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. 2021 Apr 6;6(1):22.
doi: 10.1038/s41536-021-00129-z.

Coordinated interactions between endothelial cells and macrophages in the islet microenvironment promote β cell regeneration

Diane C Saunders #  1   2 , Kristie I Aamodt #  1 , Tiffany M Richardson  1 , Alexander J Hopkirk  2 , Radhika Aramandla  2 , Greg Poffenberger  2 , Regina Jenkins  2 , David K Flaherty  3 , Nripesh Prasad  4 , Shawn E Levy  4 , Alvin C Powers  5   6   7 , Marcela Brissova  8
Affiliations

Coordinated interactions between endothelial cells and macrophages in the islet microenvironment promote β cell regeneration

Diane C Saunders et al. NPJ Regen Med. .

Abstract

Endogenous β cell regeneration could alleviate diabetes, but proliferative stimuli within the islet microenvironment are incompletely understood. We previously found that β cell recovery following hypervascularization-induced β cell loss involves interactions with endothelial cells (ECs) and macrophages (MΦs). Here we show that proliferative ECs modulate MΦ infiltration and phenotype during β cell loss, and recruited MΦs are essential for β cell recovery. Furthermore, VEGFR2 inactivation in quiescent ECs accelerates islet vascular regression during β cell recovery and leads to increased β cell proliferation without changes in MΦ phenotype or number. Transcriptome analysis of β cells, ECs, and MΦs reveals that β cell proliferation coincides with elevated expression of extracellular matrix remodeling molecules and growth factors likely driving activation of proliferative signaling pathways in β cells. Collectively, these findings suggest a new β cell regeneration paradigm whereby coordinated interactions between intra-islet MΦs, ECs, and extracellular matrix mediate β cell self-renewal.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. The RIP-rtTA; Tet-O-VEGF-A (βVEGF-A) model of β cell regeneration permits specific modulation of the islet microenvironment.
Induction of VEGF-A overexpression in β cells with doxycycline (Dox) causes rapid endothelial cell (EC) expansion, β cell death, and recruitment of circulating monocytes increasing the number of intra-islet macrophages (MΦs). Once the Dox stimulus is removed, VEGF-A levels normalize and β cells undergo self-renewal. To establish the role of MΦs in β cell proliferation, monocytes and MΦs were depleted with clodronate liposomes (scheme A). To determine whether proliferative intra-islet ECs were required for β cell loss, MΦ recruitment, and MΦ phenotype activation, an additional genetic construct was introduced to knockdown key signaling receptor VEGFR2 in ECs prior to VEGF-A induction (scheme B). To determine if VEGFR2 signaling in quiescent ECs contributes to β cell proliferation, VEGFR2 was inactivated in ECs during β cell recovery (scheme C). To identify cell-specific transcriptome changes during β cell loss and recovery, populations of ECs, MΦs, and β cells were isolated prior to and during VEGF-A induction, and after VEGF normalization (scheme D). For additional details, including mouse models utilized, see Supplementary Fig. 1.
Fig. 2
Fig. 2. Macrophages are required for β cell proliferation in βVEGF-A mice.
a To deplete macrophages (MΦs) during VEGF-A induction and normalization, βVEGF-A mice were treated with clodronate or control liposomes (150–200 μl i.v.) every other day, beginning 1 day before Dox treatment and continuing 1 week after Dox withdrawal (1wk WD). b Representative flow cytometry plots showing circulating monocytes (CD11b+ Ly6G–) of control and clodronate-treated βVEGF-A mice 24 h after single injection (No Dox). Approximately 10,000 white blood cells (WBCs) were analyzed (monocyte fraction reported as mean + s.e.m.) per each animal. c Islet architecture displayed by labeling for β cells (Insulin; blue), endothelial cells (Caveolin-1; green), and MΦs (Iba1; red) during VEGF-A induction (1wk Dox) and normalization (1wk WD). Scale bar, 50 μm. d Quantification (mean + s.e.m.) of islet MΦ area by immunohistochemistry (5.1 ± 0.7 × 105 μm2 total islet area analyzed per animal). e, f Quantification (mean + s.e.m.) of endothelial cell (EC) area (e) and β cell area (f) in βVEGF-A mice treated with control or clodronate liposomes during VEGF-A induction and normalization. g Rate of β cell proliferation (mean + s.e.m.; 1138 ± 87 β cells counted per animal) during VEGF-A normalization (1wk WD and 2wk WD) in control βVEGF-A mice was significantly reduced in clodronate-treated mice. In panels b–g, each closed circle represents one animal; asterisks indicate unpaired two-tailed t-tests of control vs. clodronate groups; *p < 0.05; **p < 0.01; ****p < 0.0001. Dashed lines in d–g depict average values in βVEGF-A mice at baseline (No Dox).
Fig. 3
Fig. 3. Inactivation of VEGFR2 signaling in endothelial cells prevents β cell loss and M2-like macrophage polarization by acute elevation of VEGF-A in the islet microenvironment.
a To inactivate VEGFR2 in endothelial cells (ECs), control (βVEGF-A; VEGFR2fl/fl) and VEGFR2iΔEC (βVEGF-A; VEGFR2iΔEC) mice were treated with Tamoxifen (Tm; 4 mg s.c.) prior to VEGF-A induction. b Islet architecture displayed by labeling for macrophages (Iba1+), ECs (CD31+), and β cells (Ins+) at baseline (No Dox) and after 3d Dox. c, d Quantification (mean + s.e.m.) of islet β cell and EC composition (10 ± 1 × 105 μm2 total islet area analyzed per animal). e Some intra-islet macrophages (MΦs) in control mice showed an “M2-like” phenotype (CD206+) after VEGF-A induction, indicated by arrowheads. Insets show representative intra-islet MΦs in each group and time point. f, g Quantification (mean + s.e.m.) of MΦ infiltration and M2-like intra-islet MΦs (percent CD206+ Iba1+ of Iba1+), 3 ± 2 × 105 μm2 total islet area analyzed per animal. Each closed circle in bar graphs represents one animal. Asterisks indicate unpaired two-tailed t-tests between genotypes; *p < 0.05; ***p < 0.001. Scale bars in b and e, 50 μm; inset, 10 μm.
Fig. 4
Fig. 4. Phenotypic and structural changes to the islet microenvironment in response to VEGFR2 inactivation in quiescent endothelial cells during β cell recovery.
a To inactivate VEGFR2 in endothelial cells (ECs) during β cell recovery, control (βVEGF-A; VEGFR2f/fl) and VEGFR2iΔEC (βVEGF-A; VEGFR2iΔEC) mice received Tamoxifen (Tm; 4 mg s.c.) after 7 days (d) of Dox withdrawal (WD). b Islet architecture visualized by labeling for macrophages (Iba1+) ECs (CD31+, green), β cells (Insulin+, blue) at 7d WD (pre-Tm) and 9d WD (2d post-Tm). Scale bar, 50 μm. c–e Area quantification (mean + s.e.m.) of islet ECs (c), MΦs (d), and β cells (e) by immunohistochemistry; 14 ± 1 × 105 μm2 total islet area analyzed per animal. Each circle represents one animal; asterisks indicate results of unpaired two-tailed t-tests between genotypes; ***p < 0.001. f β cell proliferation rates (1,947 ± 145 β cells per animal) plotted as a function of β cell loss (% β cells of total islet area) reveal a significant increase after VEGFR2 inactivation in quiescent ECs at 9d WD. Parentheses beside lines provide x- and y-intercepts derived from linear regression. At 9d WD, intercepts are significantly different; **p < 0.01. g Visualization of islet extracellular matrix by immunofluorescence (ECM; Col-IV+, red), ECs (CD31+, green), β cells (Insulin+, blue) at 7d WD (pre-Tm), and 9d WD (2d post-Tm). Scale bar, 50 μm; inset, 10 μm. Arrowhead in bottom right panel points to ECM casts where ECs have regressed.
Fig. 5
Fig. 5. Genes and pathways upregulated in β cells during recovery reflect activation of intracellular signaling to promote proliferation.
a Experimental schematic showing sorting of islet-derived β cells, endothelial cells (ECs), and macrophages (MΦs) from βVEGF-A mice at baseline (No Dox), during VEGF-A induction/β cell loss (1wk Dox), and during VEGF-A normalization/β cell recovery (1wk WD). See also Supplementary Fig. 5b–e. b Normalized expression of selected genes known to function in β cell proliferation– at No Dox (n = 4 biological replicates), 1wk Dox (n = 5), and 1wk WD (n = 3). Numbers listed in 1wk Dox and 1wk WD columns represent fold-change ≥2 or <−2 (p < 0.05) as compared to No Dox. Color scale corresponds to normalized expression values ranging from 0 (white) to ≥6000 (dark blue). c Selected differentially regulated pathways during β cell recovery (1wk WD vs. No Dox), as determined by Ingenuity Pathway Analysis (IPA). Total bar width represents z-score; colored fractions indicate percentage of pathway genes that are up (blue), down (orange), and unchanged (gray). Number of genes per category is also listed within or adjacent to bars. A full list of significantly regulated pathways (z-score ≥2 or ≤−2, p < 0.05) is provided in Supplementary Table 1. FGF fibroblast growth factor, HGF hepatocyte growth factor, ILK integrin-linked kinase, MAPK mitogen-activated protein kinase, NFAT nuclear factor of activated T cells, NF-kB nuclear factor kappa-light-chain-enhancer of activated B cells, PI3K phosphoinositide 3-kinase, PAK p21-activated kinase, TREM1 triggering receptor expressed on myeloid cells 1, VEGF vascular endothelial growth factor.
Fig. 6
Fig. 6. Model of interactions between β cells, macrophages, endothelial cells, and the extracellular matrix in β cell regeneration.
Upon VEGF-A induction, intra-islet endothelial cells (ECs) proliferate while increasing expression of cell adhesion molecules and growth factors and altering their expression of integrins and extracellular matrix (ECM) remodeling enzymes. These adhesion molecules help recruit macrophages (MΦs), which upon islet infiltration also upregulate expression of cell adhesion molecules and pro- and anti-inflammatory chemokines and cytokines, influencing further MΦ recruitment in addition to signaling through chemokine and cytokine receptors on β cells. Chemokine and cytokines become increasingly less inflammatory as VEGF-A normalizes, as MΦs produce growth factors and matrix remodeling enzymes that may promote β cell proliferation. Upon VEGF-A induction, β cells exhibit enrichment for several integrin pathways and other proteins involved in ECM remodeling and cell–matrix interactions in addition to regulating expression of chemokines known to support a regenerative (M2 or alternative) MΦ phenotype. Growth factors from all cell types act on an increased number of growth factor receptors being expressed on β cells, activating downstream signals converging on the PI3K/Akt and MAPK pathways. Other signals from cells in the microenvironment, or from the rapidly remodeling ECM, may also play a role in β cell proliferation.
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