Anna Ratajska

Cardiotoxicity of the Anticancer Therapeutic Agent Bortezomib

Recent case reports provided alarming signals that treatment with bortezomib might be associated with cardiac events. In all reported cases, patients experiencing cardiac problems were previously or concomitantly treated with other chemotherapeutics including cardiotoxic anthracyclines. Therefore, it is difficult to distinguish which components of the therapeutic regimens contribute to cardiotoxicity. Here, we addressed the influence of bortezomib on cardiac function in rats that were not treated with other drugs. Rats were treated with bortezomib at a dose of 0.2 mg/kg thrice weekly. Echocardiography, histopathology, and electron microscopy were used to evaluate cardiac function and structural changes. Respiration of the rat heart mitochondria was measured polarographically. Cell culture experiments were used to determine the influence of bortezomib on cardiomyocyte survival, contractility, Ca(2+) fluxes, induction of endoplasmic reticulum stress, and autophagy. Our findings indicate that bortezomib treatment leads to left ventricular contractile dysfunction manifested by a significant drop in left ventricle ejection fraction. Dramatic ultrastructural abnormalities of cardiomyocytes, especially within mitochondria, were accompanied by decreased ATP synthesis and decreased cardiomyocyte contractility. Monitoring of cardiac function in bortezomib-treated patients should be implemented to evaluate how frequently cardiotoxicity develops especially in patients with pre-existing cardiac conditions, as well as when using additional cardiotoxic drugs.

The Time Course of Tumor Necrosis Factor-α, Inducible Nitric Oxide Synthase and Vascular Endothelial Growth Factor Expression in an Experimental Model of Chronic Myocardial Infarction in Rats

An injury to the heart due to myocardial infarction may progress to heart failure. Among the cytokines and growth factors whose interactions promote remodeling of the heart, increased expression of tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF) has been found. However, little is known about the sequence of gene expression during the progression of heart injury. In the present study, male Sprague-Dawley rats were used for experimental myocardial infarction performed by ligation of the left anterior descending coronary artery. TNF-α, iNOS and VEGF expression was assessed by reverse transcription polymerase chain reaction. Localization of TNF-α, VEGF and iNOS protein was assessed by immunohistochemistry. An in vitro proliferation (BrdU incorporation) and differentiation (tube formation) assay of human umbilical vein endothelial cells was performed. The expression of TNF-α, iNOS, VEGF164 and VEGF188 was observed during the whole period after myocardial infarction (on days 1, 4, 11, 28 and 40), whereas VEGF120 was found only on day 1 and 4. The most intense immunostaining for TNF-α was observed at the border zone. The iNOS immunostaining was initially located in the endothelium, whereas later it was also present in the walls of larger vessels. The VEGF protein was present in the border zone. No gene expression or immunostaining was detected in sham-operated rats. The in vitro experiments showed both proangiogenic (low TNF-α concentration, short period of incubation) and antiangiogenic (high TNF-α concentration, long period of incubation) effects of TNF-α. The expression of TNF-α and iNOS genes with the concomitant occurrence of a decrease in VEGF120, VEGF188 and VEGF164 protein could be related to insufficient angiogenesis and may suggest the possible involvement of these events in remodeling after myocardial infarction.

Embryonic development of the proepicardium and coronary vessels

In the last few years, an increasing interest in progenitor cells has been noted. These cells are a source of undifferentiated elements from which cellular components of tissues and organs develop. Such progenitor tissue delivering stem cells for cardiac development is the proepicardium. The proepicardium is a transient organ which occurs near the venous pole of the embryonic heart and protrudes to the pericardial cavity. The proepicardium is a source of the epicardial epithelium delivering cellular components of vascular wall and interstitial tissue fibroblasts. It contributes partially to a fibrous tissue skeleton of the heart. Epicardial derived cells play also an inductive role in differentiation of cardiac myocytes into conductive tissue of the heart. Coronary vessel formation proceeds by vasculogenesis and angiogenesis. The first tubules are formed from blood islands which subsequently coalesce forming the primitive vascular plexus. Coronary arteries are formed by directional growth of vascular protrusions towards the aorta and establishing contact with the aortic wall. The coronary vascular wall matures by attaching smooth muscle cell precursors and fibroblast precursors to the endothelial cell wall. The cells of tunica media differentiate subsequently into vascular smooth muscle by acquiring specific contractile and cytoskeletal markers of smooth muscle cells in a proximal - distal direction. The coronary artery wall matures first before cardiac veins. Maturity of the vessel wall is demonstrated by the specific shape of the internal surface of the vascular wall.

Effects of heme oxygenase-1 on induction and development of chemically induced squamous cell carcinoma in mice

Heme oxygenase-1 (HO-1) is an antioxidative and cytoprotective enzyme, which may protect neoplastic cells against anticancer therapies, thereby promoting the progression of growing tumors. Our aim was to investigate the role of HO-1 in cancer induction. Experiments were performed in HO-1+/+, HO-1+/−, and HO-1−/− mice subjected to chemical induction of squamous cell carcinoma with 7,12-dimethylbenz[a]anthracene and phorbol 12-myristate 13-acetate. Measurements of cytoprotective genes in the livers evidenced systemic oxidative stress in the mice of all the HO-1 genotypes. Carcinogen-induced lesions appeared earlier in HO-1−/− and HO-1+/− than in wild-type animals. They also contained much higher concentrations of vascular endothelial growth factor and keratinocyte chemoattractant, but lower levels of tumor necrosis factor-α and interleukin-12. Furthermore, tumors grew much larger in HO-1 knockouts than in the other groups, which was accompanied by an increased rate of animal mortality. However, pathomorphological analysis indicated that HO-1−/− lesions were mainly large but benign papillomas. In contrast, in mice expressing HO-1, most lesions displayed dysplastic features and developed to invasive carcinoma. Thus, HO-1 may protect healthy tissues against carcinogen-induced injury, but in already growing tumors it seems to favor their progression toward more malignant forms.

Embryogenesis of the rat heart: the expression of collagenases.

Abstract Little is known about extracellular matrix (ECM) remodeling during heart development. Matrix degrading metalloproteinases are possible candidates contributing to degradation of ECM during these complex biological events. We described here different forms of MMPs, based on their substrate specificity, molecular weight, immunolocalization and in situ zymography within embryonic rat myocardium at different stages of heart development (from embryonic day – ED12 until ED 21). Murine collagenase-3 (MMP-13), stromelysin (MMP-3) and gelatinases A&B (MMP-2 & -9) were expressed in prenatal hearts, as demonstrated by quantitative zymography and immunohistochemistry. MMP-2, -3 and -9 were found within myocardium of avascular (ED12) and vascularized heart (ED14–21). An extensive immunolabeling over the heart trabeculae, epicardial tissue and a weaker labeling in the endocardial and truncoconal cushion tissue was observed at all stages of the heart development. Utilizing quantitative zymography we found that MMP-13 activity gradually increased from ED14–ED16 reaching a plateau from ED16–ED21, while MMP-2 activity demonstrated a transient increase starting at ED13, peaked at ED16 and declined thereafter. As to MMP-9 activity, it was seen only between ED16 and ED 18. In situ zymography with gelatin as a substrate represented activity of MMPs within the myocardium of the atria and the ventricles and a very strong activity in the interstitial tissue of the endocardial and the conotruncal cushion tissue. Conclusion MMPs expressed in embryonic heart correspond to all major classes of these enzymes. They may contribute to embryonic remodeling of the heart.

The anatomy of the cardiac veins in mice

Although the cardiac coronary system in mice has been the studied in detail by many research laboratories, knowledge of the cardiac veins remains poor. This is because of the difficulty in marking the venous system with a technique that would allow visualization of these large vessels with thin walls. Here we present the visualization of the coronary venous system by perfusion of latex dye through the right caudal vein. Latex injected intravenously does not penetrate into the capillary system. Murine cardiac veins consist of several principal branches (with large diameters), the distal parts of which are located in the subepicardium. We have described the major branches of the left atrial veins, the vein of the left ventricle, the caudal veins, the vein of the right ventricle and the conal veins forming the conal venous circle or the prepulmonary conal venous arch running around the conus of the right ventricle. The venous system of the heart drains the blood to the coronary sinus (the left cranial caval vein) to the right atrium or to the right cranial caval vein. Systemic veins such as the left cranial caval, the right cranial caval and the caudal vein open to the right atrium. Knowledge of cardiac vein location may help to elucidate abnormal vein patterns in certain genetic malformations.

Candidate bone-tissue-engineered product based on human-bone-derived cells and polyurethane scaffold.

Biodegradable polyurethanes (PURs) have recently been investigated as candidate materials for bone regenerative medicine. There are promising reports documenting the biocompatibility of selected PURs in vivo and the tolerance of certain cells toward PURs in vitro - potentially to be used as scaffolds for tissue-engineered products (TEPs). The aim of the present study was to take a step forward and create a TEP using human osteogenic cells and a polyurethane scaffold, and to evaluate the quality of the obtained TEP in vivo. Human-bone-derived cells (HBDCs) were seeded and cultured on polyurethane scaffolds in a bioreactor for 14 days. The TEP examination in vitro was based on the evaluation of cell number, cell phenotype and cell distribution within the scaffold. TEPs and control samples (scaffolds without cells) were implanted subcutaneously into SCID mice for 4 and 13 weeks. Explants harvested from the animals were examined using histological and immunohistochemical methods. They were also tested in mechanical trials. It was found that dynamic conditions for cell seeding and culture enable homogeneous distribution, maintaining the proliferative potential and osteogenic phenotype of the HBDCs cultured on polyurethane scaffolds. It was also found that HBDCs implanted as a component of TEP survived and kept their ability to produce the specific human bone extracellular matrix, which resulted in higher mechanical properties of the harvested explants when preseeded with HBDCs. The whole system, including the investigated PUR scaffold and the method of human cell seeding and culture, is recommended as a candidate bone TEP.

Differentiation of the smooth muscle cell phenotypes during embryonic development of coronary vessels in the rat

Abstract. Smooth muscle cell (SMC) maturation during embryonic development of coronary arteries and veins was studied in rats using different markers of the contractile phenotypes. The spatio-temporal pattern of distribution of these markers compared with the developing tunica media was examined. Alpha-smooth muscle actin (α-SMA) was the first marker of the SMC in the tunica media of coronary arteries found in ED16 hearts, followed by smooth muscle myosin heavy chain isoform which occurred on ED17. Subsequently 1E12 antigen was expressed in coronary artery wall in ED18 hearts, and finally smoothelin. The markers occur within the proximal part of the coronary arteries and deploy toward the apex. They are also found within the great vessels. None of the markers except for the α-SMA were found in coronary veins during embryonic life. We conclude that the SMC population of the developing tunica media of coronary vessels differentiates by the acquisition of particular markers and this process lasts till the end of the prenatal and early postnatal life.

Vascular endothelial growth factor is efficiently synthesized in spite of low transfection efficiency of pSG5VEGF plasmids in vascular smooth muscle cells.

The limitation of lipotransfection with plasmid vectors is its low efficiency and the short-term expression of introduced genes. This is particularly important when the synthesis of high amounts of therapeutic products is required. However, growth factors with paracrine action overcome this problem. The aim of our study was to check whether the amounts of vascular endothelial growth factor (VEGF) generated after plasmid lipotransfection into vascular smooth muscle cells (VSMC) can be sufficient to stimulate endothelial cell proliferation. Two plasmids, pSG5-VEGF121 and pSG5-VEGF165, harboring human VEGF121 and VEGF165 iso-forms were constructed and lipotransfected into COS-7 cells or to rat VSMC. The transfection efficiency, estimated by the expression of control, b-galactosidase gene, was about 50% in COS- 7 but rarely exceeded 5% in VSMC. However, despite this, the smooth muscle cells generated high amounts of VEGF protein, up to 3 ng/ml medium. The biological activity of this VEGF was confirmed by enhanced proliferation of human umbilical vein and coronary artery endothelial cells, stimulated with conditioned media of pSG5-VEGF transfected cells. Thus, the low transfection efficiency does not preclude the generation of high amounts of VEGF by VSMC. After reaching the maximum at about 48 h after transfection, the generation of VEGF decreased in the following days. Such a situation may be sufficient for the gene therapy of restenosis when the long-term expression of therapeutic gene(s) is not necessary. Thus, we suggest that the pSG5-VEGF121 and pSG5-VEGF165 plasmids can be used for therapeutic application.

Cellular phenotypes and spatio-temporal patterns of lymphatic vessel development in embryonic mouse hearts

Background: The origin of cardiac lymphatics from venous endothelial cells or from scattered lymphangioblasts has been discussed in the literature. We aimed to establish the stage when lymphatic vessels appear in the developing mouse heart, the location of the first lymphatics, and to define cellular phenotypes of growing lymphatics. Results: We found that scattered Lyve‐1‐positive cells located in the subepicardial area of developing heart expressed CD45, CD68, F4/80, or CD11b but not CD31. Prox‐1+/Lyve‐1+ cellular cords or vessels were found to invade 12.5–13.5‐dpc hearts via two routes: from the venous pole, i.e., dorsal atrioventricular sulcus, or on the dorsal atrial surface from mediastinum and from the arterial pole, i.e., along the great arteries. The Prox‐1+/Lyve‐1+ vessels were located among the Prox‐1+/Lyve‐1− cords and among the scattered Prox‐1−/Lyve‐1+ cells. The Prox‐1+/Lyve‐1− cellular cords/tubules dominate initially at the arterial pole whereas Lyve‐1+/Prox‐1− cellular cords/tubules dominate initially on the venous pole, i.e., dorsal atrioventricular sulcus. The Lyve‐1+/CD45+, Lyve‐1+/CD11b+, Lyve‐1+/F4/80+ and Lyve‐1+/CD68+ cells were subsequently found to be co‐opted to the wall of the developing lymphatic vessels while gaining Flk‐1. Conclusions: Lymphatic primordia exhibit different cellular phenotypes and different spatiotemporal pattern on the venous pole as compared with the arterial pole of the heart. Developmental Dynamics 241:1473–1486, 2012.