Regenerative capabilities of the endothelium rely on vessel-resident progenitors termed endothelial colony forming cells (ECFCs). This study aimed to investigate if these progenitors are impacted by conditions (i.e., obesity or atherosclerosis) characterized by increased serum levels of oxidized low-density lipoprotein (oxLDL), a known inducer of Endothelial-to-Mesenchymal Transition (EndMT). Our investigation focused on understanding the effects of EndMT on the self-renewal capabilities of progenitors and the associated molecular alterations. In the presence of oxLDL, ECFCs displayed classical features of EndMT, through reduced endothelial gene and protein expression, function as well as increased mesenchymal genes, contractility, and motility. Additionally, ECFCs displayed a dramatic loss in self-renewal capacity in the presence of oxLDL. RNA-sequencing analysis of ECFCs exposed to oxLDL validated gene expression changes suggesting EndMT and identified SOX9 as one of the highly differentially expressed genes. ATAC sequencing analysis identified SOX9 binding sites associated with regions of dynamic chromosome accessibility resulting from oxLDL exposure, further pointing to its importance. EndMT phenotype and gene expression changes induced by oxLDL in vitro or high fat diet (HFD) in vivo were reversed by the silencing of SOX9 in ECFCs or the endothelial-specific conditional knockout of Sox9 in murine models. Overall, our findings support that EndMT affects vessel-resident endothelial progenitor’s self-renewal. SOX9 activation is an early transcriptional event that drives the mesenchymal transition of endothelial progenitor cells. The identification of the molecular network driving EndMT in vessel-resident endothelial progenitors presents a new avenue in understanding and preventing a range of condition where this process is involved.

Targeting endothelial progenitor cells (EPCs) for clinical therapeutics, including vascularization in wound healing, has been hindered by the lack of a uniform EPC definition. Multiple putative EPC populations have been characterized using various genes/surface markers; we hypothesized that overlapping these markers would allow a uniform definition to be validated functionally. We examined the overlap of several of these markers with recently identified endovascular progenitors (EVPs; VE-Cadherin(VECad)+Lineage(Lin)negCD34+CD31lo), shown to have functional progenitor capacity, in homeostatic murine aorta and full-skin excisional wounds. Flow cytometry revealed that EVPs highly co-expressed with Protein C Receptor (PROCR) and Platelet-derived growth factor receptor alpha (PDGFRA) as compared to mature differentiated endothelial (D) cells (VECad+LinnegCD34+CD31+) in the aorta of adult C57Bl/6 mice (p<0.001). Single-cell RNA-sequencing showed clustering of PROCR and PDGFRA with EVPs, while other markers clustered with D cells. Immunofluorescent staining confirmed PROCR+ EVPs co-localize with YFP+ endothelium in the thoracic aortae of Cdh5-CreERT2/Rosa-EYFP mice (p=0.005) with increased clonogenic capacity (p<0.01). Aortic PROCR+ EVPs had greater colony forming capacity in vitro and engraftment potential in vivo than PROCRneg EVPs or PROCR+/neg D cells (p<0.05). Lineage tracing of developing PDFGRA-MerCreMer/Rosa-EYFP mice showed aortic YFP+ EVPs at day 0 (D0) differentiated into D cells by D84 (p<0.001). This result was validated in adult full-skin excisional wounds from the same mice, showing that PDGFRA+ EVPs at D0 differentiated into D cells by D5 (p<0.05), suggesting PDGFRA exclusively marks an EPC population capable of endothelial fate in both homeostasis and injury. Overall, the characteristics displayed by PROCR+ and PDGFRA+EVPs suggest these may mark a true EPC population.

As part of tissue homeostasis, endothelial progenitors (EPCs) residing within the endothelium contribute to vascular maintenance and regeneration through self-renewal and differentiation. In various stem cell niche, macrophages (MΦ) help modulate progenitor stemness and behavior and have been reported to regulate blood vessel development and remodeling. Recently, subsets of perivascular macrophage (PVMΦ) were identified in the blood vessel vicinity during tissue homeostasis and wound healing. Nevertheless, the physiological role of these PVMΦ on EPC function in homeostasis and wound re-vascularisation remains elusive. In this study, we investigated the interaction and regulatory function of PVMΦ on EPCs' stemness in the murine aorta and skin using an endothelial-specific lineage tracing model, Cdh5CreERT/Rosa-ZsGreen. Imaging flow cytometry analyses tracking remanent PVMΦ bodies on endothelial cells revealed that PVMΦ preferentially formed physical interaction with endothelial cells that displayed mesenchymal properties in the healthy aorta (18.81% ± 13.13%) and skin (10.02% ± 0.92%). In situ characterization showed that the F4/80+Lyve1+ PVMΦ were in the adventitial area of the aorta, and resided within the lower dermis of the dorsal skin. MΦ depletion using clodronate liposomes doubled aorta EPC proportion (p=0.0079) in vivo, where it displayed increased proliferative potential with reduced clonogenic capacity in vitro. Functionally, attenuating macrophage-derived Wnt activity using Wlsfl/flLysM-Cre mice elevated EPC cell number by two-fold on day 5 post cutaneous injury without affecting other endothelial subpopulations. Overall, our data indicate that PVMΦ constitute part of the vascular niche by regulating EPCs' quiescence and self-renewal, potentially via paracrine Wnt signaling.

Wnt signaling controls blood vessel growth, regression and patterning during embryonic and postnatal life. Macrophages are major producers of Wnt ligands and angiogenic growth factors. It regulates vascular development and specification during embryogenesis and wound healing. Macrophage dysregulation in wound healing impairs vessel regeneration and delay wound closure. During cutaneous wound healing, the endovascular progenitors (EVPs) proliferate and differentiate into mature endothelial (D) cells in response to signals produced by perivascular cells, including macrophages, governing blood vessels regeneration. However, the role of macrophage’s Wnt production on endothelial cells, especially the EVPs during wound healing is currently unknown. Here we used a cutaneous excisional wound model in mice with conditional deletion of Wnt secretion by myeloid cells ( Wls fl/fl LysM-Cre + ) to assess the kinetics of endothelial subpopulations (including EVP), myeloid infiltration, collagen deposition and wound closure. Deletion of Wls expression by myeloid cells did not affect wound closure and collagen deposition, indicating that myeloid Wls expression does not promote wound healing and regeneration. Myeloid-specific Wls deletion elevated the EVP population during the peak of angiogenesis, yet without affecting blood vessel density. Wounds in Wls fl/fl LysM-Cre + animals showed unperturbed myeloid infiltration and differentiation. Overall, our data indicate that macrophage Wnt production shapes EVP kinetics without major relevance to wound healing. These findings extend the knowledge of macrophage and endothelial molecular crosstalk and position myeloid-derived Wnt production as a regulator of endovascular progenitor.

Macrophages regulate cutaneous wound healing by immune surveillance, tissue repair and remodelling. The depletion of dermal macrophages during the early and middle stages of wound healing has a detrimental impact on wound closure, characterised by reduced vessel density, fibroblast and myofibroblast proliferation, delayed re-epithelization and abated post-healing fibrosis and scar formation. However, in some animal species, oral mucosa and foetal life, cutaneous wounds can heal normally and remain scarless without any involvement of macrophages. These paradoxical observations have created much controversy on macrophages' indispensable role in skin wound healing. Advanced knowledge gained by characterising macrophage subsets, their plasticity in switching phenotypes and molecular drivers provides new insights into their functional importance during cutaneous wound healing. In this review, we highlight the recent findings on skin macrophage subsets, their functional role in adult cutaneous wound healing and the potential benefits of targeting them for therapeutic use.

Elucidation of the molecular signatures that define hematopoietic tissue resident macrophage specialisation has been challenging. There are no validated markers that differentiate the specialised macrophage subsets in bone marrow (BM) that support erythropoiesis, bone homeostasis and hematopoietic stem cell (HSC) niches. We took an unbiased ex vivo approach to characterise macrophage subsets in mouse BM, spleen and lymph node using a flow cytometry marker panel which allowed analysis of all mature leucocytes, red blood cells and hematopoietic stem and progenitor cells (HSPC) in combination with in situ verified macrophage markers. Despite readily detectable F4/80 staining we were unable to identify any population in hematopoietic tissues that definitively represented intact macrophages. Imaging flow cytometry and confocal microscopy showed macrophage marker staining was derived from membrane-bound subcellular remnants associated with unrelated cell types. Remnant-restricted macrophage membrane markers, cytoplasmic reporters and mRNA were detected in non-macrophage cell populations including HSPC. Of note, HSC-associated detection of a Csf1r-reporter as well as anti-F4/80 and VCAM-1 staining were entirely attributable to membrane-bound subcellular remnants. Distinct marker expression on macrophage subsets within spleen verified that the profile of remnant binding reflected in vivo cell-cell interactions. Macrophage remnant attachment was reduced in Siglec1 deficient mice with frequency of F4/80+ BM events reduced by over 50% in HSPC and neutrophils yet unchanged in lymphocytes. Analysis of published RNA-seq data confirmed that macrophage fragmentation is a general phenomenon in disaggregated hematopoietic tissues. Overall, we have shown that abundant tissue macrophages are absent/under-represented in hematopoietic tissue cell suspensions. Detection of macrophage remnant-restricted cytoplasmic and membrane contents on other cells has confounded interpretation of ex vivo analyses and results in misattribution of macrophage-expressed genes to non-macrophage cells.

Background Prior chemotherapy and/or underlying morbidity commonly leads to poor mobilisation of hematopoietic stem cells (HSC) for transplantation in cancer patients. Increasing the number of available HSC prior to mobilisation is a potential strategy to overcome this deficiency. Resident bone marrow (BM) macrophages are essential for maintenance of niches that support HSC and enable engraftment in transplant recipients. Here we examined potential of donor treatment with modified recombinant colony-stimulating factor 1 (CSF1) to influence the HSC niche and expand the HSC pool for autologous transplantation. Methods We administered an acute treatment regimen of CSF1 Fc fusion protein (CSF1-Fc, daily injection for 4 consecutive days) to naive C57Bl/6 mice. Treatment impacts on macrophage and HSC number, HSC function and overall hematopoiesis were assessed at both the predicted peak drug action and during post-treatment recovery. A serial treatment strategy using CSF1-Fc followed by granulocyte colony-stimulating factor (G-CSF) was used to interrogate HSC mobilisation impacts. Outcomes were assessed by in situ imaging and ex vivo standard and imaging flow cytometry with functional validation by colony formation and competitive transplantation assay. Results CSF1-Fc treatment caused a transient expansion of monocyte-macrophage cells within BM and spleen at the expense of BM B lymphopoiesis and hematopoietic stem and progenitor cell (HSPC) homeostasis. During the recovery phase after cessation of CSF1-Fc treatment, normalisation of hematopoiesis was accompanied by an increase in the total available HSPC pool. Multiple approaches confirmed that CD48 − CD150 + HSC do not express the CSF1 receptor, ruling out direct action of CSF1-Fc on these cells. In the spleen, increased HSC was associated with expression of the BM HSC niche macrophage marker CD169 in red pulp macrophages, suggesting elevated spleen engraftment with CD48 − CD150 + HSC was secondary to CSF1-Fc macrophage impacts. Competitive transplant assays demonstrated that pre-treatment of donors with CSF1-Fc increased the number and reconstitution potential of HSPC in blood following a HSC mobilising regimen of G-CSF treatment. Conclusion These results indicate that CSF1-Fc conditioning could represent a therapeutic strategy to overcome poor HSC mobilisation and subsequently improve HSC transplantation outcomes.

Mouse hematopoietic tissues contain abundant tissue-resident macrophages that support immunity, hematopoiesis, and bone homeostasis. A systematic strategy to characterize macrophage subsets in mouse bone marrow (BM), spleen, and lymph node unexpectedly reveals that macrophage surface marker staining emanates from membrane-bound subcellular remnants associated with unrelated cells. Intact macrophages are not present within these cell preparations. The macrophage remnant binding profile reflects interactions between macrophages and other cell types in vivo. Depletion of CD169(+) macrophages in vivo eliminates F4/80(+) remnant attachment. Remnant-restricted macrophage-specific membrane markers, cytoplasmic fluorescent reporters, and mRNA are all detected in non-macrophage cells including isolated stem and progenitor cells. Analysis of RNA sequencing (RNA-seq) data, including publicly available datasets, indicates that macrophage fragmentation is a general phenomenon that confounds bulk and single-cell analysis of disaggregated hematopoietic tissues. Hematopoietic tissue macrophage fragmentation undermines the accuracy of macrophage ex vivo molecular profiling and creates opportunity for misattribution of macrophage-expressed genes to non-macrophage cells.

The endothelium possesses a profound regenerative capacity to adapt and reorganise in homeostasis and disease. The capacity to regenerate is increasingly attributed to a population of vessel-resident endovascular progenitor (EVP) cells that governs an endothelial hierarchy and have the ability to form vascular networks de novo. Using fate map analysis, we show that two transcription factors Sox9 and Rbpj specifically demarcate the EVP population and regulates progenitor fate choice differentiation. Conditional knock-out of Sox9 from the endothelium drove the depletion of EVP to a mature differentiated endothelial phenotype and enhanced Rbpj expression and Notch signalling. Additionally, skin wound analysis from Sox9 knock-out mice demonstrated a significant reduction in endothelial to mesenchymal transition (EndMT), reducing scar area. The converse was observed with Rbpj conditionally knocked-out from the vasculature, with enhanced Sox9 and key EndMT gene (Snail, Slug, Twist1, Twist 2, TGF-β) expression. Concurrently, vascular sonic hedgehog activation upregulates the expression Sox9 and is key in driving pathological EndMT and vascular fibrosis, resulting in over 3-fold increase in scar area in skin wound healing. In this scenario, we see EVP transitioning towards a mesenchymal fate; with increased Sox9, reduced Rbpj and enhanced EndMT gene expression. Importantly, using topical administration of siRNA against Sox9 on skin wounds significantly reduced scar area by blocking pathological EndMT. The understanding of how vascular resident EVP function opens exciting new avenues for more effective therapies in blocking fibrotic disease.

Osteal macrophages (osteomacs) support osteoblast function and promote bone anabolism, but their contribution to osteoporosis has not been explored. Although mouse ovariectomy (OVX) models have been repeatedly used, variation in strain, experimental design and assessment modalities have contributed to no single model being confirmed as comprehensively replicating the full gamut of osteoporosis pathological manifestations. We validated an OVX model in adult C3H/HeJ mice and demonstrated that it presents with human postmenopausal osteoporosis features with reduced bone volume in axial and appendicular bone and bone loss in both trabecular and cortical bone including increased cortical porosity. Bone loss was associated with increased osteoclasts on trabecular and endocortical bone and decreased osteoblasts on trabecular bone. Importantly, this OVX model was characterized by delayed fracture healing. Using this validated model, we demonstrated that osteomacs are increased post-OVX on both trabecular and endocortical bone. Dual F4/80 (pan-macrophage marker) and tartrate-resistant acid phosphatase (TRAP) staining revealed osteomacs frequently located near TRAP+ osteoclasts and contained TRAP+ intracellular vesicles. Using an in vivo inducible macrophage depletion model that does not simultaneously deplete osteoclasts, we observed that osteomac loss was associated with elevated extracellular TRAP in bone marrow interstitium and increased serum TRAP. Using in vitro high-resolution confocal imaging of mixed osteoclast-macrophage cultures on bone substrate, we observed macrophages juxtaposed to osteoclast basolateral functional secretory domains scavenging degraded bone byproducts. These data demonstrate a role for osteomacs in supporting osteoclastic bone resorption through phagocytosis and sequestration of resorption byproducts. Overall, our data expose a novel role for osteomacs in supporting osteoclast function and provide the first evidence of their involvement in osteoporosis pathogenesis. © 2021 American Society for Bone and Mineral Research (ASBMR).

Endothelial to mesenchymal transition (EndMT) is a leading cause of fibrosis and disease, however its mechanism has yet to be elucidated. The endothelium possesses a profound regenerative capacity to adapt and reorganize that is attributed to a population of vessel-resident endovascular progenitors (EVP) governing an endothelial hierarchy. Here, using fate analysis, we show that two transcription factors SOX9 and RBPJ specifically affect the murine EVP numbers and regulate lineage specification. Conditional knock-out of Sox9 from the vasculature (Sox9fl/fl/Cdh5-CreER RosaYFP) depletes EVP while enhancing Rbpj expression and canonical Notch signalling. Additionally, skin wound analysis from Sox9 conditional knock-out mice demonstrates a significant reduction in pathological EndMT resulting in reduced scar area. The converse is observed with Rbpj conditionally knocked-out from the murine vasculature (Rbpjfl/fl/Cdh5-CreER RosaYFP) or inhibition of Notch signaling in human endothelial colony forming cells, resulting in enhanced Sox9 and EndMT related gene (Snail, Slug, Twist1, Twist2, TGF-β) expression. Similarly, increased endothelial hedgehog signaling (Ptch1fl/fl/Cdh5-CreER RosaYFP), that upregulates the expression of Sox9 in cells undergoing pathological EndMT, also results in excess fibrosis. Endothelial cells transitioning to a mesenchymal fate express increased Sox9, reduced Rbpj and enhanced EndMT. Importantly, using topical administration of siRNA against Sox9 on skin wounds can substantially reduce scar area by blocking pathological EndMT. Overall, here we report distinct fates of EVPs according to the relative expression of Rbpj or Notch signalling and Sox9, highlighting their potential plasticity and opening exciting avenues for more effective therapies in fibrotic diseases.

Systemic sclerosis (SSc), also known as scleroderma is a chronic autoimmune disease where patients suffer from skin hardening and thickening as well as fibrosis of many internal organs, including the lungs. Patients also experience a loss of vasculature and an alteration of vasculature architecture. There is evidence that endothelial cells transitioning into mesenchymal cells (known as EndMT) may be one of the mechanisms that contributes to disease progression. Thus, this study aims to elucidate if EndMT does play a role in SSc using endothelial lineage tracing experiments with Cdh5-creERt2/ ROSA-YFP mice, where endothelial cells are labelled with YFP. In addition, previous data from our lab suggests that a reduction in Sox9 signalling in the endothelium can decrease the amount of fibrosis observed when mice are recovering from wounding. Thus, the second aim of our study is to determine if a vascular Sox9 knockout mouse (Sox9lox/lox Cdh5-creERt2/ROSA-YFP) will display reduced fibrosis in an SSc setting. Preliminary results indicate that EndMT may not actually be the driving force in SSc pathogenesis, as increased numbers of endothelial cells in transition (defined as YFP+/a-smooth muscle actin+/CD31+) or endothelial cells that have fully transitioned (defined as YFP+/ a-smooth muscle actin+/CD31-) were not evident in immunofluorescent staining of the skin (n=4) and the lungs (n=6). In addition, when Sox9KO mice with SSc were evaluated, there was no reduction in fibrosis observed on slides stained with Masson's Trichrome in both the skin (n=6) and the lungs (n=6-9), indicating vascular Sox9 may not function in the way we originally hypothesized. Interestingly, the Sox9KO SSc mice actually experienced more severe disease progression compared to control mice, as skin fibrosis may actually be increased in these animals (n=6, p=0.065) and mice with SSc affecting the lungs died earlier than control mice (n=6-9, p=0.11), providing an interesting opportunity to explore this unexpected phenotype.

Autologous HSC transplant is a common therapeutic strategy for some haematological malignancies but is unachievable in up to 40% of cases due to insufficient mobilisation of HSCs for successful transplantation. The monocyte-macrophage colony stimulating factor-1 (CSF1) improves HSC transplantation outcomes in preclinical models and clinical trials, but the underlying mechanism of CSF1 induced benefit is unclear. BM macrophages are a key component of the HSC niches and a primary target of granulocyte colony-stimulating factor (G-CSF) induced HSC mobilisation. We examined CSF1 impacts on HSC frequency using a modified CSF1-Fc molecule that has improved drug qualities. Haematopoietic progenitor cells (HPC), but not HSCs or multipotent progenitors (MPP), expressed the CSF1 receptor. Acute CSF1-Fc treatment (4 x daily injection) induced a robust myeloproliferative response that was evident 1 week post the initial injection. At this time, BM HSCs (17 fold) and MPP (19 fold), but not HPC, were significantly reduced. However, at 2 weeks post the first CSF1-Fc injection, after the myeloproliferative event had resolved, the overall pool of HSCs in BM (1.6 fold), and unexpectedly spleen (9.2 fold), was significantly elevated compared to controls. A mobilising regimen of G-CSF started at 2 weeks post CSF1-Fc treatment enhanced HSC mobilisation (2.5 fold) into blood when compared to mice treated with G-CSF only. This increase in mobilised HSCs was verified via competitive secondary transplantation. The data suggest that CSF1 has complex indirect effects on HSCs to increase HSC frequency and promote formation of extramedullary HSC niches. CSF1-induced increased in the total HSC pool may contribute to the reported improvement in transplantation outcomes associated with CSF1 therapy and reveals a novel strategy to increase HSCs prior to mobilisation.

The bone marrow (BM) environment is essential to successful hematopoietic stem cell (HSC) transplantation through creation of specialized niches that support HSC homeostasis and fate decisions. Within hematopoietic niches, macrophages interact directly with HSC, and with other non-hematopoietic niche cells, to regulate niche homeostasis. BM contains multiple resident macrophage subsets that contribute to discrete events during hematopoiesis but the specific identity of HSC niche macrophage remains elusive. Knowledge of macrophage functional contributions and associated molecular mechanisms that regulate HSC homeostasis is limited. We used a myeloid reporter gene (Csf1r-enhance GFP) to dissect the persistence of BM and splenic macrophage subsets following lethal irradiation and autologous HSC transplantation in a mouse model. Multiple recipient BM and splenic macrophage subsets survived post-autologous HSC transplantation with organ specific persistence kinetics. Short-term persistence (5 weeks) of recipient spleen resident macrophages paralleled the duration of extramedullary hematopoiesis post-transplant. In BM, radiation-resistant recipient CD169+ resident macrophages and erythroid-island macrophages self-repopulated long-term post transplantation via in situ autonomous cell division. Peak recovery of recipient CD169+ resident macrophage number in BM coincided with early post-transplant BM long-term engraftment of HSC. Importantly selective depletion of these recipient CD169+ macrophages in CD169-DTR recipients transplanted with wild-type HSC significantly compromised HSC engraftment. Transcriptome analysis of recipient versus donor-derived BM macrophages at 5 weeks post-transplant revealed clear difference in the functional potential of these phenotypically similar macrophages subsets, reinforcing evidence that donor-derived macrophages cannot fully reconstitute the HSC niche, at least at early post-transplantation time points. Overall, recipient BM resident macrophages are essential for optimal HSC transplantation outcomes and should be an important consideration in development of pre-transplant conditioning therapies and/or chemo-resistance approaches.

Distinct subsets of resident tissue macrophages are important in hematopoietic stem cell niche homeostasis and erythropoiesis. We used a myeloid reporter gene (Csf1r-eGFP) to dissect the persistence of bone marrow and splenic macrophage subsets following lethal irradiation and autologous hematopoietic stem cell transplantation in a mouse model. Multiple recipient bone marrow and splenic macrophage subsets survived after autologous hematopoietic stem cell transplantation with organ-specific persistence kinetics. Short-term persistence (5 weeks) of recipient resident macrophages in spleen paralleled the duration of extramedullary hematopoiesis. In bone marrow, radiation-resistant recipient CD169+ resident macrophages and erythroid-island macrophages self-repopulated long-term after transplantation via autonomous cell division. Posttransplant peak expansion of recipient CD169+ resident macrophage number in bone marrow aligned with the persistent engraftment of phenotypic long-term reconstituting hematopoietic stem cells within bone marrow. Selective depletion of recipient CD169+ macrophages significantly compromised the engraftment of phenotypic long-term reconstituting hematopoietic stem cells and consequently impaired hematopoietic reconstitution. Recipient bone marrow resident macrophages are essential for optimal hematopoietic stem cell transplantation outcomes and could be an important consideration in the development of pretransplant conditioning therapies and/or chemoresistance approaches.

Macrophages, named for their phagocytic ability, participate in homeostasis, tissue regeneration and inflammatory responses. Bone and adjacent marrow contain multiple functionally unique resident tissue macrophage subsets which maintain and regulate anatomically distinct niche environments within these interconnected tissues. Three subsets of bone–bone marrow resident tissue macrophages have been characterised; erythroblastic island macrophages, haematopoietic stem cell niche macrophages and osteal macrophages. The role of these macrophages in controlling homeostasis and repair in bone and bone marrow niches is reviewed in detail.