Midkine Controls Arteriogenesis by Regulating the Bioavailability of Vascular Endothelial Growth Factor A and the Expression of Nitric Oxide Synthase 1 and 3

Midkine is a pleiotropic factor, which is involved in angiogenesis. However, its mode of action in this process is still ill defined. The function of midkine in arteriogenesis, the growth of natural bypasses from pre-existing collateral arteries, compensating for the loss of an occluded artery has never been investigated. Arteriogenesis is an inflammatory process, which relies on the proliferation of endothelial cells and smooth muscle cells. We show that midkine deficiency strikingly interferes with the proliferation of endothelial cells in arteriogenesis, thereby interfering with the process of collateral artery growth. We identified midkine to be responsible for increased plasma levels of vascular endothelial growth factor A (VEGFA), necessary and sufficient to promote endothelial cell proliferation in growing collaterals. Mechanistically, we demonstrate that leukocyte domiciled midkine mediates increased plasma levels of VEGFA relevant for upregulation of endothelial nitric oxide synthase 1 and 3, necessary for proper endothelial cell proliferation, and that non-leukocyte domiciled midkine additionally improves vasodilation. The data provided on the role of midkine in endothelial proliferation are likely to be relevant for both, the process of arteriogenesis and angiogenesis. Moreover, our data might help to estimate the therapeutic effect of clinically applied VEGFA in patients with vascular occlusive diseases

The role of Actin-binding Rho activating protein (Abra) during arteriogenesis

Natürliches Wachstum von Kollateralarterien (Arteriogenese), die einen arteriellen Verschluss umgehen, ist entscheidend für die Gewebeerhaltung. Bedauerlicherweise bleibt es in Patienten meist unvollständig. Mit der Aufklärung der grundlegenden Mechanismen der Arteriogenese wird das Ziel verfolgt, dieses Wachstum pharmakologisch zu stimulieren, damit die präformierten Kollateralanastomosen schneller zu funktionstüchtigen Arterien umstrukturiert werden. Kürzlich konnte im Kaninchenmodell gezeigt werden, dass prinzipiell eine vollständige Wiederherstellung der vaskulären Funktion durch die Aufrechterhaltung des initialen Stimulus, der Schubspannung, erreicht werden kann. Das hierfür verwendete Modell – die chirurgische Anlage eines arteriovenösen Shunts zwischen dem distalen Ende der ligierten Femoralarterie und der benachbarten Vene – wurde im Rahmen dieser Arbeit auf die Ratte adaptiert. Ziel dieser Arbeit war es, durch die Entschlüsselung des Expressionsmusters von schubspannungsinduzierten, wachsenden Kollateralarterien, neue Gene zu identifizieren und zu charakterisieren, welche die Arteriogenese stimulieren. Dabei wurde das bis zu 16-fach hochregulierte Gen, welches für das "Actin-binding Rho activating protein" (Abra) kodiert, identifiziert. Der mRNA Expressionsverlauf korreliert mit der Einwirkung der Schubspannung. Entgegen der Literaturmeinung, in der dieses Gen als spezifisch für gestreiften Muskel beschrieben wird, wurde im Rahmen dieser Arbeit sowohl Abra Transkripte mittels in situ Hybridisierung als auch das Protein immunhistochemisch in der Gefäßwand lokalisiert. Um die funktionelle Bedeutung von Abra in der Arteriogenese zu zeigen, wurden rekombinante Adenoviren generiert. Dies waren zum einen Abra überexprimierende Viren und zum anderen solche Viren, die den „knock down” von Abra über siRNA vermittelten. Es stellte sich heraus, dass die lokale Überexpression in vivo durch intra-kollateralen adenoviralem Gentransfer von Abra im einfachen Ligaturmodell die kollaterale Konduktanz um mehr als 60% gegenüber der natürlichen Reaktion verbessert und so die Arteriogenese stimuliert. Die gleichzeitige Schubspannungsstimulation der Arteriogenese mit AV-Shunt und die siRNA-vermittelte Herunterregulierung von Abra verschlechterte dagegen das Kollateralwachstum. Es konnte weiterhin gezeigt werden, dass die Abra Überexpression in kultivierten glatten Muskelzellen deren Proliferationsaktivität steigert. Mit Abra wurde ein potentieller Mediator der Schubspannung identifiziert, der diese genbasiert – oder in Zukunft pharmakologisch aktiviert – teilweise ersetzen kann.Development of a collateral circulation (arteriogenesis) bypassing an arterial occlusion is important for tissue repair but remains functionally defective in human patients. We have previously shown that chronically elevated fluid shear stress (FSS) by arterio-venous anastomosis (AV-Shunt) in a rabbit hind limb model markedly triggers collateral growth and completely normalized blood flow. The transduction of the mechanical stimulus into a vascular growth response remains unclear. The aim of this study was a gene profiling approach in order to identify the molecular mechanism of collateral growth. The adaptation of the AV-Shunt model to rats allowed the use of whole genome Microarrays. Here we report 16-fold increased transcription of the stress mediating actin-binding Rho activating protein (Abra) during FSS-induced arteriogenesis. In contrast to previously identified restriction of endogenous Abra to cardiac and skeletal muscle it could be demonstrated that Abra is also expressed in blood vessels. mRNA and Protein expression was localized in smooth muscle cells (SMCs) as well as in endothelial cells (ECs). To address the functional implications of Abra to arteriogenesis in vivo, its expression was locally modulated by adenoviral gene transfer. Forced over-expression of Abra improved bypass-flow by more than 60 % vs. no treatment after femoral artery ligation. This beneficial effect of elevated FSS could be abolished by a simultaneous siRNA mediated knock-down of Abra. In cultivated cells Abra over-expression stimulated SMC proliferation. These findings suggest that increased FSS leads to increased Abra expression, which triggers collateral growth. Abra is not structurally related to any of the known angiogenic growth factors but turned out to be necessary and sufficient to improve arteriogenesis and thus giving rise to new therapeutic strategies for the treatment of peripheral and cardiovascular disease

Exercise-induced vascular adaptations under artificially versus pathologically reduced blood flow: a focus review with special emphasis on arteriogenesis

Background: The vascular effects of training under blood flow restriction (BFR) in healthy persons can serve as a model for the exercise mechanism in lower extremity arterial disease (LEAD) patients. Both mechanisms are, inter alia, characterized by lower blood flow in the lower limbs. We aimed to describe and compare the underlying mechanism of exercise-induced effects of disease- and external application-BFR methods. Methods: We completed a narrative focus review after systematic literature research. We included only studies on healthy participants or those with LEAD. Both male and female adults were considered eligible. The target intervention was exercise with a reduced blood flow due to disease or external application. Results: We identified 416 publications. After the application of inclusion and exclusion criteria, 39 manuscripts were included in the vascular adaption part. Major mechanisms involving exercise-mediated benefits in treating LEAD included: inflammatory processes suppression, proinflammatory immune cells, improvement of endothelial function, remodeling of skeletal muscle, and additional vascularization (arteriogenesis). Mechanisms resulting from external BFR application included: increased release of anabolic growth factors, stimulated muscle protein synthesis, higher concentrations of heat shock proteins and nitric oxide synthase, lower levels in myostatin, and stimulation of S6K1. Conclusions: A main difference between the two comparators is the venous blood return, which is restricted in BFR but not in LEAD. Major similarities include the overall ischemic situation, the changes in microRNA (miRNA) expression, and the increased production of NOS with their associated arteriogenesis after training with BFR

Effects on the profile of circulating mirnas after single bouts of resistance training with and without blood flow restriction-a three-arm, randomized crossover trial

Background: The effects of blood flow restriction (training) may serve as a model of peripheral artery disease. In both conditions, circulating micro RNAs (miRNAs) are suggested to play a crucial role during exercise-induced arteriogenesis. We aimed to determine whether the profile of circulating miRNAs is altered after acute resistance training during blood flow restriction (BFR) as compared with unrestricted low- and high-volume training, and we hypothesized that miRNA that are relevant for arteriogenesis are affected after resistance training. Methods: Eighteen healthy volunteers (aged 25 ± 2 years) were enrolled in this three-arm, randomized-balanced crossover study. The arms were single bouts of leg flexion/extension resistance training at (1) 70% of the individual single-repetition maximum (1RM), (2) at 30% of the 1RM, and (3) at 30% of the 1RM with BFR (artificially applied by a cuff at 300 mm Hg). Before the first exercise intervention, the individual 1RM (N) and the blood flow velocity (m/s) used to validate the BFR application were determined. During each training intervention, load-associated outcomes (fatigue, heart rate, and exhaustion) were monitored. Acute effects (circulating miRNAs, lactate) were determined using pre-and post-intervention measurements. Results: All training interventions increased lactate concentration and heart rate (p < 0.001). The high-intensity intervention (HI) resulted in a higher lactate concentration than both lower-intensity training protocols with BFR (LI-BFR) and without (LI) (LI, p = 0.003; 30% LI-BFR, p = 0.008). The level of miR-143-3p was down-regulated by LI-BFR, and miR-139-5p, miR-143-3p, miR-195-5p, miR-197-3p, miR-30a-5p, and miR-10b-5p were up-regulated after HI. The lactate concentration and miR-143-3p expression showed a significant positive linear correlation (p = 0.009, r = 0.52). A partial correlation (intervention partialized) showed a systematic impact of the type of training (LI-BFR vs. HI) on the association (r = 0.35 remaining after partialization of training type). Conclusions: The strong effects of LI-BFR and HI on lactate- and arteriogenesis-associated miRNA-143-3p in young and healthy athletes are consistent with an important role of this particular miRNA in metabolic processes during (here) artificial blood flow restriction. BFR may be able to mimic the occlusion of a larger artery which leads to increased collateral flow, and it may therefore serve as an external stimulus of arteriogenesis

Effects of single bouts of different endurance exercises with different intensities on microRNA biomarkers with and without blood flow restriction: a three-arm, randomized crossover trial

Purpose: Physical activity is associated with altered levels of circulating microRNAs (ci-miRNAs). Changes in miRNA expression have great potential to modulate biological pathways of skeletal muscle hypertrophy and metabolism. This study was designed to determine whether the profile of ci-miRNAs is altered after different approaches of endurance exercise. Methods: Eighteen healthy volunteers (aged 24 ± 3 years) participated this three-arm, randomized-balanced crossover study. Each arm was a single bout of treadmill-based acute endurance exercise at (1) 100% of the individual anaerobic threshold (IANS), (2) at 80% of the IANS and (3) at 80% of the IANS with blood flow restriction (BFR). Load-associated outcomes (fatigue, feeling, heart rate, and exhaustion) as well as acute effects (circulating miRNA patterns and lactate) were determined. Results: All training interventions increased the lactate concentration (LC) and heart rate (HR) (p < 0.001). The high-intensity intervention (HI) resulted in a higher LC than both lower intensity protocols (p < 0.001). The low-intensity blood flow restriction (LI-BFR) protocol led to a higher HR and higher LC than the low-intensity (LI) protocol without BFR (p = 0.037 and p = 0.003). The level of miR-142-5p and miR-197-3p were up-regulated in both interventions without BFR (p < 0.05). After LI exercise, the expression of miR-342-3p was up-regulated (p = 0.038). In LI-BFR, the level of miR-342-3p and miR-424-5p was confirmed to be up-regulated (p < 0.05). Three miRNAs and LC show a significant negative correlation (miR-99a-5p, p = 0.011, r = − 0.343/miR-199a-3p, p = 0.045, r = − 0.274/miR-125b-5p, p = 0.026, r = − 0.302). Two partial correlations (intervention partialized) showed a systematic impact of the type of exercise (LI-BFR vs. HI) (miR-99a-59: r = − 0.280/miR-199a-3p: r = − 0.293). Conclusion: MiRNA expression patterns differ according to type of activity. We concluded that not only the intensity of the exercise (LC) is decisive for the release of circulating miRNAs—as essential is the type of training and the oxygen supply

Development of an exercise training protocol to investigate arteriogenesis in a murine model of peripheral artery disease

Exercise is a treatment option in peripheral artery disease (PAD) patients to improve their clinical trajectory, at least in part induced by collateral growth. The ligation of the femoral artery (FAL) in mice is an established model to induce arteriogenesis. We intended to develop an animal model to stimulate collateral growth in mice through exercise. The training intensity assessment consisted of comparing two different training regimens in C57BL/6 mice, a treadmill implementing forced exercise and a free-to-access voluntary running wheel. The mice in the latter group covered a much greater distance than the former pre- and postoperatively. C57BL/6 mice and hypercholesterolemic ApoE-deficient (ApoE-/-) mice were subjected to FAL and had either access to a running wheel or were kept in motion-restricting cages (control) and hind limb perfusion was measured pre- and postoperatively at various times. Perfusion recovery in C57BL/6 mice was similar between the groups. In contrast, ApoE-/- mice showed significant differences between training and control 7 d postoperatively with a significant increase in pericollateral macrophages while the collateral diameter did not differ between training and control groups 21 d after surgery. ApoE-/- mice with running wheel training is a suitable model to simulate exercise induced collateral growth in PAD. This experimental set-up may provide a model for investigating molecular training effects