Capacitive Electrode-Based Electric Field Treatments on Redox-Toxic Iron Deposits in Transgenic AD Mouse Models: The Electroceutical Targeting of Alzheimer's Disease Feasibility Study.

Iron accumulation in the brain accelerates Alzheimer's disease progression. To cure iron toxicity, we assessed the therapeutic effects of noncontact transcranial electric field stimulation to the brain on toxic iron deposits in either the Aß fibril structure or the Aß plaque in a mouse model of Alzheimer's disease (AD) as a pilot study. A capacitive electrode-based alternating electric field (AEF) was applied to a suspension of magnetite (Fe3O4) to measure field-sensitized reactive oxygen species (ROS) generation. The increase in ROS generation compared to the untreated control was both exposure-time and AEF-frequency dependent. The frequency-specific exposure of AEF to 0.7-1.4 V/cm on a magnetite-bound Aß-fibril or a transgenic Alzheimer's disease (AD) mouse model revealed the degradation of the Aß fibril or the removal of the Aß-plaque burden and ferrous magnetite compared to the untreated control. The results of the behavioral tests show an improvement in impaired cognitive function following AEF treatment on the AD mouse model. Tissue clearing and 3D-imaging analysis revealed no induced damage to the neuronal structures of normal brain tissue following AEF treatment. In conclusion, our results suggest that the effective degradation of magnetite-bound amyloid fibrils or plaques in the AD brain by the electro-Fenton effect from electric field-sensitized magnetite offers a potential electroceutical treatment option for AD.

Less-Suture Vascular Anastomosis: Development of Alternative Protocols with Multifunctional Self-Wrapping, Transparent, Adhesive, and Elastic Biomaterials.

Blood vessel anastomosis by suture is a life-saving, yet time-consuming and labor-intensive operation. While suture-less alternatives utilizing clips or related devices are developed to address these shortcomings, suture anastomosis is still overwhelmingly used in most cases. In this study, practical "less-suture" strategies are proposed, rather than ideal "suture-less" methods, to reflect real-world clinical situations. In the case of rat artery (d = 0.64 mm) anastomosis, the less-suture anastomosis involves the application of thin, adhesive, transparent, and self-wrapping films to the site. This surprisingly reduces the number of stitches required from ten (without films) to four (with films), saving 27 min of operating time per vessel. Furthermore, the decreased number of stitches largely alleviates fibrosis-mediated wall-thickening. Thus, a less-suture strategy is particularly useful for anastomosis of multiple vessels in emergency conditions and small-diameter vessels.

Structural changes in the retina after implantation of subretinal three-dimensional implants in mini pigs.

The retinal structural changes after subretinal implantation of three-dimensional (3D) microelectrodes were investigated in a mini pig. Three types of electrode were implanted into the subretinal spaces of nine mini pigs: 75-µm-high 3D electrodes on a 200-µm-thick right-angled polydimethylsiloxane (PDMS) substrate (group 1); a 140-µm-thick sloped PDMS substrate without electrodes (group 2); and a 140-µm-thick sloped PDMS substrate with 20-µm-high 3D electrodes (group 3). One mini pig was used as a control. Spectral domain-optical coherence tomography (SD-OCT) images were obtained at baseline and 2, 6, and 12 weeks post-surgery. Retinal specimens were immunostained using a tissue-clearing method 3 months post-implantation. The 75-µm-high 3D electrodes progressively penetrated the inner nuclear layer (INL) and touched the inner plexiform layer (IPL) 2 weeks post-surgery. At 6 weeks post-operatively, the electrodes were in contact with the nerve-fiber layer, accompanied by a severe fibrous reaction. In the other groups, the implants remained in place without subretinal migration. Immunostaining showed that retinal ganglion and bipolar cells were preserved without fibrosis over the retinal implants in groups 2 and 3 during the 12-week implantation period. In summary, SD-OCT and immunohistology results showed differences in the extent of reactions, such as fibrosis over the implants and penetration of the electrodes into the inner retinal layer depending on different types of electrodes. A sloped substrate performed better than a right-angled substrate in terms of retinal preservation over the implanted electrodes. The 20-µm-high electrodes showed better structural compatibility than the 75-µm-high 3D electrodes. There was no significant difference between the results of sloped implants without electrodes and 20-µm-high 3D electrodes, indicating that the latter had no adverse effects on retinal tissue.

Neurotoxicity of phenylalanine on human iPSC-derived cerebral organoids.

Phenylketonuria (PKU) is a common genetic metabolic disorder that causes phenylalanine accumulation in the blood. The most serious symptoms are related to the brain, as intellectual disability, seizure, and microcephaly are commonly found in poorly treated PKU patients and the babies of maternal PKU. However, the mechanism of hyperphenylalaninemia on human neurodevelopment is still unclear. Here we utilized human induced pluripotent stem cell (iPSC)-derived cerebral organoids to investigate the neurotoxicity of hyperphenylalaninemia. Cerebral organoids at days 40 or 100 were treated with different concentrations of phenylalanine for 5 days. After phenylalanine treatments, the cerebral organoids displayed alterations in organoid size, induction of apoptosis, and depletion of neural progenitor cells. However, phenylalanine did not have an impact on neurons and glia, including astrocytes, immature oligodendrocytes, and mature oligodendrocytes. Remarkably, a reduction in the thickness of the cortical rosettes and a decrease in myelination at the intermediate zone were inspected with the elevated phenylalanine concentrations. RNA-seq of phenylalanine-treated organoids revealed that gene sets related to apoptosis, p53 signaling pathway, and TNF signaling pathway via NF-kB were enriched in upregulated genes, while those related to cell cycle and amino acid metabolism were enriched in downregulated genes. In addition, there were several microcephaly disease genes, such as ASPM, LMNB1, and CENPE, ranked at the top of the downregulated genes. These findings indicate that phenylalanine exposure may contribute to microcephaly, abnormal cortical expansion, and myelination lesions in the developing human brain.

Effect of gemigliptin on cardiac ischemia/reperfusion and spontaneous hypertensive rat models.

Diabetes is associated with an increased risk of cardiovascular complications. Dipeptidyl peptidase-4 (DPP-IV) inhibitors are used clinically to reduce high blood glucose levels as an antidiabetic agent. However, the effect of the DPP-IV inhibitor gemigliptin on ischemia/reperfusion (I/R)-induced myocardial injury and hypertension is unknown. In this study, we assessed the effects and mechanisms of gemigliptin in rat models of myocardial I/R injury and spontaneous hypertension. Gemigliptin (20 and 100 mg/kg/d) or vehicle was administered intragastrically to Sprague-Dawley rats for 4 weeks before induction of I/R injury. Gemigliptin exerted a preventive effect on I/R injury by improving hemodynamic function and reducing infarct size compared to the vehicle control group. Moreover, administration of gemigliptin (0.03% and 0.15%) powder in food for 4 weeks reversed hypertrophy and improved diastolic function in spontaneously hypertensive rats. We report here a novel effect of the gemigliptin on I/R injury and hypertension.

Topographical Visualization of the Reciprocal Projection between the Medial Septum and the Hippocampus in the 5XFAD Mouse Model of Alzheimer's Disease.

It is widely known that the degeneration of neural circuits is prominent in the brains of Alzheimer's disease (AD) patients. The reciprocal connectivity of the medial septum (MS) and hippocampus, which constitutes the septo-hippocampo-septal (SHS) loop, is known to be associated with learning and memory. Despite the importance of the reciprocal projections between the MS and hippocampus in AD, the alteration of bidirectional connectivity between two structures has not yet been investigated at the mesoscale level. In this study, we adopted AD animal model, five familial AD mutations (5XFAD) mice, and anterograde and retrograde tracers, BDA and DiI, respectively, to visualize the pathology-related changes in topographical connectivity of the SHS loop in the 5XFAD brain. By comparing 4.5-month-old and 14-month-old 5XFAD mice, we successfully identified key circuit components of the SHS loop altered in 5XFAD brains. Remarkably, the SHS loop began to degenerate in 4.5-month-old 5XFAD mice before the onset of neuronal loss. The impairment of connectivity between the MS and hippocampus was accelerated in 14-month-old 5XFAD mice. These results demonstrate, for the first time, topographical evidence for the degradation of the interconnection between the MS and hippocampus at the mesoscale level in a mouse model of AD. Our results provide structural and functional insights into the interconnectivity of the MS and hippocampus, which will inform the use and development of various therapeutic approaches that target neural circuits for the treatment of AD.

Red Ginseng Attenuates Aß-Induced Mitochondrial Dysfunction and Aß-mediated Pathology in an Animal Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disease and is characterized by neurodegeneration and cognitive deficits. Amyloid beta (Aß) peptide is known to be a major cause of AD pathogenesis. However, recent studies have clarified that mitochondrial deficiency is also a mediator or trigger for AD development. Interestingly, red ginseng (RG) has been demonstrated to have beneficial effects on AD pathology. However, there is no evidence showing whether RG extract (RGE) can inhibit the mitochondrial deficit-mediated pathology in the experimental models of AD. The effects of RGE on Aß-mediated mitochondrial deficiency were investigated in both HT22 mouse hippocampal neuronal cells and the brains of 5XFAD Aß-overexpressing transgenic mice. To examine whether RGE can affect mitochondria-related pathology, we used immunohistostaining to study the effects of RGE on Aß accumulation, neuroinflammation, neurodegeneration, and impaired adult hippocampal neurogenesis in hippocampal formation of 5XFAD mice. In vitro and in vivo findings indicated that RGE significantly improves Aß-induced mitochondrial pathology. In addition, RGE significantly ameliorated AD-related pathology, such as Aß deposition, gliosis, and neuronal loss, and deficits in adult hippocampal neurogenesis in brains with AD. Our results suggest that RGE may be a mitochondria-targeting agent for the treatment of AD.

Endocytosis of KATP Channels Drives Glucose-Stimulated Excitation of Pancreatic ß Cells.

Insulin secretion from pancreatic ß cells in response to high glucose (HG) critically depends on the inhibition of KATP channel activity in HG. It is generally believed that HG-induced effects are mediated by the increase in intracellular ATP, but here, we showed that, in INS-1 cells, endocytosis of KATP channel plays a major role. Upon HG stimulation, resting membrane potential depolarized by 30.6 mV (from -69.2 to -38.6 mV) and KATP conductance decreased by 91% (from 0.243 to 0.022 nS/pF), whereas intracellular ATP was increased by only 47%. HG stimulation induced internalization of KATP channels, causing a significant decrease in surface channel density, and this decrease was completely abolished by inhibiting endocytosis using dynasore, a dynamin inhibitor, or a PKC inhibitor. These drugs profoundly inhibited HG-induced depolarization. Our results suggest that the control of KATP channel surface density plays a greater role than ATP-dependent gating in regulating ß cell excitability.

Rac-mediated actin remodeling and myosin II are involved in KATP channel trafficking in pancreatic ß-cells.

AMP-activated protein kinase (AMPK) is a metabolic sensor activated during metabolic stress and it regulates various enzymes and cellular processes to maintain metabolic homeostasis. We previously reported that activation of AMPK by glucose deprivation (GD) and leptin increases KATP currents by increasing the surface levels of KATP channel proteins in pancreatic ß-cells. Here, we show that the signaling mechanisms that mediate actin cytoskeleton remodeling are closely associated with AMPK-induced KATP channel trafficking. Using F-actin staining with Alexa 633-conjugated phalloidin, we observed that dense cortical actin filaments present in INS-1 cells cultured in 11 mM glucose were disrupted by GD or leptin treatment. These changes were blocked by inhibiting AMPK using compound C or siAMPK and mimicked by activating AMPK using AICAR, indicating that cytoskeletal remodeling induced by GD or leptin was mediated by AMPK signaling. AMPK activation led to the activation of Rac GTPase and the phosphorylation of myosin regulatory light chain (MRLC). AMPK-dependent actin remodeling induced by GD or leptin was abolished by the inhibition of Rac with a Rac inhibitor (NSC23766), siRac1 or siRac2, and by inhibition of myosin II with a myosin ATPase inhibitor (blebbistatin). Immunocytochemistry, surface biotinylation and electrophysiological analyses of KATP channel activity and membrane potentials revealed that AMPK-dependent KATP channel trafficking to the plasma membrane was also inhibited by NSC23766 or blebbistatin. Taken together, these results indicate that AMPK/Rac-dependent cytoskeletal remodeling associated with myosin II motor function promotes the translocation of KATP channels to the plasma membrane in pancreatic ß-cells.

Ca(2+) clearance by plasmalemmal NCLX, Li(+)-permeable Na(+)/Ca(2+) exchanger, is required for the sustained exocytosis in rat insulinoma INS-1 cells.

Na(+)/Ca(2+) exchangers are key players for Ca(2+) clearance in pancreatic ß-cells, but their molecular determinants and roles in insulin secretion are not fully understood. In the present study, we newly discovered that the Li(+)-permeable Na(+)/Ca(2+) exchangers (NCLX), which were known as mitochondrial Na(+)/Ca(2+) exchangers, contributed to the Na(+)-dependent Ca(2+) movement across the plasma membrane in rat INS-1 insulinoma cells. Na(+)/Ca(2+) exchange activity by NCLX was comparable to that by the Na(+)/Ca(2+) exchanger, NCX. We also confirmed the presence of NCLX proteins on the plasma membrane using immunocytochemistry and cell surface biotinylation experiments. We further investigated the role of NCLX on exocytosis function by measuring the capacitance increase in response to repetitive depolarization. Small interfering (si)RNA-mediated downregulation of NCLX did not affect the initial exocytosis, but significantly suppressed sustained exocytosis and recovery of exocytosis. XIP (NCX inhibitory peptide) or Na(+) replacement for inhibiting Na(+)-dependent Ca(2+) clearance also selectively suppressed sustained exocytosis. Consistent with the idea that sustained exocytosis requires ATP-dependent vesicle recruitment, mitochondrial function, assessed by mitochondrial membrane potential (ΔΨ), was impaired by siNCLX or XIP. However, depolarization-induced exocytosis was hardly affected by changes in intracellular Na(+) concentration, suggesting a negligible contribution of mitochondrial Na(+)/Ca(2+) exchanger. Taken together, our data indicate that Na(+)/Ca(2+) exchanger-mediated Ca(2+) clearance mediated by NCLX and NCX is crucial for optimizing mitochondrial function, which in turn contributes to vesicle recruitment for sustained exocytosis in pancreatic ß-cells.

Protection of pancreatic ß-cells against glucotoxicity by short-term treatment with GLP-1.

Glucagon-like peptide-1 (GLP-1) reduces pancreatic ß-cell apoptosis in type 2 diabetes. Glucotoxiciy is a main cause of ß-cell apoptosis in type 2 diabetes. The aims of this study were to investigate the anti-apoptotic mechanisms of GLP-1 against glucotoxicity and whether physiological short-term treatment with GLP-1 can protect ß-cells from glucotoxicity-induced apoptosis. GLP-1 treatment for only 30 min alleviated high glucose-induced ß-cell apoptosis. The effect of GLP-1 was related with phosphoinositide 3-kinase (PI3K)/AKT-S473 phosphorylation. The increase in pAKT-S473 led to suppression of FoxO-1. GLP-1-induced AKT-S473 activation and FoxO-1 suppression were abolished by the selective inactivation of mTOR complex (mTORC) 2 using small interfering RNA directed towards the rapamycin-insensitive companion of mTOR. The protective effect of GLP-1 on ß-cell apoptosis was also abolished by the selective inactivation of mTORC2. Hence, the protective effect of GLP-1 against glucotoxicity may be mediated by FoxO-1 suppression through the PI3K/mTORC2/AKT-S473 phosphorylation. This report provides evidence that short-term treatment with GLP-1 is beneficial to protect against glucotoxicity-induced ß-cell apoptosis.

Improved application of the electrophoretic tissue clearing technology, CLARITY, to intact solid organs including brain, pancreas, liver, kidney, lung, and intestine.

BACKGROUND: Mapping of tissue structure at the cellular, circuit, and organ-wide scale is important for understanding physiological and biological functions. A bio-electrochemical technique known as CLARITY used for three-dimensional anatomical and phenotypical mapping within transparent intact tissues has been recently developed. This method provided a major advance in understanding the structure-function relationships in circuits of the nervous system and organs by using whole-body clearing. Thus, in the present study, we aimed to improve the original CLARITY procedure and developed specific CLARITY protocols for various intact organs. RESULTS: We determined the optimal conditions for reducing bubble formation, discoloration, and depositing of black particles on the surface of tissue, which allowed production of clearer organ images. We also determined the appropriate replacement cycles of clearing solution for each type of organ, and convincingly demonstrated that 250-280 mA is the ideal range of electrical current for tissue clearing. We then acquired each type of cleared organs including brain, pancreas, liver, lung, kidney, and intestine. Additionally, we determined the images of axon fibers of hippocampal region, the Purkinje layer of cerebellum, and vessels and cellular nuclei of pancreas. CONCLUSIONS: CLARITY is an innovative biochemical technology for the structural and molecular analysis of various types of tissue. We developed improved CLARITY methods for clearing of the brain, pancreas, lung, intestine, liver, and kidney, and identified the appropriate experimental conditions for clearing of each specific tissue type. These optimized methods will be useful for the application of CLARITY to various types of organs.

AMPK regulates K(ATP) channel trafficking via PTEN inhibition in leptin-treated pancreatic ß-cells.

Leptin regulates pancreatic ß-cell excitability through AMP-activated protein kinase (AMPK)-mediated ATP-sensitive potassium (KATP) channel trafficking. However, the signaling components connecting AMPK to KATP channel trafficking are not identified. In this study, we discovered that AMPK inhibits phosphatase and tensin homologue (PTEN) via glycogen synthase kinase 3ß (GSK3ß) and this signaling pathway is crucial for KATP channel trafficking in leptin-treated pancreatic ß-cells. Pharmacologic or genetic inhibition of AMPK or GSK3ß, but not casein kinase 2 (CK2), impaired leptin-induced PTEN inactivation and thereby KATP channel trafficking. The PTEN mutant lacking both protein and lipid phosphatase activity is sufficient to induce KATP channel trafficking without leptin. These results present a novel signaling mechanism that underlies leptin regulation of KATP channel trafficking in pancreatic ß-cells. Our findings assist in gaining a broader perspective on the peripheral action of leptin on pancreatic ß-cell physiology and glucose homeostasis.

Leptin promotes K(ATP) channel trafficking by AMPK signaling in pancreatic ß-cells.

Leptin is a pivotal regulator of energy and glucose homeostasis, and defects in leptin signaling result in obesity and diabetes. The ATP-sensitive potassium (K(ATP)) channels couple glucose metabolism to insulin secretion in pancreatic ß-cells. In this study, we provide evidence that leptin modulates pancreatic ß-cell functions by promoting K(ATP) channel translocation to the plasma membrane via AMP-activated protein kinase (AMPK) signaling. K(ATP) channels were localized mostly to intracellular compartments of pancreatic ß-cells in the fed state and translocated to the plasma membrane in the fasted state. This process was defective in leptin-deficient ob/ob mice, but restored by leptin treatment. We discovered that the molecular mechanism of leptin-induced AMPK activation involves canonical transient receptor potential 4 and calcium/calmodulin-dependent protein kinase kinase ß. AMPK activation was dependent on both leptin and glucose concentrations, so at optimal concentrations of leptin, AMPK was activated sufficiently to induce K(ATP) channel trafficking and hyperpolarization of pancreatic ß-cells in a physiological range of fasting glucose levels. There was a close correlation between phospho-AMPK levels and ß-cell membrane potentials, suggesting that AMPK-dependent K(ATP) channel trafficking is a key mechanism for regulating ß-cell membrane potentials. Our results present a signaling pathway whereby leptin regulates glucose homeostasis by modulating ß-cell excitability.

Sustained CaMKII activity mediates transient oxidative stress-induced long-term facilitation of L-type Ca(2+) current in cardiomyocytes.

Oxidative stress remodels Ca(2+) signaling in cardiomyocytes, which promotes altered heart function in various heart diseases. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) was shown to be activated by oxidation, but whether and how CaMKII links oxidative stress to pathophysiological long-term changes in Ca(2+) signaling remain unknown. Here, we present evidence demonstrating the role of CaMKII in transient oxidative stress-induced long-term facilitation (LTF) of L-type Ca(2+) current (I(Ca,L)) in rat cardiomyocytes. A 5-min exposure of 1mM H(2)O(2) induced an increase in I(Ca,L), and this increase was sustained for ~1h. The CaMKII inhibitor KN-93 fully reversed H(2)O(2)-induced LTF of I(Ca,L), indicating that sustained CaMKII activity underlies this oxidative stress-induced memory. Simultaneous inhibition of oxidation and autophosphorylation of CaMKII prevented the maintenance of LTF, suggesting that both mechanisms contribute to sustained CaMKII activity. We further found that sarcoplasmic reticulum Ca(2+) release and mitochondrial ROS generation have critical roles in sustaining CaMKII activity via autophosphorylation- and oxidation-dependent mechanisms. Finally, we show that long-term remodeling of the cardiac action potential is induced by H(2)O(2) via CaMKII. In conclusion, CaMKII and mitochondria confer oxidative stress-induced pathological cellular memory that leads to cardiac arrhythmia.

L-type Ca(2+) channel facilitation mediated by H(2)O(2)-induced activation of CaMKII in rat ventricular myocytes.

The Ca(2+)-dependent facilitation (CDF) of L-type Ca(2+) channels, a major mechanism for force-frequency relationship of cardiac contraction, is mediated by Ca(2+)/CaM-dependent kinase II (CaMKII). Recently, CaMKII was shown to be activated by methionine oxidation. We investigated whether oxidation-dependent CaMKII activation is involved in the regulation of L-type Ca(2+) currents (I(Ca,L)) by H(2)O(2) and whether Ca(2+) is required in this process. Using patch clamp, I(Ca)(,L) was measured in rat ventricular myocytes. H(2)O(2) induced an increase in I(Ca,L) amplitude and slowed inactivation of I(Ca)(,L). This oxidation-dependent facilitation (ODF) of I(Ca)(,L) was abolished by a CaMKII blocker KN-93, but not by its inactive analog KN-92, indicating that CaMKII is involved in ODF. ODF was not affected by replacement of external Ca(2+) with Ba(2+) or presence of EGTA in the internal solutions. However, ODF was abolished by adding BAPTA to the internal solution or by depleting sarcoplasmic reticulum (SR) Ca(2+) stores using caffeine and thapsigargin. Alkaline phosphatase, beta-iminoadenosine 5'-triphosphate (AMP-PNP), an autophosphorylation inhibitor autocamtide-2-related inhibitory peptide (AIP), or a catalytic domain blocker (CaM-KIINtide) did not affect ODF. In conclusion, oxidation-dependent facilitation of L-type Ca(2+) channels is mediated by oxidation-dependent CaMKII activation, in which local Ca(2+) increases induced by SR Ca(2+) release is required.

Glucose deprivation regulates KATP channel trafficking via AMP-activated protein kinase in pancreatic beta-cells.

OBJECTIVE: AMP-activated protein kinase (AMPK) and the ATP-sensitive K(+) (K(ATP)) channel are metabolic sensors that become activated during metabolic stress. AMPK is an important regulator of metabolism, whereas the K(ATP) channel is a regulator of cellular excitability. Cross talk between these systems is poorly understood. RESEARCH DESIGN AND METHODS: Rat pancreatic beta-cells or INS-1 cells were pretreated for 2 h at various concentrations of glucose. Maximum K(ATP) conductance (G(max)) was monitored by whole-cell measurements after intracellular ATP washout using ATP-free internal solutions. K(ATP) channel activity (NPo) was monitored by inside-out patch recordings in the presence of diazoxide. Distributions of K(ATP) channel proteins (Kir6.2 and SUR1) were examined using immunofluorescence imaging and surface biotinylation studies. Insulin secretion from rat pancreatic islets was measured using an enzyme immunoassay. RESULTS: G(max) and NPo in cells pretreated with glucose-free or 3 mmol/l glucose solutions were significantly higher than in cells pretreated in 11.1 mmol/l glucose solutions. Immunofluorescence imaging and biotinylation studies revealed that glucose deprivation induced an increase in the surface level of Kir6.2 without affecting the total cellular amount. Increases in G(max) and the surface level of Kir6.2 were inhibited by compound C, an AMPK inhibitor, and siAMPK transfection. The effects of glucose deprivation on K(ATP) channels were mimicked by an AMPK activator. Glucose deprivation reduced insulin secretion, but this response was attenuated by compound C. CONCLUSIONS: K(ATP) channel trafficking is regulated by energy status via AMPK, and this mechanism may play a key role in inhibiting insulin secretion under low energy status.

Cleavage and functional loss of human apolipoprotein E by digestion of matrix metalloproteinase-14.

By means of a degradomic approach applying proteomic techniques, we previously suggested that apolipoprotein E (apoE) is a substrate of matrix metalloproteinase-14 (MMP-14). Here we confirm that apoE is, in fact, a substrate of MMP-14 and also of MMP-7 and MMP-2 to a lesser extent. The 34 kDa apoE protein was initially processed by MMP-14 into fragments with molecular masses of 28, 23, 21, and 11 kDa. MMP-14 cleavage sites within the apoE protein were determined by C-terminal labeling of MMP-14-digested apoE fragments with isotope ((18)O/(16)O = 1:1) and identification of the doublet fragments or peptides showing 2 Da difference by MS, along with N-terminal sequencing of the fragments. It was determined that the primary MMP-14 cleavage sites were A(176)-I(177), P(183)-L(184), P(202)-L(203), and Q(249)-I(250). The MMP-14-mediated cleavage of apoE was consistent regardless of whether apoE existed in its lipid-bound or lipid-free form. Upon digestion with MMP-14, apoE loses its ability to suppress the platelet-derived growth factor-induced migration of rat vascular smooth muscle cells. Considering the important role of apoE for lipid metabolism and atherosclerosis protection, our findings suggest that MMP-14 plays an essential role for the development of hyperlipidemia and atherosclerosis as a result of degradation of apoE.

The function, composition, and particle size of high-density lipoprotein were severely impaired in an oliguric phase of hemorrhagic fever with renal syndrome patients.

BACKGROUND: Patients suffering from hemorrhagic fever with renal syndrome (HFRS) often showed strikingly reduced high-density lipoprotein (HDL)-cholesterol levels during the oliguric phase, indicating severe alterations in lipoprotein metabolism. OBJECTIVE: To compare changes in the functions and composition of HDL, lipoprotein metabolism parameters were analyzed in the sera of HFRS patients in the oliguric phase and after recovery. METHODS: The serum cholesterol, triglyceride (TG), and lipoprotein/apolipoprotein profiles of HFRS patients in the oliguric and recovery phases were compared with those of normal reference sera. The activities of HDL-associated enzymes, lecithin:cholesterol acyltransferase (LCAT), and paraoxonase (PON) were also assessed. RESULTS: In the oliguric phase, serum cholesterol was substantially decreased and serum TG was increased. As observed by electron microscopy, the sizes of the HDL particles from the HFRS patients were smaller than those seen in the reference sera, with more heterogeneous distribution. Serum amyloid A (SAA) and apolipoprotein (apo) C-III were overexpressed in the oliguric phase, particularly in the HDL fraction. However, in immunodetection, the levels of apoA-I in the HDL(2) and HDL(3) of the HFRS patients were lower than those of the reference HDL. Serum LCAT and PON activities were reduced significantly in the oliguric phase, which is associated with a reduction in HDL-cholesterol levels and HDL particle size. CONCLUSION: Overexpression of both apoC-III and apoSAA in HDL and attenuated serum LCAT and PON activity were observed during the oliguric phase in HFRS patients. These results demonstrate that structural, functional, and compositional changes of HDL occurred to a substantial degree in the oliguric phase.

A point mutant of apolipoprotein A-I, V156K, exhibited potent anti-oxidant and anti-atherosclerotic activity in hypercholesterolemic C57BL/6 mice.

In our previous study, two point mutants of apolipoprotein A-I, designated V156K and A158E, revealed peculiar characteristics in their lipid-free and lipid-bound states. In order to determine the putative therapeutic potential of these mutants, several in vitro and in vivo evaluations were conducted. In the lipid-free state, V156K showed more profound antioxidant activity against LDL oxidation than did the wildtype (WT) or A158E variants in an in vitro assay. In the lipid-bound state, V156K-rHDL showed an enhanced cholesterol delivery activity to HepG2 cells in a time-dependent manner, as compared to WT-rHDL, A158E-rHDL, and R173C-rHDL. We assessed the physiological activities of the mutants in circulation, using hypercholesterolemic mice (C57BL6/J). Palmitoyloleoyl phosphatidylcholine (POPC)-rHDL preparations containing each of the apoA-I variants were injected into the mice at a dosage of 30 mg of apoA-I/kg of body weight. Forty eight hours after injection, the sera of the V156K-rHDL injected group showed the most potent antioxidant abilities in the ferric acid removal assay. The V156K-rHDL- or R173C-rHDL-injected mice showed no atherosclerotic lesions and manifested striking increases in their serum apo-E levels, as compared to the mice injected with WT-rHDL or A158E-rHDL. In conclusion, V156K-rHDL exhibited the most pronounced antioxidant activity and anti-atherosclerotic activity, both in vitro and in vivo. These results support the notion that HDL-therapy may prove beneficial due to its capacity to induce accelerated cholesterol excretion, as well as its enhanced antioxidant and anti-inflammatory effects and lesion regression effect.