Abstract Introduction Epicardial adipose tissue (EAT) has been suggested to contribute to left atrium dysfunction via paracrine mechanisms to induce endothelial dysfunction, pro-arrhythmogenic and pro-remodeling responses thereby favoring atrial fibrillation (AF) onset. Recently, sodium-glucose co-transporter2 inhibitors (SGLT2i) have been shown to reduce AF incidence, EAT volume, differentiation and inflammation, however, the underlying mechanisms are unknown. Purpose This study investigates the impact of human EAT-derived mediators on atrial endothelial cell function and, if so, to characterize the role of SGLT2 using the inhibitor empagliflozin (EMPA). Methods Epicardial and subcutaneous adipose tissues (SAT) were collected from 70 cardiac patients at a university hospital. Conditioned medium (CM, 24 h) from fats were applied to porcine atrial endothelial cells (AECs, first passage) for 24 h. Histology of tissues was assessed by haematoxylin and eosin staining, protein expression by Western blot analysis or immunofluorescence staining, mRNA levels by RT-qPCR, formation of reactive oxygen species (ROS) and nitric oxide (NO) by fluorescent probes, and pro-inflammatory cytokine levels by ELISA. Results Compared to SAT, EATs were more vascularized with increased infiltration of M1-like macrophages, expression of proinflammatory cytokines (IL-1ß, IL-6, TNF-α), markers of ECs VCAM-1 and eNOS, fibrosis TGF-β, NADPH oxidases NOX-1 and SGLT1/2. These effects were associated with a pro-oxidant response that was inhibited by neutralizing Abs directed against IL-1ß or IL-6, and by inhibition of either NOS, NADPH oxidases, ACE, AT1R or SGLT2. Levels of SGLT2 and ROS in EATs were higher in AF compared to non-AF patients and progressively increasing with age. CM of EATs showed higher concentrations of IL-1ß, IL-6, TNF-α and MCP-1 than that of SAT. Exposure of AECs to CM of EATs increased the level of oxidative stress that was positively correlated to the level of pro-inflammatory cytokines, and reduced basal and bradykinin-induced NO formation that was prevented by EMPA. It also resulted in upregulation of p53 and SGLT2 expression, and nuclear translocation of NF-kB by CM of the top quartile inflamed EATs but not by the lowest quartile of EATs or SATs, prevented by EMPA. Conclusion The findings indicate that compared to SATs, the EATs showed higher in situ expression and release of pro-inflammatory cytokines that contributed to oxidative stress involving the AT1R/NADPH oxidases/SGLT2 pro-oxidant pathway. CMs of inflamed EATs induced AECs dysfunction, oxidative stress and activation of NF-kB involving pro-inflammatory cytokines and SGLT2. Thus, SGLT2i appear as an interesting strategy to decrease EAT inflammation and improve endothelial function contributing to prevent cardiac remodeling and fibrotic responses.

Abstract Background Takotsubo syndrome (TTS) is characterized by apical ballooning predominantly affecting post-menopausal women following extreme physical and/or emotional stress. The phenotype of TTS is associated with akinetic apex and hyperkinetic base of the left ventricle (LV). Presently, there are no targeted therapies for TTS that can effectively reduce LV impairment. Empagliflozin (EMPA), an SGLT2 inhibitor, has shown considerable clinical benefits in managing heart failure. However, its specific effects and underlying mechanisms in treating TTS remain unclear. Purpose To explore the pathophysiology of TTS in the LV of rats, with a particular focus on the potential role of SGLT2, and to elucidate the underlying mechanisms involved. Methods Female Sprague Dawley rats (250-300 g) were allocated into four groups: a control group (n=8), a group treated with EMPA (30 mg/kg/day) (n=8), a group receiving ISO (Isoproterenol) at 100 mg/kg/day (n=18), and a group receiving both ISO and EMPA (IE) at 30 mg/kg/day (n=18). EMPA was administered orally through food two weeks prior to ISO injection. Echocardiography was conducted at 6 and 24 h, and on day 3. On day 3, rats were euthanized, and their organs were collected. Gene expression of markers was assessed using RT-qPCR, protein expression via immunofluorescence (IF) staining, and oxidative stress using dihydroethidium. Results In the ISO group, the success rate of induced Takotsubo syndrome (TTS) was 53% (10 out of 18 rats), with a mortality rate of 5%. Conversely, in the EMPA-treated group, TTS occurred in 22% of rats (4 out of 18) with no mortality observed. Significant reductions in the area of left ventricular (LV) ballooning and ejection fraction were noted in the IE group compared to the ISO group. Additionally, increased levels of reactive oxygen species (ROS) were observed in the LV of ISO rats, which were normalized by EMPA treatment in the IE rats. Furthermore, the ISO group exhibited higher protein expressions of CD68, SGLT2, and VCAM-1, which were attenuated by EMPA treatment. The mRNA expression of CYBA (p22phox) and NOX4, both representing subunits of NADPH oxidases, along with VCAM1, ICAM1, NOS2 (iNOS) and SELE (e-selectin) gene, endothelial activation marker, and TGF-β1, a fibrotic marker, showed elevated levels in the apex part of the left ventricle (LV) in the ISO group; these levels were significantly lower in the IE group. Conclusions These findings suggest that TTS is associated with oxidative stress-mediated endothelial dysfunction, inflammation, and pro-fibrotic responses. Moreover, SGLT2 inhibition appears to mitigate the pathology and progression of the syndrome. Therefore, SGLT2 inhibition emerges as an appealing and novel therapeutic approach for the treatment of TTS.

Abstract Introduction In atrial fibrillation (AF), there is a complex interplay between arrhythmia burden and cardiovascular risk factors leading to left atrial remodeling, an increased risk of stroke and heart failure. Most thrombi get formed in the atrial appendage. Several studies suggested that sodium-glucose cotransporter 2 inhibitors (SGLT2i), besides showing major benefits on heart failure, might lower the risk of incident AF. Therefore, we examined the expression level of SGLT2 in human right and left atrial appendages (RAA, LAA) and determined its role in pro-oxidant, pro-fibrotic and pro-thrombotic responses. Methods Human RAA and LAA were collected from patients undergoing cardiac surgery at a university hospital. The LAA was cut into a proximal part (low stasis, high shear) and a distal part (high stasis, low shear). The level of reactive oxygen species (ROS) was determined using dihydroethidium, mRNA and protein expression levels by RT-qPCR, and Western blot analysis and immunofluorescence, respectively, and the level of fibrosis by Sirius red staining. Results The distal LAA had higher mRNA levels of ICAM-1 and p53, and protein levels of ICAM-1, AT1R, p53, p21 and tissue factor than the proximal LAA. LAA displayed higher mRNA and protein levels of SGLT2 and SGLT1, pro-adhesive, pro-thrombotic, pro-remodeling, pro-fibrotic and senescence markers, and components of the angiotensin system, in addition to IL-1 mRNA levels and p-p65 NF-kB protein levels compared to RAA. These responses were associated with higher levels of oxidative stress, nitrotyrosine and fibrosis. SGLT2 immunofluorescence signals in distal LAA were colocalized with those of CD31, CD68, TNF-α and troponin T. Oxidative stress levels in distal and proximal LAA and RAA were reduced by inhibitors of NADPH oxidases, ACE1 and SGLT2, an AT1R antagonist, and by a TNF-α neutralizing antibody whereas NG-nitro-L-Arginine methyl ester, the non-selective NO synthase inhibitor, inhibited ROS in distal and proximal LAA without affecting RAA. In addition, the level of TNF-α in LAA was positively correlated to the indexed LA volume indicating a link between inflammation and remodeling. Conclusions The distal part of the LAA of patients undergoing heart surgery showed greater impaired endothelial function and senescence associated with more pronounced pro­-oxidant, pro­-remodeling and pro-­inflammatory responses than the proximal part and the RAA. These responses were associated with an increased expression level of SGLT 2 in endothelial cells, cardiomyocytes, and macrophages in areas showing an inflammatory response. Moreover, the pro-oxidant signal was sensitive to inhibitors of the local angiotensin system, NO synthase, SGLT2 and TNF-α. Thus, the AT1R/NADPH oxidases/SGLT2 pathway appears as an interesting target to blunt the stimulatory pro-oxidant signal in cardiac tissues affected by low-grade inflammation that leads to remodeling and fibrosis.

Abstract Introduction Clinical studies showed that sodium-glucose co-transporter2 inhibitors (SGLT2i) have beneficial effects in heart failure patients regardless of ejection fraction and diabetes. Such cardiac diseases are often characterized by low-grade inflammation and impaired endothelial coronary microcirculation function. Recently, angiotensin II and TNF-α were shown to induce SGLT2 expression in endothelial cells (ECs) to sustain oxidative stress leading to endothelial dysfunction. However, the role and function of SGLT2 in the human left ventricle (LV) remain unclear. Therefore, this study evaluated the expression of SGLT2 in the LV of patients with cardiac diseases and, if so, determined the cellular localization, the underlying mechanism and the functional role. Methods Human LV biopsies were collected from 20 patients subjected to valve surgery at the Nouvel Hôpital Civil, Strasbourg, France. Expression levels of targets were determined by RT-qPCR and Western blot analysis, the in situ tissue localization by immunofluorescence (IF) staining and oxidative stress by dihydroethidium staining. Results RT-qPCR analysis of LV revealed variable SGLT1 and SGLT2 mRNA levels with no significant correlation. However, SGLT2 mRNA expression showed a positive correlation with those of pro-inflammatory markers (IL-1ß, IL-6, TNF-α, MCP-1 and CD68), AT1R, and VCAM-1. Western blot analysis confirmed the heterogenous SGLT2 protein expression level in LV with a difference up to 3.5-fold among the specimens. SGLT2 protein levels showed a positive correlation with markers of oxidative stress (phospho-p65 NF-κB, AT1R, nitrotyrosine) and markers of ECs activation (VCAM-1, ICAM-1), and a negative correlation with levels of eNOS. Further, immunofluorescence analysis of LV cryosections showed SGLT2 staining in the endothelium of the coronary microcirculation, as indicated by its co-localization with the endothelial cell marker CD31, and throughout the heart tissue. SGLT2 signals were also observed in areas showing IF signals for TNF-α, phospho-p65 NF-κB, CD68 and VCAM-1 suggesting that SGLT2 expression is localized to sites of inflammation. LV specimens with high SGLT2 protein levels showed elevated levels of oxidative stress, which were inhibited by N-acetylcysteine, VAS-2870, perindoprilat, losartan, empagliflozin, and infliximab. Conclusions The findings indicate that endothelial and cardiac SGLT2 expression in the LV of cardiac patients is associated with low-grade inflammation and oxidative stress. They further indicate that the pro-inflammatory cytokines/AT1R/NAPDH oxidases/SGLT2 crosstalk contributes to sustain a pro-oxidant state. Thus, SGLT2 inhibitors might ameliorate HF by mitigating the low-grade inflammatory-related pro-oxidant state and, hence, preserve the crucial endothelial control of the cardiomyocyte function in the LV.

Abstract Background Clinical research has demonstrated that glucagon-like peptide-1 receptor (GLP1R) agonists (GLP1RA) can decrease the occurrence of major cardiovascular events in diabetic patients, regardless of their glycemic control. However, a significant challenge in understanding the positive effects of GLP1RA has been the lack of knowledge regarding the specific cellular types that express GLP1R. Despite the established cardiovascular benefits of GLP1RA, previous studies have been unable to confirm the expression of GLP1R in the heart. Aim The study aimed to investigate the expression and function of GLP1R in heart tissue. Methods We collected human cardiac tissue samples from 30 patients who underwent left ventricle (LV) biopsies (n=21) or right atrial (RA) appendage closure (n=9) at a university hospital. Gene expression levels were assessed using RT-qPCR, protein levels by Western blot analysis, in situ tissue localization of proteins by immunofluorescence (IF) staining, and the level of oxidative stress using dihydroethidium. Results LV biopsies and RA appendages showed a substantial heterogeneity in GLP1R mRNA levels. GLP1R mRNA levels exhibited a positive correlation with IL1B, IL6, TNFα, CCL2, CD68, ACE1, AT1R, and VCAM1 mRNA levels, but a negative correlation with NOS3 mRNA levels. High GLP1R expression in both RA appendages and LV biopsies was associated with increased oxidative stress levels, which were mitigated by the antioxidant N-acetylcysteine (NAC), NADPH oxidase inhibitor (VAS-2870), ACE inhibitor (perindoprilat), AT1R antagonist (losartan), TNF-α receptor neutralizing antibody (infliximab) and by GLP1R agonists (liraglutide and semaglutide) with inhibitory effects amounting to about 50-60 %. Exendin, a GLP1R antagonist reversed the effect of liraglutide and semaglutide which accounted for a receptor-mediated antioxidant effect. H-89, a PKA inhibitor equally reversed liraglutide and semaglutide effect on ROS generation suggesting a canonical GLP1R mediated effect involving the AMPK pathway. Conclusion These findings indicate that human cardiac GLP1R expression levels are correlated to low-grade inflammation and endothelial dysfunction. Increased expression levels of GLP1R were associated with higher levels of oxidative stress sensitive to GLP1RA in a canonical GLP1R AMPK-mediated manner. Therefore, the cardiovascular beneficial effects of GLP1RA might be attributable to their antioxidant and anti-inflammatory properties, specifically within the cardiac system.

Abstract Introduction Takotsubo Syndrome (TTS) is an acute heart failure (HF) syndrome induced by both emotional and physical stressors involving a surge of catecholamines and apical ballooning. Currently, there is no specific treatment for TTS. Sodium-glucose cotransporter2 inhibitors (SGLT2i) showed major cardiovascular protective effects in patients with HF independent of glycemic control. SGLT2i have also been shown to have anti-inflammatory and anti-oxidant effects, and to improve the endothelial function in experimental models of cardiac disease. However, a possible protective role of SGLT2i in TTS is unclear. Thus, this study evaluated the effect of SGLT2i on isoprenaline-induced TTS-like syndrome in rats, and determined the underlying mechanism. Methods 32 female Sprague-Dawley rats (10 weeks old) were divided into 4 groups: control, empagliflozin (30 mg/kg/day provided in the diet for 2 weeks), isoprenaline (Iso, 100 mg/kg, i.p.) to induce a TTS-like syndrome, and Iso plus empagliflozin (IE group). After 6 h of Iso injection, echocardiography was performed to determine left ventricular (LV) ejection fraction (EF) by area and heart rate (HR). At day 3, rats were sacrificed, main mesenteric artery and microvessels were collected for vascular reactivity studies, and LV was divided into base and apex to study mRNA expression levels using RT-qPCR, macrophage infiltration by in situ CD68 immunofluorescence staining, and oxidative stress using dihydroethidium. Results The injection of Iso to rats caused at 6 h an increased LVEF and HR in the Iso and the IE groups as compared to their corresponding controls. The HR response was significantly smaller in the IE group compared to Iso group whereas LVEF was similar. In addition, in mesenteric microvessels but not the main mesenteric artery, Iso group showed significant decreased relaxations to acetylcholine and increased contractile responses to phenylephrine in intact rings both of which were not observed in the IE group. Hearts showed significantly increased mRNA levels of IL-1β, TNF-α, NOX2, VCAM-1, MMP-9, TGF-β1 and collagen-1, oxidative stress levels and CD68 staining in the apex compared to the base in the ISO group, all of which were significantly reduced in IE group. Conclusions The present findings indicate that ISO injection caused in the LV elevated levels of oxidative stress, macrophage infiltration, pro-inflammatory, pro-remodeling and pro-fibrosis responses that were more pronounced in the apex compared to the base, and also endothelial dysfunction in the systemic microcirculation, all of which were significantly prevented by the empagliflozin treatment. They further suggest that SGLT2 appears as an interesting target in TTS.

Abstract Introduction Atrial fibrillation (AF), the most common cardiac arrhythmia, is associated with increased morbidity and mortality. Inflammatory infiltrates have been observed in atrial tissues from AF patients suggesting that low-grade inflammation contributes to the pro-fibrotic, pro-remodeling and pro-senescence responses of the atrial tissue favoring AF. Although sodium glucose co-transporter2 inhibitors (SGLT2i) have shown beneficial effects in heart failure, the expression and role of SGLT2 in cardiac tissues remain unclear. Purpose This study examined the expression of SGLT1 and 2 in right atrial appendages (RAA) of cardiac patients, and, if so, to determine the underling mechanism and their functional role. Methods RAA were harvested from patients undergoing coronary artery bypass surgery or aortic valve replacement at the University Hospital of Strasbourg. Patients with comorbidities such as malignancies and chronic inflammatory diseases were excluded. The expression level of markers was assessed using RT-qPCR, Western blot analysis, and immunofluorescence staining, and the level of reactive oxygen species using dihydroethidium staining. Results Analysis of RAA samples evidenced up-to a 4-fold difference in the NOS3 mRNA level. Those with low levels of NOS3 mRNA showed high levels of ICAM1, F3, MMP9, TGF-β1, TP53, CDKN1A, CDKN2A, SLCA1, SLCA2, IL1B, IL6, TNF-a and CD68 whereas the contrary was observed in those with high levels of NOS3 mRNA. SGLT1/2 mRNA levels were negatively correlated with that of eNOS and positively with those of IL-1, IL-6, TNF- α, CD68, and the senescence marker p53. Western blot analysis of RAA indicated that those expressing low levels of eNOS protein showed high levels of ICAM-1, VCAM-1, TF, MMP-2, TGF-b, SGLT1, SGLT2, p-p65, p53, p21 and p16 proteins whereas the contrary was observed in those with high levels of eNOS protein. Immunofluorescence staining indicated that SGLT1/SGLT2 signals were colocalized with those of CD31, an endothelial cell marker, in RAA, and associated with those of CD68 and TNF-α. Samples with high levels of SGLT1 and 2 displayed higher levels of oxidative stress that were inhibited by the antioxidant NAC (N-acetylcysteine), VAS-2870 (NADPH oxidase inhibitor), perindoprilat (ACE inhibitor), losartan (AT1R antagonist), empagliflozin (selective SGLT2 inhibitor), sotagliflozin (dual SGLT1/2 inhibitor) and by infliximab (TNF-α receptor neutralizing antibody). Conclusion The findings indicate that high expression levels of SGLT1 and 2 are observed in RAA showing low eNOS levels associated with pro-inflammatory, pro-senescent, pro-remodeling and pro-fibrotic responses. They were further associated with increased levels of oxidative stress sensitive to inhibitors of either the local angiotensin system, TNF-alpha or SGLT2. Thus, inhibition of SGLT2 appears to be an interesting approach to reduce the stimulatory pro-oxidant signal in atrium related to low-grade inflammation. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Boehringer Ingelheim Pharma GmbH & Co. KG

Abstract Background Clinical research showed that glucagon-like peptide-1 receptor (GLP1R) agonists (GLP1RA) reduced the incidence of major cardiovascular events in diabetic patients. The protective effect was more noteworthy in patients with established cardiovascular disease, associated with chronic low-grade inflammation resulting in endothelial dysfunction and arterial remodeling and fibrosis. Despite the clinically proven cardiovascular benefit of GLP1RA, the expression pattern of GLP1R and its function in the human vasculature remain poorly studied. Aim This study investigated the expression of GLP1R and its role in internal thoracic arteries (ITA) from patients with coronary artery disease. Methods Human ITA were collected from patients (N=30) undergoing coronary artery bypass surgery at the University Hospital of Strasbourg. Gene expression levels were assessed using RT-qPCR, protein levels by Western blot analysis, in situ tissue localization of proteins by immunofluorescence (IF) staining, and the level of oxidative stress using dihydroethidium. Results ITA showed a substantial difference in GLP1R mRNA levels reaching up to 7-fold among patients. GLP1R mRNA levels were positively correlated with those of SLC5A1, SLC5A2, F3, AT1R, IL1B, IL6, TNFα, VCAM1 and NCF1, and negatively with NOS3 mRNA levels. High GLP1R expressing ITA showed high levels of phosphorylated p65 NF-κB, cell adhesion molecule VCAM1, members of the angiotensin system (ACE1 and AT1R), remodeling and fibrotic markers (MMP9, MMP2, TGFβ) and a low level of eNOS, whereas the contrary was observed for low GLP1R expressing ITA. IF staining showed that GLP1R signals were predominantly observed in the endothelium of low GLP1R expressing ITA and in the vascular smooth muscle of high GLP1R expressing ITA. Prominent CD68 staining associated with the endothelium and the perivascular adipose tissue were observed in high but not low GLP1R expressing ITA. High GLP1R expressing ITA showed increased levels of oxidative stress, which were inhibited by the antioxidant N-acetylcysteine (NAC), NADPH oxidase inhibitor (VAS-2870), ACE inhibitor (perindoprilat), AT1R antagonist (losartan), TNF-α receptor neutralizing antibody (infliximab), selective SGLT2 inhibitor (empagliflozin), dual SGLT1/2 inhibitor (sotagliflozin) and by GLP1R agonists (liraglutide and semaglutide) with inhibitory effects amounting to about 50 to 75%. Conclusion These findings indicate that GLP1R expression levels in human ITA are dependent on low-grade inflammation and linked to endothelial dysfunction, pro-thrombotic, pro-remodeling and pro-fibrotic responses. Furthermore, they were associated with increased level of oxidative stress sensitive to inhibitors of either the local angiotensin system, SGLT2, TNFα and agonists of GLP1R. Thus, the cardiovascular beneficial effects of GLP1R agonists might be attributable to their ability to reduce the pro-oxidant stimulatory signal related to low-grade inflammation. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): Frency Society of Vascular Medicine (SFMV)

Abstract Introduction Heart transplantation is a life-saving surgical procedure for patients with end-stage cardiac dysfunction, however, such procedures are usually at risk of rejection due to acute inflammatory activation. While such inflammatory induction leaves the newly transplanted heart at risk of functional and structural remodeling subsequent to oxidative damage, current anti-inflammatory treatment options expose the patient to an elevated risk for adverse reactions in addition to absence of cardio-protective effects. Therefore, novel therapies with anti-inflammatory and cardio-protective dual effects are needed. Recently, our group has reported that low-grade inflammation is associated with upregulation of SGLT1/2 in arteries of human with cardiovascular diseases. Yet, the role and function of SGLT2 in human cardiac tissue remains poorly understood. Aim This study focuses on the expression pattern of SGLT1/2 in cardiac biopsies of heart transplanted patients and aim to identifying their functional impact. Methods Routine endomyocardial Biopsies (23) were performed for the detection of acute rejection heart transplanted patients (less than 2 years) at our University Hospital. Gene expression levels were assessed using RT-qPCR, the in situ tissue localization of proteins by immunofluorescence staining, and the level of oxidative stress by dihydroethidium staining. Results Gene expression analysis revealed strong inflammatory reaction in 5 samples indicated by at least 20-fold higher levels of mRNA of IL1B, IL6, TNFA and CD68 compared to the other 18 samples and concomitant with high expression levels of SLC5A1, SLC5A2, AT1R, CYBA, NCF1, ICAM1, VCAM1, MMP2, MMP9 and TGFB1 in contrast to low levels of NOS3. In addition, increased levels of oxidative stress were observed in the same biopsies, which were diminished by the antioxidant N-acetylcysteine (NAC), NADPH oxidase inhibitor (VAS-2870), TNF-α receptor neutralizing antibody (infliximab), ACE inhibitor (perindoprilat), AT1R antagonist (losartan), dual SGLT1/2 inhibitor (sotagliflozin) and selective SGLT2 inhibitor (empagliflozin) with inhibitory effects reaching up to 80%. Immunofluorescence staining indicated signals for nitro-tyrosine, TNF-alpha, SGLT1 and 2 in several samples. Conclusion These findings indicate that both isoforms SGLT1 and SGLT2 are expressed in the transplanted human heart and suggest a pattern of expression associated with pro-inflammatory response. They further indicate a potential protective effect of SGLT2 inhibitors in transplanted hearts through mitigating oxidative stress and hence providing a possible novel therapy for heart transplantation recipients to preserve the heart function. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Boehringer Ingelheim Pharma GmbH & Co. KG

Abstract Background Selective sodium-glucose cotransporter 2 (SGLT2) inhibitors have shown cardiovascular protection independently of glycemic control. Angiotensin II (Ang II) and H2O2 induced the expression of SGLT1 and 2 in cultured endothelial cells and isolated arteries to promote oxidative stress and endothelial dysfunction. However, the expression level and role of SGLT1 and 2 in human arteries remain poorly studied. Purpose This study examined the expression level of SGLT1 and 2 in the human internal mammary artery (IMA) obtained from bypass surgery patients, and, if so, determined the underlying mechanism and function. Methods IMAs were obtained from 40 bypass surgery patients (age 45 to 82). The expression level of target factors was assessed by Western blot analysis, immunofluorescence staining and RT-PCR, and the level of oxidative stress using dihydroethidium staining. Human kidney was used as a control tissue known to express SGLT1 and 2. Porcine coronary artery endothelial cells (CAEC) were cultured and studied at passage 1. Results Western blot analysis of 40 IMA samples indicated a high level of both SGLT1 and 2 in 16 and 17 IMAs, an intermediate level in 8 and 6 IMAs, and a low one in 16 and 17 IMAs, respectively. Immunofluorescence staining of IMA sections indicated that SGLT1 and 2 immunofluorescence signals were observed predominantly in the intima thickening and the media. The expression levels of SGLT1 and 2 were associated with p-p65 NF-kB signals but not angiotensin-converting enzyme (ACE), AT1R, MCP-1, VCAM-1. IMAs with a high expression level of SGLT1 and 2 had a high level of ROS throughout the arterial wall including the intima thickening and endothelium, which was inhibited by the antioxidant N-acetylcysteine, the ACE inhibitor perindoprilat, the AT1R antagonist losartan, and also by the dual SGLT1 and 2 inhibitor sotagliflozin and the selective SGLT2 inhibitor empagliflozin. Pro-inflammatory cytokines mRNA levels of IL-1β, TNF-α and IL-6 were detected in IMAs. Exposure of CAEC to either TNF-α, IL-1β or IL-6 caused a concentration-dependent upregulation of SGLT1 and 2. Conclusion The present findings indicate that SGLT1 and 2 expression is observed in some but not all IMAs of bypass surgery patients predominantly in the media, the intima thickening and the endothelium. High expression levels of SGLT1 and 2 are associated with NF-kB activation and oxidative stress that is prevented by a selective SGLT2 inhibitor and by a dual SGLT1/2 inhibitor. Since pro-inflammatory cytokines triggered SGLT1 and 2 expression in endothelial cells, the inflammatory burden of patients appears to be an important trigger regulating SGLT1/2 expression and the subsequent pro-oxidant response prompting pro-inflammatory and pro-thrombotic responses. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): This work was supported by an unrestricted research grant from Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany.