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. Author manuscript; available in PMC: 2024 Mar 31.
Published in final edited form as: J Vasc Surg. 2018 Aug 17;68(6 Suppl):222S–233S.e1. doi: 10.1016/j.jvs.2018.02.055

Tumor suppressor protein p53 negatively regulates ischemia-induced angiogenesis and arteriogenesis

Miles J Pfaff 1,3,#,*, Subhradip Mukhopadhyay 2,#, Mark Hoofnagle 2, Christine Chabasse 2, Rajabrata Sarkar 2
PMCID: PMC10981785  NIHMSID: NIHMS1504194  PMID: 30126780

Abstract

Objective:

The tumor suppressor protein p53 regulates angiogenesis and is a key regulatory mediator of cellular apoptosis, proliferation, and growth. p53 expression is induced in response to ischemia; however, its role in regulating ischemia-induced angiogenesis and arteriogenesis remains undefined. The objective of this study was to define the role of p53 in regulating ischemia-induced angiogenesis and arteriogenesis, and to identify mechanisms by which this regulation occurs in vivo.

Methods:

Surgically-induced hindlimb ischemia or mesenteric artery ligation were performed in wildtype (p53+/+) and p53 knockout (p53−/−) mice. Limb perfusion and revascularization was assessed by Laser Doppler Perfusion Imaging, capillary density, and collateral artery development. Mesenteric collateral artery flow and development were determined by arterial flow measurement and by histological analysis, respectively. An in vitro aortic ring assay was performed on p53+/+ and p53−/− aortic tissue to evaluate endothelial function. The p53 inhibitor and activator, pifithrin-α and quinacrine, respectively, were used to modulate p53 activity in vivo after ischemia.

Results:

Absence of p53 in mice resulted in increased limb perfusion (P < 0.05), capillary density (P < 0.05), and collateral artery development (P < 0.05) after induction of hindlimb ischemia. In the nonischemic mesenteric artery ligation model of arteriogenesis, p53 expression was induced in collateral arteries and increased arterial blood flow in mice lacking p53 (P < 0.05). Lack of p53 decreased apoptosis in ischemic hindlimb tissue (P < 0.05) and increased proangiogenic factors hypoxia-inducible factor-1 α and vascular endothelial growth factor. Endothelial cell outgrowth in vitro increased in the absence of p53 (P < 0.05). Pharmacological augmentation of p53 expression after ischemia impaired perfusion and collateral artery formation and decreased vascular endothelial growth factor levels (P < 0.05). Conversely, inhibition of p53 with pifithrin-α augmented limb perfusion (P < 0.05), collateral artery formation (P < 0.05) and increased protein levels of hypoxia-inducible factor-1α and vascular endothelial growth factor. Pharmacologic augmentation and inhibition of p53 had no significant effect in mice lacking p53.

Conclusions:

p53 negatively regulates ischemia-induced angiogenesis and arteriogenesis. Inhibition of p53 increases ischemia-induced arteriogenesis and limb perfusion and thus represents a potential therapeutic strategy for arterial occlusive disease.

Keywords: p53, angiogenesis, arteriogenesis, neovascularization, ischemia, hypoxia-inducible factor-1α, HIF-1α, vascular endothelial growth factor, VEGF

Introduction

The normal circulatory response of the vasculature to arterial occlusion is enlargement of collateral arteries (arteriogenesis) to restore distal perfusion and relieve ischemia. Arterial occlusion creates a pressure gradient across pre-existing small muscular arteries to circumvent the blockage that increases flow causing vasodilation and eventual structural enlargement of the collateral vessel to increase distal tissue perfusion 1. Arterial occlusion also leads to downstream tissue hypoxia, activating hypoxia inducible factor 1α (HIF-1α)2 to increase expression of vascular endothelial growth factor (VEGF)3 and other genes involved in capillary angiogenesis and tissue remodeling 4, 5.

Critical limb ischemia occurs when the collateral circulation is not adequate to overcome arterial occlusive disease. Despite advances in bypass surgery and endovascular intervention, more than 150,000 major amputations are performed annually in the United States due to critical limb ischemia 6. In other vascular beds, inadequate or maladaptive collateral artery formation leads to myocardial ischemia and infarction, stroke, mesenteric ischemia and ischemic nephropathy. Thus understanding the molecular mechanisms of collateral artery enlargement to develop new therapeutic strategies is a critical area of cardiovascular investigation.

The tumor suppressor protein p53 is a highly conserved transcription factor involved in DNA repair, growth arrest, and apoptosis 7. p53 expression and activation increases in response to hypoxia, oxidative stress, and DNA damage 8, 9 p53 eliminates cancerous and precancerous cells, and inhibits tumor angiogenesis as tumors lacking p53 posses a greater vascular network and metastatic potential 10, presumably due to the numerous antiangiogenic effects of p53 11. p53 inhibits the transcription of several angiogenic genes (VEGF, fibroblast growth factor, HIF-1α)12-14, induces the expression of numerous antioangiogenic genes (semaphorin 3E, thrombospondin-1, collagen prolyl hydroxylase, and arresten) 15-18.

p53 regulates other forms of vascular remodeling, including atherosclerosis and intimal hyperplasia 19, 20. The expression of p53 is increased by hindlimb ischemia15, 21 and conditions associated with arterial occlusion such as diabetes and hypercholesterolemia independently induce p53 expression 15, 22. Moreover, treatment with atorvastatin was found to reverse the negative effects of arterial occlusion in diabetic mice, possibly through a p53-dependent mechanism 23. A recent study by Kundu et al. demonstrated improved blood flow restoration in a hindlimb ischemia model following transplantation of p53 knockout endothelial progenitor cells24. To date, the exact effect of this important stress-induced protein on these critical vascular responses to ischemia remains undefined.

The purpose of this study is to define the role of p53 in regulating ischemia-induced angiogenesis and arteriogenesis, and to identify potential mechanisms by which this regulation occurs in vivo. We identify a direct negative effect of p53 on angiogenesis, arteriogenesis, and limb perfusion as well as several potential molecular mechanisms. Further, we show that pharmacologic modulation of p53 function improves collateral artery formation (arteriogenesis) and limb perfusion after ischemia, demonstrating the potential therapeutic benefit of inhibiting p53 to improve tissue perfusion in arterial occlusive disorders.

Materials and Methods

Animal Surgical models

All experiments and animal husbandry were performed with approval from the University of California, San Francisco and University of Maryland Institutional Animal Care and Use Committees. Wild-type (p53+/+), p53 knockout (p53−/−), and heterozygoytes (p53−/+) (B6.129S2-Trp53tm1Tyj/J (The Jackson Laboratory, Bar Harbor, ME; Stock number: 002101) were maintained on a C57Bl/6J background (The Jackson Laboratory) and all experiments were done with littermate controls from the same colony to exclude effects of genetic drift. Validation of absence of p53 mRNA in p53−/−mice was confirmed by real-time PCR analysis (Supplemental Figure A and Supplemental Methods). Hindlimb ischemia was surgically induced in 8- to 12-week old male mice by excision of the unilateral femoral artery under isofluorane anesthesia as previously described21, 25. Femoral artery excision was performed on the right hindlimb of all mice. The femoral artery was isolated and ligated at three points: 1) distal to the inguinal ligament and at the proximal aspect of the 2) popliteal and 3) saphenous arteries. For the gracilis arteriogenesis analysis, animals underwent femoral artery ligation just distal to the inguinal ligament to permit perfusion through the superficial femoral artery and profunda artery For pharmacological modulation of p53 in vivo, the p53 activator quinacrine was administered intraperitoneally at 10 mg/kg/day (vehicle control: PBS), beginning 5 days prior to surgery, and continued daily post-operatively until the experimental endpoint; the p53 inhibitor pifithrin-α (PFT-α) was administered intraperitoneally at 2 mg/kg/day every other day (vehicle control: DMSO) beginning two days prior to surgery and continued until the experimental end-point. Laser Doppler perfusion imaging was performed on all experimental groups (p53+/+ vs. WT, PFT- vs. Control, and Quinacrine vs. Control) prior to induction of hindlimb ischemia, demonstrating no difference in baseline perfusion between experimental and control mice (Supplemental Figure B, C, and D).”

Mesenteric artery ligation was performed under isofluorane anesthesia on 8- to 12-week old male p53+/+ and p53−/− mice as described previously with modifications 26. A laparotomy was performed and the small intestine exposed and suffused with warmed saline. A 2nd order straight artery branching off of the superior mesenteric artery was chosen as a high flow collateral vessel (HF), after which the arteries immediately bordering it were skeletonized and ligated with 6-0 silk thread. The control artery (CTL) was considered a straight artery three arteries from the site of ligation. The peritoneum and skin were closed individually with 4-0 absorbable sutures.

Blood Flow Determination

Restoration of blood flow after hindlimb ischemia was assessed by laser Doppler perfusion imaging (LDPI) of the hindlimb paws using a PeriScan PIM 3 System (Perimed AB; Stockholm, Sweden) as previously described 21. Three successive readings were averaged and expressed as an ischemic index (ischemic paw over contralateral paw). For mesenteric artery ligation, blood flow was measured in the HF or CTL artery as volume-flow with a nanoprobe secured around the artery (0.5 mm nanoprobe, Transonic Systems Inc, Ithaca, NY) in anesthetized mice. Flow was averaged over ten minutes and expressed as an arterial index (HF artery divided by CTL artery).

Histology

Collateral arteries in the thigh adductor muscles were identified by immunostaining with α-smooth muscle actin (α-sma) (Cat#: NB600, Novus Biologicals; Littleton, CO) on formalin-fixed, paraffin-embedded sections, and visualized using the Vector ABC kit and VIP substrate (Vector Labs; Burlingame, CA). For capillary density analysis, fixed tibialis anterior (TA) muscles were frozen in an isopentane/liquid nitrogen bath, subjected to a sucrose gradient for cryoprotection, embedded in O.C.T., and cut into 5 mm frozen sections. Sections were washed briefly in Phosphate Buffered Saline (PBS) containing 0.05% Tween 20 (PBS-T), blocked for 1 hour at room temperature in PBS containing 5% BSA, and incubated with fluoresceinated isolectin B4 (lectin) (Vector). TUNEL analysis was performed on paraffin-embedded TA muscles using the ApopTag kit (Millipore; Temecula, CA). For capillary and TUNEL analysis, images were taken from two 5 μm thick sections, 1 mm apart, from each ischemic muscle in three randomly chosen fields of three areas. To quantify the arteriogenic response, two α-sma positive collateral arteries identified as being present in all animals were studied in sections cut 1 mm apart with two 5 μm sections per area and the results averaged. Independent experiments with separate animal cohorts for capillary and collateral artery analysis were performed. For the gracilis arteriogenesis assay, animals were euthanized and perfusion fixed with 10 ml PBS containing heparin (100U/L), sodium nitroprusside 10 mM, adenosine 100 mM, followed by 10% neutral-buffered formalin, and PBS containing glycine 100mM (all Sigma Aldrich, St. Louis, MO) at 37°C. The gracilis muscle was harvested and solubilized in PBS containing 0.5% triton and 1% BSA for 24 hours, then incubated with α-sma-cy3 antibody (Sigma, Cat #: C6198) at a concentration of 1:250 in 0.1% triton (Sigma) in PBS for 48 hours. The muscle was then dehydrated by successive one hour immersions in 50%, 70%, 90%, 95% and 100% ethanol, followed by clearing in a 1:1 solution of benzyl alcohol/benzyl benzoate (Sigma) prior to fluorescent whole mount imaging. To quantify the degree of arteriogenesis, the distance in pixels was taken from the superficial femoral artery to the profunda artery. This measurement was divided by 4 and labeled accordingly: distal, mid-distal, mid, mid-proximal, and proximal. Width of the vessel in pixels at each of those 5 locations from most proximal to most distal were measured. An ischemic index was calculated by dividing the ischemic vessel width by the contralateral vessel width at the same location. The average the ratios for one group at each location were calculated and compared to the corresponding control group control. For cross sections of mesenteric artery specimens, anesthetized animals were perfused with lectin (Vector) prior to fixation, followed by perfusion with Microfil (Flow Tech Inc.; Carver, MA). The HF and CTL artery were excised and the arteries embedded in OCT for frozen sectioning. Two 5 μm sections in two separate areas 0.5 mm apart were measured and the results averaged. All analyses were performed using the National Institute for Health imaging software, ImageJ (http://rsbweb.nih.gov/ij/). Fluorescent images were acquired using an epifluorescence microscope (Eclipse E800; Nikon Corporation, Tokyo, Japan) equipped with a digital camera (Qimaging Retigra EXi, Qimaging, Surrey, BC, Canada). Images were acquired using the Volocity software (PerkinElmer Inc, Shelton, CT, USA). Light images for the aortic ring assay was acquired using an inverted microscope (Eclipse TE200, Nikon Corporation, Tokyo, Japan) and the MetaFluor software (Molecular Devices, Downingtown, PA) was used for the image acquisition and analysis.

Immunoblotting

Tibialis anterior muscle from ischemic and contralateral limbs were harvested and pulverized in liquid nitrogen. Pulverized tissue were incubated in a modified RIPA buffer [50 mM Tris-HCl, pH 7.4; 1% SDS; 1% NP-40; 0.25% sodium deoxycholate; 1mM PMSF; 1 mM DTT; Complete Mini EDTA-free protease inhibitor cocktail and PhosStop phosphatase inhibitor tablets (Roche Applied Science; Indianapolis, IN)], strained, and sonicated 3 times for 10 seconds. Samples were centrifuged at 10,000 G for ten minutes at 4°C, and the supernatant preserved. The positive control for p53 was generated from the lysate of cultured endothelial cells (HUVEC) transfected with adenovirus encoding mouse p53 (Ad-p53). For mesenteric artery samples, individual first order mesenteric artery branches were dissected free of surrounding tissue, pooled to achieve sufficient protein and treated as described above. Protein concentration was measured using a BCA Protein Assay Kit. A total of 20 mg of protein was resolved on a 4-12% Bis-Tris gradiant polyacrylamide gel (Invitrogen; Carlsbad, CA) under reduced conditions and transferred to a PVDF membrane (Amersham Biosciences; Pittsburgh, PA). Membranes were blocked for 30 minutes at room temperature in StartingBlock blocking buffer (Pierce; Rockford, IL) and incubated with antibodies specific to beta-actin (SC-130656, Santa Cruz Biotechnology; Santa Cruz, CA), VEGF (AB-46154, Abcam, Cambridge, MA), p53 (PC 35, ab7 pantropic; Fisher Scientific, Pittsburg, PA), GAPDH (AB-9485; Abcam), or HIF-1α (NB-100, Novus Biologicals). Protein expression was visualized by chemiluminescence using ECL Plus (Amersham) on medical x-ray film. Densitometry was performed on selected blots using ImageJ.

Endothelial Outgrowth Aortic Ring Assay

Aortic rings (1 mm from the descending thoracic mouse aorta) were placed on a layer of solidified Matrigel (BD Bioscience; San Jose, CA) in 48-well plates and embedded with additional Matrigel. Mineral oil (Sigma) was applied to samples to achieve an hypoxic environment. All samples were incubated at 37°C in a humidified atmosphere of 5% CO2 for up to 2 weeks. Media (MCDB 131, 10% FBS, 14 mM NaHCO3, and 1% Penicillin and Streptavidin) and mineral oil were replenished twice weekly. 20 mM quinacrine or PBS-vehicle control were added to the media. Growth scores based on phase contrast microscopy were performed using a 5-point scale (5 = greatest growth, 0 = no growth) by two independent, blinded reviewers; results from both graders were averaged and used for analysis.

Statistical Analysis

Statistical analysis was performed using Microsoft Excel (Version 14.0.0, Microsoft Office 2011, Microsoft; Redmond, WA) and SPSS Statistics (Version 19, IBM; Armonk, NY). Results were expressed as the mean ± standard error of the mean (s.e.m.). A two-tailed student’s t test or Analysis of Variance test was performed for statistical comparison and an observed P value of 0.05 or less was considered statistically significant.

Results

p53 is upregulated in ischemic tissue following hindlimb ischemia and negatively regulates ischemia-induced angiogenesis and arteriogenesis

We examined p53 expression after hindlimb ischemia and noted by immunoblotting that p53 expression peaked at days 3 and 7 in ischemic tissue (Fig. 1A). Analysis of blood flow after ischemia revealed that p53−/− mice had enhanced restoration of blood flow at 2 weeks compared to p53+/+ mice, with an intermediate response observed in heterozygous p53+/− mice (Fig. 1B: Top, representative LDPI images of p53+/+ vs. p53−/−mice; Bottom, LDPI ischemic indices over time). This observed difference in blood flow in p53−/− mice compared to p53+/+ mice was sustained to day 28 (Fig. 1C). Compared to p53+/+ mice, p53−/− mice demonstrated a significant increase in both capillary density (Fig. 1D) and collateral artery enlargement by both cross-sectional and arteriogenic analysis (Fig. 1E and F) 2 weeks after hindlimb ischemia.

Figure 1. Loss of p53 potentiates angiogenesis and arteriogenesis following hindlimb ischemia.

Figure 1.

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A) p53 protein expression increased in ischemic muscle tissue up to one week following hindlimb ischemia (Day 0 refers to pre-operative time-point). B) p53−/− mice showed marked improvement in blood flow restoration compared to p53+/+ mice 2 weeks after hindlimb ischemia (n=6, p53−/−; n=9, p53−/+; n=6, p53+/+; top panel, representative LDPI images of p53+/+ and p53−/− mice; bottom panel, LDPI ischemic indices over time) that was sustained 28 days after surgery (n=6 per group) (C). D) p53−/− mice demonstrated increased capillary density (n=5/group) and E) artery area (n=5/group) and F) gracilis artery ischemic index (n=11, p53−/−; n=12, p53+/+) 2 weeks after hindlimb ischemia. Ad-p53 (HUVEC lysate expressing a recombinant adenovirus vector encoded mouse p53 protein).SFA, superficial femoral artery; D, distal; MD, mid-distal; M, mid; MP, mid-proximal; and P, proximal. Data presented as mean ± s.e.m. *p < 0.05 [Scale bar: 50 μm (D) and 1 mm (F); magnification 200X (D), and 20X (F)]

p53 suppresses fluid shear stress-mediated blood flow restoration following arterial occlusion

We observed that p53 impaired arteriogenesis after ischemia (Fig. 1E and F). Numerous components of hindlimb ischemia may induce p53 activity including necrosis, hypoxia and inflammation. To determine whether p53 directly influences flow-mediated arterial enlargement in the absence of these factors, we studied the expression and effect of p53 in a non-ischemic model of flow-mediated collateral artery formation (mesenteric ligation) (Fig. 2A) 26. In p53+/+ mice, increased flow in the collateral vessel induced accumulation of the p53 protein as compared to control vessels (Fig. 2B). Mice lacking p53 showed a statistically significant increase in blood flow after 2 weeks as compared with p53+/+ animals (Fig. 2C), and collateral arteries in mice lacking p53 showed a trend towards increased size at 2 weeks (P = 0.1) (Fig. 2D).

Figure 2. Loss of p53 improves the arteriogenic response after mesenteric artery ligation.

Figure 2.

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A) Schematic diagram depicting mesenteric artery ligation model. Secondary branches of the mesenteric artery adjacent to the high flow artery are ligated (as indicated by an X) and a control artery is chosen three arteries from the ligation site. B) p53 protein expression increased in high flow arteries seven days after mesenteric artery ligation (n=3/group; pooled). C) p53−/− mice demonstrated enhanced blood flow restoration two weeks after mesenteric artery ligation by ultrasonic volume-flow measurement compared to p53+/+ mice (n=10 per/group) and D) p53−/− mice showed an improvement in high flow arterial area two weeks after mesenteric artery ligation (n=6 per/group; [p=0.1]). HF, high flow; CTL, control. Data presented as mean ± s.e.m. *p < 0.05. [Scale bar: 100 μm (D); magnification: 100X (D)]

p53 regulates the HIF-1α/VEGF pathway, endothelial outgrowth, and apoptosis

After induction of hindlimb ischemia, p53−/− mice showed increased tissue HIF-1α and VEGF protein levels compared to p53+/+ mice (Fig. 3A), indicating that endogenous p53 suppresses the expression of these important angiogenic factors. To further define the angiogenic effect of p53 under isolated conditions, aortic rings from p53+/+ and p53−/− mice were embedded in a collagen matrix and subjected to hypoxia. p53−/− rings had an increased endothelial outgrowth compared to p53+/+ rings (Fig. 3B), indicating that endogenous p53 suppresses hypoxia-induced endothelial cell proliferation and migration, consistent with effects noted in vivo after ischemia. Analysis of apoptosis in ischemic muscle tissue demonstrated that p53−/− mice had significantly fewer apoptotic cells than p53+/+ mice (Fig. 3C), indicating that p53 increases apoptosis after ischemia.

Figure 3. Proangiogenic and anti-apoptotic effect of loss of p53.

Figure 3.

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A) Loss of p53 in ischemic tissue 3 days after hindlimb ischemia increases HIF-1α and VEGF protein expression compared to p53+/+ controls (n=3/group; densitometry results averaged per group). B) p53−/− aortic rings subjected to hypoxic conditions demonstrated increased endothelial outgrowth compared to p53+/+ aortic rings (n=4 mice/group). C) Loss of p53 resulted in a decrease in detected apoptotic cells in ischemic tissue 3 days after hindlimb ischemia (n=7/group). Data presented as mean ± s.e.m. *p < 0.05, **p < 0.01 [Scale bars: 0.5 μm (B) and 50 mm (C); magnification: 50X (B) and 200X (C)]

Pharmacological augmentation of p53 impairs ischemia-induced neovascularization

Treatment of p53+/+ mice with the p53 activator quinacrine significantly attenuated improvement in blood flow after ischemia (Fig. 4A). Morphometric analysis showed that quinacrine treatment did not affect capillary density (Fig. 4B) but did impair collateral artery enlargement (Fig. 4C). Quinacrine treatment significantly inhibited hypoxic endothelial outgrowth from aortic rings compared to vehicle-treated controls (Fig. 4D). Moreover, VEGF protein levels were suppressed in quinacrine-treated mice (Fig. 4E). In contrast, HIF-1α protein levels were moderately elevated following quinacrine treatment (Fig. 4E). Considering quinacrine has numerous intracellular targets, we confirmed that the effect of quinacrine on blood flow and arteriogenesis were mediated by activation of p53 by studying its effect in mice lacking p53. Quinacrine treatment had no effect on blood flow after ischemia in p53−/− mice (Fig. 4F) demonstrating that the effects of quinacrine were due to p53 activation.

Figure 4. Pharmacological augmentation of p53 impairs angiogenesis and arteriogenesis after hindlimb ischemia.

Figure 4.

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A) Quinacrine-treated mice showed a decrease in blood flow restoration compared to vehicle controls two weeks after hindlimb ischemia (n=7 per/group). B) Quinacrine treatment did not effect capillary density (n=5/group) C) but did negatively influence larger vessel growth (n=5/group). D) Quinacrine-treatment of aortic rings under hypoxic conditions resulted in a significant decrease in endothelial outgrowth compared to vehicle-treated controls (n=4 mice/group). E) Ischemic tissue of quinacrine-treated mice revealed decreased expression of VEGF levels and increased expression of HIF-1α seven days after hindlimb ischemia (n=3/group; densitometry results averaged per group). F) Quinacrine-treatment in p53−/− did not affect blood flow restoration by LDPI two weeks after hindlimb ischemia (n=5/group) (Day 14 time-point of quinacrine- and vehicle treated mice from panel A added to panel F for direct comparison). Data presented as mean ± s.e.m. *p<0.05, † p<0.05 between p53−/− mice treated with quinacrine or vehicle and p53+/+ quinacrine and vehicle -treated mice. [Scale bar: 50 μm (B) and 0.5 mm (D); magnification: 200X (B) and 50X (D)]

Pharmacological inhibition of p53 improves ischemia-induced neovascularization

Treatment of p53+/+ mice with the p53 inhibitor, PFT-α, enhanced blood flow restoration after ischemia compared to vehicle-treated animals (Fig. 5A). No difference in capillary growth was detected following PFT-α treatment (Fig. 5B); however, collateral vessel formation increased compared to vehicle-treated controls (Fig. 5C). PFT-α was associated with an increase in both HIF-1α and VEGF protein levels in ischemic tissue 3 days after surgery (Fig. 5D). Again, the specificity of PFT-α for the p53 pathway was examined by studying its effect in mice lacking p53. Treatment of p53−/− mice with PFT-α had no effect on blood flow restoration after ischemia (Fig. 5E), demonstrating that the beneficial effects of PFT-α on blood flow and arteriogenesis were due to inhibition of p53. The magnitude of the effect of PFT-α treatment on limb blood flow in wild type animals was similar in magnitude to genetic deletion of p53 (Fig. 5E).

Figure 5. Pharmacological inhibition of p53 improves the arteriogenic response after hindlimb ischemia.

Figure 5.

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A) PFT-α treated mice demonstrated enhanced blood flow restoration by LDPI two weeks after hindlimb ischemia compared to vehicle treated mice (n=9/group). B) No statistically significant difference in capillary counts was detected between PFT-α and vehicle treated mice (n=3 group); however, C) PFT-α treatment resulted in an increased gracilis artery index 2 weeks after hindlimb ischemia (PFT-α, n=9; vehicle-control, n=7). D) PFT-α treated animals demonstrated enhanced expression of HIF-1α and VEGF protein levels three days after surgery (n=4/group) (densitometry results averaged per group). E) Treatment of p53−/− mice with PFT-α did not affect blood flow by LDPI two weeks after hindlimb ischemia (n=4/group). Day 14 time-point of PFT-α and vehicle treated mice from panel A added to panel D for direct comparison. SFA, superficial femoral artery. D, distal; MD, mid-distal; M, mid; MP, mid-proximal; and P, proximal C) Magnification 20X. Data presented as mean ± s.e.m. *p<0.05,**p<0.05 [Scale bar: 1 mm (C); magnification 20X (C)]

Discussion

Despite indirect evidence, there is little reported on the role of p53 in regulating the response to hindlimb ischemia and no reports of utilizing p53 as a therapeutic target to pharmacologically improve perfusion after ischemia. Studies of statin treatment23, semaphorin expression15, and inhibition of retinal angiogenesis by p53 27 indirectly suggest that endogenous p53 regulates ischemia-induced angiogenesis, arteriogenesis, and limb perfusion. Direct evidence for the role of p53 includes the finding that conditional endothelial cell-specific p53 deletion increases capillary density and limb perfusion after hindlimb ischemia28, although collateral artery development was not examined. Further, transplantation of p53 deficient endothelial progenitor cells has been shown improve hindlimb revascularization following ischemic insult.24 Conversely, p53 activation with the MDM2 inhibitor Nutlin-3 decreased capillary density after hindlimb ischemia, although limb perfusion and collateral artery enlargement were again not examined27. We did not find a change in capillary angiogenesis with quinacrine activation of p53 (Fig. 4B) whereas Chavala et al. noted inhibition of angiogenesis with Nutlin-3 27. This difference may be due to the p53-independent effects of Nutlin-3, such as direct degradation of HIF-1α by MDM2 29. The effects of Nutlin-3 on capillary angiogenesis were not confirmed in p53−/− mice, as we did with quinacrine (Fig. 4F) and thus the non-p53 effects of Nutlin-3 remain a potential mechanism to account for this difference. Further, the effect of p53 activation by Nutlin-3 on HIF-1α and VEGF levels after ischemia was not examined by Chavala et al 27, 29. We found quinacrine treatment resulted in HIF-1α protein level elevation, which may be explained by the more severe hypoxic environment created by quinacrine treatment. Although quinacrine treatment had no statistically significant effect on perfusion in p53−/− mice following hindlimb ischemia, the possibility of other mechanisms independent of p53 regulating neovascularization cannot be ruled out.

Endothelial cell-specific deletion of p53 increased angiogenesis in cardiac hypertrophy and hindlimb ischemia28. This is consistent with reports of inhibition by p53 on endothelial cell migration, proliferation, apoptosis, and capillary tube formation and within the endothelial progenitor cell as well as the present data (Fig. 3B and C) 12, 24, 27, 28. Increased limb perfusion with endothelial cell-specific p53 deletion may be mediated by flow-mediated enlargement of collateral vessels due to increased capillary number. Alternatively, the lack of p53 in the endothelium lining the collateral vessels may directly enhance flow-mediated arterial enlargement 30.

Experimental hindlimb ischemia is associated with severe tissue hypoxia, necrosis, and cellular regeneration31, each of which can activate p53 expression (For review, see ref. Gudkov and Komarova32). To ensure that the effects of p53 on collateral artery enlargement observed were not dependent on these specific pathological features, we studied the effects ofp53 gene deletion in the mouse mesenteric model of collateral artery enlargement, a well-established non-ischemic model for defining roles of individual genes in arteriogenesis 26. The effects of p53 deletion paralleled those noted in hindlimb ischemia, demonstrating p53 also negatively regulates flow-mediated enlargement of collateral arteries in a non-ischemic context. The critical process in restoring limb perfusion is arteriogenesis, or flow-mediated collateral artery enlargement around an arterial occlusion rather than capillary angiogenesis 1, 33. We noted that all changes in limb perfusion with either genetic deletion or pharmacologic modulation of p53 correlated with arteriogenesis. Genetic deletion of p53 also increased capillary angiogenesis, but positive and negative modulation of p53 with quinacrine or PFT-α, respectively, altered limb perfusion and arteriogenesis without corresponding changes in capillary angiogenesis (Fig. 4B and 5B). This indicates that endogenous p53 regulates both ischemia-induced arteriogenesis and angiogenesis, but the arteriogenic response appears essential for restoration of limb perfusion.

p53 inhibited angiogenesis and expression of HIF-1α and VEGF in vivo, which may involve several mechanisms. Both p53 and HIF-1α are induced by hypoxia and several interactions between these two factors allow complex and reciprocal regulation (For review, see ref. Schmid et al.34). Ravi et al first reported that p53 binds and targets HIF-1α for degradation via the ubiquitin-proteosome pathway, which decreases HIF-1α-mediated VEGF expression and angiogenesis 35. p53 can also act as a transcriptional repressor of the VEGF gene36 and also inhibit HIF-1α transcription by competing for p300 binding34 or upregulation of the microRNA molecule miR-107 37. A number of other mechanisms have been identified for regulation of angiogenesis by p53, including generation of anti-angiogenic collagen fragments via activation of prolyl hydroxylase18 and expression of semaphorin 3E 15. The increased angiogenesis noted with loss of p53 after ischemia in vivo (Fig. 1D) correlated with changes in HIF-1α and VEGF expression as did hypoxia-induced endothelial microvessel outgrowth from isolated aortic rings (Fig. 3B). The importance of the HIF-1α pathway in regulation of angiogenesis by p53 is demonstrated by the lack of effect of p53 activation on angiogenesis in mice lacking retinal HIF-1α 27. Although overall limb perfusion after ischemia correlated more with collateral artery enlargement than angiogenesis, the ability of p53 to regulate capillary angiogenesis is important in both positive and negative roles. Interestingly, pharmacologic activation of p53 can inhibit pathologic retinal angiogenesis27, and topical p53 gene silencing can enhance capillary angiogenesis during wound healing 38.

We found that p53 gene deletion reduced cellular apoptosis in ischemic muscle (Fig. 3C). Similar regulation of apoptosis by p53 has been reported, particularly in cardiac tissue, where the effect of p53 on apoptosis appears to be the mechanism responsible for both post-infarct cardiac rupture as well the beneficial effect of p53 inhibition on post-infarct cardiac function 39-41. We did not identify significant TUNEL-positive apoptotic cells in the more proximal non-ischemic portions of the limb where collateral artery enlargement takes place (data not shown). This suggests that the effect of p53 on arteriogenesis, and consequently limb perfusion, is more likely mediated by effects on VSMC migration and proliferation during arteriogenesis 27, 42 than through changes in apoptosis.

Small molecule therapy targeting inhibition of p53 may be particularly effective in patients with diabetes and other chronic cardiovascular conditions associated with increased oxidative stress, a well-known trigger for p53 expression. In this regard, it is interesting to speculate that the decreased collateral artery formation noted in diabetes43 may be due to the increased p53 expression noted in this disease state23, presumably due to increased oxidative stress. The limited proliferative and beneficial capacity of stem cells derived from patients with cardiovascular disease is well documented, which negatively influences their therapeutic benefit for treatment of ischemic disease 44. Inhibition of normal or elevated p53 activity in such patients may be more effective than pro-angiogenic therapies, which depend on signaling pathways that are compromised in patients with atherosclerosis and cardiovascular disease.

Conclusion:

The significant findings in this study are: 1) p53 inhibits the angiogenic and arteriogenic responses to hindlimb ischemia and mesenteric arterial occlusion, 2) p53 inhibits ischemia-induced VEGF expression, 3) p53 inhibits hypoxia-induced endothelial cell growth and increases cellular apoptosis after ischemia, and 4) positive and negative pharmacologic modulation of p53 reciprocally regulates limb perfusion, collateral arterial enlargement and expression of angiogenic proteins. Future studies investigating cell-type specific p53 knockouts to define the role of p53 in individual cell types in mediating the overall effect of p53 on arteriogenesis after ischemia would be welcomed. Collectively, the present study identifies p53 as an endogenous negative regulator of ischemia-induced angiogenesis and arteriogenesis and a novel pharmacological target to improve tissue perfusion through augmented arteriogenesis after ischemia.

Supplementary Material

1

Supplemental Figure: (A) p53 mRNA expression in hindlimb muscle from p53+/+ and p53−/− animals were determined by Real-Time PCR. (B) Representative LDPI images of p53+/+ and p53−/− paw perfusion at day 0 and quantitative analysis of paw perfusion at day 0 (n = 4 for each group). (C) Representative LDPI images of PFT-treated mouse paw perfusion at day 0 and quantitative analysis of paw perfusion at day 0 (n = 4 for each group). (C) Representative LDPI images of Quinacrine-treated mouse paw perfusion at day 0 and quantitative analysis of paw perfusion at day 0 (n = 4 for each group). PFT: Pifithrin-alpha, Quin: Quinacrine, R, right paw; L, left paw; LDPI, laser Doppler perfusion imaging. All values represent the mean ± standard error of the mean.

Clinical Relevance:

The present study demonstrates that: p53 inhibits the angiogenic/arteriogenic responses to arterial occlusion; p53 inhibits ischemia-induced HIF-1α and VEGF expression; p53 inhibits hypoxia-induced endothelial cell growth and augments apoptosis; and positive and negative pharmacologic modulation of p53 reciprocally regulates limb blood flow and arteriogenesis in response to ischemia. These results identify p53 as an endogenous negative regulator of ischemia-induced angiogenesis and arteriogenesis and a novel pharmacological target to improve tissue perfusion after ischemia.

Acknowledgments

Grants

This work was supported by a grant from the National Institutes of Health (RO1-HL080584 to R.S.) and the Wylie Scholar Award from the Vascular Cures Foundation (R.S.).

Footnotes

Disclosures

All authors have no commercial associations or disclosures that may pose or create a conflict of interest with the information presented within this manuscript.

This work was presented in part at the American Heart Association’s Arteriosclerosis, Thrombosis, and Vascular Biology Annual Meeting, Washington D.C., April 2009

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References

  • 1.Schaper W, Buschmann I. Arteriogenesis, the good and bad of it. Cardiovasc Res. 1999;43(4):835–7. [DOI] [PubMed] [Google Scholar]
  • 2.Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci U S A. 1995;92(12):5510–4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Forsythe JA, Jiang BH, Iyer NV, Agani F, Leung SW, Koos RD, et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol. 1996; 16(9):4604–13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Jung F, Palmer LA, Zhou N, Johns RA. Hypoxic regulation of inducible nitric oxide synthase via hypoxia inducible factor-1 in cardiac myocytes. Circ Res. 2000;86(3):319–25. [DOI] [PubMed] [Google Scholar]
  • 5.Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer. 2003;3(10):721–32. [DOI] [PubMed] [Google Scholar]
  • 6.Ziegler-Graham K, MacKenzie EJ, Ephraim PL, Travison TG, Brookmeyer R. Estimating the prevalence of limb loss in the United States: 2005 to 2050. Arch Phys Med Rehabil. 2008;89(3):422–9. [DOI] [PubMed] [Google Scholar]
  • 7.Giaccia AJ, Kastan MB. The complexity of p53 modulation: emerging patterns from divergent signals. Genes Dev. 1998;12(19):2973–83. [DOI] [PubMed] [Google Scholar]
  • 8.Graeber TG, Osmanian C, Jacks T, Housman DE, Koch CJ, Lowe SW, et al. Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature. 1996;379(6560):88–91. [DOI] [PubMed] [Google Scholar]
  • 9.Graeber TG, Peterson JF, Tsai M, Monica K, Fornace AJ Jr., Giaccia AJ. Hypoxia induces accumulation of p53 protein, but activation of a G1-phase checkpoint by low-oxygen conditions is independent of p53 status. Mol Cell Biol. 1994;14(9):6264–77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Levine AJ. p53, the cellular gatekeeper for growth and division. Cell. 1997;88(3):323–31. [DOI] [PubMed] [Google Scholar]
  • 11.Teodoro JG, Evans SK, Green MR. Inhibition of tumor angiogenesis by p53: a new role for the guardian of the genome. J Mol Med (Berl). 2007;85(11): 1175–86. [DOI] [PubMed] [Google Scholar]
  • 12.Sano M, Minamino T, Toko H, Miyauchi H, Orimo M, Qin Y, et al. p53-induced inhibition of Hif-1 causes cardiac dysfunction during pressure overload. Nature. 2007;446(7134):444–8. [DOI] [PubMed] [Google Scholar]
  • 13.Kieser A, Weich HA, Brandner G, Marme D, Kolch W. Mutant p53 potentiates protein kinase C induction of vascular endothelial growth factor expression. Oncogene. 1994;9(3):963–9. [PubMed] [Google Scholar]
  • 14.Galy B, Creancier L, Zanibellato C, Prats AC, Prats H. Tumour suppressor p53 inhibits human fibroblast growth factor 2 expression by a post-transcriptional mechanism. Oncogene. 2001;20(14):1669–77. [DOI] [PubMed] [Google Scholar]
  • 15.Moriya J, Minamino T, Tateno K, Okada S, Uemura A, Shimizu I, et al. Inhibition of semaphorin as a novel strategy for therapeutic angiogenesis. Circ Res. 2010;106(2):391–8. [DOI] [PubMed] [Google Scholar]
  • 16.Dameron KM, Volpert OV, Tainsky MA, Bouck N. The p53 tumor suppressor gene inhibits angiogenesis by stimulating the production of thrombospondin. Cold Spring Harb Symp Quant Biol. 1994;59:483–9. [DOI] [PubMed] [Google Scholar]
  • 17.Assadian S, El-Assaad W, Wang XQ, Gannon PO, Barres V, Latour M, et al. p53 inhibits angiogenesis by inducing the production of Arresten. Cancer Res. 2012;72(5):1270–9. [DOI] [PubMed] [Google Scholar]
  • 18.Teodoro JG, Parker AE, Zhu X, Green MR. p53-mediated inhibition of angiogenesis through up-regulation of a collagen prolyl hydroxylase. Science. 2006;313(5789):968–71. [DOI] [PubMed] [Google Scholar]
  • 19.Sata M, Tanaka K, Ishizaka N, Hirata Y, Nagai R. Absence of p53 leads to accelerated neointimal hyperplasia after vascular injury. Arterioscler Thromb Vasc Biol. 2003;23(9):1548–52. [DOI] [PubMed] [Google Scholar]
  • 20.Mercer J, Bennett M. The role of p53 in atherosclerosis. Cell Cycle. 2006;5(17):1907–9. [DOI] [PubMed] [Google Scholar]
  • 21.Lee JG, Dahi S, Mahimkar R, Tulloch NL, Alfonso-Jaume MA, Lovett DH, et al. Intronic regulation of matrix metalloproteinase-2 revealed by in vivo transcriptional analysis in ischemia. Proc Natl Acad Sci U S A. 2005;102(45):16345–50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Cheng J, Cui R, Chen CH, Du J. Oxidized low-density lipoprotein stimulates p53-dependent activation of proapoptotic Bax leading to apoptosis of differentiated endothelial progenitor cells. Endocrinology. 2007;148(5):2085–94. [DOI] [PubMed] [Google Scholar]
  • 23.Morimoto Y, Bando YK, Shigeta T, Monji A, Murohara T. Atorvastatin prevents ischemic limb loss in type 2 diabetes: role of p53. J Atheroscler Thromb. 2011;18(3):200–8. [DOI] [PubMed] [Google Scholar]
  • 24.Kundu N, Domingues CC, Chou C, Ahmadi N, Houston S, Jerry DJ, et al. Use of p53-Silenced Endothelial Progenitor Cells to Treat Ischemia in Diabetic Peripheral Vascular Disease. J Am Heart Assoc. 2017;6(4). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Couffinhal T, Silver M, Zheng LP, Kearney M, Witzenbichler B, Isner JM. Mouse model of angiogenesis. Am J Pathol. 1998;152(6):1667–79. [PMC free article] [PubMed] [Google Scholar]
  • 26.Unthank JL, Fath SW, Burkhart HM, Miller SC, Dalsing MC. Wall remodeling during luminal expansion of mesenteric arterial collaterals in the rat. Circ Res. 1996;79(5):1015–23. [DOI] [PubMed] [Google Scholar]
  • 27.Chavala SH, Kim Y, Tudisco L, Cicatiello V, Milde T, Kerur N, et al. Retinal angiogenesis suppression through small molecule activation of p53. J Clin Invest. 2013;123(10):4170–81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Gogiraju R, Xu X, Bochenek ML, Steinbrecher JH, Lehnart SE, Wenzel P, et al. Endothelial p53 Deletion Improves Angiogenesis and Prevents Cardiac Fibrosis and Heart Failure Induced by Pressure Overload in Mice. J Am Heart Assoc. 2015;4(2). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Tenforde TS, Montoya VJ, Afzal SM, Parr SS, Curtis SB. Response of rat rhabdomyosarcoma tumors to split doses of mixed high- and low-let radiation. International journal of radiation oncology, biology, physics. 1989;16(6):1529–36. [DOI] [PubMed] [Google Scholar]
  • 30.Tronc F, Wassef M, Esposito B, Henrion D, Glagov S, Tedgui A. Role of NO in flow-induced remodeling of the rabbit common carotid artery. Arterioscler Thromb Vasc Biol. 1996;16(10):1256–62. [DOI] [PubMed] [Google Scholar]
  • 31.Paek R, Chang DS, Brevetti LS, Rollins MD, Brady S, Ursell PC, et al. Correlation of a simple direct measurement of muscle pO(2) to a clinical ischemia index and histology in a rat model of chronic severe hindlimb ischemia. J Vasc Surg. 2002;36(1):172–9. [DOI] [PubMed] [Google Scholar]
  • 32.Gudkov AV, Komarova EA. Pathologies associated with the p53 response. Cold Spring Harb Perspect Biol. 2010;2(7):a001180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Heil M, Eitenmuller I, Schmitz-Rixen T, Schaper W. Arteriogenesis versus angiogenesis: similarities and differences. J Cell Mol Med. 2006;10(1):45–55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Schmid T, Zhou J, Kohl R, Brune B. p300 relieves p53-evoked transcriptional repression of hypoxia-inducible factor-1 (HIF-1). Biochem J. 2004;380(Pt 1):289–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Ravi R, Mookerjee B, Bhujwalla ZM, Sutter CH, Artemov D, Zeng Q, et al. Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-inducible factor 1alpha. Genes Dev. 2000;14(1):34–44. [PMC free article] [PubMed] [Google Scholar]
  • 36.Zhang L, Yu D, Hu M, Xiong S, Lang A, Ellis LM, et al. Wild-type p53 suppresses angiogenesis in human leiomyosarcoma and synovial sarcoma by transcriptional suppression of vascular endothelial growth factor expression. Cancer Res. 2000;60(13):3655–61. [PubMed] [Google Scholar]
  • 37.Yamakuchi M, Lotterman CD, Bao C, Hruban RH, Karim B, Mendell JT, et al. P53-induced microRNA-107 inhibits HIF-1 and tumor angiogenesis. Proc Natl Acad Sci U S A. 2010;107(14):6334–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Nguyen PD, Tutela JP, Thanik VD, Knobel D, Allen RJ Jr., Chang CC, et al. Improved diabetic wound healing through topical silencing of p53 is associated with augmented vasculogenic mediators. Wound Repair Regen. 2010;18(6):553–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Liu P, Xu B, Cavalieri TA, Hock CE. Pifithrin-alpha attenuates p53-mediated apoptosis and improves cardiac function in response to myocardial ischemia/reperfusion in aged rats. Shock. 2006;26(6):608–14. [DOI] [PubMed] [Google Scholar]
  • 40.Matsusaka H, Ide T, Matsushima S, Ikeuchi M, Kubota T, Sunagawa K, et al. Targeted deletion of p53 prevents cardiac rupture after myocardial infarction in mice. Cardiovasc Res. 2006;70(3):457–65. [DOI] [PubMed] [Google Scholar]
  • 41.Zhang Y, Kohler K, Xu J, Lu D, Braun T, Schlitt A, et al. Inhibition of p53 after acute myocardial infarction: reduction of apoptosis is counteracted by disturbed scar formation and cardiac rupture. J Mol Cell Cardiol. 2011;50(3):471–8. [DOI] [PubMed] [Google Scholar]
  • 42.Mukhopadhyay UK, Eves R, Jia L, Mooney P, Mak AS. p53 suppresses Src-induced podosome and rosette formation and cellular invasiveness through the upregulation of caldesmon. Mol Cell Biol. 2009;29(11):3088–98. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Abaci A, Oguzhan A, Kahraman S, Eryol NK, Unal S, Arinc H, et al. Effect of diabetes mellitus on formation of coronary collateral vessels. Circulation. 1999;99(17):2239–42. [DOI] [PubMed] [Google Scholar]
  • 44.Loomans CJ, De Koning EJ, Staal FJ, Rabelink TJ, Zonneveld AJ. Endothelial progenitor cell dysfunction in type 1 diabetes: another consequence of oxidative stress? Antioxid Redox Signal. 2005;7(11-12):1468–75. [DOI] [PubMed] [Google Scholar]

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Supplementary Materials

1

Supplemental Figure: (A) p53 mRNA expression in hindlimb muscle from p53+/+ and p53−/− animals were determined by Real-Time PCR. (B) Representative LDPI images of p53+/+ and p53−/− paw perfusion at day 0 and quantitative analysis of paw perfusion at day 0 (n = 4 for each group). (C) Representative LDPI images of PFT-treated mouse paw perfusion at day 0 and quantitative analysis of paw perfusion at day 0 (n = 4 for each group). (C) Representative LDPI images of Quinacrine-treated mouse paw perfusion at day 0 and quantitative analysis of paw perfusion at day 0 (n = 4 for each group). PFT: Pifithrin-alpha, Quin: Quinacrine, R, right paw; L, left paw; LDPI, laser Doppler perfusion imaging. All values represent the mean ± standard error of the mean.

NIHMS1504194-supplement-1.pdf (243.2KB, pdf)

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