MK2 inhibitory peptide delivered in nanopolyplexes prevents vascular graft intimal hyperplasia

Abstract

Autologous vein grafts are commonly used for coronary and peripheral artery bypass but have a high incidence of intimal hyperplasia (IH) and failure. We present a nanopolyplex (NP) approach that efficiently delivers a mitogen-activated protein kinase (MAPK)–activated protein (MAPKAP) kinase 2 inhibitory peptide (MK2i) to graft tissue to improve long-term patency by inhibiting pathways that initiate IH. In vitro testing in human vascular smooth muscle cells revealed that formulation into MK2i-NPs increased cell internalization, endosomal escape, and intracellular half-life of MK2i. This efficient delivery mechanism enabled MK2i-NPs to sustain potent inhibition of inflammatory cytokine production and migration in vascular cells. In intact human saphenous vein, MK2i-NPs blocked inflammatory and migratory signaling, as confirmed by reduced phosphorylation of the posttranscriptional gene regulator heterogeneous nuclear ribonucleoprotein A0, the transcription factor cAMP (adenosine 3′,5′-monophosphate) element–binding protein, and the chaperone heat shock protein 27. The molecular effects of MK2i-NPs caused functional inhibition of IH in human saphenous vein cultured ex vivo. In a rabbit vein transplant model, a 30-min intraoperative graft treatment with MK2i-NPs significantly reduced in vivo IH 28 days posttransplant compared with untreated or free MK2i–treated grafts. The decrease in IH in MK2i-NP–treated grafts in the rabbit model also corresponded with decreased cellular proliferation and maintenance of the vascular wall smooth muscle cells in a more contractile phenotype. These data indicate that nanoformulated MK2 inhibitors are a promising strategy for preventing graft failure.

Mechanism of Enhanced Cellular Uptake and Cytosolic Retention of MK2 Inhibitory Peptide Nano-polyplexes

“…Anionic, pH-responsive carriers like the one described herein have been shown to reduce trafficking into acidifying compartments following CD22-dependent receptor mediated uptake 3 . While the pharmacodynamics of anionic, polymer-based nano-polyplexes (NPs) used for the delivery of both an intracellular acting MAPKAP kinase 2 inhibitory (MK2i) peptide and a peptide mimetic of phosphorylated heat shock protein 20 (p-HSP20 peptide) have been thoroughly investigated 18,19 , the mechanisms and kinetics of NP uptake and trafficking have not been rigorously studied. Because of the promise of MK2i-NPs as a prophylactic therapy to inhibit intimal hyperplasia and vasospasm of vascular grafts 18 , this specific formulation is the focus of the current studies designed to better elucidate endosomolytic NP intracellular pharmacokinetics.…”

Heparin-functionalized polymer graft surface eluting MK2 inhibitory peptide to improve hemocompatibility and anti-neointimal activity

“…The scratch-wounds were created, and then cells were incubated with samples (bare PCL, PCL–HT and PCL–HT/MK2i) in the medium containing PDGF (50 ng/mL) for 24 h. As a negative and positive control, the medium with or without MK2i peptide (200 μ g/mL) was used [7]. As shown in Figure 5a, VSMCs migrated into the scratched gap between the confluent cell areas.…”

“…VSMCs were seed within Insert 2 well at a seeding density of 1 × 10 4 cells/well, and cultured to be reached at confluency (90–95%) for 1–2 day in DMEM-supplemented with 10% FBS and 1% PS, under standard culture condition. After gently removing the Culture-Insert 2 well to create the scratch wound, the culture medium was replaced with low serum growth medium containing 1% FBS to minimize cellular growth, and incubated overnight [7, 14]. Then, the cells were covered with samples (bare PCL, PCL–HT and PCL–HT/MK2i), and incubated in the medium containing 50 ng/mL of PDGF (R&D systems, USA).…”

“…Hence, incorporating antithrombotic and antiplatelet agents ( e.g., heparin, hirudin, clopidogrel, aspirin, warfarin, and etc. ) to the surface of blood-contacting biomaterials has been tested so far [6, 7]. Up to date, bioactive heparin coating on the polymer surfaces is still recognized as a gold-standard treatment for the enhanced blood compatibility.…”

“…Various drugs that can inhibit abnormal activities of VSMCs were employed to prevent development of intimal hyperplasia [12]. In particular, we have been studying potent inhibitory effects of a cell-penetrating peptide inhibitor of Mitogen Activated Protein Kinase II (MK2i) on neointimal hyperplasia, fibrosis and inflammation [7, 13]. MK2 is a downstream regulator of the TGF-β/p38 stress-activated protein kinase pathway that is implicated in intimal hyperplasia.…”