Paola Ciceri

Production of leukotrienes in a model of focal cerebral ischaemia in the rat

The aim of this work was to evaluate the role of leukotrienes in brain damage in vivo in a model of focal cerebral ischaemia in the rat, obtained by permanent occlusion of middle cerebral artery. A significant (P<0.01) elevation of LTC4, LTD4 and LTE4 (cysteinyl‐leukotrienes) levels occurred 4 h after ischaemia induction in the ipsilateral cortices of ischaemic compared to sham‐operated animals (3998±475 and 897±170 fmol g−1 tissue, respectively, P<0.01). The NMDA receptor antagonist MK‐801 and the adenosine A2A receptor antagonist SCH 58261 were administered in vivo at doses known to reduce infarct size and compared with the leukotriene biosynthesis inhibitor MK‐886. MK‐886 (0.3 and 2 mg kg−1 i.v.) and MK‐801 (3 mg kg−1 i.p.) decreased cysteinyl‐leukotriene levels (−78%, P<0.05; −100%, P<0.01; −92%, P<0.01, respectively) 4 h after permanent occlusion of the middle cerebral artery, whereas SCH 58261 (0.01 mg kg−1 i.v.) had no significant effects. MK‐886 (2 mg kg−1 i.v.) was also able to significantly reduce the cortical infarct size by 30% (P<0.05). We conclude that cysteinyl‐leukotriene formation is associated with NMDA receptor activation, and that it represents a neurotoxic event, the inhibition of which is able to reduce brain infarct area in a focal ischaemic event.

Combined effects of ascorbic acid and phosphate on rat VSMC osteoblastic differentiation

BACKGROUND Ascorbic acid (AA) supplementation has been suggested to afford erythropoietin hyporesponsiveness and high levels of ferritin in haemodialysis (HD) patients. However, little is known about the possible side effects of this policy on vascular calcification (VC). VC, induced by a high-phosphate and uraemic milieu, is characterized by a passive deposition of calcium-phosphate (Ca-P) and an active transformation of vascular smooth muscle cells (VSMCs) in osteoblastic-like cells. The aim of these studies was to characterize the combined effects of AA and P on VC. MATERIALS AND METHODS Rat VSMCs were challenged with inorganic P (Pi) and AA, and Ca deposition analysis was performed to quantify VC. To investigate VSMC osteoblastic differentiation, we analysed α-actin protein content and core-binding factor alpha-1 (Cbfα1/RUNX2) messenger RNA (mRNA) expression. RESULTS When incubated with 5 mM Pi, VSMCs showed a significant increase in Ca deposition compared to control cells. Interestingly, the addition of AA in the calcification medium resulted in a dose-dependent increase in Pi-induced Ca deposition. At the same time, the combined effect of AA and Pi on VSMCs resulted in the reduction of α-actin protein content and in a 4-fold increase of Cbfα1/RUNX2 mRNA expression. CONCLUSIONS We demonstrated that AA combined with Pi increases Ca deposition in rat VSMCs. The role of AA as cofactor in osteoblastic differentiation was demonstrated by phenotypic changes in VSMCs and enhanced bone mineralization key gene expression. These in vitro preliminary data suggest a potential role for AA combined with Pi in worsening VC.

Pathophysiology of calcium and phosphate metabolism impairment in Chronic Kidney Disease

Secondary hyperparathyroidism (SHPT) is a classical feature of chronic kidney disease (CKD). Commonly, hypocalcemia, hyperphosphatemia, and vitamin D deficiency are involved into the pathogenesis of SHPT. Parathyroid (PT) glands are characterized by a low turnover and rarely undergo mitoses. However, in the presence of low calcium, high phosphorus, vitamin D deficiency, and uremia, PT cells leave quiescence. In the last decade, both new molecular and cellular mechanisms have been investigated in the pathophysiology of SHPT, between them the emerging role of the PT vitamin D receptor and calcium-sensing receptor. Furthermore, recent studies indicate that the fibroblast growth factor-23 may play a central role in the regulation of phosphate-vitamin D metabolism in CKD. Certainly, in the next future, these new insights into the pathogenesis of SHPT will give the possibility to improve the treatment of this condition in the CKD population.

Lanthanum Prevents High Phosphate-Induced Vascular Calcification by Preserving Vascular Smooth Muscle Lineage Markers

Vascular calcification (VC) represents a major cardiovascular risk factor in chronic kidney disease patients. High phosphate (Pi) levels are strongly associated with VC in this population. Therefore, Pi binders are commonly used to control high Pi levels. The aim of this work was to study the mechanism of action of lanthanum chloride (LaCl3) on the progression of Pi-induced VC through its direct effect on vascular smooth muscle cells (VSMCs) in vitro. High Pi induced VSCM Ca deposition. We evaluated the action of LaCl3, compared to gadolinium chloride (GdCl3), and found different effects on the modulation of VSMC lineage markers, such as α-actin and SM22α. In fact, only LaCl3 preserved the expression of both VSMC lineage markers compared to high Pi-treated cells. Interestingly, both LaCl3 and GdCl3 reduced the high Pi-induced elevations of bone morphogenic protein 2 mRNA expression, with no reduction of the high core binding factor-alpha 1 mRNA levels observed in calcified VSMCs. Furthermore, we also found that only LaCl3 completely prevented the matrix GLA protein mRNA levels and osteonectin protein expression elevations induced by high Pi compared to GdCl3. Finally, LaCl3, in contrast to GdCl3, prevented the high Pi-induced downregulation of Axl, a membrane tyrosine kinase receptor involved in apoptosis. Thus, our results suggest that LaCl3 prevents VC by preserving VSMC lineage markers and by decreasing high Pi-induced osteoblastic differentiation.

The combination of lanthanum chloride and the calcimimetic calindol delays the progression of vascular smooth muscle cells calcification

Phosphate (Pi)-binders are commonly used in dialysis patients to control high Pi levels, that associated with vascular calcification (VC). The aim of this study was to investigate the effects of lanthanum chloride (LaCl(3)) on the progression of high Pi-induced VC, in rat vascular smooth muscle cells (VSMCs). Pi-induced Ca deposition was inhibited by LaCl(3), with a maximal effect at 100μM (59.0±2.5% inhibition). Furthermore, we studied the effects on VC of calcium sensing receptor (CaSR) agonists. Gadolinium chloride, neomycin, spermine, and the calcimimetic calindol significantly inhibited Pi-induced VC (55.9±2.2%, 37.3±4.7%, 30.2±5.7%, and 63.8±5.7%, respectively). To investigate the hypothesis that LaCl(3) reduces the progression of VC by interacting with the CaSR, we performed a concentration-response curve of LaCl(3) in presence of a sub-effective concentration of calindol (10nM). Interestingly, this curve was shifted to the left (IC(50) 9.6±2.6μM), compared to the curve in the presence of LaCl(3) alone (IC(50) 19.0±4.8μM). In conclusion, we demonstrated that lanthanum chloride effectively reduces the progression of high phosphate-induced vascular calcification. In addition, LaCl(3) cooperates with the calcimimetic calindol in decreasing Ca deposition in this in vitro model. These results suggest the potential role of lanthanum in the treatment of VC induced by high Pi.

Heterogeneous platelet-activating factor (PAF) receptors and calcium increase in platelets and macrophages.

We used the increase in cytosolic Ca2+ levels, [Ca2+]i, as a way to characterize PAF (platelet-activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) receptors in human platelets and rat and human macrophages. [Ca2+] was measured by means of the fluorescent probe fura-2/acetoxymethylester. PAF recognized heterogeneous receptors in human macrophages only (curve slope <1). The PAF antagonist SCH 37370 (1-acetyl-4(8-chloro-5,6-dihydro-11H-benzo[5.6]cyclohepta[1,2-b]pyridine -11-ylidine)piperidine) abolished [Ca2+]i elevation in human platelets, while in rat and human macrophages the maximal inhibition was 76% and 85%, respectively. On the contrary, the antagonist WEB 2086 (3-[4-(2-chlorophenyl)-9-methyl-6Hthieno[3,2-f] [1,2,4]triazolo-[4,3-a] [1,4]-diazepin-2-yl]-1-(4-morpholiny)-1-propanon, apafant) totally inhibited the effect of PAF in both platelets and macrophages. The WEB 2086 concentration-response curves had a slope <1 in the three cell types, indicating interaction with heterogeneous receptors. Accordingly, 3H-WEB 2086 bound to two different classes of sites. Both phases of [Ca2+]i elevation (influx or release) were equally affected by the antagonists. These data support the notions that: 1) PAF receptors are heterogeneous; 2) the two antagonists have a different selectivity toward the receptor subtypes: WEB 2086 recognizes two different receptors both in platelets and in macrophages, while SCH 37370 does not discriminate between receptor subtypes in platelets, and only interacts with one subtype in macrophages; and 3) both SCH 37370 and WEB 2086 display different potencies in rat and human macrophages.

The calcimimetic calindol prevents high phosphate-induced vascular calcification by upregulating matrix GLA protein.

Background: High serum phosphate (Pi) levels represent a major issue in dialysis patients, because associate with secondary hyperparathyroidism, vascular calcification (VC), and cardiovascular outcomes. In this population, calcimimetics are used to control secondary hyperparathyroidism, hyperphosphatemia, and, more recently, to delay the progression of VC. The aim of this in vitro study was to investigate the direct effects of the calcimimetic calindol on the progression of high Pi-induced VC. Methods: Rat vascular smooth muscle cells (VSMCs) were incubated with high Pi concentrations, and the effects of calindol were investigated on vascular calcium deposition and VSMC osteoblastic differentiation. Results: Calindol inhibited calcium deposition concentration-dependently with a maximal inhibition of 64.0 ± 5.2% achieved at 100 nM. Furthermore, calindol was able to partially prevent the high Pi-induced bone morphogenic protein 2 (BMP-2) expression upregulation (32.4 ± 4.6% of inhibition; p < 0.01). Interestingly, the pretreatment with calindol enhanced the matrix Gla protein (MGP) gene expression significantly, compared to high Pi-treated cells (40.2 ± 6.6% of increase, p < 0.01). Conclusions: In conclusion, we demonstrated that the calcimimetic calindol prevents high Pi-induced VC by affecting osteoblastic differentiation in vitro. In particular, the inhibitory effect of calindol on VC is probably due to its stimulatory role on the calcium-sensing receptor, leading to an increase in the synthesis of MGP by VSMCs.

The Effect of Paricalcitol on Vascular Calcification and Cardiovascular Disease in Uremia: Beyond PTH Control

Secondary hyperparathyroidism is a systemic disorder that associates with bone and cardiovascular disease, including arterial calcification. Treatment with calcitriol, the active form of vitamin D, reduces parathyroid hormone levels, but may result in elevations in serum calcium and phosphorus, increasing the risk of vascular calcification in dialysis patients. New vitamin D receptor activators (VDRAs) have been developed and investigated with the rationale to treat high serum PTH levels, with a reduced risk of hypercalcemia and hyperphosphatemia. Paricalcitol is a selective VDRA that suppresses PTH secretion with minimal increases on serum calcium and phosphate. Moreover, paricalcitol prevents vascular calcification in experimental models of renal failure, compared with calcitriol.

Vascular calcification in chronic kidney disease : a changing scenario

Vascular calcification (VC) is one of the most dramatic consequences of chronic kidney disease (CKD). It has been considered a passive process, resulting essentially from mineral metabolism disorders and alterations in calcium and phosphate balance. But during the last decade, it has been elucidated how VC is not only a passive but more properly an active process, in which different factors are deeply involved. The progression of vessel wall mineralization is commonly associated with factors that promote VC, such as age, dialysis vintage and mineral metabolism abnormalities. Furthermore, many substances seem to be dynamically implicated in the regulation of the molecular mechanisms of VC. Between them, the matrix Gla protein and fetuin-A have recently been investigated in CKD. In this review, along with the most promising possible treatments, the new molecular mechanisms involved in the VC process will be elucidated.

Phosphate Control in Peritoneal Dialysis

Chronic kidney disease (CKD) is characterized by phosphorus retention and, in more advanced stages, by high serum phosphorus (P) levels. During the last decade, it has been elucidated the central role of P in the pathogenesis of CKD mineral bone disorder (CKD-MBD), determining both renal osteodystrophy and cardiovascular disease. Unfortunately, at least one third of patients on chronic dialysis have high serum P levels, with a consequent higher serum PTH levels, commonly associated with vitamin D deficiency, increased vascular calcification and the highest ratios of morbidity and mortality. In patients with CKD stage 5 on dialysis, therapeutic approaches to reduce serum P levels should include restriction of dietary phosphate intake, optimal dialysis treatment, and use of P binders. In this context, the use of P binders appears to be an essential treatment to control P overload in CKD patients. In this review, we analyzed the use of calcium-based and calcium-free P binders in peritoneal dialysis patients.