The Link Between Adipose Tissue Renin-Angiotensin-Aldosterone System Signaling and Obesity-Associated Hypertension.

Obese individuals frequently develop hypertension, which is for an important part attributable to renin-angiotensin-aldosterone system (RAAS) overactivity. This review summarizes preclinical and clinical evidence on the involvement of dysfunctional adipose tissue in RAAS activation and on the renal, central, and vascular mechanisms linking RAAS components to obesity-associated hypertension.

Urinary Sodium Excretion and Salt Intake Are Not Associated With Blood Pressure Variability in a White General Population.

Background Salt restriction may lower blood pressure variability (BPV), but previous studies have shown inconsistent results. Therefore, we investigated in an observational study and intervention trial whether urinary sodium excretion and salt intake are associated with 24-hour BPV. Methods and Results We used data from the cross-sectional population-based Maastricht Study (n=2652; 60±8 years; 52% men) and from a randomized crossover trial (n=40; 49±11 years; 33% men). In the observational study, we measured 24-hour urinary sodium excretion and 24-hour BPV and performed linear regression adjusted for age, sex, mean blood pressure, lifestyle, and cardiovascular risk factors. In the intervention study, participants adhered to a 7-day low- and high-salt diet (50 and 250 mmol NaCl/24 h) with a washout period of 14 days, 24-hour BPV was measured during each diet. We used linear mixed models adjusted for order of diet, mean blood pressure, and body mass index. In the observational study, 24-hour urinary sodium excretion was not associated with 24-hour systolic or diastolic BPV (ß, per 1 g/24 h urinary sodium excretion: 0.05 mm Hg [95% CI, -0.02 to 0.11] and 0.04 mm Hg [95% CI, -0.01 to 0.09], respectively). In the intervention trial, mean difference in 24-hour systolic and diastolic BPV between the low- and high-salt diet was not statistically significantly different (0.62 mm Hg [95% CI, -0.10 to 1.35] and 0.04 mm Hg [95% CI, -0.54 to 0.63], respectively). Conclusions Urinary sodium excretion and salt intake are not independently associated with 24-hour BPV. These findings suggest that salt restriction is not an effective strategy to lower BPV in the White general population. Registration URL: https://clinicaltrials.gov/ct2/show/NCT02068781.

Aldosterone Is Not Associated With Metabolic and Microvascular Insulin Sensitivity in Abdominally Obese Men.

Context: Impaired insulin-mediated muscle microvascular recruitment (IMMR) may add to the development of insulin resistance and hypertension. Increased aldosterone levels have been linked to these obesity-related complications in severely to morbidly obese individuals and to impaired microvascular function in experimental studies. Objectives: To investigate whether aldosterone levels are associated with IMMR, insulin sensitivity, and blood pressure in lean and moderately abdominally obese men, and to study the effect of weight loss. Design, Setting, Participants, Intervention, Main Outcome Measures: In 25 lean and 53 abdominally obese men, 24-hour blood pressure measurement was performed, and aldosterone levels were measured using ultra-performance liquid chromatography tandem mass spectrometry. Insulin sensitivity was assessed by determining whole-body glucose disposal during a hyperinsulinemic clamp. IMMR in forearm skeletal muscle was measured with contrast-enhanced ultrasonography. These assessments were repeated in the abdominally obese men following an 8-week weight loss or weight stable period. Results: Sodium excretion and aldosterone levels were similar in lean and abdominally obese participants, but sodium excretion was inversely associated with aldosterone concentration only in the lean individuals [lean, ß/100 mmol sodium excretion (adjusted for age and urinary potassium excretion) = -0.481 (95% confidence interval, -0.949 to -0.013); abdominally obese, ß/100 mmol sodium excretion = -0.081 (95% confidence interval, -0.433 to 0.271); P for interaction = 0.02]. Aldosterone was not associated with IMMR, insulin sensitivity, or blood pressure and was unaffected by weight loss. Conclusion: In moderately abdominally obese men, the inverse relationship between sodium excretion and aldosterone concentration is less than that in lean men but does not translate into higher aldosterone levels. The absolute aldosterone level does not explain differences in microvascular and metabolic insulin sensitivity and blood pressure between lean and moderately abdominally obese men.

Aldosterone-Renin Ratio and Side-Selective Renal Perfusion in Essential Hypertension.

BACKGROUND: The decrease in kidney perfusion as often observed in hypertensive individuals does not necessarily occur in a symmetrical fashion, thereby potentially introducing left-right differences in response to vasoactive agents. Increased aldosterone levels have been associated with reduced renal perfusion in normotensive and hypertensive individuals, but it is unknown whether both kidneys are equally affected in this respect and how angiotensin II is involved in this relationship. Therefore, our aim was to investigate the association of both aldosterone and the aldosterone-renin ratio with side-selective renal blood flow in essential hypertension. METHODS: We studied 146 essential hypertensive patients with patent renal arteries who had undergone renal angiography for exclusion of renal artery stenosis. Prior to contrast administration, blood samples were drawn for the determination of renin and aldosterone levels, and side-selective renal blood flow was measured using the 133Xenon washout technique. RESULTS: Left mean renal blood flow (MRBF) was significantly lower than right MRBF (227±74 vs. 250±76mL * min-1 * 100g kidney-1, P = 0.01). We could not demonstrate a correlation of ln aldosterone or ln renin with left or right kidney perfusion. Ln aldosterone-renin ratio (ARR), however, was inversely and independently associated with left MRBF (ß = -13.993, P = 0.02; fully adjusted model) but not with right MRBF. CONCLUSIONS: A higher ARR corresponds to reduced perfusion of the left kidney, yet is not associated with right kidney perfusion. Especially under circumstances of diminished right renal blood flow, this may affect blood pressure and kidney function.

Monitoring adenovirus infections with on-line and off-line methods.

Several known process monitoring methods were tested for their efficacy in the detection of adenovirus infections. The methods that we explored include several indirect indications of viral infections, including metabolic rate analysis, secondary gauges of respiration, cell size measurement, cell number and cell viability determination, and changes in capacitance. Direct indications of the adenovirus infection were also applied, including total viral particle and infectious particle measurements, as well as a flow cytometry method for detecting infected cells. All of the methods tested in the study provide some positive indication of an adenovirus infection. Many of the methods require repeated sampling, which may limit their utility in a manufacturing process. All of the indirect measures of viral infection may be limited by the fact that they do not uniquely identify an infection. The simplest monitoring methods appear to be detection of changes in respiration or the capacitance of the culture, both of which seem to provide a clear indication of an infection. Further work will be required to demonstrate that these indications are characteristic of only a successful and productive adenovirus infection.

Comparative biochemical characterization of a human IgM produced in both ascites and in vitro cell culture.

We have conducted a comparative analysis of a monoclonal human IgM obtained from cells cultured in nude-mouse ascites and from the same cells cultured in a bioreactor. We studied the glycosylation of the IgMs using lectin blotting and high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD), and we also developed reverse phase liquid chromatography (RPLC) peptide maps of the IgM samples. The HPAE-PAD data indicate that the samples differ in both the type and distribution of oligosaccharides present on the IgMs. In addition, the proteins differ in their solubility behavior and in their RPLC peptide maps. We conclude that the method of cell culture is capable of significantly altering the characteristics of the glycoprotein product.

A mathematical model of sialylation of N-linked oligosaccharides in the trans-Golgi network.

A mathematical model is developed of the compartmentalized sialylation of N-linked oligosaccharides in order to understand and predict the outcome of sialylation reactions. A set of assumptions are presented, including Michaelis-Menten-type dependency of reaction rate on the concentration of the glycoprotein substrate. The resulting model predicts the heterogeneous outcome of a posttranslational oligosaccharide biosynthesis step, a critical aspect that is not accounted for in the modeling of the cotranslational attachment of oligosaccharides to glycosylation sites (Shelikoff et al., Biotech. Bioeng., 50, 73-90, 1996) or general models of the secretion process (Noe and Delenick, J. Cell Sci., 92, 449-459, 1989). In the steady-state for the likely case where the concentration of substrate is much less than the Km of the sialyltransferase, the model predicts that the extent of sialylation, x, will depend upon the enzyme concentration, enzyme kinetic parameters and substrate residence time in the reaction compartment. The value of x predicted by the model using available literature data is consistent with the values of x that have been recently determined for the glycoproteins CD4 (Spellman et al., Biochemistry, 30, 2395-2406, 1991) and t-PA (Spellman et al., J. Biol. Chem., 264, 14100-14111, 1989) secreted by Chinese hamster ovary cells. For the unsaturated case, the model also predicts that x is independent of the concentration of secreted glycoprotein in the Golgi. The general modeling approach outlined in this article may be applicable to other glycosylation reactions and posttranslational modifications.

Characterization of the glycosylation of a human IgM produced by a human-mouse hybridoma.

We analysed the oligosaccharides of a human IgM produced by a human-human-mouse hybridoma at each of its five conserved heavy chain glycosylation sites. Consistent with previous reports, this IgM possesses sialylated oligosaccharides at Asn171, Asn332 and Asn395, and high-mannose-type oligosaccharides at Asn402. In contrast to previous reports for human IgMs, we find that Asn563 is not occupied by oligosaccharide on perhaps 25% of IgM heavy chains, while occupied Asn563 sites contain both high-mannose-type and sialylated oligosaccharides. These latter results are consistent with the glycosylation at Asn563 previously reported for the mouse MOPC 104E IgM. We demonstrate that both the human hybridoma IgM and the mouse MOPC 104E IgM are mixtures of pentamers and hexamers, raising the possibility that the unique findings concerning the glycosylation at Asn563 in this study and the previous study of the MOPC 104E IgM could be related, at least in part, to the different packing requirements of the hexameric geometry and the accessibility of oligosaccharides in the hexameric geometry for processing to complex type. In addition, we used high-pH anion-exchange (HPAE) chromatography, neutral anion-exchange chromatography, fluorophore-assisted carbohydrate electrophoresis and Western blots to compare the oligosaccharide compositions of the human hybridoma IgM, pooled human serum IgM and two mouse monoclonal IgMs (MOPC 104E and TEPC 183). Of note is the presence of N-glycolylneuraminic acid (NeuGc) and N-acetylneuraminic acid (NeuAc) at a 2:1 ratio in the oligosaccharides of the human hybridoma IgM. The presence of both NeuGc and NeuAc complicates the interpretation of HPAE chromatographs.

A high-yielding serum-free, suspension cell culture process to manufacture recombinant adenoviral vectors for gene therapy.

We have developed an efficient, reproducible, and scaleable cell culture process for a recombinant adenoviral vector expressing therapeutic transgenes for clinical trials. HEK 293 cells - which support the propagation of E1 deficient adenovirus - were first adapted to serum free media and suspension growth. Subsequent studies focused on the infection, virus production and harvest from suspension culture bioreactors. Future studies are planned to address the kinetics of adenovirus production in HEK 293 as well as in other cell lines.