IGF-1 receptor cleavage in hypertension

Abstract

Increased protease activity causes receptor dysfunction due to extracellular cleavage of different membrane receptors in hypertension. The vasodilatory effects of insulin-like growth factor-1 (IGF-1) are decreased in hypertension. Therefore, in the present study the association of an enhanced protease activity and IGF-1 receptor cleavage was investigated using the spontaneously hypertensive rats (SHRs) and their normotensive Wistar Kyoto (WKY) controls (n = 4). Matrix metalloproteinase (MMP) activities were determined using gelatin zymography on plasma and different tissue samples. WKY aorta rings were incubated in WKY or SHR plasma with or without MMP inhibitors, and immunohistochemistry was used to quantify the densities of the alpha and beta IGF-1 receptor (IGF-1R) subunits and to determine receptor cleavage. The pAkt and peNOS levels in the aorta were investigated using immunoblotting as a measure of IGF-IR function. Increased MMP-2 and MMP-9 activities were detected in plasma and peripheral tissues of SHRs. IGF-1R beta labeling was similar in both groups without plasma incubation, but the fraction of immunolabeled area for IGF-1R alpha was lower in the endothelial layer of the SHR aorta (p < 0.05). A 24-h incubation of WKY aorta with SHR plasma did not affect the IGF-1R beta labeling density, but reduced the IGF-1R alpha labeling density in the endothelium (p < 0.05). MMP inhibitors prevented this decrease (p < 0.01). Western blot analyses revealed that the pAkt and peNOS levels under IGF-1-stimulated and -unstimulated conditions were lower in SHRs (p < 0.05). A reduced IGF-1 cellular response in the aorta was associated with the decrease in the IGF-1R alpha subunit in the SHR hypertension model. Our results indicate that MMP-dependent receptor cleavage contributed to the reduced IGF-1 response in SHRs.

Fig. 1

Gelatinolytic activities in plasma and tissue samples from WKY and SHRs. Bar graphs show the means ± SD of the gelatinolytic band densities (n = 4). Gelatin zymography gels used to quantify the plasma pro-MMP-9, MMP-9, pro-MMP-2 and MMP-2 activities a. Plasma MMP activities b. MMP-9 activity in heart, kidney, spleen and liver homogenates c. *p < 0.05, **p < 0.01, ***p < 0.001 compared with WKY

Fig. 2

Micrographs of aorta sections of WKY a, c and SHRs b, d after IHC labeling with primary antibodies against IGF-1R alpha a, b and beta c, d. Arrowheads show the endothelium, and arrows indicate the smooth muscle layer. Negative controls for IGF-1 alpha and beta IHC labeling are shown as smaller images. Means ± SD of the percentage of stained area for IGF-1R alpha e and beta f in tunica intima (endothelium) and tunica media (smooth muscle cell) layers after IHC. The scale bar is 500 μm. *p < 0.05 compared with WKY (n = 4)

Fig. 3

Immunolabeling of WKY aorta sections with a primary antibody against the IGF-1R alpha subunit. Aortic rings were incubated with plasma from WKY a, b or SHRs c, d in the presence b, d or absence a, c of protease inhibitors (EDTA 10 mM, doxycycline; 11,3 μM) for 24 h. Representative images are shown a-d. Arrowheads indicate the endothelium, and arrows indicate the smooth muscle layer. The scale bar is 500 μm. Bar graphs show the means ± SD of the percentage of stained area using the same incubation conditions a-d in tunica intima (endothelium) e and tunica media (smooth muscle cell) layers f. Two to four different aorta sections per animal were labeled to determine the average per rat (n = 4). The scale bar is 500 μm. *p < 0.05 compared with WKY plasma-incubated rings; ^#p < 0.05 and ^##p < 0.01 compared with SHR plasma-incubated rings

Fig. 4

Immunostaining of the aorta sections of WKY with a primary antibody against IGF-1R beta subunit. Aortic rings were incubated with plasma from WKY a, b or SHRs c, d in the presence b,d or absence a,c of protease inhibitors (EDTA 10 mM, doxycycline; 11.3 μM) for 24 h. Representative images are shown a-d. Arrowheads show the endothelium, and arrows show the smooth muscle layer. The scale bar is 500 μm. Bar graphs show the means ± SD of the percentage of stained area under the same incubation conditions a-d in tunica intima (endothelium) e and tunica media (smooth muscle cell) layers f. Two to four different aorta sections per animal were labeled to determine the average per rat (n = 4). The scale bar is 500 μm

Fig. 5

p-AKT and peNOS levels in WKY and SHR aorta rings incubated in HEPES buffer at 37 °C for 20 min in the presence (IGF-1) or absence (NS) of 100 nM IGF-1. Representative images are shown a,c. An antibody against p-AKT recognized a band in the range of 60 kDa a, an antibody against peNOS recognized a band in the range of 140 kDa c, and the anti-beta-actin antibody recognized a band at 45 kDa a, c. The p-AKT and peNOS band densities were normalized to the respective beta-actin signal. Bar graphs show the means ± SD of the band densities for p-AKT b and peNOS d (n = 4)

Fig. 6

Schematic diagram of the loss of IGF-1 receptor intracellular signaling via the proteolytic cleavage of extracellular receptor domains