Nanotechnology Enabled Modulation of Signaling Pathways Affects Physiologic Responses in Intact Vascular Tissue

Subarachnoid hemorrhage (SAH) is associated with vasospasm that is refractory to pharmacologic intervention. SAH vasospasm is associated with decreased phosphorylation of intracellular heat shock protein (HSP)20 and increased phosphorylation of HSP27. Phosphorylated HSP20 (p-HSP20) is associated with vascular smooth muscle relaxation and phosphorylated HSP27 with contraction and impaired relaxation. This study was undertaken to modulate these key signaling molecules using intracellular delivery technologies to elucidate the effect of modulation on vasomotor tone of intact vascular tissues. Rat aorta was used as a model system to elucidate the impact of HSP20/HSP27 modulation on physiologic vasomotor responses. HSP20 expression was silenced through siRNA transfection utilizing an endosomolytic diblock copolymer. The diblock copolymer self-assembles into small (approximate to 50nm) serum stable micellar nanoparticles that effectively package siRNA and facilitate tissue penetration, cell uptake, endosomal escape, intracellular bioavailability, and tissue-level silencing of target gene expression. To increase HSP27 activity, recombinant HSP27 was fused to a cell permeant peptide domain to facilitate protein uptake and activity. Furthermore, a cell permeant peptide mimetic of p-HSP20 was delivered through formulation into nano-polyplexes using a pH-responsive endosomolytic polymer. The impact of these biomolecular modulations on physiologic vasomotor responses in intact tissue was then quantified in a muscle bath. Treatment of rat aorta with HSP20 siRNA polymeric nanocarriers decreased expression of HSP20 which was associated with enhanced contractile responses to phenylephrine (PE) and impaired relaxation responses to sodium nitroprusside. Delivery of recombinant HSP27 to the tissue was associated with increased contractile responses to PE. Treatment with a peptide mimetic of p-HSP20 resulted in decreased contractile responses to PE. These results demonstrate that manipulation of protein levels of the small heat shock phosphoproteins, HSP20 and HSP27, in intact vascular tissues is associated with changes in vasomotor responses. In particular, decreasing HSP20 expression and addition of exogenous HSP27 led to enhanced contractility and impaired relaxation. This biochemical signature is similar to the phenotype of SAH associated vasospasm and suggests that alterations in downstream signaling events may be responsible for SAH induced vasospasm being refractory to upstream, receptor-mediated pharmacologic interventions.