The long-term goal of this research is to decrease the morbidity and mortality related to diabetic vascular complications by developing bioactive therapeutic agents. Hyperglycemia-induced vascular inflammation, leading to monocyte adhesion to vascular endothelial cells (ECs), is the key factor that initiates the pathogenesis of atherosclerosis in diabetic patients. Our studies found that botanical genistein inhibits hyperglycemia-induced human monocyte-EC interaction, suggesting an anti-inflammatory action of genistein in vasculature of diabetics. Leukocyte adhesion to endothelium is mediated through both chemokines and adhesion molecules on ECs, and the expression of these molecules is critically up- regulated by nuclear factor kB (NF-kB). Further analysis showed that genistein suppressed high glucose- induced production of monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) in human aortic ECs (HAECs), two chemokines that are the key factors in the firm adhesion of monocytes to activated ECs. Our recent studies found that genistein is an activator of the cAMP signaling system in ECs. In addition, our data demonstrated that genistein can stimulate PPAR3 activity in HAECs. Interestingly, data from our animal study showed that dietary intake of genistein increased plasma cAMP but reduced MCP-1 levels in diabetic mice.
Our specific aim of this application is to determine the molecular mechanism by which genistein suppresses hyperglycemia-induced vascular inflammation. We hypothesized that genistein inhibits hyperglycemia-induced EC inflammation by activation of cAMP/PKA signaling and PPAR3 in ECs.
Aim 1 will use HAECs to investigate: 1) whether genistein inhibits high glucose-induced expression of chemokines and adhesion molecules;2) whether genistein inhibits hyperglycemia-induced NF-kB activity;3) whether the anti-inflammatory action of genistein is mediated via the cAMP/PKA and PPAR3 pathways;and 4) whether genistein also inhibits hyperglycemia-induced EC-monocyte interaction under flow.
Aim 2 will use diabetic mice to determine: 1) whether genistein activates cAMP/PKA and PPAR3 in vivo;2) whether genistein inhibits chemokine and adhesion molecule production from blood vessels;and 3) whether genistein suppresses monocyte recruitment to ECs and whether this effect is attenuated by PPAR3 deficiency. Genistein will be administered to diabetic mice through dietary supplementation approach. The protein and mRNA expression will be assessed by Western blot, ELISA or real-time PCR. Cyclic AMP levels and PKA activity will be determined using assay kits. NF-kB and PPAR3 activity will be measured with ELISA and reporter gene assays. The role of PKA and PPAR3 in the anti-inflammatory action of genistein will be determined by using chemical and molecular tools as well as gene knockout mice. The mechanistic understanding of how genistein protects against diabetes-caused vascular inflammation will facilitate the development of efficacious CAM strategies to protect diabetic patients from vascular complications.
Cardiovascular disease such as atherosclerosis is the main cause of deaths in patients with diabetes mellitus. It is increasingly recognized that vascular inflammation mediated by sustained elevation of blood glucose levels in diabetic patients plays a pivotal role in the pathogenesis of atherosclerosis. Our studies indicate that botanical genisein is a promising agent to protect against chronic high glucose- caused vascular inflammation. The results of this research will potentially lead us to develop novel, natural, and cost-effective agents to prevent and treat atherosclerosis caused by diabetes, a chronic disease that affects over 23 million Americans.
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