Reciprocal stabilization of coagulation factor XIII-A and -B subunits is a determinant of plasma FXIII concentration.

ABSTRACT: Transglutaminase factor XIII (FXIII) is essential for hemostasis, wound healing, and pregnancy maintenance. Plasma FXIII is composed of A and B subunit dimers synthesized in cells of hematopoietic origin and hepatocytes, respectively. The subunits associate tightly in circulation as FXIII-A2B2. FXIII-B2 stabilizes the (pro)active site-containing FXIII-A subunits. Interestingly, people with genetic FXIII-A deficiency have decreased FXIII-B2, and therapeutic infusion of recombinant FXIII-A2 (rFXIII-A2) increases FXIII-B2, suggesting FXIII-A regulates FXIII-B secretion, production, and/or clearance. We analyzed humans and mice with genetic FXIII-A deficiency and developed a mouse model of rFXIII-A2 infusion to define mechanisms mediating plasma FXIII-B levels. Like humans with FXIII-A deficiency, mice with genetic FXIII-A deficiency had reduced circulating FXIII-B2, and infusion of FXIII-A2 increased FXIII-B2. FXIII-A-deficient mice had normal hepatic function and did not store FXIII-B in liver, indicating FXIII-A does not mediate FXIII-B secretion. Transcriptional analysis and polysome profiling indicated similar F13b levels and ribosome occupancy in FXIII-A-sufficient and -deficient mice and in FXIII-A-deficient mice infused with rFXIII-A2, indicating FXIII-A does not induce de novo FXIII-B synthesis. Unexpectedly, pharmacokinetic/pharmacodynamic modeling of FXIII-B antigen after rFXIII-A2 infusion in humans and mice suggested FXIII-A2 slows FXIII-B2 loss from plasma. Accordingly, comparison of free FXIII-B2 vs FXIII-A2-complexed FXIII-B2 (FXIII-A2B2) infused into mice revealed faster clearance of free FXIII-B2. These data show FXIII-A2 prevents FXIII-B2 loss from circulation and establish the mechanism underlying FXIII-B2 behavior in FXIII-A deficiency and during rFXIII-A2 therapy. Our findings reveal a unique, reciprocal relationship between independently synthesized subunits that mediate an essential hemostatic protein in circulation. This trial was registered at www.ClinicalTrials.com as #NCT00978380.

The Effect of Activated FXIII, a Transglutaminase, on Vascular Smooth Muscle Cells.

Plasma factor XIII (pFXIII) is a heterotetramer of FXIII-A and FXIII-B subunits. The cellular form (cFXIII), a dimer of FXIII-A, is present in a number of cell types. Activated FXIII (FXIIIa), a transglutaminase, plays an important role in clot stabilization, wound healing, angiogenesis and maintenance of pregnancy. It has a direct effect on vascular endothelial cells and fibroblasts, which have been implicated in the development of atherosclerotic plaques. Our aim was to explore the effect of FXIIIa on human aortic smooth muscle cells (HAoSMCs), another major cell type in the atherosclerotic plaque. Osteoblastic transformation induced by Pi and Ca2+ failed to elicit the expression of cFXIII in HAoSMCs. EZ4U, CCK-8 and CytoSelect Wound Healing assays were used to investigate cell proliferation and migration. The Sircol Collagen Assay Kit was used to monitor collagen secretion. Thrombospondin-1 (TSP-1) levels were measured by ELISA. Cell-associated TSP-1 was detected by the immunofluorescence technique. The TSP-1 mRNA level was estimated by RT-qPCR. Activated recombinant cFXIII (rFXIIIa) increased cell proliferation and collagen secretion. In parallel, a 67% decrease in TSP-1 concentration in the medium and a 2.5-fold increase in cells were observed. TSP-1 mRNA did not change significantly. These effects of FXIIIa might contribute to the pathogenesis of atherosclerotic plaques.

Factor XIII-A in Diseases: Role Beyond Blood Coagulation.

Multidisciplinary research from the last few decades has revealed that Factor XIII subunit A (FXIII-A) is not only involved in blood coagulation, but may have roles in various diseases. Here, we aim to summarize data from studies involving patients with mutations in the F13A1 gene, performed in FXIII-A knock-out mice models, clinical and histological studies assessing correlations between diseases severity and FXIII-A levels, as well as from in vitro experiments. By providing a complex overview on its possible role in wound healing, chronic inflammatory bowel diseases, athe-rosclerosis, rheumatoid arthritis, chronic inflammatory lung diseases, chronic rhinosinusitis, solid tumors, hematological malignancies, and obesity, we also demonstrate how the field evolved from using FXIII-A as a marker to accept and understand its active role in inflammatory and malignant diseases.

F13A1 transglutaminase expression in human adipose tissue increases in acquired excess weight and associates with inflammatory status of adipocytes.

OBJECTIVE: F13A1/FXIII-A transglutaminase has been linked to adipogenesis in cells and to obesity in humans and mice, however, its role and associated molecular pathways in human acquired excess weight have not been explored. METHODS: We examined F13A1 expression and association to human weight gain in weight-discordant monozygotic twins (Heavy-Lean difference (ΔWeight, 16.8 kg ± 7.16 for n = 12). The twin pairs were examined for body composition (by dual-energy X-ray absorptiometry), abdominal body fat distribution (by magnetic resonance imaging), liver fat content (by magnetic resonance spectroscopy), circulating adipocytokines, leptin and adiponectin, as well as serum lipids. Affymetrix full transcriptome mRNA analysis was performed from adipose tissue and adipocyte-enriched fractions from subcutaneous abdominal adipose tissue biopsies. F13A1 differential expression between the heavy and lean co-twins was examined and its correlation transcriptome changes between co-twins were performed. RESULTS: F13A1 mRNA showed significant increase in adipose tissue (p < 0.0001) and an adipocyte-enriched fraction (p = 0.0012) of the heavier co-twin. F13A1 differential expression in adipose tissue (Heavy-Lean ΔF13A1) showed significant negative correlation with circulating adiponectin (p = 0.0195) and a positive correlation with ΔWeight (p = 0.034), ΔBodyFat (0.044) and ΔAdipocyte size (volume, p = 0.012;) in adipocyte-enriched fraction. A whole transcriptome-wide association study (TWAS) on ΔF13A1 vs weight-correlated ΔTranscriptome identified 182 F13A1-associated genes (r > 0.7, p = 0.05) with functions in several biological pathways including cell stress, inflammatory response, activation of cells/leukocytes, angiogenesis and extracellular matrix remodeling. F13A1 did not associate with liver fat accumulation. CONCLUSIONS: F13A1 levels in adipose tissue increase with acquired excess weight and associate with pro-inflammatory, cell stress and tissue remodeling pathways. This supports its role in expansion and inflammation of adipose tissue in obesity.

Distinct 3-disulfide-bonded isomers of tridegin differentially inhibit coagulation factor XIIIa: The influence of structural stability on bioactivity.

Tridegin is a 66mer cysteine-rich coagulation factor XIIIa (FXI-IIa) inhibitor from the giant amazon leech Haementeria ghilianii of yet unknown disulfide connectivity. This study covers the structural and functional characterization of five different 3-disulfide-bonded tridegin isomers. In addition to three previously identified isomers, one isomer containing the inhibitory cystine knot (ICK, knottin) motif, and one isomer with the leech antihemostatic protein (LAP) motif were synthesized in a regioselective manner. A fluorogenic enzyme activity assay revealed a positive correlation between the constriction of conformational flexibility in the N-terminal part of the peptide and the inhibitory potential towards FXI-IIa with clear differences between the isomers. This observation was supported by molecular dynamics (MD) simulations and subsequent molecular docking studies. The presented results provide detailed structure-activity relationship studies of different tridegin disulfide isomers towards FXI-IIa and reveal insights into the possibly existing native linkage compared to non-native disulfide tridegin species.

Let's cross-link: diverse functions of the promiscuous cellular transglutaminase factor XIII-A.

Essentials Plasma Factor XIII, a heterodimer of A and B subunits FXIIIA2 B2 , is a transglutaminase enzyme with a well-established role in haemostasis. Cells of bone marrow and mesenchymal lineage express the FXIII-A gene (F13A1) that encodes the cellular form of the transglutaminase, a homodimer of the A subunits, FXIII-A. FXIII-A was presumed to function intracellularly, however, several lines of evidence now indicate that FXIII-A is externalised by an as yet unknown mechanism This review describes the mounting evidence that FXIII-A is a diverse transglutaminase with many intracellular and extracellular substrates that can participate in an array of biological processes SUMMARY: Factor XIII is a tranglutaminase enzyme that catalyzes the formation of ε-(γ-glutamyl)lysyl isopeptide bonds in protein substrates. The plasma form, FXIII-A2 B2 , has an established function in hemostasis, where its primary substrate is fibrin. A deficiency in FXIII manifests as a severe bleeding diathesis, underscoring its importance in this pathway. The cellular form of the enzyme, a homodimer of the A-subunits, denoted FXIII-A, has not been studied in as extensive detail. FXIII-A was generally perceived to remain intracellular, owing to the lack of a classical signal peptide for its release. In the last decade, emerging evidence has revealed that this diverse transglutaminase can be externalized from cells, by an as yet unknown mechanism, and can cross-link extracellular substrates and participate in a number of diverse pathways. The FXIII-A gene (F13A1) is expressed in cells of bone marrow and mesenchymal lineage, notably megakaryocytes, monocytes/macrophages, dendritic cells, chrondrocytes, osteoblasts, and preadipocytes. The biological processes that FXIII-A is coupled with, such as wound healing, phagocytosis, and bone and matrix remodeling, reflect its expression in these cell types. This review describes the mounting evidence that this cellular transglutaminase can be externalized, usually in response to stimuli, and participate in extracellular cross-linking reactions. A corollary of being involved in these biological pathways is the participation of FXIII-A in pathological processes. In conclusion, the functions of this transglutaminase extend far beyond its role in hemostasis, and our understanding of this enzyme in terms of its secretion, regulation and substrates is in its infancy.

Factor XIIIA-expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking.

Lung cancer is the leading cause of cancer-related deaths worldwide, and lung squamous carcinomas (LUSC) represent about 30% of cases. Molecular aberrations in lung adenocarcinomas have allowed for effective targeted treatments, but corresponding therapeutic advances in LUSC have not materialized. However, immune checkpoint inhibitors in sub-populations of LUSC patients have led to exciting responses. Using computational analyses of The Cancer Genome Atlas, we identified a subset of LUSC tumors characterized by dense infiltration of inflammatory monocytes (IMs) and poor survival. With novel, immunocompetent metastasis models, we demonstrated that tumor cell derived CCL2-mediated recruitment of IMs is necessary and sufficient for LUSC metastasis. Pharmacologic inhibition of IM recruitment had substantial anti-metastatic effects. Notably, we show that IMs highly express Factor XIIIA, which promotes fibrin cross-linking to create a scaffold for LUSC cell invasion and metastases. Consistently, human LUSC samples containing extensive cross-linked fibrin in the microenvironment correlated with poor survival.

Coagulation F13A1 V34L, fibrinogen and homocysteine versus conventional risk factors in the pathogenesis of MI in young persons.

BACKGROUND: The pathogenesis of myocardial infarction (MI) involves environmental and genetic risk factors, with the latter putatively playing significant roles in younger patients. Genetic variability in coagulation factors comprises one such group. The coagulation factor 13 subunit A (F13A1) Val34Leu polymorphism (rs5985) has yielded variable findings in literature, with no prior South Asian data. METHODS: We studied the frequency of this polymorphism using the amplification-created restriction-enzyme site (ACRES) polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) in 101 MI patients aged below 40 years and 103 controls along with plasma fibrinogen and serum homocysteine levels. RESULTS: The distribution of Val/Val, Val/Leu and Leu/Leu genotypes was similar among cases (72.3%, 26.7% and 1.0%) and controls (78.6%, 19.4% and 1.9%, respectively). Val and Leu allele frequencies were 85.6% and 14.4% among patients and 88.3% and 11.7% among controls, respectively (p = .416). Mean plasma fibrinogen was higher in patients vis-à-vis controls (3.1 versus 3.7 g/l; p < .001) but homocysteine was elevated in both patients (52%) and controls (67%) (p = .225). Multivariate analysis revealed hypertension (p < .001, OR 6.16) and smoking (p < .001, OR 5.48) to impart strongest risk followed by positive family history, plasma fibrinogen levels and male gender. CONCLUSIONS: Despite its small sample size, this first South Asian study suggests neither protective nor deleterious effects of the F13A1 Val34Leu polymorphism on the risk of MI in young persons. The Leu allele frequency is intermediate to that reported from the West and the Far East. Traditional risk factors contribute greatly to risk even in younger MI patients in South Asia.

Transglutaminases factor XIII-A and TG2 regulate resorption, adipogenesis and plasma fibronectin homeostasis in bone and bone marrow.

Appropriate bone mass is maintained by bone-forming osteoblast and bone-resorbing osteoclasts. Mesenchymal stem cell (MSC) lineage cells control osteoclastogenesis via expression of RANKL and OPG (receptor activator of nuclear factor κB ligand and osteoprotegerin), which promote and inhibit bone resorption, respectively. Protein crosslinking enzymes transglutaminase 2 (TG2) and Factor XIII-A (FXIII-A) have been linked to activity of myeloid and MSC lineage cells; however, in vivo evidence has been lacking to support their function. In this study, we show in mice that TG2 and FXIII-A control monocyte-macrophage cell differentiation into osteoclasts as well as RANKL production in MSCs and in adipocytes. Long bones of mice lacking TG2 and FXIII-A transglutaminases, show compromised biomechanical properties and trabecular bone loss in axial and appendicular skeleton. This was caused by increased osteoclastogenesis, a cellular phenotype that persists in vitro. The increased potential of TG2 and FXIII-A deficient monocytes to form osteoclasts was reversed by chemical inhibition of TG activity, which revealed the presence of TG1 in osteoclasts and assigned different roles for the TGs as regulators of osteoclastogenesis. TG2- and FXIII-A-deficient mice had normal osteoblast activity, but increased bone marrow adipogenesis, MSCs lacking TG2 and FXIII-A showed high adipogenic potential and significantly increased RANKL expression as well as upregulated TG1 expression. Chemical inhibition of TG activity in the null cells further increased adipogenic potential and RANKL production. Altered differentiation of TG2 and FXIII-A null MSCs was associated with plasma fibronectin (FN) assembly defect in cultures and FN retention in serum and marrow in vivo instead of assembly into bone. Our findings provide new functions for TG2, FXIII-A and TG1 in bone cells and identify them as novel regulators of bone mass, plasma FN homeostasis, RANKL production and myeloid and MSC cell differentiation.

Coagulation Factor XIIIA (F13A1): Novel Perspectives in Treatment and Pharmacogenetics.

Factor XIII (FXIII) is a key molecule in the field of blood coagulation and in the last decades it has weakened attention within the field of angiogenesis and tissue repair. FXIII positively influences wound healing in several tissues by exerting multiple plasma and cellular functions. In the field of haemostasis, FXIII cross-links the neo formed fibrin fibers and supports platelet adhesion to the damaged sub-endothelium warranting a solid architecture. In addition, the pro-angiogenic functions of FXIII are directed by the interaction of vascular endothelial growth factor receptor 2 (VEGFR2) and the integrin αVß3, on the cell membrane, favouring an important step in the formation of granulation tissue at the wound site for optimal tissue healing. Conversely, the same mechanisms could lead to undesired increased neovascularisation, for example in inflammatory bowel disease or in the retinal degenerative pathologies. The classical symptoms of FXIII deficiency span from intracranial haemorrhage to delay bleeding or the staying of chronic wounds in the skin including impaired mucosal healing. In this view, FXIII bridges primary haemostasis, coagulation and definite tissue healing. Another important recently discovered function ascribed to FXIII is its ability to limit bacterial spreading from the lesion by incorporating specific macromolecules addressed to cellular infiltration, favouring in turn cell migration and survival, as observed also in fibrin-heart cultures for stem cell recruitment. In the field of the novel prognostic biomarkers, the monitoring of the residual circulating FXIII level during acute myocardial infarction has been considered predictive of the post-myocardial infarction healing. Accordingly, adequate FXIII levels can drive and predict the prognosis of complex diseases and the outcome of the associated therapies or interventions. In addition, peculiar pharmacogenetics aspects of the FXIII gene are of extraordinary interest. The present review accounts for the recognized role of FXIII in the healing process and gives some examples on how to use it as prognostic biological/ molecular marker or as potential tailored therapeutic molecule in complex diseases.

Neoplasm-induced bleeding in inherited, heterozygous FXIII-A deficiency.

UNLABELLED: Inherited mild factor XIII deficiency belongs to one of the most underdiagnosed bleeding disorders so far. This is, because most patients do not develop bleeding complications in daily life. Patient, methods: A man (age: 64 years) without a history of bleeding presented with painful swelling of neck, weight loss, anemia and episodic bleeding from the right tonsil necessitating tonsillectomy. Histologic and immunohistochemical evaluation revealed cytokeratin-positive epitheloid angiosarcoma. Blood coagulation status showed significantly elevated D-dimer and decreased FXIII levels (FXIII-activity 35%, FXIIIA-Ag 16-26%). Plasma mixing studies excluded neutralizing antibodies against FXIII. RESULTS: A novel heterozygous F13A1 gene nonsense mutation (p.Glu103Ter, c.307G>T) was found confirming heterozygous FXIII-A deficiency. The same mutation was detected in two further asymptomatic relatives. For further clinical management the patient was transfused with FXIII-concentrate and showed an adequate increase of FXIII ruling out FXIII deficiency to be induced by increased turnover. Despite this haemostatic management and antifibrinolytic treatment the patient had to undergo several revisions due to delayed, Hb relevant bleeding after cervical lymph nodes extirpation and resection of tonsil. Two chemotherapy cycles with paclitaxel and palliative radiotherapy of the neck area were performed, but the patient died unfortunately two months after diagnosis. CONCLUSIONS: It is a unique case showing the combination of a highly aggressive angiosarcoma and presence of inherited FXIII deficiency. It is also a rare example demonstrating the benefit of FXIII genotyping besides the expected acquired FXIII deficiency possibly due to neoplasm induced increased consumption by elevated crosslinking of fibrin fibers.

The activation peptide of coagulation factor XIII is vital for its expression and stability.

BACKGROUND: The human activation peptide of factor XIII (AP-FXIII) comprises the first 37 amino acids of the N-terminus and holds the FXIII in an inactive state. FXIII is activated either proteolytically by cleavage of AP-FXIII by thrombin, or non-proteolytically by high calcium concentrations. OBJECTIVE: To investigate the role of AP-FXIII in the expression and stability of FXIII. METHODS: We cloned 13 FXIII variants with progressive truncations of AP-FXIII from the N-terminus (delN-FXIII-A), expressed them in mammalian cells, and measured their thermostability, activation, and transglutaminase activity. We also used in silico calculations to analyze the stability of hypothetical delN-FXIII dimers and to identify crucial motifs within AP-FXIII. RESULTS: Variants with deletions longer than the first 10 amino acids and an R11Q point mutant were not expressed as proteins. In silico calculations indicated that the sequence (8) FGGR(12) R plays a substantial role in intersubunit interactions in FXIII-A2 homodimers. In agreement with this prediction, the temperature stability of delN-FXIII variants decreased with increasing length of deletion. These results may suggest a role of the N-terminus of AP-FXIII in dimer stability. Substantial sequence homology was found among activation peptides of vertebrate and even invertebrate (crustacean) FXIII-A orthologs, which further supports our conclusion. CONCLUSIONS: We conclude that deletion of 11 or more N-terminal amino acids disrupts intersubunit interactions, which may prevent FXIII-A2 homodimer formation. Therefore, AP-FXIII plays an important role in the stability of the FXIII-A2 dimer.

[Factor XIII-A subunit Val34Leu polymorphism and risk of venous thromboembolism in young adults].

Factor XIII (FXIII) plays an important role in formation and stabilization of the fibrin clot. It is known that FXIII-A gene G163T (Val34Leu) polymorphism leads to increased activation of this factor, however participation of the 34Leu variant in thrombogenesis remains disputable. The present work was aimed at studying peculiarities of distribution of variants of FXIII-A polymorphisms in patients with early onset of venous thromboembolism (VTE), as well as revealing associative links between the carrier state of this mutation and the character of clinical course of the disease. We examined a total of 250 patients with VTE. Of these, there were 119 (47.6%) men and 131 (52.4%) women, mean age 37.42 years (range 10-45 years). In the group of patients with VTE, the proportion of homozygous carriers of allele 163T (Leu34) turned out to be more than 1.5-fold higher as compared with the corresponding parameter in the control group (OR=1.8, 95% CI: 0.9-4.0; p=0.14). Analysing FXIII-A G163T polymorphism depending on gender revealed a considerable increase in the incidence rate (IR) of 163TT genotype in women with VTE as compared to male patients (13.0% versus 5.1 %, respectively, OR=2.8, 95% CI: 1.1-7.4; p=0.047) and to the control group (OR=2.7; 95% CI: 1.2-6.1; p=0.023). Analysing gene FXIII-A polymorphism in the groups of patients with various clinical manifestations of VTE revealed a decrease in the proportion of heterozygotes in the group of deep vein thrombosis + pulmonary artery thromboembolism (PATE) and, vice versa, an increase in the proportion of homozygotes by the Leu34 variant in patients with signs of PATE. The obtained findings make it possible to consider the genotype 163TT of FXIII-A gene as a new independent risk factor for the development of VTE in young women living in the North-West region of Russia, which is observed for the first time. Additional studies are necessary.

Prevalence of thrombophilic genetic factors among patients with retinitis pigmentosa.

PURPOSE: To determine the prevalence of thrombophilic factors in patients with retinitis pigmentosa (RP). METHODS: Fifty consecutive patients with RP and 50 controls matched by age and gender were tested for the presence of the following mutations: factor II (GA20210), factor V Leiden (GA1691), methylenetetrahydrofolate reductase (CT677), factor XIIIa (Val→Leu), ß-fibrinogen (GA455), tumor necrosis factor receptor (TNFRII) (M196R), plasminogen activator inhibitor-1 (PAI-1) (4 G/5 G), and plasminogen activator inhibitor-1 (PAI-1) (GA844). RESULTS: The following heterozygous mutations were found in patients/controls: factor V Leiden (12/14), factor XIIIa (20/30), methylenetetrahydrofolate reductase 677 TT (48/52), ß-fibrinogen GA455 (36/36), TNFRII (M196R) (40/42), PAI-1 4 G/5 G (40/48), and PAI-1 GA844 (50/52). The difference between patients with RP and the control group was not statistically significant for the prevalence of any of the studied factors (P > 0.05). CONCLUSION: In this study, thrombophilic mutations were not increased in patients with RP. Thrombophilic mutations do not seem to be risk factors for RP. Routine investigation of hereditary thrombophilia in these patients is not justified.

Effect of factor XIII-A Val34Leu polymorphism on myocardial infarction risk: a meta-analysis.

The association between factor XIII-A (FXIII-A) Val34Leu polymorphism and myocardial infarction (MI) risk remained controversial. We performed a meta-analysis. Online databases were searched. Twenty-eight studies were included. The FXIII-A Val34Leu polymorphism was significantly associated with MI risk (odds ratio (OR) = 0.83, 95% confidence interval [CI] 0.76-0.91; P < .0001). This result remained statistically significant when the adjusted ORs were combined (OR = 0.77, 95% CI 0.65-0.92; P = .004). When stratifying for race, this polymorphism showed decreased MI risk in Caucasians. In the subgroup analysis by age group, significant associations were observed in early-onset patients and in late-onset patients. In the subgroup analysis by gender, there was a significant association in women but not in men. In the subgroup analysis stratified by smoking status, MI risk was decreased in both smokers and nonsmokers. This study suggested that FXIIIA Val34Leu polymorphism was a protective factor for MI in caucasians.

Increased expression of factor XIII-A in patients with chronic rhinosinusitis with nasal polyps.

BACKGROUND: Profound edema or formation of a pseudocyst containing plasma proteins is a prominent characteristic of nasal polyps (NP). However, the mechanisms underlying NP retention of plasma proteins in the submucosa remain unclear. Recently, we reported that impairment of fibrinolysis causes excessive fibrin deposition in NP and this might be involved in the retention of plasma proteins. Although the coagulation cascade plays a critical role in fibrin clot formation at extravascular sites, the expression and role of coagulation factors in NP remain unclear. OBJECTIVE: The objective of this study was to investigate the expression of coagulation factors in patients with chronic rhinosinusitis (CRS). METHODS: Sinonasal tissues were collected from patients with CRS and control subjects. We assayed mRNA for factor XIII-A (FXIII-A) by using real-time PCR and measured FXIII-A protein by means of ELISA, immunohistochemistry, and immunofluorescence. RESULTS: FXIII-A mRNA levels were significantly increased in NP tissue from patients with CRS with NP (P < .001) compared with uncinate tissue from patients with CRS or control subjects. Similarly, FXIII-A protein levels were increased in NP. Immunofluorescence analysis revealed that FXIII-A expression in inflammatory cells and FXIII-A(+) cell numbers were significantly increased in NP. Most FXIII-A staining was observed within CD68(+)/CD163(+) M2 macrophages in NP. Levels of FXIII-A correlated with markers of M2 macrophages, suggesting that M2 macrophages are major FXIIIA-producing cells in NP. CONCLUSION: Overproduction of FXIII-A by M2 macrophages might contribute to the excessive fibrin deposition in the submucosa of NP, which might contribute to the tissue remodeling and pathogenesis of CRS with NP.

Androgen levels and metabolic parameters are associated with a genetic variant of F13A1 in women with polycystic ovary syndrome.

The polycystic ovary syndrome (PCOS), characterized by hyperandrogenism, is one of the most common hormonal disorders among premenopausal women and is associated with infertility, obesity, and insulin resistance. Accumulating evidence suggests a role of the blood coagulation factor gene F13A1 in obesity (GeneBank ID: NM_000129.3). The aim of this study was to investigate the association of intronic allelic variants of the F13A1 gene with PCOS susceptibility and metabolic parameters in lean and obese PCOS women. In a case-control study, we determined an intronic F13A1 single nucleotide polymorphism (SNP) (dbSNP ID: rs7766109) in 585 PCOS and 171 control women and tested for PCOS susceptibility and associations with anthropometric, metabolic and hormonal parameters. Genotype frequencies of the F13A1 SNP rs7766109 were equivalent in PCOS and control women. In PCOS women, F13A1 gene variants were significantly associated with body mass index (BMI) (p=0.013), systolic blood pressure (p=0.042), insulin response (AUCins) (p=0.015), triglycerides (TG) (p=0.001), and high density lipoprotein cholesterol (HDL) (p=0.012). In the subgroup of obese PCOS women free androgen index (FAI), free testosterone and sex hormone binding globulin (SHBG) as well as glucose measurements showed a significantly different pattern across F13A1 gene variants (p=0.043; p=0.039 and p=0.013, respectively). We report for the first time an association of the F13A1 SNP rs7766109 with BMI, androgens, and insulin resistance in PCOS women. Further studies are needed to confirm our findings and to evaluate whether F13A1 is causally involved in the pathogenesis of PCOS related metabolic and hormonal disturbances.

Factor XIII-A is involved in the regulation of gene expression in alternatively activated human macrophages.

Factor XIII subunit A (FXIII-A) is one of the most overrepresented genes that is expressed during the alternative activation of macrophages. Based on its substrate profile and its cellular localisation, FXIII-A is thought to function as an intracellular/intranuclear transglutaminase. Our aim was to find role for the intracellular FXIII-A by comparing the microarray profiles of alternatively activated monocyte-derived macrophages. Microarray analyses of FXIII-A-deficient patients and healthy controls were evaluated, followed by functional clustering of the differentially expressed genes. After a 48-hour differentiation in the presence of interleukin 4 (IL4), 1,017 probes out of the 24,398 expressed in macrophages from FXIII-A- deficient samples were IL4 sensitive, while only 596 probes were IL4 sensitive in wild-type samples. Of these genes, 307 were induced in both the deficient and the wild-type macrophages. Our results revealed that FXIII-A has important role(s) in mediating gene expression changes in macrophages during alternative activation. Functional clustering of the target genes carried out using Cytoscape/BiNGO and Ingenuity Pathways Analysis programs showed that, in the absence of FXIII-A, the most prominent differences are related to immune functions and to wound response. Our findings suggest that functional impairment of macrophages at the level of gene expression regulation plays a role in the wound healing defects of FXIII-A-deficient patients.

Phenotypic characterization of osteoblasts from the sclerotic zones of osteoarthritic subchondral bone.

OBJECTIVE: To determine the phenotype of osteoblasts from the sclerotic zones of human osteoarthritic (OA) subchondral bone. METHODS: Human osteoblasts were isolated from sclerotic or nonsclerotic areas of subchondral bone and cultured for 14 days in monolayer. The expression of 14 genes was investigated by real-time reverse transcription-polymerase chain reaction. The activities of alkaline phosphatase (AP) and transglutaminases (TGases) were quantified by enzymatic assays. C-terminal type I procollagen propeptide (CPI), interleukin-1beta (IL-1beta), IL-6, IL-8, transforming growth factor beta1 (TGFbeta1), osteocalcin (OC), and osteopontin (OPN) were assayed in the culture medium by immunoassay. RESULTS: Gene expression levels of matrix metalloproteinase 13, COL1A1 and COL1A2, OPN, tissue-nonspecific AP, OC, vascular endothelial growth factor, ANKH, TGase 2, factor XIIIA, and dentin matrix protein 1 were significantly up-regulated in sclerotic osteoblasts compared with nonsclerotic osteoblasts. In contrast, parathyroid hormone receptor gene expression was depressed in sclerotic osteoblasts, but bone sialoprotein levels were unchanged. The activities of AP and TGases were increased in sclerotic osteoblasts, while matrix mineralization, revealed by alizarin red staining, was decreased. In parallel, protein synthesis of CPI, OC, OPN, IL-6, IL-8, and TGFbeta1 was significantly higher in sclerotic osteoblasts than in nonsclerotic osteoblasts, while IL-1beta production was similar in both groups. CONCLUSION: These findings contribute to a better understanding of the mechanisms involved in subchondral bone sclerosis and identify osteoblasts with an altered phenotype as a potential target for future OA therapies.

Immediate mineralocorticoid receptor blockade improves myocardial infarct healing by modulation of the inflammatory response.

Mineralocorticoid receptor (MR) blockade reduces morbidity and mortality after acute myocardial infarction; however, the underlying mechanisms are still under investigation. This study examined whether MR antagonism promotes healing of the infarcted myocardium. Starting immediately after coronary ligation, male Wistar rats were treated with the selective MR antagonist eplerenone (100 mg/kg per day by gavage) or placebo for 2 to 7 days. At 7 days, eplerenone therapy versus placebo significantly reduced thinning and dilatation of the infarcted wall, improved left ventricular function, and enhanced neovessel formation in the injured myocardium. At 2 days, eplerenone-treated rats displayed lower plasma corticosterone levels, higher circulating blood monocytes, and more macrophages infiltrating the infarcted myocardium. MR blockade led to a transient upregulation (at days 2 and 3 but not at day 7) of monocyte chemoattractant protein-1, tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, interleukin-10, and interleukin-4 and an increase in factor XIIIa protein expression in the healing myocardium. Prevention of macrophage accumulation into the infarct zone by treatment with liposome-encapsulated clodronate almost abrogated the protein expression of factor XIIIa and the beneficial effects of eplerenone on infarct expansion. In conclusion, selective MR blockade immediately after myocardial infarction accelerated macrophage infiltration and transiently increased the expression of healing promoting cytokines and factor XIIIa in the injured myocardium resulting in enhanced infarct neovascularization and reduced early LV dilation and dysfunction.