Rima Sulniute

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 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.

Increased Eotaxin and MCP-1 Levels in Serum from Individuals with Periodontitis and in Human Gingival Fibroblasts Exposed to Pro-Inflammatory Cytokines

Periodontitis is a chronic inflammatory disease of tooth supporting tissues resulting in periodontal tissue destruction, which may ultimately lead to tooth loss. The disease is characterized by continuous leukocyte infiltration, likely mediated by local chemokine production but the pathogenic mechanisms are not fully elucidated. There are no reliable serologic biomarkers for the diagnosis of periodontitis, which is today based solely on the degree of local tissue destruction, and there is no available biological treatment tool. Prompted by the increasing interest in periodontitis and systemic inflammatory mediators we mapped serum cytokine and chemokine levels from periodontitis subjects and healthy controls. We used multivariate partial least squares (PLS) modeling and identified monocyte chemoattractant protein-1 (MCP-1) and eotaxin as clearly associated with periodontitis along with C-reactive protein (CRP), years of smoking and age, whereas the number of remaining teeth was associated with being healthy. Moreover, body mass index correlated significantly with serum MCP-1 and CRP, but not with eotaxin. We detected higher MCP-1 protein levels in inflamed gingival connective tissue compared to healthy but the eotaxin levels were undetectable. Primary human gingival fibroblasts displayed strongly increased expression of MCP-1 and eotaxin mRNA and protein when challenged with tumor necrosis factor-α (TNF-α and interleukin-1β (IL-1β), key mediators of periodontal inflammation. We also demonstrated that the upregulated chemokine expression was dependent on the NF-κΒ pathway. In summary, we identify higher levels of CRP, eotaxin and MCP-1 in serum of periodontitis patients. This, together with our finding that both CRP and MCP-1 correlates with BMI points towards an increased systemic inflammatory load in patients with periodontitis and high BMI. Targeting eotaxin and MCP-1 in periodontitis may result in reduced leukocyte infiltration and inflammation in periodontitis and maybe prevent tooth loss.

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.

CCL11, a novel mediator of inflammatory bone resorption

Normal bone homeostasis, which is regulated by bone-resorbing osteoclasts and bone-forming osteoblasts is perturbed by inflammation. In chronic inflammatory disease with disturbed bone remodelling, e.g. rheumatoid arthritis, patients show increased serum levels of the chemokine eotaxin-1 (CCL11). Herein, we demonstrate an inflammatory driven expression of CCL11 in bone tissue and a novel role of CCL11 in osteoclast migration and resorption. Using an inflammatory bone lesion model and primary cell cultures, we discovered that osteoblasts express CCL11 in vivo and in vitro and that expression increased during inflammatory conditions. Osteoclasts did not express CCL11, but the high affinity receptor CCR3 was significantly upregulated during osteoclast differentiation and found to colocalise with CCL11. Exogenous CCL11 was internalised in osteoclast and stimulated the migration of pre-osteoclast and concomitant increase in bone resorption. Our data pinpoints that the CCL11/CCR3 pathway could be a new target for treatment of inflammatory bone resorption.

Lack of SIRPα phosphorylation and concomitantly reduced SHP-2-PI3K-Akt2 signaling decrease osteoblast differentiation.

Normal differentiation of bone forming osteoblasts is a prerequisite for maintenance of skeletal health and is dependent on intricate cellular signaling pathways, including the essential transcription factor Runx2. The cell surface glycoprotein CD47 and its receptor signal regulatory protein alpha (SIRPα) have both been suggested to regulate bone cell differentiation. Here we investigated osteoblastic differentiation of bone marrow stromal cells from SIRPα mutant mice lacking the cytoplasmic signaling domain of SIRPα. An impaired osteoblastogenesis in SIRPα-mutant cell cultures was demonstrated by lower alkaline phosphatase activity and less mineral formation compared to wild-type cultures. This reduced osteoblastic differentiation potential in SIRPα-mutant stromal cells was associated with a significantly reduced expression of Runx2, osterix, osteocalcin, and alkaline phosphatase mRNA, as well as a reduced phosphorylation of SHP-2 and Akt2, as compared with that in wild-type stromal cells. Addition of a PI3K-inhibitor to wild-type stromal cells could mimic the impaired osteoblastogenesis seen in SIRPα-mutant cells. In conclusion, our data suggest that SIRPα signaling through SHP-2-PI3K-Akt2 strongly influences osteoblast differentiation from bone marrow stromal cells.

Non-Hematopoietic and Hematopoietic SIRPα Signaling Differently Regulates Murine B Cell Maturation in Bone Marrow and Spleen.

B lymphocyte development occurs in the bone marrow, while final differentiation and maturation can occur in both the bone marrow and the spleen. Here we provide evidence that signal regulatory protein α (SIRPα), an Ig-superfamily ITIM-receptor expressed by myeloid but not by lymphoid cells, is involved in regulating B cell maturation. Lack of SIRPα signaling in adult SIRPα-mutant mice resulted in a reduced maturation of B cells in the bone marrow, evident by reduced numbers of semi-mature IgD+IgMhi follicular type-II (F-II) and mature IgD+IgMlo follicular type-I (F-I) B cells, as well as reduced blood B cell numbers. In addition, lack of SIRPα signaling also impaired follicular B cell maturation in the spleen. Maturing BM or splenic B cells of SIRPα-mutant mice were found to express higher levels of the pro-apoptotic protein BIM and apoptosis was increased among these B cells. Bone marrow reconstitution experiments revealed that the B cell maturation defect in bone marrow and blood was due to lack of SIRPα signaling in non-hematopoietic cells, while hematopoietic SIRPα signaling was important for follicular B cell maturation in the spleen. Adding on to our previous findings of a stromal cell defect in SIRPα-mutant mice was the finding that gene expression of receptor activator of nuclear factor-ĸB ligand (RANKL) was significantly lower in cultured bone marrow stromal cells of SIRPα mutant mice. These data suggest a novel and opposite contribution of SIRPα signaling within non-hematopoietic and hematopoietic cells, respectively, to maintain B cell maturation and to prevent apoptosis in the bone marrow and spleen of adult mice.