Biological and Pro-Angiogenic Properties of Genetically Modified Human Primary Myoblasts Overexpressing Placental Growth Factor in In Vitro and In Vivo Studies

Cardiovascular diseases are a growing problem in developing countries; therefore, there is an ongoing intensive search for new approaches to treat these disorders. Currently, cellular therapies are focused on healing the damaged heart by implanting stem cells modified with pro-angiogenic factors. This approach ensures that the introduced cells are capable of fulfilling the complex requirements of the environment, including the replacement of the post-infarction scar with cells that are able to contract and promote the formation of new blood vessels that can supply the ischaemic region with nutrients and oxygen. This study focused on the genetic modification of human skeletal muscle cells (SkMCs). We chose myoblast cells due to their close biological resemblance to cardiomyocytes and the placental growth factor (PlGF) gene due to its pro-angiogenic potential. In our in vitro studies, we transfected SkMCs with the PlGF gene using electroporation, which has previously been proven to be efficient and generate robust overexpression of the PlGF gene and elevate PlGF protein secretion. Moreover, the functionality of the secreted pro-angiogenic proteins was confirmed using an in vitro capillary development assay. We have also examined the influence of PlGF overexpression on VEGF-A and VEGF-B, which are well-known factors described in the literature as the most potent activators of blood vessel formation. We were able to confirm the overexpression of VEGF-A in myoblasts transfected with the PlGF gene. The results obtained in this study were further verified in an animal model. These data were able to confirm the potential therapeutic effects of the applied treatments.

     

Vitamin D receptor binding to DNA is altered without the change in its expression in human renal clear cell cancer

Vitamin D co-regulates cell proliferation, differentiation and apoptosis, the processes that are disturbed in cancer tissues. It acts through the vitamin D nuclear receptor (VDR) that binds to DNA in the regulatory sequences of the target genes. As the kidney is one of the key organs for vitamin D metabolism and action, we analyzed VDR expression and its DNA binding activity in human renal clear cell cancer. 24 tumors, 24 controls that were excised from the opposite pole of the same kidney and 7 controls originating from kidneys without cancer were examined. Independently of tumor grading neither Northern blots nor immunoblotting demonstrated statistically significant differences of the mean VDR mRNA and protein amounts, respectively, in the cancer as compared to both control types. In contrast, the amount of VDR-DNA complexes was lower in 52.2% of the tumors in comparison to their corresponding controls. After normalization against VDR receptor protein amount in 34.8% of the tumors VDR-DNA binding was at least 3-4 times weaker than in the controls. However, the expression of vitamin D-dependent P21 gene on the mRNA level was not decreased in these cancers. It remains to be elucidated if altered VDR function due to its impaired binding to DNA contributes to the process of tumorigenesis, and what potential vitamin D-dependent mechanisms are involved in this process.     

The kinetics and apoptotic profile of circulating endothelial cells in autologous hematopoietic stem cell transplantation in patients with lymphoproliferative disorders

In neoplastic disorders, endothelial cells take part in tumor progression and also influence the recovery of hematopoiesis after high-dose chemotherapy. Measurements of circulating endothelial cells (CEC), their subsets and kinetics were taken in patients with lymphoid malignancies (37 multiple myeloma, ten lymphoma) during autologous hematopoietic stem cell transplantation (HSCT). CEC were evaluated by four-color flow cytometry at different time points. Additionally levels of angiopoietins 1 and 2 were evaluated by ELISA assay. The baseline number of CECs and their subsets in patients were higher than in the control group. The median CEC number dropped significantly after transplantation (from 9.5/μL to 6.2/μL, p?<?0.001). Apoptosis of CECs 24 h after chemotherapy was enhanced in comparison to baseline values (median apoptotic CEC number 4.15/μL vs 3.1/μL; p?<?0,001). The time for neutrophil engraftment was shorter for patients with a low apoptotic CEC count at baseline as compared to those with a high apoptotic CEC count (median time to engraftment 13 vs. 16 days respectively, p?=?0.04). We observed an adverse correlation of progenitor CEC numbers measured 1 h after transplantation with the time to neutrophil engraftment (r?=??0.49, p?=?0.008). We also found a negative correlation between the number of CECs originating from microvessels measured 1 h after transplantation, and the time to neutrophil engraftment (r?=??0.39, p?=?0.04). Baseline angiopoietins 1 and 2 concentration did not influence the post-transplant regeneration time. CEC numbers significantly change during autologous HSCT. Our results suggest that progenitor CECs and CECs derived from microvessels both take part in successful engraftment.     

Severe hyperandrogenemia in postmenopausal woman as a presentation of ovarian hyperthecosis. Case report and mini review of the literature

Ovarian hyperthecosis (OH) is characterized by the presence of abundant luteinized theca cells in ovaries that secret androgen. It typically presents as severe hyperandrogenism and/or virilization in postmenopausal woman. Here we describe a 66-year old woman with presentation of severe hirsutism, alopecia, clitoromegaly and laboratory finding of significantly elevated serum total testosterone concentration and hyperinsulinemia. Performed imaging studies revealed normal sized, homogeneous ovaries, signs of endometrial hypertrophy and normal adrenal glands. Due to severe hyperandrogenemia and signs of endometrial hypertrophy, the total abdominal hysterectomy with bilateral salpingo-oophorectomy has been performed. Pathological examination revealed OH and endometrial hyperplasia. Androgenic activity of ovarian stromal cells has been confirmed using alpha-inhibin histochemical staining. Postmenopausal hyperandrogenemia is a diagnostic and therapeutic challenge and the imaging studies often may be misleading and require careful and critical consideration.     

The polymorphisms of methionine synthase (MTR) and methionine synthase reductase (MTRR) genes in pathogenesis of preeclampsia

Purpose: The aim of the study was to determine the MTR (methionine synthase) and MTRR (mehionine synthase reductase) polymorphisms in pregnant women with preeclampsia (PE).

Materials and methods: The group of 98 women with PE and the group of 120 healthy pregnant women were analyzed. Determination of MTR 2756A?>?G and MTRR 66A?>?G polymorphisms was performed using polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) method.

Results: The study did not show any statistically significant differences in frequency of genotypes and alleles of MTR 2756A?>?G polymorphism between PE group and controls. Higher frequency of 66GG genotype and 66G allele of MTRR 66A?>?G polymorphism was observed in the women with PE compared to control group. Moreover, the 66GG genotype correlated with higher doses of methyldopa, lower birth weight and higher placenta weight in women with PE.

Conclusions: The obtained results for 66A?>?G polymorphism of MTRR gene suggest the predisposition to PE in carriers of mutated 66GG genotype and – 66G allele.     

Adiponectin and leptin gene polymorphisms in women with gestational diabetes mellitus

Purpose

Gestational diabetes mellitus (GDM) is the glucose intolerance occurring during pregnancy. The prevalence of GDM is increased in obese women. Leptin and adiponectin are adipokines that play an important role in the regulation of insulin secretion and glucose and lipid metabolism. The aim of this study was to examine the association between adiponectin and leptin gene polymorphisms and the development of GDM.

Methods

This case–control study included 204 pregnant women with GDM and 207 pregnant women with normal glucose tolerance (NGT). The diagnosis of GDM was based on a 75-g oral glucose tolerance test (OGTT) at 24–28 weeks’ gestation. To discriminate the ADIPOQ rs266729, rs1501299 and LEP rs2167270 alleles, TaqMan® Pre-Designed SNP Genotyping Assays were used.

Results

There was a statistically significant association between the ADIPOQ rs266729 gene polymorphism and GDM. Among women with GDM, a higher prevalence of the G allele was observed (GG and CG genotypes). Multivariate logistic regression analysis, taking into account age, BMI before pregnancy, past pregnancies and the ADIPOQ rs266729 gene polymorphism, revealed that the presence of a G allele is an independent risk factor for GDM. Moreover, there was the association between the LEP rs2167270 polymorphism and the requirement for daily insulin, which was significantly higher in women with the A allele (AA and GA genotypes).

Conclusions

The results of our study suggest an association between adiponectin gene rs266729 as well as leptin gene rs2167270 polymorphisms and GDM.

     

Atorvastatin affects several angiogenic mediators in human endothelial cells.

The pleiotropic effects of statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, have been recently extended to the modulation of angiogenesis. Here, to get more insight into the statins action, the authors have investigated the effect of atorvastatin on the expression of several angiogenic and inflammatory genes in human umbilical endothelial cells (HUVECs). Atorvastatin was proangiogenic at the dose of 10 nM, and antiangiogenic at the concentrations of 1 to 10 micro M. Moreover, these higher concentrations inhibited also the proliferation of HUVECs induced by vascular endothelial growth factor (VEGF). Lower doses of atorvastatin did not influence endothelial cell proliferation. Importantly, atorvastatin at the micromolar concentrations diminished the production of interleukin (IL)-8, a proinflammatory and proangiogenic chemokine, and inhibited the synthesis of urokinase plasminogen activator (uPA), a potent proinflammatory mediator. However, it decreased also the expression of plasminogen activator inhibitor-1 (PAI-1) and thrombospondin-1 (TSP-1), the inhibitors of angiogenesis. Atorvastatin stimulated the expression of angiopoietin (Ang)-2 and moderately enhanced the expression of endothelial nitric oxide synthase (eNOS), whereas heme oxygenase-1 (HO-1) was not significantly affected. In conclusion, the present findings points to other angiogenesis-related effects of atorvastatin, which may be of relevance to the beneficial influence of statins in cardiovascular system.     

Month‐of‐birth effect on height and weight in Polish rural children

This study investigated a hypothesis of dependence of child height and weight on the month of their birth. The sample comprised 1,241 subjects, 568 boys and 673 girls, age 6–20 years, from villages in Olsztyn Province, Northeast Poland. Individuals' height and weight data were standardized by sex and age to allow grouping of individuals born in the same month irrespective of their sex and age at examination. Subjects born in October to March proved to be significantly taller and heavier than those born in April to September. The magnitude of differences between the semiannual groupings equaled 13.1% of a standard deviation (SD) in height (P = 0.03) and 14.2% of SD in weight (P = 0.02). The month‐of‐birth effect was much stronger for children characterized by high socioeconomic status, where these differences amounted to 39.1% of SD in height (P = 0.02) and 49.4% of SD in weight (P = 0.01). There were no regular changes of the effect with age and no differences between the sexes were found. Fitted cosine functions identified the highest values of examined traits for individuals born in December with the lowest values being found in those born in June. Possible explanations of the month‐of‐birth effect are considered in terms of age categorizing, seasonal variety of growth rates, as well as birth‐related or conception‐related global, hemispheric, and local factors. This study rejects the first two possibilities and suggests this effect requires further research to be conducted in various geographical locations, climates, and cultures, on humans as well as on other species. Am. J. Hum. Biol. 16:31–42, 2004. © 2003 Wiley‐Liss, Inc.