Untersuchung zur quantitativen Genexpression in Primärkulturen humaner Adipocyten am Beispiel ausgewählter Gene des Renin-Angiotensin-Systems

Wie sich in den letzten Jahren gezeigt hat, ist Fettgewebe nicht nur ein inerter Fettspeicher, sondern produziert auch eine Vielzahl endokrin wirksamer Substanzen, die unter anderem auch an der Blutdruckregulation beteiligt sind. Da Adipositas ein wichtiger Risikofaktor für die Entwicklung der Hypertonie ist, sollte im Rahmen dieser Dissertation ein System zur quantitativen Untersuchung der Genexpression in Primärkulturen humaner Adipocyten entwickelt werden und dessen Funktionalität am Beispiel der hormonellen Regulation der Gene des Renin-Angiotensin-Systems demonstriert werden. Dies beinhaltete die Etablierung der Adipocytenisolierung und -kultivierung, eines Stimulationsassays, die Entwicklung einer der besonderen Größe und dem hohen Fettgehalt der Zellen angepaßten Zellzahl- und Vitalitätsbestimmungsmethode, die Untersuchung vier verschiedener RNA-Extraktionsmethoden auf ihre Eignung für Adipocyten und die Etablierung eines besonders sensitiven RT-PCR Systems zur Untersuchung der Genexpression mittels einer fluoreszenzmarkierten Sonde. Exemplarisch konnte anhand der Renin-Angiotensin-System-Gene die Funktionalität der Methoden demonstriert werden, indem nicht nur die Genexpression aller Komponenten des Renin-Angiotensin-Systems in humanen Adipocyten nachgewiesen wurden, sondern auch gezeigt werden konnte, dass Hydrocortison sowohl die Genexpression als auch die Dichte des Angiotensin II Typ 1-Rezeptors in der Adipocytenmembran stimuliert. Dieser Aspekt könnte möglicherweise nicht nur bei der besonderen Adipositasform des Cushing-Syndroms, sondern auch für die Entstehung der zentralen Adipositas von Bedeutung sein.Adipose tissue has functions above-and-beyond storing fat. It also produces a variety of different endocrine substances, some of which influence blood pressure regulation. Obesity is a well known risk factor for the development of hypertension Thus, the genes regulating expression of vasoactive molecules in adipose tissue, possibly contributing to an increase in blood pressure are of great interest. The aim of this work was to develope a system for quantitative gene expression analysis in primary cultured human adipocytes and to demonstrate its utility for studying the hormonal regulation of genes encoding the renin-angiotensin-system. We established procedures for the isolation and culture of human adipocytes, as well as a stimulatory assay. We also developed methods for the determination of cell number and vitality. Above this, four RNA extraction protocols were evaluated regarding their suitability for adipocytes, and a very sensitive RT-PCR system for gene expression analysis using fluorescent labeled probes was established. As an example for the functionality of these methods we showed that all genes of the renin-angiotensin-system are expressed in human adipocytes. We also demonstrated that hydrocortisone stimulates the gene expression as well as the density of the angiotensin II receptor type 1 on cultured human adipocytes. This finding may be of interest for the development of the obesity phenotype found in cushing syndrome, but could also contribute to the development of central obesity

Platelet glycoproteins and fibrinogen in recovery from idiopathic sudden hearing loss.

BACKGROUND: The pathomechanism and location of idiopathic sudden sensorineural hearing loss (ISSHL) is unclear. In a previous case-control study, we found elevated fibrinogen concentrations and a higher prevalence of T allele carriers of the glycoprotein (Gp) Ia C807T polymorphism in ISSHL patients. METHODOLOGY: 127 patients with ISSHL (mean age 53.3 years, 48.8% females), who underwent a standard therapy with high dose steroids, pentoxifyllin and sterofundine over 8 days were included. We examined the influence of GpIa genotype and fibrinogen (BclI-, A312-, HaeIII-) genotype and fibrinogen plasma levels on hearing recovery after 8 weeks (change from baseline: 0 dB  =  no recovery, >0 to 10 dB = moderate recovery, >10 dB = good recovery). In a subsample of 59 patients with ISSHL, we further studied the association of platelet glycoprotein GpIa, Ib and IIIa densities on hearing recovery as well as the possible effect-modification of platelet glycoproteins on hearing recovery by plasma fibrinogen. RESULTS: In univariate analysis, neither the GpIa genotype nor fibrinogen genotype (all p>0.1) but lower fibrinogen levels (p = 0.029), less vertigo (p = 0.002) and lower GpIIIa receptor density (p = 0.037, n = 59) were associated with hearing recovery. In multivariate analysis, fibrinogen significantly modified the effect of GPIa receptor density on good hearing recovery (effect-modification on multiplicative scale OR = 0.45 (95% confidence interval (0.21-0.94)), p = 0.03). GPIb receptor density below the mean was associated with a 2-fold increase in good hearing recovery both in patients with fibrinogen levels above (p = 0.04) as well as in patients with fibrinogen levels below the mean (p = 0.06). There was no indication for an effect-modification (p = 0.97). CONCLUSIONS: The findings suggest a vascular/rheological origin of ISSHL with unique features of thrombosis in the inner ear artery that may include complex interrelationships among platelet glycoproteins and plasma fibrinogen

Test calibration cytogram with cursor settings in gated fluorescence histogram (a) and platelet rich plasma cytogram with GpIa immune-labelling cursor settings (b).

<p>A single color flow cytometric analysis of the platelet glycoproteins GpIa, GpIb and GpIIIa was used (Biocytex, Marseille, France). The number of antigenic sites is determined by converting the fluorescence intensity into the corresponding number of sites per platelet based on calibrated bead standards (a). In all experiments, more than 90% of the platelets of the PRP were gated for the experiment, as shown here for GpIa (b).</p

Number of GpIa (a), GpIb (b) and GpIIIa (c) receptor molecules per platelet in patients (left) and controls (right).

<p>The values are shown as boxplots, separated for T allele carriers (phenotype TT or CT) and CC allele carriers. The boundary of the box closest to zero indicates the 25^th percentile, the line within the box marks the median, the square indicates the mean, and the boundary of the box farthest from zero indicates the 75^th percentile. Whiskers above and below the box indicate the 90^th and 10^th percentiles. The points represent the 1^st and 99^th percentiles. P indicates the level of significance according to the Mann-Whitney-U-test. *, P<0.05; ***, P<0.001.</p

Results of binary logistic regression analyses, dependent variable = hearing improvement > 10 dB, independent variables = GPIb receptor density and fibrinogen, n = 55^#.

<p>Measure of effect modification on additive scale: relative excess risk due to interaction, RERI (95% CI)  =  0.24 (−1.46 – 1.95)</p><p>Measure of effect modification on multiplicative scale: ratio of ORs (95% CI) = 0.99 (0.52– 1.86), p = 0.968</p><p>ORs are adjusted for smoking and vertigo, #  =  4 datasets missing due to missing covariates.</p

Comparison of study participants with and without data on glycoprotein receptor density, n = 127.

<p>&  =  data missing on 3 patients; &&  =  data missing on 6 patients; CC  =  wild type, TC  =  heterozygous; TT  =  homozygous; §  =  Mantel-Haenszel Chi-square test.</p

Tyramine in the assessment of regional adrenergic function

Regional adrenergic function is difficult to assess in humans. Tyramine given through a microdialysis probe may be a useful tool in this regard. However, tyramine data is hard to interpret given the drug's complex mode of action. We characterized the response to tyramine, isoproterenol, and dopamine in adipose tissue with microdialysis probes in normal subjects. We measured glycerol concentrations to follow changes in lipolysis and monitored tissue perfusion with ethanol dilution. During perfusion with tyramine, dialysate glycerol concentration increased dose-dependently from 83+/-8muM at baseline to 181+/-18muM at 3.5mM tyramine (p&lt;0.001) followed by a fall down to 121+/-9muM at 35mM tyramine (p&lt;0.001). Propranolol almost completely blocked this response. A similar lipolytic response was not observed in isolated human adipocytes. Dopamine &lt;35muM did not replicate the tyramine-induced lipolysis; however, dopamine &gt;35muM potently inhibited lipolysis. We conclude that tyramine-induced lipolysis is explained by a pre-synaptic mechanism. Tyramine applied through a microdialysis probe in concentrations up to 3.5mM can be used to assess pre- and post-synaptic mechanisms regulating lipid mobilization

Results of binary logistic regression analyses, dependent variable = hearing improvement >10 dB, independent variables = GPIa receptor density and fibrinogen, n = 49^#.

<p>Measure of effect modification on additive scale: relative excess risk due to interaction, RERI (95% CI)  =  −0.90 (−1.97 – 0.17)</p><p>Measure of effect modification on multiplicative scale: ratio of ORs (95% CI) = 0.45 (0.21 – 0.94), p = <b>0.034</b></p><p>ORs are adjusted for smoking and vertigo, #  =  4 datasets missing due to missing covariates.</p