Chromatin protein HMGB1 triggers endothelial dysfunction in diabetes via TLR4
Diabetes mellitus (DM) is at epidemic levels in the U.S. and is a major cause of cardiovascular disease. Endothelial dysfunction, a major contributor to the onset of diabetic vascular complications, is characterized by diminished nitric oxide (NO) bioavailability and increased generation of reactive oxygen species (ROS), leading to impaired endothelium-dependent vasodilation, endothelial cell (EC) apoptosis, and vascular inflammation. A critical barrier to improving survival from DM is understanding how metabolic processes interface with immune responses. The adaptive immune system is a contributing factor to the development of DM; however, the mechanisms initiating the inflammatory response is unknown. Damage-associated molecular patterns (DAMPs) are endogenous molecules released by injured cells that impact the nature and magnitude of an immune response by activating the innate immune system. High mobility group box 1 protein (HMGB1) is a DAMP released by necrotic cells that stimulates a pro-inflammatory state via Toll-like receptors (TLR). The impact of HMGB1 on EC function in DM is unknown. Based on our compelling preliminary data indicating that TLR4 signaling is upregulated in the vasculature of diabetic animals and its activation contributes to augmented ROS generation and impaired vasodilation, our central hypothesis is that HMGB1 activates TLR4 signaling in EC leading to endothelial dysfunction. Two specific aims are proposed: 1) To test the hypothesis that HMGB1 activates TLR4 signaling in human aortic EC leading to increased ROS generation, decreased NO bioavailability and NFKB activation, which will result in in EC apoptosis and inflammation. 2) To test the hypothesis that HMGB1 induces impaired endothelium-dependent vasodilatation in DM through mechanisms that involve TLR4. Streptozotocin will be used to induce diabetes in C57Bl/6 and TLR4-/- mice. Some mice will be treated with an anti-HMGB1 neutralizing antibody. Endothelium-dependent relaxation will be measured in isolated aortic rings. Elucidation of the role for HMGB1 in diabetic endothelial dysfunction has the potential to shed new insight into the development of therapeutic strategies for prevention or intervention of this disorder.
Toll-like Receptor 4 Mediates Diabetic Bladder Dysfunction
Bladder dysfunction affects approximately 50% of patients with diabetes. Increases in bladder size and alterations of bladder contractile responses are characteristics of diabetic bladder dysfunction (DBD) which underlie the clinical manifestations of this condition. One of the main hurdles in the development of therapeutic approaches for diabetic complications is a gap in the understanding of the pathogenetic mechanisms underlying them. Chronic activation of the innate immune system by endogenous molecules released from cell injury and death, such as the chromatin protein high mobility group box 1 (HMGB1) has recently been implicated in diabetes pathogenesis. Circulating levels of glucose and HMGB1 are chronically increased during diabetes. Both high glucose and HMGB1 can activate Toll like receptor 4 (TLR4), an innate immune receptor, in non-immune cells, leading to oxidative stress and activation of inflammatory pathways. We made the novel observation that the specific blockade of TLR4 abolishes the increase in reactive oxygen species (ROS) levels induced by exposure to high glucose. Additionally, our preliminary data suggest that TLR4 activation in the bladder mediates increased contractile responses in a mouse model of type I diabetes and that genetic deficiency of TLR4 protects against bladder hypertrophy and hypercontractility in diabetes. The long-term goal of our research is to determine whether and how the innate immune system plays into the development of DBD. In this project, we propose the innovative hypothesis that in diabetes, hyperglycemia and HMGB1 activate TLR4 in the bladder, leading to bladder smooth muscle hypertrophy and hypercontractility and mediating DBD. We will address this hypothesis with two specific aims, to be tested both during the early (hypercontractile) and late (hypocontractile) phases of DBD. In specific aim 1, diabetic mice will be treated with inhibitors of HMGB1 and cell death to test the hypothesis that cell death-induced release of HMGB1 leads to DBD. In specific aim 2, in vivo and in vitro effects of HMGB1 administration effects will be examined in wild type and TLR4 knock-out mice.
Toll-like receptor 4 mediates diabetic bladder dysfunction.
Diabetes mellitus (DM) is at epidemic proportions in the U.S. with 18.8 million people diagnosed and an estimated 7 million undiagnosed. Furthermore, 35% of U.S. adults are pre-diabetic. Because DM affects every organ, patients with DM suffer from several complications including lower urinary tract dysfunctions. These complications are costly and diminish the quality of life of patients with DM. Bladder dysfunction is among the most common complications of the lower urinary tract in DM. Despite significant recent advances in understanding diabetic bladder dysfunction, the underlying molecular pathways that initiate this dysfunction are poorly understood, and therefore, the current treatments of this complication are not always effective. Activation of Toll-like receptors (TLR), receptors of the innate immune system, has been shown to play a role in the development of DM. For example, hyperglycemia induces the expression and activation of TLR4, leading to inflammation and oxidative stress, both components of the pathology of DM. Furthermore, patients with DM are prone to urinary and bladder infections, which can induce the activation of TLR4 via pathogen-associated molecular patterns expressed by bacteria. Nevertheless, it is currently unknown whether activation of TLR4 leads to bladder dysfunction and whether a hyperglycemic environment potentiates the role of the innate immune system in diabetic bladder complications. Accordingly, we propose the innovative hypothesis that hyperglycemia leads to TLR4 activation, which causes bladder smooth muscle hypertrophy and hypercontractility, characteristics of the diabetic overactive bladder. Two aims are proposed for this study: Specific aim 1 will determine whether hyperglycemia activates TLR4 to increase cell proliferation and reactive oxygen species (ROS) production in mouse bladder smooth muscle cells. Specific aim 2 will determine whether bladder hypertrophy and hypercontractility in diabetes is mediated by TLR4. To test the proposed hypothesis, we will use primary bladder smooth muscle cells and bladder segments from diabetic and non-diabetic mice and from TLR4 deficient mice.
Thermosenitive TRPM8 channels and diabetic erectile dysfunction
Erectile dysfunction (ED) seriously diminishes quality of life and is a warning of undetected circulatory problems elsewhere in the body. Diabetes increases the risk of ED and the onset may be at a relatively young age. ED prevalence is expected to increase due to rising diabetes rates from obesity, population aging, and increased number of children diagnosed with type 2 diabetes. A wide variety of therapeutic options are available for the treatment of ED, including vacuum therapy, intra-cavernous and transurethral drug therapy, surgery and oral medications. Unfortunately, despite these options, ED persists in many patients. Viagra and other phosphodiesterase-5 (PDE5) inhibitors have been successful in the treatment of many forms of ED, but often, patients fail to benefit from use of these agents. This is particularly relevant to diabetic patients where it is estimated that as many as 40-60% do not respond to PDE5 inhibitors. Also, PDE5 inhibitors are often contraindicated in diabetic patients because they take nitrates for angina or myocardial infarction. Thus, there is a critical need to develop alternative therapeutic agents to treat ED in diabetic patients. Transient receptor potential (TRP) channels are typically Ca2+ permeable, polymodal and display variable gating mechanisms. They are expressed in a wide variety of cell types, where they are involved in many physiological functions. Thus, dysfunction of these channels can cause important acquired or inherited human diseases. The goal of this project is to investigate the role of thermosensitive TRPM8 channels in the internal pudendal arteries and corpus cavernosum, tissues that are critical for sexual function and known to undergo pathological changes due to the diabetic state. This research plan will also test the innovative hypothesis that in diabetes, a therapeutic approach utilizing agonists at TRPM8 will improve the erection in diabetes. Three specific aims are proposed: 1) to investigate the expression and mechanism of action of TRPM8 activation in the pudendal artery and corpus cavernosum from diabetic mice (db/db) and their nondiabetic controls; 2) to confirm the mechanism of action of TRPM8 activation in db/db mice using a knockdown strategy to reduce TRPM8 channel expression in tissues isolated form db/db mice and their controls; 3) to test the hypothesis that chronic administration of a TRPM8 agonist (menthol and icilin, osmotic minipump) in diabetic leads to the prevention and improvement of ED.