Annayya R. Aroor

Background: Excess dietary salt increases vascular stiffness in humans, especially in salt- sensitive populations. While we recently suggested that the endothelial sodium channel (EnNaC) contributes to salt-sensitivity related endothelial cell (EC) and arterial stiffening, mechanistic understanding is incomplete. This study thus aimed to explore the role of EC-serum and glucocorticoid regulated kinase 1 (SGK1), as a regulator of sodium channels, in EC and arterial stiffening. Methods and Results: A mouse model of salt sensitivity-associated vascular stiffening was produced by subcutaneous implantation of slow-release deoxycorticosterone acetate (DOCA) pellets, with salt (1% NaCl, 0.2% KCl) administered via drinking water. Preliminary data showed that global SGK1 deletion caused significantly decreased blood pressure, EnNaC activity and aortic endothelium stiffness as compared to control mice following DOCA-salt treatment. To probe EC signaling pathways, selective deletion of EC-SGK1 was performed by cross-breeding cadherin 5-Cre mice with sgk1 ^flox/flox mice. DOCA-salt treated control mice had significantly increased blood pressure, EC and aortic stiffness in vivo and ex vivo, which were attenuated by EC-SGK1 deficiency. To demonstrate relevance to humans, human aortic ECs were cultured in the absence or presence of aldosterone and high salt with or without the SGK1 inhibitor, EMD638683 (10uM or 25uM). Treatment with aldosterone and high salt increased intrinsic stiffness of ECs, which was prevented by SGK1 inhibition. Further, the SGK1 inhibitor prevented aldosterone and high salt induced actin polymerization, a key mechanism in cellular stiffening. Conclusion: EC-SGK1 mediates salt-sensitivity related EC and aortic stiffening by mechanisms appearing to involve regulation actin polymerization. Graphical

Obesity is characterized by enhanced MR (mineralocorticoid receptor) activation, vascular stiffness, and associated cardiovascular and kidney disease. Consumption of a Western-style diet (WD), high in saturated fat and refined carbohydrates, by female mice, leads to obesity and vascular stiffening. Use of ECMR (endothelial cell–specific MR) knockout mice supports that ECMR activation is critical for development of vascular and cardiac fibrosis and stiffening. However, the role of ECMR activation in kidney inflammation and fibrosis remains unknown. We hypothesized that cell-specific deletion of ECMR would prevent WD-induced central aortic stiffness and protect the kidney from endothelial dysfunction and vascular stiffening. Four-week-old female ECMR KO and wild-type mice were fed either mouse chow or WD for 16 weeks. WD feeding increased body weight and fat mass, proteinuria, as well as vascular stiffness indices (pulse wave velocity and kidney artery stiffening) and impaired endothelial-dependent vasodilatation without blood pressure changes. The WD-induced kidney arterial stiffening was associated with attenuated eNOS (endothelial NO synthase) activation, increased oxidative stress, proinflammatory immune responses, alterations in extracellular matrix degradation pathways, and fibrosis. ECMR deletion prevented these abnormalities by improving eNOS activation and reducing macrophage proinflammatory M1 polarization, expression of TG2 (transglutaminase 2), and MMP (matrix metalloproteinase)-9. Our data support the concept that ECMR activation contributes to endothelial dysfunction, increased kidney artery fibrosis/stiffening, and impaired NOS (NO synthase) activation, processes associated with macrophage infiltration and polarization, inflammation, and oxidative stress, collectively resulting in tubulointerstitial fibrosis in females consuming a WD.

Type 2 diabetes is associated with diabetic cognopathy. Anti-hyperglycemic sodium glucose transporter 2 (SGLT2) inhibitors have shown promise in reducing cognitive impairment in mice with type 2 diabetes mellitus. We recently described marked ultrastructural (US) remodeling of the neurovascular unit (NVU) in type 2 diabetic db/db female mice. Herein, we tested whether the SGLT-2 inhibitor, empagliflozin (EMPA), protects the NVU from abnormal remodeling in cortical gray and subcortical white matter. Ten-week-old female wild-type and db/db mice were divided into lean controls (CKC, n = 3), untreated db/db (DBC, n = 3), and EMPA-treated db/db (DBE, n = 3). Empagliflozin was added to mouse chow to deliver 10 mg kg⁻¹ day⁻¹ and fed for ten weeks, initiated at 10 weeks of age. Brains from 20-week-old mice were immediately immersion fixed for transmission electron microscopic study. Compared to CKC, DBC exhibited US abnormalities characterized by mural endothelial cell tight and adherens junction attenuation and/or loss, pericyte attenuation and/or loss, basement membrane thickening, glia astrocyte activation with detachment and retraction from mural cells, microglia cell activation with aberrant mitochondria, and oligodendrocyte–myelin splitting, disarray, and axonal collapse. We conclude that these abnormalities in the NVU were prevented in DBE. Empagliflozin may provide neuroprotection in the diabetic brain.

Obesity, insulin resistance, and type 2 diabetes mellitus are associated with diabetic cognopathy. In this study, we tested the hypothesis that neurovascular unit(s) (NVU), oligodendrocytes, and myelin within cerebral cortical grey matter and deeper transitional zone regions between the cortical grey matter and white matter may be abnormal. The monogenic (Lepr^db) female diabetic db/db [BKS.CgDock7^m +/+ Lepr^db/J] (DBC) mouse model was utilized for this ultrastructural study. Upon sacrifice (20 weeks of age), left-brain hemispheres of the DBC and age-matched non-diabetic wild type control C57BL/KsJ (CKC) mice were immediately immersion-fixed. We found prominent remodeling of oligodendrocytes with increased nuclear chromatin condensation and volume and increased numbers of active myelination sites of the cytoplasm in transition zones. Marked dysmyelination with outer myelin lamellae sheath splitting, separation, and ballooning with aberrant mitochondria in grey matter and similar myelin remodeling changes with marked disarray with additional axonal collapse in transitional zones in DBC as compared to CKC models.

Obesity, insulin resistance, and type 2 diabetes mellitus are associated with diabetic cognopathy. This study tested the hypothesis that neurovascular unit(s) (NVU) within cerebral cortical gray matter regions may depict abnormal cellular remodeling. The monogenic (Lepr^db) female diabetic db/db [BKS.CgDock7^m +/+Lepr^db/J] (DBC) mouse model was utilized for this ultrastructural study. Upon sacrifice (20 weeks), left-brain hemispheres of the DBC and age-matched nondiabetic control C57BL/KsJ (CKC) mice were immediately immersion-fixed. We observed an attenuation/loss of endothelial blood–brain barrier tight/adherens junctions and pericytes, thickened basement membranes, adherent red and white blood cells, neurovascular unit microbleeds and pathologic remodeling of protoplasmic astrocytes. In this second of a three-part series, we focus on the observational ultrastructural remodeling of microglia and mitochondria in relation to the NVU in leptin receptor deficient DBC models. This study identified novel ultrastructural core signature remodeling changes, which consisted of invasive activated microglia, microglial aberrant mitochondria with nuclear chromatin condensation and adhesion of white blood cells to an activated endothelium of the NVU. In conclusion, the results implicate activated microglia in NVU uncoupling and the resulting ischemic neuronal and synaptic damage, which may be related to impaired cognition and diabetic cognopathy.

Obesity, insulin resistance, and type 2 diabetes mellitus are associated with cognitive impairment, known as diabetic cognopathy. In this study, we tested the hypothesis that neurovascular unit(s) (NVU) within cerebral cortical gray matter regions display abnormal cellular remodeling. The monogenic (Lepr^db) female diabetic db/db (BKS.CgDock7^m +/+Lepr^db/J; DBC) mouse model was utilized for this ultrastructural study. Upon sacrifice (at 20 weeks of age), left-brain hemispheres of the DBC and age-matched non-diabetic wild-type control C57BL/KsJ (CKC) mice were immediately immersion-fixed. We found attenuation/loss of endothelial blood–brain barrier tight/adherens junctions and pericytes, thickening of the basement membrane, aberrant mitochondria, and pathological remodeling of protoplasmic astrocytes. Additionally, there were adherent red blood cells and NVU microbleeds (cortical layer III) in DBC mice, which were not observed in CKC animals. While this study represents only a “snapshot in time”, it does allow for cellular remodeling comparisons between DBC and CKC. In this paper, the first of a three-part series, we report the observational ultrastructural remodeling changes of the NVU and its protoplasmic astrocytes in relation to the surrounding neuropil. Having identified multiple abnormal cellular remodeling changes in the DBC as compared to CKC models, we will design future experiments to evaluate various treatment modalities in DBC mice.

Obese and diabetic individuals are at increased risk for impairments in diastolic relaxation and heart failure with preserved ejection fraction. The impairments in diastolic relaxation are especially pronounced in obese and diabetic women and predict future cardiovascular disease (CVD) events in this population. Recent clinical data suggest sodium glucose transporter-2 (SGLT2) inhibition reduces CVD events in diabetic individuals, but the mechanisms of this CVD protection are unknown. To determine whether targeting SGLT2 improves diastolic relaxation, we utilized empagliflozin (EMPA) in female db/db mice. Eleven week old female db/db mice were fed normal mouse chow, with or without EMPA, for 5 weeks. Blood pressure (BP), HbA1c and fasting glucose were significantly increased in untreated db/db mice (DbC) (P < 0.01). EMPA treatment (DbE) improved glycemic indices (P < 0.05), but not BP (P > 0.05). At baseline, DbC and DbE had already established impaired diastolic relaxation as indicated by impaired septal wall motion (>tissue Doppler derived E′/A′ ratio) and increased left ventricular (LV) filling pressure (<E/E′ ratio). Although these abnormalities persisted throughout the study period in DbC, diastolic function improved with EMPA treatment. In DbC, myocardial fibrosis was accompanied by increased expression of profibrotic/prohypertrophic proteins, serum/glucocorticoid regulated kinase 1 (SGK1) and the epithelial sodium channel (ENaC), and the development of these abnormalities were reduced with EMPA. DbC exhibited eccentric LV hypertrophy that was slightly improved by EMPA, indicated by a reduction in cardiomyocyte cross sectional area. In summary, EMPA improved glycemic indices along with diastolic relaxation, as well as SGK1/ENaC profibrosis signaling and associated interstitial fibrosis, all of which occurred in the absence of any changes in BP.