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. Author manuscript; available in PMC: 2023 May 1.
Published in final edited form as: Microvasc Res. 2022 Jan 7;141:104311. doi: 10.1016/j.mvr.2022.104311

Gene Therapy with Pellino-1 Improves Perfusion and Decreases Tissue Loss in Flk-1 Heterozygous Mice but Fails in MAPKAP KINASE-2 Knockout Murine Hind Limb Ischemia Model

Mahesh Thirunavukkarasu 1, Seetur R Pradeep 1, Gopi Ukani 1,2,*, Salim Abunnaja 1,2,*, Mark Youssef 1,2,*, Diego Accorsi 1,2,ψ, Santosh Swaminathan 1,2,ψ, Sue Ting Lim 1,2,ψ, Virginia Parker 1,2,ψ, Jacob Campbell 1, Muhammad Tipu Rishi 1,2, J Alexander Palesty 2, Nilanjana Maulik 1,#
PMCID: PMC9250804  NIHMSID: NIHMS1773237  PMID: 34999110

Abstract

Objectives:

In the United States, over 8.5 million people suffer from peripheral arterial disease (PAD). Previously we reported that Pellino-1(Peli1) gene therapy reduces ischemic damage in the myocardium and skin flaps in Flk-1 [Fetal Liver kinase receptor-1 (Flk-1)/ Vascular endothelial growth factor receptor-2/VEGFR2] heterozygous (Flk-1+/−) mice. The present study compares the angiogenic response and perfusion efficiency following hind limb ischemia (HLI) in, Flk-1+/− and, MAPKAPKINASE2 (MK2−/−) knockout (KO) mice to their control wild type (WT). We also demonstrated the use of Peli1 gene therapy to improve loss of function following HLI.

Study Design and Methods:

Femoral artery ligation (HLI) was performed in both Flk-1+/− and MK2−/− mice along with their corresponding WT. Another set of Flk-1+/− and MK2−/− were injected with either Adeno-LacZ (Ad.LacZ) or Adeno-Peli1 (Ad.Peli1) after HLI. Hind limb perfusion was assessed by laser doppler imaging at specific time points. A standardized scoring scale is used to quantify the extent of ischemia. Histology analysis performed includes capillary density, fibrosis, pro-angiogenic and anti-apoptotic proteins.

Results:

Flk-1+/− and MK2−/− had a slower recovery of perfusion efficiency in the ischemic limbs than controls. Both Flk-1+/− and MK2−/− KO mice showed decreased capillary density and capillary myocyte ratios with increased fibrosis than their corresponding wild types. Ad.Peli1 injected ischemic Flk-1+/− limb showed improved perfusion, increased capillary density, and pro-angiogenic molecules with reduced fibrosis compared to Ad.LacZ group. No significant improvement in perfusion was observed in MK2−/− ischemic limb after Ad. Peli1 injection.

Conclusion:

Deletion of Flk-1 and MK2 impairs neovascularization and perfusion following HLI. Treatment with Ad. Peli1 results in increased angiogenesis and improved perfusion in Flk-1+/− mice but fails to rectify perfusion in MK2 KO mice. Overall, Peli1 gene therapy is a promising candidate for the treatment of PAD.

Keywords: Hind limb ischemia, VEGF signaling, Perfusion, Ischemic Score, Peli1

Introduction

Peripheral arterial disease (PAD) is a prevalent condition. It is mainly caused by atherosclerosis, which results in plaque formation in the arteries and leads to impaired perfusion in the lower extremity. Over time, the disease can reduce mobility, quality of life and increase morbidity. If left untreated, it might lead to gangrene and amputation (Katwal et al., 2013). Chronic limb-threatening ischemia (CLTI) is a clinical syndrome and is due to the presence of PAD (Conte et al., 2019). Many patients are not eligible for surgical or catheter-based revascularization. Induction of collateral vessel formation or increased angiogenesis offers treatment for PAD. Generally, PAD patients are treated with lipid-lowering drugs, which could be helpful in this case. However, most drugs are still at the experimental stage and have yet to show their efficacy in PAD to increase blood flow to ischemic limbs. Again, the gene therapy approach to cure PAD also failed at the clinical trial, e.g., TAMARIS Trial failed at Phase-3 (Belch et al., 2011; Fowkes and Price, 2011). We believe that the failure of most of the clinical trials, with or without gene therapy, lies in not understanding the target molecule or molecular mechanism of PAD and, thereby, failing to rectify blood flow to save ischemic lower limbs.

Vascular endothelial growth factor (VEGF) promotes endothelial cell proliferation, microvascular permeability, vasodilation, and angiogenesis (Ferrara and Davis-Smyth, 1997; Neufeld et al., 1999). The first most important thing to understand is the molecular mechanism and downstream target proteins of vascular endothelial growth factor-mediated angiogenesis and neovascularization. VEGF is the most essential growth factor that is specific to, and necessary for, the formation of blood vessels (Dvorak et al., 1999; Eriksson and Alitalo, 1999; Spyridopoulos et al., 1997). Signals from endothelial cells’ transmembrane receptor tyrosine kinases (RTKs) and non-receptor tyrosine kinases (Src family) regulate the angiogenesis process. The two RTKs that have been shown to regulate blood vessel formation during embryogenesis when combined with their ligand VEGF are fetal liver kinase receptor-1 (Flk-1), also known as Vascular endothelial growth factor receptor-2 (VEGFR2), and Fms-related tyrosine kinase-1 (Flt-1), also known as VEGF receptor-1 (VEGFR1) (Fong et al., 1995; Takeshita et al., 1994). It has been shown that this receptor/ligand system boosts neovascularization (Asahara et al., 1998; Banai et al., 1994; Isner et al., 1996). VEGF is an endothelial cell-specific angiogenic factor and a key regulator of angiogenesis, stimulating endothelial cell proliferation, migration, and proteolytic action (Maruyama et al., 1999). However, the signaling pathways that regulate VEGF’s mitogenic effects in vascular cells are unknown (Rousseau et al., 1997).

We have recently observed that Pellino-1 (Peli1) plays an essential role in VEGF-mediated angiogenesis in MI and ischemic skin flap models (Rednam et al., 2017; Selvaraju et al., 2020; Thirunavukkarasu et al., 2018). Gene therapy with Peli1 was found to rectify impaired angiogenesis in Flk-1+/− knockout (KO) mice subjected to MI and an ischemic skin flap (Rednam et al., 2017; Thirunavukkarasu et al., 2018). Peli1 is a novel oncogene and potential therapeutic target in lung cancer (Jeon et al., 2016). Also, Peli1 functions as a critical mediator of nuclear factor kappa B (NF- κB), and mitogen-activated protein kinase (MAPKinase) activation in tolllike receptor-3 (TLR3) and toll-like receptor-4 (TLR4) signaling (Chang et al., 2009; Ji et al., 2014; Xiao et al., 2013).

Several reports recently have mentioned the involvement of endothelial nitric oxide synthase/ nitric oxide (eNOS/NO) in VEGF-mediated signaling. This involvement of VEGF in NO production is observed in leporine, porcine, bovine, and human vascular endothelial cells (Houck et al., 1991; Parenti et al., 1998; van der Zee et al., 1997). VEGF-induced NO production, when inhibited, showed loss of VEGF mediated mitogenic and angiogenic effects (Morbidelli et al., 1996). One of the primary mediators of VEGF-induced hemodynamic changes and microvascular permeability has been identified as endothelial NOS/NO (Bates, 2010). VEGF-mediated triggering of MAPK signaling and inhibition of Stress-activated protein kinases (SAPK)/Jun amino-terminal kinases (JNK) activity has been reported as a crucial step in determining if the endothelial cell survives or undergoes apoptosis (Gupta et al., 1999). However, the precise mechanism of myocardial angiogenesis is far from clear. It is imperative to explore in vivo molecular mechanism(s) of angiogenesis to understand the therapeutic strategies in ischemic tissues.

Hence, we used two genetic knockout (KO) mouse models (Flk-1+/− and MK2−/−) and exposed them to hind limb ischemia (HLI) to determine the effect on perfusion and the extent of angiogenesis when compared to their corresponding wild-type controls. The purpose of the current study was to investigate, identify and understand the molecular mechanism by using Adeno-Peli1 (Ad.Peli1) gene therapy as a rescue molecule in both the knockout mouse models after HLI. This approach will help us understand the Peli1-VEGF signaling axis further, which may hold significant therapeutic potential in the field of therapeutic angiogenesis in ischemic tissue models.

Materials and Methods

Animal studies:

This study was executed in compliance with the standards of laboratory animal care designed by the National Society for Medical Research under the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (publication No. 85–23, revised 1985). All animal experiments involved in this work have been reviewed and approved by the Institutional Animal Care and Use of Committee (IACUC) of the University of Connecticut Health Center (Farmington, CT). The approved IACUC protocol number is TE-102051–0322.

Knockout mouse models:

In order to better understand the mechanism by which Peli1 promotes angiogenesis, as well as its relationship to MAP Kinase-Activated Protein Kinase 2 (MAPKAP Kinase 2; MK2), we used two distinct knockout mouse models, Flk-1 heterozygous (Flk-1+/−) and MAPKAP Kinase 2 homozygous KO (MK2−/−) mice.

Generation of Flk-1 Heterozygous (Flk-1+/−) mice

Male ICR (CD1) mice between 8–12 weeks of age were purchased from Envigo (Indianapolis, IN, USA). Heterozygous Flk-1 mice (Strain name: B6.129-Kdrtm1Jrt/J) were purchased from Jackson Laboratory (Bar Harbor, Maine, USA) and back-crossed with the CD1/ICR mice, as mentioned earlier (Thirunavukkarasu et al., 2018), for ten generations to accomplish a 50% Flk-1gene deletion (Flk-1+/−). Littermate ICR (CD1) mice served as the wild type for this subset of animals.

Generation of MAPKAP Kinase 2 KO (MK2−/−) mice

MK2−/− mice (Kotlyarov et al., 1999) were originally obtained as a generous donation from Dr. Gaestel (Institut für Zellbiochemie, Medizinische Hochschule Hannover) (Kotlyarov et al., 1999). These mice were further bred and maintained in the University of Connecticut Health Center animal facility. C57BL/6J mice were used as the corresponding wild type (WT) for this subset of animals.

Experimental Design

Experiment 1: Effect of Flk-1 gene and MK2 gene knockout in mice subjected to HLI

The first part of our experiment aimed to assess the gene knockout effects in mice subjected to hind limb ischemia. Both Flk-1+/− and MK2−/− mice and their respective WT mice were subjected to right femoral artery ligation to create an HLI model. The animals used were grouped as follows: (i) (1) WTHLI, (2) Flk-1+/−HLI (Figure 1A); (ii) (1) WTHLI, (2) MK2−/−HLI, (Figure 2A). Functional parameters were obtained at predetermined time intervals using Doppler imaging and analysis along with limb ischemia scoring. Tissues collected from ischemic and non-ischemic limbs were examined for the extent of fibrosis, capillary density, and capillary-to-myocyte ratios. The expression of proangiogenic molecules, such as VEGF and eNOS, as well as the pro-apoptotic factor Bax, were assessed via immunofluorescence analysis of the muscle tissues.

Fig 1.

Fig 1.

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A) Graphical representation of experimental design showing laser doppler imaging (LDI) being performed on both limbs preoperatively, immediately after surgery, and on days 3, 7, 14, 21, and 28 on both WT and Flk-1+/− group. On postoperative day 28, animals were sacrificed, hind limb muscles were harvested from both limbs. B) Representative perfusion images from the ischemic limb of the WT group (n=14) and Flk-1+/− group (n=15) at the predetermined time points. C) Flk-1+/− mice subjected to HLI showed decreased perfusion ratio as early as day 3 and were most pronounced on day 28 when compared to WT mice. Values are mean ± S.E.M; [(Preop: p=0.1383, Unpaired t-test, Two-tailed); (Postop Day 0: p=0.3375, Unpaired t-test, Twotailed); (Postop Day 3: *p=0.0105, Unpaired t-test, Two-tailed); (Postop Day 7: p=0.0245, Unpaired t-test, Two-tailed); (Postop Day 14: p=0.1320, Mann–Whitney U test, Two-tailed); (Postop Day 21: *p=0.0469, Mann–Whitney U test, Two-tailed); (Postop Day 28: *p=0.0022, Mann–Whitney U test, Two-tailed)]. D) Representative picture shows Flk-1 negative control (no primary antibody) staining (GFP: Gain-18, Exposure-100; DAPI: Gain-6, Exposure-80). E) Representative picture showing Flk-1 staining in Flk-1+/− mice limb tissue compared to WT mice (GFP: Gain-18, Exposure-100; DAPI: Gain-6, Exposure-100). F) Graphical representation Flk-1 expression in both WT and Flk-1+/− mice showed decreased levels of Flk-1 expression in Flk-1+/− mice when compared to WT mice (n=5). Values are mean ± S.E.M; *p=0.0498, Unpaired t-test, Two-tailed.

Fig 2:

Fig 2:

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A) Graphical representation of experimental design showing laser doppler imaging (LDI) being performed on both limbs preoperatively, immediately after surgery, and on days 3, 7, 14, 21, and 28 both WT and MK2−/− group. At postoperative day 28, animals were sacrificed, hind limb muscles were harvested from both limbs. B) Representative perfusion images from the ischemic limb of the WT group (n=9–10) and MK2−/− group (n=12–16) at the predetermined time points. C) MK2−/− mice subjected to HLI showed decreased perfusion ratio as early as day 3 and most pronounced on day 28 when compared to WT mice. Values are mean ± S.E.M; [(Preop: p=0.1945, Unpaired t-test, Two-tailed); (Postop Day 0: *p=0.0974, Mann-Whitney U test, Two-tailed); (Postop Day 3: *p=0.0001, Unpaired t-test, Two-tailed); (Postop Day 7: *p<0.0001, Unpaired t-test, Two-tailed); (Postop Day 14: p=*0.0008, Unpaired t-test, Two-tailed); (Postop Day 21: *p=0.0001, Unpaired t-test, Two-tailed); (Postop Day 28: *p=0.0010, Unpaired t-test, Two-tailed)]. D) Representative picture shows MK2 negative control (no primary antibody) staining (GFP: Gain-18, Exposure-100; DAPI: Gain-6, Exposure-80). E) Representative picture showing MK2 staining in MK2−/− mice limb tissue compared to WT mice (GFP: Gain-18, Exposure-100; DAPI: Gain-6, Exposure-80). F) Graphical representation of MK2 expression in both WT and MK2−/− mice showed decreased levels of MK2 expression in MK2−/− mice when compared to WT mice (n=5). Values are mean ± S.E.M; *p<0.0001, Unpaired t-test, Two-tailed.

Experiment 2 – Effect of Pellino-1 gene therapy on Flk-1 heterozygous and MK2 gene knockout in mice subjected to HLI

The goal of this experiment was to observe the effect of Peli1 gene therapy using adenoviral vectors in both the knockout mice. Flk-1+/− and MK2−/− mice were subjected to right femoral artery ligation, followed by intramuscular injection with Ad. Peli1 (1×109 PFU in 50μL PBS) or Ad.LacZ (1×109 PFU in 50μL PBS) as control into the ischemic limb. The animals were grouped as follows: (i) (1) Flk-1+/−HLI + Ad.LacZ, (2) Flk-1+/−HLI + Ad.Peli1 (Figure 3A); (ii) (1) MK2−/−HLI + Ad.LacZ (2) MK2−/−HLI + Ad.Peli1 (Figure 4A). Similar experimental procedures and time intervals were followed to determine the functional parameters, tissue collection, and protein expression as discussed above for Experiment 1.

Fig 3.

Fig 3.

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A) Graphical representation of experimental design showing laser doppler imaging (LDI) being performed on both limbs preoperatively, immediately after surgery, and on days 3, 7, 14, 21, and 28 on Flk-1+/− mice subjected to HLI and further treated both Ad.LacZ and Ad.Peli1. At postoperative day 28, animals were sacrificed, hind limb muscles were harvested from both limbs. B) Representative perfusion images from the ischemic limb of Flk-1+/− mice treated with Ad.LacZ (n=9–15) and Ad.Peli1 (n=9–15) at the predetermined time points. C) Perfusion ratios in Flk-1+/− mice treated with Ad. Peli1 shows statistically significant improvement of perfusion ratio from postoperative day 7 and continues to improve until day 28 compared to WT mice. Values are mean ± S.E.M; [(Preop: p=0.5907, Mann–Whitney U test, Two-tailed); (Postop Day 0: p=0.1607, Mann–Whitney U test, Two-tailed); (Postop Day 3: p=0.0675, Mann–Whitney U test, Two-tailed); (Postop Day 7: *p=0.0015, Unpaired t-test, Two-tailed); (Postop Day 14: p=*0.0009, Unpaired t-test, Two-tailed); (Postop Day 21: *p=0.0160, Unpaired t-test, Twotailed); (Postop Day 28: *p=0.0029, Mann–Whitney U test, Two-tailed)].

Fig 4.

Fig 4.

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A) Graphical representation of experimental design showing laser doppler imaging (LDI) being performed on both limbs preoperatively, immediately after surgery, and on days 3, 7, 14, 21, and 28 on MK2−/− mice subjected to HLI and further treated both Ad.LacZ and Ad.Peli1. At postoperative day 28, animals were sacrificed, hind limb muscles were harvested from both limbs. B) Representative perfusion images from the ischemic limb of MK2−/− mice treated with Ad.LacZ (n=11) and Ad.Peli1 (n=10) at the predetermined time points. C) There is no significant difference observed between perfusion ratios in MK2−/− mice treated with Ad.LacZ and Ad. Peli1. Values are mean ± S.E.M; [(Preop: p=0.2568, Unpaired t-test, Two-tailed); (Postop Day 0: p=0.2050, Unpaired t-test, Two-tailed); (Postop Day 3: p=0.5972, Unpaired t-test, Two-tailed); (Postop Day 7: p=01.463, Unpaired t-test, Two-tailed); (Postop Day 14: p=0.5832, Unpaired ttest, Two-tailed); (Postop Day 21: p=0.3969, Mann–Whitney U test, Two-tailed); (Postop Day 28: p=0.6168, Unpaired t-test, Two-tailed)]. D) MK2−/− mice treated with Ad.Peli1 or Ad.LacZ after HLI shows no significant difference in ischemic score up to 28 days post-HLI. Values are mean ± S.E.M.; [ (Postop Day 3: p=0.7002, Unpaired t-test, Two-tailed); (Postop Day 7: p=0.5053, Unpaired t-test, Two-tailed); (Postop Day 14: p=0.4174, Unpaired t-test, Two-tailed); (Postop Day 21: p=0.7058, Unpaired t-test, Two-tailed); (Postop Day 28: p=0.4677, Unpaired ttest, Two-tailed)]. E-F) Representative photographs MK2−/− mice after HLI followed by Ad.LacZ and Ad.Peli1 treatment. MK2−/− mice injected with Ad.LacZ or Ad.Peli1 did not suffer from any limb amputations (n=10–11).

Surgical Procedure of Hind Limb Ischemia Model

The animals were placed under an operative microscope on a heating pad after being anesthetized with a rodent isoflurane vaporizer set to 2–3 percent and a continuous flow of oxygen administered at 1 L/min. Half dose of buprenorphine (0.05–0.1 mg/kg) was provided as analgesia before and after surgery (0.05–0.1 mg/kg) by subcutaneous injection. Depilatory cream was used to remove hair from both hind limbs. After cleaning the right leg with betadine, an incision was made in the mid-thigh from the groin to just above the knee. The femoral artery was separated with delicate blunt dissection after the femoral neurovascular bundle was exposed, taking care not to damage the accompanying nerve and vein. The femoral artery was then ligated at two different points using 8–0 Prolene suture, just proximal to the take-off of the deep femoral artery, and a few mm distally to the same and roughly at the take-off of the saphenous artery. Any collaterals between these two ligation points were cauterized or ligated. At this stage, adenovirus, if applicable, was injected intramuscularly at four different sites. The skin was then closed using a 5–0 vicryl suture (Rishi et al., 2015; Selvaraju et al., 2020; Thirunavukkarasu et al., 2018; Thirunavukkarasu et al., 2021). Buprenorphine (0.05–0.1 mg/kg) was also provided as postoperative analgesia twice per day for two days after surgery at 8–14-hour intervals.

Gene Therapy with Ad. Peli1 and Ad.LacZ

In Experiment 2, Flk-1+/− mice were subjected to surgical hind limb ischemia and Ad. Peli1 or Ad.LacZ (1×109 PFU in 50μL PBS) was injected equally before wound closure at four different sites of both semimembranosus and gastrocnemius muscles of the right leg. MK2−/− mice were similarly subjected to surgical hind limb ischemia followed by injection of Ad.Peli1 or Ad.LacZ (1×109 PFU in 50μL PBS) (Selvaraju et al., 2020; Thirunavukkarasu et al., 2021).

Laser Doppler Perfusion Imaging

Laser doppler imaging was performed on all animals preoperatively (baseline), immediately postoperative (day 0), and on postoperative days 3, 7, 14, 21, and 28. Mice were laid in supine condition after bilateral hind limb depilation with chemical depilatory cream and kept on a temperature-controlled heating pad for 5 min. A PeriScan blood perfusion imager (PIM 3; Perimed, Sweden) was used to measure the blood flow in both hind limbs. The left limb, not operated on, served as an internal control. A standardized region of interest (ROI) which included both the thigh and leg, was selected for both the ischemic and non-ischemic limbs of each mouse. The blood flow in the ischemic limb at any particular time point was calculated and expressed as perfusion ratio, which is the ratio of ischemic perfusion to non-ischemic perfusion within these ROIs (Perfusion ratio = Perfusion within ROI-ischemic limb/ Perfusion within ROI-nonischemic limb) (Rishi et al., 2015; Selvaraju et al., 2020; Thirunavukkarasu et al., 2018; Thirunavukkarasu et al., 2021).

Assessment of Ischemic Score

The severity of the hind limb ischemia for each animal was assessed using a modified version of the ischemic score proposed by Tormod et al. (Brenes et al., 2012; Tarlov, 1954; Westvik et al., 2009; Yu et al., 2005). A score from 0 to 8 was assigned preoperatively, immediately post-op, and on postoperative days 3, 7, 14, 21, and 28. The score was given according to the following parameters on physical examination: 0 = no ischemia, 1 = one nail discolored, 2 = two or more nails discolored, 3 = one toe discolored, 4 = two or more toe discolored, 5 = foot necrosis, 6 = autoamputation of foot/midfoot, 7 = leg necrosis, 8 = autoamputation of leg.

At the end of the study, all the animals were anesthetized with ketamine/xylazine mixture at a dose of 90–120 mg/kg + 5–10 mg/kg and were be perfused with PBS, followed by the removal of limb muscle tissue for molecular and histological analysis. This method of euthanasia was reviewed and approved by the Institutional Animal Care and Use of Committee of the University of Connecticut Health (Farmington, CT)

Capillary Density and Capillary-to-Myocytes Ratio

Gastrocnemius muscle sections were harvested from the ischemic limb of each animal on postoperative day 28. After undergoing histological processing, 5 μm thick sections were stained with Goat polyclonal anti-CD31 (Cat# AF3628; R&D Systems, Minneapolis, MN, USA) followed by ImPRESS® HRP Horse Anti-Goat IgG polymer detection kit, peroxidase (Vector Laboratories, Inc., Burlingame, CA) and were then visualized using 3,3-diaminobenzidine (DAB) substrate. Images were captured at 40x magnification using an Olympus BH2 microscope equipped with an Olympus Q-Color3 camera (Olympus). Captured images were stored in digital Tiff file format. Adobe Photoshop software was used to measure the number of capillaries after superimposing a calibrated morphometric grid on each digital image and counted manually. Capillary density was calculated as an average count per mm2 (Rishi et al., 2015; Selvaraju et al., 2020; Shaikh et al., 2017; Thirunavukkarasu et al., 2018; Thirunavukkarasu et al., 2021).

Assessment of Fibrosis

The extent of tissue fibrosis was assessed on skeletal muscle cross-sections collected from the ischemic limb on postoperative day 28. Slides were deparaffinized in Histoclear and gradually rehydrated with a decreasing ethanol gradient of concentrations ranging from 100% to 70%. Collagen staining in fibrotic areas was performed by washing the tissues with distilled water and incubating with Picrosirius Red (Cat# 365548, Sigma-Aldrich, St. Louis, MO) stain for one hour at room temperature. These sections were washed in acidified water and subsequently dehydrated with an increasing ethanol gradient of concentrations ranging from 70% to 100%. After a final wash with Histoclear, the slides were mounted. Multiple images were taken for each sample using BH-2 Olympus (Olympus, PA, USA) microscope at 40x magnification. The images taken were processed as described in the ImageJ software (Schneider et al., 2012). In brief, the images were opened in ImageJ software and split into three channels (red, blue, and green using the command “Image>Type>RGB Stack.” All the measurements were thresholded in the green channel using the command “Image>Adjust>Threshold,” and each image was manually analyzed by measuring the fibrotic area. The extent of fibrosis was calculated as an average percent area using ImageJ software (NIH, Bethesda, MD) (Rishi et al., 2015; Selvaraju et al., 2020; Shaikh et al., 2017; Thirunavukkarasu et al., 2018; Thirunavukkarasu et al., 2021).

Immunofluorescence Analysis

The muscle tissue was harvested from the ischemic limb following surgery and was embedded in paraffin. In order to study intracellular Flk-1, MK2, Peli1, VEGF, eNOS, and Bax protein expression, 5 μm-thick sections were prepared from paraffin blocks. Tissue sections were blocked in 5% BSA/PBS and incubated with primary antibodies: Anti-VEGF receptor-1/Flk-1 (Cat# ab-32152; Abcam, Boston, MA), Anti-MAPKAP-2 (Cat# 3042L; Cell Signaling, Danvers, MA), Anti-Pellino-1 (Cat# sc-31619, Santa Cruz Biotechnology, Inc., Dallas, TX), monoclonal Mouse anti-VEGF (Cat# MA5–13182; Thermofisher Scientific, Waltham, MA), polyclonal Rabbit Anti-eNOS (Cat# 610299; BD Biosciences), and polyclonal Rabbit Anti-Bax (Cat# sc493; Santa Cruz Biotechnology, Inc., Dallas, TX) followed by a secondary antibody coupled with Alexa Fluor 555 (Cat#A31570 and A21428, Invitrogen). Slides were mounted with UltraCruz Mounting Medium with DAPI (Cat# sc-24941; Santa Cruz Biotechnology, Inc., Dallas, TX), and fluorescent microscopic images were acquired at 20x magnification using a digital microscope. (IRIS Digital Microscope, Logos Biosystems, South Korea) (Campbell et al., 2021; Rishi et al., 2015; Thirunavukkarasu et al., 2021). ImageJ software was used to measure the area, and integrated mean density from the acquired images and the corrected total fluorescence (CTF) was calculated by the formula: [integrated density – (area of the selected picture (x) mean fluorescence of background reading)] (Cappell et al., 2012; Harari-Steinberg et al., 2013; Jensen, 2013; Rishi et al., 2015; Thirunavukkarasu et al., 2021).

Statistical Analysis

All results obtained from this study were reported as mean ± SEM. Statistical analyses were carried out using the GraphPad Prism 8.0 and 9.0 software. The normal distribution of the data was analyzed using Shapiro–Wilk test followed by an unpaired t-test or Mann–Whitney test to test the significant differences between two groups. Results were reported to be significant if p<0.05.

Results

Flk-1 and MK2 transgene expression in both Flk-1+/− and MK2−/− knockout mice

We performed immunohistochemical staining for Flk-1 and MK2 expression in the skeletal muscle tissue of non-ischemic limb (control limb) in the respective mice (Flk-1+/− and MK2−/−) to confirm the deletion of corresponding transgenes. Our results validated a significant reduction of Flk-1 (Figure 1E–F) and MK2 (Figure 2E–F) transgene expression in the respective genetically modified mice compared to WT.

Perfusion Ratio

Perfusion of bilateral hind limbs was measured via laser Doppler imaging preoperatively and at days 0 (immediately postoperatively), 3, 7, 14, 21, and 28. The data were expressed as perfusion ratio between ischemic and non-ischemic ROIs (perfusion ratio = perfusion to ischemic limb/perfusion to non-ischemic limb). Flk-1+/− mice subjected to HLI (Flk-1+/−HLI) showed decreased perfusion ratios as early as postoperative day 3 (0.08 ± 0.01 vs. 0.16 ± 0.03, p=0.0105, n=13), postoperative day 7 (0.08 ± 0.02 vs. 0.16 ± 0.03, p=0.0245, n=13) with the difference being most pronounced on day 28 (0.14 ± 0.06 vs. 0.44 ± 0.05, p=0.0022, n=11–15), compared to WTHLI group (Figure 1B–C). Similarly, MK2−/− mice subjected to HLI (MK2-/HLI) had decreased perfusion ratios starting on postoperative day 3 (0.1 ± 0.01 vs. 0.21 ± 0.03, p=0.0001, n=10–14), postoperative day 7 (0.14 ± 0.02 vs. 0.37 ± 0.03, p<0.0001, n=10–14), postoperative day 14 (0.26 ± 0.03 vs. 0.49 ± 0.06, p=0.0008, n=10–13) with differences also being most pronounced on postoperative day 28 (0.33 ± 0.03 vs. 0.60 ± 0.08, p=0.0010, n=10–14) compared to WTHLI group (Figure 2B–C).

In the second part of our experiment (Experiment 2), a different subset of Flk-1+/− mice subjected to right hind limb ischemia were postoperatively injected with equal concentration (1×109 PFU in 50μL PBS) of Ad. Peli1 (Flk-1+/− + Ad. Peli1) and Ad.LacZ (Flk-1+/− + Ad.LacZ, control) to investigate whether Peli1 gene therapy could reverse the effects of Flk-1 gene deletion. Flk-1+/− + Ad. Peli1 group showed a statistically significant improvement in perfusion ratios compared to the Flk-1+/− + Ad.LacZ group on postoperative days 7 (0.19 ± 0.03 vs. 0.08 ± 0.02, p=0.0015, n=15), postoperative day14 (0.41 ± 0.07 vs. 0.09 ± 0.03, p=0.0009, n=9–10), postoperative day 21 (0.33 ± 0.05 vs. 0.14 ± 0.05, p=0.0160, n=9) and postoperative day 28 (0.31 ± 0.05 vs. 0.15 ± 0.02, p=0.0029, n=10) (Figure 3B–C). This finding supports our hypothesis that Peli1 acts downstream of Flk-1, and an exogenous supplementation may partially ameliorate the effects of Flk-1 gene knockout and potentiates an angiogenic response. Treatment of MK2−/− mice with Ad.Peli1 (MK2−/− + Ad.Peli1), however, did not show any statistically significant improvement in perfusion ratio when compared to the MK2−/−+Ad.LacZ group at all time points (Figure 4B–C). This finding was supported by a similar lack of improvement in ischemic scores (Figure 4D). These results suggest that MK2 is necessary for Peli1 function, and as such, the most likely scenario is that Peli1 exerts its function upstream of MK2 but downstream of Flk-1.

Ischemia Score and Auto-amputation Rate

The level and degree of ischemia were assessed using an 8-point standardized ischemia score preoperatively, immediately postoperatively (Day 0), and on postoperative days 3, 7, 14, 21, and 28. Mean ischemic scores across the timeline were compared to values from respective WT mice. Flk-1+/−HLI mice demonstrated higher ischemic scores consistently throughout when compared to WTHLI counterparts, reaching statistical significance on postoperative day 14 (4.93 ± 0.67 vs. 2.85 ± 0.71, p=0.0417, n=13–14) and day 28 (5.14 ± 0.71 vs. 3 ± 0.75, p=0.0484, n=13–14) (Figure 5F). Similarly, MK2−/−HLI mice demonstrated higher ischemic scores with statistical significance reached at postoperative days 14 (2 ± 0.68 vs. 0 ± 0, p=0.0307, n=10–16), 21 (2 ± 0.68 vs. 0 ± 0, p=0.0401, n=9–16) and 28 (2 ± 0.68 vs. 0 ± 0, p=0.0307, n=10–16) when compared to their WTHLI (Figure 5N). These results support the notion that the observed decrease in limb perfusion seen in Flk-1+/− (Figure 1C) and MK2−/− (Figure 2C) mice is accompanied by a clinically relevant increase in the degree of tissue ischemia, tissue necrosis, and/or tissue loss, ultimately impairing limb function.

Fig 5.

Fig 5.

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A) Representative micrograph of the ischemic limb in Flk-1+/− mice showed decreased capillary density compared to WT mice. B) Graphical representation of capillary density count between two groups, showing statistically significant reduction in capillary density in Flk-1+/− mice compared to WT mice (n=4–5). Values are mean ± S.E.M; *p=0.0047, Unpaired t-test, Two-tailed. C) Graphical representation of capillary-to-myocyte ratio showing a similar reduction in Flk-1+/− mice compared to corresponding WT (n=4–5). Values are mean ± S.E.M; *p=0.0008, Unpaired t-test, Two-tailed. D-E) Picrosirius red staining demonstrates increased fibrosis in Flk-1+/− mice compared to WT mice (n=5). Values are mean ± S.E.M; *p<0.0001, Unpaired t-test, Two-tailed. F) Flk-1+/− mice showed increased ischemic scores than WT mice, reaching statistical significance on day 14 (*p=0.0417, Unpaired t-test, Two-tailed) and day 28 (*p=0.0484, Unpaired t-test, Two-tailed) (n=14). G-H) Representative photographs of WT mice and Flk-1+/−mice following HLI. The amputation rate in Flk-1+/− group is 28.6%, whereas no WT mice suffered limb loss following surgery (n=14). I) Representative micrograph of the ischemic limb in MK2−/− mice showing decreased capillary density as compared to WT mice. J) Graphical representation of capillary density count between two groups, showing statistically significant reduction in capillary density in MK2−/− mice compared to WT mice (n=5). Values are mean ± S.E.M; *p<0.0001, Unpaired t-test, Two-tailed. K) Graphical representation of capillary-to-myocyte ratio showing a similar reduction in MK2−/− mice compared to corresponding WT (n=5). Values are mean ± S.E.M; *p<0.0001, Unpaired t-test, Two-tailed. L-M) Picrosirius red staining demonstrates increased fibrosis in MK2−/− mice compared to WT (n=5). Values are mean ± S.E.M; *p=0.0003, Unpaired t-test, Two-tailed. N) MK2−/− mice show increased ischemic scores than WT mice, reaching statistical significance on days 14 (*p=0.0307, Unpaired t-test, Twotailed), 21 (*p=0.0401, Unpaired t-test, Two-tailed), and 28 (*p=0.0307, Unpaired t-test, Two-tailed), (n=10–16). O-P) Representative photographs of WT mice and MK2−/− mice following HLI. In both groups, no animals suffered limb loss. Values are mean ± S.E.M; *p<0.05.

The rescue of perfusion with Ad.Peli1 therapy was demonstrated by a concomitant decrease in ischemic scores in the Flk-1+/−group. Ischemic scores were statistically decreased in Flk-1+/− + Ad.Peli1 mice versus Flk-1+/− + Ad.LacZ mice on postoperative day 3 (1.93 ± 0.47 vs. 4.07 ± 0.53, p=0.0056, n=15), postoperative day 7 (2.20 ± 0.43 vs. 4.93 ± 0.62, p=0.0011, n=15), postoperative day 14 (2.3 ± 0.50 vs. 5.67 ± 1.16, p=0.0128, n=9–10), postoperative day 21 (2 ± 0.55 vs. 6.56 ± 0.93, p=0.0007, n=9), and postoperative day 28 (1.8 ± 0.53 vs. 5.9 ± 1.06, p=0.0028, n=10) (Figure 6E). Treatment of MK2−/− mice with Ad.Peli1 (MK2−/−+Ad.Peli1, n=10), however, failed to show any statistically significant improvement in ischemic scores at all time points, which is in concordance with the perfusion ratios obtained. (Figure 4D).

Fig 6.

Fig 6.

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A) Representative micrograph of the ischemic limb in Flk-1+/− mice showing decreased capillary density in mice treated with Ad.LacZ compared to those treated with Ad.Peli1. B) Graphical representation of capillary density count between two groups, showing a statistically significant increase in capillary density in the group treated with Ad.Peli1 (n=5–6). Values are mean ± S.E.M; *p=0.0016, Unpaired t-test, Two-tailed. C-D) Picrosirius red staining demonstrates a reduction in fibrosis in Ad.Peli1 treated mice compared to Ad.LacZ group (n=5–6). Values are mean ± S.E.M; *p=0.0389, Unpaired t-test, Two-tailed. E) Flk-1+/− mice treated with Ad.Peli1 after HLI shows significant improvement in the ischemic score as early as day 3 (*p=0.0056, Unpaired t-test, Two-tailed), with its effect lasting up to day 28 (*p=0.0028, Unpaired t-test, Two-tailed). F-G) Representative photographs Flk-1+/− mice after HLI followed by Ad.LacZ and Ad.Peli1 treatment. Flk-1+/−Ad.LacZ group has a limb loss rate of 60%, whereas none from Ad.Peli1 group suffered limb loss (n=10–15).

We also observed an interesting difference in auto-amputation rates among different subset of animals during the assessment of ischemic scores. Flk-1+/− mice after HLI exhibit an auto-amputation rate of 28.6% (n=14), whereas no autoamputations were noted in WT mice subjected to HLI (Figure 5G–H). Following the rescue of the ischemic limb with gene therapy, we noted the Flk-1+/− + Ad.Peli1 group shows no auto-amputation (n=10) while Flk-1+/− + Ad.LacZ mice sustained an auto-amputation rate of 60% (n=10), further consolidating the evidence that Ad.Peli1 therapy can reverse some of the effects of Flk-1 gene knockout (Figure 6F–G). MK2−/− mice and their corresponding WT shows no auto-amputation with (Figure 4E–F) and without (Figure 5O–P) gene therapy.

Tissue Fibrosis, Capillary Density and Capillary-to-Myocytes Ratio

Ischemic muscles were harvested at the end of 28 days following right femoral artery ligation in the Flk-1+/−HLI and MK2−/−HLI groups along with their respective WT groups. The tissues were evaluated for capillary density, capillary-to-myocyte ratio, and extent of tissue fibrosis. There was a marked decrease in capillary density (Figure 5A–B) and the capillary-tomyocyte ratio (Figure 5C) seen in the Flk-1+/−HLI group (n=4) compared to its WT (n=5) (Capillary density: 581 ± 40 counts/mm2 vs 1081 ± 104 counts/mm2, p=0.0047 and capillary-to-myocyte ratio: 0.84 ± 0.03 vs 1.64 ± 0.12, p=0.0008). This corresponds with the decreased perfusion and tissue ischemia as demonstrated earlier and suggests that loss of either Flk-1 or MK2 function can lead to hampered vessel formation. Picrosirius staining of the tissues to reveal the extent of tissue fibrosis (Figure 5D–E) showed a significant increase in fibrosis in ischemic muscles harvested from the Flk-1+/−HLI group (n=5) when compared to the WTHLI group (25.84 ± 1.42% vs. 13.57 ±0.83%, p<0.0001).

These results were paralleled by MK2−/−HLI mice versus their corresponding WT mice (n=5 each), with a significant reduction observed in the capillary density (454.1 ± 30.09 vs. 758.6 ± 28.10 counts/mm2, p<0.0001) (Figure 5I–J) and capillary-to-myocyte ratio (0.88 ± 0.02 vs. 1.39 ± 0.06, p<0.0001) (Figure 5K). Results of tissue staining with Picrosirius red revealed a marked increase in the area of fibrosis in the MK2−/−HLI group compared to the corresponding WTHLI group (n=5 each) (25.39 ± 2.26% vs. 10.40 ±0.87%, p=0.0003) (Figure 5L–M).

Significantly elevated capillary density was noted in the Flk-1+/−+Ad.Peli1 treatment group compared to the Flk-1+/−+Ad.LacZ (1626 ± 133.6 vs. 741.7 ± 146.4 counts/mm2, p=0.0016, n=5–6) indicates improved neovascularization with Peli1 gene therapy and correlates (Figure 6A–B) with the improved perfusion (Figure 3B–C) seen on vascular imaging, clinical ischemic scores, and auto-amputation rates (Figure 6E–G). Similarly, we were able to demonstrate a marked decrease in the extent of tissue fibrosis of the ischemic muscles in the Flk1+/−+Ad.Peli1 treatment group compared to the Flk-1+/−+Ad.LacZ group (5.43 ± 2.70 vs. 23.76 ± 7.73%, p=0.0389, n=5–6) (Figure 6C–D).

Transfection Efficiency:

Immunohistochemical staining for Peli1 expression in Flk-1+/− and MK2−/− mice injected with Ad.Peli1 was performed to validate the increased expression level of Peli-1 by Ad. Peli1 gene therapy. Flk-1+/−+Ad.Peli1 treated mice showed increased Peli1 expression when compared to Flk-1+/−+Ad.LacZ treatment group (Figure 7B). Similarly, MK2−/+Ad.Peli1 treated mice showed increased Peli1 expression compared to MK2−/− +Ad.LacZ treatment group (Figure 7D). Figure 7A (Peli1 staining in Flk-1+/−) and 7C (Peli1 staining in MK2−/−) shows the negative control (no primary antibody) staining.

Fig 7.

Fig 7.

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A) Representative picture shows Peli1 negative control (no primary antibody) staining in Flk-1+/− injected with Ad.LacZ or Ad.Peli1 treatment (GFP: Gain-17, Exposure-150; DAPI: Gain-7, Exposure-100). B) Representative digital micrograph showing the increased level of Peli1 expression in Flk-1+/− mice subjected to HLI followed by Ad.LacZ or Ad.Peli1 injection (GFP: Gain-17, Exposure-150; DAPI: Gain-7, Exposure-100), (n=6). C) Representative picture shows Peli1 negative control (no primary antibody) staining in MK2−/− mice injected with Ad.LacZ or Ad.Peli1 treatment (GFP: Gain-17, Exposure-150; DAPI: Gain-7, Exposure-100). D) Representative digital micrograph showing the increased level of Peli1 expression in MK2−/− mice subjected to HLI followed by Ad.LacZ or Ad.Peli1 injection (GFP: Gain-17, Exposure-150; DAPI: Gain-7, Exposure-100), (n=3).

Immunofluorescence Analysis

Immunofluorescence analysis of VEGF, eNOS, and Bax was performed in Flk-1+/− and MK2−/− mice 4 days after surgery. There is a significant reduction in proangiogenic factor VEGF (12.5-fold, p=0.0464) (Figure 8B–C) and eNOS (6.3-fold, p=0.0175) (Figure 8N–O) expression in Flk-1+/− mice compared to their WT mice (p<0.05). Similarly, there was reduced expression of VEGF (3-fold, p=0.0073) (Figure 8E–F) and eNOS (1.8-fold, p=0.0291) (Figure 8Q–R) in MK2−/− mice compared to their control (p<0.05). On the other hand, pro-apoptotic molecule Bax expression was significantly increased in both Flk-1+/− (3.2-fold, p=0.0008) (Figure 8H–IF) and MK2−/− (2.5-fold, p=0.0095) (Figure 8K–L) mice on POD4 after HLI compared to their respective controls. Figure 8A, 8D, 8G, 8J, 8M, and 8P shows the negative control staining (no primary antibody added).

Fig 8.

Fig 8.

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A) Representative picture shows VEGF negative control (no primary antibody) staining in both WT and Flk-1+/− mice (GFP: Gain-17, Exposure-250; DAPI: Gain-8, Exposure-100). (B-C) Representative digital micrograph (GFP: Gain-17, Exposure-250; DAPI: Gain-8, Exposure-100) (B) and a bar graph (C) showing the suppressed level of VEGF in Flk-1+/− mice subjected to HLI compared to WT mice (n=4). Values are mean ± S.E.M; *p=0.0464, Unpaired t-test, Two-tailed. D) Representative picture shows VEGF negative control (no primary antibody) staining in both WT and MK2−/− mice (GFP: Gain-17, Exposure-250; DAPI: Gain-8, Exposure-100). E-F) Representative digital micrograph (E) (GFP: Gain-17, Exposure-250; DAPI: Gain-8, Exposure100) and a bar graph (F) showing the suppressed level of VEGF in MK2−/− mice subjected to HLI compared to WT mice (n=3–5). Values are mean ± S.E.M; *p=0.0073, Unpaired t-test, Twotailed. G) Representative picture shows Bax negative control (no primary antibody) staining in both WT and Flk-1+/− mice (RFP: Gain-17, Exposure-200; DAPI: Gain-13, Exposure-30). H-I) Representative digital micrograph (RFP: Gain-17, Exposure-200; DAPI: Gain-13, Exposure-30) (H) and a bar graph (I) showing the increased level of Bax in Flk-1+/− mice subjected to HLI compared to WT mice (n=4). Values are mean ± S.E.M; *p=0.0008, Unpaired t-test, Two-tailed. J) Representative picture shows Bax negative control (no primary antibody) staining in both WT and MK2−/− mice (RFP: Gain-17, Exposure-200; DAPI: Gain-13, Exposure-30). (K-L) Representative digital micrograph (RFP: Gain-17, Exposure-200; DAPI: Gain-13, Exposure-30) (K) and a bar graph (L) showing the increased level of Bax in MK2−/− mice subjected to HLI compared to WT mice (n=4–5). Values are mean ± S.E.M; *p=0.0095, Unpaired t-test, Twotailed. M) Representative picture shows eNOS negative control (no primary antibody) staining in both WT and Flk-1+/− mice (GFP: Gain-17, Exposure-250; DAPI: Gain-8, Exposure-100). (N-O) Representative digital micrograph (GFP: Gain-17, Exposure-250; DAPI: Gain-8, Exposure-100) (N) and a bar graph (O) showing the suppressed level of eNOS in Flk-1+/− mice subjected to HLI compared to WT mice (n=4). Values are mean ± S.E.M; *p=0.0175, Unpaired t-test, Two-tailed. P) Representative picture shows eNOS negative control (no primary antibody) staining in both WT and MK2−/− mice (GFP: Gain-17, Exposure-250; DAPI: Gain-8, Exposure-100). (Q-R) Representative digital micrograph (GFP: Gain-17, Exposure-250; DAPI: Gain-8, Exposure-100) (Q) and a bar graph (R) showing the suppressed level of eNOS in MK2−/− mice subjected to HLI compared to WT mice (n=6–7). Values are mean ± S.E.M; *p=0.0291, Unpaired t-test, Two-tailed.

Immunofluorescence analysis was carried out on postoperative day 7 for Flk-1+/− animals in order to assess the therapeutic efficacy of Ad.Peli1 gene therapy in restoring the expression of proangiogenic factors such as VEGF and eNOS, as well as downregulation of pro-apoptotic molecule Bax. Ad.Peli1 gene therapy significantly upregulated expression of VEGF (4.7-fold, p=0.0260) (Figure 9B–C) and eNOS [(2.4-fold, p=0.2739; postoperative day 7); (8.6-fold, p=0.0156; postoperative day 28)] (Figure 9H–I), and downregulated Bax expression (1.8-fold, p=0.0216) (Figure 9E–F) in Flk-1+/− mice compared to Ad.LacZ-treated group. Figure 9A, 9D, 9G shows the negative control staining (no primary antibody added).

Fig 9.

Fig 9.

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A) Representative picture shows VEGF negative control (no primary antibody) staining in Flk-1+/− injected with Ad.LacZ or Ad.Peli1 treatment (GFP: Gain-17, Exposure-250; DAPI: Gain-8, Exposure-100). (B-C) Representative digital micrograph (GFP: Gain-17, Exposure-250; DAPI: Gain-8, Exposure-100) (B) and a bar graph (C) show the increased VEGF level in Flk-1+/− + Ad.Peli1 group compared to Flk-1+/− + Ad.LacZ group (n=5). Values are mean ± S.E.M; *p=0.0260, Unpaired t-test, Two-tailed. D) Representative picture shows Bax negative control (no primary antibody) staining in Flk-1+/− injected with Ad.LacZ or Ad.Peli1 treatment (RFP: Gain-17, Exposure-200; DAPI: Gain-13, Exposure-30). (E-F) Representative digital micrograph (RFP: Gain-17, Exposure-200; DAPI: Gain-13, Exposure-30) (E) and a bar graph (F) showing the decreased level of Bax in Flk-1+/− + Ad.Peli1 group compared to Flk-1+/− + Ad.LacZ group (n=4–5). Values are mean ± S.E.M; *p=0.0216, Unpaired t-test, Two-tailed. G) Representative picture shows eNOS negative control (no primary antibody) staining in Flk-1+/− injected with Ad.LacZ or Ad.Peli1 treatment (GFP: Gain-17, Exposure-250; DAPI: Gain-8, Exposure-100). (H-I) Representative digital micrograph (GFP: Gain-17, Exposure-250; DAPI: Gain-8, Exposure-100) (H) and a bar graph (I) showing the increased level of eNOS in Flk-1+/− + Ad.Peli1 group compared to Flk-1+/− + Ad.LacZ group (n=4–5). Values are mean ± S.E.M; p=0.2739, Unpaired t-test, Two-tailed.

Discussion:

This study reveals that the protection afforded by Peli1 overexpression in Flk-1+/− mice in the HLI model is dependent on increased vessel density and blood perfusion efficiency. However, this protection afforded by Peli1 gene therapy failed in MK2−/− KO mice subjected to HLI (Figure 10). Given the current interest in translating gene therapy to clinics, especially in the field of therapeutic angiogenesis in the ischemic hind limb model, two important knockout mouse colonies were studied: (1) Flk1 and (2) MK2 (a downstream target of VEGF/Flk1). Both of these mouse colonies have been well characterized (Kotlyarov et al., 1999; Thirunavukkarasu et al., 2018). First, we determined whether deletion of any of these two molecules can hinder vascularization and blood perfusion in HLI or not. Both KO mice and their wild-type counterparts were subjected to HLI, and we found that they cannot withstand femoral artery ligation as reflected by the significant amputation rate and tissue necrosis in these KO mice when compared to their corresponding wild-type counterparts. These studies also revealed a prominent decrease in proangiogenic molecules, VEGF, and eNOS expression and a significant increase in the pro-apoptotic molecule, Bax, when compared with their wild-type controls.

Fig 10.

Fig 10.

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Schematic diagram showing the overall effect achieved by Peli1 gene therapy to ischemic limbs of Flk-1+/− and MK2−/− mice (created with Biorender.com).

Peli1 positively affects angiogenesis, as reflected by a significant increase in capillary density on POD28 after gene therapy with Ad.Peli1 in Flk-1+/− mice subjected to HLI. These results were observed in Flk-1+/− mice compared to their wild-type counterparts suggesting that Ad.Peli1 gene therapy has a ‘rescue’ effect in Flk-1+/− knockout model (Figure 9). However, this effect was not observed in MK2−/− mice when exposed to HLI (Figure 4). Results were expressed as average counts of intact vessels per mm2, stained for CD31 at standard magnification. Sections were selectively taken from the gastrocnemius muscles. In human physiology, this muscle group would be more dependent on neovascularization in the setting of chronic ischemia compared to proximal muscle groups that rely more on arterial collateralization. An increase in capillary density, in turn, should correlate with improved perfusion following the ischemic insult. Indeed, we observed a positive and ultimately significant uptrend in perfusion in Flk-1+/− mice over time in Ad.Peli1-treated group when compared to the Ad.LacZ control and also non-treated Flk-1+/− group as measured by laser doppler imaging. Perfusion was measured for each time point, as shown in Figure 3, as a ratio between perfusion to a region of interest (ROI) in the ischemic limb and perfusion to a matching ROI in the non-ischemic limb. Improved perfusion to the ischemic limb implies improved oxygenation, which is needed to hasten recovery. These findings are likely attributable to the role of Peli1 downstream of VEGF/Flk-1, including the induction of proangiogenic molecules like eNOS and VEGF.

Also, the role of Peli1 in HLI can also be further explored in the context of tissue remodeling. Tissue remodeling is the process by which organisms attempt to heal an injury, including ischemic injury. Similar to myocardial infarction, remodeling plays a significant role in limb ischemia and ultimately determines the extent of remaining function following an insult. Remodeling takes place in multiple phases. The initial, or inflammatory phase, is characterized by massive tissue necrosis due to extrinsic factors and, unlike apoptosis, is almost always detrimental. Next is the proliferation phase, which is characterized by cell turnover as inflammatory cells are replaced with collagen-producing cells, which is aided by apoptosis. Lastly is the maturation phase, in which functional cells such as endothelial cells and myocytes repopulate the newly-created collagen matrices, but is a process that often results in excessive collagen deposition (fibrosis) that interferes with organ function. Therefore, the balance of necrosis, apoptosis, and fibrosis is a key factor that ultimately impacts tissue function.

Herein, it has been shown that treatment of Flk-1+/− mice subjected to HLI with Ad.Peli1 gene therapy results in decreased tissue necrosis and tissue loss over time compared to Flk-1+/− mice without gene therapy (Figure 6). The same treatment with Ad.Peli1 in MK2−/− HLI had no beneficial effect on ameliorating tissue necrosis and/or tissue loss (Figure 4). This was achieved by assigning an ischemic score from 0 to 8 to each animal at different time points depending on clinical and photographic examination of the ischemic limb to determine the anatomical extent of tissue necrosis. Neovascularization following acute ischemic events tends to occur later in the stages of remodeling. Necrosis, which is irreversible, tends to occur within hours of an acute insult such as femoral artery ligation; the femoral artery ligation performed in this study parallels acute limb ischemia in humans as seen in acute thromboembolic events. Current treatment modalities for acute limb ischemia in humans focus primarily on prompt surgical revascularization to minimize necrosis and subsequent inflammation. Therefore, it seems likely that Peli1, either directly or indirectly, possesses additional protective effects that limit and/or minimize necrosis early during acute limb ischemia. It will be imperative to investigate these effects in the future for Peli1 to assume a role in the management of acute limb ischemia.

Chronic limb ischemic patients rely heavily on collateralization as well as neovascularization to maintain adequate perfusion for tissue sustainability. However, if the disease progression rate is more rapid than the rate of neovascularization, then gradual tissue necrosis and chronic inflammation can eventually ensue, a process termed critical limb ischemia (CLI). CLI is currently another indication for surgical intervention, but many patients do not have anatomically favorable disease for revascularization procedures. It is here where Peli1 could prove helpful as it offers an alternative as a potentially-limb-sparing form of medical therapy by hastening angiogenesis and preventing necrosis during the transition into CLI.

Peli1 gene therapy also has beneficial effects later down the remodeling timeline. The decreased expression of Bax, an apoptosis marker, in the Ad. Peli1 treated group of Flk-1+/− compared to the Ad.LacZ group suggests decreased inflammatory cell turnover and, by extension, decreased inflammation. This is subsequently supported by decreased levels of pathologic collagen deposition in the maturation phase seen in Ad. Peli1-treated Flk-1+/− mice. The amount of fibrosis is inversely proportional to function. While a sturdy collagen network is required for cellular migration, pathological collagen deposition often lacks functional cells such as myocytes or endothelial cells. Thus the combined decrease of inflammation and fibrosis are seen after Ad.Peli1 gene therapy in Flk-1+/− but not in MK2 −/− mice subjected to HLI allows for preservation of functional skeletal muscle and formation of collagen matrices appropriate for neovascularization.

In conclusion, this study demonstrates that the induction of an angiogenic response for neovascularization in ischemic tissue requires both Flk-1 and MK2 signaling. Deletion of these molecules (Flk-1 or MK2) is proven to be deleterious enough to abruptly inhibit the angiogenic response, decreasing vessel density and reducing perfusion. However, Peli1 gene therapy can rectify perfusion efficiency in Flk-1+/− KO mice subjected to HLI compared to Flk-1+/− without treatment. The primary mechanism of action seems to be the induction of angiogenesis by increasing VEGF and eNOS expression. The same therapy with Peli1 failed to rectify the perfusion status in MK2−/− KO mice when exposed to HLI, confirming the role of Peli1 downstream of VEGF receptor Flk-1 but upstream of MK2. However, it was also established that Peli1 seems to improve function by mechanisms other than angiogenesis, namely by reducing necrosis, diminishing apoptotic cell turnover as may be seen in inflammation, and reducing tissue fibrosis, although the specific molecular mechanisms behind these observations are yet to be elucidated. Altogether, these results provide evidence that Peli1 may have a future therapeutic role in managing limb ischemia.

Highlights.

  1. Therapeutic vascularization in the hind-limb ischemia model with Pellino-1 gene therapy, an E3 ligase is promising.

  2. Pellino-1 repair and rectifies downstream abnormal VEGF signaling in ischemic tissues.

  3. Pellino-1 is more effective and appropriate in inducing angiogenic response than VEGFA.

  4. VEGF-A increases vascular permeability and thrombosis, whereas Pellino-1 bypasses these effects.

  5. The role of E3 Ligases in therapeutic angiogenesis is new.

Acknowledgments:

The study was supported by the National Institutes of Health Grant (NIH) GM 112957 to NM. We would like to thank Bryan Cernuda, MS for his help in the preparation of Figure 10.

Footnotes

Disclosures: This work was presented at the following meetings:

i. American College of Surgeons Clinical Congress, 2014, San Francisco

ii. Connecticut Chapter of the American College of Surgeons, 2013, Farmington, CT

iii. Scientific Sessions, American Heart Association, 2020, Dallas, TX

Conflict of Interest: None

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