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

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.


INTRODUCTION
While investigating how vascular endothelial growth factor-A (VEGF-A) regulates islet 13 vascularization, our group previously characterized a mouse model in which signals from the 14 local microenvironment stimulate β cell self-renewal 1 . In this model, transiently increasing 15

VEGF-A production in β cells (βVEGF-A) induces endothelial cell (EC) expansion and 16
hypervascularization that causes β cell loss; remarkably, islet morphology, capillary network, β 17 cell mass, and function normalize 6 weeks after withdrawal (WD) of the VEGF-A stimulus. This 18 regenerative response is a result of a transient but robust burst in β cell proliferation, which is 19 dependent on VEGF-A-mediated recruitment of macrophages (MΦs). These recruited cells 20 express markers of both pro-inflammatory (M1) and restorative (M2) activation, suggesting a 21 unique regenerative phenotype [1][2][3][4][5] . Further investigation into the role of various 22 microenvironmental components on β cell proliferation in this model is needed given that this 23 regenerative microenvironment promotes proliferation of human in addition to mouse β cells 1 . 24 MΦs are often perceived as damaging to islets due to their role in β cell loss during diabetes 6-8 , 25 but it is becoming increasingly appreciated that tissue-resident MΦs play important roles in 26 immune surveillance and tissue homeostasis and function in the islet. Mice with compromised 27 MΦ populations during pancreatic development exhibit reduced β cell proliferation, β cell mass, 28 2 and impaired islet morphogenesis 9,10 . Furthermore, recent studies have supported the notion 29 that MΦs contribute to β cell regeneration after several types of injury, including surgically 30 induced pancreatitis 11 and diphtheria toxin (DT)-mediated apoptosis 12 . This recent work 31 highlights the importance of understanding how MΦs contribute to β cell proliferation and further 32 defining MΦ phenotype and function in the βVEGF-A model . 33 In addition to MΦs, ECs are known to participate in tissue repair via activation of the VEGF-A-34 VEGFR2 pathway, which mediates angiocrine factor production and promotes local cell renewal 35 and regeneration [13][14][15][16] . There is a precedent for ECs facilitating tissue repair by influencing MΦ 36 activation toward a restorative, M2-like phenotype 4 . Because vasculature is essential for normal 37 islet function, understanding signals that govern EC homeostasis and the effects of ECs on 38 neighboring cell populations is crucial for maintaining and restoring islet health. Signaling 39 between ECs and the pancreatic epithelium is critical for establishing islet vasculature and β cell 40 mass during development, and in mature islets ongoing signaling between endocrine and ECs 41 is required to maintain the capillary network through which endocrine cells receive adequate 42 nutrition and oxygen and can rapidly sense and secrete hormones [17][18][19] . The vascular basement 43 membrane is also the primary component of the intra-islet extracellular matrix (ECM) and acts 44 as a reservoir for growth factors and other signaling molecules important for β cell 45 differentiation, function, and proliferation [20][21][22][23] . In the βVEGF-A model, ECs may affect the 46 regenerative process indirectly by promoting MΦ recruitment and activation, altering ECM 47 composition and signaling, and/or by directly influencing β cell proliferation. 48 Here we deconstructed the complex in vivo islet microenvironment in the βVEGF-A model and 49 show that β cell self-renewal is mediated by coordinated interactions between recruited MΦs, 50 intra-islet ECs, and the ECM (Figures 1, S1). To isolate the roles of MΦs and ECs in this model 51 we removed MΦs from the islet microenvironment (Figures 1, S1; experimental scheme A) and 52 inactivated VEGFR2 signaling in ECs to discern the effects of proliferative or quiescent ECs on 53 β cell proliferation (Figures 1, S1; experimental schemes B and C). Since the βVEGF-A islet 54 microenvironment is a complex in vivo system involving dynamic changes in islet cell 55 composition, we also identified regenerative signals by performing transcriptome analysis 56 (Figures 1, S1; experimental scheme D) of purified islet cell populations including β cells, ECs, 57 and MΦs over the course of β cell loss and recovery. Based on previous work 1, 16 we predicted 58 that either MΦ depletion (clodronate) or loss of VEGFR2 signaling in ECs would perturb MΦ 59 recruitment and polarization and impair β cell regeneration. Indeed, we found that MΦ depletion 60 suppressed the transient burst in β cell proliferation leading to reduced recovery of β cell mass. 61 demonstrating that clodronate treatment and MΦ depletion did not influence intra-islet EC 91 expansion and hypervascularization-induced β cell loss. In contrast, MΦ depletion significantly 92 impaired β cell proliferation during the recovery period (8.5 vs. 2.1%, p<0.001; Figure 2G) and 93 resulted in reduced β cell area compared to controls after 6 weeks of VEGF-A normalization 94 ( Figure 2F), demonstrating that MΦs are required for the regenerative response in βVEGF-A 95 islets. Interestingly, β cell area is slightly but significantly increased one week after Dox 96 withdrawal ( Figure 2F, 1wk WD) in clodronate-treated βVEGF-A mice before ultimately the 97 impaired β proliferation leads to reduced β cell area 6 weeks after VEGF-A normalization. This 98 finding suggests that MΦs play an important role in islet remodeling and composition in βVEGF-99 A mice separate from their effect on β cell proliferation, most likely through their function as 100 phagocytes. 101

Proliferative ECs are required for MΦ polarization and maximal MΦ recruitment 102
To investigate the contribution of ECs to β cell loss and recovery in the βVEGF-A model, we first 103 created a mouse line in which VEGFR2, which is enriched in ECs of islet capillaries and the 104 main transducer of VEGF-A signal in islets, is inactivated by tamoxifen (Tm)-inducible Cre-105 mediated excision in ECs. We initially tested the EC-SCL-CreER T transgene 24 , which contains a 106   5' endothelial enhancer for the stem cell leukemia (SCL) transcription factor, and although Cre 107 activity was confirmed in islet ECs using the Gt(ROSA)26Sor tm1Sor reporter strain 25 , VEGFR2 108 expression was unchanged in EC-SCL-CreER T ; VEGFR2 fl/fl mice (data not shown). Fortunately, 109 were able to obtain the Cad5-CreER T2 line 26 and achieved efficient EC-specific VEGFR2 110 knockdown in Cad5-CreER T2 ; VEGFR2 fl/fl (VEGFR2 iΔEC ) mice. Pancreata harvested after 3 111 doses of Tm were evaluated to confirm VEGFR2 ablation (Figures S3A and S3B) without 112 significant changes in islet capillary density or size (Figures S3C and S3D) or basal β cell 113 proliferation (Figure S3E) To determine the effect of proliferative ECs on MΦ recruitment and polarization, Tm was 122 administered to knock down VEGFR2 (R2) in ECs prior to VEGF-A induction in β cells ( Figures  123   4A and S4A). VEGF-A was induced in both control βVEGF-A; R2 fl/fl and βVEGF-A; R2 iΔEC 124 genotypes, with efficient knockdown of VEGFR2 in the latter ( Figure S4B)

VEGFR2 inactivation in quiescent ECs accelerates EC regression, enhancing β cell 142 recovery 143
To investigate the role of VEGFR2 signaling in quiescent ECs during β cell recovery, VEGF-A-144 mediated EC proliferation and β cell loss was first induced with 3-day Dox treatment in βVEGF-145 A; R2 iΔEC mice and controls, followed by 7 days of Dox withdrawal to allow VEGF-A to normalize 146 and ECs to return back to quiescence (Figures 4A, S5A). Islet phenotype and β cell 147 proliferation was assessed after 7 days of VEGF-A normalization (7d WD) and subsequently 2 148 Since VEGFR2 inactivation in quiescent ECs of βVEGF-A; R2 iΔEC mice leads to a relatively rapid 159 EC decline after Tm treatment (9d WD), we next evaluated the islet vascular regression by 160 visualization of collagen IV, a major component of the islet ECM 32,33 generated by intra-islet 161 ECs. This study revealed that regressing islet capillaries leave behind vascular "casts" of ECM 162 that are no longer associated with intact ECs (Figure 4F, 9d WD). Even more surprising was 163 our finding that this accelerated decline in islet ECs enhances β cell proliferation at 9d WD 164 ( Figure 4G). We hypothesize that in contrast to β cell homeostasis, the regression of quiescent 165 ECs during the β cell recovery phase stimulates release of growth factors from degraded ECM, 166 thereby promoting β cell proliferation ( Figure 4H). 167

Identifying interactions between β cells, ECs, and MΦs in the βVEGF-A islet
Tnf    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 Table  S1. FGF, fibroblast growth factor; HGF, hepatocyte growth factor; ILK, integrinlinked 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.
At the peak of regeneration (1wk WD), β cells highly express integrins and other molecules that 205 sense and respond to changes in the extracellular milieu ( Figure 5B). Activation of integrin-206 mediated signaling and the integrin-linked kinase pathway were accompanied by increases in 207 PI3K/Akt and MAPK signaling genes (Figures S8A and 5C), many of which are known 208 modulators of β cell proliferation [34][35][36][37] . Transcriptional activators Cdk6 and Foxm1 are initially 209 upregulated more than 3-fold compared to baseline, followed by downregulation of suppressor 210 Cdkn2c (p18) specifically during β cell recovery, consistent with previous studies of both mouse 211 and human β cells [38][39][40] . Creb5 and Ets1 are notably increased as well. Collectively, these data 212 provide evidence for a model where β cell proliferation is largely driven through MΦ-EC-β cell 213 paracrine signaling, ECM remodeling, and cell-matrix interactions (Figure 6). 214 Figure 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. increasing number of studies observing tissue-restorative effects of MΦs triggered in various β 248 cell injury models 11,12,47 . Further studies will be necessary to clarify the specific signals that 249 regulate the phenotypic shift and define the cues that govern their withdrawal. 250

Role of endothelial cells 251
Regulated EC-derived signals are necessary for pancreatic and islet development 17,48-50 , 252 optimal adult β cell function 18 , and islet revascularization after transplantation [51][52][53][54] . In addition, 253 ECs and VEGFR2 signaling have been previously implicated in organ regeneration 13,16 . survival in vitro 20,55 , we hypothesize that ECM changes associated with rapidly expanding 266 endothelium likely contribute to β cell loss in the βVEGF-A system. In addition, we show that 267 intact VEGFR2 signaling is necessary for accumulation of intra-islet MΦs expressing CD206, a 268 marker of an M2 phenotype. This observation is consistent with MΦ activation by EC-derived 269 cues in the context of acute injury, a phenomenon that has been noted in numerous tissues 56-58 . 270 Furthermore, a similar M2 phenotype (high IL-10, CD206) was shown to be required for β cell 271 regeneration after DT-mediated ablation 12 , and elsewhere CD206 has been linked to increased 272 expression of Tgfb1 and Egf 59 and promotion of β cell proliferation via a Smad7-cyclin 273 pathway 11 . 274 In contrast to lung and liver regeneration 13,16 , VEGFR2 inactivation in quiescent ECs (1wk WD) 275 resulted in accelerated β cell recovery, which is associated with rapid islet capillary regression, 276 leaving behind vascular "casts" of ECM components. We postulate that β cell proliferation is Remodeling of extracellular milieu can also influence MΦ phenotype 67,68 , though more work is 298 required to define specific signaling pathways that regulate this process in the pancreas. 299 However, our transcriptome data provides evidence that both MΦs and ECs are quite attuned to 300 their rapidly changing environment, with integrins and cell adhesion molecules being some of 301 the most dynamically regulated genes in both cell types. Changes in these molecules are likely 302 regulating MΦ recruitment and/or polarization [69][70][71][72] , which may explain the phenotypic shift that 303 happens between MΦ recruitment (occurring rapidly in response to VEGF-A) and the 304 appearance of β cell proliferation (during VEGF-A normalization). 305 It is also possible that β cells undergo intrinsic changes heightening their sensitivity to 306 extracellular signals, supported by the observation that ECs and MΦs increase expression of 307 several growth factors known to promote β cell proliferation (IGF-1, PDGF, and CTGF) 37,73-75 308 while β cells simultaneously upregulate expression of corresponding receptors (Igf1r, Pdgfr). 309 16 Signaling cascades activated by integrins and growth factors exhibit extensive downstream 310 crosstalk and protein activity that makes it difficult to determine pathway activation status based 311 solely on gene expression. Nonetheless, we did observe transcriptional changes to components 312 of the PI3K/Akt, PLC, and MAPK pathways, as well as upregulation of transcription factors 313 regulated by the MAPK pathway [34][35][36][37] suggesting that the cell-cell and cell-ECM interactions may 314 converge on the activation of these pathways leading to β cell proliferation. We therefore 315 propose a model in which coordination of growth factors -whose bioavailability is likely 316 modulated by ECs and MΦs -together with increased integrin signaling promotes activation of 317 pro-proliferative pathways in surviving β cells during VEGF-A normalization to ultimately restore 318 β cell mass (Figure 6).  Table S2.  Table S3, crosses A1-A2). Heterozygous VEGFR2 fl/wt 348 mice on a C57BL/6 background were obtained from Jackson Laboratories (stock #018977) and 349 bred to create a homozygous VEGFR2 fl/fl line. Frozen sperm from the Cd5-CreER line 26 Table S4. DNA was 371 extracted and PCR reactions were performed with tail snips from mice as described previously 1 . 372 Thermal cycler conditions listed in Table S4 were