Tamoxifen-Activated CreERT Impairs Retinal Angiogenesis Independently of Gene Deletion

Brash, James T.; Bolton, Rebecca; Rashbrook, Victoria S.; Denti, Laura; Kubota, Yoshiaki; Ruhrberg, Christiana

doi:10.1161/circresaha.120.317025

Cited by 48 publications

(33 citation statements)

References 5 publications

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“…However, obesity-induced insulin resistance is associated with reduced vascular insulin receptor expression and impaired angiogenesis ( 63 ), so it would be interesting to explore endothelial VEGFR2 internalization and ERK signal transduction in this setting. Finally, our ECInsr +/- control data come from littermates expressing Tie2-Cre, and recent data show that Cre is not biologically inert ( 64 ); however, the similar retinal vascular phenotype of Insr +/- and ECInsr +/- mice provides some reassurance that off-target Cre effects do not underpin our findings.…”

Section: Discussionmentioning

confidence: 51%

Endothelial Insulin Receptors Promote VEGF-A Signaling via ERK1/2 and Sprouting Angiogenesis

et al. 2021

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Endothelial insulin receptors (Insr) promote sprouting angiogenesis, although the underpinning cellular and molecular mechanisms are unknown. Comparing mice with whole body insulin receptor haploinsufficiency (Insr +/-) against littermate controls, we found impaired limb perfusion and muscle capillary density after inducing hind-limb ischemia; this was in spite of increased expression of the pro-angiogenic growth factor Vegfa. Insr +/- neonatal retinas exhibited reduced tip cell number and branching complexity during developmental angiogenesis, which was also found in separate studies of mice with endothelium-restricted Insr haploinsufficiency. Functional responses to VEGF-A, including in vitro angiogenesis, were also impaired in aortic rings and pulmonary endothelial cells from Insr +/- mice. Human umbilical vein endothelial cells (HUVEC) with shRNA-mediated knockdown of Insr also demonstrated impaired functional angiogenic responses to VEGF-A. VEGF-A signaling to Akt and eNOS was intact, but downstream signaling to ERK1/2 was impaired, as was VEGF receptor-2 (VEGFR-2) internalization, which is required specifically for signaling to ERK1/2. Hence, endothelial insulin receptors facilitate the functional response to VEGF-A during angiogenic sprouting and are required for appropriate signal transduction from VEGFR-2 to ERK1/2.

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Section: Discussionmentioning

confidence: 51%

Endothelial Insulin Receptors Promote VEGF-A Signaling via ERK1/2 and Sprouting Angiogenesis

et al. 2021

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“…4B); overall the blood vessel network in Sox7 iECKO mutant embryos appeared intact and comparable to sibling controls. To rule out the possibility that the observed lymphatic patterning defect is due to CreERT2 toxicity (Brash et al, 2020), we created Cdh5-CreERT2: Sox7 fl/fl ; mT/mG and Cdh5-CreERT2 ; mT/mG animals. The mT/mG double fluorescence Cre-reporter mice express the tdTomato red fluorescent protein prior to Cre-mediated excision, and green fluorescent protein after excision, therefore allowing Cdh5-CreERT2 activity to be measured and traced (Muzumdar et al, 2007).…”

Section: Resultsmentioning

confidence: 99%

The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

Ikn

Jiang

et al. 2021

Preprint

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Despite a growing catalogue of secreted factors critical for lymphatic network assembly, little is known about the mechanisms that modulate the expression level of these molecular cues in blood vascular endothelial cells (BECs). Here, we show that a BEC-specific transcription factor, SOX7, plays a crucial role in lymphatic vessel patterning by modulating the transcription of lymphangiocrine signals. While SOX7 is not expressed in lymphatic endothelial cells (LECs), loss of SOX7 function in mouse embryos causes a dysmorphic dermal lymphatic phenotype. We identify novel distant regulatory regions in mice and humans that contribute to directly repressing the transcription of a major lymphangiogenic growth factor (Vegfc) in a SOX7-dependent manner. Further, we show that SOX7 directly binds HEY1, a canonical repressor of the Notch pathway, suggesting that transcriptional repression may also be modulated by the recruitment of this protein partner at Vegfc genomic regulatory regions. Our work unveils a role for SOX7 in modulating downstream signalling events crucial for lymphatic patterning, at least in part via the transcriptional repression of VEGFC levels in the blood vascular endothelium.

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“…Importantly, since it is well described that 4-OHT is toxic and the survival of the pups following its injection can be unpredictable 43 , our protocol also describes a method to minimize the exposure of mice to 4-OHT. Generally, the higher the dosage the higher the mortality rate and frequent injections can further induce damage to the treated organ/tissue 44 , 45 . Using small easily injectable reagent volumes, our protocol defines a low dosage treatment with minimal mortality rate (1/21).…”

Section: Discussionmentioning

confidence: 99%

Activation of creER recombinase in the mouse calvaria induces local recombination without effects on distant skeletal segments

Hou

Lin

Intini

2021

Sci Rep

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Conditional creER-mediated gene inactivation or gene induction has emerged as a robust tool for studying gene functions in mouse models of tissue development, homeostasis, and regeneration. Here, we present a method to conditionally induce cre recombination in the mouse calvarial bone while avoiding systemic recombination in distal bones. To test our method, we utilized Prx1creER-egfp;td-Tomato mice and delivered 4-hydroxytamoxifen (4-OHT) to the mouse calvaria, subperiosteally. First, we showed that two calvaria subperiosteal injections of 10 µg of 4-OHT (3.3 mg of 4-OHT/kg of body weight) can induce local recombination as efficiently as two intraperitoneal systemic injections of 200 μg of tamoxifen (70 mg of tamoxifen/kg of body weight). Then, we studied the recombination efficiency of various subperiosteal calvaria dosages and found that two subperiosteal injections of 5 µg 4-OHT (1.65 mg of 4-OHT/kg of body weight) uphold the same recombination efficiency observed with higher dosages. Importantly, the result indicated that the low dosage does not induce significant systemic recombination in remote skeletal tissues. With the proposed local low dosage protocol, the recombination efficiency at the injection site (calvarial bone) reached 94%, while the recombination efficiency at the mandible and the digits was as low as the efficiency measured in control animals.

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Tamoxifen-Activated CreERT Impairs Retinal Angiogenesis Independently of Gene Deletion

Cited by 48 publications

References 5 publications

Endothelial Insulin Receptors Promote VEGF-A Signaling via ERK1/2 and Sprouting Angiogenesis

Endothelial Insulin Receptors Promote VEGF-A Signaling via ERK1/2 and Sprouting Angiogenesis

The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

Activation of creER recombinase in the mouse calvaria induces local recombination without effects on distant skeletal segments

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