Department of Ophthalmology & Visual Sciences

Vanderbilt University Medical Center

Nashville, Tennessee

BASIC RESEARCH PROJECT

The Cellular Mechanisms by which Arginine and Citrulline promote vision threatening Diabetic Retinopathy

Research Interests

Diabetic retinopathy (DR) is a leading cause of blindness throughout the world. DR is challenging to treat, and it is critical that new treatments be developed that can slow DR progression and reduce the chance of severe vision loss. Dr. Brantley’s lab recently discovered that blood levels of two molecules, arginine and citrulline, are elevated in patients with DR. To investigate this, the research team added arginine and citrulline together to retinal endothelial cells, the cells that make up the blood vessels in the retina. They found that arginine and citrulline caused the endothelial cells to become leaky and to form new blood vessel tubes, two classic features of DR. With continued support from Retina Research Foundation, Dr. Brantley is now working to determine exactly how these molecules promote diabetic retinopathy.

The purpose of this project is to understand precisely how arginine and citrulline alter the cells in the retina that are specifically involved in DR. We will determine exactly how arginine and citrulline function in retinal endothelial cells to cause retinopathy as well as how they may be able to modify current treatments for DR. Dr. Brantley believes these studies will help the development of new ways of treating, or even preventing, diabetic retinopathy.

Plans for 2024

Dr. Brantley is planning additional work to determine precisely which intracellular signaling molecules are necessary for citrulline and arginine to activate eNOS, promote cell migration and tube formation, and enhance endothelial cell permeability. His lab will continue to further define the differences between citrulline and arginine-mediated and VEGF-mediated angiogenesis and vascular permeability. This will help us to define specific therapeutic targets that can improve on the current anti-VEGF treatment for diabetic retinopathy.

Specific Aims:

Aim 1. Determine how citrulline and arginine-induced retinal endothelial cell
permeability interacts with the VEGF signaling pathway.

Aim 2. Complete experiments to determine if citrulline and arginine activate the AMPK
pathway to induce an angiogenic response and promote NO production

Progress in 2023

Dr. Brantely’s data in previous years showed that citrulline and arginine induce an angiogenic response in retinal endothelial cells by activating eNOS to produce NO with no effect on arginase activity. We also demonstrated that inhibiting eNOS blocks citrulline and arginine-induced angiogenesis. We have shown that citrulline and arginine
phosphorylate eNOS-activating signaling molecules AMPK and Akt. We have a manuscript with a portion of these data in revision at IOVS. In the 2023 grant period, Dr. Brantely’s lab team demonstrated that citrulline and arginine activate signaling molecules downstream of VEGFR in the VEGF signaling pathway, that AMPK inhibition blocks citrulline and arginine-induced angiogenesis without limiting NO production, and that citrulline plus arginine does not increase protein or gene expression of transporters LAT1 or CAT1.

The purpose in 2023 was to understand precisely how arginine and citrulline alter the cells in the retina that are specifically involved in DR. Dr. Brantley and his team proposed to determine exactly how arginine and citrulline function in retinal endothelial cells to cause retinopathy as well as how they may be able to modify current treatments for DR. He believes these studies will help develop new ways of treating, or even preventing, diabetic retinopathy.

During 2023, Dr. Brantley intends to determine precisely which intracellular signaling molecules are necessary for citrulline and arginine to activate eNOS, promote cell migration and tube formation, and enhance endothelial cell permeability. His lab team will further define the differences between citrulline and arginine-mediated and VEGF-mediated angiogenesis and vascular permeability. This will help to define specific therapeutic targets that can improve on the current anti-VEGF treatment for diabetic retinopathy.

Specific Aims: Aim 1. Clarify the specific cellular signaling mechanism through which citrulline and arginine activate eNOS; Aim 2. Determine if citrulline and arginine activate the AMPK pathway to induce an angiogenic response and promote NO production; Aim 3. Confirm the role of eNOS and NO in citrulline and arginine-induced permeability in retinal endothelial cells.

Progress in 2022

Dr. Brantley’s data in previous years showed that citrulline and arginine induce an angiogenic response in retinal endothelial cells by activating eNOS to produce NO with no effect on arginase activity. His team prepared a manuscript with these data and will submit it soon. In 2022, the lab demonstrated that inhibiting eNOS blocks citrulline and arginine-induced angiogenesis. Members showed that citrulline and arginine phosphorylate eNOS-activating signaling molecules AMPK and Akt, and that Akt inhibition ameliorates cell migration but not tube formation in retinal endothelial cells treated with citrulline and arginine. Finally, they further investigated the disruption of claudin-5 at the cell membrane and showed that citrulline and arginine increase retinal endothelial cell permeability.

Specific Aims: Aim 1. Determine the specific cellular signaling mechanism through which citrulline and arginine activate eNOS. Aim 2. Determine if citrulline and arginine activate the Akt pathway independent of VEGF signaling. Aim 3. Determine the molecular changes in tight junction structure by which arginine and citrulline increase vascular permeability in retinal endothelial cells.

Progress in 2021

Progress in 2021: Dr. Brantley’s data thus far suggest that treatment of retinal endothelial cells with arginine and citrulline leads to increased nitric oxide synthase activity and nitric oxide production. Arginine and citrulline in combination also reduce arginase-1 expression. His lab team has also shown that citrulline and arginine-induced angiogenesis is inhibited in the presence of an Akt inhibitor, suggesting that citrulline and arginine promote angiogenesis via the Akt signaling pathway. Lastly, Dr. Brantley demonstrated that citrulline plus arginine alters the association of Claudin-5 to the endothelial cell membrane, suggesting the mechanism by which citrulline and arginine increases retinal endothelial cell permeability

2021 Specific Aims: Aim 1. Confirm the cellular pathway by which arginine and citrulline induce angiogenesis in retinal endothelial cells. Aim 2. Determine the changes in membrane protein structure by which arginine and citrulline increase vascular permeability in retinal endothelial cells. Aim 3. Determine if the angiogenesis-promoting effects of arginine and citrulline are independent of VEGF in retinal endothelial cells.

Progress in 2020

The data thus far suggest that treatment of retinal endothelial cells with arginine and citrulline leads to increase nitric oxide synthase activity and nitric oxide production.  Arginine and citrulline in combination also reduce arginase activity.  These data support the hypothesis that arginine and citrulline induce new blood vessel growth through the nitric oxide pathway.