Sílvio Roberto Consonni

Hydrocephalus and arthrogryposis in an immunocompetent mouse model of ZIKA teratogeny: a developmental study

The teratogenic mechanisms triggered by ZIKV are still obscure due to the lack of a suitable animal model. Here we present a mouse model of developmental disruption induced by ZIKV hematogenic infection. The model utilizes immunocompetent animals from wild-type FVB/NJ and C57BL/6J strains, providing a better analogy to the human condition than approaches involving immunodeficient, genetically modified animals, or direct ZIKV injection into the brain. When injected via the jugular vein into the blood of pregnant females harboring conceptuses from early gastrulation to organogenesis stages, akin to the human second and fifth week of pregnancy, ZIKV infects maternal tissues, placentas and embryos/fetuses. Early exposure to ZIKV at developmental day 5 (second week in humans) produced complex manifestations of anterior and posterior dysraphia and hydrocephalus, as well as severe malformations and delayed development in 10.5 days post-coitum (dpc) embryos. Exposure to the virus at 7.5–9.5 dpc induces intra-amniotic hemorrhage, widespread edema, and vascular rarefaction, often prominent in the cephalic region. At these stages, most affected embryos/fetuses displayed gross malformations and/or intrauterine growth restriction (IUGR), rather than isolated microcephaly. Disrupted conceptuses failed to achieve normal developmental landmarks and died in utero. Importantly, this is the only model so far to display dysraphia and hydrocephalus, the harbinger of microcephaly in humans, as well as arthrogryposis, a set of abnormal joint postures observed in the human setting. Late exposure to ZIKV at 12.5 dpc failed to produce noticeable malformations. We have thus characterized a developmental window of opportunity for ZIKV-induced teratogenesis encompassing early gastrulation, neurulation and early organogenesis stages. This should not, however, be interpreted as evidence for any safe developmental windows for ZIKV exposure. Late developmental abnormalities correlated with damage to the placenta, particularly to the labyrinthine layer, suggesting that circulatory changes are integral to the altered phenotypes.

Elastic Fiber Assembly in the Adult Mouse Pubic Symphysis During Pregnancy and Postpartum

ABSTRACT Impairment of pelvic organ support has been described in mice with genetic modifications of the proteins involved in elastogenesis, such as lysyl oxidase-like 1 (LOXL1) and fibulin 5. During pregnancy, elastic fiber-enriched pelvic tissues are modified to allow safe delivery. In addition, the mouse pubic symphysis is remodeled in a hormone-controlled process that entails the modification of the fibrocartilage into an interpubic ligament (IpL) and the relaxation of this ligament. After first parturition, recovery occurs to ensure pelvic tissue homeostasis. Because ligaments are the main supports of the pelvic organs, this study aimed to evaluate elastogenesis in the IpL during mouse pregnancy and postpartum. Accordingly, virgin, pregnant, and postpartum C57BL/6 mice were studied using light, confocal, and transmission electron microscopy as well as Western blots and real-time PCR. Female mice exhibited the separation of the pubic bones and the formation, relaxation, and postpartum recovery of the IpL. By the time the IpL was formed, the elastic fibers had increased in profile length and diameter, and they consisted of small conglomerates of amorphous material distributed among the bundles of microfibrils. Our analyses also indicated that elastin/tropoelastin, fibrillin 1, LOXL1/Loxl1, and fibulin 5 were spatially and temporally regulated, suggesting that these molecules may contribute to the synthesis of new elastic fibers during IpL development. Overall, this work revealed that adult elastogenesis may be important to assure the elasticity of the pelvic girdle during preparation for parturition and postpartum recovery. This finding may contribute to our understanding of pathological processes involving elastogenesis in the reproductive tract.

αB-crystallin interacts with and prevents stress-activated proteolysis of focal adhesion kinase by calpain in cardiomyocytes.

Focal adhesion kinase (FAK) contributes to cellular homeostasis under stress conditions. Here we show that αB-crystallin interacts with and confers protection to FAK against calpain-mediated proteolysis in cardiomyocytes. A hydrophobic patch mapped between helices 1 and 4 of the FAK FAT domain was found to bind to the β4-β8 groove of αB-crystallin. Such an interaction requires FAK tyrosine 925 and is enhanced following its phosphorylation by Src, which occurs upon FAK stimulation. αB-crystallin silencing results in calpain-dependent FAK depletion and in the increased apoptosis of cardiomyocytes in response to mechanical stress. FAK overexpression protects cardiomyocytes depleted of αB-crystallin against the stretch-induced apoptosis. Consistently, load-induced apoptosis is blunted in the hearts from cardiac-specific FAK transgenic mice transiently depleted of αB-crystallin by RNA interference. These studies define a role for αB-crystallin in controlling FAK function and cardiomyocyte survival through the prevention of calpain-mediated degradation of FAK.

Morphometric-stereological and functional epididymal alterations and a decrease in fertility in rats treated with finasteride and after a 30-day post-treatment recovery period

OBJECTIVE To evaluate morphometric-stereological changes in the epididymal caput, sperm quality, and fertility parameters in rats treated with finasteride and after a 30-day post-treatment recovery period. DESIGN Experimental study in a research laboratory. SETTING Reproductive biology research laboratory. ANIMAL(S) Male and female Sprague Dawley rats. INTERVENTION(S) Treatment with finasteride (5 mg/kg/day) for 56 days followed by 30 days without treatment. MAIN OUTCOME MEASURE(S) Serum hormone analyses, morphometric-stereological and ultrastructural evaluation of the epididymal caput, sperm transit time, natural mating, in utero insemination, sperm membrane integrity, and fertility parameters. RESULT(S) Serum dihydrotestosterone levels in the finasteride group decreased by ~40% compared with that of control rats. Ultrastructural analysis revealed significant reductions in several morphometric-stereological parameters of the epididymal caput. All parameters recovered significantly in the post-treatment period. There was no alteration in daily sperm production in the finasteride group. However, significant reductions in sperm transit time, motility, sperm membrane integrity, and fertility parameters were observed in rats treated with finasteride. CONCLUSION(S) Treatment with finasteride caused morphometric-stereological and functional changes in the epididymis and in sperm function that led to a reduction in fertility parameters. A 30-day post-treatment recovery period was insufficient to restore normal sperm motility, sperm transit time, and some fertility parameters.

Defective Autophagy in Diabetic Retinopathy

PURPOSE Müller cells (MCs) are a major source of VEGF in diabetic retinopathy (DR). Vascular endothelial growth factor is the main therapeutic target for treating DR. This study aimed to investigate whether autophagy is involved in MC response under high glucose (HG). METHODS Rat retinal Müller cells (rMCs) were exposed to normal or high glucose in and out of presence of pharmacologic inhibitors and activators and small interfering RNA (siRNA) for p62/SQTSM1 for 24 hours. RESULTS High glucose induces increase of early and late autophagic markers, accumulation of p62/SQTSM1 and endoplasmic reticulum (ER) stress response associated with apoptosis augmentation (P < 0.01). The inhibition of autophagy in HG leads to higher rMC apoptotic rate (P < 0.001). By silencing the p62/SQTSM1, ER stress is ameliorated (p<0.0001), preventing apoptosis. Retinal MCs in HG treated with rapamycin (mTOR inhibitor) show autophagy machinery activation and reestablishment of cargo degradation, protecting cells from apoptosis (P < 0.0001). Rapamycin improves lysosomal proteolytic activity by improving cathepsin L activity restoring autophagic cargo degradation, and preventing increased VEGF release (P < 0.0001). In experimental model of diabetes, Beclin-1 and p62/SQTSM-1 were found to be marked increased in retinas from diabetic Wystar Kyoto rats compared with control group (P < 0.003) with reduction of cathepsin L activity. CONCLUSIONS High glucose upregulates autophagy but accumulates p62/SQTSM1 cargo due to lysosomal dysfunction, leading to massive VEGF release and cell death of rMCs. Lysosomal impairment and autophagic dysfunction are early events present in the pathogenesis of diabetic retinopathy (DR). This might be valuable for developing a novel therapeutic strategy to treat DR.

Genetics and metabolic deregulation following cancer initiation: A world to explore.

Cancer is a group of highly complex and heterogeneous diseases with several causes. According to the stochastic model, cancer initiates from mutation in somatic cells, leading to genomic instability and cell transformation. This canonical pathway of carcinogenesis is related to the discovery of important mechanisms that regulate cancer initiation. However, there are few studies describing genetic and metabolic alterations that deregulate transformed cells, resulting in epithelial-mesenchymal transition (EMT) and its most dramatic consequence, the metastasis. This review summarizes the main genetics and metabolic changes induced by reactive oxygen species (ROS) that lead to EMT.

High iNOS mRNA and protein localization during late pregnancy suggest a role for nitric oxide in mouse pubic symphysis relaxation

Remodeling and relaxation of the mouse pubic symphysis (PS) are central events in parturition. The mouse PS remodels in a hormone‐controlled process that involves the modification of the fibrocartilage into an interpubic ligament (IpL), followed by its relaxation prior to parturition. It is recognized that nitric oxide synthase (NOS) and consequently nitric oxide (NO) generation play important roles in extracellular matrix modification, and may promote cytoskeleton changes that contribute to the remodeling of connective tissue, which precedes the onset of labor. To our knowledge, no studies thus far have investigated inducible nitric oxide synthase (iNOS) expression, protein localization, and NO generation in the mouse PS during pregnancy. In this work, we used a combination of the immunolocalization of iNOS, its relative mRNA expression, and NO production to examine the possible involvement of iNOS in remodeling and relaxation of the mouse IpL during late pregnancy. The presence of iNOS was observed in chondrocytes and fibroblast‐like cells in the interpubic tissues. In addition, iNOS mRNA and NO production were higher during preterm labor on Day 19 of pregnancy (D19) than NO production on D18 or in virgin groups. The significant increase in iNOS mRNA expression and NO generation from the partially relaxed IpL at D18 to the completely relaxed IpL at D19 may indicate that NO plays an important role in late pregnancy during relaxation of the mouse IpL. Mol. Reprod. Dev. 79: 272–282, 2012.

Bovine papillomavirus productive infection in cell cultures: First evidences

Bovine papillomavirus (BPV) is the etiological agent of bovine papillomatosis (BP), infectious disease, characterized by the presence of multiples papillomas that can regress spontaneously or progress to malignances. Although recognized as mutagen, BPV action following cancer initiation remains few explored, since studies about cancer progression and metastasis are based on cell cultures. The lack of attention to in vitro models is a reflection of the papillomavirus replication paradigm, which is dependent of epithelium cell differentiation. Since 2008, we have explored the potential of cell lines derived from BPV-infected neoplasms as model to study the oncogenic process. In this study, we described BPV productive infection in cell lines derived from cutaneous papilloma, fibropapilloma and esophageal carcinoma (EC) in which BPV DNA sequences were previously detected by PCR. Considering that the immunodetection of L1 capsid protein is the main evidence of productive infection, we analyzed the expression of this protein by immunofluorescence and flow cytometry. Results showed the immunodetection of L1 protein in cell lines derived from cutaneous papilloma, fibropapilloma and EC, but not in cells derived from BPV-free normal skin. We also observed the presence of spherical and electron-dense particles, with 41.02-61.94 nm diameter in cytoplasmic vesicles of cells in the sixth passage of cutaneous papilloma, fibropapilloma and EC, being compatible with the expected BPV morphology. Cells derived from BPV-free normal skin, in turn, showed membranous particles up to 75.00 nm not compatible with BPV morphology. These results suggest the BPV productive infection in cells lines derived from BPV-infected neoplasm, reinforcing that these cells are useful models to study the viral biology and pathogenesis. Correspondence to: Rita de Cassia Stocco, Genetics Laboratory, Butantan Institute, São Paulo, 05503-900, Brazil, Tel: +55 11 2627-9701; e-mail: rita. stocco@butantan.gov.br

Human mitochondrial pyruvate carrier 2 as an autonomous membrane transporter

The active transport of glycolytic pyruvate across the inner mitochondrial membrane is thought to involve two mitochondrial pyruvate carrier subunits, MPC1 and MPC2, assembled as a 150 kDa heterotypic oligomer. Here, the recombinant production of human MPC through a co-expression strategy is first described; however, substantial complex formation was not observed, and predominantly individual subunits were purified. In contrast to MPC1, which co-purifies with a host chaperone, we demonstrated that MPC2 homo-oligomers promote efficient pyruvate transport into proteoliposomes. The derived functional requirements and kinetic features of MPC2 resemble those previously demonstrated for MPC in the literature. Distinctly, chemical inhibition of transport is observed only for a thiazolidinedione derivative. The autonomous transport role for MPC2 is validated in cells when the ectopic expression of human MPC2 in yeast lacking endogenous MPC stimulated growth and increased oxygen consumption. Multiple oligomeric species of MPC2 across mitochondrial isolates, purified protein and artificial lipid bilayers suggest functional high-order complexes. Significant changes in the secondary structure content of MPC2, as probed by synchrotron radiation circular dichroism, further supports the interaction between the protein and ligands. Our results provide the initial framework for the independent role of MPC2 in homeostasis and diseases related to dysregulated pyruvate metabolism.

Time-dependent regulation of morphological changes and cartilage differentiation markers in the mouse pubic symphysis during pregnancy and postpartum recovery

Animal models commonly serve as a bridge between in vitro experiments and clinical applications; however, few physiological processes in adult animals are sufficient to serve as proof-of-concept models for cartilage regeneration. Intriguingly, some rodents, such as young adult mice, undergo physiological connective tissue modifications to birth canal elements such as the pubic symphysis during pregnancy; therefore, we investigated whether the differential expression of cartilage differentiation markers is associated with cartilaginous tissue morphological modifications during these changes. Our results showed that osteochondral progenitor cells expressing Runx2, Sox9, Col2a1 and Dcx at the non-pregnant pubic symphysis proliferated and differentiated throughout pregnancy, giving rise to a complex osteoligamentous junction that attached the interpubic ligament to the pubic bones until labour occurred. After delivery, the recovery of pubic symphysis cartilaginous tissues was improved by the time-dependent expression of these chondrocytic lineage markers at the osteoligamentous junction. This process potentially recapitulates embryologic chondrocytic differentiation to successfully recover hyaline cartilaginous pads at 10 days postpartum. Therefore, we propose that this physiological phenomenon represents a proof-of-concept model for investigating the mechanisms involved in cartilage restoration in adult animals.