Tomotake Tokunou

Local Delivery of Protease-Resistant Stromal Cell Derived Factor-1 for Stem Cell Recruitment After Myocardial Infarction

Background— Local delivery of chemotactic factors represents a novel approach to tissue regeneration. However, successful chemokine protein delivery is challenged by barriers including the rapid diffusion of chemokines and cleavage of chemokines by proteases that are activated in injured tissues. Stromal cell–derived factor-1 (SDF-1) is a well-characterized chemokine for attracting stem cells and thus a strong candidate for promoting regeneration. However, SDF-1 is cleaved by exopeptidases and matrix metalloproteinase-2, generating a neurotoxin implicated in some forms of dementia. Methods and Results— We designed a new chemokine called S-SDF-1(S4V) that is resistant to matrix metalloproteinase-2 and exopeptidase cleavage but retains chemotactic bioactivity, reducing the neurotoxic potential of native SDF-1. To deliver S-SDF-1(S4V), we expressed and purified fusion proteins to tether the chemokine to self-assembling peptides, which form nanofibers and allow local delivery. Intramyocardial delivery of S-SDF-1(S4V) after myocardial infarction recruited CXCR4+/c-Kit+ stem cells (46±7 to 119±18 cells per section) and increased capillary density (from 169±42 to 283±27 per 1 mm2). Furthermore, in a randomized, blinded study of 176 rats with myocardial infarction, nanofiber delivery of the protease-resistant S-SDF-1(S4V) improved cardiac function (ejection fraction increased from 34.0±2.5% to 50.7±3.1%), whereas native SDF-1 had no beneficial effects. Conclusions— The combined advances of a new, protease-resistant SDF-1 and nanofiber-mediated delivery promoted recruitment of stem cells and improved cardiac function after myocardial infarction. These data demonstrate that driving chemotaxis of stem cells by local chemokine delivery is a promising new strategy for tissue regeneration.

Cardiac Progenitor Cells and Biotinylated Insulin-Like Growth Factor-1 Nanofibers Improve Endogenous and Exogenous Myocardial Regeneration After Infarction

Background— Cardiac progenitor cells (CPCs) possess the insulin-like growth factor-1 (IGF-1)-IGF-1 receptor system, and IGF-1 can be tethered to self-assembling peptide nanofibers (NF-IGF-1), leading to prolonged release of this growth factor to the myocardium. Therefore, we tested whether local injection of clonogenic CPCs and NF-IGF-1 potentiates the activation and differentiation of delivered and resident CPCs enhancing cardiac repair after infarction. Methods and Results— Myocardial infarction was induced in rats, and untreated infarcts and infarcts treated with CPCs or NF-IGF-1 only and CPCs and NF-IGF-1 together were analyzed. With respect to infarcts exposed to CPCs or NF-IGF-1 alone, combination therapy resulted in a greater increase in the ratio of left ventricular mass to chamber volume and a better preservation of +dP/dt, −dP/dt, ejection fraction, and diastolic wall stress. Myocardial regeneration was detected in all treated infarcts, but the number of newly formed myocytes with combination therapy was 32% and 230% higher than with CPCs and NF-IGF-1, respectively. Corresponding differences in the volume of regenerated myocytes were 48% and 115%. Similarly, the length density of newly formed coronary arterioles with both CPCs and NF-IGF-1 was 73% and 83% greater than with CPCs and NF-IGF-1 alone, respectively. Importantly, activation of resident CPCs by paracrine effects contributed to cardiomyogenesis and vasculogenesis. Collectively, CPCs and NF-IGF-1 therapy reduced infarct size more than CPCs and NF-IGF-1 alone. Conclusions— The addition of nanofiber-mediated IGF-1 delivery to CPC therapy improved in part the recovery of myocardial structure and function after infarction.

Peroxisome Proliferator-Activated Receptor γ Activators Downregulate Angiotensin II Type 1 Receptor in Vascular Smooth Muscle Cells

Background—Peroxisome proliferator-activated receptor γ (PPARγ) activators, such as troglitazone (Tro), not only improve insulin resistance but also suppress the neointimal formation after balloon injury. However, the precise mechanisms have not been determined. Angiotensin II (Ang II) plays crucial roles in the pathogenesis of atherosclerosis, hypertension, and neointimal formation after angioplasty. We examined the effect of PPARγ activators on the expression of Ang II type 1 receptor (AT1-R) in cultured vascular smooth muscle cells (VSMCs). Methods and Results—AT1-R mRNA and AT1-R protein levels were determined by Northern blot analysis and radioligand binding assay, respectively. Natural PPARγ ligand 15-deoxy-Δ12,14-prostaglandin J2, as well as Tro, reduced the AT1-R mRNA expression and the AT1-R protein level. The PPARγ activators also reduced the calcium response of VSMCs to Ang II. PPARγ activators suppressed the AT1-R promoter activity measured by luciferase assay but did not affect the AT1-R mRNA...

Critical Role of Rho-Kinase and MEK/ERK Pathways for Angiotensin II–Induced Plasminogen Activator Inhibitor Type-1 Gene Expression

Abstract—Plasminogen activator inhibitor type-1 (PAI-1) plays an integral role not only in the regulation of fibrinolytic activity but also in the pathogenesis of atherosclerosis and hypertension. We investigated the signaling pathways of angiotensin II (Ang II) leading to PAI-1 gene expression. Ang II increased the PAI-1 mRNA and protein levels in a time- and dose-dependent manner through the Ang II type 1 receptor in vascular smooth muscle cells. PAI-1 gene promoter activity measured by luciferase assay was significantly increased by Ang II. PAI-1 mRNA stability was also increased by Ang II. Ang II-induced PAI-1 mRNA upregulation was inhibited by BAPTA-AM, genistein, and AG1478, suggesting that intracellular calcium, tyrosine kinase, and epidermal growth factor receptor transactivation are involved. Furthermore, PD98059, an inhibitor of extracellular signal-regulated kinase (ERK) kinase (MEK), almost completely suppressed Ang II-induced PAI-1 upregulation. Adenovirus-mediated overexpression of the dominant-negative form of Rho-kinase or Y27632, a Rho-kinase inhibitor, also completely prevented PAI-1 induction by Ang II without affecting Ang II-induced ERK activation. These data suggest that activation of MEK/ERK and Rho-kinase pathways plays a pivotal role in PAI-1 gene upregulation by Ang II. The Rho-kinase pathway may be a novel target to inhibit Ang II signaling, and its inhibition may be useful in the treatment of hypertension as well as atherosclerosis.

Downregulation of Vascular Angiotensin II Type 1 Receptor by Thyroid Hormone

Abstract—Thyroid hormone has a broad effect on cardiovascular system. 3,3′,5-triiodo-l-thyronine (T3), a biologically active form of thyroid hormone, increases cardiac contractility. T3 causes arterial relaxation and reduction of systemic vascular resistance, resulting in an increase in cardiac output. However, the molecular mechanisms of vascular relaxation by T3 are incompletely characterized. We studied the effect of T3 on the angiotensin (Ang) II type 1 receptor (AT1R) expression in vascular smooth muscle cells. T3 dose-dependently decreased expression levels of AT1R mRNA, with a peak at 6 hours of stimulation. Binding assay using [125I]Sar1-Ile8-Ang II revealed that AT1R number was decreased by stimulation with T3 without changing the affinity to Ang II. T3 reduced calcium response of vascular smooth muscle cells to Ang II by 26%. AT1R promoter activity measured by luciferase assay was reduced by 50% after 9 hours of T3 administration. mRNA stability was also decreased by T3. Real-time quantitative reverse transcription–polymerase chain reaction and Western blot analysis revealed that AT1R mRNA and protein were downregulated in the aorta of T3-treated rats. These results suggest that T3 downregulates AT1R expression both at transcriptional and posttranscriptional levels, and attenuates biological function of Ang II. Our results suggest that downregulation of AT1R gene expression may play an important role for T3-induced vascular relaxation.

Apoptosis Induced by Inhibition of Cyclic AMP Response Element–Binding Protein in Vascular Smooth Muscle Cells

Background—The balance between apoptosis and proliferation of vascular smooth muscle cells (VSMCs) is believed to contribute to the vascular remodeling process. Cyclic AMP response element–binding protein (CREB) is a critical transcription factor for the survival of neuronal cells and T lymphocytes. However, the role of CREB in blood vessels is incompletely characterized. Methods and Results—Nuclear staining with Hoechst 33258 or propidium iodine showed an increase in apoptotic cells with activation of caspase-3 in VSMCs infected with adenovirus expressing the dominant-negative form of CREB (AdCREBM1). Basal expression of Bcl-2 and Bcl-2 promoter activity were decreased by infection with AdCREBM1. Immunohistochemistry revealed that CREB was mainly induced and activated in the neointimal &agr;-smooth muscle actin–positive cells of rat carotid artery after balloon injury. Infection with AdCREBM1 suppressed neointimal formation (intima-media ratio) by 33.8% after 14 days of injury, which was accompanied by an increase in apoptosis as indicated by terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling–positive cells and a decrease in bromodeoxyuridine incorporation. Conclusions—These results suggest that CRE-dependent gene transcription might play an important role in the survival and proliferation of VSMCs. CREB might be a novel transcription factor mediating the vascular remodeling process and a potential therapeutic target for atherosclerotic disease.

Cyclic AMP Response Element–Binding Protein Mediates Reactive Oxygen Species–Induced c-fos Expression

Abstract—Although the cyclic AMP response element–binding protein (CREB) plays an important role in the survival of neuronal cells and T lymphocytes, the role of CREB in vascular smooth muscle cells (VSMCs) is incompletely characterized. We examined the role of CREB in VSMCs stimulated with reactive oxygen species. Activation of CREB was examined by Western blot analysis with an antibody that specifically recognizes phosphorylation at serine 133 of CREB, which is a critical marker of activation. Hydrogen peroxide (H2O2) time-dependently induced phosphorylation of CREB, with a peak at 15 minutes. The H2O2-induced phosphorylation of CREB was partially blocked by inhibition of either extracellular signal–regulated protein kinase kinase by PD98059 or of p38 mitogen-activated protein kinase (MAPK) by SB203580. AG1478, an epidermal growth factor receptor (EGFR) inhibitor, suppressed the H2O2-induced phosphorylation of CREB and tyrosine phosphorylation of EGFR. Overexpression of the dominant-negative form of CREB by an adenovirus vector suppressed H2O2-induced c-fos expression. These findings suggest that H2O2 induces CREB phosphorylation through EGFR transactivation and mitogen-activated protein kinase pathways. CREB might be a novel redox-sensitive transcription factor involved in the regulation of VSMC gene expression.

Engineering insulin-like growth factor-1 for local delivery

Insulin‐like growth factor‐1 (IGF‐1) is a small protein that promotes cell survival and growth, often acting over long distances. Although for decades IGF‐1 has been considered to have therapeutic poten tial, systemic side effects of IGF‐1 are significant, and local delivery of IGF‐1 for tissue repair has been a long‐standing challenge. In this study, we designed and purified a novel protein, heparin‐binding IGF‐1 (Xp‐ HB‐IGF‐1), which is a fusion protein of native IGF‐1 with the heparin‐binding domain of heparin‐binding epidermal growth factor‐like growth factor. Xp‐HB‐ IGF‐1 bound selectively to heparin as well as the cell surfaces of 3T3 fibroblasts, neonatal cardiac myocytes and differentiating ES cells. Xp‐HB‐IGF‐1 activated the IGF‐1 receptor and Akt with identical kinetics and dose response, indicating no compromise of biological activ ity due to the heparin‐binding domain. Because carti lage is a proteoglycan‐rich environment and IGF‐1 is a known stimulus for chondrocyte biosynthesis, we then studied the effectiveness of Xp‐HB‐IGF‐1 in cartilage. Xp‐HB‐IGF‐1 was selectively retained by cartilage ex plants and led to sustained chondrocyte proteoglycan biosynthesis compared to IGF‐1. These data show that the strategy of engineering a “long‐distance” growth factor like IGF‐1 for local delivery may be useful for tissue repair and minimizing systemic effects.— Tokunou, T., Miller, R., Patwari, P., Davis, M. E., Segers, V. F. M., Grodzinsky, A. J., Lee, R. T. Engineering insulin‐like growth factor‐1 for local delivery. FASEB J. 22, 1886–1893 (2008)

Reactive Oxygen Species–Mediated Homologous Downregulation of Angiotensin II Type 1 Receptor mRNA by Angiotensin II

Recent studies suggest a crucial role of reactive oxygen species (ROS) for the signaling of angiotensin (Ang) II through Ang II type 1 receptor (AT1-R). However, the role of ROS in the regulation of AT1-R expression has not been explored. In this study, we examined the effect of an antioxidant on the homologous downregulation of AT1-R by Ang II. Ang II (10−6 mol/L) decreased AT1-R mRNA with a peak suppression at 6 hours of stimulation in rat aortic vascular smooth muscle cells. Preincubation of vascular smooth muscle cells with N-acetylcysteine (NAC), a potent antioxidant, almost completely inhibited the Ang II–induced downregulation of AT1-R mRNA. The effect of NAC was due to stabilization of the AT1-R mRNA that was destabilized by Ang II. The Ang II–induced AT1-R mRNA downregulation was also blocked by PD98059, an extracellular signal–regulated protein kinase (ERK) kinase inhibitor. Ang II–induced ERK activation was inhibited by NAC as well as by PD98059. Exogenous H2O2 also suppressed AT1-R mRNA. These results suggest that the production of ROS and the activation of ERK are critical for the downregulation of AT1-R mRNA. The generation of ROS through stimulation of AT1-R not only mediates signaling of Ang II but also may play a crucial role in the adaptation process of AT1-R to the sustained stimulation of Ang II.

Deletion of Phd2 in myeloid lineage attenuates hypertensive cardiovascular remodeling

BACKGROUND Hypertension induces cardiovascular hypertrophy and fibrosis. Infiltrated macrophages are critically involved in this process. We recently reported that inhibition of prolyl hydroxylase domain protein 2 (PHD2), which hydroxylates the proline residues of hypoxia-inducible factor-α (HIF-α) and thereby induces HIF-α degradation, suppressed inflammatory responses in macrophages. We examined whether myeloid-specific Phd2 deletion affects hypertension-induced cardiovascular remodeling. METHODS AND RESULTS Myeloid-specific PHD2-deficient mice (MyPHD2KO) were generated by crossing Phd2-floxed mice with LysM-Cre transgenic mice, resulting in the accumulation of HIF-1α and HIF-2α in macrophage. Eight- to ten-week-old mice were given N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, and Angiotensin II (Ang II) infusion. L-NAME/Ang II comparably increased systolic blood pressure in control and MyPHD2KO mice. However, MyPHD2KO mice showed less aortic medial and adventitial thickening, and macrophage infiltration. Cardiac interstitial fibrosis and myocyte hypertrophy were also significantly ameliorated in MyPHD2KO mice. Transforming growth factor-β and collagen expression were decreased in the aorta and heart from MyPHD2KO mice. Echocardiographic analysis showed that left ventricular hypertrophy and reduced ejection fraction induced by L-NAME/Ang II treatment in control mice were not observed in MyPHD2KO mice. Administration of digoxin that inhibits HIF-α synthesis to L-NAME/Ang II-treated MyPHD2KO mice reversed these beneficial features. CONCLUSIONS Phd2 deletion in myeloid lineage attenuates hypertensive cardiovascular hypertrophy and fibrosis, which may be mediated by decreased inflammation- and fibrosis-associated gene expression in macrophages. PHD2 in myeloid lineage plays a critical role in hypertensive cardiovascular remodeling.