Stabilization of hypoxia inducible factor-1α ameliorates acute renal neurogenic hypertension.

OBJECTIVES: Renal neurogenic hypertension (RNH) contributes to cardiovascular morbidity. Renal hypoxia may cause RNH and vice versa, leading to a vicious circle. Hypoxia adaptation is conferred through hypoxia-inducible factors (HIFs). We hypothesized that acute RNH is accompanied by increased renal vascular resistance (RVR) and that hypertension and increased RVR are countered by increasing HIF-1α by cobalt chloride (CoCl2) preconditioning. METHODS: First, we studied mean arterial pressure (MAP) and RVR in innervated or denervated contralateral kidneys in anesthetized rats before and after unilateral intrarenal injection of phenol, a manoeuvre known to elicit acute RNH. Then HIFα was induced by CoCl2 in drinking water (2 mM, 10 days) after which we compared intrarenal isotonic saline or phenol injection on MAP and RVR in CoCl2 preconditioned and control rats. HIF-1α was determined by immunohistochemistry. RESULTS: Unilateral intrarenal phenol induced immediate rise in MAP and contralateral RVR, and comparable HIF-1α upregulation in both kidneys, consistent with bi-renal hypoxia. Removing the phenol-injected kidney immediately normalized MAP. Contralateral renal denervation had no effect on the rise in MAP, but abrogated the contralateral increase in RVR, suggesting mediation by increased efferent nerve activity. Strong renal staining for HIF-1α confirmed efficacy of CoCl2 preconditioning, and time-dependent increase in heme oxygenase-1 gene expression stabilization of HIFα. CoCl2 preconditioning prior to phenol reduced both ΔMAP (+10 ± 2 vs. +20 ± 3%, P=0.015) and ΔRVR (+21 ± 11 vs. +90 ± 26%, P=0.003). CONCLUSION: Acute RNH leads to renal vasoconstriction and increased renal HIF-1α. Increasing HIF-1α by CoCl2 preconditioning ameliorates intrarenal phenol-induced RNH and renal vasoconstriction.

[Renal adverse reactions of antiretroviral medication: proximal tubular dysfunction associated with tenofovir]. / Renale bijwerking van antiretrovirale medicatie: proximale tubulaire disfunctie door tenofovir.

Two male patients with HIV, aged 61 and 58 years, presented with slowly progressive renal failure. Both had been treated with highly active antiretroviral therapy (HAART) for several years. Additional laboratory investigations showed hypophosphatemia, hypouricemia, metabolic acidosis and normoglycemic glucosuria. These results are an indication of proximal tubular dysfunction (PTD). Complete PTD is also known as Fanconi syndrome. Other laboratory abnormalities in Fanconi syndrome include aminoaciduria, phosphaturia and ß2-microglobulinuria. Fanconi syndrome is a side effect of tenofovir. Recent work demonstrates that up to 22% of tenofovir users display subtle abnormalities of the proximal tubule. Intracellular accumulation of tenofovir is toxic for the proximal tubular cell. This may be related to the concomitant use of ritonavir which inhibits the active transport of tenofovir from the proximal tubular cell to pre-urine. Possible consequences of prolonged PTD are osteomalacia and progressive renal failure. Medication-induced PTD should be considered in patients using antiretrovirals who develop electrolyte disorders, urinary sediment abnormalities or glucosuria.

Genomic profiling by array comparative genomic hybridization reveals novel DNA copy number changes in breast phyllodes tumours.

Breast phyllodes tumour (PT) is a rare fibroepithelial tumour. The genetic alterations contributing to its tumorigenesis are largely unknown. To identify genomic regions involved in pathogenesis and progression of PTs we obtained genome-wide copy number profiles by array comparative genomic hybridization (CGH).DNA was isolated from fresh-frozen tissue samples. 11 PTs and 3 fibroadenomas, a frequently occurring fibroepithelial breast tumour, were analyzed. Arrays composed of 2464 genomic clones were used, providing a resolution of ~1.4 Mb across the genome. Each clone contains at least one STS for linkage to the human genome sequence.No copy number changes were detected in fibroadenomas. On the other hand, 10 of 11 PT (91%) showed DNA copy number alterations. The mean number of chromosomal events in PT was 5.5 (range 0-16) per case. A mean of 2.0 gains (range 0-10) and 3.0 losses (range 0-9) was seen per case of PT. Three cases showed amplifications. DNA copy number change was not related to PT grade. We observed recurrent loss on chromosome 1q, 4p, 10, 13q, 15q, 16, 17p, 19 and X. Recurrent copy number gain was seen on 1q, 2p, 3q, 7p, 8q, 16q, 20.In this study we used array CGH for genomic profiling of fibroepithelial breast tumours. Whereas most PT showed chromosomal instability, fibroadenomas lacked copy number changes. Some copy number aberrations had not previously been associated with PT. Several well-known cancer related genes, such as TP53 and members of the Cadherin, reside within the recurrent regions of copy number alteration. Since copy number change was found in all benign PT, genomic instability may be an early event in PT genesis.

Egfr amplification specific gene expression in phyllodes tumours of the breast.

BACKGROUND: Recently, we were able to show that amplifications of the epidermal growth factor receptor (egfr) gene and the overexpression of EGFR were associated with the initiation and progression of phyllodes tumours. METHODS: In order to gain further insights into regulation mechanisms associated with egfr amplifications and EGFR expression in phyllodes tumours, we performed global gene expression analysis (Affymetrix A133.2) on a series of 10 phyllodes tumours, of these three with and seven without amplifications of an important regulatory repeat in intron 1 of egfr (CA-SSR I). The results were verified and extended by means of immunohistochemistry using the tissue microarray method on an extensively characterized series of 58 phyllodes tumours with antibodies against caveolin-1, eps15, EGF, TGF-alpha, pErk, pAkt and mdm2. RESULTS: We were able to show that the presence of egfr CA-SSR I amplifications in phyllodes tumours was associated with 230 differentially expressed genes. Caveolin-1 and eps15, involved in EGFR turnover and signalling, were regulated differentially on the RNA and protein level proportionally to egfr gene dosage. Further immunohistochemical analysis revealed that the expression of caveolin-1 and eps15 were also significantly correlated with the expression of pAkt (p<0.05), pERK (p<0.05), mdm2 (p<0.01) and EGF (p<0.001 for caveolin-1). Eps15 and pERK were further associated with tumour grade (p<0.01 and p<0.001, respectively). CONCLUSION: Our results show that amplifications within regulatory sequences of egfr are associated with the expression of eps15 and caveolin-1, indicating an increased turnover of EGFR. The interplay between EGFR and caveolin-1, eps15, pAkt, mdm2 and pERK therefore seems to present a major molecular pathway in carcinogenesis and progression of breast phyllodes tumours.

Amplifications of the epidermal growth factor receptor gene (egfr) are common in phyllodes tumors of the breast and are associated with tumor progression.

Phyllodes tumors of the breast are rare biphasic tumors with the potential for invasion and metastatic spread. An important role of the epidermal growth factor receptor (EGFR) in phyllodes tumors has been proposed. However, detailed pathogenetic mechanisms remained unclear. We investigated 58 phyllodes tumors of the breast (40 benign, 10 borderline and eight malignant) by means of egfr fluorescence in situ hybridization (FISH) and gene dosage PCR for a regulatory sequence within intron 1 of egfr. Immunohistochemical staining was performed for EGFR, p16, p21, p27, p53, c-myc, Cyclin A, Cyclin D1, Cyclin E, c-kit and Ki67. Immunopositivity for EGFR was detected in 19% of phyllodes tumors (75% of all malignant tumors) in stromal tumor cells but not in the epithelial component. Whole-gene amplifications were seen by FISH in 15.8% (in stromal cells only) and intron 1 amplifications by gene dosage PCR in as much as 41.8% of all phyllodes tumors. Significant correlations were seen between tumor grade on the one hand and EGFR overexpression (P=0.001) and intron 1 amplifications (P<0.05) on the other. EGFR overexpression further correlated positively with immunohistochemical staining for p53, p16, Cyclin A, Cyclin E, Ki67 and c-kit. Presence of intron 1 amplifications correlated with p16 (P<0.01), p21 (P=0.009) and p53 immunoreactivity (P<0.001). Neither EGFR overexpression nor whole-gene amplification was observed in a control series of 167 fibroadenomas and only one of 43 (2.3%) exhibited intron 1 amplification in gene dosage PCR. In conclusion, our results show for the first time that activating mutations in and overexpression of egfr are associated with the progression in grade of phyllodes tumors of the breast. The observed association between intron 1 amplification and overexpression of EGFR provides further insight into regulation mechanisms of EGFR overexpression.

Expression of hypoxia-inducible factor 1 alpha and its downstream targets in fibroepithelial tumors of the breast.

INTRODUCTION: Hypoxia-inducible factor 1 (HIF-1) alpha and its downstream targets carbonic anhydrase IX (CAIX) and vascular endothelial growth factor (VEGF) are key factors in the survival of proliferating tumor cells in a hypoxic microenvironment. We studied the expression and prognostic relevance of HIF-1alpha and its downstream targets in phyllodes tumors and fibroadenomas of the breast. METHODS: The expression of HIF-1alpha, CAIX, VEGF and p53 was investigated by immunohistochemistry in a group of 37 primary phyllodes tumors and 30 fibroadenomas with known clinical follow-up. The tumor microvasculature was visualized by immunohistochemistry for CD31. Proliferation was assessed by Ki67 immunostaining and mitotic counts. Being biphasic tumors, immunoquantification was performed in the stroma and epithelium. RESULTS: Only two fibroadenomas displayed low-level stromal HIF-1alpha reactivity in the absence of CAIX expression. Stromal HIF-1alpha expression was positively correlated with phyllodes tumor grade (P = 0.001), with proliferation as measured by Ki67 expression (P < 0.001) and number of mitoses (P < 0.001), with p53 accumulation (P = 0.003), and with global (P = 0.015) and hot-spot (P = 0.031) microvessel counts, but not with CAIX expression. Interestingly, concerted CAIX and HIF-1alpha expression was frequently found in morphologically normal epithelium of phyllodes tumors. The distance from the epithelium to the nearest microvessels was higher in phyllodes tumors as compared with in fibroadenomas. Microvessel counts as such did not differ between fibroadenomas and phyllodes tumors, however. High expression of VEGF was regularly found in both tumors, with only a positive relation between stromal VEGF and grade in phyllodes tumors (P = 0.016). Stromal HIF-1alpha overexpression in phyllodes tumors was predictive of disease-free survival (P = 0.032). CONCLUSION: These results indicate that HIF-1alpha expression is associated with diminished disease-free survival and may play an important role in stromal progression of breast phyllodes tumors. In view of the absence of stromal CAIX expression in phyllodes tumors, stromal upregulation of HIF-1alpha most probably arises from hypoxia-independent pathways, with p53 inactivation as one possible cause. In contrast, coexpression of HIF-1alpha and CAIX in the epithelium in phyllodes tumors points to epithelial hypoxia, most probably caused by relatively distant blood vessels. On the other hand, HIF-1alpha and CAIX seem to be of minor relevance in breast fibroadenomas.

Progressive deregulation of the cell cycle with higher tumor grade in the stroma of breast phyllodes tumors.

We studied cell cycle-regulating proteins in phyllodes tumor pathogenesis by immunohistochemical analysis for Ki-67, cyclin A, cyclin D1, retinoblastoma protein (pRb), p53, p16INK4A, bcl-2, and p21waf1 in the epithelium and stroma of 40 primary (benign, 21; borderline, 8; malignant, 11) and 7 recurrent tumors of different grades. In most cases, the epithelium showed no altered expression of cell cycle regulators. Stromal overexpression of p16INK4A, p53, cyclin A, pRb, and p21waf1 correlated significantly with tumor grade. The number of altered proteins in stroma increased with higher grade and was accompanied by increased proliferation. Stromal cyclin A expression was the best separating marker between tumor grades. Correlations existed between stromal overexpression of p16NK4A and p21waf1, p16INK4A and p53, and p53 and pRb. No immunostaining differences were detected between primary tumors and recurrences. Four or more altered proteins and p53 expression in the stromal component were independent negative prognosticators for disease-free survival. The stromal component of mammary phyllodes tumors displays an increasing level of cell cycle deregulation with higher tumor grade; the epithelial compartment mostly remains inconspicuous. Several combinations of aberrantly expressed cell cycle proteins seem important in the stromal progression of phyllodes tumors. The number of stromal cell cycle aberrations and stromal p53 expression might predict clinical behavior.

Analysis of the progression of fibroepithelial tumours of the breast by PCR-based clonality assay.

Fibroadenoma and phyllodes tumour of the breast are both fibroepithelial tumours. Although progression to epithelial malignancy has been described, the behaviour of most fibroadenomas is benign. Phyllodes tumours, on the other hand, can display locally destructive growth and can even metastasize. A relationship between the two tumours has been suggested in the literature. This study investigated the clonality of both the stroma and the epithelium of these fibroepithelial tumours and attempted to construct a model in which fibroadenoma can progress in both an epithelial and a stromal direction. Fibroadenomas (n=25) and phyllodes tumours (n=12) were selected for analysis. Tissue was microdissected and analysed for clonality using a polymerase chain reaction (PCR)-based assay targeted at an X-linked polymorphic marker, the human androgen receptor gene (HUMARA). Nineteen fibroadenomas and nine phyllodes tumours could be analysed. Normal-appearing epithelium, hyperplastic epithelium, and stroma removed from fibroadenomas were polyclonal. As expected, carcinoma in situ (CIS) removed from four fibroadenomas was monoclonal. Three areas of apparent stromal expansion within fibroadenoma were monoclonal, suggesting stromal progression. Mostly, the stroma of phyllodes tumours was monoclonal and the epithelium polyclonal. In two cases, however, the epithelium seemed to be monoclonal, whereas in three other cases the stromal component was polyclonal. These findings indicate that fibroadenoma can progress in an epithelial direction to CIS and in a stromal direction to phyllodes tumour.