Matthew R. Richardson

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.

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.

VEGF and IHH rescue definitive hematopoiesis in Gata-4 and Gata-6–deficient murine embryoid bodies

OBJECTIVE Murine embryonic stem cells can be differentiated into embryoid bodies (EBs), which serve as an in vitro model recapitulating many aspects of embryonic yolk sac hematopoiesis. Differentiation of embryonic stem cells deficient in either Gata-4 or Gata-6 results in EBs with disrupted visceral endoderm (VE). While lack of VE has detrimental effects on hematopoiesis in vivo, it is unclear whether lack of VE affects hematopoiesis in EBs. Therefore, we compared Gata-4 null (G4N) and Gata-6 null (G6N) EBs with wild-type EBs to assess their ability to commit to hematopoietic cells. MATERIALS AND METHODS EB VE formation was examined using cell-sorting techniques and analysis visceral endoderm gene expression. Hematopoietic progenitor potential of EBs cultured under various conditions was assessed using colony-forming assays. RESULTS Definitive erythroid, granulocyte-macrophage, and mixed colonies were significantly reduced in G4N and G6N EBs compared to wild-type EBs. Vascular endothelial growth factor (VEGF) expression and secretion were also reduced in both G4N and G6N EBs, consistent with VE serving as a site of VEGF production. Addition of exogenous VEGF(165), to EB cultures completely rescued definitive colony-forming cells in G4N and G6N EBs. This rescue response could be blocked by addition of soluble Flk-1 Fc to EB cultures. Similarly, addition of exogenous Indian hedgehog to EB cultures also recovers the diminishment in definitive hematopoiesis in a reversible manner. CONCLUSION These results suggest that the absence of VE in G4N and G6N EBs does not prevent emergence of definitive progenitors from EBs. However, the decreased level of VEGF and Indian hedgehog production in VE devoid G4N and G6N EBs attenuates definitive hematopoietic progenitor cell expansion.

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.

Differential protein expression during aging in ventricular myocardium of Fischer 344 × Brown Norway hybrid rats

The aging heart undergoes well characterized structural changes associated with functional decline, though the underlying mechanisms are not understood. The aim of this study was to determine to what extent ventricular myocardial protein expression was altered with age and which proteins underwent protein nitration. Fischer 344 x Brown Norway F1 hybrid (FBN) rats of four age groups were used, 4, 12, 24, and 34 months. Differential protein expression was determined by 2-DE and proteins were identified by peptide mass fingerprinting. Altered protein nitration with age was assessed by immunoblotting. Over 1000 protein spots per sample were detected, and 255 were found to be differentially expressed when all aged groups were compared to young rats (4 months) (p0.05). A strong positive correlation between differential protein expression and increasing age (p=0.03, R(2)=0.997) indicated a progressive, rather than abrupt, change with age. Of 46 differentially expressed proteins identified, seventeen have roles in apoptosis, ten in hypertrophy, seven in fibrosis, and three in diastolic dysfunction, aging-associated processes previously reported in both human and FBN rat heart. Protein expression alterations detected here could have beneficial effects on cardiac function; thus, our data indicate a largely adaptive change in protein expression during aging. In contrast, differential protein nitration increased abruptly, rather than progressively, at 24 months of age. Altogether, the results suggest that differential myocardial protein expression occurs in a progressive manner during aging, and that a proteomic-based approach is an effective method for the identification of potential therapeutic targets to mitigate aging-related myocardial dysfunction.

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.

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

Electroacupuncture Promotes CNS-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