Monosialoganglioside-Containing Nanoliposomes Restore Endothelial Function Impaired by AL Amyloidosis Light Chain Proteins

Abstract

Background: Light chain amyloidosis (AL) is associated with high mortality, especially in patients with advanced cardiovascular involvement. It is caused by toxicity of misfolded light chain proteins (LC) in vascular, cardiac, and other tissues. There is no treatment to reverse LC tissue toxicity. We tested the hypothesis that nanoliposomes composed of monosialoganglioside, phosphatidylcholine, and cholesterol (GM1 ganglioside-containing nanoliposomes [NLGM1]) can protect against LC-induced human microvascular dysfunction and assess mechanisms behind the protective effect.

Methods and results: The dilator responses of ex vivo abdominal adipose arterioles from human participants without AL to acetylcholine and papaverine were measured before and after exposure to LC (20 μg/mL) with or without NLGM1 (1:10 ratio for LC:NLGM1 mass). Human umbilical vein endothelial cells were exposed for 18 to 20 hours to vehicle, LC with or without NLGM1, or NLGM1 and compared for oxidative and nitrative stress response and cellular viability. LC impaired arteriole dilator response to acetylcholine, which was restored by co-treatment with NLGM1. LC decreased endothelial cell nitric oxide production and cell viability while increasing superoxide and peroxynitrite; these adverse effects were reversed by NLGM1. NLGM1 increased endothelial cell protein expression of antioxidant enzymes heme oxygenase 1 and NAD(P)H quinone dehydrogenase 1 and increased nuclear factor, erythroid 2 like 2 (Nrf-2) protein. Nrf-2 gene knockdown reduced antioxidant stress response and reversed the protective effects of NLGM1.

Conclusions: NLGM1 protects against LC-induced human microvascular endothelial dysfunction through increased nitric oxide bioavailability and reduced oxidative and nitrative stress mediated by Nrf-2-dependent antioxidant stress response. These findings point to a potential novel therapeutic approach for light chain amyloidosis.

Keywords: amyloid; endothelium; nanotechnology; oxidant stress.

Figure 1

Arteriole vasoreactivity. A, EC50 dose of acetylcholine (log mol/L; note that the y‐axis scale is inverted). B, Maximum dilator response to acetylcholine (10⁻⁴ mol/L). Treatments were compared with baseline control (paired analyses) and with other treatments (unpaired analyses). Exposure to LC resulted in reduced dilator response to acetylcholine compared with baseline control response (n=12). Cotreatment of LC with monosialoganglioside‐containing nanoliposomes (NLGM1) fully restored dilator response to acetylcholine (n=14). The NLGM1‐protective response in LC‐treated arterioles was abolished by cotreatment of L‐NAME, a specific inhibitor of nitric oxide synthase (LC with NLGM1 and L‐NAME plot, n=3). Dilator response to NLGM1 alone did not differ from baseline control (n=3). C, Dilator response to papaverine. There was no significant change in dilator response to papaverine with treatment with LC, LC with NLGM1, LC with NLGM1 and L‐NAME, or NLGM1 alone. Dilator response to papaverine did not differ significantly among the various treatments. C indicates control; EC50, half‐maximal effective concentration; LC, light chain proteins; NLGM1, GM1 ganglioside–containing nanoliposomes; Tx, treatment.

Figure 2

Endothelial cell NO production and eNOS expression. A, Human umbilical vein endothelial cells exposed to 18 to 20 hours of LC showed significant reduction in NO head gas production compared with control. Cotreatment with NLGM1 restored NO production. There was no difference in NO production between endothelial cells treated with NLGM1 and vehicle control (n=10 for C, LC, LC with NLGM1, n=7 for LC with NLGM1 and LNAME, and n=6 for NLGM1). B, Protein expression of total eNOS and peNOS (threonine 495) and phosphorylated/total eNOS ratios were not significantly different among cells treated with vehicle, LC, LC with NLGM1, or NLGM1 (n=7). C, Similar results were observed with peNOS (serine 1177) (n=4). C indicates control; eNOS,endothelial nitric oxide synthase; LC, light chain proteins; NLGM1, GM1 ganglioside–containing nanoliposomes; NO, nitric oxide; NS, not significant; peNOS, phosphorylated endothelial nitric oxide synthase; T, total.

Figure 3

Endothelial cell superoxide, peroxynitrite production, and viability. A, HUVECs treated with LC showed increased superoxide production that was reversed by cotreatment with NLGM1 (n=17). B, There was increased peroxynitrite in LC‐treated cells; this increase was abolished by cotreatment with NLGM1. NLGM1 alone showed no difference in peroxynitrite production compared with vehicle (n=11). C, Cell viability assessed using calcein acetoxymethyl fluorescence showed reduced HUVEC viability following treatment with LC. Cell viability was restored by NLGM1 cotreatment (n=14). C indicates control; HUVEC, human umbilical vein endothelial cell; LC, light chain proteins; NLGM1, GM1 ganglioside–containing nanoliposomes.

Figure 4

Endothelial cell gene and protein expression. A, Human umbilical vein endothelial cells treated with LC showed no significant increase in HO‐1 gene expression compared with control. Cotreatment with NLGM1 and NLGM1 alone resulted in significant increases in HO‐1 gene expression compared with control or LC‐treated cells (n=13 control, n=11 LC, n=17 LC plus NLGM1, and n=11 NLGM1). B, A similar pattern was seen for protein expression of HO‐1 except there was a significant difference in HO‐1 between cells treated with NLGM1 and with LC with NLGM1 (n=7 each treatment). C, NLGM1 increased NQO1 gene expression when given to cells alone or as cotreatment with LC compared with control or LC‐treated cells (n=12 control, n=11 LC, n=17 LC with NLGM1, n=10 NLGM1). D, NLGM1 also increased NQO1 protein expression when given to cells alone or as cotreatment with LC compared with control or LC‐treated cells (n=8). C indicates control; HO‐1, heme oxygenase 1; LC, light chain proteins; NLGM1, GM1 ganglioside–containing nanoliposomes; NQO1, NAD(P)H quinone dehydrogenase 1.

Figure 5

Endothelial cell Nrf‐2. A, Treatment with NLGM1 or cotreatment of LC with NLGM1 resulted in increased nuclear Nrf‐2 protein compared with vehicle control or LC‐treated cells (n=8). B through E, Endothelial cell Nrf‐2 siRNA transfection. B, There is reduced Nrf‐2 gene expression in HUVECs transfected with Nrf‐2 siRNA compared with control siRNA, showing effective suppression of gene expression (n=6). C and D, NLGM1 increased HO‐1 and NQO1 gene expression in HUVECs transfected with control siRNA; this increase was abolished in HUVECs transfected with Nrf‐2 siRNA (n=3). E and F, In HUVECs treated with control siRNA, LC increased peroxynitrite (n=5) and reduced cell viability (n=6); these effects were reversed by cotreatment with NLGM1. The protective effect of NLGM1 was reversed in HUVECs treated with Nrf‐2 siRNA. C indicates control; HO‐1, heme oxygenase 1; HUVEC, human umbilical vein endothelial cell; LC, light chain proteins; NLGM1, GM1 ganglioside–containing nanoliposomes; NQO1, NAD(P)H quinone dehydrogenase 1; Nrf‐2, nuclear factor erythroid 2 like 2.

Figure 6

Proposed schema by which NLGM1 confers protection against LC‐induced endothelial dysfunction. LC causes reduced endothelial cell NO production that leads to endothelial dysfunction. LC also causes increased oxidative and nitrative stress that leads to reduced endothelial cell viability. NLGM1 induces antioxidant stress responses (Nrf‐2, HO‐1 and NQO1) that lead to reduced nitrative stress, increased NO bioavailability, increased endothelial cell viability, and restoration of endothelial function. HO‐1, heme oxygenase 1; LC, light chain proteins; NLGM1, GM1 ganglioside–containing nanoliposomes; NO, nitric oxide; NQO1, NAD(P)H quinone dehydrogenase 1; Nrf‐2, nuclear factor erythroid 2 like 2; SO, superoxide.