In Mice With Type 2 Diabetes, a Vascular Endothelial Growth Factor (VEGF)-Activating Transcription Factor Modulates VEGF Signaling and Induces Therapeutic Angiogenesis After Hindlimb Ischemia

Li, Yongjun; Hazarika, Surovi; Xie, Donghua; Pippen, Anne M.; Kontos, Christopher D.; Annex, Brian H.

doi:10.2337/db06-0999

Cited by 109 publications

(80 citation statements)

References 37 publications

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“…Therefore, the increased levels of surface VEGFR1 on day 10 of ischemia suggests a significant inductive role for VEGFR1 in ischemic signaling outcomes. Previous studies (32,46) have reported no changes in VEGFR1 protein levels in ischemic skeletal muscle compared with nonischemic skeletal muscle on days 3 and 10 after femoral artery ligation, despite an upregulation of VEGF-A protein levels at 3 days postischemia. Although total protein levels may not directly correlate with surface receptor levels, the recruitment of intracellularly localized VEGFR1 may explain the increased VEGFR1 surface levels that we observed.…”

Section: Cd31mentioning

confidence: 98%

Endothelial cell-by-cell profiling reveals the temporal dynamics of VEGFR1 and VEGFR2 membrane localization after murine hindlimb ischemia

Imoukhuede

Dokun

Annex

et al. 2013

American Journal of Physiology-Heart and Circulatory Physiology

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Imoukhuede PI, Dokun AO, Annex BH, Popel AS. Endothelial cell-by-cell profiling reveals the temporal dynamics of VEGFR1 and VEGFR2 membrane localization after murine hindlimb ischemia. Am J Physiol Heart Circ Physiol 304: H1085-H1093, 2013. First published February 1, 2013 doi:10.1152/ajpheart.00514.2012 cell surface localization plays a critical role in transducing VEGF signaling toward angiogenic outcomes, and quantitative characterization of these parameters is critical to advancing computational models for predictive medicine. However, studies to this point have largely examined intact muscle; thus, essential data on the cellular localization of the receptors within the tissue are currently unknown. Therefore, our aims were to quantitatively analyze VEGFR localization on endothelial cells (ECs) from mouse hindlimb skeletal muscles after the induction of hindlimb ischemia, an established model for human peripheral artery disease. Flow cytometry was used to measure and compare the ex vivo surface localization of VEGFR1 and VEGFR2 on CD31 ϩ /CD34 ϩ ECs 3 and 10 days after unilateral ligation of the femoral artery. We determined that 3 days after hindlimb ischemia, VEGFR2 surface levels were decreased by 80% compared with ECs from the nonischemic limb; 10 days after ischemia, we observed a twofold increase in surface levels of the modulatory receptor, VEGFR1, along with increased proliferating cell nuclear antigen, urokinase plasminogen activator, and urokinase plasminogen activator receptor mRNA expression compared with the nonischemic limb. The significant upregulation of VEGFR1 surface levels indicates that VEGFR1 indeed plays a critical role in the ischemia-induced perfusion recovery process, a process that includes both angiogenesis and arteriogenesis. The quantification of these dissimilarities, for the first time ex vivo, provides insights into the balance of modulatory (VEGFR1) and proangiogenic (VEGFR2) receptors in ischemia and lays the foundation for systems biology approaches toward therapeutic angiogenesis.angiogenesis; peripheral artery disease; endothelial cells; skeletal muscle; quantitative flow cytometry; receptor localization; Quantibrite; hindlimb ischemia; vascular endothelial growth factor receptor THE VEGF RECEPTOR (VEGFR)-ligand family is a key regulator of angiogenesis, the growth of new blood vessels from preexisting microvasculature. VEGF binding to its receptors activates intracellular signaling pathways inducing the endothelial proliferation and migration necessary for angiogenesis. Therapeutic angiogenesis aims to treat ischemic disease through the supplementation of VEGF and/or other angiogenic growth factors (31,48). The use of VEGF has successfully stimulated neovascularization in animal models of ischemia (30, 68). However, the use of VEGFs, fibroblast growth factor, hepatocyte growth factor, and other proangiogenic molecules has not yielded successful clinical outcomes (8,31,34). These clinical setbacks have been attributed to inadequate dosing, duration, and delivery as well a...

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Section: Cd31mentioning

confidence: 98%

Endothelial cell-by-cell profiling reveals the temporal dynamics of VEGFR1 and VEGFR2 membrane localization after murine hindlimb ischemia

Imoukhuede

Dokun

Annex

et al. 2013

American Journal of Physiology-Heart and Circulatory Physiology

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“…In the ischemic and nonischemic gastrocnemius muscles, vascular density was analyzed by immunohistochemistry with a rat anti-mouse CD31 antibody by counting 3 random high-power (magnification ϫ200) fields for a minimum of 200 fibers and was expressed as the number of CD31 ϩ cells per fiber, as described previously. 16 …”

Section: Hindlimb Ischemia Perfusion Recovery Necrosis Score and Hmentioning

confidence: 99%

“…15,16 Perfusion flow in the ischemic and contralateral nonischemic limbs was measured as described previously with the use of a laser Doppler perfusion imaging system (Perimed, Stockholm, Sweden). 15,16 Perfusion was expressed as the ratio of the left (ischemic) to right (nonischemic) hindlimb and was performed immediately after surgery and weekly or biweekly up to 21 or 35 days postoperatively. Perfusion recovery was determined either by the absolute perfusion ratio at follow-up or as the ratio at follow-up minus the ratio immediately after surgery.…”

Section: Hindlimb Ischemia Perfusion Recovery Necrosis Score and Hmentioning

confidence: 99%

A Quantitative Trait Locus (LSq-1) on Mouse Chromosome 7 Is Linked to the Absence of Tissue Loss After Surgical Hindlimb Ischemia

et al. 2008

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Background— Peripheral arterial disease (PAD) caused by occlusive atherosclerosis of the lower extremity has 2 major clinical manifestations. Critical limb ischemia is characterized by rest pain and/or tissue loss and has a ≥40% risk of death and major amputation. Intermittent claudication causes pain on walking, has no tissue loss, and has amputation plus mortality rates of 2% to 4% per year. Progression from claudication to limb ischemia is infrequent. Risk factors in most PAD patients overlap. Thus, we hypothesized that genetic variations may be linked to presence or absence of tissue loss in PAD. Methods and Results— Hindlimb ischemia (murine model of PAD) was induced in C57BL/6, BALB/c, C57BL/6×BALB/c (F1), F1×BALB/c (N2), A/J, and C57BL/6J-Chr7 A/J /NaJ chromosome substitution strains. Mice were monitored for perfusion recovery and tissue necrosis. Genome-wide scanning with polymorphic markers across the 19 murine autosomes was performed on the N2 mice. Greater tissue loss and poorer perfusion recovery occurred in BALB/c than in the C57BL/6 strain. Analysis of 105 N2 progeny identified a single quantitative trait locus on chromosome 7 that exhibited significant linkage to both tissue necrosis and extent of perfusion recovery. Using the appropriate chromosome substitution strain, we demonstrate that C57BL/6-derived chromosome 7 is required for tissue preservation. Conclusions— We have identified a quantitative trait locus on murine chromosome 7 (LSq-1) that is associated with the absence of tissue loss in a preclinical model of PAD and may be useful in identifying gene(s) that influence PAD in humans.

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“…9,20,21 We therefore tested whether inhibition of miR29a expression is sufficient to improve angiogenesis when VEGF signaling is impaired using VEGFR blocking antibodies in simulated ischemia and high glucose. In HUVECs exposed to simulated ischemia and high glucose, we assessed in vitro tube formation when cells were treated with blocking antibodies against VEGFR1, VEGFR2, and both VEGFR1 and VEGFR2 with and without miR29a inhibitor.…”

Section: Tubes/sp CMmentioning

confidence: 99%

Modulation of miR29a improves impaired post-ischemic angiogenesis in hyperglycemia

Chen

Okeke

Ayalew

et al. 2017

Exp Biol Med (Maywood)

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Individuals with diabetes mellitus suffer from impaired angiogenesis and this contributes to poorer peripheral arterial disease outcomes. In experimental peripheral arterial disease, angiogenesis and perfusion recovery are impaired in mice with diabetes. We recently showed that a disintegrin and metalloproteinase domain-containing protein 12 (ADAM12) is upregulated in ischemic endothelial cells and plays a key role in post-ischemic angiogenesis and perfusion recovery following experimental peripheral arterial disease. Here we investigated the role of miR29a in the regulation of endothelial cell ADAM12 expression in ischemia and how hyperglycemia negatively affects this regulation. We also explored whether modulating miR29a can improve impaired post-ischemic angiogenesis associated with hyperglycemia. Additionally, we tested whether miR29a modulation could improve post ischemic angiogenesis in the setting of impaired vascular endothelial growth factor signaling. We forced miR29a expression in ischemic endothelial cells and assessed ADAM12 expression. We also evaluated whether hyperglycemia in vivo and in vitro impair ischemia-induced ADAM12 upregulation and miR29a downregulation. Lastly, we determined whether modulating endothelial cell miR29a expression in ischemia and hyperglycemia could improve impaired endothelial cell functions. We found under ischemic conditions where ADAM12 is upregulated in endothelial cells, miR29a is downregulated. Forced expression of miR29a in ischemic endothelial cell prevented ADAM12 upregulation . In ischemic hind limbs of mice with type 1 diabetes and in endothelial cells exposed to simulated ischemia plus hyperglycemia, ADAM12 upregulation and miR29a downregulation were blunted while angiogenesis was impaired. Knocking down miR29a with an miR29a inhibitor was sufficient to improve ADAM12 upregulation and angiogenesis in simulated ischemia plus hyperglycemia. It was also sufficient to improve perfusion recovery in type 1 diabetes mellitus mice in vivo and angiogenesis in vitro even when vascular endothelial growth factor signaling was impaired with blocking antibodies. In conclusion, MiR29a regulates endothelial cell ADAM12 upregulation in ischemia and this is impaired in hyperglycemia. Modulating miR29a improves impaired post-ischemic angiogenesis associated with hyperglycemia. Impact statement Individuals with diabetes are more likely to develop peripheral arterial disease (PAD), and when PAD is present, in those with diabetes, it is more severe and there is currently no effective medical treatment for impaired blood flow which occurs in diabetics with PAD. The current work advances the field by providing an understanding of a molecular mechanism involved in impaired post ischemic angiogenesis in diabetes. It shows for the first time that failure to downregulate miR29a in ischemic diabetic tissues is a major contributing factor to poor perfusion recovery in experimental PAD, and miR29a is elevated in skeletal muscle samples from human diabetics compared with levels in those without diabetes. Knocking down the elevated miR29a in ischemic diabetic mouse hind limbs improved perfusion recovery following experimental PAD. This shows miR29a modulation as a novel therapeutic target for improving blood flow in diabetics with PAD.

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In Mice With Type 2 Diabetes, a Vascular Endothelial Growth Factor (VEGF)-Activating Transcription Factor Modulates VEGF Signaling and Induces Therapeutic Angiogenesis After Hindlimb Ischemia

Cited by 109 publications

References 37 publications

Endothelial cell-by-cell profiling reveals the temporal dynamics of VEGFR1 and VEGFR2 membrane localization after murine hindlimb ischemia

Endothelial cell-by-cell profiling reveals the temporal dynamics of VEGFR1 and VEGFR2 membrane localization after murine hindlimb ischemia

A Quantitative Trait Locus (LSq-1) on Mouse Chromosome 7 Is Linked to the Absence of Tissue Loss After Surgical Hindlimb Ischemia

Modulation of miR29a improves impaired post-ischemic angiogenesis in hyperglycemia

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