Jinan Li

The plasminogen activator/plasmin system is essential for development of the joint inflammatory phase of collagen type II-induced arthritis.

The plasminogen activator (PA) system has been proposed to have important roles in rheumatoid arthritis. Here we have used the autoimmune collagen type II (CII)-induced arthritis (CIA) model and mice deficient for urokinase-type PA (uPA) or plasminogen to investigate the role of the PA system for development of arthritis. Our data revealed that uPA-deficient mice have a lower severity and incidence of CIA than wild-type mice. Furthermore, although >80% of wild-type control mice developed CIA, we found that none of the 50 plasminogen-deficient littermates that were tested developed CIA within a 40-day period. Antibody generation after CII immunization as well as the binding of labeled anti-CII antibodies to the surface of cartilage were similar in wild-type and plasminogen-deficient mice. No sign of inflammation was seen when plasminogen-deficient mice were injected with a mixture of monoclonal antibodies against CII. However, after daily injections of human plasminogen, these mice developed arthritis within 5 days. Our finding that infiltration of inflammatory cells into the synovial joints was impaired in plasminogen-deficient mice suggests that uPA and plasminogen are important mediators of joint inflammation. Active plasmin is therefore essential for the induction of pathological inflammatory joint destruction in CIA.

Protective Effects of Plasmin(ogen) in a Mouse Model of Staphylococcus aureus-Induced Arthritis

OBJECTIVE To assess the functional roles of plasmin in a murine model of Staphylococcus aureus-induced bacterial arthritis. METHODS Bacterial arthritis was induced in plasminogen-deficient (Plg(-/-)) and wild-type (Plg(+/+)) littermates by local injection of 1 x 10(6) colony-forming units of S aureus into the knee joints. Human plasminogen was administered to Plg(-/-) mice on days 0-7 or days 7-14. Antibiotic treatment was administered to Plg(-/-) mice on days 7-14. Bacteria counts and histologic, immunohistochemical, and Western blot analyses were performed. RESULTS In Plg(+/+) mice, S aureus counts had declined within 2 days, and by day 28 the bacteria had been completely eliminated. However, S aureus was still detectable in all injected joints from Plg(-/-) mice, and bacteria counts were 27 times higher than the amount injected on day 0. The extent of macrophage and neutrophil recruitment to the infected joints was comparable for Plg(+/+) and Plg(-/-) mice on days 1, 7, and 14. The activation of these inflammatory cells appeared to be impaired in Plg(-/-) mice, however. Treatment of Plg(-/-) mice with antibiotic (cloxacillin) resulted in successful killing of the bacteria, but the necrotic tissue remained in the infected joints. When human plasminogen was given intravenously to Plg(-/-) mice daily for 7 days, bacterial clearance was greatly improved as compared with their untreated counterparts, and the amount of necrotic tissue in the joint cavity was dramatically reduced. The expression of interleukin 6 (IL-6) and IL-10 was higher in Plg(+/+) mice than in Plg(-/-) mice during bacterial arthritis. CONCLUSION Our findings indicate that plasmin plays a pluripotent role in protecting against S aureus-induced arthritis by activating inflammatory cells, killing bacteria, removing necrotic tissue, and enhancing cytokine expression.

Plasminogen is a key proinflammatory regulator that accelerates the healing of acute and diabetic wounds

Despite decades of research on wound healing, effective biologic agents for the treatment of chronic wounds, especially diabetic wounds, are still lacking. In the present study, we report that the inert plasma protein plasminogen (plg) acts as a key regulatory molecule that potentiates wound healing in mice. Early in the healing process, plg bound to inflammatory cells is transported to the wound area, where the level of plg is increased locally, leading to the induction of cytokines and intracellular signaling events and to a potentiation of the early inflammatory response. Systemic administration of additional plg not only accelerates the healing of acute burn wounds in wild-type mice, but also improves the healing of chronic diabetic wounds in a mouse model of diabetes. Our results suggest that the administration of plg may be a novel therapeutic strategy to treat many different types of wounds, especially chronic wounds such as those caused by diabetes.

Plasmin/plasminogen is essential for the healing of tympanic membrane perforations

Plasminogen has been proposed to play an important role in different tissue remodeling processes such as wound healing and tissue regeneration after injuries. The healing of tympanic membrane perforations is a well-organized chain of inflammatory events, with an initial invasion of inflammatory cells followed by reparative and restoration phases. Here we show that the healing of tympanic membrane perforations is completely arrested in plasminogen-deficient mice, with no signs of any healing even 143 days after perforation. Inflammatory cells were recruited to the wounded area, but there were no signs of tissue debridement. In addition, removal of fibrin, keratinocyte migration and in-growth of connective tissue were impaired. This contrasts with skin wound healing, where studies have shown that, although the healing process is delayed, it reaches completion in all plasminogen-deficient mice. Our finding that keratinocyte migration and re-epithelialization were completely arrested in plasminogen-deficient mice indicates that plasminogen/plasmin plays a more profound role in the healing of tympanic membrane perforations than in the healing of other epithelial wounds.

Beneficial and Detrimental Effects of Plasmin(ogen) during Infection and Sepsis in Mice

Plasmin has been proposed to be an important mediator during inflammation/infection. In this study, by using mice lacking genes for plasminogen, tissue-type plasminogen activator (tPA), and urokinase-type PA (uPA), we have investigated the functional roles of active plasmin in infection and sepsis. Two models were used: an infection model by intravenous injection of 1×107 CFU of S. aureus, and a sepsis model by intravenous injection of 1.6×108 CFU of S. aureus. We found that in the infection model, wild-type (WT) mice showed significantly higher survival rates than plasminogen-deficient (plg-/-) mice. However, in the sepsis model, plg-/- or tPA-/-/uPA-/- mice showed the highest survival rate whereas WT and tPA+/-/uPA+/- mice showed the lowest survival rate, and plg+/-, tPA-/-, and uPA-/- mice had an intermediate survival rate. These results indicate that the levels of active plasmin are critical in determining the survival rate in the sepsis, partly through high levels of inflammatory cytokines and enhanced STAT3 activation. We conclude that plasmin is beneficial in infection but promotes the production of inflammatory cytokines in sepsis that may cause tissue destruction, diminished neutrophil function, and an impaired capacity to kill bacteria which eventually causes death of these mice.

Plasmin Is Essential in Preventing Periodontitis in Mice

Periodontitis involves bacterial infection, inflammation of the periodontium, degradation of gum tissue, and alveolar bone resorption, which eventually leads to loss of teeth. To study the role of the broad-spectrum protease plasmin in periodontitis, we examined the oral health of plasminogen (Plg)-deficient mice. In wild-type mice, the periodontium was unaffected at all time points studied; in Plg-deficient mice, periodontitis progressed rapidly, within 20 weeks. Morphological study results of Plg-deficient mice revealed detachment of gingival tissue, resorption of the cementum layer, formation of necrotic tissue, and severe alveolar bone degradation. IHC staining showed massive infiltration of neutrophils in the periodontal tissues. Interestingly, doubly deficient mice, lacking both tissue- and urokinase-type plasminogen activators, developed periodontal disease similar to that in Plg-deficient mice; however, mice lacking only tissue- or urokinase-type plasminogen activator remained healthy. Supplementation by injection of Plg-deficient mice with human plasminogen for 10 days led to necrotic tissue absorption, inflammation subsidence, and full regeneration of gum tissues. Notably, there was also partial regrowth of degraded alveolar bone. Taken together, our results show that plasminogen is essential for the maintenance of a healthy periodontium and plays an important role in combating the spontaneous development of chronic periodontitis. Moreover, reversal to healthy status after supplementation of Plg-deficient mice with plasminogen suggests the possibility of using plasminogen for therapy of periodontal diseases.

Plasminogen initiates and potentiates the healing of acute and chronic tympanic membrane perforations in mice

BackgroundMost tympanic membrane (TM) perforations heal spontaneously, but approximately 10-20% remain open as chronic TM perforations. Chronic perforations can lead to an impaired hearing ability and recurrent middle ear infections. Traditionally, these perforations must be surgically closed, which is costly and time consuming. Therefore, there is a need for simpler therapeutic strategies. Previous studies by us have shown that plasminogen (plg) is a potent pro-inflammatory regulator that accelerates cutaneous wound healing in mice. We have also shown that the healing of TM perforations is completely arrested in plg-deficient (plg-/-) mice and that these mice develop chronic TM perforations. In the present study, we investigated the therapeutic potential of local plg injection in acute and chronic TM perforation mice models.MethodsPlg-/- mice and wild-type mice were subjected to standardized TM perforations followed by local injection of plg into the soft tissue surrounding the TM. TM perforations with chronic characteristics were induced by leaving TM perforations in plg-/- mice untreated for 9 days before treatment. The healing process was observed through otomicroscope and finally confirmed by immunostaining. The quality of TM healing was evaluated based on the morphology of the TM.ResultDaily local injections of plg into the soft tissue surrounding the TM restored the ability to heal TM perforations in plg-/- mice in a dose-dependent manner, and potentiated the healing rate and quality in wild-type mice. A single local injection of plg initiated the healing of the chronic-like TM perforations in these mice, resulting in a closed TM with a continuous but rather thick outer keratinocyte layer. However, three plg injections led to a completely healed TM with a thin keratinizing squamous epithelium covering a connective tissue layer.ConclusionOur data suggests that plg is a promising drug candidate for the treatment of chronic TM perforations in humans.

Lack of plasminogen does not alter the early inflammatory response following a tympanic membrane perforation: a study in plasminogen-deficient mice.

Conclusions. The results of the present study show that the early inflammatory response in plasminogen (plg)-deficient mice is not altered compared to that in wild-type (wt) mice. Therefore the chronicity of the perforation in the long-term healing experiment cannot be explained by an impairment of the early inflammatory response, but rather by an impairment in activation of the inflammatory cells. These findings give further insight into the mechanisms resulting in a clinically seen chronic tympanic membrane (TM) perforation and thus possible therapeutic strategies to replace todays conventional surgical treatment of these perforations. Objectives. Plg has been shown to play an essential role in the healing of TM perforations. In plg-deficient mice a completely arrested healing reaction was seen, resulting in a chronic TM perforation. The mechanisms involved seem to be an abundant neutrophil recruitment, an accumulation of macrophages, an arrested keratinocyte migration, and a massive deposition of fibrin along the TM tissue. However, the exact functional role of plg in the early inflammatory response during healing of TM perforation remains unclear. This study aimed to evaluate the early inflammatory response, mainly the occurrence of macrophages and neutrophils, during the first 48 h following a perforation in the pars tensa (PT) of the TM, in mice lacking the plasminogen gene compared to the corresponding response in wt mice. Materials and methods. The TMs were perforated in 45 plg-deficient and 39 wt mice. Otomicroscopic evaluation was performed at 3, 6, 9, 12, 18, 24, and 48 h after the perforation was made. Mice were harvested at all time points and prepared for morphology including immunohistochemistry (IHC). IHC was performed with antibodies targeting macrophages, neutrophils, T and B cells, cytokeratin, and fibrin(ogen). Morphometry was performed regarding the volume percentage of TM tissue occupied by the different inflammatory cells. Results. Perforation of the TM resulted in early otomicroscopic changes of the pars flaccida (PF) in both genotypes. Infiltration of inflammatory cells to PF and the presence of edema occurred as early as 6 h after the perforation was made, in both plg-deficient and wt mice. Morphometry did not reveal any significant differences between the genotypes concerning the occurrence of inflammatory cells. In contrast to the PF, the PT showed only sparse reactions during the experimental period. Furthermore, the migration pattern of keratinocytes did not differ between the genotypes throughout the experimental period.

Plasminogen is a critical regulator of cutaneous wound healing

Wound healing is a complicated biological process that consist of partially overlapping inflammatory, proliferation and tissue remodelling phases. A successful wound healing depends on a proper activation and subsequent termination of the inflammatory phase. The failure to terminate the inflammation halts the completion of wound healing and is a known reason for formation of chronic wounds. Previous studies have shown that wound closure is delayed in plasminogen-deficient mice, and a role for plasminogen in dissection of extracellular matrix was suggested. However, our finding that plasminogen is transported to the wound by inflammatory cells early during the healing process, where it potentiates inflammation, indicates that plasminogen may also have other roles in the wound healing process. Here we report that plasminogen-deficient mice have extensive fibrin and neutrophil depositions in the wounded area long after re-epithelialisation, indicating inefficient debridement and chronic inflammation. Delayed formation of granulation tissue suggests that fibroblast function is impaired in the absence of plasminogen. Therefore, in addition to its role in the activation of inflammation, plasminogen is also crucial for subsequent steps, including resolution of inflammation and activation of the proliferation phase. Importantly, supplementation of plasminogen-deficient mice with human plasminogen leads to a restored healing process that is comparable to that in wild-type mice. Besides of being an activator of the inflammatory phase during wound healing, plasminogen is also required for the subsequent termination of inflammation. Based on these results, we propose that plasminogen may be an important future therapeutic agent for wound treatment.