Cross-talk between TGF-ß and PDGFRα signaling pathways regulates the fate of stromal fibro-adipogenic progenitors.

Fibro-adipogenic progenitors (FAPs) are tissue-resident mesenchymal stromal cells (MSCs) required for proper skeletal muscle development, regeneration and maintenance. However, FAPs are also responsible for fibro-fatty scar deposition following chronic damage. We aimed to investigate the role of functional cross-talk between TGF-ß and PDGFRα signaling pathways in the fate of FAPs. Here, we show that the number of FAPs correlates with TGF-ß levels and with extracellular matrix deposition during regeneration and repair. Interestingly, the expression of PDGFRα changed dynamically in the fibroblast lineage after injury. Furthermore, PDGFRα-dependent immediate early gene expression changed during regeneration and repair. We also found that TGF-ß signaling reduces PDGFRα expression in FAPs, mouse dermal fibroblasts and in two related mesenchymal cell lines. Moreover, TGF-ß promotes myofibroblast differentiation of FAPs but inhibits their adipogenicity. Accordingly, TGF-ß impairs the expression of PDGFRα-dependent immediate early genes in a TGFBR1-dependent manner. Finally, pharmacological inhibition of PDGFRα activity with AG1296 impaired TGF-ß-induced extracellular matrix remodeling, Smad2 signaling, myofibroblast differentiation and migration of MSCs. Thus, our work establishes a functional cross-talk between TGF-ß and PDGFRα signaling pathways that is involved in regulating the biology of FAPs and/or MSCs.This article has an associated First Person interview with the first author of the paper.

EphrinB2/EphB4 signaling regulates non-sprouting angiogenesis by VEGF.

Vascular endothelial growth factor (VEGF) is the master regulator of angiogenesis, whose best-understood mechanism is sprouting. However, therapeutic VEGF delivery to ischemic muscle induces angiogenesis by the alternative process of intussusception, or vascular splitting, whose molecular regulation is essentially unknown. Here, we identify ephrinB2/EphB4 signaling as a key regulator of intussusceptive angiogenesis and its outcome under therapeutically relevant conditions. EphB4 signaling fine-tunes the degree of endothelial proliferation induced by specific VEGF doses during the initial stage of circumferential enlargement of vessels, thereby limiting their size and subsequently enabling successful splitting into normal capillary networks. Mechanistically, EphB4 neither inhibits VEGF-R2 activation by VEGF nor its internalization, but it modulates VEGF-R2 downstream signaling through phospho-ERK1/2. In vivo inhibitor experiments show that ERK1/2 activity is required for EphB4 regulation of VEGF-induced intussusceptive angiogenesis. Lastly, after clinically relevant VEGF gene delivery with adenoviral vectors, pharmacological stimulation of EphB4 normalizes dysfunctional vascular growth in both normoxic and ischemic muscle. These results identify EphB4 as a druggable target to modulate the outcome of VEGF gene delivery and support further investigation of its therapeutic potential.

Immune Cell Therapies to Improve Regeneration and Revascularization of Non-Healing Wounds.

With the increased prevalence of chronic diseases, non-healing wounds place a significant burden on the health system and the quality of life of affected patients. Non-healing wounds are full-thickness skin lesions that persist for months or years. While several factors contribute to their pathogenesis, all non-healing wounds consistently demonstrate inadequate vascularization, resulting in the poor supply of oxygen, nutrients, and growth factors at the level of the lesion. Most existing therapies rely on the use of dermal substitutes, which help the re-epithelialization of the lesion by mimicking a pro-regenerative extracellular matrix. However, in most patients, this approach is not efficient, as non-healing wounds principally affect individuals afflicted with vascular disorders, such as peripheral artery disease and/or diabetes. Over the last 25 years, innovative therapies have been proposed with the aim of fostering the regenerative potential of multiple immune cell types. This can be achieved by promoting cell mobilization into the circulation, their recruitment to the wound site, modulation of their local activity, or their direct injection into the wound. In this review, we summarize preclinical and clinical studies that have explored the potential of various populations of immune cells to promote skin regeneration in non-healing wounds and critically discuss the current limitations that prevent the adoption of these therapies in the clinics.

Long-lasting fibrin matrices ensure stable and functional angiogenesis by highly tunable, sustained delivery of recombinant VEGF164.

Clinical trials of therapeutic angiogenesis by vascular endothelial growth factor (VEGF) gene delivery failed to show efficacy. Major challenges include the need to precisely control in vivo distribution of growth factor dose and duration of expression. Recombinant VEGF protein delivery could overcome these issues, but rapid in vivo clearance prevents the stabilization of induced angiogenesis. Here, we developed an optimized fibrin platform for controlled delivery of recombinant VEGF, to robustly induce normal, stable, and functional angiogenesis. Murine VEGF164 was fused to a sequence derived from α2-plasmin inhibitor (α2-PI1-8) that is a substrate for the coagulation factor fXIIIa, to allow its covalent cross-linking into fibrin hydrogels and release only by enzymatic cleavage. An α2-PI1-8-fused variant of the fibrinolysis inhibitor aprotinin was used to control the hydrogel degradation rate, which determines both the duration and effective dose of factor release. An optimized aprotinin-α2-PI1-8 concentration ensured ideal degradation over 4 wk. Under these conditions, fibrin-α2-PI1-8-VEGF164 allowed exquisitely dose-dependent angiogenesis: concentrations ≥25 µg/mL caused widespread aberrant vascular structures, but a 500-fold concentration range (0.01-5.0 µg/mL) induced exclusively normal, mature, nonleaky, and perfused capillaries, which were stable after 3 mo. Optimized delivery of fibrin-α2-PI1-8-VEGF164 was therapeutically effective both in ischemic hind limb and wound-healing models, significantly improving angiogenesis, tissue perfusion, and healing rate. In conclusion, this optimized platform ensured (i) controlled and highly tunable delivery of VEGF protein in ischemic tissue and (ii) stable and functional angiogenesis without introducing genetic material and with a limited and controllable duration of treatment. These findings suggest a strategy to improve safety and efficacy of therapeutic angiogenesis.

Protocol for generation of a time-resolved cellular interactome during tissue remodeling in adult mice.

Efficient skeletal muscle regeneration necessitates fine-tuned coordination among multiple cell types through an intricate network of intercellular communication. We present a protocol for generation of a time-resolved cellular interactome during tissue remodeling. We describe steps for isolating distinct cell populations from skeletal muscle of adult mice after acute damage and extracting RNA from purified cells prior to the generation of RNA sequencing data. We then detail procedures for generating and deciphering a time- and lineage-resolved model of intercellular crosstalk. For complete details on the use and execution of this protocol, please refer to Groppa et al. (2023).1.

DPPIV+ fibro-adipogenic progenitors form the niche of adult skeletal muscle self-renewing resident macrophages.

Adult tissue-resident macrophages (RMs) are either maintained by blood monocytes or through self-renewal. While the presence of a nurturing niche is likely crucial to support the survival and function of self-renewing RMs, evidence regarding its nature is limited. Here, we identify fibro-adipogenic progenitors (FAPs) as the main source of colony-stimulating factor 1 (CSF1) in resting skeletal muscle. Using parabiosis in combination with FAP-deficient transgenic mice (PdgfrαCreERT2 × DTA) or mice lacking FAP-derived CSF1 (PdgfrαCreERT2 × Csf1flox/null), we show that local CSF1 from FAPs is required for the survival of both TIM4- monocyte-derived and TIM4+ self-renewing RMs in adult skeletal muscle. The spatial distribution and number of TIM4+ RMs coincide with those of dipeptidyl peptidase IV (DPPIV)+ FAPs, suggesting their role as CSF1-producing niche cells for self-renewing RMs. This finding identifies opportunities to precisely manipulate the function of self-renewing RMs in situ to further unravel their role in health and disease.

Spatiotemporal signaling underlies progressive vascular rarefaction in myocardial infarction.

Therapeutic angiogenesis represents a promising avenue to revascularize the ischemic heart. Its limited success is partly due to our poor understanding of the cardiac stroma, specifically mural cells, and their response to ischemic injury. Here, we combine single-cell and positional transcriptomics to assess the behavior of mural cells within the healing heart. In response to myocardial infarction, mural cells adopt an altered state closely associated with the infarct and retain a distinct lineage from fibroblasts. This response is concurrent with vascular rarefaction and reduced vascular coverage by mural cells. Positional transcriptomics reveals that the infarcted heart is governed by regional-dependent and temporally regulated programs. While the remote zone acts as an important source of pro-angiogenic signals, the infarct zone is accentuated by chronic activation of anti-angiogenic, pro-fibrotic, and inflammatory cues. Together, our work unveils the spatiotemporal programs underlying cardiac repair and establishes an association between vascular deterioration and mural cell dysfunction.

Ischemic wound revascularization by the stromal vascular fraction relies on host-donor hybrid vessels.

Nonhealing wounds place a significant burden on both quality of life of affected patients and health systems. Skin substitutes are applied to promote the closure of nonhealing wounds, although their efficacy is limited by inadequate vascularization. The stromal vascular fraction (SVF) from the adipose tissue is a promising therapy to overcome this limitation. Despite a few successful clinical trials, its incorporation in the clinical routine has been hampered by their inconsistent results. All these studies concluded by warranting pre-clinical work aimed at both characterizing the cell types composing the SVF and shedding light on their mechanism of action. Here, we established a model of nonhealing wound, in which we applied the SVF in combination with a clinical-grade skin substitute. We purified the SVF cells from transgenic animals to trace their fate after transplantation and observed that it gave rise to a mature vascular network composed of arteries, capillaries, veins, as well as lymphatics, structurally and functionally connected with the host circulation. Then we moved to a human-in-mouse model and confirmed that SVF-derived endothelial cells formed hybrid human-mouse vessels, that were stabilized by perivascular cells. Mechanistically, SVF-derived endothelial cells engrafted and expanded, directly contributing to the formation of new vessels, while a population of fibro-adipogenic progenitors stimulated the expansion of the host vasculature in a paracrine manner. These data have important clinical implications, as they provide a steppingstone toward the reproducible and effective adoption of the SVF as a standard care for nonhealing wounds.

Spatial compartmentalization of signaling imparts source-specific functions on secreted factors.

Efficient regeneration requires multiple cell types acting in coordination. To better understand the intercellular networks involved and how they change when regeneration fails, we profile the transcriptome of hematopoietic, stromal, myogenic, and endothelial cells over 14 days following acute muscle damage. We generate a time-resolved computational model of interactions and identify VEGFA-driven endothelial engagement as a key differentiating feature in models of successful and failed regeneration. In addition, the analysis highlights that the majority of secreted signals, including VEGFA, are simultaneously produced by multiple cell types. To test whether the cellular source of a factor determines its function, we delete VEGFA from two cell types residing in close proximity: stromal and myogenic progenitors. By comparing responses to different types of damage, we find that myogenic and stromal VEGFA have distinct functions in regeneration. This suggests that spatial compartmentalization of signaling plays a key role in intercellular communication networks.

Genetic lineage tracing reveals poor angiogenic potential of cardiac endothelial cells.

AIMS: Cardiac ischaemia does not elicit an efficient angiogenic response. Indeed, lack of surgical revascularization upon myocardial infarction results in cardiomyocyte death, scarring, and loss of contractile function. Clinical trials aimed at inducing therapeutic revascularization through the delivery of pro-angiogenic molecules after cardiac ischaemia have invariably failed, suggesting that endothelial cells in the heart cannot mount an efficient angiogenic response. To understand why the heart is a poorly angiogenic environment, here we compare the angiogenic response of the cardiac and skeletal muscle using a lineage tracing approach to genetically label sprouting endothelial cells. METHODS AND RESULTS: We observed that overexpression of the vascular endothelial growth factor in the skeletal muscle potently stimulated angiogenesis, resulting in the formation of a massive number of new capillaries and arterioles. In contrast, response to the same dose of the same factor in the heart was blunted and consisted in a modest increase in the number of new arterioles. By using Apelin-CreER mice to genetically label sprouting endothelial cells we observed that different pro-angiogenic stimuli activated Apelin expression in both muscle types to a similar extent, however, only in the skeletal muscle, these cells were able to sprout, form elongated vascular tubes activating Notch signalling, and became incorporated into arteries. In the heart, Apelin-positive cells transiently persisted and failed to give rise to new vessels. When we implanted cancer cells in different organs, the abortive angiogenic response in the heart resulted in a reduced expansion of the tumour mass. CONCLUSION: Our genetic lineage tracing indicates that cardiac endothelial cells activate Apelin expression in response to pro-angiogenic stimuli but, different from those of the skeletal muscle, fail to proliferate and form mature and structured vessels. The poor angiogenic potential of the heart is associated with reduced tumour angiogenesis and growth of cancer cells.

Human skeletal muscle CD90+ fibro-adipogenic progenitors are associated with muscle degeneration in type 2 diabetic patients.

Type 2 diabetes mellitus (T2DM) is associated with impaired skeletal muscle function and degeneration of the skeletal muscles. However, the mechanisms underlying the degeneration are not well described in human skeletal muscle. Here we show that skeletal muscle of T2DM patients exhibit degenerative remodeling of the extracellular matrix that is associated with a selective increase of a subpopulation of fibro-adipogenic progenitors (FAPs) marked by expression of THY1 (CD90)-the FAPCD90+. We identify platelet-derived growth factor (PDGF) as a key FAP regulator, as it promotes proliferation and collagen production at the expense of adipogenesis. FAPsCD90+ display a PDGF-mimetic phenotype, with high proliferative activity, clonogenicity, and production of extracellular matrix. FAPCD90+ proliferation was reduced by in vitro treatment with metformin. Furthermore, metformin treatment reduced FAP content in T2DM patients. These data identify a PDGF-driven conversion of a subpopulation of FAPs as a key event in the fibrosis development in T2DM muscle.

Murine Tissue-Resident PDGFRα+ Fibro-Adipogenic Progenitors Spontaneously Acquire Osteogenic Phenotype in an Altered Inflammatory Environment.

Acquired heterotopic ossifications (HO) arising as a result of various traumas, including injury or surgical interventions, often result in pain and loss of motion. Though triggers for HO have been identified, the cellular source of these heterotopic lesions as well as the underlying mechanisms that drive the formation of acquired HO remain poorly understood, and treatment options, including preventative treatments, remain limited. Here, we explore the cellular source of HO and a possible underlying mechanism for their spontaneous osteogenic differentiation. We demonstrate that HO lesions arise from tissue-resident PDGFRα+ fibro/adipogenic progenitors (FAPs) in skeletal muscle and not from circulating bone marrow-derived progenitors. Further, we show that accumulation of these cells in the tissue after damage due to alterations in the inflammatory environment can result in activation of their inherent osteogenic potential. This work suggests a mechanism by which an altered inflammatory cell and FAP interactions can lead to the formation of HO after injury and presents potential targets for therapeutics in acquired HO. © 2020 American Society for Bone and Mineral Research.

Inhibition of Methyltransferase Setd7 Allows the In Vitro Expansion of Myogenic Stem Cells with Improved Therapeutic Potential.

The development of cell therapy for repairing damaged or diseased skeletal muscle has been hindered by the inability to significantly expand immature, transplantable myogenic stem cells (MuSCs) in culture. To overcome this limitation, a deeper understanding of the mechanisms regulating the transition between activated, proliferating MuSCs and differentiation-primed, poorly engrafting progenitors is needed. Here, we show that methyltransferase Setd7 facilitates such transition by regulating the nuclear accumulation of ß-catenin in proliferating MuSCs. Genetic or pharmacological inhibition of Setd7 promotes in vitro expansion of MuSCs and increases the yield of primary myogenic cell cultures. Upon transplantation, both mouse and human MuSCs expanded with a Setd7 small-molecule inhibitor are better able to repopulate the satellite cell niche, and treated mouse MuSCs show enhanced therapeutic potential in preclinical models of muscular dystrophy. Thus, Setd7 inhibition may help bypass a key obstacle in the translation of cell therapy for muscle disease.

VEGF dose regulates vascular stabilization through Semaphorin3A and the Neuropilin-1+ monocyte/TGF-ß1 paracrine axis.

VEGF is widely investigated for therapeutic angiogenesis, but while short-term delivery is desirable for safety, it is insufficient for new vessel persistence, jeopardizing efficacy. Here, we investigated whether and how VEGF dose regulates nascent vessel stabilization, to identify novel therapeutic targets. Monoclonal populations of transduced myoblasts were used to homogeneously express specific VEGF doses in SCID mouse muscles. VEGF was abrogated after 10 and 17 days by Aflibercept treatment. Vascular stabilization was fastest with low VEGF, but delayed or prevented by higher doses, without affecting pericyte coverage. Rather, VEGF dose-dependently inhibited endothelial Semaphorin3A expression, thereby impairing recruitment of Neuropilin-1-expressing monocytes (NEM), TGF-ß1 production and endothelial SMAD2/3 activation. TGF-ß1 further initiated a feedback loop stimulating endothelial Semaphorin3A expression, thereby amplifying the stabilizing signals. Blocking experiments showed that NEM recruitment required endogenous Semaphorin3A and that TGF-ß1 was necessary to start the Semaphorin3A/NEM axis. Conversely, Semaphorin3A treatment promoted NEM recruitment and vessel stabilization despite high VEGF doses or transient adenoviral delivery. Therefore, VEGF inhibits the endothelial Semaphorin3A/NEM/TGF-ß1 paracrine axis and Semaphorin3A treatment accelerates stabilization of VEGF-induced angiogenesis.

Osteogenic graft vascularization and bone resorption by VEGF-expressing human mesenchymal progenitors.

Rapid vascularisation of tissue-engineered osteogenic grafts is a major obstacle in the development of regenerative medicine approaches for bone repair. Vascular endothelial growth factor (VEGF) is the master regulator of vascular growth. We investigated a cell-based gene therapy approach to generate osteogenic grafts with an increased vascularization potential in an ectopic nude rat model in vivo, by genetically modifying human bone marrow-derived stromal/stem cells (BMSC) to express rat VEGF. BMSC were loaded onto silicate-substituted apatite granules, which are a clinically established osteo-conductive material. Eight weeks after implantation, the vascular density of constructs seeded with VEGF-BMSC was 3-fold greater than with control cells, consisting of physiologically structured vascular networks with both conductance vessels and capillaries. However, VEGF specifically caused a global reduction in bone quantity, which consisted of thin trabeculae of immature matrix. VEGF did not impair BMSC engraftment in vivo, but strongly increased the recruitment of TRAP- and Cathepsin K-positive osteoclasts. These data suggest that VEGF over-expression is effective to improve the vascularization of osteogenic grafts, but also has the potential to disrupt bone homoeostasis towards excessive degradation, posing a challenge to its clinical application in bone tissue engineering.