Karsten Peppel

Potentiation of beta-adrenergic signaling by adenoviral-mediated gene transfer in adult rabbit ventricular myocytes.

Our laboratory has been testing the hypothesis that genetic modulation of the beta-adrenergic signaling cascade can enhance cardiac function. We have previously shown that transgenic mice with cardiac overexpression of either the human beta2-adrenergic receptor (beta2AR) or an inhibitor of the beta-adrenergic receptor kinase (betaARK), an enzyme that phosphorylates and uncouples agonist-bound receptors, have increased myocardial inotropy. We now have created recombinant adenoviruses encoding either the beta2AR (Adeno-beta2AR) or a peptide betaARK inhibitor (consisting of the carboxyl terminus of betaARK1, Adeno-betaARKct) and tested their ability to potentiate beta-adrenergic signaling in cultured adult rabbit ventricular myocytes. As assessed by radioligand binding, Adeno-beta2AR infection led to approximately 20-fold overexpression of beta-adrenergic receptors. Protein immunoblots demonstrated the presence of the Adeno-betaARKct transgene. Both transgenes significantly increased isoproterenol-stimulated cAMP as compared to myocytes infected with an adenovirus encoding beta-galactosidase (Adeno-betaGal) but did not affect the sarcolemmal adenylyl cyclase response to Forskolin or NaF. beta-Adrenergic agonist-induced desensitization was significantly inhibited in Adeno-betaARKct-infected myocytes (16+/-2%) as compared to Adeno-betaGal-infected myocytes (37+/-1%, P < 0.001). We conclude that recombinant adenoviral gene transfer of the beta2AR or an inhibitor of betaARK-mediated desensitization can potentiate beta-adrenergic signaling.

Graft-Extrinsic Cells Predominate in Vein Graft Arterialization

Objective—Vein graft disease involves neointimal smooth muscle cells, the origins of which are unclear. This study sought to characterize and quantitate vein graft infiltration by cells extrinsic to the graft in a mouse model of vein graft disease. Methods and Results—Inferior vena cava-to-carotid artery interposition grafting between C57Bl/6 and congenic &bgr;-galactosidase–expressing ROSA26 mice was performed. Vein grafts were harvested 6 weeks postoperatively and stained with X-gal. More than 60% of neointimal cells derived from the recipient, and 50% of these cells expressed smooth muscle &agr;-actin. The distribution of donor and recipient-derived cells within this vein graft wall layer was distinctly focal, consistent with focal infiltration and expansion of progenitor cells. When bone marrow transplantation with congenic green fluorescent protein (GFP)-expressing cells was used in vein graft recipients 1 month before surgery, abundant GFP-expressing cells appeared in the media, but not the neointima, of mature grafts. Endothelial cells in mature grafts derived from graft-intrinsic and graft-extrinsic sources and were, in part, of bone marrow origin. Conclusions—Cells extrinsic to the graft, including bone marrow-derived cells, predominate during vein graft remodeling.

Expression of Tumor Necrosis Factor Receptor-1 in Arterial Wall Cells Promotes Atherosclerosis

Objective—Mechanisms by which tumor necrosis factor-α (TNF) contributes to atherosclerosis remain largely obscure. We therefore sought to determine the role of the arterial wall TNF receptor-1 (TNFR1) in atherogenesis. Methods and Results—Carotid artery-to-carotid artery interposition grafting was performed with tnfr1−/− and congenic (C57Bl/6) wild-type (WT) mice as graft donors, and congenic chow-fed apolipoprotein E-deficient mice as recipients. Advanced atherosclerotic graft lesions developed within 8 weeks, and had 2-fold greater area in WT than in tnfr1−/− grafts. While the prevalence of specific atheroma cells was equivalent in WT and tnfr1−/− grafts, the overall abundance of cells was substantially greater in WT grafts. WT grafts demonstrated greater MCP-1, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 expression at both early and late time points, and proliferating cell nuclear antigen expression at early time points. Aortic atherosclerosis was also reduced in 14-month-old apoe−/−/tnfr1−/− mice, as compared with cognate apoe−/− mice. In coculture with activated macrophages, smooth muscle cells expressing the TNFR1 demonstrated enhanced migration and reduced scavenger receptor activity. Conclusions—TNFR1 signaling, just in arterial wall cells, contributes to the pathogenesis of atherosclerosis by enhancing arterial wall chemokine and adhesion molecule expression, as well as by augmenting medial smooth muscle cell proliferation and migration.

Phosphorylation of the Platelet-derived Growth Factor Receptor-β and Epidermal Growth Factor Receptor by G Protein-coupled Receptor Kinase-2 MECHANISMS FOR SELECTIVITY OF DESENSITIZATION

Accumulating evidence suggests that receptor protein-tyrosine kinases, like the platelet-derived growth factor receptor-β (PDGFRβ) and epidermal growth factor receptor (EGFR), may be desensitized by serine/threonine kinases. One such kinase, G protein-coupled receptor kinase-2 (GRK2), is known to mediate agonist-dependent phosphorylation and desensitization of multiple heptahelical receptors. In testing whether GRK2 could phosphorylate and desensitize the PDGFRβ, we first found by phosphoamino acid analysis that cells expressing GRK2 could serine-phosphorylate the PDGFRβ in an agonist-dependent manner. Augmentation or inhibition of GRK2 activity in cells, respectively, reduced or enhanced tyrosine phosphorylation of the PDGFRβ but not the EGFR. Either overexpressed in cells or as a purified protein, GRK2 demonstrated agonist-promoted serine phosphorylation of the PDGFRβ and, unexpectedly, the EGFR as well. Because GRK2 did not phosphorylate a kinase-dead (K634R) PDGFRβ mutant, GRK2-mediated PDGFRβ phosphorylation required receptor tyrosine kinase activity, as does PDGFRβ ubiquitination. Agonist-induced ubiquitination of the PDGFRβ, but not the EGFR, was enhanced in cells overexpressing GRK2. Nevertheless, GRK2 overexpression did not augment PDGFRβ down-regulation. Like the vast majority of GRK2 substrates, the PDGFRβ, but not the EGFR, activated heterotrimeric G proteins allosterically in membranes from cells expressing physiologic protein levels. We conclude that GRK2 can phosphorylate and desensitize the PDGFRβ, perhaps through mechanisms related to receptor ubiquitination. Specificity of GRK2 for receptor protein-tyrosine kinases, expressed at physiologic levels, may be determined by the ability of these receptors to activate heterotrimeric G proteins, among other factors.

Ex Vivo Adenovirus-Mediated Gene Transfer To The Adult Rat Heart

OBJECTIVE The ability to transfer genes to adult myocardium may have therapeutic implications for cardiac transplantation. We investigated the feasibility of adenovirus-mediated transfer of marker genes LacZ and Luciferase, as well as the potentially therapeutic gene of the human beta2-adrenergic receptor in a rat heterotopic heart transplant model. METHODS Donor hearts were flushed with 10(12) total viral particles of one of three transgenes. Hearts were harvested at various time points after transplantation. LacZ-treated hearts were assessed by histologic staining and Luciferase-treated hearts were assayed for specific luminescence activity. Hearts treated with beta2-adrenergic receptor underwent radioligand binding assays and immunohistochemistry with the use of an antibody specific for the human beta2-adrenergic receptor. RESULTS LacZ hearts revealed diffuse myocyte staining as opposed to none within controls at 5 days. Luciferase hearts demonstrated a mean activity of 970,000 +/- 220,000 arbitrary light units versus 500 +/- 200 for the controls (p = 0.001). Total beta2-adrenergic receptor densities (fmol/mg membrane protein) for hearts that received the beta2-adrenergic receptor transgene at 3, 5, 7, 10, and 14 days after infection were as follows: right ventricle, 488.5 +/- 126.8, 519.4 +/- 81.8,* 477.1 +/- 51.8,* 183.0 +/- 6.5,* and 82.7 +/- 19.1; left ventricle, 511.0 +/- 167.6, 1206.4 +/- 321.8,* 525.3 +/- 188.7, 183.5 +/- 18.6,* and 75.9 +/- 15.2 (*p < 0.05 vs control value of 75.6 +/- 6.4). Immunohistochemical analysis revealed diffuse staining of varying intensity within myocardial sarcolemmal membranes. CONCLUSIONS We conclude that global overexpression of different transgenes is possible during cardiac transplantation and, ultimately, adenovirus-mediated gene transfer may provide a unique opportunity for genetic manipulation of the donor organ, potentially enhancing its function.

Targeting Gβγ Signaling to Inhibit Prostate Tumor Formation and Growth

Prostate cancer starts as androgen-dependent malignancy and responds initially to androgen ablative therapy. Beneficial effects of androgen ablation, however, are often temporary and the cancer reappears as androgen-independent tumor, suggesting the existence of additional factors responsible for progression of the disease. Attention has focused on receptor tyrosine kinases as the growth mediators of androgen-independent prostate cancer; overexpression of epidermal growth factor receptors or their ligand heparin-bound epidermal growth factor, for example, promotes transition to androgen independence. Emerging data demonstrate involvement of another class of cell membrane-anchored receptors, the heterotrimeric guanine-binding (G) protein-coupled receptors (GPCRs) in prostate cancer. In vitro, stimulation of many endogenous GPCRs induces mitogenic signaling and growth of prostate cancer cells. The GPCRs transduce mitogenic signals via activated G proteins in the form of Gα-GTP and Gβγ subunits. Here, we show that expression of a Gβγ inhibitor peptide derived from carboxy terminus of G protein-coupled receptor kinase 2 obliterates serum-regulated prostate cancer cell growth in vitro and prevents prostate tumor formation in vivo. We also demonstrate that inhibition of Gβγ signaling retards growth of existing prostate tumors by inducing cell death. These data establish a central role for heterotrimeric G proteins in prostate cancer and suggest targeted inhibition of Gβγ signaling may serve as specific molecular therapy tool to limit pathologic growth of advanced prostate cancer.

Overexpression of G Protein–Coupled Receptor Kinase-2 in Smooth Muscle Cells Attenuates Mitogenic Signaling via G Protein–Coupled and Platelet-Derived Growth Factor Receptors

BACKGROUND Neointimal hyperplasia involves activation of smooth muscle cells (SMCs) by several G protein-coupled receptor (GPCR) agonists, including endothelin-1, angiotensin II, thrombin, and thromboxane A(2). Signaling of many GPCRs is diminished by GPCR kinase-2 (GRK2). We therefore tested whether overexpression of GRK2 in SMCs could diminish mitogenic signaling elicited by agonists implicated in the pathogenesis of neointimal hyperplasia. METHODS AND RESULTS Overexpression of GRK2 was achieved in primary rabbit aortic SMCs with a recombinant adenovirus. Control SMCs were infected with an empty vector adenovirus. Inositol phosphate responses to endothelin-1, angiotensin II, thrombin agonist peptide, and platelet-derived growth factor (PDGF) were attenuated by 37% to 72% in GRK2-overexpressing cells (P<0.01), but the response to the thromboxane A(2) analogue U46619 was unaffected. GRK2 also inhibited SMC [(3)H]thymidine incorporation stimulated not only by these agonists (by 30% to 60%, P<0.01) but also by 10% FBS (by 35%, P<0. 05). However, GRK2 overexpression had no effect on epidermal growth factor-induced [(3)H]thymidine incorporation. Agonist-induced tyrosine phosphorylation of the PDGF-beta receptor, but not the epidermal growth factor receptor, was reduced in GRK2-overexpressing SMCs. GRK2 overexpression also reduced SMC proliferation in response to endothelin-1, PDGF, and 10% FBS by 62%, 51%, and 29%, respectively (P<0.01), without any effect on SMC apoptosis. CONCLUSIONS GRK2 overexpression diminishes SMC mitogenic signaling and proliferation stimulated by PDGF or agonists for several GPCRs. Gene transfer of GRK2 may therefore be therapeutically useful for neointimal hyperplasia.

The Adaptor Protein β-Arrestin2 Enhances Endocytosis of the Low Density Lipoprotein Receptor

Endocytosis of the low density lipoprotein (LDL) receptor (LDLR) in coated pits employs the clathrin adaptor protein ARH. Similarly, agonist-dependent endocytosis of heptahelical receptors in coated pits employs the clathrin adaptor β-arrestin proteins. In mice fed a high fat diet, we found that homozygous deficiency of β-arrestin2 increased total and LDL plus intermediate-density lipoprotein cholesterol levels by 23 and 53%, respectively (p < 0.05), but had no effect on high density lipoprotein cholesterol levels. We therefore tested whether β-arrestins could affect the constitutive endocytosis of the LDLR. When overexpressed in cells, β-arrestin1 and β-arrestin2 each associated with the LDLR, as judged by co-immunoprecipitation, and augmented LDLR endocytosis by ∼70%, as judged by uptake of fluorescent LDL. However, physiologic expression levels of only β-arrestin2, and not β-arrestin1, enhanced endogenous LDLR endocytosis (by 65%) in stably transfected β-arrestin1/β-arrestin2 double-knockout mouse embryonic fibroblasts (MEFs). Concordantly, when RNA interference was used to suppress expression of β-arrestin2, but not β-arrestin1, LDLR endocytosis was reduced. Moreover, β-arrestin2–/– MEFs demonstrated LDLR endocytosis that was 50% less than cognate wild type MEFs. In fusion protein pull-down assays, β-arrestin2 bound to the LDLR cytoplasmic tail stoichiometrically, and binding was abolished by mutation of LDLR Tyr807 to Ala. Mutation of LDLR cytoplasmic tail Ser833 to Asp enhanced both the affinity of LDLR fusion protein binding to β-arrestin2, and the efficiency of LDLR endocytosis in cells expressing β-arrestin2 physiologically. We conclude that β-arrestin2 can bind to and enhance endocytosis of the LDLR, both in vitro and in vivo, and may thereby influence lipoprotein metabolism.

Overexpression of wild-type Gαi-2 suppresses β-adrenergic signaling in cardiac myocytes

The role of Gαi‐2 overexpression in desensitization of β‐adrenergic signaling in heart failure is controversial. An adenovirus‐based approach was used to investigate whether overexpression of Gαi‐2 impairs β‐adrenergic stimulation of adenylyl cyclase (AC) activity and cAMP levels in neonatal rat cardiac myocytes (NRCM) and cell shortening of adult rat ventricular myocytes (ARVM). Infection of NRCM with Ad5Gαi‐2 increased Gαi‐2 by 50–600% in a virus dose‐dependent manner. Overexpression was paralleled by suppression of GTP‐ and isoprenaline‐stimulated AC by 10–72% (P<0.001) in a PTX‐sensitive manner. Isoprenaline‐stimulated shortening of Ad5Gαi‐2‐infected ARVM was attenuated by 34% (P<0.01). Ad5Gαi‐2/GFP (Gαi‐2, green fluorescent protein; bicistronic) was constructed to monitor transfection homogeneity and target Gαi‐2 overexpression to levels found in heart failure. At Gαi‐2 levels of 93% above control, isoprenaline‐stimulated AC activity and cAMP levels were reduced by 17% and 40% (P<0.02), respectively. β1‐ and β2‐adrenergic stimulation was reduced similarly. Our results suggest that (a) the Gαi‐2 system exhibits tonic inhibition of stimulated AC in cardiac myocytes, (b) Gαi‐2‐mediated inhibition is concentration‐dependent and occurs at Gαi‐2 levels seen in heart failure, and (c) Gαi‐2‐mediated inhibition affects both β1‐ and β2‐adrenergic stimulation of AC. The data argue for an important, independent role of the Gαi‐2 increase in heart failure.

Vein Graft Neointimal Hyperplasia Is Exacerbated by Tumor Necrosis Factor Receptor-1 Signaling in Graft-Intrinsic Cells

Objective—Vein graft remodeling and neointimal hyperplasia involve inflammation, graft-intrinsic cells, and recruitment of vascular progenitor cells. We sought to examine if the inflammatory cytokine tumor necrosis factor (TNF) affects vein graft remodeling via its p55 TNF receptor-1 (p55). Methods and Results—Inferior vena cava-to-carotid artery interposition grafting was performed between p55−/− and congenic (C57Bl/6) wild-type (WT) mice. Immunofluorescence revealed TNF in early (2-week) vein grafts. Six weeks postoperatively, luminal and medial areas were indistinguishable among all vein graft groups. However, neointimal area was reduced in p55−/− grafts: by 40% in p55−/− grafts placed in p55−/− recipients, and by 21% in p55−/− grafts placed in WT recipients, compared with WT grafts in WT recipients (P<0.05). In 2-week-old vein grafts, p55 deficiency reduced intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and monocyte chemoattractant protein-1 expression by 50% to 60%, and increased the extent of graft endothelialization. In vitro, TNF promoted chemokine expression and [3H]thymidine incorporation in vascular smooth muscle cells (SMCs) from WT, but not from p55−/− mice. However, responses of WT and p55−/− SMCs to other growth factors were equivalent. Conclusions—Signaling via p55, in vein graft-intrinsic cells, contributes to the pathogenesis of vein graft neointimal hyperplasia.