Induction of macrophage chemotaxis by aortic extracts from patients with Marfan syndrome is related to elastin binding protein

Abstract

Marfan syndrome is an autosomal dominantly inherited disorder of connective tissue with prominent skeletal, ocular, and cardiovascular manifestations. Aortic aneurysm and dissection are the major determinants of premature death in untreated patients. In previous work, we showed that extracts of aortic tissues from the mgR mouse model of Marfan syndrome showed increased chemotactic stimulatory activity related to the elastin-binding protein. Aortic samples were collected from 6 patients with Marfan syndrome and 8 with isolated aneurysms of the ascending aorta. Control samples were obtained from 11 organ donors without known vascular or connective tissue diseases. Soluble proteins extracted from the aortic samples of the two patient groups were compared against buffer controls and against the aortic samples from controls with respect to the ability to induce macrophage chemotaxis as measured using a modified Boyden chamber, as well as the reactivity to a monoclonal antibody BA4 against bioactive elastin peptides using ELISA. Samples from Marfan patients displayed a statistically significant increase in chemotactic inductive activity compared to control samples. Additionally, reactivity to BA4 was significantly increased. Similar statistically significant increases were identified for the samples from patients with idiopathic thoracic aortic aneurysm. There was a significant correlation between the chemotactic index and BA4 reactivity, and the increases in chemotactic activity of extracts from Marfan patients could be inhibited by pretreatment with lactose, VGVAPG peptides, or BA4, which indicates the involvement of EBP in mediating the effects. Our results demonstrate that aortic extracts of patients with Marfan syndrome can elicit macrophage chemotaxis, similar to our previous study on aortic extracts of the mgR mouse model of Marfan syndrome (Guo et al., Circulation 2006; 114:1855-62).

Figure 1. BA4 reactivity and chemotactic activity of human aortic extracts.

(A) BA4 reactivity was measured by competitive ELISA in aortic extracts from patients with MFS (n = 6), isolated TAA (n = 8) and controls (n = 11). A statistically significant increase in BA4 reactivity as compared to control samples was observed for the samples from individuals with MFS and isolated TAA. (B) Chemotactic activity of the same extracts was measured by a Boyden chamber. A statistically significant increase in chemotactic activity as compared to control samples was observed for the samples from individuals with MFS and isolated TAA. Red lines indicate the median levels of BA4 reactivity or chemotactic index (CI). Data are representative of three independent experiments. * 0.05, ** 0.01.

Figure 2. BA4 reactivity (g/mg) versus chemotactic index (CI) of human aortic extracts, with calculated Pearson correlation coefficient (r) and value.

Figure 3. Chemotactic index (CI) of RAW 264.7 cells upon stimulation with aortic extracts from individuals with MFS ().

(A) RAW 264.7 cells were preincubated with 1 mmol/L lactose or glucose for one hour at 37°C prior to exposure to aortic extracts. There was a statistically significant inhibition of chemotaxis. (B) RAW 264.7 cells were preincubated with 0.1 mmol/L VGVAPG hexapeptide for 1 hour incubation at 37°C before the chemotaxis assays were started. There was a statistically significant inhibition of the chemotactic response after VGVAPG pretreatment. (C) Aortic extracts were preincubated with BA4 or non-specific IgG for 30 minutes at room temperature prior to chemotaxis assays. There was a statistically significant inhibition of the chemotactic response by BA4 pretreatment. Data are representative of three independent experiments. * 0.05, ** 0.01.