This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A.
Specific Aims Specific Aim 1: Define the contribution of enhanced levels of oxidative stress, cytokine production and foam cell formation to the relationship between adiposity (lean versus obese) and pro-inflammatory status of obese African American women with metabolic syndrome.
Specific Aim 2 : Characterize the K+ channel species underlying K+ currents in macrophages from lean or obese African American (AA) women with metabolic syndrome and their impact on oxidative burst activity, cytokine production and foam cell formation. B. Studies and Results Specific Aim 1: Recruitment for DIVA study participants (lean AA females and obese AA females with metabolic syndrome) to return to Morehouse School of Medicine for a blood draw has continued to be a challenge during the final year of this developmental grant. An amendment to recruit new study participants outside of the DIVA participant pool was approved in April 2009 by MSM IRB committee. With this new recruitment initiative, we have recruited a total of 55 participants since initiating the study. Following monocyte isolation and culture of these cells to mature human macrophages, we performed experiments to quantitate basal and PMA-stimulated oxidative burst activity. We have also determined the response of the macrophages to lipopolysaccharide (LPS), another stimulator of hydrogen peroxide production. Basal and stimulated oxidative burst activity was measured using the fluorescent probe, 2', 7'-dichlorofluorescein which fluoresces when the probe is bound to intracellular hydrogen peroxide. Basal oxidative burst activity was similar in Lean IS and Obese IR PBDMs compared to control human THP-1 monocytes and macrophages (Figure 1A). Acute exposure to phorbol ester, PMA (100 nM), stimulated oxidative burst activity by 19% in THP-1 monocytes and was enhanced an additional 50% in THP-1 cells chemically differentiated to macrophages (Figure 1B). PMA-stimulated oxidative burst activity in Lean PBDMs was enhanced to 317% of basal THP-1 monocyte. Macrophages isolated from Obese AA women possessed a significantly higher oxidative burst response to PMA (533% of basal THP-1 monocytes) compared to Lean PBDMs (Figure 1B). While immature THP-1 monocytes did not respond to an LPS challenge, mature THP-1 macrophages responded to LPS with a 23% increase in hydrogen peroxide production (Figure 1C) similar to our observations with Lean PBDMs. Obese PBDMs possessed a 63% higher oxidative burst activity than Lean PBDMs (Figure 1C). These data suggest that macrophage infiltration into adipose tissue may contribute to elevated oxidative stress and development of obesity-induced inflammation in AA women with MetS. To date, our measurements of cytokines, interleukin-6 and tumor necrosis factor-? have not revealed a difference in production levels in Lean or Obese AA women macrophages. Uptake of modified low density lipoprotein into macrophages which is one of the measurements for foam cell formation has not revealed a significant difference between Lean or Obese AA women macrophages.
Specific Aim 2 : To begin to determine if there is a differential expression of K+ channel activity in Lean or Obese AA macrophages, we have collected and analyzed whole-cell currents when our cell yield allowed. Ionic currents were elicited by the patch-clamp technique using the whole-cell recording configuration. Under high KCl pipette and high NaCl bath solution conditions, cells were voltage-clamped at -40 mV and hyperpolarized or depolarized in 20 mV voltage steps (-160 mV to +60 mV). Resting membrane potential was obtained in the current-clamp mode and all current amplitude was normalized by cell size (capacitance). In the majority of Lean AA MACs an inwardly rectifying K+ current was elicited during hyperpolarizing steps while none was present in Obese AA MACs (Figure 2). During step depolarization small outward current was produced in Lean AA MACs. However, Obese AA MACs possessed large outwardly rectifying currents. This differential expression levels of ionic currents between Lean and Obese AA MACs may alter other endpoints of macrophage function like oxidative burst activity, cytokine production, and foam cell formation.
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