Emerging technologies: polymer-free phospholipid encapsulated sirolimus nanocarriers for the controlled release of drug from a stent-plus-balloon or a stand-alone balloon catheter.

Drug-eluting stents have proven to be effective in reducing the risk of late restenosis. In order to achieve a controlled and prolonged release of the antiproliferative agent, current drug-eluting stents utilise various biodegradable as well as non-erodible polymeric blends to coat the stent surface and to serve as drug carriers. The utilisation of polymeric compounds in current drug-eluting stents may eventually limit their performance as well as their clinical applicability due to the potential induction of undesirable local reactions. The development of alternative, polymer-free drug carriers has the potential to overcome some of the limitations of current drug-eluting stent formulations. Moreover, improvements in drug carriers may also result in an expansion of the technological possibilities for other intravascular drug delivery systems, such as metal-free or even implant-free solutions. This article describes the structure and the preclinical validation profile of a novel phospholipid encapsulated sirolimus nanocarrier, used as a coating in two formulations: a coronary stent-plus-balloon system and a stand-alone balloon catheter. The nanoparticles provided a stable, even and homogenous coating to the devices in both formulations. Dose-finding studies allowed the most appropriate identification of the best nanoparticle structure associated with an extremely efficient transfer of drug to all layers of the vessel wall, achieving high tissue concentrations that persisted days after the application, with low systemic drug leaks.

Cellular prion protein (PrP(C)) and superoxide dismutase (SOD) in vascular cells under oxidative stress.

UNLABELLED: The PrP(C) is expressed in several cell types but its physiological function is unknown. Some studies associate the PrP(C) with copper metabolism and the antioxidant activity of SOD. Our hypothesis was that changes in PrP(C) expression lead to abnormal copper regulation and induce SOD downregulation in the vascular wall. OBJECTIVES: to study whether the PrP(C) expression undergoes induction by agents that trigger endoplasmic reticulum stress (ERS) and, in this context, to evaluate the SOD activity. METHODS: To trigger ERS, in vitro, rabbit aortic smooth muscle cells were challenged for 4, 8 and 18 hours, with angiotensin-II, tunicamycin and 7-ketocholesterol. For in vivo studies rabbit aortic arteries were subjected to injury by balloon catheter. RESULTS: In vitro baseline SOD activity, determined through inhibition of cytochrome-c reduction, was 13.9±1.2 U/mg protein, angiotensin-II exposed for 8 hours produced an increase in SOD activity, and cellular copper concentration was about 9 times greater only under these conditions. Western blotting analysis for SOD isoenzymes showed an expression profile that was not correlated with the enzymatic activity. PrP(C) expression decreased after exposure to all agents after different incubation periods. RT-PCR assay showed increased mRNA expression for PrP(C) only in cells stimulated for 8 hours with the different stressors. The PrP(C) mRNA expression in rabbit aortic artery fragments, subjected to balloon catheter injury, showed a pronounced increase immediately after overdistension. The results obtained indicated a PrP(C) protection factor during the early part of the ERS exposure period, but did not demonstrate a SOD-like profile for the PrP(C).

Rosiglitazona e lesão vascular em coelhos hipercolesterolêmicos: avaliação da formação neointimal / Rosiglitazone and vascular injury in hypercholesterolemic rabbits: neointimal formation assessment / Rosiglitazona y lesión vascular en conejos hipercolesterolémicos

FUNDAMENTO: O uso da rosiglitazona tem sido o objeto de extensas discussões. OBJETIVO: Avaliar os efeitos da rosiglitazona nas artérias ilíacas, no local da injúria e na artéria contralateral, de coelhos hipercolesterolêmicos submetidos à lesão por cateter-balão. MÉTODOS: Coelhos brancos machos receberam uma dieta hipercolesterolêmica através de gavagem oral por 6 semanas e foram divididos em 2 grupos: grupo rosiglitazona (GR - 14 coelhos tratados com rosiglitazona por 6 semanas) e grupo controle (GC - 18 coelhos sem rosiglitazona). Os animais foram submetidos a lesão por cateter-balão na artéria ilíaca direita no 14º dia. RESULTADOS: Na artéria ilíaca contralateral, não houve diferença significante na razão entre as áreas intimal e medial (RIM) entre os grupos GR e GC. A rosiglitazona não reduziu a probabilidade de lesões tipo I, II ou III (72,73 por cento vs 92,31 por cento; p=0,30) e lesões tipo IV ou V (27,27 por cento vs 7,69 por cento; p=0,30). Na artéria ilíaca homolateral, a área intimal era significantemente menor no GR quando comparado ao GC (p = 0,024). A área luminal era maior no GR quando comparado ao GC (p < 0,0001). Houve uma redução significante de 65 por cento na IMR no GR quando comparado ao GC (p = 0,021). Nenhum dos critérios histológicos para lesões ateroscleróticas tipos I a V (American Heart Association) foram encontrados na artéria ilíaca homolateral. CONCLUSÃO: Esses achados demonstram que a administração de rosiglitazona por 6 semanas impede a aterogênese no local da lesão, mas não em um vaso distante do sítio da lesão.

BACKGROUND: Rosiglitazone has been the focus of extensive discussion. OBJECTIVE: To evaluate the effects of rosiglitazone on iliac arteries, both at the injury site and the contralateral artery, of hypercholesterolemic rabbits undergoing balloon catheter injury. METHODS: White male rabbits were fed a hypercholesterolemic diet by oral gavage for 6 weeks and divided into two groups as follows: rosiglitazone group (14 rabbits treated with rosiglitazone during 6 weeks) and the control group (18 rabbits without rosiglitazone). Animals underwent balloon catheter injury of the right iliac artery on the 14th day. RESULTS: In the contralateral iliac artery, there was no significant difference in the intima/media layer area ratio (IMR) between the control and rosiglitazone groups. Rosiglitazone did not reduce the probability of type I, II, or III lesions (72.73 percent vs 92.31 percent; p=0.30) and type IV or V lesions (27.27 percent vs 7.69 percent; p=0.30). As for the homolateral iliac artery, the intimal area was significantly lower in the rosiglitazone group, as compared to the control group (p = 0.024). The luminal layer area was higher in the rosiglitazone group vs. the control group (p < 0.0001). There was a significant reduction of 65 percent in the IMR in the rosiglitazone group vs the control group (p = 0.021). None of the histological criteria for type I-V atherosclerotic lesions (American Heart Association) were found in the homolateral iliac artery. CONCLUSION: These findings demonstrate that rosiglitazone given for 6 weeks prevents atherogenesis at the injury site, but not in a vessel distant from the injury site.

FUNDAMENTO: El uso de rosiglitazona ha estado siendo el objeto de extensas discusiones. OBJETIVO: Evaluar los efectos de la rosiglitazona en las arterias ilíacas, en el local de la injuria y en la arteria contralateral, de conejos hipercolesterolémicos sometidos a la lesión por catéter-balón. MÉTODOS: Conejos blancos machos recibieron una dieta hipercolesterolémica a través de gavage oral por 6 semanas y se los dividieron en 2 grupos: grupo rosiglitazona (GR - 14 conejos tratados con rosiglitazona por 6 semanas) y grupo control (GC - 18 conejos sin rosiglitazona). Los animales se sometieron a lesión por catéter-balón en la arteria ilíaca derecha en el 14º día. RESULTADOS: En la arteria ilíaca contralateral, no hubo diferencia significativa en la razón entre las áreas íntima y media (RIM) entre los grupos GR y GC. La rosiglitazona no redujo la probabilidad de lesiones tipo I, II ó III (72,73 por ciento vs 92,31 por ciento; p=0,30) y lesiones tipo IV ó V (27,27 por ciento vs 7,69 por ciento; p=0,30). En la arteria ilíaca homolateral, el área intima era significantemente menor en el GR cuando comparado al GC (p = 0,024). El área luminal era mayor en el GR cuando comparado al GC (p < 0,0001). Hubo una reducción significante del 65 por ciento en la IMR en el GR cuando comparado al GC (p = 0,021). Ningún de los criterios histológicos para lesiones ateroscleróticas tipos I a V (American Heart Association) se encontraron en la arteria ilíaca homolateral. CONCLUSIÓN: Estos hallazgos demuestran que la administración de rosiglitazona por 6 semanas impide la aterogénesis en el local de la lesión, pero no en un vaso distante del sitio de la lesión.

Rosiglitazone and vascular injury in hypercholesterolemic rabbits: neointimal formation assessment.

BACKGROUND: Rosiglitazone has been the focus of extensive discussion. OBJECTIVE: To evaluate the effects of rosiglitazone on iliac arteries, both at the injury site and the contralateral artery, of hypercholesterolemic rabbits undergoing balloon catheter injury. METHODS: White male rabbits were fed a hypercholesterolemic diet by oral gavage for 6 weeks and divided into two groups as follows: rosiglitazone group (14 rabbits treated with rosiglitazone during 6 weeks) and the control group (18 rabbits without rosiglitazone). Animals underwent balloon catheter injury of the right iliac artery on the 14th day. RESULTS: In the contralateral iliac artery, there was no significant difference in the intima/media layer area ratio (IMR) between the control and rosiglitazone groups. Rosiglitazone did not reduce the probability of type I, II, or III lesions (72.73% vs 92.31%; p=0.30) and type IV or V lesions (27.27% vs 7.69%; p=0.30). As for the homolateral iliac artery, the intimal area was significantly lower in the rosiglitazone group, as compared to the control group (p = 0.024). The luminal layer area was higher in the rosiglitazone group vs. the control group (p < 0.0001). There was a significant reduction of 65% in the IMR in the rosiglitazone group vs the control group (p = 0.021). None of the histological criteria for type I-V atherosclerotic lesions (American Heart Association) were found in the homolateral iliac artery. CONCLUSION: These findings demonstrate that rosiglitazone given for 6 weeks prevents atherogenesis at the injury site, but not in a vessel distant from the injury site.

[The collagen of the restenosis post angioplasty with stent: Is its origin in intima or adventitia?]. / El colágeno de la reestenosis post angioplastia con stent: se origina en la íntima o en la adventicia?

BACKGROUND: Restenosis post stenting is due to the deposit of extracellular matrix, mainly collagen in the neointima. Controversy exists regarding if collagen is generated locally or by immigration from the adventitia. AIM: To study the fibrocellular response after stent implantation in rabbit iliac arteries. To observe, by immunohistochemistry and in situ hybridization, if collagen type I mRNA is expressed in the neointima, in the media or in the adventitia. MATERIAL AND METHODS: Thirty eight white rabbits (New Zealand) of 4 kg received an hypercholesterolemic diet during 1 month. After this period, in all but 6 of them, an angioplasty with stent implantation was performed via right carotid artery in both iliac arteries, using a 1:1.3 relationship regarding the reference vessel. Angiograms were performed at day 0, 4, 21, and 40, followed by paraffin fixation of the injured segments, immunohistochemistry for alpha-actin and in situ hybridization to detect procollagen type I (alpha 1R1) mRNA. RESULTS: No hybridization was observed in non injured arteries or at day 0 (n = 6). Expression of alpha 1R1 mRNA was observed in the neointima starting at day 4 after stenting (n = 8). At day 21 (n = 8) hybridization of procollagen type I was not only observed in the neointima, but also in the media, which became equally intense in both areas. At day 40 (n = 6) hybridization was observed similarly in the media and adventitia. CONCLUSIONS: In this model, hybridization of procollagen type I started in the neointima, then involved the media and finally the adventitia. This finding might be useful for designing therapies to be delivered locally at the end of an angioplasty to prevent collagen deposition in the neointima.

Inhibition of vascular NADH/NADPH oxidase activity by thiol reagents: lack of correlation with cellular glutathione redox status.

Vascular NAD(P)H oxidase activity contributes to oxidative stress. Thiol oxidants inhibit leukocyte NADPH oxidase. To assess the role of reactive thiols on vascular oxidase, rabbit iliac/carotid artery homogenates were incubated with distinct thiol reagents. NAD(P)H-driven enzyme activity, assessed by lucigenin (5 or 250 microM) luminescence, was nearly completely (> 97%) inhibited by the oxidant diamide (1mM) or the alkylator p-chloromercuryphenylsulfonate (pCMPS, 0.5mM). Analogous inhibition was also shown with EPR spectroscopy using DMPO as a spin trap. The oxidant dithionitrobenzoic acid (0.5mM) inhibited NADPH-driven signals by 92% but had no effect on NADH-driven signals. In contrast, the vicinal dithiol ligand phenylarsine oxide (PAO, 1 microM) induced minor nonsignificant inhibition of NADPH-driven activity, but significant stimulation of NADH-triggered signals. The alkylator N-ethyl maleimide (NEM, 0.5mM) or glutathione disulfide (GSSG, 3mM) had no effect with each substrate. Coincubation of N-acetylcysteine (NAC, 3mM) with diamide or pCMPS reversed their inhibitory effects by 30-60%, whereas NAC alone inhibited the oxidase by 52%. Incubation of intact arterial rings with the above reagents disclosed similar results, except that PAO became inhibitor and NAC stimulator of NADH-driven signals. Notably, the cell-impermeant reagent pCMPS was also inhibitory in whole rings, suggesting that reactive thiol(s) affecting oxidase activity are highly accessible. Since lack of oxidase inhibition by NEM or GSSG occurred despite significant cellular glutathione depletion, change in intracellular redox status is not sufficient to account for oxidase inhibition. Moreover, the observed differences between NADPH and NADH-driven oxidase activity point to complex or multiple enzyme forms.

Oxidized thiols markedly amplify the vascular response to balloon injury in rabbits through a redox active metal-dependent pathway.

OBJECTIVE: Our aim was to assess whether exposure to oxidized thiols--a known usual consequence of oxidant stress--has the potential to affect the vascular repair response to angioplasty-induced injury. In addition, we also assessed the role of redox active metals in disulfide effects. METHODS: In 82 rabbits submitted to overdistention of iliac arteries, the following variables were analyzed: neointimal thickening, immunoreactivity to Proliferating Cell Nuclear Antigen, and cellular and collagen densities. RESULTS: A single intraarterial challenge of oxidized glutathione (GSSG, 6.5 mumol/kg) during and immediately after injury triggered a marked increase of the vascular repair reaction, as follows: (A) at day 7 after injury, there was a 2.7-fold increase in proliferation (p < 0.001 vs. control); (B) at day 14, there was increase of intimal/medial area ratio to 1.35 +/- 0.14, vs. 0.56 +/- 0.08 in controls. Proliferating cells increased to 5.5 +/- 0.8 cells/mm2, vs. 2.2 +/- 0.5 in controls (p < 0.002 for both variables). Overall cellularity was enhanced 2.2-fold; (C) at day 28, there was ongoing vessel wall proliferation, contrarily to controls. All GSSG effects were completely prevented by co-infusion of reduced glutathione (GSH) and were mimicked by cystine (6.5 mumol/kg). The uninjured artery showed no response to disulfides. To assess the role of redox active metals in GSSG action, the effects of 1,10-phenanthroline or N-CBZ-Pro-Leu-Gly hydroxamic acid (HXA), metal chelators with metalloproteinase inhibitor properties, were evaluated. Both compounds totally blocked the GSSG-induced amplification of vascular responses. In rabbits not exposed to GSSG, HXA decreased neointimal thickening by 50% (p < 0.05). CONCLUSIONS: Exposure to excess disulfide levels early after vascular balloon injury markedly amplified the late cellular response through interaction with redox active metals. These pathways can potentially mediate noxious effects of oxidative stress in vessels.