Hedgehog pathway responsiveness correlates with the presence of primary cilia on prostate stromal cells

<p>Abstract</p> <p>Background</p> <p>Hedgehog (Hh) signaling from the urogenital sinus (UGS) epithelium to the surrounding mesenchyme plays a critical role in regulating ductal formation and growth during prostate development. The primary cilium, a feature of most interphase vertebrate cell types, serves as a required localization domain for Hh signaling transducing proteins.</p> <p>Results</p> <p>Immunostaining revealed the presence of primary cilia in mesenchymal cells of the developing prostate. Cell-based assays of a urongenital sinus mesenchymal cell line (UGSM-2) revealed that proliferation-limiting (serum starvation and/or confluence) growth conditions promoted cilia formation and correlated with pathway activation associated with accumulation of Smoothened in primary cilia. The prostate cancer cell lines PC-3, LNCaP, and 22RV1, previously shown to lack demonstrable autocrine Hh signaling capacity, did not exhibit primary cilia even under proliferation-limiting growth conditions.</p> <p>Conclusion</p> <p>We conclude that paracrine Hedgehog signaling activity in the prostate is associated with the presence of primary cilia on stromal cells but that a role in autocrine Hh signaling remains speculative.</p

Establishment and characterization of immortalized Gli-null mouse embryonic fibroblast cell lines

<p>Abstract</p> <p>Background</p> <p>Hedgehog (Hh) signaling is a conserved morphogenetic pathway which plays critical roles in embryonic development, with emerging evidence also supporting a role in healing and repair processes and tumorigenesis. The Gli family of transcription factors (Gli1, 2 and 3) mediate the Hedgehog morphogenetic signal by regulating the expression of downstream target genes. We previously characterized the individual and cooperative roles of the Gli proteins in Hh target gene regulation using a battery of primary embryonic fibroblasts from Gli null mice.</p> <p>Results</p> <p>Here, we describe the establishment of spontaneously immortalized mouse embryonic fibroblast (iMEF) cell lines lacking single and multiple Gli genes. These non-clonal cell lines recapitulate the unique ligand mediated transcriptional response of primary MEFs. While loss of Gli1 had no effect on target gene induction, Gli2 null cells demonstrated reduced target gene induction while Gli3 null cells exhibited elevated basal and ligand-induced expression. Target gene response in <it>Gli1</it>^-/-<it>2</it>^-/-iMEFs was severely reduced while <it>Gli2</it>^-/-<it>3</it>^-/-iMEFs were incapable of ligand-induced transcriptional response. However, we found that both <it>Gli1</it>^-/-<it>2</it>^-/-and <it>Gli2</it>^-/-<it>3</it>^-/-iMEFs exhibited robust leukotriene synthesis-dependent migration responses to Hh ligand, demonstrating that this response is not transcriptionally-dependent.</p> <p>Conclusion</p> <p>This study provides fundamental characterizations of the transcriptional and non-transcriptional Hh responsiveness of a battery of Gli-null iMEFs. Moving forward, these cell lines should prove a valuable tool set to study the unique functional regulation of the Gli proteins in a Hh-responsive cell-type.</p

Cleft lip and palate results from Hedgehog signaling antagonism in the mouse: Phenotypic characterization and clinical implications

The Hedgehog (Hh) pathway provides inductive signals critical for developmental patterning of the brain and face. In humans as well as in animal model systems, interference with this pathway yields birth defects; among the most well-studied of which fall within the holoprosencephaly (HPE) spectrum

Downregulation of FIP200 Induces Apoptosis of Glioblastoma Cells and Microvascular Endothelial Cells by Enhancing Pyk2 Activity

The expression of focal adhesion kinase family interacting protein of 200-kDa (FIP200) in normal brain is limited to some neurons and glial cells. On immunohistochemical analysis of biopsies of glioblastoma tumors, we detected FIP200 in the tumor cells, tumor-associated endothelial cells, and occasional glial cells. Human glioblastoma tumor cell lines and immortalized human astrocytes cultured in complete media also expressed FIP200 as did primary human brain microvessel endothelial cells (MvEC), which proliferate in culture and resemble reactive endothelial cells. Downregulation of endogenous expression of FIP200 using small interfering RNA resulted in induction of apoptosis in the human glioblastoma tumor cells, immortalized human astrocytes, and primary human brain MvEC. It has been shown by other investigators using cells from other tissues that FIP200 can interact directly with, and inhibit, proline-rich tyrosine kinase 2 (Pyk2) and focal adhesion kinase (FAK). In the human glioblastoma tumor cells, immortalized human astrocytes, and primary human brain MvEC, we found that downregulation of FIP200 increased the activity of Pyk2 without increasing its expression, but did not affect the activity or expression of FAK. Coimmunoprecipitation and colocalization studies indicated that the endogenous FIP200 was largely associated with Pyk2, rather than FAK, in the glioblastoma tumor cells and brain MvEC. Moreover, the pro-apoptotic effect of FIP200 downregulation was inhibited significantly by a TAT-Pyk2-fusion protein containing the Pyk2 autophosphorylation site in these cells. In summary, downregulation of endogenous FIP200 protein in glioblastoma tumor cells, astrocytes, and brain MvECs promotes apoptosis, most likely due to the removal of a direct interaction of FIP200 with Pyk2 that inhibits Pyk2 activation, suggesting that FIP200 expression may be required for the survival of all three cell types found in glioblastoma tumors

A phantom based evaluation of vessel lumen area quantification for coronary CT angiography

Coronary computed tomography (CT) angiography is a noninvasive method for visualizing coronary artery disease. However, coronary CT angiography is limited in assessment of stenosis severity by the partial volume effect and calcification. Therefore, an accurate method for assessment of stenosis severity is needed. A 10&nbsp;cm diameter cylindrical Lucite phantom with holes in the range of 0.4-4.5&nbsp;mm diameter was fitted in a chest phantom. The holes were filled with an iodine solution of 8&nbsp;mg/mL. To simulate coronary artery disease, different levels of stenosis were created by inserting Lucite rods into the holes with diameter range of 2-4.5&nbsp;mm. The resulting lumen cross sectional areas ranged from 1.4 to 12.3 mm2. To simulate arterial calcification, calcium hydroxyapatite rods were inserted into the holes with diameter range of 2-4.5&nbsp;mm. Images of the phantoms were acquired at 100 kVp using a 320-slice CT scanner. A maual and a semi-automated technique based on integrated Hounsfield units was used to calculate vessel cross-sectional area. There was an excellent correlation between the measured and the known cross-sectional area for both normal and stenotic vessels using the manual and the semi-automated techniques. However, the overall measurement error for the manual method was more than twice as compared with the integrated HU technique. Determination of vessel lumen area using the semi-automated integrated Hounsfield unit technique yields more than a factor of two improvement in precision and accuracy as compared to the existing manual technique for vessels with and without stenosis. This technique can also be used to accurately measure arterial cross-sectional area in the presence of coronary calcification

Functional Assessment of Coronary Artery Disease Using Whole-Heart Dynamic Computed Tomographic Perfusion

Computed tomographic (CT) angiography is an important tool for the evaluation of coronary artery disease but often correlates poorly with myocardial ischemia. Current dynamic CT perfusion techniques can assess ischemia but have limited accuracy and deliver high radiation dose. Therefore, an accurate, low-dose, dynamic CT perfusion technique is needed. A total of 20 contrast-enhanced CT volume scans were acquired in 5 swine (40±10 kg) to generate CT angiography and perfusion images. Varying degrees of stenosis were induced using a balloon catheter in the proximal left anterior descending coronary artery, and a pressure wire was used for reference fractional flow reserve (FFR) measurement. Perfusion measurements were made with only 2 volume scans using a new first-pass analysis (FPA) technique and with 20 volume scans using an existing maximum slope model (MSM) technique. Perfusion (P) and FFR measurements were related by PFPA=1.01 FFR-0.03 (R2=0.85) and PMSM=1.03 FFR-0.03 (R2=0.80) for FPA and MSM techniques, respectively. Additionally, the effective radiation doses were calculated to be 2.64 and 26.4 mSv for FPA and MSM techniques, respectively. A new FPA-based dynamic CT perfusion technique was validated in a swine animal model. The results indicate that the FPA technique can potentially be used for improved anatomical and functional assessment of coronary artery disease at a relatively low radiation dose

Comprehensive Assessment of Coronary Artery Disease by Using First-Pass Analysis Dynamic CT Perfusion: Validation in a Swine Model

Purpose To retrospectively validate a first-pass analysis (FPA) technique that combines computed tomographic (CT) angiography and dynamic CT perfusion measurement into one low-dose examination. Materials and Methods The study was approved by the animal care committee. The FPA technique was retrospectively validated in six swine (mean weight, 37.3 kg ± 7.5 [standard deviation]) between April 2015 and October 2016. Four to five intermediate-severity stenoses were generated in the left anterior descending artery (LAD), and 20 contrast material-enhanced volume scans were acquired per stenosis. All volume scans were used for maximum slope model (MSM) perfusion measurement, but only two volume scans were used for FPA perfusion measurement. Perfusion measurements in the LAD, left circumflex artery (LCx), right coronary artery, and all three coronary arteries combined were compared with microsphere perfusion measurements by using regression, root-mean-square error, root-mean-square deviation, Lin concordance correlation, and diagnostic outcomes analysis. The CT dose index and size-specific dose estimate per two-volume FPA perfusion measurement were also determined. Results FPA and MSM perfusion measurements (PFPA and PMSM) in all three coronary arteries combined were related to reference standard microsphere perfusion measurements (PMICRO), as follows: PFPA_COMBINED = 1.02 PMICRO_COMBINED + 0.11 (r = 0.96) and PMSM_COMBINED = 0.28 PMICRO_COMBINED + 0.23 (r = 0.89). The CT dose index and size-specific dose estimate per two-volume FPA perfusion measurement were 10.8 and 17.8 mGy, respectively. Conclusion The FPA technique was retrospectively validated in a swine model and has the potential to be used for accurate, low-dose vessel-specific morphologic and physiologic assessment of coronary artery disease. © RSNA, 2017

The Implementation and Outcomes of a Nurse-Run Extracorporeal Membrane Oxygenation Program, a Retrospective Single-Center Study.

Due to a shortage of perfusionists and increasing utilization of extracorporeal membrane oxygenation in the United States, many programs are training nurses as bedside extracorporeal membrane oxygenation specialists (i.e., nurse-run extracorporeal membrane oxygenation). Our objective was to evaluate if a nurse-run extracorporeal membrane oxygenation program has noninferior survival to discharge and complication rates compared with a perfusionist-run extracorporeal membrane oxygenation program. Additionally, to sought to describe increases in extracorporeal membrane oxygenation capacity and the potential for cost savings by implementing a nurse-run extracorporeal membrane oxygenation program