Cleavage and reduced CD36 ectodomain density on heart and spleen macrophages in the spontaneously hypertensive rat

Abstract

Metabolic disease is accompanied by a range of cellular defects ("comorbidities") whose origin is uncertain. To investigate this pathophysiological phenomenon we used the Spontaneously Hypertensive Rat (SHR), which besides an elevated arterial blood pressure also has many other comorbidities, including a defective glucose and lipid metabolism. We have shown that this model of metabolic disease has elevated plasma matrix metalloproteinase (MMP) activity, which cleaves the extracellular domain of membrane receptors. We hypothesize here that the increased MMP activity also leads to abnormal cleavage of the scavenger receptor and fatty acid transporter CD36. To test this idea, chronic pharmaceutical MMP inhibition (CGS27023A) of the SHR and its normotensive control, the Wistar Kyoto Rat (WKY), was used to determine if inhibition of MMP activity serves to maintain CD36 receptor density and function. Surface density of CD36 on macrophages from the heart, spleen, and liver was determined in WKY, SHR, CGS-treated WKY (CGS WKY), and CGS-treated SHR (CGS SHR) by immunohistochemistry with an antibody against the CD36 ectodomain. The extracellular CD36 density was lower in SHR heart and spleen macrophages compared to that in the WKY. MMP inhibition by CGS served to restore the reduced CD36 density on SHR cardiac and splanchnic macrophages to levels of the WKY. To examine CD36 function, culture assays with murine macrophages (RAW 264.7) after incubation in fresh WKY or SHR plasma were used to test for adhesion of light-weight donor red blood cell (RBC) by CD36. This form of RBC adhesion to macrophages was reduced after incubation in SHR compared WKY plasma. Analysis of the supernatant macrophage media by Western blot shows a higher level of CD36 extracellular protein fragments following exposure to SHR plasma compared to WKY. MMP inhibition in the SHR plasma compared to untreated plasma, served to increase the RBC adhesion to macrophages and decrease the number of receptor fragments in the macrophage media. In conclusion, these studies bring to light that plasma in the SHR model of metabolic disease has an unchecked MMP degrading activity which causes cleavage of a variety of membrane receptors, including CD36, which attenuates several cellular functions typical for the metabolic disease, including RBC adhesion to the scavenger receptor CD36. In addition to other cell dysfunctions chronic MMP inhibition restores CD36 in the SHR.

Keywords: CD36; MMP; MMP inhibitor CGS27023A; Spontaneously Hypertensive Rat (SHR); TIMP.

Fig. 1

TIMP-1 levels in WKY, SHR, CGS WKY, and CGS SHR plasma. Representative TIMP-1 band at 22 kDa (the molecular weight of TIMP-1) for Western blots performed. Plasma from WKY, SHR, CGS WKY, and CGS SHR (n = 4 rats per group) were tested. One WKY plasma sample was used as normal reference between blots and included in every Western blot. Histogram shows average relative band density for TIMP-1 levels in plasma of WKY, SHR, CGS WKY, and CGS SHR. By Student’s t-test, *p < 0.05 WKY vs. SHR. By single-factor ANOVA, p < 0.01 among WKY, SHR, CGS WKY, and CGS SHR.

Fig. 2

TIMP-2 levels in WKY, SHR, CGS WKY, and CGS SHR plasma by Western blot. Representative TIMP-2 band at 21 kDa (the molecular weight of TIMP-2) for Western blots performed. Plasma from WKY, SHR, CGS WKY, and CGS SHR (n = 4 rats per group) were tested. One WKY plasma sample was used as a normalizing reference between blots and included in every Western blot. The histogram shows the average relative band density for TIMP-2 levels in plasma of WKY, SHR, CGS WKY, and CGS SHR. By Student’s t-test, *p < 0.05 WKY vs. SHR. By single-factor ANOVA, p < 0.01 among WKY, SHR, CGS WKY, and CGS SHR.

Fig. 3

WKY and SHR macrophages labeled with CD68 macrophage marker by immunohistochemistry in frozen tissue sections. A. Representative micrographs of heart, spleen, and liver macrophages labeled with CD68 antibody are shown along with respective negative controls in which the primary CD68 antibody was replaced by an irrelevant goat IgG. Scale bar = 20 μm. Single cell macrophage boundaries are outlined by dashed lines. Blue arrows in micrographs of WKY macrophages point towards areas on the cell membrane with higher CD68 label density compared to those of the SHR. Black arrows in micrographs of SHR macrophages indicate areas of reduced CD68 label. B. Average macrophage count per 0.0016 cm² area of heart, spleen, and liver sections (n = 3; average of 5 image frames per animal per tissue section) in WKY and SHR. *p < 0.05 WKY vs. SHR macrophage count in the heart. **p < 0.05 WKY vs. SHR macrophage count in the spleen.

Fig. 4

Immunolabeling of extracellular CD36 on macrophage membranes in the heart. A. Representative micrographs of WKY, SHR, CGS WKY, and CGS SHR macrophages with CD36 labeled (brown) in 5 μm frozen heart sections. Single cell macrophage boundaries are outlined by dashed lines. Negative control (normal goat IgG) has near undetectable intensity levels. Scale bar = 20 μm. Blue arrows shown on the micrograph of WKY macrophage indicate areas on the cell membrane that have higher and more evenly distributed CD36 density than that of the SHR. Black arrows point towards areas on the SHR cell membrane that have low CD36 ectodomain density. B. Histogram of average CD36 label density by light absorption where a contour of each cell was drawn. The log₁₀ of {the mean intensity within the contour of a given cell was normalized to background intensity without cells}. The light absorption was measured in 30 cells per animal and averaged between 3 rats (n = 3) per following groups of animals: WKY, SHR, CGS WKY, and CGS SHR. By Student’s t-test, *p < 0.05 WKY vs. SHR. **p < 0.05 SHR vs. CGS SHR. By single-factor ANOVA, p < 0.01 among WKY, SHR, CGS WKY, and CGS SHR.

Fig. 5

Immunolabeling of extracellular CD36 on macrophage membranes in the spleen. A. Representative micrographs of WKY, SHR, CGS WKY, and CGS SHR macrophages with CD36 labeled (brown) in frozen spleen sections. Single cell macrophage boundaries are outlined by dashed lines. Negative control with replacement of the CD36 primary antibody with normal goat IgG is shown on the right panel. Scale bar = 20 μm. Blue arrows on the micrograph of WKY macrophage indicate areas on the WKY cell membrane with higher and more evenly distributed CD36 label density compared to that of the SHR. Black arrows point towards areas on the SHR cell membrane that have low CD36 ectodomain density. B. Histogram of average CD36 label density by light absorption where a contour of each cell was drawn. The log₁₀ of {the mean intensity within the contour of a given cell was normalized to background intensity without cells}. The light absorption was measured in 30 cells per animal and averaged between rats (n = 3) in each of the following animal groups: WKY, SHR, CGS WKY, and CGS SHR. By Student’s t-test, *p < 0.05 WKY vs. SHR. **p < 0.05 SHR vs. CGS SHR. By single-factor ANOVA, p < 0.05 among WKY, SHR, CGS WKY, and CGS SHR.

Fig. 6

Immunolabeling of extracellular CD36 on macrophage membranes in the liver. A. Representative micrographs of WKY, SHR, CGS WKY, and CGS SHR macrophages with CD36 labeled (brown) in frozen liver sections. In negative controls, the CD36 primary antibody was replaced with normal goat IgG. Scale bar = 20 μm. Blue arrows in the micrograph of WKY macrophage point towards areas on the cell membrane with sparse CD36 label. Black arrows in the micrograph of SHR macrophage point towards areas on the cell membrane with higher CD36 label density compared to the WKY. B. Histogram of average CD36 label density by light absorption where a contour of each cell was drawn. The log₁₀ of {the mean intensity within the contour of a given cell was normalized to background intensity without cells}. The light absorption was measured in 30 cells per animal and averaged between 3 rats (n = 3) per following animals: WKY, SHR, CGS WKY, and CGS SHR. By Student’s t-test, *p < 0.05 WKY vs. SHR.

Fig. 7

Red blood cell attachment to macrophages after incubation in WKY, SHR, and CGS SHR plasma. A. Representative micrographs of cultured murine macrophages (black arrows) incubated in control incubation medium (DMEM) and diluted WKY, SHR and CGS-inhibited SHR plasma in the presence of light-weight red blood cells (red arrows). Panel inset shows the smaller red blood cell adhered to a larger macrophage (green arrow). Excess red blood cells were removed by mild fluid shear. Micrographs show the relative ratios of attached red blood cells to macrophages. B. Histogram of average ratio of light-weight red blood cell attachment to macrophages after DMEM, and diluted WKY, SHR and CGS-SHR plasma conditions. The ImageJ cell counter tool was used to generate the ratio of attached light-weight red blood cells over macrophages per field of view. Ratios from 10 fields of view were calculated per animal with n = 4 animals used WKY plasma group and n = 5 for the SHR and CGS SHR groups. p < 0.0001 among all groups by single-factor ANOVA ****p < 0.0001 WKY plasma vs. SHR plasma and **p < 0.01 CGS-SHR plasma vs. SHR plasma by Student’s t-test with Bonferroni correction.

Fig. 8

Soluble extracellular CD36 levels in macrophage culture supernatant after exposure to plasma of WKY, SHR, CGS WKY, and CGS SHR. A. Representative extracellular CD36 band at 53 kDa (the molecular weight of extracellular CD36) for Western blots performed. Culture supernatant from WKY, SHR, and CGS SHR (n = 3 rats per group) was tested. One WKY plasma sample was used as control reference for every Western blot. Histogram of normalized band density (relative to WKY plasma band density) for extracellular CD36 levels in culture supernatant of WKY, SHR, and CGS SHR. **p < 0.01 WKY and CGS SHR vs. SHR by Student’s t-test with Tukey post hoc correction. ***p < 0.001 among WKY, SHR, and CGS SHR by single-factor ANOVA.