Igor Prudovsky, PhD, DSc
Faculty Scientist II
Center for Molecular Medicine

Damien Carter, MD
Critical Care Surgeon
Maine Medical Center

Prudovsky Lab & Tissue Repair and Regeneration Program

The Prudovsky laboratory studies molecular mechanisms underlying tissue repair.

The Tissue Repair and Regeneration Program is a collaboration between Drs. Prudovsky and Carter.  This work brings together basic or bench science (Prudovsky Lab) and the clinical expertise in the area of burn trauma (Dr. Carter) to develop a better understanding of tissue repair and regeneration.

Mitochondria as a Target for Trauma Repair

Figure 1: Tranexamic acid (TXA) increases mitochondrial respiration and ATP production in endothelial cells: study using the Seahorse analyzer.

Severe trauma results in the dysregulation of mitochondrial respiration leading to cells damage, and systemic inflammation. The search for efficient methods to enhance mitochondria function can result in the improvement of trauma treatment methods. Tranexamic acid (TXA) is a popular anti-fibrinolytic drug widely used in hemorrhagic trauma, cardiovascular surgery and in orthopedic surgery patients. TXA binds plasminogen and prevents its maturation to the fibrinolytic enzyme plasmin. A number of studies demonstrated broad life-saving effects of TXA in trauma, superior to those of other anti-fibrinolytic agents. Besides preventing fibrinolysis and resulting blood loss, TXA has been reported to suppress post-traumatic inflammation and edema.  Although the efficiency of TXA transcends simple inhibition of fibrinolysis, very little is known about cell and molecular mechanisms underlying its extensive activities. The major events taking place after severe trauma, are metabolic shock, release to the bloodstream of pro-inflammatory Damage Associated Molecular Patterns (DAMP), and shedding of endothelial glycocalyx.

In our studies in collaboration with Drs. Damien Carter, Joseph Rappold, and Robert Kramer (all surgeons from Maine Medical Center), we have found that TXA enhances the efficiency of mitochondrial respiration in endothelial cells (Figure 1) increases the length of mitochondria (Figure 2), suppresses the burn injury-induced release of a major DAMP, mitochondrial DNA to mouse bloodstream (Figure 3), and decreases the burn-induced lung inflammation in mice (Figure 4). Interestingly, using plasminogen null mice we found that inhibition of the endotoxin-induced expression of two major proinflammatory cytokines, IL1alpha (Figure 5) and TNFalpha (not shown) is not dependent on the inhibition of plasminogen, the classical target of TXA. We suggest that enhancement of mitochondrial respiration by TXA suppresses DAMP release and metabolic stress, resulting in decreased inflammation and edema, and improved outcomes after severe trauma. (Figure 6).  We are currently studying the effects of TXA on the healing of burn wounds and on burn-induced edema. The effects of other compounds enhancing mitochondrial respiration on burn injury and other types of severe trauma are also studied in our laboratory.

Figure 2. TXA treatment increases the length of mitochondria

Figure 3

Figure 3. TXA treatment suppresses the release of mitochondrial DNA to bloodstream of mice after severe burn.

Figure 4

Figure 4.TXA suppresses macrophage invasion (macrophages are brown) to mouse lungs after severe burn.

Figure 5

Figure 5.TXA inhibits the endotoxin-induced expression of inflammatory cytokine IL1alpha in mouse hearts independently of plasminogen (Plg). RT-qPCR results.

Figure 6

Figure 6. Enhancement of mitochondrial metabolism by TXA suppresses systemic inflammatory syndrome (SIRS) and tissue edema caused by trauma. Hypothetic scheme.

CTHRC1, Protein Involved in Trauma Repair Suppresses Fat Cells Formation

Recent population studies indicate that obesity prevalence among the US adult population is nearing 40%. Obesity presents medical complications such as cardiovascular disease, diabetes, and certain types of cancer, as well as financial burden. The obesity epidemic stresses the importance of studies focused on mechanisms responsible for adiposity regulation. The laboratory of our colleague Volkhard Lindner found that mice with knockout of the CTHRC1 gene, coding for secreted protein involved in tissue repair, exhibit increased adiposity. Because of our long-term interest in the mechanisms of tissue repair, especially the repair-related decrease of fat tissue, we started a collaboration aimed to understand the molecular mechanisms of CTHRC1 activities. We have found that CTHRC1 suppresses the differentiation of preadipocytes (precursors of fat cells) to adipocytes (mature fat cells) (Figure 7). We have also found that recombinant CTHRC1 strongly decreases the expression of major transcription factors participating in adipogenesis. Our aim is to determine the mechanisms of CTHRC1 signaling underlying its anti-adipogenic effect and understand the molecular link between the suppression of adipogenesis and stimulation of trauma repair.

Figure 7. Recombinant CTHRC1 suppresses the chemically induced differentiation of 3T3-L1 preadipocytes (A,B). In comparison to wildtype cells stromal adipose cells from CTHRC1 null mice exhibit higher differentiation after chemical induction (C,D). Differentiated fat cells detected using Oil Red O staining.

Biology of FGF1, A Potent Stimulator of Trauma Repair:  Non-Classical Release and Regulated Proliferative Activity

Fibroblast growth factor 1 (FGF1), a ubiquitously expressed growth factor efficiently stimulates trauma repair. However, many aspects of its biology remain obscure which prevents more efficient exploitation of its beneficial characteristics. Unlike other secreted proteins, FGF1 lacks a signal peptide for classical secretion, and is released through Golgi-independent pathways. Cell stress induces the export of FGF1 through the limited domains of cells membrane (Figure 8). At stress, FGF1 translocates to the vicinity of cell membrane, where it co-localizes with cortical actin cytoskeleton to be further exported to the extracellular milieu. We found that the export of FGF1 relies on copper-dependent formation of a complex containing several secreted signal-peptide-less proteins: S100A13, sphingosine kinase 1 (SphK1), 40 kDa form of synaptotagmin 1 (p40 Syt1) and annexin II (Anx II). The export of FGF1 release complex depends on transmembrane translocation of acidic phospholipids (Figure 9). Our current aims are to understand how the release complex assembles and what is the molecular mechanism of its transmembrane translocation. In addition, to study the regulation of FGF1 export in vivo, we produced transgenic mice with FGF1 expression in endothelial cells and macrophages, and these animals exhibit a decrease of obesity induced by high fat diet.

Figure 8. FGF1 is secreted from stressed cells though limited domains of cell surface.

Figure 9. Stress-induced FGF1 export. Transmembrane translocation of copper-dependent FGF1 release complex is drawn by externalization of acidic phospholipids

We found that several cell types of mesenchymal origin respond to FGF1 presented as a single mitogen by a strictly limited proliferation. They undergo one cell cycle and then get blocked in the non-proliferating state characterized by dramatic accumulation of cell cycle inhibitors (Figure 10) and active FGFR/Erk signaling. We hypothesize that the restricted character of FGF-induced proliferative response prevents hyperplasia in damaged tissue, where FGF is released under stress conditions. We are currently studying the molecular mechanisms, which limit the growth of FGF-stimulated cells.

Figure 10. Unlike serum-stimulated cells, FGF1 treated fibroblasts express large amounts of cell cycle inhibitor p21 (green). Red – nuclei.