Karin Albrecht-Schgoer

The Angiogenic Factor Secretoneurin Induces Coronary Angiogenesis in a Model of Myocardial Infarction by Stimulation of Vascular Endothelial Growth Factor Signaling in Endothelial Cells

Background—Secretoneurin is a neuropeptide located in nerve fibers along blood vessels, is upregulated by hypoxia, and induces angiogenesis. We tested the hypothesis that secretoneurin gene therapy exerts beneficial effects in a rat model of myocardial infarction and evaluated the mechanism of action on coronary endothelial cells. Methods and Results—In vivo secretoneurin improved left ventricular function, inhibited remodeling, and reduced scar formation. In the infarct border zone, secretoneurin induced coronary angiogenesis, as shown by increased density of capillaries and arteries. In vitro secretoneurin induced capillary tubes, stimulated proliferation, inhibited apoptosis, and activated Akt and extracellular signal-regulated kinase in coronary endothelial cells. Effects were abrogated by a vascular endothelial growth factor (VEGF) antibody, and secretoneurin stimulated VEGF receptors in these cells. Secretoneurin furthermore increased binding of VEGF to endothelial cells, and binding was blocked by heparinase, indicating that secretoneurin stimulates binding of VEGF to heparan sulfate proteoglycan binding sites. Additionally, secretoneurin increased binding of VEGF to its coreceptor neuropilin-1. In endothelial cells, secretoneurin also stimulated fibroblast growth factor receptor-3 and insulin-like growth factor-1 receptor, and in coronary vascular smooth muscle cells, we observed stimulation of VEGF receptor-1 and fibroblast growth factor receptor-3. Exposure of cardiac myocytes to hypoxia and ischemic heart after myocardial infarction revealed increased secretoneurin messenger RNA and protein. Conclusions—Our data show that secretoneurin acts as an endogenous stimulator of VEGF signaling in coronary endothelial cells by enhancing binding of VEGF to low-affinity binding sites and neuropilin-1 and stimulates further growth factor receptors like fibroblast growth factor receptor-3. Our in vivo findings indicate that secretoneurin may be a promising therapeutic tool in ischemic heart disease.

Epicardial shock-wave therapy improves ventricular function in a porcine model of ischaemic heart disease.

Previously we have shown that epicardial shock‐wave therapy improves left ventricular ejection fraction (LVEF) in a rat model of myocardial infarction. In the present experiments we aimed to address the safety and efficacy of epicardial shock‐wave therapy in a preclinical large animal model and to further evaluate mechanisms of action of this novel therapy. Four weeks after left anterior descending (LAD) artery ligation in pigs, the animals underwent re‐thoracotomy with (shock‐wave group, n = 6) or without (control group, n = 5) epicardial shock waves (300 impulses at 0.38 mJ/mm2) applied to the infarcted anterior wall. Efficacy endpoints were improvement of LVEF and induction of angiogenesis 6 weeks after shock‐wave therapy. Safety endpoints were haemodynamic stability during treatment and myocardial damage. Four weeks after LAD ligation, LVEF decreased in both the shock‐wave (43 ± 3%, p < 0.001) and control (41 ± 4%, p = 0.012) groups. LVEF markedly improved in shock‐wave animals 6 weeks after treatment (62 ± 9%, p = 0.006); no improvement was observed in controls (41 ± 4%, p = 0.36), yielding a significant difference. Quantitative histology revealed significant angiogenesis 6 weeks after treatment (controls 2 ± 0.4 arterioles/high‐power field vs treatment group 9 ± 3; p = 0.004). No acute or chronic adverse effects were observed. As a potential mechanism of action in vitro experiments showed stimulation of VEGF receptors after shock‐wave treatment in human coronary artery endothelial cells. Epicardial shock‐wave treatment in a large animal model of ischaemic heart failure exerted a positive effect on LVEF improvement and did not show any adverse effects. Angiogenesis was induced by stimulation of VEGF receptors. Copyright

Topical secretoneurin gene therapy accelerates diabetic wound healing by interaction between heparan-sulfate proteoglycans and basic FGF

Diabetic foot ulcers represent a therapeutic problem of high clinical relevance. Reduced vascular supply, neuropathy and diminished expression of growth factors strongly contribute to wound healing impairment in diabetes. Secretoneurin, an angiogenic neuropeptide, has been shown to improve tissue perfusion in different animal models by increasing the amount of vessels in affected areas. Therefore, topical secretoneurin gene therapy was tested in a full thickness wound healing model in diabetic db/db mice. Secretoneurin significantly accelerated wound closure in these mice and immunohistochemistry revealed higher capillary and arteriole density in the wounded area compared to control mice. In-vitro, the mechanism of action of secretoneurin on human dermal microvascular endothelial cells was evaluated in normal and diabetic cells. Secretoneurin shows positive effects on in vitro angiogenesis, proliferation and apoptosis of these cells in a basic fibroblast growth factor dependent manner. A small molecular weight inhibitor revealed fibroblast growth factor receptor 3 as the main receptor for secretoneurin mediated effects. Additionally, we could identify heparan-sulfates as important co-factor of secretoneurin induced binding of basic fibroblast growth factor to human dermal endothelial cells. We suggest topical secretoneurin plasmid therapy as new tool for delayed wound healing in patients suffering from diabetes.

Secretoneurin Gene Therapy Improves Blood Flow in an Ischemia Model in Type 1 Diabetic Mice by Enhancing Therapeutic Neovascularization

Deficient angiogenesis after ischemia may contribute to worse outcome of peripheral arterial disease in patients with diabetes mellitus. Based on our previous work where we demonstrated that Secretoneurin (SN) is up-regulated under hypoxic conditions and enhances angiogenesis, we analyzed the therapeutic potential of SN gene therapy using a model of severe hind limb ischemia in streptozotocin-induced diabetic mice (STZ-DM). After induction of hind limb ischemia, blood flow was assessed by means of laser Doppler perfusion imaging (LDPI) and increased blood perfusion in the SN-treated animal group was observed. These results were complemented by the clinical observation of reduced necrosis and by an increased number of capillaries and arterioles in the SN-treated animal group. In vitro, we found that SN is capable of promoting proliferation and chemotaxis and reduces apoptosis in HUVECs cultured under hyperglycemic conditions. Additionally, SN activated ERK, eNOS and especially AKT as well as EGF-receptor in hyperglycemic HUVECs. In conclusion, we show that SN gene therapy improves post-ischemic neovascularization in diabetic mice through stimulation of angiogenesis and arteriogenesis indicating a possible therapeutic role of this factor in ischemia-related diseases in diabetic patients.

Nilotinib-induced vasculopathy: identification of vascular endothelial cells as a primary target site

The BCR/ABL1 inhibitor Nilotinib is increasingly used to treat patients with chronic myeloid leukemia (CML). Although otherwise well-tolerated, Nilotinib has been associated with the occurrence of progressive arterial occlusive disease (AOD). Our objective was to determine the exact frequency of AOD and examine in vitro and in vivo effects of Nilotinib and Imatinib on endothelial cells to explain AOD-development. In contrast to Imatinib, Nilotinib was found to upregulate pro-atherogenic adhesion-proteins (ICAM-1, E-selectin, VCAM-1) on human endothelial cells. Nilotinib also suppressed endothelial cell proliferation, migration and tube-formation and bound to a distinct set of target-kinases, relevant to angiogenesis and atherosclerosis, including angiopoietin receptor-1 TEK, ABL-2, JAK1 and MAP-kinases. Nilotinib and siRNA against ABL-2 also suppressed KDR expression. In addition, Nilotinib augmented atherosclerosis in ApoE−/− mice and blocked reperfusion and angiogenesis in a hindlimb-ischemia model of arterial occlusion, whereas Imatinib showed no comparable effects. Clinically overt AOD-events were found to accumulate over time in Nilotinib-treated patients. After a median observation-time of 2.0 years, the AOD-frequency was higher in these patients (29.4%) compared to risk factor- and age-matched controls (<5%). Together, Nilotinib exerts direct pro-atherogenic and anti-angiogenic effects on vascular endothelial cells, which may contribute to development of AOD in patients with CML.

Toll-like receptor 3 signalling mediates angiogenic response upon shock wave treatment of ischaemic muscle

AIMS Shock wave therapy (SWT) represents a clinically widely used angiogenic and thus regenerative approach for the treatment of ischaemic heart or limb disease. Despite promising results in preclinical and clinical trials, the exact mechanism of action remains unknown. Toll-like receptor 3, which is part of the innate immunity, is activated by binding double-stranded (ds) RNA. It plays a key role in inflammation, a process that is needed also for angiogenesis. We hypothesize that SWT causes cellular cavitation without damaging the target cells, thus liberating cytoplasmic RNA that in turn activates TLR3. METHODS AND RESULTS SWT induces TLR3 and IFN-β1 gene expression as well as RNA liberation from endothelial cells in a time-dependant manner. Conditioned medium from SWT-treated HUVECs induced TLR3 signalling in reporter cells. The response was lost when the medium was treated with RNase III to abolish dsRNAs or when TLR3 was silenced using siRNAs. In a mouse hind limb ischaemia model using wt and TLR3(-/-) mice (n = 6), SWT induced angiogenesis and arteriogenesis only in wt animals. These effects were accompanied by improved blood perfusion of treated limbs. Analysis of main molecules of the TLR3 pathways confirmed TLR3 signalling in vivo following SWT. CONCLUSION Our data reveal a central role of the innate immune system, namely Toll-like receptor 3, to mediate angiogenesis upon release of cytoplasmic RNAs by mechanotransduction of SWT.

Secretoneurin gene therapy improves hind limb and cardiac ischaemia in Apo E−/− mice without influencing systemic atherosclerosis

AIMS Hypercholesterolaemia is a major risk factor for cardiovascular diseases and has been shown to influence angiogenesis in the hind limb ischaemia (HLI) model. The impaired up-regulation of angiogenic factors seems to be one of the underlying mechanisms for reduced vessel formation. Since we found that secretoneurin (SN) is up-regulated in hypoxic skeletal muscle cells and exerts beneficial effects in myocardial and HLI, we hypothesized that SN therapy might improve neovascularization in hypercholesterolaemic Apo E(-/-) (Apo E knockout) mice suffering from an impaired vascular response. METHODS AND RESULTS For in vitro experiments, endothelial cells (ECs) were incubated with oxidized low-density lipoprotein (oxLDL) to mimic hypercholesterolaemia. EC function was impaired by oxLDL, but SN induced EC proliferation and in vitro tube formation under these conditions. In the HLI model, injection of SN plasmid resulted in a significant better outcome regarding blood flow recovery, amputation rate, and vessel density. In the myocardial infarction (MI) model, the SN group showed improvement in cardiac parameters. Aortic plaque area was not influenced by local SN injection. Interestingly, SN-induced recruitment of angiogenic monocytic cells was abolished under hypercholesterolaemia. CONCLUSIONS SN gene therapy exerts beneficial effects in cardiovascular animal models in Apo E(-/-) mice without influencing atherosclerosis and might qualify as a promising therapy for cardiovascular disorders.

Nanoparticular delivery system for a secretoneurin derivative induces angiogenesis in a hind limb ischemia model

ABSTRACT Common therapeutic strategies for peripheral arterial disease often fail to re‐establish sufficient blood flow within legs and feet of patients for avoiding critical limb ischemia, what is characterized by a substantial risk for amputation. The neuropeptide secretoneurin induces angiogenesis in models of limb and myocardial ischemia and might be a promising tool in the treatment of patients without the option of revascularization therapy for severe ischemia. Within this manuscript, the biologically active part of secretoneurin was identified, modified by induction of a cysteine residue to gain higher stability against enzymatic degradation and further packed into S‐protected thiolated chitosan nanoparticles, which enable intra‐muscular application of secretoneurin. Secretoneurin nanoparticles restored blood flow in a mouse hind limb ischemia model within one week, whereas control particles did not. In vitro testing also revealed the angiogenic, antiapoptotic and proliferative effects of the new secretoneurin derivate, as tested in primary human umbilical vein endothelial cells. With the work from this study we provide a new promising tool for treatment of peripheral arterial disease.