Characterization of isolated liver sinusoidal endothelial cells for liver bioengineering.

BACKGROUND: The liver sinusoidal capillaries play a pivotal role in liver regeneration, suggesting they may be beneficial in liver bioengineering. This study isolated mouse liver sinusoidal endothelial cells (LSECs) and determined their ability to form capillary networks in vitro and in vivo for liver tissue engineering purposes. METHODS AND RESULTS: In vitro LSECs were isolated from adult C57BL/6 mouse livers. Immunofluorescence labelling indicated they were LYVE-1+/CD32b+/FactorVIII+/CD31-. Scanning electron microscopy of LSECs revealed the presence of characteristic sieve plates at 2 days. LSECs formed tubes and sprouts in the tubulogenesis assay, similar to human microvascular endothelial cells (HMEC); and formed capillaries with lumens when implanted in a porous collagen scaffold in vitro. LSECs were able to form spheroids, and in the spheroid gel sandwich assay produced significantly increased numbers (p = 0.0011) of capillary-like sprouts at 24 h compared to HMEC spheroids. Supernatant from LSEC spheroids demonstrated significantly greater levels of vascular endothelial growth factor-A and C (VEGF-A, VEGF-C) and hepatocyte growth factor (HGF) compared to LSEC monolayers (p = 0.0167; p = 0.0017; and p < 0.0001, respectively), at 2 days, which was maintained to 4 days for HGF (p = 0.0017) and VEGF-A (p = 0.0051). In vivo isolated mouse LSECs were prepared as single cell suspensions of 500,000 cells, or as spheroids of 5000 cells (100 spheroids) and implanted in SCID mouse bilateral vascularized tissue engineering chambers for 2 weeks. Immunohistochemistry identified implanted LSECs forming LYVE-1+/CD31- vessels. In LSEC implanted constructs, overall lymphatic vessel growth was increased (not significantly), whilst host-derived CD31+ blood vessel growth increased significantly (p = 0.0127) compared to non-implanted controls. LSEC labelled with the fluorescent tag DiI prior to implantation formed capillaries in vivo and maintained LYVE-1 and CD32b markers to 2 weeks. CONCLUSION: Isolated mouse LSECs express a panel of vascular-related cell markers and demonstrate substantial vascular capillary-forming ability in vitro and in vivo. Their production of liver growth factors VEGF-A, VEGF-C and HGF enable these cells to exert a growth stimulus post-transplantation on the in vivo host-derived capillary bed, reinforcing their pro-regenerative capabilities for liver tissue engineering studies.

Differential SKIP expression in PTEN-deficient glioblastoma regulates cellular proliferation and migration.

Glioblastoma is the most common and lethal primary malignant brain tumor in adults. The tumor suppressor gene PTEN is deleted, mutated or hypermethylated in more than 60% of glioblastoma cases resulting in hyperactivation of the phosphoinositide 3-kinase pathway, which leads to sustained PI(3,4,5)P3 signaling, and thereby hyperactivation of Akt and other effectors. PI(3,4,5)P3 is also hydrolyzed to PI(3,4)P2 by inositol polyphosphate 5-phosphatases such as SKIP, but the role this pathway has in glioblastoma is unknown. Microarray expression profiling of SKIP in human glioblastoma has revealed both increased and decreased SKIP gene expression. Here we have screened PTEN-deficient glioblastoma for SKIP protein expression by immunohistochemistry and report that SKIP expression is increased in some cases or decreased relative to normal brain. Using the U-87MG PTEN-deficient cell line we show that SKIP knockdown did not further enhance cell proliferation or survival. However, SKIP overexpression in U-87MG cells suppressed anchorage-independent cell growth and growth factor-induced PI(3,4,5)P3/Akt signaling. Although, SKIP knockdown did not affect cell proliferation or survival, cell migration was significantly retarded, associated with significantly increased PI(4,5)P2 signals, and decreased phosphorylation of the actin-regulatory protein cofilin, a PI(4,5)P2-binding protein. Notably, overexpression of SKIP also inhibited migration of U-87MG cells to a similar degree as observed with PTEN reconstitution, however, via distinct mechanisms. PTEN reconstitution promoted sustained lamellipodia generation and focal adhesion formation. In contrast, SKIP overexpression reduced sustained lamellipodia formation, talin incorporation into focal adhesions and recruitment of PI(4,5)P2-binding proteins to the plasma membrane. Notably, analysis of two independent ONCOMINE microarray data sets revealed a significant correlation between increased SKIP mRNA expression in glioblastoma and improved long-term survival. Therefore, SKIP expression in glioblastoma may affect the local invasion of PTEN-deficient tumors.

Mammalian inositol polyphosphate 5-phosphatase II can compensate for the absence of all three yeast Sac1-like-domain-containing 5-phosphatases.

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] plays a complex role in generating intracellular signalling molecules, and also in regulating actin-binding proteins, vesicular trafficking and vacuolar fusion. Four inositol polyphosphate 5-phosphatases (hereafter called 5-phosphatases) have been identified in Saccharomyces cerevisiae: Inp51p, Inp52p, Inp53p and Inp54p. Each enzyme contains a 5-phosphatase domain which hydrolyses PtdIns(4,5)P(2), forming PtdIns4P, while Inp52p and Inp53p also express a polyphosphoinositide phosphatase domain within the Sac1-like domain. Disruption of any two yeast 5-phosphatases containing a Sac1-like domain results in abnormalities in actin polymerization, plasma membrane, vacuolar morphology and bud-site selection. Triple null mutant 5-phosphatase strains are non-viable. To investigate the role of PtdIns(4,5)P(2) in mediating the phenotype of double and triple 5-phosphatase null mutant yeast, we determined whether a mammalian PtdIns(4,5)P(2) 5-phosphatase, 5-phosphatase II, which lacks polyphosphoinositide phosphatase activity, could correct the phenotype of triple 5-phosphatase null mutant yeast and restore cellular PtdIns(4,5)P(2) levels to near basal values. Mammalian 5-phosphatase II expressed under an inducible promoter corrected the growth, cell wall, vacuolar and actin polymerization defects of the triple 5-phosphatase null mutant yeast strains. Cellular PtdIns(4,5)P(2) levels in various 5-phosphatase double null mutant strains demonstrated significant accumulation (4.5-, 3- and 2-fold for Deltainp51Deltainp53, Deltainp51Deltainp52 and Deltainp52Deltainp53 double null mutants respectively), which was corrected significantly following 5-phosphatase II expression. Collectively, these studies demonstrate the functional and cellular consequences of PtdIns(4,5)P(2) accumulation and the evolutionary conservation of function between mammalian and yeast PtdIns(4,5)P(2) 5-phosphatases.

Cloning and characterization of a 72-kDa inositol-polyphosphate 5-phosphatase localized to the Golgi network.

The inositol-polyphosphate 5-phosphatase enzyme family removes the 5-position phosphate from both inositol phosphate and phosphoinositide signaling molecules. We have cloned and characterized a novel 5-phosphatase, which demonstrates a restricted substrate specificity and tissue expression. The 3.9-kb cDNA predicts for a 72-kDa protein with an N-terminal proline rich domain, a central 5-phosphatase domain, and a C-terminal CAAX motif. The 3. 9-kilobase mRNA showed a restricted expression but was abundant in testis and brain. Antibodies against the sequence detected a 72-kDa protein in the testis in the detergent-insoluble fraction. Indirect immunofluorescence of the Tera-1 cell line using anti-peptide antibodies to the 72-kDa 5-phosphatase demonstrated that the enzyme is predominantly located to the Golgi. Expression of green fluorescent protein-tagged 72-kDa 5-phosphatase in COS-7 cells revealed that the enzyme localized predominantly to the Golgi, mediated by the N-terminal proline-rich domain, but not the C-terminal CAAX motif. In vitro, the protein inserted into microsomal membranes on the cytoplasmic face of the membrane. Immunoprecipitated recombinant 72-kDa 5-phosphatase hydrolyzed phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3, 5-bisphosphate, forming phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3-phosphate, respectively. We propose that the novel 5-phosphatase hydrolyzes phosphatidylinositol 3,4, 5-trisphosphate and phosphatidylinositol 3,5-bisphosphate on the cytoplasmic Golgi membrane and thereby may regulate Golgi-vesicular trafficking.