| Backgrounds and aimsLong-standing diabetes leads to pathogenesis of fibrotic disorders, such as diabetic cardiac fibrosis and renal fibrosis. The resultant fibrosis disrupts the normal architecture of the affected organs, ultimately leading to their dysfunction and failure 。 Fibroblasts are major contributors to extracellular matrix accumulation in tissue fibrosis. There is increasing evidence to show that a significant fraction of these interstitial fibroblasts is derived from the endothelium, a process called Endothelial-to-mesenchymal transition (EndMT). Recent studies suggest that the EndMT could contribute to the progression of diabetic cardiac fibrosis and renal fibrosis. Widyantoro showed that 15% to 20% of fibroblasts coexpressed both the endothelial marker CD31 and the fibroblast marker FSP1 in the hearts of diabetic WT mice. Zeisberg found that 30% to 50% of fibroblasts in the kidneys of diabetic mice co-expressed the endothelial marker CD31 along with FSP-1 specific and a-SMA specific markers of fibroblasts and myofibroblasts. The current studies also indicated that blockade of EndMT could prevent the progression of organ fibrosisPoly(ADP-ribose) polymerase 1 (PARP-1) is an abundant nuclear enzyme that can be activated by oxidative DNA damage, mainly reactive oxygen species (ROS). On binding to damaged DNA, PARP-1 cleaves nicotine amide adenine dinucleotide (NAD+) to produce nicotin-amide and ADP-ribose When DNA damage is mild, PARP-1 participates in the DNA repair process. However, excessive activation of PARP-1 by stimuli, such as hyperglycemia leads to intracellular depletion of NAD+ and ATP, thus resulting in cellular energy crisis, irreversible cytotoxicity and cell death. PARP-1 activation also facilitates diverse inflammatory responses by promoting inflammation-relevant gene expression, including IL-1β, TNFα, and endothelin-1.These all play a significant role in fibrotic disorders. Recently, Kessler showed that the combination of TGF-1, IL-1, and TNFa could induce EndMT in human intestinal microvascular endothelial cells. Widyantoro also demonstrated that endothelin-1 gene silencing could inhibit hyperglycemia-induced EndMT in cultured endothelial cells. However, it is still unclear whether PARP-1 inhibition could suppress hyperglycemia-induced EndMT.Glucagon-like peptide-1 (GLP-1) is an incretin hormone produced by intestinal L cells in response to food intake. Emerging evidence has shown the GLP-1’s anti-inflammatory effects on the cardiovascular system. We recently demonstrated that GLP-1 could protect microvascular endothelial cells by inactivating the PARP-1-inducible nitric oxide (NO) synthase-NO pathway. Based on these findings, we wondered whether GLP-1 could inhibit myocardial EndMT in diabetic mice and whether this was mediated by inactivating PARP-1.The study aims are as follows:1. To explore whether GLP-1 can inhibit myocardial EndMT in diabetic mice2. To explore the potential mechanisms by which GLP-1 inhibit myocardial EndMT in diabetic mice.Methods1. Cell Culture and TreatmentHAECs were treated with 5 mmol/1 d-glucose (normal glucose, NG) or 30 mmol/l d-glucose (HG) for 3 days. The medium was changed every 24 h. siPARP-1, siSnail and si-NC duplexes were transfected into HAECs by use of Lipofectamine 2000. Then cells were exposed to HG with or without 50 nmol/1 GLP-1, or 10 nmol/l N-acetyl-L-cysteine (NAC, inhibitor of ROS) after 24-h transfection for 72 h.2. Animal ModelC57BL/6J male mice were used for this study. Non-diabetic mice were used as controls. Diabetic mice were randomly divided into 2 groups for treatment:Daily saline (DM alone) and GLP-1-analog (24nmol/kg daily; DM+GLP-1). After 24 weeks of diabetes, Mice were sacrificed.3. Cardiac Function MeasurementTransthoracic echocardiography was performed 24 weeks after STZ injection.4. ROS Production AssayROS level was determined by the oxidative conversion of H2DCF-DA to fluorescent dichlorofluorescein on reaction with ROS in cells..5. Masson stainingParaformaldehyde (4%)-fixed hearts were bisected transversely at the midventricular level, embedded in paraffin and cut into 5-μm-thick sections and then stained with Masson staining. Dark green tained collagen fibers were quantified as a measure of fibrosis and examined by light microscopy in a blinded manner.6. Immunocytochemistry and immunohistochemistryFrozen heart tissues or HAECs were fixed in 4% paraformaldehyde. Then tissue sections or cells were permeabilized in 0.03% Triton X-100. After being blocked in 5% bovine serum albumin, cells were incubated with primary antibodies, then with a fluorescence dye-conjugated secondary antibody.7. Western Blot AnalysisEqual amounts of protein were separated on 10% SDS-PAGE and electro-transferred onto nitro-cellulose membranes, which were blocked with 5% non-fat milk, then washed, then incubated overnight at 4℃ with primary antibodies, then horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Blots were visualized by chemiluminescence.8. Gelatin ZymographyThe culture supernatant was harvested and mixed with a gel sample buffer. Sample was separated by SDS-PAGE with 0.1% gelatin. After electrophoresis, gels were washed with Tris buffer containing Triton X-100. Gels were incubated for an additional 24 h in incubation fluid, then stained with Coomassie blue containing 30% methanol and glacial acetic acid and destained in methanol acetic acid/H2O. White bands on a blue background indicated zones of digestion corresponding to the presence of different MMPs.Results1. GLP-1 attenuates myocardial fibrosis and cardiac dysfunction in mice with DMAt 24 weeks, cardiac function was lower in diabetic than control mice, As compared with DM, GLP-1 treatment ameliorated the altered parameter values.Masson’s trichome staining of heart sections revealed increased ECM deposition in diabetic mouse myocardium as compared to controls. In addition, diabetes increased the expression of fibrotic markers collagen I and III as compared to controls, and GLP-1 analog treatment significantly reduced the levels as compared with DM alone.2. GLP-1 inhibits HG-induced EndMT in vitro and in vivoImmunofluorescence staining of mouse hearts after 24 weeks of GLP-1 treatment revealed that DM markedly increased the proportion of cells expressing a-SMA, which was reduced with GLP-1 treatment; The proportion of cells expressing both vWF and aSMA in the cardiac tissue was lower after GLP-1 treatment, compared to DM alone.In vitro. HAECs were treated with or without GLP-1 for 72 h under high glucose condition. Fluorescence microscopy revealed that while control HAECs had the typical rounded or cobblestone shape, HG-treated HAECs acquired a spindle-shaped morphology. This change in morphology was inhibited by GLP-1 treatment. Immunofluorescence demonstrated that HG-treated cells acquired mesenchymal marker a-SMA staining and lost endothelial marker Ve-cadherin.3. GLP-1 decreased HG-induced PARP-1 expression and activity by suppressing ROS production in HAECsROS production increased under HG conditions but decreased significantly with both GLP-1 and NAC treatment in HAECs. The expression and activity of PARP-1 were significantly increased with HG as compared to controls but were decreased with GLP-1 or NAC treatment.4. GLP-1 inhibits HG-induced EndMT by suppressing PARP-1-Snail interactionNG treatment expressed the endothelial markers VE-cadherin whereas HAECs with HG treatment showed lower VE-cadherin and higher a-SMA expression. Treatment with siPARP-1, siSnail or GLP-1 markedly inhibited HG-induced a-SMA expression Incubation of HAECs with HG significantly upregulated PARP-1 expression and activity; increased Snail expression, as well as markedly increased PARP-1-Snail interaction in nuclei. Treatment with GLP-1 significantly reduced PARP-1 and Snail expression and interaction. GLP-1 and siPARP-1 treatment markedly reduced PARP-1 and PAR protein level as compared to HG treatment.5. GLP-1 Reduced HG-Induced Collagen I and III and MMP-2 and MMP-9 Expression by Inhibiting EndMTIncubating HAECs in HG greatly increased the protein expression of collagen I and III, as compared to NG. Treatment with GLP-1 or siPARP-1 or siSnail markedly decreased HG-induced collagen I and III expression. Moreover, gelatin zymography revealed that HG treatment significantly increased the activity of MMP-2 and MMP-9 as compared to NG. GLP-1, siPARP-1 or siSnail treatment markedly reduced HG-induced MMP-2 and MMP-9 activity.6. GLP-1 Inhibits HG-induced Cell Migration by Suppressing EndMTWe performed a scratch assay and found that HG increased cell migration compared to NG. After 12 h of incubation with HG, the gap distance was significantly reduced as compared with baseline control. Both GLP-1 and PARP-1 gene silencing significantly prevented the decrease in gap distances. Immunofluorescence indicated that cells with faster migration capability were the transitioned endothelial cells.Conclusion1. Chronic hyperglycemia can induce EndMT.2. GLP-1 inhibits HG-induced EndMT in vitro and in vivo.3. GLP-1 inhibits HG-induced EndMT by inactivating PARP-1.SignificanceChronic hyperglycemia can induce EndMT which contributes to cardiac fibrosis and cardiac dyfunction.GLP-1 treatment significantly improved high glucose-induced EndMT by suppressing PARP-1 activity. Our findings provide the new insight to the treatment of diabetic fibrotic disorders.Backgrounds and aimsInsulin delivery to muscle interstitial fluid is a rate-limiting step in insulin’s peripheral action. Several studies have shown that increased muscle microvascular recruitment can facilitate the insulin delivery to muscle interstitial fluid, which increases the insulin-mediated muscle glucose uptake. In insulin resistant state (obesity or type 2 diabetes), the insulin’s action on microvascluar recruitment is impaired, which inhibits insulin’s delivery to muscle interstitium, leading to the metabolic insulin resistance.The renin-angiotensin (Ang) system (RAS) plays important roles in maintaining vascular health and hemodynamic stability. Ang Ⅱ exerts its vascular actions via 2 G protein-coupled receptors, the type 1 receptor (AT1R) and type 2 receptor (AT2R). In small resistance vessels, activation of AT1Rs increases vasoconstriction and smooth muscle proliferation, whereas AT2R stimulation activates an autacoid vasodilator cascade including bradykinin, NO, and cGMP, leading to vasodilation and opposing the vasoconstrictor actions of Ang via the AT1R. The previous studies on the regulation of arterial blood flow by the RAS have been focused on conduit arteries and/or resistance arterioles. The relationship between RAS and muscle microvascular beds has not been fully developed. In our previous studies, we found that systemic infusion of angiotensin Ⅱ at either lng/kg per minute caused a significant increase in muscle microvascular perfusion. This indicates that Ang Ⅱ plays important roles in regulating muscle microvascular bed. Compound 21 is a newly developed AT2R agonist. According to recent studies, Compound 21 administered alone can not decrease blood pressure in conscious normotensive Wistar-Kyoto rats or spontaneously hypertensive rats. However, when given in combination with the AT1 receptor antagonist, Compound 21 lowered blood pressure in SHR only. Moreover, Compound 21 can increase renal blood flow without affecting blood pressure, and this effect different between male and female rats. Therefore, we hypothesized that direct stimulation of AT2R by Compound 21 can increase muscle microvascular perfusion.The mechanisms by which Compound 21 evokes vasodilator effects are quite controversial. Bonsnyak has shown that Compound 21 can increase vasorelaxation in aortic and mesenteric vessels via AT2R/NO pathway. Similarly, Brouwers also found that Compound 21 can increase renal blood flow via AT2R/NO pathway. However, Verdonk recently found that Compound 21 could induce vasorelaxation via an endothelium- and AT2R-independent mechanism in vitro. Moreover, Shao reported that Compound 21 elevated insulin levels by activating AT2R in islets. The increased insulin level can also induce vasorelaxation. Based on these findings, we wondered whether Compound 21 can increase muscle microvascular perfusion via AT2R/NO pathway.The study aims are as follows:1. To explore whether Compound 21 can increase muscle micvascular perfusion and oxygen saturation.2. To explore the potential mechanisms by which Compound 21 can increase muscle micvascular perfusion.Methods1. Animal Preparations and Experimental ProtocolsAdult male Sprague-Dawley rats weighing 220 to 320 g were studied after an overnight fast. Rats were housed at 22±2℃, on a 12-hour light-dark cycle, and fed standard laboratory chow and water before study entry. Rats were assigned to five groups to receive the following treatments:(1) group 1 received an IV infusion of saline at 5μl per minute for 120 minutes; (2)group 2 received an IV infusion of compound 21 at 300 ng/kg per minute for 120 minutes; (3) group3 received compound 21 injection 30 minutes after beginning systemic infusion of PD123319 (AT2R blocker,50μg/kg per minute); (3)group4 received compound 21injection 30 minutes after beginning systemic infusion of NG-nitro-L-arginine methylester (L-NAME; 50μg/kg per minute).2. Anesthesia and surgical procedureRats were anesthetized with Inactin (170-180mg/kg IP), placed in a supine position on a heating pad, and intubated with a polyethylene tubing to ensure a patent airway. After cannulating the carotid artery and external jugular vein through a midline neck incision and after a 60-minute baseline period to assure hemodynamic stability and a stable level of anesthesia.3. Quantitation of muscle interstitial oxygen saturationMuscle interstitial oxygen saturation was measured using a fibro-optic oxygen meter. The measurement was on the basis of the effect of dynamic luminescence quenching by molecular oxygen. Briefly, a needle housing the fibro-optic oxygen microsensor was inserted into the left hindlimb skeletal muscle. Then the glass fiber with its oxygensensitive tip inside the needle was extended into muscle interstitium by carefully pressing the syringe plunger. Measurements were taken every 10 seconds, and 30-minute average values were calculated.4. Contrast-Enhanced UltrasoundThe skin overlying the proximal hindlimb adductor muscles was shaved, and skeletal muscle microvascular blood volume (MBV), microvascular flow velocity (MFV), and microvascular blood flow (MBF) were determined using contrast-enhanced ultrasound.5. Cell Culture and TreatmentAfter serum starvation for 12 h, rat aortic endothelial cells (RAOEC) between passages 4 to 6 were exposed to Compound 21(10-9,10-8,10-7,10-6,10-5 and 10-4 mol/1) in the absence or presence of insulin (10nmol/l) for 30 min.6. Western Blot AnalysisEqual amounts of protein were separated on 10% SDS-PAGE and electro-transferred onto nitro-cellulose membranes, which were blocked with 5% non-fat milk, then washed, then incubated overnight at 4℃ with primary antibodies, then horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Blots were visualized by chemiluminescence.7. Myograph studyThe distal saphenous artery (≤250 μm) was dissected, cleaned of adhering connective tissues, and cut into segments of ≈2 mm in length. Each segment was mounted in a Multi Myograph System. The organ chamber was filled with 6 ml of physiological salt solution buffer, which was constantly bubbled with 95% O2-5% CO2 and maintained at 37℃. In some arteries, the endothelium was mechanically removed by rubbing the luminal surface of the ring with human hair. Functional removal of the endothelium was verified by the lack of relaxation response to acetylcholine. After preconstriction of the arterial ring with 2 μmol/L phenylephrine, Compound 21 at various concentrations (10-9,10-8,10-7,10-6,10-5 and 10-4 mol/l) were added into the chamber. For the inhibitor study, preconstricted arterial rings were incubated with PD123319 (10-4mol/l), L-NAME (10-5mol/l) and Losartan (10-5mol/l) for 30 minutes before Compound 21 was added. Changes in arterial tone were recorded.Results1. Compound 21 increases eNOS phosphorylation in cultured RAOECs.We first carried out a dose-response study examining whether Compound 21 exerts a direct effect on the RAOECs in vitro prior to the animal studies. We found that incubation of RAOECs with Compound 21 at concentrations ranging from 10-7,10-6 mol/1 for 30 min potently increased the phosphorylation of eNOS.2. Compound 21 at supraphysiological concentration relaxes saphneous artery rings via an AT2R-dependent and endothelium-independent mechanismUsing phenylephrine-preconstricted distal saphenous arteries, we found that Compound 21 at supraphysiological concentration(l10-4mol/l) relaxed the artery rings. While Endothelium denudement, L-NAME and Losartan did not inhibit Compound 21-induced vasorelaxation, PD123319 inhibited Compound 21-induced vasorelaxation.3. Compound 21 increases muscle microvascular recruitment and oxygen saturation without affecting blood pressure.The above results from in vitro studies prompted us to examine whether Compound 21 recruits muscle microvasculature in vivo. In the current study, Compound 21 potently increased muscle MBV within 30 min, and this effect persisted throughout the 120-min experimental period without affecting blood pressure. MFV did not change significantly during GLP-1 infusion. As a result, Compound 21 infusion led to a significant increase in MBF. After Compound 21 infusion, muscle interstitial oxygen saturation increased significantly.4. Compound 21 increases muscle microvascular recruitment and oxygen saturation via AT2R/NO pathway.To explore the potential mechanism by which Compound 21 increased muscle microvascular perfusion and oxygen saturation, we infused PD123319 or L-NAME systemically starting 30 min before Compound 21 administration. We found that in the presence of PD 123319 or L-NAME, Compound 21 failed to increase muscle microvascular perfusion and oxygen saturation. Those data indicates that Compound 21 increases muscle microvascular recruitment and oxygen saturation via AT2R/NO pathway.Conclusion1. Compound 21 increases muscle microvascular recruitment and oxygen saturation without affecting blood pressure.2. Compound 21 increases muscle microvascular recruitment and oxygen saturation via AT2R/NO pathway.SignificanceWe show that AT2R agonist C21 increases muscle microvascular perfusion and oxygenation via AT2R/NO pathway. As expansion of muscle endothelial exchange area enhances insulin action and tissue function, AT2R agonists may have a therapeutic potential in the prevention and treatment of diabetes and its associated complications. |
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