Abstract 5278: The Sonic Hedgehog Transcription Factor Gli3 Modulates Ischemia-induced Angiogenesis

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Marie-Ange Renault ◽  
Jerome Roncalli ◽  
Joern Tongers ◽  
Sol Misener ◽  
Tina Thorne ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Limb Ischemia ◽  
Capillary Density ◽  
Left Ventricular ◽  
Dominant Negative ◽  
Tube Length ◽  
Reduced Ejection Fraction ◽  
Border Zones

Gli transcription factors are mediators of hedgehog signaling and have been shown to be critical in several steps during development. We have shown that the Hedgehog pathway is reactivated in the adult cardiovascular system under ischemic conditions, however the specific role of Gli3 has not been elucidated. Adenoviral mediated overexpression of Gli3 promotes HUVEC migration (250±58% of control, p<0.001) while down regulation of Gli3 via siRNA delayed tube formation on Matrigel (total tube length after 8 hours 6.86 vs. 70.76 control), suggesting a possible role of Gli3 in angiogenesis. We next investigated the role of Gli3 in angiogenesis using Gli3 +/− (Gli3 +/XtJ ) mice, a well established model of reduced Gli3 expression. VEGF-induced corneal angiogenesis was impaired in Gli3 +/− mice compared to WT. The role of Gli3 in angiogenesis was then confirmed in two ischemia models. Hind-limb ischemia (HLI) was induced by resection of the left femoral artery. Capillary density was reduced by a mean of 48.40±12.08% in Gli3 +/− mice vs. WT 7, 14 and 28 days. Myocardial infarction (MI) was induced by ligation of the LAD. 28 days after MI, left ventricular function assessed by echo and histological analysis revealed that Gli3 +/− mice exhibit reduced ejection fraction (27.92±4.49% versus 37.56±7.02% for the WT, p=0.004), increased fibrosis area (33.65±9.73% versus 19.81±5.40% for the WT, p=0.007) and a decrease capillary density in the ischemic and border zones. These data indicate that Gli3 deficiency leads to impaired angiogenesis in both ischemic and non ischemic conditions. Moreover, the impairment in ischemia induced neovascularization is associated with more severe impairment of cardiac function after MI. The mechanism of Gli3’s effects was then investigated in vitro . Promoter reporter assays revealed that Gli3 overexpression inhibits Gli-dependent transcription, while Western analysis show increased Akt phosphorylation, activation of the ERK1/2 and increased c-Fos expression. Using a dominant negative Akt expressing virus and a MEK1/2 inhibitor, we show that Gli3 induced-EC migration is dependent on Akt and ERK1/2. These studies provide the first evidence that the Gli3 transcription factor regulates angiogenesis and EC phenotype.

scholarly journals GATA4 protects granulosa cell tumors from TRAIL-induced apoptosis

2010 ◽  
Vol 17 (3) ◽  
pp. 709-717 ◽  
Author(s):  
Antti Kyrönlahti ◽  
Marjut Kauppinen ◽  
Essi Lind ◽  
Leila Unkila-Kallio ◽  
Ralf Butzow ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Granulosa Cell ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Granulosa Cell Tumors ◽  
Trail Receptors ◽  
Apoptotic Effect ◽  
Induced Apoptosis

Disturbances in granulosa cell apoptosis have been implicated in the pathogenesis of human granulosa cell tumors (GCTs). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent cytokine that induces apoptosis in a variety of malignancies without toxic effects on benign cells. The aim of this study was to investigate the expression and functionality of the TRAIL receptors DR4 and DR5 in human GCTs. Additionally, we examined the role of GATA4, a transcription factor expressed in normal and malignant granulosa cells, in TRAIL-induced GCT apoptosis. For this purpose, a tissue microarray of 80 primary and 12 recurrent GCTs was subjected to immunohistochemistry for DR4 and DR5, and freshly isolated primary GCT cultures were utilized to evaluate the functional effects of TRAIL on GCT cells. To clarify the role of GATA4 in the regulation of TRAIL-induced apoptosis, a human GCT-derived cell line (KGN) was transduced with lentiviral vectors expressing small hairpin RNAs targeting GATA4 or transfected with adenovirus expressing either wild-type or dominant negative mutant GATA4. We found that receptors DR4 and DR5 are expressed in a vast majority of GCTs as well as in primary GCT cultures, and that TRAIL induces apoptosis in the primary GCT cultures. Moreover, we showed that overexpressing GATA4 protects GCTs from TRAIL-induced apoptosis in vitro, whereas disrupting GATA4 function induces apoptosis and potentiates the apoptotic effect of TRAIL administration. Our results demonstrate that the TRAIL pathway is functional in GCT cells, and suggest that transcription factor GATA4 may function as a survival factor in this ovarian malignancy.

Abstract 533: Shear-Induced Collateral Artery Growth Modulated by Endoglin but Not by ALK1

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Margreet R de Vries ◽  
Leonard Seghers ◽  
Imo E Hoefer ◽  
Evangelia Pardali ◽  
Beerend P Hierck ◽  
...  
Keyword(s):  
Blood Flow ◽  
Shear Stress ◽  
Hind Limb ◽  
Limb Ischemia ◽  
Capillary Density ◽  
Artery Ligation ◽  
Laser Doppler Perfusion Imaging ◽  
Collateral Artery

Introduction: TGF- β stimulates both shear stress induced arteriogenesis and ischemia induced angiogenesis, by signaling through different receptors. In this study the role of TGF-β receptors ALK1 and endoglin is assessed in a murine model for hind limb ischemia induced neovascularization. Methods and Results: Femoral artery ligation was performed in mice heterozygous for either endoglin, or ALK1 and littermate controls. Blood flow recovery, monitored by laser doppler perfusion imaging, was significantly hampered in both endoglin and ALK1 heterozygous mice compared to controls by maximal 40% and 49%, respectively. Collateral artery size was significantly reduced in endoglin heterozygous mice compared to controls, but not in ALK1 heterozygous mice. Capillary density in ischemic calf muscles was unaffected, but capillaries from endoglin and ALK1 heterozygous mice were significantly larger when compared to controls. Furthermore, both endoglin and ALK1 heterozygous mice showed significant more erythrocyte extravasation near the capillaries than their littermate controls, pointing towards less mature capillaries. Murine endothelial cells were exposed to shear stress in vitro to study the differential role of endoglin and ALK1 in shear induced neovascularization. This resulted in high levels of endoglin messengerRNA, but not of ALK1. Conclusion: Here we demonstrated that both endoglin and ALK1 play a crucial role in blood flow recovery. Importantly, endoglin is essential in both shear induced collateral artery growth and in ischemia induced angiogenesis, whereas ALK1 is only involved in ischemia induced angiogenesis.

scholarly journals Progression and Differentiation of Alveolar Rhabdomyosarcoma Is Regulated by PAX7 Transcription Factor—Significance of Tumor Subclones

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1870
Author(s):  
Klaudia Skrzypek ◽  
Grażyna Adamek ◽  
Marta Kot ◽  
Bogna Badyra ◽  
Marcin Majka
Keyword(s):  
Transcription Factor ◽  
Cell Lines ◽  
Migration Activity ◽  
Id Proteins ◽  
Rh30 Cell ◽  
Str Profile ◽  
And Migration

Rhabdomyosarcoma (RMS), is the most frequent soft tissue tumor in children that originates from disturbances in differentiation process. Mechanisms leading to the development of RMS are still poorly understood. Therefore, by analysis of two RMS RH30 cell line subclones, one subclone PAX7 negative, while the second one PAX7 positive, and comparison with other RMS cell lines we aimed at identifying new mechanisms crucial for RMS progression. RH30 subclones were characterized by the same STR profile, but different morphology, rate of proliferation, migration activity and chemotactic abilities in vitro, as well as differences in tumor morphology and growth in vivo. Our analysis indicated a different level of expression of adhesion molecules (e.g., from VLA and ICAM families), myogenic microRNAs, such as miR-206 and transcription factors, such as MYOD, MYOG, SIX1, and ID. Silencing of PAX7 transcription factor with siRNA confirmed the crucial role of PAX7 transcription factor in proliferation, differentiation and migration of RMS cells. To conclude, our results suggest that tumor cell lines with the same STR profile can produce subclones that differ in many features and indicate crucial roles of PAX7 and ID proteins in the development of RMS.

Abstract 17597: Endogenous Del-1 is a Negative Regulator of Ischemia-induced Angiogenesis by Interacting With the Leukocytic LFA-1 Integrin

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Sebastian Cremer ◽  
Anne Klotzsche-von Ameln ◽  
Alessia Orlandi ◽  
Irina Korovina ◽  
Bettina Gercken ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Hind Limb ◽  
Hematopoietic Cells ◽  
Limb Ischemia ◽  
Inflammatory Cells ◽  
Capillary Density ◽  
Negative Regulator ◽  
Hind Limb Ischemia ◽  
Autonomous Pathway

Developmental endothelial locus-1 (Del-1) is an endothelial cell-derived secreted protein circulating in blood and associated with the cell surface and the extracellular matrix. As we previously demonstrated, Del-1 restricts leukocyte recruitment by inhibiting the β2-integrin, LFA-1. Leukocytes and progenitor cells (PC) may contribute to angiogenesis. The role of endogenous Del-1 in angiogenesis is elusive. We found, that physiological angiogenesis of the developing retina was not affected in the Del-1-/- mice compared to the wildtype (WT) mice. Surprisingly, Del-1-/- mice displayed a significantly increased angiogenic response compared to WT mice after induction of hind limb ischemia (144 ± 6 % increase of capillary density) and retinal ischemia (retinopathy of prematurity model) suggesting that endogenous Del-1 is an inhibitor of ischemia-induced neovascularization. Silencing of Del-1 with siRNA did not affect the angiogenic sprouting of endothelial cell (EC) spheroids, indicating that Del-1 blocks angiogenesis in a non-endothelial cell autonomous pathway. Soluble Del-1 blocked the adhesion of inflammatory cells on EC monolayers. In line with these results, ischemic muscles and ischemic retinae from Del-1-/- mice displayed an enhanced infiltration with inflammatory cells compared to the WT mice. Since Del-1 blocks inflammatory cell homing by inhibiting the leukocytic LFA-1-integrin, we addressed the role of the Del-1/LFA-1-integrin interaction on the inhibitory function of endogenous Del-1 on angiogenesis. Indeed, Del-1/LFA-1-double deficiency reversed the pro-angiogenic phenotype of the Del-1-/- mice to the level of WT mice in the model of hind limb ischemia. Thus, the inhibitory role of Del-1 on neovascularization is mediated by the interaction of Del-1 with the LFA-1-integrin. Moreover, Del-1-deficiency led to an increased homing of intravenously injected murine fluorescence-labeled WT Lin- BM PC in ischemic muscles in comparison to WT mice after the induction of hind limb ischemia. Taken together, Del-1 acts as a negative regulator of ischemia-induced angiogenesis by interacting with the LFA-1-integrin expressed in hematopoietic cells, thereby inhibiting the homing of hematopoietic cells to ischemic tissues.

The use of siRNA as a pharmacological tool to assess a role for the transcription factor NF-IL6 in the brain under in vitro and in vivo conditions during LPS-induced inflammatory stimulation

2017 ◽  
Vol 28 (6) ◽  
Author(s):  
Jelena Damm ◽  
Joachim Roth ◽  
Rüdiger Gerstberger ◽  
Christoph Rummel
Keyword(s):  
Transcription Factor ◽  
Brain Cells ◽  
Nuclear Factor ◽  
Si Rna ◽  
The Brain ◽  
Deficient Mice ◽  
Transcription Factor Nuclear Factor

AbstractBackground:Studies with NF-IL6-deficient mice indicate that this transcription factor plays a dual role during systemic inflammation with pro- and anti-inflammatory capacities. Here, we aimed to characterize the role of NF-IL6 specifically within the brain.Methods:In this study, we tested the capacity of short interfering (si) RNA to silence the inflammatory transcription factor nuclear factor-interleukin 6 (NF-IL6) in brain cells underResults:In cells of a mixed neuronal and glial primary culture from the ratConclusions:This approach was, thus, not suitable to characterize the role NF-IL6 in the brain

scholarly journals Long-term outcomes in patients with critical limb ischemia and heart failure with preserved or reduced ejection fraction

2017 ◽  
Vol 22 (4) ◽  
pp. 307-315 ◽  
Author(s):  
Kavita B Khaira ◽  
Ellen Brinza ◽  
Gagan D Singh ◽  
Ezra A Amsterdam ◽  
Stephen W Waldo ◽  
...  
Keyword(s):  
Heart Failure ◽  
Ejection Fraction ◽  
Critical Limb Ischemia ◽  
Limb Ischemia ◽  
Left Ventricular ◽  
Term Survival ◽  
Long Term Survival ◽  
Reduced Ejection Fraction ◽  
History Of

The impact of heart failure (HF) on long-term survival in patients with critical limb ischemia (CLI) has not been well described. Outcomes stratified by left ventricular ejection fraction (EF) are also unknown. A single center retrospective chart review was performed for patients who underwent treatment for CLI from 2006 to 2013. Baseline demographics, procedural data and outcomes were analyzed. HF diagnosis was based on appropriate signs and symptoms as well as results of non-invasive testing. Among 381 CLI patients, 120 (31%) had a history of HF and 261 (69%) had no history of heart failure (no-HF). Within the HF group, 74 (62%) had HF with preserved ejection fraction (HFpEF) and 46 (38%) had HF with reduced ejection fraction (HFrEF). The average EF for those with no-HF, HFpEF and HFrEF were 59±13% vs 56±9% vs 30±9%, respectively. The likelihood of having concomitant coronary artery disease (CAD) was lowest in the no-HF group (43%), higher in the HFpEF group (70%) and highest in the HFrEF group (83%) ( p=0.001). Five-year survival was on average twofold higher in the no-HF group (43%) compared to both the HFpEF (19%, p=0.001) and HFrEF groups (24%, p=0.001). Long-term survival rates did not differ between the two HF groups ( p=0.50). There was no difference in 5-year freedom from major amputation or freedom from major adverse limb events between the no-HF, HFpEF and HFrEF groups, respectively. Overall, the combination of CLI and HF is associated with poor 5-year survival, independent of the degree of left ventricular systolic dysfunction.

scholarly journals Lineage-specific requirements of β-catenin in neural crest development

2002 ◽  
Vol 159 (5) ◽  
pp. 867-880 ◽  
Author(s):  
Lisette Hari ◽  
Véronique Brault ◽  
Maurice Kléber ◽  
Hye-Youn Lee ◽  
Fabian Ille ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Neural Crest ◽  
Neural Crest Cells ◽  
Neural Crest Stem Cells ◽  
Downstream Signaling ◽  
Neural Crest Development ◽  
Sensory Neurogenesis

β-Catenin plays a pivotal role in cadherin-mediated cell adhesion. Moreover, it is a downstream signaling component of Wnt that controls multiple developmental processes such as cell proliferation, apoptosis, and fate decisions. To study the role of β-catenin in neural crest development, we used the Cre/loxP system to ablate β-catenin specifically in neural crest stem cells. Although several neural crest–derived structures develop normally, mutant animals lack melanocytes and dorsal root ganglia (DRG). In vivo and in vitro analyses revealed that mutant neural crest cells emigrate but fail to generate an early wave of sensory neurogenesis that is normally marked by the transcription factor neurogenin (ngn) 2. This indicates a role of β-catenin in premigratory or early migratory neural crest and points to heterogeneity of neural crest cells at the earliest stages of crest development. In addition, migratory neural crest cells lateral to the neural tube do not aggregate to form DRG and are unable to produce a later wave of sensory neurogenesis usually marked by the transcription factor ngn1. We propose that the requirement of β-catenin for the specification of melanocytes and sensory neuronal lineages reflects roles of β-catenin both in Wnt signaling and in mediating cell–cell interactions.

scholarly journals Dominant Negative Dimerization of a Mutant Homeodomain Protein in Axenfeld-Rieger Syndrome

2003 ◽  
Vol 23 (6) ◽  
pp. 1968-1982 ◽  
Author(s):  
Irfan Saadi ◽  
Adisa Kuburas ◽  
Jamison J. Engle ◽  
Andrew F. Russo
Keyword(s):  
Dominant Negative ◽  
Hill Coefficient ◽  
Homeodomain Protein ◽  
Yeast Two Hybrid ◽  
Autosomal Dominant Disorder ◽  
Rieger Syndrome ◽  
A Charge ◽  
Two Hybrid

ABSTRACT Axenfeld-Rieger syndrome is an autosomal-dominant disorder caused by mutations in the PITX2 homeodomain protein. We have studied the mechanism underlying the dominant negative K88E mutation, which occurs at position 50 of the homeodomain. By using yeast two-hybrid and in vitro pulldown assays, we have documented that PITX2a can form homodimers in the absence of DNA. Moreover, the K88E mutant had even stronger dimerization ability, primarily due to interactions involving the C-terminal region. Dimerization allowed cooperative binding of wild-type (WT) PITX2a to DNA containing tandem bicoid sites in a head-to-tail orientation (Hill coefficient, 1.73). In contrast, the WT-K88E heterodimer bound the tandem sites with greatly reduced cooperativity and decreased transactivation activity. To further explore the role of position 50 in PITX2a dimerization, we introduced a charge-conservative mutation of lysine to arginine (K88R). The K88R protein had greatly reduced binding to a TAATCC element and did not specifically bind any other TAATNN motif. Like K88E, K88R formed relatively stronger dimers with WT. As predicted by our model, the K88R protein acted in a dominant negative manner to suppress WT PITX2a activity. These results suggest that the position 50 residue in the PITX2 homeodomain plays an important role in both DNA binding and dimerization activities.

Insights in the molecular mechanisms of an azole stress adapted laboratory-generated Aspergillus fumigatus strain

2021 ◽  
Author(s):  
Marion Aruanno ◽  
Samantha Gozel ◽  
Isabelle Mouyna ◽  
Josie E Parker ◽  
Daniel Bachmann ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Aspergillus Fumigatus ◽  
Molecular Mechanisms ◽  
Drug Transporters ◽  
Major Facilitator Superfamily ◽  
Ergosterol Biosynthesis ◽  
Azole Resistance ◽  
Drug Transporter

Abstract Aspergillus fumigatus is the main cause of invasive aspergillosis, for which azole drugs are the first-line therapy. Emergence of pan-azole resistance among A. fumigatus is concerning and has been mainly attributed to mutations in the target gene (cyp51A). However, azole resistance may also result from other mutations (hmg1, hapE) or other adaptive mechanisms. We performed microevolution experiment exposing an A. fumigatus azole-susceptible strain (Ku80) to sub-minimal inhibitory concentration of voriconazole to analyze emergence of azole resistance. We obtained a strain with pan-azole resistance (Ku80R), which was partially reversible after drug relief, and without mutations in cyp51A, hmg1, and hapE. Transcriptomic analyses revealed overexpression of the transcription factor asg1, several ATP-binding cassette (ABC) and major facilitator superfamily transporters and genes of the ergosterol biosynthesis pathway in Ku80R. Sterol analysis showed a significant decrease of the ergosterol mass under voriconazole exposure in Ku80, but not in Ku80R. However, the proportion of the sterol compounds was similar between both strains. To further assess the role of transporters, we used the ABC transporter inhibitor milbemycine oxime (MLB). MLB inhibited transporter activity in both Ku80 and Ku80R and demonstrated some potentiating effect on azole activity. Criteria for synergism were reached for MLB and posaconazole against Ku80. Finally, deletion of asg1 revealed some role of this transcription factor in controlling drug transporter expression, but had no impact on azole susceptibility. This work provides further insight in mechanisms of azole stress adaptation and suggests that drug transporters inhibition may represent a novel therapeutic target. Lay Summary A pan-azole-resistant strain was generated in vitro, in which drug transporter overexpression was a major trait. Analyses suggested a role of the transporter inhibitor milbemycin oxime in inhibiting drug transporters and potentiating azole activity.

Abstract 269: Cardiac Specific Disruption of Drp-1 Causes the Development of Cardiac Dysfunction via Inhibition of Autophagy and Induction of Apoptosis

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Yoshiyuki Ikeda ◽  
Junichi Sadoshima
Keyword(s):  
Mitochondrial Dysfunction ◽  
Systolic Function ◽  
Mitochondrial Fission ◽  
Mitochondrial Mass ◽  
Reduced Ejection Fraction ◽  
Tibia Length ◽  
Cardiac Systolic Function

Fission and fusion affect mitochondrial turnover in part by modulating mitophagy. This study aimed to clarify the role of mitochondrial fission in regulating cardiac function and autophagy in the heart. Dynamin-related protein 1 (Drp-1) plays an essential role in mediating mitochondrial fission. Therefore, we generated cardiac specific Drp-1 KO mice and utilized cultured cardiomyocytes transduced with adenovirus harboring short hairpin Drp-1 (Ad-shDrp-1) to test the effect of Drp-1 disruption both in vivo and in vitro. In Drp-1 KO hearts we observed a significantly greater mitochondrial mass ratio compared to control, as assessed by electron microscopy (Drp-1 KO: 3.57 ± 1.38, control: 1.18 ± 0.31, P<0.05). Mitochondrial ATP content was significantly lower (0.70 ± 0.07 vs 1.03 ± 0.10, P<0.05), while mitochondrial swelling was significantly greater (% decrease in absorbance; 8.01 ± 1.99 vs 2.01 ± 0.58, P<0.05) in Drp-1 KO hearts versus control. Mitochondrial membrane potential, assessed by JC-1 staining, was significantly reduced in myocytes with knockdown of Drp-1. Taken together, these results suggest that inhibition of fission causes mitochondrial dysfunction. We also examined the effect of Drp-1 depletion on autophagy. We found that the amount of LC-3 II was significantly less (0.47 ± 0.16 vs 1.32 ±0.34, P<0.05), whereas p62 expression was significantly greater (1.14 ± 0.16 vs 0.16 ± 0.06, P<0.01) in Drp-1 KO hearts compared to control. The number of LC3 dots in Ad-shDrp-1 transduced myocytes was lower than that of sh-scramble treatment. We investigated apoptosis and found that the amount of cleaved caspase-3 (0.62 ± 0.24 vs 0.18 ± 0.04, P<0.05) and the number of TUNEL positive cells (0.22 ± 0.12 vs 0.03 ± 0.06%, P<0.01) were higher in Drp-1 KO versus control hearts. Cardiac systolic function was reduced (ejection fraction; 44.5 ± 6.3 vs 85.4 ± 5.7%, P<0.01) and LVW/tibia length was greater (4.48 ± 0.38 vs 3.84 ± 0.58, P<0.05) in Drp-1 KO mice compared to control. Finally, we observed that the survival rate of Drp-1 KO mice was significantly reduced compared to control mice. Our results demonstrate that inhibition of mitochondrial fission via disruption of Drp-1 inhibits autophagy and causes mitochondrial dysfunction, thereby promoting cardiomyopathy.

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