Lipid core nanoparticles as vehicle for docetaxel reduces atherosclerotic lesion, inflammation, cell death and proliferation in an atherosclerosis rabbit model.

Chemotherapeutic agents used in cancer treatment associated to nanoparticles (LDE) that mimic the composition of low-density lipoprotein and buffer their toxicity can have strong anti-atherosclerosis action, as we showed in cholesterol-fed rabbits. Here, a novel preparation of docetaxel (DTX) carried in LDE was evaluated. Eighteen rabbits were fed 1% cholesterol during 8 weeks. After the first 4 weeks, 9 animals were treated for 4 weeks with intravenous LDE-DTX (1 mg/kg/week) and 9 with LDE only (controls) once a week for 4 weeks. Animals were then euthanized and the aortas were analyzed for morphometry, immunohistochemistry and Western blot. LDE-DTX treated group showed 80% reduction of atheroma area compared to controls. LDE-DTX treatment reduced in 60% the protein expression of macrophage marker CD68 and of MCP-1 in 80%. LDE-DTX pronouncedly lowered expression of pro-inflammatory markers NF-κB, TNF-α, IL-1ß, IL-6 and von Willebrand factor and elicited 40% reduction in cell proliferation marker PCNA. The presence of smooth muscle cells in the intima was 85% smaller than in controls. Pro-apoptotic caspase 3, caspase 9, Bax, and anti-apoptotic Bcl-2 all were reduced by LDE-DTX. Protein expression of MMP-2 and MMP-9, TGF-ß, and collagen 1 and 3 were also markedly lowered by the LDE-DTX treatment. Animals showed no hematological, hepatic or renal toxicity consequent to LDE-DTX treatment. In conclusion, LDE-DTX showed a wide array of strong effects on pro-inflammatory and proliferation-promoting factors that drive the lesion development. These findings and the lack of observable toxicity indicate that LDE-DTX can be a candidate for future clinical trials.

Effect of the Antioxidant Lipoic Acid in Aortic Phenotype in a Marfan Syndrome Mouse Model.

Marfan syndrome (MFS) cardiovascular manifestations such as aortic aneurysms and cardiomyopathy carry substantial morbidity/mortality. We investigated the effects of lipoic acid, an antioxidant, on ROS production and aortic remodeling in a MFS mgΔloxPneo mouse model. MFS and WT (wild-type) 1-month-old mice were allocated to 3 groups: untreated, treated with losartan, and treated with lipoic acid. At 6 months old, echocardiography, ROS production, and morphological analysis of aortas were performed. Aortic ROS generation in 6-month-old MFS animals was higher at advanced stages of disease in MFS. An unprecedented finding in MFS mice analyzed by OCT was the occurrence of focal inhomogeneous regions in the aortic arch, either collagen-rich extremely thickened or collagen-poor hypotrophic regions. MFS animals treated with lipoic acid showed markedly reduced ROS production and lower ERK1/2 phosphorylation; meanwhile, aortic dilation and elastic fiber breakdown were unaltered. Of note, lipoic acid treatment associated with the absence of focal inhomogeneous regions in MFS animals. Losartan reduced aortic dilation and elastic fiber breakdown despite no change in ROS generation. In conclusion, oxidant generation by itself seems neutral with respect to aneurysm progression in MFS; however, lipoic acid-mediated reduction of inhomogeneous regions may potentially associate with less anisotropy and reduced chance of dissection/rupture.

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.

The Effects of Diabetes Induction on the Rat Heart: Differences in Oxidative Stress, Inflammatory Cells, and Fibrosis between Subendocardial and Interstitial Myocardial Areas.

Diabetic cardiomyopathy (DCM) is characterized by cardiac remodeling and impaired diastolic function that may lead to heart failure. The aim of this study was to evaluate oxidative stress, inflammatory cells, and fibrosis in both subendocardial (SEN) and interstitial (INT) areas of the myocardium. Male Wistar rats were allocated to 2 groups of 9 animals, a control (CT) group and streptozotocin-induced diabetes (DM). After 8 weeks, echocardiography morphometry, protein expression, and confocal microscopy in SEN and INT areas of the left ventricle (LV) were performed. The echocardiographic analysis showed that diabetes induction leads to cardiac dilation, hypertrophy, and LV diastolic dysfunction. As compared to CT, the induction of diabetes increased inflammatory cells and fibrosis in both SEN and INT areas of DM myocardium and increased ROS generation only in SEN. Comparing the SEN and INT areas in the DM group, inflammatory cells and fibrosis in SEN were greater than in INT. In conclusion, diabetic myocardium SEN area, wherein oxidative stress was more pronounced, is more susceptible to cardiac dysfunction than INT area. This finding can be important for the understanding of the heart remodeling process occurring in DCM and perhaps to engender targeted therapies to attenuate or revert DCM-related diastolic dysfunction.

Methotrexate carried in lipid core nanoparticles reduces myocardial infarction size and improves cardiac function in rats.

PURPOSE: Acute myocardial infarction (MI) is accompanied by myocardial inflammation, fibrosis, and ventricular remodeling that, when excessive or not properly regulated, may lead to heart failure. Previously, lipid core nanoparticles (LDE) used as carriers of the anti-inflammatory drug methotrexate (MTX) produced an 80-fold increase in the cell uptake of MTX. LDE-MTX treatment reduced vessel inflammation and atheromatous lesions induced in rabbits by cholesterol feeding. The aim of the study was to investigate the effects of LDE-MTX on rats with MI, compared with commercial MTX treatment. MATERIALS AND METHODS: Thirty-eight Wistar rats underwent left coronary artery ligation and were treated with LDE-MTX, or with MTX (1 mg/kg intraperitoneally, once/week, starting 24 hours after surgery) or with LDE without drug (MI-controls). A sham-surgery group (n=12) was also included. Echocardiography was performed 24 hours and 6 weeks after surgery. The animals were euthanized and their hearts were analyzed for morphometry, protein expression, and confocal microscopy. RESULTS: LDE-MTX treatment achieved a 40% improvement in left ventricular (LV) systolic function and reduced cardiac dilation and LV mass, as shown by echocardiography. LDE-MTX reduced the infarction size, myocyte hypertrophy and necrosis, number of inflammatory cells, and myocardial fibrosis, as shown by morphometric analysis. LDE-MTX increased antioxidant enzymes; decreased apoptosis, macrophages, reactive oxygen species production; and tissue hypoxia in non-infarcted myocardium. LDE-MTX increased adenosine bioavailability in the LV by increasing adenosine receptors and modulating adenosine catabolic enzymes. LDE-MTX increased the expression of myocardial vascular endothelium growth factor (VEGF) associated with adenosine release; this correlated not only with an increase in angiogenesis, but also with other parameters improved by LDE-MTX, suggesting that VEGF increase played an important role in the beneficial effects of LDE-MTX. Overall effects of commercial MTX were minor, and did not improve LV function or infarction size. Both treatments did not induce any toxicity. CONCLUSION: The remarkable improvement in heart function and reduction in infarction size achieved by LDE-MTX supports future clinical trials.

Simvastatin increases the antineoplastic actions of paclitaxel carried in lipid nanoemulsions in melanoma-bearing mice.

PURPOSE: Lipid nanoemulsions (LDEs) that bind to low-density lipoprotein (LDL) receptors used as carriers of paclitaxel (PTX) can decrease toxicity and increase PTX antitumoral action. The administration of simvastatin (Simva), which lowers LDL-cholesterol, was tested as an adjuvant to commercial PTX and to PTX associated with LDE (LDE-PTX). MATERIALS AND METHODS: B16F10 melanoma-bearing mice were treated with saline solution or LDE (controls), Simva, PTX, PTX and Simva, LDE-PTX, and LDE-PTX and Simva: PTX dose 17.5 µmol/kg (three intraperitoneal injections, 3 alternate days): Simva 50 mg/kg/day by gavage, 9 consecutive days. RESULTS: Compared with saline controls, 95% tumor-growth inhibition was achieved by LDE-PTX and Simva, 61% by LDE-PTX, 44% by PTX and Simva, and 43% by PTX. Simva alone had no effect. Metastasis developed in only 37% of the LDE-PTX and Simva, 60% in LDE-PTX, and 90% in PTX and Simva groups. Survival rates were higher in LDE-PTX and Simva and in LDE-PTX groups. The LDE-PTX and Simva group presented tumors with reduced cellular density and increased collagen fibers I and III. Tumors from all groups showed reduction in immunohistochemical expression of ICAM, MCP-1, and MMP-9; LDE-PTX and Simva presented the lowest MMP-9 expression. Expression of p21 was increased in the Simva, LDE-PTX, and LDE-PTX and Simva groups. In the Simva and LDE-PTX and Simva groups, expression of cyclin D1, a proliferation and survival promoter of tumor cells, was decreased. Therapy with LDE-PTX and Simva showed negligible toxicity compared with PTX and Simva, which resulted in weight loss and myelosuppression. CONCLUSION: Simva increased the antitumor activity of PTX carried in LDE but not of PTX commercial presentation, possibly because statins increase the expression of LDL receptors that internalize LDE-PTX.

Fibrillin-1 mgΔ(lpn) Marfan syndrome mutation associates with preserved proteostasis and bypass of a protein disulfide isomerase-dependent quality checkpoint.

Fibrillin-1 mutations promote Marfan syndrome (MFS) via complex yet unclear pathways. The roles of endoplasmic reticulum (ER) and the major ER redox chaperone protein disulfide isomerase-A1 in the processing of normal and mutated fibrillin-1 and ensuing protein secretion and/or intracellular retention are unclear. Our results in mouse embryonic fibroblasts bearing the exon-skipping mgΔ(lox-P-neo) (mgΔ(lpn)) mutation, which associates in vivo with MFS and in vitro with disrupted microfibrils, indicate a preserved ER-dependent proteostasis or redox homeostasis. Rather, mutated fibrillin-1 is secreted normally through Golgi-dependent pathways and is not intracellularly retained. Similar results occurred for the C1039G point mutation. In parallel, we provide evidence that PDIA1 physically interacts with fibrillin-1 in the ER. Moreover, siRNA against PDIA1 augmented fibrillin-1 secretion rates in wild-type cells. However, fibrillin-1 with the mgΔ(lpn) mutation bypassed PDI checkpoint delay, while the C1039G mutation did not. This heretofore undisclosed PDIA1-mediated mechanism may be important to control the extracellular availability of function-competent fibrillin-1, an important determinant of disease phenotype. Moreover, our results may reveal a novel, holdase-like, PDI function associated with ER protein quality control.

Acute aortocaval fistula: role of low perfusion pressure and subendocardial remodeling on left ventricular function.

The experimental model of aortocaval fistula is a useful model of cardiac hypertrophy in response to volume overload. In the present study it has been used to investigate the pathologic subendocardial remodeling associated with the development of heart failure during the early phases (day 1, 3, and 7) following volume overload. Compared with sham treated rats, aortocaval fistula rats showed lower systemic blood pressure and higher left ventricular end-diastolic pressure This resulted in lower coronary driving pressure and left ventricular systolic and diastolic dysfunction. Signs of myocyte necrosis, leukocyte cell infiltration, fibroplasia and collagen deposition appeared sequentially in the subendocardium where remodeling was more prominent than in the non-subendocardium. Accordingly, increased levels of TNF-alpha, IL-1 beta, and IL-6, and enhanced MMP-2 activity were all found in the subendocardium of rats with coronary driving pressure ≤ 60 mmHg. The coronary driving pressure was inversely correlated with MMP-2 activity in subendocardium in all time-points studied, and blood flow in this region showed positive correlation with systolic and diastolic function at day 7. Thus the predominant subendocardial remodeling that occurs in response to low myocardial perfusion pressure during the acute phases of aortocaval fistula contributes to early left ventricular dysfunction.

Small interfering RNA targeting focal adhesion kinase prevents cardiac dysfunction in endotoxemia.

Sepsis and septic shock are associated with cardiac depression. Cardiovascular instability is a major cause of death in patients with sepsis. Focal adhesion kinase (FAK) is a potential mediator of cardiomyocyte responses to oxidative and mechanical stress. Myocardial collagen deposition can affect cardiac compliance and contractility. The aim of the present study was to determine whether the silencing of FAK is protective against endotoxemia-induced alterations of cardiac structure and function. In male Wistar rats, endotoxemia was induced by intraperitoneal injection of lipopolysaccharide (10 mg/kg). Cardiac morphometry and function were studied in vivo by left ventricular catheterization and histology. Intravenous injection of small interfering RNA targeting FAK was used to silence myocardial expression of the kinase. The hearts of lipopolysaccharide-injected rats showed collagen deposition, increased matrix metalloproteinase 2 activity, and myocyte hypertrophy, as well as reduced 24-h +dP/dt and -dP/dt, together with hypotension, increased left ventricular end-diastolic pressure, and elevated levels of FAK (phosphorylated and unphosphorylated). Focal adhesion kinase silencing reduced the expression and activation of the kinase in cardiac tissue, as well as protecting against the increased collagen deposition, greater matrix metalloproteinase 2 activity, and reduced cardiac contractility that occur during endotoxemia. In conclusion, FAK is activated in endotoxemia, playing a role in cardiac remodeling and in the impairment of cardiac function. This kinase represents a potential therapeutic target for the protection of cardiac function in patients with sepsis.

Low coronary driving pressure is associated with subendocardial remodelling and left ventricular dysfunction in aortocaval fistula.

1. The role of haemodynamic changes in left ventricular remodelling has been poorly investigated, especially in the context of volume overload cardiac hypertrophy. Low diastolic blood pressure and high left ventricular filling pressure are expected to affect coronary driving pressure negatively and thereby put in jeopardy subendocardial perfusion in particular. The consequences to global left ventricular remodelling remain undetermined. The aim of the present study was to investigate the role of coronary driving pressure in the development of subendocardial remodelling and the conceivable effects on cardiac function, using a rat model of aortocaval fistula. 2. Wistar rats, weighing 330-350 g, were submitted to aortocaval fistula (ACF group) or sham (control group) operations. Two haemodynamic measurements were determined following surgery, the initial measurement at week 1 and the final measurement at week 8. Cytokine expression, myeloperoxidase (MPO) activity, metalloproteinase expression and activity and fibrosis were assessed in two distinct left ventricular myocardial layers: the subendocardium (SE) and the non-subendocardium (non-SE). 3. The ACF group showed lower initial and final coronary driving pressure and lower final +dP/dt and -dP/dt compared with the control group. Multivariate analyses disclosed initial coronary driving pressure as the only haemodynamic parameter independently associated with SE fibrosis (R(2) = 0.76; P < 0.0001) and with +dP/dt (R(2) = 0.55; P = 0.0004) and -dP/dt (R(2) = 0.91; P < 0.0001). Matrix metalloproteinase (MMP)-2 expression and activity predominated in the SE of ACF animals, particularly in those with low coronary driving pressure. Increased levels of interleukin (IL)-6 and IL-1beta also predominated in the SE of the ACF group. Otherwise, MPO activity and levels of tumour necrosis factor-alpha and IL-10 were similar in both groups. Final coronary driving pressure correlated with both the expression and activity of MMP-2. 4. Low coronary driving pressure early in the course of ACF determines SE damage and, by this mechanism, interferes negatively in left ventricular function.