A tissue-engineered, decellularized, connective tissue membrane for allogeneic arterial patch implantation.

BACKGROUND: We have previously applied in vivo tissue-engineered vascular grafts constructed in patients' subcutaneous spaces. However, since the formation of these vascular grafts depends on host health, their application is challenging in patients with suppressed regenerative ability. Therefore, the allogeneic implantation of grafts from healthy donors needs to be evaluated. This study aimed to fabricate allogeneic cardiovascular grafts in animals. MATERIALS AND METHODS: Silicone rod molds were implanted into subcutaneous pouches in dogs; the implants, along with surrounding connective tissues, were harvested after four weeks. Tubular connective tissues were decellularized and stored before they were cut open, trimmed to elliptical sheets, and implanted into the common carotid arteries of another dog as vascular patches (n = 6); these were resected and histologically evaluated at 1, 2, and 4 weeks after implantation. RESULTS: No aneurysmal changes were observed by echocardiography. Histologically, we observed neointima formation on the luminal graft surface and graft wall cell infiltration. At 2 and 4 weeks after implantation, α-SMA-positive cells were observed in the neointima and graft wall. At 4 weeks after implantation, the endothelial lining was observed at the grafts' luminal surfaces. CONCLUSION: Our data suggest that decellularized connective tissue membranes can be prepared and stored for later use as allogeneic cardiovascular grafts.

TAT-dextran-mediated mitochondrial transfer enhances recovery from models of reperfusion injury in cultured cardiomyocytes.

Acute myocardial infarction is a leading cause of death among single organ diseases. Despite successful reperfusion therapy, ischaemia reperfusion injury (IRI) can induce oxidative stress (OS), cardiomyocyte apoptosis, autophagy and release of inflammatory cytokines, resulting in increased infarct size. In IRI, mitochondrial dysfunction is a key factor, which involves the production of reactive oxygen species, activation of inflammatory signalling cascades or innate immune responses, and apoptosis. Therefore, intercellular mitochondrial transfer could be considered as a promising treatment strategy for ischaemic heart disease. However, low transfer efficiency is a challenge in clinical settings. We previously reported uptake of isolated exogenous mitochondria into cultured cells through co-incubation, mediated by macropinocytosis. Here, we report the use of transactivator of transcription dextran complexes (TAT-dextran) to enhance cellular uptake of exogenous mitochondria and improve the protective effect of mitochondrial replenishment in neonatal rat cardiomyocytes (NRCMs) against OS. TAT-dextran-modified mitochondria (TAT-Mito) showed a significantly higher level of cellular uptake. Mitochondrial transfer into NRCMs resulted in anti-apoptotic capability and prevented the suppression of oxidative phosphorylation in mitochondria after OS. Furthermore, TAT-Mito significantly reduced the apoptotic rates of cardiomyocytes after OS, compared to simple mitochondrial transfer. These results indicate the potential of mitochondrial replenishment therapy in OS-induced myocardial IRI.

From Cell Entry to Engraftment of Exogenous Mitochondria.

Mitochondrial transfer has been recognized to play a role in a variety of processes, ranging from fertilization to cancer and neurodegenerative diseases as well as mammalian horizontal gene transfer. It is achieved through either exogeneous or intercellular mitochondrial transfer. From the viewpoint of evolution, exogeneous mitochondrial transfer is quite akin to the initial process of symbiosis between α-protobacterium and archaea, although the progeny have developed more sophisticated machinery to engulf environmental materials, including nutrients, bacteria, and viruses. A molecular-based knowledge of endocytosis, including macropinocytosis and endosomal escape involving bacteria and viruses, could provide mechanistic insights into exogeneous mitochondrial transfer. We focus on exogeneous mitochondrial transfer in this review to facilitate the clinical development of the use of isolated mitochondria to treat various pathological conditions. Several kinds of novel procedures to enhance exogeneous mitochondrial transfer have been developed and are summarized in this review.

Development of xenogeneic decellularized biotubes for off-the-shelf applications.

In earlier studies, we developed in vivo tissue-engineered, autologous, small-caliber vascular grafts, called "biotubes," which withstand systemic blood pressure and exhibit excellent performance as small-caliber vascular prostheses in animal models. However, biotube preparation takes 4 weeks; therefore, biotubes cannot be applied in emergency situations. Moreover, for responses to various types of surgery, grafts should ideally be readily available in advance. The aim of this study was to develop novel, off-the-shelf, small-caliber vascular grafts by decellularizing in vivo tissue-engineered xenogeneic tubular materials. Silicone rod molds (diameter: 2 mm, length: 70 mm) placed in subcutaneous pouches of a beagle dog for 4 weeks were harvested with their surrounding connective tissues. Tubular connective tissues were obtained after pulling out the impregnated molds. Subsequently, they were decellularized by perfusion with sodium dodecyl sulfate and Triton X-100. They were stored as off-the-shelf grafts at -20°C for 1 week. The decellularized grafts derived from the beagle dog were xenogeneically transplanted to the abdominal aortas of rats (n = 3). No signs of abnormal inflammation or immunological problems due to the xenogeneic material were observed. Echocardiography confirmed the patency of the grafts at 1 month after implantation. Histological evaluation revealed that the grafts formed neointima on the luminal surface, and that the graft walls had cell infiltration. Little accumulation of CD68-positive macrophages in the graft wall was observed. Xenogeneic decellularized tubular tissues functioned as small-caliber vascular grafts, as well as autologous biotubes. This technology enables the easy fabrication of grafts from xenogeneic animals in advance and their storage for at least a week, satisfying the conditions for off-the-shelf grafts.

Augmented neutrophil extracellular traps formation promotes atherosclerosis development in socially defeated apoE-/- mice.

Depression is an independent risk factor of cardiovascular disease (CVD); however, the causal association remains undefined. We exposed mice to repeated social defeat (RSD) to precipitate depressive-like behaviors, and investigated the effects on atherosclerosis. Eight-week-old male apoE-/- mice were exposed to RSD by housing with a larger CD-1 mouse in a shared home cage. They were subjected to vigorous physical contact daily for 10 consecutive days and fed a high-cholesterol diet (HCD) for 6 weeks. The social interaction ratio and immobility time showed dramatic social avoidance before and after HCD feeding. Defeated mice showed higher increase in atherosclerotic lesion areas in the aortic root and entire aorta than control mice. Mean blood pressure and lipid profile were equivalent in both groups. While Ly-6G- and Mac3-positive areas in the aortic root were comparable between the groups, citrullinated histone H3 (Cit-H3)- and myeloperoxidase (MPO)-positive areas, markers of neutrophil extracellular traps (NETs), were significantly increased in the defeated mice. Treatment with DNase I completely diminished the exaggerated atherosclerosis. The proportion of peripheral blood polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC), but not of inflammatory monocytes, was markedly increased. Moreover, in vitro NETs formation from bone marrow (BM) PMN-MDSC was markedly augmented, accompanied by higher expression of Nox2 gene and reactive oxygen species. Our findings demonstrate that exposure to RSD promotes atherosclerosis by augmenting NETs formation within the plaque. This provides new insight into the underlying mechanism of depression-related CVD.

Transplantation of periaortic adipose tissue inhibits atherosclerosis in apoE-/- mice by evoking TGF-ß1-mediated anti-inflammatory response in transplanted graft.

Perivascular adipose tissue (PAT) is associated with vascular homeostasis; however, its causal effect on atherosclerosis currently remains undefined. Here, we investigated the effect of experimental PAT transplantation on atherosclerosis. The thoracic periaortic adipose tissue (tPAT) was dissected from 16-week-old wild-type mice and transplanted over the infrarenal aorta of 20-week-old apoE deficient (apoE-/-) mice fed high-cholesterol diet for 3 months. Oil-red O staining after 4 weeks showed a significant 20% decrease in the atherosclerotic lesion of suprarenal aorta compared with that of sham control mice, while that of infrarenal aorta showed no difference between the two groups. TGF-ß1 mRNA expression was significantly higher in grafted tPAT than donor tPAT, accompanied by a significant increase in serum TGF-ß1 concentration, which was inversely correlated with the suprarenal lesion area (r = -0.63, P = 0.012). Treatment with neutralizing TGF-ß antibody abrogated the anti-atherogenic effect of tPAT transplantation. Immunofluorescent analysis of grafted tPAT showed that TGF-ß-positive cells were co-localized with Mac-2-positive cells and this number was significantly increased compared with donor tPAT. There was also marked increase in mRNA expression of alternatively activated macrophages-related genes. Furthermore, the percentage of eosinophils in stromal vascular fraction of donor tPAT was much higher than that in epididymal white adipose tissue, concomitant with the significantly higher protein level of IL-4. IL-4 mRNA expression levels in grafted tPAT were increased in a time-dependent manner after tPAT transplantation. Our findings show that tPAT transplantation inhibits atherosclerosis development by exerting TGF-ß1-mediated anti-inflammatory response, which may involve alternatively activated macrophages.

Kidney-specific Sonoporation-mediated Gene Transfer.

Sonoporation can deliver agents to target local organs by systemic administration, while decreasing the associated risk of adverse effects. Sonoporation has been used for a variety of materials and in a variety of organs. Herein, we demonstrated that local sonoporation to the kidney can offer highly efficient transfer of oligonucleotides, which were systemically administrated to the tubular epithelium with high specificity. Ultrasonic wave irradiation to the kidney collapsed the microbubbles and transiently affected the glomerular filtration barrier and increased glomerular permeability. Oligonucleotides were passed through the barrier all at once and were absorbed throughout the tubular epithelium. Tumor necrosis factor alpha (TNFα), which plays a central role in renal ischemia-reperfusion injury, was targeted using small interfering RNA (siRNA) with renal sonoporation in a murine model. The reduction of TNFα expression after single gene transfer significantly inhibited the expression of kidney injury markers, suggesting that systemic administration of siRNA under temporary and local sonoporation could be applicable in the clinical setting of ischemic acute kidney injury.

Internalization of isolated functional mitochondria: involvement of macropinocytosis.

In eukaryotic cells, mitochondrial dysfunction is associated with a variety of human diseases. Delivery of exogenous functional mitochondria into damaged cells has been proposed as a mechanism of cell transplant and physiological repair for damaged tissue. We here demonstrated that isolated mitochondria can be transferred into homogeneic and xenogeneic cells by simple co-incubation using genetically labelled mitochondria, and elucidated the mechanism and the effect of direct mitochondrial transfer. Intracellular localization of exogenous mitochondria was confirmed by PCR, real-time PCR, live fluorescence imaging, three-dimensional reconstruction imaging, continuous time-lapse microscopic observation, flow cytometric analysis and immunoelectron microscopy. Isolated homogeneic mitochondria were transferred into human uterine endometrial gland-derived mesenchymal cells in a dose-dependent manner. Moreover, mitochondrial transfer rescued the mitochondrial respiratory function and improved the cellular viability in mitochondrial DNA-depleted cells and these effects lasted several days. Finally, we discovered that mitochondrial internalization involves macropinocytosis. In conclusion, these data support direct transfer of exogenous mitochondria as a promising approach for the treatment of various diseases.

Pleiotropic functions of magnetic nanoparticles for ex vivo gene transfer.

Gene transfer technique has various applications, ranging from cellular biology to medical treatments for diseases. Although nonviral vectors, such as episomal vectors, have been developed, it is necessary to improve their gene transfer efficacy. Therefore, we attempted to develop a highly efficient gene delivery system combining an episomal vector with magnetic nanoparticles (MNPs). In comparison with the conventional method using transfection reagents, polyethylenimine-coated MNPs introduced episomal vectors more efficiently under a magnetic field and could express the gene in mammalian cells with higher efficiency and for longer periods. This novel in vitro separation method of gene-introduced cells utilizing the magnetic property of MNPs significantly facilitated the separation of cells of interest. Transplanted cells in vivo were detected using magnetic resonance. These results suggest that MNPs play multifunctional roles in ex vivo gene transfer, such as improvement of gene transfer efficacy, separation of cells, and detection of transplanted cells. FROM THE CLINICAL EDITOR: This study convincingly demonstrates enhanced efficiency of gene transfer via magnetic nanoparticles. The method also enables magnetic sorting of cells positive for the transferred gene, and in vivo monitoring of the process with MRI.

Peptide-encoding gene transfer to modulate intracellular protein-protein interactions.

Peptide drug discovery has great potential, but the cell membrane is a major obstacle when the target is an intracellular protein-protein interaction (PPI). It is difficult to target PPIs with small molecules; indeed, there are no intervention tools that can target any intracellular PPI. In this study, we developed a platform that enables the introduction of peptides into cells via mRNA-based gene delivery. Peptide-length nucleic acids do not enable stable ribosome binding and exhibit little to no translation into protein. In this study, a construct was created in which the sequence encoding dihydrofolate reductase (DHFR) was placed in front of the sequence encoding the target peptide, together with a translation skipping sequence, as a sequence that meets the requirements of promoting ribosome binding and rapid decay of the translated protein. This enabled efficient translation from the mRNA encoding the target protein while preventing unnecessary protein residues. Using this construct, we showed that it can inhibit Drp1/Fis1 binding, one of the intracellular PPIs, which governs mitochondrial fission, an important aspect of mitochondrial dynamics. In addition, it was shown to inhibit pathological hyperfission, normalize mitochondrial dynamics and metabolism, and inhibit apoptosis of the mitochondrial pathway.

Dystrophin conferral using human endothelium expressing HLA-E in the non-immunosuppressive murine model of Duchenne muscular dystrophy.

Human leukocyte antigen (HLA)-E is a non-classical major histocompatibility complex class I (Ib) molecule, which plays an important role in immunosuppression. In this study, we investigated the immunomodulating effect of HLA-E in a xenogeneic system, using human placental artery-derived endothelial (hPAE) cells expressing HLA-E in a mouse model. In vitro cell lysis analysis by primed lymphocytes in combination with siRNA transfection showed that HLA-E is necessary for inhibition of the immune response. Similarly, in vivo cell implantation analysis with siRNA-mediated down-regulation of HLA-E demonstrates that HLA-E is involved in immunosuppression. As hPAE cells efficiently transdifferentiate into myoblasts/myocytes in vitro, we transplanted the cells into mdx mice, a model of Duchenne muscular dystrophy. hPAE cells conferred dystrophin to myocytes of the 'immunocompetent' mdx mice with extremely high efficiency. These findings suggest that HLA-E-expressing cells with a myogenic potential represent a promising source for cell-based therapy of patients with muscular dystrophy.

N-Cadherin is a prospective cell surface marker of human mesenchymal stem cells that have high ability for cardiomyocyte differentiation.

Mesenchymal stem cells (MSCs) are among the most promising sources of stem cells for regenerative medicine. However, the range of their differentiation ability is very limited. In this study, we explored prospective cell surface markers of human MSCs that readily differentiate into cardiomyocytes. When the cardiomyogenic differentiation potential and the expression of cell surface markers involved in heart development were analyzed using various immortalized human MSC lines, the MSCs with high expression of N-cadherin showed a higher probability of differentiation into beating cardiomyocytes. The differentiated cardiomyocytes expressed terminally differentiated cardiomyocyte-specific markers such as α-actinin, cardiac troponin T, and connexin-43. A similar correlation was observed with primary human MSCs derived from bone marrow and adipose tissue. Moreover, N-cadherin-positive MSCs isolated with N-cadherin antibody-conjugated magnetic beads showed an apparently higher ability to differentiate into cardiomyocytes than the N-cadherin-negative population. Quantitative polymerase chain reaction analyses demonstrated that the N-cadherin-positive population expressed significantly elevated levels of cardiomyogenic progenitor-specific transcription factors, including Nkx2.5, Hand1, and GATA4 mRNAs. Our results suggest that N-cadherin is a novel prospective cell surface marker of human MSCs that show a better ability for cardiomyocyte differentiation.

Large-scale cell production of stem cells for clinical application using the automated cell processing machine.

BACKGROUND: Cell-based regeneration therapies have great potential for application in new areas in clinical medicine, although some obstacles still remain to be overcome for a wide range of clinical applications. One major impediment is the difficulty in large-scale production of cells of interest with reproducibility. Current protocols of cell therapy require a time-consuming and laborious manual process. To solve this problem, we focused on the robotics of an automated and high-throughput cell culture system. Automated robotic cultivation of stem or progenitor cells in clinical trials has not been reported till date. The system AutoCulture used in this study can automatically replace the culture medium, centrifuge cells, split cells, and take photographs for morphological assessment. We examined the feasibility of this system in a clinical setting. RESULTS: We observed similar characteristics by both the culture methods in terms of the growth rate, gene expression profile, cell surface profile by fluorescence-activated cell sorting, surface glycan profile, and genomic DNA stability. These results indicate that AutoCulture is a feasible method for the cultivation of human cells for regenerative medicine. CONCLUSIONS: An automated cell-processing machine will play important roles in cell therapy and have widespread use from application in multicenter trials to provision of off-the-shelf cell products.

ZLN005 improves the survival of polymicrobial sepsis by increasing the bacterial killing via inducing lysosomal acidification and biogenesis in phagocytes.

Polymicrobial sepsis still has a high mortality rate despite the development of antimicrobial agents, elaborate strategies to protect major organs, and the investment of numerous medical resources. Mitochondrial dysfunction, which acts as the center of energy metabolism, is clearly the basis of pathogenesis. Drugs that act on PGC1α, the master regulator of mitochondrial biosynthesis, have shown useful effects in the treatment of sepsis; therefore, we investigated the efficacy of ZLN005, a PGC1α agonist, and found significant improvement in overall survival in an animal model. The mode of action of this effect was examined, and it was shown that the respiratory capacity of mitochondria was enhanced immediately after administration and that the function of TFEB, a transcriptional regulator that promotes lysosome biosynthesis and mutually enhances PGC1α, was enhanced, as was the physical contact between mitochondria and lysosomes. ZLN005 strongly supported immune defense in early sepsis by increasing lysosome volume and acidity and enhancing cargo degradation, resulting in a significant reduction in bacterial load. ZLN005 rapidly acted on two organelles, mitochondria and lysosomes, against sepsis and interactively linked the two to improve the pathogenesis. This is the first demonstration that acidification of lysosomes by a small molecule is a mechanism of action in the therapeutic strategy for sepsis, which will have a significant impact on future drug discovery.

Expression of angiotensin II receptor-like 1 in the placentas of pregnancy-induced hypertension.

Angiotensin II receptor-like 1 (APJ), a G protein-coupled receptor that was identified as a homologue of angiotensin II type 1 (AT1) receptor, exerts antagonistic effects on AT1-mediated vasoconstriction. Studies on pregnancy-induced hypertension (PIH) revealed aberrant activation of AT1 downstream signaling. In contrast, little is known about APJ in the pathophysiology of human pregnancy. In this study, we investigated APJ expression in normal human and PIH placentas. mRNAs were extracted from 50 placental villous tissues of 18 cases with severe PIH (8 late-onset, 4 early-onset, and 6 superimposed PIH) and 32 control pregnancies (including 6 preterm cases). Histopathologic studies were conducted using paraffin-embedded placental tissues from 12 control placentas (from 23 to 39 wk) and 23 PIH placentas (from 24 to 41 wk). Reverse transcriptase-polymerase chain reaction showed that APJ was cooperatively expressed with its ligand apelin and AT1 in controls and in late-onset PIH placentas but was significantly downregulated in early-onset PIH placentas with poor fetal growth. Quantitative reverse transcriptase-polymerase chain reaction analysis revealed upregulated APJ in late-onset PIH placentas but significantly downregulated APJ in early-onset PIH. In immunohistochemical staining, APJ was detected strongly in villous capillary endothelial cells and trophoblasts of late-onset PIH placentas. In contrast, APJ was poorly stained in endothelial cells of hypoplastic villi of early-onset PIH placentas. Collective data indicate that the apelin-APJ system is involved in fetoplacental circulation during human pregnancy. Impaired APJ expression in early-onset PIH placentas may reflect an aggravated placental condition with poor fetal growth.

A novel mRNA decay inhibitor abolishes pathophysiological cellular transition.

In cells, mRNA synthesis and decay are influenced by each other, and their balance is altered by either external or internal cues, resulting in changes in cell dynamics. We previously reported that it is important that an array of mRNAs that shape a phenotype are degraded before cellular transitions, such as cellular reprogramming and differentiation. In adipogenesis, the interaction between DDX6 and 4E-T had a definitive impact on the pathway in the processing body (PB). We screened a library of α-helix analogs with an alkaloid-like backbone to identify compounds that inhibit the binding between DDX6 and 4E-T proteins, which occurs between the α-helix of structured and internally disordered proteins. IAMC-00192 was identified as a lead compound. This compound directly inhibited the interaction between DDX6 and 4E-T. IAMC-00192 inhibited the temporal increase in PB formation that occurs during adipogenesis and epithelial-mesenchymal transition (EMT) and significantly suppressed these cellular transitions. In the EMT model, the half-life of preexisting mRNAs in PBs was extended twofold by the compound. The novel inhibitor of RNA decay not only represents a potentially useful tool to analyze in detail the pathological conditions affected by RNA decay and how it regulates the pathological state. The identification of this inhibitor may lead to the discovery of a first-in-class RNA decay inhibitor drug.

Amelioration of Endotoxemia by a Synthetic Analog of Omega-3 Epoxyeicosanoids.

Sepsis, a systemic inflammatory response to pathogenic factors, is a difficult to treat life-threatening condition associated with cytokine and eicosanoid storms and multi-organ damage. Omega-3 polyunsaturated fatty acids, such as eicosapentaenoic (EPA) and docosahexaenoic acid, are the precursors of potent anti-inflammatory lipid mediators, including 17,18-epoxyeicosatetraenoic acid (17,18-EEQ), the main metabolite of EPA generated by cytochrome P450 epoxygenases. Searching for novel therapeutic or preventative agents in sepsis, we tested a metabolically robust synthetic analog of 17,18-EEQ (EEQ-A) for its ability to reduce mortality, organ damage, and pro-inflammatory cytokine transcript level in a mouse model of lipopolysaccharide (LPS)-induced endotoxemia, which is closely related to sepsis. Overall survival significantly improved following preventative EEQ-A administration along with decreased transcript level of pro-inflammatory cytokines. On the other hand, the therapeutic protocol was effective in improving survival at 48 hours but insignificant at 72 hours. Histopathological analyses showed significant reductions in hemorrhagic and necrotic damage and infiltration in the liver. In vitro studies with THP-1 and U937 cells showed EEQ-A mediated repression of LPS-induced M1 polarization and enhancement of IL-4-induced M2 polarization of macrophages. Moreover, EEQ-A attenuated the LPS-induced decline of mitochondrial function in THP-1 cells, as indicated by increased basal respiration and ATP production as well as reduction of the metabolic shift to glycolysis. Taken together, these data demonstrate that EEQ-A has potent anti-inflammatory and immunomodulatory properties that may support therapeutic strategies for ameliorating the endotoxemia.

Maternal high-fat diet promotes calcified atherosclerotic plaque formation in adult offspring by enhancing transformation of VSMCs to osteochondrocytic-like phenotype.

Aim: Maternal high-fat diet (HFD) is associated with the development of cardiovascular disease (CVD) in adult offspring. Atherosclerotic vascular calcification is well documented in patients with CVD. We examined the effect of maternal HFD on calcified plaque formation. Methods and results: Seven-week-old female apo-E-/- mice (C57BL6/J) were nourished either an HFD or a normal diet (ND) a week before mating, and during gestation and lactation. Offspring of both the groups were fed a high-cholesterol diet (HCD) from 8 weeks of age. Osteogenic activity of the thoracic aorta, assessed using an ex vivo imaging system, was significantly increased after 3 months of HCD in male offspring of HFD-fed dams (O-HFD) as compared with those of ND-fed dams (O-ND). Alizarin-red-positive area in the aortic root was significantly increased after 6 months of HCD in male O-HFD as compared to that of O-ND. Plaque size and Oil Red O-positive staining were comparable between the two groups. Primary cultured vascular smooth muscle cells (VSMCs) of the thoracic aorta were treated with phosphate and interleukinL-1ß (IL-1ß) to transform them into an osteochondrocytic-like phenotype. Intracellular calcium content and alkaline phosphatase activity were markedly higher in the VSMCs of O-HFD than in O-ND. IL-1ß concentration in the supernatant of bone marrow-derived macrophages was markedly higher in O-HFD than in O-ND. Conclusion: Our findings indicate that maternal HFD accelerates the expansion of atherogenic calcification independent of plaque progression. In vitro phosphate- and IL-1ß-induced osteochondrocytic transformation of VSMCs was augmented in O-HFD. Inhibition of VSMCs, skewing toward osteochondrocytic-like cells, might be a potential therapeutic strategy for preventing maternal HFD-associated CVD development.

Repeated Social Defeat Enhances CaCl2-Induced Abdominal Aortic Aneurysm Expansion by Inhibiting the Early Fibrotic Response via the MAPK-MKP-1 Pathway.

Depression is an independent risk factor for cardiovascular disease and is significantly associated with the prevalence of abdominal aortic aneurysm (AAA). We investigated the effect of repeated social defeat (RSD) on AAA development. Eight-week-old male wild-type mice were exposed to RSD by being housed with larger CD-1 mice in a shared cage. They were subjected to vigorous physical contact. After the confirmation of depressive-like behavior, calcium chloride was applied to the infrarenal aorta of the mice. At one week, AAA development was comparable between the defeated and control mice, without any differences being observed in the accumulated macrophages or in the matrix metalloproteinase activity. At two weeks, the maximum diameter and circumference of the aneurysm were significantly increased in the defeated mice, and a significant decrease in periaortic fibrosis was also observed. Consistently, the phosphorylation of the extracellular signal-regulated kinase and the incorporation of 5-bromo-2'-deoxyuridine in the primarily cultured aortic vascular smooth muscle cells were significantly reduced in the defeated mice, which was accompanied by a substantial increase in mitogen-activated protein kinase phosphatase-1 (MKP-1). The MKP-1 mRNA and protein expression levels during AAA were much higher in the defeated mice than they were in the control mice. Our findings demonstrate that RSD enhances AAA development by suppressing periaortic fibrosis after an acute inflammatory response and imply novel mechanisms that are associated with depression-related AAA development.

Efficient transfection method using deacylated polyethylenimine-coated magnetic nanoparticles.

Low efficiencies of nonviral gene vectors, such as transfection reagent, limit their utility in gene therapy. To overcome this disadvantage, we report on the preparation and properties of magnetic nanoparticles [diameter (d) = 121.32 ± 27.36 nm] positively charged by cationic polymer deacylated polyethylenimine (PEI max), which boosts gene delivery efficiency compare with polyethylenimine (PEI), and their use for the forced expression of plasmid delivery by application of a magnetic field. Magnetic nanoparticles were coated with PEI max, which enabled their electrostatic interaction with negatively charged molecules such as plasmid. We successfully transfected 81.1 ± 4.0% of the cells using PEI max-coated magnetic nanoparticles (PEI max-nanoparticles). Along with their superior properties as a DNA delivery vehicle, PEI max-nanoparticles offer to deliver various DNA formulations in addition to traditional methods. Furthermore, efficiency of the gene transfer was not inhibited in the presence of serum in the cells. PEI max-nanoparticles may be a promising gene carrier that has high transfection efficiency as well as low cytotoxicity.