Diannexin decreases inflammatory cell infiltration into the islet graft, reduces β-cell apoptosis, and improves early graft function

Abstract

Background: A major unmet challenge is to reduce the islet mass needed for insulin independence in type 1 diabetic recipients after islet transplantation. The recombinant homodimer of human annexin V, diannexin, has completed a Phase II Clinical Trial in Kidney Transplantation (NCT00615966).

Methods: We developed a marginal islet mass transplantation model (10-12 islets per gram of recipient body weight) and investigated whether diannexin prevents β-cell apoptosis and improves islet graft function. Diannexin was administered to islet cell donors shortly before pancreas harvest, added to isolation reagents, and infused into recipients at the time of transplantation and repeated daily until day 4.

Results: In the syngeneic marginal islet mass transplantation model, the median time needed to achieve normoglycemia was reduced from 17.0 days among untreated controls to 3.5 days among diannexin-treated recipients (P=0.004). Histologic analysis of islet grafts harvested on day 3 posttransplantation revealed decreased macrophage (44.7%±9.8% vs. 19.2%±3.2%, P=0.007) and T-cell infiltration (25.9%±5.5% vs. 9.1%±1.1%, P=0.004), and a lower rate of islet cell apoptosis (20.5%±2.8% vs. 7.6%±2.3%, P=0.01) with diannexin treatment. Expression profiling of the islet grafts showed significantly lower levels of mRNA for the proapoptotic molecule Bid, but higher levels of interleukin-6, interferon-γ, and immunosuppressive cytokine interleukin-10.

Conclusions: Our findings demonstrate that diannexin improves the early function of marginal mass islet grafts, and its effects are associated with reductions in inflammatory cell infiltration and β-cell death by apoptosis after islet transplantation.

Figures

Figure 1. Diannexin accelerates the reversal of hyperglycemia following marginal islet mass transplantation

Streptozotocin-induced diabetic BALB/c mice received a marginal mass of syngeneic islets under the kidney capsule. Diannexin was given to donors, added to islet isolation reagents, and injected into recipients on days 0-4 post-transplantation. (A) Reversal of diabetes, as defined by blood glucose levels <200mg/dl on three consecutive measurements, after marginal islet mass transplantation in Diannexin treatment vs. control. Two-tailed P values calculated using the log rank test. (B) Mean (±SE) blood glucose levels of Diannexin-treated and control recipients following transplantation of a marginal islet cell mass. * P<0.05 by the Mann-Whitney test.

Figure 2. Diannexin reduces inflammatory cell recruitment and beta-cell apoptosis within syngeneic islet grafts

(A) Immunostaining of syngeneic islets retrieved on day 3 post-transplantation decreased macrophage (Iba-1) and T-cell (CD3) traffic into islets grafts treated with Diannexin. (B) Apoptosis detection by TUNEL demonstrates that Diannexin treatment is associated with a reduction of islet cell apoptosis in syngeneic grafts. Five or six histologic sections taken from different locations within three islet grafts were included in the analysis. Sections were counterstained with hematoxylin and shown at x400 magnifications. Two-tailed P values calculated using the Mann-Whitney test.

Figure 3. Effect of Diannexin on islet cell apoptosis

Diannexin (400μg/kg IV) was administered to BALB/c donor mice prior to pancreas harvest and also added at a concentration of 100ng/ml to all reagents used for islet isolation. (A) TUNEL-assisted detection of apoptosis revealed a similar apoptotic rate between control (top) and Diannexin-treated (bottom) islets. Sections were counterstained with hematoxylin and shown at x500 magnifications (inset, x400). (B) Histologic analysis of mouse islet samples. Paraffin-embedded sections of freshly isolated mouse islets demonstrate comparable levels of cleaved caspase-3 expression between untreated (top) and Diannexin-treated (bottom) islets. For (A) and (B), a total of 20 islets from three or four separate isolations were included in the analysis. Sections were counterstained with hematoxylin and shown at x500 magnifications (inset, x400). (C) The islets were dissociated with trypsin/EDTA and stained with annexin V-FITC (annexin V) and TO-PRO-3 iodide (Topro 3). Representative flow cytometry analysis from two independent experiments comprised of 4 untreated (top panels) and 3 Diannexin-pretreated (bottom) islet isolates. Panels on the left show size and granularity of the dissociated islet cells, and dual-parameter analyses for annexin V and Topro 3 to categorize viable cells (annexin V/TO-PRO-3 double-negative cells), early apoptotic cells (annexin V single-positive cells), late apoptotic/necrotic cells (annexin V/TO-PRO-3 double-positive cells), and necrotic cells (TO-PRO-3 single-positive cells), as previously described (32). Two-tailed P values were calculated using the Mann-Whitney test.

Figure 4. Intragraft mRNA expression patterns of islets from Diannexin-treated mice or control mice, as measured by real-time quantitative PCR assays

Absolute levels of intragraft mRNA levels were measured with the use of real-time quantitative PCR assays. Individual and mean (±SE) mRNA levels for control (N=3) and Diannexin-treated (N=5) recipients are shown. mRNA expression levels are expressed as a ratio of mRNA copies in 1μg of RNA to18S ribosomal RNA copies in 1pg of RNA. Two-tailed P values calculated using the Mann-Whitney test.

Figure 5. mRNA expression patterns of islet isolates from Diannexin-treated mice or untreated control mice

Diannexin (400μg/kg IV) was administered to BALB/c donor mice prior to pancreas harvest and also added at a concentration of 100ng/ml to all reagents used for islet isolation. mRNA levels in fresh mouse islets were measured with the use of real-time quantitative PCR assays. Individual and mean (±SE) mRNA levels are expressed as a ratio of mRNA copies in 1μg of total RNA to 18S ribosomal RNA copies in 1pg of RNA. Two-tailed P values were calculated using the Mann-Whitney test.