Dapagliflozin attenuates diabetes-induced diastolic dysfunction and cardiac fibrosis by regulating SGK1 signaling.

BACKGROUND: Recent studies have reported improved diastolic function in patients administered sodium-glucose cotransporter 2 inhibitors (SGLT2i). We aimed to investigate the effect of dapagliflozin on left ventricular (LV) diastolic function in a diabetic animal model and to determine the molecular and cellular mechanisms underlying its function. METHODS: A total of 30 male New Zealand white rabbits were randomized into control, diabetes, or diabetes+dapagliflozin groups (n = 10/per each group). Diabetes was induced by intravenous alloxan. Cardiac function was evaluated using echocardiography. Myocardial samples were obtained for histologic and molecular evaluation. For cellular evaluation, fibrosis-induced cardiomyoblast (H9C2) cells were obtained, and transfection was performed for mechanism analysis (serum and glucocorticoid-regulated kinase 1 (SGK1) signaling analysis). RESULTS: The diabetes+dapagliflozin group showed attenuation of diastolic dysfunction compared with the diabetes group. Dapagliflozin inhibited myocardial fibrosis via inhibition of SGK1 and epithelial sodium channel (ENaC) protein, which was observed both in myocardial tissue and H9C2 cells. In addition, dapagliflozin showed an anti-inflammatory effect and ameliorated mitochondrial disruption. Inhibition of SGK1 expression by siRNA decreased and ENaC and Na+/H+ exchanger isoform 1 (NHE1) expression was confirmed as significantly reduced as siSGK1 in the diabetes+dapagliflozin group. CONCLUSIONS: Dapagliflozin attenuated left ventricular diastolic dysfunction and cardiac fibrosis via regulation of SGK1 signaling. Dapagliflozin also reduced macrophages and inflammatory proteins and ameliorated mitochondrial disruption.

Predictive value of plaque characteristics for identification of lesions causing ischemia.

BACKGROUND: Functional assessment using fractional flow reserve (FFR) and anatomical assessment using optical coherence tomography (OCT) are used in clinical practice for patients with intermediate coronary stenosis. Moreover, coronary computed tomography angiography (CTA) is a common noninvasive imaging technique for evaluating suspected coronary artery disease before being referred for angiography. This study aimed to investigate the association between FFR and plaque characteristics assessed using coronary CTA and OCT for intermediate coronary stenosis. METHODS: Based on a prospective multicenter registry, 159 patients having 339 coronary lesions with intermediate stenosis were included. All patients underwent coronary CTA before being referred for coronary angiography, and both FFR measurements and OCT examinations were performed during angiography. A stenotic lesion identified with FFR ≤0.80 was deemed diagnostic of an ischemia-causing lesion. The predictive value of plaque characteristics assessed using coronary CTA and OCT for identifying lesions causing ischemia was analyzed. RESULTS: Stenosis severity and plaque characteristics on coronary CTA and OCT differed between lesions that caused ischemia and those that did not. In multivariate analysis, low attenuation plaque on coronary CTA (odds ratio [OR]=2.78; P=0.038), thrombus (OR=5.13; P=0.042), plaque rupture (OR=3.25; P=0.017), and intimal vasculature on OCT (OR=2.57; P=0.012) were independent predictors of ischemic lesions. Increasing the number of these plaque characteristics offered incremental improvement in predicting the lesions causing ischemia. CONCLUSIONS: Comprehensive anatomical evaluation of coronary stenosis may provide additional supportive information for predicting the lesions causing ischemia.

Assessment of fractional flow reserve in intermediate coronary stenosis using optical coherence tomography-based machine learning.

Objectives: This study aimed to evaluate and compare the diagnostic accuracy of machine learning (ML)- fractional flow reserve (FFR) based on optical coherence tomography (OCT) with wire-based FFR irrespective of the coronary territory. Background: ML techniques for assessing hemodynamics features including FFR in coronary artery disease have been developed based on various imaging modalities. However, there is no study using OCT-based ML models for all coronary artery territories. Methods: OCT and FFR data were obtained for 356 individual coronary lesions in 130 patients. The training and testing groups were divided in a ratio of 4:1. The ML-FFR was derived for the testing group and compared with the wire-based FFR in terms of the diagnosis of ischemia (FFR ≤ 0.80). Results: The mean age of the subjects was 62.6 years. The numbers of the left anterior descending, left circumflex, and right coronary arteries were 130 (36.5%), 110 (30.9%), and 116 (32.6%), respectively. Using seven major features, the ML-FFR showed strong correlation (r = 0.8782, P < 0.001) with the wire-based FFR. The ML-FFR predicted wire-based FFR ≤ 0.80 in the test set with sensitivity of 98.3%, specificity of 61.5%, and overall accuracy of 91.7% (area under the curve: 0.948). External validation showed good correlation (r = 0.7884, P < 0.001) and accuracy of 83.2% (area under the curve: 0.912). Conclusion: OCT-based ML-FFR showed good diagnostic performance in predicting FFR irrespective of the coronary territory. Because the study was a small-size study, the results should be warranted the performance in further large-scale research.

A Senolytic-Eluting Coronary Stent for the Prevention of In-Stent Restenosis.

The vast majority of drug-eluting stents (DES) elute either sirolimus or one of its analogues. While limus drugs stymie vascular smooth muscle cell (VSMC) proliferation to prevent in-stent restenosis, their antiproliferative nature is indiscriminate and limits healing of the endothelium in stented vessels, increasing the risk of late-stent thrombosis. Oxidative stress, which is associated with vascular injury from stent implantation, can induce VSMCs to undergo senescence, and senescent VSMCs can produce pro-inflammatory cytokines capable of inducing proliferation of neighboring nonsenescent VSMCs. We explored the potential of senolytic therapy, which involves the selective elimination of senescent cells, in the form of a senolytic-eluting stent (SES) for interventional cardiology. Oxidative stress was modeled in vitro by exposing VSMCs to H2O2, and H2O2-mediated senescence was evaluated by cytochemical staining of senescence-associated ß-galactosidase activity and qRT-PCR. Quiescent VSMCs were then treated with the conditioned medium (CM) of H2O2-treated VSMCs. Proliferative effects of CM were analyzed by staining for proliferating cell nuclear antigen. Senolytic effects of the first-generation senolytic ABT263 were observed in vitro, and the effects of ABT263 on endothelial cells were also investigated through an in vitro re-endothelialization assay. SESs were prepared by dip coating. Iliofemoral arteries of hypercholesteremic rabbits were implanted with SES, everolimus-eluting stents (EESs), or bare-metal stents (BMSs), and the area of stenosis was measured 4 weeks post-implantation using optical coherence tomography. We found that a portion of H2O2-treated VSMCs underwent senescence, and that CM of H2O2-treated senescent VSMCs triggered the proliferation of quiescent VSMCs. ABT263 reverted H2O2-mediated senescence and the proliferative capacity of senescent VSMC CM. Unlike everolimus, ABT263 did not affect endothelial cell migration and/or proliferation. SES, but not EES, significantly reduced stenosis area in vivo compared with bare-metal stents (BMSs). This study shows the potential of SES as an alternative to current forms of DES.

Computational Fractional Flow Reserve From Coronary Computed Tomography Angiography-Optical Coherence Tomography Fusion Images in Assessing Functionally Significant Coronary Stenosis.

Background: Coronary computed tomography angiography (CTA) and optical coherence tomography (OCT) provide additional functional information beyond the anatomy by applying computational fluid dynamics (CFD). This study sought to evaluate a novel approach for estimating computational fractional flow reserve (FFR) from coronary CTA-OCT fusion images. Methods: Among patients who underwent coronary CTA, 148 patients who underwent both pressure wire-based FFR measurement and OCT during angiography to evaluate intermediate stenosis in the left anterior descending artery were included from the prospective registry. Coronary CTA-OCT fusion images were created, and CFD was applied to estimate computational FFR. Based on pressure wire-based FFR as a reference, the diagnostic performance of Fusion-FFR was compared with that of CT-FFR and OCT-FFR. Results: Fusion-FFR was strongly correlated with FFR (r = 0.836, P < 0.001). Correlation between FFR and Fusion-FFR was stronger than that between FFR and CT-FFR (r = 0.682, P < 0.001; z statistic, 5.42, P < 0.001) and between FFR and OCT-FFR (r = 0.705, P < 0.001; z statistic, 4.38, P < 0.001). Area under the receiver operating characteristics curve to assess functionally significant stenosis was higher for Fusion-FFR than for CT-FFR (0.90 vs. 0.83, P = 0.024) and OCT-FFR (0.90 vs. 0.83, P = 0.043). Fusion-FFR exhibited 84.5% accuracy, 84.6% sensitivity, 84.3% specificity, 80.9% positive predictive value, and 87.5% negative predictive value. Especially accuracy, specificity, and positive predictive value were superior for Fusion-FFR than for CT-FFR (73.0%, P = 0.007; 61.4%, P < 0.001; 64.0%, P < 0.001) and OCT-FFR (75.7%, P = 0.021; 73.5%, P = 0.020; 69.9%, P = 0.012). Conclusion: CFD-based computational FFR from coronary CTA-OCT fusion images provided more accurate functional information than coronary CTA or OCT alone. Clinical Trial Registration: [www.ClinicalTrials.gov], identifier [NCT03298282].

Relative strength of the interlayer dipole fields and the interlayer exchange coupling in nanostructured cells of synthetic ferrimagnets.

Both analytical and numerical calculations are carried out to examine the relative strength of the interlayer dipole fields in comparison to that of the interlayer exchange coupling in nanostructured cells of synthetic ferrimagnets as functions of their size, shape, and thickness. The relative strength in aligning the magnetizations antiparallel increases with decreasing cell size and with increasing cell thickness, in agreement with the size and thickness dependence of the magnetostatic interactions. As the cell shape changes from rectangle to ellipse, the relative strength decreases, due to the decreased density of free poles with the shape change. The relative strength of the interlayer dipole field is found to be not small, being approximately 10% of the strongest interlayer exchange coupling observed for the CoFeB/Ru/CoFeB structure. In an elliptical cell with the dimensions of 160 nm (long axis) x 80 nm (short axis) x 2 nm (thickness), for example, the strength is 0.016 erg/cm2.

Microneedle drug eluting balloon for enhanced drug delivery to vascular tissue.

High rates of restenosis and neointimal formation have driven increasing interest in the application of drug eluting balloons (DEB) as counteractive measures for intraluminal drug delivery. The use of DEBs eliminates the need for stents so that serious side effects including in-stent restenosis and stent thrombosis can be avoided and long-term medication of anti-platelet agent is not needed. Despite their benefits, DEBs have poor drug delivery efficiency due to short balloon inflation times (30-60 s) that limit the passive drug diffusion from the balloon surface to the luminal lesion. To increase drug delivery efficiency, a microneedle DEB (MNDEB) was developed by a conformal transfer molding process using a thin polydimethylsiloxane mold bearing a negative array of MNs of 200 µm in height. A MN array composed of UV curable resin was formed onto the surface of DEB, and drugs were coated onto the structure. The mechanical properties of the MN array were investigated and MN penetration into luminal vasculature was confirmed in vivo. An increase in drug delivery efficiency compared to a standard DEB was demonstrated in an in vivo test in a rabbit aorta. Finally, the superior therapeutic efficacy of MNDEBs was evaluated using an atherosclerosis rabbit model.

Anti-Inflammatory Effect for Atherosclerosis Progression by Sodium-Glucose Cotransporter 2 (SGLT-2) Inhibitor in a Normoglycemic Rabbit Model.

BACKGROUND AND OBJECTIVES: We sought to investigate an anti-atherosclerotic and anti-inflammatory effect of sodium-glucose cotransporter-2 (SGLT-2) inhibitors in normoglycemic atherosclerotic rabbit model. METHODS: Male New Zealand white rabbits (n=26) were fed with a 1% high-cholesterol diet for 7 weeks followed by normal diet for 2 weeks. After balloon catheter injury, the rabbits were administered with the Dapagliflozin (1mg/kg/day) or control-medium for 8 weeks (n=13 for each group). All lesions were assessed with angiography, optical coherence tomography (OCT), and histological assessment. RESULTS: Atheroma burden (38.51±3.16% vs. 21.91±1.22%, p<0.01) and lipid accumulation (18.90±3.63% vs. 10.20±2.03%, p=0.047) was significantly decreased by SGLT-2 inhibitor treatment. The SGLT-2 inhibitor group showed lower macrophage infiltration (20.23±1.89% vs. 12.72±1.95%, p=0.01) as well as tumor necrosis factor (TNF)-α expression (31.17±4.40% vs. 19.47±2.10%, p=0.025). Relative area of inducible nitric oxide synthase⁺ macrophages was tended to be lower in the SGLT-2 inhibitor-treated group (1.00±0.16% vs. 0.71±0.10%, p=0.13), while relative proportion of Arg1⁺ macrophage was markedly increased (1.00±0.27% vs. 2.43±0.64%, p=0.04). As a result, progression of atherosclerosis was markedly attenuated in SGLT-2 inhibitor treated group (OCT area stenosis, 32.13±1.20% vs. 22.77±0.88%, p<0.01). Mechanistically, SGLT-2 treatment mitigated the inflammatory responses in macrophage. Especially, Toll-like receptor 4/nuclear factor-kappa B signaling pathway, and their downstream effectors such as interleukin-6 and TNF-α were markedly suppressed by SGLT-2 inhibitor treatment. CONCLUSIONS: These results together suggest that SGLT-2 inhibitor exerts an anti-atherosclerotic effect through favorable modulation of inflammatory response as well as macrophage characteristics in non-diabetic situation.

Pharmacokinetic Study of NADPH Oxidase Inhibitor Ewha-18278, a Pyrazole Derivative.

In a previous study, the specific NOX1/2/4 inhibitor Ewha-18278 was confirmed as a possible treatment for osteoporosis both in vitro and in vivo. Here, we investigated the pharmacokinetics (PK) of the compound by intravenous (IV) and oral administrations to rats. Dimethyl sulfoxide (DMSO)-based and diazepam injection-based formulations were used to dissolve the compound. In the latter formulation applicable to humans, the changes in PK parameters were monitored at two different concentrations (1 mg/mL and 2 mg/mL). The area under the plasma concentration-time curve from zero time to infinity (AUCinf) of Ewha-18278 was highest in the DMSO-based formulation (2 mg/mL). Also, the concentration was increased 1.6-fold at the low concentration of the diazepam injection-based formulation compared to the high concentration. There was no statistical significance in the AUCinf of the compound between DMSO-based formulation (2 mg/mL) and diazepam injection-based formulation (1 mg/mL). These results suggest that Ewha-18278 can be delivered to humans by both IV and oral routes. In addition, the diazepam injection-based formulation of Ewha-18278 appears to be a suitable candidate for dosage development for future toxicity test and clinical trial.

Synergistic protective effects of a statin and an angiotensin receptor blocker for initiation and progression of atherosclerosis.

AIM: Although the atheroprotective effects of statins and angiotensin II receptor blockers (ARBs) are well-established, little is known about their additive effects, especially during the early period of atherosclerosis. The aim of this study was to investigate whether combination of a statin and an ARB exerts synergistic anti-atherosclerotic effects, and to elucidate the mechanisms of combined effects. METHODS: Atherosclerotic plaques were developed in arteries of 23 rabbits using a high-cholesterol diet (HCD) and intra-arterial balloon inflation. Rabbits received one of five different treatment strategies for 4 weeks: positive control [n = 5, HCD]; negative control [n = 3, regular chow diet]; statin [n = 5, HCD and rosuvastatin 10 mg]; ARB [n = 5, HCD and olmesartan 20 mg]; and combination [n = 5, HCD and statin+ARB]. RESULTS: Histological analysis demonstrated that development of atherosclerotic plaques was inhibited more in combination group than in statin group (P = 0.001). Although macrophage infiltration identified by RAM11 staining was not significantly different between combination and individual treatment groups (31.76±4.84% [combination] vs. 38.11±6.53% [statin; P = 0.35] or 35.14±2.87% [ARB; P = 0.62]), the relative proportion of pro-inflammatory M1-macrophages was significantly lower in combination group than in ARB group (3.20±0.47% vs. 5.20±0.78%, P = 0.02). Furthermore, M2-macrophage polarization was higher in combination group than in statin group (17.70±3.04% vs. 7.86±0.68%, P = 0.001). CONCLUSION: Combination treatment with a statin and an ARB produced synergistic protective effects for atherosclerosis initiation and progression, which may be attributed to modulation of macrophage characteristics in the early period of atherosclerosis.

Macrophage polarization and acceleration of atherosclerotic plaques in a swine model.

AIMS: Atherosclerosis is a well-known cause of cardiovascular disease and is associated with a variety of inflammatory reactions. However, an adequate large-animal model of advanced plaques to investigate the pathophysiology of atherosclerosis is lacking. Therefore, we developed and assessed a swine model of advanced atherosclerotic plaques with macrophage polarization. METHODS: Mini-pigs were fed a 2% high-cholesterol diet for 7 weeks followed by withdrawal periods of 4 weeks. Endothelial denudation was performed using a balloon catheter on 32 coronary and femoral arteries of 8 mini-pigs. Inflammatory proteins (high-mobility group box 1 [HMGB1] or tumor necrosis factor alpha (TNF-α) were injected via a micro-infusion catheter into the vessel wall. All lesions were assessed with angiography and optical coherence tomography and all tissues were harvested for histological evaluation. RESULTS: Intima/plaque area was significantly higher in the HMGB1- and TNF-α-injected groups compared to the saline-injected group (p = 0.002). CD68 antibody detection and polarization of M1 macrophages significantly increased in the inflammatory protein-injected groups (p<0.001). In addition, advanced atherosclerotic plaques were observed more in the inflammatory protein-injected groups compared with the control upon histologic evaluation. CONCLUSION: Direct injection of inflammatory proteins was associated with acceleration of atherosclerotic plaque formation with M1 macrophage polarization. Therefore, direct delivery of inflammatory proteins may induce a pro-inflammatory response, providing a possible strategy for development of an advanced atherosclerotic large-animal model in a relatively short time period.

Linear Micro-patterned Drug Eluting Balloon (LMDEB) for Enhanced Endovascular Drug Delivery.

In-stent restenosis (ISR) often occurs after applying drug eluting stents to the blood vessels suffering from atherosclerosis or thrombosis. For treatment of ISR, drug eluting balloons (DEB) have been developed to deliver anti-proliferative drugs to the lesions with ISR. However, there are still limitations of DEB such as low drug delivery efficiency and drug loss to blood flow. Although most researches have focused on alteration of drug formulation for more efficient drug delivery, there are few studies that have attempted to understand and utilize the contact modality of DEB drug delivery. Here, we developed a linear micro-patterned DEB (LMDEB) that applied higher contact pressure to enhance drug stamping to vascular tissue. Ex vivo and in vivo studies confirmed that higher contact pressure from micro-patterns increased the amount of drug delivered to the deeper regions of vessel. Finite element method simulation also showed significant increase of contact pressure between endothelium and micro-patterns. Quantitative analysis by high performance liquid chromatography indicated that LMDEBs delivered 2.3 times higher amount of drug to vascular tissue in vivo than conventional DEBs. Finally, efficacy studies using both atherosclerotic and ISR models demonstrated superior patency of diseased vessels treated with LMDEB compared to those treated with DEB.

Preclinical assessment of a modified Occlutech left atrial appendage closure device in a canine model.

BACKGROUND: LAA occlusion has a similar stroke prevention efficacy compared to anticoagulation treatment for non-valvular atrial fibrillation. OBJECTIVE: The objective of this study was to assess the feasibility and safety of a modified Occlutech® left atrial appendage (LAA) closure device in a canine model. METHODS: The device was implanted in 10 dogs (33±1kg) using fluoroscopy and transesophageal echocardiography (TEE) guidance. The modified Occlutech® LAA occlusion device was compared with the current version, the Watchman device, and the Amplazter cardiac plug (ACP). LAA occlusion and anchoring to the LAA were evaluated. All dogs were assessed using angiography, TEE, and a gross anatomy examination. RESULTS: The 10 LAA occlusion devices were to be implanted into 10 dogs (5 modified Occlutech devices, 3 current version of Occlutech devices, 1 Watchman, and 1 ACP). LAA implantation was not performed in one dog due to transeptal puncture failure. The three current version of Occlutech devices were embolized immediately after implantation, so three modified devices of the same size were implanted securely without embolization. The mean implant size was 20.1±2.0mm. The devices chosen were a mean of 23.3±10.6% larger than the measured landing zone diameters. Post-implant angiography and TEE revealed well-positioned devices without pericardial effusion or impingement on surrounding structures. CONCLUSIONS: The results of this acute animal study suggested that a modified Occlutech® LAA occlusion device was feasible and had greater anchoring performance in canines. Additional large clinical studies are needed to evaluate safety and efficacy.

Development of Advanced Atherosclerotic Plaque by Injection of Inflammatory Proteins in a Rabbit Iliac Artery Model.

PURPOSE: Appropriate animal models of atherosclerotic plaque are crucial to investigating the pathophysiology of atherosclerosis, as well as for the evaluation of the efficacy and safety of vascular devices. We aimed to develop a novel animal model that would be suitable for the study of advanced atherosclerotic lesions in vivo. MATERIALS AND METHODS: Atherosclerotic plaque was induced in 24 iliac arteries from 12 rabbits by combining a high cholesterol diet, endothelial denudation, and injection into the vessel wall with either saline (n=5), olive oil (n=6), or inflammatory proteins [n=13, high-mobility group protein B1 (HMGB1) n=8 and tumor necrosis factor (TNF)-α n=5] using a Cricket™ Micro-infusion catheter. Optical coherence tomography (OCT) was performed to detect plaque characteristics after 4 weeks, and all tissues were harvested for histological evaluation. RESULTS: Advanced plaque was more frequently observed in the group injected with inflammatory proteins. Macrophage infiltration was present to a higher degree in the HMGB1 and TNF-α groups, compared to the oil or saline group (82.1±5.1% and 94.6±2.2% compared to 49.6±14.0% and 46.5±9.6%, p-value<0.001), using RAM11 antibody staining. On OCT, lipid rich plaques were more frequently detected in the inflammatory protein group [saline group: 2/5 (40%), oil group: 3/5 (50%), HMGB1 group: 6/8 (75%), and TNF-α group: 5/5 (100%)]. CONCLUSION: These data indicate that this rabbit model of atherosclerotic lesion formation via direct injection of pro-inflammatory proteins into the vessel wall is useful for in vivo studies investigating atherosclerosis.

A novel pyrazole derivative protects from ovariectomy-induced osteoporosis through the inhibition of NADPH oxidase.

Osteoclast cells (OCs) are differentiated from bone marrow-derived macrophages (BMMs) by activation of receptor activator of nuclear factor κB (NF-κB) ligand (RANKL). Activation of NADPH oxidase (Nox) isozymes is involved in RANKL-dependent OC differentiation, implicating Nox isozymes as therapeutic targets for treatment of osteoporosis. Here, we show that a novel pyrazole derivative, Ewha-18278 has high inhibitory potency on Nox isozymes. Blocking the activity of Nox with Ewha-18278 inhibited the responses of BMMs to RANKL, including reactive oxygen species (ROS) generation, activation of mitogen-activated protein (MAP) kinases and NF-κB, and OC differentiation. To evaluate the anti-osteoporotic function of Ewha-18278, the derivative was applied to estrogen-deficient ovariectomized (OVX) ddY mice. Oral administration of Ewha-18278 (10 mg/kg/daily, 4 weeks) into the mice recovered bone mineral density, trabecular bone volume, trabecular bone length, number and thickness, compared to control OVX ddY mice. Moreover, treatment of OVX ddY mice with Ewha-18278 increased bone strength by increasing cortical bone thickness. We provide that Ewha-18278 displayed Nox inhibition and blocked the RANKL-dependent cell signaling cascade leading to reduced differentiation of OCs. Our results implicate Ewha-18278 as a novel therapeutic agent for the treatment of osteoporosis.