Neutrophils (PMN) can migrate along gradients of chemoattractants across endothelial monolayers to sites of inflammation and infection. This chemotaxis through endothelial cell borders is involved in several acute and chronic inflammatory diseases, however our understanding of the role of endothelial second messengers in the regulation of leukocyte emigration is still incomplete. We investigated this using an in vitro model of neutrophil migration across human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMECs) on cell culture inserts. We report that activation of endothelial protein kinase C (PKC) by both phorbol myristate acetate (PMA) and Bryostatin-1 (a potent PKCδ and c activator) can completely abolish neutrophil migration mediated by both endothelial TNF-α stimulation and a leukotriene B4 (LTB4) gradient. PMA protected against LTB4 induced PMN transmigration for at least 24 hours in HMECs and HUVECs. Bryostatin-1 protected PMN migration for at least 24 hours in HMECs and at least 48 hours in HUVECs. Pretreatment with Go-6983 (PKCα, β, and δ inhibitor) before the addition of Bryostatin-1 restored the loss of LTB4 induced neutrophil migration, while pretreatment with GO-6976 (PKCα and β inhibitor) did not. In addition using PKCδ and ε specific small interfering RNA, we were able to show that PKCδ, but not ε was at least mostly responsible for the loss of neutrophil migration in response to LTB4. Taken together, these observations suggest that activation of endothelial PKCδ could be therapeutic in the treatment of various inflammatory disorders characterized by enhanced neutrophil infiltration.
This invention relates to pharmaceutical compositions, particularly pharmaceutical compositions comprising bryostatin-1 and substituted derivatives of bryostatin-1, thereof as pharmaceuticals for inhibition of inflammation, and for use in combating arteriosclerosis, diseases of the cardiovascular system, of the central nervous system and prior to/following organ transplantation, ischemia. The invention relates to methods for treating leukocyte dependent injury in chronic inflammatory diseases, and injury from transplantation mediated organ stress. The method involves injecting bryostatin-1 into patients with the inflammatory condition, treating the skin with bryostatin-1, or perfusing organs with bryostatin-1 prior to transplantation/cold storage. Activation of protein kinase Cd (PKCd) results in a near complete blockade of leukocyte infiltration which is the result of stabilization of the microvascular (endothelial) barrier.