Hyperbaric oxygen therapy improves early posttransplant islet function.

OBJECTIVE: This study investigates the therapeutic potential of hyperbaric oxygen therapy (HBO) in reducing hypoxia and improving engraftment of intraportal islet transplants by promoting angiogenesis. METHODS: Diabetic BALB/c mice were transplanted with 500 syngeneic islets intraportally and received six consecutive twice-daily HBO treatments (n = 9; 100% oxygen for 1 h at 2.5 atmospheres absolute) after transplantation. Dynamic contrast-enhanced magnetic resonance imaging (DCE MRI) was used to assess new vessel formation at postoperative days (POD) 3, 7, and 14. Liver tissue was recovered at the same time points for correlative histology, including: hematoxylin and eosin, hypoxia-inducible factor (HIF1α), Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL), vascular endothelial growth factor (VEGF), and von Willebrand Factor immunohistochemistry. RESULTS: HBO therapy significantly reduced HIF-1α, TUNEL and VEGF expression in islets at POD 7. In the non-HBO transplants, liver enhancement on DCE MRI peaked at POD 7 consistent with less mature vasculature but this enhancement was suppressed at POD 7 in the HBO-treated group. The number of new peri-islet vessels at POD 7 was not significantly different between HBO and control groups. CONCLUSION: These results are consistent with a hyperbaric oxygen-mediated decrease in hypoxia that appeared to enhance vessel maturation in the critical days following intraportal islet transplantation.

In vivo imaging demonstrates a time-line for new vessel formation in islet transplantation.

Vascularization of transplanted islets must be maintained to provide long-term graft function. In vivo assessment of new vessel formation in islet grafts has been poorly documented. The purpose of this study was to investigate whether neovascularization was detectable in vivo in a Feridex-labeled murine syngeneic subcapsular islet mass using DCE MRI over 180 days. Subcapsular transplants could be visualized at post-transplant days three, seven, 14, and 28 using T2-weighted MRI and at post-transplant day 180 by immunohistochemistry. Injection of the contrast agent gadolinium (Gd)-DTPA for DCE at three, seven, and 14 days showed increased signal in the transplant area consistent with new vessel formation. Areas under contrast enhancement curves suggested peak angiogenesis at 14 days. At 180 days, there was no observable change in signal intensity after contrast injection suggesting established vascularization or islet mass reduction. Immunohistochemistry confirmed MRI and DCE findings. These data suggest that islet angiogenesis occurs early after transplantation and is likely established after one month of transplantation. This study provides an in vivo time-line of neovascularization in subcapsular islet grafts. We anticipate that contrast extravasation captured by MRI may provide useful monitoring of graft angiogenesis if reproduced in a clinically relevant intraportal model.

Partial hepatectomy improves the outcome of intraportal islet transplantation by promoting revascularization.

Revascularization of grafts is one of the important key factors for the success of islet transplantation. After partial hepatectomy, many growth factors such as hepatocyte growth factor and vascular endothelial growth factor are increased in the remnant liver. These growth factors have properties that promote angiogenesis. This might be an optimal environment for revascularization of islets transplanted intraportally. To verify this hypothesis, syngeneic islets (330 per recipient) were transplanted into the right hepatic lobes of streptozotocin-induced diabetic Balb/c mice with (hepatectomy group) or without (control group) left liver resection. Blood glucose was monitored for 28 d after transplantation. Glucose tolerance test was performed on post-operative day (POD) 30, and histological assessments were performed on POD 7 and 30 respectively. Analysis revealed that 36.7% of the control and 90.0% of the hepatectomy mice attained normoglycemia during the observation period (*p = 0.0142). Glucose tolerance was improved in the hepatectomy group (Area under the curve of intraperitoneal glucose tolerance tests on POD 30, Control; 47,700 ± 5,890 min*mg/dl, Hepatectomy; 26,000 ± 2,060 min*mg/dl: **p = 0.00314). Revascularization of grafted islets was more pronounced in the hepatectomy group (Vessel number per islet area on POD 7, Control; 3.20 ± 0.463 × 10 (-4) /µm ( 2) , Hepatectomy; 7.08 ± 0.513 × 10 (-4) /µm ( 2) : **p < 0.01). In the present study, partial hepatectomy (30%) improved the outcome of intraportal islet transplantation. Revascularization of islets transplanted into the liver may have been promoted by the induction of liver regeneration.

Nerve growth factor is associated with islet graft failure following intraportal transplantation.

Nerve growth factor (NGF) has recently been recognized as an angiogenic factor with an important regulatory role in pancreatic ß-cell function. We previously showed that treatment of pancreatic islets with NGF improved their quality and viability. Revascularization and survival of islets transplanted under the kidney capsule were improved by NGF. However, the usefulness of NGF in intraportal islet transplantation was not previously tested. To resolve this problem, we transplanted syngeneic islets (360 islet equivalents per recipient) cultured with or without NGF into the portal vein of streptozotocin-induced diabetic BALB/c mice. Analysis revealed that 44.4% (4/9) of control and 12.5% (1/8) of NGF-treated mice attained normoglycemia (≤ 200 mg/dL) (p = 0.195). NGF-treated islets led to worse graft function (area under the curve of intraperitoneal glucose tolerance tests (IPGTT) on post-operative day (POD) 30, control; 35,800 ± 3,960 min*mg/dl, NGF-treated; 47,900 ± 3,220 min*mg/dl: *p = 0.0348). NGF treatment of islets was also associated with increased graft failure [the percentage of TdT-mediated dUTP-biotin nick-end labeling (TUNEL)-positive and necrotic transplanted islets on POD 5, control; 23.8% (5/21), NGF-treated; 52.9% (9/17): p = 0.0650] following intraportal islet transplantation. Nonviable (TUNEL-positive and necrotic) islets in both groups expressed vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α). On the other hand, viable (TUNEL-negative and not necrotic) islets in both groups did not express VEGF and HIF-1α. In the present study, pre-transplant NGF treatment was associated with impaired survival and angiogenesis of intraportal islet grafts. The effect of NGF on islet transplantation may significantly vary according to the transplant site.

Bone marrow cell cotransplantation with islets improves their vascularization and function.

BACKGROUND.: To test the angiogenesis-promoting effects of bone marrow cells when cotransplanted with islets. METHODS.: Streptozotocin-induced diabetic BALB/c mice were transplanted syngeneically under the kidney capsule: (1) 200 islets, (2) 1 to 5x10 bone marrow cells, or (3) 200 islets and 1 to 5x10 bone marrow cells. All mice were evaluated for blood glucose, serum insulin, and glucose tolerance up to postoperative day (POD) 28, and a subset was monitored for 3 months after transplantation. Histologic assessment was performed at PODs 3, 7, 14, 28, and 84 for the detection of von Willebrand factor (vWF), vascular endothelial growth factor (VEGF), insulin, cluster of differentiation-34, and pancreatic duodenal homeobox-1 (PDX-1) protein. RESULTS.: Blood glucose was the lowest and serum insulin was the highest in the islet+bone marrow group at POD 7. Blood glucose was significantly lower in the islet+bone marrow group relative to the islet only group after 63 days of transplantation (P<0.05). Significantly more new periislet vessels were detected in the islet+bone marrow group compared with the islet group (P<0.05). Vascular endothelial growth factor staining was more prominent in bone marrow than in islets (P<0.05). Pancreatic duodenal homeobox-1-positive areas were identified in bone marrow cells with an increase in staining over time. However, there were no normoglycemic mice and no insulin-positive cells in the bone marrow alone group. CONCLUSIONS.: Cotransplantation of bone marrow cells with islets is associated with enhanced islet graft vascularization and function.

Bone marrow cells produce nerve growth factor and promote angiogenesis around transplanted islets.

AIM: To clarify the mechanism by which bone marrow cells promote angiogenesis around transplanted islets. METHODS: Streptozotocin induced diabetic BALB/c mice were transplanted syngeneically under the kidney capsule with the following: (1) 200 islets (islet group: n = 12), (2) 1-5 x 10(6) bone marrow cells (bone marrow group: n = 11), (3) 200 islets and 1-5 x 10(6) bone marrow cells (islet + bone marrow group: n = 13), or (4) no cells (sham group: n = 5). All mice were evaluated for blood glucose, serum insulin, serum nerve growth factor (NGF) and glucose tolerance (GTT) up to postoperative day (POD) 14. Histological assessment for insulin, von Willebrand factor (vWF) and NGF was performed at POD 3, 7 and 14. RESULTS: Blood glucose level was lowest and serum insulin was highest in the islet + bone marrow group. Serum NGF increased in islet, bone marrow, and islet + bone marrow groups after transplantation, and there was a significant difference (P = 0.0496, ANOVA) between the bone marrow and sham groups. The number of vessels within the graft area was significantly increased in both the bone marrow and islet + bone marrow groups at POD 14 as compared to the islet alone group (21.2 +/- 3.6 in bone marrow, P = 0.01, vs islet group, 22.6 +/- 1.9 in islet + bone marrow, P = 0.0003, vs islet group, 5.3 +/- 1.6 in islet-alone transplants). NGF was more strongly expressed in bone marrow cells compared with islets. CONCLUSION: Bone marrow cells produce NGF and promote angiogenesis. Islet co-transplantation with bone marrow is associated with improvement of islet graft function.

Correlation between angiogenesis and islet graft function in diabetic mice: magnetic resonance imaging assessment.

BACKGROUND/PURPOSE: Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was used to evaluate neovascularization after intravenous injection of gadolinium, where contrast leaks out of new vessels and remains within the tissues. We examined the relationship between DCE-MRI and metabolic parameters such as blood glucose, serum insulin and glucose tolerance test (GTT) after intraportal islet transplantation. METHODS: Streptozotocin-induced diabetic BALB/c mice (n = 15) received syngeneic intraportal islet transplantation (500 islet equivalent). Blood glucose, serum insulin and GTT were evaluated till postoperative day (POD) 14. Liver DCE-MRI was performed at POD 3, 7 and 14. Correlations between DCE-MRI and metabolic parameters were examined using regression analysis. RESULTS: Eight mice achieved normoglycemia after intraportal transplantation. At POD 3 a significant but moderate correlation between DCE-MRI and blood glucose was found. No DCE-MRI or metabolic parameters correlated at POD 7. However, at POD 14 strong or moderate correlations between DCE-MRIs were found: negative correlations with blood glucose (R (2) = 0.86) and GTT (R (2) = 0.48) but a positive correlation with serum insulin (R (2) = 0.32). CONCLUSION: We report that DCE-MRI can reflect the metabolic and functional condition of the transplanted islets.

MRI assessment of ischemic liver after intraportal islet transplantation.

BACKGROUND: There is a recent focus on embolization of the portal vein by transplanted islets as a major cause of early graft loss. The resultant ischemia causes necrosis or apoptosis of cells within the liver. Thus, noninvasive assessment of the liver receiving the islet transplant is important to evaluate the status islet grafts. METHODS: This study used noninvasive magnetic resonance imaging (MRI) for assessment of the posttransplant ischemic liver. Syngeneic islets in streprozotocin-induced diabetic mice were used. MRI and morphological liver assessments were performed at 0, 2, and 28 days after transplantation. Histologic assessment of insulin, hypoxia induced factor 1-alpha, and apoptosis were undertaken at similar time points. RESULTS: Ischemic/necrotic regions in the liver were detected by MRI at 2 days but not at 28 days after transplantation and were confirmed histologically. Liver injury was quantified from high intensity areas on T2-weighted images. Insulin release peaked 2 days after transplantation. CONCLUSION: Onset and reversal of liver ischemia due to intraportal islet transplantation are detectable using T2-weighted MRI. These changes coincide with periods of maximum insulin release likely due to partial islet destruction. We propose that MRI, as a noninvasive monitor of graft-related ischemia, may be useful in assessment of liver and islet engraftment after intraportal islet transplantation in a clinical setting.

Monitoring neovascularization of intraportal islet grafts by dynamic contrast enhanced magnetic resonance imaging.

Fifteen thousand youths are diagnosed yearly with type 1 diabetes mellitus. Pancreatic islet transplantation has been shown clinically to provide short-term (~1 year) insulin independence. However, challenges associated with early vascularization of transplanted islet grafts and long-term islet survival remain. We utilized dynamic contrast enhanced magnetic resonance imaging (DCE MRI) to monitor neovascularization of islets transplanted into the right lobe of the liver in a syngeneic mouse model. The left lobe received no islets and served as a control. DCE data were analyzed for temporal dynamics of contrast (gadolinium) extravasation and the results were fit to a Tofts two-compartment exchange model. We observed maximal right lobe enhancement at seven days post-transplantation. Histological examination up to 28 days was used to confirm imaging results. DCE-derived enhancement strongly correlated with immunohistochemical measures of neovascularization. To our knowledge these results are the first to demonstrate, using a FDA approved contrast agent, that DCE MRI can effectively and non-invasively monitor the progression of angiogenesis in intraportal islet grafts.