Defense Date

5-3-2003

Graduation Date

Spring 2003

Availability

Immediate Access

Submission Type

dissertation

Degree Name

PhD

Department

Chemistry and Biochemistry

School

Bayer School of Natural and Environmental Sciences

Committee Chair

Partha Basu

Committee Member

John A. Pollack

Committee Member

Mitchell E. Johnson

Committee Member

Robert W. W. Biederman

Keywords

macrophages

Abstract

Cardiovascular disease kills nearly 1 million Americans each year. While treatment at all levels has made significant advances over the past 3 decades, diagnosis is still limited to structural or hemodynamic changes associated with advanced disease. More than 60% of myocardial infarctions (MI) result from the rupture and associated acute thrombosis of atherosclerotic plaque that reduce lumen size by less that 50%. Ultra small superparamagnetic iron oxide particles (USPIO) have recently been proposed as a macrophage targeted magnetic resonance contrast agent. The goal of the present study was to determine if, and the extent to which, molecules known to alter macrophage metabolism significantly alter the extent to which USPIO's are internalized in these cells.

Murine macrophage cells (J774) known to be constitutively activated, were cultured in 8 well chamber slides. One group of 4 wells was treated for 24 hours with the test agent. Control and treated cells were then incubated for 4 hours with 0.0µL, 1.0 µL (11.2 µg Fe), 10.0 µL (112.0 µg Fe) and 100.0 µL (1.12 mg Fe) of stock USPIO (Feridex, Berlex labs). Cell density and iron uptake was quantified using spectral analysis of digital microscope images of fixed cells stained with acridine orange and counterstained with Prussian Blue.

Macrophage uptake of USPIO was significantly related to iron concentration (r2= 0.992). However there was saturation of cell uptake at higher USPIO concentration. Uptake, (iron area/total image area) expressed as the percent, became significantly greater than background by 5 minutes (0.084+ 0.001% versus 0.045 +0.029%, p=0.028) and peaked at 4 hours. At low dose (10 and 20 ng/ml) Interleukin-4 did not affect cell uptake of USPIO. However at 40 ng/ml, IL-4 produced a significant increase in iron endocytosis at 1.12 mg Fe/ml (2-way ANOVA, p=0.032). Similarly, Human Interferon gamma (IFN-γ) had no effect at the 3 lower doses (10, 100, and 500 International Units (IU)/ml, corresponding to 0.5, 5.0, 25.0 and 50 ng/ml). Treatment with 1000 IU/ml resulted in an increase in cell uptake (2-way ANOVA, p=0.045).

To test the importance of serum proteins on endocytosis, a group of cells were cultured with and without 10% fetal bovine serum (FBS). Absence of FBS resulted in a substantial reduction in USPIO uptake (p < 0.001). There was also a trend toward reduced cell density compared to control (p=0.056) which is likely the effect of the absence of growth factors in the FBS free cell cultures.

Cytochalsin-B depolarizes actin filaments within macrophage plasma membrane and thus inhibits membrane invagination and thus endocytosis. At a dose of 1.0 µg/ml it has been previously been shown to preferentially inhibit endocytosis of larger particles (> 300 nm) with little effect on small (< 300 nm). In the present study, USPIO uptake trended toward being reduced (p=0.062) indicating that although individual particle size was small (100-150 nm), the aggregation of these particles results in a larger effective size in terms of trans-membrane pathway.

The test USPIO was coated with a thin layer of dextran-10. The mannose receptor has broad affinity to polysaccharide moieties. To test the potential for this receptor to be involved in USPIO endocytosis the receptor was blocked with mannan. There was no significant inhibition of endocytosis (p=0.188) excluding this receptor as a major endocytotic pathway for the tested USPIO.

Both angiotensin converting enzyme inhibiting drugs and HMG-CoA reductase inhibiting medications are used widely for control of hypertension and lipid lowering therapy respectively. Cells were treated with Captopril at doses of 0.001, 0.01, 0.1 and 1.0mM. Even at the highest dose there was no difference between treated and control cells in terns of cell density or USPIO uptake. Treatment with the statin mevinolin resulted substantial inhibition of USPIO uptake at 1.0 and 17.5 µM (p=0.004 and p < 0.001 respectively). In addition cell density was reduced at the higher dose compared to control (p=0.033) which is in agreement with this agents known macrophage anti-proliferative properties.

This study suggests that USPIO's enter macrophages via a number of different pathways including opsonification to serum proteins, scavenger receptors and fluid phase endocytosis. The effect of the actin blocking agent cytochalasin-B indicates that the size of USPIO aggregates rather than individual particles determine the rate of endocytosis.

Finally, these data provide evidence that substantial regulation of USPIO uptake occurs by both endogenous (cytokines) and exogenous agents (HMG Co-A reductase inhibitors). Mevinolin therapy may reduce the sensitivity for USPIO agents to detect the presence of macrophages in the vessel wall. Although it is beyond the scope of this study, it would be of interest to determine if mevinolin treated macrophages recover endocytotic capacity after treatment is withdrawn or whether the effect is permanent. If USPIO enhanced MRI can detect macrophage loss of function, and if this effect is related to inflammation or plaque stability, MRI could have a role in determining the effectiveness of statin as well as other drug therapies aimed at treating atherosclerotic disease.

Format

PDF

Language

English

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