Improvement in middle cerebral artery structure and endothelial function in stroke-prone spontaneously hypertensive rats after macrophage depletion

Abstract

Background: Inflammation is involved in the pathogenesis of hypertension. Hypertensive animals have an increased number of perivascular macrophages in cerebral arteries. Macrophages might be involved in remodeling of the cerebral vasculature. We hypothesized that peripheral macrophage depletion would improve MCA structure and function in hypertensive rats.

Methods: For macrophage depletion, six-week-old stroke-prone spontaneously hypertensive rats (SHRSP) were treated with CLOD, 10 mL/kg every three or four days, i.p., or vehicle (PBS lipo). MCA structure and function were analyzed by pressure and wire myography.

Results: Blood pressure was not affected by CLOD. The number of perivascular CD163-positive cells per microscopic field was reduced in the brain of SHRSP+CLOD. CLOD treatment caused an improvement in endothelium-dependent dilation after intralumenal perfusion of ADP and incubation with Ach. Inhibition of NO production blunted the Ach response, and endothelium-independent dilation was not altered. At an intralumenal pressure of 80 mmHg, MCA from SHRSP+CLOD showed increased lumen diameter, decreased wall thickness, and wall-to-lumen ratio. Cross-sectional area of pial arterioles from SHRSP+CLOD was higher than PBS lipo.

Conclusions: These results suggest that macrophage depletion attenuates MCA remodeling and improves MCA endothelial function in SHRSP.

Keywords: endothelium-dependent vasodilation; hypertension; liposome-encapsulated clodronate; middle cerebral artery; vascular remodeling.

Figure 1

Blood pressure from 12-week-old SHRSP treated with CLOD or PBS lipo measured by tail-cuff. There was no difference in systolic and diastolic blood pressure in SHRSP after treatment with CLOD when compared to PBS lipo-treated SHRSP. Data are means ± SEM of a total of 75 cycles per rat performed in three different days (25 cycles per day).

Figure 2

Representative FCM analysis for quantification of macrophages in SHRSP treated with PBS lipo or CLOD. Upper panels represent the analysis of circulating phagocytic cells in blood that are double-positive for CD11b and CD163. Note that the number of events is higher in SHRSP-treated with PBS lipo (upper left panel) than SHRSP-treated with CLOD (upper right panel). The number of macrophages in the peritoneal cavity was also quantified by FCM (lower panels). Cell populations were separated by side-scatter and forward-scatter. Note that the number of events is smaller in CLOD-treated SHRSP (lower right panel) than PBS lipo-treated SHRSP (lower left panel).

Figure 3

Immunofluorescent (IF) staining of perivascular macrophages in the cerebral vasculature of SHRSP. Macrophages were identified as cells with immunoreactivity to CD163 (red), and only CD163-positive cells surrounding blood vessels (identified by immunoreactivity for α-SMA, green) were counted. Nuclei were identified by DAPI-staining (blue). Upper panels are representative images from SHRSP treated with PBS lipo, and lower panels are images from CLOD-treated SHRSP. Bar graph: quantification of the IF. CLOD caused a significant reduction in perivascular CD163 positive cells in SHRSP. *p < 0.05, Student's t-test.

Figure 4

Morphometric analyses of pial arterioles. CLOD treatment caused a significant increase in the cross-sectional area of pial arterioles in SHRSP (A), when compared to SHRSP treated with PBS lipo. There was a small, although not-significant, increase in the lumen (B, p = 0.14) and wall (C, p = 0.12) cross-sectional areas after six weeks of CLOD treatment. (D) Representative images of pial arterioles from PBS lipo (left) and CLOD (right)-treated SHRSP. The circled area represents the cross-sectional area of the arteriole. Bar = 25 µm. Morphometric measurements were performed in 10 µm-thick slices of the brain from perfusion-fixed rats. Images were acquired using a 40× objective and cross-sectional area measurements performed using a calibrated software by an investigator blinded to the experimental groups. Data are means ± SEM. *p = 0.05, Student's t-test.

Figure 5

CLOD treatment did not alter MCA reactivity to the constricting agent 5-HT. The ability of the MCA to respond to agonist-induced constriction was assessed by adding increasing concentrations of 5-HT in the tissue bath. CLOD treatment did not change the concentration-response curve to 5-HT, as seen by percent of maximum response (A), or the change in lumen diameter from baseline (B). The MCA was maintained in warm (37°C), oxygenated PSS at an intralumenal pressure of 80 mmHg and was allowed to equilibrate for 10 minutes before a measurement was taken and the next dose of 5-HT was added. Data are means ± SEM.

Figure 6

CLOD treatment improved endothelium-dependent dilation of the MCA to ADP. CLOD treatment increased both the raw dilation of the MCA, observed as a change in diameter from baseline (A), and the percent of passive diameter (B). *p < 0.01, two-way ANOVA. Values are means ± SEM. The MCA was mounted in a pressure myograph and kept in oxygenated warm PSS under 80 mmHg of intralumenal pressure and physiological flow. ADP was added to the intralumenal perfusate.

Figure 7

CLOD treatment improved MCA dilation to Ach. MCA rings (2 mm) were mounted on a wire myograph and pre-constricted with 10–6 mol/L 5-hydroxytriptamine (5-HT, % KCl constriction: 114 ± 4 vs. 111 ± 4, PBS lipo vs. CLOD). Concentration-response curves to Ach in absence or presence of the NOS inhibitor l-NAME were constructed. MCA relaxation to Ach was improved in SHRSP after CLOD treatment, and this effect was blunted by pre-incubation of MCA rings with l-NAME (10–5 mol/L, A). MCA response to the endothelium-independent vasodilator SNP was not different between groups (B). *p < 0.05, PBS lipo vs. CLOD, two-way ANOVA. Values are means ± SEM.

Figure 8

Peripheral phagocyte depletion improved MCA passive structure in SHRSP. CLOD treatment increased the lumen diameter (A), decreased the wall thickness (C) and wall-to-lumen ratio (D), without changing outer diameter (B). *p < 0.05, PBS lipo vs. CLOD, two-way ANOVA. Values are means ± SEM. The MCA was mounted in a pressure myograph and kept in oxygenated warm calcium-free PSS supplemented with 2 mM EGTA and 10 µM under no-flow conditions.

Figure 9

Peripheral phagocyte depletion did not improve MRA passive structure in SHRSP (p > 0.05, PBS vs. CLOD, ANOVA). CLOD treatment did not change lumen (A) and outer (B) diameters. There was a small, but insignificant, reduction in the wall thickness (C) and wall-to-lumen ratio (D) at higher intralumenal pressures. Values are means ± SEM. The MRA was mounted in a pressure myograph and kept in oxygenated warm calcium-free PSS supplemented with 2 mM EGTA and 10 µM of sodium nitroprusside and under no-flow conditions.

Figure 10

Tumor necrosis factor (TNF)-α production by PVAT. Two-weeks treatment of SHRSP with CLOD resulted in decreased expression of CD163 mRNA (A), suggesting reduction in macrophage number. In addition, this treatment regimen caused a trend towards an increase in TNF-α mRNA expression in the MRA PVAT (B), suggesting that, in the absence of macrophages, white adipose tissue can produce proinflammatory cytokines that mediate MRA hypertensive remodeling. Data are means ± SEM. *p = 0.01, Student's t-test; Ψp = 0.056, Student's t-test.