Caterina Crescimanno

Early antiangiogenic activity of SU11248 evaluated in vivo by dynamic contrast-enhanced magnetic resonance imaging in an experimental model of colon carcinoma.

Purpose: To compare two dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) techniques in terms of their ability in assessing the early antiangiogenic effect of SU11248, a novel selective multitargeted tyrosine kinase inhibitor, that exhibits direct antitumor and antiangiogenic activity via inhibition of the receptor tyrosine kinases platelet-derived growth factor receptor, vascular endothelial growth factor receptor, KIT, and FLT3. Experimental Design: A s.c. tumor model of HT29 human colon carcinoma in athymic mice was used. Two DCE-MRI techniques were used based, respectively, on macromolecular [Gd-diethylenetriaminepentaacetic acid (DTPA)-albumin] and low molecular weight (Gd-DTPA) contrast agents. The first technique provided a quantitative measurement of transendothelial permeability and fractional plasma volume, accepted surrogate markers of tumor angiogenesis. With the second technique, we quantified the initial area under the concentration-time curve, which gives information related to tumor perfusion and vascular permeability. Experiments were done before and 24 hours after a single dose administration of SU11248. Results: The early antiangiogenic effect of SU11248 was detected by DCE-MRI with macromolecular contrast agent as a 42% decrease in vascular permeability measured in the tumor rim. The effect was also detected by DCE-MRI done with Gd-DTPA as a 31% decrease in the initial area under the concentration-time curve. Histologic slices showed a statistically significant difference in mean vessel density between the treated and control groups. Conclusions: The early antiangiogenic activity of SU11248 was detected in vivo by DCE-MRI techniques using either macromolecular or low molecular weight contrast agents. Because DCE-MRI techniques with low molecular weight contrast agents can be used in clinical studies, these results could be relevant for the design of clinical trials based on new paradigms.

In Vivo Assessment of Antiangiogenic Activity of SU6668 in an Experimental Colon Carcinoma Model

Purpose: The purpose of this research was to assess in vivo by dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) the antiangiogenic effect of SU6668, an oral, small molecule inhibitor of the angiogenic receptor tyrosine kinases vascular endothelial growth factor receptor 2 (Flk-1/KDR), platelet-derived growth factor receptor, and fibroblast growth factor receptor 1. Experimental Design: A s.c. tumor model of HT29 human colon carcinoma in athymic mice was used. DCE-MRI with a macromolecular contrast agent was used to measure transendothelial permeability and fractional plasma volume, accepted surrogate markers of tumor angiogenesis. CD31 immunohistochemical staining was used for assessing microvessels density and vessels area. Experiments were performed after 24 h, and 3, 7, and 14 days of treatment. Results: DCE-MRI clearly detected the early effect (after 24 h of treatment) of SU6668 on tumor vasculature as a 51% and 26% decrease in the average vessel permeability measured in the tumor rim and core (respectively). A substantial decrease was also observed in average fractional plasma volume in the rim (59%) and core (35%) of the tumor. Histological results confirmed magnetic resonance imaging findings. After 3, 7, and 14 days of treatment, postcontrast magnetic resonant images presented a thin strip of strongly enhanced tissue at the tumor periphery; histology examination showed that this hyperenhanced ring corresponded to strongly vascularized tissue adjacent but external to the tumor. Histology also revealed a strong decrease in the thickness of peripheral viable tissue, with a greatly reduced vessel count. SU6668 greatly inhibited tumor growth, with 60% inhibition at 14 days of treatment. Conclusions: DCE-MRI detected in vivo the antiangiogenic efficacy of SU6668.

Differentiation of human trophoblast populations involves alterations in cytokeratin patterns.

Cytokeratins (CKs) are related to proliferation and differentiation of epithelial cells. Little knowledge exists about CK patterns in human trophoblast subpopulations (villous and extravillous trophoblasts). To better understand differentiation and function of trophoblast components, we studied the distribution patterns of CKs in the placenta throughout pregnancy. A panel of well-defined monoclonal antibodies against different types of cytokeratins, vimentin, and fibrin, was used on frozen and paraffin sections. CK8, 18, and 19 were expressed in all the villous and extravillous trophoblastic subsets throughout pregnancy. In the first trimester, syncytiotrophoblasts were positive for CK7 and 13 along the basal membrane. As pregnancy progressed there was an increase in intensity of the reaction product and a more diffuse positive staining of CK7 in the cytoplasm of the syncytium, with evident positivity along the apical membrane. CK13 showed similar expression as CK7, but with less intense staining along the apical membrane and less prominent staining in the cytoplasm. Villous cytotrophoblasts were also positive for CK7 and CK13. CK17 was found related to cytotrophoblastic cells in contact with or next to fibrin deposits. Extravillous cytotrophoblasts in cell islands and cell columns were positive for CK13 only in the cell layers located proximal to the villous stroma, whereas the distal and more differentiated cells were negative. CK7 was positive in all epithelial cells of cell islands and columns, but the reaction product was not present in cells deeply migrated into the decidua. Amnion was negative for anti-CK13 antibodies in the first trimester but was positive at term. CK4 and CK16 were not found in the placenta. Our study shows for the first time that the different populations of human placental trophoblast express cytokeratins in developmental, differentiative, and functional specific patterns. These findings can be useful to distinguish and classify the various trophoblastic populations and provide a foundation for studying pathological aspects of the trophoblast.

Identification and characterization of a specific sensory epithelium in the rat larynx.

A specific laryngeal sensory epithelium (SLSE), which includes arrays of solitary chemoreceptor cells, is described in the supraglottic region of the rat. Two plates of SLSE were found, one on each side of the larynx. The first plate was located in the ventrolateral wall of the larynx, and the second was located in the interarytenoidal region. In SLSE, immunoblotting showed the presence of α‐gustducin and phospholipase C β2 (PLCβ2), which are two markers of chemoreceptor cells. At immunocytochemistry, laryngeal immunoreactivity for α‐gustducin was localized mainly in solitary chemosensory cells. Double‐label immunocytochemistry using confocal microscopy demonstrated that α‐gustducin–expressing cells in large part colocalize type III IP3 receptor (IP3R3), another key molecule in bitter taste perception. However, some IP3R3–expressing cells do not colocalize α‐gustducin. At ultrastructural immunocytochemistry, these cells showed packed apical microvilli, clear cytoplasmic vesicles, and cytoneural junctions. SLSE was characterized by high permeability to a tracer due to poorly developed junctional contacts between superficial cells. Junctions were short in length and showed little contact with the terminal web. Ultrastructural analysis showed deep pits among the superficial cells. In SLSE, high density of intraepithelial nerve fibers was found. The lamina propria of the SLSE appeared thicker than that in other supraglottic regions. It was characterized by the presence of a well‐developed subepithelial nerve plexus. The immunocytochemical and ultrastructural data suggested that SLSE is a chemoreceptor located in an optimal position for detecting substances entering the larynx from the pharynx or the trachea. J. Comp. Neurol. 475:188–201, 2004.

Immunohistochemical evidence suggests intrinsic regulatory activity of human eccrine sweat glands.

Immunohistochemistry of normal eccrine sweat glands was performed on paraffin sections of human skin. Immunoreactivity (ir) for neuron specific enolase, S100 protein (S100), regulatory peptides, nitric oxide synthase type I (NOS‐I) and choline‐acetyltransferase (ChAT) was found in small nerve bundles close to sweat glands. In the glands, secretory cells were labelled with anticytokeratin antibody. Using antibodies to S100, calcitonin gene‐related peptide (CGRP) and substance P (SP) a specific distribution pattern was found in secretory cells. Granulated (dark) and parietal (clear) cells were immunopositive for CGRP, and S100 and SP, respectively. Immunoreactivity was diffuse in the cytoplasm for CGRP and S100, and peripheral for SP. Myoepithelial cells were not labelled. Electron microscopy revealed electron dense granules, probably containing peptide, in granulated cells. Using antibodies to NOS‐I and ChAT, ir was exclusively found in myoepithelial cells. Immunoreactivity for the atrial natriuretic peptide was absent in sweat glands. These results provide evidence for the presence of both regulatory peptides involved in vasodilation and key enzymes for the synthesis of nitric oxide and acetylcholine in the secretory coil of human sweat glands. It is suggested that human sweat glands are capable of some intrinsic regulation in addition to that carried out by their nerve supply.

In vivo mapping of Fractional Plasma Volume (FPV) and endothelial transfer coefficient (KPS) in solid tumors using a macromolecular contrast agent: Correlation with histology and ultrastructure

Contrast‐enhanced MRI, immunostaining and electron microscopy were used to detect areas of intense angiogenesis in experimental tumors. This work was also aimed at evaluating the possible effect of the surrounding tissues on tumor microvasculature and at studying the penetration of macromolecules in avascular areas. Human colon carcinoma cells were implanted in subcutaneous tissue of nude mice. Dynamic T1‐weigthed 3D pulse sequences were acquired before and after administration of Gd‐DTPA‐albumin to obtain parametric maps of fractional plasma volume (fpv) and transendothelial permeability (Kps). The maps suggested that tumor can be subdivided into 4 zones located in the peripheral rim (zones I–II) or in the core (zones III–IV) of the tumor itself. Significant differences (p<0.001) were found in the values of Kps and fpv of zones I–II with respect to zones III–IV. In the peripheral rim, permeability was significantly higher (p<0.01) in the muscle‐peripheral region (zone I) with respect to the skin‐peripheral region (zone II). In areas with high Kps, histological and ultrastructural examination revealed clusters of newly formed vessels and signs of intense permeability. Numerous vascular vesicular organs were visible in these areas. In the tumoral core, analysis of the microcirculatory parameters revealed regions with mild permeability (zone III) and regions with negligible permeability (zone IV). These 2 zones were discriminated by the average value of Kps (p<0.05), while their fpv was not significantly different. Upon histological examination, the tumoral core exhibited necrotic areas; CD31 immunocytochemistry exhibited that it was diffusely hypovascularized with large avascular areas. Upon ultrastructural examination, capillaries were rarely visible and exhibited signs of endothelial cell damage. The results suggest that segmentation based on microvascular parameters detects in vivo zones characterized by immunocytochemical and ultrastructural aspects of intense angiogenesis. The finding that a certain amount of contrast agent penetrates in the tumoral core suggests that high oncotic and hydrostatic pressure only partially hinders the penetration of macromolecules.

Expression pattern alterations of syndecans and glypican-1 in normal and pathological trophoblast

Syndecans (syn‐1, ‐2, ‐3, ‐4) and glypican‐1 are proteoglycans expressed during development in association with changes in tissue organization and differentiation. They participate in the modulation of growth factor actions and in cell–cell and cell–matrix adhesion. The expression of syn‐1, ‐2, ‐3, ‐4, and glypican‐1 has been studied in normal human placenta and in gestational trophoblastic disease such as hydatidiform mole, invasive mole, and choriocarcinoma, using immunohistochemistry and western blots. Syndecan‐3 was not expressed in normal or pathological tissues. During normal gestation, the other proteoglycans showed a specific staining pattern, which for some was modified during pregnancy. For instance, syn‐1 was only expressed in syncytiotrophoblast; syn‐4 was mainly localized in the villous and extravillous cytotrophoblast in the first trimester, whereas at term it was expressed in the syncytiotrophoblast. The most striking results are the altered expression patterns of syndecans and glypican‐1 in pathological tissues. These proteoglycans showed a progressive decrease of immunostaining related to the increase of severity of trophoblastic disease, in particular in invasive mole and choriocarcinoma. In addition, dysregulation in the localization of the expression patterns was observed for syn‐2 and ‐4. Because changes in syndecan expression enable cells to become more or less responsive to their micro‐environment, the down‐regulation and/or dysregulation of syndecans in relation to the degree of severity of trophoblastic diseases provides new insights into the progression of these pathologies. Copyright

Solitary chemosensory cells in the developing chemoreceptorial epithelium of the vallate papilla.

SummaryThe solitary chemosensory cells are considered typical of aquatic vertebrates and have never been described in the oral cavity of terrestrial vertebrates. We describe elements with ultrastructural characteristics of the solitary chemosensory cell in the gustatory epithelium of rats in the first week of extrauterine life. These elements appeared isolated, located among keratinocytes, and wrapped by glial-like elements. They showed a bipolar shape with a round cell body, a thin apical process, and a thicker basal one. Nerve fibers contacted the cell body and the processes. The cells showed small dense granules mainly located near nerve contacts. Small bundles of tonofilaments were visible in the perinuclear cytoplasm. Similar elements were not found in rats after the first week of extrauterine life. The present study demonstrates in a mammal that the development of taste buds is preceded by the presence of epithelial elements with ultrastructural characteristics of the solitary chemosensory cells described in lower vertebrates. This finding suggests that the oral chemoreception in the suckling rats may be mediated by three different patways (i.e., gustatory system, common chemical sense, and solitary chemosensory cell system).

Effect of tamoxifen in an experimental model of breast tumor studied by dynamic contrast-enhanced magnetic resonance imaging and different contrast agents

Objectives:The aim of this study was to compare the efficacy of gadoteridol, B22956/1 (a new protein binding blood pool contrast agent), and (Gd-DTPA)37-albumin in detecting, by dynamic contrast-enhanced magnetic resonance imaging (MRI), the effect in vivo of tamoxifen in an experimental model of breast tumor implanted in rats. Materials and Methods:Tumors were induced by subcutaneous injection of 106 mammary adenocarcinoma cells (13762 MAT B III). Treatment with tamoxifen (or vehicle) started on day 4 after implantation. On day 10 after implantation, animals were observed by MRI using B22956/1 (or gadoteridol) and, 24 hours later, using (Gd-DTPA)37–albumin. Results:Dynamic contrast-enhanced magnetic resonance imaging data showed that tamoxifen treatment decreased vascular permeability to B22956/1, whereas no difference was detectable in permeability to gadoteridol or to (Gd-DTPA)37–albumin. No effect on fractional plasma volume was detected with either of contrast agents. Conclusions:B22956/1 is superior to both small Gd chelates and macromolecular contrast agents in the assessment of the effect of tamoxifen treatment on tumor vasculature.

Ganglion cells and topographically related nerves in the vallate papilla/von Ebner gland complex.

Ganglion cells and topographically related nerves in the vallate papilla/von Ebner gland complex were investigated in rat tongue by cytochemical, immunocytochemical, and ultrastructural methods to evaluate the possible presence of different neuronal subpopulations. Immunostaining for neurofilaments and protein gene product 9.5 revealed ganglionic cell bodies and nerve fibers. A large part of the neurons were positive at immunostaining for neuronal nitric oxide synthase (NOS), vesicular acetylcholine transporter (VAChT), or vasoactive intestinal peptide (VIP). A small subset of nerve fibers revealed immunoreactivity for cholecystokinin. Axons traveling under the lingual epithelium were evidenced by their content of calcitonin gene-related peptide (CGRP) or substance P (SP). Cell bodies positive for SP or CGRP were not detected. Using methods of co-localization, three different neuronal classes were detected. The main population was composed of AChE/NADPH-diaphorase (NADPHd)-positive cells. Small groups of acetylcholine esterase (AChE)-positive/NADPHd-negative cells were visible. Isolated neurons were AChE-negative/NADPHd-positive. The results of co-localization experiments for VAChT/NOS were consistent with those obtained by cytochemical co-localization of AChE and NADPHd. Experiments of co-localization for peptidergic and nitrergic structures revealed CGRP- and SP-immunoreactive fibers in the vallate papilla/von Ebner gland ganglion. In conclusion, the results demonstrated in the VP/VEG complex peptidergic, cholinergic, and nitrergic neurons. The presence of different neuronal subclasses suggests that a certain degree of functional specialization may exist.