CD163+ macrophages promote angiogenesis and vascular permeability accompanied by inflammation in atherosclerosis

Abstract

Intake of hemoglobin by the hemoglobin-haptoglobin receptor CD163 leads to a distinct alternative non-foam cell antiinflammatory macrophage phenotype that was previously considered atheroprotective. Here, we reveal an unexpected but important pathogenic role for these macrophages in atherosclerosis. Using human atherosclerotic samples, cultured cells, and a mouse model of advanced atherosclerosis, we investigated the role of intraplaque hemorrhage on macrophage function with respect to angiogenesis, vascular permeability, inflammation, and plaque progression. In human atherosclerotic lesions, CD163+ macrophages were associated with plaque progression, microvascularity, and a high level of HIF1α and VEGF-A expression. We observed irregular vascular endothelial cadherin in intraplaque microvessels surrounded by CD163+ macrophages. Within these cells, activation of HIF1α via inhibition of prolyl hydroxylases promoted VEGF-mediated increases in intraplaque angiogenesis, vascular permeability, and inflammatory cell recruitment. CD163+ macrophages increased intraplaque endothelial VCAM expression and plaque inflammation. Subjects with homozygous minor alleles of the SNP rs7136716 had elevated microvessel density, increased expression of CD163 in ruptured coronary plaques, and a higher risk of myocardial infarction and coronary heart disease in population cohorts. Thus, our findings highlight a nonlipid-driven mechanism by which alternative macrophages promote plaque angiogenesis, leakiness, inflammation, and progression via the CD163/HIF1α/VEGF-A pathway.

Keywords: Angiogenesis; Atherosclerosis; Macrophages; Vascular Biology.

Figure 1. CD163⁺ macrophages are associated with progression of carotid atherosclerosis in humans.

(A) Representative images of human carotid arteries with PIT, fibroatheromatous, and ruptured atherosclerotic plaques. High-magnification IHC images of CD163 with low-magnification insets. H&E and Movat pentachrome stains are also shown. Scale bars: 1 mm and 5 mm (insets). (B–D) Correlation between CD163⁺ macrophages and human carotid plaque progression. (B) Human carotid plaques were classified as fibrocalcific or fibroatheroma with calcification (Fibroatheroma-Ca) (n = 19, black); intima xanthoma or PIT (n = 8, green); fibroatheroma or TCFA (n = 14, blue); and ruptured or healed rupture (n = 16, red), with the corresponding percentage of CD163⁺ macrophages per plaque area. (C) Correlation between CD163⁺ macrophages and the percentage of stenosis. The percentage of stenosis was categorized as follows: 20%–40% (n = 5); 40%–60% (n = 14); 60%–80% (n = 23); and 80%–100% (n = 28). (D) Correlation between CD163⁺ macrophages and the percentage of necrotic core area. The percentage of necrotic core area was classified as: <10% (n = 32); 10%–20% (n = 19); 20%–30% (n = 9); and >30% (n = 10). (E and F) Human plaques from CEAs were examined by histology and immunofluorescence. Images were acquired by confocal microscopy using a ×20 objective (9 tiles, E and F), with optical slicing in the z axis. In E, as explained in the text, areas from fibroatheromatous lesions containing foam cell (CD163^– [green], CD68⁺ [cyan]) macrophages (i.e., low CD163) and M(Hb) macrophages (CD163⁺, CD68⁺ [i.e., high CD163]) were immunostained using antibodies against vWF antigen for detection of microvessels. Nuclei were counterstained using DAPI (blue). Note that calcification is seen as dense areas of dark purple. Adjacent low- and high-magnification images of H&E-stained sections show the corresponding regions of angiogenesis. Scale bars: 1 mm and 200 μm. Results are presented as the mean or the mean ± SEM. For multiple group comparisons, 1-way ANOVA was used. P values shown in B–D were determined by 1-way ANOVA.

Figure 2. Alternative CD163⁺ macrophages are associated with intraplaque angiogenesis and vascular permeability and express HIF1α and VEGF-A.

Human plaques from CEA specimens were analyzed by histology and immunofluorescence. Images were acquired by confocal microscopy using ×40 (A and E) or ×60 (C, RNAscope) objectives, with optical slicing in the z axis. (A) Representative microvessels (MV) from low and high CD163 areas (green) dual immunostained for HIF1α and VEGF-A (red channels). Scale bars: 20 μm. (B) immunoblotting for CD163, HIF1α, VEGF-A, and GAPDH of protein extracted from human atheroma expressing high or low levels of CD163 (n = 4 samples each). Bar graphs show quantitation of densitometry for the indicated proteins. (C) RNAscope ISH analysis using CD163 (red) and VEGF (green) probes on human carotid plaques. H&E and CD163 immunohistochemically stained images of human carotid plaques are shown on the left to indicate the areas (red boxes) of RNAscope ISH images of CD163 and VEGF, shown on the right. Scale bars: 1 mm (H&E- and immunostained images) and 2 μm (ISH). (D) Quantitative graph of VEGF RNA punctate counts in macrophages with low (<1–4 punctates) or high (≥5 punctates) CD163 expression (total of 3 plaques used for data collection, with 5 to 6 areas per plaque). (E) representative microvessels from low and high CD163 areas (green) dual immunostained for VE-cadherin and vWF (red channels). Note that VE-cadherin expression is located exclusively along the interendothelial contacts. The fluorescence signal for VE-cadherin appeared to be attenuated in a microvessel surrounded by CD163⁺ macrophages, with diffuse expression of vWF antigen, suggestive of leaky endothelial junctions. Scale bars: 20 μm. (F) Bar graphs show quantification of fluorescence signals for VE-cadherin and vWF in low and high CD163 areas (multiple areas from a total of 10 plaques per group were examined; see Methods). (G) Laser capture microdissection and qPCR analysis of FPN expression in low CD163⁺ and high CD163⁺ macrophage areas (n = 15 plaques sampled). Results are presented as the mean ± SEM (B and F) or the mean ± SD (D and G). (B and F) *P < 0.05, by 2-sided Student’s t test. (D and G) P < 0.01 and P < 0.05, by 2-sided Student’s t test.

Figure 3. Inhibition of PHDs by relative iron deprivation within M(Hb) macrophages increases HIF1α/VEGF-A signaling and promotes angiogenesis.

(A) Intracellular free iron levels in control [M(con)] or HH-differentiated [M(Hb)] human macrophages (n = 4 per group). (B) PHD2 activity of human macrophages after HH treatment compared with control (n = 5 per group). (C and D) Immunoblotting of human macrophages (n = 4 per group) with quantitation of densitometry for HIF1α-OH and PHD2. (E) Immunoblotting of human macrophages (n = 4 per group) with quantitation of densitometry for HIF-1α. (lane 1: control macrophages; lane 2: HH-stimulated macrophages; lane 3: control macrophages + 700 nM hepcidin; lane 4: HH-stimulated macrophages + 7 00 nM hepcidin). (F) ELISA analysis of macrophage supernatants for VEGF-A (n = 5 per group). As in E, the bars represent, from left to right: control macrophages, HH-stimulated macrophages, control macrophages plus 700 nM hepcidin, and HH-stimulated macrophages plus 700 nM hepcidin. (G) Tube formation assays of HAECs with macrophage supernatant. Relative tube-forming abilities are shown with representative images on top and quantitated tube-formation index below (n = 5 per group). Scale bars: 200 μm. All error bars indicate the mean ± SEM. Comparisons between 2 groups were conducted using a 2-sided Student’s t test. For multiple group comparisons, a 1-way ANOVA was applied. If the variance ratio test (F test) was significant, a more detailed post-hoc analysis of differences between groups was made using a Tukey-Kramer honest significant difference test. *P < 0.05 versus control in A–D versus other groups in E–G.

Figure 4. M(Hb) macrophages promote vascular permeability via VEGF-A/VEGFR2 signaling.

(A) TEER measurements after treatment of scramble siRNA– (Scr) or VEGFR2 siRNA–transfected HAECs with supernatant from control macrophages [M(con)] or HH-differentiated [M(Hb)] macrophages. The relative TEER compared with control is shown (n = 4 per group). (B) FITC-dextran permeability in scramble siRNA– or VEGFR2 siRNA–transfected HAECs treated with control or HH-differentiated macrophage supernatants. Percentage change of FITC-dextran compared with control (n = 4 per group). (C) Immunofluorescence imaging of scramble siRNA– or VEGFR2 siRNA–transfected HAECs treated with control or HH-differentiated macrophage supernatants for VE-cadherin (green) and DAPI (blue) (original magnification, ×60). Scale bar: 50 μm. Note the loss of plasma membrane VE-cadherin in endothelial cells treated with M(Hb) supernatants versus M(con) supernatants and the restoration of membrane VE-cadherin in endothelial cells transfected with VEGFR2 siRNA and treated with M(Hb) supernatants. (D) Quantitation of plasma membrane VE-cadherin in the experiment shown in C (n = 10 per group). (E) Immunoblot of the membrane fraction from scramble siRNA– or VEGFR2 siRNA–transfected HAECs treated with control or HH-differentiated macrophage supernatants, with quantitation of densitometry for VE-cadherin (n = 4 per group). (F) Quantitation of plasma membrane VE-cadherin in the experiment shown in E. All error bars indicate the mean ± SEM. *P < 0.05 versus other groups. For multiple group comparisons, a 1-way ANOVA was applied. If the variance ratio test (F test) was significant, a more detailed post-hoc analysis of differences between groups was done using a Tukey-Kramer honest significant difference test.

Figure 5. Deletion of CD163 in mice reduces intraplaque neovascularization and plaque progression.

(A) Immunoblotting of mouse macrophages with quantitation of densitometry for HIF1α in macrophages isolated from WT or CD163^–/– mice stimulated with Hb (n = 4 per group). (B) Analysis of macrophage supernatant for VEGF-A by ELISA (n = 4 per group). (1: WT macrophages, 2: Hb-stimulated WT macrophages, 3: CD163^–/– macrophages, 4: Hb-stimulated CD163^–/– macrophages). (C) Representative H&E staining of BCA plaque with a of gross inset photograph of the aortic arch from 1-year-old ApoE^–/– and ApoE^–/– CD163^–/– mice. Scale bars: 100 μm. (D–G) Quantitative measurements of lesion size, percentage of stenosis, lesion pathological scores, and necrotic areas in the BCA plaque (n = 8–12 per group). (H) Representative immunofluorescence confocal microscopic images of BCA plaque stained with VE-cadherin (green), CD163 (red), and DAPI (blue). Scale bars: 50 μm and 10 μm (enlarged images of boxed areas on the left). (I) Microvessel density quantification calculated by the number of microvessels per plaque area identified by VE-cadherin immunofluorescence confocal microscopy (n = 6–7 per group). (J) Representative immunofluorescence confocal microscopic images of intraplaque FITC-dextran (green) as a marker for permeability. Scale bars: 100 μm. Total intraplaque FITC fluorescence was quantified from confocal images of BCA plaques perfused with FITC-dextran to determine permeability (n = 7 per group). Bars and plots indicate the mean ± SEM (A and B) or the mean ± SD (D–G, I, and J). (A and B) *P < 0.05 versus other groups, by 1-way ANOVA , and, if the ratio test (F test) was significant, a more detailed post-hoc analysis of differences between groups was performed using a Tukey-Kramer honest significant difference test. (D–G, I, and J) P < 0.01 and P < 0.05, by 2-sided Student’s t test.

Figure 6. CD163⁺ macrophages increase intraplaque endothelial VCAM and leukocyte recruitment via VEGF.

Activation of VCAM and NF-κB in HAECs (A–J) and association of high CD163⁺ macrophages and inflammation in human atherosclerotic lesions (K–N). In A–C, HAECs were incubated with culture media supernatants collected from human macrophages differentiated in HH [M(Hb) Sup] or control media [M(con) Sup] overnight. The expression of VCAM, p-p65 (p–NF-κB), and total NF-κB was measured by immunoblotting. LPS (100 ng/ml) was used as a positive control (n = 4 per group). (D) Immunostaining for VE-cadherin (green) and VCAM (red) in BCA plaques from 1-year-old ApoE^–/– and ApoE^–/– CD163^–/– mice. Scale bars: 100 μm. (E) The colocalization of VE-cadherin and VCAM in confocal microscopic images was analyzed for correlation coefficiency (n = 6–7 per group). (F) Total macrophage content in BCA plaques as described in H was analyzed using macrophage marker Mac3 immunohistochemical staining (n = 5 per group). (G–I) Cells were transfected with VEGFR2 siRNA for 48 hours before incubation with culture media supernatants from macrophages differentiated with HH or control media. The expression of VCAM, ICAM, p-p65 (p–NF-κB), and total NF-κB was measured by immunoblotting (n = 4 per group). Scr siRNA, scrambled siRNA. (J) Monocyte infiltration into BCA plaques from 6- to 8-month-old mice after a 4-week treatment with a control or VEGF-blocking antibody. Monocyte tracing was performed by injecting Au-nanoparticle–labeled monocytes, and labeled monocytes were counted on histological images (n = 6 per group). (K) Representative images of H&E and CD163 immunohistochemical staining showing high CD163 and low CD163 areas. Scale bars: 500 μm and 100 μm. (L) Areas of high and low expression of CD163⁺ macrophages in human atherosclerotic plaques were scored for inflammation (n = 7–8 per group). (M) CD163⁺ macrophages and CD3⁺ T cells were detected surrounding microvessels in high CD163 areas (white dashed lines show microvessels). Scale bars: 20 μm. (N) Quantitation of CD3⁺ T cells in high and low CD163⁺ macrophages surrounding intraplaque microvessels (n = 8 per group). Data represent the mean ± SEM (B, C, and H–J), the mean ± SD (E, F, J, and N), or the median for the box and whisker plot (L). (H–J) *P < 0.05, by 1-way ANOVA, and, if the variance ratio test (F test) was significant, a more detailed post-hoc analysis of differences between groups was performed using a Tukey-Kramer honest significant difference test. (B and C) *P < 0.05, by 2-sided Student’s t test. (E, F, and N) P < 0.01 and P 0.05, by 2-sided Student’s t test. (L) P < 0.01, by Mann-Whitney-Wilcoxon test.

Figure 7. CD163⁺ macrophages are associated with increased angiogenesis and microvessel permeability in human coronary artery plaques.

(A–D) Human coronary artery microvessel permeability was assessed by EBD perfusion. (A) Representative images of EBD-perfused human coronary arteries, H&E-stained images, and confocal immunofluorescence images of CD163 (red) and VE-cadherin (yellow) or VE-cadherin (red) and VCAM (green) in an EBD-negative area (top row), EBD-positive area 1 (middle row), and EBD-positive area 2 (bottom row). Positive areas 1 and 2 are shown in progressively higher-magnification H&E-stained images from left to right in the second and third rows. Red and white arrowheads point to microvessels. Confocal images of the EBD-negative areas for CD163/VE-cadherin and VE-cadherin/VCAM are shown in the top row of columns 3 and 4, respectively, while the positive area 1 is shown for CD163/VE-cadherin in the middle rows of columns 3 and 4 (higher-magnification image on the right), and positive area 2 is shown for VE-cadherin/VCAM in the bottom of row of columns 3 and 4 (higher-magnification image on the right). (B–D) Quantification of microvessel density, CD163⁺ macrophages, and VCAM in an EBD-positive area versus an EBD-negative area (n = 6–8 per group). (E) The SNP rs7136716 was associated with human coronary artery atherosclerotic plaque rupture and risk of coronary artery disease. See Table 2 for SNP analysis of the CVPath cohort. Representative images show ruptured plaques in human coronary arteries from subjects with the rs7136717 AA genotype versus the those with the GG genotype. Scale bars: 500 μm. Arrowheads point to microvessels. (F) Microvessel density per plaque area at the ruptured coronary artery plaque site from individuals with 0 copies (AA genotype, n = 22) versus 2 copies of the minor allele (GG genotype, n = 25) who died of plaque rupture. (G) Relative CD163 mRNA expression in ruptured coronary artery plaques from AA versus GG genotype groups as measured by qPCR (n = 13 per group). (H) Cox proportional hazards ratio assessment for the association of the genetic variant rs7136717 with incident MI and incident CHD in the ARIC cohort (n = 3,225). Data represent the mean ± SD (B–D, F, and G) or ORs and 95% CI (H). P < 0.01 and P < 0.05, by 2-sided Student’s t test (B–D, F, and G). In the ARIC cohort, Cox proportional hazards models were used to examine the association of the genetic variant with incident MI and incident CHD, and the analyses were adjusted for age, sex, ancestry-informative principal components, and study center (H).

Figure 8. Summary of the role of M(Hb) macrophages in plaque angiogenesis, permeability, vascular inflammation, and plaque progression.

In areas of IPH, HH complex ingestion by macrophages induces angiogenesis via activation of HIF1α, which is a consequence of intracellular Fe²⁺ deprivation and PHD2 inhibition. VEGF-A, which is secreted by macrophages via HIF1α activation, promotes angiogenesis, endothelial expression of VCAM, inflammatory cell recruitment, and vascular permeability via VEGF-A/VEGFR2 signaling. This may cause further IPH, RBC lysis, and more Hb ingestion by CD163⁺ macrophages. This vicious cycle causes plaque progression, which eventually leads to plaque rupture.