VLO5, a heterodimeric VGD/MLD-disintegrin, ligand of alpha9/alpha4-beta1 integrins inhibits neutrophil apoptosis

Saldanha-Gama RF; Moraes JA; Souza PB; Mariano-Oliveira, A; Coelho AL; De Freitas MS; ¹Marcinkiewicz C; Barja-Fidalgo C

Dept. Farmacologia, IBRAG, Universidade do Estado do Rio de Janeiro; ^*Dept. Biology, CST-Temple University, PA, USA. 

Apoptosis is an important event in the homeostatic control of circulating neutrophil (PMN) lifespan and in the resolution of inflammation. Aged PMN undergo spontaneous apoptosis prior to their removal by macrophages. During inflammation, different cytokines and adhesion molecule interactions can accelerate or delay PMN survival, interfering in the resolution of this process. Integrin-mediated downstream signals modulates survival in different cells, activating intracellular pathways, as PI3K and MAPK, which interfere with the balance between anti and pro-apoptotic molecules, as BclxL and Bad, which in turn modulate caspase activation. Recently we have shown that disintegrins, RGD or MLD peptides ligands of alpha4 or alpha9 integrins, activate integrin-coupled signaling in PMN,  protecting or increasing, respectively, the apoptotic processes in these cells. VLO5 is a heterodimeric MLD/VGD disintegrin that was shown to directly activate integrin signaling pathways in PMN, inducing Focal adhesion kinase activation, cytoskeleton mobilization and chemotaxis.  In this study, we evaluated the effect of VLO5 on human neutrophil apoptosis and the involvement of PI3K and MAPK pathways and superoxide production. Isolated human blood PMN were incubated with VLO5 (1µM) and apoptosis was evaluated morphologically (after 18h-optical microscopy), DNA fragmentation (8h-agarose gel) and Bad degradation(30 min-western blot). VLO5 potently inhibited spontaneous apoptosis, inhibiting morphological alterations, DNA fragmentation and accelerated Bad degradation.  The MLD/VGD peptide induced in PMN the activation of PI3K, increasing its tyrosine phosphorylation and ERK2 nuclear translocation. In agreement, LY294002, a PI3K inhibitor, and PD95059, an ERK2 inhibitor, reverted VLO5 effect, accelerating PMN apoptosis. Although discrete, the superoxide production induced by VLO5 might contribute to its anti-apoptotic effect since DPI, an inhibitor  of oxidative burst, partially reverted it. The data suggests that interaction of VLO5 with PMN integrin might be related with its anti-apoptotic effect, which is dependent on PI3K and ERK2 activation and superoxide production.

(FAPERJ, CNPq, SR2-UERJ, IFS-Sweden)

Correspondence to: saldanhagama@yahoo.com.br