Background: Glucocorticoids (GCs) are commonly used for long-term medication in immunosuppressive and anti-inflammatory therapy. However, the data describing gluco-and mineralo-corticoid (MC) properties of widely applied synthetic GCs are often based on diverse clinical observations and on a variety of in vitro tests under various conditions, which makes a quantitative comparison questionable. Method: We compared MC and GC properties of different steroids, often used in clinical practice, in the same in vitro test system (luciferase transactivation assay in CV-1 cells transfected with either hMR or hGRa expression vectors) complemented by a system to test the steroid binding affinities at the hMR (protein expression in T7-coupled rabbit reticulocyte lysate). Results and Conclusions: While the potency of a GC is increased by an 11-hydroxy group, both its potency and its selectivity are increased by the D1-dehydro-configuration and a hydrophobic residue in position 16 (16-methylene, 16a-methyl or 16b-methyl group). Almost ideal GCs in terms of missing MC effects, as defined by our in vitro assay, are therefore prednylidene, budesonide, beclomethasone and betamethasone. The MC potency of a steroid is increased by a 9a-or a 6a-fluoro substituent. A hydrophilic substituent in position 16 (like 16-hydroxylation in triamcinolone) decreases both MC and GC properties. As no substituent that leads to an isolated reduction of GC activity could be characterized in our experiments, 9a-fluorocortisol, the most frequently used steroid for MC substitution, seems to be the best choice of available steroids for this purpose.

Kaposi's sarcoma (KS) is an inflammatory angioproliferative lesion induced by the infection of endothelial cells with the KS-associated herpesvirus (KSHV). Infected endothelial cells assume an elongated (spindle)shape that is one of the histologic signatures of KS. In vitro, latent viral infection of primary endothelial cells (but no other cell type) strikingly recapitulates these morphological findings. Here we report that the spindling phenotype involves major rearrangement of the actin cytoskeleton and can be attributed to the expression of a single viral protein, vFLIP, a known activator of NF-B. Consistent with this, the inhibition of NF-B activation blocks vFLIP-induced spindling in cultured endothelial cells. vFLIP expression in spindle cells also induces the production of a variety of proinflammatory cytokines and cell surface adhesion proteins that likely contribute to the inflammatory component of KS lesions.Kaposi's sarcoma (KS) is a complex angioproliferative lesion that is the most common neoplasm in patients with untreated AIDS, though it also exists in a human immunodeficiency virus-independent form. All KS lesions have three histological components: proliferation, inflammation, and neoangiogenesis. The principal proliferating element in KS is the socalled spindle cell, so named because of its distinctive elongated, spindle-like shape. Spindle cells have long been thought to be the driving force of KS, responsible for the recruitment of the inflammatory and angiogenic components of the lesion; consistent with this, cultured spindle cells produce a number of proinflammatory and angiogenic factors (12,34). The inflammatory infiltrate is also felt to be an important part of KS pathogenesis, since cultured spindle cells require the secreted products of activated T cells for growth (12,28). The histogenesis of spindle cells has long been debated. Based on the expression of markers such as CD31, CD34, CD36, UEA1 lectin, and EN4 by spindle cells, it is generally believed that the cells are most likely of endothelial origin (7). However, the exact endothelial lineage from which spindle cells are derived remains unclear. A number of lines of evidence favor the notion that KS is derived from cells of lymphatic rather than vascular endothelial origin. For instance, KS tumors are never observed in tissues (e.g., brain) that are devoid of lymphatics (47). In addition, KS cells consistently stain for markers of lymphatic endothelium, e.g., VEGF-R3, LYVE-1, and podoplanin (21,36,46). However, the pathogenetic significance of the latter observations has been rendered ambiguous by recent find- ings that KS-associated herpesvirus (KSHV) infection of lymphatic or vascular endothelial cells can reprogram their expression of endothelial markers (17, 45).Central to the pathogenesis of KS is infection by KSHV (also called human herpesvirus 8). Like all herpesviruses, KSHV has two modes of infection, latent and lytic. In KS tumors, KSHV selectively infects the spindle cells (5), most of which are latently infected (37); ...

The steroid hormone aldosterone is important for salt and water homeostasis as well as for pathological tissue modifications in the cardiovascular system and the kidney. The mechanisms of action include a classical genomic pathway, but physiological relevant nongenotropic effects have also been described. Unlike for estrogens or progesterone, the mechanisms for these nongenotropic effects are not well understood, although pharmacological studies suggest a role for the mineralocorticoid receptor (MR). Here we investigated whether the MR contributes to nongenotropic effects. After transfection with human MR, aldosterone induced a rapid and dose-dependent phosphorylation of ERK1/2 and c-Jun NH2-terminal kinase (JNK) 1/2 kinases in Chinese hamster ovary or human embryonic kidney cells, which was reduced by the MR-antagonist spironolactone and involved cSrc kinase as well as the epidermal growth factor receptor. In primary human aortic endothelial cells, similar results were obtained for ERK1/2 and JNK1/2. Inhibition of MAPK kinase (MEK) kinase but not of protein kinase C prevented the rapid action of aldosterone and also reduced aldosterone-induced transactivation, most probably due to impaired nuclear-cytoplasmic shuttling of MR. Cytosolic Ca2+ was increased by aldosterone in mock- and in human MR-transfected cells to the same extend due to Ca2+ influx, whereas dexamethasone had virtually no effect. Spironolactone did not prevent the Ca2+ response. We conclude that some nongenotropic effects of aldosterone are MR dependent and others are MR independent (e.g. Ca2+), indicating a higher degree of complexity of rapid aldosterone signaling. According to this model, we have to distinguish three aldosterone signaling pathways: 1) genomic via MR, 2) nongenotropic via MR, and 3) nongenotropic MR independent.

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Abstract-The epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, contributes to parainflammatory dysregulation, possibly causing cardiovascular dysfunction and remodeling. The physiological role of cardiovascular EGFR is not completely understood. To investigate the physiological importance of EGFR in vascular smooth muscle cells and cardiomyocytes, we generated a mouse model with targeted deletion of the EGFR using the SM22 (smooth muscle-specific protein 22) promoter. While the reproduction of knockout animals was not impaired, life span was significantly reduced. Systolic blood pressure was not different between the 2 genotypes-neither in tail cuff nor in intravascular measurementswhereas total peripheral vascular resistance, diastolic blood pressure, and mean blood pressure were reduced. Loss of vascular smooth muscle cell-EGFR results in a dilated vascular phenotype with minor signs of fibrosis and inflammation. Echocardiography, necropsy, and histology revealed a dramatic eccentric cardiac hypertrophy in knockout mice (2.5-fold increase in heart weight), with increased stroke volume and cardiac output as well as left ventricular wall thickness and lumen. Cardiac hypertrophy is accompanied by an increase in cardiomyocyte volume, a strong tendency to cardiac fibrosis and inflammation, as well as enhanced NADPH-oxidase 4 and hypertrophy marker expression. Thus, in cardiomyocytes, EGFR prevents excessive hypertrophic growth through its impact on reactive oxygen species balance, whereas in vascular smooth muscle cells EGFR contributes to the appropriate vascular wall architecture and vessel reactivity, thereby supporting a physiological vascular tone.

Like all herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) can produce either latent or lytic infection. The latent v-FLIP gene is a strong activator of NFkappaB, and in primary effusion lymphoma (PEL) cells, blockade of NFkappaB activation is associated with enhanced lytic gene expression, while overexpression of p65 impairs expression of reporter genes driven by lytic promoters. This has led to the suggestion that NFkappaB activation may promote latency by suppressing lytic reactivation. Here we examine in detail the effects of NFkappaB activation on KSHV replication in several cell types. In accord with earlier work, we find that inhibition of NFkappaB signaling in PEL cells is associated with enhanced lytic reactivation of KSHV. Similarly, in de novo KSHV infection of primary endothelial cells, inhibition of NFkappaB signaling leads to an increase in lytic gene expression and enhanced virion production. By contrast, KSHV-infected human foreskin fibroblasts (HFF) show no increase in spontaneous lytic reactivation when NFkappaB is inhibited. Moreover, if NFkappaB activation is always inhibitory to lytic gene expression, one might expect its activation to be suppressed during the lytic cycle. However, we find that NFkappaB signaling is strongly and consistently activated in lytically infected cells of all lineages. Together these data indicate that (i) the relationship of NFkappaB activation to latency and lytic reactivation is not uniform, but is dependent on the cellular context; and (ii) even though NFkappaB activation is inhibitory to lytic gene expression in some contexts, such inhibition is at least partially bypassed or overridden during lytic growth.

Objective: Selective inhibitors of 11b-hydroxysteroid-dehydrogenase type I may be of therapeutical interest for two reasons: i) 9a-Fluorinated 11-dehydrosteroids like 11-dehydro-dexamethasone (DH-D) are rapidly activated by human kidney 11b-hydroxysteroid-dehydrogenase type II (11b-HSD-II) to dexamethasone (D). If the same reaction by hepatic 11b-HSD-I could be selectively inhibited, DH-D could be used for selective renal immunosuppressive therapy. ii) Reduction of cortisone to cortisol in the liver may increase insulin resistance in type 2 diabetes mellitus, and inhibition of the enzyme may lead to a decrease in gluconeogenesis.Therefore, we characterized the metabolism of DH-D by human hepatic 11b-HSD-I and tried to find a selective inhibitor of this isoenzyme. Methods: For kinetic analysis of 11b-HSD-I, we used microsomes prepared from unaffected parts of liver segments, resected because of hepatocarcinoma or metastatic disease. For inhibition experiments, we also tested 11b-HSD-II activity with human kidney cortex microsomes. The inhibitory potency of several compounds was evaluated for oxidation and reduction in concentrations from 10 ¹9 to 10 ¹5 mol/l. Results: Whereas D was not oxidized by human liver microsomes at all, cortisol was oxidized to cortisone with a maximum velocity (V max ) of 95 pmol/mg per min. The reduction of DH-D to D (V max = 742 pmol/ mg per min, Michaelis-Menten constant (K m ) = 1.6 mmol/l) was faster than that of cortisone to cortisol (V max =187 pmol/mg per min). All reactions tested in liver microsomes showed the characteristics of 11b-HSD-I: K m values in the micromolar range, preferred cosubstrate NADP(H), no product inhibition. Of the substances tested for inhibition of 11b-HSD-I and -II, chenodeoxycholic acid was the only one that selectively inhibited 11b-HSD-I (IC 50 for reduction: 2.8 × 10 ¹6 mol/l, IC 50 for oxidation: 4.4 × 10 ¹6 mol/l), whereas ketoconazole preferentially inhibited oxidation and reduction reactions catalyzed by 11b-HSD-II. Metyrapone, which is reduced to metyrapol by hepatic 11b-HSD-I, inhibited steroid reductase activity of 11b-HSD-I and -II and oxidative activity of 11b-HSD-II. These findings can be explained by substrate competition for reductase reactions and by product inhibition of the oxidation, which is a well-known characteristic of 11b-HSD-II. Conclusions: Our in vitro results may offer a new concept for renal glucocorticoid targeting. Oral administration of small amounts of DH-D (low substrate affinity for 11b-HSD-I) in combination with chenodeoxycholic acid (selective inhibition of 11b-HSD-I) may prevent hepatic first pass reduction of DH-D, thus allowing selective activation of DH-D to D by the high affinity 11b-HSD-II in the kidney. Moreover, selective inhibitors of the hepatic 11b-HSD-I, like chenodeoxycholic acid, may become useful in the therapy of patients with hepatic insulin resistance including diabetes mellitus type II, because cortisol enhances gluconeogenesis.