The vascular endothelium lines all the blood vessels found in higher organisms and as such is the largest endocrine organ of the body.  Proteins, lipids and the gas, nitric oxide, produced by the endothelium protect blood vessels from environmental stress, oxidative damage and thrombosis which in turn maintains the patency of blood vessels and ensures the precise delivery of nutrients and oxygen to tissues.   In most cardiovascular diseases, diabetes, as well as in cancer, dysregulation of the vascular endothelium contributes directly to disease progression.   Thus, our lab is generally interested in what etiologic factors or genes regulate the transition of a healthy "normal" endothelium to a a lab we integrate molecules to disease, and use a broad range of technologies and strategies to achieve our goals. 

            One particular pathway that has been a long standing interest in the lab is understanding the detailed molecular control of the enzyme endothelial nitric oxide synthase (eNOS), the NOS isoform localization and by dynamic protein-protein interactions that act as a rheostat to control the duration and magnitude of NO production.  NO subserves at least two broad functions;  as a paracrine or autocirne second messenger.  As a paracrine mediator, NO causes vasodilation, prevents platelets and leukocytes from sticking to the endothelium, regulates the remodeling of blood vessels.  As an autocrine mediator, NO regulates vascular permeability, growth and organization of endothelial cells into angiogenic sprouts.  Thus, insights into understanding how signal transduction mechanisms activate eNOS have led to potential novels therapeutics and models of human disease.

We have shown that eNOS is a peripheral membrane protein targets to plasma membrane caveolae and the Golgi complex and while in caveolae is negative regulated by its interaction with the caveolae coat protein, caveolin-1.  Caveolae are anatomical microdomains with unknown functions but are speculated to play a role in signal transduction, protein transcytosis and fluid homeostasis.  Biochemical, genetic and pharmacological approaches have shown that the interaction of caveolin-1 with eNOS regulates systemic blood pressure, vascular permeability and angiogenesis.   Thus, one of the major roles of caveolae/caveolins are to regulate vascular function.  Recent insights into the role of the eNOS-caveolin-1 interaction have been elucidated using a cell permeant peptide that blocks the in vivo interaction of caveolin-1 with eNOS and serves as an antagonist of eNOS.  Using in vivo models of inflammation and tumor progression, treatment of mice with this peptide reduces disease by blocking vascular permeability, thus providing a novel strategy for treating inflammation and cancer. Most importantly, these results illustrate the principal that non-canonical regions of protein-protein interactions can be identified in vitro and manipulated in vivo as a "proof-of-concept" to test the importance of any protein-protein interaction in a disease model.

 In the context of signaling, we have discovered that one of the major roles of the protein kinase Akt in vivo is to phosphorylate and regulate eNOS.  Phosphorylation of eNOS by Akt increases the rate of electron flux through eNOS thus increasing NO production in vivo.  Mice lacking eNOS or Akt-1 exhibit severe limb ischemia and are excellent models for peripheral vascular disease in humans.   Interestingly, endothelial cells and vascular smooth muscle cells express not only Akt-1, but Akt-2 and -3., however, the substrates or functions of these additional family members are not well understood and are being explored.  As a method to correct these gene deficiencies, we have developed a novel approach to improve therapeutic gene transduction.  Co-complexation of cell permeable peptides with viruses (AAV, adenovirus and retrovirus) improves viral delivery of therapeutically active genes in vivo such as eNOS and can rescue the loss of limb phenotype in mice lacking eNOS or Akt.   Ongoing experiments examining how these peptides improve viral uptake and the mechanisms of how eNOS or Akt regulate cellular functions are being explored in fibroblasts or vascular cells isolated from knockout mice. 

 An additional pathway that impinges upon both eNOS and Akt is hsp90.  Hsp90 is a highly conserved protein in evolution and in mammals functions in signal transduction by serving as a scaffold for kinases or substrates.  In endothelial cells, hsp90 is critical for angiogenic factors such as vascular endothelial growth factor (VEGF) to promote cell adhesion, NO production, cell migration and angiogenesis.  Thus, we have mapped the sites of interaction between the protein partners and have generated several peptides that block the docking of either eNOS or Akt onto hsp90 that will be tested in models of inflammation and cancer.  We are also embarking on using structural approaches to understand the interaction of eNOS with the negative regulator, caveolin-1, and the positive regulators Akt and hsp90. 

 A newly emerging theme in the lab is using proteomics to discover novel proteins that may regulate blood vessel function.  We have isolated caveolae from endothelial cells in culture and have identified several new proteins. As an example, we have identified Nogo-B which had no known function. Nogo-B is a member of the reticulon family of proteins including Nogo-A and -C.   Nogo-A produced in oligodendrocytes has been identified as an inhibitor of axonal growth and repair.  We discovered that Nogo-B promotes the adhesion of endothelial cells and smooth muscle cells and is a potent chemoattractant for endothelial cells.  In contrast to its motogenic properties in the endothelium, Nogo-B blocks PDGF mediated migration of smooth muscle cells.  More importantly, Nogo-B is highly expressed in most blood vessels and disappears after vascular injury.  The genetic loss of Nogo-B does not influence vascular development but is essential for post-natal vascular remodeling and responses to tissue ischemia.   Thus, a major effort is underway to clone the receptor (s) for Nogo-B and to dissect its signaling mechanisms using genetic and pharmacological strategies.  We believe that there may be a  family of receptors for Nogo-B and perhaps common regions of Nogo-A.  We are presently developing the requisite biochemical and genetic tools to dissect this pathway and apply the information to human diseases.

06/30/04