Severe burn, blast and neurologic injuries, and certain orthopaedic surgeries, can cause muscle and other soft tissues to “calcify” — harden from deposits of calcium-phosphate crystals, the same crystals found in bone. Pathologic calcification, which can induce bone formation (heterotopic ossification), has been poorly understood, and treatments for it are lacking, said Jonathan Schoenecker, M.D., Ph.D., assistant professor of Orthopaedic Surgery and Rehabilitation.
Now, Schoenecker and his colleagues have made the surprising discovery that the protease plasmin, known for its clot-busting role in the blood, also protects soft tissue from pathologic calcification. The findings, reported in the Journal of Bone and Mineral Research, could lead to new treatments to protect soft tissue from calcification after severe injury or surgery.
To understand why bone develops in soft tissues, researchers typically focus on factors that promote bone formation, such as bone morphogenetic protein (BMP). Although aberrant signaling of the BMP pathway may contribute to trauma-induced muscle calcification, attempts to stop it by blocking bone formation could impact bone biology and harm bones.
“The most ideal treatment would be one that prevents calcification in soft tissue and is also good for bone biology,” Schoenecker said. “And that’s what we have discovered. Plasmin prevents calcification in muscle, and it is also essential for fracture healing and protecting against diseases such as osteoporosis.”
The researchers used mouse models to study the impact of reduced plasmin levels in the setting of muscle injury. In mice with reduced plasmin and Duchenne muscular dystrophy — where muscle injury happens continuously — they found that muscle begins developing calcifications immediately after birth.
“If you completely remove plasmin in a mouse with Duchenne muscular dystrophy, skeletal muscle rapidly calcifies. It’s incredible,” Schoenecker said.
The researchers also demonstrated that even in wild-type (normal) animals, plasmin is required to prevent calcification and heterotopic ossification after acute muscle injury.
In a previous study, Schoenecker’s team showed that plasmin-mediated removal of the coagulation (clotting) matrix protein fibrin is essential for bone fracture healing. Plasmin, therefore, is beneficial for both bone healing and for protection of soft tissue from calcification.
“Surprisingly, unlike fracture repair, plasmin protection against calcification is not through its classical role of dissolving the coagulation matrix protein fibrin, meaning that plasmin’s mechanism in muscle is likely different than that in bone,” Schoenecker said.
“With all that we are learning about this protease (plasmin), it is becoming clear that its biological role goes well beyond degrading fibrin clots. It is a protease that is specifically activated in virtually all areas of injury and is involved in many tissue repair networks,” Schoenecker said.
“As such, we think of plasmin as a life raft for injury. It is a singular variable that affects tissue repair, and our current findings indicate that it also can influence whether injured muscle will heal or get calcified, which is super exciting. We’re working now on ways to maximize the plasmin system.”
The team has used a genetic therapy called antisense oligonucleotides (ASOs) to remove the plasmin inhibitor alpha2-antiplasmin. In mouse muscle injury models, the ASO therapy prevented soft tissue calcification and promoted muscle repair. The researchers are moving toward clinical studies with the ASO therapy, and they are working to purify recombinant plasmin for testing in animal models.
“The idea that we can control soft tissue calcification by targeting a seemingly unrelated protease is so exciting,” Schoenecker said. “Cardiologists and neurologists have been targeting this system inside blood vessels for decades — using TPA (tissue plasminogen activator) — and now we need to manipulate this system in the extravascular space, in the soft tissues, to prevent calcification.”
To explore the clinical importance of these findings, Schoenecker and his colleagues are now examining the systemic changes that happen in conditions most associated with muscle becoming bone — burn, blast, head and spinal cord injuries.
They have teamed with Edward Sherwood, M.D., Ph.D., Blair Summitt, M.D., and Lisa Rae, M.D., in the burn unit. In preliminary work, they found that burn patients commonly experience a reduction in fibrinolysis — the process that dissolves fibrin clots. Plasmin is the main protease responsible for fibrinolysis, and levels of plasmin fall after severe injury. The researchers hope the current findings will lead to clinical trials designed to improve tissue repair and prevent calcification in these patients, Schoenecker said.
Nicholas Mignemi, Ph.D., Masato Yuasa, M.D., Courtney Baker and Stephanie Moore led the research, which was supported by The Fighting Duchenne Foundation, the National Institutes of Health (grants HL007751, GM007628, AR065762, RR027631, DK007061), the Vanderbilt Institute for Clinical and Translational Research, the Howard Hughes Medical Institute, the Vanderbilt Orthopaedic Institute and the Caitlin Lovejoy Fund.
