Upregulation of inducible NO synthase by exogenous adenosine in vascular smooth muscle cells activated by inflammatory stimuli in experimental diabetes

BACKGROUND: Adenosine has been shown to induce nitric oxide (NO) production via inducible NO synthase (iNOS) activation in vascular smooth muscle cells (VSMCs). Although this is interpreted as a beneficial vasodilating pathway in vaso-occlusive disorders, iNOS is also involved in diabetic vascular dysfunction. Because the turnover of and the potential to modulate iNOS by adenosine in experimental diabetes have not been explored, we hypothesized that both the adenosine system and control of iNOS function are impaired in VSMCs from streptozotocin-diabetic rats. METHODS: Male Sprague-Dawley rats were injected with streptozotocin once to induce diabetes. Aortic VSMCs from diabetic and nondiabetic rats were isolated, cultured and exposed to lipopolysaccharide (LPS) plus a cytokine mix for 24 h in the presence or absence of (1) exogenous adenosine and related compounds, and/or (2) pharmacological agents affecting adenosine turnover. iNOS functional expression was determined by immunoblotting and NO metabolite assays. Concentrations of adenosine, related compounds and metabolites thereof were assayed by HPLC. Vasomotor responses to adenosine were determined in endothelium-deprived aortic rings. RESULTS: Treatment with adenosine-degrading enzymes or receptor antagonists increased iNOS formation in activated VSMCs from nondiabetic and diabetic rats. Following treatment with the adenosine transport inhibitor NBTI, iNOS levels increased in nondiabetic but decreased in diabetic VSMCs. The amount of secreted NO metabolites was uncoupled from iNOS levels in diabetic VSMCs. Addition of high concentrations of adenosine and its precursors or analogues enhanced iNOS formation solely in diabetic VSMCs. Exogenous adenosine and AMP were completely removed from the culture medium and converted into metabolites. A tendency towards elevated inosine generation was observed in diabetic VSMCs, which were also less sensitive to CD73 inhibition, but inosine supplementation did not affect iNOS levels. Pharmacological inhibition of NOS abolished adenosine-induced vasorelaxation in aortic tissues from diabetic but not nondiabetic animals. CONCLUSIONS: Endogenous adenosine prevented cytokine- and LPS-induced iNOS activation in VSMCs. By contrast, supplementation with adenosine and its precursors or analogues enhanced iNOS levels in diabetic VSMCs. This effect was associated with alterations in exogenous adenosine turnover. Thus, overactivation of the adenosine system may foster iNOS-mediated diabetic vascular dysfunction

Espressione di iNOS e metabolismo dell'adenosina in cellule muscolari lisce vascolari di ratti diabetici

Diabetes mellitus is a major risk factor for cardiovascular diseases. Stimulation of inducible nitric oxide synthase (iNOS) production by inflammatory cytokines causes a significant production of peroxynitrite that is involved in many vascular disorders. In the cardiovascular system, extracellular adenosine has several vasoprotective properties: e.g. it induces vasodilatation, inhibits platelet aggregation, prevents platelet adhesion, inhibits growth of vascular smooth muscle cells (VSMCs) (Ikeda et al., 1997). Several studies demonstrate that in VSMCs adenosine modulates cytokine-induced iNOS expression and thus the release of NO from these cells via A2 receptors (Dubey et al., 1998; St. Hilaire et al., 2008). Since stimulation of iNOS production by inflammatory cytokines is involved in diabetic vascular dysfunction, we investigated the potential role of adenosine in this process by determining its influence on iNOS expression by VSMCs isolated from diabetic as compared to normoglycaemic rats. Diabetes was induced in Sprague-Dawley rats by intravenous injection of streptozotocin (STZ) and VSMCs were isolated from the aorta of control and STZ-diabetic rats 4 weeks after diabetes induction; only animals with blood glucose levels above 300 mg/dl on day 28 were considered diabetic. VSMCs from normal and diabetic rat aortas were incubated for 24 hours in the presence of LPS combined with a cytokine mixture (cytomix) to mimic the in vivo environment of some vascular inflammatory events and were exposed to exogenous adenosine. Incubation of VSMCs with LPS plus cytokine mixture for 24 h induced iNOS expression, which was undetectable in unstimulated VSMCs. Exogenous adenosine (1 mM) did not change iNOS levels in control VSMCs, but potentiated the response to cytokines in diabetic VSMCs. This response was unaffected by the equilibrative nucleoside transporter inhibitor, NBTI, but was further increased (+ 45%) by EHNA, an inhibitor of adenosine deaminase, the enzyme which deaminates adenosine to inosine. Exogenous inosine was ineffective in control and diabetic cells, but the adenosine precursor, AMP, mimicked the effect of adenosine on iNOS production in diabetic cells. Inhibition by AOPCP of ecto-5’-nucleotidase/CD73 (which dephosphorylates AMP in adenosine) did not significantly change iNOS protein levels. In the absence of exogenous adenosine, iNOS expression was reduced after treatment with EHNA in control but not in diabetic VSMCs, demonstrating that adenosine deaminase is responsible for adenosine elimination under non - pathological conditions. An HPLC method was used to quantify AMP, adenosine and their metabolites in the culture medium of VSMCs. At the end of 24 h-incubation, exogenous adenosine was undetectable in the culture medium of control as well as diabetic VSMCs, being converted into inosine and hypoxantine. In control VSMCs NBTI allowed the recovery of half of added adenosine whereas the recovery was lower in diabetic cells, indicating a different contribution of equilibrative transporters to the removal of the nucleoside in diabetes. In contrast, the addition of EHNA did not cause variations in the amount of adenosine recovered in control as compared with diabetic cells. After incubation with AMP, the nucleotide was not detectable and was converted mainly into inosine and hypoxanthine. Treatment with AOPCP allowed 47% recovery of AMP in control, but only 5% recovery in the medium of diabetic VSMCs indicating that diabetes markedly reduced CD73 sensitivity to pharmacological inhibition by AOPCP. These results show that alterations in adenosine-related inflammatory pathways may be present in diabetic vascular dysfunction; in addition, at high concentrations, adenosine seems to lose its protective effect as it stimulates the formation of iNOS, an effect that might be harmful to cells. Thus, diabetes would make VSMCs more sensitive to the potential proinflammatory effect of high concentrations of adenosine in terms of iNOS protein expression.Il diabete è una malattia metabolica associata ad aumentato rischio cardiovascolare. Nel diabete inoltre, in seguito all’attivazione della proteina iNOS, si ha una notevole produzione di perossinitrito che è implicato in molti disordini vascolari. A livello cardiovascolare l’adenosina manifesta diverse proprietà vasoprotettive: induce infatti vasodilatazione, inibisce l’aggregazione piastrinica e ne previene l’adesione, inibisce la crescita delle cellule muscolari lisce vascolari (VSMC) (Ikeda et al., 1997). Diverse evidenze suggeriscono che l’adenosina prodotta dalle VSMC può indurre i suoi effetti in parte attraverso la produzione di NO a livello della parete dei vasi: il nucleoside, per interazione con i recettori A2, modula l’espressione della proteina iNOS indotta da citochine e di conseguenza il rilascio di NO dalle VSMC (Dubey et al., 1998; St. Hilaire et al., 2008). Dal momento che un’aumentata produzione di iNOS è coinvolta nella disfunzione vascolare diabetica, si è studiato il potenziale ruolo anti-infiammatorio dell’adenosina in tale processo, determinando l’effetto del nucleoside e dei composti ad esso correlati sull’espressione di iNOS in cellule muscolari lisce vascolari di ratti resi diabetici in confronto con ratti normoglicemici. A tale scopo ad una parte degli animali è stato indotto il diabete mediante iniezione nella vena caudale di streptozotocina. Il prelievo dell’aorta dei ratti per l’ottenimento delle colture cellulari è stato effettuato dopo quattro settimane dal trattamento, considerando diabetici solo gli animali con valore di glicemia superiore a 300 mg/dl. Per indurre l’espressione della proteina iNOS le cellule muscolari lisce vascolari di ratti sani e diabetici sono state stimolate per 24 h con una miscela costituita da LPS più citochine (citomix) e queste cellule sono state trattate con adenosina esogena. Dall’analisi di Western blot emerge che la purina impiegata in concentrazione 1 mM causa un aumento significativo dei livelli di iNOS nelle cellule di ratti diabetici. Questo aumento non è influenzato dal trattamento con NBTI, inibitore dei trasportatori equilibrativi per l’adenosina, ma è potenziato in seguito all’aggiunta di EHNA, inibitore dell’adenosina deaminasi, enzima che converte l’adenosina in inosina. L’inosina esogena non influenza l’espressione di iNOS nelle VSMC controllo e diabetiche, mentre il precursore dell’adenosina, AMP, mima l’effetto dell’adenosina sulla produzione di iNOS nelle cellule diabetiche. L’AOPCP, inibitore dell’enzima CD73, che promuove la defosforilazione di AMP in adenosina, non provoca variazione dell’espressione di iNOS, rispetto al trattamento con solo AMP. Stimolando le VSMC con citomix e NBTI, EHNA o AOPCP in assenza di purine esogene, si è dimostrato che solo il trattamento con EHNA causa, nelle cellule di ratti normoglicemici, una diminuzione significativa della produzione di iNOS, indicando che l’adenosina deaminasi rappresenta la principale via di eliminazione del nucleoside in condizioni non patologiche. Le quantità di adenosina rimasta nel medium di incubazione dopo 24 h e quella dei suoi metaboliti sono state monitorate con metodo HPLC. Al termine dell’incubazione l’adenosina scompare dal medium, mentre si accumulano inosina e ipoxantina. Nelle cellule controllo NBTI permette il recupero della metà dell’adenosina inizialmente aggiunta, mentre nelle cellule diabetiche il recupero è inferiore, indicando probabilmente un diverso contributo dei trasportatori equilibrativi alla rimozione del nucleoside nel diabete. Al contrario l’aggiunta di EHNA non causa variazioni nella quantità di adenosina recuperata nelle cellule controllo rispetto a quelle diabetiche. Anche l’AMP scompare dal medium dopo 24 h con produzione di inosina e ipoxantina; l’aggiunta di AOPCP permette il recupero del 47% del nucleotide nel medium di incubazione delle cellule di ratti sani e solo il 5% nel medium delle cellule di ratti diabetici, suggerendo un’alterazione dell’attività o dell’espressione dell’enzima CD73 associata al diabete. Questi risultati dimostrano che alterazioni a livello delle vie di eliminazione e formazione dell’adenosina sono presenti nella disfunzione vascolare diabetica. Inoltre, a concentrazioni elevate l’adenosina sembra perdere il suo effetto protettivo, in quanto stimola la formazione di iNOS, con conseguenze che possono essere dannose per la cellula. Il diabete rende, pertanto, le VSMC più sensibili al potenziale effetto proinfiammatorio di elevate concentrazioni di adenosina in termini di espressione della proteina iNOS

Effects of compost and mowing on the productivity and density of a purpose-sown mixture of native herbaceous species to revegetate degraded soil in anthropized areas

Disturbance and soil fertility are some of the main drivers influencing the dynamics of herbaceous communities. Such communities are among the most biodiverse and represent a model for introducing species-rich and low-input green systems into anthropized environments, at the same time creating opportunities for conservation and restoration. Trials were set up to evaluate the effects of compost and mowing on the dynamics of purpose-sown herbaceous vegetation, inspired by the phytocenosis spontaneously growing in the nearby rural areas. Both soil properties (organic carbon, total nitrogen content, bulk density and pH) and plant species characteristics (density, biomass, height, functional traits) were determined. Our results showed that the addition of compost countered the soil compaction process with a positive effect on soil bulk density. Irrespective of compost and mowing, the amount of carbon and nitrogen in the soil was greatly influenced by the vegetation. Early season mowing increased the Shannon index and decreased the Simpson index, while over the years, with the increase in productivity, biodiversity decreased. Compost and mowing had a species-specific effect on seed mass and plant height

Wildflowers: From conserving biodiversity to urban greening—A review

Species-rich herbaceous communities, such as prairies, steppes, meadows and pastures, have a high bio-diversity value. There is considerable interest in the loss of these complex ecosystems and the associatedbiodiversity, due to intensive agriculture, pasture abandonment, pollution, and climate change. Thesehabitats can constitute models, in terms of landscape management and plant community composition,that can be successfully duplicated in anthropized areas in order to mitigate the adverse effects of humanactivities in the city and enhance the biotic component. The idea is to revegetate urban degraded soilwith aesthetically pleasing wildflower meadows, while increasing biodiversity, creating a habitat andconserving the local flora, with low management cost. In urban sites seed mixtures containing a highpercentage of native and exotic herbaceous flowering plants have been successfully used. We review thefactors affecting the ecological aspects of species-rich herbaceous communities in disturbed environ-ments and urban landscape design. The review addresses the use of these communities in urban greenareas for recreation, socialization and environmental education

Le piante spontanee come risorsa per il florovivaismo e la valorizzazione del paesaggio

No abstract availableIl volume raccoglie gli atti del workshop sull\u27impiego delle piante spontanee nel florovivasimo che si ? tenuto il 18 maggio 2007 presso la Facolt? di Agraria dell\u27Universit? di Pisa. Il workshop ? stato organizzato nell\u27ambito del progetto: "Recupero e valorizzazione del patrimonio autoctono e naturalizzato: aspetti produttivi, varietali ed economici legati alla diversificazione e all\u27introduzione di innovazione di prodotto in floricoltura" (ReVFlor) coordinato dal Prof. A. Garibaldi (Agroinnova, Universit? di Torino), promosso e finanziato dal Ministero per le Politiche agricole e forestali secondo il bando emanato dalla Regione Liguria anche in nome e per conto delle Regioni facenti parte del comitato di progetto