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Macro- and microvascular complications of diabetes: Studies on NFAT (Nuclear Factor of Activated T-cells) as a novel target for the treatment of atherosclerosis and vascular dysfunction in diabetes.

Blanco, Fabiana LU (2018) In Lund University, Faculty of Medicine Doctoral Dissertation Series 2018(160).
Abstract
Diabetes is associated with devastating chronic complications including coronary heart disease and stroke (macrovascular complications) as well as microvascular disorders leading to damage of the small vessels and the development of retinopathy, nephropathy and neuropathy. The underlying pathogenesis is not clear, but hyperglycemia has been identified as an important risk factor. Previous work from our group demonstrated that hyperglycemia effectively activates the Ca2+/calcineurin transcription factor NFAT in macro and microvessels, both ex vivo and in vivo. Once activated, NFAT enhances vascular smooth muscle cell (VSMC) excitability and vasoconstriction and promotes the expression of inflammatory markers in the arterial wall. This... (More)
Diabetes is associated with devastating chronic complications including coronary heart disease and stroke (macrovascular complications) as well as microvascular disorders leading to damage of the small vessels and the development of retinopathy, nephropathy and neuropathy. The underlying pathogenesis is not clear, but hyperglycemia has been identified as an important risk factor. Previous work from our group demonstrated that hyperglycemia effectively activates the Ca2+/calcineurin transcription factor NFAT in macro and microvessels, both ex vivo and in vivo. Once activated, NFAT enhances vascular smooth muscle cell (VSMC) excitability and vasoconstriction and promotes the expression of inflammatory markers in the arterial wall. This thesis focuses on elucidating the role of NFAT in the context of diabetes-induced atherosclerosis and retinopathy. In paper I, we showed that in vivo inhibition of NFAT signalling with the NFAT blocker A-285222 for 4 weeks completely suppressed the accelerated atherosclerosis in the aortic arch of type 1 diabetic ApoE-/- mice, having no effect in non-diabetic mice. This effect was independent of changes in plasma glucose or lipid levels and was not due to systemic immunosuppression. Inhibition of NFAT resulted in reduced lipid contents in the plaques of diabetic mice, reduced expression of interleukin 6 (IL-6), osteopontin (OPN), monocyte chemotactic protein-1 (MCP-1), intracellular adhesion molecule 1 (ICAM-1), CD68 (macrophage marker) and tissue factor (TF) in the arterial wall, as well as in lowered plasma IL-6. Further, in paper II, we showed that in vivo treatment with A-285222 reduced atherosclerotic plaque area and degree of stenosis in the brachiocephalic artery of IGF-II/LDLR–/–ApoB100/100 mice. This is a mouse model characterized by mild hyperglycaemia and hyperinsulinaemia, a human-like hypercholesterolaemic lipid profile, and advanced and complex atherosclerotic lesions, hence replicating features of human type 2 diabetes disease. The effects of NFAT inhibition on atherosclerosis in this model were not only due to limited plaque progression but also to plaque regression, as assessed by comparing plaque size non-invasively using ultrasound biomicroscopy prior and after treatment with A-285222. Treatment had no impact on plaque composition and the effects on plaque size could not be explained by effects of A-285222 on plasma glucose, insulin or lipids. Interestingly, NFAT inhibition resulted in increased expression of the atherosprotective NADPH oxidase 4 (NOX4) and of the anti-oxidant enzyme catalase in aortic VSMCs. In paper III, we demonstrated that NFAT is expressed in the endothelium of retinal microvessels and is readily activated by both acute and chronic hyperglycemia. Activation seemed mediated by a mechanism involving the release of extracelllar nucleotides. In both Akita (Ins2+/−) and streptozotocin- (STZ-) induced diabetic mice, NFAT transcriptional activity was elevated in retinal vessels. In vivo inhibition of NFAT with A-285222 decreased the expression of OPN and ICAM-1 mRNA in retinal vessels, prevented a diabetes driven down-regulation of anti-inflammatory IL-10 in retina, and limited the increased vascular permeability observed in diabetic mice. Finally, in paper IV, we performed a systematic mapping of the pattern of NFAT isoform expression in 13 different regions of the mouse vascular tree using absolute qPCR quantification. We found that NFATc3 is the most predominant isoform in all vessels examined and that NFATc2 expresion was increased in the aortic wall of diabetic mice, suggesting that this isoform can be induced or enhanced in pathological situations or under certain stimulatory conditions. We also found that genetic deletion of NFATc2 or NFATc3 differentially affected the expression of Kruppel-like factor 4 (Klf4), Kruppel-like factor 5 (Klf5) and Gata4, genes that have been implicated in the regulation of VSMC phenotypic modulation. Moreover, deletion of NFATc3 resulted in increased expression of angiotensin I converting enzyme (peptidyl-dipeptidase A) 1 (Ace) and of smooth muscle marker genes calponin 1 (Cnn1) and transgelin (Tagln), as well as in higher VSMC proliferation. In summary, NFAT seems to play a role in the development of diabetic-driven atherosclerosis and retinopathy, and targeting the NFAT signalling pathway may be an attractive approach for the treatment of diabetic complications. The differential isoform expression and effects observed upon NFATc2 and NFATc3 deletion support the idea of functional non-redundancy of NFAT isoforms in the vasculature. (Less)
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author
supervisor
opponent
  • professor Perez Garcia, Maria Teresa, Valladolid
organization
publishing date
type
Thesis
publication status
published
subject
keywords
NFAT, VSMCs, Diabetes, Hyperglycemia, Aterosclerosis, Diabetic retinopathy, Oxidative stress
in
Lund University, Faculty of Medicine Doctoral Dissertation Series
volume
2018
issue
160
pages
125 pages
publisher
Lund University: Faculty of Medicine
defense location
CRC, 28-11-026, Jan Waldenströms gata 35, Skånes Universitetssjukhud i Malmö.
defense date
2018-12-06 09:30:00
ISSN
1652-8220
ISBN
978-91-7619-729-5
language
English
LU publication?
yes
id
23b29127-a3a2-4024-8cc5-7271c8121545
alternative location
https://lu.box.com/s/sjrthxgzusy047nl6wq7cc16vd2ksu6c
date added to LUP
2018-11-15 15:27:24
date last changed
2019-11-19 13:49:43
@phdthesis{23b29127-a3a2-4024-8cc5-7271c8121545,
  abstract     = {Diabetes is associated with devastating chronic complications including coronary heart disease and stroke (macrovascular complications) as well as microvascular disorders leading to damage of the small vessels and the development of retinopathy, nephropathy and neuropathy. The underlying pathogenesis is not clear, but hyperglycemia has been identified as an important risk factor. Previous work from our group demonstrated that hyperglycemia effectively activates the Ca2+/calcineurin transcription factor NFAT in macro and microvessels, both ex vivo and in vivo. Once activated, NFAT enhances vascular smooth muscle cell (VSMC) excitability and vasoconstriction and promotes the expression of inflammatory markers in the arterial wall. This thesis focuses on elucidating the role of NFAT in the context of diabetes-induced atherosclerosis and retinopathy. In paper I, we showed that in vivo inhibition of NFAT signalling with the NFAT blocker A-285222 for 4 weeks completely suppressed the accelerated atherosclerosis in the aortic arch of type 1 diabetic ApoE-/- mice, having no effect in non-diabetic mice. This effect was independent of changes in plasma glucose or lipid levels and was not due to systemic immunosuppression. Inhibition of NFAT resulted in reduced lipid contents in the plaques of diabetic mice, reduced expression of interleukin 6 (IL-6), osteopontin (OPN), monocyte chemotactic protein-1 (MCP-1), intracellular adhesion molecule 1 (ICAM-1), CD68 (macrophage marker) and tissue factor (TF) in the arterial wall, as well as in lowered plasma IL-6. Further, in paper II, we showed that in vivo treatment with A-285222 reduced atherosclerotic plaque area and degree of stenosis in the brachiocephalic artery of IGF-II/LDLR–/–ApoB100/100 mice. This is a mouse model characterized by mild hyperglycaemia and hyperinsulinaemia, a human-like hypercholesterolaemic lipid profile, and advanced and complex atherosclerotic lesions, hence replicating features of human type 2 diabetes disease. The effects of NFAT inhibition on atherosclerosis in this model were not only due to limited plaque progression but also to plaque regression, as assessed by comparing plaque size non-invasively using ultrasound biomicroscopy prior and after treatment with A-285222. Treatment had no impact on plaque composition and the effects on plaque size could not be explained by effects of A-285222 on plasma glucose, insulin or lipids. Interestingly, NFAT inhibition resulted in increased expression of the atherosprotective NADPH oxidase 4 (NOX4) and of the anti-oxidant enzyme catalase in aortic VSMCs. In paper III, we demonstrated that NFAT is expressed in the endothelium of retinal microvessels and is readily activated by both acute and chronic hyperglycemia. Activation seemed mediated by a mechanism involving the release of extracelllar nucleotides. In both Akita (Ins2+/−) and streptozotocin- (STZ-) induced diabetic mice, NFAT transcriptional activity was elevated in retinal vessels. In vivo inhibition of NFAT with A-285222 decreased the expression of OPN and ICAM-1 mRNA in retinal vessels, prevented a diabetes driven down-regulation of anti-inflammatory IL-10 in retina, and limited the increased vascular permeability observed in diabetic mice. Finally, in paper IV, we performed a systematic mapping of the pattern of NFAT isoform expression in 13 different regions of the mouse vascular tree using absolute qPCR quantification. We found that NFATc3 is the most predominant isoform in all vessels examined and that NFATc2 expresion was increased in the aortic wall of diabetic mice, suggesting that this isoform can be induced or enhanced in pathological situations or under certain stimulatory conditions. We also found that genetic deletion of NFATc2 or NFATc3 differentially affected the expression of Kruppel-like factor 4 (Klf4), Kruppel-like factor 5 (Klf5) and Gata4, genes that have been implicated in the regulation of VSMC phenotypic modulation. Moreover, deletion of NFATc3 resulted in increased expression of angiotensin I converting enzyme (peptidyl-dipeptidase A) 1 (Ace) and of smooth muscle marker genes calponin 1 (Cnn1) and transgelin (Tagln), as well as in higher VSMC proliferation. In summary, NFAT seems to play a role in the development of diabetic-driven atherosclerosis and retinopathy, and targeting the NFAT signalling pathway may be an attractive approach for the treatment of diabetic complications. The differential isoform expression and effects observed upon NFATc2 and NFATc3 deletion support the idea of functional non-redundancy of NFAT isoforms in the vasculature.},
  author       = {Blanco, Fabiana},
  isbn         = {978-91-7619-729-5},
  issn         = {1652-8220},
  language     = {eng},
  number       = {160},
  publisher    = {Lund University: Faculty of Medicine},
  school       = {Lund University},
  series       = {Lund University, Faculty of Medicine Doctoral Dissertation Series},
  title        = {Macro- and microvascular complications of diabetes: Studies on NFAT (Nuclear Factor of Activated T-cells) as a novel target for the treatment of atherosclerosis and vascular dysfunction in diabetes.},
  url          = {https://lu.box.com/s/sjrthxgzusy047nl6wq7cc16vd2ksu6c},
  volume       = {2018},
  year         = {2018},
}