Leonel García-Benavides

Alternative Interventions to Prevent Oxidative Damage following Ischemia/Reperfusion

Ischemia/reperfusion (I/R) lesions are a phenomenon that occurs in multiple pathological states and results in a series of events that end in irreparable damage that severely affects the recovery and health of patients. The principal therapeutic approaches include preconditioning, postconditioning, and remote ischemic preconditioning, which when used separately do not have a great impact on patient mortality or prognosis. Oxidative stress is known to contribute to the damage caused by I/R; however, there are no pharmacological approaches to limit or prevent this. Here, we explain the relationship between I/R and the oxidative stress process and describe some pharmacological options that may target oxidative stress-states.

Isosorbide Dinitrate Spray as Therapeutic Strategy for Treatment of Chronic Venous Ulcers

Venous ulcers are the most common form of leg ulcers, which induce lesion because of the loss of substances deposited on the damaged skin. Isosorbide dinitrate is a vasodilator with effects on both arteries and veins and induces opening of vascular layers. The objective is to study the effects of isosorbide dinitrate-spray in patients with chronic venous ulcers. Forty-five patients of both sexes with chronic venous ulcers were randomized to receive isosorbide dinitrate or placebo sprays daily for 3 months. The ulcers were measured and clinical characteristics were taken every 15 days during the treatment. Patients treated with isosorbide dinitrate showed an improvement of the ulcerated area (71.29%) compared with patients treated with placebo (54.35%). The histopathological study indicated an increment in the number of hypertrophic and hyperplasic capillaries. Macroscopically, the isosorbide dinitrate-treatment showed the best results, but it was only during the first 6 weeks of treatment. Patients with chronic venous ulcer receiving isosorbide dinitrate spray showed improvement.

Correction: Human adipose derived stem cells regress fibrosis in a chronic renal fibrotic model induced by adenine

Background and aims ADSCs transplantation had been shown in some experimental models of kidney damage that it improves kidney function and reduces fibrosis. In this study we evaluated the effect of human adipose tissue-derived stem cell (hADSC) therapy in a chronic kidney damage experimental model. Methods A chronic kidney injury was induced by daily orogastric administration of adenine (100mg/kg) to male Wistar rats for 28 days. hADSCs were isolated, expanded and characterized before transplantation. hADSC administration was performed in a tail vein at a dose of 2 x106 cells/animal. Animals were sacrificed at 7 days post-treatment. The percentage of fibrotic tissue, serum and urine levels of urea, creatinine, total protein and renal mRNA of COL1A1, TGFB1, CTGF, ACTA2, IL6, IL10, TNF were analyzed. Results hADSCs treatment significantly reduces kidney fibrosis, improves urea and creatinine serum and urine levels, and diminishes COL1A1, TGFB1, CTGF, ACTA2 mRNA kidney levels. Conclusions These results showed that cell therapy using hADSCs improves renal function and reduces fibrosis.

P0092 : IL-17 A and F isoforms and their receptors mediate liver damage in experimental cholestasis and the IL17 A/F heterodimer induces a profibrogenic profile in hepatic stellate cells in vitro

P0092 IL-17 A AND F ISOFORMS AND THEIR RECEPTORS MEDIATE LIVER DAMAGE IN EXPERIMENTAL CHOLESTASIS AND THE IL17 A/F HETERODIMER INDUCES A PROFIBROGENIC PROFILE IN HEPATIC STELLATE CELLS IN VITRO S. Zepeda-Morales, S. Del Toro-Arreola, L. Sanchez-Orozco, M. Fafutis-Morris, L. Garcia-Benavides, A.L. Pereira-Suarez, M.R. Bueno-Topete. Instituto de Enfermedades Cronico Degenerativas, Bioloǵia Molecular y Genomica, Laboratorio de Inmunoloǵia, Instituto de Terapeutica Experimental y Cĺinica, CUCS, U de G., Guadalajara, jal, Mexico E-mail: ruthmyriamtop@hotmail.com

The Renin-Angiotensin-Aldosterone System as a Therapeutic Target in Late Injury Caused by Ischemia-Reperfusion

Ischemia-reperfusion (I/R) injury is a well-known phenomenon that involves different pathophysiological processes. Connection in diverse systems of survival brings about cellular dysfunction or even apoptosis. One of the survival systems of the cells, to the assault caused by ischemia, is the activation of the renin-angiotensin-aldosterone system (also known as an axis), which is focused on activating diverse signaling pathways to favor adaptation to the decrease in metabolic supports caused by the hypoxia. In trying to adapt to the I/R event, great changes occur that unchain cellular dysfunction with the capacity to lead to cell death, which translates into a poor prognosis due to the progression of dysfunction of the cellular activity. The search for the understanding of the diverse therapeutic alternatives in molecular coupling could favor the prognosis and evolution of patients who are subject to the I/R process.

Bacterial Translocation Is Linked to Increased Intestinal IFN-γ, IL-4, IL-17, and mucin-2 in Cholestatic Rats

Background and rationale for the study. Bacterial translocation is an important triggering factor of infection and mortality in cirrhosis. In a rat model using bile duct ligation (BDL), bacterial translocation appears within 24 h after ligation. The dynamic between TH1/TH2/TH17 cytokines and the integrity of the colonic mucosa in the context of cirrhosis is little known. This study aims to determine the link between bacterial translocation and intestinal inflammation in a cholestasis model. Additionally, alterations of the colonic mucus layer and the bacterial load were also addressed. RESULTS Bacterial translocation detected by microbiological cultures and MALDI-TOF showed that Escherichia coli predominates in mesenteric lymph nodes of BDL rats. Intestinal bacterial load analyzed by qPCR indicates a dramatic Escherichia/Shigella overgrowth at 8 and 30 days post-BDL. IFN-γ, IL-4, and IL-17 evaluated by Western blotting were increased at 8 and 30 days in the small intestine. In the colon, in contrast, only IFN-γ was significantly increased. The colonic mucus layer and mucin-2 expression determined by Alcian blue staining and immunohistochemistry surprisingly showed an increase in the mucus layer thickness related to increased mucin-2 expression during the entire process of liver damage. Hepatic enzymes, as well as collagen I, collagen III, TNF-α, and IL-6 liver gene expression were increased. In conclusion, bacterial overgrowth associated with bacterial translocation is linked to the over-expression of IFN-γ, IL-4, IL-17 and mucin-2. These molecules might facilitate the intestinal permeability through exacerbating the inflammatory process and disturbing tight junctions, leading to the perpetuation of the liver damage.INTRODUCTION The present study aimed to elucidate the potential antifibrotic effects of pinocembrin (PIN), a flavanone found abundantly in honey and propolis, by studying its effect on different oxidative stress, inflammatory and fibrosis markers in an experimental model of CCl4-induced liver fibrosis. MATERIAL AND METHODS PIN (20 mg/kg) was given orally 3 times/week for 6 consecutive weeks alternating with CCl4 (0.5 mL/kg, 1:1 mixture with corn oil, i. p.) twice weekly. Different hepatotoxicity indices, oxidative stress, inflammatory and liver fibrosis markers were assessed. RESULTS PIN significantly restored liver transaminases and total cholesterol to normal levels. Also, PIN ameliorated oxidative stress injury evoked by CCl4 as evidenced by inhibition of reduced glutathione depletion and lipid peroxidation as well as elevation of antioxidant enzyme superoxide dismutase (SOD). Further, PIN up-regulated the nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2), thereby inducing the expression and activity of the cytoprotective enzyme hemeoxygenase-1 (HO-1). Moreover, PIN alleviated pro-inflammatory cytokines such as TNF-α via inhibiting nuclear factor-KB (nF-kB) activation. As markers of fibrosis, collagen and α-SMA expression increased markedly in the CCl4 group and PIN prevented these alterations. In addition, PIN down-regulated TGFβ1 and p-Smad2/3, thereby inhibiting TGFβ1/Smad signaling pathway. CONCLUSION These results suggest that PIN possess potent antifibrotic effects that can be explained on its antioxidant properties. It ameliorates oxidative stress and inflammation during induction of fibrogenesis via its ability to augment cellular antioxidant defenses, activating Nrf2-mediated HO-1 expression and modulating NF-KB and TGF-β1/Smad signaling pathway.

Simultaneous Administration of ADSCs-Based Therapy and Gene Therapy Using Ad-huPA Reduces Experimental Liver Fibrosis.

Background and Aims hADSCs transplantation in cirrhosis models improves liver function and reduces fibrosis. In addition, Ad-huPA gene therapy diminished fibrosis and increased hepatocyte regeneration. In this study, we evaluate the combination of these therapies in an advanced liver fibrosis experimental model. Methods hADSCs were expanded and characterized before transplantation. Ad-huPA was simultaneously administrated via the ileac vein. Animals were immunosuppressed by CsA 24 h before treatment and until sacrifice at 10 days post-treatment. huPA liver expression and hADSCs biodistribution were evaluated, as well as the percentage of fibrotic tissue, hepatic mRNA levels of Col-αI, TGF-β1, CTGF, α-SMA, PAI-I, MMP2 and serum levels of ALT, AST and albumin. Results hADSCs homed mainly in liver, whereas huPA expression was similar in Ad-huPA and hADSCs/Ad-huPA groups. hADSCs, Ad-huPA and hADSCs/Ad-huPA treatment improves albumin levels, reduces liver fibrosis and diminishes Collagen α1, CTGF and α-SMA mRNA liver levels. ALT and AST serum levels showed a significant decrease exclusively in the hADSCs group. Conclusions These results showed that combinatorial effect of cell and gene-therapy does not improve the antifibrogenic effects of individual treatments, whereas hADSCs transplantation seems to reduce liver fibrosis in a greater proportion.

255. Simultaneous Administration of Cell Therapy with ADSCs and Gene Therapy Using Ad-huPA Reduces Experimental Liver Fibrosis

BACKGROUND: In experimental models of cirrhosis cell therapy with hADSCs (adipose derived stromal cells) has improved liver function while reduced fibrotic tissue. Also, gene therapy using Ad-huPA has diminished liver fibrosis and increased hepatocyte regeneration. The aim of this study was to evaluate if the simultaneous administration of these therapies presents an enhanced antifibrogenic effect in cirrhotic rats.METHODS: hADSCs were isolated from fat tissue, expanded and characterized by expression of CD105+, CD73+, HLA-ABC+, CD45-, CD34-, HLA-DRII- markers and osteogenic and adipogenic differentiation. Ad-huPA vector was generated by recombination of pMH4-huPA and pJM17ΔE1 Ad-v backbone. CCl4-cirrhotic rats were administered with hADSCs (2X106 cells/rat) or Ad-huPA (3×1011 vp/rat) or both hADSCs/Ad-huPA via ileac vein. 24 hours before initiating treatment all groups were immunosuppressed with 10mg/kg of Cyclosporine A until sacrifice. After 10 days, percentage of fibrotic tissue, collagen fiber staining and mRNA levels of TGF-b1, collagen a1, CTGF, PAI-I and α-SMA were evaluated. Besides, serum levels of ALT, AST and albumin, biodistribution of hADSCs and liver levels of huPA protein were examined.RESULTS: Detection of the DAPI signal revealed that hADSCs mainly homing in liver and few of them were arrested in lung and spleen. Administration of hADSCs, Ad-huPA and Ad-huPA/hADSCs reduces (p<0.01) liver fibrosis in 78.9%, 65.2% and 72% respectively; and diminishes Collagen a1, CTGF and α-SMA mRNA liver levels (p<0.05). Furthermore, TGF-b1 and PAI-I liver mRNA levels (p<0.05) decrease in animals treated with Ad-huPA and hADSCs. Serum levels of albumin increase in the Ad-huPA, hADSCs and Ad-huPA/hADSCs groups (p<0.05) compared with control group. ALT and AST serum levels showed a significant diminish in hADSCs group (p<0.05). huPA protein was expressed in similar levels in liver homogenates of Ad-huPA and Ad-huPA/hADSCs groups. hADSCs, Ad-huPA and hADSC/Ad-huPA administration reduce percentage of collagen staining 4.3, 2.4 and 2.7 fold (p£0.001), respectively.CONCLUSIONS: Xenogenic hADSCs, Ad-huPA and Ad-huPA/hADSCs therapies decreased liver fibrosis and expression of pro-fibrogenic molecules in CCl4-cirrhotic animals. However, simultaneous administration of cell therapy and gene therapy does not appear to improve antifibrogenic effects of individual treatments.