Abstract P154: Focal Adhesion Kinase Plays a Critical Role in Upregulating Fibrogenesis in Load-Induced Cardiac Hypertrophy

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Ana Paula Dalla Costa ◽  
Carolina F Clemente ◽  
Thais H Theizen ◽  
José Roberto Souza ◽  
Leandro Cardoso ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Focal Adhesion Kinase ◽  
Myocardial Fibrosis ◽  
Focal Adhesion ◽  
Cardiac Fibroblasts ◽  
Heart Diseases ◽  
Smooth Muscle Actin ◽  
Critical Role ◽  
Matrix Metalloproteinase 2 ◽  
Activated Fibroblasts

Myocardial fibrosis is maladaptive, accelerating the evolution of diseased hearts to failure. The pathogenesis of myocardial fibrosis is critically dependent on complex processes of activation (i.e. enhanced proliferation, production and secretion of soluble factors, collagen and matrix metalloproteinases) and terminal differentiation of cardiac fibroblasts into myofibroblasts, resultant from the mobilization of numerous signaling molecules by physical and humoral stimuli. Noting that Focal Adhesion Kinase (FAK) is activated in areas of ongoing myocardial fibrosis, we sought to examine whether it is a critical mediator of fibrogenesis in load-induced hypertrophic hearts. Isolated fibroblasts from hypertrophic hearts of mice subjected to transverse aortic constriction (TAC; 1 to 8 weeks) were highly activated as recognized by markers that indicate enhanced proliferation (nuclear Ki67), production of collagen and matrix metalloproteinase-2 (MMP-2) and differentiation into myofibroblasts (expression of α-smooth muscle actin - α-SMA). In these cells, FAK was upregulated, as also were its dowstream pathways Src/ERK1/2 and PI3K/AKT/mTOR. Depletion of FAK (∼80%) after treatment with small interfering RNA (siRNA-FAK) markedly attenuated cardiac hypertrophy and fibrosis, and significantly reduced the number of activated fibroblasts harvested from overloaded hearts. Restoration of FAK function by overexpressing a full-length FAK construct in these cells, selectively enhanced the activity of the downstream PI3K/AKT/mTOR and rescued the activated phenotype of fibroblasts. Transfection with an inactive FAK mutant (Tyr397 substituted by phenylalanine) did not rescue the activated phenotype of fibroblasts harvested from overloaded hearts depleted of FAK. However, cells harvested from overloaded hearts depleted of FAK and treated with the mTOR activating aminoacid leucine showed typical phenotype of activated fibroblasts. These findings uncover a role for FAK in regulating the signaling cascade PI3K/AKT/mTOR in cardiac fibroblasts, which seems to be critical for the pathogenesis of myocardial fibrosis in hypertrophic hearts. Targeting this pathway may provide a novel strategy for treating hypertrophic heart diseases.

Matrix metalloproteinase-2 stimulates collagen-I expression through phosphorylation of focal adhesion kinase in rat cardiac fibroblasts

2012 ◽  
Vol 303 (9) ◽  
pp. C947-C953 ◽  
Author(s):  
Yasutomo Hori ◽  
Takashige Kashimoto ◽  
Tomohiro Yonezawa ◽  
Naoya Sano ◽  
Ryuta Saitoh ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Matrix Metalloproteinase ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Cardiac Fibroblasts ◽  
Primary Cultures ◽  
Collagen I ◽  
Matrix Metalloproteinase 2 ◽  
Dependent Manner ◽  
Simultaneous Treatment

Collagen-I is thought to be the main component of the extracellular matrix in cardiac fibrosis, the accumulation of which occurs with excessive activation of matrix metalloproteinase-2 (MMP-2). MMP-2 degrades the extracellular matrix; however, the relative importance of MMP-2 to collagen-I synthesis in cardiac fibroblasts remains unclear. We investigated whether extracellular activation of MMP-2 regulates collagen-I synthesis and phosphorylation of focal adhesion kinase (FAK) in rat cardiac fibroblasts. Primary cultures of rat cardiac fibroblasts were incubated with purified active MMP-2 to determine whether extracellular MMP-2 affects collagen-I synthesis and FAK phosphorylation in cardiac fibroblasts. Exogenous MMP-2 significantly stimulated FAK (Tyr397) phosphorylation and induced collagen-I expression in a time-dependent manner. Simultaneous treatment with the FAK inhibitor PF573228 abolished exogenous MMP-2-enhanced FAK (Tyr397) phosphorylation and collagen-I expression. Cells were then stimulated with norepinephrine (NE) to investigate whether endogenous MMP-2 could also induce collagen-I expression through FAK (Tyr397) phosphorylation. NE-stimulated endogenous MMP-2 activation in conditioned medium was significantly attenuated by simultaneous treatment with the MMP inhibitor PD166793. Similarly, NE-induced FAK (Tyr397) phosphorylation and collagen-I expression were significantly inhibited by simultaneous treatment with PD166793 or PF573228. Furthermore, MMP-2 knockdown induced by small interfering RNA (siRNA) significantly abolished endogenous MMP-2 expression and activation. MMP-2 siRNA significantly abolished NE-induced FAK (Tyr397) phosphorylation and collagen-I expression. These findings suggest that the extracellular activation of MMP-2 accelerated collagen-I synthesis in rat cardiac fibroblasts and that FAK phosphorylation (Tyr397) plays a pivotal role in MMP-2-stimulated collagen-I synthesis.

Endothelial paxillin and focal adhesion kinase (FAK) play a critical role in neutrophil transmigration

2012 ◽  
Vol 42 (2) ◽  
pp. 436-446 ◽  
Author(s):  
Sean A. Parsons ◽  
Ritu Sharma ◽  
Dawn L. Roccamatisi ◽  
Hong Zhang ◽  
Björn Petri ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Critical Role

scholarly journals Cardiac fibroblasts require focal adhesion kinase for normal proliferation and migration

2009 ◽  
Vol 296 (3) ◽  
pp. H627-H638 ◽  
Author(s):  
Ana Maria Manso ◽  
Seok-Min Kang ◽  
Sergey V. Plotnikov ◽  
Ingo Thievessen ◽  
Jaewon Oh ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Adult Mouse ◽  
Cardiac Fibroblasts ◽  
Focal Adhesions ◽  
Protein Levels ◽  
Tyrosine Kinase 2 ◽  
A Cell ◽  
And Migration ◽  
Proliferation And Migration

Migration and proliferation of cardiac fibroblasts (CFs) play an important role in the myocardial remodeling process. While many factors have been identified that regulate CF growth and migration, less is known about the signaling mechanisms involved in these processes. Here, we utilized Cre-LoxP technology to obtain focal adhesion kinase (FAK)-deficient adult mouse CFs and studied how FAK functioned in modulating cell adhesion, proliferation, and migration of these cells. Treatment of FAKflox/flox CFs with Ad/Cre virus caused over 70% reduction of FAK protein levels within a cell population. FAK-deficient CFs showed no changes in focal adhesions, cell morphology, or protein expression levels of vinculin, talin, or paxillin; proline-rich tyrosine kinase 2 (Pyk2) expression and activity were increased. Knockdown of FAK protein in CFs increased PDGF-BB-induced proliferation, while it reduced PDGF-BB-induced migration. Adhesion to fibronectin was not altered. To distinguish between the function of FAK and Pyk2, FAK function was inhibited via adenoviral-mediated overexpression of the natural FAK inhibitor FAK-related nonkinase (FRNK). Ad/FRNK had no effect on Pyk2 expression, inhibited the PDGF-BB-induced migration, but did not change the PDGF-BB-induced proliferation. FAK deficiency had only modest effects on increasing PDGF-BB activation of p38 and JNK MAPKs, with no alteration in the ERK response vs. control cells. These results demonstrate that FAK is required for the PDGF-BB-induced migratory response of adult mouse CFs and suggest that FAK could play an essential role in the wound-healing response that occurs in numerous cardiac pathologies.

Focal Adhesion Kinase Inactivation Reduces the Development of Acute Leukemia and Partially Rescues Hematopoietic Stem Cell Defects in Pten-Knockout Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 864-864
Author(s):  
Dewen You ◽  
Andrew Volk ◽  
Clare Sun ◽  
Junping Xin ◽  
Geunhyoung Ha ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Phosphatase Activity ◽  
Focal Adhesion ◽  
Protein Phosphatase ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Polycytidylic Acid

Abstract Abstract 864 Phosphatase and tensin homolog on chromosome 10 (Pten) is a tumor suppressor which possesses both lipid and protein phosphatase activities. Mutations and epigenetic inactivations of the Pten gene are commonly detected in a large number of tissue malignancies, including leukemias and lymphomas. Studies using Hematopoietic Pten-knockout in adult mice (Pten−/−) have demonstrated that Pten plays a critical role in maintaining the homeostasis of bone marrow (BM) hematopoiesis. Pten inactivation promotes the proliferation and peripheral mobilization of BM hematopoietic stem cells (HSCs). Pten−/− mice develop myeloproliferative disorders (MPD) within days, followed by acute leukemic transformation. Most previous studies attributed such phenotypic changes observed in Pten−/− mice to excessive activation of the PI3K/AKT/mTOR signal, a consequence of the loss of Pten's lipid phosphatase activity. However, the role of Pten's protein phosphatase activity in the regulation of HSCs and leukemogenesis is not well studied. Focal adhesion kinase (Fak) is a critical substrate for the protein phosphatase activity of Pten. Dysregulation of Fak has been observed in many cancers, including acute myeloid leukemias (AML) and acute lymphocytic leukemias (ALL). Therefore, we postulated that Fak might play a pivotal role in the development and progression of leukemia following Pten deletion. To test this hypothesis, we generated Mx1-Cre+Ptenfl/flFakfl/fl mice (an interferon-inducible Pten and Fak compound-knockout, Pten−/−Fak−/−) in which both the Pten and Fak genes in the hematopoietic system are deleted upon injection of polyinosinic-polycytidylic acid (pI-pC). Our results showed that the genetic inactivation of Fak can partially rescue HSC defects associated with Pten deficiency. We found that peripheral mobilization of HSCs in Pten−/−Fak−/− mice is significantly reduced compared to Pten−/− mice. As a consequence, more long-term HSCs (LT-HSCs) are preserved in the BM of Pten−/−Fak−/− mice compared to Pten−/− mice. Transplantation studies suggested that the hematopoietic reconstitutive capacity of Pten−/−Fak−/− HSCs is significantly improved compared to Pten−/− HSCs. Although Fak deletion fails to prevent the development of MPD in Pten−/− mice, Fak deletion does significantly reduce the frequency of AML/ALL, also significantly delays the onset of AML/ALL in comparison to Pten−/− mice. This study suggests that Fak might be a potential target for preventing the MPD-to-AML/ALL transformation and therefore blocking the Fak activity may hold a promise for a novel anti-leukemia therapy. The molecular mechanisms underlying the phenotype restoration of Pten−/− mice by Fak deletion in the hematopoietic system are actively being studied in our laboratory. Disclosures: No relevant conflicts of interest to declare.

AB23A Reduces TGF-β1-Induced Human Cardiac Fibroblasts (HCF) Proliferation and Collagen Secretion via MER/ERK1/2/CREB Pathway

2020 ◽  
Vol 10 (6) ◽  
pp. 798-803
Author(s):  
Dinghui Hu ◽  
Yanhu Wu ◽  
Jin Du ◽  
Hang Li ◽  
Zuntao Liu
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Myocardial Fibrosis ◽  
Cardiac Fibroblasts ◽  
Heart Diseases ◽  
Protein Levels ◽  
Collagen Secretion ◽  
Assay Result ◽  
Human Cardiac Fibroblasts ◽  
Creb Pathway

Background and Objectives: Myocardial fibrosis is associated with many forms of heart diseases which is characterized by the accumulation of activated cardiac fibroblasts (CFBs) and excess deposition of extracellular matrix (ECM). Natural compounds such as Alisol B 23-acetate has been proved to maintain the activation of ERK1/2, but whether it can affect cardiac fibroblasts by MER/ERK1/2/CREB signaling pathway is still unknown. Methods: The cell was identified with α-SMA protein level detected by immunofluorescence staining method. The cell proliferation was examined by CCK8 assay. Col I and Col III protein levels were examined by western blot and sirius red staining to detect the ECM level. Furthermore, p-MERK, MERK, P-ERK, ERK and CREB were examined by western blot to verify whether Alisol could activate the MERK/ERK1/2/CREB pathway in myocardial fibrosis. Results: CCK8 assay result indicated that Alisol reduced the cell viability of CFBs induced by TGF-β1. In addition, Alisol significantly decreased the ECM deposition of CFBs. Furthermore, Alisol could activate MERK/ERK1/2/CREB signaling pathway. Conclusion: These results verified that Alisol inhibited myocardial fibrosis via MERK/ERK1/2/CREB pathway.

Regulation of mouse blastocyst adhesion, outgrowth and matrix metalloproteinase-2 by focal adhesion kinase

2003 ◽  
Vol 48 (5) ◽  
pp. 475-479
Author(s):  
Guodong Tie ◽  
Yongqiang Tian ◽  
Shuyi Chen ◽  
Yujing Cao ◽  
Zelong Liu ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Matrix Metalloproteinase 2 ◽  
Mouse Blastocyst

Abstract 73: Yap Plays a Crucial Role in the Development of Cardiomyopathy in Lysosomal Storage Diseases

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
SHOHEI IKEDA ◽  
Akihiro Shirakabe ◽  
Sebastiano Sciarretta ◽  
Junichi Sadoshima
Keyword(s):  
Cardiac Hypertrophy ◽  
Crucial Role ◽  
Smooth Muscle Actin ◽  
Hippo Pathway ◽  
Critical Role ◽  
Lysosomal Storage Diseases ◽  
Heart Weight ◽  
Cardiomyocyte Proliferation ◽  
Lysosomal Storage ◽  
Storage Diseases

Rag proteins play a critical role in regulating lysosomal functions. Muscle-specific deletion of RagA and RagB (Rag A/B) results in lysosomal dysfunction, suppression of autophagy, and development of cardiomyopathy, reminiscent of lysosomal storage diseases (LSDs). We investigated the molecular mechanism by which cardiomyopathy is developed in RagA/B knockout (KO) mice. We generated cardiac-specific Rag A/BKO (RagA/BcKO) mice using αMHC-Cre. Similar to the muscle-specific KO mice, RagA/BcKO mice showed prominent cardiac hypertrophy (heart weight (HW)/ tibial length (TL): 12.35 ± 0.41 vs. 7.12 ± 0.15, p< 0.01) at 3 months of age. RagA/BcKO mice exhibited significantly greater phospho-histone H3-positive cardiomyocytes than control mice, suggesting that cardiomyocyte proliferation is stimulated in RagA/BcKO mice. However, RagA/BcKO mice exhibited a significantly smaller percentage of fractional shortening (%FS: 23.0 ± 0.82 vs. 45.8 ± 1.06, p< 0.01) and significantly greater mortality than control mice (50% vs. 0%, p<0.01). RagA/BcKO mouse hearts showed upregulation of β-MHC and α-smooth muscle actin, indicating de-differentiation. Yes-associated protein (YAP) is a transcription co-factor that controls growth and survival and is a key downstream target of the Hippo pathway. YAP associates with p62/SQSTM1 and is degraded through autophagy. YAP is significantly more accumulated (5.9-fold, p<0.05) in RagA/BcKO mouse hearts, where autophagy is inhibited and p62/SQSTM1 is accumulated, than in control hearts. In order to elucidate the role of YAP in mediating cardiomyopathy, we crossed RagA/BcKO mice with cardiac-specific heterozygous YAPKO mice. Heterozygous deletion of YAP in RagA/BcKO mice reduced the heart size (HW/ TL: 9.12 ± 0.10 vs. 11.9 ± 0.27, p< 0.05) and improved cardiac function (%FS: 35.0 ± 2.89 vs. 24.0 ± 0.58, p< 0.05) in RagA/BcKO mice. Pharmacological inhibition of YAP by verteporfin treatment also suppressed cardiac hypertrophy (HW/ TL: 8.92 ± 0.11 vs. 12.09 ± 0.30, p< 0.05) and heart failure (%FS: 33.3 ± 2.29 vs. 23.3 ± 0.63, p< 0.05) in RagA/BcKO mice. These results indicate that YAP plays a crucial role in the development of cardiomyopathy in RagA/BcKO mice, a mouse model of LSDs.

Abstract 380: Resident Cardiac Fibroblasts Give Rise to Periostin+ Myofibroblasts Which are the Primary Mediators of Cardiac Fibrosis

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Onur Kanisicak ◽  
Hadi Khalil ◽  
Jason Karch ◽  
Matthew Brody ◽  
Suh-Chin Lin ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cardiac Fibrosis ◽  
Bone Marrow Cells ◽  
Cardiac Fibroblasts ◽  
Critical Role ◽  
Cell Types ◽  
Lineage Tracing ◽  
Matricellular Protein ◽  
Mesenchymal Transition ◽  
Activated Fibroblasts

Resident cardiac fibroblasts (CFs) are potential therapeutic targets in treating or preventing heart failure since they play a critical role in cardiac remodeling and fibrosis after injury or with prolonged stress stimulation. Heterogeneity among activated fibroblasts within the heart has been noted by a number of previous studies in the literature. In addition to resident CFs, many cell types such as endothelial, perivascular and bone marrow cells have been suggested to go through a mesenchymal transition and acquire a myofibroblast-like phenotype during disease conditions. Hence, the cellular origin of the activated myofibroblast within the heart remains uncertain, in part because of a lack in reliable genetic strategies to define cellular lineage. Recent studies suggest that epicardial precursor cells expressing transcription factor 21 (Tcf21) give rise to resident CFs in the adult heart. In addition, the secreted matricellular protein periostin (Postn), appears to be expressed only within activated fibroblasts (myofibroblasts) within the heart. Here we used Tcf21-MerCreMer (Tcf21MCM) knockin mice and Postn-MerCreMer (PostnMCM) knock-in (KI) mice to lineage trace resident CFs and myofibroblasts with injury stimulation. To account for other potential cellular lineages giving rise to fibroblasts in the heart we also performed lineage tracing with the mouse genetic models including LysM-Cre (macrophage), ckit-Cre (bone marrow), Tie2CreERT2 (endothelial) and Myh11-CreERT2 (smooth muscle) in conjunction ROSA26 (R26) locus based loxP inactivated reporter alleles. Results of this study indicate that the Tcf21+ resident CFs are the predominant source for the activated periostin+ MFs which are the key mediators of extracellular matrix (ECM) production and ECM stability in heart whereas the contribution of other lineages to MFs are minimal. Additionally, we have performed single cell RNA sequencing on TCF21+ and Postn+ isolated CFs pre and post myocardial injury in order to define the fibroblast lineage itself at greater molecular depth.

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