Anthropometric measures of obesity-their association with type 2 diabetes and hypertension across ethnic groups

Clinical trials have shown that weight reduction with lifestyles can delay or prevent diabetes and reduce blood pressure. An appropriate definition of obesity using anthropometric measures is useful in predicting diabetes and hypertension at the population level. However, there is debate on which of the measures of obesity is best or most strongly associated with diabetes and hypertension and on what are the optimal cut-off values for body mass index (BMI) and waist circumference (WC) in this regard. The aims of the study were 1) to compare the strength of the association for undiagnosed or newly diagnosed diabetes (or hypertension) with anthropometric measures of obesity in people of Asian origin, 2) to detect ethnic differences in the association of undiagnosed diabetes with obesity, 3) to identify ethnic- and sex-specific change point values of BMI and WC for changes in the prevalence of diabetes and 4) to evaluate the ethnic-specific WC cutoff values proposed by the International Diabetes Federation (IDF) in 2005 for central obesity. The study population comprised 28 435 men and 35 198 women, ≥ 25 years of age, from 39 cohorts participating in the DECODA and DECODE studies, including 5 Asian Indian (n = 13 537), 3 Mauritian Indian (n = 4505) and Mauritian Creole (n = 1075), 8 Chinese (n =10 801), 1 Filipino (n = 3841), 7 Japanese (n = 7934), 1 Mongolian (n = 1991), and 14 European (n = 20 979) studies. The prevalence of diabetes, hypertension and central obesity was estimated, using descriptive statistics, and the differences were determined with the χ2 test. The odds ratios (ORs) or  coefficients (from the logistic model) and hazard ratios (HRs, from the Cox model to interval censored data) for BMI, WC, waist-to-hip ratio (WHR), and waist-to-stature ratio (WSR) were estimated for diabetes and hypertension. The differences between BMI and WC, WHR or WSR were compared, applying paired homogeneity tests (Wald statistics with 1 df). Hierarchical three-level Bayesian change point analysis, adjusting for age, was applied to identify the most likely cut-off/change point values for BMI and WC in association with previously undiagnosed diabetes. The ORs for diabetes in men (women) with BMI, WC, WHR and WSR were 1.52 (1.59), 1.54 (1.70), 1.53 (1.50) and 1.62 (1.70), respectively and the corresponding ORs for hypertension were 1.68 (1.55), 1.66 (1.51), 1.45 (1.28) and 1.63 (1.50). For diabetes the OR for BMI did not differ from that for WC or WHR, but was lower than that for WSR (p = 0.001) in men while in women the ORs were higher for WC and WSR than for BMI (both p < 0.05). Hypertension was more strongly associated with BMI than with WHR in men (p < 0.001) and most strongly with BMI than with WHR (p < 0.001), WSR (p < 0.01) and WC (p < 0.05) in women. The HRs for incidence of diabetes and hypertension did not differ between BMI and the other three central obesity measures in Mauritian Indians and Mauritian Creoles during follow-ups of 5, 6 and 11 years. The prevalence of diabetes was highest in Asian Indians, lowest in Europeans and intermediate in others, given the same BMI or WC category. The  coefficients for diabetes in BMI (kg/m2) were (men/women): 0.34/0.28, 0.41/0.43, 0.42/0.61, 0.36/0.59 and 0.33/0.49 for Asian Indian, Chinese, Japanese, Mauritian Indian and European (overall homogeneity test: p > 0.05 in men and p < 0.001 in women). Similar results were obtained in WC (cm). Asian Indian women had lower  coefficients than women of other ethnicities. The change points for BMI were 29.5, 25.6, 24.0, 24.0 and 21.5 in men and 29.4, 25.2, 24.9, 25.3 and 22.5 (kg/m2) in women of European, Chinese, Mauritian Indian, Japanese, and Asian Indian descent. The change points for WC were 100, 85, 79 and 82 cm in men and 91, 82, 82 and 76 cm in women of European, Chinese, Mauritian Indian, and Asian Indian. The prevalence of central obesity using the 2005 IDF definition was higher in Japanese men but lower in Japanese women than in their Asian counterparts. The prevalence of central obesity was 52 times higher in Japanese men but 0.8 times lower in Japanese women compared to the National Cholesterol Education Programme definition. The findings suggest that both BMI and WC predicted diabetes and hypertension equally well in all ethnic groups. At the same BMI or WC level, the prevalence of diabetes was highest in Asian Indians, lowest in Europeans and intermediate in others. Ethnic- and sex-specific change points of BMI and WC should be considered in setting diagnostic criteria for obesity to detect undiagnosed or newly diagnosed diabetes.

Mutation analysis of TGF-β signaling pathway genes among Finnish patients with primary pulmonary hypertension and hereditary hemorrhagic telangiectasia

Primary pulmonary hypertension (PPH), or according to the recent classification idiopathic pulmonary hypertension (IPAH), is a rare, progressive disease of pulmonary vasculature leading to pulmonary hypertension and right heart failure. Most of the patients are sporadic but in about 6% of cases the disease is familial (FPPH). In 2000 two different groups identified the gene predisposing to PPH. This gene, Bone morphogenetic protein receptor type 2 (BMPR2), encodes a subunit of transforming growth factor β (TGF-β) receptor complex. There is a genetic connection between PPH and hereditary hemorrhagic telangiectasia (HHT), a bleeding disorder characterized by local telangiectasias and sometimes with pulmonary hypertension. In HHT, mutations in ALK1 (activin like kinase type 1) and Endoglin, another members of the TGF-β signaling pathway are found. In this study we identified all of the Finnish PPH patients for the years 1986-1999 using the hospital discharge registries of Finnish university hospitals. During this period we found a total of 59 confirmed PPH patients: 55 sporadic and 4 familial representing 3 different families. In 1999 the prevalence of PPH was 5.8 per million and the annual incidence varied between 0.2-1.3 per million. Among 28 PPH patients studied, heterozygous BMPR2 mutations were found in 12% (3/26) of sporadic patients and in 33% of the PPH families (1/3). All the mutations found were different. Large deletions of BMPR2 were excluded by single-stranded chain polymomorphism analysis. As a candidate gene approach we also studied ALK1, Endoglin, Bone Morphogenetic Receptor Type IA (BMPR1A or ALK3), Mothers Against Decapentaplegic Homolog 4 (SMAD4) and Serotonine Transporter Gene (SLC6A4) using single-strand conformational polymorphism (SSCP) analysis and direct sequencing. Among patients and family members studied, we found two mutations in ALK1 in two unrelated samples. We also identified all the HHT patients treated at the Department of Otorhinolaryngology at Helsinki University Central Hospital between the years of 1990-2005 and 8 of the patients were studied for Endoglin and ALK1 mutations using direct sequencing. A total of seven mutations were found and all the mutations were different. The absence of a founder mutation in the Finnish population in both PPH and HHT was somewhat surprising. This suggests that the mutations of BMPR2, ALK1 and Endoglin are quite young and the older mutations have been lost due to repetitive genetic bottlenecks and/or negative selection. Also, other genes than BMPR2 may be involved in the pathogenesis of PPH. No founder mutations were found in PPH or HHT and thus no simple genetic test is available for diagnostics.

Fibroblast nemosis in malignant progression of epithelial cells

Paracrine regulation between the components of the tumour microenvironment cancer cells, activated fibroblasts, immune and endothelial cells is under intense investigation. The signals between the different cell types are mediated by soluble factors, such as growth factors, proinflammatory cytokines and proteolytic enzymes. Nemosis is an experimental in vitro model of fibroblast activation, leading to increased production of such mediators. Nemotic activation of fibroblasts occurs as they are forced to cluster thereby forming a multicellular spheroid. The aim of the present studies was to elucidate the mechanisms underlying the nemotic response of cancer-associated fibroblasts (CAF) and the role of nemosis in paracrine regulation between activated fibroblasts and benign and malignant epithelial cells. The results presented in this thesis demonstrate that the nemotic response of CAFs and normal fibroblasts differs, and inter-individual variations exist between fibroblast populations. In co-culture experiments, fibroblasts increased colony formation of squamous cell carcinoma (SCC) cells, and CAFs further augmented this, highlighting the tumour-evolving properties of CAFs. Furthermore, fibroblast monolayers in those co-cultures started to cluster spontaneously. This kind of spontaneous nemosis response might take place also in vivo, although more direct evidence of this still needs to be obtained. The HaCaT skin carcinoma progression model was used to study the effects of benign and malignant keratinocytes on fibroblast nemosis. Benign HaCaT cells inhibited fibroblast nemosis, observed as inhibition of cyclooxygenase 2 (COX-2) induction in nemotic spheroids. In contrast, malignant HaCaTs further augmented the nemotic response by increasing expression of COX-2 and the growth factors hepatocyte growth factor / scatter factor (HGF/SF) and vascular endothelial growth factor (VEGF), as well as causing a myofibroblastic differentiation of nemotic fibroblasts into fibroblasts resembling CAFs. On the other side of this reciprocal signalling, factors secreted into conditioned medium by the nemotic fibroblasts promoted proliferation and motility of the HaCaT cell lines. Notably, the nemotic fibroblast medium increased the expression of p63, a transcription factor linked to carcinogenesis, also in the highly metastatic HaCaT cells. These results emphasize the paracrine role of factors secreted by activated fibroblasts in driving tumour progression. We also investigated the epithelial-mesenchymal transition (EMT) of the HaCaT clones in response to transforming growth factor β (TGF-β), which is a well-characterized inducer of EMT. TGF-β caused growth arrest and loss of epithelial cell junctions in the HaCaT derivatives, but mesenchymal markers were not induced, suggesting a partial, but not complete EMT response. Inflammation induced by COX-2 has been proposed to be a key mechanism in EMT of benign cells. Corroborating this notion, COX-2 was induced only in benign, not in malignant HaCaT derivatives. Furthermore, in cells in which TGF-β caused COX-2 induction, migration was clearly augmented. The concept of treating cancer is changing from targeting solely the cancer cells to targeting the whole microenvironment. The results of this work emphasise the role of activated fibroblasts in cancer progression and that CAFs should also be taken into consideration in the treatment of cancer. The results from these studies suggests that nemosis could be used as a diagnostic tool to distinguish in vitro activated fibroblasts from tumour stroma and also in studying the paracrine signalling that is mediated to other cell types via soluble factors.

Molecular profiling of malignant pleural mesothelioma and pulmonary fibrosis

Malignant mesothelioma (MM) is a rare, usually incurable, disease mainly caused by former exposure to asbestos. Even though MM has a strong etiological link, genetic factors may play a role, since not all cases can be linked to former asbestos exposure. This thesis focuses on lung diseases, mainly malignant mesothelioma (MM), and idiopathic pulmonary fibrosis (IPF), which resembles asbestosis. The specific asbestos-related pathways associated with malignant as well as non-malignant lung diseases, still need to be clarified. Since most patients diagnosed with MM or asbestosis/fibrosis have a dismal prognosis and few therapeutic options are available, early diagnosis and better understanding of the disease pathogenesis are of the utmost importance. The first objective of this thesis was to identify asbestos specific differentially expressed genes. This was approached by using high-resolution gene expression arrays, and three different human lung cell lines, as well as with three different bioinformatics approaches. Since the first study aimed to elucidate potential early changes, the second study was used to screen DNA copy number changes in MM tumour samples. This was performed using genome wide microarrays for identification of DNA copy number changes characterstic for MM. Study III focused on the role of gremlin in the regulation of bone morphogenetic protein (BMPs) in IPF. Further studies were conducted in asbestos-exposed cell cultures as well as in an asbestos-induced mouse model. Furthermore, GATA-6 was studied in MM and metastatic pleural adenocarcinoma. The GATA transcription factors are important during embryonic development, but their role in cancer is still unclear. GATA-6 is a co-factor/target of thyroid transcription factor 1 (TTF-1), which is used in differential diagnostics of pleural MM and adenocarcinoma. Bioinformatics probed the genes and biological processes ordered in terms of significance, clusters, and highly enriched chromosomal regions. The study revealed several already identified targets, produced new ideas about genes which are central for asbestos exposure, as well as provided supplementary data for researchers to check their own novel findings or ideas. The analysis revealed DNA copy number changes characteristic for MM tumors. The most common regions of loss were detected in 1p, 3p, 6q, 9p, 13, 14, and 22, and gains at 17q. The histological features in asbestosis and IPF are very similar, wherefore IPF can be studied in asbestos models. The BMP antagonist gremlin was up-regulated by asbestos exposure in human epithelial cell lines, which was also observed in Study I. The transforming growth factor (TGF) -β and BMP expression and signaling activities were measured from murine and human fibrotic lungs. BMP-7 signaling was down-regulated in response to up-regulation of gremlin, and restoration of BMP-7 signaling prevented progression of fibrosis in mice. Therefore, the study suggests that the restoration of BMP-7 signaling in fibrotic lung could potentially aid in the treatment of IPF patients. Study IV revealed that GATA-6 was strongly expressed in the majority of the MM cases, and correlated statistically significant with longer survival in subgroups of MM.

Exclusion of angiotensinogen gene in molecular basis of human hypertension: Sibpair linkage and association analyses in Australian anglo-caucasians

Linkage with essential hypertension has been claimed for a microsatellite marker near the angiotensinogen gene (AGT; chromosome 1q42), as has association for the AGT variants M235T, G(-6)A and A(-20)C. To more rigorously evaluate AGT as a candidate gene for hypertension we performed sibpair analysis with multiple microsatellite markers surrounding this locus and using more sophisticated analysis programs. We also performed an association study of the AGT variants in unrelated subjects with a strong family history (two affected parents). For the linkage study, single and multiplex polymerase chain reaction (PCRs) and automated genescan analysis were conducted on DNA from 175 Australian Anglo-Celtic Caucasian hypertensives for the following markers: D1S2880-(2.1 cM)-D1S213-(2.8 cM)-D1S251-(6.5 cM)-AGT-(2.0 cM) -D1S235. Statistical evaluation of genotype data by nonparametric methods resulted in the following scores: Single-point analysis - SPLINK, P > 0.18; APM method, P > 0.25; ASPEX, MLOD < 0.28; SIB-PAIR, P > 0. 24; Multipoint analysis - MAPMAKER/SIBS, MLOD < 0.24; GENEHUNTER, P > 0.35. Exclusion scores of Lod -4.1 to -5.1 were obtained for these markers using MAPMAKER/SIBS for a lambda(s) of 1.6. The association study of G(-6)A, A(-20)C and M235T variants in 111 hypertensives with strong family history and 190 normotensives with no family history showed significant linkage disequilibrium between particular haplotypes, but we could find no association with hypertension. The present study therefore excludes AGT in the etiology of hypertension, at least in the population of Australian Anglo-Celtic Caucasians studied.

G-protein beta3 subunit gene (GNB3) variant in causation of essential hypertension

-Essential hypertensives display enhanced signal transduction through pertussis toxin-sensitive G proteins. The T allele of a C825T variant in exon 10 of the G protein beta3 subunit gene (GNB3) induces formation of a splice variant (Gbeta3-s) with enhanced activity. The T allele of GNB3 was shown recently to be associated with hypertension in unselected German patients (frequency=0.31 versus 0.25 in control). To confirm and extend this finding in a different setting, we performed an association study in Australian white hypertensives. This involved an extensively examined cohort of 110 hypertensives, each of whom were the offspring of 2 hypertensive parents, and 189 normotensives whose parents were both normotensive beyond age 50 years. Genotyping was performed by polymerase chain reaction and digestion with BseDI, which either cut (C allele) or did not cut (T allele) the 268-bp polymerase chain reaction product. T allele frequency in the hypertensive group was 0.43 compared with 0.25 in the normotensive group (chi2=22; P=0.00002; odds ratio=2.3; 95% CI=1.7 to 3.3). The T allele tracked with higher pretreatment blood pressure: diastolic=105+/-7, 109+/-16, and 128+/-28 mm Hg (mean+/-SD) for CC, CT, and TT, respectively (P=0.001 by 1-way ANOVA). Blood pressures were higher in female hypertensives with a T allele (P=0.006 for systolic and 0.0003 for diastolic by ANOVA) than they were in male hypertensives. In conclusion, the present study of a group with strong family history supports a role for a genetically determined, physiologically active splice variant of the G protein beta3 subunit gene in the causation of essential hypertension.

Influences of Allee effects in the spreading of malignant tumours

A recent study by Korolev et al. [Nat. Rev. Cancer, 14:371–379, 2014] evidences that the Allee effect—in its strong form, the requirement of a minimum density for cell growth—is important in the spreading of cancerous tumours. We present one of the first mathematical models of tumour invasion that incorporates the Allee effect. Based on analysis of the existence of travelling wave solutions to this model, we argue that it is an improvement on previous models of its kind. We show that, with the strong Allee effect, the model admits biologically relevant travelling wave solutions, with well-defined edges. Furthermore, we uncover an experimentally observed biphasic relationship between the invasion speed of the tumour and the background extracellular matrix density.

Matrix metalloproteinases as biomarkers in premalignant and malignant tumors of the human skin

The incidence of non-melanoma skin cancer is increasing worldwide. Basal cell carcinoma followed by squamous cell carcinoma and malignant melanoma are the most frequent skin tumors. Immunosuppressed patients have an increased risk of neoplasia, of which non-melanoma skin cancer is the most common. Matrix metalloproteinases (MMPs) are proteolytic enzymes that collectively are capable of degrading virtually all components of the extracellular matrix. MMPs can also process substrates distinct from extracellular matrix proteins and influence cell proliferation, differentiation, angiogenesis, and apoptosis. MMP activity is regulated by their natural inhibitors, tissue inhibitors of metallopro-teinases (TIMPs). In this study, the expression patterns of MMPs, TIMPs, and certain cancer-related molecules were investigated in premalignant and malignant lesions of the human skin. As methods were used immunohistochemisty, in situ hybridization, and reverse transcriptase polymerase chain reaction (RT-PCR) from the cell cultures. Our aim was to evaluate the expression pattern of MMPs in extramammary Paget's disease in order to find markers for more advanced tumors, as well as to shed light on the origin of this rare neoplasm. Novel MMPs -21, -26, and -28 were studied in melanoma cell culture, in primary cutaneous melanomas, and their sentinel nodes. The MMP expression profile in keratoacanthomas and well-differentiated squamous cell carcinomas was analyzed to find markers to differentiate benign keratinocyte hyperproliferation from malignantly transformed cells. Squamous cell carcinomas of immunosuppressed organ transplant recipients were compared to squamous cell carcinomas of matched immunocompetent controls to investigate the factors explaining their more aggressive nature. We found that MMP-7 and -19 proteins are abundant in extramammary Paget's disease and that their presence may predict an underlying adenocarcinoma in these patients. In melanomas, MMP-21 was upregulated in early phases of melanoma progression, but disappeared from the more aggressive tumors with lymph node metastases. The presence of MMP-13 in primary melanomas and lymph node metastases may relate to more aggressive disease. In keratoacanthomas, the expression of MMP-7 and -9 is rare and therefore should raise a suspicion of well-differentiated squamous cell carcinomas. Furthermore, MMP-19 and p16 were observed in benign keratinocyte hyperproliferation of keratoacanthomas, whereas they were generally lost from malignant keratinocytes of SCCs. MMP-26 staining was significantly stronger in squamous cell carcinomas and Bowen s disease samples of organ transplant recipients and it may contribute to the more aggressive nature of squamous cell carcinomas in immunosuppressed patients. In addition, the staining for MMP-9 was significantly stronger in macrophages surrounding the tumors of the immunocompetent group and in neutrophils of those patients on cyclosporin medication. In conclusion, based on our studies, MMP-7 and -19 might serve as biomarkers for more aggressive extramammary Paget's disease and MMP-21 for malignant transformation of melanocytes. MMP -7, -9, and -26, however, could play an important role in the pathobiology of keratinocyte derived malignancies.

Biology and Molecular Markers of Malignant Gonadal Germ Cell Tumors

Germ cell tumors occur both in the gonads of both sexes and in extra-gonadal sites during adoles-cence and early adulthood. Malignant ovarian germ cell tumors are rare neoplasms accounting for less than 5% of all cases of ovarian malignancy. In contrast, testicular cancer is the most common malignancy among young males. Most of patients survive the disease. Prognostic factors of gonadal germ cell tumors include histology, clinical stage, size of the primary tumor and residua, and levels of tumor markers. Germ cell tumors include heterogeneous histological subgroups. The most common subgroup includes germinomas (ovarian dysgerminoma and testicular seminoma); other subgroups are yolk sac tumors, embryonal carcinomas, immature teratomas and mixed tumors. The origin of germ cell tumors is most likely primordial germ cells. Factors behind germ cell tumor development and differentiation are still poorly known. The purpose of this study was to define novel diagnostic and prognostic factors for malignant gonadal germ cell tumors. In addition, the aim was to shed further light into the molecular mechanisms regulating gonadal germ cell tumorigenesis and differentiation by studying the roles of GATA transcription factors, pluripotent factors Oct-3/4 and AP-2γ, and estrogen receptors. This study revealed the prognostic value of CA-125 in malignant ovarian germ cell tumors. In addition advanced age and residual tumor had more adverse outcome. Several novel markers for histological diagnosis were defined. In the fetal development transcription factor GATA-4 was expressed in early fetal gonocytes and in testicular carcinoma precursor cells. In addition, GATA-4 was expressed in both gonadal germinomas, thus it may play a role in the development and differentiation of the germinoma tumor subtype. Pluripotent factors Oct-3/4 and AP-2γ were expressed in dysgerminomas, thus they could be used in the differential diagnosis of the germ cell tumors. Malignant ovarian germ cell tumors expressed estrogen receptors and their co-regulator SNURF. In addition, estrogen receptor expression was up-regulated by estradiol stimulation. Thus, gonadal steroid hormone burst in puberty may play a role in germ cell tumor development in the ovary. This study shed further light in to the molecular pathology of malignant gonadal germ cell tumors. In addition, some novel diagnostic and prognostic factors were defined. This data may be used in the differential diagnosis of germ cell tumor patients.

KAT5 (Tip60) is a potential therapeutic target in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is a rare aggressive cancer of the pleura. Asbestos exposure (through inhalation) is the most well established risk factor for mesothelioma. The current standard of care for patients suffering from MPM is a combination of cisplatin and pemetrexed (or alternatively cisplatin and raltitrexed). Most patients, however, die within 24 months of diagnosis. New therapies are therefore urgently required for this disease. Lysine acetyltransferases (KATs) including KAT5 have been linked with the development of cisplatin resistance. This gene may therefore be altered in MPM and could represent a novel candidate target for intervention. Using RT-PCR screening the expression of all known KAT5 variants was found to be markedly increased in malignant tumors compared to benign pleura. When separated according to histological subtype, KAT5 was significantly overexpressed in both the sarcomatoid and biphasic subgroups for all transcript variants. A panel of MPM cell lines including the normal pleural cells LP9 and Met5A was screened for expression of KAT5 variants. Treatment of cells with a small molecule inhibitor of KAT5 (MG-149) caused significant inhibition of cellular proliferation (p<0.0001), induction of apoptosis and was accompanied by significant induction of pro-inflammatory cytokines/chemokines.