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 (Δψ) 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.

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 κB 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 (Δψ) 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 (1416). 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.

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)-1α, and tumor necrosis factor-α (TNF-α) 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-1α forward (5′-GCTCATATTACAGATGGGCTAACTAAG-3′), IL-1α reverse (5′-GTCCCCTTCCTTTTGAAAATGATAGCA-3′); TNF-α forward (5′-GCTCCTCACCCACACCGTCAGCCGATTTGC-3′), TNF-α 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 × 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 × 104 cells per well in 96-well culture plates or 5 × 105 cells per well in 24-well culture plates or 2 × 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 × 105 HASMCs in 24-well culture plates were collected, lysed with 20 μl of 1 × passive lysis buffer (Promega), reconstituted in 200 μl ATP determination reaction mix containing 1 × 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 (Δψ), the ApoAlert Membrane Sensor Kit (Clontech) was used (2022). 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 Δψ. If Δψ 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.

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).

Gene delivery of Tim44 inhibits vascular superoxide production and proliferation.

Using hydroethidine, vascular superoxide production in rat carotid artery was evaluated. Vessels were labeled with the dye hydroethidine, which produces a red fluorescence when oxidized to ethidium bromide by superoxide. The gene delivery of Tim44, Mn-SOD, and UCP-1 markedly decreased endothelial as well as smooth muscle cell–derived superoxide compared with the pcDNA3.1 group as shown in Fig. 2. The proliferation of smooth muscle cells was evaluated by Ki-67 staining. The rate of Ki-67–positive cells was significantly lower in the STZ group (0.47 ± 0.22%, P < 0.01 versus STZ + INS and pcDNA groups) compared with the STZ + INS (4.73 ± 0.80%) or pcDNA group (4.98 ± 1.45%) (Fig. 3A–E). The gene delivery of Tim44 (2.4 ± 0.30%, P < 0.05 versus pcDNA), Mn-SOD (3.8 ± 0.14%, P < 0.05 versus pcDNA), and UCP-1 (3.5 ± 0.43%, P < 0.05 versus pcDNA) significantly reduced Ki-67–positive cells compared with the pcDNA group. The presence of apoptotic cells were visualized by in situ apoptosis assay. Apoptotic cells were occasionally seen in smooth muscle cells in neointima in the pcDNA group; however, no apparent apoptotic cells were seen in Tim44–, Mn-SOD–, and UCP-1 expression vector–treated groups.

Gene delivery of Tim44 reduces the expression of vascular cell adhesion molecule-1 and inflammatory cytokines.

The vascular cell adhesion molecule-1 (VCAM-1) expression in the carotid artery of the balloon injury model was assessed by immunostaining. VCAM-1 expression on endothelial cells was prominent in the pcDNA group, and immunoreactivities of VCAM-1 were apparently reduced by the treatment of Tim44, Mn-SOD, and UCP-1 expression vectors (Fig. 4A–D). mRNA expression of MCP-1, PDGF, IL-1α, and TNF-α was assessed by real-time RT-PCR. The treatment of Tim44 expression vector reduced expression levels of MCP-1, PDGF, IL-1α, and TNF-α (Fig. 4E). In contrast, the treatment with Mn-SOD expression vector failed to inhibit mRNA expression of TNF-α, and UCP-1 failed to inhibit MCP-1 and TNF-α mRNAs.

Gene delivery of Tim44 normalizes high-glucose–induced cell proliferation, ROS production, and ATP synthesis in vitro.

To give insights into the mechanism of therapeutic potential of Tim44 expression vector on neointimal formation after balloon injury, HASMCs isolated from thoracic aorta were investigated. High-glucose–induced cell proliferation was demonstrated by BrdU ELISA, and the addition of insulin further accentuated the proliferation. Transfection of pcDNA3.1/TIM44 into HASMCs significantly inhibited proliferation, while the gene delivery of Mn-SOD and UCP-1 suppressed proliferation without statistical significance (Fig. 5A). Incubation with 300 mg/dl glucose increased ROS production from 47.8 ± 3.0 (100 mg/dl glucose) to 69.8 ± 3.5 nmol/ml. The addition of 10 μg/ml insulin further increased ROS production up to 112 ± 13.5 nmol/ml. The gene delivery of Tim44, Mn-SOD, and UCP-1 significantly suppressed ROS production compared with pcDNA plasmid–treated control (Fig. 5B). ATP production was also evaluated in HASMCs. Incubation with 300 mg/dl glucose increased ATP content from 13.0 ± 1.4 (100 mg/dl glucose) to 17.7 ± 2.7 μmol/mg protein. Compared with high-glucose condition, incubation with 300 mg/dl glucose and 10 μg/ml insulin similarly increased ATP contents (18.9 ± 1.0 μmol/mg protein). The gene delivery of Tim44 and UCP-1 significantly reduced ATP contents to 8.2 ± 2.4 and 7.8 ± 2.4 μmol/mg protein, respectively. In contrast, the gene delivery of Mn-SOD did not suppress the ATP levels (Fig. 5C).

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

In HASMCs cultured in DMEM containing 100 mg/dl glucose, MitoSensor is taken up in the mitochondria, where it forms aggregates and exhibits intense red fluorescence (Fig. 6A). In high-glucose condition, MitoSensor failed to aggregate in the mitochondria because of altered mitochondrial membrane potentials, and the dye remains in monomeric form in the cytoplasm, where green was seen (Fig. 6B and C). We measured the alteration of Δψ by qualification of fluorescent intensity using the FITC channel. The gene delivery of Tim44, Mn-SOD, and UCP-1 reduced fluorescent intensity (Fig. 6D–F) and thus partially but significantly reversed high-glucose–induced alteration of Δψ.

Tim44 gene delivery facilitates the import of Mn-SOD and glutathione reductase.

HASMCs cultured in DMEM containing 100 mg/dl glucose were transfected with pcDNA3.1/TIM44, pcDNA3.1, and siRNA for Tim44. We isolated mitochondrial fractions and performed Western blot analyses to investigate the cytochrome C, Mn-SOD, glutathione peroxidase, and Tim44 protein. Cytochrome C is an inner mitochondria protein, and the import is Tim44 independent and the amount of mitochondrial cytochrome C is not altered by the treatment of pcDNA3.1/TIM44 or siRNA for Tim44 (Fig. 7). In contrast, Mn-SOD, glutathione peroxidase, and Tim44 itself are mitochondrial matrix proteins carrying mitochondrial targeting presequence and their import process reported to be Tim44 dependent. siRNA for Tim44 significantly inhibited Tim44 protein import and reduced the mitochondria matrix proteins such as Mn-SOD and glutathione peroxidase. In turn, Tim44 expression vector increased import of Mn-SOD, glutathione peroxidase, and Tim44 into mitochondria (Fig. 7).

Based on the observed interactions and activities of TIM23 complex, mtHsp70, Tim44, and Mge1 in yeast mitochondria, two models for the mechanism of protein import into mitochondrial matrix were proposed: the “Brownian ratchet ” and “power stroke” models (11). In the Brownian ratchet or “trapping ” model, the preproteins, after the initial Δψ-mediated insertion into the TIM23 channel, moves forward and backward at random due to Brownian motion, and the mtHsp70 binds to the preprotein, prevents backsliding, and favors for translocation into matrix. In the power stroke or “pulling” model, Tim44 functions as a membrane anchor of mtHsp70 to the TIM23 complex, and ATP hydrolysis on the ATPase domain of mtHsp70 induces conformational change of mtHsp70, which generates a pulling force on the preprotein and leads to the release of mtHsp70 from Tim44 (2325). Interaction with Mge1 with the preprotein-bound mtHsp70-ADP triggers ADP dissociation and rebinding of ATP to nucleotide-free mtHsp70, inducing the opening of the substrate binding pocket and release of mtHsp70 (12). Thus, mitochondrial matrix protein import via TIM23 is dependent on inner membrane potential as well as ATP hydrolysis. Mammalian homologues of a translocation motor, such as mtHsp70, Tim44, and Mge1 in yeast mitochondria, have been identified (Fig. 8). While yeast Tim44 is tightly associated with the matrix-face of the inner membrane, mammalian Tim44, hTim44, and rTim44, were detected in the mitochondrial inner membrane fraction as well as in the matrix fraction (2628). The localization on the inner membrane suggested that mammalian Tim44 is involved in the power stroke or pulling model in preprotein import (28). In mammalian mitochondria, mtHsp70 cooperates with mtGrpE and mtDnaJ (Tid), the mammalian homologues of yeast mitochondrial cochaperone. Available data suggest that the mitochondrial import system seems to be conserved from yeast to mammalian cells during the evolution.

Taken together, our data and the functional experiments related to mitochondrial transport machinery complexes reported in the literature, we would raise three major mechanisms of therapeutic potential of Tim44 in neointimal proliferation in the balloon injury animal model of STZ-induced diabetic rats. First, Tim44 may facilitate the import of various mitochondrial matrix proteins, especially antioxidative enzymes such as SOD and glutathione peroxidase. The gene delivery of Mn-SOD suppressed high-glucose–induced ROS production and proliferation of smooth muscle cells in vivo and in vitro experiments in our current study. Similarly, it could be speculated that Tim44 exerts therapeutic effects on neointimal proliferation in diabetic rats by facilitating the antioxidative mitochondrial matrix enzymes, including SOD and glutathione peroxidase, thus suppressing ROS production from mitochondria. Second, the facilitated import of positively charged mitochondrial preprotein across the inner mitochondrial membrane would reduce electron chemical gradient differences, which are enhanced by high-glucose condition. If electron chemical gradient differences are too high, mitochondria actively produce ROS. It has been reported (29) that UCP-1 uncoupled oxidative phosphorylation and thus reduced ROS production from mitochondria. As reported in the literature, the gene delivery of UCP-1 diminished high-glucose–induced membrane potential alterations in cultured HASMCs and reduced ROS production and neointimal proliferation in vivo. The gene delivery of Tim44 may reduce the inner membrane potential by preprotein translocation across the inner membrane and finally suppress the ROS production. Third, Tim44 may facilitate the utilization of ATP by mtHsp70 in the process of protein import across the inner mitochondrial membrane. UCP-1 has the beneficial effects on proliferation of HASMCs by uncoupling the link between the inner membrane potential and ATPase, which results in reversal of enhanced ATP production to normal levels. The delivery of Tim44 also normalized ATP contents in HASMC culture with high-glucose condition, and it may also be beneficial for the suppression of cell proliferation.

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 β-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.

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.

Lee AY, Chung SK, Chung SS: Demonstration that polyol accumulation is responsible for diabetic cataract by the use of transgenic mice expressing the aldose reductase gene in the lens.
Proc Natl Acad Sci U S A
Ishii H, Jirousek MR, Koya D, Takagi C, Xia P, Clermont A, Bursell SE, Kern TS, Ballas LM, Heath WF, Stramm LE, Feener EP, King GL: Amelioration of vascular dysfunctions in diabetic rats by an oral PKC beta inhibitor.
Koya D, King GL: Protein kinase C activation and the development of diabetic complications.
Makino H, Shikata K, Hironaka K, Kushiro M, Yamasaki Y, Sugimoto H, Ota Z, Araki N, Horiuchi S: Ultrastructure of nonenzymatically glycated mesangial matrix in diabetic nephropathy.
Kidney Int
Kashiwagi A, Asahina T, Nishio Y, Ikebuchi M, Tanaka Y, Kikkawa R, Shigeta Y: Glycation, oxidative stress, and scavenger activity: glucose metabolism and radical scavenger dysfunction in endothelial cells.
45 (Suppl. 3)
Ceriello A, dello Russo P, Amstad P, Cerutti P: High glucose induces antioxidant enzymes in human endothelial cells in culture: evidence linking hyperglycemia and oxidative stress.
Modlinger PS, Wilcox CS, Aslam S: Nitric oxide, oxidative stress, and progression of chronic renal failure.
Semin Nephrol
Griendling KK, Sorescu D, Ushio-Fukai M: NAD(P)H oxidase: role in cardiovascular biology and disease.
Circ Res
Nishikawa T, Edelstein D, Brownlee M: The missing link: a single unifying mechanism for diabetic complications.
Kidney Int Suppl
Nishikawa T, Edelstein D, Du XL, Yamagishi S, Matsumura T, Kaneda Y, Yorek MA, Beebe D, Oates PJ, Hammes HP, Giardino I, Brownlee M: Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage.
Pfanner N, Geissler A: Versatility of the mitochondrial protein import machinery.
Nat Rev Mol Cell Biol
Liu Q, D’Silva P, Walter W, Marszalek J, Craig EA: Regulated cycling of mitochondrial Hsp70 at the protein import channel.
Mayer MP: Timing the catch.
Nat Struct Mol Biol
Wada J, Makino H, Kanwar YS: Gene expression and identification of gene therapy targets in diabetic nephropathy.
Kidney Int
Wada J, Kanwar YS: Characterization of mammalian translocase of inner mitochondrial membrane (Tim44) isolated from diabetic newborn mouse kidney.
Proc Natl Acad Sci U S A
Wada J, Kanwar YS, Makino H: Relevance of extracellular matrix and its receptors in mammalian nephrogenesis revealed by metanephric organ culture system.
Nephrol Dial Transplant
17 (Suppl.):9
Suzuki LA, Poot M, Gerrity RG, Bornfeldt KE: Diabetes accelerates smooth muscle accumulation in lesions of atherosclerosis: lack of direct growth-promoting effects of high glucose levels.
Indolfi C, Torella D, Cavuto L, Davalli AM, Coppola C, Esposito G, Carriero MV, Rapacciuolo A, Di Lorenzo E, Stabile E, Perrino C, Chieffo A, Pardo F, Chiariello M: Effects of balloon injury on neointimal hyperplasia in streptozotocin-induced diabetes and in hyperinsulinemic nondiabetic pancreatic islet-transplanted rats.
Hink U, Li H, Mollnau H, Oelze M, Matheis E, Hartmann M, Skatchkov M, Thaiss F, Stahl RA, Warnholtz A, Meinertz T, Griendling K, Harrison DG, Forstermann U, Munzel T: Mechanisms underlying endothelial dysfunction in diabetes mellitus.
Circ Res
Grasso S, Scifo C, Cardile V, Gulino R, Renis M: Adaptive responses to the stress induced by hyperthermia or hydrogen peroxide in human fibroblasts.
Exp Biol Med (Maywood
Kluza J, Lansiaux A, Wattez N, Mahieu C, Osheroff N, Bailly C: Apoptotic response of HL-60 human leukemia cells to the antitumor drug TAS-103.
Cancer Res
Sibille B, Ronot X, Filippi C, Nogueira V, Keriel C, Leverve X: 2,4 Dinitrophenol-uncoupling effect on delta psi in living hepatocytes depends on reducing-equivalent supply.
Huang S, Ratliff KS, Schwartz MP, Spenner JM, Matouschek A: Mitochondria unfold precursor proteins by unraveling them from their N-termini.
Nat Struct Biol
Voisine C, Craig EA, Zufall N, von Ahsen O, Pfanner N, Voos W: The protein import motor of mitochondria: unfolding and trapping of preproteins are distinct and separable functions of matrix Hsp70.
Voos W, von Ahsen O, Muller H, Guiard B, Rassow J, Pfanner N: Differential requirement for the mitochondrial Hsp70-Tim44 complex in unfolding and translocation of preproteins.
Ishihara N, Mihara K: Identification of the protein import components of the rat mitochondrial inner membrane, rTIM17, rTIM23, and rTIM44.
J Biochem (Tokyo
Ishihara N, Komiya T, Sakaguchi M, Ito A, Mihara K: Characterization of the initial steps of precursor import into rat liver mitoplasts.
J Biochem (Tokyo
Bauer MF, Gempel K, Reichert AS, Rappold GA, Lichtner P, Gerbitz KD, Neupert W, Brunner M, Hofmann S: Genetic and structural characterization of the human mitochondrial inner membrane translocase.
J Mol Biol
Echtay KS, Roussel D, St-Pierre J, Jekabsons MB, Cadenas S, Stuart JA, Harper JA, Roebuck SJ, Morrison A, Pickering S, Clapham JC, Brand MD: Superoxide activates mitochondrial uncoupling proteins.