Tgfß/Alk5 signaling is required for shear stress induced klf2 expression in embryonic endothelial cells.

Endothelial cells (EC) translate biomechanical forces into functional and phenotypic responses that play important roles in cardiac development. Specifically, EC in areas of high shear stress, i.e., in the cardiac outflow tract and atrioventricular canal, are characterized by high expression of Krüppel-like factor 2 (Klf2) and by transforming growth factor-beta (Tgfß)-driven endothelial-to-mesenchymal transition. Extraembryonic venous obstruction (venous clip model) results in congenital heart malformations, and venous clip-induced alterations in shear stress-related gene expression are suggestive for an increase in cardiac shear stress. Here, we study the effects of shear stress on Klf2 expression and Tgfß-associated signaling in embryonic EC in vivo using the venous clip model and in vitro by subjecting cultured EC to fluid flow. Cellular responses were assessed by analysis of Klf2, Tgfß ligands, and their downstream signaling targets. Results show that, in embryonic EC, shear stress activates Tgfß/Alk5 signaling and that induction of Klf2 is an Alk5 dependent process.

Generation of 3 human induced pluripotent stem cell lines LUMCi005-A, B and C from a Hereditary Cerebral Hemorrhage with Amyloidosis-Dutch type patient.

Hereditary Cerebral Hemorrhage with Amyloidosis-Dutch type (HCHWA-D) is an autosomal dominant hereditary disease caused by a point mutation in exon 17 of the APP gene. We generated human induced pluripotent stem cells (hiPSCs) from a symptomatic HCHWA-D patient by using non-integrating Sendai virus (SeV). The newly generated hiPSCs express all pluripotency markers, have a normal karyotype, carry the Dutch mutation, can differentiate in the three germ layers in vitro and are SeV free.

Prognostic value of apoptosis in rectal cancer patients of the dutch total mesorectal excision trial: radiotherapy is redundant in intrinsically high-apoptotic tumors.

PURPOSE: The combination of radiotherapy and good quality surgery reduces local recurrence rate for rectal cancer patients. This study assesses the prognostic value of both intrinsic and radiotherapy-induced apoptosis and evaluates the relevance of radiotherapy for outcome of rectal cancer patients. EXPERIMENTAL DESIGN: Tumor samples (1,198) were available from the Dutch Total Mesorectal Excision trial, in which rectal cancer patients were treated with standardized surgery and randomized for preoperative short-term radiotherapy or not. Tumor samples were obtained at time of surgery. Tissue microarrays were constructed and stained with the active caspase-specific M30 antibody to determine the amount of apoptotic epithelial tumor cells. RESULTS: Nonirradiated patients with a negative circumferential margin displaying lower than median levels of apoptosis developed more local recurrences (10.5% versus 6.1%; P=0.06) and more rapidly after surgery than patients with high intrinsic apoptosis in their tumors (median time to recurrence, 13.0 versus 21.3 months; P=0.04). In multivariate analysis, intrinsic apoptosis was an independent predictor for the development of local recurrences (hazard ratio, 2.0; P=0.05). Radiotherapy increased apoptosis level (11 versus 23 apoptotic cells/mm2 tumor epithelium; P<0.001), but this apoptosis did not influence patients' prognosis. CONCLUSIONS: Rectal cancer patients with low intrinsic apoptosis will benefit from radiotherapy with respect to the development of local recurrences. Because apoptosis is an inherent characteristic of tumors, patients who do not need radiotherapy may be selected based on the apoptotic index of the primary tumor.

Readthrough-Promoting Drugs Gentamicin and PTC124 Fail to Rescue Nav1.5 Function of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes Carrying Nonsense Mutations in the Sodium Channel Gene SCN5A.

BACKGROUND: Several compounds have been reported to induce translational readthrough of premature stop codons resulting in the production of full-length protein by interfering with ribosomal proofreading. Here we examined the effect of 2 of these compounds, gentamicin and PTC124, in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes bearing nonsense mutations in the sodium channel gene SCN5A, which are associated with conduction disease and potential lethal arrhythmias. METHODS AND RESULTS: We generated hiPSC from 2 patients carrying the mutations R1638X and W156X. hiPSC-derived cardiomyocytes from both patients recapitulated the expected electrophysiological phenotype, as evidenced by reduced Na+ currents and action potential upstroke velocities compared with hiPSC-derived cardiomyocytes from 2 unrelated control individuals. While we were able to confirm the readthrough efficacy of the 2 drugs in Human Embryonic Kidney 293 cells, we did not observe rescue of the electrophysiological phenotype in hiPSC-derived cardiomyocytes from the patients. CONCLUSIONS: We conclude that these drugs are unlikely to present an effective treatment for patients carrying the loss-of-function SCN5A gene mutations examined in this study.

Macrodissection versus microdissection of rectal carcinoma: minor influence of stroma cells to tumor cell gene expression profiles.

BACKGROUND: The molecular determinants of carcinogenesis, tumor progression and patient prognosis can be deduced from simultaneous comparison of thousands of genes by microarray analysis. However, the presence of stroma cells in surgically excised carcinoma tissues might obscure the tumor cell-specific gene expression profiles of these samples. To circumvent this complication, laser microdissection can be performed to separate tumor epithelium from the surrounding stroma and healthy tissue. In this report, we compared RNAs isolated from macrodissected, of which only surrounding healthy tissue had been removed, and microdissected rectal carcinoma samples by microarray analysis in order to determine the most reliable approach to detect the expression of tumor cell-derived genes by microarray analysis. RESULTS: As microdissection yielded low tissue and RNA quantities, extra rounds of mRNA amplification were necessary to obtain sufficient RNA for microarray experiments. These second rounds of amplification influenced the gene expression profiles. Moreover, the presence of stroma cells in macrodissected samples had a minor contribution to the tumor cell gene expression profiles, which can be explained by the observation that more RNA is extracted from tumor epithelial cells than from stroma. CONCLUSION: These data demonstrate that the more convenient procedure of macrodissection can be adequately used and yields reliable data regarding the identification of tumor cell-specific gene expression profiles.

Endothelial colony-forming cells show a mature transcriptional response to shear stress.

Endothelial progenitor cells (EPC) play a central role in endothelial maintenance and repair. Endothelial colony-forming cells (ECFC) form a subpopulation of EPC. ECFC are readily attainable, can be easily isolated, possess a high proliferation potential, and are therefore a promising source of endothelial cells (EC) for future cardiovascular therapeutic applications. The extent to which these cells respond to shear stress as adult vascular EC remains to be elucidated. Here, we study the transcriptional response of ECFC induced by shear stress and compare it with the response of mature arterial and venous cells. ECFC, as well as human umbilical vein EC (HUVEC) and human umbilical artery EC (HUAEC), were subjected to low (0.5 Pa) and high (2.5 Pa) shear stress. The endothelial differentiation phenotype and transcriptional responses were analyzed using immunocytochemistry and quantitative polymerase chain reaction (Q-PCR). Performing absolute quantification of copy numbers by Q-PCR allows comparing the responses of cell types relative to each other. Our data show that isolated ECFC resemble mature EC in cobblestone morphology and endothelial marker expression. Absolute Q-PCR quantification revealed that although being truly endothelial, ECFC do not fully resemble HUVEC or HUAEC in the expression of specific differentiation markers. When subjected to shear stress, ECFC show a mature response to fluid flow, comparable to that of HUVEC and HUAEC. The capacity of endothelial progenitors to respond to fluid flow in a similar manner to HUVEC and HUAEC highlights the universal response of EC to fluid shear stress, independently of their endothelial differentiation status. This property supports the use of these cells as an EC source for tissue engineering applications.

Primary cilia sensitize endothelial cells for fluid shear stress.

Primary cilia are mechanosensors for fluid shear stress, and are involved in a number of syndromes and congenital anomalies. We identified endothelial cilia in areas of low shear stress in the embryonic heart. The objective of the present study was to demonstrate the role of primary cilia in mechanosensing. Ciliated embryonic endothelial cells were cultured from the heart, and non-ciliated cells from the arteries. Non-ciliated cells that were subjected to fluid shear stress showed significantly less induction of the shear marker Krüppel-Like Factor-2, as compared to ciliated cells. In addition, ciliated cells from which the cilia were chemically removed show a similar decrease in flow response. This shows that primary cilia sensitize endothelial cells for fluid shear stress. In addition, we targeted and stabilized the connection of the cilium to the cytoplasm by treatment with Colchicine and Taxol/Paclitaxel, respectively, and show that microtubular integrity is essential to sense shear stress.

Gene expression profiling reveals two separate mechanisms regulating apoptosis in rectal carcinomas in vivo.

The level of apoptosis in rectal carcinomas of patients treated by surgery only predicts local failure; patients with intrinsically high-apoptotic tumors develop less local recurrences than patients with low levels of apoptosis. To identify genes involved in this intrinsic apoptotic process in vivo, 47 rectal tumors with known apoptotic phenotype (24 low- and 23 high-apoptotic) were analyzed by oligonucleotide microarray technology. We identified several genes differentially expressed between low- and high-apoptotic tumors. Unsupervised clustering of the tumors based on expression levels of these genes separated the low-apoptotic from the high-apoptotic tumors, indicating a gene expression-dependent regulation. In addition, this clustering revealed two subgroups of high-apoptotic tumors. One high-apoptotic subgroup showed subtle differences in mRNA and protein expression of the known apoptotic regulators BAX, cIAP2 and ARC compared to the low-apoptotic tumors. The other subgroup of high-apoptotic tumors showed high expression of immune-related genes; predominantly HLA class II and chemokines, but also HLA class I and interferon-inducible genes were highly expressed. Immunohistochemistry revealed HLA-DR expression in epithelial tumor cells in 70% of these high-apoptotic tumors. The expression data suggest that high levels of apoptosis in rectal carcinoma patients can be the result of either slightly altered expression of known pro- and anti-apoptotic genes or high expression of immune-related genes.

Lethal invasive cestodiasis in immunosuppressed patients.

Using both traditional methods and broad-range 18S ribosomal DNA (rDNA) polymerase chain reaction, we examined 2 cases of lethal cestodiasis, in which the disease agent had been poorly identified or misidentified. In one case, involving a patient with AIDS, we identified the human dwarf tapeworm, Hymenolepis nana, as a cause of aberrant metastatic larval disease. In the second case with similar pathologic abnormalities, involving a patient with Hodgkin disease, we identified a larval cestode with a previously uncharacterized 18S rDNA sequence. A prior report of this case nearly 30 years ago, based on tissue examination, had suggested that the parasite was a sparganum.