The Caveolin-1 Scaffolding Domain Peptide Reverses Aging-Associated Deleterious Changes in Multiple Organs.

Aging is a progressive, multifactorial, degenerative process in which deleterious changes occur in the biochemistry and function of organs. We showed that angiotensin II (AngII)-induced pathologies in the heart and kidney of young (3-month-old) mice are suppressed by the caveolin-1 scaffolding domain (CSD) peptide. Because AngII mediates many aging-associated changes, we explored whether CSD could reverse pre-existing pathologies and improve organ function in aged mice. Using 18-month-old mice (similar to 60-year-old humans), we found that >5-fold increases in leakage of serum proteins and >2-fold increases in fibrosis are associated with aging in the heart, kidney, and brain. Because tyrosine phosphorylation of cell junction proteins leads to the loss of microvascular barrier function, we analyzed the activation of the receptor tyrosine kinase PDGFR and the nonreceptor tyrosine kinases c-Src and Pyk2. We observed 5-fold activation of PDGFR and 2- to 3-fold activation of c-Src and Pyk2 in aged mice. Treatment with CSD for 4 weeks reversed these pathologic changes (microvascular leakage, fibrosis, kinase activation) in all organs almost down to the levels in healthy, young mice. In studies of heart function, CSD reduced the aging-associated increase in cardiomyocyte cross-sectional area and enhanced ventricular compliance in that echocardiographic studies demonstrated improved ejection fraction and fractional shortening and reduced isovolumic relation time. These results suggest that versions of CSD may be developed as treatments for aging-associated diseases in human patients based on the concept that CSD inhibits tyrosine kinases, leading to the inhibition of microvascular leakage and associated fibrosis, thereby improving organ function. SIGNIFICANCE STATEMENT: The caveolin-1 scaffolding domain (CSD) peptide reverses aging-associated fibrosis, microvascular leakage, and organ dysfunction in the heart, kidneys, and brain via a mechanism that involves the suppression of the activity of multiple tyrosine kinases, suggesting that CSD can be developed as a treatment for a wide range of diseases found primarily in the aged.

Reversal of maladaptive fibrosis and compromised ventricular function in the pressure overloaded heart by a caveolin-1 surrogate peptide.

Chronic ventricular pressure overload (PO) results in congestive heart failure (CHF) in which myocardial fibrosis develops in concert with ventricular dysfunction. Caveolin-1 is important in fibrosis in various tissues due to its decreased expression in fibroblasts and monocytes. The profibrotic effects of low caveolin-1 can be blocked with the caveolin-1 scaffolding domain peptide (CSD, a caveolin-1 surrogate) using both mouse models and human cells. We have studied the beneficial effects of CSD on mice in which PO was induced by trans-aortic constriction (TAC). Beneficial effects observed in TAC mice receiving CSD injections daily included: improved ventricular function (increased ejection fraction, stroke volume, and cardiac output; reduced wall thickness); decreased collagen I, collagen chaperone HSP47, fibronectin, and CTGF levels; decreased activation of non-receptor tyrosine kinases Pyk2 and Src; and decreased activation of eNOS. To determine the source of cells that contribute to fibrosis in CHF, flow cytometric studies were performed that suggested that myofibroblasts in the heart are in large part bone marrow-derived. Two CD45+ cell populations were observed. One (Zone 1) contained CD45+/HSP47-/macrophage marker+ cells (macrophages). The second (Zone 2) contained CD45moderate/HSP47+/macrophage marker- cells often defined as fibrocytes. TAC increased the number of cells in Zones 1 and 2 and the level of HSP47 in Zone 2. These studies are a first step in elucidating the mechanism of action of CSD in heart fibrosis and promoting the development of CSD as a novel treatment to reduce fibrosis and improve ventricular function in CHF patients.

mTOR Complexes Repress Hypertrophic Agonist-Stimulated Expression of Connective Tissue Growth Factor in Adult Cardiac Muscle Cells.

Connective tissue growth factor (CTGF) is a fibrogenic cytokine that promotes fibrosis in various organs. In the heart, both cardiomyocytes (CM) and cardiac fibroblasts have been reported as a source of CTGF expression, aiding cardiac fibrosis. Although the mammalian target of rapamycin (mTOR) forms 2 distinct complexes, mTORC1 and mTORC2, and plays a central role in integrating biochemical signals for protein synthesis and cellular homeostasis, we explored its role in CTGF expression in adult feline CM. CM were stimulated with 10 µM phenylephrine (PE), 200 nM angiotensin (Ang), or 100 nM insulin for 24 hours. PE and Ang, but not insulin, caused an increase in CTGF mRNA expression with the highest expression observed with PE. Inhibition of mTOR with torin1 but not rapamycin significantly enhanced PE-stimulated CTGF expression. Furthermore, silencing of raptor and rictor using shRNA adenoviral vectors to suppress mTORC1 and mTORC2, respectively, or blocking phosphatidylinositol 3-kinase (PI3K) signaling with LY294002 (LY) or Akt signaling by dominant-negative Akt expression caused a substantial increase in PE-stimulated CTGF expression as measured by both mRNA and secreted protein levels. However, studies with dominant-negative delta isoform of protein kinase C demonstrate that delta isoform of protein kinase C is required for both agonist-induced CTGF expression and mTORC2/Akt-mediated CTGF suppression. Finally, PE-stimulated CTGF expression was accompanied with a corresponding increase in Smad3 phosphorylation and pretreatment of cells with SIS3, a Smad3 specific inhibitor, partially blocked the PE-stimulated CTGF expression. Therefore, a PI3K/mTOR/Akt axis plays a suppressive role on agonist-stimulated CTGF expression where the loss of this mechanism could be a contributing factor for the onset of cardiac fibrosis in the hypertrophying myocardium.

Arrestin-dependent angiotensin AT1 receptor signaling regulates Akt and mTor-mediated protein synthesis.

Control of protein synthesis is critical to both cell growth and proliferation. The mammalian target of rapamycin (mTOR) integrates upstream growth, proliferation, and survival signals, including those transmitted via ERK1/2 and Akt, to regulate the rate of protein translation. The angiotensin AT1 receptor has been shown to activate both ERK1/2 and Akt in arrestin-based signalsomes. Here, we examine the role of arrestin-dependent regulation of ERK1/2 and Akt in the stimulation of mTOR-dependent protein translation by the AT1 receptor using HEK293 and primary vascular smooth muscle cell models. Nascent protein synthesis stimulated by both the canonical AT1 receptor agonist angiotensin II (AngII), and the arrestin pathway-selective agonist [Sar(1)-Ile(4)-Ile(8)]AngII (SII), is blocked by shRNA silencing of ßarrestin1/2 or pharmacological inhibition of Akt, ERK1/2, or mTORC1. In HEK293 cells, SII activates a discrete arrestin-bound pool of Akt and promotes Akt-dependent phosphorylation of mTOR and its downstream effector p70/p85 ribosomal S6 kinase (p70/85S6K). In parallel, SII-activated ERK1/2 helps promote mTOR and p70/85S6K phosphorylation, and is required for phosphorylation of the known ERK1/2 substrate p90 ribosomal S6 kinase (p90RSK). Thus, arrestins coordinate AT1 receptor regulation of ERK1/2 and Akt activity and stimulate protein translation via both Akt-mTOR-p70/85S6K and ERK1/2-p90RSK pathways. These results suggest that in vivo, arrestin pathway-selective AT1 receptor agonists may promote cell growth or hypertrophy through arrestin-mediated mechanisms despite their antagonism of G protein signaling.

A Kinase-Independent Function of c-Src Mediates p130Cas Phosphorylation at the Serine-639 Site in Pressure Overloaded Myocardium.

Early work in pressure overloaded (PO) myocardium shows that integrins mediate focal adhesion complex formation by recruiting the adaptor protein p130Cas (Cas) and nonreceptor tyrosine kinase c-Src. To explore c-Src role in Cas-associated changes during PO, we used a feline right ventricular in vivo PO model and a three-dimensional (3D) collagen-embedded adult cardiomyocyte in vitro model that utilizes a Gly-Arg-Gly-Asp-Ser (RGD) peptide for integrin stimulation. Cas showed slow electrophoretic mobility (band-shifting), recruitment to the cytoskeleton, and tyrosine phosphorylation at 165, 249, and 410 sites in both 48 h PO myocardium and 1 h RGD-stimulated cardiomyocytes. Adenoviral mediated expression of kinase inactive (negative) c-Src mutant with intact scaffold domains (KN-Src) in cardiomyocytes did not block the RGD stimulated changes in Cas. Furthermore, expression of KN-Src or kinase active c-Src mutant with intact scaffold function (A-Src) in two-dimensionally (2D) cultured cardiomyocytes was sufficient to cause Cas band-shifting, although tyrosine phosphorylation required A-Src. These data indicate that c-Src's adaptor function, but not its kinase function, is required for a serine/threonine specific phosphorylation(s) responsible for Cas band-shifting. To explore this possibility, Chinese hamster ovary cells that stably express Cas were infected with either ß-gal or KN-Src adenoviruses and used for Cas immunoprecipitation combined with mass spectrometry analysis. In the KN-Src expressing cells, Cas showed phosphorylation at the serine-639 (human numbering) site. A polyclonal antibody raised against phospho-serine-639 detected Cas phosphorylation in 24-48 h PO myocardium. Our studies indicate that c-Src's adaptor function mediates serine-639 phosphorylation of Cas during integrin activation in PO myocardium.

Overexpression of RNA-binding protein CELF1 prevents apoptosis and destabilizes pro-apoptotic mRNAs in oral cancer cells.

CELF1 RNA-binding protein, otherwise called CUGBP1, associates and coordinates the degradation of GU-rich element (GRE) containing mRNA's encoding factors important for cell growth, migration and apoptosis. Although many substrates of CELF1 have been identified, the biological significance of CELF1-mediated mRNA decay remains unclear. As the processes modulated by CELF1 are frequently disrupted in cancer, we investigated the expression and role of CELF1 in oral squamous cancer cells (OSCCs). We determined that CELF1 is reproducibly overexpressed in OSCC tissues and cell lines. Moreover, depletion of CELF1 reduced proliferation and increased apoptosis in OSCCs, but had negligible effect in non-transformed cells. We found that CELF1 associates directly with the 3'UTR of mRNAs encoding the pro-apoptotic factors BAD, BAX and JunD and mediates their rapid decay. Specifically, 3'UTR fragment analysis of JunD revealed that the GRE region is critical for binding with CELF1 and expression of JunD in oral cancer cells. In addition, silencing of CELF1 rendered BAD, BAX and JunD mRNAs stable and increased their protein expression in oral cancer cells. Taken together, these results support a critical role for CELF1 in modulating apoptosis and implicate this RNA-binding protein as a cancer marker and potential therapeutic target.

Cytoskeletal role in protection of the failing heart by ß-adrenergic blockade.

Formation of a dense microtubule network that impedes cardiac contraction and intracellular transport occurs in severe pressure overload hypertrophy. This process is highly dynamic, since microtubule depolymerization causes striking improvement in contractile function. A molecular etiology for this cytoskeletal alteration has been defined in terms of type 1 and type 2A phosphatase-dependent site-specific dephosphorylation of the predominant myocardial microtubule-associated protein (MAP)4, which then decorates and stabilizes microtubules. This persistent phosphatase activation is dependent upon ongoing upstream activity of p21-activated kinase-1, or Pak1. Because cardiac ß-adrenergic activity is markedly and continuously increased in decompensated hypertrophy, and because ß-adrenergic activation of cardiac Pak1 and phosphatases has been demonstrated, we asked here whether the highly maladaptive cardiac microtubule phenotype seen in pathological hypertrophy is based on ß-adrenergic overdrive and thus could be reversed by ß-adrenergic blockade. The data in this study, which were designed to answer this question, show that such is the case; that is, ß(1)- (but not ß(2)-) adrenergic input activates this pathway, which consists of Pak1 activation, increased phosphatase activity, MAP4 dephosphorylation, and thus the stabilization of a dense microtubule network. These data were gathered in a feline model of severe right ventricular (RV) pressure overload hypertrophy in response to tight pulmonary artery banding (PAB) in which a stable, twofold increase in RV mass is reached by 2 wk after pressure overloading. After 2 wk of hypertrophy induction, these PAB cats during the following 2 wk either had no further treatment or had ß-adrenergic blockade. The pathological microtubule phenotype and the severe RV cellular contractile dysfunction otherwise seen in this model of RV hypertrophy (PAB No Treatment) was reversed in the treated (PAB ß-Blockade) cats. Thus these data provide both a specific etiology and a specific remedy for the abnormal microtubule network found in some forms of pathological cardiac hypertrophy.

Multiple subregions within the caveolin-1 scaffolding domain inhibit fibrosis, microvascular leakage, and monocyte migration.

The caveolin-1 scaffolding domain (CSD, amino acids 82-101 of caveolin-1) has been shown to suppress bleomycin-induced lung and skin fibrosis and angiotensin II (AngII)-induced myocardial fibrosis. To identify active subregions within CSD, we split its sequence into three slightly overlapping 8-amino acid subregions (82-89, 88-95, and 94-101). Interestingly, all three peptides showed activity. In bleomycin-treated mice, all three subregions suppressed the pathological effects on lung and skin tissue morphology. In addition, while bone marrow monocytes isolated from bleomycin-treated mice showed greatly enhanced migration in vitro toward CXCL12, treatment in vivo with CSD and its subregions almost completely suppressed this enhanced migration. In AngII-induced heart failure, both 82-89 and 88-95 significantly suppressed fibrosis (both Col I and HSP47 levels), microvascular leakage, and heart weight/ body weight ratio (HW/BW) while improving ventricular function. In contrast, while 94-101 suppressed the increase in Col I, it did not improve the other parameters. The idea that all three subregions can be active depending on the assay was further supported by experiments studying the in vitro migration of human monocytes in which all three subregions were extremely active. These studies are very novel in that it has been suggested that there is only one active region within CSD that is centered on amino acids 90-92. In contrast, we demonstrate here the presence of other active regions within CSD.

Site-specific microtubule-associated protein 4 dephosphorylation causes microtubule network densification in pressure overload cardiac hypertrophy.

In severe pressure overload-induced cardiac hypertrophy, a dense, stabilized microtubule network forms that interferes with cardiocyte contraction and microtubule-based transport. This is associated with persistent transcriptional up-regulation of cardiac alpha- and beta-tubulin and microtubule-stabilizing microtubule-associated protein 4 (MAP4). There is also extensive microtubule decoration by MAP4, suggesting greater MAP4 affinity for microtubules. Because the major determinant of this affinity is site-specific MAP4 dephosphorylation, we characterized this in hypertrophied myocardium and then assessed the functional significance of each dephosphorylation site found by mimicking it in normal cardiocytes. We first isolated MAP4 from normal and pressure overload-hypertrophied feline myocardium; volume-overloaded myocardium, which has an equal degree and duration of hypertrophy but normal functional and cytoskeletal properties, served as a control for any nonspecific growth-related effects. After cloning cDNA-encoding feline MAP4 and obtaining its deduced amino acid sequence, we characterized by mass spectrometry any site-specific MAP4 dephosphorylation. Solely in pressure overload-hypertrophied myocardium, we identified striking MAP4 dephosphorylation at Ser-472 in the MAP4 N-terminal projection domain and at Ser-924 and Ser-1056 in the assembly-promoting region of the C-terminal microtubule-binding domain. Site-directed mutagenesis of MAP4 cDNA was then used to switch each serine to non-phosphorylatable alanine. Wild-type and mutated cDNAs were used to construct adenoviruses; microtubule network density, stability, and MAP4 decoration were assessed in normal cardiocytes following an equivalent level of MAP4 expression. The Ser-924 --> Ala MAP4 mutant produced a microtubule phenotype indistinguishable from that seen in pressure overload hypertrophy, such that Ser-924 MAP4 dephosphorylation during pressure overload hypertrophy may be central to this cytoskeletal abnormality.

Basis for MAP4 dephosphorylation-related microtubule network densification in pressure overload cardiac hypertrophy.

Increased activity of Ser/Thr protein phosphatases types 1 (PP1) and 2A (PP2A) during maladaptive cardiac hypertrophy contributes to cardiac dysfunction and eventual failure, partly through effects on calcium metabolism. A second maladaptive feature of pressure overload cardiac hypertrophy that instead leads to heart failure by interfering with cardiac contraction and intracellular transport is a dense microtubule network stabilized by decoration with microtubule-associated protein 4 (MAP4). In an earlier study we showed that the major determinant of MAP4-microtubule affinity, and thus microtubule network density and stability, is site-specific MAP4 dephosphorylation at Ser-924 and to a lesser extent at Ser-1056; this was found to be prominent in hypertrophied myocardium. Therefore, in seeking the etiology of this MAP4 dephosphorylation, we looked here at PP2A and PP1, as well as the upstream p21-activated kinase 1, in maladaptive pressure overload cardiac hypertrophy. The activity of each was increased persistently during maladaptive hypertrophy, and overexpression of PP2A or PP1 in normal hearts reproduced both the microtubule network phenotype and the dephosphorylation of MAP4 Ser-924 and Ser-1056 seen in hypertrophy. Given the major microtubule-based abnormalities of contractile and transport function in maladaptive hypertrophy, these findings constitute a second important mechanism for phosphatase-dependent pathology in the hypertrophied and failing heart.

Rapamycin treatment augments both protein ubiquitination and Akt activation in pressure-overloaded rat myocardium.

Ubiquitin-mediated protein degradation is necessary for both increased ventricular mass and survival signaling for compensated hypertrophy in pressure-overloaded (PO) myocardium. Another molecular keystone involved in the hypertrophic growth process is the mammalian target of rapamycin (mTOR), which forms two distinct functional complexes: mTORC1 that activates p70S6 kinase-1 to enhance protein synthesis and mTORC2 that activates Akt to promote cell survival. Independent studies in animal models show that rapamycin treatment that alters mTOR complexes also reduces hypertrophic growth and increases lifespan by an unknown mechanism. We tested whether the ubiquitin-mediated regulation of growth and survival in hypertrophic myocardium is linked to the mTOR pathway. For in vivo studies, right ventricle PO in rats was conducted by pulmonary artery banding; the normally loaded left ventricle served as an internal control. Rapamycin (0.75 mg/kg per day) or vehicle alone was administered intraperitoneally for 3 days or 2 wk. Immunoblot and immunofluorescence imaging showed that the level of ubiquitylated proteins in cardiomyocytes that increased following 48 h of PO was enhanced by rapamycin. Rapamycin pretreatment also significantly increased PO-induced Akt phosphorylation at S473, a finding confirmed in cardiomyocytes in vitro to be downstream of mTORC2. Analysis of prosurvival signaling in vivo showed that rapamycin increased PO-induced degradation of phosphorylated inhibitor of κB, enhanced expression of cellular inhibitor of apoptosis protein 1, and decreased active caspase-3. Long-term rapamycin treatment in 2-wk PO myocardium blunted hypertrophy, improved contractile function, and reduced caspase-3 and calpain activation. These data indicate potential cardioprotective benefits of rapamycin in PO hypertrophy.

Beta3 integrin-mediated ubiquitination activates survival signaling during myocardial hypertrophy.

Identifying the molecular mechanisms activated in compensatory hypertrophy and absent during decompensation will provide molecular targets for prevention of heart failure. We have previously shown enhanced ubiquitination (Ub) during the early growth period of pressure overload (PO) hypertrophy near intercalated discs of cardiomyocytes, where integrins are important for mechanotransduction. In this study, we tested the role of integrins upstream of Ub, whether enhanced Ub contributes to survival signaling in early PO, and if loss of this mechanism could lead to decreased ventricular function. The study used a beta(3) integrin (-/-) mouse and a wild-type mouse as a control for in vivo PO by transverse aortic constriction (TAC) and for cultured cardiomyocytes in vitro, stimulated with the integrin-activating peptide RGD. We demonstrate beta(3) integrin mediates transient Ub of targeted proteins during PO hypertrophy, which is necessary for cardiomyocyte survival and to maintain ventricular function. Prosurvival signaling proceeds by initiation of NF-kappaB transcription of the E3 ligase, cIAP1. In PO beta(3)(-/-) mice, absence of this mechanism correlates with increased TUNEL staining and decreased ventricular mass and function by 4 wk. This is the first study to show that a beta(3) integrin/Ub/NF-kappaB pathway contributes to compensatory hypertrophic growth.

Lack of beta3 integrin signaling contributes to calpain-mediated myocardial cell loss in pressure-overloaded myocardium.

Although cardiac hypertrophy initially ensues as a compensatory mechanism, it often culminates in congestive heart failure. Based on our earlier studies that calpain and beta3 integrin play cell death and survival roles, respectively, during pressure-overload (PO) hypertrophy, we investigated if the loss of beta3 integrin signaling is a potential mechanism for calpain-mediated cardiomyocyte death during PO. beta3 Integrin knockout (beta3) and wild-type mice were used to induce either moderate or severe PO in vivo for short-term (72-hour) and long-term (4-week) transverse aortic constriction. Whereas wild-type mice showed no changes during moderate PO at both time points, beta3 mice exhibited both enrichment of the mu-calpain isoform and programmed cell death of cardiomyocytes after 4-week PO. However, with severe PO that caused increased mortality in both mice groups, cell death was observed in wild-type mice also. To study calpain's role, calpeptin, a specific inhibitor of calpain, was administered through an osmotic mini-pump at 2.5 mg/kg per day beginning 3 days before moderate transverse aortic constriction or sham surgery. Calpeptin administration blocked both calpain enrichment and myocardial cell death in the 4-week PO beta3 mice. Because beta3 integrin contributes to cardioprotective signaling, these studies indicate that the loss of specific integrin function could be a key mechanism for calpain-mediated programmed cell death of cardiomyocytes in PO myocardium.

Calpain inhibition preserves myocardial structure and function following myocardial infarction.

Cardiac pathology, such as myocardial infarction (MI), activates intracellular proteases that often trigger programmed cell death and contribute to maladaptive changes in myocardial structure and function. To test whether inhibition of calpain, a Ca(2+)-dependent cysteine protease, would prevent these changes, we used a mouse MI model. Calpeptin, an aldehydic inhibitor of calpain, was intravenously administered at 0.5 mg/kg body wt before MI induction and then at the same dose subcutaneously once per day. Both calpeptin-treated (n = 6) and untreated (n = 6) MI mice were used to study changes in myocardial structure and function after 4 days of MI, where end-diastolic volume (EDV) and left ventricular ejection fraction (EF) were measured by echocardiography. Calpain activation and programmed cell death were measured by immunohistochemistry, Western blotting, and TdT-mediated dUTP nick-end labeling (TUNEL). In MI mice, calpeptin treatment resulted in a significant improvement in EF [EF decreased from 67 + or - 2% pre-MI to 30 + or - 4% with MI only vs. 41 + or - 2% with MI + calpeptin] and attenuated the increase in EDV [EDV increased from 42 + or - 2 microl pre-MI to 73 + or - 4 microl with MI only vs. 55 + or - 4 microl with MI + calpeptin]. Furthermore, calpeptin treatment resulted in marked reduction in calpain- and caspase-3-associated changes and TUNEL staining. These studies indicate that calpain contributes to MI-induced alterations in myocardial structure and function and that it could be a potential therapeutic target in treating MI patients.

Graphene Oxide-A Tool for the Preparation of Chemically Crosslinking Free Alginate-Chitosan-Collagen Scaffolds for Bone Tissue Engineering.

Developing a biodegradable scaffold remains a major challenge in bone tissue engineering. This study was aimed at developing novel alginate-chitosan-collagen (SA-CS-Col)-based composite scaffolds consisting of graphene oxide (GO) to enrich porous structures, elicited by the freeze-drying technique. To characterize porosity, water absorption, and compressive modulus, GO scaffolds (SA-CS-Col-GO) were prepared with and without Ca2+-mediated crosslinking (chemical crosslinking) and analyzed using Raman, Fourier transform infrared (FTIR), X-ray diffraction (XRD), and scanning electron microscopy techniques. The incorporation of GO into the SA-CS-Col matrix increased both crosslinking density as indicated by the reduction of crystalline peaks in the XRD patterns and polyelectrolyte ion complex as confirmed by FTIR. GO scaffolds showed increased mechanical properties which were further increased for chemically crosslinked scaffolds. All scaffolds exhibited interconnected pores of 10-250 µm range. By increasing the crosslinking density with Ca2+, a decrease in the porosity/swelling ratio was observed. Moreover, the SA-CS-Col-GO scaffold with or without chemical crosslinking was more stable as compared to SA-CS or SA-CS-Col scaffolds when placed in aqueous solution. To perform in vitro biochemical studies, mouse osteoblast cells were grown on various scaffolds and evaluated for cell proliferation by using MTT assay and mineralization and differentiation by alizarin red S staining. These measurements showed a significant increase for cells attached to the SA-CS-Col-GO scaffold compared to SA-CS or SA-CS-Col composites. However, chemical crosslinking of SA-CS-Col-GO showed no effect on the osteogenic ability of osteoblasts. These studies indicate the potential use of GO to prepare free SA-CS-Col scaffolds with preserved porous structure with elongated Col fibrils and that these composites, which are biocompatible and stable in a biological medium, could be used for application in engineering bone tissues.

Suppression of angiotensin II-induced pathological changes in heart and kidney by the caveolin-1 scaffolding domain peptide.

Dysregulation of the renin-angiotensin system leads to systemic hypertension and maladaptive fibrosis in various organs. We showed recently that myocardial fibrosis and the loss of cardiac function in mice with transverse aortic constriction (TAC) could be averted by treatment with the caveolin-1 scaffolding domain (CSD) peptide. Here, we used angiotensin II (AngII) infusion (2.1 mg/kg/day for 2 wk) in mice as a second model to confirm and extend our observations on the beneficial effects of CSD on heart and kidney disease. AngII caused cardiac hypertrophy (increased heart weight to body weight ratio (HW/BW) and cardiomyocyte cross-sectional area); fibrosis in heart and kidney (increased levels of collagen I and heat shock protein-47 (HSP47)); and vascular leakage (increased levels of IgG in heart and kidney). Echocardiograms of AngII-infused mice showed increased left ventricular posterior wall thickness (pWTh) and isovolumic relaxation time (IVRT), and decreased ejection fraction (EF), stroke volume (SV), and cardiac output (CO). CSD treatment (i.p. injections, 50 µg/mouse/day) of AngII-infused mice significantly suppressed all of these pathological changes in fibrosis, hypertrophy, vascular leakage, and ventricular function. AngII infusion increased ß1 and ß3 integrin levels and activated Pyk2 in both heart and kidney. These changes were also suppressed by CSD. Finally, bone marrow cell (BMC) isolated from AngII-infused mice showed hyper-migration toward SDF1. When AngII-infused mice were treated with CSD, BMC migration was reduced to the basal level observed in cells from control mice. Importantly, CSD did not affect the AngII-induced increase in blood pressure (BP), indicating that the beneficial effects of CSD were not mediated via normalization of BP. These results strongly indicate that CSD suppresses AngII-induced pathological changes in mice, suggesting that CSD can be developed as a treatment for patients with hypertension and pressure overload-induced heart failure.

Dasatinib Attenuates Pressure Overload Induced Cardiac Fibrosis in a Murine Transverse Aortic Constriction Model.

Reactive cardiac fibrosis resulting from chronic pressure overload (PO) compromises ventricular function and contributes to congestive heart failure. We explored whether nonreceptor tyrosine kinases (NTKs) play a key role in fibrosis by activating cardiac fibroblasts (CFb), and could potentially serve as a target to reduce PO-induced cardiac fibrosis. Our studies were carried out in PO mouse myocardium induced by transverse aortic constriction (TAC). Administration of a tyrosine kinase inhibitor, dasatinib, via an intraperitoneally implanted mini-osmotic pump at 0.44 mg/kg/day reduced PO-induced accumulation of extracellular matrix (ECM) proteins and improved left ventricular geometry and function. Furthermore, dasatinib treatment inhibited NTK activation (primarily Pyk2 and Fak) and reduced the level of FSP1 positive cells in the PO myocardium. In vitro studies using cultured mouse CFb showed that dasatinib treatment at 50 nM reduced: (i) extracellular accumulation of both collagen and fibronectin, (ii) both basal and PDGF-stimulated activation of Pyk2, (iii) nuclear accumulation of Ki67, SKP2 and histone-H2B and (iv) PDGF-stimulated CFb proliferation and migration. However, dasatinib did not affect cardiomyocyte morphologies in either the ventricular tissue after in vivo administration or in isolated cells after in vitro treatment. Mass spectrometric quantification of dasatinib in cultured cells indicated that the uptake of dasatinib by CFb was greater that that taken up by cardiomyocytes. Dasatinib treatment primarily suppressed PDGF but not insulin-stimulated signaling (Erk versus Akt activation) in both CFb and cardiomyocytes. These data indicate that dasatinib treatment at lower doses than that used in chemotherapy has the capacity to reduce hypertrophy-associated fibrosis and improve ventricular function.

mTOR complex 2 mediates Akt phosphorylation that requires PKCε in adult cardiac muscle cells.

Our earlier work showed that mammalian target of rapamycin (mTOR) is essential to the development of various hypertrophic responses, including cardiomyocyte survival. mTOR forms two independent complexes, mTORC1 and mTORC2, by associating with common and distinct cellular proteins. Both complexes are sensitive to a pharmacological inhibitor, torin1, although only mTORC1 is inhibited by rapamycin. Since mTORC2 is known to mediate the activation of a prosurvival kinase, Akt, we analyzed whether mTORC2 directly mediates Akt activation or whether it requires the participation of another prosurvival kinase, PKCε (epsilon isoform of protein kinase-C). Our studies reveal that treatment of adult feline cardiomyocytes in vitro with insulin results in Akt phosphorylation at S473 for its activation which could be augmented with rapamycin but blocked by torin1. Silencing the expression of Rictor (rapamycin-insensitive companion of mTOR), an mTORC2 component, with a sh-RNA in cardiomyocytes lowers both insulin-stimulated Akt and PKCε phosphorylation. Furthermore, phosphorylation of PKCε and Akt at the critical S729 and S473 sites respectively was blocked by torin1 or Rictor knockdown but not by rapamycin, indicating that the phosphorylation at these specific sites occurs downstream of mTORC2. Additionally, expression of DN-PKCε significantly lowered the insulin-stimulated Akt S473 phosphorylation, indicating an upstream role for PKCε in the Akt activation. Biochemical analyses also revealed that PKCε was part of Rictor but not Raptor (a binding partner and component of mTORC1). Together, these studies demonstrate that mTORC2 mediates prosurvival signaling in adult cardiomyocytes where PKCε functions downstream of mTORC2 leading to Akt activation.

ß3 integrin in cardiac fibroblast is critical for extracellular matrix accumulation during pressure overload hypertrophy in mouse.

The adhesion receptor ß3 integrin regulates diverse cellular functions in various tissues. As ß3 integrin has been implicated in extracellular matrix (ECM) remodeling, we sought to explore the role of ß3 integrin in cardiac fibrosis by using wild type (WT) and ß3 integrin null (ß3-/-) mice for in vivo pressure overload (PO) and in vitro primary cardiac fibroblast phenotypic studies. Compared to WT mice, ß3-/- mice upon pressure overload hypertrophy for 4 wk by transverse aortic constriction (TAC) showed a substantially reduced accumulation of interstitial fibronectin and collagen. Moreover, pressure overloaded LV from ß3-/- mice exhibited reduced levels of both fibroblast proliferation and fibroblast-specific protein-1 (FSP1) expression in early time points of PO. To test if the observed impairment of ECM accumulation in ß3-/- mice was due to compromised cardiac fibroblast function, we analyzed primary cardiac fibroblasts from WT and ß3-/- mice for adhesion to ECM proteins, cell spreading, proliferation, and migration in response to platelet derived growth factor-BB (PDGF, a growth factor known to promote fibrosis) stimulation. Our results showed that ß3-/- cardiac fibroblasts exhibited a significant reduction in cell-matrix adhesion, cell spreading, proliferation and migration. In addition, the activation of PDGF receptor associated tyrosine kinase and non-receptor tyrosine kinase Pyk2, upon PDGF stimulation were impaired in ß3-/- cells. Adenoviral expression of a dominant negative form of Pyk2 (Y402F) resulted in reduced accumulation of fibronectin. These results indicate that ß3 integrin-mediated Pyk2 signaling in cardiac fibroblasts plays a critical role in PO-induced cardiac fibrosis.