Hiroyuki Ikezaki

A Novel Formulation of VIP in Sterically Stabilized Micelles Amplifies Vasodilation In Vivo

AbstractPurpose. To determine whether human vasoactive intestinal peptide (VlP)-poly(ethylene glycol) (PEG)-grafted distearoyl-phosphatidyleth-anolamine (DSPE) micelles elicit potent and stable vasodilation in vivo. Methods. PEG-DSPE micelles were prepared by co-precipitation. VIP was loaded into micelles by incubation at room temperature. Vasoactivity of VIP in SSM was determined by monitoring changes in diameter of resistance arterioles in the in situ hamster cheek pouch using intravital microscopy. Results. VIP easily undergoes self-assembly into small PEG-DSPE micelles (mean [±SEM] size, 18 ± 1 nm) in a time-dependent fashion. This generates a potent vasoactive matrix at nanomole concentrations of VIP as manifested by ~3-fold potentiation and prolongation of vasodilation relative to that evoked by aqueous VIP alone (p < 0.05). This response is specific and mediated by the L-arginine/nitric oxide (NO) biosynthetic pathway. Micellar VIP dispersion remains vasoactive for at least 14 days after preparation and storage at 4°C. Conclusions. A novel, self-associated, small and stable PEG-DSPE micellar formulation of VIP amplifies vasodilation in the in situ peripheral microcirculation in a specific fashion by elaborating NO. An optimized formulation could be considered for certain cardiovascular disorders associated with L-arginine/NO biosynthetic pathway dysfunction.

Conformation and vasoreactivity of VIP in phospholipids: effects of calmodulin☆

The purpose of this study was to determine the conformation and vasorelaxant effects of vasoactive intestinal peptide (VIP) self-associated with sterically stabilized phospholipid micelles (SSM) and whether calmodulin modulates both of these processes. Circular dichroism spectroscopy revealed that VIP is unordered in aqueous solution at room temperature but assumes appreciable a helix conformation in SSM. This conformational transition was amplified at 37 degrees C and by a low concentration of calmodulin (0.1 nM). Suffusion of VIP in SSM elicited significant time- and concentration-dependent potentiation of vasodilation relative to that elicited by aqueous VIP in the in situ hamster cheek pouch (P < 0.05). This response was significantly potentiated by calmodulin (0.1 nM). Collectively, these data indicate that exogenous calmodulin interacts with VIP in SSM to elicit conformational transition of VIP molecule from a predominantly random coil in aqueous environment to alpha helix in SSM. This process is associated with potentiation and prolongation of VIP-induced vasodilation in the in situ peripheral microcirculation.

Vasodilation elicited by liposomal VIP is unimpeded by anti-VIP antibody in hamster cheek pouch

The purpose of this study was to determine whether a monoclonal anti-vasoactive intestinal peptide (VIP) antibody, which binds VIP with high affinity and specificity and catalyzes cleavage of the peptide in vitro, attenuates VIP vasorelaxation in vivo and, if so, whether insertion of VIP on the surface of sterically stabilized liposomes (SSL), which protects the peptide from trypsin- and plasma-catalyzed cleavage in vitro, curtails this response. Using intravital microscopy, we found that suffusion of monoclonal anti-VIP antibody (clone c23.5, IgG2ak), but not of nonimmune antibody (myeloma cell line UPC10, IgG2ak) or empty SSL, significantly attenuates VIP-induced vasodilation in the in situ hamster cheek pouch (P < 0.05). By contrast, anti-VIP antibody has no significant effects on vasodilation elicited by isoproterenol, nitroglycerin, and calcium ionophore A-23187, agonists that activate intracellular effector systems in blood vessels that mediate, in part, VIP vasoreactivity. Suffusion of VIP on SSL, but not of empty SSL, restores the vasorelaxant effects of VIP in the presence of anti-VIP antibody. Collectively, these data suggest that VIP catalysis by high affinity and specific VIP autoantibodies displaying protease-like activity constitutes a novel mechanism whereby VIP vasoreactivity is regulated in vivo.The purpose of this study was to determine whether a monoclonal anti-vasoactive intestinal peptide (VIP) antibody, which binds VIP with high affinity and specificity and catalyzes cleavage of the peptide in vitro, attenuates VIP vasorelaxation in vivo and, if so, whether insertion of VIP on the surface of sterically stabilized liposomes (SSL), which protects the peptide from trypsin- and plasma-catalyzed cleavage in vitro, curtails this response. Using intravital microscopy, we found that suffusion of monoclonal anti-VIP antibody (clone c23.5, IgG2ak), but not of nonimmune antibody (myeloma cell line UPC10, IgG2ak) or empty SSL, significantly attenuates VIP-induced vasodilation in the in situ hamster cheek pouch ( P < 0.05). By contrast, anti-VIP antibody has no significant effects on vasodilation elicited by isoproterenol, nitroglycerin, and calcium ionophore A-23187, agonists that activate intracellular effector systems in blood vessels that mediate, in part, VIP vasoreactivity. Suffusion of VIP on SSL, but not of empty SSL, restores the vasorelaxant effects of VIP in the presence of anti-VIP antibody. Collectively, these data suggest that VIP catalysis by high affinity and specific VIP autoantibodies displaying protease-like activity constitutes a novel mechanism whereby VIP vasoreactivity is regulated in vivo.

Exogenous calmodulin potentiates vasodilation elicited by phospholipid-associated VIP in vivo.

The purpose of this study was to determine whether exogenous calmodulin potentiates vasoactive intestinal peptide (VIP)-induced vasodilation in vivo and, if so, whether this response is amplified by association of VIP with sterically stabilized liposomes. Using intravital microscopy, we found that calmodulin suffused together with aqueous and liposomal VIP did not potentiate vasodilation elicited by VIP in the in situ hamster cheek pouch. However, preincubation of calmodulin with liposomal, but not aqueous, VIP for 1 and 2 h and overnight at 4°C before suffusion significantly potentiated vasodilation ( P < 0.05). Calmodulin-induced responses were significantly attenuated by calmidazolium, trifluoperazine, and N G-nitro-l-arginine methyl ester (l-NAME) but notd-NAME. The effects ofl-NAME were reversed byl- but notd-arginine. Indomethacin had no significant effects on calmodulin-induced responses. Calmodulin had no significant effects on adenosine-, isoproterenol-, acetylcholine-, and calcium ionophore A-23187-induced vasodilation. Collectively, these data indicate that exogenous calmodulin amplifies vasodilation elicited by phospholipid-associated, but not aqueous, VIP in the in situ peripheral microcirculation in a specific, calmodulin active sites-, and nitric oxide-dependent fashion. We suggest that extracellular calmodulin, phospholipids, and VIP form a novel functionally coordinated class of endogenous vasodilators.The purpose of this study was to determine whether exogenous calmodulin potentiates vasoactive intestinal peptide (VIP)-induced vasodilation in vivo and, if so, whether this response is amplified by association of VIP with sterically stabilized liposomes. Using intravital microscopy, we found that calmodulin suffused together with aqueous and liposomal VIP did not potentiate vasodilation elicited by VIP in the in situ hamster cheek pouch. However, preincubation of calmodulin with liposomal, but not aqueous, VIP for 1 and 2 h and overnight at 4 degrees C before suffusion significantly potentiated vasodilation (P < 0.05). Calmodulin-induced responses were significantly attenuated by calmidazolium, trifluoperazine, and NG-nitro-L-arginine methyl ester (L-NAME) but not D-NAME. The effects of L-NAME were reversed by L- but not D-arginine. Indomethacin had no significant effects on calmodulin-induced responses. Calmodulin had no significant effects on adenosine-, isoproterenol-, acetylcholine-, and calcium ionophore A-23187-induced vasodilation. Collectively, these data indicate that exogenous calmodulin amplifies vasodilation elicited by phospholipid-associated, but not aqueous, VIP in the in situ peripheral microcirculation in a specific, calmodulin active sites-, and nitric oxide-dependent fashion. We suggest that extracellular calmodulin, phospholipids, and VIP form a novel functionally coordinated class of endogenous vasodilators.

Conformation-dependent effects of VIP on nociception in rats.

The purpose of this study was to determine whether intrathecal injection of aqueous (random coil) vasoactive intestinal peptide (VIP) and VIP self-associated with sterically stabilized phospholipid micelles (alpha-helix VIP) at the lower lumbar vertebral level modulates foot withdrawal latency to low and high rate noxious radiant skin heating in anesthetized rats. We found that intrathecal random coil VIP evoked a significant bimodal, concentration-dependent response, early potent antinociception followed by hyperalgesia, during exposure to low and high rates of skin heating (P<0.05). Intrathecal alpha-helix VIP elicited a qualitatively similar response to that of random coil VIP except that the rate of decay of antinociception was faster and slower at low and high rates of skin heating, respectively. In addition, a low concentration of alpha-helix VIP evoked a potent late antinociception not observed with random coil VIP. Taken together, these data indicate that VIP modulates somatosensory processing in the lumbosacral spinal cord of rats in a complex fashion, and that this response is dependent, in part, on the conformation of VIP in the vicinity of target cells in the peripheral nervous system.

Liposomal VIP attenuates phenylephrine- and ANG II-induced vasoconstriction in vivo

The purpose of this study was to determine whether vasoactive intestinal peptide (VIP) modulates vasoconstriction elicited by phenylephrine and ANG II in vivo and, if so, to begin to elucidate the mechanisms underlying this phenomenon. Using intravital microscopy, we found that suffusion of phenylephrine and ANG II elicits significant vasoconstriction in the in situ hamster cheek pouch that is potentiated by VIP-(10-28), a VIP receptor antagonist, but not by VIP-(1-12) ( P< 0.05). Aqueous VIP has no significant effects on phenylephrine- and ANG II-induced vasoconstriction. However, VIP on sterically stabilized liposomes (SSL), a formulation where VIP assumes a predominantly α-helix conformation, significantly attenuates this response. Maximal effect is observed within 30 min and is no longer seen after 60 min. Empty SSL are inactive. Indomethacin has no significant effects on responses induced by VIP on SSL. The vasodilators ACh, nitroglycerin, calcium ionophore A-23187, 8-bromo-cAMP, and isoproterenol have no significant effects on phenylephrine- and ANG II-induced vasoconstriction. Collectively, these data suggest that vasoconstriction modulates VIP release in the in situ hamster cheek pouch and that α-helix VIP opposes α-adrenergic- and ANG II-induced vasoconstriction in this organ in a reversible, prostaglandin-, NO-, cGMP-, and cAMP-independent fashion.