Acetyl-L-carnitine is an anti-angiogenic agent targeting the VEGFR2 and CXCR4 pathways

Carnitines play an important role in the energy exchange in cells, involved in the transport of fatty acids across the inner mitochondrial membrane. L-Acetylcarnitine (ALCAR) is an acetic acid ester of carnitine that has higher bioavailability than carnitine and is considered a fat-burning energizer supplement. We previously found that in serum samples from prostate cancer (PCa) patients, 3 carnitine family members were significantly decreased, suggesting a potential protective role of carnitine against PCa. Several studies support beneficial effects of carnitines on cancer, no study has investigated the activities of carnitine on tumor angiogenesis. We examined whether ALCAR act as an \u201cangiopreventive\u201d compound and studied the molecular mechanisms involved. We found that ALCAR was able to limit inflammatory angiogenesis by reducing stimulated endothelial cell and macrophage infiltration in vitro and in vivo. Molecularly, we showed that ALCAR downregulates VEGF, VEGFR2, CXCL12, CXCR4 and FAK pathways. ALCAR blocked the activation of NF-\u3baB and ICAM-1 and reduced the adhesion of a monocyte cell line to endothelial cells. This is the first study showing that ALCAR has anti-angiogenesis and anti-inflammatory properties and might be attractive candidate for cancer angiopreventionCarnitines play an important role in the energy exchange in cells, and are involved in the transport of fatty acids across the inner mitochondrial membrane. L-Acetylcarnitine (ALCAR) is an acetic acid ester of carnitine that has higher bioavailability and is considered a fat-burning energizer supplement. We previously found that in serum samples from prostate cancer (PCa) patients, 3 carnitine family members were significantly decreased, suggesting a potential protective role of carnitine against PCa. Several studies support beneficial effects of carnitines on cancer, no study has investigated the activities of carnitine on tumor angiogenesis. We examined whether ALCAR acts as an \u201cangiopreventive\u201d compound and studied the molecular mechanisms involved. We found that ALCAR was able to limit inflammatory angiogenesis by reducing stimulated endothelial cell and macrophage infiltration in vitro and in vivo. Molecularly, we show that ALCAR downregulates VEGF, VEGFR2, CXCL12, CXCR4 and FAK pathways. ALCAR blocked the activation of NF-\u3baB and ICAM-1 and reduced the adhesion of a monocyte cell line to endothelial cells. This is the first study showing that ALCAR has anti-angiogenic and anti-inflammatory properties and might be an attractive candidate for cancer angioprevention

PlGFMMP9-engineered iPS cells supported on a PEGfibrinogen hydrogel scaffold possess an enhanced capacity to repair damaged myocardium

Cell-based regenerative therapies are significantly improved by engineering allografts to express factors that increase vascularization and engraftment, such as placental growth factor (PlGF) and matrix metalloproteinase 9 (MMP9). Moreover, the seeding of therapeutic cells onto a suitable scaffold is of utmost importance for tissue regeneration. On these premises, we sought to assess the reparative potential of induced pluripotent stem (iPS) cells bioengineered to secrete PlGF or MMP9 and delivered to infarcted myocardium upon a poly(ethylene glycol)-fibrinogen scaffold. When assessing optimal stiffness of the PEG-fibrinogen (PF) scaffold, we found that the appearance of contracting cells after cardiogenic induction was accelerated on the support designed with an intermediate stiffness. Revascularization and hemodynamic parameters of infarcted mouse heart were significantly improved by injection into the infarct of this optimized PF scaffold seeded with both MiPS (iPS cells engineered to secrete MMP9) and PiPS (iPS cells engineered to secrete PlGF) cells as compared with nonengineered cells or PF alone. Importantly, allograft-derived cells and host myocardium were functionally integrated. Therefore, survival and integration of allografts in the ischemic heart can be significantly improved with the use of therapeutic cells bioengineered to secrete MMP9 and PlGF and encapsulated within an injectable PF hydrogel having an optimized stiffness

A Polyphenol-Rich Extract of Olive Mill Wastewater Enhances Cancer Chemotherapy Effects, While Mitigating Cardiac Toxicity

Cardiovascular toxicity remains one of the most adverse side effects in cancer patients receiving chemotherapy. Extra-virgin olive oil (EVOO) is rich in cancer preventive polyphenols endowed with anti-inflammatory, anti-oxidant activities which could exert protective effects on heart cells. One very interesting derivative of EVOO preparation is represented by purified extracts from olive mill waste waters (OMWW) rich in polyphenols. Here, we have investigated the anti-cancer activity of a OMWW preparation, named A009, when combined with chemotherapeutics, as well as its potential cardioprotective activities. Mice bearing prostate cancer (PCa) xenografts were treated with cisplatin, alone or in combination with A009. In an in vivo model, we found synergisms of A009 and cisplatin in reduction of prostate cancer tumor weight. Hearts of mice were analyzed, and the mitochondria were studied by transmission electron microscopy. The hearts of mice co-treated with A009 extracts along with cisplatin had reduced mitochondria damage compared to the those treated with chemotherapy alone, indicating a cardioprotective role. To confirm the in vivo results, tumor cell lines and rat cardiomyocytes were treated with cisplatin in vitro, with and without A009. Another frequently used chemotherapeutic agent 5-fluorouracil (5-FU), was also tested in this assay, observing a similar effect. In vitro, the combination of A009 with cisplatin or 5-FU was effective in decreasing prostate and colon cancer cell growth, while it did not further reduce growth of rat cardiomyocytes also treated with cisplatin or 5-FU. A009 cardioprotective effects towards side effects caused by 5-FU chemotherapy were further investigated, using cardiomyocytes freshly isolated from mice pups. A009 mitigated toxicity of 5-FU on primary cultures of mouse cardiomyocytes. Our study demonstrates that the polyphenol rich purified A009 extracts enhance the effect of chemotherapy in vitro and in vivo, but mitigates chemotherpy adverse effects on heart and on isolated cardiomyocytes. Olive mill waste water extracts could therefore represent a potential candidate for cardiovascular prevention in patients undergoing cancer chemotherapy

New Approaches on Japanese Knotweed (Fallopia japonica) Bioactive Compounds and Their Potential of Pharmacological and Beekeeping Activities: Challenges and Future Directions

Known especially for its negative ecological impact, Fallopia japonica (Japanese knotweed) is now considered one of the most invasive species. Nevertheless, its chemical composition has shown, beyond doubt, some high biological active compounds that can be a source of valuable pharmacological potential for the enhancement of human health. In this direction, resveratrol, emodin or polydatin, to name a few, have been extensively studied to demonstrate the beneficial effects on animals and humans. Thus, by taking into consideration the recent advances in the study of Japanese knotweed and its phytochemical constituents, the aim of this article is to provide an overview on the high therapeutic potential, underlining its antioxidant, antimicrobial, anti-inflammatory and anticancer effects, among the most important ones. Moreover, we describe some future directions for reducing the negative impact of Fallopia japonica by using the plant for its beekeeping properties in providing a distinct honey type that incorporates most of its bioactive compounds, with the same health-promoting properties

Angiogenesis inhibition with novel compounds targeting the key glycolytic enzyme PFKFB3

Aim Intraplaque angiogenesis is associated with progressive and unstable vascular disease. Angiogenesis signals induce endothelial cells (ECs) to switch their metabolism to being highly glycolytic enabling their growth and division. The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) is a key glycolytic activator. PFKFB3 inhibitors have been shown to reduce EC sprouting and seem promising compounds to be used in promoting plaque stability. Methods We studied in the human EC line EA.hy926 the effects of 3PO, a commercially available inhibitor of PFKFB3, and of two self-synthesized phenoxindazole analogues (PA-1 and PA-2; based on Boyd et al., 2015) on glycolysis, proliferation, migration, capillary networking, matrix metalloproteinase (MMP) activity and gene expression. Results The three compounds significantly reduced glycolysis levels, while PA-1 and PA-2 affected also capillary-like structure networking. Accordingly, the compounds markedly inhibited EC migration, proliferation and wound closing capacity which are essential for neovessel formation. Moreover, these inhibitors reduced the activity of proMMP-9 and MMP-2 up to 40-50% and 20-30% compared to control, respectively, while gelatinase genes expression was downregulated by 80%. Finally, the PA compounds downregulated PFKFB3 expression whilst 3PO did not. Similarly, markers of migration and angiogenesis, such as CCL5, VCAM, VEGFA and VEGFR2, were also markedly reduced (up to 10-fold). Conclusions These findings show that PFKFB3 inhibition with PA compounds interferes with key pro-angiogenic functions, such as endothelial migration, proliferation and capillary-like structure and molecularly exerts a multitarget anti-angiogenetic activity. This project has been funded by the European Union\u2019s Horizon 2020 Marie Sklodowska Curie grant (#67552)

Inhibition of the Key Glycolytic Enzyme PFKFB3 with Novel Compounds Suppresses Vessel Sprouting

Aim Intraplaque angiogenesis is an important contributor to atherosclerotic plaque growth and instability. Angiogenic signals induce endothelial cells (ECs) to switch their metabolism to being highly glycolytic, enabling their growth and division. Glycolytic modulation by inhibition of the glycolytic activator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) has been shown to reduce angiogenesis. The objective of this study was to identify novel anti-angiogenic compounds with a potential to efficiently modulate (inhibit) angiogenesis. Methods Using the human EC line EA.hy926, we studied the effects of PFKFB3 inhibition with 3PO, a weak competitive inhibitor of PFKFB3, and of two potent self-synthesized phenoxindazole analogues (PA-1 and PA-2) on glycolysis, proliferation, migration, matrix metalloproteinase (MMP) activity, and capillary tube formation. The latter experiment was also performed using primary human umbilical vein endothelial cells (HUVEC). Moreover, gene expression of important markers related to angiogenesis were measured at mRNA level by real-time PCR. Results PFKFB3 inhibition with all three tested compounds significantly reduced glycolytic activity. While PA-1 and PA-2 suppressed capillary tube formation in both cell groups, 3PO did not have any effect in EA.hy926 ECs and even an inducing effect in the HUVECs. Accordingly, PA-1 and PA-2 markedly inhibited EC migration, proliferation and wound closing capacity which are essential for neovessel formation. Moreover, these inhibitors downregulated gelatinase gene expression up to 6-fold, as well reduced the activity of proMMP-9 and MMP-2 up to 50% and 30% compared to control, respectively. Gene expression analysis revealed that the PA compounds downregulated PFKFB3 expression whilst 3PO did not. Similarly, markers of migration and angiogenesis, such as CCL5, VCAM-1, VEGFA and VEGFR2, were also markedly reduced (up to 10-fold) by the PA compounds. Conclusions These findings suggest that PFKFB3 inhibition with PA compounds may interfere with key pro-angiogenic functions, such as endothelial migration, proliferation and capillary-like structure formation and this exerts a multitarget anti-angiogenic activity. Hence, PFKFB3 inhibition with PA compounds is a promising therapeutic approach to promote plaque stability

Macrophage Polarization in Chronic Inflammatory Diseases: Killers or Builders?

Macrophages are key cellular components of the innate immunity, acting as the main player in the first-line defence against the pathogens and modulating homeostatic and inflammatory responses. Plasticity is a major feature of macrophages resulting in extreme heterogeneity both in normal and in pathological conditions. Macrophages are not homogenous, and they are generally categorized into two broad but distinct subsets as either classically activated (M1) or alternatively activated (M2). However, macrophages represent a continuum of highly plastic effector cells, resembling a spectrum of diverse phenotype states. Induction of specific macrophage functions is closely related to the surrounding environment that acts as a relevant orchestrator of macrophage functions. This phenomenon, termed polarization, results from cell/cell, cell/molecule interaction, governing macrophage functionality within the hosting tissues. Here, we summarized relevant cellular and molecular mechanisms driving macrophage polarization in "distant" pathological conditions, such as cancer, type 2 diabetes, atherosclerosis, and periodontitis that share macrophage-driven inflammation as a key feature, playing their dual role as killers (M1-like) and/or builders (M2-like). We also dissect the physio/pathological consequences related to macrophage polarization within selected chronic inflammatory diseases, placing polarized macrophages as a relevant hallmark, putative biomarkers, and possible target for prevention/therapy

Differentiation of Mesenchymal Stem Cells Derived from Pancreatic Islets and Bone Marrow into Islet-Like Cell Phenotype

BACKGROUND:Regarding regenerative medicine for diabetes, accessible sources of Mesenchymal Stem Cells (MSCs) for induction of insular beta cell differentiation may be as important as mastering the differentiation process itself. METHODOLOGY/PRINCIPAL FINDINGS:In the present work, stem cells from pancreatic islets (human islet-mesenchymal stem cells, HI-MSCs) and from human bone marrow (bone marrow mesenchymal stem cells, BM-MSCs) were cultured in custom-made serum-free medium, using suitable conditions in order to induce differentiation into Islet-like Cells (ILCs). HI-MSCs and BM-MSCs were positive for the MSC markers CD105, CD73, CD90, CD29. Following this induction, HI-MSC and BM-MSC formed evident islet-like structures in the culture flasks. To investigate functional modifications after induction to ILCs, ultrastructural analysis and immunofluorescence were performed. PDX1 (pancreatic duodenal homeobox gene-1), insulin, C peptide and Glut-2 were detected in HI-ILCs whereas BM-ILCs only expressed Glut-2 and insulin. Insulin was also detected in the culture medium following glucose stimulation, confirming an initial differentiation that resulted in glucose-sensitive endocrine secretion. In order to identify proteins that were modified following differentiation from basal MSC (HI-MSCs and BM-MSCs) to their HI-ILCs and BM-ILCs counterparts, proteomic analysis was performed. Three new proteins (APOA1, ATL2 and SODM) were present in both ILC types, while other detected proteins were verified to be unique to the single individual differentiated cells lines. Hierarchical analysis underscored the limited similarities between HI-MSCs and BM-MSCs after induction of differentiation, and the persistence of relevant differences related to cells of different origin. CONCLUSIONS/SIGNIFICANCE:Proteomic analysis highlighted differences in the MSCs according to site of origin, reflecting spontaneous differentiation and commitment. A more detailed understanding of protein assets may provide insights required to master the differentiation process of HI-MSCs to functional beta cells based only upon culture conditioning. These findings may open new strategies for the clinical use of BM-MSCs in diabetes