The study aims to map plasma gelsolin (pGSN) levels in diabetic humans and mice models of type II diabetes and to evaluate the efficacy of gelsolin therapy in improvement of diabetes in mice. We report that pGSN values decrease by a factor of 0.45 to 0.5 in the blood of type II diabetic humans and mice models. Oral glucose tolerance test in mice models showed that subcutaneous administration of recombinant pGSN and its F-actin depolymerizing competent versions brought down blood sugar levels comparable to Sitagliptin, a drug used to manage hyperglycemic condition. Further, daily dose of pGSN or its truncated versions to diabetic mice for a week kept sugar levels close to normal values. Also, diabetic mice treated with Sitagliptin for 7 days, showed increase in their pGSN values with the decrease in blood glucose as compared to their levels at the start of treatment. Gelsolin helped in improving glycemic control in diabetic mice. We propose that gelsolin level monitoring and replacement of F-actin severing capable gelsolin(s) should be considered in diabetic care.

Delineation of factors which affect wound healing would be of immense value to enable on-time or early healing and reduce comorbidities associated with infections or biochemical stress like diabetes. Plasma gelsolin has been identified earlier to significantly enable injury recovery compared to placebo. This study evaluates the role of rhuGSN for its antioxidant and wound healing properties in murine fibroblasts (3T3-L1 cell line). Total antioxidant capacity of rhuGSN increased in a concentration-dependent manner (0.75-200 μg/mL). Cells pretreated with 0.375 and 0.75 μg/mL rhuGSN for 24 h exhibited a significant increase in viability in a MTT assay. Preincubation of cells with rhuGSN for 24 h followed by oxidative stress induced by exposure to H2O2 for 3 h showed cytoprotective effect. rhuGSN at 12.5 and 25 μg/mL concentration showed an enhanced cell migration after 20 h of injury in a scratch wound healing assay. The proinflammatory cytokine IL-6 levels were elevated in the culture supernatant. These results establish an effective role of rhuGSN against oxidative stress induced by H2O2 and in wound healing of 3T3-L1 fibroblast cells.

This is the first report describing temperature based initiation of gelsolin's F-actin depolymerization activity, even in absence of free Ca2+ or low pH. Small angle X-ray scattering (SAXS) and circular dichroism (CD) studies revealed that temperature in the range of 30-40 °C is capable of opening the G1 domain alone, as remaining domains are held together by the Ca2+-sensitive C-tail latch without any loss in the secondary structural content. Full opening of all domains of tail-less gelsolin, and retention of closed shape for G2-G6 gelsolin merely by heating, further substantiated our findings. The Ca2+/pH independent activity of gelsolin near physiological temperature brought out a query: whether gelsolin is always active, and if not, what might deactivate it? Earlier, PIP2 has been reported to render gelsolin inactive with no structural insight. Reduction in shape parameters and modeling revealed that PIP2 reverses the temperature induced extension of g1-g2 linker leading to a compact shape seen for Ca2+-free gelsolin. Similar results for partially activated gelsolin (by low pH or Ca2+ ions below 0.1 μM) imply that inside cells, depolymerization, capping, and nucleation of F-actin by gelsolin is regulated by the culmination of local Ca2+ ion concentration, pH, temperature and PIP2 levels.

Here, we report that minimal functional gelsolin i.e. fragment 28-161 can display F-actin depolymerizing property even after heating the protein to 80 °C. Small angle X-ray scattering (SAXS) data analysis confirmed that under Ca2+-free conditions, 28-161 associates into monomer to dimer and tetramer, which later forms β-amyloids, but in presence of Ca2+, it forms dimers which proceed to non-characterizable aggregates. The dimeric association also explained the observed decrease in ellipticity in circular dichroism experiments with increase in temperature. Importantly, SAXS data based models correlated well with our crystal structure of dimeric state of 28-161. Characterization of higher order association by electron microscopy, Congo red and ThioflavinT staining assays further confirmed that only in absence of Ca2+ ions, heating transforms 28-161 into β-amyloids. Gel filtration and other experiments showed that β-amyloids keep leaching out the monomer, and the release rates could be enhanced by addition of L-Arg to the amyloids. F-actin depolymerization showed that addition of Ca2+ ions to released monomer initiated the depolymerization activity. Overall, we propose a way to compose a supramolecular assembly which releases functional protein in sustained manner which can be applied for varied potentially therapeutic interventions.

The present study provides first evidence on the role of plasma gelsolin in protecting pulmonary thromboembolism and thrombosis in a mouse model. Gelsolin is the most abundant actin depolymerizing protein in plasma and its significantly depleted values have been reported in metabolic disorders including cardiovascular diseases and myocardial infarction. Though gelsolin replacement therapy (GRT) has been shown to be effective in some animal models, no such study has been reported for thrombotic diseases that are acutely in need of bio-therapeutics for immediate and lasting relief. Here, using mice model and recombinant human gelsolin (rhuGSN), we demonstrate the antithrombotic effect of gelsolin in ferric chloride induced thrombosis in carotid artery and thrombin induced acute pulmonary thromboembolism. In thrombosis model, arterial occlusion time was significantly enhanced upon subcutaneous (SC) treatment with 8 mg of gelsolin per mice viz. 15.83 minutes vs. 8 minutes in the placebo group. Pertinently, histopathological examination showed channel formation within the thrombi in the carotid artery following injection of gelsolin. Fluorescence molecular tomography imaging further confirmed that administration of gelsolin reduced thrombus formation following carotid artery injury. In thrombin-induced acute pulmonary thromboembolism, mice pretreated with aspirin or gelsolin showed 100 and 83.33% recovery, respectively. In contrast, complete mortality of mice was observed in vehicle treated group within 5 minutes of thrombin injection. Overall, our studies provide conclusive evidence on the thrombo-protective role of plasma gelsolin in mice model of pulmonary thromboembolism and thrombosis.

Plasma gelsolin levels significantly decline in several disease conditions, since gelsolin gets scavenged when it depolymerizes and caps filamentous actin released in the circulation following tissue injury. It is well established that our body require/implement inflammatory and analgesic responses to protect against cell damage and injury to the tissue. This study was envisaged to examine analgesic and anti-inflammatory activity of exogenous gelsolin (8 mg/mouse) in mice models of pain and acute inflammation. Administration of gelsolin in acetic acid-induced writhing and tail immersion tests not only demonstrated a significant reduction in the number of acetic acid-induced writhing effects, but also exhibited an analgesic activity in tail immersion test in mice as compared to placebo treated mice. Additionally, anti-inflammatory function of gelsolin (8 mg/mouse) compared with anti-inflammatory drug diclofenac sodium (10 mg/kg)] was confirmed in the carrageenan injection induced paw edema where latter was measured by vernier caliper and fluorescent tomography imaging. Interestingly, results showed that plasma gelsolin was capable of reducing severity of inflammation in mice comparable to diclofenac sodium. Analysis of cytokines and histopathological examinations of tissue revealed administration of gelsolin and diclofenac sodium significantly reduced production of pro-inflammatory cytokines, TNF-α and IL-6. Additionally, carrageenan groups pretreated with diclofenac sodium or gelsolin showed a marked decrease in edema and infiltration of inflammatory cells in paw tissue. Our study provides evidence that administration of gelsolin can effectively reduce the pain and inflammation in mice model.