The PFKFB3 Inhibitor AZ67 Inhibits Angiogenesis Independently of Glycolysis Inhibition

Funding: This study was funded by the University of Antwerp (BOF project ID 29068, 40183 and iBOF project ID 21-053). Besa Emini Veseli is a PhD fellow of the Horizon 2020 program of the Eu-ropean Union - Marie Skłodowska-Curie Actions, Innovative Training Networks (ITN), Call: H2020-MSCA-ITN-2015, NUMBER—675527—MOGLYNET. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: The authors thank Mandy Vermont for excellent technical support and Anne-Marie Lambeir for scientific advice. The authors are grateful to Bronwen Martin for critical reading of the manuscript.Peer reviewedPublisher PD

Acetylsalicylic acid reduces passive aortic wall stiffness and cardiovascular remodelling in a mouse model of advanced atherosclerosis

Acetylsalicylic acid (ASA) is widely used in secondary prevention of cardiovascular (CV) disease, mainly because of its antithrombotic effects. Here, we investigated whether ASA can prevent the progression of vessel wall remodelling, atherosclerosis, and CV complications in apolipoprotein E deficient (ApoE(−/−)) mice, a model of stable atherosclerosis, and in ApoE(−/−) mice with a mutation in the fibrillin-1 gene (Fbn1(C1039G+/−)), which is a model of elastic fibre fragmentation, accompanied by exacerbated unstable atherosclerosis. Female ApoE(−/−) and ApoE(−/−)Fbn1(C1039G+/−) mice were fed a Western diet (WD). At 10 weeks of WD, the mice were randomly divided into four groups, receiving either ASA 5 mg/kg/day in the drinking water (ApoE(−/−) (n = 14), ApoE(−/−)Fbn1(C1039G+/−) (n = 19)) or plain drinking water (ApoE(−/−) (n = 15), ApoE(−/−)Fbn1(C1039G+/−) (n = 21)) for 15 weeks. ApoE(−/−)Fbn1(C1039G+/−) mice showed an increased neutrophil–lymphocyte ratio (NLR) compared to ApoE(−/−) mice, and this effect was normalised by ASA. In the proximal ascending aorta wall, ASA-treated ApoE(−/−)Fbn1(C1039G+/−) mice showed less p-SMAD2/3 positive nuclei, a lower collagen percentage and an increased elastin/collagen ratio, consistent with the values measured in ApoE(−/−) mice. ASA did not affect plaque progression, incidence of myocardial infarction and survival of ApoE(−/−)Fbn1(C1039G+/−) mice, but systolic blood pressure, cardiac fibrosis and hypertrophy were reduced. In conclusion, ASA normalises the NLR, passive wall stiffness and cardiac remodelling in ApoE(−/−)Fbn1(C1039G+/−) mice to levels observed in ApoE(−/−) mice, indicating additional therapeutic benefits of ASA beyond its classical use

Endothelial dysfunction and immunothrombosis in sepsis

Sepsis is a life-threatening clinical syndrome characterized by multiorgan dysfunction caused by a dysregulated or over-reactive host response to infection. During sepsis, the coagulation cascade is triggered by activated cells of the innate immune system, such as neutrophils and monocytes, resulting in clot formation mainly in the microcirculation, a process known as immunothrombosis. Although this process aims to protect the host through inhibition of the pathogen's dissemination and survival, endothelial dysfunction and microthrombotic complications can rapidly lead to multiple organ dysfunction. The development of treatments targeting endothelial innate immune responses and immunothrombosis could be of great significance for reducing morbidity and mortality in patients with sepsis. Medications modifying cell-specific immune responses or inhibiting platelet-endothelial interaction or platelet activation have been proposed. Herein, we discuss the underlying mechanisms of organ-specific endothelial dysfunction and immunothrombosis in sepsis and its complications, while highlighting the recent advances in the development of new therapeutic approaches aiming at improving the short- or long-term prognosis in sepsis

Animal models of atherosclerosis.

An ideal animal model of atherosclerosis resembles human anatomy and pathophysiology and has the potential to be used in medical and pharmaceutical research to obtain results that can be extrapolated to human medicine. Moreover, it must be easy to acquire, can be maintained at a reasonable cost, is easy to handle and shares the topography of the lesions with humans. In general, animal models of atherosclerosis are based on accelerated plaque formation due to a cholesterol-rich/Western-type diet, manipulation of genes involved in the cholesterol metabolism, and the introduction of additional risk factors for atherosclerosis. Mouse and rabbit models have been mostly used, followed by pigs and non-human primates. Each of these models has its advantages and limitations. The mouse has become the predominant species to study experimental atherosclerosis because of its rapid reproduction, ease of genetic manipulation and its ability to monitor atherogenesis in a reasonable time frame. Both Apolipoprotein E deficient (ApoE-/-) and LDL-receptor (LDLr) knockout mice have been frequently used, but also ApoE/LDLr double-knockout, ApoE3-Leiden and PCSK9-AAV mice are valuable tools in atherosclerosis research. However, a great challenge was the development of a model in which intra-plaque microvessels, haemorrhages, spontaneous atherosclerotic plaque ruptures, myocardial infarction and sudden death occur consistently. These features are present in ApoE-/-Fbn1C1039G+/- mice, which can be used as a validated model in pre-clinical studies to evaluate novel plaque-stabilizing drugs

Nitric oxide donor molsidomine favors features of atherosclerotic plaque stability and reduces myocardial infarction in mice

Nitric oxide (NO) donors are commonly used for the prevention and treatment of ischemic heart disease. Besides their effects on the heart, NO donors may also prevent hypoxic brain damage and exert beneficial effects on atherosclerosis by favoring features of plaque stability. We recently described that apolipoprotein E (ApoE) deficient mice with a mutation in the fibrillin-1 (Fbn1) gene (ApoE(-/-)Fbn1(C1039G+/-) develop accelerated atherosclerosis, plaque rupture, myocardial infarction, cerebral hypoxia and sudden death. In the present study, we evaluated the effects of chronic treatment with the NO donor molsidomine on atherosclerotic plaque stability, cardiac function, neurological symptoms and survival in the ApoE(-/-)Fbn1(C1039G+/-) mouse model. Female ApoE(-/-)Fbn1(C1039G+/-) mice were fed a Western diet (WD). After 8 weeks of WD, the mice were divided into two groups receiving either molsidomine via the drinking water (1 mg/kg/day; n = 34) or tap water (control; n = 36) until 25 weeks of WD. Survival tended to increase after molsidomine treatment (68% vs. 58% in controls). Importantly, atherosclerotic plaques of molsidomine-treated mice had a thicker fibrous cap (11.1 +/- 1.2 vs. 8.1 +/- 0.7 mu m) and showed an increased occurrence of plaque macrocalcifications (30% vs. 0%), indicative of a more stable phenotype. Molsidomine also improved cardiac function, as fractional shortening was increased (40 +/- 2% vs. 27 +/- 2%) combined with a decreased end diastolic (3.1 +/- 0.2 vs. 3.9 +/- 0.2 mm) and end systolic diameter (1.9 +/- 0.1 vs. 2.9 +/- 0.2 mm). Furthermore, perivascular fibrosis (23 +/- 2 vs. 30 +/- 2%) and the occurrence of myocardial infarctions (12% vs. 36%) was significantly reduced. Track width, a measure of the animal's hind limb base of support and representative of hypoxic brain damage, was also normalized as a result of molsidomine treatment (2.54 +/- 0.04 vs. 2.91 +/- 0.09 cm in controls). These findings demonstrate that the NO donor molsidomine improves cardiac function, reduces neurological symptoms and enhances atherosclerotic plaque stability

Small molecule 3PO inhibits glycolysis but does not bind to 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3)

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3) is a key enzyme of the glycolytic pathway, and it plays an essential role in angiogenesis. 3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) is frequently used as a glycolysis inhibitor and is thought to inhibit PFKFB3. However, this latter effect of 3PO has never been investigated in detail and was the aim of the present study. To demonstrate binding of 3PO to PFKFB3, we used isothermal titration calorimetry. However, 3PO did not bind to PFKFB3, even up to 750 µm, in contrast to 3 µm of AZ67, which is a potent and specific PFKFB3 inhibitor. Instead, 3PO accumulated lactic acid inside the cells, leading to a decrease in the intracellular pH and an inhibition of enzymatic reactions of the glycolytic pathway.status: publishe

Small molecule 3PO inhibits glycolysis but does not bind to 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3)

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3) is a key enzyme of the glycolytic pathway, and it plays an essential role in angiogenesis. 3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) is frequently used as a glycolysis inhibitor and is thought to inhibit PFKFB3. However, this latter effect of 3PO has never been investigated in detail and was the aim of the present study. To demonstrate binding of 3PO to PFKFB3, we used isothermal titration calorimetry. However, 3PO did not bind to PFKFB3, even up to 750 µm, in contrast to 3 µm of AZ67, which is a potent and specific PFKFB3 inhibitor. Instead, 3PO accumulated lactic acid inside the cells, leading to a decrease in the intracellular pH and an inhibition of enzymatic reactions of the glycolytic pathway.</p

Nitric oxide donor molsidomine favors features of atherosclerotic plaque stability and reduces myocardial infarction in mice

Nitric oxide (NO) donors are commonly used for the prevention and treatment of ischemic heart disease. Besides their effects on the heart, NO donors may also prevent hypoxic brain damage and exert beneficial effects on atherosclerosis by favoring features of plaque stability. We recently described that apolipoprotein E (ApoE) deficient mice with a mutation in the fibrillin-1 (Fbn1) gene (ApoE(-/-)Fbn1(C1039G+/-) develop accelerated atherosclerosis, plaque rupture, myocardial infarction, cerebral hypoxia and sudden death. In the present study, we evaluated the effects of chronic treatment with the NO donor molsidomine on atherosclerotic plaque stability, cardiac function, neurological symptoms and survival in the ApoE(-/-)Fbn1(C1039G+/-) mouse model. Female ApoE(-/-)Fbn1(C1039G+/-) mice were fed a Western diet (WD). After 8 weeks of WD, the mice were divided into two groups receiving either molsidomine via the drinking water (1 mg/kg/day; n = 34) or tap water (control; n = 36) until 25 weeks of WD. Survival tended to increase after molsidomine treatment (68% vs. 58% in controls). Importantly, atherosclerotic plaques of molsidomine-treated mice had a thicker fibrous cap (11.1 +/- 1.2 vs. 8.1 +/- 0.7 mu m) and showed an increased occurrence of plaque macrocalcifications (30% vs. 0%), indicative of a more stable phenotype. Molsidomine also improved cardiac function, as fractional shortening was increased (40 +/- 2% vs. 27 +/- 2%) combined with a decreased end diastolic (3.1 +/- 0.2 vs. 3.9 +/- 0.2 mm) and end systolic diameter (1.9 +/- 0.1 vs. 2.9 +/- 0.2 mm). Furthermore, perivascular fibrosis (23 +/- 2 vs. 30 +/- 2%) and the occurrence of myocardial infarctions (12% vs. 36%) was significantly reduced. Track width, a measure of the animal's hind limb base of support and representative of hypoxic brain damage, was also normalized as a result of molsidomine treatment (2.54 +/- 0.04 vs. 2.91 +/- 0.09 cm in controls). These findings demonstrate that the NO donor molsidomine improves cardiac function, reduces neurological symptoms and enhances atherosclerotic plaque stability