Milica Labudović Borović

Effects of Fullerenol C60(OH)24 Nanoparticles on a Single-dose Doxorubicin-induced Cardiotoxicity in Pigs: An Ultrastructural Study

Abstract Cardioprotective effects of fullerenol C60(OH)24 nanoparticles (FNP) were investigated in pigs after a single treatment with doxorubicin (DOX). Semithin and ultrathin sections of myocardial tissue routinely prepared for transmission electron microscopy were analyzed. Extensive intracellular damage was confirmed in cardiomyocytes of DOX-treated animals. By means of ultrastructural analysis, a certain degree of parenchymal degeneration was confirmed even in animals treated with FNP alone, including both the oral and the intraperitoneal application of the substance. The cardioprotective effects of FNP in animals previously treated with DOX were recognized to a certain extent, but were not fully confirmed at the ultrastructural level. Nevertheless, the myocardial morphology of DOX-treated animals improved after the admission of FNP. Irregular orientation of myofibrils, myofibrillar disruption, intracellular edema, and vacuolization were reduced, but not completely eliminated. Reduction of these cellular alterations was achieved if FNP was applied orally 6 h prior to DOX treatment in a dose of 18 mg/kg. However, numerous defects, including the inner mitochondrial membrane and the plasma membrane disruption of certain cells persisted. In FNP/DOX-treated animals, the presence of multinuclear cells with mitosis-like figures resembling metaphase or anaphase were observed, indicating that DOX and FNP could have a complex influence on the cell cycle of cardiomyocytes. Based on this experiment, further careful increase in dosage may be advised to enhance FNP-induced cardioprotection. These investigations should, however, always be combined with ultrastructural analysis. The FNP/DOX interaction is an excellent model for the investigation of cardiomyocyte cell death and cell cycle mechanisms.

Skin and Sural Nerve Biopsies: Ultrastructural Findings in the First Genetically Confirmed Cases of CADASIL in Serbia

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited vascular disorder caused by Notch3 gene mutations. The main histopathological hallmark is granular osmiophilic material (GOM) deposited in the close vicinity of vascular smooth muscle cells (VSMCs). The authors report the first 7 ultrastructurally and genetically confirmed cases of CADASIL in Serbia. Samples of skin and sural nerve were investigated by transmission electron microscopy. GOM deposits were observed around degenerated VSMCs in all the skin biopsies examined. Sural nerve biopsies revealed severe alterations of nerve fibers, endoneurial blood vessels with GOM deposits, endoneurial fibroblasts, and perineurial myofibroblasts. Total genomic DNA was extracted from peripheral blood leukocytes, and exons 2–6 of the Notch3 gene were amplified by PCR and subsequently sequenced. Four different mutations in exons 2 (Cys65Tyr), 3 (Gly89Cys and Arg90Cys), and 6 (Ala319Cys), which determine the CADASIL disease, were detected among all described patients. A novel missense mutation Gly89Cys involving exon 3 was detected. Due to the difficulties in the determination of the Notch3 mutations, these data suggest that electron microscopic analysis for GOMs in dermal vessel wall provides a rapid and reliable screening method for this disease.

Coronary thrombi neovascularization in patients with ST-elevation myocardial infarction - clinical and angiographic implications

INTRODUCTION Coronary artery thrombosis in ST-elevation myocardial infarction (STEMI) is a dynamic process often preceded by episodes of silent plaque rupture and subocclusive thrombosis. Thrombus organization is achieved by ingrowth of endothelial and smooth muscle cells. Clinical significance and impact of thrombus neovascularization on primary percutaneous coronary intervention (pPCI) outcome remain unclear. Therefore we investigated composition and neovascularization of thrombi aspirated during pPCI and their association with clinical and angiographic parameters of STEMI patients. METHODS Aspirated thrombi retrieved from 84 STEMI patients were classified as fresh (<1 day), lytic (1-5 days) or organized (>5 days). Thrombus neovascularization was evaluated immunohistochemically using CD34, CD31 and VEGF antibodies. CD34 and CD31 immunopositive (CD34/CD31+) cells were organized as single, clusters and microvessels. VEGF positivity was graded as low or high, based on thrombus surface immunopositive area. RESULTS CD34/CD31+ cells were present in 67% of all aspirated thrombi. Thrombus CD34/CD31 positivity was associated with previous history of angina pectoris (χ(2)=6.142, p=0.013) and lower myocardial blush grade (MBG<3, χ(2)=12.602, p<0.001). Organization of CD34/CD31+ cells showed inverse association with the extent of VEGF positivity (χ(2)=10.607, p=0.005). Fresh thrombi were associated with shorter ischemic time (U=237.5, p=0.002) and MBG 3 (χ(2)=6.379, p=0.012). CONCLUSIONS Older thrombus age and neovascularization are associated with suboptimal myocardial perfusion in STEMI patients. Thrombus VEGF expression is inversely associated with degree of CD34+ cell organization. Therefore, neovascularization of aspirated thrombi may indicate the duration of thrombosis, coronary microcirculation status and outcome in STEMI patients.

Hepatoprotective effect of fullerenol/doxorubicin nanocomposite in acute treatment of healthy rats

In our recent studies we have designed fullerenol/doxorubicin nanocomposite (FNP/DOX) as the new drug nanocarrier. This research has demonstrated that this novel nanocomposite has had better implications on the liver tissue in vivo (Wistar rats treated intraperitoneally), than treatment based only on DOX. FNP/DOX has been characterised by DLS, TEM and AFM measurements which have shown that DOX loaded onto FNP did not influence fullerenol nanoparticles size. FNP/DOX affected oxidative status in blood causing a significant decrease of catalase and SOD activity in comparison to DOX, implicating the reduction in oxidative stress. qRT-PCR results on the mRNA level of antioxidative enzymes (catalase and MnSOD) revealed that the effect of oxidative stress is significantly reduced by the treatment with FNP/DOX (p < .05). The ultrastructural analysis of the liver tissue has revealed that FNP/DOX nanocomposite generated considerably less damage in the liver tissue, than DOX applied at the same dose. Hence, our results have indicated that FNP, within FNP/DOX nanocomposite, exhibits protective effects to the liver tissue of the healthy rats.

The randomized physiologic assessment of thrombus aspiration in patients with acute ST-segment elevation myocardial infarction trial (PATA STEMI): study rationale and design.

INTRODUCTION Routine thrombus aspiration is proposed to be superior to conventional primary percutaneous coronary intervention (PCI) in terms of improved myocardial perfusion, in patients with ST-segment elevation acute myocardial infarction (STEMI). However, myocardial perfusion after thrombus aspiration has not been evaluated by a quantitative, invasive method. We intend to determine whether manual thrombus aspiration in the infarct-related coronary artery increases myocardial perfusion assessed by index of microcirculatory resistance (IMR) compared with conventional primary PCI. STUDY DESIGN PATA STEMI is a single-center, prospective, randomized trial with a planned inclusion of 128 patients with the first STEMI. Prior to coronary angiography, patients are randomly assigned to thrombus aspiration using the Eliminate aspiration catheter (Terumo Medical Supply, Japan) or to conventional primary PCI. After completion of primary PCI, IMR is determined both in infarct-related artery and in noninfarct-related arteries without critical stenoses. The primary end-point is a group mean value of IMR after thrombus aspiration compared with conventional primary PCI. Secondary end-points are myocardial perfusion grade, resolution of ST-segment elevation, enzymatic estimation of infarct size, left ventricular remodeling assessed by echocardiographic indices, and major adverse cardiac events rate at 1, 6, and 12 months. CONCLUSION If manual thrombus aspiration significantly reduces microcirculatory resistance, thereby improving myocardial perfusion, it may become the routine strategy in primary PCI.

Contractions of the whole and longitudinally cut rat's portal vein

To investigate the role of lateral interactions, we quantified spontaneous contractions of whole and longitudinally cut rat´s portal vein in vitro. The disruption of the wall had no effect on basic frequency determined from spectra and complexity index (CI) calculated by multiscale entropy analysis. Endothelium was disrupted and nonfunctional in all samples. Considering amplitude, frequency and CI we identified two modes of contractions. Neither mode of contractions nor the effect of aminopyridine (4-AP) depended on the integrity of the wall. We concluded that contractions in vitro originate in smooth muscle cells without involvement of the endothelium and lateral interactions.

Involvement of PI3K, Akt, and RhoA in oestradiol regulation of cardiac iNOS expression

BACKGROUND Oestradiol is an important regulatory factor with several positive effects on the cardiovascular (CV) system. We evaluated the molecular mechanism of the in vivo effects of oestradiol on the regulation of cardiac inducible nitric oxide (NO) synthase (iNOS) expression and activity. METHODS Male Wistar rats were treated with oestradiol (40 mg/kg, intraperitoneally) and after 24 h the animals were sacrificed. The concentrations of NO and L-Arginine (L-Arg) were determined spectrophotometrically. For protein expressions of iNOS, p65 subunit of nuclear factor-κB (NFκB-p65), Ras homolog gene family-member A (RhoA), angiotensin II receptor type 1 (AT1R), insulin receptor substrate 1 (IRS-1), p85, p110 and protein kinase B (Akt), Western blot method was used. Coimmunoprecipitation was used for measuring the association of IRS-1 with the p85 subunit of phosphatidylinositol- 3-kinase (PI3K). The expression of iNOS messenger ribonucleic acid (mRNA) was measured with the quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemical analysis of the tissue was used to detect localization and expression of iNOS in heart tissue. RESULTS Oestradiol treatment reduced L-Arg concentration (p<0.01), iNOS mRNA (p<0.01) and protein (p<0.001) expression, level of RhoA (p<0.05) and AT1R (p<0.001) protein. In contrast, plasma NO (p<0.05), Akt phosphorylation at Thr308 (p<0.05) and protein level of p85 (p<0.001) increased after oestradiol treatment. CONCLUSION Our results suggest that oestradiol in vivo regulates cardiac iNOS expression via the PI3K/Akt signaling pathway, through attenuation of RhoA and AT1R.

Myocardial Na+ K+-ATPase and SERCA: Clinical and Pathological Significance From a Cytological Perspective

Structure and functions of Na+/K+-ATPase and SERCA are described with details on their subunits, isoforms, and intracellular localization. Main regulatory mechanisms are summarized. Molecular mechanisms of cell death and heart failure are explained with the analysis of the role of Na+/K+-ATPase and SERCA in these processes. Facts are considered from a cytological, pathological, and clinical perspective with an accent to new therapeutic strategies. The aim of this contribution is an overview of functional results in a structural context.