Effects of Temperature Adaptation on the Metabolism and Physiological Properties of Sturgeon Fish Larvae Cell Line.

This study investigated how Atlantic sturgeon cells respond to elevated temperatures, shedding light on the potential impacts of climate change on fish. Atlantic sturgeon (Acipenser oxyrinchus), an IUCN (International Union for Conservation of Nature) Red List species and evolutionarily related to paleonisiform species, may have considerable physiological adaptability, suggesting that this species may be able to cope with changing climatic conditions and higher temperatures. To test this hypothesis, the AOXlar7y cell line was examined at 20 °C (control) and at elevated temperatures of 25 °C and 28 °C. Parameters including proliferation, vitality, morphology, and gene expressions related to proliferation, stemness, and stress were evaluated. Additionally, to achieve a comprehensive understanding of cellular changes, mitochondrial and metabolic activities were assessed using Seahorse XF96. AOXlar7y cells adapted to 28 °C exhibited enhanced mitochondrial adaptability, plasticity, heightened cell proliferation, and increased hsp70 expression. Increased baseline respiration indicated elevated ATP demand, which is potentially linked to higher cell proliferation and heat stress defense. Cells at 28 °C also displayed elevated reserve respiration capacity, suggesting adaptation to energy demands. At 25 °C, AOXlar7y cells showed no changes in basal respiration or mitochondrial capacity, suggesting unchanged ATP demand compared to cells cultivated at 20 °C. Proliferation and glycolytic response to energy requirements were diminished, implying a connection between glycolysis inhibition and proliferation suppression. These research results indicate sturgeon cells are capable of withstanding and adapting to an 8 °C temperature increase. This cellular analysis lays a foundation for future studies aimed at a deeper understanding of fish cell physiological adaptations, which will contribute to a better knowledge of environmental threats facing Atlantic sturgeon and fish populations amid climate change.

Overexpression of Renin-B Induces Warburg-like Effects That Are Associated with Increased AKT/mTOR Signaling.

The classical secretory renin-a is known to be involved in angiotensin generation, thereby regulating not only blood pressure, but also promoting oxidative stress as well as apoptotic and necrotic cell death. In contrast, another cytosolic renin isoform named renin-b has been described, exerting protective effects under ischemia-related conditions in H9c2 cardiomyoblasts. Using microarray-based transcriptome analyses, we aimed to identify the signaling pathways involved in mediating cardioprotection in H9c2 cells overexpressing renin-b. By transcriptome profiling, we identified increased gene expression of several genes encoding glycolytic enzymes and glucose transporters, while the transcript levels of TCA-cycle enzymes were decreased. Complementing data from metabolic analyses revealed enhanced glucose consumption and lactate accumulation due to renin-b overexpression. Renin-b overexpression further stimulated AKT/mTOR signaling, where numerous genes involved in this pathway showed altered transcript levels. For AKT, we also detected enhanced phosphorylation levels by means of Western blotting, suggesting an activation of this kinase. Moreover, analysis of the ROS levels identified an increase in ROS accumulation in renin-b-overexpressing cells. Altogether, our data demonstrate that renin-b overexpression induces the metabolic remodeling of H9c2 cells similar to that seen under oxygen deprivation. This metabolic phenotype exerting so-called aerobic glycolysis is also known as the Warburg effect.

Overexpression of Transcripts Coding for Renin-b but Not for Renin-a Reduce Oxidative Stress and Increase Cardiomyoblast Survival under Starvation Conditions.

A stimulated renin-angiotensin system is known to promote oxidative stress, apoptosis, necrosis and fibrosis. Renin transcripts (renin-b; renin-c) encoding a cytosolic renin isoform have been discovered that may in contrast to the commonly known secretory renin (renin-a) exert protective effects Here, we analyzed the effect of renin-a and renin-b overexpression in H9c2 cardiomyoblasts on apoptosis and necrosis as well as on potential mechanisms involved in cell death processes. To mimic ischemic conditions, cells were exposed to glucose starvation, anoxia or combined oxygen-glucose deprivation (OGD) for 24 h. Under OGD, control cells exhibited markedly increased necrotic and apoptotic cell death accompanied by enhanced ROS accumulation, loss of mitochondrial membrane potential and decreased ATP levels. The effects of OGD on necrosis were exaggerated in renin-a cells, but markedly diminished in renin-b cells. However, with respect to apoptosis, the effects of OGD were almost completely abolished in renin-b cells but interestingly also moderately diminished in renin-a cells. Under glucose depletion we found opposing responses between renin-a and renin-b cells; while the rate of necrosis and apoptosis was aggravated in renin-a cells, it was attenuated in renin-b cells. Based on our results, strategies targeting the regulation of cytosolic renin-b as well as the identification of pathways involved in the protective effects of renin-b may be helpful to improve the treatment of ischemia-relevant diseases.

Angiotensin dependent and angiotensin independent protective effects of renin-b in H9c2 cells after anoxia.

The renin-angiotensin system is known to regulate blood pressure as well as water- and electrolyte balance. An activated RAS is involved in the development of hypertension and hypertension-related organ damage. Thus, inhibitors of the RAS are protective and markedly increasing the life span of patients. In contrast, renin transcripts have been discovered encoding a cytoplasmatic renin isoform, termed renin-b, which is not harmful but may be even protective. Here we demonstrate that depletion of renin-b encoding transcripts by small interference RNA decreased ATP levels and increased basal necrosis as well as apoptosis rates. Furthermore, renin-b depletion potentiated the anoxia-induced increase of necrosis rates. Vice versa, overexpression of renin-b prevented the anoxia-induced increase of caspase-mediated apoptosis rates. Besides, cells overexpressing renin-b exhibited even reduced mitochondrial mediated apoptosis rates under anoxia, when compared with normoxic conditions, as indicated by Annexin V labeling. However, whereas the protective effect of renin-b on caspase-mediated apoptosis was completely blocked by the renin inhibitor CH732, the effect on mitochondrial-mediated apoptosis was not affected by CH732 at all. From these data we conclude that renin-b overexpression mediates cardioprotective effects under anoxia with respect to mitochondrial induced apoptosis angiotensin-independently, but with respect to caspase induced apoptosis likely in an angiotensin-dependent manner.

Non-secretory renin reduces oxidative stress and increases cardiomyoblast survival during glucose and oxygen deprivation.

Although the renin-angiotensin system usually promotes oxidative stress and cell death, renin transcripts have been discovered, whose transcription product may be cardioprotective. These transcripts encode a non-secretory renin isoform that is localized in the cytosol and within mitochondria. Here we tested the hypotheses that cytosolic renin [ren(2-9)] expression promotes cell survival under hypoxia and glucose depletion by preserving the mitochondrial membrane potential (∆Ψm) and mitigating the accumulation of ROS. To simulate ischemic insults, we exposed H9c2 cells to glucose deprivation, anoxia or to combined oxygen-glucose deprivation (OGD) for 24 hours and determined renin expression. Furthermore, H9c2 cells transfected with the empty pIRES vector (pIRES cells) or ren(2-9) cDNA-containing vector [ren(2-9) cells] were analyzed for cell death, ∆Ψm, ATP levels, accumulation of ROS, and cytosolic Ca2+ content. In pIRES cells, expression of ren(1A-9) was stimulated under all three ischemia-related conditions. After OGD, the cells lost their ∆Ψm and exhibited enhanced ROS accumulation, increased cytosolic Ca2+ levels, decreased ATP levels as well as increased cell death. In contrast, ren(2-9) cells were markedly protected from these effects. Ren(2-9) appears to represent a protective response to OGD by reducing ROS generation and preserving mitochondrial functions. Therefore, it is a promising new target for the prevention of ischemia-induced myocardial damage.

An alternative renin isoform is cardioprotective by modulating mitochondrial metabolism.

The renin-angiotensin system promotes oxidative stress, apoptosis, necrosis, fibrosis, and thus heart failure. Secretory renin plays a central role in these processes, initiating the generation of angiotensins. Nevertheless, alternative renin transcripts exist, which code for a cytosolically localized renin isoform (cyto-renin) that is cardioprotective. We tested the hypothesis that the protective effects are associated with a beneficial switch of metabolic and mitochondrial functions. To assess H9c2 cell mitochondrial parameters, we used the Seahorse XF analyser. Cardiac H9c2 cells overexpressing cyto-renin exhibited enhanced nonmitochondrial oxygen consumption, lactate accumulation, and LDH activity, reflecting a switch to more aerobic glycolysis known as Warburg effect. Additionally, mitochondrial spare capacity and cell respiratory control ratio were enhanced, indicating an increased potential to tolerate stress conditions. Renin knockdown induced opposite effects on mitochondrial functions without influencing metabolic parameters. Thus, the protective effects of cyto-renin are associated with an altered bioenergetic profile and an enhanced stress tolerance, which are favourable under ischaemic conditions. Therefore, cyto-renin is a promising new target for the prevention of ischaemia-induced myocardial damage.

An Alternative Promoter in Intron1 of the Renin Gene is Regulated by Glucose Starvation via Serum Response Factor.

BACKGROUND/AIMS: Renin is known as a secretory glycoprotein that ultimately leads to angiotensin II generation. In this way renin exerts pro-inflammatory effects and promotes cardiac injury. Additional transcripts have been identified encoding for a cytosolic renin isoform that - in contrast to secretory renin - exhibits cardioprotective effects under ischemic conditions. The promoter of these transcripts is unknown. METHODS: Using qRT-PCR and dual-luciferase reporter assay we examined the expression and promotor activity of cytosolic renin as well as the regulation by glucose starvation in H9c2 cardiomyoblasts. RESULTS: We identified a promoter in intron1 of the rat renin gene with two glucose starvation-sensitive regions. One region contains a binding motif for serum response factor (SRF). Under glucose depletion expression of SRF increased prior to cytosolic renin. SRF knock down selectively decreased cytosolic renin expression and attenuated the increase of cytosolic renin expression under glucose depletion. CONCLUSIONS: Transcripts encoding for secretory and cytosolic renin are differentially expressed. The low basal expression of cytosolic renin as well as its induction under ischemia-related conditions represents an efficient system regulated in accordance with its previously identified unfavorable effects under control situations but protective effects seen after myocardial infarction or glucose depletion.

(Pro)renin receptor (ATP6AP2) depletion arrests As4.1 cells in the G0/G1 phase thereby increasing formation of primary cilia.

The (pro)renin receptor [(P)RR, ATP6AP2] is a multifunctional transmembrane protein that activates local renin-angiotensin systems, but also interacts with Wnt pathways and vacuolar H+ -ATPase (V-ATPase) during organogenesis. The aim of this study was to characterize the role of ATP6AP2 in the cell cycle in more detail. ATP6AP2 down-regulation by siRNA in renal As4.1 cells resulted in a reduction in the rate of proliferation and a G0/G1 phase cell cycle arrest. We identified a number of novel target genes downstream of ATP6AP2 knock-down that were related to the primary cilium (Bbs-1, Bbs-3, Bbs-7, Rabl5, Ttc26, Mks-11, Mks-5, Mks-2, Tctn2, Nme7) and the cell cycle (Pierce1, Clock, Ppif). Accordingly, the number of cells expressing the primary cilium was markedly increased. We found no indication that these effects were dependent of V-ATPase activity, as ATP6AP2 knock-down did not affect lysosomal pH and bafilomycin A neither influenced the ciliary expression pattern nor the percentage of ciliated cells. Furthermore, ATP6AP2 appears to be essential for mitosis. ATP6AP2 translocated from the endoplasmatic reticulum to mitotic spindle poles (pro-, meta- and anaphase) and the central spindle bundle (telophase) and ATP6AP2 knock-down results in markedly deformed spindles. We conclude that ATP6AP2 is necessary for cell division, cell cycle progression and mitosis. ATP6AP2 also inhibits ciliogenesis, thus promoting proliferation and preventing differentiation.

Mitochondrial functions of THP-1 monocytes following the exposure to selected natural compounds.

The immune system is an important target of various xenobiotics, which may lead to severe adverse effects including immunosuppression or inappropriate immunostimulation. Mitochondrial toxicity is one possibility by which xenobiotics exert their toxic effects in cells or organs. In this study, we investigated the impact of three natural compounds, cyclosporine A (CsA), deoxynivalenol (DON) and cannabidiol (CBD) on mitochondrial functions in the THP-1 monocytic cell line. The cells were exposed for 24h to two different concentrations (IC10 and IC50 determined by MTT) of each compound. The cells showed concentration-dependent elevated intracellular reactive oxygen species (iROS) and induction of apoptosis (except DON) in response to the three test compounds. Mitochondrial functions were characterized by using bioenergetics profiling experiments. In THP-1 monocytes, the IC50 of CsA decreased basal and maximal respiration as well as ATP production with an impact on spare capacity indicating a mitochondrial dysfunction. Similar reaction patterns were observed following CBD exposure. The basal respiration level and ATP-production decreased in the THP-1 cells exposed to the IC50 of DON with no major impact on mitochondrial function. In conclusion, impaired mitochondrial function was accompanied by elevated iROS and apoptosis level in a monocytic cell line exposed to CsA and CBD. Mitochondrial dysfunction may be one explanation for the cytotoxicity of CBD and CsA also in other in immune cells.

Anti-necrotic and cardioprotective effects of a cytosolic renin isoform under ischemia-related conditions.

UNLABELLED: In the heart, secretory renin promotes hypertrophy, apoptosis, necrosis, fibrosis, and cardiac failure through angiotensin generation from angiotensinogen. Thus, inhibitors of the renin-angiotensin system are among the most potent drugs in the treatment of cardiac failure. Renin transcripts have been identified encoding a renin isoform with unknown targets and unknown functions that are localized to the cytosol and mitochondria. We hypothesize that this isoform, in contrast to secretory renin, exerts cardioprotective effects in an angiotensin-independent manner. Cells overexpressing cytosolic renin were generated by transfection or obtained from CX(exon2-9)renin transgenic rats. Overexpression of cytosolic renin reduced the rate of necrosis in H9c2 cardiomyoblasts and in primary cardiomyocytes after glucose depletion. These effects were not mediated by angiotensin generation since an inhibitor of renin activity did not influence the in vitro effects. siRNA-mediated knockdown of endogenous cytosolic renin increased the rate of necrosis and aggravated the pro-necrotic effects of glucose depletion. Isolated perfused hearts obtained from transgenic rats overexpressing cytosolic renin exhibited a 50% reduction of infarct size after ischemia-reperfusion injury. Cytosolic renin is essential for survival, both under basal conditions and during glucose starvation. The protective effects are angiotensin-independent and contrary to the known actions of secretory renin. KEY MESSAGES: A cytosolic isoform of renin with unknown functions is expressed in the heart. Cytosolic renin diminishes ischemia induced damage to the heart. The protective effects of cytosolic renin contradict the known function of secretory renin. The effects of cytosolic renin are not mediated via angiotensin generation. Renin-binding protein is a potential target for cytosolic renin.

Investigations on synthesis and structure elucidation of novel [1,2,4]triazolo[1,2-a]pyridazine-1-thiones and their inhibitory activity against inducible nitric oxide synthase.

The inducible nitric oxide synthase (iNOS) is a target of great research interest due to its importance in a number of diseases, for example, septic shock and inflammatory lung diseases. A variety of 3-substituted [1,2,4]triazolo[1,2-a]pyridazine derivatives was synthesized by ring closure with hexahydropyridazine-1-carbothioamide by using aliphatic and aromatic aldehydes. The activity of the new substances was tested on the insulin-secreting rat insulinoma cell line RINm5F. iNOS was expressed through exposure to interleukin-1ß (IL-1ß) and interferon-γ (IFN-γ). A number of the investigated compounds were more active than the reference inhibitor aminoguanidine (AG). Structure-activity relationships showed that a phenyl substituent in position 3 is apparently essential for inhibition.

A new transgenic rat model overexpressing the angiotensin II type 2 receptor provides evidence for inhibition of cell proliferation in the outer adrenal cortex.

This study aimed to elucidate the role of the AT(2) receptor (AT(2)R), which is expressed and upregulated in the adrenal zona glomerulosa (ZG) under conditions of increased aldosterone production. We developed a novel transgenic rat (TGR; TGRCXmAT(2)R) that overexpresses the AT(2)R in the adrenal gland, heart, kidney, brain, skeletal muscle, testes, lung, spleen, aorta, and vein. As a consequence the total angiotensin II (Ang II) binding sites increased 7.8-fold in the kidney, 25-fold in the heart, and twofold in the adrenals. The AT(2)R number amounted to 82-98% of total Ang II binding sites. In the ZG of TGRCXmAT(2)R, the AT(2)R density was elevated threefold relative to wild-type (WT) littermates, whereas AT(1)R density remained unchanged. TGRCXmAT(2)R rats were viable and exhibited normal reproduction, blood pressure, and kidney function. Notably, a slightly but significantly reduced body weight and a moderate increase in plasma urea were observed. With respect to adrenal function, 24-h urinary and plasma aldosterone concentrations were unaffected in TGRCXmAT(2)R at baseline. Three and 14 days of Ang II infusion (300 ng·min(-1)·kg(-1)) increased plasma aldosterone levels in WT and in TGR. These changes were completely abolished by the AT(1)R blocker losartan. Of note, glomerulosa cell proliferation, as indicated by the number of Ki-67-positive glomerulosa cells, was stimulated by Ang II in TGR and WT rats; however, this increase was significantly attenuated in TGR overexpressing the AT(2)R. In conclusion, AT(2)R in the adrenal ZG inhibits Ang II-induced cell proliferation but has no obvious lasting effect on the regulation of the aldosterone production at the investigated stages.

Amiloride lowers arterial pressure in cyp1a1ren-2 transgenic rats without affecting renal vascular function.

OBJECTIVE: The epithelial sodium channel (ENaC) is expressed in the renal vasculature, where it may be involved in the control of vascular tone and arterial pressure. Using a rat model with an inducible mouse renin transgene (cyp1a1ren-2 transgenic rats), we tested the hypothesis that stimulation of the renin-angiotensin-aldosterone system (RAAS) for 3 weeks is associated with an impairment of renal vascular function that is sensitive to treatment with the ENaC blocker amiloride. METHODS: Rats were randomized to control, transgene induction, or transgene induction plus amiloride treatment (n = 7-10 per group). Additional control animals were treated with amiloride. Arterial pressure was measured telemetrically. Sodium balance was determined in metabolic cages. Renal vascular function was investigated in vitro (wire myography) and in vivo (ultrasound flow probe). ENaC mRNA expression was determined by real-time PCR. RESULTS: Transgene induction caused an increase in plasma aldosterone levels associated with a sustained elevation in arterial pressure. Amiloride elicited a transient decrease in renal sodium balance and effectively lowered arterial pressure. Neither transgene induction nor amiloride treatment significantly affected phenylephrine or acetylcholine-induced renal vascular responses. Similarly, small renal artery compliance and renal vascular resistance remained unaltered. Amiloride treatment caused an increase in α-ENaC mRNA abundance in renal cortical tissue but not in intrarenal arteries. CONCLUSION: Amiloride reduces arterial pressure in cyp1a1ren-2 transgenic rats in association with increased renal sodium excretion without affecting renal vascular function. In this model of hypertension sustained activation of the RAAS does not lead to increased ENaC expression in small intrarenal arteries.

Cytosolic renin is targeted to mitochondria and induces apoptosis in H9c2 rat cardiomyoblasts.

One important goal in cardiology is to prevent necrotic cell death in the heart. Necrotic cell death attracts neutrophils and monocytes into the injured myocardium. The consequences are fibrosis, remodelling and cardiac failure. The renin-angiotensin system promotes the development of cardiac failure. Recently, alternative renin transcripts have been identified lacking the signal sequence for a cotranslational transport to the endoplasmatic reticulum. These transcripts encode for a cytosolic renin with unknown functions. The expression of this alternative transcript increases after myocardial infarction. We hypothesized that cytosolic renin plays a role in survival and death of cardiomyocytes. To test this hypothesis, we overexpressed secretory or cytosolic renin in H9c2 cardiomyblasts and determined the rate of proliferation, necrosis and apoptosis. Proliferation rate, as indicated by BrdU incorporation into DNA, was reduced by secretory and cytosolic renin (cells transfected with control vector: 0.33 +/- 0.06; secretory renin: 0.12 +/- 0.02; P < 0.05; cytosolic renin: 0.15 +/- 0.03; P < 0.05). Necrosis was increased by secretory renin but decreased by cytosolic renin (LDH release after 10 days from cells transfected with control vector: 68.5 +/- 14.9; secretory renin: 100.0 +/- 0; cytosolic renin: 25.5 +/- 5.3% of content, each P < 0.05). Mitochondrial apoptosis, as indicated by phosphatidylserin translocation to the outer membrane, was unaffected by secretory renin but increased by cytosolic renin (controls: 23.8 +/- 3.9%; secretory renin: 22.1 +/- 4.7%; cytoplasmatic renin: 41.2 +/- 3.8%; P < 0.05). The data demonstrate that a cytosolic renin exists in cardiomyocytes, which in contradiction to secretory renin protects from necrosis but increases apoptosis. Non-secretory cytosolic renin can be considered as a new target for cardiac failure.

A renin transcript lacking exon 1 encodes for a non-secretory intracellular renin that increases aldosterone production in transgenic rats.

Renin transcripts lacking exon 1 and thus the signal sequence for co-translational transport to the endoplasmatic reticulum encode for a protein (exon[2-9]renin), that is confined to the cytoplasm. The function of exon(2-9)renin is currently unknown. Mitochondrial renin increases under conditions which stimulate aldosterone production. We hypothesized that exon(2-9)renin (1) is translated into a functionally active protein in vivo, (2) is not secreted but remains within the cytoplasm and (3) stimulates aldosterone production. To test these hypotheses we generated transgenic rats overexpressing exon(2-9)renin. Four transgenic lines were obtained expressing the transcript in various tissues including the heart and the adrenal gland. Renin was enriched particularly in the cytoplasm of transgenic rats. Renin was not elevated in plasma, indicating that exon(2-9)renin is produced but not secreted. The ratio of aldosterone to renin concentrations in plasma (PAC/PRC) was elevated in all transgenic lines except line 307, which also did not exhibit elevated cytoplasmatic renin levels in the adrenal gland (PAC/PRC in controls: 2.8+/-2.3; line 307: 1.9+/-0.8; n. s.; line 284: 5.8+/-1.9; P<0.02; line 294: 5.0+/-2.3; P<0.001; line 276: 10.3+/-5.1; P<0.001). We conclude that the exon(1A-9) renin transcript (1) is translated into a functionally active intracellular protein; (2) is targeted to the cytoplasm rather than being sorted to the secretory pathways and (3) is functionally active, regulating aldosterone production. The CX-(exon2-9)renin transgenic rat appears to be a useful model to study the role and the mechanisms of action of cytoplasmatic renin derived from exon(1A-9) transcripts.

Dose-dependent titration of prorenin and blood pressure in Cyp1a1ren-2 transgenic rats: absence of prorenin-induced glomerulosclerosis.

OBJECTIVE: Prorenin has been associated with cardiovascular disease and the development of glomerulosclerosis via a renin/prorenin receptor. In cyp1a1ren-2 transgenic rats, prorenin levels and arterial pressure can be increased by oral administration of indole-3-carbinol (I3C). The transgenic strain has been used as a model of malignant hypertension. METHODS: The present study was designed to test the hypotheses that (i) low doses of I3C would result in dose-dependent sustained increases in arterial pressure without signs of malignancy, making cyp1a1ren-2 transgenic rats a useful model to study nonmalignant hypertension, and (ii) cyp1a1ren-2 transgenic rats would develop glomerulosclerosis when they were chronically exposed to 0.125% I3C in their diet. RESULTS: I3C treatment for 2 weeks resulted in increases of plasma prorenin concentrations and arterial pressure in a dose-dependent manner. Rats thrived well over a period of 12 weeks on dietary I3C concentrations (wt/wt) of 0.125%. Plasma prorenin concentration rose from 0.1 +/- 0.1 microg to 17.9 +/- 5.0 mug angiotensin I/ml per h (P < 0.01) and mean arterial pressure increased to a plateau of 170 +/- 5 mmHg (P < 0.001) between weeks 6 and 12. After 12 weeks of 0.125% I3C, rats exhibited moderate hypertensive renal vasculopathy, but no histological signs of glomerulosclerosis. CONCLUSIONS: The cyp1a1ren-2 transgenic rat model allows for chronic dose-dependent titration of arterial pressure by a simple and non-invasive intervention, making this strain a useful model to study malignant and nonmalignant arterial hypertension. High circulating prorenin levels per se do not cause glomerulosclerosis.

Ex vivo gene transfer of viral interleukin-10 to BB rat islets: no protection after transplantation to diabetic BB rats.

Allogeneic and autoimmune islet destruction limits the success of islet transplantation in autoimmune diabetic patients. This study was designed to investigate whether ex vivo gene transfer of viral interleukin-10 (vIL-10) protects BioBreeding (BB) rat islets from autoimmune destruction after transplantation into diabetic BB recipients. Islets were transduced with adenoviral constructs (Ad) expressing the enhanced green fluorescent protein (eGFP), alpha-1 antitrypsin (AAT) or vIL-10. Transduction efficiency was demonstrated by eGFP-positive cells and vIL-10 production. Islet function was determined in vitro by measuring insulin content and insulin secretion and in vivo by grafting AdvIL-10-transduced islets into syngeneic streptozotocin (SZ)-diabetic, congenic Lewis (LEW.1 W) rats. Finally, gene-modified BB rat islets were grafted into autoimmune diabetic BB rats. Ad-transduction efficiency of islets increased with virus titre and did not interfere with insulin content and insulin secretion. Ad-transduction did not induce Fas on islet cells. AdvIL-10-transduced LEW.1 W rat islets survived permanently in SZ-diabetic LEW.1 W rats. In diabetic BB rats AdvIL-10-transduced BB rat islets were rapidly destroyed. Prolongation of islet culture prior to transplantation improved the survival of gene-modified islets in BB rats. Several genes including those coding for chemokines and other peptides associated with inflammation were down-regulated in islets after prolonged culture, possibly contributing to improved islet graft function in vivo. Islets transduced ex vivo with vIL-10 are principally able to cure SZ-diabetic rats. Autoimmune islet destruction in diabetic BB rats is not prevented by ex vivo vIL-10 gene transfer to grafted islets. Graft survival in autoimmune diabetic rats may be enhanced by improvements in culture conditions prior to transplantation.

Synthesis, structural investigations and biological evaluation of novel hexahydropyridazine-1-carboximidamides, -carbothioamides and -carbothioimidic acid esters as inducible nitric oxide synthase inhibitors.

Local excess of nitric oxide (NO) has been implicated in beta-cell damage, thus, a possible approach to the treatment of autoimmune IDDM is the selective inhibition of inducible nitric oxide synthase (iNOS). A series of variously substituted hexahydropyridazine-1-carbothioamides, -carbothioimidic acid esters and -carboximidamides was synthesized and dose-dependently evaluated as potential inhibitors of iNOS. The screening of the title compounds was performed with insulin-producing RIN-5AH cells and a combination of IL1-1 beta and IFN-gamma as inducers of cellular NO production. The structure-activity analysis revealed that the variation of substituents in the position 1 of the hexahydropyridazine strongly influences the inhibitory activity to iNOS as well as being critical for RIN cell survival. Among the compounds tested, the hexahydropyridazine-1-carbothioamides showed particularly significant inhibitory effects. However, for an efficient iNOS inhibition substitution at the nitrogen of the 1-carbothioamide group is important. Thus, the introduction of aliphatic chains such as propyl or butyl and of cyclic moieties such as cyclohexyl, 3-methoxyphenyl, and 4-methoxyphenyl (IC(50): 0.5-2.1 mM), respectively, provided compounds with similar inhibitory activity to aminoguanidine (IC(50): 0.3 mM), a common standard substance used for the selective inhibition of iNOS. However, the 1-carboximidamides, which represent more structurally related semicyclic derivatives of aminoguanidine, caused only incomplete iNOS inhibition. The hexahydropyridazine-1-carbothioimidic acid esters caused dose- and substituent-dependent damage of RIN-5AH cells. The toxicity of the synthesized compounds increased markedly if aliphatic substituents at the exocyclic N atom(s) were replaced by variously substituted aromatic rings.

Aminoguanidine downregulates expression of cytokine-induced Fas and inducible nitric oxide synthase but not cytokine-enhanced surface antigens of rat islet cells.

Autoimmune beta-cell destruction occurs directly by cell-mediated cytotoxicity or indirectly by cytokines released from infiltrating lymphocytes. Cytokines (IL-1beta/IFN-gamma) modify or induce expression of MHC antigens and ICAM-1 on beta-cells which can lead to an improved binding of T-lymphocytes to beta-cells and finally to an enhanced cell-mediated cytotoxicity. Cytokines also induce Fas-expression and inducible nitric oxide synthase (iNOS) causing generation of nitric oxide (NO) which is toxic for beta-cells. The iNOS inhibitor aminoguanidine (AG) delays diabetes onset, but does not reduce diabetes incidence. We wanted to know whether AG inhibits cytokine-induced expression of Fas, MHC antigens and ICAM-1 on beta-cells of LEW.1W and BB/OK rat islets after culture with IL-1beta/IFN-gamma. NO was completely inhibited by 5.0 mmol/L AG while 0.5 mmol/L had no inhibitory effect. AG downregulated Fas-expression on the surface of beta-cells. Cytokine-induced/enhanced expression of MHC class-II and ICAM-1 was not affected by any AG concentration. AG syngergistically increased cytokine-induced enhancement of MHC class-I antigen density. AG possibly blocks the indirect pathway of beta-cell damage in vivo due to inhibition of Fas and iNOS and improves direct cell-mediated cytotoxicity due to drastic increased MHC class-I expression. Inhibition of only one pathway of beta-cell destruction is not sufficient to prevent diabetes.