Reversal of Vascular Calcification and Aneurysms in a Rat Model Using Dual Targeted Therapy with EDTA- and PGG-Loaded Nanoparticles

Abstract

Degeneration of elastic lamina and vascular calcification are common features of vascular pathology such as aortic aneurysms. We tested whether dual therapy with targeted nanoparticles (NPs) can remove mineral deposits (by delivery of a chelating agent, ethylene diamine tetraacetic acid (EDTA)) and restore elastic lamina (by delivery of a polyphenol, pentagalloyl glucose (PGG)) to reverse moderate aneurysm development. EDTA followed by PGG NP delivery led to reduction in macrophage recruitment, matrix metalloproteinase (MMP) activity, elastin degradation and calcification in the aorta as compared to delivery of control blank NPs. Such dual therapy restored vascular elastic lamina and improved vascular function as observed by improvement in circumferential strain. Therefore, dual targeted therapy may be an attractive option to remove mineral deposits and restore healthy arterial structures in moderately developed aneurysms.

Keywords: AAA; PGG.; calcification; chelation therapy; elastin.

Figure 1

Schematic representation of the experiment. The experiment consisted of five groups (n=6 animals per group) with different NP treatments viz. DIR NPs, Blank NPs, EDTA+PGG NPs, BB-94 NPs and PGG NPs. Calcium chloride injury to the aorta was done on Day 0 following which the animals were allowed to develop aneurysm for 4 weeks. After 4 weeks, they were treated with NPs and euthanized at various chosen time points as shown in the legend. DIR NPs were administered once after 4 weeks, Blank and PGG NPs were injected twice biweekly after 6 and 8 weeks, and BB-94 NPs were injected one weekly starting at 6 weeks and through 9 weeks after injury. The EDTA+PGG NPs group was given two EDTA NP injections every week starting at 4 weeks and through 5 weeks after injury, immediately followed by biweekly PGG NP injections through 9 weeks after injury. All rats were euthanized at week 12.

Figure 2

In situ zymography for MMPs (A1), alizarin red staining for calcium (A2) and VVG staining for elastin (fibers stained black) (A3) for aortic samples 30 days after injury. In situ zymography confirmed the high activity of MMPs (-9 and-2). Alizarin red confirmed calcification in the media and adventitia. VVG showed the elastin damage. (B) NP accumulation after intravenous injection of EL-NP-DIR, 30 days after injury at the site of elastin damage (squares show sections of the abdominal aorta where CaCl₂ was applied); image taken 24 hrs after injection. (C) Organ distribution of fluorescent NPs 24 hrs after injection of EL-NP-DIR. (D) Cross-section of abdominal aorta showing NPs (EL-NP-DIR) targeting from the adventitial side and accumulating more in the degraded elastic lamina; DIR fluorescence (purple) and elastin (green autofluorescence). * indicates lumen.

Figure 3

Calcium content of aorta 30 days after injury and before any treatment compared to EL-NP-Blank group, EL-NP-BB94 group, EL-NP-PGG group and EL-NP-EDTA+EL-NP-PGG groups (#P < 0.05, Tukey's test,) (n=6). Alizarin red staining (calcium) with a Light Green SF Yellowish counterstain for the EL-NP-Blank group (B1), EL-NP-BB94 group (B2), EL-NP-PGG group (B3), and EL-NP-EDTA+EL-NP-PGG group (B4) shows high calcification (B1, B2); mild calcification (B3); minimal calcification (B4). OPN IHC revealed higher OPN expression in the EL-NP-Blank and EL-NP-BB94 groups (C1, C2); less OPN expression in the EL-NP-PGG group and EL-NP-EDTA+EL-NP-PGG groups (C3, C4). For histological confirmation of PGG binding to elastic lamina, all groups' sections were stained with a phenol-specific stain (black indicates PGG) and counterstained with Light Green SF Yellowish, showing no staining for EL-NP-Blank (D1) and EL-NP-BB94 (D2); mild phenol staining close to the elastic lamina in the EL-NP-PGG group (D3); very strong staining in the EL-NP-EDTA+EL-NP-PGG group (D4). * indicates lumen. Scale bar is 100 µm.

Figure 4

Comparison of aortic external diameter change shows a significant suppression of AAA expansion in the EL-NP-EDTA+EL-NP-PGG group compared to all other groups (A). 30 days represents aortic diameter at the initiation of treatments. Representative images of the aorta at sacrifice (B). Wooden sticks were kept as reference for external diameter change measurement. *, P < 0.05 represents statistical significance, Tukey's test (pairwise comparison). #, P < 0.05, Exact permutation test. (n=6).

Figure 5

(A) MMP activity (-2 and -9) as measured by a fluorogenic substrate assay showing increase in MMP activity in the EL-NP-Blank group abdominal aorta compared to all other groups. MMPs were not inhibited in the abdominal aorta by blank NPs, while they were suppressed in other groups. (B) MMP activity in abdominal aortic sections shown with in situ zymography. Green signal: active MMPs; blue signal: DAPI staining for cell nuclei. Only the EL-NP-Blank group shows MMPs activity. * Indicates lumen. Scale bar = 400 μm. (#P < 0.05, Tukey's test,) (n=6).

Figure 6

Hematoxylin and eosin (H&E) staining showed inflammation in the EL-NP-Blank (a), EL-NP-BB94 (b), and EL-NP-PGG (c) groups, while inflammation was minimal in the EL-NP-EDTA+EL-NP-PGG (d) group. Masson trichrome staining showed excess collagen deposition in the EL-NP-Blank (e) and EL-NP-BB94 groups (f) groups, while it was minimal in the EL-NP-PGG (g) and EL-NP-EDTA+EL-NP-PGG (h) groups. CD68 macrophage IHC was positive for the inflammatory capsule in the EL-NP-Blank (i) and EL-NP-BB94 (j) groups. CD68 expression was minimal in the EL-NP-PGG (k) and EL-NP-EDTA+EL-NP-PGG (l) groups. VSMC expression was higher in the EL-NP-EDTA+NP-PGG (p) group compared to the EL-NP-Blank (m), EL-NP-BB94 (n), and EL-NP-PGG (o) groups. * Indicates lumen. Red scale bar is 100 µm. Black scale bar is 50 µm.

Figure 7

(A) LOX activity in the abdominal part (CaCl₂ injured) over the thoracic aorta (non-injured, healthy), showing that LOX activity was reduced in the EL-NP-Blank and EL-NP-BB94 groups. The EL-NP-PGG and EL-NP-EDTA+EL-NP-PGG groups showed increased LOX; both are significantly higher than the EL-NP-Blank and EL-NP-BB94 groups. (B) Desmosine content of aorta 30 days post injury and in the four NP groups was compared to healthy non injured abdominal aorta. CaCl₂ injury caused a substantial decrease in desmosine. Desmosine content was not improved after delivery of blank or BB-94 loaded NPs (EL-NP-Blank or EL-NP-BB94). The EL-NP-EDTA+EL-NP-PGG group showed the highest desmosine content among the four groups.

Figure 8

Circumferential strain as measured by ultrasound of all four groups at week 4 after injury and before any treatment compared to week 12 after treatments. No differences (NS) were observed in the EL-NP-Blank and EL-NP-BB94 groups while in both the EL-NP-PGG and EL-NP-EDTA+EL-NP-PGG groups there was a significant enhancement. (*P < 0.05, Tukey's test,) (n=6). Dashed line shows the circumferential strain in a healthy rat with no injury.