Phosphorus and uremic serum up-regulate osteopontin expression in vascular smooth muscle cells

Phosphorus and uremic serum up-regulate osteopontin expression in vascular smooth muscle cells.BackgroundDialysis patients have accelerated atherosclerosis, with extensive calcification of both the intima and media. Cross-sectional studies have implicated hyperphosphatemia in this process, but the mechanism is unclear.MethodsTo test the hypothesis that hyperphosphatemia and/or uremia induces vascular calcification, bovine vascular smooth muscle cells (BVSMC) were treated with increasing concentrations of β-glycerophosphate, a phosphate donor, in the presence or absence of inhibitors for sodium/phosphate (Na/Pi) co-transport (foscarnet) or alkaline phosphatase (levamisole) for 48 hours. BVSMC also were incubated for various times with DMEM plus 15% pooled uremic sera from patients with low (LP) or high serum phosphorus (HP), or from pooled healthy control serum. Calcification in BVSMC was examined by quantitation of calcium deposition. Osteopontin expression and alkaline phosphatase activity were assessed by Western blotting and a colorimetric assay.Resultsβ-glycerophosphate increased osteopontin expression and alkaline phosphatase activity in BVSMC. Inhibition of either alkaline phosphatase activity or Na/Pi co-transport abolished this effect. Compared to incubation with control human serum, BVSMC cultured with uremic sera had increased mineral deposition. Uremic sera also increased alkaline phosphatase activity and osteopontin expression in BVSMC. The addition of β-glycerophosphate to uremic HP or LP sera did not further augment osteopontin expression. Blocking Na/Pi co-transport or alkaline phosphatase activity only partially inhibited uremic sera-induced osteopontin expression, indicating that other non-Na/Pi co-transport dependent mechanisms also are involved.Conclusionβ-glycerophosphate and uremic sera induce calcification and osteopontin expression in BVSMC. The uremic sera-induced osteopontin expression in BVSMC is partially mediated through alkaline phosphatase activity and a Na/Pi co-transporter dependent mechanism. However, other non-Na/Pi dependent mechanisms also contribute to accelerated vascular calcification in patients with ESRD

Role of calcification inhibitors in the pathogenesis of vascular calcification in chronic kidney disease (CKD)

Role of calcification inhibitors in the pathogenesis of vascular calcification in chronic kidney disease (CKD).BackgroundThe majority of patients with chronic kidney disease (CKD) have excessive vascular calcification; however, most studies demonstrate that a subset of CKD patients do not have, nor develop, vascular calcification despite similar exposure to the uremic environment. This suggests protective mechanisms, or naturally occurring inhibitors, of calcification may be important.MethodsIn order to determine the role of three inhibitors, fetuin-A, matrix gla protein (MGP), and osteoprotegerin (OPG) in the vascular calcification observed in patients with CKD-5, we (1) measured serum levels of these inhibitors and compared the levels to calcification assessed by computed tomography (CT); (2) examined arteries from CKD-5 patients by immunostaining for these inhibitors; and (3) examined the expression and effect of these inhibitors in cultured bovine vascular smooth muscle cells (BVSMCs) incubated in serum pooled from uremic patients compared to healthy controls.ResultsThere was a negative correlation of coronary artery calcification scores with serum fetuin-A levels (r=-0.30, P = 0.034) and a positive association with OPG levels (r = 0.29, P = 0.045). There was increasing immunostaining for both fetuin-A and MGP in arteries with increasing calcification graded semiquantitatively (P < 0.003). In vitro, fetuin-A added to mineralizing BVSMCs inhibited mineralization (P < 0.001). Compared to normal serum, BVSMCs incubated with uremic serum had a progressive increase in MGP expression with mineralization (P < 0.001) and increased expression of OPG in BVSMCs (P < 0.04).ConclusionThese data demonstrate that fetuin-A, OPG, and MGP play an important role in the pathogenesis of uremic vascular calcification

Heterologous immunity triggered by a single, latent virus in Mus musculus: combined costimulation- and adhesion- blockade decrease rejection.

The mechanisms underlying latent-virus-mediated heterologous immunity, and subsequent transplant rejection, especially in the setting of T cell costimulation blockade, remain undetermined. To address this, we have utilized MHV68 to develop a rodent model of latent virus-induced heterologous alloimmunity. MHV68 infection was correlated with multimodal immune deviation, which included increased secretion of CXCL9 and CXCL10, and with the expansion of a CD8(dim) T cell population. CD8(dim) T cells exhibited decreased expression of multiple costimulation molecules and increased expression of two adhesion molecules, LFA-1 and VLA-4. In the setting of MHV68 latency, recipients demonstrated accelerated costimulation blockade-resistant rejection of skin allografts compared to non-infected animals (MST 13.5 d in infected animals vs 22 d in non-infected animals, p<.0001). In contrast, the duration of graft acceptance was equivalent between non-infected and infected animals when treated with combined anti-LFA-1/anti-VLA-4 adhesion blockade (MST 24 d for non-infected and 27 d for infected, p = n.s.). The combination of CTLA-4-Ig/anti-CD154-based costimulation blockade+anti-LFA-1/anti-VLA-4-based adhesion blockade led to prolonged graft acceptance in both non-infected and infected cohorts (MST>100 d for both, p<.0001 versus costimulation blockade for either). While in the non-infected cohort, either CTLA-4-Ig or anti-CD154 alone could effectively pair with adhesion blockade to prolong allograft acceptance, in infected animals, the prolonged acceptance of skin grafts could only be recapitulated when anti-LFA-1 and anti-VLA-4 antibodies were combined with anti-CD154 (without CTLA-4-Ig, MST>100 d). Graft acceptance was significantly impaired when CTLA-4-Ig alone (no anti-CD154) was combined with adhesion blockade (MST 41 d). These results suggest that in the setting of MHV68 infection, synergy occurs predominantly between adhesion pathways and CD154-based costimulation, and that combined targeting of both pathways may be required to overcome the increased risk of rejection that occurs in the setting of latent-virus-mediated immune deviation

MHV68 results in the expansion of a unique T cell population, CD8^dim.

<p>A) Flow cyotmetric plots showing CD8 versus CD3 staining after gating on lymphocytes by forward and side scatter followed by gating on CD3+/CD8+ cells. Flow cytometry was performed on peripheral blood samples drawn 6 weeks post-infection or in similarly aged non-infected animals. Each plot shows the combined results from equal numbers of cells from 5 animals. B) Longitudinal analysis in MHV68-infected animals of the absolute numbers of total CD8 (dotted black line), CD8^bright (dashed blue line), and CD8^dim (solid red line) T cells following infection. Data include 10 mice from 2 independent experiments except at 1 week where 5 mice from a single experiment are displayed. Error bars represent 1 standard deviation. C) Longitudinal analysis in non-infected B6 mice of the percentage of CD8 T cells that are CD8^dim after placement of allogeneic skin grafts. The solid black line represents mice not receiving immunosuppressive therapy (n = 4). The dashed gray line shows mice receiving CTLA-4-Ig+anti-CD154 therapy (n = 6). Error bars show 1 standard deviation. D) Flow cytometric analysis at 6 weeks after infection comparing the relative expression of CD44, KLRG1, Bcl2, and CD127 with histograms between CD8 T cells from non-infected animals (gray filled area), CD8^bright (solid black line) from MHV68-infected animals, and CD8^dim (solid red line) from MHV68-infected animals. The plot in the bottom right shows the expression of CD127 versus KLRG1 for CD8^dim in MHV68-infected animals. Each cell type for each plot shows the combination of 5 individual mice.</p

The addition of adhesion blockade to costimulation blockade increases viral replication.

<p>MHV68 viral loads were measured>100 days after treatment with CoB +/− adhesion blockade by PCR for MHV68 ORF50 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071221#pone.0071221-Moorman1" target="_blank">[19]</a>. Recipients treated with CoB alone (red data points) showed a median value of 313 copies/mL (n = 5). Recipients treated with CoB+anti-LFA-1 alone (gray data points) showed a median value of 1027/mL (n = 4, p = n.s. compared to CoB alone). Recipients treated with CoB+anti-VLA-4 alone (blue data points) showed a median value of 9255/mL (n = 5, p = n.s. compared to CoB alone). Recipients treated with CoB+anti-LFA-1+ anti-VLA-4 (green data points) showed a median value of 20278/mL (n = 5, p<.05 compared to CoB alone). Plots for each group show the median viral load with range from a single experiment.</p

Comparative Effect of CTLA-4-Ig and anti-CD154+ adhesion blockade in MHV68-infected and non-infected mice.

<p>A) Kaplan-Meier survival curves comparing skin graft survival between the following groups of MHV68-infected mice: MHV68-infected mice treated with CoB (solid red line, MST 13.5 d, n = 40, 7 independent experiments); MHV68-infected mice treated with anti-LFA-1+ anti-VLA-4 (solid orange line, MST 27 d, n = 14, 2 independent experiments); MHV68-infected mice treated with anti-CD154+ anti-LFA-1/anti-VLA-4 (solid violet line, MST>100 d, n = 8, one experiment); MHV68-infected mice treated with CTLA-4-Ig+anti-LFA-1/anti-VLA-4 (solid magenta line, MST 41 d, n = 8, one experiment); MHV68-infected mice treated with CoB+anti-LFA-1/anti-VLA-4 (solid green line, MST>100 d, n = 25, 3 independent experiments). Skin graft survival between groups was compared using the log-rank test. In infected animals, the combination of anti-LFA-1 and anti-VLA-4 was compared to CoB alone resulting in p = .0023. CTLA-4-Ig+anti-LFA-1/anti-VLA-4 compared to CoB alone yielded p = .0002. The combination of anti-CD154+ anti-LFA-1/anti-VLA-4 was significantly different from CoB alone (p<.0001) and not significantly different from dual CoB (anti-CD154+ CTLA-4-Ig)+dual adhesion blockade (p = .73). B) Kaplan-Meier survival curves comparing skin graft survival between the following groups of non-infected mice: non-infected mice treated with CoB (dotted red line, MST 22 d, n = 48, 8 independent experiments); non-infected mice treated with anti-LFA-1+ anti-VLA-4 (dotted orange line, MST 24, n = 12, 2 independent experiments); non-infected mice treated with anti-CD154+ anti-LFA-1/anti-VLA-4 (dotted violet line, MST>100 d, n = 8, one experiment); non-infected mice treated with CTLA-4-Ig+anti-LFA-1/anti-VLA-4 (dotted magenta line, MST>100 d, n = 8, one experiment); non-infected mice treated with CoB+anti-LFA-1/anti-VLA-4 (dotted green line, MST>100 d, n = 26, 3 independent experiments). Graft survival between groups in non-infected animals was compared with the log-rank method. Survival in the CoB only and anti-LFA-1+ anti-VLA-4 groups was not significantly different (p = .87). Relative to CoB alone, survival was significantly prolonged in non-infected animals treated with anti-CD154+ anti-LFA-1/anti-VLA-4 (p<.0001), CTLA-4-Ig+anti-LFA-1/anti-VLA-4 (p<.0001), or dual CoB (anti-CD154+ CTLA-4-Ig)+anti-LFA-1/anti-VLA-4 (p<.0001).</p