that decreased VDAC expression contributes to PPHN. In PAECs derived from an ovine model of PPHN: (1) there is eNOS activity, but not expression; and (2) VDAC1 and -2 proteins are decreased. Immunocytochemistry, coimmunoprecipitation, and in situ proximity ligation assays in human PAECs (hPAECs) demonstrate binding between eNOS and both VDAC1 and -2, which increased upon stimulation with NO agonists. The ability of agonists to increase the eNOS/VDAC interaction was significantly blunted in hypertensive, compared with normotensive, ovine PAECs. Depletion of VDAC2, but not VDAC1, blocked the agonist-induced increase in eNOS activity in hPAECs. Overexpression of VDAC2 in hypertensive PAECs increased eNOS activity. Binding of VDAC2 enhances eNOS activity in the pulmonary circulation, and diminished VDAC2 constrains eNOS in PAECs derived from fetal lambs with chronic intrauterine pulmonary hypertension. We speculate that decreases in VDAC2 may contribute to the limited eNOS activity that characterizes pulmonary hypertension.

View details for DOI 10.1165/rcmb.2011-0436OC

View details for PubMedID 22842492

Abstract

Previously, we observed that hypoxia increases the expression of the 1-subunit (KCNMB1) of the calcium-sensitive potassium channel (BK(Ca)). Herein, we elucidate the mechanism whereby hypoxia increases KCNMB1 expression in human pulmonary artery smooth muscle cells (hPASMC). In response to hypoxia, the expression of both the transcription factor hypoxia-inducible factor 1- (HIF-1) and KCNMB1 are increased. Knockdown of HIF-1 using a shRNA plasmid blocked the hypoxic induction of KCNMB1 expression. Chromatin immunoprecipitation (ChIP) demonstrated HIF-1 binding to three discrete regions of the human KCNMB1 promoter known to contain hypoxia response elements (HREs). A KCNMB1 promoter reporter assay combined with site-directed mutagenesis identified two adjacent HREs located between -3,540 bp and -3,311 bp that are essential for the hypoxic induction of KCNMB1 promoter activity. Furthermore, additional ChIP assays demonstrated recruitment of the HIF-1 transcriptional coactivator, p300, to this same promoter region. Treatment of hPASMC with the histone deacetylase inhibitor, trichostatin, prolonged the increase in KCNMB1 observed with hypoxia, suggesting that alterations in chromatin remodeling function to limit the hypoxic induction of KCNMB1. Finally, KCNMB1 knockdown potentiated the hypoxia-induced increase in cytosolic calcium in hPASMC, highlighting the contribution of the 1-subunit in modulating vascular SMC tone in response to acute hypoxia. In conclusion, HIF-1 increases KCNMB1 expression in response to hypoxia in hPASMC by binding to two HREs located at -3,540 to -3,311 of the KCNMB1 promoter. We speculate that selective modulation of KCNMB1 expression may serve as a novel therapeutic approach to address diseases characterized by an increase in vascular tone.

View details for DOI 10.1152/ajplung.00302.2011

View details for Web of Science ID 000300245600009

View details for PubMedID 22114151

View details for PubMedCentralID PMC3289270

Abstract

Marfan syndrome (MFS) is a systemic connective tissue disorder notable for the development of aortic root aneurysms and the subsequent life-threatening complications of aortic dissection and rupture. Underlying fibrillin-1 gene mutations cause increased transforming growth factor- (TGF-) signaling. Although TGF- blockade prevents aneurysms in MFS mouse models, the mechanisms through which excessive TGF- causes aneurysms remain ill-defined.We investigated the role of microRNA-29b (miR-29b) in aneurysm formation in MFS.Using quantitative polymerase chain reaction, we discovered that miR-29b, a microRNA regulating apoptosis and extracellular matrix synthesis/deposition genes, is increased in the ascending aorta of Marfan (Fbn1(C1039G/+)) mice. Increased apoptosis, assessed by increased cleaved caspase-3 and caspase-9, enhanced caspase-3 activity, and decreased levels of the antiapoptotic proteins, Mcl-1 and Bcl-2, were found in the Fbn1(C1039G/+) aorta. Histological evidence of decreased and fragmented elastin was observed exclusively in the Fbn1(C1039G/+) ascending aorta in association with repressed elastin mRNA and increased matrix metalloproteinase-2 expression and activity, both targets of miR-29b. Evidence of decreased activation of nuclear factor B, a repressor of miR-29b, and a factor suppressed by TGF-, was also observed in Fbn1(C1039G/+) aorta. Furthermore, administration of a nuclear factor B inhibitor increased miR-29b levels, whereas TGF- blockade or losartan effectively decreased miR-29b levels in Fbn1(C1039G/+) mice. Finally, miR-29b blockade by locked nucleic acid antisense oligonucleotides prevented early aneurysm development, aortic wall apoptosis, and extracellular matrix deficiencies.We identify increased miR-29b expression as key to the pathogenesis of early aneurysm development in MFS by regulating aortic wall apoptosis and extracellular matrix abnormalities.

View details for DOI 10.1161/CIRCRESAHA.111.253740

View details for PubMedID 22116819

Abstract

Previously, we reported that murine gammaherpesvirus-68 (M1-MHV-68) induces pulmonary artery (PA) neointimal lesions in S100A4-overexpressing, but not in wild-type (C57), mice. Lesions were associated with heightened lung elastase activity and PA elastin degradation. We now investigate a direct relationship between elastase and PA neointimal lesions, the nature and source of the enzyme, and its presence in clinical disease. We found an association exists between the percentage of PAs with neointimal lesions and elastin fragmentation in S100A4 mice 6 months after viral infection. Confocal microscopy documented the heightened susceptibility of S100A4 versus C57 PA elastin to degradation by elastase. A transient increase in lung elastase activity occurs in S100A4 mice, 7 days after M1-MHV-68, unrelated to inflammation or viral load and before neointimal lesions. Administration of recombinant elafin, an elastase-specific inhibitor, ameliorates early increases in serine elastase and attenuates later development of neointimal lesions. Neutrophils are the source of elevated elastase (NE) in the S100A4 lung, and NE mRNA and protein levels are greater in PA smooth muscle cells (SMC) from S100A4 mice than from C57 mice. Furthermore, elevated NE is observed in cultured PA SMC from idiopathic PA hypertension versus that in control lungs and localizes to neointimal lesions. Thus, PA SMC produce NE, and heightened production and activity of NE is linked to experimental and clinical pulmonary vascular disease.

View details for DOI 10.1016/j.ajpath.2011.05.051

View details for PubMedID 21763677

Abstract

Kawasaki disease (KD) is an acute inflammatory illness marked by coronary arteritis. However, the factors increasing susceptibility to coronary artery lesions are unknown. Because transforming growth factor (TGF) increases elastin synthesis and suppresses proteolysis, we hypothesized that, in contrast to the benefit observed in aneurysms forming in those with Marfan syndrome, inhibition of TGF- would worsen inflammatory-induced coronary artery lesions. By using a murine model of KD in which injection of Lactobacillus casei wall extract (LCWE) induces coronary arteritis, we show that LCWE increased TGF- signaling in the coronary smooth muscle cells beginning at 2 days and continuing through 14 days, the point of peak coronary inflammation. By 42 days, LCWE caused fragmentation of the internal and external elastic lamina. Blocking TGF- by administration of a neutralizing antibody accentuated the LCWE-mediated fragmentation of elastin and induced an overall loss of medial elastin without increasing the inflammatory response. We attributed these increased pathological characteristics to a reduction in the proteolytic inhibitor, plasminogen activator inhibitor-1, and an associated threefold increase in matrix metalloproteinase 9 activity compared with LCWE alone. Therefore, our data demonstrate that in the coronary arteritis associated with KD, TGF- suppresses elastin degradation by inhibiting plasmin-mediated matrix metalloproteinase 9 activation. Thus, strategies to block TGF-, used in those with Marfan syndrome, are unlikely to be beneficial and could be detrimental.

View details for DOI 10.1016/j.ajpath.2010.11.054

View details for PubMedID 21356372

Abstract

At birth, pulmonary vasodilation occurs concomitant with the onset of air-breathing life. Whether and how Rho kinase (ROCK) modulates the perinatal pulmonary vascular tone remains incompletely understood. To more fully characterize the separate and interactive effects of ROCK signaling, we hypothesized that ROCK has discrete effects on both pulmonary artery (PA): 1) endothelial cell (PAEC) nitric oxide (NO) production and contractile state; and 2) smooth muscle cell tone independent of endothelial NO synthase (eNOS) activity. To test these hypotheses, NO production and endothelial barrier function were determined in fetal PAEC under baseline hypoxia and following exposure to normoxia with and without treatment with Y-27632, a specific pharmacological inhibitor of ROCK. In acutely instrumented, late-gestation ovine fetuses, eNOS was inhibited by nitro-l-arginine infusion into the left PA (LPA). Subsequently, fetal lambs were mechanically ventilated (MV) with 100% oxygen in the absence (control period) and presence of Y-27632. In PAEC, treatment with Y-27632 had no effect on cytosolic calcium but did increase normoxia-induced NO production. Moreover, acute normoxia increased PAEC barrier function, an effect that was potentiated by Y-27632. In fetal lambs, MV during the control period had no effect on LPA flow. In contrast, MV after Y-27632 increased LPA flow and fetal arterial P(O) (Pa(O)) and decreased PA pressure. In conclusion, ROCK activity modulates vascular tone in the perinatal pulmonary circulation via combined effects on PAEC NO production, barrier function, and smooth muscle tone. ROCK inhibition may represent a novel treatment strategy for neonatal pulmonary vascular disease.

View details for DOI 10.1152/ajplung.00199.2010

View details for Web of Science ID 000284941600016

View details for PubMedID 20709731

View details for PubMedCentralID PMC3006275

Abstract

Defective lung septation and angiogenesis, quintessential features of neonatal chronic lung disease (CLD), typically result from lengthy exposure of developing lungs to mechanical ventilation (MV) and hyperoxia. Previous studies showed fewer alveoli and microvessels, with reduced VEGF and increased transforming growth factor-beta (TGFbeta) signaling, and excess, scattered elastin in lungs of premature infants and lambs with CLD vs. normal controls. MV of newborn mice with 40% O(2) for 24 h yielded similar lung structural abnormalities linked to impaired VEGF signaling, dysregulated elastin production, and increased apoptosis. These studies could not determine the relative importance of cyclic stretch vs. hyperoxia in causing these lung growth abnormalities. We therefore studied the impact of MV for 24 h with air on alveolar septation (quantitative lung histology), angiogenesis [CD31 quantitative-immunohistochemistry (IHC), immunoblots], apoptosis [TdT-mediated dUTP nick end labeling (TUNEL), active caspase-3 assays], VEGF signaling [VEGF-A, VEGF receptor 1 (VEGF-R1), VEGF-R2 immunoblots], TGFbeta activation [phosphorylated Smad2 (pSmad2) quantitative-IHC], and elastin production (tropoelastin immunoblots, quantitative image analysis of Hart's stained sections) in lungs of 6-day-old mice. Compared with unventilated controls, MV caused a 3-fold increase in alveolar area, approximately 50% reduction in alveolar number and endothelial surface area, >5-fold increase in apoptosis, >50% decrease in lung VEGF-R2 protein, 4-fold increase of pSmad2 protein, and >50% increase in lung elastin, which was distributed throughout alveolar walls rather than at septal tips. This study is the first to show that prolonged MV of developing lungs, without associated hyperoxia, can inhibit alveolar septation and angiogenesis and increase apoptosis and lung elastin, findings that could reflect stretch-induced changes in VEGF and TGFbeta signaling, as reported in CLD.

View details for DOI 10.1152/ajplung.00251.2009

View details for Web of Science ID 000272827900005

View details for PubMedID 19854954

View details for PubMedCentralID PMC2806196

Abstract

Peroxisome proliferator-activated receptor (PPAR)-gamma is reduced in pulmonary arteries (PAs) of patients with PA hypertension (PAH), and we reported that deletion of PPARgamma in smooth muscle cells (SMCs) of transgenic mice results in PAH. However, the sequelae of loss of PPARgamma in PA endothelial cells (ECs) are unknown. Therefore, we bred Tie2-Cre mice with PPARgamma(flox/flox) mice to induce EC loss of PPARgamma (Tie2 PPARgamma(-/-)), and we assessed PAH by right ventricular systolic pressure (RVSP), RV hypertrophy (RVH), and muscularized distal PAs in room air (RA), after chronic hypoxia (CH), and after 4 wk of recovery in RA (Rec-RA). The Tie2 PPARgamma(-/-) mice developed spontaneous PAH in RA with increased RVSP, RVH, and muscularized PAs vs. wild type (WT); both genotypes exhibited a similar degree of PAH following chronic hypoxia, but Tie2 PPARgamma(-/-) mice had more residual PAH compared with WT mice after Rec-RA. The Tie2 PPARgamma(-/-) vs. WT mice in RA had increased platelet-derived growth factor receptor-beta (PDGF-Rbeta) expression and signaling, despite an elevation in the PPARgamma target apolipoprotein E, an inhibitor of PDGF signaling. Inhibition of PDGF-Rbeta signaling with imatinib, however, was sufficient to reverse the PAH observed in the Tie2 PPARgamma(-/-) mice. Thus the disruption of PPARgamma signaling in EC is sufficient to cause mild PAH and to impair recovery from CH-induced PAH. Inhibition of heightened PDGF-Rbeta signaling is sufficient to reverse PAH in this genetic model.

View details for DOI 10.1152/ajplung.00199.2009

View details for Web of Science ID 000272017900009

View details for PubMedID 19801450

View details for PubMedCentralID PMC2793182

View details for DOI 10.1016/j.ajog.2009.10.710

View details for Web of Science ID 000279559500686

View details for Web of Science ID 000271831503789

Abstract

Previously, we related fibronectin (Fn1) mRNA translation to an interaction between an AU-rich element in the Fn1 3' UTR and light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. Since human fibrosarcoma (HT1080) cells produce little fibronectin and LC3, we used these cells to investigate how LC3-mediated Fn1 mRNA translation might alter tumor growth. Transfection of HT1080 cells with LC3 enhanced fibronectin mRNA translation. Using polysome analysis and RNA-binding assays, we show that elevated levels of translation depend on an interaction between a triple arginine motif in LC3 and the AU-rich element in Fn1 mRNA. Wild-type but not mutant LC3 accelerated HT1080 cell growth in culture and when implanted in SCID mice. Comparison of WT LC3 with vector-transfected HT1080 cells revealed increased fibronectin-dependent proliferation, adhesion and invasion. Microarray analysis of genes differentially expressed in WT and vector-transfected control cells indicated enhanced expression of connective tissue growth factor (CTGF). Using siRNA, we show that enhanced expression of CTGF is fibronectin dependent and that LC3-mediated adhesion, invasion and proliferation are CTGF dependent. Expression profiling of soft tissue tumors revealed increased expression of both LC3 and CTGF in some locally invasive tumor types.

View details for DOI 10.1242/jcs.025957

View details for PubMedID 19366727

View details for Web of Science ID 000262104500180

View details for Web of Science ID 000262104500467

Abstract

Loss-of-function mutations in bone morphogenetic protein receptor II (BMP-RII) are linked to pulmonary arterial hypertension (PAH); the ligand for BMP-RII, BMP-2, is a negative regulator of SMC growth. Here, we report an interplay between PPARgamma and its transcriptional target apoE downstream of BMP-2 signaling. BMP-2/BMP-RII signaling prevented PDGF-BB-induced proliferation of human and murine pulmonary artery SMCs (PASMCs) by decreasing nuclear phospho-ERK and inducing DNA binding of PPARgamma that is independent of Smad1/5/8 phosphorylation. Both BMP-2 and a PPARgamma agonist stimulated production and secretion of apoE by SMCs. Using a variety of methods, including short hairpin RNAi in human PASMCs, PAH patient-derived BMP-RII mutant PASMCs, a PPARgamma antagonist, and PASMCs isolated from PPARgamma- and apoE-deficient mice, we demonstrated that the antiproliferative effect of BMP-2 was BMP-RII, PPARgamma, and apoE dependent. Furthermore, we created mice with targeted deletion of PPARgamma in SMCs and showed that they spontaneously developed PAH, as indicated by elevated RV systolic pressure, RV hypertrophy, and increased muscularization of the distal pulmonary arteries. Thus, PPARgamma-mediated events could protect against PAH, and PPARgamma agonists may reverse PAH in patients with or without BMP-RII dysfunction.

View details for DOI 10.1172/JCI32503

View details for PubMedID 18382765

Abstract

S100A4/Mts-overexpressing mice have thick elastic laminae and mild pulmonary arterial hypertension (PAH), and the occasional older mouse develops occlusive neointimal lesions and perivascular inflammation. We hypothesized that a vasculotropic virus could induce neointimal lesions in the S100A4/Mts1 mouse by facilitating breakdown of elastin and migration and proliferation of smooth muscle cells. To test this hypothesis, we infected S100A4/Mts1 mice with gammaherpesvirus 68 (gammaHV68). We observed, 6 mo after gammaHV68 [4 x 10(3) plaque-forming units (PFU)], perivascular inflammation in 10/15 S100A4/Mts1 mice and occlusive neointimal formation in 3/10 mice, accompanied by striking degradation of elastin. We then compared the early response after high-dose gammaHV68 (4 x 10(6) PFU) in C57Bl/6 and S100A4/Mts1 mice. In S100A4/Mts1 mice only, significant PAH, muscularization of distal vessels, and elastase activity were observed 6 wk after gammaHV68. These features resolved by 3 mo without neointimal formation. We therefore infected mice with the M1-gammaHV68 strain that reactivates from latency with higher efficiency and observed neointimal lesions at 3 mo in 2/5 C57Bl/6 (5-9% of vessels) and in 5/5 S100A4/Mts1 mice (13-40% of vessels) accompanied by mild PAH, heightened lung elastase activity, and intravascular viral expression. This suggested that enhanced generation of elastin peptides in S100A4/Mts1 mice may promote increased viral entry in the vessel wall. Using S100A4/Mts1 PA organ culture, we showed, in response to elastase activity, heightened production of elastin peptides associated with invasion of inflammatory cells and intravascular viral antigen. We therefore propose that early viral access to the vessel wall may be a critical determinant of the extent of vascular pathology following reactivation.

View details for DOI 10.1152/ajplung.00414.2007

View details for PubMedID 18083765

Abstract

Prolonged mechanical ventilation (MV) with O2-rich gas inhibits lung growth and causes excess, disordered accumulation of lung elastin in preterm infants, often resulting in chronic lung disease (CLD). Using newborn mice, in which alveolarization occurs postnatally, we designed studies to determine how MV with either 40% O2 or air might lead to dysregulated elastin production and impaired lung septation. MV of newborn mice for 8 h with either 40% O2 or air increased lung mRNA for tropoelastin and lysyl oxidase, relative to unventilated controls, without increasing lung expression of genes that regulate elastic fiber assembly (lysyl oxidase-like-1, fibrillin-1, fibrillin-2, fibulin-5, emilin-1). Serine elastase activity in lung increased fourfold after MV with 40% O2, but not with air. We then extended MV with 40% O2 to 24 h and found that lung content of tropoelastin protein doubled, whereas lung content of elastin assembly proteins did not change (lysyl oxidases, fibrillins) or decreased (fibulin-5, emilin-1). Quantitative image analysis of lung sections showed that elastic fiber density increased by 50% after MV for 24 h, with elastin distributed throughout the walls of air spaces, rather than at septal tips, as in control lungs. Dysregulation of elastin was associated with a threefold increase in lung cell apoptosis (TUNEL and caspase-3 assays), which might account for the increased air space size previously reported in this model. Our findings of increased elastin synthesis, coupled with increased elastase activity and reduced lung abundance of proteins that regulate elastic fiber assembly, could explain altered lung elastin deposition, increased apoptosis, and defective septation, as observed in CLD.

View details for DOI 10.1152/ajplung.00362.2007

View details for Web of Science ID 000252398600002

View details for PubMedID 17934062

View details for Web of Science ID 000250394301086

View details for Web of Science ID 000220591100351