Human microvascular dysfunction and apoptotic injury induced by AL amyloidosis light chain proteins

Abstract

Light chain amyloidosis (AL) involves overproduction of amyloidogenic light chain proteins (LC) leading to heart failure, yet the mechanisms underlying tissue toxicity remain unknown. We hypothesized that LC induces endothelial dysfunction in non-AL human microvasculature and apoptotic injury in human coronary artery endothelial cells (HCAECs). Adipose arterioles (n = 34, 50 ± 3 yr) and atrial coronary arterioles (n = 19, 68 ± 2 yr) from non-AL subjects were cannulated. Adipose arteriole dilator responses to acetylcholine/papaverine were measured at baseline and 1 h exposure to LC (20 μg/ml) from biopsy-proven AL subjects (57 ± 11 yr) without and with antioxidant cotreatment. Coronary arteriole dilation to bradykinin/papaverine was measured post-LC exposure. HCAECs were exposed to 1 or 24 h of LC. LC reduced dilation to acetylcholine (10(-4) M: 41.6 ± 7 vs. 85.8 ± 2.2% control, P < 0.001) and papaverine (81.4 ± 4.6 vs. 94.8 ± 1.3% control, P < 0.01) in adipose arterioles and to bradykinin (10(-6) M: 68.6 ± 6.2 vs. 90.9 ± 1.6% control, P < 0.001) but not papaverine in coronary arterioles. There was an increase in superoxide and peroxynitrite in arterioles treated with LC. Adipose arteriole dilation was restored by cotreatment with polyethylene glycol-superoxide dismutase and tetrahydrobiopterin but only partially restored by mitoquinone (mitochondria-targeted antioxidant) and gp91ds-tat (NADPH oxidase inhibitor). HCAECs exposed to LC showed reduced NO and increased superoxide, peroxynitrite, annexin-V, and propidium iodide compared with control. Brief exposure to physiological amounts of LC induced endothelial dysfunction in human adipose and coronary arterioles and increased apoptotic injury in coronary artery endothelial cells likely as a result of oxidative stress, reduced NO bioavailability, and peroxynitrite production. Microvascular dysfunction and injury is a novel mechanism underlying AL pathobiology and is a potential target for therapy.

Fig. 1.

Adipose arteriole vasoreactivity and reactive species fluorescence. A: there is a reduced dilator response to acetylcholine (endothelium-dependent) compared with baseline control following infusion of 20 μg/ml light chain proteins (LC). There is a smaller but significant reduction in the dilator response to papaverine (nonendothelium dependent). Coadministration of polyethylene glycol-superoxide dismutase (PEG-SOD) restores the dilator response. V, vehicle. B: there is increased superoxide production (hydroethidine fluorescence) in adipose arterioles treated with LC, a response attenuated by PEG-SOD. C: there is reduced nitric oxide (NO) production [4,5-diaminofluorescein (DAF)-2 fluorescence] in LC-treated adipose arterioles compared with control. C, control. D: there is increased peroxynitrite production (dihydrorhodamine fluorescence) in LC-treated adipose arterioles vs. control; this increase is attenuated by cotreatment with PEG-SOD.

Fig. 2.

Coronary arteriole vasoreactivity and reactive species production. A: there is a reduced dilator response to bradykinin when coronary arterioles are exposed to LC vs. baseline control but preserved dilator response to papaverine. B and C: there is increased superoxide and peroxynitrite production in coronary arterioles treated with LC, similar to the response of adipose arterioles.

Fig. 3.

Human coronary artery endothelial cell (HCAEC) assays. A and B: following 1 h of exposure to LC, there is increased superoxide (SO) and peroxynitrite in HCAEC vs. vehicle control. Cotreatment with mitoquinone, gp91ds-tat, or tetrahydrobiopterin prevented the increase in superoxide and peroxynitrite induced by LC. C and D: following 24 h of exposure to LC, there is no difference in total endothelial nitric oxide synthase (eNOS) and the ratio of phosphorylated eNOS to eNOS between LC-treated cells and control. MitoQ, mitoquinone. *P < 0.05 and ***P < 0.001 vs. vehicle control. +P < 0.05, ++P < 0.01, and +++P < 0.001 vs. LC.

Fig. 4.

HCAEC flow cytometry. A–C demonstrate flow cytometry of HCAECs treated with vehicle control, LC, and LC with PEG-SOD. D shows increased cells with annexin-V staining, and E shows increased cells with propidium iodide staining following 24 h LC exposure vs. vehicle control. Cotreatment with PEG-SOD reversed the increase in annexin-V and propidium iodide.

Fig. 5.

Proposed schema of light chain protein-induced microvascular dysfunction and apoptotic injury.