Optimized method for isolating highly purified and functional porcine aortic endothelial and smooth muscle cells.

Numerous protocols exist for isolating aortic endothelial and smooth muscle cells from small animals. However, establishing a protocol for isolating pure cell populations from large animal vessels that are more elastic has been challenging. We developed a simple sequential enzymatic approach to isolate highly purified populations of porcine aortic endothelial and smooth muscle cells. The lumen of a porcine aorta was filled with 25 U/ml dispase solution and incubated at 37°C to dissociate the endothelial cells. The smooth muscle cells were isolated by mincing the tunica media of the treated aorta and incubating the pieces in 0.2% and then 0.1% collagenase type I solution. The isolated endothelial cells stained positive for von Willebrand factor, and 97.2% of them expressed CD31. Early and late passage endothelial cells had a population doubling time of 38 hr and maintained a capacity to take up DiI-Ac-LDL and form tubes in Matrigel®. The isolated smooth muscle cells stained highly positive for alpha-smooth muscle actin, and an impurities assessment showed that only 1.8% were endothelial cells. Population doubling time for the smooth muscle cells was ∼70 hr at passages 3 and 7; and the cells positively responded to endothelin-1, as shown by a 66% increase in the intracellular calcium level. This simple protocol allows for the isolation of highly pure populations of endothelial and smooth muscle cells from porcine aorta that can survive continued passage in culture without losing functionality or becoming overgrown by fibroblasts.

Inhibition of Wnt6 by Sfrp2 regulates adult cardiac progenitor cell differentiation by differential modulation of Wnt pathways.

Wnt signaling has recently emerged as an important regulator of cardiac progenitor cell proliferation and differentiation, but the exact mechanisms by which Wnt signaling modulates these effects are not known. Understanding these mechanisms is essential for advancing our knowledge of cardiac progenitor cell biology and applying this knowledge to enhance cardiac therapy. Here, we explored the effects of Sfrp2, a canonical Wnt inhibitor, in adult cardiac progenitor cell (CPC) differentiation and investigated the molecular mechanisms involved. Our data show that Sfrp2 treatment can promote differentiation of CPCs after ischemia-reperfusion injury. Treatment of CPCs with Sfrp2 inhibited CPC proliferation and primed them for cardiac differentiation. Sfrp2 binding to Wnt6 and inhibition of Wnt6 canonical pathway was essential for the inhibition of CPC proliferation. This inhibition of Wnt6 canonical signaling by Sfrp2 was important for activation of the non-canonical Wnt/Planar Cell Polarity (PCP) pathway through JNK, which in turn induced expression of cardiac transcription factors and CPC differentiation. Taken together, these results demonstrate a novel role of Sfrp2 and Wnt6 in regulating the dynamic process of CPC proliferation and differentiation, as well as providing new insights into the mechanisms of Wnt signaling in cardiac differentiation.

C3orf58, a novel paracrine protein, stimulates cardiomyocyte cell-cycle progression through the PI3K-AKT-CDK7 pathway.

RATIONALE: The regenerative capacity of the heart is markedly diminished shortly after birth, coinciding with overall withdrawal of cardiomyocytes from cell cycle. Consequently, the adult mammalian heart has limited capacity to regenerate after injury. The discovery of factors that can induce cardiomyocyte proliferation is, therefore, of high interest and has been the focus of extensive investigation throughout the past years. OBJECTIVE: We have recently identified C3orf58 as a novel hypoxia and Akt induced stem cell factor (HASF) secreted from mesenchymal stem cells, which can promote cardiac repair through cytoprotective mechanisms. Here, we tested the hypothesis that HASF can also contribute to cardiac regeneration by stimulating cardiomyocyte division and proliferation. METHODS AND RESULTS: Neonatal ventricular cardiomyocytes were stimulated in culture for 7 days with purified recombinant HASF protein. Compared with control untreated cells, HASF-treated neonatal cardiomyocytes exhibited 60% increase in DNA synthesis as measured by bromodeoxyuridine incorporation. These results were confirmed by immunofluorescence confocal microscopy showing a 50% to 100% increase in the number of cardiomyocytes in the mitotic and cytokinesis phases. Importantly, in vivo cardiac overexpression of HASF in a transgenic mouse model resulted in enhanced level of DNA synthesis and cytokinesis in neonatal and adult cardiomyocytes. These proliferative effects were modulated by a phosphoinositide 3-kinase-protein kinase B-cycle-dependent kinase 7 pathway as revealed by the use of phosphoinositide 3-kinase -pathway-specific inhibitors and silencing of the Cdk7 gene. CONCLUSIONS: Our studies support the hypothesis that HASF induces cardiomyocyte proliferation via a phosphoinositide 3-kinase-protein kinase B-cycle-dependent kinase 7 pathway. The implications of this finding may be significant for cardiac regeneration biology and therapeutics.

Dynamic denitrosylation via S-nitrosoglutathione reductase regulates cardiovascular function.

Although protein S-nitrosylation is increasingly recognized as mediating nitric oxide (NO) signaling, roles for protein denitrosylation in physiology remain unknown. Here, we show that S-nitrosoglutathione reductase (GSNOR), an enzyme that governs levels of S-nitrosylation by promoting protein denitrosylation, regulates both peripheral vascular tone and ß-adrenergic agonist-stimulated cardiac contractility, previously ascribed exclusively to NO/cGMP. GSNOR-deficient mice exhibited reduced peripheral vascular tone and depressed ß-adrenergic inotropic responses that were associated with impaired ß-agonist-induced denitrosylation of cardiac ryanodine receptor 2 (RyR2), resulting in calcium leak. These results indicate that systemic hemodynamic responses (vascular tone and cardiac contractility), both under basal conditions and after adrenergic activation, are regulated through concerted actions of NO synthase/GSNOR and that aberrant denitrosylation impairs cardiovascular function. Our findings support the notion that dynamic S-nitrosylation/denitrosylation reactions are essential in cardiovascular regulation.

HASF is a stem cell paracrine factor that activates PKC epsilon mediated cytoprotection.

Despite advances in the treatment of acute tissue ischemia significant challenges remain in effective cytoprotection from ischemic cell death. It has been documented that injected stem cells, such as mesenchymal stem cells (MSCs), can confer protection to ischemic tissue through the release of paracrine factors. The study of these factors is essential for understanding tissue repair and the development of new therapeutic approaches for regenerative medicine. We have recently shown that a novel factor secreted by MSCs, which we called HASF (Hypoxia and Akt induced Stem cell Factor), promotes cardiomyocyte proliferation. In this study we show that HASF has a cytoprotective effect on ischemia induced cardiomyocyte death. We assessed whether HASF could potentially be used as a therapeutic agent to prevent the damage associated with myocardial infarction. In vitro treatment of cardiomyocytes with HASF protein resulted in decreased apoptosis; TUNEL positive nuclei were fewer in number, and caspase activation and mitochondrial pore opening were inhibited. Purified HASF protein was injected into the heart immediately following myocardial infarction. Heart function was found to be comparable to sham operated animals one month following injury and fibrosis was significantly reduced. In vivo and in vitro HASF activated protein kinase C ε (PKCε). Inhibition of PKCε blocked the HASF effect on apoptosis. Furthermore, the beneficial effects of HASF were lost in mice lacking PKCε. Collectively these results identify HASF as a protein of significant therapeutic potential, acting in part through PKCε.

Deficient ryanodine receptor S-nitrosylation increases sarcoplasmic reticulum calcium leak and arrhythmogenesis in cardiomyocytes.

Altered Ca(2+) homeostasis is a salient feature of heart disease, where the calcium release channel ryanodine receptor (RyR) plays a major role. Accumulating data support the notion that neuronal nitric oxide synthase (NOS1) regulates the cardiac RyR via S-nitrosylation. We tested the hypothesis that NOS1 deficiency impairs RyR S-nitrosylation, leading to altered Ca(2+) homeostasis. Diastolic Ca(2+) levels are elevated in NOS1(-/-) and NOS1/NOS3(-/-) but not NOS3(-/-) myocytes compared with wild-type (WT), suggesting diastolic Ca(2+) leakage. Measured leak was increased in NOS1(-/-) and NOS1/NOS3(-/-) but not in NOS3(-/-) myocytes compared with WT. Importantly, NOS1(-/-) and NOS1/NOS3(-/-) myocytes also exhibited spontaneous calcium waves. Whereas the stoichiometry and binding of FK-binding protein 12.6 to RyR and the degree of RyR phosphorylation were not altered in NOS1(-/-) hearts, RyR2 S-nitrosylation was substantially decreased, and the level of thiol oxidation increased. Together, these findings demonstrate that NOS1 deficiency causes RyR2 hyponitrosylation, leading to diastolic Ca(2+) leak and a proarrhythmic phenotype. NOS1 dysregulation may be a proximate cause of key phenotypes associated with heart disease.

Cardiac nitric oxide synthase-1 localization within the cardiomyocyte is accompanied by the adaptor protein, CAPON.

The mechanism(s) regulating nitric oxide synthase-1 (NOS1) localization within the cardiac myocyte in health and disease remains unknown. Here we tested the hypothesis that the PDZ-binding domain interaction between CAPON (carboxy-terminal PDZ ligand of NOS1), a NOS1 adaptor protein and NOS1, contribute to NOS1 localization in specific organelles within cardiomyocytes. Ventricular cardiomyocytes and whole heart homogenates were isolated from sham and post-myocardial infarction (MI) wild-type (C57BL/6) and NOS1(-/-) female mice for quantification of CAPON protein expression levels. NOS1, CAPON, xanthine oxidoreductase and Dexras1, a CAPON binding partner, were all present and enriched in isolated cardiac sarcoplasmic reticulum (SR) fractions. CAPON co-immunoprecipitated with the mu and alpha isoforms of NOS1 in whole heart lysates, and co-localization of CAPON and NOS1 was demonstrated in the SR and mitochondria with dual immuno-gold electron microscopy. Following MI, CAPON and NOS1 both redistributed to caveolae and colocalized with caveolin-3. In addition, following MI, expression level of CAPON remained unchanged and Dexras1 was reduced, CAPON binding to xanthine oxidoreductase was augmented and the plasma membrane calcium ATPase (PMCA) increased. In NOS1 deficient myocytes, CAPON abundance in the SR was reduced, and redistribution to caveolae and PMCA binding after MI was absent. Together these findings support the hypothesis that NOS1 redistribution in injured myocardium requires the formation of a complex with the PDZ adaptor protein CAPON.

Nitric oxide and cardiobiology-methods for intact hearts and isolated myocytes.

The cross talk between reactive oxygen species (ROS) and reactive nitrogen species (RNS) plays a pivotal role in the regulation of myocardial and vascular function. Both nitric oxide and redox-based signaling involve the posttranslational modification of proteins through S-nitrosylation and oxidation of specific cysteine residues. Disruption of this cross talk between ROS and RNS contributes to the pathogenesis of heart failure. Therefore, the elucidation of these complex chemical interactions may improve our understanding of cardiovascular pathophysiology. This chapter discusses the significant role of spatial confinement of nitric oxide synthases, NADPH oxidase, and xanthine oxidoreductase in the regulation of myocardial excitation-contraction coupling. This chapter describes techniques for assessing oxidative and nitrosative stress. A variety of assays have been developed that quantify S-nitrosylated proteins. Among them, the biotin-switch method directly evaluates endogenously nitrosylated proteins in a reproducible way. Identification of the biotinylated or S-nitrosylated proteins subjected to the biotin-switch assay are described and evaluated with a one-dimensional gel (Western blot) or with the newly developed two-dimensional fluorescence difference gel electrophoresis proteomic analysis. Quantifying the number of free thiols with the monobromobimane assay in a protein of interest allows estimation of cysteine oxidation and, in turn, the state of nitroso-redox balance of effector molecules. In summary, this chapter reviews the biochemical methods that assess the impact of nitroso/redox signaling in the cardiovascular system.

Enantioselective separation and online affinity chromatographic characterization of R,R- and S,S-fenoterol.

BACKGROUND: rac-Fenoterol is a beta2-adrenoceptor agonist (beta2-AR) used in the treatment of asthma. It has two chiral centers and is marketed as a racemic mixture of R,R'- and S,S'-fenoterol (R-F and S-F). Here we report the separation of the R-F and S-F enantiomers and the evaluation of their binding to and activation of the beta2-AR. METHODS: R-F and S-F were separated from the enantiomeric mixture by chiral chromatography and absolute configuration determined by circular dichroism. Beta2-AR binding was evaluated using frontal affinity chromatography with a stationary phase containing immobilized membranes from HEK-293 cells that express human beta2-AR and standard membrane binding studies using the same membranes. The effect of R-F and S-F on cardiomyocyte contractility was also investigated using freshly isolated adult rat cardiomyocytes. RESULTS: Chiral chromatography of rac-fenoterol yielded separated peaks with an enantioselectivity factor of 1.21. The less retained peak was assigned the absolute configuration of S-F and the more retained peak R-F. Frontal chromatography using membrane-bound beta2-AR as the stationary phase and rac-3H-fenoterol as a marker ligand showed that addition of increasing concentrations of R-F to the mobile phase produced concentration-dependent decreases in rac-3H-fenoterol retention, while similar addition of S-F produced no change in rac-3H-fenoterol retention. The calculated dissociation constant of R-F was 472 nM and the number of available binding sites 176 pmol/column, which was consistent with the results from the membrane binding study 460 +/- 55 nM (R-F) and 109,000 +/- 10,400 nM (S-F). In the cardiomyocytes, R-F increased maximum contractile response from (265 +/- 11.6)% to (306 +/- 11.8)% of resting cell length (P < 0.05) and reduced EC50 from -7.0 +/- 0.270 to -7.1 +/- 0.2 log[M] (P < 0.05), while S-F had no significant effect. DISCUSSION: Previous studies have shown that rac-fenoterol acts as an apparent beta2-AR/G(s) selective agonist and fully restores diminished beta2-AR contractile response in cardiomyocytes from failing hearts of spontaneously hypertensive rats (SHR). Here we report the separation of the enantiomers of rac-fenoterol and that R-F is the active component of rac-fenoterol. Further evaluation of R-F will determine if it has enhanced selectivity and specificity for beta2-AR/G(s) activation and if it can be used in the treatment of congestive heart failure.

Syntheses of immobilized G protein-coupled receptor chromatographic stationary phases: characterization of immobilized mu and kappa opioid receptors.

Opioid receptors are members of the superfamily of G protein-coupled receptors (GPCRs). Liquid chromatographic stationary phases containing either the human mu or kappa opioid receptor subtypes have been developed to study the binding between the opioid receptors and their ligands. The receptors were obtained from Chinese hamster ovary cells stably transfected with human mu or kappa receptor subtypes. The receptors were isolated through partial solubilization with sodium cholate detergent, and the partially purified receptor complex was immobilized in the phospholipid analogue monolayer of an immobilized artificial membrane liquid chromatographic stationary phase. The resulting phase was packed into a glass column (1.8 x 0.5 (i.d.) cm) and used in the on-line chromatographic determination of drug/ligand binding affinities to the immobilized opioid receptors. Preliminary on-line binding studies employing frontal chromatographic techniques were conducted with the known mu antagonist (naloxone) and a kappa agonist (U69593). The calculated dissociation constants (Kd) were 110 nM for naloxone and 84 nM for U69593. The results indicate that the immobilized receptors retained their ability to specifically bind ligands and were active for 1200 column volumes applied over two weeks. Zonal chromatographic experiments were also conducted, and competitive displacements of the marker ligands were observed. The results suggest that the immobilized opioid receptor stationary phases can be used to qualitatively assess the binding affinities of compounds to the immobilized receptors and represent the first example of the use of immobilized GPCRs in a chromatographic system.

G-protein-coupled receptor chromatographic stationary phases. 2. Ligand-induced conformational mobility in an immobilized beta2-adrenergic receptor.

Membranes from a HEK-293 cell line expressing the beta(2)-adrenergic receptor (beta(2)-AR) have been immobilized on an artificial membrane liquid chromatographic stationary phase. The resulting phase was packed into a glass column (1.8 x 0.5 (i.d.) cm) and used in on-line chromatographic system. Frontal displacement affinity chromatography was used to determine the dissociation constants (K(d)) of CGP 12177A (552.6 nM) and (S)-propranolol (84.3 nM). Zonal displacement chromatography using CGP 12177A as the marker and racemic mixtures of the antagonists nadolol and propranolol demonstrated that the immobilized beta(2)-AR retained its ability to specifically bind these compounds. Similar experiments with (R)- and (S)-propranolol demonstrated that the immobilized receptor retained its enantioselectivity as (S)-propranolol displaced the CGP 12177 marker to a great extent that the (R)-enantiomer. The addition of the agonist butoxamine to the mobile phase increased the retention of the CGP-12177A as did the addition of the agonist fenoterol. These results indicate that the immobilized beta(2)-AR retained its ability to undergo ligand-induced conformational changes. The data from this study suggest that the immobilized beta(2)-AR can be used to screen for ligand binding interactions in both the resting and active states of the receptor.