Fibroblast Growth Factor 23 (FGF23) and Alpha-Klotho Stimulate Osteoblastic MC3T3.E1 Cell Proliferation and Inhibit Mineralization

Elevated serum levels of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23) are found in patients with phosphate wasting diseases and chronic kidney disease-mineral and bone disorder (CKD-MBD). These diseases are associated with rickets and renal osteodystrophy, respectively. FGF23 is secreted from osteoblastic cells and signals through FGFRs, membrane coreceptor alpha-Klotho (Klotho), and, possibly, a circulating form of Klotho. Despite the absence of detectable Klotho on osteoblastic cells, studies have suggested that forced FGF23 expression in osteoblasts inhibited mineralization. Thus, we examined the effects of exogenously applied FGF23 on osteoblastic MC3T3.E1 cell proliferation and differentiation, with and without soluble Klotho. MC3T3.E1 cells were cultured in osteoblast differentiation medium, supplemented with FGF23 (0.1–1,000 ng/mL), Klotho (50 ng/mL), the combination FGF23 + Klotho, and FGF2 (100 ng/mL) as a control. Neither FGF23 nor Klotho exposure affected proliferation of day 4 growth phase cells or mineralization of day 14 cultures. In contrast, FGF23 + Klotho resulted in inhibition of mineralization and osteoblast activity markers at day 14, and a slight, reproducible induction of proliferation. Inhibition of FGFR1, but not FGFR2 or FGFR3, completely restored FGF23 + Klotho-induced inhibition of alkaline phosphatase (ALP) activity at day 7. ALP activity was partially restored by the MAPK inhibitor U0126 but not inhibitors p38 and P13K. Thus, soluble Klotho enables FGF23 signaling in MC3T3.E1 cells, likely through FGFR 1(IIIc). Elevated FGF23 actions, in part, appear to parallel FGF2 with lower potency. In addition to affecting bone via indirect phosphate wasting pathways, supraphysiological FGF23 and soluble Klotho may directly impact bone in diseases with elevated FGF23 levels

Depletion of homeostatic antibodies against malondialdehyde-modified low-density lipoprotein correlates with adverse events in major vascular surgery

We aimed to investigate if major vascular surgery induces LDL oxidation, and whether circulating antibodies against malondialdehyde-modified LDL (MDA-LDL) alter dynamically in this setting. We also questioned relationships between these biomarkers and post-operative cardiovascular events. Major surgery can induce an oxidative stress response. However, the role of the humoral immune system in clearance of oxidized LDL following such an insult is unknown. Plasma samples were obtained from a prospective cohort of 131 patients undergoing major non-cardiac vascular surgery, with samples obtained preoperatively and at 24- and 72 h postoperatively. Enzyme-linked immunoassays were developed to assess MDA-LDL-related antibodies and complexes. Adverse events were myocardial infarction (primary outcome), and a composite of unstable angina, stroke and all-cause mortality (secondary outcome). MDA-LDL significantly increased at 24 h post-operatively (p < 0.0001). Conversely, levels of IgG and IgM anti-MDA-LDL, as well as IgG/IgM-MDA-LDL complexes and total IgG/IgM, were significantly lower at 24 h (each p < 0.0001). A smaller decrease in IgG anti-MDA-LDL related to combined clinical adverse events in a post hoc analysis, withstanding adjustment for age, sex, and total IgG (OR 0.13, 95% CI [0.03–0.5], p < 0.001; p value for trend <0.001). Major vascular surgery resulted in an increase in plasma MDA-LDL, in parallel with a decrease in antibody/complex levels, likely due to antibody binding and subsequent removal from the circulation. Our study provides novel insight into the role of the immune system during the oxidative stress of major surgery, and suggests a homeostatic clearance role for IgG antibodies, with greater reduction relating to downstream adverse events

Pathophysiological lessons from rare associations of immunological disorders

Rare associations of immunological disorders can often tell more than mice and rats about the pathogenesis of immunologically mediated human kidney disease. Cases of glomerular disease with thyroiditis and Graves’ disease and of minimal change disease with lymphoepithelioma-like thymic carcinoma and lymphomatoid papulosis were recently reported in Pediatric Nephrology. These rare associations can contribute to the unraveling of the pathogenesis of membranous nephropathy (MN) and minimal change disease (MCD) and lead to the testing of novel research hypotheses. In MN, the target antigen may be thyroglobulin or another thyroid-released antigen that becomes planted in the glomerulus, but other scenarios can be envisaged, including epitope spreading, polyreactivity of pathogenic antibodies, and dysregulation of T regulatory cells, leading to the production of a variety of auto-antibodies with different specificities [immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX syndrome)]. The occurrence of MCD with hemopathies supports the role of T cells in the pathogenesis of proteinuria, although the characteristics of those T cells remain to be established and the glomerular permeability factor(s) identified

Factors associated with infrainguinal bypass graft patency at 1-year; a retrospective analysis of a single centre experience

Introduction: The aim of this study was to identify factors associated with primary graft patency 1 year following open lower limb revascularisation (LLR) at a tertiary referral vascular service. Methods: A retrospective analysis of patients undergoing infra-inguinal bypass surgery between January 2016 and May 2017 at a tertiary vascular centre (St Mary’s Hospital, London) was performed. Data regarding patient demographics, comorbidities, type of operation and post-operative anti-thrombotic strategy were collected. Quality of run-off score was assessed from pre-operative imaging. Results: Seventy-seven cases were included in the analysis. Overall, the primary patency rate at 1-year was 63.6% (n = 49/77) and the secondary patency rate was 67.5% (n = 52/77). Independent variables with statistically significant inferior patency rates at 1-year were (1) bypasses with below knee targets (p = 0.0096), (2) chronic limb threatening ischaemia indication (p = 0.038), (3) previous ipsilateral revascularisation (p < 0.001) and (4) absence of hypertension history (p = 0.041). There was also a trend towards significance for American Society of Anesthesiologists (ASA) grade (p = 0.06). Independent variables with log-rank test p values of <0.1 were included in a Cox proportional hazards model. The only variable with a statistically significant impact on primary patency rates was previous open or endovascular ipsilateral revascularisation (HR 2.44 (1.04–5.7), p = 0.04). Conclusion: At 1-year follow-up, previous ipsilateral revascularisation was the most significant factor in affecting patency rates. Patients in this subgroup should therefore be deemed high-risk, which should be reflected in the informed consent and peri-operative management

Cellular mechanisms of bone resorption in breast carcinoma

The cellular mechanisms that account for the increase in osteoclast numbers and bone resorption in skeletal breast cancer metastasis are unclear. Osteoclasts are marrow-derived cells which form by fusion of mononuclear phagocyte precursors that circulate in the monocyte fraction. In this study we have determined whether circulating osteoclast precursors are increased in number or have an increased sensitivity to humoral factors for osteoclastogenesis in breast cancer patients with skeletal metastases (± hypercalcaemia) compared to patients with primary breast cancer and age-matched normal controls. Monocytes were isolated and cocultured with UMR 106 osteoblastic cells in the presence of 1,25 dihydroxyvitamin D3[1,25(OH)2D3] and human macrophage colony stimulating factor (M-CSF) on coverslips and dentine slices. Limiting dilution experiments showed that there was no increase in the number of circulating osteoclast precursors in breast cancer patients with skeletal metastases (± hypercalcaemia) compared to controls. Osteoclast precursors in these patients also did not exhibit increased sensitivity to 1,25(OH)2D3 or M-CSF in terms of osteoclast formation. The addition of parathyroid hormone-related protein and interleukin-6 did not increase osteoclast formation. The addition of the supernatant of cultured breast cancer cell lines (MCF-7 and MDA-MB-435), however, significantly increased monocyte-osteoclast formation in a dose-dependent fashion. These results indicate that the increase in osteoclast formation in breast cancer is not due to an increase in the number/nature of circulating osteoclast precursors. They also suggest that tumour cells promote osteoclast formation in the bone microenvironment by secreting soluble osteoclastogenic factor(s). © 2001 Cancer Research Campaign http://www.bjcancer.co

Vascular endothelial growth factor acts as an osteolytic factor in breast cancer metastases to bone

Vascular endothelial growth factor (VEGF) is a proangiogenic cytokine that is expressed highly in many solid tumours often correlating with a poor prognosis. In this study, we investigated the expression of VEGF and its receptors in bone metastases from primary human breast tumours and further characterised its effects on osteoclasts in vitro. Breast cancer metastases to bone were immunohistochemically stained for VEGF, its receptors VEGFR1 and 2 (vascular endothelial growth factor receptor 1 and 2), demonstrating that breast cancer metastases express VEGF strongly and that surrounding osteoclasts express both VEGFR1 and VEGFR2. RAW 264.7 cells (mouse monocyte cell line) and human peripheral blood mononuclear cells (PBMCs) were cultured with VEGF, RANKL and M-CSF. VEGF and RANKL together induced differentiation of multinucleated, tartrate-resistant acid phophatase (TRAP)-positive cells in similar numbers to M-CSF and RANKL. The PBMCs were also able to significantly stimulate resorption of mineralised matrix after treatment with M-CSF with RANKL and VEGF with RANKL. We have shown that VEGF in the presence of RANKL supports PBMC differentiation into osteoclast-like cells, able to resorb substrate. Vascular endothelial growth factor may therefore play a role in physiological bone resorption and in pathological situations. Consequently, VEGF signalling may be a therapeutic target for osteoclast inhibition in conditions such as tumour osteolysis

Interleukin-32 Promotes Osteoclast Differentiation but Not Osteoclast Activation

Background: Interleukin-32 (IL-32) is a newly described cytokine produced after stimulation by IL-2 or IL-18 and IFN-γ. IL-32 has the typical properties of a pro-inflammatory mediator and although its role in rheumatoid arthritis has been recently reported its effect on the osteoclastogenesis process remains unclear. Methodology/principal findings: In the present study, we have shown that IL-32 was a potent modulator of osteoclastogenesis in vitro, whereby it promoted the differentiation of osteoclast precursors into TRAcP+ VNR+ multinucleated cells expressing specific osteoclast markers (up-regulation of NFATc1, OSCAR, Cathepsin K), but it was incapable of inducing the maturation of these multinucleated cells into bone-resorbing cells. The lack of bone resorption in IL-32-treated cultures could in part be explain by the lack of F-actin ring formation by the multinucleated cells generated. Moreover, when IL-32 was added to PBMC cultures maintained with soluble RANKL, although the number of newly generated osteoclast was increased, a significant decrease of the percentage of lacunar resorption was evident suggesting a possible inhibitory effect of this cytokine on osteoclast activation. To determine the mechanism by which IL-32 induces such response, we sought to determine the intracellular pathways activated and the release of soluble mediators in response to IL-32. Our results indicated that compared to RANKL, IL-32 induced a massive activation of ERK1/2 and Akt. Moreover, IL-32 was also capable of stimulating the release of IL-4 and IFN-γ, two known inhibitors of osteoclast formation and activation. Conclusions/significance: This is the first in vitro report on the complex role of IL-32 on osteoclast precursors. Further clarification on the exact role of IL-32 in vivo is required prior to the development of any potential therapeutic approach

Laparoscopy in management of appendicitis in high-, middle-, and low-income countries: a multicenter, prospective, cohort study.

BACKGROUND: Appendicitis is the most common abdominal surgical emergency worldwide. Differences between high- and low-income settings in the availability of laparoscopic appendectomy, alternative management choices, and outcomes are poorly described. The aim was to identify variation in surgical management and outcomes of appendicitis within low-, middle-, and high-Human Development Index (HDI) countries worldwide. METHODS: This is a multicenter, international prospective cohort study. Consecutive sampling of patients undergoing emergency appendectomy over 6 months was conducted. Follow-up lasted 30 days. RESULTS: 4546 patients from 52 countries underwent appendectomy (2499 high-, 1540 middle-, and 507 low-HDI groups). Surgical site infection (SSI) rates were higher in low-HDI (OR 2.57, 95% CI 1.33-4.99, p = 0.005) but not middle-HDI countries (OR 1.38, 95% CI 0.76-2.52, p = 0.291), compared with high-HDI countries after adjustment. A laparoscopic approach was common in high-HDI countries (1693/2499, 67.7%), but infrequent in low-HDI (41/507, 8.1%) and middle-HDI (132/1540, 8.6%) groups. After accounting for case-mix, laparoscopy was still associated with fewer overall complications (OR 0.55, 95% CI 0.42-0.71, p < 0.001) and SSIs (OR 0.22, 95% CI 0.14-0.33, p < 0.001). In propensity-score matched groups within low-/middle-HDI countries, laparoscopy was still associated with fewer overall complications (OR 0.23 95% CI 0.11-0.44) and SSI (OR 0.21 95% CI 0.09-0.45). CONCLUSION: A laparoscopic approach is associated with better outcomes and availability appears to differ by country HDI. Despite the profound clinical, operational, and financial barriers to its widespread introduction, laparoscopy could significantly improve outcomes for patients in low-resource environments. TRIAL REGISTRATION: NCT02179112