Mouse Portal Vein Research Articles

Establishment of a Transplantation Model of PDAC-Derived Liver Metastases

BackgroundThe highly metastatic nature of pancreatic ductal adenocarcinoma (PDAC) and the difficulty to achieve favorable patient outcomes emphasize the need for novel therapeutic solutions. For preclinical evaluations, genetically engineered mouse models are often used to mimic human PDAC but frequently fail to replicate synchronous development and metastatic spread. This study aimed to develop a transplantation model to achieve synchronous and homogenous PDAC growth with controlled metastatic patterns in the liver.MethodsTo generate an orthotopic PDAC model, the DT6606 cell line was injected into the pancreas head of C57BL/6 mice, and their survival was monitored over time. To generate a heterotopic transplantation model, growing doses of three PDAC cell lines (DT6606, DT6606lm, and K8484) were injected into the portal vein of mice. Magnetic resonance imaging (MRI) was used to monitor metastatic progression, and histologic analysis was performed.ResultsOrthotopically injected mice succumbed to the tumor within an 11-week period (average survival time, 78.2 ± 4.45 days). Post-mortem examinations failed to identify liver metastasis. In the intraportal model, 2 × 105 DT6606 cells resulted in an absence of liver metastases by day 21, whereas 5 × 104 DT6606lm cells and 7 × 104 K8484 cells resulted in steady metastatic growth. Higher doses caused significant metastatic liver involvement. The use of K8484 cells ensured the growth of tumors closely resembling the histopathologic characteristics of human PDAC.ConclusionsThis report details the authors’ efforts to establish an “optimal” murine model for inducing metastatic PDAC, which is critical for advancing our understanding of the disease and developing more effective treatments.

Application of Ganoderma lucidum extract for constructing an oral docetaxel delivery system

Docetaxel is one of the most valuable chemotherapeutic drugs, but it has extremely low oral bioavailability. In this study, an oral docetaxel delivery system was constructed based on the extract of Ganoderma lucidum, a well-known medicinal mushroom. First, a G. lucidum extract-docetaxel complex was prepared by stirring and freeze-drying. Results demonstrated that, compared to the docetaxel group, the exposure levels of docetaxel in the complex group were significantly increased in the mouse portal vein (Cmax, 599.7 vs. 58.5 ng/mL; AUC0–12 h, 631.2 vs. 185.9 ng·h/mL), systemic circulation (Cmax, 310.4 vs. 34.9 ng/mL; AUC0–12 h, 467.6 vs. 124.7 ng·h/mL), liver (Cmax, 431.2 vs. 58.4 ng/g liver; AUC0–12 h, 2007.3 vs. 473.0 ng·h/g liver), and lungs (Cmax, 1711.3 vs. 362.7 ng/g lung; AUC0–12 h, 5188.5 vs. 1329.5 ng·h/g lung). Moreover, in the complex group, except for metabolic stability (p > 0.05), transportation from the mucosal side of gut sacs to the serosal side (p < 0.01), solubility (52.6 vs. 4.2 μg/mL), and release of docetaxel in water were significantly improved (15 min, 5.5% vs. 2.3%; 8 h, 35.0% vs. 15.1%). Furthermore, scanning electron microscope (SEM), differential scanning calorimetry (DSC), and powder X-ray diffraction (PXRD) analysis demonstrated the amorphous existing form of docetaxel. In conclusion, the pharmacokinetics of oral docetaxel in mice was improved by the G. lucidum extract-docetaxel complex, which changed the existing form of docetaxel from crystalline to amorphous. This study provides a simple solution to docetaxel’s low oral bioavailability.

L-amino acid oxidase-1 is involved in the gut-liver axis by regulating 5-aminolevulinic acid production in mice.

L-amino acid oxidase (LAAO) is a metabolic enzyme that converts L-amino acids into ketoacids, ammonia, and hydrogen peroxide (H2O2). The generated H2O2 has previously been shown to have antibacterial and gut microbiota-modulatory properties in LAO1 knock-out (KO) mice. Since most microbial metabolites reach the liver through the portal vein, we examined gut-liver interactions in LAO1 KO mice. We found lower total cholesterol levels, higher glutamic pyruvic transaminase (GPT) levels in the serum, and higher pro-inflammatory cytokine mRNA expression in the liver tissue. In wild-type (WT) mice, LAO1 was expressed in gut tissues (ileum and colon). Microbiome analysis revealed that the abundance of some bacteria was altered in LAO1 KO mice. However, short-chain fatty acid (SCFAs) levels in cecal feces and gut permeability did not change. Fecal microbiota transplantation (FMT) revealed that feces from LAO1 KO mice slightly stimulated pro-inflammatory cytokine expression in the liver. During metabolomic analysis, 5-Aminolevulinic acid (5-ALA) was the only metabolite found to be significantly upregulated in the portal and abdominal veins of the LAO1 KO mice. Intraperitoneal administration of 5-ALA to WT mice significantly increased IL-6 mRNA expression in the liver. These observations suggest that gut LAO1 plays a role in regulating 5-ALA production and that a high level of 5-ALA stimulates the liver to increase pro-inflammatory cytokine expression by disrupting LAO1 in mice.

Propionic Acid, Induced in Gut by an Inulin Diet, Suppresses Inflammation and Ameliorates Liver Ischemia and Reperfusion Injury in Mice.

Liver ischemia and reperfusion injury (IRI) is one of the obstacles in liver surgery such as liver resection and transplantation. In this study, we investigated the preventive effect on mouse liver IRI by feeding mice with inulin, which is a heterogeneous blend of indigestible fructose polymer. Mice were fed either a control ordinary diet (CD) or an inulin diet (ID) containing 5% inulin in the CD, for 14 days before the ischemia and reperfusion (IR) maneuver. IR induced-liver damages were significantly ameliorated in the ID group, compared with those in the CD group. Feeding mice with an ID, but not a CD, elevated levels of Bacteroidetes among gut microbiota, and especially increased Bacteroides acidifaciens in mouse feces, which resulted in significant elevation of short-chain fatty acids (SCFAs) in the portal vein of mice. Among SCFAs, propionic acid (PA) was most significantly increased. The microbial gene functions related to PA biosynthesis were much higher in the fecal microbiome of the ID group compared to the CD. However, the action of PA on liver IRI has not been yet clarified. Direct intraperitoneal administration of PA alone prior to the ischemia strongly suppressed liver cell damages as well as inflammatory responses caused by liver IR. Furthermore, PA suppressed the secretion of inflammatory cytokines from peritoneal macrophages stimulated in vitro through TLR-4 with high-mobility group box 1 protein (HMGB-1), known to be released from apoptotic liver cells during the IR insult. The present study shows that PA may play a key role in the inulin-induced amelioration of mouse liver IRI.

STING Induces Liver Ischemia-Reperfusion Injury by Promoting Calcium-Dependent Caspase 1-GSDMD Processing in Macrophages.

Objectives Although a recent study reported that stimulator of interferon genes (STING) in macrophages has an important regulatory effect on liver ischemia-reperfusion injury (IRI), the underlying mechanism of STING-dependent innate immune activation in liver macrophages (Kupffer cells, KCs) remains unclear. Here, we investigated the effect of STING on liver macrophage pyroptosis and the associated regulatory mechanism of liver IRI. Methods Clodronate liposomes were used to block liver macrophages. AAV-STING-RNAi-F4/80-EGFP, an adenoassociated virus (AAV), was transfected into the portal vein of mice in vivo, and the liver IRI model was established 14 days later. In vitro, liver macrophages were treated with STING-specific siRNA, and a hypoxia-reoxygenation (H/R) model was established. The level of STING was detected via Western blotting (WB), RT-PCR, and immunostaining. Liver tissue and blood samples were collected. Pathological changes in liver tissue were detected by hematoxylin and eosin (H&E) staining. Macrophage pyroptosis was detected by WB, confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and enzyme-linked immunosorbent assay (ELISA). The calcium concentration was measured by immunofluorescence and analyzed with a fluorescence microplate reader. Results The expression of STING increased with liver IRI but decreased significantly after the clodronate liposome blockade of liver macrophages. After knockdown of STING, the activation of caspase 1-GSDMD in macrophages and liver IRI was alleviated. More interestingly, hypoxia/reoxygenation (H/R) increased the calcium concentration in liver macrophages, but the calcium concentration was decreased after STING knockdown. Furthermore, after the inhibition of calcium in H/R-induced liver macrophages by BAPTA-AM, pyroptosis was significantly reduced, but the expression of STING was not significantlydecreased. Conclusions Knockdown of STING reduces calcium-dependent macrophage caspase 1-GSDMD-mediated liver IRI, representing a potential therapeutic approach in the clinic.

Natural Nano-Drug Delivery System in Coptidis Rhizoma Extract with Modified Berberine Hydrochloride Pharmacokinetics.

PurposeThis study aimed to evaluate the pharmaceutical and pharmacokinetic effects of the natural nanoparticles (Nnps) isolated from Coptidis Rhizoma extract on berberine hydrochloride (BBR) and systematically explore the related mechanisms.MethodsFirstly, Nnps were isolated from Coptidis Rhizoma extract and then an Nnps-BBR complex was prepared. After qualitative and quantitative analysis in terms of size, Zeta potential, morphology, and composition of the Nnps and the Nnps-BBR complex, the effects of the Nnps on the crystallization of BBR were characterized. The effects of the Nnps on the solubility and dissolution of BBR were then evaluated. In addition, the effects of the Nnps on BBR in terms of cellular uptake, transmembrane transport, metabolic stability, and pharmacokinetics in mice were studied.ResultsThe Nnps had an average size of 166.6 ± 1.3 nm and Zeta potential of −12.5 ± 0.2 mV. The Nnps were formed by denaturation of co-existing plant proteins with molecular weight < 30 kDa. The Nnps adsorbed or dispersed BBR, thereby promoting BBR transformation from crystal to amorphous form and improving its solubility and dissolution. The Nnps carried and promoted BBR uptake by human colonic adenocarcinoma (Caco-2) cells via caveolae-mediated endocytosis, reducing P-gp-mediated efflux of BBR in mice gut sacs and Madin-Darby canine kidney cells stably expressing the transporter P-gp (MDCK-MDR1) cells. Moreover, the Nnps improved BBR metabolic stability in mouse intestinal S9, promoting BBR intestinal absorption in mice, as shown by increased peak BBR concentration (Cmax, 1182.3 vs 310.2 ng/mL) and exposure level (AUC0–12 h, 2842.8 vs 1447.0 ng·h/mL) in mouse portal vein. In addition, the Nnps increased BBR exposure level in mouse livers (95,443.2 vs 43,586.2 ng·h/g liver).ConclusionThe proteinaceous nanoparticles isolated from Coptidis Rhizoma extract can form a natural nano-drug delivery system with BBR, thereby significantly improving the pharmacokinetics of oral BBR.

Effects of sodium butyrate on intestinal barrier of severe scald mice and the related mechanism

Objective: To investigate the effects of sodium butyrate on intestinal barrier of the severe scald mice and the related mechanism. Methods: Eighteen C57BL/6 female mice, aged eight to twelve weeks, were divided into sham scald group, pure scald group, and scald+ sodium butyrate group according to random number table, with 6 mice in each group. Back of each mouse in pure scald group and scald+ sodium butyrate group were immersed into 90 ℃ water for 9 s, causing full-thickness scald of 30% total body surface area, while back of each mouse in sham scald group were immersed into 37 ℃ water for 9 s, causing sham injury. All of the mice in 3 groups were intraperitoneally injected with 1 mL sterile lactated Ringer's solution immediately after injury. Besides, mice in scald+ sodium butyrate group were intraperitoneally injected with 300 mg/kg sodium butyrate at 30 min before injury and immediately after injury, while mice in sham scald group and pure scald group were intraperitoneally injected with the same volume of sterile phosphate buffer solution. At post injury hour (PIH) 24, portal vein of mice in 3 groups was harvested, intestinal permeability was measured by fluorescin isothiocyanate-dextran fluorescence probe tracing method, then lileal tissue of mice in 3 groups was harvested, protein expressions of zonula occludens l (ZO-1), occludin, claudin-1, claudin-2, nucleotide-binding oligomerization domain-containing protein-like receptor family pyrin domain containing 3 (NLRP3), interleukin-1β (IL-1β), and IL-18 were detected by Western blotting, and distribution of ZO-1 in intestinal mucosa was observed by indirect immunofluorescence. Data were processed with one-way analysis of variance, least-significant difference test, and Bonferroni correction. Results: (1) At PIH 24, the intestinal permeability of mice in sham scald group, pure scald group, and scald+ sodium butyrate group was 0.88±0.19, 2.62±0.48, 1.23±0.16, respectively. Compared with that in sham scald group, the intestinal permeability of mice in pure scald group was significantly elevated (P<0.01), while the intestinal permeability of mice in scald+ sodium butyrate group showed no obvious change (P>0.05). Compared with that in pure scald group, the intestinal permeability of mice in scald+ sodium butyrate group was significantly decreased (P<0.01). (2) At PIH 24, compared with those in sham scald group, the protein expressions of ZO-1, occludin, and claudin-1 of mice in pure scald group and scald+ sodium butyrate group were significantly decreased (P<0.05), while the protein expression of claudin-2 was significantly increased (P<0.05). At PIH 24, compared with those of pure scald group, the protein expressions of ZO-1 and occludin of mice in scald+ sodium butyrate group were significantly elevated (P<0.05), while the protein expression of claudin-2 was significantly decreased (P<0.05), the protein expression of claudin-1 showed no significant difference (P>0.05). (3) At PIH 24, compared with those in sham scald group, the protein expressions of NLRP3, IL-1β, and IL-18 of mice in pure scald group and scald+ sodium butyrate group were significantly increased (P<0.05). Compared with those of pure scald group, the protein expressions of NLRP3, IL-1β, and IL-18 of mice in scald+ sodium butyrate group were significantly decreased (P<0.05). (4) At PIH 24, ZO-1 in intestinal mucosa of mice in sham scald group was distributed smoothly, continuously and homogeneously along the membrane. ZO-1 in intestinal mucosa of mice in pure scald group was distributed unsmoothly with breaks. The distribution of ZO-1 in intestinal mucosa of mice in scald+ sodium butyrate group was ameliorated compared with that in pure scald group. Conclusions: Sodium butyrate can inhibit the activation of NLRP3 inflammasome and decrease the production of IL-1β and IL-18 in intestinal mucosa of severe scald mice, which protects the intestinal barrier function by alleviating the alteration of tight junction protein expression and localization.

Automated In Vivo Navigation of Magnetic-Driven Microrobots Using OCT Imaging Feedback.

The application of in vivo microrobot navigation has received considerable attention from the field of precision therapy, which uses microrobots in living organisms. This study investigates the navigation of microrobots in vivo using optical coherence tomography (OCT) imaging feedback. The electromagnetic gradient field generated by a home-made electromagnetic manipulation system is magnetically modeled. With this model, the magnetic force acting on the microrobot is calculated, and the relationship between this force and the velocity of the microrobot is characterized. Results are verified through in vitro experiments wherein microrobots are driven in three types of fluid, namely, normal saline, gastric juice, and mouse urine. In vivo experiments are performed to navigate the microrobot in a mouse portal vein in which the OCT imaging system tracks the microrobot in vivo. Experimental results demonstrate the effectiveness of the proposed approach. The microrobots can be magnetically driven in the in vivo environment using the OCT imaging feedback. The significance of this study lies in providing a new method of driving microrobots in vivo.

English

Nanoparticles can act as drug carriers that can modulate pharmacokinetics, increase bioavailability and target release with minimal toxic effects. The present work aimed to assess the therapeutic effect and electron microscopic changes of Nigella sativa loaded Chitosan Nanoparticles (NSLCN) on adult Schistosoma mansoni in vitro. Adult worms were removed from the portal and mesenteric veins of infected mice after 90 days, and then three to five mature worms including both sexes were cultured. Schistosoma adult was exposed to NSLCN at concentrations of (10, 20, 40, 60, 80, and 100 &mu;g/ml) for 24, and 48 h. Examination for worm viability was done after 24, and 48 h using a stereomicroscope comparing with control negative and control positive groups. The mortality rate in worms reached 88.9% in the group treated with 100 &mu;g and 80 and 84.6% in groups treated with 80 and 60 &mu;g respectively (p-value &lt;0.001). After 48 h of incubation with the same concentration, there were variable effects on motility and death of worms, the death rate reached 100% in all groups treated with nanoparticles. After 24 h incubation, the live worms have sluggish motility and reached dead score at 48 h of incubation. By (SEM) there were tegumental changes of both dead male and female in the form of loss of spines, swollen suckers and swollen inter tubercular ridges in male and loss of smooth architecture of female tegument with multiple pores. In conclusion, NSLCN appears as a new potential candidate drug against schistosomiasis. We successfully applied nanoemulsion preparation against the adult stage of S. mansoni in vitro. Key words: Schistosoma mansoni, Nigella sativa, chitosan nanoparticles, scanning electron microscope. &nbsp

Purification of Hepatocyte and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

The liver is primarily composed of hepatocytes and the nonparenchymal cells which include stellate cells, Kupffer cells, sinusoidal endothelial cells and various other cell populations in very low numbers such as transient leukocytes. Cell isolation with high purity is required for many experiments related to the specific biology and biochemistry of these very different cell types. Here, we outline a relatively simple procedure to obtain a high yield of live hepatocytes and sinusoidal endothelial cells. Additionally, this procedure is relatively simple and does not require specialized equipment other than a refrigerated table top centrifuge and peristatic pump. The mouse portal vein is used as the catheterization site rather than the vena cava, limiting contamination of other cell types during the isolation process. Access to the vein is quick minimizing ischemia and reperfusion of the liver which reduces hepatic cell viability. Dead hepatocytes are also easily identified during centrifugation steps due to differences in cell density. This process allows for isolation of hepatocytes with 99% cell purity and isolation of sinusoidal endothelial cells with approximately 89.1% cell purity. This method will help to answer key questions in the hepatotology field such as determining specific cellular functions with in vitro assays or assessing individual cells after an in vivo treatment.Support or Funding InformationFunding is provided in part by the NIH from grant R01HL130864.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Subunit composition of native ATP-sensitive K+ channels in mouse portal vein smooth muscle

Subunit composition of native ATP-sensitive K+ channels in mouse portal vein smooth muscle

Evaluation of the pharmacokinetic-pharmacodynamic relationship of praziquantel in the Schistosoma mansoni mouse model.

After more than 40 years of use, Praziquantel (PZQ) still remains the drug of choice for the treatment of intestinal and urogenital schistosomiasis. Its anti-parasitic activity resides primarily in the (R)-enantiomer. Hitherto neither the molecular target nor the pharmacokinetic-pharmacodynamic relationship have been fully elucidated. Here we investigated the efficacy and pharmacokinetics of PZQ in the Schistosoma mansoni mouse model to determine the key factors that drive its efficacy. Dose-response studies with racemic PZQ with or without addition of an irreversible pan-cytochrome P450 (CYP) inhibitor, 1-aminobenzotriazole (ABT), were performed. In addition, efficacy of PZQ in the presence of the CYP inducer, dexamethasone (DEX), was determined. Plasma samples were obtained by tail vein bleeding at 4 time points. The (R)-PZQ levels were determined using a LC-MS/MS method. Non-compartmental pharmacokinetic analysis was performed using PKsolver. In addition, experiments using an enhanced in vitro assay were conducted. We found that the use of ABT increased (R)-PZQ plasma exposures in the systemic circulation by ~10 to 20 fold but the latter were not predictive of efficacy. The use of DEX decreased plasma exposures of (R)-PZQ in the systemic circulation by ~10 fold without reducing efficacy. We extrapolated the (R)-PZQ concentrations in mouse portal vein / mesenteric veins from the systemic exposures and found that a free exposure of (R)-PZQ of ~ 20 μM*h in the portal vein was needed to obtain a worm burden reduction >60%. It is suggested that the high (R)-PZQ concentrations available before the hepatic first pass metabolism drive the efficacy against S. mansoni adult worms residing in the mesenteric veins. It is then possible that the current dosing regimen of 40 mg/kg in preventive chemotherapy programs may provide suboptimal concentrations in low-weight patients such as children, due to smaller total amounts of drug administered, and may consequently result in lower cure rates.

ZD0947, a sulphonylurea receptor modulator, detects functional sulphonylurea receptor subunits in murine vascular smooth muscle ATP-sensitive K+ channels

In order to identify functional sulphonylurea receptor (SUR.x) subunits of native ATP-sensitive K+ channels (KATP channels) in mouse portal vein, the effects of ZD0947, a SUR.x modulator, were investigated on spontaneous portal vein contractions, macroscopic membrane currents and unitary currents recorded (using patch-clamp techniques) in freshly dispersed mouse portal vein myocytes. Spontaneous contractions in mouse portal vein were reversibly reduced by ZD0947 in a concentration-dependent manner (Ki =293nM). The relaxation elicited by 3µM ZD0947 was antagonized by the additional application of glibenclamide (300nM), but not gliclazide (100–300nM). In the conventional whole-cell configuration, 100µM ZD0947 elicited inward glibenclamide-sensitive currents at a holding potential of −60mV that demonstrated selectivity for K+(i.e. KATP currents). The peak amplitude of the membrane current elicited by 30µM or 100µM ZD0947 was smaller than that elicited by 100µM pinacidil at −60mV. In the cell-attached mode, 100µM ZD0947 activated glibenclamide-sensitive K+ channels with a conductance (35 pS) similar to that of recombinant Kir6.1/SUR2B channels that were expressed in HEK293 cells and activated by 100µM ZD0947. These results demonstrate that ZD0947 caused a significant vascular relaxation through the activation of KATP channels and that SUR2B may be the major functional subunit of SUR.x in mouse portal vein KATP channels, based on its pharmacological selectivity.

Molecular analysis of ATP-sensitive K+ channel subunits expressed in mouse portal vein

BackgroundSeveral combinations of inwardly rectifying K+ channel 6.x family pore-forming (KIR6.x) subunits associated with sulphonylurea receptor (SUR.x) subunits have been detected among ATP-sensitive K+ (KATP) channels. It remains to be established which of these is expressed in native vascular smooth muscle. MethodsPharmacological and electrophysiological properties of KATP channels in mouse portal vein were investigated using tension measurements and patch-clamp techniques. Molecular biological analyses were also performed to investigate the structural properties of these channels. ResultsSpontaneous contractions in mouse portal vein were reversibly reduced by pinacidil and MCC-134, and the pinacidil-induced relaxation was antagonized by glibenclamide and U-37883A. In cell-attached mode, pinacidil activated glibenclamide-sensitive K+ channels with a conductance (35 pS) similar to that of KIR6.1. RT-PCR analysis revealed the expression of KIR6.1, KIR6.2 and SUR2B transcripts. Using real-time PCR methods, the quantitative expression of KIR6.1 was much greater than that of KIR6.2. Immunohistochemical studies indicated the presence of KIR6.1 and SUR2B proteins in the smooth muscle layers of mouse portal vein and in single smooth muscle cells dispersed from mouse portal vein. ConclusionsThe results indicate that native KATP channels in mouse portal vein are likely to be composed of a heterocomplex of KIR6.1 and SUR2B subunits.

Marked Antitumor Effect of NK012, a SN-38-incorporating Micelle formulation, in a Newly Developed Mouse Model of Liver Metastasis Resulting From Gastric Cancer

Gastric cancer with liver metastasis (LM) is associated with poor prognosis due to rapid progression. It is, therefore, important to develop a quantitative and highly reproducible animal model of LM using human gastric cancer cells. Cells of a human gastric cancer cell line, HSC-57, were injected into the portal vein to produce LMs. Cells from some of these metastatic foci were expanded in vitro and subsequently implanted into the portal veins of mice. This procedure was repeated nine times. The antitumor effects of CPT-11 and NK012 were compared using the LM model. The potent metastatic clone 57L9 was obtained. NK012 exerted a stronger antitumor effect than CPT-11 against 57L9 cells integrated with the luciferase gene (57L9Luc). The survival rates on day 131 in the 57L9Luc mouse model were 100% and 0% for the NK012 and CPT-11 groups, respectively. This 57L9Luc LM model was found to be useful for monitoring the responses to NK012 and CPT-11.

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

In organs, the correct architecture of vascular and ductal structures is indispensable for proper physiological function, and the formation and maintenance of these structures is a highly regulated process. The analysis of these complex, 3-dimensional structures has greatly depended on either 2-dimensional examination in section or on dye injection studies. These techniques, however, are not able to provide a complete and quantifiable representation of the ductal or vascular structures they are intended to elucidate. Alternatively, the nature of 3-dimensional plastic resin casts generates a permanent snapshot of the system and is a novel and widely useful technique for visualizing and quantifying 3-dimensional structures and networks. A crucial advantage of the resin casting system is the ability to determine the intact and connected, or communicating, structure of a blood vessel or duct. The structure of vascular and ductal networks are crucial for organ function, and this technique has the potential to aid study of vascular and ductal networks in several ways. Resin casting may be used to analyze normal morphology and functional architecture of a luminal structure, identify developmental morphogenetic changes, and uncover morphological differences in tissue architecture between normal and disease states. Previous work has utilized resin casting to study, for example, architectural and functional defects within the mouse intrahepatic bile duct system that were not reflected in 2-dimensional analysis of the structure(1,2), alterations in brain vasculature of a Alzheimer's disease mouse model(3), portal vein abnormalities in portal hypertensive and cirrhotic mice(4), developmental steps in rat lymphatic maturation between immature and adult lungs(5), immediate microvascular changes in the rat liver, pancreas, and kidney in response in to chemical injury(6). Here we present a method of generating a 3-dimensional resin cast of a mouse vascular or ductal network, focusing specifically on the portal vein and intrahepatic bile duct. These casts can be visualized by clearing or macerating the tissue and can then be analyzed. This technique can be applied to virtually any vascular or ductal system and would be directly applicable to any study inquiring into the development, function, maintenance, or injury of a 3-dimensional ductal or vascular structure.

P63RhoGEF Couples G αq/11 -Mediated Signaling to Ca 2+ Sensitization of Vascular Smooth Muscle Contractility

In normal and diseased vascular smooth muscle (SM), the RhoA pathway, which is activated by multiple agonists through G protein-coupled receptors (GPCRs), plays a central role in regulating basal tone and peripheral resistance. This occurs through inhibition of myosin light chain phosphatase, leading to increased phosphorylation of the myosin regulatory light chain. Although it is thought that specific agonists and GPCRs may couple to distinct RhoA guanine nucleotide exchange factors (GEFs), thus raising the possibility of selective targeting of specific GEFs for therapeutic use, this notion is largely unexplored for SM contraction. We examine whether p63RhoGEF, known to couple specifically to Gα(q/11) in vitro, is functional in blood vessels as a mediator of RhoA activation and if it is selectively activated by Gα(q/11) coupled agonists. We find that p63RhoGEF is present across SM tissues and demonstrate that silencing of the endogenous p63RhoGEF in mouse portal vein inhibits contractile force induced by endothelin-1 to a greater extent than the predominantly Gα(12/13)-mediated thromboxane analog U46619. This is because endothelin-1 acts on Gα(q/11) as well as Gα(12/13). Introduction of the exogenous isolated pleckstrin-homology (PH) domain of p63RhoGEF (residues 331-580) into permeabilized rabbit portal vein inhibited Ca2+ sensitized force and activation of RhoA, when phenylephrine was used as an agonist. This reinforces the results based on endothelin-1, because phenylephrine is thought to act exclusively through Gα(q/11). We demonstrate that p63RhoGEF selectively couples Gα(q/11) but not Gα(12/13), to RhoA activation in blood vessels and cultured cells and thus mediates the physiologically important Ca2+ sensitization of force induced with Gα(q/11)-coupled agonists. Our results suggest that signaling through p63RhoGEF provides a novel mechanism for selective regulation of blood pressure.

Small and intermediate conductance Ca2+‐activated K+channels regulate spontaneous contraction of mouse portal vein myocytes

Ca2+‐activated K+ channels (KCa) are key components of vascular myocytes EC coupling through their control of smooth muscle cell membrane potential. Whilst BKCa is expressed ubiquitously in smooth muscle cells, only proliferative VSMCs express KCa3.1 channels. However, mouse portal vein (mPV) is an atypical blood vessel showing spontaneous and rhythmic contractions. Since KCa channels are involved in the regulation of excitability of other cell types like neurons, the contribution of KCa2.x and KCa3.1 channels to the control of mPV excitability was investigated. Expression of KCa2.x and KCa3.1 was detected in mPV by PCR and immunohistochemistry. In single cell patch clamp studies, the RMP of isolated mPV myocytes was hyperpolarized (≈15 mV) by exposure to NS309 (10 μM), a KCa2.x and KCa3.1 channel activator. Inhibition of KCa2.x channels with UCL1689 (3 μM) reversed the effect of NS309 whilst inhibition of KCa3.1 channels (TRAM‐34; 10 μM) had no effect. NS309 significantly decreased the amplitude and increased the frequency of spontaneous contractions of intact mPV. These effects were reversed by application of UCL1689, suggesting that the NS309‐induced changes in spontaneous mPV contractions is carried out by KCa2.x channels. This study suggest that KCa2.x and KCa3.1 channels are expressed in mouse portal vein myocytes and play an important role in regulation of spontaneous contractions.Supported by FICM

Functional role of voltage‐gated Na+ channels in rat mesenteric arteries

We have previously reported that voltage-gated Na+ channels (NaV) are expressed in freshly dissociated smooth muscle cells from the mouse portal vein, which displays spontaneous, rhythmic contractions. However, the physiological function of NaV in smooth muscle is controversial. Here, we explored the role of NaV in arterial vascular contractility. Isolated mesenteric arteries from male Wistar rats (200–300g) were mounted in a wire-myograph for tension recording. The NaV activator veratridine elicited concentration-dependent (1–30μM), but endothelium-independent, contractions. These contractions (at 10μM, 8.8±0.7mN; n=8) were greatly inhibited by 1μM tetrodotoxin (TTX), a NaV blocker (1.3±0.9mN, n=4) and abolished in Na+-free physiological salt solution (−0.2±0.1mN, n=4). To probe the expression of NaV isoforms (NaV1.1–1.9), we used conventional reverse transcriptase polymerase chain reaction (RT-PCR) with mRNA extracted from the isolated vessels. The presence of TTX-sensitive NaV isoforms (1.2, 1.3, 1.6 and 1.7) was evident. Based on these data, we propose that functional NaV are expressed in arteries without spontaneous rhythmic contractions and may play a role in the regulation of vascular tone. We acknowledge the financial support from St George's University of London and British Heart Foundation

Distinct Effects of Voltage- and Store-dependent Calcium Influx on Stretch-induced Differentiation and Growth in Vascular Smooth Muscle

Stretch of the vascular wall stimulates smooth muscle hypertrophy by activating the MAPK and Rho/Rho kinase (ROK) pathways. We investigated the role of calcium in this response. Stretch-stimulated expression of contractile and cytoskeletal proteins in mouse portal vein was inhibited at mRNA and protein levels by blockade of voltage-dependent Ca(2+) entry (VDCE). In contrast, blockade of store-operated Ca(2+) entry (SOCE) did not affect smooth muscle marker expression but decreased global protein synthesis. Activation of VDCE caused membrane translocation of RhoA followed by phosphorylation of its downstream effectors LIMK-2 and cofilin-2. Stretch-activated cofilin-2 phosphorylation depended on VDCE but not on SOCE. VDCE was associated with increased mRNA expression of myocardin, myocyte enhancer factor (MEF) -2A and -2D, and smooth muscle marker genes, all of which depended on ROK activity. SOCE increased ERK1/2 phosphorylation and c-Fos expression but had no effect on phosphorylation of LIMK-2 and cofilin-2 or on myocardin and MEF2 expression. Knockdown of MEF2A or -2D eliminated the VDCE-induced activation of myocardin expression and increased basal c-Jun and c-Fos mRNA levels. These results indicate that MEF2 mediates VDCE-dependent stimulation of myocardin expression via the Rho/ROK pathway. In addition, SOCE activates the expression of immediate-early genes, known to be regulated by MEF2 via Ca(2+)-dependent phosphorylation of histone deacetylases, but this mode of Ca(2+) entry does not affect the Rho/ROK pathway. Compartmentation of Ca(2+) entry pathways appears as one mechanism whereby extracellular and membrane signals influence smooth muscle phenotype regulation, with MEF2 as a focal point.