Regeneration of the damaged myocardium is one of the most challenging fronts in the field of tissue engineering due to the limited capacity of adult heart tissue to heal and to the mechanical and structural constraints of the cardiac tissue. In this study we demonstrate that an engineered acellular scaffold comprising type I collagen, endowed with specific physiomechanical properties, improves cardiac function when used as a cardiac patch following myocardial infarction. Patches were grafted onto the infarcted myocardium in adult murine hearts immediately after ligation of left anterior descending artery and the physiological outcomes were monitored by echocardiography, and by hemodynamic and histological analyses four weeks post infarction. In comparison to infarcted hearts with no treatment, hearts bearing patches preserved contractility and significantly protected the cardiac tissue from injury at the anatomical and functional levels. This improvement was accompanied by attenuated left ventricular remodeling, diminished fibrosis, and formation of a network of interconnected blood vessels within the infarct. Histological and immunostaining confirmed integration of the patch with native cardiac cells including fibroblasts, smooth muscle cells, epicardial cells, and immature cardiomyocytes. In summary, an acellular biomaterial with specific biomechanical properties promotes the endogenous capacity of the infarcted myocardium to attenuate remodeling and improve heart function following myocardial infarction.
cardiac tissue engineering; scaffold; collagen; heart; cardiomyocyte; angiogenesis
Beta adrenergic receptor (β-AR) subtypes act through diverse signaling cascades to modulate cardiac function and remodeling. Previous in vitro studies suggest that β1-AR signaling is cardiotoxic whereas β2-AR signaling is cardioprotective, and may be the case during ischemia/reperfusion in vivo. The objective of this study was to assess whether β2-ARs also played a cardioprotective role in the pathogenesis of non-ischemic forms of cardiomyopathy.
Methods and Results
To dissect the role of β1 vs β2-ARs in modulating MLP (Muscle LIM Protein) cardiomyopathy, we crossbred MLP−/− with β1−/− or β2−/− mice. Deletion of the β2-AR improved survival, cardiac function, exercise capacity and myocyte shortening; in contrast haploinsufficency of the β1-AR reduced survival. Pathologic changes in Ca2+ handling were reversed in the absence of β2-ARs: peak Ca2+ and SR Ca2+ were decreased in MLP−/− and β1+/− /MLP−/− but restored in β2−/−MLP−/−. These changes were associated with reversal of alterations in troponin I and phospholamban phosphorylation. Gi inhibition increased peak and baseline Ca2+, recapitulating changes observed in the β2−/−/MLP−/−. The L-type Ca2+ blocker verapamil significantly decreased cardiac function in β2−/−MLP−/− vs WT. We next tested if the protective effects of β2-AR ablation were unique to the MLP model using TAC-induced heart failure. Similar to MLP, β2−/− mice demonstrated delayed progression of heart failure with restoration of myocyte shortening and peak Ca2+ and Ca2+ release.
Deletion of β2-ARs prevents the development of MLP−/− cardiomyopathy via positive modulation of Ca2+ due to removal of inhibitory Gi signaling and increased phosphorylation of troponin I and phospholamban. Similar effects were seen after TAC. Unlike previous models where β2-ARs were found to be cardioprotective, in these two models, β2-AR signaling appears to be deleterious, potentially through negative regulation of Ca2+ dynamics.
Adrenergic receptors; cardiomyopathy; excitation-contraction coupling; signal transduction
This study determined the role of angiotensin-converting enzyme (ACE) on the development of angiotensin I (AngI)-induced atherosclerosis and the contribution of leukocyte-specific expression of this enzyme.
Approach and Results
To define the contribution of ACE-dependent activity to AngII synthesis in atherosclerotic development, male LDL receptor -/- mice were fed a fat-enriched diet and infused with either AngI or AngII. The same infusion rate of these peptides had equivalent effects on atherosclerotic development. Co-infusion of an ACE inhibitor, enalapril, ablated AngI augmented atherosclerosis, but had no effect on AngII-induced lesion development. ACE protein was detected in several cell types in atherosclerotic lesions, with a predominance in macrophages. This cell type secreted AngII, which was ablated by ACE inhibition. To study whether leukocyte ACE contributed to atherosclerosis, irradiated male LDL receptor -/- mice were repopulated with bone marrow-derived cells from either ACE +/+ or -/- mice and fed the fat-enriched diet for 12 weeks. Chimeric mice with ACE deficiency in bone marrow-derived cells had modestly reduced atherosclerotic lesions in aortic arches, but had no effects in aortic roots.
ACE mediates AngI-induced atherosclerosis, and ACE expression in leukocytes modestly contributes to atherosclerotic development in hypercholesterolemic mice.
atherosclerosis; angiotensin-converting enzyme; angiotensin; macrophages; renin; hypercholesterolemia
Pulmonary hypertension (PH) is characterized by progressive elevation in pulmonary pressure and loss of small pulmonary arteries. As bone morphogenetic proteins (BMPs) promote pulmonary angiogenesis by recruiting the Wnt/βcatenin pathway, we proposed that βcatenin activation could reduce loss and/or induce regeneration of small PAs and attenuate PH.
This study aims to establish the role of β–catenin in protecting the pulmonary endothelium and stimulating compensatory angiogenesis following injury.
Methods and Results
To assess the impact of β-catenin activation on chronic hypoxia-induced PH, we used the adenomatous polyposis coli (ApcMin/+) mouse, where reduced APC causes constitutive β–catenin elevation. Surprisingly, hypoxic ApcMin/+ mice displayed greater PH and small PA loss compared to control C57Bl6J (C57) littermates. Pulmonary artery endothelial cells (PAECs) isolated from ApcMin/+ demonstrated reduced survival and angiogenic responses along with a profound reduction in adhesion to laminin. The mechanism involved failure of APC to interact with the cytoplasmic domain of the α3 integrin, to stabilize focal adhesions and activate integrin-linked kinase (ILK-1) and pAkt. We found that PAECs from lungs of patients with idiopathic PH have reduced APC expression, decreased adhesion to laminin and impaired vascular tube formation. These defects were corrected in the cultured cells by transfection of APC.
We show that APC is integral to PAEC adhesion and survival and is reduced in PAECs from PH patient lungs. The data suggest that decreased APC may be a cause of increased risk or severity of PH in genetically susceptible individuals.
Adenomatous poliposis coli; Wnt signaling; integrin signaling; angiogenesis; pulmonary hypertension
Dysfunctional bone morphogenetic protein receptor-2 (BMPR2) signaling is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We used a transcriptional high-throughput luciferase reporter assay to screen 3,756 FDA-approved drugs and bioactive compounds for induction of BMPR2 signaling. The best response was achieved with FK506 (tacrolimus), via a dual mechanism of action as a calcineurin inhibitor that also binds FK-binding protein-12 (FKBP12), a repressor of BMP signaling. FK506 released FKBP12 from type I receptors activin receptor-like kinase 1 (ALK1), ALK2, and ALK3 and activated downstream SMAD1/5 and MAPK signaling and ID1 gene regulation in a manner superior to the calcineurin inhibitor cyclosporine and the FKBP12 ligand rapamycin. In pulmonary artery endothelial cells (ECs) from patients with idiopathic PAH, low-dose FK506 reversed dysfunctional BMPR2 signaling. In mice with conditional Bmpr2 deletion in ECs, low-dose FK506 prevented exaggerated chronic hypoxic PAH associated with induction of EC targets of BMP signaling, such as apelin. Low-dose FK506 also reversed severe PAH in rats with medial hypertrophy following monocrotaline and in rats with neointima formation following VEGF receptor blockade and chronic hypoxia. Our studies indicate that low-dose FK506 could be useful in the treatment of PAH.
An accumulation of milk fat globule EGF-8 protein (MFG-E8) occurs within the context of arterial wall inflammatory remodeling during aging, hypertension, diabetes mellitus, or atherosclerosis. MFG-E8 induces VSMC invasion, but whether it effects VSMC proliferation, a salient feature of arterial inflammation, is unknown. Here, we show that in the rat arterial wall in vivo, PCNA and Ki67, markers of cell cycle activation, increase with age between 8 and 30-mo. In fresh or early passage VSMC isolated from old aortae, an increase in CDK4 and PCNA, and cell cycle with acceleration of S and G2 phases and reduction of the G1/G0 phase, and an increase in PDGF and its receptors, confer elevated proliferative capacity, compared to young VSMC. Increased co-expression and physical interaction of MFG-E8 and integrin αvβ5 occur with aging in both the rat aortic wall in vivo and in VSMC in vitro. In young VSMC in vitro, MFG-E8 added exogenously, or over-expressed endogenously, triggers phosphorylation of ERK1/2, augmented levels of PCNA and CDK4, increased BrdU incorporation and promotes proliferation, via αvβ5 integrins. MFG-E8 silencing, or its receptor inhibition, or the blockade of ERK1/2 phosphorylation in these cells reduces PCNA and CDK4 levels and decelerates the cell cycle S phase, conferring a reduction in proliferative capacity. Collectively, these results indicate that MFG-E8 in a dose-dependent manner, coordinates the expression of cell cycle molecules and facilitates VSMC proliferation via integrin/ ERK1/2 signaling. Thus, an increase in MFG-E8 signaling is a mechanism of the age-associated increase in aortic VSMC proliferation.
MFG-E8; Aging; VSMC proliferation; cell cycle; vascular remodeling
β-adrenergic receptors (β-ARs) modulate cardiotoxicity/cardioprotection through crosstalk with multiple signaling pathways. We have previously shown that β2-ARs are cardioprotective during exposure to oxidative stress induced by doxorubicin (DOX). DOX cardiotoxicity is mediated in part through a Ca2+-dependent opening of the mitochondrial permeability transition (MPT), however the signals linking a cell surface receptor like the β2-AR to regulators of mitochondrial function are not clear. The objective of this study was to assess mechanisms of crosstalk between β2-ARs and mitochondrial cell death pathways.
Methods and Results
DOX administered to WT mice resulted in no acute mortality, however 85% of β2-/- mice died within 30 min. Several pro- and anti-survival pathways were altered. The pro-survival kinase, εPKC, was decreased by 64% in β2-/- after DOX vs WT (p<0.01); the εPKC activator ψεRACK partially rescued these mice (47% reduction in mortality). Activity of the pro-survival kinase Akt decreased by 76% in β2-/- after DOX vs WT (p<0.01). The α1-antagonist prazosin restored Akt activity to normal and also partially reversed the mortality (45%). Deletion of the β2-AR increased rate of Ca2+ release by 75% and peak [Ca2+]i by 20% respectively in isolated cardiomyocytes; the Ca2+ channel blocker verapamil also partially rescued the β2-/- (26%). Mitochondrial architecture was disrupted and complex I and II activities decreased by 40.9% and 34.6% respectively after DOX only in β2-/-. The MPT blocker cyclosporine reduced DOX mortality by 41% and prazosin plus cyclosporine acted synergistically to decrease mortality by 85%.
β2-ARs activate pro-survival kinases and attenuate mitochondrial dysfunction during oxidative stress; absence of β2-ARs enhances cardiotoxicity via negative regulation of survival kinases and enhancement of intracellular Ca2+, thus predisposing the mitochondria to opening of the MPT.
Adrenergic receptors; cardiomyopathy; mitochondria; signal transduction; protein kinases
β-adrenergic receptor blockers have demonstrated significant survival benefit and have become standard therapy for adults with dilated cardiomyopathy, although their efficacy in pediatric patients is still unproven. Recent data suggests that the two major cardiac β-adrenergic receptor subtypes (β1 and β2) couple differentially to intracellular signaling pathways regulating contractility and remodeling. This has led some to suggest that the β1 receptor is the “cardiotoxic subtype” whereas the β2 receptor is “cardioprotective.” Given this paradigm, there could be situations where subtype selective β-blockade or even subtype selective β-stimulation might be beneficial. However, since most of these studies have been performed in isolated cardiomyocytes, their application to clinical practice is unclear. To better understand the roles of β1- vs. β2-receptors in the pathogenesis of clinical cardiomyopathy, we and others have taken advantage of several well-characterized murine models of cardiovascular disease. These studies demonstrate that β-receptor regulation of the balance between cardioprotection and cardiotoxicity is even more complex than previously appreciated: the role of each β-receptor subtype may vary depending on the specific cardiac stressor involved (e.g. ischemia, pressure overload, genetic mutation, cardiotoxin). Furthermore, the remodeling effects of β-receptor signaling have a temporal component, depending on whether a cardiac stress is acute vs. chronic.
Cardiomyopathy; adrenergic receptor; cell signaling; β-blocker; heart failure
β-Adrenoceptor (β-AR) subtypes act through different signaling pathways to regulate cardiac function and remodeling. Previous in vivo data show a markedly enhanced cardiotoxic response to doxorubicin in β2−/− mice, which is rescued by the additional deletion of the β1-AR. We determined whether this differential response was myocyte specific by examining the effects of doxorubicin in myocytes and fibroblasts from WT and β1, β2 and β1/β2−/− mice. Cells were exposed to doxorubicin at 1–50 µM and viability and apoptosis assessed at 6, 24 and 48 h. WT myocytes showed a time and dose-dependent decrease in viability (42% decrease at 1 µM after 24 h). β2−/− Myocytes showed a greater decrease in viability vs. WT (20.8% less at 6 h; 14% less at 24 h, P < 0.05); β1−/− and β1/β2−/− myocytes showed enhanced survival (β1−/− 11%; β1/β2−/− 18% greater than WT, P < 0.05). TUNEL staining demonstrated a similar differential susceptibility (WT 26% apoptotic nuclei, β2−/− 45.9%, β1/β2−/− 16.8%, P < 0.05). β2−/− Fibroblasts also showed enhanced toxicity. Pertussis toxin pretreatment of WT cells decreased survival similar to the β2−/−, suggesting a role for Gi signaling. JNK was differentially activated in β2−/− myocytes after doxorubicin and its inhibition increased cardiotoxicity. In conclusion, the differential cardioprotective/cardiotoxic effects mediated by β1 vs. β2-AR subtypes in knockout mice are recapitulated in myocytes isolated from these mice. β2-ARs appear to play a cardioprotective role, whereas β1-ARs a cardiotoxic role.
Cardiomyopathy; Signal transduction; Apoptosis; Adrenergic receptors; Anthracyclines
The Dok adaptor proteins play key regulatory roles in receptor and non-receptor kinase-initiated signaling pathways. Dok-1, the prototype member of this family, negatively regulates cell proliferation elicited by numerous growth factors, including platelet-derived growth factor (PDGF). However, how Dok-1 exerts its negative effect on mitogenesis has remained elusive. Using Dok-1 knockout cells and Dok-1 mutants deficient in binding to specific Dok-1-interacting proteins, we show that Dok-1 interferes with PDGF-stimulated c-myc induction and Ras/mitogen-activated protein kinase (MAPK) activation by tethering different signaling components to the cell membrane. Specifically, Dok-1 attenuates PDGF-elicited c-myc induction by recruiting Csk to active Src kinases, whereupon their activities and consequent c-myc induction are diminished. On the other hand, Dok-1 negatively regulates PDGF-induced MAPK activation by acting on Ras-GAP and at least one other Dok-1-interacting protein. Importantly, we demonstrate that Dok-1's actions on both of these signaling pathways contribute to its inhibitory effect on mitogenesis. Our data suggest a mechanistic basis for the inhibitory effect of Dok-1 on growth factor-induced mitogenesis and its role as a tumor suppressor.
Chronic myelogenous leukemia (CML) is characterized by the presence of the chimeric p210bcr/abl oncoprotein that shows elevated and constitutive protein tyrosine kinase activity relative to the normal c-abl tyrosine kinase. Although several p210bcr/abl substrates have been identified, their relevance in the pathogenesis of the disease is unclear. We have identified a family of proteins, Dok (downstream of tyrosine kinase), coexpressed in hematopoietic progenitor cells. Members of this family such as p62dok(Dok-1) and p56dok-2(Dok-2) associate with the p120 rasGTPase-activating protein (rasGAP) upon phosphorylation by p210bcr/abl as well as receptor and nonreceptor tyrosine kinases. Here, we report the generation and characterization of single and double Dok-1 or Dok-2 knockout (KO) mutants. Single KO mice displayed normal steady-state hematopoiesis. By contrast, concomitant Dok-1 and Dok-2 inactivation resulted in aberrant hemopoiesis and Ras/MAP kinase activation. Strikingly, all Dok-1/Dok-2 double KO mutants spontaneously developed transplantable CML-like myeloproliferative disease due to increased cellular proliferation and reduced apoptosis. Furthermore, Dok-1 or Dok-2 inactivation markedly accelerated leukemia and blastic crisis onset in Tec-p210bcr/abl transgenic mice known to develop, after long latency, a myeloproliferative disorder resembling human CML. These findings unravel the critical and unexpected role of Dok-1 and Dok-2 in tumor suppression and control of the hematopoietic compartment homeostasis.
cell proliferation; apoptosis; knockout; CML leukemogenesis; signal transduction
A major pathway by which growth factors, such as platelet-derived growth factor (PDGF), regulate cell proliferation is via the receptor tyrosine kinase/Ras/mitogen-activated protein kinase (MAPK) signaling cascade. The output of this pathway is subjected to tight regulation of both positive and negative regulators. One such regulator is p62dok, the prototype of a newly identified family of adaptor proteins. We recently provided evidence, through the use of p62dok-deficient cells, that p62dok acts as a negative regulator of growth factor–induced cell proliferation and the Ras/MAPK pathway. We show here that reintroduction of p62dok into p62dok−/− cells can suppress the increased cell proliferation and prolonged MAPK activity seen in these cells, and that plasma membrane recruitment of p62dok is essential for its function. We also show that the PDGF-triggered plasma membrane translocation of p62dok requires activation of phosphoinositide 3-kinase (PI3-kinase) and binding of its pleckstrin homology (PH) domain to 3′-phosphorylated phosphoinositides. Furthermore, we demonstrate that p62dok can exert its negative effect on the PDGFR/MAPK pathway independently of its ability to associate with RasGAP and Nck. We conclude that p62dok functions as a negative regulator of the PDGFR/Ras/MAPK signaling pathway through a mechanism involving PI3-kinase–dependent recruitment of p62dok to the plasma membrane.
growth factors; cell proliferation; membrane lipids; signal transduction; protein-serine-threonine kinase
p62dok has been identified as a substrate of many oncogenic tyrosine kinases such as the chronic myelogenous leukemia (CML) chimeric p210bcr-abl oncoprotein. It is also phosphorylated upon activation of many receptors and cytoplamic tyrosine kinases. However, the biological functions of p62dok in normal cell signaling as well as in p210bcr-abl leukemogenesis are as yet not fully understood. Here we show, in hemopoietic and nonhemopoietic cells derived from p62dok−/− mice, that the loss of p62dok results in increased cell proliferation upon growth factor treatment. Moreover, Ras and mitogen-activated protein kinase (MAPK) activation is markedly sustained in p62dok−/− cells after the removal of growth factor. However, p62dok inactivation does not affect DNA damage and growth factor deprivation–induced apoptosis. Furthermore, p62dok inactivation causes a significant shortening in the latency of the fatal myeloproliferative disease induced by retroviral-mediated transduction of p210bcr-abl in bone marrow cells. These data indicate that p62dok acts as a negative regulator of growth factor–induced cell proliferation, at least in part through downregulating Ras/MAPK signaling pathway, and that p62dok can oppose leukemogenesis by p210bcr-abl.
cell proliferation; signal transduction; knockout; mast cells; thymocytes