Enhanced microglial pro-inflammatory response to lipopolysaccharide correlates with brain infiltration and blood-brain barrier dysregulation in a mouse model of telomere shortening.

Microglia are a proliferative population of resident brain macrophages that under physiological conditions self-renew independent of hematopoiesis. Microglia are innate immune cells actively surveying the brain and are the earliest responders to injury. During aging, microglia elicit an enhanced innate immune response also referred to as 'priming'. To date, it remains unknown whether telomere shortening affects the proliferative capacity and induces priming of microglia. We addressed this issue using early (first-generation G1 mTerc(-/-) )- and late-generation (third-generation G3 and G4 mTerc(-/-) ) telomerase-deficient mice, which carry a homozygous deletion for the telomerase RNA component gene (mTerc). Late-generation mTerc(-/-) microglia show telomere shortening and decreased proliferation efficiency. Under physiological conditions, gene expression and functionality of G3 mTerc(-/-) microglia are comparable with microglia derived from G1 mTerc(-/-) mice despite changes in morphology. However, after intraperitoneal injection of bacterial lipopolysaccharide (LPS), G3 mTerc(-/-) microglia mice show an enhanced pro-inflammatory response. Nevertheless, this enhanced inflammatory response was not accompanied by an increased expression of genes known to be associated with age-associated microglia priming. The increased inflammatory response in microglia correlates closely with increased peripheral inflammation, a loss of blood-brain barrier integrity, and infiltration of immune cells in the brain parenchyma in this mouse model of telomere shortening.

Genetic variation and gender determine bradykinin type 1 receptor responses in human tissue: implications for the ACE-inhibitor-induced effects in patients with coronary artery disease.

The efficacy of the ACE (angiotensin-converting enzyme) inhibitor perindopril in coronary artery disease [EUROPA (European trial on reduction of cardiac events with perindopril in stable coronary artery disease) study] is associated with the rs12050217 A/G single nucleotide polymorphism in the B1 receptor (bradykinin type 1 receptor) gene. To investigate the underlying mechanism, we examined the effect of this polymorphism on B1-receptor-mediated coronary artery dilation and peripheral blood mononuclear cell activation. Vasorelaxant responses of human coronary microarteries from subjects without coronary disease to des-Arg(9)-bradykinin and to bradykinin were studied in organ bath experiments. Des-Arg9-bradykinin responses were endothelium-dependent and exclusively mediated by B1 receptors, whereas responses to bradykinin were induced through B2 receptors (bradykinin type 2 receptors). The presence of the G allele reduced the response to 3 × 10(-8) mol/l des-Arg(9)-bradykinin by 29% [AA (n=13) compared with AG/GG (n=8); P<0.03], and tended to lower concentration-related responses (P=0.065) to this agonist, whereas the responses to bradykinin were unaffected by the rs12050217 genotype. In freshly obtained human mononuclear cells 1 µmol/l des-Arg(9)-bradykinin increased expression of the pro-inflammatory factors CXCL5 (CXC chemokine ligand 5) and IL6 (interleukin-6). These responses were not affected by genotype and exclusively occurred in blood cells from women, correlating (in the case of CXCL5) with their plasma 17ß-oestradiol levels (r(2)=0.32, P=0.02; n=17). IL-1ß (interleukin-1ß) increased CXCL5 and IL6 expression in both genders, and this response was not associated with 17ß-oestradiol levels. The gender difference in responses to B1 receptor stimulation in blood mononuclear cells implies possible gender differences in the response to ACE inhibitor therapy, which needs to be studied more comprehensively. The observed decrease in coronary vasodilator response might contribute to the impaired treatment response to perindopril of G allele carriers found in the EUROPA study.

Nucleotide excision DNA repair is associated with age-related vascular dysfunction.

BACKGROUND: Vascular dysfunction in atherosclerosis and diabetes mellitus, as observed in the aging population of developed societies, is associated with vascular DNA damage and cell senescence. We hypothesized that cumulative DNA damage during aging contributes to vascular dysfunction. METHODS AND RESULTS: In mice with genomic instability resulting from the defective nucleotide excision repair genes ERCC1 and XPD (Ercc1(d/-) and Xpd(TTD) mice), we explored age-dependent vascular function compared with that in wild-type mice. Ercc1(d/-) mice showed increased vascular cell senescence, accelerated development of vasodilator dysfunction, increased vascular stiffness, and elevated blood pressure at a very young age. The vasodilator dysfunction was due to decreased endothelial nitric oxide synthase levels and impaired smooth muscle cell function, which involved phosphodiesterase activity. Similar to Ercc1(d/-) mice, age-related endothelium-dependent vasodilator dysfunction in Xpd(TTD) animals was increased. To investigate the implications for human vascular disease, we explored associations between single-nucleotide polymorphisms of selected nucleotide excision repair genes and arterial stiffness within the AortaGen Consortium and found a significant association of a single-nucleotide polymorphism (rs2029298) in the putative promoter region of DDB2 gene with carotid-femoral pulse wave velocity. CONCLUSIONS: Mice with genomic instability recapitulate age-dependent vascular dysfunction as observed in animal models and in humans but with an accelerated progression compared with wild-type mice. In addition, we found associations between variations in human DNA repair genes and markers for vascular stiffness, which is associated with aging. Our study supports the concept that genomic instability contributes importantly to the development of cardiovascular disease.

Red wine extract protects against oxidative-stress-induced endothelial senescence.

Red wine polyphenols may preserve endothelial function during aging. Endothelial cell senescence enhances age-related endothelial dysfunction. We investigated whether RWE (red wine extract) prevents oxidative-stress-induced senescence in HUVECs (human umbilical-vein endothelial cells). Senescence was induced by exposing HUVECs to tBHP (t-butylhydroperoxide), and quantified by senescence-associated ß-galactosidase staining. RWE (0-50 µg/ml) concentration dependently decreased senescence by maximally 33±7.1%. RWE prevented the senescence-associated increase in p21 protein expression, inhibited tBHP-induced DNA damage of endothelial cells and induced relaxation of PCAs (porcine coronary arteries). Inhibition of SIRT1 (sirtuin 1) by sirtinol partially reversed the effect of RWE on tBHP-induced senescence, whereas both the NOS (nitric oxide synthase) inhibitor L-NMMA (NG-monomethyl-L-arginine) and the COX (cyclo-oxygenase) inhibitor indomethacin fully inhibited it. Furthermore, incubation of HUVECs with RWE increased eNOS (endothelial NOS) and COX-2 mRNA levels as well as phosphorylation of eNOS at Ser1177. RWE protects endothelial cells from tBHP-induced senescence. NO and COX-2, in addition to activation of SIRT1, play a critical role in the inhibition of senescence induction in human endothelial cells by RWE.

Glucagon-like peptide 1 prevents reactive oxygen species-induced endothelial cell senescence through the activation of protein kinase A.

OBJECTIVE: Endothelial cell senescence is an important contributor to vascular aging and is increased under diabetic conditions. Here we investigated whether the antidiabetic hormone glucagon-like peptide 1 (GLP-1) could prevent oxidative stress-induced cellular senescence in endothelial cells. METHODS AND RESULTS: In Zucker diabetic fatty rats, a significant 2-fold higher level of vascular senescence was observed compared with control lean rats. Dipeptidyl-peptidase 4 (DPP-4) inhibition significantly increased GLP-1 levels in these animals and reduced senescence almost to lean animal levels. In vitro studies with human umbilical vein endothelial cells showed that GLP-1 had a direct protective effect on oxidative stress (H(2)O(2))-induced senescence and was able to attenuate oxidative stress-induced DNA damage and cellular senescence. The GLP-1 analogue exendin-4 provided similar results, whereas exendin fragment 9-39, a GLP-1 receptor antagonist, abolished this effect. Intracellular signaling by the phosphoinositide 3-kinase (PI3K)/Akt survival pathway did not appear to be involved. Further analysis revealed that GLP-1 activates the cAMP response element-binding (CREB) transcription factor in a cAMP/protein kinase A (PKA)-dependent manner, and inhibition of the cAMP/PKA pathway abolished the GLP-1 protective effect. Expression analysis revealed that GLP-1 can induce the oxidative defense genes HO-1 and NQO1. CONCLUSIONS: Dipeptidyl-peptidase 4 inhibition protects against vascular senescence in a diabetic rat model. In vitro studies with human umbilical vein endothelial cells showed that reactive oxygen species-induced senescence was attenuated by GLP-1 in a receptor-dependent manner involving downstream PKA signaling and induction of antioxidant genes.

Telomere biology in healthy aging and disease.

Aging is a biological process that affects most cells, organisms and species. Telomeres have been postulated as a universal biological clock that shortens in parallel with aging in cells. Telomeres are located at the end of the chromosomes and consist of an evolutionary conserved repetitive nucleotide sequence ranging in length from a few hundred base pairs in yeast till several kilo base pairs in vertebrates. Telomeres associate with shelterin proteins and form a complex protecting the chromosomal deoxyribonucleic acid (DNA) from recognition by the DNA damage-repair system. Due to the "end-replication problem" telomeres shorten with each mitotic cycle resulting in cumulative telomere attrition during aging. When telomeres reach a critical length the cell will not further undergo cell divisions and become senescent or otherwise dysfunctional. Telomere shortening has not only been linked to aging but also to several age associated diseases, including tumorigenesis, coronary artery disease, and heart failure. In the current review, we will discuss the role of telomere biology in relation to aging and aging associated diseases.

Telomere biology in cardiovascular disease: the TERC-/- mouse as a model for heart failure and ageing.

Atherosclerosis and heart failure are major causes of morbidity and mortality in Western countries. Recent studies are suggesting involvement of telomere biology in the development and progression of age-associated conditions, including hypertension, atherosclerosis, and heart failure. Whether any of these reported associations are based on causal relationships remains to be elucidated. The construction of telomerase-deficient (telomerase RNA component, TERC(-/-)) mice might provide a potential instrumental model to study the involvement of telomere biology in cardiovascular disease. Here, we review the current available information from all studies performed in TERC(-/-) mice providing information on the cardiovascular phenotypic characteristics. Although this mouse model has proven its value in the understanding of the role of telomere biology in cancer, stem cell, and basic telomere research, only few studies were specifically designed to answer cardiovascular-related questions. The TERC(-/-) mice provide exciting opportunities to expand our knowledge of telomere biology in cardiovascular disease and the potential identification of novel targets of treatment.

Bradykinin protects against oxidative stress-induced endothelial cell senescence.

Premature aging (senescence) of endothelial cells might play an important role in the development and progression of hypertension and atherosclerosis. We hypothesized that bradykinin, a hormone that mediates vasoprotective effects of angiotensin-converting enzyme inhibitors, protects endothelial cells from oxidative stress-induced senescence. Bradykinin treatment (0.001 to 1 nmol/L) dose-dependently decreased senescence induced by 25 micromol/L of H(2)O(2) in cultured bovine aortic endothelial cells, as witnessed by a complete inhibition of increased senescent cell numbers and a 34% reduction of the levels of the senescence-associated cell cycle protein p21. Because H(2)O(2) induces senescence through superoxide-induced DNA damage, single-cell DNA damage was measured by comet assay. Bradykinin reduced DNA damage to control levels. The protective effect of bradykinin also resulted in a significant increase in the migration of H(2)O(2)-treated bovine aorta endothelial cells in an in vitro endothelial injury model, or "scratch" assay. The protective effect of bradykinin was abolished by the bradykinin B2 receptor antagonist HOE-140 and the NO production inhibitor N(omega)-methyl-L-arginine acetate salt. Therefore, we conclude that bradykinin protects endothelial cells from superoxide-induced senescence through bradykinin B2 receptor- and NO-mediated inhibition of DNA damage.

Erythropoietin stimulates normal endothelial progenitor cell-mediated endothelial turnover, but attributes to neovascularization only in the presence of local ischemia.

PURPOSE: We aimed to evaluate whether ischemia is required for erythropoietin (EPO) induced stimulation of endothelial progenitor cells (EPCs) and their related effects on endothelial and cardiac function. METHODS: Bone marrow of rats was replaced by transgenic cells to allow tracking of EPCs. Ischemic heart failure was induced by left coronary artery ligation to induce myocardial infarction (MI) and control rats received a sham procedure. Three weeks after surgery, rats were randomized to receive EPO (darbepoetin alfa 40 microg/kg per 3 weeks) or vehicle and were sacrificed 9 weeks after surgery. RESULTS: In all treated groups, EPO significantly increased circulating EPCs and their incorporation into the endothelium of the ischemic and non-ischemic hearts as well as in the control organs; kidney and liver. This was associated with significantly improved endothelial function, which was strongly correlated with circulating EPCs (R = 0.7, p < 0.01). However, additional EPCs preferentially homed to the ischemic MI borderzone (p < 0.01) resulting in specific EPO-induced improvement of cardiac microvascularization and performance only in ischemic hearts (all p < 0.05). The differential stimulation of neovascularization by EPO was associated with increased EPO-receptor and VEGF expression in ischemic hearts only. CONCLUSIONS: In general, EPO stimulates normal endothelial progenitor cell-mediated endothelial turnover, but improves cardiac microvascularization and function only in the presence of ischemia.

Erythropoietin improves cardiac function through endothelial progenitor cell and vascular endothelial growth factor mediated neovascularization.

AIMS: Erythropoietin (EPO) improves cardiac function and induces neovascularization in chronic heart failure (CHF), although the exact mechanism has not been elucidated. We studied the effects of EPO on homing and incorporation of endothelial progenitor cells (EPC) into the myocardial microvasculature and myocardial expression of angiogenic factors. METHODS AND RESULTS: CHF was induced in rats by coronary artery ligation resulting in myocardial infarction (MI) after bone marrow had been replaced by human placental alkaline phosphatase (hPAP) transgenic cells. We studied the effects of darbepoetin alfa treatment (EPO, 40 microg/kg, every 3 weeks, starting 3 weeks after MI) on longitudinal changes in left ventricular (LV) function, circulating EPC, myocardial histology, and expression of vascular endothelial growth factor (VEGF) determined 9 weeks after MI. EPO prevented LV-dilatation and improved cardiac function (all P < 0.05), which was associated with 42% increased capillary growth (P < 0.01). EPO-induced mobilization of EPC from the bone marrow (P < 0.01), which resulted in a three-fold increased homing of EPC into the cardiac microvasculature. The percentage of the endothelium that consisted of bone marrow derived cells was significantly increased (3.9 +/- 0.5 vs. 11.4 +/- 1%, P < 0.001) comprising 30% of the newly formed capillaries. In addition, EPO treatment resulted in a 4.5-fold increased myocardial expression of VEGF, which correlated strongly with neovascularization (r = 0.67; P < 0.001). VEGF was equally expressed by endothelial cells of myocardial and bone marrow origin. CONCLUSION: EPO-induced neovascularization in post-MI heart failure is mediated through a combination of EPC recruitment from the bone marrow and increased myocardial expression of VEGF.