Association of endothelial function and lower extremity perfusion in peripheral artery disease.

Background: The current study aims to investigate the association between endothelial function and lower extremity perfusion in patients with peripheral artery disease (PAD). Patients and methods: In total 229 patients with PAD (Rutherford stage 0-3) were enrolled in the current study. Endothelial function was assessed by measuring flow-mediated dilation (FMD) and endothelial cell proliferation capacity (ECPC). Lower extremity perfusion was assessed by measuring oscillometry-based ankle brachial index (oABI) and pulse wave index (PWI). In addition, carotid intima-media-thickness (cIMT) was also measured as a surrogate marker for atherosclerosis. Correlations between FMD, ECPC, oABI, PWI, and cIMT were analysed using Pearson correlation coefficient. The relationship between the above variables and the severity of PAD was investigated using ordinal logistic regression analysis. Results: Correlation analysis showed that FMD negatively associated with PWI (r = -0.183, p = 0.005), ECPC positively associated with oABI (r = 0.162, p = 0.014), and oABI negatively associated with PWI (r = -0.264, p < 0.001). Ordinal logistic regression analysis showed that ECPC (ß = -0.009, p = 0.048), oABI (ß = -5.290, p < 0.001), and age (ß = -0.058, p = 0.002) negatively associated with the PAD Rutherford stages. In addition, PWI (ß = 0.006, p < 0.001), cIMT (ß = 18.363, p = 0.043) positively associated with the PAD Rutherford stages. Conclusions: Endothelial function significantly associates with lower extremity perfusion in patients with PAD, and both are related to the severity of PAD.

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.

Impact of chemotherapeutic effects on the pathophysiology of the arterial wall - insights from peripheral arterial disease.

Systemic antineoplastic treatment agents represent one of the fastest developing medical fields. Oncological treatment is becoming increasingly individualized and new targets with corresponding agents, are constantly being developed. In tandem with this progress, new combinations and algorithms have evolved and patient's outcome have improved. Expanding tumors rely on a growing neovascular network to maintain their increased metabolism, which is caused by an accelerated reproduction rate. Accordingly, interrupting this supply mechanism is a major component of antineoplastic pharmaceutics and is a hallmark of cancer treatment. With advances in cancer treatment, long-term side effects have become an important consideration, especially in cases of neoplasia in young patients. While neuropathy and cardiotoxicity are well documented, vascular adverse events remain poorly understood. The mutual risk factors, like smoking and increased age, complicate the association between the vascular pathology and the earlier antineoplastic therapy. A deeper understanding of the effects of chemotherapy on peripheral arterial disease could lead to more detailed pathophysiological insight into both maladies and to new treatment options.

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.

PCSK9 regulates the chemokine receptor CCR2 on monocytes.

Monocyte migration is a key element in atherosclerosis. LDL-C facilitates monocyte migration via induction of CCR2. PCSK9 regulates cell surface expression of the LDL-R and is expressed in vascular smooth muscle cells (VSMCs). The present study was done to investigate the regulation of PCSK9 in VSMCs and its impact on monocyte function. METHODS AND RESULTS: PCSK9 mRNA and protein levels were upregulated in VSMCs by the TLR-4 ligand LPS, whereas TGF-ß or angiotensin II had no effect. Induction of PCSK9 was selectively inhibited by TLR-4 blockade and further downstream by the SAPK/JNK-inhibitor SP600125, whereas inhibitors of ERK1/2, p38 or PI3-kinase pathways had no effect. Incubation of monocytes in conditioned media from LPS-stimulated VSMCs resulted in a significant reduction of LDL-R levels on monocytes, comparable to the effects of recombinant PCSK9. LDL-C increased monocyte CCR2 expression, which augmented monocyte migration towards MCP-1. This LDL-C dependent monocyte chemotaxis was inhibited by supernatants from LPS-stimulated VSMCs, similar to recombinant PCSK9 and a specific LDL-R blocking antibody. CONCLUSION: PCSK9 is regulated in VSMCs by TLR-4 - SAPK/JNK signaling, a pathway important in inflammation and metabolism. VSMC-derived PCSK9 reduces monocyte LDL-R expression, affecting LDL-C/LDL-R-mediated CCR2-expression on monocytes, which is crucial to cell motility and atherogenesis.

Porcine arteriogenesis based on vasa vasorum in a novel semi-acute occlusion model using high-resolution imaging.

Bridging collaterals (BC) develop in several chronic total artery occlusion diseases, and can prevent extensive myocardial necrosis. Yet, their origin, growth process, and histo-morphology are still unclear. Since vasa vasorum (VV) may take part in collateralization, we hypothesized that VV are the basis for BCs. To comprehensively investigate this arteriogenesis process, we used high-resolution imaging, including corrosion casts, post-mortem angiography with stereoscopy, micro-CT, and immunohistology, in combination with a novel semi-acute vessel occlusion model. This porcine model was produced by implanting a copper stent minimally invasively into the left anterior descending coronary artery. To define the kinetics of arteriogenesis, pigs (n = 11) were assigned to one of the five euthanasia timepoints: day 0.5 (D0.5, n = 2), D3 (n = 2), D5 (n = 1), D7 (n = 3), or D12 (n = 3) after stent implantation. We found that (1) BCs originate from longitudinally running type 1 VV, mainly VV interna, partially also from VV externa; (2) the growth of VV to BC is rapid, occurring within 7 days; and (3) porcine BCs are likely functionally relevant, considering an observed 102% increase in the number of smooth muscle cell layers in their vascular wall. High-resolution imaging in a minimally invasive non-acute vessel occlusion model is an innovative technique that allowed us to provide direct evidence that porcine BCs develop from the VV. These data may be crucial for further studies on the treatment of angina pectoris and thromboangiitis obliterans through therapeutic stimulation of BC development.

Long-term individual shear rate therapy counterpulsation enhances plasma nitrite release in patients with PAD.

BACKGROUND: Individual shear rate therapy (ISRT) has been designed as a novel non-invasive treatment option for peripheral artery disease (PAD) patients and has been shown to improve endothelial function and walking distance. The aim of this study was to elucidate the impact of ISRT on the level of nitric oxide in patient blood plasma and the expression of related molecular markers in peripheral blood mononuclear cells (PBMCs). Molecular diagnostic tests were performed for two ISRT trials. PATIENTS AND METHODS: In ISRT-1 26 healthy subjects underwent one session of treadmill training and one session of ISRT respectively in a cross-over design. In ISRT-2 14 PAD patients with a stable intermittent claudication underwent a 30 hours long-term treatment. Plasma nitrite release as well as the mRNA expression of NOS2 and key regulators of the kallikrein-kinin system were measured in PBMCs at different time points. RESULTS: Short-term ISRT revealed significantly decreased NOS2 expression in PBMCs of healthy volunteers and PAD patients. Long-term ISRT, in turn, demonstrated a significant plasma nitrite increase in PAD patients. CONCLUSIONS: We verified that long-term ISRT stimulates the vascular system and exerts a comparable effect to physical exercise in regards to NO release, which coincide with recent findings regarding an improvement of endothelial function. However, further studies are necessary to investigate the role for circulating leukocytes.
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Adaptation of external counterpulsation based on individual shear rate therapy improves endothelial function and claudication distance in peripheral artery disease.

BACKGROUND: External counterpulsation therapy enhances blood flow and was shown to improve endothelial function and quality of life in coronary artery disease patients. However, high pressures of up to 300 mmHg may lead to malperfusion of the ischaemic limb. To improve the clinical outcome of patients with peripheral artery disease (PAD), we adjusted external counterpulsation and developed a novel non-invasive approach termed individual shear rate therapy (ISRT). PATIENTS AND METHODS: In the present study, 14 patients with a Fontaine stage IIb and femoral-popliteal PAD underwent 30 hours of ISRT over 5 weeks. For ISRT, individual treatment pressures that do not exceed 160 mmHg were assessed by Doppler flow parameters during counterpulsation (individual shear rate diagnosis) in order to enhance and maximise peripheral perfusion. The study aimed to enhance peripheral perfusion and evaluate the primary clinical endpoint endothelial function, as well as to perform preliminary analysis of the ankle brachial index (ABI) and walking distance. RESULTS: Doppler flow measurements in the lower limb (ankle) validated that maximum blood flow velocity during systole and acceleration doubled during ISRT. Study results demonstrated that long-term ISRT significantly increased flow-mediated dilation (FMD) in the brachial artery (0.13+/- 0.09 mm to 0.38+/- 0.05 mm; p < 0.05), while nitromediated dilation (0.36+/- 0.10 mm to 0.45+/- 0.08 mm) remained and common femoral artery FMD did not reach statistical significance (0.38+/- 0.08 mm to 0.67+/- 0.19 mm; p<0.05). Initial claudication distance considerably improved for all patients after 30 hours of ISRT (92.6 +/- 8.2 metres to 280+/- 101.3 metres, p<0.05), just like the absolute claudication distance, which showed a more than 2.5-fold increase (167.8+/- 18.1 metres to 446.7+/- 133.3 metres; p<0.05). The ABI did not improve (0.58+/- 0.03 to 0.65+/- 0.04). CONCLUSIONS: Our data demonstrate that long-term ISRT is a potential novel non-invasive treatment to improve endothelial function and absolute pain-free walking distance for PAD patients.

Lipoprotein(a) as a risk factor for atherosclerotic cardiovascular disease in patients in non-metropolitan areas of Brandenburg, Germany.

Background and aims: In the non-metropolitan region of Brandenburg (Germany), which is characterized by high rates of cardiovascular diseases and underserved medical care, there is a lack of awareness regarding lipoprotein(a) [Lp(a)] as a risk factor. In addition, data from patients with atherosclerotic cardiovascular disease (ASCVD) in diverse regional backgrounds, including the understudied Brandenburg cohort, and various healthcare statuses remain insufficient. Methods: In this WalkByLab study, Lp(a) levels were monitored in a non-metropolitan cohort (n = 850) in Brandenburg, Germany, comprising 533 patients at high cardiovascular risk and 317 healthy controls. Patients underwent a comprehensive angiological screening, which included blood serum analysis, assessment of medical and family history, cardiovascular risk, and disease status, and evaluation of lifestyle and quality of life. All parameters were evaluated with regard to two groups based on Lp(a) levels: low (<50 mg/dl) and high (≥50 mg/dl). Results: Brandenburg patients with cardiovascular diseases showed higher Lp(a) levels than healthy controls (24.2% vs. 14.8%, p = 0.001). Logistic regression analysis with different characteristics revealed that Lp(a) was an independent risk factor significantly associated with ASCVD (OR 2.26, 95% CI 1.32-3.95, p = 0.003). The high-Lp(a) group showed a higher proportion of patients with coronary artery disease, peripheral artery disease, or cerebrovascular disease compared to the low-Lp(a) group (50% vs. 36.8%; 57.7% vs. 45.8%; 17.6% vs. 9.2%; p = 0.004); also, a higher percentage of patients in the high-Lp(a) group had heart failure (72.8% vs. 53.2%, p = 0.014) and myocardial infarction (24.7% vs. 13.9%, p = 0.001). The high-Lp(a) group exhibited higher rates of statins (63.1% vs. 50.4%, p = 0.003), ezetimibe (14.8% vs. 5.5.%, p = 0.001), and beta-blockers (55.7% vs. 40.7%, p = 0.001) use. Lp(a) levels were found to be independent of physical activity or smoking behavior and did not change over time (12 months). Conclusions: Our study highlights the significance of elevated Lp(a) levels in Brandenburg cardiovascular patients and identifies them as an independent risk factor for ASCVD, which has implications for addressing cardiovascular health of non-metropolitan populations.

Oxidized high-density lipoprotein associates with atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) is the most common heart arrhythmia and considered to be a progressive chronic disease associated with increased morbidity and mortality. Recent data suggest a link between inflammation, oxidative stress, and AF, although the underlying mechanisms are not fully understood. Because oxidized lipoproteins cause structural damage and electrophysiologic changes in cardiomyocytes, it is feasible that the transformation of atheroprotective high-density lipoprotein (HDL) into dysfunctional HDL contributes to the development of AF. OBJECTIVE: The purpose of this study was to determine whether a reduced antioxidant function of HDL is associated with the presence of AF. METHODS: In this multicenter cross-sectional cohort study, we assessed HDL function in sera of 1206 participants. Patients were divided into groups according to the presence of AF (n = 233) or no AF (n = 973). A validated cell-free biochemical assay was used to determine reduced HDL antioxidant function as assessed by increased normalized HDL lipid peroxide content (nHDLox). RESULTS: Participants with AF had a 9% higher mean relative nHDLox compared to persons without AF (P = .025). nHDLox was strongly associated with AF in all models of logistic regression, including the analysis adjusted for age, sex, and risk factors for AF (all P ≤.01). CONCLUSION: Reduced antioxidant HDL function is associated with the presence of AF, which supports growing evidence that impaired lipoprotein function is linked to electrophysiological changes in cardiomyocytes. nHDLox is one of several contributors to the initiation and perpetuation of AF.

Pulsatile shear and Gja5 modulate arterial identity and remodeling events during flow-driven arteriogenesis.

In the developing chicken embryo yolk sac vasculature, the expression of arterial identity genes requires arterial hemodynamic conditions. We hypothesize that arterial flow must provide a unique signal that is relevant for supporting arterial identity gene expression and is absent in veins. We analyzed factors related to flow, pressure and oxygenation in the chicken embryo vitelline vasculature in vivo. The best discrimination between arteries and veins was obtained by calculating the maximal pulsatile increase in shear rate relative to the time-averaged shear rate in the same vessel: the relative pulse slope index (RPSI). RPSI was significantly higher in arteries than veins. Arterial endothelial cells exposed to pulsatile shear in vitro augmented arterial marker expression as compared with exposure to constant shear. The expression of Gja5 correlated with arterial flow patterns: the redistribution of arterial flow provoked by vitelline artery ligation resulted in flow-driven collateral arterial network formation and was associated with increased expression of Gja5. In situ hybridization in normal and ligation embryos confirmed that Gja5 expression is confined to arteries and regulated by flow. In mice, Gja5 (connexin 40) was also expressed in arteries. In the adult, increased flow drives arteriogenesis and the formation of collateral arterial networks in peripheral occlusive diseases. Genetic ablation of Gja5 function in mice resulted in reduced arteriogenesis in two occlusion models. We conclude that pulsatile shear patterns may be central for supporting arterial identity, and that arterial Gja5 expression plays a functional role in flow-driven arteriogenesis.

Arteriogenesis is modulated by bradykinin receptor signaling.

RATIONALE: Positive outward remodeling of pre-existing collateral arteries into functional conductance arteries, arteriogenesis, is a major endogenous rescue mechanism to prevent cardiovascular ischemia. Collateral arterial growth is accompanied by expression of kinin precursor. However, the role of kinin signaling via the kinin receptors (B1R and B2R) in arteriogenesis is unclear. OBJECTIVE: The purpose of this study was to elucidate the functional role and mechanism of bradykinin receptor signaling in arteriogenesis. METHODS AND RESULTS: Bradykinin receptors positively affected arteriogenesis, with the contribution of B1R being more pronounced than B2R. In mice, arteriogenesis upon femoral artery occlusion was significantly reduced in B1R mutant mice as evidenced by reduced microspheres and laser Doppler flow perfusion measurements. Transplantation of wild-type bone marrow cells into irradiated B1R mutant mice restored arteriogenesis, whereas bone marrow chimeric mice generated by reconstituting wild-type mice with B1R mutant bone marrow showed reduced arteriogenesis after femoral artery occlusion. In the rat brain 3-vessel occlusion arteriogenesis model, pharmacological blockade of B1R inhibited arteriogenesis and stimulation of B1R enhanced arteriogenesis. In the rat, femoral artery ligation combined with arterial venous shunt model resulted in flow-driven arteriogenesis, and treatment with B1R antagonist R715 decreased vascular remodeling and leukocyte invasion (monocytes) into the perivascular tissue. In monocyte migration assays, in vitro B1R agonists enhanced migration of monocytes. CONCLUSIONS: Kinin receptors act as positive modulators of arteriogenesis in mice and rats. B1R can be blocked or therapeutically stimulated by B1R antagonists or agonists, respectively, involving a contribution of peripheral immune cells (monocytes) linking hemodynamic conditions with inflammatory pathways.

Tolerability safety and effectiveness of enhanced external counterpulsation versus individual shear rate therapy in patients with lower extremity atherosclerotic disease: A prospective pilot clinical trial.

AIMS: We aimed to investigate the tolerability, safety, and effectiveness of enhanced external counterpulsation therapy (EECP) versus individual shear rate therapy (ISRT) in patients with lower extremity atherosclerotic disease (LEAD). METHODS: Eighteen patients (age: 73.1 ± 6 years) underwent EECP and ISRT, each daily over five consecutive days in a cross-over design with a 1 week resting period in between the two regimens. A quality-of-life questionnaire was used to assess the therapy experience. Oxygen saturation (SO2 ), relative hemoglobin amount (rHb) and blood flow (Flow) in the capillary-venous-system (microcirculation) of the skin were monitored continuously during all therapy sessions using the micro-lightguide spectrophotometer, also known as oxygen to see (O2C). The effects of EECP and ISRT on the renal function and skeletal muscles were evaluated using serial blood and urine tests. RESULTS: EECP therapy had to be terminated early before the end of the 5th session in 10 patients (55.6%) because of discomfort. Four patients (22.2%) experienced signs of critical limb ischaemia under EECP. The total score of the quality-of-life questionnaire was significantly higher (= better tolerated) post-ISRT compared with EECP. Microcirculation monitoring revealed that ISRT significantly increased the SO2 , blood flow and rHb during the therapy. All three parameters remained significantly increased in the observation period after ISRT. The serum levels of creatin kinase and myoglobin increased significantly under EECP. CONCLUSIONS: ISRT significantly improves tolerability, safety, and effectiveness over EECP in patients with LEAD.

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.

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.

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.

Angiotensin-converting enzyme inhibitors stimulate cerebral arteriogenesis.

AIM: Arteriogenesis constitutes the most efficient endogenous rescue mechanism in cases of cerebral ischaemia. The aim of this work was to investigate whether angiotensin-converting enzyme inhibitors (ACEi) stimulates, and angiotensin II receptor type 1 blockers (ARB) inhibits cerebral collateral growth by applying a three-vessel occlusion (3-VO) model in rat. METHODS: Cerebral collateral growth was measured post 3-VO (1) by assessing blood flow using the cerebrovascular reserve capacity (CVRC) technique, and (2) by assessing vessel diameters in the posterior cerebral artery (PCA) via the evaluation of latex angiographies. A stimulatory effect on arteriogenesis was investigated for ACEi administration ± bradykinin receptor 1 (B1R) and 2 (B2R) blockers, and an inhibitory effect was analysed for ARB administration. Results were validated by immunohistochemical analysis and mechanistic data were collected by human umbilical vein endothelial cell (HUVEC) viability or scratch assay and monocyte (THP-1) migration assay. RESULTS: An inhibitory effect of ARB on arteriogenesis could not be demonstrated. However, collateral growth measurements demonstrated a significantly increased CVRC and PCA diameters in the ACEi group. ACEi stimulates cell viability and migration, which could be partially reduced by additional administration of bradykinin receptor 1 inhibitor (B1Ri). ACEi inhibits the degradation of pro-arteriogenic bradykinin derivatives, but combined ACEi + B1Ri + B1Ri (BRB) treatment did not reverse the stimulatory effect. Yet, co-administration of ACEi + BRB enhances arteriogenesis and cell migration. CONCLUSION: We demonstrate a potent stimulatory effect of ACEi on cerebral arteriogenesis in rats, presumable via B1R. However, results imply a pleiotropic and compensatory effect of ACEi on bradykinin receptor-stimulated arteriogenesis.

Risk Factor Management in Non-Metropolitan Patients with Coronary and Peripheral Artery Disease - A Protocol of a Prospective, Multi-Center, Quality Improvement Strategy.

INTRODUCTION: Coronary artery disease (CAD) and peripheral arterial disease (PAD) account for significant morbidity and mortality in Germany and are more prevalent in rural, non-metropolitan areas. The goal of this study is to screen patients for their current atherosclerotic status, initiate treatment according to the latest scientific findings using a standardised multimodal approach and track their atherosclerotic status over one year. METHODS AND ANALYSIS: This manuscript describes the study protocol of a prospective, multicentre registry of 500 sequential patients with CAD and/or PAD in rural, non-metropolitan regions of Germany. Patients, who visit the "WalkByLab" at the Brandenburg Medical School, Brandenburg, Germany, will be assessed by using our structured, multimodal risk factor management (SMART) tool to evaluate cardiovascular morbidity data, collect information on care and deliver multimodal therapy. The study's primary objective is a cross-sectional examination of the risk profile, diagnostic and therapeutic status in this patient group. Secondary objectives include the assessment of risk factor correlations as well as changes in risk-factor profile and therapy adherence. Patients will be examined at baseline and followed up at three-monthly intervals for one year. Over this time, atherosclerotic risk factors and patient adherence to defined therapeutic strategies will be evaluated. Study completion is estimated to be December 2021. An expansion of this concept into other rural, non-metropolitan neighbouring regions is planned. ETHICS AND DISSEMINATION: This registry was assessed and approved by the ethics committee of the Brandenburg State Medical Association, Brandenburg, Germany, and conducted in accordance with the Declaration of Helsinki. The study findings will be disseminated through usual academic channels including meeting presentations and peer-reviewed publications. PROTOCOL VERSION: 1.0.