Use of paclitaxel carried in solid lipid nanoparticles to prevent peritoneal fibrosis in rats.

BACKGROUND: Progressive fibrous thickening of peritoneal membrane (PM) is a major complication of long-term peritoneal dialysis. TGF-ß/SMAD pathway activation, inflammation and neoangiogenesis have an important role in PM changes induced by peritoneal dialysis. Here, we investigated the effects of paclitaxel (PTX) carried in lipid core nanoparticles (LDE) on the development of peritoneal fibrosis (PF) in rats. METHODS: To induce PF, 21 male Wistar rats (300-350g) were injected with chlorhexidine gluconate for 15 consecutive days and randomly assigned to three groups: 1)PF, n = 5: no treatment; 2)LDE, n = 8: treated with LDE only, 3/3 days during 15 days; 3)LDE-PTX, n = 8: treated with PTX (4mg/kg) associated with LDE, 3/3 days during 15 days. A Control group without PF induction (n = 5) was designed, received saline solution, 3/3 days. Peritoneum function tests were performed, and anterior abdominal wall samples of the PM were collected for analyses of peritoneal thickness, immunohistochemitry, and gene expression. RESULTS: LDE-PTX treatment preserved the membrane function, maintaining the ultrafiltration rate and mass transfer of glucose at normal levels. LDE-PTX also prevented PM thickening induced by chlorhexidine gluconate injections. LDE-PTX treatment reduced the number of myofibroblasts infiltrating PM and inhibited the cell proliferation. Gene expression of fibronectin, FSP-1, VEGF, TGF-ß, and SMAD3 were reduced by LDE-PTX. CONCLUSIONS: LDE-PTX was effective to prevent development of PF and preserve the PM filtration capacity in this rat model, with clear-cut actions on pro-fibrotic mechanisms. Thus, LDE-PTX can be candidate for future clinical trials as adjuvant to peritoneal dialysis to prevent PF development, since this preparation is devoid of toxicity as shown previously.

Nanotechnology for the treatment of deep endometriosis: uptake of lipid core nanoparticles by LDL receptors in endometriotic foci.

OBJECTIVE: Rapidly dividing cells in multiple types of cancer and inflammatory diseases undergo high low density lipoprotein (LDL) uptake for membrane synthesis, and coupling an LDL-like nanoemulsion, containing lipid nanoparticles (LDE) to a chemotherapeutic agent efficiently targets these cells without significant systemic effects. This was a prospective exploratory study that evaluated the uptake of a radioactively labeled LDE emulsion by receptors of endometriotic foci and the capacity of the LDE for cellular internalization. METHODS: The lipid profile of each patient was determined before surgery, and labeled LDE were injected into fourteen patients with intestinal or nonintestinal endometriosis. The radioactivity of each tissue sample (intestinal endometriosis, nonintestinal endometriosis, healthy peritoneum, or topical endometrium) was measured. RESULTS: The group with intestinal endometriosis presented higher levels of plasma LDL but lower LDE uptake by foci than the nonintestinal group, suggesting less cell division and more fibrosis. The uptake of LDE was highest in the topical endometrium, followed by the healthy peritoneum, and lowest in the endometriotic lesion. Since the endometriotic foci showed significant LDE uptake, there was likely increased consumption of LDL by these cells, similar to cells in cancers and inflammatory diseases. Plasma cholesterol levels had no influence on LDE uptake, which showed that the direct delivery of the nanoemulsion to target tissues was independent of serum lipoproteins. There were no significant differences in the parameters (p>0.01) because of the small sample size, but the findings were similar to those of previous studies. CONCLUSION: Nanotechnology is a promising therapeutic option for surgery and hormonal blockage for deep endometriosis, with a lower complication rate and no systemic side effects.

Nanotechnology for the treatment of deep endometriosis: uptake of lipid core nanoparticles by LDL receptors in endometriotic foci

OBJECTIVE: Rapidly dividing cells in multiple types of cancer and inflammatory diseases undergo high low density lipoprotein (LDL) uptake for membrane synthesis, and coupling an LDL-like nanoemulsion, containing lipid nanoparticles (LDE) to a chemotherapeutic agent efficiently targets these cells without significant systemic effects. This was a prospective exploratory study that evaluated the uptake of a radioactively labeled LDE emulsion by receptors of endometriotic foci and the capacity of the LDE for cellular internalization. METHODS: The lipid profile of each patient was determined before surgery, and labeled LDE were injected into fourteen patients with intestinal or nonintestinal endometriosis. The radioactivity of each tissue sample (intestinal endometriosis, nonintestinal endometriosis, healthy peritoneum, or topical endometrium) was measured. RESULTS: The group with intestinal endometriosis presented higher levels of plasma LDL but lower LDE uptake by foci than the nonintestinal group, suggesting less cell division and more fibrosis. The uptake of LDE was highest in the topical endometrium, followed by the healthy peritoneum, and lowest in the endometriotic lesion. Since the endometriotic foci showed significant LDE uptake, there was likely increased consumption of LDL by these cells, similar to cells in cancers and inflammatory diseases. Plasma cholesterol levels had no influence on LDE uptake, which showed that the direct delivery of the nanoemulsion to target tissues was independent of serum lipoproteins. There were no significant differences in the parameters (p>0.01) because of the small sample size, but the findings were similar to those of previous studies. CONCLUSION: Nanotechnology is a promising therapeutic option for surgery and hormonal blockage for deep endometriosis, with a lower complication rate and no systemic side effects.

Cholesteryl ester transfer protein (CETP), HDL capacity of receiving cholesterol and status of inflammatory cytokines in patients with severe heart failure.

BACKGROUND: Heart failure (HF) courses with chronic inflammatory process and alterations in lipid metabolism may aggravate the disease. The aim was to test whether the severity of HF, using brain natriuretic peptide (BNP) as a marker, is associated with alterations in functional aspects of HDL, such as lipid transfer, cholesterol ester transfer protein (CETP) and lecithin-cholesterol acyltransferase (LCAT) concentration. METHODS: Twenty-five HF patients in NYHA class I/II and 23 in class III/IV were enrolled. Plasma lipids, apolipoproteins, CETP, LCAT, oxidized-LDL (oxLDL) and paraoxonase-1 (PON-1) activity were determined. Lipid transfer from a donor artificial nanoparticle to HDL was measured by in vitro assay. RESULTS: Total cholesterol (p = 0.049), LDL-C (p = 0.023), non-HDL-C (p = 0.029) and CETP, that promotes lipid transfer among lipoproteins (p = 0.013), were lower in III/IV than in I/II group. Triglycerides, HDL-C, apo A-I, apo B, oxLDL, LCAT, enzyme that catalyzes serum cholesterol esterification, PON-1 activity, and in vitro transfers of cholesterol, triglycerides and phospholipids to HDL, important steps in HDL metabolism, were equal. IL-8 was higher in III/IV (p = 0.025), but TNFα, IL-1ß, IL-6 and MCP-1 were equal. BNP was negatively correlated with CETP (r = - 0.294; p = 0.042) and positively correlated with IL-8 (r = 0.299; p = 0.039). CONCLUSIONS: Our results disclosed the relationship between CETP levels and HF severity, by comparing two HF groups and by correlation analysis. Lower CETP levels may be a marker of HF aggravation and possibly of worse prognosis. Practical applications of this initial finding, as the issue whether CETP could be protective against HF aggravation, should be explored in larger experimental and clinical studies.

Regression of Atherosclerotic Plaques of Cholesterol-Fed Rabbits by Combined Chemotherapy With Paclitaxel and Methotrexate Carried in Lipid Core Nanoparticles.

In previous studies, it was demonstrated that lipid core nanoparticles (LDE) resemble the low-density lipoprotein structure and carrying the antiproliferative agent paclitaxel (PTX) strongly reduced atherosclerosis lesions induced in rabbits by cholesterol feeding. Currently, the aim was to verify whether combining LDE-PTX treatment with methotrexate (MTX) associated with LDE (LDE-MTX) could accelerate the atherosclerosis regression attained with single LDE-PTX treatment, after withdrawing the cholesterol feeding. Thirty-eight rabbits were fed 1% cholesterol chow for 8 weeks. Six of these rabbits were then euthanized for analyses of the aorta (controls). In the remaining rabbits, cholesterol feeding was withdrawn, and those 32 animals were allocated to 3 groups submitted to different 8-week intravenous treatments, all once/week: LDE-PTX (n = 10; 4 mg/kg), LDE-PTX + LDE-MTX (n = 11; 4 mg/kg), and LDE-alone (n = 11). Rabbits were then euthanized and aortas were excised for morphometric, immunohistochemical, and gene expression analyses. After cholesterol feeding withdrawal, in comparison with LDE-alone group, both LDE-PTX and LDE-PTX + LDE-MTX treatments had the ability to increase the regression of plaque areas: -49% in LDE-PTX and -59% for LDE-PTX + LDE-MTX. However, only LDE-PTX + LDE-MTX treatment elicited reduction in the intima area, estimated in -57%. Macrophage presence in aortic lesions was reduced 48% by LDE-PTX and 43% by LDE-PTX + LDE-MTX treatment. Matrix metalloproteinase 9 was reduced by either LDE-PTX (74%) or LDE-PTX + LDE-MTX (78%). Tumor necrosis factor α gene expression was reduced 65% by LDE-PTX and 79% by LDE-PTX + LDE-MTX. In conclusion, treatment with LDE-PTX indeed accelerated plaque reduction after cholesterol feeding; LDE-PTX + LDE-MTX further increased this effect, without any observed toxicity. These results pave the way for the use of combined chemotherapy to achieve stronger effects on aggravated, highly inflamed atherosclerotic lesions.

The Expression of Lipoprotein Receptors Is Increased in the Infarcted Area After Myocardial Infarction Induced in Rats With Cardiac Dysfunction.

Left ventricular (LV) remodeling after myocardial infarction constitutes the structural basis for ventricular dysfunction and heart failure. The characterization underlying the expression of lipoprotein receptors in cardiac dysfunction is scarcely explored. The aim of this study was to analyze the status of lipoprotein receptors on the infarcted and noninfarcted areas of LV and to verify whether nanoparticles that mimic the lipid structure of low-density lipoprotein (LDL) and have the ability to bind to LDL receptors (LDE) are taken up more avidly by the noninfarcted LV. 13 male Wistar rats with left coronary artery ligation (myocardial infarction [MI]) and 12 animals with SHAM operation (SHAM) were used in this study. 6 weeks after the procedure, the quantification of low-density lipoprotein receptor (LDLR), LDL receptor-related protein 1 (LRP1), scavenger receptor-class B type I (SR-BI) lipoprotein receptors, and PCNA proliferation marker, and tissue uptake of radioactively labeled LDE were performed. Immunohistochemistry and Western blot analysis showed that LDLR, LRP1, SR-BI, and PCNA, expression in infarcted area of MI was remarkably higher than SHAM and noninfarcted subendocardial (SEN) and interstitial (INT) areas. In addition, in SEN noninfarcted area of MI, the presence of LDLR was about threefold higher than in SHAM SEN and INT noninfarcted areas. The LDE uptake of noninfarcted LV of MI group was about 30% greater than that of SHAM group. In conclusion, these findings regarding the status of lipoprotein receptors after MI induction could help to establish mechanisms on myocardial repairing. In conclusion, infarcted rats with LV dysfunction showed increased expression of lipoprotein receptors mainly in the infarcted area.

Influence of Drugs Carried in Lipid Nanoparticles in Coronary Disease of Rabbit Transplanted Heart.

BACKGROUND: Coronary allograft vasculopathy is an inflammatory-proliferative process that compromises the long-term success of heart transplantation and currently has no effective prevention and treatment. Lipid nanoparticles, termed LDE can carry chemotherapeutic agents in the circulation and concentrates them in the heart. METHODS: Twenty-eight rabbits fed a cholesterol-rich diet and submitted to heterotopic heart transplantation were treated with cyclosporine A (10 mg/kg daily) and allocated to four groups of 7 animals treated with intravenous LDE-methotrexate (MTX; 4 mg/kg weekly), with LDE-paclitaxel (PACLI; 4 mg/kg weekly), or with LDE-PACLI (4 mg/kg weekly) and LDE-MTX (4 mg/kg weekly). A control group was treated with only weekly intravenous saline solution. Animals were euthanized 6 weeks later for morphometric, histologic, immunohistochemical, and gene expression analysis of the graft and native hearts. RESULTS: Compared with controls, grafts of rabbits treated with LDE-PACLI showed 50% reduction of coronary stenosis, and in the LDE-MTX and LDE-MTX/PACLI stenosis was approximately 18% less than in control, but this difference was not statistically significant. In the three treatment groups, macrophage infiltration was decreased. In the LDE-MTX group, gene expression of proinflammatory factors tumor necrosis factor-α, monocyte chemoattractant protein 1, interleukin 18, vascular cellular adhesion molecule 1, and matrix metalloproteinase 12 was strongly diminished, whereas expression of antiinflammatory interleukin 10 increased. In the LDE-PACLI and LDE-PACLI/MTX groups, proinflammatory and antiinflammatory gene expressions were not consistently changed by the treatments. CONCLUSIONS: LDE-PACLI promoted strong improvement of cardiac allograft vasculopathy, but the decrease in coronary stenosis by LDE-MTX and LDE-MTX/PACLI was not significant. All three treatments decreased macrophage infiltration in the graft. These results may encourage future clinical trials to test this new therapeutic approach to coronary allograft vasculopathy.

mRNA levels of low-density lipoprotein receptors are overexpressed in the foci of deep bowel endometriosis.

STUDY QUESTION: Is mRNA expression of LDL receptors altered in deep bowel endometriotic foci? SUMMARY ANSWER: mRNA expression of LDL receptors is up-regulated in deep bowel endometriotic foci of patients with endometriosis. WHAT IS KNOWN ALREADY: Several studies have demonstrated the overexpression of low-density lipoprotein receptors in various tumour cell lines and endometriosis has similar aspects to cancer, mainly concerning the pathogenesis of both diseases. This is the first study we know of to investigate lipoprotein receptors expression in deep endometriosis with bowel involvement. STUDY DESIGN, SIZE, DURATION: During 2014-2015, an exploratory case-control study was conducted with 39 patients, including 20 women with a histological diagnosis of deep endometriosis compromising the bowel and 19 women without endometriosis who underwent laparoscopic tubal ligation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Peripheral blood samples were collected on the day of surgery for lipid profile analysis, and samples of endometrial tissue and of bowel endometriotic lesions were also collected. The tissue samples were sent for histopathological analysis and for LDL-R and LRP-1 gene expression screening using quantitative real-time PCR. MAIN RESULTS AND THE ROLE OF CHANCE: Patients with deep endometriosis had lower LDL-cholesterol than patients without the disease (119 ± 23 versus 156 ± 35; P = 0.001). Gene expression analysis of LDL receptors revealed that LDL-R was more highly expressed in endometriotic lesions when compared to the endometrium of the same patient but not more than in the endometrium of women without endometriosis (0.027 ± 0.022 versus 0.012 ± 0.009 versus 0.019 ± 0.01, respectively; P < 0.001). LRP-1 was more highly expressed in endometriotic lesions, both when compared with the endometrium of the same patient and when compared with the endometrium of patients without the disease (0.307 ± 0.207 versus 0.089 ± 0.076 and versus 0.126 ± 0.072, respectively; P < 0.001). The study also showed that LDL-R gene expression in the endometrium of women with endometriosis was higher during the secretory phase of the menstrual cycle (P = 0.001). LRP-1 gene expression was increased during the secretory phase in the endometrium of women without the disease (P = 0.008). LIMITATIONS, REASONS FOR CAUTION: In the endometriotic lesions, the presence of fibrosis is substantial, restricting access to the stromal and glandular components of the lesion. Despite that, we found that LDL receptor mRNA was overexpressed. Future studies may perform laser microdissection to isolate the area of interest in the target tissue, excluding fibrosis contamination. WIDER IMPLICATIONS OF THE FINDINGS: This study supports the feasibility of LDL-R targeted therapy in the treatment of deep endometriosis. STUDY FUNDING/COMPETING INTERESTS: This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP #2011/17245-0). The authors have no conflicts of interest to declare.

Activation of EGFR signaling from pilocytic astrocytomas to glioblastomas.

INTRODUCTION: EGFR analyses allow for better correlation between genotype and phenotype in astrocytomas and represent an attractive therapeutic target. Most studies emphasize analyses of EGFR in glioblastomas (GBMs) but do not analyze all grades of astrocytomas (from pilocytic to GBM). The purpose of our study was to evaluate the status of EGFR (expression, deletion, and amplification) and EGFR protein expression in all grades of astrocytomas. PATIENTS AND METHODS: We analyzed a total of 145 surgical tumor specimens that included: 22 pilocytic astrocytomas, 22 grade II astrocytomas, 17 grade III astrocytomas and 84 GBMs. The specimens were compared to 17 non-neoplastic brain tissues obtained from epilepsy surgery. EGFR expression, EGFR amplification and EGFRvIII analyses were performed by quantitative real-time PCR, and protein expression was evaluated by immunohistochemistry. RESULTS: EGFR relative overexpression and EGFR amplification were observed, respectively, in 50% and 20% of astrocytomas, while EGFRvIII was only found in GBMs (34.5%, p=0.005). Amongst EGFR-amplified GBM cases, 59% also presented EGFRvIII (p<0.001). Cytoplasmic accumulation of EGFR protein was detected in 75% of astrocytomas, and 21% of the astrocytomas showed nuclear localization (p=0.003). CONCLUSIONS: EGFR alterations were found in all grades of astrocytomas, from pilocytic to GBMs, while EGFRvIII was exclusively found in GBMs. These findings provide important information on the mechanisms involved in the progression of astrocytomas for determining whether EGFR status can be used for effective and specific therapy.