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 study a functional cross-talk between TGF-β and PDGFRα signaling pathways in FAPs’ fate. Here, we show that the number of FAPs correlates with TGF-β levels and with extracellular matrix deposition during regeneration and repair. Interestingly, the expression of PDGFRα changed dynamically in the stromal/fibroblast lineage after injury. Furthermore, PDGFRα-dependent immediate early gene expression changed during regeneration and repair. We also found that TGF-β signaling reduces PDGFRα expression in FAPs, mouse dermal fibroblasts, and in two related mesenchymal/fibroblast cell lines. Moreover, TGF-β promotes myofibroblast differentiation of FAPs but inhibits their adipogenicity. Accordingly, TGF-β impairs the expression of PDGFRα-dependent immediate early genes in a TGF-BR1-dependent manner. Finally, pharmacological inhibition of PDGFRα activity with AG1296 impaired TGF-β-induced extracellular matrix remodeling, Smad2 signaling, myofibroblast differentiation, and migration of MSCs. Thus, our work establishes a functional cross-talk between TGF-β and PDGFRα signaling pathways that is involved in regulating the biology of FAPs/MSCs.

Significance Inducing the growth of new blood vessels by specific factors is an attractive strategy to restore blood flow in ischemic tissues. Vascular endothelial growth factor (VEGF) is the master regulator of angiogenesis, yet clinical trials of VEGF gene delivery failed. Major challenges include the need to control the tissue distribution of factor dose and the duration of expression. Here, we developed a highly tunable fibrin-based platform to precisely control the dose and duration of VEGF protein delivery in tissues. Optimized delivery of fibrin-bound VEGF ensured normal, stable, and functional angiogenesis and improved perfusion of ischemic tissues, without genetic modification and with limited duration of VEGF delivery. These findings suggest a strategy to improve both safety and efficacy of therapeutic angiogenesis.

No abstract

VEGF induces normal or aberrant angiogenesis depending on its dose in the microenvironment around each producing cell in vivo. This transition depends on the balance between VEGF-induced endothelial stimulation and PDGF-BB-mediated pericyte recruitment, and co-expression of PDGF-BB normalizes aberrant angiogenesis despite high VEGF doses. We recently found that VEGF over-expression induces angiogenesis in skeletal muscle through an initial circumferential vascular enlargement followed by longitudinal splitting, rather than sprouting. Here we investigated the cellular mechanism by which PDGF-BB co-expression normalizes VEGF-induced aberrant angiogenesis. Monoclonal populations of transduced myoblasts, expressing similarly high levels of VEGF alone or with PDGF-BB, were implanted in mouse skeletal muscles. PDGF-BB co-expression did not promote sprouting and angiogenesis that occurred through vascular enlargement and splitting. However, enlargements were significantly smaller in diameter, due to a significant reduction in endothelial proliferation, and retained pericytes, which were otherwise lost with high VEGF alone. A time-course of histological analyses and repetitive intravital imaging showed that PDGF-BB co-expression anticipated the initiation of vascular enlargement and markedly accelerated the splitting process. Interestingly, quantification during in vivo imaging suggested that a global reduction in shear stress favored the initiation of transluminal pillar formation during VEGF-induced splitting angiogenesis. Quantification of target gene expression showed that VEGF-R2 signaling output was significantly reduced by PDGF-BB co-expression compared to VEGF alone. In conclusion, PDGF-BB co-expression prevents VEGF-induced aberrant angiogenesis by modulating VEGF-R2 signaling and endothelial proliferation, thereby limiting the degree of circumferential enlargement and enabling efficient completion of vascular splitting into normal capillary networks despite high VEGF doses.

No abstract

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.

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 β-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.

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.