The Uremic Toxin Indoxyl Sulfate Impairs Angiogenesis Through Aryl Hydrocarbon Receptor Activation

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Introduction The aryl hydrocarbon receptor (AHR) is a ligand‐activated transcription factor that coordinates expression of genes involved in xenobiotic metabolism of a variety of environmental and endogenous toxins. Several metabolites, including indoxyl sulfate (IS) and kynurenine, that are derived from tryptophan metabolism are known AHR ligands. Aberrant tryptophan metabolism in the intestinal microbiota combined with impaired kidney function results in accumulation of these metabolites, defined as uremia. Previous studies have suggested that kidney disease results in impaired angiogenesis, however the molecular mechanisms are unknown. The goal of this study was to examine the impact of AHR activation on endothelial cell biology.Methods and ResultsTo accomplish this goal, assessments of endothelial cell proliferation, migration, and tube formation assays in human umbilical vein endothelial cells (HUVECs) and assessment of endothelial cell sprouting in aortic rings from adult C57BL/6J mice (N = 8). HUVECs and aortic rings were exposed to 500μM IS, which did not induce cell death, but activated the AHR by significantly increasing CYP1A1 mRNA levels (~50‐fold in HUVECs and ~8‐fold in aortic rings). IS treatment significantly impaired tube formation in HUVECs (IS: 13.5 ± 1.76 vs. Vehicle: 24 ± 1.9; P < 0.001) and endothelial sprouting in aortic rings (IS: 6.1 ± 0.9 vs. Vehicle: 20.7 ± 0.9; P < 0.001). Additional experiments in HUVECs demonstrated that IS treatment did not impair endothelial cell migration at 24 hours (IS: 100 ± 0 vs Vehicle: 100 ± 0; P = 0.32) but completely ablated the cells ability to proliferate (IS: 1317.8 ± 67.5 vs. Vehicle: 4388.7 ± 371.8; P < 0.001). To examine the role of the AHR, we used shRNA targeting the AHR (shAHR) and chemical AHR antagonists, resveratrol and CH223191. Both chemical antagonism and genetic knockdown of the AHR prevented AHR‐mediated increases in CYP1A1 mRNA with IS‐treatment. Moreover, chemical antagonism and shAHR treatments rescued cell proliferation (shAHR: 2910 ± 282.1 vs. Scramble: 1035 ± 41.7; P = 0.001), tube formation (shAHR: 10.6 ± 1.0 vs. Scramble: 1.1 ± 0.38; P < 0.001), and sprouting (shAHR: 22.9 ± 0.86 vs. Scramble: 7.9 ± 1.26; P < 0.001) following IS treatment. To isolate the AHR, we generated a plasmid encoding a constitutively active AHR (CAAHR) by deletion of the ligand binding domain. Transfection with CAAHR increased both AHR and CYP1A1 expression, confirming non‐ligand dependent AHR activity. Consistent with IS treatment, expression of the CAAHR impaired endothelial cell tube formation (CAAHR: 5.6 ± 1.5 vs. GFP: 20.2.8 ± 1.2; P < 0.001) and sprouting (CAAHR: 18.5 ± 1.3 vs. GFP: 25.8 ± 2.9, P = 0.028). CAAHR impairments in angiogenesis were driven by significant decreases in endothelial cell proliferation, however endothelial cell migration was not affected.ConclusionAHR activation impairs angiogenesis by compromising endothelial cell proliferation. Genetic or pharmaceutical treatments to prevent AHR activation rescued endothelial cell proliferation and angiogenesis. Taken together, these findings establish the role of the AHR in vascular biology and highlight the potential for AHR antagonism to improve angiogenesis in ischemic cardiovascular diseases.Support or Funding InformationSupported by a seed grant from the UF Office of Research