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Reconstituted High-Density Lipoprotein Containing Human Growth Hormone-1 Shows Potent Tissue Regeneration Activity with Enhancement of Anti-Oxidant and Anti-Atherosclerotic Activities.

Rejuvenation research (2015-01-28)
Eun-Young Lee, So-Hee Kim, Kyung-Hyun Cho
ABSTRACT

Human growth hormone-1 (GH-1), somatotropin, is a peptide hormone that stimulates cell division in tissues such as bone and cartilage. To compare physiological activities in lipid-free and lipid-bound states, we expressed and incorporated GH-1 in reconstituted high-density lipoprotein (rHDL). GH-1 was expressed and purified using the pET30(a) vector and an Escherichia coli expression system. Purified GH-1 (at least 98% purity, 23 kD) was characterized and synthesized with apolipoproteinA-I (apoA-I), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and cholesterol. Secondary structure analysis of GH-1 revealed 54% α-helical content in a lipid-free state and 65% α-helical content in a lipid-bound state along with blue-shifted tryptophan movement (around 2 nm). GH-1 caused less uptake of oxidized low-density lipoprotein (oxLDL) into macrophages and inhibited senescence of dermal cells in a dose-dependent manner. GH-1 in a lipid-bound state exerted stronger inhibitory activity than in a lipid-free state, indicating improved anti-atherosclerotic activity due to the lipid formulation. In a fin regeneration experiment using zebrafish (17 weeks old, n=9), GH-1 showed its highest regeneration speed without any side effects. GH-1-rHDL containing apoA-I showed 2.3-fold and 1.6-fold higher regeneration speeds than lipid-free GH-1 (in native state) and lipid-bound GH-1, respectively. Incorporation of GH-1 and apoA-I in HDL enhanced tissue regeneration activity of amputated tail fin, indicating a synergetic effect between GH-1 and apoA-I in a lipid-bound state.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
DL-Tryptophan, ≥99% (HPLC)
Sigma-Aldrich
DL-Tryptophan, ≥99% (HPLC)
Sigma-Aldrich
Suberic acid bis(3-sulfo-N-hydroxysuccinimide ester) sodium salt, ≥95% (H-NMR), powder
Sigma-Aldrich
Cholesterol, Sigma Grade, ≥99%
Sigma-Aldrich
Cholesterol, powder, BioReagent, suitable for cell culture, ≥99%
Sigma-Aldrich
Cholesterol, from sheep wool, ≥92.5% (GC), powder
SAFC
Cholesterol, from sheep wool, Controlled origin, meets USP/NF testing specifications
Sigma-Aldrich
SyntheChol® NS0 Supplement, 500 ×, synthetic cholesterol, animal component-free, aqueous solution, sterile-filtered, suitable for cell culture
Supelco
Cholesterol solution, certified reference material, 10 mg/mL in chloroform
Avanti
16:0-18:1 PC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, powder
Avanti
16:0-18:1 PC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, chloroform
Sigma-Aldrich
Sodium cholate hydrate, from bovine and/or ovine bile, ≥99%
SAFC
Cholesterol, Plant-Derived, SyntheChol®, NF, Ph. Eur., JP
Avanti
14:0 PC (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine, powder
Avanti
14:0 PC (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine, chloroform
Sigma-Aldrich
Ethylene glycol monophenyl ether, ≥90%