Sharon Chu

Efficacy of a Non-Hypercalcemic Vitamin-D2 Derived Anti-Cancer Agent (MT19c) and Inhibition of Fatty Acid Synthesis in an Ovarian Cancer Xenograft Model

Background Numerous vitamin-D analogs exhibited poor response rates, high systemic toxicities and hypercalcemia in human trials to treat cancer. We identified the first non-hypercalcemic anti-cancer vitamin D analog MT19c by altering the A-ring of ergocalciferol. This study describes the therapeutic efficacy and mechanism of action of MT19c in both in vitro and in vivo models. Methodology/Principal Finding Antitumor efficacy of MT19c was evaluated in ovarian cancer cell (SKOV-3) xenografts in nude mice and a syngenic rat ovarian cancer model. Serum calcium levels of MT19c or calcitriol treated animals were measured. In-silico molecular docking simulation and a cell based VDR reporter assay revealed MT19c–VDR interaction. Genomewide mRNA analysis of MT19c treated tumors identified drug targets which were verified by immunoblotting and microscopy. Quantification of cellular malonyl CoA was carried out by HPLC-MS. A binding study with PPAR-Y receptor was performed. MT19c reduced ovarian cancer growth in xenograft and syngeneic animal models without causing hypercalcemia or acute toxicity. MT19c is a weak vitamin-D receptor (VDR) antagonist that disrupted the interaction between VDR and coactivator SRC2-3. Genome-wide mRNA analysis and western blot and microscopy of MT19c treated xenograft tumors showed inhibition of fatty acid synthase (FASN) activity. MT19c reduced cellular levels of malonyl CoA in SKOV-3 cells and inhibited EGFR/phosphoinositol-3kinase (PI-3K) activity independently of PPAR-gamma protein. Significance Antitumor effects of non-hypercalcemic agent MT19c provide a new approach to the design of vitamin-D based anticancer molecules and a rationale for developing MT19c as a therapeutic agent for malignant ovarian tumors by targeting oncogenic de novo lipogenesis.

The human fetal lung xenograft: validation as model of microvascular remodeling in the postglandular lung.

Coordinated remodeling of epithelium and vasculature is essential for normal postglandular lung development. The value of the human‐to‐rodent lung xenograft as model of fetal microvascular development remains poorly defined.

Intussusceptive-like angiogenesis in human fetal lung xenografts: Link with bronchopulmonary dysplasia-associated microvascular dysangiogenesis?

ABSTRACT Background: Human fetal lung xenografts display an unusual pattern of non-sprouting, plexus-forming angiogenesis that is reminiscent of the dysmorphic angioarchitecture described in bronchopulmonary dysplasia (BPD). The aim of this study was to determine the clinicopathological correlates, growth characteristics and molecular regulation of this aberrant form of graft angiogenesis. Methods: Fetal lung xenografts, derived from 12 previable fetuses (15 to 22 weeks’ gestation) and engrafted in the renal subcapsular space of SCID-beige mice, were analyzed 4 weeks posttransplantation for morphology, vascularization, proliferative activity and gene expression. Results: Focal plexus-forming angiogenesis (PFA) was observed in 60/230 (26%) of xenografts. PFA was characterized by a complex network of tortuous nonsprouting vascular structures with low endothelial proliferative activity, suggestive of intussusceptive-type angiogenesis. There was no correlation between the occurrence of PFA and gestational age or time interval between delivery and engraftment. PFA was preferentially localized in the relatively hypoxic central subcapsular area. Microarray analysis suggested altered expression of 15 genes in graft regions with PFA, of which 7 are known angiogenic/lymphangiogenic regulators and 5 are known hypoxia-inducible genes. qRT-PCR analysis confirmed significant upregulation of SULF2, IGF2, and HMOX1 in graft regions with PFA. Conclusion: These observations in human fetal lungs ex vivo suggest that postcanalicular lungs can switch from sprouting angiogenesis to an aberrant intussusceptive-type of angiogenesis that is highly reminiscent of BPD-associated dysangiogenesis. While circumstantial evidence suggests hypoxia may be implicated, the exact triggering mechanisms, molecular regulation and clinical implications of this angiogenic switch in preterm lungs in vivo remain to be determined.

RESILIENCE OF THE HUMAN FETAL LUNG FOLLOWING STILLBIRTH: Potential relevance for pulmonary regenerative medicine.

ABSTRACT Recent advances in pulmonary regenerative medicine have increased the demand for alveolar epithelial progenitor cells. Fetal lung tissues from spontaneous pregnancy losses may represent a neglected, yet ethically and societally acceptable source of alveolar epithelial cells. The aim of this study was to determine the regenerative capacity of fetal lungs obtained from second trimester stillbirths. Lung tissues were harvested from 11 stillborn fetuses (13 to 22 weeks’ gestation) at postdelivery intervals ranging from 10 to 41 hours and grafted to the renal subcapsular space of immune-suppressed rats to provide optimal growth conditions. Histology, epithelial and alveolar type II cell proliferation, and surfactant protein-C mRNA expression were studied in preimplantation lung tissues and in xenografts at posttransplantation week 2. All xenografts displayed advanced architectural maturation compared with their respective preimplantation tissues, regardless of gestational age and postdelivery interval. The proliferative activity of the grafts was significantly higher than that of the preimplantation tissues (mean Ki-67 labeling index 26.7% ± 7.7% versus 14.7% ± 10.5%; P < .01). The proliferative activity of grafts obtained after a long (>36 hours) postdelivery interval was significantly higher than that of the corresponding preimplantation tissue, and equivalent to that of grafts obtained after a short postdelivery interval (<14 hours). The regenerative capacity of fetal lung tissue was greater at younger (13 to 17 weeks) than at older (19 to 22 weeks) gestational ages. The presence of inflammation/chorioamnionitis did not appear to affect graft regeneration. All grafts studied displayed robust surfactant protein-C mRNA expression. In conclusion, fetal lung tissues from second trimester stillbirths can regain their inherent high regenerative potential following short-term culture, even if harvested more than 36 hours after delivery.

Correlation between cord insertion type and chorionic villus vascularization of the co-twin in diamniotic–monochorionic twin pregnancies

BACKGROUND Recent evidence suggests that cord insertion type of one twin correlates with chorionic plate vascularization of the monochorionic co-twin. Specifically, for twins with paracentral cords, chorionic plate vascularization is significantly greater when the co-twin has a velamentous, rather than paracentral cord insertion. AIMS To determine whether this correlation between cord insertion type and vascularization of the co-twin also extends to the deeper chorionic villus tree. STUDY DESIGN Morphometric analysis of chorionic villus vascularization in CD31-immunostained sections of a retrospective cohort of gestational age-matched third trimester monochorionic placentas with discordant paracentral/velamentous (PC/V) or concordant paracentral/paracentral (PC/PC) cord insertions. OUTCOME MEASURES Vascular numerical density (number of vascular profiles per unit villus stromal area) of intermediate villi (>80 μm diameter) and terminal villi (<80 μm). RESULTS For twins with paracentral cord insertion, the vascular numerical density of intermediate villi was significantly higher for twins in a discordant PC/V relationship than for those in a concordant PC/PC relationship (P<0.05), thus replicating previous findings in superficial chorionic vessels. For terminal villi, in contrast, the vascular numerical density of twins with paracentral cords in a PC/V combination was significantly lower than of those in a PC/PC combination, and similar to that of their co-twins with velamentous cord insertion. CONCLUSIONS Early placental angiogenesis in monochorionic twin gestations may be influenced by implantation and cord localization of the co-twin. The regulation of terminal villus angiogenesis appears to be dissociated from more proximal villus angiogenesis and independent of cord insertion of the co-twin.

Long-term outcome of human cord blood-derived hematopoietic progenitor cells in murine lungs.

ABSTRACT Intranasally delivered human cord blood-derived CD34+ hematopoietic progenitor cells have the capacity to engraft and undergo transdifferentiation to surfactant-containing alveolar epithelial type II cell-like cells in lungs of newborn mice. The aim of this study was to determine the long-term fate of such transplanted cells as well as their effects on alveolar development in neonatally injured lungs. Double transgenic CCSP+/FasL+ mice with inducible lung-specific FasL expression, targeted to induce respiratory epithelial apoptosis in the perinatal period, served as model of neonatal lung injury. Non-injured single transgenic CCSP+/FasL- littermates served as controls. Freshly isolated umbilical cord blood CD34+ cells (0.5 to 1.0 × 106) were administered at postnatal day 5 by intranasal inoculation; sham controls received equal volume PBS. Engraftment, alveolar epithelial differentiation, lung growth, and alveolarization were evaluated one year after transplantation. Engrafted cord blood-derived cells, detected by human-specific FISH (fluorescent in situ hybridization) analysis, and cord blood-derived alveolar type II-like cells, detected by double immunofluorescence analysis, while sparse, were seen in all conditions and more frequent in double than single transgenic recipients. The total lung volume and volume of air-exchanging parenchyma, assessed by stereological volumetry, were significantly greater in CD34-treated double transgenic animals than in PBS-treated double transgenic controls. Alveolarization, assessed by histomorphometry, was equivalent in these groups. These results suggest that transdifferentiated alveolar epithelial cells, derived from cord blood CD34+ cells, can persist up to one year after intrapulmonary delivery. Cord blood-CD34+ cell administration appears to have growth-promoting effects in injured newborn lungs, without affecting alveolar development in this model.

Placental endoglin levels in diamniotic-monochorionic twin gestations: Correlation with clinical and placental characteristics

OBJECTIVE While endoglin has been implicated in the pathogenesis of various complications in singleton pregnancies, its potential contribution to complications of monochorionic twinning remains largely undetermined. The aim of this study was to determine the correlation between relevant clinical and pathological variables and placental endoglin levels in diamniotic-monochorionic twin pregnancies. METHODS Endoglin expression was studied by immunohistochemistry and Western blot in a prospective cohort of 68 non-TTTS and 7 TTTS monochorionic twin placentas. Placental endoglin levels were correlated with clinical and placental characteristics associated with twin-to-twin transfusion syndrome (TTTS) and selective growth restriction, including birth weight discordance, uneven placental sharing, peripheral cord insertion and choriovascular anatomy. RESULTS In non-TTTS gestations discordant for these criteria, placental endoglin levels were significantly higher for the twin with smaller birth weight, intrauterine growth restriction, and/or abnormal ultrasound Doppler studies than for the more normal co-twin. Similarly, placental endoglin levels were significantly higher in the placental territory with smaller share and/or peripheral cord insertion in cases discordant for these placental characteristics. In TTTS gestations, placental endoglin levels tended to be higher for donor twins than for recipients. There was no correlation between endoglin levels and superficial choriovascular anastomoses. CONCLUSIONS While the exact functional implications remain to be determined, our findings suggest a strong correlation between unbalanced placental endoglin levels and intertwin growth discordance in monochorionic twins.

Effects of human umbilical cord blood mononuclear cells on respiratory system mechanics in a murine model of neonatal lung injury

ABSTRACT Background: Mononuclear cells (MNCs) have well-documented beneficial effects in a wide range of adult pulmonary diseases. The effects of human umbilical cord blood-derived MNCs on neonatal lung injury, highly relevant for potential autologous application in preterm newborns at risk for bronchopulmonary dysplasia (BPD), remain incompletely established. The aim of this study was to determine the long-term morphologic and functional effects of systemically delivered MNCs in a murine model of neonatal lung injury. Materials and Methods: MNCs from cryopreserved cord blood (1 × 106 cells per pup) were given intravenously to newborn mice exposed to 90% O2 from birth; controls received cord blood total nucleated cells (TNCs) or granular cells, or equal volume vehicle buffer (sham controls). In order to avoid immune rejection, we used SCID mice as recipients. Lung mechanics (flexiVent™), engraftment, growth, and alveolarization were evaluated eight weeks postinfusion. Results: Systemic MNC administration to hyperoxia-exposed newborn mice resulted in significant attenuation of methacholine-induced airway hyperreactivity, leading to reduction of central airway resistance to normoxic levels. These bronchial effects were associated with mild improvement of alveolarization, lung compliance, and elastance. TNCs had no effects on alveolar remodeling and were associated with worsened methacholine-induced bronchial hyperreactivity. Granular cell administration resulted in a marked morphologic and functional emphysematous phenotype, associated with high mortality. Pulmonary donor cell engraftment was sporadic in all groups. Conclusions: These results suggest that cord blood MNCs may have a cell type-specific role in therapy of pulmonary conditions characterized by increased airway resistance, such as BPD and asthma. Future studies need to determine the active MNC subtype(s), their mechanisms of action, and optimal purification methods to minimize granular cell contamination.

Pathology of Neonatal Non-albicans Candidiasis: Autopsy Study and Literature Review

Introduction/Objectives Non-albicans Candida species such as Candida parapsilosis and Candida glabrata have emerged as prevalent pathogens in premature infants. The aim of this study was to systematically delineate the histopathologic findings in neonatal non-albicans candidiasis. Methods We performed a retrospective clinicopathologic analysis of extremely premature (23–28 weeks’ gestation) infants diagnosed with invasive candidiasis. Archival autopsy tissues were subjected to periodic acid-Schiff, methenamine-silver and anti-Candida (immuno)histochemical stains, as well as dual anti-Candida and anti-cytokeratin or anti-CD31 immunofluorescence assays. In addition, we studied the prevalence of intestinal Candida colonization in a consecutive autopsy series of extremely premature infants. Results Based on positive postmortem blood and/or lung cultures, invasive candidiasis (3 non-albicans and 11 Candida albicans) was diagnosed in 14 of the 187 extremely premature infants examined between 1995 and 2017. In contrast to the well-known inflammatory and tissue-destructive phenotype of congenital C. albicans infection, invasive non-albicans candidiasis/candidemia caused by C. parapsilosis and C. glabrata was inconspicuous by routine hematoxylin-eosin-based histopathologic analysis despite a heavy fungal presence detected in intestines, lungs, and blood by targeted (immuno)histochemical assays. Intestinal colonization by Candida species was identified in 16 of the 26 (61%) extremely premature neonates who had lived for at least 1 week, as assessed by anti-Candida immunostaining. Conclusion Invasive neonatal non-albicans candidiasis/candidemia appears to have no distinct histopathologic signature. Based on the notoriously low sensitivity of fungal blood cultures and the observed high frequency of Candida intestinal colonization (>50%), it is likely that non-albicans candidiasis/candidemia may be underdiagnosed in (deceased) preterm infants. Routine inclusion of targeted (immuno)histochemical fungal detection strategies in the perinatal autopsy may lead to deeper insight into the prevalence and clinical relevance of neonatal non-albicans candidiasis.