A systems pharmacology model for gene therapy in sickle cell disease.

We developed a mathematical model for autologous stem cell therapy to cure sickle cell disease (SCD). Experimental therapies using this approach seek to engraft stem cells containing a curative gene. These stem cells are expected to produce a lifelong supply of red blood cells (RBCs) containing an anti-sickling hemoglobin.

Gene Therapy as the New Frontier for Sickle Cell Disease.

Sickle Cell Disease (SCD) is one of the most common monogenic disorders caused by a point mutation in the β-globin gene. This mutation results in polymerization of hemoglobin (Hb) under reduced oxygenation conditions, causing rigid sickle-shaped RBCs and hemolytic anemia. This clearly defined fundamental molecular mechanism makes SCD a prototypical target for precision therapy.

S100A6 Regulates Endothelial Cell Cycle Progression by Attenuating Antiproliferative Signal Transducers and Activators of Transcription 1 Signaling.

Objective: S100A6, a member of the S100 protein family, has been described as relevant for cell cycle entry and progression in endothelial cells. The molecular mechanism conferring S100A6's proliferative actions, however, remained elusive.

Approach And Results: Originating from the clinically relevant observation of enhanced S100A6 protein expression in proliferating endothelial cells in remodeling coronary and carotid arteries, our study unveiled S100A6 as a suppressor of antiproliferative signal transducers and activators of transcription 1 signaling.

Cardiomyocytes, endothelial cells and cardiac fibroblasts: S100A1's triple action in cardiovascular pathophysiology.

Over the past decade, basic and translational research delivered comprehensive evidence for the relevance of the Ca(2+)-binding protein S100A1 in cardiovascular diseases. Aberrant expression levels of S100A1 surfaced as molecular key defects, driving the pathogenesis of chronic heart failure, arterial and pulmonary hypertension, peripheral artery disease and disturbed myocardial infarction healing. Loss of intracellular S100A1 renders entire Ca(2+)-controlled networks dysfunctional, thereby leading to cardiomyocyte failure and endothelial dysfunction.

S100A1 DNA-based Inotropic Therapy Protects Against Proarrhythmogenic Ryanodine Receptor 2 Dysfunction.

Restoring expression levels of the EF-hand calcium (Ca(2+)) sensor protein S100A1 has emerged as a key factor in reconstituting normal Ca(2+) handling in failing myocardium. Improved sarcoplasmic reticulum (SR) function with enhanced Ca(2+) resequestration appears critical for S100A1's cyclic adenosine monophosphate-independent inotropic effects but raises concerns about potential diastolic SR Ca(2+) leakage that might trigger fatal arrhythmias. This study shows for the first time a diminished interaction between S100A1 and ryanodine receptors (RyR2s) in experimental HF.

S100A1 is released from ischemic cardiomyocytes and signals myocardial damage via Toll-like receptor 4.

Members of the S100 protein family have been reported to function as endogenous danger signals (alarmins) playing an active role in tissue inflammation and repair when released from necrotic cells. Here, we investigated the role of S100A1, the S100 isoform with highest abundance in cardiomyocytes, when released from damaged cardiomyocytes during myocardial infarction (MI). Patients with acute MI showed significantly increased S100A1 serum levels.

Induction of microRNA-138 by pro-inflammatory cytokines causes endothelial cell dysfunction.

Exposure to pro-inflammatory cytokines, such as Angiotensin II, endothelin-1 or TNF leads to endothelial dysfunction, characterized by the reduced production of nitric oxide via endothelial nitric oxide synthase (eNOS). We recently identified the Ca(2+) binding protein S100A1 as an essential factor required for eNOS activity. Here we report that pro-inflammatory cytokines down-regulate expression of S100A1 in primary human microvascular endothelial cells (HMVECs) via induction of microRNA-138 (miR-138), in a manner that depends on the stabilization of HIF1-α.

MicroRNA-138 regulates hypoxia-induced endothelial cell dysfunction by targeting S100A1.

The Ca(2+) sensor S100A1 is essential for proper endothelial cell (EC) nitric oxide (NO) synthase (eNOS) activation. S100A1 levels are greatly reduced in primary human microvascular ECs subjected to hypoxia, rendering them dysfunctional. However mechanisms that regulate S100A1 levels in ECs are unknown.

S100A1 deficiency impairs postischemic angiogenesis via compromised proangiogenic endothelial cell function and nitric oxide synthase regulation.

Rationale: Mice lacking the EF-hand Ca2+ sensor S100A1 display endothelial dysfunction because of distorted Ca2+ -activated nitric oxide (NO) generation.

Objective: To determine the pathophysiological role of S100A1 in endothelial cell (EC) function in experimental ischemic revascularization.

Methods And Results: Patients with chronic critical limb ischemia showed almost complete loss of S100A1 expression in hypoxic tissue.

G protein-coupled receptor kinase-5 attenuates atherosclerosis by regulating receptor tyrosine kinases and 7-transmembrane receptors.

Objective: G protein-coupled receptor kinase-5 (GRK5) is a widely expressed Ser/Thr kinase that regulates several atherogenic receptors and may activate or inhibit nuclear factor-κB (NF-κB). This study sought to determine whether and by what mechanisms GRK5 affects atherosclerosis.

Methods And Results: Grk5(-/-)/Apoe(-/-) mice developed 50% greater aortic atherosclerosis than Apoe(-/-) mice and demonstrated greater proliferation of macrophages and smooth muscle cells (SMCs) in atherosclerotic lesions.

Myocardial injury after ischemia-reperfusion in mice deficient in Akt2 is associated with increased cardiac macrophage density.

Akt2 protein kinase has been shown to promote cell migration and actin polymerization in several cell types, including macrophages. Because migrating macrophages constitute an important inflammatory response after myocardial ischemia, we determined cardiac macrophage expression after ischemia-reperfusion (I/R) injury and cryo-injury in mice lacking Akt2 (Akt2-KO). At 7 days post-I/R, Akt2-KO cardiac tissues showed an increase in immunohistochemical staining for macrophage markers (Galectin 3 and F4/80) compared with wild-type (WT) mice, indicating macrophage density was increased in the injured Akt2-KO myocardium.

The inotropic peptide βARKct improves βAR responsiveness in normal and failing cardiomyocytes through G(βγ)-mediated L-type calcium current disinhibition.

Rationale: The G(βγ)-sequestering peptide β-adrenergic receptor kinase (βARK)ct derived from the G-protein-coupled receptor kinase (GRK)2 carboxyl terminus has emerged as a promising target for gene-based heart failure therapy. Enhanced downstream cAMP signaling has been proposed as the underlying mechanism for increased β-adrenergic receptor (βAR) responsiveness. However, molecular targets mediating improved cardiac contractile performance by βARKct and its impact on G(βγ)-mediated signaling have yet to be fully elucidated.

Impaired neoangiogenesis in β₂-adrenoceptor gene-deficient mice: restoration by intravascular human β₂-adrenoceptor gene transfer and role of NFκB and CREB transcription factors.

Background And Purpose: There is much evidence supporting the role of β₂-adrenoceptors (β₂AR) in angiogenesis but the mechanisms underlying their effects have not been elucidated. Hence, we studied post-ischaemic angiogenesis in the hindlimb (HL) of β₂AR knock-out mice (β₂AR-/-) in vivo and explored possible molecular mechanisms in vitro.

Experimental Approach: Femoral artery resection (FAR) was performed in wild-type and β₂AR-/- mice and adaptive responses to chronic HL ischaemia were explored; blood flow was measured by ultrasound and perfusion of dyed beads, bone rarefaction, muscle fibrosis and skin thickness were evaluated by immunoflourescence and morphometric analysis.

Human umbilical cord blood endothelial progenitor cells decrease vein graft neointimal hyperplasia in SCID mice.

Aims: Vein graft endothelial damage is a key step in the development of neointimal hyperplasia, leading to vein graft failure. We sought to determine whether exogenous endothelial progenitor cells could promote vein graft re-endothelialization, and thereby ameliorate neointimal hyperplasia.

Methods And Results: Carotid artery interposition grafting was performed with syngeneic inferior vena cavae in mice with severe combined immunodeficiency (SCID).

Aging-related atherosclerosis is exacerbated by arterial expression of tumor necrosis factor receptor-1: evidence from mouse models and human association studies.

Aging is believed to be among the most important contributors to atherosclerosis, through mechanisms that remain largely obscure. Serum levels of tumor necrosis factor (TNF) rise with aging and have been correlated with the incidence of myocardial infarction. We therefore sought to determine whether genetic variation in the TNF receptor-1 gene (TNFR1) contributes to aging-related atherosclerosis in humans and whether Tnfr1 expression aggravates aging-related atherosclerosis in mice.

Beta-arrestins regulate atherosclerosis and neointimal hyperplasia by controlling smooth muscle cell proliferation and migration.

Atherosclerosis and arterial injury-induced neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins beta-arrestin1 and -2 might regulate this pathological process. Deficiency of beta-arrestin2 in ldlr(-/-) mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that beta-arrestin2 promotes atherosclerosis through effects on SMCs.

Tumor necrosis factor receptor-2 signaling attenuates vein graft neointima formation by promoting endothelial recovery.

Objective: Inflammation appears intricately linked to vein graft arterialization. We have previously shown that tumor necrosis factor (TNF) receptor-1 (TNFR1, p55) signaling augments vein graft neointimal hyperplasia (NH) and remodeling through its effects on vascular smooth muscle cells (SMCs). In this study we examined the role of TNFR2 (p75) signaling in vein graft arterialization.

Substrate specificities of g protein-coupled receptor kinase-2 and -3 at cardiac myocyte receptors provide basis for distinct roles in regulation of myocardial function.

The closely related G protein-coupled receptor kinases GRK2 and GRK3 are both expressed in cardiac myocytes. Although GRK2 has been extensively investigated in terms of regulation of cardiac beta-adrenergic receptors, the substrate specificities of the two GRK isoforms at G protein-coupled receptors (GPCR) are poorly understood. In this study, the substrate specificities of GRK2 and GRK3 at GPCRs that control cardiac myocyte function were determined in fully differentiated adult cardiac myocytes.

Expression of tumor necrosis factor receptor-1 in arterial wall cells promotes atherosclerosis.

Objective: Mechanisms by which tumor necrosis factor-alpha (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.

Regulation of the platelet-derived growth factor receptor-beta by G protein-coupled receptor kinase-5 in vascular smooth muscle cells involves the phosphatase Shp2.

Smooth muscle cell (SMC) proliferation and migration are substantially controlled by the platelet-derived growth factor receptor-beta (PDGFRbeta), which can be regulated by the Ser/Thr kinase G protein-coupled receptor kinase-2 (GRK2). In mouse aortic SMCs, however, we found that prolonged PDGFRbeta activation engendered down-regulation of GRK5, but not GRK2; moreover, GRK5 and PDGFRbeta were coordinately up-regulated in SMCs from atherosclerotic arteries. With SMCs from GRK5 knock-out and cognate wild type mice (five of each), we found that physiologic expression of GRK5 increased PDGF-promoted PDGFRbeta seryl phosphorylation by 3-fold and reduced PDGFRbeta-promoted phosphoinositide hydrolysis, thymidine incorporation, and overall PDGFRbeta tyrosyl phosphorylation by approximately 35%.

The platelet-derived growth factor receptor-beta phosphorylates and activates G protein-coupled receptor kinase-2. A mechanism for feedback inhibition.

G protein-coupled receptor kinase-2 (GRK2) serine-phosphorylates the platelet-derived growth factor receptor-beta (PDGFRbeta), and thereby diminishes signaling by the receptor. Because activation of GRK2 may involve phosphorylation of its N-terminal tyrosines by c-Src, we tested whether the PDGFRbeta itself could tyrosine-phosphorylate and activate GRK2. To do so, we used wild type (WT) and Y857F mutant PDGFRbetas in HEK cells, which lack endogenous PDGFRs.

Activation of vascular smooth muscle cells by TNF and PDGF: overlapping and complementary signal transduction mechanisms.

Objective: Because tumor necrosis factor-alpha (TNF) has been implicated in the pathogenesis of vein graft neointimal hyperplasia, we sought to determine mechanisms by which TNF could induce proliferative and migratory responses in smooth muscle cells (SMCs).

Methods And Results: In rabbit jugulocarotid interposition vein grafts, SMCs expressed TNF as early as four days postoperatively. In rabbit aortic SMCs, TNF and platelet-derived growth factor (PDGF) elicited comparable migration (1.

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.

Phosphorylation of the platelet-derived growth factor receptor-beta by G protein-coupled receptor kinase-2 reduces receptor signaling and interaction with the Na(+)/H(+) exchanger regulatory factor.

G protein-coupled receptor kinase-2 (GRK2) can phosphorylate and desensitize the platelet-derived growth factor receptor-beta (PDGFRbeta) in heterologous cellular systems. To determine whether GRK2 regulates the PDGFRbeta in physiologic systems, we examined PDGFRbeta signaling in mouse embryonic fibroblasts from GRK2-null and cognate wild type mice. To discern a mechanism by which GRK2-mediated phosphorylation can desensitize the PDGFRbeta, but not the epidermal growth factor receptor (EGFR), we investigated effects of GRK2-mediated phosphorylation on the association of the PDGFRbeta with the Na(+)/H(+) exchanger regulatory factor (NHERF), a protein shown to potentiate dimerization of the PDGFRbeta, but not the EGFR.

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 beta-galactosidase-expressing ROSA26 mice was performed.