Final Report Summary - NRFDIABVASC (Role of Nrf2 transcription factor in diabetic vasculopathy, oxidative stress and inflammation)
Perhaps the most challenging aspect of this proposal in terms of achieving the goals will be a successful establishment of type 1 diabetes in the proposed animal model and to obtain indications of an altered vascular reactivity in these mice. After my arrival to the Universidad Autonoma de Madrid, it was much of a priority to be able to initially establish a good experimental model of diabetes. Thus, after the initial period of quarantine and the establishment of a good animal colony by serial crossing and confirmation of the genotype of the offspring at the animal facility at the university, several experimental tests were performed to achieve a good diabetic experimental model through the injection of streptozotocin (STZ) into mice. Nrf2 knockout mice do not show any apparent phenotype compared to littermate matched control mice unless challenged, which present an impairment of the antioxidant status, leading to more severe inflammation in these mice depending on the experimental model used. As an example in terms of the cardiovascular system that we are currently developing, Nrf2 deficiency did not caused any apparent structural defects and functional abnormalities in the heart and the hemodynamic parameters (e.g. blood pressure) remain normal compared to wild-type mice, suggesting that Nrf2 is not an essential mediator for normal cardiac development or functional integrity at non-stressed physiological conditions. But Nrf2 knockout mice display an impaired adaptative response to cardiac pressure overload when challenging Nrf2 knockout mice to transverse aortic constriction (TAC) and developed enhanced cardiac hypertrophy.
In the vasculature, Nrf2 gene transfer reduces neointima area in a wire-injury model of vascular lesion.
In our diabetic experimental model through the injection of streptozotocin (STZ) into mice, we chose to perform serial i.p injections to these animals to avoid an initial overdose of STZ on these animals which cause an increased mortality in Nrf2 knockout mice. Under these conditions, a successful diabetic animal model was achieved by monitoring blood glucose levels at a weekly basis after STZ injection compared to vehicle control (citrate buffer). Mice were considered diabetic with blood glucose concentrations in the upper threshold of 300 mg/dl for two consecutive weeks. To confirm the results, we evaluated glucose tolerance by performing an intraperitoneal glucose tolerance test (GTT). The plasma glucose levels during the GTT were clearly lower in mice injected with citrate buffer compared to STZ-mice. Moreover, the ability to metabolise glucose is severely impaired in STZ-mice compared to control citrate buffer mice. These results were indicative of a well-established STZ-triggered type 1 diabetic model in mice.
As stated in the description of the research methodology for aim 1 I initially investigated the mechanisms by which cytokines and glucose, both elevated in the circulation of diabetic patients, regulate antioxidant gene expression in vascular endothelial and smooth muscle cells. To analyse the importance of this approach, initial studies were performed to address the question whether Nrf2 expression were regulated in our experimental model for diabetes. Mice subjected to STZ or citrate, were allowed to develop high blood glucose levels as described above for 6-8 weeks. Aortic vessels were then isolated and mounted for immunofluorescence against Nrf2 antibody. Nrf2 expression increased both in the endothelial and smooth muscle cell layers in the aortas of STZ-versus citrate-mice. The effects of glucose and pro-inflammatory cytokines alone as well as their potential to synergise in the induction of antioxidant stress proteins were evaluated both in vivo and in vitro. First, Nrf2 knockout mice show a reduced antioxidant gene expression as determined by immunohistochemistry against heme oxygenase-1 (HO-1). And second, gain-and loss of Nrf2 function by means of adenoviral expression of Nrf2 and Keap-1 respectively, regulates antioxidant stress proteins such as HO-1 and superoxide dismutase (SOD) (data not shown) in endothelial cells.
In order to determine whether high glucose and/or pro-inflammatory cytokines play a role under these conditions we evaluatedthe expression of inducible nitric oxide synthase (iNOS) as a surrogate marker of inflammation. High glucose levels increased modestly the iNOS expression in endothelial cells in culture (HUVEC), whereas the pro-inflammatory cytokine interleukin-1 (IL-1) significantly increased (4-fold) iNOS expression. Both high glucose levels and pro-inflammatory stimuli synergistically increased iNOS expression. Of importance Nrf2 gene transfer markedly attenuated iNOS expression, indicating that Nrf2 may protect against a hyperglycemic and pro-inflammatory status in endothelial cells. These series of experiments were described in aim 1 of the proposal and are directed to define the regulation of antioxidant gene expression by glucose and pro-inflammatory cytokines in vitro.
Regarding aim 2 of the current proposal, we are in the process to systematically analyse the vascular reactivity to define whether Nrf2 plays a critical role in the vasculature by contributing to maintenance of vascular homeostasis and protecting vascular dysfunction upon hyperglycemia and pro-inflammatory signals as occurs in diabetes. We proposed to monitor both endothelial-dependent and endothelial-independent vasodilation. These experiments are currently in process and not absent of experimental challenges. For instance preservation of an intact endothelium while mounting the vessels is a critical step and requires an elevated experimental n number. On the other hand, preliminary data from isometric tension recordings on endothelium-independent vasodilation monitored by the administration of sodium nitroprusside (SNP) to preconstricted vessels with noradrenaline, might initially indicate a putative role of Nrf2 on the vascular tone in diabetic conditions.
Perspective
Overall, during this first year of we were able to establish the diabetic animal model and started to evaluate the vascular reactivity in this experimental model. In vitro studies already suggest an important role of Nrf2 and the endogenous antioxidant system in the vasculature of diabetic mice. We will further pursue together with Dr Carlos F. Sanchez-Ferrer in a collaborative manner the successfully complexion of this project. As mentioned in the accompanying letter, due to personal reason I am entitled to resign my contract at the Universidad Autonoma de Madrid, but this project is an important part of the analysis of the role of the antioxidant system in the cardiovascular function and will be continued. Part of these result are currently under revision for publication in Atherosclerosis, Thrombosis and Vascular Biology MS ID: ATVB/2009/189480, and the funding by the Marie Curie International Reintegration Grant has been appropriately acknowledged. In case any relevant outcome of this project is considered for publication in the future, it will be further acknowledge.
In the vasculature, Nrf2 gene transfer reduces neointima area in a wire-injury model of vascular lesion.
In our diabetic experimental model through the injection of streptozotocin (STZ) into mice, we chose to perform serial i.p injections to these animals to avoid an initial overdose of STZ on these animals which cause an increased mortality in Nrf2 knockout mice. Under these conditions, a successful diabetic animal model was achieved by monitoring blood glucose levels at a weekly basis after STZ injection compared to vehicle control (citrate buffer). Mice were considered diabetic with blood glucose concentrations in the upper threshold of 300 mg/dl for two consecutive weeks. To confirm the results, we evaluated glucose tolerance by performing an intraperitoneal glucose tolerance test (GTT). The plasma glucose levels during the GTT were clearly lower in mice injected with citrate buffer compared to STZ-mice. Moreover, the ability to metabolise glucose is severely impaired in STZ-mice compared to control citrate buffer mice. These results were indicative of a well-established STZ-triggered type 1 diabetic model in mice.
As stated in the description of the research methodology for aim 1 I initially investigated the mechanisms by which cytokines and glucose, both elevated in the circulation of diabetic patients, regulate antioxidant gene expression in vascular endothelial and smooth muscle cells. To analyse the importance of this approach, initial studies were performed to address the question whether Nrf2 expression were regulated in our experimental model for diabetes. Mice subjected to STZ or citrate, were allowed to develop high blood glucose levels as described above for 6-8 weeks. Aortic vessels were then isolated and mounted for immunofluorescence against Nrf2 antibody. Nrf2 expression increased both in the endothelial and smooth muscle cell layers in the aortas of STZ-versus citrate-mice. The effects of glucose and pro-inflammatory cytokines alone as well as their potential to synergise in the induction of antioxidant stress proteins were evaluated both in vivo and in vitro. First, Nrf2 knockout mice show a reduced antioxidant gene expression as determined by immunohistochemistry against heme oxygenase-1 (HO-1). And second, gain-and loss of Nrf2 function by means of adenoviral expression of Nrf2 and Keap-1 respectively, regulates antioxidant stress proteins such as HO-1 and superoxide dismutase (SOD) (data not shown) in endothelial cells.
In order to determine whether high glucose and/or pro-inflammatory cytokines play a role under these conditions we evaluatedthe expression of inducible nitric oxide synthase (iNOS) as a surrogate marker of inflammation. High glucose levels increased modestly the iNOS expression in endothelial cells in culture (HUVEC), whereas the pro-inflammatory cytokine interleukin-1 (IL-1) significantly increased (4-fold) iNOS expression. Both high glucose levels and pro-inflammatory stimuli synergistically increased iNOS expression. Of importance Nrf2 gene transfer markedly attenuated iNOS expression, indicating that Nrf2 may protect against a hyperglycemic and pro-inflammatory status in endothelial cells. These series of experiments were described in aim 1 of the proposal and are directed to define the regulation of antioxidant gene expression by glucose and pro-inflammatory cytokines in vitro.
Regarding aim 2 of the current proposal, we are in the process to systematically analyse the vascular reactivity to define whether Nrf2 plays a critical role in the vasculature by contributing to maintenance of vascular homeostasis and protecting vascular dysfunction upon hyperglycemia and pro-inflammatory signals as occurs in diabetes. We proposed to monitor both endothelial-dependent and endothelial-independent vasodilation. These experiments are currently in process and not absent of experimental challenges. For instance preservation of an intact endothelium while mounting the vessels is a critical step and requires an elevated experimental n number. On the other hand, preliminary data from isometric tension recordings on endothelium-independent vasodilation monitored by the administration of sodium nitroprusside (SNP) to preconstricted vessels with noradrenaline, might initially indicate a putative role of Nrf2 on the vascular tone in diabetic conditions.
Perspective
Overall, during this first year of we were able to establish the diabetic animal model and started to evaluate the vascular reactivity in this experimental model. In vitro studies already suggest an important role of Nrf2 and the endogenous antioxidant system in the vasculature of diabetic mice. We will further pursue together with Dr Carlos F. Sanchez-Ferrer in a collaborative manner the successfully complexion of this project. As mentioned in the accompanying letter, due to personal reason I am entitled to resign my contract at the Universidad Autonoma de Madrid, but this project is an important part of the analysis of the role of the antioxidant system in the cardiovascular function and will be continued. Part of these result are currently under revision for publication in Atherosclerosis, Thrombosis and Vascular Biology MS ID: ATVB/2009/189480, and the funding by the Marie Curie International Reintegration Grant has been appropriately acknowledged. In case any relevant outcome of this project is considered for publication in the future, it will be further acknowledge.
