Mechanisms Underlying Anti-fibrotic And Inflammatory Effects Of Glucagon-like Peptide-1 On Angiotensin Ⅱ-induced Hypertensive Rats

IntroductionMany basic studies and clinical observations have shown that angiotensin II(Ang II)derived from the renin-angiotensin-aldosterone system plays an important role in development of hypertension,myocardial fibrosis and heart failure.Through understanding in deleterious effects of Ang II on cardiovascular injury and development in anti-Ang II therapy,it has well been reorganized that Ang II plays a major role in pathogenesis of a variety of cardiovascular diseases.Pharmacological interventions using angiotensin converting enzyme(ACE)inhibitors to reduce the conversion of angiotensin(Ang)I to Ang II or Ang II type 1 receptor(AT1)antagonists to block the action of Ang II have become one of the most successful therapeutic approaches in treatment of different cardiovascular disease.However,data from clinical observations have also revealed that the use of ACE inhibitors is associated with higher rate of renin sensitivity,dry cough angioedema and limited usage in renal stenosis,while hypotensive symptoms and different personal response are often identified with AT1 receptor antagonists.Therefore,adjunctive therapies to avoid these unfavorable actions and reduce morbidity and mortality of cardiovascular diseases by inhibiting Ang II/AT1 system reserve significance of translational medicine in hospital.Glucagon like peptide-1(GLP-1),an incretin hormone released from the intestinal endocrine L-cells,plays an important role in glucose homestasis in response to food intake.GLP-1 stimulates the adenylate cyclease by activating G protein-coupled receptors expressed in pancreatic β-cells,results in an enhancement in glucose-dependent stimulation of insulin synthesis/release,and further attenuates post-prandial gastrointestinal motility and secretion.GLP-1 has a short plasma half-life(< 2 minutes)and is rapidly degraded by the enzyme dipeptidyl peptidase-4(DPP-4)once it is released into the circulation.Recently,more attention has been placed on development in long-acting GLP-1 agonist to enhance insulin release or efficient DPP-4 inhibitor to reduce cleavage of endogenous GLP-1 for the treatment of type 2 diabetes.Liraglutide,marketed under the brand nameVictoza,is a long-acting GLP-1 receptor agonist for the treatment of type 2 diabetes.Unlike native GLP-1,liraglutide is stable against metabolic degradation by DPP-4 with a plasma half-life of 13 hours after subcutaneous administration.It has been shown that both compounds also have cardiovascular effects involved in regulations of lipid,endothelial and cardiac function.However,it is unknown whether GLP-1 has effect on Ang II induced cardiovascular injury,particularly for Ang II induced hypertension,inflammatory response and myocardial fibrosis.We do not know whether preservation GLP-1 level through exogenous supply of GLP-1 or reducing degradation of endogenous GLP-1 could reduce Ang II mediated deleterious effects on cardiovascular system.Therefore,we selected a well-established rat model of Ang II infusion-induced tissue fibrosis to examine whether subcutaneous administration of liraglutide inhibits tissue fibrosis.To further gain insight into a functional role of GLP-1 in attenuating cardiac fibrosis,we also selected the DPP-4 inhibitor,linagliptin to determine whether inhibition in degradation of endogenous GLP-1 is associated with reduction of cardiac fibrosis.The entire study was divided into three parts as follows:Study part 1: Action of GLP-1 on angiotensin II induced hypertension in ratsObjective:To demonstrate the time-and action-related characteristics of Ang II mediated blood pressure elevation;illustrate and compare protective effects and mechanisms by increasing GLP-1 level with liraglutide and reducing degradation of endogenous GLP-1 with linagliptin.Methods:Male Sprague-Dawley rats weighing 200±10g were purchased from the Harlan laboratories and housed for at least one week before all experimental protocols.The rats were anesthetized with an intraperitoneal injection of a mixture of ketamine(90 mg/kg)and zylaxine(10 mg/kg)at the day for a minipump implantation.A small incision on the skin was made between the scapulae and small pocket was produced using a hemostat to spread the subcutaneous connective tissue.An osmotic minipump was then inserted into the pocket and the incision was closed with sutures.All osmotic minipumps(Models 2002 and2004,ALZET Corp,CA,USA)were prefilled in sterile 0.9% saline only or saline containing Ang II(Sigma-Aldrich,MO,USA)at 37o C for overnight prior to implantation.Non-invasive blood pressure(NIBP)was measured from all animals in an awake condition using a Power Lab coupled to a NIBP system(ML125 AD Instruments NIBP controller,CO,USA)with a pulse transducer at baseline and during the observational points of the study,respectively.The pulse transducer then intermittently measures blood pressure based on the periodic occlusion of blood flow in the tail.Two study protocols(i.e.,2 and 4 weeks of Ang II infusion)were selected for all experimental groups.Animals were randomized into four groups(n=6,for each observational period): 1)Sham control: rats were infused with a saline pump;2)Control: rats received Ang II infusion at a rate of 500 ng/kg/min;3)Ang II + liraglutide(Lira,Novo Nordisk,Denmark): rats received a subcutaneous injection of liraglutide at a dose of 0.3 mg/kg twice daily during Ang II infusion;4)Ang II + linagliptin(Lina,Boehringer Ingelheim Pharmaceuticals Inc.CT,USA): rats were orally fed with linagliptin at a dose of 8 mg/kg daily during Ang II infusion.Results:We found that arterial blood pressure was significantly increased over time with Ang II infusion relative to the Sham control,reaching to a peak at 21 days.Liraglutide reduced blood pressure(i.e.,systolic,diastolic and mean pressure)significantly during entire period of study.Immunohistochemical staining showed a significant reduction in expression of e NOS and an increased in ET-1 on endothelium with Ang II infusion.Along with reduction in blood pressure with liraglutide,expression of e NOS was increased and ET-1 was reduced by liraglutide.Oral administration of linagliptin did not alter blood pressure,but enhanced expression of e NOS and reduced ET-1 to a comparable level with liraglutide.Conclusion:These results suggest that exogenous supply of GLP-1 receptor analog,liraglutide exerts a significant effect on Ang II induced elevation of blood pressure.This beneficial effect with liraglutide is largely mediated by altering release of dilatory and contracting substances from endothelium,i.e.,by increasing e NOS and reducing ET-1.Although linagliptin has no effect on blood pressure,it also increases e NOS and reduces ET-1,suggesting that there are some other fectors involved in liraglutide-mediated reduction inblood pressure.These data provide experimental evidence for application of liraglutide in treatment of hypertension.Study part 2: Preservation of GLP-1 inhibits Ang II induced fibrosis and inflammation in ratObjective:To demonstrate the pathogenesis and characteristics of cardiac fibrosis and inflammatory response with Ang II infusion induced hypertension,and to investigate the anti-fibrosis and anti-inflammation with liraglutide and linagliptin.Methods:The osmotic minipumps containing Ang II at a dose of(500 ng/kg/min)were implanted into the male rats.Liraglutide was subcutaneously injected at a dose of 0.3 mg/kg twice daily and linagliptin was orally fed at a dose of 8 mg/kg daily during Ang II infusion.At the end of 2 or 4 weeks of Ang II infusion,the hearts were isolated and tissue blocks were saved for histological analysis.Masson’s trichrome staining was used for detection of tissue fibrosis;Western blot assay was used to measure tissue protein levels;immunohistochemical staining was used to detect inflammatory cell accumulation and adhesion molecules.Results:We found that there are significant changes in myocardial fibrosis and inflammation at the end of 4 weeks of Ang II infusion,evidenced as expanded peri-vascular fibrotic space and inter-myocardium fibrotic area;enhanced accumulation of macrophages and α-SMA expressing myofibroblasts;increased deposition of type I collagen.Relative to these changes in the control animals,treatment with liraglutide linagliptin for 4 weeks significantly reduces fibrotic tissue formation,inflammatory cell accumulation and collagen deposition.Furthermore,liraglutide and linagliptin also comparatively reduce the tissue levels of IL-6 and TNF α as well as expression of p-selectin on endothelium.Conclusion:These results suggest that Ang II has significant effects on fibrotic tissue formation and macrophages/myofibroblast accumulation that are largely associated with collagen deposition seen at 4 weeks of Ang II infusion.Preservation of GLP-1 with liraglutide and linagliptin reduces myocardial fibrosis and inflammation.These results were consistent with their protective effects on cardiovascular system as shown in previous studies and provide evidence showing a direct effect on the heart after Ang II stimulation.Study part 3: Potential mechanisms of action underlying GLP-1 inhibited myocardial fibrosis and inflammation in ratObjective:To demonstrate mechanisms of action underlying Ang II induced myocardial fibrosis and preservation of GLP-1 mediated protection.Methods:The osmotic minipumps containing Ang II at a dose of(500 ng/kg/min)were implanted into the male rats.Liraglutide was subcutaneously injected at a dose of 0.3 mg/kg twice daily and linagliptin was orally fed at a dose of 8 mg/kg daily during Ang II infusion.At the end of 2 or 4 weeks of Ang II infusion,the hearts were isolated and tissue blocks were saved for histological analysis.Western blot assay was selected to detect expression of AT1,AT2,GLP-1,TGFβ1,p-Smad2,3,Smad 7,TLR2 and TLR4.Enzymatic assay was used to measure ACE2 activity and immunohistochemical staining was selected to detect expression of receptors.Results:We found that there are no significant statistical differences in all parameters measured at 2 weeks of Ang II infusion.However,at the end of 4 weeks,relative to the sham control,Ang II caused significant changes in all parameters analyzed,as manifested as enhanced expression of AT1 receptor and reduced expression of AT2 receptor,which are further confirmed by immunohistochemical staining at peri-vascular space and inter-myocardium area.Consistent with these changes,expression of TGFβ1,p-Smad2 and Smad3 was increased and expression of Smad7 was reduced.TLR2 expression was enhanced and IkB expression was reduced.Furthermore,expression of GLP-1 receptor and ACE2 activitywere attenuated.Treatment with liraglutide and linagliptin for 4 weeks reversed all parameters measured,evidenced by inhibited expression of TGFβ1,p-Smad2 and p-Smad3,up-regulated Smad7,and enhanced expression of GLP-1 receptor and ACE2 activity.On the other hand,both drugs reduced TLR2 expression and increased IkB.Conclusion:These results suggest that Ang II induces myocardial fibrosis through Ang II AT1 receptor/TGFβ1/Smads mediated signaling pathways.Meanwhile,enhanced expression of TLR2/ NFkB further augmented vascular and myocardial inflammatory responses.We demonstrated that injection of liraglutide reduces AT1 receptor protein level,up-regulates AT2 receptor,and therefore,re-balances expression between AT1 and AT2 receptors,as evidenced by a reduced ratio of AT1 to AT2 receptor.Liraglutide also activated GLP-1 receptor and increased ACE2 activity in myocardium.In accord with these findings,the production of macrophages and expression of TGFβ1/Smads were attenuated and proliferation of myofibroblasts was inhibited.Consistent with the modulatory effects of liraglutide on Ang II receptors and TGFβ1/Smads signaling pathways,collagen synthesis and cardiac fibrosis were attenuated.Furthermore,liraglutide lowered Ang II-elevated blood pressure during the observational periods.Comparative inhibition between administration of linagliptin and injection of liraglutide on induction of cardiac fibrosis suggested that preservation of endogenous GLP-1 is as potent as exogenous supply of GLP-1 receptor agonist to protect the heart against Ang II-induced tissue injury.Study prospectOur studies demonstrated for the first time exogenous supply of GLP-1 with liraglutide or inhibition of degradation of endogenous with linagliptin has a significant anti-fibrotic effect on Ang II induced myocardial fibrosis.These data provide direct experimental evidence to explain relationship between GLP-1 and Ang II induced myocardial injury.A review of the existing literatures appears to hold promise that GLP-1 receptor agonists and DPP-4 inhibitors exert physiological effects beyond glucose control on the cardiovascular system.Our data showed that both liraglutide and linagliptin at doses selected for the first-line treatment in controlling blood glucose are effectively to reduce Ang II-induced cardiac fibrosis.Currently,since there is no unique therapy for cardiovascular diseases toimprove final outcome in patient with fibrosis-induced heart failure,the GLP-1 receptor agonist or DPP-4 inhibitor might be selected as an adjunctive therapeutic compound with other commonly used cardiovascular drugs,such as ACE inhibitor,AT1 receptor blocker or beta receptor blocker,to help lowing blood pressure and better protect the heart against cardiac dysfunction in addition to its effect on the glycemic control in diabetic patients.