Leukocyte telomere length and mitochondrial DNA copy number associate with endothelial function in aging-related cardiovascular disease.

Background: We investigated the association between leukocyte telomere length, mitochondrial DNA copy number, and endothelial function in patients with aging-related cardiovascular disease (CVD). Methods: In total 430 patients with CVD and healthy persons were enrolled in the current study. Peripheral blood was drawn by routine venipuncture procedure. Plasma and peripheral blood mononuclear cells (PBMCs) were collected. Cell-free genomic DNA (cfDNA) and leukocytic genomic DNA (leuDNA) were extracted from plasma and PBMCs, respectively. Relative telomere length (TL) and mitochondrial DNA copy number (mtDNA-CN) were analyzed using quantitative polymerase chain reaction. Endothelial function was evaluated by measuring flow-mediated dilation (FMD). The correlation between TL of cfDNA (cf-TL), mtDNA-CN of cfDNA (cf-mtDNA), TL of leuDNA (leu-TL), mtDNA-CN of leuDNA (leu-mtDNA), age, and FMD were analyzed based on Spearman's rank correlation. The association between cf-TL, cf-mtDNA, leu-TL, leu-mtDNA, age, gender, and FMD were explored using multiple linear regression analysis. Results: cf-TL positively correlated with cf-mtDNA (r = 0.1834, P = 0.0273), and leu-TL positively correlated with leu-mtDNA (r = 0.1244, P = 0.0109). In addition, both leu-TL (r = 0.1489, P = 0.0022) and leu-mtDNA (r = 0.1929, P < 0.0001) positively correlated with FMD. In a multiple linear regression analysis model, both leu-TL (ß = 0.229, P = 0.002) and leu-mtDNA (ß = 0.198, P = 0.008) were positively associated with FMD. In contrast, age was inversely associated with FMD (ß = -0.426, P < 0.0001). Conclusion: TL positively correlates mtDNA-CN in both cfDNA and leuDNA. leu-TL and leu-mtDNA can be regarded as novel biomarkers of endothelial dysfunction.

Autophagy-related genes analysis reveals potential biomarkers for prediction of the impaired walking capacity of peripheral arterial disease.

BACKGROUND: The role of autophagy and autophagy-related genes in peripheral arterial disease (PAD) remains unknown and may be of diagnostic and prognostic value. The aim of this study is to investigate the relationship between autophagy and PAD, and identify potential diagnostic or prognostic biomarkers for medical practice. METHODS: Differentially expressed autophagy-related genes in PAD were explored from GSE57691 and validated in our WalkByLab registry participants by quantitative real-time polymerase chain reaction (qRT-PCR). The level of autophagy in peripheral blood mononuclear cells (PBMCs) of WalkByLab participants was assessed by analyzing autophagic marker proteins (beclin-1, P62, LC3B). Single sample gene set enrichment analysis (ssGSEA) was used to evaluate the immune microenvironment within the artery wall of PAD patients and healthy persons. Chemokine antibody array and enzyme-linked immunosorbent assay were used to assess the chemokines in participants' plasma. Treadmill testing with Gardner protocol was used to evaluate participants' walking capacity. Pain-free walking distance, maximum walking distance, and walking time were recorded. Finally, a nomogram model based on logistic regression was built to predict impaired walking performance. RESULTS: A total of 20 relevant autophagy-related genes were identified, and these genes were confirmed to be expressed at low levels in our PAD participants. Western blotting demonstrated that the expression of autophagic marker proteins beclin-1 and LC3BII were significantly reduced in PAD patients' PBMCs. ssGSEA revealed that most of the autophagy-related genes were strongly correlated with immune function, with the largest number of associated genes showing interaction between cytokine-and-cytokine receptors (CCR). In this context, the chemokines growth-related oncogene (GRO) and neutrophil activating protein2 (NAP2) are highly expressed in the plasma of WalkByLab PAD patients and were significantly negatively correlated with the walking distance assessed by Gardner treadmill testing. Finally, the plasma NAP2 level (AUC: 0.743) and derived nomogram model (AUC: 0.860) has a strong predictive potential to identify a poor walking capacity. CONCLUSIONS: Overall, these data highlight both the important role of autophagy and autophagy-related genes in PAD and link them to vascular inflammation (expression of chemokines). In particular, chemokine NAP2 emerged as a novel biomarker that can be used to predict the impaired walking capacity in PAD patients.

Angiotensin receptor-neprilysin inhibitor improves coronary collateral perfusion.

Background: We investigated the pleiotropic effects of an angiotensin receptor-neprilysin inhibitor (ARNi) on collateral-dependent myocardial perfusion in a rat model of coronary arteriogenesis, and performed comprehensive analyses to uncover the underlying molecular mechanisms. Methods: A rat model of coronary arteriogenesis was established by implanting an inflatable occluder on the left anterior descending coronary artery followed by a 7-day repetitive occlusion procedure (ROP). Coronary collateral perfusion was measured by using a myocardial particle infusion technique. The putative ARNi-induced pro-arteriogenic effects were further investigated and compared with an angiotensin-converting enzyme inhibitor (ACEi). Expression of the membrane receptors and key enzymes in the natriuretic peptide system (NPS), renin-angiotensin-aldosterone system (RAAS) and kallikrein-kinin system (KKS) were analyzed by quantitative polymerase chain reaction (qPCR) and immunoblot assay, respectively. Protein levels of pro-arteriogenic cytokines were measured by enzyme-linked immunosorbent assay, and mitochondrial DNA copy number was assessed by qPCR due to their roles in arteriogenesis. Furthermore, murine heart endothelial cells (MHEC5-T) were treated with a neprilysin inhibitor (NEPi) alone, or in combination with bradykinin receptor antagonists. MHEC5-T proliferation was analyzed by colorimetric assay. Results: The in vivo study showed that ARNis markedly improved coronary collateral perfusion, regulated the gene expression of KKS, and increased the concentrations of relevant pro-arteriogenic cytokines. The in vitro study demonstrated that NEPis significantly promoted MHEC5-T proliferation, which was diminished by bradykinin receptor antagonists. Conclusion: ARNis improve coronary collateral perfusion and exert pro-arteriogenic effects via the bradykinin receptor signaling pathway.

Effect of ACEI and ARB treatment on nitric oxide-dependent endothelial function.

Background: Angiotensin-converting-enzyme inhibitors (ACEI) and angiotensin II receptor blockers (ARB) are widely used as a first-line therapy for the treatment of cardiovascular disease. Here, ACEI modulate the bradykinin receptor (BDKRB1 and BDKRB2) system and NO-dependent endothelial function, thus determining cardiovascular health and regenerative arteriogenesis. The current study aims at evaluating nitric oxide-dependent endothelial function, and gene expression of bradykinin receptors in peripheral blood mononuclear cells (PBMC) from patients with ACEI or ARB treatment. Patients and methods: The WalkByLab has been established to screen cardiovascular patients for peripheral artery disease and coronary artery disease. In total 177 patients from WalkByLab with heterogenous disease and risk status were randomly selected, divided according to their medication history into the following groups: 1. ACEI group, 2. ARB group or 3. non-ACE/ARB group. Total plasma nitrite/nitrate (NO) levels were measured, endothelial function was evaluated by assessing flow meditated dilation (FMD). PBMC were isolated from peripheral whole blood, and gene expression (qRT-PCR) of bradykinin receptors and angiotensin converting enzyme were assessed. Results: Plasma total NO concentration in the ACEI group (24.66±16.28, µmol/l) was increased as compared to the ARB group (18.57±11.58, µmol/l, P=0.0046) and non-ACE/ARB group (16.83±8.64, µmol/l, P=0.0127) in patients between 40 to 90 years of age. However, FMD values (%) in the ACEI group (7.07±2.40, %) were similar as compared to the ARB (6.35±2.13, %) and non-ACE/ARB group (6.51±2.15, %), but significantly negatively correlated with age. Interestingly, BDKRB1 mRNA level was significantly higher and BDKRB2 mRNA level lower in the ACEI group (BDKRB1 3.88-fold±1.05, BDKRB2 0.22-fold±0.04) as compared to the non-ACE/ARB group (BDKRB1 1.00-fold±0.39, P<0.0001, BDKRB2 1.00-fold±0.45, P=0.0136). Conclusions: ACEI treatment enhances total nitrite/nitrate concentration, furthermore, upregulates BDKRB1 in PBMC, but downregulates BDKRB2 mRNA expression. FMD is a strong determinant of vascular aging and is sensitive to underlying heterogenous cardiovascular diseases.

The Wilms tumor protein WT1 stimulates transcription of the gene encoding insulin-like growth factor binding protein 5 (IGFBP5).

Insulin-like growth factor (IGF) binding proteins (IGFBPs) constitute a family of six secreted proteins that regulate the signaling of insulin-like growth factors (IGFs). IGFBP5 is the most conserved family member in vertebrates and the major IGF binding protein in bone. IGFBP5 is required for normal development of the musculoskeletal system, and various types of cancer frequently express high levels of IGFP5. Here we identify the gene encoding IGFBP5 as a novel downstream target of the Wilms tumor protein WT1. IGFBP5 and WT1 are expressed in an overlapping pattern in the condensing metanephric mesenchyme of embryonic murine kidneys. Down-regulation of WT1 by transfection with antisense vivo-morpholino significantly decreased Igfbp5 transcripts in murine embryonic kidney explants. Likewise, silencing of Wt1 in a mouse mesonephros-derived cell line reduced Igfbp5 mRNA levels by approximately 80%. Conversely, induction of the WT1(-KTS) isoform, whose role as transcriptional regulator has been firmly established, significantly increased IGFBP5 mRNA and protein levels in osteosarcoma cells. IGFBP5 expression was not significantly changed by WT1(+KTS) protein, which exhibits lower DNA binding affinity than the WT1(-KTS) isoform and has a presumed role in post-transcriptional gene regulation. Luciferase reporter constructs harboring 0.8 and 1.6 kilobases of the murine Igfbp5 promoter, respectively, were stimulated approximately 5-fold by co-transfection of WT1(-KTS). The WT1(+KTS) variant had no significant effect on IGFBP5 promoter activity. Binding of WT1(-KTS), but not of WT1(+KTS) protein, to the IGFBP5 promoter in human osteosarcoma cells was proven by chromatin immunoprecipitation (ChIP) and confirmed by electrophoretic mobility shift assay. These findings demonstrate that WT1 activates transcription of the IGFBP5 gene with possible implications for kidney development and bone (patho)physiology.

Amine oxidase copper-containing 1 (AOC1) is a downstream target gene of the Wilms tumor protein, WT1, during kidney development.

Amine oxidase copper-containing 1 (AOC1; formerly known as amiloride-binding protein 1) is a secreted glycoprotein that catalyzes the degradation of putrescine and histamine. Polyamines and their diamine precursor putrescine are ubiquitous to all organisms and fulfill pivotal functions in cell growth and proliferation. Despite the importance of AOC1 in regulating polyamine breakdown, very little is known about the molecular mechanisms that control its expression. We report here that the Wilms tumor protein, WT1, which is necessary for normal kidney development, activates transcription of the AOC1 gene. Expression of a firefly luciferase reporter under control of the proximal AOC1 promoter was significantly enhanced by co-transfection of a WT1 expression construct. Binding of WT1 protein to a cis-regulatory element in the AOC1 promoter was confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Antisense inhibition of WT1 protein translation strongly reduced Aoc1 transcripts in cultured murine embryonic kidneys and gonads. Aoc1 mRNA levels correlated with WT1 protein in several cell lines. Double immunofluorescent staining revealed a co-expression of WT1 and AOC1 proteins in the developing genitourinary system of mice and rats. Strikingly, induced changes in polyamine homeostasis affected branching morphogenesis of cultured murine embryonic kidneys in a developmental stage-specific manner. These findings suggest that WT1-dependent control of polyamine breakdown, which is mediated by changes in AOC1 expression, has a role in kidney organogenesis.

Induction of cerebral arteriogenesis in mice.

Unilateral common carotid artery occlusion (CCAO) is a standardized method to initiate collateral artery growth (arteriogenesis) in mouse brain. After CCAO is induced, blood circulation in the circle of Willis is changed and increases shear stress, which triggers increased arterial diameter and improvements in cerebrovascular reserve capacity. Functional improvement can be quantified after experimentally induced stroke by external middle cerebral artery occlusion (MCAO). Stroke volume is evaluated by standard tetrazolium chloride (TTC) staining. Here, we describe in vivo methods of CCAO and MCAO in detail and also the evaluation of stroke volume by TTC staining.

Iodinated contrast media cause endothelial damage leading to vasoconstriction of human and rat vasa recta.

Contrast-induced acute kidney injury is an important clinical event with a worldwide increasing number of cases. Medullary hypoperfusion and hypoxia due to constriction of vasa recta are main factors in the pathophysiology of acute kidney injury. However, the mechanism of contrast media (CM)-induced vessel constriction is not known. We tested the hypothesis that vasa recta constriction is a consequence of endothelial dysfunction due to the cytotoxicity of CM. Human and rat descending vasa recta (DVR) were isolated and perfused with CM, and the luminal diameter was analyzed. For morphological analysis of the endothelium, renal arteries were CM perfused and then processed for electron microscopy. Transcellular electrical resistance was used to estimate CM-induced changes in the permeability of human umbilical vein endothelial cell (HUVEC) layers. Perfusion with CM constricted human and rat DRV (to 54.3 and 50.9% of initial diameter, respectively). This was blunted by adrenomedullin (77.7 and 77.1%, respectively). The ANG II response was enhanced by CM in rat DVR (reduction to 15.6 and 35.0% of initial diameter, respectively). Adrenomedullin blunted this effect (67.5%). CM led to endothelial damage of renal arteries characterized by a ragged surface, with sharply protruding intimal folds, spindle-like shape, and bulging in the lumen. These phenomena were reduced by adrenomedullin. The permeability of HUVEC cell layers was increased by CM, and this went along with increased myosin light chain phosporylation. Again, adremonedullin reduced the CM effect. Our study suggests that the constrictor effect of CM on the renal medullary microvasculature is a consequence of endothelial cell damage and the resulting endothelial dysfunction.

Multilevel regulation of HIF-1 signaling by TTP.

Hypoxia-inducible factor-1 (HIF-1) is a well-studied transcription factor mediating cellular adaptation to hypoxia. It also plays a crucial role under normoxic conditions, such as in inflammation, where its regulation is less well understood. The 3'-untranslated region (UTR) of HIF-1α mRNA is among the most conserved UTRs in the genome, hinting toward posttranscriptional regulation. To identify potential trans factors, we analyzed a large compilation of expression data. In contrast to its known function of being a negative regulator, we found that tristetraprolin (TTP) positively correlates with HIF-1 target genes. Mathematical modeling predicts that an additional level of posttranslational regulation of TTP can explain the observed positive correlation between TTP and HIF-1 signaling. Mechanistic studies revealed that TTP indeed changes its mode of regulation from destabilizing to stabilizing HIF-1α mRNA upon phosphorylation by p38 mitogen-activated protein kinase (MAPK)/MAPK-activated protein kinase 2. Using a model of monocyte-to-macrophage differentiation, we show that TTP-driven HIF-1α mRNA stabilization is crucial for cell migration. This demonstrates the physiological importance of a hitherto-unknown mechanism for multilevel regulation of HIF-1α in normoxia.

Granulocyte colony-stimulating factor improves cerebrovascular reserve capacity by enhancing collateral growth in the circle of Willis.

BACKGROUND AND PURPOSE: Restoration of cerebrovascular reserve capacity (CVRC) depends on the recruitment and positive outward remodeling of preexistent collaterals (arteriogenesis). With this study, we provide functional evidence that granulocyte colony-stimulating factor (G-CSF) augments therapeutic arteriogenesis in two animal models of cerebral hypoperfusion. We identified an effective dosing regimen that improved CVRC and stimulated collateral growth, thereby improving the outcome after experimentally induced stroke. METHODS: We used two established animal models of (a) cerebral hypoperfusion (mouse, common carotid artery ligation) and (b) cerebral arteriogenesis (rat, 3-vessel occlusion). Following therapeutic dose determination, both models received either G-CSF, 40 µg/kg every other day, or vehicle for 1 week. Collateral vessel diameters were measured following latex angiography. Cerebrovascular reserve capacities were assessed after acetazolamide stimulation. Mice with left common carotid artery occlusion (CCAO) were additionally subjected to middle cerebral artery occlusion, and stroke volumes were assessed after triphenyltetrazolium chloride staining. Given the vital role of monocytes in arteriogenesis, we assessed (a) the influence of G-CSF on monocyte migration in vitro and (b) monocyte counts in the adventitial tissues of the growing collaterals in vivo. RESULTS: CVRC was impaired in both animal models 1 week after induction of hypoperfusion. While G-CSF, 40 µg/kg every other day, significantly augmented cerebral arteriogenesis in the rat model, 50 or 150 µg/kg every day did not show any noticeable therapeutic impact. G-CSF restored CVRC in mice (5 ± 2 to 12 ± 6%) and rats (3 ± 4 to 19 ± 12%). Vessel diameters changed accordingly: in rats, the diameters of posterior cerebral arteries (ipsilateral: 209 ± 7-271 ± 57 µm; contralateral: 208 ± 11-252 ± 28 µm) and in mice the diameter of anterior cerebral arteries (185 ± 15-222 ± 12 µm) significantly increased in the G-CSF groups compared to controls. Stroke volume in mice (10 ± 2%) was diminished following CCAO (7 ± 4%) and G-CSF treatment (4 ± 2%). G-CSF significantly increased monocyte migration in vitro and perivascular monocyte numbers in vivo. CONCLUSION: G-CSF augments cerebral collateral artery growth, increases CVRC and protects from experimentally induced ischemic stroke. When comparing three different dosing regimens, a relatively low dosage of G-CSF was most effective, indicating that the common side effects of this cytokine might be significantly reduced or possibly even avoided in this indication.

Acetylsalicylic acid, but not clopidogrel, inhibits therapeutically induced cerebral arteriogenesis in the hypoperfused rat brain.

This study investigated the effects of acetylsalicylic acid (ASA) and clopidogrel, standardly used in the secondary prevention of vascular occlusions, on cerebral arteriogenesis in vivo and in vitro. Cerebral hypoperfusion was induced by three-vessel occlusion (3-VO) in rats, which subsequently received vehicle, ASA (6.34 mg/kg), or clopidogrel (10 mg/kg). Granulocyte colony-stimulating factor (G-CSF), which enhanced monocyte migration in an additional cell culture model, augmented cerebrovascular arteriogenesis in subgroups (40 µg/kg). Cerebrovascular reactivity and vessel diameters were assessed at 7 and 21 days. Cerebrovascular reserve capacity was completely abolished after 3-VO and remained severely compromised after 7 (-14±14%) and 21 (-5±11%) days in the ASA groups in comparison with controls (4±5% and 10±10%) and clopidogrel (4±13% and 10±8%). It was still significantly decreased when ASA was combined with G-CSF (1±4%) compared with G-CSF alone (20±8%). Posterior cerebral artery diameters confirmed these data. Monocyte migration into the vessel wall, improved by G-CSF, was significantly reduced by ASA. Acetylsalicylic acid, but not clopidogrel, inhibits therapeutically augmented cerebral arteriogenesis.

Vascular growth in health and disease.

Vascular growth forms the first functional organ system during development, and continues into adult life, wherein it is often associated with disease states. Genetically determined vasculogenesis produces a primary vascular plexus during ontogenesis. Angiogenesis, occurring, e.g., in response to metabolic stress within hypoxic tissues, enhances tissue capillarization. Arteriogenesis denotes the adaptive outgrowth of pre-existent collateral arteries to bypass arterial stenoses in response to hemodynamic changes. It has been debated whether vasculogenesis occurs in the adult, and whether or not circulating progenitor cells structurally contribute to vessel regeneration. Secondly, the major determinants of vascular growth - genetic predisposition, metabolic factors (hypoxia), and hemodynamics - cannot be assigned in a mutually exclusive fashion to vasculogenesis, angiogenesis, and arteriogenesis, respectively; rather, mechanisms overlap. Lastly, all three mechanisms of vessel growth seem to contribute to physiological embryogenesis as well as adult adaptive vascularization as occurs in tumors or to circumvent arterial stenosis. Thus, much conceptual and terminological confusion has been created, while therapies targeting neovascularization have yielded promising results in the lab, but failed randomized studies when taken to the bedside. Therefore, this review article aims at providing an exact definition of the mechanisms of vascular growth and their contribution to embryonic development as well as adult adaptive revascularization. We have been looking for potential reasons for why clinical trials have failed, how vitally the application of appropriate methods of measuring and assessment influences study outcomes, and how relevant, e.g., results gained in models of vascular occlusive disease may be for antineoplastic strategies, advocating a reverse bedside-to-bench approach, which may hopefully yield successful approaches to therapeutically targeting vascular growth.

Wilms' tumor protein Wt1 regulates the Interleukin-10 (IL-10) gene.

We identified the Wilms' tumor protein, Wt1, as a novel transcriptional activator of the immunosuppressant cytokine interleukin-10 (IL-10). Silencing of Wt1 by RNA interference reduced IL-10 mRNA levels by approximately 90%. IL-10 transcripts were increased more than 15-fold upon forced expression of Wt1. Electrophoretic mobility shift assay and chromatin immunoprecipitation revealed a cis-element that was responsible for activation of the IL-10 promoter by Wt1 in murine macrophages. Mutation of the Wt1 binding motif abrogated stimulation of the IL-10 promoter by tumor necrosis factor-α (TNFα). These results suggest a novel immune regulatory function of Wt1 in controlling IL-10 gene expression.