Anti-angiogenic VEGF₁₆₅b regulates Macrophage-polarization via S100A8/S100A9 in Peripheral Artery Disease

Abstract

Background:

Atherosclerotic occlusions decrease blood flow to the lower limbs causing ischemia and tissue loss in patients with peripheral artery disease (PAD). Currently, no effective medical therapies are available to induce angiogenesis and promote perfusion recovery in patients with severe PAD. Clinical trials aimed at inducing VEGF-A levels, a potent pro-angiogenic growth factor to induce angiogenesis and perfusion recovery were not successful. Alternate splicing in the exon-8 of VEGF-A results in the formation of VEGFxxxa (VEGF₁₆₅a) and VEGFxxxb (VEGF₁₆₅b) isoforms with existing literature focusing on VEGF₁₆₅b’s role in inhibiting VEGFR2 dependent angiogenesis. However, we have recently shown that VEGF₁₆₅b blocks VEGF-A induced endothelial VEGFR1 activation in ischemic muscle to impair perfusion recovery. Since macrophage secreted VEGF₁₆₅b has been shown to decrease angiogenesis in peripheral artery disease and macrophages were well known to play important roles in regulating ischemic muscle vascular remodeling, we examined the role of VEGF₁₆₅b in regulating macrophage function in PAD.

Methods:

Femoral artery ligation and resection was used as an in vivo preclinical PAD model and hypoxia serum starvation was used as an in vitro model for PAD. Experiments including laser-doppler perfusion imaging, adoptive cell transfer to ischemic muscle, immunoblot analysis, enzyme-linked immunosorbent assays, Immunostainings, flow cytometry, qPCR analysis and RNA-Seq analysis were performed to determine a role of VEGF₁₆₅b in regulating macrophage phenotype and function in PAD.

Results:

First, we found increased VEGF₁₆₅b-expression with increased M1-like-macrophages in PAD vs. non-PAD (controls) muscle-biopsies. Next, using in vitro hypoxia serum starvation (HSS), in vivo pre-clinical PAD models and adoptive-transfer of VEGF₁₆₅b-expressing bone marrow-derived macrophages (BMDM) or VEGFR1^+/− BMDM (M1-like-phenotype), we demonstrate that VEGF₁₆₅b inhibits VEGFR1-activation to induce an M1-like-phenotype that impairs ischemic-muscle neovascularization. Subsequently, we found S100A8/S100A9 as VEGFR1 downstream regulators of macrophage-polarization by RNA-Seq analysis of HSS-VEGFR1^+/+ vs. HSS-VEGFR1^+/− BMDM.

Conclusion:

In our current study, we demonstrate that increased VEGF₁₆₅b-expression in macrophages induces an anti-angiogenic M1-like-phenotype that directly impairs angiogenesis. VEGFR1 inhibition by VEGF₁₆₅b results in S100A8/S100A9 mediated calcium influx to induce an M1-like-phenotype that impairs ischemic-muscle revascularization and perfusion recovery.

Introduction

In patients with peripheral arterial disease (PAD), atherosclerotic-occlusions in the blood vessels leads to tissue ischemia in the lower limbs with leg pain while walking, rest and tissue loss. Severe PAD (critical limb ischemia-CLI) often results in tissue necrosis and limb amputation¹. Approximately 500–1000 patients per million PAD patients over 5–8 years develop CLI² resulting in approximately 200,000 amputations in the US alone. In the frequent setting of complete blockades in the blood vessels that supply the leg, perfusion to the limb becomes dependent on the extent of vascular remodeling from angiogenesis and arteriogenesis in the ischemic-muscle³. Currently, no therapies can directly increase the blood flow to the ischemic-muscle in PAD patients and new therapies are being actively sought⁴.

VEGF-A, a potent angiogenic molecule is upregulated in ischemic-muscle and its upregulation can contribute to tissue revascularization^5–8. The role of VEGF-A function and signaling in angiogenesis grew more complex by the discovery of alternative splicing in its 8^th exon that results in the occurrence of pro-angiogenic VEGFxxxa (xxx for the number of amino acids) and anti-angiogenic VEGFxxxb (VEGF₁₆₅b) isoform families^9–11. We and others have demonstrated that VEGF₁₆₅b can impair perfusion-recovery in experimental-PAD^12,13. However, contrary to expectations, we showed that expression of VEGF₁₆₅b impaired the extent of perfusion-recovery following experimental-PAD by blocking a novel VEGFR1-STAT3 signaling in ischemic endothelial cells but not through inhibiting VEGFR2-activation¹².

Previous publications have demonstrated that VEGFR1 regulates the mobilization of bone marrow cells of macrophage lineage to stimulate solid tumor growth¹⁴; and VEGFR1-signaling in metastasis-associated macrophages induces inflammatory signaling that promotes breast cancer metastasis¹⁵. In human and experimental-PAD, macrophages were described to play important roles in vascular remodeling^16,17. Macrophages that infiltrate the ischemic-muscle attain a pro-inflammatory, cytotoxic and anti-angiogenic M1-phenotype due to the ischemic-muscle environment and aggravate tissue damage by severely hindering reparative/regenerative process and/or adaptive processes^18,19. We have recently demonstrated that increased M1-like-macrophages in the distal ischemic leg (where the bulk of angiogenesis occur) decrease angiogenic-remodeling²⁰. So far, macrophage-specific function of VEGFR1-activation and the downstream signaling in PAD is not known. Based on our recent report demonstrating an enhanced VEGFR1-activation post VEGF₁₆₅b-inhibition promotes perfusion-recovery in experimental-PAD⁸, we investigated the mechanistic role of VEGF₁₆₅b-VEGFR1 signaling in regulating macrophage-polarization and ischemic-muscle revascularization in experimental-PAD.

Methods

Expanded methods can be found in the online-only Data supplement. The data, analytic methods, and study materials will be made available to other researchers for purposes of reproducing/replicating the procedure/results. Requests can be made to the corresponding author who manages the information.

Animal Model of Hind-Limb Ischemia (HLI):

Unilateral femoral artery ligation and excision was performed on age- and sex-matched 12–16-week-old animals^12,20,21. All animal experiments were approved and performed according to University of Virginia Institutional Animal Care Committee and conformed to the Guide for the Care and Use of Laboratory Animals published by the NIH.

Human Samples:

The Institutional Review boards at Duke University, University of Colorado, and the University of Virginia approved the research protocols with human samples. All subjects gave informed consent.

Cell Transfections:

Plasmid transfections were performed by Lipofectamine-3000 according to the manufacturers’ instructions.

Statistics:

GraphPad Prism 7 was used to analyze the statistical significance of the data. Statistical test accompanied each figure legend. P<0.05 considered significant for all experiments. Data are presented as Mean ± Standard Error.

Results

1. Macrophage-specific VEGF₁₆₅b-expression inversely correlates with VEGFR1-activation and directly correlates with M1-like-phenotype in ischemic-muscle.

A recent study by Kikuchi et al¹³ suggested that monocyte/macrophage secreted VEGF₁₆₅b increased circulating VEGF₁₆₅b levels in PAD patients’ serum and inhibited endothelial angiogenic potential by inhibiting VEGFR2-activation. In order to asses a quantitative correlation between VEGF₁₆₅b levels and PAD severity, we performed VEGF₁₆₅b ELISA (lower detection limit 62.5pg/ml) in plasma samples from PAD patients, and age and gender-matched controls. We found that VEGF₁₆₅b levels were detectable only in ~25% (5 out of total 22 samples) of control human plasma samples (range-13.2 to 25.2pg/ml), ~10% (2 in 20 samples) in intermittent claudication (IC, 18.8pg/ml) and none in (0 out of 19 samples) CLI (Fig-1A).

Figure-1:

Increased VEGF₁₆₅b-expression inversely correlates with decreased VEGFR1-activation and directly with M1-like macrophage phenotype. A) VEGF₁₆₅b ELISA in human normal, intermittent claudication (IC) and critical limb ischemia (CLI) patients’ plasma samples. Control n=24, IC n=20, CLI n=19. Non-parametric one-way ANOVA Kruskal-Wallis test with Dunn’s multiple comparisons test and Geisser-Greenhouse correction was used to determine statistical significance. B, C) VEGF₁₆₅b levels in conditioned-medium (medium) and lysates from control and hypoxia serum starved (HSS) (B) human peripheral blood monocyte derived macrophages (HPBMDM) and (C) mouse bone marrow derived macrophages (BMDM). VEGF₁₆₅b levels in conditioned-medium was normalized to the corresponding cell lysate total protein levels. n=4. A-C) One way ANOVA with Dunnett’s multiple test correction was used to determine statistical significance. D) VEGF₁₆₅b levels in conditioned-medium and lysates from bone marrow monocytes from non-ischemic leg (NIL-Mo) and monocytes from ischemic leg (IL-Mo). VEGF₁₆₅b levels in conditioned-medium was normalized to the corresponding cell lysate total protein levels. n=4. E-F) Flowcytometric analysis of E) VEGF₁₆₅b and F) pVEGFR1_Y1333 expression in macrophages from non-ischemic gastrocnemius (NGA) and ischemic gastrocnemius (IGA) muscle. n=4. G-H) Flowcytometric analysis of pVEGFR1 expression in G) VEGF₁₆₅b^+ve (VEGF₁₆₅b positive) and H) VEGF₁₆₅b^−ve (VEGF₁₆₅b negative) macrophages from NGA and IGA. n=4. I-J) Flowcytometric analysis of I) CD80^high M1-like macrophages and J) Arg-1⁺ M2-like macrophages from NGA and IGA. n=4. D-J) Unpaired T-test was used to determine statistical significance. Increased VEGF₁₆₅b-expression correlate with higher CD68⁺CD80⁺ M1-like macrophages in human PAD. Immunostaining VEGF₁₆₅b, CD68, CD206 and CD80 in normal (NL) and peripheral arterial disease (PAD) muscle-biopsies. Quantification of K) VEGF₁₆₅b expressing (VEGF₁₆₅b^+ve) cells, L) total macrophages (CD68⁺), M) macrophages expressing CD80 (CD68⁺CD80⁺) and N) macrophages expressing CD206 (CD68⁺CD206⁺). Normal n=5, PAD n=7. K-N) Unpaired T-test was used to determine statistical significance. *P<0.05 considered significant.

We sought to determine to what extent, or whether, monocytes/macrophages contribute to circulating VEGF₁₆₅b levels. We isolated human peripheral blood monocyte-derived macrophages (HPBMDMs) as previously described²⁰ and cultured them under normal or hypoxia serum-starvation (HSS). HPBMDM cell-lysates and conditioned media were examined for VEGF₁₆₅b levels. While we did not observe any detectable levels of VEGF₁₆₅b in conditioned media from control or HSS-challenged HPBMDMs, a significantly higher VEGF₁₆₅b levels was observed in HSS-challenged HPBMDMs cell-lysates vs. controls (control 32.8±2.5pg/mg vs. 6hr-HSS 78.8±1.8pg/mg (P<0.05 vs. control), 12hr-HSS 58.7±7.3pg/mg and 24hr-HSS 23.8±2.2pg/mg, Fig-1B).

We also isolated bone marrow-derived macrophages (BMDMs) from mice using methods previously described²⁰. Similar to the data from HPBMDMs, VEGF₁₆₅b was not detectable from conditioned-medium from BMDMs from Balb/c mice under HSS or control conditions (Fig-1C). However, in cell-lysates a significant increase in VEGF₁₆₅b levels was observed in BMDM under HSS vs. control (control 39.6±1.2pg/mg vs. 6hr-HSS 40.9±5.1pg/mg, 12hr-HSS 52.7±4.4pg/mg vs. 24hr-HSS 67.7±2.9pg/mg (P<0.05 vs. Control), Fig-1C). To confirm whether there is a difference in monocyte vs. macrophage-specific VEGF₁₆₅b secretion pattern, we isolated bone marrow (BM) monocytes from non-ischemic and ischemic Balb/c legs at day-3 post-HLI and examined for VEGF₁₆₅b secretion into culture-medium. Consistent with the data from HPBMDMs and Balb/c-BMDM, VEGF₁₆₅b was not detected in culture-medium from non-ischemic or ischemic-leg BM (Fig-1D). However, in cell-lysates, a significant increase in VEGF₁₆₅b levels was observed in ischemic vs. non-ischemic leg BM monocytes (Non-ischemic leg BM 8.8±1.7pg/mg vs. Ischemic-leg BM 18.5±3.2pg/mg, P=0.03, Fig-1D).

Knowing that ischemic monocytes/macrophages express but do not secrete VEGF₁₆₅b protein and higher VEGF₁₆₅b levels are linked to a poorer response to experimental-PAD¹³, we hypothesized that ‘VEGF₁₆₅b induction serves an autocrine role in macrophages that have the ability to impair ischemic-muscle tissue perfusion in PAD’. To confirm our hypothesis, we next examined macrophage-specific VEGF₁₆₅b-expression in Balb/c ischemic-muscle. Flow cytometry analysis (gating strategy presented in Supplemental-Figure-1^12,20) showed significantly higher macrophage-specific VEGF₁₆₅b-expression in ischemic (IGA) vs. non-ischemic (NGA) gastrocnemius muscle (NGA:0.8±0.7% vs. IGA:12.7±1.7%, P=0.0007, Fig-1E). We have recently shown that ischemia-induced VEGF₁₆₅b-expression inversely correlated with VEGFR1-activation in human and experimental-PAD muscle^12,22. Hence, we next examined whether increased macrophage-specific VEGF₁₆₅b-expression in ischemic-muscle correlates with changes in the extent of VEGFR1-activation. Flow cytometry analysis showed that VEGF₁₆₅b-induction inversely correlated with significantly lower VEGFR1-activation in ischemic-muscle macrophages vs. non-ischemic groups (P<0.03, Fig-1F). Furthermore, VEGF₁₆₅b-expression was 12.7% in 15.5% of total VEGF-A expression in ischemic-muscle macrophages (VEGF₁₆₅a=~2.8%, Supplemental-Figure-2) indicating that a significant increase in VEGF₁₆₅b-expression over VEGF₁₆₅a contributes to lower VEGFR1-activation in ischemic-muscle macrophages vs. non-ischemic

We next gated macrophages from non-ischemic and ischemic-muscle as VEGF₁₆₅b-expressing (CD11b⁺F4/80⁺VEGF₁₆₅b^+ve) and VEGF₁₆₅b non-expressing (CD11b⁺F4/80⁺VEGF₁₆₅b^−ve) populations and examined the extent of VEGFR1-activation within these populations¹². Flow cytometry analysis showed that while VEGF₁₆₅b^+ve macrophages in ischemic-muscle have significantly lower VEGFR1-activation (~30X) vs. non-ischemic (P=0.001, Fig-1G), VEGF₁₆₅b^−ve macrophages showed no significant differences in VEGFR1-activation vs. non-ischemic (Fig-1H). Since VEGFR1-activity has been associated with changes in macrophage-phenotype²³, we hypothesized that VEGF₁₆₅b inhibits VEGFR1-activation to induce a pro-inflammatory phenotype. Consistent with our hypothesis, increased VEGF₁₆₅b-expression in macrophages correlated with a significant increase (~6X) in M1-like-phenotype²⁴ (CD45⁺CD11b⁺F4/80⁺CD80^high, P=0.001, Fig-1I) but not with M2-like-phenotype²⁴ (CD45⁺CD11b⁺F4/80⁺Arg1⁺, Fig-1J) in IGA vs. NGA. These data showed that VEGF₁₆₅b regulates macrophage-phenotype by modulating the extent of VEGFR1-activation in distal ischemic-muscle.

2. VEGF₁₆₅b-expression in human-PAD correlates with increased M1-like-macrophages in PAD muscle-biopsies

To examine the translational relevance of our murine findings, we next examined age- and gender-matched, human, normal and PAD muscle-biopsies¹² that were used in our previous publications^12,20,22,25 and correlated VEGF₁₆₅b-expression with the number of CD80⁺ and CD206⁺ cells. Immunohistochemistry of VEGF₁₆₅b in normal (n=5) vs. PAD (n=7) muscle-biopsies showed a significant increase (~2.5X, NL-7.6±0.8 vs. PAD-19.7±1.1) in VEGF₁₆₅b^+ve cells in PAD muscle-biopsies vs. normal (P<0.0001, Fig-1K). We next performed immunofluorescence analysis to determine differences in CD206⁺ (M2-like) and CD80⁺ (M1-like) macrophages in normal and PAD muscle-biopsies. A significant increase in total macrophages (CD68⁺, a pan-macrophage marker) was observed (~2X, NL-16±2.1 vs. PAD-33.8±1.6) in PAD muscle-biopsies vs. normal (Fig-1L). Subsequent analysis of CD80⁺ and CD206⁺ macrophages in total CD68⁺-macrophages showed a significant increase (~3X) in CD80⁺-macrophages in PAD-muscle vs. normal (NL-9±3.1 vs. PAD-27.5±2.4, P=0.0004, Fig-1M); but no significant difference in CD206⁺-macrophages in total CD68⁺-macrophages (NL-7.7±2.9, PAD-12.7±1.0, Fig-1N). Immunostaining with CD163⁺ (M2-marker) also showed no significant difference in CD163⁺-macrophages between PAD-muscle vs. normal (Supplemental-Figure-3).

We next quantified CD133⁺ cells to determine whether immature progenitor cells contributed to changes in macrophage subsets in PAD muscle biopsies. While total CD133⁺ cells are significantly lower in PAD-muscle vs. normal (indicating that increased total or M1-like-macrophages are not due to CD133⁺ progenitors in PAD-muscle, Supplemental-Figure-4), no significant difference between VEGF₁₆₅b⁺CD133⁺, CD68⁺CD133⁺ or VEGF₁₆₅b⁺CD133⁺CD68⁺ cells was observed between PAD-muscle vs. normal (data not shown).

We have previously shown that higher VEGF₁₆₅b protein in PAD muscle-biopsies correlated with decreased VEGFR1-activation¹² and our current data show that VEGF₁₆₅b-induction correlates with increased M1-like-phenotype in PAD muscle-biopsies. Hence, we conclude that VEGF₁₆₅b-induction in ischemic-muscle decreases macrophage-specific VEGFR1-activation to induce an M1-like-phenotype in PAD patients.

3. VEGF₁₆₅b skews macrophage phenotype to M1-like in preclinical-PAD

To determine the causal role of VEGF₁₆₅b in modulating macrophage-phenotype, we performed loss of function experiments by intramuscular delivery of VEGF₁₆₅b-Ab (or isotype-matched IgG (control)) in ischemic-muscle; and gain of function experiments by intramuscular delivery of VEGF₁₆₅b-expressing plasmid (or negative-plasmid (control), Supplemental-Figure-5) in non-ischemic-muscle and examined differences in macrophage-phenotype based on CD80 or Arg-1-expression²⁴

Loss of VEGF₁₆₅b function:

VEGF₁₆₅b-expression in ischemic-muscle was inhibited by intramuscular delivery of VEGF₁₆₅b-Ab into ischemic-muscle immediately after HLI as previously described¹². Flow cytometry analysis showed no significant difference in the total macrophage numbers between VEGF₁₆₅b-Ab vs. IgG treated ischemic-muscle (IGA) at day-3 post-HLI (Fig-2A). VEGF₁₆₅b-inhibition in IGA significantly increased Arg-1⁺-macrophages (~2X, IgG-10.2±1.8 vs. VEGF₁₆₅b-Ab-20±3.3%, P<0.05, Fig-2B) with a concomitant decrease in CD80^high-macrophages (IgG-36.6±7.7 vs. VEGF₁₆₅b-Ab-11.4±3.7%, P<0.03, Fig-2C) vs. IgG.

Figure-2.

VEGF₁₆₅b regulates macrophage phenotype. A-C) Flowcytometric analysis of A) total (F4/80⁺), B) Arg-1⁺ M2-like (F4/80⁺Arg-1⁺) and C) CD80^high M1-like (F4/80⁺CD80^high) macrophages from ischemic gastrocnemius muscle (IGA) treated with IgG or VEGF₁₆₅b-antibody (VEGF₁₆₅b-Ab). n=4. Unpaired T-test was used to determine statistical significance. D-F) Flowcytometric analysis of D) total (F4/80⁺), E) Arg-1⁺ M2-like (F4/80⁺Arg-1⁺) and F) CD80^high M1-like (F4/80⁺CD80^high) macrophages from non-ischemic gastrocnemius muscle (NGA) treated with control plasmid (Neg Pld) or VEGF₁₆₅b-expressing plasmid (V₁₆₅b pld). n=4. Unpaired T-test was used to determine statistical significance. G) Flowcytometric analysis of Arg-1 expression in bone marrow derived macrophages (BMDM) treated with IgG (10ug/ml), V₁₆₅b (VEGF₁₆₅b, 50ng/ml) or VEGF₁₆₅b-Ab (10ug/ml). n=3. One Way ANOVA with Dunnett’s multiple test correction was used to determine statistical significance. *P<0.05 considered significant.

Gain of VEGF₁₆₅b function:

VEGF₁₆₅b overexpression in non-ischemic leg did not induce differences in total macrophage numbers in NGA (Fig-2D). However, a significant decrease in Arg-1⁺-macrophages (control-plasmid-9.9±0.4 vs. VEGF₁₆₅b-plasmid-5.9±1.1%, P=0.02, Fig-2E) and a significant increase in CD80^high-macrophages (control-plasmid-17.1±2.5 vs. VEGF₁₆₅b-plasmid-24.6±1.5%, P=0.04, Fig-2F) was observed in NGA treated with VEGF₁₆₅b-expressing vs. control-plasmid.

We next confirmed our in vivo results in vitro using Balb/c-BMDMs. In vitro, VEGF₁₆₅b significantly decreased Arg-1-expression (~2X) and VEGF₁₆₅b-inhibition significantly induced Arg-1-expression (~1.5X) vs. IgG in BMDMs (P<0.05, Fig-2G). Thrombospondin-1 is a known Anti-angiogenic molecule that has been shown to induce M1-like-macrophages²⁶. We next determined whether increased M2-like-macrophages post VEGF₁₆₅b-inhibition is due to decreased thrombospondin-1 levels. Immunoblot analysis showed no significant difference in thrombospondin-1-expression either in ischemic muscle or HSS BMDMs treated with IgG or VEGF₁₆₅b-Ab (Supplemental-Figure-6).

4. VEGF₁₆₅b-expressing M1-like-macrophages impair perfusion in preclinical-PAD in a paracrine manner.

Since C57Bl6 mice exhibit a striking ability to favorably recover from HLI (i.e. a better recovery mouse strain than the Balb/c mouse strain in preclinical-PAD)^27,28, we examined whether VEGF₁₆₅b-expressing macrophages can impair angiogenesis and perfusion-recovery in C57Bl/6 ischemic-muscle. First, we collected conditioned-medium from 1) BMDMs transfected with negative-plasmid or VEGF₁₆₅b-expressing plasmid (transfection efficiency presented in Supplemental-Figure-2) and 2) HSS-challenged BMDMs treated with IgG or VEGF₁₆₅b-Ab in vitro. While conditioned-medium from C57Bl/6-BMDM transfected with VEGF₁₆₅b-expressing plasmid significantly decreased capillary-like tube formation on growth factor reduced matrigel (GFRM) vs. C57Bl/6-BMDM transfected with negative-plasmid (P<0.05, Fig-3A), conditioned-medium from HSS-challenged C57Bl/6-BMDM treated with VEGF₁₆₅b-Ab increased capillary-like tube formation on GFRM vs. IgG (~3X, P<0.05, Fig-3B).

Figure-3.

VEGF₁₆₅b expressing macrophages inhibit angiogenesis and perfusion recovery. A, B) In vitro angiogenesis assay of endothelial cells treated with A) conditioned-medium (CM) from C57Bl/6-bone marrow derived macrophages transfected with negative plasmid (Neg Pld) or VEGF₁₆₅b-expressing plasmid (VEGF₁₆₅b-Pld). B) Conditioned-medium from HSS-challenged BMDM treated with IgG or VEGF₁₆₅b-Ab. n=6. Unpaired T-test to determine statistical significance. C) Laser Doppler perfusion imaging of C57Bl/6 ischemic-muscle treated with BMDM-transfected with negative plasmid or VEGF₁₆₅b-expressing plasmid. n≥5. Repeated measures ANOVA with Dunnett’s multiple test correction was used to determine statistical significance. *P<0.05 considered significant from day 0, ^#P<0.05 considered significant between groups at that specific day (Unpaired T-test was used to determine statistical significance). D) CD31 immunostaining in ischemic-muscle treated with BMDM transfected with negative plasmid or VEGF₁₆₅b-expressing plasmid. n≥5. Unpaired T-test was used to determine statistical significance. *P<0.05 considered significant.

We next adoptively transferred C57Bl6-BMDM transfected with control or VEGF₁₆₅b-expressing plasmid into C57Bl/6 ischemic-muscle immediately after HLI and examined angiogenesis and perfusion-recovery. Laser Doppler imaging showed a significant decrease in the perfusion-recovery in the ischemic-muscle treated with VEGF₁₆₅b-expressing macrophages vs. control (d14: Negative-plasmid BMDM 86.5±5.8 vs. VEGF₁₆₅b-plasmid BMDM 66.9±6.1, Fig-3C). Immunohistochemistry showed a significant decrease in CD31⁺ (endothelial) cells in the ischemic-muscle treated with VEGF₁₆₅b-expressing BMDM vs. control (Fig-3D). These results indicated that increased VEGF₁₆₅b-expression in macrophages induces an M1-like-phenotype to impair angiogenesis and perfusion-recovery in preclinical-PAD.

5. VEGF₁₆₅b induces an M1-like-macrophage phenotype by inhibiting VEGFR1-activation in preclinical-PAD.

We next examined the role of VEGF₁₆₅b-VEGFR1 interactions in regulating macrophage-polarization. Flow cytometry analysis showed that VEGF₁₆₅b-inhibition in ischemic-muscle significantly induced pVEGFR1_Y1333-activation vs. IgG (P=0.004, Fig-4A, left panel) and immunofluorescence analysis of F4/80 and pVEGFR1_Y1333 visually confirmed greater VEGFR1_Y1333-activation in VEGF₁₆₅b-Ab treated ischemic-muscle macrophages (Supplemental-Figure-7). Subsequently, flow cytometry analysis showed that VEGF₁₆₅b-overexpression in non-ischemic-muscle significantly decreased pVEGFR1_Y1333-activation (P=0.01, Fig-4A, right panel) in total macrophages vs. negative-plasmid controls. Correlating with increased Arg-1⁺-macrophages in VEGF₁₆₅b-Ab treated ischemic-muscle (Fig-2B), VEGF₁₆₅b-inhibition was associated with significantly greater pVEGFR1_Y1333-activation in Arg-1⁺-macrophages (P=0.005, Fig-4B, left-panel); which correlated with decreased Arg-1⁺-macrophages in VEGF₁₆₅b-plasmid treated non-ischemic-muscle (Fig-2E), VEGF₁₆₅b-overexpression significantly decreased pVEGFR1_Y1333-activation in Arg-1⁺-macrophages (P=0.03, Fig-4B, right-panel) vs. respective controls. VEGF₁₆₅b-inhibition or VEGF₁₆₅b-overexpression did not change pVEGFR1_Y1333-activation in CD80^high-macrophages vs. respective controls (Fig-4C). Placental growth factor (PLGF), a VEGFR1 specific ligand has shown to induce M2-like-macrophage polarization^{29, 30}. Hence, we determined whether increased Arg-1⁺-macrophages post VEGF₁₆₅b-inhibition is due to increased PGLF levels. Immunoblot analysis showed no significant difference in PLGF levels either in ischemic-muscle or HSS BMDMs treated with IgG or VEGF₁₆₅b-Ab (Supplemental-Figure-8).

Figure-4.

VEGF₁₆₅b inhibits macrophage pVEGFR1_Y1333 activation. A-C) Flowcytometric analysis of pVEGFR1_Y1333 activation in A) total, B) Arg-1⁺ M2-like and C) CD80^high M1-like macrophages in ischemic gastrocnemius muscle (IGA) treated with IgG or VEGF₁₆₅b-antibody (VEGF₁₆₅b-Ab); or non-ischemic-muscle treated with control plasmid (Neg Pld) or VEGF₁₆₅b-expressing plasmid (V₁₆₅b pld). n=4. Unpaired T-test was used to determine statistical significance. D-F) Flowcytometric analysis of D) total, E) Arg-1⁺ M2-like and F) CD80^high M1-like macrophages in the IGA of wild type littermates (WT, VEGFR1^+/+), VEGFR1^+/− + IgG and VEGFR1^+/− + VEGF₁₆₅b-Ab. n=4, One way ANOVA with Dunnett’s multiple test correction was used to determine statistical significance. *P<0.05 considered significant.

To confirm the role of VEGFR1 in regulating macrophage-polarization, we used VEGFR1^+/− mice (VEGFR1^−/− are embryonic lethal) backcrossed to Balb/c background that cannot up-regulate VEGFR1 in ischemic-muscle to the same extent as VEGFR1^+/+ mice (~50% reduction in VEGFR1 induction in VEGFR1^+/− ischemic-muscle vs. VEGFR1^+/+ ischemic-muscle¹²). Flow cytometry analysis showed a significant decrease (~3X) in total macrophage numbers in VEGFR1^+/− ischemic-muscle (IGA:VEGFR1^+/+ 19.9±1.0 vs. VEGFR1^+/− 5.4±0.9%, P=0.0002, Fig-4D) vs. VEGFR1^+/+. Furthermore, a significant decrease (~2X) in Arg-1⁺-macrophages (IGA: VEGFR1^+/+ 15.8±1.8 vs. VEGFR1^+/− 8.2±1.2%, P<0.03, Fig-4E) with a concomitant increase in CD80^high-macrophages (IGA: VEGFR1^+/+ 19.4±5.9 vs. VEGFR1^+/− 48.0±7.8%, P<0.04, Fig-4F) was observed in VEGFR1^+/− ischemic-muscle vs. VEGFR1^+/+. VEGF₁₆₅b-Ab did not significantly change the numbers of total, M1 or M2-like-macrophages in VEGFR1^+/− ischemic-muscle vs. IgG treated VEGFR1^+/− ischemic-muscle (Figure-4). These results confirmed that VEGFR1 plays an important role in VEGF₁₆₅b induced M1-like-polarization in preclinical-PAD.

We next examined VEGF₁₆₅b-VEGFR1 interactions in macrophages in vitro. While VEGF₁₆₅b treatment significantly decreased pVEGFR1_Y1333-activation, VEGF₁₆₅b-inhibition significantly induced pVEGFR1_Y1333 activation vs. IgG-treated controls (P<0.05, Supplemental-Figure-9A), correlating with their M1- or M2-like-phenotypes indicating that VEGF₁₆₅b induced M1-like-polarization involves pVEGFR1_Y1333-inhibition.

VEGF₁₆₅b has been predicted to be a competitive inhibitor of VEGF₁₆₅a for VEGFR2 in endothelial cells¹¹. However, in general, macrophages primarily express VEGFR1. Hence, to understand the molecular interactions that result in VEGFR1-activation post VEGF₁₆₅b-inhibition, we pulled down VEGFR1 from normal or HSS-BMDM that were treated with IgG of VEGF₁₆₅b-Ab and examined the bound fractions of VEGF-A and VEGF₁₆₅b. immunoblot analysis of VEGFR1-pull-down complexes showed a significant increase in the bound fraction of VEGF₁₆₅a to VEGFR1 post VEGF₁₆₅b-inhibition under normal (P=0.005, Supplemental-Figure-9B) and HSS conditions (P=0.0004, Supplemental-Figure-9C) indicating that increased VEGF₁₆₅b binding, over VEGF₁₆₅a, to VEGFR1 decreases VEGFR1-activation.

To confirm our results, we treated BMDM with VEGF₁₆₅a, VEGF₁₆₅b or a combination of VEGF₁₆₅a and VEGF₁₆₅b at equimolar concentrations and examined the changes in VEGFR1_Y1333-activation. Flow cytometry analysis showed that while VEGF₁₆₅a significantly induced VEGFR1-activation in BMDM, VEGF₁₆₅b significantly decreased VEGF₁₆₅a induced VEGFR1-activation under HSS conditions (Supplemental-Figure-9D, E), indicating that VEGF₁₆₅b is a competitive inhibitor of VEGFR1 at Y1333 site.

6. VEGFR1^+/− macrophages impair perfusion vs. VEGFR1^+/+ macrophages in preclinical-PAD

Since VEGF₁₆₅b induced VEGFR1 dependent M1-like-polarization, we next examined the role of macrophage-specific VEGFR1 in regulating perfusion-recovery in experimental-PAD. qPCR analysis showed that while HSS significantly induced VEGFR1-expression in VEGFR1^+/+ BMDM vs. control (~5.5X), no significant difference in VEGFR1-expression was observed in VEGFR1^+/− under HSS vs. control (Supplemental-Figure-10). We next examined BMDMs from VEGFR1^+/+ and VEGFR1^+/− for their relative M1 and M2-like-states. At baseline, no significant difference in either Arg-1 or iNos-expression was observed between VEGFR1^+/+ and VEGFR1^+/− BMDM. However, under HSS, qPCR analysis showed that VEGFR1^+/− BMDM express significantly lower Arg-1 (6h-HSS ~16.5X, P<0.05) and significantly higher iNos (6h-HSS~3X, 12h-HSS~3X, 24h-HSS~2.4X, P<0.05) levels vs. VEGFR1^+/+ (Fig-5A) indicating that VEGFR1 plays an important role in regulating macrophage-polarization. Functional analysis of VEGFR1^+/− BMDM showed that conditioned-medium from normal VEGFR1^+/− BMDMs did not change endothelial capillary-like tube formation on GFRM (Fig-5B), however, HSS-VEGFR1^+/− BMDMs significantly decreased endothelial capillary-like tube formation on GFRM vs. conditioned-medium from HSS-VEGFR1^+/+ BMDMs (P=0.02, Fig-5C). After confirming that HSS induces M1-like-phenotype in VEGFR1^+/− BMDM that can inhibit endothelial angiogenic potential, in a paracrine manner, in vitro, we next examined their functional role in regulating perfusion in vivo. BMDM from VEGFR1^+/− and VEGFR1^+/+ were adoptively transferred into ischemic-muscle of WT mice immediately after HLI. Laser Doppler imaging showed that ischemic-muscle that received VEGFR1^+/− BMDM have significantly lower perfusion vs. VEGFR1^+/+-BMDM (d14: VR1^+/+ BMDM+VR1^+/+ ischemic-muscle 63.4+/−1.9 vs. VR1^+/− BMDM+VR1^+/+ ischemic-muscle 48.8+/−3.5 Fig-5D). Immunohistochemistry showed a significant decrease in vascular density (CD31⁺) in ischemic-muscle that received VEGFR1^+/− BMDM vs. VEGFR1^+/+ BMDM (Fig-5E). This data indicated that VEGF₁₆₅b-inhibition of VEGFR1 plays a role in regulating macrophage paracrine effects on ischemic vasculature.

Figure-5.

VEGFR1^+/− macrophages inhibit angiogenesis and perfusion recovery. A) qPCR analysis of temporal changes in Arg-1 and iNos expression in VEGFR1^+/+ and VEGFR1^+/− BMDM under normal and hypoxia serum starvation (HSS) conditions. n=6. One way ANOVA with Dunnett’s multiple test correction was used to determine statistical significance. B-C) In vitro angiogenesis assay of endothelial cells treated with conditioned-medium (CM) from B) normal or C) HSS-challenged VEGFR1^+/+ (VR1^+/+) or VEGFR1^+/− (VR1^+/−) bone marrow derived macrophages. n=6. Unpaired T-test was used to determine statistical significance. D) Laser Doppler perfusion imaging of WT (VR1^+/+) ischemic-muscle treated with VEGFR1^+/+ BMDM or VEGFR1^+/− BMDM. n≥5. Repeated measures ANOVA with Dunnett’s multiple test correction was used to determine statistical significance. *P<0.05 considered significant from day 0, ^#P<0.05 considered significant between groups at that specific day (Unpaired T-test was used to determine statistical significance). E) CD31 immunostaining in WT ischemic-muscle treated with VEGFR1^+/+ or VEGFR1^+/− BMDM. n≥5. Unpaired T-test was used to determine statistical significance. *P<0.05 considered significant.

7. VEGFR1-STAT3 interactions regulate macrophage-phenotype in preclinical-PAD

We next wanted to determine the signaling processes/intermediates downstream of VEGFR1_Y1333-activation that plays a role in macrophage-polarization. We have previously shown that VEGFR1 can regulate STAT3-activation¹². Hence, we examined whether STAT3 plays a role in VEGFR1 downstream signaling in regulating macrophage-polarization.

Flow cytometry analysis showed that VEGF₁₆₅b-inhibition induced a significant increase in pSTAT3-activation in total (IgG 8.3±1.7 vs. VEGF₁₆₅b-Ab 14.4±1.2, P=0.02, Fig-6A, left panel) and Arg-1⁺-macrophages (IgG 10.9±1.2 vs. VEGF₁₆₅b-Ab 16.4±1.3, P=0.02, Fig-6B, left panel) in Balb/c-IGA vs. IgG. No significant difference in pSTAT3-activation was observed in CD80^high-macrophages from Balb/c-IGA treated with VEGF₁₆₅b-Ab vs. IgG

Figure-6.

VEGF₁₆₅b inhibits VEGFR1-STAT3 signaling. A) Flowcytometric analysis of pSTAT3 activation in A) total, B) Arg-1⁺ M2-like and C) CD80^high M1-like macrophages in ischemic gastrocnemius muscle (IGA) treated with IgG or VEGF₁₆₅b-Ab; or non-ischemic gastrocnemius muscle (NGA) treated with control plasmid (Neg Pld) or VEGF₁₆₅b-expressing plasmid (VEGF₁₆₅b Pld). n=4. Unpaired T-test was used to determine statistical significance. D-F) Flowcytometric analysis of pSTAT3 activation in D) total, E) Arg-1⁺ M2-like and F) CD80^high M1-like macrophages in IGA of wild type littermates (WT, VEGFR1^+/+), VEGFR1^+/−+ IgG and VEGFR1^+/−+ VEGF₁₆₅b-Ab. n=4, One way ANOVA with Dunnett’s multiple test correction was used to determine statistical significance. *P<0.05 considered significant.

VEGF₁₆₅b-overexpression (by VEGF₁₆₅b-plasmid) significantly decreased pSTAT3-activation in total (control plasmid 1.0±0.01 vs. VEGF₁₆₅b-plasmid 0.6±0.08, P=0.03, Fig-6A, right panel) and Arg-1⁺-macrophages (control plasmid 0.6±0.07 vs. VEGF₁₆₅b-plasmid 0.4±0.01, P=0.01, Fig-6B, right panel) in Balb/c-NGA vs. control plasmid. No significant differences in pSTAT3-activation was observed in CD80^high-macrophages from Balb/c-NGA treated with VEGF₁₆₅b-expressing plasmid vs. control-plasmid (Fig-6C).

We next examined the causal role of VEGFR1 in regulating STAT3-activation to modulate macrophage-polarization using VEGFR1^+/− mice. Flow cytometry analysis showed a significant decrease in STAT3-activation in total macrophages from VEGFR1^+/− ischemic-muscle (IGA~2X, P<0.03, Fig-6D) vs. VEGFR1^+/+. A significant decrease in Arg-1⁺-macrophages in VEGFR1^+/− ischemic-muscle was associated with decreased STAT3-activation (IGA~10X, P=0.001, Fig-6E). A significant increase in CD80^high-macrophages in VEGFR1^+/− ischemic-muscle was associated with decreased STAT3-activation (IGA~3X, P=0.01, Fig-6F) when vs. VEGFR1^+/+.

Based on our recent data that show VEGFR1 when activated by VEGF₁₆₅a interacts with STAT3 to induce STAT3-activation in endothelial cells¹², we next examined the role of VEGF₁₆₅b in regulating VEGFR1-STAT3 interactions in macrophages. In vitro, immunoblotting of VEGFR1-immunoprecipitated complexes from VEGF₁₆₅b-Ab treated HSS-challenged BMDMs showed a significant increase in STAT3 binding vs. IgG (P=0.03, Supplemental-Figure-11A). This data indicated that VEGF₁₆₅b competes with VEGF₁₆₅a to bind and inhibit VEGFR1-STAT3 signaling activation in ischemic macrophages.

Flow cytometry analysis of BMDM treated with VEGF₁₆₅b had significantly decreased pVEGFR1⁺pSTAT3⁺ vs. controls correlating with CD80^high-macrophages vs. control; and BMDM treated with VEGF₁₆₅b-Ab treatment significantly induced pVEGFR1⁺pSTAT3⁺ correlating with Arg-1⁺macrophages vs. IgG in vitro (Supplemental-Figure-11B). We next examined whether pVEGFR1⁺pSTAT3⁺ distinguishes M1 vs. M2-like-macrophages post VEGF₁₆₅b-inhibition in vivo. Flow cytometry analysis showed a significant increase in pVEGFR1⁺pSTAT3⁺ expression in total macrophages (~3X, IgG 1.3±0.4 vs. VEGF₁₆₅b-Ab 3.7±0.7%, P<0.04, Supplemental-Figure-11C), in ischemic-muscle treated with VEGF₁₆₅b-Ab vs. IgG. This increase correlated with Arg-1⁺-macrophages (~2X, IgG 23.2±2.5 vs. VEGF₁₆₅b-Ab 54.4±5.5%, P=0.0009, Supplemental-Figure-11D) but not with CD80^high-macrophages (Supplemental-Figure-11E). These results indicated that VEGF₁₆₅b-inhibition induced pVEGFR1⁺pSTAT3⁺ double positive Arg-1⁺-macrophages in ischemic-muscle vs. IgG.

8. S100A8/S100A9 downstream of VEGFR1 regulate macrophage-polarization.

Unlike VEGFR2, information on genes that regulate VEGFR1 downstream processes is limited^31,32. Hence, to identify the VEGFR1 downstream signaling genes that regulate macrophage-polarization we performed RNA-Seq analysis of HSS-challenged VEGFR1^+/− and VEGFR1^+/+ BMDMs (Supplemental-table-1, 2). Volcano plots, principal component analysis (PCA) analysis³³ and hallmark pathway analysis³⁴ (GSEA) of the RNA-Seq data is presented in Supplemental-Figure-12. RNA-Seq analysis showed that S100A8 and S100A9, members of S100 family that regulate intracellular calcium levels^35,36 are significantly upregulated in HSS-challenged VEGFR1^+/− BMDM vs. VEGFR1^+/+ (within top 5 genes, q<0.05, Supplemental-Figure-12, Supplemental-Table-1). qPCR analysis of S100A8 and S100A9 in normal and HSS-challenged VEGFR1^+/+ and VEGFR1^+/− BMDM showed no significant difference in S100A8 or S100A9-expression between normal VEGFR1^+/− and VEGFR1^+/+ BMDM, but a significant increase in HSS-VEGFR1^+/− BMDM vs. HSS- VEGFR1^+/+ BMDM confirming the RNA-Seq data (Fig-7A, B). Furthermore, a significant increase in S100A8 and S100A9-expression was also observed in VEGFR1^+/− ischemic-muscle (Fig-7C, D) and HSS VEGFR1^+/− skeletal muscle endothelial cells (Supplemental-Figure-13) vs. respective controls. No significant difference in S100A8 and S100A9-expression was observed between VEGFR1^+/+ vs. VEGFR1^+/− non-ischemic-muscle (Fig-7C, D) or normal VEGFR1^+/+ vs. VEGFR1^+/− skeletal muscle endothelial cells (Supplemental-Figure-13).

Figure-7.

VEGFR1 regulates S100A8/S100A9 expression in macrophages. A-B) qPCR analysis of A) S100A8 and B) S100A9 expression in VEGFR1^+/+ (VR1^+/+), VEGFR1^+/− (VR1^+/−) bone marrow derived macrophages (BMDM) under normal or HSS conditions. n=6. One way ANOVA with Bonferroni select pair comparison was used to determine statistical significance. C-D) qPCR analysis of C) S100A8 and D) S100A9 expression in VEGFR1^+/+ (VR1^+/+), VEGFR1^+/− (VR1^+/−) non-ischemic (NGA) and ischemic gastrocnemius muscle (NGA). n=5. One way ANOVA with Bonferroni select pair comparison was used to determine statistical significance. E-H) qPCR analysis of arginase-1 (Arg-1) and inducible nitric oxide synthase (iNos) expression in macrophages (Raw264.7) transfected with (E-F) S100A8 or (G-H) S100A9 under normal or HSS conditions. n=6. Unpaired T-test was used to determine statistical significance. I-J) qPCR analysis Arg-1:iNos expression in I-K) S100A8 or J-L) S100A9 transfected macrophages treated with BAPTA-AM under normal or HSS conditions. n=6. Unpaired T-test was used to determine statistical significance. M-N) In vitro angiogenesis assay of endothelial cells treated with conditioned-medium (CM) from M) normal or N) HSS-challenged macrophages transfected with control plasmid (Neg Pld), S100A8 plasmid (S100A8 Pld) or S100A9 plasmid (S100A9 Pld). n=4. One way ANOVA with Dunnett’s multiple test correction was used to determine statistical significance. *P<0.05 considered significant.

We next examined S100A8 and S100A9 function in regulating macrophage-polarization and angiogenesis in vitro. qPCR analysis of Arg-1 and iNos in macrophages overexpressing S100A8 or S100A9 showed a significant decrease in Arg-1-expression (S100A8: Nor ~9X (P=0.051), HSS ~1X (P=0.041); S100A9: Nor ~7X (P=0.0087), HSS ~5.5X (P<0.0001)) with a concomitant increase in iNos-expression (S100A8: Nor ~3.5X (P=0.003), HSS ~2X (P=0.002); S100A9: Nor ~4X (P=0.001) HSS (P=0.02)) under normal and in HSS conditions (Fig-7E-H). Transfection efficacy of S100A8 presented in Supplemental-Figure-14. Ct values of S100A9 in negative-plasmid transfected macrophages (Raw264.7) was higher than 50 cycles and S100A9 plasmid transfection decreased the Ct values to a mean of 22±0.17.

Since S100A8 and S100A9 were shown to induce calcium influx in neutrophils³⁷, we next examined whether calcium influx due to increased S100A8 and S100A9-expression drive M1-like-phenotype. To confirm, we overexpressed S100A8 or S100A9 in macrophages and treated them with or without BAPTA-AM (1,2-Bis (2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester), a cell-permeable calcium chelator³⁸ under normal or HSS conditions. Interestingly, BAPTA-AM induced greater Arg1:iNos expression in macrophages under normal conditions but no significant difference in Arg1:iNos expression was observed under HSS conditions (Supplemental-Figure-15). However, BAPTA-AM not only blocked S100A8 and S100A9 induced M1-like-phenotype but also promoted an M2-like-phenotype both under normal (Arg1:iNos: S100A8 ~1X (P=0.03), S100A9 ~3X (P=0.0003), Fig-7I, J) and HSS (Arg1:iNos: S100A8 (P=0.03), S100A9 (P=0.01), Fig-7K, L) conditions. These data suggested that while HSS induces an M1-like-phenotype, this phenotype may not be due to increased calcium levels; but S100A8 and S100A9 induced M1-like-phenotype is via increased calcium influx.

We next examined the role of S100A8 and S100A9 in endothelial cells. Overexpressing either S100A8 or S100A9 showed no significant difference in capillary-like tube formation on GFRM in vitro (Supplemental-Figure-16). However, conditioned-medium from macrophages transfected with S100A8 or S100A9 under normal and HSS conditions significantly decreased endothelial tube-like formation on GFRM (Fig-7M, N). These data suggested that increased S100A8 and S100A9-expression in macrophages inhibit endothelial angiogenic potential in a paracrine manner.

Since earlier reports have indicated that TLR-signaling regulates S100A8-expression via STAT3 pathway in macrophages³⁹, we next examined whether STAT3 regulates S100A8 in macrophages under HSS conditions. qPCR analysis of S100A8 in macrophages treated with or without STAT3 inhibitor (100uM) showed no significant difference in S100A8-expression (Supplemental-Figure-17) indicating that in macrophages STAT3 is not a regulator of S100A8. Furthermore, our data also indicate that VEGFR1-activation induces distinct signaling independent of STAT3-activation to inhibit S100A8 and S100A9-expression in macrophages.

9. VEGF₁₆₅b regulates S100A8/S100A9 expression in preclinical-PAD

We next wanted to obtain a direct evidence of VEGF₁₆₅b in regulating macrophage-specific S100A8 and S100A9-expression. Since S100A8 and S100A9-expression is significantly higher in ischemic vs. non-ischemic-muscle (Supplemental-Figure-18, correlating with increased VEGF₁₆₅b levels), we performed flow cytometry analysis of ischemic-muscle treated with IgG or VEGF₁₆₅b-Ab. Flow cytometry analysis showed that VEGF₁₆₅b-inhibition significantly decreased ischemia-induced total tissue (Fig-8A), endothelial (Supplemental-Figure-19) and macrophage (Fig-8B) specific S100A8/S100A9-expression vs. IgG. Furthermore, gating on S100A8/S100A9+ macrophages showed that VEGF₁₆₅b-inhibition significantly decreased S100A8/S100A9⁺ CD80⁺-macrophages (Fig-8C) in ischemic-muscle vs. IgG. No significant difference in S100A8/S100A9⁺ Arg-1⁺-macrophages was observed.

Figure-8.

VEGF₁₆₅b regulates S100A8/S100A9 expression in ischemic-muscle. A-B) Flowcytometric analysis S100A8/S100A9 expression in A) non-ischemic (NGA), ischemic-muscle treated with IgG or ischemic-muscle treated with VEGF₁₆₅b-Ab, B) macrophages from NGA, IGA+IgG or IGA+VEGF₁₆₅b-Ab. C) CD80^high M1-like macrophages in NGA, IGA+IgG or IGA+VEGF₁₆₅b-Ab. A-C) n=5, One way ANOVA with Dunnett’s multiple test correction was used to determine statistical significance. D-E) qPCR analysis of D) Arg-1 and E) iNos expression in macrophages (Raw264.7) transfected with S100A8 or S100A9 plasmid and treated with IgG or VEGF₁₆₅b-Ab under normal or HSS conditions. n=6. Unpaired T-test was used to determine statistical significance. *P<0.05 considered significant. F) Schematic of VEGF165b induced macrophage M1 like polarization that inhibits perfusion recovery in PAD.

To confirm the in vivo data, we next overexpressed S100A8 or S100A9 in macrophages and treated them with IgG or VEGF₁₆₅b-Ab under normal or HSS conditions. qPCR analysis showed that VEGF₁₆₅b-inhibition significantly induced Arg-1-expression (S100A8: Nor ~6X, HSS ~9X; S100A9: Nor ~6.5X, HSS~14.5X, Fig-8D) in macrophages both under normal and HSS conditions. No significant difference in iNos-expression was observed between IgG or VEGF₁₆₅b-Ab treated S100A8 or S100A9 transfected macrophages under normal or HSS conditions (Fig-8E). This data indicated that VEGFR1-activation post VEGF₁₆₅b-inhibition decreases S100A8/S100A9-expression to induce M2-like-polarization in pre-clinical PAD. Schematic of the VEGF₁₆₅b signaling that regulates macrophage-polarization in ischemic-muscle is presented in Fig-8F.

Discussion

The results of our study demonstrate for the first time that VEGF₁₆₅b, a hereunto unrecognized splice variant within a family of critical growth factors, is capable and sufficient to alter macrophage-polarization in whole or in part through S100A8/S100A9. The effects of VEGF₁₆₅b on macrophages have not been previously reported. We showed that VEGF₁₆₅b binds to VEGFR1 on macrophages and this binding has two functions. First, this polarizes macrophages toward an M1 (inflammatory)-like-phenotype and this binding prevents the binding of VEGFR1-activating ligands, which can promote an M2 (reparative)-like-phenotype. Our findings in the area of ischemic-muscle revascularization including human-PAD-biopsies; mouse preclinical-PAD models that utilized VEGF₁₆₅b-depletion and over-expression strategies, adoptive cell transfer experiments; complimentary in vitro experiments and RNA-Seq experiments showed that increased anti-angiogenic VEGF₁₆₅b isoform expression in macrophages directly and competitively inhibits VEGFR1-S100A8/S100A9 signaling to induce an M1-like-phenotype that inhibits ischemic-muscle angiogenesis and perfusion-recovery in PAD.

Our understanding of the function of the alternatively spliced VEGF₁₆₅b isoform is expanding but studies to date have largely focused on its role in blocking VEGFR2-activation in endothelial cells. Our current data show that in ischemic macrophages VEGF₁₆₅b induces an autocrine effect that inhibits VEGFR1_Y1333-activation that results in an M1-like-phenotype which in-turn exerts a paracrine effect on hypoxia-dependent angiogenesis. We have recently shown that VEGF₁₆₅b exhibits differential effects on VEGFR1 and VEGFR2-activation i.e. it functions as an inhibitory isoform for VEGFR1_Y1333, and functions as an activator of VEGFR2_Y1175 in endothelial cells¹² (contrary to the existing paradigm that VEGF₁₆₅b largely functions as a competitive blocker of VEGF₁₆₅a induced VEGFR2-activation^{9,11,31,40–43}). While VEGFR2-expression is largely confined to endothelial cells, VEGFR1 is expressed on non-endothelial cell types^44,45, including progenitor cells, macrophages, and inflammatory cells suggesting that the function of VEGF₁₆₅b may well extend beyond endothelial cells. However, unlike extensive literature that exists on the VEGFR2-signaling networks, far less is known of VEGFR1-signaling and function in cardiovascular diseases^44,46. Our finding of an increased M1-like-phenotype with VEGFR1-inhibition suggested that VEGF₁₆₅b may function as more than an ‘inert blocker’.

We have previously shown that VEGF₁₆₅b modulates VEGFR1-STAT3 activation in endothelial cells to increase angiogenesis. However, in macrophages, VEGF₁₆₅b not only regulates VEGFR1-STAT3 signaling but also a VEGFR1-S100A8/S100A9 signaling to regulate an M1-like-phenotype. We identified S100A8/S100A9 as downstream signaling intermediates of VEGFR1 by RNA-Seq analysis of HSS VEGFR1^+/+ BMDM vs. VEGFR1^+/− BMDM. We used HSS VEGFR1^+/+ and HSS VEGFR1^+/− BMDM because no difference in VEGFR1-expression was observed between VEGFR1^+/+ and VEGFR1^+/− BMDM under normal conditions. Even though we identified S100A8/S100A9 downstream of VEGFR1 in macrophages, we observed significantly higher S100A8/S100A9 levels in HSS VEGFR1^+/− ECs vs. HSS VEGFR1^+/+ ECs, indicating that VEGFR1 regulates S100A8/S100A9-expression in endothelial cells also. Based on the limited amount of literature that exists on VEGFR1-signaling intermediates this data should advance the study of VEGFR1-biology.

Recent literature has shown that STAT3 can bind to the promoter of S100A8 in macrophages to induce S100A8-expression³⁹; and increased S100A8-expression can induce STAT3 levels in lung injury models by LPS or cPG-DNA⁴⁷ indicating a potentially positive feedback loop between S100A8 and STAT3 to regulate macrophage-phenotype. However, in culture, STAT3-inhibition did not change S100A8 levels, indicating that STAT3 regulation of S100A8 is cell and context dependent. These data expanded our recent findings and showed that a novel VEGFR1 dependent but STAT3 independent signaling regulates S100A8/S100A9-expression in macrophages that converge on regulating M2-like-phenotype. These data demonstrate that removal of VEGF₁₆₅b inhibitory effect on VEGFR1 regulates M2-like-phenotype in (at least) two distinct ways. One involves VEGFR1-activation of STAT3, another signaling involves VEGFR1-inhibition of S100A8/S100A9, which converges on inducing an M2-like-phenotype that promotes angiogenesis and tissue recovery in distal ischemic-muscle. Additional work will be required to delineate the molecular signaling that connects VEGFR1-S100A8/S100A9 signaling.

Interestingly, while S100A8 or S100A9 overexpression did not directly modulate endothelial angiogenic potential under normal or HSS conditions; conditioned-medium from S100A8 or S100A9 transfected macrophages significantly decreased endothelial angiogenic potential. Even though S100A8 or S100A9 did not directly modulate endothelial angiogenic potential in vitro, it is possible that S100A8/S100A9 can modulate other endothelial properties such as vascular integrity/permeability, adhesion molecule expression and/or immune cells migration^48,49. Furthermore, while conditioned-medium from VEGF₁₆₅b transfected and HSS-VEGFR1^+/− BMDM significantly decreased endothelial angiogenic capacity, conditioned-medium from BMDM treated with VEGF₁₆₅b-Ab significantly induced endothelial angiogenic capacity indicating that increased VEGF₁₆₅b-expression in ischemic macrophages inhibits angiogenesis in a paracrine manner. In addition, adoptive transfer of VEGF₁₆₅b transfected BMDM and VEGFR1^+/− BMDM significantly decreased angiogenesis and perfusion in the ischemic-muscle in vivo indicating that macrophage-specific VEGFR1-signaling plays a major role in regulating PAD muscle perfusion-recovery. Consistent with our murine findings, our recently published human data from PAD and control (age and gender matched) biopsies showed that greater VEGF₁₆₅b binding to VEGFR1 correlated with decreased VEGFR1-activation¹²; and in our current work we show that VEGF₁₆₅b induction correlated with increased CD80⁺-macrophages in human-PAD. This data showed that VEGF₁₆₅b inhibits VEGFR1 to induce M1-like-phenotype in human-PAD. However, the markers used to distinguish M1 vs. M2-macrophages²⁴ are not binary. While flow cytometry analysis will provide a better delineation of these cell populations with a more restricted marker expression, muscle availability from human muscle-biopsies limited us for studies such as double immunofluorescence analysis of CD68 and CD80 or CD206.

Kikuchi et al¹³ have previously shown that VEGF₁₆₅b levels are significantly higher in the serum of PAD vs normal patients by western blot analysis. Serum preparation involves platelet activation to form a blood clot and it is possible that VEGF₁₆₅b is released by circulating monocytes or perhaps activated platelets in this process in PAD patients. Furthermore, HSS macrophages expressed higher VEGF₁₆₅b levels but this remains cell-associated and is not secreted, suggesting an autocrine effect. Unlike the freely circulating VEGF₁₂₁a isoform, VEGF₁₆₅a is predominantly membrane-bound and indeed Guzman-Hernandez et al⁵⁰ showed that VEGF₁₆₅a is prominently present on the outer surface of the cells. The membrane binding properties of VEGF₁₆₅a is due to the heparin-binding sites in exon-7, which is same in VEGF₁₆₅b isoforms. As such, we predict that VEGF₁₆₅b similar to VEGF₁₆₅a is inherently membrane bound. Whether and to what extent the 6-aminoacid switch in VEGF₁₆₅b isoforms confer increased membrane binding properties resulting in decreased secretion is not clear. Such studies will advance our understanding of the mechanism of VEGF₁₆₅b action.

The novelty of these findings on VEGF₁₆₅b and its role on macrophages are relevant not only for PAD but also across other human cardiovascular diseases.

Clinical Perspective.

What is new?

• While known functions of VEGF₁₆₅b, an anti-angiogenic VEGF-A isoform has been confined to endothelial cells, we show that VEGF₁₆₅b induces an anti-angiogenic, cytotoxic M1-like phenotype in macrophages that inhibits ischemic muscle angiogenesis in Peripheral artery disease (PAD)

• In ischemic macrophages, VEGF₁₆₅b silences VEGFR1 by an autocrine mechanism to induce an M1-like phenotype that inhibits angiogenesis by a paracrine mechanism in ischemic muscle

• We identified S100A8/A9 as a novel VEGF₁₆₅b-VEGFR1 downstream signaling intermediate that induces M1-like phenotype in ischemic macrophages in PAD

What are the clinical implications?

• Therapies that can induce and sustain an M2-phenotype in ischemic-muscle were not yet successful due to the reversible nature of macrophage-polarization states.

• Our current work showed that targeting anti-angiogenic VEGF₁₆₅b isoforms have the translational potential to induce and sustain M2-like-macrophage phenotype in ischemic-muscle to promote therapeutic angiogenesis and enhance tissue recovery