Coupling growth-factor engineering with nanotechnology for therapeutic angiogenesis

Abstract

Therapeutic angiogenesis is an emerging paradigm for the management of ischemic pathologies. Proangiogenic Therapy is limited, however, by the current inability to deliver angiogenic factors in a sustained manner at the site of pathology. In this study, we investigated a unique nonglycosylated active fragment of hepatocyte growth factor/scatter factor, 1K1, which acts as a potent angiogenic agent in vitro and in a zebrafish embryo and a murine matrigel implant model. Furthermore, we demonstrate that nanoformulating 1K1 for sustained release temporally alters downstream signaling through the mitogen activated protein kinase pathway, and amplifies the angiogenic outcome. Merging protein engineering and nanotechnology offers exciting possibilities for the treatment of ischemic disease, and furthermore allows the selective targeting of downstream signaling pathways, which translates into discrete phenotypes.N eovascularization in the adult holds the potential for ameliorating ischemic disease, which is the leading cause of morbidity and mortality in the United States (1). Ferrara and Kerbel recently articulated that "therapeutic angiogenesis" is an emerging and exciting frontier of cardiovascular medicine (2). However, while early preclinical studies and phase I clinical trials exhibited promising results with recombinant proteins or gene therapy, more rigorous phase II and III studies have reported conflicting outcomes (3-6). A recent review of these clinical trials highlighted vector "washout" leading to inadequate duration of exposure to the angiogenic agent as a key factor that may have impaired the effectiveness of therapy (7). Indeed, it is now established that sustained activation of downstream signal pathways promotes cellular processes critical for angiogenesis (8). We rationalized that harnessing a nanotechnology-based approach can enable a temporal control over the presentation of the angiogenic factors to the cellular target, thereby altering the activation of downstream signal transduction.While nanoformulation of angiogenic proteins is an attractive strategy, the synthesis and instability of these large complex glycosylated structures pose a significant barrier (9). Hepatocyte growth factor/scatter factor (HGF/SF) is a potent angiogenic agent (10), signaling through the Met receptor and downstream MAPK and the PI3K pathways (11-12). Interestingly, the Met receptor is upregulated during hypoxia, suggesting that the ischemic tissue will be primed for activation by HGF/SF (13). These results indicate that HGF/SF could emerge as an attractive therapy for angiogenesis. However, HGF/SF is a heparin-binding protein with a complex multidomain structure consisting of an N-terminal (N) domain, four copies of the kringle domain (K1 to K4) and a C-terminal domain homologous to that of serine proteineases (SP) (Fig. 1A) (14). Biologically active HGF/SF is produced by proteolytic cleavage of the linker connecting the fourth kringle and the SP domain yielding a two-chain (α/β)-heterodimer, in which both the α-and the...

Engineering hepatocyte growth factor fragments with high stability and activity as Met receptor agonists and antagonists

“…The improved NK1 mutants described here could support further development of other Met agonists that are comprised partially or wholly from the unstable wild-type NK1 fragment (33)(34)(35), and given their enhanced stability have potential to be readily incorporated into biomaterials for therapeutic applications (36). In addition, these engineered NK1 agonists and antagonists will be important tools for further elucidating determinants of Met binding and activation.…”

Semi-synthesis of a HGF/SF kringle one (K1) domain scaffold generates a potent in vivo MET receptor agonist

“…The biological data accumulated for the NK1 molecule led most investigators to consider this structure as the minimal unit for MET agonist design. Indeed, several NK1 derivatives having either altered HS binding properties 25 , 27 or improved dimeric stability 28 , 29 were produced with the goal to promote tissue diffusion capacities and MET agonistic potential. A similar strategy was applied for the design of a MET antagonist with potential applications in cancer targeted chemotherapy.…”

Growth factors, aging and age-related diseases

“…In yeast, amino acid scarcity which increases lifespan, acts through the Tor/Sch9 pathway [37], and amino acid withdrawal and repletion studies point to the TORC1 complex as a major amino acid transducer in mammalian cells [38, 39] which include the essential amino acids, leucine and methionine and to a lesser extent the non-essential amino acid arginine [40]. In yeast, we recently showed that methionine restriction does not extend lifespan and instead threonine and valine can promote stress sensitization by activating the mTOR pathway.…”