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Raul C Maranhão,1,2 Maria C Guido,1 Aline D de Lima,1 Elaine R Tavares,1 Alyne F Marques,1 Marcelo D Tavares de Melo,3 Jose C Nicolau,3 Vera MC Salemi,3 Roberto Kalil-Filho3 1Laboratory of Metabolism and Lipids, 2Faculty of Pharmaceutical Sciences, 3Heart Failure Unit, Clinical Cardiology Division, Heart Institute (InCor), Medical School Hospital, University of São Paulo, São Paulo, Brazil 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. Keywords: lipid particle, drug delivery, adenosine, VEGF, myocardial infarction

Iara F Kretzer,1,2 Durvanei A Maria,3 Maria C Guido,1 Thaís C Contente,1 Raul C Maranhão1,2 1Laboratory of Metabolism and Lipids, Heart Institute of the Medical School Hospital, 2Department of Clinical Chemistry, Faculty of Pharmaceutical Sciences, University of São Paulo, 3Biochemistry and Biophysics Laboratories, Butantan Institute, São Paulo, Brazil 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. Keywords: lipid nanoparticles, drug delivery, statins, cancer treatment, B16F10 melanoma

Skin flaps are employed to cover cutaneous denuded surfaces, but ensuing flap necrosis often occurs. Previously, rats with myocardial infarction treated with lipid-core nanoparticles (LDE) loaded with methotrexate (MTX) improved myocardial irrigation and reduced necrosis. Here, the aim was to investigate the efficacy of LDE-MTX to preserve the viability of cutaneous flaps and its implications for surgical wound healing. Twenty-eight male rats were divided into 4 groups: (1) LDE, injected intraperitoneally with LDE only; (2) MTX (1 mg/Kg commercial MTX): (3) LDE-MTX (1 mg/Kg MTX associated with LDE), and controls without treatment. LDE, MTX or LDE-MTX were repeated after 2 days. Then, flap surgery (9x3cm) was performed on the dorsal region. Injections were continued every other day until day 7 when animals were euthanized. LDE-MTX treatment improved the total viable area of the flaps with a fourfold increase in blood flow and reduced inflammatory cell number ( p  < 0.001), accompanied by decreased protein expression of pro-inflammatory factors. SOD-1 was higher in LDE-MTX-treated rats ( p  < 0.05). In conclusion, LDE-MTX treatment achieved total viability of cutaneous flaps, with increased irrigation and diminished local inflammation. LDE-MTX may offer efficient and cost-effective prevention of cutaneous flaps and treatment for wounds from surgical procedures to be tested in future clinical studies.

Rabies, one of the most lethal global zoonoses, affects all mammals. It remains circulating worldwide in sylvatic cycles through terrestrial and airborne reservoirs, and in Brazil, bats are currently the main reservoirs and source of transmission. Wild boars, an important invasive alien species in Brazil, are a proven food source for hematophagous bats and may participate in the Brazilian sylvatic cycle of rabies. We evaluated the presence of this pathogen in hunted wild boars from the São Paulo state using histopathology, the direct fluorescent antibody test (DFA), viral isolation in cell culture (VICC), the rapid fluorescent focus inhibition test (RFFIT), and quantitative reverse transcription polymerase chain reaction (RT-qPCR). The results of histopathological, DFA, VICC, and RT-qPCR analysis were negative for all samples; seven serum samples tested positive in the RFFIT, and titers ranged from 0.13 IU/mL to 0.5 IU/mL. The presence of rabies virus-neutralizing antibodies in the studied wild boars suggests the circulation of the virus in these animals. Educative actions directed at hunters should include information on the prevention of this important zoonosis.

Calciphylaxis is a rare disease characterized by calcification of the middle layer of small arteries and arterioles, causing secondary cuta-neous ischemia. The diagnosis is clinical but may be confirmed by histological examination. The optimal treatment is not exactly known, although there is consensus that a multifactorial approach is required. This report is regarding the case of a female patient with a kidney transplant requiring peritoneal dialysis, in the late postoperative period of partial parathyroidectomy due to severe hyperparathyroidism, with refractory hypocalcemia and severe calciphylaxis, subsequently treated with intralesional sodium thiosulfate due to initial intoler-ance to intravenous thiosulfate treatment. J Drugs Dermatol. 2023;22(12):e28-e30.&nbsp; &nbsp;&nbsp; doi:10.36849/JDD.7362e.

In Marfan syndrome (MFS), dilation, dissection, and rupture of the aorta occur. Inflammation can be involved in the pathogenicity of aortic defects and can thus be a therapeutic target for MFS. Previously, we showed that the formulation of methotrexate (MTX) associated with lipid nanoparticles (LDE) has potent anti-inflammatory effects without toxicity. To investigate whether LDEMTX treatment can prevent the development of aortic lesions in the MFS murine model. MgΔloxPneo MFS ( n = 40) and wild-type (WT, n = 60) mice were allocated to 6 groups weekly injected with IP solutions of: (1) only LDE; (2) commercial MTX; (3) LDEMTX (dose = 1mg/kg) between 3rd and 6th months of life. After 12 weeks of treatments, animals were examined by echocardiography and euthanatized for morphometric and molecular studies. MFS mice treated with LDEMTX showed narrower lumens in the aortic arch, as well as in the ascending and descending aorta. LDEMTX reduced fibrosis and the number of dissections in MFS but not the number of elastic fiber disruptions. In MFS mice, LDEMTX treatment lowered protein expression of pro-inflammatory factors macrophages (CD68), T-lymphocytes (CD3), tumor necrosis factor-α (TNF-α), apoptotic factor cleaved-caspase 3, and type 1 collagen and lowered the protein expression of the transforming growth factor-β (TGF-β), extracellular signal-regulated kinases ½ (ERK1/2), and SMAD3. Protein expression of CD68 and CD3 had a positive correlation with an area of aortic lumen ( r 2 = 0.36; p < 0.001), suggesting the importance of inflammation in the causative mechanisms of aortic dilation. Enhanced adenosine availability by LDEMTX was suggested by higher aortic expression of an anti-adenosine A2a receptor (A2a) and lower adenosine deaminase expression. Commercial MTX had negligible effects. LDEMTX prevented the development of MFS-associated aortic defects and can thus be a candidate for testing in clinical studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Guido, Lopes, Albuquerque, Tavares, Jensen, Carvalho, Tavoni, Dias, Pereira, Laurindo and Maranhão.)

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-1β, 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-β, 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., (Copyright © 2019. Published by Elsevier Inc.)

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 mgΔ loxPneo 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.

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., (© 2018 AOCS.)

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.

Development of cervical cancer is associated with persistent infections by high-risk human papillomavirus (HPV). Although current HPV L1-based prophylactic vaccines prevent infection, they do not help to eliminate prevalent infections or lesions. Our aims were (i) to generate a vaccine combining prophylactic and therapeutic properties by producing chimeric capsomers after fusion of the L1 protein to different fragments of E2 from HPV 16, and (ii) to evaluate their capacity to generate an antitumoral cellular response, while conserving L1 neutralizing epitopes. Chimeric proteins were produced in Escherichia coli and purified by glutathione S-transferase (GST)-affinity chromatography. Their structure was characterized using size exclusion chromatography, sucrose gradient centrifugation, electron microscopy, and anti-L1 enzyme-linked immunosorbent assay. All chimeric proteins form capsomers and heterogeneous aggregates. One, containing part of the carboxy-terminal domain of E2 and its hinge region (L1Δ+E2H/NC, aa 206-307), conserved the neutralizing epitope H16.V5. We then evaluated the capacity of this chimeric protein to induce a cytotoxic T-cell response against HPV 16 E2. In (51)Cr release cytotoxicity assays, splenocytes from C57BL/6 immunized mice recognized and lysed TC-1/E2 cells, which express and present endogenously processed E2 peptides. Moreover, this E2-specific cytotoxic response inhibited the growth of tumors of TC-1/E2 cells in mice. Finally, we identified an epitope (aa 292-301) of E2 involved in this cytotoxic response. We conclude that the L1Δ+E2H/NC chimeric protein produced in bacteria can be an effective and economically interesting candidate for a combined prophylactic and therapeutic vaccine that could help eliminating HPV16-positive low-grade cervical lesions and persistent viral infections, thus preventing the development of lesions and, at the same time, the establishment of new infections.

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objective: To test the hypothesis that intravenous use of methotrexate associated with lipid nanoemulsions can achieve superior anti-inflammatory effects in the joints of rabbits with antigen-induced arthritis compared with commercial methotrexate., Methods: Arthritis was induced in New Zealand rabbits sensitized with methylated bovine serum albumin and subsequently intra-articularly injected with the antigen. A nanoemulsion of methotrexate labeled with 3H-cholesteryl ether (4 mg/kg methotrexate) was then intravenously injected into four rabbits to determine the plasma decaying curves and the biodistribution of the methotrexate nanoemulsion by radioactive counting. Additionally, the pharmacokinetics of the methotrexate nanoemulsion were determined by high-pressure liquid chromatography. Twenty-four hours after arthritis induction, the animals were allocated into three groups, with intravenous injection with saline solution (n=9), methotrexate nanoemulsion (0.5 µmol/kg methotrexate, n=7), or commercial methotrexate (0.5 µmol/kg, n=4). The rabbits were sacrificed 24 h afterward. Synovial fluid was then collected for protein leakage and cell content analyses and synovial membranes were collected for histopathological analysis., Results: The methotrexate nanoemulsion was taken up mainly by the liver and the uptake by arthritic joints was two-fold greater than that by control joints. The methotrexate nanoemulsion treatment reduced leukocyte influx into the synovial fluid by nearly 65%; in particular, mononuclear and polymorphonuclear cells were reduced by 47 and 72%, respectively. In contrast, cell influx was unaffected following treatment with commercial methotrexate. Protein leakage into the arthritic knees of the rabbits was also more limited following methotrexate nanoemulsion treatment than following commercial methotrexate treatment., Conclusions: The intravenous methotrexate nanoemulsion showed anti-inflammatory effects on the synovia of arthritic joints that were clearly superior to the effects of a commercial methotrexate preparation. This result is conceivably due to greater methotrexate uptake by the joints when the drug is associated with a nanoemulsion.

Sepsis impairs the autoregulation of myocardial microcirculatory blood flow, but whether this impairment is correlated with myocardial remodeling is unknown. This study investigated the role of coronary driving pressure (CDP) as a determinant of microcirculatory blood flow and myocardial fibrosis in endotoxemia and sepsis. The study is composed of two parts: a prospective experimental study and an observational clinical study. The experimental study was performed on male Wistar rats weighing 300 to 320 g. Endotoxemia was induced in rats by lipopolysaccharide (LPS) injection (10 mg·kg intraperitoneally). Hemodynamic evaluation was performed 1.5 to 24 h after LPS injection by measuring the mean arterial pressure, CDP, left ventricular end-diastolic pressure, dP/dtmax, and dP/dtmin. Microspheres were also infused into the left ventricle to measure myocardial blood flow, and myocardial tissue was histologically assessed to analyze collagen deposition. The CDP, mean arterial pressure, and myocardial blood flow were reduced by 55%, 30%, and 70%, respectively, in rats 1.5 h after LPS injection compared with phosphate buffer saline injection (P < 0.05). The CDP was significantly correlated with subendocardial blood flow (r = 0.73) and fibrosis (r = 0.8). Left ventricular function was significantly impaired in the LPS-treated rats, as demonstrated by dP/dtmax (6,155 ± 455 vs. 3,746 ± 406 mmHg·s, baseline vs. LPS; P < 0.05) and dP/dtmin (-5,858 ± 236 vs. -3,516 ± 436 mmHg·s, baseline vs. LPS; P < 0.05). The clinical study was performed on 28 patients with septic shock analyzed for CDP. The CDP data and histological slices were collected from septic patients. In addition, the clinical data demonstrated fibrosis and 45% CDP reduction in nonsurvivors compared with survivors. In conclusion, the left ventricular subendocardial blood flow was positively correlated with CDP, and higher CDP was negatively correlated with myocardial collagen deposition. Thus, early reductions in myocardial blood flow and CDP facilitate late myocardial fibrosis in rats and likely in humans.

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., (© 2013 The Authors. International Journal of Experimental Pathology © 2013 International Journal of Experimental Pathology.)

Despite significant advances in the care of critically ill patients, acute lung injury continues to be a complex problem with high mortality. The present study was designed to characterize early lipopolysaccharide (LPS)-induced pulmonary injury and small interfering RNA targeting focal adhesion kinase (FAK) as a possible therapeutic tool in the septic lung remodeling process. Male Wistar rats were assigned into endotoxemic group and control group. Total collagen deposition was performed 8, 16, and 24 h after LPS injection. Focal adhesion kinase expression, interstitial and vascular collagen deposition, and pulmonary mechanics were analyzed at 24 h. Intravenous injection of small interfering RNA targeting FAK was used to silence expression of the kinase in pulmonary tissue. Focal adhesion kinase, total collagen deposition, and pulmonary mechanics showed increased in LPS group. Types I, III, and V collagen showed increase in pulmonary parenchyma, but only type V increased in vessels 24 h after LPS injection. Focal adhesion kinase silencing prevented lung remodeling in pulmonary parenchyma at 24 h. In conclusion, LPS induced a precocious and important lung remodeling. There was fibrotic response in the lung characterized by increased amount in total and specific-type collagen. These data may explain the frequent clinical presentation during sepsis of reduced lung compliance, oxygen diffusion, and pulmonary hypertension. The fact that FAK silencing was protective against lung collagen deposition underscores the therapeutic potential of FAK targeting by small interfering RNA.

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.

Background: The activation of the members of the myocyte enhancer factor-2 family (MEF2A, B, C and D) of transcription factors promotes cardiac hypertrophy and failure. However, the role of its individual components in the pathogenesis of cardiac hypertrophy remains unclear., Methodology/principal Findings: In this study, we investigated whether MEF2C plays a role in mediating the left ventricular hypertrophy by pressure overload in mice. The knockdown of myocardial MEF2C induced by specific small interfering RNA (siRNA) has been shown to attenuate hypertrophy, interstitial fibrosis and the rise of ANP levels in aortic banded mice. We detected that the depletion of MEF2C also results in lowered levels of both PGC-1alpha and mitochondrial DNA in the overloaded left ventricle, associated with enhanced AMP:ATP ratio. Additionally, MEF2C depletion was accompanied by defective activation of S6K in response to pressure overload. Treatment with the amino acid leucine stimulated S6K and suppressed the attenuation of left ventricular hypertrophy and fibrosis in the aforementioned aortic banded mice., Conclusion/significance: These findings represent new evidences that MEF2C depletion attenuates the hypertrophic responses to mechanical stress and highlight the potential of MEF2C to be a target for new therapies to cardiac hypertrophy and failure.

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.

Unlabelled: Left ventricular hypertrophy following volume overload is regarded as an example of cardiac remodeling without increased fibrosis accumulation. However, infarction is associated with increased fibrosis within the noninfarcted, hypertrophied myocardium, particularly in the subendocardial regions. It is conceivable to suppose that, as also occurs postinfarction, low coronary driving pressure may also interfere with accumulation of myocardial fibrosis following aortocaval fistula., Purpose: To investigate the role of acute hemodynamic changes in subsequent deposition of cardiac fibrosis in response to aortocaval fistula., Method: Aortocaval fistula were created in 4 groups of Wistar rats that were followed over 4 and 8 weeks: aortocaval fistula 4 and aortocaval fistula 8 (10 rats each) and their respective controls (sham-operated controls - Sh), Sh4 and Sh8 (8 rats each). Hemodynamic measurements were performed 1 week after surgery. Hypertrophy and fibrosis were quantified by myocyte diameter and collagen volume fraction at the end of follow up., Result: Compared with Sh4 and Sh8, pulse pressure, left ventricular end-diastolic pressure, and +dP/dt were higher in aortocaval fistula 4 and aortocaval fistula 8, but -dP/dt was similar. Coronary driving pressure (mm Hg), used as an estimate of perfusion pressure, was lower in aortocaval fistula 8 (52.6 +/- 4.1) than in Sh8 (100.8 +/- 1.3), but comparable between aortocaval fistula 4 (50.0 +/- 8.9) and Sh4 (84.8 +/- 2.3). Myocyte diameter was greater in aortocaval fistula 8, whereas interstitial and subendocardial fibrosis were greater in aortocaval fistula 4 and aortocaval fistula 8. Coronary driving pressure correlated inversely and independently with subendocardial fibrosis (r(2) = .86, P <.001), whereas left ventricular systolic pressure (r(2) = 0.73, P = .004) and end-diastolic pressure (r(2) = 0.55, P = 012) correlated positively and independently with interstitial fibrosis., Conclusion: Coronary driving pressure falls and ventricular pressures increase early after aortocaval fistula and are associated with subsequent myocardial fibrosis deposition.

Human papillomavirus (HPV) DNA sequences are found in most carcinomas originating from the uterine cervix. HPV E6 and E7 oncogenes have been shown to cooperate with ras oncogenes to fully transform human epithelial cells. We investigated the effect of the Ha-ras oncogene on the transcriptional activity of HPV-18 and found that it induced the transcriptional activity of the viral promoter, whereas the normal gene had only a minimal effect. However, transfection of the normal Ha-ras gene and simultaneous inhibition of protein phosphatase sensitive to okadaic acid (OA) resulted in a cooperative transactivation of the viral promoter. When cloned upstream of a minimal promoter, the AP-1 binding sites present in the viral promoter conferred transcriptional responsiveness to Ha-ras and OA. Furthermore, HeLa cell clones permanently expressing the Ha-ras oncogene or high levels of the normal gene exhibited a twofold to threefold increase in E6*E7/E1 and E6*E7 transcripts. We propose that both Ha-ras and a protein phosphatase sensitive to OA regulate HPV oncogene expression through modulation of AP-1 activity and suggest that increased levels of E6 and E7, resulting from activated viral transcription in the presence of ras oncogenes, may in part explain the observed cooperation between these viral and cellular oncogenes in the transformation of human cells.

The physical state of the human papillomavirus (HPV) genome is usually different in malignant lesions of the skin, in which it is generally found in episomal form, and genital mucosa, in which it is frequently integrated with disruption of the E2 gene. Using chimeric or natural HPV promoters in the presence of the bovine papillomavirus type 1 E2 gene product, we observed transcription activation or repression, depending on the distance of E2-binding motifs from the start site. We found a clear difference in the positions of E2-binding motifs in cutaneous and genital HPVs that may partly explain the selective pressure for genome integration of genital HPV types in malignant lesions.