Utility of pVHL, maspin, IMP3, S100P and Ki67 in the distinction of autoimmune pancreatitis from pancreatic ductal adenocarcinoma.

Morphology plays an important role in the distinction of autoimmune pancreatitis (AIP) from pancreatic ductal adenocarcinoma (PDAC). However, we aimed to determine the utility of immunohistochemical tumor markers to contribute in the distinction of these entities. In surgical specimens with AIP (n = 20), PDAC (n = 20) and normal pancreas (n = 20), the expression of pVHL, maspin, IMP3, S100P and Ki67 was examined. We evaluated intralobular reactive ducts / acinoductal metaplasia (ILDs) and extralobular ducts (ELDs) in AIP, neoplastic glands in PDAC, and ductal epithelium in the normal pancreas, using a five-tiered scoring system. The Ki67 hot spot index (Ki67-HSPI) was determined manually and using automated digital imaging analysis of virtual double stains of Ki67 and CK8. Besides, sequential dual-immunohistochemical staining of maspin/pVHL, maspin/IMP3 and Ki67/maspin was performed in a subset of the specimens. Strong overexpression of IMP3, maspin, S100P and Ki67 and loss of pVHL was observed in PDAC compared to AIP and normal pancreas. In AIP however, focal and weak aberrant expression was observed with the following proportions in ILDs/ELDs: pVHL in 45 %/85 %, maspin in 30 %/70 %, IMP3 in 55 %/5%, S100P in 10 %/35 % and Ki67-HSPI >20 % in 15 %/70 %. At least two markers were aberrantly expressed in ILDs/ELDs in 45 %/60 %. The aberrant expression was more pronounced in type 2 AIP compared to type 1. In conclusion, our data indicate that pVHL, maspin, IMP3, S100P and Ki67 can be focal and weak aberrantly expressed in AIP. However, when used as a panel, these markers seem to be useful for the differentiation of AIP from PC.

Transcriptomic expression levels of the VHL, TIMP-3, and RASSF1A genes in renal tumors.

OBJECTIVE: In this study, we aimed to investigate the relation between the mRNA expression levels of VHL, TIMP-3 and RASSF1A genes, and the histopathological and clinical characteristics of patients with renal tumors. PATIENTS AND METHODS: Radical nephrectomy specimens of cases presented without neoadjuvant treatment were confirmed to be cancerous, non-cancerous, benign, and healthy after removal from separate localizations. A total of 69 patients with kidney tumors (138 tissue samples) were included in the study group. RNA isolation, reverse transcriptase PCR (RT-PCR), and quantitative real time PCR (qPCR) were performed, and the GAPDH gene was used to normalize mRNA levels. RESULTS: In the RCC cancerous tissue, TIMP-3 levels increased 1.3 times and RASSF1A levels increased 1.4 times compared to the corresponding levels in non-cancerous tissues, and there was no statistically significant difference in these values. On the other hand, VHL gene expression levels in cancerous tissue were 2.8 times higher than in matched adjacent non-cancerous tissues (p < 0.05). In the case of oncocytomas, TIMP-3 levels were found to be 3.2 times higher, RASSF1A levels 3.8 times higher, and VHL levels 2.2 times lower than the corresponding levels in healthy tissues (p < 0.05). CONCLUSIONS: The roles of VHL, TIMP-3, and RASSF1A mRNA expression in contributing to the development of renal tumors could not be clearly established. Further studies are therefore required to elucidate the mechanisms underlying renal tumors.

Small activating RNA induced expression of VHL gene in renal cell carcinoma.

Recent studies have reported that chemically synthesized double-stranded RNAs (dsRNAs), also known as small activating RNA (saRNAs), can specifically induce gene expression by targeting promoter sequences by a mechanism termed RNA activation (RNAa). In the present study, we designed 4 candidate saRNAs targeting the Von Hippel-Lindau (VHL) gene promoter. Among these saRNAs, dsVHL-821 significantly inhibited cell growth by up-regulating VHL at both the mRNA and protein levels in renal cell carcinoma 769-P cells. Functional analysis showed that dsVHL-821 induced apoptosis by increasing p53, decreasing Bcl-xL, activating caspase 3/7 and poly-ADP-ribose polymerase in a dose-dependent manner. Chromatin immunoprecipitation analysis revealed that dsVHL-821 increased the enrichment of Ago2 and RNA polymerase II at the dsVHL-821 target site. In addition, Ago2 depletion significantly suppressed dsVHL-821-induced up-regulation of VHL gene expression and related effects. Single transfection of dsVHL-821 caused long-lasting (14 days) VHL up-regulation. Furthermore, the activation of VHL by dsVHL-821 was accompanied by an increase in dimethylation of histone 3 at lysine 4 (H3K4me2) and acetylation of histone 4 (H4ac) and a decrease in dimethylation of histone 3 at lysine 9 (H3K9me2) and lysine 27 (H3K27me2) in the dsVHL-821 target region. Taken together, these results demonstrate that dsVHL-821, a novel saRNA for VHL, induces the expression of the VHL gene by epigenetic changes, leading to inhibition of cell growth and induction of apoptosis, and suggest that targeted activation of VHL by dsVHL-821 may be explored as a novel treatment of renal cell carcinoma.

MIR-92 stimulates VEGF by inhibiting von Hippel-Lindau gene product in epithelial ovarian cancer.

The molecular mechanisms underlying regulation of vascular endothelial growth factor (VEGF) in epithelial ovarian cancer (EOC) remain poorly defined. VEGF, a potent angiogenic factor, is up-regulated in a variety of cancers and contributes to angiogenesis in tumor tissues. The level of VEGF correlates with progression of malignancy. We previously reported that miR-92 is abnormally elevated in the plasma of EOC patients. Here, we tested the hypothesis that miR-92 inhibits von Hippel-Lindau gene product (VHL), a tumor suppressor gene, and in turn de-represses HIF-1α, a known key transcription factor for VEGF, to stimulate VEGF expression. Using a variety of biomedical methods including Western blot, RT-PCR, gene silencing, luciferase assay, and chromatin immunoprecipitation in both surgically-resected specimens and EOC cell culture, we established that EOC cells have elevated levels of HIF-1α and miR-92 expression, but the expression of VHL is reduced. We further demonstrated that miR-92 can target the VHL transcript to repress its expression. We also found that stabilized HIF-1α can form an active complex with transcriptional coactivator p300 and phosphorylated-STAT3 at the VEGF promoter to stimulate its expression. In addition, matrix metalloproteinases MMP-2 and MMP-9 are positively regulated by HIF-1α. These results suggest that miR-92 can potentially be considered as a novel therapeutical target in treatment of EOS.

Expression of von Hippel-Lindau tumor suppressor protein (pVHL) characteristic of tongue cancer and proliferative lesions in tongue epithelium.

BACKGROUND: Patients with tongue cancer frequently show loss of heterozygosity (LOH) of the von Hippel-Lindau (VHL) tumor suppressor gene. However, expression of VHL protein (pVHL) in tongue cancer has rarely been investigated and remains largely unknown. We performed immunohistochemical staining of pVHL in tongue tissues and dysplasia, and examined the association with LOH and its clinical significance. METHODS: Immunohistochemical staining of pVHL in formalin-fixed, paraffin-embedded sections of cancerous and other tissues from 19 tongue cancer patients showed positivity for LOH of VHL in four samples, negativity in four samples, and was non-informative in 11 samples. The staining pattern of pVHL was also compared with those of cytokeratin (CK) 13 and CK17. RESULTS: In normal tongue tissues, pVHL staining was localized to the cytoplasm of cells in the basal layer and the area of the spinous layer adjacent to the basal layer of stratified squamous epithelium. Positive staining for pVHL was observed in the cytoplasm of cancer cells from all 19 tongue cancer patients. No differences as a result of the presence or absence of LOH were found. Notably, cytoplasm of poorly differentiated invasive cancer cells was less intensely stained than that of well and moderately differentiated invasive cancer cells. pVHL staining was also evident in epithelial dysplasia lesions with pVHL-positive cells expanding from the basal layer to the middle of the spinous layer. However, no CK13 staining was noted in regions of the epithelium, which were positive for pVHL. In contrast, regions with positive staining for CK17 closely coincided with those positive for pVHL. CONCLUSIONS: Positive staining for pVHL was observed in cancerous areas but not in normal tissues. pVHL expression was also detected in lesions of epithelial dysplasia. These findings suggest that pVHL may be a useful marker for proliferative lesions.

Forkhead Transcription Factor 3a (FOXO3a) Modulates Hypoxia Signaling via Up-regulation of the von Hippel-Lindau Gene (VHL).

FOXO3a, a member of the forkhead homeobox type O (FOXO) family of transcriptional factors, regulates cell survival in response to DNA damage, caloric restriction, and oxidative stress. The von Hippel-Lindau (VHL) tumor suppressor gene encodes a component of the E3 ubiquitin ligase complex that mediates hypoxia-inducible factor α degradation under aerobic conditions, thus acting as one of the key regulators of hypoxia signaling. However, whether FOXO3a impacts cellular hypoxia stress remains unknown. Here we show that FOXO3a directly binds to the VHL promoter and up-regulates VHL expression. Using a zebrafish model, we confirmed the up-regulation of vhl by foxo3b, an ortholog of mammalian FOXO3a Furthermore, by employing the clustered regularly interspaced short palindromic repeats (CRISPR)-associated RNA-guided endonuclease Cas9 (CRISPR/Cas9) technology, we deleted foxo3b in zebrafish and determined that expression of hypoxia-inducible genes was affected under hypoxia. Moreover, foxo3b-null zebrafish exhibited impaired acute hypoxic tolerance, resulting in death. In conclusion, our findings suggest that, by modulating hypoxia-inducible factor activity via up-regulation of VHL, FOXO3a (foxo3b) plays an important role in survival in response to hypoxic stress.

FAT1 is a novel upstream regulator of HIF1α and invasion of high grade glioma.

The hypoxic microenvironment is an important contributor of glioblastoma (GBM) aggressiveness via HIF1α, while tumour inflammation is profoundly influenced by FAT Atypical Cadherin (FAT1). This study was designed to explore the functional interaction and significance of FAT1 and HIF1α under severe hypoxia-mimicking tumour microenvironment in primary human tumours. We first identified a positive correlation of FAT1 with HIF1α and its target genes in GBM tumour specimens, revealing the significance of the FAT1-HIF1α axis in glioma cells. We found that severe hypoxia leads to an increased expression of FAT1 and HIF1α in U87MG and U373MG cells. To reveal the relevance of FAT1 under hypoxic conditions, we depleted endogenous FAT1 under hypoxia and found a substantial reduction in the expression of HIF1α and its downstream target genes like CA9, GLUT1, VEGFA, MCT4, HK2, BNIP3 and REDD1, as well as a significant reduction in the invasiveness in GBM cells. At the molecular level, under hypoxia the FAT1 depletion-associated reduction in HIF1α was due to compromised EGFR-Akt signaling as well as increased VHL-dependent proteasomal degradation of HIF1α. In brief, for the first time, these results reveal an upstream master regulatory role of FAT1 in the expression and role of HIF1α under hypoxic conditions and that FAT1-HIF1α axis controls the invasiveness of GBM. Hence, FAT1 represents a novel potential therapeutic target for GBM.

Structural Basis for Modulation of Quality Control Fate in a Marginally Stable Protein.

The human von Hippel-Lindau (VHL) tumor suppressor is a marginally stable protein previously used as a model substrate of eukaryotic refolding and degradation pathways. When expressed in the absence of its cofactors, VHL cannot fold and is quickly degraded by the quality control machinery of the cell. We combined computational methods with in vivo experiments to examine the basis of the misfolding propensity of VHL. By expressing a set of randomly mutated VHL sequences in yeast, we discovered a more stable mutant form. Subsequent modeling suggested the mutation had caused a conformational change affecting cofactor and chaperone interaction, and this hypothesis was then confirmed by additional knockout and overexpression experiments targeting a yeast cofactor homolog. These findings offer a detailed structural basis for the modulation of quality control fate in a model misfolded protein and highlight burial mode modeling as a rapid means to detect functionally important conformational changes in marginally stable globular domains.

Targeting FAK scaffold functions inhibits human renal cell carcinoma growth.

Human conventional renal cell carcinoma (CCC) remains resistant to current therapies. Focal Adhesion Kinase (FAK) is upregulated in many epithelial tumors and clearly implicated in nearly all facets of cancer. However, only few reports have assessed whether FAK may be associated with renal tumorigenesis. In this study, we investigated the potential role of FAK in the growth of human CCC using a panel of CCC cell lines expressing or not the von Hippel-Lindau (VHL) tumor suppressor gene as well as normal/tumoral renal tissue pairs. FAK was found constitutively expressed in human CCC both in culture cells and freshly harvested tumors obtained from patients. We showed that CCC cell growth was dramatically reduced in FAK-depleted cells or after FAK inhibition with various inhibitors and this effect was obtained through inhibition of cell proliferation and induction of cell apoptosis. Additionally, our results indicated that FAK knockdown decreased CCC cell migration and invasion. More importantly, depletion or pharmacological inhibition of FAK substantially inhibited tumor growth in vivo. Interestingly, investigations of the molecular mechanism revealed loss of FAK phosphorylation during renal tumorigenesis impacting multiple signaling pathways. Taken together, our findings reveal a previously uncharacterized role of FAK in CCC whereby FAK exerts oncogenic properties through a non canonical signaling pathway involving its scaffolding kinase-independent properties. Therefore, targeting the FAK scaffold may represent a promising approach for developing innovative and highly specific therapies in human CCC.

The von Hippel-Lindau tumor suppressor regulates programmed cell death 5-mediated degradation of Mdm2.

Functional loss of the von Hippel-Lindau (VHL) tumor suppressor protein (pVHL), which is part of an E3-ubiquitin ligase complex, initiates most inherited and sporadic clear-cell renal cell carcinomas (ccRCC). Genetic inactivation of the TP53 gene in ccRCC is rare, suggesting that an alternate mechanism alleviates the selective pressure for TP53 mutations in ccRCC. Here we use a zebrafish model to describe the functional consequences of pVHL loss on the p53/Mdm2 pathway. We show that p53 is stabilized in the absence of pVHL and becomes hyperstabilized upon DNA damage, which we propose is because of a novel in vivo interaction revealed between human pVHL and a negative regulator of Mdm2, the programmed cell death 5 (PDCD5) protein. PDCD5 is normally localized at the plasma membrane and in the cytoplasm. However, upon hypoxia or loss of pVHL, PDCD5 relocalizes to the nucleus, an event that is coupled to the degradation of Mdm2. Despite the subsequent hyperstabilization and normal transcriptional activity of p53, we find that zebrafish vhl(-/-) cells are still as highly resistant to DNA damage-induced cell cycle arrest and apoptosis as human ccRCC cells. We suggest this is because of a marked increase in expression of birc5a, the zebrafish homolog of Survivin. Accordingly, when we knock down Survivin in human ccRCC cells we are able to restore caspase activity in response to DNA damage. Taken together, our study describes a new mechanism for p53 stabilization through PDCD5 upon hypoxia or pVHL loss, and reveals new clinical potential for the treatment of pathobiological disorders linked to hypoxic stress.

von Hippel-Lindau exonic methylation analysis using MALDI-TOF mass spectrometry.

PURPOSE: Aberrant promoter methylation turns off gene expression and is involved in human malignancy. Studies show that first exon methylation has a tighter association with gene silencing compared to promoter methylation or gene mutation. However, to our knowledge the clinical importance of exonic methylation in renal cell carcinoma is unknown. We analyzed renal cell carcinoma for VHL gene exonic methylation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. MATERIALS AND METHODS: In 48 institutionally banked renal cell carcinoma patient tissue samples VHL exon sequencing was done as well as methylation analysis of promoter and exon 1 by mass spectrometry or conventional bisulfite analysis. Methylated human lymphocytic DNA (0% and 100%), nontemplate distilled H2O, and the UOK121 and UOK171 human renal cell carcinoma cell lines served as assay controls. Samples were considered hypermethylated if a CpG site showed greater than 50% methylation. RESULTS: Nine of the 43 patient samples read by our exon 1 assay had methylated VHL exon 1 sites, including 3 showing hypermethylation. The exon 1 methylation assay was robust and reproducible. Samples with exon 1 hypermethylation showed no exonic mutations. All samples assayed at VHL exon 2 were hypermethylated. CONCLUSIONS: To assay renal cell carcinoma tumors for VHL methylation matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is robust and reproducible, and capable of quantifying the methylation status of individual DNA bases. Exon 1 methylation may be an alternate mechanism of VHL gene silencing in renal cell carcinoma in addition to mutation and promoter methylation. Applying this assay in patient populations may allow enhanced diagnosis or tumor typing in the future.

Upregulation of miRNA-155 promotes tumour angiogenesis by targeting VHL and is associated with poor prognosis and triple-negative breast cancer.

MicroRNA-155 (miR-155) is frequently upregulated in various types of human cancer; however, its role in cancer angiogenesis remains unknown. Here, we demonstrate the role of miR-155 in angiogenesis through targeting von Hippel-Lindau (VHL) tumour suppressor in breast cancer. Ectopic expression of miR-155 induced whereas knockdown of miR-155 inhibited human umbilical vein endothelial cell network formation, proliferation, invasion and migration. Furthermore, mammary fat pad xenotransplantation of ectopically expressed miR-155 resulted in extensive angiogenesis, proliferation, tumour necrosis and recruitment of pro-inflammatory cells such as tumour-associated macrophages. Expression of VHL abrogated these miR-155 effects. Moreover, miR-155 expression inversely correlates with VHL expression level and is associated with late-stage, lymph node metastasis and poor prognosis, as well as triple-negative tumour in breast cancer. These findings indicate that miR-155 has a pivotal role in tumour angiogenesis by downregulation of VHL, and provide a basis for miR-155-expressing tumours to embody an aggressive malignant phenotype, and therefore miR-155 is an important therapeutic target in breast cancer.

The von Hippel-Lindau protein pVHL inhibits ribosome biogenesis and protein synthesis.

pVHL, the product of von Hippel-Lindau (VHL) tumor suppressor gene, functions as the substrate recognition component of an E3-ubiquitin ligase complex that targets hypoxia inducible factor α (HIF-α) for ubiquitination and degradation. Besides HIF-α, pVHL also interacts with other proteins and has multiple functions. Here, we report that pVHL inhibits ribosome biogenesis and protein synthesis. We find that pVHL associates with the 40S ribosomal protein S3 (RPS3) but does not target it for destruction. Rather, the pVHL-RPS3 association interferes with the interaction between RPS3 and RPS2. Expression of pVHL also leads to nuclear retention of pre-40S ribosomal subunits, diminishing polysomes and 18S rRNA levels. We also demonstrate that pVHL suppresses both cap-dependent and cap-independent protein synthesis. Our findings unravel a novel function of pVHL and provide insight into the regulation of ribosome biogenesis by the tumor suppressor pVHL.

The VHL tumor suppressor protein regulates tumorigenicity of U87-derived glioma stem-like cells by inhibiting the JAK/STAT signaling pathway.

The signal transducer and activator of transcription 3 (STAT3) factor plays an important role in the tumorigenicity of cancer stem cells. The purpose of this study was to investigate the inhibitory mechanism of this pathway acting through the tumor suppressor von Hippel-Lindau (VHL) protein in glioma cancer stem cells. We isolated floating neurosphere-forming CD133+ cells as glioma stem-like cells (GSLCs) by the MACS method. Furthermore, we examined these cells for their growth rate, ability to form colonies and neurospheres in soft agar, capacity for implantation into SCID mice and expression of CD133, STAT3, JAK2, Elongin A, PTEN and VHL. Furthermore, we transferred the VHL gene, an inhibitor of STAT3, into GSLCs using an adenovirus vector and compared these transfectants with control vector-transfected GSLCs. GSLCs proved to be implantable and formed a tumor in the subcutaneous tissue of SCID mice, the histology of which was similar to that of human glioblastomas. In addition, GSLCs exhibited a high capacity for soft agar colony and neurosphere formation, nearly all of which were CD133 positive. The majority of GSLCs were immunopositive for STAT3, JAK2 and Elongin A, but immunonegative for PTEN and VHL. When the VHL gene was transferred to GSLCs and these cells were transplanted into SCID mice, they did not result in tumor formation. Their capacity for soft agar colony and neurosphere formation was significantly inhibited, although their proliferation was only moderately inhibited. Regarding the expression of various factors, that of CD133 was decreased in the VHL transfectants and those of STAT3, JAK2 and Elongin A were eliminated. However, the expression of PTEN and of VHL was upregulated. These findings suggest that VHL regulated the tumorigenicity and self-renewal ability of glioma cancer stem cells by inhibiting the JAK/STAT signaling pathway.

Exposure to cigarette smoke induces overexpression of von Hippel-Lindau tumor suppressor in mouse skeletal muscle.

Cigarette smoke (CS) is a well-established risk factor in the development of chronic obstructive pulmonary disease (COPD). In contrast, the extent to which CS exposure contributes to the development of the systemic manifestations of COPD, such as skeletal muscle dysfunction and wasting, remains largely unknown. Decreased skeletal muscle capillarization has been previously reported in early stages of COPD and might play an important role in the development of COPD-associated skeletal muscle abnormalities. To investigate the effects of chronic CS exposure on skeletal muscle capillarization and exercise tolerance, a mouse model of CS exposure was used. The 129/SvJ mice were exposed to CS for 6 mo, and the expression of putative elements of the hypoxia-angiogenic signaling cascade as well as muscle capillarization were studied. Additionally, functional tests assessing exercise tolerance/endurance were performed in mice. Compared with controls, skeletal muscles from CS-exposed mice exhibited significantly enhanced expression of von Hippel-Lindau tumor suppressor (VHL), ubiquitin-conjugating enzyme E2D1 (UBE2D1), and prolyl hydroxylase-2 (PHD2). In contrast, hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression was reduced. Furthermore, reduced muscle fiber cross-sectional area, decreased skeletal muscle capillarization, and reduced exercise tolerance were also observed in CS-exposed animals. Taken together, the current results provide evidence linking chronic CS exposure and induction of VHL expression in skeletal muscles leading toward impaired hypoxia-angiogenesis signal transduction, reduced muscle fiber cross-sectional area, and decreased exercise tolerance.

Increased expression of the von Hippel-Lindau gene in the implantation site of human tubal pregnancy.

The objective of this study was to investigate the expression of the von Hippel-Lindau (VHL) gene in tissues of human fallopian tube and tubal pregnancy. Twenty patients undergoing salpingectomy for tubal pregnancy were recruited into the study group. Tissues of tubal pregnancy were separated into both the implantation and non-implantation sites as the implantation group and the non-implantation group, respectively. Samples of ampullary fallopian tube during mid-secretory phase were collected from twenty patients with benign uterine disease as the control group. Immunohistochemistry, real-time reverse transcription polymerase chain reaction, and Western blotting analysis were performed to detect expressions of VHL mRNA and protein. The results showed that VHL immunostaining appeared in the cytoplasm of tubal epithelial cells. Expression of VHL mRNA in the implantation group was higher than that in the non-implantation group or the control group (P < 0.01). Intensity of VHL protein in the implantation group was increased compared with that in the non-implantation group (P < 0.05) or in the control group (P < 0.01). There was no difference on expressions of VHL mRNA and protein between the non-implantation group and the control group (P > 0.05). In conclusion, VHL mRNA and protein are present in human tubal tissues. The VHL gene expression is increased in the implantation site of tubal pregnancy, and locally elevated expression of the VHL gene might be associated with human tubal pregnancy.

Increased expression of HIF-1A and its implication in the hypoxia pathway in primary advanced uterine cervical carcinoma.

The development of cervical cancer exhibits some unique differences compared to other solid tumors. Normal cervical stratified epithelia have characteristics of hypoxic tissue. Lack of oxygen (hypoxia) induces the HIF-1 (hypoxia-inducible factor-1) transcription factor, which is a heterodimer composed of a constitutively expressed ß subunit and a hypoxia-inducible α-subunit. HIF-1A targets the transcription of over 70 genes involved in many aspects of cancer biology. In well-oxygenated environments, the HIF-1A subunit is hydroxylated, recognized and marked for proteosomal destruction by an E3 ubiquitin ligase, the von Hippel-Lindau protein (pVHL) complex. Under hypoxic stress, proline hydroxylase (PHD) activity is diminished, and stabilized HIF-1A is involved in the activation of the tissue response to hypoxia. Here, we examined the HIF-1A and VHL transcript levels and HIF-1A protein levels in cancerous tissue (n=30) and non-cancerous, normal uterine cervical tissue (n=30). We also compared the methylation status of HIF-1A and of the VHL promoter regions in cancerous and normal tissue samples. Significantly higher levels of HIF-1A and VHL transcripts (p<0.0001 and p=0.0042, respectively) and of HIF-1A protein (p=0.0037) were detected in cancerous tissue compared to normal samples. We did not observe DNA methylation in the HIF-1A and VHL promoter region in either control or cancerous tissue samples. VHL has a functional hypoxia response element (HRE) in the promoter region, and the induction of this HRE acts within a negative feedback loop to limit the hypoxic HIF-1A response. Our findings may suggest that HIF-1A could promote its own degradation by the induction of VHL gene expression (Spearman correlation coefficient, 0.515; p=0.003). Our study shows for the first time that this increase in VHL expression could be HIF-1A-dependent and serves within a negative feedback pathway during hypoxia to regulate the cell-specific oxygen threshold for HIF-1A activation.

Reversal of experimental renovascular hypertension restores coronary microvascular function and architecture.

BACKGROUND: Hypertension (HTN) may lead to left ventricular hypertrophy and vascular dysfunction, which are independent factors for adverse cardiovascular outcomes. We hypothesized that decreased blood pressure by percutaneous transluminal renal angioplasty (PTRA) would improve the function and architecture of coronary microvessels, in association with decreased inflammation and fibrosis. METHODS: Three groups of pigs were studied: normal, HTN, and HTN+PTRA. After 6 weeks of renovascular HTN, induced by placing a local-irritant coil in the renal artery, pigs underwent PTRA or sham. Four weeks later multidetector-computed tomography (CT) was used to assess systolic, diastolic, and microvascular function, and responses to adenosine. Microvascular architecture, oxygen sensors, inflammation, and fibrosis were then explored in cardiac tissue. RESULTS: PTRA successfully decreased blood pressure and left ventricular hypertrophy. Basal fractional vascular volume (FVV) was similar among the groups, but its response to adenosine was significantly attenuated in HTN, whereas microvascular permeability (MP) and response to adenosine were greater than normal. Both were restored by PTRA. These were accompanied by increased myocardial expression of hypoxia-inducible factor (HIF)-1α, inflammation, and microvascular remodeling, including increased density of epicardial microvessels (20-200 µm), as well as cardiac diastolic dysfunction, all of which improved by reversal of HTN. However, PTRA only partially decreased myocardial fibrosis. CONCLUSIONS: Reversal of early renovascular HTN improved coronary microvascular function and architecture and reversed myocardial hypertrophy and diastolic dysfunction, in association with decreased levels of myocardial ischemia and inflammation markers, underscoring the benefits of blood pressure normalization for preservation of cardiovascular function and structure.

Hypoxia and cell cycle regulation of the von Hippel-Lindau tumor suppressor.

Inactivation of von Hippel-Lindau tumor-suppressor protein (pVHL) is associated with von Hippel-Lindau disease, an inherited cancer syndrome, as well as the majority of patients with sporadic clear cell renal cell carcinoma (RCC). Although the involvement of pVHL in oxygen sensing through targeting hypoxia-inducible factor-α subunits to ubiquitin-dependent proteolysis has been well documented, less is known about pVHL regulation under both normoxic and hypoxic conditions. We found that pVHL levels decreased in hypoxia and that hypoxia-induced cell cycle arrest is associated with pVHL expression in RCC cells. pVHL levels fluctuate during the cell cycle, paralleling cyclin B1 levels, with decreased levels in mitosis and G1. pVHL contains consensus destruction (D) box sequences, and pVHL associates with Cdh1, an activator of the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. We show that pVHL has a decreased half-life in G1, Cdh1 downregulation results in increased pVHL expression, whereas Cdh1 overexpression results in decreased pVHL expression. Taken together, these results suggest that pVHL is a novel substrate of APC/C(Cdh1). D box-independent pVHL degradation was also detected, indicating that other ubiquitin ligases are also activated for pVHL degradation.

The relationship of erythropoietin overexpression with von Hippel-Lindau tumour suppressor gene mutations between hypoxia-inducible factor-1α and -2α in sporadic clear cell renal carcinoma.

Decreased levels of von Hippel-Lindau (VHL) tumour suppressor protein are associated with up-regulation of hypoxia-inducible factor (HIF), leading to increased tumour proliferation, angiogenesis and progression. The role of erythropoietin (EPO), a target gene for HIF, remains unknown for sporadic clear cell renal cell carcinoma (sCCRCC). In this study, we determined expression levels of EPO, and its correlation with VHL mutations and HIF-1α and HIF-2α expression in 82 patients identified with sCCRCC following nephrectomy. We identified VHL gene alterations using multiplex polymerase chain reaction, purifying products of polymerase chain reaction, and direct sequencing. Immunohistochemical staining for HIF-1α, HIF-2α and EPO was performed for tumour and corresponding normal tissues. Data were analyzed with respect to clinicopathological factors. EPO was detected in 87.8% of sCCRCC tumours versus 7.3% for normal tissues. EPO expression was related to tumours demonstrating VHL gene abnormalities. Of specimens with VHL alterations 95.6% tested positive for EPO, versus 78.3% when VHL gene expression was normal (P<0.01). EPO was identified in 96.2 and 94.2% of HIF-1α and HIF-2α positive specimens, respectively, compared to 72.4 and 53.8% for HIF-1α and HIF-2α negative groups (p<0.01). Moreover, EPO expression correlated significantly with increasing nuclear grade (p<0.05). HIF-2α was identified in 84.1% of sCCRCC, compared to 64.6% for HIF-1α. Expression of HIF-1α, HIF-2α and EPO is common in sCCRCC. Although both forms of HIF up-regulate expression of EPO, the relationship to HIF-2α appears to be more pronounced. The VHL-HIF-EPO pathway requires further study, as it may represent a potential molecular target for therapy of sCCRCC.