Functional Antagonism of Junctional Adhesion Molecule-A (JAM-A), Overexpressed in Breast Ductal Carcinoma In Situ (DCIS), Reduces HER2-Positive Tumor Progression.

Breast ductal carcinoma in situ (DCIS) is clinically challenging, featuring high diagnosis rates and few targeted therapies. Expression/signaling from junctional adhesion molecule-A (JAM-A) has been linked to poor prognosis in invasive breast cancers, but its role in DCIS is unknown. Since progression from DCIS to invasive cancer has been linked with overexpression of the human epidermal growth factor receptor-2 (HER2), and JAM-A regulates HER2 expression, we evaluated JAM-A as a therapeutic target in DCIS. JAM-A expression was immunohistochemically assessed in patient DCIS tissues. A novel JAM-A antagonist (JBS2) was designed and tested alone/in combination with the HER2 kinase inhibitor lapatinib, using SUM-225 cells in vitro and in vivo as validated DCIS models. Murine tumors were proteomically analyzed. JAM-A expression was moderate/high in 96% of DCIS patient tissues, versus 23% of normal adjacent tissues. JBS2 bound to recombinant JAM-A, inhibiting cell viability in SUM-225 cells and a primary DCIS culture in vitro and in a chick embryo xenograft model. JBS2 reduced tumor progression in in vivo models of SUM-225 cells engrafted into mammary fat pads or directly injected into the mammary ducts of NOD-SCID mice. Preliminary proteomic analysis revealed alterations in angiogenic and apoptotic pathways. High JAM-A expression in aggressive DCIS lesions and their sensitivity to treatment by a novel JAM-A antagonist support the viability of testing JAM-A as a novel therapeutic target in DCIS.

Leydig Cell Hyperplasia Mimicking a Testicular Tumour in a Patient with Klinefelter Syndrome.

BACKGROUND: Klinefelter syndrome (KS) is the most common sex-chromosomal disorder in males. Frequently under-recognized, it occurs in 1 in 500-600 male births. It is caused by the inheritance of at least one additional X chromosome from either parent. Patients often have uncommon or atypical malignancies. PATIENT: We describe the case of a 35-year-old man with 47XXY KS and previous cryptorchidism, presenting with a painful testicular mass. Histology confirmed Leydig cell hyperplasia. DISCUSSION: Cryptorchidism is an established risk factor for testicular tumours and occurs six times more commonly in KS than in the general population. Despite this, large epidemiological studies have shown a reduced burden of testicular cancer in these patients. The presentation of a hypoechoic lesion on ultrasound will prompt consideration of testicular tumours, however orchalgia represents an atypical presentation. In patients with KS, Leydig cell hyperplasia is a much more common entity and should be considered early in the differential diagnosis. LEARNING POINTS: The differential diagnosis of a testicular mass in Klinefelter syndrome includes malignancy and nodular Leydig cell hyperplasia.Diagnosis can be challenging, both radiologically and histologically.Orchalgia is atypical in Leydig cell hyperplasia.

Marked hyperandrogenicity in a 60-year-old woman.

Markedly elevated androgen levels can lead to clinical virilization in females. Clinical features of virilization in a female patient, in association with biochemical hyperandrogenism, should prompt a search for an androgen-producing tumor, especially of ovarian or adrenal origin. We herein report the case of a 60-year-old woman of Pakistani origin who presented with the incidental finding of male pattern baldness and hirsutism. Her serum testosterone level was markedly elevated at 21 nmol/L (normal range: 0.4-1.7 nmol/L), while her DHEAS level was normal, indicating a likely ovarian source of her elevated testosterone. Subsequently, a CT abdomen-pelvis was performed, which revealed a bulky right ovary, confirmed on MRI of the pelvis as an enlarged right ovary, measuring 2.9 × 2.2 cm transaxially. A laparoscopic bilateral salpingo-oophorectomy was performed, and histopathological examination and immunohistochemistry confirmed the diagnosis of a Leydig cell tumor, a rare tumor accounting for 0.1% of ovarian tumors. Surgical resection led to normalization of testosterone levels. LEARNING POINTS: Hirsutism in postmenopausal women should trigger suspicion of androgen-secreting tumorExtremely elevated testosterone level plus normal DHEAS level point toward ovarian sourceLeydig cell tumor is extremely rare cause of hyperandrogenicity.

High-throughput oncogene mutation profiling shows demographic differences in BRAF mutation rates among melanoma patients.

Because of advances in targeted therapies, the clinical evaluation of cutaneous melanoma is increasingly based on a combination of traditional histopathology and molecular pathology. Therefore, it is necessary to expand our knowledge of the molecular events that accompany the development and progression of melanoma to optimize clinical management. The central objective of this study was to increase our knowledge of the mutational events that complement melanoma progression. High-throughput genotyping was adapted to query 159 known single nucleotide mutations in 33 cancer-related genes across two melanoma cohorts from Ireland (n=94) and Belgium (n=60). Results were correlated with various clinicopathological characteristics. A total of 23 mutations in 12 genes were identified, that is--BRAF, NRAS, MET, PHLPP2, PIK3R1, IDH1, KIT, STK11, CTNNB1, JAK2, ALK, and GNAS. Unexpectedly, we discovered significant differences in BRAF, MET, and PIK3R1 mutations between the cohorts. That is, cases from Ireland showed significantly lower (P<0.001) BRAF(V600E) mutation rates (19%) compared with the mutation frequency observed in Belgian patients (43%). Moreover, MET mutations were detected in 12% of Irish cases, whereas none of the Belgian patients harbored these mutations, and Irish patients significantly more often (P=0.027) had PIK3R1-mutant (33%) melanoma versus 17% of Belgian cases. The low incidence of BRAF(V600E)(-) mutant melanoma among Irish patients was confirmed in five independent Irish cohorts, and in total, only 165 of 689 (24%) Irish cases carried mutant BRAF(V600E). Together, our data show that melanoma-driving mutations vary by demographic area, which has important implications for the clinical management of this disease.

Effects of endothelin-1 on endothelial progenitor cell function.

BACKGROUND: Circulating endothelial progenitor cells (EPCs) contribute to vascular endothelial repair. Endothelin (ET)-1 is associated with endothelial damage and atherogenesis. The experimental aim of this study was to determine, in vitro, the effects of ET-1 on the ability of EPCs to form colonies, migrate, release angiogenic growth factors and resist apoptosis. METHODS: Peripheral blood samples were collected from 10 healthy adult humans. Cells with phenotypic EPC characteristics were isolated and EPC colony-forming capacity (CFU assay), migratory activity (Boyden chamber), release of angiogenic growth factors (enzyme immunoassay) and apoptosis (TUNEL assay) were determined in the absence and presence of ET-1 (100 pmol). RESULTS: EPC colony-forming units (42±12 vs. 39±11), migratory capacity (910±146 vs. 936±148 AU) and release of vascular endothelial growth factor (202.8±68.1 vs. 204.8±69.8 pg/mL) and granulocyte-colony stimulating factor (1294.4±378.3 vs. 1136.1±310.3 pg/mL) were not significantly affected by ET-1. EPCs treated with ET-1 demonstrated a 20% increase (p<0.05) in cellular apoptosis. The proapoptotic effect of ET-1 was abolished with ET receptor blockade as well as with apocynin, a nicotinamide adenine dinucleotide phosphate (NADPH) inhibitor. CONCLUSIONS: These results indicate that ET-1 does not affect EPC colony formation, migratory capacity or angiogenic growth factor release, but does increase EPC susceptibility to apoptosis through an NADPH-dependent mechanism. Increased EPC apoptosis may contribute to the proatherogenic effects of ET-1.

Habitual short sleep duration and circulating endothelial progenitor cells.

Chronic short sleep duration has been linked to endothelial dysfunction and increased risk of cardiovascular disease. Circulating endothelial progenitor cells (EPCs) are vital to endogenous vascular repair processes and cardiovascular health. We tested the hypothesis that habitual short sleep duration is associated with impairment in EPC number and function. Cells with phenotypic EPC characteristics were isolated from 37 healthy, sedentary adults: 20 with normal sleep duration (13M/7F; age: 59±1 years; sleep duration: 7.7±0.1 h/night) and 17 with short sleep duration (9M/8F; 56±2 years; 6.0±0.2 h/night). EPC number was assessed by flow cytometric analysis of the percentage of peripheral blood mononuclear cells negative for CD45 and positive for CD34, VEGFR-2, and CD133 antigens. EPC colony-forming capacity was determined by colony-forming unit (CFU) assay; migration by Boyden chamber; and intracellular caspase-3 concentrations by immunoassay. There were no significant differences between groups in EPC number (0.001±0.0004 vs. 0.001±0.0003 %), colony-forming capacity (6.1±1.5 vs. 5.4±1.7 CFUs), or migration to VEGF (1410.1±151.2 vs. 1334.3±111.1 AU). Furthermore, there were no group differences in basal and staurosporine-stimulated intracellular concentrations of active caspase-3 (0.3±0.03 vs. 0.5±0.1 ng/mL; and 2.9±0.4 vs. 2.7±0.3 ng/mL), a marker of apoptotic susceptibility. Taken together, these data indicate that short sleep duration is not associated with EPC dysfunction in healthy adults. Numerical and functional impairment in circulating EPCs may not contribute to the increased cardiovascular risk with habitual short sleep duration.

Aging is associated with a proapoptotic endothelial progenitor cell phenotype.

The aim of this study was to determine if aging is associated with enhanced endothelial progenitor cell (EPC) sensitivity to apoptosis. Cells with phenotypic EPC characteristics were isolated from healthy, nonobese young (age 25 ± 1 years) and older (61 ± 1 years) men. Intracellular active caspase-3 concentrations in response to staurosporine stimulation were approximately 35% higher (p < 0.05) in EPCs from older (3.15 ± 0.29 pg/ml) compared with young (2.33 ± 0.24 pg/ml) men. Protein expression of Akt, p70 S6-kinase and Bcl-2 was markedly lower (approx. 35, 75 and 60%, respectively, all p < 0.05) in EPCs from older compared with young men, whereas there were no age-related differences in either 14-3-3ε or Bax expression. Additionally, EPC telomerase activity was 57% lower (p < 0.05) in older (0.18 ± 0.11 AU) versus young (0.43 ± 0.11 AU) men. These results indicate that aging is associated with a proapoptotic EPC phenotype characterized by decreased expression of key antiapoptotic proteins associated with the PI-3-kinase signaling pathway and reduced telomerase activity. These age-related changes likely contribute, in part, to the diminished ability of EPCs to resist an apoptotic stimulus in older men. Increased susceptibility to apoptosis may contribute to the numerical and functional impairments observed in EPCs with aging.

Human aging and CD31+ T-cell number, migration, apoptotic susceptibility, and telomere length.

CD31(+) T cells, or so-called "angiogenic T cells," have been shown to demonstrate vasculoprotective and neovasculogenic qualities. The influence of age on CD31(+) T-cell number and function is unclear. We tested the hypothesis that circulating CD31(+) T-cell number and migratory capacity are reduced, apoptotic susceptibility is heightened, and telomere length is shortened with advancing age in adult humans. Thirty-six healthy, sedentary men were studied: 12 young (25 ± 1 yr), 12 middle aged (46 ± 1 yr), and 12 older (64 ± 2 yr). CD31(+) T cells were isolated from peripheral blood samples by magnetic-activated cell sorting. The number of circulating CD31(+) T cells (fluorescence-activated cell sorting analysis) was lower (P < 0.01) in older (24% of CD3(+) cells) compared with middle-aged (38% of CD3(+) cells) and young (40% of CD3(+) cells) men. Migration (Boyden chamber) to both VEGF and stromal cell-derived factor-1α was markedly blunted (P < 0.05) in cells harvested from middle-aged [306.1 ± 45 and 305.6 ± 46 arbitrary units (AU), respectively] and older (231 ± 65 and 235 ± 62 AU, respectively) compared with young (525 ± 60 and 570 ± 62 AU, respectively) men. CD31(+) T cells from middle-aged and older men demonstrated greater apoptotic susceptibility, as staurosporine-stimulated intracellular caspase-3 activation was ∼ 40% higher (P < 0.05) than young. There was a progressive age-related decline in CD31(+) T-cell telomere length (young: 10,706 ± 220 bp; middle-aged: 10,179 ± 251 bp; and older: 9,324 ± 192 bp). Numerical and functional impairments in this unique T-cell subpopulation may contribute to diminished angiogenic potential and greater cardiovascular risk with advancing age.

Endothelial progenitor cell function, apoptosis, and telomere length in overweight/obese humans.

Excess adiposity is associated with increased cardiovascular morbidity and mortality. Endothelial progenitor cells (EPCs) play an important role in vascular repair. We tested the hypothesis that increased adiposity is associated with EPC dysfunction, characterized by diminished capacity to release angiogenic cytokines, increased apoptotic susceptibility, reduced cell migration, and shorter telomere length. A total of 67 middle-aged and older adults (42-67 years) were studied: 25 normal weight (normal weight; BMI: 18.5-24.9 kg/m(2)) and 42 overweight/obese (overweight/obese; BMI: 25.0-34.9 kg/m(2)). Cells with phenotypic EPC characteristics were isolated from peripheral blood. EPC release of vascular endothelial growth factor (VEGF) and granulocyte colony-stimulating factor (G-CSF) was determined in the absence and presence of phytohemagglutinin (10 microg/ml). Intracellular active caspase-3 and cytochrome c concentrations were determined by immunoassay. Migratory activity of EPCs in response to VEGF (2 ng/ml) and stromal cell-derived factor-1alpha (SDF-1alpha; 10 ng/ml) was determined by Boyden chamber. Telomere length was assessed by Southern hybridization. Phytohemagglutinin-stimulated release of VEGF (90.6 +/- 7.6 vs. 127.2 +/- 11.6 pg/ml) and G-CSF (896.1 +/- 77.4 vs. 1,176.3 +/- 126.3 pg/ml) was ~25% lower (P < 0.05) in EPCs from overweight/obese vs. normal weight subjects. Staurosporine induced a ~30% greater (P < 0.05) increase in active caspase-3 in EPCs from overweight/obese (2.8 +/- 0.2 ng/ml) compared with normal weight (2.2 +/- 0.2) subjects. There were no significant differences in EPC migration to either VEGF or SDF-1alpha. Telomere length did not differ between groups. These results indicate that increased adiposity adversely affects the ability of EPCs to release proangiogenic cytokines and resist apoptosis, potentially compromising their reparative potential.

CD31+ T cells represent a functionally distinct vascular T cell phenotype.

In contrast to CD3(+)/CD31(-) cells, CD3(+)/CD31(+) cells aid in endothelial repair and revascularization. There are limited data regarding the functional differences between circulating CD3(+)/CD31(+) and CD3(+)/CD31(-) cells that may contribute to their divergent cardiovascular effects. The aim of the present study was to characterize functional differences between CD3(+)/CD31(+) and CD3(+)/CD31(-) cells. To address this aim, migratory capacity, proangiogenic cytokine release and apoptotic susceptibility of CD3(+)/CD31(+) and CD3(+)/CD31(-) cells were determined. Human CD3(+)/CD31(+) and CD3(+)/CD31(-)cells from peripheral blood were isolated using magnetic-activated cell sorting. CD3(+)/CD31(+) cells demonstrated significantly higher ( approximately 60%) migratory capacity to the chemokines SDF-1alpha (655+/-99 vs. 273+/-54 AU) and VEGF (618+/-99 vs. 259+/-57 AU) vs. CD3(+)/CD31(-) cells. Release of angiogenic cytokines G-CSF, interleukin-8 and matrix metallopeptidase-9 were all approximately 100% higher (P<0.05) in CD3(+)/CD31(+) than CD3(+)/CD31(-) cells. CD3(+)/CD31(+) cells exhibited significantly higher intracellular concentrations of active caspase-3 (2.61+/-0.60 vs. 0.34+/-0.09 ng/mL) and cytochrome-c (21.8+/-1.4 vs. 13.7+/-1.0 ng/mL). In summary, CD3(+)/CD31(+) cells have greater migratory and angiogenic cytokine release capacity, but are more susceptible to apoptosis compared with CD3(+)/CD31(-) cells. Enhanced migratory capacity and angiogenic cytokine release may contribute to the vasculogenic properties of this unique T cell subpopulation.

Effect of prior exercise on postprandial lipemia and markers of inflammation and endothelial activation in normal weight and overweight adolescent boys.

Postprandial lipemia (PPL) is associated with impaired endothelial function and inflammation. Acute exercise reduces PPL in adults. This investigation examined the effect of an acute bout of exercise on postprandial changes in triglycerides (TG), glucose, insulin, inflammation [white blood cell count (WBC), interleukin-6 (IL-6) tumor necrosis factor-alpha, C-reactive protein (CRP)] and endothelial activation [soluble intercellular adhesion molecule-1 (sICAM-1), vascular adhesion molecule-1 (sVCAM-1)] following a high-fat meal in adolescents. Ten normal weight (NW) (BMI, 20.9 +/- 1.7 kg m(-2); 15.6 +/- 0.7 years) and eight overweight (OW) (BMI, 28.3 +/- 3.6 kg m(-2); 15.9 +/- 0.4 years) adolescent boys underwent two 6-h oral fat tolerance tests (OFTT) separated by 7-10 days. On the evening prior to each OFTT, subjects either rested or completed a treadmill exercise bout (65% V(O)(2max); 600 kcal expended). Exercise reduced (P < 0.01) the postprandial TG area under the curve by approximately 20% in the NW and OW groups. The postprandial glucose and insulin response did not differ between the control and exercise trials or between the NW and OW groups. Circulating leukocytes and plasma IL-6 levels increased (P < 0.01) in the NW and OW groups 6 h following the OFTT in both experimental conditions. There were no changes in CRP, sVCAM-1 or sICAM-1 following the OFTT and there were no differences between experimental condition or NW and OW groups. In conclusion, a moderate exercise bout prior to a high-fat meal effectively reduces postprandial TG concentrations to a similar degree in both NW and OW adolescents, but does not reduce the concomitant postprandial increase in WBC or IL-6.

Aging and endothelial progenitor cell telomere length in healthy men.

BACKGROUND: Telomere length declines with age in mature endothelial cells and is thought to contribute to endothelial dysfunction and atherogenesis. Bone marrow-derived circulating endothelial progenitor cells (EPCs) are critical to vascular health as they contribute to both reendothelialization and neovascularization. We tested the hypothesis that EPC telomere length decreases with age in healthy adult humans. METHODS: Peripheral blood samples were collected from 40 healthy, non-obese, sedentary men: 12 young (age 21-34 years), 12 middle-aged (43-55 years) and 16 older (57-68 years). Putative EPCs were isolated from peripheral blood mononuclear cells and telomere length was determined using genomic DNA preparation and Southern hybridization techniques. RESULTS: EPC telomere length (base pairs) was approximately 20% (p=0.01) lower in the older (8492+523 bp) compared to the middle-aged (10,565+572 bp) and young (10,205+501 bp) men. Of note, there was no difference in EPC telomere length between the middle-aged and young men. CONCLUSIONS: These results demonstrate that EPC telomere length declines with age in healthy, sedentary men. Interestingly, telomere length was well preserved in the middle-aged compared to young men, suggesting that EPC telomere shortening occurs after the age of 55 years.

Gender and Endothelial Progenitor Cell Number in Middle-Aged Adults.

BACKGROUND: Between the ages of 45 and 65 years, the prevalence of cardiovascular disease is significantly lower in women compared with men. Circulating bone marrow-derived endothelial progenitor cells (EPCs) play an important role in vascular repair. Reduced EPC number is predictive of more cardiovascular events. It is currently unknown whether there is a sex-difference in EPC number in middle-aged adults. OBJECTIVE: We tested the hypothesis that circulating EPC number is higher in middle-aged women than men. METHODS: Peripheral blood samples were collected from 58 sedentary adults, 29 men (57 ± 1 yr) and 29 women (58 ± 1 yr). Mononuclear cells were isolated and fluorescence-activated cell sorting (FACS) analysis of cells negative for CD45 was performed for those positive for CD34, and triple positive for CD34, VEGFR-2, and CD133 according to the recommendations of the International Society for Hematotherapy and Graft Engineering. RESULTS: The number of CD45(-)/CD34(+) and CD45(-)/CD34(+)/ VEGFR-2(+)/CD133(+) were not significantly different between women and men (0.055 ± 0.006% vs 0.069 ± 0.008% and 0.0013 ± 0.0003% vs 0.0018 ± 0.0004%, respectively). CONCLUSIONS: These results demonstrate no sex-difference in EPC number in middle-age adults. Therefore, it is unlikely that differences in EPC number contribute to the gender-related differences in the prevalence of cardiovascular events in this population.

Gender differences in circulating endothelial progenitor cell colony-forming capacity and migratory activity in middle-aged adults.

Middle-aged women have a lower prevalence and incidence of cardiovascular events compared with men. The mechanisms responsible for this gender-specific difference are unclear. Numeric and functional impairments of bone marrow-derived circulating endothelial progenitor cells (EPCs) are associated with increased cardiovascular and cerebrovascular morbidity and mortality. It is currently unknown whether there are gender-related differences in EPC number and function in middle-aged adults. We tested the hypothesis that EPCs isolated from middle-aged women demonstrate greater colony-forming capacity and migratory activity compared with men of similar age. Peripheral blood samples were collected from 50 sedentary adults, 25 men (59 +/- 1 years of age) and 25 women (58 +/- 1 years of age). Mononuclear cells were isolated and preplated for 2 days, and nonadherent cells were further cultured for 7 days to determine EPC colony-forming units. Migratory activity of EPCs was determined using a modified Boyden chamber. The number of EPC colony-forming units was significantly higher (approximately 150%) in samples collected from women (16 +/- 3) compared with that collected from men (7 +/- 1). In addition, EPC migration (relative fluorescent units) was approximately 40% greater in women (729 +/- 74) than in men (530 +/- 67). In conclusion, these results demonstrate that EPC colony-forming capacity and migratory activity are higher in middle-aged women than in men.