Inflammatory neovascularization during graft-versus-host disease is regulated by α_v integrin and miR-100

The connection between inflammation and neovascularization has been demonstrated for different diseases^1,2  including graft-versus-host disease (GvHD).^3,4  However, the molecular and cellular mechanisms mediating neovascularization and the contribution to inflammation following allogeneic hematopoietic cell transplantation (allo-HCT) remain unresolved.

A central event in the early phase of GvHD following allo-HCT is the toxic damage of different recipient tissues.⁵  We have recently demonstrated a GvHD promoting role for the release of intracellular adenosine triphosphate,⁶  which is converted by ecto-5′-nucleotidase⁷  to metabolites such as adenosine diphosphate with known proangiogenic function.⁸  During this early phase, endothelial cells (ECs) are injured by chemotherapy,⁹  irradiation,¹⁰  and immunosuppressive drugs such as mammalian target of rapamycin (mTOR) inhibitors¹¹  and calcineurin inhibitors.¹²  Toxic damage is followed by inflammatory neovascularization that contributes to GvHD³  by a yet unknown mechanism. In a variety of disease models, the process of neovascularization was shown to be determined by different micro RNAs (miRs)^13,14  that have been identified as important regulators of gene expression in a wide range of organisms and biological systems.¹⁵  miRs are short, noncoding, single-strand RNA molecules that are transcribed as precursor molecules and are later processed into mature miRs. They control the expression of genes predominantly by translational repression and can negatively^13,16  and positively¹⁷  regulate neovascularization by impacting signaling in ECs. miRs are therefore potential targets for therapies that aim to enhance or suppress neovascularization.

Another regulatory checkpoint of neovascularization is the interaction of ECs with extracellular matrix molecules (ECMs) via integrins.¹⁸  Basement membrane deposition and mechanical cues from the ECMs are transmitted via integrins to coordinate vessel sprouting and remodeling.¹⁹  Because of their transmembrane structure and their ability to bind to extracellular ligands, integrins function as central mediators during the neoangiogenic process.²⁰ 

By using positron emission tomography (PET) imaging of α_vβ₃ integrin activity as a novel technique to localize and quantify ECs during murine GvHD, we could visualize neovascularization kinetics in the intestinal tract (IT) following allo-HCT. We show that inflammatory neovascularization can be inhibited by targeting α_v integrin, which reduced GvHD severity. By using expression analysis of candidate miRs in the inflamed IT of mice developing GvHD, we identified miR-100 downregulation and demonstrated that inactivation of miR-100 enhanced GvHD. Therefore, we provide the first evidence that intestinal neovascularization during GvHD shares pathomechanistic features with adaptive blood vessel growth as it occurs in response to ischemia after arterial occlusion, where miR-100 functions as an endogenous repressor.

We collected all samples after approval by the ethics committee of the Albert-Ludwigs-University, Freiburg, Germany (protocol number: 267/11), and after written informed consent in accordance with the Declaration of Helsinki. Intestinal tissue biopsies were collected in a prospective manner from individuals undergoing allo-HCT with or without GvHD (supplemental Table 1; see the Blood Web site). GvHD grading was performed on the basis of histopathology. Human intestinal biopsies were stained for CD31 (clone: 1A10; Leica Microsystems) and α_v integrin (polyclonal; Sigma-Aldrich).

C57BL/6 (H-2^b, Thy-1.2) and BALB/c (H-2^d, Thy-1.2) mice were purchased either from Charles River Laboratory (Sulzburg, Germany) or from the local stock of the animal facility at Freiburg University. Recipients between 6 and 12 weeks of age and only gender-matched combinations were used for transplant experiments. The luciferase (luc⁺) transgenic C57BL/6 mice have been previously described.²¹  The animal protocols (G-10/62, X-10/13H) were approved by the University Committee on the Use and Care of Laboratory Animals at Albert-Ludwigs-University, Freiburg, Germany.

BMT experiments were performed as previously described.²²  Briefly, recipients were injected intravenously with 5 × 10⁶ bone marrow (BM) cells after lethal irradiation with 900 centi Gray (cGy). To induce acute GvHD (aGvHD), 3 × 10⁵ CD4⁺/CD8⁺ T cells (Tc’s) were given (C57BL/6→BALB/c) intravenously on day 0. Slides of small- and large-intestine samples collected on day 7 were stained with hematoxylin/eosin and scored by experienced pathologists (U.V.G., A.S.G.) blinded to the treatment groups on the basis of a published histopathology scoring system.²³ 

BM-derived DCs were prepared as described,²⁴  except that interleukin (IL) 4 was not included in the culture.

Tc proliferation in vitro and in vivo was performed as previously described.²² 

To investigate graft-versus-leukemia (GvL) activity of transferred donor Tc’s, we employed A20^luc B-cell leukemia.²⁵  Animals were injected with A20^luc cells (BALB/c background, 5 × 10⁵) 2 days prior to administration of the Tc’s from C57BL/6 donors.

Cilengitide (EMD 121974) [cyclo Arg-Gly-Asp-D-Phe-(N-methyl)-Val; EMD 121974] is an antagonist selective for α_vβ₃ and α_vβ₅ integrins, with 50% inhibitory concentration values in the low nanomolar range for isolated α_vβ₃ integrins and was provided by Merck KGaA. The dosage administered intraperitoneally was 75 mg/kg twice daily from day –1 to day +10 after allo-HCT according to previous studies.²⁶  Phosphate-buffered saline (PBS) was administered to the animals as control group.

In vivo BLI was performed as previously described.²²  Imaging data were analyzed and quantified with Living Image 3.0 Software (Calipers).

[⁶⁸Ga]NODAGA-cyclo(Arg-Gly-Asp-D-Phe-Lys) was synthesized and labeled in the Department of Nuclear Medicine, Freiburg University Medical Center, as previously described.²⁷  This peptide is structurally similar to cilengitide and has a comparable affinity to α_v integrins as cilengitide.²⁸ 

Six to 12 MBq/0.6 nmol per 0.1 mL of [⁶⁸Ga]NODAGA-c(RGDfK) was injected via lateral tail vein. One hour after injection, mice were scanned for 30 minutes using the small animal PET scanner microPET Focus 120 (Concorde Microsystems). PET images were reconstructed with the 2D Ordered Subset Expectation Maximization algorithm provided by the scanner software. The resolution of the images ranges between 1.1 and 2.5 mm depending on the distance from the center of the field of view. Image counts per pixel per second were calibrated to activity concentrations (Bq/mL) by measuring a ⁶⁸Ga cylindrical phantom filled with a known concentration of radioactivity.

To determine tracer concentration in various tissues, cylindrical regions of interest were placed in the abdominal region that exhibited the highest radioactivity using amide Software 0.9.2. Tracer uptake in the investigated mice is expressed as percent of the decay-corrected injected dose per gram of tissue. Previous studies have shown that uptake of [⁶⁸Ga]NODAGA-c(RGDfK) correlates closely with the expression of activated α_vβ₃ integrins.²⁷ 

Effector Tc’s were isolated from BALB/c recipients treated with cilengitide or PBS as control on day 14 after allo-HCT by depletion of non-CD3⁺ Tc’s (Miltenyi Biotec) and cocultured for 6 hours at 37°C with L1210 target cells at the indicated ratio of target:effector cells. Afterward cells were stained with annexin V–fluorescein isothiocyanate/propidium iodide (PI), and cytotoxicity was analyzed by flow cytometry.

RNA was isolated from intestines of untreated mice on day 14 after allo-HCT. A more detailed description can be found in the supplemental data (ArrayExpress accession number: E-MEXP-3807).

Antagomirs were designed as previously described¹³  and custom synthesized (Biozym). Antagomir sequences were as follows: antagomir-100 (antag-100), 5′-cacaaguucggaucuacggguu-3′; antagomir control 2 (antag-cont2), 5′-caccaguuaggcucuacggauu-3′. Antagomirs were administered via tail vein injection of 8 mg/kg antagomir on days 0, 3, and 9 after allo-HCT.

Total RNA was isolated from the small intestine of BALB/c recipients on days 0, 2, 6, and 12 after allo-HCT using miRNeasy MiniKit (50) (Qiagen). MiR-100 expression was measured by quantitative stem-loop PCR technology (TaqMan MicroRNA Assays; Applied Biosystems) as previously described.¹³ 

We adapted the protocol as previously described²⁹  with modifications. After surgically removing and flushing the intestine, the tissue was next incubated in 1 mM dithiothreitol (Sigma-Aldrich). After washing with PBS, pieces were incubated 4 times in 5 mM EDTA (Merck) prior to a digestion step in 0.1% collagenase (Invitrogen), 2.5 U/mL dispase (Invitrogen), and 1 mg/mL DNaseI (Sigma-Aldrich) for 1 hour at 37°C.

Flow cytometry analysis was performed with a CyanADP (Beckman Coulter) and analyzed with FlowJo 7/8 software (Tree Star).

The following monoclonal antibodies purchased from BD Bioscience, BioLegend, eBiosciences, or AbD Serotec were used: B220 (RA3-6B2), CD4 (GK 1.5/RM4-5), CD8 (53-6.7), CD11c (HL3/N418), CD11b (M1/70), CD19 (6D5), CD45 (30-F11), CD31 (390), CD34 (MEC14.7) (HM34), CD40 (3_23), CD44 (IM7), CD51 (RMV7), CD62L (MEL-14), CD80 (16-10A1), CD86 (GL-1), CD107a (1D4B), epithelial cell adhesion molecule (EPCAM) (G8.8), F4/80 (CI:A3-1), Foxp3 (FJK-16s), Gr-1 (RB6-8C5), H-2K^b (AF6-88.5), H-2K^d (SF1-1.1), I-A^d (SF1-1.1), interferon γ (IFN-γ) (XMG1.2), IL-4 (11B11), IL-17A (TC11-18H10), and NK1.1 (PK136). The intracellular staining for Foxp3 and cytokines and for effector memory Tc’s (T_EM’s), central memory Tc’s (T_CM’s), and naive Tc’s (T_N’s) was performed as previously described.²² 

The murine tissues were stained with anti-CD34 (clone: MEC14.7; BioLegend). Streptavidin-conjugated horseradish peroxidase and the diaminobenzidine-system (Dako) were used for visualization. For quantification of vessel density, murine small intestines were stained with biotin rat anti-mouse antibody CD31 (clone: MEC13.3; BD Biosciences) and visualized with streptavidin-conjugated AlexaFluor488 (Invitrogen). Ten sections per sample were investigated, and vessel density was determined by quantification of CD31⁺ area/total area using a Zeiss LSM 710 laser scanning microscope and Adobe Photoshop CS5 software.

Sixty milligrams per kilogram of pimonidazole HCl (Hypoxyprobe Inc.), which selectively binds to oxygen-starved cells,³⁰  was administered orally to mice. After 1 hour, animals were euthanized and intestine was isolated and fixed with 4% formaldehyde (Riedel-de Haen). Immunohistochemistry of paraffin sections was performed according to the manufacturer’s protocol.

The serum of BALB/c recipients was collected on day 7 after allo-HCT, and the levels of the indicated cytokines were analyzed with the Cytometric Bead Array (CBA) Inflammation kit (BD Bioscience).

Data are reported as mean values ± standard deviation (SD). We compared pairs by the Student t test. Differences in animal survival were analyzed by log-rank test. A P value < .05 was considered statistically significant.

We first aimed to determine the kinetics and anatomical localization of neovascularization at serial time points following allo-HCT. Flow cytometry–based evaluation of the inflamed IT revealed that allo-HCT itself caused increased numbers of CD34⁺ and CD31⁺ ECs, which was further enhanced when donor Tc’s were added to the graft and GvHD evolved (Figure 1A). We found no significant increase of CD31⁺ cells in the IT of syngeneic BMT recipients compared with untreated mice or animals undergoing allo-HCT without Tc’s (Figure 1A). Compatible with the observation that neovascularization took place in the IT following allo-HCT, we detected increased RNA transcript levels of vascular endothelial growth factor and fibroblast growth factor in the IT of mice with GvHD as compared with BM controls (Figure 1B). [⁶⁸Ga]NODAGA-c(RGDfK)–based PET imaging demonstrated a significantly higher signal in mice that had undergone allo-HCT with or without Tc’s (BM or BM/Tc) as compared with untreated animals (Figure 1C), indicating increased expression of activated α_v integrin. Regions of the higher PET signal projected over the abdominal area, which indicated EC accumulation in the IT (Figure 1C). Collectively, these data indicate that the IT is a central location for neovascularization that occurs during the development of GvHD.

The interaction of ECs with ECMs can be targeted by cilengitide, a cyclic arginine–glycine–aspartic acid pentapeptide that selectively binds the cell surface receptors α_vβ₃ and α_vβ₅ integrin²⁸  expressed on activated ECs during angiogenesis. The drug induces programmed cell death in angiogenic blood vessels by preventing interaction of their cell surface α_v integrins with specific matrix ligands, such as vitronectin, tenascin, and fibronectin.³¹  To evaluate if intestinal ECs represent a target for cilengitide, we quantified α_v integrin expression on ECs isolated from the IT of mice developing GvHD. The α_v integrin subunit was expressed by a significant number of ECs (Figure 2A), whereas enterocytes were largely negative (supplemental Figure 1A). We found α_v integrin expression on Tc’s, B cells, natural killer cells, and DCs (supplemental Figure 1B). The α_v integrin expression on ECs was not different in untreated animals as compared with mice developing GvHD (supplemental Figure 1C).

In a next step, we studied if the PET-based α_v integrin signal could be saturated by cilengitide treatment. We observed significantly reduced signal intensity, indicating effective saturation of α_v integrin in the whole body by cilengitide (Figure 2B). Cilengitide also reduced the numbers of ECs in the small and large intestines as assessed by immunohistochemistry of the small and large intestines separately (Figure 2C), flow cytometry of the total IT (Figure 2D), and immunofluorescence-based vessel density measurement (Figure 2E). Because hypoxia was shown to trigger neovascularization,³²  we next determined the levels of hypoxia in the IT via pimonidazole hydrochloride, a small-molecule hypoxia marker that selectively binds to oxygen-starved cells.³⁰  The levels of hypoxia in the small bowel were higher as compared with the large bowel (Figure 2F), which may explain why the inhibition of neovascularization via cilengitide was more pronounced at this site. These data indicate that cilengitide reduced intestinal neovascularization during the development of GvHD. Comparable inhibitory effects on the vasculature were also seen in vivo in the solid tumor setting with reduced relative blood flow in tumor tissue after cilengitide therapy.³³ 

Because Tc’s are the major effector cells that cause GvHD, we next aimed to determine the impact of cilengitide on Tc^luc expansion in vivo. Allogeneic Tc^luc’s migrated to secondary lymphoid organs and expanded without a significant inhibition by cilengitide treatment as shown for representative time points (Figure 3A, left panel) or as quantified over time (Figure 3A, right panel). Compatible with the BLI data, cilengitide had no impact on the expansion of carboxyfluorescein diacetate succinimidyl ester (CFSE)–positive Tc’s in vivo (Figure 3B) and in vitro (supplemental Figure 2A). Survival studies in a major histocompatibility complex disparate GvHD model indicated that cilengitide prolonged survival following allo-HCT as compared with PBS treatment (Figure 3C) without an impact on Tc viability (Figure 3D; supplemental Figure 2B). Cilengitide did not affect the frequency of CD4⁺ T_N’s, T_CM’s, or T_EM’s (Figure 3E). There was also no difference in the abundance of T helper (Th)1, Th2, or Th17 Tc’s (Figure 3F); Foxp3⁺ Tc’s (supplemental Figure 2C); or intestinal granulocytes, macrophages, or monocytes (supplemental Figure 2D) between cilengitide- and PBS-treated groups. The absolute numbers of Tc’s in the spleen were not different in vehicle-treated compared with cilengitide-treated recipients (supplemental Figure 2E). In addition, the phenotype of Tc’s exposed to allogeneic DCs in vitro was independent of α_v integrin inhibition by cilengitide (supplemental Figure 2F). There was no impact of cilengitide on the expression of costimulatory molecules on DCs in vitro (supplemental Figure 2G) or in vivo (supplemental Figure 2H) nor the ability of DCs to stimulate proliferation of allogeneic Tc’s (supplemental Figure 2I).

Consistent with its impact on survival, cilengitide treatment led to reduced GvHD severity by decreasing the apoptosis and inflammation-based GvHD histopathology score for the small and large intestines and liver (Figure 3G) and the production of proinflammatory tumor necrosis factor (TNF) (Figure 3H). Because previous studies had shown that inhibition of angiogenesis by anti–vascular endothelial growth factor receptor (VEGFR) 1/anti-VEGFR2 antibodies impaired hematopoietic reconstitution,³  we next studied engraftment in mice that received cilengitide, which was independent of cilengitide treatment of different lymphoid and myeloid lineages (Figure 3I).

Effector function of cytotoxic T lymphocytes is required to reject tumor cells following allo-HCT. To probe in vitro cytolytic activity of Tc’s against L1210 target cells, Tc’s were isolated from PBS- or cilengitide-treated BALB/c recipients on day 14 after allo-HCT. Cytolytic activity of Tc’s was found to be independent of cilengitide treatment (Figure 4A). Expression of CD107a in CD8⁺ Tc’s isolated after allo-HCT was comparable in recipients treated with cilengitide vs PBS (Figure 4B-C). In a further step, the A20^luc lymphoma rejection model³⁴  was used to study if in vivo antitumor activity was maintained during cilengitide treatment. Where indicated, continuous PBS or cilengitide treatment was given. Serial BLI images demonstrated leukemic BM and organ infiltration at different time points after transplantation in the BM/A20^luc and BM/A20^luc+cilengitide group. Animals that received BM/A20^luc+Tc, independent of treatment with PBS or cilengitide, achieved tumor control as determined by loss of tumor signal (Figure 4D) and quantified by flow cytometry for B220^high A20 leukemic cells in BM and lymph nodes (LNs) (Figure 4E), which is indicative of an intact in vivo effector function of CD8⁺ Tc’s. The survival of A20 lymphoma-bearing mice was significantly prolonged when cilengitide treatment was given (Figure 4F). The improved survival was due to the impact on GvHD, whereas there was no evidence that GvL effects were enhanced by cilengitide. Cilengitide treatment had no direct effect on the survival of animals receiving BM/A20^luc without Tc’s (Figure 4E). These data indicate a significant impact of cilengitide on GvHD severity without impacting Tc expansion, antitumor immunity, and immune reconstitution after allo-HCT.

To clarify if the observed neovascularization in mice developing GvHD could also be detected in human GvHD tissues, we investigated patient samples with or without GvHD of individuals having undergone allo-HCT and receiving diagnostic colonoscopy (supplemental Table 1). Conditioning and GvHD prophylaxis were restricted to 2 major protocols previously reported.^35,36  The endothelial marker CD31 was used to determine the level of neovascularization. We found that the severity of intestinal GvHD determined on the basis of histology was strongly correlated with the number of ECs in GvHD lesions as shown for representative cases (Figure 5A) and when all patients were analyzed (Figure 5B). Importantly, the intestinal human ECs displayed high α_v integrin expression (Figure 5C), and α_v integrin expression was significantly correlated with the severity of GvHD (Figure 5D). These data indicate that neovascularization in inflamed lesions is a feature of human acute intestinal GvHD and based on their α_v integrin expression could be targeted by cilengitide, which is already in use for the treatment of glioblastoma in humans.³⁷ 

Because tissue ischemia due to atherosclerotic arterial occlusion or narrowing, which is regulated by miRs,¹³  and a hypoxic microenvironment of the inflamed IT may share common features, we next studied the role of different miRs in the small bowel by miR array to identify candidates for further analysis (data not shown). MiRs isolated from the small bowel of untreated mice vs mice developing GvHD showed that miR-100 was downregulated on days 2, 6, and 12 after allo-HCT as quantified by qRT-PCR (Figure 6A), compatible with reduced suppression of neovascularization by miR-100 when GvHD evolved. MiR-100 is a previously reported negative regulator of neovascularization in murine vascular ligation–induced ischemia models.¹³  By using miR-100–specific antagomir treatment in a model of delayed GvHD, we observed that blocking the function of miR-100 enhanced GvHD with reduced survival (Figure 6B) and increased neovacularization in the small and large intestines (Figure 6C). The group that received miR-100 antagomir and cilengitide had a significantly improved survival as compared with the group that received miR-100 antagomir only (P < .001) (Figure 6B). In line with reduced survival, the blockade of miR-100 led to higher GvHD histopathology scores (Figure 6D) and increased levels of proinflammatory IL-6, IFN-γ, monocyte chemoattractant protein 1, and TNF (Figure 6E) as compared with the group that received the nonfunctional antag-cont2. These data support the concept that neovascularization is not only a bystander effect of intestinal inflammation but significantly contributes to the pathophysiology of the disease and that antagonizing its negative regulator miR-100 enhances disease severity.

aGvHD is a major complication of allo-HCT and significantly contributes to mortality and morbidity associated with this treatment option. A better understanding of the pathomechanistic features of GvHD is critical to improve the outcome of allo-HCT. Recent studies had identified neovascularization to occur during aGvHD.³  Here we report that inflammatory neovascularization observed during GvHD is regulated by α_v integrin and miR-100 and can be targeted by α_v integrin blocking or enhanced by miR-100 antagonism.

We first clarified when and where neovascularization takes place during GvHD. To monitor this process under real-time in vivo conditions, we used PET-based whole body imaging and observed that following transplantation, neovascularization was most abundant in the IT, occurred already on day 2 after allo-HCT, and continued for the following weeks until the death of the animals. The results were verified by more conventional methods using immunohistochemistry and flow cytometry. As the interaction of ECs with ECMs via integrins is crucial during neovascularization,^18-20  we used cilengitide, an inhibitor already in clinical use for the inhibition of angiogenesis in anticancer therapy.³⁷  Our studies showed that development of aGvHD required α_ν integrin function because the survival of mice developing GvHD was significantly improved by cilengitide treatment independent of Tc expansion. The advantage of this preventive approach over conventional immunosuppressive therapy may be that Tc function is not targeted. Compatible with this concept, we observed intact GvL effects and immune reconstitution when α_ν integrin was inhibited. Previous work has identified different integrins such as β₂³⁸  and β₇ integrin³⁹  as critical mediators in the ability of Tc’s to induce GvHD. Conversely, α_ν integrin was never found to be involved in allogeneic Tc function despite a well-described role in neovascularization.¹⁸ 

To apply the concept of α_ν integrin inhibition to patients development aGvHD, it was important to study neovascularization in the IT of humans undergoing allo-HCT. We observed a strong correlation between the number of CD31⁺ ECs in the intestines of individuals that had undergone allo-HCT and the extent of aGvHD. We found that comparable to the mouse, human ECs located in intestinal GvHD lesions displayed high α_v integrin expression, indicating that these cells could be targeted by cilengitide.

To better understand the regulation of neovascularization during GvHD, we tried to identify pathomechanistic parallels with other conditions causing neovascularization. Reactive neovascularization is a key feature of ischemia found during coronary artery disease and peripheral arterial disease. Recent studies have shown that miRs negatively^13,16  or positively¹⁷  regulate ischemia-induced neovascularization by impacting signaling in ECs. We found that miR-100 was downregulated when aGvHD evolved, suggesting its role as a negative regulator that is degraded after transplantation and then permits the development of GvHD. Functional inactivation of miR-100 with antagomir treatment enhanced GvHD severity, indicating a protective role for miR-100 by blocking inflammatory neovascularization during GvHD. The role of miR-100 had only been previously reported for adaptive neovascularization following arterial occlusion¹³  but not for inflammatory neovascularization. This common feature may be explained by hypoxia, which occurs during ischemia and which is also a key feature of the inflamed microenvironment in different inflammatory diseases.^40,41  The presence of hypoxia in the human synovium of patients with active rheumatoid arthritis (RA) was shown using microelectrodes,⁴²  and a comparable finding was made in inflamed joints when using a rat model of RA.⁴³  Another inflammatory process connected to hypoxia is wound healing. Wounds exhibit areas of marked hypoxia due to lack of perfusion caused by vascular damage and the intense metabolic activity of cells infiltrating the wound.⁴¹  Oxygen tension measurements in a rodent model showed that immediately postinjury, wound oxygen tension was significantly reduced,⁴⁴  indicating that hypoxia in inflamed areas and production of hypoxia-regulated factors promote angiogenesis during inflammatory wound healing.⁴⁵  These studies support our concept for common regulatory mechanisms of neovascularization by hypoxia during ischemia and inflammation related to GvHD. Importantly, the inflammatory neovascularization had a negative impact on GvHD-related mortality and proinflammatory cytokine production, which proves its functional contribution to the disease.

Our findings on the occurrence of neovascularization during GvHD in conjunction with data derived from a variety of inflammatory diseases including asthma,⁴⁶  RA,^47-49  skin inflammation,⁵⁰  and inflammatory bowel disease⁵¹  suggest that inflammation and neovascularization are functionally connected. Another study identified α_ν integrin to be required in DCs for Th17 cell differentiation in experimental autoimmune encephalomyelitis,⁵²  whereas we observed no effect of α_ν integrin inhibition on Tc’s and DCs in the GvHD model. This observation could be due to model-specific differences in particular alloreactivity vs autoimmunity and the fact that the other group used a genetic model,⁵²  whereas we applied a pharmacologic α_ν integrin inhibitor. We could expand on the previous findings by demonstrating that neovascularization can be targeted via α_νβ₃ integrin antagonism, and we identified miR-100 as a central negative regulator of this process.

In summary, we provide the first in vivo evidence that α_v integrin is upregulated on ECs participating in intestinal neovascularization during GvHD in the mouse and corresponding expression in human GvHD lesions. Our findings indicate that inflammatory neovascularization is a central event during GvHD and can be inhibited by targeting α_v integrin–expressing ECs. Mechanistically, the process of neovascularization is negatively regulated by miR-100, which indicates pathophysiological similarities between ischemic and inflammatory neovascularization.

The authors thank Sophie Krüger and Volker Schmidt for excellent technical assistance, and Merck Serono AG for valuable suggestions and for providing cilengitide.

Merck Serono has reviewed the publication, and the views and opinions described in the publication do not necessarily reflect those of Merck Serono.

This work was supported by the Deutsche Forschungsgemeinschaft, Germany (SFB850 [R.Z. and W.A.W.], Z2 project; Heisenberg Fellowship, DFG ZE 872/2-1 [R.Z.]; and Gr3459/2-1 [S.G.]), and by a Comprehensive Cancer Center Freiburg seeding grant (R.A.D., R.Z., W.A.W.).

Contribution: F.L. helped to design the experiments, performed experiments, and helped to write the manuscript; S.G. and F.B. helped to design and perform miR-100–related experiments and helped with the manuscript; M.B., R.A.D., and F.B. planned and performed PET imaging experiments; M.F. synthesized and labeled [⁶⁸Ga]NODAGA-c(RGDfK); G.P. and A.K.H. helped with mouse experiments and flow cytometry; K.R. and O.P. analyzed the vessel density in the IT of mice by microscopy; H.D. helped with immunohistochemistry; U.V.G. and A.S.G. performed histologic analysis; J.F. and W.A.W. helped to design experiments and discussed data; and R.Z. developed the overall concept, designed experiments, analyzed data, and wrote the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interests. A patent for the application of cilengitide for GvHD prophylaxis or treatment is pending (Merck AG).

Correspondence: Robert Zeiser, Department of Hematology and Oncology, Freiburg University Medical Center, Albert-Ludwigs-University, Hugstetterstr. 55, 79106 Freiburg, Germany; e-mail: robert.zeiser@uniklinik-freiburg.de.