Faculty Bibliography

BACKGROUND: Statins are widely used cholesterol-lowering drugs that act by inhibiting HMGCoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. Recent evidence suggests that statin use may be associated with a decreased risk for Alzheimer disease, although the mechanisms underlying this apparent risk reduction are poorly understood. One popular hypothesis for statin action is related to the drugs' ability to activate alpha-secretase-type shedding of the alpha-secretase-cleaved soluble Alzheimer amyloid precursor protein ectodomain (sAPP(alpha)). Statins also inhibit the isoprenoid pathway, thereby modulating the activities of the Rho family of small GTPases-Rho A, B, and C-as well as the activities of Rac and cdc42. Rho proteins, in turn, exert many of their effects via Rho-associated protein kinases (ROCKs). Several cell-surface molecules are substrates for activated alpha-secretase-type ectodomain shedding, and regulation of shedding typically occurs via activation of protein kinase C or extracellular-signal-regulated protein kinases, or via inactivation of protein phosphatase 1 or 2A. However, the possibility that these enzymes play a role in statin-stimulated shedding has been excluded, leading us to investigate whether the Rho/ROCK1 protein phosphorylation pathway might be involved. METHODS AND FINDINGS: We found that both atorvastatin and simvastatin stimulated sAPP(alpha) shedding from a neuroblastoma cell line via a subcellular mechanism apparently located upstream of endocytosis. A farnesyl transferase inhibitor also increased sAPP(alpha) shedding, as did a dominant negative form of ROCK1. Most conclusively, a constitutively active ROCK1 molecule inhibited statin-stimulated sAPP(alpha) shedding. CONCLUSION: Together, these data suggest that statins exert their effects on shedding of sAPP(alpha) from cultured cells, at least in part, by modulation of the isoprenoid pathway and ROCK1

Hematopoietic cells have been reported to convert into a number of non-hematopoietic cells types after transplantation/injury. Here, we have used a lineage tracing approach to determine whether hematopoietic plasticity is relevant for the normal development of hepatocytes and endothelial cells, both of which develop in close association with blood cells. Two mouse models were analyzed: vav ancestry mice, in which essentially all hematopoietic cells, including stem cells, irreversibly express yellow fluorescent protein (YFP); and lysozyme ancestry mice, in which all macrophages, as well as a small subset of all other non-myeloid hematopoietic cells, are labeled. Both lines were found to contain YFP+ hepatocytes at similar frequencies, indicating that macrophage to hepatocyte contributions occur in unperturbed mice. However, the YFP+ hepatocytes never formed clusters larger than three cells, suggesting a postnatal origin. In addition, the frequency of these cells was very low (approximately 1 in 75,000) and only increased two- to threefold after acute liver injury. Analysis of the two mouse models revealed no evidence for a hematopoietic origin of endothelial cells, showing that definitive HSCs do not function as hemangioblasts during normal development. Using endothelial cells and hepatocytes as paradigms, our study indicates that hematopoietic cells are tightly restricted in their differentiation potential during mouse embryo development and that hematopoietic plasticity plays at best a minor role in adult organ maintenance and regeneration

OBJECTIVE: Studies in vitro and in vivo of macrophage foam cells have shown evidence of cytotoxicity after acyl-CoA:cholesterol acyltransferase (ACAT) inhibition. Foam cells of smooth muscle origin are also found in human and animal atherosclerotic lesions. METHODS AND RESULTS: To study whether cytotoxicity from ACAT inhibition is independent of cell type, we first established a protocol to conveniently induce aortic smooth muscle foam cell formation using cholesterol-cyclodextrin complexes (CCC). Rat aortic smooth muscle cells (ASMCs) treated for 48 hours with CCC (20 microg/mL) became foam cells by morphological (oil-red-O staining) and biochemical (approximately 1200% and approximately 180% increase in cellular esterified and free cholesterol, respectively) criteria. ACAT activity increased 500% (P<0.01 versus untreated). Similar results were obtained in human ASMC, but ACAT activity increased to an even greater extent (3200%; P<0.01 versus untreated). Western blots indicated that CCC treatment increased human (to 380+/-20% of untreated, P<0.001), but not rat, ACAT protein expression. ACAT inhibition by Fujirebio compound F1394 suppressed CCC-induced foam cell formation in rat and human ASMC, but, notably, did not induce significant cytotoxicity. CONCLUSIONS: ASMC might be more resistant to FC-induced adverse effects than are macrophages

The mammary gland reaches a fully differentiated phenotype at lactation, a stage characterized by the abundant expression of beta-casein. We have investigated the expression and regulation of gap junction proteins (connexins, Cx) during the various developmental stages of mouse mammary gland. Immunohistochemical analysis, with specific antibodies, reveals that Cx26 and Cx32 are expressed and confined to the cell borders of luminal epithelial cells in all developmental stages of the gland. Cx26 and Cx32 expression, at the mRNA and protein levels, increases in pregnancy and peaks in lactation. Whereas Cx43 mRNA decreases in pregnancy and lactation, the functional activity of Cx43 protein, which has been localized to myoepithelial cells, is regulated (through phosphorylation) during pregnancy and peaks during lactation. Cx30 mRNA and proteins have, for the first time, been detected in mammary gland epithelia. Using reverse transcription/polymerase chain reaction and sequencing techniques, we show that Cx30 is abundant in pregnant and lactating mammary gland. Cx30 protein levels have not been detected in the mammary gland prior to day 15 of pregnancy, whereas maximum expression occurs at the onset of lactation. In mouse mammary cells in culture, Cx30 is epithelial-cell-specific and is induced by lactogenic hormones. These data identify a novel player in mammary differentiation and suggest a potential role for Cx30 in the fully differentiated gland

PURPOSE: The rat has been a cost-effective model for the evaluation of bladder development, cancer and stromal-epithelial interactions. Serial cultivation of rat urothelium has been difficult. We developed a reliable protocol for the harvest, serial cultivation and cryopreservation of rat urothelium. We investigated the differentiation markers of in vivo bladder urothelium compared with cells reconstituted onto an acellular bladder matrix. MATERIALS AND METHODS: Epithelial harvest techniques using trypsin and collagenase were compared. Medium and conditions were optimized for serial culture and growth characteristics were calculated. Cultured cells were cryopreserved, and then recovered and grown on acellular bladder matrices. Morphology and markers of differentiation were compared between normal bladder and engineered grafts using scanning electron microscopy (SEM) and immunohistochemistry. RESULTS: Atraumatic enzymatic removal of urothelium with trypsin yielded more cells with greater viability than collagenase. Cells could be reliably grown beyond 10 passages using fibroblast conditioned medium and a 3T3 feeder layer during initial passages. Cryopreserved cells were successfully recovered and incorporated onto acellular matrices. Immunostaining and SEM of engineered grafts demonstrated early markers of differentiation, such as surface microvilli and cytokeratin 17, on polygonal cells with typical tight junctions. CONCLUSIONS: Rat urothelium can be reliably grown using fibroblast conditioned medium and a 3T3 feeder layer during primary culture. Serially passaged cells can survive cryopreservation and they are able to reconstitute epithelium on an acellular bladder matrix. Cells that are incorporated into the matrix express markers of early differentiation and demonstrate typical morphological characteristics by SEM. These culture techniques and this in vitro organoid model should facilitate the use of rat urothelium

Sonic hedgehog (Shh) signaling controls many aspects of ontogeny, orchestrating congruent growth and patterning. During brain development, Shh regulates early ventral patterning while later on it is critical for the regulation of precursor proliferation in the dorsal brain, namely in the neocortex, tectum and cerebellum. We have recently shown that Shh also controls the behavior of cells with stem cell properties in the mouse embryonic neocortex, and additional studies have implicated it in the control of cell proliferation in the adult ventral forebrain and in the hippocampus. However, it remains unclear whether it regulates adult stem cell lineages in an equivalent manner. Similarly, it is not known which cells respond to Shh signaling in stem cell niches. Here we demonstrate that Shh is required for cell proliferation in the mouse forebrain's subventricular zone (SVZ) stem cell niche and for the production of new olfactory interneurons in vivo. We identify two populations of Gli1+ Shh signaling responding cells: GFAP+ SVZ stem cells and GFAP- precursors. Consistently, we show that Shh regulates the self-renewal of neurosphere-forming stem cells and that it modulates proliferation of SVZ lineages by acting as a mitogen in cooperation with epidermal growth factor (EGF). Together, our data demonstrate a critical and conserved role of Shh signaling in the regulation of stem cell lineages in the adult mammalian brain, highlight the subventricular stem cell astrocytes and their more abundant derived precursors as in vivo targets of Shh signaling, and demonstrate the requirement for Shh signaling in postnatal and adult neurogenesis

The effects of estradiol (E(2)) and progesterone (P(4)) on fluid and sodium regulation may have important clinical implications with respect to cardiovascular and renal disease as well as reproductive syndromes such as preeclampsia and ovarian hyperstimulation syndrome. We tested the hypothesis that sodium excretion is reduced in response to a sodium load during combined P(4)-E(2) treatment, but P(4) administration alone has little effect on sodium regulation. Fifteen women (22 +/- 2 yr) used a GnRH antagonist to suppress endogenous E(2) and P(4) for 9 d; for d 4-9, eight subjects used P(4) (200 mg/d), and seven subjects used P(4) with E(2) (two E(2) patches, 0.1 mg/d each). On d 3 and 9, isotonic saline (0.9% NaCl) was infused [120 min at 0.1 ml/kg body weight (BW).min], followed by 120 min of rest. Compared with GnRH antagonist alone, P([P4]) increased from 1.6 +/- 0.8 to 9.4 +/- 2.3 ng/ml (5.1 +/- 2.5 to 29.9 +/- 7.3 nmol/liter, P < 0.05) in the P(4) treated group, with no change in P([E2]). In the P(4)-E(2) treated group P([P4]) increased from 1.6 +/- 0.5 to 6.7 +/- 0.6 ng/ml (5.1 +/- 1.6 to 21.3 +/- 1.6 nmol/liter, P < 0.05 and P([E2]) increased from 17.9 +/- 6.3 to 200 +/- 41 pg/ml (65.7 +/- 23 to 734.6 +/- 150.0 pmol/liter, P < 0.05). Before isotonic saline infusion, renal sodium and water excretion were similar under all conditions, but during isotonic saline infusion, cumulative sodium excretion was lower in the P(4)-E(2) treated women (34.1 +/- 5.1 mEq) compared with GnRH antagonist (50.2 +/- 11.4 mEq). Sodium excretion was unaffected by P(4) treatment (48.0 +/- 8.2 and 41.2 +/- 5.1 mEq, for GnRH antagonist and P(4)). Compared with GnRH antagonist alone, P(4)-E(2) treatment increased distal sodium reabsorption and transiently decreased proximal sodium reabsorption, whereas P(4) treatment did not alter either distal or proximal sodium reabsorption. Before isotonic saline infusion, the plasma aldosterone (Ald) concentration was greater during P(4) treatment (153 +/- 25 pg/ml; 3883 +/- 1102 pmol/liter) and P(4)-E(2) treatment (242 +/- 47 pg/ml; 6373 +/- 1390 pmol/liter) than during their respective GnRH antagonist alone treatments [96 +/- 13 and 148 +/- 47 pg/ml (2598 +/- 475 and 3284 +/- 973 pmol/liter) for P(4) and combined P(4)-E(2), respectively]. Compared with GnRH antagonist alone treatments, preisotonic saline infusion plasma renin activity was greater only with P(4)-E(2) treatment, whereas the plasma atrial natriuretic peptide concentration was lower only with P(4) treatment. Isotonic saline infusion suppressed plasma Ald under all conditions, but decreased plasma renin activity only with P(4)-E(2) treatment (average decrease, 1.3 +/- 0.5 ng/ml angiotensin I.h; P < 0.05). In summary, we found that P(4)-E(2) treatment decreased sodium excretion via either renin-angiotensin-Ald system stimulation or direct effects on kidney tubules. P(4) treatment at these plasma concentrations had no independent effect on the renal response to acute sodium loading. These data suggest that E(2) is the more powerful reproductive hormone involved in sodium retention relative to P(4), and that estrogen-induced up-regulation of P(4) receptors is required for the effects of P(4) on sodium regulation

Mineralization of growth plate cartilage is a critical event during endochondral bone formation, which allows replacement of cartilage by bone. Ankylosis protein (Ank), which transports intracellular inorganic pyrophosphate (PP(i)) to the extracellular milieu, is expressed by hypertrophic and, especially highly, by terminally differentiated mineralizing growth plate chondrocytes. Blocking Ank transport activity or ank expression in terminally differentiated mineralizing growth plate chondrocytes led to increases of intra- and extracellular PP(i) concentrations, decreases of alkaline phosphatase (APase) expression and activity, and inhibition of mineralization, whereas treatment of these cells with the APase inhibitor levamisole led to an increase of extracellular PP(i) concentration and inhibition of mineralization. Ank-overexpressing hypertrophic nonmineralizing growth plate chondrocytes showed decreased intra- and extracellular PP(i) levels; increased mineralization-related gene expression of APase, type I collagen, and osteocalcin; increased APase activity; and mineralization. Treatment of Ank-expressing growth plate chondrocytes with a phosphate transport blocker (phosphonoformic acid [PFA]) inhibited uptake of inorganic phosphate (P(i)) and gene expression of the type III Na(+)/P(i) cotransporters Pit-1 and Pit-2. Furthermore, PFA or levamisole treatment of Ank-overexpressing hypertrophic chondrocytes inhibited APase expression and activity and subsequent mineralization. In conclusion, increased Ank activity results in elevated intracellular PP(i) transport to the extracellular milieu, initial hydrolysis of PP(i) to P(i), P(i)-mediated upregulation of APase gene expression and activity, further hydrolysis and removal of the mineralization inhibitor PP(i), and subsequent mineralization

Macrophages perform both injury-inducing and repair-promoting tasks in different models of inflammation, leading to a model of macrophage function in which distinct patterns of activation have been proposed. We investigated macrophage function mechanistically in a reversible model of liver injury in which the injury and recovery phases are distinct. Carbon tetrachloride---induced liver fibrosis revealed scar-associated macrophages that persisted throughout recovery. A transgenic mouse (CD11b-DTR) was generated in which macrophages could be selectively depleted. Macrophage depletion when liver fibrosis was advanced resulted in reduced scarring and fewer myofibroblasts. Macrophage depletion during recovery, by contrast, led to a failure of matrix degradation. These data provide the first clear evidence that functionally distinct subpopulations of macrophages exist in the same tissue and that these macrophages play critical roles in both the injury and recovery phases of inflammatory scarring.

Although the stroma in which carcinomas arise has been previously regarded as a bystander to the clonal expansion and acquisition of malignant characteristics of tumor cells, it is now generally acknowledged that stromal changes are required for the establishment of cancer. In the present article, we discuss three recent publications that highlight the complex role the stroma has during the development of cancer and the potential for targeting the stroma by therapeutic approaches