The mitochondrial signaling peptide MOTS-c improves myocardial performance during exercise training in rats.

Cardiac remodeling is a physiological adaptation to aerobic exercise and which is characterized by increases in ventricular volume and the number of cardiomyocytes. The mitochondrial derived peptide MOTS-c functions as an important regulator in physical capacity and performance. Exercise elevates levels of endogenous MOTS-c in circulation and in myocardium, while MOTS-c can significantly enhance exercise capacity. However, the effects of aerobic exercise combined with MOTS-c on cardiac structure and function are unclear. We used pressure-volume conductance catheter technique to examine cardiac function in exercised rats with and without treatment with MOTS-c. Surprisingly, MOTS-c improved myocardial mechanical efficiency, enhanced cardiac systolic function, and had a tendency to improve the diastolic function. The findings suggest that using exercise supplements could be used to modulate the cardiovascular benefits of athletic training.

Mitochondrial Fusion Promoter Alleviates Brain Damage in Rats with Cardiac Ischemia/Reperfusion Injury.

BACKGROUND: Cardiac ischemia/reperfusion (I/R) injury induces brain damage through increased blood-brain barrier (BBB) breakdown, microglial hyperactivity, pro-inflammatory cytokines, amyloid-ß deposition, loss of dendritic spines, brain mitochondrial dysfunction, and imbalanced mitochondrial dynamics. Previous studies demonstrated that mitochondrial fusion promoter reduced cardiac damage from cardiac I/R injury; however, following cardiac I/R injury, the roles of mitochondrial dynamics on the brain have not been investigated. OBJECTIVE: To investigate the effects of pharmacological modulation using mitochondrial fusion promoter (M1) in the brain of rats following cardiac I/R injury. METHODS: Twenty-four male Wistar rats were separated into two groups; 1) sham-operation (n = 8) and 2) cardiac I/R injury (n = 16). Rats in the cardiac I/R injury group were randomly received either normal saline solution as a vehicle or a mitochondrial fusion promoter (M1, 2 mg/kg) intravenously. Both treatments were given to the rats 15 minutes before cardiac I/R injury. At the end of the reperfusion protocol, the brain was rapidly removed to investigate brain mitochondrial function, mitochondrial dynamics proteins, microglial activity, and Alzheimer's disease (AD) related proteins. RESULTS: Cardiac I/R injury induced brain mitochondrial dynamics imbalance as indicated by reduced mitochondrial fusion proteins expression without alteration in mitochondrial fission, brain mitochondrial dysfunction, BBB breakdown, increased macrophage infiltration, apoptosis, and AD-related proteins. Pretreatment with M1 effectively increased the expression of mitofusin 2, a mitochondrial outer membrane fusion protein, reduced brain mitochondrial dysfunction, BBB breakdown, macrophage infiltration, apoptosis, and AD-related proteins in rats following cardiac I/R injury. CONCLUSION: This mitochondrial fusion promoter significantly protected rats with cardiac I/R injury against brain damage.

Apoptosis-inducing peptide loaded in PLGA nanoparticles induces anti-tumor effects in vivo.

Induction of apoptosis in tumor cells specifically within the complex tumor microenvironment is highly desirable to kill them efficiently and to enhance the effects of chemotherapy. Second mitochondria-derived activator of caspase (Smac) is a key pro-apoptotic pathway which can be activated with a Smac mimetic peptide. However, in vivo application of peptides is hampered by several limitations such as poor pharmacokinetics, rapid elimination, enzymatic degradation, and insufficient intracellular delivery. In this study, we developed a nanosystem to deliver a Smac peptide to tumor by passive targeting. We first synthesized a chimeric peptide that consists of the 8-mer Smac peptide and a 14-mer cell penetrating peptide (CPP) and then encapsulated the Smac-CPP into polymeric nanoparticles (Smac-CPP-NPs). In vitro, Smac-CPP-NPs were rapidly internalized by 4T1 mammary tumor cells and subsequently released Smac-CPP into the cells, as shown with fluorescence microscopy. Furthermore, Smac-CPP-NPs induced apoptosis in tumor cells, as confirmed with cell viability and caspase 3/7 assays. Interestingly, combination of Smac-CPP-NPs with doxorubicin (dox), a clinically used cytostatic drug, showed combined effects in vitro in 4T1 cells. The effect was significantly better than that of SMAC-CPP-NPs alone as well as empty nanoparticles and dox. In vivo, co-treatment with Smac-CPP-NPs and free dox reduced the tumor growth to 85%. Furthermore, the combination of Smac-CPP-NPs and free dox showed reduced proliferating tumor cells (Ki-67 staining) and increased apoptotic cells (cleaved caspase-3 staining) in tumors. In conclusion, the present study demonstrates that the intracellular delivery of Smac-mimetic peptide using nanoparticle system can be an interesting strategy to attenuate the tumor growth and to potentiate the therapeutic efficacy of chemotherapy in vivo.

Heat Shock Protein 60 (HSP60) Serves as a Potential Target for the Sensitization of Chemoresistant Ovarian Cancer Cells.

HSP60 is a mitochondrial chaperone protein that is associated with decreased overall survival of ovarian cancer patients. We determined whether targeting HSP60 with its monoclonal antibody would induce cytotoxicity in sensitive and chemoresistant ovarian cancer cells and whether it is synergistic when combined with chemotherapeutic drugs. Epithelial ovarian cancer (EOC) cells and their docetaxel- or cisplatin-resistant counterparts were utilized. HSP60 mRNA levels were determined by real-time RT-PCR. Cytotoxicity of HSP60 antibody (0.5 or 1.5 µg/ml) alone and in combination with chemotherapy were assessed by MTT Cell Proliferation Assay. Unpaired t tests were used to compare groups for real-time RT-PCR. One-way ANOVA followed by Tukey's post hoc tests with Bonferroni correction was performed for cytotoxicity comparisons. Significant synergistic effects of the antibody combined with chemotherapy were determined by the CompuSyn Software. Basal HSP60 mRNA levels were increased in chemoresistant EOC cells as compared with their sensitive counterparts (p < 0.05). There was no significant difference in cytotoxicity between EOC cell types; however, treatment with the HSP60 antibody for 24 h showed a dose response (0.5 and 1.5 µg/ml) cytotoxic effect to both sensitive and chemoresistant EOC cells as compared with the isotype control (p < 0.05). Importantly, treatment with both doses of HSP60 antibody was not cytotoxic to normal macrophages. Combination of the HSP60 antibody with docetaxel or cisplatin was significantly synergistic in both sensitive and chemoresistant EOC cells. Here, we identify a novel target that may serve not only for ovarian cancer treatment but also for sensitization of patients to chemotherapy. The cytotoxic effect of HSP60 monoclonal antibody and its synergism with chemotherapeutic agents highlight HSP60 as a promising target for therapy and chemosensitization in ovarian cancer treatment.

The intraperitoneal administration of MOTS-c produces antinociceptive and anti-inflammatory effects through the activation of AMPK pathway in the mouse formalin test.

The activation of the AMP activated protein kinase (AMPK) exerts antinociceptive effects in acute and neuropathic pain models. Mitochondrial open-reading-frame of the twelve S rRNA-c (MOTS-c), a mitochondrial-derived peptide, regulates many biological activities via activating AMPK. However, the role of MOTS-c in the formalin-induced inflammatory nociception remains unclear. In this study, we investigated the role of MOTS-c in the formalin-induced inflammatory nociception. The antinociceptive effect of MOTS-c was assessed by recording the time spent licking paw. The anti-inflammatory effect of MOTS-c was evaluated by detecting the inflammatory cytokine level changes in the mouse serum. Western blot was used to detect the changes of protein phosphorylation levels in the mouse spinal cord. Changes of c-fos expression in the spinal cord were assessed by immunohistochemistry. Our results showed that the intraperitoneal administration of MOTS-c reduced the time spent on licking in phase 2 in a dose-dependent manner in the formalin test. The antinociceptive effects of MOTS-c (50 mg/kg, i.p.) were attenuated by the AMPK antagonist compound C (10 mg/kg, i.p.). MOTS-c (50 mg/kg, i.p.) significantly reduced pro-inflammatory cytokine levels and elevated the level of anti-inflammatory cytokine in mouse serum. In addition, MOTS-c treatment significantly increased AMPKα phosphorylation level and suppressed formalin-induced extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinases (JNK), and P38 activation and c-fos expression in the mouse spinal cord. These results suggest that systemic administration of MOTS-c exerts antinociceptive and anti-inflammatory effects, at least partially, through activating AMPK pathway and inhibiting MAP kinases-c-fos signaling pathway in the mouse formalin test.

Mitochondrial-Derived Peptide MOTS-c Attenuates Vascular Calcification and Secondary Myocardial Remodeling via Adenosine Monophosphate-Activated Protein Kinase Signaling Pathway.

INTRODUCTION: Vascular calcification (VC) is a complex, regulated process involved in many disease entities. So far, there are no treatments to reverse it. Exploring novel strategies to prevent VC is important and necessary for VC-related disease intervention. OBJECTIVE: In this study, we evaluated whether MOTS-c, a novel mitochondria-related 16-aa peptide, can reduce vitamin D3 and nicotine-induced VC in rats. METHODS: Vitamin D3 plus nicotine-treated rats were injected with MOTS-c at a dose of 5 mg/kg once a day for 4 weeks. Blood pressure, heart rate, and body weight were measured, and echocardiography was performed. The expression of phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and the angiotensin II type 1 (AT-1) and endothelin B (ET-B) receptors was determined by Western blot analysis. RESULTS: Our results showed that MOTS-c treatment significantly attenuated VC. Furthermore, we found that the level of phosphorylated AMPK was increased and the expression levels of the AT-1 and ET-B receptors were decreased after MOTS-c treatment. CONCLUSIONS: Our findings provide evidence that MOTS-c may act as an inhibitor of VC by activating the AMPK signaling pathway and suppressing the expression of the AT-1 and ET-B receptors.

The mitochondrial-derived peptide MOTS-c is a regulator of plasma metabolites and enhances insulin sensitivity.

MOTS-c is an exercise mimetic and improves insulin sensitivity in aged and diet-induced obese mice. Although plasma markers are good markers for the metabolic condition, whether MOTS-c changes plasma markers in diet-induced obese mice has not been examined. Here, we used an unbiased metabolomics approach to examine the effect of MOTS-c on plasma markers of metabolic dysfunction. We found that three pathways - sphingolipid metabolism, monoacylglycerol metabolism, and dicarboxylate metabolism - were reduced in MOTS-c-injected mice. Interestingly, these pathways are upregulated in obese and T2D models. MOTS-c improves insulin sensitivity and increases beta-oxidation to prevent fat accumulation in DIO mice through these pathways. These results provide us a better understanding of the mechanism of how MOTS-c improves insulin sensitivity and reduces the body weight and fatty liver and opens a new venue for further study.

Single-domain antibody fusion proteins can target and shuttle functional proteins into macrophage mannose receptor expressing macrophages.

The tumor microenvironment of numerous prevalent cancer types is abundantly infiltrated with tumor-associated macrophages (TAMs). Macrophage mannose receptor (MMR or CD206) expressing TAMs have been shown to be key promoters of tumor progression and major opponents of successful cancer therapy. Therefore, depleting MMR+ TAMs is an interesting approach to synergize with current antitumor therapies. We studied the potential of single-domain antibodies (sdAbs) specific for MMR to target proteins to MMR+ TAMs. Anti-MMR sdAbs were genetically coupled to a reporter protein, mWasabi (wasabi green, WG), generating sdAb "drug" fusion proteins (SFPs), referred to as WG-SFPs. The resulting WG-SFPs were highly efficient in targeting MMR+ macrophages both in vitro and in vivo. As we showed that second mitochondria-derived activator of caspase (SMAC) mimetics modulate MMR+ macrophages, we further coupled the anti-MMR sdAb to an active form of SMAC, referred to as tSMAC. The resulting tSMAC-SFPs were able to bind and upregulate caspase3/7 activity in MMR+ macrophages in vitro. In conclusion, we report the proof-of-concept of an elegant approach to conjugate anti-MMR sdAbs to proteins, which opens new avenues for targeted manipulation of MMR+ tumor-promoting TAMs.

MOTS-c improves osteoporosis by promoting osteogenic differentiation of bone marrow mesenchymal stem cells via TGF-ß/Smad pathway.

OBJECTIVE: To explore whether MOTS-c could improve osteoporosis by promoting osteogenic differentiation of rat bone mesenchymal stem cells (BMSCs) via transforming growth factor-ß (TGF-ß)/Smad pathway. MATERIALS AND METHODS: Rat BMSCs were isolated and cultured, followed by osteogenic and lipid differentiation. CCK-8 (cell counting kit-8) assay was performed to detect the highest treatment dose of MOTS-c that did not affect cell proliferation. Expressions of osteogenesis-related genes (ALP, Bglap, and Runx2) were detected by qRT-PCR (quantitative Real-Time Polymerase Chain Reaction) and Western blot, respectively. Alizarin red staining and alkaline phosphatase (ALP) cytochemical staining were carried out to evaluate the effect of MOTS-c on BMSCs osteogenesis. TGF-ß/Smad pathway-related genes (TGF-ß1, TGF-ß2, and Smad7) in BMSCs treated with MOTS-c were detected. Finally, TGF-ß1 was knocked down to investigate the regulatory effect of MOTS-c on BMSCs osteogenesis. RESULTS: BMSCs exhibited an elongated morphology and was identified with a high purity by flow cytometry. After osteogenic differentiation, alizarin red staining and ALP staining were all positive. MOTS-c treatment could remarkably stimulate the formation of calcified nodules in BMSCs. Besides, TGF-ß/Smad pathway-related genes were significantly upregulated after BMSCs were treated with MOTS-c. Promoted osteogenesis by MOTS-c treatment was reversed by the TGF-ß1 knockdown. CONCLUSIONS: MOTS-c promotes cell differentiation of BMSCs to osteoblasts via TGF-ß/Smad pathway.

RNA-seq analyses reveal that cervical spinal cords and anterior motor neurons from amyotrophic lateral sclerosis subjects show reduced expression of mitochondrial DNA-encoded respiratory genes, and rhTFAM may correct this respiratory deficiency.

Amyotrophic lateral sclerosis (ALS) is a generally fatal neurodegenerative disease of adults that produces weakness and atrophy due to dysfunction and death of upper and lower motor neurons. We used RNA-sequencing (RNA-seq) to analyze expression of all mitochondrial DNA (mtDNA)-encoded respiratory genes in ALS and CTL human cervical spinal cords (hCSC) and isolated motor neurons. We analyzed with RNA-seq mtDNA gene expression in human neural stem cells (hNSC) exposed to recombinant human mitochondrial transcription factor A (rhTFAM), visualized in 3-dimensions clustered gene networks activated by rhTFAM, quantitated their interactions with other genes and determined their gene ontology (GO) families. RNA-seq and quantitative PCR (qPCR) analyses showed reduced mitochondrial gene expression in ALS hCSC and ALS motor neurons isolated by laser capture microdissection (LCM), and revealed that hNSC and CTL human cervical spinal cords were similar. Rats treated with i.v. rhTFAM showed a dose-response increase in brain respiration and an increase in spinal cord mitochondrial gene expression. Treatment of hNSC with rhTFAM increased expression of mtDNA-encoded respiratory genes and produced one major and several minor clusters of gene interactions. Gene ontology (GO) analysis of rhTFAM-stimulated gene clusters revealed enrichment in GO families involved in RNA and mRNA metabolism, suggesting mitochondrial-nuclear signaling. In postmortem ALS hCSC and LCM-isolated motor neurons we found reduced expression of mtDNA respiratory genes. In hNSC's rhTFAM increased mtDNA gene expression and stimulated mRNA metabolism by unclear mechanisms. rhTFAM may be useful in improving bioenergetic function in ALS.

[Radiosensitization Induced by ANTP-SmacN7 Fusion Peptide in H460 Cell Line].

BACKGROUND: The curative effect of radiotherapy may be limited by the radioresistance of tumor. Mimetic compounds of Second mitochondria-derived activator of caspase (Smac) were hopeful to become new drugs of radiosensitization for tumor because they can increase radiation induced apoptosis in tumor cells. The aim of present study is to observe the radiosensitization effect of a new Smac mimetic ANTP-SmacN7 fusion peptide in H460 cell line. METHODS: In order to observe if the fusion peptide can enter into tumor cell, ANTP-SmacN7 fusion peptide was synthesized and linked by FITC. H460 cell was divided into control, radiation only, ANTP-SmacN7 only and ANTP-SmacN7 combined with radiation group. The cells were exposed by 0, 2, 4 and 6 Gy and the concentration of ANTP-SmacN7 was 20 µmol/L. Proliferation of H460 tumor cell was detected by WST-1 assay. There are four groups in the present study: control group, radiation group, ANTP-SmacN7 group and ANTP-SmacN7 combined with radiation group. Apoptosis was detected by flow cytometry at 24 and 48 hours after the treatment of all the groups. The level of caspase3 and cleaved caspase3 were detected by Western blot assay. RESULTS: ANTP-SmacN7 can enter into cells and promote the radiosensitization of H460 cell obviously (F=25.1, P<0.01, sensitivity enhancement ratio was 1.86). The treatment of ANTP-SmacN7 combined with radiation decreased the cloning forming efficiency (χ2=45.2, P<0.01; χ2=40.3, P<0.01), activated caspase3 by promoting the expression of cleaved caspase3 and increased the apoptosis of H460 cell line. CONCLUSIONS: ANTP-SmacN7 fusion peptide had remarkably radiosensitization effect on H460 cell line. ANTP-SmacN7 fusion peptide might be hopeful to be applied in radiosensitization therapy as a new Smac mimetic polypeptide in the future.

Enhanced Antitumor Activity of EGFP-EGF1-Conjugated Nanoparticles by a Multitargeting Strategy.

Tumor stromal cells have been increasingly recognized to interact with tumor parenchyma cells and promote tumor growth. Therefore, we speculated that therapeutics delivery to both parenchyma cells and stromal cells simultaneously might treat a tumor more effectively. Tissue factor (TF) was shown to be extensively located in a tumor and was abundantly sited in both tumor parenchyma cells and stromal cells including neo-vascular cells, tumor-associated fibroblasts, and tumor-associated macrophages, indicating it might function as a favorable target for drug delivery to multiple cell types simultaneously. EGFP-EGF1 is a fusion protein derived from factor VII, the natural ligand of TF. It retains the specific TF binding capability but does not cause coagulation. In the present study, a nanoparticle modified with EGFP-EGF1 (ENP) was constructed as a multitargeting drug delivery system. The protein binding experiment showed EGFP-EGF1 could bind well to A549 tumor cells and other stromal cells including neo-vascular cells, tumor-associated fibroblasts, and tumor-associated macrophages. Compared with unmodified nanoparticles (NP), ENP uptake by A549 cells and those stromal cells was significantly enhanced but inhibited by excessive free EGFP-EGF1. In addition, ENP induced more A549 tumor cell apoptosis than Taxol and NP when paclitaxel (PTX) was loaded. In vivo, ENP accumulated more specially in TF-overexpressed A549 tumors by in vivo imaging, mainly regions unoccupied by factor VII and targeted tumor parenchyma cells as well as different types of stromal cells by immunofluorescence staining. Treatment with PTX-loaded ENP (ENP-PTX) significantly reduced the A549 tumor growth in nude mice while NP-PTX- and Taxol-treated mice had lower response to the therapy. Furthermore, H&E and TUNEL staining revealed that ENP-PTX induced more severe tumor necrosis and more extensive cell apoptosis. Altogether, the present study demonstrated that ENP could target multiple key cell types in tumors through TF, which could be utilized to improve the therapeutic effect of anticancer drugs.

New p32/gC1qR Ligands for Targeted Tumor Drug Delivery.

Cell surface p32, the target of LyP-1 homing peptide, is upregulated in tumors and atherosclerotic plaques and has been widely used as a receptor for systemic delivery of payloads. Here, we identified an improved LyP-1-mimicking peptide (TT1, CKRGARSTC). We used this peptide in a fluorescence polarization-based high-throughput screening of a 50,000-compound chemical library and identified a panel of compounds that bind p32 with low micromolar affinity. Among the hits identified in the screen, two compounds were shown to specifically bind to p32 in multiple assays. One of these compounds was chosen for an in vivo study. Nanoparticles surface-functionalized with this compound specifically adhered to surfaces coated with recombinant p32 and, when injected intravenously, homed to p32-expressing breast tumors in mice. This compound provides a lead for the development of p32-targeted affinity ligands that circumvent some of the limitations of peptide-based probes in guided drug delivery.

Smac mimetic with TNF-α targets Pim-1 isoforms and reactive oxygen species production to abrogate transformation from blebbishields.

Cancer cells are capable of sphere formation (transformation) through reactive oxygen species (ROS) and glycolysis shift. Transformation is linked to tumorigenesis and therapy resistance, hence targeting regulators of ROS and glycolysis is important for cancer therapeutic candidates. Here, we demonstrate that Smac mimetic AZ58 in combination with tumour necrosis factor-α (TNF-α) was able to inhibit the production of ROS, inhibit glycolysis through Pim-1 kinase-mediated Ser-112 phosphorylation of BAD, and increase depolarization of mitochondria. We also identified mitochondrial isoforms of Pim-1 kinase that were targeted for degradation by AZ58 in combination with TNF-α or AZ58 in combination with Fas ligand (FasL) plus cycloheximide (CHX) through caspase-3 to block transformation. Our study demonstrates that Smac mimetic in combination with TNF-α is an ideal candidate to target Pim-1 expression, inhibit ROS production and to block transformation from blebbishields.

Complex I subunit gene therapy with NDUFA6 ameliorates neurodegeneration in EAE.

PURPOSE: To address the permanent disability induced by mitochondrial dysfunction in experimental autoimmune encephalomyelitis (EAE). METHODS: Mice sensitized for EAE were rescued by intravitreal injection of adeno-associated viral vector serotype 2 with the complex I subunit gene scAAV-NDUFA6Flag. Controls were injected with a mitochondrially targeted red fluorescent protein (scAAV-COX8-cherry). Another group received scAAV-COX8-cherry, but was not sensitized for EAE. Serial pattern electroretinograms (PERGs) and optical coherent tomography (OCT) evaluated visual function and structure of the retina at 1, 3, and 6 months post injection (MPI). Treated mice were killed 6 MPI for histopathology. Immunodetection of cleaved caspase 3 gauged apoptosis. Complex I activity was assessed spectrophotometrically. Expression of NDUFA6Flag in the retina and optic nerve were evaluated between 1 week to 1 month post injection by RT-PCR, immunofluorescence and immunoblotting. RESULTS: Reverse transcription-PCR and immunoblotting confirmed NDUFA6Flag overexpression with immunoprecipitation and blue native PAGE showing integration into murine complex I. Overexpression of NDUFA6Flag in the visual system of EAE mice rescued retinal complex I activity completely, axonal loss by 73%, and retinal ganglion cell (RGC) loss by 88%, RGC apoptosis by 66%, and restored the 33% loss of complex I activity in EAE to normal levels; thereby, preventing loss of vision indicated by the 43% reduction in the PERG amplitudes of EAE mice. CONCLUSIONS: NDUFA6 gene therapy provided long-term suppression of neurodegeneration in the EAE animal model suggesting that it may also ameliorate the mitochondrial dysfunction associated with permanent disability in optic neuritis and MS patients.

Oral dosing with multi-antigenic construct induces atheroprotective immune tolerance to individual peptides in mice.

Inflammatory immune response to self-antigens plays an important role in the development of atherosclerosis. Restoring immune tolerance to self-proteins reduces the pro-inflammatory response. We previously showed that oral tolerance to a combination of two peptides is atheroprotective. In the present study we expressed epitopes from apolipoprotein B 100 (ApoB), human heat shock protein (HSP60) and Chlamydia pneumonia outer membrane protein (Cpn) in a single protein scaffold and used this multi-antigenic construct to induce tolerance to individual peptides by oral route in ApoBtm2Sgy/Ldlrtm1Her/J mice. Antigen specific tolerance to individual peptides was observed in treated animals as seen by an increase in regulatory T cells. Tolerance to the peptides resulted in a 46.5% (p=0.002) reduction in the development of atherosclerosis compared with control. Atheroprotection was associated with a significant (p<0.05) decrease in plaque inflammation and an increase in the expression of immune regulatory markers in the aorta. CD11c+ cells coexpressing CD11b and CD103 increased in lymphoid organs and were found to activate regulatory T cells and reduce effector T-cell response. Adoptive transfer of CD11c+ cells was atheroprotective. Our results suggest that atheroprotection by oral tolerance to a multi-antigenic construct is mediated by antigen specific regulatory T cells and CD11c+ cells with immune regulatory properties.

PGC1α-mediated mitofusin-2 deficiency in female rats and humans with pulmonary arterial hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is a lethal, female-predominant, vascular disease. Pathologic changes in PA smooth muscle cells (PASMC) include excessive proliferation, apoptosis-resistance, and mitochondrial fragmentation. Activation of dynamin-related protein increases mitotic fission and promotes this proliferation-apoptosis imbalance. The contribution of decreased fusion and reduced mitofusin-2 (MFN2) expression to PAH is unknown. OBJECTIVES: We hypothesize that decreased MFN2 expression promotes mitochondrial fragmentation, increases proliferation, and impairs apoptosis. The role of MFN2's transcriptional coactivator, peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1α), was assessed. MFN2 therapy was tested in PAH PASMC and in models of PAH. METHODS: Fusion and fission mediators were measured in lungs and PASMC from patients with PAH and female rats with monocrotaline or chronic hypoxia+Sugen-5416 (CH+SU) PAH. The effects of adenoviral mitofusin-2 (Ad-MFN2) overexpression were measured in vitro and in vivo. MEASUREMENTS AND MAIN RESULTS: In normal PASMC, siMFN2 reduced expression of MFN2 and PGC1α; conversely, siPGC1α reduced PGC1α and MFN2 expression. Both interventions caused mitochondrial fragmentation. siMFN2 increased proliferation. In rodent and human PAH PASMC, MFN2 and PGC1α were decreased and mitochondria were fragmented. Ad-MFN2 increased fusion, reduced proliferation, and increased apoptosis in human PAH and CH+SU. In CH+SU, Ad-MFN2 improved walking distance (381 ± 35 vs. 245 ± 39 m; P < 0.05); decreased pulmonary vascular resistance (0.18 ± 0.02 vs. 0.38 ± 0.14 mm Hg/ml/min; P < 0.05); and decreased PA medial thickness (14.5 ± 0.8 vs. 19 ± 1.7%; P < 0.05). Lung vascularity was increased by MFN2. CONCLUSIONS: Decreased expression of MFN2 and PGC1α contribute to mitochondrial fragmentation and a proliferation-apoptosis imbalance in human and experimental PAH. Augmenting MFN2 has therapeutic benefit in human and experimental PAH.

Smac mimetic SM-164 potentiates APO2L/TRAIL- and doxorubicin-mediated anticancer activity in human hepatocellular carcinoma cells.

BACKGROUND: The members of inhibitor of apoptosis proteins (IAPs) family are key negative regulators of apoptosis. Overexpression of IAPs are found in hepatocellular carcinoma (HCC), and can contribute to chemotherapy resistance and recurrence of HCC. Small-molecule Second mitochondria-derived activator of caspases (Smac) mimetics have recently emerged as novel anticancer drugs through targeting IAPs. The specific aims of this study were to 1) examine the anticancer activity of Smac mimetics as a single agent and in combination with chemotherapy in HCC cells, and 2) investigate the mechanism of anticancer action of Smac mimetics. METHODS: Four HCC cell lines, including SMMC-7721, BEL-7402, HepG2 and Hep3B, and 12 primary HCC cells were used in this study. Smac mimetic SM-164 was used to treat HCC cells. Cell viability, cell death induction and clonal formation assays were used to evaluate the anticancer activity. Western blotting analysis and a pancaspase inhibitor were used to investigate the mechanisms. RESULTS: Although SM-164 induced complete cIAP-1 degradation, it displayed weak inhibitory effects on the viability of HCC cells. Nevertheless, SM-164 considerably potentiated Apo2 ligand or TNF-related apoptosis-inducing ligand (APO2L/TRAIL)- and Doxorubicin-mediated anticancer activity in HCC cells. Mechanistic studies demonstrated that SM-164 in combination with chemotherapeutic agents resulted in enhanced activation of caspases-9, -3 and cleavage of poly ADP-ribose polymerase (PARP), and also led to decreased AKT activation. CONCLUSIONS: Smac mimetics can enhance chemotherapeutic-mediated anticancer activity by enhancing apoptosis signaling and suppressing survival signaling in HCC cells. This study suggests Smac mimetics are potential therapeutic agents for HCC.

Mitochondrial transcription factor A is a proinflammatory mediator in hemorrhagic shock.

Endogenous molecules released by dying cells [i.e., damage-associated molecular patterns (DAMPs)] after trauma and severe blood loss can activate pattern recognition receptors, leading to a cascade of inflammatory responses and organ injury. Mitochondrial transcription factor A (TFAM) is a transcription factor for mitochondrial DNA. TFAM is structurally related to high mobility group box 1 (HMGB1), an important member of DAMPs. We, therefore, hypothesized that TFAM can be released into the circulation after hemorrhage to initiate inflammatory responses. In order to examine this hypothesis, male Sprague-Dawley rats were bled to and maintained at a mean arterial pressure of 40 mmHg for 90 min. They were then resuscitated with an equal volume of shed blood in the form of Ringer's lactate (i.e., low-volume resuscitation) over 60 min. TFAM levels in the serum were measured at 4 h after hemorrhage and resuscitation. Our results showed that serum levels of TFAM were more than doubled after hemorrhage and resuscitation. To further characterize TFAM's biological activity, we expressed recombinant rat TFAM with a GST-tag (GST-TFAM) in an E. coli expression system. The purity of GST-TFAM was over 99% and it was immunoreactive for specific anti-TFAM antibodies. Using RAW 264.7 cells and primary rat peritoneal macrophages, we showed that GST-TFAM dose-dependently increased TNF-α release. To determine the biological activity of GST-TFAM in vivo, GST-TFAM was intravenously injected in healthy male adult rats. Our results demonstrated that intravenous injection of GST-TFAM, not GST alone, upregulated circulating levels of pro-inflammatory cytokines, increased neutrophil infiltration to the lungs and caused organ injury in healthy animals. Thus, TFAM can act as a DAMP and may contribute to the initiation of inflammatory responses in hemorrhagic shock.

Recombinant human mitochondrial transcription factor A stimulates mitochondrial biogenesis and ATP synthesis, improves motor function after MPTP, reduces oxidative stress and increases survival after endotoxin.

Recombinant human mitochondrial transcription factor A protein (rhTFAM) was evaluated for its acute effects on cultured cells and chronic effects in mice. Fibroblasts incubated with rhTFAM acutely increased respiration in a chloramphenicol-sensitive manner. SH-SY5Y cells showed rhTFAM concentration-dependent reduction of methylpyridinium (MPP(+))-induced oxidative stress and increases in lowered ATP levels and viability. Mice treated with weekly i.v. rhTFAM showed increased mitochondrial gene copy number, complex I protein levels and ATP production rates; oxidative damage to proteins was decreased ~50%. rhTFAM treatment improved motor recovery rate after treatment with MPTP and dose-dependently improved survival in the lipopolysaccharide model of endotoxin sepsis.