Biliverdin Protects against Liver Ischemia Reperfusion Injury in Swine

Ischemia reperfusion injury (IRI) in organ transplantation remains a serious and unsolved problem. Organs that undergo significant damage during IRI, function less well immediately after reperfusion and tend to have more problems at later times when rejection can occur. Biliverdin has emerged as an agent that potently suppress IRI in rodent models. Since the use of biliverdin is being developed as a potential therapeutic modality for humans, we tested the efficacy for its effects on IRI of the liver in swine, an accepted and relevant pre-clinical animal model. Administration of biliverdin resulted in rapid appearance of bilirubin in the serum and significantly suppressed IRI-induced liver dysfunction as measured by multiple parameters including urea and ammonia clearance, neutrophil infiltration and tissue histopathology including hepatocyte cell death. Taken together, our findings, in a large animal model, provide strong support for the continued evaluation of biliverdin as a potential therapeutic in the clinical setting of transplantation of the liver and perhaps other organs

Worsening of Cardiomyopathy Using Deflazacort in an Animal Model Rescued by Gene Therapy

We have previously demonstrated that gene therapy can rescue the phenotype and extend lifespan in the delta-sarcoglycan deficient cardiomyopathic hamster. In patients with similar genetic defects, steroids have been largely used to slow down disease progression. Aim of our study was to evaluate the combined effects of steroid treatment and gene therapy on cardiac function. We injected the human delta-sarcoglycan cDNA by adeno-associated virus (AAV) 2/8 by a single intraperitoneal injection into BIO14.6 Syrian hamsters at ten days of age to rescue the phenotype. We then treated the hamsters with deflazacort. Treatment was administered to half of the hamsters that had received the AAV and the other hamsters without AAV, as well as to normal hamsters. Both horizontal and vertical activities were greatly enhanced by deflazacort in all groups. As in previous experiments, the AAV treatment alone was able to preserve the ejection fraction (70±7% EF). However, the EF value declined (52±14%) with a combination of AAV and deflazacort. This was similar with all the other groups of affected animals. We confirm that gene therapy improves cardiac function in the BIO14.6 hamsters. Our results suggest that deflazacort is ineffective and may also have a negative impact on the cardiomyopathy rescue, possibly by boosting motor activity. This is unexpected and may have significance in terms of the lifestyle recommendations for patients

Acute arterial mesenteric ischemia and reperfusion: macroscopic and MRI findings, preliminary report

AIM: To explore the physiopathology and magnetic resonance imaging (MRI) findings in an animal model of acute arterial mesenteric ischemia (AAMI) with and without reperfusion.METHODS: In this study, 8 adult Sprague-Dawley rats underwent superior mesenteric artery (SMA) ligation and were then randomly divided in two groups of 4. In group., the ischemia was maintained for 8 h. In group., 1-h after SMA occlusion, the ligation was removed by cutting the thread fixed on the back of the animal, and reperfusion was monitored for 8 h. MRI was performed using a 7-T system.RESULTS: We found that, in the case of AAMI without reperfusion, spastic reflex ileus, hypotonic reflex ileus, free abdominal fluid and bowel wall thinning are present from the second hour, and bowel wall hyperintensity in T2-W sequences are present from the fourth hour. The reperfusion model shows the presence of early bowel wall hyperintensity in T2-W sequences after 1 h and bowel wall thickening from the second hour.CONCLUSION: Our study has shown that MRI can assess pathological changes that occur in the small bowel and distinguish between the presence and absence of reperfusion after induced acute arterial ischemia. (C) 2013 Baishideng. All rights reserved

Hydrogen sulfide regulates the redox state of soluble guanylate cyclase in CSE-/- mice corpus cavernosum microcirculation

: The corpus cavernosum (CC) is a highly vascularized tissue and represents an excellent example of microcirculation. Indeed, erectile dysfunction is considered an early index of cardiovascular disease. Hydrogen sulfide (H2S) at the vascular level is endogenously produced from L-cysteine mainly by the action of cystathionine-γ-lyase (CSE) and plays a role in CC vascular homeostasis. Here we have evaluated the involvement of the endogenous H2S in the regulation of the soluble guanylate cyclase (sCG) redox state. The lack of CSE-derived endogenous H2S, in CSE-/- mice, disrupted the eNOS/NO/sGC/PDE pathway. Indeed, the absence of CSE-derived endogenous H2S caused a significant reduction of the relaxant response to riociguat, an sGC redox-dependent stimulator. Conversely, the response to cinaciguat, an sGC redox-independent activator, was not modified. The relevance of the role played at the redox level of the endogenous H2S was confirmed by the findings that in CC harvested from CSE-/- mice there was a significant reduction of GCβ1 expression coupled with a decrease in CYP5R3, a reductase involved in the regulation of the redox state of sGC. These molecular changes driven by the lack of endogenous H2S translate into a significant reduction in cGMP levels. The replenishment of the lack of H2S with an H2S donor rescued the relaxant response to riociguat in CC of CSE-/- mice. In conclusion, the endogenous CSE-derived H2S plays a physiological key role in the regulation of the redox state of sGC in CC microcirculation

Induction of VX2 para-renal carcinoma in rabbits: generation of animal model for loco-regional treatments of solid tumors

BACKGROUND: Animal models of para-renal cancer can provide useful information for the evaluation of tumor response to loco-regional therapy experiments in solid tumors. The aim of our study was to establish a rabbit para-renal cancer model using locally implanted VX2 tumors. METHODS: In order to generate a rabbit model of para-renal cancer, we established four hind limb donor rabbits by using frozen VX2 tumor samples. Following inoculation, rabbits were monitored for appetite and signs of pain. Viable tumors appeared as palpable nodules within 2 weeks of inoculation. Tumor growth was confirmed in all rabbits by high-resolution ultrasound analysis and histology. Once tumor growth was established, hind limb tumors extraction was used for tumor line propagation and para-renal tumor creation. Twenty-one rabbit models bearing para-renal cancer were established by implanting VX2 tumor into the para-renal capsula. Tumors developed into discreet 2-3 cm nodules within 1-3 weeks of implantation. Serial renal ultrasonography follow-up, starting 1 week after tumor implantation, was performed. Two weeks after tumor implantation, rabbits were euthanized and tumors and other organs were collected for histopathology. RESULTS: Tumor growth after VX2 tumor fragment implantation was confirmed in all rabbits by high-resolution ultrasound (US) imaging examinations of the para-renal regions and was measured with digital caliper. The para-renal injection of VX2 tumor fragments, achieved tumor growth in 100% of cases. All data were confirmed by histological analysis. CONCLUSIONS: We generated for the first time, a model of para-renal cancer by surgical tumor implantation of VX2 frozen tumor fragments into rabbit's para-renal region. This method minimizes the development of metastases and the use of non-necrotic tumors and will optimize the evaluation of tumor response to loco-regional therapy experiments.Background: Animal models of para-renal cancer can provide useful information for the evaluation of tumor response to loco-regional therapy experiments in solid tumors. The aim of our study was to establish a rabbit para-renal cancer model using locally implanted VX2 tumors. Methods: In order to generate a rabbit model of para-renal cancer, we established four hind limb donor rabbits by using frozen VX2 tumor samples. Following inoculation, rabbits were monitored for appetite and signs of pain. Viable tumors appeared as palpable nodules within 2 weeks of inoculation. Tumor growth was confirmed in all rabbits by high-resolution ultrasound analysis and histology. Once tumor growth was established, hind limb tumors extraction was used for tumor line propagation and para-renal tumor creation. Twenty-one rabbit models bearing para-renal cancer were established by implanting VX2 tumor into the para-renal capsula. Tumors developed into discreet 2-3 cm nodules within 1-3 weeks of implantation. Serial renal ultrasonography follow-up, starting 1 week after tumor implantation, was performed. Two weeks after tumor implantation, rabbits were euthanized and tumors and other organs were collected for histopathology. Results: Tumor growth after VX2 tumor fragment implantation was confirmed in all rabbits by high-resolution ultrasound (US) imaging examinations of the para-renal regions and was measured with digital caliper. The para-renal injection of VX2 tumor fragments, achieved tumor growth in 100% of cases. All data were confirmed by histological analysis. Conclusions: We generated for the first time, a model of para-renal cancer by surgical tumor implantation of VX2 frozen tumor fragments into rabbit's para-renal region. This method minimizes the development of metastases and the use of non-necrotic tumors and will optimize the evaluation of tumor response to loco-regional therapy experiments

Hydrogen sulfide donor AP123 restores endothelial nitric oxide-dependent vascular function in hyperglycemia via a CREB-dependent pathway

Diabetes is associated with severe vascular complications involving the impairment of endothelial nitric oxide synthase (eNOS) as well as cystathionine γ-lyase (CSE) activity. eNOS function is suppressed in hyperglycaemic conditions, resulting in reduced NO bioavailability, which is paralleled by reduced levels of hydrogen sulfide (H2S). Here we have addressed the molecular basis of the interplay between the eNOS and CSE pathways. We tested the impact of H2S replacement by using the mitochondrial-targeted H2S donor AP123 in isolated vessels and cultured endothelial cells in high glucose (HG) environment, at concentrations not causing any vasoactive effect per se. Aorta exposed to HG displayed a marked reduction of acetylcholine (Ach)-induced vasorelaxation that was restored by the addition of AP123 (10 nM). In HG condition, bovine aortic endothelial cells (BAEC) showed reduced NO levels, downregulation of eNOS expression, and suppression of CREB activation (p-CREB). Similar results were obtained by treating BAEC with propargylglycine (PAG), an inhibitor of CSE. AP123 treatment rescued eNOS expression, as well as NO levels, and restored p-CREB expression in both the HG environment and the presence of PAG. This effect was mediated by a PI3K-dependent activity since wortmannin (PI3K inhibitor) blunted the rescuing effects operated by the H2S donor. Experiments performed in the aorta of CSE−/− mice confirmed that reduced levels of H2S not only negatively affect the CREB pathway but also impair Ach-induced vasodilation, significantly ameliorated by AP123. We have demonstrated that the endothelial dysfunction due to HG involves H2S/PI3K/CREB/eNOS route, thus highlighting a novel aspect of the H2S/NO interplay in the vasoactive response

Biliverdin Treatment Increases Hepatocyte Proliferation After IRI.

<p><b>A.</b> Representative immunostaining images for Ki-67 as a marker of cell proliferation in liver sections harvested pre-ischemia, immediately post-ischemia and 12 hrs after reperfusion ± BV treatment. Effects of biliverdin on Ki-67 expression as an indicator of cell (primarily hepatocyte) proliferation. <b>B.</b> Ki-67 positive cells were counted and expressed as a percentage of positive cells among the total number of cells present in each field. Data are expressed as mean ± SD of n = 3 pigs/group where a total number of cells counted was at least 500. Magnification = 400×, Bar represents 50 µm. (*p = 0.046).</p

Effects of Biliverdin to Reduce Neutrophil Influx into the Liver.

<p><b>A.</b> Representative H&E staining of liver sections harvested pre-ischemia, immediately post-ischemia and 12 hrs after reperfusion ± BV treatment. <b>B.</b> Neutrophils were counted based on morphology and expressed as a percentage of neutrophils among the total number of cells present in each field. Data are expressed as mean ± SD of n = 3 pigs/group where a total number of cells counted was at least 500. Magnification = 400×, Bar represents 50 µm. (*p = 0.013).</p

Kinetics of serum bilirubin levels in pigs in response to intravenous biliverdin administration.

<p>Biliverdin was administered as a single i.v. bolus of 50 µmol/kg. Results are mean ± SD of 3 pigs/treatment group. The black bars correspond to the donors and the open bars to the recipient.</p

Effects of biliverdin on hepatocyte cell death.

<p><b>A</b>. Representative immunostained liver sections for TUNEL and caspase 3 from liver sections harvested pre-ischemia, immediately post-ischemia and 12 hrs after reperfusion ± BV treatment. <b>B</b>. Quantitation of the number of positive cells in each stained sections as described in the methods. The degree of apoptosis and caspase 3 positivity was quantified by counting the number of positive cells among the total cells present in at least 10 selected fields with a minimum of 500 total positive cells counted. There is a statistically significant difference after 12 hrs of reperfusion *p = 0.01 versus ischemia alone and between biliverdin preconditioned animals after 12 hrs of reperfusion compared to controls (*p = 0.017). Results represent mean ± SD of 10 fields from 3 pigs/group where a total number of cells counted was at least 500. Magnification = 400×, Bar represents 50 µm.</p