Fatima R. Freitas

The Thyroid Hormone Receptor β‐Specific Agonist GC‐1 Selectively Affects the Bone Development of Hypothyroid Rats

We investigated the effects of GC‐1, a TRβ‐selective thyromimetic, on bone development of hypothyroid rats. Whereas T3 reverted the IGF‐I deficiency and the skeletal defects caused by hypothyroidism, GC‐1 had no effect on serum IGF‐I or on IGF‐I protein expression in the epiphyseal growth plate of the femur, but induced selective effects on bone development. Our findings indicate that T3 exerts some essential effects on bone development that are mediated by TRβ1.

Double disruption of α2A- and α2C -adrenoceptors results in sympathetic hyperactivity and high-bone-mass phenotype

Evidence demonstrates that sympathetic nervous system (SNS) activation causes osteopenia via β2‐adrenoceptor (β2‐AR) signaling. Here we show that female mice with chronic sympathetic hyperactivity owing to double knockout of adrenoceptors that negatively regulate norepinephrine release, α2A‐AR and α2C‐AR (α2A/α2C‐ARKO), present an unexpected and generalized phenotype of high bone mass with decreased bone resorption and increased formation. In α2A/α2C‐ARKO versus wild‐type (WT) mice, micro–computed tomographic (µCT) analysis showed increased, better connected, and more plate‐shaped trabeculae in the femur and vertebra and increased cortical thickness in the vertebra, whereas biomechanical analysis showed increased tibial and femoral strength. Tibial mRNA expression of tartrate‐resistant acid phosphatase (TRACP) and receptor activator of NF‐κB (RANK), which are osteoclast‐related factors, was lower in knockout (KO) mice. Plasma leptin and brain mRNA levels of cocaine amphetamine–regulated transcript (CART), which are factors that centrally affect bone turnover, and serum levels of estradiol were similar between mice strains. Tibial β2‐AR mRNA expression also was similar in KO and WT littermates, whereas α2A‐, α2B‐ and α2C‐AR mRNAs were detected in the tibia of WT mice and in osteoblast‐like MC3T3‐E1 cells. By immunohistochemistry, we detected α2A‐, α2B‐, α2C‐ and β2‐ARs in osteoblasts, osteoclasts, and chondrocytes of 18.5‐day‐old mouse fetuses and 35‐day‐old mice. Finally, we showed that isolated osteoclasts in culture are responsive to the selective α2‐AR agonist clonidine and to the nonspecific α‐AR antagonist phentolamine. These findings suggest that β2‐AR is not the single adrenoceptor involved in bone turnover regulation and show that α2‐AR signaling also may mediate the SNS actions in the skeleton.

Breakdown of the blood-ocular barrier as a strategy for the systemic use of nanosystems.

Several drug delivery systems have been proposed to overcome physiological barriers, improving ocular bioavailability. Systemic routes are seldom used due to the blood-ocular barrier. Novel drug delivery systems based on nanotechnology techniques have been developed to overcome ocular physiological barriers. This non-systematic review suggests the utilization of a transitory blood-ocular breakdown to allow the access of drugs by nanotechnology drug delivery systems via the systemic route. We discuss the possible ways to cause the breakdown of the blood-ocular barrier: acute inflammation caused by intraocular surgery, induced ocular hypotony, and the use of inflammatory mediators. The suitability of use of the systemic route and its toxic effects are also discussed in this article.

reduction of atherosclerotic lesions in rabbits treated with etoposide associated with cholesterol-rich nanoemulsions

Objectives Cholesterol-rich nanoemulsions (LDE) bind to low-density lipoprotein (LDL) receptors and after injection into the bloodstream concentrate in aortas of atherosclerotic rabbits. Association of paclitaxel with LDE markedly reduces the lesions. In previous studies, treatment of refractory cancer patients with etoposide associated with LDE had been shown devoid of toxicity. In this study, the ability of etoposide to reduce lesions and inflammatory factors in atherosclerotic rabbits was investigated. Methods Eighteen New Zealand rabbits were fed a 1% cholesterol diet for 60 days. Starting from day 30, nine animals were treated with four weekly intravenous injections of etoposide oleate (6 mg/kg) associated with LDE, and nine control animals were treated with saline solution injections. Results LDE-etoposide reduced the lesion areas of cholesterol-fed animals by 85% and intima width by 50% and impaired macrophage and smooth muscle cell invasion of the intima. Treatment also markedly reduced the protein expression of lipoprotein receptors (LDL receptor, LDL-related protein-1, cluster of differentiation 36, and scavenger receptor class B member 1), inflammatory cytokines (interleukin-1β and tumor necrosis factor-α), matrix metallopeptidase-9, and cell proliferation markers (topoisomerase IIα and tubulin). Conclusion The ability of LDE-etoposide to strongly reduce the lesion area and the inflammatory process warrants the great therapeutic potential of this novel preparation to target the inflammatory-proliferative basic mechanisms of the disease.

An artificial nanoemulsion carrying paclitaxel decreases the transplant heart vascular disease: A study in a rabbit graft model

OBJECTIVE In previous studies cholesterol-rich nanoemulsions (LDE) resembling low-density lipoprotein were shown to concentrate in atherosclerotic lesions of rabbits. Lesions were pronouncedly reduced by treatment with paclitaxel associated with LDE. This study aimed to test the hypothesis of whether LDE-paclitaxel is able to concentrate in grafted hearts of rabbits and to ameliorate coronary allograft vasculopathy after the transplantation procedure. METHODS Twenty-one New Zealand rabbits fed 0.5% cholesterol were submitted to heterotopic heart transplantation at the cervical position. All rabbits undergoing transplantation were treated with cyclosporin A (10 mg · kg(-1) · d(-1) by mouth). Eleven rabbits were treated with LDE-paclitaxel (4 mg/kg body weight paclitaxel per week administered intravenously for 6 weeks), and 10 control rabbits were treated with 3 mL/wk intravenous saline. Four control animals were injected with LDE labeled with [(14)C]-cholesteryl oleate ether to determine tissue uptake. RESULTS Radioactive LDE uptake by grafts was 4-fold that of native hearts. In both groups the coronary arteries of native hearts showed no stenosis, but treatment with LDE-paclitaxel reduced the degree of stenosis in grafted hearts by 50%. The arterial luminal area in grafts of the treated group was 3-fold larger than in control animals. LDE-paclitaxel treatment resulted in a 7-fold reduction of macrophage infiltration. In grafted hearts LDE-paclitaxel treatment reduced the width of the intimal layer and inhibited the destruction of the medial layer. No toxicity was observed in rabbits receiving LDE-paclitaxel treatment. CONCLUSIONS LDE-paclitaxel improved posttransplantation injury to the grafted heart. The novel therapeutic approach for heart transplantation management validated here is thus a promising strategy to be explored in future clinical studies.

HDL metabolism and atheroprotection: predictive value of lipid transfers.

High-density lipoprotein (HDL) intravascular metabolism is complex, and the major HDL functions are esterification of cholesterol and reverse cholesterol transport, in which cholesterol from cells is excreted in bile. HDL has also several other antiatherogenic functions: antioxidative, vasodilatatory, anti-inflammatory, antiapoptotic, anti thrombotic, and anti infectious. Low HDL cholesterol is a major risk factor for cardiovas cular disease (CVD) and high HDL cholesterol may favor the many protective abilities of HDL. However, aspects of HDL function can be independent of HDL cholesterol levels, including the efflux of cholesterol from cells to HDL. Some populations show low incidence of CVD despite their low HDL cholesterol. Lipid exchange between HDL and other lipoproteins and cells is fundamental in HDL metabolism and reverse cholesterol transport. By determining HDL composition, lipid transfers can also affect HDL functions that depend on proteins that anchor on HDL particle surface. Cholesteryl ester protein (CETP) and phospholipid transfer protein facilitate lipid transfers among lipoprotein classes, but the role of the lipid transfers and transfer proteins in atherosclerosis and other diseases is not well established. CETP has become a therapeutic target because CETP inhibitors increase HDL cholesterol, but to date the clinical trials failed to show benefits for the patients. Recently, we introduced a practical in vitro assay to evaluate the simultaneous transfer from a donor nanoemulsion to HDL of unesterified and esterified cho lesterol, phospholipids, and triglycerides. Groups of subjects at different clinical, nutritional, and training conditions were tested, and among other findings, lower transfer ratios of unesterified cholesterol to HDL were predictors of the presence of CVD.

Lipid transfers to HDL are predictors of precocious clinical coronary heart disease

BACKGROUND High-density-lipoprotein (HDL) has several antiatherogenic properties and, although the concentration of HDL-cholesterol negatively correlates with incidence of coronary artery disease (CAD), this is not sufficient to evaluate the overall HDL protective role. The aim was to investigate whether precocious CAD patients show abnormalities in lipid transfers to HDL, a fundamental step in HDL metabolism and function. METHODS Thirty normocholesterolemic CAD patients aged <50 y and 30 controls paired for sex, age and B.M.I. were studied. Fasting blood samples were collected for the in vitro lipid transfer assay and plasma lipid determination. A donor nanoemulsion labeled with radioactive free-cholesterol, cholesteryl esters, phospholipids and triglycerides was incubated with whole plasma and after chemical precipitation of non-HDL fractions, supernatant was counted for radioactivity in HDL. RESULTS LDL and HDL-cholesterol and triglycerides were equal in both groups. Transfers of free-cholesterol (3.8±1.2%vs 7.0±3.3%,p<0.0001) and triglycerides (3.7±1.7%vs 4.9±1.9%, p=0.0125) were diminished in CAD patients whereas cholesteryl ester transfer increased (6.5±1.9%vs 4.8±1.8%, p=0.0008); phospholipid transfer was equal (17.8±3.5% vs 19.5±3.9%). CONCLUSION Alterations in the transfer of lipids to HDL may constitute a new marker for precocious CAD and relation of this metabolic alteration with HDL antiatherogenic function should be investigated in future studies.

Alveolar osseous defect in rat for cell therapy: preliminary report

PURPOSE To study were to reproduce an alveolar bone defect model in Wistar rats to be used for testing the efficacy of stem cell therapies. Additionally, we also aimed to determine the osteogenesis process of this osseous defect in the 1 month period post-surgery. METHODS The animals were randomly divided into two groups of 7 animals each. A gingivobuccal incision was made, and a bone defect of 28 mm(2) of area was performed in the alveolar region. Animals were killed at 2 weeks after surgery (n=7) and 4 weeks after surgery (n=7). RESULTS The average area of the alveolar defect at time point of 2 weeks was 22.27 +/- 1.31 mm(2) and the average area of alveolar defect at time point of 4 weeks was 9.03 +/- 1.17 mm(2). The average amount of bone formation at time point of 2 weeks was 5.73 +/- 1.31 mm(2) and the average amount of bone formation at time point of 4 weeks was 19 +/- 1.17 mm(2). Statistically significant differences between the amount of bone formation at 2 weeks and 4 weeks after surgery were seen (p=0.003). CONCLUSION The highest rate of ossification occurred mostly from 2 to 4 weeks after surgery. This observation suggests that 4 weeks after the bone defect creation should be a satisfactory timing to assess the potential of bone inductive stem cells to accelerate bone regeneration in Wistar rats.

Paclitaxel Associated With Lipid Nanoparticles as a New Antiscarring Agent in Experimental Glaucoma Surgery.

PURPOSE To investigate the effects of paclitaxel associated with lipid nanoemulsions (LDE-PTX) on postoperative scarring in rabbits undergoing trabeculectomy. METHODS Thirty-four rabbits that underwent trabeculectomy were allocated to four groups: LDE-PTX/SC (n = 9), treated with LDE-PTX (1.5 mg, intraoperative subconjunctival injection); LDE-PTX/IV (n = 9), treated with LDE-PTX (4 mg/kg per day intravenously) at the end of the surgery and once per week for 3 weeks; MMC (n = 9), treated with intraoperative 0.4 mg/mL mitomycin-C for 3 minutes; and control group (CTL, n = 7), without treatment. Bleb characteristics and IOP were evaluated over 4 weeks. Animals were killed on day 28. Histologic analyses were performed to assess the amount of scarring and toxicity to the conjunctiva and ciliary body. RESULTS Groups were similar with respect to IOP and anterior chamber depth during the 28-day observation period. The LDE-PTX/SC, LDE-PTX/IV, and MMC groups showed greater bleb height than CTL on days 14 and 21 (P < 0.001). The LDE-PTX/SC, LDE-PTX/IV, and MMC groups showed longer bleb survival time than CTL (P < 0.001). The LDE-PTX/SC, LDE-PTX/IV, and MMC groups were equally effective in reducing fibrosis (P < 0.001), number of blood vessels (P < 0.001), and chronic inflammatory cells (P < 0.01) at the surgical site. However, LDE-PTX/SC and LDE-PTX/IV treatments had lower conjunctival (P < 0.001) and ciliary body toxicity (P < 0.01), compared with MMC. CONCLUSIONS The LDE-PTX/SC was effective in reducing the scarring process following trabeculectomy to the same extent as MMC, but with considerably less toxicity to the conjunctiva and ciliary body. The LDE-PTX/IV was somewhat less effective than LDE-PTX/SC or MMC, but could have potential as a postoperative adjuvant treatment. Therefore, the LDE-PTX preparation in both administration routes may offer promising options for wound-healing modulation in the surgical treatment of glaucoma.

Altered Expression in Patients with Heart Failure of Circulating MicroRNAs Related to Lipoprotein Metabolism

Objective: This study aimed to investigate, for the first time, the expression of circulating miRNAs (microRNAs) related to lipoprotein metabolism in patients with HF (heart failure). Methods: Twenty patients with HF and 10 controls without HF were included. BNP (brain natriuretic peptide), a marker of HF severity, plasma lipid parameters and the expression of circulating miRNAs were determined. Key findings: Total, LDL-, non-HDLand HDL-cholesterol, triglycerides, and apo A-I did not differ between both groups, but apo B was lower in the HF group compared to controls (p = 0.007). In respect to miRNAs, miR-33a, miR-144, miR-125, miR-30c, miR-122, miR-26a, miR-185, miR-758 and miR-106b were higher, from tento 25-fold, and miR-10b was lower about 4-fold, in HF group compared to controls. In HF patients a negative correlation between miR-26a and BNP, the marker of disease severity, was found (r = -0.552; p = 0.041). Conclusions: Plasma levels of miRNAs involved in HDL and LDL metabolism regulation were strikingly changed in HF patients. The negative correlation between miR-26a and BNP values may suggest the possibility of the rise of a novel biomarker or therapeutic target in HF.