BACKGROUND:This study examines the impact of dietary fatty acids on regulation of gene expression in mammary epithelial cells before and during puberty.METHODS:Diets primarily consisted of n-9 monounsaturated fatty acids (olive oil), n-6 polyunsaturated fatty acids (safflower), saturated acids (butter), and the reference AIN-93G diet (soy oil). The dietary regimen mimics the repetitive nature of fatty acid exposure in Western diets. Diet-induced changes in gene expression were examined in laser capture microdissected mammary ductal epithelial cells at day of weaning and end of puberty. PCNA immunohistochemistry analysis compared proliferation rates between diets.RESULTS:Genes differentially expressed between each test diets and the reference diet were significantly enriched by cell cycle genes. Some of these genes were involved in activation of the cell cycle pathway or the G2/M check point pathway. Although there were some differences in the level of differential expression, all diets showed qualitatively the same pattern of differential expression compared to the reference diet. Cluster analysis identified an expanded set of cell cycle as well as immunity and sterol metabolism related clusters of differentially expressed genes.CONCLUSION:Fatty acid-enriched diets significantly upregulated proliferation above normal physiological levels during puberty. Higher cellular proliferation during puberty caused by enriched fatty acid diets poses a potential increase risk of mammary cancer in later life. The human homologs of 27 of 62 cell cycle rat genes are included in a human breast cancer cluster of 45 cell cycle genes, further emphasizing the importance of our findings in the rat model.

BACKGROUND Haemodialysis vascular access dysfunction (due to venous stenosis and thrombosis) is a leading cause of hospitalization and morbidity. The aim of the current study was to identify the specific cell types present within stenotic tissue samples from patients with AV fistula and graft failure. METHODS Discarded tissue segments were collected from the stenotic portions (usually near the graft-vein anastomosis or the AV anastomosis) of 23 dialysis grafts and 20 AV fistulae, and examined for expression of smooth muscle alpha actin, desmin, vimentin and a macrophage marker. RESULTS The majority of cells within the venous neointima (both grafts and fistulae) were myofibroblasts, with a smaller number of desmin positive smooth muscle cells. The graft neointima had a similar cellular phenotype, albeit without any desmin positive contractile smooth muscle cells. The majority of cells within the PTFE graft material were macrophages. Analysis of sequential sections revealed the presence of fibroblasts within the venous neointima and intragraft region. CONCLUSIONS Our results demonstrate that contractile smooth muscle cells, myofibroblasts, fibroblasts and macrophages all play a role in the pathogenesis of dialysis access dysfunction (grafts and fistulae). Targeting these specific cell types might result in the development of novel therapeutic paradigms for haemodialysis vascular access dysfunction.

Developmental stages of mammary glands influence their susceptibility to initiating events related to carcinogenesis. The "window of susceptibility" to mammary carcinogenesis is classically defined as the time in early puberty when the mammary gland morphology is most sensitive to initiation events. Administration of the polyaromatic hydrocarbon, 7,12-dimethylbenz(a)anthracene (DMBA), in a single oral dose yields maximal mammary tumor formation when administered in this "window". We examined the DMBA treated mammary glands, precursor lesions, and morphology of the uninvolved mammary epithelium for the first 100 days of life for Charles River Sprague Dawley CD(R) IGS. Our goal was to determine the DMBA dose at which 50% of the rats (IC50) developed carcinoma in situ (CIS) within three months of dosing. Here we demonstrate, rather than the classical U-shaped dose curve in which there is maximum sensitivity for DMBA at 50 days, there is an increasing degree of sensitivity with age in the CD(R) IGS rat. Additionally, we report that vehicle-treated animals developed mammary CIS without any known initiator, and 100 day virgin animals demonstrated lactational changes, independent of DMBA exposure or dose. Lastly, we demonstrate this strain of virgin female rats has elevated pituitary prolactin immunoreactivity independent of the level of mammary differentiation. We conclude this strain of Charles River Sprague Dawley rats has prolactin-induced pituitary stimulation, and therefore, the window of susceptibility for mammary tumorigenesis is absent.

Hemodialysis vascular access dysfunction as a result of venous neointimal hyperplasia in dialysis access grafts and fistulae is currently a huge clinical problem. The aim of this study was to assess the effects of paclitaxel and radiation, both singly and in combination on the proliferation of cell types present within the lesion of venous neointimal hyperplasia (vascular smooth muscle cells, fibroblasts and endothelial cells within the neointimal microvessels). Vascular smooth muscle cells, fibroblasts and endothelial cells were plated onto 96-well plates and exposed to different concentrations and doses of paclitaxel and radiation, respectively (both individually and in combination). Growth inhibition was assessed with an MTT assay. Both paclitaxel and radiation resulted in significant growth inhibition of all three cell types. However, even small doses of paclitaxel appeared to attenuate the antiproliferative effect of radiation on these cell types. Further experiments to elucidate the mechanism behind these findings could result in a better understanding of combination antiproliferative therapies.

Decades of experimentation on angiotensin and bradykinin have focused on macrovascular systemic effects. However, angiotensin II and bradykinin are both angiogenic agents, highlighting their ability to also effect the microvascular circulation. Not surprisingly, inhibition of angiotensin converting enzyme, which inhibits angiotensin II synthesis and bradykinin degradation, would have different impacts on angiogenesis in vivo dependent upon what factors were present in the system. Several pathological states in which angiogenesis is important, including peripheral ischemia, stroke, retinopathy, and cancer are examined in this review with respect to activity of angiotensin II and bradykinin and the impact of angiotensin converting enzyme inhibition. Although generalizations are not without legitimate criticism, one can think about peripheral ischemia and stroke as being more dependent upon bradykinin signaling and retinopathy and cancer as more dependent upon angiotensin II signaling to drive angiogenesis. Many exceptions are found that are specific to individual animal model systems. Furthermore, cancer systems that have been examined at any depth are few. However, published data on in vitro cultures and animal models present interesting predictions about how the renin angiotensin and bradykinin systems may function in humans. Since angiotensin converting enzyme inhibitors have been widely utilized pharmaceuticals for many years, we are now accumulating epidemiological data that test our predictions. The importance of understanding which agent, angiotensin and/or bradykinin, appears to be the more important regulator of angiogenesis in a given pathology will become increasing evident as more specific angiotensin II and bradykinin receptor blocking drugs make their way into clinical use.

There is significant evidence that both angiotensin I converting enzyme inhibitors (ACEI) and type 1 and type 2 angiotensin 2 (A2) receptor blockers may inhibit tumor growth. The finding is supported by many reports where these two classes of drugs showed cytostatic effects on the cultures of several lines of both normal and neoplastic cells. These drugs often transformed the cellular biochemical structures, especially in neoplastic cell lines. The same drugs also delayed the growth of different types of tumors in a variety of experimental animals (breast and lung carcinoma in mice; sarcomas, squamous cell carcinomas and hepatocellular carcinomas in rats), and there are a few reports of successful treatment of a limited number of cases of Kaposi sarcoma and gliomas with these drugs. Retrospective studies in hypertensive subjects treated with ACEI or A2 receptor blockers also seem to indicate that the incidence and growth of different neoplasms was delayed when these patients were compared to hypertensive patients receiving alternate medications. There is strong indication that the pharmacologic effect of these drugs may be exerted by reduction or inhibition of the synthesis of angiotensin 2. A2 is a powerful mitogen and its effect on cellular growth is exerted through stimulation of many factors, including transforming growth factor beta (TGFbeta), epidermal growth factor (EGF), smooth muscle actin (SMA), and tyrosine kinase. A2 also regulates apoptotic mechanisms and angiogenesis. The pharmacologic action of most of these drugs, however, is not necessarily limited to downregulaton of A2. Many ACEI, especially those containing the sulfhydryl (SH group), possess antioxidant or metalloprotease inhibitory properties per se. These experimental and retrospective data justify clinical testing of these drugs in appropriate randomized trials. Several such trials are currently in process. If these trials confirm the experimental and retrospective studies, these agents will provide a significant contribution to the therapeutic treatment of many malignancies in humans.

Hemodialysis vascular access dysfunction (HVAD) is currently a huge clinical problem. The major cause of HVAD is venous stenosis (as a result of venous neointimal hyperplasia) which leads to thrombosis in polytetrafluoroethylene dialysis access grafts and fistulae. Despite the magnitude of the clinical problem there are currently no effective therapeutic interventions for this condition. In an attempt to reduce the morbidity associated with HVAD, we have developed and validated a local perivascular paclitaxel release system for use in a pig model of arteriovenous graft stenosis. Ethylene vinyl acetate polymers with 5% paclitaxel were formulated. The release profile of paclitaxel was then manipulated to maximize its biological impact in the in vivo situation. In vitro experiments were performed to confirm that the paclitaxel released from the polymer was biologically active against cell types that were similar to those present in the in vivo lesion of neointimal hyperplasia. Our results demonstrate that the paclitaxel polymer wraps which we have developed are mechanically stable with a burst release phase followed by a slower continuous release phase. The paclitaxel released from these polymeric wraps retains its physicochemical and biological properties and is able to inhibit the proliferation of smooth muscle cells, endothelial cells and fibroblasts in vitro. We believe that these paclitaxel-loaded polymeric wraps could be ideally suited for perivascular drug delivery in the context of dialysis access grafts and fistulae.

Obesity is an increasing worldwide epidemic. With an increased prevalence of obesity there is an increase in obesity-related cancers, such as breast cancer. Although reproductive and lifestyle choices are among the best-recognized risk factors for breast cancer, few of these can be modified readily by the individual. Obesity is unlike these other risk factors since it can be modified and controlled. Breast cancer prognosis is worse in patients who are obese, and epidemiological data suggests that obesity is a significant risk factor for postmenopausal breast cancer. Addressing the obesity epidemic, at both an individual and public health level, is expected to have a significant impact on breast cancer incidence and mortality.

BACKGROUND:Haemodialysis vascular access dysfunction is currently a huge clinical problem. In an attempt to reduce the morbidity associated with haemodialysis vascular access dysfunction, we have previously developed and validated a local perivascular paclitaxel release system that has been shown to release paclitaxel for at least 3 weeks. The aim of the current study was to evaluate the in vivo use of these perivascular wraps (for both safety and efficacy) at different time points in our pig model of arteriovenous graft stenosis.METHODS:Paclitaxel-loaded ethylene vinyl acetate wraps were placed around the graft-vein anastomosis on one side, with control polymers being placed on the contralateral side in our pig model of arteriovenous graft stenosis. Animals were sacrificed at early (10-11 days), middle (23-24 days) and late (32-38 days) time points. The entire graft-vein anastomosis was removed at the time of sacrifice and assessed for the extent of luminal stenosis using histomorphometric techniques.RESULT:Graft-vein anastomoses treated with the paclitaxel-loaded polymers had an almost complete absence of luminal stenosis at the middle (23-24 days) and late (32-38 days) time points (when one would expect the development of neointimal hyperplasia) as compared with the contralateral control graft-vein anastomoses (37.90% luminal stenosis in the controls vs 0.10% in the paclitaxel group). There were minimal local side effects from this procedure.CONCLUSIONS:Our results demonstrate the safety and efficacy of paclitaxel-loaded perivascular wraps in the setting of a pig model of arteriovenous graft stenosis. We believe that such a local approach which could be easily applied at the time of surgery is ideally suited for use in the clinical setting of haemodialysis vascular access dysfunction. It is likely that this novel approach could result in a significant reduction in the huge economic and health morbidity costs currently associated with this recalcitrant clinical problem.

Hormone responsive breast cancer is usually determined by the presence of estrogen receptors (ER) or progesterone receptors (PR) on primary invasive breast cancers. Adjuvant and metastatic hormone therapy are recommended based on primary ER and PR determination. Little information is available to determine if primary hormone receptors correlate with metastatic disease and if survival is influenced by metastatic receptor status. We retrospectively compared primary to metastatic tumor ER and PR content from 200 metastatic breast cancer patients. ER and PR analyses were available in both primary and metastatic disease in 200 and 173 patients, respectively. There was a correlation between both the ER and PR in the primary and metastatic lesion (p < 0.001). However, in 60 of 200 (30%) patients, discordance between primary and metastatic ER was noted. Tumors from 68 of 173 (39.3%) showed discordance for PR. In 39 (19.5%) patients, the ER primary status was positive and metastatic status was negative and in 21 (10.5%) patients, the primary status was negative and metastatic status was positive. Survival from the time of metastatic diagnosis was calculated. Those patients with ER positive primary and metastatic tumors (Positive/Positive) or only the metastatic lesion (Negative/Positive) had similar median survival (1131 and 1111 days, respectively). However, patients with tumors that changed from positive primary to negative metastasis (Positive/Negative) experienced significantly shorter median survival (669 days, p < 0.05). Likewise, median survival (580 days) was significantly shorter for patients with primary and metastasis ER negative (Negative/Negative, p < 0.001) compared to Positive/Positive (p < 0.001) or compared to Negative/Positive (p < 0.02). The changes in PR status were not associated with a change in survival. We found a significant discordance between hormone receptor content of primary versus metastatic breast cancer. The ER status of the metastatic lesion was a better predictor of survival. Therefore, optimal metastatic treatment cannot be determined solely on primary ER and PR analysis.

Hemodialysis vascular access dysfunction is a major cause of morbidity in the hemodialysis population and contributes significantly to the overall cost of end-stage renal disease programs. At a histological level, most hemodialysis vascular access dysfunction (in both native arteriovenous fistulae and PTFE dialysis access grafts) is due to venous stenosis and thrombosis, secondary to venous neointimal hyperplasia. However, despite a wealth of experimental and clinical data on the use of novel therapeutic interventions that target neointimal hyperplasia in the setting of coronary artery disease, there are unfortunately no effective therapeutic interventions for hemodialysis vascular access dysfunction at the present time. This is particularly unfortunate, since neointimal hyperplasia in the setting of hemodialysis vascular access fistulae and grafts could be the ideal clinical model to test novel therapeutic interventions for neointimal hyperplasia.

This article (1) identifies the types of hemodialysis access, (2) summarizes the clinical standard of care for dialysis access grafts and fistulae, (3) describes the pathology and pathogenesis of venous stenosis in dialysis access grafts and fistulae, (4) tabulates avail-able therapies for hemodialysis vascular access dysfunction and speculates on the rea-sons for the lack of effective therapies, and (5) discusses the development and application of novel therapeutic interventions for this difficult clinical problem. The possibility that dialysis access grafts and fistulae could be the ideal clinical model for testing novel local therapies to block neointimal hyperplasia is discussed.

Preinvasive mammary pathologies in humans and rat chemical carcinogenesis model systems have an increased microvascular density relative to normal tissue. This suggests the possibility of preventing invasive breast cancer by inhibiting angiogenesis. Vascular endothelial cell growth factor (VEGF) is a potent angiogenic growth factor, commonly involved in tumor-induced angiogenesis. Here, we show that both VEGF and VEGFR2 expression increase with histological progression to invasive disease in the rat 7,12-dimethylbenz[a]anthracene (DMBA) model. Other VEGF receptors, VEGFR1, neuropilin 1 and neuropilin 2, are constitutively expressed throughout progression. To examine whether VEGF signaling is functionally relevant to tumor-induced endothelial tubule formation in vitro and for tumor formation in vivo, we utilized the VEGFR2 inhibitor, ZD6474. In vitro endothelial cell tubulogenesis induced by isolated mammary organoids or carcinoma in situ from DMBA-treated rats is inhibited by ZD6474, in a dose-dependent fashion. The administration of ZD6474 to DMBA-treated rats inhibits the formation of atypical ductal hyperplasia and carcinoma in situ by greater than 95% (P < 0.05), when administered 1 week or 6 weeks post-DMBA initiation. Invasive disease was absent in all ZD6474 cohorts. These data support the hypothesis that progression of DMBA-induced preinvasive mammary pathologies to palpable disease requires angiogenesis via a VEGF-dependent mechanism.

Objective: In the 7,12-dimethylbenz[a]anthracene (DMBA) model of rat mammary carcinogenesis, microvascular density and angiogenic potential increase with progression from normal to invasive disease, but the mechanisms involved are unknown. Using RT-PCR, we determined the expression of angiogenic regulators in DMBA-induced intraductal hyperplasia (IDP), carcinoma in situ (CIS), invasive tumors (INV), as well as normal tissue. Methods: RT-PCR was performed on frozen tissue sections of each type of pathology for factors known to regulate angiogenesis in other systems. Results: MMP-2, MMP-9, uPA, PAI-1, IGF-2, BFGF, VEGF, ANG-1, IRS-1, and TSP-1 were significantly (p ≤ 0.05) upregulated in CIS and INV, whereas TIMP-1, ANG-2, MASPIN, IGF1-R and HBEGF were unchanged. IGF-1 was uniquely elevated in IDP. SPARC was downregulated in CIS. Inhibition of IGF-1R by the tyrphostin, AG1024, blocked endothelial tubulogenesis in vitro, confirming that IGF-1 functions as a regulator of angiogenesis. Conclusions: These data support the involvement of specific angiogenic mediators in mammary tumor formation. Angiogenesis at different stages of tumorigenesis may be regulated by unique factors.

Hemodialysis vascular access dysfunction is a major cause of morbidity in the hemodialysis population and contributes significantly to the overall cost of end-stage renal disease programs. At a histological level, most hemodialysis vascular access dysfunction (in both native arteriovenous fistulae and PTFE dialysis access grafts) is due to venous stenosis and thrombosis, secondary to venous neointimal hyperplasia. However, despite a wealth of experimental and clinical data on the use of novel therapeutic interventions that target neointimal hyperplasia in the setting of coronary artery disease, there are unfortunately no effective therapeutic interventions for hemodialysis vascular access dysfunction at the present time. This is particularly unfortunate, since neointimal hyperplasia in the setting of hemodialysis vascular access fistulae and grafts could be the ideal clinical model to test novel therapeutic interventions for neointimal hyperplasia.