Patophysiology of atherosclerosis based on research on apoE-knockout mice and their usefulness in checking new antiatherosclerotic agents

Obecnie wiadomo, że miażdżyca jest chorobą o podłożu zapalnym. Długo brakowało jednoznacznego dowodu na istotny wpływ zapalenia na rozwój miażdżycy. Uzyskano go dzięki nowemu zwierzęcemu modelowi miażdżycy - myszy, u której zastosowano technikę celowania genowego (apoE-knockout mice). Kluczowym etapem rozwoju miażdżycy jest prezentacja limfocytom T antygenu przez komórki dendrytyczne. Antygenem tym może być fragment "strawionej" przez makrofag, utlenionej lipoproteiny o małej dogęstości (LDL), białko szoku cieplnego 60 (HSP60), β2 glikoproteina I lub fragmenty antygenów bakteryjnych. W wyniku oddziaływania tych komórek następuje odpowiedź immunologiczna typu T helper 1 (komórkowa) lub typu T helper 2 (humoralna). Obecnie uważa się, że odpowiedź typu Th1 i jej mediatory: interferon γ, czynnik martwicy nowotworów α, interleukina 1, interleukina 12 oraz interleukina 18 działają przyspieszająco na rozwój miażdżycy, podczas gdy odpowiedź typu Th2 i jej mediatory: interleukina 4, interleukina 5, interleukina 10 oraz interleukina 13 hamują rozwój miażdżycy. Koncepcja miażdżycy jako zapalenia ukształtowała się dopiero w ostatnich latach, lecz obecnie jej wartość jest niekwestionowana, co wiąże się z określonymi konsekwencjami terapeutycznymi. W ostatnich latach oczywiste stało się więc, że szlak produktów 5-lipooksygenazy może odgrywać istotną rolę patogenezie miażdżycy. Zaczęto rozważać leki przeciwleukotrienowe jako potencjalną terapię miażdżycy. W niniejszej pracy omówiono wyniki badań własnych przeprowadzonych na eksperymentalnym modelu miażdżycy: myszach z podwójnie wyłączonymi genami dla apolipoproteiny E oraz dla receptora LDL. Wykazano, że inhibitory białka aktywującego 5-lipooksygenazę (FLAP) oraz bloker receptora dla leukotrienów cysteinylowych zmniejszają miażdżycę u zmienionych genetycznie myszy.Although atherosclerosis was previously thought to be primarily a degenerative disease, it is now well ascertained that its pathogenesis is inflammatory. This review describes the history of the new atherogenetic concept, including the pivotal role of apoE-knockout mice in understanding the inflammatory background of atherosclerosis. The pivotal stage of atherogenesis is antigen presentation by macrophages to T lymphocytes. This antigen could be a fragment of oxidized LDL"digested" by macrophage, heat shock protein 60, β2 glycoprotein I, or fragments of bacterial antigens. During interaction between these cells, an immunological response of type T helper 1 (cellular) or T helper 2 (humoral) arises. Th1 response and its mediators: (IFN-γ, TNF-α, interleukin 1, interleukin 12, and interleukin 18) increase atherogenesis, whereas Th2 response and its mediators: (interleukin 4, interleukin 5, and interleukin 10) decrease the development of atherosclerosis. The concept of atherosclerosis as an inflammatory disease is quite fresh; however, it is already considered an undisputable achievement of science, bringing particular therapeutic consequences. Since inflammation plays an important role in atherogenesis, during recent years it has become apparent that the 5-lipoxygenase (5-LO) pathway may play an important role in modifying the pathogenesis of atherosclerosis. These data raised the possibility that antileukotriene drugs may be an effective treatment regimen in atherosclerosis. In fact, we have found that among apoE and LDLR-double knockout mice the inhibition of FLAP, as well as cysteinyl leukotriene receptor blockade, was able to significantly prevent the development of atherosclerosis in gene-targeted mice

Triple immunofluorescence labeling of atherosclerotic plaque components in apoE/LDLR ^{-/-} mice

This paper presents a simple and reliable method of triple immunofluorescence staining that allows simultaneous detection of various cell types present in atherosclerotic plaque of apolipoprotein E and LDL receptor-double knockout (apoE/LDLR -/-) mice. We used combined direct and indirect procedures applying commercially available primary antibodies raised in different species to detect smooth muscle cells (Cy3-conjugated mouse anti-smooth muscle actin, SMA), macrophages (rat anti-CD68) and T lymphocytes (rabbit anti-CD3). Fixation of the material in acetone and modified incubation protocol employing nonfat dry milk in preincubation and incubation media significantly increased the intensity of labeling and effectively quenched the background. Our method offers an efficient way to detect qualitative as well as quantitative changes of macrophages, T lymphocytes and smooth muscle cells in atherosclerotic plaque of apoE/LDLR -/- mice during atherosclerosis development or in response to pharmacological treatment

Triple immunofluorescence labeling of atherosclerotic plaque components in apoE/LDLR -/- mice.

This paper presents a simple and reliable method of triple immunofluorescence staining that allows simultaneous detection of various cell types present in atherosclerotic plaque of apolipoprotein E and LDL receptor-double knockout (apoE/LDLR -/-) mice. We used combined direct and indirect procedures applying commercially available primary antibodies raised in different species to detect smooth muscle cells (Cy3-conjugated mouse anti-smooth muscle actin, SMA), macrophages (rat anti-CD68) and T lymphocytes (rabbit anti-CD3). Fixation of the material in acetone and modified incubation protocol employing nonfat dry milk in preincubation and incubation media significantly increased the intensity of labeling and effectively quenched the background. Our method offers an efficient way to detect qualitative as well as quantitative changes of macrophages, T lymphocytes and smooth muscle cells in atherosclerotic plaque of apoE/LDLR -/- mice during atherosclerosis development or in response to pharmacological treatment

Exonic deletions of mismatch repair genes MLH1 and MSH2 correlate with prognosis and protein expression levels in malignant melanomas

The mutations of MLH1 and MSH2 have been reported to be responsible for malignant transformation and tumour progression in several sporadic tumours. Eighty-six primary malignant melanomas with known follow-up were investigated. Point mutations of DNA mismatch repair MLH1 and MSH2 in malignant melanomas were not found. Exon 12 (MSH2) was not present in 26 out of the 86 melanomas and exon 13 (MSH2) was lost in 25 of the tumours. The loss of exon 15 (MLH1) was observed in 22 out of the 86 tumours and the loss of exon 16 (MLH1) in 24 melanomas. The loss of exons correlated strongly with the loss of MLH1 and MSH2 protein expression. In multivariate analysis, including all 4 exons and expressions of MLH1 and MSH2, prognostic significance was found only for loss of exon 12 (MSH2) and loss of exon 15 (MLH1)

Inactivation of heparin by cationically modified chitosan

This study was performed to evaluate the ability of N-(2-hydroxypropyl)-3- tri methylammonium chitosan chloride (HTCC), the cationically modified chitosan, to form biologically inactive complexes with unfractionated heparin and thereby blocking its anticoagulant activity. Experiments were carried out in rats in vivo and in vitro using the activated partial thromboplastin time (APTT) and prothrombin time (PT) tests for evaluation of heparin anticoagulant activity. For the first time we have found that HTCC effectively neutralizes anticoagulant action of heparin in rat blood in vitro as well as in rats in vivo. The effect of HTCC on suppression of heparin activity is dose-dependent and its efficacy can be comparable to that of protamine-the only agent used in clinic for heparin neutralization. HTCC administered i.v. alone had no direct effect on any of the coagulation tests used. The potential adverse effects of HTCC were further explored using rat experimental model of acute toxicity. When administered i.p. at high doses (250 and 500 mg/kg body weight), HTCC induced some significant dose-dependent structural abnormalities in the liver. However, when HTCC was administered at low doses, comparable to those used for neutralization of anticoagulant effect of heparin, no histopathological abnormalities in liver were observed

The current state of knowledge on the prevention of venous thromboembolism in orthopaedic and trauma surgery — selected topics

Profilaktyka żylnej choroby zakrzepowo-zatorowej w ortopedii i traumatologii narządu ruchu należy do zagadnień najlepiej zbadanych i opracowanych w formie szczegółowych zaleceń i protokołów postępowania oraz do najszybciej rozwijających się w całym zakresie problematyki zakrzepowej. Najnowsze doniesienia naukowe dotyczące tego zagadnienia zmuszają do nieustannej modyfikacji podglądów oraz aktualizacji zasad jej stosowania. Zmiany te mogą być publikowane jako opracowania indywidualne lub prace zespołów badawczych powoływanych w tym celu przez stowarzyszenia naukowe. Takim przedsięwzięciem było wyodrębnienie interdyscyplinarnej grupy specjalistów, której celem było określenie aktualnego stanu wiedzy i opracowanie propozycji zaleceń dotyczących wybranych zagadnień z zakresu profilaktyki żylnej choroby zakrzepowo-zatorowej. Grupa ta działała pod auspicjami Prezesa Polskiego Towarzystwa Ortopedycznego i Traumatologicznego oraz Prezesa Polskiej Fundacji do Walki z Zakrzepicą THROMBOSIS. Wyniki prac zespołu zawarto w poniższym opracowaniu. Acta Angiol 2011; 17, 1: 1–36Prevention of venous thromboembolism in orthopaedic and trauma surgery is one of the best-studied topics, with respect to which the most detailed recommendations and management protocols have been drawn up. It is also the fastest-developing area of the study of thrombotic conditions. The most recent reports make us continually modify our views and update the principles of its use. These changes may be published by individual authors or by research teams appointed for this particular purpose by scientific societies. An example of such efforts was the appointment of a multidisciplinary group of specialists whose aim was to determine the current state of knowledge on the prevention of VTE and to propose recommendations on selected aspects of this topic. The group carried out its activities under the auspices of the Chair of the Polish Society of Orthopaedic and Trauma Surgeons and the Chair of the Polish Foundation Against Thrombosis. This paper presents the results of this team’s efforts. Acta Angiol 2011; 17, 1: 1–3