Integrated miRNA-mRNA networks underlie attenuation of chronic ß-adrenergic stimulation-induced cardiac remodeling by minocycline.

Adverse cardiac remodeling contributes to heart failure development and progression, partly due to inappropriate sympathetic nervous system activation. Although ß-adrenergic receptor (ß-AR) blockade is a common heart failure therapy, not all patients respond, prompting exploration of alternative treatments. Minocycline, an FDA-approved antibiotic, has pleiotropic properties beyond antimicrobial action. Recent evidence suggests it may alter gene expression via changes in miRNA expression. Thus, we hypothesized that minocycline could prevent adverse cardiac remodeling induced by the ß-AR agonist isoproterenol, involving miRNA-mRNA transcriptome alterations. Male C57BL/6J mice received isoproterenol (30 mg/kg/day sc) or vehicle via osmotic minipump for 21 days, along with daily minocycline (50 mg/kg ip) or sterile saline. Isoproterenol induced cardiac hypertrophy without altering cardiac function, which minocycline prevented. Total mRNA sequencing revealed isoproterenol altering gene networks associated with inflammation and metabolism, with fibrosis activation predicted by integrated miRNA-mRNA sequencing, involving miR-21, miR-30a, miR-34a, miR-92a, and miR-150, among others. Conversely, the cardiac miRNA-mRNA transcriptome predicted fibrosis inhibition in minocycline-treated mice, involving antifibrotic shifts in Atf3 and Itgb6 gene expression associated with miR-194 upregulation. Picrosirius red staining confirmed isoproterenol-induced cardiac fibrosis, prevented by minocycline. These results demonstrate minocycline's therapeutic potential in attenuating adverse cardiac remodeling through miRNA-mRNA-dependent mechanisms, especially in reducing cardiac fibrosis. NEW & NOTEWORTHY We demonstrate that minocycline treatment prevents cardiac hypertrophy and fibrotic remodeling induced by chronic ß-adrenergic stimulation by inducing antifibrotic shifts in the cardiac miRNA-mRNA transcriptome.

Multi-omic analysis of the cardiac cellulome defines a vascular contribution to cardiac diastolic dysfunction in obese female mice.

Coronary microvascular dysfunction (CMD) is associated with cardiac dysfunction and predictive of cardiac mortality in obesity, especially in females. Clinical data further support that CMD associates with development of heart failure with preserved ejection fraction and that mineralocorticoid receptor (MR) antagonism may be more efficacious in obese female, versus male, HFpEF patients. Accordingly, we examined the impact of smooth muscle cell (SMC)-specific MR deletion on obesity-associated coronary and cardiac diastolic dysfunction in female mice. Obesity was induced in female mice via western diet (WD) feeding alongside littermates fed standard diet. Global MR blockade with spironolactone prevented coronary and cardiac dysfunction in obese females and specific deletion of SMC-MR was sufficient to prevent obesity-associated coronary and cardiac diastolic dysfunction. Cardiac gene expression profiling suggested reduced cardiac inflammation in WD-fed mice with SMC-MR deletion independent of blood pressure, aortic stiffening, and cardiac hypertrophy. Further mechanistic studies utilizing single-cell RNA sequencing of non-cardiomyocyte cell populations revealed novel impacts of SMC-MR deletion on the cardiac cellulome in obese mice. Specifically, WD feeding induced inflammatory gene signatures in non-myocyte populations including B/T cells, macrophages, and endothelium as well as increased coronary VCAM-1 protein expression, independent of cardiac fibrosis, that was prevented by SMC-MR deletion. Further, SMC-MR deletion induced a basal reduction in cardiac mast cells and prevented WD-induced cardiac pro-inflammatory chemokine expression and leukocyte recruitment. These data reveal a central role for SMC-MR signaling in obesity-associated coronary and cardiac dysfunction, thus supporting the emerging paradigm of a vascular origin of cardiac dysfunction in obesity.

Endothelial cell-specific mineralocorticoid receptor activation promotes diastolic dysfunction in diet-induced obese male mice.

Widespread consumption of diets high in fat and fructose (Western diet, WD) has led to increased prevalence of obesity and diastolic dysfunction (DD). DD is a prominent feature of heart failure with preserved ejection fraction (HFpEF). However, the underlying mechanisms of DD are poorly understood, and treatment options are still limited. We have previously shown that deletion of the cell-specific mineralocorticoid receptor in endothelial cells (ECMR) abrogates DD induced by WD feeding in female mice. However, the specific role of ECMR activation in the pathogenesis of DD in male mice has not been clarified. Therefore, we fed 4-wk-old ECMR knockout (ECMRKO) male mice and littermates (LM) with either a WD or chow diet (CD) for 16 wk. WD feeding resulted in DD characterized by increased left ventricle (LV) filling pressure (E/e') and diastolic stiffness [E/e'/LV inner diameter at end diastole (LVIDd)]. Compared with CD, WD in LM resulted in increased myocardial macrophage infiltration, oxidative stress, and increased myocardial phosphorylation of Akt, in concert with decreased phospholamban phosphorylation. WD also resulted in focal cardiomyocyte remodeling, characterized by areas of sarcomeric disorganization, loss of mitochondrial electron density, and mitochondrial fragmentation. Conversely, WD-induced DD and associated biochemical and structural abnormalities were prevented by ECMR deletion. In contrast with our previously reported observations in females, WD-fed male mice exhibited enhanced Akt signaling and a lower magnitude of cardiac injury. Collectively, our data support a critical role for ECMR in obesity-induced DD and suggest critical mechanistic differences in the genesis of DD between males and females.

Empagliflozin Reverses Oxidized LDL-Induced RECK Suppression, Cardiotrophin-1 Expression, MMP Activation, and Human Aortic Smooth Muscle Cell Proliferation and Migration.

Persistent oxidative stress and inflammation contribute causally to smooth muscle cell (SMC) proliferation and migration, the characteristic features of vascular proliferative diseases. Oxidatively modified low-density lipoproteins (OxLDL) elevate oxidative stress levels, inflammatory responses, and matrix metallopeptidase (MMP) activation, resulting ultimately in SMC migration, proliferation, and phenotype change. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a membrane-anchored MMP inhibitor. Empagliflozin is an SGLT2 inhibitor and exerts pleiotropic cardiovascular protective effects, including antioxidant and anti-inflammatory effects. Here, we investigated (i) whether OxLDL regulates RECK expression, (ii) whether ectopic expression of RECK reverses OxLDL-induced SMC migration and proliferation, and (iii) whether pretreatment with empagliflozin reverses OxLDL-induced RECK suppression, MMP activation, and SMC migration, proliferation, and differentiation. Indeed, results show that OxLDL at pathophysiological concentration promotes SMC migration and proliferation via NF-κB/miR-30b-dependent RECK suppression. Moreover, OxLDL changed the SMC phenotype to a more pro-inflammatory type, and this effect is blunted by RECK overexpression. Further, treatment with empagliflozin reversed OxLDL-induced miR-30b induction, RECK suppression, MMP activation, SMC migration, proliferation, and proinflammatory phenotype changes. OxLDL-induced cardiotrophin (CT)-1 expression and CT-1 stimulated SMC proliferation and migration in part via leukemia inhibitory factor receptor (LIFR) and glycoprotein 130 (gp130). Ectopic expression of RECK inhibited these effects by physically associating with LIFR and gp130, as evidenced by immunoprecipitation/immunoblotting and double immunofluorescence. Importantly, empagliflozin inhibited CT-1-induced mitogenic and migratory effects. Together, these results suggest the therapeutic potential of sustaining RECK expression or empagliflozin in vascular diseases characterized by SMC proliferation and migration.

Inhibition of sphingomyelinase attenuates diet - Induced increases in aortic stiffness.

Sphingomyelinases ensure ceramide production and play an integral role in cell turnover, inward budding of vesicles and outward release of exosomes. Recent data indicate a unique role for neutral sphingomyelinase (nSMase) in the control of ceramide-dependent exosome release and inflammatory pathways. Further, while inhibition of nSMase in vascular tissue attenuates the progression of atherosclerosis, little is known regarding its role on metabolic signaling and arterial vasomotor function. Accordingly, we hypothesized that nSMase inhibition with GW4869, would attenuate Western diet (WD) - induced increases in aortic stiffness through alterations in pathways which lead to oxidative stress, inflammation and vascular remodeling. Six week-old female C57BL/6L mice were fed either a WD containing excess fat (46%) and fructose (17.5%) for 16 weeks or a standard chow diet (CD). Mice were variably treated with GW4869 (2.0 µg/g body weight, intraperitoneal injection every 48 h for 12 weeks). WD feeding increased nSMase2 expression and activation while causing aortic stiffening and impaired vasorelaxation as determined by pulse wave velocity (PWV) and wire myography, respectively. Moreover, these functional abnormalities were associated with aortic remodeling and attenuated AMP-activated protein kinase, Sirtuin 1, and endothelial nitric oxide synthase activation. GW4869 treatment prevented the WD-induced increases in nSMase activation, PWV, and impaired endothelium dependent/independent vascular relaxation. GW4869 also inhibited WD-induced aortic CD36 expression, lipid accumulation, oxidative stress, inflammatory responses, as well as aortic remodeling. These findings indicate that targeting nSMase prevents diet - induced aortic stiffening and impaired vascular relaxation by attenuating oxidative stress, inflammation and adverse vascular remodeling.

Cystamine reduces vascular stiffness in Western diet-fed female mice.

Consumption of diets high in fat, sugar, and salt (Western diet, WD) is associated with accelerated arterial stiffening, a major independent risk factor for cardiovascular disease (CVD). Women with obesity are more prone to develop arterial stiffening leading to more frequent and severe CVD compared with men. As tissue transglutaminase (TG2) has been implicated in vascular stiffening, our goal herein was to determine the efficacy of cystamine, a nonspecific TG2 inhibitor, at reducing vascular stiffness in female mice chronically fed a WD. Three experimental groups of female mice were created. One was fed regular chow diet (CD) for 43 wk starting at 4 wk of age. The second was fed a WD for the same 43 wk, whereas a third cohort was fed WD, but also received cystamine (216 mg/kg/day) in the drinking water during the last 8 wk on the diet (WD + C). All vascular stiffness parameters assessed, including aortic pulse wave velocity and the incremental modulus of elasticity of isolated femoral and mesenteric arteries, were significantly increased in WD- versus CD-fed mice, and reduced in WD + C versus WD-fed mice. These changes coincided with respectively augmented and diminished vascular wall collagen and F-actin content, with no associated effect in blood pressure. In cultured human vascular smooth muscle cells, cystamine reduced TG2 activity, F-actin:G-actin ratio, collagen compaction capacity, and cellular stiffness. We conclude that cystamine treatment represents an effective approach to reduce vascular stiffness in female mice in the setting of WD consumption, likely because of its TG2 inhibitory capacity.NEW & NOTEWORTHY This study evaluates the novel role of transglutaminase 2 (TG2) inhibition to directly treat vascular stiffness. Our data demonstrate that cystamine, a nonspecific TG2 inhibitor, improves vascular stiffness induced by a diet rich in fat, fructose, and salt. This research suggests that TG2 inhibition might bear therapeutic potential to reduce the disproportionate burden of cardiovascular disease in females in conditions of chronic overnutrition.

Sacubitril/valsartan inhibits obesity-associated diastolic dysfunction through suppression of ventricular-vascular stiffness.

OBJECTIVE: Cardiac diastolic dysfunction (DD) and arterial stiffness are early manifestations of obesity-associated prediabetes, and both serve as risk factors for the development of heart failure with preserved ejection fraction (HFpEF). Since the incidence of DD and arterial stiffness are increasing worldwide due to exponential growth in obesity, an effective treatment is urgently needed to blunt their development and progression. Here we investigated whether the combination of an inhibitor of neprilysin (sacubitril), a natriuretic peptide-degrading enzyme, and an angiotensin II type 1 receptor blocker (valsartan), suppresses DD and arterial stiffness in an animal model of prediabetes more effectively than valsartan monotherapy. METHODS: Sixteen-week-old male Zucker Obese rats (ZO; n = 64) were assigned randomly to 4 different groups: Group 1: saline control (ZOC); Group 2: sacubitril/valsartan (sac/val; 68 mg•kg-1•day-1; ZOSV); Group 3: valsartan (31 mg•kg-1•day-1; ZOV) and Group 4: hydralazine, an anti-hypertensive drug (30 mg•kg-1•day-1; ZOH). Six Zucker Lean (ZL) rats that received saline only (Group 5) served as lean controls (ZLC). Drugs were administered daily for 10 weeks by oral gavage. RESULTS: Sac/val improved echocardiographic parameters of impaired left ventricular (LV) stiffness in untreated ZO rats, without altering the amount of food consumed or body weight gained. In addition to improving DD, sac/val decreased aortic stiffness and reversed impairment in nitric oxide-induced vascular relaxation in ZO rats. However, sac/val had no impact on LV hypertrophy. Notably, sac/val was more effective than val in ameliorating DD. Although, hydralazine was as effective as sac/val in improving these parameters, it adversely affected LV mass index. Further, cytokine array revealed distinct effects of sac/val, including marked suppression of Notch-1 by both valsartan and sac/val, suggesting that cardiovascular protection afforded by both share some common mechanisms; however, sac/val, but not val, increased IL-4, which is increasingly recognized for its cardiovascular protection, possibly contributing, in part, to more favorable effects of sac/val over val alone in improving obesity-associated DD. CONCLUSIONS: These studies suggest that sac/val is superior to val in reversing obesity-associated DD. It is an effective drug combination to blunt progression of asymptomatic DD and vascular stiffness to HFpEF development in a preclinical model of obesity-associated prediabetes.

Western diet induces renal artery endothelial stiffening that is dependent on the epithelial Na+ channel.

Consumption of a Western diet (WD) induces central aortic stiffening that contributes to the transmittance of pulsatile blood flow to end organs, including the kidney. Our recent work supports that endothelial epithelial Na+ channel (EnNaC) expression and activation enhances aortic endothelial cell stiffening through reductions in endothelial nitric oxide (NO) synthase (eNOS) and bioavailable NO that result in inflammatory and oxidant responses and perivascular fibrosis. However, the role that EnNaC activation has on endothelial responses in the renal circulation remains unknown. We hypothesized that cell-specific deletion of the α-subunit of EnNaC would prevent WD-induced central aortic stiffness and protect the kidney from endothelial dysfunction and vascular stiffening. Twenty-eight-week-old female αEnNaC knockout and wild-type mice were fed either mouse chow or WD containing excess fat (46%), sucrose, and fructose (17.5% each). WD feeding increased fat mass, indexes of vascular stiffening in the aorta and renal artery (in vivo pulse wave velocity and ultrasound), and renal endothelial cell stiffening (ex vivo atomic force microscopy). WD further impaired aortic endothelium-dependent relaxation and renal artery compliance (pressure myography) without changes in blood pressure. WD-induced renal arterial stiffening occurred in parallel to attenuated eNOS activation, increased oxidative stress, and aortic and renal perivascular fibrosis. αEnNaC deletion prevented these abnormalities and support a novel mechanism by which WD contributes to renal arterial stiffening that is endothelium and Na+ channel dependent. These results demonstrate that cell-specific EnNaC is important in propagating pulsatility into the renal circulation, generating oxidant stress, reduced bioavailable NO, and renal vessel wall fibrosis and stiffening.

The combination of a neprilysin inhibitor (sacubitril) and angiotensin-II receptor blocker (valsartan) attenuates glomerular and tubular injury in the Zucker Obese rat.

OBJECTIVE: Diabetic nephropathy (DN) is characterized by glomerular and tubulointerstitial injury, proteinuria and remodeling. Here we examined whether the combination of an inhibitor of neprilysin (sacubitril), a natriuretic peptide-degrading enzyme, and an angiotensin II type 1 receptor blocker (valsartan), suppresses renal injury in a pre-clinical model of early DN more effectively than valsartan monotherapy. METHODS: Sixty-four male Zucker Obese rats (ZO) at 16 weeks of age were distributed into 4 different groups: Group 1: saline control (ZOC); Group 2: sacubitril/valsartan (sac/val) (68 mg kg-1 day-1; ZOSV); and Group 3: valsartan (val) (31 mg kg-1 day-1; ZOV). Group 4 received hydralazine, an anti-hypertensive drug (30 mg kg-1 day-1, ZOH). Six Zucker Lean (ZL) rats received saline (Group 5) and served as lean controls (ZLC). Drugs were administered daily for 10 weeks by oral gavage. RESULTS: Mean arterial pressure (MAP) increased in ZOC (+ 28%), but not in ZOSV (- 4.2%), ZOV (- 3.9%) or ZOH (- 3.7%), during the 10 week-study period. ZOC were mildly hyperglycemic, hyperinsulinemic and hypercholesterolemic. ZOC exhibited proteinuria, hyperfiltration, elevated renal resistivity index (RRI), glomerular mesangial expansion and podocyte foot process flattening and effacement, reduced nephrin and podocin expression, tubulointerstitial and periarterial fibrosis, increased NOX2, NOX4 and AT1R expression, glomerular and tubular nitroso-oxidative stress, with associated increases in urinary markers of tubular injury. None of the drugs reduced fasting glucose or HbA1c. Hypercholesterolemia was reduced in ZOSV (- 43%) and ZOV (- 34%) (p < 0.05), but not in ZOH (- 13%) (ZOSV > ZOV > ZOH). Proteinuria was ameliorated in ZOSV (- 47%; p < 0.05) and ZOV (- 30%; p > 0.05), but was exacerbated in ZOH (+ 28%; p > 0.05) (ZOSV > ZOV > ZOH). Compared to ZOC, hyperfiltration was improved in ZOSV (p < 0.05 vs ZOC), but not in ZOV or ZOH. None of the drugs improved RRI. Mesangial expansion was reduced by all 3 treatments (ZOV > ZOSV > ZOH). Importantly, sac/val was more effective in improving podocyte and tubular mitochondrial ultrastructure than val or hydralazine (ZOSV > ZOV > ZOH) and this was associated with increases in nephrin and podocin gene expression in ZOSV (p < 0.05), but not ZOV or ZOH. Periarterial and tubulointerstitial fibrosis and nitroso-oxidative stress were reduced in all 3 treatment groups to a similar extent. Of the eight urinary proximal tubule cell injury markers examined, five were elevated in ZOC (p < 0.05). Clusterin and KIM-1 were reduced in ZOSV (p < 0.05), clusterin alone was reduced in ZOV and no markers were reduced in ZOH (ZOSV > ZOV > ZOH). CONCLUSIONS: Compared to val monotherapy, sac/val was more effective in reducing proteinuria, renal ultrastructure and tubular injury in a clinically relevant animal model of early DN. More importantly, these renoprotective effects were independent of improvements in blood pressure, glycemia and nitroso-oxidative stress. These novel findings warrant future clinical investigations designed to test whether sac/val may offer renoprotection in the setting of DN.

Targeting TRAF3IP2 by Genetic and Interventional Approaches Inhibits Ischemia/Reperfusion-induced Myocardial Injury and Adverse Remodeling.

Re-establishing blood supply is the primary goal for reducing myocardial injury in subjects with ischemic heart disease. Paradoxically, reperfusion results in nitroxidative stress and a marked inflammatory response in the heart. TRAF3IP2 (TRAF3 Interacting Protein 2; previously known as CIKS or Act1) is an oxidative stress-responsive cytoplasmic adapter molecule that is an upstream regulator of both IκB kinase (IKK) and c-Jun N-terminal kinase (JNK), and an important mediator of autoimmune and inflammatory responses. Here we investigated the role of TRAF3IP2 in ischemia/reperfusion (I/R)-induced nitroxidative stress, inflammation, myocardial dysfunction, injury, and adverse remodeling. Our data show that I/R up-regulates TRAF3IP2 expression in the heart, and its gene deletion, in a conditional cardiomyocyte-specific manner, significantly attenuates I/R-induced nitroxidative stress, IKK/NF-κB and JNK/AP-1 activation, inflammatory cytokine, chemokine, and adhesion molecule expression, immune cell infiltration, myocardial injury, and contractile dysfunction. Furthermore, Traf3ip2 gene deletion blunts adverse remodeling 12 weeks post-I/R, as evidenced by reduced hypertrophy, fibrosis, and contractile dysfunction. Supporting the genetic approach, an interventional approach using ultrasound-targeted microbubble destruction-mediated delivery of phosphorothioated TRAF3IP2 antisense oligonucleotides into the LV in a clinically relevant time frame significantly inhibits TRAF3IP2 expression and myocardial injury in wild type mice post-I/R. Furthermore, ameliorating myocardial damage by targeting TRAF3IP2 appears to be more effective to inhibiting its downstream signaling intermediates NF-κB and JNK. Therefore, TRAF3IP2 could be a potential therapeutic target in ischemic heart disease.

TRAF3IP2 mediates high glucose-induced endothelin-1 production as well as endothelin-1-induced inflammation in endothelial cells.

Hyperglycemia-induced production of endothelin (ET)-1 is a hallmark of endothelial dysfunction in diabetes. Although the detrimental vascular effects of increased ET-1 are well known, the molecular mechanisms regulating endothelial synthesis of ET-1 in the setting of diabetes remain largely unidentified. Here, we show that adapter molecule TRAF3 interacting protein 2 (TRAF3IP2) mediates high glucose-induced ET-1 production in endothelial cells and ET-1-mediated endothelial cell inflammation. Specifically, we found that high glucose upregulated TRAF3IP2 in human aortic endothelial cells, which subsequently led to activation of JNK and IKKß. shRNA-mediated silencing of TRAF3IP2, JNK1, or IKKß abrogated high-glucose-induced ET-converting enzyme 1 expression and ET-1 production. Likewise, overexpression of TRAF3IP2, in the absence of high glucose, led to activation of JNK and IKKß as well as increased ET-1 production. Furthermore, ET-1 transcriptionally upregulated TRAF3IP2, and this upregulation was prevented by pharmacological inhibition of ET-1 receptor B using BQ-788, or inhibition of NADPH oxidase-derived reactive oxygen species using gp91ds-tat and GKT137831. Notably, we found that knockdown of TRAF3IP2 abolished ET-1-induced proinflammatory and adhesion molecule (IL-1ß, TNF-α, monocyte chemoattractant protein 1, ICAM-1, VCAM-1, and E-selectin) expression and monocyte adhesion to endothelial cells. Finally, we report that TRAF3IP2 is upregulated and colocalized with CD31, an endothelial marker, in the aorta of diabetic mice. Collectively, findings from the present study identify endothelial TRAF3IP2 as a potential new therapeutic target to suppress ET-1 production and associated vascular complications in diabetes. NEW & NOTEWORTHY This study provides the first evidence that the adapter molecule TRAF3 interacting protein 2 mediates high glucose-induced production of endothelin-1 by endothelial cells as well as endothelin-1-mediated endothelial cell inflammation. The findings presented herein suggest that TRAF3 interacting protein 2 may be an important therapeutic target in diabetic vasculopathy characterized by excess endothelin-1 production.

The role of dipeptidylpeptidase-4 inhibitors in management of cardiovascular disease in diabetes; focus on linagliptin.

Multiple population based analyses have demonstrated a high incidence of cardiovascular disease (CVD) and cardiovascular (CV) mortality in subjects with T2DM that reduces life expectancy by as much as 15 years. Importantly, the CV system is particularly sensitive to the metabolic and immune derangements present in obese pre-diabetic and diabetic individuals; consequently, CV dysfunction is often the initial CV derangement to occur and promotes the progression to end organ/tissue damage in T2DM. Specifically, diabetic CVD can manifest as microvascular complications, such as nephropathy, retinopathy, and neuropathy, as well as, macrovascular impairments, including ischemic heart disease, peripheral vascular disease, and cerebrovascular disease. Despite some progress in prevention and treatment of CVD, mainly via blood pressure and dyslipidemia control strategies, the impact of metabolic disease on CV outcomes is still a major challenge and persists in proportion to the epidemics of obesity and diabetes. There is abundant pre-clinical and clinical evidence implicating the DPP-4-incretin axis in CVD. In this regard, linagliptin is a unique DPP-4 inhibitor with both CV and renal safety profiles. Moreover, it exerts beneficial CV effects beyond glycemic control and beyond class effects. Linagliptin is protective for both macrovascular and microvascular complications of diabetes in preclinical models, as well as clinical models. Given the role of endothelial-immune cell interactions as one of the key events in the initiation and progression of CVD, linagliptin modulates these cell-cell interactions by affecting two important pathways involving stimulation of NO signaling and potent inhibition of a key immunoregulatory molecule.

Glycemic control by the SGLT2 inhibitor empagliflozin decreases aortic stiffness, renal resistivity index and kidney injury.

BACKGROUND: Arterial stiffness is emerging as an independent risk factor for the development of chronic kidney disease. The sodium glucose co-transporter 2 (SGLT2) inhibitors, which lower serum glucose by inhibiting SGLT2-mediated glucose reabsorption in renal proximal tubules, have shown promise in reducing arterial stiffness and the risk of cardiovascular and kidney disease in individuals with type 2 diabetes mellitus. Since hyperglycemia contributes to arterial stiffness, we hypothesized that the SGLT2 inhibitor empagliflozin (EMPA) would improve endothelial function, reduce aortic stiffness, and attenuate kidney disease by lowering hyperglycemia in type 2 diabetic female mice (db/db). MATERIALS/METHODS: Ten-week-old female wild-type control (C57BLKS/J) and db/db (BKS.Cg-Dock7m+/+Leprdb/J) mice were divided into three groups: lean untreated controls (CkC, n = 17), untreated db/db (DbC, n = 19) and EMPA-treated db/db mice (DbE, n = 19). EMPA was mixed with normal mouse chow at a concentration to deliver 10 mg kg-1 day-1, and fed for 5 weeks, initiated at 11 weeks of age. RESULTS: Compared to CkC, DbC showed increased glucose levels, blood pressure, aortic and endothelial cell stiffness, and impaired endothelium-dependent vasorelaxation. Furthermore, DbC exhibited impaired activation of endothelial nitric oxide synthase, increased renal resistivity and pulsatility indexes, enhanced renal expression of advanced glycation end products, and periarterial and tubulointerstitial fibrosis. EMPA promoted glycosuria and blunted these vascular and renal impairments, without affecting increases in blood pressure. In addition, expression of "reversion inducing cysteine rich protein with Kazal motifs" (RECK), an anti-fibrotic mediator, was significantly suppressed in DbC kidneys and partially restored by EMPA. Confirming the in vivo data, EMPA reversed high glucose-induced RECK suppression in human proximal tubule cells. CONCLUSIONS: Empagliflozin ameliorates kidney injury in type 2 diabetic female mice by promoting glycosuria, and possibly by reducing systemic and renal artery stiffness, and reversing RECK suppression.

Endothelial Mineralocorticoid Receptor Mediates Diet-Induced Aortic Stiffness in Females.

RATIONALE: Enhanced activation of the mineralocorticoid receptors (MRs) in cardiovascular tissues increases oxidative stress, maladaptive immune responses, and inflammation with associated functional vascular abnormalities. We previously demonstrated that consumption of a Western diet (WD) for 16 weeks results in aortic stiffening, and that these abnormalities were prevented by systemic MR blockade in female mice. However, the cell-specific role of endothelial cell MR (ECMR) in these maladaptive vascular effects has not been explored. OBJECTIVE: We hypothesized that specific deletion of the ECMR would prevent WD-induced increases in endothelial sodium channel activation, reductions in bioavailable nitric oxide, increased vascular remodeling, and associated increases in vascular stiffness in females. METHODS AND RESULTS: Four-week-old female ECMR knockout and wild-type mice were fed either mouse chow or WD for 16 weeks. WD feeding resulted in aortic stiffness and endothelial dysfunction as determined in vivo by pulse wave velocity and ex vivo by atomic force microscopy, and wire and pressure myography. The WD-induced aortic stiffness was associated with enhanced endothelial sodium channel activation, attenuated endothelial nitric oxide synthase activation, increased oxidative stress, a proinflammatory immune response and fibrosis. Conversely, cell-specific ECMR deficiency prevented WD-induced aortic fibrosis and stiffness in conjunction with reductions in endothelial sodium channel activation, oxidative stress and macrophage proinflammatory polarization, restoration of endothelial nitric oxide synthase activation. CONCLUSIONS: Increased ECMR signaling associated with consumption of a WD plays a key role in endothelial sodium channel activation, reduced nitric oxide production, oxidative stress, and inflammation that lead to aortic remodeling and stiffness in female mice.

Sodium glucose transporter 2 (SGLT2) inhibition with empagliflozin improves cardiac diastolic function in a female rodent model of diabetes.

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 (

Dipeptidyl peptidase-4 (DPP-4) inhibition with linagliptin reduces western diet-induced myocardial TRAF3IP2 expression, inflammation and fibrosis in female mice.

BACKGROUND: Diastolic dysfunction (DD), a hallmark of obesity and primary defect in heart failure with preserved ejection fraction, is a predictor of future cardiovascular events. We previously reported that linagliptin, a dipeptidyl peptidase-4 inhibitor, improved DD in Zucker Obese rats, a genetic model of obesity and hypertension. Here we investigated the cardioprotective effects of linagliptin on development of DD in western diet (WD)-fed mice, a clinically relevant model of overnutrition and activation of the renin-angiotensin-aldosterone system. METHODS: Female C56Bl/6 J mice were fed an obesogenic WD high in fat and simple sugars, and supplemented or not with linagliptin for 16 weeks. RESULTS: WD induced oxidative stress, inflammation, upregulation of Angiotensin II type 1 receptor and mineralocorticoid receptor (MR) expression, interstitial fibrosis, ultrastructural abnormalities and DD. Linagliptin inhibited cardiac DPP-4 activity and prevented molecular impairments and associated functional and structural abnormalities. Further, WD upregulated the expression of TRAF3IP2, a cytoplasmic adapter molecule and a regulator of multiple inflammatory mediators. Linagliptin inhibited its expression, activation of its downstream signaling intermediates NF-κB, AP-1 and p38-MAPK, and induction of multiple inflammatory mediators and growth factors that are known to contribute to development and progression of hypertrophy, fibrosis and contractile dysfunction. Linagliptin also inhibited WD-induced collagens I and III expression. Supporting these in vivo observations, linagliptin inhibited aldosterone-mediated MR-dependent oxidative stress, upregulation of TRAF3IP2, proinflammatory cytokine, and growth factor expression, and collagen induction in cultured primary cardiac fibroblasts. More importantly, linagliptin inhibited aldosterone-induced fibroblast activation and migration. CONCLUSIONS: Together, these in vivo and in vitro results suggest that inhibition of DPP-4 activity by linagliptin reverses WD-induced DD, possibly by targeting TRAF3IP2 expression and its downstream inflammatory signaling.

Dipeptidyl peptidase-4 inhibition with linagliptin prevents western diet-induced vascular abnormalities in female mice.

BACKGROUND: Vascular stiffening, a risk factor for cardiovascular disease, is accelerated, particularly in women with obesity and type 2 diabetes. Preclinical evidence suggests that dipeptidylpeptidase-4 (DPP-4) inhibitors may have cardiovascular benefits independent of glycemic lowering effects. Recent studies show that consumption of a western diet (WD) high in fat and simple sugars induces aortic stiffening in female C57BL/6J mice in advance of increasing blood pressure. The aims of this study were to determine whether administration of the DPP-4 inhibitor, linagliptin (LGT), prevents the development of aortic and endothelial stiffness induced by a WD in female mice. METHODS: C56Bl6/J female mice were fed a WD for 4 months. Aortic stiffness and ex vivo endothelial stiffness were evaluated by Doppler pulse wave velocity (PWV) and atomic force microscopy (AFM), respectively. In addition, we examined aortic vasomotor responses and remodeling markers via immunohistochemistry. Results were analyzed via 2-way ANOVA, p < 0.05 was considered as statistically significant. RESULTS: Compared to mice fed a control diet (CD), WD-fed mice exhibited a 24 % increase in aortic PWV, a five-fold increase in aortic endothelial stiffness, and impaired endothelium-dependent vasodilation. In aorta, these findings were accompanied by medial wall thickening, adventitial fibrosis, increased fibroblast growth factor 23 (FGF-23), decreased Klotho, enhanced oxidative stress, and endothelial cell ultrastructural changes, all of which were prevented with administration of LGT. CONCLUSIONS: The present findings support the notion that DPP-4 plays a role in development of WD-induced aortic stiffening, vascular oxidative stress, endothelial dysfunction, and vascular remodeling. Whether, DPP-4 inhibition could be a therapeutic tool used to prevent the development of aortic stiffening and the associated cardiovascular complications in obese and diabetic females remains to be elucidated.

Regional variation in arterial stiffening and dysfunction in Western diet-induced obesity.

Increased central vascular stiffening, assessed in vivo by determination of pulse wave velocity (PWV), is an independent predictor of cardiovascular event risk. Recent evidence demonstrates that accelerated aortic stiffening occurs in obesity; however, little is known regarding stiffening of other disease-relevant arteries or whether regional variation in arterial stiffening occurs in this setting. We addressed this gap in knowledge by assessing femoral PWV in vivo in conjunction with ex vivo analyses of femoral and coronary structure and function in a mouse model of Western diet (WD; high-fat/high-sugar)-induced obesity and insulin resistance. WD feeding resulted in increased femoral PWV in vivo. Ex vivo analysis of femoral arteries revealed a leftward shift in the strain-stress relationship, increased modulus of elasticity, and decreased compliance indicative of increased stiffness following WD feeding. Confocal and multiphoton fluorescence microscopy revealed increased femoral stiffness involving decreased elastin/collagen ratio in conjunction with increased femoral transforming growth factor-ß (TGF-ß) content in WD-fed mice. Further analysis of the femoral internal elastic lamina (IEL) revealed a significant reduction in the number and size of fenestrae with WD feeding. Coronary artery stiffness and structure was unchanged by WD feeding. Functionally, femoral, but not coronary, arteries exhibited endothelial dysfunction, whereas coronary arteries exhibited increased vasoconstrictor responsiveness not present in femoral arteries. Taken together, our data highlight important regional variations in the development of arterial stiffness and dysfunction associated with WD feeding. Furthermore, our results suggest TGF-ß signaling and IEL fenestrae remodeling as potential contributors to femoral artery stiffening in obesity.

Mineralocorticoid receptor blockade prevents Western diet-induced diastolic dysfunction in female mice.

Overnutrition/obesity predisposes individuals, particularly women, to diastolic dysfunction (DD), an independent predictor of future cardiovascular disease. We examined whether low-dose spironolactone (Sp) prevents DD associated with consumption of a Western Diet (WD) high in fat, fructose, and sucrose. Female C57BL6J mice were fed a WD with or without Sp (1 mg·kg(-1)·day(-1)). After 4 mo on the WD, mice exhibited increased body weight and visceral fat, but similar blood pressures, compared with control diet-fed mice. Sp prevented the development of WD-induced DD, as indicated by decreased isovolumic relaxation time and an improvement in myocardial performance (

Pleiotropic effects of the dipeptidylpeptidase-4 inhibitors on the cardiovascular system.

Dipeptidylpeptidase-4 (DPP-4) is a ubiquitously expressed transmembrane protein that removes NH2-terminal dipeptides from various substrate hormones, chemokines, neuropeptides, and growth factors. Two known substrates of DPP-4 include the incretin hormones glucagon-like peptide-1 (GLP-1) and gastric inhibitory peptide, which are secreted by enteroendocrine cells in response to postprandial hyperglycemia and account for 60­70% of postprandial insulin secretion. DPP-4 inhibitors (DPP-4i) block degradation of GLP-1 and gastric inhibitory peptide, extend their insulinotropic effect, and improve glycemia. Since 2006, several DPP-4i have become available for treatment of type 2 diabetes mellitus. Clinical trials confirm that DPP-4i raises GLP-1 levels in plasma and improves glycemia with very low risk for hypoglycemia and other side effects. Recent studies also suggest that DPP-4i confers cardiovascular and kidney protection, beyond glycemic control, which may reduce the risk for further development of the multiple comorbidities associated with obesity/type 2 diabetes mellitus, including hypertension and cardiovascular disease (CVD) and kidney disease. The notion that DPP-4i may improve CVD outcomes by mechanisms beyond glycemic control is due to both GLP-1-dependent and GLP-1-independent effects. The CVD protective effects by DPP-4i result from multiple factors including insulin resistance, oxidative stress, dyslipidemia, adipose tissue dysfunction, dysfunctional immunity, and antiapoptotic properties of these agents in the heart and vasculature. This review focuses on cellular and molecular mechanisms mediating the CVD protective effects of DPP-4i beyond favorable effects on glycemic control.