Mammalian cell transfection is a powerful technique commonly used in molecular biology to express exogenous DNA or RNA in cells and study gene and protein function. Although several transfection strategies have been developed, there is a wide variation with regards to transfection efficiency, cell toxicity and reproducibility. Thus, a sensitive and robust method that can optimize transfection efficiency based not only on expression of the target protein of interest but also on the uptake of the nucleic acids, can be an important tool in molecular biology. Herein, we present a simple, rapid and robust flow cytometric method that can be used as a tool to optimize transfection efficiency while overcoming limitations of prior established methods that quantify transfection efficiency.

Endothelial progenitor cells (EPCs) repair damaged vascular endothelium, and low circulating EPC levels have been associated with cardiovascular disease (CVD). CD34⁺/KDR⁺ EPCs are commonly reported in the literature and CD34⁺/CD133⁺/KDR⁺ EPCs are rare in circulation but highly specific for endothelial lineage. HIV-infected (HIV+) adults have chronic inflammation and increased CVD risk, but the relationship between CVD, vascular inflammation, and EPCs in HIV remains unclear. In a pilot study, EPCs were measured in 57 HIV+ men [≥50 years old, HIV-1 RNA <50 copies/ml on antiretroviral therapy (ART)] by real-time flow cytometry using cellular immaturity (CD34 and/or CD133) and endothelial commitment (KDR) markers. Fasting inflammatory biomarker levels were measured by ELISA. Median age was 57 years; CD4⁺ T lymphocyte count was 570 cells/mm³. Prevalent CVD risk factors included 16% diabetes, 28% hypertension, 53% dyslipidemia, and 33% smoking. Median (interquartile range) EPC values were CD34⁺/KDR⁺ 0.1 (0.0-0.9) cells/10⁵ peripheral blood mononuclear cells (PBMCs) and CD34⁺/CD133⁺/KDR⁺ 0.1 (0.0-0.9) cells/10⁵ PBMCs. We observed a high prevalence of undetectable CD34⁺/KDR⁺ (40%) and CD34⁺/CD133⁺/KDR⁺ EPCs (44%). Men with undetectable EPCs were more likely to have ≥2 CVD risk factors, lower interleukin-6 (IL-6), and higher sCD163 levels. In these older HIV+ men on suppressive ART, CD34⁺/KDR⁺ and CD34⁺/CD133⁺/KDR⁺ EPC levels were low and often undetectable. Undetectable EPC levels were associated with greater CVD risk factor burden, lower IL-6 (consistent with decreased EPC production stimulus), and higher sCD163 (consistent with monocyte activation and prior CVD associations) levels, suggesting a potential relationship between EPCs and atherosclerotic burden in this population.

AbstractBackgroundFluorescence-based cell-free assays offer an attractive alternative to current cell-based assays for measuring the redox activity of High-Density Lipoprotein (HDL). We have recently developed a biochemical assay that assesses the effect of HDL on the oxidation rate of dihydrorhodamine 123 (DHR), reflected by increasing fluorescence over time. However, an immediate reduction in the fluorescence signal is observed after addition of HDL to DHR, due to fluorescence quenching from lipid-probe interactions. Understanding this process is important for interpretation of the results of all fluorescence-based cell-free assays that measure oxidative properties of lipids.MethodsWe determined the effect of quenchers (proteins or lipids) on the fluorescence signal of two fluorescence-based cell-free assays: the rhodamine 123 (RHD)-based assay, and a previously described assay based on dichlorodihydrofluorescein (DCF) in patients with systemic inflammation or atherosclerosis versus healthy subjects.ResultsWe found lipid-probe interactions between the non-fluorescent substrate and the lipid, which affect the observed rate of change of fluorescence after addition of lipids to DHR and DCFH. These interactions depended on: sample collection and storage, types and concentrations of lipid and fluorescent probe, method of HDL isolation, diluents and matrices, and pH. The RHD-based assay yielded reproducible measurements despite fluorescence quenching, while the DCF-based assay displayed more experimental variability. Furthermore, the lipid-probe interactions varied according to the setting of systemic inflammation when using apolipoprotein (apo) B-depleted plasma. However, under fixed conditions the rhodamine assay could reliably detect similar mean relative differences in the redox activity of HDL samples between different groups of patients using either purified HDL or apo-B depleted plasma.ConclusionsLipid-probe interactions should be considered when interpreting the results of fluorescence assays for measuring lipid oxidative state. Ideally, samples should be freshly obtained and purified HDL should be utilized rather than Apo B-depleted serum. Assay variability can be reduced by strict standardization of conditions (particularly sample collection, storage, lipid isolation method). Data comparisons between different studies similarly require strict standardization of conditions between studies and this caveat must be considered when using these assays to study the role of HDL function in the development of atherosclerosis in vivo.

Objective

The role of high-density lipoprotein (HDL) function in HIV-related atherosclerotic cardiovascular disease (CVD) is unclear. HDLs isolated from HIV [HIV(+)HDL] and HIV-uninfected individuals [HIV(-)HDL] were assessed for HDL function and ability to promote monocyte-derived foam cell formation (MDFCF; a key event in HIV-related CVD) ex vivo.

Design/methods

Using an established in-vitro model of atherogenesis and plasma samples from an established cross-sectional study of virologically suppressed HIV men on stable effective antiretroviral therapy and with low CVD risk (median age: 42 years; n = 10), we explored the impact of native HDL [HIV(+)HDL] on MDFCF. In this exploratory study, we selected HIV(+)HDL known to be dysfunctional based on two independent measures of impaired HDL function: antioxidant (high HDLox) ability of HDL to release apolipoprotein A-I (ApoA-I) (low HDL-ApoA-I exchange). Five healthy men matched by age and race to the HIV group were included. Given that oxidation of HDL leads to abnormal HDL function, we also compared proatherogenic effects of HIV(+)HDL vs. chemically derived HDLox. The ex-vivo atherogenesis assay was performed using lipoproteins (purchased or isolated from plasma using ultracentrifugation) and monocytes purified via negative selection from healthy donors.

Results

HIV(+)HDL known to have reduced antioxidant function and rate of HDL/ApoAI exchange promoted MDFCF to a greater extent than HDL (33.0 vs. 26.2% foam cells; P = 0.015). HDL oxidized in vitro also enhanced foam cell formation as compared with nonoxidized HDL (P < 0.01).

Conclusion

Dysfunctional HDL in virologically suppressed HIV individuals may potentiate atherosclerosis in HIV infection by promoting MDFCF.The role of HDL function in HIV-related atherosclerotic CVD is unclear. HDL isolated from HIV [HIV(+)HDL] and HIV-uninfected individuals [HIV(-)HDL] were assessed for HDL function and ability to promote foam cell formation ex vivo. HIV(+)HDL known to have reduced antioxidant function and rate of HDL/ApoA1 exchange promoted MDFCF to a greater extent than HDL(-)HDL (33.0 vs. 26.2% foam cells.Subject codes: Inflammation, Lipids and Cholesterol, Vascular Biology, Oxidant Stress, Atherosclerosis.

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).

Current cell-based assays for determining the functional properties of high-density lipoproteins (HDL) have limitations. We report here the development of a new, robust fluorometric cell-free biochemical assay that measures HDL lipid peroxidation (HDLox) based on the oxidation of the fluorochrome Amplex Red. HDLox correlated with previously validated cell-based (r = 0.47, p<0.001) and cell-free assays (r = 0.46, p<0.001). HDLox distinguished dysfunctional HDL in established animal models of atherosclerosis and Human Immunodeficiency Virus (HIV) patients. Using an immunoaffinity method for capturing HDL, we demonstrate the utility of this novel assay for measuring HDLox in a high throughput format. Furthermore, HDLox correlated significantly with measures of cardiovascular diseases including carotid intima media thickness (r = 0.35, p<0.01) and subendocardial viability ratio (r = -0.21, p = 0.05) and physiological parameters such as metabolic and anthropometric parameters (p<0.05). In conclusion, we report the development of a new fluorometric method that offers a reproducible and rapid means for determining HDL function/quality that is suitable for high throughput implementation.

Transfection is one of the most frequently used techniques in molecular biology that is also applicable for gene therapy studies in humans. One of the biggest challenges to investigate the protein function and interaction in gene therapy studies is to have reliable monospecific detection reagents, particularly antibodies, for all human gene products. Thus, a reliable method that can optimize transfection efficiency based on not only expression of the target protein of interest but also the uptake of the nucleic acid plasmid, can be an important tool in molecular biology. Here, we present a simple, rapid and robust flow cytometric method that can be used as a tool to optimize transfection efficiency at the single cell level while overcoming limitations of prior established methods that quantify transfection efficiency. By using optimized ratios of transfection reagent and a nucleic acid (DNA or RNA) vector directly labeled with a fluorochrome, this method can be used as a tool to simultaneously quantify cellular toxicity of different transfection reagents, the amount of nucleic acid plasmid that cells have taken up during transfection as well as the amount of the encoded expressed protein. Finally, we demonstrate that this method is reproducible, can be standardized and can reliably and rapidly quantify transfection efficiency, reducing assay costs and increasing throughput while increasing data robustness.