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Liver Sinusoidal Endothelial Cells Escape Senescence by Loss of p19ARF.

Koudelkova P, Weber G, Mikulits W - PLoS ONE (2015)

Bottom Line: In line, treatment with small molecule inhibitors against VEGFR-2 caused cell death, demonstrating the sustained ability of p19ARF-/- LSECs to respond to anti-angiogenic therapeutics.From these data we conclude that loss of p19ARF overcomes senescence of LSECs, allowing immortalization of cells without losing endothelial characteristics.Thus, p19ARF-/- LSECs provide a novel cellular model to study endothelial cell biology.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine I, Division: Institute of Cancer Research, Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria.

ABSTRACT
Liver sinusoidal endothelial cells (LSECs) represent a highly differentiated cell type that lines hepatic sinusoids. LSECs form a discontinuous endothelium due to fenestrations under physiological conditions, which are reduced upon chronic liver injury. Cultivation of rodent LSECs associates with a rapid onset of stress-induced senescence a few days post isolation, which limits genetic and biochemical studies ex vivo. Here we show the establishment of LSECs isolated from p19ARF-/- mice which undergo more than 50 cell doublings in the absence of senescence. Isolated p19ARF-/- LSECs display a cobblestone-like morphology and show the ability of tube formation. Analysis of DNA content revealed a stable diploid phenotype after long-term passaging without a gain of aneuploidy. Notably, p19ARF-/- LSECs express the endothelial markers CD31, vascular endothelial growth factor receptor (VEGFR)-2, VE-cadherin, von Willebrand factor, stabilin-2 and CD146 suggesting that these cells harbor and maintain an endothelial phenotype. In line, treatment with small molecule inhibitors against VEGFR-2 caused cell death, demonstrating the sustained ability of p19ARF-/- LSECs to respond to anti-angiogenic therapeutics. From these data we conclude that loss of p19ARF overcomes senescence of LSECs, allowing immortalization of cells without losing endothelial characteristics. Thus, p19ARF-/- LSECs provide a novel cellular model to study endothelial cell biology.

No MeSH data available.


Related in: MedlinePlus

Endothelial integrity of mLSECs.(A) Western blot analysis of early and late passaged mLSECs using anti-CD31, anti-VE-cadherin and anti-VEGFR-2 antibodies. The EC phenotype of mLSECs were compared to blood endothelial cells (BECs) and hHSECs. Immortalized p19ARF-deficient hepatocytes, termed MIM1-4, were used as a negative control. Actin is shown as a loading control. (B) qPCR analyses of Stab-1, Stab-2, vWF, Lyve-1, CD32b and CD146 in mLSECs. MIM1-4 cells served as a negative control. RhoA was used as a housekeeping gene. (C) Migratory abilities of mLSECs, BECs and HSECs as shown by wound healing assay in medium containing ECGS. Quantification of phase contrast images of cells after wounding (0 hours) and after wound closure (20 hours). (D) Tube formation of mLSECs as detected after cytokine stimulation.
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pone.0142134.g002: Endothelial integrity of mLSECs.(A) Western blot analysis of early and late passaged mLSECs using anti-CD31, anti-VE-cadherin and anti-VEGFR-2 antibodies. The EC phenotype of mLSECs were compared to blood endothelial cells (BECs) and hHSECs. Immortalized p19ARF-deficient hepatocytes, termed MIM1-4, were used as a negative control. Actin is shown as a loading control. (B) qPCR analyses of Stab-1, Stab-2, vWF, Lyve-1, CD32b and CD146 in mLSECs. MIM1-4 cells served as a negative control. RhoA was used as a housekeeping gene. (C) Migratory abilities of mLSECs, BECs and HSECs as shown by wound healing assay in medium containing ECGS. Quantification of phase contrast images of cells after wounding (0 hours) and after wound closure (20 hours). (D) Tube formation of mLSECs as detected after cytokine stimulation.

Mentions: Next we analyzed more closely the EC phenotype of mLSECs. Immunoblotting showed that mLSECs express CD31, VE-cadherin and VEGFR-2 similarly to hHSECs and the human TERT-immortalized blood endothelial cells (BECs, Fig 2A). Interestingly, mLSECs retained expression of these markers during cell passaging. Furthermore, we analyzed other published markers for their expression in mLSECs. vWF, Stab-2 and CD146 which are reported as characteristic markers of rodent liver endothelium were detected in both, early and late passaged cells. In contrast, Stab-1, Lyve-1 and CD32b expression were not increased in comparison to immortalized MIM1-4 hepatocytes (Fig 2B). In addition, endothelial cells are endowed with a high migratory capacity which precedes angiogenesis in vivo and which could be observed by wound healing assays upon stimulation with ECGS. Notably, mLSECs displayed a capability of wound closure which is comparable to established cultures of BECs and hHSECs (Fig 2C). To further demonstrate the endothelial integrity of isolated mLSECs, we performed tube formation assays on growth factor-reduced Matrigel. Formation of vessel-like structures upon stimulation with pro-angiogenic mitogens is a particular feature of all ECs [32–34]. Isolated mLSECs generated tube-like structures upon stimulation with VEGF-A, ECGS or a combination of both, whereas no tubes were detected without stimulation (Fig 2D). From these data we concluded that mLSECs exhibit characteristics that are typical for ECs.


Liver Sinusoidal Endothelial Cells Escape Senescence by Loss of p19ARF.

Koudelkova P, Weber G, Mikulits W - PLoS ONE (2015)

Endothelial integrity of mLSECs.(A) Western blot analysis of early and late passaged mLSECs using anti-CD31, anti-VE-cadherin and anti-VEGFR-2 antibodies. The EC phenotype of mLSECs were compared to blood endothelial cells (BECs) and hHSECs. Immortalized p19ARF-deficient hepatocytes, termed MIM1-4, were used as a negative control. Actin is shown as a loading control. (B) qPCR analyses of Stab-1, Stab-2, vWF, Lyve-1, CD32b and CD146 in mLSECs. MIM1-4 cells served as a negative control. RhoA was used as a housekeeping gene. (C) Migratory abilities of mLSECs, BECs and HSECs as shown by wound healing assay in medium containing ECGS. Quantification of phase contrast images of cells after wounding (0 hours) and after wound closure (20 hours). (D) Tube formation of mLSECs as detected after cytokine stimulation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4631446&req=5

pone.0142134.g002: Endothelial integrity of mLSECs.(A) Western blot analysis of early and late passaged mLSECs using anti-CD31, anti-VE-cadherin and anti-VEGFR-2 antibodies. The EC phenotype of mLSECs were compared to blood endothelial cells (BECs) and hHSECs. Immortalized p19ARF-deficient hepatocytes, termed MIM1-4, were used as a negative control. Actin is shown as a loading control. (B) qPCR analyses of Stab-1, Stab-2, vWF, Lyve-1, CD32b and CD146 in mLSECs. MIM1-4 cells served as a negative control. RhoA was used as a housekeeping gene. (C) Migratory abilities of mLSECs, BECs and HSECs as shown by wound healing assay in medium containing ECGS. Quantification of phase contrast images of cells after wounding (0 hours) and after wound closure (20 hours). (D) Tube formation of mLSECs as detected after cytokine stimulation.
Mentions: Next we analyzed more closely the EC phenotype of mLSECs. Immunoblotting showed that mLSECs express CD31, VE-cadherin and VEGFR-2 similarly to hHSECs and the human TERT-immortalized blood endothelial cells (BECs, Fig 2A). Interestingly, mLSECs retained expression of these markers during cell passaging. Furthermore, we analyzed other published markers for their expression in mLSECs. vWF, Stab-2 and CD146 which are reported as characteristic markers of rodent liver endothelium were detected in both, early and late passaged cells. In contrast, Stab-1, Lyve-1 and CD32b expression were not increased in comparison to immortalized MIM1-4 hepatocytes (Fig 2B). In addition, endothelial cells are endowed with a high migratory capacity which precedes angiogenesis in vivo and which could be observed by wound healing assays upon stimulation with ECGS. Notably, mLSECs displayed a capability of wound closure which is comparable to established cultures of BECs and hHSECs (Fig 2C). To further demonstrate the endothelial integrity of isolated mLSECs, we performed tube formation assays on growth factor-reduced Matrigel. Formation of vessel-like structures upon stimulation with pro-angiogenic mitogens is a particular feature of all ECs [32–34]. Isolated mLSECs generated tube-like structures upon stimulation with VEGF-A, ECGS or a combination of both, whereas no tubes were detected without stimulation (Fig 2D). From these data we concluded that mLSECs exhibit characteristics that are typical for ECs.

Bottom Line: In line, treatment with small molecule inhibitors against VEGFR-2 caused cell death, demonstrating the sustained ability of p19ARF-/- LSECs to respond to anti-angiogenic therapeutics.From these data we conclude that loss of p19ARF overcomes senescence of LSECs, allowing immortalization of cells without losing endothelial characteristics.Thus, p19ARF-/- LSECs provide a novel cellular model to study endothelial cell biology.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine I, Division: Institute of Cancer Research, Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria.

ABSTRACT
Liver sinusoidal endothelial cells (LSECs) represent a highly differentiated cell type that lines hepatic sinusoids. LSECs form a discontinuous endothelium due to fenestrations under physiological conditions, which are reduced upon chronic liver injury. Cultivation of rodent LSECs associates with a rapid onset of stress-induced senescence a few days post isolation, which limits genetic and biochemical studies ex vivo. Here we show the establishment of LSECs isolated from p19ARF-/- mice which undergo more than 50 cell doublings in the absence of senescence. Isolated p19ARF-/- LSECs display a cobblestone-like morphology and show the ability of tube formation. Analysis of DNA content revealed a stable diploid phenotype after long-term passaging without a gain of aneuploidy. Notably, p19ARF-/- LSECs express the endothelial markers CD31, vascular endothelial growth factor receptor (VEGFR)-2, VE-cadherin, von Willebrand factor, stabilin-2 and CD146 suggesting that these cells harbor and maintain an endothelial phenotype. In line, treatment with small molecule inhibitors against VEGFR-2 caused cell death, demonstrating the sustained ability of p19ARF-/- LSECs to respond to anti-angiogenic therapeutics. From these data we conclude that loss of p19ARF overcomes senescence of LSECs, allowing immortalization of cells without losing endothelial characteristics. Thus, p19ARF-/- LSECs provide a novel cellular model to study endothelial cell biology.

No MeSH data available.


Related in: MedlinePlus