Preserving the Precious Gift of Sight

Lih Kuo, PhD

GUEYMARD RESEARCH PROJECT

Systems Biology & Translational Medicine and Ophthalmology

Scott & White Memorial Hospital

Texas A & M Health Science Center

Temple, TX

 

Dr. Kuo’s Research Project

Activation of Endothelin-dependent RhoA/ROCK by C-Reactive Protein Elicits Retinal Arteriolar Dysfunction
 

Current Research Interests

The proper function of the retina depends upon the sufficient oxygen and nutrients supply to the neuronal tissues via the microvascular system. Blood flow regulation by these microvessels is critically important. Since chronic retinal vascular dysfunction leads to tissue ischemia, early surrogate clinical markers are needed to diagnose and quantitate the presence of preclinical lesions of vascular retinopathy that would allow the institution of treatments at the beginning stages of the disease. The vascular alteration and its mechanism leading to the dysregulation of blood flow in retinal circulation remains unknown. Recent studies have implicated that elevation of the potent vasoconstrictor and inflammatory agent endothelin-1 (ET-1) is a major risk factor for ischemic vascular disorder during the development of cardiovascular disease including diabetes. In diabetes, the upregulated vascular endothelial growth factor (VEGF) is known to be a major culprit for the development of diabetic retinopathy. Dr. Kuo will address whether ET-1, VEGF, and the associated signaling molecules related to inflammation and oxidative stress play adverse roles in retinal arteriolar function in diabetes and by delineating the involved mechanisms under this pathophysiology.

Progress in 2016

Dr. Kuo’s data are the first to demonstrate the adverse effect of diabetes on retinal microcirculation in the pig model relevant to human physiology and pathophysiology. Recent studies supported by RRF showed that hyperglycemia compromises endothelium-dependent nitric oxide (NO)-mediated vasodilator function in retinal arterioles via increased activation of JNK, a proinflammatory stress kinase. In the pig animal model, he subsequently demonstrated that simvastatin elicits mainly an endothelium-dependent, NO-mediated dilation of retinal arterioles by inhibiting Rho kinase-ROCK pathway. These results strongly suggest that statins have a mechanistic action and therapeutic potential in improving endothelium-dependent vasomotor function in retinal vascular disorders.

Plans for 2017

Dr. Kuo has planned a series of experiments to elucidate the signaling pathway for vascular pathogenesis, in relation to ET and VEGF system activation and oxidative stress, exerted by diabetes. Aim 1) Diabetes exert detrimental action on retinal arteriolar function by activating ECE for ET-1 release and promoting VEGF synthesis from endothelial cells and leading to superoxide protection via RhoA/ROCK activation; 2) Determine whether enhanced RhoA/ROCK-dependent or arginase-dependent phosphorylation of c-Jun N-terminal kinase (JNK)-interacting protein-1 (JIP1) by ET-1 contributes to diabetes-induced dysfunction of retinal arterioles by increasing oxidative stress; 3) Examination of the therapeutic potential of intravitreal administration of inhibitors for ET-1, VEGF, RhoA/ROCK, and arginase upon vascular dysfunction elicited by diabetes.

Progress in 2015

Dr. Kuo accomplished several projects to elucidate the cellular mechanism responsible for the activation of eNOS by increased flow in association with VEGFR2 signaling. He found that the NO-mediated vasocilation is not compromised by the vasoconstrictor activity of ET-1, and will look for an alternative explanation for the observed clinical outcome of ET-1-asociated retinal disease. The striking result of this year’s study is the involvement of VEGF receptors in mediating flow-induced dilation of retinal arterioles. It appears that VEGFR2 acts as a mechanical sensor for vasodilation to increased flow.

Progress in 2014

Dr. Kuo accomplished Specific Aims focusing on the possible counter-interaction between endothelium-released vasodilator NO and the vasoconstrictor ET-1. The results of this study were published in the Invest Ophthalmol Vis Sci. He further examined the cellular signaling mechanism of activation of NO synthesis by the enzyme NO synthase (NOS) during elevated flow (shear stress). Unfortunately, the mechanism by which NOS is activated by the increased flow for vasodilation remains unknown. The results of this study not only help our understanding on how NOS is activated by flow elevation but also provide useful information on how to protect NOS from the insult of the disease related to retinal ischemia.

Progress in 2013

Dr. Kuo’s project is to understand the pathophysiology of inflammation and diabetes-associated retinal vascular dysfunction, at molecular, cellular and intact tissue levels and to develop a therapeutic approach for disease treatment. He addresses whether cardiovascular risk factors C-reactive protein (CRP) and endothelin-1 (ET-1), in association with oxidative stress, play adverse role in retinal arteriolar function in diabetes. Dr. Kuo has recently established a pig model of retinal microvascular dysfunction induced by type-1 diabetes, which he has shown to resemble human in retinal vascular physiology and pathophysiology. He continues to utilize the pig model to test central hypothesis that CRP/diabetes can activate endothelin converting enzyme activity leading to endothelial dysfunction and impaired vasodilation in retinal arterioles.

Progress in 2012

Dr. Kuo’s laboratory reported that the retinal arteriolar function can be impaired by an acute retinal ischemia due to nitric oxide deficiency and oxidative stress in the endothelium by activating NADP(H) oxidase. This pathophysiology is similar to that elicited by C-reactive protein (CRP) via Rho kinase (ROCK) signaling. In addition they also found that ROCK is a key signaling molecule responsible for the vasoconstriction and oxidative stress evoked by the endothelin-1. Therefore, the endothelin-1 system can be the major linkage between ROCk activation and vascular pathophysiology exerted by inflammation (i.e., CRP) and ischemia. The retinal arteriolar dysfunction can be produced by acute diabetes in the pig, which they have recently shown to resemble human in retinal arteriolar physiology and pathophysiology.

Preliminary data suggest that the retinal vascular dysfunction induced by diabetes might be related to the activation of ROCK via the endothelin system in the vascular wall. This newly developed animal disease model could allow investigation of the early phase of diabetic retinopathy at the mechanistic levels.The purpose of this project is to understand the pathophysiology of inflammation-and ischemia-associated retinal vascular dysfunction at molecular, cellular and intact tissue levels and to develop a therapeutic approach for disease treatment.

Retinal vascular diseases, e.g., diabetic retinopathy, acute angle-closure glaucoma and retinal vascular occlusion are the leading causes of visual impairment in the USA, but the etiology and development of this vasculature-related ocular disease are not fully understood. Because the proper function of retina neuronal tissues depends upon the sufficient oxygen and nutrients supply via the microvascular system, the blood flow regulation by these microvessels is critically important and closely related to the disease development.  Early surrogate clinical markers are needed to diagnose and quantitate the presence of preclinical lesions of vascular retinopathy that would allow the institution of treatments at the beginning stages of the disease.

The cellular/molecular mechanisms in regulation of retinal blood flow during disease states remain largely unknown. In this study, Dr. Kuo’s laboratory uses a novel approach (i.e., isolated vessel preparation) to directly address this issue.

The results derived from this study will have significant impact on the design and treatment of vascular disorder in the eye related to inflammatory and ischemia disease. They test the preventive and therapeutic effects of statins, as well as endothelin system blockade and RhoA/ROCK signaling inhibition, on retinal arteriolar dysfunction induced by pathogenic factor CRP/ischema.

Nitric oxide (NO) released from the vascular endothelium plays an important role in vasodilation, and the deficiency of its synthesis from the enzyme NO synthase (eNOS) contributes to the development of various ocular diseases, including diabetic retinopathy and ischemia-related retinal vascular dysfunction. Although protein kinase C (PKC) activation is also known to participate in the pathogenesis of vascular disease related to ischemia and diabetes, the involved isoform in disease development remains unclear. We investigated the location and distribution of eNOS and PKCβII isoform in porcine retinal arterioles (40-80 µm) using immunohistochemical tools. The eNOS (green) was detected in the endothelial layer of inner arteriolar wall. The PKCβII (red) is located in both smooth muscle and endothelial cells with prominent expression in the endothelium. The close proximity of eNOS and PKCβII in the endothelium may highlight the importance of PKCβII in the regulation of NO production by the endothelium.

Progress in 2011

Dr. Kuo’s laboratory has developed a clinically relevant animal model for retinal ischemia to support the critical role of ET-1 system and RhoA kinase/ROCK in the development of vascular pathophysiology. The proposed works on the adverse effect of ischemia on retinal arteriolar function has been accomplished and the results were just accepted for publication.

Retinal vascular disease such as diabetic retinopathy is one of the leading causes of blindness in the USA, but the etiology and development of vascular and visual pathology in this disease is not fully understood. Elevated plasma level of inflammatory marker C-reactive protein (CRP) is associated with patients with diabetes and various cardiovascular diseases.

Interestingly, Dr. Kuo’s laboratory has found that CRP elicits retinal vascular disorder by losing endothelium-dependent vasodilatory function. However, the mechanistic action of CRP on retinal vasomotor function remains elusive. Since the plasma level of both CRP and endothelin-1 (ET-1), a potent vasoconstrictor, is elevated in the patients with diabetic retinopathy, they test the hypothesis that enhanced endothelin-converting enzyme activity (for ET-1 production) and the subsequent RhoA kinase (ROCK) activation are responsible for the adverse action of CRP.

Dr. Kuo also examines the therapeutic potential of statins in the protection and treatment of vascular dysfunction elicited by CRP. His laboratory uses an isolated vessel approach to directly assess retinal microvascular function and uses molecular tools to address the signaling pathways leading to vascular dysfunction by CRP.

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Mission of RRF

The mission of the Retina Research Foundation is to reduce retinal blindness worldwide by funding programs in research and education. As a public charity, RRF raises funds from the private sector and the investment of its endowment funds.

RRF’s 48th Anniversary

October 1, 2017