Bone marrow derived cells in collateral formation : studies toward therapeutic arteriogenesis
Hellingman, A.A.
Citation
Hellingman, A. A. (2011, September 15). Bone marrow derived cells in collateral formation : studies toward therapeutic arteriogenesis. Retrieved from https://hdl.handle.net/1887/17838
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Chapter 6
Protease-Activated Receptor-2 rather than Protease-Activated Receptor-1 plays a crucial role in arteriogenesis
A.A.Hellingman1,2 , L.G. van den Hengel1, H.C. de Boer, A.J.N.M. Bastiaansen1,2 , A.M. van Oeveren-Rietdijk1,3, C.A.
Spek4, A.J. van Zonneveld1,3, P.H. Reitsma1, J.F.Hamming2, P.H.A. Quax1,2, H.H. Versteeg1
1Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden
2Dept. of Vascular Surgery, Leiden University Medical Center, Leiden
3Department of Nephrology, University Medical Center, Leiden
4Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
Submitted for publication
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ABSTRACT
Aim
Protease-activated receptors (PARs) constitute a family of receptors and can be stimu- lated by specific proteases. Some clues point towards a role of PAR-2 in arteriogenesis, but the role of PAR-1 in arteriogenesis has not been studied before. In the present study, the role of PAR-1 and PAR-2 in arteriogenesis is studied in a murine model of hind limb ischemia using genetic knockout mice lacking PAR-1 and PAR-2.
Methods and Results
PAR-1-/-, PAR-2-/- and age-matched control mice underwent unilateral electro-coagu- lation of the femoral artery. Blood flow recovery was monitored using Laser Doppler Perfusion Imaging (LDPI) and collaterals were visualized by immunohistochemistry and angiography. Differences in monocytes number and subpopulation composition were characterized for PAR-2-/- mice and control mice by FACS analysis for specific surface markers. No difference in ischemia-induced blood flow recovery in PAR-1-/- mice was observed compared to controls. In contrast, PAR-2-/- mice showed significantly impaired blood flow recovery. Immunohistochemical stainings and angiographs confirmed these differences. FACS-analysis showed less Ly6Clo reparative monocytes in PAR-2-/- mice compared to controls. Importantly, Ly6Clo monocytes from PAR-2-/- mice showed higher CD115 expression indicating a decreased capacity to become activated after hind limb ischemia.
Conclusion
The present study indicates a pro-arteriogenic role of PAR-2 in vivo. In contrast, the involvement of PAR-1 in arteriogenesis has not been demonstrated. Therefore, PAR-2 seems more attractive as a new target for therapeutic arteriogenesis for peripheral arte- rial disease.
INTRODUCTION
Protease activated receptors (PARs) are a family of transmembrane receptors, which can be activated by serine protease-induced proteolytic cleavage of the N terminus. Four PARs have been characterized, PAR-1-PAR-41-3. The physiological activator of PAR-1 is thrombin1, 2, whereas PAR-2, which is resistant to thrombin, can be activated by tissue factor (TF)/FactorVIIa (FVII) complex4, 5, trypsin1, 2, mast cell tryptase6 and factor Xa7.
Several clues suggest that PAR-1 and PAR-2 are involved in neovascularization, although evidence for a direct physiological role is scarce. Thrombin, a key player in the blood coagulation cascade, stimulates many vascular cells such as endothelial cells, smooth muscle cells and platelets8. Furthermore, it regulates the release, expression and activation of a variety of angiogenic mediators such as vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2)9-11 principally through activation of PAR-18, 12. In addition, PAR-2 is widely expressed by the cardiovascular system13-19, mediates endo- thelial cell proliferation17 and promotes vasodilatation18-22 suggesting that it is involved in neovascularization. PAR-2 expression is up regulated by cytokines such as TNF_, IL1`
and LPS that are implicated in neovascularization23. Milia et al.24 assessed the role of PAR-2 on angiogenesis in a hind limb ischemia mouse model. They reported that PAR-2 activation with daily injections of trypsin or PAR-2 activating peptide results in increased capillary density and perfusion recovery in the post-ischemic hind limb.
PAR-1 and PAR-2 are expressed on various immune cells like T-lymphocytes and monocytes25. Although a role for PAR-2 in inflammatory processes has been well docu- mented, the exact mechanism by which it contributes to the inflammatory response is not fully understood. Recent studies indicate that PAR-2 activation may have detri- mental or protective effects on inflammation in various infectious and non-infectious disease models26-34. For example, PAR-2 deficiency resulted in a significant reduction in inflammation and infarct size in a model of cardiac ischemia/reperfusion injury35. Recently, it has been reported36 that neointimal formation after vascular injury showed enhanced leukocyte adhesion that was PAR-2 dependent. Thus, PAR-2 appears to exert pro-inflammatory effects in the injured vessels, resulting in neointimal hyperplasia.
Besides their involvement in atherogenesis, leukocytes play an important and beneficial role in vascular regeneration. This is illustrated by the reduced blood flow recovery and collateral artery outgrowth upon hind limb ischemia in various animal models deficient for T-lymphocytes37-39, monocytes40 and NK cells37. The contribution of PAR-1 and PAR-2 to leukocyte-induced neovascularization is unknown.
These considerations prompted us to study PAR-1 and PAR-2 in neovascularization more into detail and in particular in relation to their role in monocyte activation. In the present study, the contribution of PAR-1 and PAR-2 in post-ischemic arteriogenesis was studied in a hind limb ischemia mouse model using genetic knockout mice lacking PAR-
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1 and PAR-2. Differences in monocyte subsets and activation status were characterized for PAR-2-/- mice, to study the mechanism by which PAR-2 is involved in arteriogenesis.
MATERIAL AND METHODS
Experimental animals
The committee on animal welfare of our institute approved all experiments. Our institu- tion’s ethical policy is strictly confirmed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. For all experiments male mice were used. C57Bl6 PAR-1-/- mice (Jackson Laboratory) and C57Bl6 PAR-2-/- mice (Jackson Laboratory) were used as well as age-matched C57Bl6 mice as controls
Surgical procedure
Before surgery, mice were anesthetized with an intraperitoneal injection of a combina- tion of Midazolam (5mg/kg, Roche), Medetomidine (0.5mg/kg, Orion) and Fentanyl (0.05 mg/kg, Janssen). A small skin incision was made in the left inguinal region. After dissec- tion of the artery from the nerve and vein, ischemia was induced by unilateral single electro-coagulation of the left femoral artery, proximal to the superficial epigastric artery as described before41. After surgery the skin was closed with 6-0 Ethilon sutures.
Laser Doppler Perfusion Imaging
Blood flow was measured before ischemia induction, immediately after ischemia induc- tion and 3, 7, 14, 21 and 28 days after surgery in the ischemic and non-ischemic paws, using Laser Doppler Perfusion Imaging (Moor Instruments). Each animal served as its own control. Perfusion was expressed as a ratio of the ischemic to non-ischemic limb, as described previously41.
Histological analysis
Animals were sacrificed 7 days and 28 days after ischemia induction by cervical disloca- tion and calf and adductor muscles (from ischemic and non-ischemic paw) were removed and fixed with 4% formaldehyde and paraffin-embedded. Serial 5µm cross-sections were generated. Sections were re-hydrated and endogenous peroxidase activity was blocked for 20 minutes in methanol containing 0.3% hydrogen peroxide. Capillaries and collaterals were visualized using antibodies recognizing CD31 (endothelial cells) or α-smooth muscle actin (SMA) (smooth muscle cells) respectively.
For CD31 labeling, sections were pre-incubated with trypsin (30 minutes/ 37°C), incubated overnight with primary antibody (rat anti-mouse CD31Ab, BD Biosciences, dilution 1:200) followed by a biotin-conjugated secondary antibody (goat anti-rat,
AbCam, dilution 1:300). The reaction was enhanced by tyramine amplification and the avidin-biotin-horseradish-peroxidase system (HRP) (DakoCytomation) and visualized by NovaRED (Vector laboratories). For SMA labeling, tissue sections were incubated overnight with anti-αSMA (mouse anti-human, DAKO, dilution 1:800) without antigen retrieval. Labeling was followed by an HRP-conjugated secondary antibody (rabbit anti- mouse, DAKO, dilution 1:300). Anti-mouse CD45 was used to identify monocytes in the ischemic adductor muscle. Sections were pre-incubated with trypsin (30 minutes/ 37°C), incubated overnight with primary antibody (rat anti-mouse CD45Ab, BD Biosciences, di- lution 1:200) followed by a biotin-conjugated secondary antibody (goat anti-rat, AbCam, dilution 1:300). The reaction was enhanced by the avidin-biotin-horseradish-peroxidase system (DakoCytomation) and visualized by DAB (Vector laboratories).
All sections were counterstained with hematoxylin. Isotype control antibodies were used as controls. Quantification of labeled tissue sections was performed using ImageJ (9 section per mouse were analyzed to obtain the mean per animal, 10 animals per group were measured).
Imaging
To visualize collateral vessel development, post mortem angiography of both hind limbs was performed using microfill contrast agent. After thoracotomy, contrast fluid was in- jected into the left ventricle of the mouse heart. Five minutes before contrast injection, mice were intravenously injected with papaverine (50mg/ml) for vasodilatation. The skin of both hind limbs was removed and X-rays were made.
FACS staining of monocytes in PAR-2-/- peripheral blood
Mouse blood was withdrawn from the tail vein in EDTA-containing tubes and incubated with antibodies against Ly6C-FITC (kindly provided by Dr P.J. Leenen, Erasmus University, Rotter- dam, The Netherlands), Ly6G-PE, CD115-biotin and CD11b-APC (all from Pharmingen). In be- tween all steps, cells were washed with PBS containing 1% BSA and 0.05% Na-azide. Binding of CD115-biotin was detected with streptavidin conjugated with PerCP-Cy5.5 (Pharmingen).
Red blood cells were lysed with shock-buffer, cells were fixed with 1% paraformaldehyde (PFA) and measured with an LSRII (Becton Dickinson, Erembodegem, Belgium).
To obtain leukocyte profiles, the following gating strategy was applied: debris was gated out in a forward (FSC)/ side scatter (SSC) plot (Supplemental Figure 1, plot A) and leukocytes minus debris were divided according to their CD11b expression (plot B:
myeloid lineage: CD11b-pos; lymphoid lineage: CD11b-neg). CD11b-pos cells were then selected and using ly6G expression and side scatter (SSC) (plot C), cells were divided in neutrophilic granulocytes (Ly6Ghi/ SSC-hi), eosinophilic granulocytes (Ly6G-neg/
SSC-hi) and a non-granulo population (Ly6G-neg/SSC-low). The non-granulo population was then selected and using their CD11b and CD115 expression (plot D), monocytes
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(CD11b-pos/ CD115-pos) and NK cells (CD11b-med/ CD115-neg) were identified. Next, the monocyte population was selected and using Ly6C expression and forward scatter (FSC) (plot E), classical monocytes (Ly6C-hi/FSC-low), no-classical monocytes (Ly6C-low/
FSC-low) and intermediate monocytes (Ly6C-med/FSC-low) were identified. Going back to the lymphoid lineage (CD11b-neg cell population), using Ly6C-expression (Plot F), lymphocytes (Ly6C-low/FSC-low) and lymphoblasts (Ly6-ly6C-med/ FSC-hi) were identi- fied. For quantification, the percentages of the parent population were used (panel F) or mean fluorescence intensity (MFI) of sub-populations was measured.
Stimulation of monocytes with lipopolysaccharide (LPS)
Peripheral blood of control mice (n=5) was incubated with LPS (50ng/ml) for 3 hours.
FACS staining was performed as described above.
Figure 1.
A. The graph depicts the mean±SEM of blood flow restoration in hind limb of C57Bl6 mice (n=10) and PAR-1-/- mice (n=10). Blood flow was monitored by LDPI and expressed as ratio between operated and non-operated limb. Images on the right depict representative LDPI images of paws of C57Bl6 mice (upper) and PAR-1-/- mice (lower) at 7 days after hind limb ischemia induction.
B. The graph depicts the mean±SEM of blood flow restoration in hind limb of C57Bl6 mice (n=10) and PAR-2-/- mice (n=7). Blood flow was monitored by LDPI and expressed as ratio between operated and non- operated limb. *P<0.05. Images on the right depict representative LDPI images of paws of C57Bl6 mice (upper) and PAR-2-/- mice (lower) at 7 days after hind limb ischemia induction.
C. Histograms depict mean±SEM of the number of collaterals in the post-ischemic adductor muscle of PAR1-/- mice as quantified by smooth muscle actin (SMA) labelling.
D. The histogram depicts mean±SEM of the number of capillaries in the post-ischemic calf muscle of PAR- 1-/- mice as quantified by anti-CD31 labelling.
E. Histograms depict mean±SEM of the number of collaterals in the post-ischemic adductor muscle of PAR-2-/- mice as quantified by smooth muscle actin (SMA) labelling. *P<0.05. Representative photographs of SMA-labelling are shown 7 days after ischemia of C57Bl6 mice (upper) and PAR-2-/- mice (lower).
F. The histogram depicts mean±SEM of the number of capillaries in the post-ischemic calf muscle as quantified by anti-CD31 labelling. *P<0.05. Representative photographs of CD31-labelling are shown 7 days after hind limb ischemia induction in C57Bl6 mice (upper) and Par-2-/- mice (lower).
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Statistical analysis
Results are expressed as mean ± sem. Comparisons between means were performed using an independent T-test. P-values <0.05 were considered statistically significant. All calculations were performed in SPSS 16.0.
RESULTS
PAR-2 rather than PAR-1 contributes to post-ischemic blood flow recovery The role of PAR-1 and PAR-2 on arteriogenesis in a physiological mouse model lacking PAR- 1 and PAR-2 was unknown. This prompted us to study arteriogenesis in genetic knockout mice lacking PAR-1 and PAR-2. PAR-1-/- mice showed fast blood flow recovery up to 70%
at day 14 after hind limb ischemia induction. No differences in blood flow restoration between PAR-1-/- mice and controls were observed during 28 days after ischemia (Figure 1A). In contrast, PAR-2-/- mice showed significantly attenuated blood flow recovery from day 3 until day 28 after ischemia as compared to control mice. Seven days after surgery, restoration of blood flow was already 70% recovered in control mice, whereas only 30%
restoration of blood flow was observed in the treated hind limb of PAR-2-/- mice (Figure 1B).
Post-ischemic neovascularization of PAR-1-/- and PAR-2-/- mice at the tissue level In line with laser doppler perfusion imaging (LDPI)-measurements, PAR-1-/- mice showed no differences in number of collaterals, expressed as SMA positive structures in the post-isch- emic adductor muscle as compared to controls (Figure 1C). In addition, PAR-1-/- mice did not show differences in capillary density of the post-ischemic calf muscles expressed as CD31+ structures as compared to controls (Figure 1D). Analyses of collateral- and capillary density of the non-operated paw did not show differences between groups (data not shown).
Conversely, PAR-2 seemed to be heavily involved in arteriogenesis (collateral artery formation) and angiogenesis (formation of new capillaries) at the tissue level. PAR-2- /- mice showed a significantly lower number of collaterals (SMA+) in the post-ischemic adductor muscle as compared to controls (Figure 1E). Furthermore, PAR-2-/- mice showed a significantly decrease of the capillary density (CD31+) of the post-ischemic calf muscles as compared to controls (Figure 1F). Again, analyses of collateral- and capillary density of the non-operated paw did not show differences between the groups (data not shown).
To visualize collateral growth in the post-ischemic hind limb of PAR-2-/- mice, angio- graphs were made 7 days after hind limb ischemia induction. Solid collaterals were ob- served at the angiographs of post-ischemic adductor muscles of control mice, whereas no collaterals were formed in the post-ischemic adductor muscle of PAR-2-/- mice. This is in line with the results of the SMA-staining (Figure 2).
Less reparative Ly6Clo monocytes in PAR2-/- mice
To assess the impact of PAR-2 deficiency on the composition of circulating myeloid subsets, peripheral blood (PB) monocytes of PAR-2-/- mice and controls were examined by FACS at 7 days after ischemia-induction. At that moment, PAR-2-/- mice showed a trend towards less CD11b+CD115+ monocytes in the PB as compared to controls (not significant) (Figure 3B).
Significant less Ly6Clo monocytes were present in PAR-2-/- mice, indicating that there were less reparative monocytes in PAR-2-/- mice as compared to controls (Figure 3C). In addition, there was a trend towards more Ly6Chi monocytes in PAR-2-/- mice (not sig- nificant), suggesting that monocytes of PAR-2-/- mice showed a classical early monocyte phenotype as compared to controls (Figure 3D).
Activation of PAR-2-/- Ly6Clo monocytes
CD115 expression inversely correlated with the activation of Ly6Clo monocytes as is demonstrated in Figure 3A where the CD115 expression on Ly6Clo monocytes either or not activated by LPS is demonstrated. Activation with LPS leads to a significant down- regulation of CD115. The activation of monocytes was studied using CD115 as marker.
Seven days after hind limb ischemia induction, a strong trend towards increased CD115 surface expression of monocytes of PAR-2-/- mice as compared to controls was reported (P=0.0556), suggesting that monocytes of PAR-2-/- mice had hampered capac- ity to become activated after hind limb ischemia (Figure 3E). CD115 was significantly higher in Ly6Clo monocytes of PAR-2-/- mice, indicating less activation of pro-angiogenic reparative monocytes in PAR-2-/- mice (Figure 3F). For Ly6Chi monocytes, a higher cell surface expression of CD115 was observed too, although not significant as compared to control (Figure 3G).
Figure 2.
Representative angiographs were made 7 days after the surgical procedure of post-ischemic paws of C57Bl6 mice (left) and PAR-2-/- mice (right). In contrast to C57Bl6 mice, the adductor muscle of PAR-2-/- mice did not show any typical cork-screw like collateral as is indicated with the white arrow.
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Presence of monocytes in post-ischemic muscle tissue of PAR-2-/- mice
To demonstrate the actual presence of monocytes in the tissue of the adductor muscle in which collateral formation occurs, the CD45 positive cells were analysed using im- muno-histochemistry with an anti-CD45 antibody. CD45+ cells could be detected near collateral arteries 7 days after ischemia-induction in the adductor muscle of PAR2-/- mice
Figure 3.
A. FACS analyses of peripheral blood of C57Bl6 control mice (n=5) showing CD115 expression and CD11b expression either with or without LPS incubation. CD115 on all subsets of monocytes was significantly downregulated after activation with LPS.
B. Percentage of monocytes (CD11b+CD115+) in non-granulated cell population for C57Bl6 control mice (n=5) and PAR-2-/- mice (n=5).
C. Percentage of Ly6Clo monocytes in monocyte cell population for C57Bl6 control mice (n=5) and PAR-2-/- mice (n=5).
D. Percentage of Ly6Chi monocytes in monocyte cell population for C57Bl6 control mice (n=5) and PAR-2-/- mice (n=5).
E. CD115 expression of Ly6Clo monocytes in C57Bl6 control mice (n=5) and PAR-2-/- mice (n=5).
F. CD115 expression of Ly6Chi monocytes in C57Bl6 control mice (n=5) and PAR-2-/- mice (n=5).
and controls (Figure 4). However, no differences in the number of monocytes present around collateral arteries were found, which is in line with the non-significant differ- ences in monocyte number found by FACS analyses.
DISCUSSION
Although the role of PAR signalling already has been described in the coagulation cas- cade4, 5 and inflammatory and immune responses25, their importance in neovasculariza- tion is poorly understood. In the present study, the contribution of PAR-1 and PAR-2 to post-ischemic neovascularization was studied using a hind limb ischemia model with genetic knockout mouse lacking PAR-1 and PAR-2. This study provides evidence that PAR-2, but not PAR-1 is critical involved in post-ischemic neovascularization.
PAR-2-/- mice, but not PAR-1-/- mice, showed heavily impaired post-ischemic blood flow restoration measured with Laser Doppler Perfusion Imaging (LDPI) indicating that PAR-2 plays an important role in arteriogenesis. A three-fold lower collateral density in the post-ischemic adductor muscle of PAR-2-/- mice confirmed this. These results are in line with the previously reported improved blood flow restoration following PAR-2 activa- tion with PAR-2 activated peptide in a hind limb ischemia mouse model24.
There is ample evidence that monocytes play a key role in collateral artery formation42-44. Recent studies demonstrated that classically activated Ly-6Chi monocytes dominate in tissue in the first four days following ischemia45, 46. In contrast, 5-10 days after ischemia reparative Ly-6Clo monocytes that promote neovascularization make up the major mono- cyte subfraction in the healing tissue45, 47, 48. For PAR-2, a growing body of evidence sug- gests a regulatory role in inflammatory responses26-34. PAR-2 dependent signalling plays an important role in various animal models of infectious and non-infectious diseases leading to inflammation such as arthritis27, inflammatory pain28, skin disorders31, allergic inflammation30, and ischemic brain injury33. In particular the recently reported role in the regulation of cardiac remodelling after ischemia/reperfusion injury35 is interesting. The
Figure 4.
Representative pictures of anti-CD45 staining of adductor muscle 7 days after hind limb ischemia induction. Monocytes are present near collateral arteries in both C57Bl6 mice (left) and PAR-2-/- mice (right).
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question was raised whether altered characteristics of inflammatory cells in PAR2-/- mice may explain the impaired revascularization capacity of PAR2-/- mice. To assess the impact of PAR-2 deficiency on the composition of circulating myeloid subsets, peripheral blood (PB) monocytes of PAR-2-/- mice and controls were examined by FACS at 7 days after ischemia-induction. The present study showed significantly less Ly-6Clo monocytes in PAR2-/- mice as compared to wild type mice one week after surgery. Ly6Clo monocytes propagate repair by the expression of pro-angiogenic growth factors and cytokines45. The reduced number of Ly6Clo monocytes in PB of PAR-2-/- mice after hind limb ischemia induction, may explain the impaired blood flow recovery in PAR-2-/- mice. The activation of the Ly6Clo monocytes can be monitored by the expression of CD115 (Figure 3A). In PAR-2-/- mice, Ly6Clo monocytes had an impaired capacity to become activated 7 days after hind limb ischemia. Previous studies showed that proper pre-stimulation of naïve monocytes is critical to exploit its full vascular regenerative capacity42, 43, 49. Although the present study showed that monocytes are present near collaterals in the post-ischemic adductor muscle of PAR2-/- mice, impaired activated Ly6Clo monocytes in PAR-2-/- mice could probably not assist in post-ischemic neovascularization in the ischemic muscle tissue. Arteriogenesis involves the enlargement of small pre-existent arterioles and shares many mechanistic similarities with inflammatory processes. Several studies have investigated the possible connections between PAR-2 and TLR4-mediated signalling pathways, synergistic cytokine production in inflammatory responses and the TLR/IL-1 receptor domain of TLR4 interacts with the cytoplasmic C-terminus of PAR-250-52. Recent data suggest a unique interaction between these two distinct innate immune response receptors and support receptor cooperatively in inflammatory responses53. For example, concurrent activation of PAR-2 and TLR4 by PAR-2 activating peptide or LPS respectively, amplifies NF-gB activation and IL-6 production in endothelial cells51. Thus, crosstalk between PAR-2 and TLR4 actual can augment inflammatory responses when both re- ceptors are accessible. The disturbance of this crosstalk in PAR-2-/- mice probably also contributes to the observed impairment in arteriogenesis in PAR-2-/- mice. This relates closely to the recently reported impaired arteriogenesis in TLR4-/- mice54.
The pro-arteriogenic action of PAR-1 has not been studied before. The present study showed that PAR-1-/- mice did not have any effect on post-ischemic blood flow recovery and collateral and capillary formation in the post-ischemic skeletal muscle. This was remarkable since PAR-1 is involved in the generation of monocyte chemoattractant protein-1 (MCP-1)55, 56, which is necessary for the recruitment of monocytes and natural killer cells (NK-cells), key players in collateral artery formation37, 40, 42. However, it should be noted that also in cardiac repair, unlike PAR-2, PAR-1 did not seem to be involved in infarct size after ischemic/reperfusion injury35, 57.
In conclusion, the present study supports a critical role of PAR-2 in post-ischemic neo- vascularization. Mice deficient for PAR-2 showed severely impaired blood flow recovery
after hind limb ischemia induction and the formation of collaterals and capillaries was heavily reduced in post-ischemic muscle tissue. Our results points toward a hampered monocyte activation in PAR-2-/- mice that affects the impaired post-ischemic neovascu- larization. Mice lacking PAR-1 did not show any effect on post-ischemic neovasculariza- tion, suggesting that there is no important role of PAR-1 in this process in vivo.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the financial support of the Translational Excellence in Regenerative Medicine (TeRM) Smart Mix Program of the Netherlands Ministry of Economic Affairs and the Netherlands Ministry of Education, Culture and Science.
Conflict-of-interest disclosure
The authors declare that they have no competing financial interests.
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SUPPLEMENTAL FIGURES
Supplemental Figure 1
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