Brett K. Levay-Young

Opioids heal ischemic wounds in the rat

Opioids are sometimes used to treat pain in ulcerative wounds, and it is speculated that pain interferes with the healing process. Because the direct effect of opioids on this process remains unknown, we examined the effect of topically applied opioids on the healing of open ischemic wounds in rats. Topically applied opioids hastened wound closure, particularly in the first 4 days when no healing was initiated in phosphate buffered saline solution‐treated wounds. After 1 week of application, fentanyl, hydromorphone, and morphine resulted in 66%, 55%, and 42% wound closure, respectively, as compared to only 15% in control wounds. Opioid‐induced healing was accompanied by a 1.5‐ to 2.5‐fold increase in nuclear density in the granulation tissue and 45–87% increase in angiogenesis as compared to phosphate buffered saline solution‐treated wounds. Fentanyl showed significantly improved healing compared to morphine and hydromorphone (p < 0.05, fentanyl vs. others). Fentanyl‐induced healing was inhibited by the opioid receptor antagonist naloxone, suggesting that peripheral opioid receptor(s) mediate the healing process. Opioids accelerate healing by up‐regulating both endothelial and inducible nitric oxide synthase and the vascular endothelial‐derived growth factor receptor Flk1 in the wounds. We envision that opioids can be used topically to accelerate wound healing in diverse clinical conditions ranging from surgical incisions to nonhealing ischemic ulcers in pathophysiological conditions and in hospice patients.

Development of Adrenal Zonation in Fetal Rats Defined by Expression of Aldosterone Synthase and 11β-Hydroxylase

The adult rat adrenal cortex is comprised of three concentric steroidogenic zones that are morphologically and functionally distinguishable: the zona glomerulosa, zona intermedia, and the zona fasciculata/reticularis. Expression of the zone-specific steroidogenic enzymes, cytochrome P450 aldosterone synthase (P450aldo), and P450 11beta hydroxylase (P45011beta), produced by the zona glomerulosa and zona fasciculata/reticularis, respectively, can be used to define the adrenal cortical cell phenotype of these two zones. In this study, immunohistochemistry and in situ hybridization were used to determine the ontogeny of expression of P450aldo and P45011beta to monitor the pattern of development of the rat adrenal cortex. RIA was used to measure adrenal content of aldosterone and corticosterone, the resulting products of the two enzymatic pathways. Double immunofluorescent staining for both enzymes at gestational day 16 (E16) showed P45011beta protein expressed in cells distributed throughout most of the adrenal intermixed with a separate, but smaller, population of cells expressing P450aldo protein. Whereas expression of P45011beta protein retained a similar pattern of distribution from E16 to adulthood (ignoring distribution of SA-1 positive, presumptive medullary cells), P450aldo protein changed its pattern of distribution by E19, becoming localized in a discontinuous ring of cells adjacent to the capsule. By postnatal day 1, P450aldo protein distribution was similar to that observed in adult glands; P450aldo-positive cells formed a continuous zone underlying the capsule. In situ hybridization showed that the pattern of P45011beta messenger RNA expression paralleled protein expression at all times, whereas P450aldo messenger RNA paralleled protein at E19 and after, but was undetectable before E19. However, adrenal aldosterone and corticosterone, as measured by RIA, were detected by E16, supporting the functional capacity of both phenotypes for all ages studied. These data suggest that the development of the adrenal zona glomerulosa occurs in two distinct phases; initial expression of the glomerulosa phenotype in scattered cells of the inner cortex before E17, followed by a change in distribution to the outer cortex between E17 and E19. It is hypothesized that this change in distribution occurs via cell differentiation, rather than cell migration, and that a possible regulator of these events is the fetal renin-angiotensin system.

IL-4 and IL-13 Induce Protection of Porcine Endothelial Cells from Killing by Human Complement and from Apoptosis through Activation of a Phosphatidylinositide 3-Kinase/Akt Pathway

Vascular endothelial cells (EC) perform critical functions that require a balance of cell survival and cell death. EC death by apoptosis and EC activation and injury by the membrane attack complex of complement are important mechanisms in atherosclerosis and organ graft rejection. Although the effects of various cytokines on EC apoptosis have been studied, little is known about their effects on complement-mediated EC injury. Therefore, we studied the abilities of various cytokines to induce protection of porcine aortic EC against apoptosis and killing by human complement, a model of pig-to-human xenotransplantation. We found that porcine EC incubated with IL-4 or IL-13, but not with IL-10 or IL-11, became protected from killing by complement and apoptosis induced by TNF-α plus cycloheximide. Maximal protection required 10 ng/ml IL-4 or IL-13, developed progressively from 12 to 72 h of incubation, and lasted 48–72 h after cytokine removal. Protection from complement was not associated with reduced complement activation, C9 binding, or changes in CD59 expression. Inhibition of PI3K prevented development of protection; however, inhibition of p38 MAPK or p42/44 MAPK had no effect. IL-4 and IL-13 induced rapid phosphorylation of Akt. Although protection was inhibited by an Akt inhibitor and a dominant negative Akt mutant transduced into EC, it was induced by transduction of EC with the constitutively active Akt variant, myristylated Akt. We conclude that IL-4 and IL-13 can induce protection of porcine EC against killing by apoptosis and human complement through activation of the PI3K/Akt signaling pathway.

Inducing tolerance to MHC-matched allogeneic islet grafts in diabetic NOD mice by simultaneous islet and bone marrow transplantation under nonirradiative and nonmyeloablative conditioning therapy.

Background. Human type 1 diabetes is associated with defects in the hematopoietic stem cells. Simultaneous donor islet and bone marrow transplantation may be an ideal therapeutic approach for inducing tolerance to islet allogeneic antigens and restoring self-tolerance to islet autoimmune antigens. Methods. Using a nonobese diabetic (NOD) mouse model of human type 1 diabetes, we investigated whether tolerance to MHC-matched allogeneic islet grafts from male nonobese diabetes-resistant (NOR) donors can be induced in female NOD recipients by simultaneous islet and bone marrow transplantation under fludarabine phosphate-based nonmyeloablative and irradiation-free conditioning therapy. Donor-specific chimerism in the peripheral blood of tolerant mice (n=7) was measured by semiquantitative polymerase chain reaction for a male-specific marker (SRY). Results. Donor-specific tolerance to NOR islet grafts was induced in all diabetic NOD mice after simultaneous islet and bone marrow transplantation and treated with fludarabine phosphate, cyclophosphamide, anti-mouse lymphocyte serum, and rapamycin. At 100 days and 200 days after transplantation, the average percentage of male NOR marker in DNA derived from the peripheral blood of NOD recipients that had long-term islet graft survival was over 10%. Conclusion. Our data suggest that this approach may induce donor-specific tolerance in clinical islet transplantation and living-related donor solid organ transplantation.

Differential gene expression of cytochrome P450 11β-hydroxylase in rat adrenal cortex after in vivo activation

In situ hybridization histochemistry was used to monitor the expression of 3β-hydroxysteroid dehydrogenase,Δ 4-isomerase (3βHSD) and cytochrome P450 11β-hydroxylase (P45011β) messenger RNA (mRNA) in adult rat adrenals after stimulation in vivo. In Exp 1, adrenals were collected from rats injected with saline or ACTH for 1, 2, 3, or 4 days. Adrenal sections from saline-treated rats showed uniform expression of 3βHSD mRNA that extended from the adrenal capsule to the medullary border. In contrast, P45011β mRNA showed high levels in the outer fasciculata and low levels in the inner fasciculata/reticularis. In response to ACTH, the integrated density of 3βHSD hybridization did not increase until 4 days. The integrated density of P45011β hybridization increased in ACTH-treated rats between 1–4 days due to increased hybridization in the inner fasciculata/reticularis. In Exp 2, rats were treated with ACTH or saline, and adrenals were harvested at 4, 8, or 24 h. The hybridization density of 3βHSD did not change a...

Changes in the glomerulosa cell phenotype during adrenal regeneration in rats

In situ hybridization was used to examine cellular differentiation during rat adrenal regeneration, defining zona glomerulosa [cytochrome P-450 aldosterone synthase (P-450aldo) mRNA positive], zona fasciculata [cytochrome P-450 11beta-hydroxylase (P-45011beta) mRNA positive], or zona intermedia [negative for both but 3beta-hydroxysteroid dehydrogenase (3beta-HSD) mRNA positive]. After unilateral adrenal enucleation with contralateral adrenalectomy (ULE/ULA), the expression of all mRNA was reduced at 2 days. From 5 to 10 days, P-45011beta and 3beta-HSD mRNA increased while P-450aldo remained low; at 20 days, all mRNA were increased. From 2 to 10 days, cells adjacent to the capsule showed intermedia cell differentiation; by 20 days, the subcapsular glomerulosa cells reappeared. This suggests that after enucleation the glomerulosa dedifferentiates to zona intermedia. The experiment was repeated in rats where the postenucleation ACTH rise was prevented. Rats underwent ULE with sham ULA (ULE/SULA) or ULE/SULA with ACTH treatment. Adrenals from ULE/SULA rats expressed increased P-450aldo mRNA at 10 days and reduced P-45011beta mRNA and adrenal weight at 30 days. ACTH treatment reversed the pattern toward that seen in ULE/ULA. These findings show that the enucleation-induced dedifferentitation of the glomerulosa cell may result in part from elevated plasma ACTH and that prevention of dedifferentiation may result in impaired regeneration.In situ hybridization was used to examine cellular differentiation during rat adrenal regeneration, defining zona glomerulosa [cytochrome P-450 aldosterone synthase ( P-450aldo) mRNA positive], zona fasciculata [cytochrome P-450 11β-hydroxylase ( P-45011β) mRNA positive], or zona intermedia [negative for both but 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA positive]. After unilateral adrenal enucleation with contralateral adrenalectomy (ULE/ULA), the expression of all mRNA was reduced at 2 days. From 5 to 10 days, P-45011β and 3β-HSD mRNA increased while P-450aldo remained low; at 20 days, all mRNA were increased. From 2 to 10 days, cells adjacent to the capsule showed intermedia cell differentiation; by 20 days, the subcapsular glomerulosa cells reappeared. This suggests that after enucleation the glomerulosa dedifferentiates to zona intermedia. The experiment was repeated in rats where the postenucleation ACTH rise was prevented. Rats underwent ULE with sham ULA (ULE/SULA) or ULE/SULA with ACTH treatment. Adrenals from ULE/SULA rats expressed increased P-450aldo mRNA at 10 days and reduced P-45011β mRNA and adrenal weight at 30 days. ACTH treatment reversed the pattern toward that seen in ULE/ULA. These findings show that the enucleation-induced dedifferentitation of the glomerulosa cell may result in part from elevated plasma ACTH and that prevention of dedifferentiation may result in impaired regeneration.

Porcine endothelial cells and iliac arteries transduced with AdenoIL-4 are intrinsically protected, through Akt activation, against immediate injury caused by human complement.

Vascular endothelial cells (ECs) can be injured in a variety of pathologic processes that involve activated complement. We reported previously that porcine ECs incubated with exogenous IL-4 or IL-13 are protected from cytotoxicity by human complement and also from apoptosis by TNF-α. The resistance to complement consists of an intrinsic mechanism that is lost a few days after cytokine removal. In our current study, we investigated whether transfer of the IL-4 gene into porcine ECs in vitro and into porcine vascular tissues in vivo would induce efficient and durable protection from human complement. We found that ECs transduced with adenoIL-4 or adenoIL-13 exhibited continuous production of the cytokine and prolonged protection from complement-mediated killing. IL-4 also protected ECs from activation: ECs incubated with IL-4 did not develop cell retraction and intercellular gaps upon stimulation with sublytic complement. The endothelium and subendothelium of pig iliac arteries that were transduced with the IL-4 gene were effectively protected from complement-dependent immediate injury after perfusion with human blood. However, after similar perfusion, the endothelium was immediately lost from arteries that were transduced with a control adenovirus. The protection was not due to up-regulation of the complement regulators decay accelerating factor, membrane cofactor protein, and CD59, or to reduced complement activation, but required the participation of Akt. Although our studies model protection in pig-to-primate xenotransplantation, our findings of IL-4 induction of Akt-mediated protection may be more broadly applicable to EC injury as manifested in ischemia-reperfusion, allotransplantation, and various vascular diseases.

Expression of cytochrome p450 aldosterone synthase and 11β-hydroxylase mRNA during adrenal regeneration

In situ hybridization histochemistry was used to monitor the expression of cytochrome P450 aldosterone synthase (P450aldo) and cytochrome P450 11 beta-hydroxylase (P45011 beta) mRNA in regenerating rat adrenals. Comparisons were made between regenerating adrenals from unilateral enucleated/unilateral adrenalectomized (ULE/ULA) and bilateral enucleated (BLE) rats. During the first week after enucleation, P45011 beta mRNA was expressed in all adrenals reflecting the presence of fasciculata cells; however, P450aldo mRNA was detected only in adrenals from ULE/ULA rats suggesting that the glomerulosa cell phenotype was absent after BLE. These findings suggest that the expression of glomerulosa cells during the early period of regeneration is influenced by the presence of a second regenerating adrenal.

Comparison of Tolerated and Rejected Islet Grafts: A Gene Expression Study:

Recently we showed that donor-specific tolerance to MHC-matched islet allografts in diabetic NOD mice could be induced by simultaneous islet and bone marrow transplantation. Mononuclear cell infiltration surrounding the islets was also found in tolerated grafts. In this study, we compared gene expression in the tolerated and rejected islet grafts by using Affymetrix Murine U74A oligonucleotide arrays. To confirm the results of microarray analysis, we performed real-time PCR and RNase protection assay on selected genes. Of over 12,000 genes studied, 57 genes were expressed at consistently higher levels in tolerated islet grafts, and 524 genes in rejected islet grafts. Genes from a variety of functional clusters were found to be different between rejected and tolerated grafts. In the rejected islet grafts, a number of T-cell surface markers and of cytotoxicity-related genes were highly expressed. Also in the rejected grafts, a number of cytokines and chemokines and their receptors were highly expressed. The differential expression of selected genes found by microarray analysis was also confirmed by real-time PCR and RNase protection assay. Our results indicated that gene microarray analysis can help us to detect gene expression differences representative of the biologic mechanisms of tolerance and rejection.

IL-4 induces protection of vascular endothelial cells against killing by complement and melittin through lipid biosynthesis

We have shown previously that cytokines IL‐4 and IL‐13 induce protection in porcine vascular endothelial cells (EC) against killing by the membrane attack complex (MAC) of human complement. This protection is intrinsic, not due to changes in complement regulatory proteins, and requires activation of Akt and sterol receptor element binding protein‐1 (SREBP‐1), which regulates fatty acid and phospholipid synthesis. Here we report that, compared to EC incubated in medium, IL‐4‐treated EC had a profound reduction in complement‐mediated ATP loss and in killing assessed by vital dye uptake, but only a slight reduction in permeability disruption measured by calcein release. While controls exposed to complement lost mitochondrial membrane potential and subsequently died, protected EC maintained mitochondrial morphology and membrane potential, and remained alive. SREBP‐1 and fatty acid synthase activation were required for protection and fatty acid and phospholipid synthesis, including cardiolipin, were increased after IL‐4 stimulation, without increase in cholesterol content or cell proliferation. IL‐4 also induced protection of EC from killing by the channel forming protein melittin, similar to protection observed for the MAC. We conclude that IL‐4 induced activation of Akt/SREBP‐1/lipid biosynthesis in EC, resulting in protection against MAC and melittin, in association with mitochondrial protection.