PCSK9 inhibitors suppress oxidative stress and inflammation in atherosclerotic development by promoting macrophage autophagy.

OBJECTIVES: Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, a novel class of cholesterol-lowering drugs, can reduce atherosclerosis independent of systemic lipid changes. However, the mechanism by which PCSK9 inhibition protects against arteriosclerosis has not been fully elucidated. Recent evidence has demonstrated a correlation between PCSK9 inhibitors and oxidative stress, which accelerates atherosclerotic development. Moreover, an increasing number of studies have shown that autophagy protects the vasculature against atherosclerosis. Therefore, the aims of this study were to investigate the effect of PCSK9 inhibition on oxidative stress and autophagy in atherosclerosis and determine whether autophagy regulates PCSK9 inhibition-mediated oxidative stress and inflammation in macrophages. METHODS: Male apolipoprotein E (ApoE)-/- mice were fed a high-fat diet (HFD) for 8 weeks and then received the PCSK9 inhibitor (evolocumab), vehicle, or evolocumab plus chloroquine (CQ) for another 8 weeks. ApoE-/- mice in the control group were fed a regular (i.e., non-high-fat) diet for 16 weeks. Additional in vitro experiments were performed in oxidized low-density lipoprotein (ox-LDL)-treated human acute monocytic leukemia cell line THP-1-derived macrophages to mimic the pathophysiologic process of atherosclerosis. RESULTS: PCSK9 inhibitor treatment reduced oxidative stress, lipid deposition, and plaque lesion area and induced autophagy in HFD-fed ApoE-/- mice. Most importantly, the administration of chloroquine (CQ), an autophagy inhibitor, significantly reduced the beneficial effects of PCSK9-inhibitor treatment on oxidative stress, lipid accumulation, inflammation, and atherosclerotic lesions in HFD-fed ApoE-/- mice. The in vitro experiments further showed that the PCSK9 inhibitor enhanced autophagic flux in ox-LDL-treated THP-1-derived macrophages, as indicated by increases in the numbers of autophagosomes and autolysosomes. Moreover, the autophagy inhibitor CQ also reduced PCSK9 inhibition-mediated protection against oxidative stress, generation of reactive oxygen species (ROS) and inflammation in ox-LDL-treated THP-1-derived macrophages. CONCLUSIONS: This study reveals a novel protective mechanism by which PCSK9 inhibition enhances autophagy and thereby reduces oxidative stress and inflammation in atherosclerosis.

Correlative analysis of lung CT findings in patients with Birt-Hogg-Dubé Syndrome and the occurrence of spontaneous pneumothorax: a preliminary study.

BACKGROUND: The diagnosis of patients with Birt-Hogg-Dubé (BHD) syndrome is always delayed (even for more than 10 years). Improving the understanding and diagnosis of this disease is vital for clinicians and radiologists. In this study we presented the chest computed tomography (CT) findings of BHD syndrome and offered suggestions for BHD cases with spontaneous pneumothorax. METHODS: Twenty-six BHD patients from 11 families (10 men, 16 women; mean age: 46 ± 12 years, 20-68 years) were included. The clinical features of the patients included pneumothorax, renal lesions, and skin lesions. Twenty-three patients underwent chest CT imaging. The cyst condition of each patient derived from reconstructed chest CT imaging was recorded, including the cyst number, size, volume, pattern, and distribution. RESULTS: Pneumothorax occurred in 54% (14/26) of patients. Among them, 43% (6/14) had pneumothorax more than twice. However, typical skin and renal lesions were absent. Four patients had renal hamartoma. CT showed that 23 (100%) patients had lung cysts. Pulmonary cysts were bilateral and multiple, round, irregular, or willow-like. And 93.6% of the large cysts (long-axis diameter ≥ 20 mm) were under the pleura, and near the mediastinum and spine. The long-axis diameter, short-axis diameter and volume of the largest cysts were associated with the occurrence of pneumothorax (all P < 0.05). CONCLUSIONS: Chest CT imaging can reveal some characteristic features of BHD syndrome. The occurrence of pneumothorax in BHD patients is closely related to their pulmonary cystic lesions.

Single-Injection Midpoint Transverse Process-to- Pleura Block Versus Thoracic Paravertebral Block for Postoperative Analgesia After Uniportal Video-Assisted Thoracoscopic Surgery: A Randomized Controlled Trial.

OBJECTIVES: The effect of midpoint transverse process-to-pleura block (MTPB) is unclear. The authors compared the analgesic characteristics of MTPB with those of thoracic paravertebral nerve block (TPVB) in patients undergoing uniportal video-assisted thoracoscopic surgery (uVATS) and examined the 2 types of blocks for noninferiority. DESIGN: A randomized, controlled trial. SETTING: A single-institution, university hospital. PARTICIPANTS: The study comprised 82 patients between 18 and 75 years of age. Eighty-one patients were included for final analysis. INTERVENTIONS: Patients were randomly allocated to either the MTPB group (Group M) or the TPVB group (Group P). MEASUREMENTS AND MAIN RESULTS: The primary outcome was the mean difference in the postoperative visual analog scale (VAS) score between Group M and Group P at rest and at 12 hours. The secondary outcomes included VAS scores during rest and coughing, nerve block performance, intraoperative consumption of anesthetics and vasoactive medication, time at first use of patient-controlled intravenous analgesia (PCIA), number of uses of PCIA, consumption of sufentanil in PCIA, and side effects. The mean difference in VAS score in the MTPB and TPVB group at rest and 12 hours postoperatively was 0.5 (95% confidence interval, -0.26 to 0.36). There was no significant difference in the time at first use of PCIA, which was 12 (10, 12) hours and 11 (10, 12) hours in Group M and Group P, respectively. The depth of puncture was shallower, and the time to perform block was shorter in Group M compared with Group P (p < 0.001). CONCLUSIONS: The analgesic effect of single-level-injection MTPB is noninferior to that of TPVB in patients undergoing uVATS. The target point of the needle in MTPB is shallower and farther away from the pleura compared with TPVB. For these reasons, it is quicker and safer and, thus, may be preferable.

Lysophosphatidic Acid Is Associated With Cardiac Dysfunction and Hypertrophy by Suppressing Autophagy via the LPA3/AKT/mTOR Pathway.

Background: Lysophosphatidic acid (LPA), as a phospholipid signal molecule, participates in the regulation of various biological functions. Our previous study demonstrated that LPA induces cardiomyocyte hypertrophy in vitro; however, the functional role of LPA in the post-infarct heart remains unknown. Growing evidence has demonstrated that autophagy is involved in regulation of cardiac hypertrophy. The aim of the current work was to investigate the effects of LPA on cardiac function and hypertrophy during myocardial infarction (MI) and determine the regulatory role of autophagy in LPA-induced cardiomyocyte hypertrophy. Methods: In vivo experiments were conducted in Sprague-Dawley rats subjected to MI surgery or a sham operation, and rats with MI were assigned to receive an intraperitoneal injection of LPA (1 mg/kg) or vehicle for 5 weeks. The in vitro experiments were conducted in H9C2 cardiomyoblasts. Results: LPA treatment aggravated cardiac dysfunction, increased cardiac hypertrophy, and reduced autophagy after MI in vivo. LPA suppressed autophagy activation, as indicated by a decreased LC3II-to-LC3I ratio, increased p62 expression, and reduced autophagosome formation in vitro. Rapamycin, an autophagy enhancer, attenuated LPA-induced autophagy inhibition and H9C2 cardiomyoblast hypertrophy, while autophagy inhibition with Beclin1 siRNA did not further enhance the hypertrophic response in LPA-treated cardiomyocytes. Moreover, we demonstrated that LPA suppressed autophagy through the AKT/mTOR signaling pathway because mTOR and PI3K inhibitors significantly prevented LPA-induced mTOR phosphorylation and autophagy inhibition. In addition, we found that knockdown of LPA3 alleviated LPA-mediated autophagy suppression in H9C2 cardiomyoblasts, suggesting that LPA suppresses autophagy through activation of the LPA3 and AKT/mTOR pathways. Conclusion: These findings suggest that LPA plays an important role in mediating cardiac dysfunction and hypertrophy after a MI, and that LPA suppresses autophagy through activation of the LPA3 and AKT/mTOR pathways to induce cardiomyocyte hypertrophy.

Lysophosphatidic Acid Pretreatment Attenuates Myocardial Ischemia/Reperfusion Injury in the Immature Hearts of Rats.

The cardioprotection of the immature heart during cardiac surgery remains controversial due to the differences between the adult heart and the newborn heart. Lysophosphatidic acid (LPA) is a small bioactive molecule with diverse functions including cell proliferation and survival via its receptor: LPA1-LPA6. We previously reported that the expressions of LPA1 and LPA3 in rat hearts were much higher in immature hearts and then declined rapidly with age. In this study, we aimed to investigate whether LPA signaling plays a potential protective role in immature hearts which had experienced ischemia/reperfusion (I/R) injury. The results showed that in Langendorff-perfused immature rat hearts (2 weeks), compared to I/R group, LPA pretreatment significantly enhanced the cardiac function, attenuated myocardial infarct size and CK-MB release, decreased myocardial apoptosis and increased the expression of pro-survival signaling molecules. All these effects could be abolished by Ki16425, an antagonist to LPA1 and LPA3. Similarly, LPA pretreatment protected H9C2 from hypoxia-reoxygenation (H/R) induced apoptosis and necrosis in vitro. The mechanisms underlying the anti-apoptosis effects were related to activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinas B (AKT) signaling pathways as well as phosphorylation of the downstream effector of AKT, glycogen synthase kinase 3 beta (GSK3ß), through LPA1 and/or LPA3. What's more, we found that LPA preconditioning increased glucose uptake of H9C2 subjected to H/R by the activation of AMP-Activated Protein Kinase (AMPK) but not the translocation of GLUT4. In conclusion, our study indicates that LPA is a potent survival factor for immature hearts against I/R injuries and has the potential therapeutic function as a cardioplegia additive for infantile cardiac surgery.

[Cardiac protective effect of the autoantibody against ß3-adrenoceptor in rats with experimental heart failure].

OBJECTIVE: To explore the effect of the autoantibody against the ß3-adrenoceptor on rats with experimental heart failure. METHOD: The peptide corresponding to the sequence of ß3 adrenoceptor was synthesized to actively immunize the rats, ELISA was used to detect the serum level of autoantibody against the ß3-adrenoceptor (ß3AA). Total IgGs were extracted from the serum containing ß3AA in immunized rats. Aortic banding surgery was used to establish the heart failure model in male Wistar rats and rats were divided into the sham group (n = 8), heart failure group(n = 8),ß3AA-immunized heart failure group (HF+ß3AA, n = 8) and corresponding negative IgG-immunized heart failure group (HF+ IgG, n = 8).In 6 weeks and 8 weeks after aortic banding surgery, the serum levels of NT-pro brain natriuretic peptide (NT-proBNP) were assayed with ELISA assay and cardiac function was assessed by echocardiography. RESULTS: ß3AA was used to immunize rat with heart failure, the serum level of ß3AA was stable at 50 days post immunization. At 8 weeks after aortic banding surgery, heart failure group showed significantly increased LVEDD [(6.92 ± 0.22) mm vs.(5.62 ± 0.19) mm, P < 0.001], LVESD [(4.63 ± 0.23) mm vs.(3.50 ± 0.20) mm, P < 0.01] and IVS [(2.44 ± 0.06) mm vs.(2.28 ± 0.05) mm, P < 0.05], and decreased LVEF[(62.07 ± 3.99)% vs.(79.63 ± 3.02)%, P < 0.01] and LVFS [(31.46 ± 3.22)% vs.(43.65 ± 2.68) %, P < 0.05] compared with the sham group.HF+ß3AA IgG group showed decreased LVEDD [(6.07 ± 0.30) mm vs.(6.92 ± 0.24) mm, P < 0.05] and LVESD [(3.92 ± 0.22) mm vs.(4.68 ± 0.23) mm, P < 0.05], and higher LVEF [(70.29 ± 1.78)% vs.(61.95 ± 3.03)%, P < 0.05] and LVFS [(38.08 ± 2.32)% vs.(30.50 ± 1.82)%, P < 0.05] compared to the HF+ IgG group.In addition, compared with the HF+ IgG group, HF+ß3AA IgG group showed decreased serum levels of NT-proBNP [(196.43 ± 6.56) pg/ml vs.(242.13 ± 7.86) pg/ml, P < 0.01]. CONCLUSION: Our results demonstrate that ß3AA can improve cardiac function and reduce the serum levels of NT-proBNP in rat with heart failure.

Reciprocal regulation of miR-23a and lysophosphatidic acid receptor signaling in cardiomyocyte hypertrophy.

Earlier, our study demonstrated that lysophosphatidic acid (LPA) receptor mediated cardiomyocyte hypertrophy. However, the subtype-specific functions for LPA1 and LPA3 receptors in LPA-induced hypertrophy have not been distinguished. Growing evidence indicates that microRNAs (miRNAs) are involved in the pathogenesis of cardiac hypertrophy by down-regulating target molecules. The present work therefore aimed at elucidating the functions mediated by different subtypes of LPA receptors and investigating the modulatory role of miRNAs during LPA induced hypertrophy. Experiments were done with cultured neonatal rat cardiomyocytes (NRCMs) exposed to LPA and we showed that knockdown of LPA1 by small interfering RNA (siRNA) enhanced LPA-induced cardiomyocyte hypertrophy, whereas LPA3 silencing repressed hypertrophy. miR-23a, a pro-hypertrophic miRNA, was up-regulated by LPA in cardiomyocytes and its down-regulation reduced LPA-induced cardiomyocyte hypertrophy. Importantly, luciferase reporter assay confirmed LPA1 to be a target of miR-23a, indicating that miR-23a is involved in mediating the LPA-induced cardiomyocyte hypertrophy by targeting LPA1. In addition, knockdown of LPA3, but not LPA1, eliminated miR-23a elevation induced by LPA. And PI3K inhibitor, LY294002, effectively prevented LPA-induced miR-23a expression in cardiomyocytes, suggesting that LPA might induce miR-23a elevation by activating LPA3 and PI3K/AKT pathway. These findings identified opposite subtype-specific functions for LPA1 and LPA3 in mediating cardiomyocyte hypertrophy and indicated LPA1 to be a target of miR-23a, which discloses a link between miR-23a and the LPA receptor signaling in cardiomyocyte hypertrophy.

Lysophosphatidic acid promotes secretion of VEGF by increasing expression of 150-kD Oxygen-regulated protein (ORP150) in mesenchymal stem cells.

We previously reported that transplantation of lysophosphatidic acid (LPA) treated mesenchymal stem cells (MSCs) enhances capillary density in the myocardium and improves myocardial function in the ischemic heart. This effect may be mediated through the release of paracrine factors by MSC and potentially involves pro-angiogenic molecules such as vascular endothelial growth factor (VEGF). In this study, we examined the pharmacological and molecular regulation of VEGF secretion induced by LPA in rat MSCs. We showed that LPA stimulated VEGF secretion in MSCs but not in cardiomyocytes or cardiac fibroblasts. LPA-induced VEGF secretion occurred at the post-transcriptional levels and was mediated through the classical ER/Golgi-dependent protein secretory route. LPA also increased ORP150 protein expression. Inhibition of ORP150 upregulation by siRNA knockdown attenuated LPA-induced VEGF secretion. On the other hand, diazoxide, an activator of KATP channel, markedly inhibited LPA-induced ORP150 expression and VEGF secretion. Meanwhile, ATP concentration dependently increased VEGF secretion. In addition, l-Glutamate and NH4Cl significantly reduced VEGF secretion. Furthermore, inhibition of two major subtypes of LPA receptors by Ki16425 and specific siRNA for LPA receptors prevented LPA-induced VEGF secretion and ORP150 expression. Lastly, inhibition of Gi protein that couples with LPA receptors by PTX and siRNA knockdown had no effect on LPA-induced VEGF secretion. Taken together, our findings demonstrate that LPA promotes VEGF secretion at the post-translation level by up-regulating ORP150 expression. Both LPA1 and LPA3 are involved in the LPA-induced VEGF secretion that is independent of Gi protein coupling but associated with the inactivation of KATP channels and inhibition of Na(+)/K(+)-ATPase activity.

Pathogenic cellular phenotypes are germline transmissible in a transgenic primate model of Huntington's disease.

A transgenic primate model for Huntington's Disease (HD) first reported by our group that (HD monkeys) carry the mutant Huntingtin (HTT) gene with expanded polyglutamine (CAG) repeats and, develop chorea, dystonia, and other involuntary motor deficiencies similar to HD [ 1 ]. More recently, we have found that longitudinal magnetic resonance imaging of the HD monkey brain revealed significant atrophy in regions associated with cognitive deficits symptomatic in HD patients, providing the first animal model which replicates clinical phenotypes of diagnosed humans. Here we report germline transmission of the pathogenic mutant HTT in HD monkey by the production of embryos and subsequent derivation of HD monkey embryonic stem cells (rHD-ESCs) using HD monkey sperm. rHD-ESCs inherit mutant HTT and green fluorescent protein (GFP) genes through the gametes of HD monkey. rHD-ESCs express mutant HTT and form intranuclear inclusion, a classical cellular feature of HD. Notably, mosaicism of the pathogenic polyQ region in the sperm as well as derived ESCs were also observed, consistent with intraindividual and intergenerational reports of mosaic CAG repeats [ 2 , 3 ]and CAG expansion in HD patients [ 4-7 ]. The confirmation of transgene inheritability and development of pathogenic HD phenotype in derived rHD-ESCs reported in this study is a milestone in the pursuit of a transgenic primate model with inherited mutant HTT for development of novel disease biomarkers and therapeutics.

Characterization of dental pulp stem/stromal cells of Huntington monkey tooth germs.

BACKGROUND: Dental pulp stem/stromal cells (DPSCs) are categorized as adult stem cells (ASCs) that retain multipotent differentiation capabilities. DPSCs can be isolated from individuals at any age and are considered to be true personal stem cells, making DPSCs one of the potential options for stem cell therapy. However, the properties of DPSCs from individuals with an inherited genetic disorder, such as Huntington's disease (HD), have not been fully investigated. RESULTS: To examine if mutant huntingtin (htt) protein impacts DPSC properties, we have established DPSCs from tooth germ of transgenic monkeys that expressed both mutant htt and green fluorescent protein (GFP) genes (rHD/G-DPSCs), and from a monkey that expressed only the GFP gene (rG-DPSCs), which served as a control. Although mutant htt and oligomeric htt aggregates were overtly present in rHD/G-DPSCs, all rHD/G-DPSCs and rG-DPSCs shared similar characteristics, including self-renewal, multipotent differentiation capabilities, expression of stemness and differentiation markers, and cell surface antigen profile. CONCLUSIONS: Our results suggest that DPSCs from Huntington monkeys retain ASC properties. Thus DPSCs derived from individuals with genetic disorders such as HD could be a potential source of personal stem cells for therapeutic purposes.

Identification of MicroRNAs involved in hypoxia- and serum deprivation-induced apoptosis in mesenchymal stem cells.

The use of bone marrow mesenchymal stem cell- (MSC) transplantation therapy for cardiac diseases is limited due to poor survival of implanted cells. MicroRNAs (miRNAs) have been reported to be involved in regulating almost all cellular processes, including apoptosis. In this study, we found that the miRNA profile was altered during apoptosis induced by hypoxia and serum deprivation (hypoxia/SD). We further revealed that over-expression of miR-21, miR-23a and miR-210 could promote the survival of MSCs exposed to hypoxia/SD. In contrast, down-regulation of miR-21, miR-23a and miR-503 aggravated apoptosis of MSCs. It was indicated that these miRNAs may play important roles during MSC apoptosis induced by hypoxia/SD.

Reprogramming Huntington monkey skin cells into pluripotent stem cells.

Induced pluripotent Huntington's disease monkey stem cells (rHD-iPSCs) were established by the overexpression of rhesus macaque transcription factors (Oct4, Sox2, and Klf4) in transgenic Huntington's monkey skin fibroblasts. The rHD-iPSCs were pluripotent and capable of differentiating into neuronal cell types in vitro and developed teratoma in immune compromised mice. We also demonstrated the upregulation of endogenous Oct4 and Sox2 after successful reprogramming to pluripotency in rHD-iPSCs, which was not expressed in skin fibroblasts. rHD-iPSCs also developed cellular features comparable to Huntington's disease (HD), including the accumulation of mutant huntingtin (htt) aggregate and the formation of intranuclear inclusions (NIs) paralleling neural differentiation in vitro. Induced pluripotent stem cells from transgenic HD monkeys open a new era of nonhuman primate modeling of human diseases. rHD-iPSCs that develop key HD cellular features and parallel neural differentiation can be a powerful platform for investigating the developmental impact on HD pathogenesis and developing new therapies, which can be evaluated in HD monkeys from whom the rHD-iPSCs were derived.

Noninvasive monitoring of embryonic stem cells in vivo with MRI transgene reporter.

Reporter gene-based magnetic resonance imaging (MRI) offers unique insights into behavior of cells after transplantation, which could significantly benefit stem cell research and translation. Several candidate MRI reporter genes, including one that encodes for iron storage protein ferritin, have been reported, and their potential applications in embryonic stem (ES) cell research have yet to be explored. We have established transgenic mouse ES (mES) cell lines carrying human ferritin heavy chain (FTH) as a reporter gene and succeeded in monitoring the cell grafts in vivo using T(2)-weighted MRI sequences. FTH generated MRI contrast through compensatory upregulation of transferrin receptor (Tfrc) that led to increased cellular iron stored in ferritin-bound form. At a level sufficient for MRI contrast, expression of FTH posed no toxicity to mES cells and did not interfere with stem cell pluripotency as observed in neural differentiation and teratoma formation. The compatibility and functionality of ferritin as a reporter in mES cells opens up the possibility of using MRI for longitudinal noninvasive monitoring of ES cell-derived cell grafts at both molecular and cellular levels.

Towards a transgenic model of Huntington's disease in a non-human primate.

Non-human primates are valuable for modelling human disorders and for developing therapeutic strategies; however, little work has been reported in establishing transgenic non-human primate models of human diseases. Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor impairment, cognitive deterioration and psychiatric disturbances followed by death within 10-15 years of the onset of the symptoms. HD is caused by the expansion of cytosine-adenine-guanine (CAG, translated into glutamine) trinucleotide repeats in the first exon of the human huntingtin (HTT) gene. Mutant HTT with expanded polyglutamine (polyQ) is widely expressed in the brain and peripheral tissues, but causes selective neurodegeneration that is most prominent in the striatum and cortex of the brain. Although rodent models of HD have been developed, these models do not satisfactorily parallel the brain changes and behavioural features observed in HD patients. Because of the close physiological, neurological and genetic similarities between humans and higher primates, monkeys can serve as very useful models for understanding human physiology and diseases. Here we report our progress in developing a transgenic model of HD in a rhesus macaque that expresses polyglutamine-expanded HTT. Hallmark features of HD, including nuclear inclusions and neuropil aggregates, were observed in the brains of the HD transgenic monkeys. Additionally, the transgenic monkeys showed important clinical features of HD, including dystonia and chorea. A transgenic HD monkey model may open the way to understanding the underlying biology of HD better, and to the development of potential therapies. Moreover, our data suggest that it will be feasible to generate valuable non-human primate models of HD and possibly other human genetic diseases.