Alexandre Bruni-Cardoso

On the Road to Understanding of the Osteoblast Adhesion : Cytoskeleton Organization Is Rearranged by Distinct Signaling Pathways

Pre‐osteoblast adhesion attracts increasing interest in both medicine and dentistry. However, how this physiological event alters osteoblast phenotype is poorly understood. We therefore attempted to address this question by investigating key biochemical mechanism that governs pre‐osteoblast adhesion on polystyrene surface. Importantly, we found that cofilin activity was strongly modulated by PP2A (Ser/Thr phosphatase), while cell‐cycle was arrested. Accordingly, we observed that the profile of cofilin phosphorylation (at Ser03) was similar to phospho‐PP2A (at Tyr307). Also, it is plausible to suggest during pre‐osteoblast adhesion that PP2A phosphorylation at Y307 was executed by phospho‐Src (Y416). In addition, it was observed that MAPKp38, but not MAPK‐erk, played a key role on pre‐osteoblast adhesion by phosphorylating MAPKAPK‐2 and ATF‐2 (also called CRE‐BP1). Also, the up‐modulation of RhoA reported here suggests its involvement at the beginning of osteoblast attachment, while Akt remained active during all periods. Altogether, our results clearly showed that osteoblast adhesion is under an intricate network of signaling molecules, which are responsible to guide their interaction with substrate mainly via cytoskeleton rearrangement. J. Cell. Biochem. 108: 134–144, 2009.

Osteoclast-derived matrix metalloproteinase-9 directly affects angiogenesis in the prostate tumor-bone microenvironment.

In human prostate to bone metastases and in a novel rodent model that recapitulates prostate tumor–induced osteolytic and osteogenic responses, we found that osteoclasts are a major source of the proteinase, matrix metalloproteinase (MMP)-9. Because MMPs are important mediators of tumor-host communication, we tested the effect of host-derived MMP-9 on prostate tumor progression in the bone. To this end, immunocompromised mice that were wild-type or null for MMP-9 received transplants of osteolytic/osteogenic-inducing prostate adenocarcinoma tumor tissue to the calvaria. Surprisingly, we found that that host MMP-9 significantly contributed to prostate tumor growth without affecting prostate tumor–induced osteolytic or osteogenic change as determined by microcomputed tomography, microsingle-photon emission computed tomography, and histomorphometry. Subsequent studies aimed at delineating the mechanism of MMP-9 action on tumor growth focused on angiogenesis because MMP-9 and osteoclasts have been implicated in this process. We observed (a) significantly fewer and smaller blood vessels in the MMP-9 null group by CD-31 immunohistochemistry; (b) MMP-9 null osteoclasts had significantly lower levels of bioavailable vascular endothelial growth factor-A164; and (c) using an aorta sprouting assay, conditioned media derived from wild-type osteoclasts was significantly more angiogenic than conditioned media derived from MMP-9 null osteoclasts. In conclusion, these studies show that osteoclast-derived MMP-9 affects prostate tumor growth in the bone microenvironment by contributing to angiogenesis without altering prostate tumor–induced osteolytic or osteogenic changes. Mol Cancer Res; 8(4); 459–70. ©2010 AACR.

Lobe Identity in the Mongolian Gerbil Prostatic Complex: A New Rodent Model for Prostate Study

Knowledge of structural and physiological differences among the prostatic lobes (PL) is the basis for development of experimental studies in traditional laboratory rodents. Although Mongolian gerbil reproductive organs have been increasingly investigated, its prostate structure is far from being properly known, and investigations of this organ focused on the ventral lobe (VL). Thus, the present study provides a thorough morphological description of prostatic complex in the male adult gerbil on the basis of topographic, histological, and ultrastructural analysis and ductal branching. Like other rodents, four pairs of PL were observed. However, in contrast to the rat and mouse, the VL is the least voluminous component and the dorsolateral lobe (DLL) is the most prominent and spatially isolated from remaining PL. The occurrence of a dorsal lobe (DL), hidden between bladder and insertion of seminal vesicles, has not been mentioned in previous reports with Mongolian gerbil. Collagenase digestion followed by microdissection revealed that, except for DL, which has a tubular‐acinar organization, all PL exhibit tubular organization and variable ductal branching. Distinct histological and ultrastructural features such as secretory epithelium, aspect of luminal secretion and stromal organization are reported for each PL and are confirmed by morphometric and stereological methods. Histological sections showed at least three intralobar segments in VL and DL. Ultrastructural analysis evidenced that, although luminal epithelial cells of PL share typical features of exocrine secretory cells, there are striking lobe phenotypical variations. Both merocrine and apocrine pathways are observed in variable rates in all PL, with the predominance of the former in the DLL and the latter in the CG. The morphological observations presented herein point to distinct structural identities for each PL, which probably reflects specific functional compromise of seminal fluid secretion. These data also point to the gerbil as a good model for investigations concerning the regulation of prostate development and homeostasis, mainly with regard to the dorsal and dorsolateral PL. Anat Rec, 290:1233‐1247, 2007.

Localized matrix metalloproteinase (MMP)-2 and MMP-9 activity in the rat ventral prostate during the first week of postnatal development

The initial events in prostatic morphogenesis involve cell proliferation, epithelial canalization and outgrowth toward the stroma. We have hypothesized that stromal rearrangement takes place at the sites of epithelial growth and branching and that this rearrangement involves the action of gelatinases matrix metalloproteinase (MMP)-2 and MMP-9. Thus, the purpose of the present study was to characterize structural aspects of epithelial growth during the first week of postnatal development of the rat ventral prostate and to investigate the expression, localization and activity of MMP-2 and MMP-9 during this period by histological, ultrastructural and immunocytochemical analysis, in addition to gel zymography, in situ zymography and Western blotting. An increasing complexity of prostatic architeture was observed within the first postnatal week. Concurrently, the stroma became more organized and some cells differentiated into smooth muscle cells. Reticulin fibers appeared in a basket-like arrangement around both growing tips and epithelial sprouts, associated with a fainter staining for laminin. MMP-2 and MMP-9 activities were detected. MMP-2/MMP-9 expression decreased during the first week. Developing epithelial cords showed strong and difuse gelatinolytic activity. This activity coincided with the distribution of MMP-2 as determined by immunocytochemistry. On the other hand, MMP-9 was rather concentrated at the epithelial tips. These results suggest that gelatinolytic activity (with contribution of both MMP-2 and MMP-9) in the epithelium and at the epithelium–stroma interface are at least in part responsible for the tissue remodeling that allows epithelial growth and its projection into the surrounding stroma.

Stromal remodelling is required for progressive involution of the rat ventral prostate after castration: Identification of a matrix metalloproteinase-dependent apoptotic wave

Prostate epithelial-cell apoptosis occurs in response to androgen deprivation. We have hypothesized that continued regression would require stromal changes. Studying apoptosis kinetics up to the 14th day after castration, we identified successive waves of apoptosis, with a prominent peak on day 11. This peak was associated with caspase-3 activity, nuclear translocation of apoptosis-inducing factor and clusterin expression. The apoptosis peak on day 11 was preceded by increased MMP-2 and MMP-7 activation, and MMP-9 expression on days 9 and 10. Treatment with the matrix metalloproteinases inhibitors doxycyclin, hydrocortisone, or GM6001 caused significant reduction in the apoptosis rate on day 11. The present data demonstrate that prostatic epithelial-cell deletion at the 11th day after castration was induced by focal degradation of the extracellular matrix associated with stromal remodelling.

MMP-2 Regulates Rat Ventral Prostate Development In Vitro

We have hypothesized that epithelial growth, branching, and canalization in the rodent ventral prostate (VP) would require matrix remodeling, and hence matrix metalloproteinase (MMP) activity. Therefore, the aim of this study was to evaluate the impact of blocking MMP‐2, using whole organ culture. siRNA was employed to inhibit MMP‐2 expression, and this was compared to GM6001s (a broad‐spectrum MMP inhibitor) inhibition of general MMPs. These blocks impaired VP morphogenesis. MMP‐2 silencing reduced organ size, epithelial area, and the number of tips, as well as caused a dilation of the distal parts of the epithelium. Histology, 3‐D reconstruction, biochemistry, and second harmonic generation (SHG) revealed that MMP‐2 silencing affected VP architecture by interfering in epithelial cell proliferation, lumen formation, and cellular organization of both epithelium and stroma, besides intense accumulation of collagen fibers. These data suggest that MMP‐2 plays important roles in prostate growth, being directly involved with epithelial morphogenesis. Developmental Dynamics 239:737–746, 2010.

MMP-2 contributes to the development of the mouse ventral prostate by impacting epithelial growth and morphogenesis

Epithelial growth, branching, and canalization are important morphogenetic events of the rodent ventral prostate (VP) that take place during the first postnatal week. In this study, we evaluated the effect of knocking out MMP‐2 (MMP‐2−/−), by examining developmental and structural aspects of the VP in MMP‐2−/− mice. Neonate (day 6) MMP‐2−/− mice showed fewer epithelial tips, a lower epithelial cell proliferation rate, and also reticulin fiber accumulation. The VP of adult MMP‐2−/− mice showed lower relative weight, smaller epithelial and smooth‐muscle cell volume, and a larger amount of thicker reticulin fibers. No differences in cell proliferation or apoptotic index were noted between adult MMP‐2−/− and wild‐type mice. MMP‐9 was found in the adult MMP‐2−/−, but not in the wild‐type. In conclusion, MMP‐2 function is essential for the epithelial morphogenesis of the mouse VP, and expression of MMP‐9 is not sufficient for acquisition of the normal adult histology. Developmental Dynamics 239:2386–2392, 2010.

The anti-oestrogen fulvestrant (ICI 182,780) reduces the androgen receptor expression, ERK1/2 phosphorylation and cell proliferation in the rat ventral prostate.

This study proposed to investigate further the role of oestrogens during pubertal growth of rat ventral prostate, by analysing the effect of anti-oestrogen fulvestrant (ICI 182,780) on the expression of androgen (AR) and oestrogen receptors (ESR1 and ESR2), mitogen-activated protein kinase (ERK1/2) phosphorylation, and expression of Ki-67, a biomarker for cell proliferation. Ventral prostates were obtained from 90-day-old rats treated once a week for 2 months with vehicle (control) or ICI 182,780 (10 mg/rat, s.c.). Transcripts for AR, ESR1 and ESR2 were evaluated by quantitative real-time polymerase chain reaction. Expression of AR, ESR1, ESR2, total and phospho-ERK1/2 was analysed by Western blot or immunofluorescence. Ki-67-positive cells and myosin heavy chain were detected by immunohistochemistry. Cylindrical epithelial cells slightly taller, epithelial dysplasia and an increase in smooth muscle layer were observed in the ventral prostate from ICI 182,780-treated rats. ICI 182,780 did not change the mRNA, but decreased the protein levels for AR in the ventral prostate. The expression of ESR1 (mRNA and protein) was upregulated by ICI 182,780, but no changes were observed on ESR2 expression (mRNA and protein). ICI 182,780 decreased the phosphorylation state of ERK1/2, with no changes in total ERK1/2 levels. Ki-67-positive cells in the ventral prostate were also decreased by ICI 182,780. In conclusion, ICI 182,780 induces downregulation of AR expression and may block the translocation of ESR1 and ESR2 from the nucleus to the plasma membrane, decreasing ERK1/2 phosphorylation and prostatic epithelial cell proliferation. These findings provide a basis for physiological roles of oestrogen in the ventral prostate. Further studies with fulvestrant are necessary in benign prostate hyperplasia and prostatic cancer models.

Dynamics of the Epithelium During Canalization of the Rat Ventral Prostate

Outgrowth and branching of solid cords are the initial events in postnatal prostatic morphogenesis. These processes involve cell proliferation and their projection into the stroma and precede epithelial canalization. The purpose of the present study was to examine the dynamics of the prostate epithelium during canalization of the rat ventral prostate in the first week of postnatal development using histological, stereological, and ultrastructural analyses. The terminal deoxynucleotidyltransferase [TdT]‐mediated deoxy‐UTP nick end labeling assay was used to investigate the occurrence of DNA fragmentation. Our results demonstrate that canalization of the prostate epithelium starts as early as on day 1 (24 hr after birth) and progresses thereafter. By the end of the first week (day 6), luminal volume density reached ∼3% (P < 0.05) of the organ. Canalization was the result of epithelial cell differentiation and apoptosis. The former involved organization of the epithelial cells into a single layer sitting on the basement membrane, polarization, enlargement of secretory organelles and accumulation of secretory vesicles, microvilli formation, and establishment of the adult pattern of cell junctions. The latter was observed to occur mostly to epithelial cells not in contact with the basement membrane. Structures of variable electron density were observed in the developing lumen. In conclusion, different phenomena seem to be involved in the canalization of the rat ventral prostate. However, it was evident from the present results that complex epithelial cell fate decisions take place during this process. Anat Rec, 290:1223–1232, 2007.

Cardioprotective mechanism of S-nitroso-N-acetylcysteine via S-nitrosated betadrenoceptor-2 in the LDLr-/- mice.

Previous studies from our group have demonstrated the protective effect of S-nitroso-N-acetylcysteine (SNAC) on the cardiovascular system in dyslipidemic LDLr-/- mice that develop atheroma and left ventricular hypertrophy after 15 days on a high fat diet. We have shown that SNAC treatment attenuates plaque development via the suppression of vascular oxidative stress and protects the heart from structural and functional myocardial alterations, such as heart arrhythmia, by reducing cardiomyocyte sensitivity to catecholamines. Here we investigate the ability of SNAC to modulate oxidative stress and cell survival in cardiomyocytes during remodeling and correlation with β₂-AR signaling in mediating this protection. Ventricular superoxide (O₂⁻) and hydrogen peroxide (H₂O₂) generation was measured by HPLC methods to allow quantification of dihydroethidium (DHE) products. Ventricular histological sections were stained using terminal dUTP nick-end labeling (TUNEL) to identify nuclei with DNA degradation (apoptosis) and this was confirmed by Western blot for cleaved caspase-3 and caspase-7 protein expression. The findings show that O₂⁻ and H₂O₂ production and also cell apoptosis were increased during left ventricular hypertrophy (LVH). SNAC treatment reduced oxidative stress during on cardiac remodeling, measured by decreased H₂O₂ and O₂⁻ production (65% and 52%, respectively), and a decrease in the ratio of p-Ser1177 eNOS/total eNOS. Left ventricle (LV) from SNAC-treated mice revealed a 4-fold increase in β₂-AR expression associated with coupling change to Gi; β₂-ARs-S-nitrosation (β₂-AR-SNO) increased 61%, while apoptosis decreased by 70%. These results suggest that the cardio-protective effect of SNAC treatment is primarily through its anti-oxidant role and is associated with β₂-ARs overexpression and β₂-AR-SNO via an anti-apoptotic pathway.