Type IV collagen drives alveolar epithelial-endothelial association and the morphogenetic movements of septation

Background: Type IV collagen is the main component of the basement membrane that gives strength to the blood-gas barrier (BGB). In mammals, the formation of a mature BGB occurs primarily after birth during alveologenesis and requires the formation of septa from the walls of the saccule. In contrast, in avians, the formation of the BGB occurs rapidly and prior to hatching. Mutation in basement membrane components results in an abnormal alveolar phenotype; however, the specific role of type IV collagen in regulating alveologenesis remains unknown. Results: We have performed a microarray expression analysis in late chick lung development and found that COL4A1 and COL4A2 were among the most significantly upregulated genes during the formation of the avian BGB. Using mouse models, we discovered that mutations in murine Col4a1 and Col4a2 genes affected the balance between lung epithelial progenitors and differentiated cells. Mutations in Col4a1 derived from the vascular component were sufficient to cause defects in vascular development and the BGB. We also show that Col4a1 and Col4a2 mutants displayed disrupted myofibroblast proliferation, differentiation and migration. Lastly, we revealed that addition of type IV collagen protein induced myofibroblast proliferation and migration in monolayer culture and increased the formation of mesenchymal-epithelial septal-like structures in co-culture. Conclusions: Our study showed that type IV collagen and, therefore the basement membrane, play fundamental roles in coordinating alveolar morphogenesis. In addition to its role in the formation of epithelium and vasculature, type IV collagen appears to be key for alveolar myofibroblast development by inducing their proliferation, differentiation and migration throughout the developing septum

Serum oxidized low-density lipoprotein is inversely correlated to telomerase activity in peripheral blood mononuclear cells of haemodialysis patients

Background. Telomerase preserves telomeres’ function and structure preventing cellular senescence. Its activity is reduced in peripheral blood mononuclear cells (PBMC) of haemodialysis (HD) patients. The purpose of this study is to investigate the potential correlation between increased oxidative stress/inflammation and telomerase activity in PBMC of HD patients. Methods: Telomerase activity was measured by PCR-ELISA in PBMC isolated from a group of 42 HD patients and 39 subjects with estimated glomerular filtration rate >= 80 mL/min (control group). Serum oxidized low-density lipoprotein (ox-LDL), tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) were also measured in both groups by ELISA. Results: Ox-LDL was negatively correlated to percentage telomerase activity in PBMC (r = -0.506, P = 0.000 in the whole group of 81 HD and normal subjects and r = -0.559, P < 0.001 in HD patients). TNF was also inversely associated with percentage telomerase activity in the whole group studied (r= -0.492, P= 0.000) while IL-10 was not. In stepwise multiple linear regression, taking into consideration the most important characteristics of the HD patients and control group, the only significant predictors for percentage telomerase activity in PBMC were ox-LDL and TNF (beta = -0.421, t= -4.083, P= 0.000 and beta= -0.381, t= -3.691, P= 0.000, respectively) while examining separately HD patients, the predictors for the same parameter were ox-LDL and HD duration (beta = -0.671, t = -4.709, P = 0.000 and beta= -0.349, t = -2.447, P = 0.023, respectively). Conclusion: Ox-LDL serum level is inversely correlated to telomerase activity in PBMC of HD patients. Our study proposes a new consequence of increased oxidative stress in HD patients: the premature cellular senescence potentially related to atherosclerosis through LDL oxidation

Type IV collagen drives alveolar epithelial-endothelial association and the morphogenetic movements of septation.

BackgroundType IV collagen is the main component of the basement membrane that gives strength to the blood-gas barrier (BGB). In mammals, the formation of a mature BGB occurs primarily after birth during alveologenesis and requires the formation of septa from the walls of the saccule. In contrast, in avians, the formation of the BGB occurs rapidly and prior to hatching. Mutation in basement membrane components results in an abnormal alveolar phenotype; however, the specific role of type IV collagen in regulating alveologenesis remains unknown.ResultsWe have performed a microarray expression analysis in late chick lung development and found that COL4A1 and COL4A2 were among the most significantly upregulated genes during the formation of the avian BGB. Using mouse models, we discovered that mutations in murine Col4a1 and Col4a2 genes affected the balance between lung epithelial progenitors and differentiated cells. Mutations in Col4a1 derived from the vascular component were sufficient to cause defects in vascular development and the BGB. We also show that Col4a1 and Col4a2 mutants displayed disrupted myofibroblast proliferation, differentiation and migration. Lastly, we revealed that addition of type IV collagen protein induced myofibroblast proliferation and migration in monolayer culture and increased the formation of mesenchymal-epithelial septal-like structures in co-culture.ConclusionsOur study showed that type IV collagen and, therefore the basement membrane, play fundamental roles in coordinating alveolar morphogenesis. In addition to its role in the formation of epithelium and vasculature, type IV collagen appears to be key for alveolar myofibroblast development by inducing their proliferation, differentiation and migration throughout the developing septum

Verteporfin-induced formation of protein cross-linked oligomers and high molecular weight complexes is mediated by light and leads to cell toxicity

Verteporfin (VP) was first used in Photodynamic therapy, where a non-thermal laser light (689 nm) in the presence of oxygen activates the drug to produce highly reactive oxygen radicals, resulting in local cell and tissue damage. However, it has also been shown that Verteporfin can have non-photoactivated effects such as interference with the YAP-TEAD complex of the HIPPO pathway, resulting in growth inhibition of several neoplasias. More recently, it was proposed that, another non-light mediated effect of VP is the formation of cross-linked oligomers and high molecular weight protein complexes (HMWC) that are hypothesized to interfere with autophagy and cell growth. Here, in a series of experiments, using human uveal melanoma cells (MEL 270), human embryonic kidney cells (HEK) and breast cancer cells (MCF7) we showed that Verteporfin-induced HMWC require the presence of light. Furthermore, we showed that the mechanism of this cross-linking, which involves both singlet oxygen and radical generation, can occur very efficiently even after lysis of the cells, if the lysate is not protected from ambient light. This work offers a better understanding regarding VP’s mechanisms of action and suggests caution when one studies the non-light mediated actions of this drug

Cross-Packaging and Capsid Mosaic Formation in Multiplexed AAV Libraries

International audienceGeneration and screening of libraries of adeno-associated virus (AAV) variants have emerged as a powerful method for identifying novel capsids for gene therapy applications. For the majority of libraries, vast population diversity requires multi-plexed production, in which a library of inverted terminal repeat (ITR)-containing plasmid variants is transfected together into cells to generate the viral library. This process has the potential to be confounded by cross-packaging and mosaicism, in which particles are comprised of genomes and capsid monomers derived from different library members. Here, we investigate the prevalence of cross-packaging and mosaicism in simplified, minimal libraries using novel assays designed to assess capsid composition and packaging fidelity. We show that AAV library variants are prone to cross-packaging and capsid mosaic formation when produced at high plasmid levels, although to a lesser extent than in a recombi-nant context. We also provide experimental evidence that dilution of input library DNA significantly increases capsid monomer homogeneity and increases capsid:genome correlation in AAV libraries. Lastly, we determine that similar dilution methods yield higher-quality libraries when used for in vivo screens. Together, these findings quantitatively characterized the prevalence of cross-packaging and mosaicism in AAV libraries and established conditions that minimize related noise in subsequent screens

Retinoblastoma cells are inhibited by aminoimidazole carboxamide ribonucleotide (AICAR) partially through activation of AMP-dependent kinase

5-Aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR), an analog of AMP, is widely used as an activator of AMP-kinase (AMPK), a protein that regulates the responses of the cell to energy change. We studied the effects of AICAR on the growth of retinoblastoma cell lines (Y79, WERI, and RB143). AICAR inhibited Rb cell growth, induced apoptosis and S-phase cell cycle arrest, and led to activation of AMPK. These effects were abolished by treatment with dypiridamole, an inhibitor that blocks entrance of AICAR into cells. Treatment with the adenosine kinase inhibitor 5-iodotubericidin to inhibit the conversion of AICAR to ZMP (the direct activator of AMPK) reversed most of the growth-inhibiting effects of AICAR, indicating that some of the antiproliferative effects of AICAR are mediated through AMPK activation. In addition, AICAR treatment was associated with inhibition of the mammalian target of rapamycin pathway, decreased phosphorylation of ribosomal protein-S6 and 4E-BP1, down-regulation of cyclins A and E, and decreased expression of p21. Our results indicate that AICAR-induced activation of AMPK inhibits retinoblastoma cell growth. This is one of the first descriptions of a nonchemotherapeutic drug with low toxicity that may be effective in treating Rb patients.—Theodoropoulou, S., Kolovou, P. E., Morizane, Y., Kayama, M., Nicolaou, F., Miller, J. W., Gragoudas, E., Ksander, B. R., Vavvas, D. G. Retinoblastoma cells are inhibited by aminoimidazole carboxamide ribonucleotide (AICAR) partially through activation of AMP-dependent kinase