Low Oxygen Tension Enhances Expression of Myogenic Genes When Human Myoblasts Are Activated from G0 Arrest

Most cell culture studies have been performed at atmospheric oxygen tension of 21%, however the physiological oxygen tension is much lower and is a factor that may affect skeletal muscle myoblasts. In this study we have compared activation of G0 arrested myoblasts in 21% O2 and in 1% O2 in order to see how oxygen tension affects activation and proliferation of human myoblasts.Human myoblasts were isolated from skeletal muscle tissue and G0 arrested in vitro followed by reactivation at 21% O2 and 1% O2. The effect was assesses by Real-time RT-PCR, immunocytochemistry and western blot.We found an increase in proliferation rate of myoblasts when activated at a low oxygen tension (1% O2) compared to 21% O2. In addition, the gene expression studies showed up regulation of the myogenesis related genes PAX3, PAX7, MYOD, MYOG (myogenin), MET, NCAM, DES (desmin), MEF2A, MEF2C and CDH15 (M-cadherin), however, the fraction of DES and MYOD positive cells was not increased by low oxygen tension, indicating that 1% O2 may not have a functional effect on the myogenic response. Furthermore, the expression of genes involved in the TGFβ, Notch and Wnt signaling pathways were also up regulated in low oxygen tension. The differences in gene expression were most pronounced at day one after activation from G0-arrest, thus the initial activation of myoblasts seemed most sensitive to changes in oxygen tension. Protein expression of HES1 and β-catenin indicated that notch signaling may be induced in 21% O2, while the canonical Wnt signaling may be induced in 1% O2 during activation and proliferation of myoblasts

Spatial and phenotypic characterization of pancreatic cancer-associated fibroblasts after neoadjuvant treatment

Pancreatic ductal adenocarcinoma (PC) is characterized by a highly fibrotic desmoplastic stroma. Subtypes of cancer-associated fibroblasts (CAFs) have been identified in chemotherapy-naïve PC (CTN-PC), but their precise functions are still unclear. Our knowledge regarding the properties of CAFs in the regressive stroma after neoadjuvant treatment (NAT) of PC (NAT-PC) is particularly limited. We aimed to examine the marker phenotypic properties of CAFs in the regressive stroma of PC. Surgical specimens from patients with CTN-PC (n=10) and NAT-PC (n=10) were included. Juxtatumoural, peripheral, lobular, septal, peripancreatic, and regressive stromal compartments were manually outlined using digital imaging analysis (DIA) for area quantification. The compartment-specific expression of CD271, cytoglobin, DOG-1, miR-21, osteonectin, PDGF-Rβ, and tenascin C was evaluated by immunohistochemistry or in situ hybridization, using manual scoring and automated DIA. The area fraction of the regressive stroma was significantly higher in NAT-PC than in CTN-PC (P=0.0002). CD271 (P<0.01), cytoglobin (P<0.05), DOG1 (P<0.05), miR-21 (P<0.05), and tenascin C (P<0.05) exhibited significant differences in their expression profiles between the juxtatumoural compared to the peripheral and regressive stroma. PDGF-Rβ expression was significantly higher in juxtatumoural than in peripheral CAFs (P<0.05). Our data provide further support of the concept of stromal heterogeneity and phenotypic different CAF subtypes in PC. CAFs in the regressive stroma of NAT- PC show a marker phenotype similar to some (namely, peripheral) and different from other (namely, juxtatumoural) previously defined CAF subtypes. It may be hypothesized that phenotypic CAF subtypes, at least in part, also may share functional properties. Studies examining the precise functional characteristics of CAF subtypes in PC are neede

The expression of <i>HIF1A</i> was down regulated in myoblasts cultured in 1% O₂ compared to 21% O₂.

<p>The genes <i>TGF-β1</i>, <i>SMAD3</i>, <i>SMAD7</i>, <i>DCN</i> and <i>INHBA</i> are all part of the TGF-β signalling pathway and were all up regulated in myoblasts cultured in 1% O₂ for all three days. Filled symbols: 1% O₂; Empty symbols: 21% O₂. Significance level: * p<0.05, ** p<0.01.</p

Myoblast purity and proliferation rate.

<p>The purity of the isolated myoblasts was tested by desmin stainings and differentiation assays (A). Almost all of the isolated cells were desmin positive confirming a high level of myoblast purity and the myoblasts were able to form large multinucleated myofiber when cultured in differentiation medium. Proliferation rate of myoblasts in 1% O₂ and 21% O₂, respective, was measured by proliferation assays (three days) and colony forming assays (14 days). The short-term proliferation rate demonstrated no difference in myoblast proliferation (B). The colony forming assays (crystal violet staining) (C) demonstrated no difference in the number of colonies formed by myoblasts in 1% and 21% O₂, however, the colonies formed in 1% O₂ were bigger and had a higher cell density, thus demonstrating an induced proliferation. Scale bar: 400 μm.</p

Immunostainings for NCAM and DES were performed on myoblasts activated from G₀ arrest under hypoxic and normoxic conditions.

<p>We found large number of cells expressing both NCAM and DES, however, when quantitating the fraction of NCAM and DES positive cells, we found no consistent result pointed towards an up regulation in 1% O2, as seen in the <i>NCAM</i> and <i>DES</i> gene expressions. Scale bar: 50 μm.</p

Low Oxygen Tension Enhances Expression of Myogenic Genes When Human Myoblasts Are Activated from G₀ Arrest

<div><p>Objectives</p><p>Most cell culture studies have been performed at atmospheric oxygen tension of 21%, however the physiological oxygen tension is much lower and is a factor that may affect skeletal muscle myoblasts. In this study we have compared activation of G₀ arrested myoblasts in 21% O₂ and in 1% O₂ in order to see how oxygen tension affects activation and proliferation of human myoblasts.</p><p>Materials and Methods</p><p>Human myoblasts were isolated from skeletal muscle tissue and G₀ arrested <i>in vitro</i> followed by reactivation at 21% O₂ and 1% O₂. The effect was assesses by Real-time RT-PCR, immunocytochemistry and western blot.</p><p>Results and Conclusions</p><p>We found an increase in proliferation rate of myoblasts when activated at a low oxygen tension (1% O₂) compared to 21% O₂. In addition, the gene expression studies showed up regulation of the myogenesis related genes <i>PAX3</i>, <i>PAX7</i>, <i>MYOD</i>, <i>MYOG</i> (myogenin), <i>MET</i>, <i>NCAM</i>, <i>DES</i> (desmin), <i>MEF2A</i>, <i>MEF2C</i> and <i>CDH15</i> (M-cadherin), however, the fraction of DES and MYOD positive cells was not increased by low oxygen tension, indicating that 1% O₂ may not have a functional effect on the myogenic response. Furthermore, the expression of genes involved in the TGFβ, Notch and Wnt signaling pathways were also up regulated in low oxygen tension. The differences in gene expression were most pronounced at day one after activation from G₀-arrest, thus the initial activation of myoblasts seemed most sensitive to changes in oxygen tension. Protein expression of HES1 and β-catenin indicated that notch signaling may be induced in 21% O₂, while the canonical Wnt signaling may be induced in 1% O₂ during activation and proliferation of myoblasts.</p></div

Immunostainings for PAX7 and MYOD were performed on myoblasts activated from G₀ arrest in 1% O₂ and 21% O₂.

<p>We observed an up regulation of MYOD when myoblasts were activated from G₀ arrest. However, no difference in the number of PAX7 and MYOD positive cells were found in 1% O₂ compared to 21% O₂. Scale bar: 50 μm.</p

The Notch and Wnt signalling pathways were affected by hypoxic conditions when human myoblasts were activated from G₀ arrest.

<p>The expression of <i>NOTCH1</i>, <i>NOTCH3</i> and <i>HES1</i> were up regulated in 1% O₂ for all three days, while <i>DLL1</i>, <i>JAG1</i> and <i>NUMB</i> were up regulated on day one only. However, the HES1 protein expression was induced in 21% O₂ on day 2 and 3. In the Wnt signalling pathway we observed an up regulation of <i>FZD7</i>, <i>LRP6</i> and <i>SFRP1</i> on day 1–3 in 1% O₂, whereas <i>WNT5B</i>, <i>AXIN2</i> and <i>CTNNB1</i> was up regulated only on day one. Accordingly, the protein expression of total and active non-phosphorylated β-catenin was up regulated in 1% O₂ in particular on day 1. Filled symbols: 1% O₂; Empty symbols: 21% O₂. Significance level: * p<0.05, ** p<0.01.</p

The expression of <i>PAX3</i>, <i>PAX7</i>, MRFs and genes involved in myogenesis were studied in human myoblasts re-activation from G₀-arrest in hypoxic and normoxic culture conditions.

<p>The up regulation of <i>PAX7</i> was highly significant in 1% O₂ for all three days in hypoxic conditions, while <i>PAX3</i> was increased only on day one. <i>MYOD</i> and <i>MYOG</i> were up regulated in 1% O₂, whereas <i>MYF5</i> and <i>MYF6</i> had similar expression levels at 1% O₂ and 21% O₂. The myogenesis related genes <i>C-MET</i>, <i>MEF2A</i>, <i>MEF2C</i>, <i>NCAM</i>, <i>DES</i> and <i>CDH15</i> all had increased expression levels in 1% O₂. Thus, most of the genes involved in the myogenic programme were induced at 1% O₂. Filled symbols: 1% O₂; Empty symbols: 21% O₂. Significance level: * p<0.05, ** p<0.01.</p