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Original Basic Science—General

Engraftment Site and Effectiveness of the Pan-Caspase Inhibitor F573 to Improve Engraftment in Mouse and Human Islet Transplantation in Mice

Pepper, Andrew R. PhD1,2; Bruni, Antonio BSc1,2; Pawlick, Rena BSc1; Wink, John BSc1; Rafiei, Yasmin1; Gala-Lopez, Boris MD, PhD1,2; Bral, Mariusz MD1,2; Abualhassan, Nasser MD, MSc1,2; Kin, Tatsuya MD, PhD1; Shapiro, A.M. James MD, PhD1,2

Author Information
doi: 10.1097/TP.0000000000001638
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Islet transplantation has recently been proven to stabilize glycemic control in select patients with type 1 diabetes complicated by hypoglycemia in a phase 3 trial,1 extending the initial success of the Edmonton Protocol popularized by Shapiro and colleagues in 2000.2 Within the last 16 years, islet transplantation success rates have improved substantially, with insulin-independence rates in at least 50% of recipients achieved out to 5 years post transplant in 6 International Centers.3 Yet despite these clear advances in clinical islet transplantation, achievement of single-donor engraftment success has been difficult to establish routinely, with most recipients requiring at least 2 donors to achieve insulin independence.4,5 Numerous factors contribute to islet loss in the acute and peritransplant period which results in an estimated 70% loss of transplanted β-cell mass.6 The instant blood-mediated inflammatory reaction is largely responsible for immediate cell loss especially in the intraportal hepatic site, and associated with platelet activation and triggering of biochemical cell death pathways including both apoptosis and necrosis.7,8 Potent cell death inhibitors, and exploration of nonblood exposed extrahepatic sites, are being actively explored to improve islet viability and engraftment outcomes in the early transplant period.

The use of early generation anti-apoptotic agents have previously demonstrated improved in vitro islet viability outcomes.9 Further studies revealed marked improvements in islet engraftment in preclinical experimental models. For example, using short course Benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone (zVAD-FMK) therapy in islet culture and within the acute transplant period, Emamaullee et al10 demonstrated enhanced long-term in vivo function up to 1 year posttransplant in mice transplanted with a marginal islet dose under the renal capsule and via the intraportal route The next generation pan-caspase inhibitors, including EP1013 and IDN-6556, demonstrated augmented long-term engraftment using a marginal islet dose capable of effectively restoring euglycemia in transplant recipients in both small and large animal models.11

In an alternative approach to avoid the intravascular site, recent studies in our laboratory have also examined extrahepatic transplant sites that could permit the engraftment of islets and alternative β-cell sources, including insulin-producing stem cells or xenogeneic sources. Alternative transplant sites with potential clinical feasibility, should accommodate a sufficient transplant mass and be readily retrievable if they are to be considered as prospective sites.12 The deviceless (DL) transplant technique, which modifies the subcutaneous space through temporary implantation of a commercially approved angiocatheter, in routine clinical use for other indications, has demonstrated successful restoration of euglycemia using mouse and human islets in a preclinical animal model.13 Moreover, the DL technique was also effective in reversing hyperglycemia when transplanted with a marginal islet dose in a syngeneic mouse model.14 Thus far, incorporating therapeutic strategies to augment islet engraftment in this alternative transplant site have yet to be elucidated.

Herein, we sought to determine whether the potent pan-caspase inhibitor, F573, could effectively reduce apoptosis in murine and human islets. Furthermore, we evaluated whether F573 treatment could differentially augment islet engraftment in standard and alternative transplant sites using full and marginal islet transplant doses. We reasoned that alternative sites would have differential susceptibility to oxygen delivery and metabolic exchange, and therefore initially nonvascularized implanted cells may have increased susceptibility to hypoxia-mediated cell death signaling.

MATERIALS AND METHODS

Caspase Inhibitor F573

The pan-caspase inhibitor F573 (Molecular weight: 382.38 g/mol) was obtained from Shanghai Genomics Inc. (Lot: 20141203, Shanghai, China). Stock preparations of F573 were prepared by dissolving 30 mg (lyophilized white powder) in 1 mL dimethyl sulphoxide (DMSO). For in vitro and in vivo studies, stock solutions were diluted with sterile saline to a final working concentration of 1 mg/mL.

Human Islets

Human islets were prepared by the Clinical Islet Laboratory at Alberta Health Services. Deceased donor pancreata were processed for islet isolation with appropriate ethical approval and consent obtained from next-of-kin of the donor. Islets were isolated from 2 donor pancreata, implementing a modified Ricordi technique.15,16 Permission for these studies was granted by the Health Research Ethics Board of the University of Alberta, Edmonton, Alberta, Canada. Upon receiving the human islet preparation, islet aliquots (±10%) were counted and randomly distributed into standard culture media ± 100 μM F573 supplementation for 24 hours at 37°C and 5% CO2 before transplantation. Standard culture media (CMRL-1066, Mediatech, Manassas, VA) contains fetal bovine serum (10%), L-glutamine (100 mg/L), penicillin (112 kU/L), streptomycin (112 mg/L) and HEPES (25 mmol/L) at pH 7.4. The average human islet purity for the transplant preparations used in this study was 43.8% (n = 2).

Mouse Islet Isolation and Culture

Pancreatic islets were isolated from 8- to 12-week-old male C57BL/6 mice (Jackson Laboratories, Canada). Animals were housed under conventional conditions having access to food and water ad libitum. Mouse care was in accordance with the guidelines approved by the Canadian Council on Animal Care. Before pancreatectomy, the common bile duct was cannulated with a 30G needle and the pancreas was distended with 0.125 mg/mL cold Liberase TL Research Grade enzyme (Roche Diagnostics, Laval, QC, CA) in Hank Balanced Salt Solution (Sigma-Aldrich Canada Co., Oakville, ON, CA). Islets were isolated by digesting the pancreases in a 50 mL tube placed in a 37°C water bath for 14 minutes with light agitation. After the pancreatic digestion phase, islets were purified using histopaque-density gradient centrifugation (1.108, 1.083, and 1.069 g/mL, Sigma-Aldrich Canada Co., Oakville, ON, Canada). Isolated mouse islet aliquots (±10%) were then counted and randomly distributed into standard culture media ± 100 μM F573 supplementation for 2 hours at 37°C and 5% CO2 before transplantation. Standard culture media (CMRL-1066, Mediatech, Manassas, VA) contains fetal bovine serum (10%), L-glutamine (100 mg/L), penicillin (112 kU/L), streptomycin (112 mg/L) and HEPES (25 mmol/L) at pH 7.4.

Apoptosis Analysis

Mouse and human islet apoptosis was measured before transplantation and subsequent to culture (2 and 24 hours, respectively) ± F573 media supplementation. Apoptosis was assayed in all islets groups using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (DeadEnd Apoptosis Detection System, Promega, Madison, WI), after formalin fixation (10%), agar embedding, processing, paraffinizing and sectioning. In addition to TUNEL staining, islets were costained with insulin and DAPI. Subsequent to deparaffinization and antigen heat retrieval, islet sections were washed with phosphate-buffered saline (PBS) supplemented with 1% goat serum, followed by blocking with 20% goat serum in PBS for 30 minutes. The sections were treated with a primary antibody of guinea pig anti-pig insulin (Dako A0564) diluted 1:100 (PBS with 1% goat serum) for 2 hours at 4°C. Samples were rinsed with PBS with 1% goat serum followed by secondary antibody treatment consisting of goat anti-guinea pig (Alexa 568) diluted 1:500 (PBS with 1% goat serum) for 1 hour at room temperature. Samples were rinsed with PBS and counter-stained with DAPI in anti-fade mounting medium (ProLong; LifeTechnologies). Using a fluorescent microscope, the resulting microphotographs were taken using the appropriate filter with AxioVision imaging software.

Islet apoptosis was quantified as a percentage of positive TUNEL staining nuclei per islet (+TUNEL/total nuclei) using ImageJ software (freeware ImageJ v1.33 and Cell Counter plug-in, both downloaded from the NIH website [http://rsb.info.nih.gov/ij]).

To further evaluate the degree of apoptosis in both mouse and human islet preparations before transplantation, cytosolic cleaved caspase-3 activation was quantified from the lysates of frozen islet samples (3 × 100-300 islets per group, n = 2 isolations). Briefly, islet lysates from both control and F573 cultured mouse and human islets were tested, according to manufactures specifications, for protease activity by the addition of a caspase-specific peptide that is conjugated to the color reporter substrate p-nitroaniline (BF3100: R & D Systems, Minneapolis, MN). Caspase activity was quantified spectrophotometrically at 405 nm. Results are expressed as absorbance normalized to 100 islets.

In Vitro Islet Viability Assessment

Human and mouse islet viability was assessed postculture ± F573 media supplementation, (24 and 2 hours, respectively) at the time of transplantation, Islet viability was determined by simultaneous staining of live and dead cells using a membrane integrity fluorescence assay (SytoGreen 13 and ethidium bromide; Invitrogen, Oregon). The percentage of viable and dead cells was determined for both control and F573-treated islets.

Concurrently, static glucose-stimulated insulin secretion assay was performed on both human and mouse islets postculture (3 × 50 islets per group, n = 2 isolations). Islets were incubated in RPMI-1640 containing low (2.8 mmol/L) glucose for 1 hour, followed by high (16.7 mmol/L) glucose for an additional hour. Cell-free supernatants were harvested postglucose incubation and insulin levels were measured by enzyme-linked immunosorbent assay (ELISA) (Mercodia, Uppsala, Sweden). Human insulin data Are represented as U/L and as a stimulation index (insulin secreted high glucose/low glucose). Mouse insulin data Are represented as μg/L and as a stimulation index.

Diabetes Induction and Islet Transplantation

One week before transplantation, recipient mice either C57BL/6 (syngeneic studies) or B6-RAG−/− (B6.129S7-Rag1tm1Mom/J) (human islet studies) were rendered diabetic by chemical induction with intraperitoneal streptozotocin (Sigma-Aldrich Canada Co., Oakville, ON, Canada), at 180 mg/kg in acetate phosphate buffer, pH 4.5. Diabetes was confirmed when blood glucose levels exceeded 15 mmol/L for 2 consecutive daily readings.

For human islet transplants, postculture ± F573 media supplementation, islets were counted and transplanted under kidney capsule (KC) at a marginal islet dose of 500 islet equivalents per diabetic recipient. Syngeneic, mouse islets, postculture ± F573 media supplementation were transplanted into 3 groups: (1) under KC17 at a marginal islet dose (150 islets), (2) intrahepatic portal vein (PV) infusion11 at a full dose (500 islets), and (3) into the prevascularized subcutaneous DL site13,14 at a marginal islet dose (150 islets). Nonsupplemented F573 islet transplants served as controls for each transplant group. Before recovery, all recipients received a 0.1 mg/kg subcutaneous bolus of buprenorphine.

Transplant recipients were administered a subcutaneous injection of either F573 (3 mg/kg: KC, PV, DL groups and 10 mg/kg: PV group) for the treatment groups or vehicle (saline) for the control recipients on the day of transplant and for 5 days thereafter.

Evaluation of Islet Graft Function

Transplant efficacy was assessed 3 times per week in recipients through nonfasting blood glucose measurements, using a portable glucometer (OneTouch Ultra 2; LifeScan, Canada) in all groups tested. Graft function and reversal of diabetes was defined as 2 consecutive readings less than 11.1 mmol/L and maintained until study completion. In addition, intraperitoneal glucose tolerance tests (IPGTTs) were conducted posttransplant to further assess metabolic capacity by mimicking postprandial stimulation. Mice were fasted overnight before receiving an intraperitoneal glucose bolus (3 g/kg). Blood glucose levels were evaluated at baseline (time 0), 15, 30, 60, 90, and 120 minutes postinjection. Blood glucose area under the curve (AUC-blood glucose) was calculated and analyzed between transplant groups.

Both KC and DL islet transplants were retrieved by nephrectomy or subcutaneous graft excision to confirm graft dependent euglycemia, as previously described.14 Nonfasting blood glucose measurements were monitored for 7 days after graft removal to confirm hyperglycemia and thus posttransplant graft function.

Statistical Analysis

Nonfasting blood glucose, AUC-blood glucose, mouse and human insulin, caspase-3 activation, and percent apoptosis data are represented as the mean ± SEM. In vitro and in vivo data analysis between treatment groups were conducted by unpaired 2-tailed t test. Kaplan-Meyer survival function curves were compared using the log-rank statistical method (Mantel-Cox). P less than 0.05 was considered significant.

RESULTS

Pan-caspase Inhibitor F573 Abrogates Human Islet Apoptosis Postculture

Human islets were cultured in media supplemented with or without F573. After in vitro culture, control islets exhibited a significantly higher rate of apoptosis compared with F573-treated islets (control islets: 3.06 ± 1.10% vs F573 islets: 0.60 ± 0.19%, P < 0.05, unpaired 2-tailed t-test) (Figures 1A, B). Furthermore, F573-treated human islets displayed significantly less activated caspase-3 compared with controls (F573 islets: 15.02 ± 1.18 vs control islets: 20.15 ± 2.23 Abs.405 nm/100 Islets, P < 0.05, unpaired 2-tailed t-test) (Figure 1C).

F1-16
FIGURE 1:
Assessment of human islet apoptosis before transplantation and subsequent to culture with or without F573 supplementation. A, Representative fluorescent microphotographs of human islets stained for insulin (red), TUNEL (apoptosis) (green) and nuclei (blue) ± F573. B, Percentage of TUNEL positive cells in both groups as an expression of apoptosis postculture. C, Concentration of cleaved caspase-3 expressed in islets postculture ± F573 supplementation. Data points represent mean ± SEM, n = 2 donor, *P < 0.05, unpaired 2-tailed t test.

F573 Culture Supplementation Improves Human Islet In Vitro Viability and Function

Dual-fluorescence membrane integrating staining (SytoEB) of human islets revealed a significant increase in viability in islets cultured for 24 hours in the presence of F573 compared with controls (F573: 96.3 ± 2.2% vs control: 86.0 ± 4.2%, P < 0.05, unpaired t-test, n = 2 isolations) (Figure 2A). Similarly, glucose static challenge revealed that F573-treated human islets had a greater insulin secretory capacity in response to glucose (Stimulation index: 1.87 ± 0.05) compared with control islets (Stimulation index: 1.66 ± 0.03, P < 0.05, unpaired t test, n = 2 human islet preparations tested in triplicate) (Figures 2B, C).

F2-16
FIGURE 2:
In vitro viability assessment of control and F573-treated human islets 24 hours postculture. A, F573-treated islets exhibited improved islet viability as assessed by dual-fluorescence staining compared to control islets (*P < 0.05, t-test). B, C, Islet function, as assessed by static glucose-stimulated insulin secretion, demonstrated that F573-treated islets have an improved insulin secretory capacity compared to control (*P < 0.05, t-test)(n = 2 isolations, triplicate samples per isolation).

F573 Enhances Diabetes Reversal in Marginal Mass Transplantation of Human Islets in Immunodeficient Mice

A marginal human islet mass (500 islet equivalents per recipient) transplanted beneath the murine KC was evaluated in the presence or absence of F573 (3 mg/kg). Of the diabetic mice recipients transplanted with control human islets, 2 (25%) of 8 became euglycemic. In contrast, F573 supplementation in culture plus subcutaneous F573 therapy significantly improved human islet engraftment efficacy and diabetes reversal in 6 (75%) of 8 recipients (P < 0.05, long-rank, compared with control transplants) (Figure 3A). As such, F573 recipients presented with an overall reduced daily nonfasting blood glucose profile compared to controls (Figure 3B). Euglycemic mice in both transplant cohorts maintained glucose homeostasis until islet-bearing kidney grafts were retrieved; reverting to hyperglycemia thus proving graft-dependent function (Figure 3B). Before graft retrieval, mice in the F573 groups (n = 7) displayed a superior physiological response to IPGTT 30 days posttransplant compared with control mice (n = 7) (Figure 3C), as demonstrated by a significantly lower mean AUC-blood glucose ± SEM (F573: 1998 ± 237 mmol/L /120 min vs control: 2678 ± 290 mmol/L/120 min, P < 0.05, unpaired 2-tailed t test) (Figure 3D).

F3-16
FIGURE 3:
F573 therapy improves marginal human islet mass engraftment transplanted under the kidney capsule (KC) of mice. A, Reversal of diabetes rates, percent euglycemia, in the F573 3 mg/kg recipients group (red, n = 8) were higher than the control transplant recipients (blue, n = 8) recipients 60 days posttransplant (*P < 0.001, log-rank). B, Nonfasting blood glucose measurements of recipients posttransplant. All euglycemic recipient of marginal human islet grafts maintained glycemic control until graft retrieval (arrow). C, Blood glucose profile during IPGTT of F573 recipients (red, n = 7) and control recipients (blue, n = 7), 30 days posttransplant. D, Mean AUC-blood glucose was significantly lower in the F573 treatment group (*P < 0.05, unpaired 2-tailed t test). Mice were administered 3 mg/kg 50% dextrose i.p. Blood glucose measurements were monitored at t = 0, 15, 30, 60, 90 and 120 minutes. i.p., intraperitoneal.

F573 Inhibits Mouse Islet Apoptosis in Vitro

Immediately postisolation and before syngeneic islet transplantation, murine islets were cultured with or without F573 media supplementation. The percentage of apoptotic cells in the control cells was significantly greater than the F573-treated islets when assessed by TUNEL assay (F573: 0.49 ± 0.18% vs Control: 2.34 ± 1.08%, P < 0.05, unpaired 2-tailed t test) (Figures 4A, B). Likewise, the quantity of active caspase-3 was significantly elevated postculture in control mouse islets compared to F573-treated islets (control islets: 10.20 ± 1.17 vs F573 islets: 7.60 ± 0.37 Abs. 405 nm/100 Islets, P < 0.05, unpaired 2-tailed t test) (Figure 4C).

F4-16
FIGURE 4:
Evaluation of mouse islet apoptosis before transplantation and subsequent to culture with or without F573 supplementation. A, Representative fluorescent microphotographs of mouse islets stained for insulin (red), TUNEL (apoptosis) (green) and nuclei (blue) ± F573. B, Percentage of TUNEL positive cells in both groups as an expression of apoptosis postculture. C, Caspase-3 activation expressed in islets postculture ± F573 supplementation. Data points represent mean ± SEM, n = 7/group, *P < 0.05, unpaired 2-tailed t test.

F573 Culture Supplementation Does Not Improve Mouse Islet in Vitro Viability

Membrane integrity dual-florescence staining did not differ between control and F573-treated mouse islets, 2 hours postculture (Control: 93.4 ± 0.8% vs F573: 93.7 ± 0.6%, P > 0.05, unpaired t-test, n = 2 isolations) (Figure 5A). Similarly, glucose static challenge revealed that F573-treated and control mouse islets did not difference in their insulin secretory capacity in response to glucose postculture (F573 stimulation index: 1.56 ± 0.16 vs control stimulation index 1.87 ± 0.16, P > 0.05, unpaired t-test, n = 2 mouse islet preparations tested in triplicate) (Figures 5B, C).

F5-16
FIGURE 5:
In vitro viability assessment of control and F573-treated human islets 24 hours postculture. A, F573-treated islets exhibited improved islet viability as assessed by dual-fluorescence staining compared to control islets (P > 0.05, t-test). B, C, Islet function, as assessed by static glucose stimulated insulin secretion, demonstrated that F573-treated islets have an improved insulin secretory capacity compared to control (P > 0.05, t test)(n = 2 isolations, triplicate samples per isolation).

F573 Does Not Improve Efficacy of Mouse Marginal Islet Mass Grafts Transplanted Under the KC

The ability of F573 to enhance islet engraftment efficacy was evaluated using the KC site (F573: 3 mg/kg: n = 11), in a marginal islet transplant mass model (150 islets per recipient). As a means to compare engraftment efficiency, a control group (no F573 supplementation or therapy) of diabetic recipients was also transplanted with 150 islets, under the KC (control: n = 11). F573 therapy did not enhance rates of euglycemia postmarginal islet engraftment compared with controls [F573 3 mg/kg: 64% (7 of 11) vs control: 55% (6 of 11), P > 0.05, log-rank] (Figure 6A). IPGTTs were performed on recipients 45 days posttransplant. Mice from both F573 (n = 7) and control (n = 6) groups demonstrated a robust physiological response to the glucose challenge with a prompt restoration of normoglycemia (Figure 6B). Furthermore, there was no difference in mean AUC ± SEM (F573: 1932 ± 238 mmol/L/120 min vs control: 1890 ± 141 mmol/L per120 minutes, P > 0.05, unpaired 2-tailed t-test) (Figure 6C).

F6-16
FIGURE 6:
Posttransplant efficacy of mouse syngeneic islet transplants ± F573 therapy using standard and alternative engraftment sites. A, F573 therapy did not improve marginal mouse islet engraftment efficacy (control: n = 11, 3 mg/kg F573: n = 11)(P > 0.05 log-rank), (B) glucose clearance in response to IPGTT and (C) AUC-blood glucose (P > 0.05, unpaired 2-tailed t test), when islet were transplanted under the KC. D, Reversal of diabetes rates were significantly greater in 10 mg/kg F573 therapy recipients (red, n = 11) compared to control transplants (blue, n = 13) (*P < 0.01, log-rank), when islets were transplanted into the PV at a full islet dose. E, Blood glucose profiles and (F) AUC-blood glucose in response to an IPGTT was improved in the F573 transplant group compared to controls (*P < 0.05, unpaired 2-tailed t test). G, Therapeutic intervention with F573 (red, n = 12) improved the rate of euglycemia postmarginal islet mass transplants into the DL site compared to control islet recipients (blue, n = 22)(*P < 0.05). H, Blood glucose profile during IPGTTs of F573 recipients (red, n = 9) and control recipients (blue, n = 14), 100 days posttransplant. I, F573 recipients demonstrated a significantly improved response to glucose challenge compared to control recipients as demonstrated by markedly reduced AUC-blood glucose (***P < 0.001, unpaired, 2-tailed t test). Mice were administered 3 mg/kg 50% dextrose i.p. Blood glucose measurements were monitored at t = 0, 15, 30, 60, 90, and 120 minutes.

F573 Enhances the Rate of Diabetes Reversal Postfull Mouse Islet Mass Transplanted Into PV

Full islet mass (500 islets per recipient) engraftment efficacy posttransplant into the PV was evaluated ± F573 therapy at 3 and 10 mg/kg. Posttransplant intervention with 3 mg/kg of F573 in a small cohort of recipients had no beneficial effect on engraftment (data not shown). Subsequently, a dose of 10 mg/kg of F573 was examined. Of the recipients transplanted with control islets into the PV, 4 of 13 (31%) became euglycemic. In contrast, 8 of 11 (72%) recipients receiving F573 supplemented islets and exogenous F573 (10 mg/kg) therapy post-PV transplant, reversed diabetes; a significant improvement compared to control recipients (P < 0.05, long-rank) (Figure 6D). Recipients in the F573 transplant groups (n = 6) demonstrated an improved glucose clearance in response to an IPGTT 45 days posttransplant compared with control mice (n = 8) (Figure 6E), as corroborated by a markedly reduced mean AUC-blood glucose ± SEM (F573: 1980 ± 242 mmol/L per 120 minutes vs control: 2617 ± 199 mmol/L per 120 minutes, P < 0.05, unpaired 2-tailed t test) (Figure 6F).

F573 Improves and Accelerates Marginal Islet Mass Engraftment Into a Prevascularized Subcutaneous DL Site

The effect of F573 islet supplementation and recipient administration (3 mg/kg) was evaluated using a prevascularized subcutaneous site with a marginal islet transplant dose (150 islet per recipient). Of the recipients transplanted with control islets into the DL site 16 of 22 (72%) became euglycemic, whereas in the F573 experimental group, a significantly higher rate of diabetes reversal was observed, as 10 (83%) of 12 became euglycemic posttransplant (P < 0.05, log-rank) (Figure 6G). Furthermore, F573 significantly reduced the time to euglycemia from 41.1 ± 4.3 days posttransplant in control recipients to 20.1 ± 5.4 days posttransplant in the F573 experimental group (P < 0.001, unpaired 2-tailed t test). As a means to assess long-term function of mice posttransplant with or without F573 therapy, IPGTTs were conducted 100 days posttransplant. Recipients in the F573 group (n = 9) rapidly became normoglycemic after glucose challenge, demonstrating superior glucose clearance profiles compared to control DL transplants (n = 14) (Figure 6H). As a result, blood glucose AUCs ± SEM for glucose clearance were significantly elevated in the control group compared to the F573 DL islet transplant recipient (Control DL: 2861 ± 119 mmol/L per 120 minutes vs F573 DL: 2012 ± 137 mmol/L per 120 minutes, P > 0.001, unpaired 2-tailed t test) (Figure 6I).

DISCUSSION

Islet viability is compromised in the acute and peritransplant period, which accounts for substantial cell death, leading to failed engraftment and impaired islet function. Strategies aimed to reduce islet death and promote engraftment have included the utility of therapeutic agents capable of reducing apoptosis through pan-caspase inhibition, and alternatively through exploration of alternative transplant sites of potential clinical relevance, which do not involve direct introduction of freshly transplanted cells within the vascular space. One such strategy was recently described by Giovannoni et al,18 who demonstrated that Toll-like receptor 4 blockage was efficacious in reducing islet apoptosis and improving both syngeneic and allogeneic islet transplant outcomes in mice. In the current study, we sought to evaluate whether the administration of a pan-caspase inhibitor, F573, in culture and in the acute posttransplant period could reduce islet death and enhance engraftment in various transplant sites in preclinical rodent models.

The culture of murine and human islets with F573 resulted in reduced TUNEL-positive nuclei and caspase-3 activity when compared to islets in standard culture media alone. These findings demonstrate F573's caspase-specific inhibition of islet death, confirming previously established findings from our laboratory with earlier generations of caspase inhibitors.7,8,10,11 Our in vivo results demonstrated varying degrees of efficacy between murine and human islets transplanted beneath the renal capsule, as well as murine islets transplanted in alternative transplant sites. Notably, we found that a marginal mass of human islets transplanted beneath the kidney capsule of immunodeficient mice were highly protected by F573 treatment, but there was less measurable difference with murine islets. It is routine for us to culture human islets for periods exceeding 24 hours, but that we routinely transplant mouse islets within 2 hours of isolation. Therefore, the window of F573 exposure for the human islet studies (24 hours) was considerably longer than that of the mouse islet experiments (2 hours). Nonetheless, we see significant protection from apoptosis in both settings, and of greater magnitude in human versus murine islets. Furthermore, by the nature of human organ procurement, long cold ischemic transport times, a more intense isolation, purification, and culture conditions, by necessity human islets are exposed to more cumulative stressing events than the murine islets. This likely also explains the differential susceptibility of human islets to apoptosis and protection from F573 in the kidney capsule setting. In vitro viability data within the present study supports this hypothesis as the distressed human islets demonstrated improved function when cultured in the presence of F573 compared with the robust freshly isolated mouse islets. The cytoprotective effect of F573 in mouse islets was not apparent until a more inhospitable transplant site was implemented.

Further accounting for the in vivo difference is the impurity of human islet preparations in comparison to murine islets, with the former containing a greater percentage of acinar tissue. When contained in the renal subcapsular space and placed in close proximity to one another, the release of acinar enzymes may inflict significant death on neighboring islets.19 Drognitz and colleagues20 previously demonstrated that cold ischemia and reperfusion of the pancreas induces acinar apoptosis, whereas endocrine tissue was less susceptible. It is plausible that acinar tissue-specific apoptosis was preserved in the presence of F573, thus protecting neighboring islets from enzymatic lysis and improving engraftment outcomes.

Early generations of pan-caspase inhibitors have demonstrated improved PV islet engraftment at doses of 10 mg/kg.10 Initially, we sought to evaluate if a lower dose (3 mg/kg) would yield improved islet engraftment; however, this therapeutic strategy did not prove to be efficacious as it did when human islets were transplanted under the KC. Therefore, we increased the dose of F573 to 10 mg/kg for the PV transplant recipients. When administered via the intraportal route, murine islets transplanted at a full therapeutic dose exhibited significant engraftment in F573-treated (10 mg/kg) recipients as compared to control islet recipients. Robust islet loss occurs in the early transplant period, culture and administration of F573 likely preserved a sufficient islet mass subsequent to transplant, allowing for reversal of diabetes in a greater number of recipients. This has been confirmed in previous studies using earlier generation pan-caspase inhibitors in which islets were preincubated with the inhibitor and recipients were treated up to 5 days posttransplant.11 The inherent inflammatory cascades evoked by this route of islet delivery, such as instant blood-mediated inflammatory reaction,21 may indeed explain, in part, the requirement of a larger therapeutic dose of F573 to demonstrate improved transplant efficacy. Several antioxidants have been shown to optimize islet engraftment in mice such as the antioxidant cyaniding-3-O-glucoside.22 Our results support these findings, further suggesting the therapeutic benefit of this novel inhibitor, F573, in the clinical islet transplant setting.

Additional sources of insulin-producing cells are becoming available for potential future β-cell replacement therapy, including insulin-producing stem cells and xenogeneic islet sources. The establishment of an optimal alternative transplant site should consider its ability to accommodate a sufficient transplant mass, its clinically feasibility and whether it can be easily retrieved should complications arise.13 We previously reported that modification of the subcutaneous space using the DL technique could adequately support islet engraftment and restore euglycemia to a greater extent than the unmodified space while having the capacity to safely retrieve the graft.13 Moreover, the DL technique was shown to be efficacious in restoring euglycemia when transplanted with a marginal islet mass in a murine diabetes model.14 Our data in the current study demonstrates that F573 administration augments islet engraftment outcomes relative to control recipients in the DL site suggesting that this technique may exert some degree of apoptosis on the transplanted graft. Similarly, Espes et al23 demonstrated induced apoptosis in intramuscular islet grafts that could be reduced with the co-transplantation of low dose polymerized hemoglobin. Furthermore, our observations reflect the efficiency of F573 to improve prevascularized subcutaneous DL engraftment while expediting the restoration of glycemic control in a regulated physiological manner. Should this transplant technique be translated to the clinical setting, it may be of benefit to incorporate pan-caspase treatment in vitro and in the acute transplant period as an added therapy to promote early engraftment.

This study supports the utility of the pan-caspase inhibitor, F573, in islet transplantation. The differences in engraftment efficacy observed in murine and human islets transplanted under the renal capsule strongly endorse the potential benefit of this therapy in clinical transplantation. Should extrahepatic transplant sites be incorporated in the clinical setting, pan-caspase therapy may also prove beneficial in the acute posttransplant period to expedite engraftment outcomes. The ability of F573 to restore euglycemia with subtherapeutic, marginal islet doses is of added benefit, because donor availability impedes the number of potential patients that may be treated.

ACKNOWLEDGMENTS

The pan-caspase inhibitor F573 was generously supplied by Dr. Luo of Shanghai Genomics.

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