Dapagliflozin improves the dysfunction of HUVECs by downregulating high glucose/high fat-induced autophagy through inhibiting SGLT-2

doi:10.21203/rs.3.rs-1958565/v1

Download PDF

Research Article

Dapagliflozin improves the dysfunction of HUVECs by downregulating high glucose/high fat-induced autophagy through inhibiting SGLT-2

https://doi.org/10.21203/rs.3.rs-1958565/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Objective: To investigate the effect of Dapagliflozin (Da) on the disorders of human umbilical vein endothelial cells (HUVECs) induced by high glucose and high fat (HG/HF).

Methods: Immunohistochemistry and immunofluorescence were used to detect the SGLT-2 expression in thoracic aortic tissues. After transfected with overexpressed plasmid SLC5A2, autophagy and cell functions of HUVECs were detected with the treatment of autophagy inhibitor 3-MA (5 mM). HUVECs were exposed to mannitol (MAN), glucose/palmitate (Hg/PA), and Hg/PA/Da for 24 h, and the proliferation of HUVECs was detected by CCK-8. The protein expression levels, endothelial cell functions (cell proliferation, migration, tubular formation, apoptosis, and autophagy) in endothelial cells were evaluated.

Results: The SGLT-2 expression was found in atherosclerotic human thoracic aorta tissues and HG/PA induced HUVECs (P<0.05). After the overexpression of SGLT-2 in HUVECs, the proliferation, migration and tubule formation ability of HUVECs were inhibited, and autophagy and apoptosis were increased, which were reversed by 3-MA (P<0.05). After the addition of Sodium-glucose co-transporters 2 inhibitor, Dapagliflozin, the proliferation of HUVECs, the tubule formation, autophagy, apoptosis and migration ability of cells inhibited by HG/PA were significantly improved (P<0.05). Moreover, the increased protein expression levels of autophagy and apoptosis in HG/PA induced HUVECs were also decreased by the treatment of Dapagliflozin (P<0.05).

Conclusions: Dapagliflozin can improve the dysfunction of high glucose/high fat-induced human umbilical vein endothelial cells by downregulate autophagy through inhibiting SGLT-2.

SGLT-2

Dapagliflozin

Autophagy

Apoptosis

HUVECs

Diabetic macroangiopathy is one of the common and serious chronic complications of Type 2 diabetes mellitus (T2DM), and it is an important cause of disability and even death in T2DM patients. Atherosclerosis (AS) is the most pivotal characteristic of macroangiopathy in T2DM patients, and endothelial cell dysfunction is the early manifestation and initiation factor of diabetic macroangiopathy [2]. Hyperglycemia and hyperlipidemia can induce structural damage and dysfunction of vascular endothelial cells and smooth muscle cells, thus inducing lipoprotein deposition and accelerating AS formation. Therefore, effective drugs that correct hyperglycemia and hyperlipidemia-induced endothelial dysfunction are particularly important for delaying the occurrence and development of diabetic macroangiopathy.

In recent years, sodium-glucose co-transporters 2 inhibitor (SGLT-2i) has attracted more attentions due to its capability to decrease the risk of death in patients with cardiovascular disease. CVD-REAL research results have suggested that Dapagliflonzin, a SGLT-2i, could significantly reduce the incidence of primary compound endpoint events and produce cardiovascular gains in patients with T2DM ^[4]. It may be associated with SGLT-2i to lower glucose toxicity, alleviate the dysfunction of endothelial cells and reduce oxidative stress ^[5]. However, the mechanism of its direct cardiovascular benefits is not well understood.

Our previous researches have shown that autophagy plays a role in the occurrence and development of diabetic macrovascular disease. Excessive autophagy in vascular endothelial cells induces cell and tissue dysfunction and even large vascular lesions in patients with T2DM ^{[6, 7]}. It has been reported that SGLT-2i could induce the upregulation of both AMPK and SIRT1, thereby regulating autophagy and reducing cellular stress and tissue damage ^[8]. It suggests that darpagliflozin has cardiovascular benefits and may be associated with autophagy. Therefore, we aimed to explore the role of SGLT-2 in endothelial cells and the possible mechanisms of dapagliflozin on cardiovascular benefits.

2.1 Materials

HUVECs cells were donated by the Institute of Cardiovascular Disease, University of South China (Hengyang, China). SLC5A2 eukaryotic plasmid strains and empty vector strains (kanamycin resistant, GFP labeled) were purchased from The Gikai gene (Shanghai, China). Dapagliflozin was purchased from Astrazeneca China; 3-MA (MCE Company, China); Glucose powder, mannitol and palmitic acid were purchased from Sigma, USA; Bovine serum albumin (BSA), MTT kit, Hoechst 33258 staining solution and PMSF were purchased from Solebo (Beijing, China); Sodium hydroxide (Taishan Chemical); Lipofectamine®2000 Liposome reagent (Thermo Fisher); Endotoxin-free Plasmid Small Extraction medium dose kit and BCA Protein quantitative kit were purchased from TianGen (Beijing, China); Rabbit anti-human SGLT-2 antibody, rabbit anti-human LC3 mab and rabbit anti-human Beclin 1 antibody were obtained from Wuhan Sanying Proteintech (Wuhan, China); Rabbit anti-human P62 antibody (Cell Signaling Technology, USA); Rabbit anti-human ATG5 antibody, rabbit anti-human Casepase 3 antibody, rabbit anti-human Bax antibody, mouse anti-human Bcl-2 antibody were obtained from Cell Signaling Technology (USA); Rabbit anti-human SGLT-2 antibody, mouse anti-human GAPDH antibody and mouse anti-human β-actin antibody were bought from Nanjing Baode (Nanjing, China); Green fluorescent goat anti-rabbit secondary antibody (Jackson ImmunoResearch, USA); Red fluorescent goat anti-rat secondary antibody (Invitroge, USA); FluoroshieldTM with DAPI and BD Matrigel™ Basement Membrane Matirx glue were obtained from Sigma-Aldrich (Germany).

2.2 Reagent configuration

(1) High-glucose solution (HG) configuration: 50% glucose injection was diluted into five different concentrations of 11, 22, 33, 44 and 55 mmol/L, respectively; (2) High-fat solution (PA) configuration: 0.5 mM PA stock solution was diluted to five different concentrations of 0.125, 0.25, 0.5, 1.0 and 2.0 mmol/L with complete medium; (3) Control group solution (MAN) configuration: The 20% mannitol solution was prepared into a mannitol working solution with a concentration of 16.5 mmol/L in a complete medium, and mixed with the corresponding amount of 10% palmitic acid-free BSA in HG/PA induced group.

2.3. Clinical tissue specimen collection

Patients with aortic dissection who underwent surgical treatment in the Second Affiliated Hospital of Nanhua University from September 2019 to September 2020 were selected, and chest aortic vascular tissue samples were collected after the informed consent of the patients and their families. This study was approved by the Ethics Committee of the Second Affiliated Hospital of the University of South China.

Inclusion criteria (referring to previous research criteria [9]): Aortic dissection was diagnosed in combination with the patient's age, previous history, clinical diagnosis, thoracic and abdominal aortic CTA, and surgical treatment of aortic dissection was performed. Exclusion criteria: (1) damage of thoracic aortic wall caused by exogenous injury; (2) damage to the wall of the thoracic aorta caused by infectious diseases, such as tuberculosis, bacterial endocarditis, or treponema pallidum; (3) thoracic aorta injury caused by non-infectious arteritis, such as giant cell arteritis, multiple arteritis, etc. (4) combined with malignant tumors.

2.4 Immunohistochemistry

Tissue specimens were collected. The tissue slides were placed in xylene, gradient alcohol, running water and distilled water successively after embedding, sectioning and pasting. The tissue slides were then placed in 3% H₂O₂, clean water and distilled water. Finally, it was immersed in the prepared citric acid antigen repair solution. Adding the blocking solution, brewing with PBS, adding the secondary antibody at the labeled place, and standing in the oven at room temperature. DAB chromogenic agent was dropped at the marks to develop color and clean the mark. After stopping the chromogenic process, hematoxylin was redyed, dried and prepared, and observed under microscope.

2.5 Cell culture

HUVECs were cultured in low-sugar DMEM (NG, 5.5 mmol/L) medium containing 10% fetal bovine serum (FBS). The humidification was stimulated for 24 h with glucose/palmitic acid concentration gradients (11 mM/0.125 mM, 22 mM/0.25 mM, 33 mM/0.5 mM, 44 mM/1.0 mM, 55 mM/2.0 mM, and Dapa (1 µM/2 µM/5 µM/10 µM), respectively. At the same time, the control group (16.5 mM mannitol + 10% BSA + 0.1 M sodium hydroxide) was used to exclude the cytotoxic effects caused by HA/PA.

2.6 CCK-8 assay

HUVECs were cultured in 96-well plates and treated with corresponding drugs for 24 h. CCK-8 solution was added into each well and incubated away from light for 4 h. The OD value of each well was measured at 450 nm with a microplate reader.

2.7 Scratch test

Three parallel lines were drawn on the back of the 6-well plate, and HUVECs were inoculated in the 6-well plate and treated with corresponding drugs, and 1–2 multiple holes were set. 200 µL sterile spear head was used to mark each well with 2–3 stripes, which were perpendicular to the back line. The migration of HUVECs at 0 h, 24 h and 48 h after scratches was recorded by inverted fluorescence microscope.

2.8 Tubular experiments

200 µl of matrigel was added to each well of a 48-well plate and incubated in an incubator. Then, the drug-treated cell suspension was added after the matrigel solidified. After HUVECs were formed into tubes, the number of HUVECs and the size of the tube lumen were observed by using MetaMorph software system to quantify the ability of cells to form tubes.

2.9 Hoechst staining

HUVECs were inoculated in the 6-well plates and treated with corresponding drugs. Hoechst 33258 working solution was added to each well. After continued incubation for 24 h, images were collected under an inverted fluorescence microscope.

2.10 Autophagy bodies detecting by electron microscopy

HUVECs were inoculated in the 6-well plates and treated with corresponding drugs. After washed with PBS, cells were gently scraped off and transferred to a 1.5 ml centrifuge tube. electron microscope fixative was added. After sealed, the cells were sent to Wuhan Sewell Company (Wuhan, China) for electron microscope treatment and take pictures.

2.11 Cellular immunofluorescence analysis

HUVECs was inoculated into the cell slides of 24-well plates and treated with corresponding drugs. 4% paraformaldehyde solution was added dropwise and fixed at 4°C. After washed, cells were blocked with 5% BSA serum at 4°C overnight. Primary antibody was incubated overnight at 4°C. After washed, anti-rabbit secondary antibody with green fluorescence (Alexa Fluor® 488)-conjugated was diluted at a ratio of 1:250 and incubated for 1 h at room temperature in the dark. The slides were mounted in FluoroshieldTM with DAPI mounting medium, left to dry in a dark room. Images were collected under an inverted fluorescence microscope.

2.12 Western blotting

The proteins were extracted by lysis solution, and sample protein concentrations was measured by BCA kits. Protein was denatured and placed at -20°C for short-term storage. SDS-PAGE was used to perform electrophoresis. The proteins were transferred to PVDF membrane and blocked with BSA at room temperature for 2 h. Rabbit anti-human SGLT-2, rabbit anti-human LC3-I/II, rabbit anti-human P62, rabbit anti-human ATG5, rabbit anti-human Beclin-1, Rabbit anti-human Bcl-2, rabbit anti-human Caspase 3, rabbit anti-human Bax, mouse anti-human GAPDH antibody, and mouse anti-human β-actin antibodies were incubated at 4°C overnight. The secondary antibody was incubated at room temperature for 1 h. After washing with TBST solution, ECL chemoluminescence solution was developed to collect pictures. Analysis was determined with Image J image analysis software.

2.13 Transfection with overexpression and empty plasmid

HUVECs were inoculated into a 6-well plate and transfected with 50–80% confluence. The SGLT-2 overexpression plasmid and Lipofectamine 2000 liposome were transfected with 1:1.5 (optimal transfection ratio). The transfection was performed according to Lipofectamine 2000 instruction. The transfection efficiency and the fluorescent expression were observed by Fluorescence microscope.

2.14 Statistical analysis

SPSS 25.0 software were used for statistical analysis. All experiments were replicated three times independently and the data were represented by Mean ± standard deviation (SD). The results of the Western blot experiment were analyzed by Image J software for gray value analysis of the stripe chart, and the single-factor analysis of variance (ANVOA) was followed and Tukey's post-hoc intra-group difference statistics were performed. P < 0.05 was statistically significant for the difference.

3.1 SGLT-2 expression level in the endothelial tissue of the human thoracic aorta.

A total of 9 cases of thoracic aortic tissue, 7 males and 2 females, were collected in this study, including 1 case of diabetes mellitus and 8 cases of hypertension. The average age was 49.8 ± 4.0 years. The difference between the above indicators was not statistically significant (P > 0.05), and the collected data were clinically true. Experimental group included vascular tissue specimens of thoracic aorta (n = 9); Control group included adjacent thoracic aorta vascular tissue specimens (n = 2). Double fluorescent staining of collected human thoracic aortic tissue specimens were performed to detect the markers, CD31 and SGLT-2 ^[10]. As shown in Fig. 1A, 1B, SGLT-2 was expressed in the thoracic aortic endothelium, and SGLT-2 positive cells and fluorescent spots in the diseased thoracic aortic tissue were elevated in the lesion tissues compared to the lesion side tissues.

3.2 SGLT-2 overexpression induced HUVECs dysfunction and upregulated autophagy

HUVECs were transferred with SGLT-2 overexpressed plasmid (Figs. 2A, 2B). When SGLT-2 was overexpressed, the proliferation of HUVECs was reduced (Fig. 2C), the migration of HUVECs (Fig. 2D, 2E), the tubular capacity (Fig. 2F, 2G) were inhibited, and the apoptosis of cells was enhanced compared with the control group and the vector group (P < 0.05). The migration (Fig. 2D, 2E, 2F) and apoptosis of HUVECs (Fig. 2H, 2I, 2J, 2K) were enhanced in the HUVECs were transferred with SGLT-2 overexpressed plasmid in comparison with the control group (P < 0.05). After transferred with SGLT-2 overexpressed plasmid, the LC3-II to LC3-I ratio was significantly higher than that of the control group (Fig. 2L, 2P) (P < 0.05). The protein expression levels of Beclin1 and ATG5 were upregulated in comparison with the control group (Fig. 2L, 2M, 2N), and the p62 protein expression was significantly reduced (Fig. 2L, 2O), and the number of autophagosomes in HUVECs was also increased (Fig. 3) (P < 0.05).

3.3 SGLT-2-regulated autophagy improved endothelial cell dysfunction

To elucidate the role of SGLT-2-regulated autophagy in dapagliflozin improved endothelial disorders, we added the autophagy inhibitor, 3-methyladenine (3-MA), to observe its effect on endothelial cell function. With the addition of 3-MA, the proliferation of HUVECs (Fig. 4A), migration (Fig. 4E, 4F) and tubing capacity (Fig. 3G, 3H) were restored (P < 0.05). At the same time, SGLT-2-induced endothelial cell apoptosis was reduced (Fig. 4B, 4C, 4D). These results suggested that SGLT-2- regulated autophagy played an important role in improving endothelial dysfunction.

3.4 Effect of dapagliflozin on HG/PA-induced HUVECs dysfunction

HUVECs were stimulated with different concentrations of HG/PA for 24 h, and higher concentrations of HG/PA were found to significantly reduce cell proliferation (Fig. 5A). When HG/PA was combined with different concentrations of dapagliflozin, dapagliflozin upregulated HG/PA-reduced cell proliferation (Fig. 5B), excluding the cytotoxic effects caused by high osmotic pressure and palmitate lysate (P < 0.05).

HUVECs were cultured in the normal control group (Control, 5.5 mM), osmotic solvent group (MAN, 16.5 mM mannitol + palmitic acid solute), HG/PA concentration (HG + PA, 22 mM/0.25 mM), and HG/PA combined with optimal dagliflozin action concentration (HG + PA + Da, 22 mM/0.25 mM + 5 µM). The SGLT-2 protein expression in the HG/PA group was significantly increased compared with the Control group, and the fluorescent spots were also significantly increased, and the expression (Fig. 6A, 6B) and the formation of fluorescent spots were reduced after the addition of dapagliflozin (Fig. 6C).

In addition, compared with the Control group, the HG/PA group significantly reduced the migration and tube-forming capacity of HUVECs, while the addition of dapagliflozin restored the migration (Fig. 5C, 5D) and tube-forming (Fig. 5E, 5F) capacities of HG/PA decline (P < 0.05). Similarly, HG/PA significantly increased the apoptosis of HUVECs, while the addition of dapagliflozin significantly reduced HG/PA-induced apoptosis (Fig. 6G, 6H, 6I, 6J, 6K) (P < 0.05). In summary, HG/PA can induce the upregulated SGLT-2 protein expression, while dapagliflozin had a direct protective effect on HG/PA-induced HUVECs dysfunction by inhibiting SGLT-2.

3.5 Dapagliflozin recovered HG/PA-upregulated autophagy in HUVECs by inhibiting SGLT-2

We further explored whether dapagliflozin improved endothelial dysfunction by inhibiting SGLT-2 was associated with autophagy. Compared with the control group, the ratio of LC3-II to LC3-I in the HG/PA group was significantly increased, while the ratio of LC3-II to LC3-I was reduced after the addition of dapagliflozin (P < 0.05, Fig. 7A, 7E). The protein expression trends of Beclin1 and ATG5 were similar to that of LC3 (P < 0.05, Fig. 7A, 7B, 7C). However, the p62 expression was significantly increased in the HG/PA group compared with the control group, and the p62 expression was restored after the addition of dapagliflozin (P < 0.05, Fig. 7A, 7B, 7D). Whether the autophagic stream was activated or inhibited, the LC3 protein level changed rapidly, and P62, as the substrate of autophagy, tended to take a long time to degrade, so the P62 protein change had a certain lag ^[11]. To this end, we extended the stimulation time of HG/PA, and LC3-II protein expression gradually increased with the prolongation of HG/PA stimulation time, while P62 protein peaked at 48 h and decreased at 72 h (Fig. 7F, 7G, 7H). Meantime, electron microscopy showed that HG/PA significantly increased the number of autophagosomes in HUVECs, while the addition of dapagliflozin reduced the number of autophagosomes stimulated by HG/PA (Fig. 8).

As the most important glucose transporters, SGLT-2 is mainly distributed in the S1 and S2 segments of the proximal tubules of the kidneys, which is responsible for reabsorbing 90% of the glucose in the proximal tubular fluid of the kidneys ^{[12, 13]}. Recent studies have shown that SGLT-2 can be highly expressed in highly differentiated lung adenocarcinoma, pancreatic cancer, and nonalcoholic steatohepatitis (NASH)-associated hepatocellular carcinoma (HCC) ^[14–16]. In addition, high glucose not only increases the expression level of SGLT-2 in endothelial cells, but also upregulates the expression of SGLT-2 in pigs' coronary arteries ^[17]. However, whether SGLT-2 can be expressed on lesion human vascular endothelial cells is not fully determined. In this study, we found decreased SGLT-2 protein expression in the diseased human thoracic aortic tissue and HG/HA-induced HUVECs, and the SGLT-2 expression was up-regulated, and the SGLT-2 fluorescent spots was increased in HG/PA-stimulated HUVECs. This suggested that SGLT-2 may be associated with the development of large vascular lesions in patients with T2DM.

In in vitro experiments, over-expressing SGLT-2 could significantly reduce the cell proliferation, angiogenesis and migration capacity of HUVECs, and increase the apoptosis of HUVECs. Long-term exposure to HG/PA has been shown to significantly inhibit cellular and vascular functions ^{[18, 19]}. Both endothelial cells isolated from the aorta of high-fat-fed diabetic animals and HUVECs exposed to HG/PA stimulation had significantly increased cell apoptosis compared with normal cells ^[20]. Our experiments also had shown that HG/PA not only changed the morphology and quantity of HUVECs, but also significantly reduced the proliferation ability, angiogenesis and migration ability of HUVECs, and significantly increased the apoptosis and apoptosis-related proteins of HUVECs. As a result, both over-expressed SGLT-2 and HG/PA interfere with the function of endothelial cells. Of course, it has been reported that lower concentrations of glucose and palmitic acid can be used as nutrients to promote cell survival and maintain cell function, while high concentrations can damage cells ^[21]. The reason for the difference may be related to the minimum action concentration level and the related mode of action, but the specific mechanism and reason still need to be further confirmed and explored.

Autophagy is a pervasive stress response in vascular cells and also thought to be a process that copes with environmental stress, which plays an important role in cell survival and promoting plaque stabilization ^[22]. From the perspective of plaque stability in patients with diabetic macrovascular disease, excessive autophagy is harmful to the body ^[23], which can damage endothelial cells ^[24] block angiogenesis, and promote endothelial dysfunction ^[7]. As confirmed in our experiments, HG/PA could significantly up-regulate the autophagy level in HUVECs, and induce cellular dysfunction, such as weakening proliferation, inhibiting migration, reducing tubularization, and increasing apoptosis. By detecting autophagy- related proteins and autophagosomes, we confirmed that SGLT-2 could significantly up-regulate the autophagic protein levels in HUVECs and significantly increase the number of autophagosomes. The autophagy inhibitor, 3-MA, could partially restore the dysfunction of HUVECs and reduce the apoptosis of cells. Accordingly, SGLT-2-regulated autophagy plays an important role in improving endothelial disorders. However, whether SGLT-2 has the ability to bind to or directly interact with autophagy-related proteins still needs to be further studied.

As a novel hypoglycemic drug, SGLT-2i can reduce the re-absorption of glucose by the kidney proximal tubules through the non-dependent mechanism of action of insulin, increase the amount of urine glucose excreted, and thereby reduce the concentration of blood glucose ^[25]. There is growing clinical evidence that SGLT-2i has become an important means of preventing and treating cardiovascular disease in patients with T2DM ^{[26, 27]}. In our study, we found that dapagliflozin could significantly restore the proliferation, angiogenesis and migration of HIVECs inhibited by HG/PA, reduce HG/PA-induced apoptosis, and significantly improve the dysfunction of HUVECs. However, the mechanism by which SGLT-2i regulates endothelial function in diabetic conditions remains complex. In addition, the SGLT-2 inhibitor Empagliflozin significantly improves its LC3II/I ratio, reduces p62 expression, and not only improves the autophagy level of diabetic rats, but also exerts anti-inflammatory effects and protects cardiac metabolism ^[28]. And similar results were found in our study, SGLT-2i, dapagliflozin, can significantly reduce the HG/PA-induced HUVECs autophagy levels by down-regulating autophagic protein expression such as LC3-II, Beclin1, ATG5, restoring the p62 protein expression, and reducing the number of autophagosomes.

In conclusion, dapagliflozin may regulate autophagy by directly affecting SGLT-2 on vascular endothelial cells, thereby improving endothelial cell dysfunction and alleviating AS formation.

Compliance with ethical standards

Conflict of interest:

The authors declare that they have no conflict of interests.

Funding Information:

Natural Science Foundation of Hunan Province (2019JJ40262)

Huang D, Refaat M, Mohammedi K, et al. Macrovascular Complications in Patients with Diabetes and Prediabetes. BioMed Research International. 2017; 2017(1):1-9.
Erusalimsky JD. Vascular endothelial senescence: from mechanisms to pathophysiology. J Appl Physiol. 2009; 106: 326-332.
Xiang L, Mittwede P N, Clemmer J S. Glucose homeostasis and cardiovascular alterations in diabetes. Compr Physiol. 2015; 5(4): 1815-39
Kosiborod M, Cavender MA, Fu AZ. Lower risk of heart failure and death in patients initiated on sodium-glucose co-transporter-2 inhibitors versus other glucose-lowering drugs: the CVD-REAL study (comparative effectiveness of cardiovascular out-comes in new users of sodium-glucose) cotransporter-2 inhibitors. Circulation. 2017; 136(3): 249-259.
Han JH, Oh TJ, Lee G, et al. The beneficial effects of empagliflozin, an SGLT2 inhibitor, on atherosclerosis in ApoE −/− mice fed a western diet. Diabetologia. 2017; 60(2): 364-376.
De Meyer GR, Grootaert MO, Michiels CF, et al. Autophagy in vascular disease. Circulation research. 2015; 116(1): 468-479.
Rezabakhsh A, Rahbarghazi R, Malekinejad H, et al. Quercetin alleviates high glucose-induced damage on human umbilical vein endothelial cells by promoting autophagy. Phytomedicine. 2019; 56(1):183-193.
Milton Packer, M.D. Interplay of AMPK/SIRT1 activation and sodium influx inhibition mediates the renal benefits of SGLT2 inhibitors in type 2 diabetes: a novel conceptual framework. Diabetes Obes Metab. 2020; 2020(1): 1-9.
YD Sun, Y Xiao, HY Sun, et al. miR-27a regulates vascular remodeling by targeting endothelial cells' apoptosis and interaction with vascular smooth muscle cells in aortic dissection.Theranostics. 2019; 9(25): 7961-7975.
GY Guan, JJ Xie, YM Dai, et al. TFPI2 suppresses the interaction of TGF-β2 pathway regulators to promote endothelial-mesenchymal transition in diabetic nephropathy. J Biol Chem. 2022; 101725.
Q Zheng, H Su, MJ Ranek, et al. Autophagy and p62 in cardiac proteinopathy. CircRes. 2011; 109: 296−308.
EG Lander, LD Raquel, Y Gautham, et al. Receptor for Advanced Glycation End Products (RAGE) and Mechanisms and Therapeutic Opportunities in Diabetes and Cardiovascular Disease: Insights from Human Subjects and Animal Models. Front Cardiovasc Med. 2020; 7: 37.
Anastasia Poznyak, Andrey V Grechko, Paolo Poggio, et al. The Diabetes Mellitus–Atherosclerosis Connection: The Role of Lipid and Glucose Metabolism and Chronic Inflammation. Int J Mol Sci. 2022; 21(5): 1835.
Scafoglio CR, Villegas B, Abdelhady G, et al. Sodium-glucose transporter 2 is a diagnostic and therapeutic target for early-stage lung adenocarcinoma. Sci Transl Med. 2018; 10(467): 5933-47.
Di Franco A, Cantini G, Tani A, et al. Sodium-dependent glucose transporters (SGLT) in human ischemic heart: A new potential pharmacological target. Int J Cardiol. 2017; 243(243): 86-90.
Teruo Jojima, Sho Wakamatsu, Masato Kase, et al. The SGLT2 Inhibitor Canagliflozin Prevents Carcinogenesis in a Mouse Model of Diabetes and Non-Alcoholic Steatohepatitis-Related Hepatocarcinogenesis: Association with SGLT2 Expression in Hepatocellular Carcinoma. Int J Mol Sci. 2019; 20(20): 5237.
Sonia Khemais-Benkhiat, Eugenia Belcastro, Noureddine Idris-Khodja, et al. Angiotensin II‐induced redox‐sensitive SGLT1 and 2 expression promotes high glucose‐induced endothelial cell senescence. J Cell Mol Me. 2020; 24(3): 2109-2122.
Jingqi Fu, Qi Cui, Bei Yang, et al. The impairment of glucose-stimulated insulin secretion in pancreatic β-cells caused by prolonged glucotoxicity and lipotoxicity is associated with elevated adaptive antioxidant response. Food Chem Toxicol. 2017; 100: 161-167.
Anil Bhatta, Lin Yao, Zhimin Xu, et al. Obesity-induced vascular dysfunction and arterial stiffening requires endothelial cell arginase 1. Cardiovasc Res. 2017; 113(13): 1664-1676.
J Liu, ZJ Meng, L Gan, et al. C1q/TNF-related protein 5 contributes to diabetic vascular endothelium dysfunction through promoting Nox-1 signaling. Redox Biol. 2020; 34: 101476.
Y Zhang, RX Zhan, JQ Chen, et al. Pharmacological activation of PPAR gamma ameliorates vascular endothelial insulin resistance via a non-canonical PPAR gamma-dependent nuclear factor-kappa B trans-repression pathway. Eur J Pharmacol. 2015; 754: 41-51.
Crusio WE, Dong H, Radeke, HH, et al. Autophagy: Biology and Diseases. Advances in Experimental Medicine and Biology. 2009: 213-270.
Xie Y, You SJ, Zhang YL, et al. Protective role of autophagy in AGE-induced early injury of human vascular endothelial cells. Mol Med Rep. 2011; 4(3): 459-64.
Liu J, Wu J, Sun A, et al. Hydrogen sulfide decreases high glucose/palmitate-induced autophagy in endothelial cells by the Nrf2-ROS- AMPK signaling pathway. Cell Biosci. 2016; 6:33.
YG Niu, R Liu, CC Guan, et al. Structural basis of inhibition of the human SGLT2-MAP17 glucose transporter. Nature. 2022; 601(7892): 280-284.
Kosiborod M, Lam CSP, Kohsaka S, et al. CVD-REAL Investigators and Study Group. Cardiovascular Events Associated with SGLT-2 Inhibitors Versus Other Glucose-Lowering Drugs: The CVD-REAL 2 Study. J Am Coll Cardiol. 2018; 71(23): 2628-2639.
Nyström T, Bodegard J, Nathanson D, et al. Novel oral glucose-lowering drugs are associated with lower risk of all-cause mortality, cardiovascular events and severe hypoglycaemia compared with insulin in patients with type 2 diabetes. Diabetes Obes Metab. 2017; 19(6): 831-41.
Aragón-Herrera A, Feijóo-Bandín S, Otero Santiago M, et al. Empagliflozin reduces the levels of CD36 and cardiotoxic ipids while improving autophagy in the hearts of zucker diabetic fatty rats. Biochem. Pharmacol 2019; 170(1): 1136-77.

Download PDF

Version 1

posted

You are reading this latest preprint version

Dapagliflozin improves the dysfunction of HUVECs by downregulating high glucose/high fat-induced autophagy through inhibiting SGLT-2

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Materials

2.2 Reagent configuration

2.3. Clinical tissue specimen collection

2.4 Immunohistochemistry

2.5 Cell culture

2.6 CCK-8 assay

2.7 Scratch test

2.8 Tubular experiments

2.9 Hoechst staining

2.10 Autophagy bodies detecting by electron microscopy

2.11 Cellular immunofluorescence analysis

2.12 Western blotting

2.13 Transfection with overexpression and empty plasmid

2.14 Statistical analysis

3. Results

3.1 SGLT-2 expression level in the endothelial tissue of the human thoracic aorta.

3.2 SGLT-2 overexpression induced HUVECs dysfunction and upregulated autophagy

3.3 SGLT-2-regulated autophagy improved endothelial cell dysfunction

3.4 Effect of dapagliflozin on HG/PA-induced HUVECs dysfunction

3.5 Dapagliflozin recovered HG/PA-upregulated autophagy in HUVECs by inhibiting SGLT-2

4. Discussion

5. Conclusion

Declarations

References

Status:

Version 1