In vivo imaging of autologous islet grafts in the liver and under the kidney capsule in non-human primates.

OBJECTIVE: As islet transplantation begins to show promise as a clinical method, there is a critical need for reliable, noninvasive techniques to monitor islet graft survival. Previous work in our laboratory has shown that human islets labeled with a superparamagnetic iron oxide contrast agent and transplanted into mice could be detected by magnetic resonance imaging (MRI). The potential translation of these findings to the clinical situation requires validation of our methodology in a non-human primate model, which we have now carried out in baboons (Papio hamadryas) and reported here. RESEARCH DESIGN AND METHODS: For islet labeling, we adapted the Food and Drug Administration-approved superparamagnetic iron oxide contrast agent, Feridex, which is used clinically for liver imaging. After partial pancreatectomy, Feridex-labeled islets were prepared and autotransplanted underneath the renal capsule and into the liver. Longitudinal in vivo MRI at days 1, 3, 8, 16, 23, and 30 after transplantation was performed to track the islet grafts. RESULTS: The renal subcapsular islet graft was easily detectable on T2*-weighted MR images as a pocket of signal loss disrupting the contour of the kidney at the transplantation site. Islets transplanted in the liver appeared as distinct signal voids dispersed throughout the liver parenchyma. A semiautomated computational analysis of our MRI data established the feasibility of monitoring both the renal and intrahepatic grafts during the studied posttransplantation period. CONCLUSION: This study establishes a method for the noninvasive, longitudinal detection of pancreatic islets transplanted into non-human primates using a low-field clinical MRI system.

Multifunctional magnetic nanocarriers for image-tagged SiRNA delivery to intact pancreatic islets.

BACKGROUND: With the ultimate hope of finding a cure for diabetes, researches are looking into altering the genetic profile of the beta cell as a way to manage metabolic dysregulation. One of the most powerful new approaches for the directed regulation of gene expression uses the phenomenon of RNA interference. METHODS: Here, we establish the feasibility of a novel technology centered around multifunctional magnetic nanocarriers, which concurrently deliver siRNA to intact pancreatic islets and can be detected by magnetic resonance and optical imaging. RESULTS: In the proof-of-principle studies described here, we demonstrate that, after in vitro incubation, magnetic nanoparticles carrying siRNA designed to target the model gene for enhanced green fluorescent protein are efficiently taken up by murine pancreatic islets, derived from egfp transgenic animals. This uptake can be visualized by magnetic resonance imaging and near-infrared fluorescence optical imaging and results in suppression of the target gene. CONCLUSIONS: These results illustrate the value of our approach in overcoming the challenges associated with genetic modification of intact pancreatic islets in a clinically acceptable manner. Furthermore, an added advantage of our technology derives from the combined capability of our magnetic nanoparticles for siRNA delivery and magnetic labeling of pancreatic islets.

Effects of glucose toxicity and islet purity on in vivo magnetic resonance imaging of transplanted pancreatic islets.

BACKGROUND: Pancreatic islet transplantation has recently emerged as a powerful clinical modality to restore normoglycemia in diabetic patients. Despite the success of the Edmonton protocol, these patients still experience a significant islet loss immediately after transplantation. Noninvasive magnetic resonance imaging (MRI) allows for longitudinal monitoring of graft loss providing that islets are labeled with a magnetically "visible" contrast agent. To fully interpret the imaging data, it is critical to investigate factors normally present during clinical transplantation and influencing MRI of transplanted islets. METHODS: Here, we focused on both the effect of hyperglycemia and the effect of contaminating nonendocrine tissue, which is always present in islet preparations, on MRI imaging of islet grafts. Human pancreatic islets labeled with Feridex were transplanted in diabetic and healthy animals. Separate groups of animals were transplanted with Feridex-labeled pure and nonpure (50% islets and 50% nonendocrine tissue) preparations. The fate of the graft in all groups was monitored by in vivo MRI. RESULTS: We found that diabetic animals with transplanted islets showed a significantly higher rate of islet death than their healthy counterparts on in vivo MR images. Interestingly, transplantation of islets contaminated with nonendocrine tissue did not have any significant influence on MR images, presumably because of a low labeling rate of this tissue and a fast rate of its disappearance after transplantation. CONCLUSIONS: We believe that this study serves as yet another step on our way to clinical use of in vivo imaging of islet transplantation.

In vivo imaging of a diabetogenic CD8+ T cell response during type 1 diabetes progression.

Type 1 diabetes is preceded by a long, protracted period of pancreatic islet inflammation by autoreactive lymphocytes. Noninvasive imaging of islet inflammation prior to the onset of hyperglycemia might have diagnostic and therapeutic implications, but this is not currently possible. Here, MRI is used to track, noninvasively, the accumulation diabetogenic CD8+ T-cells during type 1 diabetes progression in nonobese diabetic (NOD) mice. The contrast agent is an MRI probe (MN-NRP-V7) that specifically labels CD8+ T-cells recognizing residues 206-214 of islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP(206-214)) in the context of the major histocompatibility complex (MHC) class I molecule H-2K(d). This probe consists of superparamagnetic iron oxide nanoparticles (MN) coated with K(d) molecules presenting NRP-V7, a high-avidity mimotope of IGRP(206-214). NOD mice of different ages (5, 8, 15, and 24 weeks) were imaged by MRI before and after a single intravenous injection of MN-NRP-V7 or unmodified MN nanoparticles. MN-NRP-V7 accumulation, as determined by semiquantitative MRI analysis of pancreas-associated T(2) relaxation time, was antigen-specific, age-dependent, and well correlated with the numbers of MN-NRP-V7-labeled CD8+ T-cells recovered from the pancreata of the treated mice. Antigen/MHC-coupled nanoparticles represent a promising new avenue for noninvasive imaging of lymphocyte inflammation in organ-specific autoimmunity and transplantation.

Inhaled agonists of soluble guanylate cyclase induce selective pulmonary vasodilation.

RATIONALE: Nitric oxide-independent agonists of soluble guanylate cyclase (sGC) have been developed. OBJECTIVES: We tested whether inhalation of novel dry-powder microparticle formulations containing sGC stimulators (BAY 41-2272, BAY 41-8543) or an sGC activator (BAY 58-2667) would produce selective pulmonary vasodilation in lambs with acute pulmonary hypertension. We also evaluated the combined administration of BAY 41-8543 microparticles and inhaled nitric oxide (iNO). Finally, we examined whether inhaling BAY 58-2667 microparticles would produce pulmonary vasodilation when the response to iNO is impaired. METHODS: In awake, spontaneously breathing lambs instrumented with vascular catheters and a tracheostomy tube, U-46619 was infused intravenously to increase mean pulmonary arterial pressure to 35 mm Hg. MEASUREMENTS AND MAIN RESULTS: Inhalation of microparticles composed of either BAY 41-2272, BAY 41-8543, or BAY 58-2667 and excipients (dipalmitoylphosphatidylcholine, albumin, lactose) produced dose-dependent pulmonary vasodilation and increased transpulmonary cGMP release without significant effect on mean arterial pressure. Inhalation of microparticles containing BAY 41-8543 or BAY 58-2667 increased systemic arterial oxygenation. The magnitude and duration of pulmonary vasodilation induced by iNO were augmented after inhaling BAY 41-8543 microparticles. Intravenous administration of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), which oxidizes the prosthetic heme group of sGC, markedly reduced the pulmonary vasodilator effect of iNO. In contrast, pulmonary vasodilation and transpulmonary cGMP release induced by inhaling BAY 58-2667 microparticles were greatly enhanced after treatment with ODQ. CONCLUSIONS: Inhalation of microparticles containing agonists of sGC may provide an effective novel treatment for patients with pulmonary hypertension, particularly when responsiveness to iNO is impaired by oxidation of sGC.

In vivo imaging of immune rejection in transplanted pancreatic islets.

As islet transplantation becomes an acceptable clinical modality for restoring normoglycemia in type 1 diabetic patients, there is a crucial need for noninvasive assessment of the fate of the grafts. In spite of the success of the Edmonton Protocol, a significant graft loss occurs due to immunological and nonimmunological events immediately after transplantation. Allogeneic rejection in graft recipients is one of the major reasons for islet death and graft failure. Therefore, monitoring the islet rejection using reliable noninvasive methods would significantly aid in clinical assessment of graft success. We have previously developed a method to detect transplanted islets noninvasively using magnetic resonance imaging (MRI). For this procedure, human pancreatic islets are labeled with an MRI contrast agent that enables their visualization on magnetic resonance images. In our present study, we not only detected labeled human islets in a preclinical intrahepatic model of human islet transplantation in mice but also showed that islet rejection can be monitored noninvasively and repeatedly in real time by MRI. In addition, in this study, we have adapted, for islet cell labeling, a Food and Drug Administration-approved commercially available contrast agent, Feridex, that is used clinically for liver imaging. We believe that this agent, in combination with our preclinical model of islet transplantation, will facilitate the transition of imaging immune rejection to clinical trials.

In vivo imaging of islet transplantation.

Type 1 diabetes mellitus is characterized by the selective destruction of insulin-producing beta cells, which leads to a deficiency in insulin secretion and, as a result, to hyperglycemia. At present, transplantation of pancreatic islets is an emerging and promising clinical modality, which can render individuals with type 1 diabetes insulin independent without increasing the incidence of hypoglycemic events. To monitor transplantation efficiency and graft survival, reliable noninvasive imaging methods are needed. If such methods were introduced into the clinic, essential information could be obtained repeatedly and noninvasively. Here we report on the in vivo detection of transplanted human pancreatic islets using magnetic resonance imaging (MRI) that allowed noninvasive monitoring of islet grafts in diabetic mice in real time. We anticipate that the information obtained in this study would ultimately result in the ability to detect and monitor islet engraftment in humans, which would greatly aid the clinical management of this disease.

Inhibition of phosphodiesterase 1 augments the pulmonary vasodilator response to inhaled nitric oxide in awake lambs with acute pulmonary hypertension.

Phosphodiesterase 1 (PDE1) modulates vascular tone and the development of tolerance to nitric oxide (NO)-releasing drugs in the systemic circulation. Any role of PDE1 in the pulmonary circulation remains largely uncertain. We measured the expression of genes encoding PDE1 isozymes in the pulmonary vasculature and examined whether or not selective inhibition of PDE1 by vinpocetine attenuates pulmonary hypertension and augments the pulmonary vasodilator response to inhaled NO in lambs. Using RT-PCR, we detected PDE1A, PDE1B, and PDE1C mRNAs in pulmonary arteries and veins isolated from healthy lambs. In 13 lambs, the thromboxane A(2) analog U-46619 was infused intravenously to increase mean pulmonary arterial pressure to 35 mmHg. Four animals received an intravenous infusion of vinpocetine at incremental doses of 0.3, 1, and 3 mg.kg(-1).h(-1). In nine lambs, inhaled NO was administered in a random order at 2, 5, 10, and 20 ppm before and after an intravenous infusion of 1 mg.kg(-1).h(-1) vinpocetine. Administration of vinpocetine did not alter pulmonary and systemic hemodynamics or transpulmonary cGMP or cAMP release. Inhaled NO selectively reduced mean pulmonary arterial pressure, pulmonary capillary pressure, and pulmonary vascular resistance index, while increasing transpulmonary cGMP release. The addition of vinpocetine enhanced pulmonary vasodilation and transpulmonary cGMP release induced by NO breathing without causing systemic vasodilation but did not prolong the duration of pulmonary vasodilation after NO inhalation was discontinued. Our findings demonstrate that selective inhibition of PDE1 augments the therapeutic efficacy of inhaled NO in an ovine model of acute chemically induced pulmonary hypertension.

In vivo multimodal imaging of transplanted pancreatic islets.

Interest is increasing in the transplantation of pancreatic islets as a means to achieve insulin independence in individuals with type I diabetes. The success of this approach is hampered by the absence of methods to follow the fate of transplanted islets non-invasively. In vivo imaging seems to be the most appropriate technique to achieve this goal in small animals and eventually in humans. Here we describe a protocol for labeling and subsequent imaging of transplanted islets in vivo using magnetic resonance imaging (MRI) and optical imaging. The whole series of experiments can be carried out in roughly 48 h. We believe that our approach can significantly advance the current ability to determine islet distribution, and possibly survival, after transplantation. This information would be essential not only for the long-term monitoring of graft function but also for the design of improved transplantation and immunomodulatory methods.

Soluble guanylate cyclase activator reverses acute pulmonary hypertension and augments the pulmonary vasodilator response to inhaled nitric oxide in awake lambs.

BACKGROUND: Inhaled nitric oxide (NO) is a potent and selective pulmonary vasodilator, which induces cGMP synthesis by activating soluble guanylate cyclase (sGC) in ventilated lung regions. Carbon monoxide (CO) has also been proposed to influence smooth muscle tone via activation of sGC. We examined whether direct stimulation of sGC by BAY 41-2272 would produce pulmonary vasodilation and augment the pulmonary responses to inhaled NO or CO. METHODS AND RESULTS: In awake, instrumented lambs, the thromboxane analogue U-46619 was intravenously administered to increase mean pulmonary arterial pressure to 35 mm Hg. Intravenous infusion of BAY 41-2272 (0.03, 0.1, and 0.3 mg x kg(-1) x h(-1)) reduced mean pulmonary arterial pressure and pulmonary vascular resistance and increased transpulmonary cGMP release in a dose-dependent manner. Larger doses of BAY 41-2272 also produced systemic vasodilation and elevated the cardiac index. N(omega)-nitro-l-arginine methyl ester abolished the systemic but not the pulmonary vasodilator effects of BAY 41-2272. Furthermore, infusing BAY 41-2272 at 0.1 mg x kg(-1) x h(-1) potentiated and prolonged the pulmonary vasodilation induced by inhaled NO (2, 10, and 20 ppm). In contrast, inhaled CO (50, 250, and 500 ppm) had no effect on U-46619-induced pulmonary vasoconstriction before or during administration of BAY 41-2272. CONCLUSIONS: In lambs with acute pulmonary hypertension, BAY 41-2272 is a potent pulmonary vasodilator that augments and prolongs the pulmonary vasodilator response to inhaled NO. Direct pharmacological stimulation of sGC, either alone or in combination with inhaled NO, may provide a novel approach for the treatment of pulmonary hypertension.

Parenteral administration of glipizide sodium salt, an inhibitor of adenosine triphosphate-sensitive potassium channels, prolongs short-term survival after severe controlled hemorrhage in rats.

OBJECTIVE: Recent experimental evidence suggests that activation of adenosine triphosphate (ATP)-sensitive potassium channels contributes to vascular failure and early mortality after hemorrhagic shock. The present investigation evaluated the effects of the water-soluble sodium salt of glipizide, an inhibitor of ATP-sensitive potassium channels, in anesthetized and awake rats subjected to severe controlled hemorrhage. DESIGN: Prospective, randomized, controlled study. SETTING: Animal research laboratory. SUBJECTS: Male Wistar rats. INTERVENTIONS: Anesthetized rats were subjected to bleeding to reduce mean arterial pressure to either 40 or 35 mm Hg, which was maintained constant for 60 mins. In addition, awake rats underwent blood withdrawal of 4.25 mL/100 g over 20 mins. At the end of the hemorrhage period and 30 mins later, the animals received intravenous (5 and 20 mg/kg) or intramuscular (10 and 40 mg/kg) injections of glipizide sodium salt or vehicle. MEASUREMENTS AND MAIN RESULTS: In anesthetized rats subjected to pressure-controlled hemorrhage, glipizide sodium salt improved mean arterial pressure in a dose-dependent manner. Compared with the vehicle-treated animals, mean arterial pressure increased from 41.6 +/- 4.6 to 63.1 +/- 3.1 mm Hg in the 20 mg/kg intravenous group and from 33.2 +/- 4.9 to 54.0 +/- 4.7 mm Hg in the 40 mg/kg intramuscular group 60 mins after a 40-mm Hg shock. Furthermore, the drug did not affect the hemorrhage-induced changes in blood glucose concentrations. However, the higher doses of glipizide sodium salt attenuated the increments in plasma concentrations of lactate, alanine aminotransferase, creatinine, and amylase. Moreover, the higher doses markedly improved short-term survival after pressure- and volume-controlled bleeding. Overall, the intramuscular injections of the drug exerted salutary effects that were comparable to the intravenous administration. CONCLUSIONS: In rats, parenteral administration of the water-soluble glipizide sodium salt attenuates vascular and end-organ dysfunction associated with severe hemorrhagic shock and prolongs short-term survival. The intramuscular administration provides comparable benefits as obtained by the intravenous injection.