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1.  Alpha1-adrenergic-mediated eNOS phosphorylation in intact arteries 
Vascular pharmacology  2012;58(1-2):112-117.
Activation of arterial smooth muscle alpha1-adrenergic receptors results in vasoconstriction, as well as a secondary release of nitric oxide and slow vasodilation, presumably through gap junction communication from smooth muscle to endothelium. We hypothesized that this slow vasodilation is due to activation of eNOS through phosphorylation at Ser1179 and dephosphorylation at Thr495. Phosphorylation was measured by western blot using mouse mesenteric arteries that were cannulated and pressurized (75 mmHg) and treated either by 1) 5 min of phenylephrine superfusion (10−5 M) (PE5), 2) 15 minutes of phenylephrine (PE15), 3) 15 min phenylephrine followed by acetylcholine (10−4 M) (PE+ACh), or 4) 20 min time control with no treatment (NT) [4–5 arteries pooled per treatment per blot; 5 blots performed]. These treatments allowed correlation between vasomotor changes, namely maximal constriction (PE5), slow vasodilation (PE15), and maximal dilation (PE+ACh), and relative phosphorylation changes. Phosphorylation of eNOS at Ser1179 was increased relative to NT by more than 2-fold at PE5 and remained similarly increased at PE15 and PE+ACh. Phosphorylation of eNOS at Thr495 was less in all treatments relative to NT, but not significantly. Treatment with L-NAME (10−4 M) or endothelial denudation indicated that the slow dilation in response to phenylephrine was completely due to nitric oxide synthase and was endothelial dependent. These results indicate that eNOS phosphorylation at Ser1179 occurs before the slow dilation and is not actively involved in this vasodilation or dilation to acetylcholine, but may play a permissive role in eNOS activation by other mechanisms. It is not yet known what mechanism is responsible for Ser1179 phosphorylation with phenylephrine stimulation.
doi:10.1016/j.vph.2012.09.003
PMCID: PMC3534816  PMID: 22982055
mouse; mesenteric; myoendothelial junctions; vasodilation; phenylephrine; acetylcholine
2.  Genetic Ancestry, Skin Reflectance and Pigmentation Genotypes in Association with Serum Vitamin D Metabolite Balance 
Background
Lower serum vitamin D (25(OH)D) among individuals with African ancestry is attributed primarily to skin pigmentation. However, the influence of genetic polymorphisms controlling for skin melanin content has not been investigated. Therefore, we investigated differences in non-summer serum vitamin D metabolites according to self-reported race, genetic ancestry, skin reflectance and key pigmentation genes (SLC45A2 and SLC24A5).
Materials and Methods
Healthy individuals reporting at least half African American or half European American heritage were frequency matched to one another on age (+/− 2 years) and sex. 176 autosomal ancestry informative markers were used to estimate genetic ancestry. Melanin index was measured by reflectance spectrometry. Serum vitamin D metabolites (25(OH)D3, 25(OH)D2 and 24,25(OH)2D3) were determined by high performance liquid chromatography (HPLC) tandem mass spectrometry. Percent 24,25(OH)2D3 was calculated as a percent of the parent metabolite (25(OH)D3). Stepwise and backward selection regression models were used to identify leading covariates.
Results
Fifty African Americans and 50 European Americans participated in the study. Compared with SLC24A5 111Thr homozygotes, individuals with the SLC24A5 111Thr/Ala and 111Ala/Ala genotypes had respectively lower levels of 25(OH)D3 (23.0 and 23.8 nmol/L lower, p-dominant=0.007), and percent 24,25(OH)2D3 (4.1 and 5.2 percent lower, p-dominant=0.003), controlling for tanning bed use, vitamin D/fish oil supplement intake, race/ethnicity, and genetic ancestry. Results were similar with melanin index adjustment, and were not confounded by glucocorticoid, oral contraceptive, or statin use.
Conclusions
The SLC24A5 111Ala allele was associated with lower serum vitamin 25(OH)D3 and lower percent 24,25(OH)2D3, independently from melanin index and West African genetic ancestry.
doi:10.1515/HMBCI.2011.021
PMCID: PMC3606023  PMID: 23525585
African Continental Ancestry Group; European Continental Ancestry Group; SLC24A5; 25-hydroxyvitamin D; 24,25-Dihydroxyvitamin D 3
3.  Lead, calcium uptake, and related genetic variants in association with renal cell carcinoma risk in a cohort of male Finnish smokers 
Background
Lead (Pb) is classified as a probable human carcinogen. However, its role in renal cell cancer (RCC) has not been established. Calcium and vitamin D may off-set toxicity in vivo.
Methods
In this nested case-control study, whole blood lead (Pb), total serum calcium, and serum 25-hydroxyvitamin D were measured in blood drawn prior to diagnosis among male smokers participating in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study. Single nucleotide polymorphisms (SNPs) in five genes (CALB1, TRPV5, TRPV6, VDR, and ALAD) related to lead toxicity or calcium transport were genotyped. Logistic and linear regression were used to determine RCC risk and time to diagnosis (respectively), adjusting for other risk factors.
Results
Among 154 newly diagnosed cases and 308 matched controls, RCC was associated with higher whole blood lead (OR=2.0, 95% CI:1.0,3.9; quartile 4 (Q4) v. Q1, Ptrend=0.022) and CALB1 rs1800645 (Ptrend=0.025, minor ‘T’ allele frequency=0.34). Higher total serum calcium (Ptrend=<0.001) was associated with reduced RCC risk. Total serum calcium and 25-hydroxyvitamin D levels did not alter the association observed with lead. Time from enrollment to RCC diagnosis was positively associated with serum calcium (Ptrend=0.002) and 25-hydroxyvitamin D (Ptrend=0.054) among cases.
Conclusions
Higher blood lead concentrations, below the 10 ug/dL level of concern, were associated with RCC, independent from serum calcium and CALB1 promoter polymorphism.
Impact
Increased risk of RCC is associated with lower serum calcium and higher whole blood lead in smokers. The clinical prognostic value of serum calcium and vitamin D in RCC should be further investigated.
doi:10.1158/1055-9965.EPI-11-0670
PMCID: PMC3601826  PMID: 22086884
calcium; lead; vitamin D; renal cell carcinoma; polymorphism; CALB1
4.  Cancers of the Urinary Tract Among American Indians and Alaska Natives in the United States, 1999–2004 
Cancer  2008;113(5 Suppl):1213-1224.
BACKGROUND
Assessment of the kidney parenchyma (“kidney”) and urinary bladder (“bladder”) cancer burden among American Indians and Alaska Natives (AI/AN) has been limited. Using a database with improved classification for AI/AN, the authors described patterns of these 2 cancers among AI/AN and non-Hispanic whites (NHW) in the United States.
METHODS
Cases diagnosed during 1999 to 2004 were identified through National Program of Cancer Registries and the Surveillance, Epidemiology and End Results program and linked to the Indian Health Service (IHS) registration records. Age-adjusted incidence rates, rate ratios (RR), annual percent change, and stage at diagnosis were stratified by IHS Contract Health Service Delivery Area (CHSDA) counties to adjust for misclassification.
RESULTS
Kidney cancer incidence among AI/AN in CHSDA counties exceeded that among NHW (RR, 1.51; 95% confidence interval [CI], 1.42-1.61), and was highest among AI/AN in the Northern Plains, Southern Plains, Alaska, and Southwest. Average annual increases were highest among AI/AN (5.9%) and NHW (5.9%) males aged 20 to 49 years, although statistically significant only among NHW. Conversely, bladder cancer incidence was significantly lower among AI/AN than NHW (RR, 0.40; 95% CI, 0.37-0.44). For both sites, AI/AN were significantly less likely to be diagnosed at an earlier stage than NHW.
CONCLUSIONS
AI/AN have about 50% greater risk of kidney cancer and half the risk of bladder cancer than NHW. Although reasons for these enigmatic patterns are not known, sustained primary prevention efforts through tobacco cessation and obesity prevention are warranted.
doi:10.1002/cncr.23733
PMCID: PMC3587982  PMID: 18720377
cancer; incidence; American Indian; Alaska Native; misclassification; NPCR; SEER; United States; health disparity
5.  Breast and Cervical Cancer Screening Patterns Among American Indian Women at IHS Clinics in Montana and Wyoming 
Public Health Reports  2011;126(6):806-815.
SYNOPSIS
Objectives
We investigated factors associated with primary and secondary breast and cervical cancer screening among American Indian (AI) women receiving care from the Indian Health Service (IHS) in Montana and Wyoming.
Methods
Rates of primary screening (i.e., screening without evidence of a prior abnormal) and secondary screening during a three-year period (2004–2006) were determined in an age- and clinic-stratified random sample of 1,094 women at six IHS units through medical record review.
Results
Three-year mammography prevalence rates among AI women aged ≥45 years were 37.7% (95% confidence interval [CI] 34.1, 41.3) for primary and 58.7% (95% CI 43.9, 73.5) for secondary screening. Among women aged ≥18 years, three-year Pap test prevalence rates were 37.8% (95% CI 34.9, 40.6) for primary and 53.2% (95% CI 46.0, 60.4) for secondary screening. Primary mammography screening was positively associated with number of visits and receiving care at an IHS hospital (both p<0.001). Primary Pap test screening was inversely associated with age and positively associated with the number of patient visits (both p<0.001). Secondary mammography screening was inversely associated with driving distance to an IHS facility (p=0.035).
Conclusion
Our results are consistent with other surveys among AI women, which report that Healthy People 2010 goals for breast (90%) and cervical (70%) cancer screening have not been met. Improvements in breast and cervical cancer screening among AI women attending IHS facilities are needed.
PMCID: PMC3185316  PMID: 22043096
6.  Pannexin1 regulates α1-adrenoreceptor-mediated vasoconstriction 
Circulation research  2011;109(1):80-85.
Rationale
The coordination of vascular smooth muscle cell (VSMC) constriction plays an important role in vascular function such as regulation of blood pressure. However, the mechanism responsible for VSMC communication is not clear in the resistance vasculature. Pannexins (Panx) are purine releasing channels permeable to the vasoconstrictor ATP and thus may play a role in the coordination of VSMC constriction.
Objective
We investigated the role of pannexins in phenylephrine (PE) and KCl mediated constriction of resistance arteries.
Methods and Results
Western blot, immunohistochemistry and immunogold labeling coupled to scanning and transmission electron microscopy revealed the presence of Panx1 but not Panx2 or Panx3 in thoracodorsal resistance arteries (TDA). Functionally, the contractile response of pressurized TDA to PE was significantly decreased by multiple Panx inhibitors (mefloquine, probenecid and 10Panx1), ectonucleotidase (apyrase) and purinergic receptor inhibitors (suramin and reactive-blue-2). Electroporation of TDA with either Panx1-GFP or Panx1 siRNA showed enhanced and decreased constriction respectively in response to PE. Lastly, the Panx inhibitors did not alter constriction in response to KCl. This result is consistent with co-immunoprecipitation experiments from TDA, which suggested an association between Panx1 and α1D-adrenoreceptor.
Conclusions
Our data demonstrate for the first time a key role for Panx1 in resistance arteries, by contributing to the coordination of VSMC constriction and possibly regulation of blood pressure.
doi:10.1161/CIRCRESAHA.110.237594
PMCID: PMC3135971  PMID: 21546608
pannexins; phenylephrine; adrenergic receptor; smooth muscle cells; vasoconstriction
7.  Formulation and Characterization of Echogenic Lipid–Pluronic Nanobubbles 
Molecular Pharmaceutics  2010;7(1):49-59.
The advent of microbubble contrast agents has enhanced the capabilities of ultrasound as a medical imaging modality and stimulated innovative strategies for ultrasound-mediated drug and gene delivery. While the utilization of microbubbles as carrier vehicles has shown encouraging results in cancer therapy, their applicability has been limited by a large size which typically confines them to the vasculature. To enhance their multifunctional contrast and delivery capacity, it is critical to reduce bubble size to the nanometer range without reducing echogenicity. In this work, we present a novel strategy for formulation of nanosized, echogenic lipid bubbles by incorporating the surfactant Pluronic, a triblock copolymer of ethylene oxide copropylene oxide coethylene oxide into the formulation. Five Pluronics (L31, L61, L81, L64 and P85) with a range of molecular weights (Mw: 1100 to 4600 Da) were incorporated into the lipid shell either before or after lipid film hydration and before addition of perfluorocarbon gas. Results demonstrate that Pluronic–lipid interactions lead to a significantly reduced bubble size. Among the tested formulations, bubbles made with Pluronic L61 were the smallest with a mean hydrodynamic diameter of 207.9 ± 74.7 nm compared to the 880.9 ± 127.6 nm control bubbles. Pluronic L81 also significantly reduced bubble size to 406.8 ± 21.0 nm. We conclude that Pluronic is effective in lipid bubble size control, and Pluronic Mw, hydrophilic–lipophilic balance (HLB), and Pluronic/ lipid ratio are critical determinants of the bubble size. Most importantly, our results have shown that although the bubbles are nanosized, their stability and in vitro and in vivo echogenicity are not compromised. The resulting nanobubbles may be better suited for contrast enhanced tumor imaging and subsequent therapeutic delivery.
doi:10.1021/mp9001816
PMCID: PMC3285380  PMID: 19957968
Ultrasound Contrast Agent; Pluronic; Nanobubbles; Lipid; Surfactant
8.  The number of women who would need to be screened regularly by mammography to prevent one death from breast cancer 
Journal of Medical Screening  2011;18(4):210-212.
The number of women who would need to be screened regularly by mammography to prevent one death from breast cancer depends strongly on several factors, including the age at which regular screening starts, the period over which it continues, and the duration of follow-up after screening. Furthermore, more women would need to be INVITED for screening than would need to be SCREENED to prevent one death, since not all women invited attend for screening or are screened regularly. Failure to consider these important factors accounts for many of the major discrepancies between different published estimates. The randomised evidence indicates that, in high income countries, around one breast cancer death would be prevented in the long term for every 400 women aged 50–70 years regularly screened over a ten-year period.
doi:10.1258/jms.2011.011134
PMCID: PMC3266234  PMID: 22184734
9.  Compartmentalized Connexin 43 S-Nitrosylation/Denitrosylation Regulates Heterocellular Communication in the Vessel Wall 
Objective
To determine whether S-nitrosylation of connexins (Cxs) modulates gap junction communication between endothelium and smooth muscle.
Methods and Results
Heterocellular communication is essential for endothelium control of smooth muscle constriction; however, the exact mechanism governing this action remains unknown. Cxs and NO have been implicated in regulating heterocellular communication in the vessel wall. The myoendothelial junction serves as a conduit to facilitate gap junction communication between endothelial cells and vascular smooth muscle cells within the resistance vasculature. By using isolated vessels and a vascular cell coculture, we found that Cx43 is constitutively S-nitrosylated on cysteine 271 because of active endothelial NO synthase compartmentalized at the myoendothelial junction. Conversely, we found that stimulation of smooth muscle cells with the constrictor phenylephrine caused Cx43 to become denitrosylated because of compartmentalized S-nitrosoglutathione reductase, which attenuated channel permeability. We measured S-nitrosoglutathione breakdown and NOx concentrations at the myoendothelial junction and found S-nitrosoglutathione reductase activity to precede NO release.
Conclusion
This study provides evidence for compartmentalized S-nitrosylation/denitrosylation in the regulation of smooth muscle cell to endothelial cell communication.
doi:10.1161/ATVBAHA.110.215939
PMCID: PMC3056333  PMID: 21071693
NO; GSNO-R; connexin; myoendothelial junction; nitrosylation
10.  Fish, Vitamin D, and Flavonoids in Relation to Renal Cell Cancer Among Smokers 
American Journal of Epidemiology  2009;170(6):717-729.
Fish, vitamin D, flavonoids, and flavonoid-containing foods may have cardiovascular benefits and therefore may also reduce the risk of renal cell cancer. Risk was prospectively assessed in the Alpha-Tocopherol Beta-Carotene Cancer Prevention Study (1985–2002) cohort (N = 27,111; 15.2 mean person-years of follow-up). At enrollment, demographic, health, and dietary history information was recorded. Individuals who smoked less than 5 cigarettes/day, with chronic renal insufficiency or prior cancer, were excluded. Hazard ratios and 95% confidence intervals from Cox regression were used to compare upper quartiles (quartiles 2–4) with the lowest quartile (quartile 1) of dietary intake. Among 228 cases, risk (quartile 4 vs. quartile 1) was associated with consumption of the flavonoid quercetin (hazard ratio = 0.6, 95% confidence interval: 0.4, 0.9; Ptrend = 0.015) and Baltic herring (hazard ratio = 2.0, 95% confidence interval: 1.4, 3.0; Ptrend < 0.001), with adjustment for age, body mass index, smoking, blood pressure, alcohol use, physical activity, urban residence, and education. In geographically stratified models, the risks associated with herring and total fish intake appeared to be highest in the urban coast region, although the interaction was not statistically significant. These results suggest that the flavonoid quercetin may prevent renal cell cancer among male smokers. The possible risk associated with fish intake warrants further investigation before conclusions may be drawn.
doi:10.1093/aje/kwp178
PMCID: PMC2768520  PMID: 19651663
carcinoma (renal cell); fish products; flavonoids; vitamin D
11.  Evidence that congenital hydrocephalus is a precursor to idiopathic normal pressure hydrocephalus in only a subset of patients 
Objective
To identify the proportion of patients in a large idiopathic normal pressure hydrocephalus (INPH) cohort with large head circumference (HC) who presumably have congenital hydrocephalus that has not become clinically apparent until late in life.
Methods
HC was measured in 158 patients diagnosed with communicating INPH and assigned to HC centile range adjusted for height and sex. The proportion of patients with INPH and HC above the 97th, 90th or 50th centiles was compared with the proportion expected in a normal population.
Results
The proportion of patients with HC >90th centile was significantly larger than would be expected in a normal distribution (19.6% vs 10%, p = 0.0001), as was the proportion of patients with HC >97th centile (8.9% vs 3%, p = 0.0001). The relative association between INPH and HC >97th centile was nearly tripled (relative association 2.95; CI 1.36 to 6.41), but the relative association between INPH and HC >50th centile was not significantly higher than predicted (relative association 1.13; CI 0.95 to 1.34).
Conclusion
A significantly larger proportion of elderly adults with INPH have a HC greater than the 90th or 97th centile than predicted by population norms, supporting the concept that compensated congenital hydrocephalus that does not become symptomatic until late in life is one aetiology of INPH, but is not responsible for all INPH. The mechanism that leads to the development of INPH in most patients remains elusive.
doi:10.1136/jnnp.2006.108761
PMCID: PMC2117818  PMID: 17202229
12.  Plasminogen activator inhibitor-1 regulates myoendothelial junction formation 
Circulation research  2010;106(6):1092-1102.
Rationale
Plasminogen activator inhibitor-1 (PAI-1) is a biomarker for several vascular disease states; however, its target of action within the vessel wall is undefined.
Objective
Determine the ability of PAI-1 to regulate myoendothelial junction (MEJ) formation.
Methods and Results
Myoendothelial junctions are found throughout the vasculature linking endothelial cells (EC) and vascular smooth muscle cells (VSMC). Using a vascular cell co-culture (VCCC) we isolated MEJ fractions and performed two-dimensional differential gel electrophoresis. Mass spectrometry identified PAI-1 as being enriched within MEJ fractions, which we confirmed in vivo. In the VCCC, recombinant PAI-1 (rPAI-1) added to the EC monolayer significantly increased MEJs. Conversely, addition of a PAI-1 monoclonal antibody to the EC monolayer reduced the number of MEJs. This was also observed in vivo where mice fed a high fat diet had increased PAI-1 and MEJs and the number of MEJs in coronary arterioles of PAI-1−/− mice was significantly reduced when compared to C57Bl/6 mice. The presence of MEJs in PAI-1−/− coronary arterioles was restored when their hearts were transplanted into and exposed to the circulation of C57Bl/6 mice. Application of biotin-conjugated PAI-1 to the EC monolayer in vitro confirmed the ability of luminal PAI-1 to translocate to the MEJ. Functionally, phenylephrine-induced heterocellular calcium communication in the VCCC was temporally enhanced when rPAI-1 was present, and prolonged when PAI-1 was absent.
Conclusion
Our data implicate circulating PAI-1 as a key regulator of MEJ formation and a potential target for pharmacological intervention in diseases with vascular abnormalities (e.g., diabetes mellitus).
doi:10.1161/CIRCRESAHA.109.215723
PMCID: PMC2848897  PMID: 20133900
myoendothelial junction; plasminogen activator inhibitor-1; endothelial cell; smooth muscle cell
13.  Shared Occupational Risks for Transitional Cell Cancer of the Bladder and Renal Pelvis among Men and Women in Sweden 
Background:
Unlike cancer of the bladder, cancer of the renal pelvis is not considered an occupational cancer and little is known about risks among women.
Methods:
Using the Swedish national census and cancer registry-linked data (1971-1989), we identified transitional cell cancers of the renal pelvis (N=1374) and bladder (N=21,591). Correlation between cancer sites for the Standardized Incidence Ratios (SIR) were determined using Pearson's coefficient of the log SIR. Relative risks of job exposure matrix variables were calculated using Poisson regression.
Results:
Both cancer sites were significantly elevated among women and men employed in the machine/electronics industry, sedentary work, and indoor work, as well as among men employed in the shop and construction metal industry, contributing 10-14% of cases among men. Risks by industry were more highly correlated among women (r=0.49, p=0.002) than men (r=0.24, p=0.04).
Conclusion:
Cancers of the renal pelvis and bladder share common occupational risk factors that may be more frequent among women. In addition, there may be several jobs that pose an increased risk specifically for cancer of the renal pelvis but not bladder.
doi:10.1002/ajim.20522
PMCID: PMC2800169  PMID: 18067176
epidemiology; bladder neoplasms; kidney neoplasms; occupational exposure; industry
14.  A Novel SNP in a Vitamin D Response Element of the CYP24A1 Promoter Reduces Protein Binding, Transactivation, and Gene Expression 
The active form of vitamin D (1α,25(OH)2D3) is known to have antiproliferative effects and has been implicated in cancers of the colon, breast, and prostate. These cancers occur more frequently among African Americans than Caucasians, and individuals with African ancestry are known to have approximately two-fold lower levels of serum vitamin D (25(OH)D) compared with individuals of European ancestry. However, epidemiological studies of the vitamin D receptor (VDR) have shown inconsistent associations with cancer risk, suggesting that differences in other genes in the pathway may be important. We sought to identify functionally significant polymorphic variants in CYP24A1, a gene that is highly inducible by 1α,25(OH)2D3 and that encodes the primary catabolic enzyme in the pathway. Here we report the identification of six novel SNPs in the human CYP24A1 promoter, including one at nucleotide -279 occurring within the distal vitamin D response element (VDRE2). Our experiments demonstrate that the VDRE2 variant results in decreased protein binding and transactivation in vitro, and reduced expression of CYP24A1 in cultured primary human lymphocytes provides evidence for an effect in vivo. This variant was only observed in our African American population, and represents a first step toward understanding differences in disease risk among racial/ethnic groups.
doi:10.1016/j.jsbmb.2008.08.009
PMCID: PMC2749287  PMID: 18824104
CYP24A1; 24-hydroxylase; promoter; 1α,25(OH)2D3; vitamin D receptor; single nucleotide polymorphism; VDRE
15.  The phosphorylation state of eNOS modulates vascular reactivity and outcome of cerebral ischemia in vivo 
Journal of Clinical Investigation  2007;117(7):1961-1967.
NO plays critical roles in vascular function. We show that modulation of the eNOS serine 1179 (S1179) phosphorylation site affects vascular reactivity and determines stroke size in vivo. Transgenic mice expressing only a phosphomimetic (S1179D) form of eNOS show greater vascular reactivity, develop less severe strokes, and have improved cerebral blood flow in a middle cerebral artery occlusion model than mice expressing an unphosphorylatable (S1179A) form. These results provide a molecular mechanism by which multiple diverse cardiovascular risks, such as diabetes and obesity, may be centrally integrated by eNOS phosphorylation in vivo to influence blood flow and cardiovascular disease. They also demonstrate the in vivo relevance of posttranslational modification of eNOS in vascular function.
doi:10.1172/JCI29877
PMCID: PMC1884686  PMID: 17557122

Results 1-16 (16)