Electroacupuncture Promotes Central Nervous System-Dependent Release of Mesenchymal Stem Cells.

Electroacupuncture (EA) performed in rats and humans using limb acupuncture sites, LI-4 and LI-11, and GV-14 and GV-20 (humans) and Bai-hui (rats) increased functional connectivity between the anterior hypothalamus and the amygdala and mobilized mesenchymal stem cells (MSCs) into the systemic circulation. In human subjects, the source of the MSC was found to be primarily adipose tissue, whereas in rodents the tissue sources were considered more heterogeneous. Pharmacological disinhibition of rat hypothalamus enhanced sympathetic nervous system (SNS) activation and similarly resulted in a release of MSC into the circulation. EA-mediated SNS activation was further supported by browning of white adipose tissue in rats. EA treatment of rats undergoing partial rupture of the Achilles tendon resulted in reduced mechanical hyperalgesia, increased serum interleukin-10 levels and tendon remodeling, effects blocked in propranolol-treated rodents. To distinguish the afferent role of the peripheral nervous system, phosphoinositide-interacting regulator of transient receptor potential channels (Pirt)-GCaMP3 (genetically encoded calcium sensor) mice were treated with EA acupuncture points, ST-36 and LIV-3, and GV-14 and Bai-hui and resulted in a rapid activation of primary sensory neurons. EA activated sensory ganglia and SNS centers to mediate the release of MSC that can enhance tissue repair, increase anti-inflammatory cytokine production and provide pronounced analgesic relief. Stem Cells 2017;35:1303-1315.

Angiopoietin-like protein 2 regulates endothelial colony forming cell vasculogenesis.

Angiopoietin-like 2 (ANGPTL2) has been reported to induce sprouting angiogenesis; however, its role in vasculogenesis, the de novo lumenization of endothelial cells (EC), remains unexplored. We sought to investigate the potential role of ANGPTL2 in regulating human cord blood derived endothelial colony forming cell (ECFC) vasculogenesis through siRNA mediated inhibition of ANGPTL2 gene expression. We found that ECFCs in which ANGPTL2 was diminished displayed a threefold decrease in in vitro lumenal area whereas addition of exogenous ANGPTL2 protein domains to ECFCs lead to increased lumen formation within a 3 dimensional (3D) collagen assay of vasculogenesis. ECFC migration was attenuated by 36 % via ANGPTL2 knockdown (KD) although proliferation and apoptosis were not affected. We subsequently found that c-Jun NH2-terminal kinase (JNK), but not ERK1/2, phosphorylation was decreased upon ANGPTL2 KD, and expression of membrane type 1 matrix metalloproteinase (MT1-MMP), known to be regulated by JNK and a critical regulator of EC migration and 3D lumen formation, was decreased in lumenized structures in vitro derived from ANGPTL2 silenced ECFCs. Treatment of ECFCs in 3D collagen matrices with either a JNK inhibitor or exogenous rhTIMP-3 (an inhibitor of MT1-MMP activity) resulted in a similar phenotype of decreased vascular lumen formation as observed with ANGPTL2 KD, whereas stimulation of JNK activity increased vasculogenesis. Based on gene silencing, pharmacologic, cellular, and biochemical approaches, we conclude that ANGPTL2 positively regulates ECFC vascular lumen formation likely through its effects on migration and in part by activating JNK and increasing MT1-MMP expression.

Endothelial progenitor cells: quo vadis?

The term endothelial progenitor cell (EPC) was coined to refer to circulating cells that displayed the ability to display cell surface antigens similar to endothelial cells in vitro, to circulate and lodge in areas of ischemia or vascular injury, and to facilitate the repair of damaged blood vessels or augment development of new vessels as needed by a tissue. More than 10 years after the first report, the term EPC is used to refer to a host of circulating cells that display some or all of the qualities indicated above, however, essentially all of the cells are now known to be members of the hematopoietic lineage. The exception is a rare viable circulating endothelial cell with clonal proliferative potential that displays the ability to spontaneously form inosculating human blood vessels upon implantation into immunodeficient murine host tissues. This paper will review the current lineage relationships among all the cells called EPC and will propose that the term EPC be retired and that each of the circulating cell subsets be referred to according to the terms already existent for each subset. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".

Alterations in the aqueous humor proteome in patients with a glaucoma shunt device.

PURPOSE: To investigate whether implantation of a glaucoma shunt device leads to inappropriate accumulation of plasma derived proteins in the aqueous humor. METHODS: Aqueous humor samples were collected from 11 patients (study group) with a glaucoma shunt device undergoing either cataract surgery or a corneal transplant and 11 patients (control) with senile cataract undergoing routine cataract extraction. Of the study group, 9 had an Ahmed valve implant and 2 eyes had a Baerveldt implant. Tryptic digests of the mixture of proteins in aqueous humor (AH) were analyzed using Liquid Chromatography/Mass Spectrometry (LC-MS/MS). Proteins were identified with high confidence using stringent criteria and compared quantitatively using a label-free platform (IdentiQuantXL™). RESULTS: We identified 135 proteins in the albumin-depleted fraction in both the study and control group AH. Using stringent criteria, 13 proteins were detected at a significantly higher level compared to controls. These proteins are known to play a role in oxidative stress, apoptosis, inflammation and/or immunity and include gelsolin (p=0.00005), plasminogen (p=0.00009), angiotensinogen (p=0.0001), apolipoprotein A-II (p=0.0002), beta-2-microglobulin (p=0.0002), dickkopf-3 (DKK-3; p=0.0002), pigment epithelium-derived factor (p=0.0002), RIG-like 7-1 (p=0.0002), afamin (p=0.0003), fibronectin 1 (FN1; p=0.0003), apolipoprotein A-I (p=0.0004), activated complement C4 protein (C4a; p=0.0004) and prothrombin (p=0.0004). Many of the identified proteins were novel proteins that have not been associated with glaucoma in prior studies. All but C4a (complement C4 is a plasma protein but not in an activated form) are known plasma proteins and the elevated levels of these proteins in the aqueous humor suggests a breach in the blood-aqueous barrier with passive influx into the anterior chamber of the eye. CONCLUSIONS: The presence of these proteins in the aqueous humor suggests that glaucoma shunt device causes either a breach in blood-aqueous barrier or chronic trauma, increasing influx of oxidative, apoptotic and inflammatory proteins that could potentially cause corneal endothelial damage.

Venous and arterial endothelial proteomics: mining for markers and mechanisms of endothelial diversity.

Endothelial cells (ECs) line the inside of arterial and venous blood vessels in a continuous monolayer and have the important function of responding to environmental cues to regulate vascular tone and new blood vessel formation. They also have well-defined roles in supporting tumorigenesis, and alterations in their function lead to cardiovascular disease. Consequently, ECs have been studied extensively as a cellular model of both normal and abnormal physiology. Despite their importance and the increased utility of proteomic tools in medical research, there are relatively few publications on the topic of vascular endothelial proteomics. A thorough search of the literature mined 52 publications focused exclusively on arterial and/or venous endothelial proteomics. These studies mostly relied upon examination of whole-cell lysates from cultured human umbilical vein ECs to investigate in vitro effects of various molecules, such as VEGF in the context of altering human umbilical vein EC functions related to angiogenesis. Only a few of these publications focused solely on a proteomic characterization of ECs and our analysis further revealed a lack of published studies incorporating proteomic analysis of freshly isolated ECs from tissues or in vitro conditions that mimic in vivo variables, such as oxygen tension and shear stress. It is the purpose of this article to account for the diversity of vascular EC proteomic investigations and comment on the issues that have been and should be addressed in future work.

Alterations in the aqueous humor proteome in patients with Fuchs endothelial corneal dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is a progressive disorder characterized by corneal endothelial decompensation leading to corneal edema, clouding, and vision impairment. Despite improved understanding over the last century since its first description, the exact mechanism(s) behind the pathogenesis of FECD remain unknown, and surgical correction is the only effective treatment available. Previous studies have suggested a role for changes in aqueous humor (AH) composition in FECD pathogenesis, so to explore this possibility, we probed the AH proteome for alterations correlating with end-stage corneal disease. Following albumin depletion we performed label-free quantitative tandem mass spectrometry on proteins isolated from patients with and without FECD who were scheduled to undergo routine cataract extraction. We identified 64 proteins, most of which were identified in previous AH proteomic studies of patients with cataracts, in the albumin-depleted fraction. The levels of five of these were significantly lower (afamin, complement C3, histidine-rich glycoprotein, immunoglobulin heavy [IgH], and protein family with sequence similarity 3, member C [FAM3C]), while the levels of one (suprabasin) was significantly higher in patients with FECD compared to controls (p≤0.01). We also identified 34 proteins in the albumin-bound fraction, four of which were significantly elevated in patients with FECD including a hemoglobin fragment, immunoglobulin kappa (IgK), immunoglobulin lambda (IgL), and uncharacterized protein albumin (ALB), (p≤0.01). Although it has been reported that females have a greater extent of disease than males, we were unable to detect any significant differences in protein levels due to gender. Because FECD is a progressive disorder, regression analyses were performed to determine any significant correlations with age, and of interest retinol-binding protein 3 was significantly correlated with age in patients with FECD (p≤0.01), whereas no proteins in the control group correlated with age. This is the first report indicating alterations in the AH proteome with FECD, and taken together this study suggests several novel hypotheses regarding AH proteins role in FECD pathogenesis.

Diabetic dyslipidemia and exercise alter the plasma low-density lipoproteome in Yucatan pigs.

Although low-density lipoprotein (LDL) plays a predominant role in atherogenesis, the low-density lipoproteome has not been fully characterized. Moreover, alterations from a Western diet, diabetes, and physical inactivity on this proteome have yet to be determined. Accordingly, relative quantification was determined in LDL proteins from male Yucatan diabetic dyslipidemic (DD) swine in the early stages of atherosclerosis compared to healthy control (C) and non-diabetic hyperlipidemic (H) swine. Importantly, coronary vascular dysfunction was prevented by aerobic exercise training in these animals (DDX) without altering total LDL concentration. Using 2-DE, Western blot, label-free quantitative MS, and selected reaction monitoring, alterations in the abundance of apolipoproteins A-I, B, C-III, D, E, and J and noncovalently associated proteins were determined in LDL isolated using fast protein liquid chromatography. At least 28 unique proteins, many of which were novel, were identified with high confidence. An apolipoprotein E isoform demonstrated stronger correlation to disease (percent of coronary artery segments with intimal thickening) than some traditional risk factors (total cholesterol, LDL cholesterol, and LDL/HDL cholesterol). Taken together, this work identifies new possible biomarkers, potential therapeutic targets for atherosclerosis, and generates new hypotheses regarding the role of LDL in atherogenesis.

Proteomic analysis of human aqueous humor using multidimensional protein identification technology.

Aqueous humor (AH) supports avascular tissues in the anterior segment of the eye, maintains intraocular pressure, and potentially influences the pathogenesis of ocular diseases. Nevertheless, the AH proteome is still poorly defined despite several previous efforts, which were hindered by interfering high abundance proteins, inadequate animal models, and limited proteomic technologies. To facilitate future investigations into AH function, the AH proteome was extensively characterized using an advanced proteomic approach. Samples from patients undergoing cataract surgery were pooled and depleted of interfering abundant proteins and thereby divided into two fractions: albumin-bound and albumin-depleted. Multidimensional Protein Identification Technology (MudPIT) was utilized for each fraction; this incorporates strong cation exchange chromatography to reduce sample complexity before reversed-phase liquid chromatography and tandem mass spectrometric analysis. Twelve proteins had multi-peptide, high confidence identifications in the albumin-bound fraction and 50 proteins had multi-peptide, high confidence identifications in the albumin-depleted fraction. Gene ontological analyses were performed to determine which cellular components and functions were enriched. Many proteins were previously identified in the AH and for several their potential role in the AH has been investigated; however, the majority of identified proteins were novel and only speculative roles can be suggested. The AH was abundant in anti-oxidant and immunoregulatory proteins as well as anti-angiogenic proteins, which may be involved in maintaining the avascular tissues. This is the first known report to extensively characterize and describe the human AH proteome and lays the foundation for future work regarding its function in homeostatic and pathologic states.

Using ensemble models to classify the sentiment expressed in suicide notes.

In 2007, suicide was the tenth leading cause of death in the U.S. Given the significance of this problem, suicide was the focus of the 2011 Informatics for Integrating Biology and the Bedside (i2b2) Natural Language Processing (NLP) shared task competition (track two). Specifically, the challenge concentrated on sentiment analysis, predicting the presence or absence of 15 emotions (labels) simultaneously in a collection of suicide notes spanning over 70 years. Our team explored multiple approaches combining regular expression-based rules, statistical text mining (STM), and an approach that applies weights to text while accounting for multiple labels. Our best submission used an ensemble of both rules and STM models to achieve a micro-averaged F(1) score of 0.5023, slightly above the mean from the 26 teams that competed (0.4875).

Sample complexity reduction for two-dimensional electrophoresis using solution isoelectric focusing prefractionation.

Despite its excellent resolving power, 2-DE is of limited use when analyzing cellular proteomes, especially in differential expression studies. Frequently, fewer than 2000 protein spots are detected on a single 2-D gel (a fraction of the total proteome) regardless of the gel platform, sample, or detection method used. This is due to the vast number of proteins expressed and their equally vast dynamic range. To exploit 2-DE unique ability as both an analytical and a preparative tool, the significant sample prefractionation is necessary. We have used solution isoelectric focusing (sIEF) via the ZOOM IEF Fractionator (Invitrogen) to generate sample fractions from complex bacterial lysates, followed by parallel 2-DE, using narrow-range IPG strips that bracket the sIEF fractions. The net result of this process is a significant enrichment of the bacterial proteome resolved on multiple 2-D gels. After prefractionation, we detected 5525 spots, an approximate 3.5-fold increase over the 1577 spots detected in an unfractionated gel. We concluded that sIEF is an effective means of prefractionation to increase depth of field and improve the analysis of low-abundance proteins.

Two-dimensional gels for toxicological drug discovery applications.

Two-dimensional gel electrophoresis (2DGE) continues to be a useful approach to study protein expression. Although liquid chromatographic and mass spectrometric approaches that overcome some of the limitations and labour intensity of 2DGE are increasingly popular, this electrophoretic approach still has exceptional relevance in toxicology. Despite the technical challenges, pharmacologists/toxicologists continue to use gel-based proteomics to assess the biological and health effects of chemical treatment and exposure. This brief review addresses the use of 2DGE-based proteomics in drug development and toxicology, emphasising its unique strengths and weaknesses, and considers recent developments in this strategy that have evolved to directly confront the issues of dynamic range and reproducibility that have previously limited the overall use of 2D electrophoresis.