Dependence of Induced Biological Damage on the Energy Distribution and Intensity of Clinical Intra-Operative Radiotherapy Electron Beams.

The survival fraction of epithelial HaCaT cells was analysed to assess the biological damage caused by intraoperative radiotherapy electron beams with varying energy spectra and intensities. These conditions were achieved by irradiating the cells at different depths in water using nominal 6 MeV electron beams while consistently delivering a dose of 5 Gy to the cell layer. Furthermore, a Monte Carlo simulation of the entire irradiation procedure was performed to evaluate the molecular damage in terms of molecular dissociations induced by the radiation. A significant agreement was found between the molecular damage predicted by the simulation and the damage derived from the analysis of the survival fraction. In both cases, a linear relationship was evident, indicating a clear tendency for increased damage as the averaged incident electron energy and intensity decreased for a constant absorbed dose, lowering the dose rate. This trend suggests that the radiation may have a more pronounced impact on surrounding healthy tissues than initially anticipated. However, it is crucial to conduct additional experiments with different target geometries to confirm this tendency and quantify the extent of this effect.

Selective miRNA inhibition in CD8+ cytotoxic T lymphocytes enhances HIV-1 specific cytotoxic responses.

miRNAs dictate relevant virus-host interactions, offering new avenues for interventions to achieve an HIV remission. We aimed to enhance HIV-specific cytotoxic responses-a hallmark of natural HIV control- by miRNA modulation in T cells. We recruited 12 participants six elite controllers and six patients with chronic HIV infection on long-term antiretroviral therapy ("progressors"). Elite controllers exhibited stronger HIV-specific cytotoxic responses than the progressors, and their CD8+T cells showed a miRNA (hsa-miR-10a-5p) significantly downregulated. When we transfected ex vivo CD8+ T cells from progressors with a synthetic miR-10a-5p inhibitor, miR-10a-5p levels decreased in 4 out of 6 progressors, correlating with an increase in HIV-specific cytotoxic responses. The effects of miR-10a-5p inhibition on HIV-specific CTL responses were modest, short-lived, and occurred before day seven after modulation. IL-4 and TNF-α levels strongly correlated with HIV-specific cytotoxic capacity. Thus, inhibition of miR-10a-5p enhanced HIV-specific CD8+ T cell capacity in progressors. Our pilot study proves the concept that miRNA modulation is a feasible strategy to combat HIV persistence by enhancing specific cytotoxic immune responses, which will inform new approaches for achieving an antiretroviral therapy-free HIV remission.

Biomarkers Associated with Regorafenib First-Line Treatment Benefits in Metastatic Colorectal Cancer Patients: REFRAME Molecular Study.

First-line treatment with regorafenib in frail metastatic colorectal cancer (mCRC) patients has shown some benefit. To accurately identify such patients before treatment, we studied blood biomarkers and primary tumor molecules. We unveiled serum microRNAs (miRNAs), single-nucleotide polymorphisms (SNPs) in angiogenic-related genes, and Notch 1 expression as biomarkers associated with response or toxicity. MicroRNA array profiling and genotyping of selected SNPs were performed in the blood of fragile mCRC patients treated with regorafenib. Notch 1 and CRC-associated miRNA expression was also analyzed in tumors. High levels of miR-185-5p in serum, rs7993418 in the vascular endothelial growth factor receptor 1 (VEGFR1) gene, and Notch 1 expression in biopsies were associated with a favorable response to treatment. Serum levels of miR-126-3p and miR-152-3p and tumor expression of miR-92a-1-5p were associated with treatment toxicity, particularly interesting in patients exhibiting comorbidities, and high levels of miR-362-3p were associated with asthenia. Additionally, several miRNAs were associated with the presence of metastasis, local recurrence, and peritoneal metastasis. Besides, miRNAs determined in primary tumors were associated with tumor-node-metastasis (TNM) staging. The rs2305948 and rs699947 SNPs in VEGFR2 and VEGFA, respectively, were markers of poor prognosis correlating with locoregional relapse, a higher N stage, and metastatic shedding. In conclusion, VEGF and VEGFR SNPs, miRNAs, and Notch 1 levels are potential useful biomarkers for the management of advanced CRC under regorafenib treatment.

Cyclooxygenase 2 Effector Genes as Potential Inflammation-Related Biomarkers for Colorectal Cancer Circulating Tumor Cells Detection by Liquid Biopsy.

Cyclooxygenase 2 (COX2) has been implicated in cancer development and metastasis. We have identified several COX2-regulated inflammation-related genes in human colorectal cancer cells and shown that some of them play important roles in tumor progression. In this work, we have studied the COX2-regulated genes in the mouse colorectal cancer cell line CT26, to find that many are also regulated by COX2 over-expression. On the other hand, we generated a CT26 cell line expressing Gfp and Luciferase, to study tumor growth and metastasis in immunocompetent Balb/c mice. We then collected solid tissue, and blood samples, from healthy and tumor-bearing mice. Using the Parsortix® cell separation system and taking advantage of the fact that the tumor cells expressed Gfp, we were able to identify circulating tumor cells (CTCs) in some of the mice. We compared the mRNA expression levels of Ptgs2 and effector genes in the samples obtained from tumor-bearing or healthy mice, namely, tumor or healthy colon, Ficoll purified buffy coat, and Parsortix-isolated cells to find different patterns between healthy, tumor-bearing mice with or without CTCs. Although for genes like Il15 we did not observe any difference between healthy and tumor-bearing mice in Ficoll or Parsortix samples; others, such as Egr1, Zc3h12a, Klf4, or Nfat5, allowed distinguishing for cancer or CTC presence. Gene expression analysis in Ficoll or Parsortix processed samples, after liquid biopsy, may offer valuable diagnostic and prognostic information and thus should be further studied.

Epigenetic Landscape in Pancreatic Ductal Adenocarcinoma: On the Way to Overcoming Drug Resistance?

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive solid malignancies due to the rapid rate of metastasis and high resistance to currently applied cancer therapies. The complex mechanism underlying the development and progression of PDAC includes interactions between genomic, epigenomic, and signaling pathway alterations. In this review, we summarize the current research findings on the deregulation of epigenetic mechanisms in PDAC and the influence of the epigenome on the dynamics of the gene expression changes underlying epithelial-mesenchymal transition (EMT), which is responsible for the invasive phenotype of cancer cells and, therefore, their metastatic potential. More importantly, we provide an overview of the studies that uncover potentially actionable pathways. These studies provide a scientific basis to test epigenetic drug efficacy in synergy with other anticancer therapies in future clinical trials, in order to reverse acquired therapy resistance. Thus, epigenomics has the potential to generate relevant new knowledge of both a biological and clinical impact. Moreover, the potential, hurdles, and challenges of predictive biomarker discoveries will be discussed, with a special focus on the promise of liquid biopsies.

BRAF Mutated Colorectal Cancer: New Treatment Approaches.

Colon cancer is one of the most frequently diagnosed malignancies in adults, considering both its incidence and prevalence. Anatomically, the right colon is considered as being from the cecum to the splenic flexure, and the left colon is from the splenic flexure to the rectum. Sidedness is a surrogate of a wide spectrum of colorectal cancer (CRC) biology features (embryology, microbiome, methylation, microsatellite instability (MSI), BRAF, aging, KRAS, consensus molecular subtypes (CMS), etc.), which result in prognostic factors. Different molecular subtypes have been identified, according to genomic and transcriptomic criteria. A subgroup harboring a BRAF mutation has been described, and represents approximately 10% of the patients diagnosed with colon cancer. This subgroup has morphological, clinical, and therapeutic characteristics that differ substantially from patients who do not carry this genetic alteration. Unfortunately, there is no established standard of care for this particular cohort of patients. This manuscript aims to study the biology of this subgroup of colon cancer, to understand the current approach in clinical research.

Prostaglandin F2α-induced Prostate Transmembrane Protein, Androgen Induced 1 mediates ovarian cancer progression increasing epithelial plasticity.

The role of prostaglandin (PG) F2α has been scarcely studied in cancer. We have identified a new function for PGF2α in ovarian cancer, stimulating the production of Prostate Transmembrane Protein, Androgen Induced 1 (PMEPA1). We show that this induction increases cell plasticity and proliferation, enhancing tumor growth through PMEPA1. Thus, PMEPA1 overexpression in ovarian carcinoma cells, significantly increased cell proliferation rates, whereas PMEPA1 silencing decreased proliferation. In addition, PMEPA1 overexpression buffered TGFß signaling, via reduction of SMAD-dependent signaling. PMEPA1 overexpressing cells acquired an epithelial morphology, associated with higher E-cadherin expression levels while ß-catenin nuclear translocation was inhibited. Notwithstanding, high PMEPA1 levels also correlated with epithelial to mesenchymal transition markers, such as vimentin and ZEB1, allowing the cells to take advantage of both epithelial and mesenchymal characteristics, gaining in cell plasticity and adaptability. Interestingly, in mouse xenografts, PMEPA1 overexpressing ovarian cells had a clear survival and proliferative advantage, resulting in higher metastatic capacity, while PMEPA1 silencing had the opposite effect. Furthermore, high PMEPA1 expression in a cohort of advanced ovarian cancer patients was observed, correlating with E-cadherin expression. Most importantly, high PMEPA1 mRNA levels were associated with lower patient survival.

Intracellular Delivery of an Antibody Targeting Gasdermin-B Reduces HER2 Breast Cancer Aggressiveness.

PURPOSE: Gasdermin B (GSDMB) overexpression/amplification occurs in about 60% of HER2 breast cancers, where it promotes cell migration, resistance to anti-HER2 therapies, and poor clinical outcome. Thus, we tackle GSDMB cytoplasmic overexpression as a new therapeutic target in HER2 breast cancers. EXPERIMENTAL DESIGN: We have developed a new targeted nanomedicine based on hyaluronic acid-biocompatible nanocapsules, which allow the intracellular delivery of a specific anti-GSDMB antibody into HER2 breast cancer cells both in vitro and in vivo. RESULTS: Using different models of HER2 breast cancer cells, we show that anti-GSDMB antibody loaded to nanocapsules has significant and specific effects on GSDMB-overexpressing cancer cells' behavior in ways such as (i) lowering the in vitro cell migration induced by GSDMB; (ii) enhancing the sensitivity to trastuzumab; (iii) reducing tumor growth by increasing apoptotic rate in orthotopic breast cancer xenografts; and (iv) diminishing lung metastasis in MDA-MB-231-HER2 cells in vivo. Moreover, at a mechanistic level, we have shown that AbGB increases GSDMB binding to sulfatides and consequently decreases migratory cell behavior and may upregulate the potential intrinsic procell death activity of GSDMB. CONCLUSIONS: Our findings portray the first evidence of the effectiveness and specificity of an antibody-based nanomedicine that targets an intracellular oncoprotein. We have proved that intracellular-delivered anti-GSDMB reduces diverse protumor GSDMB functions (migration, metastasis, and resistance to therapy) in an efficient and specific way, thus providing a new targeted therapeutic strategy in aggressive HER2 cancers with poor prognosis.

Targeting Tyrosine kinases in Renal Cell Carcinoma: "New Bullets against Old Guys".

Clear cell renal cell carcinoma (ccRCC) is the seventh most frequently diagnosed tumor in adults in Europe and represents approximately 2.5% of cancer deaths. The molecular biology underlying renal cell carcinoma (RCC) development and progression has been a key milestone in the management of this type of tumor. The discovery of Von Hippel Lindau (VHL) gene alterations that arouse in 50% of ccRCC patients, leads the identification of an intracellular accumulation of HIF and, consequently an increase of VEGFR expression. This change in cell biology represents a new paradigm in the treatment of metastatic renal cancer by targeting angiogenesis. Currently, there are multiple therapeutic drugs available for advanced disease, including therapies against VEGFR with successful results in patients´ survival. Other tyrosine kinases' pathways, including PDGFR, Axl or MET have emerged as key signaling pathways involved in RCC biology. Indeed, promising new drugs targeting those tyrosine kinases have exhibited outstanding efficacy. In this review we aim to present an overview of the central role of these tyrosine kinases' activities in relevant biological processes for kidney cancer and their usefulness in RCC targeted therapy development. In the immunotherapy era, angiogenesis is still an "old guy" that the medical community is trying to fight using "new bullets".

Novel Molecular Characterization of Colorectal Primary Tumors Based on miRNAs.

microRNAs (miRNA) expression in colorectal (CR) primary tumours can facilitate a more precise molecular characterization. We identified and validated a miRNA profile associated with clinical and histopathological features that might be useful for patient stratification. In situ hybridization array using paraffin-embedded biopsies of CR primary tumours were used to screen 1436 miRNAs. 17 miRNAs were selected for validation by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (n = 192) and were further correlated with clinical and histopathological data. We demonstrated that miRNAs associated to Colorectal Cancer (CRC) diagnosis age (over 50s and 60s) included miR-1-3p, miR-23b-3p, miR-27b-3p, miR-143-3p, miR-145-5p and miR-193b-5p. miR-23b-3p and miR-24-3p discriminated between Lynch Syndrome and sporadic CRC. miR-10a-5p, miR-20a-5p, miR-642b and Let-7a-5p were associated to stroma abundance. miR-642b and Let-7a-5p were associated with to peritumoral inflammation abundance. miR-1-3p, miR-143-3p and miR-145-5p correlated with mucinous component. miR-326 correlated with tumour location (right or left sided). miR-1-3p associated with tumour grade. miR-20a-5p, miR-193b-5p, miR-320a, miR-326 and miR-642b-3p associated to tumour stage and progression. Remarkably, we also demonstrated that miR-1-3p and miR-326 expression significantly associated with patient overall survival (OS). Hierarchical clustering and bioinformatics analysis indicated that selected miRNAs could re-classify the patients and work cooperatively, modulating common target genes involved in colorectal cancer key signalling pathways. In conclusion, molecular characterization of CR primary tumours based on miRNAs could lead to more accurate patient reclassification and may be useful for efficient patient management.

VHL promotes immune response against renal cell carcinoma via NF-κB-dependent regulation of VCAM-1.

Vascular cell adhesion molecule 1 (VCAM-1) is an adhesion molecule assigned to the activated endothelium mediating immune cells adhesion and extravasation. However, its expression in renal carcinomas inversely correlates with tumor malignancy. Our experiments in clear cell renal cell carcinoma (ccRCC) cell lines demonstrated that von Hippel Lindau (VHL) loss, hypoxia, or PHD (for prolyl hydroxylase domain-containing proteins) inactivation decreased VCAM-1 levels through a transcriptional mechanism that was independent of the hypoxia-inducible factor and dependent on the nuclear factor κB signaling pathway. Conversely, VHL expression leads to high VCAM-1 levels in ccRCC, which in turn leads to better outcomes, possibly by favoring antitumor immunity through VCAM-1 interaction with the α4ß1 integrin expressed in immune cells. Remarkably, in ccRCC human samples with VHL nonmissense mutations, we observed a negative correlation between VCAM-1 levels and ccRCC stage, microvascular invasion, and symptom presentation, pointing out the clinical value of VCAM-1 levels as a marker of ccRCC progression.

HIF-1α induction during reperfusion avoids maladaptive repair after renal ischemia/reperfusion involving miR127-3p.

Ischemia/reperfusion (I/R) leads to Acute Kidney Injury. HIF-1α is a key factor during organ response to I/R. We previously demonstrated that HIF-1α is induced during renal reperfusion, after ischemia. Here we investigate the role of HIF-1α and the HIF-1α dependent mechanisms in renal repair after ischemia. By interference of HIF-1α in a rat model of renal I/R, we observed loss of expression and mis-localization of e-cadherin and induction of α-SMA, MMP-13, TGFß, and collagen I. Moreover, we demonstrate that HIF-1α inhibition promotes renal cell infiltrates by inducing IL-1ß, TNF-α, MCP-1 and VCAM-1, through NFkB activity. In addition, HIF-1α inhibition induced proximal tubule cells proliferation but it did not induce compensatory apoptosis, both in vivo. In vitro, HIF-1α knockdown in HK2 cells subjected to hypoxia/reoxygenation (H/R) promote cell entry into S phase, correlating with in vivo data. HIF-1α interference leads to downregulation of miR-127-3p and induction of its target gene Bcl6 in vivo. Moreover, modulation of miR-127-3p in HK2 cells subjected to H/R results in EMT regulation: miR127-3p inhibition promote loss of e-cadherin and induction of α-SMA and collagen I. In conclusion, HIF-1α induction during reperfusion is a protector mechanism implicated in a normal renal tissue repair after I/R.

Prostaglandins induce early growth response 1 transcription factor mediated microsomal prostaglandin E2 synthase up-regulation for colorectal cancer progression.

Cyclooxygenase2 (COX2) has been associated with cell growth, invasiveness, tumor progression and metastasis of colorectal carcinomas. However, the downstream prostaglandin (PG)-PG receptor pathway involved in these effects is poorly characterized.We studied the PG-pathway in gene expression databases and we found that PTGS2 (prostaglandin G/H synthase and cyclooxygenase) and PTGES (prostaglandin E synthase) are co-expressed in human colorectal tumors. Moreover, we detected that COX2 and microsomal Prostaglandin E2 synthase 1 (mPGES1) proteins are both up-regulated in colorectal human tumor biopsies.Using colon carcinoma cell cultures we found that COX2 overexpression significantly increased mPGES1 mRNA and protein. This up-regulation was due to an increase in early growth response 1 (EGR1) levels and its transcriptional activity. EGR1 was induced by COX2-generated PGF2α. A PGF2α receptor antagonist, or EGR1 silencing, inhibited the mPGES1 induction by COX2 overexpression. Moreover, using immunodeficient mice, we also demonstrated that both COX2- and mPGES1-overexpressing carcinoma cells were more efficient forming tumors.Our results describe for the first time the molecular pathway correlating PTGS2 and PTGES in colon cancer progression. We demonstrated that in this pathway mPGES1 is induced by COX2 overexpression, via autocrine PGs release, likely PGF2α, through an EGR1-dependent mechanism. This signaling provides a molecular explanation to PTGS2 and PTGES association and contribute to colon cancer advance, pointing out novel potential therapeutic targets in this oncological context.

miR-9-5p suppresses pro-fibrogenic transformation of fibroblasts and prevents organ fibrosis by targeting NOX4 and TGFBR2.

Uncontrolled extracellular matrix (ECM) production by fibroblasts in response to injury contributes to fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). Reactive oxygen species (ROS) generation is involved in the pathogenesis of IPF. Transforming growth factor-ß1 (TGF-ß1) stimulates the production of NADPH oxidase 4 (NOX4)-dependent ROS, promoting lung fibrosis (LF). Dysregulation of microRNAs (miRNAs) has been shown to contribute to LF. To identify miRNAs involved in redox regulation relevant for IPF, we performed arrays in human lung fibroblasts exposed to ROS. miR-9-5p was selected as the best candidate and we demonstrate its inhibitory effect on TGF-ß receptor type II (TGFBR2) and NOX4 expression. Increased expression of miR-9-5p abrogates TGF-ß1-dependent myofibroblast phenotypic transformation. In the mouse model of bleomycin-induced LF, miR-9-5p dramatically reduces fibrogenesis and inhibition of miR-9-5p and prevents its anti-fibrotic effect both in vitro and in vivo. In lung specimens from patients with IPF, high levels of miR-9-5p are found. In omentum-derived mesothelial cells (MCs) from patients subjected to peritoneal dialysis (PD), miR-9-5p also inhibits mesothelial to myofibroblast transformation. We propose that TGF-ß1 induces miR-9-5p expression as a self-limiting homeostatic response.

A Pilot Study Identifying a Set of microRNAs As Precise Diagnostic Biomarkers of Acute Kidney Injury.

In the last decade, Acute Kidney Injury (AKI) diagnosis and therapy have not notably improved probably due to delay in the diagnosis, among other issues. Precocity and accuracy should be critical parameters in novel AKI biomarker discovery. microRNAs are key regulators of cell responses to many stimuli and they can be secreted to the extracellular environment. Therefore, they can be detected in body fluids and are emerging as novel disease biomarkers. We aimed to identify and validate serum miRNAs useful for AKI diagnosis and management. Using qRT-PCR arrays in serum samples, we determined miRNAs differentially expressed between AKI patients and healthy controls. Statistical and target prediction analysis allowed us to identify a panel of 10 serum miRNAs. This set was further validated, by qRT-PCR, in two independent cohorts of patients with relevant morbi-mortality related to AKI: Intensive Care Units (ICU) and Cardiac Surgery (CS). Statistical correlations with patient clinical parameter were performed. Our results demonstrated that the 10 selected miRNAs (miR-101-3p, miR-127-3p, miR-210-3p, miR-126-3p, miR-26b-5p, miR-29a-3p, miR-146a-5p, miR-27a-3p, miR-93-3p and miR-10a-5p) were diagnostic biomarkers of AKI in ICU patients, exhibiting areas under the curve close to 1 in ROC analysis. Outstandingly, serum miRNAs estimated before CS predicted AKI development later on, thus becoming biomarkers to predict AKI predisposition. Moreover, after surgery, the expression of the miRNAs was modulated days before serum creatinine increased, demonstrating early diagnostic value. In summary, we have identified a set of serum miRNAs as AKI biomarkers useful in clinical practice, since they demonstrate early detection and high diagnostic value and they recognize patients at risk.

Hypoxia inducible factor 1-alpha (HIF-1 alpha) is induced during reperfusion after renal ischemia and is critical for proximal tubule cell survival.

Acute tubular necrosis (ATN) caused by ischemia/reperfusion (I/R) during renal transplantation delays allograft function. Identification of factors that mediate protection and/or epithelium recovery could help to improve graft outcome. We studied the expression, regulation and role of hypoxia inducible factor 1-alpha (HIF-1 α), using in vitro and in vivo experimental models of I/R as well as human post-transplant renal biopsies. We found that HIF-1 α is stabilized in proximal tubule cells during ischemia and unexpectedly in late reperfusion, when oxygen tension is normal. Both inductions lead to gene expression in vitro and in vivo. In vitro interference of HIF-1 α promoted cell death and in vivo interference exacerbated tissue damage and renal dysfunction. In pos-transplant human biopsies, HIF-1 α was expressed only in proximal tubules which exhibited normal renal structure with a significant negative correlation with ATN grade. In summary, using experimental models and human biopsies, we identified a novel HIF-1 α induction during reperfusion with a potential critical role in renal transplant.

Mutual regulation of hypoxic and retinoic acid related signalling in tubular proximal cells.

Hypoxia-inducible factor-1α (HIF-1α) and all-trans retinoic acid (ATRA) afford protection in several experimental models of kidney disease. HIF-1α protein is degraded under normoxia but stabilized by hypoxia, which activates its transcription factor function. ATRA activates another set of transcription factors, the retinoic acid receptors (RAR) α, ß and γ, which mediate its effects on target genes. ATRA also up-regulates the expression of RAR α, ß and γ at the transcriptional level. Here we demonstrate the presence of mutual regulation of hypoxic and retinoic acid related signalling in tubular proximal cells. In human proximal tubular HK-2 cells we have found that: (i) ATRA treatment induces HIF-1α under normoxic conditions and also synergizes with hypoxia leading to the over-expression of HIF-1α and vascular endothelial growth factor-A, a HIF-1α-regulated renal protector. ATRA-induced HIF-1α expression involved stabilization of HIF-1α mRNA but not of HIF-1α protein. (ii) Expression of HIF-1α is an absolute requirement for the transcriptional up-regulation of RARß by ATRA. Transfection with HIF-1α siRNA abolished the induction by ATRA of the expression of both RARß mRNA and protein while treatment with HIF-1α inhibitor YC-1 results in the abolishment of ATRA-induced activity of a retinoic acid-response element (RARE) construct from the RARß promoter. (iii) Hypoxia up-regulates RARß through HIF-1α since this effect was inhibited by HIF-1α knockdown. In contrast to ATRA-induced RARß up-regulation, induction of RARß expression by ATRA did not involve transcriptional up-regulation as hypoxia did not increase the expression of RARß mRNA or the activity of the RARE construct. These results suggest the presence of crosstalk between hypoxia/HIF-1α and ATRA/RARß that may be physiologically and pharmacologically relevant.

Differential resolution of inflammation and recovery after renal ischemia-reperfusion injury in Brown Norway compared with Sprague Dawley rats.

To investigate mechanisms conferring susceptibility or resistance to renal ischemia, we used two rat strains known to exhibit different responses to ischemia-reperfusion. We exposed proximal tubule cells isolated from Sprague Dawley or Brown Norway rats, to a protocol of hypoxia, followed by reoxygenation in vitro. The cells isolated from both rat strains exhibited comparable responses in the disruption of intercellular adhesions and cytoskeletal damage. In vivo, after 24 h of reperfusion, both strains showed similar degrees of injury. However, after 7 days of reperfusion, renal function and tubular structure almost completely recovered and inflammation resolved, but only in Brown Norway rats. Hypoxia-inducible factor-dependent gene expression, ERK1/2, and Akt activation were different in the two strains. Inflammatory mediators MCP-1, IL-10, INF-gamma, IL-1beta, and TNF-alpha were similarly induced at 24 h in both strains but were downregulated earlier in Brown Norway rats, which correlated with shorter NFkappaB activation in the kidney. Moreover, VLA-4 expression in peripheral blood lymphocytes and VCAM-1 expression in kidney tissues were initially similar at 24 h but reached basal levels earlier in Brown Norway rats. The faster resolution of inflammation in Brown Norway rats suggests that this strain might be a useful experimental model to determine the mechanisms that promote repair of renal ischemia-reperfusion injury.

15-Deoxy-Delta(12,14)-prostaglandin-J(2) reveals a new pVHL-independent, lysosomal-dependent mechanism of HIF-1alpha degradation.

Hypoxia-inducible factor-1alpha (HIF-1alpha) protein is degraded under normoxia by its association to von Hippel-Lindau protein (pVHL) and further proteasomal digestion. However, human renal cells HK-2 treated with 15-deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)) accumulate HIF-1alpha in normoxic conditions. Thus, we aimed to investigate the mechanism involved in this accumulation. We found that 15d-PGJ(2) induced an over-accumulation of HIF-1alpha in RCC4 cells, which lack pVHL and in HK-2 cells treated with inhibitors of the pVHL-proteasome pathway. These results indicated that pVHL-proteasome-independent mechanisms are involved, and therefore we aimed to ascertain them. We have identified a new lysosomal-dependent mechanism of HIF-1alpha degradation as a target for 15d-PGJ(2) based on: (1) HIF-1alpha colocalized with the specific lysosomal marker Lamp-2a, (2) 15d-PGJ(2) inhibited the activity of cathepsin B, a lysosomal protease, and (3) inhibition of lysosomal activity did not result in over-accumulation of HIF-1alpha in 15d-PGJ(2)-treated cells. Therefore, expression of HIF-1alpha is also modulated by lysosomal degradation.