Anoop K. Enjeti

Detection and Measurement of Microparticles: An Evolving Research Tool for Vascular Biology

Microparticles are small, membrane-bound vesicles that are generated from cells of different origin. It now appears that all circulating blood cells as well as endothelial cells release membranous fragments ~1 mum in size or smaller bearing at least some characteristics of the parent cell. Elevated levels of microparticles have been described in cardiovascular states, thrombotic conditions, and cancer. Various methods of detection for microparticles include flow cytometry, enzyme-linked immunoassays, and functional assays. Flow cytometry has several advantages including its ability to quantitate and identify microparticles of different cellular origin. However, the standardization of preanalytical and analytical variables for enumeration of microparticles remains a significant challenge. Newer approaches are being investigated, and an international collaboration is working on standardization of detection as well as quantitation of microparticles by flow cytometry. Although it has evolved as an important vascular biology research tool, microparticle detection needs further evaluation and refinement before it becomes truly useful as a clinical tool.

Microparticles in health and disease.

Microparticles (MPs) are small fragments of membrane-bound cytoplasm that are shed from the surface of an activated or apoptotic cell. Recently, their function as vectors of transcellular exchange of biologic information, in addition to better described forms of intercellular communication such as growth factors, cytokines, and chemokines, has become well recognized. Circulating levels of MPs are thought to reflect a balance between cell stimulation, proliferation, and death. The production of MPs is thought to predominately occur by vesiculation or blebbing of the cell membrane. The mechanisms governing the remodeling of the plasma membrane are complex, involving cytoskeletal changes and a shift from normal phospholipid asymmetry. Increased intracellular calcium subsequent to cell activation leads to intracellular increases in several proteins including gelsolin and calpain, as well as the activity of enzymes such as translocase, floppase, and scramblase, which play important roles in the homeostasis of the cell membrane. The membrane vesiculation and phospholipids asymmetry leading to the production of MPs occurs by the complex interplay of the proteins involved. There are several clinical conditions associated with elevated MPs, and most are also associated with an increased risk of thrombosis. Apart from cardiovascular disease and venous thromboembolism, MPs are also elevated in solid tumors with metastatic disease. The measurement of MPs is being regarded as a potential biomarker, given the range of conditions in which they are elevated and the association with prothrombotic states. The utility of measuring MPs as a diagnostic and prognostic marker is currently a subject of intense investigation. The further development of the various methods for the detection and measurement of MPs and prospective clinical trials establishing the utility of such tests will be critical prior to the routine measurement of MPs in the diagnostic laboratory.

The putative diabetic plasma marker, soluble CD36, is non-cleaved, non-soluble and entirely associated with microparticles

Summary.  Background: CD36 is a widely expressed cell surface receptor that binds lipoproteins, and its function has been implicated in many complications of the metabolic syndrome. A cell‐free form of CD36, soluble CD36 (sCD36), has been reported in human plasma, found to be elevated in obesity and diabetes, and claimed as a marker of insulin resistance. Objective: To determine the nature of sCD36; in particular, whether sCD36 is truly soluble or, as hypothesized, is found as a component of circulating microparticles (MPs). Methods: Lipoproteins were fractionated by density gradient centrifugation, and plasma MPs were isolated by ultracentrifugation, size exclusion, and immunoprecipitation with CD36 detected by immunoblotting. MPs from plasma and activated platelets were analyzed by multicolor flow cytometry, with a DyLight‐488 anti‐CD36 conjugate in combination with antibodies against different cellular markers. Results: Cell‐free plasma CD36 was not observed associated with lipoproteins and was not a proteolytic fragment; rather, it was associated with the plasma MP fraction, suggesting that sCD36 in the plasma of normal subjects is a product of circulating MPs. Cytometric and immunoblotting analyses of plasma from normal donors showed that these MPs were derived mainly from platelets. Analysis of in vitro activated platelets also showed that CD36 to be secreted in the form of MPs. Conclusions: sCD36 is not a proteolytic product, but rather is associated with a specific subset of circulating MPs that can readily be analysed. This finding will enable more specific investigations into the cellular source of the increased levels of plasma CD36 found in subjects with diabetes.

The origin of circulating CD36 in type 2 diabetes

Objective:Elevated plasma levels of the fatty acid transporter, CD36, have been shown to constitute a novel biomarker for type 2 diabetes mellitus (T2DM). We recently reported such circulating CD36 to be entirely associated with cellular microparticles (MPs) and aim here to determine the absolute levels and cellular origin(s) of these CD36+MPs in persons with T2DM.Design:An ex vivo case-control study was conducted using plasma samples from 33 obese individuals with T2DM (body mass index (BMI)=39.9±6.4 kg m−2; age=57±9 years; 18 male:15 female) and age- and gender-matched lean and obese non-T2DM controls (BMI=23.6±1.8 kg m−2 and 33.5±5.9 kg m−2, respectively). Flow cytometry was used to analyse surface expression of CD36 together with tissue-specific markers: CD41, CD235a, CD14, CD105 and phosphatidyl serine on plasma MPs. An enzyme-linked immunosorbent assay was used to quantify absolute CD36 protein concentrations.Results:CD36+MP levels were significantly higher in obese people with T2DM (P<0.00001) and were primarily derived from erythrocytes (CD235a+=35.8±14.6%); although this did not correlate with haemoglobin A1c. By contrast, the main source of CD36+MPs in non-T2DM individuals was endothelial cells (CD105+=40.9±8.3% and 33.9±8.3% for lean and obese controls, respectively). Across the entire cohort, plasma CD36 protein concentration varied from undetectable to 22.9 μg ml−1 and was positively correlated with CD36+MPs measured by flow cytometry (P=0.0006) but only weakly associated with the distribution of controls and T2DM (P=0.021). Multivariate analysis confirmed that plasma CD36+MP levels were a much better biomarker for diabetes than CD36 protein concentration (P=0.009 vs P=0.398, respectively).Conclusions:Both the levels and cellular profile of CD36+MPs differ in T2DM compared with controls, suggesting that these specific vesicles could represent distinct biological vectors contributing to the pathology of the disease.

Bio-maleimide as a generic stain for detection and quantitation of microparticles

Microparticles (MP) are small fragments of cytoplasm shed from a cell surface and their role in the pathophysiology of disease is being extensively investigated. A novel staining technique for quantifying total MP in peripheral blood was evaluated in this study. Evaluation of Bodipy‐maleimide (or bio‐maleimide) as a stain for quantifying total MP in peripheral blood by flow cytometry. Samples were obtained from 10 healthy donors after informed consent. Plasma was prepared by sequential centrifugation at 1500 g followed by 13 000 g and stained with Annexin V and bio‐maleimide. Enumeration beads were added after 15 min of incubation with the stain and samples analyzed on a FACS Canto flow cytometer. Detection and quantification of MP by bio‐maleimide staining was comparable with that by Annexin V. The total mean MP level with bio‐maleimide staining was 34 ± 19.7/μl (range of 11.6–68.1/μl) and with Annexin V staining it was 38.9 ± 29.8/μl (range of 10.6 to 112.9/μl). There was no significant difference using a paired t‐test and methods were comparable using a Bland–Altman plot. Bio‐maleimide is a useful and inexpensive stain to measure total MP levels in peripheral blood by flow cytometry. This technique could be employed to study thrombotic risks in a variety of disease states.

Spontaneous major bleeding in acquired factor X deficiency secondary to AL‐amyloidosis

Summary.  Abnormal bleeding may be seen in up to a third of patients with amyloidosis. Factor X deficiency is the commonest acquired coagulopathy in amyloidosis. While mild intracutaneous bleeding is common, spontaneous life‐threatening haemorrhage is rare. We report a case of acquired FX deficiency due to amyloid light chain (AL)‐amyloidosis presenting with spontaneous retroperitoneal bleeding. The diagnostic and management issues in this patient are discussed.

Primary lymphoma of the uterus and cervix: two case reports and review of the literature

Primary lymphoma of the uterus and cervix is rarely encountered. We present two cases of diffuse large B‐cell lymphoma of the cervix and uterus that were treated with R‐CHOP chemotherapy followed by pelvic radiotherapy. The women are disease free 20 and 19 months after the diagnosis respectively. Seventy‐two cases of primary uterine and cervical lymphoma reported in the English literature in the last 10 years from 2000 to 2010 are reviewed.

Circulating microparticles are elevated in carriers of Factor V Leiden

INTRODUCTION Microparticles (MP) are small membrane bound cellular particles that play an important role in thrombosis. This study was carried out to evaluate if increased numbers or procoagulant potential of circulating MP contribute to the heterogeneity in occurrence of thrombosis in heterozygotes carrying Factor V Leiden (FVL) mutation. METHODS Levels of circulating platelet (CD41a), endothelial (CD62e) as well as leukocyte (CD45) derived MP from 45 FVL heterozygous individuals were enumerated by flow cytometry and compared with normal controls. Functional studies included enzyme linked immunoassay based prothrombinase activity (ELISA) and modified dilute Russell Viper venom test (DRVVT). RESULTS Circulating MP were significantly higher in the FVL cohort compared to the controls (median=2100 vs. 1508 MP/microl, respectively p=0.0021).All subsets of MP (platelet, endothelial and leukocyte) were significantly elevated in the FVL group, the most striking disparity seen in the number of CD45 positive leukocyte MP. Despite the differences in the number of MP between the controls and FVL cohorts, there was no significant difference in the prothrombinase activity recorded by the ELISA (2.0 vs 2.4 PS equivalents; p=0.7374) or clotting time assessed by the DRVVT (47 vs 46 sec, p=0.8118). When the FVL cohort was considered alone there was no significant difference in MP parameters between FVL subjects with or without a history of thrombosis. CONCLUSIONS This is the first study on circulating MP levels in subjects who are heterozygote for factor V Leiden. We report that circulating platelet and leukocyte MP are elevated in carriers of this mutation and may be important contributors to risk of thrombosis.

Genomic profiling of plasma cell disorders in a clinical setting: integration of microarray and FISH, after CD138 selection of bone marrow

Aim To evaluate the role of whole genome comparative genomic hybridisation microarray (array-CGH) in detecting genomic imbalances as compared to conventional karyotype (GTG-analysis) or myeloma specific fluorescence in situ hybridisation (FISH) panel in a diagnostic setting for plasma cell dyscrasia (PCD). Methods A myeloma-specific interphase FISH (i-FISH) panel was carried out on CD138 PC-enriched bone marrow (BM) from 20 patients having BM biopsies for evaluation of PCD. Whole genome array-CGH was performed on reference (control) and neoplastic (test patient) genomic DNA extracted from CD138 PC-enriched BM and analysed. Results Comparison of techniques demonstrated a much higher detection rate of genomic imbalances using array-CGH. Genomic imbalances were detected in 1, 19 and 20 patients using GTG-analysis, i-FISH and array-CGH, respectively. Genomic rearrangements were detected in one patient using GTG-analysis and seven patients using i-FISH, while none were detected using array-CGH. I-FISH was the most sensitive method for detecting gene rearrangements and GTG-analysis was the least sensitive method overall. All copy number aberrations observed in GTG-analysis were detected using array-CGH and i-FISH. Conclusions We show that array-CGH performed on CD138-enriched PCs significantly improves the detection of clinically relevant and possibly novel genomic abnormalities in PCD, and thus could be considered as a standard diagnostic technique in combination with IGH rearrangement i-FISH.

Activation of protein phosphatase 2A in FLT3+ acute myeloid leukemia cells enhances the cytotoxicity of FLT3 tyrosine kinase inhibitors

Constitutive activation of the receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3), via co-expression of its ligand or by genetic mutation, is common in acute myeloid leukemia (AML). In this study we show that FLT3 activation inhibits the activity of the tumor suppressor, protein phosphatase 2A (PP2A). Using BaF3 cells transduced with wildtype or mutant FLT3, we show that FLT3-induced PP2A inhibition sensitizes cells to the pharmacological PP2A activators, FTY720 and AAL(S). FTY720 and AAL(S) induced cell death and inhibited colony formation of FLT3 activated cells. Furthermore, PP2A activators reduced the phosphorylation of ERK and AKT, downstream targets shared by both FLT3 and PP2A, in FLT3/ITD+ BaF3 and MV4-11 cell lines. PP2A activity was lower in primary human bone marrow derived AML blasts compared to normal bone marrow, with blasts from FLT3-ITD patients displaying lower PP2A activity than WT-FLT3 blasts. Reduced PP2A activity was associated with hyperphosphorylation of the PP2A catalytic subunit, and reduced expression of PP2A structural and regulatory subunits. AML patient blasts were also sensitive to cell death induced by FTY720 and AAL(S), but these compounds had minimal effect on normal CD34+ bone marrow derived monocytes. Finally, PP2A activating compounds displayed synergistic effects when used in combination with tyrosine kinase inhibitors in FLT3-ITD+ cells. A combination of Sorafenib and FTY720 was also synergistic in the presence of a protective stromal microenvironment. Thus combining a PP2A activating compound and a FLT3 inhibitor may be a novel therapeutic approach for treating AML.