Decreased mechanical properties of heart valve tissue constructs cultured in platelet lysate as compared to fetal bovine serum.

In autologous heart valve tissue engineering, there is an ongoing search for alternatives of fetal bovine serum (FBS). Human platelet-lysate (PL) might be a promising substitute. In the present article, we aimed to examine the tissue formation, functionality, and mechanical properties of engineered three-dimensional tissue constructs cultured in PL as a substitute for FBS. Our results show that tissue constructs that were cultured in PL and FBS produce similar amounts of collagen, glycosoaminoglycans, and collagen crosslinks, and that the cellular phenotype remains unchanged. Nevertheless, mechanical testing showed that the ultimate tensile strength in PL constructs was on average approximately three times lower as compared to FBS (0.25 vs. 0.74 MPa, respectively, p<0.01), and also the elastic modulus was almost three times lower (1.33 MPa of PL constructs vs. 3.94 MPa of FBS constructs, p<0.01). Additional tests indicated that this difference might be explained by different collagen fiber architecture possibly due to increased production of matrix-degrading proteases by cells cultured in PL. In summary, our results indicate that PL is not preferred for the culture of strong heart valve tissue constructs.

Mobility of the von Hippel-Lindau tumour suppressor protein is regulated by kinesin-2.

The von Hippel-Lindau tumour suppressor protein (pVHL) participates in many cellular processes including oxygen sensing, microtubule stability and primary cilia regulation. Recently, we identified ATP-dependent motor complex kinesin-2 to endogenously bind the full-length variant of VHL (pVHL30) in primary kidney cells, and mediate its association to microtubules. Here we show that pVHL also endogenously binds the neuronal kinesin-2 complex, which slightly differs from renal kinesin-2. To investigate the role of kinesin-2 in pVHL mobility, we performed fluorescence recovery after photobleaching (FRAP) experiments in neuroblastoma cells. We observe that pVHL30 is a highly mobile cytoplasmic protein, which becomes an immobile centrosomal protein after ATP-depletion in living cells. This response to ATP-depletion is independent of GSK3beta-dependent phosphorylation of pVHL30. Furthermore, VHL variant alleles with reduced binding to kinesin-2 fail to respond to ATP-depletion. Accordingly, interfering with pVHL30-KIF3A interaction by either overexpressing a dominant negative construct or by reducing endogenous cellular levels of KIF3A by RNAi abolishes pVHL's response to ATP-depletion. From these data we suggest that mobility of a subcellular pool of pVHL is regulated by the ATP-dependent kinesin-2 motor. Kinesin-2 driven mobility of cytoplasmic pVHL might enable pVHL to function as a tumour suppressor.

The von Hippel-Lindau tumour suppressor interacts with microtubules through kinesin-2.

Synthesis and maintenance of primary cilia are regulated by the von Hippel-Lindau (VHL) tumour suppressor protein. Recent studies indicate that this regulation is linked to microtubule-dependent functions of pVHL such as orienting microtubule growth and increasing plus-end microtubule stability, however little is known how this occurs. We have identified the kinesin-2 motor complex, known to regulate cilia, as a novel and endogenous pVHL binding partner. The interaction with kinesin-2 facilitates pVHL binding to microtubules. These data suggest that microtubule-dependent functions of pVHL are influenced by kinesin-2.

Polarisation, key to good localisation.

Polarisation of cells is crucial for vectorial transport of ions and solutes. In literature, however, proteins specifically targeted to the apical or basolateral membrane are often studied in non-polarised cells. To investigate whether these data can be extrapolated to expression in polarised cells, we studied several membrane-specific proteins. In polarised MDCK cells, the Aquaporin-2 water channel resides in intracellular vesicles and apical membrane, while the vasopressin-type 2 receptor, anion-exchanger 1 (AE1) protein and E-Cadherin mainly localise to the basolateral membrane. In non-polarised MDCK cells, however, Aquaporin-2 localises, besides plasma membrane, mainly in the Golgi complex, while the others show a dispersed staining throughout the cell. Moreover, while AQP2 mutants in dominant nephrogenic diabetes insipidus are missorted to different organelles in polarised cells, they all predominantly localise to the Golgi complex in non-polarised MDCK cells. Additionally, the maturation of V2R, and likely its missorting, is affected in transiently-transfected compared to stably-transfected cells. In conclusion, we show that the use of stably-transfected polarised cells is crucial in interpreting the processing and the localisation of membrane targeted proteins.

The von Hippel-Lindau tumor suppressor protein influences microtubule dynamics at the cell periphery.

The von Hippel-Lindau (VHL) protein protects microtubules (MTs) from destabilization by nocodazole treatment. Based on this fixed-cell assay with static end points, VHL has been reported to directly stabilize the MT cytoskeleton. To investigate the dynamic changes in MTs induced by VHL in living cells, we measured the influence of VHL on tubulin turnover using fluorescence recovery after photobleaching (FRAP). To this end, we engineered VHL-deficient renal cell carcinoma cells to constitutively incorporate fluorescently labeled tubulin and to inducibly express VHL. Induction of VHL in these cells resulted in a decrease of tubulin turnover as measured by FRAP at the cell periphery, while minimally influencing MT dynamics around the centrosome. Our data indicates that VHL changes the behavior of MTs dependent on their subcellular localization implying a role for VHL in cellular processes such as migration, polarization, and cell-cell interactions. Here we propose a complementary method to directly measure VHL-induced subcellular changes in microtubule dynamics, which may serve as a tool to study the effect of MT binding proteins such as VHL.

Beta-catenin up-regulates the expression of the urokinase plasminogen activator in human colorectal tumors.

Expression of the urokinase plasminogen activator (uPA) increases during the progression of colorectal tumors from adenomas to carcinomas. The highest amounts of uPA are found at the invasion front of carcinomas, which also displays a strong expression of nuclear beta-catenin and is therefore a region expressing beta-catenin target genes at high levels. Here we show that beta-catenin contributes to the transactivation of uPA. Therefore, beta-catenin might have an impact on the capacity of colorectal tumors for invasion and metastasis, as well as dormancy, which are hallmarks of cancer.

Fibronectin is a hypoxia-independent target of the tumor suppressor VHL.

The von Hippel-Lindau (VHL) tumor suppressor gene regulates the extracellular matrix by controlling fibronectin deposition. To identify novel VHL target genes, we subjected mRNA from VHL-deficient RCC cells (786-0-pRC) and a transfectant re-expressing wildtype VHL (786-0-VHL) to differential expression profiling. Among the differentially expressed genes, we detected that fibronectin is upregulated in the presence of VHL, while it is not affected by hypoxia. Thus regulation of fibronectin deposition by VHL occurs at the transcriptional level, irrespective of oxygen levels.