Linnéa Eriksson

Exendin-4 protects endothelial cells from lipoapoptosis by PKA, PI3K, eNOS, p38 MAPK, and JNK pathways.

Experimental studies have indicated that endothelial cells play an important role in maintaining vascular homeostasis. We previously reported that human coronary artery endothelial cells (HCAECs) express the glucagon-like peptide 1 (GLP1) receptor and that the stable GLP1 mimetic exendin-4 is able to activate the receptor, leading to increased cell proliferation. Here, we have studied the effect of exendin-4 and native GLP1 (7-36) on lipoapoptosis and its underlying mechanisms in HCAECs. Apoptosis was assessed by DNA fragmentation and caspase-3 activation, after incubating cells with palmitate. Nitric oxide (NO) and reactive oxidative species (ROS) were analyzed. GLP1 receptor activation, PKA-, PI3K/Akt-, eNOS-, p38 MAPK-, and JNK-dependent pathways, and genetic silencing of transfection of eNOS were also studied. Palmitate-induced apoptosis stimulated cells to release NO and ROS, concomitant with upregulation of eNOS, which required activation of p38 MAPK and JNK. Exendin-4 restored the imbalance between NO and ROS production in which ROS production decreased and NO production was further augmented. Incubation with exendin-4 and GLP1 (7-36) protected HCAECs against lipoapoptosis, an effect that was blocked by PKA, PI3K/Akt, eNOS, p38 MAPK, and JNK inhibitors. Genetic silencing of eNOS also abolished the anti-apoptotic effect afforded by exendin-4. Our results support the notion that GLP1 receptor agonists restore eNOS-induced ROS production due to lipotoxicity and that such agonists protect against lipoapoptosis through PKA-PI3K/Akt-eNOS-p38 MAPK-JNK-dependent pathways via a GLP1 receptor-dependent mechanism.

A characteristic electrophoretic pattern of cytosolic polypeptides from hepatocyte nodules generated during liver carcinogenesis in several models

The cytosolic polypeptides of hepatocyte nodules in six models of liver carcinogenesis were analysed by SDS-polyacrylamide gel electrophoresis and their patterns compared with these of control and variously treated livers. The amount of a polypeptide of Mr 21,000 was about tenfold elevated in the cytosol of five of the six types of nodules and moderately elevated in the sixth. Certain other polypeptides, particularly one of Mr 26,000, also varied in amount, so that all of the nodules analysed could be distinguished from liver by their electrophoretic patterns. Some possible identities of the two polypeptides are discussed. Their study may have mechanistic as well as diagnostic importance.

Effects of some anti-diabetic and cardioprotective agents on proliferation and apoptosis of human coronary artery endothelial cells

BackgroundThe leading cause of death for patients suffering from diabetes is macrovascular disease. Endothelial dysfunction is often observed in type 2 diabetic patients and it is considered to be an important early event in the pathogenesis of atherogenesis and cardiovascular disease. Many drugs are clinically applied to treat diabetic patients. However, little is known whether these agents directly interfere with endothelial cell proliferation and apoptosis. This study therefore aimed to investigate how anti-diabetic and cardioprotective agents affect human coronary artery endothelial cells (HCAECs).MethodsThe effect of anti-diabetic and cardioprotective agents on HCAEC viability, proliferation and apoptosis was studied. Viability was assessed using Trypan blue exclusion; proliferation in 5 mM and 11 mM of glucose was analyzed using [3H]thymidine incorporation. Lipoapoptosis of the cells was investigated by determining caspase-3 activity and the subsequent DNA fragmentation after incubation with the free fatty acid palmitate, mimicking diabetic lipotoxicity.ResultsOur data show that insulin, metformin, BLX-1002, and rosuvastatin improved HCAEC viability and they could also significantly increase cell proliferation in low glucose. The proliferative effect of insulin and BLX-1002 was also evident at 11 mM of glucose. In addition, insulin, metformin, BLX-1002, pioglitazone, and candesartan significantly decreased the caspase-3 activity and the subsequent DNA fragmentation evoked by palmitate, suggesting a protective effect of the drugs against lipoapoptosis.ConclusionOur results suggest that the anti-diabetic and cardioprotective agents mentioned above have direct and beneficial effects on endothelial cell viability, regeneration and apoptosis. This may add yet another valuable property to their therapeutic effect, increasing their clinical utility in type 2 diabetic patients in whom endothelial dysfunction is a prominent feature that adversely affect their survival.

Activation of AMP-activated protein kinase by metformin protects human coronary artery endothelial cells against diabetic lipoapoptosis

BackgroundThe prevalence of type 2 diabetes (T2D) among adults worldwide is rapidly increasing, and in patients with diabetes the major cause of death is macrovascular disease. Endothelial cells play an important role in maintaining vascular homeostasis. Free fatty acids, which are elevated in T2D, have previously been shown to induce endothelial dysfunction and apoptosis of endothelial cells, which is considered as an important and early factor in the onset of atherosclerosis and cardiovascular disease. Metformin, which is used as first line treatment of T2D patients, is believed to exert its pharmacological effects through activation of AMP-activated protein kinase, which has emerged as a new potential target in reversing endothelial dysfunction.MethodsHere we studied the protective effect of metformin against free fatty acid-induced apoptosis of human coronary artery endothelial cells (HCAECs) by assessing DNA fragmentation and cleaved caspase 3 levels. We also attempted to elucidate the underlying mechanisms by investigating the involvement of AMP-activated protein kinase, p38 MAPK and eNOS. Generation of reactive oxygen species by free fatty acid exposure was also examined.ResultsOur results suggest that metformin protects HCAECs from lipoapoptosis, an effect that involves eNOS and p38 MAPK, downstream of AMPK signaling, but not as previously suggested through suppression of reactive oxygen species.ConclusionThe protective effect of metformin against free fatty acid induced apoptosis is potentially clinically relevant as metformin is first line treatment for patients with T2D, a patient group which is rapidly increasing and carries a high burden of cardiovascular disease.

Glucagon-Like Peptide-1 Receptor Activation Does Not Affect Re-Endothelialization but Reduces Intimal Hyperplasia via Direct Effects on Smooth Muscle Cells in a Nondiabetic Model of Arterial Injury

Diabetic patients have an increased risk of restenosis and late stent thrombosis after angioplasty, i.e. complications that are related to a defective re-endothelialization. Exendin-4, a stable glucagon-like peptide (GLP)-1 receptor agonist, has been suggested to influence the formation of intimal hyperplasia and to increase endothelial cell proliferation in vitro. Thus, the aim of this study was to investigate the mechanisms by which treatment with exendin-4 could influence re-endothelialization and intimal hyperplasia after vascular injury. Methods: Sprague-Dawley rats were subjected to balloon injury of the left common carotid artery and treated for 4 weeks with exendin-4 or vehicle. Intimal hyperplasia and vessel wall elasticity were monitored noninvasively by high-frequency ultrasound, and re-endothelialization was evaluated upon sacrifice using Evans blue dye. Results and Conclusion: Exendin-4 selectively reduced the proliferation of smooth muscle cells (SMCs) and intimal hyperplasia in vivo without affecting the re-endothelialization process, but treatment with exendin-4 improved arterial wall elasticity. Our data also show that exendin-4 significantly decreased the proliferation and increased the apoptosis of SMCs in vitro, effects that appear to be mediated through cAMP signaling and endothelial nitric oxide synthase following GLP-1 receptor activation. Together, these effects of exendin-4 are highly desirable and may lead to an improved outcome for patients undergoing vascular interventions.

Exendin-4 restores glucolipotoxicity-induced gene expression in human coronary artery endothelial cells

Exendin-4, a stable GLP-1 receptor agonist, has been shown to stimulate insulin secretion. It has also been shown to exert beneficial effects on endothelial function that are independent of its glycemic effects. The molecular mechanisms underlying the protective actions of exendin-4 against diabetic glucolipotoxicity in endothelial cells largely remain elusive. We have investigated the long-term in vitro effect of palmitate or high glucose (simulating the diabetic milieu) and the role of exendin-4 on gene expression in human coronary artery endothelial cells. Gene expression profiling in combination with Western blotting revealed that exendin-4 regulates expression of a number of genes involved in angiogenesis, inflammation and thrombogenesis under glucolipotoxic conditions. Our results indicate that exendin-4 may improve endothelial cell function in diabetes through regulating expression of the genes, whose expression was disrupted by glucolipotoxicity. As endothelial dysfunction appears to be an early indicator of vascular damage, and predicts both progression of atherosclerosis and incidence of cardiovascular events, exendin-4 and possibly other incretin-based strategies may confer additional cardiovascular benefit beyond improved glycemic control.

Perlecan heparan sulfate deficiency impairs pulmonary vascular development and attenuates hypoxic pulmonary hypertension.

AIMS Excessive vascular cell proliferation is an important component of pulmonary hypertension (PH). Perlecan is the major heparan sulfate (HS) proteoglycan in the vascular extracellular matrix. It binds growth factors, including FGF2, and either restricts or promotes cell proliferation. In this study, we have explored the effects of perlecan HS deficiency on pulmonary vascular development and in hypoxia-induced PH. METHODS AND RESULTS In normoxia, Hspg2(Δ3/Δ3) mice, deficient in perlecan HS, had reduced pericytes and muscularization of intra-acinar vessels. Pulmonary angiography revealed a peripheral perfusion defect. Despite these abnormalities, right ventricular systolic pressure (RVSP) and myocardial mass remained normal. After 4 weeks of hypoxia, increases in the proportion of muscularized vessels, RVSP, and right ventricular hypertrophy were significantly less in Hspg2(Δ3/Δ3) compared with wild type. The early phase of hypoxia induced a significantly lower increase in fibroblast growth factor receptor-1 (FGFR1) protein level and receptor phosphorylation, and reduced pulmonary artery smooth muscle cell (PASMC) proliferation in Hspg2(Δ3/Δ3). At 4 weeks, FGF2 mRNA and protein were also significantly reduced in Hspg2(Δ3/Δ3) lungs. Ligand and carbohydrate engagement assay showed that perlecan HS is required for HS-FGF2-FGFR1 ternary complex formation. In vitro, proliferation assays showed that PASMC proliferation is reduced by selective FGFR1 inhibition. PASMC adhesion to fibronectin was higher in Hspg2(Δ3/Δ3) compared with wild type. CONCLUSIONS Perlecan HS chains are important for normal vascular arborization and recruitment of pericytes to pulmonary vessels. Perlecan HS deficiency also attenuates hypoxia-induced PH, where the underlying mechanisms involve impaired FGF2/FGFR1 interaction, inhibition of PASMC growth, and altered cell-matrix interactions.

PC196 Effects of Linagliptin on Impaired Vessel Wall Healing After Arterial Injury in the Rat Diabetic Model

Objectives: Diabetic patients suffer an increased risk of restenosis and late stent thrombosis after angioplasty, complications that are related to a defective re-endothelialization. Dipeptidyl peptidase-4 inhibitors have been suggested to exert direct effect on endothelial and smooth muscle cells (SMCs). Therefore, the objective was to study whether the dipeptidyl peptidase-4 inhibitor linagliptin could influence vascular repair and accelerate re-endothelialization after arterial injury in healthy and diabetic animals. Methods: Diabetic Goto-Kakizaki and healthy Wistar rats were subjected to arterial injury and treated with linagliptin or vehicle. Vessel wall healing was monitored noninvasively using ultrasound imaging and upon sacrifice with Evans blue staining and immunohistochemistry. The effect of linagliptin on SMCs was also studied in vitro. Results: We observed a delay in the healing response in the diabetic animals compared with the Wistar controls. Goto-Kakizaki rats had more pronounced intimal hyperplasia, which affected the lumen diameter. We found that linagliptin reduced the proliferation and dedifferentiation of SMCs in vitro and modulated the inflammatory response in the SMCs after arterial injury in vivo. However, these effects of linagliptin did not affect the neointima formation or the re-endothelialization under normal and diabetic conditions. Conclusions: Although linagliptin did not influence vessel wall healing, it appears to possess a desirable antiproliferative influence on SMCs in vitro and an anti-inflammatory effect in vivo. These pharmacologic properties might carry a potential significance for favorable outcome after vascular interventions in diabetic patients.

Effects of linagliptin on vessel wall healing in the rat model of arterial injury under normal and diabetic conditions.

Abstract: Diabetic patients suffer an increased risk of restenosis and late stent thrombosis after angioplasty, complications which are related to a defective reendothelialization. Dipeptidyl peptidase-4 inhibitors have been suggested to exert a direct effect on endothelial and smooth muscle cells (SMCs). Therefore, the objective was to study if the dipeptidyl peptidase-4 inhibitor linagliptin could influence vascular repair and accelerate reendothelialization after arterial injury in healthy and diabetic animals. Diabetic Goto-Kakizaki and healthy Wistar rats were subjected to arterial injury and treated with linagliptin or vehicle. Vessel wall healing was monitored noninvasively using ultrasound, and on sacrifice, with Evans blue staining and immunohistochemistry. The effect of linagliptin on SMCs was also studied in vitro. We found that linagliptin reduced the proliferation and dedifferentiation of SMCs in vitro, and modulated the inflammatory response in the SMCs after arterial injury in vivo. However, these effects of linagliptin did not affect the neointima formation or the reendothelialization under normal and diabetic conditions. Although linagliptin did not influence vessel wall healing, it seems to possess a desirable antiproliferative influence on SMCs in vitro and an antiinflammatory effect in vivo. These pharmacological properties might carry a potential significance for favorable outcome after vascular interventions in diabetic patients.