Despite the considerable progress in vascular biology and development, there is a lack of technologies preserving endothelial function, as well as deficits in understanding how stable and mature neovasculature is formed. The main goal of these studies is to dissect the detailed molecular mechanisms and identify novel targets regulating endothelial function and neoangiogenesis. This is a resubmission of our project focused on the role of integrin-Akt signaling axis in vascular biology. We have demonstrated that integrin activation on endothelium is essential for angiogenesis and vascular homeostasis and have identified key molecules regulating this process. Mechanistically, we have identified integrin/Akt axis as an important driver of vascular growth and maturation, suggesting that this pathway can act as an on/off switch for tissue specific angiogenesis. Our preliminary data demonstrate that Akt in endothelium controls vascular functions and angiogenesis. We will focus on the role of Akt in endothelial migration and integrin function and will determine molecular interactions controlling this interplay. The main hypothesis is: endothelial Akt1 is recruited to integrins via heterotrimeric complex of PINCH, ILK, Parvin, and serves as an essential regulator of integrin inside-out signaling, which, in turn, controls endothelial migratio and angiogenesis in vivo. While other parallel pathways might mediate cell survival and proliferation in the absence of Akt, it is indispensable for the endothelial attachment and migration. The following specific Aims are proposed:
Aim 1. To establish the role for endothelial Akt in integrin-dependent functions in angiogenesis: We will determine the levels of Akt activity which are sufficient to support integrin functions in vivo during angiogenic responses. We will assess how and why Akt deletion in endothelium affects the key characteristics of neovasculature.
Aim 2. To determine the levels of Akt activity and Akt isoforms required for integrin inside-out vs. outside-in signaling in EC. We will compare the direct involvement of individual Akt isoforms in the regulation of integrin inside-out signaling triggered either by VEGFR or by TLR2 activation (as an example of a novel VEGF- independent pathway in endothelium). Rescue experiments with constitutively active integrins or forced integrin activation will be performed.
Aim 3. To establish a detailed molecular mechanism of Akt1 recruitment to integrin complex and the role for this complex in integrin function in endothelium. We will focus on the direct recruitment of Akt1 to integrin-associated complex (ILK/ PINCH/Parvin and Kindlin) and determine how this complex coordinates integrin functions on endothelium. To this end, we will determine the structural determinants of this complex (residues within Akt1 required to interact with Parvin, which is a prerequisite for the formation o the entire complex) and show how disruption of the individual interactions affects bidirectional integrin signaling. Together, our studies will show how the Akt-integrin axis regulates vascular homeostasis and angiogenesis in a complexity of in vivo settings, and reveal the molecular determinants connecting Akt signaling to integrin function. These studies will identify novel cellular and molecular mechanisms underlying endothelial functions in health and disease.

Public Health Relevance

Ischemic heart disease, stroke, and peripheral vascular disease together are responsible for deaths of more than 16 million people each year. It is well recognized that the only way to adapt to ischemia and to rescue tissues is the development of new functional vasculature. Unfortunately, at present, there is a lack of technologies facilitating growth of properly organized and functional vasculature in ischemic tissues. Thus, there is a need to understand how tissue vascularization is regulated during development and in postnatal life so that innovative approaches for the control vascular growth in patients with distinct therapeutic needs can be identified. The main goal of these studies is to dissect the detailed molecular mechanisms and to identify novel targets regulating endothelial function in adults and neo-angiogenesis in pathological conditions. This is a second renewal of our project focused on the role of Akt and integrin signaling in angiogenesis. We identified a series of new targets for this pathway which mechanistically connect endothelial adhesion, migration, and survival to in vivo functions. This proposal will take our studies to the next level, and we will test a new hypothesis to establish how a main pro-survival molecule, Akt kinase controls endothelial cell adhesion and migration in angiogenesis. Using new models and approaches we will establish the key molecular pathways responsible for re-vascularization of tissues as well as the mechanisms supporting stable and healthy vasculature in adults.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
2R01HL071625-10A1
Application #
8696096
Study Section
Cardiovascular Differentiation and Development Study Section (CDD)
Program Officer
Kindzelski, Andrei L
Project Start
Project End
Budget Start
Budget End
Support Year
10
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
City
Cleveland
State
OH
Country
United States
Zip Code
44195
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