Paricalcitol Has a Potent Anti-Inflammatory Effect in Rat Endothelial Denudation-Induced Intimal Hyperplasia

Abstract

Neointimal hyperplasia is the main cause of vascular graft failure in the medium term. Vitamin D receptor activation modulates the biology of vascular smooth muscle cells and has been reported to protect from neointimal hyperplasia following endothelial injury. However, the molecular mechanisms are poorly understood. We have now explored the impact of the selective vitamin D receptor activator, paricalcitol, on neointimal hyperplasia, following guidewire-induced endothelial cell injury in rats, and we have assessed the impact of paricalcitol or vehicle on the expression of key cell stress factors. Guidewire-induced endothelial cell injury caused neointimal hyperplasia and luminal stenosis and upregulated the expression of the growth factor growth/differentiation factor-15 (GDF-15), the cytokine receptor CD74, NFκB-inducing kinase (NIK, an upstream regulator of the proinflammatory transcription factor NFκB) and the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Immunohistochemistry confirmed the increased expression of the cellular proteins CD74 and NIK. Paricalcitol (administered in doses of 750 ng/kg of body weight, every other day) had a non-significant impact on neointimal hyperplasia and luminal stenosis. However, it significantly decreased GDF-15, CD74, NIK and MCP-1/CCL2 mRNA expression, which in paricalcitol-injured arteries remained within the levels found in control vehicle sham arteries. In conclusion, paricalcitol had a dramatic effect, suppressing the stress response to guidewire-induced endothelial cell injury, despite a limited impact on neointimal hyperplasia and luminal stenosis. This observation identifies novel molecular targets of paricalcitol in the vascular system, whose differential expression cannot be justified as a consequence of improved tissue injury.

Keywords: inflammation; intimal hyperplasia; paricalcitol; peripheral vascular disease; vitamin D receptor.

Figure 1

Increased intima–media ratio and stenosis following endothelial injury in rat femoral arteries. Endothelial injury was induced by scraping (injury artery) and a sham control without scraping was obtained from the contralateral artery. One group of rats received paricalcitol (750 ng/kg) and the other received the vehicle for 14 days, and artery samples were evaluated at 14 days. Images represent 20X (A–D) and 40X (E–H) magnification of hematoxylin eosin staining representative samples from the different groups. In panels E–H, intimal hyperplasia delimited by the internal elastic lamina can be better appreciated. Table 1 presents quantitative results for intima–media ratio and % stenosis.

Figure 2

Increased femoral artery gene expression for key molecules: impact of paricalcitol. Arterial injury was associated with an increased mRNA expression of growth factors and inflammatory mediators, and this was prevented by paricalcitol. mRNA expression was studied using RT-qPCR. Data expressed as % increase over the sham artery, which was assigned a value of 100%. (A) GDF15. (B) CD74. (C) NIK. (D) MCP-1. * p < 0.01 vs. injured vehicle; ** p < 0.05 vs. injured vehicle.

Figure 3

Increased femoral artery CD74 protein expression: impact of paricalcitol. Expression of CD74 in rat femoral arteries. Constitutive CD74 expression is observed in vehicle sham arteries (A,E) and is lower in paricalcitol sham arteries (B,F). CD74 expression increased in injured arteries, both in the media (C) and endothelium (G). Treatment with paricalcitol in injured arteries reduced CD74 expression to basal levels (D,H).

Figure 4

Increased femoral artery NIK protein expression: impact of paricalcitol. NIK expression in rat femoral arteries. Constitutive NIK expression is observed in vehicle sham arteries (A,E). In sham arteries of rats that received paricalcitol (B,F), NIK expression is lower in all layers of the artery. Injured arteries developing intimal hyperplasia (C,G) express NIK in endothelium and media. Paricalcitol decreased NIK expression in injured arteries (D,H).

Figure 5

Increased femoral artery infiltration by CD43-expressing leukocytes: impact of paricalcitol. CD43 protein expressing leukocytes in rat femoral arteries (×20), either sham (A,B) or injured (C,D). CD43 staining showed leukocyte infiltrates in vehicle injured arteries (C). This infiltrate was lower in paricalcitol-treated injured (D) arteries.

Figure 6

Experimental design. (A) Experimental model in rats. In each rat, the endothelium of the left femoral artery was injured by a vascular-guided scraping technique (injury artery). A similar procedure without scraping in the right femoral artery yielded sham artery controls. One group of rats received paricalcitol (750 ng/kg) and the other the vehicle for 2 weeks. Artery samples were evaluated 14 days after the intervention. Samples for mRNA, immunohistochemistry and histomorphometry were obtained from each arterial bundle. (B) Processing of arterial samples. After euthanasia, both femoral arteries were removed. The most distal third was used for molecular biology and protein studies. Histomorphometry and immunohistochemistry were performed in the proximal two thirds of the vascular bundle. To assess the degree of endothelial injury throughout the entire artery, this was divided into 10 consecutive sections that were stained and results averaged to yield the result of the sample.