Blancas‐Mejía, Luis M.

Light chain (LC) amyloidosis (AL) is a systemic amyloidosis associated with significant morbidity and mortality. Current treatment involves depletion of LC producing cells but patients with advanced disease are usually intolerant of chemotherapy and autologous stem cell transplantation, with no treatment available to directly address the tissue pathology of LC. Microvascular injury may be important in it spathobiology. Our overall objective is to discover strategies to ameliorate LC‐mediated injury at the level of endothelial cell death and dysfunction. We tested the hypothesis that nanoliposomes containing monosialoganglioside, phosphatidylcholine and cholesterol (NLGM1) are capable of protecting human endothelial cells from LC‐mediated injury by hormetic induction of the nuclear factor (erythroid‐derived 2)‐like (Nrf2) oxidative defense pathway. Human umbilical vein endothelial cells (HUVEC) were exposed to LC (purified from the urine of 2 AL patients with cardiac involvement, 20μg/ml) for 18–20 hours either with or without co‐treatment with NLGM1 (given at 1:10 mass ratio to LC). Using a fluorescent probe for peroxynitrite, we found that LC caused a 51.2% (p<0.01) increase in peroxynitrite production with a coincident 33% (p<0.01) decrease in cell viability as measured by Calcein‐AM fluorescence. However, in samples that were given NLGM1 and LC, peroxynitrite was diminished and cell viability was preserved to near control levels. Interestingly, in HUVEC that were treated with NLGM1 alone, we noticed a4‐fold increase in Nrf2 gene expression (p<0.05) and in antioxidant enzymes such as heme oxygenase 1 (HO‐1) and NAD(P)H quinone oxioreductase 1 (NQO1)(119‐fold, p<0.05; and 5‐fold, p<0.01 respectively). Similarly, we found significant upregulation of Nrf2, HO‐1 and NQO1 proteins in response to NLGM1 treatment. To determine if induction of HO‐1 and NQO1 by NLGM1 were dependent on Nrf2 activation, we knocked down Nrf2 using specific siRNAs. We were able to reduce Nrf2 gene expression in HUVEC by 41%. HUVEC treated with control siRNA demonstrated a 9.4‐fold increase in HO‐1 (p<0.05)and a 3.8‐fold increase in NQO1 (p<0.05) gene expression, whereas cell streated with Nrf2‐specific siRNAs had blunted NLGM1‐mediated increases to 3.0‐fold and 1.3‐fold for HO‐1 and NQO1 ( p<0.001 and p<0.05 vs. control siRNA ), respectively. Nrf2 knockdown abolished the protective effect of NLGM1 on HUVEC viability; Nrf‐2 siRNA treated cells exposed to LC and NLGM1 demonstrated 30% reduction in viability (p<0.05) similar to the 28% reduction in control siRNA‐treated cells exposed to LC; control siRNA‐treated cells exposed to LC and NLGM1 demonstrated no difference in viability versus those exposed to vehicle. Presently, we show that NLGM1 preserve endothelial cell viability and reduce peroxynitrite production in HUVEC exposed to LC proteins. We further demonstrate that NLGM1 has the potential to modulate Nrf2 antioxidant defense pathway and is likely a relevant protective mechanism against nitrative stress generated by LC exposure. When considered together, these findings hint at a novel potential therapeutic approach to mitigate the pathobiology of AL. Support or Funding Information Funding: Veterans Affairs Merit grant (I01BX007080), National Institutes Health (NIA R21AG044723, GM RO1 071514), Amyloidosis Foundation, American Heart Association 0855683G

Light chain (AL) amyloidosis is a devastating, complex, and incurable protein misfolding disease.

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 [ NLGM 1]) 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 NLGM 1 (1:10 ratio for LC : NLGM 1 mass). Human umbilical vein endothelial cells were exposed for 18 to 20 hours to vehicle, LC with or without NLGM 1, or NLGM 1 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 NLGM 1. LC decreased endothelial cell nitric oxide production and cell viability while increasing superoxide and peroxynitrite; these adverse effects were reversed by NLGM 1. NLGM 1 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 NLGM 1. 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.

AbstractLight chain (AL) amyloidosis is an incurable human disease, where the amyloid precursor is a misfolding‐prone immunoglobulin light‐chain. Here, we identify the role of somatic mutations in the structure, stability and in vitro fibril formation for an amyloidogenic AL‐12 protein by restoring four nonconservative mutations to their germline (wild‐type) sequence. The single restorative mutations do not affect significantly the native structure, the unfolding pathway, and the reversibility of the protein. However, certain mutations either decrease (H32Y and H70D) or increase (R65S and Q96Y) the protein thermal stability. Interestingly, the most and the least stable mutants, Q96Y and H32Y, do not form amyloid fibrils under physiological conditions. Thus, Q96 and H32 are key residues for AL‐12 stability and fibril formation and restoring them to the wild‐type residues preclude amyloid formation. The mutants whose equilibrium is shifted to either the native or unfolded states barely sample transient partially folded states, and therefore do not form fibrils. These results agree with previous observations by our laboratory and others that amyloid formation occurs because of the sampling of partially folded states found within the unfolding transition (Blancas‐Mejia and Ramirez‐Alvarado, Ann Rev Biochem 2013;82:745–774). Here we provide a new insight on the AL amyloidosis mechanism by demonstrating that AL‐12 does not follow the established thermodynamic hypothesis of amyloid formation. In this hypothesis, thermodynamically unstable proteins are more prone to amyloid formation. Here we show that within a thermal stability range, the most stable protein in this study is the most amyloidogenic protein.