Exploring the expression and potential function of follicle stimulating hormone receptor in extragonadal cells related to abdominal aortic aneurysm.

INTRODUCTION: Follicle stimulating hormone (FSH) is identified to play a role in postmenopausal disease and hypothesized to affect abdominal aortic aneurysm (AAA) onset/progression in postmenopausal women. We aimed to detect FSHR gene expression in AAA tissue and cell types involved in AAA formation. METHODS: FSH stimulation of human umbilical cord endothelial cells (HUVECs), smooth muscle cells (HUCs) and PMA-differentiated macrophages to assess gene expression of FSHR and various markers. Human macrophages activated with various stimuli were assessed for FSHR gene expression. AAA dataset, AAA tissue samples and AAA-derived smooth muscle cells (SMC) obtained from elderly female donors were assessed for FSHR gene expression. AAA-SMCs were stimulated with FSH to assess its effect on gene expression. Lastly, oxidized low-density-lipoprotein (ox-LDL) uptake and abundance of cell surface protein markers were assessed by flow cytometry after FSH stimulation of human monocytes. RESULTS: FSH stimulation showed similar levels of gene expression in HUVECs and HUCs. Only ACTA2 was downregulated in HUCs. In PMA-differentiated macrophages, gene expression of inflammation markers was unchanged after FSH stimulation. FSHR gene expression was found to be low in the AAA datasets. Female AAA-SMCs show occasional FSHR gene expression at a very low level, yet stimulation with FSH did not affect gene expression of SMC- or inflammation markers. FSH stimulation did not impact ox-LDL uptake or alter cell surface protein expression in monocytes. While FSHR gene expression was detected in human testis tissue, it was below quantification level in all other investigated cell types, even upon activation of macrophages with various stimuli. CONCLUSION: Despite previous reports, we did not detect FSHR gene expression in various extragonadal cell types, except in occasional female AAA-SMCs. No clear effect on cell activation was observed upon FSH stimulation in any cell type. Our data suggest that a direct effect of FSH in AAA-related extragonadal cells is unlikely to influence AAA.

The effect of losartan therapy on ventricular function in Marfan patients with haploinsufficient or dominant negative FBN1 mutations.

BACKGROUND: Mild biventricular dysfunction is often present in patients with Marfan syndrome. Losartan has been shown to reduce aortic dilatation in patients with Marfan syndrome. This study assesses the effect of losartan on ventricular volume and function in genetically classified subgroups of asymptomatic Marfan patients without significant valvular regurgitation. METHODS: In this predefined substudy of the COMPARE study, Marfan patients were classified based on the effect of their FBN1 mutation on fibrillin-1 protein, categorised as haploinsufficient or dominant negative. Patients were randomised to a daily dose of losartan 100 mg or no additional treatment. Ventricular volumes and function were measured by magnetic resonance imaging at baseline and after 3 years of follow-up. RESULTS: Changes in biventricular dimensions were assessed in 163 Marfan patients (48 % female; mean age 38 ± 13 years). In patients with a haploinsufficient FBN1 mutation (n = 43), losartan therapy (n = 19) increased both biventricular end diastolic volume (EDV) and stroke volume (SV) when compared with no additional losartan (n = 24): left ventricular EDV: 9 ± 26 ml vs. -8 ± 24 ml, p = 0.035 and right ventricular EDV 12 ± 23 ml vs. -18 ± 24 ml; p < 0.001 and for left ventricle SV: 6 ± 16 ml vs. -8 ± 17 ml; p = 0.009 and right ventricle SV: 8 ± 16 ml vs. -7 ± 19 ml; p = 0.009, respectively. No effect was observed in patients with a dominant negative FBN1 mutation (n = 92), or without an FBN1 mutation (n = 28). CONCLUSION: Losartan therapy in haploinsufficient Marfan patients increases biventricular end diastolic volume and stroke volume, furthermore, losartan also appears to ameliorate biventricular filling properties.

The revised role of TGF-ß in aortic aneurysms in Marfan syndrome.

BACKGROUND: Recently, we demonstrated that losartan reduced the aortic root dilatation rate (AoDR) in adults with Marfan syndrome (MFS); however, responsiveness was diverse. The aim was to determine the role of transforming growth factor-ß (TGF-ß) as therapeutic biomarker for effectiveness of losartan on AoDR. METHODS: Baseline plasma TGF-ß levels of 22 healthy controls and 99 MFS patients, and TGF-ß levels after 1 month of losartan treatment in 42 MFS patients were measured. AoDR was assessed by magnetic resonance imaging at baseline and after 3 years of follow-up. RESULTS: Patients with MFS had higher TGF-ß levels compared with healthy controls (121 pg/ml versus 54 pg/mL, p = 0.006). After 1 month of therapy, losartan normalised the TGF-ß level in 15 patients (36%); the other 27 patients (64%) showed a significant increase of TGF-ß. After 3 years of losartan therapy, patients with a decrease in TGF-ß had significantly higher AoDR compared with patients with increased TGF-ß (1.5 mm/3 years versus 0.5 mm/3 years, p = 0.04). Patients showing a decrease in TGF-ß after losartan therapy had significantly elevated baseline TGF-ß levels compared with patients with increased TGF-ß (189 pg/ml versus 94 pg/ml, p = 0.05). CONCLUSION: Patients responding to losartan therapy with a reduction of the plasma TGF-ß level had higher baseline TGF-ß levels and a higher AoDR. Most likely, TGF-ß levels may be considered to be a readout of the disease state of the aorta. We propose that increased angiotensin II is the initiator of aorta dilatation and is responsible for increased TGF-ß levels in MFS. The concept of TGF-ß as initiator of aortic dilatation in MFS patients should be nuanced.

The role of plasminogen activator inhibitor type 1 in the inflammatory response to local tissue injury.

BACKGROUND: The plasma levels of the plasminogen activator-inhibitor type 1 (PAI-1) are consistently elevated in patients with sterile tissue injury, often accompanied by a systemic acute phase protein response. It remains unknown, however, whether and to what extent PAI-1 affects the host response to trauma. METHODS AND RESULTS: By using the well-established murine model of turpentine-induced tissue injury we compared local and systemic inflammatory responses in PAI-1 gene-deficient (PAI-1-/-) and normal wild-type (Wt) mice. Subcutaneous turpentine injection elicited strong increases in PAI-1 protein concentration in plasma and at the site of injury, but not in liver. PAI-1 mRNA was locally increased and expressed mainly by macrophages and endothelial cells. PAI-1 deficiency greatly enhanced the early influx of neutrophils to the site of inflammation, which was associated with increased edema and necrosis at 8 h after injection. Furthermore, PAI-1-/- mice showed a reduced early interleukin (IL)-6 induction with subsequently lower acute phase protein levels and a much slower recovery of body weight loss. CONCLUSION: These findings suggest that PAI-1 is not merely a marker of tissue injury but plays a functional role in the local and systemic host response to trauma.

Transduction of the E6 and E7 genes of epidermodysplasia-verruciformis-associated human papillomaviruses alters human keratinocyte growth and differentiation in organotypic cultures.

Epidermodysplasia-verruciformis-associated human papilloma virus DNA has been detected in skin cancers, in premalignant and benign skin lesions, and in plucked hairs from immunocompetent and immunosuppressed patients. The role of epidermodysplasia-verruciformis-associated human papilloma virus in the pathogenesis of nonmelanoma skin cancer is still enigmatic. In organotypic cultures we investigated the effects of retroviral transduction of the E6 and E7 genes of epidermodysplasia-verruciformis-associated human papilloma virus types 5, 12, 15, 17, 20, and 38 on the growth and differentiation of human keratinocytes. Differentiation was disturbed to different degrees as revealed by histology and by the expression patterns of differentiation markers keratin 10 and small proline rich protein 2. Conversely, proliferating cell nuclear antigen was induced in some of the suprabasal, differentiated cells to varying extent. No unscheduled DNA synthesis was detected in these cells, however, as probed by 5'-bromo-2'-deoxyuridine incorporation. Most intriguingly, when the E6 and E7 genes of epidermodysplasia-verruciformis-associated human papilloma virus types 15 and 17 were transduced, a broadening layer of basal cells and an accelerated differentiation were observed. In addition, "papilla-like structures" comprising basal-like keratinocytes arose from the basal layer into the differentiated layers. These cells did not express the differentiation markers keratin 10 and small proline rich protein 2, but did actively replicate DNA. These observations warrant further research by using this system to elucidate the replication strategy of epidermodysplasia-verruciformis-associated human papilloma virus types in keratinocytes and to shed light on the role of these human papilloma virus types in the pathogenesis of skin cancer.

Serial analysis of gene expression to assess the endothelial cell response to an atherogenic stimulus.

Activation of human, arterial endothelial cells (ECs) is an early event in the pathogenesis of atherosclerosis. To identify the repertoire of genes that are differentially expressed after activation, we used serial analysis of gene expression (SAGE) to compare the mRNA spectrum of quiescent ECs with that of ECs activated for 6h with a strong atherogenic stimulus. SAGE methodology generates concatenated 'tags' of 10bp that are derived from a specific mRNA. About 5% of over 12000 tags analyzed is derived from genes that are differentially expressed (at least 5-fold up- or downregulated). These transcript tags are derived from only 56 genes, close to 1% of the total number of analyzed genes. Among these 56 differentially expressed genes are 42 known genes, including the hallmark endothelial cell activation markers interleukin 8 (IL-8), monocyte chemoattractant protein 1 (MCP-1), vascular cell adhesion molecule 1 (VCAM-1), plasminogen activator inhibitor 1 (PAI-1), Gro-alpha, Gro-beta and E-selectin. Differential transcription of a selection of the upregulated genes was confirmed by Northern blot analysis. A novel observation is the upregulation of activin betaA mRNA, a member of the transforming growth factor beta family. Apparent discrepancies between this novel technology and conventional methods are discussed. In conclusion, we demonstrate that for the application of SAGE, a moderate number of analyzed transcript tags suffices to reveal the significant alterations of EC transcription that results from a strong atherogenic stimulus.

Tissue distribution and regulation of murine von Willebrand factor gene expression in vivo.

von Willebrand factor (vWF) is frequently used as a biochemical marker for endothelial cells (ECs). Despite this, little is known about the relative level of expression and regulation of this hemostatic factor in ECs in different vascular beds in vivo. In the present study, we used quantitative reverse transcription polymerase chain reaction and in situ hybridization analysis to study vWF gene expression in murine tissues. Large differences in the level of vWF mRNA were observed when comparing highly vascularized tissues, with the lung and brain containing 5 to 50 times higher concentrations of vWF mRNA than the kidney and liver. In this regard, ECs of small vessels and some microvessels in the lung and brain expressed abundant vWF mRNA, whereas ECs of similar sized vessels in the liver and kidney expressed relatively low levels. In general, significantly higher levels of vWF mRNA and antigen were demonstrated in ECs of larger vessels compared with microvessels and in venous ECs compared with arterial ECs. Although intraperitoneal administration of endotoxin (or tumor necrosis factor-alpha) increased plasma vWF levels, it had variable effects on the steady-state level of vWF mRNA in murine tissues (ie, it decreased vWF mRNA in many tissues, increased it in others, and had little effect on still others). These results indicate that vWF is differentially expressed and regulated in ECs present in different tissues and within the same vascular bed.

Lack of gradual regulation of tetracycline-controlled gene expression by the tetracyclin-repressor/VP16 transactivator (tTA) in cultured cells.

Von Willebrand factor (vWF) is an essential multimeric protein for adhesion of platelets to an injured vessel wall. Endothelial cells secrete vWF by either a constitutive or a regulated pathway. It is unknown whether the secretory partitioning of vWF is dependent on the level of vWF synthesis. We employed the widely applied tetracycline-controlled transactivator system (tTA) to study the regulation of vWF mRNA synthesis in stably transfected Madin Darby kidney (MDCK-II) cells in a quantitative manner. Immunofluorescence staining with anti-vWF antibodies revealed that increasing the concentration of tetracycline resulted in a decreased number of MDCK-II cells that synthesize vWF. Apparently, tTA-regulated gene expression in an individual cell functions as an 'on/off' system rather than regulating the level of gene expression in a dose-response manner, as reported previously.