Milica Labudovic-Borovic

In vivo effects of 17β-estradiol on cardiac Na(+)/K(+)-ATPase expression and activity in rat heart.

In this study the in vivo effects of estradiol in regulating Na(+)/K(+)-ATPase function in rat heart was studied. Adult male Wistar rats were treated with estradiol (40μg/kg, i.p.) and after 24h the animals were sacrificed and the heart excised. Following estradiol administration, cardiac Na(+)/K(+)-ATPase activity, expression of the α1 subunit, and phosphorylation of the α1 subunit were significantly increased. These animals also had significantly decreased levels of digoxin-like immunoreactive factor(s). Na(+) levels were also significantly reduced but to a level that was still within the normal physiological range, highlighting the ability of the Na(+)/K(+)-ATPase to balance the ionic composition following treatment with estradiol. Estradiol treated rats also showed increased phosphorylation of protein kinase B (Akt), and extracellular-signal-regulated kinase 1/2 (ERK1/2). We therefore suggest a role for Akt and/or ERK1/2 in estradiol-mediated regulation of cardiac Na(+)/K(+)-ATPase expression and activity in rat heart.

Estradiol In Vivo Induces Changes in Cardiomyocytes Size in Obese Rats

We studied the in vivo effects of estradiol on size and biochemical parameters of cardiomyocytes in pathophysiological conditions such as obesity and insulin resistance. Male Wistar rats were normally fed (controls, n = 7) or fed with high-fat diet (obese, n = 14). Half of the obese rats (obese + estradiol, n = 7) were treated with a single dose of estradiol (40 μg/kg, intraperitoneally) and 24 hours after treatment all the rats were killed. Estradiol in vivo in obese rats resulted in a significant increase in protein kinase B (Akt) activation (P < .05) and decrease in heart mass (P < .05), ratio of the heart mass/body mass (P < .05), transverse diameters of cardiomyocytes (P < .001), concentration of serum high-sensitivity C-reactive protein (P < .001), and total cholesterol (P < .01) compared with obese nontreated rats. Our results suggest that estradiol in obese/IR rats affects the size of cardiomyocytes and its actions lead in vivo to a reduction in obesity-induced cardiac hypertrophy, via Akt.

Metallophilic macrophages are lacking in the thymus of lymphotoxin-β receptor-deficient mice

Lymphotoxin-β receptor (LTβR) axis plays a crucial role in development and compartmentalization of peripheral lymphatic organs. But, it is also required for the appropriate function and maintenance of structural integrity of the thymus: in LTβR-deficient animals the clonal deletion of autoreactive lymphocytes is impaired and differentiation of thymic medullary epithelial cells is disturbed. In this study, using several markers, we showed that thymic metallophilic macrophages were lacking in LTβR-deficient mice. In tumor necrosis factor receptor-I (p55)-deficient mice (which we used as positive control) thymic metallophilic cells were located, similarly as in normal mice, in the thymic cortico-medullary zone at the junction of cortex and medulla. These findings show that LTβR is necessary for maintenance of metallophilic macrophages in the thymus and provide further evidence that these cells may represent a factor involved in thymic negative selection.

Uric acid metabolism in pre-hypertension and the metabolic syndrome.

In humans uric acid (UA) is the end product of degradation of purines. The handling of UA by the renal system is a complex process which is not fully understood. To date, several urate transporters in the renal proximal tubule have been identified. Among them, urate transporter 1 (URAT1) and a glucose transporter 9 (GLUT9) are considered of greater importance, as potential targets for treatment of hyperuricemia and the potential associated cardio-metabolic risk. Therefore, the recognition of the metabolic pathway of UA and elucidation of occurrence of hyperuricemia may provide important insights about the relationship between UA, pre-hypertension (preHT) and the metabolic syndrome (MetS). We also review the available clinical studies in this field, including experimental studies dealing with the mechanisms of UA transport via different transporters, as well as current treatment options for hyperuricemia in patients with MetS, preHT or cardiovascular risk factors.

Metallophilic macrophages are fully developed in the thymus of autoimmune regulator (Aire)-deficient mice

Thymic metallophilic macrophages represent a significant component in the thymus physiology. Recently, we showed their presence to be dependent on functional lymphotoxin-β receptor (LTβR) signaling pathway. However, it is unknown whether the development of metallophilic macrophages also requires the Autoimmune regulator (Aire) transcription factor, as suggested by some studies for medullary thymic epithelial cells, or perhaps the presence of Aire-expressing thymic epithelial cells themselves. Therefore, we investigated the presence of metallophilic macrophages in Aire-deficient thymus. Our study shows that the metallophilic macrophages are fully developed in the Aire-deficient thymus; their development is not regulated via Aire transcription factor and does not require the presence of Aire-expressing epithelial cells. On the contrary, in alymphoplasia (ALY) mice (deficient in nuclear factor-kappaB-inducing kinase, NIK), which we used as negative control, thymic metallophilic macrophages are completely lacking, similarly as in LTβR-deficient animals. Together, these results show that the development/maintenance of thymic metallophilic macrophages is executed via LTβR circumventing the Aire transcription factor. Thus, we shed a new light on the molecular requirements for development of these cells and also show that LTβR pathway is a common developmental regulator of metallophilic macrophages in different lymphatic organs (i.e., thymus and spleen).

Effects of 17β-estradiol on cardiac Na(+)/K(+)-ATPase in high fat diet fed rats.

The aim of this study was to investigate in vivo effects of estradiol on Na(+)/K(+)-ATPase activity/expression in high fat (HF) diet fed rats. Adult male Wistar rats were fed normally (Control, n = 7) or with a HF diet (Obese, n = 14) for 10 weeks. After 10 weeks, half of the obese rats were treated with estradiol (Obese + Estradiol, n = 7, 40 μg/kg, i.p.) as a bolus injection and 24 h after treatment all the rats were sacrificed. Estradiol in vivo in obese rats in comparison with obese non-treated rats led to a statistically significant increase in concentration of serum Na(+) (p < 0.05), Na(+)/K(+)-ATPase activity (p < 0.01), expression of α1 (p < 0.01) and α2 (p < 0.05) subunit of Na(+)/K(+)-ATPase, both PI3K subunits p85 (p < 0.01), p110 (p < 0.05), and association of IRS-1 with p85 (p < 0.05), while significantly decrease expression of AT1 (p < 0.05) and Rho A (p < 0.01) proteins. Our results suggest that estradiol in vivo in pathophysiological conditions, such as obesity accompanied with insulin resistance stimulates activity and expression of Na(+)/K(+)-ATPase by a mechanism that involves the participation of IRS-1/PI3K/Akt signaling. In addition, the decreasing level of AT1 and Rho A proteins estradiol probably attenuates the detrimental effect of obesity to decreased IRS-1/PI3K association and consequently reduce Na(+)/K(+)-ATPase activity/expression.

Micro- and nanostructures of iridescent wing scales in purple emperor butterflies (Lepidoptera: Apatura ilia and A. iris).

Apatura ilia (Denis and Schiffermüller, 1775) and A. iris (Linnaeus, 1758) are fascinating butterflies found in the Palaearctic ecozone (excepting the north of Africa). The wings of these insects are covered with a great number of two types of scales positioned like roof tiles. Type I scales are on the surface, while type II scales are situated below them. The structural color of the type I scales is recognized only on the dorsal side of both the fore and hind wings of the males of the aforementioned species. Both types of scales are responsible for pigment color of the wings, but iridescence is observed only in the type I scales. The brilliant structural color is due to a multilayer structure. The features of the scales, their dimensions and fine structure were obtained using scanning electron microscopy. Cross sections of the scales were then analyzed by transmission electron microscopy. The scales of the “normal” and clytie forms of A. ilia have a different nanostructure, but are of the same type. A similar type of structure, but with a different morphology, was also noticed in A. iris. The scales of the analyzed species resemble the scales of tropical Morpho butterflies. Microsc. Res. Tech., 2012.

Endothelial cell markers from clinician's perspective

Endothelial cell markers are membrane-bound or cytoplasmic molecules expressed by endothelial cells, which help their easier identification and discrimination from other cell types. During vasculogenesis, endothelial cells differentiate from hemangioblasts to form new blood vessels. With the discovery of endothelial progenitor cells (EPC) and their ability to form new blood vessels, the term vasculogenesis is not only reserved for the embryonic development. Possibility of de novo blood vessel formation from EPC is now widely explored in different ischemic conditions, especially in cardiovascular medicine. Numerous clinical trials have tested enhancing tissue vascularization by delivering hematopoietic cells that expressed endothelial markers. This therapeutic approach proved to be challenging and promising, particularly for patients who have exhausted all conventional therapeutic modalities. Angiogenesis, which refers to the formation of new blood vessels from existing vasculature, is indispensable process during tumor progression and metastasis. Blockage of tumor angiogenesis by targeting and inhibiting endothelial cell has emerged as novel safe and efficacious method to control many advanced malignant diseases. Numerous clinical studies are currently testing new antiangiogenic drugs which target and inhibit endothelial cell markers, receptors or molecules which transmit receptor-mediated signals, therefore inhibiting endothelial cell proliferation, migration and vascular tube formation. Many of these drugs are now widely used in clinical settings as first- or second-line chemotherapy in advanced malignant conditions. So far, these therapeutic approaches gave modest, yet encouraging clinical improvements, prolonging survival and improving functional capacity and quality of life for many terminally ill patients. Here we present the most commonly used endothelial cell markers along with their applicability in contemporary clinical practice.

Role of CCL19/21 and its possible signaling through CXCR3 in development of metallophilic macrophages in the mouse thymus

We have already shown that metallophilic macrophages, which represent an important component in the thymus physiology, are lacking in lymphotoxin-β receptor-deficient mice. However, further molecular requirements for the development and correct tissue positioning of these cells are unknown. To this end, we studied a panel of mice deficient in different chemokine ligand or receptor genes. In contrast to normal mice, which have these cells localized in the thymic cortico-medullary zone (CMZ) as a distinct row positioned between the cortex and medulla, in plt/plt (paucity of lymph node T cells) mice lacking the functional CCL19/CCL21 chemokines, metallophilic macrophages are not present in the thymic tissue. Interestingly, in contrast to the CCL19/21-deficient thymus, metallophilic macrophages are present in the CCR7-deficient thymus. However, these cells are not appropriately located in the CMZ, but are mostly crowded in central parts of thymic medulla. The double staining revealed that these metallophilic macrophages are CCR7-negative and CXCR3-positive. In the CXCL13-deficient thymus the number, morphology and localization of metallophilic macrophages are normal. Thus, our study shows that CCL19/21 and its possible signaling through CXCR3 are required for the development of thymic metallophilic macrophages, whereas the CXCL13–CXCR5 signaling is not necessary.

Co-expression of vascular and lymphatic endothelial cell markers on early endothelial cells present in aspirated coronary thrombi from patients with ST-elevation myocardial infarction.

INTRODUCTION Angiogenesis is the growth of both new vascular and lymphatic blood vessels from the existing vasculature. During this process, blood endothelial cells (BECs) and lymphatic endothelial cells (LECs) express specific markers, which help their discrimination and easier identification. Since the coronary thrombi material aspirated from patients with ST-elevation myocardial infarction (STEMI) proved as good angiogenesis model, we investigated the expression of CD34 and CD31 as BECs markers, and D2-40, LYVE-1 and VEGFR3 as LEC markers in this material. MATERIALS AND METHODS Aspirated thrombi were stained immunohistochemically for CD34, CD31, D2-40, LYVE-1 and VEGFR3. Organizational patterns of immunopositive cells were graded as single cells, clusters or microvessels. Double immunofluorescence for CD31, D2-40, LYVE-1 and VEGRF3 was done. Thrombi were also graded as fresh (<1day old), lytic (1-5days old) and organized (>5days old). RESULTS Serial sections of aspirated thrombi showed concordant BEC and LEC markers immunopositivity. Double immunoflorescence proved co-expression of CD31 and LEC markers on the same cells. Cells expressing LEC markers organized in clusters and microvessels were mainly present in lytic and organized thrombi. CONCLUSION Co-expression of BEC and LEC markers on the same non-tumorous cell during thrombus neovascularization indicates existing in vivo plasticity of endothelial cells under non-tumorous pathological conditions. It also points that CD34 and CD31 on one hand, and D2-40, LYVE-1 and VEGFR3 immunostaining on the other hand, cannot solely be a reliable indicators whether vessel is lymphatic or not.