Co‐expression of glycosylated aquaporin‐1 and transcription factor NFAT5 contributes to aortic stiffness in diabetic and atherosclerosis‐prone mice

Abstract Increased stiffness characterizes the early change in the arterial wall with subclinical atherosclerosis. Proteins inducing arterial stiffness in diabetes and hypercholesterolaemia are largely unknown. This study aimed at determining the pattern of protein expression in stiffening aorta of diabetic and hypercholesterolaemic mice. Male Ins2+/Akita mice were crossbred with ApoE−/− (Ins2+/Akita: ApoE−/−) mice. Relative aortic distension (relD) values were determined by ultrasound analysis and arterial stiffness modulators by immunoblotting. Compared with age‐ and sex‐matched C57/BL6 control mice, the aortas of Ins2+/Akita, ApoE−/− and Ins2+/Akita:ApoE−/− mice showed increased aortic stiffness. The aortas of Ins2+/Akita, ApoE−/− and Ins2+/Akita:ApoE−/− mice showed greater expression of VCAM‐1, collagen type III, NADPH oxidase and iNOS, as well as reduced elastin, with increased collagen type III‐to‐elastin ratio. The aorta of Ins2+/Akita and Ins2+/Akita:ApoE−/− mice showed higher expression of eNOS and cytoskeletal remodelling proteins, such as F‐actin and α‐smooth muscle actin, in addition to increased glycosylated aquaporin (AQP)‐1 and transcription factor NFAT5, which control the expression of genes activated by high glucose‐induced hyperosmotic stress. Diabetic and hypercholesterolaemic mice have increased aortic stiffness. The association of AQP1 and NFAT5 co‐expression with aortic stiffness in diabetes and hypercholesterolaemia may represent a novel molecular pathway or therapeutic target.


| BACKG ROU N D
Compared with non-diabetic individuals, diabetic patients suffer more aggressive, rapidly progressive atherosclerosis often with earlier complications, such as arterial stiffness. 1,2 This is because altered glucose metabolism in diabetes can modify and increase the impact of comorbidities, such as hypercholesterolaemia. 3 In these patients at high risk of cardiovascular events, the identification of clinical and molecular markers that allow an early diagnosis of atherosclerosis is essential. Arterial stiffening is also an established clinical marker of early subclinical atherosclerosis. 2,4,5 The arterial stiffness has been shown to be associated with hypertension 6 and kidney disease. 7 The underlying mechanisms may be attributed to (a) endothelial dysfunction leading to vasoconstriction; (b) oxidative stress and inflammatory factors; 8 and (c) vascular calcifications. 9 However, proteins that promote the arterial stiffening in diabetes and hypercholesterolaemia are less known. Pathogenetic mechanisms and biomarkers of early-stage vascular abnormalities, such as arterial stiffening, can be identified through animal models of diabetes and hypercholesterolaemia. 10 Cytoskeletal reorganization and arterial remodelling are adaptive responses to altered biomechanical stress. Reorganization of the vascular cytoskeleton is causally connected with arterial stiffening and contractile dysfunction. 11 It can be favoured by an increase in wall stress or biomechanical stretching caused by high blood pressure. 11 In diabetes, arterial remodelling can occur as the result of a chronic increase in wall stress or biomechanical stretching due to high glucose-induced hyperosmolar stress, often fluctuating in uncontrolled diabetes. We have previously shown that concentrations of glucose up to 30.5 mmol/L, attainable under hyperglycaemic conditions, induce an up-regulation of F-actin, α-smooth muscle actin (ASMA) and cytoskeletal remodelling in human-induced pluripotent stem (iPS) cells. 12 These effects appeared to be mediated through an aquaporin isoform 1 (AQP1)-and transcription factor nuclear factor of activated T cells 5 (NFAT5)-dependent hyperosmolar stress which, through this mechanism, promotes cell migration. 12 Thus, hyperosmolar stress can act as a most important biophysical factor that promotes cytoskeletal remodelling and the migration of vascular cells, such as vascular smooth muscle cells (VSMCs), within the arterial media. Directed migration of VSMCs requires a polarized reorganization of the cytoskeleton actin. From the six mammalian actin genes, the expression of VSMC-specific cytoskeleton actin ASMA appears to be regulated by both CArG promoter elements, such as the transcriptional coactivator myocardin, 13 and NFAT5. 14 NFAT5, a rel/NF-κB family member, is a transcription factor activated by hyperosmolar stress, which regulates the expression of osmosensing genes, including those involved in cell migration and cytoskeletal remodelling. 12,[15][16][17][18] In the present research, we sought to determine the impact of hypercholesterolaemia and diabetes on arterial wall stiffness in the aortas of genetic diabetes and hypercholesterolaemia mice models. We also analysed gene expression underlying arterial stiffening at the protein level, including the product of genes responsive to biomechanical stretch, such as high glucose-induced hyperosmolar stress. Understanding changes in protein expression and molecular circuits activated by concomitantly present risk factors, such as hypercholesterolaemia and diabetes, may indicate new targets for diagnostic and therapeutic strategies aimed at early atherosclerosis in high-risk patients.

| Materials
All chemicals were purchased from Sigma St Louis MO, unless otherwise specified.
Two-dimensional and M-mode echocardiographic images were recorded and analysed using a portable ultrasound apparatus (Esaote) equipped with a 21-MHz linear probe. Images were obtained in the parasternal long-axis view. Aortic diameter instantaneous values were derived from B-mode images and were recorded in late systole and late diastole using edge detection. 21 Mean diameter (Dm) and relative distension (relD) values (this latter considered as a surrogate marker for arterial stiffness) were evaluated from the obtained diameter waveforms; relD was calculated as (D s − D d )/D d and expressed as a percentage (where D s is the diameter in systole and D d the diameter in diastole). After measurements, mice were killed, and their aorta excised for protein extraction.

| Statistical analysis
Groups were compared by one-way analysis of variance followed by Scheffé's test for multiple comparisons. Statistical significance was defined as P < .05.

| Structural proteins
Collagen and elastin are the main structural proteins related to aortic stiffness. Abnormalities in the quantity and quality of collagen and elastin contribute to aortic stiffening. 23 The aortas of 3-month-old apoE −/− and Ins2 +/Akita mice had significantly higher levels of collagen type III ( Figure 2A) and lower levels of elastin ( Figure 2B), with increased collagen type III-to-elastin ratio ( Figure 2C) than non-diabetic, non-hypercholesterolaemic control mice. The combination of the two comorbidities further modified the expression of these proteins and their ratio, as levels of collagen type III and elastin were higher and lower in Ins2 +/Akita :apoE −/− mice compared with Ins 2+/Akita mice and apoE −/− mice, respectively (Figure 2).

| VCAM-1
An increased expression of pro-inflammatory cytokines and adhesion molecules has been suggested to contribute to collagen quality abnormalities, which can destabilize collagen fibres in the vascular wall. 24,25 While there were no apparent changes in the expression of monocyte chemotactic protein-1, intercellular adhesion molecule (ICAM-1), tumour necrosis factor α and interleukin-1 in the aortas of apoE −/− and Ins2 +/Akita mice ( Figure 3B-D), the expression of vascular cell adhesion molecule (VCAM)-1 was significantly higher in Ins2 +/ Akita mice and apoE −/− mice than in non-diabetic non-hypercholesterolaemic control mice ( Figure 3A). The combination of the two comorbidities further increased the expression of VCAM-1, as the levels of this early pro-inflammatory adhesion protein were higher in Ins2 +/Akita :apoE −/− mice compared with Ins 2+/Akita mice and apoE −/− mice ( Figure 3A).    Figure 5A) and F-actin ( Figure 5B). In parallel, the aorta of Ins 2+/Akita mice and Ins 2+/Akita : ApoE −/− mice showed a higher expression of glycosylated AQP1 ( Figure 6A) and NFAT5 ( Figure 6B). abnormalities in the quality of collagen fibres and a disarray of elastic fibres, due to elastolysis, which in turn impairs the elastic properties and the structural integrity of the aortic wall, leading to arterial stiffening. 33 Our results imply that diabetes and hypercholesterolaemia can both contribute to arterial stiffening at least in part through inflammation and increased oxidative stress in the aortic wall, leading to abnormalities in the quality of elastic and collagen fibres. These effects might be aggravated in ageing, although in our study we did not analyse the influence of ageing as a comorbidity.

| D ISCUSS I ON
Here, we provide the first evidence that NFAT5 and AQP1 are involved in the hypertonicity-related induction of arterial stiffening, since NFAT5 and AQP1 were increased in the aortas of diabetic Ins 2+/ Akita mice exposed to high glucose-induced hyperosmolar stress, independent of the presence of hypercholesterolaemia. AQP1 is a water channel protein expressed widely in vascular endothelia and smooth muscle cells, where it increases cell membrane water permeability, as well as cell motility and migration, and regulates cellular homeostasis during osmolarity changes. 12

| CON CLUS ION
Diabetic and hypercholesterolaemic Ins 2+/Akita : ApoE −/− mice develop aortic stiffening, associated with up-regulation of hypertonicityresponsive gene such as AQ1 and NFAT5, along with genes implicated in early inflammation and atherosclerosis, such as VCAM-1; cytoskeletal remodelling, such as F-actin and ASMA; endothelial dysfunction, such as iNOS and eNOS; and ROS generation such as NADPH oxidase. These changes in gene expression may be important mechanisms leading to the development of arterial stiffening in diabetes and hypercholesterolaemia.

CO N FLI C T O F I NTE R E S T
PF is the founder and CEO of Pharmahungary Group, a group of R&D companies.

DATA AVA I L A B I L I T Y S TAT E M E N T
Original data are available upon request from the journal.