Aims

Ultrasound-guided injection techniques are expected to enhance therapeutic efficacy for skeletal muscle injuries and disorders, but basic knowledge is lacking. The purpose of this study was to examine the diagnostic accuracy of ultrasound for abnormal skeletal muscle lesions, and to examine the distribution patterns of solution and cells injected into abnormal muscle lesions under ultrasound guidance.

Aims

Dystrophic calcification (DC) is the abnormal appearance of calcified deposits in degenerating tissue, often associated with injury. Extensive DC can lead to heterotopic ossification (HO), a pathological condition of ectopic bone formation. The highest rate of HO was found in combat-related blast injuries, a polytrauma condition with severe muscle injury. It has been noted that the incidence of HO significantly increased in the residual limbs of combat-injured patients if the final amputation was performed within the zone of injury compared to that which was proximal to the zone of injury. While aggressive limb salvage strategies may maximize the function of the residual limb, they may increase the possibility of retaining non-viable muscle tissue inside the body. In this study, we hypothesized that residual dead muscle tissue at the zone of injury could promote HO formation.

Background. There are currently no effective treatments for skeletal muscle fibrosis. Myofibroblasts are the major cellular effectors of fibrosis but their origin in muscle is unknown. We report that PDGFRβ (platelet derived growth factor receptor beta) Cre inactivates genes in murine PDGFRβ+ cells and myofibroblasts in muscle with high efficiency. We used this system to delete the integrin αv subunit because of the suggested role of multiple αv integrins as central mediators of fibrosis in multiple organs. Methods. Muscle fibrosis was induced by intramuscular cardiotoxin (CTX) injection. The contribution of PDGFRβ+ cells to fibrosis was assessed in double-flourescent reporter (mTmG) mice under PDGFRβ-Cre control. Itgavflox/flox;PDGFRβ-Cre mice were used to investigate whether loss of αv integrins on PDGFRβ+ cells influences fibrosis development. A small-molecule inhibitor of αv integrins (CWHM12) was used to determine whether pharmacological blockade of αv integrins could attenuate fibrosis. Results. At 21 days following injury PDGFRβ+ cells in mTmG;PDGFRβ-Cre mice were distributed in a manner characteristic of myofibroblasts. PDGFRβ+ cells sorted from injured muscles of mTmG;PDGFRβ-Cre mice showed induction of genes associated with myofibroblastic transition. Itgavflox/flox;PDGFRβ-Cre mice were protected from CTX induced fibrosis, as determined by picrosirius red staining for collagen (p<0.01). Sorted and culture activated αv-null PDGFRβ+ cells demonstrated significant reduction in collagen1 over controls (p<0.05). CWHM12 significantly reduced muscle fibrosis when delivered from the time of injury (prophylactic model: p<0.01) and from day 10 post injury (therapeutic model: p<0.01). Furthermore, CWHM12 inhibited collagen1 expression by PDGFRβ+ cells ex-vivo (p<0.05). Conclusions. PDGFRβ-Cre labels profibrotic cells in skeletal muscle and depletion of αv integrins in these cells reduces muscle fibrosis. Most importantly from a treatment standpoint, pharmacologic inhibition of αv integrins using a small molecule inhibitor may have utility in the prevention and treatment of skeletal muscle fibrosis. Level of Evidence. Basic Science

Scar tissue formation secondary to acute muscle injury, surgical wounding and compartment syndrome can result in significant functional impairment and predispose to further injury. The source of fibroblasts, and the molecular mechanisms driving their activation and persistence in skeletal muscle fibrosis are not known. We hypothesized that cells expressing PDGFRβ become fibroblasts in response to injury and that targeting αv integrins in these cells reduces skeletal muscle fibrosis. We used double-fluorescent reporter mice to demonstrate that cells expressing PDGFRβ become activated myofibroblasts in response to cardiotoxin (CTX) induced skeletal muscle injury. Following injury, PDGFRβ+ cells moved from perivascular locations into the interstitium in a distribution characteristic of fibroblasts, and showed marked induction of fibroblastic genes including αSMA and collagen1 (all p<0.0001). To confirm that αv integrins present on PDGFRβ cells critically regulate skeletal muscle fibrosis we used Itgavflox/flox;PDGFRβ-Cre mice (transgenic mice in which αv integrins are ‘knocked-down’ in PDGFRβ+ cells). These mice were significantly protected from CTX induced fibrosis (p<0.01). To demonstrate potential clinical utility of targeting αv integrins, we used a small molecule inhibitor of αv integrins (CWHM12). Treatment with CWHM12 significantly reduced fibrosis when delivered from the time of injury (p<0.01) and when delivered after the fibrotic response had become established (p<0.01). We have identified a core pathway regulating fibrosis in skeletal muscle. Pharmacologic inhibition of αv integrins has potential clinical utility in the treatment and prevention of skeletal muscle fibrosis