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Gene Delivery of Tim44 Reduces Mitochondrial Superoxide Production and Ameliorates Neointimal Proliferation of Injured Carotid Artery in Diabetic Rats

¹ Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
² Department of Brain Science, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
³ Department of Pathology, Northwestern University, Feinberg School of Medicine, Chicago, Illinois

	ABSTRACT

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Hyperglycemia induces the production of reactive oxygen species (ROS) from mitochondria, which is closely related to diabetic vascular complications. Mammalian translocase of inner mitochondrial membrane (Tim)44 was identified by upregulation in streptozotocin (STZ)-induced diabetic mouse kidneys; Tim44 functions as a membrane anchor of mtHsp70 to TIM23 complex and is involved in the import of preproteins with mitochondria-targeted presequence into mitochondrial matrix. The process is dependent on inner membrane potential () and ATP hydrolysis on ATPase domain of mtHsp70. Here, we show that the gene delivery of Tim44 using pcDNA3.1 vector (pcDNA3.1/TIM44) into the balloon injury model of STZ-induced diabetic rats ameliorated neointimal proliferation. ROS production, inflammatory responses, and cell proliferation in injured carotid artery were diminished by delivery of pcDNA3.1/TIM44. In vitro experiments using human aortic smooth muscle cells (HASMCs) revealed that the gene delivery of Tim44 normalized high-glucose–induced enhanced ROS production and increased ATP production, alterations in inner membrane potential, and cell proliferation. Transfection of siRNA and pcDNA3.1/TIM44 using HASMC culture clarified that import of antioxidative enzymes such as superoxide dismutase and glutathione peroxidase was facilitated by Tim44. Tim44 and its related molecules in mitochondrial import machinery complex are novel targets in the therapeutic interventions for diabetes and its vascular complications.

Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; HASMC, human aortic smooth muscle cell; HVJ, hemagglutination virus of Japan; IL, interleukin; MCP-1, monocyte chemoattractant protein-1; Mn-SOD, manganese-containing SOD; PDGF, platelet-derived growth factor; ROS, reactive oxygen species; SOD, superoxide dismutase; STZ, streptozotocin; TIM, translocase of inner mitochondrial membrane; TNF-, tumor necrosis factor-; UCP-1, uncoupling protein-1; VCAM-1, vascular cell adhesion molecule-1

Diabetic hyperglycemia evokes a wide variety of metabolic and signaling pathways, such as increased aldose reductase activity (1), enhanced activity of protein kinase C isoforms (2,3), and increased formation of advanced glycation end products (4). Hyperglycemia also increases the production of reactive oxygen species (ROS), and an imbalance between ROS production and host protection mechanisms against ROS critically contributes to the development of micro- and macrovascular complications of diabetes (5,6). Indeed, high glucose activates the membrane-bound NADPH oxidase pathway, generating O₂^.–, and is operational in endothelial cells, smooth muscle cells, and mesangial cells, while hyperglycemia leads to reduction in antioxidative defense, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (7,8). Another major site for the ROS production is mitochondria (9,10). Hyperglycemia induces intracellular glucose oxidation generating NADH and pyruvate and enhances influx of pyruvate into mitochondria. Pyruvate is oxidized by the tricarboxylic acid cycle to produce NADH and FADH₂ (flavin adenine dinucleotide), which donate electrons that flow through the mitochondrial electron transport chain formed by inner membrane–associated enzyme complexes. The electron transfer through complex I, III, and IV generates a proton gradient across the inner mitochondrial membrane. If the electrochemical potential difference is too high under high-glucose conditions, ROS is generated at complex I and the interface between ubiquinone and complex III. The inhibitor of complex II, manganese SOD, and uncoupling protein-1 (UCP-1) prevent the high-glucose–induced ROS production in bovine endothelial cells and inhibits the subsequent activation of protein kinase C, formation of advanced glycation end products, sorbitol accumulation, and nuclear factor B activation (9,10).

Mitochondria consist of 1,000 different proteins, and most of them 10–20% of cellular proteins, are encoded by the nuclear genome (11). The vast majority of mitochondrial proteins are synthesized in the cytosol and imported into mitochondria by protein machineries located in the mitochondrial outer and inner membranes. It becomes clear that hydrophobic as well as hydrophilic preproteins use a common translocase in the outer mitochondrial membranes, TOM (translocase of the outer mitochondrial membrane) complex, while there are two distinct translocase of inner mitochondrial membrane (TIM) complexes, TIM23 and TIM22 complexes, which are involved in translocation across the inner membrane of matrix protein with mitochondrial target presequence and insertion of inner membrane protein with integral signals, respectively. TIM23 translocase is formed by a 90-kDa complex, consisting of two essential proteins, Tim23 and Tim17, forming channels for translocation of preproteins. Protein translocation across the inner mitochondrial membrane requires two driving forces: an inner membrane potential () and an ATP-dependent import motor, consisting of mtHsp70, the translocase subunit Tim44, and the cochaperone Mge1. mtHsp70 binds to extended segments of preproteins emerging on the matrix side of the TIM23 channel powered by binding and hydrolysis of ATP. Tim44 functions as a membrane anchor for the ATPase domain of mtHsp70, while Mge1 is a nucleotide exchange factor and promotes the reaction cycle or rebinding of ATP to mtHsp70 (12,13). The functional analysis in yeast mitochondria clearly revealed that all three components of the import motor are essential for mitochondria matrix proteins with target sequence.

Mammalian Tim44 was first identified by upregulation in streptozotocin (STZ)-induced diabetic mouse kidney during the process of PCR-based differential screening (14–16). Upregulation of Tim44 as a response to elevated blood glucose may lead to efficient import of nuclear-encoded mitochondrial matrix proteins that are required for maintenance of mitochondrial functions. Here, we hypothesize that Tim44 may have a protective role in diabetic milieus by importing various mitochondrial matrix proteins, especially antioxidative defense enzymes such as SOD and glutathione peroxidase. It has been reported that hyperglycemia causes the accelerated progression of atherosclerosis in diabetic patients, and the arterial smooth muscle cell proliferation and accumulation contribute to formation of advanced atherosclerosis lesions (17). We used the balloon injury model of carotid artery in rats with STZ-induced diabetes, where exogenous insulin administration enhances neointimal hyperplasia (18). Both hyperglycemia and hyperinsulinemia may facilitate the glucose influx into smooth muscle cells by insulin-independent as well as insulin-dependent mechanisms, and it may lead to excess of oxidative phosphorylation, inner membrane electrochemical potential, and ROS production. The aims of this study were to investigate 1) the therapeutic potential of gene delivery of Tim44 into balloon injury model of diabetic rats; 2) the effects of Tim44 on high-glucose–induced ROS production, inflammatory response, and cell proliferation in balloon-injured carotid arteries; and 3) the effects of Tim44 on ROS production, ATP production, inner membrane potential, and import of mitochondrial matrix antioxidative enzymes in cultured smooth muscle cells.

	RESEARCH DESIGN AND METHODS

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Animal model of diabetes.
Forty-eight male Wistar rats at 14 weeks of age (Charles River, Yokohama, Japan) were treated with a single injection of 100 mg/kg STZ (Sigma, St. Louis, MO) in citrate buffer at pH 4.6. Three days after STZ injection, hyperglycemia (>15 mmol/l) was confirmed and daily administration of NPH insulin, from 10 to 20 IU according to plasma glucose levels determined, was started in 40 rats and continued up to day 21. At day 7, angioplasty of the carotid artery and gene delivery using hemagglutination virus of Japan (HVJ)-envelope vector (GenomeONE-Neo; Ishihara Sangyo, Osaka, Japan) were performed.

Generation of eukaryotic expression constructs.
Expression of Tim44 (accession no. U69898), manganese-containing SOD (Mn-SOD; Y00985), and UCP-1 (M11814) was carried out using the pcDNA3.1/V5-His TOPO Expression Kit (Invitrogen). cDNAs containing full-coding region without stop codon were generated by RT-PCR and subcloned into pcDNA3.1/V5-His TOPO to prepare Tim44 sense (pcDNA3.1/TIM44), Mn-SOD sense (pcDNA3.1/Mn-SOD), and UCP-1 sense (pcDNA3.1/UCP) orientation vectors. All plasmids were sequenced with an automated DNA sequencer (ABI PRISM 310 Genetic Analyzer; Applied Biosystems, Foster City, CA). Primers were 5'-AACATGGCGGCGGCACGTCT-3' and 5'-GAGGATCTGCTCTGTGCTGG-3' for Tim44, 5'-ACCAGCACTAGCAGCATGGCGA-3' and 5'-CTTTTTGCAAGCCATGTATCTTTCAG-3' for Mn-SOD, and 5'-AAGATGGTGAGTTCGACAACTTCC-3' and 5'-TGTGGTGCAGTCCACTGTCTG-3' for UCP-1. pcDNA3.1/CT–green fluorescent protein (GFP) (Invitrogen) was used to test the efficiency of gene transfer to arterial walls.

Balloon injury of rat carotid artery and gene transfer.
Balloon catheter (2F Fogarty; Edwards Lifesciences, Irvine, CA) was introduced through the right external carotid artery into the aorta, and the balloon was inflated (18). The carotid artery was damaged by passing an inflated balloon through the lumen three times. After balloon injury, 2 AU of HVJ-envelope vector carrying 20 µg plasmid DNA was suspended in 100 µl suspension buffer supplied in the kit and incubated in the carotid artery for 20 min.

Study design of in vivo experiments.
The animals with STZ-induced diabetes were subjected to balloon injury at 7 days after the induction of diabetes, and they were randomized into seven groups: the nondiabetic control group (CON group, n = 8), STZ group without insulin (STZ group, n = 8), STZ group treated with insulin (STZ + INS group, n = 8), STZ + INS treated with pcDNA3.1/TIM44 (TIM44 group, n = 8), STZ + INS treated with pcDNA3.1/Mn-SOD (Mn-SOD group, n = 8), and STZ + INS treated with pcDNA3.1/UCP-1 (UCP group, n = 8). pcDNA3.1 was used as a control plasmid. At 14 days after the balloon injury, rats were killed, and carotid arteries were subjected to the following studies.

Light microscopic examinations and immunohistochemistry.
Carotid arteries were fixed in 10% formaldehyde and embedded in paraffin, and 4-µm-thick sections were prepared. The sections were stained with elastica van Gieson staining. The areas of intima and media were measured with image analysis software (Optimas, version 6.5; Media Cybernetics, Silver Spring, MD). Four-micrometer-thick sections of formalin-fixed paraffin-embedded tissues were deparaffinized and rehydrated, and endogenous peroxidase was blocked by incubation in 3% hydrogen peroxide and methanol. Sections were pretreated by microwave for 20 min in citrate buffer for antigen retrieval. Nonspecific binding was blocked by incubation for 30 min in 10% rabbit serum. The tissues were then incubated with anti-Ki67 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). After PBS wash, sections were incubated with a biotinylated secondary antibody, ABC-Elite Reagent (Vector Laboratories, Burlingame, CA). Apoptotic cells were visualized by in situ transferase-mediated dUTP nick-end labeling method using fluorescein isothiocyanate (FITC)-dUTP (In situ Cell Death Detection Kit; Roche, Basel, Switzerland). Four-micrometer-thick cryostat sections were prepared and fixed with 4% paraformaldehyde for 20 min at 22°C. Sections were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice. The sections were then immersed in transferase-mediated dUTP nick-end labeling reaction mixture and incubated in a humidified chamber for 60 min in the dark.

Real-time RT-PCR.
For quantitative real-time PCR analysis, cDNA synthesized from 2 µg total RNA was analyzed in a sequence detector (model 7900; Applied Biosystems) with specific primers and SYBR Green PCR Master (Perkin Elmer Life Sciences). The relative abundance of mRNAs was standardized with 36B4 mRNA as the invariant control. Gene-specific primers for MCP-1, platelet-derived growth factor (PDGF), interleukin (IL)-1, and tumor necrosis factor- (TNF-) were as follows: MCP-1 forward (5'-CAGGTCTCTGTCACGCTTCT-3'), monocyte chemoattractant protein-1 (MCP-1) reverse (5'-TGTAGTTCTCCAGCCGACTC-3'); PDGF forward (5'-CAAAGGGAAGCACCGAAAGTT-3'), PDGF reverse (5'-ACCATTGGCCGTCCGAATCAG-3'); IL-1 forward (5'-GCTCATATTACAGATGGGCTAACTAAG-3'), IL-1 reverse (5'-GTCCCCTTCCTTTTGAAAATGATAGCA-3'); TNF- forward (5'-GCTCCTCACCCACACCGTCAGCCGATTTGC-3'), TNF- reverse (5'-CAATGACTCCAAAGTAGACCTGCCCGGACT-3'); and 36B4 forward (5'-GACAATGGCAGCATCTACAG-3'), and 36B4 reverse (5'-CAACAGTCGGGTAGC-3').

Oxidant fluorescent microtopography.
Unfixed frozen rings of carotid arteries were cut into 30-µm-thick sections and placed onto a glass slide. Hydroethidine (2 x 10^–6 mol/l) was applied to each tissue section and coverslipped. After adjusting the basal settings, images were obtained with a confocal laser scanning microscope (Carl Zeiss Laser Scanning System LSM 510; Carl Zeiss, Jena, Germany) (19).

Human aortic smooth muscle cell culture.
Human aortic smooth muscle cells (HASMCs) isolated from thoracic aorta were purchased from Kurabo (Osaka, Japan). HASMCs were grown in HuMedia-EB2 medium (Kurabo) in the presence of 5% fetal bovine serum (FBS), 100 mg/dl D-glucose, 0.5 ng/ml human epidermal growth factor, 2 ng/ml human basic fibroblast growth factor, 5 µg/ml insulin, 50 µg/ml gentamicin, and 50 ng/ml amphotericin B. The cells, up to six passages, were stocked and used for the following experiments. HASMCs, 1 x 10⁴ cells per well in 96-well culture plates or 5 x 10⁵ cells per well in 24-well culture plates or 2 x 10⁵ Lab-Tek16 chamber slides (Nalge Nunc International), were cultured and incubated with Dulbecco’s modified Eagle’s medium (DMEM) containing 2% FBS for 24 h. HASMCs were transfected with pcDNA3.1, pcDNA3.1/TIM44, pcDNA3.1/Mn-SOD, and pcDNA3.1/UCP-1 using lipofectamine reagent (Invitrogen) in serum-free DMEM. As controls, HASMCs were treated with lipofectamine only. After 3 h, HASMCs were further cultured in complete DMEM containing 2% FBS for 12 h and then stimulated with DMEM containing high glucose (HG; 300 mg/dl) supplemented with 10 µg/ml insulin (HG + INS) for 12 h. As controls, HASMCs were incubated with DMEM containing normal glucose (NG; 100 mg/dl) or high glucose (300 mg/dl). Finally, HASMCs were subjected to the following experiments.

Cell proliferation enzyme-linked immunosorbent assay.
HASMCs in 96-well plates were subjected to cell proliferation enzyme-linked immunosorbent assay (ELISA) using 5-bromo-2'-deoxyuridine (BrdU) (Roche). BrdU labeling solution was added into culture media and reincubated for an additional 6 h. The culture media were removed and serially incubated with FixDenat, anti–BrdU-POD, washing solution, and substrate solution. Finally, we measured the absorbance of the samples in an ELISA plate reader (BioRad) at 370 nm (reference wavelength 492 nm).

Intracellular ROS.
HASMCs in 96-well plates were loaded with 10 µmol/l CM-H₂ DCFDA (5- [and -6]-chloromethyl-2', 7'-dichloridihydrofluorescein diacetate, acetyl ester) (Molecular Probe), incubated for 1 h at 37°C, and analyzed with an HTS 7000 Bio Assay Fluorescent Plate Reader (Perkin Elmer, Norwalk, CT). ROS production was determined from an H₂O₂ standard curve (10).

ATP determination.
1 x 10⁵ HASMCs in 24-well culture plates were collected, lysed with 20 µl of 1 x passive lysis buffer (Promega), reconstituted in 200 µl ATP determination reaction mix containing 1 x reaction buffer, 1 mmol/l dithiothreitol, 0.5 mmol/l luciferin, and 1.25 µg/ml luciferase, and subjected to a luminometer measurement (AB-2200; ATTO, Tokyo, Japan).

Mitochondrial membrane potential integrity measurement.
To measure mitochondrial membrane potential (), the ApoAlert Membrane Sensor Kit (Clontech) was used (20–22). We added the MitoSensor fluorescence reagent directly into HASMCs in 96-well culture plates and chamber slides. Fluorescence was measured with an HTS 7000 Bio Assay Fluorescent Plate Reader using the FITC channel to measure the green fluorescence produced when the MitoSensor reagent remains in monomeric form in the cytoplasm because of alteration of . If is not altered, MitoSensor is taken up in the mitochondria, where it forms aggregates that exhibit red fluorescence. Fluorescence micrographic images were obtained with a confocal laser scanning microscope (Zeiss).

Mitochondrial import of Mn-SOD and glutathione peroxidase in HASMCs transfected with pcDNA3.1/TIM44 and siRNA.
HASMCs were transfected with pcDNA3.1/TIM44, pcDNA3.1, and siRNA for Tim44 (SiGENOME; Dharmacon, Lafayette, CO) using Lipofectamine 2000 reagent (Invitrogen) in serum-free DMEM. After 3 h, HASMCs were further cultured in complete DMEM containing 2% FBS with 100 mg/dl glucose for 24 h. Mitochondria fractions were isolated with the ApoAlert Cell Fraction Kit (Clontech). The preparation of mitochondira fraction was ascertained with Western blot analysis using cytochome C antibody supplied in the kit. Five micrograms of protein were loaded onto SDS-PAGE, and Western blot analyses using gout polyclonal anti-human Mn-SOD (Santa Cruz), sheep polyclonal anti-human glutathione peroxidase (Abcam, Cambridge, U.K.), and goat polyclonal anti-human Tim44 (Santa Cruz) were performed.

Statistical evaluation.
Data are presented as means ± SE. Statistical analysis was performed by a standard ANOVA test with post hoc analysis. A P value of <0.05 was considered significant.

	RESULTS

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Gene delivery of Tim44 ameliorates neointimal hyperplasia in the balloon injury model.
As reported earlier (18), a significant reduction of the neointima-to-media ratio was observed in the STZ group (neointima-to-media ratio 0.606 ± 0.153) compared with the CON group (1.023 ± 0.085, P < 0.05). The treatment of STZ-induced diabetic Wistar rats with insulin (STZ + INS group) significantly increased the neointima-to-media ratio compared with the STZ group (1.205 ± 0.112, P < 0.01 versus STZ group) (Fig. 1A–H). HVJ-envelope vector–mediated gene transfer of pcDNA3.1 into carotid artery of STZ-induced diabetic Wistar rats treated with insulin (pcDNA group) induced more prominent neointimal proliferation (1.514 ± 0.088) compared with the STZ + INS group. HVJ-envelope–mediated gene transfer of Tim44 (0.766 ± 0.133, P < 0.01 versus pcDNA group), Mn-SOD (1.047 ± 0.103, P < 0.05 versus pcDNA group), and UCP-1 (1.01 ± 0.159, P < 0.05 versus pcDNA group) significantly reduced neointimal proliferation. The body weight of the STZ group was severely reduced compared with the CON and STZ + INS groups (Fig. 1K), and postprandial plasma glucose levels in the STZ + INS group were still higher than in the CON group (Fig. 1L). The efficiency of HVJ-envelope vector–mediated gene transfer was ascertained by pcDNA3.1/CT-GFP (Fig. 1K–L).

View larger version (74K): [in this window] [in a new window]   FIG. 1. Neointimal proliferation of balloon-injured carotid arteries in STZ-induced diabetic rats. A: Control rats (CON). B: Rats treated with STZ. C: STZ-induced diabetic rats treated with insulin (STZ + INS). D–G: STZ-induced diabetic rats treated with insulin and pcDNA3.1 (pcDNA) (D), pcDNA/Tim44 (TIM44) (E), pcDNA/Mn-SOD (Mn-SOD) (F), and pcDNA/UCP-1 (UCP-1) (G). H: Intimal-to-media ratio. I and J: Body weight (I) and postprandial plasma glucose levels (J). K and L: STZ-induced diabetic rats treated with pcDNA3.1/CT-GFP. GFP-positive cells are seen in medial (K) and intimal (L) cells. *P < 0.05, **P < 0.01 vs. pcDNA group, P < 0.05, P < 0.01 vs. CON group.

View larger version (43K): [in this window] [in a new window]   FIG. 2. Superoxide production in injured carotid arteries in STZ-induced diabetic rats detected with hydroethidine. STZ-induced diabetic rats treated with insulin and pcDNA3.1 (pcDNA) (A), pcDNA/Tim44 (TIM44) (B), pcDNA/Mn-SOD (Mn-SOD) (C), and pcDNA/UCP-1 (UCP-1) (D).

View larger version (47K): [in this window] [in a new window]   FIG. 3. Cell proliferation and apoptosis in injured carotid arteries in STZ-induced diabetic rats. A–D: Ki-67 staining in STZ-induced diabetic rats treated with insulin and plasmids, i.e., pcDNA3.1 (pcDNA) (A), pcDNA/Tim44 (TIM44) (B), pcDNA/Mn-SOD (Mn-SOD) (C), and pcDNA/UCP-1 (UCP-1) (D). E: Count of Ki-67–positive cells (%) in neointima. *P < 0.05, **P < 0.01 vs. pcDNA group. F–I: In situ transferase-mediated dUTP nick-end labeling assay using FITC-dUTP. Apoptotic cells are indicated by arrows.

View larger version (80K): [in this window] [in a new window]   FIG. 4. Expression of VCAM-1 and inflammatory cytokines in injured carotid arteries in STZ-induced diabetic rats. VCAM-1 staining in rats treated with insulin and pcDNA3.1 (pcDNA) (A), pcDNA/Tim44 (TIM44) (B), pcDNA/Mn-SOD (Mn-SOD) (C), and pcDNA/UCP-1 (UCP-1) (D). E: mRNA expression of MCP-1, PDGF, IL-1, and TNF- measured with real-time RT-PCR. The pcDNA group is used as standard. *P < 0.05, **P < 0.01 vs. pcDNA group.

View larger version (36K): [in this window] [in a new window]   FIG. 5. Cell proliferations, ROS production, and ATP levels in HASMCs cultured under high-glucose condition. HASMCs treated with normal glucose (100 mg/dl) (NG), high glucose (300 mg/dl) (HG), and high glucose plus insulin (10 µg/ml insulin) (HG + INS). HASMCs under HG + INS conditions were transfected with various plasmids. A: Cell proliferation BrdU ELISA. B: Intracellular ROS measured with CM-H2DCFFDA. C: Cellular ATP contents. *P < 0.05, **P < 0.01 vs. pcDNA group.

High-glucose–induced alteration of was reversed by gene delivery of Tim44.

View larger version (31K): [in this window] [in a new window]   FIG. 6. Mitochondrial membrane potential () in HASMCs cultured under high-glucose condition. HASMCs treated with normal glucose (100 mg/dl) (NG), high glucose (300 mg/dl) (HG), and high glucose plus insulin (10 µg/ml insulin) (HG + INS). HASMCs under HG + INS condition were transfected with various plasmids. A–F: Red fluorescence shows MitoSensor taken up in the mitochondria under unaltered . Green color shows MitoSensor that remains in cytoplasm because of alteration of . G: The gene delivery of Tim44 and UCP-1 significantly reduces green fluorescent intensity and partially reverses the alteration of . *P < 0.05, **P < 0.01 vs. pcDNA group.

View larger version (51K): [in this window] [in a new window]   FIG. 7. Mitochondrial import of Mn-SOD and glutathione peroxidase in HASMCs transfected with pcDNA3.1/TIM44 and siRNA. HASMCs were transfected with pcDNA3.1/TIM44, pcDNA3.1, and siRNA for Tim44. Mitochondria fractions were isolated and subjected to Western blot.

	DISCUSSION

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

View larger version (44K): [in this window] [in a new window]   FIG. 8. Two main pathways of protein import into mitochondrial matrix and inner membrane.

To normalize mitochondrial ROS production in diabetic states, an uncoupler of oxidative phosphorylation by UCP-1, Mn-SOD, and an inhibitor of electron transport chain complex II has been reported (10). We would like to add Tim44 as one of the therapeutic targets to sustain mitochondrial function and normalize ROS production in the diabetic state by facilitating the mitochondrial matrix protein import. In clinical settings, the gene delivery of Mn-SOD, UCP-1, or Tim44 using plasmid vectors may be beneficial at the time of pericutaneous coronary interventions in diabetic patients, since an exaggerated intimal hyperplasia both in the presence and in the absence of stent development has been reported. The gene delivery of Tim44 may be beneficial in microvascular complications such as neuropathy, retinopathy, and nephropathy; however, local delivery of eukaryotic expression plasmid vector may be difficult in these microvascular complications. Instead of overexpression of Tim44 by mammalian expression plasmid vector, screening of low–molecular weight compounds that stimulate protein import machinery by interacting with Tim44, mtHsp70, mtGrpE, mtDnaJ, or other components of inner mitochondrial import machinery complex may be necessary. The development of such agents may be beneficial in prevention of micro- and macrovascular diabetes complications. Furthermore, it may be beneficial for treating glucose toxicity toward pancreatic ß-cells, where increased influx of glucose causes mitochondrial ROS production, impairs mitochondria functions, and reduces insulin secretion.

In conclusion, the gene delivery of Tim44 into the balloon injury model of diabetic rats ameliorated neointimal proliferations by reducing ROS production, inflammatory responses, and cell proliferation in the carotid artery. In vitro experiments using HASMCs revealed that the gene delivery of Tim44 normalized high-glucose–induced enhanced ROS production and increased ATP production, alterations in inner membrane potential, and cell proliferation. siRNA and expression vector for Tim44 using HASMC culture clarified that import of antioxidative enzymes such as SOD and glutathione peroxidase was facilitated by Tim44. Tim44 and its related molecules in the mitochondrial import machinery complex are novel targets in the therapeutic interventions for diabetes and its vascular complications.

	ACKNOWLEDGMENTS

 
This work was supported by the Cell Science Research Foundation, Yamanouchi Foundation for Research on Metabolic Disorders Grant-in-Aid for Scientific Research (C), the Ministry of Education, Science and Culture, Japan (14571025, 17590829) to J.W.; the Uehara Memorial Foundation Grant-in-Aid for Scientific Research (B), Ministry of Education, Science and Culture, Japan (14370319) to H.M.; and National Institutes of Health Grants to Y.S.K.

Received for publication March 20, 2005 and accepted in revised form July 6, 2005

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