Study Of Mechanisms In Restenosis After PTA In Diabetic Lower Extremity Peripheral Arterial Disease And Sulfonylureas’ Extrapancreatic Hypoglycemic Effects

BackgroundLower extremity peripheral arterial disease in diabetes (Diabetic LEAD), one of the major clinical manifestations of atherosclerosis, is highly prevalent in diabetic patients, and may cause intermittent claudication and critical limb ischemia. Due to some advantages, such as good effect, little trauma, and fast recovery, percutaneous transluminal angioplasty (PTA) is gradually becoming the main treatment strategies for Diabetic LEAD. However, the application and long-term effect of PTA to Diabetic LEAD is largely limited due to the high restenosis (RS) rate. RS of larger blood vessels can be prevent or improved through the endovascular stenting implantation, but the effect of artery stent in Diabetic LEAD in which the lesions were located mainly in infrapopliteal arteries, remains to be seen. PTA is still the main treatment of Diabetic LEAD. RS caused by PTA still remains to be a tough problem for the treatment of Diabetic LEAD. Exploring hazards and the mechanism of the RS lesions is particularly important for the intervention treatment.RS vascular tissue in human is hard to obtain, efforts to elucidate the mechanisms of RS as well as the studies to prevention and treatment would be greatly aided by in vitro and animal models study. In vitro studies cannot fully explain the mechanism of RS and at present in most in vivo studies, balloon injury surgery was used, through which, the animal model established has limitations and couldn’t mimic human restenosis lesions well. An ideal animal model of restenosis mimicking human lesions is still lacking, which has limited the progress of basic research in restenosis. In this study, a double-injury surgery was explored to mimic the process of restenosis in human, and to establish a restenosis animal model. Based on this model, hyperglycemia’s effect on and the mechanism of restenosis were also detected.Objectives(1) To establish a restenosis animal model which is identical to humans, and elucidate the mechanisms of RS.(2) By comparing RS with atherosclerosis to explain the difference between them and assess hyperglycemia’s effects on restenosis after PTA, then further elucidate the mechanisms of RS.Materials and methods1. Animals model and grouping(1) Diabetic restenosis versus non-diabetic restenosis;(2) Diabetic atherosclerosis versus non-diabetic atherosclerosis.Three-to-four-months-old male New Zealand white rabbits were obtained. The rabbits were allowed to acclimatize for at least 7 days, and then were fed with a high-cholesterol diet.All rabbits were randomly divided into four groups:① The diabetic restenosis group:i) In the first week of the experiment, rabbits received one injection of alloxan (80 mg/kg) via ear vein to induce type 1 diabetes, ii) In the second week of the experiment, balloon injury surgery was taken to establish atherosclerotic vessel, iii) In the sixth week of the experiment, Doppler ultrasonography demonstrated atherosclerosis stenosis in the injured vessel and PTA surgery was taken.② Non-diabetic restenosis group:ⅰ) In the first week of the experiment, rabbits received injection of normal saline via ear vein to induce type 1 diabetes.ⅱ) In the second week of the experiment, balloon injury surgery was taken to establish atherosclerotic vessel.ⅲ) In the sixth week of the experiment, Doppler ultrasonography demonstrated atherosclerosis stenosis in the injured vessel and PTA surgery was taken.③ The diabetic atherosclerosis group:ⅰ) In the first week of the experiment, rabbits received one injection of alloxan (80 mg/kg) via ear vein to induce type 1 diabetes.ⅱ) In the sixth week of the experiment, balloon injury surgery was taken to establish atherosclerotic vessel.④ Non-diabetic atherosclerosis group:ⅰ) In the first week of the experiment, rabbits received injection of normal saline via ear vein to induce type 1 diabetes.ⅱ) In the sixth week of the experiment, balloon injury surgery was taken to establish atherosclerotic vessel.2. Glycosylated hemoglobin test:before killing the animals, blood sample was obtained and glycosylated hemoglobin was tested.3. Tissue harvesting and histological processingThe animals were killed under anesthesia on day 28 after the last surgical procedure. Restenosis and atherosclerotic vessels were harvested, fixed in 4% paraformaldehyde and embedded in paraffin. The sections (5 mm) were stained with hematoxylin and eosin (HE) for general appearance, Masson’s trichrome for collagen, and elastic van-Gieson dye for elastin and observed by microscopy. Total vessel area and the area of lumen, media, and neointima were measured using Image Pro-Plus Software. PCNA, α-actin and CD68 antibody were used to exam the proliferative cells, smooth muscle cells and macrophages in the plaque, respectively.4. Statistical analysesAll data are presented as mean±S.D. Comparisons were assessed by unpaired t-test with SPSS Software (version 20.0, SPSS China). A value of P<0.05 was considered statistically significant.Results1. AnimalsNonfasting blood glucose levels were kept identical in all animals. Of the 24 alloxan treated rabbits,16 developed hyperglycemia. After all surgical procedures, three animals (two in the diabetic atherosclerosis group and one in the diabetic restenosis group) died due to severe hyperglycemia. Three rabbits (two in the non-diabetic atherosclerosis group and one in the non-diabetic restenosis group) died during ultrasound examination and three (one in the non-diabetic restenosis group and two in the diabetic atherosclerosis group) died of thrombosis.2. Body mass and blood glucoseThe differences in body mass in rabbits between four groups, before and after the experiment weren’t significant. Baseline glucose levels in the four groups were the same. One week after the injection of alloxan, blood glucose in rabbits in diabetic restenosis and atherosclerosis groups were markedly increased and maintained in a high level throughout the experiment. Glycosylated hemoglobin levels were consistent with the blood glucose condition.3. Doppler ultrasonography imagingIn the sixth week of the experiment, balloon injury surgery was taken. Atherosclerosis plaques were established at sites of vascular injury. Vessels in the diabetic and non-diabetic atherosclerosis groups were normal. Stenoses were established in all rabbits in four groups at the end the experiment.4. Stenosis rate and intima/media ratioThe rate of stenosis in the diabetic restenosis group was not significantly different from the non-diabetic restenosis group. Whereas the diabetic atherosclerosis group showed a higher stenosis rate than the non-diabetic atherosclerosis group. Tunica media in rabbits in restenosis groups were thicker than that in atherosclerosis groups, in which in the diabetic restenosis group was the thinnest and the non-diabetic atherosclerosis group was the thickest. The calculated intima/media ratio results were consistent with stenosis rates.5. Plaque components and PCNA indexImmunohistochemical analysis revealed a significantly higher smooth muscle cell content (α-actin) in restenotic plaques while atherosclerotic plaques contained significantly more macrophages (CD68). In samples from both diabetic and non-diabetic models, large numbers of SMCs were observed in the intima of restenotic plaques, while fewer SMCs were found in the atherosclerotic plaques. The content of macrophages in plaques in diabetic atherosclerosis group was significantly higher than that in non-diabetic atherosclerosis group.The PCNA index in the diabetic atherosclerosis group was significantly higher than that in the non-diabetic atherosclerosis group. In contrast, in both the diabetic and non-diabetic restenosis groups, PCNA index was both markedly increased in the restenotic plaques and no significant difference was found between these two groups.Conclusions(1) This study successfully established restenosis animal model mimic the process of restenosis after PTA in human.(2) Hyperglycemia is an independent risk factor for atherosclerosis, but it has no evident effect on restenosis. The main reason is that the processes of atherosclerosis and restenosis may involve different pathological mechanisms.(3) Unlike atherosclerotic plaques in which inflammation are major mediators, restenotic plaques is typically hypercellular with foci of vascular SMCs and extracellular matrix. The migration of vascular SMCs from the media to the intima may be the principal step in the development of restenosis.BackgroundPercutaneous transluminal angioplasty (PTA) is widely used in the treatment of lower extremity peripheral arterial disease in diabetes (Diabetic LEAD) in recent years.However, restenosis occurred in dilated position after PTA restricts its long-term efficacy. To know the factors influencing the development of restenosis is a matter of great urgency. Hypoglycemic agents are routine treatment for diabetic mellitus, while researches showed that these agents were concerned with restenosis, in which the effect of sulfonylureas (SUs) on restenosis has been found, but the mechanism is unclear.SUs are one of the most common oral hypoglycemic drugs and have been applied to clinic for several decades. As we known, SUs act by binding to the SUR subunit of the ATP-sensitive potassium (KATP) channel on pancreatic 13-cells, inducing channel closure, stimulating insulin secretion and reducing the blood glucose. Recently, the SUs has received widespread attention for its’ extrapancreatic effect. While actually, studies showed that SUR exists not only in the pancreatic 13-cells but also in peripheral tissues (SUR2A in heart and SUR2B in VSMCs). And SUs can not only bind SUR receptors on pancreatic 13-cells, but also act on peripheral SURs and close the KATP channel in extrapancreatic tissues which had been demonstrated to affect the physiological and pathological process of cellar function and metabolism.In part I, we have demonstrated that the migration and proliferation of vascular smooth muscle cells (VSMCs) were the main mechanisms in the development of restenosis. Blocking this process is of great effect on restenosis prevention and cure. SUs act by binding to the SUR subunit of the KATP channel, and a subunit of SUR—--SUR2B was widely expressed on VSMCs, these indicated that SUs may involve in the regulation of vascular smooth muscle cells’s physiological function by interaction with SUR2B. Whether could SUs regulate VSMCs’ proliferation and migration or not still remains unknown. The present study was carded out to answer this question.Objectives(1) To investigate the effects of different SUs on VSMCs’ migration and proliferation in vitro study.(2) To clarify the mechanism of SUs’ effect on VSMCs’ migration and proliferation.(3) To analysis the relationship between the media of SUs on VSMCs’ migration and proliferation with their effect on the process of restenosis.Materials and methods1. Cell culture and groupingHA-VSMC cell lines are bought from ScienCell companies (United States). The 4th and 6th generations were use in subsequent experiment.Grouping:① Control group; ② Glimepiride group; ③ Glibenclamide group; ④ Gliclazide group.2. Immunofluorescence assay: SUR2 antibody was used to detect SUR2 expression in HA-VSMC.3. Dose selectionThe effects of the three SUs (glimepiride, glibenclamide and gliclazide) on HA-VSMC’ vitality were determined. Inhibition rate of cell vitality in all groups was calculated. Suitable doses, under which three SUs has the same dynamic effect on HA-VSMC, were selected for the follow-up study.4. Cell proliferation and migration assay① CCK-8 cell proliferation assay was used to detect the influence of HA-VSMC’s proliferation ability by glimepiride, glibenclamide and gliclazide.② Transwell assay was used to measure the cell migration ability in all groups.③ For further research, 100μM diazoxide (KATP channel activator) was added, and the cell proliferation and migration ability were examined again.5. Western blotWestern blot was used to detect the expression ofp-NF-r,B-p65.6. Statistical analysisAll data are presented as mean±S.E.. Comparisons were assessed by unpaired t-test with SPSS Software (version20.0, SPSS China). A value of P < 0.05 was considered statistically significant.Results1. Immunofluorescence assay: SUR2 was widely expressed in the cell lines of HA-VSMC.2. Dose selection resultsThe select dose of glimepiride, glibenclamide and gliclazide is 208 ummol/l, 21 ummol/l and 2000ummol/l, respectively.2. Cell proliferation assayCompared with the control group, HA-VSMC’s proliferation ability was enhanced by glibenclamide and glimepiride, and inhibited by gliclazide. After adding with diazoxide, the promoting effect on HA-VSMC’s proliferation was reversed in either glimepiride or glibenclamide groups. The inhibition effect of gliclazide on HA-VSMC’s proliferation was further enhanced.3. Cell migration assayTranswell assay showed that compared with the control group, HA-VSMC’s migration ability was enhanced by glibenclamide and glimepiride, and inhibited by gliclazide. After adding with diazoxide, HA-VSMC’s migration was inhibited in both glimepiride and glibenclamide groups.4. Western blotWestern blot analysis showed that after the treatment of gliclazide, compared with the control group the expression of p-NF-κB-p65 in HA-VSMC was greatly decreased. After adding with PMA (NF-κB activator), gliclazide’s inhibitory effect on HA-VSMC’s proliferation and migration could be reversed.Conclusion (1) Glimepiride, glibenclamide and gliclazide had different effects on HSMCs’ migration and proliferation. Glimepiride and glibenclamide could promote HSMCs’ proliferation and migration which is more effective in glibenclamide group, and the function of KATP channel was involved in this process.(2) Gliclazide manifested as inhibiting effect on HSMCs’ migration and proliferation. NF-κ,B was involved in the mechanism.BackgroundSulfonylureas (SUs) have been introduced into the therapy of type 2 diabetes with great success. It is generally accepted that SUs reduce blood glucose by stimulating the release of insulin from pancreatic beta cells. SUs stimulate insulin secretion by combined with their receptor--SUR1, the ATP-sensitive K channels, in pancreatic beta cells. Now, an increasing number of investigations at home or broad have indicated that SUs possess extrapancreatic effect. Involving in regulation of blood glucose, besides stimulating insulin secretion, SUs can also inhibit glucagon release, reduce the removal of insulin by the liver, and increase the insulin sensitivity of peripheral target cells and so on. This means that SUs possess extrapancreatic hypoglycemic effect. Patients using SUs benefit a lot. So, even the guidelines emphasize that SUs shouldn’t be used when starting insulin intensive treatment, but quite a number of clinicians prefer to combine insulin intensive therapy with the SUs, which was recognized do "more good than bad" in clinical work.Now the confirmatory studies on SUs’ extrapancreatic hypoglycemic effects lacking of in vivo evidences. Moreover, the in vivo studies demonstrating the extrapancreatic hypoglycemic effects of SUs used somatostains to inhibit SUs induced insulin secretion or KK-Ay mice. However, the secretions of insulin stimulated by SUs were not completely blocked via such measures. Namely, all above evidences are not sufficient to confirm the existence of SUs’ extrapancreatic hypoglycemic effects. Therefore, up to now, the existence of the so called "extrapancreatic hypoglycemic effects" of SUs in vivo remains unknown.There are differences in extrapancreatic effect in different kinds of SUs. Gliclazide is a second-generation of SUs. The unique feature of the gliclazide molecule is its azabicyclo-octyl ring, grafted to a sulfonylurea group. This feature is responsible for the ability of gliclazide to reduce oxidative stress and gliclazide’s many other specific functions (extrapancreatic effect). Nowadays, researches showed that gliclazide possess extrapancreatic effect in terms of glucose improving. To confirm this, in order to guide clinical. We established SUR1-/- rat model with type 2 diabetes, thus gliclazide completely lost its ability to stimulate islet beta cells’ insulin secretion, then gliclazide was applied to these rats, in order to clarify the function and mechanism of this tradition hypoglycemic drug on extrapancreatic tissues. Metformin, which decrease blood glucose not by combining with SUR1 was used as an standard drug.Aim:(1) To definite the extrapancreatic hypoglycemic effects of gliclazide(2) To clarify the mechanism of gliclazide’s extrapancreatie hy~glycemic effects.Materials and methods1. Construction of the SUR1 knock-out rat model.TALEN-mediated gene targeting technology was used to the construction of the SUR1 knock-out rat model. Through screening and breeding, SUR1-/- rats can be obtained.2. Generation of type 2 diabetes and grouping.Male Sprague-Dawley (SD) rats (-120 g) were chosen, and were fed with a high-fat diet. Four weeks later, intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed. Diabetes mellitus was induced by a single intraperitoneal injection of STZ to rats with insulin resistance. Two week after STZ administration, rats with FBG > 11.1 mmol/1 in 2 consecutive analyses were considered the diabetic rat model.Then the diabetic rats were randomized into 3 groups: gliclazide (10mg/kg/d),metformin (212.5mg/kg/d) and control (normal saline). IPGTT and IPITT were performed again. After 14 days of drugs treatment, rats were sacrificed.3. Blood glucose and body mass: Blood glucose and body mass were measured before treatment and on 7, 14 days after treatment.4. IPGTT and IPITTGlucose tolerance was assessed by IPGTT after rats fasted for 12 h. A bolus of glucose (1 g/kg i.p.) was injected, and blood samples were collected sequentially from the tail vein at 0, 15, 30, 60, and 120 min. Plasma glucose was measured with a One-Touch Glucometer (Ascensia Breeze, Bayer, Germany). The mean area under the receiver operating characteristic curve (AUC) was calculated for glucose.To evaluate insulin tolerance, IPITT was performed after rats fasted for 4 h. A bolus of insulin (1 unit/kg ip) was administered, and blood samples were taken for glucose measurement as described above.5. Hyperinsulinemic-euglycemic clampsSix rats from each group were used in this experiment. Rats were anesthetized and catheters were implanted in the left femoral artery one week before clamps and tunneled to the back of the neck where the animal was unable to reach them.After fasted for 16 h, a 2 hour hyperinsulinemic-euglycemic clamp was performed in conscious, catheterized rats. Venous indwelling needle (26G) was inserted into the tail vein, and three-way stopcocks were used. A continuous infusion of insulin (10mU/kg/min) and a variable infusion of 20% glucose were taken to maintain the plasma glucose concentration at about 6mmol/1. Blood glucose was tested at an interval of every 5 minute. Glucose infusion rate was recorded.6. Tissue harvestingAt the end of the clamp, the rats were anesthetized, and tissue samples (liver, quadriceps muscle and epididymal fat) were fixed with 10% neutral formalin and embedded in paraffin or stored at -80~C for subsequent analysis.7. Glycogen analysis and immunofluorescence stainingPeriodic acid-Schiff (PAS) staining was used to determined glycogen content in liver. Muscle and fat tissues were pressed for immunofluorescence staining to observe the expression and location of glucose transporter 4 (GLUT4).8. Western blotProteins in liver, muscle and fat tissue were obtained. Western blot was used to detect the changes of phosphorylation activation of Akt, AMPK in liver in the four groups. In muscle and fat, the expression of total and membrane GLUT4, PPAR-γ and phosphorylation activation ofAkt, AMPK were detected.9. Statistical analysisAll data were analyzed with Graphpad Prism 5 and presented as means±S.E.M. One-way or two-way ANOVA was performed where appropriate and differences were considered statistically significant at P < 0.05.Results1. Construction of the SUR1 knock-out rat modelAbcc8 (SUR1) knockout vector was constructed and injected to embryonic stem cells. Transfected cells were selected, embryonic stem cells were injected into SD blastocysts using standard techniques. Chimeric males were selected and crossed with wild type SD females to generate SUR1+/- heterozygotes, which were bred to obtain SUR1+/+, SUR1+/-, and SURF-/- animals. PCR, Sanger sequencing and western blot were used, and SURF-/- rat model was obtained successfully.2. AnimalAfter 4 weeks’ HF diet, insulin resistance was established in all rats. 27 rats developed hyperglycemia after the injection of STZ. Before the end of the experiment,one rat in gliclazide group and one rat in control group died.3. Body mass and blood glucoseBody mass: No changes were observed in each group.Fasting blood glucose: Baseline fasting blood glucose levels were the same. Fourteen days after treatment, compared with the control group, fasting blood glucose in rats in gliclazide group was decreased (P<0.05), but not significantly as the decrease in metformin group (P<0.01). Gliclazide’s extrapancreatic hypoglycemic effect is identified.IPGTT: Baseline IPGTT levels were the same. After treatment for 14 days, both gliclazide and metformin groups showed relieved glucose tolerance on IPGTT compared with the control group, area under the curve (AUC) across the time for glucose level was lower. But the differences of AUC in gliclazide and control groups were not significant (P>0.05).IPITT: Impaired insulin sensitivity was revealed by IPITT. After treatment for 14 days, the control group showed the higher mean AUC, AUC in gliclazide group was decreased and compared with the control group, the difference is significant (P <0.05), but not significantly as the metformin group.4. Hyperinsulinemic-euglycemic clampsHyperinsulinemic-euglycemic clamps were used and GIR in each groups were calculated to reflect the whole body glucose utilization.Compared with the metformin group, GIR in gliclazide group was lower (P <0.05), however, compared with the control group, GIR in gliclazide group was increased (P<0.05). This further indicated that gliclazide possess extrapancreatic insulin resistance relieving effect.5. Insulin resistance in liverPAS staining: The liver plays a unique role in the regulation of glucose homeostasis. Hepatic insulin resistance manifested as increased hepatic glucose production and decreased glycogen synthesis. PAS staining results showed that in gliclazide group, hepatic glycogen content was mildly increased compared with the control group. Hepatic glycogen content was markedly increased in metformin group.Phosphorylation activation ofAkt, AMPK:Western blot analysis showed that gliclazide could increase the activation of Akt compared with the control group (P<0.05). However, the phosphorylation of Akt was lower in the gliclazide group than the metformin group (P < 0.05).Different from the activation of Akt, AMPK could not be activated by gliclazide(vs. control group, P>0.05).6. Insulin resistance in muscle and fat tissueTotal and membrane GLUT4 expression: In states of insulin resistance, in peripheral tissue the cellular effect of insulin is impaired, which leads to a reduced glucose uptake in muscle and fat tissue. Glucose uptake is mediated by GLUTs and in muscle and fat, GLUT4 is the predominant transporter.Immunofluorescent staining and western blot analysis revealed that the protein expression of both total and membrane GLUT4 were markedly increased in the gliclazide group in muscle and fat tissue, as compared with the control and metformin groups.In metformin group, membrane but not total GLUT4 expression was increased in muscle tissue, as compared with the control group (P < 0.01). In fat tissue, neither membrane no total GLUT4 expression in metformin group was increased (P > 0.05).Activation of Akt phosphorylation: Western blot analysis showed that gliclazide significantly increased the activation of Akt in muscle and fat tissue, compared with the control group (P < 0.01). In metformin group, Akt was activated in muscle (vs. control group, P < 0.01) but not in fat (vs. control group, P > 0.05).Activation of AMPK phosphorylation: Different from Akt activation, AMPK could not be activated by gliclazide (vs. control group, P > 0.05).PPARγ expression was also examined in muscle and fat tissue. Compared with both the control and metformin group, PPAR-γ expression was markedly elevated in muscle and fat tissues in rats of gliclazide group (vs. control group, P < 0.01; vs. metformin group, P < 0.05).Conclusions(1) In addition to the traditional glucose lowering effect of gliclazide on promoting insulin secretion, it also has extrapancreatic hypoglycemic functions.(2) Type 2 diabetes is associated with decreased insulin sensitivity, which led to the insulin resistance (IR). Gliclazide can improve IR directly, but compared withmetformin, its effect is weaker. The mechanisms of the two drugs in improving IR is quite different. The main target organ of metformin is liver, while the gliclazide’s are muscle and fat tissues, disregulation of glucose mechanism in these organs inducing IR.(3) Glucose uptake is mediated by GLUTs in most cells, and it is correlated with the expression of GLUTs on cell membrane. In muscle and fat tissue, GLUT4 is the predominant transporter. Gliclazide could remarkably promote the expression of total and membrane GLUT4 in muscle and fat tissue. And the up-regulation of GLUT4 expression and translocation by gliclazide seems to mediated via PPAR-γ/AKT pathway.The mechanism of SUs’ hypoglycemic effect is promoting the insulin secretion.Sulfonylureas stimulate insulin secretion by inhibiting ATP sensitive potassium (KATP) channels in pancreatic cells. It is now well established that these channels consist of a regulatory sulfonylurea receptor (SUR) and a pore-forming subunit (Kir). In our study, SUR1 in pancreas β cell was knocked out, SUs’ insulin stimulating promotion effect was blocked. Gliclazide, as the second generation SUs, can still decrease blood glucose level and act on the peripheral tissue, extrapancreatic hypoglycemic functions was comformed.