Liposome-encapsulated statins reduce hypertrophic scarring through topical application.

Hypertrophic scar is an important clinical problem with limited therapeutic options. Aside from their roles as 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, statins have also been demonstrated to decrease scarring by reducing connective tissue growth factor (CTGF) expression. However, poor penetrative ability limits their utility as topical treatments for hypertrophic scar. Here, we aim to develop novel statin formulations using liposomes to enhance dermal penetrative ability and to evaluate their efficacy against formation of hypertrophic scar utilizing our validated rabbit ear hypertrophic scar model. Liposomal simvastatin or pravastatin were compounded using a novel, flexible liposomal formulation and applied topically to rabbit ear hypertrophic scars daily from postoperation day (POD) 14 until POD 25. Scar color, including erythema and melanin, was measured using reflectance spectrophotometry on POD 28, and scar tissue was harvested for evaluation of scar elevation index as well as gene and protein expression. Human foreskin fibroblasts were also treated with statin formulations and CCN2 expression was determined by quantitative PCR. Both simvastatin and pravastatin were efficiently encapsulated in liposomes, forming nanometer-scale particles possessing highly negative charges. Topical treatment with liposomal simvastatin and pravastatin at 6.5% concentration significantly reduced scar elevation index and decreased type I/III collagen content and myofibroblast persistence in the wound. The erythema/vascularity of scars was reduced by liposomal statin treatment, with concomitant decrease of CD31 expression as measured histologically. Expression levels of transcripts encoding CTGF, collagen I, and collagen III collagen in scar tissue were also decreased by liposomal pravastatin treatment, as were myofibroblast persistence and the type I/III collagen ratio as assessed by immunofluorescence and picrosirus red staining, respectively. Treatment of human foreskin fibroblasts with simvastatin or with liposome-encapsulated pravastatin resulted in decreased expression of transcript encoding CTGF. Overall, our novel statin formulations encapsulated in liposomes were successfully delivered through topical application, significantly reducing hypertrophic scarring in a rabbit ear model.

Berberine-induced Inactivation of Signal Transducer and Activator of Transcription 5 Signaling Promotes Male-specific Expression of a Bile Acid Uptake Transporter.

Sodium-taurocholate co-transporting polypeptide (Ntcp/NTCP) is the major uptake transporter of bile salts in mouse and human livers. In certain diseases, including endotoxemia, cholestasis, diabetes, and hepatocarcinoma, Ntcp/NTCP expression is markedly reduced, which interferes with enterohepatic circulation of bile salts, impairing the absorption of lipophilic compounds. Therefore, normal Ntcp/NTCP expression in the liver is physiologically important. Berberine is an herbal medicine used historically to improve liver function and has recently been shown to repress STAT signaling. However, berberine effects on Ntcp/NTCP expression are unknown, prompting use to investigate this possible connection. Our results showed that berberine dose-dependently increased Ntcp expression in male mouse liver and decreased taurocholic acid levels in serum but increased them in the liver. In mouse and human hepatoma cells, berberine induced Ntcp/NTCP mRNA and protein expression and increased cellular uptake of [3H] taurocholate. Mechanistically, berberine decreased nuclear protein levels of phospho-JAK2 and phospho-STAT5, thus disrupting the JAK2-STAT5 signaling. Moreover, berberine stimulated luciferase reporter expression from the mouse Ntcp promoter when one putative STAT5 response element (RE) (-1137 bp) was deleted and from the human NTCP promoter when three putative STAT5REs (-2898, -2164, and -691 bp) were deleted. Chromatin immunoprecipitation demonstrated that berberine decreased binding of phospho-STAT5 protein to the-2164 and -691 bp STAT5REs in the human NTCP promoter. In summary, berberine-disrupted STAT5 signaling promoted mouse and human Ntcp/NTCP expression, resulting in enhanced bile acid uptake. Therefore, berberine may be a therapeutic candidate compound for maintaining bile acid homeostasis.

Hormonal and Chemical Regulation of the Glut9 Transporter in Mice.

Glucose transporter (Glut) 9 plays an important role in maintaining the homeostasis of uric acid in the body. Although the physiologic functions of Glut9 have been well established, the regulation of Glut9 expression is less well understood. In this study, we showed that the mRNA and protein expression of Glut9 in mouse liver and kidney are female predominant. Ontogeny studies further revealed that the female-predominant Glut9 expression in mouse liver only occurs in adult mice, which is primarily attributable to the fact that Glut9 expression sustains in females but gradually decreases in males after it reaches the peak level at day 22. Hormone replacement studies in gonadectomized mice, lit/lit mice, and hypophysectomized mice demonstrated that female-predominant Glut9 expression in mouse liver and kidney are primarily due to the inhibitory effects of male-pattern growth hormone secretion, but not sex hormones. In silico analysis of DNA sequences revealed that conserved response elements of signal transducer and activator of transcription 5b, which is an established relay molecule in the growth hormone signaling pathway, are present in mouse and human Glut9/GLUT9 gene promoters, suggesting that Glut9/GLUT9 is a potential target gene of growth hormone. Analysis of mice treated with a panel of chemicals revealed that agonists of the aryl hydrocarbon receptor and the peroxisome proliferator-activated receptor α induced Glut9 mRNA expression in the liver, which is further supported by the presence of conserved xenobiotic response elements and direct repeat 1 DNA motifs in the mouse Glut9 gene promoter. In summary, Glut9 expression is downregulated by male-pattern growth hormone secretion but is upregulated by activation of aryl hydrocarbon receptor and peroxisome proliferator-activated receptor α signaling in mice.

Decreased bile-acid synthesis in livers of hepatocyte-conditional NADPH-cytochrome P450 reductase-null mice results in increased bile acids in serum.

NADPH-cytochrome P450 reductase (Cpr) is essential for the function of microsomal cytochrome P450 monooxygenases (P450), including those P450s involved in bile acid (BA) synthesis. Mice with hepatocyte-specific deletion of NADPH-cytochrome P450 reductase (H-Cpr-null) have been engineered to understand the in vivo function of hepatic P450s in the metabolism of xenobiotics and endogenous compounds. However, the impact of hepatic Cpr on BA homeostasis is not clear. The present study revealed that H-Cpr-null mice had a 60% decrease in total BA concentration in liver, whereas the total BA concentration in serum was almost doubled. The decreased level of cholic acid (CA) in both serum and livers of H-Cpr-null mice is likely due to diminished enzyme activity of Cyp8b1 that is essential for CA biosynthesis. Feedback mechanisms responsible for the reduced liver BA concentrations and/or increased serum BA concentrations in H-Cpr-null mice included the following: 1) enhanced alternative BA synthesis pathway, as evidenced by the fact that classic BA synthesis is diminished but chenodeoxycholic acid still increases in both serum and livers of H-Cpr-null mice; 2) inhibition of farnesoid X receptor activation, which increased the mRNA of Cyp7a1 and 8b1; 3) induction of intestinal BA transporters to facilitate BA absorption from the intestine to the circulation; 4) induction of hepatic multidrug resistance-associated protein transporters to increase BA efflux from the liver to blood; and 5) increased generation of secondary BAs. In summary, the present study reveals an important contribution of the alternative BA synthesis pathway and BA transporters in regulating BA concentrations in H-Cpr-null mice.

Adaptive hepatic and intestinal alterations in mice after deletion of NADPH-cytochrome P450 Oxidoreductase (Cpr) in hepatocytes.

Cytochrome P450 enzymes (P450) play an important role in first-pass metabolism in both the intestine and liver. NADPH-cytochrome P450 oxidoreductase (Cpr) is an essential electron transfer protein required for microsomal P450 activity. Mice with conditional knockout of Cpr in hepatocytes develop normally and survive even with complete loss of liver microsomal P450 activity. Our current studies were performed to determine whether alternative drug-metabolizing pathways increase in an attempt to maintain whole-body homeostasis. In addition to the liver, Cpr is mainly expressed in tissues such as lung, kidney, and gastrointestinal tract. In livers of H-Cpr-null mice, there is a marked increase in mRNA expression of phase I enzymes (Aldh1a1, 1a7, 3a2; Ces1b2, 2a6, and 2a12), antioxidant enzymes (Ho-1, Nqo1, and epoxide hydrolase), phase II enzymes (Ugt1a9; Gsta1/2, m3, m4, m6, t1, and t3; and Sult1a1 and 1d1), and drug transporters (Oatp1a4, Oct3, Mate1, Mdr1a, and Mrp3 and 4). In addition, glucuronide-conjugated bilirubin concentrations are doubled in serum of H-Cpr-null mice. Both constitutive androstane receptor (CAR) and nuclear factor erythroid 2-related factor 2 (Nrf2) protein in nuclei are higher in the livers of H-Cpr-null mice, indicating that CAR and Nrf2 are activated. In the small intestine of H-Cpr-null mice, mRNA expression of Cyp3a11 and Mdr1a, two genes critical for intestinal first-pass metabolism, are markedly up-regulated. In addition, nutrient (Pept1) and cholesterol (Npc1l1) transporters are induced in the small intestine of H-Cpr-null mice. In conclusion, in H-Cpr-null mice, adaptive regulation of alternative detoxification genes in liver and small intestine appear to partially compensate for the loss of microsomal P450 function in liver.

Age- and sex-related differences of organic anion-transporting polypeptide gene expression in livers of rats.

Organic anion-transporting polypeptides (Oatps) play important roles in transporting endogenous substances and xenobiotics into the liver and are implicated in drug-drug interactions. Many factors could influence their expression and result in alterations in drug disposition, efficacy and toxicity. This study was aimed to examine the development-, aging-, and sex-dependent Oatps expression in livers of rats. The livers from SD rats during development (-2, 1, 7, 14, 21, 28, 35, and 60 d) and aging (60, 180, 540 and/or 800 d) were collected and total RNAs were extracted, purified, and subjected to real-time PCR analysis. Total proteins were extracted for western-blot analysis. Results showed that Oatp1a1, Oatp1a4, Oatp1a5 and Oatp1b2 were all hardly detectable in fetal rat livers, low at birth, rapidly increased after weaning (21 d), and reached the peak at 60 d. The Oatps remained stable during the age between 60-180 d, and decreased at elderly (540 and/or 800 d). After birth, Oatp1a1, Oatp1a4, and Oatp1b2 were all highly expressed in liver, in contrast, Oatp1a5 expression was low. Oatp expressions are male-predominant in rat livers. In the livers of aged rats, the Oatp expression decreased and shared a consistent ontogeny pattern at the mRNA and protein level. In conclusion, this study showed that in rat liver, Oatp1a1, Oatp1a4, Oatp1a5 and Oatp1b2 gene expressions are influenced by age and gender, which could provide a basis of individual variation in drug transport, metabolism and toxicity in children, elderly and women.

Fibroblast growth factor (Fgf) 21 is a novel target gene of the aryl hydrocarbon receptor (AhR).

The toxic effects of dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), mainly through activation of the aryl hydrocarbon receptor (AhR) are well documented. Fibroblast growth factor (Fgf) 21 plays critical roles in metabolic adaptation to fasting by increasing lipid oxidation and ketogenesis in the liver. The present study was performed to determine whether activation of the AhR induces Fgf21 expression. In mouse liver, TCDD increased Fgf21 mRNA in both dose- and time-dependent manners. In addition, TCDD markedly increased Fgf21 mRNA expression in cultured mouse and human hepatocytes. Moreover, TCDD increased mRNA (in liver) and protein levels (in both liver and serum) of Fgf21 in wild-type mice, but not in AhR-null mice. Chromatin immunoprecipitation assays showed that TCDD increased AhR protein binding to the Fgf21 promoter (-105/+1 base pair). Fgf21-null mice administered 200µg/kg of TCDD died within 20days, whereas wild-type mice receiving the same treatment were still alive at one month after administration. This indicates that TCDD-induced Fgf21 expression protects against TCDD toxicity. Diethylhexylphthalate (DEHP) pretreatment attenuated TCDD-induced Fgf21 expression in mouse liver and white adipose tissue, which may explain a previous report that DEHP pretreatment decreases TCDD-induced wasting. In conclusion, Fgf21 appears to be a target gene of AhR-signaling pathway in mouse and human liver.

Distinct bile acid alterations in response to a single administration of PFOA and PFDA in mice.

Per- and polyfluoroalkyl substances (PFASs), a group of synthetic chemicals that were once widely used for industrial purposes and in consumer products, are widely found in the environment and in human blood due to their extraordinary resistance to degradation. Once inside the body, PFASs can activate nuclear receptors such as PPARα and CAR. The present study aimed to investigate the impact of perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA) on liver structure and functions, as well as bile acid homeostasis in mice. A single administration of 0.1 mmole/kg of PFDA, not PFOA, elevated serum ALT and bilirubin levels and caused cholestasis in WT mice. PFDA increased total and various bile acid species in serum but decreased them in the liver. Furthermore, in mouse livers, PFDA, not PFOA, down-regulated mRNA expression of uptake transporters (Ntcp, Oatp1a1, 1a4, 1b2, and 2b1) but induced efflux transporters (Bcrp, Mdr2, and Mrp2-4). In addition, PFDA, not PFOA, decreased Cyp7a1, 7b1, 8b1, and 27a1 mRNA expression in mouse livers with concomitant hepatic accumulation of cholesterol. In contrast, in PPARα-null mice, PFDA did not increase serum ALT, bilirubin, or total bile acids, but produced prominent hepatosteatosis; and the observed PFDA-induced expression changes of transporters and Cyps in WT mice were largely attenuated or abolished. In CAR-null mice, the observed PFDA-induced bile acid alterations in WT mice were mostly sustained. These results indicate that, at the dose employed, PFDA has more negative effects than PFOA on liver function. PPARα appears to play a major role in mediating most of PFDA-induced effects, which were absent or attenuated in PPARα-null mice. Lack of PPARα, however, exacerbated hepatic steatosis. Our findings indicate separated roles of PPARα in mediating the adaptive responses to PFDA: protective against hepatosteatosis but exacerbating cholestasis.

Hormonal and chemical regulation of paraoxonases in mice.

In humans and rodents, paraoxonase (PON/Pon) 1 expression and activity in livers and serum are higher in females than in males, and some drugs increase paraoxonase's expression. However, the underlining mechanisms of gender-divergent expression and chemical regulation of Pon1 remain largely unknown. The present study determined the regulatory mechanisms contributing to gender-divergent and chemically altered Pon expression in mouse livers. Pon1 mRNA was much more abundant in the livers of mice than other tissues, with higher levels in female livers than male livers at mRNA and protein levels. Pon2 mRNA was ubiquitously expressed in mouse tissues, but minimally in mouse liver. Pon3 mRNA was most abundant in mouse lung and liver and less abundant in other tissues. Pon1 mRNA was lowest in fetal liver, markedly increased at parturition, and remained relatively constant thereafter. Pon2 and Pon3 mRNA are highly expressed in fetal liver and decreased after birth. Male-pattern growth hormone (GH) administration in hypophysectomized and lit/lit mice decreased Pon1 expression. Sex hormones and female-pattern GH administration had no effect on Pon1 expression, indicating the importance of male-pattern GH in regulating Pon1. Aryl hydrocarbon receptor, pregnane X receptor, and NF-E2-related factor activators had no effect on Pon1 mRNA. A constitutive androstane receptor (CAR) activator decreased Pon1 expression in wild-type but not CAR-null mice. In conclusion, Pon1 mRNA was most abundant in adult mouse livers, whereas Pon2 and Pon3 mRNAs were most abundant in fetal mouse livers. Female-predominant Pon1 expression in mouse livers is caused by the inhibitory effects of male-pattern GH secretion, and CAR activation decreases Pon1 expression.

Transcription factor-mediated regulation of carboxylesterase enzymes in livers of mice.

The induction of drug-metabolizing enzymes by chemicals is one of the major reasons for drug-drug interactions. In the present study, the regulation of mRNA expression of one arylacetamide deacetylase (Aadac) and 11 carboxylesterases (Cess) by 15 microsomal enzyme inducers (MEIs) was examined in livers of male C57BL/6 mice. The data demonstrated that Aadac mRNA expression was suppressed by three aryl hydrocarbon receptor (AhR) ligands, two constitutive androstane receptor (CAR) activators, two pregnane X receptor (PXR) ligands, and one nuclear factor erythroid 2-related factor 2 (Nrf2) activator. Ces1 subfamily mRNA expression was not altered by most of the MEIs, whereas Ces2 subfamily mRNA was readily induced by the activators of CAR, PXR, and Nrf2 but not by peroxisome proliferator-activated receptor α activators. Studies using null mice demonstrated that 1) AhR was required for the 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated suppression of Aadac and Ces3a; 2) CAR was involved in the 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene-mediated induction of Aadac, Ces2c, Ces2a, and Ces3a; 3) PXR was required for the pregnenolone-16α-carbonitrile-mediated induction of Aadac, Ces2c, and Ces2a; 4) Nrf2 was required for the oltipraz-mediated induction of Ces1g and Ces2c; and 5) PXR was not required for the DEX-mediated suppression of Cess in livers of mice. In conclusion, the present study systematically investigated the regulation of Cess by MEIs in livers of mice and demonstrated that MEIs modulated mRNA expression of mouse hepatic Cess through the activation of AhR, CAR, PXR, and/or Nrf2 transcriptional pathways.

CYP2D plays a major role in berberine metabolism in liver of mice and humans.

Berberine is a widely used plant extract for gastrointestinal infections, and is reported to have potential benefits in treatment for diabetes and hypercholesterolemia. It has been suggested that interactions between berberine-containing products and cytochromes P450 (CYPs) exist, but little is known about which CYPs mediate the metabolism of berberine in vivo. In this study, berberine metabolites in urine and feces of mice were analyzed, and the role that CYPs play in producing these metabolites were characterized in liver microsomes from mice (MLM) and humans (HLM), as well as recombinant human CYPs. Eleven berberine metabolites were identified in mice, including 5 unconjugated metabolites, mainly in feces, and 6 glucuronide and sulfate conjugates, predominantly in urine. Three novel berberine metabolites were observed. Three unconjugated metabolites of berberine were produced by MLM, HLM, and recombinant human CYPs. CYP2D6 was the primary recombinant human CYP producing these metabolites, followed by CYP1A2, 3A4, 2E1 and CYP2C19. The metabolism of berberine in MLM and HLM was decreased the most by a CYP2D inhibitor, and moderately by inhibitors of CYP1A and 3A. CYP2D plays a major role in berberine biotransformation, therefore, CYP2D6 pharmacogenetics and potential drug-drug interactions should be considered when berberine is used.

Activation of activator protein-1-fibroblast growth factor 21 signaling attenuates Cisplatin hepatotoxicity.

Fibroblast growth factor (Fgf/FGF) 21, which plays important roles in sugar, lipid and energy metabolism, has been accepted as a mito-stress marker gene. We recently reported that FGF21 expression can be up-regulated via activation of aryl hydrocarbon receptor (AhR) or glucocorticoid receptor (GR) and that FGF21 plays important cytoprotective roles. Cisplatin (cis-diamminedichloroplatinum, CDDP) is a widely used chemotherapeutic drug. Numerous adverse effects including hepatotoxicity have been noted during CDDP therapy. It is known that CDDP can induce mitochondrial dysfunction. The studies were designed to determine the regulation of Fgf/FGF21 expression by CDDP, and to characterize the underlying mechanisms of its regulation, as well as to determine the impact of gain or loss of Fgf/FGF21 function on the progression of CDDP hepatotoxicity. Our results showed that CDDP and phorbol ester induced mRNA and protein expression of Fgf/FGF21 and ß-Klotho, two essential components of Fgf21 signaling, in mouse livers and cultured mouse/human hepatocytes. Luciferase reporter assays and ChIP-qPCR assays demonstrated that the cJun-AP-1 activation is responsible for CDDP- and phorbol ester-induced Fgf/FGF21 expression. Such induction is abolished after cotreated with AP-1 inhibitor SR11302. In addition, CDDP produces more severe liver injury in Fgf21-null than wild-type mice. Pre-treatment of GR activator dexamethasone or AhR activator ß-Naphthoflavone, both of which can induce Fgf21 expression, attenuated CDDP-induced hepatotoxicity in vivo and in vitro. In conclusion, Fgf/FGF21-ß-Klotho signaling can be activated via AP-1 activation. Gain of Fgf/FGF21 function attenuates the progression of CDDP hepatotoxicity, which may be considered clinically to improve CDDP therapy.

Modulation of Immune Response to Chlamydia muridarum by Host miR-135a.

Previously, our laboratory established the role of small, noncoding RNA species, i.e., microRNA (miRNA) including miR-135a in anti-chlamydial immunity in infected hosts. We report here chlamydial infection results in decreased miR-135a expression in mouse genital tissue and a fibroblast cell line. Several chemokine and chemokine receptor genes (including CXCL10, CCR5) associated with chlamydial pathogenesis were identified in silico to contain putative miR-135a binding sequence(s) in the 3' untranslated region. The role of miR-135a in the host immune response was investigated using exogenous miR-135a mimic to restore the immune phenotype associated with decreased miR-135a following Chlamydia muridarum (Cm) infection. We observed miR-135a regulation of Cm-primed bone marrow derived dendritic cells (BMDC) via activation of Cm-immune CD4+ T cells for clonal expansion and CCR5 expression. Using a transwell cell migration assay, we explore the role of miR-135a in regulation of genital tract CXCL10 expression and recruitment of CXCR3+ CD4+ T cells via the CXCL10/CXCR3 axis. Collectively, data reported here support miR-135a affecting multiple cellular processes in response to chlamydial infection.

Regulation of Hox and ParaHox genes by perfluorochemicals in mouse liver.

Homeobox (Hox) genes encode homeodomain proteins, which play important roles in the development and morphological diversification of organisms including plants and animals. Perfluorinated chemicals (PFCs), which are well recognized industrial pollutants and universally detected in human and wildlife, interfere with animal development. In addition, PFCs produce a number of hepatic adverse effects, such as hepatomegaly and dyslipidemia. Homeodomain proteins profoundly contribute to liver regeneration. Hox genes serve as either oncogenes or tumor suppressor genes during target organ carcinogenesis. However, to date, no study investigated whether PFCs regulate expression of Hox genes. This study was designed to determine the regulation of Hox (including Hox-a to -d subfamily members) and paraHox [including GS homeobox (Gsx), pancreatic and duodenal homeobox (Pdx), and caudal-related homeobox (Cdx) family members] genes by PFCs including perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorodecanoic acid (PFDA) in mouse liver. 46.4 mg/kg PFNA induced mRNA expression of Hoxa5, b7, c5, d10 and Pdx1 in wild-type and CAR-null mouse livers, but not in PPARα-null mouse livers, indicating a PPARα-dependent manner. PFOA, PFNA, and PFDA all induced mRNA expression of Hoxa5, b7, c5, d10, Pdx1 and Zeb2 in wild-type but not PPARα-null mouse livers. In addition, in Nrf2-null mouse livers, PFNA continued to increase mRNA expression of Hoxa5 and Pdx1, but not Hoxb7, c5 or d10. Furthermore, Wy14643, a classical PPARα agonist, induced mRNA expression of Hoxb7 and c5 in wild-type but not PPARα-null mouse livers. However, Wy14643 did not induce mRNA expression of Hoxa5, d10 or Pdx1 in either wild-type or PPARα-null mouse livers. TCPOBOP, a classical mouse CAR agonist, increased mRNA expression of Hoxb7, c5 and d10 but not Hoxa5 or Pdx1 in mouse livers. Moreover, PFNA decreased cytoplasmic and nuclear Hoxb7 protein levels in mouse livers. However, PFNA increased cytoplasmic Hoxc5 protein level but decreased nuclear Hoxc5 protein level in mouse livers. In conclusion, PFCs induced mRNA expression of several Hox genes such as Hoxb7, c5 and d10, mostly through the activation of PPARα and/or Nrf2 signaling.

Evaluation of Cytotoxicity of Self-Emulsifying Formulations Containing Long-Chain Lipids Using Caco-2 Cell Model: Superior Safety Profile Compared to Medium-Chain Lipids.

Medium-chain (MC) and long-chain (LC) lipids are used for development of self-emulsifying drug delivery systems (SEDDS). MC lipids are often preferred because of their ability to form stable microemulsions with relatively high drug solubilization capacity. On the other hand, LC lipids could be more biocompatible as most endogenous and dietary lipids are LC glycerides. They also maintain high drug solubilization capacity after digestion. The present study was undertaken to determine the cytotoxicity of LC lipids and their formulations on Caco-2 cells of 1-day, 5-day, and 21-day maturity. The results were compared with the cytotoxicity profiles of MC lipids reported previously from our laboratory. The cell viability and cell membrane integrity were, respectively, determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the lactate dehydrogenase assay. The cytotoxicity was partially due to lipid surfactant-induced membrane rupture, and it was influenced by cell maturity and formulation composition. The lipid-surfactant combinations showed greater tolerance than surfactants alone, and LC-SEDDS were well-tolerated at almost 10-fold higher concentration than corresponding MC-SEDDS. Furthermore, the cytotoxicity of digestion end products of both LC and MC triglycerides in the presence of 3 mM sodium taurocholate was compared on 21-day Caco-2 cultures by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The LC lipid formulations showed better tolerance than MC lipid formulations after digestion. Thus, although MC and LC lipids are well-tolerated at doses normally administered to humans, LC lipids show much better safety than MC lipids in a cell-culture model.

Tissue distribution, ontogeny, and hormonal regulation of xenobiotic transporters in mouse kidneys.

Kidneys play important roles in the elimination of numerous endogenous and exogenous chemicals. In recent years, at least 37 xenobiotic transporters have been identified in mammalian kidneys. Although much progress has been made, information on 14 of these transporters (ATP-binding cassette [Abc] a1, apical sodium bile acid transporter [Asbt], breast cancer resistance protein, concentrative nucleoside transporter 1, equilibrative nucleoside transporter [Ent] 2, Ent3, sodium-phosphate cotransporter [Npt] 1, Npt2a, Npt2b, Npt2c, organic anion transporter [Oat] 5, organic anion-transporting polypeptide [Oatp] 4c1, peptide transporter 2, and uric acid transporter [Urat] 1) in kidneys is quite limited. Therefore, the purpose of the present study was to examine the tissue distribution, ontogeny, and hormonal regulation of these 14 transporters in kidneys of mice. Other than in kidneys, Npt2b is also highly expressed in liver and lung, Npt2c in liver and colon, Asbt in ileum, and Abca1 in liver, lung, testis, ovary, and placenta of mice. Most of these (13 of 14) transporters are lowly expressed in mouse kidneys until 15 days of age, which in part contributes to the immaturity of excretory function in fetal and newborn kidneys. One exception is Ent2, which is highly expressed before birth and gradually decreases after birth until reaching adult levels at 15 days of age. Gender-divergent expression of male-predominant (Urat1 and Oatp4c1) and female-predominant (Oat5) transporters in mouse kidneys is primarily due to stimulatory effects of androgens and estrogens, respectively. In conclusion, the mRNA expression of xenobiotic transporters in kidneys is determined by tissue, age, and sex hormones.

Tissue distribution, gender-divergent expression, ontogeny, and chemical induction of multidrug resistance transporter genes (Mdr1a, Mdr1b, Mdr2) in mice.

Multidrug resistance (Mdr) transporters are ATP-binding cassette transporters that efflux amphipathic cations from cells and protect tissues from xenobiotics. Unfortunately, Mdr transporters also efflux anticancer drugs from some tumor cells, resulting in multidrug resistance. There are two groups of Mdrs in mice: group I includes Mdr1a and Mdr1b that transport xenobiotics, whereas group II is Mdr2, a flipase that facilitates phospholipid excretion into bile. Little is known about the regulation of Mdr genes in vivo. The purpose of this study was to determine tissue distribution, gender differences, ontogeny, and chemical induction of Mdrs in mice. The mRNA of Mdr1a is highest in gastrointestinal tract, Mdr1b in ovary and placenta, and Mdr2 in liver. Both Mdr1a and Mdr1b in kidney show female-predominant expression patterns due to repression by androgens. The ontogeny of mouse Mdr1a in duodenum and brain as well as Mdr1b in brain, kidney, and liver all share a similar developmental pattern: low expression at birth, followed by a gradual increase to mature levels at approximately 30 days of age. In contrast, Mdr2 mRNA in liver is markedly up-regulated at birth, which returns to low levels by 5 days of age and then gradually increases to mature levels. None of the Mdrs in liver are readily inducible by any class of microsomal enzyme inducers. In conclusion, the three Mdr transporters in mice are expressed in a tissue-specific and age-dependent pattern, there are gender differences in expression, and Mdr transporters are inducible by only a few microsomal enzyme inducers.

Analysis of crystals leading to joint arthropathies by Raman spectroscopy: comparison with compensated polarized imaging.

The current study assessed the feasibility of the application of Raman spectroscopy toward the diagnosis of gout and pseudogout. First, the lowest concentrations of monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD) crystals detectable by Raman spectroscopy were investigated by mixing known amounts of synthetic crystals with synovial fluid in the concentration range of 1 to 100 microg/mL. Second, a digestion protocol was developed for clinical samples to improve crystal extraction. The ensuing centrifugation of the digest congregated crystals at a well-defined point and allowed for point-and-shoot Raman analysis without having to conduct an extensive search for individual crystals. Finally, synovial fluid samples obtained from patients (n = 35) were cross-analyzed by polarized light microscopy (PLM) and the Raman method to compare and contrast the diagnoses of the two methods. It was found that Raman spectroscopy can detect MSUM and CPPD crystals with good sensitivity and specificity at concentrations as low as 5 microg/mL and 2.5 microg/mL, respectively, using the current method. This detection limit of Raman analysis is lower than that reported for PLM. Raman and PLM diagnoses of clinical samples agreed in 32 out of 35 samples in the entire sample pool. However, the rate of disagreement between PLM-based and Raman-based diagnoses was noteworthy within the subset of diseased samples (3 out of 10), indicating that PLM has limitations and that the confirmation by a secondary method is essential for a reliable outcome. The proposed protocol of sample preparation and Raman analysis ascribes baseline feasibility to the diagnosis of gout and pseudogout by Raman spectroscopy, thus justifying further studies using a larger clinical sample set for obtaining sensitivity and specificity.

An electrochemical fabrication process for the assembly of anisotropically oriented collagen bundles.

Controlled assembly of collagen molecules in vitro remains a major challenge for fabricating the next generation of engineered tissues. Here we present a novel electrochemical alignment technique to control the assembly of type-I collagen molecules into highly oriented and densely packed elongated bundles at the macroscale. The process involves application of electric currents to collagen solutions which in turn generate a pH gradient. Through an isoelectric focusing process, the molecules migrate and congregate within a plane. It was possible to fabricate collagen bundles with 50-400 microm diameter and several inches length via this process. The current study assessed the orientational order, and the presence of fibrillar assembly in such electrochemically oriented constructs by polarized optical microscopy, small angle X-ray scattering, second harmonic generation, and electron microscopy. The mechanical strength of the aligned crosslinked collagen bundles was 30-fold greater than its randomly oriented-crosslinked counterpart. Aligned crosslinked collagen bundles had about half the strength of the native tendon. Tendon-derived fibroblast cells were able to migrate and populate multiple macroscopic bundles at a rate of 0.5mm/day. The anisotropic order within biocompatible collagenous constructs was conferred upon the nuclear morphology of cells as well. These results indicate that the electrochemically oriented collagen scaffolds carry baseline characteristics to be considered for tendon/ligament repair.

Tissue distribution, ontogeny and induction of the transporters Multidrug and toxin extrusion (MATE) 1 and MATE2 mRNA expression levels in mice.

Transporters are expressed in a wide variety of tissues where they perform the critical function of enabling anionic and cationic chemicals of exogenous and endogenous origin to cross otherwise impermeable cell membranes. The Multidrug and toxin extrusion (MATE) transporters mediate cellular efflux of a variety of organic cations, including many drugs. The purpose of the current study was to determine (1) constitutive expression levels of MATE mRNA in various tissues, (2) whether there are gender differences in the expression of MATEs, (3) the ontogenic expression pattern of MATE1 in kidney and (4) whether MATEs are pharmacologically inducible in liver via activation of known transcription factors. In both male and female mice, MATE1 mRNA levels were highest in the kidney, where male expression was higher than female. MATE2 mRNA expression levels were the highest in the testis, where high expression was localized to Sertoli cells, a critical cell type of the blood testis barrier. In female mice, MATE2 mRNA levels were expressed most highly in the colon. The ontogenic pattern of expression of MATE1 mRNA in the kidneys of both males and females was gradual, with levels increasing steadily from prenatal day -2 to 45 days of age, and a gender difference appearing at day 30. Of the transcription factor activators examined (AhR, CAR, Nrf2, PPARalpha and PXR), none were capable of altering MATE1 or MATE2. The current findings support a potential role for MATE1 and MATE2 in a wide range of tissues and, notably, a unique role for MATE2 in the blood-testis barrier.