Michael H. Creer

19F magnetic resonance imaging for stem/progenitor cell tracking with multiple unique perfluorocarbon nanobeacons

MRI has been employed to elucidate the migratory behavior of stem/progenitor cells noninva‐sively in vivo with traditional proton (1H) imaging of iron oxide nanoparticle‐labeled cells. Alternatively, we demonstrate that fluorine (19F) MRI of cells labeled with different types of liquid perfluorocarbon (PFC) nanoparticles produces unique and sensitive cell markers distinct from any tissue background signal. To define the utility for cell tracking, mononuclear cells harvested from human umbilical cord blood were grown under proendothelial conditions and labeled with nanoparticles composed of two distinct PFC cores (perfluorooctylbromide and perfluoro‐15‐crown‐5 ether). The sensitivity for detecting and imaging labeled cells was defined on 11.7T (research) and 1.5T (clinical) scanners. Stem/progenitor cells (CD34+CD133+CD31+) readily internalized PFC nanoparticles without aid of adjunctive labeling techniques, and cells remained functional in vivo. PFC‐labeled cells exhibited distinct 19F signals and were readily detected after both local and intravenous injection. PFC nanoparticles provide an unequivocal and unique signature for stem/progenitor cells, enable spatial cell localization with 19F MRI, and permit quantification and detection of multiple fluorine signatures via 19F MR spectroscopy. This method should facilitate longitudinal investigation of cellular events in vivo for multiple cell types simultaneously.—Partlow, K. C., Chen, J., Brant, J. A., Neubauer, A. M., Meyerrose, T. E., Creer, M. H., Nolta, J. A., Caruthers, S. D., Lanza, G. M., Wickline, S. A. 19F magnetic resonance imaging for stem/progenitor cell tracking with multiple unique perfluorocarbon nanobeacons. FASEB J. 21, 1647–1654 (2007)

Prophylaxis of early ventricular fibrillation by inhibition of acylcarnitine accumulation.

Hypoxia in isolated myocytes results in accumulation of long-chain acylcarnitines (LCA) in sarcolemma. Inhibition of carnitine acyltransferase I (CAT-I) with sodium 2-[5-(4-chlorophenyl)-pentyl]-oxirane-2-carboxylate (POCA) prevents both the accumulation of LCA in the sarcolemma and the initial electrophysiologic derangements associated with hypoxia. Another amphiphilic metabolite, lysophosphatidylcholine (LPC), accumulates in the ischemic heart in vivo, in part because of inhibition of its catabolism by accumulating LCA. It induces electrophysiologic alterations in vitro analogous to early changes induced by ischemia in vivo. The present study was performed to determine whether POCA could prevent accumulation of both LCA and LPC induced by ischemia in vivo and if so, whether attenuation of early arrhythmogenesis would result. LAD coronary artery occlusions were induced for 5 min in chloralose-anesthetized cats. Coronary occlusion in untreated control animals elicited prompt, threefold increases of LCA (73 +/- 8 to 286 +/- 60 pmol/mg protein) and twofold increase of LPC (3.3 +/- 0.4 to 7.5 +/- 0.9 nmol/mg protein) selectively in the ischemic zone, associated with ventricular tachycardia (VT) or ventricular fibrillation (VF) occurring within the 5-min interval before acquisition of myocardial samples in 64% of the animals. POCA prevented the increase of both LCA and LPC. It also prevented the early occurrence of VT or VF (within 5 min of occlusion) in all animals studied. The antiarrhythmic effect of POCA was not attributable to favorable hemodynamic changes or to changes in myocardial perfusion measured with radiolabeled microspheres. Thus, inhibition of CAT-I effectively reduced the incidence of lethal arrhythmias induced early after the onset of ischemia. Accordingly, pharmacologic inhibition of this enzyme provides a promising approach for prophylaxis of sudden cardiac death, that typically occurs very soon after the onset of acute ischemia, in man.

Amphipathic lipid metabolites and their relation to arrhythmogenesis in the ischemic heart

Myocardial ischemia is associated with profound electrophysiologic derangements which occur within minutes and are rapidly reversible with reperfusion, suggesting that subtle and reversible biochemical alterations within or near the sarcolemma contribute. Our efforts have concentrated on two structurally similar amphipathic metabolites, long-chain acylcarnitine and lysophosphatidylcholine. Studies performed in vitro in isolated tissue indicate that incorporation of either metabolite into the sarcolemma at concentrations of 1-2 mole %, as verified using electron microscopic (EM) autoradiography, elicits profound electrophysiologic derangements analogous to those seen in the ischemic heart in vivo. In isolated myocytes in vitro, the electrophysiologic derangements elicited by hypoxia are associated with a marked 70-fold increase in the endogenous sarcolemmal accumulation of long-chain acylcarnitine. Inhibition of carnitine acyltransferase I (CAT-I) not only prevents the accumulation of long-chain acylcarnitine in isolated myocytes exposed to severe hypoxia, but also markedly attenuates the electrophysiologic alterations. Several lines of experimental evidence, including measurements in venous effluents as well as cardiac lymph, indicate that lysophosphatidylcholine (LPC) accumulates to a large extent in the extracellular space during ischemia. This extracellular accumulation may be secondary to release from vascular endothelium, smooth muscle or blood cell elements. In crude homogenates of myocardial tissue, the total enzymic activity for catabolism of LPC far exceeds the total activity for synthesis of LPC mediated by phospholipase A2 (PLA2) catalyzed hydrolysis of phosphatidylcholine (PC). Therefore, inhibition of catabolism would be required for net accumulation of LPC to occur. Three enzymes responsible for the catabolism of LPC are inhibited by either long-chain acylcarnitine or acidic pH. Thus, accumulation of long-chain acylcarnitine and acidosis contribute to the increase in LPC observed in ischemic tissue. In this report, we provide evidence that accumulation of long-chain acylcarnitine occurs very rapidly in ischemic myocardium in vivo, coincident with the development of electrophysiologic alterations leading to malignant arrhythmias as verified using 3-dimensional cardiac mapping procedures. Following a brief, 2-min period of ischemia, long-chain acylcarnitine content increased four-fold in the ischemic region, concomitant with the development of electrophysiologic abnormalities observed during this period. Additionally, we demonstrate that modification of intracellular lipolysis by beta-adrenergic receptor stimulation or blockade does not influence long-chain acylcarnitine accumulation following this 2-min interval of ischemia. These results suggest that production of long-chain acylcarnitine is not limited by the intracellular free fatty acid concentration early in ischemia.(ABSTRACT TRUNCATED AT 400 WORDS)

Widespread nonhematopoietic tissue distribution by transplanted human progenitor cells with high aldehyde dehydrogenase activity

Transplanted adult progenitor cells distribute to peripheral organs and can promote endogenous cellular repair in damaged tissues. However, development of cell‐based regenerative therapies has been hindered by the lack of preclinical models to efficiently assess multiple organ distribution and difficulty defining human cells with regenerative function. After transplantation into β‐glucuronidase (GUSB)‐deficient NOD/SCID/mucopolysaccharidosis type VII mice, we characterized the distribution of lineage‐depleted human umbilical cord blood‐derived cells purified by selection using high aldehyde dehydrogenase (ALDH) activity with CD133 coexpression. ALDHhi or ALDHhiCD133+ cells produced robust hematopoietic reconstitution and variable levels of tissue distribution in multiple organs. GUSB+ donor cells that coexpressed human leukocyte antigen (HLA‐A,B,C) and hematopoietic (CD45+) cell surface markers were the primary cell phenotype found adjacent to the vascular beds of several tissues, including islet and ductal regions of mouse pancreata. In contrast, variable phenotypes were detected in the chimeric liver, with HLA+/CD45+ cells demonstrating robust GUSB expression adjacent to blood vessels and CD45−/HLA− cells with diluted GUSB expression predominant in the liver parenchyma. However, true nonhematopoietic human (HLA+/CD45−) cells were rarely detected in other peripheral tissues, suggesting that these GUSB+/HLA−/CD45− cells in the liver were a result of downregulated human surface marker expression in vivo, not widespread seeding of nonhematopoietic cells. However, relying solely on continued expression of cell surface markers, as used in traditional xenotransplantation models, may underestimate true tissue distribution. ALDH‐expressing progenitor cells demonstrated widespread and tissue‐specific distribution of variable cellular phenotypes, indicating that these adult progenitor cells should be explored in transplantation models of tissue damage.

Role of cytosolic calcium-independent plasmalogen-selective phospholipase A2 in hypoxic injury to rabbit proximal tubules

Although the activation of calcium-independent phospholipase A2 (PLA2) enzymes has been described in the heart, the pathogenetic role of this enzyme(s) in hypoxic cell injury has not been previously examined in any tissue. Therefore, we characterized the time course of activation of calcium-independent PLA2 using both plasmalogen and diacylglycerophospholipid substrates during hypoxia in rabbit proximal tubules and examined whether inhibition of calcium-independent PLA2 activity is associated with a cytoprotective effect. Subjecting rabbit proximal tubules to hypoxia for 5 min resulted in at least a threefold increase in cytosolic calcium-independent PLA2, which was selective for plasmalogen substrates (control 444 +/- 69 vs hypoxia 1,675 +/- 194 pmol.mg protein-1.min-1, n = 5). In contrast, no changes in PLA2 activity were observed in the presence of 4 mM EGTA in the membrane fraction using plasmenylcholine substrates. 20 min of hypoxia resulted in an increase in arachidonate from 3 +/- 1 to 28 +/- 4 ng/mg protein and lactate dehydrogenase release from 7.5 +/- 2% to 38 +/- 5%, n = 4. Pretreatment of proximal tubules with 10 microM Compound I, a specific inhibitor of calcium-independent PLA2, resulted in reduction in the magnitude of both hypoxia-induced arachidonic acid release (11 +/- 3 ng/mg protein) and lactate dehydrogenase release (18 +/- 4%). Our data indicate that a significant fraction of PLA2 activity in the proximal tubule is calcium-independent and selective for plasmalogen substrates. Furthermore, the activation of this enzyme plays an important role in the pathogenesis of membrane injury during hypoxia in the proximal tubule.

Selective hydrolysis of plasmalogen phospholipids by Ca2+-independent PLA2 in hypoxic ventricular myocytes

Accelerated phospholipid catabolism occurs early after the onset of myocardial ischemia and is likely to be mediated by the activation of one or more phospholipases in ischemic tissue. We hypothesized that hypoxia increases phospholipase A2 (PLA2) activity in isolated ventricular myocytes, resulting in increased lysophospholipid and arachidonic acid production, contributing to arrhythmogenesis in ischemic heart disease. The majority of ventricular myocyte arachidonic acid was found in plasmalogen phospholipids. Hypoxia increased membrane-associated, Ca2+-independent, plasmalogen-selective PLA2 activity, resulting in increased arachidonic acid release and lysoplasmenylcholine production. Pretreatment with the specific Ca2+-independent PLA2 inhibitor bromoenol lactone blocked hypoxia-induced increases in PLA2 activity, arachidonic acid release, and lysoplasmenylcholine production. Lysoplasmenylcholine produced action potential derangements, including shortening of action potential duration, and induced early and delayed afterdepolarizations in normoxic myocytes. The electrophysiological alterations induced by lysoplasmenylcholine would likely contribute to the initiation of arrhythmogenesis in the ischemic heart.Accelerated phospholipid catabolism occurs early after the onset of myocardial ischemia and is likely to be mediated by the activation of one or more phospholipases in ischemic tissue. We hypothesized that hypoxia increases phospholipase A2(PLA2) activity in isolated ventricular myocytes, resulting in increased lysophospholipid and arachidonic acid production, contributing to arrhythmogenesis in ischemic heart disease. The majority of ventricular myocyte arachidonic acid was found in plasmalogen phospholipids. Hypoxia increased membrane-associated, Ca2+-independent, plasmalogen-selective PLA2activity, resulting in increased arachidonic acid release and lysoplasmenylcholine production. Pretreatment with the specific Ca2+-independent PLA2 inhibitor bromoenol lactone blocked hypoxia-induced increases in PLA2 activity, arachidonic acid release, and lysoplasmenylcholine production. Lysoplasmenylcholine produced action potential derangements, including shortening of action potential duration, and induced early and delayed afterdepolarizations in normoxic myocytes. The electrophysiological alterations induced by lysoplasmenylcholine would likely contribute to the initiation of arrhythmogenesis in the ischemic heart.

Development and Testing of New Screening Method for Keratan Sulfate in Mucopolysaccharidosis IVA

Mucopolysaccharidosis IVA (MPS IVA), a progressive lysosomal storage disease, causes skeletal dysplasia through excessive storage of keratan sulfate (KS). We developed an ELISA-sandwich assay that used a MAb specific to KS. Forty-five blood and 59 urine specimens from MPS IVA patients (ages 1–65 y) were analyzed to determine whether KS concentration is a suitable marker for early diagnosis and longitudinal assessment of disease severity. Blood specimens were obtained from patients categorized as phenotypically severe (n = 36) and milder (n = 9). Urine specimens were also analyzed from patients categorized as severe (n = 56) and milder (n = 12), respectively. Blood KS levels (101–1525 ng/mL) in MPS IVA patients were two to eight times higher than those in age-matched controls (15–323 ng/mL). It was found that blood KS level varied with age and clinical severity. Blood KS levels in both MPS IVA and controls peaked between 5 and 10 y of age (mean, 776 versus 234 ng/mL, respectively). Blood levels in severe MPS IVA were 1.5 times higher than in the milder form. In contrast to blood, urine KS levels in both MPS IVA and controls peaked between 1 and 5 y (15.3 versus 0.26 mg/g creatinine), and thereafter declined with age. Urine KS level also varied with age and clinical severity, and the severe MPS IVA phenotype was associated with 6.7 times greater urine KS excretion than the milder one. These findings indicate that the new assay for blood or urine KS may be suitable for early diagnosis and longitudinal assessment of disease severity in MPS IVA.

Separation of isomeric lysophospholipids by reverse phase HPLC.

A reverse-phase high performance liquid chromatography (HPLC) method was developed which resolved isomers of lysophosphatidylcholine (LPC) differing in the location of the aliphatic chain (sn-1 orsn-2 position) and the position (Δ6 or Δ9) or geometric configuration (cis ortrans) of the olefin group in monounsaturated species. LPC isomers containing an acyl substituent at thesn-2 position eluted before their 1-acyl-sn-glycero-3-phosphocholine (1-acyl LPC) counterparts. The retention times of both thesn-1 andsn-2 isomers of monounsaturated species increased in the order Δ9-cis < Δ9-trans < Δ6-cis. The integrated ultraviolet absorbance (203 nm) in binary mixtures of the Δ9-cis and Δ6-cis 2-acyl lysophospholipid isomers correlated with the lipid phosphorus content of corresponding column eluates (r-0.994). Thus, the present method will facilitate synthesis of isomerically pure diradylphospholipids by providing homogeneous lysophospholipid precursors and help simplify the quantitative analysis of unsaturated lysophospholipid species.

Lysophosphoglycerides and ventricular fibrillation early after onset of ischemia

Lysophosphoglycerides accumulate in ischemic myocardium and induce electrophysiologic alterations in normoxic tissue in vitro closely analogous to those seen during ischemia in vivo. The present study was performed to define the temporal alterations of myocardial phospholipids during the first 3 minutes of ischemia in anesthetized cats and to determine whether the magnitude of the increase in lysophosphoglycerides correlates with the severity of ventricular arrhythmias. Fast-frozen transmural biopsies were obtained simultaneously from the ischemic and non-ischemic zones of the left ventricle. In control animals, values of lysophosphatidylcholine (LPC) did not differ in anterior (2.1 +/- 0.2 nmol/mg protein) compared with lateral (2.2 +/- 0.2 nmol/mg protein) regions of the left ventricular wall. The values for LPC in the anterior and lateral regions were also identical when expressed as % of total phospholipid phosphorus (1.4 +/- 0.1%). Comparing these values to those of all other animals biopsied within 3 minutes of ischemia, no significant increase in LPC was seen (1.7 +/- 0.2%). However, stratification of the animals based on the severity of ventricular arrhythmias showed striking differences. In animals without arrhythmias, no significant change occurred in LPC (1.2 +/- 0.2% phospholipid phosphorus or 2.0 +/- 0.3 nmol/mg protein) compared with the non-ischemic tissue control values (1.4 +/- 0.1% phospholipid phosphorus or 2.1 +/- 0.2 nmol/mg protein). In contrast, in animals with arrhythmias, a striking and significant increase in LPC (to 2.0 +/- 0.2% phospholipid phosphorus or 3.1 +/- 0.3 nmol/mg protein) was seen.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of local reference populations on upper limits of normal for serum alanine aminotransferase levels.

Chronic liver disease can progress to cirrhosis if not detected early and appropriate interventions taken when possible. Serum levels of alanine aminotransferase (ALT, SGPT) and aspartate aminotransferase (AST, SGOT) are commonly measured during routine health care to detect such unsuspected liver disease. Care providers often use the reporting laboratory’s ALT upper reference limit (upper limit of normal, ULN) or a multiple thereof (e.g., 1.5 × ULN) to trigger further evaluation. Such evaluation can be expensive and invasive, yet ignoring aminotransferase elevations can allow life-threatening liver disease to progress if not recognized and treated appropriately. Therefore, how clinical laboratories define their own ALT ULN values is critically important in determining the risk benefit ratio of further evaluation. For technical reasons related to sample stability, validated standards are not used to establish a ULN for ALT and AST1. Instead, laboratories use locally-defined reference populations to establish their own reference ranges for these tests. The criteria used to include and exclude individuals from this important cohort directly determine the value of the test in identifying the presence of disease2. As obesity increases in the general population, such reference populations could increasingly include individuals with unsuspected nonalcoholic fatty liver disease (NAFLD) which would skew the upper reference limit to inappropriately high levels. One recent population study excluded people at risk for NAFLD and concluded that the “healthy range” for serum ALT should be up to 30 U/L for men and 19 U/L for women3. Although some have argued that this would lead to unnecessary medical expenditures and further burden our healthcare system4, 5, large population studies in Korea have demonstrated increased prevalence of NAFLD6 and increased liver-related mortality in middle aged adults with ALT levels between 20 and 40 U/L compared to those with ALT < 20 U/L7. The Nonalcoholic Steatohepatitis (NASH) Clinical Research Network (CRN), a group of eight academic institutions assembled by and in collaboration with the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) to study fatty liver disease, began to design clinical studies in 2002 and considered using an ALT value greater than the ULN as an entry criterion for its major pediatric treatment trial8. However, variability was found in the ALT ULN values reported by clinical laboratories at CRN clinical centers that could confound the inclusion of homogeneous patient cohorts in this and other studies. The study described here was undertaken to establish the causes of the variability reported by laboratories of their self-defined ULN using results of analyses of samples distributed to clinical laboratories as part of annual accreditation by the College of American Pathologists (CAP).