Factor XII and Upar Upregulate Neutrophil Functions to Influence Wound Healing

Coagulation factor XII (FXII) deficiency is associated with decreased neutrophil migration, but the mechanisms remain uncharacterized. Here, we examine how FXII contributes to the inflammatory response. In 2 models of sterile inflammation, FXII-deficient mice (F12–/–) had fewer neutrophils recruited than WT mice. We discovered that neutrophils produced a pool of FXII that is functionally distinct from hepatic-derived FXII and contributes to neutrophil trafficking at sites of inflammation. FXII signals in neutrophils through urokinase plasminogen activator receptor–mediated (uPAR-mediated) Akt2 phosphorylation at S474 (pAktS474). Downstream of pAkt2S474, FXII stimulation of neutrophils upregulated surface expression of αMβ2 integrin, increased intracellular calcium, and promoted extracellular DNA release. The sum of these activities contributed to neutrophil cell adhesion, migration, and release of neutrophil extracellular traps in a process called NETosis. Decreased neutrophil signaling in F12–/– mice resulted in less inflammation and faster wound healing. Targeting hepatic F12 with siRNA did not affect neutrophil migration, whereas WT BM transplanted into F12–/– hosts was sufficient to correct the neutrophil migration defect in F12–/– mice and restore wound inflammation. Importantly, these activities were a zymogen FXII function and independent of FXIIa and contact activation, highlighting that FXII has a sophisticated role in vivo that has not been previously appreciated

Non-Adherence in Patients on Peritoneal Dialysis: A Systematic Review

Background: It has been increasingly recognized that non-adherence is an important factor that determines the outcome of peritoneal dialysis (PD) therapy. There is therefore a need to establish the levels of non-adherence to different aspects of the PD regimen (dialysis procedures, medications, and dietary/fluid restrictions). Methods: A systematic review of peer-reviewed literature was performed in PubMed, PsycINFO and CINAHL databases using PRISMA guidelines in May 2013. Publications on non-adherence in PD were selected by two reviewers independently according to predefined inclusion and exclusion criteria. Relevant data on patient characteristics, measures, rates and factors associated with non-adherence were extracted. The quality of studies was also evaluated independently by two reviewers according to a revised version of the Effective Public Health Practice Project assessment tool. Results: The search retrieved 204 studies, of which a total of 25 studies met inclusion criteria. Reported rates of nonadherence varied across studies: 2.6 1353% for dialysis exchanges, 3.9 1385% for medication, and 14.4 1367% for diet/fluid restrictions. Methodological differences in measurement and definition of non-adherence underlie the observed variation. Factors associated with non-adherence that showed a degree of consistency were mostly socio-demographical, such as age, employment status, ethnicity, sex, and time period on PD treatment. Conclusion: Non-adherence to different dimensions of the dialysis regimen appears to be prevalent in PD patients. There is a need for further, high-quality research to explore these factors in more detail, with the aim of informing intervention designs to facilitate adherence in this patient populatio

IGF-I enhances cell growth by suppressing TGF-β autocrine signaling.

<p>(A–C) TGF-β receptor kinase inhibitors stimulate the growth of NRP-152 cells. NRP-152 cells plated were treated with 5 µM of SB431542 (SB) (A) or with various concentrations of HTS466284 or TKDI and cell growth was measured 6 days later by counting cell number (A) or by crystal violet staining of fixed cells (B,C). (D) Growth of NRP-152-Sh-Smad2+3 cells versus NRP-152-Sh-LacZ cells in GM3 medium. (E) NRP-152-sh-LacZ and NRP-152-sh-Smad2+3 cell lines were incubated in the presence or absence of LR³-IGF-I (2 nM) for 5 days and cell growth was monitored daily for 5 days (D,E). Percent of growth inhibition by rapamycin in NRP-152-sh-LacZ and NRP-152-sh-Smad2+3 cell lines. Cell numbers were measured using Coulter Electronics Counter. Data shown are the average of triplicate determinations ± S.E. (*p<0.01). Statistical significance (*p<0.01) was assessed by two-way Anova analysis of variance.</p

Role of mTORC1 and mTORC2 in control of Survivin suppression and Rb activation by autocrine TGF-β.

<p>(A) Inhibition of autocrine TGF-β signaling reverses suppression of the Survivin promoter by antagonists of PI3K, Akt, mTOR or MEK. NRP-152 cells were transiently transfected with WT rSur-pro-Luc#2 promoter reporter construct, cultured overnight in GM2.1, and treated with 200 nM TKDI for 4 h prior to the indicated kinase inhibitors; dual luciferase activity was measured 24 h later. (B,C) NRP152 cells were plated overnight at a density of 50,000 cells/well/2 ml GM2.1 in six-well dishes, and were infected lentiviruses expressing with sh-LacZ, sh-mTOR, sh-Rictor and sh-Raptor for 24 h. Viral supernatants were replaced with GM2.1, cells were treated with 200 nM TKDI for 72 h, and cells then were harvested for Western blotting analysis of mTOR, Rictor, Raptor, Survivin, P-Rb^807/811, P-Akt^Ser473, P-S6^Ser235/236 and P-Smad2^Ser465/467 (B) and cell growth by a BCA protein assay (C). Error bars represent S.E. or triplicate determinations, and statistical significance (*p<0.01) was assessed by two-way Anova. Schematic model depicting a central role of TGF-β as regulator/mediator of Survivin expression by IGF-I/PI3K/Akt/mTOR signaling.</p

Transcriptional regulation by IGF-I of Survivin.

<p>(A) NRP-152 cells were co-transfected with full length (FL) or truncations (Trunc-1 to -4) of rat Survivin promoter-Firefly luciferase reporter constructs, along with CMV-Renilla control reporter one day before a 24 h treatment with LR³-IGF-I or vehicle, and cells were then analyzed for dual luciferase reporter activity. (B) NRP-152 cells were co-transfected with Trunc-2 Survivin promoter-luciferase construct (rSur-pro-Luc#2) and CMV-Renilla as in A, and next day cells were treated with various kinase inhibitors (LY: 10 µM LY294004; Rap: 200 pM rapamycin; 10 µM of either SB431542, SB202190, SP600125 or U0126) or DMSO vehicle for 2 h before 24 h treatment with 2 nM LR³-IGF-I or vehicle. (C) NRP-152 cells were co-transfected with rSur-pro-Luc#2 (WT) or rSur-pro-Luc#2 mutated at CDE and CHR (CDE/CHR Mut) along with CMV-Renilla, next day cells were treated with 2 nM LR³-IGF-I or vehicle, and harvested for dual luciferase activity. Data shown are relative values of Firefly luciferase normalized to Renilla luciferase, and expressed as relative luciferase units (R.L.U.). Each bar represents the average of triplicate determinations ± S.E. Statistical significance (*p<0.01) was assessed by two-way Anova analysis of variance. D) IGF-I reverses the ability of TGF-β to suppress Survivin mRNA and such reversal is mitigated by rapamycin. NRP-152 cells plated in GM3 overnight were pre-treated for 2 h with either 200 nM rapamycin or vehicle, followed by overnight with 2 nM LR³-IGF-I or vehicle, and then treated with 10 ng/ml rhTGF-β1 for 24 h. RNA was extracted and processed for RT-PCR of Survivin, Osteopontin (Ost-1) and β-actin.</p

IGF-I decreases cell doubling time and induces the expression of Survivin at both mRNA and protein levels in the NRP-152 rat prostatic epithelial cell line.

<p>(A) Growth of NRP-152 cells in response to 2 nM LR3-IGF-I or vehicle in GM3 was monitored by changes in cell number daily for 5 days. (B) Effect of IGF-I on Survivin expression and activation of Smads 2 and 3 in NRP-152 cells. Cells were treated with LR3-IGF-I at the indicated concentrations for 24 h (Top) or with 2 nM of LR3-IGF-I for the indicated times from 2 to 72 h (Bottom). Survivin, P-Smad3, P-Smad2 were measured by Western blot analysis. (C) Time dependent induction of Survivin mRNA expression by 2 nM LR3-IGF-I, as demonstrated by semi-quantitative RT-PCR and real-time q-PCR. (D) IGF-I induces the expression of Survivin in malignant and non-tumorigenic prostate epithelial cell lines. Cells were cultured in DMEM/F12 medium supplemented with 1% FBS (LNCaP, VCaP, DU145), DMEM/F12 alone (RWPE-1) or GM3 (NRP-152, RWPE-1), treated with or without 2 nM LR3-IGF-I for 24 h prior to Western blot analysis. Data shown are representative of two or three independent experiments (A–D). Data in A and C are the average of triplicate determination ± S.E.; *p<0.01 by two-way Anova analysis of variance.</p

IGF-I induces Survivin through a Smad dependent mechanism.

<p>(A) NRP-152 cells plated overnight in GM3 were treated at various times with LR³-IGF-I for up to 72 h, and cell lysates were analyzed for Western blot expression of Survivin, and P-Smads 2 and 3. (B) NRP-152 cells stably expressing sh-Smads 2, 3, and 2+3 or lentiviral sh-LacZ (control) were treated with 2 nM LR³-IGF-I or vehicle for 24 h prior to Western blot analysis for Survivin and Smads 2 and 3. (C) NRP-152 cells stably expressing sh-Smads 2+3 or lentiviral sh-LacZ (control) were treated with DMSO vehicle or 10 µM SB431542 for 2 h prior to treatment with 2 nM LR³-IGF-I or vehicle for 24 h, and changes in Survivin expression was assessed by Western blot analysis. (D) NRP-152 cells were treated with either 10 µM HTS466284 or 10 µM SB43152 for 2 h prior to treatment with 2 nM LR³-IGF-I or vehicle for 24 h, and changes in Survivin expression were assessed by Western blot analysis. (E,F) RWPE-1 and VCaP cells plated in GM3 were treated with LR³-IGF-I and the TGF-β receptor kinase inhibitors HTS466284 (HTS) or TKDI for 24 h prior to lysing cells for Western blot analysis of Survivin expression. Results are representative of two to three separate experiments.</p

Role of TGF-β signaling in suppression of cell growth, Survivin expression and activation of Rb by antagonists of mTOR, Akt, PI3K and MEK in prostate epithelial cells.

<p>The ability of the mTORC1 inhbitor rapamycin (A) and the mTORC1 and mTORC2 dual inhibitor Ku-0063794 (B) to inhibit growth of NRP-152 cells is revered by silencing Smads 2 and 3 (sh-S2+3) or by the TβRI/TβRII kinase dual inhibitor (TKDI), as assessed by cell growth after 6 days in GM3 medium. Although the Akt inhibitor MK2206 (0.5 µM) effectively represses LR3-IGF-I-induced cell growth in GM3 (C), TKDI reverses such suppression (D) as well as growth suppression (E) and Survivin suppression (F) by LY294002, rapamycin, U0126, MK2206 and Ku-0063794. D,E,F) Cells were plated in GM2.1 and pre-treated with TKDI or vehicle 4 h prior to treatment with the other kinase inhibitors. Cells were examined for cell growth after 5 days (by crystal violet staining) (D,E) or for expression of Survivin, XIAP, P-Smad3, P-Smad1/5/8, P-Rb, and ID-1 (by Western blot) after 24 h treatment (F). Data shown are the average of triplicate determinations ± S.E; Statistical significance (*p<0.01) was determined by two-way Anova.</p

Glycated albumin modulates the contact system with implications for the kallikrein-kinin and intrinsic coagulation systems

Background: Human serum albumin (HSA) is the most abundant plasma protein and is sensitive to glycation in vivo. The chronic hyperglycemic conditions in patients with diabetes mellitus (DM) induce a nonenzymatic Maillard reaction that denatures plasma proteins and forms advanced glycation end products (AGEs). HSA-AGE is a prevalent misfolded protein in patients with DM and is associated with factor XII activation and downstream proinflammatory kallikrein-kinin system activity without any associated procoagulant activity of the intrinsic pathway. Objectives: This study aimed to determine the relevance of HSA-AGE toward diabetic pathophysiology. Methods: The plasma obtained from patients with DM and euglycemic volunteers was probed for activation of FXII, prekallikrein (PK), and cleaved high-molecular-weight kininogen by immunoblotting. Constitutive plasma kallikrein activity was determined via chromogenic assay. Activation and kinetic modulation of FXII, PK, FXI, FIX, and FX via in vitro-generated HSA-AGE were explored using chromogenic assays, plasma-clotting assays, and an in vitro flow model using whole blood. Results: Plasma obtained from patients with DM contained increased plasma AGEs, activated FXIIa, and resultant cleaved cleaved high-molecular-weight kininogen. Elevated constitutive plasma kallikrein enzymatic activity was identified, which positively correlated with glycated hemoglobin levels, representing the first evidence of this phenomenon. HSA-AGE, generated in vitro, triggered FXIIa-dependent PK activation but limited the intrinsic coagulation pathway activation by inhibiting FXIa and FIXa-dependent FX activation in plasma. Conclusion: These data indicate a proinflammatory role of HSA-AGEs in the pathophysiology of DM via FXII and kallikrein-kinin system activation. A procoagulant effect of FXII activation was lost through the inhibition of FXIa and FIXa-dependent FX activation by HSA-AGEs.</p