Integrin-specific bioactive peptides increase the potency of
BMSCs.
Since both FN-adhesion-promoting peptide- and cyclic RGD-primed BMSCs
stimulated hematopoiesis, I examined whether the peptides specific to
FN-interacting integrins also would do the same. Several integrins form
the receptors for FN, out of which α5β1 and αIIbβ3 are known to play
essential roles in hematopoiesis [36,37]. Hence, I examined whether
priming the BMSCs with peptides specific to α5β1, and αIIbβ3 would have
any effects on the hematopoiesis-supportive ability of BMSCs. Since
α4β1-mediated interactions are essential for the interaction of
HSCs/HSPCs with the niche cells [37], I used α4β1
[1-adamantaneacetyl-Cys-Gly-Arg-Gly-Asp-Ser-Pro-Cys (disulfide bridge
between residues 1-8); α4β1-ada] to examine whether α4β1-mediated
interactions are involved in the BMSC-HSC/HSPC crosstalk.
I found that the BMSCs primed with cyclized peptides to α5β1 and αIIbβ3
stimulated significantly higher colony formation from the MNCs briefly
interacted with them (Fig. 3c, 2nd and
3rd bars). On the other hand, α4β1-ada peptide-primed
BMSCs exerted a potent dose-dependent inhibitory effect on colony
formation (Fig. 3c, last bar; Fig. 3d). These data underscore the
importance of integrin-mediated interactions in the BMSC-mediated
regulation of hematopoiesis. In the future, I propose to examine the
effect of other FN-specific integrin peptides, such as αvβ3, αvβ5, etc.,
on the hematopoiesis-supportive ability of BMSCs.
Considering the dose-dependent potent negative effect exerted by
α4β1-ada peptide, here I examined whether its effect is dominant over
the salutary effect of the α5β1
peptide by treating the BMSCs with α5β1peptide alone or in combination
with α4β1-ada (both used at
10µg/ml, overnight). As seen in Fig. 3e, BMSCs treated with a
combination of α5β1 and α4β1-ada peptides exerted an inhibitory effect
on the colony formation from the MNCs interacted with them. The CFU
formed in this set were significantly fewer as compared to those
obtained in BMSC*α5β1 and control BMSC sets, showing that α4β1-ada
peptide acts dominantly. Since Ada-peptide inhibits the binding of the
integrin α4β1to the FN connecting segment (CS-1) and to the vascular
cell adhesion molecule 1(VCAM1) [38], the data also show that
α4β1-CS-1 domain of FN-VCAM1 axis is crucial in the BMSC-HSC/HSPC
interaction and subsequent development of hematopoiesis.
In my next set of experiments, I made differential scoring of the CFU
formed by the MNCs to examine whether the effect of peptide-primed BMSCs
was specific to any particular lineage. I observed that the
peptide-primed BMSCs, both α5β1,
and αIIbβ3, stimulated the formation of all types of colonies, including
the GEMM ones formed by primitive HSCs (Fig. 3f). These data were
further supported by the results obtained in the co-culture assays. As
seen in the Fig. 3g, the CD34+cells co-cultured with
BMSCs primed with α5β1 or αIIbβ3 peptide proliferated luxuriantly, as
compared to their control counterparts. Several
CD34+cells showed an expression of Ki67, indicating
their proliferative state. Although the CD34+ cells in
the control sets were very few, they expressed Ki67, indicating they
would divide eventually.