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To describe the use of hydroxychloroquine (HCQ), azithromycin (AZI) and low-molecular-weight-heparins (LMWH) by sex among elderly residents in nursing homes (NHs) of Tuscany region (Italy) during the first pandemic peak.A population-based drug utilization study was performed linking the Tuscany Administrative Databases (TAD) to the regional COVID-19 registry. Among elderly patients (≥ 65 years old) registered in TAD on 1st December 2019, new users of HCQ, AZI and LMWH that started the treatment during NHs stay (i.e., no dispensing of the same drug during the previous 6 months) were respectively identified in each month of observation period (December 2019-May 2020). The percentage of subjects diagnosed with COVID-19 (COVID-19+) on, or before, the date of first dispensing of the drug of interest was described. Results were stratified by sex (male = M; female = F).New users of HCQ, AZI and LMWH were 62, 300, and 1215, respectively. About 95% of HCQ new users received the drug in April (M = 20; F = 40) of which 11 of men (55%) and 25 of women (62%) were COVID-19+; AZI new users in April (M = 32; F = 83) were two-fold compared to February, of which 10 of men (31%) and 30 of women (36%) were COVID-19+; LMWH new users showed a peak in April (M = 74; F = 142), when 31% of men and 34% of women were COVID-19 + .New users showed a peak during the first pandemic wave and females were over two-fold compared to men. HCQ was often prescribed in absence of COVID-19 diagnosis. During future global emergencies drug prescriptions should better be monitored in frail populations, especially in case of drugs with uncertain efficacy and safety.

cope We studied the effects and mechanism of 2-(3,4-dihydroxyphenil)ethanol (or hydroxytyrosol, HT), a polyphenol from extra virgin olive oil, investigating the regulation of epidermal growth factor receptor (EGFR) expression in colon tumour cells. Methods and results We demonstrate that HT significantly downregulates EGFR expression in human colorectal adenocarcinoma cells HT-29, CaCo2, and WiDr, and in HT-29 xenografts. HT accelerates EGFR degradation by reducing its half-life. Specifically, HT induces EGFR ubiquitination that is mediated by phosphorylation at pY1045, the docking site for Cbl, thereby enabling receptor ubiquitination and degradation. Pretreatment with either the lysosomal inhibitor chloroquine, or the proteasomal inhibitor MG132 blocks HT-induced EGFR downregulation. In colon cancer cells, EGFR downregulation by HT is associated with reduced cell proliferation. Tumour growth and EGFR expression levels are also decreased by HT treatment in HT-29 xenograft. Conclusion We conclude that HT downregulates EGFR expression via lysosomal and proteasomal degradation, activated by HT-induced EGFR phosphorylation at pY1045 and increased Cbl activity. Cbl activation induces, in turn, EGFR ubiquitination. Our results reveal a new mechanism for HT's antitumour effects that may be important for colon tumour prevention and treatment.

Background and Purpose Hydrogen sulfide (H2S), an endogenous volatile mediator with pleiotropic functions, promotes vasorelaxation, exerts anti-inflammatory actions and regulates angiogenesis. Previously, the SH-containing angiotensin-converting enzyme inhibitor (ACEI), zofenopril, was identified as being effective in preserving endothelial function and inducing angiogenesis among ACEIs. Based on the H2S donor property of its active metabolite zofenoprilat, the objective of this study was to evaluate whether zofenoprilat-induced angiogenesis was due to increased H2S availability. Experimental Approach HUVECs were used for in vitro studies of angiogenesis, whereas the Matrigel plug assay was used for in vivo assessments. Key Results Zofenoprilat-treated HUVECs showed an increase in all functional features of the angiogenic process in vitro. As zofenoprilat induced the expression of CSE (cystathionine-γ-lyase) and the continuous production of H2S, CSE inhibition or silencing blocked the ability of zofenoprilat to induce angiogenesis, both in vitro and in vivo. The molecular mechanisms underlying H2S/zofenoprilat-induced angiogenesis were dependent on Akt, eNOS and ERK1/2 cascades. ATP-sensitive potassium (KATP) channels, the molecular target that mediates part of the vascular functions of H2S, were shown to be involved in the upstream activation of Akt and ERK1/2. Moreover, the up-regulation of fibroblast growth factor-2 was dependent on CSE-derived H2S response to H2S and KATP activation. Conclusions and Implications Zofenoprilat induced a constant production of H2S that stimulated the angiogenic process through a KATP channel/Akt/eNOS/ERK1/2 pathway. Thus, zofenopril can be considered as a pro-angiogenic drug acting through H2S release and production, useful in cardiovascular pathologies where vascular functions need to be re-established and functional angiogenesis induced.

Prostaglandin E2 (PGE2), a key mediator of immunity, inflammation, and cancer, acts through 4 G-protein-coupled E-prostanoid receptors (EPs 1-4). Crosstalk between EPs and receptor tyrosine kinases also occurs. Colony-stimulating factor-1 receptor (CSF-1R) is an RTK that sustains the survival, proliferation, and motility of monocytes/macrophages, which are an essential component of innate immunity and cancer development. The aim of this study was to investigate on a possible crosstalk between EP and CSF-1R. In BAC1.2F5 and RAW264.7 murine macrophages, CSF-1 (EC₅₀ = 18.1 and 10.2 ng/ml, respectively) and PGE2 (EC₅₀ = 1.5 and 5.5 nM, respectively) promoted migration. PGE2 induced rapid CSF-1R phosphorylation that was dependent on Src family kinases (SFKs). CSF-1R inhibition reduced PGE2-elicited ERK1/2 phosphorylation and macrophage migration, indicating that CSF-1R plays a role in PGE2-mediated immunoregulation. EP4 appeared responsible for functional PGE2/CSF-1R crosstalk. Furthermore, PGE2 synergized with CSF-1 in inducing ERK1/2 phosphorylation and macrophage migration. ERK1/2 inhibition completely blocked migration induced by the combination CSF-1/PGE2. CSF-1/PGE2 functional interaction with respect to migration also occurred in bone marrow-derived murine macrophages (EC₅₀ CSF-1, 6.7 ng/ml; EC₅₀ PGE2, 16.7 nM). These results indicated that PGE2 transactivates CSF-1R and synergizes with its signaling at ERK1/2 level in promoting macrophage migration.

In a previous article, we demonstrated the existence of fucosyl-containing O-glycans forms of nucleolin in bovine post-capillary venular endothelial cells (CVEC) and malignant cultured human A431 cells. The tool for this discovery was an antibody found to interact strongly and exclusively with nucleolin in total protein extracts. The antibody was originally raised against a mollusc glycoprotein and was demonstrated to be directed against its O-glycans, recently found to belong prevalently to the blood group H-antigen type with fucose linked in alpha1, 2 to galactose. Here, we show that si-RNA induced down-regulation of the expression of FUT1 and FUT2, the fucosyltransferases required for the biosynthesis of the terminal glycan motif Fucα-2-Galβ-R, reduced expression of the fucosylated nucleolin glycoforms and their exposure at the cell surface in CVEC. Treatment of the cells with FUT1/2 siRNA also reduced their ability to bind and internalize endostatin and their adhesion efficiency and inhibited cell growth. Expression of FUT1, FUT2, and FUT6 was also analyzed in serum-stimulated versus serum-starved cells and in cells treated with FUT1 and FUT2 siRNA. A reduced expression of fucosylated nucleolin and inhibition of cell growth by suppressing FUT1/2 expression was also tested and shown to be exhibited in human A431 cells. J. Cell. Biochem. 111: 229–238, 2010. © 2010 Wiley-Liss, Inc.

Cerebral amyloid angiopathy (CAA) caused by amyloid beta (Abeta) deposition around brain microvessels results in vascular degenerative changes. Antiangiogenic Abeta properties are known to contribute to the compromised cerebrovascular architecture. Here we hypothesize that Abeta peptides impair angiogenesis by causing endothelial cells to enter senescence at an early stage of vascular development. Wild-type (WT) Abeta and its mutated variant E22Q peptide, endowed with marked vascular tropism, were used in this study. In vivo, in zebrafish embryos, the WT or E22Q peptides reduced embryo survival with an IC(50) of 6.1 and 4.7 microM, respectively. The 2.5 microM concentration, showing minimal toxicity, was chosen. Alkaline phosphatase staining revealed disorganized vessel patterning, narrowing, and reduced branching of vessels. Beta-galactosidase staining and the cyclin-dependent kinase inhibitor p21 expression, indicative of senescence, were increased. In vitro, WT and E22Q reduced endothelial cell survival with an IC(50) of 12.3 and 8.8 microM, respectively. The 5 microM concentration, devoid of acute effects on the endothelium, was applied chronically to long-term cultured human umbilical vein endothelial cells (HUVECs). We observed reduced cumulative population doubling, which coincided with beta-galactosidase accumulation, down-regulation of telomerase reverse-transcriptase mRNA expression, decreased telomerase activity, and p21 activation. Senescent HUVECs showed marked angiogenesis impairment, as Abeta treatment reduced tube sprouting. The endothelial injuries caused by the E22Q peptide were much more aggressive than those induced by the WT peptide. Premature Abeta-induced senescence of the endothelium, producing progressive alterations of microvessel morphology and functions, may represent one of the underlying mechanisms for sporadic or heritable CAA.

1. Bradykinin (BK) contributes to the inflammatory response inducing vasodilation of postcapillary venules and has been demonstrated to induce neovascular growth in subcutaneous rat sponges. 2. In this study the ability of BK to stimulate cell growth and migration in cultured endothelium from coronary postcapillary venules (CVEC) has been investigated. 3. [3H]-thymidine incorporation in subconfluent and synchronised CVEC was used to monitor DNA synthesis over 24 h. BK promoted a concentration-dependent increase of DNA synthesis with maximal activity at 100 nM. At this concentration BK also induced 18 fold accumulation of c-Fos protein immunoreactivity in the nucleus within 1 h from peptide exposure. 4. The total number of cells recovered after 48 h exposure to BK was increased in a concentration-dependent manner. Maximal effect was produced by 100 nM concentration of the peptide which produced 50% increase in cell number. The selective B1 receptor agonist Des-Arg9-BK mimicked the proliferative effect of BK, while the B2 receptor agonist kallidin was devoid of any activity. The proliferation induced by BK was abolished in a concentration-dependent manner by the addition of the B1 selective antagonist Des-Arg9-Leu8-BK, while the selective B2 receptor antagonist HOE140 did not modify BK-induced growth. 5. DNA synthesis and growth promoted by a threshold concentration of fibroblast growth factor-2 (FGF-2) (0.25 nM) were potentiated by increasing concentrations of BK and Des-Arg9-BK. 6. Endothelial cell migration assessed by the Boyden Chamber procedure was not promoted by BK or the selective B1 and B2 receptor agonists. 7. These data are the first demonstration that BK promotes growth of endothelial cells from postcapillary venules. The mitogenic activity of BK involves c-Fos expression and potentiates the growth promoting effect of FGF-2. Only the B1 receptor appears to be responsible for the proliferation induced by BK and suggests that this type of receptor might be implicated in favouring angiogenesis of coronary venules.

1. Linomide (N-phenylmethyl-1,2-dihydro-4-hydroxyl-1-methyl-2-oxoquinoline-3-carb oxa mide) inhibits vascular proliferation and has been proposed as an antiangiogenic drug. We have investigated the vascular effect of linomide in rabbit aortic and saphenous vein ring preparations and in rat cultured vascular smooth muscle cells (VSMCs). 2. Linomide (25-300 micrograms ml-1) did not alter the basal tone of the preparations. The drug induced a concentration-dependent relaxant effect in aortic rings with endothelium, preconstricted by noradrenaline (NA), 5-hydroxytryptamine (5-HT) and by the thromboxane mimetic U46619. 3. The degree of relaxation induced by linomide was significantly reduced by exposure to the cyclooxygenase inhibitors indomethacin (3 microM) and acetylsalicylic acid (500 microM), and was not influenced by pretreatment with the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (100 microM) in aortic rings with endothelium, preconstricted with NA. 4. Endothelium removal significantly reduced the relaxant response to linomide in aortic ring preparations. 5. A concentration-dependent relaxant response was observed also in rabbit saphenous vein preparations deprived of endothelium and preconstricted either by NA or U46619. The degree of relaxation obtained in a high potassium solution was consistently smaller than that observed in NA-pretreated venous preparations. 6. The vasorelaxant effect of linomide was consistently blunted by the adenylate cyclase inhibitor SQ 22536 (50 microM), both in intact aortic rings and in those deprived of endothelium. 7. In rat cultured vascular smooth muscle cells, linomide (100-200 micrograms ml-1) induced a significant increase in cyclic AMP levels, which was blocked by exposure to 50 microM SQ 22536. 8. In endothelium-deprived aortic ring preparations, the linomide-induced relaxant effect was greatly reduced in high potassium medium (KCl = 25 mM). Pretreatment with the ATP potassium channel inhibitor glibenclamide (3 microM) significantly reduced the linomide-induced relaxation. 9. The results show that linomide possesses a vasorelaxant effect which is attributable to both endothelium-dependent and -independent properties. While the former component of the drug's activity is apparently due to the release of a prostanoid from endothelial cells, the endothelium-independent mechanism involved in linomide relaxation is linked to cyclic AMP accumulation and to ATP-sensitive potassium channel activation in VSMCs.

Conventional medical knowledge predicates that inflammation and angiogenesis are closely associated phenomena. For decades, angiogenesis, previously described as erythema or redness, has been viewed as one of the cardinal signs of inflammation, the two phenomena being inextricably interwoven. This firmly established notion has been challenged by the discovery of mediators of inflammation endowed with a wide repertoire of angiogenic actions ranging from potent stimulators to suppressors of angiogenesis. The most cogent examples of molecules, which may exert opposing actions on angiogenesis, while invariably contributing to inflammation, are found in the interleukin and cytokine class of mediators (Cozzolino et al., 1990; Szekanecz & Koch 2001; Hatzi et al., 2002). For example, IL-1, IL-6 and IL-10 exert both angiogenic and angiostatic action depending on the experimental conditions (Cervenak et al., 2000; Kohno et al., 2003; Wei et al., 2003; Bar et al., 2004). Thus, the relationship between inflammation and angiogenesis appears less unequivocal than previously thought. Reports on tumor angiogenesis and the prognostic significance of the inflammatory infiltrate are a good example of conflicting evidence (Griffioen et al., 1996; Offersen et al., 2002). Certainly the extrapolation of observations in isolated cell populations to in vivo experimental models designed either for inflammation or angiogenesis contribute to the ambiguity on angiogenic properties of inflammatory mediators (Gullino, 1981; Auerbach et al., 2003). In the current issue of the journal, Ferreira et al. (2004) have revisited the role of endogenous PAF in chronic inflammation, in angiogenesis, and other sequela of inflammation such as cell recruitment and in the production of cytokines. To study endogenous PAF, they used either PAF receptor-deficient mice (PAFR-KO) or wild-type mice orally treated with a PAF receptor antagonist. Chronic inflammation was induced by a sponge implant, a condition that promotes granolomatous tissues closely reproducing that occurring in human pathology. In the study of Ferreira et al. (2004), the absence of the effects of endogenous PAF induced a significant stimulation of angiogenesis, and attenuation of the inflammatory response. This finding sharply contrasts with a wealth of literature data describing the abundance of PAF, rather than its absence, as a potent stimulus for the activation of endothelial cells leading to the formation of new vessels (Brizzi et al., 1999; Montrucchio et al., 2000; Deo et al., 2002). However, the portrayal of PAF as a proangiogenic molecule has been drawn from in vitro experiments on cultured endothelial cells exposed to exogenous PAF (Brizzi et al., 1999), or from in vivo studies in which angiogenesis was inherently primed by matrigel implants (Montrucchio et al., 2000), or a combination of PAF and angiogenic stimulator (Montrucchio et al., 2000). An emerging concept is that the extent of angiogenic response greatly depends on the innate angiogenic tone. Thus, it appears that the activity of PAF, a ubiquitous lipid mediator of inflammation, is strongly influenced by the experimental conditions giving rise to opposing effects. The decreased inflammation observed by Fereira et al. (2004) in PAFR-KO mice was accompanied by a reduced level of inflammatory cell infiltrate and no modification of known angiogenic factors such as VEGF and TNF-a, but unexpectedly by a sustained rise of chemokines CXCL2 (KC) and CCL2 (MCP-1/JE). The enhanced level of these chemokines indicates that loss of PAF's effects does not indiscriminately suppress all inflammatory signals, which perhaps compensates for the absence of an important molecule in maintaining immunogical surveillance. Moreover, the elevation of CCL2 (MCP-1/JE), particularly significant in PAFR-KO mice, may account for the increased vascularization, since this cytokine possesses consistent proangiogenic activity (Goede et al., 1999). In summary, the article by Ferreira et al. (2004) contributes to the better definition of the role of PAF in inflammation and in angiogenesis in conditions that mimic mild chronic inflammation in man. PAF appears to be a physiological mediator of inflammation, which negatively regulates angiogenesis rather than promoting it, a profile that is also shared by other mediators of inflammation. In physiological angiogenesis the interplay among endogenous stimulators and inhibitors, although highly redundant, is tightly regulated to achieve neovascular growth in maintaining tissue integrity. While some molecules have been selectively assigned to either function, others have been shown to possess opposing actions. Whether PAF belongs to the latter class of molecules, as suggested by the report of Ferreira et al. (2004), needs to be substantiated in other models.